Antibody targeting PD-1 and VEGF and application thereof

Abstract

Provided are an antibody targeting PD-1 and VEGF and an application thereof. The invention also provides an antigen binding protein which comprises a first targeting part and a second targeting part, the first targeting part has the function of specifically binding to PD-1, and the second targeting part has the function of specifically binding to VEGF.

Description

Antibody targeting PD-1 and VEGF and its application Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an antibody targeting PD-1 and VEGF and its application. Background Art

[0002] Malignant tumors are a serious threat to human health worldwide and the leading cause of death. With the aging of the Chinese population, the incidence of tumors is increasing, and effective therapeutic drugs are urgently needed.

[0003] The development of immune checkpoint drugs has been a research hotspot in recent years. Programmed death receptor 1 (PD-1), abbreviated as PD-1, is widely expressed on immune cells and is an important immunosuppressive molecule. The main ligand of PD-1 is PD-L1, which is mainly expressed on the surface of tumor cells. After the ligand PD-L1 binds to the receptor PD-1, it inhibits the activation of T cells in the tumor microenvironment, resulting in the inability of T cells and other immune systems to kill tumor cells normally, thereby achieving immune escape. The mechanism of action of PD-1 or PD-L1 immunotherapy is to design specific monoclonal antibodies against PD-1 or PD-L1, prevent the recognition of PD-1 and PD-L1, restore the normal function of T cells, and thus enable T cells to effectively kill tumor cells. Currently, a number of therapeutic antibodies targeting this signaling pathway have been developed, such as pembrolizumab and nivolumab, but low response rates and easy development of drug resistance are common during the treatment process.

[0004] Furthermore, during tumor growth, epidermal growth factor (VEGF) can specifically stimulate endothelial cell proliferation and play a key role in angiogenesis in various tumor types. VEGF binds to the epidermal growth factor receptor (VEGFR), mediating the transcriptional expression of related intracellular protein genes through downstream signaling pathways, thereby promoting endothelial cell proliferation. Currently, several humanized monoclonal antibodies targeting VEGF, such as bevacizumab, have been developed, but treatment still suffers from low response rates and the development of drug resistance.

[0005] At the same time, the vast majority of currently marketed therapeutic antibody drugs are monoclonal antibodies, which are specific to a single target and are more prone to low response rates and drug resistance during treatment. Therefore, there is an urgent need for a multispecific antibody targeting PD-1 and VEGF that is structurally stable, highly effective, and suitable for large-scale industrial production.

[0006] Summary of the Invention

[0007] The antigen binding protein described in the present application has at least one characteristic selected from the following group: easy expression and purification, high affinity binding activity with VEGF, ability to effectively regulate PD-1 and PD-L1 signals, and inhibition of tumor growth.

[0008] In one aspect, the present application provides an antigen-binding protein comprising a first targeting moiety and a second targeting moiety, wherein the first targeting moiety comprises an antibody or an antigen-binding fragment thereof that specifically binds to PD-1, and the second targeting moiety comprises an antibody or an antigen-binding fragment thereof that specifically binds to VEGF;

[0009] wherein the first targeting moiety comprises HCDR1, HCDR2, and HCDR3 of an antibody or antigen-binding fragment thereof, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 5 or 11, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 6 or 12, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7 or 13;

[0010] wherein the first targeting moiety comprises LCDR1, LCDR2, and LCDR3 of an antibody or antigen-binding fragment thereof, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8 or 14, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9 or 15, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10 or 16;

[0011] Wherein, the second targeting portion comprises HCDR1, HCDR2 and HCDR3 of the nanobody, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 26, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 27, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 28.

[0012] In one aspect, the present application provides a nucleic acid encoding the antigen-binding protein of the present application.

[0013] In one aspect, the present application provides a vector comprising the nucleic acid of the present application.

[0014] In one aspect, the present application provides an immunoconjugate comprising the antigen-binding protein of the present application.

[0015] In one aspect, the present application provides a cell comprising the antigen-binding protein, the nucleic acid, the vector, and / or the immunoconjugate of the present application.

