Note: Descriptions are shown in the official language in which they were submitted.
WO 2022/217048
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ONCOLYTIC VIRUSES EXPRESSING
ANTI-ROR1/ANTI-CD3 BISPECIFIC ANTIBODIES
[0001] This application claims the benefit of priority under 35 U.S.C. 119 to
U.S.
provisional application No. 63/173,205, filed April 9, 2021, the entire
contents of which are
incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] The present disclosure provides bispecific antibodies that bind to both
ROR1 and
CD3 simultaneously. The present disclosure provides anti-ROR1/anti-CD3
bispecific
antibodies, nucleic acids encoding the anti-ROR1/anti-CD3 bispecific
antibodies, oncolytic
viruses that include constructs encoding anti-ROR1/anti-CD3 bispecific
antibodies, and
methods of use in treating cancer.
BACKGROUND
[0003] Receptor tyrosine kinase orphan receptors-1 and -2 (ROR1 and ROF,.2)
have been
described as being specifically associated with particular cancers (RehaYay et
al., 2012, Froni
Oncol., 2(34)), while being largely absent in. expression on healthy tissue
with few exceptions
(Balakrishnan at al., 2017, (7/in. Cancer Res., 23(12), 3061-3071). Due to the
very tumor--
selective expression of the ROR lamily members, they represent relevant
targets for targeted
cancer therapies.
[0004] Receptor tyrosine kinase orphan receptor-1. (ROR1) is aberrantly
expressed in B-
cell chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (3,4CL). ROM
exhibits
nearly 100% association with chronic lymphocytic leukemia (CLL.) (Cm et al.,
2016,
Mood, 128(25), 2931) and has been established as a. marker for some
acuteNnwhoblastic
leukemias (ALL), mantle cell lymphomas, and some other blood malignancies.
ROR1 is also
expressed in certain solid tumors, such as those of lung and breast cancer
(Ballakrishnan et al.,
2017, (7/in. Cancer Res., 23(12), :3061-3071). ROR1 has been found to he
involved in
progression of a number of solid tumors, such as rieuroblastoma, sarcoma,
renal cell
carcinoma, breast cancer, lung cancer, colon cancer, head and neck cancer, and
melanoma
and has been shown to inhibit apoptosis, potentiate -EGFR signaling, induce
epithelial-
ITICS C In al transition t:All), and contribute to CaVCOlae format/
[0005] ROR1 is mainly detectable in embryonic tissue and generally absent in
adult tissue,
making the protein an ideal drug target for cancer therapy. ROR1 has therefore
been
recognized as a target for the development of ROR1 specific antibodies.
However, due to the
hi2.1-i homology of ROR1 between different mammalian species, which is 100%
conserved on
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the amino acid level between humans and cynomolgus monkeys, 96.7% homologous
between
human and mouse, and 96.3% homologous between human and rabbit, it has been
difficult to
raise high affinity antibodies against this target by standard technologies,
such as animal
immunizations
[0006] Oncolytic viruses are viruses that selectively infect and lyse cancer
cells. Oncolytic
viruses have been the subject of clinical trials for the treatment various
cancers, including
melanoma, glioma, head and neck cancer, ovarian cancer, lung cancer, liver
cancer, bladder
cancer, prostate cancer, and pancreatic cancer (Aghi & Martuza (2005) Oncogene
24:7802-
7816). Multiple clinical trials have demonstrated the safety of oncolytic
herpes simplex
viruses (HSVs) attenuated in their ability to replicate in normal cells by
deletion of at least
one copy of the gene encoding ICP34.5 (Rampling et al. (2000) Gene Therapy
7:859-866;
Papanastassiou etal. (2002) Gene Therapy 9:398-406; Makie et al. (2001) Lancet
357:525-
526; Marken etal. (2000) Gene Therapy 7:867-874; Marken etal. (2009) Molecular
Therapy 17:199-207; Senzer etal. (2009) J Clin Oncol 27:5763-5771).
[0007] In addition to directly attacking the tumor by lysing cancer cells,
oncolytic HSVs
can induce an anti-tumor immune response in the patient (Papanastassiou et at.
(2002);
Marken et al. (2009); Senzer et al. (2009)) as viral antigens are expressed on
infected cancer
cells and tumor antigens are released when cancer cells are lysed. Viruses
also engage
mediators of the innate immune response as part of the host recognition of -
viral infection
resulting in an inflammatory response (Hu et al. (2006) Clin Cancer Res.
12:6737-6747).
These immune responses to treatment with oncolytic viruses may provide a
systemic benefit
to cancer patients resulting in the suppression of tumors which have not been
infected with
the virus, including metastatic tumors, and may prevent disease rec-urrence.
SUMMARY
[0008] The present application ("ascribes bispecific antibodies that
simultaneously bind
RORI and CD3 (aROR1/aCD3 Bsp Abs). Constructs encoding aROR1/aCD3 Bsp Abs as
described herein were cloned into an oncolytic HSV-1 virus ("Seprehvec")
derived from
HSV 17 that does not include a functional RL-1 gene. Virus-infected cells were
used to
produce Virus Free Cell Media (VFCMs) that include bispecific antibodies which
were tested
for their ability to enhance cytotoxicity of T cells toward ROR1-expressing
tumor cells. The
aROR 1 /aCD3 BspAbs disclosed herein demonstrated potent T cell targeting with
specific
anti-tumor activity in preclinical studies. The expression of an aROR1/aCD3
BspAb by the
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oncolytic Seprehvec HSV significantly increased anti-tumor activity of viral
treatment in an
antigen-dependent manner.
[0009] Provided herein in a first aspect is a bispecific antibody comprising a
first single
chain variable fragment antibody (ScFv) that binds ROR1 and a second single
chain variable
fragment antibody (ScFv) that binds CD3, wherein the anti-RORI scFv and the
anti-CD3
scFv are joined via a linker. The linker can be, for example a GS linker such
as but not
limited to a (G4S)n linker, where n can be an integer from 1-20, for example,
from 1-8. The
anti-ROR1/anti-CD3 bispecific antibody (aROR1/aCD3 BspAb) can be an isolated
protein,
and in some examples is partially or substantially purified.
[0010] The anti-ROR1 scFv of the bispecific antibody can have a heavy chain
variable
domain (VH) sequence and a light chain variable domain (VL) sequence connect
by a linker,
such as a (G4S)n linker, and the VH and VL sequences can be derived from a
monoclonal
antibody that binds ROR1, for example, binds the human ROR1 protein. For
example, the
anti-ROR1 scFv of the bispecific antibody can include a VH domain sequence
having at least
95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ
ID NO:1, and a
VH domain sequence having at least 95%, at least 96%, at least 97%, at least
98%, or at least
99% identity to SEQ ID NO:5. In another example, the anti-ROR1 scFv of the
bispecific
antibody can include a VH domain sequence having at least 95%, at least 96%,
at least 97%,
at least 98%, or at least 99% identity to SEQ ID NO:10, and a VH domain
sequence having at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity
to SEQ ID NO:14.
In a further example, the anti-ROR1 scFv of the bispecific antibody can
include a VH domain
sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99%
identity to SEQ ID NO:19, and a VH domain sequence having at least 95%, at
least 96%, at
least 97%, at least 98%, or at least 99% identity to SEQ ID NO:23.
[0011] In further examples, the anti-ROR1 scFv of the bispecific antibody can
include a
VH domain sequence having at least 95%, at least 96%, at least 97%, at least
98%, or at least
99% identity to SEQ ID NO:52, and a VH domain sequence having at least 95%, at
least
96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:56; or
the anti-ROR1
scFv of the bispecific antibody can include a VH domain sequence having at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID
NO:60, and a VH
domain sequence having at least 95%, at least 96%, at least 97%, at least 98%,
or at least
99% identity to SEQ ID NO:64.
[0012] In various embodiments, the anti-ROR1 scFv of anti-ROR1/anti-CD3
bispecific
antibody (aROR1/aCD3 BspAb) as provided herein can have an amino acid sequence
having
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at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
identity to SEQ ID
NO:9, SEQ ID NO:18, or SEQ ID NO:27.
100131 The anti-CD3 scFv of the aROR1/aCD3 BspAb provided herein can be, in
nonlimiting embodiments, an anti-CD3 scFv that comprises a heavy chain
variable domain
having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
identity to SEQ
ID NO:32 and a light chain variable domain having at least 95%, at least 96%,
at least 97%,
at least 98%, or at least 99% identity to SEQ ID NO:33. In some embodiments
the anti-CD3
scFv comprises an amino acid sequence having at least 95%, at least 96%, at
least 97%, at
least 98%, or at least 99% identity to SEQ ID NO:34.
100141 A further aspect provided herein are nucleic acid constructs encoding
any of the
aROR1/aCD3 BspAbs disclosed herein. The aROR1/aCD3 BspAb encoded by the
nucleic
acid construct can include a signal peptide at the N-terminus of the
bispecific antibody
construct, for example, the signal peptide of SEQ ID NO:28, or any suitable
signal peptide.
The nucleic acid construct can be a DNA construct that includes a promoter
operably linked
to the aROR1/aCD3 BspAb encoding sequence. The promoter can be, as nonlimiting
examples, an EF1 a promoter, a CMV promoter (e.g., SEQ ID NO:42), a JeT
promoter, an
RSV promoter, an SV40 promoter, a CAG promoter, a beta-actin promoter, an HTLV
promoter, or an EFla/HTLV hybrid promoter (e.g., SEQ ID NO:41). The nucleic
acid
construct can further include a polyadenylation sequence 3' of the BspAb-
encoding sequence,
such as, for example, an SV40 3' sequence. The nucleic acid construct can be
provided in a
vector, and in some examples may be cloned into a recombinant viral genome.
100151 A further aspect provided herein is a recombinant oncolytic virus
comprising a
nucleic acid construct comprising a nucleic acid sequence encoding an
aROR1/aCD3 BspAb
according to any disclosed herein. In various embodiments the recombinant
oncolytic virus is
a recombinant herpes simplex virus (HSV), for example, and HSV-1 virus such as
a virus
derived from HSV-1 strain 17, HSV-1 strain F, HSV-1 strain KOS, or HSV-1
strain JS1. In
some embodiments, a recombinant oncolytic HSV that includes a genetic
construct for
expressing an aROR1/aCD3 BspAb as provided herein does not include a
functional
ICP34.5-encoding gene, and in some examples, all or a portion of the ICP34.5-
encoding gene
may be deleted. For example, the recombinant oncolytic HSV may be derived from
the HSV
17 strain, and the nucleic acid construct encoding an aROR1/aCD3 BspAb may be
inserted
into the ICP34.5-encoding gene locus.
[0016] In certain embodiments a recombinant oncolytic virus comprising a
nucleic acid
construct comprising a nucleic acid sequence encoding an aROR1/aCD3 BspAb can
further
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include a nucleic acid sequence encoding a cytokine. For example, an oncolytic
virus can
include a gene encoding an aROR1/aCD3 BspAb and a gene encoding IL-12. In
particular
examples disclosed herein an oncolytic virus includes a gene encoding an
aROR1/aCD3
BspAb, such as any disclosed herein and a gene encoding IL-12, such as, for
example, a
human IL-12 having at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99%
identity to SEQ ID NO:46. In some examples, an oncolytic virus can include a
gene
encoding an aROR1/aCD3 BspAb and a gene encoding a different antibody, for
example, an
scFv that binds a growth factor or growth factor receptor, such as VEGFR2. In
particular
examples disclosed herein an oncolytic virus includes a gene encoding an
aROR1/aCD3
BspAb, such as any disclosed herein and a gene encoding an anti-VEGFR2 scFv,
such as, for
example, an VEGFR2 scFV having at least 95%, at least 96%, at least 97%, at
least 98%, or
at least 99% identity to SEQ ID NO:49. In some embodiments, an oncolytic virus
as
disclosed herein encodes 1) an aROR1/aCD3 BspAb; 2) an IL-12 polypeptide, and
3) an
anti-VEGFR2 scFv.
[0017] Further included is a pharmaceutical composition comprising a
recombinant
oncolytic virus, which may be a recombinant oncolytic HSV, that includes a
genetic construct
for expressing an aROR1/aCD3 BspAb, and, optionally, one or more additional
transgenes,
and a pharmaceutically acceptable excipient. The oncolytic virus can be
provided in a saline
solution, for example, such as PBS, Ringer's, or HBSS, and the formulation can
optionally
further include, as nonlimiting examples, one or more preservatives, or
cryoprotectants (e.g.,
DMSO or glycerol). In some embodiments, the concentration of virus in the
pharmaceutical
composition is at least 106 per ml, at least 107 per ml, at least 5 x 107 per
ml, or at least 108
per ml.
[0018] Yet another aspect is a method of treating cancer by administering an
oncolytic
virus encoding an aROR1/aCD3 BspAb as provided herein, including a
pharmaceutical
composition as provided herein. The oncolytic virus can include one or more
additional
transgenes, such as but not limited to a gene encoding an IL-12 polypeptide
and/or a gene
encoding an antibody that binds VEGFR2. The subject can be a subject diagnosed
with
cancer which may be a hematological cancer or a solid tumor. The subject can
be, as
nonlimiting examples, a dog, horse, or primate, and may be a human subject.
The oncolytic
virus can be an oncolytic HSV, and administration may be for example,
intravenous, intra-
arterial, intracavitary, intraperitoneal, intratumoral, or peritumoral
delivery. For example, the
oncolytic virus can be delivered by injection, by infusion, or by means of a
catheter. The
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methods can include multiple administrations, where dosings can be separated
by days,
weeks, or months.
[0019] Also provided are methods of treating a subject using a VFCM produced
by
culturing cells infected with any of the oncolytic viruses disclosed herein.
[0020] Also provided herein are host cells infected with an oncolytic virus
that includes a
genetic construct for expressing an aROR1/aCD3 BspAb as provided herein. The
host cells
can be, for example, mammalian host cells and can be of a cell line. In some
embodiments
the host cells are Vero cells, BHK cells, or HEK293 cells. Also provided are
methods of
treating a subject having cancer using a VFCM produced by culturing cells
infected with any
of the oncolytic viruses disclosed herein. The VCFM can be prepared by for
example,
centrifugation and filtration of the cell supernatant, where the VFCM can
comprise one or
more recombinant polypeptides encoded by the oncolytic virus, such as, for
example, an
aROR1/aCD3 BspAb as provided herein, and optionally IL-12 and/or an anti-
VEGFR2
antibody. The subject to be treated in some embodiments can be a nonhuman
subject.
[0021] Provided in a further aspect are methods for producing a bispecific
antibody,
including producing any of the aROR1/aCD3 bispecific antibodies disclosed
herein, by
culturing a host cell infected with an oncolytic virus that includes a genetic
construct for
expressing a bispecific antibody to produce a virus free conditioned cell
medium (VFCM)
that includes bispecific antibodies and isolating bispecific antibodies from
the VFCM. The
VFCM can include one or more additional polypeptides or antibodies, such as
but not limited
to an IL-12 polypeptide and/or an antibody that binds VEGFR2. Further included
are
pharmaceutical compositions including aROR1/aCD3 bispecific antibodies as
disclosed
herein and methods of treating a subject with cancer by administering an
aROR1/aCD3
bispecific antibody as disclosed herein to the subject. In some embodiments
the methods
include treating a subject, such as but not limited to a non-human subject,
with a VFCM that
may be prepared from cell culture using for example, centrifugation and
filtration.
DESCRIPTION OF THE FIGURES
[0022] Figure 1 is a schematic showing an example of a construct encoding an
anti-
ROR1/anti-CD3 bispecific antibody (transcribed from right to left).
[0023] Figure 2A illustrates the format of an EL1SA detection assay for anti-
ROR1/anti-
CD3 bispecific antibodies.
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[0024] Figure 2B provides binding curves of anti-ROR1/anti-CD3 bispecific
antibodies
produced by cells infected with HSVs SepGI-189, SepGI-201, and SepGI-203.
VFCMs of
cell cultures were assayed.
[0025] Figure 3A illustrates the format of a cell binding assay for anti-
ROR1/anti-CD3
bispecific antibodies. Wild type A549 cells express ROR1; A549 cells knocked
out for the
ROR1 gene were also tested as controls.
[0026] Figure 3B provides the results cell binding assay for anti-ROR1/anti-
CD3
bispecific antibodies. VFCMs of cultures of cells infected with anti-ROR1/anti-
CD3 BspAb-
encoding viruses SepGI-189, SepGI-201, and SepGI-203 were assayed.
[0027] Figure 4A illustrates the format of a T cell-tumor cell interaction
assay.
[0028] Figure 4B provides the results of flow cytometry analysis of T cell-
tumor cell
interaction as mediated by aROR1/aCD3 BsAbs present in VFCMS of cultures
infected with
HSVs SepGI-189, SepGI-201, and SepGI-203.
[0029] Figure 5A illustrates the format of a luciferase-based cell signaling
assay for anti-
ROR1/anti-CD3 bispecific antibodies.
[0030] Figure 5B provides the results of the cell signaling assay using VFCMS
of cultures
infected with HSVs SepGI-189, SepGI-201, and SepGI-203.
[0031] Figure 6 provides percent killing in cytotoxicity assays that included
T cells and
VFCMS of cultures infected with HSVs SepGI-189, SepGI-201, and SepGI-203.
Interferon
gamma (IFNy) secretion by the T cells is also provided in the graphs on the
right.
[0032] Figure 7A provides a graph of binding of anti-ROR1 antibody to A549,
A549/ROR1 KO, MCF-7, and HepG2 tumor cells.
[0033] Figure 7B provides a graph of percent killing in cytotoxicity assays
using A549,
MCF-7, and HepG2 tumor cells as targets that included T cells and VFCM of
cultures
infected with the SepGI-201 HSV that expresses an aROR1/aCD3 BsAb. Controls
included
assays in the absence of T cells and assays of VFCM produced from cells
infected with a
SepGI-Null virus that did not express an aROR1/aCD3 BsAb. Also provided are
the results
of IFNy assays of the co-cultures.
[0034] Figure 8A provides the procedure for assays for killing of A549 tumor
cells by
aROR1/aCD3 BsAb-expressing HSVs.
100351 Figure 8B provides graphs demonstrating enhanced killing of ROR1-
positive tumor
cells and ROR1-knockout cells by virus used to infect the cultures at various
MOIs.
[0036] Figure 9A illustrates the format of an ELISA detection assay for
binding of mouse
ROR1 by anti-ROR1/anti-CD3 bispecific antibodies.
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[0037] Figure 9B provides the binding curves for antibodies slO and j1v1011
against
mouse RORI.
[0038] Figure 10A provides the tumor inoculation and treatment schedule for an
in vivo
study of treatment of tumors with HSVs SepGI-189 and SepGI-201.
[0039] Figure 10B provides graphs of tumor volumes of A549 tumor-inoculated
mice
treated with HSVs SepGI-189 and SepGI-201.
[0040] Figure 10C provides a graph of the percent tumor growth inhibition of
mice treated
with HSVs SepGI-Null, SepGI-189, and SepGI-201.
[0041] Figure 10D provide graphs of body weights over the course of the study
shown in
Figures 10A, B and C.
[0042] Figure 11A is a schematic showing an example of a construct encoding an
anti-
ROR1/anti-CD3 bispecific antibody (transcribed from right to left) and a human
IL-12
polypeptide (transcribed from left to right).
[0043] Figure 11B is a schematic showing an example of a construct encoding an
anti-
ROR1/anti-CD3 bispecific antibody (transcribed from right to left), and an
anti-VEGFR2
scFv and human 1L-12 polypeptide. The anti-VEGFR2 scFv and human 1L-12
polypeptide
are transcribed from left to right by the same promoter and their coding
sequences are
connected via a T2A self-cleaving peptide-encoding sequence.
[0044] Figure 12A provides the results of ELISAs for detecting the anti-RSV
antibody in
VFCMs of cells infected with different HSVs. The graph shows that the SepGI-
207 and
SepGI-218 VFCMs included the anti-RSV antibody.
[0045] Figure 12B provides the results of ELISAs for detecting the anti-RORI
antibody in
VFCMs of cells infected with different HSVs. The graph shows that the SepGI-
201, SepGI-
212, and SepGI-216 VFCMs included the anti-ROR1 antibody.
[0046] Figure 12C provides the results of ELISAs for detecting human IL-12 in
VFCMs of
cells infected with different HSVs. The graph shows that the SepGI-212, SepGI-
216, and
SepGI-218 VFCMs included human IL-12.
[0047] Figure 13 provides the results of ELISAs for detecting the anti-VEGFR2
antibody
in VFCMs of cells infected with different HSVs. The graph shows that the VFCM
of an
isolate of SepGI-212 included the anti-VEGFR2 antibody.
100481 Figure 14 is a bar graph providing the results of assays to detect the
activity of IL-
12 in the VFCMs of cells infected with HSVs SepGI-Null, SepGI-201, SepGI-207,
SepGI-
212, SepGI-214, SepGI-216, and SepGI-218.
[0049] Figure 15A provides the results of flow cytometry of unlabeled tumor
cells.
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[0050] Figure 15B provides the results of flow cytometry of tumor cells
labeled with
eFluor 450.
[0051] Figure 15C provides the results of flow cytometry of human T cells
labeled with
eFluor 670.
[0052] Figure 15D provides the results of flow cytometry of labeled tumor
cells and
labeled T cells co-incubated with VFCM that included an anti-ROR1-anti-CD3
bispecific
antibody.
[0053] Figure 16A provides a bar graph of the results of flow cytometry assays
for tumor
cell-T cell interaction mediated by SepGI-218 VFCM (aRSV-aCD3 bsp antibody
plus IL-12)
as percentages of analyzed cells when the tumor cells were Hepa 1-6, A549, and
A549 ROR1
knockout cells.
[0054] Figure 16B provides a bar graph of the results of flow cytometry assays
for tumor
cell-T cell interaction mediated by SepGI-201 VFCM (aROR1-aCD3 bsp antibody)
as
percentages of analyzed cells when the tumor cells were Hepa 1-6 and A549
cells.
[0055] Figure 16C provides a bar graph of the results of flow cytometry assays
for tumor
cell-T cell interaction mediated by SepG1-216 VFCM (aROR1-aCD3 bsp antibody
plus IL-
12 and VEGFR2 antibody) as percentages of analyzed cells when the tumor cells
were Hepa
1-6 and A549 cells.
[0056] Figure 17A is a graph showing the percentages of live CD3+ T cells used
in T cell
activation assays over 3 days, where the T cells have been incubated in the
presence of ROR1
knockout tumor target cells (for bars proceeding from left to right for each
day): VFCM of
uninfected cells, VFCM of cells infected with SepGI-Null, VFCM of cells
infected with
SepGI-207, VFCM of cells infected with SepGI-201, and CD3/CD28 beads. The
first of each
pair of bars provides the value for the assays performed at a 10:1 E:T ratio,
and the second of
each pair of bars provides the value for the assays performed at a 5:1 E:T
ratio.
100571 Figure 17B is a graph providing the CD3+CD4+ cell count in each of the
T cell
activation assays. Assay VFCMs and E:T ratios are as in Figure 17A.
[0058] Figure 17C provides the CD25+ T cells as percentages of CD3+CD4+ cells
in the
activation assays in which the T cells have been incubated in the presence of
ROR1+ tumor
target cells (for bars proceeding from left to right for each day): VFCM of
uninfected cells,
VFCM of cells infected with SepGI-Null, VFCM of cells infected with SepG1-207,
VFCM of
cells infected with SepGI-201, and CD3/CD28 beads. The first of each pair of
bars provides
the value for the assays performed at a 10:1 E:T ratio, and the second of each
pair of bars
provides the value for the assays performed at a 5:1 E:T ratio.
