Note: Descriptions are shown in the official language in which they were submitted.
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EFFICACY OF ANTI-HLA-DR ANTIBODY DRUG CONJUGATE IMMU-140
(hL243-CL2A-SN-38) IN HLA-DR POSITIVE CANCERS
INVENTORS:
SERENGULAM V. GOVINDAN
THOMAS M. CARDILLO
AND
DAVID M. GOLDENBERG
ASSIGNEE: IMMUNOMEDICS, INC.
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CROSS REFERENCE TO RELATED APPLICATIONS
[01] This application is a continuation-in-part of U.S. Patent Application
Serial No.
15/281,453, filed September 30, 2016, which was a divisional of U.S. Patent
Application
Serial No. 14/667,982 (now U.S. Patent No. 9,493,573), filed March 25, 2015,
which was a
divisional of U.S. Patent Application Serial No. 13/948,732 (now U.S. Patent
No. 9,028,833),
filed July 23, 2013, which claimed the benefit under 35 U.S.C. 119(e) of
Provisional U.S.
Patent Application Serial Nos. 61/749,548, filed January 7, 2013, and
61/736,684, filed
December 13, 2012. This application is a continuation-in-part of U.S. Patent
application
Serial No. 15/484,308, filed April 11, 2017, which claimed the benefit under
35 U.S.C.
119(e) of Provisional U.S. Patent Application Serial Nos. 62/373,591, filed
August 11, 2016,
and 62/322,441, filed April 14, 2016. This application claims the benefit
under 35 U.S.C.
119(e) of Provisional U.S. Patent Application Serial Nos. 62/373,591, filed
August 11, 2016,
and 62/428,231, filed November 30, 2016. The text of each priority application
incorporated
herein by reference in its entirety.
SEQUENCE LISTING
[02] The instant application contains a Sequence Listing which has been
submitted in ASCII
format via EFS-Web and is hereby incorporated by reference in its entirety.
Said ASCII
copy, created on July 25, 2017, is named IMM369W01 SL.txt and is 9,661 bytes
in size.
FIELD OF THE INVENTION
[03] The present invention relates to therapeutic use of immunoconjugates of
antibodies or
antigen-binding antibody fragments and camptothecins, such as SN-38, with
improved ability
to target various cancer cells in human subjects. Preferably, the antibodies
or fragments are
anti-HLA-DR antibodies or fragments, such as hL243. In other preferred
embodiments, the
antibodies and therapeutic moieties are linked via an intracellularly-
cleavable linkage that
increases therapeutic efficacy. In more preferred embodiments, the
immunoconjugates are
administered at specific dosages and/or specific schedules of administration
that optimize the
therapeutic effect. The optimized dosages and schedules of administration of
SN-38-
conjugated anti-HLA-DR antibodies for human therapeutic use disclosed herein
show
unexpected superior efficacy that could not have been predicted from animal
model studies,
allowing effective treatment of cancers that are resistant to standard anti-
cancer therapies,
including the parental compound, irinotecan (CPT-11). The immunoconjugates may
be
administered alone or in combination with one or more other therapeutic
agents, administered
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before, concurrently with, or after the immunoconjugate. Exemplary therapeutic
agents that
may be used in combination with anti-HLA-DR include, but are not limited to
proteosome
inhibitors such as bortezomib, Bruton kinase inhibitors such as ibrutinib, or
phosphoinositide-
3-kinase inhibitors such as idelalisib. In preferred embodiments, the cancer
to be treated is an
HLA-DR positive cancer, such as B-cell lymphoma, B-cell leukemia, skin,
esophageal,
stomach, colon, rectal, pancreatic, lung, breast, ovarian, bladder,
endometrial, cervical,
testicular, melanoma, kidney, or liver cancer. More preferably, the cancer is
AML (acute
myelocytic leukemia), ALL (acute lymphocytic leukemia) or MM (multiple
myeloma). Most
preferably, the patient to be treated has relapsed from or shown resistance to
at least one
standard anti-cancer therapy, prior to treatment with the immunoconjugate.
However, the
person of ordinary skill will realize that in some embodiments the
immunoconjugate may be
employed in a front-line therapy.
BACKGROUND OF THE INVENTION
[04] For many years it has been an aim of scientists in the field of
specifically targeted drug
therapy to use monoclonal antibodies (MAbs) for the specific delivery of toxic
agents to
human cancers. Conjugates of tumor-associated MAbs and suitable toxic agents
have been
developed, but have had mixed success in the therapy of cancer in humans, and
virtually no
application in other diseases, such as autoimmune diseases. The toxic agent is
most
commonly a chemotherapeutic drug, although particle-emitting radionuclides, or
bacterial or
plant toxins, have also been conjugated to MAbs, especially for the therapy of
cancer
(Sharkey and Goldenberg, CA Cancer J Cl/n. 2006 Jul-Aug;56(4):226-243) and,
more
recently, with radioimmunoconjugates for the preclinical therapy of certain
infectious
diseases (Dadachova and Casadevall, Q J Nucl Med Mol Imaging 2006;50(3):193-
204).
[05] The advantages of using MAb-chemotherapeutic drug conjugates are that (a)
the
chemotherapeutic drug itself is structurally well defined; (b) the
chemotherapeutic drug is
linked to the MAb protein using very well-defined conjugation chemistries,
often at specific
sites remote from the MAbs' antigen binding regions; (c) MAb-chemotherapeutic
drug
conjugates can be made more reproducibly and usually with less immunogenicity
than
chemical conjugates involving MAbs and bacterial or plant toxins, and as such
are more
amenable to commercial development and regulatory approval; and (d) the MAb-
chemotherapeutic drug conjugates are orders of magnitude less toxic
systemically than
radionuclide MAb conjugates, particularly to the radiation-sensitive bone
marrow.
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[06] Camptothecin (CPT) and its derivatives are a class of potent antitumor
agents.
Irinotecan (also referred to as CPT-11) and topotecan are CPT analogs that are
approved
cancer therapeutics (Iyer and Ratain, Cancer Chemother. Phamacol. 42: S31-S43
(1998)).
CPTs act by inhibiting topoisomerase I enzyme by stabilizing topoisomerase I-
DNA complex
(Liu, et at. in The Camptothecins: Unfolding Their Anticancer Potential, Liehr
J.G.,
Giovanella, B.C. and Verschraegen (eds), NY Acad Sci., NY 922:1-10 (2000)).
CPTs
present specific issues in the preparation of conjugates. One issue is the
insolubility of most
CPT derivatives in aqueous buffers. Second, CPTs provide specific challenges
for structural
modification for conjugating to macromolecules. For instance, CPT itself
contains only a
tertiary hydroxyl group in ring-E. The hydroxyl functional group in the case
of CPT must be
coupled to a linker suitable for subsequent protein conjugation; and in potent
CPT
derivatives, such as SN-38, the active metabolite of the chemotherapeutic CPT-
11, and other
C-10-hydroxyl-containing derivatives such as topotecan and 10-hydroxy-CPT, the
presence
of a phenolic hydroxyl at the C-10 position complicates the necessary C-20-
hydroxyl
derivatization. Third, the lability under physiological conditions of the 6-
lactone moiety of
the E-ring of camptothecins results in greatly reduced antitumor potency.
Therefore, the
conjugation protocol is performed such that it is carried out at a pH of 7 or
lower to avoid the
lactone ring opening. However, conjugation of a bifunctional CPT possessing an
amine-
reactive group such as an active ester would typically require a pH of 8 or
greater. Fourth, an
intracellularly-cleavable moiety preferably is incorporated in the
linker/spacer connecting the
CPTs and the antibodies or other binding moieties.
[07] The human leukocyte antigen¨DR (HLA-DR) is one of three isotypes of the
major
histocompatibilty complex (MHC) class II antigens. HLA-DR is highly expressed
on a
variety of hematologic malignancies and has been actively pursued for antibody-
based
lymphoma therapy (Brown et al., 2001, Clin Lymphoma 2:188-90; DeNardo et al.,
2005, Clin
Cancer Res 11:7075s-9s; Stein et al., 2006, Bl000d 108:2736-44). The human HLA-
DR
antigen is expressed in non-Hodgkin lymphoma (NHL), chronic lymphocytic
leukemia
(CLL), and other B-cell malignancies at significantly higher levels than
typical B-cell
markers, including CD20. Preliminary studies indicate that anti-HLA-DR mAbs
are markedly
more potent than other naked mAbs of current clinical interest in in vitro and
in vivo
experiments in lymphomas, leukemias, and multiple myeloma (Stein et al.,
unpublished
results).
[08] HLA-DR is also expressed on a subset of normal immune cells, including B
cells,
monocytes/macrophages, Langerhans cells, dendritic cells, and activated T
cells (Dechant et
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al., 2003, Semin Oncol 30:465-75). Thus, it is perhaps not surprising that
prior attempts to
develop anti-HLA-DR antibodies have been hampered by toxicity, notably
infusion-related
toxicities that are likely related to complement activation (Lin et al, 2009,
Leuk Lymphoma
50:1958-63; Shi et al., 2002, Leuk Lymphoma 43:1303-12).
[09] The L243 antibody (hereafter mL243) is a murine IgG2a anti-HLA-DR
antibody. This
antibody may be of potential use in the treatment of diseases such as
autoimmune disease or
cancer, particularly leukemias or lymphomas, by targeting the D region of HLA.
mL243
demonstrates potent suppression of in vitro immune function and is monomorphic
for all
HLA-DR proteins. However, problems exist with the administration of mouse
antibodies to
human patients, such as the induction of a human anti-mouse antibody (HAMA)
response. A
need exists for more effective compositions and methods of use of anti-HLA-DR
antibodies,
with improved efficacy and decreased toxicity. A further need exists for more
effective
methods of preparing and administering antibody-CPT conjugates, such as anti-
HLA-DR-
SN-38 conjugates. Preferably, the methods comprise optimized dosing and
administration
schedules that maximize efficacy and minimize toxicity of the antibody-CPT
conjugates for
therapeutic use in human patients.
SUMMARY OF THE INVENTION
[010] As used herein, the abbreviation "CPT" may refer to camptothecin or any
of its
derivatives, such as SN-38, unless expressly stated otherwise. The present
invention resolves
an unfulfilled need in the art by providing improved methods and compositions
for preparing
and administering CPT-antibody immunoconjugates. Preferably, the camptothecin
is SN-38.
The disclosed methods and compositions are of use for the treatment of a
variety of diseases
and conditions which are refractory or less responsive to other forms of
therapy, and can
include diseases against which suitable antibodies or antigen-binding antibody
fragments for
selective targeting can be developed, or are available or known, such as
cancer.
[011] Preferably, the targeting moiety is an antibody, antibody fragment,
bispecific or other
multivalent antibody, or other antibody-based molecule or compound. More
preferably, the
antibody or fragment is an anti-HLA-DR antibody or fragment. The antibody can
be of
various isotypes, preferably human IgGl, IgG2, IgG3 or IgG4, more preferably
comprising
human IgG1 hinge and constant region sequences. Most preferably, the antibody
is a human
IgG4. The antibody or fragment thereof can be a chimeric human-mouse, a
chimeric human-
primate, a humanized (human framework and murine hypervariable (CDR) regions),
or fully
human antibody, as well as variations thereof, such as half-IgG4 antibodies
(referred to as
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"unibodies"), as described by van der Neut Kolfschoten etal. (Science 2007;
317:1554-
1557). More preferably, the antibody or fragment thereof may be designed or
selected to
comprise human constant region sequences that belong to specific allotypes,
which may
result in reduced immunogenicity when the immunoconjugate is administered to a
human
subject. Preferred allotypes for administration include a non-Glml allotype
(nG1m1), such
as G1m3, G1m3,1, G1m3,2 or G1m3,1,2. More preferably, the allotype is selected
from the
group consisting of the nGlml, G1m3, nG1m1,2 and Km3 allotypes.
[012] Where the disease state is cancer, many antigens expressed by or
otherwise associated
with tumor cells are known in the art, including but not limited to, carbonic
anhydrase IX,
alpha-fetoprotein (AFP), a-actinin-4, A3, antigen specific for A33 antibody,
ART-4, B7, Ba
733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8/mõ CCL19, CCL21, CD1,
CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20,
CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD4OL,
CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD7OL,
CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147, CD154,
CDC27, CDK-4/m, CDKN2A, CTLA-4, CXCR4, CXCR7, CXCL12, HIF-la, colon-specific
antigen-p (CSAp), CEA (CEACAM5), CEACAM6, c-Met, DAM, DLL3, DLL4, EGFR,
EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, fibroblast growth factor
(FGF),
Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-0, HLA-DR,
HM1.24,
human chorionic gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxia
inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-y, IFN-a, IFN-f3,
IFN-k, IL-
4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-
17, IL-18, IL-
23, IL-25, insulin-like growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen,
KS1-4, Le-Y,
LDR/FUT, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1,
MART-2, mesothelin, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF,
MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3, NCA66,
NCA95, NCA90, PAM4 antigen, pancreatic cancer mucin, PD-1 receptor, placental
growth
factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, ILGF,
ILGF-
1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC,
TAG-72,
tenascin, TRAIL receptors, TNF-a, Tn antigen, Thomson-Friedenreich antigens,
tumor
necrosis antigens, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complement
factors C3,
C3a, C3b, C5a, C5, an angiogenesis marker, bc1-2, bc1-6, Kras, an oncogene
marker and an
oncogene product (see, e.g., Sensi etal., Clin Cancer Res 2006, 12:5023-32;
Parmiani etal., J
Immunol 2007, 178:1975-79; Novellino etal. Cancer Immunol Immunother 2005,
54:187-
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207). Preferably, the antibody binds to CEACAM5, CEACAM6, EGP-1 (TROP-2), MUC-
16, AFP, MUC5ac, CD74, CD19, CD20, CD22 or HLA-DR. The preferred anti-HLA-DR
antibody may be utilized alone, or in combination with another anti-TAA (tumor-
associated
antigen) antibody.
[013] Exemplary antibodies that may be utilized include, but are not limited
to, hR1 (anti-
IGF-1R, U.S. Patent No. 9,441,043), hPAM4 (anti-mucin, U.S. Patent No.
7,282,567), hA20
(anti-CD20, U.S. Patent No. 7,151,164), hA19 (anti-CD19, U.S. Patent No.
7,109,304),
hIMMU31 (anti-AFP, U.S. Patent No. 7,300,655), hLL1 (anti-CD74, U.S. Patent
No.
7,312,318), hLL2 (anti-CD22, U.S. Patent No. 5,789,554), hMu-9 (anti-CSAp,
U.S. Patent
No. 7,387,772), hL243 (anti-HLA-DR, U.S. Patent No. 7,612,180), hMN-14 (anti-
CEACAM5, U.S. Patent No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Patent No.
8,287,865), hRS7 (anti-EGP-1, U.S. Patent No. 7,238,785), hMN-3 (anti-CEACAM6,
U.S.
Patent No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Patent No.
7,138,496), the
Examples section of each cited patent or application incorporated herein by
reference. More
preferably, the antibody is IMMU-31 (anti-AFP), hRS7 (anti-TROP-2), hMN-14
(anti-
CEACAM5), hMN-3 (anti-CEACAM6), hMN-15 (anti-CEACAM6), hLL1 (anti-CD74),
hLL2 (anti-CD22), hL243 or IMMU-114 (anti-HLA-DR), hA19 (anti-CD19) or hA20
(anti-
CD20). Most preferably, the antibody is hL243. As used herein, the terms
epratuzumab and
hLL2 are interchangeable, as are the terms veltuzumab and hA20 and the terms
hL243g4P,
hL243gamma4P and IMMU-114.
[014] Alternative antibodies of use include, but are not limited to, abciximab
(anti-
glycoprotein IIb/IIIa), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF),
cetuximab
(anti-EGFR), gemtuzumab (anti-CD33), ibritumomab (anti-CD20), panitumumab
(anti-
EGFR), rituximab (anti-CD20), tositumomab (anti-CD20), trastuzumab (anti-
ErbB2),
pembrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab
(anti-
CTLA-4), abagovomab (anti-CA-125), adecatumumab (anti-EpCAM), atlizumab (anti-
IL-6
receptor), benralizumab (anti-CD125), obinutuzumab (GA101, anti-CD20), CC49
(anti-
TAG-72), AB-PG1-XG1-026 (anti-PSMA, U.S. Patent Application 11/983,372,
deposited as
ATCC PTA-4405 and PTA-4406), D2/B (anti-PSMA, WO 2009/130575), tocilizumab
(anti-
IL-6 receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab
(anti-CD ii a),
GA101 (anti-CD20; Glycart Roche), muromonab-CD3 (anti-CD3 receptor),
natalizumab
(anti-a4 integrin), omalizumab (anti-IgE); anti-TNF-a antibodies such as
CDP571 (Ofei et
al., 2011, Diabetes 45:881-85), MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B,
M303 (Thermo Scientific, Rockford, IL), infliximab (Centocor, Malvern, PA),
certolizumab
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pegol (UCB, Brussels, Belgium), anti-CD4OL (UCB, Brussels, Belgium),
adalimumab
(Abbott, Abbott Park, IL), and Benlysta (Human Genome Sciences).
[015] In a preferred embodiment, the chemotherapeutic moiety is selected from
camptothecin (CPT) and its analogs and derivatives and is more preferably SN-
38. However,
other chemotherapeutic moieties that may be utilized include taxanes (e.g,
baccatin III, taxol),
epothilones, anthracyclines (e.g., doxorubicin (DOX), epirubicin,
morpholinodoxorubicin
(morpholino-DOX), cyanomorpholino-doxorubicin (cyanomorpholino-DOX), 2-
pyrrolinodoxorubicin (2-PDOX) or a prodrug form of 2-PDOX (pro-2-PDOX); see,
e.g.,
Priebe W (ed.), ACS symposium series 574, published by American Chemical
Society,
Washington D.C., 1995 (332pp) and Nagy et at., Proc. Natl. Acad. Sci. USA
93:2464-2469,
1996), benzoquinoid ansamycins exemplified by geldanamycin (DeBoer et at.,
Journal of
Antibiotics 23:442-447, 1970; Neckers et al., Invest. New Drugs /7:361-373,
1999), and the
like. Preferably, the antibody or fragment thereof links to at least one
chemotherapeutic
moiety; preferably 1 to about 5 chemotherapeutic moieties; more preferably 6
or more
chemotherapeutic moieties, more preferably 7 to 8, most preferably about 6 to
about 12
chemotherapeutic moieties.
[016] An example of a water soluble CPT derivative is CPT-11. Extensive
clinical data are
available concerning CPT-11's pharmacology and its in vivo conversion to the
active SN-38
(Iyer and Ratain, Cancer Chemother Pharmacol. 42:S31-43 (1998); Mathijssen et
at., Clin
Cancer Res. 7:2182-2194 (2002); Rivory, Ann 1VY Acad Sci. 922:205-215, 2000)).
The active
form SN-38 is about 2 to 3 orders of magnitude more potent than CPT-11. In
specific
preferred embodiments, the immunoconjugate may be an hMN-14-SN-38, hMN-3-SN-
38,
hMN-15-SN-38, IMMU-31-SN-38, hRS7-SN-38, hA20-SN-38, hL243-SN-38, hLL1-SN-38
or hLL2-SN-38 conjugate.
[017] Various embodiments may concern use of the subject methods and
compositions to
treat a cancer, including but not limited to non-Hodgkin's lymphomas, B-cell
acute and
chronic lymphatic leukemias, Burkitt lymphoma, Hodgkin's lymphoma, acute large
B-cell
lymphoma, hairy cell leukemia, acute myeloid leukemia, chronic myeloid
leukemia, T-cell
lymphomas and leukemias, multiple myeloma, Waldenstrom's macroglobulinemia,
carcinomas, melanomas, sarcomas, gliomas, bone, and skin cancers. The
carcinomas may
include carcinomas of the oral cavity, esophagus, gastrointestinal tract,
pulmonary tract, lung,
stomach, colon, breast, ovary, prostate, uterus, endometrium, cervix, urinary
bladder,
pancreas, bone, brain, connective tissue, liver, gall bladder, urinary
bladder, kidney, skin,
central nervous system and testes.
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[018] In certain embodiments involving treatment of cancer, the drug
conjugates may be
used in combination with surgery, radiation therapy, chemotherapy,
immunotherapy with
naked antibodies, radioimmunotherapy, immunomodulators, vaccines, and the
like. These
combination therapies can allow lower doses of each therapeutic to be given in
such
combinations, thus reducing certain severe side effects, and potentially
reducing the courses
of therapy required. When there is no or minimal overlapping toxicity, full
doses of each can
also be given.
[019] Preferred optimal dosing of immunoconjugates may include a dosage of
between 3
mg/kg and 18 mg/kg, more preferably between 4 and 16 mg/kg, more preferably
between 6
and 12 mg/kg, most preferably between 8 and 10 mg/kg, preferably given either
weekly,
twice weekly or every other week. The optimal dosing schedule may include
treatment
cycles of two consecutive weeks of therapy followed by one, two, three or four
weeks of rest,
or alternating weeks of therapy and rest, or one week of therapy followed by
two, three or
four weeks of rest, or three weeks of therapy followed by one, two, three or
four weeks of
rest, or four weeks of therapy followed by one, two, three or four weeks of
rest, or five weeks
of therapy followed by one, two, three, four or five weeks of rest, or
administration once
every two weeks, once every three weeks or once a month. Treatment may be
extended for
any number of cycles, preferably at least 2, at least 4, at least 6, at least
8, at least 10, at least
12, at least 14, or at least 16 cycles. Exemplary dosages of use may include 1
mg/kg, 2
mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10
mg/kg, 11
mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, and 18
mg/kg. The
person of ordinary skill will realize that a variety of factors, such as age,
general health,
specific organ function or weight, as well as effects of prior therapy on
specific organ
systems (e.g., bone marrow) may be considered in selecting an optimal dosage
of
immunoconjugate, and that the dosage and/or frequency of administration may be
increased
or decreased during the course of therapy. The dosage may be repeated as
needed, with
evidence of tumor shrinkage observed after as few as 4 to 8 doses. The
optimized dosages
and schedules of administration disclosed herein show unexpected superior
efficacy and
reduced toxicity in human subjects, which could not have been predicted from
animal model
studies. Surprisingly, the superior efficacy allows treatment of tumors that
were previously
found to be resistant to one or more standard anti-cancer therapies, including
the parental
compound, CPT-11, from which SN-38 is derived in vivo.
