The present disclosure generally relates to bispecific anti-PD-L1 x VEGF antibodies, a
method for preparing the same and uses thereof
BACKGROUND
Angiogenesis is essential for the growth of tumor and the development of metastasis.
Controlling tumor-associated angiogenesis is a promising strategy for cancer therapy. Vascular
endothelial growth factor (VEGF) is the key mediator of angiogenesis, and has been validated in
20 various types of human cancers. Tumor cells release growth factors such as VEGF that bind to
nearby endothelial cells, which initiates a signaling cascade that stimulates endothelial cells to
divide and form new blood vessels. VEGF signaling through its receptors VEGFR plays a
critical role in angiogenesis and growth of many solid tumors. Antiangiogenic drugs, such as
Avastin (Bevacizumab) which targets the VEGF pathway, have achieved a success in clinic.
25 On the other hand, targeting immune checkpoint molecules, such as programmed death
ligand 1 (PD-L1) or its receptor, programmed death 1 (PD-1), has shown promising clinical
success. PD-L1 expression strongly correlates with unfavorable prognosis in various types of
cancers. Anti-PD-L1 antibody can target PD-L1 expressed on tumor cells and tumor-infiltrating
immune cells, and prevent binding to PD-1 and B7.1 on the surface ofT cells, and also enable
30 the activation ofT cells as well as recruit other T cells to attack the tumor, then empower the
immune system to fight multiple types of cancer.
Anti-VEGF therapy, in addition to its established anti-angiogenic effects, may further
enhance anti-PD-1/PD-L1 therapy's ability to restore anti-cancer immunity, by inhibiting VEGFrelated
immunosuppression, promoting T -cell tumor infiltration and enabling priming and
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activation ofT-cell responses against tumor antigens. Therefore, developing VEGF and PD-L1
bispecific antibodies which combine anti-angiogenesis therapy and immune checkpoint
inhibition together could achieve promising results in cancer therapy.
Despite the obvious benefit of targeting VEGF and also targeting PD-1/PD-L1 therapies,
5 there is still a significant unmet need. 15%-20% patients do not respond to anti-VEGF treatment,
and increasing evidence has indicated that prolonged use of anti-VEGF agents for cancer therapy
promotes tumor resistance. 3%-9% patients develop immunogenicity of the treatment. And also
there exists the limited overall survival time extension and limited safety issue, including
changes in bone morphology, glomerulopathy with inflammation in kidney, and decreased
10 vacuolation with inflammation in adrenal gland. Immune checkpoint inhibitors blocking the PD-
1/PD-L1 pathway, such as nivolumab, pembrolizumab and atezolizumab, represent a standard
treatment option for patients with multiple cancers. However, with a response rate of 14-23% in
unselected populations and 16-48% in patients with PD-L1-expressing tumors, these drugs offer
improved outcomes in some patients, but not all.
15 Therefore, there is great need to develop novel anti-PD-L1/anti-VEGF bispecific antibodies.
20
In the present disclosure, a bispecific antibody that could simultaneously bind to human PD-L1
and VEGF with high affinity, block both PD-1/PD-L1 and VEGF/VEGFR signaling, and display
superior anti-tumor efficacy has been generated.
SUMMARY
These and other objectives are provided for by the present disclosure which, in a broad
sense, is directed to compounds, methods, compositions and articles of manufacture that provide
antibodies with improved efficacy. The benefits provided by the present disclosure are broadly
applicable in the field of antibody therapeutics and diagnostics and may be used in conjunction
25 with antibodies that react with a variety of targets.
In one aspect, the present disclosure provides a bispecific antibody or antigen-binding
portion thereof, comprising a PD-L1 antigen-binding moiety associated with a VEGF antigenbinding
moiety, wherein:
the PD-L1 antigen-binding moiety comprises: a complementarity determining region (CDR)
30 1 comprising SEQ ID NO: 1, a CDR2 comprising SEQ ID NO: 2, and a CDR3 comprising SEQ
IDNO: 3, and
the VEGF antigen-binding moiety comprises: a heavy chain complementarity determining
region (HCDR) 1 comprising SEQ ID NO: 4, a HCDR2 comprising SEQ ID NO: 5, a HCDR3
comprising SEQ ID NO: 6, a light chain complementarity determining region (LCDR) 1
35 comprising SEQ ID NO: 7, a LCDR2 comprising SEQ ID NO: 8, and a LCDR3 comprising SEQ
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IDNO: 9.
PCT/CN2021/072584
In certain embodiements, the PD-L1 antigen-binding moiety as disclosed herein comprises:
a variable domain comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid
sequence at least 85%, 90%, or 95% identical to SEQ ID NO: 10.
5 In certain embodiements, the VEGF antigen-binding moiety as disclosed herein comprises:
a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 11 or
an amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NO: 11; and
a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12 or an
amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NO: 12.
10 In certain embodiements, the PD-L1 antigen-binding moiety is fused to theN terminal of
the VEGF antigen-binding moiety. In some other embodiements, the PD-L1 antigen-binding
moiety is fused to the C terminal of the VEGF antigen-binding moiety.
In certain embodiements, the PD-L1 antigen-binding moiety is from a single-domain
antibody (sdAb ), such as a VHH antibody. The VHH may be derived from a camelid animal,
15 comprising an alpaca or a llama. Preferably, the VHH is a humanized VHH.
In certain embodiements, the PD-L1 antigen-binding moiety is operably linked to the N
terminal of the light chain or heavy chain of the VEGF antigen-binding moiety, optionally via a
linker. The linker may comprise or consist of 1 to 4 copies of GGGGS (G4S), for example, the
linker may be (G4S)2.
20 In certain embodiements, the bispecific antibody or antigen-binding portion thereof as
disclosed herein comprises a heavy chain and a light chain, wherein:
the heavy chain comprises domains operably linked as in VH-CH1-hinge-Fc, wherein the
VH-CH1 is from the VEGF antigen binding moiety; and
the light chain comprises domains operably linked as in VHH-VL-CL, wherein the VHH is
25 from the PD-L1 antigen binding moiety and the VL-CL is from the VEGF antigen binding
moiety.
