Antibodies targeting vegf and pd-1 / pd-l1 and uses thereof

Abstract

Disclosed herein are bispecific antibodies targeting human VEGF and PD-1 / PD-L1. Also provided herein are polynucleotides encoding the bispecific antibodies, pharmaceutical compositions comprising the bispecific antibodies, and methods of making the bispecific antibodies. Also disclosed herein are medical uses of the bispecific antibodies described herein.

Description

[0001] This application claims priority to PCT patent application number PCT / CN2024 / 122922, filed on September 30, 2024, the entire contents of which are incorporated herein by reference. 1. References to sequence lists submitted electronically

[0002] This application incorporates a sequence list as an XML file, titled "720A008WO02_SL.XML", created on September 29, 2025, and with a size of 158,261 bytes. Technical Field

[0003] This invention relates to molecular biology, immunology, and tumor biology. This document provides bispecific antibodies targeting VEGF and PD-1 / PD-L1, and their use in the treatment of human diseases such as cancer. Background Technology

[0004] The programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) pathways are important mechanisms in the immune system, particularly relevant to cancer immunotherapy. PD-1 is a receptor present on the surface of T cells, which are essential for the immune system's ability to recognize and attack pathogens or abnormal cells, including cancer cells. When PD-1 binds to its ligand, it transmits an inhibitory signal to T cells. PD-L1 is one of the major ligands of PD-1 and is expressed on the surface of a variety of cells, including cancer cells. When PD-L1 binds to PD-1 on T cells, it effectively suppresses the immune response and inhibits the ability of T cells to attack tumors. By expressing high levels of PD-L1, tumors effectively evade immune detection. Immune checkpoint inhibitors are a class of drugs designed to block the PD-1 / PD-L1 interaction and restore the immune system's ability to recognize and eliminate cancer cells. Notable examples include pembrolizumab (Keytruda) and nivolumab (Opdivo), which target PD-1, and atezolizumab (Tecentriq) and durvalumab (Imfinzi), which target PD-L1. The PD-1 / PD-L1 pathway is an important area of ​​research and development in cancer therapy.

[0005] Meanwhile, the vascular endothelial growth factor (VEGF) pathway plays a crucial role in tumor angiogenesis. By promoting angiogenesis, VEGF not only ensures adequate nutrition and oxygen supply to the tumor but also promotes its metastatic spread. Therefore, strategies aimed at inhibiting VEGF signaling have gained significant attention in cancer treatment, leading to the development of several successful anti-angiogenic agents.

[0006] However, the complexity of cancer biology often renders monotherapy insufficient for durable responses. Agents targeting VEGF and PD-1 / PD-L1 could potentially overcome these limitations by simultaneously disrupting key pathways involved in tumor growth, angiogenesis, and immune escape. However, current treatment options targeting VEGF and PD-1 / PD-L1 are limited. Therefore, there is an unmet need for additional treatment options for cancer patients, particularly dual-targeting agents against VEGF and PD-1 / PD-L1. The compositions and methods presented in this article address these needs and offer other relevant advantages. Summary of the Invention

[0007] In some embodiments, this document provides a bispecific antibody comprising: (i) a first peptide chain (HC) comprising, from the N-terminus to the C-terminus, a first heavy chain variable domain (VH1), a heavy chain constant (CH) region, a linker, and a single-chain variable fragment (scFv), wherein the scFv comprises a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2); and (ii) a second peptide chain (LC) comprising, from the N-terminus to the C-terminus, a first light chain variable domain (VL1) and a light chain constant (CL) region; wherein, (1) the VL1 / VH1 pair specifically binds to human VEGF, and the VL1 comprises VL CDR1, VLCDR2, and VL CDR3, the amino acid sequences of which are SEQ ID NOs:1, 2, and 3, respectively; and the VH1 comprises VH CDR1, VHCDR2, and VH CDR3, the amino acid sequences of which are SEQ ID NOs:4, 5, and 6, respectively; and (2) The VL2 / VH2 pair specifically binds to human PD-L1 or human PD-1.

[0008] In some embodiments of the bispecific antibody disclosed herein, the amino acid sequence of the HC has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NOs:61-64, and the amino acid sequence of the LC has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:60. In some embodiments, the HC has an amino acid sequence selected from the group consisting of SEQ ID NOs:56-59. In some embodiments, the bispecific antibody: (1) inhibits VEGF signaling; (2) inhibits vascular endothelial cell proliferation; (3) inhibits tumor angiogenesis; (4) blocks PD-1 / PD-L1 binding; (5) reduces immunosuppression; or (6) promotes T cell activation and / or proliferation; or any combination of (1)-(6).

[0009] This article also provides pharmaceutical compositions comprising a therapeutically effective amount of the bispecific antibody disclosed herein and a pharmaceutically acceptable carrier.

[0010] This document also provides polynucleotides encoding peptide chains of the bispecific antibodies disclosed herein, and vectors comprising the polynucleotides disclosed herein.

[0011] This document also provides cells comprising the polynucleotides or vectors disclosed herein. In some embodiments, this document provides a method for preparing the bispecific antibodies disclosed herein, which includes culturing the cells disclosed herein under conditions that allow expression of said bispecific antibodies.

[0012] This document also provides methods for reducing immune cell suppression, comprising contacting the immune cells with an effective amount of the bispecific antibody disclosed herein. This document also provides methods for inducing or stimulating immune cell activation and / or proliferation in a subject in need, comprising administering an effective amount of the bispecific antibody disclosed herein to the subject.

[0013] This article also provides methods for inhibiting cancer angiogenesis in subjects in need, comprising administering an effective amount of the bispecific antibody disclosed herein to the subject. This article also provides methods for treating cancer in subjects in need, comprising administering a therapeutically effective amount of the bispecific antibody disclosed herein to the subject. Attached Figure Description

[0014] Figure 1 A schematic diagram is provided to illustrate the candidate bispecific antibody targeting VEGF and PD-1 / PD-L1 disclosed herein. The candidate bispecific antibody is in "IgG-scFv" format, comprising two distinct peptide chains, namely a first peptide chain (HC) and a second peptide chain (LC), with the following configuration: HC:(N)-VH1-CH-(L)-scFv-(C) LC:(N)-VL1-CL-(C) (N): N-terminus; (C): C-terminus; (L): Linker; VH1: First heavy chain variable domain; VL1: First light chain variable domain; CH: Heavy chain constant region; CL: Light chain constant region. scFv includes a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2) connected by a second linker (L).

[0015] Figures 2A-2D Provides representative results from ELISA assays, demonstrating the binding affinity and blocking ability of candidate bispecific antibodies. Figure 2A The binding of HX016-7 / 8 / 9 / 10 to VEGF is shown; Figure 2BThis shows the combination of HX016-7 / 8 and PD-L1; Figure 2C This shows the combination of HX016-9 / 10 and PD-1; Figure 2D The HX016-9 / 10 demonstrates the blocking effect of HX016-9 / 10 on the binding of PD-1 and PD-L1.

[0016] Figures 3A-3B Representative results from FACS binding assays are provided, showing the binding of candidate bispecific antibodies to PD-1 positive and PD-L1 positive cells. Figure 3A The binding of HX016-9 / 10 to PD-1 positive cells was shown; Figure 3B The binding of HX016-7 / 8 to PD-L1 positive cells was shown.

[0017] Figures 4A-4C It provides representative results based on reporter gene bioassays, demonstrating the activity of candidate bispecific antibodies in blocking the PD-1 / PD-L1 and VEGF pathways. Figure 4A HX016-7 / 8 / 9 / 10 showed that it inhibited the PD-1 / PD-L1 axis, thereby triggering downstream T cell activation pathways. Figure 4B HX016-7 / 8 / 9 / 10 showed inhibition of VEGF165-mediated downstream pathways. Figure 4C HX016-9 was shown to induce enhanced PD-1 blocking activity in the PD-1-PD-L1 pathway. rhVEGF165: recombinant human VEGF165.

[0018] Figure 5 Representative results from HUVEC cell assays are provided, showing that HX016-7 / 8 / 9 / 10 effectively inhibits HUVEC growth.

[0019] Figure 6 We provide representative results from our study of candidate bispecific antibodies in the MC38-hPD-L1-hVEGFA model.

[0020] Figures 7A-7D Representative results are provided to demonstrate the in vivo activity of the candidate bispecific antibody in xenograft models. Figure 7A The results showed that HX016-7 and HX016-9 effectively inhibited tumor growth in the A549 xenograft model. Figure 7B The results showed that HX016-7 and HX016-9 effectively inhibited tumor growth in the HCC827 xenograft model. Figure 7C The results showed that HX016-7 and HX016-9 effectively inhibited tumor growth in the NCI-H1975 humanized xenograft model. Figure 7D This shows the results of a single dose of the candidate antibody. BIW: Twice a week. Detailed Implementation

[0021] This disclosure provides bispecific antibodies that specifically bind to human VEGF and human PD-1 / PD-L1. It also provides pharmaceutical compositions comprising a therapeutically effective amount of such bispecific antibodies, and methods for using the bispecific antibodies or pharmaceutical compositions disclosed herein to treat cancers, such as cancers expressing VEGF and / or PD-L1.

[0022] Vascular endothelial growth factor (VEGF), also known as VEGFA, belongs to a family of growth factors that also includes VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PIGF), and plays a crucial role in angiogenesis. In cancer, VEGF expression is often dysregulated, leading to excessive angiogenesis within the tumor microenvironment. This abnormal angiogenesis is essential for tumor growth and metastasis because it provides oxygen and nutrients to the tumor while promoting the spread of cancer cells to distant sites.

[0023] VEGF promotes angiogenesis through several mechanisms. It stimulates endothelial cell proliferation, migration, and survival, leading to new blood vessel formation. In addition, VEGF increases vascular permeability, allowing plasma proteins and cells to leak into surrounding tissues—a process known as vascular leakage or vascular permeability—which can further promote tumor growth and metastasis.

[0024] Given its central role in tumor angiogenesis and progression, VEGF has become an important target in cancer therapy. Several strategies have been developed to inhibit VEGF signaling and disrupt tumor-associated angiogenesis, including, for example, monoclonal antibodies that block its interaction with receptors on endothelial cells, and VEGFR tyrosine kinase inhibitors (TKIs) that target the intracellular kinase domain of the VEGF receptor (VEGFR). Although VEGF-targeted therapy has shown efficacy in various cancer types, including colorectal cancer, lung cancer, breast cancer, and renal cell carcinoma, challenges such as the development of resistance and adverse reactions remain significant concerns.

[0025] The term "VEGF" as used in this article refers to VEGFA. Human VEGF has several isoforms produced by the use of alternative promoters, alternative splicing, and alternative initiation. Some isoforms are produced by using alternative upstream CUG codons, resulting in long isoforms with an N-terminal extension compared to the classic, shorter AUG-initiated form. These longer forms are post-translational processed to produce an N-terminal N-VEGF chain and a C-terminal VEGFA chain. VEGF165 is the major and biologically active isoform of VEGF-A. It consists of 165 amino acids and is known for its role in promoting angiogenesis by binding to VEGF receptors (VEGFR-1 and VEGFR-2) on endothelial cells. VEGF165 possesses mitogenic and angiogenic properties.

[0026] Exemplary classic sequences for human VEGF are available via accession number Uniprot Accession No. P15692-13. More information about human VEGF is available in public databases with the following IDs: HGNC: 12680; NCBIGene: 7422; Ensembl: ENSG00000112715; OMIM®: 192240; UniProtKB / Swiss-Prot: P15692.

[0027] Programmed cell death 1 (PD-1), also known as DCD1, SystemicLupus Erythematosus Susceptibility 2, Protein PD-1, HPD-1, CD279 Antigen, HPD-L, HSLE1, or SLEB2, is a cell surface receptor belonging to the CD28 receptor family and expressed on T cells and pro-B cells. PD-1 functions as an immune checkpoint, playing a crucial role in downregulating the immune system by inhibiting T cell activation, thereby reducing autoimmunity and promoting self-tolerance. The inhibitory effect of PD-1 is believed to be achieved through a dual mechanism: promoting apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis of regulatory T cells (suppressive T cells). PD-1 is known to bind two ligands: programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2). PD-L1 is a member of the B7 family of proteins and plays a role in suppressing immune responses during specific events. PD-L1 is expressed on a variety of cells, including normal tissues, immune cells, and various types of cancer cells. PD-L1 is widely recognized for its role in tumor immune escape. High expression of PD-L1 on tumors is often associated with poor prognosis because it allows cancer cells to evade immune surveillance. The binding of PD-L1 to PD-1 transmits inhibitory signals, thereby reducing the proliferation of PD-1-expressing T cells. PD-L1 is expressed by various cancer cells, and its expression is thought to at least partially contribute to a weakened immune response against cancer cells. Drugs that block the PD-1 / PD-L1 interaction are used as immune checkpoint inhibitors in cancer immunotherapy. Several antibodies that block PD-1 or PD-L1 activity can activate the immune system to attack tumors and have therefore been successfully used to treat certain types of cancer.

[0028] As used herein, the term "PD-1" includes any variant or isoform of PD-1. The term "PD-L1" as used herein includes any variant or isoform of PD-L1. An exemplary sequence of human PD-1 is available via UniProt: Q15116. Human PD-1 consists of 288 amino acids, comprising an extracellular region (amino acid residues 24-170), a transmembrane domain (amino acid residues 171-191), and a cytoplasmic domain (amino acid residues 192-288). The sequence of human PD-1 can be further processed into a mature form. Exemplary sequences of various isoforms of human PD-1 are also available via accession numbers such as ENSG00000188389; OMIM: 600244; and NCBI: NP_005009.2. An exemplary sequence of human PD-L1 is available via UniProt: Q9NZQ7. Human PD-L1 consists of 290 amino acids, including an extracellular domain (amino acid residues 19-238), a transmembrane domain (amino acid residues 239-259), and a cytoplasmic domain (amino acid residues 260-290). The sequence of human PD-L1 can be further processed into a mature form. Exemplary sequences of various isoforms of human PD-L1 are also available through accession numbers, such as ENSG00000120217; OMIM: 605402; and NCBI: NP_054862.1.

[0029] This article presents bispecific antibodies that simultaneously bind to VEGF and either PD-1 or PD-L1. By targeting VEGF, the bispecific antibodies presented in this article inhibit tumor angiogenesis and tumor growth. This effect also promotes the penetration of immune cells and other therapeutic agents into the tumor. By simultaneously targeting PD-1 or PD-L1, the bispecific antibodies presented in this article block the PD-1 / PD-L1 pathway and restore the activity of the immune system in attacking cancer cells. The bispecific antibodies presented in this article have a synergistic effect in inhibiting angiogenesis and anti-tumor responses.

[0030] Before further describing this disclosure, it should be understood that this disclosure is not limited to the specific embodiments set forth herein, and it should also be understood that the terminology used herein is intended to describe specific embodiments and is not intended to constitute a limitation. 6.1 Terminology Definition

[0031] Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural forms, and plural terms shall include singular forms. Generally, the nomenclature and techniques described herein relating to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are well-known and commonly used in the art.

[0032] The term “a” refers to one or more of the same entity; for example, “an antibody” should be understood to mean one or more antibodies.

[0033] As used herein, the term “and / or” should be considered as a specific disclosure of each of the two features or components, with or without the other. Therefore, the term “and / or” as used in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0034] The terms “polypeptide,” “peptide,” “protein,” “polypeptide chain,” “peptide chain,” and their grammatical equivalents, used interchangeably in this document, refer to an amino acid polymer of any length, which may be linear or branched. It may include non-natural or modified amino acids, or be interrupted by non-amino acid molecules. Polypeptides, peptides, polypeptide chains, peptide chains, or proteins may also be modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other treatment or modification.

[0035] The terms “polynucleotide,” “nucleotide,” and their grammatical equivalents, used interchangeably herein, refer to nucleotide polymers of any length, including DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogues, or any substrate that can be incorporated into the polymer by DNA or RNA polymerases.

[0036] In this document, the term "variant" as used in connection with a protein or polypeptide having a specific sequence characteristic ("reference protein" or "reference polypeptide") refers to a different protein or polypeptide compared to the reference protein or reference polypeptide with one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and / or additions. Changes to the amino acid sequence can be amino acid substitutions. Changes to the amino acid sequence can be conserved amino acid substitutions. Functional fragments or functional variants of the protein or polypeptide retain the basic structural and functional properties of the reference protein or reference polypeptide.

[0037] As used herein, the term "specific binding" refers to a more frequent, faster, longer-lasting, or more affinity interaction between a polypeptide or molecule and an epitope, protein, or target molecule, or any combination thereof, relative to interactions with surrogate substances, including related and unrelated proteins. The binding moiety (e.g., antibody) that specifically binds to a target molecule (e.g., an antigen) can be identified, for example, by immunoassay, ELISA, biolayer interference (“BLI”), SPR (e.g., Biacore), or other techniques known to those skilled in the art. Typically, the specific response is at least twice the background signal or noise, and can exceed 10 times the background. For example, a discussion of antibody specificity can be found in Paul (ed.), 1989, Fundamental Immunology Second Edition, Raven Press, New York, pp. 332-336. The binding moiety that specifically binds to a target molecule can bind to the target molecule with an affinity higher than its affinity for different molecules. In some embodiments, the binding portion of the specific target molecule can bind the target molecule with an affinity at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than its affinity for different molecules. In some embodiments, the binding portion of the specific target molecule binds to different molecules with such low affinity that the binding is undetectable using assays described herein or known in the art. In some embodiments, "specific binding" refers, for example, to a binding portion with an affinity of about 0.1 mM or lower. D Binding to a molecular target. In some embodiments, "specific binding" refers to the peptide or molecule binding at a concentration of approximately 10 µM or less, or approximately 1 µM or less, of K+. D Binding to the target. In some implementations, "specific binding" refers to the peptide or molecule binding at a K+ level of about 0.1 µM or less, about 0.01 µM or less, or about 1 nM or less. DTarget binding. Due to sequence identity between homologous proteins in different species, specific binding can include peptides or molecules that recognize proteins or targets in more than one species. Similarly, due to homology in certain regions of the polypeptide sequences of different proteins, specific binding can include peptides or molecules that recognize more than one protein or target. It should be understood that in some embodiments, the binding portion (e.g., an antibody) that specifically binds to a first target may or may not specifically bind to a second target. Therefore, “specific binding” does not necessarily require (although it may include) exclusive binding, i.e., binding to only a single target. Thus, in some embodiments, the binding portion (e.g., an antibody) may specifically bind to more than one target. For example, in some cases, an antibody may contain two identical antigen-binding sites, each of which specifically binds to the same epitopes on two or more proteins. In some alternative embodiments, the antibody may be bispecific and contain at least two antigen-binding sites with different specificities.

[0038] The term "binding affinity" as used in this article generally refers to the sum of all non-covalent interactions between the binding moiety and the target molecule (e.g., antigen). Binding of the binding moiety to the target molecule is a reversible process, and the affinity of this binding is typically expressed as a function of the equilibrium dissociation constant (K0). D () indicates. K D dissociation rate (k) off or k d ) and association rate (k on or k a The ratio of ) to. Combined with the pair K D The lower the value, the higher the affinity. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure. Specific exemplary embodiments include the following. In some embodiments, “K…” D "or "K D The "value" can be measured by methods known in the art, such as by binding assays. D It can be measured in radiolabeled antigen binding assays (RIA) (Chen, et al., (1999) J. Mol Biol 293:865-881). K D or K D The value can also be measured using biological layer interferometry (BLI), for example, using the Gator system (Probe Life) or the Octet-96 system (Sartorius AG). D or K DThe value can also be measured using Biacore's surface plasmon resonance (SPR) assays, such as the BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). Binding affinity can also be measured using EC. 50 To quantify, EC 50 It is the ligand concentration when half of the target is in the bound state during the binding assay.

[0039] The terms “identical,” “percentage of identity,” and their grammatical equivalents, used herein in the context of two or more polynucleotides or peptides, refer to two or more identical sequences or subsequences, or sequences or subsequences having a specified percentage of identical nucleotide or amino acid residues, when compared and aligned (introducing gaps where necessary) to obtain maximum correspondence, without considering any conserved amino acid substitutions as part of sequence identity. Percentage of identity can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software known in the art for obtaining amino acid or nucleotide sequence alignments are available. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two polynucleotides or peptides provided herein are substantially identical, i.e., when compared and aligned to obtain maximum correspondence, they are measured to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide or amino acid residue identity, measured by sequence comparison algorithms or by visual inspection. In some embodiments, identity exists in amino acid sequence regions of at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues, or any integer value therebetween. In some embodiments, identity exists in regions longer than 60-80 residues, for example, at least about 80-100 residues, and in some embodiments, the sequences are substantially identical in length across the entire length of the compared sequences, such as the coding region of a target protein or antibody. In some embodiments, identity exists in nucleotide sequence regions of at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases, or any integer value therebetween. In some embodiments, identity exists in regions longer than 60-80 bases, for example, at least about 80-100 bases or more, and in some embodiments, the sequences are substantially identical in length across the entire length of the compared sequences, such as the nucleotide sequence encoding a target protein.

[0040] As used herein, the term "vector" and its grammatical equivalents refer to a vector for carrying genetic material (e.g., a polynucleotide sequence) that can be introduced into a host cell, where it can be replicated and / or expressed. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selectable sequences or markers operable for stable integration into the host cell chromosome. Furthermore, the vector may include one or more selectable marker genes and appropriate expression regulatory sequences. For example, the selectable marker genes may provide resistance to antibiotics or toxins, compensate for auxotrophic deficiencies, or provide critical nutrients absent in the culture medium. Expression regulatory sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., as are well known in the art. When two or more polynucleotides are to be co-expressed, for example, the two polynucleotides may be inserted into a single expression vector or into separate expression vectors. For single-vector expression, the encoding polynucleotide may be operably linked to a common expression regulatory sequence or to different expression regulatory sequences, such as an inducible promoter and a constitutive promoter. The introduction of polynucleotides into host cells can be confirmed using methods known in the art. Those skilled in the art will understand that polynucleotides are expressed in an amount sufficient to produce the desired product, and will also understand that expression levels can be optimized using methods known in the art to obtain sufficient expression.

[0041] "Isolated" polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions refer to polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions that exist in a form not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions include those that have been purified to the point that they no longer exist in their natural form. In some embodiments, the isolated polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions are substantially pure.

[0042] The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refer to materials suitable for individual administration together with an active agent, which do not cause adverse biological effects and do not interact with any other component of the pharmaceutical composition in a harmful manner. In some embodiments, the pharmaceutical compositions disclosed herein may comprise one or more of buffer systems, preservatives, isotonic agents, chelating agents, stabilizers, and / or surfactants, as well as various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those skilled in the art. See Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0043] The term “treatment” as used herein, and its grammatical equivalents, when relating to a disease or condition, or a subject suffering from a disease or condition, refers to an action that inhibits, eliminates, reduces, and / or improves symptoms, the severity of symptoms, and / or the frequency of symptoms associated with the disease or disorder being treated.

[0044] As used herein, the term "administration" and its grammatical equivalents refer to the act of delivering, or facilitating the delivery of, a therapeutic or pharmaceutical composition to a subject by the methods described herein or other methods known in the art. The therapeutic may be a compound, peptide, antibody, cell, or cell population. Administration of a therapeutic or pharmaceutical composition includes prescribing for delivery of the therapeutic or pharmaceutical composition to a subject. Exemplary forms of administration include oral dosage forms such as tablets, capsules, syrups, and suspensions; injectable dosage forms such as intravenous (IV), intramuscular (IM), or intraperitoneal (IP); transdermal dosage forms including creams, gels, powders, or patches; buccal mucosal dosage forms; inhaled powders, sprays, suspensions, and rectal suppositories.

