Note: Descriptions are shown in the official language in which they were submitted.
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ANTIBODIES TO M(H)DM2/4 AND THEIR USE IN DIAGNOSING AND TREATING
CANCER
1. CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of International Patent
Application No.
PCT/US2018/043908, filed July 26, 2018, which claims the benefit of U.S.
Provisional Patent
Application No. 62/537,914, filed July 27, 2017, which are incorporated herein
by reference in
their entireties.
2. REFERENCE TO SEOUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] This application incorporates by reference a Sequence Listing filed
with this
application as an ASCII text file entitled "14160-007-228 SEQ LISTING" created
on January
30, 2019 and having a size of 170,676 bytes.
3. FIELD
[0003] The present invention relates, inter alia, to certain anti-M(H)DM2/4
antibodies
(including chimeric and humanized antibodies) or antigen- binding fragments
thereof, pharmaceutical
compositions comprising anti-M(H)DM2/4 antibodies or antigen-binding fragments
thereof,
antibody-drug conjugates comprising anti-M(H)DM2/4 antibodies or antigen-
binding fragments
thereof bound to a cytotoxic drug, and the use of such antibodies, fragments,
compositions and
conjugates for treating cancer and/or for preventing metastases.
4. BACKGROUND
[0004] The MDM2 (MDM2 is a mouse homologue of HDM2) protein is composed of
489
amino acids and contains a p53 binding domain, two nuclear localization
signals (amino acids
176-182 and 464-471), and zinc-finger motifs (amino acids 297-326 and 436-477)
(see UniProt
website, at UniProt Accession No. P23804). Its human homologue, HDM2, is
composed of 491
amino acids and contains a p53 binding domain, two nuclear localization
signals (amino acids
179-185 and 466-473) and zinc-finger motifs (amino acids 299-328 and 438-479)
(see UniProt
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website, at UniProt Accession No. Q00987). Mouse protein MDM4 (also identified
as MDMX)
is a homologue of the MDM2 protein (see UniProt website, at UniProt Accession
No. 035618),
and both MDM2 and MDM4 are major negative regulators of p53 (Wade et al.,
2013, Nat Rev.
Cancer 13:83-96; Marine et al., 2004, Cell Cycle 3:900-904; Momand et al.,
2011, Gene 486:23-
30). HDM4 (also identified as HDMX) is a human homologue of MDM4 (see UniProt
website,
at UniProt Accession No. 015151). The most conserved domain within all M(H)DM2
and
M(H)DM4 proteins is the RING domain which is responsible for ubiquitination of
its target
proteins, including p53 protein, and heterodimerization between M(H)DM2 and
M(H)DM4.
M(H)DM4 is required for M(H)DM2-mediated polyubiquitination of p53. A
distinctive feature
of M(H)DM2 and M(H)DM4 are their very complex expression pattern. Multiple-
sized
transcripts and protein products of M(H)DM2 have been identified in cancer
cells by a number
of groups (Olson et al., 1993, Oncogene 8:2353-2360; Bartel et al., 2002,
Cancer Cell 2:9-15
("Bartel 2002"), Sigalas et al., 1996, Nat. Med. 2:912-917 ("Sigalas 1996"),
Iwakuma & Lozano,
2003, Mol. Cancer Res. 1:993-1000 ("Iwakuma & Lozano 2003")). Many types of
human
cancers overexpress MDM2 protein and a common characteristic among these
cancers is an
associated increase in mdm2 splice variants. These M(H)DM2 variants have been
shown to be
expressed in a variety of tumors such as human ovarian, bladder, breast and
astrocytic
neoplasms, glioblastomas, leukemia and pediatric Rhabdomyosarcoma tumors
(reviewed by
Iwakuma & Lozano 2003; Rosso et al., 2014, Subcell Biochem. 85:247-61 ("Rosso
2014")).
Most interestingly they have been found to be more frequent in tumors of
advanced stage (Bartel
2002). The multiple-sized M(H)DM2 transcripts that have been shown to be
splice variants
forms of the M(H)DM2 mRNA have been reported to be expressed more frequently
in tumor
cells than in normal cells (Bartel et al., 2004, Mol. Cancer Res. 2:29
("Bartel 2004")). It has been
proposed that a mRNA surveillance system exists in untransformed cells, which
degrades spliced
transcripts and protects the cells from errors of transcription, mRNA
processing, or mRNA
transport whereas in transformed cells this system may not be functioning
correctly (Bartel
2004). Moreover, some of these variants in cancer cells encode protein
products, which have
been shown to transform NIH3T3 cells in vitro, and some promoted tumor
formation in mouse
model (Sigalas 1996; Volk el. al., 2009, Mol Cancer Res. 7(6): 863-869).
[0005] To date more than 70 different M(H)DM2 splice variants have been
identified (Bartel
2002; Bartel 2004; Rosso 2014). Some of the variants, for example MDM2-A and
MDM2-B, are
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common to several tumor types (Sigalas 1996). Others have only been found in
specific tumors,
for example MDM2-FB25 and MDM2-FB26 in pediatric rhabdomyosarcoma. Several
short
forms of these alternatively spliced MDM2 transcripts correlate with high-
grade malignancy in
human ovarian tumors, bladder carcinomas and astrocytic tumors (Sigalas 1996;
Matsumoto et
al., 1998, Cancer Res. 58:609-613; Tamborini et al., 2001, Int. J. Cancer
92:790-796; Steinman
et al., 2004, JBC 279:4877-4886). It has also been shown that aberrant mdm2
(281-, 254-, and
219-bp) and alternative mdm2 (653-bp) splice products strongly associated with
shorter overall
patient survival in breast cancer (Lukas et al., 2001, Cancer Res. 61:3212).
[0006] Several studies have evaluated the cellular localization of various
human and murine
M(H)DM2 protein variants to predict potential activities. In 25% of non-small
cell lung
carcinomas, M(H)DM2 variants were aberrantly localized to the cytoplasm (Evans
et al., 2001,
Oncogene 20:4041-4049). This result led to the discovery that the cytoplasmic
compartmentalization of the full-length M(H)DM2 was due to binding and
sequestration by an
alternative-spliced M(H)DM2 product (HDM2ALT1). In another study, MDM2-D150-
230
localized to the cytoplasm of U205 cells (Schuster et al., 2007, Mol. Cancer
Res. 5:403-412
("Schuster 2007")). Both of these M(H)DM2 protein variants lacked part of the
NH2-terminal
region that contains a nuclear localization signal (NLS), suggesting that loss
of this signal
prevented the nuclear entry of these two proteins (Schuster 2007). Taken
together, these data
show that cellular localization of M(H)DM2 protein variants is highly complex.
[0007] Various M(H)DM4 protein variants have also been characterized,
including splicing
variant MDMX-S (Lenos and Jochemsen, 2011, J. Biomed Biotechnol.,
doi:10.1155/2011/876173).
[0008] HDM2 was found to be expressed in the plasma membrane of cancer
cells (Sarafraz-
Yazdi et al., 2010, PNAS 107:1918-1923 ("Sarafraz-Yazdi 2010"). Further, anti-
cancer
peptides, PNC-27 and PNC-28, which bind to HDM2 expressed in the cancer cell
membranes
and kill cancer cells by inducing necrosis, have been developed (Sarafraz-
Yazdi 2010; Davitt et
al., 2014, Annals Clin. Lab. Sci. 44:241248) (the amino acid sequences of PNC-
27 and PNC-28
are provided in Table I of U.S. Patent Application Publication No.
2012/0177566). PNC-27 has
been reported to bind within amino acids 25-109 of HDM2 (Do et al., 2003,
Oncogene
22(10):1431-1444 ("Do 2003"); Chene, 2003, Nat. Rev. Cancer 3(2):102-109).
Also, U.S.
Patent Application Publication No. 2012/0177566 discloses methods of
selectively necrosing
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cells by administering to the cells a compound (such as PNC-27 and PNC-28),
including an
HDM-2 targeting component and a cytotoxic component attached to the HDM-2
targeting
component, wherein said compound comprises a membrane-active form. The
membrane active
function of PNC-27 and PNC-28 peptides (which comprise a membrane resident
peptide
("MRP") and a p53 sequence) is only achieved if the cargo (i.e., the p53
sequence component) is
attached to the MRP component so as to form a cytotoxic structure (Kanovsky et
al, 2001, PNAS
98:12438- 12443 ("Kanovsky 2001"); Bowne et al., 2008, Ann Surg Oncol. 15:3588-
3600
"Bowne 2008")). When either the MRP component or the HDM-2 targeting component
(i.e. p53
component) were used separately, they were found to be non-cytotoxic to cancer
cells (Kanovsky
2001; Do 2003), demonstrating lack of activity of the MRP component alone and
the HDM-2
targeting component alone. When recombinantly expressed inside the cell, the
HDM-2 targeting
component was observed to cause apoptosis (Bowne 2008). U.S. Patent No.
9,765,117 discloses
HDM2 targeting peptides and fusion peptides comprising an HDM2 targeting
peptide and a
transmembrane penetrating sequence, such as MRP; it is disclosed that MRP is
required for
induction of cell necrosis (see col. 4, lines 27-28). U.S. Patent No.
9,765,117 indicates that
expression of the p53 HDM2 targeting sequence in the absence of the MRP in
cancer cells
causes p53-dependent apoptosis and not tumor necrosis (see col. 4, lines 29-
32).
[0009] The cell-penetrating peptides ("CPPs") (such as MRPs, Membrane
Transduction
Domain of Antennapedia, trans-activating transcriptional activator ("TAT") and
Penetratin
peptides) enable cellular membrane delivery of the peptides, and molecules
attached to the
peptides, to plasma membrane lipid bilayers, including those of normal healthy
cells. These
peptides were shown to efficiently transport various biologically active
molecules inside living
cells (Bechara et al., 2013, FEBS Lett. 587:1693-1702, Dupont et al., 2015,
Methods Mol. Biol.
1324:29-37). The use of these CPPs (such as MRPs, Membrane Transduction Domain
of
Antennapedia, TAT and Penetratin peptides) linked to various cargos (such as
other peptides,
DNA, RNA, small molecules, antibodies or fragments thereof) have been
demonstrated to
improve pharmacokinetics, bio-distribution, retention, uptake and delivery of
these cargos to
various tumors in vitro and in vivo (Torchilin et al., 2003, PNAS 100:1972-
1977; Shin et al.,
2014, J. Biomed. Mater Res. A. 102:575-587; Kleemann et al., 2005, J. Control
Release 109:299-
316; Olson et al., 2009, Integr. Biol (Camb) 1:382-393; Jain et al., 2005,
Cancer Res. 65:7840-
7846; William et al., 2002, PNAS 99:10423-10428; Bolhassani, 2011, Biochim
Biophys. Acta
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1816:232-246). While most of these CPPs are used as cargo-delivery entities,
Penetratin and
MRP peptides have been reported to form a unique cytotoxic membrane-active
structure upon
linkage to their cargo (Rosal et al., 2005, Adv Drug Deliv Rev 57:653-60
("Rosal 2005"), Bowne
2008; Kanovsky 2001). PNC-27 and PNC-28 peptides are examples of Penetratin-
/MRP-cargo
conjugates that exhibit a cytotoxic function that is dependent on the
attachment and linkage of
their cargo to the MRP, which is required for the formation of their membrane
active structure,
and hence, cytotoxic function (Kanovsky 2001; Rosal 2005; Bowne 2008).
[0010] There is long-standing unmet need in the art to effectively treat
cancer, such as
metastatic cancer, and to prevent metastasis in patients.
5. SUMMARY OF THE INVENTION
[0011] In one aspect, described herein is an antibody or a fragment thereof
that specifically
binds to an extracellularly accessible epitope of M(H)DM2/4. In particular,
described herein are
antibodies or fragments thereof that specifically bind to an extracellularly
accessible epitope of
M(H)DM2/4, wherein said antibodies or fragments inhibit tumor growth in vivo
(or inhibit tumor
cell proliferation in vivo). In certain embodiments, the antibodies or
fragments described herein
are not bound to a cell-penetrating peptide (e.g., a membrane resident
peptide). In certain
embodiments, the antibodies or fragments described herein are not bound to a
cytotoxic
component (i.e., not bound to a cytotoxic agent).
[0012] In one aspect, described herein are antibodies or fragments thereof
that specifically
bind to an extracellularly accessible epitope of M(H)DM2/4, wherein the
antibody or fragment
specifically binds to a peptide, wherein the sequence of the peptide consists
of
MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or
CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In particular, described herein are
antibodies or
fragments that specifically bind to M(H)DM2/4 (e.g., HDM2), wherein the
antibody or fragment
specifically binds to an extracellularly accessible epitope of M(H)DM2/4,
wherein the antibody
or fragment specifically binds to a peptide, wherein the sequence of the
peptide consists of
MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or
CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In one embodiment, the antibody or fragment
binds
to an extracellularly accessible epitope of M(H)DM2/4, wherein the antibody or
fragment
specifically binds to a peptide, wherein the sequence of the peptide is
MCNTNMSVPTDGAVT
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(SEQ ID NO:1). In one embodiment, the antibody or fragment binds to an
extracellularly
accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically
binds to a
peptide, wherein the sequence of the peptide is TTSQIPASEQE (SEQ ID NO:2). In
one
embodiment, the antibody or fragment binds to an extracellularly accessible
epitope of
M(H)DM2/4, wherein the antibody or fragment specifically binds to a peptide,
wherein the
sequence of the peptide is CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In certain
embodiments,
described herein is an antibody or a fragment thereof that specifically binds
to an extracellularly
accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:1, SEQ
ID NO:2,
or SEQ ID NO:3. In one embodiment, described herein is an antibody or a
fragment thereof that
specifically binds to an extracellularly accessible epitope of M(H)DM2/4
contained within a
peptide of SEQ ID NO: 1. In one embodiment, described herein is an antibody or
a fragment
thereof that specifically binds to an extracellularly accessible epitope of
M(H)DM2/4 contained
within a peptide of SEQ ID NO:2. In one embodiment, described herein is an
antibody or a
fragment thereof that specifically binds to an extracellularly accessible
epitope of M(H)DM2/4
contained within a peptide of SEQ ID NO:3.
[0013] In
one aspect, described herein is a humanized antibody or a fragment thereof
that
specifically binds to HDM2, said antibody or fragment comprising: (i) a heavy
chain variable
region (VH) comprising VH complementarity determining region ("CDR") 1, VH CDR
2, and
VH CDR3; said VH CDR 1, VH CDR 2 and VH CDR 3 being the CDRs of a VH that has
an
amino acid sequence selected from the group consisting of SEQ ID NO:36, SEQ ID
NO:38, and
SED ID NO:40, or (ii) a light chain variable region (VL) comprising VL CDR 1,
VL CDR 2 and
VL CDR 3 being the CDRs of a VL that has an amino acid sequence selected from
the group
consisting of SEQ ID NO:37, SEQ ID NO:39, and SEQ ID NO:41. In one embodiment,
the
humanized antibody or a fragment that specifically binds to HDM2 comprises a
VH wherein VH
CDR 1, VH CDR 2 and VH CDR 3 are of a VH having the amino acid sequence of SEQ
ID
NO:36. In one embodiment, the humanized antibody or a fragment that
specifically binds to
HDM2 comprises a VH wherein VH CDR 1, VH CDR 2 and VH CDR 3 are of a VH having
the
amino acid sequence of SEQ ID NO:38. In one embodiment, the humanized antibody
or a
fragment that specifically binds to HDM2 comprises a VH wherein VH CDR 1, VH
CDR 2 and
VH CDR 3 are of a VH having the amino acid sequence of SEQ ID NO:40. In one
embodiment,
the humanized antibody or a fragment that specifically binds to HDM2 comprises
a VL wherein
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VL CDR 1, VL CDR 2 and VL CDR 3 are of a VL having the amino acid sequence of
SEQ ID
NO:37 (and, optionally, a VH wherein VH CDR 1, VH CDR 2 and VH CDR 3 are of a
VH
having the amino acid sequence of SEQ ID NO:36). In one embodiment, the
humanized
antibody or a fragment that specifically binds to HDM2 comprises a VL wherein
VL CDR 1, VL
CDR 2 and VL CDR 3 are of a VL having the amino acid sequence of SEQ ID NO:39
(and,
optionally, a VH wherein VH CDR 1, VH CDR 2 and VH CDR 3 are of a VH having
the amino
acid sequence of SEQ ID NO:38). In one embodiment, the humanized antibody or a
fragment
that specifically binds to HDM2 comprises a VL wherein VL CDR 1, VL CDR 2 and
VL CDR 3
are of a VL having the amino acid sequence of SEQ ID NO:41 (and, optionally,
the a VH
wherein VH CDR 1, VH CDR 2 and VH CDR 3 are of a VH having the amino acid
sequence of
SEQ ID NO:40).
[0014] In one aspect, described herein is an antibody or a fragment thereof
that specifically
binds to M(H)DM2/4,said antibody or fragment comprising a heavy chain variable
region (VH)
comprising VH complementarity determining region ("CDR") 1, VH CDR 2, and VH
CDR 3,
wherein:
(i) the VH CDR 1 has the amino acid sequence GFTFTHY (SEQ ID NO:18), the VH
CDR 2 has
the amino acid sequence RNKAKGYT (SEQ ID NO:19), and the VH CDR 3 has the
amino acid
sequence DIGDN (SEQ ID NO:20);
(ii) the VH CDR 1 has the amino acid sequence GFTFTHYYMS (SEQ ID NO:42), the
VH
CDR 2 has the amino acid sequence FIRNKAKGYTAE (SEQ ID NO:45), and the VH CDR
3
has the amino acid sequence DIGDN (SEQ ID NO:20);
(iii) the VH CDR 1 has the amino acid sequence HYYMS (SEQ ID NO:43), the VH
CDR 2 has
the amino acid sequence FIRNKAKGYTAEYSASVKG (SEQ ID NO:46), and the VH CDR 3
has the amino acid sequence DIGDN (SEQ ID NO:20);
(iv) the VH CDR 1 has the amino acid sequence THYYMS (SEQ ID NO:44), the VH
CDR 2 has
the amino acid sequence WLGFIRNKAKGYTAE (SEQ ID NO:47), and the VH CDR 3 has
the
amino acid sequence ARDIGD (SEQ ID NO:48); or
(v) the VH CDR 1 has the amino acid sequence FTFTHYY (SEQ ID NO:144), the VH
CDR 2
has the amino acid sequence IRNKAKGYTA (SEQ ID NO:145), and the VH CDR 3 has
the
amino acid sequence ARDIGDN (SEQ ID NO:146).
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[0015] In one aspect, described herein is an antibody or a fragment thereof
that specifically
binds to M(H)DM2/4 (e.g., HDM2), said antibody or fragment comprising a heavy
chain
variable region (VH) comprising VH complementarity determining region ("CDR")
1, VH CDR
2, and VH CDR 3, wherein:
(i) the VH CDR 1 has the amino acid sequence GDTLSGS (SEQ ID NO:24), the VH
CDR 2 has
the amino acid sequence HLNRGT (SEQ ID NO:25), and the VH CDR 3 has the amino
acid
sequence SPGFAY (SEQ ID NO:26);
(ii) the VH CDR 1 has the amino acid sequence GDTLSGSWIVIR (SEQ ID NO:52), the
VH
CDR 2 has the amino acid sequence EIHLNRGTTN (SEQ ID NO:55), and the VH CDR 3
has
the amino acid sequence SPGFAY (SEQ ID NO:26);
(iii) the VH CDR 1 has the amino acid sequence GSWM1-1 (SEQ ID NO:53), the VH
CDR 2 has
the amino acid sequence EIHLNRGTTNYNEKFKG (SEQ ID NO:56), and the VH CDR 3 has
the amino acid sequence SPGFAY (SEQ ID NO:26);
(iv) the VH CDR 1 has the amino acid sequence SGSWMH (SEQ ID NO:54), the VH
CDR 2
has the amino acid sequence WIGEIHLNRGTTN (SEQ ID NO:57), and the VH CDR 3 has
the
amino acid sequence ARSPGFA (SEQ ID NO:58); or
(v) the VH CDR 1 has the amino acid sequence GDTLSGSW (SEQ ID NO:148), the VH
CDR 2
has the amino acid sequence IHLNRGTT (SEQ ID NO:143), and the VH CDR 3 has the
amino
acid sequence ARSPGFA (SEQ ID NO:58).
[0016] In one aspect, provided herein is an antibody or a fragment thereof
that specifically
binds to M(H)DM2/4 (e.g., HDM2), said antibody or fragment comprising a heavy
chain
variable region (VH) comprising VH complementarity determining region ("CDR")
1, VH CDR
2, and VH CDR 3, wherein:
(i) the VH CDR 1 has the amino acid sequence GYTFTSY (SEQ ID NO:30), the VH
CDR 2 has
the amino acid sequence NPRNGG (SEQ ID NO:31), and the VH CDR 3 has the amino
acid
sequence SGYYAMDY (SEQ ID NO:32);
(ii) the VH CDR 1 has the amino acid sequence GYTFTSYYMY (SEQ ID NO:62), the
VH CDR
2 has the amino acid sequence GINPRNGGTN (SEQ ID NO:65), and the VH CDR 3 has
the
amino acid sequence SGYYAMDY (SEQ ID NO:32);
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(iii) the VH CDR 1 has the amino acid sequence SYYMY (SEQ ID NO:63), the VH
CDR 2 has
the amino acid sequence GINPRNGGTNFNEKFKN (SEQ ID NO:66), and the VH CDR 3 has
the amino acid sequence SGYYAMDY (SEQ ID NO:32); or
(iv) the VH CDR 1 has the amino acid sequence TSYYMY (SEQ ID NO:64), the VH
CDR 2 has
the amino acid sequence WIGGINPRNGGTN (SEQ ID NO:67), and the VH CDR 3 has the
amino acid sequence TRSGYYAMD (SEQ ID NO:68)..
[0017] In
one aspect, described herein is a humanized antibody or a fragment thereof
that
specifically binds to HDM2, said antibody or fragment comprising a heavy chain
variable region
(VH) comprising VH complementarity determining region ("CDR") 1, VH CDR 2, and
VH
CDR 3, wherein:
(i) the VH CDR 1 has the amino acid sequence GFTFTHY (SEQ ID NO:18), the VH
CDR 2 has
the amino acid sequence RNKAKGYT (SEQ ID NO:19), and the VH CDR 3 has the
amino acid
sequence DIGDN (SEQ ID NO:20);
(ii) the VH CDR 1 has the amino acid sequence GFTFTHYYMS (SEQ ID NO:42), the
VH
CDR 2 has the amino acid sequence FIRNKAKGYTAE (SEQ ID NO:45), and the VH CDR
3
has the amino acid sequence DIGDN (SEQ ID NO:20);
(iii) the VH CDR 1 has the amino acid sequence HYYMS (SEQ ID NO:43), the VH
CDR 2 has
the amino acid sequence FIRNKAKGYTAEYSASVKG (SEQ ID NO:46), and the VH CDR 3
has the amino acid sequence DIGDN (SEQ ID NO:20);
(iv) the VH CDR 1 has the amino acid sequence THYYMS (SEQ ID NO:44), the VH
CDR 2 has
the amino acid sequence WLGFIRNKAKGYTAE (SEQ ID NO:47), and the VH CDR 3 has
the
amino acid sequence ARDIGD (SEQ ID NO:48); or
(v) the VH CDR 1 has the amino acid sequence FTFTHYY (SEQ ID NO:144), the VH
CDR 2
has the amino acid sequence IRNKAKGYTA (SEQ ID NO:145), and the VH CDR 3 has
the
amino acid sequence ARDIGDN (SEQ ID NO:146).
[0018] In
one aspect, described herein is a humanized antibody or a fragment thereof
that
specifically binds to HDM2, said antibody or fragment comprising a VH, wherein
the VH
comprises VH CDR1, VH CDR2, and VH CDR 3, wherein:
(i) the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20);
(ii) the VH CDR 3 has the amino acid sequence ARDIGD (SEQ ID NO:48); or
(iii) the VH CDR 3 has the amino acid sequence ARDIGDN (SEQ ID NO:146).
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[0019] In one aspect, described herein is a humanized antibody or a
fragment thereof that
specifically binds to HDM2, said antibody or fragment comprising a light chain
variable region
(VL) comprising VL complementarity determining region ("CDR") 1, VL CDR 2, and
VL CDR
3, wherein:
(i) the VL CDR 1 has the amino acid sequence RSSKNLLHSNGITYLY (SEQ ID NO:21),
the
VL CDR 2 has the amino acid sequence RVSNLAS (SEQ ID NO:22), and the VL CDR 3
has
the amino acid sequence AQLLELPYT (SEQ ID NO:23);
(ii) the VL CDR 1 has the amino acid sequence LHSNGITYLYWY (SEQ ID NO:49), the
VL
CDR 2 has the amino acid sequence LLISRVSNLA (SEQ ID NO:50), and the VL CDR 3
has the
amino acid sequence AQLLELPY (SEQ ID NO:51); or
[0020] (iii) the VL CDR 1 has the amino acid sequence KNLLHSNGITY (SEQ ID
NO:147),
the VL CDR 2 has the amino acid sequence RVS, and the VL CDR 3 has the amino
acid
sequence AQLLELPYT (SEQ ID NO:23),In one aspect, described herein is a
humanized
antibody or a fragment thereof that specifically binds to HDM2, said antibody
or fragment
comprising a light chain variable region (VL) comprising VL complementarity
determining
region ("CDR") 1, VL CDR 2, and VL CDR 3, wherein:
(i) the VL CDR 1 has the amino acid sequence RSSKNLLHSNGITYLY (SEQ ID NO:21),
the
VL CDR 2 has the amino acid sequence RVSNRAS (SEQ ID NO:236), and the VL CDR 3
has
the amino acid sequence AQLLELPYT (SEQ ID NO:23); or
(ii) the VL CDR 1 has the amino acid sequence LHSNGITYLYWY (SEQ ID NO:49), the
VL
CDR 2 has the amino acid sequence LLISRVSNRAS (SEQ ID NO:237), and the VL CDR
3 has
the amino acid sequence AQLLELPY (SEQ ID NO:51).
[0021] In one aspect, described herein is an antibody or a fragment
comprising:
(a) a light chain variable region (VL) comprising VL complementarity
determining region
("CDR") 1, VL CDR 2, and VL CDR 3, wherein:
(i) the VL CDR 1 has the amino acid sequence RSSKNLLHSNGITYLY (SEQ ID NO:21),
the
VL CDR 2 has the amino acid sequence RVSNRAS (SEQ ID NO:236), and the VL CDR 3
has
the amino acid sequence AQLLELPYT (SEQ ID NO:23); or
(ii) the VL CDR 1 has the amino acid sequence LHSNGITYLYWY (SEQ ID NO:49), the
VL
CDR 2 has the amino acid sequence LLISRVSNRAS (SEQ ID NO:237), and the VL CDR
3 has
the amino acid sequence AQLLELPY (SEQ ID NO:51); and
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(b) a heavy chain variable region (VH) comprising VH complementarity
determining region
("CDR") 1, VH CDR 2, and VH CDR 3, wherein:
(i) the VH CDR 1 has the amino acid sequence GFTFTHY (SEQ ID NO:18), the VH
CDR 2 has
the amino acid sequence RNKAKGYT (SEQ ID NO:19), and the VH CDR 3 has the
amino acid
sequence DIGDN (SEQ ID NO:20);
(ii) the VH CDR 1 has the amino acid sequence GFTFTHYYMS (SEQ ID NO:42), the
VH
CDR 2 has the amino acid sequence FIRNKAKGYTAE (SEQ ID NO:45), and the VH CDR
3
has the amino acid sequence DIGDN (SEQ ID NO:20);
(iii) the VH CDR 1 has the amino acid sequence HYYMS (SEQ ID NO:43), the VH
CDR 2 has
the amino acid sequence FIRNKAKGYTAEYSASVKG (SEQ ID NO:46), and the VH CDR 3
has the amino acid sequence DIGDN (SEQ ID NO:20);
(iv) the VH CDR 1 has the amino acid sequence THYYMS (SEQ ID NO:44), the VH
CDR 2 has
the amino acid sequence WLGFIRNKAKGYTAE (SEQ ID NO:47), and the VH CDR 3 has
the
amino acid sequence ARDIGD (SEQ ID NO:48); or
(v) the VH CDR 1 has the amino acid sequence FTFTHYY (SEQ ID NO:144), the VH
CDR 2
has the amino acid sequence IRNKAKGYTA (SEQ ID NO:145), and the VH CDR 3 has
the
amino acid sequence ARDIGDN (SEQ ID NO:146).
[0022] In
one aspect, described herein is a humanized antibody or a fragment thereof
that
specifically binds to HDM2, said antibody or fragment comprising:
(a) a heavy chain variable region (VH) comprising VH complementarity
determining region
("CDR") 1, VH CDR 2, and VH CDR 3, wherein:
(i) the VH CDR 1 has the amino acid sequence GFTFTHY (SEQ ID NO:18), the VH
CDR 2 has
the amino acid sequence RNKAKGYT (SEQ ID NO:19), and the VH CDR 3 has the
amino acid
sequence DIGDN (SEQ ID NO:20);
(ii) the VH CDR 1 has the amino acid sequence GFTFTHYYMS (SEQ ID NO:42), the
VH
CDR 2 has the amino acid sequence FIRNKAKGYTAE (SEQ ID NO:45), and the VH CDR
3
has the amino acid sequence DIGDN (SEQ ID NO:20);
(iii) the VH CDR 1 has the amino acid sequence HYYMS (SEQ ID NO:43), the VH
CDR 2 has
the amino acid sequence FIRNKAKGYTAEYSASVKG (SEQ ID NO:46), and the VH CDR 3
has the amino acid sequence DIGDN (SEQ ID NO:20);
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(iv) the VH CDR 1 has the amino acid sequence THYYMS (SEQ ID NO:44), the VH
CDR 2 has
the amino acid sequence WLGFIRNKAKGYTAE (SEQ ID NO:47), and the VH CDR 3 has
the
amino acid sequence ARDIGD (SEQ ID NO:48); or
(v) the VH CDR 1 has the amino acid sequence FTFTHYY (SEQ ID NO:144), the VH
CDR 2
has the amino acid sequence IRNKAKGYTA (SEQ ID NO:145), and the VH CDR 3 has
the
amino acid sequence ARDIGDN (SEQ ID NO:146); and
(b) a light chain variable region (VL) comprising VL complementarity
determining region
("CDR") 1, VL CDR 2, and VL CDR 3, wherein:
(i) the VL CDR 1 has the amino acid sequence RSSKNLLHSNGITYLY (SEQ ID NO:21),
the
VL CDR 2 has the amino acid sequence RVSNLAS (SEQ ID NO:22), and the VL CDR 3
has
the amino acid sequence AQLLELPYT (SEQ ID NO:23);
(ii) the VL CDR 1 has the amino acid sequence LHSNGITYLYWY (SEQ ID NO:49), the
VL
CDR 2 has the amino acid sequence LLISRVSNLA (SEQ ID NO:50), and the VL CDR 3
has the
amino acid sequence AQLLELPY (SEQ ID NO:51); or
(iii) the VL CDR 1 has the amino acid sequence KNLLHSNGITY (SEQ ID NO:147),
the VL
CDR 2 has the amino acid sequence RVS, and the VL CDR 3 has the amino acid
sequence
AQLLELPYT (SEQ ID NO:23).
[0023] In one aspect, described herein is an antibody or a fragment thereof
that specifically
binds to HDM2, said antibody or fragment comprising a heavy chain variable
region (VH)
comprising VH complementarity determining region ("CDR") 1, VH CDR 2, and VH
CDR 3,
wherein:
(i) the VH CDR 1 has the amino acid sequence GDTLSGS (SEQ ID NO:24), the VH
CDR 2 has
the amino acid sequence HLNRGT (SEQ ID NO:25), and the VH CDR 3 has the amino
acid
sequence SPGFAY (SEQ ID NO:26);
(ii) the VH CDR 1 has the amino acid sequence GDTLSGSWMH (SEQ ID NO:52), the
VH
CDR 2 has the amino acid sequence EIHLNRGTTN (SEQ ID NO:55), and the VH CDR 3
has
the amino acid sequence SPGFAY (SEQ ID NO:26);
(iii) the VH CDR 1 has the amino acid sequence GSWMH (SEQ ID NO:53), the VH
CDR 2 has
the amino acid sequence EIHLNRGTTNYNEKFKG (SEQ ID NO:56), and the VH CDR 3 has
the amino acid sequence SPGFAY (SEQ ID NO:26);
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(iv) the VH CDR 1 has the amino acid sequence SGSWMH (SEQ ID NO:54), the VH
CDR 2
has the amino acid sequence WIGEIHLNRGTTN (SEQ ID NO:57), and the VH CDR 3 has
the
amino acid sequence ARSPGFA (SEQ ID NO:58); or
(v) the VH CDR 1 has the amino acid sequence GDTLSGSW (SEQ ID NO:148), the VH
CDR 2
has the amino acid sequence IHLNRGTT (SEQ ID NO:143), and the VH CDR 3 has the
amino
acid sequence ARSPGFA (SEQ ID NO:58).
[0024] In one aspect, described herein is an antibody or a fragment thereof
that specifically
binds to HDM2, said antibody or fragment comprising a VH, wherein the VH
comprises VH
CDR1, VH CDR2, and VH CDR 3, wherein:
(i) the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO:26); or
(ii) the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO:58).
[0025] In one aspect, described herein is an antibody or a fragment thereof
that specifically
binds to HDM2, said antibody or fragment comprising a light chain variable
region (VL)
comprising a VL complementarity determining region ("CDR") 1, VL CDR 2, and VL
CDR 3,
wherein:
(i) the VL CDR 1 has the amino acid sequence RSSKSLLHSNGNSYLY (SEQ ID NO:27),
the
VL CDR 2 has the amino acid sequence RMSNLAS (SEQ ID NO:28), and the VL CDR 3
has
the amino acid sequence MQHLEYPFT (SEQ ID NO:29);
(ii) the VL CDR 1 has the amino acid sequence LHSNGNSYLYWF (SEQ ID NO:59), the
VL
CDR 2 has the amino acid sequence LLIYRMSNLA (SEQ ID NO:60), and the VL CDR 3
has
the amino acid sequence MQHLEYPF (SEQ ID NO:61); or
(iii) the VL CDR 1 has the amino acid sequence KSLLHSNGNSY (SEQ ID NO:141),
the VL
CDR 2 has the amino acid sequence RMS, and the VL CDR 3 has the amino acid
sequence
MQHLEYPFT (SEQ ID NO:29).
[0026] In one aspect, described herein is an antibody or a fragment thereof
that specifically
binds to HDM2, said antibody or fragment comprising:
(a) a heavy chain variable region (VH) comprising VH complementarity
determining region
("CDR") 1, VH CDR 2, and VH CDR 3, wherein:
(i) the VH CDR 1 has the amino acid sequence GDTLSGS (SEQ ID NO:24), the VH
CDR 2 has
the amino acid sequence HLNRGT (SEQ ID NO:25), and the VH CDR 3 has the amino
acid
sequence SPGFAY (SEQ ID NO:26);
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(ii) the VH CDR 1 has the amino acid sequence GDTLSGSWIVIE1 (SEQ ID NO:52),
the VH
CDR 2 has the amino acid sequence EIHLNRGTTN (SEQ ID NO:55), and the VH CDR 3
has
the amino acid sequence SPGFAY (SEQ ID NO:26);
(iii) the VH CDR 1 has the amino acid sequence GSWMH (SEQ ID NO:53), the VH
CDR 2 has
the amino acid sequence EIHLNRGTTNYNEKFKG (SEQ ID NO:56), and the VH CDR 3 has
the amino acid sequence SPGFAY (SEQ ID NO:26);
(iv) the VH CDR 1 has the amino acid sequence SGSWMH (SEQ ID NO:54), the VH
CDR 2
has the amino acid sequence WIGEIHLNRGTTN (SEQ ID NO:57), and the VH CDR 3 has
the
amino acid sequence ARSPGFA (SEQ ID NO:58); or
(v) the VH CDR 1 has the amino acid sequence GDTLSGSW (SEQ ID NO:148), the VH
CDR 2
has the amino acid sequence IHLNRGTT (SEQ ID NO:143), and the VH CDR 3 has the
amino
acid sequence ARSPGFA (SEQ ID NO:58); and
(b) a light chain variable region (VL) comprising VL complementarity
determining region
("CDR") 1, VL CDR 2, and VL CDR 3, wherein:
(i) the VL CDR 1 has the amino acid sequence RSSKSLLHSNGNSYLY (SEQ ID NO:27),
the
VL CDR 2 has the amino acid sequence RMSNLAS (SEQ ID NO:28), and the VL CDR 3
has
the amino acid sequence MQHLEYPFT (SEQ ID NO:29);
(ii) the VL CDR 1 has the amino acid sequence LHSNGNSYLYWF (SEQ ID NO:59), the
VL
CDR 2 has the amino acid sequence LLIYRMSNLA (SEQ ID NO:60), and the VL CDR 3
has
the amino acid sequence MQHLEYPF (SEQ ID NO:61); or
(iii) the VL CDR 1 has the amino acid sequence KSLLHSNGNSY (SEQ ID NO:141),
the VL
CDR 2 has the amino acid sequence RMS, and the VL CDR 3 has the amino acid
sequence
MQHLEYPFT (SEQ ID NO:29).
[0027] In one aspect, described herein is an antibody or a fragment thereof
that specifically
binds to HDM2, said antibody or fragment comprising a heavy chain variable
region (VH)
comprising VH complementarity determining region ("CDR") 1, VH CDR 2, and VH
CDR 3,
wherein:
(i) the VH CDR 1 has the amino acid sequence GYTFTSY (SEQ ID NO:30), the VH
CDR 2 has
the amino acid sequence NPRNGG (SEQ ID NO:31), and the VH CDR 3 has the amino
acid
sequence SGYYAMDY (SEQ ID NO:32);
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(ii) the VH CDR 1 has the amino acid sequence GYTFTSYYMY (SEQ ID NO:62), the
VH CDR
2 has the amino acid sequence GINPRNGGTN (SEQ ID NO:65), and the VH CDR 3 has
the
amino acid sequence SGYYAMDY (SEQ ID NO:32);
(iii) the VH CDR 1 has the amino acid sequence SYYMY (SEQ ID NO:63), the VH
CDR 2 has
the amino acid sequence GINPRNGGTNFNEKFKN (SEQ ID NO:66), and the VH CDR 3 has
the amino acid sequence SGYYAMDY (SEQ ID NO:32); or
(iv) the VH CDR 1 has the amino acid sequence TSYYMY (SEQ ID NO:64), the VH
CDR 2 has
the amino acid sequence WIGGINPRNGGTN (SEQ ID NO:67), and the VH CDR 3 has the
amino acid sequence TRSGYYAMD (SEQ ID NO:68).
[0028] In one aspect, described herein is an antibody or a fragment thereof
that specifically
binds to HDM2, said antibody or fragment comprising a VH, wherein the VH
comprises VH
CDR1, VH CDR2, and VH CDR 3, wherein:
(i) the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO:32); or
(ii) the VH CDR 3 has the amino acid sequence TRSGYYAMD (SEQ ID NO:68).
[0029] In one aspect, described herein is an antibody or fragment further
comprises a light
chain variable region (VL) comprising VL complementarity determining region
("CDR") 1, VL
CDR 2, and VL CDR 3, wherein:
(i) the VL CDR 1 has the amino acid sequence RASQDISNFLN (SEQ ID NO:33), the
VL CDR
2 has the amino acid sequence YTSRLHS (SEQ ID NO:34), and the VL CDR 3 has the
amino
acid sequence QQGNTLPRT (SEQ ID NO:35); or
(ii) the VL CDR 1 has the amino acid sequence SNFLNWY (SEQ ID NO:69), the VL
CDR 2
has the amino acid sequence LLIYYTSRLH (SEQ ID NO:70), and the VL CDR 3 has
the amino
acid sequence QQGNTLPR (SEQ ID NO:71).
[0030] In one aspect, described herein is an antibody or a fragment thereof
that specifically
binds to HDM2, said antibody or fragment comprising:
(a) a heavy chain variable region (VH) comprising VH complementarity
determining region
("CDR") 1, VH CDR 2, and VH CDR 3, wherein:
(i) the VH CDR 1 has the amino acid sequence GYTFTSY (SEQ ID NO:30), the VH
CDR 2 has
the amino acid sequence NPRNGG (SEQ ID NO:31), and the VH CDR 3 has the amino
acid
sequence SGYYAMDY (SEQ ID NO:32);
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(ii) the VH CDR 1 has the amino acid sequence GYTFTSYYMY (SEQ ID NO:62), the
VH CDR
2 has the amino acid sequence GINPRNGGTN (SEQ ID NO:65), and the VH CDR 3 has
the
amino acid sequence SGYYAMDY (SEQ ID NO:32);
(iii) the VH CDR 1 has the amino acid sequence SYYMY (SEQ ID NO:63), the VH
CDR 2 has
the amino acid sequence GINPRNGGTNFNEKFKN (SEQ ID NO:66), and the VH CDR 3 has
the amino acid sequence SGYYAMDY (SEQ ID NO:32); or
(iv) the VH CDR 1 has the amino acid sequence TSYYMY (SEQ ID NO:64), the VH
CDR 2 has
the amino acid sequence WIGGINPRNGGTN (SEQ ID NO:67), and the VH CDR 3 has the
amino acid sequence TRSGYYAMD (SEQ ID NO:68); and
(b) a light chain variable region (VL) comprising VL complementarity
determining region
("CDR") 1, VL CDR 2, and VL CDR 3, wherein:
(i) the VL CDR 1 has the amino acid sequence RASQDISNFLN (SEQ ID NO:33), the
VL CDR
2 has the amino acid sequence YTSRLHS (SEQ ID NO:34), and the VL CDR 3 has the
amino
acid sequence QQGNTLPRT (SEQ ID NO:35); or
(ii) the VL CDR 1 has the amino acid sequence SNFLNWY (SEQ ID NO:69), the VL
CDR 2
has the amino acid sequence LLIYYTSRLH (SEQ ID NO:70), and the VL CDR 3 has
the amino
acid sequence QQGNTLPR (SEQ ID NO:71).
[0031] In
one aspect, described herein is an antibody or fragment thereof that
specifically
binds to HDM2 comprising a VH having the amino acid sequence of SEQ ID NO:36,
or a VH
having at least 90%, at least 95%, at least 98%, or at least 99% sequence
identity to the amino
acid sequence of SEQ ID NO:36.
[0032] In
one aspect, described herein is an antibody or fragment thereof that
specifically
binds to HDM2 comprising a VL having the amino acid sequence of SEQ ID NO:37,
or a VL
having at least 90%, at least 95%, at least 98%, or at least 99% sequence
identity to the amino
acid sequence of SEQ ID NO:37 (and, optionally, comprising a VH having the
amino acid
sequence of SEQ ID NO:36, or a VH having at least 90%, at least 95%, at least
98%, or at least
99% sequence identity to the amino acid sequence of SEQ ID NO:36).
[0033] In
one aspect, described herein is an antibody or fragment thereof that
specifically
binds to HDM2 comprising a VH having the amino acid sequence of SEQ ID NO:38,
or a VH
having at least 90%, at least 95%, at least 98%, or at least 99% sequence
identity to the amino
acid sequence of SEQ ID NO:38.
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[0034] In
one aspect, described herein is an antibody or fragment thereof that
specifically
binds to HDM2 comprising a VL having the amino acid sequence of SEQ ID NO:39,
or a VL
having at least 90%, at least 95%, at least 98%, or at least 99% sequence
identity to the amino
acid sequence of SEQ ID NO:39 (and, optionally, comprising a VH having the
amino acid
sequence of SEQ ID NO:38, or a VH having at least 90%, at least 95%, at least
98%, or at least
99% sequence identity to the amino acid sequence of SEQ ID NO:38).
[0035] In
one aspect, described herein is an antibody or fragment thereof that
specifically
binds to HDM2 comprising a VH having the amino acid sequence of SEQ ID NO:40,
or a VH
having at least 90%, at least 95%, at least 98%, or at least 99% sequence
identity to the amino
acid sequence of SEQ ID NO:40.
[0036] In
one aspect, described herein is an antibody or fragment thereof that
specifically
binds to HDM2 comprising a VL having the amino acid sequence of SEQ ID NO:41,
or a VL
having at least 90%, at least 95%, at least 98%, or at least 99% sequence
identity to the amino
acid sequence of SEQ ID NO:41 (and, optionally, comprising a VH having the
amino acid
sequence of SEQ ID NO:40, or a VH having at least 90%, at least 95%, at least
98%, or at least
99% sequence identity to the amino acid sequence of SEQ ID NO:40).
[0037] In
one aspect, described herein is a humanized antibody or a fragment thereof
that
specifically binds to HDM2 or MDM2, said antibody or fragment comprising:
(a) a heavy chain variable region (VH) comprising a VH having an amino acid
sequence
selected from the group consisting of SEQ ID NO:287, SEQ ID NO:291, SEQ ID
NO:295, SEQ
ID NO:299, SEQ ID NO:305, and SEQ ID NO:309, or a VH having at least 95%
sequence
identity to the amino acid sequence selected from the group consisting of SEQ
ID NO:287, SEQ
ID NO:291, SEQ ID NO:295, SEQ ID NO:299, SEQ ID NO:305, and SEQ ID NO:309,
and/or
(b) a light chain variable region (VL) comprising a VL having an amino acid
sequence
selected from the group consisting of SEQ ID NO:289, SEQ ID NO:293, SEQ ID
NO:297, SEQ
ID NO:301, SEQ ID NO:303; SEQ ID NO:307, and SEQ ID NO:311, or a VL having at
least
95% sequence identity to the amino acid sequence selected from the group
consisting of SEQ ID
NO:289, SEQ ID NO:293, SEQ ID NO:297, SEQ ID NO:301, SEQ ID NO:303; SEQ ID
NO:307, and SEQ ID NO:311.
[0038] In
one aspect, described herein is a humanized antibody or a fragment thereof
that
specifically binds to HDM2 or MDM2, said antibody or fragment comprising:
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(a) a heavy chain variable region (VH) comprising a VH having an amino acid
sequence selected
from the group consisting of SEQ ID NO:287, SEQ ID NO:291, SEQ ID NO:295, SEQ
ID
NO:299, SEQ ID NO:305, and SEQ ID NO:309, and
(ii) a light chain variable region (VL) comprising a VL having an amino acid
sequence selected
from the group consisting of SEQ ID NO:289, SEQ ID NO:293, SEQ ID NO:297, SEQ
ID
NO:301, SEQ ID NO:303; SEQ ID NO:307, and SEQ ID NO:311.
[0039] In one aspect, described herein is a humanized antibody or fragment
thereof that
specifically binds to HDM2 or MDM2 comprising a VH having the amino acid
sequence of SEQ
ID NO:287, or a VH having at least 90%, at least 95%, at least 98%, or at
least 99% sequence
identity to the amino acid sequence of SEQ ID NO:287.
[0040] In one aspect, described herein is a humanized antibody or fragment
thereof that
specifically binds to HDM2 or MDM2 comprising a VH having the amino acid
sequence of SEQ
ID NO:291, or a VH having at least 90%, at least 95%, at least 98%, or at
least 99% sequence
identity to the amino acid sequence of SEQ ID NO:291.
[0041] In one aspect, described herein is a humanized antibody or fragment
thereof that
specifically binds to HDM2 or MDM2 comprising a VH having the amino acid
sequence of SEQ
ID NO:295, or a VH having at least 90%, at least 95%, at least 98%, or at
least 99% sequence
identity to the amino acid sequence of SEQ ID NO:295.
[0042] In one aspect, described herein is a humanized antibody or fragment
thereof that
specifically binds to HDM2 or MDM2 comprising a VH having the amino acid
sequence of SEQ
ID NO:299, or a VH having at least 90%, at least 95%, at least 98%, or at
least 99% sequence
identity to the amino acid sequence of SEQ ID NO:299.
[0043] In one aspect, described herein is a humanized antibody or fragment
thereof that
specifically binds to HDM2 or MDM2 comprising a VH having the amino acid
sequence of SEQ
ID NO:305, or a VH having at least 90%, at least 95%, at least 98%, or at
least 99% sequence
identity to the amino acid sequence of SEQ ID NO:305.
[0044] In one aspect, described herein is a humanized antibody or fragment
thereof that
specifically binds to HDM2 or MDM2 comprising a VH having the amino acid
sequence of SEQ
ID NO:309, or a VH having at least 90%, at least 95%, at least 98%, or at
least 99% sequence
identity to the amino acid sequence of SEQ ID NO:309.
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[0045] In one aspect, described herein is a humanized antibody or fragment
thereof that
specifically binds to HDM2 or MDM2 comprising a VL having the amino acid
sequence of SEQ
ID NO:289, or a VL having at least 95% sequence identity to the amino acid
sequence of SEQ
ID NO:289.
[0046] In one aspect, described herein is a humanized antibody or fragment
thereof that
specifically binds to HDM2 or MDM2 comprising a VL having the amino acid
sequence of SEQ
ID NO:293, or a VL having at least 90%, at least 95%, at least 98%, or at
least 99% sequence
identity to the amino acid sequence of SEQ ID NO :293.
[0047] In one aspect, described herein is a humanized antibody or fragment
thereof that
specifically binds to HDM2 or MDM2 comprising a VL having the amino acid
sequence of SEQ
ID NO:297, or a VL having at least 90%, at least 95%, at least 98%, or at
least 99% sequence
identity to the amino acid sequence of SEQ ID NO:297.
[0048] In one aspect, described herein is a humanized antibody or fragment
thereof that
specifically binds to HDM2 or MDM2 comprising a VL having the amino acid
sequence of SEQ
ID NO:301, or a VL having at least 90%, at least 95%, at least 98%, or at
least 99% sequence
identity to the amino acid sequence of SEQ ID NO:301.
[0049] In one aspect, described herein is a humanized antibody or fragment
thereof that
specifically binds to HDM2 or MDM2 comprising a VL having the amino acid
sequence of SEQ
ID NO:303, or a VL having at least 90%, at least 95%, at least 98%, or at
least 99% sequence
identity to the amino acid sequence of SEQ ID NO:303.
[0050] In one aspect, described herein is a humanized antibody or fragment
thereof that
specifically binds to HDM2 or MDM2 comprising a VL having the amino acid
sequence of SEQ
ID NO:307, or a VL having at least 90%, at least 95%, at least 98%, or at
least 99% sequence
identity to the amino acid sequence of SEQ ID NO:307.
[0051] In one aspect, described herein is a humanized antibody or fragment
thereof that
specifically binds to HDM2 or MDM2 comprising a VL having the amino acid
sequence of SEQ
ID NO:311, or a VL having at least 90%, at least 95%, at least 98%, or at
least 99% sequence
identity to the amino acid sequence of SEQ ID NO:311.
[0052] In one aspect, described herein is a humanized antibody or fragment
thereof that
specifically binds to HDM2 or MDM2 comprising: (i) a VH having the amino acid
sequence of
SEQ ID NO:299, and (ii) a VL having the amino acid sequence of SEQ ID NO:297.
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[0053] In one aspect, described herein is a humanized antibody or fragment
thereof that
specifically binds to HDM2 or MDM2 comprising: (a) a heavy chain variable
region (VH)
comprising a VH having an amino acid sequence selected from the group
consisting of SEQ ID
NO:287, SEQ ID NO:291, SEQ ID NO:295, and SEQ ID NO:299, and (ii) a light
chain variable
region (VL) comprising a VL having an amino acid sequence selected from the
group consisting
of SEQ ID NO:289, SEQ ID NO:293, SEQ ID NO:297, SEQ ID NO:301, and SEQ ID
NO:303.
[0054] In one aspect, described herein is a humanized antibody or fragment
thereof that
specifically binds to HDM2 or MDM2 comprising: (a) a heavy chain variable
region (VH)
comprising a VH having an amino acid sequence selected from the group
consisting of SEQ ID
NO:299, SEQ ID NO:305, and SEQ ID NO:309, and (ii) a light chain variable
region (VL)
comprising a VL having an amino acid sequence selected from the group
consisting of SEQ ID
NO:297, SEQ ID NO:307, and SEQ ID NO:311.
[0055] In one aspect, described herein is an antibody or a fragment thereof
that specifically
binds to HDM2 or MDM2, said antibody or fragment comprising:
(a) a heavy chain variable region (VH) having an amino acid sequence of SEQ ID
NO:283, or a
VH having at least 95% sequence identity to the amino acid sequence of SEQ ID
NO:283, and
(b) a light chain variable region (VL) having an amino acid sequence of SEQ ID
NO:285, or a
VL having at least 95% sequence identity to the amino acid sequence of SEQ ID
NO:285.
[0056] In one aspect, described herein is an antibody or a fragment thereof
that specifically
binds to HDM2 or MDM2, said antibody or fragment comprising:
(a) a heavy chain variable region (VH) having an amino acid sequence of SEQ ID
NO:283, and
(b) a light chain variable region (VL) having an amino acid sequence of SEQ ID
NO:285.
[0057] In one aspect, described herein is an antibody or fragment thereof
that specifically
binds to HDM2 or MDM2 comprising a VH having the amino acid sequence of SEQ ID
NO:283, or a VH having at least 90%, at least 95%, at least 98%, or at least
99% sequence
identity to the amino acid sequence of SEQ ID NO:283.
[0058] In one aspect, described herein is an antibody or fragment thereof
that specifically
binds to HDM2 or MDM2 comprising a VL having the amino acid sequence of SEQ ID
NO:285,
or a VL having at least 90%, at least 95%, at least 98%, or at least 99%
sequence identity to the
amino acid sequence of SEQ ID NO:285.
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[0059] In one aspect, described herein is an antibody or fragment thereof
that specifically
binds to HDM2 or MDM2, said antibody comprising:
(a) a heavy chain having an amino acid sequence of SEQ ID NO:312, or a heavy
chain having at
least 95% sequence identity to the amino acid sequence of SEQ ID NO:312,
and/or
(b) a light chain having an amino acid sequence of SEQ ID NO:313, or a light
chain having at
least 95% sequence identity to the amino acid sequence of SEQ ID NO:313.
[0060] In one aspect, described herein is an antibody or fragment thereof
that specifically
binds to HDM2 or MDM2, said antibody comprising:
(a) a heavy chain having an amino acid sequence of SEQ ID NO:312, and
(b) a light chain having an amino acid sequence of SEQ ID NO:313, or a light
chain having at
least 95% sequence identity to the amino acid sequence of SEQ ID NO:313.
[0061] In one aspect, described herein is an antibody or fragment thereof
that specifically
binds to HDM2 or MDM2 comprising a heavy chain having the amino acid sequence
of SEQ ID
NO:312, or a heavy chain having at least 90%, at least 95%, at least 98%, or
at least 99%
sequence identity to the amino acid sequence of SEQ ID NO:312.
[0062] In one aspect, described herein is an antibody or fragment thereof
that specifically
binds to HDM2 or MDM2 comprising a light chain having the amino acid sequence
of SEQ ID
NO:313, or a light chain having at least 90%, at least 95%, at least 98%, or
at least 99%
sequence identity to the amino acid sequence of SEQ ID NO: 313.
[0063] In one aspect, described herein is an antibody or fragment thereof
that specifically
binds to HDM2 or MDM2, which inhibits tumor cell proliferation in vivo.
[0064] In preferred embodiments, the anti-M(H)DM2/4 antibody described
herein is a
monoclonal antibody. In certain embodiments, the anti-M(H)DM2/4 antibody
described herein
is a human, humanized, or a chimeric antibody (e.g., a human, humanized or
chimeric
monoclonal antibody). In one embodiment, the anti-M(H)DM2/4 antibody described
herein is a
human antibody. In one embodiment, the anti-M(H)DM2/4 antibody described
herein is a
humanized antibody. In one embodiment, the anti-M(H)DM2/4 antibody described
herein is a
chimeric antibody.
[0065] In certain embodiments, the anti-M(H)DM2/4 antibody described herein
is a purified
antibody.
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[0066] In certain embodiments, the anti-M(H)DM2/4 antibody described herein
is an
immunoglobulin (e.g., IgG or IgM). In one embodiment, the immunoglobulin is an
IgG. In one
embodiment, the immunoglobulin is an IgM. In certain embodiments, the
immunoglobulin is of
IgG1 isotype. In other embodiments, the immunoglobulin is of IgG3 isotype. In
other
embodiments, the immunoglobulin is of IgG2 isotype. In other embodiments, the
immunoglobulin
is of IgG4 isotype. In certain embodiments, the anti- M(H)DM2/4 antibody
described herein
comprises an Fc region, wherein the Fc region is a human IgG Fc region or a
human IgM Fc
region. In specific embodiments, the anti- M(H)DM2/4 antibody described herein
comprises an
Fc region, which is a human IgG1 Fc region, a human IgG2 Fc region, or a human
IgG3 Fc
region, or a human IgG4 Fc region. In one embodiment, the anti- M(H)DM2/4
antibody
described herein comprises a human IgG1 Fc region. In one embodiment, the anti-
M(H)DM2/4
antibody described herein comprises a human IgG3 Fc region. In one embodiment,
the anti-
M(H)DM2/4 antibody described herein comprises a human IgG4 Fc region. In one
embodiment,
the anti- M(H)DM2/4 antibody described herein comprises a human IgG2 Fc
region. In one
embodiment, the anti- M(H)DM2/4 antibody described herein comprises a human
IgM Fc region.
In one embodiment, the anti-M(H)DM2/4 antibody described herein comprises a
human IgE Fc
region.
[0067] In certain embodiments, the anti-M(H)DM2/4 antibody or fragment
described herein
is an antigen-binding fragment of an anti-M(H)DM2/4 antibody. In certain
embodiments, the
antibody or fragment described herein is an Fv fragment, a Fab fragment, a
Fab' fragment, a
F(ab1)2 fragment, a single chain antibody molecule, or a single chain Fv
(scFv). In one
embodiment, the antibody or fragment described herein is an Fv fragment. In
one embodiment,
the antibody or fragment described herein is a Fab fragment. In one
embodiment, the antibody
or fragment described herein is a Fab' fragment. In one embodiment, the
antibody or fragment
described herein is a F(ab1)2 fragment. In one embodiment, the antibody or
fragment described
herein is a single chain antibody molecule. In one embodiment, the antibody or
fragment
described herein is a single chain Fv (scFv).
[0068] In certain embodiments, the anti-M(H)DM2/4 antibody or antigen-
binding fragment
described herein mediates complement-dependent cytotoxicity (CDC) or antibody-
dependent
cell-mediated cytoxicity (ADCC). In one embodiment, the anti- M(H)DM2/4
antibody or
antigen-binding fragment mediates complement-dependent cytotoxicity (CDC).
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[0069] In certain embodiments, the anti-M(H)DM2/4 antibody described herein
is a
bispecific antibody that also specifically binds to a cell surface antigen of
an effector cell (e.g., a
T cell, a B lymphocyte, a neutrophil, a macrophage, a natural killer cell, or
a dendritic cell).
[0070] In one embodiment, the antibody or fragment described herein
specifically binds to
an extracellularly accessible epitope of M(H)DM2 (e.g., HDM2) and does not
bind to M(H)DM4
(e.g., HDM4). In another embodiment, the antibody or fragment described herein
specifically
binds to extracellularly accessible epitopes of both M(H)DM2 (e.g., HDM2) and
M(H)DM4
(e.g., HDM4). In one embodiment, the antibody or fragment described herein
specifically binds
to an extracellularly accessible epitope of HDM2, and optionally, may also
bind to an
extracellularly accessible epitope of MDM2.
[0071] In a specific embodiment, the anti-HDM2 antibody or fragment
described herein
specifically binds HDM2 within amino acids of SEQ ID NO: 1 (which are amino
acids 1 to 15 of
HDM2 (SEQ ID NO:4)). In another specific embodiment, the anti-HDM2 antibody
described
herein specifically binds HDM2 within amino acids of SEQ ID NO: 2 (which are
amino acids 15
to 25 of HDM2 (SEQ ID NO:4)). In another specific embodiment, the anti-HDM2
antibody
described herein specifically binds HDM2 within amino acids of SEQ ID NO: 3
(which are
amino acids 475-491 of HDM2 (SEQ ID NO:4)). In another specific embodiment,
the anti-
HDM2 antibody described herein specifically binds within amino acids 19 to 50
of SEQ ID NO:
4. In another specific embodiment, the anti-HDM2 antibody described herein
specifically binds
within amino acids 154 to 167 of SEQ ID NO: 4. In yet another specific
embodiment, the anti-
HDM2 antibody described herein specifically binds within amino acids 1 to 60
of SEQ ID NO:
4. In yet another specific embodiment, the anti- HDM2 antibody described
herein specifically
binds within amino acids 1 to 100 of SEQ ID NO: 4. In another specific
embodiment, the anti-
HDM2 antibody described herein specifically binds within amino acids 100 to
110 of SEQ ID
NO: 4. In another specific embodiment, the anti-HDM2 antibody described herein
specifically
binds within amino acids 50 to 60 of SEQ ID NO: 4. In yet another specific
embodiment, the
anti-HDM2 antibody described herein specifically binds within amino acids 1 to
109 of SEQ ID
NO: 4. In another specific embodiment, the anti-HDM2 antibody described herein
specifically
binds within amino acids 26 to 60 of SEQ ID NO: 4. In one specific embodiment,
the anti-
HDM2 antibody described herein specifically binds within the terminal 60 amino
acids at the C-
terminus of the HDM2 on the plasma membrane of the cancer cells. In another
specific
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embodiment, the anti-HDM2 antibody described herein specifically binds within
the terminal
100 amino acids at the C-terminus of the HDM2 on the plasma membrane of the
cancer cells.
[0072] In one embodiment, the anti- M(H)DM2/4 antibody described herein
does not bind
within amino acids 101 to 200 of SEQ ID NO:4. In one embodiment, the anti-
M(H)DM2/4
antibody described herein does not bind to the epitope of HDM2 or MDM2 to
which "MDM2
monoclonal antibody (M01), clone 1A7" (Abnova, Cat. No. H00004193-M01) binds.
In one
embodiment, the anti- M(H)DM2/4 antibody described herein does not compete
with "MDM2
monoclonal antibody (M01), clone 1A7" (Abnova, Cat. No. H00004193-M01) for
binding to
HDM2.
[0073] In another embodiment, the anti- M(H)DM2/4 antibody described herein
binds within
amino acids 101 to 200 of SEQ ID NO:4. In one embodiment, the anti- M(H)DM2/4
antibody
described herein binds to the epitope of HDM2 or MDM2 to which "MDM2
monoclonal
antibody (M01), clone 1A7" (Abnova, Cat. No. H00004193-M01) binds. In one
embodiment,
the anti- M(H)DM2/4 antibody described herein competes with "MDM2 monoclonal
antibody
(M01), clone 1A7" (Abnova, Cat. No. H00004193-M01) for binding to HDM2.
[0074] In one embodiment, the anti- M(H)DM2/4 antibody described herein
does not bind
within amino acids 153 to 222 of SEQ ID NO:4. In one embodiment, the anti-
M(H)DM2/4
antibody described herein does not bind within amino acids 26 to 169 of SEQ ID
NO:4. In a
specific embodiment, the anti- M(H)DM2/4 antibody described herein does not
bind within
amino acids 26 to 222 of SEQ ID NO:4.
[0075] In certain embodiments, the M(H)DM2/4 exposed on the surface of
cancer cells being
targeted by the antibodies or fragments described herein is an M(H)DM2/4
variant that lacks one
or more nuclear localization signal domains. In specific embodiments, the HDM2
exposed on the
surface of cancer cells being targeted by the antibodies or fragments
described herein is an
HDM2 variant that lacks the sequence of amino acids 179 to 185 of SEQ ID NO: 4
and/or the
sequence of amino acids 464 to 471 of SEQ ID NO: 4. In one embodiment, the
HDM2 exposed
on the surface of cancer cells being targeted by the antibodies or fragments
described herein is an
HDM2 variant that lacks the sequence of amino acids 181 to 185 of SEQ ID NO:
4.
[0076] In one aspect, provided herein are antibodies or fragments thereof
that compete for
binding to M(H)DM2/4 with an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or
fragment
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thereof described herein. Preferably, such antibodies or fragments that
compete for binding are
monoclonal antibodies or fragments thereof.
[0077] In one aspect, provided herein are antibodies or fragments thereof
that compete for
binding to M(H)DM2/4 with a mouse anti-HDM2 immunoglobulin (preferably IgG)
antibody
selected from the group consisting of: (i) an antibody comprising a heavy
chain variable region
(VH) having the amino acid sequence of SEQ ID NO:36, and a light chain
variable region (VL)
having the amino acid sequence of SEQ ID NO:37; (ii) an antibody comprising a
VH having the
amino acid sequence of SEQ ID NO:38, and a VL having the amino acid sequence
of SEQ ID
NO:39; and (iii) an antibody comprising a VH having the amino acid sequence of
SEQ ID
NO:40, and a VL having the amino acid sequence of SEQ ID NO:41.
[0078] In one aspect, provided herein are antibodies or fragments thereof
that: (i) compete
for binding to a peptide of sequence SEQ ID NO:1 with a mouse anti-HDM2 IgG1
antibody
comprising a heavy chain variable region (VH) having the amino acid sequence
of SEQ ID
NO:36, and a light chain variable region (VL) having the amino acid sequence
of SEQ ID
NO:37; or (ii) compete for binding to a peptide of SEQ ID NO:2 with a mouse
anti-HDM2 IgG3
antibody comprising a VH having the amino acid sequence of SEQ ID NO:38, and a
VL having
the amino acid sequence of SEQ ID NO:39; or (iii) compete for binding to a
peptide of SEQ ID
NO:3 with a mouse IgM antibody comprising a VH having the amino acid sequence
of SEQ ID
NO:40, and a VL having the amino acid sequence of SEQ ID NO:41.
[0079] In one aspect, provided herein are antibody-drug conjugates
comprising any antibody
or fragment described herein (e.g., an antibody-drug conjugate in which an
anti-M(H)DM2/4
antibody or fragment thereof described herein is covalently bound to a
cytotoxic drug).
[0080] In one aspect, provided herein are pharmaceutical compositions
comprising a
therapeutically effective amount of any antibody or fragment described herein.
[0081] In one aspect, provided herein are pharmaceutical compositions
comprising a
therapeutically effective amount of any chimeric or humanized anti-M(H)DM2/4
antibody or
fragment described herein.