[020] A surprising result with the instant claimed compositions and methods is
the
unexpected tolerability of high doses of antibody-drug conjugate, even with
repeated
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infusions, with only relatively low-grade toxicities of nausea and vomiting
observed, or
manageable neutropenia. A further surprising result is the lack of
accumulation of the
antibody-drug conjugate, unlike other products that have conjugated SN-38 to
albumin, PEG
or other carriers. The lack of accumulation is associated with improved
tolerability and lack
of serious toxicity even after repeated or increased dosing. These surprising
results allow
optimization of dosage and delivery schedule, with unexpectedly high
efficacies and low
toxicities. The claimed methods provide for shrinkage of solid tumors, in
individuals with
previously resistant cancers, of 15% or more, preferably 20% or more,
preferably 30% or
more, more preferably 40% or more in size (as measured by longest diameter).
The person of
ordinary skill will realize that tumor size may be measured by a variety of
different
techniques, such as total tumor volume, maximal tumor size in any dimension or
a
combination of size measurements in several dimensions. This may be with
standard
radiological procedures, such as computed tomography, ultrasonography, and/or
positron-
emission tomography. The means of measuring size is less important than
observing a trend
of decreasing tumor size with immunoconjugate treatment, preferably resulting
in elimination
of the tumor.
[021] While the immunoconjugate may be administered as a periodic bolus
injection, in
alternative embodiments the immunoconjugate may be administered by continuous
infusion
of antibody-drug conjugates. In order to increase the Cmax and extend the PK
of the
immunoconjugate in the blood, a continuous infusion may be administered for
example by
indwelling catheter. Such devices are known in the art, such as HICKMAN ,
BROVIAC
or PORT-A-CATH catheters (see, e.g., Skolnik et al., Ther Drug Monit 32:741-
48, 2010)
and any such known indwelling catheter may be used. A variety of continuous
infusion
pumps are also known in the art and any such known infusion pump may be used.
More
preferably, these immunoconjugates can be administered by intravenous
infusions over
relatively short periods of 2 to 5 hours, more preferably 2-3 hours.
[022] In particularly preferred embodiments, the immunoconjugates and dosing
schedules
may be efficacious in patients resistant to standard therapies. For example,
an hL243-SN-38
immunoconjugate may be administered to a patient who has not responded to
prior therapy
with irinotecan, the parent agent of SN-38. Surprisingly, the irinotecan-
resistant patient may
show a partial or even a complete response to hL243-SN-38. The ability of the
immunoconjugate to specifically target the tumor tissue may overcome tumor
resistance by
improved targeting and enhanced delivery of the therapeutic agent.
Combinations of different
SN-38 immunoconjugates, or SN-38-antibody conjugates in combination with an
antibody
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conjugated to a radionuclide, toxin or other drug, may provide even more
improved efficacy
and/or reduced toxicity.
BRIEF DESCRIPTION OF THE DRAWINGS
[023] The following drawings form part of the present specification and are
included to
further demonstrate certain embodiments of the present invention. The
embodiments may be
better understood by reference to one or more of these drawings in combination
with the
detailed description of specific embodiments presented herein.
[024] FIG. 1. Structure of IMMU-140 (hL243-CL2A-SN-38).
[025] FIG. 2. Comparative binding of IMMU-114 and IMMU-140. Binding curves
were
obtained for the SN-38 conjugated (IMMU-140) and naked (IMMU-1140 forms of
hL243. A
control non-specific antibody (h679) showed no binding to HLA-DR+ cells.
[026] FIG. 3. In vivo efficacy of IMMU-140 vs. IMMU-114 in MOLM-14 AML
xenografts.
[027] FIG. 4. In vivo efficacy of IMMU-140 vs. IMMU-114 in MN-60 ALL
xenografts.
[028] FIG. 5. In vivo efficacy of IMMU-140 vs. IMMU-114 in CAG MM xenografts.
[029] FIG. 6. In vivo efficacy of IMMU-140 vs. IMMU-114 in JVM-3 CLL
xenografts.
[030] FIG. 7. Binding of hL243-y4P to human melanoma cells in vitro.
[031] FIG. 8. Efficacy of IMMU-140 in human melanoma xenografts in vivo.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[032] In the description that follows, a number of terms are used and the
following
definitions are provided to facilitate understanding of the claimed subject
matter. Terms that
are not expressly defined herein are used in accordance with their plain and
ordinary
meanings.
[033] Unless otherwise specified, a or an means "one or more."
[034] The term about is used herein to mean plus or minus ten percent (10%) of
a value. For
example, "about 100" refers to any number between 90 and 110.
[035] An antibody, as used herein, refers to a full-length (i.e., naturally
occurring or formed
by normal immunoglobulin gene fragment recombinatorial processes)
immunoglobulin
molecule (e.g., an IgG antibody) or an antigen-binding portion of an
immunoglobulin
molecule, such as an antibody fragment. An antibody or antibody fragment may
be
conjugated or otherwise derivatized within the scope of the claimed subject
matter. Such
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antibodies include but are not limited to IgGl, IgG2, IgG3, IgG4 (and IgG4
subforms), as
well as IgA isotypes. As used below, the abbreviation "MAb" may be used
interchangeably to
refer to an antibody, antibody fragment, monoclonal antibody or multispecific
antibody.
[036] An antibody fragment is a portion of an antibody such as F(ab')2,
F(ab)2, Fab', Fab, Fv,
scFv (single chain Fv), single domain antibodies (DABs or VHHs) and the like,
including the
half-molecules of IgG4 cited above (van der Neut Kolfschoten et al. (Science
2007; 317(14
Sept):1554-1557). Regardless of structure, an antibody fragment of use binds
with the same
antigen that is recognized by the intact antibody. The term "antibody
fragment" also includes
synthetic or genetically engineered proteins that act like an antibody by
binding to a specific
antigen to form a complex. For example, antibody fragments include isolated
fragments
consisting of the variable regions, such as the "Fv" fragments consisting of
the variable
regions of the heavy and light chains and recombinant single chain polypeptide
molecules in
which light and heavy variable regions are connected by a peptide linker
("scFv proteins").
The fragments may be constructed in different ways to yield multivalent and/or
multispecific
binding forms.
[037] A naked antibody is generally an entire antibody that is not conjugated
to a therapeutic
agent. A naked antibody may exhibit therapeutic and/or cytotoxic effects, for
example by Fc-
dependent functions, such as complement fixation (CDC) and ADCC (antibody-
dependent
cell cytotoxicity). However, other mechanisms, such as apoptosis, anti-
angiogenesis, anti-
metastatic activity, anti-adhesion activity, inhibition of heterotypic or
homotypic adhesion,
and interference in signaling pathways, may also provide a therapeutic effect.
Naked
antibodies include polyclonal and monoclonal antibodies, naturally occurring
or recombinant
antibodies, such as chimeric, humanized or human antibodies and fragments
thereof. In some
cases a "naked antibody" may also refer to a "naked" antibody fragment. As
defined herein,
"naked" is synonymous with "unconjugated," and means not linked or conjugated
to a
therapeutic agent.
[038] A chimeric antibody is a recombinant protein that contains the variable
domains of
both the heavy and light antibody chains, including the complementarity
determining regions
(CDRs) of an antibody derived from one species, preferably a rodent antibody,
more
preferably a murine antibody, while the constant domains of the antibody
molecule are
derived from those of a human antibody.
[039] A humanized antibody is a recombinant protein in which the CDRs from an
antibody
from one species; e.g., a murine antibody, are transferred from the heavy and
light variable
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chains of the murine antibody into human heavy and light variable domains
(framework
regions). The constant domains of the antibody molecule are derived from those
of a human
antibody. In some cases, specific residues of the framework region of the
humanized
antibody, particularly those that are touching or close to the CDR sequences,
may be
modified, for example replaced with the corresponding residues from the
original murine,
rodent, subhuman primate, or other antibody.
[040] A human antibody is an antibody obtained, for example, from transgenic
mice that
have been "engineered" to produce human antibodies in response to antigenic
challenge. In
this technique, elements of the human heavy and light chain loci are
introduced into strains of
mice derived from embryonic stem cell lines that contain targeted disruptions
of the
endogenous heavy chain and light chain loci. The transgenic mice can
synthesize human
antibodies specific for various antigens, and the mice can be used to produce
human
antibody-secreting hybridomas. Methods for obtaining human antibodies from
transgenic
mice are described by Green et at., Nature Genet. 7:13 (1994), Lonberg et at.,
Nature
368:856 (1994), and Taylor et at., Int. Immun. 6:579 (1994). A fully human
antibody also can
be constructed by genetic or chromosomal transfection methods, as well as
phage display
technology, all of which are known in the art. See for example, McCafferty et
at., Nature
348:552-553 (1990) for the production of human antibodies and fragments
thereof in vitro,
from immunoglobulin variable domain gene repertoires from unimmunized donors.
In this
technique, human antibody variable domain genes are cloned in-frame into
either a major or
minor coat protein gene of a filamentous bacteriophage, and displayed as
functional antibody
fragments on the surface of the phage particle. Because the filamentous
particle contains a
single-stranded DNA copy of the phage genome, selections based on the
functional properties
of the antibody also result in selection of the gene encoding the antibody
exhibiting those
properties. In this way, the phage mimics some of the properties of the B
cell. Phage display
can be performed in a variety of formats, for their review, see e.g. Johnson
and Chiswell,
Current Opinion in Structural Biology 3:5564-571 (1993). Human antibodies may
also be
generated by in vitro activated B cells. See U.S. Patent Nos. 5,567,610 and
5,229,275, the
Examples section of each of which is incorporated herein by reference.
[041] A therapeutic agent is an atom, molecule, or compound that is useful in
the treatment
of a disease. Examples of therapeutic agents include, but are not limited to,
antibodies,
antibody fragments, immunoconjugates, drugs, cytotoxic agents, pro-apopoptotic
agents,
toxins, nucleases (including DNAses and RNAses), hormones, immunomodulators,
chelators,
boron compounds, photoactive agents or dyes, radionuclides, oligonucleotides,
interference
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RNA, siRNA, RNAi, anti-angiogenic agents, chemotherapeutic agents, cyokines,
chemokines, prodrugs, enzymes, binding proteins or peptides or combinations
thereof
[042] An immunoconjugate is an antibody, antigen-binding antibody fragment,
antibody
complex or antibody fusion protein that is conjugated to a therapeutic agent.
Conjugation
may be covalent or non-covalent. Preferably, conjugation is covalent.
[043] As used herein, the term antibody fusion protein is a recombinantly-
produced antigen-
binding molecule in which one or more natural antibodies, single-chain
antibodies or
antibody fragments are linked to another moiety, such as a protein or peptide,
a toxin, a
cytokine, a hormone, etc. In certain preferred embodiments, the fusion protein
may comprise
two or more of the same or different antibodies, antibody fragments or single-
chain
antibodies fused together, which may bind to the same epitope, different
epitopes on the same
antigen, or different antigens.
[044] An immunomodulator is a therapeutic agent that when present, alters,
suppresses or
stimulates the body's immune system. Typically, an immunomodulator of use
stimulates
immune cells to proliferate or become activated in an immune response cascade,
such as
macrophages, dendritic cells, B-cells, and/or T-cells. However, in some cases
an
immunomodulator may suppress proliferation or activation of immune cells. An
example of
an immunomodulator as described herein is a cytokine, which is a soluble small
protein of
approximately 5-20 kDa that is released by one cell population (e.g., primed T-
lymphocytes)
on contact with specific antigens, and which acts as an intercellular mediator
between cells.
As the skilled artisan will understand, examples of cytokines include
lymphokines,
monokines, interleukins, and several related signaling molecules, such as
tumor necrosis
factor (TNF) and interferons. Chemokines are a subset of cytokines. Certain
interleukins and
interferons are examples of cytokines that stimulate T cell or other immune
cell proliferation.
Exemplary interferons include interferon-a, interferon-0, interferon-y and
interferon-k.
[045] CPT is an abbreviation for camptothecin, and as used in the present
application CPT
represents camptothecin itself or an analog or derivative of camptothecin,
such as SN-38. The
structures of camptothecin and some of its analogs, with the numbering
indicated and the
rings labeled with letters A-E, are given in formula 1 in Chart 1 below.
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[046] Chart 1
CPT: Ri = R2 = R3 = H
R R3 R2 10-Hydroxy-CPT: Ri = OH; R 2 = R3 = H
1
7
B C N 0
CPT-11: R1 =
O¨NO ; R2 = ethyl; R3 = H
N Dz 0
E 0
0 SN-38: R 1= OH; R2 = ethyl; R3 =H
OH
(1) Topotecan: R1 = OH; R2 = H; R3 = CH 2-N(CH 3)2
Camptothecin Conjugates
[047] Non-limiting methods and compositions for preparing immunoconjugates
comprising a
camptothecin therapeutic agent attached to an antibody or antigen-binding
antibody fragment
are described below. In preferred embodiments, the solubility of the drug is
enhanced by
placing a defined polyethyleneglycol (PEG) moiety (i.e., a PEG containing a
defined number
of monomeric units) between the drug and the antibody, wherein the defined PEG
is a low
molecular weight PEG, preferably containing 1-30 monomeric units, more
preferably
containing 1-12 monomeric units, most preferably 7-8 monomeric units.
[048] Preferably, a first linker connects the drug at one end and may
terminate with an
acetylene or an azide group at the other end. This first linker may comprise a
defined PEG
moiety with an azide or acetylene group at one end and a different reactive
group, such as
carboxylic acid or hydroxyl group, at the other end. Said bifunctional defined
PEG may be
attached to the amine group of an amino alcohol, and the hydroxyl group of the
latter may be
attached to the hydroxyl group on the drug in the form of a carbonate.
Alternatively, the non-
azide(or acetylene) moiety of said defined bifunctional PEG is optionally
attached to the N-
terminus of an L-amino acid or a polypeptide, with the C-terminus attached to
the amino
group of amino alcohol, and the hydroxy group of the latter is attached to the
hydroxyl group
of the drug in the form of carbonate or carbamate, respectively.
[049] A second linker, comprising an antibody-coupling group and a reactive
group
complementary to the azide (or acetylene) group of the first linker, namely
acetylene (or
azide), may react with the drug-(first linker) conjugate via acetylene-azide
cycloaddition
reaction to furnish a final bifunctional drug product that is useful for
conjugating to disease-
targeting antibodies. The antibody-coupling group is preferably either a thiol
or a thiol-
reactive group.
[050] Methods for selective regeneration of the 10-hydroxyl group in the
presence of the C-
20 carbonate in preparations of drug-linker precursor involving CPT analogs
such as SN-38
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are provided below. Other protecting groups for reactive hydroxyl groups in
drugs such as the
phenolic hydroxyl in SN-38, for example t-butyldimethylsilyl or t-
butyldiphenylsilyl, may
also be used, and these are deprotected by tetrabutylammonium fluoride prior
to linking of
the derivatized drug to an antibody-coupling moiety. The 10-hydroxyl group of
CPT analogs
is alternatively protected as an ester or carbonate, other than 'WC', such
that the
bifunctional CPT is conjugated to an antibody without prior deprotection of
this protecting
group. The protecting group is readily deprotected under physiological pH
conditions after
the bioconjugate is administered.
[051] In the acetylene-azide coupling, referred to as 'click chemistry', the
azide part may be
on L2 with the acetylene part on L3. Alternatively, L2 may contain acetylene,
with L3
containing azide. 'Click chemistry' refers to a copper (+1)-catalyzed
cycloaddition reaction
between an acetylene moiety and an azide moiety (Kolb HC and Sharpless KB,
Drug Discov
Today 2003; 8: 1128-37), although alternative forms of click chemistry are
known and may
be used. Click chemistry takes place in aqueous solution at near-neutral pH
conditions, and is
thus amenable for drug conjugation. The advantage of click chemistry is that
it is
chemoselective, and complements other well-known conjugation chemistries such
as the
thiol-maleimide reaction.
[052] While the present application focuses on use of antibodies or antibody
fragments as
targeting moieties, the skilled artisan will realize that where a conjugate
comprises an
antibody or antibody fragment, another type of targeting moiety, such as an
aptamer, avimer,
affibody or peptide ligand, may be substituted.
[053] An exemplary preferred embodiment is directed to a conjugate of a drug
derivative and
an antibody of the general formula 2,
MAb-[L2]-[L1]-[AA]AA']-Drug (2)
where MAb is a disease-targeting antibody; L2 is a component of the cross-
linker comprising
an antibody-coupling moiety and one or more of acetylene (or azide) groups; Li
comprises a
defined PEG with azide (or acetylene) at one end, complementary to the
acetylene (or azide)
moiety in L2, and a reactive group such as carboxylic acid or hydroxyl group
at the other end;
AA is an L-amino acid; m is an integer with values of 0, 1, 2, 3, or 4; and A'
is an additional
spacer, selected from the group of ethanolamine, 4-hydroxybenzyl alcohol, 4-
aminobenzyl
alcohol, or substituted or unsubstituted ethylenediamine. The L amino acids of
'AA' are
selected from alanine, arginine, asparagine, aspartic acid, cysteine,
glutamine, glutamic acid,
glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
proline, serine,
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threonine, tryptophan, tyrosine, and valine. If the A' group contains
hydroxyl, it is linked to
the hydroxyl group or amino group of the drug in the form of a carbonate or
carbamate,
respectively.
[054] In a preferred embodiment of formula 2, A' is a substituted ethanolamine
derived from
an L-amino acid, wherein the carboxylic acid group of the amino acid is
replaced by a
hydroxymethyl moiety. A' may be derived from any one of the following L-amino
acids:
alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic
acid, glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline,
serine, threonine,
tryptophan, tyrosine, and valine.
[055] A preferred embodiment, referred to as MAb-CL2A-SN-38, is shown below.
N=N 0
H
0 /
0 8 8 0
MAb-CL2A-SN-38 OH
NH2 (as amine salt)
[056] Other embodiments are possible within the context of 10-hydroxy-
containing
camptothecins, such as SN-38. In the example of SN-38 as the drug, the more
reactive 10-
hydroxy group of the drug is derivatized leaving the 20-hydroxyl group
unaffected. Within
the general formula 2, A' is a substituted ethylenediamine.
[057] In another preferred embodiment, the Li component of the conjugate
contains a
defined polyethyleneglycol (PEG) spacer with 1-30 repeating monomeric units.
In a further
preferred embodiment, PEG is a defined PEG with 1-12 repeating monomeric
units. The
introduction of PEG may involve using heterobifunctionalized PEG derivatives
which are
available commercially. The heterobifunctional PEG may contain an azide or
acetylene
group. An example of a heterobifunctional defined PEG containing 8 repeating
monomeric
units, with 'NHS' being succinimidyl, is given below in formula 3:
0 0
0 ONHS
7
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(3)
[058] In a preferred embodiment, L2 has a plurality of acetylene (or azide)
groups, ranging
from 2-40, but preferably 2-20, and more preferably 2-5, and a single antibody-
binding
moiety.
[059] A representative SN-38 conjugate of an antibody containing multiple drug
molecules
and a single antibody-binding moiety is shown below. The 12' component of this
structure
is appended to 2 acetylenic groups, resulting in the attachment of two azide-
appended SN-38
molecules. The bonding to MAb is represented as a succinimide.
MA4H0
N R
H NO
0
Where R residue is:
0
0 0
N=1\1, Fi 0
H2C¨
NH2 (salt)
OH
[060] In preferred embodiments, when the bifunctional drug contains a thiol-
reactive moiety
as the antibody-binding group, the thiols on the antibody are generated on the
lysine groups
of the antibody using a thiolating reagent. Methods for introducing thiol
groups onto
antibodies by modifications of MAb's lysine groups are well known in the art
(Wong in
Chemistry of protein conjugation and cross-linking, CRC Press, Inc., Boca
Raton, FL (1991),
pp 20-22). Alternatively, mild reduction of interchain disulfide bonds on the
antibody
(Willner et al., Bioconjugate Chem. 4:521-527 (1993)) using reducing agents
such as
dithiothreitol (DTT) can generate 7-to-10 thiols on the antibody; which has
the advantage of
incorporating multiple drug moieties in the interchain region of the MAb away
from the
antigen-binding region. In a more preferred embodiment, attachment of SN-38 to
reduced
disulfide sulfhydryl groups results in formation of an antibody-SN-38
immunoconjugate with
6 to 8 SN-38 moieties covalently attached per antibody molecule. Other methods
of providing
cysteine residues for attachment of drugs or other therapeutic agents are
known, such as the
use of cysteine engineered antibodies (see U.S. Patent No. 7,521,541, the
Examples section
of which is incorporated herein by reference.)
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[061] In alternative preferred embodiments, the chemotherapeutic moiety is
selected from
the group consisting of doxorubicin (DOX), epirubicin, morpholinodoxorubicin
(morpholino-
DOX), cyanomorpholino-doxorubicin (cyanomorpholino-DOX), 2-pyrrolino-
doxorubicin (2-
PDOX), Pro-2PDOX, CPT, 10-hydroxy camptothecin, SN-38, topotecan, lurtotecan,
9-
aminocamptothecin, 9-nitrocamptothecin, taxanes, geldanamycin, ansamycins, and
epothilones. In a more preferred embodiment, the chemotherapeutic moiety is SN-
38.
Preferably, in the conjugates of the preferred embodiments, the antibody links
to at least one
chemotherapeutic moiety; preferably 1 to about 12 chemotherapeutic moieties;
more
preferably about 6 to about 12 chemotherapeutic moieties, most preferably
about 6 to 8
chemotherapeutic moieties.
[062] Furthermore, in a preferred embodiment, the linker component 12'
comprises a thiol
group that reacts with a thiol-reactive residue introduced at one or more
lysine side chain
amino groups of said antibody. In such cases, the antibody is pre-derivatized
with a thiol-
reactive group such as a maleimide, vinylsulfone, bromoacetamide, or
iodoacetamide by
procedures well described in the art.