[0045] As used herein, the terms "effective amount," "therapeutic effective amount," and their grammatical equivalents refer to the amount of an agent administered to a subject, which may be administered alone or as part of a pharmaceutical composition, and may be administered in a single dose or as part of a series of doses; the amount administered to the subject is capable of producing any detectable positive effect on any symptom, aspect, or characteristic of a disease, disorder, or condition. The therapeutic effective amount can be determined by measuring the relevant physiological effects. The exact amount required varies from subject to subject, depending on the subject's age, weight and general condition, the severity of the condition being treated, the clinician's judgment, etc. In any individual case, the appropriate "effective amount" can be determined by a person skilled in the art through routine experiments.

[0046] As used herein, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, canines, felines, rodents, etc., that will be the recipient of a specific reagent (e.g., a therapeutic or diagnostic reagent). Subjects may be human. Subjects may have a specific disease or condition. Subjects may be at risk of developing a specific disease or condition.

[0047] Scope: In this disclosure, various aspects of the invention may be presented in a scope format. It should be understood that the scope format is used for convenience and brevity purposes only and should not be construed as a rigid limitation on the scope of the invention. Therefore, a description of a scope should be considered as specifically disclosing all possible sub-scopes within that scope as well as individual numerical values. For example, a description of a scope of 1 to 6 should be considered as specifically disclosing sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that scope, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. The above principles apply regardless of the width of the scope.

[0048] This document describes exemplary genes and peptides with reference to GenBank numbers, GI numbers, and / or SEQ ID NOS. It should be understood that those skilled in the art can readily identify homologous sequences using reference sequence sources (including, but not limited to, Uniprot (https: / / www.uniprot.org / ), GenBank (ncbi.nlm.nih.gov / genbank / ), and EMBL (embl.org / )). 6.2 Bispecific antibodies

[0049] This document provides bispecific antibodies capable of specifically binding to human VEGF and human PD-1 or PD-L1. As used herein, the terms "PD-(L)1" and "PD-1 / PD-L1" refer to PD-1 or PD-L1. In some embodiments, the bispecific antibodies provided herein are monoclonal antibodies. In some embodiments, the bispecific antibodies provided herein are isolated. In some embodiments, the bispecific antibodies provided herein are substantially pure.

[0050] As used herein and as understood in the art, an "antibody" is an immunoglobulin molecule that recognizes and specifically binds to a target (e.g., a protein) through at least one antigen-binding fragment (which is typically located within a variable region of an immunoglobulin molecule). Antibodies can have a variety of different types and structures. For example, an antibody can be a polyclonal antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, a monospecific antibody, a monovalent antibody, or any other modified immunoglobulin molecule containing an antigen-binding site. Antibodies also include, but are not limited to, mouse antibodies, camel antibodies, chimeric antibodies, humanized antibodies, and human antibodies. An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or a subclass (isotype) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) based on the identity of its heavy chain constant domain, referred to as α, δ, ε, γ, and μ, respectively. Unless otherwise expressly indicated, the term "antibody" as used herein includes the "antigen-binding fragment" of a complete antibody. As used herein, the term "antigen-binding fragment" refers to a portion or fragment of a complete antibody, which is the antigen-determining variable region of the complete antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, linear antibodies, single-chain antibody molecules (e.g., scFv), heavy chain antibodies (HCAbs), light chain antibodies (LCAbs), disulfide-linked scFv (dsscFv), diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD), single variable domain antibodies (sdAbs; e.g., camelid antibodies, alpaca antibodies), and the single variable domain (VHH) of heavy chain antibodies.

[0051] As used herein and as understood in the art, a "bispecific" antibody is an artificial hybrid antibody having two different antigen-binding fragments. In some embodiments, the two different antigen-binding fragments specifically bind to two different target antigens. In some embodiments, the two different antigen-binding fragments specifically bind to two different epitopes on the same target antigen. In some embodiments, the bispecific antibodies provided herein comprise an antigen-binding fragment that specifically binds to human VEGF and an antigen-binding fragment that specifically binds to human PD-1 / PD-L1. Bispecific antibodies can be formed from antibody fragments.

[0052] The structure of immunoglobulins has been well characterized (see, for example, Chapter 7 of Fundamental Immunology (Paul, W., ed., 2nd ed., Raven Press, New York (1989))). Typically, immunoglobulins consist of two pairs of polypeptide chains, one pair of light (L; low molecular weight) chains and one pair of heavy (H; high molecular weight) chains, all four chains being interconnected by disulfide bonds.

[0053] Each light chain of an immunoglobulin typically comprises a variable region (“VL region”) and a constant region (“CL region”). There are two distinct types of light chains, designated κ (kappa) or λ (lambda) based on the amino acid sequence of the CL region. The amino acid sequence of the CL region is well-known in the art.

[0054] Each heavy chain typically comprises a variable region (“VH region”) and a constant region (“CH region”). Based on the amino acid sequence, the VH region can be one of five different types, referred to as α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu). When bound to a light chain, these different types of heavy chains produce five known classes of antibodies: IgA, IgD, IgE, IgG, and IgM. IgG has four subclasses: IgG1, IgG2, IgG3, and IgG4. The amino acid sequences of the CH regions of different antibody classes are well known in the art.

[0055] The CH region of immunoglobulins contains multiple domains. For example, the CH region of IgG antibodies consists of three domains: heavy chain constant domain 1 (CH1), heavy chain constant domain 2 (CH2), and heavy chain constant domain 3 (CH3). The highly flexible region between the CH1 and CH2 domains is called the "hinge region." The disulfide bonds in the hinge region are part of the interaction between the two heavy chains in an immunoglobulin. The "Fc region" refers to the C-terminal region of the immunoglobulin heavy chain, which contains at least a portion of the constant region. In the IgG, IgA, and IgD isotypes, the Fc region consists of the hinge region, the CH2 domain, and the CH3 domain; the Fc region of IgM and IgE contains three heavy chain constant domains (CH domains 2–4). The amino acid sequences of the Fc regions of human IgG, IgA, IgD, IgM, and IgE, as well as the subtypes IgG1, IgG2, IgG3, and IgG4, are known to those skilled in the art. In some embodiments, the Fc region of the IgG heavy chain can extend from the hinge region to the carboxyl terminus of the heavy chain. The native Fc region can be modified. Modifications to the Fc region are further described below.

[0056] Unless otherwise stated or the context contradicts, references to amino acid positions in constant regions are made to EU numbers (Edelman et al., PNAS. 1969; 63:78-85, Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242). An exemplary list of amino acid sequences for the constant domains / regions of human IgG antibodies is provided below. Several exemplary variants are also included, and further variants are disclosed in the following sections.

[0057] Table 1A. Constant regions / domains of natural human IgG.

[0058] Table 1B. Exemplary variants of human IgG constant regions / domains.

[0059] The term "variable region" refers to a portion of the light or heavy chain of an immunoglobulin, typically located at the amino terminus of the light or heavy chain, and is responsible for the binding and specificity of each particular antibody to its specific antigen. The variable region of the light chain is called the "light chain variable region" or "VL region," and it includes at least one, typically one, "light chain variable domain" or "VL." The variable region of the heavy chain is called the "heavy chain variable region" or "VH region," and it includes at least one, typically one, "heavy chain variable domain" or "VH." Variable domains vary widely in sequence between different antibodies. A "VL / VH pair," a "VL / VH pair," or a "VH / VL pair" can associate with each other to form a binding site that specifically binds to the target antigen or epitope.

[0060] The VH and VL regions can be further subdivided into hypervariable regions (or hypervariable areas that may be highly variable in sequence and / or in the form of structurally defined loops), also known as complementarity-determining regions (CDRs), interspersed with more conserved regions called frame regions (FRs). Sequence variability is concentrated in the CDRs, while the less variable portions of the variable domains are called frame regions (FRs). The CDRs of the light and heavy chains are primarily responsible for antibody-antigen interactions. Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk, J Mol Biol. 1987;196:901-17).

[0061] A CDR (Constant Variable Domain) refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the VH β-sheet framework of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of the VL β-sheet framework of an antibody. CDR regions are well known to those skilled in the art and have been defined using various methods / systems. These systems and / or definitions include, for example, Kabat, Chothia, IMGT, AbM, and Contact. For instance, Kabat defines the region with the highest variability within the variable (V) domain of an antibody (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, Adv.Prot. Chem. 32: 1-75 (1978)). Software programs (e.g., abYsis) for analyzing antibody sequences and determining CDRs are known and available to those skilled in the art.

[0062] Single-chain Fv (“scFv”) polypeptides are covalently linked VL / VH heterodimers, typically expressed via gene fusion, whereby the gene fusion comprises a VL-coding gene and a VH-coding gene linked by a peptide linker. The scFv fragment comprises CDRs maintained in a suitable conformation, particularly achieved through the use of gene recombination techniques. In some embodiments of scFv, the N-terminus of VL is linked to the C-terminus of VH via a linker. In some embodiments of scFv, the N-terminus of VH is linked to the C-terminus of VL via a linker.

[0063] When a test VH or VL contains CDR1, CDR2, and CDR3 that are identical to or variants of the corresponding CDRs of a reference VH or VL, the test VH or VL is considered “derived” from the reference VH or VL, wherein the total number of amino acid substitutions, insertions, and / or deletions in the “variant” within the CDR1–CDR3 range of that chain does not exceed about five (5), while retaining specific binding to the same antigens recognized by the reference VH or VL. Framework residues may be modified without restriction (e.g., humanization, reversion, deimmunization, affinity maturation) as long as the aforementioned CDR criteria and antigen specificity are maintained. When a test VH and / or VL (or VH / VL pair) contains CDR1, CDR2, and CDR3 that are identical to or variants of the corresponding CDRs of the VH and / or VL of the reference antibody, the test VH and / or VL (or VH / VL pair) is considered “derived” from the reference antibody, wherein the “variant” has no more than about five (5) amino acid substitutions, insertions, and / or deletions in the CDR1–CDR3 range of that chain, while retaining specific binding to the same antigen as the reference antibody. Framework residues may be modified without restriction as described above.

[0064] As used herein, the term "linker" refers to one or more amino acid residues inserted between domains (e.g., immunoglobulin domains) to provide sufficient mobility for said domains. Linkers can be inserted at the sequence level between variable domains or at transitions between variable and constant domains. Some exemplary linkers are provided below. Those skilled in the art will understand that the bispecific antibodies disclosed herein are not limited to the specific linkers illustrated herein. Any peptide linker of suitable length and flexibility that allows the VL / VH pair to correctly form antigen-binding sites can be used.

[0065] Table 1C. Exemplary connectors.

[0066] As used herein, the term "humanized antibody" refers to a form of non-human (e.g., mouse) antibody that is a specific immunoglobulin chain, a chimeric immunoglobulin, or a fragment thereof, and contains a minimal non-human sequence. Typically, humanized antibodies are human immunoglobulins. In some cases, the variable region residues of a human immunoglobulin are replaced with corresponding residues from an antibody derived from a non-human species. In other cases, residues of CDRs are replaced with residues from CDRs of non-human species (e.g., mice, rats, hamsters, camels) possessing the desired specificity, affinity, and / or binding capacity. The humanized antibody can also be further modified by replacing additional residues within the variable region and / or within the replaced non-human residues to refine and optimize the antibody's specificity, affinity, and / or binding capacity.

[0067] This article provides a bispecific antibody having a first VL / VH pair (VL1 / VH1) that specifically binds to human VEGF, and a second VL / VH pair (VL2 / VH2) that specifically binds to human PD-1 or human PD-L1.

[0068] In some embodiments, the bispecific antibody provided herein may have a VL1 / VH1 pair derived from the anti-human VEGF antibody Nevegimab (HX006, disclosed in CN105330739B, which is incorporated herein by reference in its entirety). In some embodiments, the bispecific antibody provided herein may have VL / VH CDRs of Nevegimab. In some embodiments, VL1 comprises VL CDR1, VL CDR2, and VLCDR3, or variants thereof, from a reference VL having the amino acid sequence of SEQ ID NO:21, wherein the VL CDRs have up to about 5 amino acid substitutions, additions, and / or deletions; and VH1 comprises VH CDR1, VH CDR2, and VH CDR3, or variants thereof, from a reference VH having the amino acid sequence of SEQ ID NO:22, wherein the VH CDRs have up to about 5 amino acid substitutions, additions, and / or deletions. In some embodiments, the first VL / VH pair (VL1 / VH1) of the bispecific antibody provided herein binds to human VEGF, wherein VL1 comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ IDNOs:1, 2, and 3, respectively; and VH1 comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ IDNOs:4, 5, and 6, respectively.

[0069] In some embodiments of the VL1 / VH1 pair that specifically binds to human VEGF, the amino acid sequence of VL1 has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:21. In some embodiments of the bispecific antibody disclosed herein, the amino acid sequence of VH1 has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:22. In some embodiments, VL1 has the amino acid sequence of SEQ ID NO:21 and has up to 10 amino acid substitutions, additions, and / or deletions. In some embodiments, VL1 has the amino acid sequence of SEQ ID NO:21 and has up to 5 amino acid substitutions, additions, and / or deletions. In some embodiments, VL1 has the amino acid sequence of SEQ ID NO:21. In some embodiments, VL1 has the amino acid sequence of SEQ ID NO:21 and has up to 3 amino acid substitutions, additions, and / or deletions. In some embodiments, VL1 has the amino acid sequence of SEQ ID NO:21 and has up to 10, up to 5, or up to 3 amino acid substitutions. In some embodiments, VL1 has the amino acid sequence of SEQ ID NO:21 and has up to 10, up to 5, or up to 3 conserved amino acid substitutions. In some embodiments, VL1 has the amino acid sequence of SEQ ID NO:21. In some embodiments, VH1 has the amino acid sequence of SEQ ID NO:22 and has up to 5 amino acid substitutions, additions, and / or deletions. In some embodiments, VH1 has the amino acid sequence of SEQ ID NO:22. In some embodiments, VH1 has the amino acid sequence of SEQ ID NO:22 and has up to 3 amino acid substitutions, additions, and / or deletions. In some embodiments, VH1 has the amino acid sequence of SEQ ID NO:22 and has up to 10, up to 5, or up to 3 amino acid substitutions. In some embodiments, VH1 has the amino acid sequence of SEQ ID NO:22 and has up to 10, up to 5, or up to 3 conserved amino acid substitutions. In some embodiments, VH1 has the amino acid sequence of SEQ ID NO:22. In some embodiments, the VL1 / VH1 pair has the amino acid sequences of SEQ ID NO:21 and 22, respectively.

[0070] The bispecific antibodies provided herein have a second VL / VH pair (VL2 / VH2) that binds to human PD-1 or human PD-L1. In some embodiments, the VL2 / VH2 pair may bind to any anti-PD-1 / PD-L1 antibody disclosed herein or other antibodies known in the art. In some embodiments, the VL2 / VH2 of the bispecific antibody provided herein binds to human PD-1. In some embodiments, the VL2 / VH2 of the bispecific antibody provided herein binds to human PD-L1. In some embodiments, the VL2 / VH2 pair is derived from an anti-PD1 or PD-L1 antibody selected from: putelimab, nivolumab, pembrolizumab, cimiprimab, dotalimab, retivalimab, toripalimab, tislelizumab, camrelizumab, sintilimab, penaplimab, cepalimab, slulimab, fenolinumab, durvalumab, atezolizumab, avelumab, cosibelimab, sugemalimab, envorimab, adebenone, socarzolimab, and tagorimab. The sequences of the above exemplary antibodies are known in the art; some of them are provided in Table 2 below.

[0071] In some embodiments, the second VL / VH pair (VL2 / VH2) of the bispecific antibody provided herein binds to human PD-L1. In some embodiments, the bispecific antibody provided herein may have a VL2 / VH2 pair derived from the anti-human PD-L1 antibody durvalumab (see US. Patent No. 8,779,108, which is incorporated herein by reference in its entirety). In some embodiments, VL2 comprises VLCDR1, VL CDR2, and VL CDR3, or variants thereof, from a reference VL having the amino acid sequence of SEQ ID NO:67, wherein the VL CDRs have up to about 5 amino acid substitutions, additions, and / or deletions; and VH2 comprises VH CDR1, VH CDR2, and VH CDR3, or variants thereof, from a reference VH having the amino acid sequence of SEQ ID NO:68, wherein the VH CDRs have up to about 5 amino acid substitutions, additions, and / or deletions. In some embodiments, VL2 comprises VL CDR1, VL CDR2, and VLCDR3 having amino acid sequences of SEQ ID NOs:7, 8, and 9, respectively; and VH2 comprises VH CDR1, VH CDR2, and VHCDR3 having amino acid sequences of SEQ ID NOs:10, 11, and 12, respectively.

[0072] In some embodiments of the bispecific antibody disclosed herein, VL2 / VH2 specifically binds to human PD-L1, and the amino acid sequence of VL2 has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:67. In some embodiments, VL2 has the amino acid sequence of SEQ ID NO:67 and has up to 10 amino acid substitutions, additions, and / or deletions. In some embodiments, VL2 has the amino acid sequence of SEQ ID NO:67 and has up to 5 amino acid substitutions, additions, and / or deletions. In some embodiments, VL2 has the amino acid sequence of SEQ ID NO:67 and has up to 3 amino acid substitutions, additions, and / or deletions. In some embodiments, VL2 has the amino acid sequence of SEQ ID NO:67 and has up to 10, up to 5, or up to 3 amino acid substitutions. In some embodiments, VL2 has the amino acid sequence of SEQ ID NO:67 and has up to 10, up to 5, or up to 3 conserved amino acid substitutions. In some embodiments, VL2 has the amino acid sequence of SEQ ID NO:67. In some embodiments of the bispecific antibody disclosed herein, the amino acid sequence of VH2 has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:68. In some embodiments, VH2 has the amino acid sequence of SEQ ID NO:68 and has up to 10 amino acid substitutions, additions, and / or deletions. In some embodiments, VH2 has the amino acid sequence of SEQ ID NO:68 and has up to 5 amino acid substitutions, additions, and / or deletions. In some embodiments, VH2 has the amino acid sequence of SEQ ID NO:68 and has up to 3 amino acid substitutions, additions, and / or deletions. In some embodiments, VH2 has the amino acid sequence of SEQ ID NO:68 and has up to 10, up to 5, or up to 3 amino acid substitutions. In some embodiments, VH2 has the amino acid sequence of SEQ ID NO:68 and has up to 10, up to 5, or up to 3 conserved amino acid substitutions. In some embodiments, VH2 has the amino acid sequence of SEQ ID NO:68.

[0073] In some embodiments, VL2 has the amino acid sequence of SEQ ID NO:67 and has an amino acid substitution at Q101. In some embodiments, Q101 is replaced with Q101C. In some embodiments, VL2 has the amino acid sequence of SEQ ID NO:23. In some embodiments, the amino acid sequence of VL2 has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:23. In some embodiments, VH2 has the amino acid sequence of SEQ ID NO:68 and has an amino acid substitution at G44. In some embodiments, G44 is replaced with G44C. In some embodiments, VH2 has the amino acid sequence of SEQ ID NO:24. In some embodiments, the amino acid sequence of VH2 has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:24.

[0074] In some embodiments, the VL2 / VH2 pair has amino acid sequences of SEQ ID NOs:67 and 68, respectively. In some embodiments, the VL2 / VH2 pair has amino acid sequences of SEQ ID NOs:67 and 24, respectively. In some embodiments, the VL2 / VH2 pair has amino acid sequences of SEQ ID NOs:23 and 68, respectively. In some embodiments, the VL2 / VH2 pair has amino acid sequences of SEQ ID NOs:23 and 24, respectively.

[0075] In some embodiments, the VL2 / VH2 pair of the bispecific antibody provided herein binds to human PD-1. In some embodiments, the bispecific antibody provided herein may have a VL2 / VH2 pair derived from the anti-human PD-1 antibody putelimab (HX008, see CN107286242B, which is incorporated herein by reference in its entirety). In some embodiments, the VL2 / VH2 pair specifically binds to human PD-1, and wherein the VL2 comprises VL CDR1, VL CDR2, and VL CDR3, or variants thereof, from a reference VL having the amino acid sequence of SEQ ID NO:69, wherein the VL CDRs have up to about 5 amino acid substitutions, additions, and / or deletions; and the VH2 comprises VH CDR1, VHCDR2, and VH CDR3, or variants thereof, from a reference VH having the amino acid sequence of SEQ ID NO:70, wherein the VH CDRs have up to about 5 amino acid substitutions, additions, and / or deletions. In some embodiments, VL2 comprises VL CDR1, VLCDR2, and VL CDR3 having amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively; and VH2 comprises VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively.

[0076] In some embodiments of the bispecific antibody disclosed herein, VL2 / VH2 specifically binds to human PD-1. In some embodiments, the amino acid sequence of VL2 has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:69. In some embodiments, VL2 has the amino acid sequence of SEQ ID NO:69 and has up to 10 amino acid substitutions, additions, and / or deletions. In some embodiments, VL2 has the amino acid sequence of SEQ ID NO:69 and has up to 5 amino acid substitutions, additions, and / or deletions. In some embodiments, VL2 has the amino acid sequence of SEQ ID NO:69 and has up to 3 amino acid substitutions, additions, and / or deletions. In some embodiments, VL2 has the amino acid sequence of SEQ ID NO:69 and has up to 10, up to 5, or up to 3 amino acid substitutions. In some embodiments, VL2 has the amino acid sequence of SEQ ID NO:69 and has up to 10, up to 5, or up to 3 conserved amino acid substitutions. In some embodiments, VL2 has the amino acid sequence of SEQ ID NO:69. In some embodiments of the bispecific antibody disclosed herein, the amino acid sequence of VH2 has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:70. In some embodiments, VH2 has the amino acid sequence of SEQ ID NO:70 and has up to 10 amino acid substitutions, additions, and / or deletions. In some embodiments, VH2 has the amino acid sequence of SEQ ID NO:70 and has up to 5 amino acid substitutions, additions, and / or deletions. In some embodiments, VH2 has the amino acid sequence of SEQ ID NO:70 and has up to 3 amino acid substitutions, additions, and / or deletions. In some embodiments, VH2 has the amino acid sequence of SEQ ID NO:70 and has up to 10, up to 5, or up to 3 amino acid substitutions. In some embodiments, VH2 has the amino acid sequence of SEQ ID NO:70 and has up to 10, up to 5, or up to 3 conserved amino acid substitutions. In some embodiments, VH2 has the amino acid sequence of SEQ ID NO:70.

[0077] In some embodiments, VL2 has the amino acid sequence of SEQ ID NO:69, with an amino acid substitution at N85, G104, or both. In some embodiments, N85 is replaced with N85E. In some embodiments, G104 is replaced with G104C. In some embodiments, VL2 has the amino acid sequence of SEQ ID NO:25. In some embodiments, the amino acid sequence of VL2 has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:25. In some embodiments, VH2 has the amino acid sequence of SEQ ID NO:70, with an amino acid substitution at G44. In some embodiments, G44 is replaced with G44C. In some embodiments, VH2 has the amino acid sequence of SEQ ID NO:26. In some embodiments, the amino acid sequence of VH2 has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:26.

[0078] In some embodiments, the VL2 / VH2 pair has amino acid sequences of SEQ ID NOs:69 and 70, respectively. In some embodiments, the VL2 / VH2 pair has amino acid sequences of SEQ ID NOs:69 and 26, respectively. In some embodiments, the VL2 / VH2 pair has amino acid sequences of SEQ ID NOs:25 and 70, respectively. In some embodiments, the VL2 / VH2 pair has amino acid sequences of SEQ ID NOs:25 and 26, respectively.