[0016] In one aspect, the present application provides a pharmaceutical composition comprising the antigen-binding protein of the present application, the nucleic acid of the present application, the vector of the present application, the immunoconjugate of the present application, and / or the cell of the present application, and optionally a pharmaceutically acceptable carrier.

[0017] In one aspect, the present application provides a kit comprising the antigen-binding protein of the present application, the nucleic acid of the present application, the vector of the present application, the immunoconjugate of the present application, the cell of the present application, and / or the pharmaceutical composition of the present application.

[0018] In one aspect, the present application provides a method for preparing the antigen-binding protein of the present application, comprising culturing the cell of the present application under conditions that allow the antigen-binding protein to be expressed.

[0019] On the one hand, the present application provides the use of the antigen-binding protein, nucleic acid, vector, immunoconjugate, cell, pharmaceutical composition, and / or kit of the present application in the preparation of a drug for preventing, alleviating and / or treating tumors.

[0020] In one aspect, the present application provides an antigen-binding protein, a nucleic acid, a vector, an immunoconjugate, a cell, a pharmaceutical composition, and / or a kit of the present application for use in treatment.

[0021] On the one hand, the present application provides an antigen-binding protein, nucleic acid, vector, immunoconjugate, cell, pharmaceutical composition, and / or kit for preventing, alleviating, and / or treating tumors.

[0022] On the one hand, the present application provides a method for preventing, alleviating and / or treating tumors, comprising administering to a subject an effective amount of the antigen-binding protein of the present application, the nucleic acid of the present application, the vector of the present application, the immunoconjugate of the present application, the cell of the present application, the pharmaceutical composition of the present application, and / or the kit of the present application.

[0023] In one aspect, the present application provides a method of inhibiting the binding of PD-1 protein to PD-L1 protein and / or PD-L2 protein, and / or inhibiting the binding of VEGF protein to VEGFR protein, the method comprising administering the antigen-binding protein of the present application, the nucleic acid of the present application, the vector of the present application, the immunoconjugate of the present application, the cell of the present application, the pharmaceutical composition of the present application, and / or the kit of the present application.

[0024] A method for regulating an immune response, the method comprising administering the antigen-binding protein, nucleic acid, vector, immunoconjugate, cell, pharmaceutical composition, and / or kit of the present application.

[0025] The bispecific antibody molecular structure of the present application can be IgG-VHH. The use of this form of bispecific antibody targeting PD-1 or PD-L1 and VEGF can effectively avoid mispairing of the antibody light and heavy chains, improve molecular stability, and be more suitable for industrial production. At the same time, the antigen-binding molecule of the present application can block two signaling pathways and has tumor microenvironment targeting, which can have better safety and therapeutic effects.

[0026] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0028] FIG1 shows that the antigen binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described in the present application bind to the human PD-1 protein on the surface of CHOK1 cells.

[0029] Figure 2 shows that the antigen binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described in the present application bind to the cynomolgus monkey PD-1 protein on the surface of CHOK1 cells.

[0030] Figure 3 shows that the antigen binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described in the present application block the binding of human PD-L1 to the human PD-1 protein on the surface of CHOK1.

[0031] Figure 4 shows that the antigen binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described in the present application block human PD-L2 and human PD-1 protein on the surface of CHOK1 cells.

[0032] FIG5 shows the results that both humanized antibodies 1910h3hzL3H4 and 41D2HzL4H3 can stimulate lymphocytes to secrete IL2.

[0033] FIG6 shows a scheme for constructing a single domain antibody displayed on the yeast cell wall surface.

[0034] FIG7 shows the results of blocking the binding of VEGF165 and human VEGFR2 by a single domain antibody.

[0035] FIG8 shows the blocking results of Ab1910VE18 and Ab1910VE21 antibodies.

[0036] FIG9 shows the binding results of bispecific antibodies Kab5 and Kab6 to the cell surface antigen PD-1.

[0037] FIG10 shows the results of bispecific antibodies blocking the binding of cell surface antigens PD-1 and PD-L1.