In certain embodiements, the Fe region is a human IgG Fe region, preferably a human IgG1
Fe region.
In certain embodiements, the bispecific antibody or antigen-binding portion thereof as
30 disclosed herein comprise a heavy chain comprising SEQ ID NO: 13 and a light chain
comprising SEQ ID NO: 14.
In certain embodiements, the bispecific antibody or antigen-binding portion thereof as
disclosed herein is a humanized antibody.
In one aspect, the present disclosure provides an isolated nucleic acid molecule, comprising
35 a nucleic acid sequence encoding the bispecific antibody or the antigen-binding portion thereof
as disclosed herein.
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In one aspect, the present disclosure provides a vector comprising the nucleic acid molecule
as disclosed herein. In one aspect, the present disclosure provides a host cell comprising the
nucleic acid molecule or the vector as disclosed herein.
In one aspect, the present disclosure provides a pharmaceutical composition comprising the
5 bispecific antibody or the antigen-binding portion thereof as disclosed herein and a
pharmaceutically acceptable carrier.
In one aspect, the present disclosure provides a method for producing the bispecific
antibody or the antigen-binding portion thereof as disclosed herein, comprising the steps of:
- expressing the antibody or the antigen-binding portion thereof in the host cell; and
10 -isolating the antibody or antigen-binding portion thereof from the host cell.
In one aspect, the present disclosure provides a method for modulating an immune response
in a subject, comprising administering to the subject the bispecific antibody or the antigenbinding
portion thereof or the pharmaceutical composition as disclosed herein to the subject.
In one aspect, the present disclosure provides a method for inhibiting growth of tumor cells
15 in a subject, comprising administering an effective amount of the bispecific antibody or the
antigen-binding portion thereof or the pharmaceutical composition as disclosed herein to the
subject.
In one aspect, the present disclosure provides a method for preventing or treating cancer in a
subject, comprising administering an effective amount of the bispecific antibody or the antigen-
20 binding portion thereof or the pharmaceutical composition to the subject. The cancer may be
selected from colon cancer, colorectal cancer, breast cancer, lung cancer, cervical cancer, renal
cancer, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, esophageal cancer,
gastric cancer, lymphoma, melanoma, liver cancer, and head and neck cancer. In certain
embodiements, the cancer is colon cancer or colorectal cancer.
25 In certain embodiements, the bispecific antibody or antigen-binding portion thereof as
disclosed herein may be administered in combination with a chemotherapeutic agent, radiation
and/or other agents for use in cancer immunotherapy.
In one aspect, the present disclosure provides a bispecific antibody or antigen-binding
portion thereof as disclosed herein for use
30 i) in the modulation ofPD-L1/VEGF related immune responses;
ii) in enhancing T cell proliferation and cytokine production; and/or
iii) in stimulating an immune response or function, such as boosting the immune response
against cancer cells.
In one aspect, the present disclosure provides the bispecific antibody or antigen-binding
35 portion thereof as disclosed herein for use in diagnosing, preventing or treating cancers.
In one aspect, the present disclosure provides use of the bispecific antibody or antigen-
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binding portion thereof as disclosed herein in the manufacture of a medicament for modulating
an immune response or inhibiting growth of tumor cells in a subject.
In one aspect, the present disclosure provides use of the bispecific antibody or antigenbinding
portion thereof as disclosed herein in the manufacture of a medicament for treating or
5 preventing cancers.
In one aspect, the present disclosure provides a kit comprising the bispecific antibody or
antigen-binding portion thereof as disclosed herein. The kit can be used for detection, diagnosis,
prognosis, or treatment of a disease or condition, such as cancer.
The foregoing is a summary and thus contains, by necessity, simplifications, generalizations,
10 and omissions of detail; consequently, those skilled in the art will appreciate that the summary is
illustrative only and is not intended to be in any way limiting. Other aspects, features, and
advantages of the methods, compositions and/or devices and/or other subject matter described
herein will become apparent in the teachings set forth herein. The summary is provided to
introduce a selection of concepts in a simplified form that are further described below in the
15 Detailed Description. This summary is not intended to identify key features or essential features
of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of
the claimed subject matter. Further, the contents of all references, patents and published patent
applications cited throughout this application are incorporated herein in entirety by reference.
20 BRIEF DESCFRIPTION OF FIGURES
Figure 1 shows a schematic representation of the format ofW3256 antibody.
Figures 2 shows SDS-PAGE of W3256 antibody (lane 1). NR: Non-reducing status, R:
reducing status in NuPAGE (Novex 4-12% Bis-Tris) gel. M, PageRuler™ Unstained Protein
Ladder.
25 Figures 3 shows the result ofHPLC-SEC ofW3256 antibody.
Figure 4 shows the DSF profile ofW3256 antibody.
Figure 5 shows the ELISA binding result of W3256 antibody to human VEGF (same as
cyno VEGF). WBP332-1.80.12 X Ab.hlgG1 is an isotype control.
Figure 6 shows the FACS binding result ofW3256 antibody to human PD-Ll.
30 Figure 7 shows the dual binding result ofW3256 antibody to VEGF and then PD-Ll.
Figure 8 shows the dual binding ofW3256 antibody to PD-L1 and then VEGF.
Figure 9 shows the FACS binding result ofW3256 antibody to cynomolgus PD-Ll.
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Figure 10 shows the ELISA binding result ofW3256 antibody to mouse VEGF.
Figure 11 shows the FACS binding result ofW3256 antibody to mouse PD-Ll.
Figures 12-13 show the SPR sensorgram ofW3256 antibody binding to human PD-L1 (Fig.
12) and VEGF (Fig. 13).
5 Figures 14-15 show the competition of W3256 antibody to human VEGFR1 on human
VEGF binding (Fig. 14), and the competition ofW3256 antibody to human PD-1 on human PDL1
binding (Fig. 15).
Figures 16-17 show the competition of W3256 antibody to mouse VEGFR1 on human
VEGF binding (Fig. 16), and the competition ofW3256 antibody to mouse PD-1 on mouse PD-
10 L1 binding (Fig. 17).
Figure 18 shows the inhibition of antibodies on HUVEC cells proliferation.
