Bispecific anti-PD-L1 / VEGF antibody and its use

Abstract

Bispecific anti-VEGFxPD-L1 antibodies, or antigen-binding portions thereof, methods of producing the bispecific antibodies, or antigen-binding portions thereof, and methods of treating diseases or conditions using the bispecific antibodies, or antigen-binding portions thereof, are provided.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to International Application No. PCT / CN2020 / 073497, filed on January 21, 2020, the entire content of which is incorporated herein by reference.

[0002] Sequence Listing This application is filed with an electronic sequence listing. The entire content of the sequence listing is incorporated herein by reference.

[0003] The present disclosure generally relates to bispecific anti - PD - L1xVEGF antibodies, methods for their preparation, and their use.

Background Art

[0004] Angiogenesis is essential for tumor growth and the occurrence of metastasis. The control of tumor - associated angiogenesis is a promising strategy for cancer treatment. Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis and has been verified in various types of human cancers. Tumor cells release growth factors such as VEGF that bind to nearby endothelial cells, initiating a signaling cascade that stimulates endothelial cells to divide and form new blood vessels. VEGF signaling via its receptor VEGFR plays an important role in angiogenesis and the growth of many solid tumors. Anti - angiogenic drugs such as Avastin (bevacizumab) that target the VEGF pathway have achieved clinical success.

[0005] On the one hand, targeting immune checkpoint molecules such as programmed death ligand 1 (PD-L1) and its receptor programmed death 1 (PD-1) has shown promising clinical success. The expression of PD-L1 is strongly correlated with poor prognosis in various types of cancer. Anti-PD-L1 antibodies can target PD-L1 expressed on tumor cells and tumor-infiltrating immune cells, prevent binding to PD-1 and B7.1 on the surface of T cells, activate T cells, recruit other T cells to attack tumors, and also empower the immune system to fight multiple types of cancer.

[0006] In addition to its established anti-angiogenic effect, anti-VEGF therapy inhibits VEGF-related immunosuppression, promotes T cell tumor infiltration, and enables priming and activation of T cell responses against tumor antigens, thereby further enhancing the ability to restore anti-cancer immunity of anti-PD-1 / PD-L1 therapy. Therefore, the development of VEGF and PD-L1 bispecific antibodies combining anti-angiogenic therapy and immune checkpoint inhibition may achieve promising results in cancer treatment.

[0007] Targeting VEGF and targeting PD-1 / PD-L1 therapy has obvious advantages, but there are still important unmet needs. 15% - 20% of patients do not respond to anti-VEGF treatment, and there is increasing evidence that long-term use of anti-VEGF agents for cancer treatment promotes tumor resistance. 3% - 9% of patients develop immunogenicity of the treatment. Also, the extension of overall survival is limited, such as changes in bone morphology, glomerulopathy with kidney inflammation, and decreased vacuolization with adrenal inflammation, and safety issues are also limited. Immune checkpoint inhibitors that block the PD-1 / PD-L1 pathway, such as nivolumab, pembrolizumab, atezolizumab, etc., are standard treatment options for multiple cancer patients. However, with response rates of 14 - 23% in the unselected population and 16 - 48% in patients with PD-L1-expressing tumors, these drugs improve the outcomes of some patients, but not all.

[0008] Therefore, there is a great need to develop a novel anti-PD-L1 / anti-VEGF bispecific antibody. In the present disclosure, a bispecific antibody that binds to human PD-L1 and VEGF simultaneously with high affinity, blocks both PD-1 / PD-L1 and VEGF / VEGFR signaling, and can exhibit excellent anti-tumor efficacy has been generated.

SUMMARY OF THE INVENTION

[0009] These and other objects are provided by the present disclosure directed, in broad terms, to compounds, methods, compositions, and articles that provide antibodies having improved efficacy. The advantages provided by the present disclosure are widely applicable in the fields of antibody therapy and diagnosis and can be used in combination with antibodies that react with various targets.

[0010] In one aspect, the present disclosure provides a bispecific antibody or an antigen-binding portion thereof comprising a PD-L1 antigen-binding portion associated with a VEGF antigen-binding portion, The PD-L1 antigen-binding portion comprises complementarity-determining region (CDR) 1 comprising SEQ ID NO: 1, CDR2 comprising SEQ ID NO: 2, and CDR3 comprising SEQ ID NO: 3, The VEGF antigen-binding portion comprises heavy chain complementarity-determining region (HCDR) 1 comprising SEQ ID NO: 4, HCDR2 comprising SEQ ID NO: 5, HCDR3 comprising SEQ ID NO: 6, light chain complementarity-determining region (LCDR) 1 comprising SEQ ID NO: 7, LCDR2 comprising SEQ ID NO: 8, and LCDR3 comprising SEQ ID NO: 9.

[0011] In certain embodiments, the PD-L1 antigen-binding portion disclosed herein comprises a variable domain comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 10.

[0012] In certain embodiments, the VEGF antigen-binding portion disclosed herein comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 11, and It includes a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 12.

[0013] In certain embodiments, the PD-L1 antigen-binding portion is fused to the N-terminus of the VEGF antigen-binding portion. In some other embodiments, the PD-L1 antigen-binding portion is fused to the C-terminus of the VEGF antigen-binding portion.

[0014] In certain embodiments, the PD-L1 antigen-binding portion is derived from a single domain antibody (sdAb) such as a VHH antibody. The VHH can be derived from an animal of the camelid family including alpaca or llama. Preferably, the VHH is a humanized VHH.

[0015] In certain embodiments, the PD-L1 antigen-binding portion is operably linked to the N-terminus of the light or heavy chain of the VEGF antigen-binding portion, optionally via a linker. The linker may comprise or consist of 1 to 4 copies of GGGGS (G4S), for example, the linker can be (G4S)2.

[0016] In certain embodiments, the bispecific antibody or its antigen-binding portion disclosed herein comprises a heavy chain and a light chain, the heavy chain comprises domains operably linked like VH-CH1-hinge-Fc, where VH-CH1 is derived from the VEGF antigen-binding portion, the light chain comprises domains operably linked like VHH-VL-CL, where VHH is derived from the PD-L1 antigen-binding portion, and VL-CL is derived from the VEGF antigen-binding portion.

[0017] In certain embodiments, the Fc region is a human IgG Fc region, preferably a human IgG1 Fc region.

[0018] In certain embodiments, the bispecific antibody or its antigen-binding portion disclosed herein comprises a heavy chain comprising SEQ ID NO: 13 and a light chain comprising SEQ ID NO: 14.

[0019] In certain embodiments, the bispecific antibodies or antigen-binding portions thereof disclosed herein are humanized antibodies.

[0020] In one aspect, the present disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a bispecific antibody or antigen-binding portion thereof disclosed herein.

[0021] In one aspect, the present disclosure provides a vector comprising a nucleic acid molecule disclosed herein. In one aspect, the present disclosure provides a host cell comprising a nucleic acid molecule or vector disclosed herein.

[0022] In one aspect, the present disclosure provides a pharmaceutical composition comprising a bispecific antibody or antigen-binding portion thereof disclosed herein and a pharmaceutically acceptable carrier.

[0023] In one aspect, the present disclosure - expressing an antibody or antigen-binding portion thereof in a host cell; and - isolating the antibody or antigen-binding portion thereof from the host cell, and provides a method for producing a bispecific antibody or antigen-binding portion thereof disclosed herein.

[0024] In one aspect, the present disclosure provides a method for modulating an immune response in a subject, comprising administering to the subject a bispecific antibody or antigen-binding portion thereof, or a pharmaceutical composition, disclosed herein.

[0025] In one aspect, the present disclosure provides a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject an effective amount of a bispecific antibody or antigen-binding portion thereof, or a pharmaceutical composition, disclosed herein.

[0026] In one aspect, the present disclosure provides a method for preventing or treating cancer in a subject, the method comprising administering to the subject an effective amount of a bispecific antibody or an antigen-binding portion thereof or a pharmaceutical composition. The cancer can be selected from colorectal cancer, colorectal cancer, breast cancer, lung cancer, cervical cancer, kidney cancer, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, esophageal cancer, gastric cancer, lymphoma, melanoma, liver cancer, and head and neck cancer. In certain embodiments, the cancer is colorectal cancer or colorectal cancer.

[0027] In certain embodiments, the bispecific antibody or antigen-binding portion thereof disclosed herein can be administered in combination with a chemotherapeutic agent, radiation, and / or other agents for use in cancer immunotherapy.

[0028] In one aspect, the present disclosure i) in the regulation of PD-L1 / VEGF-related immune responses, ii) in promoting T cell proliferation and cytokine production, and / or iii) provides the bispecific antibody or antigen-binding portion thereof disclosed herein for use in stimulating an immune response or function, such as enhancing the immune response against cancer cells.

[0029] In one aspect, the present disclosure provides the bispecific antibody or antigen-binding portion thereof disclosed herein for use in the diagnosis, prevention or treatment of cancer.

[0030] In one aspect, the present disclosure provides the use of the bispecific antibody or antigen-binding portion thereof disclosed herein in the manufacture of a medicament for regulating an immune response in a subject or inhibiting the growth of tumor cells.

[0031] In one aspect, the present disclosure provides the use of the bispecific antibody or antigen-binding portion thereof disclosed herein in the manufacture of a medicament for treating or preventing cancer.

[0032] In one aspect, the present disclosure provides a kit comprising a bispecific antibody or an antigen-binding portion thereof disclosed herein. This kit can be used for the detection, diagnosis, prognosis, or treatment of diseases or conditions such as cancer.

[0033] The above is an overview, and thus, if necessary, includes simplification, generalization, and omission of details. Accordingly, those skilled in the art will understand that the overview is for illustrative purposes only and is not intended to be limiting in any way. The methods, compositions and / or devices, and / or other aspects, features, and advantages of the other subject matter described herein will become apparent in the teachings described herein. The overview is provided to introduce, in a simplified form, a selected choice of concepts that are further described in the following detailed description. This overview is not intended to identify the 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. The contents of all figures and all references, patents, and published patent applications cited throughout this application are hereby expressly incorporated herein by reference.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0035] The present disclosure may be embodied in many different forms, and specific exemplary embodiments that illustrate the principles of the present disclosure are disclosed herein. It should be emphasized that the present disclosure is not limited to the specific embodiments illustrated. Further, any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter being described.

[0036] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include singulars. More specifically, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of proteins. Reference to "a cell" includes, for example, a mixture of cells. In this application, the use of "or" means "and / or" unless stated otherwise. Further, the use of the term "comprising," as well as other forms such as "comprises" and "comprised," is not limiting. Further, the ranges provided in the specification and the appended claims include both endpoints and all points between the endpoints.

[0037] In general, the nomenclature and techniques used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. The methods and techniques of the present disclosure are generally carried out according to conventional methods well known in the art, unless otherwise indicated, and as described in various general and more specific references cited and discussed throughout this specification. For example, Abbas et al., Cellular and Molecular Immunology, 6 th ed., W.B. Saunders Company (2010), Sambrook J. & Russell D.Molecular Cloning: A Laboratory Manual, 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 nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as their experimental procedures and techniques, are well known and commonly used in the art.

[0038] Definitions To better understand the disclosure, definitions and explanations of related terms are provided below.

[0039] As used herein, the terms "antibody" or "Ab" are used in the broadest sense to encompass various antibody structures, including polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies). A native intact antibody is generally a Y-shaped tetrameric protein composed of two heavy (H) and two light (L) polypeptide chains linked by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be classified as κ and λ light chains. The heavy chains can be classified as μ, δ, γ, α, and ε, which define the isotypes of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. In both the light and heavy chains, the variable regions are linked to the constant regions via a "J" region of about 12 or more amino acids, and the heavy chains further contain a "D" region of about 3 or more amino acids. Each heavy chain generally consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain generally consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementarity-determining regions (CDRs)), which are separated by relatively conserved regions (called framework regions (FRs)). VH and VL each consist of three CDRs and four FRs in the following order, from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (V H and V LThey each form an antigen-binding site. The distribution of amino acids in various regions or domains follows the definitions of 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. The antibody can be a different antibody isotype, such as an IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

[0040] The terms "antigen-binding portion" or "antigen-binding fragment" of an antibody can be used interchangeably in the context of this application and refer to a polypeptide that includes a fragment of a full-length antibody, retains the ability to specifically bind to an antigen to which the full-length antibody specifically binds, and / or competes 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)). An antigen-binding fragment of an antibody can be derived from a complete antibody molecule using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding the antibody variable domain and optionally the constant domain. Such DNA is known and / or can be readily obtained, for example, from commercial sources, DNA libraries (including phage antibody libraries), or synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable domains and / or constant domains in an appropriate configuration, introduce codons, create cysteine residues, or modify, add or delete amino acids.