[0079] Table 2: Exemplary sequences of VL1 / VH1 and VL2 / VH2 and their CDRs.

[0080] Therefore, in some embodiments of the bispecific antibodies disclosed herein, VL1 and VH1 specifically binding to human VEGF have the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:22, respectively; and VL2 and VH2 specifically binding to human PD-L1 have the amino acid sequences of SEQ ID NO:67 and SEQ ID NO:68, respectively. VL1 and VH1 specifically binding to human VEGF have the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:22, respectively; and VL2 and VH2 specifically binding to human PD-L1 have the amino acid sequences of SEQ ID NO:67 and SEQ ID NO:24, respectively. VL1 and VH1 specifically binding to human VEGF have the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:22, respectively; and VL2 and VH2 specifically binding to human PD-L1 have the amino acid sequences of SEQ ID NO:23 and SEQ ID NO:68, respectively. VL1 and VH1, which specifically bind to human VEGF, have amino acid sequences of SEQ ID NO:21 and SEQ ID NO:22, respectively; and VL2 and VH2, which specifically bind to human PD-L1, have amino acid sequences of SEQ ID NO:23 and SEQ ID NO:24, respectively.

[0081] In some embodiments, the VL2 / VH2 pair is derived from the anti-PD-L1 antibody atezolizumab, avelumab, cocibelimab, sugemalimab, envorimab, adebelimab, socarzolimab, or tagorimab. In some embodiments, the VL2 / VH2 pair is derived from atezolizumab. In some embodiments, VL2 and VH2 have the amino acid sequences of SEQ ID NOs:103 and 104, respectively. In some embodiments, the VL2 / VH2 pair is derived from avelumab. In some embodiments, VL2 and VH2 have the amino acid sequences of SEQ ID NOs:105 and 106, respectively. In some embodiments, the VL2 / VH2 pair is derived from cocibelimab. In some embodiments, VL2 and VH2 have the amino acid sequences of SEQ ID NOs:107 and 108, respectively. In some embodiments, the VL2 / VH2 pair is derived from sugemalimab. In some embodiments, VL2 and VH2 have the amino acid sequences of SEQ ID NOs:109 and 110, respectively. In some embodiments, the VL2 / VH2 pair is derived from adebelimumab. In some embodiments, VL2 and VH2 have the amino acid sequences of SEQ ID NOs:111 and 112, respectively. In some embodiments, the VL2 / VH2 pair is derived from socarzolizumab. In some embodiments, VL2 and VH2 have the amino acid sequences of SEQ ID NOs:113 and 114, respectively. In some embodiments, the VL2 / VH2 pair is derived from tagorimab. In some embodiments, VL2 and VH2 have the amino acid sequences of SEQ ID NOs:115 and 116, respectively.

[0082] In some embodiments of the bispecific antibodies disclosed herein, VL1 and VH1 specifically binding to human VEGF have amino acid sequences of SEQ ID NO:21 and SEQ ID NO:22, respectively; and VL2 and VH2 specifically binding to human PD-1 have amino acid sequences of SEQ ID NO:69 and SEQ ID NO:70, respectively. VL1 and VH1 specifically binding to human VEGF have amino acid sequences of SEQ ID NO:21 and SEQ ID NO:22, respectively; and VL2 and VH2 specifically binding to human PD-1 have amino acid sequences of SEQ ID NO:69 and SEQ ID NO:26, respectively. VL1 and VH1 specifically binding to human VEGF have amino acid sequences of SEQ ID NO:21 and SEQ ID NO:22, respectively; and VL2 and VH2 specifically binding to human PD-1 have amino acid sequences of SEQ ID NO:25 and SEQ ID NO:70, respectively. VL1 and VH1, which specifically bind to human VEGF, have amino acid sequences of SEQ ID NO:21 and SEQ ID NO:22, respectively; and VL2 and VH2, which specifically bind to human PD-1, have amino acid sequences of SEQ ID NO:25 and SEQ ID NO:26, respectively.

[0083] In some embodiments, the VL2 / VH2 pair is derived from the anti-PD-1 antibody nivolumab, pembrolizumab, cimiprimab, dotalimab, retivalimab, toripalimab, tislelizumab, camrelizumab, sintilimab, penaprilimab, cepalimumab, slulilimab, or fenolinimab. In some embodiments, the VL2 / VH2 pair is derived from nivolumab. In some embodiments, VL2 and VH2 have the amino acid sequences of SEQ ID NOs:77 and 78, respectively. In some embodiments, the VL2 / VH2 pair is derived from pembrolizumab. In some embodiments, VL2 and VH2 have the amino acid sequences of SEQ ID NOs:79 and 80, respectively. In some embodiments, the VL2 / VH2 pair is derived from toripalimab. In some embodiments, VL2 and VH2 have the amino acid sequences of SEQ ID NOs:81 and 82, respectively. In some embodiments, the VL2 / VH2 pair is derived from sintilimab. In some embodiments, VL2 and VH2 have amino acid sequences of SEQ ID NOs:83 and 84, respectively. In some embodiments, the VL2 / VH2 pair is derived from tislelizumab. In some embodiments, VL2 and VH2 have amino acid sequences of SEQ ID NOs:85 and 86, respectively. In some embodiments, the VL2 / VH2 pair is derived from camrelizumab. In some embodiments, VL2 and VH2 have amino acid sequences of SEQ ID NOs:87 and 88, respectively. In some embodiments, the VL2 / VH2 pair is derived from slulilimab. In some embodiments, VL2 and VH2 have amino acid sequences of SEQ ID NOs:89 and 90, respectively. In some embodiments, the VL2 / VH2 pair is derived from cepallimab. In some embodiments, VL2 and VH2 have amino acid sequences of SEQ ID NOs:91 and 92, respectively. In some embodiments, the VL2 / VH2 pair is derived from cimiprimab. In some embodiments, VL2 and VH2 have amino acid sequences of SEQ ID NOs:93 and 94, respectively. In some embodiments, the VL2 / VH2 pair is derived from dotalimab. In some embodiments, VL2 and VH2 have amino acid sequences of SEQ ID NOs:95 and 96, respectively. In some embodiments, the VL2 / VH2 pair is derived from retivalimab. In some embodiments, VL2 and VH2 have amino acid sequences of SEQ ID NOs:97 and 98, respectively. In some embodiments, the VL2 / VH2 pair is derived from penamprimab. In some embodiments, VL2 and VH2 have amino acid sequences of SEQ ID NOs:99 and 100, respectively. In some embodiments, the VL2 / VH2 pair is derived from fenolinumab.In some embodiments, VL2 and VH2 have amino acid sequences of SEQ ID NOs:101 and 102, respectively.

[0084] In addition to the specific anti-human VEGF and anti-human PD-1 / PD-L1 VL / VH pairs illustrated in this article, variants of these VL / VH pairs that retain their ability to bind to their respective target antigens are also explicitly covered.

[0085] The bispecific antibody component can be configured in any format capable of presenting two pairs of VL / VH to bind to two targets. For example, a heterodimeric full-length IgG carrying two different Fab arms (each arm containing its own VL / VH pair; heterodimerization of the heavy chain can be forced and proper light chain pairing maintained through techniques such as knots-into-holes, chain exchange / seed-like frames, electrostatic guidance, CrossMab domain exchange, common light chain design, or orthogonal Fab interfaces). In another embodiment, an IgG-scFv “2+1” format is used (a standard bivalent IgG against one antigen, with a single scFv—a linked VL / VH pair—fused to the end of either the heavy or light chain to provide a second specificity). In a related embodiment, an IgG-Fab “2+1” format is used (a full-length IgG with an additional Fab—a second pair of VL / VH—fused to the end of the heavy chain, thereby providing two binding sites for one antigen and one binding site for another). In some implementations, DVD-Ig (dual variable domain IgG) is used (two variable domains are tandemly arranged on each heavy and light chain, providing two pairs of VL / VH per arm, thus providing tetravalent binding overall). Alternatives include the CODV-Ig architecture (a cross-arrangement of dual variable domains that positions two pairs of VL / VH on each arm while maintaining homologous pairing) and the diabody / DART basic format with or without Fc (two pairs of VL / VH are arranged in a cross geometry to form two discrete antigen-binding sites; engineered linkers / disulfide bonds stabilize the assembly, and if Fc is present, half-life and effector function options are restored). In some embodiments, a tandem scFv (BiTE-like) construct is used (two scFv modules, each a pair of VL / VH, joined end-to-end by a flexible linker to achieve compact, Fc-free bispecificity); and in other embodiments, an IgG-(scFv)2 “2+2” design is employed (full-length IgG with two scFv modules appended, thereby achieving tetravalentity on two targets). The historical quadroma / “Triomab”-like IgG format is also covered (full-length IgG with two distinct Fab arms generated through controlled Fab arm exchange or interspecies heavy / light chain pairing preference). In a further embodiment, an EpimAb FIT-Ig (Fabs-in-Tandem Immunoglobulin) conformation is used (two complete Fabs fused in a cross / tandem orientation to produce a symmetrical, tetravalent, IgG-like bispecific antibody that maintains proper association of two pairs of VL / VH without the need for framework mutations or peptide linkers).

[0086] In some implementations, the bispecific antibody provided herein has Figure 1The “IgG-scFv” structure is shown. As shown, the bispecific antibody comprises anti-VEGF IgG, with two anti-PD-1 / PD-L1 scFvs linked to the C-terminus of each of the two heavy chains of the IgG. Specifically, the IgG-scFv bispecific antibody comprises two peptide chains: (1) a first peptide chain (HC) comprising, from the N-terminus to the C-terminus, a first heavy chain variable domain, a heavy chain constant (CH) region, a linker, and a single-chain variable fragment (scFv), wherein the scFv comprises a second light chain variable domain and a second heavy chain variable domain; and (2) a second peptide chain (LC) comprising, from the N-terminus to the C-terminus, a first light chain variable domain and a light chain constant (CL) region. In some embodiments of the bispecific antibody provided herein, the scFv comprises, from the N-terminus to the C-terminus, VL2, a second linker, and VH2. In some embodiments of the bispecific antibody provided herein, the scFv comprises, from the N-terminus to the C-terminus, VH2, a second linker, and VL2.

[0087] In some embodiments, the bispecific antibody provided herein has: (1) a first peptide chain (HC) comprising, from the N-terminus to the C-terminus, VH1, a heavy chain constant (CH) region, a linker, and a single-chain variable fragment (scFv), wherein the scFv comprises, from the N-terminus to the C-terminus, VL2, a second linker, and VH2; and (2) a second peptide chain (LC) comprising, from the N-terminus to the C-terminus, VL1 and a light chain constant (CL) region.

[0088] In some embodiments, the bispecific antibody provided herein has: (1) a first peptide chain (HC) comprising, from the N-terminus to the C-terminus, VH1, a heavy chain constant (CH) region, a linker, and a single-chain variable fragment (scFv), wherein the scFv comprises, from the N-terminus to the C-terminus, VH2, a second linker, and VL2; and (2) a second peptide chain (LC) comprising, from the N-terminus to the C-terminus, VL1 and a light chain constant (CL) region.

[0089] like Figure 1 As shown, in some embodiments, the bispecific antibody provided herein contains two identical HC / LC pairs, thereby forming two VEGF binding sites and two PD-1 / PD-L1 binding sites.

[0090] The IgG-scFv format bispecific antibodies provided herein also include CL and CH regions. The amino acid sequences of the CL and CH regions of the bispecific antibodies disclosed herein can be derived from any suitable source, such as the constant region of an antibody, such as the constant region of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the constant region of the bispecific antibodies provided herein is derived from human IgG. In some embodiments, the constant region of the bispecific antibodies provided herein is derived from human IgG1. In some embodiments, the constant region of the bispecific antibodies provided herein is derived from human IgG2. In some embodiments, the constant region of the bispecific antibodies provided herein is derived from human IgG3. In some embodiments, the constant region of the bispecific antibodies provided herein is derived from human IgG4. In some embodiments, the amino acid sequences of the CL and CH regions of the bispecific antibodies disclosed herein may contain one or more amino acid substitutions different from those of wild-type immunoglobulins, such as one or more amino acid substitutions in wild-type IgG1 or IgG4. Such substitutions are known in the art (see, for example, US7704497, US7083784, US6821505, US8323962, US6737056, US7416727 and CN108299560B).

[0091] In some embodiments of the bispecific antibody provided herein, (1) the CL region is Cκ (SEQ ID NO:29) or Cλ (SEQ ID NO:30), or a variant thereof having up to ten amino acid substitutions, additions and / or deletions; or (2) the CH region is human IgG1 CH region (SEQ ID NO:31), IgG2 CH region (SEQ ID NO:32), IgG3 CH region (SEQ ID NO:33) or IgG4 CH region (SEQ ID NO:34), or a variant thereof having up to ten amino acid substitutions, additions and / or deletions; or both (1) and (2).

[0092] In some embodiments of the bispecific antibody provided herein, the CL region is Cκ (SEQ ID NO:29) or a variant thereof having up to ten amino acid substitutions, additions, and / or deletions. In some embodiments of the bispecific antibody provided herein, the CL region is Cλ (SEQ ID NO:30) or a variant thereof having up to ten amino acid substitutions, additions, and / or deletions. In some embodiments, the CL region may be κ CL (Cκ; SEQ ID NO:29). In some embodiments, the CL region may be λ CL (Cλ; SEQ ID NO:30).

[0093] In some embodiments, the CH region of the bispecific antibody provided herein may be selected from the group consisting of the human IgG1 CH region (SEQ ID NO:31), the human IgG2 CH region (SEQ ID NO:32), the human IgG3 CH region (SEQ ID NO:33), and the human IgG4 CH region (SEQ ID NO:34), or a variant thereof having up to ten amino acid substitutions, additions, and / or deletions. In some embodiments, the CH region is the human IgG1 CH region (SEQ ID NO:31) or a variant thereof having up to ten amino acid substitutions, additions, and / or deletions. In some embodiments, the CH region is the human IgG2 CH region (SEQ ID NO:32) or a variant thereof having up to ten amino acid substitutions, additions, and / or deletions. In some embodiments, the CH region is the human IgG3 CH region (SEQ ID NO:33) or a variant thereof having up to ten amino acid substitutions, additions, and / or deletions. In some embodiments, the CH region is the human IgG4 CH region (SEQ ID NO:34) or a variant thereof having up to ten amino acid substitutions, additions, and / or deletions.

[0094] In some embodiments, the CH region of the bispecific antibody provided herein is the human IgG1 CH region (SEQ ID NO: 31). In some embodiments, the CH region also has an M252Y substitution. In some embodiments, the CH region also has an S254T substitution. In some embodiments, the CH region also has a T256E substitution. In some embodiments, the CH region also has an N297A, N297Q, or N297G mutation. In some embodiments, the CH region also has an N297A mutation. In some embodiments, the CH region also has a T307A, E380A, or N434A mutation. In some embodiments, the CH region also has a T307A mutation. In some embodiments, the CH region also has an E380A mutation. In some embodiments, the CH region also has an N434A mutation. In some embodiments, the CH region also has T307A, E380A, and N434A mutations. In some embodiments, the CH region is the human IgG1 CH region having any or any combination of mutations described herein. In some embodiments, the CH region of the bispecific antibody provided herein is a human IgG1 CH region with L234A and L235A substitutions (SEQ ID NO:47). In some embodiments, the CH region is a human IgG1 CH region with L234A, L235A, and M252Y substitutions. In some embodiments, the CH region is a human IgG1 CH region with L234A, L235A, and S254T substitutions. In some embodiments, the CH region is a human IgG1 CH region with L234A, L235A, and T256E substitutions. In some embodiments, the CH region is a human IgG1 CH region with L234A, L235A, M252Y, and S254T substitutions. In some embodiments, the CH region is a human IgG1 CH region with L234A, L235A, M252Y, and T256E substitutions. In some embodiments, the CH region is a human IgG1 CH region with L234A, L235A, S254T, and T256E substitutions. In some embodiments, the CH region is a human IgG1 CH region with L234A, L235A, M252Y, S254T, and T256E substitutions (SEQ ID NO:48). In some embodiments, the CH region is a human IgG1 CH region with T307A, E380A, and N434A mutations. In some embodiments, the human IgG1 CH region also has N297A, N297Q, or N297G mutations. In some embodiments, the human IgG1 CH region also has an N297A mutation.

[0095] In some embodiments, the CH region is the human IgG4 CH region (SEQ ID NO:34). In some embodiments, the CH region is the human IgG4 CH region with S228P substitution (SEQ ID NO:49). In some embodiments, the CH region also has S254T substitution. In some embodiments, the CH region also has V308P substitution. In some embodiments, the CH region also has N434A substitution. In some embodiments, the CH region is the human IgG4 CH region with S228P and S254T substitutions. In some embodiments, the CH region is the human IgG4 CH region with S228P and V308P substitutions. In some embodiments, the CH region is the human IgG4 CH region with S228P and N434A substitutions. In some embodiments, the CH region is the human IgG4 CH region with S228P, S254T, and V308P substitutions. In some embodiments, the CH region is the human IgG4 CH region with S228P, S254T, and N434A substitutions. In some embodiments, the CH region is a human IgG4 CH region with S228P, V308P, and N434A substitutions. In some embodiments, the CH region is a human IgG4 CH region with S228P, S254T, V308P, and N434A substitutions (SEQ ID NO:50).

[0096] In some embodiments of the bispecific antibody provided herein, the CL region is Cκ (SEQ ID NO:29), and the CH region is a human IgG1 CH region with L234A and L235A substitutions (SEQ ID NO:47). In some embodiments of the bispecific antibody provided herein, the CL region is Cκ (SEQ ID NO:29), and the CH region is a human IgG1 CH region with L234A, L235A, M252Y, S254T, and T256E substitutions (SEQ ID NO:48). In some embodiments of the bispecific antibody provided herein, the CL region is Cκ (SEQ ID NO:29), and the CH region is a human IgG4 CH region with S228P substitutions (SEQ ID NO:49). In some embodiments of the bispecific antibody provided herein, the CL region is Cκ (SEQ ID NO:29), and the CH region is a human IgG4 CH region with S228P, S254T, V308P, and N434A substitutions (SEQ ID NO:50).

[0097] The IgG-scFv bispecific antibody provided herein includes a linker connecting the CH region to the scFv, specifically connecting the C-terminus of the CH region to the N-terminus of the scFv region. This linker can be any suitable linker disclosed herein or otherwise known in the art. For example, the linker can be selected from the linkers identified in Table 1C. In some embodiments, the linker can be selected from the linkers identified in Table 1C. The linker can be (GGGGS)n; n = 1, 2, 3, 4, or 5 (SEQ ID NO: 53). The linker can be (GSGGS)n; n = 1, 2, 3, 4, or 5 (SEQ ID NO: 54). The linker can be (GGGS)n; n = 1, 2, 3, 4, or 5 (SEQ ID NO: 55). The linker can be GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 51). The linker can be (EAAAK)n, n = 1, 2, 3, 4, or 5 (SEQ ID NO: 52).

[0098] In some embodiments of the scFv, VL and VH are also connected by a second connector. This second connector can be any suitable connector disclosed herein or otherwise known in the art. For example, the second connector can be selected from the connectors identified in Table 1C. In some embodiments, the second connector can be selected from the connectors identified in Table 1C. The second connector can be (GGGGS)n; n = 1, 2, 3, 4, or 5 (SEQ ID NO: 53). The second connector can be (GSGGS)n; n = 1, 2, 3, 4, or 5 (SEQ ID NO: 54). The second connector can be (GGGS)n; n = 1, 2, 3, 4, or 5 (SEQ ID NO: 55). The second connector can be GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 51). The second connector can be (EAAAK)n, n = 1, 2, 3, 4, or 5 (SEQ ID NO: 52).

[0099] Table 3A: Exemplary Bispecific Antibodies (IgG-scFv) Note: Nevegimab VH: VH of Nevegimab (e.g., SEQ ID NO: 22); Nevegimab VL: VL of Nevegimab (e.g., SEQ ID NO: 21); durvalumab VL: VL of durvalumab (e.g., SEQ ID NOs: 23 and 67); durvalumab VH: VH of durvalumab (e.g., SEQ ID NOs: 24 and 68); potelimab VL: VL of potelimab (e.g., SEQ ID NOs: 25 and 69); potelimab VH: VH of potelimab (e.g., SEQ ID NOs: 26 and 70); CL (e.g., Cκ: SEQ ID NO: 29; or Cλ: SEQ ID NO: 30); (L): linker (e.g., SEQ ID NOs: 51-55); IgG1 CH (e.g., SEQ ID NOs: 31 and 47-48).

[0100] Table 3A provides a schematic diagram of two peptide chains of an exemplary bispecific antibody that specifically binds to VEGF and PD-1 / PD-L1 in IgG-scFv format. For illustrative purposes, as shown in the figure, antibody number 1 has two peptide chains HC and LC, wherein (1) HC contains, from the N-terminus to the C-terminus, the VH of Nevegimab (e.g., SEQ ID NO:22), the CH region (e.g., SEQ ID NO:48), the linker (e.g., SEQ ID NO:51), and the scFv, which contains, from the N-terminus to the C-terminus, the VL of durvalumab (e.g., SEQ ID NO:23), the second linker (e.g., SEQ ID NO:51), and the VH of durvalumab (e.g., SEQ ID NO:24); and (2) LC contains, from the N-terminus to the C-terminus, the VL of Nevegimab (e.g., SEQ ID NO:21) and the CL region (e.g., SEQ ID NO:29).

[0101] As another example, antibody number 4 has two peptide chains HC and LC, wherein (1) HC contains, from the N-terminus to the C-terminus, the VH of Nevegimab (e.g., SEQ ID NO:22), the CH region (e.g., SEQ ID NO:48), the linker (e.g., SEQ ID NO:51), and the scFv, which contains, from the N-terminus to the C-terminus, the VH of patelimab (e.g., SEQ ID NO:26), the second linker (e.g., SEQ ID NO:51), and the VL of patelimab (e.g., SEQ ID NO:25); and (2) LC contains, from the N-terminus to the C-terminus, the VL of Nevegimab (e.g., SEQ ID NO:21) and the CL region (e.g., SEQ ID NO:29).

[0102] Table 3B: Sequences of Exemplary Bispecific Antibodies (IgG-scFv)

[0103] In some embodiments, this document provides a bispecific antibody that specifically binds to human VEGF and human PD-1 / PD-L1, wherein the bispecific antibody has a first peptide chain (HC) and a second peptide chain (LC), wherein the amino acid sequence of the HC has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 60, and the amino acid sequence of the LC has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the HC has an amino acid sequence selected from the group consisting of SEQ ID NOs:56-59.

[0104] In some embodiments, this document provides a bispecific antibody that specifically binds to human VEGF and human PD-L1, wherein the bispecific antibody has a first peptide chain (HC) and a second peptide chain (LC), wherein the amino acid sequence of the HC has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 60, and the amino acid sequence of the LC has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 61. In some embodiments, the amino acid sequence of the HC has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 61. The amino acid sequence of HC has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:61. The amino acid sequence of HC has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:61. The amino acid sequence of HC has at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO:61. The amino acid sequence of HC has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:61. HC may have the amino acid sequence of SEQ ID NO:61. In some embodiments, HC has the amino acid sequence of SEQ ID NO:56. In some embodiments, the amino acid sequence of LC has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The amino acid sequence of LC has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The amino acid sequence of LC has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The amino acid sequence of LC has at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The amino acid sequence of the LC can have at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The LC can have the amino acid sequence of SEQ ID NO:60. In some embodiments, this document provides a bispecific antibody that specifically binds to human VEGF and human PD-L1, having a first peptide chain (HC) and a second peptide chain (LC), wherein the HC and LC have the amino acid sequences of SEQ ID NOs:61 and 60, respectively.In some embodiments, this document provides a bispecific antibody that specifically binds to human VEGF and human PD-L1, having a first peptide chain (HC) and a second peptide chain (LC), wherein the HC and LC have amino acid sequences of SEQ ID NOs:56 and 60, respectively. In some embodiments, the bispecific antibody has four peptide chains, including two identical HC chains and two identical LC chains.