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[0059] Figure 17D provides the CD69+ T cells as percentages of CD3+CD4+ cells
in the
activation assays in which the T cells have been incubated in the presence of
ROR1+ tumor
target cells (for bars proceeding from left to right for each day): VFCM of
uninfected cells,
VFCM of cells infected with SepGI-Null, VFCM of cells infected with SepGI-207,
VFCM of
cells infected with SepGI-201, and CD3/CD28 beads. The first of each pair of
bars provides
the value for the assays performed at a 10:1 E:T ratio, and the second of each
pair of bars
provides the value for the assays performed at a 5:1 E:T ratio
[0060] Figure 17E is a graph showing the percentages of live CD3+ T cells used
in T cell
activation assays over 3 days, where the T cells have been incubated in the
presence of
ROR1+ tumor target cells (for bars proceeding from left to right for each
day): VFCM of
uninfected cells, VFCM of cells infected with SepGI-Null, VFCM of cells
infected with
SepGI-207, VFCM of cells infected with SepGI-201, and CD3/CD28 beads. The
first of each
pair of bars provides the value for the assays performed at a 10:1 E:T ratio,
and the second of
each pair of bars provides the value for the assays performed at a 5:1 E:T
ratio.
[0061] Figure 17F is a graph providing the CD3+CD4+ cell count in each of the
T cell
activation assays. Assay VFCMs and E:T ratios are as in Figure 17E.
[0062] Figure 17G provides the CD25+ T cells as percentages of CD3+CD4+ cells
in the
activation assays in which the T cells have been incubated in the presence of
ROR1+ tumor
target cells (for bars proceeding from left to right for each day): VFCM of
uninfected cells,
VFCM of cells infected with SepGI-Null, VFCM of cells infected with SepGI-207,
VFCM of
cells infected with SepGI-201, and CD3/CD28 beads. The first of each pair of
bars provides
the value for the assays performed at a 10:1 E:T ratio, and the second of each
pair of bars
provides the value for the assays performed at a 5:1 E:T ratio.
[0063] Figure 17H provides the CD69+ T cells as percentages of CD3+CD4+ cells
in the
activation assays in which the T cells have been incubated in the presence of
ROR1+ tumor
target cells (for bars proceeding from left to right for each day): VFCM of
uninfected cells,
VFCM of cells infected with SepGI-Null, VFCM of cells infected with SepGI-207,
VFCM of
cells infected with SepGI-201, and CD3/CD28 beads. The first of each pair of
bars provides
the value for the assays performed at a 10:1 E:T ratio, and the second of each
pair of bars
provides the value for the assays performed at a 5:1 E:T ratio
100641 Figure 18A are graphs showing the percentages of live CD3+ T cells used
in T cell
activation assays over 3 days, where the T cells have been incubated in the
presence of A549
wild type (ROR1+) tumor target cells (for bars proceeding from left to right
for each day):
VFCM of cells infected with SepGI-Null, VFCM of cells infected with SepGI-207,
VFCM of
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cells infected with SepGI-201, VFCM of cells infected with SepGI-218,and VFCM
of cells
infected with SepGI-216. Assays performed at a5:1 E:T ratio.
[0065] Figure 18B are graphs showing the percentages of live CD3+ T cells used
in T cell
activation assays over 3 days, where the T cells have been incubated in the
presence of A549
ROR1 knockout tumor target cells (for bars proceeding from left to right for
each day):
VFCM of cells infected with SepGI-Null, VFCM of cells infected with SepGI-207,
VFCM of
cells infected with SepGI-201, VFCM of cells infected with SepGI-218,and VFCM
of cells
infected with SepGI-216. Assays performed at a 5:1 E:T ratio.
[0066] Figure 19A is a graph showing the results of luciferase-based toxicity
assays in the
absence and presence of T cells where the targets were A549 wild-type cells
expressing
luciferase and the assays were performed in the presence of VFCMs of
uninfected cells or
cells infected with SepGI-Null, SepGI-201, SepGI-207, SepGI-212, SepGI-214,
SepGI-216,
and SepGI-218.
[0067] Figure 19B is a graph showing the results of luciferase-based toxicity
assays in the
absence and presence of T cells where the targets were A549 ROR1 knockout
cells
expressing luciferase and the assays were performed in the presence of VFCMs
of uninfected
cells or cells infected with SepGI-Null, SepGI-201, SepGI-207, SepGI-212,
SepGI-214,
SepGI-216, and SepGI-218.
[0068] Figure 19C is a graph providing the percentage killing of the assays of
Figure 17C.
[0069] Figure 19D is a graph providing the percentage killing of the assays of
Figure 17B.
[0070] Figure 20 shows the cell index over time of cells in impedance-based
cytotoxicity
assays using A549 wild type cells as targets. See Example 18.
[0071] Figure 21 shows the cell index over time of cells in impedance-based
cytotoxicity
assays using A549 knockout cells as targets. See Example 18.
DETAILED DESCRIPTION
[0072] Throughout this application various publications, patents, and/or
patent applications
are referenced. The disclosures of the publications, patents and/or patent
applications are
hereby incorporated by reference in their entireties into this application in
order to more fully
describe the state of the art to which this disclosure pertains.
Definitions:
[0073] Unless defined otherwise, technical and scientific terms used herein
have meanings
that are commonly understood by those of ordinary skill in the art unless
defined otherwise.
Generally, terminologies pertaining to techniques of cell and tissue culture,
molecular
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biology, immunology, microbiology, genetics, transgenic cell production,
protein chemistry
and nucleic acid chemistry and hybridization described herein are well known
and commonly
used in the art. The methods and techniques provided herein are generally
performed
according to conventional procedures well known in the art and as described in
various
general and more specific references that are cited and discussed herein
unless otherwise
indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual,
2d ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et
al.,
Current Protocols in Molecular Biology, Greene Publishing Associates (1992). A
number of
basic texts describe standard antibody production processes, including,
Borrebaeck
(ed) Antibody Engineering, 2nd Edition Freeman and Company, NY, 1995;
McCafferty et
al. Antibody Engineering, A Practical Approach IRL at Oxford Press, Oxford,
England,
1996; and Paul (1995) Antibody Engineering Protocols Humana Press, Towata,
N.J., 1995;
Paul (ed.), Fundamental Immunology, Raven Press, N.Y, 1993; Coligan (1991)
Current
Protocols in Immunology Wiley/Greene, NY; Harlow and Lane (1989)Antibodies: A
Laboratory Manual Cold Spring Harbor Press, NY; Stites et al. (eds.) Basic and
Clinical
Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif, and
references cited
therein; Coding Monoclonal Antibodies: Principles and Practice (2nd ed.)
Academic Press,
New York, N.Y., 1986, and Kohler and Milstein Nature 256: 495-497, 1975. All
of the
references cited herein are incorporated herein by reference in their
entireties. Enzymatic
reactions and enrichment/purification techniques are also well known and are
performed
according to manufacturer's specifications, as commonly accomplished in the
art or as
described herein. The terminology used in connection with, and the laboratory
procedures
and techniques of, analytical chemistry, synthetic organic chemistry, and
medicinal and
pharmaceutical chemistry described herein are well known and commonly used in
the art.
Standard techniques can be used for chemical syntheses, chemical analyses,
pharmaceutical
preparation, formulation, and delivery, and treatment of patients.
[0074] The headings provided herein are not limitations of the various aspects
of the
disclosure, which aspects can be understood by reference to the specification
as a whole.
[0075] Unless otherwise required by context herein, singular
terms shall include
pluralities and plural terms shall include the singular. Singular forms -a-,
"an- and "the-,
and singular use of any word, include plural referents unless expressly and
unequivocally
limited on one referent.
[0076] It is understood the use of the alternative (e.g., "or")
herein is taken to mean either
one or both or any combination thereof of the alternatives.
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[0077] The term "and/or" used herein is to be taken mean specific
disclosure of each of
the specified features or components with or without the other. For example,
the term
"and/or" as used in a phrase such as "A and/or B" herein is intended to
include "A and B,"
"A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in
a phrase such
as "A, B, and/or C- is intended to encompass each of the following aspects: A,
B, and C; A,
B, or C; A or C, A or B; B or C; A and C, A and B; B and C; A (alone); B
(alone); and C
(alone).
[0078] As used herein, terms -comprising", -including", -having" and -
containing", and
their grammatical variants, as used herein are intended to be non-limiting so
that one item or
multiple items in a list do not exclude other items that can be substituted or
added to the listed
items. It is understood that wherever aspects are described herein with the
language
-comprising," otherwise analogous aspects described in terms of -consisting
of' and/or
"consisting essentially of" are also provided.
[0079] As used herein, the term -about" refers to a value or
composition that is within an
acceptable error range for the particular value or composition as determined
by one of
ordinary skill in the art, which will depend in part on how the value or
composition is
measured or determined, i.e., the limitations of the measurement system. For
example,
"about- or "approximately- can mean within one or more than one standard
deviation per the
practice in the art. Alternatively, "about" or "approximately" can mean a
range of up to 10%
(i.e., 10%) or more depending on the limitations of the measurement system.
For example,
about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore,
particularly
with respect to biological systems or processes, the terms can mean up to an
order of
magnitude or up to 5-fold of a value. When particular values or compositions
are provided in
the instant disclosure, unless otherwise stated, the meaning of "about" or
"approximately"
should be assumed to be within an acceptable error range for that particular
value or
composition.
[0080] The terms "peptide", "polypeptide" and "protein" and other related
terms used
herein are used interchangeably and refer to a polymer of amino acids and are
not limited to
any particular length. Polypeptides may comprise natural and non-natural amino
acids.
Polypeptides include recombinant or chemically-synthesized forms. Polypeptides
also
include precursor molecules that have not yet been subjected to cleavage, for
example
cleavage by a secretory signal peptide or by non-enzymatic cleavage at certain
amino acid
residues. Polypeptides include mature molecules that have undergone cleavage.
These terms
encompass native and artificial proteins, protein fragments and polypeptide
analogs (such as
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muteins, variants, chimeric proteins and fusion proteins) of a protein
sequence as well as
post-translationally, or otherwise covalently or non-covalently, modified
proteins. Two or
more polypeptides (e.g., 3 polypeptide chains) can associate with each other,
via covalent
and/or non-covalent association, to form a polypeptide complex. Association of
the
polypeptide chains can also include peptide folding. Thus, a polypeptide
complex can be
dimeric, trimeric, tetrameric, or higher order complexes depending on the
number of
polypeptide chains that form the complex.
[0081] The terms -nucleic acid", "polynucleotide" and "oligonucleotide" and
other related
terms used herein are used interchangeably and refer to polymers of
nucleotides and are not
limited to any particular length. Nucleic acids include recombinant and
chemically-
synthesized forms. Nucleic acids include DNA molecules (cDNA or genomic DNA),
RNA
molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide
analogs
(e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs),
and hybrids
thereof Nucleic acid molecule can be single-stranded or double-stranded. In
one
embodiment, the nucleic acid molecules of the disclosure comprise a contiguous
open
reading frame encoding an antibody, or a fragment or scFv, derivative, mutein,
or variant
thereof In one embodiment, nucleic acids comprise one type of polynucleotide
or a mixture
of two or more different types of polynucleotides. Nucleic acids encoding
bispecific
antibodies are described herein.
[0082] The term "recover" or -recovery" or "recovering", and
other related terms, refers
to obtaining a protein (e.g., an antibody or an antigen binding portion
thereof), from host cell
culture medium or from host cell lysate or from the host cell membrane. In one
embodiment,
the protein is expressed by the host cell as a recombinant protein fused to a
secretion signal
peptide (leader peptide sequence) sequence which mediates secretion of the
expressed protein
from a host cell (e.g., from a mammalian host cell). The secreted protein can
be recovered
from the host cell medium. In one embodiment, the protein is expressed by the
host cell as a
recombinant protein that lacks a secretion signal peptide sequence which can
be recovered
from the host cell lysate. In one embodiment, the protein is expressed by the
host cell as a
membrane-bound protein which can be recovered using a detergent to release the
expressed
protein from the host cell membrane. In one embodiment, irrespective of the
method used to
recover the protein, the protein can be subjected to procedures that remove
cellular debris
from the recovered protein. For example, the recovered protein can be
subjected to
chromatography, gel electrophoresis and/or dialysis. In one embodiment, the
chromatography comprises any one or any combination or two or more procedures
including
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affinity chromatography, hydroxyapatite chromatography, ion-exchange
chromatography,
reverse phase chromatography and/or chromatography on silica. In one
embodiment, affinity
chromatography comprises protein A or G (cell wall components from
Staphylococcus
aureus).
[0083] The term "isolated" refers to a protein (e.g., an antibody or an
antigen binding
portion thereof) or polynucleotide that is substantially free of other
cellular material. A
protein may be rendered substantially free of naturally associated components
(or
components associated with a cellular expression system or chemical synthesis
methods used
to produce the antibody) by isolation, using protein purification techniques
well known in the
art. The term isolated also refers to protein or polynucleotides that are
substantially free of
other molecules of the same species, for example other protein or
polynucleotides having
different amino acid or nucleotide sequences, respectively. The purity of
homogeneity of the
desired molecule can be assayed using techniques well known in the art,
including low
resolution methods such as gel electrophoresis and high resolution methods
such as HPLC or
mass spectrometry. In various embodiments bispecific antibodies of the present
disclosure are
isolated.
[0084] The term "signal peptide", "[peptide] signal sequence", "leader
sequence", -leader
peptide-, or "secretion signal peptide- refers to a peptide sequence that is
located at the N-
terminus of a polypeptide. A leader sequence directs a polypeptide chain to a
cellular
secretory pathway and can direct integration and anchoring of a membrane
protein into the
lipid bilayer of the cellular membrane. Typically, a leader sequence is about
10-60 amino
acids in length, more commonly 15-50 amino acids in length. A leader sequence
can direct
transport of a precursor polypeptide from the cytosol to the endoplasmic
reticulum. In various
embodiments, a leader sequence includes signal sequences comprising CD8a,
CD28, or
CD16 leader sequences or a mouse or human Ig gamma secretion signal peptide.
In one
embodiment, a leader sequence comprises a mouse Ig gamma leader peptide
sequence
MEWSWVFLFFLSVTTGVHS (SEQ ID NO:).
[0085] An "antigen binding protein" and related terms used herein
refers to a protein
comprising a portion that binds to an antigen and, optionally, a scaffold or
framework portion
that allows the antigen binding portion to adopt a conformation that promotes
binding of the
antigen binding protein to the antigen. Examples of antigen binding proteins
include
antibodies, antibody fragments (e.g., an antigen binding portion of an
antibody), antibody
derivatives, and antibody analogs. The antigen binding protein can comprise,
for example, an
alternative protein scaffold or artificial scaffold with grafted CDRs or CDR
derivatives. Such
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scaffolds include, but are not limited to, antibody-derived scaffolds
comprising mutations
introduced to, for example, stabilize the three-dimensional structure of the
antigen binding
protein as well as wholly synthetic scaffolds comprising, for example, a
biocompatible
polymer. See, for example, Korndorfer et al., 2003, Proteins: Structure,
Function, and
Bioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004, Biotechnol.
Prog. 20:639-
654. In addition, peptide antibody mimetics ("PAMs") can be used, as well as
scaffolds based
on antibody mimetics utilizing fibronection components as a scaffold.
[0086] An antigen binding protein can have, for example, the
structure of a naturally
occurring immunoglobulin. In one embodiment, an "immunoglobulin" refers to a
naturally-
occurring tetrameric molecule composed of two identical pairs of polypeptide
chains, each
pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70
kDa). The amino-
terminal portion of each chain includes a variable region of about 100 to 110
or more amino
acids primarily responsible for antigen recognition. The carboxy-terminal
portion of each
chain defines a constant region primarily responsible for effector function.
Human light
chains are classified as kappa or lambda light chains. Heavy chains are
classified as mu,
delta, gamma, alpha, or epsilon, and define the antibody's isotype as 1gM,
1gD, IgG, IgA, and
IgE, respectively. Within light and heavy chains, the variable and constant
regions are joined
by a "J" region of about 12 or more amino acids, with the heavy chain also
including a "D"
region of about 10 more amino acids. See generally, Fundamental Immunology Ch.
7 (Paul,
W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its
entirety for all
purposes). The heavy and/or light chains may or may not include a leader
sequence for
secretion. The variable regions of each light/heavy chain pair form the
antibody binding site
such that an intact immunoglobulin has two antigen binding sites. In one
embodiment, an
antigen binding protein can be a synthetic molecule having a structure that
differs from a
tetrameric immunoglobulin molecule but still binds a target antigen or binds
two or more
target antigens. For example, a synthetic antigen binding protein can comprise
antibody
fragments, 1-6 or more polypeptide chains, asymmetrical assemblies of
polypeptides, or other
synthetic molecules. In various embodiments, bispecific antibodies of the
present disclosure
exhibit immunoglobulin-like properties and bind specifically to two different
target antigens
(ROR1 and CD3).
100871 The variable regions of immunoglobulin chains exhibit the
same general structure
of relatively conserved framework regions (FR) joined by three hypervariable
regions, also
called complementarily determining regions or CDRs. From N-terminus to C-
terminus, both
light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3
and FR4.
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[0088] One or more CDRs may be incorporated into a molecule
either covalently or
noncovalently to make it an antigen binding protein. An antigen binding
protein may
incorporate the CDR(s) as part of a larger polypeptide chain, may covalently
link the CDR(s)
to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The
CDRs
permit the antigen binding protein to specifically bind to a particular
antigen of interest.
[0089] The assignment of amino acids to each domain is in
accordance with the
definitions of Kabat et al. in Sequences of Proteins of Immunological
Interest, 5th Ed., US
Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242,
1991 (-Kabat
numbering"). Other numbering systems for the amino acids in immunoglobulin
chains
include IMGT® (international ImMunoGeneTics information system; Lefranc et
al, Dev.
Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, I Mol. Biol.
309(3):657-670; 2001); Chothia (Al-Lazikani et al., 1997 Journal of Molecular
Biology
273:927-948; Contact (Maccallum et al., 1996 Journal of Molecular Biology
262:732-745,
and Aho (Honegger and Pluckthun 2001 Journal of Molecular Biology 309:657-670.
[0090] An "antibody" and "antibodies" and related terms used
herein refers to an intact
immunoglobulin or to an antigen binding portion thereof that binds
specifically to an antigen.
Antigen binding portions may be produced by recombinant DNA techniques or by
enzymatic
or chemical cleavage of intact antibodies. Antigen binding portions include,
inter alio, Fab,
Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarily determining
region (CDR)
fragments. single-chain antibodies (scFv), chimeric antibodies, diabodies,
triabodies,
tetrabodies, and polypeptides that contain at least a portion of an
immunoglobulin that is
sufficient to confer specific antigen binding to the polypeptide.
[0091] Antibodies include recombinantly produced antibodies and
antigen binding
portions. Antibodies include non-human, chimeric, humanized and fully human
antibodies.
Antibodies include monospecific, multispecific (e.g., bispecific, trispecific
and higher order
specificities). Antibodies include tetrameric antibodies, light chain
monomers, heavy chain
monomers, light chain dimers, heavy chain dimers. Antibodies include F(ab')2
fragments,
Fab' fragments and Fab fragments. Antibodies include single domain antibodies,
monovalent
antibodies, single chain antibodies, single chain variable fragment (scFv),
camelized
antibodies, affibodies, disulfide-linked Fvs (sdFv), anti-idiotypic antibodies
(anti-Id),
minibodies. Antibodies include monoclonal and polyclonal populations. In some
embodiments described herein, bispecific antibodies include two single chain
variable
fragment antibodies, which may be described as "scFv moieties" or simply -
scFvs" of the
bispecific antibody molecule, joined by a linker.
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[0092] An "antigen binding domain," "antigen binding region," or
"antigen binding site"
and other related terms used herein refer to a portion of an antigen binding
protein that
contains amino acid residues (or other moieties) that interact with an antigen
and contribute
to the antigen binding protein's specificity and affinity for the antigen. For
an antibody that
specifically binds to its antigen, this will include at least part of at least
one of its CDR
domains. Antigen binding domains from monoclonal antibodies and bispecific
antibodies are
provided herein.
[0093] The terms "specific binding", "specifically binds" or
"specifically binding" and
other related terms, as used herein in the context of an antibody or antigen
binding protein
(e.g., heterodimeric antibody) or antibody fragment, refer to non-covalent or
covalent
preferential binding to an antigen relative to other molecules or moieties
(e.g., an antibody
specifically binds to a particular antigen relative to other available
antigens). In one
embodiment, an antibody specifically binds to a target antigen if it binds to
the antigen with a
dissociation constant KD of 10-5 M or less, or 10' M or less, or 10-7 M or
less, or 10' M or
less, or 10 M or less, or 10-ll) M or less, or 10-'' M or less. Bispecific
antibodies that
specifically bind ROR1 and CD3 are described herein.
[0094] In one embodiment, binding specificity can be measure by
ELISA, radioimmune
assay (MA), electrochemiluminescence assays (ECL), immunoradiometric assay
(IRMA), or
enzyme immune assay (ETA).
[0095] In one embodiment, a dissociation constant (KD) can be
measured using a
BIACORE surface plasmon resonance (SPR) assay. Surface plasmon resonance
refers to an
optical phenomenon that allows for the analysis of real-time interactions by
detection of
alterations in protein concentrations within a biosensor matrix, for example
using the
BIACORE system (Biacore Life Sciences division of GE Healthcare, Piscataway,
NJ).
[0096] An "epitope" and related terms as used herein refers to a
portion of an antigen that
is bound by an antigen binding protein (e.g., by an antibody or an antigen
binding portion
thereof). An epitope can comprise portions of two or more antigens that are
bound by an
antigen binding protein. An epitope can comprise non-contiguous portions of an
antigen or
of two or more antigens (e.g., amino acid residues that are not contiguous in
an antigen's
primary sequence but that, in the context of the antigen's tertiary and
quaternary structure, are
near enough to each other to be bound by an antigen binding protein).
Generally, the variable
regions, particularly the CDRs, of an antibody interact with the epitope.
Bispecific antibodies
that bind an epitope of a ROR1 polypeptide and that bind an epitope of a CD3
polypeptide
are described herein.
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[0097] An "antibody fragment", "antibody portion", "antigen-
binding fragment of an
antibody", or "antigen-binding portion of an antibody" and other related terms
used herein
refer to a molecule other than an intact antibody that comprises a portion of
an intact antibody
that binds the antigen to which the intact antibody binds. Examples of
antibody fragments
include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(a131)2; Fd; and Fv
fragments, as well
as dAb; diabodies; linear antibodies; single-chain antibody molecules (e.g.
scFv);
polypeptides that contain at least a portion of an antibody that is sufficient
to confer specific
antigen binding to the polypeptide. Antigen binding portions of an antibody
may be produced
by recombinant DNA techniques or by enzymatic or chemical cleavage of intact
antibodies.
Antigen binding portions include, inter alia, Fab, Fab', F(a13)2, Fv, domain
antibodies (dAbs),
and complementarily determining region (CDR) fragments, chimeric antibodies,
diabodies,
triabodies, tetrabodies, and polypeptides that contain at least a portion of
an immunoglobulin
that is sufficient to confer antigen binding properties to the antibody
fragment.
[0098] The terms -Fab", -Fab fragment" and other related terms
refers to a monovalent
fragment comprising a variable light chain region (VL), constant light chain
region (CL),
variable heavy chain region (VII), and first constant region (Cm). A Fab is
capable of
binding an antigen. An F(ab')2 fragment is a bivalent fragment comprising two
Fab fragments
linked by a disulfide bridge at the hinge region. A F(Ab')2 has antigen
binding capability.
An Fd fragment comprises Vti and CHI regions. An Fv fragment comprises VL and
Vit
regions. An FAT can bind an antigen. A dAb fragment has a Vfi domain, a VL
domain, or an
antigen-binding fragment of a VH or VL domain (U.S. Patents 6,846,634 and
6,696,245; U.S.
published Application Nos. 2002/02512, 2004/0202995, 2004/0038291,
2004/0009507,
2003/0039958; and Ward et al., Nature 341:544-546, 1989).