[0041] Non-limiting examples of antigen-binding fragments include the following: (i) Fab fragments, (ii) F(ab’)2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, (vii) hypervariable regions of antibodies (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides) or minimal recognition units consisting of amino acid residues that mimic constrained FR3-CDR3-FR4 peptides. Other modified molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, minibodies, nanobodies (such as monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed by the expression "antigen-binding fragment" as used herein. In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently attached to at least one constant domain. The variable and constant domains may be directly linked to each other or may be linked by a full or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule.

[0042] As used herein, the term "variable domain" with respect to an antibody refers to the variable region of the antibody or a fragment thereof that includes one or more CDRs. The variable domain may include an intact variable region (such as HCVR or LCVR), but may also include less than an intact variable region, yet still retain the ability to bind to an antigen or form an antigen-binding site.

[0043] As used herein, the term "antigen-binding portion" refers to an antibody fragment formed from a portion of an antibody that includes one or more CDRs, or any other antibody fragment that binds an antigen but does not include an intact native antibody structure. Unlike the term "antigen-binding site," which generally refers to the variable domain, an antigen-binding portion may include a constant domain in addition to the variable domain. Examples of antigen-binding portions include, without limitation, variable domains, variable regions, bispecific antibodies, Fab, Fab’, F(ab’) 2 , Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv’), disulfide-stabilized bispecific antibodies (ds bispecific antibodies), multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding portion can bind the same antigen to which the parent antibody binds. In certain embodiments, the antigen-binding portion can be a Fab fragment or a VHH antibody. In some embodiments, the antigen-binding portion can include one or more CDRs from a particular human antibody transplanted into framework regions from one or more different human antibodies. More detailed forms of antigen-binding portions 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), the entireties of which are incorporated herein by reference.

[0044] "Fab" with respect to an antibody refers to the portion of the antibody consisting of a single light chain (both variable and constant regions) associated by a disulfide bond to the variable region and the first constant region of a single heavy chain.

[0045] "Fc" with respect to an antibody refers to the portion of the antibody that includes the second (CH2) and third (CH3) constant regions of the first heavy chain bound via disulfide bonds to the second and third constant regions of the second heavy chain. When referring to the Fc region, depending on the context, it may refer to one or both chains of the Fc region. The Fc portion of an antibody is responsible for various effector functions such as ADCC and CDC, but does not function in antigen binding. The ability of an antibody to initiate and regulate effector functions via the Fc domain is an important factor in in vivo protective activity. It was previously thought that the neutralizing activity of antibodies was only the result of Fab-antigen interactions, but it has been revealed that their in vivo activity largely depends on the interaction between the IgG Fc domain and its cognate receptor, the Fcγ receptor (FcγR), expressed on the surface of effector leukocytes.

[0046] The term "PD-L1", also known as programmed death ligand 1, is a 40 kDa type I transmembrane protein and is presumed to play a major role in the suppression of the adaptive arm of the immune system. PD-L1 is the major ligand of programmed death 1 (PD-1) and is a co-inhibitory receptor constitutively expressed or induced in myeloid, lymphoid, normal epithelial cells and cancer. As used herein, the term "PD-L1" when referring to the amino acid sequence of the PD-L1 protein includes the full-length PD-L1 protein, or the extracellular domain of PD-L1 (PD-L1 ECD) or a fragment containing the PD-L1 ECD; fusion proteins of the PD-L1 ECD, for example, fragments fused to IgG Fc (mFc or hFc) derived from mouse or human are also included. Further, as will be understood by those skilled in the art, the PD-L1 protein also includes those in which amino acid sequence variations have been introduced naturally or artificially (including but not limited to substitutions, deletions and / or additions) without affecting the biological function.

[0047] As used herein, the terms "antibody that binds to PD-L1" or "anti-PD-L1 antibody" include antibodies that specifically recognize PD-L1 and antigen-binding fragments thereof. The antibodies and antigen-binding fragments of the present disclosure are capable of binding to soluble PD-L1 protein and / or cell surface-expressed PD-L1. Soluble PD-L1 includes not only native PD-L1 protein but also recombinant PD-L1 protein variants that lack a transmembrane domain or are otherwise not associated with the cell membrane. As used herein, the expression "anti-PD-L1 antibody" includes both monovalent antibodies having a single specificity and bispecific antibodies that include a first antigen-binding site that binds to PD-L1 and a second antigen-binding site that binds to a second (target) antigen, and the anti-PD-L1 antigen-binding site includes either an HCVR / LCVR or CDR sequence as shown 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, for example, bispecific antibodies.

[0048] The term "VEGF" (vascular endothelial growth factor, also known as VEGF-A) is a signaling protein produced by cells that stimulate the formation of blood vessels. VEGF is a subfamily of growth factors and is a 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 an existing vascular system). The VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF (placental growth factor), VEGF-E (Orf-VEGF), and Trimelissus flavoviridis svVEGF.

[0049] As used herein, the terms "VEGF receptor" or "VEGFR" refer to receptors for vascular endothelial growth factor (VEGF). There are three main subtypes of VEGFR, numbered 1, 2, and 3. VEGF receptors can be membrane-bound or soluble, depending on alternative splicing. Among the VEGF receptors, VEGFR-1 binds to VEGF-A, PlGF, and VEGF-B.

[0050] As used herein, "bispecific antibody" refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and can bind to two different epitopes. The two epitopes may be present on the same antigen or on two different antigens.

[0051] The term "bispecific antigen-binding molecule" means a protein, polypeptide, or molecular complex that includes at least a first antigen-binding domain (also referred to herein as the first antigen-binding site) and a second antigen-binding domain (also referred to herein as the second antigen-binding site). In some embodiments, the "bispecific antigen-binding molecule" is a "bispecific antibody". Each antigen-binding domain within a bispecific antibody includes at least one CDR that specifically binds to a particular antigen, either alone or in combination with one or more additional CDRs and / or FRs. 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).

[0052] The terms "anti-PD-L1 / anti-VEGF antibody", "anti-PD-L1 / anti-VEGF bispecific antibody", "antibody against PD-L1 and VEGF", "anti-PD-L1×VEGF bispecific antibody", "PD-L1×VEGF antibody", when used interchangeably herein, refer to a bispecific antibody that specifically binds to PD-L1 and VEGF.

[0053] As used herein, the term "monoclonal antibody" or "mAb" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope.

[0054] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, for example, 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.

[0055] The term "humanized antibody" is intended to refer to an antibody in which CDR sequences derived from the germline of another mammalian species such as a mouse have been transplanted into human framework sequences. Additional framework region modifications can be made within the human framework sequences.

[0056] The term "operably linked" refers to the juxtaposition of two or more biological sequences of interest such that, with or without a spacer or linker, they are in a relationship that enables them to function in the intended manner. When used with respect to a polypeptide, it is intended to mean that the polypeptide sequences are linked in such a way as to enable the linked product to have the intended biological function. For example, an antibody variable region can be operably linked to a constant region to provide a stable product having antigen-binding activity. This term can also be used with respect to polynucleotides. As an example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), it is intended to mean that the polynucleotide sequences are linked in such a way as to enable regulated expression of the polypeptide from the polynucleotide.

[0057] As used herein, the term "Ka" is intended to refer to the association rate of a particular antibody-antigen interaction, and the term "Kd" as used herein is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The Kd value of an antibody can be determined using methods well established in the art. As used herein, the term "K D " 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 molar concentration (M). A preferred method for determining the Kd of an antibody is by using surface plasmon resonance, preferably by using a biosensor system such as a Biacore® system.

[0058] As used herein, the term "high affinity" for an IgG antibody refers to an antibody having a value of 1×10 -7 M or less, more preferably 5×10 -8 M or less, even more preferably 1×10 -8 M or less, even more preferably 5×10 -9 M or less, even more preferably 1×10 -9 M or less for the target antigen.

[0059] As used herein, the term "EC 50 " is also referred to as "half maximal effective concentration" and refers to the concentration of a drug, antibody, or toxic substance that elicits a response midway between the baseline and the maximum value after a specific exposure time. In the context of this application, EC 50 is expressed in units of "nM".

[0060] As used herein, the term "IC 50 " is also referred to as "half maximal inhibitor concentration" and is a measure of the potency of a substance that inhibits a particular biological or biochemical function. In the context of this application, IC 50 is expressed in units of "nM".

[0061] As used herein, the ability to "inhibit binding" refers to the ability of an antibody or antigen-binding fragment thereof to inhibit the binding of two molecules (e.g., human PD-L1 / VEGF and human PD-1 / VEGFR) at any detectable level. In certain embodiments, the binding of the two molecules is inhibited by the antibody with an IC 50 of 50 nM or less, 30 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or less.

[0062] As used herein, the term "epitope" refers to the portion on an antigen to which an immunoglobulin or antibody specifically binds. An "epitope" is also known as an "antigenic determinant". An 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 specific charge characteristics. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0063] As used herein, the term "isolated" refers to a state obtained from its natural state by artificial means. When a particular "isolated" substance or component exists in nature, its natural environment may be changed, the substance may be isolated from its natural environment, or both may cause it to exist. For example, a particular unisolated polynucleotide or polypeptide naturally exists in the body of a particular living animal, and a high-purity identical polynucleotide or polypeptide isolated from such a natural state is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixed artificial or synthetic substances or other impurities that do not affect the activity of the isolated substance.

[0064] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to a PD-L1 / VEGF protein is substantially free of antibodies that specifically bind to antigens other than the PD-L1 / VEGF protein). However, an isolated antibody that specifically binds to a human PD-L1 / VEGF protein may be cross-reactive with other antigens such as PD-L1 / VEGF proteins from other species. Further, an isolated antibody may be substantially free of other cellular materials and / or chemical substances.

[0065] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can have a carried genetic element that is expressed within a host cell by transformation, transduction, or transfection into the host cell. Vectors include, but are not limited to, artificial chromosomes such as plasmids, phages, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), which are well known to those of skill in the art; phages such as λ phage and M13 phage, and animal viruses, including but not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector can contain multiple elements for controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Further, a vector may contain an origin of replication.

[0066] As used herein, the term "host cell" refers to a cell line that can be engineered to produce a protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells such as mammalian cultured cells or hybridoma cells derived from rodents (rat, mouse, guinea pig, or hamster) or human tissue such as CHO, BHK, NSO, SP2 / 0, YB2 / 0, yeast cells, insect cells, and cells contained within transgenic animals or cultured tissues. The term encompasses not only the particular subject cells but also the progeny of such cells. Such progeny may not be identical to the parental cells due to mutations or environmental influences that may occur in the next generation, but are still included within the scope of the term "host cell".

[0067] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, which is determined by aligning and comparing the sequences. "Percent identity" means the percentage of identical residues between amino acids or nucleotides in the compared molecules and is calculated based on the smallest size of the molecules being compared. In these calculations, gaps in the alignment (if any) are preferably handled by a particular mathematical model or computer program (i.e., "algorithm"). Methods that can be used to calculate the identity of 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.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, SIAM J. Applied Math. 48:1073.

[0068] As used herein, the term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It not only refers to the property that an antigen stimulates specific immune cells to activate, proliferate, differentiate, and ultimately generate immune effector substances such as antibodies and sensitized lymphocytes, but also refers to the specific immune response that after an organism is stimulated with an antigen, antibodies or sensitized T lymphocytes can be formed in the immune system of the organism. 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: the properties of the antigen, the reactivity of the host, and the means of immunization.

[0069] As used herein, the term "transfection" refers to the process by which nucleic acids are introduced into eukaryotic cells, particularly mammalian cells. Transfection protocols and techniques include, but are not limited to, chemical and physical methods such as lipid transfection and electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra, Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier, Chu et al, 1981, Gene 13:197. In certain embodiments of the present disclosure, the human PD-L1 / VEGF gene was transfected into 293F cells.

[0070] As used herein, the terms "SPR" or "surface plasmon resonance" refer to and include an optical phenomenon that enables the analysis of real-time biospecific interactions, for example, by detecting changes in protein concentration within a biosensor matrix using a BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further explanation, see Example 5 and Jonsson, U., et al. (1993) Ann. Biol. 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.

[0071] As used herein, the term "fluorescence-activated cell sorting" or "FACS" refers to a special type of flow cytometry. This provides a method for sorting a heterogeneous mixture of biological cells one cell at a time into two or more containers based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence Activated Cell Sorting". Retrieved 2017-11-09). Instruments for performing FACS are known to those of skill in the art and are generally commercially available. Examples of such instruments include the FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, Calif.), the Epics C from Coulter Epics Division (Hialeah, Fla.), and the MoFlo from Cytomation (Colorado Springs, Colo.).

[0072] The term "subject" includes any human or non-human animal, preferably a human.

[0073] As used herein, the term "cancer" refers to tumors or the growth, proliferation, or metastatic solid tumors and non-solid tumors such as leukemia of malignant cells, and initiates the disease state.