[0105] In some embodiments, this document provides a bispecific antibody that specifically binds to human VEGF and human PD-L1, wherein the bispecific antibody has a first peptide chain (HC) and a second peptide chain (LC), wherein the amino acid sequence of the HC has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 60, and the amino acid sequence of the LC has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the amino acid sequence of the HC has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 62. The amino acid sequence of HC has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:62. The amino acid sequence of HC has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:62. The amino acid sequence of HC has at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO:62. The amino acid sequence of HC has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:62. HC may have the amino acid sequence of SEQ ID NO:62. In some embodiments, HC has the amino acid sequence of SEQ ID NO:57. In some embodiments, the amino acid sequence of LC has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The amino acid sequence of LC has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The amino acid sequence of LC has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The amino acid sequence of LC has at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The amino acid sequence of the LC can have at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The LC can have the amino acid sequence of SEQ ID NO:60. In some embodiments, this document provides a bispecific antibody that specifically binds to human VEGF and human PD-L1, having a first peptide chain (HC) and a second peptide chain (LC), wherein the HC and LC have the amino acid sequences of SEQ ID NOs:62 and 60, respectively.In some embodiments, this document provides a bispecific antibody that specifically binds to human VEGF and human PD-L1, having a first peptide chain (HC) and a second peptide chain (LC), wherein the HC and LC have amino acid sequences of SEQ ID NOs:57 and 60, respectively. In some embodiments, the bispecific antibody has four peptide chains, including two identical HC chains and two identical LC chains.

[0106] In some embodiments, this document provides a bispecific antibody that specifically binds to human VEGF and human PD-1, wherein the bispecific antibody has a first peptide chain (HC) and a second peptide chain (LC), wherein the amino acid sequence of the HC has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 60, and the amino acid sequence of the LC has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 63. In some embodiments, the amino acid sequence of the HC has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 63. The amino acid sequence of HC has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:63. The amino acid sequence of HC has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:63. The amino acid sequence of HC has at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO:63. The amino acid sequence of HC has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:63. HC may have the amino acid sequence of SEQ ID NO:63. In some embodiments, HC has the amino acid sequence of SEQ ID NO:58. In some embodiments, the amino acid sequence of LC has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The amino acid sequence of LC has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The amino acid sequence of LC has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The amino acid sequence of LC has at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The amino acid sequence of the LC can have at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The LC can have the amino acid sequence of SEQ ID NO:60. In some embodiments, this document provides a bispecific antibody that specifically binds to human VEGF and human PD-1, having a first peptide chain (HC) and a second peptide chain (LC), wherein the HC and LC have the amino acid sequences of SEQ ID NOs:63 and 60, respectively.In some embodiments, this document provides a bispecific antibody that specifically binds to human VEGF and human PD-L1, having a first peptide chain (HC) and a second peptide chain (LC), wherein the HC and LC have amino acid sequences of SEQ ID NOs:58 and 60, respectively. In some embodiments, the bispecific antibody has four peptide chains, including two identical HC chains and two identical LC chains.

[0107] In some embodiments, this document provides a bispecific antibody that specifically binds to human VEGF and human PD-1, wherein the bispecific antibody has a first peptide chain (HC) and a second peptide chain (LC), wherein the amino acid sequence of the HC has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 60, and the amino acid sequence of the LC has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 64. In some embodiments, the amino acid sequence of the HC has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 64. The amino acid sequence of HC has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:64. The amino acid sequence of HC has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:64. The amino acid sequence of HC has at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO:64. The amino acid sequence of HC has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:64. HC may have the amino acid sequence of SEQ ID NO:64. In some embodiments, HC has the amino acid sequence of SEQ ID NO:59. In some embodiments, the amino acid sequence of LC has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The amino acid sequence of LC has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The amino acid sequence of LC has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The amino acid sequence of LC has at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The amino acid sequence of the LC can have at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:60. The LC can have the amino acid sequence of SEQ ID NO:60. In some embodiments, this document provides a bispecific antibody that specifically binds to human VEGF and human PD-1, having a first peptide chain (HC) and a second peptide chain (LC), wherein HC and LC have the amino acid sequences of SEQ ID NOs:64 and 60, respectively.In some embodiments, this document provides a bispecific antibody that specifically binds to human VEGF and human PD-L1, having a first peptide chain (HC) and a second peptide chain (LC), wherein the HC and LC have the amino acid sequences of SEQ ID NOs:59 and 60, respectively. In some embodiments, the bispecific antibody has four peptide chains, including two identical HC chains and two identical LC chains.

[0108] In some embodiments, the bispecific antibody provided herein inhibits VEGF signaling. In some embodiments, the bispecific antibody provided herein inhibits vascular endothelial cell proliferation. In some embodiments, the bispecific antibody provided herein inhibits tumor angiogenesis. In some embodiments, the bispecific antibody provided herein blocks PD-1 / PD-L1 binding. In some embodiments, the bispecific antibody provided herein reduces immunosuppression. In some embodiments, the bispecific antibody provided herein promotes T cell activation and / or proliferation. In some embodiments, the bispecific antibody provided herein has any combination of the functions described above.

[0109] This disclosure also covers other variants and equivalents substantially homologous to the bispecific antibodies described herein. In some embodiments, it is desirable to enhance the binding affinity of the antibody. In some embodiments, it is desirable to modulate the biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector function, glycosylation, immunogenicity, and / or solubility. Those skilled in the art will understand that amino acid alterations can modify the post-translational processes of an antibody, such as changing the number or location of glycosylation sites or altering membrane anchoring properties.

[0110] This document also provides antibodies comprising functional variants of the heavy chain, light chain, VL region, VH region, or one or more CDRs of the antibodies described in this document. Functional variants of the heavy chain, light chain, VL, VH, or CDRs used in the antibody context still enable the antibody to retain at least a substantial proportion (at least about 90%, 95%, or higher) of the functional characteristics of the “reference” and / or “parent” antibody, including affinity and / or specificity / selectivity, Fc inertness, and PK parameters such as half-life, Tmax, and Cmax. Such functional variants typically retain significant sequence identity with the parent antibody and / or have substantially similar heavy and light chain lengths. Exemplary variants include variants that differ primarily from the parent antibody sequence in the heavy chain and / or light chain, VH and / or VL and / or CDR regions through conserved substitutions, for example, in the 10 substitutions in such a variant, substitutions such as 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitution may be a conserved amino acid residue substitution.

[0111] Mutations can be substitutions, deletions, or insertions of one or more nucleotides encoding the antibody or polypeptide, resulting in an amino acid sequence change compared to the native antibody or polypeptide sequence. In some embodiments, amino acid substitutions are produced by replacing one amino acid with another amino acid having similar structure and / or chemical properties, such as replacing leucine with serine, i.e., a conserved amino acid substitution. Insertions or deletions can range from about 1 to 5 amino acids. In some embodiments, the substitutions, deletions, or insertions relative to the parent molecule include substitutions of fewer than 25 amino acids, fewer than 20 amino acids, fewer than 15 amino acids, fewer than 10 amino acids, fewer than 5 amino acids, fewer than 4 amino acids, fewer than 3 amino acids, or fewer than 2 amino acids. In some embodiments, biologically useful and / or relevant amino acid sequence variations can be identified by systematically performing insertions, deletions, or substitutions in the sequence and testing the activity of the resulting variant protein relative to the parent protein.

[0112] In some embodiments, variants of the bispecific antibodies described herein are provided. In some embodiments, the variants include 1 to 30 amino acid substitutions, additions, and / or deletions in the parent antibody or antigen-binding fragment. In some embodiments, the variants include 1 to 25 amino acid substitutions, additions, and / or deletions in the parent antibody or antigen-binding fragment. In some embodiments, the variants include 1 to 20 substitutions, additions, and / or deletions in the parent antibody or antigen-binding fragment. In some embodiments, the variants include 1 to 15 substitutions, additions, and / or deletions in the parent antibody or antigen-binding fragment. In some embodiments, the variants include 1 to 10 substitutions, additions, and / or deletions in the parent antibody or antigen-binding fragment. In some embodiments, the variants include 1 to 5 amino acid substitutions, additions, and / or deletions in the parent antibody or antigen-binding fragment. In some embodiments, the variants include 1 to 3 amino acid substitutions, additions, and / or deletions in the parent antibody or antigen-binding fragment. In some embodiments, the amino acid substitutions are located in the CDR of the antibody or antigen-binding fragment. In some embodiments, the amino acid substitutions are not located in the CDR of the antibody or antigen-binding fragment. In some embodiments, the amino acid substitution is located within the framework region of the antibody or antigen-binding fragment. In some embodiments, the amino acid substitution, addition, and / or deletion are conserved amino acid substitutions.

[0113] The variant antibodies or antigen-binding fragments described herein can be generated using methods known in the art, including but not limited to site-directed mutagenesis, alanine scan mutagenesis, and PCR mutagenesis. Methods of mutagenesis and nucleotide sequence alteration are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Methods Enzymol. 54:367-382 (1987); Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor, NY); US Pat. No. 4,873,192; and the references cited therein, which are incorporated herein by reference. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the peptide of interest can be found in the model of Dayhoff et al. (1978) in *Atlas of Protein Sequence and Structure* (Natl. Biomed. Res. Found., Washington, DC), pp. 345–352, which is incorporated herein by reference in its entirety. Dayhoff et al.'s model uses a Point Accepted Mutation (PAM) amino acid similarity matrix (PAM 250 matrix) to determine appropriate conserved amino acid substitutions. Conserved substitutions, such as exchanging one amino acid for another with similar properties, may be beneficial.

[0114] In some embodiments, the variant may have at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher identity in the amino acid sequence with the parental antibody or antigen-binding fragment. In some embodiments, variants of the bispecific antibody disclosed herein comprise the amino acid sequence of the parental bispecific antibody disclosed herein and have one or more conserved amino acid substitutions. Conserved amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties.

[0115] In some embodiments, variants of the bispecific antibodies disclosed herein comprise the amino acid sequence of the parent antibody and have one or more non-conserved amino acid substitutions. In some embodiments, variants of the bispecific antibodies disclosed herein comprise the amino acid sequence of the parent binding antibody and have one or more non-conserved amino acid substitutions, wherein said one or more non-conserved amino acid substitutions do not interfere with or inhibit one or more biological activities of the variant. In some embodiments, said one or more conserved amino acid substitutions and / or said one or more non-conserved amino acid substitutions can enhance the biological activity of the variant, such that the biological activity of the functional variant is increased relative to the parent antibody.

[0116] In some embodiments, the variants may have 1, 2, 3, 4 or 5 amino acid substitutions in the CDRs of the binding portion (e.g., VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2 and VL CDR3).

[0117] In some embodiments, the variant may include the addition of amino acid residues to the amino and / or carboxyl termini of the antibody or peptide. The length of the added amino acid residues may range from one residue to one hundred or more residues. In some embodiments, the variant contains an N-terminal methionine residue. In some embodiments, the variant contains additional peptide / protein (e.g., an Fc region) to form a fusion protein. In some embodiments, the variant is engineered to be detectable and may contain a detectable marker and / or protein (e.g., a fluorescent tag or enzyme).

[0118] In some embodiments, the bispecific antibodies disclosed herein can be chemically modified naturally or through intervention. In some embodiments, the bispecific antibodies are chemically modified by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, and / or linking to cellular ligands or other proteins. Any of these numerous chemical modifications can be implemented using known techniques. The bispecific antibodies provided herein may comprise one or more amino acid analogs (including, for example, non-natural amino acids), as well as other modifications known in the art.

[0119] The bispecific antibodies disclosed herein can be analyzed for their physical, chemical, and / or biological properties using various methods known in the art. In some embodiments, the ability of the antibody to bind to VEGF and PD-1 / PD-L1 is tested. Binding assays include, but are not limited to, BLI, SPR (e.g., Biacore), ELISA, and FACS. Furthermore, the antibody's solubility, stability, thermal stability, viscosity, expression level, expression quality, and / or purification efficiency can be evaluated. In some embodiments, variants of the bispecific antibodies disclosed herein may retain their ability to bind to human VEGF and / or PD-1 / PD-L1 to a similar, equal, or greater extent than that of the parental bispecific antibody.

[0120] In some embodiments, the bispecific antibodies provided herein include modifications in their Fc regions. In some embodiments, the modified antibodies (e.g., the modified Fc region) provide altered effector functions, thereby affecting the antibody's biological characteristics. For example, in some embodiments, the deletion or inactivation of the constant region (through point mutation or other means) reduces the Fc receptor binding of the modified antibody during circulation. In some embodiments, constant region modifications reduce the immunogenicity of the antibody. In some embodiments, constant region modifications increase the serum half-life of the antibody. In some embodiments, constant region modifications decrease the serum half-life of the antibody. In some embodiments, constant region modifications reduce or eliminate the antibody's ADCC and / or complement-dependent cytotoxicity (CDC). In some embodiments, specific amino acid substitutions in the human IgG1 Fc region with corresponding IgG2 or IgG4 residues reduce the effector functions (e.g., ADCC and CDC) of the modified antibody. In some embodiments, the antibody does not have one or more effector functions (e.g., a "no-effect" antibody). In some embodiments, the antibody does not have ADCC activity and / or does not have CDC activity. In some embodiments, the antibody does not bind to Fc receptors and / or complement factors. In some embodiments, the antibody does not have effector function. In some embodiments, constant region modifications increase or enhance the antibody's ADCC and / or CDC. In some embodiments, the constant region is modified to eliminate disulfide bonds or oligosaccharide moieties. In some embodiments, the constant region is modified to add / replace one or more amino acids, thereby providing one or more cytotoxin, oligosaccharide, or carbohydrate linkage sites.

[0121] In some embodiments of the bispecific antibodies provided herein, the Fc domain comprises one or more amino acid substitutions that reduce binding to the Fc receptor. The Fc receptor may be a human Fc receptor. The Fc receptor may be an Fcγ receptor. The Fc receptor may be an activating Fc receptor. The Fc receptor may be an activating human Fcγ receptor, such as human Fcγ RIIIa, Fcγ RI, or Fcγ RIIa. In some embodiments of the bispecific antibodies provided herein, the Fc domain comprises one or more amino acid substitutions that reduce effector function. The effector function may be complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, or any combination thereof. In some embodiments, the effector function is ADCC.

[0122] In some embodiments of the bispecific antibody provided herein, the same one or more amino acid substitutions are present in each of the two subunits of the Fc region. In one aspect, the one or more amino acid substitutions reduce the binding affinity of the Fc region to the Fc receptor. In another aspect, the one or more amino acid substitutions reduce the binding affinity of the Fc region to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold.

[0123] Variants with reduced effector function are known in the art and can be incorporated into the bispecific antibodies disclosed herein. For example, amino acid substitutions are known to reduce effector function. The hIgG1 L235A / G237A / E318A antibody cannot bind to human cell lines expressing FcγRs, resulting in reduced ADCC. hIgG1 and hIgG4 antibodies with L234A / L235A Fc domains do not have detectable binding to low-affinity FcγRs and C1q, and ADCC and CDC are significantly reduced. Mutations at specific residues in hIgG1 known to interact with both FcγRs and C1q, such as the amino acid substitution L234F / L235E / P331S, can reduce binding to low-affinity FcγRs and result in undetectable binding to FcγRIs. The G236R / L328R mutation reduces or completely eliminates binding to FcγRs. S267E substitution also reduces binding to all low-affinity hFcγRs. S267K substitution combines with a series of mutations in the hIgG2 lower hinge (E233P / L234V / L235A mutations and deletion of residue G236) and incorporates into the hIgG1 background, resulting in a lack of binding to all hFcγRs. P329G disrupts the interaction between hIgG and hFcγRs. The triple mutant L234A / L235A / P329G shows undetectable binding to either C1q or FcγRs and leads to the elimination of ADCC when introduced into hIgG1. The combined point mutations N297Q, L234F, L235E, D265A, and P331S eliminate Fc function. The combined potent silencing of the Fc region by L234F / L235E / D265A resulted in undetectable binding to FcγRI, reduced binding to low-affinity FcγRs, and reduced binding to C1q. From a site-saturated mutation library centered on the Fc C′ / E loop, the S298G / T299A mutation eliminated or significantly reduced binding to C1q and most FcγRs except for FcγRIIA-R131 and FcγRIIB.

[0124] Furthermore, glycosylation techniques can be used to generate antibodies with reduced effector functions. The N297 glycan is the core of the binding between hIgG1 and FcγRs and C1q. Therefore, removing amino acid mutations at this site, including N297A, N297Q, and N297G, can reduce binding to all FcγRs and C1q, resulting in reduced ADCC and CDC.

[0125] For hIgG4, which exhibits low affinity for all FcγRs, the serine residue at position 228 plays a crucial role in F(ab) arm exchanges. S228P substitution provides homogeneous hIgG4 and is commonly incorporated into therapeutic hIgG4 antibodies. Due to its inherent lack of effector function, the human γ4 constant region can be used for Fc silencing strategies. For example, replacing the human γ1 region with the human γ4 region reduces effector function. The mouse IgG2b isotype also exhibits low FcγR binding activity, differing from hIgG4 at position 235. Incorporating a mouse IgG2b residue (glutamate) into this position in an hIgG4 antibody further minimizes Fc effector function, resulting in an antibody (with the S228P / L235E mutation) that exhibits significantly reduced binding to all FcγRs and C1q (if any) and has no measurable ADCC. Furthermore, rather than replacing the entire constant region of hIgG1 with hIgG4, it is preferable to introduce specific amino acids from human γ4 into antibodies against other IgG isotypes. For example, when the amino acid mutation combination H268Q / V309L / A330S / P331S (IgG2m4) is introduced into the hIgG2 backbone, compared to WT hIgG2 antibodies, it results in undetectable binding to hFcγRI, hFcγRIIIA, or C1q, reduced binding to hFcγRIIB, and no change in binding to FcγRIIA-H131. As another example, the V234A / G237A / P238S / H268A / V309L / A330S / P331S (IgG2c4d) mutation (in which multiple residues in the hIgG2 constant region are replaced by IgG4 residues) results in undetectable binding to any FcγRs or C1q, and no measurable ADCC, ADCP, or CDC, compared to WT hIgG2 counterparts.

[0126] Several Fc mutations have been engineered to improve the half-life of therapeutic antibodies by enhancing their interaction with the neonatal Fc receptor (FcRn). Among these, YTE mutations (M252Y, S254T, and T256E) in the Fc region of human IgG1 have been shown to significantly increase antibody half-life. These mutations reduce the dissociation constant (KD) of the Fc-FcRn complex by 10-fold, resulting in more stable binding and prolonged cycling time. Furthermore, LS mutations, specifically the combination of M428L and N434S, also contribute to enhanced serum half-life. These mutations reduce the KD of the FcRn-IgG1 complex, resulting in a three-fold increase in serum half-life. LS modification does not significantly alter ADCC, making it suitable for applications requiring sustained effector function. LA mutations (involving M428L and N434A) also prolong antibody half-life. The T307A / E380A / N434A(AAA) mutation is another modification shown to improve antibody half-life. This triple mutation significantly enhances binding to FcRn at endosomal pH, increasing FcRn binding by 11.8-fold compared to wild-type human IgG1. This enhanced binding allows for more efficient recovery and prevents degradation, resulting in a 2.5-fold increase in half-life in a humanized FcRn transgenic mouse model. Mutation combinations including M252Y, S254T, T256E, H433K, and N434F have been shown to effectively increase antibody half-life.

[0127] Therefore, for illustrative purposes, the antibodies disclosed herein may include at least one of the following Fc mutations: L234A, L235A, G237A, M252Y, S254T, T256E, N297A, N297Q, N297G, T307A, E318A, E380A, M428L, N434S, N434A, or any combination thereof. In some implementations, the variants include: deglycosylation (N297A / Q / G; or "NA"), L235A / G237A / E318A ("LAGAEA"), T307A / E380A / N434A (AAA), L234A / L235A ("LALA"), M252Y / S254T / T256E ("YTE"), M428L and N434S ("LS"), M428L and N434A ("LA"), S228P / L235E ("IgG4"), and so on. PE"), G236R / L328R ("RR"), S298G / T299A ("GA"), L234F / L235E / P331S ("FES"), H268Q / V309L / A330S / P331S ("IgG2m4"), E233P / L234V / L235A / G 236 deletion / S267K, L234A / L235A / P329G (“LALAPG”), V234A / G237A / P238S / H268A / V309L / A330S / P331S (“IgG2c4d”), and L234F / L235E / D265A (“FEA”). (See Liu et al., Antibodies 9.4 (2020): 64; Delidakis et al., Annual review of biomedical engineering 24 (2022): 249-274, both incorporated herein by reference in their entirety.) In some embodiments, the bispecific antibodies disclosed herein contain an NA mutation. In some embodiments, the bispecific antibodies disclosed herein contain an AAA mutation. In some embodiments, the bispecific antibodies disclosed herein contain an LALA mutation. In some embodiments, the bispecific antibodies disclosed herein contain an RR mutation. In some embodiments, the bispecific antibodies disclosed herein contain a GA mutation. In some embodiments, the bispecific antibodies disclosed herein contain an FES mutation. In some embodiments, the bispecific antibodies disclosed herein contain a LALAPG mutation. In some embodiments, the bispecific antibodies disclosed herein contain a FEA mutation. In some embodiments, the bispecific antibodies disclosed herein contain an IgG2m4 mutation. In some embodiments, the bispecific antibodies disclosed herein contain an IgG2-PE mutation.In some embodiments, the bispecific antibodies disclosed herein contain an IgG2c4d mutation. As will be understood by those skilled in the art, the bispecific antibodies disclosed herein are not limited to specific Fc modifications, and any combination and arrangement of Fc modifications disclosed herein or otherwise known in the art that can reduce effector function or reduce affinity for FcγR binding can be used in the bispecific antibodies disclosed herein.

[0128] In some embodiments, the Fc region of the bispecific antibody provided herein may include amino acid substitutions at positions selected from the group consisting of E233, L234, L235, N297, P331, M252, S254, and T256. In some embodiments, the Fc region includes amino acid substitutions at positions selected from the group consisting of L234, L235, M252, S254, and T256. In some embodiments, the Fc region includes amino acid substitutions L234A and L235A. In some embodiments, the Fc region is an IgG1 Fc region, particularly a human IgG1 Fc region. In some embodiments, the Fc region includes amino acid substitutions L234A and L235A, and further amino acid substitutions at positions selected from the group consisting of E233, N297, P331, M252, S254, and T256. In some embodiments, the further amino acid substitutions are E233P, N297A, N297D, P331S, M252Y, S254T, or T256E. In some embodiments, the Fc region includes amino acid substitutions at positions L234, L235, M252, S254, and T256. In some embodiments, the Fc region includes amino acid mutations L234A, L235A, M252Y, S254T, and T256E. In some embodiments, the Fc region is an IgG4 Fc region, particularly a human IgG4 Fc region. In some embodiments, the Fc region includes amino acid substitutions at positions selected from the group consisting of S228, S254, V308, and N434. In some embodiments, the Fc region includes the amino acid substitution S228P. In some embodiments, the Fc region includes the amino acid substitution S228P and further amino acid substitutions at positions selected from the group consisting of S254, V308, and N434. In some embodiments, the further amino acid substitutions are S254T, V308P, or N434A. In some embodiments, the Fc region includes amino acid substitutions at the positions of S228, S254, V308, and N434. In some embodiments, the Fc region contains amino acid mutations S228P, S254T, V308P, and N434A. All amino acid residues are numbered according to the EU index.