[0099] A single-chain antibody (scFv) is an antibody in which a
VL and a VII region are
joined via a linker (e.g., a synthetic sequence of amino acid residues) to
form a continuous
protein chain. Preferably the linker is long enough to allow the protein chain
to fold back on
itself and form a monovalent antigen binding site (see, e.g., Bird et al.,
1988, Science
242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).
Single chain
antibodies that specifically bind ROR1 and single chain antibodies that
specifically bind CD3
are described herein.
1001001 Diabodies are bivalent antibodies comprising two polypeptide chains,
wherein
each polypeptide chain comprises VH and V. domains joined by a linker that is
too short to
allow for pairing between two domains on the same chain, thus allowing each
domain to pair
with a complementary domain on another polypeptide chain (see, e.g., Holliger
et al., 1993,
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Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al., 1994, Structure
2:1121-23). If the
two polypeptide chains of a diabody are identical, then a diabody resulting
from their pairing
will have two identical antigen binding sites. Polypeptide chains having
different sequences
can be used to make a diabody with two different antigen binding sites.
Similarly, tribodies
and tetrabodies are antibodies comprising three and four polypeptide chains,
respectively, and
forming three and four antigen binding sites, respectively, which can be the
same or different.
[00101] The term "human antibody- refers to antibodies that have one or more
variable
and constant regions derived from human immunoglobulin sequences. In one
embodiment,
all of the variable and constant domains are derived from human immunoglobulin
sequences
(e.g., a fully human antibody). These antibodies may be prepared in a variety
of ways,
examples of which are described below, including through recombinant
methodologies or
through immunization with an antigen of interest of a mouse that is
genetically modified to
express antibodies derived from human heavy and/or light chain-encoding genes.
1001021 A -humanized" antibody refers to an antibody having a sequence that
differs from
the sequence of an antibody derived from a non-human species by one or more
amino acid
substitutions, deletions, and/or additions, such that the humanized antibody
is less likely to
induce an immune response, and/or induces a less severe immune response, as
compared to
the non-human species antibody, when it is administered to a human subject. In
one
embodiment, certain amino acids in the framework and constant domains of the
heavy and/or
light chains of the non-human species antibody are mutated to produce the
humanized
antibody. In another embodiment, the constant domain(s) from a human antibody
are fused to
the variable domain(s) of a non-human species. In another embodiment, one or
more amino
acid residues in one or more CDR sequences of a non-human antibody are changed
to reduce
the likely immunogenicity of the non-human antibody when it is administered to
a human
subject, wherein the changed amino acid residues either are not critical for
immunospecific
binding of the antibody to its antigen, or the changes to the amino acid
sequence that are
made are conservative changes, such that the binding of the humanized antibody
to the
antigen is not significantly worse than the binding of the non-human antibody
to the antigen.
Examples of how to make humanized antibodies may be found in U.S. Pat. Nos.
6,054,297,
5,886,152 and 5,877,293.
1001031 The term -chimeric antibody" and related terms used herein refers to
an antibody
that contains one or more regions from a first antibody and one or more
regions from one or
more other antibodies. In one embodiment, one or more of the CDRs are derived
from a
human antibody. In another embodiment, all of the CDRs are derived from a
human
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antibody. In another embodiment, the CDRs from more than one human antibody
are mixed
and matched in a chimeric antibody. For instance, a chimeric antibody may
comprise a
CDR1 from the light chain of a first human antibody, a CDR2 and a CDR3 from
the light
chain of a second human antibody, and the CDRs from the heavy chain from a
third antibody.
In another example, the CDRs originate from different species such as human
and mouse, or
human and rabbit, or human and goat. One skilled in the art will appreciate
that other
combinations are possible.
[00104] Further, the framework regions may be derived from one of the same
antibodies,
from one or more different antibodies, such as a human antibody, or from a
humanized
antibody. In one example of a chimeric antibody, a portion of the heavy and/or
light chain is
identical with, homologous to, or derived from an antibody from a particular
species or
belonging to a particular antibody class or subclass, while the remainder of
the chain(s) is/are
identical with, homologous to, or derived from an antibody (-ies) from another
species or
belonging to another antibody class or subclass. Also included are fragments
of such
antibodies that exhibit the desired biological activity (i.e., the ability to
specifically bind a
target antigen).
[00105] As used herein, the term "variant" polypeptides and "variants" of
polypeptides
refers to a polypeptide comprising an amino acid sequence with one or more
amino acid
residues inserted into, deleted from and/or substituted into the amino acid
sequence relative to
a reference polypeptide sequence. Polypeptide variants include fusion
proteins. In the same
manner, a variant polynucleotide comprises a nucleotide sequence with one or
more
nucleotides inserted into, deleted from and/or substituted into the nucleotide
sequence relative
to another polynucleotide sequence. Polynucleotide variants include fusion
polynucleotides.
[00106] As used herein, the term "derivative" of a polypeptide is
a polypeptide (e.g.,
an antibody) that has been chemically modified, e.g., via conjugation to
another chemical
moiety such as, for example, polyethylene glycol, albumin (e.g., human serum
albumin),
phosphorylation, and glycosylation. Unless otherwise indicated, the term
"antibody"
includes, in addition to antibodies comprising two full-length heavy chains
and two full-
length light chains, derivatives, variants, fragments, and muteins thereof,
examples of which
are described below.
1001071 The term -Fc" or -Fc region" as used herein refers to the portion of
an antibody
heavy chain constant region beginning in or after the hinge region and ending
at the C-
terminus of the heavy chain. The Fc region comprises at least a portion of the
CH and CH3
regions, and may or may not include a portion of the hinge region. Two
polypeptide chains
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each carrying a half Fc region can dimerize to form an Fc region. An Fc region
can bind Fe
cell surface receptors and some proteins of the immune complement system. An
Fc region
exhibits effector function, including any one or any combination of two or
more activities
including complement-dependent cytotoxicity (CDC), antibody-dependent cell-
mediated
cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), opsonization
and/or cell
binding. An Fc region can bind an Fc receptor, including FcyRI (e.g., CD64),
FcyRII
CD32) and/or FcyRIII (e.g., CD16a).
[00108] The term -labeled antibody" or related terms as used herein refers to
antibodies
and their antigen binding portions thereof that are unlabeled or joined to a
detectable label or
moiety for detection, wherein the detectable label or moiety is radioactive,
colorimetric,
antigenic, enzymatic, a detectable bead (such as a magnetic or electrodense
(e.g., gold) bead),
biotin, streptavidin or protein A. A variety of labels can be employed,
including, but not
limited to, radionuclides, fluorescers, enzymes, enzyme substrates, enzyme
cofactors, enzyme
inhibitors and ligands (e.g., biotin, haptens). Any of the bispecific
antibodies described herein
can be unlabeled or can be joined to a detectable label or moiety.
[00109] The -percent identity" or "percent homology" and related terms used
herein refers
to a quantitative measurement of the similarity between two polypeptide or
between two
polynucleotide sequences. The percent identity between two polypeptide
sequences is a
function of the number of identical amino acids at aligned positions that are
shared between
the two polypeptide sequences, taking into account the number of gaps, and the
length of
each gap, which may need to be introduced to optimize alignment of the two
polypeptide
sequences. In a similar manner, the percent identity between two
polynucleotide sequences is
a function of the number of identical nucleotides at aligned positions that
are shared between
the two polynucleotide sequences, taking into account the number of gaps, and
the length of
each gap, which may need to be introduced to optimize alignment of the two
polynucleotide
sequences. A comparison of the sequences and determination of the percent
identity between
two polypeptide sequences, or between two polynucleotide sequences, may be
accomplished
using a mathematical algorithm. For example, the "percent identity" or
"percent homology"
of two polypeptide or two polynucleotide sequences may be determined by
comparing the
sequences using the GAP computer program (a part of the GCG Wisconsin Package,
version
10.3 (Accelrys, San Diego, Calif.)) using its default parameters. Expressions
such as
"comprises a sequence with at least X% identity to Y" with respect to a test
sequence mean
that, when aligned to sequence Y as described above, the test sequence
comprises residues
identical to at least X% of the residues of Y.
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[00110] In one embodiment, the amino acid sequence of a test antibody may be
similar but
not identical to any of the amino acid sequences of the polypeptides that make
up the
bispecific antibodies described herein. The similarities between the test
antibody and the
polypeptides can be at least 95%, or at or at least 96% identical, or at least
97% identical, or
at least 98% identical, or at least 99% identical, to any of the polypeptides
that make up the
bispecific antibodies described herein. In one embodiment, similar
polypeptides can contain
amino acid substitutions within a heavy and/or light chain. In one embodiment,
the amino
acid substitutions comprise one or more conservative amino acid substitutions.
A
"conservative amino acid substitution" is one in which an amino acid residue
is substituted by
another amino acid residue having a side chain (R group) with similar chemical
properties
(e.g., charge or hydrophobicity). In general, a conservative amino acid
substitution will not
substantially change the functional properties of a protein. In cases where
two or more amino
acid sequences differ from each other by conservative substitutions, the
percent sequence
identity or degree of similarity may be adjusted upwards to correct for the
conservative nature
of the substitution. Means for making this adjustment are well-known to those
of skill in the
art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein
incorporated by
reference in its entirety. Examples of groups of amino acids that have side
chains with
similar chemical properties include (1) aliphatic side chains: glycine,
alanine, valine, leucine
and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3)
amide-containing
side chains: asparagine and glutamine; (4) aromatic side chains:
phenylalanine, tyrosine, and
tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic
side chains:
aspartate and glutamate, and (7) sulfur-containing side chains are cysteine
and methionine.
[00111] Antibodies can be obtained from sources such as serum or plasma that
contain
immunoglobulins having varied antigenic specificity. If such antibodies are
subjected to
affinity purification, they can be enriched for a particular antigenic
specificity. Such enriched
preparations of antibodies usually are made of less than about 10% antibody
having specific
binding activity for the particular antigen. Subjecting these preparations to
several rounds of
affinity purification can increase the proportion of antibody having specific
binding activity
for the antigen. Antibodies prepared in this manner are often referred to as
"monospecific."
Monospecific antibody preparations can be made up of about 10%, 20%, 30%, 40%,
50%,
60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.9% antibody having specific
binding activity for the particular antigen. Antibodies can be produced using
recombinant
nucleic acid technology as described below.
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[00112] A "vector" and related terms used herein refers to a nucleic acid
molecule (e.g.,
DNA or RNA) which can be operably linked to foreign genetic material (e.g.,
nucleic acid
transgene). Vectors can be used as a vehicle to introduce foreign genetic
material into a cell
(e.g., host cell). Vectors can include at least one restriction endonuclease
recognition
sequence for insertion of the transgene into the vector. Vectors can include
at least one gene
sequence that confers antibiotic resistance or a selectable characteristic to
aid in selection of
host cells that harbor a vector-transgene construct. Vectors can be single-
stranded or double-
stranded nucleic acid molecules. Vectors can be linear or circular nucleic
acid molecules. A
donor nucleic acid used for gene editing methods employing zinc finger
nuclease, TALEN or
CRISPR/Cas can be a type of a vector. One type of vector is a "plasmid," which
refers to a
linear or circular double stranded extrachromosomal DNA molecule which can be
linked to a
transgene, and is capable of replicating in a host cell, and transcribing
and/or translating the
transgene. A viral vector typically contains viral RNA or DNA backbone
sequences which
can be linked to the transgene. The viral backbone sequences can be modified
to disable
infection but retain insertion of the viral backbone and the co-linked
transgene into a host cell
genome. Examples of viral vectors include retroviral, lentiviral, adenoviral,
adeno-associated,
baculoviral, papovaviral, vaccinia viral, herpes simplex viral and Epstein
Barr viral vectors.
Certain vectors are capable of autonomous replication in a host cell into
which they are
introduced (e.g., bacterial vectors comprising a bacterial origin of
replication and episomal
mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are
integrated
into the genome of a host cell upon introduction into the host cell, and
thereby are replicated
along with the host genome.
[00113] An "expression vector is a type of vector that can contain one or more
regulatory
sequences, such as inducible and/or constitutive promoters and enhancers.
Expression
vectors can include ribosomal binding sites and/or polyadenylation sites.
Expression vectors
can include one or more origin of replication sequence. Regulatory sequences
direct
transcription, or transcription and translation, of a transgene linked to the
expression vector
which is transduced into a host cell. The regulatory sequence(s) can control
the level, timing
and/or location of expression of the transgene. The regulatory sequence can,
for example,
exert its effects directly on the transgene, or through the action of one or
more other
molecules (e.g., polypeptides that bind to the regulatory sequence and/or the
nucleic acid).
Regulatory sequences can be part of a vector. Further examples of regulatory
sequences are
described in, for example, Goeddel, 1990, Gene Expression Technology: Methods
in
Enzymology 185, Academic Press, San Diego, Calif and Baron et al., 1995,
Nucleic Acids
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Res. 23:3605-3606. An expression vector can comprise nucleic acids that encode
at least a
portion of any of the bispecific antibodies described herein.
[00114] A transgene is -operably linked" to a vector when there is linkage
between the
transgene and the vector to permit functioning or expression of the transgene
sequences
contained in the vector. In one embodiment, a transgene is "operably linked"
to a regulatory
sequence when the regulatory sequence affects the expression (e.g., the level,
timing, or
location of expression) of the transgene.
[00115] The terms "transfected" or "transformed" or "transduced" or other
related terms
used herein refer to a process by which exogenous nucleic acid (e.g.,
transgene) is transferred
or introduced into a host cell. A "transfected" or "transformed" or
"transduced" host cell is
one which has been transfected, transformed or transduced with exogenous
nucleic acid
(transgene). The host cell includes the primary subject cell and its progeny.
Exogenous
nucleic acids encoding at least a portion of any of the bispecific antibodies
described herein
can be introduced into a host cell. Expression vectors comprising at least a
portion of any of
the bispecific antibodies described herein can he introduced into a host cell,
and the host cell
can express polypeptides comprising at least a portion of the bispecific
antibodies.
[00116] The terms "host cell" or "or a population of host cells" or related
terms as used
herein refer to a cell (or a population thereof) into which foreign (exogenous
or transgene)
nucleic acids have been introduced. The foreign nucleic acids can include an
expression
vector operably linked to a transgene, and the host cell can be used to
express the nucleic acid
and/or polypeptide encoded by the foreign nucleic acid (transgene). A host
cell (or a
population thereof) can be a cultured cell or can be extracted from a subject.
The host cell (or
a population thereof) includes the primary subject cell and its progeny
without any regard for
the number of passages. Progeny cells may or may not harbor identical genetic
material
compared to the parent cell. Host cells encompass progeny cells. In one
embodiment, a host
cell describes any cell (including its progeny) that has been modified,
transfected, transduced,
transformed, and/or manipulated in any way to express an antibody, as
disclosed herein. In
one example, the host cell (or population thereof) can be introduced with an
expression
vector operably linked to a nucleic acid encoding the desired antibody, or an
antigen binding
portion thereof, described herein. Host cells and populations thereof can
harbor an expression
vector that is stably integrated into the host's genome or can harbor an
extrachromosomal
expression vector. In one embodiment, host cells and populations thereof can
harbor an
extrachromosomal vector that is present after several cell divisions or is
present transiently
and is lost after several cell divisions.
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[00117] Transgenic host cells can be prepared using non-viral methods,
including well-
known designer nucleases including zinc finger nucleases, TALENS or
CRISPR/Cas. A
transgene can be introduced into a host cell's genome using genome editing
technologies
such as zinc finger nuclease. A zinc finger nuclease includes a pair of
chimeric proteins each
containing a non-specific endonuclease domain of a restriction endonuclease
(e.g., FokI )
fused to a DNA-binding domain from an engineered zinc finger motif The DNA-
binding
domain can be engineered to bind a specific sequence in the host- s genome and
the
endonuclease domain makes a double-stranded cut. The donor DNA carries the
transgene,
for example any of the nucleic acids encoding a CAR or DAR construct described
herein, and
flanking sequences that are homologous to the regions on either side of the
intended insertion
site in the host cell's genome. The host cell's DNA repair machinery enables
precise
insertion of the transgene by homologous DNA repair. Transgenic mammalian host
cells
have been prepared using zinc finger nucleases (U.S. patent Nos. 9,597,357,
9,616,090,
9A416,074 and 8.)45,863). A transgenic host cell can be prepared using TALEN
(Transcription Activator-Like Effector Nucleases) which are similar to zinc
finger nucleases
in that they include a non-specific endonuclease domain fused to a DNA-binding
domain
which can deliver precise transgene insertion. Like zinc finger nucleases,
TALEN also
introduce a double-stranded cut into the host's DNA. Transgenic host cells can
be prepared
using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats).
CRISPR
employs a Cas endonuclease coupled to a guide RNA for target specific donor
DNA
integration. The guide RNA includes a conserved multi-nucleotide containing
protospacer
adjacent motif (PAM) sequence upstream of the gRNA-binding region in the
target DNA and
hybridizes to the host cell target site where the Cas endonuclease cleaves the
double-stranded
target DNA. The guide RNA can be designed to hybridize to a specific target
site. Similar to
zinc finger nuclease and TALEN, the CRISPR/Cas system can be used to introduce
site
specific insertion of donor DNA having flanking sequences that have homology
to the
insertion site. Examples of CRISPRJCas systems used to modify genomes are
described for
example in U.S. Pat. Nos. 8,697,359, 10,000,772, 9,790,490, and U. S. Patent
Application
Publication No. US 2018/0346927. In one embodiment, transgenic host cells can
be prepared
using zinc finger nuclease, TALEN or CRISPR/Cas system, and the host target
site can be a
TRAC gene (T Cell Receptor Alpha Constant). The donor DNA can include for
example any
of the nucleic acids encoding a CAR or DAR construct described herein.
Electroporation,
nucleofection or lipofection can be used to co-deliver into the host cell the
donor DNA with
the zinc finger nuclease, TALEN or CRISPR/Cas system.
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1001181 A host cell can be a prokaryote, for example, E. coil, or it can be a
eukaryote, for
example, a single-celled eukaryote (e.g., a yeast or other fungus), a plant
cell (e.g., a tobacco
or tomato plant cell), an mammalian cell (e.g., a human cell, a monkey cell, a
hamster cell, a
rat cell, a mouse cell, or an insect cell) or a hybridoma. In one embodiment,
a host cell can be
introduced with an expression vector operably linked to a nucleic acid
encoding a desired
antibody thereby generating a transfected/transformed host cell which is
cultured under
conditions suitable for expression of the antibody by the
transfected/transformed host cell,
and optionally recovering the antibody from the transfected/transformed host
cells (e.g.,
recovery from host cell lysate) or recovery from the culture medium. In one
embodiment,
host cells comprise non-human cells including CHO, BHK, NSO, SP2/0, and YB2/0.
In one
embodiment, host cells comprise human cells including HEK293, HT-1080, Huh-7
and
PER.C6. Examples of host cells include the COS-7 line of monkey kidney cells
(ATCC CRL
1651) (see Gluzman et al., 1981, Cell 23: 175), L cells, C127 cells, 3T3 cells
(ATCC CCL
163), Chinese hamster ovary (CHO) cells or their derivatives such as Veggie
CHO and
related cell lines which grow in serum- free media (see Rasmussen et al.,
1998,
Cytotechnology 28:31) or CHO strain DX-B 11, which is deficient in DHFR (see
Urlaub et
al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20), HeLa cells, BHK (ATCC CRL
10) cell
lines, the CV1/EBNA cell line derived from the African green monkey kidney
cell line CV1
(ATCC CCL 70) (see McMahan et al., 1991, EMBO J. 10:2821), human embryonic
kidney
cells such as 293, 293 EBNA or MSR 293, human epidermal A431 cells, human Colo
205
cells, other transformed primate cell lines, normal diploid cells, cell
strains derived from in
vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat
cells. In one
embodiment, host cells include lymphoid cells such as YO, NSO or Sp20. In one
embodiment, a host cell is a mammalian host cell, but is not a human host
cell. Typically, a
host cell is a cultured cell that can be transformed or transfected with a
polypeptide-encoding
nucleic acid, which can then be expressed in the host cell. The phrase -
transgenic host cell"
or "recombinant host cell" can be used to denote a host cell that has been
transformed or
transfected with a nucleic acid to be expressed. A host cell also can be a
cell that comprises
the nucleic acid but does not express it at a desired level unless a
regulatory sequence is
introduced into the host cell such that it becomes operably linked with the
nucleic acid. It is
understood that the term host cell refers not only to the particular subject
cell but also to the
progeny or potential progeny of such a cell. Because certain modifications may
occur in
succeeding generations due to, e.g., mutation or environmental influence, such
progeny may
not, in fact, be identical to the parent cell, but are still included within
the scope of the term as
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used herein. A host cell, or a population of host cells, harboring a vector
(e.g., an expression
vector) operably linked to at least one nucleic acid encoding one or more
bispecific
antibodies are described herein.
[00119] Polypeptides of the present disclosure (e.g., antibodies and antigen
binding
proteins) can be produced using any method known in the art. In one example,
the
polypeptides are produced by recombinant nucleic acid methods by inserting a
nucleic acid
sequence (e.g., DNA) encoding the polypeptide into a recombinant expression
vector which
is introduced into a host cell and expressed by the host cell under conditions
promoting
expression.
[00120] General techniques for recombinant nucleic acid manipulations are
described for
example in Sambrook et al., in Molecular Cloning: A Laboratory Manual, Vols. 1-
3, Cold
Spring Harbor Laboratory Press, 2 ed., 1989, or F. Ausubel et al., in Current
Protocols in
Molecular Biology (Green Publishing and Wiley-Interscience: New York, 1987)
and periodic
updates, herein incorporated by reference in their entireties. The nucleic
acid (e.g., DNA)
encoding the polypeptide is operably linked to an expression vector carrying
one or more
suitable transcriptional or translational regulatory elements derived from
mammalian, viral,
or insect genes. Such regulatory elements include a transcriptional promoter,
an optional
operator sequence to control transcription, a sequence encoding suitable mRNA
ribosomal
binding sites, and sequences that control the termination of transcription and
translation. The
expression vector can include an origin or replication that confers
replication capabilities in
the host cell. The expression vector can include a gene that confers selection
to facilitate
recognition of transgenic host cells (e.g., transformants).
[00121] The recombinant DNA can also encode any type of protein tag sequence
that may
be useful for purifying the protein. Examples of protein tags include but are
not limited to a
histidine tag, a FLAG tag, a myc tag, an HA tag, or a GST tag. Appropriate
cloning and
expression vectors for use with bacterial, fungal, yeast, and mammalian
cellular hosts can be
found in Cloning Vectors: A Laboratory Manual, (Elsevier, N.Y., 1985).
[00122] The expression vector construct can be introduced into the host cell
using a
method appropriate for the host cell. A variety of methods for introducing
nucleic acids into
host cells are known in the art, including, but not limited to,
electroporation; transfection
employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-
dextran, or other
substances; viral transfection; non-viral transfection; microprojectile
bombardment;
lipofection; and infection (e.g., where the vector is an infectious agent).
Suitable host cells
include prokaryotes, yeast, mammalian cells, or bacterial cells.
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[00123] Suitable bacteria include gram negative or gram positive organisms,
for
example, E. coil or Bacillus spp. Yeast, preferably from the Saccharom.yces
species, such
as S. cerevisiae, may also be used for production of polypeptides. Various
mammalian or
insect cell culture systems can also be employed to express recombinant
proteins.
Baculovirus systems for production of heterologous proteins in insect cells
are reviewed by
Luckow and Summers, (Bio/Technology, 6:47, 1988). Examples of suitable
mammalian host
cell lines include endothelial cells, COS-7 monkey kidney cells, CV-1, L
cells, C127, 3T3,
Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 2931,
and BHK
cell lines. Purified polypeptides are prepared by culturing suitable
host/vector systems to
express the recombinant proteins. For many applications, the small size of
many of the
polypeptides disclosed herein would make expression in E. coil as the
preferred method for
expression. The protein is then purified from culture media or cell extracts.