[0074] As used herein, the terms "treatment", "treating", or "being treated" in the context of treating a symptom generally relate to treatments and therapies in which any desired therapeutic effect is achieved, for example, inhibition of the progression of the symptom, reduction in the rate of progression, cessation of the rate of progression, regression of the symptom, improvement of the symptom, and cure of the symptom. Treatments as preventive measures (i.e., prevention, prophylaxis) are also included. In the case of cancer, "treating" may refer to weakening or delaying the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. In the case of tumors, "treatment" includes removal of all or part of the tumor, inhibition or delay of tumor growth and metastasis, prevention or delay of tumor occurrence, or a combination thereof.

[0075] As used herein, the term "effective amount" relates to an active compound, or a material, composition or dosage containing the active compound, which, when administered according to a desired treatment regimen, is effective to produce a desired therapeutic effect commensurate with a reasonable benefit / risk ratio. For example, an "effective amount", when used in connection with the treatment of a PD-L1 / VEGF-related disease or condition, refers to an amount or concentration of an antibody or antigen-binding portion thereof effective to treat the disease or condition.

[0076] As used herein, the terms "prevent", "prevention", or "preventing" refer to preventing or delaying the onset of a disease, or preventing the manifestation of its clinical or asymptomatic signs, with respect to a particular disease state in a mammal.

[0077] As used herein, the term "pharmaceutically acceptable" means that a vehicle, diluent, excipient and / or salts thereof are chemically and / or physically compatible with the other ingredients in the formulation and physiologically compatible with the recipient.

[0078] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent, and these are well known in the art (see, for example, Remington’s Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), including, but not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride.

[0079] As used herein, the term "adjuvant" refers to a non-specific immune enhancer that, when delivered to an organism with or prior to an antigen, can enhance the immune response to the antigen or change the type of immune response in the organism. There are various adjuvants, including, but not limited to, aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. Freund's adjuvants are the most commonly used adjuvants in current animal experiments. Aluminum hydroxide adjuvants are more commonly used in clinical trials.

[0080] Bispecific antibodies and antigen-binding portions thereof In certain embodiments, the antibodies and antigen-binding portions thereof provided herein are bispecific. In some embodiments, the bispecific antibodies and antigen-binding portions thereof provided herein have a first specificity for PD-L1 and a second specificity for VEGF.

[0081] According to certain exemplary embodiments, the present disclosure includes a bispecific antibody or an antigen-binding portion thereof that includes a first antigen-binding portion that specifically binds to PD-L1 and a second antigen-binding portion that specifically binds to VEGF. Such antibodies may be referred to herein, for example, as "anti-VEGF / anti-PD-L1" or "anti-PD-L1 / VEGF", or "anti-PD-L1xVEGF" or "PD-L1xVEGF" bispecific antibodies or other similar terms.

[0082] The bispecific antibodies of the present disclosure were able to bind to human PD-L1 and human VEGF with high affinity. The binding of the antibodies of the present disclosure to PD-L1 or VEGF can be evaluated using one or more techniques well established in the art, such as ELISA. The binding specificity of the antibodies of the present disclosure can also be determined by monitoring the binding of the antibodies to cells expressing the PD-L1 protein or VEGF protein, for example, by flow cytometry. For example, the antibodies can be tested by a flow cytometry assay in which the antibodies are reacted with cell lines expressing human PD-L1, such as CHO cells transfected to express PD-L1 on the cell surface. Further, or alternatively, the binding of the antibodies, including the binding kinetics (e.g., K D value), can be tested in a BIAcore binding assay. Still other suitable binding assays include, for example, ELISA or FACS assays using recombinant PD-L1 protein.

[0083] For example, the antibodies of the present disclosure have a K -7 of 1×10 D M or less, a K -8 of 5×10 D M or less, a K -8 of 2×10 D M or less, a K -8 of 1×10 D M or less, a K -9 of 5×10 D M or less, a K -9 of 4×10 D M or less, a K -9K that is less than or equal to M D、2×10-9 K that is less than or equal to M D、1×10-9 K that is less than or equal to M D、5×10-10 K that is less than or equal to M D、1×10-10 K that is less than or equal to M D and binds to human PD-L1 protein or human VEGF protein.

[0084] As shown in the Examples section, the bispecific antibodies of the present disclosure can bind to human PD-L1 and human VEGF (the same as cyno VEGF) with high affinity, can bind to cyno and mouse PD-L1, can effectively block both the PD-1 / PD-L1 and VEGFR / VEGF signaling pathways (e.g., IC50 in the nM grade), can block VEGF-induced HUVEC proliferation, and can produce a strong agonistic effect on cytokine secretion.

[0085] PD-L1 antigen-binding portion The PD-L1 binding portion as defined herein can have various forms (e.g., VHH, scFv, Fab) as long as it can specifically bind to the antigen. Generally, the PD-L1 binding portion included in the bispecific antibody is derived from a monospecific anti-PD-L1 antibody, which can be an antibody known in the art or a newly developed antibody. In some embodiments according to the present application, the PD-L1 binding portion may be derived from a parent single-domain antibody (sdAb) such as a VHH antibody, which generally refers to an antibody consisting of a single variable domain. Similar to the whole antibody, a single-domain antibody can selectively bind to a specific antigen. In some other embodiments, the PD-L1 binding portion can be derived from a heavy-chain antibody lacking a light chain.

[0086] The term "single variable domain" or "heavy-chain variable region of a heavy-chain antibody" is interchangeably used with terms such as "VHH", "VHH antibody", "VHH domain", "VHH antibody fragment", "V HH ", or "nanobody", etc. The V HHThe molecule is one of the smallest known intact antigen-binding domains (about 15 kDa, or 10-fold smaller than conventional IgG), and is thus suitable for delivery to dense tissues and access to the limited space between macromolecules.

[0087] The parent VHH antibodies disclosed herein can be made by one of ordinary skill in the art according to methods known in the art or any future methods. For example, VHHs can be obtained by immunizing camels and obtaining hybridomas therefrom using methods known in the art, or by cloning a library of VHHs of the present invention using molecular biology techniques known in the art and subsequently selecting using phage display.

[0088] For example, VHH antibodies can be obtained by immunizing llamas or alpacas with the desired antigen, followed by isolation of the mRNA encoding the heavy chain antibody. Reverse transcription and polymerase chain reaction generate a gene library of single-domain antibodies containing millions of clones. Screening techniques such as phage display and ribosome display are useful for identifying clones that bind to the antigen. One technique is phage display, in which a library of (e.g., human) antibodies is synthesized on phage, the library is screened with the antigen of interest or its antibody-binding portion, and phage that bind to the antigen are isolated, from which immunoreactive fragments can be obtained. 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., Pharmacia Recombinant Phage Antibody System, catalog number 27-9400-01, and Stratagene SurfZAP™ phage display kit, catalog number 240612). There are also other methods and reagents that can be used for generating and screening antibody display libraries (see, e.g., Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991)).

[0089] Humanization of VHH antibodies can be achieved by a number of well-established methods in the art, for example, the amino acid sequence of the VHH framework region can be blasted against the human germline V gene database, and the humanized VHH sequence can be generated by replacing the top hit human CDR sequences with the VHH CDR sequences using Kabat's CDR definition. Further, specific residues within the framework region can be mutated to maintain affinity.

[0090] In some embodiments, the PD-L1 antigen-binding portion (i) CDR1 comprising SEQ ID NO: 1, or an amino acid sequence different from SEQ ID NO: 1 by an addition, deletion, or substitution of one or two amino acids, (ii) CDR2 comprising SEQ ID NO: 2, or an amino acid sequence different from SEQ ID NO: 2 by an addition, deletion, or substitution of one or two amino acids, and (iii) one or more CDRs selected from the group consisting of CDR3 comprising SEQ ID NO: 3, or an amino acid sequence different from SEQ ID NO: 3 by an addition, deletion, or substitution of one or two amino acids.

[0091] In some embodiments, the PD-L1 antigen-binding portion is a VHH antibody comprising (i) CDR1 comprising or consisting of SEQ ID NO: 1, (ii) CDR2 comprising or consisting of SEQ ID NO: 2, and (iii) CDR3 comprising or consisting of SEQ ID NO: 3.

[0092] In some embodiments, the VHH of the PD-L1 antigen-binding portion comprises (i) the amino acid sequence of SEQ ID NO: 10, (ii) an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 10, or (iii) an amino acid sequence having one or more (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 10.

[0093] VEGF antigen-binding portion Similarly, the VEGF antigen-binding portion provided herein may be derived from a parental anti-VEGF monoclonal antibody. In some embodiments according to the present application, the VEGF antigen-binding portion is a Fab fragment of an anti-VEGF full antibody, i.e., it includes the VH and CH1 regions of the heavy chain, and the VL and CL regions of the light chain.

[0094] The anti-VEGF antibody used as the parental antibody may be a monoclonal antibody known in the art (such as bevacizumab) or a newly developed monoclonal antibody. Preferably, the anti-VEGF antibody is a fully human antibody or a humanized antibody.

[0095] In some embodiments, the VH region of the VEGF antigen-binding portion (i) comprises HCDR1 consisting of SEQ ID NO: 4, or HCDR1 whose amino acid sequence differs from SEQ ID NO: 4 by an addition, deletion, or substitution of one or two amino acids, (ii) comprises HCDR2 consisting of SEQ ID NO: 5, or HCDR2 whose amino acid sequence differs from SEQ ID NO: 5 by an addition, deletion, or substitution of one or two amino acids, (iii) comprises one or more heavy chain CDRs (HCDRs) selected from the group consisting of HCDR3 consisting of SEQ ID NO: 6, or HCDR3 whose amino acid sequence differs from SEQ ID NO: 6 by an addition, deletion, or substitution of one or two amino acids, and / or the VL region (i) comprises LCDR1 consisting of SEQ ID NO: 7, or LCDR1 whose amino acid sequence differs from SEQ ID NO: 7 by an addition, deletion, or substitution of one or two amino acids, (ii) comprises LCDR2 consisting of SEQ ID NO: 8, or LCDR2 whose amino acid sequence differs from SEQ ID NO: 8 by an addition, deletion, or substitution of one or two amino acids, (iii) comprises one or more light chain CDRs (LCDRs) selected from the group consisting of LCDR3 consisting of SEQ ID NO: 9, or LCDR3 whose amino acid sequence differs from SEQ ID NO: 9 by an addition, deletion, or substitution of one or two amino acids.

[0096] In some embodiments, VH comprises (i) an HCDR1 that comprises or consists of SEQ ID NO: 4, (ii) an HCDR2 that comprises or consists of SEQ ID NO: 5, (iii) an HCDR3 that comprises or consists of SEQ ID NO: 6, and VL comprises (i) an LCDR1 that comprises or consists of SEQ ID NO: 7, (ii) an LCDR2 that comprises or consists of SEQ ID NO: 8, (iii) an LCDR3 that comprises or consists of SEQ ID NO: 9.

[0097] In some embodiments, the VH of the VEGF antigen-binding portion comprises (i) the amino acid sequence of SEQ ID NO: 11, (ii) an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 11, or (iii) an amino acid sequence having one or more (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 11.

[0098] In some embodiments, the VL of the VEGF antigen-binding portion comprises (i) the amino acid sequence of SEQ ID NO: 12, (ii) an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 12, or (iii) an amino acid sequence having one or more (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 12.

[0099] Unless otherwise specified, the amino acid assignment to each CDR is according to Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5 th th Ed.), U.S. Department of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242; Chothia et al., 1987, PMID: 3681981; Chothia et al., 1989, PMID: 2687698; MacCallum et al., 1996, PMID: 8876650; or Dubel, Ed. (2007) Handbook of Therapeutic Antibodies, 3 rdIt can follow one of the numbering schemes provided by Ed., Wily-VCH Verlag GmbH and Co.

[0100] The variable regions and CDRs in an antibody sequence can be identified according to general rules developed in the art (e.g., such as the Kabat numbering system as shown above), or by aligning the sequence against a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences can be accessed at the “Abysis” website at www.bioinf.org.uk / abs (managed by A.C. Martin at the Department of Biochemistry & Molecular Biology University College London, London, England) and the VBASE2 website at www.vbase2.org as described by Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005). Preferably, the sequences are analyzed using the Abysis database that integrates sequence data from Kabat, IMGT, and the Protein Data Bank (PDB) with the structural data from the PDB. See Dr. Andrew C.R. Martin’s book chapter Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available at the website bioinforg.uk / abs). The Abysis database website further includes general rules developed to identify CDRs that can be used in accordance with the teachings herein.Unless otherwise indicated, all CDRs described in this specification were derived according to the Kabat Abysis database website.

[0101] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Further, according to the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), either a Blossum62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6 can be used to determine the percent identity between two amino acid sequences.

[0102] Additionally or alternatively, the protein sequences of the present disclosure can be further used, for example, as "query sequences" to perform searches against public databases in order to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. The BLAST protein search can be carried out using the XBLAST program with a score = 50 and word length = 3 to obtain amino acid sequences homologous to the antibody molecules of the present disclosure. To obtain a gapped alignment for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0103] In other embodiments, the CDR amino acid sequences can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to each of the above sequences. In other embodiments, the amino acid sequences of the variable regions can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to each of the above sequences.