[063] In the context of this work, a process was surprisingly discovered by
which CPT drug-
linkers can be prepared wherein CPT additionally has a 10-hydroxyl group. This
process
involves, but is not limited to, the protection of the 10-hydroxyl group as a
t-
butyloxycarbonyl (BOC) derivative, followed by the preparation of the
penultimate
intermediate of the drug-linker conjugate. Usually, removal of BOC group
requires treatment
with strong acid such as trifluoroacetic acid (TFA). Under these conditions,
the CPT 20-0-
linker carbonate, containing protecting groups to be removed, is also
susceptible to cleavage,
thereby giving rise to unmodified CPT. In fact, the rationale for using a
mildly removable
methoxytrityl (MMT) protecting group for the lysine side chain of the linker
molecule, as
enunciated in the art, was precisely to avoid this possibility (Walker et at.,
2002). It was
discovered that selective removal of phenolic BOC protecting group is possible
by carrying
out reactions for short durations, optimally 3-to-5 minutes. Under these
conditions, the
predominant product was that in which the 'BOC' at 10-hydroxyl position was
removed,
while the carbonate at '20' position was intact.
[064] An alternative approach involves protecting the CPT analog's 10-hydroxy
position
with a group other than 'BOC', such that the the final product is ready for
conjugation to
antibodies without a need for deprotecting the 10-0H protecting group. The 10-
hydroxy
protecting group, which converts the 10-0H into a phenolic carbonate or a
phenolic ester, is
readily deprotected by physiological pH conditions or by esterases after in
vivo
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administration of the conjugate. The faster removal of a phenolic carbonate at
the 10 position
vs. a tertiary carbonate at the 20 position of 10-hydroxycamptothecin under
physiological
condition has been described by He et al. (He et al., Bioorganic & Medicinal
Chemistry 12:
4003-4008 (2004)). A 10-hydroxy protecting group on SN-38 can be `COR' where R
can be
a substituted alkyl such as "N(CH3)2-(CH2)õ¨" where n is 1-10 and wherein the
terminal
amino group is optionally in the form of a quaternary salt for enhanced
aqueous solubility, or
a simple alkyl residue such as "CH3-(CH2)õ¨" where n is 0-10, or it can be an
alkoxy moiety
such as "CH3-(CH2)n-0¨" where n is 0-10, or "N(CH3)2-(CH2)õ-0¨" where n is 2-
10, or
"R10-(CH2-CH2-0)-CH2-CH2-0¨" where R1 is ethyl or methyl and n is an integer
with
values of 0-10. These 10-hydroxy derivatives are readily prepared by treatment
with the
chloroformate of the chosen reagent, if the final derivative is to be a
carbonate. Typically, the
10-hydroxy-containing camptothecin such as SN-38 is treated with a molar
equivalent of the
chloroformate in dimethylformamide using triethylamine as the base. Under
these conditions,
the 20-0H position is unaffected. For forming 10-0-esters, the acid chloride
of the chosen
reagent is used.
[065] In a preferred process of the preparation of a conjugate of a drug
derivative and an
antibody of the general formula 2, wherein the descriptors L2, Li, AA and A-X
are as
described in earlier sections, the bifunctional drug moiety, [L2]-[Li]-[AA]-[A-
X]-Drug is
first prepared, followed by the conjugation of the bifunctional drug moiety to
the antibody
(indicated herein as "MAb").
[066] In a preferred process of the preparation of a conjugate of a drug
derivative and an
antibody of the general formula 2, wherein the descriptors L2, Li, AA and A-OH
are as
described in earlier sections, the bifunctional drug moiety is prepared by
first linking A-OH
to the C-terminus of AA via an amide bond, followed by coupling the amine end
of AA to a
carboxylic acid group of Li. If AA is absent (i.e. m =0), A-OH is directly
attached to Li via
an amide bond. The cross-linker, [L1]-[AA]-[A-OH], is attached to drug's
hydroxyl or
amino group, and this is followed by attachment to the Li moiety, by taking
recourse to the
reaction between azide (or acetylene) and acetylene (or azide) groups in Li
and L2 via click
chemistry.
[067] In one embodiment, the antibody is a monoclonal antibody (MAb). In other
embodiments, the antibody may be a multivalent and/or multispecific MAb. The
antibody
may be a murine, chimeric, humanized, or human monoclonal antibody, and said
antibody
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may be in intact, fragment (Fab, Fab', F(ab)2, F(ab')2), or sub-fragment
(single-chain
constructs) form, or of an IgGl, IgG2a, IgG3, IgG4, IgA isotype, or
submolecules therefrom.
[068] In a preferred embodiment, the antibody binds to an antigen or epitope
of an antigen
expressed on a cancer or malignant cell. The cancer cell is preferably a cell
from a
hematopoietic tumor, carcinoma, sarcoma, melanoma or a glial tumor. A
preferred
malignancy to be treated according to the present invention is a malignant
solid tumor or
hematopoietic neoplasm.
[069] In a preferred embodiment, the intracellularly-cleavable moiety may be
cleaved after it
is internalized into the cell upon binding by the MAb-drug conjugate to a
receptor thereof,
and particularly cleaved by esterases and peptidases.
Anti-HLA-DR Antibodies
[070] In preferred embodiments, the immunoconjugate comprises an anti-HLA-DR
antibody,
such as a humanized L243 antibody. Humanized L243 antibodies bind to the same
epitope
on HLA-DR as the parental murine L243 antibody, but have reduced
immunogenicity.
mL243 is a monoclonal antibody previously described by Lampson & Levy (1
Immunol,
1980, 125:293), which has been deposited at the American Type Culture
Collection,
Rockville, MD, under Accession number ATCC HB55.
[071] The humanized L243 antibodies comprise the L243 heavy chain CDR
sequences
CDR1 (NYGMN, SEQ ID NO:1), CDR2 (WINTYTREPTYADDFKG, SEQ ID NO:2) and
CDR3 (DITAVVPTGFDY, SEQ ID NO:3) and the light chain CDR sequences CDR1
(RASENIYSNLA, SEQ ID NO:4), CDR2 (AASNLAD, SEQ ID NO:5), and CDR3
(QHFWTTPWA, SEQ ID NO:6), attached to human antibody FR and constant region
sequences. In more preferred embodiments, one or more murine FR amino acid
residues are
substituted for the corresponding human FR residues, particularly at locations
adjacent to or
nearby the CDR residues. Exemplary murine VH residues that may be substituted
in the
humanized design are at positions: F27, K38, K46, A68 and F91. Exemplary
murine VL
residues that may be substituted in the humanized design are at positions R37,
K39, V48,
F49, and Gl.
[072] A particularly preferred form of hL243 antibody is illustrated in U.S.
Patent No.
7,612,180, incorporated herein by reference, which incorporates FR sequences
from the
human RF-T53, NEWM and REI antibodies. However, in other embodiments, the FR
residues may be derived from any suitable human immunoglobulin, provided that
the
humanized antibody can fold such that it retains the ability to specifically
bind HLA-DR.
Preferably the type of human framework (FR) used is of the same/similar
class/type as the
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donor antibody. More preferably, the human FR sequences are selected to have a
high degree
of sequence homology with the corresponding murine FR sequences, particularly
at positions
spatially close or adjacent to the CDRs. In accordance with this embodiment,
the frameworks
(le, FR1-4) of the humanized L243 VH or VL may be derived from a combination
of human
antibodies. Examples of human frameworks which may be used to construct CDR-
grafted
humanized antibodies are LAY, POM, TUR, TEI, KOL, NEWM, REI, RF and EU.
Preferably human RF-TS3 FR1-3 and NEWM FR4 are used for the heavy chain and
REI
FR1-4 are used for the light chain. The variable domain residue numbering
system used
herein is described in Kabat et al, (1991), Sequences of Proteins of
Immunological Interest,
5th Edition, United States Department of Health and Human Services
[073] The light and heavy chain variable domains of the humanized antibody
molecule may
be fused to human light or heavy chain constant domains. The human constant
domains may
be selected with regard to the proposed function of the antibody. In one
embodiment, the
human constant domains may be selected based on a lack of effector functions.
The heavy
chain constant domains fused to the heavy chain variable region may be those
of human IgA
(al or a2 chain), IgG (yl, y2, y3 or y4 chain) or IgM (II chain). The light
chain constant
domains which may be fused to the light chain variable region include human
lambda and
kappa chains.
[074] In one particular embodiment of the present invention, a yl chain is
used. In yet another
particular embodiment, a y4 chain is used. The use of the y4 chain may in some
cases
increase the tolerance to hL243 in subjects (decreased side effects and
infusion reactions,
etc).
[075] In one embodiment, analogues of human constant domains may be used.
These include
but are not limited to those constant domains containing one or more
additional amino acids
than the corresponding human domain or those constant domains wherein one or
more
existing amino acids of the corresponding human domain have been deleted or
altered. Such
domains may be obtained, for example, by oligonucleotide directed mutagenesis.
[076] In a particular embodiment, an anti-HLA-DR antibody or fragment thereof
may be a
fusion protein. The fusion protein may contain one or more anti-HLA-DR
antibodies or
fragments thereof. In various embodiments, the fusion protein may also
comprise one or
more additional antibodies against a different antigen, or may comprise a
different effector
protein or peptide, such as a cytokine. For example, the different antigen may
be a tumor
marker selected from a B cell lineage antigen, (eg, CD19, CD20, or CD22) for
the treatment
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of B cell malignancies. In another example, the different antigen may be
expressed on other
cells that cause other types of malignancies. Further, the cell marker may be
a non-B cell
lineage antigen, such as selected from the group consisting of HLA-DR, CD3,
CD33, CD52,
CD66, MUC1 and TAC.
[077] In one embodiment, an anti-HLA-DR antibody may be combined with other
antibodies
and used to treat a subject having or suspected of developing a disease. In
accordance with
this embodiment, an anti-HLA-DR antibody or fragment thereof may be combined
with an
anticancer monoclonal antibody such as a humanized monoclonal antibody (eg
hA20, anti-
CD20 Mab) and used to treat cancer. It is contemplated herein that an anti-HLA-
DR antibody
may be used as a separate antibody composition in combination with one or more
other
separate antibody compositions, or used as a bi-functional antibody
containing, for example,
one anti-HLA-DR and one other anti-tumor antibody, such as hA20. In another
particular
embodiment, the antibody may target a B cell malignancy. The B cell malignancy
may
consist of indolent forms of B cell lymphomas, aggressive forms of B cell
lymphomas,
chronic lymphatic leukemias, acute lymphatic leukemias, Waldenstrom's
macroglobulinemia,
and multiple myeloma. Other non-malignant B cell disorders and related
diseases that may be
treated with the subject antibodies include many autoimmune and immune
dysregulatory
diseases, such as septicemia and septic shock.
General Antibody Techniques
[078] Techniques for preparing monoclonal antibodies against virtually any
target antigen
are well known in the art. See, for example, Kohler and Milstein, Nature 256:
495 (1975),
and Coligan et at. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages
2.5.1-2.6.7 (John Wiley & Sons 1991). Briefly, monoclonal antibodies can be
obtained by
injecting mice with a composition comprising a human antigen, such as human
HLA-DR,
removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with
myeloma cells
to produce hybridomas, cloning the hybridomas, selecting positive clones which
produce
antibodies to the human antigen, culturing the clones that produce antibodies
to the antigen,
and isolating the antibodies from the hybridoma cultures.
[079] MAbs can be isolated and purified from hybridoma cultures by a variety
of well-
established techniques. Such isolation techniques include affinity
chromatography with
Protein-A or Protein-G Sepharose, size-exclusion chromatography, and ion-
exchange
chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and pages
2.9.1-2.9.3.
Also, see Baines et at., "Purification of Immunoglobulin G (IgG)," in METHODS
IN
MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).
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[080] After the initial raising of antibodies to the immunogen, the antibodies
can be
sequenced and subsequently prepared by recombinant techniques. Humanization
and
chimerization of murine antibodies and antibody fragments are well known to
those skilled in
the art, as discussed below.
[081] The skilled artisan will realize that the claimed methods and
compositions may utilize
any of a wide variety of antibodies known in the art. Antibodies of use may be
commercially
obtained from a wide variety of known sources. For example, a variety of
antibody secreting
hybridoma lines are available from the American Type Culture Collection (ATCC,
Manassas,
VA). A large number of antibodies against various disease targets, including
but not limited
to tumor-associated antigens, have been deposited at the ATCC and/or have
published
variable region sequences and are available for use in the claimed methods and
compositions.
See, e.g., U.S. Patent Nos. 7,312,318; 7,282,567; 7,151,164; 7,074,403;
7,060,802;
7,056,509; 7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018;
7,037,498;
7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863;
6,965,018;
6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244; 6,946,129;
6,943,020;
6,939,547; 6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475;
6,905,681;
6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812;
6,875,580;
6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226; 6,838,282;
6,835,549;
6,835,370; 6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450;
6,767,711;
6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155;
6,716,966;
6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355; 6,682,737;
6,682,736;
6,682,734; 6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833;
6,610,294;
6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745; 6,572;856; 6,566,076;
6,562,618;
6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227; 6,518,404;
6,511,665;
6,491,915; 6,488,930; 6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529;
6,465,173;
6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310; 6,444,206' 6,441,143;
6,432,404;
6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091; 6,395,276;
6,395,274;
6,387,350; 6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481;
6,355,444;
6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175;
6,306,393;
6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289; 6,077,499;
5,922,302;
5,874,540; 5,814,440; 5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595;
5,677,136;
5,587,459; 5,443,953, 5,525,338, the Examples section of each of which is
incorporated
herein by reference. These are exemplary only and a wide variety of other
antibodies and
their hybridomas are known in the art. The skilled artisan will realize that
antibody
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sequences or antibody-secreting hybridomas against almost any disease-
associated antigen
may be obtained by a simple search of the ATCC, NCBI and/or USPTO databases
for
antibodies against a selected disease-associated target of interest. The
antigen binding
domains of the cloned antibodies may be amplified, excised, ligated into an
expression
vector, transfected into an adapted host cell and used for protein production,
using standard
techniques well known in the art. Isolated antibodies may be conjugated to
therapeutic
agents, such as camptothecins, using the techniques disclosed herein.
Chimeric and Humanized Antibodies
[082] A chimeric antibody is a recombinant protein in which the variable
regions of a human
antibody have been replaced by the variable regions of, for example, a mouse
antibody,
including the complementarity-determining regions (CDRs) of the mouse
antibody. Chimeric
antibodies exhibit decreased immunogenicity and increased stability when
administered to a
subject. Methods for constructing chimeric antibodies are well known in the
art (e.g., Leung
et al., 1994, Hybridoma 13:469).
[083] A chimeric monoclonal antibody may be humanized by transferring the
mouse CDRs
from the heavy and light variable chains of the mouse immunoglobulin into the
corresponding variable domains of a human antibody. The mouse framework
regions (FR) in
the chimeric monoclonal antibody are also replaced with human FR sequences. To
preserve
the stability and antigen specificity of the humanized monoclonal, one or more
human FR
residues may be replaced by the mouse counterpart residues. Humanized
monoclonal
antibodies may be used for therapeutic treatment of subjects. Techniques for
production of
humanized monoclonal antibodies are well known in the art. (See, e.g., Jones
et al., 1986,
Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen et al.,
1988, Science,
239:1534; Carter et al., 1992, Proc. Nat'l Acad. Sci. USA, 89:4285; Sandhu,
Crit. Rev.
Biotech., 1992, 12:437; Tempest et al., 1991, Biotechnology 9:266; Singer et
al., I Immun.,
1993, 150:2844.)
[084] Other embodiments may concern non-human primate antibodies. General
techniques
for raising therapeutically useful antibodies in baboons may be found, for
example, in
Goldenberg et al., WO 91/11465 (1991), and in Losman et al., Int. 1 Cancer 46:
310 (1990).
In another embodiment, an antibody may be a human monoclonal antibody. Such
antibodies
may be obtained from transgenic mice that have been engineered to produce
specific human
antibodies in response to antigenic challenge, as discussed below.
Human Antibodies
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[085] Methods for producing fully human antibodies using either combinatorial
approaches
or transgenic animals transformed with human immunoglobulin loci are known in
the art
(e.g., Mancini et al., 2004, New Microbiol. 27:315-28; Conrad and Scheller,
2005, Comb.
Chem. High Throughput Screen. 8:117-26; Brekke and Loset, 2003, Curr. Op/n.
Phamacol.
3:544-50; each incorporated herein by reference). Such fully human antibodies
are expected
to exhibit even fewer side effects than chimeric or humanized antibodies and
to function in
vivo as essentially endogenous human antibodies. In certain embodiments, the
claimed
methods and procedures may utilize human antibodies produced by such
techniques.
[086] In one alternative, the phage display technique may be used to generate
human
antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res. 4:126-40,
incorporated herein
by reference). Human antibodies may be generated from normal humans or from
humans
that exhibit a particular disease state, such as cancer (Dantas-Barbosa et
al., 2005). The
advantage to constructing human antibodies from a diseased individual is that
the circulating
antibody repertoire may be biased towards antibodies against disease-
associated antigens.
[087] In one non-limiting example of this methodology, Dantas-Barbosa et al.
(2005)
constructed a phage display library of human Fab antibody fragments from
osteosarcoma
patients. Generally, total RNA was obtained from circulating blood lymphocytes
(Id.)
Recombinant Fab were cloned from the II., y and lc chain antibody repertoires
and inserted
into a phage display library (Id.) RNAs were converted to cDNAs and used to
make Fab
cDNA libraries using specific primers against the heavy and light chain
immunoglobulin
sequences (Marks et al., 1991, 1 Mol. Biol. 222:581-97, incorporated herein by
reference).
Library construction was performed according to Andris-Widhopf et al. (2000,
In: Phage
Display Laboratory Manual, Barbas et al. (eds), 1st edition, Cold Spring
Harbor Laboratory
Press, Cold Spring Harbor, NY pp. 9.1 to 9.22, incorporated herein by
reference). The final
Fab fragments were digested with restriction endonucleases and inserted into
the
bacteriophage genome to make the phage display library. Such libraries may be
screened by
standard phage display methods. The skilled artisan will realize that this
technique is
exemplary only and any known method for making and screening human antibodies
or
antibody fragments by phage display may be utilized.
[088] In another alternative, transgenic animals that have been genetically
engineered to
produce human antibodies may be used to generate antibodies against
essentially any
immunogenic target, using standard immunization protocols as discussed above.
Methods for
obtaining human antibodies from transgenic mice are described by Green et al.,
Nature
Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al.,
Int. Immun.
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6:579 (1994). A non-limiting example of such a system is the XENOMOUSE (e.g.,
Green
et al., 1999, 1 Immunol. Methods 231:11-23, incorporated herein by reference)
from Abgenix
(Fremont, CA). In the XENOMOUSE and similar animals, the mouse antibody genes
have
been inactivated and replaced by functional human antibody genes, while the
remainder of
the mouse immune system remains intact.
[089] The XENOMOUSE was transformed with germline-configured YACs (yeast
artificial chromosomes) that contained portions of the human IgH and Ig kappa
loci,
including the majority of the variable region sequences, along accessory genes
and regulatory
sequences. The human variable region repertoire may be used to generate
antibody producing
B cells, which may be processed into hybridomas by known techniques. A
XENOMOUSE
immunized with a target antigen will produce human antibodies by the normal
immune
response, which may be harvested and/or produced by standard techniques
discussed above.
A variety of strains of XENOMOUSE are available, each of which is capable of
producing
a different class of antibody. Transgenically produced human antibodies have
been shown to
have therapeutic potential, while retaining the pharmacokinetic properties of
normal human
antibodies (Green et al., 1999). The skilled artisan will realize that the
claimed compositions
and methods are not limited to use of the XENOMOUSE system but may utilize
any
transgenic animal that has been genetically engineered to produce human
antibodies.
Production of Antibody Fragments
[090] Some embodiments of the claimed methods and/or compositions may concern
antibody
fragments. Such antibody fragments may be obtained, for example, by pepsin or
papain
digestion of whole antibodies by conventional methods. For example, antibody
fragments
may be produced by enzymatic cleavage of antibodies with pepsin to provide a
5S fragment
denoted F(ab')2. This fragment may be further cleaved using a thiol reducing
agent and,
optionally, a blocking group for the sulfhydryl groups resulting from cleavage
of disulfide
linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an
enzymatic cleavage
using pepsin produces two monovalent Fab fragments and an Fc fragment.
Exemplary
methods for producing antibody fragments are disclosed in U.S. Pat. No.
4,036,945; U.S. Pat.
No. 4,331,647; Nisonoff et al., 1960, Arch. Biochem. Biophys., 89:230; Porter,
1959,
Biochem. J., 73:119; Edelman et al., 1967, METHODS IN ENZYMOLOGY, page 422
(Academic Press), and Coligan et al. (eds.), 1991, CURRENT PROTOCOLS IN
IMMUNOLOGY, (John Wiley & Sons).
[091] Other methods of cleaving antibodies, such as separation of heavy chains
to form
monovalent light-heavy chain fragments, further cleavage of fragments or other
enzymatic,
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chemical or genetic techniques also may be used, so long as the fragments bind
to the antigen
that is recognized by the intact antibody. For example, Fv fragments comprise
an association
of VH and VL chains. This association can be noncovalent, as described in
Inbar et al., 1972,
Proc. Nat'l. Acad. Sci. USA, 69:2659. Alternatively, the variable chains may
be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde. See
Sandhu, 1992, Crit. Rev. Biotech., 12:437.
[092] Preferably, the Fv fragments comprise VH and VL chains connected by a
peptide linker.
These single-chain antigen binding proteins (scFv) are prepared by
constructing a structural
gene comprising DNA sequences encoding the VH and VL domains, connected by an
oligonucleotides linker sequence. The structural gene is inserted into an
expression vector
that is subsequently introduced into a host cell, such as E. coli. The
recombinant host cells
synthesize a single polypeptide chain with a linker peptide bridging the two V
domains.
Methods for producing scFvs are well-known in the art. See Whitlow et al.,
1991, Methods:
A Companion to Methods in Enzymology 2:97; Bird et al., 1988, Science,
242:423; U.S. Pat.
No. 4,946,778; Pack et al., 1993, Bio/Technology, 11:1271, and Sandhu, 1992,
Crit. Rev.
Biotech., 12:437.
[093] Another form of an antibody fragment is a single-domain antibody (dAb),
sometimes
referred to as a single chain antibody. Techniques for producing single-domain
antibodies are
well known in the art (see, e.g., Cossins et al., Protein Expression and
Purification, 2007,
51:253-59; Shuntao et al., Molec Immunol 2006, 43:1912-19; Tanha et al., I
Biol. Chem.