[0129] In some embodiments, the variant may include the addition of amino acid residues to the amino and / or carboxyl termini of the antibody. The length of the added amino acid residues may range from one residue to one hundred or more residues. In some embodiments, the variant contains an N-terminal methionine residue. In some embodiments, the variant is engineered to be detectable and may contain a detectable tag and / or protein (e.g., a fluorescent tag or enzyme).

[0130] The variant antibodies described herein can be generated using methods known in the art, including but not limited to site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

[0131] In some embodiments, the bispecific antibodies disclosed herein may be chemically modified naturally or through intervention. In some embodiments, the bispecific antibodies are chemically modified by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization via known protecting / blocking groups, proteolytic cleavage, and / or linking to cellular ligands or other proteins. Any of these numerous chemical modifications can be implemented using known techniques. The bispecific antibodies provided herein may comprise one or more amino acid analogs (including, for example, non-natural amino acids), as well as other modifications known in the art.

[0132] The bispecific antibodies disclosed herein can be analyzed for their physical, chemical, and / or biological properties using various methods known in the art. In some embodiments, the bispecific antibodies provided herein are tested for their ability to bind to human VEGF and / or human PD-1 / PD-L1. Binding assays include, but are not limited to, BLI, SPR (e.g., Biacore), ELISA, and FACS. Furthermore, the bispecific antibodies can also be evaluated for solubility, stability, thermal stability, viscosity, expression level, expression quality, and / or purification efficiency.

[0133] In some embodiments, the bispecific antibody disclosed herein may be conjugated to a detectable substance or molecule that allows the reagent to be used for detection. The detectable substance may include, but is not limited to, enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; prosthetic groups such as biotin and flavin; fluorescent materials such as umbelliferone, luciferin, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazineamine luciferin, dansyl chloride, anthocyanin (Cy3), and phycoerythrin; bioluminescent materials such as luciferase; and radioactive materials such as... 212 Bi、 14 C 57 Co、 51 Cr 67 Cu、 18 F,68 Ga、 67 Ga、 153 Gd, 159 Gd, 68 Ge 3 H, 166 Ho、 131 I, 125 I, 123 I, 121 I, 115 In、 113 In、 112 In、 111 In、 14 0La、 177 Lu、 54 Mn, 99 Mo、 32 P, 103 Pd, 149 Pm, 142 Pr、 18 6Re、 188 Re、 105 Rh、 97 Ru、 35 S, 47 Sc、 75 Se、 153 Sm、 113 Sn、 117 Sn、 85 Sr、 99 mTc, 201 Ti、 133 Xe, 90 Y、 69 Yb, 1 75 Yb、 65 Zn; positron-emitting metals; and magnetic metal ions.

[0134] The anti-VEGF and PD-1 / PD-L1 bispecific antibodies disclosed herein can be attached to a solid-phase support. Such solid-phase supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, the immobilized bispecific antibodies are used for immunoassays. In some embodiments, the immobilized bispecific antibodies are used for purification. 6.3 Polynucleotides, Carriers, and Cells

[0135] This document provides polynucleotides encoding at least one peptide chain of the bispecific antibodies disclosed herein. In some embodiments, the polynucleotides provided herein encode one peptide. In some embodiments, the polynucleotides provided herein encode more than one peptide. In some embodiments, the polynucleotides provided herein may, for example, encode two peptide chains of the bispecific antibodies provided herein. In some embodiments, this document provides polynucleotides encoding HC, LC, or both of the anti-VEGF and anti-PD-1 / PD-L1 bispecific antibodies disclosed herein. For example, this document provides polynucleotides encoding HC, LC, or both of the bispecific antibodies illustrated in Tables 3A and 3B.

[0136] Cistrons can be separated, for example, by internal ribosome entry sites (IRES) or 2A elements. As understood in the art, an IRES refers to a nucleotide sequence in the expression cassette that, upon transcription into mRNA, can directly recruit ribosomes without requiring pre-scanning of the untranslated regions of the mRNA by the ribosomes. As understood in the art, a 2A element encodes a self-cleaving short peptide (approximately 20 amino acids) that provides a mechanism for the subsequent separation of the target polypeptide produced in equimolar amounts. Exemplary 2A self-cleaving peptides include P2A, E2A, F2A, and T2A.

[0137] As used herein, the term “encoding” and its syntactic equivalents refer to the inherent properties of a specific nucleotide sequence in a polynucleotide or nucleic acid (e.g., gene, cDNA, or mRNA), namely, its role as a template in biological processes for the synthesis of other polymers and macromolecules, processes having a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the resulting biological properties. Thus, if the transcription and translation of the mRNA corresponding to a gene produces the protein, then the gene encodes that protein. Unless otherwise stated, “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns.

[0138] The term "polynucleotide encoding a polypeptide" encompasses polynucleotides that include only the coding sequence of the polypeptide, as well as polynucleotides that include additional coding and / or non-coding sequences. The polynucleotides disclosed herein can be in RNA or DNA form. The DNA can be cDNA, genomic DNA, or synthetic DNA, and can be double-stranded or single-stranded. Single-stranded DNA can be a coding strand or a non-coding (antisense) strand. The polynucleotides disclosed herein can be mRNA.

[0139] This disclosure also provides variants of the polynucleotides described herein, wherein said variants have a polynucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the polynucleotide sequence encoding at least one peptide chain of the bispecific antibody described herein. As used herein, the phrase “polynucleotide having a nucleotide sequence that is at least about 95% identical to the polynucleotide sequence” means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, but said polynucleotide sequence may include up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced by another nucleotide, or up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence can occur at the 5' or 3' end of the reference nucleotide sequence or anywhere between these end positions, and can be scattered individually among the nucleotides of the reference sequence or located within the reference sequence in the form of one or more consecutive groups.

[0140] Polynucleotide variants may contain alterations in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that produce silent substitutions, additions, or deletions, but do not change the properties or activity of the encoded polypeptide. In some embodiments, polynucleotide variants contain silent substitutions that result in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can be prepared for a variety of reasons, such as optimizing codon expression for a specific host (e.g., changing codons in human mRNA to codons preferred by a bacterial host such as E. coli). In some embodiments, polynucleotide variants contain at least one silent mutation in a non-coding or coding region of the sequence.

[0141] In some embodiments, polynucleotide variants are prepared to modulate or alter the expression (or expression level) of the encoded polypeptide. In some embodiments, polynucleotide variants are prepared to increase the expression of the encoded polypeptide. In some embodiments, polynucleotide variants are prepared to decrease the expression of the encoded polypeptide. In some embodiments, the polynucleotide variant has increased expression of the encoded polypeptide compared to the parental polynucleotide sequence. In some embodiments, the polynucleotide variant has decreased expression of the encoded polypeptide compared to the parental polynucleotide sequence.

[0142] In some embodiments, the polynucleotide contains a coding sequence for a polypeptide (e.g., an antibody or a fusion protein), which is fused within the same reading frame to a polynucleotide that facilitates the expression and secretion of the polypeptide from the host cell (e.g., a guide sequence that functions as a secretion sequence to control polypeptide transport). The polypeptide can be excised by the host cell from the guide sequence to form the “mature” form of the polypeptide.

[0143] In some embodiments, the polynucleotide contains a coding sequence for a peptide (e.g., an antibody) fused with a marker or tag sequence within the same reading frame. For example, in some embodiments, the marker sequence is a hexahistidine tag (HIS-tag), which allows for efficient purification of the peptide fused with said marker. In some embodiments, when using a mammalian host (e.g., COS-7 cells), the marker sequence is a hemagglutinin (HA) tag derived from influenza hemagglutinin protein. In some embodiments, the marker sequence is a FLAG™ tag. In some embodiments, the marker may be used in combination with other markers or tags.

[0144] In some implementations, the polynucleotides are isolated. In some implementations, the polynucleotides are essentially pure.

[0145] In some embodiments, this document also provides vectors containing the polynucleotides disclosed herein. As used herein, the term "vector" and its grammatical equivalents refer to a tool for carrying genetic material (e.g., polynucleotide sequences) that can be introduced into a host cell, where it can be replicated and / or expressed. Suitable vectors for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selectable sequences or markers operable for stable integration into the host cell chromosome. Furthermore, the vector may include one or more selectable marker genes and appropriate expression control sequences. For example, the selectable marker genes may provide resistance to antibiotics or toxins, compensate for auxotrophic deficiencies, or provide critical nutrients absent in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and similar elements known in the art. When two or more polynucleotides are to be co-expressed, for example, the two polynucleotides may be inserted into a single expression vector or into different expression vectors. For single-vector expression, the encoding polynucleotide may be operably linked to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of polynucleotides into host cells can be confirmed using methods known in the art. Those skilled in the art will understand that polynucleotides are expressed in amounts sufficient to produce the desired product, and will also understand that expression levels can be optimized using methods known in the art to achieve adequate expression.

[0146] In some embodiments, the vectors provided herein may be expression vectors. In some embodiments, the vectors provided herein contain a polynucleotide encoding at least one peptide chain of the bispecific antibody described herein. In some embodiments, a recombinant expression vector is also provided herein, which can be used to amplify and express a polynucleotide encoding at least one peptide chain of the bispecific antibody described herein. For example, the recombinant expression vector may be a reproducible DNA construct comprising a synthetic or cDNA-derived DNA fragment encoding at least one peptide chain of the bispecific antibody described herein, and operatively linked to a suitable transcriptional and / or translational regulatory element derived from a mammalian, microbial, viral, or insect gene. In some embodiments, a viral vector is used. When DNA regions are functionally related to each other, they are referred to as being “operatively linked.” For example, if a promoter controls transcription of the sequence, the promoter is operatively linked to the coding sequence; or, if a ribosome binding site is positioned to allow translation, the ribosome binding site is operatively linked to the coding sequence. In some embodiments, structural elements intended for use in certain expression systems include a guide sequence that enables the host cell to secrete the translated protein extracellularly. In some implementations, where the recombinant protein is expressed without a guide or transport sequence, the polypeptide may contain an N-terminal methionine residue.

[0147] Examples of vectors include plasmids, autonomously replicating sequences, and transposable elements. Useful expression vectors suitable for bacterial hosts include known bacterial plasmids, such as those from E. coli, including pCR1, pBR322, pMB9, and their derivatives, as well as plasmids with a broader host range, such as M13 and other filamentous single-stranded DNA phages. Other exemplary vectors include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes (e.g., yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC)), bacterial phages (e.g., λ phage or M13 phage), and animal viruses. Examples of animal virus classes useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and polyomaviruses (e.g., SV40). Examples of expression vectors include the pClneo vector (Promega) for expression in mammalian cells; and pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Exemplary transposon systems (e.g., Sleeping Beauty and PiggyBac) can be used, which can be stably integrated into the genome (e.g., Ivics et al., Cell, 91 (4): 501–510 (1997); Cadiñanos et al., (2007) Nucleic Acids Research. 35(12): e87).

[0148] In some embodiments, the vector is an episome vector or a vector maintained extrachromosomally. As used herein, the term "epitaphone" refers to a vector capable of replication without integration into the host chromosomal DNA and without gradual loss from dividing host cells; it also means that the vector replicates extrachromosomally or as an episome. The vector is engineered to carry a sequence encoding a DNA replication origin, or "ori," derived from a lymphoherpesvirus or gamma herpesvirus, adenovirus, SV40, bovine papillomavirus, or yeast, specifically an episome of a lymphoherpesvirus or gamma herpesvirus corresponding to oriP of EBV. In some embodiments, the lymphoherpesvirus may be Epstein Barr virus (EBV), Kaposi's sarcoma herpes virus (KSHV), Herpes virus saimiri (HS), or Marek's disease virus (MDV). Epstein Barr virus (EBV) and Kaposi's sarcoma herpes virus (KSHV) are also examples of gamma herpesviruses. Typically, host cells contain viral replication transactivation proteins that activate replication.

[0149] The "expression control sequences," "control elements," or "regulatory sequences" present in expression vectors are those untranslated regions of the vector—origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno or Kozak sequences), introns, polyadenylated sequences, and the 5' and 3' untranslated regions—that interact with host cell proteins to carry out transcription and translation. These elements can vary in strength and specificity. Depending on the vector system and host used, any number of suitable transcriptional and translational elements can be used, including ubiquitous and inducible promoters.

[0150] Exemplary ubiquitous expression control sequences that can be used in this disclosure include, but are not limited to, the immediate early promoter of cytomegalovirus (CMV), the promoter of viral simian virus 40 (SV40) (e.g., early or late), the Moloney mouse leukemia virus (MoMLV) LTR promoter, the Rous sarcoma virus (RSV) LTR, the herpes simplex virus (HSV) (thymidine kinase) promoter, the H5, P7.5, and P11 promoters of vaccinia virus, the elongation factor 1-α (EF1a) promoter, the early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock protein 70kDa 5 (HSPA5), heat shock protein 90kDa β subunit member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinin (β-KIN), and human ROSA 26 site (Irions). et al., Nature Biotechnology 25, 1477 - 1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter and β-actin promoter.

[0151] Examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters of genes encoding glucocorticoids or estrogen receptors (induced by treatment with the corresponding hormone), metallothionein promoters (induced by treatment with various heavy metals), MX-1 promoters (induced by interferon), the "GeneSwitch" mifepristone-regulated system (Sirin et al., 2003, Gene, 323:67), kumaate-induced gene switches (WO 2002 / 088346), tetracycline-dependent regulatory systems, etc. The bispecific antibodies described herein can be prepared by any method known in the art, including chemical synthesis and recombinant expression techniques. Unless otherwise stated, the present invention is practiced using conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields known to those skilled in the art.

[0152] This disclosure also provides cells comprising the polynucleotides disclosed herein, said polynucleotides encoding at least one peptide chain of the bispecific antibody disclosed herein. In some embodiments, the cells provided herein comprise polynucleotides encoding HC and LC of the bispecific antibody disclosed herein. In some embodiments, the cells provided herein comprise a first polynucleotide encoding HC and a second polynucleotide encoding LC of the bispecific antibody disclosed herein.

[0153] This document also covers cells comprising the vectors disclosed herein. In some embodiments, this document provides a host cell comprising a vector containing the polynucleotides disclosed herein. In some embodiments, the host cell provided herein comprises one or more vectors, the vectors collectively containing a polynucleotide encoding a polypeptide chain encoding the bispecific antibody described herein. In some embodiments, the host cell provided herein produces the bispecific antibody described herein.

[0154] Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (derived from monkey kidney), L-929 (derived from mouse fibroblasts), C127 (derived from mouse mammary tumors), 3T3 (derived from mouse fibroblasts), CHO (derived from Chinese hamster ovary), HeLa (derived from human cervical cancer), BHK (derived from hamster kidney fibroblasts), HEK-293 (derived from human embryonic kidney) cell lines and their variants. Mammalian expression vectors may contain non-transcriptional elements, such as origin of replication, appropriate promoters and enhancers linked to the gene to be expressed, and other 5' or 3' flanking non-transcriptional and 5' or 3' untranslated sequences, such as essential ribosome binding sites, polyadenylation sites, splicing donor and acceptor sites, and transcription termination sequences. Expression of recombinant proteins in insect cell culture systems (e.g., baculoviruses) also provides a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for producing heterologous proteins in insect cells are well known to those skilled in the art. 6.4 Production Method

[0155] This document also provides methods for producing the bispecific antibodies disclosed herein. In some embodiments, the bispecific antibodies disclosed herein consist of more than one polypeptide chain, which may be produced individually or together. In some embodiments, the methods provided herein produce at least one polypeptide chain of the bispecific antibodies disclosed herein. In some embodiments, the methods provided herein produce all polypeptide chains of the bispecific antibodies disclosed herein.

[0156] The bispecific antibodies or peptides described herein can be produced and isolated using methods known in the art. Peptides can be synthesized wholly or partially using chemical methods (see, for example, Caruthers (1980). Nucleic Acids Res. Symp. Ser. 215; Horn (1980); and Banga, AK, Therapeutic Peptides And Proteins, Formulation, Processing And Delivery Systems (1995) Technomic Publishing Co., Lancaster, PA). Peptide synthesis can be performed using various solid-phase techniques (see, for example, Roberge, Science 269:202 (1995); Merrifield, Methods. Enzymol. 289:3 (1997)), and can be automated, for example, using the ABI 431A Peptide Synthesizer (Perkin Elmer) according to the manufacturer's instructions. Peptides can also be synthesized using combinatorial methodologies. Synthetic residues and peptides can be synthesized using a variety of procedures and methodologies known in the art (see, for example, Organic Syntheses Collective Volumes, Gilman, et al. (eds.), John Wiley & Sons, Inc., NY). Modified peptides can be prepared by chemical modification methods (see, for example, Belousov, Nucleic Acids Res. 25:3440 (1997); Frenkel, Free Radic. Biol. Med. 19:373 (1995); and Blommers, Biochemistry 33:7886 (1994)). Peptide sequence variants, derivatives, substitutions, and modifications can also be prepared using methods such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR-based mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res. 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA 317:415 (1986)), and other techniques can be implemented on cloned DNA to produce the peptide sequences, variants, fusions, and chimeras of the present invention, as well as their variants, derivatives, substitutions, and modifications.

[0157] A variety of host-expression vector systems can be used to recombinantly express the bispecific antibodies described herein or one or more polypeptide chains thereof. Suitable host cells for expression include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters. Appropriate cloning and expression vectors suitable for bacterial, fungal, yeast, and mammalian cell hosts, as well as protein production methods (including antibody production), are well known in the art. Such host-expression systems represent vectors that can be used to generate and subsequently purify the encoding sequences of the bispecific antibodies described herein, but also represent cells that can express the bispecific antibodies described herein in situ when transformed or transfected with appropriate polynucleotide encoding sequences. These include, but are not limited to: microorganisms (e.g., bacteria such as E. coli and B. subtilis) transformed with recombinant phage DNA, plasmid DNA, or copious DNA expression vectors containing sequences encoding the compounds described herein; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing sequences encoding the compounds described herein; insect cell systems infected with recombinant viral expression vectors containing sequences encoding the compounds described herein (e.g., baculovirus); plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) and containing sequences encoding the molecular compounds described herein; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphoid cells (see U.S. Patent No. 5,807,715), Per... C.6 cells (human retinal cells developed by Crucell) carry recombinant expression constructs containing promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or from mammalian viruses (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters).

[0158] In bacterial systems, a number of expression vectors can be advantageously selected based on the intended use of the expressed protein. For example, when it is necessary to produce large quantities of such proteins for use in pharmaceutical compositions of the bispecific antibodies described herein, a vector capable of directing high-level expression and readily purified protein products may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al. (1983), EMBO J. 2: 1791-1794); the pIN vector (Inouye et al. (1985), Nucleic Acids Res. 13:3101-3110; Van Heeke et al. (1989), J. Biol. Chem. 24:5503-5509); and analogues. The pGEX vector can also be used to express peptides as fusion proteins with glutathione S-transferase (GST). Generally, these proteins are soluble and can be readily purified from lysed cells by adsorption and binding to a glutathione-agarose bead matrix, followed by elution in the presence of free glutathione. The pGEX vector is designed to contain thrombin or factor Xa protease cleavage sites, allowing the cloned target gene product to be released from the GST moiety.

[0159] Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. In mammalian host cells, a variety of virus-based expression systems can be utilized. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (derived from monkey kidney), L-929 (derived from mouse fibroblasts), C127 (derived from mouse mammary tumors), 3T3 (derived from mouse fibroblasts), CHO (derived from Chinese hamster ovary), HeLa (derived from human cervical cancer), BHK (derived from hamster kidney fibroblasts), HEK-293 (derived from human embryonic kidney) cell lines and their variants. Mammalian expression vectors may contain non-transcriptional elements, such as origin of replication, appropriate promoters and enhancers linked to the gene to be expressed, and other 5' or 3' flanking non-transcriptional and 5' or 3' non-translational sequences, such as essential ribosome binding sites, polyadenylation sites, splicing donor and acceptor sites, and transcription termination sequences. Expressing recombinant proteins in insect cell culture systems (e.g., baculoviruses) also provides a robust method for producing correctly folded proteins with biological functions. Baculovirus systems for producing heterologous proteins in insect cells are well known to those skilled in the art. Autographa californica nuclear polyhedrosis virus (AcNPV) has been used as a vector for expressing foreign genes.

[0160] Furthermore, a host cell line can be selected that regulates the expression of the inserted sequence or modifies and processes the gene product in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) may be important for protein function. For example, in some embodiments, the bispecific antibody described herein can be expressed as a single gene product (e.g., as a single polypeptide chain, i.e., as a multi-protein precursor), which requires proteolytic cleavage via natural or recombinant cellular mechanisms to form the isolated polypeptide of the bispecific antibody described herein. Therefore, this disclosure covers multi-protein precursor molecules engineered with nucleic acid sequences to encode polypeptides comprising the bispecific antibody described herein, including coding sequences capable of directing post-translational cleavage of the multi-protein precursor. Post-translational cleavage of the multi-protein precursor results in the formation of the polypeptide of the bispecific antibody described herein. Post-translational cleavage of precursor molecules containing the compounds described herein can occur in vivo (i.e., within host cells via natural or recombinant cellular systems / mechanisms, such as furin cleavage at appropriate sites) or in vitro (e.g., incubating the polypeptide chain in a composition containing a protease or peptidase of known activity and / or in a composition containing conditions or reagents known to promote the desired proteolytic activity). The purification and modification of recombinant proteins are well known in the art, and therefore the design of multi-protein precursors can include several embodiments readily understood by those skilled in the art. Any protease or peptidase known in the art can be used for the modification of the precursor molecule.

[0161] Different host cells possess characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure proper modification and processing of expressed exogenous proteins. For this purpose, eukaryotic host cells with cellular machinery for appropriate processing of primary transcripts and glycosylation and phosphorylation of gene products can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20, T47D, CRL7030, and Hs578Bst.

[0162] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines stably expressing the compounds described herein can be constructed. Instead of using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. After introducing the exogenous DNA, engineered cells can be grown in enrichment media for 1–2 days, then switched to selective media. Selectable markers in the recombinant plasmid confer resistance to selection and enable cells to stably integrate the plasmid into their chromosomes and grow to form clonal foci, which can then be cloned and expanded into cell lines. This method can be advantageously used to construct cell lines expressing the compounds described herein. Such engineered cell lines may be particularly useful in screening and evaluating compounds that interact directly or indirectly with the compounds described herein.

[0163] A variety of screening systems can be used, including but not limited to the herpes simplexvirus thymidine kinase (Wigler et al., (1977), Cell 11: 223-232), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al., (1992) Bioessays 14: 495-500), and adenine phosphoribosyltransferase (Lowy et al., (1980), Cell 22: 817-823) genes, which can be used in tk-, hgprt-, or aprt- cells, respectively. Furthermore, antimetabolite resistance can be used as a basis for screening for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., (1980) PNAS 77:3567-3570; O'Hare et al., (1981) PNAS, 78: 1527-1531); gpt, which confers resistance to mycophenolic acid (Mulligan et al., (1981) PNAS, 78: 2072-2076); neo, which confers resistance to the aminoglycoside G-418 (Tolstoshev (1993), Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993), Science 260:926-932; and Morgan et al., (1993), Ann. Rev. Biochem. 62: 191-217); and hygro, which confers resistance to hygromycin (Santerre et al., (1984) Gene 30: 147-156).Well-known methods in the field of recombinant DNA technology are described in Chapters 12 and 13 of Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and Dracopoli et al. (eds.), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.