[0082] In one aspect, provided herein are methods for treating cancer in a
subject in need
thereof, said method comprising administering to the subject: (i) any anti-
M(H)DM2/4 antibody
described herein; (ii) an antibody or a fragment thereof that specifically
binds to an
extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or
fragment is not
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bound to a cell-penetrating peptide (e.g., a membrane resident peptide); or an
antibody-drug
conjugate comprising the antibody or fragment (i.e., said antibody or fragment
that is not bound
to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any
pharmaceutical composition
described herein, or (iv) any antibody-drug conjugate described herein. In one
aspect, provided
herein are methods for treating cancer in a subject in need thereof, said
method comprising
administering to the subject: an antibody or a fragment thereof that
specifically binds to an
extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said
antibody or
fragment is not bound to a cytotoxic component. In one aspect, provided herein
are methods for
treating cancer in a subject in need thereof, said method comprising
administering to the subject:
(i) any anti-HDM2 antibody or fragment described herein; (ii) an antibody or a
fragment thereof
that specifically binds to an extracellularly accessible epitope of HDM2,
wherein said antibody
or fragment is not bound to a cell-penetrating peptide (e.g., a membrane
resident peptide), or an
antibody-drug conjugate comprising the antibody or fragment (i.e., said
antibody or fragment
that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug,
(iii) any
pharmaceutical composition described herein, or (iv) any antibody-drug
conjugate described
herein. In one embodiment, the method comprises administering to the subject
an antibody-drug
conjugate comprising the antibody or a fragment thereof that specifically
binds to an
extracellularly accessible epitope of M(H)DM2/4 bound to a cytotoxic drug,
wherein said
antibody or fragment is not bound to a cell-penetrating peptide. In one
embodiment, provided
herein is a method for treating cancer in a subject in need thereof, said
method comprising
administering to the subject an anti-M(H)DM2/4 antibody or fragment thereof
that specifically
binds to an extracellularly accessible epitope of M(H)DM2/4, wherein the
antibody or fragment
specifically binds to a peptide, wherein the sequence of the peptide consists
of
MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or
CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In one embodiment, provided herein is a
method for
treating cancer in a subject in need thereof, said method comprising
administering to the subject
an anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an
extracellularly
accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:1, SEQ
ID NO:2,
or SEQ ID NO:3.
[0083] In
one embodiment, provided herein is a method for treating cancer in a subject
in
need thereof, said method comprising administering to the subject any chimeric
or humanized
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anti-M(H)DM2/4 antibody or fragment described herein. In one aspect, provided
herein are
methods for inhibiting tumor growth in a subject in need thereof, said method
comprising
administering to the subject: (i) any anti-M(H)DM2/4 antibody or fragment
described herein; (ii)
an antibody or a fragment thereof that specifically binds to an
extracellularly accessible epitope
of M(H)DM2/4, wherein said antibody or fragment is not bound to a cell-
penetrating peptide
(e.g., a membrane resident peptide), or an antibody-drug conjugate comprising
the antibody or
fragment (i.e., said antibody or fragment that is not bound to a cell-
penetrating peptide) bound to
a cytotoxic drug, (iii) any pharmaceutical composition described herein, or
(iv) any antibody-
drug conjugate described herein. In one aspect, provided herein are methods
for inhibiting
tumor growth in a subject in need thereof, said method comprising
administering to the subject:
an antibody or a fragment thereof that specifically binds to an
extracellularly accessible epitope
of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a
cytotoxic
component. In one aspect, provided herein are methods for inhibiting tumor
growth in a subject
in need thereof, said method comprising administering to the subject: (i) any
anti-HDM2
antibody or fragment described herein; (ii) an antibody or a fragment thereof
that specifically
binds to an extracellularly accessible epitope of HDM2, wherein said antibody
or fragment is not
bound to a cell- penetrating peptide(e.g., a membrane resident peptide), or an
antibody-drug
conjugate comprising the antibody or fragment (i.e., said antibody or fragment
that is not bound
to a cell-penetraitng peptide) bound to a cytotoxic drug, (iii) any
pharmaceutical composition
described herein, or (iv) any antibody-drug conjugate described herein. In one
embodiment, the
method comprises administering to the subject an antibody-drug conjugate
comprising the
antibody or a fragment thereof that specifically binds to an extracellularly
accessible epitope of
M(H)DM2/4 bound to a cytotoxic drug, wherein said antibody or fragment is not
bound to a cell-
penetrating peptide. In one embodiment, provided herein is a method for
inhibiting tumor
growth in a subject in need thereof, said method comprising administering to
the subject an anti-
M(H)DM2/4 antibody or fragment thereof that specifically binds to an
extracellularly accessible
epitope of M(H)DM2/4, wherein the antibody or fragment specifically binds to a
peptide,
wherein the sequence of the peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1),
TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In one
embodiment, provided herein is a method for inhibiting tumor growth in a
subject in need
thereof, said method comprising administering to the subject an anti-M(H)DM2/4
antibody or
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fragment thereof that specifically binds to an extracellularly accessible
epitope of M(H)DM2/4
contained within a peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In one
embodiment, provided herein is a method for inhibiting tumor growth in a
subject in need
thereof, said method comprising administering to the subject any chimeric or
humanized anti-
M(H)DM2/4 antibody or fragment described herein.In one aspect, provided herein
are methods
for inhibiting tumor progression in a subject in need thereof, said method
comprising
administering to the subject: (i) any anti-M(H)DM2/4 antibody or fragment
described herein; (ii)
an antibody or a fragment thereof that specifically binds to an
extracellularly accessible epitope
of M(H)DM2/4, wherein said antibody or fragment is not bound to a cell-
penetrating peptide
(e.g., a membrane resident peptide), or an antibody-drug conjugate comprising
the antibody or
fragment (i.e., said antibody or fragment that is not bound to a cell-
penetrating peptide) bound to
a cytotoxic drug, (iii) any pharmaceutical composition described herein, or
(iv) any antibody-
drug conjugate described herein. In one aspect, provided herein are methods
for inhibiting tumor
progression in a subject in need thereof, said method comprising administering
to the subject: an
antibody or a fragment thereof that specifically binds to an extracellularly
accessible epitope of
M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a
cytotoxic
component. In one aspect, provided herein are methods for inhibiting tumor
progression in a
subject in need thereof, said method comprising administering to the subject:
(i) any anti-HDM2
antibody or fragment described herein; (ii) an antibody or a fragment thereof
that specifically
binds to an extracellularly accessible epitope of HDM2, wherein said antibody
or fragment is not
bound to a cell- penetrating peptide (e.g., a membrane resident peptide), or
an antibody-drug
conjugate comprising the antibody or fragment (i.e., said antibody or fragment
that is not bound
to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any
pharmaceutical composition
described herein, or (iv) any antibody-drug conjugate described herein. In one
embodiment, the
method comprises administering to the subject an antibody-drug conjugate
comprising the
antibody or a fragment thereof that specifically binds to an extracellularly
accessible epitope of
M(H)DM2/4 bound to a cytotoxic drug, wherein said antibody or fragment is not
bound to a cell-
penetrating peptide. In one embodiment, provided herein is a method for
inhibiting tumor
progression in a subject in need thereof, said method comprising administering
to the subject an
anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an
extracellularly
accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically
binds to a
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peptide, wherein the sequence of the peptide consists of MCNTNMSVPTDGAVT (SEQ
ID
NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In one
embodiment, provided herein is a method for inhibiting tumor progression in a
subject in need
thereof, said method comprising administering to the subject an anti-M(H)DM2/4
antibody or
fragment thereof that specifically binds to an extracellularly accessible
epitope of M(H)DM2/4
contained within a peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
[0084] In one aspect, provided herein is a method for preventing cancer
recurrence or relapse
in a subject in need thereof (e.g., a subject who is in remission from
cancer), said method
comprising administering to the subject: (i) any anti-M(H)DM2/4 antibody or
fragment
described herein; (ii) an antibody or a fragment thereof that specifically
binds to an
extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or
fragment is not
bound to a cell- penetrating peptide (e.g., a membrane resident peptide), or
an antibody-drug
conjugate comprising the antibody or fragment (i.e., said antibody or fragment
that is not bound
to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any
pharmaceutical composition
described herein, or (iv) any antibody-drug conjugate described herein. In one
aspect, provided
herein is a method for preventing cancer recurrence or relapse in a subject in
need thereof (e.g., a
subject who is in remission from cancer), said method comprising administering
to the subject:
an antibody or a fragment thereof that specifically binds to an
extracellularly accessible epitope
of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a
cytotoxic
component. In one aspect, provided herein is a method for preventing cancer
recurrence or
relapse in a subject in need thereof (e.g., a subject who is in remission from
cancer), said method
comprising administering to the subject: (i) any anti-HDM2 antibody or
fragment described
herein; (ii) an antibody or a fragment thereof that specifically binds to an
extracellularly
accessible epitope of HDM2, wherein said antibody or fragment is not bound to
a cell-
penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug
conjugate
comprising the antibody or fragment (i.e., said antibody or fragment that is
not bound to a cell-
penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical
composition described
herein, or (iv) any antibody-drug conjugate described herein. In one
embodiment, the method
comprises administering to the subject an antibody-drug conjugate comprising
the antibody or a
fragment thereof that specifically binds to an extracellularly accessible
epitope of M(H)DM2/4
bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a
cell-penetrating
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peptide. In one embodiment, provided herein is a method for preventing cancer
recurrence or
relapse in a subject in need thereof (e.g., a subject who is in remission from
cancer), said method
comprising administering to the subject an anti-M(H)DM2/4 antibody or fragment
thereof that
specifically binds to an extracellularly accessible epitope of M(H)DM2/4,
wherein the antibody
or fragment specifically binds to a peptide, wherein the sequence of the
peptide consists of
MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or
CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In one embodiment, provided herein is a
method for
preventing cancer recurrence or relapse in a subject in need thereof (e.g., a
subject who is in
remission from cancer), said method comprising administering to the subject an
anti-
M(H)DM2/4 antibody or fragment thereof that specifically binds to an
extracellularly accessible
epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:1, SEQ ID NO:2,
or SEQ ID
NO:3. In one embodiment, provided herein is a method for preventing cancer
recurrence or
relapses in a subject in need thereof, said method comprising administering to
the subject any
chimeric or humanized anti-M(H)DM2/4 antibody or fragment described herein.
[0085] In one aspect, provided herein is a method for increasing survival
in a subject having
a cancer (e.g., relative to a subject not treated with anti-M(H)DM2/4 antibody
or fragment), said
method comprising administering to the subject: (i) any anti-M(H)DM2/4
antibody or fragment
described herein; (ii) an antibody or a fragment thereof that specifically
binds to an
extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or
fragment is not
bound to a cell- penetrating peptide (e.g., a membrane resident peptide), or
an antibody-drug
conjugate comprising the antibody or fragment (i.e., said antibody or fragment
that is not bound
to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any
pharmaceutical composition
described herein, or (iv) any antibody-drug conjugate described herein. In one
aspect, provided
herein is a method for increasing survival in a subject having a cancer (e.g.,
relative to a subject
not treated with anti-M(H)DM2/4 antibody or fragment), said method comprising
administering
to the subject: an antibody or a fragment thereof that specifically binds to
an extracellularly
accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or
fragment is not bound
to a cytotoxic component. In one aspect, provided herein is a method for
increasing survival in a
subject a cancer (e.g., relative to a subject not treated with anti-M(H)DM2/4
antibody or
fragment), said method comprising administering to the subject: (i) any anti-
HDM2 antibody or
fragment described herein; (ii) an antibody or a fragment thereof that
specifically binds to an
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extracellularly accessible epitope of HDM2, wherein said antibody or fragment
is not bound to a
cell- penetrating peptide (e.g., a membrane resident peptide), or an antibody-
drug conjugate
comprising the antibody or fragment (i.e., said antibody or fragment that is
not bound to a cell-
penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical
composition described
herein, or (iv) any antibody-drug conjugate described herein. In one
embodiment, the method
comprises administering to the subject an antibody-drug conjugate comprising
the antibody or a
fragment thereof that specifically binds to an extracellularly accessible
epitope of M(H)DM2/4
bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a
cell-penetrating
peptide. In one embodiment, provided herein is a method for increasing
survival in a subject
having a cancer (e.g., relative to a subject not treated with anti-M(H)DM2/4
antibody or
fragment), said method comprising administering to the subject an anti-
M(H)DM2/4 antibody or
fragment thereof that specifically binds to an extracellularly accessible
epitope of M(H)DM2/4,
wherein the antibody or fragment specifically binds to a peptide, wherein the
sequence of the
peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2),
or CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In one embodiment, provided herein is a
method
for increasing survival in a subject having a cancer (e.g., relative to a
subject not treated with
anti-M(H)DM2/4 antibody or fragment), said method comprising administering to
the subject an
anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an
extracellularly
accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:1, SEQ
ID NO:2,
or SEQ ID NO:3. In one embodiment, provided herein is a method for increasing
survival in a
subject having a cancer (e.g., relative to a subject not treated with anti-
M(H)DM2/4 antibody or
fragment thereof described herein), said method comprising administering to
the subject any
chimeric or humanized anti-M(H)DM2/4 antibody or fragment described herein.
[0086] In one aspect, provided herein are methods for preventing metastasis
in a subject
[0087] having a cancer, said method comprising administering to the
subject: (i) any anti-
M(H)DM2/4 antibody or fragment described herein; (ii) an antibody or a
fragment thereof that
specifically binds to an extracellularly accessible epitope of M(H)DM2/4,
wherein said antibody
or fragment is not bound to a cell-penetrating peptide (e.g., a membrane
resident peptide), or an
antibody-drug conjugate comprising the antibody or fragment (i.e., said
antibody or fragment
that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug,
(iii) any
pharmaceutical composition described herein, or (iv) any antibody-drug
conjugate described
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herein. In one aspect, provided herein are methods for preventing metastasis
in a subject in need
thereof, said method comprising administering to the subject: an antibody or a
fragment thereof
that specifically binds to an extracellularly accessible epitope of M(H)DM2/4
(e.g., HDM2),
wherein said antibody or fragment is not bound to a cytotoxic component. In
one aspect,
provided herein are methods for preventing metastasis in a subject having a
cancer, said method
comprising administering to the subject: (i) any anti-HDM2 antibody or
fragment described
herein; (ii) an antibody or a fragment thereof that specifically binds to an
extracellularly
accessible epitope of HDM2, wherein said antibody or fragment is not bound to
a cell-
penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug
conjugate
comprising the antibody or fragment (i.e., said antibody or fragment that is
not bound to a cell-
penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical
composition described
herein, or (iv) any antibody-drug conjugate described herein. In one
embodiment, the method
comprises administering to the subject an antibody-drug conjugate comprising
the antibody or a
fragment thereof that specifically binds to an extracellularly accessible
epitope of M(H)DM2/4
bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a
cell-penetrating
peptide. In one embodiment, provided herein is a method for preventing
metastasis in a subject
having a cancer, said method comprising administering to the subject an anti-
M(H)DM2/4
antibody or fragment thereof that specifically binds to an extracellularly
accessible epitope of
M(H)DM2/4, wherein the antibody or fragment specifically binds to a peptide,
wherein the
sequence of the peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE
(SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In one embodiment, provided
herein is a method for preventing metastasis in a subject having a cancer,
said method
comprising administering to the subject an anti-M(H)DM2/4 antibody or fragment
thereof that
specifically binds to an extracellularly accessible epitope of M(H)DM2/4
contained within a
peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In one embodiment,
provided herein
is a method for preventing metastasis in a subject having a cancer, said
method comprising
administering to the subject any chimeric or humanized anti-M(H)DM2/4 antibody
or fragment
described herein.In one aspect, provided herein are methods for inhibiting
metastasis (e.g.,
reducing the number, size or invasiveness of metastases) in a subject having a
metastatic cancer,
said method comprising administering to the subject: (i) any anti-M(H)DM2/4
antibody or
fragment described herein; (ii) an antibody or a fragment thereof that
specifically binds to an
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extracellularly accessible epitope of M(H)DM2/4 exposed, wherein said antibody
or fragment is
not bound to a cell-penetrating peptide (e.g., a membrane resident peptide),
or an antibody-drug
conjugate comprising the antibody or fragment (i.e., said antibody or fragment
that is not bound
to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any
pharmaceutical composition
described herein, or (iv) any antibody-drug conjugate described herein. In one
aspect, provided
herein are methods for inhibiting metastasis in a subject in need thereof,
said method comprising
administering to the subject: an antibody or a fragment thereof that
specifically binds to an
extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said
antibody or
fragment is not bound to a cytotoxic component. In one aspect, provided herein
are methods for
inhibiting metastasis (e.g., reducing the number, size or invasiveness of
metastases) in a subject
having a metastatic cancer, said method comprising administering to the
subject: (i) any anti-
HDM2 antibody or fragment described herein; (ii) an antibody or a fragment
thereof that
specifically binds to an extracellularly accessible epitope of HDM2, wherein
said antibody or
fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident
peptide), or an
antibody-drug conjugate comprising the antibody or fragment (i.e., said
antibody or fragment
that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug,
(iii) any
pharmaceutical composition described herein, or (iv) any antibody-drug
conjugate described
herein. In one embodiment, the method comprises administering to the subject
an antibody-drug
conjugate comprising the antibody or a fragment thereof that specifically
binds to an
extracellularly accessible epitope of M(H)DM2/4 bound to a cytotoxic drug,
wherein said
antibody or fragment is not bound to a cell-penetrating peptide. In one
embodiment, provided
herein is a method for inhibiting metastasis (e.g., reducing the number, size
or invasiveness of
metastases) in a subject having a metastatic cancer, said method comprising
administering to the
subject an anti-M(H)DM2/4 antibody or fragment thereof that specifically binds
to an
extracellularly accessible epitope of M(H)DM2/4, wherein the antibody or
fragment specifically
binds to a peptide, wherein the sequence of the peptide consists of
MCNTNMSVPTDGAVT
(SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID
NO:3). In one embodiment, provided herein is a method for inhibiting
metastasis (e.g., reducing
the number, size or invasiveness of metastases) in a subject having a
metastatic cancer, said
method comprising administering to the subject an anti-M(H)DM2/4 antibody or
fragment
thereof that specifically binds to an extracellularly accessible epitope of
M(H)DM2/4 contained
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within a peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In one
embodiment,
provided herein is a method for inhibiting metastasis (e.g., reducing the
number, size or
invasiveness of metastases) in a subject having a metastatic cancer, said
method comprising
administering to the subject any chimeric or humanized anti-M(H)DM2/4 antibody
or fragment
described herein.
[0088] In
one aspect, provided herein are methods of treating cancer in a subject who
has
experienced an accelerated rate of cancer growth in response to administration
to the subject of
an inhibitor of one or more inhibitory checkpoint molecules, said method
comprising
administering to the subject:
(i) an antibody or a fragment thereof that specifically binds to an
extracellularly accessible
epitope of M(H)DM2/4, or an antibody-drug conjugate comprising the antibody or
fragment
bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a
cell-penetrating
peptide,
(ii) an antibody or a fragment thereof that specifically binds to an
extracellularly accessible
epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a
cytotoxic
component,
(iii) any anti-M(H)DM2/4 antibody or fragment described herein,
(iv) an antibody-drug conjugate comprising any anti-M(H)DM2/4 antibody or
fragment
described herein, bound to a cytotoxic drug, or
(v) a pharmaceutical composition comprising a therapeutically effective
amount of any anti-
M(H)DM2/4 antibody or fragment described herein or an antibody-drug conjugate
comprising
any anti-M(H)DM2/4 antibody or fragment described herein, bound to a cytotoxic
drug.
[0089] In
one aspect, provided herein are methods of treating cancer in a subject who
has
experienced an accelerated rate of cancer growth in response to administration
to the subject of
an inhibitor of one or more inhibitory checkpoint molecules, wherein the one
or more inhibitory
checkpoint molecules are selected from the group consisting of: CTLA-4, PD-1,
PD-L1, and PD-
L2.
[0090] In
one aspect, provided herein are methods of treating cancer in a subject who
has
experienced an accelerated rate of cancer growth in response to administration
to the subject of
an inhibitor of one or more inhibitory checkpoint molecules, wherein the one
or more inhibitory
checkpoint molecules is PD-1.
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[0091] In one aspect, provided herein are methods of treating cancer in a
subject who has
experienced an accelerated rate of cancer growth in response to administration
to the subject of
an inhibitor of one or more inhibitory checkpoint molecules, wherein the
inhibitor of one or more
inhibitory checkpoint molecules is an inhibitory antibody to PD-1.
[0092] In one aspect, provided herein are methods of treating cancer in a
subject who has
experienced an accelerated rate of cancer growth in response to administration
to the subject of
an inhibitor of one or more inhibitory checkpoint molecules, wherein said
method comprises
administering to the subject any anti-M(H)DM2/4 antibody or fragment described
herein.
[0093] In one aspect, provided herein are methods of treating cancer in a
subject who has
experienced an accelerated rate of cancer growth in response to administration
to the subject of
an inhibitor of one or more inhibitory checkpoint molecules, wherein said
method comprises
administering to the subject any anti-M(H)DM2/4 antibody or fragment described
herein,
wherein the anti-M(H)DM2/4 antibody or fragment is not bound to a cell-
penetrating peptide.
[0094] In one aspect, provided herein are methods of treating cancer in a
subject who has
experienced an accelerated rate of cancer growth in response to administration
to the subject of
an inhibitor of one or more inhibitory checkpoint molecules, said methods
comprising
administering to the subject any pharmaceutical composition comprising a
therapeutically
effective amount of any anti-M(H)DM2/4 antibody or fragment described herein
or an antibody-
drug conjugate comprising any anti-M(H)DM2/4 antibody or fragment described
herein, bound
to a cytotoxic drug, wherein the antibody or fragment is not bound to a cell-
penetrating peptide.
[0095] In one aspect, provided herein is any method described herein,
wherein the method
comprises administering an anti-M(H)DM2/4 antibody or fragment which competes
for binding
to M(H)DM2/4 with a humanized antibody or a fragment that specifically binds
to HDM2 or
MDM2, said humanized antibody or fragment comprising:
(a) a heavy chain variable region (VH) comprising a VH having an amino acid
sequence
selected from the group consisting of SEQ ID NO:287, SEQ ID NO:291, SEQ ID
NO:295, and
SEQ ID NO:299, and
(b) a light chain variable region (VL) comprising a VL having an amino acid
sequence
selected from the group consisting of SEQ ID NO:289, SEQ ID NO:293, SEQ ID
NO:297, SEQ
ID NO:301, and SEQ ID NO:303.
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[0096] In one aspect, provided herein is any method described herein,
wherein the method
comprises administering an anti-M(H)DM2/4 antibody or fragment which competes
for binding
to M(H)DM2/4 with a humanized antibody or a fragment that specifically binds
to HDM2 or
MDM2, said humanized antibody or fragment comprising:
(a) a heavy chain variable region (VH) comprising a VH having an amino acid
sequence
selected from the group consisting of SEQ ID NO:299, SEQ ID NO:305, and SEQ ID
NO:309,
and
(b) a light chain variable region (VL) comprising a VL having an amino acid
sequence
selected from the group consisting of SEQ ID SEQ ID NO:297, SEQ ID NO:307, and
SEQ ID
NO:311.
[0097] In one aspect, provided herein is any method described herein,
wherein the method
comprises administering an anti-M(H)DM2/4 antibody or fragment which competes
for binding
to M(H)DM2/4 with an antibody or a fragment that specifically binds to HDM2 or
MDM2, said
antibody or fragment comprising:
(a) a heavy chain variable region (VH) having an amino acid sequence of SEQ
ID NO:283,
and
(b) a light chain variable region (VL) having an amino acid sequence of SEQ
ID NO:285.
[0098] In one aspect, provided herein are any method described herein,
wherein the method
comprises administering an anti-M(H)DM2/4 antibody or fragment which competes
for binding
to M(H)DM2/4 with a chimeric antibody that specifically binds to HDM2 or MDM2,
said
chimeric antibody comprising:
(a) a heavy chain having an amino acid sequence of SEQ ID NO:312, and
(b) alight chain having an amino acid sequence of SEQ ID NO:313.
[0099] In one aspect, provided herein is any method described herein,
wherein the anti-
M(H)DM2/4 antibody or fragment is purified.
[00100] In one aspect, provided herein are any method described herein,
wherein the anti-
M(H)DM2/4 antibody or fragment is not bound to a cytotoxic drug.
[00101] In one aspect, provided herein are methods of selecting and
treating a subject (e.g., a
human) having a cancer, said method comprising: (a) identifying a subject
having a cancer
wherein an antibody or a fragment thereof (e.g., a labeled antibody or
fragment) that specifically
binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) binds
to the surface
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of an intact cell of the cancer; and (b) administering to the subject (i) any
M(H)DM2/4 (e.g., anti-
HDM2) antibody or fragment described herein or an antibody-drug conjugate
comprising said
antibody or fragment (e.g., an anti-M(H)DM2/4 antibody or fragment thereof
that specifically
binds to an extracellularly accessible epitope of M(H)DM2/4, wherein the
antibody or fragment
specifically binds to a peptide, wherein the sequence of the peptide consists
of SEQ ID NO:1,
SEQ ID NO:2, or SEQ ID NO:3); (ii) an antibody or a fragment thereof that
specifically binds to
an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said
antibody or
fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident
peptide), or an
antibody-drug conjugate comprising the antibody or fragment (i.e., said
antibody or fragment
that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug,
(iii) an antibody or a
fragment thereof that specifically binds to an extracellularly accessible
epitope of M(H)DM2/4
(e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic
component, (iv) any
pharmaceutical composition described herein, or (v) any antibody-drug
conjugate described
herein. The antibody or fragment thereof in step (b) can be the same or
different from the
antibody or fragment thereof in step (a). In certain embodiments, provided
herein are methods
that further comprise, before step (b), a step of determining whether the
antibody or fragment
binds to the surface of the intact cell of the cancer (e.g., using FACS or
cell-based ELISA
analysis) (using any anti-M(H)DM2/4 antibody described herein). In one
embodiment, provided
herein are methods that further comprise, before the determining step, the
step of obtaining intact
cells of the cancer (e.g., by biopsy of the cancerous tumor in the subject, or
by obtaining a blood
sample with circulating cancer cells from the subject). In one embodiment, the
method
comprises administering to the subject an antibody-drug conjugate comprising
the antibody or a
fragment thereof that specifically binds to an extracellularly accessible
epitope of M(H)DM2/4
bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a
cell-penetrating
peptide.
[00102] In one aspect, provided herein are methods for selecting a subject
(e.g., a human) for
treatment and treating cancer in the subject, said method comprising: (a)
selecting a subject
having a cancer for treatment by: (i) obtaining an intact cancer cell from the
subject (e.g., by
biopsy of the cancerous tumor in the subject, or by obtaining a blood sample
with circulating
cancer cells from the subject), and (ii) determining whether an antibody or a
fragment thereof
(e.g., a labeled antibody or fragment) that specifically binds to M(H)DM2/4
(e.g., an antibody or
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fragment that specifically binds to an extracellularly accessible epitope of
M(H)DM2/4, such as
any anti-M(H)DM2/4 antibody or fragment described herein) binds to the surface
of the intact
cancer cell obtained from the subject (e.g., using FACS or cell-based ELISA
analysis), and (b) if
the binding is detected in step (a), administering to the subject said
antibody or fragment,
wherein said antibody or fragment is not bound to a cell-penetrating peptide.
In one aspect,
provided herein are methods for selecting a subject for treatment and treating
cancer in the
subject, said method comprising: (a) selecting a subject having a cancer for
treatment by: (i)
obtaining an intact cancer cell from the subject (e.g., by biopsy of the
cancerous tumor in the
subject, or by obtaining a blood sample with circulating cancer cells from the
subject), and (ii)
determining whether an antibody or a fragment thereof that specifically binds
to HDM2 (e.g., an
antibody or fragment that specifically binds to an extracellularly accessible
epitope of HDM2,
such as any anti-M(H)DM2/4 antibody or fragment described herein) binds to the
surface of the
intact cancer cell obtained from the subject (e.g., using FACS or cell-based
ELISA analysis), and
(b) if the binding is detected in step (a), administering to the subject said
antibody or fragment,
wherein said antibody or fragment is not bound to a cell-penetrating peptide.
[00103] In certain aspects, provided herein are methods of treating cancer in
a subject who has
experienced an accelerated rate of cancer growth in response to administration
to the subject of
an inhibitor of one or more inhibitory checkpoint molecules, said method
comprising:
(a) identifying a subject who (i) has experienced accelerated rate of
cancer growth in
response to administration to the subject of an inhibitor of one or more
inhibitory checkpoint
molecules, and (ii) has a cancer wherein an antibody or a fragment thereof
that specifically binds
to an extracellularly accessible epitope of M(H)DM2/4 binds to the surface of
intact cells of the
cancer; and (b) administering to the subject: (i) an antibody or a fragment
thereof that
specifically binds to an extracellularly accessible epitope of M(H)DM2/4, or
an antibody-drug
conjugate comprising the antibody or fragment bound to a cytotoxic drug,
wherein said antibody
or fragment is not bound to a cell-penetrating peptide, (ii) an antibody or a
fragment thereof that
specifically binds to an extracellularly accessible epitope of M(H)DM2/4,
wherein said antibody
or fragment is not bound to a cytotoxic component, (iii) any anti- M(H)DM2/4
antibody or
fragment described herein, (iv) any antibody-drug conjugate described herein,
or (v) any
pharmaceutical composition described herein. In certain embodiments of the
methods, the
method further comprises before step (b), a step of determining whether any
antibody or
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fragment thereof described herein that specifically binds to an
extracellularly accessible epitope
of M(H)DM2/4 binds to the surface of intact cells of the cancer. In certain
embodiments of the
method, step (a) further comprises selecting a subject who has a gene
amplification of
M(H)DM2/4. In certain embodiments of the methods, step (a) further comprises
selecting a
subject who has an increased protein expression of M(H)DM2/4 in the cells of
the cancer relative
to the level of protein expression of M(H)DM2/4 in normal cells. In certain
embodiments of the
methods, step (a) further comprises selecting a subject who has a cancer
wherein there is an
increased binding of an antibody or a fragment thereof that specifically binds
to an
extracellularly accessible epitope of M(H)DM2/4 to the surface of intact cells
of the cancer
relative to the binding of the antibody or fragment thereof to the surface of
intact normal cells.
[00104] In certain aspects, provided herein are methods of treating cancer in
a subject who is a
hyper-progressor in response to administration of an inhibitor of one or more
inhibitory
checkpoint molecules, said methods comprising: (a) identifying a subject,
wherein the subject
has (i) a cancer wherein an antibody or a fragment thereof that specifically
binds to an
extracellularly accessible epitope of M(H)DM2/4 binds to the surface of intact
cells of the
cancer, and (ii) a gene amplification of M(H)DM2/4, or an increased protein
expression of
M(H)DM2/4 in the cells of the cancer relative to the level of protein
expression of M(H)DM2/4
in normal cells; and (b) administering to the subject: (i) an antibody or a
fragment thereof that
specifically binds to an extracellularly accessible epitope of M(H)DM2/4, or
an antibody-drug
conjugate comprising the antibody or fragment bound to a cytotoxic drug,
wherein said antibody
or fragment is not bound to a cell-penetrating peptide, (ii) an antibody or a
fragment thereof that
specifically binds to an extracellularly accessible epitope of M(H)DM2/4,
wherein said antibody
or fragment is not bound to a cytotoxic component, (iii) any anti- M(H)DM2/4
antibody or
fragment thereof described herein, (iv) any antibody-drug conjugate described
herein, or (v) any
pharmaceutical composition described herein. In certain embodiments of the
methods, the
method further comprises before step (b) a step of determining whether the
antibody or fragment
thereof that specifically binds to an extracellularly accessible epitope of
M(H)DM2/4 binds to
the surface of intact cells of the cancer. In certain embodiments of the
methods, step (a) further
comprises selecting a subject who has a gene amplification of M(H)DM2/4. In
certain
embodiments of the methods, step (a) further comprises selecting a subject who
has an increased
binding of an antibody or a fragment thereof that specifically binds to an
extracellularly
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accessible epitope of M(H)DM2/4 to the surface of intact cells of the cancer
relative to its
binding to the surface of intact normal cells.
[00105] In certain embodiments, the cancer treated in accordance with the
methods described
herein is a type of cancer that is known to metastasize. In some embodiments,
the cancer treated
in accordance with the methods described herein is an advanced stage cancer.
In other
embodiments, the cancer treated in accordance with the methods described
herein is an early
stage cancer. In specific embodiments, the cancer treated in accordance with
the methods
described herein is a metastatic cancer. The cancer being treated can be a
solid cancer or a non-
solid cancer (e.g., leukemia or lymphoma).
[00106] In certain embodiments, the cancer treated in accordance with the
methods described
herein is a cervical cancer, an endometrial cancer, an ovarian cancer, a
pancreatic cancer, a
melanoma (e.g., a uveal melanoma), a breast cancer, a triple negative breast
cancer, a colorectal
cancer (e.g. a colon cancer), a bladder cancer, an astrocytic neoplasm, a
glioblastoma, a pediatric
Rhabdomyosarcoma, or a lung cancer (e.g., a non-small cell lung carcinoma). In
specific
embodiments, the cancer treated in accordance with the methods described
herein is a melanoma,
a pancreatic cancer, a breast cancer, or an ovarian cancer. In one embodiment,
the cancer treated
in accordance with the methods described herein is a lung cancer. In one
embodiment, the
cancer treated in accordance with the methods described herein is a colorectal
cancer. In one
embodiment, the cancer treated in accordance with the methods described herein
is a colon
cancer. In one embodiment, the cancer treated in accordance with the methods
described herein
is a melanoma. In one embodiment, the cancer treated in accordance with the
methods described
herein is a pancreatic cancer. In one embodiment, the cancer treated in
accordance with the
methods described herein is a breast cancer. In one embodiment, the cancer
treated in
accordance with the methods described herein is an ovarian cancer.
[00107] The subject treated in accordance with the methods described herein
can be a human
or a non-human animal (such as a mammal). In a preferred embodiment, the
subject is a human.
[00108] In certain embodiments, the anti-M(H)DM2/4 antibodies or fragments
described
herein are administered intravenously, intraperitoneally, intramuscularly,
subcutaneously, or
intratumorally. In other embodiments, the anti-M(H)DM2/4 antibodies or
fragments described
herein are administered orally.
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[00109] In certain embodiments, the subject being treated in accordance with
the methods
described herein is further administered an additional anti-cancer therapy
that is different from
said antibody or fragment or antibody-drug conjugate (e.g., vaccine, targeted
therapy,
chemotherapy, radiotherapy, surgery, or immunotherapy). In one embodiment, the
additional
therapy is a vaccine. In one embodiment, the additional therapy is a targeted
therapy. In one
embodiment, the additional therapy is a chemotherapy (e.g., gemcitabine,
paclitaxel, nab-
paclitaxel, or a combination of gemcitabine and nab-paclitaxel). In one
embodiment, the
additional therapy is an immunotherapy. In one embodiment, the additional
therapy is a
radiotherapy. In one embodiment, the additional therapy is a surgery (e.g., to
remove part or all
of the cancerous tumor being treated). In specific embodiments, the additional
therapy is an
inhibitor of the function of one or more checkpoint inhibitory molecules
(e.g., an inhibitor, such
as an inhibitory antibody to, one or more of: CTLA-4, PD-1, PD-L1, PD-L2, TIM-
3, 0X40, and
LAG-3). In certain embodiments, the additional therapy is not a cell cycle
inhibitor. In a specific
embodiment, the subject treated using the methods described herein is not
administered a cell
cycle inhibitor during the course of treatment with said antibody or fragment.
In certain
embodiments, the anti-M(H)DM2/4 antibodies or fragments described herein are
administered
alone, without any additional anti-cancer therapy (e.g., the subject treated
using the methods
described herein is not administered an additional anti-cancer therapy during
the course of
treatment with said antibody or fragment).
[00110] In certain embodiments, the subject being treated in accordance with
the methods
described herein is further administered an additional cancer therapy that is
different from an
anti-M(H)DM2/4 antibody or fragment or antibody-drug conjugate, wherein the
additional
cancer therapy is an inhibitor of one or more of: EGFR, KRAS, STK11, ALK,
BRAF, ERBB2,
RET, ROS1, B2M, HLA, POLE, IGF-1, ERK/MAPK, PI3K/AKT, TGF-B, DNMT3A, IFN0,
JAK1/JAK2/JAK3, CD274, PTEN, ART, AND CDK.
[00111] In certain embodiments, the subject being treated in accordance with
the methods
described herein is further administered an additional cancer therapy that is
different from an
anti-M(H)DM2/4 antibody or fragment or antibody-drug conjugate, wherein the
additional
cancer therapy is a peptide inhibitor of p53-M(H)DM2/4 interaction.or a small
molecule
inhibitor of p53-M(H)DM2/4 interaction.
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[00112] In certain embodiments, the subject being treated in accordance with
the methods
described herein is further administered chemotherapy, wherein the
chemotherapy is cisplatin.
In certain embodiments, the subject being treated in accordance with the
methods described
herein is further administered chemotherapy, wherein the chemotherapy is 5-FU.
In certain
embodiments, the subject being treated in accordance with the methods
described herein is
further administered chemotherapy, wherein the chemotherapy is paclitaxel. In
certain
embodiments, the subject being treated in accordance with the methods
described herein is
further administered chemotherapy, wherein the chemotherapy is paclitaxel
formulated as
albumin-bound particles (e.g., ABRAXANE ). In certain embodiments, the subject
being
treated in accordance with the methods described herein is further
administered chemotherapy,
wherein the chemotherapy is gemcitabine (e.g., where the cancer being treated
is a pancreatic
cancer). In certain embodiments, the subject being treated in accordance with
the methods
described herein is further administered chemotherapy, wherein the
chemotherapy is nab-
paclitaxel (e.g., where the cancer being treated is a pancreatic cancer). In
certain embodiments,
the cancer is a pancreatic cancer, and the subject being treated in accordance
with the methods
described herein is further administered chemotherapy, wherein the
chemotherapy is a
combination of gemcitabine and nab-paclitaxel. In certain embodiments, the
gemcitabine and/or
nab-paclitaxel are administered in doses that are lower than doses used when
gemcitabine and/or
nab-paclitaxel are administered not in combination with an anti-cancer
antibody (such as an anti-
M(H)DM2/4 antibody or fragment described herein). In certain embodiments,
wherein the
subject is human, gemcitabine is administered in a dose that is less than
1,500 mg/m2, and/or
nab-paclitaxel is administered in a dose that is less than 300 mg/m2. In one
embodiment,
wherein the subject is human, gemcitabine is administered in a dose that is
equal to or less than
1,000 mg/m2 and/or the nab-paclitaxel is administered in a dose that is equal
to or less than 125
mg/m2. In one embodiment, wherein the subject is human, gemcitabine is
administered in a dose
that is equal to or less than 500 mg/m2 and/or the nab-paclitaxel is
administered in a dose that is
equal to or less than 62.5 mg/m2. In certain embodiments, the combination of
gemcitabine and
nab-paclitaxel is administered with a frequency of every 2 weeks or less.
[00113] In certain embodiments, the subject being treated in accordance with
the methods
described herein is resistant to other cancer therapies (e.g., vaccine,
targeted therapy,
chemotherapy, radiotherapy, surgery, or immunotherapy). In specific
embodiments, the subject
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being treated in accordance with the methods described herein is resistant to
chemotherapy. In
one embodiment, the subject being treated in accordance with the methods
described herein has a
chemotherapy-resistant ovarian cancer. In other embodiments, the subject being
treated in
accordance with the methods described herein is resistant to one or more
inhibitor of an
inhibitory immune checkpoint molecule. In other embodiments, the subject being
treated in
accordance with the methods described herein is resistant to radiotherapy.
[00114] In a specific embodiment, the anti-M(H)DM2/4 antibody or fragment used
in the
methods described herein specifically binds within amino acids 19 to 50 of SEQ
ID NO:4. In
another specific embodiment, the anti-M(H)DM2/4 antibody used in the methods
described
herein specifically binds within amino acids 154 to 167 of SEQ ID NO:4. In yet
another specific
embodiment, the anti-M(H)DM2/4 antibody used in the methods described herein
specifically
binds within amino acids 1 to 60 of SEQ ID NO:4. In yet another specific
embodiment, the anti-
M(H)DM2/4 antibody used in the methods described herein specifically binds
within amino
acids 1 to 100 of SEQ ID NO:4. In yet another specific embodiment, the anti-
M(H)DM2/4
antibody used in the methods described herein specifically binds within amino
acids 1 to 109 of
SEQ ID NO:4. In another specific embodiment, the anti-M(H)DM2/4 antibody used
in the
methods described herein specifically binds within amino acids 26 to 60 of SEQ
ID NO: 4. In
one specific embodiment, the anti-M(H)DM2/4 antibody used in the methods
described herein
specifically binds within the terminal 60 amino acids at the C-terminus of the
HDM2 on the
plasma membrane of cancer cells. In another specific embodiment, the anti-
M(H)DM2/4
antibody used in the methods described herein specifically binds within the
terminal 100 amino
acids at the C-terminus of the HDM2 on the plasma membrane of cancer cells. In
one specific
embodiment, the anti-M(H)DM2/4 antibody used in the methods described herein
specifically
binds within amino acids 101 to 200 of SEQ ID NO:4.
[00115] In particular embodiments, the anti-M(H)DM2/4 antibody or fragment
used in the
methods described herein competes for binding to M(H)DM2/4 with mouse anti-
HDM2 antibody
OP145 (which is described herein, see, e.g., Table 10). In other particular
embodiments, the
anti- M(H)DM2/4 antibody or fragment used in the methods described herein
competes for
binding to M(H)DM2/4 with mouse anti-HDM2 antibody 965 (SMP14) (which is
described
herein, see, e.g., Tables 3 and 10). In yet other particular embodiments, the
anti- M(H)DM2/4
antibody or fragment used in the methods described herein competes for binding
to M(H)DM2/4
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with rabbit anti-HDM2 antibody sc-813 (N-20) (which is described herein, see,
e.g., Table 10).
In another embodiment, the anti- M(H)DM2/4 antibody or fragment used in the
methods
described herein competes for binding to M(H)DM2/4 with rabbit anti-HDM2
antibody sc-812
(C-18) (which is described herein, see, e.g., Table 10). In another
embodiment, the anti-
M(H)DM2/4 antibody or fragment used in the methods described herein competes
for binding to
M(H)DM2/4 with mouse anti-HDM2 antibody M01, clone 1A7 (which is described
herein, see,
e.g., Table 3).
[00116] In certain aspects, provided herein are methods for treating cancer or
preventing
metastases in a subject in need thereof, said method comprising administering
to the subject any
anti-M(H)DM2/4 antibody described herein (such as an antibody that
specifically binds to an
extracellularly accessible epitope of M(H)DM2/4 on the surface of cells of
said cancer), wherein
the antibody comprises a human IgG Fc region that mediates complement-
dependent
cytotoxicity (CDC) and/or antibody-dependent cell-mediated cytotoxicity
(ADCC). In some of
these embodiments, the extracellular region of HDM2 targeted by the anti-HDM2
antibodies or
fragments used herein is within one of the following amino acid regions of
HDM2: amino acids
of SEQ ID NO: 1 (which are amino acids 1 to 15 of SEQ ID NO:4), amino acids of
SEQ ID NO:
2 (which are amino acids 15 to 25 of SEQ ID NO:4), amino acids of SEQ ID NO: 3
(which are
amino acids 475 to 491 of SEQ ID NO:4), amino acids 19 to 50 of SEQ ID NO: 4,
amino acids
50 to 60 of SEQ ID NO: 4, amino acids 100 to 110 of SEQ ID NO: 4, amino acids
154 to 167 of
SEQ ID NO: 4, amino acids 1 to 60 of SEQ ID NO: 4, or the terminal 60 amino
acids at the C-
terminus of the HDM2 on the plasma membrane of the cancer cells. In specific
embodiments of
the methods described in this paragraph, the cancer is a leukemia, a lung
cancer, a colon cancer,
a melanoma, a pancreatic cancer, a breast cancer, or an ovarian cancer.
[00117] In one aspect, provided herein are methods of diagnosing cancer in a
subject (e.g., a
human), said method comprising: (a) detecting whether an antibody or a
fragment thereof (e.g., a
labeled antibody or fragment) that specifically binds to M(H)DM2/4 (e.g.,
HDM2) binds to the
surface of intact cells of the subject, wherein the antibody or fragment is
any anti- M(H)DM2/4
antibody or fragment described herein (in a preferred example, wherein the
antibody or fragment
is any anti- M(H)DM2/4 antibody or fragment that specifically binds to a
peptide of SEQ ID
NO"1, SED ID NO:2, or SEQ ID NO:3); and (b) diagnosing the subject with cancer
if binding is
detected in step (a). In one embodiment, the method of diagnosing is an ex
vivo method. In one
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embodiment, the method of diagnosing further comprises, before step (a),
obtaining intact cells
from the subject. In one embodiment, the method of diagnosing comprises
administering the
antibody or fragment to the subject before the detecting in step (a), and
wherein the detecting is
performed by in vivo imaging of the subject.
[00118] In certain aspects, provided herein are methods of diagnosing a hyper-
progressive
disease in a subject who has cancer, said method comprising: (a) determininig
whether a gene
amplification of M(H)DM2/4 is present in the cells of the cancer of the
subject; (b) determining
whether an antibody or a fragment thereof that specifically binds to an
extracellularly accessible
epitope of M(H)DM2/4 binds to the surface of intact cells of the subject; and
(c) diagnosing the
subject with the hyper-progressive disease if the gene amplification is
determined to be present
in step (a) and binding is detected in step (b). In certrain aspects, provided
herein are methods of
diagnosing a hyper-progressive disease in a subject who has cancer, said
methods are ex vivo
methods. In certrain aspects, provided herein are methods of diagnosing a
hyper-progressive
disease in a subject who has cancer, said methods further comprising obtaining
intact cells from
the subject before step (b). In certrain aspects, provided herein are methods
of diagnosing a
hyper-progressive disease in a subject who has cancer, said methods comprising
administering
the antibody or fragment to the subject before the detecting in step (b), and
wherein the detecting
is performed by in vivo imaging of the subject. In certrain aspects, provided
herein are methods
of diagnosing a hyper-progressive disease in a subject who has cancer,
wherein, in step (b), the
antibody or fragment is labeled. In certrain aspects, provided herein are
methods of diagnosing a
hyper-progressive disease in a subject who has cancer, wherein any anti-
M(H)DM2/4 antibody
or fragment thereof described herein is used. In certrain aspects of the
methods, the subject is a
human.
[00119] In one aspect, provided herein are vaccine compositions comprising:
(i) an
immunogenic amount of a peptide, wherein the amino acid sequence of the
peptide is
MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or
CPVCRQPIQMIVLTYFP (SEQ ID NO:3), or a polynucleotide encoding the peptide; and
(ii) a
pharmaceutically acceptable carrier. In certain aspects, the peptide in the
vaccine compositions
is purified. In certain aspects, vaccine compositions further comprise an
adjuvant. In one aspect,
provided herein are methods of vaccinating a subject at risk for developing
cancer or a subject
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who has been dignosed with cancer by administering to the subject the vaccine
composition
described herein.
5.1 Terminology
[00120] As used herein, the term "HDM2" refers to the human E3 ubiquitin-
protein ligase of
UniProt Accession Number Q00987 (SEQ ID NO:4) (i.e., full-length HDM2 protein)
or a protein
product of any splice variant of the full-length HDM2 protein known in the art
or described
herein. The amino acid sequences of exemplary splice variants of the full-
length HDM2 protein
are shown as SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID N011, SEQ ID
NO:12,
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17.
[00121] As used herein, the term "MDM2" refers to the mouse E3 ubiquitin-
protein ligase of
UniProt Accession Number P23804 (SEQ ID NO:5) (i.e., full-length MDM2 protein)
or a protein
product of any splice variant of the full-length MDM2 protein known in the art
or described
herein.
[00122] As used herein, the term "M(H)DM2" refers to HDM2, MDM2, or an E3
ubiquitin-
protein ligase from species other than human and mouse that is a homolog of
HDM2 or MDM2.
[00123] As used herein, the term "HDM4" refers to the human protein of UniProt
Accession
Number 015151 (SEQ ID NO :6) (i.e., full-length HDM4 protein) or a protein
product of any
splice variant of the full-length HDM4 protein known in the art or described
herein.
[00124] As used herein, the term "MDM4" refers to the mouse protein of UniProt
Accession
Number 035618 (i.e., full-length MDM4 protein) or a protein product of any
splice variant of
the full-length MDM4 protein known in the art or described herein. The amino
acid sequence of
an exemplary splice variant of the full-length MDM4 protein is shown as SEQ ID
NO:6. Other
splice variants of the full length MDM4 protein known in the art include,
without limitation
MDM4-S, MDM4-A, MDM4-G, MDM4-XALT1/XALT2 and MDM4-211.
[00125] As used herein, the term "M(H)DM4" refers to HDM4 (also called HDMX),
MDM4
(also called MDMX), or a protein from a species other than human and mouse
that is a homolog
of HDM4 or MDM4.
[00126] As used herein, the term "M(H)DM2/4" refers to HDM2, MDM2, HDM4, MDM4,
or
a protein from a species other than human and mouse that is a homolog of HDM2,
MDM2,
HDM4 or MDM4.
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[00127] As used herein, the term "about," when used to modify a numeric value,
indicate that
deviations of up to 10% above and below the numeric value remain within the
intended meaning
of the recited value.
[00128] As used herein, the term "intact" with reference to a cell refers
to a cell that is viable
or fixed but not permeabilized.
[00129] As used herein, the term "extracellularly accessible" with reference
to an epitope of
M(H)DM2/4, refers to an epitope of M(H)DM2/4 that, when the M(H)DM2/4 is
expressed by an
intact cell, the epitope is available for binding with an extracellular
antibody (without a need for
intracellular transport of the antibody). An antibody or a fragment thereof
can be determined to
bind to an extracellularly accessible epitope of M(H)DM2/4, when the antibody,
when
extracellular, binds to M(H)DM2/4 expressed by an intact cell. As will be
clear to one skilled in
the art, the M(H)DM2/4 expressed by an intact cell in the foregoing definition
is the form of the
M(H)DM2/4 retained on the plasma membrane (i.e., as a transmembrane protein).
[00130] "Membrane-bound M(H)DM2/4" or "plasma membrane-bound M(H)DM2/4" refers
to a transmembrane M(H)DM2/4 protein variant.
[00131] As used herein, the term "VL" refers to the light chain variable
region of an antibody.
[00132] As used herein, the term "VK" refers to the kappa isotype of the light
chain variable
region of an antibody.
[00133] As used herein, the term"VH" refers to the heavy chain variable region
of an
antibody.
[00134] As used herein, the term "hyper-progression" refers to an accelerated
rate of tumor or
cancer growth therapy after administration of a therapy compared to prior to
the administration
of the therapy.
[00135] As used herein, the term "hyper-progressor" refers to a subject who
exhibits an
accelerated rate of tumor or cancer growth after administration of a therapy
compared to prior to
the administration of the therapy.
[00136] As used herein, the term "percent (%) amino acid sequence identity" or
"percent
sequence identity" with respect to a reference polypeptide sequence is defined
as the percentage
of amino acid residues in a candidate sequence that are identical with the
amino acid residues in
the reference polypeptide sequence, after aligning the sequences and
introducing gaps, if
necessary, to achieve the maximum percent sequence identity. Alignment for
purposes of
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determining percent amino acid sequence identity can be achieved in various
ways that are
known in the art, for instance, using publicly available computer software
such as BLASTp,
BLAST-2, ALIGN (e.g., ALIGN-2) or Megalign (DNASTAR) software.
6. BRIEF DESCRIPTION OF FIGURES
[00137] Figures 1A-D show that monoclonal antibodies NMC-103, NMC-204 and NMC-
303
specifically bound to NMC-P1 (SEQ ID NO:1), NMC-P2 (SEQ ID NO:2) and NMC-P3
(SEQ
ID NO:3) peptide antigens, respectively, in peptide-ELISA experiments. (A) NMC-
103 bound
to NMC-Pl peptide while NMC-204 did not show binding to NMC-Pl. (B) NMC-204
bound to
NMC-P2 peptide while NMC-103 did not bind to NMC-P2. (C) NMC-303 bound to NMC-
P3
peptide while NMC-204 did not bind to NMC-P3. (D) While NMC-103 bound to NMC-
P1, pre-
incubation of NMC-P1 peptide with NMC-103 abolished the binding of NMC-103 to
NMC-P1
peptide on the plate. In contrast, pre-incubation of NMC-103 with either NMC-
P2 or NMC-P3
did not affect the binding of NMC-103 to NMC-P1.
[00138] Figure 2 shows that monoclonal antibodies NMC-103, NMC-204, NMC-303
bound
to HDM2 recombinant protein.
[00139] Figures 3A-B show that monoclonal antibody NMC-103 bound to an
extracellularly
accessible epitope of HDM2 on intact human (A) and murine (B) cancer cells.
[00140] Figures 4A-B show that monoclonal antibody NMC-204 bound to an
extracellularly
accessible epitope of HDM2 on intact human (A) and murine (B) cancer cells.
[00141] Figures 5A-B show that monoclonal antibody NMC-303 bound to an
extracellularly
accessible epitope of HDM2 on intact human (A) and murine (B) cancer cells.
[00142] Figure 6 shows that monoclonal antibody NMC-204 bound to an
extracellularly
accessible epitope of HDM2 on intact human cancer cells but did not bind to
intact normal
human peripheral blood mononuclear cells.
[00143] Figures 7A-B depict the binding curves of the binding of monoclonal
antibodies
NMC-103 (A) and NMC-204 (B) to intact MIA PaCa-2 cells.
[00144] Figures 8A-C show that the binding of monoclonal antibody NMC-103 to
its
extracellularly accessible epitope of HDM2 on the plasma membrane of intact
human pancreatic
cancer MIA PaCa-2 cells was competed by the NMC-P1 peptide (A), the binding of
monoclonal
antibody NMC-204 to its extracellularly accessible epitope of HDM2 on the
plasma membrane
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of intact human pancreatic cancer MIA PaCa-2 cells was competed by the NMC-P2
peptide (B),
and the binding of monoclonal antibody NMC-303 to its extracellularly
accessible epitope of
HDM2 on the plasma membrane of intact human pancreatic cancer MIA PaCa-2 cells
was
competed by the NMC-P3 peptide (C).
[00145] Figure 9 shows that the binding of monoclonal antibody NMC-103 to its
extracellularly accessible epitope of HDM2 on the plasma membrane of intact
human pancreatic
cancer MIA PaCa-2 cells was competed by the full-length recombinant HDM2
protein.
[00146] Figure 10 shows the cell-ELISA binding results of monoclonal antibody
NMC-103,
monoclonal antibody NMC-204, an antibody against E-cadherin, and an antibody
against
Cytochrome-C to intact human pancreatic MiaPaCa-2 cells.
[00147] Figures 11A-C present flow cytometry data on % cells stained with
monoclonal
antibodies NMC-103 (A), NMC-204 (B), and anti-Na+/K+ ATPase a-1 (C),
respectively.
[00148] Figures 12A-D show that monoclonal antibodies NMC-103 (A and D) and
NMC-204
(B and D), but not an anti-Cytochrome-C antibody (C and D), inhibited cell
proliferation of
intact human pancreatic MIAPaCa-2 cells.
[00149] Figures 13A-C show that monoclonal antibody NMC-103 (B and C) in the
presence
of normal human serum induces complement-mediated cytotoxicity against human
pancreatic
MIAPaCa-2 cells as compared with cells treated with normal human serum in the
absence of any
antibody (A).
[00150] Figures 14A-B show the lack of binding of many commercially available
monoclonal
antibodies to either NMC-P1 (A) or NMC-P2 (B).
[00151] Figures 15A-B show that an anti-HDM2 antibody termed "MDM2 monoclonal
antibody (M01), clone 1A7" (Abnova, Cat. No. H00004193-M01) reacted with
intact cancer
cells (A), but an anti-HDM2 antibody termed "Anti-MDM2 (Ab-4) Mouse mAb
(2A9C1.18)"
(EMD Millipore, Cat. No. 0P144) and an anti-HDM2 antibody termed "Anti-MDM2
(Ab-1)
Mouse mAb (IF2)" (EMD Millipore, Cat. No. 0P46) did not react with intact
cancer cells (B).
[00152] Figure 16 depicts the effect of monoclonal antibody NMC-204 on tumor
volume of
the LL/2 syngeneic mouse model of lung cancer.
[00153] Figures 17A-B depict the effect of monoclonal antibody NMC-103 on
tumor volume
(A) and tumor cell proliferation (B) of the MC-38 syngeneic mouse model of
colon cancer.
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[00154] Figures 18A-B depict the effect of monoclonal antibody NMC-204 on
tumor volume
(A) and tumor cell proliferation (B) of the MC-38 syngeneic mouse model of
colon cancer.
[00155] Figure 19 depicts the effect of monoclonal antibody NMC-103 alone (2
mg/kg), a
combination of low dose Gemcitabine (25 mg/kg) and nab-Paclitaxel (5 mg/kg), a
combination
of low dose Gemcitabine (25 mg/kg), nab-Paclitaxel (5 mg/kg) and NMC-103
(2mg/kg), and
isotype control mouse IgG1 (2 mg/kg), respectively, on tumor volume of the
Panc-2 syngeneic
mouse model of pancreatic cancer. Treatment started when tumors in mice
reached
approximately 70 mm3.
[00156] Figure 20 depicts the DNA sequence and protein sequence of the heavy
chain
variable region and the light chain variable region, respectively, of
monoclonal antibody NMC-
103.
[00157] Figure 21 depicts the DNA sequence and protein sequence of the heavy
chain
variable region and the light chain variable region, respectively, of
monoclonal antibody NMC-
204.
[00158] Figure 22 depicts the DNA sequence and protein sequence of the heavy
chain
variable region and the light chain variable region, respectively, of
monoclonal antibody NMC-
303. The leader sequence before the DNA and protein sequences of the heavy
chain variable
region and the light chain variable region is in bold (but not underlined).
[00159] Figure 23 shows the tumor size of mice treated with the anti-HDM2
antibody termed
"MDM2 monoclonal antibody (M01), clone 1A7" (Abnova, Cat. No. H00004193-M01),
the
tumor size of mice treated with NMC-103, the tumor size of mice treated with
NMC-204, and
the tumor size of mice treated with isotype control.
[00160] Figures 24A-F. Anti-HDM2-specific antibodies stain the surface of
cancer cells but
not normal cells. Intact cells released either with either EDTA or Trypsin
were blocked with 5%
human serum albumin. Cells were then incubated with either polyclonal N-20
M(H)DM2-
specific antibody (sc-813, N-20, rabbit IgG; from Santa Cruz; "N-20") or
monoclonal
M(H)DM2-specific OP145 antibody (0P145, mouse IgGl; from Calbiochem; "0P145")
for 90
min. on ice. Another set of cells prepared under the same conditions were
incubated with the
same antibodies that were pre-incubated with their corresponding blocking
peptides before
incubation with cells. Following primary antibody incubation, cells were
washed 3 times with
ice-cold PBS followed by FITC-secondary antibody incubation for 60 min. Cells
were then
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washed 3 times with PBS and were subjected to FACS analyzer. Human melanoma
cells (24A,
24B, and 24C), primary human ovarian cancer cells (24D and 24E), and normal
mouse
splenocytes (24F). Figure 24A: area under curve #1 represents cells incubated
with goat anti-
rabbit secondary antibody only; area under curve #2 represents cells incubated
with anti-HDM2
polyclonal antibody N-20 pre-incubated with its blocking peptide followed by
goat anti-rabbit
secondary antibody; area under curve #3 represents cells incubated with anti-
HDM2 polyclonal
antibody N-20 followed by goat anti-rabbit secondary antibody. Figure 24B:
area under curve
#1 represents cells incubated with goat anti-mouse secondary antibody only;
area under curve #2
represents cells incubated with anti-HDM2 monoclonal antibody OP145 pre-
incubated with its
blocking peptide followed by goat anti-rabbit secondary antibody; area under
curve #3 represents
cells incubated with anti-HDM2 monoclonal antibody OP145 followed by goat anti-
mouse
secondary antibody. Figure 24C: area under curve #1 represents cells incubated
with goat anti-
rabbit secondary antibody only; area under curve #2 represents trypsin-
released cells incubated
with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary
antibody; area
under curve #3 represents EDTA-released cells incubated with anti-HDM2
polyclonal antibody
N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit
secondary antibody;
area under curve #4 represents EDTA-released cells incubated with anti-HDM2
polyclonal
antibody N-20 followed by goat anti-rabbit secondary antibody. Figures 24D &
E: area under
curve #1 represents cells incubated with goat anti-rabbit secondary antibody
only; area under
curve #2 represents cells incubated with anti-HDM2 polyclonal antibody N-20
pre-incubated
with its blocking peptide followed by goat anti-rabbit secondary antibody;
area under curve #3
represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by
goat anti-
rabbit secondary antibody. Figure 24F: area under curve #1 represents cells
incubated with goat
anti-rabbit secondary antibody only; area under curve #2 represents trypsin-
released cells
incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit
secondary
antibody; area under curve #3 represents EDTA-released cells incubated with
anti-HDM2
polyclonal antibody N-20 pre-incubated with its blocking peptide followed by
goat anti-rabbit
secondary antibody; area under curve #4 represents EDTA-released cells
incubated with anti-
HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody.
[00161] Figures 25A-C. (A) Human pancreatic or ovarian cancer cells and normal
human
fibroblasts were treated with normal human serum (NETS) alone, NHS + anti-HDM2
OP145
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monoclonal antibody (mouse IgGl, from Calbiochem, "0P145") or control antibody
(NHS +
Cytochrome C). Extensive cell death as evident by Propidium Iodide (PI)
staining was observed
when cancer cells were treated with the OP145 antibody (see panels b and e) in
the presence of
NHS, whereas the same antibody had no effect on the viability of normal human
fibroblasts (see
panel g). Control antibody to Cytochrome C shows no cytotoxicity (see panel c)
beyond that
observed in untreated cells (see panel a). Lack of cell death is manifested by
no or little PI
staining in panels a, c, d, f and g. The cell death marker PI was visualized
using Olympus
FluoView FV1000 Confocal Laser Scanning Biological Microscope built on the
Olympus IX81
Inverted Microscope. (B) Rodent pancreatic cancer cells were treated with anti-
HDM2
antibodies and control cytochrome C antibody. Extensive cell death as evident
by Propidium
Iodide (PI) staining was observed when cells were treated with anti-HDM2
antibodies, N-20
(polyclonal, sc-813 N-20, rabbit IgG, from Santa Cruz, "N-20") or C-18
(polyclonal, sc-812 C-
18, rabbit IgG; from Santa Cruz; "C-18") (see panels b and e), whereas, no
cytotoxicity was
observed when cells were treated with anti-HDM2 monoclonal 0P46 antibody (0P46
(Ab-1);
mouse IgGl; from Calbiochem; "0P46") (see panel d) or control Cytochrome C
antibody (see
panel e). (C) M(H)DM2-specific antibodies are cytotoxic to pancreatic cancer
MiaPaCa-2 cells
in the presence of NHS. Quantitative representations of M(H)DM2-specific
antibody-dependent
complement cytotoxicity against human pancreatic cancer cells. Cells treated
with anti-
M(H)DM2 (C-18) antibody in the presence of NHS demonstrated cytotoxicity over
15-30 min.
post-treatment, whereas anti-HDM2 0P46 shows no cytotoxic effect beyond that
observed when
cells were treated with control anti-Cytochrome C antibody or when cells were
treated with NHS
in the absence of anti-M(H)DM2 antibodies.
[00162] Figure 26 shows the tumor size of mice treated with anti-HDM2 antibody
OP145 and
the tumor size of mice treated with PBS control (in Panc02 syngeneic mouse
model of pancreatic
cancer). The x axis shows days after tumor cell injection into the mice. The y
axis shows tumor
volume in mm3. The arrow shows the day on which the treatment was started.
[00163] Figure 27 depicts the effect of monoclonal antibody NMC-103 alone (10
mg/kg), a
combination of low dose Gemcitabine (25 mg/kg) and nab-Paclitaxel (5 mg/kg), a
combination
of low dose Gemcitabine (25 mg/kg), nab-Paclitaxel (5 mg/kg) and NMC-103 (10
mg/kg), and
isotype control mouse IgG1 (10 mg/kg), respectively, on tumor volume of the
Panc-2 syngeneic
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mouse model of pancreatic cancer (number of mice/group = 8). Treatment in this
study started
when tumors in mice reached approximately 80-100 mm3.
[00164] Figure 28 depicts a Kaplan Meier survival analysis demonstrating
survival benefit in
mice that received NMC-103 alone or in combination with chemotherapy when
compared to
chemotherapy alone or control antibody under the experimental conditions
described in Figure
27.
[00165] Figure 29 shows that mice previously treated with NMC-103 as described
in Figure
27, become immune to tumor re-challenging after drug withdrawal. To evaluate
the long-term
anti-tumor effect of NMC-103, at 62 days after the start of the study, mice
that had previously
received with a combination of G + nP, mice in group C (mice that had been
previously treated
with NMC-103) and group D (mice that had been previously treated with a
combination of
NMC-103 + G + nP) were re-challenged by a second round of Panc-2 inoculation
(subcutaneous
injection of 2x106 cells/mouse), on the left dorsal flank. Tumor growth was
monitored for 10
days at which point, a tumor of 90 mm3 was measured in the mice from group B.
No tumor was
observed in mice from the two groups that had previously received NMC-103
antibody (Groups
C and D).
[00166] Figure 30 depicts the effect of a single dose of monoclonal antibody
NMC-103 (10
mg/kg) or isotype control mouse IgG1 (10 mg/kg), when added to a treatment
regimen of a
combination of low dose Gemcitabine (25 mg/kg) and nab-Paclitaxel (5 mg/kg),
in the treatment
of large size tumors (i.e. advanced cancers) on tumor volume of the Panc-2
syngeneic mouse
model of pancreatic cancer. Mice were treated with pancreatic cancer standard
of care
(Gemcitabine (25 mg/kg) + nab-Paclitaxel (5 mg/kg)) for 19 days at which point
they reached a
tumor size of approximately 450 mm3. Mice were then randomly divided in 2
groups that
received a single dose of an isotype control mouse IgG1 (10 mg/kg) or NMC-103
(10 mg/kg).
As shown in this figure, a single i.p. injection of NMC-103 reduced the tumor
size by almost half
6 days post treatment (from 438 mm3 to 233 mm3).
[00167] Figure 31 depicts the effect of monoclonal antibody NMC-103 on tumor
volume of
the MC-38 syngeneic mouse model of colon cancer. As shown in this figure, mice
treated with
NMC-103 at 10 mg/kg, 2 times per week for 2 weeks reached an average tumor
size of 210 mm3,
while mice in the group that weretreated with isotype control antibody at 10
mg/kg grew rapidly
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and reached 1168 mm3 by day 12. When compared to mice treated with NMC-103 at
0.4 mg/kg
(Figure 17), these data support the dose-dependent anti-tumor effect of NMC-
103 antibody.