2001, 276:24774-780). Other types of antibody fragments may comprise one or
more
complementarity-determining regions (CDRs). CDR peptides ("minimal recognition
units")
can be obtained by constructing genes encoding the CDR of an antibody of
interest. Such
genes are prepared, for example, by using the polymerase chain reaction to
synthesize the
variable region from RNA of antibody-producing cells. See Larrick et al.,
1991, Methods: A
Companion to Methods in Enzymology 2:106; Ritter et al. (eds.), 1995,
MONOCLONAL
ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, pages
166-179 (Cambridge University Press); Birch et al., (eds.), 1995, MONOCLONAL
ANTIBODIES: PRINCIPLES AND APPLICATIONS, pages 137-185 (Wiley-Liss, Inc.)
Antibody Variations
[094] In certain embodiments, the sequences of antibodies, such as the Fc
portions of
antibodies, may be varied to optimize the physiological characteristics of the
conjugates, such
as the half-life in serum. Methods of substituting amino acid sequences in
proteins are widely
known in the art, such as by site-directed mutagenesis (e.g. Sambrook et al.,
Molecular
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Cloning, A laboratory manual, 2nd Ed, 1989). In preferred embodiments, the
variation may
involve the addition or removal of one or more glycosylation sites in the Fe
sequence (e.g.,
U.S. Patent No. 6,254,868, the Examples section of which is incorporated
herein by
reference). In other preferred embodiments, specific amino acid substitutions
in the Fe
sequence may be made (e.g., Hornick et al., 2000, J Nucl Med 41:355-62; Hinton
et al., 2006,
Immunol 176:346-56; Petkova et al. 2006, Int Immunol 18:1759-69; U.S. Patent
No.
7,217,797; each incorporated herein by reference).
Known Antibodies
[095] Antibodies of use may be commercially obtained from a wide variety of
known sources.
For example, a variety of antibody secreting hybridoma lines are available
from the American
Type Culture Collection (ATCC, Manassas, VA). A large number of antibodies
against
various disease targets, including but not limited to tumor-associated
antigens, have been
deposited at the ATCC and/or have published variable region sequences and are
available for
use in the claimed methods and compositions. See, e.g., U.S. Patent Nos.
7,312,318;
7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132;
7,041,803;
7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598; 6,998,468;
6,994,976;
6,994,852; 6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813;
6,956,107;
6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645;
6,921,533;
6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625; 6,887,468;
6,887,466;
6,884,594; 6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062;
6,861,511;
6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778;
6,812,206;
6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681; 6,764,679;
6,743,898;
6,733,981; 6,730,307; 6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908;
6,689,607;
6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104;
6,652,852;
6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279; 6,596,852;
6,592,868;
6,576,745; 6,572;856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058;
6,528,625;
6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598;
6,482,408;
6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356; 6,455,044;
6,455,040;
6,451,310; 6,444,206; 6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726;
6,406,694;
6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484;
6,376,654;
6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244; 6,346,246;
6,344,198;
6,340,571; 6,340,459; 6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744;
6,129,914;
6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229;
5,789,554;
5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953, 5,525,338.
These are
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exemplary only and a wide variety of other antibodies and their hybridomas are
known in the
art. The skilled artisan will realize that antibody sequences or antibody-
secreting hybridomas
against almost any disease-associated antigen may be obtained by a simple
search of the
ATCC, NCBI and/or USPTO databases for antibodies against a selected disease-
associated
target of interest. The antigen binding domains of the cloned antibodies may
be amplified,
excised, ligated into an expression vector, transfected into an adapted host
cell and used for
protein production, using standard techniques well known in the art.
[096] Exemplary antibodies that may be utilized include, but are not limited
to, hR1 (anti-
IGF-1R, U.S. Patent No. 9,441,043), hPAM4 (anti-mucin, U.S. Pat. No.
7,282,567), hA20
(anti-CD20, U.S. Pat. No. 7,151,164), hA19 (anti-CD19, U.S. Pat. No.
7,109,304),
hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1 (anti-CD74, U.S. Pat. No.
7,312,318),
hLL2 (anti-CD22, U.S. Pat. No. 5,789,554), hMu-9 (anti-CSAp, U.S. Pat. No.
7,387,772),
hL243 (anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Pat.
No.
6,676,924), hMN-15 (anti-CEACAM6, U.S. Pat. No. 8,287,865), hRS7 (anti-EGP-1,
U.S.
Pat. No. 7,238,785), hMN-3 (anti-CEACAM6, U.S. Pat. No. 7,541,440), Ab124 and
Ab125
(anti-CXCR4, U.S. Pat. No. 7,138,496), the Examples section of each cited
patent or
application incorporated herein by reference. More preferably, the antibody is
IMMU-31
(anti-AFP), hRS7 (anti-TROP-2), hMN-14 (anti-CEACAM5), hMN-3 (anti-CEACAM6),
hMN-15 (anti-CEACAM6), hLL1 (anti-CD74), hLL2 (anti-CD22), hL243 or IMMU-114
(anti-HLA-DR), hA19 (anti-CD19) or hA20 (anti-CD20). As used herein, the terms
epratuzumab and hLL2 are interchangeable, as are the terms veltuzumab and
hA20, and the
terms hL243g4P, hL243gamma4P and IMMU-114.
[097] Alternative antibodies of use include, but are not limited to, abciximab
(anti-
glycoprotein IIb/IIIa), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF),
cetuximab
(anti-EGFR), gemtuzumab (anti-CD33), ibritumomab (anti-CD20), panitumumab
(anti-
EGFR), rituximab (anti-CD20), tositumomab (anti-CD20), trastuzumab (anti-
ErbB2),
pembrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab
(anti-
CTLA-4), abagovomab (anti-CA-125), adecatumumab (anti-EpCAM), atlizumab (anti-
IL-6
receptor), benralizumab (anti-CD125), obinutuzumab (GA101, anti-CD20), CC49
(anti-
TAG-72), AB-PG1-XG1-026 (anti-PSMA, U.S. patent application Ser. No.
11/983,372,
deposited as ATCC PTA-4405 and PTA-4406), D2/B (anti-PSMA, WO 2009/130575),
tocilizumab (anti-IL-6 receptor), basiliximab (anti-CD25), daclizumab (anti-
CD25),
efalizumab (anti-CD11 a), GA101 (anti-CD20; Glycart Roche), natalizumab (anti-
a4
integrin), omalizumab (anti-IgE); anti-TNF-a antibodies such as CDP571 (Ofei
et al., 2011,
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Diabetes 45:881-85), MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303
(Thermo Scientific, Rockford, Ill.), infliximab (Centocor, Malvern, Pa.),
certolizumab pegol
(UCB, Brussels, Belgium), anti-CD4OL (UCB, Brussels, Belgium), adalimumab
(Abbott,
Abbott Park, Ill.), or Benlysta (Human Genome Sciences).
[098] A comprehensive analysis of suitable antigen (Cluster Designation, or
CD) targets on
hematopoietic malignant cells, as shown by flow cytometry and which can be a
guide to
selecting suitable antibodies for drug-conjugated immunotherapy, is Craig and
Foon, Blood
prepublished online Jan. 15, 2008; DOL 10.1182/blood-2007-11-120535.
[099] The CD66 antigens consist of five different glycoproteins with similar
structures,
CD66a-e, encoded by the carcinoembryonic antigen (CEA) gene family members,
BCG,
CGM6, NCA, CGM1 and CEA, respectively. These CD66 antigens (e.g., CEACAM6) are
expressed mainly in granulocytes, normal epithelial cells of the digestive
tract and tumor cells
of various tissues. Also included as suitable targets for cancers are cancer
testis antigens, such
as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007; 120(11):2411-7), as well
as CD79a in
myeloid leukemia (Kozlov et al., Cancer Genet. Cytogenet. 2005; 163(1):62-7)
and also B-
cell diseases, and CD79b for non-Hodgkin's lymphoma (Poison et al., Blood
110(2):616-
623). A number of the aforementioned antigens are disclosed in U.S.
Provisional Application
Ser. No. 60/426,379, entitled "Use of Multi-specific, Non-covalent Complexes
for Targeted
Delivery of Therapeutics," filed Nov. 15, 2002. Cancer stem cells, which are
ascribed to be
more therapy-resistant precursor malignant cell populations (Hill and Penis,
J. Natl. Cancer
Inst. 2007; 99:1435-40), have antigens that can be targeted in certain cancer
types, such as
CD133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos. Proc.
2006; 5:155-
79), non-small-cell lung cancer (Donnenberg et al., J. Control Release 2007;
122(3):385-91),
and glioblastoma (Beier et al., Cancer Res. 2007; 67(9):4010-5), and CD44 in
colorectal
cancer (Dalerba er al., Proc. Natl. Acad. Sci. USA 2007; 104(24)10158-63),
pancreatic
cancer (Li et al., Cancer Res. 2007; 67(3):1030-7), and in head and neck
squamous cell
carcinoma (Prince et al., Proc. Natl. Acad. Sci. USA 2007; 104(3)973-8).
[0100] For multiple myeloma therapy, suitable targeting antibodies have been
described
against, for example, CD38 and CD138 (Stevenson, Mol Med 2006; 12(11-12):345-
346;
Tassone et al., Blood 2004; 104(12):3688-96), CD74 (Stein et al., ibid.), CS1
(Tai et al.,
Blood 2008; 112(4):1329-37, and CD40 (Tai et al., 2005; Cancer Res.
65(13):5898-5906).
[0101] Macrophage migration inhibitory factor (MIF) is an important regulator
of innate and
adaptive immunity and apoptosis. It has been reported that CD74 is the
endogenous receptor
for MIF (Leng et al., 2003, J Exp Med 197:1467-76). The therapeutic effect of
antagonistic
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anti-CD74 antibodies on MIF-mediated intracellular pathways may be of use for
treatment of
a broad range of disease states, such as cancers of the bladder, prostate,
breast, lung, colon
and chronic lymphocytic leukemia (e.g., Meyer-Siegler et al., 2004, BMC Cancer
12:34;
Shachar & Haran, 2011, Leuk Lymphoma 52:1446-54); autoimmune diseases such as
rheumatoid arthritis and systemic lupus erythematosus (Morand & Leech, 2005,
Front Biosci
10:12-22; Shachar & Haran, 2011, Leuk Lymphoma 52:1446-54); kidney diseases
such as
renal allograft rejection (Lan, 2008, Nephron Exp Nephrol. 109:e79-83); and
numerous
inflammatory diseases (Meyer-Siegler et al., 2009, Mediators Inflamm epub Mar.
22, 2009;
Takahashi et al., 2009, Respir Res 10:33; Milatuzumab (hLL1) is an exemplary
anti-CD74
antibody of therapeutic use for treatment of MIF-mediated diseases.
[0102] Anti-TNF-a antibodies are known in the art and may be of use to treat
immune
diseases, such as autoimmune disease, immune dysfunction (e.g., graft-versus-
host disease,
organ transplant rejection) or diabetes. Known antibodies against TNF-a
include the human
antibody CDP571 (Ofei et al., 2011, Diabetes 45:881-85); murine antibodies
MTNFAI,
M2TNFAI, M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford,
Ill.);
infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,
Belgium); and
adalimumab (Abbott, Abbott Park, Ill.). These and many other known anti-TNF-a
antibodies
may be used in the claimed methods and compositions. Other antibodies of use
for therapy of
immune dysregulatory or autoimmune disease include, but are not limited to,
anti-B-cell
antibodies such as veltuzumab, epratuzumab, milatuzumab or hL243; tocilizumab
(anti-IL-6
receptor); basiliximab (anti-CD25); daclizumab (anti-CD25); efalizumab (anti-
CD11a);
muromonab-CD3 (anti-CD3 receptor); anti-CD4OL (UCB, Brussels, Belgium);
natalizumab
(anti-a4 integrin) and omalizumab (anti-IgE).
[0103] Studies with checkpoint inhibitor antibodies for cancer therapy have
generated
unprecedented response rates in cancers previously thought to be resistant to
cancer treatment
(see, e.g., Ott & Bhardwaj, 2013, Frontiers in Immunology 4:346; Menzies &
Long, 2013,
Ther Adv Med Oncol 5:278-85; Pardoll, 2012, Nature Reviews 12:252-264; Mavilio
& Lugli,
). Therapy with antagonistic checkpoint blocking antibodies against CTLA-4, PD-
1 and PD-
Li are one of the most promising new avenues of immunotherapy for cancer and
other
diseases. In contrast to the majority of anti-cancer agents, checkpoint
inhibitor do not target
tumor cells directly, but rather target lymphocyte receptors or their ligands
in order to
enhance the endogenous antitumor activity of the immune system (Pardoll, 2012,
Nature
Reviews 12:252-264). Because such antibodies act primarily by regulating the
immune
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response to diseased cells, they may be used in combination with other
therapeutic
modalities, such as the subject anti-HLA-DR antibodies, to enhance their anti-
tumor effect.
[0104] Programmed cell death protein 1 (PD-1, also known as CD279) encodes a
cell surface
membrane protein of the immunoglobulin superfamily, which is expressed in B
cells and NK
cells (Shinohara et al., 1995, Genomics 23:704-6; Blank et al., 2007, Cancer
Immunol
Immunother 56:739-45; Finger et al., 1997, Gene 197:177-87; Pardoll, 2012,
Nature Reviews
12:252-264). Anti-PD1 antibodies have been used for treatment of melanoma, non-
small-cell
lung cancer, bladder cancer, prostate cancer, colorectal cancer, head and neck
cancer, triple-
negative breast cancer, leukemia, lymphoma and renal cell cancer (Topalian et
al., 2012, N
Engl J Med 366:2443-54; Lipson et al., 2013, Clin Cancer Res 19:462-8; Berger
et al., 2008,
Clin Cancer Res 14:3044-51; Gildener-Leapman et al., 2013, Oral Oncol 49:1089-
96;
Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85).
[0105] Exemplary anti-PD1 antibodies include pembrolizumab (MK-3475, MERCK),
nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), and pidilizumab (CT-011,
CURETECH LTD.). Anti-PD1 antibodies are commercially available, for example
from
ABCAM (AB137132), BIOLEGEND (EH12.2H7, RMP1-14) and AFFYMETRIX
EBIOSCIENCE (J105, J116, MIH4).
[0106] Programmed cell death 1 ligand 1 (PD-L1, also known as CD274) is a
ligand for PD-
1, found on activated T cells, B cells, myeloid cells and macrophages. The
complex of PD-1
and PD-Li inhibits proliferation of CD8+ T cells and reduces the immune
response (Topalian
et al., 2012, N Engl J Med 366:2443-54; Brahmer et al., 2012, N Eng J Med
366:2455-65).
Anti-PDL1 antibodies have been used for treatment of non-small cell lung
cancer, melanoma,
colorectal cancer, renal-cell cancer, pancreatic cancer, gastric cancer,
ovarian cancer, breast
cancer, and hematologic malignancies (Brahmer et al., N Eng J Med 366:2455-65;
Ott et al.,
2013, Clin Cancer Res 19:5300-9; Radvanyi et al., 2013, Clin Cancer Res
19:5541; Menzies
& Long, 2013, Ther Adv Med Oncol 5:278-85; Berger et al., 2008, Clin Cancer
Res
14:13044-51).
[0107] Exemplary anti-PDL1 antibodies include MDX-1105 (MEDAREX), MEDI4736
(MEDIMMUNE) MPDL3280A (GENENTECH) and BMS-936559 (BRISTOL-MYERS
SQUIBB). Anti-PDL1 antibodies are also commercially available, for example
from
AFFYMETRIX EBIOSCIENCE (MIH1).
[0108] Cytotoxic T-lymphocyte antigen 4 (CTLA-4, also known as CD152) is also
a member
of the immunoglobulin superfamily that is expressed exclusively on T-cells.
CTLA-4 acts to
inhibit T cell activation and is reported to inhibit helper T cell activity
and enhance regulatory
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T cell immunosuppressive activity (Pardo11, 2012, Nature Reviews 12:252-264).
Anti-CTL4A
antibodies have been used in clinical trials for treatment of melanoma,
prostate cancer, small
cell lung cancer, non-small cell lung cancer (Robert & Ghiringhelli, 2009,
Oncologist
14:848-61; Ott et al., 2013, Clin Cancer Res 19:5300; Weber, 2007, Oncologist
12:864-72;
Wada et al., 2013, J Transl Med 11:89).
[0109] Exemplary anti-CTLA-4 antibodies include ipilimumab (Bristol-Myers
Squibb) and
tremelimumab (PFIZER). Anti-PD1 antibodies are commercially available, for
example from
ABCAM (AB134090), SINO BIOLOGICAL INC. (11159-H03H, 11159-H08H), and
THERMO SCIENTIFIC PIERCE (PAS-29572, PAS-23967, PAS-26465, MA1-12205, MA1-
35914). Ipilimumab has recently received FDA approval for treatment of
metastatic
melanoma (Wada et al., 2013, J Transl Med 11:89).
[0110] These and other known checkpoint inhibitor antibodies may be used in
combination
with anti-HLA-DR antibodies alone or in further combination with an
interferon, such as
interferon-a, for improved cancer therapy.
[0111] The person of ordinary skill will be aware that it is possible to
generate any number of
antibodies against a known and well characterized target antigen, such as
human HLA-DR.
The human HLA-DR antigen has been well characterized in the art, for example
by its amino
acid sequence (see, e.g., GenBank Accession No. ADM15723.1).
Bispecific and Multispecific Antibodies
[0112] Bispecific antibodies are useful in a number of biomedical
applications. For instance,
a bispecific antibody with binding sites for a tumor cell surface antigen and
for a T-cell
surface receptor can direct the lysis of specific tumor cells by T cells.
Bispecific antibodies
recognizing gliomas and the CD3 epitope on T cells have been successfully used
in treating
brain tumors in human patients (Nitta, et al. Lancet. 1990; 355:368-371). A
preferred
bispecific antibody is an anti-CD3 X anti-CD19 antibody. In alternative
embodiments, an
anti-CD3 antibody or fragment thereof may be attached to an antibody or
fragment against
another B-cell associated antigen, such as anti-CD3 X anti-HLA-DR. In certain
embodiments, the techniques and compositions for therapeutic agent conjugation
disclosed
herein may be used with bispecific or multispecific antibodies as the
targeting moieties.
[0113] Numerous methods to produce bispecific or multispecific antibodies are
known, as
disclosed, for example, in U.S. Patent No. 7,405,320, the Examples section of
which is
incorporated herein by reference. Bispecific antibodies can be produced by the
quadroma
method, which involves the fusion of two different hybridomas, each producing
a monoclonal
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antibody recognizing a different antigenic site (Milstein and Cuello, Nature,
1983; 305:537-
540).
[0114] Another method for producing bispecific antibodies uses
heterobifunctional cross-
linkers to chemically tether two different monoclonal antibodies (Staerz, et
al. Nature, 1985;
314:628-631; Perez, et al. Nature, 1985; 316:354-356). Bispecific antibodies
can also be
produced by reduction of each of two parental monoclonal antibodies to the
respective half
molecules, which are then mixed and allowed to reoxidize to obtain the hybrid
structure
(Staerz and Bevan. Proc Natl Acad Sci USA. 1986; 83:1453-1457). Another
alternative
involves chemically cross-linking two or three separately purified Fab'
fragments using
appropriate linkers. (See, e.g., European Patent Application 0453082).
[0115] Other methods include improving the efficiency of generating hybrid
hybridomas by
gene transfer of distinct selectable markers via retrovirus-derived shuttle
vectors into
respective parental hybridomas, which are fused subsequently (DeMonte, et al.
Proc Natl
Acad Sci USA. 1990, 87:2941-2945); or transfection of a hybridoma cell line
with
expression plasmids containing the heavy and light chain genes of a different
antibody.
[0116] Cognate VH and VL domains can be joined with a peptide linker of
appropriate
composition and length (usually consisting of more than 12 amino acid
residues) to form a
single-chain Fv (scFv) with binding activity. Methods of manufacturing scFvs
are disclosed
in U.S. Pat. No. 4,946,778 and U.S. Pat. No. 5,132,405, the Examples section
of each of
which is incorporated herein by reference. Reduction of the peptide linker
length to less than
12 amino acid residues prevents pairing of VH and VL domains on the same chain
and forces
pairing of VH and VL domains with complementary domains on other chains,
resulting in the
formation of functional multimers. Polypeptide chains of VH and VL domains
that are joined
with linkers between 3 and 12 amino acid residues form predominantly dimers
(termed
diabodies). With linkers between 0 and 2 amino acid residues, trimers (termed
triabody) and
tetramers (termed tetrabody) are favored, but the exact patterns of
oligomerization appear to
depend on the composition as well as the orientation of V-domains (VH-linker-
VL or VL-
linker-VH), in addition to the linker length.
[0117] These techniques for producing multispecific or bispecific antibodies
exhibit various
difficulties in terms of low yield, necessity for purification, low stability
or the labor-
intensiveness of the technique. More recently, a technique known as DOCK-AND-
LOCK
(DNL ) has been utilized to produce combinations of virtually any desired
antibodies,
antibody fragments and other effector molecules (see, e.g., U.S. Patent Nos.
7,521,056;
7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070; 7,871,622; 7,906,121;
7,906,118;
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8,163,291; 7,901,680; 7,981,398; 8,003,111 and 8,034,352, the Examples section
of each of
which incorporated herein by reference). The technique utilizes complementary
protein
binding domains, referred to as anchoring domains (AD) and dimerization and
docking
domains (DDD), which bind to each other and allow the assembly of complex
structures,
ranging from dimers, trimers, tetramers, quintamers and hexamers. These form
stable
complexes in high yield without requirement for extensive purification. The
DNL technique
allows the assembly of monospecific, bispecific or multispecific antibodies.
Any of the
techniques known in the art for making bispecific or multispecific antibodies
may be utilized
in the practice of the presently claimed methods.
[0118] In various embodiments, a conjugate as disclosed herein may be part of
a composite,
multispecific antibody. Such antibodies may contain two or more different
antigen binding
sites, with differing specificities. The multispecific composite may bind to
different epitopes
of the same antigen, or alternatively may bind to two different antigens.