[0164] The expression levels of the bispecific antibodies or their polypeptide chains described in this article can be increased by vector amplification (see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987) for a review). When the marker in the vector system described in this article is amplifiable, increasing the level of the inhibitor in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the nucleotide sequence of the target protein, the yield of the target protein will also increase (Crouse et al., (1983) Mol. Cell. Biol. 3:257-266).

[0165] Host cells may be co-transfected with more than one expression vector, each vector encoding one polypeptide chain of the bispecific antibody described herein. The vectors may contain the same selectable marker, thereby allowing all polypeptides to be expressed in equal amounts. Alternatively, a single vector encoding two or more polypeptides may be used. The coding sequence of the polypeptide of the compounds described herein may include cDNA or genomic DNA.

[0166] Once the bispecific antibody or peptide described herein has been recombinantly expressed, it can be purified by any method known in the art for purifying peptides, multiproteins, or antibodies (e.g., similar to antigen-selectivity-based antibody purification protocols), such as by chromatography (e.g., ion exchange, affinity chromatography, especially by affinity for a specific antigen (optionally performed after Protein A screening when the compound contains an Fc domain (or a portion thereof), and molecular sieve column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying peptides or antibodies.

[0167] This document provides a method for preparing the bispecific antibody described herein or a polypeptide chain of the bispecific antibody described herein, the method comprising obtaining the cells described herein and expressing the polynucleotide described herein in the cells. In some embodiments, the method further comprises isolating and purifying the bispecific antibody or polypeptide chain described herein.

[0168] The bispecific antibody described herein can be detected, for example, by a standard ELISA, for its binding to human VEGF and / or PD-1 / PD-L1. Briefly, a microtiter plate is coated with purified antigen and then blocked with bovine serum albumin. Antibody dilution is added to each well and incubated. After washing, the plate is incubated with a secondary reagent conjugated to horseradish peroxidase (HRP) (e.g., for human antibodies, a goat anti-human IgG Fc-specific polyclonal reagent). After washing, color development can be performed and analyzed using a spectrophotometer. The antibody can also be further detected by flow cytometry for its binding to cell lines expressing human VEGF and / or PD-1 / PD-L1, without binding to control cell lines not expressing the target antigen. Briefly, antibody binding can be assessed by incubating VEGF-expressing CHO cells, PD-1-expressing 293T cells, and / or PD-L1-expressing CHO cells with the bispecific antibody provided herein. Cells can be washed, and binding can be detected using anti-human IgG Ab. Flow cytometry analysis can be performed using a FACS scanning flow cytometer (Becton Dickinson, San Jose, CA).

[0169] The bispecific antibodies described herein can also be detected for their reactivity with target antigens by Western blotting. Furthermore, other methods known in the art for analyzing the binding affinity, cross-reactivity, and binding kinetics of the various bispecific antibodies described herein include, for example, biolayer interferometry (BLI), such as using the Gator system (Probe Life) or the Octet-96 system (Sartorius AG), or BIACORE™ surface plasmon resonance (SPR) analysis using a BIACORE™ 2000SPR instrument (Biacore AB, Uppsala, Sweden).

[0170] Typically, the bioactivity of the bispecific antibodies disclosed herein can be characterized using any method used to test the bioactivity of agents that affect immune responses.

[0171] Unless otherwise stated, the present invention is practiced using conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields that are well-known to those skilled in the art. These techniques are described in the references cited herein and are fully elaborated therein. See, for example, Maniatis et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Borrebaeck (ed.) (1995); Each of the above references is incorporated herein by reference in its entirety. 6.5 Pharmaceutical Composition

[0172] This document also provides pharmaceutical compositions comprising the bispecific antibodies disclosed herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the bispecific antibody disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is useful in immunotherapy. In some embodiments, the pharmaceutical composition is useful in tumor immunotherapy. In some embodiments, the pharmaceutical composition is useful in inhibiting tumor growth in a subject (e.g., a human patient). In some embodiments, the pharmaceutical composition is useful in inhibiting tumor angiogenesis in a subject (e.g., a human patient). In some embodiments, the pharmaceutical composition is useful in treating cancer in a subject (e.g., a human patient).

[0173] The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refer to a material suitable for administration to an individual together with an active agent without causing undesirable biological effects and without interacting with any other component of the pharmaceutical composition in a harmful manner. In some embodiments, the pharmaceutical compositions disclosed herein may comprise one or more buffer systems, preservatives, isotonic agents, chelating agents, stabilizers, and / or surfactants, as well as various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those skilled in the art. See Remington: The Science and Practice of Pharmacy, 19 th edition, 1995.

[0174] In some embodiments, the pharmaceutical compositions provided herein comprise the bispecific antibody provided herein. Pharmaceutically acceptable carriers that can be used in the compositions provided herein include any and all physiologically compatible solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient (i.e., the bispecific antibody) may be encapsulated in a material to protect the active ingredient from acids and other natural conditions that can inactivate it.

[0175] This document also provides kits for preparing pharmaceutical compositions comprising the bispecific antibodies disclosed herein. In some embodiments, the kit comprises the bispecific antibody disclosed herein and a pharmaceutically acceptable carrier in one or more containers. In another embodiment, the kit may comprise the bispecific antibody disclosed herein for administration to a subject. In a particular embodiment, the kit includes instructions for the preparation and / or administration of the bispecific antibody disclosed herein.

[0176] In some embodiments, this document provides a pharmaceutical composition comprising a bispecific antibody disclosed herein, wherein the composition is suitable for local administration. In some embodiments, local administration includes intratumoral injection, peritumoral injection, adjacent tumor injection, intralesional injection and / or injection into tumor draining lymph nodes, or substantially any tumor-targeting injection, wherein the antitumor agent is expected to leak into the primary lymph node adjacent to the target solid tumor.

[0177] This document also provides pharmaceutical compositions or formulations that improve the stability of the bispecific antibodies disclosed herein to allow for long-term storage. In some embodiments, the pharmaceutical compositions or formulations disclosed herein include: (a) the bispecific antibody disclosed herein; (b) a buffer; (c) a stabilizer; (d) a salt; (e) a filler; and / or (f) a surfactant. In some embodiments, the pharmaceutical compositions or formulations are stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years, or longer. In some embodiments, the pharmaceutical compositions or formulations are stable when stored at 4°C, 25°C, or 40°C.

[0178] The buffers useful in the pharmaceutical compositions or formulations disclosed herein may be weak acids or weak bases used to maintain the acidity (pH) of the solution at a near-selected value after the addition of another acid or base. Suitable buffers can maximize the stability of the pharmaceutical formulation by maintaining pH control. Suitable buffers can also ensure physiological compatibility or optimize solubility. Rheological properties, viscosity, and other properties may also depend on the pH of the formulation. Common buffers include, but are not limited to, histidine, citrate, succinate, acetate, and phosphate. In some embodiments, the buffer comprises histidine (e.g., L-histidine) with an isotonic agent, and pH adjustment can be performed using acids or bases known in the art. In some embodiments, the buffer is L-histidine. In some embodiments, the pH of the formulation is maintained between about 2 and about 10, or between about 4 and about 8.

[0179] Stabilizers are added to pharmaceutical products to stabilize them. These agents can stabilize proteins in various ways. Common stabilizers include, but are not limited to, amino acids (e.g., glycine, alanine, lysine, arginine, or threonine), carbohydrates (e.g., glucose, sucrose, trehalose, raffinose, or maltose), polyols (e.g., glycerol, mannitol, sorbitol, cyclodextrin, or any type and molecular weight of dextran), or PEG. In some embodiments, stabilizers are selected to maximize the stability of the FIX peptide in the lyophilized formulation. In some embodiments, the stabilizer is sucrose and / or arginine.

[0180] Fillers can be added to pharmaceutical compositions or formulations to increase the volume and mass of the product, thereby facilitating its precise measurement and handling. Common fillers include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

[0181] Surfactants are amphiphilic substances having both soluble and soluble groups. Surfactants can be anionic, cationic, amphoteric, or nonionic. Examples of nonionic surfactants include, but are not limited to, alkyl ethoxylates, nonylphenol ethoxylates, amine ethoxylates, polyethylene oxide, polypropylene oxide, fatty alcohols (e.g., cetyl alcohol or oleyl alcohol), cocoamide MEA, cocoamide DEA, polysorbate, or dodecyl dimethylamine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0182] The pharmaceutical compositions disclosed herein may further comprise one or more buffer systems, preservatives, isotonic agents, chelating agents, stabilizers, and / or surfactants, as well as various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those skilled in the art. See Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0183] In some embodiments, the pharmaceutical composition is an aqueous formulation. Such formulations are typically solutions or suspensions, but may also include colloids, dispersions, emulsions, and multiphase materials. The term "aqueous formulation" is defined as a formulation containing at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution containing at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension containing at least 50% w / w water.

[0184] In some embodiments, the pharmaceutical compositions disclosed herein are lyophilized preparations, which are added by a physician or patient with solvents and / or diluents prior to use.

[0185] The pharmaceutical compositions disclosed herein may also include pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0186] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions or formulations described herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). For example, appropriate flowability can be maintained by using coating materials (e.g., lecithin), maintaining the desired particle size in the case of dispersions, and using surfactants.

[0187] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. The presence of microorganisms can be prevented by the sterilization procedures described above and by the addition of various antimicrobial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). It may also be desirable to add isotonic agents (e.g., sugars, sodium chloride, etc.) to the compositions. Furthermore, the absorption of injectable drug formulations can be prolonged by adding absorption-retarding agents (e.g., aluminum monostearate and gelatin).

[0188] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, as well as sterile powders for the preparation of sterile injectable solutions or dispersions. The use of such media and reagents is known in the art for pharmaceutically active substances. In some embodiments, this document provides a pharmaceutical composition comprising the bispecific antibody or cells provided herein, wherein said composition is suitable for topical administration.

[0189] Pharmaceutical compositions or formulations must generally remain sterile and stable under manufacturing and storage conditions. The compositions can be prepared as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium, such as comprising water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. For example, appropriate flowability can be maintained by using coatings such as lecithin, maintaining the desired particle size in the case of dispersions, and using surfactants. In many cases, the composition may include isotonic agents such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride. The absorption of injectable compositions can be prolonged by adding absorption-retarding agents (e.g., monostearate and gelatin).

[0190] A sterile injectable solution can be prepared by incorporating the desired amount of the active compound into a suitable solvent, adding one or more of the components listed above as needed, followed by sterile microfiltration. Typically, a dispersion is prepared by incorporating the active compound into a sterile carrier containing a basic dispersion medium and any other desired components listed herein. For sterile powders used to prepare sterile injectable solutions, some preparation methods include vacuum drying and lyophilization (freeze-drying), which produce a powder containing the active ingredient and any additional desired components from a previously sterile filtered solution.

[0191] In the pharmaceutical compositions or formulations disclosed herein, the amount of active ingredient that can bind to a carrier material can vary. In some embodiments, the amount of active ingredient that can bind to a carrier material is the amount that produces a therapeutic effect. Typically, this amount, in percentage terms, will be about 0.01% to about 99% of the active ingredient, about 0.1% to about 70% of the active ingredient, or about 1% to about 30% of the active ingredient bound to a pharmaceutically acceptable carrier.

[0192] The pharmaceutical compositions disclosed herein can be prepared using carriers that protect the active ingredient from rapid release, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers can be used, such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyortho-esters, and polylactic acid. Many methods for preparing such formulations are patented or are well known to those skilled in the art. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, ed. JR Robinson, Marcel Dekker, Inc., New York, 1978.

[0193] This document also provides kits for preparing pharmaceutical compositions comprising the anti-VEGF and anti-PD-1 / PD-L1 bispecific antibodies disclosed herein. In some embodiments, the kit comprises the anti-VEGF and anti-PD-1 / PD-L1 bispecific antibodies disclosed herein and a pharmaceutically acceptable carrier in one or more containers. In another embodiment, the kit may comprise the anti-VEGF and anti-PD-1 / PD-L1 bispecific antibodies disclosed herein for administration to a subject. In a particular embodiment, the kit comprises instructions for the preparation and / or administration of the anti-VEGF and anti-PD-1 / PD-L1 bispecific antibodies. 6.6 Methods and Applications

[0194] The bispecific antibody compositions and methods described herein have a variety of in vitro and in vivo uses, such as those relating to enhancing immune responses, for example by inhibiting (or antagonizing) PD-1 / PD-L1-mediated immunosuppression, inhibiting tumor angiogenesis, and / or treating cancer.

[0195] In some embodiments, the bispecific antibodies described herein can be administered to cells in culture (in vitro or ex vivo) or to human subjects (e.g., in vivo) to enhance immunity in a variety of diseases. Therefore, this document provides a method for modulating an immune response in a subject, comprising administering the bispecific antibody or pharmaceutical composition described herein to the subject, thereby modulating the immune response in the subject. In some embodiments, the response is enhanced, stimulated, or upregulated.

[0196] In some embodiments, this document provides a method for inducing or stimulating immune cell activation using an effective amount of the bispecific antibody described herein. In some embodiments, this document provides a method for inducing or stimulating immune cell proliferation using an effective amount of the bispecific antibody described herein. In some embodiments, this document provides a method for reducing PD-1 / PD-L1-mediated inhibition of immune cell proliferation and / or activation using an effective amount of the bispecific antibody described herein. In some embodiments, this document provides a method for inhibiting the interaction between PD-1 and PD-L1 on tumor cells using an effective amount of the bispecific antibody described herein.

[0197] In some embodiments, this document provides a method for reducing VEGF-mediated angiogenesis using an effective amount of the bispecific antibody described herein. In some embodiments, this document provides a method for inhibiting the interaction between VEGF and VEGF receptors on tumor cells using an effective amount of the bispecific antibody described herein.

[0198] In some embodiments, this document provides a method for increasing the production of cytokines (e.g., IFN-γ) by immune cells, which includes contacting the immune cells with an effective amount of the bispecific antibody described herein.

[0199] The immune cells may be, for example, T cells, such as CD4 cells. + T cells, CD8 + T cells, helper T (Th) cells (e.g., Th1 cells), cytotoxic T (Tc) cells, or TILs. The immune cells may also be NK cells, NKT cells, or myeloid cells. The myeloid cells may be macrophages. The myeloid cells may be dendritic cells.

[0200] Subjects suitable for this method include human patients who wish to enhance their immune response. The method is particularly suitable for treating human patients with diseases treatable by enhancing immune responses, such as T-cell-mediated immune responses, or antigen-specific T-cell responses. In some embodiments, the method is particularly suitable for in vivo treatment of cancer. To achieve antigen-specific immune enhancement, the bispecific antibody described herein may be administered together with the target antigen, or the antigen may already be present in the subject to be treated (e.g., a subject carrying a tumor or a virus). When the bispecific antibody disclosed herein is administered together with another agent, the two may be administered separately or simultaneously.

[0201] Given that the bispecific antibodies disclosed herein can stimulate or co-stimulate T cell responses, such as antigen-specific T cell responses, for example by inhibiting the negative effects of PD-1 / PD-L1, this article provides methods for stimulating, enhancing, or upregulating antigen-specific T cell responses (e.g., anti-tumor T cell responses) using the bispecific antibodies disclosed herein in vitro and in vivo.

[0202] In some embodiments, CD3 stimulation is also provided (e.g., by co-incubation with cells expressing membrane CD3), which can be provided simultaneously, before, or after stimulation with the bispecific antibody disclosed herein. For example, this document provides a method for stimulating an antigen-specific T cell response, comprising contacting the T cells with the bispecific antibody described herein and optionally with an anti-CD3 antibody, thereby stimulating an antigen-specific T cell response.

[0203] Antigen-specific T-cell responses can be measured using any suitable indicator of antigen-specific T-cell response. Non-limiting examples of such suitable indicators include increased T-cell proliferation in the presence of the bispecific antibody and / or increased cytokine production in the presence of the bispecific antibody. In some embodiments, antigen-specific T cells are stimulated to produce interleukin-2 and / or interferon-γ.

[0204] It also covers a method for stimulating an immune response (e.g., an antigen-specific T-cell response) in a subject, comprising administering the bispecific antibody described herein to the subject, thereby stimulating an immune response (e.g., an antigen-specific T-cell response) in the subject. In some embodiments, the subject is a tumor-bearing subject, and an immune response against the tumor is stimulated. The tumor may be a solid tumor or a liquid tumor, such as a hematologic malignancy. In some embodiments, the tumor is an immunogenic tumor. In some embodiments, the tumor is a non-immunogenic tumor. In some embodiments, the tumor is VEGF-positive. In some embodiments, the tumor is PD-L1-positive. The subject may also be a virus-bearing subject, and an immune response against the virus is stimulated.

[0205] A method for inhibiting tumor cell growth in a subject is also provided, comprising administering the bispecific antibody described herein to the subject, thereby inhibiting tumor growth in the subject. A method for treating a viral infection in a subject is also provided, comprising administering the bispecific antibody described herein to the subject, thereby treating the viral infection in the subject.

[0206] This disclosure also provides methods of using the bispecific antibodies disclosed herein, polynucleotides encoding such bispecific antibodies, carriers comprising such polynucleotides, or pharmaceutical compositions containing such bispecific antibodies in the treatment of cancer. In some embodiments, the bispecific antibody is capable of specifically targeting VEGF-expressing cancer cells in vivo, thereby inhibiting tumor angiogenesis and tumor growth. In some embodiments, the bispecific antibody is capable of specifically targeting PD-L1-expressing cancer cells in vivo, thereby delivering its therapeutic effects of eliminating, lysing, and / or killing cancer cells. In some embodiments, the bispecific antibody is capable of reducing immunosuppression mediated by the PD-1 / PD-L1 signaling pathway, thereby promoting the activity of immune cells in eliminating, lysing, and / or killing cancer cells.

[0207] In some embodiments, the method includes administering a therapeutically effective amount of the bispecific antibody disclosed herein to a subject in need. In some embodiments, this document provides a method for treating a tumor or cancer in a subject in need, comprising administering a therapeutically effective amount of the bispecific antibody disclosed herein to the subject. In some embodiments, this document provides the use of the bispecific antibody disclosed herein in the treatment of tumors or cancer. In some embodiments, this document provides the use of the bispecific antibody provided herein in the preparation of a medicament for treating tumors or cancer. In some embodiments, this document provides a method for treating a tumor or cancer in a subject in need, comprising administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to the subject. In some embodiments, this document provides the use of the pharmaceutical composition disclosed herein in the treatment of tumors or cancer. In some embodiments, this document provides the use of the pharmaceutical composition disclosed herein in the preparation of a medicament for treating tumors or cancer.

[0208] In some embodiments, the bispecific antibodies disclosed herein do not exhibit significant toxicity. For example, as determined, for instance, in clinical trials, the bispecific antibodies disclosed herein do not exhibit significant toxicity to human organs, such as the liver, kidneys, brain, lungs, and heart. In some embodiments, the bispecific antibodies disclosed herein do not significantly trigger undesirable immune responses, such as autoimmunity or inflammation.

[0209] In some embodiments, treatment of a subject with the bispecific antibody disclosed herein does not cause overstimulation of the immune system to the point that the subject's immune system subsequently attacks the subject itself (e.g., an autoimmune response) or, for example, causes anaphylactic shock. Therefore, in some embodiments, the bispecific antibody provided herein does not cause anaphylactic shock.

[0210] In some embodiments, treatment of subjects with the bispecific antibodies described herein does not induce significant inflammatory responses, such as immune-mediated pneumonia, immune-mediated colitis, immune-mediated hepatitis, immune-mediated nephritis or renal dysfunction, immune-mediated hypopituitarism, immune-mediated hypothyroidism and hyperthyroidism, or other immune-mediated adverse reactions. In some embodiments, the bispecific antibodies provided herein induce limited inflammatory responses, such as immune-mediated pneumonia, immune-mediated colitis, immune-mediated hepatitis, immune-mediated nephritis or renal dysfunction, immune-mediated hypopituitarism, immune-mediated hypothyroidism and hyperthyroidism, anaphylactic shock, or other immune-mediated adverse reactions. In some implementations, treatment of subjects with the bispecific antibodies disclosed herein does not cause significant cardiac disease, such as ventricular arrhythmias; eye diseases, such as iridocyclitis; infusion-related reactions; elevated amylase or lipase levels; neurological disorders, such as dizziness, peripheral neuropathy, and sensory neuropathy; skin and subcutaneous tissue disorders, such as rash, pruritus, exfoliative dermatitis, erythema multiforme, vitiligo, or psoriasis; respiratory, thoracic, and mediastinal disorders, such as cough; fatigue; nausea; decreased appetite; constipation; arthralgia; or diarrhea.

[0211] The actual dose level of the active ingredient (i.e., the bispecific antibody described herein) in the pharmaceutical compositions described herein can be varied to obtain an amount of active ingredient effective for a specific patient, composition, and route of administration to achieve the desired therapeutic response without causing toxicity to the patient. The selected dose level will depend on a variety of pharmacokinetic factors, including the activity of the specific composition described herein, route of administration, time of administration, excretion rate, duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition used, the age, sex, weight, condition, general health status, and medical history of the patient being treated, as well as similar factors known in the medical field.

[0212] This bispecific antibody can be administered in a sustained-release formulation, in which case a lower frequency of dosing is required. The dosage and frequency vary depending on the half-life of the bispecific antibody in the patient. In therapeutic applications, sometimes a relatively high dose needs to be administered at relatively short intervals until disease progression is reduced or stopped, and until the patient shows partial or complete remission of disease symptoms.

[0213] The bispecific antibody or pharmaceutical composition provided herein may be administered to a subject by any method known in the art, including but not limited to pleural administration, intravenous administration, subcutaneous administration, intralymphatic administration, intratumoral administration, intramuscular administration, intradermal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, intracranial administration, spinal administration, or other parenteral routes, such as by injection or infusion, or direct administration to the thymus. The term "parenteral administration" as used herein refers to a route of administration other than enteral and local administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subcutaneous layer, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. In some embodiments, subcutaneous administration is used. In some embodiments, intravenous administration is used. In some embodiments, oral administration is used. In one embodiment, the bispecific antibody provided herein can be regionally delivered to a tumor using known methods, including but not limited to hepatic or aortic pumps; limb, lung, or liver perfusion; via portal vein; via venous shunt; intracavitary or intravenous delivery near the tumor, etc. In another embodiment, the bispecific antibody provided herein can be administered systemically. In some embodiments, the bispecific antibody is administered regionally at the tumor site. The bispecific antibody can also be administered intratumorally, for example by direct injection at the tumor site and / or into the tumor vascular system. For example, in the case of malignant pleural diseases, mesothelioma, or lung cancer, intrapleural administration is preferred (see Adusumilli et al., Science Translational Medicine 6(261):261ra151 (2014)). Those skilled in the art can select an appropriate route of administration based on the type of cancer to be treated and / or the location of the tumor. The bispecific antibody can be introduced by injection or catheter. In one embodiment, the bispecific antibody is administered pleurally to a subject in need, for example using an intrapleural catheter.

[0214] Cancers or tumors treated with the bispecific antibody or pharmaceutical compositions provided herein include cancers that typically respond to immunotherapy and cancers that typically do not respond to immunotherapy. Treatable cancers also include VEGF-positive cancers. Treatable cancers also include PD-L1-positive cancers. In some embodiments, the cancer exhibits high microsatellite instability.