Figure 19 shows the effect of antibodies on reporter gene assay.
Figure 20 shows the effect of antibodies on hCD4+T cell IFN-y secretion in MLR. "Combo"
refers to the combination ofWBP325-BMK3.ulgG1 and W315-BMK8.ulgG1K(RKNA).
15 Figure 21 shows mouse PK analysis of W3256 antibody in total IgG binding assay after a
single intravenous injection of equal molar dose of antibody. "mpk" refers to the abbreviation of
"mg/kg".
Figure 22 shows mouse PK analysis ofW3256 antibody in dual-antigen binding assay after
a single intravenous injection of equal molar dose of antibody.
20 Figure 23 shows the efficacy of antibodies in PBMC-RKO cancer model in mice.
DETAILED DESCRIPTION
While the present disclosure may be embodied in many different forms, disclosed herein are
specific illustrative embodiments thereof that exemplify the principles of the disclosure. It
25 should be emphasized that the present disclosure is not limited to the specific embodiments
illustrated. Moreover, any section headings used herein are for organizational purposes only and
are not to be construed as limiting the subject matter described.
Unless otherwise defined herein, scientific and technical terms used in connection with the
present disclosure shall have the meanings that are commonly understood by those of ordinary
30 skill in the art. Further, unless otherwise required by context, singular terms shall include
pluralities and plural terms shall include the singular. More specifically, as used in this
specification and the appended claims, the singular forms "a," "an" and "the" include plural
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referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein"
includes a plurality of proteins; reference to "a cell" includes mixtures of cells, and the like. In
this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of
the term "comprising," as well as other forms, such as "comprises" and "comprised," is not
5 limiting. In addition, ranges provided in the specification and appended claims include both end
points and all points between the end points.
Generally, nomenclature used in connection with, and techniques of, cell and tissue culture,
molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry
and hybridization described herein are those well-known and commonly used in the art. The
10 methods and techniques of the present disclosure are generally performed according to
conventional methods well known in the art and as described in various general and more
specific references that are cited and discussed throughout the present specification unless
otherwise indicated. See, e.g., Abbas et al., Cellular and Molecular Immunology, 6th ed., W.B.
Saunders Company (2010); Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual,
15 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); Ausubel et al.,
Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in
Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998);
and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The
20 nomenclature used in connection with, and the laboratory procedures and techniques of,
analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry
described herein are those well-known and commonly used in the art.
Definitions
25 In order to better understand the disclosure, the definitions and explanations of the relevant
terms are provided as follows.
The term "antibody" or "Ab," herein is used in the broadest sense, which encompasses
various antibody structures, including polyclonal antibodies, monospecific and multispecific
antibodies (e.g. bispecific antibodies). A native intact antibody generally is a Y-shaped
30 tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together
by covalent disulfide bonds and non-covalent interactions. Light chains of an antibody may be
classified into K and 'A light chain. Heavy chains may be classified into !l, 8, y, a and £,
which define isotypes of an antibody as IgM, IgD, IgG, IgA and IgE, respectively. In a
light chain and a heavy chain, a variable region is linked to a constant region via a "J"
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region of about 12 or more amino acids, and a heavy chain further comprises a "D" region
of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region
(VH) and a heavy chain constant region (CH). A heavy chain constant region consists of 3
domains (CHI, CH2 and CH3). Each light chain consists of a light chain variable region
5 (VL) and a light chain constant region (CL). VH and VL region can further be divided into
hypervariable regions (called complementary determining regions (CDR)), which are
interspaced by relatively conservative regions (called framework region (FR)). Each VH
and VL consists of 3 CDRs and 4 FRs in the following order: FRI, CDRI, FR2, CDR2,
FR3, CDR3, FR4 from N-terminal to C-terminal. The variable region (VH and VL) of each
10 heavy/light chain pair forms antigen binding sites, respectively. Distribution of amino acids
in various regions or domains follows the definition in Kabat Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991 )), or
Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:878-
883. Antibodies may be of different antibody isotypes, for example, IgG (e.g., IgG 1, IgG2,
15 IgG3 or IgG4 subtype), IgAl, IgA2, IgD, IgE or IgM antibody.
The term "antigen-binding portion" or "antigen-binding fragment" of an antibody,
which can be interchangeably used in the context of the application, refers to polypeptides
comprising fragments of a full-length antibody, which retain the ability of specifically
binding to an antigen that the full-length antibody specifically binds to, and/or compete
20 with the full-length antibody for binding to the same antigen. Generally, see Fundamental
Immunology, Ch. 7 (Paul, W., ed., the second edition, Raven Press, N.Y. (1989), which is
incorporated herein by reference for all purposes. Antigen-binding fragments of an
antibody may be derived, e.g., from full antibody molecules using any suitable standard
techniques such as proteolytic digestion or recombinant genetic engineering techniques
25 involving the manipulation and expression of DNA encoding antibody variable and
optionally constant domains. Such DNA is known and/or is readily available from, e.g.,
commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be
synthesized. The DNA may be sequenced and manipulated chemically or by using
molecular biology techniques, for example, to arrange one or more variable and/or constant
30 domains into a suitable configuration, or to introduce codons, create cysteine residues,
modify, add or delete amino acids, etc.
Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii)
F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv)
molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino
35 acid residues that mimic the hypervariable region of an antibody (e.g., an isolated
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complementarity determining regwn (CDR) such as a CDR3 peptide), or a constrained
FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies,
single domain antibodies, domain- deleted antibodies, chimeric antibodies, CDR-grafted
antibodies, diabodies, triabodies, tetrabodies, mini bodies, nanobodies (e.g. monovalent
5 nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs),
and shark variable IgNAR domains, are also encompassed within the expression "antigenbinding
fragment," as used herein. In certain embodiments, an antigen-binding fragment of an
antibody may contain at least one variable domain covalently linked to at least one constant
domain. The variable and constant domains may be either directly linked to one another or may
10 be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g.,
5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage
between adjacent variable and/or constant domains in a single polypeptide molecule.