[0119] Such antibodies need not only be used in combination, but can be
combined as fusion
proteins of various forms, such as IgG, Fab, scFv, and the like, as described
in U.S. Patent
Nos. 6,083,477; 6,183,744 and 6,962,702 and U.S. Patent Application
Publication Nos.
20030124058; 20030219433; 20040001825; 20040202666; 20040219156; 20040219203;
20040235065; 20050002945; 20050014207; 20050025709; 20050079184; 20050169926;
20050175582; 20050249738; 20060014245 and 20060034759, the Examples section of
each
incorporated herein by reference.
DOCK AND LOCK (DNLO)
[0120] In certain embodiments, the anti-HLA-DR antibodies or fragments may be
incorporated into a multimeric complex, for example using a technique referred
to as DOCK-
AND-LOCK0 (DNLO). The method exploits specific protein/protein interactions
that occur
between the regulatory (R) subunits of cAMP-dependent protein kinase (PKA) and
the
anchoring domain (AD) of A-kinase anchoring proteins (AKAPs) (Baillie et at.,
FEBS
Letters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5:
959). PKA,
which plays a central role in one of the best studied signal transduction
pathways triggered by
the binding of the second messenger cAMP to the R subunits, was first isolated
from rabbit
skeletal muscle in 1968 (Walsh et al., J. Biol. Chem. 1968;243:3763). The
structure of the
holoenzyme consists of two catalytic subunits held in an inactive form by the
R subunits
(Taylor, J. Biol. Chem. 1989;264:8443). Isozymes of PKA are found with two
types of R
subunits (RI and Rh), and each type has a and l isoforms (Scott, Pharmacol.
Ther.
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1991;50:123). The R subunits have been isolated only as stable dimers and the
dimerization
domain has been shown to consist of the first 44 amino-terminal residues
(Newlon et at., Nat.
Struct. Biol. 1999;6:222). Binding of cAMP to the R subunits leads to the
release of active
catalytic subunits for a broad spectrum of serine/threonine kinase activities,
which are
oriented toward selected substrates through the compartmentalization of PKA
via its docking
with AKAPs (Scott et al., J. Biol. Chem. 1990;265;21561).
[0121] Since the first AKAP, microtubule-associated protein-2, was
characterized in 1984
(Lohmann et at., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs
that
localize to various suB cellular sites, including plasma membrane, actin
cytoskeleton,
nucleus, mitochondria, and endoplasmic reticulum, have been identified with
diverse
structures in species ranging from yeast to humans (Wong and Scott, Nat. Rev.
Mol. Cell
Biol. 2004;5:959). The AD of AKAPs for PKA is an amphipathic helix of 14-18
residues
(Carr et at., J. Biol. Chem. 1991;266:14188), with binding affinities reported
for RII dimers
ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.
2003;100:4445).
Interestingly, AKAPs will only bind to dimeric R subunits. For human RIIa, the
AD binds to
a hydrophobic surface formed by the 23 amino-terminal residues (Colledge and
Scott, Trends
Cell Biol. 1999; 6:216). Thus, the dimerization domain and AKAP binding domain
of human
RIIa are both located within the same N-terminal 44 amino acid sequence
(Newlon et at.,
Nat. Struct. Biol. 1999;6:222; Newlon et al., EMBO J. 2001;20:1651), which is
termed the
DDD herein.
DDD of Human Rila and AD of AKAPs as Linker Modules
[0122] We have developed a platform technology to utilize the DDD of human
RIIa and the
AD of AKAP proteins as an excellent pair of linker modules for docking any two
entities,
referred to hereafter as A and B, into a noncovalent complex, which could be
further locked
into a stably tethered structure through the introduction of cysteine residues
into both the
DDD and AD at strategic positions to facilitate the formation of disulfide
bonds. The general
methodology of the "dock-and-lock" approach is as follows. Entity A is
constructed by
linking a DDD sequence to a precursor of A, resulting in a first component
hereafter referred
to as a. Because the DDD sequence would effect the spontaneous formation of a
dimer, A
would thus be composed of a2. Entity B is constructed by linking an AD
sequence to a
precursor of B, resulting in a second component hereafter referred to as b.
The dimeric motif
of DDD contained in a2 will create a docking site for binding to the AD
sequence contained
in b, thus facilitating a ready association of a2 and b to form a binary,
trimeric complex
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composed of a2b. This binding event is made irreversible with a subsequent
reaction to
covalently secure the two entities via disulfide bridges, which occurs very
efficiently based
on the principle of effective local concentration because the initial binding
interactions should
bring the reactive thiol groups placed onto both the DDD and AD into proximity
(Chimura et
at., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically.
[0123] In preferred embodiments, the anti-HLA-DR MAb DNL constructs may be
based on a
variation of the az b structure, in which an IgG immunoglobulin molecule
(e.g., hL243) is
attached at its C-terminal end to two copies of an AD moiety. Preferably the
AD moiety is
attached to the C-terminal end of each light chain. Each AD moiety is capable
of binding to
two DDD moieties in the form of a dimer. By attaching a cytokine or other
therapeutic
protein or peptide to each DDD moiety, four copies of cytokine or other
therapeutic moiety
are conjugated to each IgG molecule.
[0124] By attaching the DDD and AD away from the functional groups of the two
precursors, such site-specific ligations are also expected to preserve the
original activities of
the two precursors. This approach is modular in nature and potentially can be
applied to link,
site-specifically and covalently, a wide range of substances. The DNL method
was disclosed
in U.S. Patent Nos. 7,550,143; 7,521,056; 76,534,866; 7,527,787 and 7,666,400,
the
Examples section of each incorporated herein by reference.
[0125] In preferred embodiments, the effector moiety is a protein or peptide,
more preferably
an antibody, antibody fragment or cytokine, which can be linked to a DDD or AD
unit to
form a fusion protein or peptide. A variety of methods are known for making
fusion proteins,
including nucleic acid synthesis, hybridization and/or amplification to
produce a synthetic
double-stranded nucleic acid encoding a fusion protein of interest. Such
double-stranded
nucleic acids may be inserted into expression vectors for fusion protein
production by
standard molecular biology techniques (see, e.g. Sambrook et al., Molecular
Cloning, A
laboratory manual, 2' Ed, 1989). In such preferred embodiments, the AD and/or
DDD moiety
may be attached to either the N-terminal or C-terminal end of an effector
protein or peptide.
However, the skilled artisan will realize that the site of attachment of an AD
or DDD moiety to
an effector moiety may vary, depending on the chemical nature of the effector
moiety and the
part(s) of the effector moiety involved in its physiological activity. Site-
specific attachment of a
variety of effector moieties may be performed using techniques known in the
art, such as the use
of bivalent cross-linking reagents and/or other chemical conjugation
techniques.
DDD and AD Sequence Variants
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[0126] In certain embodiments, the AD and DDD sequences incorporated into the
anti-HLA-
DR MAb DNL complex comprise the amino acid sequences of DDD1 (SEQ ID NO:7) and
AD1 (SEQ ID NO:9) below. In more preferred embodiments, the AD and DDD
sequences
comprise the amino acid sequences of DDD2 (SEQ ID NO:8) and AD2 (SEQ ID
NO:10),
which are designed to promote disulfide bond formation between the DDD and AD
moieties.
DDD/
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID
NO:7)
DDD2
CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID
NO:8)
AD]
QIEYLAKQIVDNAIQQA (SEQ ID NO:9)
AD2
CGQIEYLAKQIVDNAIQQAGC (SEQ ID NO:10)
[0127] However, in alternative embodiments sequence variants AD and/or DDD
moieties
may be utilized in construction of the anti-HLA-DR MAb DNL complexes. The
structure-
function relationships of the AD and DDD domains have been the subject of
investigation.
(See, e.g., Burns-Hamuro et al., 2005, Protein Sci 14:2982-92; Carr et al.,
2001, J Biol Chem
276:17332-38; Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50;
Hundsrucker et al.,
2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J 400:493-99; Gold
et al., 2006,
Mol Cell 24:383-95; Kinderman et al., 2006, Mol Cell 24:397-408, the entire
text of each of
which is incorporated herein by reference.)
[0128] For example, Kinderman et al. (2006) examined the crystal structure of
the AD-DDD
binding interaction and concluded that the human DDD sequence contained a
number of
conserved amino acid residues that were important in either dimer formation or
AKAP
binding, underlined below in SEQ ID NO:7. (See Figure 1 of Kinderman et al.,
2006,
incorporated herein by reference.) The skilled artisan will realize that in
designing sequence
variants of the DDD sequence, one would desirably avoid changing any of the
underlined
residues, while conservative amino acid substitutions might be made for
residues that are less
critical for dimerization and AKAP binding. Conservative amino acid
substitutions are
discussed in more detail below, but could involve for example substitution of
an aspartate
residue for a glutamate residue, or a leucine or valine residue for an
isoleucine residue, etc.
Such conservative amino acid substitutions are well known in the art.
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Human DDD sequence from protein kinase A
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO:7)
[0129] Alto et al. (2003) performed a bioinformatic analysis of the AD
sequence of various
AKAP proteins to design an Rh selective AD sequence called AKAP-IS (SEQ ID
NO:9),
with a binding constant for DDD of 0.4 nM. The AKAP-IS sequence was designed
as a
peptide antagonist of AKAP binding to PKA. Residues in the AKAP-IS sequence
where
substitutions tended to decrease binding to DDD are underlined in SEQ ID NO:9.
AKAP-IS sequence
QIEYLAKQIVDNAIQQA (SEQ ID NO:9)
[0130] Similarly, Gold (2006) utilized crystallography and peptide screening
to develop a
SuperAKAP-IS sequence (SEQ ID NO:11), exhibiting a five order of magnitude
higher
selectivity for the Rh isoform of PKA compared with the RI isoform. Underlined
residues
indicate the positions of amino acid substitutions, relative to the AKAP-IS
sequence, that
increased binding to the DDD moiety of RIIa. In this sequence, the N-terminal
Q residue is
numbered as residue number 4 and the C-terminal A residue is residue number
20. Residues
where substitutions could be made to affect the affinity for Mkt were residues
8, 11, 15, 16,
18, 19 and 20 (Gold et al., 2006). It is contemplated that in certain
alternative embodiments,
the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moiety
sequence to
prepare anti-HLA-DR MAb DNL constructs. It is anticipated that, as with the
AKAP-IS
sequence shown in SEQ ID NO:9, the AD moiety may also include the additional N-
terminal
residues cysteine and glycine and C-terminal residues glycine and cysteine, as
shown in SEQ
ID NO:10.
SuperAKAP-IS
QIEYVAKQIVDYAIHQA (SEQ ID NO:11)
[0131] Hundsrucker et al. (2006) developed still other peptide competitors for
AKAP binding
to PKA, with a binding constant as low as 0.4 nM to the DDD of the RII form of
PKA. The
sequences of various AKAP antagonistic peptides is provided in Table 1 of
Hundsrucker et
al. (incorporated herein by reference). Residues that were highly conserved
among the AD
domains of different AKAP proteins are indicated below by underlining with
reference to the
AKAP IS sequence (SEQ ID NO:9). The residues are the same as observed by Alto
et al.
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(2003), with the addition of the C-terminal alanine residue. (See FIG. 4 of
Hundsrucker et al.
(2006), incorporated herein by reference.)
AKAP -IS
QIEYLAKQIVDNAIQQA (SEQ ID NO:9)
[0132] Can et al. (2001) examined the degree of sequence homology between
different
AKAP-binding DDD sequences from human and non-human proteins and identified
residues
in the DDD sequences that appeared to be the most highly conserved among
different DDD
moieties. These are indicated below by underlining with reference to the human
PKA RIIa
DDD sequence of SEQ ID NO:7. Residues that were particularly conserved are
further
indicated by italics. The residues overlap with, but are not identical to
those suggested by
Kinderman et al. (2006) to be important for binding to AKAP proteins.
SHIQ/PPGL TELLQ GY TV EVLRQOPPDLVEFA VEYFIRLREARA (SEQ ID NO :7)
[0133] The skilled artisan will realize that in general, those amino acid
residues that are
highly conserved in the DDD and AD sequences from different proteins are ones
that it may
be preferred to remain constant in making amino acid substitutions, while
residues that are
less highly conserved may be more easily varied to produce sequence variants
of the AD
and/or DDD sequences described herein.
Antibody Allotypes
[0134] Immunogenicity of therapeutic antibodies is associated with increased
risk of infusion
reactions and decreased duration of therapeutic response (Baert et al., 2003,
N Engl J Med
348:602-08). The extent to which therapeutic antibodies induce an immune
response in the host
may be determined in part by the allotype of the antibody (Stickler et al.,
2011, Genes and
Immunity 12:213-21). Antibody allotype is related to amino acid sequence
variations at specific
locations in the constant region sequences of the antibody. The allotypes of
IgG antibodies
containing a heavy chain y-type constant region are designated as Gm allotypes
(1976, J
Immunol 117:1056-59).
[0135] For the common IgG1 human antibodies, the most prevalent allotype is
Glml (Stickler
et al., 2011, Genes and Immunity 12:213-21). However, the G1m3 allotype also
occurs
frequently in Caucasians (Id.). It has been reported that Glml antibodies
contain allotypic
sequences that tend to induce an immune response when administered to non-Glml
(nG1m1)
recipients, such as G1m3 patients (Id.). Non-Glml allotype antibodies are not
as immunogenic
when administered to Glml patients (Id.).
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[0136] The human Glml allotype comprises the amino acids D12 (Kabat position
356) and L14
(Kabat position 358) in the CH3 sequence of the heavy chain IgGl. The nGlml
allotype
comprises the amino acids E12 and M14 at the same locations. Both Glml and
nGlml allotypes
comprise an E13 residue in between the two variable sites and the allotypes
are sometimes
referred to as DEL and EEM allotypes. A non-limiting example of the heavy
chain constant
region sequences for Glml and nGlml allotype antibodies is shown for the
exemplary
antibodies rituximab (SEQ ID NO:12) and veltuzumab (SEQ ID NO:13).
Rituximab heavy chain variable region sequence (SEQ ID NO. 12)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Veltuzumab heavy chain variable region (SEQ ID NO. 13)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0137] With regard to therapeutic antibodies, veltuzumab and rituximab are,
respectively,
humanized and chimeric IgG1 antibodies against CD20, of use for therapy of a
wide variety of
hematological malignancies and/or autoimmune diseases. Table 1 compares the
allotype
sequences of rituximab vs. veltuzumab. As shown in Table 1, rituximab
(G1m17,1) is a DEL
allotype IgGl, with an additional sequence variation at Kabat position 214
(heavy chain CH1) of
lysine in rituximab vs. arginine in veltuzumab. It has been reported that
veltuzumab is less
immunogenic in subjects than rituximab (see, e.g., Morchhauser et al., 2009, J
Clin Oncol
27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak & Robak, 2011,
BioDrugs
25:13-25), an effect that has been attributed to the difference between
humanized and chimeric
antibodies. However, the difference in allotypes between the EEM and DEL
allotypes likely
also accounts for the lower immunogenicity of veltuzumab.
Table 1. Allotypes of Rituximab vs. Veltuzumab
Heavy chain position and associated allotypes
Complete allotype 214 (allotype) 356/358 (allotype) 431
(allotype)
Rituximab G1m17,1 K 17 D/L 1 A
Veltuzumab G1m3 R 3 E/M A
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[0138] In order to reduce the immunogenicity of therapeutic antibodies in
individuals of
nGlml genotype, it is desirable to select the allotype of the antibody to
correspond to the EEM
allotype, with a glutamate residue at Kabat position 356, a methionine at
Kabat position 358, and
preferably an arginine residue at Kabat position 214. Surprisingly, it was
found that repeated
subcutaneous administration of G1m3 antibodies over a long period of time did
not result in a
significant immune response.
Amino Acid Substitutions
[0139] In alternative embodiments, the disclosed methods and compositions may
involve
production and use of proteins or peptides with one or more substituted amino
acid residues.
For example, the DDD and/or AD sequences used to make DNL constructs may be
modified as discussed above.
[0140] The skilled artisan will be aware that, in general, amino acid
substitutions typically
involve the replacement of an amino acid with another amino acid of relatively
similar
properties (i.e., conservative amino acid substitutions). The properties of
the various amino
acids and effect of amino acid substitution on protein structure and function
have been the
subject of extensive study and knowledge in the art.
[0141] For example, the hydropathic index of amino acids may be considered
(Kyte &
Doolittle, 1982,1 Mol. Biol., 157:105-132). The relative hydropathic character
of the amino
acid contributes to the secondary structure of the resultant protein, which in
turn defines the
interaction of the protein with other molecules. Each amino acid has been
assigned a
hydropathic index on the basis of its hydrophobicity and charge
characteristics (Kyte &
Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);
phenylalanine
(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-
0.4); threonine (-
0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6);
histidine (-3.2); glutamate
(-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9);
and arginine (-4.5).
In making conservative substitutions, the use of amino acids whose hydropathic
indices are
within 2 is preferred, within 1 are more preferred, and within 0.5 are
even more
preferred.
[0142] Amino acid substitution may also take into account the hydrophilicity
of the amino
acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicity values have been
assigned to
amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0);
glutamate (+3.0); serine
(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4);
proline (-0.5 ±1);
alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-
1.5); leucine (-1.8);
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isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
Replacement of
amino acids with others of similar hydrophilicity is preferred.
[0143] Other considerations include the size of the amino acid side chain. For
example, it
would generally not be preferred to replace an amino acid with a compact side
chain, such as
glycine or serine, with an amino acid with a bulky side chain, e.g.,
tryptophan or tyrosine.
The effect of various amino acid residues on protein secondary structure is
also a
consideration. Through empirical study, the effect of different amino acid
residues on the
tendency of protein domains to adopt an alpha-helical, beta-sheet or reverse
turn secondary
structure has been determined and is known in the art (see, e.g., Chou &
Fasman, 1974,
Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,
Biophys. J.,
26:367-384).
[0144] Based on such considerations and extensive empirical study, tables of
conservative
amino acid substitutions have been constructed and are known in the art. For
example:
arginine and lysine; glutamate and aspartate; serine and threonine; glutamine
and asparagine;
and valine, leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg
(R) gln, asn, lys;
Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys (C) ala, ser; Gln (Q)
glu, asn; Glu (E)
gln, asp; Gly (G) ala; His (H) asn, gln, lys, arg; Ile (I) val, met, ala, phe,
leu; Leu (L) val, met,
ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F) leu, val,
ile, ala, tyr; Pro (P)
ala; Ser (S), thr; Thr (T) ser; Trp (W) phe, tyr; Tyr (Y) trp, phe, thr, ser;
Val (V) ile, leu, met,
phe, ala.
[0145] Other considerations for amino acid substitutions include whether or
not the residue is
located in the interior of a protein or is solvent exposed. For interior
residues, conservative
substitutions would include: Asp and Asn; Ser and Thr; Ser and Ala; Thr and
Ala; Ala and
Gly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr; Tyr and
Trp (See,
e.g., PROWL website at rockefeller.edu). For solvent exposed residues,
conservative
substitutions would include: Asp and Asn; Asp and Glu; Glu and Gln; Glu and
Ala; Gly and
Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and
Arg; Val and
Leu; Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have been
constructed to
assist in selection of amino acid substitutions, such as the PAM250 scoring
matrix, Dayhoff
matrix, Grantham matrix, McLachlan matrix, Doolittle matrix, Henikoff matrix,
Miyata
matrix, Fitch matrix, Jones matrix, Rao matrix, Levin matrix and Risler matrix
(Idem.)
[0146] In determining amino acid substitutions, one may also consider the
existence of
intermolecular or intramolecular bonds, such as formation of ionic bonds (salt
bridges)
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between positively charged residues (e.g., His, Arg, Lys) and negatively
charged residues
(e.g., Asp, Glu) or disulfide bonds between nearby cysteine residues.
[0147] Methods of substituting any amino acid for any other amino acid in an
encoded
protein sequence are well known and a matter of routine experimentation for
the skilled
artisan, for example by the technique of site-directed mutagenesis or by
synthesis and
assembly of oligonucleotides encoding an amino acid substitution and splicing
into an
expression vector construct.
Conjugation Protocols
[0148] In certain embodiments, the anti-HLA-DR antibody or fragment may be
conjugated to
one or more therapeutic or diagnostic agents. The therapeutic agents do not
need to be the
same but can be different, e.g. a drug and a radioisotope. For example, 131I
can be
incorporated into a tyrosine of an antibody or fusion protein and a drug
attached to an epsilon
amino group of a lysine residue. Therapeutic and diagnostic agents also can be
attached, for
example to reduced SH groups and/or to carbohydrate side chains. Many methods
for making
covalent or non-covalent conjugates of therapeutic or diagnostic agents with
antibodies or
fusion proteins are known in the art and any such known method may be
utilized.
[0149] A therapeutic or diagnostic agent can be attached at the hinge region
of a reduced
antibody component via disulfide bond formation. Alternatively, such agents
can be attached
using a heterobifunctional cross-linker, such as N-succinyl 3-(2-
pyridyldithio)propionate
(SPDP). Yu et at., Int. I Cancer 56: 244 (1994). General techniques for such
conjugation are
well-known in the art. See, for example, Wong, CHEMISTRY OF PROTEIN
CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et at.,
"Modification of Antibodies by Chemical Methods," in MONOCLONAL ANTIBODIES:
PRINCIPLES AND APPLICATIONS, Birch et at. (eds.), pages 187-230 (Wiley-Liss,
Inc.
1995); Price, "Production and Characterization of Synthetic Peptide-Derived
Antibodies," in
MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL
APPLICATION, Ritter et at. (eds.), pages 60-84 (Cambridge University Press
1995).
Alternatively, the therapeutic or diagnostic agent can be conjugated via a
carbohydrate moiety
in the Fc region of the antibody. The carbohydrate group can be used to
increase the loading
of the same agent that is bound to a thiol group, or the carbohydrate moiety
can be used to
bind a different therapeutic or diagnostic agent.
[0150] Methods for conjugating peptides to antibody components via an antibody
carbohydrate moiety are well-known to those of skill in the art. See, for
example, Shih et at.,
Int.' Cancer 41: 832 (1988); Shih et al., Int.' Cancer 46: 1101 (1990); and
Shih et al.,U U.S.