[0215] In some embodiments, the cancers or tumors treatable with the bispecific antibodies or pharmaceutical compositions disclosed herein are hematologic malignancies. In some embodiments, the cancers or tumors treatable with the bispecific antibodies or pharmaceutical compositions disclosed herein are solid tumors. In some embodiments, the cancers or tumors treatable with the bispecific antibodies or pharmaceutical compositions disclosed herein include hepatocellular carcinoma, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), renal cell carcinoma, endometrial cancer, cervical cancer, colorectal cancer, breast cancer (e.g., triple-negative breast cancer), biliary tract cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, gynecological tumors, thymic malignancies, and other solid tumors. In some embodiments, the bispecific antibodies or pharmaceutical compositions provided herein can treat non-small cell lung cancer (NSCLC). In some embodiments, the bispecific antibodies or pharmaceutical compositions provided herein can treat renal cell carcinoma (RCC). In some embodiments, the bispecific antibodies or pharmaceutical compositions provided herein can treat hepatocellular carcinoma (HCC). In some embodiments, the bispecific antibodies or pharmaceutical compositions provided herein can treat endometrial cancer. In some embodiments, the bispecific antibodies or pharmaceutical compositions provided herein can treat cervical cancer.

[0216] In cancer treatment, cancer or tumor cells in a subject may be eliminated, but any clinical improvement constitutes a benefit. Antitumor effects can be manifested by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with the cancer state. Antitumor effects can also be manifested by the ability of the bispecific antibodies or pharmaceutical compositions provided herein to prevent tumorigenesis in the first place. In some embodiments, an “antitumor effect” can be manifested by a reduction in cancer-induced immunosuppression. Clinical improvement includes a reduction in the risk or rate of progression or a reduction in the pathological consequences of the cancer or tumor. It should also be understood that methods of treating cancer may include any effect that improves cancer-related signs or symptoms. Such signs or symptoms include, but are not limited to, a reduction in tumor burden, including inhibition of tumor growth, slowing of tumor growth rate, reduction of tumor size, reduction of tumor number, and elimination of tumor, all of which can be measured using conventional tumor imaging techniques known in the art. Other cancer-related signs or symptoms include, but are not limited to, fatigue, pain, weight loss, and other signs or symptoms associated with various cancers.

[0217] In some embodiments, the methods or uses provided herein can reduce tumor burden. Therefore, administration of the bispecific antibody or pharmaceutical composition disclosed herein can reduce the number of tumor cells in a subject, reduce tumor size, and / or eradicate the tumor. Methods for monitoring a patient's response to administration of the bispecific antibody or pharmaceutical composition disclosed herein are known in the art and can be employed in accordance with the methods disclosed herein. In some embodiments, treatment of a subject with cancer using the bispecific antibody or pharmaceutical composition disclosed herein can result in, for example, disease stabilization, partial remission, increased overall survival, increased disease-free survival, or improved progression-free survival.

[0218] In some embodiments, antitumor effects have been observed in subjects with tumors or cancer who have been treated with the bispecific antibody or pharmaceutical composition described herein as a monotherapy (i.e., not in combination with another treatment). In some embodiments, tumor burden has been reduced in subjects with tumors or cancer who have been treated with the bispecific antibody or pharmaceutical composition described herein as a monotherapy (i.e., not in combination with another treatment).

[0219] In the methods disclosed herein, a therapeutically effective amount of the bispecific antibody or pharmaceutical composition disclosed herein is administered to a subject in need of cancer treatment. The subject may be a mammal. In some embodiments, the subject is a human. In some embodiments, such individuals do not have clinically measurable tumors. However, they are suspected of having a risk of disease progression, either near the original tumor site or through metastasis. This group can be further subdivided into high-risk and low-risk individuals. This subdivision is based on characteristics observed before or after initial treatment. These characteristics are known in the clinical field and are appropriately defined for different types of cancer. A typical characteristic of the high-risk subgroup is that the tumor has invaded adjacent tissues or shows lymph node involvement.

[0220] The bispecific antibody or pharmaceutical compositions provided herein can be administered in conjunction with medical devices known in the art. For example, in some embodiments, needle-free subcutaneous injection devices, such as those disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556, can be used. Examples of known implants and modules for the uses described herein include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for transdermal drug administration; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are also known to those skilled in the art.

[0221] In some embodiments, a method of treating cancer in a subject includes first determining whether the subject is VEGF-positive, for example, the subject has tumor cells or TILs expressing VEGF, and if the subject has VEGF-positive cancer or TIL cells, administering the bispecific antibody or pharmaceutical composition described herein to the subject. A method of treating a subject with cancer using the bispecific antibody or pharmaceutical composition described herein may include administering a therapeutically effective amount of the bispecific antibody or pharmaceutical composition described herein to a subject having cancer cells or TIL cells expressing VEGF. This document also provides a method for predicting whether a subject will respond to treatment using the bispecific antibody or pharmaceutical composition described herein, wherein the method includes determining the VEGF level in the patient's cancer cells or TIL cells, and if the subject's cancer cells or TIL cells are VEGF-positive, the subject may respond to treatment using the bispecific antibody or pharmaceutical composition described herein.

[0222] In some embodiments, a method of treating cancer in a subject includes first determining whether the subject is PD-1 / PD-L1 positive, for example, that the subject has tumor cells or TILs expressing PD-1 or PD-L1, and if the subject has PD-1 / PD-L1 positive cancer or TIL cells, administering the bispecific antibody or pharmaceutical composition described herein to the subject. A method of treating a subject with cancer using the bispecific antibody or pharmaceutical composition described herein may include administering a therapeutically effective amount of the bispecific antibody or pharmaceutical composition described herein to a subject having cancer cells or TIL cells expressing PD-1 / PD-L1. This document also provides a method for predicting whether a subject will respond to treatment using the bispecific antibody or pharmaceutical composition described herein, wherein the method includes determining the PD-1 / PD-L1 level in a patient's cancer cells or TIL cells, and if the subject's cancer cells or TIL cells are PD-1 / PD-L1 positive, the subject may respond to treatment using the bispecific antibody or pharmaceutical composition described herein.

[0223] In some embodiments, the cancer treated by the bispecific antibody or pharmaceutical composition disclosed herein is a VEGF-expressing cancer (i.e., a VEGF-positive cancer). In some embodiments, the cancer treated by the bispecific antibody or pharmaceutical composition disclosed herein is a PD-L1-expressing cancer (i.e., a PD-L1-positive cancer).

[0224] The bispecific antibody or pharmaceutical composition described herein can be administered concurrently with standard treatment. The bispecific antibody or pharmaceutical composition described herein can also be administered as maintenance therapy, for example, in treatments aimed at preventing tumor development or recurrence.

[0225] The bispecific antibody or pharmaceutical composition described herein can be administered concurrently with another treatment, such as radiotherapy, surgery, or chemotherapy. For example, the bispecific antibody or pharmaceutical composition described herein can be administered as adjunctive therapy when there is a risk of micrometastases and / or to reduce the risk of recurrence. This adjunctive therapy can be administered before, simultaneously with, or after the administration of the bispecific antibody or pharmaceutical composition described herein. Co-administration can include co-administration (whether in a single pharmaceutical formulation or using separate formulations) or sequential administration in any order, but generally aims to allow all active agents to exert their biological activity simultaneously over a period of time. Those skilled in the art can readily determine appropriate regimens for co-administering the bispecific antibody or pharmaceutical composition described herein with adjunctive therapy based on the needs of the treated subject, including the timing and dosage of the adjunct for co-administration. 6.7 Reagent Kit

[0226] In some embodiments, this disclosure provides a variety of kits to facilitate and / or effectively implement the methods of this disclosure. In some embodiments, this document provides kits comprising the anti-VEGF / PD-(L)1 bispecific antibodies (e.g., HX016-07, HX016-08, HX016-09, HX016-010) disclosed herein. In some embodiments, this document provides kits comprising anti-VEGF / PD-(L)1 bispecific antibodies (e.g., HX016-07, HX016-08, HX016-09, HX016-010) for cancer treatment. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is hepatocellular carcinoma, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), renal cell carcinoma, endometrial cancer, cervical cancer, colorectal cancer, breast cancer (e.g., triple-negative breast cancer), biliary tract cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, gynecological tumors, thymic malignancies, and other solid tumors.

[0227] Typically, the kit will include sufficient amounts and / or quantities of components to enable the user to perform multiple treatments and / or multiple experiments on one or more subjects.

[0228] Any pharmaceutical composition disclosed herein may be included in the kit. In some embodiments, the kit may further include reagents and / or instructions for preparing and / or synthesizing the compounds and / or pharmaceutical compositions disclosed herein. In some embodiments, the kit may also include one or more buffer solutions.

[0229] In some embodiments, the kit components may be packaged in an aqueous medium or lyophilized form. The kit's container device will typically include at least one vial, tube, flask, bottle, syringe, or other container device into which the components can be placed and suitably aliquoted. When more than one kit component is present (labeling reagents and labels may be packaged together), the kit may also include second, third, or other additional containers into which additional components can be placed separately. In some embodiments, the kit may also include a second container device for containing sterile, pharmaceutically acceptable buffers and / or other diluents. In some embodiments, various component combinations may be contained in one or more vials. The kits of this disclosure may also typically include means for containing the compounds and / or pharmaceutical compositions (e.g., proteins) of this disclosure, as well as any other reagent containers, for close packaging for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the desired vials are held.

[0230] In some embodiments, the reagent kit components are provided in the form of one and / or more liquid solutions. In some embodiments, the liquid solution is an aqueous solution, particularly a sterile aqueous solution. In some embodiments, the reagent kit components may be provided in the form of a dry powder. When the reagents and / or components are provided in dry powder form, the powder can be reconstituted by adding an appropriate volume of solvent. In some embodiments, it is also envisioned that the solvent may also be provided in a separate container.

[0231] In some implementations, the kit may include instructions for using the kit components, as well as instructions for using any other reagents not included in the kit. These instructions may include implementable variations. 6.8 Exemplary Implementation Scheme

[0232] Implementation Scheme 1. A bispecific antibody comprising a first light chain variable domain (VL1), a first heavy chain variable domain (VH1), a second light chain variable domain (VL2), and a second heavy chain variable domain (VH2); wherein (1) VL1 / VH1 specifically binds to human VEGF, and wherein VL1 comprises VLCDR1, VL CDR2, and VL CDR3 of a reference VL having the amino acid sequence of SEQ ID NO:21, or variants thereof, having up to about 5 amino acid substitutions, additions, and / or deletions in said VL CDRs; and VH1 comprises VH CDR1, VH CDR2, and VH CDR3 of a reference VH having the amino acid sequence of SEQ ID NO:22, or variants thereof, having up to about 5 amino acid substitutions, additions, and / or deletions in said VH CDRs; and (2) VL2 / VH2 specifically binds to human PD-L1 or human PD-1.

[0233] Implementation Scheme 2. The bispecific antibody of Implementation Scheme 1, wherein VL1 comprises VL CDR1, VL CDR2 and VL CDR3 having amino acid sequences of SEQ ID NOs:1, 2 and 3 respectively; and VH1 comprises VH CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NOs:4, 5 and 6 respectively.

[0234] Implementation Scheme 3. The bispecific antibody of Implementation Scheme 2, wherein the amino acid sequences of VL1 and VH1 independently have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown in SEQ ID NO:21 and SEQ ID NO:22, respectively.

[0235] Implementation Scheme 4. A bispecific antibody from any one of Implementation Schemes 1 to 3, wherein VL2 / VH2 is derived from antibodies selected from the group consisting of: putelimab, nivolumab, pembrolizumab, cimiprimab, dotalimumab, retivalimumab, toripalimab, tislelizumab, camrelizumab, sintilimab, penaplimumab, cepalimumab, slulimumab, fenolinumab, durvalumab, atezolizumab, avelumab, cosibelimab, sugmalimab, envorimab, adebenone, socazolimumab, and tagorimab.

[0236] Implementation Scheme 5. A bispecific antibody of any one of Implementation Schemes 1 to 3, wherein VL2 / VH2 specifically binds to human PD-L1; wherein VL2 comprises VL CDR1, VL CDR2 and VLCDR3 of a reference VL having the amino acid sequence of SEQ ID NO:67, or a variant thereof, having up to about 5 amino acid substitutions, additions and / or deletions in said VL CDRs; and VH2 comprises VH CDR1, VH CDR2 and VH CDR3 of a reference VH having the amino acid sequence of SEQ ID NO:68, or a variant thereof, having up to about 5 amino acid substitutions, additions and / or deletions in said VH CDRs.

[0237] Implementation Scheme 6. The bispecific antibody of Implementation Scheme 5, wherein VL2 comprises VL CDR1, VL CDR2 and VL CDR3 having amino acid sequences of SEQ ID NOs:7, 8 and 9, respectively; and VH2 comprises VH CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NOs:10, 11 and 12, respectively.

[0238] Implementation Scheme 7. The bispecific antibody of Implementation Scheme 6, wherein the amino acid sequences of VL2 and VH2 independently have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the following: (1) SEQ ID NOs:23 and 24, respectively; (2) SEQ ID NOs:23 and 68, respectively; (3) SEQ ID NOs:67 and 24, respectively; or (4) SEQ ID NOs:67 and 68, respectively.

[0239] Implementation Scheme 8. The bispecific antibody of Implementation Scheme 7, wherein VL1 and VH1 have amino acid sequences of SEQ ID NOs:21 and 22; and VL2 and VH2 have amino acid sequences of SEQ ID NOs:23 and 24, respectively.

[0240] Implementation Scheme 9. A bispecific antibody of any one of Implementation Schemes 1 to 3, wherein VL2 / VH2 specifically binds to human PD-1, and wherein VL2 comprises VL CDR1, VL CDR2 and VL CDR3 of a reference VL having the amino acid sequence of SEQ ID NO:69, or a variant thereof, having up to about 5 amino acid substitutions, additions and / or deletions in said VL CDRs; and VH2 comprises VH CDR1, VH CDR2 and VH CDR3 of a reference VH having the amino acid sequence of SEQ ID NO:70, or a variant thereof, having up to about 5 amino acid substitutions, additions and / or deletions in said VH CDRs.

[0241] Implementation Scheme 10. The bispecific antibody of Implementation Scheme 9, wherein VL2 comprises VL CDR1, VL CDR2 and VL CDR3 having amino acid sequences of SEQ ID NOs:13, 14 and 15, respectively; and VH2 comprises VH CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NOs:16, 17 and 18, respectively.

[0242] Implementation Scheme 11. The bispecific antibody of Implementation Scheme 10, wherein the amino acid sequences of VL2 and VH2 independently have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the following: (1) SEQ ID NOs:25 and 26, respectively; (2) SEQ ID NOs:25 and 70, respectively; (3) SEQ ID NOs:69 and 26, respectively; or (4) SEQ ID NOs:69 and 70, respectively.

[0243] Implementation Scheme 12. The bispecific antibody as described in Implementation Scheme 11, wherein VL1 and VH1 have amino acid sequences of SEQ ID NOs:21 and 22; and VL2 and VH2 have amino acid sequences of SEQ ID NOs:25 and 26, respectively.

[0244] Implementation Scheme 13. A bispecific antibody as described in any one of Implementation Schemes 1 to 12, comprising: (i) a first peptide chain (HC) comprising, from the N-terminus to the C-terminus, VH1, a heavy chain constant (CH) region, a linker, and a single-chain variable fragment (scFv), wherein the scFv comprises VL2 and VH2; and (ii) a second peptide chain (LC) comprising, from the N-terminus to the C-terminus, VL1 and a light chain constant (CL) region.

[0245] Implementation Scheme 14. The bispecific antibody as described in Implementation Scheme 13, wherein the linker has an amino acid sequence selected from the group consisting of SEQ ID NOs:51-55.

[0246] Implementation Scheme 15. The bispecific antibody as described in Implementation Scheme 13 or 14, wherein the scFv comprises VL2, a second linker, and VH2 sequentially from the N-terminus to the C-terminus.

[0247] Implementation Scheme 16. The bispecific antibody as described in Implementation Scheme 13 or 14, wherein the scFv comprises VH2, a second linker, and VL2 sequentially from the N-terminus to the C-terminus.

[0248] Implementation Scheme 17. The bispecific antibody as described in Implementation Scheme 15 or 16, wherein the second linker has an amino acid sequence selected from the group consisting of SEQ ID NOs:51-55.

[0249] Implementation Scheme 18. A bispecific antibody as described in any one of Implementation Schemes 13 to 17, wherein (1) the CL region is Cκ (SEQ ID NO:29) or Cλ (SEQ ID NO:30), or a variant thereof having up to ten amino acid substitutions, additions and / or deletions; or (2) the CH region is a human IgG1 CH region (SEQ ID NO:31), IgG2 CH region (SEQ ID NO:32), IgG3 CH region (SEQ ID NO:33) or IgG4 CH region (SEQ ID NO:34), or a variant thereof having up to ten amino acid substitutions, additions and / or deletions; or both (1) and (2) are satisfied.

[0250] Implementation Scheme 19. The bispecific antibody as described in Implementation Scheme 18, wherein the CL region is Cκ (SEQ ID NO: 29).

[0251] Implementation Scheme 20. A bispecific antibody as described in Implementation Scheme 18 or 19, wherein the CH region is a human IgG1 CH region with L234A and L235A substitutions.

[0252] Implementation Scheme 21. The bispecific antibody as described in Implementation Scheme 20, wherein the CH region further has an M252Y, S254T or T256E substitution, or any combination thereof.

[0253] Implementation Scheme 22. The bispecific antibody as described in Implementation Scheme 21, wherein the CH region has L234A, L235A, M252Y, S254T and T256E substitutions.

[0254] Implementation Scheme 23. The bispecific antibody as described in Implementation Scheme 13, wherein the amino acid sequence of HC has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NOs:61-64, and the amino acid sequence of LC has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:60.

[0255] Implementation Scheme 24. The bispecific antibody as described in Implementation Scheme 23, wherein the HC and LC have amino acid sequences of SEQ ID NOs:61 and 60, respectively.

[0256] Implementation Scheme 25. The bispecific antibody as described in Implementation Scheme 23, wherein the HC and LC have amino acid sequences of SEQ ID NOs:62 and 60, respectively.

[0257] Implementation Scheme 26. The bispecific antibody as described in Implementation Scheme 23, wherein the HC and LC have amino acid sequences of SEQ ID NOs:63 and 60, respectively.

[0258] Implementation Scheme 27. The bispecific antibody as described in Implementation Scheme 23, wherein the HC and LC have amino acid sequences of SEQ ID NOs:64 and 60, respectively.

[0259] Implementation Scheme 28. The bispecific antibody as described in Implementation Scheme 23, wherein the HC has an amino acid sequence selected from the group consisting of SEQ ID NOs:56-59, and the LC has an amino acid sequence consisting of SEQ ID NO:60.

[0260] Implementation Scheme 29. A bispecific antibody as described in any one of Implementation Schemes 1 to 28, wherein the bispecific antibody (1) inhibits VEGF signaling; (2) inhibits vascular endothelial cell proliferation; (3) inhibits tumor angiogenesis; (4) blocks PD-1 / PD-L1 binding; (5) reduces immunosuppression; or (6) promotes T cell activation and / or proliferation; or any combination of (1)-(6).

[0261] Implementation Scheme 30. A pharmaceutical composition comprising a therapeutically effective amount of any one of Implementation Schemes 1 to 29, and a pharmaceutically acceptable carrier.

[0262] Implementation Scheme 31. A polynucleotide encoding a peptide chain of a bispecific antibody as described in any one of Implementation Schemes 1 to 29.

[0263] Implementation Scheme 32. The polynucleotide as described in Implementation Scheme 31, which encodes the two peptide chains of the bispecific antibody.

[0264] Implementation Scheme 33. A plurality of polynucleotides as described in Implementation Scheme 31, which collectively encode the two peptide chains of the bispecific antibody.

[0265] Implementation Scheme 34. A vector comprising the polynucleotide described in Implementation Scheme 31 or 32.

[0266] Implementation Scheme 35. A cell comprising one or more polynucleotides as described in any one of Implementation Schemes 31 to 33, or the vector described in Implementation Scheme 34.

[0267] Implementation Scheme 36. A method for preparing a bispecific antibody that specifically binds to human VEGF and human PD-1 / PD-L1, comprising culturing the cells described in Implementation Scheme 35 under conditions that allow expression of the bispecific antibody.

[0268] Implementation Scheme 37. The method as described in Implementation Scheme 36, comprising isolating the bispecific antibody from the culture.

[0269] Implementation Scheme 38. A method for reducing immune cell suppression, comprising contacting the immune cells with an effective amount of the bispecific antibody according to any one of Implementation Schemes 1 to 29.

[0270] Implementation Scheme 39. The method as described in Implementation Scheme 38, wherein the immune cells are T cells, NK cells, NKT cells, or myeloid cells.

[0271] Implementation Scheme 40. The method as described in Implementation Scheme 39, wherein the immune cells are T cells.

[0272] Implementation Scheme 41. A method for inducing or stimulating the activation and / or proliferation of immune cells in a subject in need, comprising administering to the subject an effective amount of the bispecific antibody as described in any one of Implementation Schemes 1 to 29.

[0273] Implementation Scheme 42. A method for inhibiting cancer angiogenesis in a subject in need, comprising administering to the subject an effective amount of the bispecific antibody as described in any one of Implementation Schemes 1 to 29.

[0274] Implementation Scheme 43. The method as described in Implementation Scheme 41 or 42, wherein the subject is a human being.

[0275] Implementation Scheme 44. A method for treating cancer in a subject in need, comprising administering to the subject a therapeutically effective amount of the bispecific antibody as described in any one of Implementation Schemes 1 to 29.

[0276] Implementation Scheme 45. The method of implementation scheme 44, further comprising administering additional therapy to the subject.

[0277] Implementation Scheme 46. The method as described in Implementation Scheme 44 or 45, wherein the subject is a human being.

[0278] Implementation Scheme 47. Use of the bispecific antibody as described in any one of Implementation Schemes 1 to 29 in cancer treatment.

[0279] Implementation Scheme 48. Use of the bispecific antibody described in any one of Implementation Schemes 1 to 29 for the preparation of a medicament for treating cancer.

[0280] Implementation Scheme 49. The method or use as described in any one of Implementation Schemes 44 to 48, wherein the cancer is a hematologic cancer.

[0281] Implementation Scheme 50. The method or use as described in any one of Implementation Schemes 44 to 50, wherein the cancer is a solid tumor.

[0282] Implementation Scheme 51. The method or use as described in any one of Implementation Schemes 44 to 50, wherein the cancer is hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, endometrial cancer, cervical cancer, colorectal cancer, breast cancer, biliary tract cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, gynecological tumor, or thymic malignancy.

[0283] Implementation Scheme 52. The method or use as described in any one of Implementation Schemes 44 to 51, wherein the cancer is a VEGF-expressing cancer.

[0284] Implementation Scheme 53. The method or use as described in any one of Implementation Schemes 44 to 52, wherein the cancer is a PD-L1-expressing cancer. 6.9 Example

[0285] The embodiments provided below are for illustrative purposes only and are not intended to be limiting unless expressly stated otherwise. Therefore, the invention should not be construed in any way as limited to the embodiments described below, but should be construed as covering any and all variations that may be apparent from the teachings provided herein.