[0104] Preferably, the CDRs of the isolated antibody or its antigen-binding portion contain no more than two amino acids, or conservative substitutions of no more than one amino acid. As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the essential properties of a protein / polypeptide containing the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of an amino acid residue with another amino acid residue having a similar side chain, e.g., a residue that is physically or functionally similar to the corresponding amino acid residue (having chemical properties such as similar size, shape, charge, ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (such as lysine, arginine, histidine, etc.), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, the corresponding amino acid residue is preferably substituted with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993), Kobayashi et al., Protein Eng. 12(10):879-884 (1999), and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0105] Generation of Bispecific Antibodies To construct the anti-PD-L1 / VEGF bispecific antibody, the above PD-L1 antigen-binding portion and VEGF antigen-binding portion can be fused in various forms. In certain embodiments, the PD-L1 antigen-binding portion is fused to the N-terminus of the VEGF antigen-binding portion. When the PD-L1 antigen-binding portion is a VHH, the single chain of the PD-L1 antigen-binding portion can be operably linked to the heavy or light chain of the VEGF antigen-binding portion, optionally via a linker. Preferably, the PD-L1 antigen-binding portion is linked to the light chain of the VEGF antigen-binding portion. The linker can be a peptide linker containing 1 to 4 copies of GGGGS (G4S). In one embodiment, the linker is (G4S)2.

[0106] The bispecific antibodies and antigen-binding portions provided herein can be made by any suitable method known in the art. In conventional approaches, two immunoglobulin heavy chain-light chain pairs are co-expressed in a host cell and the bispecific antibody can be produced by recombinant methods (see, e.g., Milstein and Cuello, Nature, 305:537 (1983)), followed by purification by affinity chromatography. Sequences encoding the antibody heavy chain variable domains for the two specificities are also each fused to an immunoglobulin constant domain sequence and subsequently inserted into an expression vector for co-transfection with an expression vector for the light chain sequence into a suitable host cell for recombinant expression of the bispecific antibody (see, e.g., WO94 / 04690; Suresh et al., Methods in Enzymology, 121:210 (1986)).

[0107] Fc region In certain embodiments, the bispecific antibody comprises an Fc region operably linked to the VEGF antigen-binding portion. The Fc region of the bispecific antibodies disclosed herein can be a human IgG Fc region. The IgG Fc region can be of any isotype including, but not limited to, IgG1, IgG2, IgG3 or IgG4. In certain embodiments, the Fc region is of the IgG1 isotype.

[0108] In the context of the bispecific antibodies of the present disclosure, the Fc region can include one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a specific chimeric version of the Fc region. For example, the present disclosure includes bispecific antigen-binding molecules that include one or more modifications in the Fc region that result in a modified Fc region having an altered binding interaction with Fc and FcRn or FcγR.

[0109] The "EU numbering system" or "EU index" is generally used when referring to residues in the constant region of an immunoglobulin heavy chain (e.g., the EU index reported in Kabat et al. supra). "EU numbering in Kabat" or "EU index in Kabat" refers to the residue numbering of a human IgG1 EU antibody. Unless otherwise indicated herein, references to residue numbers in the constant domain of an antibody mean residue numbering according to the EU numbering system.

[0110] In certain embodiments, the Fc region is operably linked to the VEGF-binding portion via a hinge region. Optionally, the hinge region can be derived from human IgG1, IgG2, or IgG4. In certain embodiments, the hinge region is derived from human IgG1.

[0111] A nucleic acid molecule encoding an antibody of the present disclosure In some aspects, the present disclosure is directed to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a bispecific antibody or antigen-binding portion disclosed herein. For example, the nucleic acid sequence can encode the heavy and / or light chains of a bispecific antibody. Alternatively, the nucleic acid sequence can encode the heavy or light chain variable region of a PD-L1 antigen-binding portion, or a VEGF antigen-binding portion. The nucleic acid sequence can further encode the Fc region of a bispecific antibody.

[0112] In some embodiments, the present disclosure is directed to vectors comprising the nucleic acid sequences disclosed herein. In further embodiments, the expression vector further comprises a nucleotide sequence encoding a constant region of a bispecific antibody, such as a humanized bispecific antibody.

[0113] A vector in the context of the present disclosure can be any suitable vector, including chromosomal, episomal, and synthetic nucleic acid vectors (nucleic acid sequences comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding a PD-L1 or VEGF antibody is included in a naked DNA or RNA vector, such as a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a compacted nucleic acid vector (e.g., as described in US6,077,835 and / or WO00 / 70087), a plasmid vector such as pBR322, pUC 19 / 18, or pUC 118 / 119, a "mini" minimal size nucleic acid vector (e.g., as described in Schakowski et al., Mol Ther 3, 793-800 (2001)), or a precipitated nucleic acid vector construct such as a CaP04 precipitate (a. e.g., as described in WO2000 / 46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such nucleic acid vectors and methods of using them are well known in the art (see, e.g., US5,589,466 and US5,973,972).

[0114] In one embodiment, the vector is suitable for the expression of anti-PD-L1 antibody and / or anti-VEGF antibody in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vector (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989), pET vector (Novagen, Madison WI), etc.). The vector may be a vector suitable for expression in a yeast system or, alternatively, instead thereof. Any vector suitable for expression in a yeast system can be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH (F. Ausubel et al., ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), and Grant et al., Methods in Enzymol 153, 516-544 (1987)).

[0115] The vector may be a vector suitable for expression in mammalian cells, for example, a vector as a selection marker such as the vector described in Bebbington (1992) Biotechnology (NY) 10:169-175.

[0116] The nucleic acid and / or vector may also include a nucleic acid sequence encoding a secretion / localization sequence that can target a polypeptide such as a nascent polypeptide chain to the periplasmic space or the cell culture medium. Such sequences are known in the art and include secretion leaders or signal peptides.

[0117] The vector may contain or be associated with any suitable promoter, enhancer, and other expression-promoting elements. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter / enhancer, RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly(A) termination sequences, origins of replication of E. coli plasmid products, antibiotic resistance genes as selection markers, and / or convenient cloning sites (e.g., polylinkers). The nucleic acid may also include an inducible promoter, as opposed to a constitutive promoter such as CMV IE.

[0118] In a further aspect, the present disclosure relates to a host cell comprising the vector specified above herein. Thus, the present disclosure also relates to recombinant eukaryotic or prokaryotic host cells, such as transfectomas, that produce the bispecific antibodies of the present disclosure.

[0119] The PD-L1-specific antibody can be expressed in recombinant eukaryotic or prokaryotic host cells, such as transfectomas, that produce the antibody of the present disclosure as defined herein or the bispecific antibody of the present disclosure as defined herein. Similarly, the VEGF-specific antibody can be expressed in recombinant eukaryotic or prokaryotic host cells, such as transfectomas, that produce the antibody of the present disclosure as defined herein or the bispecific antibody of the present disclosure as defined herein.

[0120] Examples of host cells include yeast, bacteria, plants, and mammalian cells such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6, or NSO cells or lymphocyte cells. For example, in one embodiment, the host cell may contain first and second nucleic acid constructs stably integrated into the cell genome. In another embodiment, the present disclosure provides a cell containing non-integrated nucleic acids, such as plasmids, cosmids, phagemids, or linear expression elements, comprising the first and second nucleic acid constructs specified above.

[0121] In a further aspect, the disclosure relates to a transgenic non-human animal or plant comprising a nucleic acid encoding one or two sets of human heavy and human light chains, wherein the animal or plant produces a bispecific antibody of the disclosure.

[0122] In a further aspect, the disclosure relates to a hybridoma that produces an antibody for use in a bispecific antibody of the disclosure as defined herein.

[0123] In one aspect, the disclosure is (i) a nucleic acid sequence encoding a PD-L1 antigen-binding portion, (ii) a nucleic acid sequence encoding a heavy chain and / or a light chain of a VEGF antigen-binding portion, (iii) a nucleic acid sequence encoding an Fc region, or (iv) relates to an expression vector comprising a nucleic acid sequence encoding a heavy chain or a light chain of a bispecific antibody.

[0124] In one aspect, the disclosure relates to a nucleic acid construct encoding one or more amino acid sequences shown in the sequence listing.

[0125] In one aspect, the disclosure relates to a method for producing a bispecific antibody according to any one of the embodiments disclosed herein, comprising culturing a host cell disclosed herein comprising an expression vector or a plurality of expression vectors disclosed herein that express the bispecific antibody disclosed herein, and purifying the antibody from the culture medium. In one aspect, the disclosure relates to a host cell comprising the expression vector defined above. In one embodiment, the host cell is a recombinant eukaryotic, recombinant prokaryotic, or recombinant microbial host cell.

[0126] Pharmaceutical composition In some aspects, the disclosure is directed to a pharmaceutical composition comprising at least one bispecific antibody or an antigen-binding portion thereof disclosed herein and a pharmaceutically acceptable carrier.

[0127] Components of the composition The pharmaceutical composition may optionally contain one or more additional pharmaceutically active components such as another antibody or drug. The pharmaceutical compositions of the present disclosure may also be administered in combination therapies with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, such that the anti-PD-L1 / anti-VEGF bispecific antibody enhances the immune response to the vaccine. Pharmaceutically acceptable carriers can include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antibacterial agents, isotonic agents, buffering agents, antioxidants, anesthetics, suspension / dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art or various combinations thereof.

[0128] Suitable components can include, for example, antioxidants, bulking agents, binders, disintegrants, buffering agents, preservatives, lubricants, flavoring agents, thickening agents, coloring agents, emulsifying agents, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercapto glycerol, thioglycolic acid, mercapto sorbitol, butyl methyl anisole, butylated hydroxytoluene and / or propyl gallact. As disclosed in the present disclosure, compositions are disclosed that contain one or more antioxidants such as methionine in a solvent containing an antibody or antigen-binding fragment of the present disclosure, and the reduced antibody or its antigen-binding fragment can be oxidized. Oxidation-reduction can prevent or reduce a decrease in binding affinity, thereby increasing the stability of the antibody and extending its shelf life. Thus, in some embodiments, the present disclosure provides compositions comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine. The present disclosure further provides various methods by which an antibody or antigen-binding fragment thereof can be mixed with one or more antioxidants such as methionine to prevent oxidation of the antibody or antigen-binding fragment thereof, extend their shelf life, and / or increase their activity.

[0129] More specifically, pharmaceutically acceptable carriers include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, fixed oils of plant origin, cottonseed oil, corn oil, sesame oil, or peanut oil, bacteriostatic or bactericidal concentrations of antibacterial agents, isotonic agents such as sodium chloride and dextrose, buffer solutions such as phosphate buffer or citrate buffer, antioxidant substances such as sodium bisulfite, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, or polyvinyl pyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN-80), metal ion sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. The antibacterial agents used as carriers can be added to the pharmaceutical composition in multi-dose containers, including phenol or cresol, mercury agents, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoate esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients can include, for example, water, physiological saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances can include, for example, wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, or drugs such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0130] Usage, Prescription and Dosage The pharmaceutical compositions of the present disclosure can be administered in vivo to a subject in need thereof by various routes including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, intraoral, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or alternatively by implantation or inhalation. The compositions of the subject can be formulated into preparations in solid, semi-solid, liquid, or gaseous form, which include, but are not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, aerosols. Appropriate formulations and routes of administration can be selected according to the intended application and treatment regimen.

[0131] Formulations suitable for enteral administration include hard or soft gelatin capsules, pills, tablets including coated tablets, elixirs, suspensions, syrups or inhalants, and their controlled release forms.

[0132] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free sterile liquids in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles) (e.g., solutions, suspensions). Such liquids may further contain other pharmaceutically acceptable components such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspending agents, thickening agents, and solutes that render the formulation isotonic with the recipient's blood (or other relevant body fluid). Examples of excipients include, for example, water, alcohol, polyols, glycerol, vegetable oils, etc. Examples of isotonic carriers suitable for use in such formulations include sodium chloride injection solution, Ringer's solution, or lactated Ringer's injection solution. Similarly, specific dosing regimens, including dose, timing, and repetition, will depend on empirical considerations such as the particular individual and their medical history, as well as pharmacokinetics (e.g., half-life, clearance rate, etc.).

[0133] The dosing frequency can be determined and adjusted during the course of treatment and is based on a decrease in the number of proliferating cells or tumorigenic cells, maintenance of such a decrease in tumor cells, a decrease in the proliferation of tumor cells, or a delay in the occurrence of metastasis. In some embodiments, the dose administered can be adjusted or reduced to manage potential side effects and / or toxicities. Alternatively, a sustained continuous release formulation of the subject therapeutic composition may be appropriate.