The term "variable domain" with respect to an antibody as used herein refers to an antibody
variable region or a fragment thereof comprising one or more CDRs. Although a variable domain
15 may comprise an intact variable region (such as HCVR or LCVR), it is also possible to comprise
less than an intact variable region yet still retain the capability of binding to an antigen or
forming an antigen-binding site.
The term "antigen-binding moiety" as used herein refers to an antibody fragment formed
from a portion of an antibody comprising one or more CDRs, or any other antibody fragment
20 that binds to an antigen but does not comprise an intact native antibody structure. Different from
the term "antigen binding site" which generally refers to the variable domains, an antigenbinding
moiety may comprise constant domains in addition to variable domains. Examples of
antigen-binding moiety include, without limitation, a variable domain, a variable region, a
diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a
25 (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a
multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and
a bivalent domain antibody. An antigen-binding moiety is capable of binding to the same antigen
to which the parent antibody binds. In certain embodiments, an antigen-binding moiety may be a
Fab fragment or a VHH antibody. In some embodiments, an antigen-binding moiety may
30 comprise one or more CDRs from a particular human antibody grafted to a framework region
from one or more different human antibodies. For more and detailed formats of antigen-binding
moiety are described in Spiess et al., Molecular Immunology, 67(2), pp.95-106 (2015), and
Brinkman et al., mAbs, 9(2), pp.182-212 (2017), which are incorporated herein by their entirety.
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"Fab" with regard to an antibody refers to that portion of the antibody consisting of a single
light chain (both variable and constant regions) associating to the variable region and first
constant region of a single heavy chain by a disulfide bond.
"Fe" with regard to an antibody refers to that portion of the antibody comprising the second
5 (CH2) and third (CH3) constant regions of a first heavy chain bound to the second and third
constant regions of a second heavy chain via disulfide bonding. When referring to a Fe region,
depending on the context, it may refer to one chain or both chains of the Fe region. The Fe
portion of the antibody is responsible for various effector functions such as ADCC, and CDC,
but does not function in antigen binding. The capacity of antibodies to initiate and regulate
10 effector functions through their Fe domain is a key component of their in vivo protective activity.
Although the neutralizing activity of antibodies has been previously considered to be solely the
outcome of Fab-antigen interactions, it has become apparent that their in vivo activity is highly
dependent on interactions of the IgG Fe domain with its cognate receptors, Fey receptors (FcyRs),
expressed on the surface of effector leukocytes.
15 The term "PD-L1", also known as programmed death-ligand 1, is a 40 kDa type 1
transmembrane protein that has been speculated to play a major role in suppressing the adaptive
arm of immune system. PD-L1 is the principal ligand of programmed death 1 (PD-1), a
coinhibitory receptor that can be constitutively expressed or induced in myeloid, lymphoid,
normal epithelial cells and in cancer. The term "PD-L1" as used herein, when referring to the
20 amino acid sequence of PD-L1 protein, including full-length PD-L1 protein, or the extracellular
domain of PD-L1 (PD-L1 ECD) or fragment containing PD-L1 ECD; Fusion protein of PD-L1
ECD, for example, fragment fused with IgG Fe from mice or human (mFc or hFc) is also
included. Moreover, as understood by a person skilled in the art, PD-L1 protein would also
include those into which mutations of amino acid sequence are naturally or artificially introduced
25 (including but not limited to replacement, deletion and/or addition) without affecting the
biological functions.
The term "an antibody that binds PD-L1" or an "anti-PD-L1 antibody" as used herein
includes antibodies and antigen-binding fragments thereof that specifically recognize PD-Ll.
The antibodies and antigen-binding fragments of the present disclosure may bind soluble PD-L1
30 protein and/or cell surface expressed PD-Ll. Soluble PD-L1 includes natural PD-L1 proteins as
well as recombinant PD-L1 protein variants that lack a transmembrane domain or are otherwise
unassociated with a cell membrane .As used herein, the expression "anti-PD-L1 antibody"
includes both monovalent antibodies with a single specificity, as well as bispecific antibodies
comprising a first antigen-binding site that binds PD-L1 and a second antigen-binding site that
35 binds a second (target) antigen, wherein the anti-PD-L1 antigen-binding site comprises any of
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the HCVR/LCVR or CDR sequences as set forth in Table A herein. Examples of anti-PD-L1
bispecific antibodies are described elsewhere herein. The term "antigen-binding molecule"
includes antibodies and antigen-binding fragments of antibodies, including, e.g., bispecific
antibodies.
5 The term "VEGF" (vascular endothelial growth factor, also known as VEGF-A), is a signal
protein produced by cells that stimulates the formation of blood vessels. VEGF is a sub-family of
growth factors, the platelet-derived growth factor family of cystine-knot growth factors. They are
important signaling proteins involved in both vasculogenesis (the de novo formation of the
embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing
10 vasculature). The VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF
(placental growth factor), VEGF-E (Orf-VEGF), and Trimeresurusjlavoviridis svVEGF.
The term "VEGF receptor" or "VEGFR" as used herein refers to receptors for vascular
endothelial growth factor (VEGF). There are three main subtypes ofVEGFR, numbered 1, 2 and
3. The VEGF receptors may be membrane-bound or soluble, depending on alternative splicing.
15 Among the VEGF receptors, VEGFR-1 binds VEGF-A, PlGF, and VEGF-B.
As used herein, a "bispecific antibody" refers to an artificial antibody, which has
fragments derived from two different monoclonal antibodies and is capable of binding to
two different epitopes. The two epitopes may present on the same antigen, or they may
present on two different antigens.
20 The term "bispecific antigen-binding molecule" means a protein, polypeptide or
molecular complex comprising at least a first antigen-binding domain (also referred to as a
first antigen-binding site herein) and a second antigen-binding domain (also referred to as a
second antigen-binding site herein). In some embodiments, the "bispecific antigen-binding
molecule" is a "bispecific antibody". Each antigen-binding domain within the bispecific
25 antibody comprises at least one CDR that alone, or in combination with one or more
additional CDRs and/or FRs, specifically binds to a particular antigen. In the context of the
present disclosure, the first antigen-binding site specifically binds to a first antigen (e.g.,
PD-L1), and the second antigen-binding site specifically binds to a second, distinct antigen
(e.g., VEGF).