[00168] Figures 32A and 32B depict the effect of a chimeric version of
monoclonal antibody
NMC-303. Isotype class-switching was performed on a mouse NMC-303 to convert
it from a
mouse IgM to a chimeric IgGl. The mouse Heavy and Light chain variable regions
were cloned
into a human Ig gamma-1 chain and human Ig kappa chain as constant region. A
total of eight
(8) BALB/c mice were injected subcutaneously with CT-26. Mice were then
divided into two
groups (n=4) that received: A) control antibody (10 mg/kg) or B) chimeric
version of NMC-303
antibody (10 mg/kg) two times a week for 3 weeks. Figure 32A shows that by day
24 post tumor
inoculation, mice treated with chimeric version of NMC-303 (10 mg/kg) reached
an average
tumor size of 726 mm3, while mice treated with control antibody (10 mg/kg) had
an average
tumor size of 1746 mm3. Furthermore, Figure 32B shows the individual mouse
tumor sizes on
day 24 post tumor inoculation.
[00169] Figure 33 shows the cell-based-ELISA results of the binding of
humanized
monoclonal NMC-H103 (VH4/VK3) and chimeric monoclonal (NMC-C303) antibodies to
membrane-bound MDM2 on intact mouse Lewis Lung (LL/2) cancer cells. An
antibody to
plasma membrane marker E-Cadherin also binds to intact un-permeabilized mouse
LL/2 cancer
cells, while antibodies to cytoplasmic proteins cytochrome C, cyclin D1 and
Bc1-2 do not bind to
intact un-permeabilized cells. All primary antibodies were used at 51.tg/mL.
Anti-mouse and
anti-human secondary HRP conjugated antibodies were used at 1:2000 to 1:4000
range.
[00170] Figures 34A-B show the cell-based ELISA binding results demonstrating
the binding
of humanized NMC-H103 (VH4/VK3) and chimeric (NMC-C303) antibodies to a panel
of
intact, un-permeabilized human (34A) and murine (34B) cancer cells. No binding
beyond
background staining was shown using anti-human secondary antibody.
[00171] Figures 35A-B. Figure 35A shows the cell-based ELISA results
demonstrating the
binding of humanized monoclonal antibody NMC-H103 (VH4/VK3) to intact, un-
permeabilized
mouse LL/2 cancer cells, while pre-incubation of humanized monoclonal antibody
NMC-H103
(VH4/VK3) (51.tg/mL) with NMC-P1, reduces the binding efficiency of humanized
monoclonal
antibody NMC-H103 (VH4/VK3) to its target. Pre-incubation of chimeric
monoclonal antibody
NMC-C303 with NMC-Pi has no effect on its binding to mouse LL/2 cancer cells.
Figure 35B
shows flow cytometry results on live (7AAD negative) cells showing the binding
of humanized
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monoclonal antibody NMC-H103 (VH4/VK3) (left panel; solid black curve) and
chimeric
monoclonal antibody NMC-C303 (right panel; solid black curve) to live OVCAR-3
(upper
panel) and mouse LL/2 cancer cells (lower panel). Dashed line curves show the
reduced binding
of humanized monoclonal NMC-H103 (VH4/VK3) and chimeric monoclonal NMC-C303
antibodies to OVCAR-3 and mouse LL/2 cancer cells, when cells are pre-treated
with trypsin.
[00172] Figures 36A-B show results from in vivo experiments utilizing NOD scid
gamma
mice (NSG: NOD.Cg-Prkdc"1dIl2rg"lwil/SzJ) immune-deficient mice demonstrating
that the
anti-cancer activity of the humanized monoclonal NMC-H103 (VH4/VK3) and the
chimeric
monoclonal NMC-C303 antibodies is significantly more effective in presence of
normal human
peripheral blood monocytes (Hu-PBMCs) than in the absence of Hu-PBMCs. In
Figure 36A,
mouse colon cancer MC-38 cells subcutaneously inoculated in NSG mice (n=12)
and allowed to
reach a tumor volume of 400 mm3 at which point they were randomly divided into
3 groups (n=4
mouse/group). Hu-PBMCs (7x106Hu-PBMCs/mouse) were injected in tail vein of
mice in
group 1 (filled triangle) and group 3 (filled circle), whereas mice in group 2
(filled square) did
not receive any Hu-PBMCs. A week later (tumor volume of 600 mm3), mice in
group 1 received
intraperitoneal (i.p.) injection of isotype control antibody (10 mg/kg), while
mice in groups 2 and
3 received i.p. injection of chimeric monoclonal NMC-C303 (10 mg/kg). In
Figure 36B, human
lung cancer A549 cells were subcutaneously injected into NSG mice (n=12) and
allowed to reach
a tumor volume of 300 mm3, at which time mice were randomly divided into 3
groups (n=4 per
group). Hu-PBMCs (7x106Hu-PBMCs/mouse) were injected in tail vein of mice in
group 1 (left
bar) & 3 (right bar), whereas mice in group 2 (middle bar) did not receive any
Hu-PBMCs. Mice
in group 1 received a single i.p.injection of isotype control antibody
(10mg/kg), while mice in
groups 2 and 3 received a single i.p. injection of humanized monoclonal
antibody NMC-H103
(VH4/VK3) (10mg/kg). Subcutaneous tumors were measured 4 days post antibody
treatment.
[00173] Figures 37A-C show in vitro cell proliferation MTT assay results of
M109 cells
treated with anti-PD1 antibody (A), results of the in vivo effect of anti-PD1
antibody on the
growth of M109 tumor in NSG mice (B), and flow cytometry results on freshly
excised tumors
from mice treated with isotype control or anti-PD1 antibodies. Figure 37A
shows MTT cell
proliferation assay results of M109 cells treated with 751.tg/mL (middle bar)
or 10011g/mL (right
bar) of anti-PD1 antibody for 48 hours. As control, M109 cell proliferation
was also measured in
presence of isotype control antibody at 10011g/mL (left bar). Figure 37B
depicts the effect of
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anti-PD1 antibody on the growth of M109 tumor in mice. NSG mice (n=8) were
subcutaneously
inoculated with M109 cells (50,000 cells/mouse). Two weeks later, mice were
randomly divided
in two groups. Mice in group 1 (solid curve) received isotype control antibody
(12.5 mg/kg;
xl/week), while mice in group 2 (dashed curve) received anti-PD1 antibody
(12.5 mg/kg;
xl/week). In Figure 37C, tumors from mice in groups 1 and 2 (n=4) were
assessed for the
expression of plasma membrane-bound M(H)DM2 by flow cytometry with humanized
monoclonal antibody NMC-H103 (VH4/VK3). Binding of humanized monoclonal
antibody
NMC-H103 (VH4/VK3) to cells of tumors from mice in group 1 (left curve) and
group 2 (right
curve) is shown.
[00174] Figures 38A-B show single cycle (A) and multiple cycle (B) sensorgrams
data and
fitted curves (1:1 binding model) for binding to NMC-P1 of chimeric monoclonal
antibody
NMC-C103 (VHO/VKO) and humanized monoclonal antibody NMC-H103 variants having
VH/
and VK combinations identified in the figure. The sequences of the respective
VH and VK of
humanized monoclonal antibody NMC-H103 variants are provided in Tables 15-27
and Section
11.
7. DETAILED DESCRIPTION
[00175] Provided herein are antibodies or antigen-binding fragments thereof
that specifically
bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2). In a
specific
embodiment, the extracellularly accessible epitope is contained within SEQ ID
NO:1, SEQ ID
NO:2, or SEQ ID NO:3. Antibodies provided herein are described in Section 5.1,
below. Also
provided herein are antibody-drug conjugates comprising an antibody or
fragment that
specifically binds to an extracellularly accessible epitope of M(H)DM2/4
(e.g., HDM2)
described herein bound (e.g., covalently) to a cytotoxic drug. Also provided
herein are
antibodies or antigen-binding fragments thereof that specifically bind to an
extracellularly
accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibodies or
fragments are not
bound to a cytotoxic component.
[00176] Also provided herein are pharmaceutical compositions comprising an
antibody or
fragment that specifically binds to an extracellularly accessible epitope of
M(H)DM2/4 (e.g.,
HDM2) described herein. In certain embodiments, such pharmaceutical
compositions comprise
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a therapeutically effective amount of such antibody or fragment (i.e., an
amount that can be used
to treat a cancer in a subject, e.g. by achieving one or more anti-tumor
effects described herein).
[00177] Also provided herein are nucleic acids encoding the antibodies and
antigen-binding
fragments described herein. In certain embodiments, provided herein are
vectors and cells
comprising nucleic acids encoding such antibodies or antigen-binding fragments
thereof Cells
recombinantly producing the antibodies or antigen-binding fragments thereof
described herein
are also provided.
[00178] Chimeric antigen receptors (CARs) are engineered receptors that
provide both antigen
binding and immune cell activation functions (Sadelain et al., 2013, Cancer
Discovery 3:388-
398). Also provided herein are CARs comprising a single-chain variable
fragment (scFv) that
specifically binds to an extracellularly accessible epitope of M(H)DM2/4, such
as a scFv
comprising the VH and VL of an anti-M(H)DM2/4 antibody described herein, fused
via a linker
to a transmembrane domain (e.g., of CD3 zeta) fused to an intracellular T cell
activation domain
such as CD3 zeta intracellular domain, optionally further fused to a co-
stimulatory domain (e.g.,
CD28 intracellular domain). T cells expressing such CARs are also provided.
[00179] Also provided herein is a peptide, the amino acid sequence of which is
MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or
CPVCRQPIQMIVLTYFP (SEQ ID NO:3). The peptide can be, for example, synthetic or
recombinant. In some embodiments, the peptide is purified. In some
embodiments, the peptide
islabeled with a detectable marker (e.g., a fluorescent marker or an isotope).
In some
embodiments, the peptide is tagged (e.g., with a GST, His, Strep, myc, FLAG,
or HA tag). In
some embodiments, a cysteine is added at one of the ends of the peptide(which
may allow for
linkage to a carrier protein). In some embodiments, the peptide islinked to a
carrier protein (e.g.,
linked to Keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA),
ovalbumin,
thyroglobulin, tetanus toxoid, or diphtheria toxoid). Also provided herein are
nucleic acids
encoding a peptide described herein. Also provided herein are vectors and
cells comprising a
nucleic acid encoding a peptide described herein. Cells recombinantly
producing a peptide
described herein are also provided. Also provided herein are uses of the
peptides described
herein as immunogens. The peptides of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID
NO:3
described herein contain extracellularly accessible epitopes of MDM2 and HDM2.
In particular,
provided herein are methods of making an anti-M(H)DM2/4 antibody (e.g., an
antibody that
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specifically binds to M(H)DM2/4) by immunizing an animal (e.g., a mouse or a
rabbit) with a
peptide described herein. Also provided herein are methods of screening
antibodies for binding
to one or more of thepeptides described herein (for example, using ELISA with
a plate-bound
peptide). Also provided herein are methods of identifying an anti-M(H)DM2/4
antibody suitable
for therapeutic use in treating cancer or preventing metastasis, or suitable
for use in diagnosis of
cancer, by contacting an anti-M(H)DM2/4 antibody with a peptide described
herein under
conditions suitable for binding between the antibody and the peptide, and
detecting or measuring
binding between the antibody and the peptide that occurs, where the detection
of binding
between the antibody and the peptide indicates that the antibody is suitable
for the therapeutic or
diagnostic use. For example, provided herein are methods of identifying an
anti-M(H)DM2/4
antibody suitable for therapeutic use by contacting an anti-M(H)DM2/4 antibody
with a peptide
described herein under conditions suitable for binding between the antibody
and the peptide, and
detecting or measuring binding between the antibody and the peptide that
occurs, and, if the
binding between the antibody and the peptide is detected, using the antibody
in the methods of
treating cancer described herein. In another example, provided herein are
methods of identifying
an anti-M(H)DM2/4 antibody suitable for diagnostic use by contacting an anti-
M(H)DM2/4
antibody with a peptide described herein under conditions suitable for binding
between the
antibody and the peptide, and detecting or measuring binding between the
antibody and the
peptide that occurs, and, if the binding between the antibody and the peptide
is detected, using
the antibody in the methods of diagnosing cancer described herein.
[00180] Also provided herein are methods for treating cancer, inhibiting tumor
growth or
proliferation, inhibiting tumor progression, and/or preventing metastases in a
subject by
administering to the subject an anti-M(H)DM2/4 antibody or fragment described
herein, in
particular, an antibody or a fragment thereof that specifically binds to an
extracellularly
accessible epitope of M(H)DM2/4 (in particular, a region exposed on the plasma
membrane
surface of cancer cells). Preferably the antibody or fragment thereof
specifically binds to an
extracellularly accessible epitope of HDM2 (in particular, a region exposed on
the plasma
membrane surface of cancer cells). In certain embodiments, the antibody or
fragment (e.g., for
use in the methods described herein) does not bind or only minimally binds to
the plasma
membrane surface of normal cells of the tissue type from which the cancer in
the subject
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originates. In a specific embodiment, the anti-M(H)DM2/4 antibody or fragment
is any chimeric
or humanized anti-M(H)DM2 antibody described herein.
[00181] Also provided herein are methods for treating cancer in a subject who
has
experienced an accelerated rate of cancer growth in response to administration
of an inhibitor of
one or more inhibitory checkpoint molecules by administering to the subject an
anti-M(H)DM2/4
antibody or fragment described herein, in particular, an antibody or a
fragment thereof that
specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (in
particular, a region
exposed on the plasma membrane surface of cancer cells). Preferably the
antibody or fragment
thereof specifically binds to an extracellularly accessible epitope of HDM2
(in particular, a
region exposed on the plasma membrane surface of cancer cells). In certain
embodiments, the
antibody or fragment (e.g., for use in the methods described herein) does not
bind or only
minimally binds to the plasma membrane surface of normal cells of the tissue
type from which
the cancer in the subject originates. In a specific embodiment, the anti-
M(H)DM2/4 antibody or
fragment is any chimeric or humanized anti-M(H)DM2 antibody described herein.
[00182] Where the subject being treated is a human, in certain embodiments, an
antibody or a
fragment thereof used herein specifically binds to an extracellularly
accessible epitope of HDM2
and/or HDM4 (a region exposed on the plasma membrane surface of cancer cells).
In one
embodiment of treating a human, an antibody or a fragment thereof used herein
specifically
binds to an extracellularly accessible epitope of HDM2 (a region exposed on
the plasma
membrane surface of cancer cells) (optionally, such antibody or fragment that
does not bind to
HDM4). In one embodiment of treating a human, an antibody or a fragment
thereof used herein
specifically binds to an extracellularly accessible epitope of HDM4 (a region
exposed on the
plasma membrane surface of cancer cells) (optionally, such antibody or
fragment that does not
bind to HDM2).
[00183] Where the subject is a non-human animal (e.g., a mammal such as a dog
or a cat), an
antibody or a fragment thereof used herein binds to an extracellularly
accessible epitope of
M(H)DM2/4 (a region exposed on the plasma membrane surface of cancer cells),
where the
M(H)DM2/4 is a homologue of HDM2 and/or HDM4 expressed in such animal. In one
embodiment, an antibody or a fragment thereof used herein binds to an
extracellularly accessible
epitope of M(H)DM2 (a region exposed on the plasma membrane surface of cancer
cells), where
the M(H)DM2 is a homologue of HDM2 expressed in such animal (optionally, such
antibody or
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fragment does not bind to M(H)DM4). In one embodiment, an antibody or a
fragment thereof
used herein binds to an extracellularly accessible of M(H)DM4 (a region
exposed on the plasma
membrane surface of cancer cells), where the M(H)DM4 is a homologue of HDM4
expressed in
such animal (optionally, such antibody or fragment does not bind to M(H)DM2).
[00184] The description of the invention that follows is largely in terms of
HDM2 and
antibodies and antibody fragments thereto, which shall be understood to be for
use in treating a
human; it will be clear to one skilled in the art that the description also
should be deemed
applicable to: (i) HDM4 and antibodies and antibody fragments thereto, and use
thereof for
treatment of humans (unless indicated otherwise explicitly or by context), and
(ii) M(H)DM2/4
and antibodies and antibody fragments thereto, and use thereof for treatment
of non-human
animals, e.g., mammals (unless indicated otherwise explicitly or by context).
In preferred
embodiments, the patients or subjects being treated using the methods
described herein are
human.
[00185] In preferred embodiments, the anti-HDM2 antibody or a fragment thereof
used in
accordance with the methods described herein mediates complement-dependent
cytotoxicity
(CDC), mediates antibody-dependent cell-mediated cytotoxicity (ADCC), and/or
is bound to a
cytotoxic drug or drugs (e.g., is an antibody-drug conjugate). In preferred
embodiments, the
invention provides for the use of antibodies that mediate complement-dependent
cytotoxicity
(CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).
[00186] In certain embodiments, the anti-HDM2 antibody or a fragment thereof
used in
accordance with the methods described herein is not bound to a cell-
penetrating peptide. Cell
penetrating peptides can insert into a cell plasma membrane and transport
molecules to which
they are attached into the cell. Such cell-penetrating peptides include,
without limitation, a
membrane resident peptide (MRP), Membrane Transduction Domain of Antennapedia,
trans-
activating transcriptional activator (TAT), and a Penetratin peptide. In
certain embodiments, the
anti-HDM2 antibody or a fragment thereof used in accordance with the methods
described herein
is not attached to a membrane resident peptide (MRP), Membrane Transduction
Domain of
Antennapedia, TAT, and/or a Penetratin peptide. In certain embodiments, the
anti-HDM2
antibody or a fragment thereof used in accordance with the methods described
herein is not
attached to any peptide sequence that can insert into the lipid bilayer of the
plasma membrane of
cells. In one embodiment, the anti-HDM2 antibody or a fragment thereof used in
accordance
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with the methods described herein is not attached to an MRP. In one
embodiment, the anti-
HDM2 antibody or a fragment thereof used in accordance with the methods
described herein is
not attached to a Penetratin peptide.
[00187] The examples set forth herein demonstrate that HDM-2 targeting
antibodies alone are
selectively cytotoxic to cancer cells. Further, as set forth in the examples
herein, it has been
demonstrated that extracellularly accessible epitopes of HDM2 are appropriate
therapeutic
targets for anti-HDM2 antibodies, and that cancer cells expressing HDM2 on
their surface can be
successfully targeted and destroyed with antibodies to such extracellular
regions of HDM2. In
particular, the data presented in the examples demonstrate that select HDM2-
specific antibodies
can bind to the extracellularly accessible sequences of HDM2 on the surface
membrane of intact
cancer cells, while exhibiting minimal binding to the surface membrane of
normal human blood
mononuclear cells. In addition, the data presented in the examples show that
such HDM2-
specific antibodies can inhibit the growth of cancer cells in vitro and in
vivo, strongly suggesting
that they can be used as therapeutic agents in vivo. Further, the data
presented in the examples
show that such HDM2-specific antibodies can have a synergistic anti-tumor
effect when
combined with chemotherapeutic drugs. In addition, the data presented in the
examples show
that certain chimeric and humanized HDM2-specific antibodies described herein
have anti-tumor
effect. Further, the data presented in the examples show that subjects whose
tumors undergo
hyper-progression in response to treatment with an inhibitor of an inhibitory
immune checkpoint
molecule have an increased level of plasma-membrane-bound M(H)DM2 that could
be targeted
with HDM2-specific antibodies that bind to the extracellularly accessible
epitopes of M(H)DM2
(e.g., certain chimeric and humanized HDM2-specific antibodies described
herein).
7.1 Antibodies
[00188] Provided herein are antibodies or antigen-binding fragments thereof
that (immuno)
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) (a region
exposed on the plasma membrane surface of cells). "Specifically
bind[s]/binding" as those terms
are used herein does not exclude cross-reactivity of the antibody or antigen-
binding fragment;
thus, for example, antibodies or antigen-binding fragments thereof that
(immuno) specifically
bind to an extracellularly accessible epitope of HDM2 exposed on the plasma
membrane surface
of cells may also specifically bind to (cross-react with) MDM2. In particular,
provided herein
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are anti-M(H)DM2/4 antibodies and fragments thereof that (immuno) specifically
bind to an
extracellularly accessible epitope of M(H)DM2/4 and that have an anti-tumor
effect (e.g., inhibit
tumor growth in vivo). In specific embodiments, an antibody or an antigen-
binding fragment
thereof specifically binds an epitope of M(H)DM2/4 that is extracellularly
accessible on cancer
cells but not on non-cancer cells (e.g., non-cancerous cells of the same organ
type or tissue type as
the cancer cells). In other specific embodiments, an antibody or an antigen-
binding fragment
thereof specifically binds an epitope of M(H)DM2/4, exposure or accessibility
of which on the
plasma membrane surface of cancer cells is increased relative to its exposure
or accessibility on
the plasma membrane surface of non-cancer cells (e.g., non-cancerous cells of
the organ or
tissues of the host). Also provided herein are antibodies or antigen-binding
fragments thereof
that (immuno) specifically bind to an extracellularly accessible epitope of
M(H)DM2/4 (e.g.,
HDM2), which are not bound to a cell-penetrating peptide (e.g., a membrane
resident peptide).
[00189] Also provided herein are antibodies or antigen-binding fragments
thereof that
(immuno) specifically bind to M(H)DM2/4 (e.g., HDM2), in particular to an
extracellularly
accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically
binds to a
peptide the sequence of which peptide consists of MCNTNMSVPTDGAVT (SEQ ID
NO:1),
TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In certain
embodiments, provided herein are antibodies or antigen-binding fragments
thereof that
(immuno) specifically bind to M(H)DM2/4 (e.g., HDM2), in particular to an
extracellularly
accessible epitope of M(H)DM2/4wherein the antibody or fragment specifically
binds to a
peptide the sequence of which peptide consists of MCNTNMSVPTDGAVT (SEQ ID
NO:1),
TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3); and wherein
such antibodies or fragments have an anti-tumor effect in vivo, and/or wherein
such antibodies or
fragments are not bound to a cell-penetrating peptide.
[00190] Also provided herein are anti- M(H)DM2/4 antibodies and fragments
having heavy
chain variable regions and/or light chain variable regions described herein
(see, e.g., having
sequences of heavy chain variable regions and/or light chain variable regions
of antibodies
NMC-103, NMC-204 and NMC-303 provided herein, see, e.g., Section 8 and Figures
20-22).
Also provided herein are anti-M(H)DM2/4 antibodies and fragments having one or
more
complementarity determining regions (CDRs) described herein (see, e.g., CDRs
provided in
Tables 4-9 and Figures 20-22).
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[00191] Also provided herein are chimeric and humanized anti- M(H)DM2/4
antibodies and
fragments having heavy chain variable regions and/or light chain variable
regions described
herein (e.g., having sequences of heavy chain variable regions and/or light
chain variable regions
of antibodies NMC-C303, NMC-C103 and variants of NMC-H103 provided herein,
see, e.g.,
Sections 9 and 11, and Tables 11-27). Also provided herein are anti-M(H)DM2/4
antibodies and
fragments having one or more complementarity determining regions (CDRs)
described herein
(see, e.g., CDRs provided in Tables 11-27) (e.g., chimeric or humanized
antibodies). Also
provided herein are chimeric and humanized anti-M(H)DM2/4 antibodies and
fragments having
one or more framework regions (FR) described herein (see, e.g., heavy chain
framework regions
(HFR) and light chain framework regions (LFR) provided in Tables 11-27).
[00192] CDRs are defined in various ways in the art, including the Kabat,
Chothia, AbM,
Contact, and IMGT. In certain aspects, the CDRs of an antibody can be defined
according to the
Kabat system, which is based on sequence variability (see, e.g., Kabat EA & Wu
TT (1971) Ann
NY Acad Sci 190: 382-391; Kabat EA et al., (1991) Sequences of Proteins of
Immunological
Interest, Fifth Edition, U.S. Department of Health and Human Services, NIE
Publication No. 91-
3242. In a specific embodiment, with respect to the Kabat system, (i) the VH
CDR1 is present at
amino acid positions 31 to 35 of the heavy chain; (ii) the VH CDR2 is present
at amino acid
positions 50 to 68 or 50 to 66 of the heavy chain; and (iii) the VH CDR3 is
present at amino acid
positions 101 to 105 or 99 to 104 or 99 to 106 of the heavy chain. In a
specific embodiment,
with respect to the Kabat system, (i) the VL CDR1 is present at amino acid
positions 24 to 39 or
24 to 34 of the light chain; (ii) the VH CDR2 is present at amino acid
positions 55 to 61 or 50 to
56 of the light chain; and (iii) the VH CDR3 is present at amino acid
positions 94 to 102 or 89 to
97 of the light chain. As is well known to those of skill in the art, with
respect to the Kabat
system, the actual linear amino acid sequence of the antibody variable domain
can contain fewer
or additional amino acids due to a shortening or lengthening of a framework
region (FR) and/or
CDR and, as such, an amino acid's Kabat number is not necessarily the same as
its linear amino
acid number. The Kabat CDR positions may vary depending on the antibody, and
may be
determined according to methods known in the art. In a specific embodiment,
the CDRs of the
antibodies described herein are determined using the Kabat system.
[00193] In certain aspects, the CDRs of an antibody can be defined according
to the Chothia
system, which is based on the location of immunoglobulin structural loop
regions (see, e.g.,
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Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et at.,
(1997) J Mol Biol
273: 927-948; Chothia C et at., (1992) J Mol Biol 227: 799-817; Tramontano A
et at., (1990) J
Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226). The term "Chothia
CDRs," and like
terms are recognized in the art and refer to antibody CDR sequences as
determined according to
the method of Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917, which will
be referred to
herein as the "Chothia CDRs" (see also, e.g., U.S. Patent No. 7,709,226 and
Martin, A., "Protein
Sequence and Structure Analysis of Antibody Variable Domains," in Antibody
Engineering,
Kontermann and Dilbel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin
(2001)). In a
specific embodiment, with respect to the Chothia system, using the Kabat
numbering system of
numbering amino acid residues in the VH region, (i) the VH CDR1 is present at
amino acid
positions 26 to 32 of the heavy chain; (ii) the VH CDR2 is present at amino
acid positions 52 to
59 or 52 to 57 of the heavy chain; and (iii) the VH CDR3 is present at amino
acid positions 101
to 105 or 99 to 104 or 99 to 106 of the heavy chain. In a specific embodiment,
with respect to
the Chothia system, using the Kabat numbering system of numbering amino acid
residues in the
VL region, (i) the VL CDR1 is present at amino acid positions 24 to 39 or 24
to 34 of the light
chain; (ii) the VL CDR2 is present at amino acid positions 55 to 61 or 50 to
56 of the light chain;
and (iii) the VL CDR3 is present at amino acid positions 94 to 102 or 89 to 97
of the light chain.
The Chothia CDR positions may vary depending on the antibody, and may be
determined
according to methods known in the art. In a specific embodiment, the CDRs of
the antibodies
described herein are determined using the Chothia system.
[00194] In certain aspects, the CDRs of an antibody can be defined according
to the AbM
system, which is based on AbM hypervariable regions that represent a
compromise between the
Kabat CDRs and Chothia structural loops, and where CDRs are determined using
Oxford
Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In
a specific
embodiment, with respect to the AbM system, using the Kabat numbering system
of numbering
amino acid residues in the VH region, (i) the VH CDR1 is present at amino acid
positions 26 to
35 of the heavy chain; (ii) the VH CDR2 is present at amino acid positions 50
to 61 or 50 to 59
of the heavy chain; and (iii) the VH CDR3 is present at amino acid positions
101 to 105 or 99 to
104 or 99 to 106 of the heavy chain. In a specific embodiment, with respect to
the AbM system,
using the Kabat numbering system of numbering amino acid residues in the VL
region, (i) the
VL CDR1 is present at amino acid positions 24 to 39 or 24 to 34 of the light
chain; (ii) the VH
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CDR2 is present at amino acid positions 55 to 61 or 50 to 56 of the light
chain; and (iii) the VH
CDR3 is present at amino acid positions 94 to 102 or 89 to 97 of the light
chain. The AbM CDR
positions may vary depending on the antibody, and may be determined according
to methods
known in the art. In a specific embodiment, the CDRs of the antibodies
described herein are
determined using the AbM numbering system.
[00195] In certain aspects, the CDRs of an antibody can be defined according
to the IMGT
system (see "IMGT , the international ImMunoGeneTics information system
website
imgt.org, founder and director: Marie-Paule Lefranc, Montpellier, France; see,
e.g., Lefranc, M.-
P., 1999, The Immunologist, 7:132-136 and Lefranc, M.-P. et al., 1999, Nucleic
Acids Res.,
27:209-212, both of which are incorporated herein by reference in their
entirety). In a specific
embodiment, with respect to the IMGT system, (i) the VH CDR1 is present at
amino acid
positions 27 to 33 or 26 to 33 of the heavy chain; (ii) the VH CDR2 is present
at amino acid
positions 51 to 60 or 51 to 58 of the heavy chain; and (iii) the VH CDR3 is
present at amino acid
positions 99 to 105 or 97 to 103 of the heavy chain. In a specific embodiment,
with respect to
the IMGT system, (i) the VL CDR1 is present at amino acid positions 27 to 37
of the light chain;
(ii) the VH CDR2 is present at amino acid positions 55 to 57 of the light
chain; and (iii) the VH
CDR3 is present at amino acid positions 94 to 102 of the light chain. The IMGT
CDR positions
may vary depending on the antibody, and may be determined according to methods
known in the
art. In a specific embodiment, the CDRs of the antibodies described herein are
determined using
the IMGT system.
[00196] In certain aspects, the CDRs of an antibody can be defined according
to the Contact
system. The Contact definition is based on an analysis of the available
complex crystal
structures (bioinf. org.uk/abs) (see MacCallum RM et al., (1996) J Mol Biol 5:
732-745; see also,
e.g., Martin A. "Protein Sequence and Structure Analysis of Antibody Variable
Domains," in
Antibody Engineering, Kontermann and Dithel, eds., Chapter 31, pp. 422-439,
Springer-Verlag,
Berlin (2001)). In a specific embodiment, with respect to the Contact system,
using the Kabat
numbering system of numbering amino acid residues in the VH region, (i) the VH
CDR1 is
present at amino acid positions 30 to 35 of the heavy chain; (ii) the VH CDR2
is present at
amino acid positions 47 to 61 or 47 to 59 of the heavy chain; and (iii) the VH
CDR3 is present at
amino acid positions 99 to 104 or 97 to 103 or 97 to 105 of the heavy chain.
In a specific
embodiment, with respect to the Contact system, using the Kabat numbering
system of
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numbering amino acid residues in the VL region, (i) the VL CDR1 is present at
amino acid
positions 30 to 41 or 30 to 36 of the light chain; (ii) the VH CDR2 is present
at amino acid
positions 51 to 60 or 46 to 55 of the light chain; and (iii) the VH CDR3 is
present at amino acid
positions 94 to 101 or 89 to 96 of the light chain. The Contact CDR positions
may vary
depending on the antibody, and may be determined according to methods known in
the art. In a
specific embodiment, the CDRs of the antibodies described herein are
determined using the
Contact system.
[00197] In a particular embodiment, provided herein are antibodies or
fragments thereof that
specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise CDRs of any one of
the antibodies
described herein (any one of antibodies NMC-103, NMC-204, and NMC-303), which
are
defined according to any of the above-described systems.
[00198] In certain embodiments, provided herein are antibodies or antigen-
binding fragments
thereof that specifically bind to an extracellularly accessible epitope of
M(H)DM2/4 (e.g.,
HDM2) and comprise a heavy chain variable region (VH) having one, two or all
three VH CDRs
(preferably all three VH CDRs) of any anti-HDM2 antibody described herein
(such as NMC-
103, NMC-204, or NMC-303). As is known in the art, VHs contain VH CDRs
surrounded by
framework regions (the CDR and FR sequences appear in the following sequence
in the VH:
FR1-VH CDR 1-FR2-VH CDR 2-FR3-VH CDR 3-FR4), optionally the framework regions
are
human framework regions. In certain embodiments, provided herein are
antibodies or antigen-
binding fragments thereof that specifically bind to an extracellularly
accessible epitope of
M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having
one, two or
all three VH CDRs of a VH having the amino acid sequence of SEQ ID NO:36
(which is the VH
of NMC-103). In certain embodiments, provided herein are antibodies or antigen-
binding
fragments thereof that specifically bind to an extracellularly accessible
epitope of M(H)DM2/4
(e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two
or all three VH
CDRs of a VH having the amino acid sequence of SEQ ID NO:38 (which is the VH
of NMC-
204). In certain embodiments, provided herein are antibodies or antigen-
binding fragments
thereof that specifically bind to an extracellularly accessible epitope of
M(H)DM2/4 (e.g.,
HDM2) and comprise a heavy chain variable region (VH) having one, two or all
three VH CDRs
of a VH having the amino acid sequence of SEQ ID NO:40 (which is the VH of NMC-
303). In
certain embodiments, such antibody or fragment is a humanized antibody or
fragment.
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[00199] In certain embodiments, provided herein are antibodies or antigen-
binding fragments
thereof that specifically bind to an extracellularly accessible epitope of
M(H)DM2/4 (e.g.,
HDM2) and comprise a light chain variable region (VL) having one, two or all
three VL CDRs
(preferably all three VL CDRs) of any anti-HDM2 antibody described herein
(such as NMC-103,
NMC-204, or NMC-303). As is known in the art, VLs contain VL CDRs surrounded
by
framework regions (the CDR and FR sequences appear in the following sequence
in the VL:
FR1-VL CDR 1-FR2-VL CDR 2-FR3-VL CDR 3-FR4); optionally the framework regions
are
human framework regions. In certain embodiments, provided herein are
antibodies or antigen-
binding fragments thereof that specifically bind to an extracellularly
accessible epitope of
M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having
one, two or
all three VL CDRs of a VL having the amino acid sequence of SEQ ID NO:37
(which is the VL
of NMC-103). In certain embodiments, provided herein are antibodies or antigen-
binding
fragments thereof that specifically bind to an extracellularly accessible
epitope of M(H)DM2/4
(e.g., HDM2) and comprise a light chain variable region (VL) having one, two
or all three VL
CDRs of a VL having the amino acid sequence of SEQ ID NO:39 (which is the VL
of NMC-
204). In certain embodiments, provided herein are antibodies or antigen-
binding fragments
thereof that specifically bind to an extracellularly accessible epitope of
M(H)DM2/4 (e.g.,
HDM2) and comprise a light chain variable region (VL) having one, two or all
three VL CDRs
of a VL having the amino acid sequence of SEQ ID NO:40 (which is the VL of NMC-
303). In
certain embodiments, such antibody or fragment is a humanized antibody or
fragment.
[00200] In certain embodiments, provided herein are antibodies or antigen-
binding fragments
thereof that specifically bind to an extracellularly accessible epitope of
M(H)DM2/4 (e.g.,
HDM2) and comprise a heavy chain variable region (VH) having one, two or all
three VH CDRs
(preferably all three VH CDRs) of any anti-HDM2 antibody described herein
(such as NMC-
103, NMC-204, or NMC-303) and comprise a light chain variable region (VL)
having one, two
or all three VL CDRs (preferably all three VL CDRs) of such anti-HDM2
antibody.
[00201] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two
or all three VH CDRs identified in Table 4 (providing VH CDRs of NMC-103). In
one
embodiment, provided herein is an antibody or a fragment thereof that
specifically binds to
HDM2 and comprises a VH CDR3 ("CDR-H3") identified in Table 4. In certain
embodiments,
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provided herein is an antibody or a fragment thereof that specifically binds
to HDM2 and
comprises a light chain variable region (VL) having one, two or all three VL
CDRs identified in
Table 5 (providing VL CDRs of NMC-103). In certain embodiments, provided
herein is an
antibody or a fragment thereof that specifically binds to HDM2 and comprises a
haeavy chain
variable region (VH) having one, two or all three VH CDRs identified in Table
4; and comprises
a light chain variable region (VL) having one, two or all threeVL CDRs
identified in Table 5.
[00202] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two
or all three VH CDRs identified in Table 6 (providing VH CDRs of NMC-204). In
one
embodiment, provided herein is an antibody or a fragment thereof that
specifically binds to
HDM2 and comprises a VH CDR3 ("CDR-H3") identified in Table 6. In certain
embodiments,
provided herein is an antibody or a fragment thereof that specifically binds
to HDM2 and
comprises a light chain variable region (VL) having one, two or all three VL
CDRs identified in
Table 7 (providing VL CDRs of NMC-204). In certain embodiments, provided
herein is an
antibody or a fragment thereof that specifically binds to HDM2 and comprises a
heavy chain
variable region (VH) having one, two or all three VH CDRs identified in Table
6; and comprises
a light chain variable region (VL) having one, two or all three VL CDRs
identified in Table 7.
[00203] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two
or all three VH CDRs identified in Table 8 (providing VH CDRs of NMC-303). In
one
embodiment, provided herein is an antibody or a fragment thereof that
specifically binds to
HDM2 and comprises a VH CDR3 ("CDR-H3") identified in Table 8. In certain
embodiments,
provided herein is an antibody or a fragment thereof that specifically binds
to HDM2 and
comprises a light chain variable region (VL) having one, two or all three VL
CDRs identified in
Table 9 (providing VL CDRs of NMC-303). In certain embodiments, provided
herein is an
antibody or a fragment thereof that specifically binds to HDM2 and comprises a
heavy chain
variable region (VH) having one, two or all three VH CDRs identified in Table
8; and comprises
a light chain variable region (VL) having one, two or all three VL CDRs
identified in Table 9.
[00204] In certain embodiments described herein referencing an antibody or
fragment thereof
comprising a sequence (e.g., VH or VL sequence) or sequences having a certain
percent identity
(which is less than 100%) to a sequence, the antibody or fragment thereof
comprises the
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sequence (e.g., VH or VL sequence) and not the sequences having a certain
percent identity
(which is less than 100%) to the sequence. In certain embodiments, described
herein referencing
an antibody or fragment thereof comprising sequences having a certain percent
identity (which is
less than 100%) to a sequence, substitutions, insertions, or deletions in
these sequences occur in
regions outside the CDRs (i.e., in the FRs).
[00205] In certain embodiments, provided herein is an antibody or fragment
thereof that
specifically binds to HDM2 comprising a VH of any antibody described herein,
such as a VH of
any antibody provided in Section 8 or Figures 20-22 (e.g., the VH of NMC-103,
the VH of
NMC-204, or the VH of NMC-303), or a VH having at least 75%, 80%, 85%, 90%,
95%, 98%,
or 99% sequence identity thereto. In certain embodiments, provided herein is
an antibody or
fragment thereof that specifically binds to HDM2 comprising a VL of any
antibody described
herein, such as a VL of any antibody provided in Section 8 or Figures 20-22
(e.g., the VL of
NMC-103, the VL of NMC-204, or the VL of NMC-303), or a VL having at least
75%, 80%,
85%, 90%, 95%, 98%, or 99% sequence identity thereto. In certain embodiments,
substitutions,
insertions, or deletions in these sequences occur in regions outside the CDRs
(i.e., in the FRs).
[00206] In certain embodiments, provided herein is an antibody or fragment
thereof that
specifically binds to HDM2 comprising a VH and a VL of any antibody described
herein, such
as a VH and VL of any antibody provided in Section 8 or Figures 20-22 (e.g.,
the VH and VL of
NMC-103, the VH and VL of NMC-204, or the VH and VL of NMC-303), or a VHand VL
having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity
thereto.
[00207] In certain embodiments, provided herein is an antibody or fragment
thereof that
specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence
of SEQ ID
NO:36, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence
identity
thereto; and/or (ii) a VL having the amino acid sequence of SEQ ID NO:37, or a
VL having at
least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto.
[00208] In certain embodiments, provided herein is an antibody or fragment
thereof that
specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence
of SEQ ID
NO:38, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence
identity
thereto; and/or (ii) a VL having the amino acid sequence of SEQ ID NO:39, or a
VL having at
least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto.
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[00209] In certain embodiments, provided herein is an antibody or fragment
thereof that
specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence
of SEQ ID
NO:40, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence
identity
thereto; and/or (ii) a VL having the amino acid sequence of SEQ ID NO:41, or a
VL having at
least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto.
[00210] In certain aspects, provided herein are antibodies or fragments
thereof that
specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more Kabat VL
CDRs of a
VL of any one of the antibodies described herein (any one of NMC-103, NMC-204,
and NMC-
303) and/or one or more Kabat VH CDRs of a VH of any one of the antibodies
described herein
(any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment,
provided
herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4
(e.g., HDM2)
and comprise Kabat VH CDR 3 of a VH of any one of the antibodies described
herein (any one
of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided
herein are
antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g.,
HDM2) and comprise
three Kabat VL CDRs of a VL of any one of the antibodies described herein (any
one of NMC-
103, NMC-204, and NMC-303) and/or three Kabat VH CDRs of a VH of any one of
the
antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-
303).
[00211] In certain aspects, provided herein are antibodies or fragments
thereof that
specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more Chothia
VL CDRs of a
VL of any one of the antibodies described herein (any one of NMC-103, NMC-204,
and NMC-
303) and/or one or more Chothia VH CDRs of a VH of any one of the antibodies
described
herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one
embodiment,
provided herein are antibodies or fragments thereof that specifically bind to
M(H)DM2/4 (e.g.,
HDM2) and comprise Chothia VH CDR 3 of a VH of any one of the antibodies
described herein
(any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment,
provided
herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4
(e.g., HDM2)
and comprise three Chothia VL CDRs of a VL of any one of the antibodies
described herein (any
one of NMC-103, NMC-204, and NMC-303) and/or three Chothia VH CDRs of a VH of
any one
of the antibodies described herein (any one of antibodies NMC-103, NMC-204,
and NMC-303).
[00212] In certain aspects, provided herein are antibodies or fragments
thereof that
specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more AbM VL
CDRs of a
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VL of any one of the antibodies described herein (any one of NMC-103, NMC-204,
and NMC-
303) and/or one or more AbM VH CDRs of a VH of any one of the antibodies
described herein
(any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment,
provided
herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4
(e.g., HDM2)
and comprise AbM VH CDR 3 of a VH of any one of the antibodies described
herein (any one
of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided
herein are
antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g.,
HDM2) and comprise
three AbM VL CDRs of a VL of any one of the antibodies described herein (any
one of NMC-
103, NMC-204, and NMC-303) and/or three AbM VH CDRs of a VH of any one of the
antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-
303).
[00213] In certain aspects, provided herein are antibodies or fragments
thereof that
specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more Contact
VL CDRs of a
VL of any one of the antibodies described herein (any one of NMC-103, NMC-204,
and NMC-
303) and/or one or more Contact VH CDRs of a VH of any one of the antibodies
described
herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one
embodiment,
provided herein are antibodies or fragments thereof that specifically bind to
M(H)DM2/4 (e.g.,
HDM2) and comprise Contact VH CDR 3 of a VH of any one of the antibodies
described herein
(any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment,
provided
herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4
(e.g., HDM2)
and comprise three Contact VL CDRs of a VL of any one of the antibodies
described herein (any
one of NMC-103, NMC-204, and NMC-303) and/or three Contact VH CDRs of a VH of
any one
of the antibodies described herein (any one of antibodies NMC-103, NMC-204,
and NMC-303).
[00214] In certain aspects, provided herein are antibodies or fragments
thereof that
specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more IMGT VL
CDRs of a
VL of any one of the antibodies described herein (any one of NMC-103, NMC-204,
and NMC-
303) and/or one or more IMGT VH CDRs of a VH of any one of the antibodies
described herein
(any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment,
provided
herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4
(e.g., HDM2)
and comprise IMGT VH CDR 3 of a VH of any one of the antibodies described
herein (any one
of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided
herein are
antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g.,
HDM2) and comprise
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three IMGT VL CDRs of a VL of any one of the antibodies described herein (any
one of NMC-
103, NMC-204, and NMC-303) and/or three IMGT VH CDRs of a VH of any one of the
antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-
303).
[00215] In a particular embodiment, provided herein are antibodies or
fragments thereof that
specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise CDRs of any one of
the antibodies
described herein (such as NMC-C303, NMC-C103 and variants of NMC-H103), which
are
defined according to any of the above-described systems.
[00216] In certain embodiments, provided herein are antibodies (e.g., chimeric
or humanized
antibodies) or antigen-binding fragments thereof that specifically bind to an
extracellularly
accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain
variable region
(VH) having one, two or all three VH CDRs (preferably all three VH CDRs) of
any anti-HDM2
antibody described herein (such as NMC-C303, NMC-C103 and variants of NMC-
H103). As is
known in the art, VHs contain VH CDRs surrounded by framework regions (the CDR
and FR
sequences appear in the following sequence in the VH: FR1-VH CDR 1-FR2-VH CDR
2-FR3-
VH CDR 3-FR4), optionally the framework regions are humanized or human
framework regions.
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a heavy chain variable region (VH) having one, two or all three VH
CDRs of a VH
having the amino acid sequence of SEQ ID NO:40 (which is the VH of NMC-C303).
In certain
embodiments, provided herein are antibodies or antigen-binding fragments
thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a heavy chain variable region (VH) having one, two or all three VH
CDRs of a VH
having the amino acid sequence of SEQ ID NO:283 (which is the VH of NMC-C103).
In certain
embodiments, provided herein are antibodies or antigen-binding fragments
thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a heavy chain variable region (VH) having one, two or all three VH
CDRs of a VH
having the amino acid sequence of SEQ ID NO:287 (which is the VH1 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a heavy chain variable region (VH) having one, two or all three VH
CDRs of a VH
having the amino acid sequence of SEQ ID NO:291 (which is the VH2 variant of
NMC-H103).
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In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a heavy chain variable region (VH) having one, two or all three VH
CDRs of a VH
having the amino acid sequence of SEQ ID NO:295 (which is the VH3 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a heavy chain variable region (VH) having one, two or all three VH
CDRs of a VH
having the amino acid sequence of SEQ ID NO:299 (which is the VH4 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a heavy chain variable region (VH) having one, two or all three VH
CDRs of a VH
having the amino acid sequence of SEQ ID NO:305 (which is the VH6 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a heavy chain variable region (VH) having one, two or all three VH
CDRs of a VH
having the amino acid sequence of SEQ ID NO:309 (which is the VH7 variant of
NMC-H103).
[00217] In certain embodiments, provided herein are chimeric or humanized
antibodies or
antigen-binding fragments thereof that specifically bind to an extracellularly
accessible epitope
of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH)
having one, two,
three or all four VH FRs (preferably all fourVH FRs) of any anti-HDM2 antibody
described
herein (such as NMC-C303, NMC-C103 and variants of NMC-H103). As is known in
the art,
VHs contain VH CDRs surrounded by framework regions (the CDR and FR sequences
appear in
the following sequence in the VH: FR1-VH CDR 1-FR2-VH CDR 2-FR3-VH CDR 3-FR4),
optionally the framework regions are humanized or human framework regions. In
certain
embodiments, provided herein are antibodies or antigen-binding fragments
thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a heavy chain variable region (VH) having one, two, three or all four
VH FRs of a VH
having the amino acid sequence of SEQ ID NO:40 (which is the VH of NMC-C303).
In certain
embodiments, provided herein are antibodies or antigen-binding fragments
thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a heavy chain variable region (VH) having one, two, three or all four
VH FRs of a VH
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having the amino acid sequence of SEQ ID NO:283 (which is the VH of NMC-C103).
In certain
embodiments, provided herein are antibodies or antigen-binding fragments
thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a heavy chain variable region (VH) having one, two, three or all four
VH FRs of a VH
having the amino acid sequence of SEQ ID NO:287 (which is the VH1 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a heavy chain variable region (VH) having one, two, three or all four
VH FRs of a VH
having the amino acid sequence of SEQ ID NO:291 (which is the VH2 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a heavy chain variable region (VH) having one, two, three or all four
VH FRs of a VH
having the amino acid sequence of SEQ ID NO:295 (which is the VH3 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a heavy chain variable region (VH) having one, two, three or all four
VH FRs of a VH
having the amino acid sequence of SEQ ID NO:299 (which is the VH4 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a heavy chain variable region (VH) having one, two, three or all
fourVH FRs of a VH
having the amino acid sequence of SEQ ID NO:305 (which is the VH6 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a heavy chain variable region (VH) having one, two, three or all four
VH FRs of a VH
having the amino acid sequence of SEQ ID NO:309 (which is the VH7 variant of
NMC-H103).
[00218] In certain embodiments, provided herein are antibodies (e.g., chimeric
or humanized
antibodies) or antigen-binding fragments thereof that specifically bind to an
extracellularly
accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain
variable region (VL)
having one, two or all three VL CDRs (preferably all three VL CDRs) of any
anti-HDM2
antibody described herein (such as NMC-C303, NMC-C103 and variants of NMC-
H103). As is
known in the art, VLs contain VL CDRs surrounded by framework regions (the CDR
and FR
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sequences appear in the following sequence in the VL: FR1-VL CDR 1-FR2-VL CDR
2-FR3-
VL CDR 3-FR4); optionally the framework regions are human or humanized
framework regions.
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two or all three VL
CDRs of a VL
having the amino acid sequence of SEQ ID NO:41 (which is the VL of NMC-C303).
In certain
embodiments, provided herein are antibodies or antigen-binding fragments
thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two or all three VL
CDRs of a VL
having the amino acid sequence of SEQ ID NO:285 (which is the VL of NMC-C103).
In certain
embodiments, provided herein are antibodies or antigen-binding fragments
thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two or all three VL
CDRs of a VL
having the amino acid sequence of SEQ ID NO:289 (which is the VK1 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two or all three VL
CDRs of a VL
having the amino acid sequence of SEQ ID NO:293 (which is the VK2 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two or all three VL
CDRs of a VL
having the amino acid sequence of SEQ ID NO:297 (which is the VK3 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two or all three VL
CDRs of a VL
having the amino acid sequence of SEQ ID NO:301 (which is the VK4 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two or all three VL
CDRs of a VL
having the amino acid sequence of SEQ ID NO:303 (which is the VK5 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
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specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two or all three VL
CDRs of a VL
having the amino acid sequence of SEQ ID NO:307 (which is the VK6 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two or all three VL
CDRs of a VL
having the amino acid sequence of SEQ ID NO:311 (which is the VK7 variant of
NMC-H103).
[00219] In certain embodiments, provided herein are chimeric or humanized
antibodies or
antigen-binding fragments thereof that specifically bind to an extracellularly
accessible epitope
of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL)
having one, two,
three or all four VL FRs (preferably all fourVL FRs) of any anti-HDM2 antibody
described
herein (such as NMC-C303, NMC-C103 and variants of NMC-H103). As is known in
the art,
VLs contain VL CDRs surrounded by framework regions (the CDR and FR sequences
appear in
the following sequence in the VL: FR1-VL CDR 1-FR2-VL CDR 2-FR3-VL CDR 3-FR4),
optionally the framework regions are humanized or human framework regions. In
certain
embodiments, provided herein are antibodies or antigen-binding fragments
thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two, three or all four
VL FRs of a VL
having the amino acid sequence of SEQ ID NO:41 (which is the VL of NMC-C303).
In certain
embodiments, provided herein are antibodies or antigen-binding fragments
thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two, three or all four
VL FRs of a VL
having the amino acid sequence of SEQ ID NO:285 (which is the VL of NMC-C103).
In certain
embodiments, provided herein are antibodies or antigen-binding fragments
thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two, three or all four
VL FRs of a VL
having the amino acid sequence of SEQ ID NO:289 (which is the VL1 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two, three or all four
VL FRs of a VL
having the amino acid sequence of SEQ ID NO:293 (which is the VL2 variant of
NMC-H103).
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In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two, three or all four
VL FRs of a VL
having the amino acid sequence of SEQ ID NO:297 (which is the VL3 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two, three or all four
VL FRs of a VL
having the amino acid sequence of SEQ ID NO:301 (which is the VL4 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two, three or all four
VL FRs of a VL
having the amino acid sequence of SEQ ID NO:303 (which is the VL5 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two, three or all
fourVL FRs of a VL
having the amino acid sequence of SEQ ID NO:307 (which is the VL6 variant of
NMC-H103).
In certain embodiments, provided herein are antibodies or antigen-binding
fragments thereof that
specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g.,
HDM2) and
comprise a light chain variable region (VL) having one, two, three or all four
VL FRs of a VL
having the amino acid sequence of SEQ ID NO:311 (which is the VL7 variant of
NMC-H103).
[00220] In certain embodiments, provided herein are chimeric or humanized
antibodies or
antigen-binding fragments thereof that specifically bind to an extracellularly
accessible epitope
of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH)
having one, two
or all three VH CDRs (preferably all three VH CDRs) of any chimeric or
humanized anti-HDM2
antibody described herein (such as NMC-C103, NMC-C303, or variants of NMC-
H103) and
comprise a light chain variable region (VL) having one, two or all three VL
CDRs (preferably all
three VL CDRs) of such anti-HDM2 antibody.
[00221] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two
or all three VH CDRs identified in Table 11 (providing VH CDRs of NMC-C303).
In one
embodiment, provided herein is an antibody or a fragment thereof that
specifically binds to
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HDM2 and comprises a VH CDR3 ("CDR-H3") identified in Table 11. In certain
embodiments,
provided herein is an antibody or a fragment thereof that specifically binds
to HDM2 and
comprises a light chain variable region (VL) having one, two or all three VL
CDRs identified in
Table 12 (providing VL CDRs of NMC-C303). In certain embodiments, provided
herein is an
antibody or a fragment thereof that specifically binds to HDM2 and comprises a
haeavy chain
variable region (VH) having one, two or all three VH CDRs identified in Table
11; and
comprises a light chain variable region (VL) having one, two or all threeVL
CDRs identified in
Table 12.
[00222] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two
or all three VH CDRs identified in Table 13 (providing VH CDRs of NMC-C103).
In one
embodiment, provided herein is an antibody or a fragment thereof that
specifically binds to
HDM2 and comprises a VH CDR3 ("CDR-H3") identified in Table 13. In certain
embodiments,
provided herein is an antibody or a fragment thereof that specifically binds
to HDM2 and
comprises a light chain variable region (VL) having one, two or all three VL
CDRs identified in
Table 14 (providing VL CDRs of NMC-C103). In certain embodiments, provided
herein is an
antibody or a fragment thereof that specifically binds to HDM2 and comprises a
heavy chain
variable region (VH) having one, two or all three VH CDRs identified in Table
13; and
comprises a light chain variable region (VL) having one, two or all three VL
CDRs identified in
Table 14. In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two,
three or all four VH FRs identified in Table 13 (providing VH FRs of NMC-
C103). In certain
embodiments, provided herein is an antibody or a fragment thereof that
specifically binds to
HDM2 and comprises a light chain variable region (VL) having one, two, three
or all four VL
FRs identified in Table 14 (providing VL FRs of NMC-C103). In certain
embodiments,
provided herein is an antibody or a fragment thereof that specifically binds
to HDM2 and
comprises a heavy chain variable region (VH) having one, two, three or all
four VH FRs
identified in Table 13; and comprises a light chain variable region (VL)
having one, two, three or
all fourVL FRs identified in Table 14.
[00223] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two
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or all three VH CDRs identified in Table 15 (providing VH1 variant CDRs of NMC-
H103). In
one embodiment, provided herein is an antibody or a fragment thereof that
specifically binds to
HDM2 and comprises a VH CDR3 ("CDR-H3") identified in Table 15. In certain
embodiments,
provided herein is an antibody or a fragment thereof that specifically binds
to HDM2 and
comprises a light chain variable region (VL) having one, two or all three VL
CDRs identified in
Table 16 (providing VK1 variant CDRs of NMC-H103). In certain embodiments,
provided
herein is an antibody or a fragment thereof that specifically binds to HDM2
and comprises a
heavy chain variable region (VH) having one, two or all three VH CDRs
identified in Table 15;
and comprises a light chain variable region (VL) having one, two or all three
VL CDRs
identified in Table 16.
[00224] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two,
three or all four VH FRs identified in Table 15 (providing VH1 variant FRs of
NMC-H103). In
certain embodiments, provided herein is an antibody or a fragment thereof that
specifically binds
to HDM2 and comprises a light chain variable region (VL) having one, two,
three or all four VL
FRs identified in Table 16 (providing VK1 variant CDRs of NMC-H103).
[00225] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two
or all three VH CDRs identified in Table 17 (providing VH2 variant CDRs of NMC-
H103). In
one embodiment, provided herein is an antibody or a fragment thereof that
specifically binds to
HDM2 and comprises a VH CDR3 ("CDR-H3") identified in Table 17. In certain
embodiments,
provided herein is an antibody or a fragment thereof that specifically binds
to HDM2 and
comprises a light chain variable region (VL) having one, two or all three VL
CDRs identified in
Table 18 (providing VK2 variant CDRs of NMC-H103). In certain embodiments,
provided
herein is an antibody or a fragment thereof that specifically binds to HDM2
and comprises a
heavy chain variable region (VH) having one, two or all three VH CDRs
identified in Table 17;
and comprises a light chain variable region (VL) having one, two or all three
VL CDRs
identified in Table 18.
[00226] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two,
three or all four VH FRs identified in Table 17 (providing VH2 variant FRs of
NMC-H103). In
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certain embodiments, provided herein is an antibody or a fragment thereof that
specifically binds
to HDM2 and comprises a light chain variable region (VL) having one, two,
three or all four VL
FRs identified in Table 18 (providing VK2 variant CDRs of NMC-H103).
[00227] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two
or all three VH CDRs identified in Table 19 (providing VH3 variant CDRs of NMC-
H103). In
one embodiment, provided herein is an antibody or a fragment thereof that
specifically binds to
HDM2 and comprises a VH CDR3 ("CDR-H3") identified in Table 19. In certain
embodiments,
provided herein is an antibody or a fragment thereof that specifically binds
to HDM2 and
comprises a light chain variable region (VL) having one, two or all three VL
CDRs identified in
Table 20 (providing VK3 variant CDRs of NMC-H103). In certain embodiments,
provided
herein is an antibody or a fragment thereof that specifically binds to HDM2
and comprises a
heavy chain variable region (VH) having one, two or all three VH CDRs
identified in Table 19;
and comprises a light chain variable region (VL) having one, two or all three
VL CDRs
identified in Table 20.
[00228] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two,
three or all four VH FRs identified in Table 19 (providing VH3 variant FRs of
NMC-H103). In
certain embodiments, provided herein is an antibody or a fragment thereof that
specifically binds
to HDM2 and comprises a light chain variable region (VL) having one, two,
three or all four VL
FRs identified in Table 20 (providing VK3 variant CDRs of NMC-H103).
[00229] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two
or all three VH CDRs identified in Table 21 (providing VH4 variant CDRs of NMC-
H103). In
one embodiment, provided herein is an antibody or a fragment thereof that
specifically binds to
HDM2 and comprises a VH CDR3 ("CDR-H3") identified in Table 21. In certain
embodiments,
provided herein is an antibody or a fragment thereof that specifically binds
to HDM2 and
comprises a light chain variable region (VL) having one, two or all three VL
CDRs identified in
Table 22 (providing VK4 variant CDRs of NMC-H103). In certain embodiments,
provided
herein is an antibody or a fragment thereof that specifically binds to HDM2
and comprises a
heavy chain variable region (VH) having one, two or all three VH CDRs
identified in Table 21;
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and comprises a light chain variable region (VL) having one, two or all three
VL CDRs
identified in Table 22.
[00230] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two,
three or all four VH FRs identified in Table 21 (providing VH4 variant FRs of
NMC-H103). In
certain embodiments, provided herein is an antibody or a fragment thereof that
specifically binds
to HDM2 and comprises a light chain variable region (VL) having one, two,
three or all four VL
FRs identified in Table 22 (providing VK4 variant CDRs of NMC-H103).
[00231] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a light chain variable region (VL)
having one, two or
all three VL CDRs identified in Table 23 (providing VK5 variant CDRs of NMC-
H103).
[00232] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a light chain variable region (VL)
having one, two,
three or all four VL FRs identified in Table 23 (providing VK5 variant CDRs of
NMC-H103).
[00233] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two
or all three VH CDRs identified in Table 24 (providing VH6 variant CDRs of NMC-
H103). In
one embodiment, provided herein is an antibody or a fragment thereof that
specifically binds to
HDM2 and comprises a VH CDR3 ("CDR-H3") identified in Table 24. In certain
embodiments,
provided herein is an antibody or a fragment thereof that specifically binds
to HDM2 and
comprises a light chain variable region (VL) having one, two or all three VL
CDRs identified in
Table 25 (providing VK6 variant CDRs of NMC-H103). In certain embodiments,
provided
herein is an antibody or a fragment thereof that specifically binds to HDM2
and comprises a
heavy chain variable region (VH) having one, two or all three VH CDRs
identified in Table 24;
and comprises a light chain variable region (VL) having one, two or all three
VL CDRs
identified in Table 25.
[00234] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two,
three or all four VH FRs identified in Table 24 (providing VH6 variant FRs of
NMC-H103). In
certain embodiments, provided herein is an antibody or a fragment thereof that
specifically binds
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to HDM2 and comprises a light chain variable region (VL) having one, two,
three or all four VL
FRs identified in Table 25 (providing VK6 variant CDRs of NMC-H103).
[00235] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two
or all three VH CDRs identified in Table 26 (providing VH7 variant CDRs of NMC-
H103). In
one embodiment, provided herein is an antibody or a fragment thereof that
specifically binds to
HDM2 and comprises a VH CDR3 ("CDR-H3") identified in Table 26. In certain
embodiments,
provided herein is an antibody or a fragment thereof that specifically binds
to HDM2 and
comprises a light chain variable region (VL) having one, two or all three VL
CDRs identified in
Table 27 (providing VK7 variant CDRs of NMC-H103). In certain embodiments,
provided
herein is an antibody or a fragment thereof that specifically binds to HDM2
and comprises a
heavy chain variable region (VH) having one, two or all three VH CDRs
identified in Table 26;
and comprises a light chain variable region (VL) having one, two or all three
VL CDRs
identified in Table 27.
[00236] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two,
three or all four VH FRs identified in Table 26 (providing VH7 variant FRs of
NMC-H103). In
certain embodiments, provided herein is an antibody or a fragment thereof that
specifically binds
to HDM2 and comprises a light chain variable region (VL) having one, two,
three or all four VL
FRs identified in Table 27 (providing VK7 variant CDRs of NMC-H103).
[00237] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two,
three or all four VH FRs identified in any one of Tables 15, 17, 19, 21, 24,
and 26; and
comprises a light chain variable region (VL) having one, two, three or all
four VL FRs identified
in any one of Tables 16, 18, 20, 22, 23, 25, and 27.
[00238] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two,
or all three or VH CDRs identified in any one of Tables 15, 17, 19, 21, 24,
and 26; and
comprises a light chain variable region (VL) having one, two, or all three VL
CDRs identified in
any one of Tables 16, 18, 20, 22, 23, 25, and 27.
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[00239] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two,
three or all four VH FRs identified in any one of Tables 15, 17, 19, and 21;
and comprises a light
chain variable region (VL) having one, two, three or all four VL FRs
identified in any one of
Tables 16, 18, 20, 22, and 23.
[00240] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two,
or all three VH CDRs identified in any one of Tables 15, 17, 19, and 21; and
comprises a light
chain variable region (VL) having one, two, or all three VL CDRs identified in
any one of Tables
16, 18, 20, 22, and 23.
[00241] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two,
three or all four VH FRs identified in any one of Tables 21, 24, and 26; and
comprises a light
chain variable region (VL) having one, two, three or all four VL FRs
identified in any one of
Tables 20, 25, and 27.
[00242] In certain embodiments, provided herein is an antibody or a fragment
thereof that
specifically binds to HDM2 and comprises a heavy chain variable region (VH)
having one, two,
or all three VH CDRs identified in any one of Tables 21, 24, and 26; and
comprises a light chain
variable region (VL) having one, two, or all three VL CDRs identified in any
one of Tables 20,
25, and 27.
[00243] In certain embodiments, provided herein is an antibody or fragment
thereof that
specifically binds to HDM2 comprising a VH of any antibody described herein,
such as a VH of
any antibody provided in Sections 9 and 11 (e.g., the VH of NMC-C103, the VH
of NMC-C303,
or the VH of any of NMC-H103 variants), or a VH having at least 75%, 80%, 85%,
90%, 95%,
98%, or 99% sequence identity thereto. In certain embodiments, provided herein
is an antibody
or fragment thereof that specifically binds to HDM2 comprising a VL of any
antibody described
herein, such as a VL of any antibody provided in Sections 9 and 11 (e.g., the
VL of NMC-C103,
the VL of NMC-C303, or the VL of any of NMC-H103 variants), or a VL having at
least 75%,
80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto. In certain
embodiments,
substitutions, insertions, or deletions in these sequences occur in regions
outside the CDRs (i.e.,
in the FRs).
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[00244] In certain embodiments, provided herein is an antibody or fragment
thereof that
specifically binds to HDM2 comprising a VH and a VL of any antibody described
herein, such
as a VH and VL of any antibody provided in Sections 9 and 11 (e.g., the VH and
VL of NMC-
C103, the VH and VL of NMC-C303, or the VH and VL of any NMC-H103 variant,
e.g., the VH
and VL of any NMC-H103 variant tested for binding to NMC-P1 as described in
Section 9.6,
Example 18 and Figures 38A and 38B), or a VH and VL having at least 75%, 80%,
85%, 90%,
95%, 98%, or 99% sequence identity thereto. In certain embodiments,
substitutions, insertions,
or deletions in these sequences occur in regions outside the CDRs (i.e., in
the FRs).
[00245] In certain embodiments, provided herein is an antibody or fragment
thereof that
specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence
of SEQ ID
NO:40, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence
identity
thereto; and/or (ii) a VL having the amino acid sequence of SEQ ID NO:41, or a
VL having at
least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto. In
certain
embodiments, provided herein is an antibody or fragment thereof that
specifically binds to
HDM2 comprising: (i) a VH having the amino acid sequence of SEQ ID NO:40, or a
VH having
at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and
(ii) a VL
having the amino acid sequence of SEQ ID NO:41, or a VL having at least 70%,
75%, 80%,
85%, 90%, 95%, 98%, 99% sequence identity thereto. In certain embodiments,
substitutions,
insertions, or deletions in these sequences occur in regions outside the CDRs
(i.e., in the FRs).
[00246] In certain embodiments, provided herein is an antibody or fragment
thereof that
specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence
of SEQ ID
NO:283, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%
sequence identity
thereto; and/or (ii) a VL having the amino acid sequence of SEQ ID NO:285, or
a VL having at
least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto. In
certain
embodiments, provided herein is an antibody or fragment thereof that
specifically binds to
HDM2 comprising: (i) a VH having the amino acid sequence of SEQ ID NO:283, or
a VH
having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity
thereto; and (ii) a
VL having the amino acid sequence of SEQ ID NO:285, or a VL having at least
70%, 75%, 80%,
85%, 90%, 95%, 98%, 99% sequence identity thereto. In certain embodiments,
substitutions,
insertions, or deletions in these sequences occur in regions outside the CDRs
(i.e., in the FRs).