[0134] One of ordinary skill in the art will understand that the appropriate dosage can vary from patient to patient. To determine the optimal dosage, generally a balance of the level of therapeutic effect against risk or adverse side effects needs to be taken. The dosage level selected will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and / or materials used in combination, the severity of the symptoms, and the species, sex, age, weight, condition, general health, and prior medical history of the patient. The amount and route of administration of the compound are ultimately at the discretion of the physician, veterinarian, or clinician, but generally the dosage is selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial adverse or harmful side effects.

[0135] Generally, the antibodies or antigen-binding portions thereof of the present disclosure can be administered in a variety of ranges. These include from about 5 μg / kg body weight to about 100 mg / kg body weight per dose, from about 50 μg / kg body weight to about 5 mg / kg body weight per dose, from about 100 μg / kg body weight to about 10 mg / kg body weight per dose. Other ranges include from about 100 μg / kg body weight to about 20 mg / kg body weight, and from about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In certain embodiments, the dosage is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, 5 mg / kg body weight, at least about 10 mg / kg body weight per dose.

[0136] In any case, the antibody or antigen-binding portion thereof of the present disclosure is preferably administered as needed to a subject in need thereof. The determination of the dosing frequency can be made by a person skilled in the art, such as a attending physician, taking into account the symptoms being treated, the age of the subject being treated, the severity of the symptoms being treated, and the general health status of the subject being treated, etc.

[0137] In certain preferred embodiments, a series of treatments comprising the antibody or antigen-binding portion thereof of the present disclosure includes multiple administrations of a selected pharmaceutical agent over a period of weeks or months. More specifically, the antibody or antigen-binding portion thereof of the present disclosure can be administered once daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, every six weeks, every two months, every ten weeks, or every three months. It should be understood in this regard that the dosage can be changed or the interval adjusted based on the patient's response and clinical practice.

[0138] The dosage and regimen can also be determined empirically for the disclosed therapeutic compositions in individuals who have received one or more administrations. For example, an individual can be given a gradually increasing dosage of a therapeutic composition produced as described herein. In selected embodiments, the dosage can be gradually increased, decreased, or thinned, respectively, based on side effects or toxicities that are determined or observed empirically. To evaluate the effectiveness of a selected composition, markers of a particular disease, disorder, or condition can be followed as described above. In the case of cancer, these include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by x-ray or other imaging techniques; improvement evaluated by direct tumor biopsy and microscopic examination of tumor samples; measurement of indirect tumor markers (e.g., PSA for prostate cancer) or tumorigenic antigens identified according to the methods described herein; reduction of pain or paralysis; improvement of conversations, vision, breathing, or other disorders associated with the tumor; increased appetite; or improvement in quality of life measured by accepted tests or extension of survival period. It will be apparent to those skilled in the art that the dosage will vary depending on the individual, the type of neoplastic condition, the stage of the neoplastic condition, whether the neoplastic condition has begun to metastasize to other locations in the individual, and past and current treatments being used.

[0139] Formulations suitable for parenteral administration (e.g., intravenous injection) will contain the antibodies or antigen-binding portions thereof disclosed herein at a concentration of about 10 μg / ml to about 100 mg / ml. In certain selected embodiments, the concentration of the antibody or antigen-binding portion thereof will include 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, or 1 mg / ml. In other preferred embodiments, the concentration of the antibody or antigen-binding portion thereof will include 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 8 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg / ml, 16 mg / ml, 18 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, or 100 mg / ml.

[0140] Disclosure applications In some aspects, the present disclosure provides a method of treating a disorder in a subject (e.g., a human) in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding portion thereof disclosed herein. For example, the disorder is cancer.

[0141] A variety of cancers involving PD-L1 and / or VEGF, whether malignant or benign, primary or secondary, can be treated or prevented by the methods provided by the present disclosure. The cancer can be a solid cancer or a hematological malignancy. Examples of such cancers include lung cancers such as bronchogenic carcinoma (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondromatous hamartoma (non-cancerous), and sarcoma (cancerous); heart cancers such as myxoma, fibroma, and rhabdomyoma; bone cancers such as osteochondroma, chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing tumor (Ewing sarcoma), and reticulum cell sarcoma; brain tumors such as glioma (e.g., glioblastoma multiforme), anaplastic astrocytoma, astrocytoma, oligodendroglioma, medulloblastoma, chordoma, schwannoma, epithelioma, meningioma, pituitary adenoma, pinealoma, osteoma, hemangioblastoma, craniopharyngioma, chordoma, germinoma, teratoma, dermoid cyst, and hemangioma; digestive system cancers such as colon cancer, leiomyoma, epidermoid cancer, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, polyps of the large intestine, and colorectal cancer; liver cancers such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma, and angiosarcoma; kidney cancers such as adrenal cancer, renal cell carcinoma, hypernephroma, and transitional cell carcinoma of the renal pelvis; bladder cancer; blood cancers such as acute lymphoblastic (lymphoblastic) leukemia, acute myeloid (myeloid, myelogenous, myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sezary syndrome and hairy cell leukemia), chronic myeloid (myeloid, myelogenous, granulocytic) leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, mycosis fungoides, myeloproliferative disorders (including myeloproliferative disorders such as polycythemia vera, myelofibrosis, thrombocythemia, and chronic myeloid leukemia); skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi sarcoma, and Paget's disease; head and neck cancers; eye-related cancers such as retinoblastoma and intraocular melanoma; male genital system cancers such as benign prostatic hyperplasia, prostate cancer, and testicular cancer (such as seminoma, teratoma, fetal cancer, and choriocarcinoma); breast cancer;Female genital cancers such as uterine cancer (endometrial cancer), cervical cancer (cervical carcinoma), ovarian cancer, vulvar cancer, vaginal cancer, fallopian tube cancer, and choriocarcinoma; thyroid cancer (including papillary cancer, follicular cancer, anaplastic cancer, or medullary cancer); pheochromocytoma (adrenal gland); non-cancerous proliferation of the parathyroid gland; pancreatic cancer; and blood cancers such as leukemia, myeloma, non-Hodgkin lymphoma, and Hodgkin lymphoma are included. In certain embodiments, the cancer is colon cancer.;

[0142] In some embodiments, examples of cancers include B cell lymphomas (including low grade / follicular non-Hodgkin lymphoma (NHL)), B cell cancers; small lymphocytic (SL) NHL; intermediate / follicular NHL; intermediate diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myelogenous leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as hemangiomatosis, edema (such as those associated with brain tumors), B cell proliferative disorders, and abnormal angiogenesis associated with Meigs syndrome, but are not limited thereto. More specific examples include relapsed or refractory NHL, frontline low grade NHL, stage III / IV NHL, chemotherapy-resistant NHL, precursor B lymphoblastic leukemia and / or lymphoma, small lymphocytic lymphoma, B cell chronic lymphocytic leukemia and / or prolymphocytic leukemia and / or small lymphocytic lymphoma, B cell prolymphocytic lymphoma, immunocytoma and / or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone-MALT lymphoma, nodal marginal zone lymphoma, hairy cell leukemia, plasmacytoma and / or multiple myeloma, low grade / follicular lymphoma, intermediate / follicular NHL, mantle cell lymphoma, follicular center lymphoma (follicular), intermediate grade diffuse NHL, diffuse large cell type B cell lymphoma, aggressive NHL (including aggressive frontline NHL and aggressive relapsed NHL), NHL relapse or refractory after autologous stem cell transplantation, primary mediastinal large cell type B cell lymphoma, primary pleural effusion lymphoma, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, Burkitt lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and / or Sézary syndrome, cutaneous (skin) lymphoma, anaplastic large cell lymphoma, angiocentric lymphoma, but are not limited thereto.

[0143] In some embodiments, examples of cancers further include, but are not limited to, B cell proliferative disorders including lymphoma (e.g., B cell non-Hodgkin lymphoma (NHL)) and lymphocytic leukemia. Such lymphomas and lymphocytic leukemias include, for example, a) follicular lymphoma, b) small non-cleaved cell lymphoma / Burkitt lymphoma (including endemic Burkitt lymphoma, sporadic Burkitt lymphoma, and non-Burkitt lymphoma), c) marginal zone lymphoma (including extranodal marginal zone B cell lymphoma (mucosa-associated lymphoid tissue lymphoma, MALT), nodal marginal zone B cell lymphoma, and splenic marginal zone lymphoma), d) mantle cell lymphoma (MCL), e) large cell lymphoma (including B cell diffuse large cell lymphoma (DLCL), diffuse mixed cell lymphoma, immunoblastic lymphoma, primary mediastinal B cell lymphoma, angiocentric lymphoma - pulmonary B cell lymphoma), f) hairy cell leukemia, g) lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, h) acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, i) plasmacytoma, plasma cell myeloma, multiple myeloma, plasmacytosis, and / or j) Hodgkin's disease.

[0144] In some other embodiments, the disorder is an autoimmune disease. Examples of autoimmune diseases that can be treated with an antibody or an antigen-binding portion thereof include autoimmune encephalomyelitis, lupus erythematosus, and rheumatoid arthritis. The antibody or an antigen-binding portion thereof can also be used to treat or prevent infectious diseases, inflammatory diseases (such as allergic asthma), and chronic graft-versus-host disease.

[0145] Combination with chemotherapy The antibody or an antigen-binding portion thereof can be used in combination with an anti-cancer agent, a cytotoxic agent, or a chemotherapeutic agent.

[0146] The term "anticancer agent" or "antiproliferative agent" means any agent that can be used to treat cell proliferative disorders such as cancer, including but not limited to cytotoxic agents, cytostatic agents, antiangiogenic agents, antiangiogenic agents, cytoreductive agents, chemotherapeutic agents, radiation therapy and radiotherapeutic agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiation therapy and antimetastatic agents and immunotherapeutic agents. In the selected embodiments described above, it is understood that such anticancer agents may include conjugates and may bind to site-specific antibodies disclosed prior to administration. More specifically, in certain embodiments, the selected anticancer agent is linked to engineered antibody mismatched cysteines to provide the engineered conjugates described herein. Thus, such engineered conjugates are clearly contemplated to be within the scope of the present disclosure. In other embodiments, the disclosed anticancer agents are administered in combination with site-specific conjugates containing the different therapeutic agents described above.

[0147] As used herein, the term "cytotoxic agent" means a substance that is toxic to cells, reduces or inhibits cell function, and / or causes cell destruction. In certain embodiments, the substance is a naturally occurring molecule derived from an organism. Examples of cytotoxic agents include small molecule toxins or enzymatically active toxins derived from bacteria (e.g., diphtheria toxin, Pseudomonas endotoxin and exotoxins, staphylococcal enterotoxin A), fungi (e.g., α-sarcin, restrictocin), plants (e.g., abrin, ricin, modeccin, viscumine, American yam antiviral protein, saporin, gelonin, momoridin, trichosanthin, barley toxin, allulight forditoxin, dianthin toxin, Japanese yam protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, curcin, crocin, saponaria inhibitor, gelonin, mitogellin, restrictocin, phenomycin, neomycin, and trichothecene) or animals (e.g., cytotoxic Rnases such as extracellular pancreatic Rnase; including Dnase I, fragments and / or variants thereof), but are not limited to these.

[0148] For the purposes of the present disclosure, a "chemotherapeutic agent" includes compounds (e.g., cytotoxic agents or cytostatic agents) that non-specifically decrease or inhibit the growth, proliferation, and / or survival of cancer cells. Such chemicals are often directed at intracellular processes necessary for cell growth or division and are thus generally particularly effective against cancer cells that rapidly grow and divide. For example, vincristine depolymerizes microtubules and inhibits cells from entering mitosis. In general, chemotherapeutic agents can include any chemical substance that inhibits, or is designed to inhibit, cancer cells or cells that have the potential to become cancerous or tumorigenic progeny (e.g., TIC). Such agents are often administered in combination, for example, in regimens such as CHOP or FOLFIRI, and are often most effective in many cases.