Any of the
bispecific antibodies disclosed herein can be expressed by transgenic host
cells.
[00124] Antibodies and antigen binding proteins disclosed herein can also be
produced
using cell-translation systems. For such purposes the nucleic acids encoding
the polypeptide
must be modified to allow in vitro transcription to produce mRNA and to allow
cell-free
translation of the mRNA in the particular cell-free system being utilized
(eukaryotic such as a
mammalian or yeast cell-free translation system or prokaryotic such as a
bacterial cell-free
translation system.
[00125] Nucleic acids encoding any of the various polypeptides disclosed
herein may be
synthesized chemically. Codon usage may be selected so as to improve
expression in a cell.
Such codon usage will depend on the cell type selected. Specialized codon
usage patterns
have been developed for E. coli and other bacteria, as well as mammalian
cells, plant cells,
yeast cells and insect cells. See for example: Mayfield et al., Proc. Natl.
Acad. Sci.
USA. 2003 100(2):438-42; Sinclair et al. Protein Expr. Puff. 2002 (1):96-105,
Connell N
D. Curr. Opin. Biotechnol. 2001 12(5):446-9; Makrides et al. Microbiol. Rev.
1996
60(3):512-38; and Sharp et al. Yeast. 1991 7(7):657-78.
[00126] Antibodies and antigen binding proteins described herein can also be
produced by
chemical synthesis (e.g., by the methods described in Solid Phase Peptide
Synthesis, 2nd ed.,
1984, The Pierce Chemical Co., Rockford, Ill.). Modifications to the protein
can also be
produced by chemical synthesis.
[00127] Antibodies and antigen binding proteins described herein
can be purified by
isolation/purification methods for proteins generally known in the field of
protein chemistry.
Non-limiting examples include extraction, recrystallization, salting out
(e.g., with ammonium
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sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration,
adsorption chromatography,
ion exchange chromatography, hydrophobic chromatography, normal phase
chromatography,
reversed-phase chromatography, gel filtration, gel permeation chromatography,
affinity
chromatography, electrophoresis, countercurrent distribution or any
combinations of these.
After purification, polypeptides may be exchanged into different buffers
and/or concentrated
by any of a variety of methods known to the art, including, but not limited
to, filtration and
dialysis.
[00128] The purified antibodies and antigen binding proteins described herein
are
preferably at least 65% pure, at least 75 % pure, at least 85% pure, more
preferably at least
95% pure, and most preferably at least 98% pure. Regardless of the exact
numerical value of
the purity, the polypeptide is sufficiently pure for use as a pharmaceutical
product. Any of
the bispecific antibodies described herein can be expressed by transgenic host
cells and then
purified to about 65-98% purity or high level of purity using any art-known
method.
[00129] In certain embodiments, the antibodies and antigen binding proteins
herein can
further comprise post-translational modifications. Exemplary post-
translational protein
modifications include phosphorylation, acetylation, methylation, ADP-
ribosylation,
ubiquitination, glycosylation, carbonylation, sumoylation, biotinylation or
addition of a
polypeptide side chain or of a hydrophobic group. As a result, the modified
polypeptides may
contain non-amino acid elements, such as lipids, poly- or mono-saccharide, and
phosphates.
A preferred form of glycosylation is sialylation, which conjugates one or more
sialic acid
moieties to the polypeptide. Sialic acid moieties improve solubility and serum
half-life while
also reducing the possible immunogenicity of the protein. See Raju et al.
Biochemistry. 2001
31; 40(30):8868-76.
[00130] In one embodiment, the antibodies and antigen binding proteins
described herein
can be modified to become soluble polypeptides which comprises linking the
Antibodies and
antigen binding proteins to non-proteinaceous polymers. In one embodiment, the
non-
proteinaceous polymer comprises polyethylene glycol ("PEG--), polypropylene
glycol, or
polyoxyalkylenes, in the manner as set forth in U.S. Pat. Nos. 4,640,835;
4,496,689;
4,301,144; 4,670,417; 4,791,192 or 4,179,337.
1001311 The present disclosure provides therapeutic compositions comprising
any of the
bispecific antibodies described herein in an admixture with a pharmaceutically-
acceptable
excipient. An excipient encompasses carriers, stabilizers, and excipients.
Examples of
pharmaceutically acceptable excipients includes for example inert diluents or
fillers (e.g.,
sucrose and sorbitol), lubricating agents, glidants, and anti-adhesives (e.g.,
magnesium
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stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils,
or talc). Additional
examples include buffering agents, stabilizing agents, preservatives, non-
ionic detergents,
anti-oxidants, and isotonifiers.
[00132] Therapeutic compositions and methods for preparing them are well known
in the
art and are found, for example, in "Remington: The Science and Practice of
Pharmacy" (20th
ed., ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins,
Philadelphia, Pa.).
Therapeutic compositions can be formulated for parenteral administration may,
and can for
example, contain excipients, sterile water, saline, polyalkylene glycols such
as polyethylene
glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible,
biodegradable
lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-
polyoxypropylene
copolymers may be used to control the release of the antibody (or antigen
binding protein
thereof) described herein. Nanoparticulate formulations (e.g., biodegradable
nanoparticles,
solid lipid nanoparticles, liposomes) may be used to control the
biodistribution of the
antibody (or antigen binding protein thereof). Other potentially useful
parenteral delivery
systems include ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable
infusion systems, and liposomes. The concentration of the antibody (or antigen
binding
protein thereof) in the formulation varies depending upon a number of factors,
including the
dosage of the drug to be administered, and the route of administration.
[00133] Any of the bispecific antibodies (or antigen binding protein thereof)
may be
optionally administered as a pharmaceutically acceptable salt. such as non-
toxic acid addition
salts or metal complexes that are commonly used in the pharmaceutical
industry. Examples of
acid addition salts include organic acids such as acetic, lactic, pamoic,
maleic, citric, malic,
ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric,
methanesulfonic,
toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as
tannic acid,
carboxymethyl cellulose, or the like; and inorganic acid such as hydrochloric
acid,
hydrobromic acid, sulfuric acid phosphoric acid, or the like. Metal complexes
include zinc,
iron, and the like. In one example, the antibody (or antigen binding protein
thereof) is
formulated in the presence of sodium acetate to increase thermal stability.
[00134] Any of the bispecific antibodies (or antigen binding protein thereof)
may be
formulated for oral use include tablets containing the active ingredient(s) in
a mixture with
non-toxic pharmaceutically acceptable excipients. Formulations for oral use
may also be
provided as chewable tablets, or as hard gelatin capsules wherein the active
ingredient is
mixed with an inert solid diluent, or as soft gelatin capsules wherein the
active ingredient is
mixed with water or an oil medium.
31
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[00135] The term "subject" as used herein refers to human and non-human
animals,
including vertebrates, mammals and non-mammals. In one embodiment, the subject
can be
human, non-human primates, simian, ape, murine (e.g., mice and rats), bovine,
porcine,
equine, canine, feline, caprine, lupine, ranine or piscine.
[00136] The term "administering-, "administered- and grammatical variants
refers to the
physical introduction of an agent to a subject, using any of the various
methods and delivery
systems known to those skilled in the art. Exemplary routes of administration
for the
formulations disclosed herein include intravenous, intramuscular,
subcutaneous,
intraperitoneal, spinal or other parenteral routes of administration, for
example by injection or
infusion. The phrase "parenteral administration" as used herein means modes of
administration other than enteral and topical administration, usually by
injection, and
includes, without limitation, intravenous, intramuscular, intraarterial,
intrathecal,
intralymphatic, intralesional, intracapsular, intraorbital, intracardiac,
intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular,
subcapsular,
subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as
well as in vivo
electroporation. In one embodiment, the formulation is administered via a non-
parenteral
route, e.g., orally. Other non-parenteral routes include a topical, epidermal
or mucosal route
of administration, for example, intranasally, vaginally, rectally,
sublingually or topically.
Administering can also be performed, for example, once, a plurality of times,
and/or over one
or more extended periods. Any of the bispecitic antibodies described herein
(or antigen
binding protein thereof) can be administered to a subject using art-known
methods and
delivery routes.
[00137] The terms "effective amount", "therapeutically effective amount" or
"effective
dose" or related terms may be used interchangeably and refer to an amount of
antibody or an
antigen binding protein (e.g., bispecific antibodies) that when administered
to a subject, is
sufficient to effect a measurable improvement or prevention of a disease or
disorder
associated with tumor or cancer antigen expression. Therapeutically effective
amounts of
antibodies provided herein, when used alone or in combination, will vary
depending upon the
relative activity of the antibodies and combinations (e.g. , in inhibiting
cell growth) and
depending upon the subject and disease condition being treated, the weight and
age and sex
of the subject, the severity of the disease condition in the subject, the
manner of
administration and the like, which can readily be determined by one of
ordinary skill in the
art.
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[00138] In one embodiment, a therapeutically effective amount will depend on
certain
aspects of the subject to be treated and the disorder to be treated and may be
ascertained by
one skilled in the art using known techniques. In general, the polypeptide is
administered at
about 0.01 g/kg to about 50 mg/kg per day, preferably 0.01 mg/kg to about 30
mg/kg per day,
most preferably 0.1 mg/kg to about 20 mg/kg per day. The polypeptide may be
administered
daily (e.g., once, twice, three times, or four times daily) or preferably less
frequently (e.g.,
weekly, every two weeks, every three weeks, monthly, or quarterly). In
addition, as is known
in the art, adjustments for age as well as the body weight, general health,
sex, diet, time of
administration, drug interaction, and the severity of the disease may be
necessary.
[00139] The present disclosure provides methods for treating a subject having
a disease
associated with expression of one or more tumor-associated antigens. The
disease comprises
cancer or tumor cells expressing the tumor-associated antigens, such as for
example CD38
and/or CD3 antigen. In one embodiment, the cancer or tumor includes cancer of
the prostate,
breast, ovary, head and neck, bladder, skin, colorectal, anus, rectum,
pancreas, lung
(including non-small cell lung and small cell lung cancers), leiomyoma, brain,
glioma,
glioblastoma, esophagus, liver, kidney, stomach, colon, cervix, uterus,
endometrium, vulva,
larynx, vagina, bone, nasal cavity, paranasal sinus, nasopharynx, oral cavity,
oropharynx,
larynx, hypolarynx, salivary glands, ureter, urethra, penis and testis.
[00140] In one embodiment, the cancer comprises hematological cancers,
including
leukemias, lymphomas, myelomas and B cell lymphomas. Hematologic cancers
include
multiple myeloma (MM), non-Hodgkin's lymphoma (NHL) including Burkitt's
lymphoma
(BL), B chronic lymphocytic leukemia (B-CLL), systemic lupus erythematosus
(SLE), B and
T acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic
lymphocytic
leukemia (CLL), diffuse large B cell lymphoma, chronic myelogenous leukemia
(CML),
hairy cell leukemia (HCL), follicular lymphoma, Waldenstrom's
Macroglobulinemia, mantle
cell lymphoma, Hodgkin's Lymphoma (HL), plasma cell myeloma, precursor B cell
lymphoblastic leukemia/lymphoma, plasmacytoma, giant cell myeloma, plasma cell
myeloma, heavy-chain myeloma, light chain or Bence-Jones myeloma,
lyinphornatoid
granitioinatosis, post-transplant lyinphoproliferative disorder, an
imritunorgulatory disorder,
dietimatoid ;arthritis, myasthenia. gra.vis, idiopathic .thrombocytopenia
purpura, anti-
phospholipi d syndrome. Chagas' disease, Grave's disease, 'Wegener's
grartulomatosis, pol.!yr-
arter4is nodosa, Sjogren's syndrome, pernphigus vulgaris, scleroderrna,
multiple sclerosis,
anti-phospholipid syndrome, .ANCA associated vaseulitis. Goodpasture's
disease, Kawasaki
disease, autoimmurie heinelytie anemia and rapidly progressive
glomerulonephritis, heavy-
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chain disease, primary or immunocyte-associated arayloidosis, and monoclonal
garnitiopailly
of undetermined significance.
Recombinant Oncolytic Viruses encoding anti-RORlianti-CD3 Bispecific
Antibodies
[00141] The present disclosure provides, inter alia, oncolytic
viruses that express
bispecific antibodies that bind ROR1 and CD3, cells infected with such
viruses, and methods
of treating cancer using the viruses expressing bispecific antibodies. Also
provided is virus-
free conditioned culture media (VFCM) produced by the infected cells and
methods of
producing pharmaceutical formulations using VFCMs.
[00142] Oncolytic viruses provide a targeted approach to cancer therapy, as
they
selectively replicate in and lyse tumor cells. Various types of oncolytic
viruses are known in
the art, including include parvoviruses, myxoma virus, Reovirus, Newcastle
disease virus
(NDV), Seneca Valley virus (SVV), poliovirus (PV), measles virus (MV),
vaccinia virus
(VACV), adenovirus, vesicular stomatitis virus (VSV), and herpes simplex virus
(HSV).
These viruses replicate in tumor cells and cause cell lysis and/or induce an
immune response
to the tumor cells they infect. This disclosure provides recombinant oncolytic
viruses that
include a heterologous gene construct that encodes an anti-ROR1/anti-CD3
bispecific
antibody (aROR1/aCD3 BspAb) such as any disclosed herein. The construct can
include a
promoter active in a mammalian cell operably linked to the aROR1/aCD3 BspAb-
encoding
sequence and the construct can be inserted into the genome of the oncolytic
virus.
[00143] In various embodiments, an oncolytic virus modified for
expression of an
aROR1/aCD3 BspAb can be a herpes simplex virus (Human alphaherpesvirus; HSV),
such
as an HSV-1. HSV-2, or a recombinant HSV having sequences of both HSV-1 or HSV-
2. For
example, a laboratory strain or clinical isolate of an HSV-1 or HSV-2 strain
can be used.
Multiple isolated and modified strains of HSV-1 and HSV-2 are known in the art
and can be
considered for use in the compositions and methods disclosed herein,
including, as
nonlimiting examples, HSV-1 strain A44. HSV-1 strain Angelotti, HSV-1 strain
CL101,
HSV-1 strain CVG-2, HSV-1 strain H129, HSV-1 strain HFEM, HSV-1 strain HZT,
HSV-1
strain JS1, HSV-1 strain MGH10, HSV-1 strain MP, HSV-1 strain Patton, HSV-1
strain R15,
HSV-1 strain R19, HSV-1 strain RH2, HSV-1 strain SC16, HSV-1 strain KOS, HSV-1
strain
F, and HSV-1 strain 17, HSV-2 strain 186, HSV-2 strain 333, HSV-2 strain
B4327UR, HSV-
2 strain G, HSV-2 strain G, HSV-2 strain HG52, HSV-2 strain SA8, HSV-2 strain
SD90,
HSV-2 strain SN03, HSV-2 strain SS01, and HSV-2 strain ST04. Also considered
for use in
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the compositions and methods provided herein are derivates or mutants of these
strains or
others that may be known in the art or isolated.
[00144] Derivatives of viral strains include, without
limitation, viruses that may have
one or more endogenous genes that is mutated, including one or more endogenous
genes that
is partially or entirely deleted, may have a transgene (heterologous gene)
inserted into the
viral genome (including but not limited to one or more selectable markers,
negative selectable
markers ("suicide genes-), and/or detectable markers (e.g., a gene encoding a
fluorescent
protein or a gene encoding an enzyme that produces a detectable product)),
and/or may have
one or more modifications such as but not limited to restriction sites,
recombination sites or
"landing pads", exogenous promoters, etc. A derivative may have other
modifications such as
but not limited to deletion or mutation of non-gene sequences, such as for
example gene
regulatory regions such as promoters or non-coding sequences such as but not
limited to
direct or inverted repeat sequences. Derivatives of viral strains may be
viruses that
alternatively or in addition to other modifications include one or more
transgenes supporting
or regulating viral growth or viability, one or more genes affecting host cell
functions, or one
or more transgenes encoding therapeutic proteins, as nonlimiting examples.
1001451 In some nonlimiting embodiments the HSV is an HSV-1 such
as HSV-1 strain
17, HSV-1 strain KOS, or HSV-1 strain F, or a derivative of any of HSV-1
strain 17, HSV-1
strain KOS, or HSV-1 strain F. For example, a strain used for the introduction
of an ScFA,r-Fc-
TGF[3trap construct can be HSV-1 strain 17 mutant 1716. HSV-1 strain F mutant
R3616
(Chou & Roizman (1992) Proc. Natl. Acad Sc. 89: 3266-3270), HSV-1 strain F
mutant
G207 (Toda etal. (1995) Human Gene Therapy 9:2177-2185), HSV-1 strain F mutant
G474
(Todo etal. (2001) Proc Natl Acad Sci USA 98:6396-6401), HSV-1 mutant NV1020
(Geevarghese et at. (2010) Human Gene Therapy 21:1119-28), RE6 (Thompson et
at. (1983)
Virology 131:171-179), KeM34.5 (Manservigi et al. (2010) The Open Virology
Journal
4:123-156), M032 (Campadelli-Fiume et al. (2011) Rev Med. Virol 21:213-226),
Baco (Fu et
al. (2011) Int. J. Cancer 129:1503-10), M032 or C134 (Cassady etal. (2010) The
Open
Virology Journal 4:103-108), or Talimogenelaherparepvec ("TVec", formerly
OncoVexk;
Liu et al. (2003) Gene Therapy 10:292-303), or a further derivative or mutant
of any of these.
1001461 Mutation of endogenous viral genes can include mutation
or deletion of genes
that affect replication or propagation of the virus in non-cancerous cells or
the ability of
viruses to avoid host defenses. For example, an HSV that includes an
aROR1/aCD3 BspAb
can be deleted in any of the ICP34.5-encoding gene, the ICP6-encoding gene,
the ICP0-
encoding gene, the vhs-encoding gene, or the ICP27-encoding gene. Mutants that
do not
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produce a functional protein encoded by a gene or genes (where the gene is
multicopy) are
refen-ed to herein as having a functionally deleted gene. Functional deletion
of one or more of
the ICP34.5-encoding gene, the ICP6-encoding gene, the 'CPO-encoding gene, and
the vhs-
encoding gene can result in an HSV impaired in replication in noncancerous
cells.
[00147] The ICP34.5-encoding gene RL1 is located in the long
repeat (RL) of the HSV-
1 genome and is present in two copies. In some embodiments one or both copies
of the
ICP34.5-encoding genes is mutated or is partially or entirely deleted such
that no functional
protein is made. In preferred embodiments, an oncolytic HSV that includes a
transgene
encoding an ScFv-Fc-TGFf3trap protein and, optionally, an IL12 gene, is
functionally deleted
for the ICP34.5-encoding gene responsible for neurovirulence (Chou etal.
(1990) Science
250:1262-1266), e.g., both copies of the ICP34.5-encoding gene of the HSV
viral genome are
inactivated. For example the oncolytic HSV used for introduction of an ScFv-Fc-
TGFI3trap
construct can be a mutant of HSV-1 strain 17 and may be HSV1716 (Brown etal.
(1994)
Journal of General Virology 75: 2367-2377; MacLean et al. (1991) Journal of
General
Virology 72:631-639) or a mutant or derivative thereof, or may be SeprehvecTM
or a
derivative or mutant thereof HSV1716 and SeprehvecTm both have deletions in
both copies
of the ICP34.5-encoding gene such that they do not produce a functional gene
product, but
each otherwise has a genome substantially similar to that of HSV strain 17,
which has been
completely sequenced (Pfaff et al. (2016) J Gen Virol 97:2732-2741;
ncbi.nlm.nih.gov/genome, Accession number JN555585).
[00148] Recombinant HSVs as provided herein can have one or more
transgenes
inserted into the ICP34.5 locus, the ICP6 locus, the 'CPO locus, or the vhs
locus. In some
preferred embodiments a recombinant oncolytic HSV as provided herein can have
an
ctROR1/aCD3 BspAb gene inserted into a deleted ICP34.5-encoding gene locus. In
some
preferred embodiments a recombinant oncolytic HSV as provided herein is
functionally
deleted for ICP34.5 (i.e., is ICP34.5 null) and has an aROR1/aCD3 BspAb gene
inserted into
both copies of the ICP34.5-encoding gene locus.
[00149] The recombinant oncolytic viruses provided herein, which
are able to infect
many tumor cell types, include expression constructs that encode novel
bispecific antibodies
that bind ROR1, a protein expressed on many tumor cells, and CD3, expressed on
T cells,
where the bispecific antibodies can be expressed and secreted by cells
infected by the
recombinant viruses that encode them. The RORI scFv moiety of the aROR1/aCD3
BspAb
specifically binds an immune checkpoint protein and the CD3 scFv moiety binds
T cells,
bringing T cells into proximity with target tumor cells to enhance killing of
tumor cells.
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[00150] Exemplary constructs encoding the ctROR1/aCD3 BspAbs described herein
use
scFvs derived from ROR1 monoclonal antibody oil, having a variable heavy chain
region of
SEQ ID NO:1 or sequences having at least 95% identity thereto, with heavy
chain variable
region CDRs of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, and a variable light
chain
region of SEQ ID NO:5 or sequences having at least 95% identity thereto, with
light chain
variable region CDRs of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
[00151] Additional exemplary constructs encoding the aROR1/aCD3 BspAbs
described
herein use scFvs derived from ROR1 monoclonal antibody s10, having a variable
heavy
chain region of SEQ ID NO:10 or sequences having at least 95% identity
thereto, with heavy
chain variable region CDRs of SEQ ID NO: ii, SEQ ID NO:12, and SEQ ID NO:13,
and a
variable light chain region of SEQ ID NO:14 or sequences having at least 95%
identity
thereto, with light chain variable region CDRs of SEQ ID NO:15, SEQ ID NO:16,
and SEQ
ID NO:17.
[00152] Further exemplary constructs encoding the aROR1/aCD3 BspAbs described
herein use scFvs derived from ROR1 monoclonal antibody jlvl 011, having a
variable heavy
chain region of SEQ ID NO:19 or sequences having at least 95% identity
thereto, with heavy
chain variable region CDRs of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22,
and a
variable light chain region of SEQ ID NO:23 or sequences having at least 95%
identity
thereto, with light chain variable region CDRs of SEQ ID NO:24, SEQ ID NO:25,
and SEQ
ID NO:26.
[00153] In particular examples an ctROR1/aCD3 BspAbs can have the sequence of
SEQ
ID NO:36, SEQ ID NO:38, or SEQ ID NO:40, or can have an amino acid sequence
having at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to any of
SEQ ID NO:36,
SEQ ID NO:38, or SEQ ID NO:40.
[00154] The aROR1/aCD3 BspAbs can have the format: heavy chain
variable region -
linker-light chain variable region or light chain variable region -linker-
heavy chain variable
region. The anti-ROR1 scFv of the aROR1/aCD3 BspAbs can be N-terminal to the
anti-CD3
scFv moiety or vice versa.
[00155] A construct encoding an aROR1/aCD3 BspAb, an IL-12 polypeptide, or an
anti-
VEGFR antibody (e.g., an anti-VEGFR scFv) can be operably linked to a promoter
for
expression in a eukaryotic cell. Examples of promoters that can be used in a
recombinant
virus for expression of an ctROR1/aCD3 BspAb include, without limitation, a
Cytomegalovirus (CMV) promoter (e.g., SEQ ID NO:33), a hybrid CMV promoter
(e.g., U.S
9,777,290), an HTLV promoter, an EFla promoter, a hybrid EF1ct/HTLV promoter
(e.g.,
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SEQ ID NO:32), a JeT promoter (US Patent No. 6,555,674), a SPARC promoter
(e.g., US
8,436,160), an RSV promoter, an SV40 promoter, or a retroviral LTR promoter
such as an
MMLV promoter, or a promoter derived from any of these. The construct can also
include a
polyadenylation sequence, such as, for example, a BGH, 5V40, HGH, or RBG
polyadenylation sequence. In some embodiments the polyadenylation sequence has
the
sequence of SEQ ID NO:38.