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Patent No. 5,057,313, incorporated herein in their entirety by reference. The
general method
involves reacting an antibody component having an oxidized carbohydrate
portion with a
carrier polymer that has at least one free amine function. This reaction
results in an initial
Schiff base (imine) linkage, which can be stabilized by reduction to a
secondary amine to
form the final conjugate.
[0151] The Fc region may be absent if the antibody used as the antibody
component of the
immunoconjugate is an antibody fragment. However, it is possible to introduce
a carbohydrate
moiety into the light chain variable region of a full length antibody or
antibody fragment.
See, for example, Leung et al., I Immunol. 154: 5919 (1995); Hansen et al.,U
U.S. Patent No.
5,443,953 (1995), Leung et at., U.S. patent No. 6,254,868, incorporated herein
by reference
in their entirety. The engineered carbohydrate moiety is used to attach the
therapeutic or
diagnostic agent.
[0152] In some embodiments, a chelating agent may be attached to an antibody,
antibody
fragment or fusion protein and used to chelate a therapeutic or diagnostic
agent, such as a
radionuclide. Exemplary chelators include but are not limited to DTPA (such as
Mx-DTPA),
DOTA, TETA, NETA or NOTA. Methods of conjugation and use of chelating agents
to attach
metals or other ligands to proteins are well known in the art (see, e.g., U.S.
Patent Application
Serial No. 12/112,289, incorporated herein by reference in its entirety).
[0153] In certain embodiments, radioactive metals or paramagnetic ions may be
attached to
proteins or peptides by reaction with a reagent having a long tail, to which
may be attached a
multiplicity of chelating groups for binding ions. Such a tail can be a
polymer such as a
polylysine, polysaccharide, or other derivatized or derivatizable chains
having pendant
groups to which can be bound chelating groups such as, e.g.,
ethylenediaminetetraacetic acid
(EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines,
crown ethers,
bis-thiosemicarbazones, polyoximes, and like groups known to be useful for
this purpose.
[0154] Chelates may be directly linked to antibodies or peptides, for example
as disclosed in
U.S. Patent 4,824,659, incorporated herein in its entirety by reference.
Particularly useful
metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and
cyclohexyl
analogs, used with diagnostic isotopes in the general energy range of 60 to
4,000 keV, such
as 1251, 1311, 1231, 1241, 62cti, 64cti, 18F, "In, 67Ga, 68-a,
G 99mTc, 94mTc, "C, 13N, 150, 76Br , for
radioimaging. The same chelates, when complexed with non-radioactive metals,
such as
manganese, iron and gadolinium are useful for MM. Macrocyclic chelates such as
NOTA,
DOTA, and TETA are of use with a variety of metals and radiometals, most
particularly with
radionuclides of gallium, yttrium and copper, respectively. Such metal-chelate
complexes can
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be made very stable by tailoring the ring size to the metal of interest. Other
ring-type chelates
such as macrocyclic polyethers, which are of interest for stably binding
nuclides, such as
223Ra for RAIT are encompassed.
[0155] More recently, methods of 18F-labeling of use in PET scanning
techniques have been
disclosed, for example by reaction of F-18 with a metal or other atom, such as
aluminum.
The 18F-Al conjugate may be complexed with chelating groups, such as DOTA,
NOTA or
NETA that are attached directly to antibodies or used to label targetable
constructs in pre-
targeting methods. Such F-18 labeling techniques are disclosed in U.S. Patent
Application
Serial No. 12/112,289, filed 4/30/08, the entire text of which is incorporated
herein by
reference.
[0156] In preferred embodiments, the conjugation protocol is based on a thiol-
maleimide, a
thiol-vinylsulfone, a thiol-bromoacetamide, or a thiol-iodoacetamide reaction
that is facile at
neutral or acidic pH. This obviates the need for higher pH conditions for
conjugations as, for
instance, would be necessitated when using active esters. Further details of
exemplary
conjugation protocols are described below in the Examples section.
Therapeutic Treatment
[0157] In another aspect, the invention relates to a method of treating a
subject, comprising
administering a therapeutically effective amount of a therapeutic conjugate as
described
herein to a subject. Diseases that may be treated with the therapeutic
conjugates described
herein include, but are not limited to B-cell malignancies (e.g., non-
Hodgkin's lymphoma,
mantle cell lymphoma, multiple myeloma, Hodgkin's lymphoma, diffuse large B
cell
lymphoma, Burkitt lymphoma, follicular lymphoma, acute lymphatic leukemia,
chronic
lymphatic leukemia, hairy cell leukemia) using an anti-HLA-DR immunoconjugate.
More
preferably, the cancer is AML (acute myelocytic leukemia), ALL (acute
lymphocytic
leukemia) or MM (multiple myeloma). However, any HLA-DR positive tumor may be
treated with the subject immunoconjugates, such as skin, esophageal, stomach,
colon, rectal,
pancreatic, lung, breast, ovarian, bladder, endometrial, cervical, testicular,
melanoma, kidney,
or liver cancer. Such therapeutics can be given once or repeatedly, depending
on the disease
state and tolerability of the conjugate, and can also be used optionally in
combination with
other therapeutic modalities, such as surgery, external radiation,
radioimmunotherapy,
immunotherapy, chemotherapy, anti sense therapy, interference RNA therapy,
gene therapy,
and the like. Each combination will be adapted to the tumor type, stage,
patient condition and
prior therapy, and other factors considered by the managing physician.
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[0158] As used herein, the term "subject" refers to any animal (i.e.,
vertebrates and
invertebrates) including, but not limited to mammals, including humans. It is
not intended
that the term be limited to a particular age or sex. Thus, adult and newborn
subjects, as well
as fetuses, whether male or female, are encompassed by the term. Doses given
herein are for
humans, but can be adjusted to the size of other mammals, as well as children,
in accordance
with weight or square meter size.
[0159] In an exemplary embodiment, an hL243 antibody is a humanized antibody
comprising
the heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:1), CDR2
(WINTYTREPTYADDFKG, SEQ ID NO:2), and CDR3 (DITAVVPTGFDY, SEQ ID
NO:3) and light chain CDR sequences CDR1 (RASENIYSNLA, SEQ ID NO:4), CDR2
(AASNLAD, SEQ ID NO:5), and CDR3 (QHFWTTPWA, SEQ ID NO:6).
[0160] In a preferred embodiment, the antibodies that are used in the
treatment of human
disease are human or humanized (CDR-grafted) versions of antibodies; although
murine and
chimeric versions of antibodies can be used. Same species IgG molecules as
delivery agents
are mostly preferred to minimize immune responses. This is particularly
important when
considering repeat treatments. For humans, a human or humanized IgG antibody
is less
likely to generate an anti-IgG immune response from patients. Antibodies such
as hLL1 and
hLL2 rapidly internalize after binding to internalizing antigen on target
cells, which means
that the chemotherapeutic drug being carried is rapidly internalized into
cells as well.
However, antibodies that have slower rates of internalization can also be used
to effect
selective therapy.
[0161] In another preferred embodiment, the therapeutic conjugates can be used
to treat
autoimmune disease or immune system dysfunction (e.g., graft-versus-host
disease, organ
transplant rejection). Antibodies of use to treat autoimmune/immune
dysfunction disease may
bind to exemplary antigens including, but not limited to, BCL-1, BCL-2, BCL-6,
CD1a, CD2,
CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19,
CD20, CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40, CD4OL, CD41a, CD43,
CD45, CD55, CD56, CCD57, CD59, CD64, CD71, CD74, CD79a, CD79b, CD117, CD138,
FMC-7 and HLA-DR. Antibodies that bind to these and other target antigens,
discussed
above, may be used to treat autoimmune or immune dysfunction diseases.
Autoimmune
diseases that may be treated with immunoconjugates may include acute
idiopathic
thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura,
dermatomyositis,
Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus
nephritis,
rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetes
mellitus, Henoch-
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Schonlein purpura, post-streptococcal nephritis, erythema nodosum, Takayasu's
arteritis,
ANCA-associated vasculitides, Addison's disease, rheumatoid arthritis,
multiple sclerosis,
sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,
polyarteritis nodosa,
ankylosing spondylitis, Goodpasture's syndrome, thromboangitis obliterans,
Sjogren's
syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis,
scleroderma,
chronic active hepatitis, polymyositis/dermatomyositis, polychondritis,
bullous pemphigoid,
pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,
amyotrophic
lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia,
pernicious anemia, rapidly
progressive glomerulonephritis, psoriasis or fibrosing alveolitis.
[0162] In another preferred embodiment, a therapeutic agent used in
combination with the
camptothecin conjugate of this invention may comprise one or more isotopes.
Radioactive
isotopes useful for treating diseased tissue include, but are not limited to-
1771,,u, 212Bi,
213Bi, niAt, 62cu, 67cti, 90y, 1251, 1311, 32p, 33p, 47se, 111Ag, 67Ga, 142pr,
153sm, 161Tb,
166Dy, 166H0, 186Re, 188Re, 189Re, 212pb, 223Ra, 225Ac, 59Fe, 75Se, 77As,
89Sr, 99M0,
105Rb, 109pd, 143pr, 149pm, 169Er, 194k 198Au, 'Au, 227Th and 211pb. The
therapeutic
radionuclide preferably has a decay-energy in the range of 20 to 6,000 keV,
preferably in the
ranges 60 to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter,
and 4,000-
6,000 keV for an alpha emitter. Maximum decay energies of useful beta-particle-
emitting
nuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, and most
preferably
500-2,500 keV. Also preferred are radionuclides that substantially decay with
Auger-emitting
particles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111,
Sb-119, I-
125, Ho-161, Os-189m and Ir-192. Decay energies of useful beta-particle-
emitting nuclides
are preferably <1,000 keV, more preferably <100 keV, and most preferably <70
keV. Also
preferred are radionuclides that substantially decay with generation of alpha-
particles. Such
radionuclides include, but are not limited to: Dy-152, At-211, Bi-212, Ra-223,
Rn-219, Po-
215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, Th-227 and Fm-255. Decay energies
of useful
alpha-particle-emitting radionuclides are preferably 2,000-10,000 keV, more
preferably
3,000-8,000 keV, and most preferably 4,000-7,000 keV. Additional potential
radioisotopes
of use include
HC, 13N, 'so, 75Br, 198Au, 224Ac, 1261, 1331, 77Br, 113m-n,
95RU, 97RU, 1 3RU,
105Ru, io7Hg, 203Hg, iilmTe, 122mTe, 125mTe, 1651,m, 1671,m, 168Tm, 197pt,
109pd, 105Rb,
142pr, 143pr, 161Tb, 166H0, 'Au, 57Co, 58Co, 51Cr, 59Fe, 755e, 201T1, 225Ac,
76Br, 169yb,
and the like.
[0163] Radionuclides and other metals may be delivered, for example, using
chelating groups
attached to an antibody or conjugate. Macrocyclic chelates such as NOTA, DOTA,
and
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TETA are of use with a variety of metals and radiometals, most particularly
with
radionuclides of gallium, yttrium and copper, respectively. Such metal-chelate
complexes can
be made very stable by tailoring the ring size to the metal of interest. Other
ring-type
chelates, such as macrocyclic polyethers for complexing 223Ra, may be used.
[0164] Therapeutic agents of use in combination with the camptothecin
conjugates described
herein also include, for example, chemotherapeutic drugs such as vinca
alkaloids,
anthracyclines, epipodophyllotoxins, taxanes, antimetabolites, tyrosine kinase
inhibitors,
alkylating agents, antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic
and proapoptotic
agents, particularly doxorubicin, methotrexate, taxol, other camptothecins,
and others from
these and other classes of anticancer agents, and the like. Other cancer
chemotherapeutic
drugs include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes,
folic acid analogs,
pyrimidine analogs, purine analogs, platinum coordination complexes, hormones,
and the
like. Suitable chemotherapeutic agents are described in REMINGTON'S
PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in
GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF
THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as well as revised
editions of
these publications. Other suitable chemotherapeutic agents, such as
experimental drugs, are
known to those of skill in the art.
[0165] Exemplary drugs of use include, but are not limited to, 5-fluorouracil,
afatinib,
aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291,
bendamustine,
bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin,
camptothecin,
carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil,
cisplatin
(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatinum,
cladribine,
camptothecans, crizotinib, cyclophosphamide, cytarabine, dacarbazine,
dasatinib, dinaciclib,
docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine
(2P-DOX),
cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide,
erlotinib,
estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptor
binding agents,
etoposide (VP16), etoposide glucuronide, etoposide phosphate, exemestane,
fingolimod,
floxuridine (FUdR), 3',5'-0-dioleoyl-FudR (FUdR-d0), fludarabine, flutamide,
farnesyl-
protein transferase inhibitors, flavopiridol, fostamatinib, ganetespib, GDC-
0834, GS-1101,
gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib,
ifosfamide, imatinib, L-
asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,
mechlorethamine,
melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,
mithramycin,
mitomycin, mitotane, navelbine, neratinib, nilotinib, nitrosurea, olaparib,
plicomycin,
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procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene,
semustine, sorafenib,
streptozocin, SU11248, sunitinib, tamoxifen, temazolomide, transplatinum,
thalidomide,
thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vatalanib,
vinorelbine,
vinblastine, vincristine, vinca alkaloids and ZD1839. Such agents may be part
of the
conjugates described herein or may alternatively be administered in
combination with the
described conjugates, either prior to, simultaneously with or after the
conjugate.
Alternatively, one or more therapeutic naked antibodies as are known in the
art may be used
in combination with the described conjugates. Exemplary therapeutic naked
antibodies are
described above.
[0166] In preferred embodiments, a therapeutic agent to be used in combination
with a DNA-
breaking antibody conjugate (e.g., an SN-38-ADC) is a microtubule inhibitor,
such as a vinca
alkaloid, a taxanes, a maytansinoid or an auristatin. Exemplary known
microtubule inhibitors
include paclitaxel, vincristine, vinblastine, mertansine, epothilone,
docetaxel, discodermolide,
combrestatin, podophyllotoxin, CI-980, phenylahistins, steganacins, curacins,
2-methoxy
estradiol, E7010, methoxy benzenesuflonamides, vinorelbine, vinflunine,
vindesine,
dolastatins, spongistatin, rhizoxin, tasidotin, halichondrins, hemiasterlins,
cryptophycin 52,
MMAE and eribulin mesylate.
[0167] In an alternative preferred embodiment, a therapeutic agent to be used
in combination
with a DNA-breaking ADC, such as an SN-38-antibody conjugate, is a PARP
inhibitor, such
as olaparib, talazoparib (BMN-673), rucaparib, veliparib, CEP 9722, MK 4827,
BGB-290,
ABT-888, AG014699, BSI-201, CEP-8983 or 3-aminobenzamide.
[0168] In another alternative, a therapeutic agent used in combination with an
antibody or
immunoconjugate is a Bruton kinase inhibitor, such as such as ibrutinib (PCI-
32765), PCI-
45292, CC-292 (AVL-292), ONO-4059, GDC-0834, LFM-A13 or RN486.
[0169] In yet another alternative, a therapeutic agent used in combination
with an antibody or
immunoconjugate is a PI3K inhibitor, such as idelalisib, Wortmannin,
demethoxyviridin,
perifosine, PX-866, IPI-145 (duvelisib), BAY 80-6946, BEZ235, RP6530, TGR1202,
SF1126,
INK1117, GDC-0941, BKM120, XL147, XL765, Palomid 529, G5K1059615, Z5TK474,
PWT33597, IC87114, TG100-115, CAL263, PI-103, GNE477, CUDC-907, AEZS-136 or
LY294002.
[0170] Therapeutic agents that may be used in concert with the camptothecin
conjugates also
may comprise toxins conjugated to targeting moieties. Toxins that may be used
in this regard
include ricin, abrin, ribonuclease (RNase), ranpirnase, DNase I,
Staphylococcal enterotoxin-
A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas
exotoxin, and
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Pseudomonas endotoxin. (See, e.g., Pastan. et al., Cell (1986), 47:641, and
Sharkey and
Goldenberg, CA Cancer J Cl/n. 2006 Jul-Aug;56(4):226-43.) Additional toxins
suitable for
use herein are known to those of skill in the art and are disclosed in U.S.
6,077,499.
[0171] Yet another class of therapeutic agent may comprise one or more
immunomodulators.
Immunomodulators of use may be selected from a cytokine, a stem cell growth
factor, a
lymphotoxin, an hematopoietic factor, a colony stimulating factor (CSF), an
interferon (IFN),
erythropoietin, thrombopoietin and a combination thereof Specifically useful
are
lymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors, such
as interleukin
(IL), colony stimulating factor, such as granulocyte-colony stimulating factor
(G-CSF) or
granulocyte macrophage-colony stimulating factor (GM-CSF), interferon, such as
interferons-a, -13, -y or -X., and stem cell growth factor, such as that
designated "51 factor".
Included among the cytokines are growth hormones such as human growth hormone,
N-
methionyl human growth hormone, and bovine growth hormone; parathyroid
hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones
such as follicle
stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing
hormone
(LH); hepatic growth factor; prostaglandin, fibroblast growth factor;
prolactin; placental
lactogen, OB protein; tumor necrosis factor-a and - B; mullerian-inhibiting
substance; mouse
gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth
factor;
integrin; thrombopoietin (TP0); nerve growth factors such as NGF-B; platelet-
growth factor;
transforming growth factors (TGFs) such as TGF- a and TGF- B; insulin-like
growth factor-I
and -II; erythropoietin (EPO); osteoinductive factors; interferons such as
interferon-a, -13, and
-y; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
interleukins (ILs)
such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-
11, IL-12; IL-13,
IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand or FLT-3,
angiostatin,
thrombospondin, endostatin, tumor necrosis factor and lymphotoxin (LT). As
used herein, the
term cytokine includes proteins from natural sources or from recombinant cell
culture and
biologically active equivalents of the native sequence cytokines.
[0172] Chemokines of use include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-
10.
[0173] The person of ordinary skill will realize that the subject
immunoconjugates,
comprising a camptothecin conjugated to an antibody or antibody fragment, may
be used
alone or in combination with one or more other therapeutic agents, such as a
second antibody,
second antibody fragment, second immunoconjugate, radionuclide, toxin, drug,
chemotherapeutic agent, radiation therapy, chemokine, cytokine,
immunomodulator, enzyme,
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hormone, oligonucleotide, RNAi or siRNA. Such additional therapeutic agents
may be
administered separately, in combination with, or attached to the subject
antibody-drug
immunoconjugates.
Formulation and Administration
[0174] Suitable routes of administration of the conjugates include, without
limitation, oral,
parenteral, subcutaneous, rectal, transmucosal, intestinal administration,
intramuscular,
intramedullary, intrathecal, direct intraventricular, intravenous,
intravitreal, intraperitoneal,
intranasal, or intraocular injections. The preferred routes of administration
are parenteral.
Alternatively, one may administer the compound in a local rather than systemic
manner, for
example, via injection of the compound directly into a solid tumor.
[0175] Immunoconjugates can be formulated according to known methods to
prepare
pharmaceutically useful compositions, whereby the immunoconjugate is combined
in a
mixture with a pharmaceutically suitable excipient. Sterile phosphate-buffered
saline is one
example of a pharmaceutically suitable excipient. Other suitable excipients
are well-known to
those in the art. See, for example, Ansel et at., PHARMACEUTICAL DOSAGE FORMS
AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro
(ed.),
REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing
Company 1990), and revised editions thereof.
[0176] In a preferred embodiment, the immunoconjugate is formulated in Good's
biological
buffer (pH 6-7), using a buffer selected from the group consisting of N-(2-
acetamido)-2-
aminoethanesulfonic acid (ACES); N-(2-acetamido)iminodiacetic acid (ADA); N,N-
bis(2-
hydroxyethyl)-2-aminoethanesulfonic acid (BES); 4-(2-hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES); 2-(N-morpholino)ethanesulfonic acid (IVIES); 3-(N-
morpholino)propanesulfonic acid (MOPS); 3-(N-morpholiny1)-2-
hydroxypropanesulfonic
acid (MOP SO); and piperazine-N,N'-bis(2-ethanesulfonic acid) [Pipes]. More
preferred
buffers are IVIES or MOPS, preferably in the concentration range of 20 to 100
mM, more
preferably about 25 mM. Most preferred is 25 mM MES, pH 6.5. The formulation
may
further comprise 25 mM trehalose and 0.01% v/v polysorbate 80 as excipients,
with the final
buffer concentration modified to 22.25 mM as a result of added excipients. The
preferred
method of storage is as a lyophilized formulation of the conjugates, stored in
the temperature
range of -20 C to 2 C, with the most preferred storage at 2 C to 8 C.
[0177] The immunoconjugate can be formulated for intravenous administration
via, for
example, bolus injection, slow infusion or continuous infusion. Preferably,
the antibody of
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the present invention is infused over a period of less than about 4 hours, and
more preferably,
over a period of less than about 3 hours. For example, the first 25-50 mg
could be infused
within 30 minutes, preferably even 15 min, and the remainder infused over the
next 2-3 hrs.
Formulations for injection can be presented in unit dosage form, e.g., in
ampoules or in multi-
dose containers, with an added preservative. The compositions can take such
forms as
suspensions, solutions or emulsions in oily or aqueous vehicles, and can
contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active
ingredient can be in powder form for constitution with a suitable vehicle,
e.g., sterile
pyrogen-free water, before use.