[0149] Examples of anticancer agents that can be used in combination with the site-specific constructs of the present disclosure (either as components of site-specific conjugates or in an unconjugated state) include alkylating agents, alkyl sulfonates, aziridines, ethyleneimines and methylmelamines, acetogenins, camptothecin, bryostatin, calistatin, CC-1065, cryptophycin, dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustard, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esperamicin, chromoprotein enediyne antibiotic chromophore, aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, calminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, keramycin, rhodomycin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogs, purine analogs, androgens, antiadrenal, folic acid supplements such as folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defamin, dexamethasone, diacron, elfomithine, elliptinium acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoid, mitoguazone, mitoxantrone, mopidanmol, nitralin, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, PSK® polysaccharide complex (JHS Natural Products, Eugene, OR), razoxane; lyzoxime; sizofiran; spirogermanium;Tenuazonic acid; Triazicon; 2,2’,2”-trichloro-triethylamine; Trichothecene (especially T-2 toxin, Verracurin A, Lolitrem A and Angidin); Urethane; VinDesine; Dacarbazine; Manomustine; Mitobronitol; Mitolactol; Pipobroman; Gacitabine; Arabinoside (“Ara-C”); Cyclophosphamide; Thiotepa; Taxoid, Chlorambucil; GEMZAR (registered trademark) Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analog, Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; NAVELBINE (registered trademark) Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11), Topoisomerase inhibitor RFS2000; Difluoromethylhydroxylnitine; Retinoid; Capecitabine; Combretastatin; Leucovorin; Oxaliplatin; Inhibitor of PKC-alpha, Raf, H-Ras, EGFR and VEGF-A that suppress cell proliferation and pharmaceutically acceptable salts, and acids or derivatives of any of the above, but not limited thereto. This definition includes antihormonal agents that act to regulate or inhibit the hormonal action on tumors such as antiestrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit the enzyme aromatase that regulates estrogen production in the adrenal gland, and anti-androgens; Troxacitabine (1,3-dioxolane nucleoside cytosine analog); Antisense oligonucleotides, ribozymes such as VEGF expression inhibitors, HER2 expression inhibitors; Vaccines, PROLEUKIN (registered trademark) rIL-2; LURTOTECAN (registered trademark) Topoisomerase 1 inhibitor; ABARELIX (registered trademark) rmRH; Vinorelbine and Esperamicin, and pharmaceutically acceptable salts, acids or derivatives of any of the above are also included.;

[0150] Combined with radiotherapy The present disclosure also provides combinations of antibodies or antigen-binding portions thereof with radiation therapy (i.e., any mechanism for locally inducing DNA damage within tumor cells, such as gamma irradiation, X-rays, UV irradiation, microwaves, electron emission, etc.). Combinatorial therapies using the targeted delivery of radioisotopes to tumor cells are also contemplated, and the disclosed conjugates can be used in connection with a targeted anti-cancer agent or other targeting means. Typically, radiation therapy is administered in pulses over a period of about one to about two weeks. Radiation therapy can be administered to a subject having head and neck cancer for about 6 - 7 weeks. Optionally, radiation therapy can be administered as a single dose or multiple consecutive doses.

[0151] Pharmaceutical Packs and Kits Also provided are pharmaceutical packs and kits comprising one or more containers containing one or more doses of an antibody or antigen-binding portion thereof. In certain embodiments, unit doses are provided, and the unit dose comprises, for example, a predetermined amount of a composition comprising an antibody or antigen-binding portion thereof, with or without one or more additional agents. In other embodiments, such unit doses are provided in single-use pre-filled syringes for injection. In yet other embodiments, the composition included in the unit dose may include, for example, saline, sucrose, etc.; a buffer such as a phosphate, and / or is formulated within a stable and effective pH range. Alternatively, in certain embodiments, the composition can be provided as a lyophilized powder that can be reconstituted by adding an appropriate liquid, such as sterile water or saline. In certain preferred embodiments, the composition includes one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. The label on or associated with the container indicates that the enclosed composition is to be used for treating the symptoms of a selected neoplastic disease.

[0152] The present disclosure also provides a unit dosage of a single or multiple dosages of an antibody, and optionally a kit for generating one or more anti-cancer agents. The kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from various materials such as glass or plastic and may contain a pharmaceutically effective amount of the disclosed antibody in either conjugate or non-conjugate form. In other preferred embodiments, the container includes a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper through which a subcutaneous injection needle can penetrate). Such kits generally contain a pharmaceutically acceptable formulation of the antibody in a suitable container and optionally one or more anti-cancer agents in the same or different containers. The kit may also include other pharmaceutically acceptable formulations for either diagnostic or combination therapy. For example, in addition to the antibodies or antigen-binding portions thereof of the present disclosure, such kits may include any one or more of a series of anti-cancer agents such as chemotherapeutic or radiotherapeutic agents; anti-angiogenic agents; anti-metastatic agents; targeted anti-cancer agents; cytotoxic agents; and / or other anti-cancer agents.

[0153] More specifically, the kit may have a single container that contains the disclosed antibody or antigen-binding portion thereof, with or without additional components, or may have separate containers for each desired agent. If a combined therapeutic agent is provided for combination, a single solution can be pre-mixed in molar equivalents of the combination or with one component in excess of the other. Alternatively, the antibody of the kit and any anti-cancer agent can be maintained separately in separate containers prior to administration to the patient. The kit may also include second / third container means for containing a sterile pharmaceutically acceptable buffer or other diluent such as bacteriostatic water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution, and dextrose solution.

[0154] If the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, and a sterilized aqueous solution or physiological saline is particularly preferred. However, the components of the kit may be provided as dry powders. When a reagent or component is provided as a dry powder, the powder can be reconstituted by adding an appropriate solvent. It is assumed that the solvent may also be provided in a separate container.

[0155] As briefly shown above, the kit may also include means for administering the antibody or antigen-binding portion thereof and any components to a patient, such as one or more needles, I.V. bags or syringes, and further an eye dropper, pipette, or other similar devices, from which the formulation can be injected or introduced into an animal or applied to an affected part of the body. The kits of the present disclosure will also typically include vials, or the like, and means for hermetically containing other components for commercial sale, such as injection or blow-molded plastic containers in which the desired vials and other implements are positioned and held.

[0156] Summary of the Sequence Listing This application is accompanied by a sequence listing containing a number of amino acid sequences. Table A below provides an overview of the sequences included.

[0157] One exemplary antibody disclosed herein, which is an anti-VEGF / anti-PD-L1 bispecific antibody, is designated W3256-U15T2.G6-1.uIgG1 (abbreviated as "W3256" throughout the disclosure). [Table A] [Examples]

[0158] The present disclosure, generally described as such, will be more readily understood by reference to the following examples. These are provided by way of illustration and are not intended to limit the present disclosure. The examples are not intended to represent that the following experiments were all or the only experiments performed.

[0159] Example 1 Preparation of Materials, Benchmark Antibodies, and Cell Lines 1.1 Preparation of Materials Information on commercially available materials used in the examples is provided in Table 1. [Table 1]

[0160] 1.2 Preparation of Antigens The DNA sequences encoding the sequences of human VEGF (UniProt number: P15692), mouse VEGF (UniProt number: Q00731), and the extracellular domain sequences of human PD-L1 (UniProt number: Q9NZQ7), mouse PD-L1 (UniProt number: Q9EP73), human PD-1 (UniProt number: Q15116), and mouse PD-1 (UniProt number: Q02242) were synthesized by Sangon Biothech (Shanghai, China) and subcloned into a modified pcDNA3.3 expression vector with different tags (such as 6xhis, human Fc, or mouse Fc) at the C-terminus.

[0161] Expi293 cells (Thermo Fisher Scientific, A14527) were transfected with the purified expression vector. The cells were cultured for 5 days, and the supernatant was collected for protein purification using a Ni-NTA column (GE Healthcare, 175248), a protein A column (GE Healthcare, 175438), or a protein G column (GE Healthcare, 170618). The obtained human VEGF, human PD-L1, mouse PD-L1, human PD-1, and mouse PD-1 were QC'd by SDS-PAGE and SEC and stored at -80 °C.

[0162] 1.3 Preparation of Benchmark Antibodies (BMK Abs) The DNA sequence encoding the fragment of the anti-VEGF antibody, bevacizumab (the sequence from Drug Bank, named WBP325-BMK3 or WBP325-BMK3.uIgG1, Drug Bank number: DB00112), was subcloned into a modified pcDNA3.3 expression vector with the Fc region of human IgG1 by Sangon Biothech (Shanghai, China) or Genewiz (Suzhou, China).

[0163] The anti-PD-L1 VHH antibody (W3156-AP3R2-1A3-z12) was generated by alternately immunizing alpacas with the extracellular domains of human and mouse PD-L1, and PBMCs were isolated for phage library construction. After panning, screening, and sequencing, one unique positive VHH fragment was identified (SEQ ID NO: 4 disclosed in Patent No. PCT / CN2020 / 117351).

[0164] Atezolizumab, an anti-PD-L1 antibody developed by Roche (named W315-BMK8.uIgG1K(RKNA) or abbreviated as W315-BMK8), was used as a control antibody.

[0165] To produce WBP325-BMK3, plasmids containing recombinant VH and VL genes were co-transfected into Expi293 cells. The cells were cultured for 5 days, and the supernatant was collected for protein purification using a Protein A column (GE Healthcare, 175438) or a Protein G column (GE Healthcare, 170618). The obtained antibody was analyzed by SDS-PAGE and SEC and then stored at -80°C.

[0166] 1.4 Establishment of stable cell lines / cell pools PD-L1-expressing cell line Using Lipofectamine 2000 (Thermo Fisher Scientific, 11668019) or PlasFect (Bioline, 46026), CHO-K1 or 293F cells were transfected with an expression vector containing the gene encoding full-length human PD-L1, mouse PD-L1, or cynomolgus monkey PD-L1. Cells were cultured in a medium containing an appropriate selection marker. Human PD-L1 high-expression stable cell line (WBP315.CHO-K1.hPro1.C11), mouse PD-L1 high-expression stable cell line (WBP315.293F.mPro1.C1), and cynomolgus monkey PD-L1 high-expression stable cell line (WBP315.293F.cPro1.2A) were obtained by the limiting dilution method.

[0167] The genes for human PD-L1, mouse PD-L1, or cynomolgus monkey PD-L1 were each inserted into the expression vector pcDNA 3.3. Then, the plasmids were transfected into CHO-K1 cells or 293 cells, respectively. Briefly, for CHO-K1 cells, one day before transfection, 5×10 5 CHO-K1 cells were plated into one well of a 6-well tissue culture plate and incubated at 5% CO 2 and 37 °C. The cells were supplied with 3 ml of fresh non-selective medium (F12-K, 10% FBS). The transfection reagent was prepared in a 1.5 ml tube containing 4 μg of DNA mixed with 10 μg of Lipofectamine 2000, and the final volume was adjusted to 200 μl with Opti-MEM medium. The solution in the tube pipette was added drop by drop to the cells. Six to eight hours after transfection, the cells were washed with PBS and 3 ml of fresh non-selective medium was added. Twenty-four to forty-eight hours after transfection, the expressing cells were harvested with trypsin and plated into a T75 flask in selective medium (F12-K, 10% FBS, 10 μg / ml blasticidin). For 293F cells, 20 μg of DNA was mixed with 50 μl of PlasFect and the final volume was adjusted to 200 μl with Opti-MEM medium. This was added to 20 ml of 293F cells (1×10 6In addition to ( / ml), it was cultured in Freestyle 293 medium in a 125 ml flask. 48 hours after transfection, blasticidin was added as a selection marker. After 2 or 3 passages of selection, the cells were detected by an anti-PD-L1 antibody. Stable single cell clones were isolated by the limiting dilution method and screened by FACS using an anti-PD-L1 antibody.

[0168] Target expression cell line Human umbilical vein endothelial cells (HUVEC) were purchased from ScienceCell (Cat: 8000) and cultured in endothelial cell medium (ECM, ScienceCell, Cat: 1001) containing basal medium, 5% FBS, and 1% endothelial cell growth supplement (ECGS, ScienceCell, 1052). The cells were cultured in an incubator at 37 °C and 5% CO 2 for cell culture. For long-term storage, the cells were frozen in complete growth medium supplemented with 5% (v / v) DMSO and stored in the liquid nitrogen vapor phase.

[0169] Example 2 Generation of PD-L1 / VEGF bispecific antibody 2.1 Construction of expression vectors The DNA sequence encoding VHH antibody W3156-AP3R2-1A3-z12, which was linked by a flexible G4S linker, was placed at the N-terminus of the light chain of WBP325-BMK3 (bevacizumab). The heavy chain was constructed using the same sequence as that of WBP325-BMK3 (bevacizumab). The recombinant DNA sequences were cloned into modified pcDNA3.3 expression vectors, respectively. The constructed antibody was named W3256-U15T2.G6-1.uIgG1 (abbreviated as "W3256" throughout the disclosure).

[0170] As described above, the heavy chain of W3256-U15T2.G6-1.uIgG1 contains the variable heavy chain region of WBP325-BMK3 (bevacizumab) and the constant heavy chain region (CH1-CH3) of human IgG1. As shown in Figure 1, the light chain of W3256-U15T2.G6-1.uIgG1 is composed of the variable light chain region of WBP325-BMK3 (bevacizumab) and the constant light chain region (CL) of human IgG1, and the VHH antibody W3156-AP3R2-1A3-z12 is at the N-terminus. The specific sequences of the W3256 antibody are shown in Tables 2 to 4 below.

Table 2

Table 3

Table 4

[0171] 2.2 Transfection, Expression, and Purification The Expi293 expression system kit (ThermoFisher-A14635) was used according to the manufacturer's instructions to co-transfect the heavy chain and light chain expression plasmids into Expi293 cells. Five days after transfection, the supernatant was collected and used for protein purification using a protein A column (GE Healthcare-17543802). The antibody concentration was measured by NanoDrop. The protein purity was evaluated by SDS-PAGE and HPLC-SEC. The bispecific antibody containing W3256-U15T2.G6-1.uIgG1 was obtained after expression and purification.