30 The term "anti-PD-Lllanti-VEGF antibody", "anti-PD-Lllanti-VEGF bispecific
antibody", "antibody against PD-L1 and VEGF", "anti-PD-L1 xVEGF bispecific antibody",
"PD-L1 xVEGF antibody", as used herein interchangeably, refers to a bispecific antibody
that specifically binds to PD-L1 and VEGF.
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The term "monoclonal antibody" or "mAb", as used herein, refer to a preparation of
antibody molecules of single molecular composition. A monoclonal antibody displays a
single binding specificity and affinity for a particular epitope.
The term "chimeric antibody", as used herein, refers to antibodies in which the
5 variable region sequences are derived from one species and the constant region sequences
are derived from another species, such as an antibody in which the variable region
sequences are derived from a mouse antibody and the constant region sequences are
derived from a human antibody.
The term "humanized antibody" is intended to refer to antibodies in which CDR sequences
10 derived from the germline of another mammalian species, such as a mouse, have been grafted
onto human framework sequences. Additional framework region modifications may be made
within the human framework sequences.
The term "operably linked" refers to a juxtaposition, with or without a spacer or linker, of
two or more biological sequences of interest in such a way that they are in a relationship
15 permitting them to function in an intended manner. When used with respect to polypeptides, it is
intended to mean that the polypeptide sequences are linked in such a way that permits the linked
product to have the intended biological function. For example, an antibody variable region may
be operably linked to a constant region so as to provide for a stable product with antigen-binding
activity. The term may also be used with respect to polynucleotides. For one instance, when a
20 polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g.,
promoter, enhancer, silencer sequence, etc.), it is intended to mean that the polynucleotide
sequences are linked in such a way that permits regulated expression of the polypeptide from the
polynucleotide.
The term "Ka," as used herein, is intended to refer to the association rate of a particular
25 antibody-antigen interaction, whereas the term "Kd" as used herein, is intended to refer to the
dissociation rate of a particular antibody-antigen interaction. Kd values for antibodies can be
determined using methods well established in the art. The term "Kn" as used herein, is intended
to refer to the dissociation constant of a particular antibody-antigen interaction, which is
obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M).
30 A preferred method for determining the Kd of an antibody is by using surface plasmon resonance,
preferably using a biosensor system such as a Biacore® system.
The term "high affinity" for an IgG antibody, as used herein, refers to an antibody having a
Kn of 1 x 10"7 M or less, more preferably 5 x 10"8 M or less, even more preferably lxl0-8 M or
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less, even more preferably 5 x 1 o-9 M or less and even more preferably 1 x 1 o-9 M or less for a
target antigen.
The term "EC50," as used herein, which IS also termed as "half maximal effective
concentration" refers to the concentration of a drug, antibody or toxicant which induces a
5 response halfway between the baseline and maximum after a specified exposure time. In the
context of the application, EC50 is expressed in the unit of "nM".
The term "IC50", as used herein, which is also termed as "half maximal inhibitory
concentration" is a measure of the potency of a substance in inhibiting a specific biological or
biochemical function. In the context of the application, IC50 is expressed in the unit of "nM".
10 The ability of "inhibit binding," as used herein, refers to the ability of an antibody or
antigen-binding fragment thereof to inhibit the binding of two molecules ( eg, human PDL1/
VEGF and human PD-1/VEGFR) to any detectable level. In certain embodiments, the
binding of the two molecules can be inhibited by the antibodies at an IC50 of no more than 50 nM,
no more than 30 nM, no more than 10 nM, no more than 5 nM, no more than 1 nM or even less.
15 The term "epitope," as used herein, refers to a portion on antigen that an
immunoglobulin or antibody specifically binds to. "Epitope" is also known as "antigenic
determinant". Epitope or antigenic determinant generally consists of chemically active
surface groups of a molecule such as amino acids, carbohydrates or sugar side chains, and
generally has a specific three-dimensional structure and a specific charge characteristic.
20 See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.
E. Morris, Ed. (1996).
The term "isolated," as used herein, refers to a state obtained from natural state by
artificial means. If a certain "isolated" substance or component is present in nature, it is
possible because its natural environment changes, or the substance is isolated from natural
25 environment, or both. For example, a certain un-isolated polynucleotide or polypeptide
naturally exists in a certain living animal body, and the same polynucleotide or polypeptide
with a high purity isolated from such a natural state is called isolated polynucleotide or
polypeptide. The term "isolated" excludes neither the mixed artificial or synthesized
substance nor other impure substances that do not affect the activity of the isolated
30 substance.
The term "isolated antibody," as used herein, is intended to refer to an antibody that is
substantially free of other antibodies having different antigenic specificities (e.g., an isolated
antibody that specifically binds a PD-L1/VEGF protein is substantially free of antibodies that
specifically bind antigens other than PD-L1/VEGF proteins). An isolated antibody that
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specifically binds a human PD-Ll/VEGF protein may, however, have cross- reactivity to other
antigens, such as PD-Ll/VEGF proteins from other species. Moreover, an isolated antibody can
be substantially free of other cellular material and/or chemicals.
The term "vector," as used herein, refers to a nucleic acid vehicle which can have a
5 polynucleotide inserted therein. When the vector allows for the expression of the protein
encoded by the polynucleotide inserted therein, the vector is called an expression vector.
The vector can have the carried genetic material elements expressed in a host cell by
transformation, transduction, or transfection into the host cell. Vectors are well known by a
person skilled in the art, including, but not limited to plasmids, phages, cosmids, artificial
10 chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome
(BAC) or PI-derived artificial chromosome (PAC); phage such as 'A phage or M13 phage
and animal virus. The animal viruses that can be used as vectors, include, but are not
limited to, retrovirus (including lentivirus), adenovirus, adena-associated virus, herpes
virus (such as herpes simplex virus), pox virus, baculovirus, papillomavirus, papovavirus
15 (such as SV40). A vector may comprise multiple elements for controlling expression,
including, but not limited to, a promoter sequence, a transcription initiation sequence, an
enhancer sequence, a selection element and a reporter gene. In addition, a vector may
comprise origin of replication.