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[00247] In certain embodiments, provided herein is an antibody or fragment
thereof that
specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence
of SEQ ID
NO:287, 291, 295, 299, 305, or 309, or a VH having at least 70%, 75%, 80%,
85%, 90%, 95%,
98%, 99% sequence identity thereto; and/or (ii) a VL having the amino acid
sequence of SEQ ID
NO:289, 293, 297, 301, 303, 307, or 311, or a VL having at least 70%, 75%,
80%, 85%, 90%,
95%, 98%, 99% sequence identity thereto. In certain embodiments, provided
herein is an
antibody or fragment thereof that specifically binds to HDM2 comprising: (i) a
VH having the
amino acid sequence of SEQ ID NO:287, 291, 295, 299, 305, or 309, or a VH
having at least
70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and (ii) a
VL having the
amino acid sequence of SEQ ID NO:289, 293, 297, 301, 303, 307, or 311, or a VL
having at
least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto. In
certain
embodiments, substitutions, insertions, or deletions in these sequences occur
in regions outside
the CDRs (i.e., in the FRs).
[00248] In certain embodiments, provided herein is an antibody or fragment
thereof that
specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence
of SEQ ID
NO:287, 291, 295, or 299, or a VH having at least 70%, 75%, 80%, 85%, 90%,
95%, 98%, 99%
sequence identity thereto; and/or (ii) a VL having the amino acid sequence of
SEQ ID NO:289,
293, 297, 301, or 303, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%,
98%, 99%
sequence identity thereto. In certain embodiments, provided herein is an
antibody or fragment
thereof that specifically binds to HDM2 comprising: (i) a VH having the amino
acid sequence of
SEQ ID NO:287, 291, 295, or 299, or a VH having at least 70%, 75%, 80%, 85%,
90%, 95%,
98%, 99% sequence identity thereto; and (ii) a VL having the amino acid
sequence of SEQ ID
NO:289, 293, 297, 301, or 303, or a VL having at least 70%, 75%, 80%, 85%,
90%, 95%, 98%,
99% sequence identity thereto. In certain embodiments, substitutions,
insertions, or deletions in
these sequences occur in regions outside the CDRs (i.e., in the FRs).
[00249] In certain embodiments, provided herein is an antibody or fragment
thereof that
specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence
of SEQ ID
NO: 299, 305, or 309, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%,
98%, 99%
sequence identity thereto; and/or (ii) a VL having the amino acid sequence of
SEQ ID NO:297,
307, or 311, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%
sequence
identity thereto. In certain embodiments, provided herein is an antibody or
fragment thereof that
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specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence
of SEQ ID
NO: 299, 305, or 309, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%,
98%, 99%
sequence identity thereto; and (ii) a VL having the amino acid sequence of SEQ
ID NO:297, 307,
or 311, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%
sequence identity
thereto. In certain embodiments, substitutions, insertions, or deletions in
these sequences occur
in regions outside the CDRs (i.e., in the FRs).
[00250] In certain aspects, provided herein are antibodies or fragments
thereof that
specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more Kabat VL
CDRs and
VL FRs of a VL of any one of the chimeric or humanized antibodies described
herein (such as
NMC-C103, NMC-C303, and variants of NMC-H103), and/or one or more Kabat VH
CDRs and
VH FRs of a VH of any one of the chimeric or humanized antibodies described
herein (such as
NMC-C103, NMC-C303, and variants of NMC-H103). In one embodiment, provided
herein are
antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g.,
HDM2) and comprise
three Kabat VL CDRs and four Kabat VL FRs of a VL of any one of the chimeric
or humanized
antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-
H103),
and/or three Kabat VH CDRs and four Kabat VH FRs of a VH of any one of the
chimeric or
humanized antibodies described herein (such as NMC-C103, NMC-C303, and
variants of NMC-
H103).
[00251] In certain aspects, provided herein are antibodies or fragments
thereof that
specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more Chothia
VL CDRs and
VL FRs of a VL of any one of the chimeric or humanized antibodies described
herein (such as
NMC-C103, NMC-C303, and variants of NMC-H103), and/or one or more Chothia VH
CDRs
and VH FRs of a VH of any one of the chimeric or humanized antibodies
described herein (such
as NMC-C103, NMC-C303, and variants of NMC-H103). In one embodiment, provided
herein
are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g.,
HDM2) and
comprise three Chothia VL CDRs and four Chothia VL FRs of a VL of any one of
the chimeric
or humanized antibodies described herein (such as NMC-C103, NMC-C303, and
variants of
NMC-H103), and/or three Chothia VH CDRs and four Chothia VH FRs of a VH of any
one of
the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-
C303, and
variants of NMC-H103).
[00252] In certain aspects, provided herein are antibodies or fragments
thereof that
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specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more AbM VL
CDRs and
VL FRs of a VL of any one of the chimeric or humanized antibodies described
herein (such as
NMC-C103, NMC-C303, and variants of NMC-H103), and/or one or more AbM VH CDRs
and
VH FRs of a VH of any one of the chimeric or humanized antibodies described
herein (such as
NMC-C103, NMC-C303, and variants of NMC-H103). In one embodiment, provided
herein are
antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g.,
HDM2) and comprise
three AbM VL CDRs and four AbM VL FRs of a VL of any one of the chimeric or
humanized
antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-
H103),
and/or three AbM VH CDRs and four AbM VH FRs of a VH of any one of the
chimeric or
humanized antibodies described herein (such as NMC-C103, NMC-C303, and
variants of NMC-
H103).
[00253] In certain aspects, provided herein are antibodies or fragments
thereof that
specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more Contact
VL CDRs and
VL FRs of a VL of any one of the chimeric or humanized antibodies described
herein (such as
NMC-C103, NMC-C303, and variants of NMC-H103), and/or one or more Contact VH
CDRs
and VH FRs of a VH of any one of the chimeric or humanized antibodies
described herein (such
as NMC-C103, NMC-C303, and variants of NMC-H103). In one embodiment, provided
herein
are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g.,
HDM2) and
comprise three Contact VL CDRs and four Contact VL FRs of a VL of any one of
the chimeric
or humanized antibodies described herein (such as NMC-C103, NMC-C303, and
variants of
NMC-H103), and/or three Contact VH CDRs and four Contact VH FRs of a VH of any
one of
the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-
C303, and
variants of NMC-H103).
[00254] In certain aspects, provided herein are antibodies or fragments
thereof that
specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more IMGT VL
CDRs and
VL FRs of a VL of any one of the chimeric or humanized antibodies described
herein (such as
NMC-C103, NMC-C303, and variants of NMC-H103), and/or one or more IMGT VH CDRs
and
VH FRs of a VH of any one of the chimeric or humanized antibodies described
herein (such as
NMC-C103, NMC-C303, and variants of NMC-H103). In one embodiment, provided
herein are
antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g.,
HDM2) and comprise
three IMGT VL CDRs and four IMGT VL FRs of a VL of any one of the chimeric or
humanized
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antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-
H103),
and/or three IMGT VH CDRs and four IMGT VH FRs of a VH of any one of the
chimeric or
humanized antibodies described herein (such as NMC-C103, NMC-C303, and
variants of NMC-
H103).
[00255] In certain embodiments, provided herein are antibodies or fragments
thereof that
specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise combinations of Kabat
CDRs,
Chothia CDRs, AbM CDRs, IMGT CDRs, and Contact CDRs (or a combination of CDRs
defined by any two, three, four or five of these CDR defining systems).
[00256] In a specific embodiment, the position of one or more CDRs along the
VH (e.g.,
CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) region of an
antibody
described herein may vary by one, two, three, four, five, or six amino acid
positions so long as
immunospecific binding to M(H)DM2/4 (e.g., HDM2) is maintained (e.g.,
substantially
maintained, for example, at least 50%, at least 60%, at least 70%, at least
80%, at least 90%, at
least 95%). For example, in one embodiment, the position defining a CDR of any
of antibody
described herein (any one of antibodies NMC-103, NMC-204, and NMC-303) may
vary by
shifting the N-terminal and/or C-terminal boundary of the CDR by one, two,
three, four, five, or
six amino acids, so long as immunospecific binding to M(H)DM2/4 (e.g., HDM2)
is maintained
(e.g., substantially maintained, for example, at least 50%, at least 60%, at
least 70%, at least
80%, at least 90%, at least 95%). In another embodiment, the length of one or
more CDRs along
the VH (e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3)
region of an
antibody described herein may vary (e.g., be shorter or longer) by one, two,
three, four, five, or
more amino acids, so long as immunospecific binding to M(H)DM2/4 (e.g., HDM2)
is
maintained (e.g., substantially maintained, for example, at least 50%, at
least 60%, at least 70%,
at least 80%, at least 90%, at least 95%).
[00257] In specific embodiments, an anti-M(H)DM2/4 (e.g., HDM2) antibody
described
herein is a humanized immunoglobulin (e.g., an IgG) that comprises the 3 VH
CDRs and the 3
VL CDRs (i.e., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) of
any
of the antibodies described herein (any one of murine antibodies NMC-103, NMC-
204, and
NMC-303), respectively, human or human-derived framework regions, and human or
human-
derived constant regions; antigen-binding fragments of such humanized
antibodies are also
provided by the present invention. Non-limiting examples of human framework
regions are
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described in the art, e.g., see Kabat et al. (1991) Sequences of Proteins of
Immunological
Interest, Fifth Edition, U.S. Department of Health and Human Services, NIE
Publication No. 91-
3242). In certain embodiments, a humanizedanti-M(H)DM2/4 (e.g., HDM2) antibody
or
antigen-binding fragment thereof comprises a VH with VH CDR1, VH CDR2, and VH
CDR3 as
described herein (e.g., those of MNC-103, NMC-204, or NMC-303), surrounded by
VH
framework regions that are human framework regions or derived from human
framework
regions. In certain embodiments, an anti-M(H)DM2/4 (e.g., HDM2) antibody or
antigen-binding
fragment thereof comprises a VL with VL CDR1, VL CDR2, and VL CDR3 as
described herein
(e.g., those of MNC-103, NMC-204, or NMC-303), surrounded by VL framework
regions that
are human framework regions or derived from human framework regions. In
certain
embodiments, an anti-M(H)DM2/4 (e.g., HDM2) antibody or antigen-binding
fragment thereof
comprises (i) a VH with VH CDR1, VH CDR2, and VH CDR3 as described herein
(e.g., those of
MNC-103, NMC-204, or NMC-303), surrounded by VH framework regions that are
human
framework regions or derived from human framework regions; and (ii) a VL with
VL CDR1, VL
CDR2, and VL CDR3 as described herein (e.g., those of MNC-103, NMC-204, or NMC-
303),
surrounded by VL framework regions that are human framework regions or derived
from human
framework regions.
[00258] Human framework regions that may be used include, without limitation:
(i)
framework regions selected using the "best-fit" method (see, e.g., Sims et al.
J. Immunol.
151:2296 (1993)); (ii) framework regions derived from the consensus sequence
of human
antibodies of a particular subgroup of light or heavy chain variable regions
(see, e.g., Carter et al.
Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol.,
151:2623 (1993));
(iii) human mature (somatically mutated) framework regions or human germline
framework
regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008));
and (iv)
framework regions derived from screening FR libraries (see, e.g., Baca et al.,
J. Biol. Chem.
272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618
(1996)). See, e.g.,
Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human
framework regions.
[00259] In a specific embodiment, using routine recombinant DNA techniques,
one or more of
the CDRs of an anti-M(H)DM2/4 (e.g., HDM2) or antigen-binding fragment thereof
described
herein may be inserted within known framework regions. The framework regions
may be
naturally occurring or consensus framework regions, and preferably human
framework regions.
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[00260] In certain embodiments, described herein are polynucleotides
comprising
combinations of the framework regions and CDRs that encode an anti-M(H)DM2/4
(e.g.,
HDM2) or antigen-binding fragment thereof that specifically binds M(H)DM2/4
(e.g., HDM2).
One or more (e.g., one or two or three) amino acid substitutions may be made
within the
framework regions, preferably, one or more (e.g, one or two or three) amino
acid substitutions
may be made that improve binding of the antibody to M(H)DM2/4 (e.g., HDM2).
[00261] In an alternative embodiment wherein the antibody or fragment thereof
is not
humanized, the framework regions in the variable domains can be those of the
native (e.g.,
murine) antibody).
[00262] Antibodies provided herein include immunoglobulin molecules and
immunologically
active fragments of immunoglobulin molecules (i.e., molecules that possess an
antigen-binding
site) that specifically bind to an extracellular region (epitope) of M(H)DM2/4
accessible on the
plasma membrane surface of cancer cells (for example, an epitope that is
expressed or exposed
on the plasma membrane of cancer cells at greater levels than on non-cancer
cells (e.g., when
such cancer and non-cancer cells originated from the same tissue)).
[00263] In a preferred embodiment, anti- M(H)DM2/4 antibodies described herein
are
monoclonal antibodies or fragments thereof. The antibodies and fragments
described herein are
preferably human, humanized or chimeric. A human antibody can be a human
immunoglobulin,
which may be isolated from a human immunoglobulin library or isolated from
mice or other
animals that express antibodies from human genes. In one embodiment, an
antibody provided
herein is human (or a fragment of a human antibody). In one embodiment, an
antibody provided
herein is humanized (or a fragment of a humanized antibody). In one
embodiment, an antibody
provided herein is chimeric (or a fragment of a chimeric antibody) (where a
chimeric antibody is
an antibody with a variable region of one species (e.g., murine) and a
constant region of another
species (e.g., human)). Preferably, an antibody provided herein is a human,
humanized or
chimeric monoclonal antibody (which is particularly suitable for treatment of
human subjects).
In one embodiment, an antibody provided herein is a synthetic antibody. In one
embodiment, an
antibody provided herein is a multi-specific antibody (e.g., a bi-specific
antibody). In one
embodiment, an antibody provided herein is a single chain antibody, e.g., a
single chain Fv
(scFv). In one embodiment, an antigen-binding fragment of an anti- M(H)DM2/4
antibody is
provided herein wherein the fragment can be, without limitation, an Fv
fragment, a Fab
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fragment, a F(ab') fragment, a F(ab1)2fragment, or a disulfide-linked FIT
(sdFv). In one
embodiment, an antigen-binding fragment provided herein is an FIT fragment. In
one
embodiment, an antigen-binding fragment provided herein is a Fab fragment. In
one
embodiment, an antigen-binding fragment provided herein is a F(ab1)2fragment.
In one
embodiment, an antigen- binding fragment provided herein is a F(ab') fragment.
[00264] In a specific embodiment, an antibody provided herein is a
multispecific antibody
(such as a bi-specific antibody) that specifically binds to an extracellularly
accessible epitope of
M(H)DM2/4 exposed on the plasma membrane surface of cancer cells and
specifically binds to a
second antigen, wherein such binding allows re-targeting of effector cells
towards tumor cells (as
an example of such engineered bi-specific antibodies directed to a different
target see Chames et
al., 2009, MAbs 1:539-547, describing an antibody termed catumaxomab, a T-cell
targeting
agent). In one embodiment, an antibody provided herein is a multispecific
antibody (such as a bi-
specific antibody) that specifically binds to an extracellularly accessible
epitope of M(H)DM2/4
exposed on the plasma membrane surface of cancer cells, and also binds to an
antigen exposed
on the plasma membrane surface of an effector cell. Effector cells include but
are not limited to
T cells, natural killer cells, neutrophils, macrophages, dendritic cells and B
lymphocytes. In one
embodiment, an antibody provided herein is a multispecific antibody (e.g., a
bi-specific
antibody) that specifically binds to an extracellularly accessible epitope of
M(H)DM2/4 exposed
on the plasma membrane surface of cancer cells, and also specifically binds to
an antigen
exposed on the surface of T cells (e.g., cytotoxic T cells). In one
embodiment, an antibody
provided herein is a multispecific antibody (e.g., a bi-specific antibody)
that specifically binds to
an extracellularly accessible epitope of M(H)DM2/4 exposed on the plasma
membrane surface of
cancer cells, and also specifically binds to CD3. In specific embodiments, an
antibody provided
herein is a multispecific antibody (e.g., a bi-specific antibody) that
specifically binds to an
extracellularly accessible epitope of M(H)DM2/4 exposed on the plasma membrane
surface of
cancer cells, and also specifically binds to an antigen exposed on the surface
of natural killer
cells, neutrophils, macrophages, dendritic cells, and/or B-lymphocytes. In
specific embodiments,
an antibody provided herein is a multispecific antibody (e.g., a bi-specific
antibody) that
specifically binds to an extracellularly accessible epitope of M(H)DM2/4
exposed on the plasma
membrane surface of cancer cells, and also specifically binds to an antigen
exposed on the
surface of neutrophils, macrophages, dendritic cells, and/or B-lymphocytes. In
specific
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embodiments, an antibody provided herein is a multispecific antibody (e.g., a
bi-specific
antibody) that specifically binds to an extracellularly accessible epitope of
M(H)DM2/4 exposed
on the plasma membrane surface of cancer cells, and also specifically binds to
an antigen
exposed on the surface of natural killer cells, macrophage and/or dendritic
cells.
[00265] In a preferred embodiment, wherein a human subject is treated, the
antibody used is a
monoclonal antibody or an antigen-binding fragment thereof that is human,
humanized or
chimeric.
[00266] In certain embodiments, for example where non-human subjects are being
treated,
such as cats, dogs, cows, and other domestic, farm and wild animals, the
antibody can be an
antibody or fragment appropriate for use in the treated species (i.e., of that
species). The
antibodies described herein can be from any animal species, such as mammals
(e.g., mouse,
donkey, sheep, rabbit, goat, guinea pig, camel, horse, dog, cat) or birds
(e.g., chicken).
[00267] In specific embodiments, wherein the antibody is an immunoglobulin,
the
immunoglobulin molecules that can be used are of any type (e.g., IgG, IgE,
IgM, IgD, IgA, IgY),
class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2) or subclass of immunoglobulin
molecule. In a
preferred embodiment, the antibody is an immunoglobulin, and, in particular,
an IgG. In another
embodiment, the antibody is an IgM.
[00268] In preferred embodiments, the anti- M(H)DM2/4 antibodies or fragments
described
herein are antibodies or fragments that mediate complement-dependent
cytotoxicity (CDC),
antibody-dependent cell-mediated cytoxicity (ADCC), and/or cytotoxicity due to
a cytotoxic
drug bound to the antibody or fragment.
[00269] In specific embodiments, the anti- M(H)DM2/4 antibodies or fragments
described
herein are antibodies or fragments that are capable of inducing cytotoxicity
against the cancer
cells being targeted by such antibodies or fragments, where the cytotoxicity
can be due to
complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated
cytotoxicity
(ADCC), or due to cytotoxicity of a drug bound to the antibody (where the
antibody used is in a
form of an antibody-drug conjugate).
[00270] In a specific embodiment, the anti- M(H)DM2/4 antibodies or fragments
described
herein are antibodies or fragments that mediate complement-dependent
cytotoxicity (CDC).
Methods of making an antibody that has CDC function are known in the art. In
some
embodiments, in which the CDC activity is desired, the Fc region of the
antibody described
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herein is of a human IgG (e.g., IgGl, IgG2, IgG3, IgG4) type or a human IgM
type. In some
embodiments, in which the CDC activity is desired, the Fc region of the
antibody described
herein is of a mouse IgG (e.g., IgGl, IgG2a, IgG2b, IgG3) or mouse IgM type.
In one
embodiment, the Fc region of the antibody described herein is of a human IgG1
isotype. In one
embodiment, the Fc region of the antibody described herein is of a human IgG3
isotype. In one
embodiment, the Fc region of the antibody described herein is of a human IgG2
isotype. In one
embodiment, the Fc region of the antibody described herein is bioengineered
(e.g., mutated) to
increase its CDC activity. In one embodiment, the antibody or fragment is a
bispecific antibody
or fragment that specifically binds to two distinct extracellular epitopes on
M(H)DM2/4 (which
may, e.g., lead to the amplification of complement activation, the increased
deposition of
fragments (C3b, iC3b, C3d, C3g, C4b) on the cancer cell surface membrane,
and/or the increased
cancer cell killing by the MAC). In another embodiment, the antibody or
fragment is a bispecific
antibody or fragment that specifically binds to an extracellular epitope of
M(H)DM2/4 and
specifically binds to an extracellular epitope of a complement regulatory
protein (CRP) (which
may, e.g., prevent the degradation of the freshly deposited immunologically
active fragments
(C3b, iC3b, C3d, C3g, C4b) by CRPs, amplify the activation of the complement
cascade, and/or
amplify MAC induced cancer cell lysis). Such bispecific anti-M(H)DM2/4
antibodies or
fragments may increase CDC activity, increase ADCC activity, increase antibody-
dependent
cellular phagocytosis (ADCP) by neutrophils and macrophages, or increase CDC,
ADCC and
ADCP.
[00271] In a specific embodiment, the anti-M(H)DM2/4 antibodies or fragments
described
herein are antibodies or fragments that mediate antibody-dependent cell-
mediated cytoxicity
(ADCC) and/or antibody-dependent cellular phagocytosis (ADCP).
[00272] Methods of making an antibody that has ADCC function are known in the
art.
Methods of making an antibody that has ADCP function are known in the art.
Generally, the Fc
region of the antibody mediates its binding to an Fc receptor, FcR, on
neutrophils, macrophages,
natural killer cells, eosinophils and mast cells, which leads to ADCC and on
neutrophils,
macrophages and dendritic cells resulting in ADCP. In some embodiments, in
which the ADCC
and/or ADCP activity is desired, the Fc region of the antibody described
herein is of a human
IgG (e.g., IgGl, IgG2, IgG3) type or a human IgE type. In one embodiment, the
Fc region of the
antibody described herein is of a human IgG1 isotype.
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[00273] In one embodiment, the Fe region of the antibody described herein is
bioengineered
(e.g., via cross-linking, via di-sulfide bond formation, via oligosaccharide
addition, or via
mutation) to increase its ADCC and/or ADCP activity. In one embodiment, the Fe
region of the
antibody described herein is mutated to increase the lifespan of the intact
antibody (e.g., in
accordance with the methods described in Vaccaro et al., 2005, Nat Biotechnol.
23:1283-8128,
the disclosure of which is incorporated by reference herein). In another
embodiment, amino acid
substitutions in the Fe CH2 and CH3 domains can be employed to direct the
efficacy towards
ADCC/ADCP and away from CDC or to increase the efficiency of all three
cytotoxic activities
(i.e., ADCC, ADCP and CDC). In another embodiment, CH2 and/or CH3 domains of
the Fe
region of the antibody described herein are modified at their glycosylation
sites to remove/reduce
fucose residues in order to improve ADCC and/or ADCP function, e.g., in
accordance with the
methods described in, e.g., Satoh et al., 2006, Expert Opin Biol Ther. 6:1161-
1173 and/or Liu et
al., 2015, Ca Immunol Res. 3:173-183, the disclosures of which are
incorporated by reference
herein).
[00274] In a specific embodiment, the anti-M(H)DM2/4 antibodies described
herein having an
IgG Fe region are bioengineered at their Fe region to change the N-glycan
structure at their
glycosylation site to the GO glycan type terminating in GlcNAc (N-
acetylglucosamine), and
without fucose and sialic acid residues (which may result in, e.g., the
activation of both the
classic and alternate pathway of complement pathways and in increased binding
to lectins
including the mannose-binding lectins secreted during inflammatory responses).
[00275] In one embodiment, the Fe region of the antibody described herein is
of a human
IgG1 isotype and has alanine substitution at position 333 of its CH2 domain.
In one embodiment,
the Fe region of the antibody described herein is of a human IgG1 isotype and
has a triple
mutation S239D/I332E/A330L (which leads to a higher affinity for FcyRIIIa and
a lower affinity
for FcyRIIb resulting in enhanced ADCC) (such Fe modification can be made,
e.g., in
accordance with the methods described in Lazar et al., 2006, PNAS 103:4005-
4010). In one
embodiment, the Fe region of the antibody described herein is of a human IgG1
isotype and has a
triple mutation S239D/I332E/G236A (which leads to improved FcyRIIIa affinity
and
FcyRIIa/FcyRIIb ratio that mediates enhanced phagocytosis of target cells by
macrophages)
(such Fe modification can be made, e.g., in accordance with the methods
described in Richards et
al., 2008, Mol.Cancer Ther. 7:2517-27).
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[00276] In a specific embodiment, the anti-M(H)DM2/4 antibodies or fragments
described
herein are antibodies or fragments that mediate both complement-dependent
cytotoxicity (CDC)
and antibody-dependent cell-mediated cytoxicity (ADCC). In a specific
embodiment, the anti-
M(H)DM2/4 antibodies or fragments described herein are antibodies or fragments
that mediate
complement-dependent cytotoxicity (CDC), antibody- dependent cell-mediated
cytoxicity
(ADCC) and antibody-dependent cellular phagocytosis (ADCP). In other
embodiments,
contemplated herein is an antibody or fragment that mediates only CDC or only
ADCC activity.
The CDC and ADCC function of the antibodies described herein can be tested by
any in vitro
and/or in vivo cytotoxicity assays known in the art. In certain embodiments,
an antibody or
fragment used herein comprises one or more amino acid mutations or
substitutions in the Fc
region that improve its CDC or ADCC activity (e.g., any mutations or
substitutions described
herein or known in the art (see, e.g., Idusogie et al., 2001. J Immunol.
166(4):2571-5; Strohl,
2009, Curr Opin Biotechnol. 20(6):685-91; Lazar et al., 2006, PNAS 103(11):
4005-4010, the
disclosures of which are incorporated by reference herein)).
[00277] In a specific embodiment, the anti-M(H)DM2/4 antibodies or fragments
are
unconjugated, for example, are not conjugated to a cytotoxic drug).
[00278] In certain embodiments, the anti-M(H)DM2/4 antibodies or fragments
described
herein are not bound (e.g., not conjugated) to a drug (e.g., to a cytotoxic
drug). In some of these
embodiments, the anti-M(H)DM2/4 antibodies or fragments mediate CDC and/or
ADCC.
[00279] In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof
specifically
binds to an extracellularly accessible segment (i.e. epitope) within amino
acids 1 to 15, 15 to 25
or 475 to 491 of HDM2 (SEQ ID NO:4).
[00280] In one embodiment, an anti-HDM2 antibody or fragment thereof
specifically binds to
HDM2 within amino acids of SEQ ID NO:1 (which are amino acids 1 to 15 of HDM2
(SEQ ID
NO:4)). Amino acids of SEQ ID NO:1 (which are amino acids 1 to 15 of SEQ ID
NO:4) are in
an extracellularly accessible epitope of HDM2.
[00281] In one embodiment, an anti- HDM2 antibody or fragment thereof
specifically binds to
HDM2 within amino acids of SEQ ID NO:2 (which are amino acids 15 to 25 of HDM2
(SEQ ID
NO:4)). Amino acids of SEQ ID NO:2 (which are amino acids 15 to 25 of SEQ ID
NO:4) are in
an extracellularly accessible epitope of HDM2.
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[00282] In one embodiment, an anti-HDM2 antibody or fragment thereof
specifically binds to
HDM2 within amino acids of SEQ ID NO:3 (which are amino acids 475 to 491 of
HDM2 (SEQ
ID NO:4)). Amino acids of SEQ ID NO:3 (which are amino acids 475 to 491 of SEQ
ID NO:4)
are in an extracellularly accessible epitope of HDM2.
[00283] In one embodiment, anti-M(H)DM2/4 antibodies or fragments thereof
specifically
bind to an extracellularly accessible epitope of HDM2 within amino acids 50 to
60 of
M(H)DM2/4 (SEQ ID NO:4 or SEQ ID NO:6).
[00284] In one embodiment, anti-M(H)DM2/4 antibodies or fragments thereof
specifically
bind to an extracellularly accessible epitope of M(H)DM2/4 within amino acids
100 to 110 of
M(H)DM2/4 (SEQ ID NO:4 or SEQ ID NO:6).
[00285] In one embodiment, anti-M(H)DM2/4 antibodies or fragments thereof
specifically
bind to an extracellular epitope of M(H)DM2/4 within amino acids 1 to 126 of
M(H)DM2/4
(SEQ ID NO:4 or SEQ ID NO:6).
[00286] In one embodiment, anti-M(H)DM2/4 antibodies or fragments thereof
specifically
bind to an extracellularly accessible epitope of M(H)DM2/4 within amino acids
436 to 482 of
M(H)DM2/4 (SEQ ID NO:4 or SEQ ID NO:6).
[00287] In one embodiment, anti-M(H)DM2/4 antibodies or fragments thereof
specifically
bind to an extracellularly accessible epitope of M(H)DM2/4 within the terminal
100 amino acids
at the C- terminus of the M(H)DM2/4 (e.g., M(H)DM2/4 protein variant (splice
variant) known
or expected to be expressed on the plasma membrane of cells of the cancer type
being treated, or
M(H)DM2/4 protein variant (splice variant) determined to be expressed on the
plasma membrane
of cancer cells of the subject being treated).
[00288] The invention also provides anti-M(H)DM2/4 antibodies or fragments
thereof that
compete for binding to HDM2 with an antibody that specifically binds to HDM2
within the
amino acid sequence of SEQ ID NO:1 (e.g., NMC-103 antibody described herein,
or any
antibody or fragment having the VH of NMC-103 (i.e., the VH of SEQ ID NO:36)
and the VL of
NMC-103 (i.e., the VL of SEQ ID NO:37), or any antibody or fragment having the
VH and VL
CDRs of NMC-103). The invention also provides anti-M(H)DM2/4 antibodies or
fragments
thereof that compete for binding to HDM2 with an antibody that specifically
binds to HDM2
within the amino acid sequence of SEQ ID NO:2 (e.g., NMC-204 antibody
described herein, or
any antibody or fragment having the VH of NMC-204 (i.e., the VH of SEQ ID
NO:38) and the
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VL of NMC-204 (i.e., the VL of SEQ ID NO:39), or any antibody or fragment
having the VH
and VL CDRs of NMC204). The invention also provides anti-M(H)DM2/4 antibodies
or
fragments thereof that compete for binding to HDM2 with an antibodythat
specifically binds to
HDM2 within the amino acid sequence of SEQ ID NO:3 (e.g., NMC-303 antibody
described
herein, or any antibody or fragment having the VH of NMC-303 (i.e., the VH of
SEQ ID NO:40)
and the VL of NMC-303 (i.e., the VL of SEQ ID NO:41), or any antibody or
fragment having
the VH and VL CDRs of NMC-303).
[00289] Any competition assay known in the art can be used to identify an
antibody that
competes with an antibody described herein for binding to M(H)DM2/4 (see
Harlow and Lane
(1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory,
Cold Spring
Harbor, NY)). In an exemplary competition assay, immobilized M(H)DM2/4 (e.g.,
immobilized
on a microtiter plate or well) is incubated in a solution comprising a first
labeled antibody that
binds to M(H)DM2/4 and a second unlabeled antibody that is being tested for
its ability to
compete with the first antibody for binding to M(H)DM2/4. As a control,
immobilized
M(H)DM2/4 can be incubated in a solution comprising the first labeled antibody
but without the
second unlabeled antibody. After incubation, excess unbound antibody is
removed, and the
amount of label associated with immobilized M(H)DM2/4 is measured. The
substantial reduction
of the amount of label in the test sample relative to the control sample
indicates that the second
antibody is competing with the first antibody for binding to M(H)DM2/4.
[00290] In specific embodiments, an antibody that competes with an antibody
described
herein (e.g., antibodies having the VH and VL of NMC-103, NMC-204 or NMC-303)
for
binding to M(H)DM2/4 also binds to the same peptide derived from M(H)DM2/4
that is bound
by such antibody (e.g., the peptide of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID
NO:3). In
specific embodiments, an antibody that competes with an antibody described
herein (e.g.,
antibodies having the VH and VL of NMC-103, NMC-204 or NMC-303) for binding to
M(H)DM2/4 also binds to the same epitope in M(H)DM2/4 that is bound by such
antibody.
[00291] In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof
binds to the
same epitope of M(H)DM2/4 as an antibody or fragment having a VH of SEQ ID
NO:36 and a
VL of SEQ ID NO:37. In one embodiment, an anti-M(H)DM2/4 antibody or fragment
thereof
described herein binds to the same epitope of M(H)DM2/4 as an antibody or
fragment having a
VH of SEQ ID NO:38 and a VL of SEQ ID NO:39 . In one embodiment, an anti-
M(H)DM2/4
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antibody or fragment thereof described herein binds to the same epitope of
M(H)DM2/4 as an
antibody or fragment having a VH of SEQ ID NO:40 and a VL of SEQ ID NO:41.
[00292] In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for
use in the
methods described herein specifically binds to an extracellularly accessible
epitope within amino
acids 19 to 50 of HDM2 (SEQ ID NO:4).
[00293] In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for
use in the
methods described herein specifically binds to an extracellularly accessible
epitope within amino
acids 19 to 108 of HDM2 (SEQ ID NO:4).
[00294] In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for
use in the
methods described herein specifically binds to an extracellularly accessible
epitope within amino
acids 154 to 167 of HDM2 (SEQ ID NO:4).
[00295] In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for
use in the
methods described herein specifically binds to an extracellularly accessible
epitope within amino
acids 1 to 60 of HDM2 (SEQ ID NO:4).
[00296] In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for
use in the
methods described herein specifically binds to an extracellularly accessible
epitope within amino
acids 1 to 100 of HDM2 (SEQ ID NO:4).
[00297] In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for
use in the
methods described herein specifically binds to an extracellularly accessible
epitope within amino
acids 1 to 108 of HDM2 (SEQ ID NO:4).
[00298] In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for
use in the
methods described herein specifically binds to an extracellularly accessible
epitope within amino
acids 26 to 60 of HDM2 (SEQ ID NO:4).
[00299] In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for
use in the
methods described herein specifically binds to an extracellularly accessible
epitope within the
terminal 60 amino acids at the C-terminus of the HDM2 (e.g., HDM2 protein
variant (splice
variant) known or expected to be expressed on the plasma membrane of cells of
the cancer type
being treated, or HDM2 protein variant (splice variant) determined to be
expressed on the plasma
membrane of cancer cells of the subject being treated).
[00300] In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for
use in the
methods described herein specifically binds to an extracellularly accessible
epitope within the
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terminal 100 amino acids at the C-terminus of the HDM2 (e.g., HDM2 protein
variant (splice
variant) known or expected to be expressed on the plasma membrane of cells of
the cancer type
being treated, or HDM2 protein variant (splice variant) determined to be
expressed on the plasma
membrane of cancer cells of the subject being treated).
[00301] In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for
use in the
methods described herein specifically binds to an extracellularly accessible
epitope within amino
acids 101 to 200 of HDM2 (SEQ ID NO:4).
[00302] In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for
use in the
methods described herein competes for binding to HDM2 with antibody OP145
(monoclonal
antibody commercially available from Calbiochem, Catalogue No. 0P145-100UG;
see Table 10,
below, for further details regarding OP145). In one embodiment, an anti-
M(H)DM2/4 antibody
or fragment thereof for use in the methods described herein competes for
binding to HDM2 with
antibody 965 (SMP14) (monoclonal antibody commercially available from Santa
Cruz,
Catalogue No. Sc-965; see Tables 3 and 10, below, for further details
regarding 965 (SMP14)).
In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in
the methods
described herein competes for binding to HDM2 with antibody sc-813 (N-20)
(polyclonal
antibody commercially available from Santa Cruz, Catalogue No. Sc-813; see
Table 10, below,
for further details regarding sc-813 (N-20)). In one embodiment, an anti-
M(H)DM2/4 antibody
or fragment thereof for use in the methods described herein competes for
binding to HDM2 with
antibody sc-812 (C-18) (polyclonal antibody commercially available from Santa
Cruz, Catalogue
No. Sc-812; see Table 10, below, for further details regarding sc-812 (C-18)).
In one
embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the
methods described
herein competes for binding to HDM2 with antibody M01, clone 1A7 (monoclonal
antibody
commercially available from Abnova, Catalogue No. H00004193-M01; see Table 3,
below, for
further details regarding M01, clone 1A7).
[00303] Any competition assay known in the art can be used to identify an
antibody that
competes with antibody 0P145, SMP14, N-20, C-18, or M01, clone 1A7 for binding
to
M(H)DM2/4 (e.g., HDM2) (see Harlow and Lane (1988) Antibodies: A Laboratory
Manual
ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)).
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[00304] In specific embodiments, an antibody that competes with antibody
0P145, SMP14,
N-20, C-18, or MOL clone 1A7 for binding to M(H)DM2/4 also binds to the same
epitope that is
bound by such antibodies.
[00305] In one embodiment, an anti- M(H)DM2/4 antibody or fragment thereof for
use in the
methods described herein binds to the same epitope of HDM2 as antibody OP145
(see Table 10,
below, for further details regarding OP145). In one embodiment, an anti-
M(H)DM2/4 antibody
or fragment thereof for use in the methods described herein binds to the same
epitope of HDM2
as antibody 965 (SMP14) (see Tables 3 and 10, below, for further details
regarding 965
(SMP14)). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof
for use in the
methods described herein binds to one of the same epitope(s) of HDM2 as
polyclonal antibody
sc-813 (N-20) (see Table 10, below, for further details regarding 813 (N-20)).
In one
embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the
methods described
herein binds to one of the same epitope(s) of HDM2 as polyclonal antibody sc-
812 (C-18) (see
Table 10, below, for further details regarding sc-812 (C-18)). In one
embodiment, an anti-
M(H)DM2/4 antibody or fragment thereof for use in the methods described herein
binds to the
same epitope of HDM2 as antibody MOL clone 1A7 (i.e., monoclonal antibody
commercially
available from Abnova, Catalogue No. H00004193-M01; see Table 3, below, for
further details
regarding M01, clone 1A7).
[00306] In a specific embodiment, the anti-M(H)DM2/4 antibody or fragment
thereof
described herein is purified. In certain embodiments, an antibody or fragment
is purified to
greater than 95% or 99% purity as determined by, for example, electrophoretic
(e.g., SDS-
PAGE, isoelectric focusing (IEF), capillary electrophoresis) or
chromatographic (e.g., ion
exchange or reverse phase HPLC) methods (see Flatman et al., I Chromatogr. B
848:79-87
(2007) for review of methods for assessment of antibody purity).
[00307] The anti-M(H)DM2/4 antibody or fragment described herein can be fused
or
conjugated (e.g., covalently or non-covalently linked) to a detectable label
or substance. Such
labeled antibodies or fragments can be used to detect M(H)DM2/4 on the plasma
membrane
surface of cells.
[00308] Examples of detectable labels or substances include enzyme labels,
radioisotopes
(e.g., iodine, carbon, sulfur, tritium, indium, and technetium), luminescent
labels, fluorescent
labels, and biotin. This methodology can be used to determine whether cells of
a certain cancer
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(e.g., cells of cancer in a patient) express M(H)DM2/4, or a certain splice
variant of
M(H)DM2/4, on the plasma membrane, where the detection of M(H)DM2/4 using the
antibody
(or fragment) may indicate that the antibody (or fragment) (with or without
the detectable label
or substance) can be used in the diagnosis and treatment of the cancer or
preventing metastases
of the cancer.
7.2 Antibody-drug conjugates
[00309] In a specific embodiment, the invention provides antibody-drug
conjugates
comprising an anti-M(H)DM2/4 antibody or fragment described herein bound
(e.g., covalently
bound) to a cytotoxic drug. In such embodiments, the antibody-drug conjugates
are intended to
mediate cytotoxicity by delivery of a cytotoxic drug to the cells of the
cancer.
[00310] Accordingly, an anti-M(H)DM2/4 antibody or an antigen-binding fragment
described
herein can be bound or conjugated to one or more cytotoxic agents. The
cytotoxic agent can be
any agent that inhibits or prevents a vital cellular function (e.g., cell
division) and/or causes cell
death or destruction. The cytotoxic agents that can be bound or conjugated to
an anti-
M(H)DM2/4 antibody or fragment include, without limitation, chemotherapeutic
agents (e.g.,
any chemotherapeutic agent known in the art or described herein), toxins
(e.g., protein toxins,
enzymatically active toxins of bacterial, fungal, plant or animal origin, or
fragments thereof),
radioactive isotopes, growth inhibitory agents, and nucleolytic enzymes. The
antibody-drug
conjugates and their methods of making (including the types of antibodies that
can be used in
such conjugates, drugs that can be used in such conjugates, and linkers that
can be used to link
the antibody to the drug) are known in the art (see, e.g., Peters & Brown,
2015, Biosci. Rep. 35,
e00225, doi:10.1042/BSR20150089).
[00311] Examples of the cytotoxic agents that can be conjugated to an anti-
M(H)DM2/4
antibody or fragment described herein include, without limitation,
anthracyclin, doxorubicin,
methotreaxate, an anti-metabolite agent, an anti-folate agent, an auristatin
(e.g., MMAE or
MMAF), a maytansine, a calicheamicin, a duocarymucin, and a
pyrrolobenzodiazepine (PBD)
dimer.
[00312] In specific embodiments, an M(H)DM2/4 antibody or fragment described
herein is
conjugated to one or more of the following drugs: a maytansinoid, an
auristatin (such as
monomethylauristatin drug moieties DE and DF (MMAE and MMAF)), a dolastatin, a
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calicheamicin or derivative thereof, an anthracycline (such as daunomycin or
doxorubicin),
methotrexate, vindesine, a taxane (such as docetaxel), paclitaxel, larotaxel,
tesetaxel, ortataxel,
and a trichothecene.
[00313] In another embodiment, an M(H)DM2/4 antibody or fragment described
herein is
conjugated to a toxin or a fragment thereof (e.g., diphtheria A chain,
nonbinding active
fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain,
modeccin A chain,
alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca
americana proteins,
momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis
inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin, or a tricothecene).
[00314] In another embodiment, an M(H)DM2/4 antibody or fragment described
herein is
conjugated to a radioactive isotope (e.g., At211, 1131, 1125, y90, Re186,
Re188, sm153,j22, P32,
pb212,
or a radioactive isotope of Lu).
[00315] In another embodiment, an M(H)DM2/4 antibody or fragment thereof
described
herein is conjugated to nanoparticles or other targeting tools to promote
concentrated delivery to
and retention of the antibodies at the tumor site.
7.3 Making of antibodies
[00316] The anti-M(H)DM2/4 antibodies or fragments described herein can be
produced by
any method known in the art.
[00317] Methods for producing polyclonal antibodies are known in the art (see,
for example,
Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel
FM et at., eds.,
John Wiley and Sons, New York).
[00318] Methods for producing monoclonal antibodies are also known in the art,
and include
the use of hybridoma, recombinant and phage display technologies, and the use
of humanized
mice. For example, monoclonal antibodies can be produced using hybridoma
techniques as
taught, for example, in Harlow E & Lane D, Antibodies: A Laboratory Manual,
(Cold Spring
Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ et at., in: Monoclonal
Antibodies and
T-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981), or in Kohler G & Milstein C
(1975) Nature
256: 495. In another example, human monoclonal antibodies can be produced
using humanized
mice as taught, for example, in Laffleur et at., 2012, Methods Mol. Biol.
901:149-59.
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[00319] Methods for producing specific antibodies using hybridoma technology
are routine
and well known in the art. In particular, a mouse or another appropriate host
animal can be
immunized to elicit lymphocytes that produce or are capable of producing
antibodies that will
specifically bind to the target protein (i.e., extracellular region of
M(H)DM2/4) used for
immunization. Lymphocytes then are fused with myeloma cells to form a
hybridoma cell (see
Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press,
1986); see also Kozbor D (1984) J Immunol 133: 3001-5; Brodeur et at.,
Monoclonal Antibody
Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New
York, 1987)).
The hybridoma cells are then grown in a suitable culture medium, which can be
assayed for
production of monoclonal antibodies directed against M(H)DM2/4. The binding
specificity of
monoclonal anti- M(H)DM2/4 antibodies produced by this method can be
determined by
methods known in the art, e.g., immunoprecipitation or an in vitro assay, such
as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). The
hybridoma
clones thus selected are then grown by standard methods (see Goding JW (Ed),
Monoclonal
Antibodies: Principles and Practice, supra). The monoclonal antibodies can
then be separated
from the culture medium and purified.
[00320] Further, the antibodies or fragments described herein can also be made
using various
phage display technologies known in the art (see Brinkman U et at., (1995) J
Immunol Methods
182: 41-50; Ames RS et at., (1995) J Immunol Methods 184: 177-186;
Kettleborough CA et al.,
(1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene 187: 9-18; and
Burton DR &
Barbas CF (1994) Advan Immunol 57: 191-280).
[00321] Methods for producing chimeric antibodies (i.e., antibody with a
variable region of
one species (e.g., murine) and a constant region of another species (e.g.,
human)) are known in
the art (see Morrison SL (1985) Science 229: 1202-7; Oi VT & Morrison SL
(1986)
BioTechniques 4: 214-221; Gillies SD et al., (1989) J Immunol Methods 125: 191-
202; and U.S.
Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415).
[00322] Methods of making antibody fragments are known in the art. For
example, Fab and
F(ab1)2 fragments can be produced by proteolytic cleavage of immunoglobulin
molecules, using
enzymes such as papain (to produce Fab fragments) or pepsin (to produce
F(ab1)2 fragments).
[00323] Methods of making humanized antibodies are also known in the art (see
International
Publication No. WO 91/09967; Padlan EA (1991) Mol Immunol 28(4/5): 489-498;
Studnicka
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GM et at., (1994) Prot Engineering 7(6): 805-814; and Roguska MA et at.,
(1994) PNAS 91:
969-973; International Publication No. WO 93/17105; Tan P et at., (2002) J
Immunol 169: 1119-
25; Caldas C et al., (2000) Protein Eng. 13(5): 353-60; Morea V et al.,
(2000), Methods 20(3):
267-79; Baca M et at., (1997) J Biol Chem 272(16): 10678-84; Roguska MA et
at., (1996)
Protein Eng 9(10): 895 904; Couto JR et at., (1995) Cancer Res. 55 (23 Supp):
5973s-5977s;
Couto JR et at., (1995) Cancer Res 55(8): 1717-22; Sandhu JS (1994) Gene
150(2): 409- 10 and
Pedersen JT et at., (1994) J Mol Biol 235(3): 959-73). In a specific
embodiment, a humanized
antibody is made by CDR grafting.
[00324] Methods of making human antibodies are known in the art and include
phage display
methods using antibody libraries derived from human immunoglobulin sequences
(see U.S.
Patent Nos. 4,444,887, 4,716,111, and 5,885,793; and International Publication
Nos. WO
98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and
WO
91/10741). In some embodiments, human antibodies can be produced using mouse-
human
hybridomas (see Shinmoto H et at., (2004) Cytotechnology 46: 19-23; Naganawa Y
et at., (2005)
Human Antibodies 14: 27-31).
[00325] Methods of making single domain antibodies, for example, antibodies
lacking the
light chains, are also known in the art (see Riechmann L & Muyldermans S
(1999) J Immunol
231: 25-38; Nuttall SD et at., (2000) Curr Pharm Biotechnol 1(3): 253-263;
Muyldermans S,
(2001) J Biotechnol 74(4): 277-302).
[00326] Methods of making single chain Fv (scFv) antibodies are also known in
the art (see
Ahmad et al., 2012, Clinical and Developmental Immunology,
doi:10.1155/2012/980250; Wang
et al., 2006, Anal. Chem. 78, 997-1004; Pansri et al., 2009, BMC Biotechnology
9:6). For
example, scFv antibodies can be constructed by fusing variable domains of
heavy and light
chains of immunoglobulins via short polypeptide linkers (using recombinant
expression
techniques), and scFv antibodies having desired antigen-binding properties can
be selected by
phage display technology.
[00327] Methods of producing bispecific antibodies are well known in the art
(Konterman,
2012, MAbs 4:182-197; Gramer et al., 2013, MAbs 5:962-973).
[00328] Methods of conferring CDC or ADCC activity on an antibody (such as an
antibody
that does not have CDC or ADCC activity to begin with) are known in the art
(see Kellner et al.,
2014, Methods 65:105-113; International Publication No. WO 2012010562; Natsume
et al.,
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2009, Drug Design, Development and Therapy 3(3):7-16). Such methods include,
without
limitation, Fc region isotype shuffling, amino acid mutations in the Fc region
conferring
enhanced or optimized CDC and/or ADCC activity, and changes in the Fc region
glycosylation
profile conferring enhanced or optimized CDC and/or ADCC activity).
7.4 Antibody Selection
[00329] If a candidate antibody or fragment for use in the therapeutic and
diagnostic methods
provided herein is not yet known or has not yet been demonstrated to bind to a
region of
M(H)DM2/4 exposed on the surface of cancer cells, the antibody or fragment may
optionally be
tested by any of the following methods:
[00330] In certain aspects, provided herein is a method for identifying an
anti- M(H)DM2/4
antibody or a fragment thereof for use in the methods described herein (e.g.,
for diagnosis of
cancer, treating cancer or for preventing metastases) comprising: (a)
contacting intact cancer
cells (e.g., cancer cells expected, known, or determined to express M(H)DM2/4)
with an anti-
M(H)DM2/4 antibody or a fragment thereof; and (b) determining whether the
antibody or
fragment binds to the intact cancer cells, in particular on the extracellular
surface of the cancer
cells (e.g., relative to intact cancer cells not contacted by said antibody or
fragment), wherein the
binding of the anti-M(H)DM2/4 antibody or fragment to the intact cancer cells
contacted with
such antibody or fragment indicates that said antibody or fragment is suitable
for use in the
methods described herein. In a specific embodiment, the cancer cells are from
the patient
proposed to be treated with the antibody or fragment thereof.
[00331] In certain aspects, provided herein is a method for identifying an
anti- M(H)DM2/4
antibody or a fragment thereof for use in the methods described herein (e.g.,
for diagnosis of
cancer, treating cancer or for preventing metastases) comprising: (a)
contacting intact cancer
cells (e.g., cancer cells expected, known, or determined to express M(H)DM2/4)
with an anti-
M(H)DM2/4 antibody or a fragment thereof; and (b) determining whether the
antibody or
fragment binds to the intact cancer cells (in particular on the extracellular
surface of the cancer
cells) at an increased level relative to binding to intact normal cells (e.g.,
non-cancerous cells of
the same tissue or organ type as the cancer cells), wherein increased binding
of the anti-
M(H)DM2/4 antibody or fragment to the intact cancer cells relative to normal
cells indicates that
said antibody or fragment is suitable for use in the methods described herein.
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[00332] In certain aspects, provided herein is a method for identifying an
anti-M(H)DM2/4
antibody or a fragment thereof for use in the methods described herein (e.g.,
for treating cancer
or for preventing metastases) comprising: (a) contacting intact cancer cells
(e.g., cancer cells
known, expected or determined to express M(H)DM2/4) with an anti-M(H)DM2/4
antibody or a
fragment thereof; and (b) determining whether the contacting step results in
an increased amount
of cell death or destruction of the intact cancer cells (e.g., as determined
by cell-death markers
such as Propidium Iodide staining) relative to the amount of death or
destruction of intact cancer
cells not contacted by said antibody or fragment and/or relative to the amount
of death or
destruction of intact normal cells (e.g., non-cancerous cells of the same
tissue or organ as the
cancer cells) contacted by said antibody or fragment, wherein increased amount
of cell death or
destruction of the intact cancer cells contacted with the antibody or fragment
indicates that said
antibody or fragment is suitable for use in the methods described herein.
[00333] In certain aspects, provided herein is a method for identifying an
anti-M(H)DM2/4
antibody or a fragment thereof for use in the methods described herein (e.g.,
for treating cancer
or for preventing metastases) comprising: (a) contacting intact cancer cells
(e.g., cells known,
expected or determined to express M(H)DM2/4) with an anti- M(H)DM2/4 antibody
or
fragment; and (b) determining whether the contacting step results in an
increased level of
complement-dependent cytotoxicity (CDC) or antibody-dependent cell- mediated
cytotoxicity
(ADCC) (as determined by one or more cytotoxicity assays) towards the intact
cancer cells
relative to the level of CDC or ADCC towards intact cancer cells not contacted
by said antibody
or fragment and/or relative to the level of CDC or ADCC towards intact normal
cells (e.g., non-
cancerous cells of the same tissue or organ as the cancer cells) contacted by
said antibody or
fragment, wherein increased level of CDC or ADCC towards the intact cancer
cells indicates that
said antibody or fragment is suitable for use in the methods described herein.
[00334] In certain aspects, provided herein is a method for identifying an
anti-M(H)DM2/4
antibody or a fragment thereof for use in the methods described herein (e.g.,
for treating cancer
or for preventing metastases) comprising: (a) contacting intact cancer cells
(e.g., cells known,
expected or determined to express M(H)DM2/4) with an anti-M(H)DM2/4 antibody
or a
fragment thereof; and (b) determining whether the contacting step results in
increased inhibition
of growth and proliferation of the intact cancer cells relative to the
inhibition of growth and
proliferation of intact cancer cells not contacted by said antibody or
fragment and/or relative to
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the inhibition of growth and proliferation of intact normal cells (e.g., non-
cancerous cells of the
same tissue or organ as the cancer cells) contacted by said antibody or
fragment, wherein
increased inhibition of growth and proliferation of the intact cancer cells
contacted with the
antibody or fragment indicates that said antibody or fragment is suitable for
use in the methods
described herein.
7.5 Pharmaceutical compositions
[00335] Provided herein are pharmaceutical compositions comprising an anti-
M(H)DM2/4
antibody or an antigen-binding fragment thereof and a pharmaceutically
acceptable carrier. Also
provided herein are pharmaceutical compositions comprising an antibody-drug
conjugate as
described herein and a pharmaceutically acceptable carrier. Appropriate
pharmaceutically
acceptable carriers including, but not limited to, excipients and stabilizers)
are known in the art
(see, e.g., Remington's Pharmaceutical Sciences (1990) Mack Publishing Co.,
Easton, PA). The
anti-M(H)DM2/4 antibody or fragment, or antibody-drug conjugate, in the
pharmaceutical
compositions described herein can be purified.
[00336] Pharmaceutically acceptable carriers may include an isotonic agent, a
buffer, a
suspending agent, a dispersing agent, an emulsifying agent, a wetting agent, a
sequestering or
chelating agent, a pH buffering agent, a solubility enhancer, an antioxidant,
an anesthetic, and/or
an antimicrobial agent. Suitable excipients include, without limitation,
water, saline, glycerol,
ethanol, starch, lactose, sucrose, gelatin, malt, propylene, silica gel,
sodium stearate, base cream
and dextrose. If administered parenterally, suitable pharmaceutically
acceptable carriers may
include physiological saline or phosphate buffered saline (PBS), and solutions
containing such
agents as glucose, polyethylene glycol, polypropylene glycol or other agents.
[00337] In specific embodiments, pharmaceutical compositions provided herein
comprise an
anti-M(H)DM2/4 antibody or an antigen-binding fragment thereof, or antibody-
drug conjugate,
described herein, and optionally one or more other therapeutic (e.g., anti-
cancer) agents, in a
pharmaceutically acceptable carrier.
[00338] A pharmaceutical composition may be formulated for any route of
administration to a
subject. Formulations for injections can be prepared as liquid solutions,
suspensions, emulsions,
or solid forms suitable for making into a solution or suspension prior to
injection.
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[00339] The anti-M(H)DM2/4 antibody or fragment thereof, or antibody-drug
conjugate, can
be used or present in the pharmaceutical composition in a therapeutically
effective amount. The
therapeutically effective amount of the antibody or conjugate can be
determined by standard
clinical techniques.
[00340] In a specific embodiment, a pharmaceutical composition contemplated
for use in the
therapeutic methods described herein comprises an anti-M(H)DM2/4 antibody or
an antigen-
binding fragment thereof, or antibody-drug conjugate described herein, and
does not comprise
any additional anti-cancer agent or therapy. In another specific embodiment, a
pharmaceutical
composition contemplated for use in the therapeutic methods described herein
comprises an anti-
M(H)DM2/4 antibody or an antigen-binding fragment thereof, or antibody-drug
conjugate
described herein, and further comprises an additional anti-cancer agent or
therapy (e.g., any one,
two or more additional anti-cancer agents or therapies described herein).
[00341] In one aspect, provided herein is a vaccine composition comprising:
(i) an
immunogenic amount of a peptide, wherein the amino acid sequence of the
peptide is
MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or
CPVCRQPIQMIVLTYFP (SEQ ID NO:3), or a polynucleotide encoding the peptide; and
(ii) a
pharmaceutically acceptable carrier. In a certain embodiment, provided herein
is a vaccine
composition comprising: (i) an immunogenic amount of a peptide, wherein the
amino acid
sequence of the peptide is MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID
NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3); and (ii) a pharmaceutically
acceptable
carrier. In one embodiment of the vaccine compositions described herein, the
peptide is purified.
In one embodiment of the vaccine compositions described herein, the
composition further
comprises an adjuvant (such as an immune-stimulating adjuvant). In one
embodiment of the
vaccine compositions described herein, the pharmaceutically acceptable carrier
is a liposome. In
one aspect, provided herein is a method of vaccinating a subject at risk for
developing cancer or
a subject who has been dignosed with cancer by administering to the subject
the vaccine
composition described herein. A vaccine composition can be produced using any
method known
in the art (see, e.g., Li et al., 2014, Vaccines 2:515-536).
7.6 Therapeutic Methods
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[00342] In one aspect, the invention provides for treating cancer (e.g.,
inhibiting cancer
proliferation, inhibiting cancer progression, and/or increasing survival) in a
subject in need
thereof comprising administering to the subject any anti-M(H)DM2/4 (e.g., anti-
HDM2)
antibody or fragment described herein. In a specific embodiment, the invention
provides a
method of treating cancer in a subject in need thereof, said method comprising
administering to
the subject: an antibody or a fragment thereof that specifically binds to an
extracellularly
accessible epitope of M(H)DM2/4. In another specific embodiment, the invention
provides a
method of preventing metastasis in a subject in need thereof, said method
comprising
administering to the subject: an antibody or a fragment thereof that
specifically binds to an
extracellularly accessible epitope of M(H)DM2/4. In a specific embodiment of
the methods
described herein, the antibody or fragment is not bound to a cell-penetrating
peptide.
[00343] In one aspect, the invention provides for treating cancer (e.g.,
inhibiting cancer
proliferation, inhibiting cancer progression, and/or increasing survivial) in
a subject in need
thereof comprising administering to the subject an antibody or a fragment
thereof that
specifically binds to an extracellularly accessible epitope of HDM2 exposed on
the surface of
cancer cells (e.g., where the cells of the type of cancer being treated are
known or expected to
have such extracellular region of HDM2 exposed on their plasma membrane
surface), preferably
wherein the antibody or fragment is not bound to a cell-penetrating peptide.
In one embodiment,
the method of treating cancer encompasses preventing metastasis of the cancer.
In one
embodiment, the method of treating cancer is a method for reducing tumor size
(as measured,
e.g., by tumor volume or diameter), inhibiting the growth of the tumor,
reducing the growth of
the tumor, or eradicating the tumor.
[00344] In another aspect, the invention provides for preventing metastases in
a subject that
has cancer comprising administering to the subject any anti-M(H)DM2/4 (e.g.,
anti-HDM2)
antibody or fragment described herein. In another aspect, the invention
provides for preventing
metastases in a subject that has cancer comprising administering to the
subject an antibody or a
fragment thereof that specifically binds to an extracellular region of HDM2
exposed on the
surface of cancer cells (e.g., where the cells of the type of cancer being
treated are known or
expected to have such extracellular region of HDM2 exposed on their plasma
membrane
surface).
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[00345] In another aspect, the invention provides for reducing the number,
size or
invasiveness of metastases, or eradicating metastases, in a subject that has a
metastatic cancer
comprising administering to the subject any anti-M(H)DM2/4 (e.g., anti-HDM2)
antibody or
fragment described herein. In another aspect, the invention provides for
reducing the number,
size or invasiveness of metastases, or eradicating metastases, in a subject
that has a metastatic
cancer comprising administering to the subject an antibody or a fragment
thereof that specifically
binds to an extracellularly accessible epitope of HDM2 exposed on the surface
of cancer cells
(e.g., where the cells of the type of cancer being treated are known or
expected to have such
extracellularly accessible epitope of HDM2 exposed on their plasma membrane
surface).
[00346] In one aspect, the invention provides for treating cancer (e.g.,
inhibiting cancer
proliferation, inhibiting cancer progression, and/or increasing survival) in a
subject who has
experienced an accelerated rate of cancer growth in response to administration
of an inhibitor of
one or more inhibitory checkpoint molecules, comprising administering to the
subject any anti-
M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein. In one
aspect, the
invention provides for treating cancer (e.g., inhibiting cancer proliferation,
inhibiting cancer
progression, increasing survival) in a subject who has experienced hyper-
progression in response
to administration of an inhibitor of one or more inhibitory checkpoint
molecules, comprising
administering to the subject any anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or
fragment
described herein. In one aspect, the invention provides for treating cancer
(e.g., inhibiting cancer
proliferation, inhibiting cancer progression, increasing survival) in a
subject who is at risk for
hyper-progression in response to administration of an inhibitor of one or more
inhibitory
checkpoint molecules, comprising administering to the subject any anti-
M(H)DM2/4 (e.g., anti-
HDM2) antibody or fragment described herein. In a specific embodiment, the
methods
described herein comprise administering to the subject: an antibody or a
fragment thereof that
specifically binds to an extracellularly accessible epitope of M(H)DM2/4. In a
specific
embodiment of the methods described herein, the antibody or fragment is not
bound to a cell-
penetrating peptide. In certain embodiments of the methods described herein,
the inhibitor of
one or more inhibitory checkpoint molecules is an inhibitor, such as an
inhibitory antibody to, of
one or more of: CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, 0X40, and LAG-3. In a
specific
embodiment of the methods described herein, the inhibitor of one or more
inhibitory checkpoint
molecules is an antibody that is nivolumab (Opdivo, Bristol-Myers Squibb),
pembrolizumab
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(Keytruda, Merck), avelumab (Bavencio; EMD Serono, Pfizer), atezolizumab
(Tecentriq,
Genentech), durvalumab (Imfinzi, AstraZeneca), cemiplimab (Libtayo, Regeneron)
or
ipilimumab (Yervoy, Bristol-Myers Squibb). In one embodiment, the inhibitor of
one or more
inhibitory checkpoint molecules is an inhibitor of, such as an inhibitory
antibody to, PD-1. In
certain embodiments, the methods described herein further comprise, before
administering an
anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein,
identifying the
subject as a hyper-progressor in response to administration of an inhibitor of
one or more
inhibitory checkpoint molecules (and, e.g., administering any anti-M(H)DM2/4
(e.g., anti-
HDM2) antibody or fragment described herein if the subject is a hyper-
progressor). In certain
embodiments, the methods described herein further comprise, before
administering an anti-
M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein, determining
whether any
anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein binds
to the surface
of intact cells of the cancer (e.g., intact cancer cells isolated from the
subjects) (and, e.g.,
administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment
described herein if
an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein
binds to the
surface of intact cells of the cancer). In certain embodiments, the methods
described herein
further comprise, before administering an anti-M(H)DM2/4 (e.g., anti-HDM2)
antibody or
fragment described herein, determining whether the subject has a gene
amplification of
M(H)DM2/4 (and, e.g., administering an anti-M(H)DM2/4 (e.g., anti-HDM2)
antibody or
fragment described herein if the subject has a gene amplification of
M(H)DM2/4). In certain
embodiments, the methods described herein further comprise, before
administering an anti-
M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein, determining
how many
copies of the M(H)DM2/4 gene are present in the cancer cells of the subject,
and, e.g.,
administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment
described herein if
the cancer cells of the subject have 8 or more than 8 copies of M(H)DM2/4
gene. In certain
embodiments, the methods described herein further comprise, before
administering an anti-
M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein, determining
the degree
of M(H)DM2/4 gene amplification in the cancer cells of the subject, and, e.g.,
administering an
anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein if the
cancer cells of
the subject have 50% or more than 50% amplification of the M(H)DM2/4 gene
relative to the
normal cells of the subject (or relative to the cancer cells of the subject
prior to administration of
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an inhibitor of an immune checkpoint molecule). In one embodiment, the gene
amplification of
M(H)DM2/4 is gene amplification of HDM2. In one embodiment, the gene
amplification of
M(H)DM2/4 is gene amplification of HDM4. In one embodiment, the gene
amplification of
M(H)DM2/4 is gene amplification of HDM2 and HDM4. In certain embodiments, the
methods
described herein further comprise, before administering an anti-M(H)DM2/4
(e.g., anti-HDM2)
antibody or fragment described herein, determining whether the subject has an
increased protein
expression of M(H)DM2/4 in the cells of the cancer relative to the level of
protein expression of
M(H)DM2/4 in normal cells (and, e.g., administering an anti-M(H)DM2/4 (e.g.,
anti-HDM2)
antibody or fragment described herein if the subject has an increased protein
expression of
M(H)DM2/4 in the cells of the cancer relative to the level of protein
expression of M(H)DM2/4
in normal cells). In certain embodiments, the methods described herein further
comprise, before
administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment
described herein,
determining whether the subject has an increased protein expression of
M(H)DM2/4 in the cells
of the cancer relative to the level of protein expression of M(H)DM2/4 in
cancer cells prior to
administration of the inhibitor of a checkpoint molecule (and, e.g.,
administering an anti-
M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein if the
subject has an
increased protein expression of M(H)DM2/4 in the cells of the cancer relative
to the level of
protein expression of M(H)DM2/4 in cancer cells prior to administration of the
inhibitor of a
checkpoint molecule). In certain embodiments, the methods described herein
further comprise,
before administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment
described
herein, determining whether the subject has an increased binding of an
antibody or a fragment
thereof that specifically binds to an extracellularly accessible epitope of
M(H)DM2/4 to the
surface of intact cells of the cancer relative to its binding to the surface
of intact normal cells (or
relative to its binding to the surface of intact cells of the cancer prior to
administration of an
inhibitor of an immune checkpoint molecule). In certain embodiments, the
methods described
herein comprise administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or
fragment
described herein if the subject has an increased binding of an antibody or a
fragment thereof that
specifically binds to an extracellularly accessible epitope of M(H)DM2/4 to
the surface of intact
cells of the cancer relative to its binding to the surface of intact normal
cells (or relative to its
binding to the surface of intact cells of the cancer prior to administration
of an inhibitor of an
immune checkpoint molecule). In certain embodiments of the methods described
herein, the
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cancer is a bladder cancer, a breast cancer (e.g., a triple negative breast
cancer), a lung cancer, a colon
cancer, a melanoma, a sarcoma (e.g., an endometrial stromal sarcoma), an
ovarian cancer, a glioma, a
head and neck cancer, an urothelial cancer, a pancreatic cancer, or a squamous
cell carcinoma of the
hypopharynx. In certain embodiments of the methods described herein, an anti-
M(H)DM2/4 (e.g.,
anti-HDM2) antibody or fragment described herein is administered in
combination with another
therapy, such as immunotherapy (e.g., an inhibitor of an inhibitory checkpoint
molecule), a
chemotherapy, a small molecule inhibitor (such as an inhibitor of one or more
of: EGFR, KRAS,
STK11, ALK, BRAF, ERBB2, RET, ROS1, B2M, HLA, POLE, IGF-1, ERK/MAPK,
PI3K/AKT, TGF-f3, DNMT3A, IFNy, JAK1/JAK2/JAK3, CD274, PTEN, ART, CDK), or an
inhibitor of p53-H(M)DM2 interaction (e.g., a small molecule inhibitor or a
peptide inhibitor).