[00156] Oncolytic viruses, such as for example those described herein, that
include
trangenes encoding an aROR1/aCD3 BspAb, IL-12, and/or an anti-VEGFR antibody,
can be
used to infect host cells that can be cultured for the production of VFCMs,
and optionally
bispecific antibodies or other recombinant polypeptides that may be used for
therapeutic
purposes. VFCMs can be produced using, for example, centrifugation of cell
supernatants
followed by filtration using, for example, 0.22, 0.2, and/or 0.1 micron
filters. A subject, such
as a subject having cancer, can be treated with a VFCM that includes, for
example, an
aROR1/aCD3 BspAb. The subject in some embodiments can be a non-human animal,
and
may be, as nonlimiting examples, a dog, horse, cat, monkey, ape, farm animal,
or member of
an endangered species.
[00157] The disclosure provides methods of treating cancer using a recombinant
HSV that
encodes an aROR1/aCD3 BspAb. The method can include administering a
recombinant HSV
that comprises a nucleic acid construct encoding an aROR1/aCD3 BspAb as
provided herein
to a subject having cancer. In some embodiments the cancer may be a solid
tumor. The
recombinant HSV can be any disclosed herein, such as, for example, any that
encodes an
aROR1/aCD3 BspAb. The subject may be a human or may be a non-human animal such
as,
for example, a dog, cat, cow, bull, or horse. The cancer can be without
limitation, bladder,
bone, breast, eye, stomach, head and neck, kidney, liver, lung, ovarian,
pancreatic, prostate,
skin, or uterine cancer, a mesothelioma, a glioma, a neurocytoma, or a
chondrosarcoma. The
administering can be by any means and can be, as nonlimiting examples,
parenteral,
systemic, intracavitary (e.gõ intrapleural, intraperitoneal), peritumoral, or
intratumoral, and
may be by injection, intravenous or intra-arterial infusion, or other delivery
means. Injection
can be, for example, parenteral, subcutaneous, intramuscular, intravenous,
intra-arterial,
intratumoral, or peritumoral. The treatment regimen may include more than one
administration of the virus and can include multiple dosings over a period of
days, weeks, or
months.
[00158] In some embodiments the aROR1/aCD3 BspAb encoded by the HSV used in
the
methods is an aROR1/aCD3 BspAb having at least 95%, at least 96%, at least
97%, at least
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98%, or at least 99% identity to SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40
(or
otherwise homologous aROR1/aCD3 BspAbs having different signal peptides or
lacking
signal peptides). The HSV can further include one or more additional
transgenes that may
encode, as nonlimiting examples, an IL-12 polypeptide having at least 95%, at
least 96%, at
least 97%, at least 98%, or at least 99% identity to SEQ ID NO:47 or an anti-
VEGFR scFV
having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
identity to SEQ
ID NO:49 (or otherwise homologous polypeptideshaving different signal peptides
or lacking
signal peptides).
EXAMPLES
Example 1. ot-ROR1/a-CD3 BspAb Herpes Simplex Virus (HSV) constructs.
[00159] Three constructs were designed for expressing a bispecific antibody
(BspAb) that
included, proceeding from the N-terminus to the C-terminus: a signal peptide
(SEQ ID
NO:28), an scFv antibody that specifically binds ROR1, a GS linker (SEQ ID
NO:29), and an
anti-CD3 scFv antibody (hum291, SEQ ID NO:34). Figure 1 provides a general
diagram
representing the constructs encoding aR0R1/aCD3 bispecific antibodies
(aR0R1/aCD3
BspAbs). All constructs included the EFla/HTLV hybrid promoter (SEQ ID NO:41)
operably linked to the BspAb-encoding sequence. Constructs encoding three
different
aR0R1/aCD3BspAbs were made that differed only in the ROR1 scFv: an aCD3/aR0R1
BspAb that included an oil ROR1 scFv (SEQ ID NO:9), an aCD3/aR0R1 BspAb that
included an sl 0 ROR1 scFv (SEQ ID NO:18), and an aCD3/ aR0R1 BspAb that
included a
j1v1011 ROR1 scFv (SEQ ID N0:27).
[00160] To clone these constructs into a viral genome, BspAb constructs
flanked by attL
sites were generated by PCR cloning and inserted into the internally deleted
RL1 locus of the
HSV-1 Seprehvec genome. Seprehvec is an HSV-1 vector derived from HSV strain
17 in
which both copies of the RL1 gene that encodes the y34.5 kd (ICP34.5)
polypeptide
responsible for neurovirulence are disrupted by a 695 bp deletion (nucleotides
125975 to
125221 within the RL1 sequence) that inactivates the RL1 gene. The RL1
deletion site
includes attR recombination sites for insertion of any gene or construct of
interest flanked by
attL sequences. The anti-CD3/anti-ROR1 BspAb constructs flanked by attL
sequences were
inserted into both RL1 loci at the deletion sites using in vitro
recombinational cloning that
used the LR ClonaseTM Plus enzyme mixture of Integrase and Integration Host
Factor
(ThermoFisher, Carlsbad, CA) essentially according to the manufacturer's
instructions.
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[00161] Following the recombination reaction, viral genomic DNA was
transfected into
BHK (Baby Hamster Kidney fibroblast) cells for the production of recombinant
virus. Virus
was harvested from transfected BHK cells, then used to infect Vero (African
Green Monkey
(Chlorocebus sp.) kidney epithelial) cells. Individual plaques from infected
Vero cells were
collected and passaged to new Vero cells. This process was repeated for a
total of four rounds
of plaque isolation. Virus stocks were then generated by infection of ¨3.2 x
107 BHK cells
with ¨3.2 x 105 plaque forming units (PFU) of virus and culturing for three
days. After three
days, supernatants were spun twice at 2,100g to pellet cells and debris. After
pelleting the
cells, the supernatant containing virus was spun at 17,200g to pellet virus.
Virus was
resuspended, filtered, and titered on Vero cells. Viral seed stocks and
research stocks were
produced from purified aROR1/aCD3 BspAb viruses SepGI-189 (oil aROR1 scFv),
SepGI-
201 (s10 aROR1 scFv), and SepGI-203 (j1v1011 aROR1 scFv).
Table 1. ROR1 antibodies, scFvs, Bispecific antibodies, and Engineered
viruses.
ROR1 VH VL aROR1 aROR1/aCD3
Oncolytic HSV
antibody domain domain scFv BspAbBspAb expressing
(precursor) aROR1/aCD3
SEQ ID SEQ ID SEQ ID SEQ ID BspAb
NO NO NO NO
oil 1 5 9 36 SepGI-
189
slO 10 14 18 38 SepGI-
201
j1v1011 19 23 27 40 SepGI-
203
Example 2. Production of Virus-Free Conditioned Media (VFCM) from HSVs
expressing citROR1icitCD3 BspAb constructs.
[00162] To generate virus-free conditioned medium (VFCM) from aROR1/a-CD3BspAb
viruses SepGI-189, SepGI-201, and SepGI-203, 12-well plates were seeded with 3
x 105
A431 cells, or, in separate plates, HepG2 cells, in 1 mL of medium at 37 C,
5% CO2. The
next day, the A431 cells and HepG2 cells were infected with recombinant HSVs
at MOI
(Multiplicity of Infection) 0.5 and incubated for 3 days in 1.25 mL of medium.
After 3 days,
cell supernatants were removed and filtered through 0.1j.tm membranes (Pall
Acrodisc
Syringe filter part #4611) to remove virus. The VFCMs were then aliquoted and
stored at -80
'C. VFCMs of SepGI-Null, the Seprehveck HSV vector not including an exogenous
transgene, was also prepared as a control.
Example 3. Detection of (FROR1icr,CD3 bispecific antibodies in VFCMs.
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[00163] A 96-well plate was coated with 50 .it/well of recombinant human RORI
Fc
fusion protein at 2 pg/mL (R&D Systems, Cat No. 9490-RO-050). The plate was
then sealed
and incubated overnight at 4 C. The next day, the plate was washed with 150
pi/well of
wash buffer (Dulbecco's phosphate-buffered saline lx with 0.05% VN Tween20).
Non-
specific binding was blocked using 80 4/well of blocking buffer (Dulbecco's
phosphate-
buffered saline with 2% BSA + 0.05% Tween20) and the plate was incubated at 37
C for 1
hour. After three washes, aROR1/aCD3 BspAb-containing virus-free culture
medium
(VFCMs) of SepGI-189, SepGI-201, or SepGI-203 as well as control VFCM (SepGI-
Null)
were serially diluted in blocking buffer and 50 u.L/well incubated for 2 hours
at room
temperature (RT) under slow shaking conditions. The plate was washed three
times with
washing buffer and 50 4/wel1 of anti-CD3 Hum291 anti-idiotype clone 5A2
diluted in
blocking buffer (1:80 dilution) was added. The plate was incubated for I hour
at 37 C. After
three washings 50 4/well of goat anti-rabbit IgG HRP antibody (Abcam; Cat. No.
ab6721)
diluted in blocking buffer at 1:120,000 dilution was added. The plate was
incubated for 1
hour at 37 C in the dark. The plate was washed three times with washing
buffer and
SureBlue Reserve TMB 1-Component Microwell Peroxidase Substrate Solution
(SeraCare,
Cat No. 5120-0082) was added to the wells (80 uL/well). The plate was
incubated for 10-15
minutes at RT in the dark. Signal development was stopped by adding 50 pt/well
of TMB
Blue STOP Solution (SeraCare, Cat No. 5150-0022), subsequently the signal was
read at 450
nm (specific to SeraCare TMB BlueSTOP Solution) using TecanSpark or other
devices.
Figure 2A provides a schematic of the assay. Figure 2B shows that all three
bispecific
constructs were expressed by engineered SepGI oncolytic viruses SepGI-189,
SepGI-201,
and SepGI-203 and were able to bind ROR1.
Example 4. Binding of oR0R1/uCD3 BspAbs from VFCM of virus-infected cultures
to
ROTH-positive tumor cells.
1001641 A549 human alveolar adenocarcinoma (non-small cell lung cancer) cells
were
knocked out for the RORI gene using CRISPR/Cas-9 methods. A549/ROR1-knockout
(A549/ROR1-KO) cells or A549 wild-type (WT) cells were transferred into a V-
bottom 96-
well plate (80,000 cells per well). Preparations of virus-free culture medium
(VFCMs)
produced as described in Example 2 were serially diluted (1:5 to 1:3,125
dilution) in FACS
buffer (PBS IX + 2% FCS/FBS), added to the wells (100 mt/well), and incubated
for I hour
at room temperature with the cells. After three washes, the cells were
resuspended in 100
u.L/well of monoclonal rabbit anti-CD3 Hum291 anti-idiotype antibody (clone
5A2) diluted
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at 10 [ig/mL in FACS buffer. The plate was covered with plate seal and
incubated 1 hour at
37 C.
[00165] The cells were then resuspended in 100 [IL/well of FACS buffer
containing
donkey anti-rabbit-APC (Southern Biotech ; Cat No. 6441-31-31, Lot No. K2916-
Z779B)
diluted 1:1000 and the plate was incubated for 1 hour at 37 C in the dark.
Cells were finally
resuspended in 120 [tL/well of FACS buffer and the signal was analyzed on an
AttuneNxt
flow cytometer. Figure 3A provides the assay format, where A549 WT cells
expressing
ROR1 bind the BspAb present in the VFCM which in turn is recognized by the
anti-idiotypic
anti-CD3 antibody. The complex is revealed using an Allophycocyanin (APC)-
labelled
donkey anti-rabbit antibody. No binding of A549/ROR1-K0 cells by the BspAbs
present in
the VFCM is expected to occur. Figure 3B provides the flow cytometry results
showing that
all VFCMs of viruses that included bispecific constructs contained aROR1/aCD3
bispecific
antibodies that bound ROR1-expressing A549 tumor cells but failed to bind
A549/ROR1-K0
cells. The VFCM prepared from the culture of cells infected with the control
virus that did
not include a bispecific construct (SepGI-Null) did not contain antibodies
that were able to
bind the cells and anti-idiotypic CD3 antibody.
Example 5. Binding of uROR1/uCD3 bispecific antibodies from VFCM of virus-
infected
cultures to ROR1-positive tumor cells.
[00166] A549-WT and A549-ROR1 KO cells were stained with eFluor450 dye (Thermo
Fisher Scientific; Cat. No. 50-246-096) as recommended by the manufacturer.
Purified
human T cells were freshly isolated from healthy blood donor using the PAN T-
Cell isolation
kit, human (Miltenyi Biotec; Cat No. 130-096-535) and stained with eFluor670
dye (Thermo
Fisher Scientific; Cat. No. 65-0840-85) as recommended by the manufacturer.
The cells were
resuspended at 1.0E+07 cells/mL in Dulbecco's 1X phosphate-buffered saline
(DPBS) at 37
C. eFluor450-labelled A549-WT or A549-ROR1 KO tumor cells were mixed with
purified
eFluor670-labelled T cells at a 1:1 ratio (30,000 tumor cells and 30,000 T
cells/well) in a U-
bottom low adherence 96-well plate (in 100 [iL/well of complete RPMI 1640
media
containing 10% FBS). Cells were centrifuged 3 min at 1,500 rpm and the
supernatant
removed by quickly flicking the plate. The cell pellets were resuspended in
501AL of
undiluted virus-free culture medium (VFCM) containing an aCD3/aROR1 bispecific
construct (SepGI-189, SepGI-201, or SepGI-203). Cells were incubated for 1
hour at 37 'C.
Subsequently, cells were fixed with 100 [IL of fixation buffer (Biolegend;
Cat. No. 420801,
Lot No. B295965) added directly to the wells and the cells plus antibody were
incubated for
20 min at room temperature in the dark without disrupting the cells or
pipetting. Samples
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were immediately analyzed on the Attune NxT cytometer without washing or
pipetting.
Figure 4A shows the assay design, where a BspAb that binds both ROR1
(expressed on WT
A549 cells) and CD3 (expressed on T cells) is able to bind both eFluor 450-
labeled WT A549
cells and eFluor 670-labeled T cells (but is not able to bind A549/ROR1-K0
cells).
[00167] Figure 4B shows the fluorescence quadrants where labeled T cells
(alone) and
labeled WT A549 cells (alone) are found after flow cytomety. The rightmost
plot shows that
when the cells are mixed in the presence of VFCM, cell appear in a new region
of the plot
indicating that both fluorophores have spatially come in close contact. The
graph shows the
percent interaction of T cells with WT A549 cells and A549/ROR1-K0 cells for
VFCMs
made from cultures infected with the SepGI-189 virus, the SepGI-201 virus, and
the SepGI-
203 virus, each of which includes a aROR1/aCD3 BspAb construct. In each case,
the
interaction of WT A549 cells and CD3 cells is dramatically higher than the
interaction of
A549/ROR1-K0 cells and CD3 cells, showing the all three bispecific antibodies
are able to
simultaneously bind to tumor and T cells in an antigen-specific manner.
Example 6. Functional activity uROR1/uCD3 bispecific antibodies of VFCMs.
[00168] To test the ability of a bispecific aROR1/aCD3 antibody to induce
signaling in T
cells, an assay using Jurkat cells having a luciferase gene under the control
of a NFAT
response element (Jurkat-NFAT-Luc) was used. Figure 5A depicts the assay set-
up where
BspAb bound to ROR1-expressing A549 cells also binds CD3 on Jurkat cells,
resulting in
signaling that leads to luciferase expression and a luminescent signal.
A549/ROR1-K0 cells
that do not bind the aROR1/aCD3 BspAb do not stimulate Jurkat cell signaling.
[00169] To perform the assay, 20,000 A549-WT or A549-ROR1 KO cells were plated
in a
white opaque, flat-bottom 96-well assay plates (Corning Cat.# 3917) in 100 [IL
of complete
RPMI-1640 (RPMI-1640 containing 10% FCS). The plate was spun 1 mm at 1,500 rpm
and
incubated overnight at 37 C to let the cells adhere. The plate was then spun
for 3 mm at
1,500 rpm and the supernatant was discarded by quickly flicking the plate.
Jurkat cells
expressing luciferase under the control of a NFAT response element (Jurkat-
NFAT-Luc)
were then plated in the wells (30,000 cells in 50 [IL of complete RPMI-1640
medium/well).
Cell activation was induced by adding 50 pt/well of either aROR1/aCD3 (SepGI-
189,
SepGI-201, or SepGI-203) or negative control VFCM (SepGI-Null) diluted
1:1,000. Purified
anti-CD3 clone Hum291 antibody was added in separated wells at 2 pg/mL as a
positive
control for T cell activation. The plate was incubated for 5 hours at 37 C in
a humidified cell
incubator. Luminescent signal was revealed by adding 100 pt/well of Bio-Glo
Luciferase
Assay substrate (Promega; Cat No. G7940; Lot. No. 0000422404) as recommended
by
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manufacturer and the plate was incubated at room temperature for 5 min in the
dark under
slow shaking conditions. The luminescent signal was read with the TECAN Device
(integration time: 500ms). Figure 5B shows that all three VFCMs induced T cell
activation,
with SepGI-201 and SepGI-203 VFCMs inducing potent T cell activation in a ROR1-
dependent manner.
Example 7. Cytotoxicity assays using VFCMs containing uROR1/uCD3 bispecific
antibodies.
[00170] To test the killing of ROR1-expressing tumor cells by T cells in the
presence of
the aROR1/aCD3 bispecific antibodies, cytotoxicity assays were performed as
follows. On
day 0, 10,000 A549-FLuc WT and A549-FLuc ROR1 KO cells (target cells) were
plated in
100 [IL of complete RPMI1640 (RPMI1640 supplemented with 10% FCS) in white
opaque,
flat-bottom 96-well assay plates (Corning Cat.# 3917). The plate was spun 1
min at 1,500
rpm and incubated overnight at 37 C to let the cells adhere.
[00171] On day 1, human peripheral blood mononuclear cells (hPBMCs) were
isolated
from human healthy whole blood and human T cells were isolated from hPBMCs
using a pan
T cell isolation kit (Miltenyi Biotec; Cat. no. 130-096-535, lot 519115439) as
recommended
by the manufacturer.
[00172] The supernatants from the 96-well plate containing target cells were
removed by
quickly flicking the plate, and VFCMs containing a-ROR1/a-CD3 bispecific
antibodies were
diluted in complete RPM11640 and added to the target cells at 100 L/well.
Subsequently,
100 [it/well of purified human T cells (effector cells) were added (5,000
cells/well) on top of
the target cells to reach an E:T ratio of 0.5:1. As a control, some wells did
not receive effector
cells. Cells were gently mixed, spun for 1 min at 1,500 rpm and incubated for
3 days at 37
C. On day 4, The supernatants (100 pt/well) were collected to measure IF1\17
expression
levels for each condition using the proinflammatory panel 1 (human) kit from
Meso Scale
Discovery (MSD; Cat. No. K15049D) by following the manufacturer's
recommendations.
The killing activity was evaluated by measuring the luminescent signal which
was revealed
by adding 100 4/well of Bio-Gle Luciferase Assay substrate (Promega; Cat No.
G7940;
Lot. No. 0000422404) as recommended by the manufacturer and incubated at room
temperature for 8 min in the dark under slow shaking conditions. Luminescent
signal was
read with the TECAN Device (integration time: 500ms). Percent killing of the
samples was
calculated as follows: 100 ¨ (IlLuminescence sample / Baseline Luminescence no
VFC'M control]) X
100.
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[00173] The results provided in Figure 6 show that VFCMs of cells infected
with SepGI-
189 and SepGI-201 were able to stimulate killing of ROR1 -expressing tumor
cells by T cells,
and that this efficient killing was specific for tumor cells expressing ROR1.
T cells co-
cultured with target cells in the presence of the aROR1/aCD3 bispecific
antibodies also
secreted significant amounts of interferon gamma.
Example 8. Cytotoxicity assays including VFCMs containing uROR1/uCD3
bispecific
antibodies using tumor lines with different levels of ROR1 expression.
[00174] ROR1 expression on tumor cell lines A549-Fluc WT, A459-Fluc/ROR1 KO,
MCF-7-Fluc and HepG2-Fluc expressing firefly luciferase (Fluc) was evaluated
by flow
cytometry. Briefly, cells were plated at 80,000 cells/well in a V-bottom 96-
well plate and
washed twice using 170 4/well of FACS buffer (PBS 1X + 2% FCS/FBS + 0.1%
sodium
azide). A purified human anti-human ROR1 antibody was diluted in FACS buffer
at various
concentrations (ranging from 10 to 0.00061 Rg/mL; dilution 1:4), then cells
were
resuspended in 100 4/well of diluted antibody and incubated for 30 min at 4 C.
After 2
washes in 170 jiL/well of FACS buffer, cells were incubated with an AF647-
conjugated goat
anti-human IgG secondary antibody (Southern Biotech; Cat. no. 2040-31, lot.
K471X873C;
dilution 1:2,000 in FACS buffer) at 80 4/well for 20 mm at 4 C. Cell pellets
were washed
twice, and subsequently resuspended with 120 4 of fixation buffer (Biolegend;
Cat No.
420801, Lot No. B306498) and incubated for 15 min at room temperature in the
dark. Then,
cells were centrifuged at 1,500 rpm for 2 min and the supernatant removed by
quickly
flicking the plate. Cells were washed twice, resuspended in 150 4/well of FACS
buffer and
acquired by flow cytometry on the Attune NxT. Data were analyzed by using
FlowJo v10.
Figure 7A shows that of the human tumor cell lines, A549 (alveolar
adenocarcinoma) has the
highest level of ROR1 expression, and HepG2 (liver cancer) express little
ROR1, with the
detected labeling with ROR1 antibody being comparable to that of A549-ROR1
knockout
cells. MCF-7 (breast cancer) cells expressed an intermediate level of ROR1.
[00175] To perform the killing assay, human peripheral blood mononuclear cells
(hPBMCs) were isolated from human healthy whole blood, then human T cells were
isolated
from hPBMCs using the EasySep human T cell Isolation kit (StemCell Technology;
Cat. No.
17951, lot 1000024139) as recommended by the manufacturer. A549-Fluc WT, A459-
Fluc/ROR1 KO, MCF-7-FLuc and HepG2-Fluc target cells were plated at 10,000
cells/well
in 100 RI. of complete culture medium in white opaque, flat-bottom 96-well
assay plates
(Corning Cat.# 3917). The plate was spun 1 min at 1,500 rpm and incubated
overnight at 37
C to let the cells adhere. The supernatants from the 96-well plate containing
target cells were
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removed by quickly flicking the plate. Purified effector T cells (1001dt/well)
were plated on
top of target cells at a 2:1 E:T ratio. As controls, some wells did not
receive effector cells.
VFCM of SepGI-201-infected cultures or SepGI-Null-infected cultures were
diluted in
complete RPMI1640 and added to the cells at 100 ut/well. The SepGI-201 virus
includes the
aROR1/aCD3 BspAb construct and the SepGI-Null virus does not include a BspAb
construct. Cells were gently mixed, spun for 1 min at 1,500 rpm and incubated
for 3 days at
37 'V, after which the supernatants (100 pi/well) were collected. IFNy levels
present in the
supernatants were measured using the proinflammatory panel 1 (human) kit from
Meso Scale
Discovery (MSD; Cat. No. K15049D) by following the manufacturer's
recommendations.
Killing activity was evaluated by measuring the luminescent signal from the
wells by adding
100 4/well of Bio-Glo Luciferase Assay substrate (Promega; Cat No. G7940;
Lot. No.