[0178] Additional pharmaceutical methods may be employed to control the
duration of action
of the therapeutic conjugate. Control release preparations can be prepared
through the use of
polymers to complex or adsorb the immunoconjugate. For example, biocompatible
polymers
include matrices of poly(ethylene-co-vinyl acetate) and matrices of a
polyanhydride
copolymer of a stearic acid dimer and sebacic acid. Sherwood et at.,
Bio/Technology 10:
1446 (1992). The rate of release of an immunoconjugate from such a matrix
depends upon
the molecular weight of the immunoconjugate, the amount of immunoconjugate
within the
matrix, and the size of dispersed particles. Saltzman et al., Biophys. 1 55:
163 (1989);
Sherwood et at., supra. Other solid dosage forms are described in Ansel et
at.,
PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th
Edition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL
SCIENCES, 18th Edition (Mack Publishing Company 1990), and revised editions
thereof
[0179] Generally, the dosage of an administered immunoconjugate for humans
will vary
depending upon such factors as the patient's age, weight, height, sex, general
medical
condition and previous medical history. A dosage of 1-20 mg/kg for a 70 kg
patient, for
example, is 70-1,400 mg, or 41-824 mg/m2 for a 1.7-m patient. The dosage may
be repeated
as needed, for example, once per week for 4-10 weeks, once per week for 8
weeks, or once
per week for 4 weeks. It may also be given less frequently, such as every
other week for
several months, or monthly or quarterly for many months, as needed in a
maintenance
therapy. Preferred dosages may include, but are not limited to, 1 mg/kg, 2
mg/kg, 3 mg/kg, 4
mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12
mg/kg, 13
mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, and 18 mg/kg. The dosage is
preferably
administered multiple times, once or twice a week. A minimum dosage schedule
of 4 weeks,
more preferably 8 weeks, more preferably 16 weeks or longer may be used. The
schedule of
administration may comprise administration once or twice a week, on a cycle
selected from
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the group consisting of: (i) weekly; (ii) every other week; (iii) one week of
therapy followed
by two, three or four weeks off; (iv) two weeks of therapy followed by one,
two, three or four
weeks off; (v) three weeks of therapy followed by one, two, three, four or
five week off; (vi)
four weeks of therapy followed by one, two, three, four or five week off;
(vii) five weeks of
therapy followed by one, two, three, four or five week off; and (viii)
monthly. The cycle may
be repeated 4, 6, 8, 10, 12, 16 or 20 times or more.
[0180] Alternatively, an immunoconjugate may be administered as one dosage
every 2 or 3
weeks, repeated for a total of at least 3 dosages. Or, twice per week for 4-6
weeks. If the
dosage is lowered to approximately 200-300 mg/m2(340 mg per dosage for a 1.7-m
patient),
it may be administered once or even twice weekly for 4 to 10 weeks.
Alternatively, the
dosage schedule may be decreased, namely every 2 or 3 weeks for 2-3 months. It
has been
determined, however, that even higher doses, such as 12 mg/kg once weekly or
once every 2-
3 weeks can be administered by slow iv. infusion, for repeated dosing cycles.
The dosing
schedule can optionally be repeated at other intervals and dosage may be given
through
various parenteral routes, with appropriate adjustment of the dose and
schedule.
[0181] In certain preferred embodiments, the SN-38 conjugated anti-HLA-DR may
be
administered subcutaneously. For subcutaneous administration, dosages of ADCs
such as
IMMU-140 (hL243-CL2A-SN-38) may be limited by the ability to concentrate the
ADC
without precipitation or agregation, as well as the volume of administration
that may be given
subcutaneously (preferably, 1, 2, or 3 ml or less). Consequently, for
subcutaneous
administration the ADC may be given at 2 to 4 mg/kg, given daily for 1 week,
or 3 times
weekly for 2 weeks, or twice weekly for two weeks, followed by rest.
Maintenance doses of
ADC may be administered iv. or s.c. every two to three weeks or monthly after
induction.
Alternatively, induction may occur with two to four cycles of iv.
administration at 8-10
mg/kg (each cycle with ADC administration on Days 1 and 8 of two 21-day cycles
with a
one-week rest period in between), followed by s.c. administration as active
dosing one or
more times weekly or as maintenance therapy. Dosing may be adjusted based on
interim
tumor scans and/or by analysis of Trop-2 positive circulating tumor cells.
[0182] In preferred embodiments, the immunoconjugates are of use for therapy
of cancer.
Examples of cancers include, but are not limited to, carcinoma, lymphoma,
glioblastoma,
melanoma, sarcoma, and leukemia, myeloma, or lymphoid malignancies. More
particular
examples of such cancers are noted below and include: squamous cell cancer
(e.g., epithelial
squamous cell cancer), Ewing sarcoma, Wilms tumor, astrocytomas, lung cancer
including
small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung
and squamous
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carcinoma of the lung, cancer of the peritoneum, gastric or stomach cancer
including
gastrointestinal cancer, pancreatic cancer, glioblastoma multiforme, cervical
cancer, ovarian
cancer, liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma,
neuroendocrine
tumors, medullary thyroid cancer, differentiated thyroid carcinoma, breast
cancer, ovarian
cancer, colon cancer, rectal cancer, endometrial cancer or uterine carcinoma,
salivary gland
carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer, anal
carcinoma, penile
carcinoma, as well as head-and-neck cancer. The term "cancer" includes primary
malignant
cells or tumors (e.g., those whose cells have not migrated to sites in the
subject's body other
than the site of the original malignancy or tumor) and secondary malignant
cells or tumors
(e.g., those arising from metastasis, the migration of malignant cells or
tumor cells to
secondary sites that are different from the site of the original tumor).
[0183] Other examples of cancers or malignancies include, but are not limited
to: Acute
Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute
Lymphocytic
Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary)
Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic
Leukemia,
Adult Acute Myeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic
Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft
Tissue
Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, Anal Cancer,
Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma,
Brain
Tumors, Breast Cancer, Cancer of the Renal Pelvis and Ureter, Central Nervous
System
(Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma,
Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary) Hepatocellular
Cancer,
Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,
Childhood
Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Childhood Cerebellar
Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell
Tumors,
Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood
Hypothalamic
and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood
Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal and
Supratentorial Primitive Neuroectodermal Tumors, Childhood Primary Liver
Cancer,
Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual
Pathway
and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, Chronic Myelogenous
Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet
Cell
Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal
Cancer,
Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial
Germ Cell
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Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye
Cancer, Female
Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer,
Gastrointestinal
Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational
Trophoblastic
Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer,
Hodgkin's
Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,
Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell Pancreatic Cancer,
Kaposi's Sarcoma,
Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer,
Lung Cancer,
Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer,
Malignant
Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma, Mesothelioma,
Metastatic Occult Primary Squamous Neck Cancer, Metastatic Primary Squamous
Neck
Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple
Myeloma/Plasma
Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia, Myeloid
Leukemia,
Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer,
Nasopharyngeal
Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-
Small
Cell Lung Cancer, Occult Primary Metastatic Squamous Neck Cancer,
Oropharyngeal
Cancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous
Histiocytoma,
Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial
Cancer, Ovarian
Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer,
Paraproteinemias, Polycythemia vera, Parathyroid Cancer, Penile Cancer,
Pheochromocytoma, Pituitary Tumor, Primary Central Nervous System Lymphoma,
Primary
Liver Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis
and Ureter
Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis
Sarcomas,
Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer,
Soft Tissue
Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive
Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer,
Thymoma,
Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,
Transitional Renal
Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell
Cancer,
Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual
Pathway and
Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Wilms'
tumor,
and any other hyperproliferative disease, besides neoplasia, located in an
organ system listed
above.
[0184] The methods and compositions described and claimed herein may be used
to treat
malignant or premalignant conditions and to prevent progression to a
neoplastic or malignant
state, including but not limited to those disorders described above. Such uses
are indicated in
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conditions known or suspected of preceding progression to neoplasia or cancer,
in particular,
where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or
most particularly,
dysplasia has occurred (for review of such abnormal growth conditions, see
Robbins and
Angell, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79
(1976)).
[0185] Dysplasia is frequently a forerunner of cancer, and is found mainly in
the epithelia. It
is the most disorderly form of non-neoplastic cell growth, involving a loss in
individual cell
uniformity and in the architectural orientation of cells. Dysplasia
characteristically occurs
where there exists chronic irritation or inflammation. Dysplastic disorders
which can be
treated include, but are not limited to, anhidrotic ectodermal dysplasia,
anterofacial dysplasia,
asphyxiating thoracic dysplasia, atriodigital dysplasia, bronchopulmonary
dysplasia, cerebral
dysplasia, cervical dysplasia, chondroectodermal dysplasia, cleidocranial
dysplasia,
congenital ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal
dysplasia,
craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,
ectodermal dysplasia,
enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia epiphysialis
hemimelia,
dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata, epithelial
dysplasia,
faciodigitogenital dysplasia, familial fibrous dysplasia of j aws, familial
white folded
dysplasia, fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous
dysplasia,
hereditary renal-retinal dysplasia, hidrotic ectodermal dysplasia,
hypohidrotic ectodermal
dysplasia, lymphopenic thymic dysplasia, mammary dysplasia, mandibulofacial
dysplasia,
metaphysial dysplasia, Mondini dysplasia, monostotic fibrous dysplasia,
mucoepithelial
dysplasia, multiple epiphysial dysplasia, oculoauriculovertebral dysplasia,
oculodentodigital
dysplasia, oculovertebral dysplasia, odontogenic dysplasia,
opthalmomandibulomelic
dysplasia, periapical cemental dysplasia, polyostotic fibrous dysplasia,
pseudoachondroplastic spondyloepiphysial dysplasia, retinal dysplasia, septo-
optic dysplasia,
spondyloepiphysial dysplasia, and ventriculoradial dysplasia.
[0186] Additional pre-neoplastic disorders which can be treated include, but
are not limited
to, benign dysproliferative disorders (e.g., benign tumors, fibrocystic
conditions, tissue
hypertrophy, intestinal polyps or adenomas, and esophageal dysplasia),
leukoplakia,
keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar
keratosis.
[0187] In preferred embodiments, the method of the invention is used to
inhibit growth,
progression, and/or metastasis of cancers, in particular those listed above.
[0188] Additional hyperproliferative diseases, disorders, and/or conditions
include, but are
not limited to, progression, and/or metastases of malignancies and related
disorders such as
leukemia (including acute leukemias; e.g., acute lymphocytic leukemia, acute
myelocytic
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leukemia [including myeloblastic, promyelocytic, myelomonocytic, monocytic,
and
erythroleukemia]) and chronic leukemias (e.g., chronic myelocytic
[granulocytic] leukemia
and chronic lymphocytic leukemia), polycythemia vera, lymphomas (e.g.,
Hodgkin's disease
and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia,
heavy
chain disease, and solid tumors including, but not limited to, sarcomas and
carcinomas such
as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,
leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian
cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland
carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas,
cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell
carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma,
Wilm's
tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung
carcinoma, bladder
carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma,
ependymoma, pinealoma, emangioblastoma, acoustic neuroma, oligodendroglioma,
meningioma, melanoma, neuroblastoma, and retinoblastoma.
[0189] Autoimmune diseases that may be treated with immunoconjugates may
include acute
and chronic immune thrombocytopenias, dermatomyositis, Sydenham's chorea,
myasthenia
gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever,
polyglandular
syndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura,
post-
streptococcal nephritis, erythema nodosum, Takayasu's arteritis, ANCA-
associated
vasculitides, Addison's disease, rheumatoid arthritis, multiple sclerosis,
sarcoidosis,
ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis
nodosa, ankylosing
spondylitis, Goodpasture's syndrome, thromboangitis obliterans, Sjogren's
syndrome, primary
biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma,
chronic active
hepatitis, polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,
pemphigus
vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic
lateral sclerosis,
tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly
progressive
glomerulonephritis, psoriasis or fibrosing alveolitis.
Kits
[0190] Various embodiments may concern kits containing components suitable for
treating
diseased tissue in a patient. Exemplary kits may contain at least one
conjugated antibody or
other targeting moiety as described herein. If the composition containing
components for
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administration is not formulated for delivery via the alimentary canal, such
as by oral
delivery, a device capable of delivering the kit components through some other
route may be
included. One type of device, for applications such as parenteral delivery, is
a syringe that is
used to inject the composition into the body of a subject. Inhalation devices
may also be used.
[0191] The kit components may be packaged together or separated into two or
more
containers. In some embodiments, the containers may be vials that contain
sterile,
lyophilized formulations of a composition that are suitable for
reconstitution. A kit may also
contain one or more buffers suitable for reconstitution and/or dilution of
other reagents. Other
containers that may be used include, but are not limited to, a pouch, tray,
box, tube, or the
like. Kit components may be packaged and maintained sterilely within the
containers.
Another component that can be included is instructions to a person using a kit
for its use.
EXAMPLES
[0192] Various embodiments of the present invention are illustrated by the
following
examples, without limiting the scope thereof
General
[0193] Abbreviations used below are: DCC, dicyclohexylcarbodiimide; NHS, N-
hydroxysuccinimide, DMAP, 4-dimethylaminopyridine; EEDQ, 2-ethoxy-1-
ethoxycarbonyl-
1,2-dihydroquinoline; MNIT, monomethoxytrityl; PABOH, p-aminobenzyl alcohol;
PEG,
polyethylene glycol; SMCC, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-
carboxylate; TBAF, tetrabutylammonium fluoride; TBDMS, tert-butyldimethylsilyl
chloride.
[0194] Chloroformates of hydroxy compounds in the following examples were
prepared
using triphosgene and DMAP according to the procedure described in Moon et al.
(J.
Medicinal Chem. 51:6916-6926, 2008). Extractive work-up refers to extraction
with
chloroform, dichloromethane or ethyl acetate, and washing optionally with
saturated
bicarbonate, water, and with saturated sodium chloride. Flash chromatography
was done
using 230-400 mesh silica gel and methanol-dichloromethane gradient, using up
to 15% v/v
methanol-dichloromethane, unless otherwise stated. Reverse phase HPLC was
performed by
Method A using a 7.8 x 300 mm C18 HPLC column, fitted with a precolumn filter,
and using
a solvent gradient of 100% solvent A to 100% solvent B in 10 minutes at a flow
rate of 3 mL
per minute and maintaining at 100% solvent B at a flow rate of 4.5 mL per
minute for 5 or 10
minutes; or by Method B using a 4.6x30 mm Xbridge C18, 2.5 m, column, fitted
with a
precolumn filter, using the solvent gradient of 100% solvent A to 100% of
solvent B at a flow
rate of 1.5 mL per minutes for 4 min and 100% of solvent B at a flow rate of 2
mL per
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minutes for 1 minutes. Solvent A was 0.3% aqueous ammonium acetate, pH 4.46
while
solvent B was 9:1 acetonitrile-aqueous ammonium acetate (0.3%), pH 4.46. HPLC
was
monitored by a dual in-line absorbance detector set at 360 nm and 254 nm.
Example 1. Efficacy of Anti-HLA-DR Antibody Drug Conjugate IMMU-140
(hL243-CL2A-SN-38) in HLA-DR+ Cancers In vitro and In vivo
[0195] Relapsed AML (acute myelocytic leukemia), ALL (acute lymphocytic
leukemia) and
MM (multiple myeloma) continue to be a therapy challenge. IMMU-114 (hL243) is
a
humanized anti-HLA-DR Igai monoclonal antibody engineered to lack effector-
cell
functions, but retains HLA-DR binding and a broad range of antitumor effects
in diverse
hematological neoplasms (Stein R et at., Blood. 2010;115:5180-90). When given
subcutaneously, it demonstrated efficacy in an initial Phase I clinical trial
in relapsed or
refractory NHL and CLL, with a good safety profile (ClinicalTrials.gov,
NCT01728207).
[0196] In vitro, AML has proven to be resistant to the antitumor effects of
IMMU-114,
despite high expression levels of HLA-DR. Likewise, in several different human
ALL, CLL,
and MM cell lines, IMMU-114 demonstrated a range of antitumor effects from a
low of 9%
to a high of 69%.
[0197] In an effort to improve the antitumor activity of IMMU-114, an antibody-
drug
conjugate (ADC), termed IMMU-140, was made in which IMMU-114 was conjugated
with
the active metabolite of irinotecan, SN-38. Another ADC utilizing SN-38
(sacituzumab
govitecan) being studied in solid tumors has been well tolerated, with
clinically significant
objective responses in patients given multiple cycles over >6 months, with
manageable
neutropenia being the major toxicity. Thus, our goal was to determine if SN-
38, a drug not
commonly used in hematopoietic cancers, would prove to be an effective and
safe therapeutic
when targeted with the IMMU-114 antibody.
[0198] In this current study, the in vitro and in vivo activity of hL243-SN-38
(IMMU-140)
versus parental IMMU-114 was examined in human AML, ALL, MM, and CLL
xenografts.
Methods
[0199] The method used for the conjugation of SN-38 to hL243 Igai has been
previously
described and resulted in a drug-to-antibody-ratio range of 6.1 to 6.6 (Moon
SJ et at. I Med.
Chem. 2008;51:6916-26). The SN-38 linker (below, on left) contains a short
polyethylene
glycol (PEG) moiety to confer aqueous solubility. A maleimide group was
incorporated for
fast thiol-maleimide conjugation to mildly reduced antibody. A benzylcarbonate
site provided
a pH-mediated cleavage site to release the drug from the linker. The cross-
linker was attached
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to SN-38's 20-hydroxy position, to keep the lactone ring of the drug from
opening to the less
active carboxylic acid form under physiological conditions. The structure of
the ADC is
shown in FIG. 1.
[0200] The conjugate was characterized by size-exclusion HPLC (not shown).
Unmodified
IMMU-114 and IMMU-140 conjugate with a drug/antibody molar substitution of 6.1
were
compared (not shown). Unmodified IMMU-114 was detected at 280 nm, and the
conjugate
was detected at the absorbance wavelength of SN-38, namely 360 nm. The
conjugate was >
98 % monomeric (not shown).
[0201] There was no evidence of any loss of binding specificity of the ADC, as
demonstrated
by comparable binding of IMMU-140 and IMMU-114 to an HLA-DR positive human
melanoma cell line (A-374) via a cell-based ELISA (FIG. 2). Both recognize the
a-chain
when it associates with the b-chain. KD-values are shown in the Table 2 below.
Antibody
control (h679) is a humanized anti-histamine-succinyl-glycine (HSG) IgG.
Table 2. Binding affinities of IMMU-140 vs. IMMU-114.
KD (nM) 95% C.I. R2
IMMU-140 0.77 0.62 to 0.91 0.97
IMMU-114 0.65 0.53 to 0.77 0.97
[0202] For in vitro cytotoxicity assays, cells were plated in 96-well plates
(1x104 cells/well)
followed by addition of each test agent: Free SN-38 (2.5x10-7 to 3.8x10'2 M),
IMMU-140
(2.5x10-7 to 3.8x10'2 M, SN-38 equivalents), and IMMU-114 (4x10-8 to 6.2x10'3
M). Plates
were incubated for 96 h before cell viability determined by MTS. Inhibition
determined as
percent viable cells in treated wells compared to untreated controls.
[0203] Apoptosis signaling was determined in cells (2x106) treated with 10 nM
protein
concentration of either IMMU-114 or IMMU-140 before being lysed and proteins
(25 mg)
resolved via SDS-PAGE. Proteins were transferred to PVD membranes for Western
blotting.
[0204] For AML and MM disease models, NSG/SCID and C.B.-17 SCID mice received
2 Gy
irradiation 24 h prior to an i.v. injection of MOLM-14 (2x106) or CAG cells
(1x107),
respectively.
[0205] ALL (MN-60) and CLL (JVM-3) were established in C.B-17 SCID mice
injected i.v.
with 1x107 cells.
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[0206] All therapies began 5 days post-tumor-cell injection. Test agents,
including non-
targeting control SN-38-ADCs, were administered at doses indicated in the
figures (100 to
500 mg), twice-weekly for 4 wks. Animals were sacrificed at disease
progression,
characterized by the onset of hind-limb paralysis or loss of more than 15%
body weight.
Results
[0207] HLA-DR expression was examined in various human cancer cell lines.
Human ALL,
MM, CLL, and AML cell lines were harvested from tissue culture and analyzed by
FACS for
HLA-DR (alpha chain) expression using AlexaFluor-647-labeled IMMU-114. Mean
fluorescence intensity (MFI) of IMMU-114 and non-targeting control h679
antibody
demonstrated high expression of HLA-DR in all four cell lines, with MFI values
for IMMU-
114 of 6.58 x 104 (MN-60 ALL cell line), 8.07 x 104 (CAG MM cell line), 7.87 x
104 (JVM-3
CLL cell line) and 5.75 x 103(MOLM-14 AML cell line). By comparison, CAG, MN-
60, and
JVM-3 exhibit >10-fold higher expression compared to the AML cell line, MOLM-
14.
[0208] In vitro, IMMU-140 achieved IC50-values at low nM concentrations
(ranging from 0.8
to 7.1 nM) in all four hematopoietic tumor types (ALL, MM, CLL, and AML), as
shown in
Table 3 below. In vitro, JVNI-3 demonstrated the most sensitivity to both IMMU-
140 and
IMMU-114. The remaining three cell lines only exhibited 50% or greater growth-
inhibition
in the presence of SN-38 and IMMU-140. While 50% inhibition was not achieved
in those
cell lines with IMMU-114, the unconjugated antibody mediated significant
growth inhibition
in both CAG (MM) and MN-60 (ALL) at 40 nM when compared to a non-targeting
control
antibody. As was reported previously with two other ANIL cell lines (Stein R
et al., Blood.
2010;115:5180-90), MOLM-14 also was resistant to IMMU-114, but was sensitive
to
IMMU-140.
Table 3. In vitro cytotoxicity of IMMU-140 vs. IMMU-114 in AML, ALL, CLL and
MM
cells.
IMMU-114
Maximum
Free SN-38 IMMU-1401 % Inhibition
Cell Line Disease IC50 (nM) IC50 (nM) IC50 (nM) at 40 nM
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JVM-3 CLL 0.51 0.18 0.77 0.15 1.52 0.84
1.65 11
CAG MM 7.02 1.77 7.05 2.73 >40 1.28
6
MN-60 ALL 0.86 0.09 1.29 0.27 >40 1.37
10
MOLM- *090
14
AM 0.13
L 1.21 0.08 >40 11 5
IConcentration of IMMU-140 shown as SN-38 equivalents
*IC50 of free SN-38 is significantly different compared to IMMU-140 in MOLM-14
(P=0.0266)
tSignificant inhibition compared to control antibody (P<0.0061)
[0209] IMMU-140 demonstrated dual-apoptosis signaling pathways mediated
through its
anti-HLA-DR binding of target cells and delivery of SN-38. IMMU-114 signals
apoptosis via
p-ERK-1/2 and apoptosis-inducing factor (AIF) in NHL, ALL, MM, and CLL, but
not ANIL
(Stein R et al. Blood. 2010;115(25):5180-5190). Here we demonstrated that both
IMMU-114
and IMMU-140 are capable of mediating the phosphorylation of ERK1/2 and up-
regulating
AIF in three different hematopoietic cell lines (not shown), including the
ANIL cell line
MOLM-14 (not shown), suggesting defects in other signaling components of this
pathway in
AML since it is insensitive to IMMU-114 both in vitro and in vivo.