In certain embodiments of the methods described herein, an anti-M(H)DM2/4
(e.g., anti-HDM2)
antibody or fragment described herein is administered as a monotherapy.
[00347] In one aspect, the invention provides for treatment of a cancer that
is resistant to
another cancer therapy or therapies (e.g., vaccine, targeted therapy (such as
small molecule
targeted therapy), chemotherapy, radiotherapy, or immunotherapy (such as
treatment with
another monoclonal antibody). In one embodiment, the invention provides for
treating a cancer
resistant to chemotherapy (i.e., resistant to one or more chemotherapeutic
drugs). In one
embodiment, the invention provides for treating a cancer resistant to
treatment with another
monoclonal antibody or antibodies. In one embodiment, the invention provides
for treating a
cancer resistant to radiotherapy. In one embodiment, the invention provides
for treating a cancer
resistant to small molecule targeted therapy or therapies.
[00348] Disclosed herein is therapeutic use of an anti-M(H)DM2/4 (e.g., anti-
HDM2)
antibody or antigen-binding fragment thereof in a patient who has cancer
(e.g., has been
diagnosed with cancer). In preferred embodiments, disclosed herein is
therapeutic use of an anti-
HDM2 antibody (or a fragment thereof) in a patient who has cancer that is
known to metastasize
(i.e., is a type of cancer that is commonly known to become metastatic
cancer). In some
embodiments, the patient being treated has metastatic cancer. In other
embodiments, a patient
with a cancer that has not metastasized is treated in accordance with a method
described herein
in order to prevent metastasis of the cancer. In some embodiments, the patient
being treated has
been previously treated with other cancer therapies (e.g. vaccine, targeted
therapy,
chemotherapy, immunotherapy). In a specific embodiment, the patient with a
cancer that has
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metastasized is treated in accordance with a method described herein in order
to reduce, slow
down or stop metastases, or decrease the number or size of metastases of the
cancer.
[00349] The methods described herein are suitable for treating cancers that
are expected,
known or determined to express anti-M(H)DM2/4 (e.g., HDM2) on the surface of
their cells. The
HDM2 on the surface of cancer cells targeted by the methods described herein
can be one of the
splice variants of HDM2 protein known in the art or described herein. Without
intending to be
bound by a mechanism of action, it is believed that HDM2 on the surface of
cancer cells is
usually a splice variant of the HDM2 protein that lacks at least one or all
nuclear localization
signals (e.g., the nuclear localization sequence at the N-terminal portion of
HDM2, the nuclear
localization signal at the C-terminal portion of HDM2, or both nuclear
localization signals). In a
specific embodiment, HDM2 on the surface of cancer cells is a splice variant
of the HDM2
protein that lacks at least one or all nuclear localization signals, and
further lacks a nuclear
export signal. In one embodiment, the HDM2 on the surface of cancer cells is
HDM2 that lacks
the nuclear localization signal at amino acids 179-185 of SEQ ID NO: 4 (i.e.,
lacks amino acids
179 to 185 of SEQ ID NO: 4). In one embodiment, the HDM2 on the surface of
cancer cells is
HDM2 that lacks the nuclear localization signal at amino acids 466-473 of SEQ
ID NO: 4 (i.e.,
lacks amino acids 466 to 473 of SEQ ID NO: 4). In one embodiment, the HDM2 on
the surface
of cancer cells is HDM2 that lacks the nuclear localization signals at amino
acids 179-185 and
amino acids 466-473 of SEQ ID NO: 4 (i.e., lacks amino acids 179 to 185 and
466 to 473 of
SEQ ID NO: 4). In some embodiments, the HDM2 on the surface of cancer cells is
HDM2 that
lacks the nuclear export signal, such as the nuclear export signal at amino
acids 190-202 of SEQ
ID NO: 4 (i.e., lacks amino acids 190 to 202 of SEQ ID NO: 4).
[00350] In certain embodiments, the HDM2 on the surface of cancer cells
targeted by the
methods described herein is a splice variant of the HDM2 protein, such as one
of the splice
variants known in the art or described herein (see Table 1 and Table 2, and
Section 8, for the list
of HDM2 variants). In some embodiments, the HDM2 on the surface of cancer
cells targeted by
the methods described herein is a splice variant of the HDM2 protein known as
MDM2-A (SEQ
ID NO: 8), which lacks amino acids 28-222 of SEQ ID NO: 4. In some
embodiments, the
HDM2 on the surface of cancer cells targeted by the methods described herein
is a splice variant
of the HDM2 protein known as MDM2-A1 (SEQ ID NO: 9), which lacks amino acids
28-222
and 275-300 of SEQ ID NO: 4. In some embodiments, the HDM2 on the surface of
cancer cells
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targeted by the methods described herein is a splice variant of the HDM2
protein known as
MDM2-B (SEQ ID NO: 10), which lacks amino acids 28-300 of SEQ ID NO: 4. In
some
embodiments, the HDM2 on the surface of cancer cells targeted by the methods
described herein
is a splice variant of the HDM2 protein known as MDM2-C (SEQ ID NO: 11), which
lacks
amino acids 53-222 of SEQ ID NO: 4. In some embodiments, the HDM2 on the
surface of
cancer cells targeted by the methods described herein is a splice variant of
the HDM2 protein
known as MDM2-D (SEQ ID NO:12 ), which lacks amino acids 30-388 of SEQ ID NO:
4. In
some embodiments, the HDM2 on the surface of cancer cells targeted by the
methods described
herein is a splice variant of the HDM2 protein known as MDM2-E (SEQ ID NO:
13), which
lacks amino acids 76-102 and 103-491 of SEQ ID NO: 4. In some embodiments, the
HDM2 on
the surface of cancer cells targeted by the methods described herein is a
splice variant of the
HDM2 protein known as MDM2-F (SEQ ID NO: 14), which lacks amino acids 53-97 of
SEQ ID
NO: 4. In some embodiments, the HDM2 on the surface of cancer cells targeted
by the methods
described herein is a splice variant of the HDM2 protein known as MDM2-G (SEQ
ID NO: 15),
which lacks amino acids 115-169 of SEQ ID NO: 4. In some embodiments, the HDM2
on the
surface of cancer cells targeted by the methods described herein is a splice
variant of the HDM2
protein known as MDM2-11 (SEQ ID NO: 16), in which amino acid M has been
substituted with
M MVRSRQM in SEQ ID NO: 4. In some embodiments, the HDM2 on the surface of
cancer
cells targeted by the methods described herein is a splice variant of the HDM2
protein known as
MDM2-KB2 (SEQ ID NO: 17), which lacks amino acids 157-248 of SEQ ID NO: 4. In
some
embodiments, the methods described herein target a splice variant of M(H)DM4
on the surface
of cancer cells, for example, target one or more of the following splice
variants: MDMX-S,
MDM4-S, MDM4-A, MDM4-G, MDM4-XALT1/XALT2 and MDM4-211 (or a human
equivalent of the listed splice variants).
[00351] In a specific embodiment, the anti-M(H)DM2/4 (e.g., anti-HDM2)
antibody or a
fragment thereof for use in the methods described herein has been tested and
determined to be
expected to bind to HDM2 exposed on the surface of the cells of the cancer to
be treated. This
binding to HDM2 can be shown by, for example, the ability of the anti-HDM2
antibody or
fragment to bind to an intact cancer cell of the tissue type of the tissue of
origin of the cancer
being treated (which can be but does not need to be from the subject being
treated). In one
embodiment, an anti-HDM2 antibody or a fragment thereof is tested and
determined to be
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expected to bind to the intact cells of the cancer of a subject, before
administering the antibody
or fragment to the subject. This testing can be done, e.g., by showing binding
of the anti-HDM2
antibody or fragment to the surface of intact cancer cells obtained by biopsy
or to a cancer cell
line of the appropriate tissue type.
[00352] Optionally, the cancer cells of the prospective patient to be
treated can be tested for
expression of M(H)DM2/4 (e.g., HDM2) on their surface using techniques known
in the art in
order to determine whether the subject is an appropriate candidate for anti-
HDM2 therapy
described herein; however, such ordinarily would not be deemed necessary if
the patient has a
cancer of a tissue type that is known or expected to have an extracellularly
accessible epitope of
M(H)DM2/4. In a specific embodiment, the cells of the cancer in the subject
being treated have
been tested and determined to have an extracellularly accessible epitope of
HDM2 (targeted by
the anti-HDM2 antibody or fragment thereof) exposed on their plasma membrane
surface (e.g.,
determined to express a variant of HDM2 that is known to have this
extracellular region exposed
on the plasma membrane surface). In certain embodiments, the cancer being
treated using the
methods described herein is a cancer that is known or determined to express a
splice variant of
HDM2 (for example, MDM2-A (SEQ ID NO: 8), MDM2-A1 (SEQ ID NO: 9), MDM2-B (SEQ
ID NO: 10), MDM2-C (SEQ ID NO: 11), MDM2-D (SEQ ID NO:12 ), MDM2-E (SEQ ID NO:
13), MDM2-F (SEQ ID NO: 14), MDM2-G (SEQ ID NO: 15), MDM2-11 (SEQ ID NO: 16)
or
MDM-KB2 (SEQ ID NO: 17)), on the plasma membrane surface of its cells. Such
can be
detected using techniques known in the art (e.g., RT-PCR on a nucleic acid
sample from cancer
biopsy).
[00353] In specific embodiments, the administration of an anti-M(H)DM2/4
(e.g., anti-
HDM2) antibody or an antigen-binding fragment thereof in accordance with the
methods
described herein can be carried out to achieve, or found to result in
achieving, at least one, two,
three, four or more of the following effects (e.g., in a subject with a
cancerous tumor): (i) a
decrease in tumor size (e.g., volume or diameter), (ii) a reduction in the
growth of the tumor, (iii)
inhibition of the progression of tumor growth, (iv) the regression of the
tumor, (v) inhibition of a
recurrence of the tumor, (vi) eradication of the tumor (e.g., primary tumor or
metastatic tumor),
(vii) prevention of metastasis of the tumor, (vii) reduction in the number,
size or invasiveness of
the metastases of the tumor, (viii) reduction or amelioration of the severity
or duration of one or
more symptoms of the tumor, (ix) the inhibition of the development or onset of
one or more
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symptoms associated with cancer, (x) the enhancement or improvement of the
therapeutic effect
of another therapy, (xi) reduction in hospitalization (e.g., length of the
hospitalization) in the
subject, (xii) improvement in subject's quality of life, (xiii) a reduction in
mortality, (xiv) an
increase in a relapse free survival or the length of remission in the subject.
Any of these effects
can be assessed by any method known in the art. For example, the tumor size
can be assessed
using magnetic resonance imaging (MM), dynamic contrast-enhanced MM (DCE-MRI),
X-ray,
computed tomography (CT) scan, or positron emission tomography (PET) scan.
[00354] In certain embodiments, the administration of an anti-M(H)DM2/4 (e.g.,
anti-HDM2)
antibody or an antigen-binding fragment thereof in accordance with the methods
described herein
is effective to treat cancer in a subject (e.g., reduces tumor volume or
diameter, reduces tumor
growth, reduce tumor proliferation, eradicates the tumor, or improves one or
more symptoms of
cancer), when used alone or in combination with another therapy. In certain
embodiments, the
administration of an anti-HDM2 antibody or an antigen-binding fragment thereof
in accordance
with the methods described herein is effective to prevent metastases in a
subject that has cancer,
when used alone or in combination with another therapy. In certain
embodiments, the
administration of an anti-HDM2 antibody or an antigen-binding fragment thereof
in accordance
with the methods described herein is effective to treat a metastatic cancer
(e.g., reduces the
number, size or invasiveness of metastases, or eradicates metastases), when
used alone or in
combination with another therapy.
[00355] In particular embodiments, the administration of an anti-M(H)DM2/4
(e.g., anti-
HDM2) antibody or an antigen-binding fragment thereof in accordance with the
methods
described herein is effective to treat cancer or prevent metastasis in a
subject when used alone
(i.e., without an additional therapy). In other particular embodiments, the
administration of an
anti-HDM2 antibody or an antigen-binding fragment thereof in accordance with
the methods
described herein is effective to treat cancer or prevent metastasis in a
subject when used in
combination with one or more of the additional therapies described herein.
[00356] The effectiveness of therapies described herein can be assessed by
evaluating a
parameter (e.g., tumor size) before and after administration of the therapies
described herein to
the subject being treated. Alternatively, the effectiveness of therapy can be
assessed by
evaluating a parameter (e.g., tumor size) before and after administration of
the therapies
described herein to an animal model (e.g., in an animal model, such as a mouse
model, a rat
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model, or a hamster model, of the cancer being treated). Any assay known in
the art can be used
to evaluate the therapeutic effectiveness of the therapies described herein.
[00357] In the therapeutic methods described herein using an anti-M(H)DM2/4
(e.g., anti-
HDM2) antibody or an antigen-binding fragment thereof, it will be understood
that an antibody-
drug conjugate described herein can alternatively be used.
7.6.1 Cancers to be treated
[00358] Examples of cancers that can be treated in accordance with the methods
described
herein include, but are not limited to, breast cancer, cervical cancer,
ovarian cancer, endometrial
cancer, uterine cancer, pancreatic cancer, skin cancer (e.g., melanoma),
prostate cancer (e.g.,
hormone refractory, such as castration resistant, prostate cancer), lung
cancer (e.g., small-cell
lung cancer, or non-small cell lung cancer), colorectal cancer (e.g., colon
cancer, or rectal
cancer), gastrointestinal cancer, stomach cancer, small bowel cancer, appendix
cancer,
esophageal cancer, gastric cancer, renal cancer, bladder cancer, gallbladder
cancer, kidney cancer
(e.g., renal cell carcinoma, or Wilms tumor)), liver cancer (e.g., hepatic
carcinoma, or
hepatoma), central nervous system cancer (e.g., brain cancer), peripheral
nervous system cancer,
bronchial cancer, cancer of the oral cavity or pharynx (e.g., oropharyngeal
cancer, laryngeal
cancer), thyroid cancer, biliary tract cancer, salivary gland cancer, thyroid
gland cancer, adrenal
gland cancer, vulvar cancer, testicular cancer, urethral cancer, vaginal
cancer, penile cancer,
bone cancer, eye cancer (e.g. retinoblastoma or uveal melanoma), and head and
neck cancer
(e.g., head and neck squamous cell carcinoma). In specific embodiments, the
cancer is cervical
cancer, endometrial cancer, ovarian cancer, pancreatic cancer, melanoma,
breast cancer, or colon
cancer. In one embodiment, the cancer is a pancreatic cancer. In one
embodiment, the cancer is a
melanoma. In one embodiment, the cancer is a breast cancer. In one embodiment,
the cancer is
an ovarian cancer.
[00359] In certain embodiments, the cancer that can be treated in accordance
with the methods
described herein is resistant to another cancer therapy or therapies (e.g.,
vaccine, targeted therapy
(such as small molecule targeted therapy), chemotherapy, radiotherapy, or
immunotherapy (such
as treatment with another monoclonal antibody)). In one embodiment, the cancer
that can be
treated in accordance with the methods described herein is resistant to
chemotherapy. In one
embodiment, the cancer that can be treated in accordance with the methods
described herein is
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resistant to treatment with another monoclonal antibody or antibodies. In one
embodiment, the
cancer that can be treated in accordance with the methods described herein is
resistant to
radiation. In one embodiment, the cancer that can be treated in accordance
with the methods
described herein is resistant to small molecule targeted therapy.
[00360] In a specific embodiment, the cancer treated in accordance with the
methods
described herein is a solid cancer. In another specific embodiment, the cancer
treated in
accordance with the methods described herein is a non-solid cancer (e.g.,
hematologic cancer).
[00361] In specific embodiments, the cancer treated in accordance with the
invention is
leukemia (e.g., acute leukemia (such as acute lymphocytic leukemia or acute
myelocytic
leukemia), chronic leukemia (such as chronic myelocytic leukemia or chronic
lymphocytic
leukemia), or hairy cell leukemia), lymphoma (e.g., Hodgkin's lymphoma, non-
Hodgkin's
lymphoma, B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma,
lymphoblastic
lymphoma, mantle cell lymphoma, or T-cell lymphoma).
[00362] In specific embodiments, the cancer treated in accordance with the
invention is
carcinoma (e.g., adenocarcinoma, basal cell carcinoma, renal cell carcinoma,
squamous cell
carcinoma, osteocarcinoma, thyoma/thymic carcinoma, or choriocarcinoma),
blastoma, sarcoma
(e.g., soft tissue sarcoma, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, or
synovia
sarcoma), lymphoma, leukemia, a germ cell tumor, myeloma (e.g., multiple
myeloma),
squamous cell cancer, mesothelioma, glioblastoma (e.g., glioblastoma
multiforme), glioma,
neuroblastoma, melanoma, astrocytoma, medulloblastoma, hepatoma, seminoma,
craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, neuroma,
oligodendroglioma,
meningioma, or retinoblastoma.
[00363] In specific embodiments, the cancer treated in accordance with the
methods described
herein is a sarcoma or carcinoma, e.g., fibrosarcoma, myxosarcoma,
liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,
Ewing's tumor,
leiomyosarcoma, rhabdomyosarcoma, endometrial stromal sarcoma, mast cell
sarcoma, adult
soft tissue sarcoma, uterine sarcoma, Kaposi sarcoma, merkel cell carcinoma,
urothelial
carcinoma, colon carcinoma, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma,
papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic
carcinoma, renal
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cell carcinoma, bile duct carcinoma, embryonal carcinoma, lung carcinoma
(e.g., small cell lung
carcinoma), bladder carcinoma, or epithelial carcinoma.
[00364] In certain embodiments, the cancer treated in accordance with the
methods of the
invention is metastatic. In some embodiments, the cancer treated in accordance
with the methods
described herein is a metastatic melanoma, a metastatic ovarian cancer, a
metastatic cervical
cancer, a metastatic endometrial cancer, a metastatic pancreatic cancer, a
metastatic breast
cancer, a metastatic colon cancer, or a metastatic brain cancer.
7.6.2 Methods of Administration
[00365] The anti-M(H)DM2/4 (e.g., anti-HDM2) antibodies or fragments described
herein
(and pharmaceutical compositions comprising such antibodies) can be
administered to a subject
by any suitable means which include, but are not limited to, parenteral (e.g.,
intravenous,
intraarterial, intramuscular, intraosseous, intracerebral,
intracerebroventricular, intrathecal,
subcutaneous), intraperitoneal, intratumoral, intrapulmonary, intradermal,
transdermal,
conjunctival, intraocular, intranasal, intratracheal, oral and local
intralesional routes of
administration. In certain embodiments, the anti-HDM2 antibodies or fragments
described herein
are administered intravenously, intraarterially, intramuscularly,
intraperitoneally, intratumorally,
or subcutaneously. In one embodiment, the anti-HDM2 antibodies or fragments
described herein
are administered intravenously. In one embodiment, the anti-HDM2 antibodies or
fragments
described herein are administered intraperitoneally. In one embodiment, the
anti-HDM2
antibodies or fragments described herein are administered intramuscularly. In
one embodiment,
the anti-HDM2 antibodies or fragments described herein are administered
subcutaneously. In
one embodiment, the anti-HDM2 antibodies or fragments described herein are
administered
intratumorally (such as by an injection into the tumor of the cancer being
treated). In particular
embodiments, the anti-HDM2 antibodies or fragments described herein are
administered
intravenously, intraperitoneally, or intratumorally.
[00366] In a specific embodiment, nano-particles coated with an anti-M(H)DM2/4
(e.g., anti-
HDM2) antibody or a fragment thereof described herein are used for tumor
targeting and
treatment (see, for example, Cardoso et al., 2012, Curr. Med. Chem.
19(19):3103-27 and
Arruebo et al., 2009, J. of Nanomater. 2009:Article ID 439389, regarding nano-
particle coating
with antibodies). In one embodiment, provided herein are methods for treating
cancer or
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preventing metastasis in a subject having a cancer comprising administering to
the subject nano-
particles coated with an an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or a
fragment thereof
[00367] Various dosing schedules of the anti-M(H)DM2/4 (e.g., anti-HDM2)
antibodies or
fragments described herein (and pharmaceutical compositions comprising such
antibodies) are
contemplated including single administration or multiple administrations over
a period of time.
The methods of administration include, without limitation, bolus
administration, pulse infusions,
and continuous infusions.
[00368] The therapeutic regimen for use in the methods described herein may
include
administration of anti-M(H)DM2/4 (e.g., anti-HDM2) antibodies or fragments
thereof (and
compositions comprising such antibodies) once every week, once every two
weeks, once every
three weeks, once every four weeks, once every six weeks, once every eight
weeks or once every
twelve weeks (e.g., such that the subject receives from at least two, at least
three, at least four, at
least five, at least six, at least eight, or at least ten doses of the
antibody, or from two to twenty
doses of the antibody). In certain embodiments, anti-HDM2 antibodies or
fragments thereof (and
compositions comprising such antibodies) are administered daily, every other
day, or two, three,
or four times a week (e.g., for a period of time, such as one week, two weeks,
three weeks, four
weeks, six weeks, two months or three months). The treatment regimens
contemplated herein
include regimens wherein the initial higher dose of the antibody may be
followed by one or more
lower doses, or wherein the initial lower dose of the antibody is followed by
one or more higher
doses. An exemplary treatment course (in which the anti-HDM2 antibody or
fragment is
administered) may last for one week, two weeks, three weeks, four weeks, six
weeks, two
months, three months, four months, five months, six months, one year, or over
several years.
[00369] In some embodiments, the initial treatment period (where the antibody
is
administered, e.g., once a month, once in two weeks, once a week, twice a week
or three times a
week) is followed by a withdrawal period in which the antibody is not
administered (for, e.g., a
week, two weeks, three weeks, four weeks, six weeks, two months, three months,
four months,
six months or one year), and then followed by a second treatment period (where
the antibody is
administered, e.g., once a month, once in two weeks, once a week, twice a week
or three times a
week). Such initial treatment and such second treatment periods can last, for
example, two
weeks, three weeks, four weeks, six weeks, two months, three months, four
months, or six
months (where the initial treatment period can be the same or different from
the second treatment
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period). This course of treatment (having the initial treatment period, a
withdrawal period and a
second treatment period) can be repeated twice, three times, four times, five
times, six times, ten
times or more than ten times.
[00370] In some embodiments, two or more antibodies or fragments thereof with
different
binding specificities for M(H)DM2/4 (e.g., HDM2) are administered
simultaneously or
sequentially to the subject being treated.
[00371] The appropriate dosage of anti-M(H)DM2/4 (e.g., anti-HDM2) antibodies
or
fragments for use in the methods described herein will depend on the type of
antibody used, the
type of cancer being treated, the severity of the cancer being treated, the
route of administration,
the target site, the condition of the patient (e.g., age, body weight,
health), the responsiveness of
the patient to the antibody, other medications used by the patient, and other
factors to be
considered at the discretion of the medical practitioner performing the
treatment.
[00372] In certain embodiments, the dosage of an anti-M(H)DM2/4 (e.g., anti-
HDM2)
antibody or fragment described herein which is administered to the subject can
be from about 1
[tg/kg to 200 mg/kg of the patient's body weight. In certain embodiments, the
dosage of an anti-
M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein which is
administered to
the subject can be from about 1 [tg/kg to 100 mg/kg of the patient's body
weight (e.g., from
about 0.01 mg/kg to about 100 mg/kg, from about 0.05 mg/kg to about 100 mg/kg,
or from about
0.5 mg/kg to about 100 mg/kg). In certain embodiments, the dosage of an anti-
M(H)DM2/4
(e.g., anti-HDM2) antibody or fragment described herein which is administered
to the subject
can be from about 1 mg/kg to 200 mg/kg of the patient's body weight. In one
embodiment, the
dosage of an anti-HDM2 antibody or fragment described herein which is
administered to the
subject is from 0.025 mg/kg to about 5 mg/kg. In one embodiment, the dosage of
an anti-
HDM2 antibody or fragment described herein which is administered to the
subject is from 0.05
mg/kg to about 2 mg/kg. In one embodiment, the dosage of an anti-HDM2 antibody
or fragment
described herein which is administered to the subject is from 5 mg/kg to about
30 mg/kg. In
specific embodiments, doses (e.g., one or more doses) of about 0.025 mg/kg,
0.05 mg/kg, 0.1
mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg,
4 mg/kg, 5
mg/kg, 7.5 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 75 mg/kg,
or 100 mg/kg
of an anti-HDM2 antibody or fragment described herein can be administered to
the subject being
treated. In one embodiment, a dose (e.g., one or more doses) of about 0.1
mg/kg of an anti-
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HDM2 antibody or fragment described herein can be administered to the subject
being treated
(e.g., when the antibody is administered intratumorally).
7.7 Diagnostic, Companion Diagnostic and Prognostic Methods
[00373] In one aspect, provided herein are methods of diagnosing cancer in a
subject (e.g., a
human), said method comprising: (a) detecting whether an antibody or a
fragment thereof (e.g., a
labeled antibody or fragment) that specifically binds to M(H)DM2/4 (e.g.,
HDM2) binds to the
surface of an intact cell of the subject, wherein the antibody or fragment is
any anti-M(H)DM2/4
antibody or fragment described herein (in particular, any antibody or fragment
that specifically
binds to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3); and (b) diagnosing the
subject with
cancer if the binding is detected in step (a). In one embodiment, the method
of diagnosing
further comprises, before the detecting in step (a), obtaining the intact cell
from the subject, and
then performing the detecting by, e.g., determining whether a labeled antibody
or fragment binds
to the intact cell from the subject using, e.g., FACS or cell-based ELISA
analysis. In one
embodiment, the method of diagnosing comprises administering the antibody or
fragment to the
subject before the detecting in step (a), and wherein the detecting is
performed by in vivo
imaging of the subject.
[00374] In one aspect, a patient is selected for treatment using an anti-
M(H)DM2/4 (e.g., anti-
HDM2) antibody or antibody fragment described herein based on the detection of
binding of
such antibody or fragment to the surface of intact cancer cells obtained from
the patient. In
certain embodiments, provided herein is a method of selecting a patient having
a cancer for
treatment with an antibody or fragment that specifically binds to an
extracellularly accessible
epitope of HDM2 comprising: obtaining an intact cancer cell from the patient
(e.g., by biopsy of
the cancerous tumor in the patient, or by obtaining a blood sample with
circulating cancer cells
from the patient), and determining whether the antibody or fragment binds to
the surface of the
intact cancer cell of the patient (using any method known in the art or
described herein, e.g.,
using cell-based ELISA or FACS analysis), wherein the detection of binding
indicates that the
patient can be treated with the antibody or fragment. In specific embodiments,
provided herein is
a method of selecting a patient having a cancer for treatment with an antibody
or fragment that
specifically binds to an extracellularly accessible epitope of HDM2
comprising: obtaining an
intact cancer cell from the patient (e.g., by biopsy of the cancerous tumor in
the patient, or by
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obtaining a blood sample with circulating cancer cells from the patient),
determining whether the
antibody or fragment binds to the surface of the intact cancer cell of the
patient (using any
method known in the art or described herein, e.g., using cell-based ELISA or
FACS analysis),
and, if the binding is detected, administering the antibody or fragment to the
patient. The
antibody or fragment administered to the patient can be the same or different
from the antibody
or fragment used for selection of the patient to be treated. In one
embodiment, provided herein is
a method of selecting a patient having an ovarian cancer for treatment with an
antibody or
fragment that specifically binds to an extracellularly accessible epitope of
HDM2 comprising:
obtaining an intact ovarian cancer cell from the patient, determining whether
the antibody or
fragment binds to the surface of the intact cancer cell of the patient, and,
if the binding is
detected, administering the antibody or fragment to the patient. In one
embodiment, any
antibody or a fragment thereof that specifically binds to SEQ ID NO:1, SEQ ID
NO:2, or SEQ
ID NO:3 is used for such patient selection and/or treatment. In one
embodiment, any antibody or
a fragment thereof that specifically binds to an extracellularly accessible
epitope of M(H)DM2/4,
which is not bound to a cell-penetrating peptide is used for such patient
selection and/or
treatment. In one embodiment, any antibody or a fragment thereof that
specifically binds to an
extracellularly accessible epitope of M(H)DM2/4, which is not bound to a cell-
penetrating
peptide is used for such patient selection, and any antibody or a fragment
thereof that specifically
binds to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 is used for the treatment of
the
patient.In one aspect, provided herein are methods of diagnosing a hyper-
progressive disease
(such as diagnosing a subject as exhibiting hyper-progression, or likely to
exhibit hyper-
progression, of a cancer in response to administration of an inhibitor of an
inhibitory checkpoint
molecule) in subject (e.g., a human) who has a cancer, said method comprising:
(a) optionally,
determininig whether a gene amplification of M(H)DM2/4 is present in the cells
of the cancer of
the subject; (b) determining whether an antibody or a fragment thereof that
specifically binds to
an extracellularly accessible epitope of M(H)DM2/4 binds to the surface of
intact cells of the
cancer (e.g., determining whether there is an increased binding of an antibody
or a fragment
thereof that specifically binds to an extracellularly accessible epitope of
M(H)DM2/4 to the
surface of intact cells of the cancer relative to the binding of the antibody
or fragment to the
surface of intact normal cells; or determining whether there is an increased
binding of an
antibody or a fragment thereof that specifically binds to an extracellularly
accessible epitope of
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M(H)DM2/4 to the surface of intact cells of the cancer relative to the binding
of the antibody or
fragment to the surface of intact cells of the cancer prior to administration
of an inhibitor of an
inhibitory checkpoint molecule); and (c) diagnosing the subject with the hyper-
progressive
disease if binding is detected in step (b), and, optionally if the gene
amplification is determined
to be present in step (a). In one embodiment, the method of diagnosing further
comprises, before
step (b), obtaining (e.g., isolating) the intact cell from the subject, and
then performing the
determining in step (b) by, e.g., determining whether a labeled antibody or
fragment binds to the
intact cell from the subject using, e.g., FACS or cell-based ELISA analysis.
In one embodiment,
the method of diagnosing comprises administering the antibody or fragment to
the subject before
step (b), and wherein the determining in step (b) is performed by in vivo
imaging of the subject.
In one embodiment, the determining in step (a) is performed using PCR, Next
Generation
Sequencing or RNA sequencing. In certain embodiments, the gene amplification
is determined
to be present in step (a) if the cancer cells of the subject have 8 or more
than 8 copies of
M(H)DM2/4 gene. In certain embodiments, the gene amplification is determined
to be present in
step (a) if the cancer cells of the subject have 50% or more than 50%
amplification of the
M(H)DM2/4 gene relative to the normal cells of the subject (or relative to the
cells of the subject
prior to administration of an inhibitor of an inhibitory checkpoint molecule).
In one
embodiment, the gene amplification of M(H)DM2/4 is gene amplification of HDM2.
In one
embodiment, the gene amplification of M(H)DM2/4 is gene amplification of HDM4.
In one
embodiment, the gene amplification of M(H)DM2/4 is gene amplification of HDM2
and HDM4.
[00375] Non-limiting exemplary samples that can be used for in diagnostic or
patient selection
methods using an anti-M(H)DM2/4 antibody (e.g., anti-HDM2 antibody) or
fragment thereof
described herein include: tissue biopsies, intact cells obtained from
malignant tissues, and
circulating cancer cells isolated from blood. For example, a tissue sample can
be obtained from
a patient and immunohistochemistry can be performed to detect whether a
labeled anti-
M(H)DM2/4 antibody (e.g., anti-HDM2 antibody) or fragment thereof binds to the
tissue sample.
Alternatively, intact cells (known or suspected to be malignant) can be
isolated from a patient
and FACS or cell-based ELISA analysis can be performed to detect whether a
labeled anti-
M(H)DM2/4 antibody (e.g., anti-HDM2 antibody) or fragment thereof binds to
such cell. In yet
another example, a blood sample with circulating cancer cells can be obtained
from a patient and
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FACS or cell-based ELISA analysis can be performed to detect whether a labeled
anti-
M(H)DM2/4 antibody (e.g., anti-HDM2 antibody) or fragment thereof binds to
such cells.
[00376] In one aspect, the duration of treatment and/or dosage of an anti-
M(H)DM2/4 (e.g.,
anti-HDM2) antibody or antibody fragment described herein to be used in the
treatment of a
patient is determined based on the detection of binding of such antibody or
fragment to the
surface of intact cancer cells obtained from the patient. In certain
embodiments, provided herein
is a method of determining whether to continue the treatment of a patient
having a cancer with an
antibody or fragment that specifically binds to an extracellularly accessible
epitope of HDM2
comprising: administering the antibody or fragment to the patient for a first
period of time (e.g.,
where the patient had been selected for treatment as described above),
obtaining an intact cancer
cell from the patient (e.g., by biopsy of the cancerous tumor in the patient,
or by obtaining a
blood sample with circulating cancer cells from the patient), and determining
whether the
antibody or fragment binds to the surface of the intact cancer cell of the
patient (using any
method known in the art or described herein, e.g., using cell-based ELISA or
FACS analysis),
and, if the binding is detected, continuing administering the antibody or
fragment to the patient
for a second period of time (but, e.g., if the binding is not detected,
discontinuing the treatment).
In certain embodiments, provided herein is a method of determining whether to
increase the dose
of an antibody or fragment that specifically binds to an extracellularly
accessible epitope of
HDM2 for use in the treatment of a patient having a cancer comprising:
administering a dose the
antibody or fragment to the patient for a period of time (e.g., where the
patient had been selected
for treatment as described above), obtaining an intact cancer cell from the
patient (e.g., by biopsy
of the cancerous tumor in the patient, or by obtaining a blood sample with
circulating cancer
cells from the patient), and determining whether the antibody or fragment
binds to the surface of
the intact cancer cell of the patient (using any method known in the art or
described herein, e.g.,
using cell-based ELISA or FACS analysis), and, if the binding is detected,
administering a dose
of the antibody or fragment to the patient for a second period of time,
wherein the dose
administered during the second period of time is higher than the dose
administered during the
first period of time (e.g., two times, or three times higher) (but, e.g., if
the binding is not
detected, discontinuing the treatment or administering a lower dose of the
antibody or fragment
during the second period of time).
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[00377] In another specific embodiment, treatment with an anti-M(H)DM2/4
(e.g., anti-
HDM2) antibody or an antibody fragment described herein is monitored in a
patient by
determining the amount of HDM2 expressed on the surface of cancer cells
obtained from the
patient, before and after treatment, wherein a decrease in the amount is a
positive prognosis.
7.8 Patient populations
[00378] The patients or subjects being treated in accordance with the methods
described
herein include, but are not limited to, humans and non-human vertebrates. In
certain
embodiments, the subject being treated is a mammal, e.g., a human, a dog, a
cat, a monkey, a
rabbit, a cow, a horse, a goat, a sheep, or a pig. In a preferred embodiment,
the subject being
treated is a human.
[00379] In certain embodiments, the subject being treated in accordance with
the methods
described herein has been diagnosed with a cancer (e.g., using a biopsy or any
another method
known in the art). In particular embodiments, the subject being treated has
been diagnosed with
an early stage cancer. In other embodiments, the subject being treated has
been diagnosed with
an advanced stage cancer. In particular embodiments, the subject being treated
has been
diagnosed with a high-grade tumor. In other embodiments, the subject being
treated has been
diagnosed with a low-grade tumor. In certain embodiments, the subject being
treated has been
diagnosed with a cancer that can metastasize. In specific embodiments, the
subject being treated
has been diagnosed with a metastatic cancer.
[00380] In specific embodiments, the subject being treated in accordance with
the methods
described herein has been diagnosed with a cervical cancer, an endometrial
cancer, an ovarian
cancer, a pancreatic cancer, a melanoma, a breast cancer, a colorectal cancer
(e.g., a colon
cancer), a bladder cancer, an astrocytic neoplasm, a glioblastoma, a pediatric
Rhabdomyosarcoma, or a lung cancer (e.g., non-small cell lung carcinoma). In
specific
embodiments, the subject being treated in accordance with the methods
described herein has
been diagnosed with a melanoma, a pancreatic cancer, a breast cancer, or an
ovarian cancer. In
one embodiment, the subject being treated in accordance with the methods
described herein has
been diagnosed with a melanoma. In one embodiment, the subject being treated
in accordance
with the methods described herein has been diagnosed with a pancreatic cancer.
In one
embodiment, the subject being treated in accordance with the methods described
herein has been
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diagnosed with a breast cancer. In one embodiment, the subject being treated
in accordance with
the methods described herein has been diagnosed with an ovarian cancer. In one
embodiment,
the subject being treated in accordance with the methods described herein has
been diagnosed
with a lung cancer.
[00381] In certain embodiments, the subject being treated is a hyper-
progressor in response to
treatment with an inhibitor of an inhibitory immune checkpoint molecule (e.g.,
the subject has
been diagnosed with hyper-progression in response to treatment with an
inhibitor of an inhibitory
immune checkpoint molecule). In certain embodiments, the subject being treated
is at risk of
being a hyper-progressor in response to treatment with an inhibitor of an
inhibitory immune
checkpoint molecule (e.g., the subject has a gene amplification of M(H)DM2/4,
and/or the
subject has an increased binding of an antibody or a fragment thereof that
specifically binds to an
extracellularly accessible epitope of M(H)DM2/4 to the surface of intact cells
of the cancer
relative to its binding to the surface of intact normal cells or relative to
its binding to the surface
of intact cells of the cancer prior to administration of the inhibitor).
[00382] In certain embodiments, the subject being treated has previously
undergone one or
more other cancer therapies (e.g., vaccine, targeted therapy (such as small
molecule targeted
therapy), chemotherapy, radiotherapy, or immunotherapy (such as treatment with
another
monoclonal antibody)), and the subject's cancer has developed resistance to
the one or more
other cancer therapies. In one embodiment, the subject being treated is
resistant to
chemotherapy. In one embodiment, the subject being treated is resistant to
radiotherapy. In one
embodiment, the subject being treated is resistant to a small molecule
targeted therapy. In one
embodiment, the subject being treated is resistant to treatment with another
monoclonal
antibody.
[00383] In certain embodiments, the subject being treated has a type of a
cancer that is known
or expected to have M(H)DM2/4 (e.g., HDM2) on the surface of its cells. In
specific
embodiments, the subject being treated has a type of cancer, the cells of
which express one or
more of splice variants of HDM2 on their cell surface, for example (and
without limitation), one
or more of the following splice variants: MDM2-A (SEQ ID NO: 8), MDM2-A1 (SEQ
ID NO:
9), MDM2-B (SEQ ID NO: 10), MDM2-C (SEQ ID NO: 11), MDM2-D (SEQ ID NO:12 ),
MDM2-E (SEQ ID NO: 13), MDM2-F (SEQ ID NO: 14), MDM2-G (SEQ ID NO: 15), MDM2-
11 (SEQ ID NO: 16) or MDM-KB2 (SEQ ID NO: 17).
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[00384] In certain embodiments, the subject being treated has a cancer that
has been tested and
determined (using any assay known in the art) to carry M(H)DM2/4 (e.g., HDM2)
on the plasma
membrane of its cells. In particular embodiments, the subject being treated
has a cancer, the
cells of which have been tested and determined (by any method known in the
art) to expose on
their plasma membrane surface an extracellular region of HDM2 that can be
targeted by an anti-
HDM2 antibody or fragment (and such antibody can then be administered to the
subject). In
specific embodiments, the subject being treated has a cancer, the cells of
which have been tested
and determined (by any method known in the art) to express one or more of
splice variants of
HDM2 on their cell surface, for example (and without limitation), one or more
of the following
splice variants: MDM2-A (SEQ ID NO: 8), MDM2-A1 (SEQ ID NO: 9), MDM2-B (SEQ ID
NO: 10), MDM2-C (SEQ ID NO: 11), MDM2-D (SEQ ID NO:12 ), MDM2-E (SEQ ID NO:
13), MDM2-F (SEQ ID NO: 14), MDM2-G (SEQ ID NO: 15), MDM2-11 (SEQ ID NO: 16)
or
MDM-KB2 (SEQ ID NO: 17).
7.9 Combination therapies and kits
[00385] In certain embodiments, an anti-M(H)DM2/4 antibody or fragment thereof
described
herein is administered to a subject in combination with one or more anti-
cancer therapies
different from said antibody or fragment, e.g., a chemotherapy, a surgery, a
radiation therapy,
another antibody with an anti-cancer activity, a cytokine, a T cell therapy, a
vaccine (e.g., a
cellular vaccine), a small molecule with an anti-cancer activity, an anti-
hormonal agent, or any
other anti-cancer therapy known in the art.
[00386] In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment
thereof
described herein is administered to a subject in combination with
chemotherapy. Examples of
types of chemotherapeutic agents that can be used in the methods described
herein include,
without limitation, an alkylating agent, a nitrosourea agent, an
antimetabolite, a topoisomerase
inhibitor, an aromatase inhibitor, an antitumor antibiotic, an alkaloid
derived from a plant, a
hormone antagonist, a P-glycoprotein inhibitor, and a platimum complex
derivative. Specific
examples of chemotherapeutic drugs that can be used in the methods described
herein include,
without limitation, taxol, paclitaxel, nab-paclitaxel, 5-fluorouracil (5-FU),
gemcitabine,
doxorubicin, daunorubicin, colchicin, mitoxantrone, tamoxifen,
cyclophosphamide,
mechlorethamine, melphalan, chlorambucil, busulfan, uramustine, mustargen,
ifosamide,
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bendamustine, carmustine, lomustine, semustine, fotemustine, streptozocin,
thiotepa, mitomycin,
diaziquone, tetrazine, altretamine, dacarbazine, mitozolomide, temozolomide,
procarbazine,
hexamethylmelamine, altretamine, hexalen, trofosfamide, estramustine,
treosulfan, mannosulfan,
triaziquone, carboquone, nimustine, ranimustine, azathioprine, sulfanilamide,
fluoropyrimidine,
thiopurine, thioguanine, mercaptopurine, cladribine, capecitabine, pemetrexed,
fludarabine,
methotrexate, hydroxyurea, nelarabine or clofarabine, cytarabine, decitabine,
pralatrexate,
floxuridine, thioquanine, azacitidine, cladribine, pentostatin,
mercaptopurine, imatinib,
dactinomycin, cerubidine, bleomycin, actinomycin, luteomycin, epirubicin,
idarubicin,
plicamycin, vincristin, vinblastine, vinorelbine, vindesine, vinflunine,
paclitaxel, docetaxel,
etoposide, teniposide, periwinkle, via, taxane, irinotecan, topotecan,
camptothecin, teniposide,
pirarubicin, novobiocin, merbarone, aclarubicin, amsacrine, antiandrogen, anti-
estrogen,
bicalutamide, medroxyprogesterone, fluoxymesterone, diethylstilbestrol,
estrace, octreotide,
megestrol, raloxifene, toremifene, fulvestrant, prednisone, flutamide,
leuprolide, goserelin,
aminoglutethimide, testolactone, anastrozole, letrozole, exemestane, vorozole,
formestane,
fadrozole, androstene, resveratrol, myosmine, catechin, apigenin eriodictyol
isoliquiritigenin,
mangostin, amiodarone, azithromycin, captopril, clarithromycin, cyclosporine,
piperine,
quercetine, quinidine, quinine, reserpine, ritonavir, tariquidar, verapamil,
cisplatin, carboplatin,
oxaliplatin, transplatin, nedaplatin, satraplatin, triplatin and carboplatin.
In specific
embodiments, an anti- M(H)DM2/4 antibody or fragment thereof described herein
is
administered to a subject in combination with one or more of the following
chemotherapeutic
agents: gemcitabine, nab-paclitaxel, capecitabine, irinotecan, and celecoxib.
In specific
embodiments, an anti- M(H)DM2/4 antibody or fragment thereof described herein
is
administered to a subject in combination with one or more of the following
chemotherapeutic
agents: gemcitabine, nab-paclitaxel, cisplatin, 5-FU, and paclitaxel (e.g.,
paclitaxel formulated as
albumin-bound particles such as ABRAXANEg). In specific embodiments, the
cancer treated
using a combination therapy described herein is a pancreatic cancer, a breast
cancer, a lung
cancer or an ovarian cancer.
[00387] In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof
described
herein is administered to a subject in combination with gemcitabine (e.g., for
treatment of a non-
small cell lung cancer, a pancreatic cancer or an ovarian cancer). In one
embodiment, an anti-
M(H)DM2/4 antibody or fragment thereof described herein is administered to a
subject in
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combination with capecitabine. In one embodiment, an anti-M(H)DM2/4 antibody
or fragment
thereof described herein is administered to a subject in combination with
irinotecan. In one
embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is
administered
to a subject in combination with celecoxib. In one embodiment, an anti-
M(H)DM2/4 antibody
or fragment thereof described herein is administered to a subject in
combination with paclitaxel
(e.g., paclitaxel formulated as albumin-bound particles such as ABRAXANEg)
(e.g., for
treatment of a metasatic breast cancer). In one embodiment, an anti- M(H)DM2/4
antibody or
fragment thereof described herein is administered to a subject in combination
with nab-
paclitaxel. In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof
described
herein is administered to a subject in combination with cisplatin. In one
embodiment, an anti-
M(H)DM2/4 antibody or fragment thereof described herein is administered to a
subject in
combination with 5-FU (e.g., for treatment of a colorectal cancer such as a
colon cancer). In one
embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is
administered
to a subject in combination with carboplatin. In one embodiment, an anti-
M(H)DM2/4 antibody
or fragment thereof described herein is administered to a subject in
combination with
gemcitabine and nab-paclitaxel (e.g., for treatment of a pancreatic cancer).
In one embodiment,
an anti- M(H)DM2/4 antibody or fragment thereof described herein is
administered to a subject
in combination with gemcitabine and carboplatin (e.g., for treatment of an
ovarian cancer). In
one embodiment, an anti- M(H)DM2/4 antibody or fragment thereof described
herein is
administered to a subject in combination with paclitaxel (e.g., paclitaxel
formulated as albumin-
bound particles such as ABRAXANEg) and gemcitabine (e.g., for treatment of a
breast cancer,
or a pancreatic cancer such as adenocarcinoma of the pancreas or metastatic
adenocarcinoma of
the pancreas). In one embodiment, an anti- M(H)DM2/4 antibody or fragment
thereof described
herein is administered to a subject in combination with gemcitabine and
cisplatin (e.g., for
treatment of anon-small cell lung cancer). In one embodiment, an anti-
M(H)DM2/4 antibody or
fragment thereof described herein is administered to a subject in combination
with gemcitabine
and 5-FU. In one embodiment, an anti- M(H)DM2/4 antibody or fragment thereof
described
herein is administered to a subject in combination with paclitaxel (e.g.,
paclitaxel formulated as
albumin-bound particles such as ABRAXANE ) and carboplatin (e.g., for
treatment of a non-
small cell lung cancer).
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[00388] In certain embodiments, wherein the subject is human, gemcitabine is
administered in
a dose of 1,500 mg/m2. In certain embodiments, wherein the subject is human,
nab-paclitaxel is
administered in a dose of 300 mg/m2. In certain embodiments, wherein the
subject is human,
gemcitabine is administered in a dose of 1,000 mg/m2. In certain embodiments,
wherein the
subject is human, nab-paclitaxel is administered in a dose of 125 mg/m2.
[00389] In certain embodiments, the gemcitabine and/or nab-paclitaxel are
administered in
doses that are lower than doses used when gemcitabine and/or nab-paclitaxel
are administered
not in combination with an anti-cancer antibody (such as an anti-M(H)DM2/4
antibody or
fragment described herein). In certain embodiments, wherein the subject is
human, gemcitabine
is administered in a dose that is less than 1,500 mg/m2, and/or nab-paclitaxel
is administered in a
dose that is less than 300 mg/m2. In certain embodiments, wherein the subject
is human,
gemcitabine is administered in a dose that is less than 1,000 mg/m2, and/or
nab-paclitaxel is
administered in a dose that is less than 125 mg/m2. In one embodiment, wherein
the subject is
human, gemcitabine is administered in a dose that is equal to or less than 500
mg/m2, 400 mg/m2,
300 mg/m2 or 200 mg/m2, and/or the nab-paclitaxel is administered in a dose
that is equal to or
less than 62.5 mg/m2, 50 mg/m2, 40 mg/m2, 30 mg/m2, or 20 mg/m2. In one
embodiment,
wherein the subject is human, gemcitabine is administered in a dose that is
equal to or less than
900 mg/m2, 800 mg/m2, 700 mg/m2 or 600 mg/m2, and/or the nab-paclitaxel is
administered in a
dose that is equal to or less than 110 mg/m2, 100 mg/m2, 90 mg/m2, 80 mg/m2,
or 70 mg/m2. In
certain embodiments, gemcitabine and/or nab-paclitaxel are administered with a
frequency of
every 2 weeks or less (e.g., every 3 weeks, every 4 weeks, every 6 weeks, or
every 8 weeks, or
less). In certain embodiments, gemcitabine is administered with a frequency of
once a day, 4
times per week, 3 times per week, 2 times per week, or once per week. In
certain embodiments,
nab-paclitaxel is administered with a frequency of once a day, 4 times per
week, 3 times per
week, 2 times per week, or once per week. In certain embodiments, gemcitabine
and nab-
paclitaxel are administered with a frequency of once a day, 4 times per week,
3 times per week, 2
times per week, or once per week. In one embodiment, gemcitabine and/or nab-
paclitazel is
administered once a week. In certain embodiments, the total duration of
treatment with
gemcitabine and/or nab-paclitaxel is, or is more than, 2 weeks, 3 weeks, 4
weeks, 5 weeks, 6
weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks.
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[00390] In certain embodiments, an anti-M(H)DM2/4 antibody or fragment thereof
described
herein is administered to a subject with a cancer in combination with the
chemotherapy drug(s)
indicated for said cancer, which chemotherapy drug(s) can be optionally
administered in the
dosage and/or regime of administration indicated for said cancer. Non-limiting
examples of
chemotherapy drugs as well as their dosage and regime of administration
indicated for various
cancers are provided below.
[00391] Ovarian Cancer: The following information is taken from Gemzar
(gemcitabine for
injection), Eli Lilly and Company, Highlights of Prescribing Information,
revised March 2017,
http://pi.lilly.com/us/gemzar.pdf (last accessed on July 27, 2017).
Gemcitabine (Gemzar ) in
combination with carboplatin is indicated for the treatment of patients with
advanced ovarian
cancer that has relapsed at least 6 months after completion of platinum-based
therapy. The
recommended dose of Gemzar is 1000 mg/m2 as an intravenous infusion over 30
minutes on
Days 1 and 8 of each 21-day cycle, in combination with carboplatin (AUC 4)
intravenously after
Gemzar administration on Day 1 of each 21-day cycle.
[00392] Breast Cancer: The following information is taken from Gemzar
(gemcitabine for
injection), Eli Lilly and Company, Highlights of Prescribing Information,
revised March 2017,
http://pi.lilly.com/us/gemzar.pdf (last accessed on July 27, 2017).
Gemcitabine (Gemzar ) in
combination with paclitaxel is indicated for the first-line treatment of
patients with metastatic
breast cancer after failure of prior anthracycline-containing adjuvant
chemotherapy, unless
anthracyclines were clinically contraindicated. The recommended dose of Gemzar
is 1250
mg/m2 intravenously over 30 minutes on Days 1 and 8 of each 21-day cycle that
includes
paclitaxel. Paclitaxel can be administered at 175 mg/m2 on Day 1 as a 3 hour
intravenous
infusion before Gemzar administration.
[00393] Non-Small Cell Lung Cancer: The following information is taken from
Gemzar
(gemcitabine for injection), Eli Lilly and Company, Highlights of Prescribing
Information,
revised March 2017, http://pi.lilly.com/us/gemzar.pdf (last accessed on July
27, 2017).
Gemcitabine (Gemzar ) is indicated in combination with cisplatin for the first-
line treatment of
patients with inoperable, locally advanced (Stage IIIA or IIIB), or metastatic
(Stage IV) non-
small cell lung cancer. Every 4-week schedule: the recommended dose of Gemzar
is 1000
mg/m2 intravenously over 30 minutes on Days 1, 8, and 15 in combination with
cisplatin
therapy; cisplatin can be administered intravenously at 100 mg/m2 on Day 1
after the infusion of
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Gemzar . Every 3-week schedule: the recommended dose of Gemzar is 1250 mg/m2
intravenously over 30 minutes on Days 1 and 8 in combination with cisplatin
therapy; cisplatin
can be administered intravenously at 100 mg/m2 on Day 1 after the infusion of
Gemzar .
[00394] Pancreatic Cancer: The following information is taken from Gemzar
(gemcitabine
for injection), Eli Lilly and Company, Highlights of Prescribing Information,
revised March
2017, http://pi.lilly.com/us/gemzar.pdf (last accessed on July 27, 2017).
Gemcitabine (Gemzar )
is indicated as first-line treatment for patients with locally advanced
(nonresectable Stage II or
Stage III) or metastatic (Stage IV) adenocarcinoma of the pancreas. Gemzar is
indicated for
patients previously treated with 5-FU. The recommended dose of Gemzar is 1000
mg/m2 over
30 minutes intravenously. The recommended treatment schedule is as follows:
weeks 1-8 --
weekly dosing for the first 7 weeks followed by one week rest; after week 8 --
weekly dosing on
Days 1, 8, and 15 of 28-day cycles.
[00395] Metastatic Breast Cancer: The following information is taken from
ABRAXANE
(paclitaxel protein-bound particles for injectable suspension, albumin-bound),
Celgene
Corporation, Highlights of Prescribing Information, revised July 2015,
http://www.abraxane.com/wp-content/pi/prescribing-info.html (last accessed
July 27, 2017).
ABRAXANE is indicated for the treatment of breast cancer after failure of
combination
chemotherapy for metastatic disease or relapse within 6 months of adjuvant
chemotherapy. Prior
therapy should have included an anthracycline unless clinically
contraindicated. After failure of
combination chemotherapy for metastatic breast cancer or relapse within 6
months of adjuvant
chemotherapy, the recommended regimen for ABRAXANE is 260 mg/m2 administered
intravenously over 30 minutes every 3 weeks.
[00396] Non-Small Cell Lung Cancer: The following information is taken from
ABRAXANE (paclitaxel protein-bound particles for injectable suspension,
albumin-bound),
Celgene Corporation, Highlights of Prescribing Information, revised July 2015,
http://www.abraxane.com/wp-content/pi/prescribing-info.html (last accessed
July 27, 2017).
ABRAXANE is indicated for the first-line treatment of locally advanced or
metastatic non-
small cell lung cancer, in combination with carboplatin, in patients who are
not candidates for
curative surgery or radiation therapy. The recommended dose of ABRAXANE is
100 mg/m2
administered as an intravenous infusion over 30 minutes on Days 1, 8, and 15
of each 21-day
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cycle. Carboplatin can be administered on Day 1 of each 21 day cycle
immediately after
ABRAXANE .
[00397] Adenocarcinoma of the Pancreas: The following information is taken
from
ABRAXANE (paclitaxel protein-bound particles for injectable suspension,
albumin-bound),
Celgene Corporation, Highlights of Prescribing Information, revised July 2015,
http://www.abraxane.com/wp-content/pi/prescribing-info.html (last accessed
July 27, 2017).
ABRAXANE is indicated for the first-line treatment of patients with
metastatic
adenocarcinoma of the pancreas, in combination with gemcitabine. The
recommended dose of
ABRAXANE is 125 mg/m2 administered as an intravenous infusion over 30-40
minutes on
Days 1, 8 and 15 of each 28-day cycle. Gemcitabine can be administered
immediately after
ABRAXANE on Days 1, 8 and 15 of each 28-day cycle.
[00398] In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment
thereof
described herein is administered to a subject in combination with an
immunomodulator (e.g., a
cytokine, an antigen, or a checkpoint targeting agent). In one embodiment, an
anti-M(H)DM2/4
antibody or fragment thereof described herein is administered to a subject in
combination with a
checkpoint targeting agent such as, without limitation, an antagonist of PD-1,
an antagonist of
PD-L1, an antagonist of PD-L2, an antagonist of CTLA-4, an antagonist of TIM-
3, an antagonist
of GITR, an antagonist of 0X40, an antagonist of LAG-3 (e.g., the antagonist
of any of the
above- mentioned checkpoint molecules can be an antibody, such as an
inhibitory antibody to
these molecules, an antibody fragment, or a small molecule). In one
embodiment, an anti-
M(H)DM2/4 antibody or fragment thereof described herein is administered to a
subject in
combination with an inhibitor of PD-1, an inhibitor of PD-L1, or an inhibitor
of CTLA-4 (where
the inhibitor can be an antagonistic antibody, an antibody fragment, or a
small molecule).
[00399] In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment
thereof
described herein is administered to a subject in combination with radiation
therapy (e.g., x-rays,
gamma- rays or another source of radiation).
[00400] In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment
thereof
described herein is administered to a subject in combination with surgery
(such as a surgery to
remove part or all of the cancerous tumor being treated).
[00401] In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment
thereof
described herein is administered to a subject in combination with a Treg-
inhibitory agent.
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[00402] In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment
thereof
described herein is administered to a subject in combination with a T-cell
therapy.
[00403] In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment
thereof
described herein is administered to a subject in combination with a tumor
vaccine.
[00404] In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment
thereof
described herein is administered to a subject in combination with an EGFR
inhibitor (e.g.,
erlotinib).
[00405] In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment
thereof
described herein is administered to a subject in combination with an inhibitor
of one or more of:
EGFR, KRAS, STK11, ALK, BRAF, ERBB2, RET, ROS1, B2M, HLA, POLE, IGF-1,
ERK/MAPK, PI3K/AKT, TGF-13, DNMT3A, IFNy, JAK1/JAK2/JAK3, CD274, PTEN, ART,
and
CDK).
[00406] In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment
thereof
described herein is administered to a subject in combination with an inhibitor
of p53-H(M)DM2
interaction (e.g., a small molecule inhibitor or a peptide inhibitor).
[00407] In certain embodiments, an anti-M(H)DM2/4 antibody or fragment thereof
described
herein is used to treat a subject that is not treated with a cell cycle
inhibitor (i.e., the additional
therapy is not an agent that inhibits cell cycle). In one embodiment, an anti-
M(H)DM2/4
antibody or fragment thereof described herein is used to treat a subject that
is not concurrently
(during the same treatment period) treated with a cell cycle inhibitor (i.e.,
the subject is not
treated with an anti-M(H)DM2/4 antibody or fragment thereof and a cell cycle
inhibitor during
the same period of time, e.g., day or week). In one embodiment, an anti-
M(H)DM2/4 antibody
or fragment thereof described herein is used to treat a subject that has not
been previously treated
and is not concurrently treated with a cell cycle inhibitor.
[00408] In particular embodiments, an anti-M(H)DM2/4 antibody or fragment
thereof
described herein can be used before, during, or after the second therapy
(e.g., a chemotherapy, a
radiation therapy, a surgery, or any other therapy described herein or known
in the art).
[00409] In certain embodiments, the subject being treated in accordance with
the methods
described herein has not received an anti-cancer therapy prior to the
administration of an anti-
M(H)DM2/4 antibody or fragment thereof. In other embodiments, an anti-
M(H)DM2/4 antibody
or fragment thereof is administered to a subject that has received an anti-
cancer therapy prior to
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administration of the antibody or fragment. In particular, embodiments, anti-
M(H)DM2/4
antibody or fragment thereof is administered to a subject recovering from or
receiving an
immunosuppressive therapy.
[00410] In certain embodiments, provided herein are kits comprising an anti-
M(H)DM2/4
antibody or a fragment thereof, and one or more additional anti-cancer agents.
In one
embodiment, provided herein are kits comprising (i) an anti-M(H)DM2/4 antibody
or a fragment
thereof (e.g., in a therapeutically effective amount), and (ii) one or more of
chemotherapeutic
drugs, for example, gemcitabine, paclitaxel, or gemcitabine and nab-paclitaxel
(e.g., in
therapeutically effective amounts, such as any amounts described herein, which
may be less than
the therapeutically effective amount of the drug or drugs when the drug or
drugs are used without
the anti-M(H)DM2/4 antibody or fragment).
[00411] The following examples are offered by way of illustration and not by
way of
limitation. Various other embodiments of the invention may be practiced, given
the general
description provided herein.
8. EXAMPLES
[00412] The data presented herein demonstrate that specific segments (i.e.,
epitopes) of
HDM2 are extracellularly accessible on the plasma membrane surface of intact
(i.e., viable and
non-permeabilized) cancer cells. Three (3) different extracellularly
accessible epitopes have been
identified. Specific segments of HDM2 that are extracellularly accessible
include but are not
limited to epitopes present in the NMC-P1, NMC-P2 and NMC-P3 peptide sequences
(SEQ ID
NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively). These extracellularly
accessible
sequences are appropriate therapeutic and diagnostic targets for anti-HDM2
antibodies.
Therefore, cancer cells expressing HDM2 on their surface membrane can be
targeted with
antibodies to HDM2 for diagnostic and therapeutic (i.e., anti-tumor cytotoxic
and inhibitory
effect) purposes.
[00413] In particular, the data presented herein demonstrated that select HDM2-
specific
antibodies bound to the extracellularly accessible sequences of M(H)DM2/4 on
the surface
membrane of intact cells of several rodent and human cancer cell lines as well
as primary tumor
cells from human patients. In contrast, the same HDM2-specific antibodies
exhibited minimal
binding to the surface membrane of normal human blood mononuclear cells. It
was found that
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these HDM2-specific antibodies selectively bound to various cancer cells such
as: intact human
melanoma, uveal melanoma, pancreatic, breast, colon, lung and ovarian cancer
cells in vitro. In
addition, the data presented herein showed that HDM2-specific antibodies
inhibited the growth
of cancer cells in vitro and in vivo, strongly indicating that they can be
used as therapeutic agents
in vivo. Data from in vivo studies and described herein showed that select
HDM2-specific
antibodies inhibited tumor growth and were cytotoxic against tumors in rodent
tumor models.
The examples below demonstrated that select HDM2-specific antibodies were not
only cytotoxic
to tumor cells but also inhibited tumor growth in mouse models of pancreatic
cancer, lung cancer
and colon cancer. Data herein further demonstrated that only select antibodies
recognized
extracellularly accessible epitopes of HDM2.
Methods of Making of Antibodies used in the Examples:
[00414] Anti-HDM2 antibodies that specifically bind to (i) NMC-P1, i.e., the
peptide of SEQ
ID NO:1 ("NMC-100s series of monoclonal antibodies)", (ii) NMC-P2, i.e., the
peptide of SEQ
ID NO:2 ("NMC-200s series of monoclonal antibodies"), and (iii) NMC-P3, i.e.,
the peptide of
SEQ ID NO:3 ("NMC-300s series of monoclonal antibodies") were generated using
the
hybridoma approach. NMC-P1, NMC-P2 and NMC-P3 peptides were conjugated to
Keyhole
limpet hemocyanin (KLH) using Sulfo-SMCC method (Thermo Scientific, Cat. No.
22122).
Briefly, Protein-NH2 was made in Conjugation Buffer (provided by
manufacturer). Twenty-fold
molar excess of crosslinker was added to the protein solution and the reaction
mixture was
incubated for 30 minutes at room temperature. Excess cross linker was then
removed using
desalting column equilibrated with Conjugation Buffer. Protein-SH and desalted
Protein-NH2
were then combined, mixed and incubated for 30 minutes at room temperature.
The conjugation
reaction was then stopped by addition of buffer containing reduced cysteine at
a concentration
several times greater than the sulfhydryls of Protein-SH. Following peptide
conjugation, mice
(BALB/c female) were immunized with the peptide NMC-P1, NMC-P2 or NMC-P3 by
intraperitoneal injection at 10011g/mouse. Boosters were injected 5-7 times to
provoke immune
response. Specific-antibody production was then evaluated by peptide-ELISA of
the mice serum
for antibody titration. Spleens of mice with high antibody titer were then
harvested from each
mice and single cell suspension of splenocytes were prepared. Splenocytes were
then fused with
5P2/0 myeloma cells (1:5 ratio). Briefly, 250 pi of EDTA was added to the
mixture of
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splenocytes and myeloma cells. Cells were then span down and supernatant was
removed. Cell
pellet was then loosened and 1 mL of PEG was dispensed alongside the tube
slowly over 1 min
and the mixture was incubated for 5 min in 37 C water bath. 1 mL of 100 x FBS
and 10 mL of
IMDM medium (10% FBS) was then added and kept in the incubator for 1 hour.
Cells were then
centrifuged and supernatant was removed. Cell pellet was then re-suspended in
IMDM (20%
FBS)-containing HAT Fusion medium (hypoxanthine-aminopterin-thymidine medium).
Cells
were then plated in 96-well dishes and incubated for roughly 10 to 14 days.
After 10-14 days
when clones became visible, media supernatant from each well was tested by
ELISA for its
binding to their corresponding specific immunogenic peptide (NMC-P1, NMC-P2 or
NMC-P3).
ELISA-positive wells were then selected for further clone selection, single-
cell sub-cloning and
monoclonal antibody purification. Monoclonal antibody selection was done by
peptide-ELISA
using NMC-P1, NMC-P2 or NMC-P3 peptide antigen. To further select antibodies
that react
with the peptide antigen as well as with plasma membrane HDM2, binding assays
were also
performed for mAb selection by cell-ELISA on intact cancer cells.
Predicted reactivities of the generated antibodies:
[00415] Predicted reactivities of the NMC-100s series, NMC-200s series, and
NMC-300s
series of monoclonal antibodies with different HDM2 isoforms/variants are
presented in Table 1
and Table 2. Predictions were made based on whether the respective isoform
contains the
sequence corresponding to the sequence of Pl, P2 or P3 (and knowledge of to
which peptide (P1,
P2, or P3) the respective NMC series mAb binds, based on which peptide was
used as
immunogen for which series).
[00416] Table 1. Predicted reactivities of the NMC-100s series, NMC-200s
series, and NMC-
300s series of monoclonal antibodies with different HDM2 isoforms/variants.