0000422404) as recommended by the manufacturer and incubating at room
temperature for 5
min in the dark under slow shaking conditions. The luminescent signal was read
with the
TECAN Device (integration time: 500ms). Percent killing of the samples was
calculated as
follows: 100 ¨ (ILuminescence sample / Baseline Luminescence no VF CM
control]) X 100. Figure
7B demonstrates that while A549 and MCF-7 cells were killed by T cells in the
presence of
SepGI-201 VCFM, HepG2 cells were preserved (due to low ROR1 expression) even
if T
cells were activated at high VFCM concentration, as demonstrated by increased
IFNy
expression.
Example 9. Killing activity of oncolytic viruses SepGI-189, SepGI-201, and
SepGI-203
expressing bispecific antibodies.
[00176] Figure 8A provides the experimental plan for evaluating killing of
A549 tumor
cells by oncolytic viruses SepGI-189, SepGI-201, and SepGI-203, expressing the
"oil"
aROR1/aCD3, "s10" aROR1/aCD3 and "j1v1011" aROR1/aCD3 BspAb constructs,
respectively (Table 1). On day 0, A549-Fluc WT and A549-Fluc ROR1 KO target
cells were
plated at 10,000 cells/well in 100 pt of complete RPMI-1640 + 10% FCS in white
opaque,
flat-bottom 96-well assay plates (Corning Cat.# 3917). The plate was spun 1
min at 1,500
rpm and incubated overnight at 37 C to let the cells adhere. On day 1, the
target cells were
infected with either an ctRORI/aCD3 virus (SepGI-189, SepGI-201, or SepGI-203,
see Table
1) or the negative control SepGI-Null virus at multiplicities of infection
(MOI) of 1, 0.33,
0.11, 0.04, and 0.01. On day 2, T cells were purified from freshly isolated
PBMCs using a
pan T cell isolation kit (Miltenyi Biotec; Cat. no. 130-096-535, lot
519115439) as
recommended by the manufacturer. The supernatants were removed from target
cells by
quickly flicking the 96-well plate and 20,000 effector T cells plated in 100
pt/well to reach
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an E:T ratio of 2:1. Cells were gently mixed, spun for 1 min at 1,500 rpm, and
incubated for 4
days at 37 'C. On day 6, the killing activity was evaluated by measuring the
luminescent
signal using 100 uL/well of Bio-Glo Luciferase Assay substrate (Promega; Cat
No. G7940;
Lot. No. 0000422404) as recommended by the manufacturer and incubated at room
temperature for 5 min in the dark under slow shaking conditions. Luminescent
signal was
read with the TECAN Device (integration time: 500ms). Percent killing of the
samples was
calculated as follows: 100 ¨ (Luminescence sample / Baseline Luminescence no
virus]) X 100.
[00177] Figure 8B shows that tumor cells infected with each of the BspAb-
expressing
viruses at MOIs as low as 0.11 were killed at significantly percentages than
tumor cells
infected with the SepGI-null virus. Infection of tumor cells with SepGI-201
led to
significantly higher killing of tumor cells at an MOI of 0.04, while infection
with of tumor
cells with SepGI-203 led to significantly higher killing of tumor cells at an
MOI of 0.01. The
same effect was not seen when ROR1 knockout tumor cells were infected with
viruses and
used as targets. In this case, only infection with the SepGI-203 virus led to
significantly
higher killing demonstrating that SepGI-203 showed some degree of non-specific
killing at
MOI higher than 0.11. All together these data showedthat oncolytic activity
combined with
aROR1/aCD3 BspAb significantly increased anti-tumor activity in an antigen-
specific
dependent manner.
Example 10. Mouse cross-reactivity of slO and j1v1011 monoclonal antibodies.
[00178] To test whether the slO (R06D8-s10) and j1v1011 (RO6D8-j1v1011)
monoclonal
antibodies used in engineering the aROR1/aCD3 bispecific antibodies recognized
mouse
ROR1 in addition to human ROR1, the assay depicted in Figure 9A was employed.
A 96-
well plate was coated with 50 uL/well of recombinant mouse ROR1 IgG2-Fc fusion
protein at
2 ug/mL (R&D Systems, Cat No. 9910-RO-050, Lot No. DIWM0120121), the plate was
then
sealed and incubated overnight at 4 C. The next day, the plate was washed with
150 pL/well
of wash buffer (Dulbecco's phosphate-buffered saline lx with 0.05% VN
Tween20). Non-
specific binding was blocked by using 80 iL/well of blocking buffer
(Dulbecco's phosphate-
buffered saline with 2% BSA + 0.05% Tween20) and the plate was incubated at 37
C for 1
hour. After three washes, two anti- human ROR1 antibodies (s10 and j1v1011)
were serially
diluted in blocking buffer (80 uL/well) and incubated for 2 hours at room
temperature (RT)
with slow shaking. The plate was washed thrice with wash buffer and then 80
L/well of
secondary HRP-labelled goat anti-human IgG Fc (SouthernBiotech; Cat No. 2081-
05; Lot
No. L5311-TE40) diluted in blocking buffer (1:2,000 dilution) was added. The
plate was
incubated for 1 hour at 37 C in the dark. The plate was washed thrice with
washing buffer
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and SureBlue Reserve TMB 1-Component Microwell Peroxidase Substrate Solution
(SeraCare, Cat No. 5120-0082) was added to the wells (80 [it/well). The plate
was incubated
for 10-15 minutes at RT in the dark. The signal development was stopped by
adding 50
rat/well of TMB Blue STOP Solution (SeraCare, Cat No. 5150-0022), and
subsequently the
signal was read at 450 nm (specific to SeraCare TMB BlueSTOP Solution) using
TecanSpark
or other devices. Figure 9B shows that both anti-RORI antibodies used to
generate the
BspAb constructs of SepGI-201 and SepGI-203 exhibit mouse-cross-reactivity.
Example 11. In vivo study of tumor treatment with virus expressing BspAb.
[00179] Figure 10A provides a diagram of the inoculation and treatment
schedule of mice
used to test the effectiveness of oncolytic viruses expressing aROR1/aCD3
bispecific
antibodies. On day ¨6 (D-6), Female NSG-Tg(Hu-IL-15) mice (6 weeks of age)
were injected
intraperitoneally (I.P.) with 1.0E+07 freshly purified human peripheral blood
mononuclear
cells (PBMCs) in Dulbecco's phosphate-buffered saline (DPBS) lx. On day 0
(DO), mice
were injected subcutaneously (S.C.) in the right flank with 5.0E+06 A549-WT
tumor cells
diluted in 100 [IL of DPBS 1X. On day 6 (D6), mice were randomized into four
groups (three
'viral treatment' groups and one 'no viral treatment' control group) and viral
treatments were
initiated: 50 .it/mouse/injection of either SepG1-189, SepG1-201, SepGI-Null,
or no virus
was delivered peri-tumorally (P.T.) on days 6, 10 and 12. Tumor growth and
body weight
were monitored twice weekly. Tumor volume was measured using a caliper and
calculated
using the formula V=(LengthxWidth2)/2. The study was terminated on day 31 and
percent
tumor growth inhibition (TGI) was calculated as follows: [1 ¨ (Relative tumor
volume of the
treated group)/(Relative tumor volume of the control group)] x 100.
[00180] Figures 10B and 10C provide the tumor volumes and calculated tumor
growth
inhibition (TGI) for the treatment groups and non-treatment group and Figure
10D provides
mouse body weights over the course of the experiment. Treatment with viruses
expressing
aROR1/aCD3 bispecific antibodies (SepGI-189 and SepGI-201) led to greater
inhibition of
tumor growth than either no treatment or treatment with a virus (SepGI-Null)
that did not
express an aROR1/aCD3 BspAb.
Example 12. Constructs for expressing ROR1/CD3 Bispecific antibodies together
with
additional gene encoding IL-12 or IL-12 plus an anti-VEGFR antibody.
1001811 Additional constructs were made for the synthesis of HSVs encoding a
ROR1-
CD3 bispecific antibody and in addition, the cytokine IL-12. The SepGI-216
construct (a
double gene construct, Figure 11A) included the aROR1(s10)-aCD3 bispecific
antibody
(SEQ ID NO:18) under the control of the EF1a/HTLV promoter (SEQ ID NO :41) and
a gene
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encoding human IL-12 (SEQ ID NO:46) under the control of the CMV promoter (SEQ
ID
NO:42). The human IL-12 gene encoded a single polypeptide (SEQ ID NO:47)
encompassing both the p40 and p35 subunits of IL-12 connected by a 2x elastin
linker (SEQ
ID NO:66). The SepGI-212 construct (a triple gene construct, Figure 11B)
included a gene
encoding the aROR1(s10)-aCD3 bispecific antibody (SEQ ID NO:37) under the
control of
the EFla/HTLV promoter (SEQ ID NO:41) and a gene encoding an anti-VEGFR2 scFv
linked to an Fcl region (SEQ ID NO:50) and the human IL-12 gene (SEQ ID
NO:46), where
the sequence encoding the aVEGFR2 scFv-Fcl (SEQ ID NO:48) and the sequence
encoding
human IL-12 (SEQ ID NO:46) were separated by a sequence encoding a T2A self-
cleavage
peptide (SEQ ID NO:51) and the continuous open reading frame encompassing
sequences
encoding the human IL-12 polypeptide and the VEGFR2 scFv-Fcl polypeptide was
under the
control of the CMV promoter (SEQ ID NO:42) (see, Figure 11B).
1001821 In addition, for use as controls, analogous constructs were designed
in which the
gene encoding the aROR1(s10)-aCD3 bispecific antibody was replaced by a gene
encoding a
bispecific antibody that included an scFv that bound the F protein of
Respiratory Syncyti al
Virus (SEQ ID NO:43) and CD3 (referred to herein as the -aRSV-aCD3 bispecific
antibody"). Cloning of these constructs and production and isolation of
recombinant viruses
was performed essentially as set forth in Example 1.
Table 2. SepGI HSV constructs.
Virus Transgenes
SepGI-Null none
SepGI-201 aROR1(s10)-aCD3
SepGI-207 aRSV-aCD3
SepGI-212 aROR1(s10)-aCD3; aVEGFR2-Fc; hulL-12
SepGI-214 aRSV(s10)-aCD3; aVEGFR2-Fc; huIL-12
SepGI-216 aROR1(s10)-aCD3; huIL-12
SepGI-218 aRSV(s10)-aCD3, huIL-12
[00183] VFCMs produced from the viruses (see Example 2) was tested to assess
by
ELISA for the expression of the transgenes essentially as described in Example
3, where the
wells of the plates were coated with either ROR1, the RSV protein, or human
VEGFR.
[00184] The results of the ELISAs are provide in Figures 12A, B, and C. The
first graph
(Figure 12A) shows that all three viruses that included the gene encoding the
RSV protein
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antibody (SepGI-207, SepGI-214, and SepGI-218) did produce the RSV antibody,
whereas
none of the other viruses (lacking the RSV protein antibody) did. Figure 12B
shows that
viruses SepGI-201, SepGI-216, and SepGI-212 all expressed the ROR1 antibody,
as
expected, whereas the control viruses lacking the gene encoding the aROR1(s10)-
aCD3
bispecific antibody did not. Figure 12C shows the results of the ELISA used to
detect IL-12.
In this case, VFCMs from cells infected with SepGI-212, SepGI-216, and SepGI-
218 were all
found to contain IL-12 protein, whereas the two isolates of cells infected
with SepGI-214 did
not. (Subsequent isolates of SepGI-214 were later found to produce IL-12.) As
expected, IL-
12 was not detected in VFCMs of uninfected cultures or cultures infected with
the SepGI
Null virus or the SepGI-207 virus.
[00185] The results of an ELISA for detecting the VEGFR2 scFv antibody are
shown in
Figure 13, where the wells of the 96 well ELISA plates were coated with
recombinant human
VEGFR2 (VEGFR2/KDR Protein (ECD, His Tag) (Sino Biological). After washing the
antigen-coated wells, VFCMs were 8-fold serially diluted in blocking buffer
and added at 50
uL/well and the plate was incubated for 2 h at room temperature on a shaker.
The plate was
washed 3X with wash buffer and 50 L/well of Goat anti Human IgG (H+L)
Secondary
Antibody, HRP (diluted 1:5,000x in blocking buffer) was added (Invitrogen) and
the plate
was incubated for 1 h at 37 C. After the plate was washed 3X with wash buffer,
the signal
was detected by using 50 pt/well of SureBlue Reserve TMB 1-Component Microwell
Peroxidase Substrate Solution (Cat No. 5120-0082, SeraCare). The plate was
incubated 10-12
min at room temperature in the dark. Then 50 L/well of TMB BlueSTOP Solution
(Cat No.
5150-0022, SeraCare) was added and the absorbance read at 450 nm (specific to
SeraCare
TMB BlueSTOP Solution) using TecanSpark. The graph of Figure 13 shows that the
VFCM
of one isolate of the triple gene virus SepGI-212 demonstrated expression of
the uVEGFR2-
Fc antibody, whereas there was no binding of any of the VCFMs of viruses that
did not
include the aVEGFR2-Fc antibody gene (SepGI-Null, SepGI-201, SepGI-207, SepGI-
216,
and SepGI-218).
Example 13. IL-12 Activity Assays.
[00186] Assays for the activity of IL-12 were performed essentially as
described in
Example 9, where the VFCMs of cells infected with SepGI-Null, SepGI-201, SepGI-
207,
SepGI-212, SepGI-214, SepGI-216, and SepG1-218 were tested in luciferase-based
assays. A
cell-based assay was used in which cells having a heterodimeric IL-12 receptor
and
engineered to have a luciferase gene under the control of an IL-12-responsive
promoter
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(iLite IL-12 Assay Ready Cells (Eagle Biosciences (Amherst, NH)) were
incubated with
lysates of cells infected with the recombinant HSVs. Promega Corporations One-
Glo
Luciferase system was used for detection.
[00187] Briefly, the assay was performed by adding diluted VFCM
from uninfected cells
or cells infected with various HSVs to the wells of a 96 well plate. The
lysates of infected cell
cultures (VFCMs) were produced as described in Example 2. A dilution series of
recombinant IL-12 (R&D Systems) was added to additional wells to generate a
standard
curve. The IL-12 reporter cells were used essentially according to the
manufacturer's
instructions. 40K iLite Cells were thawed, diluted, and 40 1 was added to each
well of a 96-
well plate. 40 1 of a dilution series of the VFCMs was then added to the assay
wells, the
contents of the wells were mixed, and the plate was incubated for five hours
at 37 C, 5%
CO2. Recombinant IL-12 was added in dilution series to separate wells for
generating a
standard curve. The One-Glo luciferase reagent (Promega Corp., Madison, WI)
was then
added to each well (40 L) and after 10 min at room temperature, firefly
luciferase
luminescence was measured using a Tecan Spark plate reader. The results are
shown in
Figure 14 which provides a graph of the luminescence from assays using
uninfected cell
conditioned media, conditioned media from cells infected with a virus that did
not include
exogenous transgenes (SepGI-Null), and conditioned media from cells infected
with the IL12
gene-containing viruses SepGI-201 and SepGI-207 (no IL12 gene), SepGI-212 and
SepGI-
214 (triple gene viruses with IL-12 gene). and SepGI-216 and SepGI-216 (double
gene
viruses with IL-12 gene). Notably, all cells infected with viruses that
included the IL-12 gene
expressed functional IL-12, with the exception of isolates of the triple gene
virus SepGI-214
that also did not show production of the IL-12 protein in the ELISA (Example
12).
Example 14. Cell-Cell interaction assays.
1001881 To assess the ability of the aROR1-aCD3 bispecific antibodies encoded
by the
engineered HSVs to conjugate target ROR1-expressing tumor cells and T cells,
mouse tumor
cells and human T cells were separately labeled with fluorophores. Hepa 1-6
cells and A549
cells, both of which express ROR1, were labeled with eFluor 450 (ThermoFisher)
(Figure
15B) using and human T cells isolated from PBMCs were pre-labled with eFluor
670 (Figure
15C) and cell-cell interactions were assayed and analyzed by flow cytometry
essentially as
described in Example 5. Figure 15D shows an example of the flow cytometry
results, where
conjugated cells (fluorescing at both wavelengths) are seen in the upper right
quadrant of the
plot.
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[00189] The results of flow cytometric assays for aROR1-aCD3 bispecific
antibody-
mediated conjugation of T cells with ROR1-expressing tumor cells is presented
graphically in
Figure 16A, B, and C. Figure 16A shows, from left to right, the percentage of
Hepa 1-6
cells, A549 wild type cells, and A549 ROR1 knockout cells that were conjugated
to T cells
after co-incubation in the presence of SepGI-218 VFCM which expresses a
construct that
encodes an aRSV-aCD3 bispecific antibody as well as IL-12. Figure 16B shows,
from left to
right, the percentage of Hepa 1-6 cells and then A549 wild type cells after co-
incubation in
the presence of SepGI-201 VFCM which expresses a construct that encodes an
aROR1-
aCD3 bispecific antibody. Figure 16C shows, from left to right, the percentage
of Hepa 1-6
cells and then A549 wild type cells after co-incubation in the presence of
SepGI-216 VFCM
which expresses a construct that encodes an aROR1-aCD3 bispecific antibody as
well as IL-
12. Minmal cell-cell interaction is observed in the presence of the SROR1+
Tumor cell- T
cell interaction is observed in the presence of VFCM of SepGI-201-infected
cells and SepGI-
216-infected cells, with no significant differences observed between SepGI-201-
infected cells
and SepGI-216-infected cells.
Example 15. T-Cell Activation Assays.
[00190] To determine the effect of the aROR1-aCD3 bispecific antibodies on T
cell
activation, assays were performed in which ROR1-expressing tumor cells were
incubated
with T cells in the presence of VFCMs of cells infected with viruses encoding
the aROR1-
aCD3 bispecific antibodies, after which activation markers on the surfaces of
the T cells were
assessed. Briefly, wild type A549 cells, or as controls, ROR1 knockout A549
cells, were
plated in the wells of 96 well plates at 104 cells per well. The next day,
freshly isolated CD3+
T cells, stained with CFSE, were added to the wells at E:T ratios of 10:1 or
5:1. VFCM was
added to the wells at a 1,000 fold dilution, or, as positive controls,
CD3/CD28 beads were
added to the wells (bead:cell ratio of 1:20). One, two, and three days later
supernatant was
removed for staining of T cells for the expression of activation markers and
analysis by flow
cytometry.
[00191] Figure 17A-D provide the results of assays with ROR1 knockout A549
cells as
targets. Figure 17A shows that the viability of the CD3+ T cells on days 1, 2,
and 3 of the
assay was close to 100% regardless of whether the cells were cultured with
VFCM of
uninfected cultures (first two bars) or with VFCM of cells infected with the
SepGI-Null virus
(second two bars), the SepGI-207 virus (second two bars), the SepGI-201 virus
(third two
bars), or CD3/CD28 beads (fourth two bars). Figure 17B provides the CD3+ CD4+
cell
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count for each assay group on successive days of the assay. Figure 17C
provides the
percentage of CD25+ cells for each assay group on successive days of the assay
based on
flow cytometry, and Figure 17D provides the percentage of CD69+ cells for each
assay
group on successive days of the assay. Although CD3/CD38 beads resulted in
activation of
the T cells as evidenced by increased expression of both CD25 and CD69 over
the course of
the assay, when ROR1 knockout cells were used as targets, no activation of the
T cells was
observed as assessed by expression of CD25 and CD69, regardless of the
presence of VFCM.
[00192] Figure 17E-H provide the results of assays with wild type A549 cells
that express
ROR1 as targets. Figure 17E shows that the viability of the CD3+ T cells on
days 1, 2, and 3
of the assay was close to 100% regardless of whether the cells were cultured
with VFCM of
uninfected cultures (first two bars) or with VFCM of cells infected with the
SepGI-Null virus
(second two bars), the SepGI-207 virus (second two bars), the SepGI-201 virus
(third two
bars), or CD3/CD28 beads (fourth two bars). Figure 17F provides the CD3+ CD4+
cell
count for each assay group on successive days of the assay. Figure 17G
provides the
percentage of CD25+ cells for each assay group on successive days of the assay
based on
flow cytometry, and Figure 17H provides the percentage of CD69+ cells for each
assay
group on successive days of the assay. Notably, the presence of VFCM of
cultures infected
with the SepGI-201 virus that was engineered to express the aROR1-aCD3
bispecific
antibody resulted in expression of both CD25 and CD69 by the T cells in the co-
culture. This
induced expression was not observed for co-cultures that instead included the
VFCM of
SepGI-207 infected cells, with the VFCM of SepGI-207 infected cells, or with
the VFCM of
uninfected cells. Thus, using target cells that expressed ROR1, the activation
of T cells in co-
cultures could be attributed to the presence of the aROR1-aCD3 bispecific
antibody, which
can engage the T cells leading to their activation.
Example 16. T cell proliferation/activation assays.
[00193] An additional cell culture assay was performed with single and double
gene
expressing viruses. In these assays A549 wild type or A549 ROR1 knockout cells
were plated
in the wells of 96 well plates at 104 cells per well. Purified human T cells,
stained with
celltrace violet (CTV) dye, were added to the wells at 10:1 and 5:1
effector:target ratios, and
VFCMs at 1:1,000 dilution were added to the wells. The VFCMs were of cells
infected with
SepGI-207 (aRSV-aCD3 bispecific antibody gene), SepGI-201 (aROR1-aCD3
bispecific
antibody gene), SepGI-216 (aROR1-aCD3 bispecific antibody gene plus IL-12
gene), and
SepGI-218 (aROR1-aCD3 bispecific antibody gene plus IL-12 gene). The plates
were
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incubated for 3 days, with flow cytometry performed after 1, 2, or 3 days to
determine cell
proliferation by the percentage of CTV+ T cells. Figure 18 shows the results
of assays at a
5:1 effector to target ratio, where specific T cell proliferation was observed
only for ROR+
target cells and only when the aROR1-aCD3 bispecific antibody-containing VFCMs
of
SepGI-201 and SepGI-216 infected cultures were included in the cultures.
Example 17. Luciferase-based killing assay using VFCMs of cells infected with
single,
double, and triple gene HSVs engineered to express aROR1-uCD3 bispecific
antibodies.
[00194] Assays were performed to assess the effects of VFCMs of cells infected
with
HSVs engineered to express aROR1-aCD3 bispecific antibodies on the killing of
ROR1+
tumor cells by T cells. For these assays, target cells (A549 wild type cells
or A549 ROR1
knockout cells for use as controls) were labeled by transducing the cells with
a retrovirus for
expressing GFP and firefly luciferasel. The luciferase-expressing target cells
were plated at
104 cells per well in 100 1RPMI-1640+10% FCS in 96 well plates and cultured
for two days
at 37 C. Freshly isolated human T cells freshly isolated from PBMCs were then
added to the
wells at a ratio of 0.5:1 and VFCMs at dilutions of 1,000 or 1:8,000 were
added to each assay
well. The plates were incubated for four days at 37 C, after which the number
of
luceriferase-expressing cells was assessed by adding 80 ill of Bio-Glo
Luciferase Assay
reagent (Promega), incubating the plate in the dark for 5 min, and reading
luminescence with
a TECAN device (integration time, 500 ms). Figure 19A shows that in the
absence of T cells
(effectors) the number of A549 wild type cells is close to 107 regardless of
the presence or
type of VCFM added to the culture. In the presence of T cells however, a
reduction in A549
wild type target cells is evident in cultures that included VFCMs of viruses
engineered to
express the aROR1-aCD3 bispecific antibody: SepGI-201, SepGI-212, and SepGI-
216 (see
Table 2). Killing of target cells was not observed in cultures that included
VFCMs of the
SepGI-207, SepGI-214, and SepGI218 viruses that were not engineered to express
the
aROR1-aCD3 bispecific antibody, as also seen in the graph providing the
percentage of
killing, shown in Figure 19C. Figures 19B and 1911 provide the results when
ROR1
knockout A549 target cells were used, demonstrating lack of killing of cells
that did not
express ROR1 by the T cell effectors regardless of the VCFM (or bispecific
antibody) in the
co-culture.