The term "host cell," as used herein, refers to a cellular system which can be
20 engineered to generate proteins, protein fragments, or peptides of interest. Host cells
include, without limitation, cultured cells, e.g., mammalian cultured cells derived from
rodents (rats, mice, guinea pigs, or hamsters) such as CHO, BHK, NSO, SP2/0, YB2/0; or
human tissues or hybridoma cells, yeast cells, and insect cells, and cells comprised within a
transgenic animal or cultured tissue. The term encompasses not only the particular subject
25 cell but also the progeny of such a cell. Because certain modifications may occur in
succeeding generations due to either mutation or environmental influences, such progeny
may not be identical to the parent cell, but are still included within the scope of the term
"host cell."
The term "identity," as used herein, refers to a relationship between the sequences of
30 two or more polypeptide molecules or two or more nucleic acid molecules, as determined
by aligning and comparing the sequences. "Percent identity" means the percent of identical
residues between the amino acids or nucleotides in the compared molecules and is
calculated based on the size of the smallest of the molecules being compared. For these
calculations, gaps in alignments (if any) are preferably addressed by a particular
35 mathematical model or computer program (i.e., an "algorithm"). Methods that can be used
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to calculate the identity of the aligned nucleic acids or polypeptides include those described
in Computational Molecular Biology, (Lesk, A. M., ed.), 1988, New York: Oxford
University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.),
1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.
5 M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987,
Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis
Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and
Carillo et al, 1988, SIAMJ. Applied Math. 48:1073.
The term "immunogenicity," as used herein, refers to ability of stimulating the
10 formation of specific antibodies or sensitized lymphocytes in organisms. It not only refers
to the property of an antigen to stimulate a specific immunocyte to activate, proliferate and
differentiate so as to finally generate immunologic effector substance such as antibody and
sensitized lymphocyte, but also refers to the specific immune response that antibody or
sensitized T lymphocyte can be formed in immune system of an organism after stimulating
15 the organism with an antigen. Immunogenicity is the most important property of an antigen.
Whether an antigen can successfully induce the generation of an immune response in a host
depends on three factors, properties of an antigen, reactivity of a host, and immunization
means.
The term "transfection," as used herein, refers to the process by which nucleic acids
20 are introduced into eukaryotic cells, particularly mammalian cells. Protocols and
techniques for transfection include but not limited to lipid transfection and chemical and
physical methods such as electroporation. A number of transfection techniques are well
known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456;
Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986,
25 Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13: 197. In a specific
embodiment of the disclosure, human PD-L1/VEGF gene was transfected into 293F cells.
The term "SPR" or "surface plasmon resonance," as used herein, refers to and includes an
optical phenomenon that allows for the analysis of real-time biospecific interactions by detection
of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore
30 system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further
descriptions, see Example 5 and Jonsson, U., et al. (1993)Ann. Bioi. Clin. 51:19-26; Jonsson, U.,
et al. (1991) Biotechniques 11 :620-627; Johnsson, B., et al. (1995) J Mol. Recognit. 8: 125-131;
and Johnnson, B., et al. (1991)Anal. Biochem. 198:268-277.
The term "fluorescence-activated cell sorting" or "F ACS," as used herein, refers to a
35 specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of
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biological cells into two or more containers, one cell at a time, based upon the specific light
scattering and fluorescent characteristics of each cell (FlowMetric. "Sorting Out Fluorescence
Activated Cell Sorting". Retrieved 2017-11-09.). Instruments for carrying out FACS are known
to those of skill in the art and are commercially available to the public. Examples of such
5 instruments include F ACS Star Plus, F ACScan and F ACSort instruments from Becton Dickinson
(Foster City, Calif) Epics C from Coulter Epics Division (Hialeah, Fla.) and MoFlo from
Cytomation (Colorado Springs, Colo.).
The term "subject" includes any human or nonhuman animal, preferably humans.
The term "cancer," as used herein, refers to any or a tumor or a malignant cell growth,
10 proliferation or metastasis-mediated, solid tumors and non-solid tumors such as leukemia and
initiate a medical condition.
The term "treatment," "treating" or "treated," as used herein in the context of treating a
condition, pertains generally to treatment and therapy, whether of a human or an animal, in
which some desired therapeutic effect is achieved, for example, the inhibition of the progress of
15 the condition, and includes a reduction in the rate of progress, a halt in the rate of progress,
regression of the condition, amelioration of the condition, and cure of the condition. Treatment
as a prophylactic measure (i.e., prophylaxis, prevention) is also included. For cancer, "treating"
may refer to dampen or slow the tumor or malignant cell growth, proliferation, or metastasis, or
some combination thereof For tumors, "treatment" includes removal of all or part of the tumor,
20 inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a
tumor, or some combination thereof
The term "an effective amount," as used herein, pertains to that amount of an active
compound, or a material, composition or dosage from comprising an active compound, which is
effective for producing some desired therapeutic effect, commensurate with a reasonable
25 benefit/risk ratio, when administered in accordance with a desired treatment regimen. For
instance, the "an effective amount," when used in connection with treatment of PD-L1/VEGFrelated
diseases or conditions, refers to an antibody or antigen-binding portion thereof in an
amount or concentration effective to treat the said diseases or conditions.
The term "prevent," "prevention" or "preventing," as used herein, with reference to a
30 certain disease condition in a mammal, refers to preventing or delaying the onset of the disease,
or preventing the manifestation of clinical or subclinical symptoms thereof
The term "pharmaceutically acceptable," as used herein, means that the vehicle, diluent,
excipient and/or salts thereof, are chemically and/or physically is compatible with other
ingredients in the formulation, and the physiologically compatible with the recipient.
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As used herein, the term "a pharmaceutically acceptable carrier and/or excipient" refers to a
carrier and/or excipient pharmacologically and/or physiologically compatible with a subject and
an active agent, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences.
Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes,
5 but is not limited to pH adjuster, surfactant, adjuvant and ionic strength enhancer. For example,
the pH adjuster includes, but is not limited to, phosphate buffer; the surfactant includes, but is
not limited to, cationic, anionic, or non-ionic surfactant, e.g., Tween-80; the ionic strength
enhancer includes, but is not limited to, sodium chloride.
As used herein, the term "adjuvant" refers to a non-specific immunopotentiator, which can
10 enhance immune response to an antigen or change the type of immune response in an organism
when it is delivered together with the antigen to the organism or is delivered to the organism in
advance. There are a variety of adjuvants, including, but not limited to, aluminium adjuvants (for
example, aluminum hydroxide), Freund's adjuvants (for example, Freund's complete adjuvant
and Freund's incomplete adjuvant), coryne bacterium parvum, lipopolysaccharide, cytokines,
15 and the like. Freund's adjuvant is the most commonly used adjuvant in animal experiments now.
Aluminum hydroxide adjuvant is more commonly used in clinical trials.
Bispecific Antibodies and Antigen-Binding Portions thereof
In certain embodiments, the antibodies and antigen-binding portions thereof provided herein
20 are bispecific. In some embodiments, the bispecific antibodies and antigen-binding portions
thereof provided herein have a first specificity for PD-Ll, and a second specificity for VEGF.
According to certain exemplary embodiments, the present disclosure includes a bispecific
antibody or the antigen-binding portion thereof, comprising a first antigen-binding moiety that
specifically binds to PD-Ll and a second antigen-binding moiety that specifically binds to VEGF.
25 Such antibodies may be referred to herein as, e.g., "anti-VEGF/anti-PD-Ll" or "anti-PDLl/
VEGF," or "anti-PD-LlxVEGF" or "PD-LlxVEGF" bispecific antibodies, or other similar
terminology.
The bispecific antibodies of the disclosure could bind to human PD-Ll and human VEGF
with high affinity. The binding of an antibody of the disclosure to PD-Ll or VEGF can be
30 assessed using one or more techniques well established in the art, for instance, ELISA The
binding specificity of an antibody of the disclosure can also be determined by monitoring
binding of the antibody to cells expressing a PD-Ll protein or a VEGF protein, e.g., flow
cytometry. For example, an antibody can be tested by a flow cytometry assay in which the
antibody is reacted with a cell line that expresses human PD-Ll, such as CHO cells that have
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been transfected to express PD-L1 on their cell surface. Additionally or alternatively, the binding
of the antibody, including the binding kinetics (e.g., Kn value) can be tested in BIAcore binding
assays. Still other suitable binding assays include ELISA or F ACS assays, for example using a
recombinant PD-L1 protein.
5 For instance, an antibody of the disclosure binds to a human PD-L1 protein or human
VEGF protein with a Kn of 1 x 1o-7M or less, a Kn of 5 x 1o-8M or less, a Kn of 2x 1o-8M or less,
a Kn of 1 x 10"8 M or less, a Kn of 5 x 10"9 M or less, a Kn of 4x 10"9 M or less, a Kn of 3 x 10"9 M
or less, a Kn of 2x 1 o-9 M or less, a Kn of 1 x 1 o-9 M or less, a Kn of 5 x 1 o-10 M or less, or a Kn of
1 x 1 0"10 M or less, as measured by Surface Plasmon Resonance.
10 As demonstrated in the Example section, the bispecific antibodies of the disclosure could
15
bind to human PD-L1 and human VEGF (same as cyno VEGF) with a high affinity; bind to cyno
and mouse PD-L1; effectively block both PD-1/PD-L1 and VEGFR/VEGF signaling pathways,
e.g. with an IC50 of nM grade; block VEGF induced HUVEC proliferation; and produce strong
agonistic effect on cytokine secretion.
The PD-Ll antigen-binding moiety
The PD-L1 binding moiety as defined herein may have various formats (e.g. VHH, scFv,
Fab), as long as it can specifically bind to the antigen. Generally, the PD-L1 binding moiety
comprised in the bispecific antibody is derived from a monospecific anti-PD-L1 antibody, which
20 may be a known antibody in the art or an antibody developed de novo. In some embodiments
according to the present application, the PD-L1 binding moiety may be derived from a parental
single-domain antibody (sdAb ), such as a VHH antibody, which generally refers to an antibody
consisting of a single variable domain. Like a whole antibody, a single-domain antibody is able
to bind selectively to a specific antigen. In some other embodiments, the PD-L1 binding moiety
25 may be derived from a heavy chain antibody, which is devoid of light chains.
The term "single variable domain" or "heavy chain variable region of a heavy chain
antibody" is interchangeably used with the terms "VHH", "VHH antibody", "VHH domain",
"VHH antibody fragment", "VHH" or "Nanobody," etc. VHH molecules derived from Camelidae
antibodies are among the smallest intact antigen-binding domains known (approximately 15 kDa,
30 or 10 times smaller than a conventional IgG) and hence are well suited towards delivery to dense
tissues and for accessing the limited space between macromolecules.
The parental VHH antibody as disclosed herein may be made by the skilled artisan
according to methods known in the art or any future method. For example, VHHs may be
obtained using methods known in the art such as by immunizing a camel and obtaining
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hybridoma's therefrom, or by cloning a library of VHHs of the invention using molecular biology
techniques known in the art and subsequent selection by using phage display.
For instance, a VHH antibody can be obtained by immunization of llamas or alpacas with
the desired antigen and subsequent isolation of the mRNA coding for heavy-chain antibodies.
5 By reverse transcription and polymerase chain reaction, a gene library of single-domain
antibodies containing several million clones is produced. Screening techniques like phage
display and ribosome display help to identify the clones binding the antigen. One technique is
phage display in which a library of (e.g., human) antibodies is synthesized on phages, the library
is screened with the antigen of interest or an antibody-binding portion thereof, and the phage that
10 binds the antigen is isolated, from which one may obtain the immunoreactive fragments.