Reactivity
Reactivity Reactivity
Uniprot with mAb with mAb
with mAb
Accession Amino acid residues of HDM2 (SEQ ID NMC-100s
NMC-200s NMC-300s
Isoform NO:4) that are missing in the isoform series
series series
Q00987-2 Mdm2-A 28-222
28-222;
Q00987-3 Mdm2-A1 275-300
Q00987-4 Mdm2-B 28-300
Q00987-5 Mdm2-C 53-222
Q00987-6 Mdm2-D 30-388
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76-102:
YCSNDLLGDLF GVP SF SVKEHRKIYTM
¨> NDCANLFPLVDL SIRELYISNYITL GI;
Q00987-7 Mdm2-E 103-491 + + -
Q00987-9 Mdm2-F 53-97 + +
+
Q00987-10 Mdm2-G 115-169 + +
+
Q00987-11 Isoform 11 1-1: M ¨> MVRSRQM +
+ +
MDM2-
KB2 157-248 + + +
Mdm2-
Q00987-8 alpha 1-61 - -
+
Q00987 Canonical 1-491 + +
+
[00417] Table 2. Predicted reactivities of the NMC-100s series, NMC-200s
series, and NIVIC-
300s series of monoclonal antibodies with different HDM2 isoforms/variants.
Reactivity Reactivity Reactivity
with mAb with mAb with mAb
NMC-100 NMC-200 NMC-300
NCBI Accession # series series series
EAW97204.1 + + -
EAW97206.1 + + +
EAW97213.1 + + +
EAW97210.1 + + +
EAW97201.1 + + -
EAW97202.1 + + +
EAW97203.1 + + +
EAW97212.1 + + +
EAW97209.1 + + +
EAW97205.1 + + +
AFM80534.1 - - +
CAP16722.1 + + -
CAP16731.1 + + -
AAA75518.1 + + -
CAD36961.1 + + -
CAP16717.1 + + -
AFM80529.1 + + +
AFM80530.1 + + +
CAC07811.1 + + +
AAA75517.1 + + +
AFM80531.1 + + +
CAP16743.1 + + -
CAC07810.1 + - +
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CAP16730.1 + + -
AAL13247.1 + + +
AFM80533.1 + + +
CAD36959.1 + + -
CAP16712.1 + + -
CAD23252.1 + + +
CAP16721.1 + + -
CAP16716.1 + + -
AFM80535.1 + + +
CAP16728.1 + + -
CAP16734.1 + + -
AAL13243.1 + + -
AFM80536.1 + + +
AFM80537.1 + + +
AAA75515.1 + + +
AFM80538.1 + + -
AFM80539.1 + + +
CAC07809.1 + + +
CAD23251.1 + + +
NP 001138812.1 + + +
AAA75514.1 + + +
CAP16729.1 + + -
CAP16733.1 + + -
AFM80540.1 + + +
XP_006719463.1 - - +
CAP16735.1 + + -
CAP16708.1 + + +
XP_006719462.1 - - +
AAL40179.1 + + +
NP 001138811.1 + + +
NP 001138809.1 + + +
AAL40180.1 + + +
CAP16705.1 + + +
ACX31156.1 + + +
CAP16732.1 + + +
CAP16703.1 + + -
CAP16738.1 + + -
XP_005268929.1 + + +
NP 002383.2 + + +
CAD36962.1 + + -
CAP16704.1 + + +
CAP16715.1 + + -
CA1D79459.1 + + -
CAP16707.1 + + -
AAL13242.1 + + -
CAP16718.1 + + -
CAP16727.1 + + -
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CAD79456.1
AAL13244.1
CAP16739.1
CAD79457.1
CAD36960.1
CAD79458.1
CAP16725.1
CAP16719.1
CAP16740.1
CAP16736.1
AFM80527.1
AAL13245.1
CAP16737.1
AFM80528.1
AAL13246.1
CAC07812.1
AAF42995.1
Antibodies used in the Examples:
[00418] Monoclonal antibody NMC-103 is an antibody that binds to NMC-P1 (SEQ
ID NO:1)
(it is one of the NMC-100s series of antibodies). Monoclonal antibody NMC-204
is an antibody
that binds to NMC-P2 (SEQ ID NO:2) (it is one of the NMC-200s series of
antibodies).
Monoclonal antibody NMC-303 is an antibody that binds to NMC-P3 (SEQ ID NO:3)
(it is one
of the NMC-300s series of antibodies). The heavy chain/light chain frame work
region
sequences, complementarity determining region (CDR) sequences, and variable
region
sequences of these antibodies are listed in Section 8, below.
[00419] The following anti-HDM2 antibodies were used in the experiments
described in
Examples 1-9: (i) purified NMC-103 mouse monoclonal antibody (mAb) of the IgG1
isotype
(NMC-103 mAbs produced by single-cell cloned hybridoma cells were purified on
protein G/A
columns), (ii) purified NMC-204 mouse mAb of the IgG3 isotype (NMC-204 mAbs
produced by
single-cell cloned hybridoma cells were purified on protein G/A columns);
(iii) purified NMC-
303 mouse mAb of the IgM isotype (NMC-303 mAbs produced by single-cell cloned
hybridoma
cells were purified on protein G/A columns); (iv) an anti-HDM2 antibody termed
"MDM2
monoclonal antibody (M01), clone 1A7" (Abnova, Cat. No. H00004193-M01); (v) an
anti-
HDM2 antibody termed "MDM2 Antibody (D-7)" (Santa Cruz, Cat. No. sc-13161);
(vi) an anti-
HDM2 antibody termed "p-MDM2 Antibody (2G2)" (Santa Cruz, Cat. No. sc-53368);
(vii) an
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anti-HDM2 antibody termed "MDM2 Antibody (SPM344)" (Santa Cruz, Cat. No. sc-
56430);
(viii) an anti-HDM2 antibody termed "MDM2 Antibody (SMP14)" (Santa Cruz, Cat.
No. sc-
965); (ix) an anti-HDM2 antibody termed "Anti-MDM2 clone 4B2C1.11" (EMD
Millipore, Cat.
No. MABE331); (x) an anti-HDM2 antibody termed "Anti-MDM2 clone 3G9" (EMD
Millipore,
Cat. No. 04-1530); (xi) an anti-HDM2 antibody termed "Anti-MDM2 clone 2A10"
(EMD
Millipore, Cat. No. MABE281); (xii) an anti-HDM2 antibody termed "Anti-MDM2
(Ab-1)
Mouse mAb (IF2)" (EMD Millipore, Cat. No. 0P46); (xiii) an anti-HDM2 antibody
termed
"Anti-MDM2 (Ab-3) Mouse mAb (4B11)" (EMD Millipore, Cat. No. OP143); (xiv) an
anti-
HDM2 antibody termed "Anti-MDM2 (Ab-4) Mouse mAb (2A9C1.18)" (EMD Millipore,
Cat.
No. 0P144); (xv) an anti-HDM2 antibody termed "Anti-MDM2 (Ab-5) Mouse mAb
(4B2C1.11)" (EMD Millipore, Cat. No. 0P145); (xvi) an anti-HDM2 antibody
termed "MDM2
Antibody (C-18): sc-812" (Santa Cruz, polyclonal, Cat No. sc-812); and (xvii)
n anti-HDM2
antibody termed "MDM2 Antibody (N-20): sc-813" (Santa Cruz, polyclonal, Cat
No. sc-813).
8.1 Example 1: Selection of HDM2-specific mAbs to extracellularly
accessible
epitopes of HDM2.
[00420] Utilizing enzyme-linked immunosorbent assay (ELISA) the data presented
herein
showed that mAb NMC-103 specifically bound to peptide NMC-P1 (SEQ ID NO:1)
corresponding to amino acids 1-15 of HDM2, while mAb NMC-204 specifically
bound to NMC-
P2 peptide (SEQ ID NO:2) corresponding to amino acids 15-25 of HDM2, and mAb
NMC-303
specifically bound to NMC-P3 peptide (SEQ ID NO:3) corresponding to amino
acids 475-491 of
HDM2. Moreover, immunoblot analysis presented here shows that mAbs NMC-103,
NMC-204
and NMC-303 recognized the full-length recombinant HDM2 protein.
[00421] Peptide-ELISA Methodology: 5 g/m1 of NMC-P1, NMC-P2 or NMC-P3 peptide
antigen was dried onto a 96-well ELISA plate overnight. Plates were then
blocked with 5% BSA
in lx phosphate-buffered saline (PBS) (100 l/well) for 2 hours at room
temperature. Microplate
wells were then washed 5 times with 300 1 of ice cold lx PBS. Appropriate
dilutions of
monoclonal antibodies NMC-103, NMC-204 or NMC-303 in 1% bovine serum albumin
(BSA)
in PBS solution were then incubated with their corresponding peptides (NMC-P1,
NMC-P2 or
NMC-P3, respectively) at room temperature. After 2 hours, wells were washed 5
times with 300
1 ice-cold PBS/well and 100 1 secondary antibody (HRP-labeled Goat anti-Mouse
F(ab')2
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H&L cross-adsorbed secondary antibody (HRP-GaM F(ab')2), ThermoFisher, Cat.
No. A24524)
diluted 1:2500 to 1:5000 in PBS-1%B SA solution were added and the incubation
continued at
room temperature. After 1 hour, the wells were washed again 5 times with 200
1 of ice-cold
PBS and 50 [EL of the TMB Substrate Solution (1-StepTM Ultra TMB-ELISA,
ThermoFisher,
Cat. No. 34028) were added to each well and color was allowed to develop for
30 minutes at
room temperature. The reaction was stopped by addition of 50 [IL of stop
solution
(ThermoFisher,Cat. No. SS04) to each well and absorbance of each well was read
immediately
for optical density (OD) at 450 nm.
[00422] Competition assays: As for peptide antigen binding competition
experiments, 1 1/m1
of mAb NMC-103 was pre-incubated with 10 1/m1 of NMC-P1, NMC-P2 or NMC-P3 for
1
hour at room temperature. MAb NMC-103 was then incubated with ELISA plates
coated with
NMC-P1 as described above.
[00423] Western Blot: As for immunoblots, 1 g/well of recombinant HDM2
protein (GST
tagged, Abcam, Cat. No. ab188727) was separated on 8-16% sodium dodecyl
sulfate (SDS)
polyacrylamide gel and proteins were then transferred to polyvinylidene
difluoride (PVDF)
membrane. The membrane was blocked with 5% milk followed by incubation with
monoclonal
antibody NMC-103, NMC-204 or NMC-303 at 1 g/m1 in 1% milk in PBS for 2 hours.
The
membrane was then washed x 3 times (10 minutes each) and incubated for 1 hour
with
corresponding HRP-conjugated secondary antibody (Goat anti-Mouse IgG (H&L),
F(ab')2 Frag
Cross-adsorbed HRP (HRP-GaM F(ab')2), ThermoFisher, Cat. No. A24524) diluted
1:5000 in
1%BSA-PBS. The membrane was then washed x 3 times and incubated with Pierce
ECL Plus
Western Blotting Substrate (ThermoFisher, Cat. No. #32132) for 10 min before
developing on a
LICOR Scanner.
[00424] The peptide-ELISA experiments shown in Figure 1 demonstrated selective
and
specific binding of monoclonal antibodies (mAb) NMC-103, NMC-204 and NMC-303
to their
corresponding peptide antigens. Figure 1A shows that NMC-103 bound to NMC-P1
peptide
while NMC-204 did not bind to NMC-P1. Figure 1B shows that mAb NMC-204 bound
to NMC-
P2 peptide while mAb NMC-103 did not bind to NMC-P2. Figure 1C shows that mAb
NMC-
303 bound to NMC-P3 peptide while NMC-204 did not bind to NMC-P3. Thus, the
Peptide-
ELISA results demonstrated that each of the mAbs NMC-103, NMC-204 and NMC-303
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selectively bound to their corresponding antigen peptides on the ELISA-plate
and not to other
peptides. Figure 1D shows that while mAb NMC-103 bound to NMC-P1, pre-
incubation of
NMC-P1 peptide with mAb NMC-103 abolished the binding of NMC-103 to NMC-P1
peptide
on the plate. In contrast, pre-incubation of mAb NMC-103 with either NMC-P2 or
NMC-P3 did
not affect the binding of mAb NMC-103 to NMC-Pl. These experiments demonstrate
the
specificity of mAbs NMC-103, NMC-204 and NMC-303 for their corresponding
peptide
antigens.
[00425] The results of the immunoblot experiments in Figure 2 demonstrated
that mAbs
NMC-103, NMC-204 and NMC-303 reacted with the recombinant full-length HDM2
protein.
The recombinant protein has a GST tag at its N-terminal which brings the
molecular weight to
approximately 83 kD. Lane 2 of the immunoblot shows the reactivity of a
commercially
available antibody raised against amino acids 100-320 of MDM2 of human origin
(MDM2 (D-
7); Santa Cruz, Cat. No. sc-13161) to the full-length recombinant HDM-2
protein. Lane 3 of the
immunoblot shows the reactivity of mAb NMC-103 with a single band at
approximately 83 kD,
corresponding to the recombinant HDM2-GST protein. Lane 4 of the immunoblot
shows the
binding of mAb NMC-204 to a single band at approximately 83 kD, corresponding
to the
recombinant HDM2-GST protein. Lane 5 of the immunoblot shows reactivity of mAb
NMC-303
with the recombinant HDM2-GST protein at 83 kD. In contrast, in Lane 1,
control mouse IgG
(Abcam, Cat. No. ab18447) did not react with the recombinant HDM2-GST protein.
The data
presented here further demonstrated that mAbs NMC-103, NMC-204 and NMC-303
recognized
full-length HDM2.
8.2 Example 2: HDM2-specific antibodies bound to extracellularly
accessible
epitopes of M(H)DM2/4 on intact cancer cells from different rodent and
human cancer cell lines and freshly isolated primary human cancer cells but
not to normal cells.
[00426] General description: Utilizing ELISA, the data presented herein
demonstrated the
binding of mAbs NMC-103, NMC-204 and NMC-303 to extracellularly accessible
epitopes of
M(H)DM2/4 within the NMC-P1, NMC-P2 and NMC-P3 sequences, respectively, on the
plasma
membrane of human breast cancer cells, human triple negative breast cancer
cells, human
melanoma cells, chemo-resistant human ovarian cancer cells as well as primary
patient-derived
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human ovarian cancer cells, human and mouse pancreatic cancer cells, mouse
colon cancer cells
and mouse lung cancer cells. Data presented herein also demonstrated the
selective binding of
these mAb to cancer cells and not to normal untransformed cells. Moreover,
peptide-antigen
competition results showed that each of these mAbs specifically bound to their
corresponding
NMC-P1, NMC-P2 or NMC-P3 regions of the plasma membrane M(H)DM2/4.
[00427] Cell-ELISA Methodology: 8,000-10,000 cells/well of a 96-well
microplate were
grown overnight. The next day, unbound cells were washed off with sterile lx
PBS. The cells in
each well were fixed with freshly prepared 4% buffered paraformaldehyde (pH
7.2) for 1 hour
followed by 3 washes with lx PBS. The wells were then blocked with 5% BSA in
PBS (100
l/well) for 2 hours at room temperature. Microplate wells were then washed 5
times with 300 1
of ice cold lx PBS. MAbs NMC-103, NMC-204 or NMC-303 at 1 [tg/mL in 1%BSA/PBS
were
then incubated with various cancer or normal untransformed cells for 2 hours
at room
temperature. Wells were then washed with 300 1 of ice-cold lx PBS for 5 times
and
corresponding secondary antibody HRP-GaM F(ab')2 diluted 1:2500 or 1:5000 in
PBS with 1%
BSA were added at100 l/well for 1 hour at room temperature followed by
washing 5 times with
300 1 of ice-cold lx PBS. TMB Substrate Solution (1-StepTM Ultra TMB-ELISA,
ThermoFisher, Cat. No. 34028) was then added at 50 pL to each microplate well
and incubated
at room temperature for 30 minutes. The reaction was stopped by addition of 50
pL of stop
solution (ThermoFisher, Cat. No. SS04) to each well and absorbance of each
well was measured
at 0D450 nm. The absorbance value of each experimental well was corrected for
the absorbance
value obtained from wells treated with isotype-identical mAbs included in each
experiment. The
results are thus expressed as "relative binding".
[00428] Figures 3-5 show the relative binding of mAbs NMC-103, NMC-204 and NMC-
303
to intact cells of different types of human (A) and rodent (B) cancers. Figure
3 shows reactivity
of mAb NMC-103 to human breast cancer MCF-7 cells, human triple negative
breast cancer
HCC1806 cells, human pancreatic cancer MIA PaCa-2 cells, human ovarian cancer
OVCAR-3
cells that are resistant to adriamycin, melphalan, and cisplatin, primary
patient-derived human
ovarian cancer OVCA4 cells, human melanoma A2058 cells, human uveal melanoma
92.1 cells,
mouse colon cancer MC-38 cells, mouse Lewis Lung LL/2 cells and mouse
pancreatic Panc02
cells. Figure 4 presents the reactivity of NMC-204 monoclonal antibody to
human breast cancer
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MCF-7 cells, human triple negative breast cancer HCC1806 cells, human
pancreatic cancer MIA
PaCa-2 cells, human ovarian cancer OVCAR-3 cells, primary patient-derived
human ovarian
cancer OVCA.4 cells, human melanoma A2058 cells, human uveal melanoma 92.1
cells, mouse
colon cancer MC-38 cells, mouse Lewis Lung LL/2 cells and mouse pancreatic
Panc02 cells.
Figure 5 shows reactivity of NMC-303 monoclonal antibody to human breast
cancer MCF-7
cells, human triple negative breast cancer HCC1806 cells, human pancreatic
cancer MIA PaCa-2
cells, human ovarian cancer OVCAR-3 cells, primary patient-derived human
ovarian cancer
OVCA.4 cells, human melanoma A2058 cells, human uveal melanoma 92.1 cells,
mouse colon
cancer MC-38 cells, mouse Lewis Lung LL/2 cells and mouse pancreatic Panc02
cells.
[00429] Figure 6 demonstrates that NMC-204 did not react with normal human
PBMCs. In
contrast to cancer cells, neither mAb NMC-103, NMC-204 nor NMC-303 bound to
normal intact
cells (the data for NMC-103 and NMC-303 is not shown). Data presented herein
show that,
while mAb NMC-204 reacted with human pancreatic cancer MIA PaCa-2 cells, no
binding was
seen above the background when mAb NMC-204 was incubated with freshly isolated
normal
human peripheral blood mononuclear cells (PBMCs). Moreover, Figure 6
demonstrates that
while mAb NMC-204 did not react with normal human PBMCs (Figure 6 left; white
bar graph),
these cells showed strong reactivity with an mAb against CD3e, a cell surface
marker for T cells
(Figure 6 left; shaded bar graph).
[00430] Figure 7 depicts mAb NMC-103 and NMC-204 saturation curves. Figure 7A
shows
that cell-ELISA binding of mAb NMC-103 to intact MIA PaCa-2 cells increased as
the
concentration of the antibody increased. However, this binding reached a
plateau at
concentrations of above 501.tg/mL of mAb NMC-103, demonstrating the saturation
of antigen
binding sites by the specific antibody NMC-103. Moreover, Figure 7B depicts
the binding
saturation curve of mAb NMC-204 to intact MIA PaCa-2 cells. The binding of mAb
NMC-204
to its antigen on the intact MIA PaCa-2 cells reached saturation at
concentrations above 50
1.tg/mL of mAb NMC-204, demonstrating the binding saturation of antigen sites
for mAb NMC-
204.
8.3 Example 3: mAb NMC-103. NMC-204 and NMC-303 were specific for
extracellularly accessible NMC-P1. NMC-P2 and NMC-P3 sequences of
HDM2 on intact cancer cells. respectively.
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[00431] The data shown in Figure 8 demonstrated the specificity of mAbs NMC-
103, NMC-
204 and NMC-303 for NMC-P1, NMC-P2 and NMC-P3 sequences of HDM2 that are
extracellularly accessible on the membrane of cancer cells. Figure 8A shows
the binding of mAb
NMC-103 to intact human pancreatic cancer MIA PaCa-2 cells (left, solid bar).
The binding of
monoclonal antibody NMC-103 to its epitope of HDM2 that is accessible on the
surface plasma
membrane of MIA PaCa-2 cells was completely abolished when mAb NMC-103 was
competed
with soluble NMC-P1 (middle column, solid bar). In contrast, competition with
NMC-P2 did not
have any effect on the reactivity of NMC-103 with the epitope of HDM2 to which
it binds and
which is accessible on the cell surface of the MIA PaCa-2 cells (solid, filled
bar).
[00432] On the other hand, as shown in Figure 8B, mAb NMC-204's reactivity
with intact
MIA PaCa-2 cells was competed with NMC-P2 peptide and not with NMC-P1,
demonstrating
epitope specificity for another extracellularly accessible epitope/peptide of
HDM2 expressed on
the plasma membrane of MIA PaCa-2 cells.
[00433] The observation of the specificity of the newly generated mAbs for
certain epitopes
on HDM2 was further extended by the observation that mAb NMC-303's binding to
intact MIA
PaCa-2 was competed with NMC-P3 peptide and not with NMC-P2(Figure 8C). Taken
together,
these data demonstrate the specificity of mAbs NMC-103, NMC-204 and NMC-303
for 3
different extracellularly accessible sequences of HDM2, namely NMC-P1, NMC-P2
and NMC-
P3, respectively.
[00434] As demonstrated in Figure 9, when mAb NMC-103 was pre-incubated with
full-
length recombinant HDM2, the binding of the antibody to intact MIA PaCa-2
cells was reduced.
This result further confirms the specific binding of NMC antibodies to HDM2.
8.4 Example 4: A plasma membrane marker antibody. antibody NMC-103 and
antibody NMC-204 stain plasma membrane surface of intact cells. while an
intracellular marker antibody does not.
[00435] The data presented herein (Figure 10) showed that select HDM2-specific
antibodies
bound to the extracellularly accessible HDM2 on the plasma membrane of intact
cancer cells
(human pancreatic MiaPaCa-2 cells) that were treated with EDTA (10 mmols;
pH7.2; 5 min,
37 C). EDTA treatment therefore, appeared to have little or no effect on the
expression of
HDM2 on the cancer cells' surface membrane, strongly suggesting that the HDM2
protein
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antigen is an integral membrane protein. Additional data presented here show
that antibodies to
plasma membrane markers such as E-Cadherin (Transduction Lab, Cat. No. C37020)
bound to
intact cells, while antibodies specific for markers that are located
intracellularly such as
Cytochrome-C (Santa Cruz, Cat. No. sc-13156) were unable to bind to their
targets due to the
inaccessibility of the intracellular compartments in the intact cells.
8.5 Example 5: Extracellular protease digestion of intact cancer cells
removed
extracellularly accessible sequences of M(H)DM2 on the plasma membrane
of intact cancer cells.
[00436] MAbs NMC-103, NMC-204 and NMC-303 were raised against amino acids 1-15
(NMC-P1), 15-25 (NMC-P2) and 475-491 (NMC-P3) of HDM2, respectively, and were
highly
specific for HDM2. As shown in the immunoblot of Figure 2, all three mAbs NMC-
103, NMC-
204 and NMC-303 recognized and bound to purified HDM2 protein, providing
evidence that the
unique antigenic epitopes recognized by the three mAbs in NMC-P1, NMC-P2 and
NMC-P3,
respectively, are de facto structures of intact HDM2. The results of Cell-
ELISAs in Figures 3-5
and 8 took the observations one step further by providing evidence that the
M(H)DM2 epitopes
are expressed extracellularly on the surface membrane of intact rodent and
human cancer cells:
(1) mAbs NMC-103, NMC-204 and NMC-303 bound to intact cancer cells (Figure 3-
5); (2) each
mAb's binding was effectively competed with by its specific peptide but not by
the other
peptides (Figure 8); (3) NMC- mAb's binding was also effectively competed by
the intact
HDM2-protein (Figure 9); (4) virtually no binding could be seen of mAbs NMC-
103, NMC-204
or NMC-303 to intact normal human PBMCs (Figure 6).
[00437] To further solidify the finding of the expression of M(H)DM2 on the
surface
membrane of cancer cells, binding by indirect immunofluorescence of mAbs NMC-
103, NMC-
204 and NMC-303, respectively, to cancer cells was examined before and after
protease
treatment of the cells. Extracellular protease digestion has been validated as
a powerful means
for evaluation of a cell surface localization of plasma membrane proteins
(Besingi and Clark,
2015, Nat. Protoc. 10(12): 2074-2080; Schillein et al., 1996, J. Biol. Chem.
271(46):28844-
28852): loss of binding of a cell surface antigen-specific antibody to cells
after protease
treatment indicates not only that the antigen is accessible to proteolysis but
also, and most
importantly, that the antigen is exposed on the extracellular surface of the
cells. The
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accessibility, therefore, of extracellularly displayed M(H)DM2 protein antigen
and particularly
of the amino acid sequences of NMC-P1, NMC-P2 and NMC-P3, was evaluated by the
antigen's
susceptibility to digestion by trypsin. The effect of trypsin on the binding
of mAbs NMC-103,
NMC-204 and NMC-303, was then examined using cell ELISA and HRP reactivity at
0D450
nm as described above.
[00438] Flow Cytometry Methodology: Cells that had been allowed to grow to
about 80%
confluency in 25cm2 tissue culture flasks were released with either EDTA (10
mmols; pH7.2; 5
min, 37 C) or Trypsin (Gibco TrypLE Express), fixed with freshly made 4%
buffered (pH 7.4)
paraformaldehyde for 1 h at room temperature, washed several times with a
large volume of ice-
cold PBS, and blocked for 30 minutes at room temperature with 5% human serum
albumin in
PBS (intact, non-permeabilized cells). After establishing viability, the cell
number was adjusted
to 106 cells/ml and kept in ice-cold PBS until staining. Another set of the
same cells was released
with either EDTA or Trypsin and fixed with 4% buffered paraformaldehyde as
above, then
washed and blocked with 5% human serum albumin in PBS, and was treated
separately with
Triton X-100 (0.1% in PBS) (International Biotechnologies Inc. 07100) for 5
minutes at room
temperature for membrane permeabilization. After washing off the Triton X-100
solution with
PBS the cells were adjusted to 106 cells/ml and stored in ice-cold PBS. Cells
(106/m1) from each
preparation were then incubated either with 51.tg/m1 of mAb to Na+/K+ ATPase a-
1 (Abcam,
Cat. No. ab2826), 51.tg/m1 of mAb NMC-103, or 51.tg/m1 of mAb NMC-204 for 90
min at room
temperature. Following primary antibody incubation, cells were washed 3 times
with ice-cold
PBS followed by incubation with rabbit anti-mouse PE-Cy5 labeled secondary
antibody (PE-
Cy5-RaM IgG (H&L); Invitrogen, Cat. No. M35018) at room temperature. Sixty
minutes later
the cells were washed 3 times with PBS and subjected to flow cytometry
analysis (BD Canto
Flow Cytometry). Controls to establish background fluorescence included the
analysis of
samples of unstained cells as well as cells that had been processed with
secondary antibody
under the same conditions as used in the experimental samples.
[00439] To evaluate the extracellular versus intracellular accessibility of
antibodies on an
intact cell, the binding of an antibody to a plasma membrane marker, E-
Cadherin (Transduction
Lab C37020) and the binding of an antibody to an intracellular protein,
Cytochrome-C (santa
cruz sc-13156) were tested using intact human pancreatic cancer MiaPaCa-2
cells. The cell-
ELISA results in Figure 10 demonstrate the binding of E-Cadherin antibody to
intact, EDTA-
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released MiaPaCa-2 cells (first bar from left). In contrast, mAb to Cytochrome-
C showed only
minimal binding to EDTA-released cells (second bar from left), which is
indicative of the
inaccessibility of intracellular targets in intact cells to large molecules
such as antibodies. On the
other hand, both mAbs NMC-103 and NMC-204 bound to intact, EDTA-released
MIAPaCa-2
cells, establishing strong evidence for their interaction with their epitopes
present in the
extracellularly accessible sequences of HDM2.
[00440] To further validate that the epitopes of the plasma membrane M(H)DM2
are in fact
extracellularly accessible sequences on cancer cells, binding of mAbs NMC-103,
NMC-204 and
NMC-303 was performed on mouse Lewis Lung LL/2 cancer cells under treatment
conditions of
EDTA versus trypsin. Both of these conditions were also tested on intact
versus permeabilized
cells. Figure 11 panels A, B and C present flow cytometry data on % cells
stained with mAbs
NMC-103, NMC-204 and anti-Na+/K+ ATPase a-1, respectively. Each antibody was
reacted
with cells from four treatment conditions: EDTA-treated intact cells, EDTA-
treated
permeabilized cells, trypsin-treated intact cells, trypsin-treated
permeabilized cells. EDTA-
treated intact cells showed staining with mAb NMC-103 (70.4%), NMC-204 (51.2%)
and anti-
Na+/K+ ATPase a-1 antibody (29.9%). When EDTA-treated cells were
permeabilized, staining
with NMC-103, NMC-204 and anti-Na+/K+ ATPase a-1 increased to 75.8%, 52.85 and
63.8%,
respectively. However, when compared with EDTA-treated cells, the binding of
mAbs NMC-
103, NMC-204 and anti-Na+/K+ ATPase a-1 to intact cells that were treated with
trypsin
reduced to 36.8%, 27% and 20.5%, respectively, due to extracellular protease
digestion of
antibody recognition epitopes. However, when trypsin-treaded cells were
permeabilized, the
above reduction in binding was compensated by reactivity of the antibodies to
intracellular
MDM2 or Na+/K+ ATPase a-1. Taken together, results presented herein further
demonstrate
the accessibility of extracellular sequences of transmembrane M(H)DM2 on
intact cancer cells
which can be cleaved by extracellular protease treatment.
8.6 Example 6: Specific HDM2 antibodies inhibited the growth of intact
cancer
cells in vitro.
[00441] Data presented herein demonstrate that antibodies that recognized
extracellularly
accessible sequences of HDM2 were able to inhibit the growth of cancer cells
in vitro.
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[00442] Methodology used: Human pancreatic cancer MIAPaCa-2 cells (8,000
cells/well)
were pre-incubated with mAb NMC-103, NMC-204 and anti-Cytochrome-C antibody
(Santa
Cruz, Cat No. sc-13156) at 1, 5 and 101.tg/mL, respectively. Cells in the
presence of one of the
antibodies at various concentrations were then plated in triplicate in 96-well
cell culture plates
overnight. The next day, each plate well was analyzed morphologically and a
picture was taken
from each well using EVOS FL microscope. Cell count was also performed and the
number of
living cells was graphed as % of control.
[00443] Figure 12 (A-D) demonstrates that human pancreatic cancer MIAPaCa-2
cells that
were treated with mAb NMC-103 or NMC-204 exhibited a concentration-dependent
growth
inhibition, while an antibody against an intracellular marker (i.e.,
Cytochrome C) had no effect
on the growth of these cancer cells. As demonstrated in Figure 12A, MIAPaCa-2
cells treated
with 11.tg/mL (top panel, left image), 51.tg/mL (to panel, middle image) and
101.tg/mL (top
panel, left image) mAb NMC-103 showed an increasing effect in inhibition of
their growth.
Figure 12B presents images of the cells treated with 1, 10 and 201.tg/mL of
mAb NMC-204,
showing a concentration-dependent inhibition of cell growth. In contract,
Figure 12C
demonstrates that treatment of MIAPaCa-2 cells with an antibody against
Cytochrome-C, which
was used as an intracellular marker control, had no effect on the growth of
these cells at any of
the 1, 10 and 201.tg/mL concentration. Figure 12D quantifies the growth
inhibitory effect of mAb
NMC-103 at 11.tg/mL (65%), 5 1.tg/mL (84%) and 101.tg/mL (91%) (solid black
line) and of
NMC-204 at 11.tg/mL (65%), 5 1.tg/mL (73%) and 101.tg/mL (77%) (dashed black
line). In
contrast, treatment with an antibody to Cytochrome C (an intracellular target;
Santa Cruz, Cat.
No. sc-13156) showed no sign of growth inhibition at 11.tg/mL (100%), 51.tg/mL
(100%) and 10
1.tg/mL (104%) (solid gray line).
8.7 Example 7: HDM2-specific antibodies initiated complement-dependent
cvtotoxicitv (CDC) against cancer cells.
[00444] The data presented herein show the ability of HDM2-specific antibodies
to not only
bind to the extracellularly accessible sequences of HDM2 on the surface
membrane of cancer
cells but also to initiate a potent cytotoxic effect in the presence of fresh
normal human serum
(NETS). The cytotoxic effect was measured in pancreatic cancer cells as well
as in normal human
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fibroblasts. Data presented herein demonstrated one such cytotoxic effect when
human
pancreatic cancer MIAPaCa-2 cells were treated with mAb NMC-103 in the
presence of NHS.
[00445] Methodology used: Cells seeded in 24-well dishes were grown overnight
at 37 C in a
humidified atmosphere supplied with 5% CO2 ¨ 95% air mixture. The following
day the cells
were thoroughly washed with ice-cold PBS and incubated with HDM2-specific mAb
NMC-103
diluted in culture medium containing 1% BSA to a final antibody concentration
of 301.tg/ml.
After 30 min, NHS was added to the cells at a final concentration of 1:10 in
the culture medium
with 1% BSA together with 1% propidium iodide (PI) solution. Cells were then
incubated with
NHS for 2 hours and images were taken using EVOS FL fluorescent microscope
(Life
Technologies).
[00446] Figure 13 demonstrates the cytotoxic effect of HDM2-specific mAb NMC-
103
against human pancreatic cells. mAb NMC-103 in the presence of NHS triggered
complement-
mediated cytotoxicity in cancer cells, resulting in the death of the cancer
cells as evident by the
nuclear uptake of the cell-death marker Propidium Iodide (PI). Figure 13C
provides a
quantitative representation of HDM2-specific antibody complement-dependent-
cytotoxicity
(CDC) against human pancreatic cancer cells. Cells treated with mAb NMC-103
(Figure 13B) in
the presence of NHS demonstrated cytotoxicity over 2 hours post-treatment as
compared with
cells treated with NHS in the absence of any antibody (Figure 13A). Control
experiement was
performed with anti-Cytochrome C antibody or with cells left untreated in the
presence of NHS.
8.8 Example 8: Evaluation of other anti-HDM2 mAb antibodies in their
binding
to NMC-P1. NMC-P2 and NMC-P3 and to intact cancer cells.
[00447] There are a number of HDM2-specific monoclonal antibodies that are
commercially
available. To evaluate the binding of commercially available mAbs to HDM2,
utilizing peptide-
and cell-ELISA, we tested a number of such mAb for their binding to intact
cancer cells. These
mAbs included antibodies that were raised against various regions from the N-
terminus or C-
terminus, or segments in the middle of the HDM2 protein, and were tested for
their binding to
newly identified extracellularly accessible NMC-P1 and NMC-P2 sequences as
well as to intact
cancer cells.
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[00448] Table 3 summarizes the commercially available mAbs that were tested,
the
companies that generated these mAbs, and amino acid residues of HDM2 against
which they had
been raised.
[00449] Table 3. Commercially available monoclonal antibodies that were
tested.
Antigen Residues on
Antibody Company Cat. No. HDM2
MDM2 monoclonal antobpdy (M01), clone 1A7 ABNOVA H00004193-
M01 101-200
MDM2 Antibody (D-7) SANTA CRUZ SC-13161 100-320
p-MDM2 Antibody (2G2) SANTA CRUZ SC-53368 180-190
MDM2 Antibody (5PM344) SANTA CRUZ SC-56430 154-167
MDM2 Antibody (SMP14) SANTA CRUZ SC-965 154-167
Recombinant human
Anti-MDM2 clone 462C1.11 MILLIPORE MABE331 MDM2
Anti-MDM2 clone 3G9 MILLIPORE 04-1530
His-tagged
recombinant human
Anti-MDM2 clone 2A10 MILLIPORE MABE281 MDM2
Anit-MDM2 (Ab-1) Mouse mAb (IF2) CALBIOCHEM 0P46 26-169
Anti-MDM2 (Ab-3) Mouse mAb (4611) CALBIOCHEM 0P143 383-491
Anti-MDM2 (Ab-4) Mouse mAb (2A9C1.18) CALBIOCHEM 0P144 153-222
Anti-MDM2 (Ab-5) Mouse mAb (462C1.11) CALBIOCHEM 0P145 19-50
[00450] Utilizing Peptide-ELISA, Figures 14A and 14B demonstrated the lack of
binding of
any of these mAbs to either NMC-P1 or NMC-P2. In contrast, NMC-103 and NMC-204
showed
strong binding to NMC-P1 and NMC-P2, respectively.
[00451] In these experiments, it was further evaluated whether HDM-2 binding
components
such as peptides, for example PNC-27, interfered with the binding of mAb
antibodies to NNW-
P1 and NMC-P2. Even though it is not known where exactly the HDM2-binding
component of
PNC-27 and PNC-28 peptides binds on HDM2, it has been reported to bind within
amino acids
25-109 of HDM2 (Do et al., 2003, Oncogene 22(10):1431-1444 ("Do 2003"); Chene,
2003, Nat.
Rev. Cancer 3(2):102-109). To rule out that PNC-27 and PNC-28 peptides bind to
NMC-P1 or
NMC-P2, the peptides' binding to these HDM2 sequences was evaluated by peptide-
ELISA. As
shown in Figure 14A and 14B, PNC-27 (10 [tg/mL) did not compete with the
binding of NMC-
103 and NMC-204 (1 [tg/mL) to NMC-P1 and NMC-P2, respectively. It was found
that PNC-27
does not bind to NMC-P1 or NMC-P2 regions of HDM2). Overall, these
observations
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demonstrate that the HDM2-binding component of PNC-27 does not react with NMC-
P1 or
NMC-P2. Moreover, it has been demonstrated that the HDM-2 binding components
of PNC-27
and PNC-28 have no anti-cancer activity by themselves and are only active when
attached to a
membrane resident peptide (MRP or Penetratin sequence) (see Kanovsky 2001, Do
2003, and
Bowne 2008).
[00452] In the analysis of mAbs by cell-ELISA, despite the fact that none of
the tested
commercially available antibodies bound to either NMC-P1 or NMC-P2, an anti-
HDM2
antibody raised against amino acid residues 19-50 of HDM2, termed "Anti-MDM2
(Ab-5)
Mouse mAb (4B2C1.11)" (EMD Millipore, Cat. No. 0P145), and an anti-HDM2
antibody
termed "MDM2 monoclonal antibody (M01), clone 1A7" (Abnova, Cat. No. H00004193-
M01)
were identified as reactive with intact cancer cells (see Figure 15A, showing
data for MDM2
monoclonal antibody (M01), clone 1A7). MDM2 monoclonal antibody (M01), clone
1A7 was
raised against amino acid 101 to 200 of full-length HDM2. Thus, the data
demonstrate that
epitopes of HDM2 other than NMC-P1, NMC-P2 and NMC-P3 may be extracellularly
accessible
on cancer cells for binding. In similar experiments, two HDM2-specific
monoclonal antibodies
that did not react with intact cancer cells were identified (Figure 15B). One
mAb was an anti-
HDM2 antibody termed "Anti-MDM2 (Ab-4) Mouse mAb (2A9C1.18)" (EMD Millipore,
Cat.
No. OP144), which is raised against amino acid 153 to 222 of full length HDM2
(SEQ ID NO:4)
that includes one Nuclear Localization Signal (NLS) and one Nuclear Export
Signal (NES).
Another HDM2-specific mAb that did not react with intact cancer cell membrane
was an anti-
HDM2 antibody termed "Anti-MDM2 (Ab-1) Mouse mAb (IF2)" (EMD Millipore, Cat.
No.
0P46), which is raised against amino acids 26-169 of HDM2 (Figure 15B).
8.9 Example 9: In vivo anti-tumor effect of antibodies that target the
extracellularly accessible sequences of HDM2 in cancer cells.
[00453] As demonstrated above, there are select epitopes of HDM2 that are
extracellularly
accessible on cancer cells. Described herein are three (3) such
extracellularly accessible
segments of HDM2, namely NMC-P1 (SEQ ID NO:1), NMC-P2 (SEQ ID NO: 2) and NMC-
P3
(SEQ ID NO: 3). Data herein demonstrated that select antibodies raised against
these 3 segments
selectively and specifically bound to various types of cancers but not normal
health cells (Figures
3-9). Moreover, results presented herein demonstrated the extracellular
accessibility of these 3
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segments of HDM2 on cancer cells (Figure 10 and 11). Furthermore, data herein
show that
HDM2-specific antibodies had both growth inhibitory and cytotoxic effect
against cancer cells in
vitro. Finally, the anti-tumor activity of HDM2-specific antibodies was
evaluated in vivo. As an
example, efficacy of mAb NMC-103 and NMC-204 on lung, colon and pancreatic
cancer was
tested.
[00454] Methodology used (Syngeneic Mouse Models of lung, colon and pancreatic
cancer):
To investigate the anti-tumor effects of HDM2-specific antibodies on lung,
colon and pancreatic
tumors in vivo, three well-known syngeneic mouse models of lung, colon and
pancreatic cancer
were used (Sharma et al., 1999, J. Immunol. 163 (9):5020-5028; McIntyre, 2015,
Bioassays
37(8):909-920; Li, et al., 2015, Sci. Rep. 5:7856). Subcutaneous tumors were
prepared by
implanting either mouse Lewis Lung cancer LL/2 cells (2.5x105 cells/mouse),
mouse colon
cancer MC-38 cells (5x105 cells/mouse) or mouse pancreatic cancer Panc-2 cells
(2x106
cells/mouse) in the right flanks of 8-week-old female C57BL/6 mice (n=6-7).
The mice were
returned to their respective cages after tumor implantation. From day 7 after
tumor implantation,
the tumor volumes were measured 2 times/week with a digital caliper. In the
case of MC-38 and
Panc-2 study, treatment started when the tumors reached an average of 70-80
mm3. In the case of
LL/2 study, tumor cell inoculation and treatment were initiated
simultaneously. Mice were
micro-chipped and registered following tumor implantation. Tumor volume
measurements and
body weights were recorded and mice were randomized into groups that received:
A) mAb
NMC-103 at 0.4 mg/kg (3 times a week for 3 weeks), 2 mg/kg (2 times a week for
3 weeks), or 4
mg/kg (2 times a week for 3 weeks); B) NMC-204 at 0.4 mg/kg (3 times a week
for 3 weeks); C)
isotype control mouse IgG1 (Abcam, Cat. No. ab18447) or IgG3 (Abcam, Cat. No.
ab18392) at
0.4 mg/kg (3 times a week for 3 weeks) or 4mg/kg (2 times a week for 3 weeks);
D) Gemcitabine
(25 mg/kg) and nab-paclitaxel (5 mg/kg) 2 times a week for 3 weeks; or E) a
combination of
gemcitabine (25 mg/kg), nab-paclitaxel (5 mg/kg) and mAb NMC-103 (2mg/kg; 2
times a week
for 3 weeks) or (10mg/kg; 2 times a week for 2 weeks). All tumors were
injected subcutaneously
and all treatments were performed by intraperitoneal injection. Any mouse
displaying prolonged
adverse clinical signs, or with body weight loss exceeding 15% relative to
body weight at day 0,
was euthanized. Tumor measurements from post-treatment with HDM2-specific
antibodies
versus isotype control mouse antibodies versus standard of care treatment
(gemcitabine and nab-
paclitaxel) were made, and results are presented in Figures 16-19.
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[00455] Figure 16 demonstrates the anti-tumor efficacy of mAb NMC-204 (0.4
mg/kg 3 times
a week for 3 weeks; dashed line), in mice that were simultaneously inoculated
with LL/2 Lewis
Lung cancer cells. As presented in the tumor volume measurement graph, by day
21, treatment
with NMC-204 reduced tumor volume (1446 mm3) as compared with treatment with
isotype
control mouse IgG3 (2138 mm3). These results demonstrate the efficacy of an
anti-HDM2-
specific antibody (i.e., mAb NMC-204) that targets extracellularly accessible
epitopes of HDM2
on lung cancer cells. For future patient treatment, the dosing and method of
administration can
be further expanded, e.g., to oral or intravenous delivery of the drug for
optimum effect.
[00456] Figures 17 and 18 demonstrate the efficacy of mAb NMC-103 and NMC-204
against
MC-38 syngeneic mouse model of colon cancer, respectively. As presented in
Figure 17A, mice
treated for 18 days with mAb NMC-103 (0.4mg/kg; 3 times a week for 2.5 weeks;
dashed line)
grew to 1268 mm3 while mice treated with isotype control mouse IgG1 reached a
tumor volume
of 2205 mm3 (solid line). Moreover, immunohistochemical staining for Ki67
protein, a well-
established cell proliferation marker (Li et al., 2015, Mol. Med. Rep.
11(3):1566-72), revealed
that mice treated with mAb NMC-103 had only 5% of tumor cells that stained
positive for Ki67,
while 80% of tumors in mice treated with isotype control antibody stained
positive for Ki67
(Figure 17B). These results further confirm and are consistent with the growth
inhibition result
observed in vitro (Figure 12). Furthermore, the anti-tumor efficacy of another
HDM2-specific
antibody, mAb NMC-204, that recognizes a different extracellularly accessible
segment of
membrane HDM2, namely NMC-P2 (SEQ ID NO:2), was evaluated. As shown in Figure
18A,
tumor volume in mice treated with mAb NMC-204 (0.4mg/kg; 3 times a week for 3
weeks) grew
to 1670 mm3 while tumor in the control antibody-treated group reached 2555 mm3
in volume.
Furthermore, Figure 18B shows that mice treated with NMC-204 had 30% of their
tumor cells
stained positively for Ki67 while mice in the control group had approximately
80% of their
tumor cells staining positively for Ki67, demonstrating the anti-proliferative
effect of NMC-204
treatment on tumor growth.
[00457] The efficacy of HDM2-specific antibody treatment and standard of care
chemotherapy was further compared. Utilizing the Panc-2 syngeneic mouse model
of pancreatic
cancer, Figure 19 demonstrates the synergistic effect of NMC-103 in
combination with
pancreatic cancer standard-of-care treatment drugs: Gemcitabine (G) + nab-
Paclitaxel (nP). As
shown in the tumor volume graph of Figure 19, when tumors in mice reached
approximately 70
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mm3, mice were divided into 4 groups (n=6) that received: A) isotype control
mouse IgG1 (2
mg/kg); B) low dose Gemcitabine (25 mg/kg) and nab-Paclitaxel (5 mg/kg); C)
NMC-103
(2mg/kg); or D) a combination of low dose Gemcitabine (25 mg/kg), nab-
Paclitaxel (5 mg/kg)
and NMC-103 (2mg/kg). All drugs were injected intraperitoneally 2 times a week
for 3 weeks.
After receiving the 6th dose on day 19, treatments were stopped for 2 weeks.
By day 35, mice in
groups that received either isotype control IgG (group A) or a combination of
low dose
Gemcitabine and nab-Paclitaxel (group B) reached an average of 2175 mm3 (open
triangle) and
2314 mm3 (open circle), respectively. Both of these groups had reached
morbidity criteria and
were terminated. By day 35, tumors in mice of group C that received mAb NMC-
103 (2mg/kg)
only reached on average 1523 mm3 (filled triangle), whereas tumors in mice of
group D that had
received a combination of NMC-103 (2mg/kg), Gemcitabine and nab-Paclitaxel
were measured
at an average of 797 mm3 (filled circle). These two groups (groups C and D) of
mice were then
treated with a combination of NMC-103 (10mg/kg), Gemcitabine and nab-
Paclitaxel two times a
week for 2 weeks. As demonstrated in Figure 19, within 2 weeks tumors in both
groups (C and
D) reached the point where no measurable tumor was found.
[00458] The anti-tumor efficacy of NMC-103 and of its combination with
pancreatic cancer
standard of care treatment Gemcitabine (G) and nab-Paclitaxel (nP) was further
evaluated in vivo
utilizing the Panc-2 syngeneic mouse model of pancreatic cancer discussed
above. In this Panc-
2 study, treatment started after Panc-2 cell inoculation and when the tumors
reached 80-100
mm3, with mAb NMC-103 administered at 10 mg/kg, two times a week in a larger
number of
mice (n=8/group) than that used for the study of Figure 19.
[00459] Figure 27 shows the results of a study wherein when Panc-02 tumors
reached 80-100
mm3 in size, mice were randomly divided into 4 groups (n=8/group) that
received: A) isotype
control mouse IgG1 (10 mg/kg); B) low dose Gemcitabine (25 mg/kg) and nab-
Paclitaxel (5
mg/kg); C) NMC-103 (10 mg/kg); or D) a combination of low dose Gemcitabine (25
mg/kg),
nab-Paclitaxel (5 mg/kg) and NMC-103 (10 mg/kg). All drugs were injected
intraperitoneally 2
times a week for 4 weeks. By day 30, mice in group A that had received isotype
control
antibody reached an average tumor size of 2028 mm3 and were terminated. By day
34, half of
the mice in group B that had received low dose of the standard of care G & nP
chemotherapy had
died (n=4) and the tumor in the other half (n=4) had reached an average size
of 1654 mm3. In
contrast, all mice (n=8) in group C that had received NMC-103 were alive with
average
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measurements of 230 mm3 that was due to scard and inflammation at the site of
initial tumor
inoculation. On the other hand, in group D that received NMC-103 in
combination with G + nP,
one mouse had died and the average measurements for the rest of the mice in
that group (n=7)
was 117 mm3 that was due to scars and inflammation at the siste of tumor
inoculation. Both
mice in groups C (n=8) and D (n=7) were then kept for another 4 weeks (62 days
since the start
of the study) with no further drug treatments during which time no sign of
tumor growth beyond
the initial scar size was observed, which demonstrated the lack of tumor in
these mice.
[00460] As shown in Figure 28, a Kaplan Meier survival analysis demonstrated
significant
survival benefit in mice that received NMC-103 alone or in combination with
chemotherapy
when compared to chemotherapy alone or control antibody under the experimental
conditions
described in Figure 27. This study confirms the observations discussed above
in connection with
Figure 19 and the ability of NMC-103 antibody to be used as single arm or in
combination with
chemotherapy.
[00461] To further evaluate the underlying immunological response of the long-
term anti-
tumor effect of NMC-103, at 62 days after the start of the study, mice that
had previously
received a combination of G + nP, mice in group C (mice that had previously
been treated with
NMC-103) and group D (mice that had been treated with a combination of NMC-103
+ G + nP),
as described in Figure 27, were re-challenged by a second round of Panc-2
inoculation
(subcutaneous injection of 2x106 cells/mouse), on the left dorsal flank. Tumor
growth was
monitored for 10 days at which point, a tumor of 90 mm3 (Figure 29) was
measured in the mice
from group B. In contrast, as shown in Figure 29, no tumor was observed in
mice from the two
groups that had previously received NMC-103 antibody (Groups C and D). These
studies
demonstrate the ability of antibodies that target the extracellularly
accessible epitopes of
M(H)DM2/4 to robustly activate the host immune system and provide long-term
anti-tumor
immunity against cancer.
[00462] To further establish the effectiveness of NMC-103 antibody in the
treatment of large
size tumors (i.e. advanced cancers), in another Panc-2 study (with results
shown in Figure 30),
mice were treated with pancreatic cancer standard of care (Gemcitabine (25
mg/kg) + nab-
Paclitaxel (5 mg/kg)) for 19 days at which point the tumor reached a size of
approximately 450
mm3. The mice were then randomly divided in 2 groups that received a single
dose of: A)
isotype control mouse IgG1 (10 mg/kg) or B) NMC-103 (10 mg/kg). As shown in
Figure 30, a
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single i.p. injection of NMC-103 reduced the tumor size by almost half 6 days
post treatment
(from 438 mm3 to 233 mm3).
[00463] To further evaluate the NMC-103 dose-dependent anti-tumor effect, in a
separate
study, the MC-38 syngeneic tumor in mice (n=5) was re-established and
treatment carried out
similarly to the study of Figure 17 with the following modification: increase
of antibody
concentration from 0.4 mg/kg to 10 mg/kg. As shown in Figure 31, mice treated
with NMC-103
at 10 mg/kg, two times per week for three weeks resulted in a greater tumor
reduction when
compared to mice treated with NMC-103 at 0.4 mg/kg (Figure 17). These data
support the dose-
dependent anti-tumor effect of antibodies that bind to extracellularly
accessible epitopes of
M(H)DM2/4.
[00464] Utilizing another syngeneic mouse model of colon cancer, CT-26 (Selby
et al., 2016,
PLoS One. 9;11(9): e0161779), the anti-tumor efficacy of a chimeric version of
monoclonal
antibody NMC-303 was assessed. To create the chimeric version of monoclonal
antibody NMC-
303, isotype class-switching was performed on a mouse NMC-303 (having heavy
variable
regions of SEQ ID NO:40 and light chain variable regions of SEQ ID NO:41) to
convert it from
a mouse IgM to a chimeric IgGl. The mouse Heavy and Light chain variable
regions (SEQ ID
NO:40 and SEQ ID NO:41, respectively) were cloned into a human Ig gamma-1
chain and
human Ig kappa chain as constant region. A total of eight (8) BALB/c mice were
injected
subcutaneously with CT-26 (800,000 cells/mouse). Mice were then divided into
two groups
(n=4) that received: A) control antibody (10 mg/kg) or B) the chimeric version
of monoclonal
antibody NMC-303 (10 mg/kg) two times a week for 3 weeks. Figure 32A shows
that by day 24
post tumor inoculation, mice treated with the chimeric version of monoclonal
antibody NMC-
303 (10 mg/kg) reached an average tumor size of 726 mm3, while mice treated
with control
antibody (10 mg/kg) had an average tumor size of 1746 mm3. Furthermore, Figure
32B shows
the individual mouse tumor sizes on day 24 post tumor inoculation. Due to the
human constant
region of the chimeric version of the monoclonal antibody NMC-303, the anti-
tumor efficacy of
this antibody might be improved if tested in mouse models with human immune
background.
The above in vivo results further support the anti-tumor efficacy of
antibodies raised against
NMC-P3 immunogen that target the extracellularly accessible epitopes of the
M(H)DM2/4 on
cancer cells.
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[00465] Taken together, these results demonstrate two important therapeutic
aspects of
M(H)DM2/4-specific antibodies: First, anti-M(H)DM2/4 antibodies that target
extracellularly
accessible sequences of M(H)DM2/4 are in themselves effective anti-cancer
agents. Second,
M(H)DM2/4 antibodies used in combination with low concentrations of
chemotherapeutics have
a potently synergistic anti-tumor effect. This is particularly important,
considering the frequently
observed side-effects and limitations of chemotherapy used at their clinically
effective
concentrations. In addition, these results demonstrate that M(H)DM2/4
antibodies lead to
development of long-term anti-tumor immunity against cancer as demonstrated by
prevention of
recurrence of cancer and long-term survival of animals previously treated with
M(H)DM2/4
antibodies.
[00466] The in vitro data showed an increase in plasma membrane M(H)DM2/4 when
cells
are treated with various types of chemotherapeutic agents such as Gemcitabine
and Paclitaxel
(data not shown). This treatment potentially makes those cells more
susceptible to anti-HDM2
antibody therapy, which further explains the synergistic effect of their
combinatorial
administration (Figure 19).
8.10 Conclusions based on Examples 1-9:
[00467] Taken together, the data presented herein demonstrate that select
M(H)DM2/4-
specific antibodies that recognize extracellularly accessible segments of
M(H)DM2/4 in cancers
cell have growth inhibitory and cytotoxic effect against a variety of cancers
while sparing normal
untransformed cells. The selective anti-tumor effect of these antibodies is
believed to be due at
least in part to their recognition of extracellularly accessible epitopes of
M(H)DM2/4 protein
variants that are expressed on the surface of cancer cells while the
expression levels of these
M(H)DM2/4 variants on the cell surface of normal cells are low or absent.
[00468] Examples 10-12 below describe data obtained using other anti-HDM2
antibodies that
bind to segments of HDM2 that are extracellularly accessible on cancer cells.
8.11 Example 10: Other HDM2-specific antibodies that also bind to intact cells
from different human cancer cell lines and freshly isolated primary human
cancer cells but not to normal cells.
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[00469] To further extend the therapeutic and diagnostic potentials of other
antibodies raised
against M(H)DM2/4 we evaluated several other available antibodies for their
binding and anti-
tumor activity. As described above, some of these antibodies (i.e.
Calbiochem/Millipore OP-46
and OP-144) did not react with extracellularly accessible epitopes of
M(H)DM2/4,
demonstrating lack of epitope availability on cancer cell membranes,
indicating that not all anti-
M(H)DM2/4 antibodies can be used for the treatment of cancer. However, several
other anti-
M(H)DM2/4 antibodies were shown to not only interact with extracellularly
accessible epitopes
on the cancer cell membrane, but also to have in vitro and/or in vivo anti-
tumor activity.
[00470] The following anti-HDM2 antibodies were used in the experiments
described in
Examples 10-12: (i) polyclonal sc-813, N-20, rabbit IgG, from Santa Cruz
(abbreviated
throughout the specification as "N-20" or "sc-813 (N-20)"); (ii) monoclonal
OP145, mouse
IgGl, from Calbiochem (abbreviated throughout the specification as "OP145");
(iii) monoclonal
0P46 (Ab-1), mouse IgGl, from Calbiochem (abbreviated throughout the
specification as
"0P46"); (iv) monoclonal 0P144 (Ab-4), mouse IgGl, from Calbiochem
(abbreviated
throughout the specification as "OP144"); (v) polyclonal sc-812, C-18, rabbit
IgG, from Santa
Cruz (abbreviated throughout the specification as "C-18" or "sc-812 (C-18)");
and (vi)
monoclonal 965 (SMP14), mouse IgGl, from Santa Cruz (abbreviated throughout
the
specification as "SMP14" or "965 (SMP14)"). Table 10, below, provides
information regarding
the HDM2 recognition sites of these antibodies (i.e., amino acids of HDM2
recognized by these
antibodies), and whether or not these antibodies are cytotoxic to cancer
cells. OP145, N-20, C-
18 and SMP14 were cytotoxic to cancer cells tested, and 0P46 and OP144 were
not cytotoxic to
cancer cells tested.
[00471] Utilizing Fluorescence-activated cell sorting (FACS), the data
presented herein show
that select HDM2-specific antibodies bind to the surface membrane of live
human cancer cells,
human melanoma A2058 cells maintained in culture, and two primary patient-
derived ovarian
cancer cells OVCA-1 and OVCA-4 that had been freshly isolated from ovarian
cancer tissues. In
contrast, FACS analysis of live normal mouse spleenocytes demonstrated the
absence of plasma
membrane staining with the same HDM2-specific antibodies.
[00472] Methodology used: Intact cells released either with either EDTA or
Trypsin were
blocked with 5% human serum albumin. Cells were then incubated with the either
polyclonal
(N20) or monoclonal (0P145) HDM2-specific antibodies for 90 min on ice.
Another set of cells
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prepared under the same conditions were incubated with the same antibodies
that were pre-
incubated with their corresponding blocking peptides before incubation with
cells. Following
primary antibody incubation, cells were washed 3 times with ice-cold PBS
followed by FITC-
secondary antibody incubation for 60 min. Cells were then washed 3 times with
PBS and were
subjected to FACS analyzer.
[00473] Figure 24 presents results of the FACS analysis of human melanoma,
primary ovarian
cancer, and normal mouse spleenocytes. Figure 24A: area under curve #1
represents cells
incubated with goat anti-rabbit secondary antibody only; area under curve #2
represents cells
incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its
blocking peptide
followed by goat anti-rabbit secondary antibody; area under curve #3
represents cells incubated
with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary
antibody.
Figure 24B: area under curve #1 represents cells incubated with goat anti-
mouse secondary
antibody only; area under curve #2 represents cells incubated with anti-HDM2
monoclonal
antibody OP145 pre-incubated with its blocking peptide followed by goat anti-
rabbit secondary
antibody; area under curve #3 represents cells incubated with anti-HDM2
monoclonal antibody
OP145 followed by goat anti-mouse secondary antibody. Figure 24C: area under
curve #1
represents cells incubated with goat anti-rabbit secondary antibody only; area
under curve #2
represents trypsin-released cells incubated with anti-HDM2 polyclonal antibody
N-20 followed
by goat anti-rabbit secondary antibody; area under curve #3 represents EDTA-
released cells
incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its
blocking peptide
followed by goat anti-rabbit secondary antibody; area under curve #4
represents EDTA-released
cells incubated with anti-HDM2 polyclonal antibody N20 followed by goat anti-
rabbit secondary
antibody. Figures 24D & E: area under curve #1 represents cells incubated with
goat anti-rabbit
secondary antibody only; area under curve #2 represents cells incubated with
anti-HDM2
polyclonal antibody N-20 pre-incubated with its blocking peptide followed by
goat anti-rabbit
secondary antibody; area under curve #3 represents cells incubated with anti-
HDM2 polyclonal
antibody N-20 followed by goat anti-rabbit secondary antibody. Figure 24F:
area under curve #1
represents cells incubated with goat anti-rabbit secondary antibody only; area
under curve #2
represents trypsin-released cells incubated with anti-HDM2 polyclonal antibody
N-20 followed
by goat anti-rabbit secondary antibody; area under curve #3 represents EDTA-
released cells
incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its
blocking peptide
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followed by goat anti-rabbit secondary antibody; area under curve #4
represents EDTA-released
cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-
rabbit
secondary antibody.
[00474] EDTA-released intact human melanoma A2058 cells incubated with either
an anti-
HDM2 polyclonal N-20 antibody (Figure 24A; area under the curve #3) or
monoclonal OP145
antibody (Figure 24B; area under the curve #3) show cells that are stained
positive for HDM2 on
their cell surface. To control for epitope-specificity, no specific staining
beyond background was
observed when either one of these antibodies was pre-incubated with its
corresponding blocking
peptide prior to incubation with the cells (see Figures 24A and 24B; area
under the curve #2).
Interestingly, in the case of human melanoma cells released using trypsin,
which cuts the
external portion of cell surface proteins, no cell surface staining of HDM2
was observed (see
Figure 24C; area under the curve #3 when compared with cells released with
EDTA, area under
the curve #4), further indicating the presence of at least parts of the HDM2
protein on the
external face of the plasma membrane. Moreover, freshly isolated tumors from
two patients with
ovarian cancer (OVCA-1 and OVCA-4) showed extensive surface staining when
incubated with
the polyclonal N-20 antibody (see Figures 24D and 24E; area under the curve
#3). This staining
was completely blocked when the antibody was pre-incubated with its blocking
peptide prior to
incubation with the cells (see Figures 24D and 24E; area under the curve #2).
However, intact
mouse normal spleenocytes did not stain over the background with the same
antibodies (see
Figure 24F; compare staining when incubated with HDM2-specific N-20 antibody
in area under
the curve #4, with staining in samples treated with the antibody pre-incubated
with N-20
blocking peptide in area under the curve #3). Taken together, the results of
FACS analysis
strongly indicate the presence of extracellularly accessible epitopes of
M(H)DM2 on the plasma
membrane of the intact cancer cells and its absence in normal untransformed
cells.
8.12 Example 11: Certain HDM2-specific antibodies initiate complement-
mediated cytotoxiciIT against different types of cancers.
[00475] The data presented herein show the ability of select HDM2-specific
antibodies to not
only bind to the surface membrane of cancer cells but also to initiate a
cytotoxic effect in the
presence of fresh normal human serum (NHS). The cytotoxic effect was measured
in various
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cancer cells such as human melanoma, pancreatic, breast and ovarian cancer
cells as well as in
normal human fibroblasts and blood cells.
[00476] Methodology used: Cells seeded in 24-well dishes were grown over night
at 37 C in
a humidified atmosphere supplied with 5% CO2¨ 95% air mixture. The following
day the cells
were thoroughly washed with ice-cold PBS and incubated with various HDM2-
specific
antibodies (i.e., 0P145, N-20 and C-18 antibodies; see description of the
antibodies in Table 10)
or control antibodies (directed to the intra-cellular protein Cytochrome C)
diluted in culture
medium containing 1% BSA to a final antibody concentration of 5-10 ug/ml.
After 30 min, NHS
was added to the cells to make a final concentration of at 1:30 in the culture
medium with 1%
BSA together with 1% propidium iodide (PI) solution. As controls for the role
of complement in
the fresh human serum, parallel cultures treated with HDM2-specific antibodies
were exposed to
fresh human serum that had been incubated for 30 min at 56 C (HiNHS), a
process that is known
to disenable complement activity. Images were taken at 15, 30, 45 and 60 min
post incubation
using Olympus FluoView FV1000 Confocal Laser Scanning Biological Microscope
built on the
Olympus IX81 Inverted Microscope.
[00477] Figure 25 demonstrates the cytotoxic effect of HDM2-specific
antibodies against
human pancreatic and ovarian cancer cells (Figure 25A), and rodent pancreatic
cells (Figure
25B). 0P145, N-20 and C-18 antibodies in the presence of NHS trigger
complement-mediated
cytotoxicity in cancer cells incubated with such antibodies, resulting in the
death of the cancer
cells as evident by the nuclear uptake of the cell-death marker Propidium
Iodide (PI) (see Figure
25A, panels b and e, for ovarian cancer cells incubated with OP145 and Figure
25B, panels b and
c, for pancreatic cancer cells incubated with N-20 and C-18, respectively). In
the presence of
control antibodies (i.e., anti-cytochrome C antibody, see Figure 25A, panel c,
and Figure 25B,
panel e) cancer cell death is similar to that of control NHS alone, without
any antibodies (see
Figure 25A, panels a and d, and Figure 25B, panel a) or normal cells
(Fibroblasts, Figure 25A,
panel g) treated with HDM2-specific antibodies (i.e., there is no or minimal
cell death as
indicated by lack of PI staining, se Figures 25A, panels a, c, d, f and g, and
Figure 25B, panel 1).
Further, no cytotoxicity was observed when cells were treated with anti-HDM2
monoclonal
0P46 antibody (see Figure 25B, panel d). Figure 25C provides quantitative
representations of
HDM2-specific antibody-dependent complement cytotoxicity against human
pancreatic cancer
cells. Cells treated with anti-HDM2 (C-18) antibody in the presence of NHS
demonstrated
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cytotoxicity over 15-30 min. post-treatment, whereas anti-HDM2 0P46 shows no
cytotoxic
effect beyond that observed when cells were treated with control anti-
Cytochrome C antibody or
when cells were left untreated in the presence of NHS.
[00478] Moreover, HDM2-specific antibodies in the presence of heat-inactivated
human
serum (i.e., fresh human serum that had been incubated for 30 min at 56 C
(HiNHS)) did not
have any cytotoxic effect on cancer cells (i.e., did not result in the death
of the cancer cells as
was evident by PI staining), demonstrating that the cytotoxic effect is due to
complement
activity.