[0286] The antibodies involved in the research described below include: HX006: monoclonal anti-VEGF IgG1 antibody (VL and VH: SEQ ID NOs: 21 and 22); HX008: monoclonal anti-PD-1 IgG4 antibody (VL and VH: SEQ ID NOs: 69 and 70); AK112: bispecific antibody targeting PD-1 and VEGF (HC and LC: SEQ ID NOs: 73 and 74); HX541: “AK112-like” bispecific antibody targeting PD-1 and VEGF (SEQ ID NOs: 73 and 74), which has the same structure and sequence as AK112 and is prepared by recombinant synthesis; PM8002: bispecific antibody targeting PD-L1 and VEGF (HC and LC: SEQ ID NOs: 75 and 76); HX542: “PM8002-like” bispecific antibody targeting PD-L1 and VEGF (SEQ ID NOs: 75 and 76). NOs:75 and 76), which have the same structure and sequence as PM8002 and are prepared by recombination; Tecentriq (atelizumab): monoclonal anti-PD-L1 IgG1 antibody (VL and VH: SEQ ID NOs:71 and 72). 6.9.1 Example 1: Binding of HX016 candidate bispecific antibody to its respective protein and ligand blocking

[0287] method :

[0288] HX016-7, HX016-8, HX016-9, and HX016-10, as well as reference antibodies HX006, HX008, HX541, and HX542, were prepared as follows: Recombinant expression of the two peptide chains of each antibody was achieved using a single vector system encoding the two peptide chains. The encoding genes for each peptide chain were cloned into the vectors, respectively. The vectors were prepared using an endotoxin-free plasmid DNA purification method (EndoFree Plasmid kit, TransGen Biotech). After preparing the DNA vectors, transient expression was performed using CHO suspension cells. The DNA was mixed with PEI solution, and this mixture was added to the cells. After 7 days of culture, the cells were harvested by centrifugation.

[0289] result :like Figures 2A-2D As shown, the HX016 candidate bispecific antibody exhibits strong binding affinity and blocking ability against the target protein. Figure 2A As shown, HX016-7 / 8 / 9 / 10 exhibits a similar binding affinity to VEGF as HX006 and HX542. Figure 2B As shown, HX016-7 / 8 has a higher affinity for binding to PD-L1 protein than HX542, but lower than Tecentriq. Figure 2C As shown, HX016-9 / 10 exhibits a binding affinity for PD-1 protein comparable to that of HX008 and HX541. Figure 2D As shown, the PD-1 ligand blocking ability of HX016-9 / 10 is higher than that of HX541, but slightly lower than that of HX008. 6.9.2 Example 2: Binding of HX016 candidate bispecific antibody to PD-1 positive cells and PD-L1 positive cells

[0290] method :

[0291] 293T-PD1 cells were constructed to express human PD-1. These cells were incubated at 4 °C for 1 hour with reference antibodies (HX008 and HX541) and candidate bsAbs (HX016-9 and HX016-10) provided herein at serially diluted titrated doses. Similarly, CHO-PDL1 cells were constructed to express human PD-L1. These cells were incubated at 4 °C for 1 hour with reference antibodies (Tecentriq and HX542) and bsAbs (HX016-7 and HX016-8) provided herein at serially diluted titrated doses. The bound antibodies were detected by Alexa Fluor® 647 (AF647) conjugated AffiniPure Goat Anti-Human IgG (H+L) secondary antibody (Jackson Immuno Research-109-605-003) and analyzed by flow cytometry. EC 50 The values ​​were calculated using GraphPad Prism software based on the best-fit curve.

[0292] result :like Figure 3A As shown, compared to HX008, HX016-9 / 10 exhibited slightly lower binding affinity to PD-1 positive cells, but showed similar binding affinity to HX541. Figure 3B As shown, HX016-7 / 8 exhibited similar binding affinity to PD-L1 positive cells compared to Tecentriq and HX542. 6.9.3 Example 3: Effects of HX016 candidate bispecific antibody on PD-1 / PD-L1 and VEGF function

[0293] method :

[0294] Perform reporter gene assays to measure T cell activation. Use 5 x 10 4CHO-PDL1-CD3L cells (CHO cells constructed to express human PD-L1 and CD3 agonists) were seeded in 96-well Solid White Flat Bottom Polystyrene TC-treated Microplates and cultured for 18 h at 26 °C and 5% CO2. 1 x 10 5 Jurkat-NFAT-PD1 cells (Jurkat cells constructed to express human PD-1 and luciferase reporter gene under NFAT regulation) were incubated with a titrated dose of the test antibody. Subsequently, both cell populations were co-cultured at 37 °C and 5% CO2 for 18 h in the presence of the antibody. Afterward, 100 μL / well of Stable-Lite™ Luciferase Assay System reagent (Vazyme-DD1202-02) was added to each well, and the cells were incubated at room temperature for 10 min to stabilize the luminescence signal. The luminescence signal was then recorded on a 2104 EnVision microplate reader. EC 50 The values ​​were calculated based on the best-fit binding curve using GraphPad Prism software. Parallel assays were also performed, in which cells were incubated with a titration of 100 ng / mL rhVEGF165 and the antibody being tested.

[0295] Similarly, another reporter gene assay was performed to measure downstream regulation of VEGF. 5 x 10 4 293T-VEGFR2 cells (constructed to stably express human VEGFR2) were seeded in 96-well Solid White FlatBottom Polystyrene TC-treated Microplates and cultured at 37°C and 5% CO2 for 18 h. The cells were then incubated with 100 ng / mL VEGF165 and a titrated dose of the test antibody. The cells were then co-cultured at 37°C and 5% CO2 in the presence of the antibody for 18 h. Afterward, 100 μL / well Stable-Lite™ Luciferase Assay System reagent (Vazyme-DD1202-02) was added to each well and incubated at room temperature for 10 min to stabilize the luminescence signal. The luminescence signal was then recorded on a 2104 EnVision microplate reader. 50 The values ​​were calculated using GraphPad Prism software based on the best-fit curve.

[0296] result :like Figure 4AAs shown, compared with HX541 and HX542, HX016-7 / 8 / 9 / 10 exhibited similar biological activity in blocking the PD-1-PD-L1 pathway, but its activity was slightly lower than that of HX008. Figure 4B As shown, HX016-7 / 8 / 9 / 10 exhibited similar biological activity to HX541 and HX542 in blocking the VEGF-VEGFR2 pathway. Figure 4C As shown, the bispecific antibodies HX016-9 and AK112 exhibited a clear increase in potency upon the addition of VEGF165, while the monospecific anti-PD-1 antibody HX008 showed almost no change, thus confirming that VEGF binding provides an activity advantage beyond simple PD-1 blockade. 6.9.4 Example 4: Effect of HX016 candidate bispecific antibody on HUVEC growth inhibition

[0297] method :

[0298] HUVEC cell proliferation was measured to quantify downstream regulation by VEGF. 5 x 10⁻⁶ cells were used. 3 HUVEC cells (Human Umbilical Vein Endothelial Cells) were seeded in 96-well Solid White Flat Bottom Polystyrene TC-treated Microplates and cultured at 37 °C and 5% CO2 for 18 h. Subsequently, the titration dose of the candidate antibody was... 50 Incubate with ng / mL VEGF165 for 2 h, discard the cell supernatant, and then further incubate with the mixed antibody at 37 °C and 5% CO2 for 72 h. Afterward, add 10 μL / well Alamar Blue reagent (INVITROGEN) to each well and incubate at 37 °C for 4 h. The luminescence signal is then recorded on a SpectraMax Mini microplate reader. EC 50 The values ​​were calculated using GraphPad Prism software based on the best-fit curve.

[0299] result :like Figure 5 As shown, HX016-7 / 8 / 9 / 10 exhibited similar HUVEC growth inhibition compared to HX541 and HX542, indicating its anti-angiogenic activity. 6.9.5 Example 5: Tumor-suppressive effect of HX016 candidate bispecific antibody in vivo

[0300] method :

[0301] MC38-hPD-L1-hVEGFA tumor cells were generated by overexpressing human PD-L1 and VEGFA in parental MC38 colorectal cancer cells. The tumor cells were subcutaneously inoculated into the right inguinal region of 8-9 week old female hPD-1 HuGEMM mice, in which the mouse PD-1 gene was replaced by its human counterpart. Tumor-bearing mice were randomly assigned to eight groups and received the specified treatments as follows: PBS, ip, BIW x 3 weeks; HX006, 3.6 mg / kg, ip, BIW x 3 weeks; HX008, 3.6 mg / kg, ip, BIW x 3 weeks; HX016-9, 5 mg / kg, ip, BIW x 3 weeks; HX541, 5 mg / kg, ip, BIW x 3 weeks; durvalumab, 3.6 mg / kg, ip, BIW x 3 weeks; HX016-7, 5 mg / kg, ip, BIW x 3 weeks; HX542, 4.3 mg / kg, ip, BIW x 3 weeks. Tumor volume and body weight were measured twice weekly. Tumor growth inhibition (TGI) was used as an indicator of the antitumor activity of each assay.

[0302] result :like Figure 6 As shown in the table below, the HX016 candidate bispecific antibody effectively inhibited tumor growth in the MC38-hPD-L1-hVEGFA model, demonstrating its potent in vivo antitumor activity. 6.9.6 Example 6: Tumor Suppressive Effect of HX016 Candidate Bispecific Antibody in Xenograft Model

[0303] method :

[0304] A549 xenograft: A549 tumor cells were subcutaneously inoculated into the right inguinal region of 6-7 week old female Balb / c nude mice to establish tumors. Tumor-bearing mice were randomly assigned to 6 groups and received their designated treatments as follows: PBS (carrier), HX006 (5 mg / kg three times and 7.25 mg / kg three times), HX016-7 (6.9 mg / kg three times and 10 mg / kg three times), HX016-9 (6.9 mg / kg three times and 10 mg / kg three times), AK112 (6.9 mg / kg three times and 10 mg / kg three times), and HX542 (5.9 mg / kg three times and 8.6 mg / kg three times). The treatments were administered twice weekly. Tumor volume was measured twice weekly. Tumor growth inhibition rate (TGI) was used as an indicator of the antitumor efficacy of each test item.

[0305] HCC827 Xenograft: HCC827 NSCLC tumor cells were subcutaneously inoculated into the right inguinal region of 6-8 week old female Balb / c nude mice. Tumor-bearing mice were randomly assigned to receive their designated treatments as follows: PBS (carrier), HX006 (2 mg / kg), HX016-7 (2.8 mg / kg), HX016-9 (2.8 mg / kg), HX541 (2.8 mg / kg), and HX542 (2.4 mg / kg). These treatments were administered twice weekly. Tumor volume was measured twice weekly. Tumor growth inhibition rate (TGI) was used as an indicator of the antitumor efficacy of each test item.

[0306] NCI-H1975 Humanized Xenograft: Human PBMCs were implanted into 6-8 week old female NCG mice. Six days later, NCI-H1975 NSCLC tumor cells were subcutaneously inoculated into the right inguinal region of the mice reconstructed by human PBMCs. Tumor-bearing mice were randomly assigned to 6 groups and received their designated treatments as follows: PBS (carrier), HX006 (7.25 mg / kg), HX016-7 (10 mg / kg), HX016-9 (10 mg / kg), HX541 (10 mg / kg), and HX542 (8.6 mg / kg). These treatments were administered twice weekly. Tumor volume was measured twice weekly, and tumor weight was measured at the end of the experiment (day 20). Tumor growth inhibition rate (TGI) was used as an indicator of the antitumor efficacy of each test item. In addition, in a separate study, PBS (carrier), HX016-9 (10 mg / kg), or HX541 (10 mg / kg) were administered as a single dose. Tumor volume was measured twice weekly.

[0307] result :like Figure 7A As shown, the HX016 candidate bispecific antibody effectively inhibited tumor growth in vivo in the A549 xenograft model. Figure 7B As shown, the HX016 candidate bispecific antibody also effectively inhibited tumor growth in vivo in the HCC827 xenograft model, with efficacy comparable to AK112 / HX542. Figure 7C As shown, the HX016 candidate bispecific antibody also effectively inhibited tumor growth in vivo in the NCI-H1975 humanized xenograft model, and its effect was comparable to or better than AK112 / HX542. Figure 7D As shown, unexpectedly, a single dose of the HX016 candidate antibody demonstrated significantly stronger antitumor efficacy than AK112. This result suggests that HX016 may offer superior therapeutic characteristics in terms of antitumor efficacy, potentially making it a more effective candidate for cancer immunotherapy (e.g., with reduced dose requirements or better tolerability). * * *

[0308] Although the invention has been described in considerable detail by way of examples and instances for the purpose of clarity, it will be readily understood by those skilled in the art, based on the teachings of the invention, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0309] Therefore, the foregoing only illustrates the principles of the invention. Those skilled in the art will understand that, although not explicitly described or shown herein, various arrangements embodying the principles of the invention and encompassing its spirit and scope are conceived. Furthermore, all instances and conditional language described herein are primarily intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to advance the art, and should be interpreted as not constituting limitation on such specific instances and conditions. Moreover, all statements herein recounting the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to cover both structural and functional equivalents. Additionally, the term "equivalent" is intended to include both currently known equivalents and those developed in the future, i.e., any element developed that performs the same function, regardless of its structure. Furthermore, nothing disclosed herein is intended to be offered to the public, whether or not such disclosure is expressly stated in the claims.

[0310] All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually designated to be incorporated by reference, and are incorporated herein by reference to disclose and describe methods and / or materials relating to the referenced publications. References to any publication refer to its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to precede that publication by prior art. Furthermore, the publication dates provided may differ from the actual publication dates, which may require independent verification.

Claims

1. A bispecific antibody comprising a first light chain variable domain (VL1), a first heavy chain variable domain (VH1), a second light chain variable domain (VL2), and a second heavy chain variable domain (VH2); wherein: (1) The VL1 / VH1 pair specifically binds to human VEGF, and the VL1 comprises VL CDR1, VL CDR2 and VL CDR3 of a reference VL having the amino acid sequence shown in SEQ ID NO:21, or variants thereof, having up to about 5 amino acid substitutions, additions and / or deletions in the VL CDRs; and the VH1 comprises VH CDR1, VH CDR2 and VH CDR3 of a reference VH having the amino acid sequence shown in SEQ ID NO:22, or variants thereof, having up to about 5 amino acid substitutions, additions and / or deletions in the VH CDRs; and (2) The VL2 / VH2 pair specifically binds to human PD-L1 or human PD-1.

2. The bispecific antibody as described in claim 1, wherein, The VL1 includes VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences shown in SEQ ID NOs:1, 2, and 3, respectively; and the VH1 includes VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NOs:4, 5, and 6, respectively.

3. The bispecific antibody as described in claim 2, wherein, The amino acid sequences of VL1 and VH1 independently have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs:21 and 22, respectively.

4. The bispecific antibody according to any one of claims 1 to 3, wherein, The VL2 / VH2 pairs are derived from antibodies selected from the group consisting of: putelimab, nivolumab, pembrolizumab, cimiprimab, dotalimab, retivalimab, toripalimab, tislelizumab, camrelizumab, sintilimab, penaprilimab, cepalimumab, slulimab, fenolinumab, durvalumab, atezolizumab, avelumab, cosibelimab, sugmalimab, envorimab, adebenone, socazolimumab, and tagorimab.

5. The bispecific antibody according to any one of claims 1 to 3, wherein, The VL2 / VH2 specifically binds to human PD-L1; wherein the VL2 comprises VL CDR1, VL CDR2, and VL CDR3 of a reference VL having the amino acid sequence shown in SEQ ID NO:67, or variants thereof, having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs; and the VH2 comprises VH CDR1, VH CDR2, and VHCDR3 of a reference VH having the amino acid sequence shown in SEQ ID NO:68, or variants thereof, having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs.

6. The bispecific antibody as described in claim 5, wherein, The VL2 comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences shown in SEQ ID NOs:7, 8, and 9, respectively; and the VH2 comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NOs:10, 11, and 12, respectively.

7. The bispecific antibody as described in claim 6, wherein, The amino acid sequences of the VL2 and the VH2 are independently identical to the following sequences by at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% respectively: (1) SEQ ID NOs:23 and 24 respectively; (2) SEQ ID NOs:23 and 68 respectively; (3) SEQ ID NOs:67 and 24 respectively; or (4) SEQ ID NOs:67 and 68 respectively.

8. The bispecific antibody as described in claim 7, wherein, The VL1 and VH1 have the amino acid sequences shown in SEQ ID NOs:21 and 22; and the VL2 and VH2 have the amino acid sequences shown in SEQ ID NOs:23 and 24, respectively.

9. The bispecific antibody according to any one of claims 1 to 3, wherein, The VL2 / VH2 specifically binds to human PD-1, and the VL2 comprises VL CDR1, VL CDR2, and VL CDR3 of a reference VL having the amino acid sequence shown in SEQ ID NO:69, or variants thereof, having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs; and the VH2 comprises VH CDR1, VH CDR2, and VH CDR3 of a reference VH having the amino acid sequence shown in SEQ ID NO:70, or variants thereof, having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs.

10. The bispecific antibody as described in claim 9, wherein, The VL2 comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences shown in SEQ ID NOs: 13, 14, and 15, respectively; and the VH2 comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, respectively.

11. The bispecific antibody of claim 10, wherein, The amino acid sequences of the VL2 and the VH2 are independently identical to the following sequences by at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% respectively: (1) SEQ ID NOs:25 and 26 respectively; (2) SEQ ID NOs:25 and 70 respectively; (3) SEQ ID NOs:69 and 26 respectively; or (4) SEQ ID NOs:69 and 70 respectively.

12. The bispecific antibody as described in claim 11, wherein, The VL1 and VH1 have the amino acid sequences shown in SEQ ID NOs:21 and 22, respectively; and the VL2 and VH2 have the amino acid sequences shown in SEQ ID NOs:25 and 26, respectively.

13. The bispecific antibody according to any one of claims 1 to 12, comprising: (i) a first peptide chain (HC) comprising, from the N-terminus to the C-terminus, the VH1, a heavy chain constant (CH) region, a linker, and a single-chain variable fragment (scFv) comprising the VL2 and the VH2; and (ii) a second peptide chain (LC) comprising, from the N-terminus to the C-terminus, the VL1 and a light chain constant (CL) region.

14. The bispecific antibody as described in claim 13, wherein, The linker has an amino acid sequence selected from the group consisting of SEQ ID NOs:51-55.

15. The bispecific antibody as described in claim 13 or 14, wherein, The scFv includes VL2, a second connector, and VH2 sequentially from the N-terminus to the C-terminus.

16. The bispecific antibody as described in claim 13 or 14, wherein, The scFv includes VH2, a second connector, and VL2 sequentially from the N-terminus to the C-terminus.

17. The bispecific antibody as described in claim 15 or 16, wherein, The second linker has an amino acid sequence selected from the group consisting of SEQ ID NOs:51-55.

18. The bispecific antibody according to any one of claims 13 to 17, wherein: (1) The CL region is Cκ (SEQ ID NO:29) or Cλ (SEQ ID NO:30), or a variant thereof, wherein there are up to ten amino acid substitutions, additions and / or deletions; or (2) The CH region is a human IgG1 CH region (SEQ ID NO:31), IgG2 CH region (SEQ ID NO:32), IgG3 CH region (SEQ ID NO:33) or IgG4 CH region (SEQ ID NO:34), or a variant thereof, wherein there are up to ten amino acid substitutions, additions and / or deletions; or both (1) and (2) are satisfied.

19. The bispecific antibody as described in claim 18, wherein, The CL region is Cκ (SEQ ID NO:29).

20. The bispecific antibody as described in claim 18 or 19, wherein, The CH region is the human IgG1 CH region with L234A and L235A substitutions.

21. The bispecific antibody of claim 20, wherein, The CH region also has M252Y, S254T or T256E replacements, or any combination thereof.

22. The bispecific antibody of claim 21, wherein, The CH region has L234A, L235A, M252Y, S254T and T256E replacements.

23. The bispecific antibody as described in claim 13, wherein, The amino acid sequence of the HC has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NOs:61-64, and the amino acid sequence of the LC has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

60.

24. The bispecific antibody as described in claim 23, wherein, The HC and the LC have the amino acid sequences shown in SEQ ID NOs:61 and 60, respectively.

25. The bispecific antibody as described in claim 23, wherein, The HC and the LC have the amino acid sequences shown in SEQ ID NOs:62 and 60, respectively.

26. The bispecific antibody as described in claim 23, wherein, The HC and the LC have the amino acid sequences shown in SEQ ID NOs:63 and 60, respectively.

27. The bispecific antibody as described in claim 23, wherein, The HC and the LC have the amino acid sequences shown in SEQ ID NOs:64 and 60, respectively.

28. The bispecific antibody as described in claim 23, wherein, The HC has an amino acid sequence selected from the group consisting of SEQ ID NOs:56-59, and the LC has the amino acid sequence shown in SEQ ID NO:

60.

29. The bispecific antibody according to any one of claims 1 to 28, wherein, The bispecific antibody (1) inhibits VEGF signaling; (2) inhibits vascular endothelial cell proliferation; (3) inhibits tumor angiogenesis; (4) blocks PD-1 / PD-L1 binding; (5) reduces immunosuppression; or (6) promotes T cell activation and / or proliferation; or any combination of (1)-(6).

30. A pharmaceutical composition comprising a therapeutically effective amount of the bispecific antibody as described in any one of claims 1 to 29 and a pharmaceutically acceptable carrier.

31. A polynucleotide encoding a peptide chain of the bispecific antibody according to any one of claims 1 to 29.

32. The polynucleotide of claim 31, wherein the polynucleotide encodes the two peptide chains of the bispecific antibody.

33. A plurality of polynucleotides as described in claim 31, which collectively encode the two peptide chains of the bispecific antibody.

34. A carrier comprising the polynucleotide of claim 31 or 32.

35. A cell comprising one or more polynucleotides as described in any one of claims 31 to 33, or the carrier as described in claim 34.

36. A method for preparing a bispecific antibody that specifically binds to human VEGF and human PD-1 / PD-L1, comprising culturing the cells of claim 35 under conditions that allow expression of the bispecific antibody.

37. The method of claim 36, further comprising isolating the bispecific antibody from the culture.

38. A method for reducing immune cell suppression, comprising contacting the immune cells with an effective amount of a bispecific antibody as described in any one of claims 1 to 29.

39. The method of claim 38, wherein, The immune cells are T cells, NK cells, NKT cells, or myeloid cells.

40. The method of claim 39, wherein, The immune cells mentioned are T cells.

41. A method for inducing or stimulating the activation and / or proliferation of immune cells in a subject in need, comprising administering to the subject an effective amount of the bispecific antibody as described in any one of claims 1 to 29.

42. A method for inhibiting cancer angiogenesis in a subject in need, comprising administering to the subject an effective amount of the bispecific antibody as described in any one of claims 1 to 29.

43. The method of claim 41 or 42, wherein the subject is a human being.

44. A method of treating cancer in a subject in need, comprising administering to the subject a therapeutically effective amount of the bispecific antibody as described in any one of claims 1 to 29.

45. The method of claim 44, further comprising administering additional therapy to the subject.

46. ​​The method of claim 44 or 45, wherein the subject is a human being.

47. Use of a bispecific antibody as described in any one of claims 1 to 29 in cancer treatment.

48. Use of a bispecific antibody as described in any one of claims 1 to 29 in the preparation of a medicament for treating cancer.

49. The method or use as claimed in any one of claims 44 to 48, wherein the cancer is a hematologic cancer.

50. The method or use as claimed in any one of claims 44 to 48, wherein the cancer is a solid tumor.

51. The method or use according to any one of claims 44 to 50, wherein the cancer is hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, endometrial cancer, cervical cancer, colorectal cancer, breast cancer, biliary tract cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, gynecological tumor, or thymic malignancy.

52. The method or use as claimed in any one of claims 44 to 51, wherein the cancer is a VEGF-expressing cancer.

53. The method or use as described in any one of claims 44 to 52, wherein the cancer is a PD-L1-expressing cancer.

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