Example 18. Xcelligence Killing Assay using 549 WT cells, with Single, Double,
Triple
gene expressors (VFCMs).
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[00195] Killing assays were also performed using the xCELLigence Real Time
Cell
Analyzer (Acea Biosciences, San Diego, CA). For these experiments, A549 wild
type and
A549 ROR1 knockout cells were seeded into the wells of 96 well E-plates (Acea
Biosciences) at 10,000 cells/well in 50 n1 of RPMI-1640+10% FCS. T cells were
added at a
0.5:1 effector to target ratio, and 1:1,000 dilutions of VFCMs of cell
cultures infected with
the HSVs SepGI-Null, SepGI-207, SepGI-212, SepGI-216, and SepGI-218 were
added. The
plates were read continously for three days. Figure 20 shows that assays that
included
VFCMs of HSVs that did not include aROR1-aCD3 bispecific antibody constructs:
SepGI-
207, SepGI-214, SepGI-218, and SepGI-123 (IL-12 gene only), proliferated to
essentially the
same extent and with the same pattern as cultures that lacked VFCM altogether.
On the other
hand, assays that included VFCMs of HSVs engineered to express aROR1-aCD3
bispecific
antibody constructs: SepGI-201, SepGI-212, and SepGI-216, demonstrated reduced
proliferation, indicating killing of the ROR1+ target cells in these cultures.
Figure 21 shows
the results of parallel assay in which the target cells were ROR1 knockout
cells. In this case
no impairment of proliferation was observed.
Example 19. In vivo study of anti-tumor activity of SepGI-201 VFCM in NOD/Scid
pseudo-humanized mouse model.
[00196] A study was designed to assess the effect of treating of tumor-bearing
NOD/Scid
pseudo-humanized mice with VFCM of cells infected with the SepGI-201 HSV
engineered to
express an aROR1-aCD3 bispecific antibody. As controls, some tumor-bearing
mice are
treated with VFCM of cells infected with the SepGI-207 HSV engineered to
express an
aRSV-aCD3 bispecific antibody. Six groups of eight mice are established with
the treatment
regimens shown in Table 3.
Table 3. Groups of mice for VFCM treatment study.
Group Tumor Cells PBMCs VFCM
1 4 x 106 A549 wt 4 x 106 hPBMCs None
2 4 x 106 A549 wt 4 x 106 hPBMCs aRSV-
aCD3 (SepGI-207)
3 4 x 106 A549 wt 4 x 106 hPBMCs aROR1-
aCD3 (SepGI-
201)
4 4 x 106 A549 ROR1 KO 4 x
106 hPBMCs None
4 x 106 A549 ROR1 KO 4 x 106 hPBMCs aRSV-aCD3 (SepGI-207)
6 4 x 106 A549 ROR1 KO 4 x
106 hPBMCs aROR1-aCD3 (SepGI-
201)
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[00197] Tumor cells, either A549 wild type or A549 ROR1 knockout, are
subcutaneously
coinjected with human PBMCs in all mice. Four weeks later, treatments begin,
in which mice
of groups 2-6 are injected peri-tumorally with 50 1 of VFCM every four to
five days for a
total of five treatments. Tumor growth and body weight are monitored twice
weekly. Tumor
volume is measured using a caliper and on termination of the study at
approximately 9 weeks
tumor growth inhibition (TGI) is calculated as follows: [1 ¨ (Relative tumor
volume of the
treated group)/(Relative tumor volume of the control group)] x 100.
Example 20. In vivo study of anti-tumor activity of SepGI-201 VFCM in NSG-132m
KO
pseudo-humanized mouse model.
[00198] A study was designed to assess the effect of treating of tumor-bearing
NSG-B2m
knockout pseudo-humanized mice with the SepGI-201 HSV engineered to express an
aROR1-aCD3 bispecific antibody. As controls some mouse groups are treated with
the
SepGI-207 HSV engineered to express an aRSV-aCD3 bispecific antibody. Six
groups of
eight mice are established with the treatment shown in Table 4.
Table 4. Groups of mice for VFCM treatment study.
Group Tumor Cells PBMCs Virus
1 5 x 106 A549 wt 5 x 106 hPBMCs None
2 5 x 106 A549 wt 5 x 106 hPBMCs SepGI-
207 (aRSV-aCD3)
3 5 x 106 A549 wt 5 x 106 hPBMCs SepGI-
201 (aROR1-
aCD3)
4 5 x 106 A549 ROR1 KO 5 x
106 hPBMCs SepGI-207 (aRSV-aCD3)
5 x 106 A549 ROR1 KO 5 x 106 hPBMCs SepGI-201 (aROR1-
aCD3)
[00199] Tumor cells, either A549 wild type or A549 ROR1 knockout, are
subcutaneously
coinjected with human PBMCs in all mice. Four weeks later, treatments begin,
in which mice
of groups 2-5 are injected peri-tumorally with 50 1 of oncolytic virus every
four to five days
for a total of three treatments. Tumor growth and body weight ae monitored
twice weekly.
Tumor volume is measured using a caliper and on termination of the study at
approximately 9
weeks tumor growth inhibition (TGI) is calculated as follows: [1 ¨ (Relative
tumor volume of
the treated group)/(Relative tumor volume of the control group)] x 100.
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SEQUENCES
SEQ ID NO:1
Protein
Artificial
oil anti-ROR1 antibody Heavy Chain Variable Region
QVQLVQSGAEVKKPGASVKVSCKAS GYT FTNYYMHWVRQAPGQGLEWMGI INPTSGRTSYAQKFQGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARDSSSWYSGWYFDLWGQCTTVTVSS
SEQ ID NO:2
Protein
Artificial
oil anti-ROR1 antibody Heavy Chain Variable Region CDR1:
NiP11-1
SEQ ID NO:3
Protein
Artificial
oil anti-ROR1 antibody Heavy Chain Variable Region CDR2:
I II,:
SEQ ID NO:4
Protein
Artificial
oil anti-ROR1 antibody Heavy Chain Variable Region CDR3:
DSSSWYSGWYFDL
SEQ ID NO:5
Protein
Artificial
oil anti-ROR1 antibody ROR1 antibody Light Chain Variable Region
AIQMTQSPS SL SASVGDRVT I TCRASQGIRTDLAWYQQKPGKAPKL LIYAAS SLQSGVPSRFSGSGSG
TDFTLT IS SLQPEDFATYYCQQYYGYP IAFGQGTRLE IK
SEQ ID NO:6
Protein
Artificial
oil anti-ROR1 antibody Light Chain Variable Region CDR1
RAS Q. GIRT D.LA
SEQ ID NO:7
Protein
Artificial
oil anti-ROR1 antibody Light Chain Variable Region CDR2
ASSLCS
SEQ ID NO:8
Protein
Artificial
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oil anti-ROR1 antibody Light Chain Variable Region CDR3
QQYYGYP IA
SEQ ID NO:9
Protein
Artificial
oil anti-ROR1 single chain antibody (scFv)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGI INPTSGRTSYAQKFQGRV
TMTRDT ST S TVYMELS SLRSEDTAVYYCARDS SSWYSGWYFDLWGQGTTVTVS S GGGGSGGGGSGGGG
SGGGGSAIQMTQSPSSLSASVGDRVT I TCRASQGI RTDLAWYQQKP GKAPKLL IYAAS SLQSGVPSRF
SGSGSGTDFTLT S SLQPEDFATYYCQQYYGYP IAFGQGTRLE K
SEQ ID NO:10
Protein
Artificial
slO anti-ROR1 antibody Heavy Chain Variable Region
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGI INPSGGSTSYAQKFQGRV
TMTRDT ST S TVYMELS SLRSEDTAVYYCARSSRS SYYLWVLDLWGQGTTVTVS S
SEQ ID NO: II
Protein
Artificial
slO anti-ROR1 antibody Heavy Chain Variable Region CDR1
SEQ ID NO:12
Protein
Artificial
slO anti-ROR1 antibody Heavy Chain Variable Region CDR2
I P (.,_= 'GS '1' K
SEQ ID NO:13
Protein
Artificial
slO anti-ROR1 antibody Heavy Chain Variable Region CDR3
SSRSSYYLWVLDL
SEQ ID NO:14
Protein
Artificial
slO anti-ROR1 antibody Light chain Variable Region
AIQMTQSPSSLSASVGDRVTITCRASQGVSTEIAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSG
TDFTLT I S SLQPEDFATYYCQQFNSYP I T FGQGTRLE I K
SEQ ID NO:15
Protein
Artificial
slO anti-ROR1 antibody Light chain Variable Region CDR1
58
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RAS QGVS T E IA
SEQ ID NO:16
Protein
Artificial
slO anti-ROR1 antibody Light chain Variable Region CDR2
AASSLQS.
SEQ ID NO:17
Protein
Artificial
slO anti-ROR1 antibody Light chain Variable Region CDR3
QQFNSYPIT
SEQ ID NO:1 8
Protein
Artificial
slO anti-ROR1 single chain antibody (scFy)
QVQLVQSGAEVKKPGASVKVSCKAS GYT FTNYYMHWVRQAPGQGLEWMGI INPSGGSTSYAQKFQGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARSSRSSYYLWVLDLWGQCTTVTVSSCGGGSGGGCSGGCG
SGGGGSAIQLTQSPSSLSASVGDRVT I TCRASQGVSTE IAWYQQKPGKAPKLL IYAAS SLQSGVPS RF
SGSGSGTDFTLT I S SLQPEDFATYYCQQFNSYP I TFGQGTRLE IK
SEQ ID NO:19
Protein
Artificial
j1v1011 anti-ROR1 antibody Heavy chain Variable region:
QVQLVQSGAEVKKPGASVKVSCKAS GYT FT SKYYHWVRQAPGQGLEWMGI INPTSGSTSYAQKFQGRV
TMTRDTSTSTVYMELS SLRSEDTAVYYCARDS SRYSGWYFDLWGQGTTVTVS S
SEQ ID NO:20
Protein
Artificial
j1v1011 anti-ROR1 antibody Heavy chain Variable region CDR1:
SKYYI-i
SEQ ID NO:21
Protein
Artificial
j1v1011 anti-ROR1 antibody Heavy chain Variable region CDR2:
IN TS GS S ..rAQ '<EQ. G
59
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SEQ ID NO:22
Protein
Artificial
j1v1011 anti-RORI antibody Heavy chain Variable region CDR3:
DSSRYSGWYFDL
SEQ ID NO:23
Protein
Artificial
j1v1011 anti-ROR1 antibody Light chain Variable region:
AIQLTQSPS SL SASVGDRVT I TCRASQGVSTE IAWYQQKPGKAPKL LIYAAS SLQSGVPSRFSGSGSG
TDFTLT IS SLQPEDFATYYCQQYYGYP IAFGQGTRLE IK
SEQ ID NO:24
Protein
Artificial
j1v1011 anti-ROR1 antibody Light chain Variable region CDR1
RA SQGVST .E a A
SEQ ID NO:25
Protein
Artificial
j1v1011 anti-ROR1 antibody Light chain Variable region CDR2
24ASS.LQS
SEQ ID NO:26
Protein
Artificial
jlvl 011 anti-ROR1 antibody Light chain Variable region CDR3
QQYYGYPIA
SEQ ID NO:27
Protein
Artificial
j1v1011 single chain antibody (scFv)
QVQLVQSGAEVKKPGASVKVSCKAS GYT FT SKYYHWVRQAPGQGLEWMGI INPTSGSTSYAQKFQGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARDSSRYSGWYFDLWGQGTTVTVSSGGGGSGGGGSGGGGS
GGGGSAIQLTQS PS SLSASVGDRVTI TCRASQGVSTE IAWYQQKPGKAPKLL IYAASS LQSGVPSRFS
GSGSGTDFTLT IS SLQPEDFATYYCQQYYGYP IAFGQGTRLE IK
SEQ ID NO:28
Protein
Artificial
Signal peptide
MEWSWVFLFFLSVTTGVHS
CA 03214641 2023- 10-5
WO 2022/217048
PCT/US2022/024017
SEQ ID NO:29
Protein
Artificial
(G4S)4 linker
GGGGSGGGGSGGGGSGGGGS
SEQ ID NO:30
Protein
Artificial
Alternative GS Linker
GGGSGGGSGGGSGGGSG
SEQ ID NO:31
Protein
Artificial
(G4S)1 Linker
GGGGS
SEQ ID NO:32
Protein
Artificial
Hum291 anti-CD3 antibody Heavy chain Variable region:
QVQLVQSGAEVKKPGASVKVSCKASGYTFI SYTMHWVRQAPGQGLEWIGYINPRSGYTHYNQKLKDRA
TLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSS
SEQ ID NO:33
Protein
Artificial
Hum291 anti-CD3 antibody Light chain Variable region:
DIQMTQSP S SL SASVGDRVT I TCSAS S SVSYMNWYQQKPGKAPKRL IYDT SKLASGVP SRFS GSGS
GT
DFTLT I SSLQPEDFATYYCQQWSSNP PT FGGGTKVE IK
SEQ ID NO:34
Protein
Artificial
Hum291 anti-CD3 single chain antibody (scFv)
OVOLVOSGAEVKKPGASVKVSCKAS GYT F I SYTMHWVRQAPGQGLEWIGYINPRSGYTHYNOKLKDRA
TLTADKSAS TAYMELS SLRSEDTAVYYCARSAYYDYDGFAYWGQGT LVTVS SGGGSGGGSGGGSGGGS
GDIQMTQS P S SL SASVGDRVT I TCSAS S SVSYMNWYQQKPGKAPKRL IYDT SKLASGVP SRF
SGSGSG
TDFTLT IS SLQPEDFATYYCQQWS SNP PT FGGGTKVE IK
SEQ ID NO:35
DNA
Artificial
61
CA 03214641 2023- 10-5
WO 2022/217048
PCT/US2022/024017
Encodes oil anti-ROR1/anti-CD3 Bispecific Antibody precursor having
signal peptide - anti-ROR1 clone 011 scFv ¨ linker - anti-CD3 clone hum291
scFv
ATGGAATGGAGT TGGGTT T TCT TGTT T T TCCT TAGT GTCACCACGGGAGTCCACAGCCAAGTACAACT
GGT GCAGAGT GGTGCAGAAGT CAAGAAACCTGGCGCTAGCGT GAAGGT CT CCT GTAAAGCAAGT GGAT
ATACGTTCACAAATTATTACATGCACTGGGTCCGCCAAGCTCCCGGTCAAGGGCTGGAATGGATGGGC
ATTATAAACCCCACGTCAGGCCGAACCTCCTATGCACAAAAATTCCAGGGTAGAGTGACCATGACCAG
GGATACGTCCACAAGTACAGT T TACAT GGAGCT T TCT TCACTCCGGTCT GAAGACACT GCT GTT TAT
T
ATT GCGCCCGCGATAGCTCAAGCT GGTACTCT GGAT GGTACT T T GACCT GT GGGGACAGGGGACCACC
GTGACAGTATCTTCAGGAGGCGGCGGTTCAGGTGGCGGTGGAAGCGGGGGAGGAGGCTCCGGAGGCGG
CGGATCCGCGAT TCAGAT GACGCAATCCCCAAGCAGCCTCAGT GCAAGT GTAGGCGACCGCGTTACCA
TCACTTGCCGAGCCAGTCAAGGAATACGAACCGACCTCGCCTGGTATCAGCAGAAACCTGGGAAGGCG
CCCAAACrt Crl'Arrl'ACGCCGCGT CCT CT CT CCAGAGCGGAGT GC C_;GAGT CGArl"I'T
CAGGAAGT GG
ATCT GGGACCGATT TCACACT TACAAT T TCAAGTCT TCAGCCCGAGGACT TCGCGACGTAT TAT T GCC
AACAATAT TAT GGCTATCCTATAGCAT T CGGACAAGGAACCAGGCT CGAGAT TAAAGGCGGGGGGGGC
TCTCAAGT T CAACT TGTT CAAT CT GGAGCAGAGGTAAAGAAGCCCGGCGCGAGC GTAAAGGT CT CAT G
TAAAGCCT CAGGTTATACAT T CAT TT CCTACACAAT GCACT GGGT C CGGCAGGCACCC GGT CAAGGT
C
TCGAAT GGATAGGATATAT CAATCCT CGCAGT GGCTATACT CACTATAACCAGAAGCT CAAGGAT C GA
GCCACGTT GACT GCAGATAAGT CT GCAAGTACCGCATATAT GGAAC TT T CCT CC CT CC GCT
CAGAGGA
CACT GCAGT GTACTACTGT GCACGGT CAGCATAT TACGAT TAT GAC GGAT T CGC CTAC T
GGGGACAAG
GTACACTGGT GACCGTAAGTAGTGGT GGCGGTAGT GGT GGT GGAAGCGGT GGGGGT TCCGGAGGCGGT
TCAGGT GACAT CCAAATGACT CAGAGCCCAAGCT CACT TT CCGCCT CAGTAGGGGAT C GCGT TACAAT
AACGTGCAGT GCCT CCTCAT CCGT GAGCTATAT GAACT GGTACCAACAGAAACC TGGTAAAGCT CC GA
AGCGCTTGATATATGACACGTCAAAGCTGGCTAGTGGAGTACCCAGTAGGTTTAGTGGGAGCGGGAGC
GGTACAGATTTCACTCTGACAATATCATCACTGCAACCTGAGGACT TT GCTACC TACTAT T GCCAGCA
ATGGAGTAGTAATCCGCCGACGTT TGGT GGGGGAACGAAGGT GGAGAT CAAA
SEQ ID NO:36
Protein
Artificial
oil anti-ROR1/anti-CD3 Bispecific Antibody precursor:
signal peptide - anti-ROR1 clone 011 scFv ¨ linker - anti-CD3 clone hum291
scFv
MEW SWVFL FEL SVT TGVHSQVQLVQS GAEVKKP GASVKVS CKAS GY TFTNYYMHWVRQAP GQGL
EWMG
I INP TS GRT SYAQKEQGRVTMTRDTS T S TVYMEL S SLRSEDTAVYYCARDS S
SWYSGWYEDLWGQGTT
VTVS SGGGGSGGGGSGGGGSGGGGSAIQMTQS PS SL SASVGDRVT I TCRASQGI RTDLAWYQQKPGKA
PKLL I YAAS S LQS GVP S RFS GS GS GTDFTL TI SS LQPEDFATYYCQQYYGYP IAFGQGTRLE I
KGGGG
SQVQLVQSGAEVKKPGASVKVSCKASGYTF SYTMHWVRQAP GQGL EW GY INP RS GY THYNQKLKDR
ATLTADKSASTAYMELS SLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVS SGGGSGGGSGGGSGGG
S GD I QMTQS PS SL SASVGDRVT I TCSAS SSVSYMNWYQQKPGKAPKRL IYDT S KLASGVP S RFS
GS GS
GTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIK
SEQ ID NO:37
DNA
Artificial
Encodes slO anti-ROR1/anti-CD3 Bispecific Antibody precursor having
signal peptide - anti-ROR1 clone s10 scFv ¨ linker - anti-CD3 clone hum291
scFv
ATGGAATGGTCCTGGGTGTTCCTGTTCTTCCTGAGCGTGACCACAGGCGTGCACTCTCAGGTTCAGCT
GGT T CAGT CT GGCGCCGAAGT GAAGAAACCTGGCGCCT CT CT GAAGGT CT CCT
GCAAGGCCAGCGGCT
62
CA 03214641 2023- 10-5
WO 2022/217048
PCT/US2022/024017
ACACCTTTACCAACTACTACATGCACTGGGTCCGACAGGCCCCTGGACAAGGACTTGAGTGGATGGGC
ATCATCAACCCTAGCGGCGGCAGCACAAGCTACGCCCAGAAATTCCAGGGCAGAGTGACCATGACCAG
AGACACCAGCACCTCCACCGTGTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACT
ACTGCGCCAGAAGCAGCAGATCCAGCTACTACCTGTGGGTGCTCGATCTGTGGGGCCAGGGAACAACC
GTGACAGTCTCTTCTGGTGGCGGAGGATCTGGCGGAGGTGGAAGCGGCGGAGGCGGTAGCGGAGGTGG
TGGATCTGCAATTCAGCTGACACAGAGCCCCAGCAGCCTGTCTGCCTCTGTGGGAGACAGAGTGACAA
TCACCTGTAGAGCCAGCCAGGGCGTGTCCACAGAGATCGCTTGGTATCAGCAGAAGCCCGGCAAGGCC
CCTAAGCT GCT GAT CTAT GCT GCCTCCAGT CT GCAGAGCGGCGT CC CAT CTAGATT T T CT
GGCAGC CC
CTCCGGCACCGACTTCACCCTGACAATATCTAGCCTGCAGCCAGAGGACTTCGCCACCTACTACTGCC
AGCAGTTCAACAGCTACCCCATCACCTTCGGCCAGGGCACCAGACTGGAAATCAAAGGTGGTGGTGGC
AGCCAGGT GCAGCT CGTT CAAAGCGGAGCT GAAGT GAAAAAGCCAGGGCCCACC CT CAAACT CT CT TG
CAAAGCCTCTGGCTACACATTCATCAGCTACACCATGCATTGGGTTCGCCAGGCTCCAGGCCAGGGAC
TCGAATGGATCGGCTACATCAATCCCAGAAGCGGCTATACCCACTACAACCAGAAGCTGAAGGACCGG
GCCACACTGACCGCCGATAAGTCTGCCAGCACCGCCTATATGGAACTGTCCTCTCTGCGGAGCGAAGA
TACAGCCGTGTATTATTGTGCCCGCAGCGCCTACTACGACTACGACGGCTTTGCCTAT TGGGGACAGG
GCACCCTGGTCACCGTTTCTTCTGGCGGAGGAAGTGGCGGCGGAAGCGGTGGTGGTTCTGGCGGTGGT
AGTGGCGACATCCAGATGACCCAGTCTCCAAGCTCTCTGAGCGCCAGCGTGGGCGATAGAGTCACCAT
CACATGTAGCGCCT CCAGCAGCGT GT CCTACAT GAACT GGTAT CAACAAAAGCC TGGGAAAGCT CC CA
AGCGCCTGATCTACGACACAAGCAAACTGGCCAGCGGAGTGCCCAGCAGATTTTCCGGATCTGGCAGT
GGCACAGACTTTACACTCACCATAAGCTCACTGCAGCCCGAAGATT TT GCCACGTACTAT T GTCAGCA