[00479] Table 10 lists antibodies tested by the inventors and summarizes
results obtained
relating to the in vitro cytotoxic effects of various anti-HDM2 antibodies
against cancer cells.
Table 10: Antibodies used and their ability to induce a cytotoxic effect in
cancer cells in vitro.
M(H)DM2 Abs HDM2 Cytotoxicity Species Host Antibody TypE
Company
Recognition Site Reactivity* Purchased
Amino Acid Nos. From
of SEQ ID NO:4
0P46 (Ab-1) 26-169 Mouse IgG1 Monoclonal
Calbiochem
0P144 (Ab-4) 153-222 H & M Mouse IgG1 Monoclonal
Calbiochem
0P145 19-50 H & M Mouse IgG1 Monoclonal
Calbiochem
sc-813 (N-20) N-terminus H & M Rabbit IgG Polyclonal
Santa Cruz
sc-812 (C-18) C-terminus H, M, R Rabbit IgG Polyclonal
Santa Cruz
965 (SMP14) 154-167 H, M, R Mouse IgG1 Monoclonal Santa
Cruz
Control
Antibodies
Cytochrome C N/A Mouse IgG1 Monoclonal Santa
Cruz
Histone H4 N/A Broad Mouse Monoclonal Santa
Cruz
Species IgG2,
Beta-Tubulin N/A H, M, R Rabbit IgG Polyclonal
Santa Cruz
* H¨binds to human protein; M¨binds to murine protein; R¨binds to rabbit
protein
8.13 Example 12: Results regarding in vivo anti-tumor effect of other
antibodies
that bind to extracellularlv accessible epitopes of HDM2.
[00480] To further evaluate the anti-cancer activity of mAbs raised against
extracellularly
accessible epitopes of HDM2 (other than NMC-P1, NMC-P2 and NMC-P3) on cancer
cells,
studies of the anti-tumor efficacy of anti-HDM2 monoclonal antibody OP-145
(Calbiochem) and
"MDM2 monoclonal antibody (M01), clone 1A7" (Abnova, Cat. No. H00004193-M01)
were
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conducted. As demonstrated above, neither one of these antibodies showed
reactivity to NMC-
P1 and NMC-P2 (see Figures 14A-B), but both of these antibodies showed
reactivity with intact
cancer cells (see Figure 15A and 24B).
[00481] Evaluation of anti-tumor effects of antibody 0P145 on pancreatic tumor
growth
in vivo: Subcutaneous tumors were prepared by implanting Panc02 cells (2x106
per mouse) in
the right flanks of female 8-week-old C57BL/6 mice. The mice were returned to
their respective
cages after tumor implantation. From day 7 after tumor implantation, the tumor
volumes were
measured 2 times/week with a digital caliper. Treatment started when the
tumors reached an
average of 200 mm3 (see Figure 26, day 14). Mice were micro-chipped and
registered following
tumor implantation. Tumor volume measurements and body weights were recorded
and mice
were randomized into two groups that received either OP145 antibody (by intra-
tumoral injection
of 0.1 mg/kg of mouse monoclonal antibody 0P145,3 times per week) or PBS.
Three mice were
treated with 0P145, and five mice were treated with PBS. Any mouse displaying
prolonged
adverse clinical signs, or with body weight loss exceeding 15% from Day 0 body
weight, was
euthanized. Tumor measurements from the first 10 days post treatment with
OP145 antibody
versus PBS were made, and results are represented in Figure 26.
[00482] It was found that subcutaneous tumors in mice administered the OP145
antibody had
a size of 260 mm3 at 21 days post tumor cell injection, whereas subcutaneous
tumors in mice
administered PBS had a size of 375 mm3 (see Figure 26).
[00483] In vivo studies of the effect of "MDM2 monoclonal antibody (MOO, clone
1A7"
against MC-38 syngeneic mouse model of colon cancer (using 0.4 mg/kg of the
MO1, clone
1A7 antibody; 3 times a week for 3 weeks, intraperitoneally; n=4) demonstrated
that while the
antibody-treated mice initially responded to the treatment (average tumor size
after 6 doses was
at 1588 mm3 in antibody-treated group vs 1999 mm3 in isotype control-treated
group), tumors in
both control and antibody-treated mice reached approximately 2500 mm3 by third
week (Figure
23). During the same time period, mAb NMC-103 and NMC-204 given to mice at the
same dose
(0.4 mg/kg; 3 times a week for 3 weeks, intraperitoneally) demonstrated a
significant (p<0.005)
reduction in tumor volume with lasting anti-tumor activity (Figure 19; open
circle and open
square, respectively; also see Figure 17A and 18A). This finding indicates
that although mAb
MO1 had strong binding to cells in vitro, the anti-tumor effect of NMC-103 and
NMC-204 was
superior to that of mAb MO1 (Figure 23).
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[00484] It must be noted that these results relating to in vivo anti-tumor
efficacy of
monoclonal antibodies OP-145 and MO1 must be interpreted with caution since
the commercial
antibody preparations used may not necessarily have the desired clonality,
purity or
pharmaceutically acceptable formulation for in vivo efficacy testing.
9. EXAMPLES 13-18
[00485] The data presented in Examples 13-18 herein shows the binding of
humanized and
chimeric antibodies to extracellularly accessible epitopes NMC-P1 and NMC-P3
of plasma
membrane-bound M(H)DM2 to intact, non-permeabilized cancer cells, as
demonstrated by the
binding epitope availability of plasma membrane markers (i.e. E-Cadherin) and
the absence of
binding of antibodies to intracellular markers (Cytochrome-C, Cyclin D1 and
Bc12).
[00486] In addition, data presented herein shows reactivity of humanized and
chimeric
antibodies to extracellularly accessible epitopes NMC-P1 and NMC-P3 of plasma
membrane-
bound M(H)DM2 in several human and rodent cancer cell lines as well as primary
human
tumors.
[00487] Furthermore, the binding of humanized and chimeric antibodies to
extracellularly
accessible epitopes NMC-P1 and NMC-P3 of plasma membrane-bound M(H)DM2 is
reduced
when the binding of the antibodies is competed with their respective
immunogenic peptides.
Moreover, antibody-antigen binding is abolished when surface proteins are
enzymatically
digested in otherwise intact cells, demonstrating the extracellular
availability of the binding
epitopes.
[00488] The in vivo anti-tumor activity of the humanized and chimeric
antibodies was also
tested. Data presented here demonstrates the in vivo anti-tumor activity of
humanized and
chimeric antibodies to plasma membrane-bound M(H)DM2 and show that the anti-
cancer
efficacy is dependent on stimulation of the immune system.
[00489] Finally, in vitro and in vivo data presented here demonstrate, for the
first time, the
correlation between MDM2 gene amplification observed in hyper-progressive
patients under
immunotherapy and increased expression of plasma membrane-bound M(H)DM2 on the
surface
of the hyper-progressive tumors. This observation further demonstrates the
rationale for the
therapeutic utilization of antibodies that target extracellularly accessible
epitopes of plasma
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membrane-bound M(H)DM2 for treatment of patients who exhibit hyper-progression
of cancer
in response to administration of an immunotherapy.
Antibodies used in Examples 9-13
[00490] The following anti-HDM2 antibodies were used in the experiments
described in
Examples 13-18: (i) chimeric NMC-103 ("NMC-C103.VHO/VKO" or "NMC-C103")
monoclonal antibodies, (ii) humanized variants of NMC-C103 ("Humanized NMC-
103" or
"NMC-H103"), and (iii) chimeric NMC-303 ("NMC-C303") monoclonal antibodies as
described
below. Chimeric NMC-103 ("NMC-C103.VHO/VKO" or "NMC-C103") monoclonal
antibodies
were generated by replacing the constant region of mouse IgG1 monoclonal
antibody NMC-103
with a human IgG1 constant region. As described above, a monoclonal antibody
NMC-103 is an
antibody that binds to NMC-P1 (SEQ ID NO:1) (it is one of the NMC-100s series
of antibodies).
Humanized variants of NMC-C103 ("Humanized NMC-103" or "NMC-H103") were then
designed comprising combinations of the following variable heavy and light
chains: VHO, VH1,
VH2, VH3, VH4, VH6, VH7 and VKO, VKl, VK2, VK3, VK4, VK5, VK6, VK7,
correspondingly (see Tables 13-27 and SEQ ID NOs: 283, 287, 291, 295, 299,
305, 309 and 285,
289, 293, 297, 301, 303, 307, 311, respectively). Immunoglobulin class
switching was used to
generate chimeric NMC-303 ("NMC-C303") monoclonal antibodies, where the
constant region
of NMC-303 mouse IgM monoclonal antibody underwent isotype-switching with
human IgG1
constant region (Tables 11 and 12 and SEQ ID Nos: 40 and 41). As discussed
above, a
monoclonal antibody NMC-303 is an antibody that binds to NMC-P3 (SEQ ID NO:3)
(it is one
of the NMC-300s series of antibodies). The heavy chain/light chain framework
region
sequences, complementarity determining region (CDR) sequences, and variable
region
sequences of these antibodies are listed in Section 11, below.
Methods of Making of Antibodies used in Examples 9-13
[00491] Generation of humanized and chimeric antibodies: Design of Composite
Human
AntibodyTM Variable Region Structural models of the murine antibody variable
(V) regions were
produced using Swiss PDB and analyzed in order to identify important
"constraining" amino
acids in the V regions that were likely to be essential for the binding
properties of the antibody.
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Most residues contained within the CDRs (using both Kabat and Chothia
definitions) together
with a number of framework residues were considered to be important. The VH
and VK
sequences of murine mAb contain typical framework residues and the CDR 1, 2
and 3 motifs are
comparable to many murine antibodies. From the above analysis, it was
considered that
Composite Human sequences of mAbs could be created with a wide latitude for
alternative
residues outside of the CDRs but with only a narrow menu of possible residues
within the CDR
sequences. Preliminary analysis indicated that corresponding sequence segments
from several
human antibodies could be combined to create CDRs similar or identical to
those in the murine
sequences. For regions outside of, and flanking the CDRs, a wide selection of
human sequence
segments were identified as possible components of the novel humanized V
regions.
[00492] CD4+ T Cell Epitope Avoidance: Based upon the structural analysis, a
large
preliminary set of sequence segments were identified that could be used to
create humanized
variants. These segments were selected and analyzed using iTopeTm technology
for in silico
analysis of peptide binding to human MEW class II alleles (Perry et al., 2008)
and using the
TCEDTm of known antibody sequence-related T cell epitopes (Bryson et al.,
2010). Sequence
segments that were identified as significant non-human germline binders to
human MHC class II
or that scored significant hits against the TCEDTm were discarded. This
resulted in a reduced set
of segments, and combinations of these were again analyzed, as above, to
ensure that the
junctions between segments did not contain potential T cell epitopes. Selected
sequence
segments were assembled into complete V region sequences that were reduced in
significant T
cell epitopes. Six heavy chain (VH1 to VH4, VH6, and VH7) and seven light
chain (VK1 to
VK7) sequences were chosen for gene expression in mammalian cells.
[00493] Construction, Transfection and expression of Chimeric Antibody and
Humanized variants: The VH and VK sequences of murine mAb together with the
humanized
variants were synthesized with flanking restriction enzyme sites for cloning
into expression
vector system for human IgG1 heavy and kappa light chains. All constructs were
confirmed by
sequencing. DNA constructs encoding chimeric antibodies and humanized antibody
variants
having combinations of various heavy and light chain sequences were initially
transiently
transfected into CHO cells using a MaxCyte STX. Electroporation system
(MaxCyte Inc.,
Gaithersburg, USA). Transfections were undertaken for each antibody using OC-
400 processing
assemblies. Following cell recovery, cells were pooled and diluted to 3 x106
cells/mL in CD
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Opti-CHO medium (ThermoFisher, Loughborough, UK) containing 8 mM LGlutamine
(ThermoFisher, Loughborough, UK) and lx Hypoxanthine-Thymidine (ThermoFisher,
Loughborough, UK). Twenty four hours post transfection, the culture
temperature was reduced
to 32AdC and 1 mM sodium butyrate (Sigma, Dorset, UK), 30% volume of CD-
Efficient Feed B
(ThermoFisher, Loughborough, UK), and 3.3% Volume of FunctionMax Titre
Enhancer was
added. An additional 15% (of the total volume) of CD Efficient Feed B and
1.65% FunctionMax
titre enhancer was added at day 7 post transfection. Culture supernatants were
harvested around
10-12 days post transfection.
[00494] Purification of humanized and chimeric antibodies: Humanized and
chimeric
variant antibodies were purified from cell culture supernatant using a 1 mL
Mab Select Sure
column (GE Healthcare, Little Chalfont, UK). The column was washed using 1 x
DPBS and
protein eluted using 0.1 M sodium citrate pH 3Ø Collected fractions were
then pooled, buffer
exchanged into PBS pH 7.4 and filter sterilized. Purified antibodies were then
quantified by
OD280nm using an extinction coefficient (Ec (0.1%)) based on the predicted
amino acid
sequence and analyzed by reducing SDS-PAGE.
Antibodies and reagents used in cell-based ELISA, Flow cytometry and in-vivo
studies
[00495] The following commercial primary antibodies were used in the cell-
based ELISA
experiments: Cytochrome C (Santa-Cruz 13156), a-Bc12 N19 (SC-492), a-cyclin D1
(SC- 717),
E-cadherin G10 (SC-8426). Secondary anti-mouse (A24524, Thermo-fisher) and
Secondary anti-
human (62-8420, Thermo-fisher) with HRP conjugate were used as secondary
antibodies. TMB
substrate (34028, Thermo scientific) and Stop solution (cat. No SS04, Life
Technologies) was
used in ELISA assay for development of chemoluminescence.
[00496] For flow cytometry, an anti-mouse fluorescently labeled Secondary
antibody
(Biolegend 405307), Anti-human fluorescently labeled Secondary antibody (Life
technologies
H10104) and 7-AAD (420404, Biolegend,) were used.
[00497] For in-vivo studies, the following antibodies were used: anti-mouse PD-
1 antibody
(BioXCell, Cat. No. BE0146) and mouse isotype control antibody (BioXCell
BP0083) and
human isotype control (BioXCell, Cat. No. BP0297). Human PBMCs were purchased
from Stem
Cell Technologies (Cat. No. 70025).
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Cell-Based ELISA
[00498] In a 96-well microplate, 15,000 cells/well were cultured overnight.
The next day,
unbound cells were washed off with sterile lx PBS. The cells in each well were
fixed with
freshly prepared 4% buffered paraformaldehyde (pH 7.2) for 1 hour, followed by
3 washes with
lx PBS. The wells were then blocked with 5% BSA in PBS (100W/well) for 2 hours
at room
temperature. Microplate wells were then washed 5 times with 3001_11 of ice
cold lx PBS.
Monoclonal antibodies NMC-H103 (VH4/VK3) or NMC-C303 at 51.tg/mL in 1% BSA/PBS
were then incubated with various cancer cells for 2 hours at room temperature.
Wells were then
washed with 3001_11 of ice-cold lx PBS for 5 times and corresponding secondary
antibody HRP-
GaH F(ab')2 diluted 1:2000 or 1:4000 in PBS with 1% BSA were added at100
1/well for 1 hour
at room temperature followed by washing 5 times with 3001_11 of ice-cold lx
PBS. TMB
Substrate Solution (1-StepTM Ultra TMB-ELISA, ThermoFisher, Cat. No. 34028)
was then
added at 50 [IL to each microplate well and incubated at room temperature for
30 minutes. The
reaction was stopped by addition of 50 [IL of stop solution (ThermoFisher,
Cat. No. SS04) to
each well and absorbance of each well was measured at 0D450 nm. The absorbance
value of
each experimental well was corrected for the absorbance value obtained from
wells treated with
isotype-identical mAbs included in each experiment.
Flow cytometry
[00499] Cells that had been allowed to grow to about 80% confluency in
75cm2 tissue culture
flasks were released with either EDTA (10 mmols; pH 7.2; 5 min, 37 C) or
Trypsin (Gibco
TrypLE Express), washed several times with ice-cold PBS, and blocked for 20
minutes on ice
with 5% human serum albumin in PBS (intact, non-permeabilized cells). After
establishing
viability, the cell number was adjusted to 106 cells/ml and kept in ice-cold
PBS until staining.
Cells were then incubated with primary antibodies NMC-H103 (VH4/VK3) or NMC-
C303
(11.tg/m1) for 30 minutes on ice. Following primary antibody incubation, cells
were washed 3
times with ice-cold PBS, followed by incubation with goat anti-human PE
labeled secondary
antibody (Life technologies H10104) on ice for 20 minutes. Cells were then
washed 3 times
with PBS and incubated with 7AAD (5 1/500mL of buffer) before flow cytometry
analysis using
Fortessa 5.
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MTT Cell Proliferation assay
[00500] Cells were seeded in a 96-well plate at a density of 1000 cells/well
in Serum free
Media (Sigma Lifesciences Cat. No. S9388) and were simultaneously treated with
anti-mouse
PD-1 (BioXcell Cat. No. CD279) at the concentration of 75 g/m1 and 100 g/m1
and IgG control
(Abcam Cat. No. ab18443) at 100 g/ml. Cells were grown at 37 C for 48 hours in
a humidified
CO2 incubator. On the 2nd day of incubation, 15111 of MTT Dye Solution
(Promega Cat. No.
G402A) was added to each well. The plate was incubated at 37 C for 3 hours in
a humidified
CO2 incubator. 100111 of Solubilization/Stop Solution (Promega Cat. No. G401A)
was then
added to each well. Plates were then read at absorbance at 570nm using a 96-
well plate reader
(BioTek).
In vivo studies
[00501] Subcutaneous tumors were prepared by implanting either mouse colon MC-
38 cells
(5x105 cells/mouse), human lung cancer A549 cells (5x106 cells/mouse) or mouse
lung cancer
M109 cells (0.5x105 cells/mouse) in the right flanks of NOD scid gamma mice
(NSG: NOD.Cg-
Prkdcsc1dIl2rg"iwil/SzJ) purchased from Jacksons Laboratory. From day 7 after
tumor
implantation, the tumor volumes were measured 2 times/week with a digital
caliper. In the case
of mouse colon MC-38 cells and human lung cancer A549 cells study in NSG mice,
once tumors
reached an average volume of 300-400 mm3, mice were randomly divided into 3
groups (n=4)
and 7x106 human PBMCs (Stem Cell Technologies Cat # 70025) was injected into
the tail vein
of mice in groups 1 and 3. Seven days later, in the case of mouse colon MC-38
cells study, mAb
NMC-C303 antibody (10mg/kg) was injected intraperitoneally (i.p.) to mice in
groups 2 and 3
and human isotype control antibody (BioXCell BP02) into mice in group 1 on
days 33 and 35
post tumor inoculation. In the case of human lung cancer A549 cells study,
seven days post
PBMC injection, mice in group 2 and 3 received 1 intraperitoneal injection of
mAb NMC-H103
(VH4/VK3) (10mg/kg) while mice in group 1 received 1 injection of human
isotype control
antibody (BioXCell BP02) and tumors were measure 5 days later. In the case of
mouse lung
cancer M109 cells study, post subcutaneous injection of M109 cells, NSG mice
were randomly
divided into 2 groups. Group 1 mice received mouse isotype control antibody
(12.5 mg/kg; once
a week), while mice in group 2 received mouse anti-PD1 antibody (12.5 mg/kg;
once a week)
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intraperitoneally or 5 weeks at which point tumors from mice in both groups
were harvested for
flow cytometry analysis.
Preparation of single-cell suspension of tumors
[00502] Preparation of single-cell suspension of the tumors harvested from
mice was
performed using MACS Tumor Dissociation Kit Mouse (Cat. no. 130-096-730) and
manufacturer's protocol. Briefly, enzyme mix was prepared by adding 2.35 mL of
RPMI, 100
[IL of Enzyme D, 50 [IL of Enzyme R, and 12.5 [IL of Enzyme A into a gentle-
MACS C Tube.
Tumors were cut into pieces of 2-4 mm and transferred to gentle-MACS C Tune
containing the
enzyme mix and attached to gentle-MACS Dissociator. A pre-fix program was ran
and samples
were incubated for 40 minutes at 37 C with continuous rotation using the
MACSmix Tube
Rotator. Tubes were re-suspended and the cell suspension was transferred to a
MACS
SmartStrainer (70 Ilm) placed on a 15mL tube. Samples were washed with 10mL of
RPMI 1640
followed by centrifugation at 300xg for 7 minutes. Supernatant was then
completely removed
and the resulting single cells were re-suspended into Flow Cytometry staining
buffer to be
stained with desired antibodies.
9.1 Example 13: Humanized and Chimeric antibodies to extracellularly
accessible epitopes NMC-P1 and NMC-P3 of the membrane-bound
M(111DM2 bind to the surface of intact cancer cells.
[00503] In Example 13, data of cell-based enzyme-linked immunosorbent assay
(ELISA)
experiments are presented showing that humanized mAb NMC-103 (NMC-H103;
VH4/VK3)
and chimeric mAb NMC-303 (NMC-C303) (both at 5 g/mL) bind to intact mouse
LL/2 cancer
cells (average OD reading of 3.51m and 3.59nm, respectively) (Figure 33).
[00504] Results of the cell-based ELISA experiments showed that antibodies
against intra-
cellular proteins such as cytochrome-C, cyclin D1 and Bc1-2 (all at 51.tg/mL)
showed no binding
to intact mouse LL/2 cancer cells beyond background staining with secondary
antibody only
(OD < 0.5nm) (Figure 33), while an antibody against the plasma membrane
protein E-Cadherin
(51.tg/mL) bound to its target (average OD reading of 1.49 nm) (Figure 33),
demonstrating intra-
cellular target inaccessibility to antibodies in intact cells. The large size
and ionic nature of
antibodies preclude them from gaining intra-cellular access without membrane
permeabilization
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(Muller S. et al, 2005). Considering the fact that antibodies are unable to
cross the cell membrane
of intact, un-permeabilized cells, antibodies to intra-cellular proteins are
unable to bind their
intra-cellular targets, while antibodies to surface proteins can bind their
targets.
[00505] As shown in Figure 33, similarly to the results shown in connection
with plasma
membrane protein E-Cadherin, mAb NMC-H103 (VH4/VK3) and mAb NMC-C303 (NMC-
C303) bound to intact mouse LL/2 cancer cells, demonstrating that both of
their target epitopes
(i.e. NMC-Pi and NMC-P3) are extracellularly accessible on intact mouse LL/2
cancer cells.
9.2 Example 14: Humanized and Chimeric antibodies to extracellularly
accessible epitopes NMC-P1 and NMC-P3 of the plasma membrane-bound
M(H)DM2 bind to various intact. un-permeabilized human and murine
cancer cells.
[00506] Example 14 presents data of cell-based ELISA demonstrating that
humanized mAb
NMC-H103 (VH4/VK3) and chimeric mAb NMC-C303, which were raised against
extracellularly accessible NMC-P1 and NMC-P3 epitopes of plasma membrane-bound
M(H)DM2, respectively, bind to intact, un-permeabilized human (Figure 34A) and
murine
(Figure 34B) cancer cells.
[00507] As shown in Figure 34A, cell-based ELISA experiments demonstrated the
binding of
mAb NMC-H103 (VH4/VK3) (5 g/mL) and mAb NMC-C303 (5 g/mL) to following human
cancer cells: A2085 melanoma cells, MCF-7 breast cancer cells, MIAPaCa-2
pancreatic cancer
cells, HeLa cervical cancer cells, OVCA-4 ovarian cancer cells,and OVCAR-3
ovarian cancer
cells. In addition, as shown in Figure 34B, cell-based ELISA experiments
demonstrated the
binding of mAb NMC-H103 (VH4/VK3) (5 g/mL) and mAb NMC-C303 (5 g/mL) to the
following mouse cancer cells: MC-38 colon cancer cells and Panc-2 pancreatic
cancer cells. No
binding beyond background staining was shown using anti-human secondary
antibody.
[00508] These data show the ability of mAb NMC-H103 (VH4/VK3) and NMC-C303 to
react
with extracellularly accessible epitopes of the plasma membrane-bound M(H)DM2
on intact
human and rodent cancer cells.
9.3 Example 15: Humanized and Chimeric antibody's binding to
extracellularly
accessible epitopes NMC-P1 and NMC-P3 of the plasma membrane-bound
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M(H)DM2 on human and murine cancer cells is specific and can be
competed with epitope-specific peptides and reduced by enzymatic digestion
of surface target antigen.
[00509] To evaluate the specificity of humanized mAb NMC-H103 (VH4/VK3) to
plasma
membrane-bound M(H)DM2, a competition experiment with the mAb's immunogenic
peptide
was performed. Figure 35A shows data from cell-based ELISA experiments
demonstrating the
binding of NMC-H103 (VH4/VK3) to intact, un-permeabilized mouse Lewis Lung
LL/2 cancer
cells with an OD reading of 3.48nm, while pre-incubation of NMC-H103 (VH4/VK3)
(5 g/mL)
with its specific immunogenic peptide (NMC-P1 at x10 the concentration of
mAbs), reduced the
binding efficiency of mAb NMC-H103 (VH4/VK3) to its target (OD = 1.09nm). In
contrast,
pre-incubation of NMC-C303 with NMC-P1 had no effect on its binding to LL/2
cells, as mAb
NMC-C303 has a different immunogenic peptide sequence (i.e. NMC-P3).
[00510] To distinguish live intact cells from permeabilized or dead cells,
7-
Aminoactinomycin D (7-AAD), a fluorescent chemical compound with a strong
affinity for
DNA, was used (Paterson AM et al, 2011). Figure 35B shows results from flow
Cytometry
experiments on live (7AAD negative) cells demonstrating the binding of mAb NMC-
H103
(VH4/VK3) and NMC-C303 (solid black curve) to live OVCAR-3 (upper panel) and
LL/2 cells
(lower panel). In contrast, when OVCAR-3 and LL/2 cells were briefly treated
with digestive
enzyme Trypsin, while remaining alive (7AAD negative), the binding of mAb NMC-
H103
(VH4/VK3) and NMC-C303 was reduced (Dashed line shifted back to that of
background
secondary Ab only), indicating loss of antibody binding epitope. These data
demonstrate the
specificity of these antibodies to their extracellularly accessible epitopes
of the plasma
membrane-bound M(H)DM2 on human ovarian cancer OVCAR-3 and mouse lung cancer
LL/2
cells.
9.4 Example 16: Anti-tumor effect of antibodies to the plasma membrane-
bound M(H1DM2 on cancer cells. which is in part dependent on activation
of the immune system.
[00511] Anti-cancer activity of humanized and chimeric antibodies to
extracellularly
accessible epitopes NMC-P1 and NMC-P3 of the plasma membrane-bound M(H)DM2 on
cancer
cells, was investigated utilizing NOD scid gamma mice (NSG: NOD.Cg-
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PrkdcscidIl2rgtmlWjl/SzJ). As described by the manufacturer (The Jackson
Laboratory), these
mice lack B-cell, T-cells, NK cells and complement and have defective
dendritic cells and
macrophages (Sultz LD et al, 2005). Utilization of NSG immune-deficient mice
allows for
engraftment of human peripheral monocytes (Hu-PBMSCs), which would enable
testing of
humanized and chimeric antibodies.
[00512] As shown in Figure 36A, mouse colon cancer MC-38 cells were
subcutaneously
inoculated in NSG mice (n=12) and allowed to reach a tumor volume of 400 mm3
at which point
they were randomly divided into 3 groups (n=4 mouse/group). Normal Hu-PBMCs
(7x106
PBMCs/mouse) were injected in tail vein of groups 1 and 3 mice, whereas mice
in group 2 did
not receive any Hu-PBMCs. A week later (tumors volume of 600 mm3), mice in
group 1
received intraperitoneal injection of isotype control antibody (10 mg/kg),
while mice in groups 2
and 3 received NMC-C303 (10 mg/kg). As shown in Figure 36, mAb NMC-C303 was
most
effective in group 3 mice, which had PBMC (tumor volume of 1,492 mm3) as
compared to mice
in group 2, which received NMC-C303 but had received no Hu-PBMCs (tumor volume
of 2,722
mm3). These observations demonstrate an immunological role for antibodies
against plasma
membrane-bound M(H)DM2 on cancer cells in suppression of tumor growth.
[00513] Figure 36B demonstrates the immune-system's involvement in the anti-
tumor activity
of humanized NMC-H103 (VH4/VK3). As shown in Figure 36B, human lung cancer
A549 cells
were subcutaneously injected into NSG mice (n=12) and were allowed to reach a
tumor volume
of 300 mm3 at which time mice were randomly divided into 3 groups (n=4 per
group). Normal
Hu-PBMCs (7x106PBMC/mouse) were injected in tail vein of mice in groups 1 and
3, whereas
mice in group 2 did not receive any Hu-PBMCs. Mice in group 1 received 1
intraperitoneal
injection of isotype control antibody (10mg/kg), while mice in groups 2 and 3
received 1 intra-
peritoneal injection of NMC-H103 (VH4/VK3) (10mg/kg). As shown in Figure 36B,
mice
treated with 1 dose of mAb NMC-H103 (VH4/VK3) in presence of Hu-PBMCs had an
average
tumor volume of 784 mm3, while mice treated with 1 dose of mAb NMC-H103
(VH4/VK3) in
the absence of Hu-PBMCs had an average of tumor volume of 1367 mm3,
demonstrating that the
anti-cancer activity of NMC-H103 (VH4/VK3) is significantly more effective in
presence of Hu-
PBMCs (Figure 36B, black bar) than in the absence of Hu-PBMCs (Figure 36B,
middle bar),
indicating an immune-assisted mechanism of action.
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9.5 Example 17: Tumor hyper-progression under immunotherapy results in
an
increase in the expression of plasma membrane-bound M(H)DM2 on the
surface of the hyper-progressive tumors.
[00514] Recent observations in patients undergoing immune checkpoint inhibitor
(ICI)
treatment has identified a patient sub-population whose tumors hyper-
progressed during or after
treatment (Champiat, et al., Clin. Cancer Res. 23, 1920-1928 (2017)). Lung
cancer M109 cells
exhibit similar phenotype upon treatment with ICI (Shisuo Du et at.,
Oncoimmunology 2018;
7(4): e1408747).
[00515] Figure 37A demonstrates the in vitro MTT cell proliferation assay of
M109 cells
treated with 75 ug/mL or 100 ug/mL of anti-PD1 antibody for 48 hours as
described above. As
shown in the bar graphs, a dose-dependent increase in cell proliferation is
observed when cells
are incubated with anti-PD1 antibody as compared to cells incubated with
isotype control
antibody at 100 ug/mL, demonstrating M109 cell proliferation in presence of
anti-PD1 antibody.
[00516] Figure 37B demonstrates the in vivo effect of anti-PD1 antibody on the
growth of
M109 tumor. NSG mice (n=8) were subcutaneously inoculated with M109 cells
(50,000
cells/mouse). Two weeks later, mice were randomly divided in two groups. Mice
in group 1
received isotype control antibody (12.5 mg/kg; xl/week), while mice in group 2
received anti-
PD1 antibody (12.5 mg/kg; xl/week). As shown in Figure 37B, after 3 weeks of
treatment mice
treated with anti-PD1 antibody (group 2) start to experience an increase in
their tumor growth as
compared to mice that received isotype control antibody (group 1), further
confirming the results
observed in vitro (Figure 37A). Tumors from mice in both group 1 and 2 were
harvested from
sacrificed mice and assessed for the expression of plasma membrane-bound
M(H)DM2.
[00517] Figure 37C demonstrates results from flow cytometry experiments on
freshly excised
tumors from mice treated with isotype control or anti-PD1 antibodies (groups 1
and 2 discussed
above). Single-cell suspension of tumors was performed as described above.
Flow cytometry
data has demonstrated more than 25% increase in the binding of NMC-H103
(VH4/VK3) and
NMC-C303 to hyper-progressive tumors. As shown in Figure 37C, an increased
binding of
NMC-H103 (VH4/VK3) was observed in hyper-progressive tumors that received anti-
PD1 as
compared to isotype control treated mice in group 1.
[00518] These observations demonstrate the correlation between the previously
reported
MDM2 gene amplification in hyper-progressive patients under immunotherapy
(Kato et at., Clin.
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Cancer Res. 23, 4242-4250 (2017) and increased plasma membrane-bound MDM2 that
could be
targeted with antibodies to key accessible epitopes.
9.6 Example 18: Binding of Several Humanized mAb NMC-H103 Variants to
NMC-P1.
[00519] Example 38 presents data demonstrating the binding of multiple
humanized mAb
NMC-H103 variants to NMC-P1. In this Example, purified humanized variants of
NMC-H103
antibody were tested by Biacore single cycle kinetic analysis for binding to
NMC-P1, loading to
a lower immobilization level (RL) of - 3600 RU, the theoretical value to
obtain an Rmax of
about 75 RU to reduce the amount of antibody used. The surface was then
allowed to stabilize.
A three point, two-fold dilution range from 18.75 nM to 75 nM NMC-P.1 without
regeneration
between each concentration was used. The association phase for the three
injections of
increasing concentrations of NMC-P1 was monitored for 200 seconds each time
and a single
dissociation phase was measured for 200 seconds following the last injection.
Regeneration of
the Protein A surface was conducted using two injections of 10 mM glycine-HCL
pH 1.5. Data
was fitted using a 1-to-1 binding model. The sensorgrams and fitted data for
the single cycle
kinetics are shown in Figure 38A and for multi-cycle kinetic analysis in
Figure 38B.
[00520] Figure 38A presents the single cycle binding kinetics of humanized
antibodies to
NMC-P1, wherein the humanized antibodies comprise a combination of VH1, VH2,
VH3, or
VH4 humanized heavy chain variable region with VKl, VK2, VK3, VK4, or VK5
humanized
light chain variable region. Figure 38B presents multi-cycle binding kinetics
of humanized
antibodies to NMC-P1, wherein the humanized antibodies comprise a combination
of VH4,
VH6, or VH7 humanized heavy chain variable region with VK3, VK6, or VK7
humanized light
variable region. The sequences of these humanized heavy and light chains are
provided in Tables
15-27 and Section 11 (see SEQ ID NOs: 287, 291, 295, 299, 305, and 309 for
VH1, VH2, VH3,
VH4, VH6, and VH7 sequences, respectively; and see SEQ ID Nos: 289, 293, 297,
301, 303,
307, 311, for VKl, VK2, VK3, VK4, VK5, VK6, and VK7 sequences, respectively).
9.7 Conclusions based on Examples 13-18:
[00521] In Examples 1-12, key segments of plasma membrane M(H)DM2 (PM-HDM2)
that
are extracellularly available for drug targeting were identified. Three
examples of such
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extracellularly available regions of membrane-bound M(H)DM2 are: NMC-P1, NMC-
P2 and
NMC-P3 (SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3). Mouse monoclonal
antibodies
were developed against these key epitopes and their binding to a variety of
cancer cells and their
lack of binding or significantly lower binding ability to untransformed cells
was demonstrated. In
addition, it was demonstrated that not all antibodies to M(H)DM2 are able to
bind to plasma
membrane-bound HDM2. It was also demonstrated that select antibodies against
plasma
membrane-bound HDM2 that bind to extra-cellulalry accessible epitopes have
anti-cancer
activity in vitro by various mechanisms including CDC and ADCC. Moreover, it
was shown that
select antibodies against extra-cellulalry accessible regions of plasma
membrane-bound HDM2
demonstrate in vivo anti-tumor activity in mouse models including but not
limited to lung, colon,
and pancreatic cancers.
[00522] In Examples 9-13, humanized and chimeric monoclonal antibodies were
developed
against these key epitopes and their binding to intact, un-permeabilized
cancer cells was
demonstrated to be similar to that of plasma membrane markers (Figure 33). It
was also shown
that these antibodies are specific for M(H)DM2 and it was shown that their
binding to intact, un-
permeabilized cancer cells can be competed with their epitope-specific
peptides (Figure 35A).
[00523] In addition, it was shown that the tested humanized and chimeric
antibodies against
extracellulalry accessible epitopes of plasma membrane-bound HDM2 have anti-
tumor effect,
and the anti-tumor effect of these antibodies is in part immune-mediated, as
their in-vivo efficacy
is reduced in in the immune-deficient NSG mice (Figure 36A and 36B).
[00524] Immune checkpoint inhibitors (IC's) such as anti-PD-1/PDL-1 and CTLA-4
have
demonstrated long lasting effects in various cancers and have become standard
of care for some
patients. However, recent observations have identified a sub-population of
patients who
experienced accelerated tumor progression under ICI (Champiat et al., Clin.
Cancer Res. 23,
1920-1928 (2017); Kato et al., Clin. Cancer Res. 23,4242-4250 (2017); Saada-
Bouzid et al.,
Ann. Oncol. 28,1605-1611 (2017); Ferrara et al., JAMA Oncol. (2018); and Zuazo-
Ibarra et al.,
2018). According to these observations, hyperprogressive disease was not
limited to any
particular cancer type. Hyper-progression under ICI was observed in non-small
cell lung cancer
(NSCLC) with an incident ranging from 8-21% (Kato et al., Clin. Cancer Res.
23,4242-4250
(2017); Ferrara et al., JAMA Oncol. (2018); and Zuazo- Ibarra et al., (2018)),
in melanoma with
an incident of 9% (Champiat et al., Clin. Cancer Res. 23,1920-1928 (2017)) and
poor median
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overall survivals (<3 months) was observed in patients with hyperprogressive
disease (Champiat
et al., Clin. Cancer Res. 23,1920-1928 (2017); and Ferrara et al., JAMA Oncol.
(2018)). These
observations were supported by Phase III clinical trials involving patients
with NSCLC
(Borghaei et al., N. Engl. J. Med. 373,1627-1639 (2015)), HNSCC (Ferris et
al., N. Engl. J.
Med. 375,1856-1867 (2016)) and Urethrial carcinoma (Bellmunt et al,. N. Engl.
J. Med. 376,
1015-1026 (2017)). MDM2/4 gene amplification and EGFR alterations were found
to be
associated with time to treatment failure (TTF) (Kato et al., Clin. Cancer
Res. 23,4242-4250
(2017)). In two studies, around two third of the patients with MDM2/MDM4 gene
amplification
were found to be hyperprogressors (Kato et al., Clin. Cancer Res. 23,4242-4250
(2017) A.K.
Singavi et al., Ann Oncol 2017;28).
[00525] A mouse model of lung cancer (M109) has demonstrated phenotypic
similarities to
that of hyperprogressive patients under ICI (Shisuo Oncoimmunology. 2018;
7(4): e1408747).
Data presented here demonstrates an increase in the binding of antibodies to
extracellularly
accessible regions of plasma membrane-bound M(H)DM2 of ICI-treated hyper-
progressive
tumors as compared to untreated tumors (Figure 37). Therefore, data presented
here
demonstrates a correlation between MDM2 gene amplification and an increase in
the expression
of plasma membrane-bound M(H)DM2. Taken together, data presented herein
demonstrate the
rationale for using antibodies to plasma membrane-bound M(H)DM2 in combination
with ICI
(before, during or after administraton of ICI).
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to the nucleoplasm mediated by a COOH-terminal nuclear localization signal",
Mol Cancer Res.
5(4):403-12.
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11. SEOUENCES
SEQ ID NO:1 (NMC-P1)
MCNTNMSVPTDGAVT
SEQ ID NO:2 (NMC-P2)
TTSQIPASEQE
SEQ ID NO:3 (NMC-P3)
CPVCRQPIQMIVLTYFP
SEQ ID NO:4 (Human HDM2 Protein):
MCNTNMSVPT DGAVTTSQIP ASEQETLVRP KPLLLKLLKS VGAQKDTYTM
KEVLFYLGQY IMTKRLYDEK QQHIVYCSND LLGDLFGVPS FSVKEHRKIY
TMIYRNLVVV NQQESSDSGT SVSENRCHLE GGSDQKDLVQ ELQEEKPSSS
HLVSRPSTSS RRRAISETEE NSDELSGERQ RKRHKSDS IS LSFDESLALC
VIREICCERS SSSESTGTPS NPDLDAGVSE HSGDWLDQDS VSDQFSVEFE
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VESLDSEDYS LSEEGQELSD EDDEVYQVTV YQAGESDTDS FEEDPE ISLA
DYWKCTSCNE MNPPLPSHCN RCWALRENWL PE DKGKDKGE I S EKAKLENS
TQAEEGFDVP DCKKT IVNDS RESCVEENDD KI TQAS QS QE SEDYSQPSTS
SSI I YS S QED VKEFEREETQ DKEESVESSL PLNAIEPCVI CQGRPKNGC I
VHGKTGHLMA CFTCAKKLKK RNKPCPVCRQ P I QMIVL TYF P
SEQ ID NO:5 (Mouse MDM2 Protein):
MCNTNMSVST EGAASTSQIP ASEQETLVRP KPLLLKLLKS VGAQNDTYTM
KE I I FY I GQY IMTKRLYDEK QQHIVYCSND LLGDVFGVPS FSVKEHRKIY
AMIYRNLVAV SQQDSGTSLS ESRRQPEGGS DLKDPLQAPP EEKPS S SDL I
SRLSTSSRRR S I SE TEENTD ELPGERHRKR RRSLS FDPSL GLCELREMCS
GGSSSSSSSS SESTETPSHQ DLDDGVSEHS GDCLDQDSVS DQFSVEFEVE
SLDSEDYSLS DEGHELSDED DEVYRVTVYQ TGESDTDS FE GDPE I SLADY
WKCTSCNEMN PPLPSHCKRC WTLRENWLPD DKGKDKVE IS EKAKLENSAQ
AEEGLDVPDG KKLTENDAKE PCAEEDSEEK AEQTPLSQES DDYSQPSTSS
S IVYSSQESV KELKEETQDK DESVESS FSL NAIEPCVICQ GRPKNGCIVH
GKTGHLMSCF TCAKKLKKRN KPCPVCRQP I QMIVLTYFN
SEQ ID NO:6 (Human HDM4 protein):
MIS FS T SAQCS T SDSACRI S PGQ INQVRPKLPLLKI LHAAGAQGEMFTVKEVMHYLGQ
YIMVKQLYDQQEQHMVYCGGDLLGELLGRQS FSVKDPS PLYDMLRKNLVTLATATTDAAQT
LALAQDHSMDI PS QDQLKQSAEES S T SRKRT TEDDI PTLPT SEHKC IHSREDEDL IEN
LAQDE T SRLDLGFEEWDVAGLPWWFLGNLRSNYT PRSNGS TDLQTNQDVGTAIVSDT T
DDLWFLNESVSEQLGVGIKVEAADTEQT SEEVGKVSDKKVIEVGKNDDLEDSKSLSDD
TDVEVT SEDEWQCTECKKFNS PSKRYCFRCWALRKDWYSDCSKL THSLS T SDI TAI PE
KENEGNDVPDCRRT I SAPVVRPKDAY IKKENSKL FDPCNSVE FLDLAHS SES QE T ISS
MGEQLDNLSEQRTDTENMEDCQNLLKPCSLCEKRPRDGNI I HGRTGHLVTC FHCARRL
KKAGASCP I CKKE I QLVIKVFIA
SEQ ID NO:7 (Mouse MDMX-S protein):
MT SHS T SAQCSASDSACRI S SEQ I S QVRPKLQLLKI LHAAGAQGEVFTMKEVMHYLGQY IMVK
189
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QLYDQQEQHMVYCGGDLLGDLLGCQS FSVKDPS PLYDMLRKNLVT SAS INTARC
NRILQSQKKN
M(H)DM2/4 variants:
SEQ ID NO:8 (HDM2 variant MDM2-A which lacks amino acids residues 28-222 of
SEQ ID
NO:4 as indicated in the below sequence by "(28-222)"):
MCNTNMSVPTDGAVTTSQIPASEQETLD (28-222)
LDAGVSEHSGDWLDQDSVSDQFSVEFEVESLDSEDYSLSEEGQELSDEDDEVYQVTVYQAG
ESDTDS FEEDPE I SLADYWKCT SCNEMNPPLPSHCNRCWALRENWLPEDKGKDKGE I SEKA
KLENSTQAEEGFDVPDCKKT IVNDSRESCVEENDDKI TQASQSQESEDYSQPS TSSS I IYS
SQEDVKEFEREETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHGKTGHLMACFTCAKK
LKKRNKPCPVCRQP I QMIVLTYFP
SEQ ID NO:9 (HDM2 variant MDM2-A1 which lacks amino acids residues 28-222 and
275-
300 of SEQ ID NO:4 as indicated in the below sequence by "(28-222)" and "(275-
300)"):
MCNTNMSVPT DGAVTTSQIP ASEQETLD (28-222)
LDAGVSEH SGDWLDQDSV SDQFSVEFEV ESLDSEDYSL SEEGQELSDE DDEDY
(275-300) WKCTS CNEMNPPLPS HCNRCWALRE NWLPEDKGKD
KGEISEKAKL ENSTQAEEGF DVPDCKKTIV NDSRESCVEE NDDKITQASQ
SQESEDYSQP STSSSIIYSS QEDVKEFERE ETQDKEESVE SSLPLNAIEP
CVICQGRPKN GCIVHGKTGH LMACFTCAKK LKKRNKPCPV CRQPIQMIVL
TYFP
SEQ ID NO:10 (HDM2 variant MDM2-B which lacks amino acids residues 28-300 of
SEQ ID
NO:4 as indicated in the below sequence by "(28-300)"):
MCNTNMSVPTDGAVTTSQIPASEQETLD (28-300)
YWKCT S CNEMNPPLPSHCNRCWALRENWLPEDKGKDKGE I SEKAKLENS TQAEEGFDVPDCKK
T IVNDSRESCVEENDDKI TQASQSQESEDYSQPS TSSS I IYSSQEDVKEFEREETQDKEESVE
SSLPLNAIEPCVICQGRPKNGCIVHG
KTGHLMACFTCAKKLKKRNKPCPVCRQP I QMIVLTYFP
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SEQ ID NO:11 (HDM2 variant MDM2-C which lacks amino acids residues 53-222 of
SEQ ID
NO:4 as indicated in the below sequence by "(53-222)"):
MCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKED ( 53-222 )
LDAGVSEHSGDWLDQDSVSDQFSVEFEVESLDSEDYSLSEEGQELSDEDDEVYQVTVYQAGESD
TDS FEEDPE I SLADYWKCT SCNEMNPPLPSHCNRCWALRENWLPEDKGKDKGE I SEKAKLENS
TQAEEGFDVPDCKKT IVNDSRESCVEENDDKI TQAS QS QESEDYS QPS TSSS I IYS S QED
VKEFEREETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHGKTGHLMACFTCAKKLKK
RNKPCPVCRQP I QMIVL TYFP
SEQ ID NO:12 (HDM2 variant MDM2-D which lacks amino acids residues 30-388 of
SEQ ID
NO:4 as indicated in the below sequence by "(30-388)"):
MCNTNMSVPTDGAVTTSQIPASEQETLVRQ ( 30-38 8 )
ESEDYS QPS TSSS I IYSSQEDVKEFEREETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHG
KTGHLMACFTCAKKLKKRNKPCPVCRQP I QMIVL TYFP
SEQ ID NO:13 (HDM2 variant MDM2-E which lacks amino acids residues 76-
102 and 103-491 of SEQ ID NO:4 as indicated in the below sequence by "(76-
102)" and "(103-491)"):
MCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLF
YLGQY IMTKRLYDEKQQHIVN ( 76-102 ) (103-491)
DCANLFPLVDLS IRELY I SNY I TLGI
SEQ ID NO:14 (HDM2 variant MDM2-F which lacks amino acids residues 53-97 of
SEQ
ID NO:4 as indicated in the below sequence by "(53-97)"):
MCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKE ( 53-97 )
KIYTMIYRNLVVVNQQES SDSGT SVSENRCHLEGGSDQKDLVQELQEEKPS S SHLVSRPS
T S SRRRAI SE TEENSDELSGERQRKRHKSDS I SLS FDESLALCVIRE I CCERS S S SES TG
TPSNPDLDAGVSEHSGDWLDQDSVSDQFSVEFEVESLDSEDYSLSEEGQELSDEDDEVYQ
VTVYQAGESDTDS FEEDPE I SLADYWKCT SCNEMNPPLPSHCNRCWALRENWLPEDKGKD
KGE I SEKAKLENS TQAEEGFDVPDCKKT IVNDSRESCVEENDDKI TQAS QS QESEDYS QP
191
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STSSS I I YS S QEDVKE FEREE TQDKEESVES SLPLNAIEPCVI CQGRPKNGC IVHGKTGH
LMACFTCA KKLKKRNKPCPVCRQP I QMIVL TYFP
SEQ ID NO:15 (HDM2 variant MDM2-G which lacks amino acids residues 115-169 of
SEQ
ID NO:4 as indicated in the below sequence by "(115-169)"):
MCNTNMSVPTDGAVT TSQI PASEQE TLVRPKPLLLKLLKSVGAQKDTYTMKEVL FYLGQY
IMTKRLYDEKQQHIVYCSNDLLGDLFGVPS FSVKEHRKIYTMIYRNLVVVNQQEE ( 115-
169 )
NSDELSGERQRKRHKSDS I SLS FDESLALCVIRE I CCERS S S SES TGT PSNPDLDAGVSEHS
GDWLDQDSVSDQFSVEFEVESLDSEDYSLSEEGQELSDEDDEVYQVTVYQAGESDTDS FEED
PE I S LADYWKCT S CNEMNPPLPSHCNRCWALRENWLPEDKGKDKGE I SEKAKLENS TQAEE
GFDVPDCKKT IVNDSRESCVEENDDKI TQAS QS QESEDYS QPS TSSS I I YS S QEDVKE FE
REETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHGKTGHLMACFTCAKKLKKRNKPC
PVCRQP I QMIVL TYFP
SEQ ID NO:16 (HDM2 variant MDM2-11):
MVRSRQMCNTNMSVPTDGAVT TSQI PASEQE TLVRPKPLLLKLLKSVGAQKDTYTMKEVL
FYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPS FSVKEHRKIYTMIYRNLVVVNQQE
S SDSGT SVSENRCHLEGGSDQKDLVQELQEEKPS S SHLVSRPS T S SRRRAI SE TEENSDE
LSGERQRKRHKSDS I SLS FDESLALCVIRE I CCERS S S SES TGT PSNPDLDAGVSEHSGD
WLDQDSVSDQFSVEFEVESLDSEDYSLSEEGQELSDEDDEVYQVTVYQAGESDTDS FEED
PE I SLADYWKCT S CNEMNP PL P S HCNRCWALRENWL PE DKGKDKGE I SEKAKLENS TQAE
EGFDVPDCKKT IVNDSRESCVEENDDKI TQAS QS QESEDYS QPS TSSS I I YS S QEDVKE F
EREETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHGKTGHLMACFTCAKKLKKRNKP
CPVCRQP I QMIVL TYFP
SEQ ID NO:17 (HDM2 variant MDM2-KB2 which lacks amino acids residues 157-248
of
SEQ ID NO:4 as indicated in the below sequence by "(157-248)"):
MCNTNMSVPTDGAVT TSQI PASEQE TLVRPKPLLLKLLKSVGAQKDTYTMKEDYWKCT SC
NEMNPPLPSHCNRCWALRENWLPEDKGKDKGE I SEKAKLENS TQAEEGFDVPDCKKT IVN
DSRESCVEENDDKI TQAS QS QESEDYS QPS TSSS I I ( 157-
192
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2 4 8 ) YS SQEDVKE FEREE TQDKEESVES SLPLNAIEPCVI CQGRPKNGC IVHGKTGHLMACF
TCAKKLKKRNKPCPVCRQP I QMIVLTYFP
193
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Table 4. rnAb NMC-103 Heavy Chain CDR and HFR Sequences using
Chothia, AbM, Kabat, Contact and IMGT CDR definitions:
Region Definition Sequence Fragment Residues' Length
SEQ
NO:ID
Chothia EVQLQESGGGLVQPGGSLRLSCTTS 1 - 25
25 72
AbM EVQLQESGGGLVQPGGSLRLSCTTS 1 - 25
25 72
HFR1
Kabat EVQLQESGGGLVQPGGSLRLSCTTSGFTFT 1 - 30
30 73
Contact EVQLQESGGGLVQPGGSLRLSCTTSGFTF- 1 - 29
29 74
Chothia GFTFTHY--- 26 - 32
7 18
AbM GFTFTHYYMS 26 - 35
10 42
CDR-H1 Kabat HYYMS 31 - 35
5 43
Contact THYYMS 30 - 35
6 44
IMGT -FTFTHYY-- 27 - 33
7 144
Chothia YMSWVRQPPGKALEWLGFI 33 - 51
19 75
AbM WVRQPPGKALEWLG-- 36 - 49
14 76
HFR2
Kabat WVRQPPGKALEWLG-- 36 - 49
14 76
Contact ---WVRQPPGKALE 36 - 46
11 77
Chothia RNKAKGYT 52 - 59
8 19
AbM ---FIRNKAKGYTAE 50 - 61
12 45
CDR-H2 Kabat FIRNKAKGYTAEYSASVKG 50 - 68
19 46
Contact WLGFIRNKAKGYTAE 47 - 61
15 47
IMGT IRNKAKGYTA 51 - 60
10 145
Chothia AEYSASVKGRFTISRDNSQSILYLQMNTLRPEDSATYYCAR 60 - 100 41
78
AbM --YSASVKGRFTISRDNSQSILYLQMNTLRPEDSATYYCAR 62 - 100 39
79
HFR3
Kabat RFTISRDNSQSILYLQMNTLRPEDSATYYCAR 69 - 100 32
80
Contact --YSASVKGRFTISRDNSQSILYLQMNTLRPEDSATYYC-- 62 - 98 37 81
Chothia --DIGDN 101 - 105 5
20
AbM --DIGDN 101 - 105 5
20
CDR-H3 Kabat --DIGDN 101 - 105 5
20
Contact ARDIGD- 99 - 104 6
48
IMGT ARDIGDN 99 - 105 7
146
Chothia -WGQGTLVTVSS 106 - 116 11
82
AbM -WGQGTLVTVSS 106 - 116 11
82
HFR4
Kabat -WGQGTLVTVSS 106 - 116 11
82
Contact NWGQGTLVTVSS 105 - 116 12
83
1 The listed residues are residue numbers of the heavy chain variable region
(SEQ ID NO:36) of NMC-103.
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Table 5. rnAb NMC-103 Light Chain CDR and HFR Sequences using
Chothia, AbM, Kabat, Contact and IMGT CDR definitions:
Region Definition Sequence Fragment Residues2 Length
SEQ
NO:ID
Chothia DIVMTQAAFSNPVTLGTSASISC 1 - 23
23 84
AbM DIVMTQAAFSNPVTLGTSASISC 1 - 23
23 84
LFR1
Kabat DIVMTQAAFSNPVTLGTSASISC 1 - 23
23 84
Contact DIVMTQAAFSNPVTLGTSASISCRSSKNL 1 - 29
29 85
Chothia RSSKNLLHSNGITYLY-- 24 - 39
16 21
AbM RSSKNLLHSNGITYLY-- 24 - 39
16 21
CDR-L1 Kabat RSSKNLLHSNGITYLY-- 24 - 39
16 21
Contact LHSNGITYLYWY 30 - 41
12 49
IMGT KNLLHSNGITY 27 - 37
11 147
Chothia WYLQRPGQSPQLLIS 40 - 54
15 86
AbM WYLQRPGQSPQLLIS 40 - 54
15 86
LFR2
Kabat WYLQRPGQSPQLLIS 40 - 54
15 86
Contact --LQRPGQSPQ 42 - 50
9 87
Chothia RVSNLAS 55 - 61
7 22
AbM RVSNLAS 55 - 61
7 22
CDR-L2 Kabat RVSNLAS 55 - 61
7 22
Contact LLISRVSNLA- 51 - 60
10 50
IMGT ----RVS---- 55 - 57
3
Chothia -GVPNRFSGSESGTDFTLRISRVEAEDVGVYFC 62 - 93
32 88
AbM -GVPNRFSGSESGTDFTLRISRVEAEDVGVYFC 62 - 93
32 88
LFR3
Kabat -GVPNRFSGSESGTDFTLRISRVEAEDVGVYFC 62 - 93
32 88
Contact SGVPNRFSGSESGTDFTLRISRVEAEDVGVYFC 61 - 93
33 89
Chothia AQLLELPYT 94 - 102 9
23
AbM AQLLELPYT 94 - 102 9
23
CDR-L3 Kabat AQLLELPYT 94 - 102 9
23
Contact AQLLELPY- 94 - 101 8
51
IMGT AQLLELPYT 94 - 102 9
23
Chothia -FGGGTKLEIK 103 - 112 10
90
AbM -FGGGTKLEIK 103 - 112 10
90
LFR4
Kabat -FGGGTKLEIK 103 - 112 10
90
Contact TFGGGTKLEIK 102 - 112 11
91
2. The listed residues are residue numbers of the light chain variable region
(SEQ ID NO:37) of NIVIC-103.
195
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Table 6. rnAb NMC-204 Heavy Chain CDR and HFR Sequences using
Chothia, AbM, Kabat, Contact and IMGT CDR defintions:
Region Definition Sequence Fragment Residues3 Length
SEQ
NO:ID
Chothia EVQLQESGSVLVRPGASVKLSCKAS 1 - 25
25 92
AbM EVQLQESGSVLVRPGASVKLSCKAS 1 - 25
25 92
HFR1
Kabat EVQLQESGSVLVRPGASVKLSCKASGDTLS 1 - 30
30 93
Contact EVQLQESGSVLVRPGASVKLSCKASGDTL- 1 - 29
29 94
Chothia GDTLSGS--- 26 - 32
7 24
AbM GDTLSGSWMH 26 - 35
10 52
CDR-H1 Kabat GSWMH 31 - 35
5 53
Contact SGSWMH 30 - 35
6 54
IMGT GDTLSGSW-- 26 - 33
8 148
Chothia WMHWAMQRPGQGLEWIGEI 33 - 51
19 95
AbM WAMQRPGQGLEWIG-- 36 - 49
14 96
HFR2
Kabat WAMQRPGQGLEWIG-- 36 - 49
14 96
Contact ---WAMQRPGQGLE 36 - 46
11 97
Chothia HLNRGT 52 - 57
6 25
AbM ---EIHLNRGTTN 50 - 59
10 55
CDR-H2 Kabat EIHLNRGTTNYNEKFKG 50 - 66
17 56
Contact WIGEIHLNRGTTN 47 - 59
13 57
IMGT IHLNRGTT 51 - 58
8 143
Chothia TNYNEKFKGKATVTVDTSSSTAYVDLSSLTSEDSAVYYCAR 58 - 98 41 98
AbM --
YNEKFKGKATVTVDTSSSTAYVDLSSLTSEDSAVYYCAR 60 - 98 39 99
HFR3
Kabat
KATVTVDTSSSTAYVDLSSLTSEDSAVYYCAR 67 - 98 32 100
Contact --YNEKFKGKATVTVDTSSSTAYVDLSSLTSEDSAVYYC-- 60 - 96 37 101
Chothia --SPGFAY 99 - 104 6
26
AbM --SPGFAY 99 - 104 6
26
CDR-H3 Kabat --SPGFAY 99 - 104 6
26
Contact ARSPGFA- 97 - 103 7
58
IMGT ARSPGFA- 97 - 103 7
58
Chothia -WGQGTLVTVSA 105 - 115 11
102
AbM -WGQGTLVTVSA 105 - 115 11
102
HFR4
Kabat -WGQGTLVTVSA 105 - 115 11
102
Contact YWGQGTLVTVSA 104 - 115 12
103
3 The listed residues are residue numbers of the heavy chain variable region
(SEQ ID NO:38) of NMC-204.
196
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Table 7. rnAb NMC-204 Light Chain CDR and HFR Sequences using
Chothia, AbM, Kabat, Contact and IMGT CDR defintions
Region Definition Sequence Fragment Residues4 Length
SEQ
NO:ID
Chothia GIVMTQAAPSVPVTPGESVSISC 1 - 23
23 104
AbM GIVMTQAAPSVPVTPGESVSISC 1 - 23
23 104
LFR1
Kabat GIVMTQAAPSVPVTPGESVSISC 1 - 23
23 104
Contact GIVMTQAAPSVPVTPGESVSISCRSSKSL 1 - 29
29 105
Chothia RSSKSLLHSNGNSYLY-- 24 - 39
16 27
AbM RSSKSLLHSNGNSYLY-- 24 - 39
16 27
CDR-L1 Kabat RSSKSLLHSNGNSYLY-- 24 - 39
16 27
Contact LHSNGNSYLYWF 30 - 41
12 59
IMGT KSLLHSNGNSY 27 - 37
11 141
Chothia WFLQRPGQSPQLLIY 40 - 54
15 106
AbM WFLQRPGQSPQLLIY 40 - 54
15 106
LFR2
Kabat WFLQRPGQSPQLLIY 40 - 54
15 106
Contact --LQRPGQSPQ 42 - 50
9 107
Chothia RMSNLAS 55 - 61
7 28
AbM RMSNLAS 55 - 61
7 28
CDR-L2 Kabat RMSNLAS 55 - 61
7 28
Contact LLIYRMSNLA- 51 - 60
10 60
IMGT ----RNS---- 55 - 57
3
Chothia -GVPDRFSGSGSGTAFTLRITRVEAEDVGVYYC 62 - 93
32 108
AbM -GVPDRFSGSGSGTAFTLRITRVEAEDVGVYYC 62 - 93
32 108
LFR3
Kabat -GVPDRFSGSGSGTAFTLRITRVEAEDVGVYYC 62 - 93
32 108
Contact SGVPDRFSGSGSGTAFTLRITRVEAEDVGVYYC 61 - 93
33 109
Chothia MQHLEYPFT 94 - 102 9
29
AbM MQHLEYPFT 94 - 102 9
29
CDR-L3 Kabat MQHLEYPFT 94 - 102 9
29
Contact MQHLEYPF- 94 - 101 8
61
IMGT MQHLEYPFT 94 - 102 9
29
Chothia -FGSGTKLEIK 103 - 112 10
110
AbM -FGSGTKLEIK 103 - 112 10
110
LFR4
Kabat -FGSGTKLEIK 103 - 112 10
110
Contact TFGSGTKLEIK 102 - 112 11
111
4 The listed residues are residue numbers of the light chain variable region
(SEQ ID NO:39) of NIVIC-204.
197
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Table 8. rnAb NMC-303 Heavy Chain CDR and HFR Sequences using
Chothia, AbM, Kabat and Contact CDR definitions:
Region Definition Sequence Fragment Residues5 Length
SEQ
NO:ID
Chothia QVQLQQPGAELVKPGASVKLSCKAS 1 - 25
25 112
AbM QVQLQQPGAELVKPGASVKLSCKAS 1 - 25
25 112
HFR1
Kabat QVQLQQPGAELVKPGASVKLSCKASGYTFT 1 - 30
30 113
Contact QVQLQQPGAELVKPGASVKLSCKASGYTF- 1 - 29
29 114
Chothia GYTFTSY--- 26 - 32
7 30
AbM GYTFTSYYMY 26 - 35
10 62
CDR-H1
Kabat SYYMY 31 - 35
5 63
Contact TSYYMY 30 - 35
6 64
Chothia YMYWVKQRPGQGLEWIGGI 33 - 51
19 115
AbM WVKQRPGQGLEWIG-- 36 - 49
14 116
HFR2
Kabat WVKQRPGQGLEWIG-- 36 - 49
14 116
Contact ---WVKQRPGQGLE 36 - 46
11 117
Chothia NPRNGG 52 - 57
6 31
AbM ---GINPRNGGTN 50 - 59
10 65
CDR-H2
Kabat ---GINPRNGGTNFNEKFKN 50 - 66
17 66
Contact WIGGINPRNGGTN 47 - 59
13 67
Chothia TNFNEKFKNKATLTADKSSTTAYMQLSSLTSEDSAVYYCTR 58 - 98 41 118
AbM --
FNEKFKNKATLTADKSSTTAYMQLSSLTSEDSAVYYCTR 60 - 98 39 119
HFR3
Kabat
KATLTADKSSTTAYMQLSSLTSEDSAVYYCTR 67 - 98 32 120
Contact --FNEKFKNKATLTADKSSTTAYMQLSSLTSEDSAVYYC-- 60 - 96 37 121
Chothia --SGYYAMDY 99 - 106 8
32
AbM --SGYYAMDY 99 - 106 8
32
CDR-H3
Kabat --SGYYAMDY 99 - 106 8
32
Contact TRSGYYAMD- 97 - 105 9
68
Chothia -WGQGTSVTVSS 107 - 117 11
122
AbM -WGQGTSVTVSS 107 - 117 11
122
HFR4
Kabat -WGQGTSVTVSS 107 - 117 11
122
Contact YWGQGTSVTVSS 106 - 117 12
123
The listed residues are residue numbers of the heavy chain variable region
(SEQ ID NO:40) of NMC-303.
198
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Table 9. rnAb NMC-303 Light Chain CDR and HFR Sequences using
Chothia, AbM, Kabat and Contact CDR definitions:
Region Definition Sequence Fragment Residues6 Length
SEQ
NO:ID
Chothia DIQMTQTTSSLSASLGDRVTISC 1 - 23
23 124
AbM DIQMTQTTSSLSASLGDRVTISC 1 - 23
23 124
LFR1
Kabat DIQMTQTTSSLSASLGDRVTISC 1 - 23
23 124
Contact DIQMTQTTSSLSASLGDRVTISCRASQDI 1 - 29
29 125
Chothia RASQDISNFLN-- 24 - 34
11 33
AbM RASQDISNFLN-- 24 - 34
11 33
CDR-L1
Kabat RASQDISNFLN-- 24 - 34
11 33
Contact SNFLNWY 30 - 36
7 69
Chothia WYQQKPDGTVKLLIY 35 - 49
15 126
AbM WYQQKPDGTVKLLIY 35 - 49
15 126
LFR2
Kabat WYQQKPDGTVKLLIY 35 - 49
15 126
Contact --QQKPDGTVK 37 - 45
9 127
Chothia YTSRLHS 50 - 56
7 34
AbM YTSRLHS 50 - 56
7 34
CDR-L2
Kabat YTSRLHS 50 - 56
7 34
Contact LLIYYTSRLH- 46 - 55
10 70
Chothia -GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC 57 - 88
32 128
AbM -GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC 57 - 88
32 128
LFR3
Kabat -GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC 57 - 88
32 128
Contact SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC 56 - 88
33 129
Chothia QQGNTLPRT 89 - 97
9 35
AbM QQGNTLPRT 89 - 97
9 35
CDR-L3
Kabat QQGNTLPRT 89 - 97
9 35
Contact QQGNTLPR- 89 - 96
8 71
Chothia -FGGGTKLEIK 98 - 107 10
130
AbM -FGGGTKLEIK 98 - 107 10
130
LFR4
Kabat -FGGGTKLEIK 98 - 107 10
130
Contact TFGGGTKLEIK 97 - 107 11
131
6 The listed residues are residue numbers of the light chain variable region
(SEQ ID NO:41) of NIVIC-303.