[0210] Importantly, IMMU-140, through its SN-38 payload, also mediated PARP
cleavage in
all three cell lines (not shown), including MOLM-14. The resulting double-
stranded DNA
(dsDNA) breaks, as evidenced by increased p-H2A.X levels, were most evident in
the cells
treated with IMMU-140 (not shown).
[0211] In experimental MOLM-14 AML, saline control and IMMU-114 treated mice
succumbed to disease progression quickly, with a median survival time (MST) of
only 14 and
15 days, respectively (FIG. 3). Conversely, mice treated with IMMU-140 had a
greater than
1.5-fold increase in survival (MST=37 d, P=0.0031) (FIG. 3). Further, a dose-
reduction to
250 mg IMMU-140 still provided a statistically significant, greater than 80%
improvement in
survival compared to saline and control ADC (anti-CEA-SN-38 IMMU-130)
administered at
the same dose (MST=21 d, P=0.0031) (FIG. 3).
[0212] In mice bearing MN-60 ALL xenografts (FIG. 4), IMMU-114 provided a >60%
improvement in survival compared to saline control (MST = 37 d vs. 22.5 d,
respectively;
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P<0.0001), whereas IMMU-140 increased this by another 80% (MST=66.5 d) which
was
significantly better than all other treatments, including IMMU-114 (P<0.0001)
(FIG. 4).
Therapy with IMMU-140 was well tolerated by the mice with no appreciable loss
in body
weight.
[0213] Mice with CAG MM xenografts (FIG. 5) had a greater than 151-d MST when
treated
with IMMU-140 compared to 32 d for saline control (P<0.0001). This survival
benefit also
was significantly higher than for mice treated with bortezomib (0.89 mg/kg) or
control ADC
+ bortezomib (MST=32.5 d for both; P<0.0001) (FIG. 5). While not significant,
IMMU-140
does provide a >60% improvement in survival when compared to IMMU-114 therapy
(MST=94.5 d, P=0.0612) (FIG. 5). Bortezomib therapy combined with IMMU-114 or
IMMU-140 did not improve survival above monotherapy (FIG. 5).
[0214] Mice bearing JVM-3 CLL xenografts (FIG. 6) demonstrated similar
sensitivity to
both IMMU-140 and IMMU-114. There was a significant survival benefit in mice
treated
with either the high (500 mg) or low (100 mg) dose of IMMU-140 vs. saline or
control ADC
treated mice (P<0.0002) (FIG. 6). Likewise, mice treated with either dose of
IMMU-114 had
>96-d MSTs (P<0.0001 vs. saline and P<0.0003 vs. control ADC) (FIG. 6). There
are no
significant differences between mice treated with IMMU-140 and IMMU-114 at the
doses
administered to the mice (FIG. 6). These results demonstrate that efficacy may
be achieved at
doses less than 100 mg which suggest a wide therapeutic window clinically for
IMMU-140 in
this disease.
[0215] In all experiments, therapy with IMMU-140 was well tolerated, as
evidenced by no
significant loss in body weight
Conclusions
[0216] IMMU-114 is an Igai Mab that lacks immune functions, eliminating known
adverse
events of prior HLA-DR Mabs. HLA-DR, as recognized by IMMU-114, is expressed
on a
wide range of human hematopoietic and solid cancer types. Conjugating 6-8 SN-
38
molecules via a cleavable linker to IMMU-114 (hL243-SN-38) did not alter its
binding to
HLA-DR positive cells.
[0217] hL243-SN-38 (IMMU-140) provides an added benefit of a dual-therapeutic
through
the direct antitumor activity mediated by the IMMU-114 HLA-DR-binding moiety
(p-
ERK1/2 and AIF signaling) and the added cytotoxic effect of SN-38 delivery to
the cells
(caspase cascade and PARP cleavage). IMMU-140 antibody-drug conjugate showed
higher
potency than naked IMMU-114 pre-clinically in ALL and AML and an added, if not
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significant, survival benefit in experimental MM and CLL. Overall, the dual-
therapeutic
potential of SN-38-conjugated IMMU-114 (IMMU-140) allows for the ability to
treat a
range of HLA-DR-positive hematopoietic and solid cancers.
[0218] Therapy with the IMMU-140 ADC proved to be superior to IMMU-114 (which
is
active clinically in NHL and CLL) in both AML and ALL xenografts, and
beneficial in MM
and CLL. Most importantly, in IMMU-114-refractive AML, IMMU-140 demonstrated a
significant antitumor effect without any undue toxicity. The data show that
this new ADC is
of use in these intractable malignancies.
Example 2. Efficacy of IMMU-140 in HLA-DR+ Human Melanoma
[0219] Expression of the HLA-DR antigen is not limited to hematopoietic
cancer, but rather
is also found in skin, esophageal, stomach, colon, rectal, pancreatic, lung,
breast, ovarian,
bladder, endometrial, cervical, testicular, melanoma, kidney, and liver
cancers. The present
study was conducted to examine the efficacy of IMMU-140 in non-hematopoietic
HLA-DR+
tumors.
Cell Binding Studies
[0220] LUMIGLO chemiluminescent substrate system was used to detect antibody
binding
to cells. Briefly, A-375 human melanoma cells were plated into a 96 black-
well, flat-clear-
bottom plate overnight. The hL243-y4P antibody was added to triplicate wells
(2 g/mL final
concentration in the well). As a control for non-specific binding, a humanized
anti-CD22
antibody was likewise added to another set of triplicate wells. Two plates
were set up in
which one was incubated at room temperature (RT) and one at 4 C. After
incubating for 1 h
the media was removed and the cells washed with fresh, cold media followed by
the addition
of a 1:20,000 dilution of goat-anti-human horseradish peroxidase-conjugated
secondary
antibody for 1 h at 4 C. The plates were again washed before the addition of
the
LUMIGLO reagent.
[0221] Plates were read for luminescence using an ENVISIONTM plate reader.
Mean
luminescent values were determined and graphed as shown in FIG. 7. Mean
luminescence of
hL243y4 on A-375 cells, at both 4 C and RT, was greater than 48-fold higher
than
background and 25-fold higher than non-specific control, consistent with high
expression of
HLA-DR on this human melanoma cell line. In all, 4 of 4 human melanoma cell
lines tested
(A-375, SK-MEL-28, SK-MEL-5, and SK-MEL-2) were positive for hL243-y4P binding
(48-
25-, 12-, and 2-fold above background, respectively).
IN VIVO EFFICACY IN A-375 TUMOR XENOGRAFTS.
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[0222] Athymic NCr flu/flu nude mice were injected s.c. with 5x106 A-375 cells
per mouse.
Once tumors reached approximately 0.3 cm3 in size, the animals were divided
into six
different treatment groups of 10 mice each. Mice received 250 jig i.p.
injections of IMMU-
140 (hL243-SN-38) (DAR=5.05) twice a week for four weeks. An ADC control group
consisted of mice receiving the same doses of non-tumor targeting anti-CD20
ADC (hA20-
CL2A-SN-38; DAR=6.08) on the same schedule. Additionally, one group of mice
received
naked hL243-y4P alone (250 g) and one group hL243-y4P plus irinotecan at
doses
equivalent to the ADC dose (250 jig MAb + 7.5 jig irinotecan). A final group
received only
irinotecan at 10-fold higher doses than the amount of SN-38 carried by hL243-
SN-38 (i.e., 75
g). All irinotecan injections were administered as i.v. injections. A final
group of mice
received only saline (100 I, i.p.). Treatment groups are summarized in Table4
below.
Table 4. Treatment Groups for Melanoma-Bearing Nude Mice
hL243-CL2A-SN-38 Therapy of Mice Bearing Human
Melanoma Tumors (A-375)
Group (N) Amount Injected Schedule
Saline
1 10 Twice weekly x 4 wks
(100 I, i.p.)
hL243-CL2A-SN-38
2 10 Twice weekly x 4 wks
(250 jig i.p.)
hA20-CL2A-SN-38
3 10 Twice weekly x 4 wks
(250 jig i.p.)
hL243-y4P + Irinotecan
4 10 Twice weekly x 4 wks
(250 jig i.p. + 7.5 g i.v.)
hL243-y4P Alone
10 Twice weekly x 4 wks
(250 jig i.p.)
Irinotecan Alone
6 10 Twice weekly x 4 wks
10-fold excess (75 g i.v.)
[0223] Tumors were measured and mice weighed weekly. Animals were euthanized
for
disease progression if their tumor volume exceeded 2.0 cm3 in size. A partial
response was
defined as shrinking the tumor >30% from initial size. Stable disease was when
the tumor
volume remains between 70% and 120% of initial size. Time-to-tumor progression
(TTP)
was determined as time when tumor grew more than 20% from its nadir.
[0224] Statistical analysis for the tumor growth data was based on area under
the curve
(AUC) and TTP. Profiles of individual tumor growth were obtained through
linear curve
modeling. An F-test was employed to determine equality of variance between
groups prior to
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statistical analysis of growth curves. A two-tailed t-test was used to assess
statistical
significance between all the various treatment groups and controls except for
the saline
control in which a one-tailed t-test was used in the analysis. As a
consequence of
incompleteness of some of the growth curves (due to deaths), statistical
comparisons of AUC
was only performed up to the time at which the first animal within a group was
sacrificed. A
two-tailed t-test was used to compare TTP values between groups.
[0225] Mean tumor volume for all the groups when therapy began was 0.314
0.078 cm3.
Mean tumor growth curves are shown in FIG. 8. This disease model proved to be
very
aggressive with saline control tumors progressing rapidly (TTP=7 days; Table
5). While
tumors likewise progressed in the control groups, all treatments were able to
slow tumor
growth relative to saline control (P<0.0142, AUC) (FIG. 8 and Table 5).
However, only
mice treated with hL243-SN-38 demonstrated a significant antitumor effect when
compared
to all other groups (P<0.0244; AUC) (FIG. 8 and Table 5). All the mice in this
group were
partial responders with two mice tumor-free when the experiment ended on
therapy day 70.
This resulted in a greater than 3-fold delay in tumor progression when
compared to all the
other non-ADC control groups (P<0.0005) (FIG. 8 and Table 5). Even though this
tumor
was sensitive to the non-specific ADC, treatment with hL243-SN-38 imparted an
80% greater
delay in TTP compared to mice treated with the control ADC (28 9.9 days vs.
15.6 7.7
days, respectively; P=0.012) (FIG. 8 and Table 5). These data demonstrate that
even in a
mouse disease-model of an aggressive human melanoma tumor, therapy with hL243-
SN-38
resulted in significant tumor regressions and delay in disease progression
(FIG. 8 and Table
5).
Table 5. Time-to-tumor progression for A-375 tumor-bearing
mice treated with hL243-SN-38.
hL243-SN-38 vs.
% PR TTP Controls
Treatment N (TF) (days) (P-value)
hL243-SN-38 10 100 28.0 9.9 N.A.
(2)
Control ADC 10 30 15.6 7.0 0.0120
(1)
CTP-11 10 0 8.4 4.4 0.0005
(0)
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Naked hL243 + CPT-11 10 0 7.0 0.0 0.0005
(0)
Naked hL243 10 0 7.0 0.0 0.0005
(0)
Saline 9* 0 7.0 0.0 0.0003
(0)
N = Number of mice per group
*One mouse censored as an outlier (Critical-Z test).
% PR = Percent of mice that exhibited a positive response to treatment
TF = Number of mice tumor-free when experiment ended.
TTP=Time to Tumor Progression for mice not tumor-free
N.A. = Not Applicable
Example 3. Preparation of CL2A-SN-38
[0226] To the mixture of commercially available Fmoc-Lys(MMT)-OH (0.943g), p-
aminobenzyl alcohol (0.190g) in methylene choloride (10 mL) was added EEDQ
(0.382g) at
room temperature and stirred for 4 h. Extractive work up followed by flash
chromatograph
yielded 1.051 g of material as white foam. All HPLC analyses were performed by
Method B
as stated in 'General' in section 0148. HPLC ret. time : 3.53 min.,
Electrospray mass
spectrum showed peaks at m/e 745.8 (M+H) and m/e 780.3 (M+C1-), consistent
with
structure. This intermediate (0.93 g) was dissolved in diethylamine (10 mL)
and stirred for 2
h. After solvent removal, the residue was washed in hexane to obtain 0.6 g of
the
intermediate ((2) in Scheme-3) as colorless precipitate (91.6% pure by HPLC).
HPLC ret.
time : 2.06 min. Electrospray mass spectrum showed peaks at m/e 523.8 (M+H),
m/e 546.2
(M+Na) and m/e 522.5 (M-H).
[0227] This crude intermediate (0.565g) was coupled with commercially
available 042-
azidoethyl)-0'-(N-diglycoly1-2-aminoethyl)heptaethyleneglycol (`PEG-N3',
0.627g) using
EEDQ in methylene chloride (10 mL). Solvent removal and flash chromatography
yielded
0.99 g of the product ((3) in Scheme-3; light yellow oil; 87% yield). HPLC
ret. time : 2.45
min. Electrospray mass spectrum showed peaks at m/e 1061.3 (M+H), m/e 1082.7
(M+Na)
and m/e 1058.8(M-H), consistent with structure. This intermediate (0.92 g) was
reacted with
10-0-TBDMS-SN-38-20-0-chloroformate in methylene chloride (10 mL) for 10 min
under
argon. The mixture was purified by flash chromatography to obtain 0.944g as
light yellow
oil ((6) in Scheme-3; yield = 68%). HPLC ret. time : 4.18 min. To this
intermediate (0.94 g)
in methylene chloride (10 mL) was added the mixture of TBAF ( 1M in THF, 0.885
mL) and
acetic acid (0.085 mL) in methylene chloride (3 mL), then stirred for 10 min.
The mixture
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was diluted with methylene chloride (100 mL), washed with 0.25 M sodium
citrate and brine.
The solvent removal yielded 0.835g of yellow oily product. HPLC ret. time :
2.80 min.,
(99% purity). Electrospray mass spectrum showed peaks at m/e 1478 (M+H), m/e
1500.6
(M+Na), m/e 1476.5 (M-H), m/e 1590.5 (M+TFA), consistent with structure.
Scheme-3: Preparartion of CL2A-SN-38
0 0 OH
Fmoc' OH gpi
dik NH2
¨
(i) EEDQ/CH2Cl2 H2N
JL
+ HO
(ii) Diethylamine
(2)
(1
HN, HN, FENQN3 ) MIVIT MMT
CH2Cl2
0
0 0
triphosgene, 0 /
ci 0 OH
10-0-TBDMS-SN-38 DMAP/CH2C12
40
N N
(4)
N/ 0 0
(5) (3)
0¨TBD HN,
MMT
0
N3OO
0 0
0 N
N N 0
N/
8 0 0
(6)
(i) TBAF/AcOH/ O¨TBDMS
CH2Cl2 HN,
MMT
(ii) 0
N .
, CuBr
0
(Click Cycloadditon)
DCA/anisole/
CH2Cl2 0 0
0
0 /
0 0¨lc
0
0 \ N
N/
0 8 0 0
(
CL2A-SN-38 7)
OH
NH2 (as amine salt)
[0228] This azido-derivatized SN-38 intermediate (0.803g) was reacted with 4-
(N-
maleimidomethyl)-N-(2-propynyl)cyclohexane-1- carboxamide ( 0.233 g) in
methylene
chloride (10 mL) in presence of CuBr ( 0.0083 g,), DIEA (0.01 mL) and
triphenylphosphine
( 0.015 g), for 18 h. Extractive work up, including washing with and 0.1M EDTA
( 10 mL),
and flash chromatography yielded 0.891 g as yellow foam. (yield = 93%), HPLC
ret. time :
2.60 min. Electrospray mass spectrum showed peaks at m/e 1753.3 (M+H), m/e
1751.6 (M-
H), 1864.5 (M+TFA), consistent with structure. Finally, deprotection of the
penultimate
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intermediate ( 0.22g ) with a mixture of dichloroacetic acid ( 0.3 mL) and
anisole (0.03 mL)
in methylene chloride (3 mL), followed by precipitation with ether yielded
0.18 g (97%
yield) of CL2A-SN-38; (7) in Scheme-3) as light yellow powder. HPLC ret. time:
1.88 min.
Electrospray mass spectrum showed peaks at m/e 1480.7 (M+H), 1478.5 (M-H),
consistent
with structure.
Example 4. Conjugation of bifunctional SN-38 products to mildly reduced
antibodies
[0229] Each antibody was reduced with dithiothreitol (DTT), used in a 50-to-70-
fold molar
excess, in 40 mM PBS, pH 7.4, containing 5.4 mM EDTA, at 37 C (bath) for 45
min. The
reduced product was purified by size-exclusion chromatography and/or
diafiltration, and was
buffer-exchanged into a suitable buffer at pH 6.5. The thiol content was
determined by
Ellman's assay, and was in the 6.5-to-8.5 SH/IgG range. Alternatively, the
antibodies were
reduced with Tris (2-carboxyethyl) phosphine (TCEP) in phosphate buffer at pH
in the range
of 5-7, followed by in situ conjugation. The reduced MAb was reacted with CL2A-
SN-38
using DMSO at 7-15 % v/v as co-solvent, and incubating for 20 min at ambient
temperature.
The conjugate was purified by centrifuged SEC, passage through a hydrophobic
column, and
finally by ultrafiltration-diafiltration. The product was assayed for SN-38 by
absorbance at
366 nm and correlating with standard values, while the protein concentration
was deduced
from absorbance at 280 nm, corrected for spillover of SN-38 absorbance at this
wavelength.
This way, the SN-38/MAb substitution ratios were determined. The purified
conjugates were
stored as lyophilized formulations in glass vials, capped under vacuum and
stored in a ¨20 C
freezer. SN-38 molar substitution ratios (MSR) obtained for some of these
conjugates, which
were typically in the 5-to-7 range, are shown in Table 6.
Table 6. SN-38/1VL4b Molar substitution ratios (MSR) in some conjugates
MAb Conjugate MSR
hMN-14 hMN-14-[CL2A-SN-38] 6.1
hRS7 hRS7-CL2A-SN-38 using drug-linker of Example 10 5.8
hA20 hA20-CL2A-SN-38 using drug-linker of Example 10 5.8
hLL2 hLL2-CL2A-SN-38 using drug-linker of Example 10 5.7
hPAM4 hPAM4-CL2A-SN-38 using drug-linker of Example 10 5.9
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Example 5. Use of hL243-SN-38 to treat therapy-refractive metastatic colonic
cancer (mCRC)
[0230] The patient is a 67-year-old man who presents with metastatic colon
cancer.
Following transverse colectomy shortly after diagnosis, the patient then
receives 4 cycles of
FOLFOX chemotherapy in a neoadjuvant setting prior to partial hepatectomy for
removal of
metastatic lesions in the left lobe of the liver. This is followed by an
adjuvant FOLFOX
regimen for a total of 10 cycles of FOLFOX.
[0231] CT shows metastases to liver. His target lesion is a 3.0-cm tumor in
the left lobe of
the liver. Non-target lesions included several hypo-attenuated masses in the
liver. Baseline
CEA is 685 ng/mL.
[0232] After the patient signs the informed consent, hL243-SN-38 (10 mg/kg) is
given every
other week for 4 months. The patient experiences nausea (Grade 2) and fatigue
(Grade 2)
following the first treatment and continues the treatment without major
adverse events. The
first response assessment done (after 8 doses) shows shrinkage of the target
lesion by 26% by
computed tomography (CT) and his CEA level decreases to 245 ng/mL. In the
second
response assessment (after 12 doses), the target lesion has shrunk by 35%. His
overall health
and clinical symptoms are considerably improved.
Example 6. Treatment of relapsed follicular lymphoma with IMMU-140 (anti-
HLA-DR-SN-38)
[0233] After receiving R-CHOP chemotherapy for follicular lymphoma presenting
with
extensive disease in various regional lymph nodes (cervical, axillary,
mediastinal, inguinal,
abdominal) and marrow involvement, this 68-year-old man is given the
experimental agent,
IMMU-114-SN-38 (anti-HLA-DR-SN-38) at a dose of 10 mg/kg weekly for 3 weeks,
with a
rest of 3 weeks, and then a second course for another 3 weeks. He is then
evaluated for
change in index tumor lesions by CT, and shows a 23% reduction according
CHESON
criteria. The therapy is repeated for another 2 courses, which then shows a
55% reduction of
tumor by CT, which is a partial response.
Example 7. Treatment of relapsed chronic lymphatic leukemia with IMMU-140
[0234] A 67-year-old man with a history of CLL, as defined by the
International Workshop
on Chronic Lymphocytic Leukemia and World Health Organization classifications,
presents
with relapsed disease after prior therapies with fludarabine, dexamethasone,
and rituximab, as
well as a regimen of CVP. He now has fever and night sweats associated with
generalized
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lymph node enlargement, a reduced hemoglobin and platelet production, as well
as a rapidly
rising leukocyte count. His LDH is elevated and the beta-2-microglobulin is
almost twice
normal. The patient is given therapy with IMMU-114-SN-38 conjugate at a dosing
scheme of
8 mg/kg weekly for 4 weeks, a rest of 2 weeks, and then the cycle repeated
again. Evaluation
shows that the patient's hematological parameters are improving and his
circulating CLL
cells appear to be decreasing in number. The therapy is then resumed for
another 3 cycles,
after which his hematological and lab values indicate that he has a partial
response.
Example 8. Immunoconjugate storage
[0235] The conjugates described in above were purified and buffer-exchanged
with 2-(N-
morpholino)ethanesulfonic acid (IYMS), pH 6.5, and further formulated with
trehalose (25
mM final concentration) and polysorbate 80 (0.01% v/v final concentration),
with the final
buffer concentration becoming 22.25 mM as a result of excipient addition. The
formulated
conjugates were lyophilized and stored in sealed vials, with storage at 2 C ¨
8 C. The
lyophilized immunoconjugates were stable under the storage conditions and
maintained their
physiological activities.
[0236] From the foregoing description, one skilled in the art can easily
ascertain the essential
characteristics of this invention, and without departing from the spirit and
scope thereof, can
make various changes and modifications of the invention to adapt it to various
usage and
conditions without undue experimentation. All patents, patent applications and
publications
cited herein are incorporated by reference.
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