ATGGTCCAGCAATCCTCCTACCTTCGGAGGCGGCACCAAGGTCGAGATCAAG
SEQ ID NO:38
Protein
Artificial
slO anti-ROR1/anti-CD3 Bispecific Antibody precursor
signal peptide - anti-ROR1 clone slO scFv ¨ linker - anti-CD3 clone hum291
scFv
MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMG
I INPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELS SLRSEDTAVYYCARS S RS SYYLWVLDLWGQGTT
VTVS SGGGGSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVT I TCRASQGVSTEIAWYQQKPGKA
PKLL I YAAS S LQS GVP S RFS GS GS GTDFTL TISS LQPEDFATYYCQQFNSYP IT
FGQGTRLEIKGGGG
SQVQLVQSGAEVKKPGASVKVSCKASGYTF I SYTMHWVRQAPGQGL EW I GY INP RS GY THYNQKLKDR
ATLTADKSASTAYMELS SLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVS SGGGSGGGSGGGSGGG
S GD I QMTQS P S SLSASVGDRVT I TCSAS SSVSYMNWYQQKPGKAPKRL IYDT SKLASGVP S RFS
GS GS
GTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIK
SEQ ID NO:39
DNA
Artificial
Encodes j1v1011 anti-ROR1/anti-CD3 Bispecific Antibody precursor having
signal peptide - anti-ROR1 clone j1v1011 scFv¨ linker - anti-CD3 clone hum291
scFv
ATGGAATGGTCCTGGGTGT TCCTGTTCT TCCTGAGCGTGACCACAGGCGTGCACTCTCAGGT TCAGCT
GGT T CAGT CT GGCGCCGAAGT GAAGAAACCTGGCGCCT CT CT GAAGGT CT CCT
GCAAGGCCAGCGGCT
ACACCTTTACCAGCAAGTACTACCACTGGGTCCGACAGGCCCCTGGACAAGGACTTGAGTGGATGGGC
ATCATCAACCCCACCAGCGGCAGCACAAGCTACGCCCAGAAATTCCAGGGCAGAGTGACCATGACCAG
AGACACCAGCACCTCCACCGTGTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACT
ACTGCGCCAGAGACAGCTCTAGATACAGCGGCTGGTACTTCGACCT GTGGGGCCAGGGAACAACCGTG
63
CA 03214641 2023- 10-5
WO 2022/217048
PCT/US2022/024017
ACAGTTTCTTCTGGCGGCGGAGGATCTGGCGGAGGTGGAAGCGGAGGCGGAGGAAGCGGTGGCGGCGG
ATCTGCTAT TCAGCTGACACAGAGCCCTAGCAGCCTGTCTGCCTCT GTGGGCGACAGAGTGACAAT CA
CCT GTAGAGCCT CT CAGGGCGT GT CCACAGAGAT CGCCTGGTAT CAGCAGAAGC CT GGCAAGGCCC CT
AAGCTGCTGATCTATGCCGCTAGCTCTCTGCAGTCCGGCGTGCCATCTAGATTTTCCGGCTCTGGCAG
CGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCAGAGGACTTCGCCACCTACTACTGTCAGC
AGTACTACGGCTACCCTATCGCCTTCGGCCAGGGCACCAGACTGGAAATCAAAGGTGGCGGTGGCAGC
CAGGTGCAGCT CGT TCAAAGCGGAGCT GAAGT GAAAAAGCCAGGGGCCAGCGT GAAAGT GT C TT GCAA
AGCCTCTGGCTACACATTCATCAGCTACACCATGCAT TGGGT TCGCCAGGCTCCAGGCCAGGGACT CG
AATGGATCGGCTACATCAATCCCAGAAGCGGCTATACCCACTACAACCAGAAGCTGAAGGACCGGGCC
ACACTGACCGCCGATAAGTCTGCCAGCACCGCCTATATGGAACTGTCCTCTCTGCGGAGCGAAGATAC
AGCCGTGTATTATTCTGCCCGCAGCGCCTACTACCACTACCACGGCTTTGCCTATTGGGCACAGGGCA
CCCTGGTCACCGTTTCTTCTGGCGGAGGAAGTGGCGGCGGAAGCGGTGGTGGTTCTGGCGGTGGTAGT
GGCGACAT CCAGAT GACCCAGT CT CCAAGCTCT CT GAGCGCCAGCGTGGGCGATAGAGT CAC CAT CAC
ATGTAGCGCCTCCAGCAGCGTGTCCTACATGAACTGGTATCAACAAAAGCCTGGGAAAGCTCCCAAGC
GCCTGATCTACGACACAAGCAAACTGGCCAGCGGAGTGCCCAGCAGATTTTCCGGATCTGGCAGTGGC
ACAGACTT TACACT CACCATAAGCTCACT GCAGCCCGAAGAT T T T GCCACGTAC TAT T GT CAGCAAT
G
GTCCAGCAATCCTCCTACCTTCGGAGGCGGCACCAAGGTCGAGATCAAG
SEQ ID NO:40
Protein
Artificial
j1v1011 anti-ROR1ianti-CD3 Bispecific Antibody precursor:
signal peptide - anti-ROR1 clone j1v1011 scFv ¨ linker - anti-CD3 clone hum291
scFv
MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSKYYHWVRQAPGQGLEWMG
I INP TS GS T SYAQKFQGRVTMTRDTS T S TVYMEL S SLRSEDTAVYYCARD S SRY SGWY
FDLWGQGT TV
TVS S GGGGS GGGGS GGGGS GGGGSAI QL TQ SP SSL SASVGDRVT T CRASQGVS
TEIAWYQQKPGKAP
KLL I YAAS SLQS GVPSRFS GSGSGTDFTL T I S SLQPEDFATYYCQQYYGY P IAFGQGT RLE I
KGGGGS
QVQLVQSGAEVKKPGASVKVSCKAS GYT F I SYTMHWVRQAPGQGLEWIGYINPRSGYTHYNQKLKDRA
TLTADKSAS TAYMELS SLRSEDTAVYYCARSAYYDYDGFAYWGQGT LVTVS S GGGS GGGS GGGS GGGS
GDIQMTQSPSSLSASVGDRVT I TCSAS S SVSYMNWYQQKPGKAPKRL I YDT SKLAS GVP SRF SGS GS
G
TDFTLT IS SLQPEDFATYYCQQWS SNP P T FGGGTKVE 1K
SEQ ID NO:41
DNA
Artificial
EF1 ct/HTLV promoter
AAGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGT
TGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGAT
GTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGT
GAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTC
CTT CACGCGCCCGCCGCCCTACCT GAGGCCGCCAT CCACGCCGGT T TACT CGCGTT CT GCCGCCTC CC
GCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGT T TAAAGCTCAGGTCGAGACCGGGCCT
TTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTG
CTCAACTCTACGTCTT TGT T T CGT TT T CT GTT CT GCGCCGT TACAGAT C
SEQ ID NO:42
DNA
64
CA 03214641 2023- 10-5
WO 2022/217048
PCT/US2022/024017
Cytomegalovirus
CMV promoter
AAGCTT GGGAGT TCCGCGT TACATAACT TACGGTAAAT GGCCCGCC TGGCT GACCGCCCAACGACCCC
CGCCCATT GACGTCAATAAT GACGTAT GT TCCCATAC TAACGCCAATACGGACT TTCCAT T GACC T CA
ATGGGT GGAGTATT TACGGTAAACTGCCCACT T GGCAGTACAT CAAGT GTAT CATAT GCCAAGTAC GC
CCCCTATT GACGTCAATGACGGTAAAT GGCCCGCCT GGCAT TAT GCCCAGTACATGACCT TATGGGAC
TTTCCTACT T GGCAGTACATCTACGTAT TAGTCATCGCTAT TACCATGGT GAT GCGGT TTTGGCAGTA
CATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATG
GGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG
CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGAT
CGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGACTCTACTAGA
SEQ ID NO:43
DNA
Artificial
Encodes Respiratory Syncytial Virus protein F (RSV) scFv Antibody precursor:
signal
peptide, VH chain ¨ linker ¨ VL chain
ATGGAATGGTCCTGGGTGTTCCTGTTCTTCCTGAGCGTGACCACAGGCGTGCACAGCCAAGTGACACT
GAGAGAGTCTGGCCCCGCTCTGGTCAAGCCTACACAGACCCTGACACTGACCTGCACCTTCAGCGGCT
TTAGCCTGAGCACAAGCGGCATGAGCGTCGGCTGGATTAGACAGCCTCCTGGCAAAGCCCTGGAATGG
CTGGCCGACAT T TGGT GGGACGACAAGAAGGACTACAACCCCAGCC TGAAGT CC CGGC T GAC CAT CAG
CAAGGACACCAGCAAGAACCAGGT GGT GCT GAAAGT GACCAACAT GGACCCT GC CGACACCGCCAC CT
ACTACT GT GCCAGATCCAT GAT CACCAACT GGTACT T CGACGT GT GGGGAGCCGGCAC CACAGT
GACA
GTTTCTAGCGGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCTGATATCCAGATGAC
ACAGAGCCCCAGCACACT GT CT GCCAGCGT GGGAGACAGAGT GACCAT CACAT GCAAGT GCCAGCT GA
GCGT GGGCTACATGCACT GGTATCAGCAGAAGCCT GGCAAGGCCCC TAAGCT GC TGAT CTACGACACA
AGCAAGCT GGCCTCTGGCGT GCCCAGCAGATT T TCT GGCAGCGGCAGCGGAACCGAGT TCACCCTGAC
CAT CTCAAGCCT GCAGCCT GACGACT T CGCTACGTACTACT GCT TC CAAGGCAGCGGC TACCCCT T
CA
CAT T TGGAGGCGGCACCAAGCT GGAAAT CAAG
SEQ ID NO:44
Protein
Artificial
Respiratory Syncytial Virus protein F (RSV) scFv antibody precursor (signal
peptide-VH
chain¨ linker¨VL chain)
MEWSWVFLFFLSVTTGVHSQVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPC_;KALEW
LADIWWDDKKDYNPSLKSRLT I SKDT S KNQVVLKVTNMDPADTATYYCARSMI TNWYFDVWGAGT TVT
VS S GGGGS GGGGS GGGGS DIQMTQSPS TL SASVGDRVT TCKCQL SVGYMHWYQQKP GKAPKLL IYDT
SKLASGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCFQGSGYPFT FGGGTKLEIK
SEQ ID NO:45
Protein
Artificial
Respiratory Syncytial Virus protein F (RSV) scFv antibody: (VH chain¨
linker¨VL chain)
CA 03214641 2023- 10-5
WO 2022/217048
PCT/US2022/024017
QVTLRE SGPALVKP TQTL TL TCTFS GFS L S TS GMSVGW IRQP PGKALEWLAD IWWDDKKDYN PS
LKS R
LT I SKDTSKNQVVLKVTNMDPADTATYYCARSMI TNWYFDVWGAGT TVTVSSGGGGSGGGGS GGGGSD
I QMTQS PS TL SASVGDRVT I TCKCQLSVGYMHWYQQKPGKAPKLL I YDT S KLAS GVP S RFS
GSGS GTE
FTLT I SSLQPDDFATYYCFQGSGYPFTFGGGTKLEIK
SEQ ID NO:46
DNA
Artificial
Encodes human IL-12 (p40-2x elastin-p35)
ATGTGCCACCAGCAGCTGGTCATCAGCTGGTTTAGCCTGGTGTTTCTGGCCTCTCCACTGGTGGCCAT
CTGGGAGCTGAAGAAAGACGT a TACGTGGTGGAACTGGACTGGTAT CCCGATGCTCCT GGCGAGAT GG
TGGTGCTGACCTGCGATACCCCTGAGGAAGAT GGCAT CACCT GGAC TCT GGACCAGT C CT CT GAGGTG
CTCGGAAGCGGCAAGACCCT GACCAT CCAAGT GAAAGAGT T T GGCGACGCCGGC CAGTACAC CT GT CA
CAAAGGCGGAGAAGTGCT GAGCCACAGCCT GCT GCT GCTCCACAAGAAAGAGGACGGCAT CT GGT C CA
CCGACATCCTGAAGGACCAGAAAGAGCCTAAGAACAAGACCTTCCTGCGCTGCGAGGCCAAGAACTAC
AGCGGCAGATTCACCTGTTGGTGGCTGACCACAATCAGCACCGACCTGACCTTCTCCGTGAAGTCTAG
CAGGGGCAGCAGTGATCCTCAGGGCGT TACATGTGGCGCCGCTACACTGTCTGCCGAAAGAGTGCGGG
GCGACAACAAAGAATACGAGTACAGCGT GGAAT GCCAAGAGGACAGCGCCT GT C CAGC CGCC GAAGAG
TCTCTGCCTATCGAAGTGATGGTGGACGCCGTGCACAAGCTGAAGTACGAGAACTACACCTCCAGCTT
TTT CAT CCGGGACATCAT CAAGCCCGAT CCTCCAAAGAACCT GCAGCT CAAGCC CCT GAAGAACAGCA
GACAGGTGGAAGTGTCTTGGGAGTACCCCGACACCTGGTCTACCCCTCACtcCTACTTCAGCCTGACC
TTT T GCGT GCAAGT GCAGGGCAAGTCCAAGCGCGAGAAAAAGGACC GGGT GT T CACCGATAAGACCAG
CGCCACCGT GAT CT GCcGAAAGAACGCCAGCAT CAGCGTCAGAGCC CAGGACCGGTAC TACAGCAGCT
CTTGGAGCGAATGGGCCAGCGTGCCATGTTCTGTGCCTGGCGTTGGAGTTCCTGGCGTGGGCAGAAAT
CTGCCAGTGGCCACGCCTGATCCTGGCATGTTTCCTTGTCTGCACCACtcCCAGAACCTGCTGAGAGC
CGTGTCCAATATGCTGCAGAAGGCCCGGCAGACACTGGAATTCTACCCCTGCACCAGCGAGGAAATCG
ACCACGAGGATATCACCAAGGACAAGACCAGCACCGTGGAAGCCTGCCTGCCTCTGGAACTGACAAAG
AACGAGAGCT GCCT GAACAGCCGGGAAACCAGCT T CAT CACCAACGGCT CT T GC CT GGCCT C
CAGAAA
GACCTCCT TCATGATGGCCCTGTGCCTGAGCAGCATCTACGAGGACCTGAAGAT GTACCAGGTGGAAT
TCAAGACCAT GAACGCCAAGCT GCTGAT GGACCCCAAGAGACAGAT CT T CCT GGACCAGAACAT GC T G
GCCGTGATCGATGAGCTGATGCAGGCCCTGAACTTCAACAGCGAGACAGTGCCCCAGAAGTCCAGCCT
GGAAGAACCCGACT TCTATAAGACCAAGAT CAAGCT GT GCAT CCT GCT GCACGC CT T C
CGGATCAGAG
CCGTGACCATCGACAGAGTGATGAGCTACCTGAACGCCTCC
SEQ ID NO:47
Protein
Artificial
Human IL-12 (p40-2x elastin-p35)
MCHQQLVI SWFS LVFLAS PLVAIWELKKDVYVVELDWY PDAPGEMVVL TCDT PE EDGI TWTL DQS S
EV
LGSGKTLT I QVKEFGDAGQYTCHKGGEVL SHS LLLLHKKEDG IWST DI LKDQKE PKNKT FLRCEAKNY
S GRFTCWWL TT I S TDL TFSVKS SRGS S DPQGVTCGAATL SAERVRGDNKEYEYSVECQEDSACPAAEE
SLPIEVMVDAVHKLKYENYTSSFFIRDI IKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLT
FCVQVQGKS KREKKDRVFTDKT SATVI CRKNAS I SVRAQDRYYSSSWSEWASVPCSVPGVGVPGVGRN
LPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLE FY PCT S EE IDHED TKDKTSTVEACL PLEL TK
NES CLNSRET S F I TNGSCLASRKTSFMMALCLSS I YEDLKMYQVE FKTMNAKLLMDPKRQ I FLDQNML
AVI DELMQALNFNS ETVPQKS S LEEPDFYKTKI KLC ILLHAFRI RAVT IDRVMSYLNAS
SEQ ID NO:48
DNA
Artificial
encodes anti-VEGFR-2 VKl38 scFv antibody precursor (signal peptide-VH chain¨
linker¨VL
chain-IgG1 Fc region)
66
CA 03214641 2023- 10-5
WO 2022/217048
PCT/US2022/024017
ATGGAATGGTCCTGGGTGTTCCTGTTCTTCCTGAGCGTGACCACAGGCGTGCACTCTGAAGTGCAGCT
GGTTGAGTCTGGCGCCGAAGTGAAGAAACCTGGGAGCAGGGTGAAGGTGTCGTGCAAGGCTTAGGGCG
GCACCTTTGGCTCTTATGGCGTGTCCTGGGTTCGCAGAGCACCTGGACAAGGCCTGGAATGGATGGGC
AGACTGAT CCCCAT CT TCGGCACCAGAGACTACGCCCAGAAAT T CCAGGGCAGAGT GACCCT GACAGC
CGACGAGTCTACCAACACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACT
ACTGTGCCAGAGATGGCGACTACTACGGCAGCGGCAGCTACTATGGCATGGATGTGTGGGGCCAGGGC
ACCCTGGT TACAGT TT CT T CT GGT GGCGGAGGAT CT GGCGGAGGT GGAAGCGGC GGAGGCGGAT CT
GA
AACAACACTGACACAGAGCCCCGCCACACTGAGTGTGTCTCCAGGCGAAAGGGCCACCGTGT CT TGT c
GAGCCTCTCAGAGCCTGGGCAGCAACCTCGGATGGTTCCAGCAGAAACCAGGACAGGCCCCTCGGCTG
CTGATCTATGGCGCTTCTACAAGAGCCACAGGCATCCCCGCCAGAT TT TCTGGCTCTGGCAGCGGAAC
CGAGTTCACCCTGACAATCTCTAGCCTGCAGTCCGAGGACTTCGCTGTGTACTTCTGCCAGCAGTACA
AGGACTGGCCCATGAGATTCGGCCAGGGGACCAAGGTGGAAATCAAAGAGCCCAAGAGCAGGGACAAG
ACCCACACCTGTCCTCCATGTCCTGCTCCt GAACTGCTCGGCGGACCT TCCGTGTT TCTGT T CCCT CC
AAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCC
ACGAGGACCCAGAAGT GAAGT T cAAc T GGTAt GT gGACGGCGT GGAAGT GCACAACGC CAAGACCAAG
CCTAGAGAGGAACAGTACAACAGCACCTACAGAGT GGT GT CCGT GC TGACCGT GCT GCACCAGGAT TG
GCT GAACGGCAAAGAGTACAAGTGCAAGGT GT CCAACAAGGCCCT GCCT GCT CC TAT C GAGAAAAC CA
TCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTG
ACCAAGAATCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATG
GGAGAGCAAT GGCCAGCCAGAGAACAACTACAAGACCACT CCT CCT GTGCTGGACAGCGACGGCTCAT
TCTTCCTGTACtcCAAGCTGACAGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGC
GTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGTCCCTGT CTCTGAGCCCCGGCAAG
SEQ ID NO:49
Protein
Artificial
anti-VEGFR-2 VKB8 scFv-Fcl antibody precursor (signal peptide-VH chain¨
linker¨VL
chain- IgG1 Fc region)
MEWSWVFL FFL SVT TGVHS EVQLVQS GAEVKKPGS SVKVS CKAYGGTFGSYGVSWVRRAPGQGLEWMG
RL I P1 FGTRDYAQKFQGRVTL TADES TNTAYMEL S S LRSEDTAVYYCARDGDYYGS GSYYGMDVWGQG
TLVTVS SGGGGS GGGGS GGGGS ET TL TQS PATL SVS PGERATVS CRASQS L GSNLGWFQQKP
GQAP RL
LIYGASTRATGI PARFSGSGSGTEFTLT S SLQSEDFAVYFCQQYNDWP ITFGQGTKLEIKEPKSSDK
THTCPPCPAPELLGGP SVFL FP PKPKDTLM I S RT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLP PS RDEL
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CS
VMHEALHNHYTQKS LS LS PGK
SEQ ID NO:50
DNA
Artificial
anti-VEGFR-2 VKB8 scFv-Fcl antibody (VH chain¨ linker¨VL chain- IgG1 Fc
region)
EVQLVQSGAEVKKPGS SVKVSCKAYGGT FGSYGVSWVRRAPGQGLEWMGRL I P I FGTRDYAQKFQGRV
TLTADESTNTAYMELSSLRSEDTAVYYCARDGDYYGSGSYYGMDVWGQGTLVTVSSGGGGSGGGGS GC
GGSETTLTQSPATLSVSPGERATVSCRASQSLGSNLGWFQQKPGQAPRLL I YGASTRATGI PARFS GS
GSGTEFTLTISSLQSEDFAVYFCQQYNDWP I T FGQGTKLE I KE PKS SDKTHTCP PCPAPELLGGPSVF
LFPPKPKDTLMI SRTPEVTGVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREFOYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKAL PAP IEKT I SKAKGQPREPQVYTL P P SRDEL TKNQVSL TCLVKGFYP S D
I
AVEWESNGQPENNYKT TP PVLDSDGS FFLY SKL TVDKS RWQQGNVF SCSVMHEALHNHYTQKSL SLSP
GK
SEQ ID NO:51
Protein
T2A self-cleaving peptide
67
CA 03214641 2023- 10-5
WO 2022/217048
PCT/US2022/024017
GS GE GRGS LL T CGDVE ENP GP
SEQ ID NO:52
Protein
Artificial
A7 anti-ROR1 antibody Heavy Chain Variable Region
QVQLVE SGGGLVKPGGS LRL SCAAS GET FS DYYMTW I RQAPGKGLEWVSY I S GS
SAYSNYADSVKGRF
T I S RDNSKNTLYLQMNS LRAEDTAVYYCARDPLLYGWL TDWGQGTLVTVS S
SEQ ID NO:53
Protein
Artificial
A7 anti-ROR1 antibody Heavy Chain CDR1
DYYMT
SEQ ID NO:54
Protein
Artificial
A7 anti-ROR1 antibody Heavy Chain CDR2
Y I S GS SAYSNYADSVKG
SEQ ID NO:55
Protein
Artificial
A7 anti-RORI antibody Heavy Chain CDR3
DPLLYGWLTD
SEQ ID NO:56
Protein
Artificial
A7 anti-ROR1 antibody Light Chain Variable Region
QSAL TQPASVS GS PGQS I T I SCTGTS SVSWYQQHPGKAPKLM I YEVSKRP SGVSNRFS GS KS
GNTASL
TI S GLQAEDEADYYCS SY INDAVFEGGGTKLTVL
SEQ ID NO:57
Protein
Artificial
A7 anti-ROR1 antibody Light Chain CDR1
TGTS S
SEQ ID NO:58
Protein
Artificial
A7 anti-ROR1 antibody Light Chain CDR2
EVSKRPS
SEQ ID NO:59
Protein
Artificial
68
CA 03214641 2023- 10-5
WO 2022/217048
PCT/US2022/024017
A7 anti-ROR1 antibody Light Chain CDR3
S SY INDAVF
SEQ ID NO:60
Protein
Artificial
A8 anti-ROR1 antibody Heavy Chain Variable Region
QVQLVE SGGGLVKPGGS LRL SCAAS GET FS DYYMTW I RQAPGKGLEWVSY I S GS
SAYSNYADSVKGRF
T I SRDNSKNTLYLQMNSLRAEDTAVYYCARDPLLYGWL TDWGQGTLVTVS S
SEQ ID NO:61
Protein
Artificial
A8 anti-ROR1 antibody Heavy Chain CDR1
DYYMT
SEQ ID NO:62
Protein
Artificial
A8 anti-ROR1 antibody Heavy Chain CDR2
Yl S GS SAYSNYADSVKG
SEQ ID NO:63
Protein
Artificial
anti-ROR1 antibody Heavy Chain CDR3
DPLLYGWLTD
SEQ ID NO:64
Protein
Artificial
A8 anti-ROR1 antibody Light Chain Variable Region
QSAL TQPASVS GS PGQS I T I SCTGTS SDGGGYDSVSWYQQHPGKAP KLMI YDVNKRP S GVS GRES
GSK
GNTASLT I 5 GLQAEDEADYYCS 5 FT SDVMVFGGGTKL TVL
SEQ ID NO:65
Protein
Artificial
anti-ROR1 antibody Light Chain CDR1
TGTSSDGGGYDSVS
SEQ ID NO:66
Protein
Artificial
A8 anti-ROR1 antibody Light Chain CDR2
DVNKRPS
69
CA 03214641 2023- 10-5
WO 2022/217048
PCT/US2022/024017
SEQ ID NO:67
Protein
Artificial
A8 anti-ROR1 antibody Light Chain CDR3
SSFTSDVMV
SEQ ID NO:68
Protein
Artificial
2x elastin linker
VPGVGVPGVG
SEQ ID NO:69
Protein
Artificial
(GGGGS)3 linker
GGGGS GGGGS GGGGS
CA 03214641 2023- 10-5