199
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Table 11. rnAb NMC-C303 Heavy Chain(hu IgG1)CDR and HFR
Sequences using Chothia, AbM, Kabat,and Contact CDR definitions:
Region Definition Sequence Fragment Residues7 Length
SEQ
NO:ID
Chothia QVQLQQPGAELVKPGASVKLSCKAS 1 - 25
25 112
AbM QVQLQQPGAELVKPGASVKLSCKAS 1 - 25
25 112
HFR1
Kabat QVQLQQPGAELVKPGASVKLSCKASGYTFT 1 - 30
30 113
Contact QVQLQQPGAELVKPGASVKLSCKASGYTF- 1 - 29
29 114
Chothia GYTFTSY--- 26 - 32
7 30
AbM GYTFTSYYMY 26 - 35
10 62
CDR-H1
Kabat SYYMY 31 - 35
5 63
Contact TSYYMY 30 - 35
6 64
Chothia YMYWVKQRPGQGLEWIGGI 33 - 51
19 115
AbM WVKQRPGQGLEWIG-- 36 - 49
14 116
HFR2
Kabat WVKQRPGQGLEWIG-- 36 - 49
14 116
Contact ---WVKQRPGQGLE 36 - 46
11 117
Chothia NPRNGG 52 - 57
6 31
AbM ---GINPRNGGTN 50 - 59
10 65
CDR-H2
Kabat ---GINPRNGGTNFNEKFKN 50 - 66
17 66
Contact WIGGINPRNGGTN 47 - 59
13 67
Chothia TNFNEKFKNKATLTADKSSTTAYMQLSSLTSEDSAVYYCTR 58 - 98 41 118
AbM --
FNEKFKNKATLTADKSSTTAYMQLSSLTSEDSAVYYCTR 60 - 98 39 119
HFR3
Kabat
KATLTADKSSTTAYMQLSSLTSEDSAVYYCTR 67 - 98 32 120
Contact --FNEKFKNKATLTADKSSTTAYMQLSSLTSEDSAVYYC-- 60 - 96 37 121
Chothia --SGYYAMDY 99 - 106 8
32
AbM --SGYYAMDY 99 - 106 8
32
CDR-H3
Kabat --SGYYAMDY 99 - 106 8
32
Contact TRSGYYAMD- 97 - 105 9
68
Chothia -WGQGTSVTVSS 107 - 117 11
122
AbM -WGQGTSVTVSS 107 - 117 11
122
HFR4
Kabat -WGQGTSVTVSS 107 - 117 11
122
Contact YWGQGTSVTVSS 106 - 117 12
123
7 7 The listed residues are residue numbers of the heavy chain variable region
(SEQ ID NO:40) of NNIC-C303.
200
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Table 12. rnAb NMC-C303 Light Chain (hu IgG1) CDR and HFR
Sequences using Chothia, AbM, Kabat and Contact CDR definitions:
Region Definition Sequence Fragment Residues8 Length
SEQ
NO:ID
Chothia DIQMTQTTSSLSASLGDRVTISC 1 - 23
23 124
AbM DIQMTQTTSSLSASLGDRVTISC 1 - 23
23 124
LFR1
Kabat DIQMTQTTSSLSASLGDRVTISC 1 - 23
23 124
Contact DIQMTQTTSSLSASLGDRVTISCRASQDI 1 - 29
29 125
Chothia RASQDISNFLN-- 24 - 34
11 33
AbM RASQDISNFLN-- 24 - 34
11 33
CDR-L1
Kabat RASQDISNFLN-- 24 - 34
11 33
Contact SNFLNWY 30 - 36
7 69
Chothia WYQQKPDGTVKLLIY 35 - 49
15 126
AbM WYQQKPDGTVKLLIY 35 - 49
15 126
LFR2
Kabat WYQQKPDGTVKLLIY 35 - 49
15 126
Contact --QQKPDGTVK 37 - 45
9 127
Chothia YTSRLHS 50 - 56
7 34
AbM YTSRLHS 50 - 56
7 34
CDR-L2
Kabat YTSRLHS 50 - 56
7 34
Contact LLIYYTSRLH- 46 - 55
10 70
Chothia -GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC 57 - 88
32 128
AbM -GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC 57 - 88
32 128
LFR3
Kabat -GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC 57 - 88
32 128
Contact SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC 56 - 88
33 129
Chothia QQGNTLPRT 89 - 97
9 35
AbM QQGNTLPRT 89 - 97
9 35
CDR-L3
Kabat QQGNTLPRT 89 - 97
9 35
Contact QQGNTLPR- 89 - 96
8 71
Chothia -FGGGTKLEIK 98 - 107 10
130
AbM -FGGGTKLEIK 98 - 107 10
130
LFR4
Kabat -FGGGTKLEIK 98 - 107 10
130
Contact TFGGGTKLEIK 97 - 107 11
131
88 The listed residues are residue numbers of the light chain variable region
(SEQ ID NO:41) of NIVIC-C303.
201
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Table 13. rnAb NMC-C103.VHO Heavy Chain (hu IgG1) CDR and HFR
Sequences using Chothia, AbM, Kabat and Contact CDR definitions:
Region Definition Sequence Fragment
Residues9 Length SE:
ID
Chothia EVKLVESGGGLVQPGGSLRLSCTTS 1-25
25 149
AbM EVKLVESGGGLVQPGGSLRLSCTTS 1-25 25
149
HFR1
Kabat EVKLVESGGGLVQPGGSLRLSCTTSGFTFT
1-30 30 150
Contact EVKLVESGGGLVQPGGSLRLSCTTSGFTF- 1-29
29 151
Chothia GFTFTHY--- 26-32 7
18
AbM GFTFTHYYMS 26-35 10
42
CDR-H1 Kabat -HYYMS
31-35 5 43
Contact THYYMS 30-35 6
44
Chothia YMSWVRQPPGKALEWLGFI 33-51 19
75
AbM WVRQPPGKALEWLG-- 36-49 14
76
HFR2
Kabat WVRQPPGKALEWLG-- 36-49 14
76
Contact ---WVRQPPGKALE 36-46 11
77
Chothia RNKAKGYT 52-59 8
19
AbM ---FIRNKAKGYTAE 50-61 12
45
CDR-H2 Kabat FIRNKAKGYTAEYSASVKG 50-68 19
46
Contact WLGFIRNKAKGYTAE 47-61 15
47
Chothia AEYSASVKGRFTISRDNSQSILYLQMNTLRPEDSATYYCAR 60-100 41 78
AbM --YSASVKGRFTISRDNSQSILYLQMNTLRPEDSATYYCAR 62-100 39 79
HFR3
Kabat
RFTISRDNSQSILYLQMNTLRPEDSATYYCAR 69-100 32 80
Contact --YSASVKGRFTISRDNSQSILYLQMNTLRPEDSATYYC-- 62-98 37 81
Chothia --DIGDN 101-
105 5 20
AbM --DIGDN 101-105 5
20
CDR-H3 Kabat --DIGDN
101-105 5 20
Contact ARDIGD- 99-104 6
48
Chothia -WGQGTLVTVSS 106-
116 11 82
AbM -WGQGTLVTVSS 106-116 11
82
HFR4
Kabat -WGQGTLVTVSS
106-116 11 82
Contact NWGQGTLVTVSS 105-
116 12 83
9 The listed residues are residue numbers of the heavy chain variable region
(SEQ ID NO:283) of NIVIC-C103.VHO.
202
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Table 14. rnAb NMC-C103.VKO Light Chain (hu IgG1) CDR and HFR
Sequences using Chothia, AbM, Kabat,and Contact CDR definitions:
Region Definition Sequence Fragment Residues Length
SEQ
NO:ID
io
Chothia DIVMTQAAFSNPVTLGTSASISC 1-23 23
84
LFR1
AbM DIVMTQAAFSNPVTLGTSASISC 1-23 23
84
Kabat DIVMTQAAFSNPVTLGTSASISC
1-23 23 84
Contact DIVMTQAAFSNPVTLGTSASISCRSSKNL 1-29 29
85
Chothia RSSKNLLHSNGITYLY-- 24-39 16
21
AbM RSSKNLLHSNGITYLY-- 24-39 16
21
CDR-L1 Kabat RSSKNLLHSNGITYLY--
24-39 16 21
Contact LHSNGITYLYWY 30-41 12
49
Chothia WYLQRPGQSPQLLIS 40-54 15
86
AbM WYLQRPGQSPQLLIS 40-54 15
86
LFR2
Kabat WYLQRPGQSPQLLIS
40-54 15 86
Contact --LQRPGQSPQ 42-50 9
87
Chothia RVSNLAS 55-61 7
22
AbM RVSNLAS 55-61 7
22
CDR-L2
Kabat RVSNLAS 55-61 7
22
Contact LLISRVSNLA- 51-60 10
50
Chothia -GVPNRFSGSESGTDFTLRISRVEAEDVGVYFC 62-93 32
88
AbM -GVPNRFSGSESGTDFTLRISRVEAEDVGVYFC 62-93 32
88
LFR3
Kabat -GVPNRFSGSESGTDFTLRISRVEAEDVGVYFC
62-93 32 88
Contact SGVPNRFSGSESGTDFTLRISRVEAEDVGVYFC 62-93 33
89
Chothia AQLLELPYT 94-102 9
23
AbM AQLLELPYT 94-102 9
23
CDR-L3 Kabat AQLLELPYT
94-102 9 23
Contact AQLLELPY- 94-102 8
51
Chothia -FGGGTKLEIK 103-
112 10 90
AbM -FGGGTKLEIK 103-112
10 90
LFR4
Kabat -FGGGTKLEIK
103-112 10 90
Contact TFGGGTKLEIK 102-
112 11 91
' The listed residues are residue numbers of the light chain variable region
(SEQ ID NO:285) of NMC-C103.VKO.
203
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Table 15. rnAb NMC-H103.VH1 Heavy Chain (hu IgG1) CDR and HFR
Sequences using Chothia, AbM, Kabat,and Contact CDR definitions:
Region Definition Sequence Fragment Residues Length
SEQ
NO:ID
ii
Chothia EVKLVESGGGLVQPGPSLRLSCTTS 1-25
25 152
AbM EVKLVESGGGLVQPGPSLRLSCTTS 1-25 25
152
HFR1
Kabat EVKLVESGGGLVQPGPSLRLSCTTSGFTFT
1-30 30 153
Contact EVKLVESGGGLVQPGPSLRLSCTTSGFTF- 1-29
29 154
Chothia GFTFTHY--- 26-32 7
18
AbM GFTFTHYYMS 26-35 10
42
CDR-H1 Kabat HYYMS
31-35 5 43
Contact THYYMS 30-35 6
44
Chothia YMSWVRQAPGKGLEWLGFI 33-51
19 155
AbM WVRQAPGKGLEWLG-- 36-49 14
156
HFR2
Kabat WVRQAPGKGLEWLG-- 36-49 14
156
Contact ---WVRQAPGKGLE 36-46
11 157
Chothia RNKAKGYT 52-59 8
19
AbM ---FIRNKAKGYTAE 50-61 12
45
CDR-H2
Kabat ---FIRNKAKGYTAEYSASVKG
50-68 19 46
Contact WLGFIRNKAKGYTAE 47-61 15
47
Chothia AEYSASVKGRFTISRDNSKSTLYLQMNTLRAEDSATYYCAR 60-100 41
158
AbM --YSASVKGRFTISRDNSKSTLYLQMNTLRAEDSATYYCAR 62-100 39
159
HFR3
Kabat RFTISRDNSKSTLYLQMNTLRAEDSATYYCAR 69-100 32
160
Contact --YSASVKGRFTISRDNSKSTLYLQMNTLRAEDSATYYC-- 62-98
37 161
Chothia --DIGDN 101-
105 5 20
AbM --DIGDN 101-105 5
20
CDR-H3 Kabat --DIGDN
101-105 5 20
Contact ARDIGD- 99-104 6
48
Chothia -WGQGTLVTVSS
106-116 11 162
AbM -WGQGTLVTVSS 106-116 11
162
HFR4
Kabat -WGQGTLVTVSS
106-116 11 162
Contact NWGQGTLVTVSS
105-116 12 163
11 The listed residues are residue numbers of the heavy chain variable region
(SEQ ID NO:287) of NIVIC-H103.VH1.
204
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Table 16. rnAb NMC-H103.VK1 Light Chain (hu IgG1) CDR and HFR
Sequences using Chothia, AbM, Kabat,and Contact CDR definitions:
Region Definition Sequence Fragment Residues Length
SEQ
NO:ID
12
Chothia DIVMTQAAFSNPVTLGQPASISC 1-23
23 164
AbM DIVMTQAAFSNPVTLGQPASISC 1-23 23
164
LFR1
Kabat DIVMTQAAFSNPVTLGQPASISC
1-23 23 164
Contact DIVMTQAAFSNPVTLGQPASISCRSSKNL 1-29
29 165
Chothia RSSKNLLHSNGITYLY-- 24-39 16
21
AbM RSSKNLLHSNGITYLY-- 24-39 16
21
CDR-L1 Kabat RSSKNLLHSNGITYLY--
24-39 16 21
Contact LHSNGITYLYWY 30-41 12
49
Chothia WYLQKPGQSPQLLIS 40-54 15
166
AbM WYLQKPGQSPQLLIS 40-54 15
166
LFR2
Kabat WYLQKPGQSPQLLIS
40-54 15 166
Contact --LQKPGQSPQ 42-50 9
167
Chothia RVSNLAS 55-61 7
22
AbM RVSNLAS 55-61 7
22
CDR-L2
Kabat RVSNLAS 55-61 7
22
Contact LLISRVSNLA- 51-60 10
50
Chothia -GVPNRFSGSESGTDFTLKISRVEAEDVGVYFC 62-93
32 168
AbM -GVPNRFSGSESGTDFTLKISRVEAEDVGVYFC 62-93 32
168
LFR3
Kabat -GVPNRFSGSESGTDFTLKISRVEAEDVGVYFC
62-93 32 168
Contact SGVPNRFSGSESGTDFTLKISRVEAEDVGVYFC 62-93
33 169
Chothia AQLLELPYT 94-102 9
23
AbM AQLLELPYT 94-102 9
23
CDR-L3 Kabat AQLLELPYT
94-102 9 23
Contact AQLLELPY- 94-102 8
51
Chothia -FGGGTKVEIK
103-112 10 170
AbM -FGGGTKVEIK 103-112 10
170
LFR4
Kabat -FGGGTKVEIK
103-112 10 170
Contact TFGGGTKVEIK
102-112 11 171
12. The listed residues are residue numbers of the light chain variable region
(SEQ ID NO:289) of NMC-H103.VK1.
205
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Table 17. rnAb NMC-H103.VH2 Heavy Chain (hu IgG1) CDR and HFR
Sequences using Chothia, AbM, Kabat,and Contact CDR definitions:
Region Definition Sequence Fragment Residues Length
SEQ
NO:ID
13
Chothia EVKLVESGGGLVQPGPSLRLSCTTS 1-25
25 172
AbM EVKLVESGGGLVQPGPSLRLSCTTS 1-25 25
172
HFR1
Kabat EVKLVESGGGLVQPGPSLRLSCTTSGFTFT
1-30 30 173
Contact EVKLVESGGGLVQPGPSLRLSCTTSGFTF- 1-29
29 174
Chothia GFTFTHY--- 26-32 7
18
AbM GFTFTHYYMS 26-35 10
42
CDR-H1 Kabat HYYMS
31-35 5 46
Contact THYYMS 30-35 6
44
Chothia YMSWVRQAPGKGLEWLGFI 33-51
19 175
AbM WVRQAPGKGLEWLG-- 36-49 14
176
HFR2
Kabat WVRQAPGKGLEWLG-- 36-49 14
176
Contact ---WVRQAPGKGLE 36-46
11 177
Chothia RNKAKGYT 52-59 8
19
AbM ---FIRNKAKGYTAE 50-61 12
45
CDR-H2
Kabat ---FIRNKAKGYTAEYSASVKG
50-68 19 46
Contact WLGFIRNKAKGYTAE 47-61 15
47
Chothia AEYSASVKGRFTISRDNSKSTLYLQMNSLRAEDTATYYCAR 60-100 41 178
AbM --YSASVKGRFTISRDNSKSTLYLQMNSLRAEDTATYYCAR 62-100 39 179
HFR3
Kabat
RFTISRDNSKSTLYLQMNSLRAEDTATYYCAR 69-100 32 180
Contact --YSASVKGRFTISRDNSKSTLYLQMNSLRAEDTATYYC-- 62-98 37 181
Chothia --DIGDN 101-
105 5 20
AbM --DIGDN 101-105 5
20
CDR-H3 Kabat --DIGDN
101-105 5 20
Contact ARDIGD- 99-104 6
48
Chothia -WGQGTLVTVSS
106-116 11 182
AbM -WGQGTLVTVSS 106-116 11
182
HFR4
Kabat -WGQGTLVTVSS
106-116 11 182
Contact NWGQGTLVTVSS
105-116 12 183
' The listed residues are residue numbers of the heavy chain variable region
(SEQ ID NO:291) of NIVIC-H103.VH2.
206
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Table 18. rnAb NMC-H103.VK2 Light Chain (hu IgG1) CDR and HFR
Sequences using Chothia, AbM, Kabat,and Contact CDR definitions:
Region Definition Sequence Fragment Residues Length
SEQ
NO:ID
14
Chothia DIVMTQTPLSSPVTLGQPASISC 1-23
23 184
AbM DIVMTQTPLSSPVTLGQPASISC 1-23 23
184
LFR1
Kabat DIVMTQTPLSSPVTLGQPASISC
1-23 23 184
Contact DIVMTQTPLSSPVTLGQPASISCRSSKNL 1-29
29 185
Chothia RSSKNLLHSNGITYLY-- 24-39 16
21
AbM RSSKNLLHSNGITYLY-- 24-39 16
21
CDR-L1 Kabat RSSKNLLHSNGITYLY--
24-39 16 21
Contact LHSNGITYLYWY 30-41 12
49
Chothia WYLQKPGQSPQLLIS 40-54
15 186
AbM WYLQKPGQSPQLLIS 40-54 15
186
LFR2
Kabat WYLQKPGQSPQLLIS
40-54 15 186
Contact --LQKPGQSPQ 42-50
9 187
Chothia RVSNLAS 55-61 7
22
AbM RVSNLAS 55-61 7
22
CDR-L2
- Kabat RVSNLAS 55-61 7
22
Contact LLISRVSNLA- 51-60 10
50
Chothia -GVPNRFSGSESGTDFTLKISRVEAEDVGVYFC 62-93
32 188
AbM -GVPNRFSGSESGTDFTLKISRVEAEDVGVYFC 62-93 32
188
LFR3
Kabat -GVPNRFSGSESGTDFTLKISRVEAEDVGVYFC
62-93 32 188
Contact SGVPNRFSGSESGTDFTLKISRVEAEDVGVYFC 62-93
33 189
Chothia AQLLELPYT 94-102 9
23
AbM AQLLELPYT 94-102 9
23
CDR-L3 Kabat AQLLELPYT
94-102 9 23
Contact AQLLELPY- 94-102 8
51
Chothia -FGGGTKVEIK
103-112 10 190
AbM -FGGGTKVEIK 103-112 10
190
LFR4
Kabat -FGGGTKVEIK
103-112 10 190
Contact TFGGGTKVEIK
102-112 11 191
" The listed residues are residue numbers of the light chain variable region
(SEQ ID NO:293) of NMC-H103.VK2.
207
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Table 19. rnAb NMC-H103.VH3 Heavy Chain (hu IgG1) CDR and HFR
Sequences using Chothia, AbM, Kabat,and Contact CDR definitions:
Region Definition Sequence Fragment Residues Length
SEQ
NO:ID
Chothia EVQLVESGGGLVQPGPSLRLSCTTS 1-25
25 192
AbM EVQLVESGGGLVQPGPSLRLSCTTS 1-25 25
192
HFR1
Kabat EVQLVESGGGLVQPGPSLRLSCTTSGFTFT
1-30 30 193
Contact EVQLVESGGGLVQPGPSLRLSCTTSGFTF- 1-29
29 194
Chothia GFTFTHY--- 26-32 7
18
AbM GFTFTHYYMS 26-35 10
42
CDR-H1 Kabat HYYMS
31-35 5 43
Contact THYYMS 30-35 6
44
Chothia YMSWVRQAPGKGLEWLGFI 33-51
19 195
AbM WVRQAPGKGLEWLG-- 36-49 14
196
HFR2
Kabat WVRQAPGKGLEWLG-- 36-49 14
196
Contact ---WVRQAPGKGLE 36-46
11 197
Chothia RNKAKGYT 52-59 8
19
AbM ---FIRNKAKGYTAE 50-61 12
45
CDR-H2
Kabat ---FIRNKAKGYTAEYSASVKG
50-68 19 46
Contact WLGFIRNKAKGYTAE 47-61 15
47
Chothia AEYSASVKGRFTISRDNSKSTLYLQMNSLRAEDTATYYCAR 60-100 41 198
AbM --YSASVKGRFTISRDNSKSTLYLQMNSLRAEDTATYYCAR 62-100 39 199
HFR3
Kabat
RFTISRDNSKSTLYLQMNSLRAEDTATYYCAR 69-100 32 200
Contact --YSASVKGRFTISRDNSKSTLYLQMNSLRAEDTATYYC-- 62-98 37 201
Chothia --DIGDN 101-
105 5 20
AbM --DIGDN 101-105 5
20
CDR-H3 Kabat --DIGDN
101-105 5 20
Contact ARDIGD- 99-104 6
48
Chothia -WGQGTLVTVSS
106-116 11 202
AbM -WGQGTLVTVSS 106-116 11
202
HFR4
Kabat -WGQGTLVTVSS
106-116 11 202
Contact NWGQGTLVTVSS
105-116 12 203
15 The listed residues are residue numbers of the heavy chain variable region
(SEQ ID NO:295) of NIVIC-H103.VH3.
208
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Table 20. rnAb NMC-H103.VK3 Light Chain (hu IgG1) CDR and HFR
Sequences using Chothia, AbM, Kabat,and Contact CDR definitions:
Region Definition Sequence Fragment Residues Length
SEQ
NO:ID
16
Chothia DIVMTQTPLSSPVTLGQPASISC 1-23
23 204
AbM DIVMTQTPLSSPVTLGQPASISC 1-23 23
204
LFR1
Kabat DIVMTQTPLSSPVTLGQPASISC
1-23 23 204
Contact DIVMTQTPLSSPVTLGQPASISCRSSKNL 1-29
29 205
Chothia RSSKNLLHSNGITYLY-- 24-39 16
21
AbM RSSKNLLHSNGITYLY-- 24-39 16
21
CDR-L1 Kabat RSSKNLLHSNGITYLY--
24-39 16 21
Contact LHSNGITYLYWY 30-41 12
49
Chothia WYLQKPGQSPQLLIS 40-54
15 206
AbM WYLQKPGQSPQLLIS 40-54 15
206
LFR2
Kabat WYLQKPGQSPQLLIS
40-54 15 206
Contact --LQKPGQSPQ 42-50
9 207
Chothia RVSNLAS 55-61 7
22
AbM RVSNLAS 55-61 7
22
CDR-L2
Kabat RVSNLAS 55-61 7
22
Contact LLISRVSNLA- 51-60 10
50
Chothia -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC 62-93
32 208
AbM -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC 62-93 32
208
LFR3
Kabat -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC
62-93 32 208
Contact SGVPDRFSGSESGTDFTLKISRVEAEDVGVYFC 62-93
33 209
Chothia AQLLELPYT 94-102 9
23
AbM AQLLELPYT 94-102 9
23
CDR-L3 Kabat AQLLELPYT
94-102 9 23
Contact AQLLELPY- 94-102 8
51
Chothia -FGGGTKVEIK
103-112 10 210
AbM -FGGGTKVEIK 103-112 10
210
LFR4
Kabat -FGGGTKVEIK
103-112 10 210
Contact TFGGGTKVEIK
102-112 11 211
' The listed residues are residue numbers of the light chain variable region
(SEQ ID NO:297) of NMC-H103.VK3.
209
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Table 21. rnAb NMC-H103.VH4 Heavy Chain (hu IgG1) CDR and HFR
Sequences using Chothia, AbM, Kabat,and Contact CDR definitions:
Region Definition Sequence Fragment Residues Length
SEQ
NO:ID
17
Chothia EVQLVESGGGLVQPGPSLRLSCTTS 1-25
25 212
AbM EVQLVESGGGLVQPGPSLRLSCTTS 1-25 25
212
HFR1
Kabat EVQLVESGGGLVQPGPSLRLSCTTSGFTFT
1-30 30 213
Contact EVQLVESGGGLVQPGPSLRLSCTTSGFTF- 1-29
29 214
Chothia GFTFTHY--- 26-32 7
18
AbM GFTFTHYYMS 26-35 10
42
CDR-H1 Kabat HYYMS
31-35 5 43
Contact THYYMS 30-35 6
44
Chothia YMSWVRQAPGKGLEWLGFI 33-51
19 215
AbM WVRQAPGKGLEWLG-- 36-49 14
216
HFR2
Kabat WVRQAPGKGLEWLG-- 36-49 14
216
Contact ---WVRQAPGKGLE 36-46
11 217
Chothia RNKAKGYT 52-59 8
19
AbM ---FIRNKAKGYTAE 50-61 12
45
CDR-H2
Kabat ---FIRNKAKGYTAEYSASVKG
50-68 19 46
Contact WLGFIRNKAKGYTAE 47-61 15
47
Chothia AEYSASVKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYCAR 60-100 41 218
AbM --YSASVKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYCAR 62-100 39 219
HFR3
Kabat
RFTISRDNSKSTLYLQMNSLRAEDTAVYYCAR 69-100 32 220
Contact --YSASVKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYC-- 62-98 37 221
Chothia --DIGDN 101-
105 5 20
AbM --DIGDN 101-105 5
20
CDR-H3 Kabat --DIGDN
101-105 5 20
Contact ARDIGD- 99-104 6
48
Chothia -WGQGTLVTVSS
106-116 11 222
AbM -WGQGTLVTVSS 106-116 11
222
HFR4
Kabat -WGQGTLVTVSS
106-116 11 222
Contact NWGQGTLVTVSS
105-116 12 223
' The listed residues are residue numbers of the heavy chain variable region
(SEQ ID NO:299) of NIVIC-H103.VH4.
210
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Table 22. rnAb NMC-H103.VK4 Light Chain (hu IgG1) CDR and HFR
Sequences using Chothia, AbM, Kabat,and Contact CDR definitions:
Region Definition Sequence Fragment Residues Length
SEQ
NO:ID
18
Chothia DIVMTQTPLSSPVTLGQPASISC 1-23
23 224
AbM DIVMTQTPLSSPVTLGQPASISC 1-23 23
224
LFR1
Kabat DIVMTQTPLSSPVTLGQPASISC
1-23 23 224
Contact DIVMTQTPLSSPVTLGQPASISCRSSKNL 1-29
29 225
Chothia RSSKNLLHSNGITYLY-- 24-39 16
21
AbM RSSKNLLHSNGITYLY-- 24-39 16
21
CDR-L1 Kabat RSSKNLLHSNGITYLY--
24-39 16 21
Contact LHSNGITYLYWY 30-41 12
49
Chothia WYLQKPGQSPQLLIS 40-54
15 226
AbM WYLQKPGQSPQLLIS 40-54 15
226
LFR2
Kabat WYLQKPGQSPQLLIS
40-54 15 226
Contact --LQKPGQSPQ 42-50
9 227
Chothia RVSNLAS 55-61 7
22
AbM RVSNLAS 55-61 7
22
CDR-L2
Kabat RVSNLAS 55-61 7
22
Contact LLISRVSNLA- 51-60 10
50
Chothia -GVPDRFSGSESGTDFTLKISRVEAEDVGVYYC 62-93
32 228
AbM -GVPDRFSGSESGTDFTLKISRVEAEDVGVYYC 62-93 32
228
LFR3
Kabat -GVPDRFSGSESGTDFTLKISRVEAEDVGVYYC
62-93 32 228
Contact SGVPDRFSGSESGTDFTLKISRVEAEDVGVYYC 62-93
33 229
Chothia AQLLELPYT 94-102 9
23
AbM AQLLELPYT 94-102 9
23
CDR-L3 Kabat AQLLELPYT
94-102 9 23
Contact AQLLELPY- 94-102 8
51
Chothia -FGGGTKVEIK
103-112 10 230
AbM -FGGGTKVEIK 103-112 10
230
LFR4
Kabat -FGGGTKVEIK
103-112 10 230
Contact TFGGGTKVEIK
102-112 11 231
18 The listed residues are residue numbers of the light chain variable region
(SEQ ID NO:301) of NMC-H103.VK4.
211
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Table 23. rnAb NMC-H103.VK5 Light Chain (hu IgG1) CDR and HFR
Sequences using Chothia, AbM, Kabat,and Contact CDR definitions:
Region Definition Sequence Fragment Residues Length
SEQ
NO:ID
19
Chothia DIVMTQTPLSSPVTLGQPASISC 1-23
23 232
AbM DIVMTQTPLSSPVTLGQPASISC 1-23 23
232
LFR1
Kabat DIVMTQTPLSSPVTLGQPASISC
1-23 23 232
Contact DIVMTQTPLSSPVTLGQPASISCRSSKNL 1-29
29 233
Chothia RSSKNLLHSNGITYLY-- 24-39 16
21
AbM RSSKNLLHSNGITYLY-- 24-39 16
21
CDR-L1 Kabat RSSKNLLHSNGITYLY--
24-39 16 21
Contact LHSNGITYLYWY 30-41 12
49
Chothia WYLQKPGQSPQLLIS 40-54
15 234
AbM WYLQKPGQSPQLLIS 40-54 15
234
LFR2
Kabat WYLQKPGQSPQLLIS
40-54 15 234
Contact --LQKPGQSPQ 42-50
9 235
Chothia RVSNRAS 55-61 7
236
AbM RVSNRAS 55-61 7
236
CDR-L2
Kabat RVSNRAS 55-61 7
236
Contact LLISRVSNRAS 51-60
10 237
Chothia -GVPDRFSGSESGTDFTLKISRVEAEDVGVYYC 62-93
32 238
AbM -GVPDRFSGSESGTDFTLKISRVEAEDVGVYYC 62-93 32
238
LFR3
Kabat -GVPDRFSGSESGTDFTLKISRVEAEDVGVYYC
62-93 32 238
Contact SGVPDRFSGSESGTDFTLKISRVEAEDVGVYYC 62-93
33 239
Chothia AQLLELPYT 94-102 9
23
AbM AQLLELPYT 94-102 9
23
CDR-L3 Kabat AQLLELPYT
94-102 9 23
Contact AQLLELPY- 94-102 8
51
Chothia -FGGGTKVEIK
103-112 10 240
AbM -FGGGTKVEIK 103-112 10
240
LFR4
Kabat -FGGGTKVEIK
103-112 10 240
Contact TFGGGTKVEIK
102-112 11 241
19 The listed residues are residue numbers of the light chain variable region
(SEQ ID NO:303) of NMC-H103.VK5.
212
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Table 24. rnAb NMC-H103.VH6 Heavy Chain (hu IgG1) CDR and HFR
Sequences using Chothia, AbM, Kabat,and Contact CDR definitions:
Region Definition Sequence Fragment
Residues Length SEQ
NO:ID
Chothia EVQLVESGGGLVQPGGSLRLSCTTS 1-25
25 242
AbM EVQLVESGGGLVQPGGSLRLSCTTS
1-25 25 242
HFR1
Kabat EVQLVESGGGLVQPGGSLRLSCTTSGFTFT
1-30 30 243
Contact EVQLVESGGGLVQPGGSLRLSCTTSGFTF- 1-29
29 244
Chothia GFTFTHY--- 26-32
7 18
AbM GFTFTHYYMS 26-35 10
42
CDR-H1 Kabat HYYMS
31-35 5 43
Contact THYYMS 30-35 6
44
Chothia YMSWVRQAPGKGLEWLGFI 33-51
19 245
AbM WVRQAPGKGLEWLG--
36-49 14 246
HFR2
Kabat WVRQAPGKGLEWLG--
36-49 14 246
Contact ---WVRQAPGKGLE 36-46
11 247
Chothia RNKAKGYT 52-59 8
19
AbM ---FIRNKAKGYTAE 50-61 12
45
CDR-H2
Kabat ---FIRNKAKGYTAEYSASVKG
50-68 19 46
Contact WLGFIRNKAKGYTAE 47-61
15 47
Chothia AEYSASVKGRFTISRDNSQSILYLQMNSLRAEDTAVYYCAR 60-100 41 248
AbM --YSASVKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYCAR 62-100 39 249
HFR3
Kabat
RFTISRDNSKSTLYLQMNSLRAEDTAVYYCAR 69-100 32 250
Contact --YSASVKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYC-- 62-98
37 251
Chothia --DIGDN
101-105 5 20
AbM --DIGDN 101-105 5
20
CDR-H3 Kabat --DIGDN
101-105 5 20
Contact ARDIGD- 99-104
6 48
Chothia -WGQGTLVTVSS
106-116 11 252
AbM -WGQGTLVTVSS
106-116 11 252
HFR4
Kabat -WGQGTLVTVSS
106-116 11 252
Contact NWGQGTLVTVSS
105-116 12 253
' The listed residues are residue numbers of the heavy chain variable region
(SEQ ID NO:305) of NIVIC-H103.VH6.
213
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Table 25. rnAb NMC-H103.VK6 Light Chain (hu IgG1) CDR and HFR
Sequences using Chothia, AbM, Kabat,and Contact CDR definitions:
Region Definition Sequence Fragment Residues Length
SEQ
NO:ID
21
Chothia DIVMTQTPLSLPVTLGQPASISC 1-23
23 254
AbM DIVMTQTPLSLPVTLGQPASISC 1-23 23
254
LFR1
Kabat DIVMTQTPLSLPVTLGQPASISC
1-23 23 254
Contact DIVMTQTPLSLPVTLGQPASISCRSSKNL 1-29
29 255
Chothia RSSKNLLHSNGITYLY 24-39 10
21
AbM RSSKNLLHSNGITYLY 24-39 10
21
CDR-L1 Kabat RSSKNLLHSNGITYLY
24-39 10 21
Contact LHSNGITYLYWY-- 30-41 12
49
Chothia WYLQKPGQSPQLLIS 40-54
15 256
AbM WYLQKPGQSPQLLIS 40-54 15
256
LFR2
Kabat WYLQKPGQSPQLLIS
40-54 15 256
Contact --LQKPGQSPQ 42-50
9 257
Chothia RVSNLAS 55-61 7
22
AbM RVSNLAS 55-61 7
22
CDR-L2
Kabat RVSNLAS 55-61 7
22
Contact LLISRVSNLA- 51-60 10
50
Chothia -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC 62-93
32 258
AbM -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC 62-93 32
258
LFR3
Kabat -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC
62-93 32 258
Contact SGVPDRFSGSESGTDFTLKISRVEAEDVGVYFC 62-93
33 259
Chothia AQLLELPYT 94-102 9
23
AbM AQLLELPYT 94-102 9
23
CDR-L3 Kabat AQLLELPYT
94-102 9 23
Contact AQLLELPY- 94-102 8
51
Chothia FGGGTKVEIK-
103-112 10 260
AbM FGGGTKVEIK- 103-112 10
260
LFR4
Kabat FGGGTKVEIK- 103-112 10
260
Contact TFGGGTKVEIK
102-112 11 261
2.1 The listed residues are residue numbers of the light chain variable region
(SEQ ID NO:307) of NMC-H103.VK6.
214
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Table 26. rnAb NMC-H103.VH7 Heavy Chain (hu IgG1) CDR and HFR
Sequences using Chothia, AbM, Kabat,and Contact CDR definitions:
Region Definition Sequence Fragment Residues Length
SEQ
NO:ID
22
Chothia EVQLVESGGGLVQPGPSLRLSCTTS 1-25
25 262
AbM EVQLVESGGGLVQPGPSLRLSCTTS 1-25 25
262
HFR1
Kabat EVQLVESGGGLVQPGPSLRLSCTTSGFTFT
1-30 30 263
Contact EVQLVESGGGLVQPGPSLRLSCTTSGFTF- 1-29
29 264
Chothia GFTFTHY--- 26-32 7
18
AbM GFTFTHYYMS 26-35 10
42
CDR-H1 Kabat HYYMS
31-35 5 43
Contact THYYMS 30-35 6
44
Chothia YMSWVRQAPGKGLEWLGFI 33-51
19 265
AbM WVRQAPGKGLEWLG-- 36-49 14
266
HFR2
Kabat WVRQAPGKGLEWLG-- 36-49 14
266
Contact ---WVRQAPGKGLE 36-46
11 267
Chothia RNKAKGYT 52-59 8
19
AbM ---FIRNKAKGYTAE 50-61 12
45
CDR-H2
Kabat ---FIRNKAKGYTAEYSASVKG
50-68 19 46
Contact WLGFIRNKAKGYTAE 47-61 15
47
Chothia AEYSASVKGRFTISRDNSQSILYLQMNSLRAEDTAVYYCAR 60-100 41 268
AbM --YSASVKGRFTISRDNSQSILYLQMNSLRAEDTAVYYCAR 62-100 39 269
HFR3
Kabat
RFTISRDNSQSILYLQMNSLRAEDTAVYYCAR 69-100 32 270
Contact --YSASVKGRFTISRDNSQSILYLQMNSLRAEDTAVYYC-- 62-98 37 271
Chothia --DIGDN 101-
105 5 20
AbM --DIGDN 101-105 5
20
CDR-H3 Kabat --DIGDN
101-105 5 20
Contact ARDIGD- 99-104 6
48
Chothia -WGQGTLVTVSS
106-116 11 272
AbM -WGQGTLVTVSS 106-116 11
272
HFR4
Kabat -WGQGTLVTVSS
106-116 11 272
Contact NWGQGTLVTVSS
105-116 12 273
22. The listed residues are residue numbers of the heavy chain variable region
(SEQ ID NO:309) of NIVIC-H103.VH7.
215
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Table 27. rnAb NMC-H103.VK7 Light Chain (hu IgG1) CDR and HFR
Sequences using Chothia, AbM, Kabat,and Contact CDR definitions:
Region Definition Sequence Fragment
Residues Length SE:
ID
23
Chothia DIVMTQTPLSSPVTLGQSASISC 1-23
23 274
LFR1 AbM DIVMTQTPLSSPVTLGQSASISC 1-23 23
274
Kabat DIVMTQTPLSSPVTLGQSASISC 1-23 23
274
Contact DIVMTQTPLSSPVTLGQSASISCRSSKNL
1-29 29 275
Chothia RSSKNLLHSNGITYLY-- 24-39 10
21
AbM RSSKNLLHSNGITYLY-- 24-39 10
21
CDR-L1 Kabat RSSKNLLHSNGITYLY-- 24-39 10
21
Contact LHSNGITYLYWY 30-41 12 49
Chothia WYLQKPGQSPQLLIS 40-54 15
276
LFR2 AbM WYLQKPGQSPQLLIS 40-54 15
276
Kabat WYLQKPGQSPQLLIS 40-54 15
276
Contact --LQKPGQSPQ 42-50 9 277
Chothia RVSNLAS 55-61 7 22
AbM RVSNLAS 55-61 7
22
CDR-L2
Kabat RVSNLAS 55-61 7
22
Contact LLISRVSNLA- 51-60 10 50
Chothia -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC
62-93 32 278
AbM -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC 62-93 32
278
LFR3
Kabat -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC 62-93 32
278
Contact SGVPDRFSGSESGTDFTLKISRVEAEDVGVYFC
62-93 33 279
Chothia AQLLELPYT 94-102 9 23
AbM AQLLELPYT 94-102 9
23
CDR-L3 Kabat AQLLELPYT 94-102 9
23
Contact AQLLELPY- 94-102 8 51
Chothia -FGGGTKVEIK 103-112 10 280
AbM -FGGGTKVEIK 103-112 10
280
LFR4
Kabat -FGGGTKVEIK 103-112 10
280
Contact TFGGGTKVEIK 102-112 11 281
' The listed residues are residue numbers of the light chain variable region
(SEQ ID NO:311) of NIVIC-H103.VK7.
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mAb NMC-103 Heavy Chain Variable Region DNA Sequence (SEQ ID NO:
132) :
gaggtgcagctgcaggagtctggaggaggcttggtacagcctgggggttctctgagactctcctg
tacaacttctgggttcaccttcactcattactacatgagctgggtccgccagcctccaggcaagg
cacttgagtggttgggctttattagaaataaagctaagggttacacagcagagtacagtgcatct
gtgaagggtcggttcaccatctccagagataattcccaaagcatcctctatcttcaaatgaacac
cctgagacctgaggacagtgccacttattactgtgcaagagatattggggacaactggggtcaag
gaaccttagtcaccgtctcctcag
mAb NMC-103 Heavy Chain Variable Region Protein Sequence (SEQ ID
NO: 36)(Complementarity determining regions (CDRs) determined
according to the IMGT numbering system are underlined):
EVQLQESGGGLVQPGGSLRLSCTTSGFTFTHYYMSWVRQPPGKALEWLGFIRNKAKGYTAEYSAS
VKGRFTISRDNSQSILYLQMNTLRPEDSATYYCARDIGDNWGQGTLVTVSS
mAb NMC-103 Light Chain Variable Region DNA Sequence (SEQ ID NO:
134):
gatattgtgatgacgcaggctgccttctccaatccagtcactcttggaacatcagcttccatctc
ctgcaggtctagtaagaatctcctacatagtaatggcatcacttatttgtattggtatctgcaga
ggccaggccagtctcctcagctcctgatatctcgggtgtccaatctggcctcaggagtcccaaac
aggttcagtggcagtgagtcaggaactgatttcacactgagaatcagcagagtggaggctgagga
tgtgggtgtttatttctgtgctcaactgctagaactcccgtacacgttcggaggggggaccaagt
tggaaataaaac
mAb NMC-103 Light Chain Variable Region Protein Sequence (SEQ ID
NO: 37)(Complementarity determining regions (CDRs) determined
according to the IMGT numbering system are underlined):
DIVMTQAAFSNPVTLGTSASISCRSSKNLLHSNGITYLYWYLQRPGQSPQLLISRVSNLASGVPN
RFSGSESGTDFTLRISRVEAEDVGVYFCAQLLELPYTFGGGTKLEIK
mAb NMC-204 Heavy Chain Variable Region DNA Sequence (SEQ ID NO:
136):
217
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gaggtgcagctgcaggagtctgggtctgtgctggtgaggcctggagcttcagtgaagctgtcctg
caaggcttctggcgacaccctcagcggctcctggatgcactgggcgatgcagaggcctggacaag
gccttgagtggattggagagattcatcttaatagaggtactactaactacaatgagaagttcaag
ggcaaggccacagtgactgtggacacatcctccagcacagcctacgtggatctcagcagcctgac
atctgaggactctgcggtctattactgtgcaagaagcccggggtttgcttactggggccaaggga
ctctggtcactgtctctgcag
mAb NMC-204 Heavy Chain Variable Region Protein Sequence (SEQ ID
NO: 38)(Complementarity determining regions (CDRs) determined
according to the IMGT numbering system are underlined):
EVQLQESGSVLVRPGASVKLSCKASGDTLSGSWMHWAMQRPGQGLEWIGEIHLNRGTTNYNEKFK
GKATVTVDTSSSTAYVDLSSLTSEDSAVYYCARSPGFAYWGQGTLVTVSA
mAb NMC-204 Light Chain Variable Region DNA Sequence (SEQ ID NO:
138):
ggcattgtgatgacccaggctgcaccctctgtacctgtcactcctggagagtcagtatccatctc
ctgcaggtctagtaagagtctcctgcatagtaatggcaacagttacttgtattggttcctgcaga
ggccaggccagtctcctcagctcctgatatatcggatgtccaaccttgcctcaggagtcccagac
aggttcagtggcagtgggtcaggaactgctttcacactgagaatcactagagtggaggctgagga
tgtgggtgtttattactgtatgcaacatctagaatatcctttcacgttcggctcggggacaaagt
tggaaataaaac
mAb NMC-204 Light Chain Variable Region Protein Sequence (SEQ ID
NO: 39)(Complementarity determining regions (CDRs) determined
according to the IMGT numbering system are underlined):
GIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNSYLYWFLQRPGQSPQLLIYRMSNLASGVPD
RFSGSGSGTAFTLRITRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK
mAb NMC-303 Heavy Chain Variable Region DNA Sequence (SEQ ID NO:
140):
caggtccaactgcagcagcctggggctgaactggtgaagcctggggcttcagtgaagttgtcctg
caaggcttctggctacaccttcaccagctactatatgtactgggtgaagcagaggcctggacaag
218
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gccttgagtggattggggggattaatcctaggaatggtggtactaacttcaatgagaagttcaag
aacaaggccacactgactgcagacaaatcctccaccacagcctacatgcaactcagtagcctgac
atctgaggactctgcggtctattactgtacaagatctggttactatgctatggactattggggtc
aaggaacctcagtcaccgtctcctca
mAb NMC-303 Heavy Chain Variable Region Protein Sequence (SEQ ID
NO: 40)( Complementarity determining regions (CDRs) determined
according to the Kabat numbering system are underlined):
QVQLQQPGAELVKPGASVKLSCKASGYTFTSYYMYWVKQRPGQGLEWIGGINPRNGGTNFNEKFK
NKATLTADKSSTTAYMQLSSLTSEDSAVYYCTRSGYYAMDYWGQGTSVTVSS
mAb NMC-303 Light Chain Variable Region DNA Sequence (SEQ ID NO:
142):
gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcag
ttgcagggcaagtcaggacattagcaattttttaaactggtatcagcagaaaccagatggaactg
ttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagt
gggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttactt
ttgccaacagggtaatacgcttcctcggacgttcggtggaggcaccaagctggaaatcaaa
mAb NMC-303 Light Chain Variable Region Protein Sequence (SEQ ID
NO: 41)( Complementarity determining regions (CDRs) determined
according to the Kabat numbering system are underlined):
DIQMTQTTSSLSASLGDRVTISCRASQDISNFLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGS
GSGTDYSLTISNLEQEDIATYFCQQGNTLPRTFGGGTKLEIK
mAb NMC-C303 Heavy Chain Variable Region DNA Sequence (SEQ ID NO:
140):
caggtccaactgcagcagcctggggctgaactggtgaagcctggggcttcagtgaagttgtcctg
caaggcttctggctacaccttcaccagctactatatgtactgggtgaagcagaggcctggacaag
gccttgagtggattggggggattaatcctaggaatggtggtactaacttcaatgagaagttcaag
aacaaggccacactgactgcagacaaatcctccaccacagcctacatgcaactcagtagcctgac
219
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atctgaggactctgcggtctattactgtacaagatctggttactatgctatggactattggggtc
aaggaacctcagtcaccgtctcctca
mAb NMC-C303 Heavy Chain Variable Region Protein Sequence (SEQ ID
NO: 40)(CDRs determined according to the Kabat numbering system
are underlined):
QVQLQQPGAELVKPGASVKLSCKASGYTFTSYYMYWVKQRPGQGLEWIGGINPRNGGTNFNEKFK
NKATLTADKSSTTAYMQLSSLTSEDSAVYYCTRSGYYAMDYWGQGTSVTVSS
mAb NMC-C303 Light Chain Variable Region DNA Sequence (SEQ ID NO:
142):
gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcag
ttgcagggcaagtcaggacattagcaattttttaaactggtatcagcagaaaccagatggaactg
ttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagt
gggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttactt
ttgccaacagggtaatacgcttcctcggacgttcggtggaggcaccaagctggaaatcaaa
mAb NMC-C303 Light Chain Variable Region Protein Sequence (SEQ ID
NO: 41)(CDRs determined according to the Kabat numbering system
are underlined):
DIQMTQTTSSLSASLGDRVTISCRASQDISNFLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGS
GSGTDYSLTISNLEQEDIATYFCQQGNTLPRTFGGGTKLEIK
mAb NMC-C103.VE0 Heavy Chain Variable Region DNA Sequence (SEQ ID
NO: 282):
GAGGTCAAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGTTCTCTGAGACTCTCCTG
TACAACTTCTGGGTTCACCTTCACTCATTACTACATGAGCTGGGTCCGCCAGCCTCCAGGCAAGG
CACTTGAGTGGITGGGCTITATTAGAAATAAAGCTAAGGGITACACAGCAGAGTACAGTGCATCT
GTGAAGGGICGGITCACCATCTCCAGAGATAATTCCCAAAGCATCCTCTATCTICAAATGAACAC
CCTGAGGCCTGAGGACAGTGCCACTTATTACTGTGCAAGAGATATTGGGGACAACTGGGGTCAAG
GAACCTTAGTCACCGTCTCCTCA
220
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mAb NMC-C103.VHO Heavy Chain Variable Region Protein Sequence
(SEQ ID NO: 283) (CDRs determined according to the Kabat numbering
system are underlined):
EVKLVESGGGLVQPGGSLRLSCTTSGFTFTHYYMSWVRQPPGKALEWLGFIRNKAKGYTAaYSAS
VKGRFTISRDNSQSILYLQMNTLRPEDSATYYCARDIGDNWGQGTLVTVSS
mAb NMC-C103.VKO Light Chain Variable Region DNA Sequence (SEQ ID
NO: 284):
GATATTGTGATGACGCAGGCTGCCTTCTCCAATCCAGTCACTCTTGGAACATCAGCTTCCATCTC
CTGCAGGTCTTCCAAGAATCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGA
GGCCAGGCCAGICTCCTCAGCTCCTGATATCTCGGGIGTCCAATCTGGCCTCAGGAGTCCCAAAC
AGGTTCAGTGGCTCCGAGTCAGGAACTGATTTCACACTGAGAATCAGCAGAGTGGAGGCTGAGGA
TGTGGGTGTTTATTTCTGTGCTCAACTGCTAGAACTCCCGTACACGTTCGGAGGGGGGACCAAGC
TGGAAATAAAA
mAb NMC-C103.VKO Light Chain Variable Region Protein Sequence
(SEQ ID NO: 285)(CDRs determined according to the Kabat numbering
system are underlined):
DIVMTQAAFSNPVTLGTSASISCRSSKNLLHSNGITYLYWYLQRPGQSPQLLISRVSNLASGVPN
RFSGSESGTDFTLRISRVEAEDVGVYFCAQLLELPYTFGGGTKLEIK
mAb NMC-H103.VE1 Heavy Chain Variable Region DNA Sequence (SEQ ID
NO: 286):
GAGGTCAAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGCCGTCTCTGAGACTCTCCTG
TACAACTTCTGGGTTCACCTTCACTCATTACTACATGAGCTGGGTCCGCCAGGCTCCAGGCAAGG
GGCTIGAGTGGITGGGCTITATTAGAAATAAAGCTAAGGGITACACAGCAGAGTACAGTGCATCT
GTGAAGGGICGGTICACCATCTCCAGAGATAATTCCAAGAGCACGCTCTATCTICAAATGAACAC
CCTGAGAGCTGAGGACAGTGCCACTTATTACTGTGCAAGAGATATTGGGGACAACTGGGGCCAAG
GAACCCTGGTCACCGTCTCCTCA
mAb NMC-H103.VE1 Heavy Chain Variable Region Protein Sequence
(SEQ ID NO: 287)(CDRs determined according to the Kabat numbering
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system are underlined):
EVKLVESGGGLVQPGPSLRLSCTTSGFTFTHYYMSWVRQAPGKGLEWLGFIRNKAKGYTAaYSAS
VKGRFTISRDNSKSTLYLQMNTLRAEDSATYYCARDIGDNWGQGTLVTVSS
mAb NMC-H103.VK1 Light Chain Variable Region DNA Sequence (SEQ ID
NO: 288):
GATATTGTGATGACGCAGGCTGCCTTCTCCAATCCAGTCACTCTTGGACAGCCGGCTTCCATCTC
CTGCAGGTCTTCCAAGAATCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGA
AGCCAGGCCAGICTCCTCAGCTCCTGATATCTCGGGIGTCCAATCTGGCCTCAGGAGTCCCAAAT
AGGTICAGTGGCTCCGAGTCAGGAACTGATTICACACTGAAAATCAGCAGAGTGGAGGCTGAGGA
TGTGGGTGTTTATTTCTGTGCTCAACTGCTAGAACTCCCGTACACGTTCGGAGGGGGGACCAAGG
TGGAAATCAAA
mAb NMC-H103.VK1 Light Chain Variable Region Protein Sequence
(SEQ ID NO: 289)(CDRs determined according to the Kabat numbering
system are underlined):
DIVMTQAAFSNPVTLGQPASISCRSSKNLLHSNGITYLYWYLQKPGQSPQLLISRVSNLASGVPN
RFSGSESGTDFTLKISRVEAEDVGVYFCAQLLELPYTFGGGTKVEIK
mAb NMC-H103.VE2 Heavy Chain Variable Region DNA Sequence (SEQ ID
NO: 290):
GAGGTCAAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGCCGTCTCTGAGACTCTCCTG
TACAACTTCTGGGTTCACCTTCACTCATTACTACATGAGCTGGGTCCGCCAGGCTCCAGGCAAGG
GGCTIGAGTGGITGGGCTITATTAGAAATAAAGCTAAGGGITACACAGCAGAGTACAGTGCATCT
GTGAAGGGICGGTICACCATCTCCAGAGATAATTCCAAGAGCACGCTCTATCTICAAATGAACAG
CCTGAGAGCTGAGGACACGGCCACTTATTACTGTGCAAGAGATATTGGGGACAACTGGGGCCAAG
GAACCCTGGTCACCGTCTCCTCA
mAb NMC-H103.VE2 Heavy Chain Variable Region Protein Sequence
(SEQ ID NO: 291)(CDRs determined according to the Kabat numbering
system are underlined):
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EVKLVESGGGLVQPGPSLRLSCTTSGFTFTHYYMSWVRQAPGKGLEWLGFIRNKAKGYTAaYSAS
VKGRFTISRDNSKSTLYLQMNSLRAEDTATYYCARDIGDNWGQGTLVTVSS
mAb NMC-H103.VK2 Light Chain Variable Region DNA Sequence (SEQ ID
NO: 292):
GATATTGTGATGACGCAGACTCCACTCTCCTCACCAGTCACTCTTGGACAGCCGGCTTCCATCTC
CTGCAGGTCTTCCAAGAATCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGA
AGCCAGGCCAGICTCCTCAGCTCCTGATATCTCGGGIGTCCAATCTGGCCTCAGGAGTCCCAAAT
AGGTICAGTGGCTCCGAGTCAGGAACTGATTICACACTGAAAATCAGCAGAGTGGAGGCTGAGGA
TGTGGGTGTTTATTTCTGTGCTCAACTGCTAGAACTCCCGTACACGTTCGGAGGGGGGACCAAGG
TGGAAATCAAA
mAb NMC-H103.VK2 Light Chain Variable Region Protein Sequence
(SEQ ID NO: 293)(CDRs determined according to the Kabat numbering
system are underlined):
DIVMTQTPLSSPVTLGQPASISCRSSKNLLHSNGITYLYWYLQKPGQSPQLLISRVSNLASGVPN
RFSGSESGTDFTLKISRVEAEDVGVYFCAQLLELPYTFGGGTKVEIK
mAb NMC-H103.VE3 Heavy Chain Variable Region DNA Sequence (SEQ ID
NO: 294):
GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGCCGTCTCTGAGACTCTCCTG
TACAACTTCTGGGTTCACCTTCACTCATTACTACATGAGCTGGGTCCGCCAGGCTCCAGGCAAGG
GGCTIGAGTGGITGGGCTITATTAGAAATAAAGCTAAGGGITACACAGCAGAGTACAGTGCATCT
GTGAAGGGICGGTICACCATCTCCAGAGATAATTCCAAGAGCACGCTCTATCTICAAATGAACAG
CCTGAGAGCTGAGGACACGGCCACTTATTACTGTGCAAGAGATATTGGGGACAACTGGGGCCAAG
GAACCCTGGTCACCGTCTCCTCA
mAb NMC-H103.VE3 Heavy Chain Variable Region Protein Sequence
(SEQ ID NO: 295)(CDRs determined according to the Kabat numbering
system are underlined):
EVQLVESGGGLVQPGPSLRLSCTTSGFTFTHYYMSWVRQAPGKGLEWLGFIRNKAKGYTAaYSAS
VKGRFTISRDNSKSTLYLQMNSLRAEDTATYYCARDIGDNWGQGTLVTVSS
223
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mAb NMC-H103.VK3 Light Chain Variable Region DNA Sequence (SEQ ID
NO: 296) :
GATATTGTGATGACGCAGACTCCACTCTCCTCACCAGTCACTCTTGGACAGCCGGCTTCCATCTC
CTGCAGGTCTTCCAAGAATCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGA
AGCCAGGCCAGTCTCCTCAGCTCCTGATATCTCGGGTGTCCAATCTGGCCTCAGGAGTCCCAGAC
AGGTICAGTGGCTCCGAGTCAGGAACTGATTICACACTGAAAATCAGCAGAGTGGAGGCTGAGGA
TGTGGGTGTTTATTTCTGTGCTCAACTGCTAGAACTCCCGTACACGTTCGGAGGGGGGACCAAGG
TGGAAATCAAA
mAb NMC-H103.VK3 Light Chain Variable Region Protein Sequence
(SEQ ID NO: 297)(CDRs determined according to the Kabat numbering
system are underlined):
DIVMTQTPLSSPVTLGQPASISCRSSKNLLHSNGITYLYWYLQKPGQSPQLLISRVSNLASGVPD
RFSGSESGTDFTLKISRVEAEDVGVYFCAQLLELPYTFGGGTKVEIK
mAb NMC-H103.VE4 Heavy Chain Variable Region DNA Sequence (SEQ ID
NO: 298):
GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGCCGTCTCTGAGACTCTCCTG
TACAACTTCTGGGTTCACCTTCACTCATTACTACATGAGCTGGGTCCGCCAGGCTCCAGGCAAGG
GGCTIGAGTGGITGGGCTITATTAGAAATAAAGCTAAGGGITACACAGCAGAGTACAGTGCATCT
GTGAAGGGICGGTICACCATCTCCAGAGATAATTCCAAGAGCACGCTCTATCTICAAATGAACAG
CCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCAAGAGATATTGGGGACAACTGGGGCCAAG
GAACCCTGGTCACCGTCTCCTCA
mAb NMC-H103.VE4 Heavy Chain Variable Region Protein Sequence
(SEQ ID NO: 299)(CDRs) determined according to the Kabat
numbering system are underlined):
EVQLVESGGGLVQPGPSLRLSCTTSGFTFTHYYMSWVRQAPGKGLEWLGFIRNKAKGYTAaYSAS
VKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYCARDIGDNWGQGTLVTVSS
224
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mAb NMC-H103.VK4 Light Chain Variable Region DNA Sequence (SEQ ID
NO: 300) :
GATATTGTGATGACGCAGACTCCACTCTCCTCACCAGTCACTCTTGGACAGCCGGCTTCCATCTC
CTGCAGGTCTTCCAAGAATCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGA
AGCCAGGCCAGTCTCCTCAGCTCCTGATATCTCGGGTGTCCAATCTGGCCTCAGGAGTCCCAGAC
AGGTICAGTGGCTCCGAGTCAGGAACTGATTICACACTGAAAATCAGCAGAGTGGAGGCTGAGGA
TGTGGGTGTTTATTACTGTGCTCAACTGCTAGAACTCCCGTACACGTTCGGAGGGGGGACCAAGG
TGGAAATCAAA
mAb NMC-H103.VK4 Light Chain Variable Region Protein Sequence
(SEQ ID NO: 301)(CDRs) determined according to the Kabat
numbering system are underlined):
DIVMTQTPLSSPVTLGQPASISCRSSKNLLHSNGITYLYWYLQKPGQSPQLLISRVSNLASGVPD
RFSGSESGTDFTLKISRVEAEDVGVYYCAQLLELPYTFGGGTKVEIK
mAb NMC-H103.VK5 Light Chain Variable Region DNA Sequence (SEQ ID
NO: 302):
GATATTGTGATGACGCAGACTCCACTCTCCTCACCAGTCACTCTTGGACAGCCGGCTTCCATCTC
CTGCAGGTCTTCCAAGAATCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGA
AGCCAGGCCAGTCTCCTCAGCTCCTGATATCTCGGGTGTCCAATCGGGCCTCAGGAGTCCCAGAC
AGGTICAGTGGCTCCGAGTCAGGAACTGATTICACACTGAAAATCAGCAGAGTGGAGGCTGAGGA
TGTGGGTGTTTATTACTGTGCTCAACTGCTAGAACTCCCGTACACGTTCGGAGGGGGGACCAAGG
TGGAAATCAAA
mAb NMC-H103.VK5 Light Chain Variable Region Protein Sequence
(SEQ ID NO: 303)(CDRs determined according to the Kabat numbering
system are underlined):
DIVMTQTPLSSPVTLGQPASISCRSSKNLLHSNGITYLYWYLQKPGQSPQLLISRVSNRASGVPD
RFSGSESGTDFTLKISRVEAEDVGVYYCAQLLELPYTFGGGTKVEIK
225
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mAb NMC-H103.VH6 Heavy Chain Variable Region DNA Sequence (SEQ ID
NO: 304) :
GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGGTCTCTGAGACTCTCCTG
TACAACTTCTGGGTTCACCTTCACTCATTACTACATGAGCTGGGTCCGCCAGGCTCCAGGCAAGG
GGCTIGAGTGGITGGGCTITATTAGAAATAAAGCTAAGGGITACACAGCAGAGTACAGTGCATCT
GTGAAGGGICGGTICACCATCTCCAGAGATAATTCCAAGAGCACGCTCTATCTICAAATGAACAG
CCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCAAGAGATATTGGGGACAACTGGGGCCAAG
GAACCCTGGTCACCGTCTCCTCA
mAb NMC-H103.VH6 Heavy Chain Variable Region Protein Sequence
(SEQ ID NO: 305)(CDRs determined according to the Kabat numbering
system are underlined):
EVQLVESGGGLVQPGGSLRLSCTTSGFTFTHYYMSWVRQAPGKGLEWLGFIRNKAKGYTAaYSAS
VKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYCARDIGDNWGQGTLVTVSS
mAb NMC-H103.VK6 Light Chain Variable Region DNA Sequence (SEQ ID
NO: 306):
GATATTGTGATGACGCAGACTCCACTCTCCCTGCCAGTCACTCTTGGACAGCCGGCTTCCATCTC
CTGCAGGTCTTCCAAGAATCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGA
AGCCAGGCCAGTCTCCTCAGCTCCTGATATCTCGGGTGTCCAATCTGGCCTCAGGAGTCCCAGAC
AGGTICAGTGGCTCCGAGTCAGGAACTGATTICACACTGAAAATCAGCAGAGTGGAGGCTGAGGA
TGTGGGTGTTTATTTCTGTGCTCAACTGCTAGAACTCCCGTACACGTTCGGAGGGGGGACCAAGG
TGGAAATCAAA
mAb NMC-H103.VK6 Light Chain Variable Region Protein Sequence
(SEQ ID NO: 307)(CDRs determined according to the Kabat numbering
system are underlined):
DIVMTQTPLSLPVTLGQPASISCRSSKNLLHSNGITYLYWYLQKPGQSPQLLISRVSNLASGVPD
RFSGSESGTDFTLKISRVEAEDVGVYFCAQLLELPYTFGGGTKVEIK
226
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mAb NMC-H103.VH7 Heavy Chain Variable Region DNA Sequence (SEQ ID
NO: 308) :
GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGCCGTCTCTGAGACTCTCCTG
TACAACTTCTGGGTTCACCTTCACTCATTACTACATGAGCTGGGTCCGCCAGGCTCCAGGCAAGG
GGCTIGAGTGGITGGGCTITATTAGAAATAAAGCTAAGGGITACACAGCAGAGTACAGTGCATCT
GTGAAGGGICGGTICACCATCTCCAGAGATAATTCCCAAAGCATCCTCTATCTICAAATGAACAG
CCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCAAGAGATATTGGGGACAACTGGGGCCAAG
GAACCCTGGTCACCGTCTCCTCA
mAb NMC-H103.VE7 Heavy Chain Variable Region Protein Sequence
(SEQ ID NO: 309)(CDRs determined according to the Kabat numbering
system are underlined):
EVQLVESGGGLVQPGPSLRLSCTTSGFTFTHYYMSWVRQAPGKGLEWLGFIRNKAKGYTAaYSAS
VKGRFTISRDNSQSILYLQMNSLRAEDTAVYYCARDIGDNWGQGTLVTVSS
mAb NMC-H103.VK7 Light Chain Variable Region DNA Sequence (SEQ ID
NO: 310):
GATATTGTGATGACGCAGACTCCACTCTCCTCACCAGTCACTCTTGGACAGTCAGCTTCCATCTC
CTGCAGGTCTTCCAAGAATCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGA
AGCCAGGCCAGTCTCCTCAGCTCCTGATATCTCGGGTGTCCAATCTGGCCTCAGGAGTCCCAGAC
AGGTICAGTGGCTCCGAGTCAGGAACTGATTICACACTGAAAATCAGCAGAGTGGAGGCTGAGGA
TGTGGGTGTTTATTTCTGTGCTCAACTGCTAGAACTCCCGTACACGTTCGGAGGGGGGACCAAGG
TGGAAATCAAA
mAb NMC-H103.VK7 Light Chain Variable Region Protein Sequence
(SEQ ID NO: 311)(CDRs determined according to the Kabat numbering
system are underlined):
DIVMTQTPLSSPVTLGQSASISCRSSKNLLHSNGITYLYWYLQKPGQSPQLLISRVSNLASGVPD
RFSGSESGTDFTLKISRVEAEDVGVYFCAQLLELPYTFGGGTKVEIK
mAb NMC-C303 Heavy Chain Protein Sequence (SEQ ID NO: 312):
CA 03127776 2021-07-23
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MGWSCIILFLVATATGVHSQVQLQQPGAELVKPGASVKLSCKASGYTFTSYYMYWVKQRPGQGLE
WIGGINPRNGGTNFNEKFKNKATLTADKSSTTAYMQLSSLTSEDSAVYYCTRSGYYAMDYWGQGT
SVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK
mAb NMC-C303 Light Chain Protein Sequence (SEQ ID NO: 313):
MGWSCIILFLVATATGVHSDIQMTQTTSSLSASLGDRVTISCRASQDISNFLNWYQQKPDGTVKL
LIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPRTFGGGTKLEIKRTVA
APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
INCORPORATION BY REFERENCE
[00600] Various references such as patents, patent applications, and
publications are cited
herein, the disclosures of which are hereby incorporated by reference herein
in their entireties.
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