[0172] 2.3 Preparation of Bispecific Antibodies for In Vivo Studies (Including Endotoxin Control and Testing) The W3256-U15T2.G6-1.uIgG1 expression plasmid was transfected into Expi293 cells according to the manufacturer's instructions using the Expi293 Expression System Kit (ThermoFisher - A14635). Five days after transfection, the supernatant was harvested and used for protein purification using a Protein A column (GE Healthcare - 17543802) under endotoxin-controlled conditions. By using an endotoxin detection kit (GenScript - L00350), low endotoxin levels (less than 10 EU / mg) were confirmed.

[0173] When the antibody concentration was measured by NanoDrop, the yield of W3256-U15T2.G6-1.uIgG1 was 44.8 mg / l. The protein purity was evaluated by SDS-PAGE (Figure 2) and HPLC-SEC (Figure 3). According to HPLC-SEC, the purity of W3256-U15T2.G6-1.uIgG1 after one-step purification with Protein A was 93.43%.

[0174] Example 3 In Vitro Characterization Evaluation of Bispecific Antibodies 3.1 Differential Scanning Fluorimetry (DSF) The DSF assay was performed using a 7500Fast Real-Time PCR System (Applied Biosystems). Briefly, 19 μl of the bispecific antibody solution was mixed with 1 μl of 62.5X SYPRO Orange solution (ThermoFisher - S6650) and added to a 96-well plate. The plate was heated from 26°C to 95°C at a rate of 2°C / min, and the resulting fluorescence data was collected. The data was automatically analyzed by its operating software, and Th was calculated by taking the maximum value of the negative derivative of the fluorescence data obtained with respect to temperature. on T can be roughly determined as the temperature of the negative derivative plot when it begins to decrease from the baseline before the transition.

[0175] As shown in Figure 4, the T of the W3256 antibody h1 The value is 65.7 °C.

[0176] 3.2 Human / Cynomolgus VEGF Binding (ELISA) The binding of the antibody to human VEGF antigen (WBP325-hPro1, Sino Biological, 11066-HNAB) was tested by a binding ELISA assay. Since the amino acid sequence of cynomolgus VEGF is the same as that of human VEGF, the binding results represent both human VEGF binding and cynomolgus VEGF binding. Briefly, a 96-well ELISA plate (Nunc MaxiSorp, ThermoFisher, 442404) was coated overnight at 4 °C with 0.25 μg / ml of human VEGF in carbonate-bicarbonate buffer (20 mM Na 2 CO 3 , 180 mM NaHCO 3 , pH 9.2). After a 1-hour blocking step with 2% (w / v) bovine serum albumin (Pierce) dissolved in PBS, serial dilutions of W3256-U15T2.G6-1.UIgG1 were incubated on the plate for 2 hours at room temperature. After incubation, the plate was washed 3 times with 300 μL per well of PBS containing 0.5% (v / v) Tween 20. 100 ng / ml of goat anti-human IgG Fc-HRP (SouthernBiotech, #A80-304P) was added and incubated on the plate for 1 hour at room temperature. After washing 6 times, tetramethylbenzidine (TMB) substrate (Sigma-860336-5G) was added for detection. The reaction was stopped after about 8 minutes by adding 100 μl per well of 2 M HCl. The absorbance of the wells was measured at 450 nm using a multi-well plate reader (SpectraMax® M5 e ).

[0177] W3256 showed a binding ability comparable to that of WBP325-BMK3.uIgG1 on human VEGF, and the EC 50 was about 0.095 nM (Figure 5).

[0178] 3.3 Human PD-L1 Binding (FACS) The operated human PD-L1-expressing cells (WBP315.CHO-K1.hPro1.C11) were plated in a U-bottom 96-well plate at 1x10 5 cells / well. Serial dilution of W3256-U15T2.G6-1. UIgG1 was added to the cells. The plate was incubated at 4°C for 1 hour. After washing, FITC-labeled goat anti-human IgG (Jackson ImmunoResearch, 109-095-008) was added to each well and the plate was incubated at 4°C for 1 hour. The binding of the antibody to the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

[0179] W3256 also showed a binding ability comparable to that of W3156-AP3R2-1A3-z12-hIgG1 on human PD-L1, and the EC 50 was approximately 0.128 nM (Figure 6).

[0180] 3.4 Human VEGF / Human PD-L1 Dual Binding (ELISA) To test whether the bispecific antibody can bind to both VEGF and PD-L1, an ELISA assay was developed as follows. A 96-well ELISA plate (Nunc MaxiSorp, ThermoFisher) was coated overnight at 4°C with 1 μg / ml antigen-1 (VEGF, WBP325-hPro1, Sino Biological) or 1 μg / ml antigen-2 (PD-L1.ECD.mFc, W3153-hPro1.ECD.mFc) in carbonate-bicarbonate buffer. After a 1-hour blocking step, serial dilutions of W3256-U15T2.G6-1.UIgG1 in casein buffer were incubated on the plate with UIgG1 in casein buffer for 1 hour at room temperature. After incubation, the plate was washed three times with 300 μL per well of PBS containing 0.5% (v / v) Tween20. 0.5 μg / ml antigen-2.biotin (PD-L1-ECD, WBP315-hPro1.ECD.mFc) or 0.5 μg / ml antigen-1.biotin (VEGF, WBP325-hPro1.his) was added to the plate and incubated for 1 hour. After the plate was washed three times, an HRP-labeled secondary detection antibody was added and incubated on the plate for 1 hour at room temperature. After the plate was washed six times, tetramethylbenzidine (TMB) substrate (Sigma-860336-5G) was added for detection. The reaction was stopped after approximately 10 minutes by adding 100 μL of 2M HCl per well. The absorbance of the wells was measured at 450 nm using a multi-well plate reader (SpectraMax® M5 e )

[0181] The results showed that the binding of VEGF to W3256 did not affect the subsequent binding of PD-L1 (Figure 7), and vice versa (Figure 8).

[0182] 3.5 Cross-species binding (FACS / ELISA) Cyno PD-L1 binding (FACS) Engineered cynomolgus PD-L1-expressing cells (WBP315.293F.cPro1.2A) were seeded at 1×10 5Plated in cells / wells. Serial dilution of W3256-U15T2.G6-1. UIgG1 was added to the cells. The plates were incubated at 4°C for 1 hour. After washing, FITC-labeled goat anti-human IgG (Jackson ImmunoResearch, 109-095-008) was added to each well and the plates were incubated at 4°C for 1 hour. Binding of the antibody to the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

[0183] Mouse VEGF Binding (ELISA) Binding of the antibody to mouse VEGF antigen (WBP325-mPro1, Sino Biological, 50159-MNAB) was tested by the same ELISA assay as the human VEGF binding above, except that the coating protein was 100 μL of 0.25 μg / mL mouse VEGF.

[0184] Mouse PD-L1 Binding (FACS) Engineered mouse PD-L1-expressing cells (WBP315.293F.mPro1.C1) were plated at 1x10 5 in cells / wells of a U-bottom 96-well plate. Serial dilutions of different antibodies were added to the cells. The plates were incubated at 4°C for 1 hour. After washing, FITC-labeled goat anti-human IgG (Jackson ImmunoResearch, 109-095-008) was added to each well and the plates were incubated at 4°C for 1 hour. Binding of W3256-U15T2.G6-1. UIgG1 to the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

[0185] Since the amino acid sequence of cynomolgus monkey VEGF is the same as that of human VEGF, W3256 also has binding activity against cynomolgus monkey VEGF. The cross-species binding activities of W3256 against cynomolgus monkey PD-L1, mouse VEGF, and mouse PD-L1 were also evaluated. As shown in FIGS. 9, 10, and 11, binding activity against VEGF or PD-L1 was detected. W3256 exhibited binding ability equivalent to that of the parental antibody against cynomolgus monkey PD-L1, and the EC 50 was approximately 2.618 nM (FIG. 9). W3256 was unable to bind to mouse VEGF (FIG. 10). W3256 also exhibited binding ability equivalent to that of the parental antibody against mouse PD-L1, and the EC 50 was approximately 1.687 nM (FIG. 11).

[0186] 3.6 Binding affinity to VEGF and PD-L1 (SPR) Using SPR technology, the on-rate constant (ka) and off-rate constant (kd) of the antibody against VEGF or ECD of PD-L1 were measured. As a result, the affinity constant (KD) was determined.

[0187] Biacore T200, Series S sensor chip CM5, amine coupling kit, and 10x HBS-EP were purchased from GE Healthcare. The antibody was captured on the surface of anti-human Fc IgG (Jackson, 109-005-098) immobilized on a CM5-biosensor chip (GE Healthcare Inc.). The assay was performed at 25 °C using HBS-EP+ buffer (GE Healthcare Inc.) as the running and dilution buffer. Serial diluted PD-L1 antigen or VEGF antigen (W315-hPro1.ECD.his or W325-hPro1.his) and the running buffer were injected for the binding phase, and dissociation phase detection was performed. Regeneration of the chip surface was achieved by injection of 10 mM glycine, pH 1.5.

[0188] The affinity constant (KD) of W3256 was measured based on SPR technology. Meanwhile, the on-rate constant (ka) and off-rate constant (kd) were also measured. The final data for each interaction was subtracted from the data of the reference channel and the buffer channel. The experimental data was analyzed as shown in Figures 12 and 13. The results of the kinetic affinity of the antibody are shown in Table 5. [Table 5]

[0189] 3.7 Ligand Competition Assay (ELISA) Human VEGFR1 Competition Assay Inhibition of VEGF binding to human VEGF receptor 1 (W325-hpro1R1.ECD.hFc, Sino Biological, 10136-H02H) was determined by competitive ELISA. A 96-well ELISA plate (Nunc MaxiSorp, ThermoFisher, 442404) was coated with carbonate-bicarbonate buffer (20 mM Na 2 CO 3 , 180 mM NaHCO 3, coated overnight at 4 °C with 2 μg / mL of W325-hpro1R1.ECD.His in carbonate buffer (pH 9.2). All wells were washed three times with 300 μL per well of PBS / 0.5‰ Tween-20 (v / v), and all subsequent washing steps in the assay were performed similarly. Next, the wells were blocked for 1 hour with 200 μL per well of 50% casein (Thermo SCIENTIFIC, 37528). After the washing step, a mixture of serially diluted antibody and 0.02 μg / ml of biotinylated human VEGF (W325-hPro1.Biotin, ACRO Biosystems, VE5-H8210) was added to the wells and incubated at 25 °C for 2 hours. The plate was washed three times before adding peroxidase-conjugated streptavidin (Jackson, 016-030-084) diluted 1:10,000 in PBS / 50% casein. After incubating the plate at 25 °C for 1 hour, it was washed six times as before. 100 μL / well of tetramethylbenzidine (TMB) substrate (Sigma, 860336) was added to all wells for 8 minutes, and then the reaction was stopped with 100 μL of 2M HCl. The degree of human VEGF-biotin binding to W325-hpro1R1.ECD.hFc was determined by measuring the OD e ) using a multi-well plate reader (SpectraMax® M5 450 . The IC 50 value of the binding was obtained by four-parameter non-linear regression analysis using GraphPad Prism5 software.

[0190] W3256 showed a competitive ability comparable to that of the parental antibody against hVEGFR1 and bound to VEGF with an IC 50 of approximately 3.86 nM (Figure 14).

[0191] Mouse VEGFR1 Competition Assay The effect of an antibody that blocks the binding of VEGF to mouse VEGF receptor 1 (W325-mpro1R1.ECD.hFc.his (R&D, 471-F1)) was evaluated by competitive ELISA. The mouse VEGFR1 competitive ELISA method is the same as the human VEGFR1 competitive ELISA, except that the protein pre-coated on the 96-well ELISA plate is 2 μg / ml of W325-mpro1R1.ECD.hFc.his, the blocking buffer is PBS / 2% BSA, and the concentration of W325-hPro1.Biotin is 0.44 μg / ml.

[0192] W3256 showed a competitive ability comparable to that of the parental antibody against mVEGFR1 (Figure 16), binding to VEGF at an IC 50 of approximately 31 nM.

[0193] Human PD-1 competitive assay The competition of W3256 against human PD-1 was measured by a FACS-based competition assay. Human PD-L1-expressing CHO-K1 cells (WBP315.CHO-K1.hPro1.C11) were plated at 1×10 5 cells / well in a 96-well U-bottom plate. Serial dilutions of the test antibody were pre-mixed with 5 μg / ml of mouse Fc-tagged human PD-1 and added to the cells, followed by incubation at 4°C for 1 hour. After washing, PE-labeled anti-mouse IgG was added and the cells were incubated with it at 4°C for 45 minutes. The cells were washed twice, and the MFI of the cells was measured with a flow cytometer and analyzed with FlowJo.

[0194] W3256 showed a competitive ability comparable to that of the parental antibody, blocking the binding of hPD-1 to PD-L1 (Figure 15) at an IC 50 of approximately 0.048 nM.