Methods for preparing and screening such libraries are well known in the art and kits for
generating phage display libraries are commercially available (e.g., the Pharmacia Recombinant
Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SurtZAP™ phage display
kit, catalog no. 240612). There also are other methods and reagents that can be used in
15 generating and screening antibody display libraries (see, e.g., Barbas et al., Proc. Natl. Acad Sci.
USA 88:7978-7982 (1991)).
Humanization of the VHH antibody can be achieved via a number of well-established
methods in the art, for example, amino acid sequences of VHH framework regions can be blasted
against human germline V -gene database, and humanized VHH sequences can be generated by
20 replacing human CDR sequences in the top hit with VHH CDR sequences using Kabat CDR
definition. Further, certain residues in the framework region may be back mutated to maintain
affinity.
In some embodiments, the PD-L1 antigen binding moiety comprises one or more CDRs
selected from the group consisting of:
25 (i) a CDR1 comprising SEQ ID NO: 1 or an amino acid sequence that differsfrom SEQ ID
NO: 1 by an amino acid addition, deletion or substitution of not more than 2 amino acids;
(ii) a CDR2 comprising SEQ ID NO: 2 or an amino acid sequence that differs from SEQ ID
NO: 2 by an amino acid addition, deletion or substitution of not more than 2 amino acids; and
(iii) a CDR3 comprising SEQ ID NO: 3 or an amino acid sequence that differs from SEQ
30 ID NO: 3 by an amino acid addition, deletion or substitution of not more than 2 amino acids.
In some embodiments, the PD-L1 antigen binding moiety is a VHH antibody that comprises
(i) a CDR1 comprising or consisting of SEQ ID NO: 1; (ii) a CDR2 comprising or consisting of
SEQ ID NO: 2; and (iii) a CDR3 comprising or consisting of SEQ ID NO: 3.

WHAT IS CLAIMED
1. A bispecific antibody or antigen-binding portion thereof, comprising a PD-L1 antigen-binding
moiety associated with a VEGF antigen-binding moiety, wherein:
the PD-L1 antigen-binding moiety comprises:
a complementarity determining region (CDR) 1 comprising SEQ ID NO: 1, a
CDR2 comprising SEQ ID NO: 2, and a CDR3 comprising SEQ ID NO: 3; and
the VEGF antigen-binding moiety comprises:
a heavy chain complementarity determining region (HCDR) 1 comprising SEQ
ID NO: 4, a HCDR2 comprising SEQ ID NO: 5, a HCDR3 comprising SEQ ID
NO: 6, a light chain complementarity determining region (LCDR) 1 comprising
SEQ ID NO: 7, a LCDR2 comprising SEQ ID NO: 8, and a LCDR3 comprising
SEQIDNO: 9.
2. The bispecific antibody or antigen-binding portion thereof of claim 1, wherein the PD-L1
antigen-binding moiety comprises:
a variable domain comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid
sequence at least 85%, 90%, or 95% identical to SEQ ID NO: 10.
3. The bispecific antibody or antigen-binding portion thereof of claim 1 or 2, wherein the VEGF
antigen-binding moiety comprises:
a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 11 or
an amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NO: 11; and
a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12 or an
amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NO: 12.
4. The bispecific antibody or antigen-binding portion thereof of any of the preceding claims,
wherein the PD-L1 antigen-binding moiety is fused to the N terminal of the VEGF antigenbinding
moiety.
5. The bispecific antibody or antigen-binding portion thereof of any of the preceding claims,
wherein the PD-L1 antigen-binding moiety is from a single-domain antibody (sdAb ), such as a
VHH antibody.
6. The bispecific antibody or antigen-binding portion thereof of claim 5, wherein the VHH is
derived from a camelid animal, comprising an alpaca or a llama.
7. The bispecific antibody or antigen-binding portion thereof of claim 5 or 6, wherein the PD-L1
antigen-binding moiety is operably linked to the N terminal of the light chain or heavy chain of
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the VEGF antigen-binding moiety, optionally via a linker.
8. The bispecific antibody or antigen-binding portion thereof of claim 7, wherein the linker
comprises or consists of 1 to 4 copies ofGGGGS (G4S).
9. The bispecific antibody or antigen-binding portion thereof of any of the preceding claims,
comprising a heavy chain and a light chain, wherein:
the heavy chain comprises domains operably linked as in VH-CH1-hinge-Fc, wherein the
VH-CH1 is from the VEGF antigen binding moiety; and
the light chain comprises domains operably linked as in VHH-VL-CL, wherein the VHH is
from the PD-L1 antigen binding moiety and the VL-CL is from the VEGF antigen binding
moiety.
10. The bispecific antibody or antigen-binding portion thereof of claim 9, wherein the Fe region
is a human IgG Fe region, preferably a human IgG 1 Fe region.
11. The bispecific antibody or antigen-binding portion thereof of any of the preceding claims,
wherein the heavy chain comprises SEQ ID NO: 13, and the light chain comprises SEQ ID NO:
14.
12. The bispecific antibody or antigen-binding portion thereof of any of the preceding claims,
wherein the bispecific antibody is a humanized antibody.
13. An isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the
bispecific antibody or the antigen-binding portion thereof of any of claims 1-12.
14. A vector comprising the nucleic acid molecule of claim 13.
15. A host cell comprising the nucleic acid molecule of claim 13 or the vector of claim 14.
16. A pharmaceutical composition comprising the bispecific antibody or the antigen-binding
portion thereof of any of claims 1-12 and a pharmaceutically acceptable carrier.
17. A method for producing the bispecific antibody or the antigen-binding portion thereof of
any of claims 1-12, comprising the steps of:
- expressing the antibody or the antigen-binding portion thereof of any of claims 1-12 in
the host cell of claim 15; and
-isolating the antibody or antigen-binding portion thereof from the host cell.
18. A method for modulating an immune response in a subject, comprising administering to the
subject the bispecific antibody or the antigen-binding portion thereof as defined in any of claims
1-12 or the pharmaceutical composition of claim 16 to the subject.
19. A method for inhibiting growth of tumor cells in a subject, comprising administering an
effective amount of the bispecific antibody or the antigen-binding portion thereof as defined in
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