[0195] Mouse PD-1 competitive assay Mouse PD-L1-expressing CHO-K1 cells (WBP315.293F.mPro1.C1) were plated at 1×10 5Cells were plated in wells. Serial dilutions of the test antibody were pre-mixed with 5 μg / ml of mouse Fc-tagged mouse PD-1 and added to the cells, which were then incubated at 4 °C for 1 hour. After washing, PE-labeled anti-mouse IgG was added and the cells were incubated with it at 4 °C for 1 hour. The cells were washed twice, and the mean fluorescence intensity (MFI) of the cells was measured using a flow cytometer and analyzed with FlowJo.

[0196] W3256 showed a competitive ability comparable to that of the parental antibody and blocked the binding of mouse PD-1 (Figure 17) to PD-L1 with an IC 50 of approximately 1.687 nM.

[0197] 3.8 HUVEC Cell Proliferation Assay The biological activity of W3256 in VEGF-induced HUVEC proliferation was evaluated. HUVEC cells were routinely cultured in ECM + 5% FBS + 1% ECGS. Sub-confluent cells were harvested with trypsin and diluted to 1 × 10 5 cells / mL in ECM + 1% FBS + 0.05% ECGS. The cells were plated in 96-well clear-bottom black plates (Greiner, 655090) at a density of 5000 cells / well. Serial dilutions of the antibody were added together with 50 ng / mL of human VEGF (WBP325-hPro1, Sino Biological, 11066-HNAB). The plates were returned to the incubator for 4 days before evaluating cell viability using CellTiter Glo (Promega, G7573). Wells without added ligand served as controls for ligand-stimulated cell proliferation. The effect of the test antibody on ligand-stimulated cell proliferation inhibition was calculated by comparing the luminescence values with or without antibody addition (ligand only) after subtracting the background (no ligand) luminescence. Four-parameter non-linear regression analysis was used to obtain the proliferation inhibition IC 50 values using GraphPad Prism5 software.

[0198] W3256 had an IC 50And blocked VEGF-induced HUVEC proliferation in a concentration-dependent manner with a maximum inhibition rate of 101% effectively (Figure 18).

[0199] 3.9 Reporter gene assay Normal cultured CHOK1-OKT3-PD-L1 cells were harvested with trypsin and plated at a density of 20,000 cells / well in 96-well clear-bottom black plates (Greiner, 655090). Serial diluted antibodies were added together with the reporter signal incorporated with NFAT-RE-Luc2p and full-length PD-1-expressing Jurkat cells. The plates were returned to the incubator for 4 hours, and 50 μl of One-glo luciferase assay buffer / substrate mixture was added. Luminescence was measured with a multi-well plate reader M5e.

[0200] W3256 showed functionality in the reporter gene assay (Figure 19), indicating that the antibody induced the PD-1 signaling pathway.

[0201] 3.10 Mixed lymphocyte reaction (MLR) assay The MLR was used to test the agonist effect of the PD-L1 antibody on cytokine, human IFN-γ secretion, and the proliferation of activated human CD4 + T cells.

[0202] Human peripheral blood mononuclear cells (PBMCs) were freshly isolated from healthy donors using Ficoll-Paque PLUS gradient centrifugation. The isolated PBMCs were cultured in complete RPMI-1640 (containing 10% FBS and 1% PS) supplemented with 100 U of recombinant human IL-2 (SL PHARM). Monocytes were isolated using a human monocyte enrichment kit according to the manufacturer's instructions. The cell concentration was 2 × 10 6Adjusted to cells / ml. The cells were cultured for 5 - 7 days to differentiate into dendritic cells (DCs). The cytokines were replenished every 2 - 3 days by replacing half of the medium with fresh medium supplemented with cytokines. 1 μg / ml of LPS was added to the culture 18 - 24 hours before the MLR to induce DC maturation. According to the manufacturer's protocol, human CD4 + Using a human CD4 + T cell enrichment kit, human CD4

[0203] The MLR was set up in a 96-well round-bottom plate (Nunc, 163320) using complete RPMI-1640 medium. CD4 + T cells, various concentrations of antibody, and mature DCs were added to the plate. The plate was incubated at 37 °C, 5% CO 2 . IFN-γ production was measured on day 5.

[0204] Human IFN-γ was measured by enzyme-linked immunosorbent assay (ELISA) using the corresponding antibody pair. Recombinant human IFN-γ (PeproTech, 300-02) was used as a standard. The plates were pre-coated with a capture antibody specific for human IFN-γ (Pierce, M700A). After blocking, the standard or samples were pipetted into each well and incubated for 2 hours at ambient temperature. After removing unbound substances, a biotin-conjugated detection antibody specific for IFN-γ (Pierce, M701B) was added to the wells and incubated for 1 hour each. Then, streptavidin-conjugated horseradish peroxidase (HRP) (Invitrogen, SNN1004) was added to the wells and incubated for 30 minutes at ambient temperature. TMB substrate was dispensed to develop color, and the color development was stopped with 2 M HCl. The absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer.

[0205] Figure 20 shows the effect of the antibody on hCD4+ T cell IFN-γ secretion in the mixed lymphocyte reaction assay. As a result, it was shown that W3256 could induce IFN-γ secretion in a concentration-dependent manner in the MLR.

[0206] Example 4 In vivo antitumor efficacy study 4.1 Pharmacokinetic study in mice Male C57BL / 6 mice (6 - 8 weeks old, 18 - 22 g) were purchased from Shanghai SLAC or BK Laboratory Co., LTD., and housed in the facilities of WuXi Biologics (Shanghai, China) under specific pathogen - free conditions, allowing the animals to freely access food and water.

[0207] Blood samples were collected from male C57BL / 6 mice (n = 5) at 0, 0.5, 6, 24, 48, 72, 120, and 168 hours after a single intravenous injection of equimolar concentration of W3256 antibody. The concentration of serum antibody was measured using human IgG ELISA quantification method and double - antigen - binding ELISA method.

[0208] According to the results of total IgG binding (Figure 21), the half - life of W3256 was 129 hours via the i.v. injection route at a dose of 11.5 mg / kg, and 104 hours via the i.v. injection route at a dose of 11.5 mg / kg in the double - antigen - binding PK assay (Figure 22).

[0209] 4.2 Efficacy in PBMC - RKO mouse model RKO tumor cells were maintained in vitro as monolayer cultures in a culture medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in an atmosphere of 5% CO 2 in air. The tumor cells were sub - cultured twice a week by trypsin - EDTA treatment. Cells in the exponential growth phase were harvested and counted for tumor inoculation.

[0210] Female NCG mice (6 - 10 weeks old) were purchased from Shanghai SLAC or BK Laboratory Co., LTD., and housed in the animal facilities of WuXi Biologics (Shanghai, China) under specific pathogen - free conditions allowing free access to food and water.

[0211] Each mouse was inoculated with RKO tumor cells (2 × 10 cells) in 0.2 ml of PBS containing 50% matrix gel for tumor development. 6 ) was subcutaneously inoculated with 4 × 10 6 PBMC (Hemacare) were injected intraperitoneally. Treatment was initiated on day 7.

[0212] The primary endpoint was to determine whether tumor growth could be slowed or whether the mice could be cured. Tumor size was measured twice a week using calipers and volume was calculated using the formula: V = 0.5 a × b 2 Using mm 3 This is expressed as:

[0213] Summary statistics including the mean and standard error of the mean (SEM) are provided for tumor volumes in each group at each time point. Statistical analysis of tumor volume differences between groups and analysis of drug interactions were performed on data obtained at the best treatment time point after the last dose (day 28 after starting dosing). Two-way ANOVA was performed to compare tumor volumes between groups, followed by post-hoc multiple comparisons of Dunnett's t test (all compared to the IgG group). All data were analyzed using SPSS 17.0 or Prism 5. p<0.05 was considered statistically significant.

[0214] W3256 demonstrated antitumor efficacy in a PBMC-RKO colorectal cancer model in NCG mice that was significantly superior to W315-BMK8 and comparable to W325-BMK3 (Figure 23).

[0215] Those skilled in the art will further appreciate that the present disclosure may be embodied in other specific forms without departing from its spirit or central attributes. It should be understood that the foregoing description of the present disclosure discloses only exemplary embodiments thereof, and that other variations are contemplated within the scope of the present disclosure. Accordingly, the present disclosure is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims as indicating the scope and content of the disclosure.

Claims

**Claim 1** A bispecific antibody or an antigen-binding fragment thereof, wherein the bispecific antibody or the antigen-binding fragment thereof comprises a PD-L1 antigen-binding portion fused or operably linked to a VEGF antigen-binding portion, wherein the PD-L1 antigen-binding portion, comprises complementarity-determining region (CDR) 1 comprising SEQ ID NO: 1, CDR2 comprising SEQ ID NO: 2, and CDR3 comprising SEQ ID NO: 3, wherein the VEGF antigen-binding portion, comprises heavy-chain complementarity-determining region (HCDR) 1 comprising SEQ ID NO: 4, HCDR2 comprising SEQ ID NO: 5, HCDR3 comprising SEQ ID NO: 6, light-chain complementarity-determining region (LCDR) 1 comprising SEQ ID NO: 7, LCDR2 comprising SEQ ID NO: 8, and LCDR3 comprising SEQ ID NO: 9, a bispecific antibody or an antigen-binding fragment thereof. **Claim 2** wherein the PD-L1 antigen-binding portion, comprises a variable domain comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 10, the bispecific antibody or the antigen-binding fragment thereof according to claim 1. **Claim 3** wherein the VEGF antigen-binding portion, comprises a heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that is 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 that is at least 85%, 90%, or 95% identical to SEQ ID NO: 12, the bispecific antibody or the antigen-binding fragment thereof according to claim 1 or 2. **Claim 4** wherein the PD-L1 antigen-binding portion is fused to the N-terminus of the VEGF antigen-binding portion, the bispecific antibody or the antigen-binding fragment thereof according to any one of claims 1 to 3. **Claim 5** wherein the PD-L1 antigen-binding portion is derived from a single-domain antibody (sdAb), the bispecific antibody or the antigen-binding fragment thereof according to any one of claims 1 to 4. **Claim 6** The bispecific antibody or the antigen-binding fragment thereof according to claim 5, wherein the single-domain antibody (sdAb) is a VHH antibody. **Claim 7** The bispecific antibody or the antigen-binding fragment thereof according to claim 6, wherein the VHH antibody is derived from a camelid animal including alpaca or llama. **Claim 8** The bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 to 7, wherein the PD-L1 antigen-binding portion is operably linked to the N-terminus of the light chain or heavy chain of the VEGF antigen-binding portion. **Claim 9** The bispecific antibody or antigen-binding fragment thereof according to claim 8, wherein the PD-L1 antigen-binding portion is operably linked to the N-terminus of the light chain or heavy chain of the VEGF antigen-binding portion via a linker. **Claim 10** The bispecific antibody or antigen-binding fragment thereof according to claim 9, wherein the linker comprises or consists of 1 to 4 copies of GGGGS (G4S). **Claim 11** Comprising a heavy chain and a light chain, wherein the heavy chain comprises a domain operably linked like VH-CH1-hinge-Fc, and the VH-CH1 is derived from the VEGF antigen-binding portion, the light chain comprises a domain operably linked like VHH-VL-CL, the VHH is derived from the PD-L1 antigen-binding portion, and the VL-CL is derived from the VEGF antigen-binding portion, the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 10. **Claim 12** The bispecific antibody or antigen-binding fragment thereof according to claim 11, wherein the Fc region is a human IgG Fc region. **Claim 13** The bispecific antibody or antigen-binding fragment thereof according to claim 12, wherein the Fc region is a human IgG1 Fc region. **Claim 14** The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the heavy chain comprises SEQ ID NO: 13 and the light chain comprises SEQ ID NO:

14. **Claim 15** The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein the bispecific antibody is a humanized antibody. **Claim 16** An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 15. **Claim 17** A vector comprising the nucleic acid molecule according to claim 16. **Claim 18** A host cell comprising the nucleic acid molecule according to claim 16 or the vector according to claim 17. **Claim 19** A pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier. **Claim 20** A method for producing a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 15, comprising: - expressing the antibody or an antigen-binding fragment thereof according to any one of claims 1 to 15 in a host cell according to claim 18; - isolating the antibody or an antigen-binding fragment thereof from the host cell. **Claim 21** i) modulation of a PD-L1 / VEGF-related immune response; ii) promotion of T cell proliferation and cytokine production; and / or iii) stimulation of an immune response or function. A medicament comprising a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 15 for use in **Claim 22** A medicament comprising a bispecific antibody or an antigen-binding fragment thereof according to claim 21, wherein the stimulation of the immune response or function is enhancement of the immune response against cancer cells. **Claim 23** A medicament comprising a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 15 for use in the diagnosis, treatment or prevention of cancer. **Claim 24** A kit comprising a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 15.

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