[0038] FIG11 shows the results of bispecific antibodies blocking the binding of cell surface antigens PD-1 and PD-L2.

[0039] FIG12 shows the results of the bispecific antibody competing with VEGFR2 for binding to the antigen VEGF.

[0040] FIG13 shows the results of bispecific antibodies stimulating mixed lymphocytes to secrete cytokine IL2. DETAILED DESCRIPTION

[0041] The following describes the implementation of the present invention through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0042] In this application, the terms "first targeting moiety" and "second targeting moiety" generally refer to the portion of the antigen-binding protein of the present application that is capable of targeting a specific molecule. For example, the targeting moiety may include, but is not limited to, antibodies or fragments thereof, peptides, hormones, growth factors, cytokines, and any other natural or non-natural ligands.

[0043] In this application, the term "linker" generally refers to a portion that joins or connects two or more discrete, separate monomer domains. A linker can be a peptide linker, which comprises a polypeptide of two or more amino acid residues connected by a peptide bond and is used to connect one or more antigen-binding portions. For example, the linker can be (GxS)n, where G = glycine, S = serine, x = 3 and n = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or x = 4 and n = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. For example, the linker can be ADIEGRMD.

[0044] In this application, the term "VEGF" generally refers to vascular endothelial growth factor, including its naturally occurring allelic forms and processed forms. The VEGF may include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and / or P1GF. In this application, the VEGF may be VEGF-A, also referred to as VEGF165. VEGF-A can bind to receptor tyrosine kinases, namely VEGFR-1 (Flt-1), VEGFR-2 (Flk-1 / KDR), VEGFR3 and Neuropilin-1 (NRP-1). After VEGF165 binds to VEGFR2, it mediates the transcriptional expression of intracellular related protein genes through the downstream PLC-γ-PKC-Raf-MEK-MAPK signaling pathway, thereby promoting the proliferation of vascular endothelial cells. The term "VEGF" may also refer to VEGF from non-human species such as mice, rats or primates. In this application, VEGF from a particular species may be indicated as follows: hVEGF refers to human VEGF, and mVEGF refers to mouse VEGF. Generally, VEGF may refer to human VEGF. The term "VEGF" is also used to refer to truncated forms or fragments of intact VEGF, and also includes functional variants, isoforms, species homologs, derivatives, analogs of VEGF, and analogs having at least one common epitope with VEGF. VEGF sequences are known in the art. For example, exemplary full-length human VEGFA protein sequences can be found under NCBI accession numbers NP_001020537, NP_001020538, NP_001020539, NP_001020540, or NP_001020541.

[0045] In this application, the term "PD-1" generally refers to the programmed death 1 receptor, which may also be referred to as "programmed death 1", "CD279", "cluster of differentiation 279", "PD1", "PDCD1" or "CD297". The PD-1 protein generally includes an extracellular IgV domain, a transmembrane region and an intracellular tail. PD-1 is generally expressed on T cells, B cells, natural killer T cells, activated monocytes and dendritic cells (DCs). PD-1 can bind to its ligands PD-L1 and PD-L2. The term "PD-1" encompasses any natural PD-1 or modified PD-1 of any vertebrate origin, including mammals, such as primates (e.g., humans or monkeys) and rodents (e.g., mice or rats). The term encompasses "full length", unprocessed PD-1 as well as any form of PD-1 produced by processing in cells. PD-1 may exist as a transmembrane protein or as a soluble protein. "PD-1" includes complete PD-1 and fragments thereof, and also includes functional variants, isoforms, species homologs, derivatives, analogs, and analogs having at least one common epitope with PD-1. PD-1 sequences are known in the art. For example, an exemplary full-length human PD-1 protein sequence can be found under NCBI accession number NP_005009.2, and an exemplary full-length cynomolgus monkey PD-1 protein sequence can be found under NCBI accession number NP_001271065 or Uniprot accession number B0LAJ3.

[0046] In this application, the term "PD-L1" generally refers to programmed death ligand 1 protein. PD-L1 is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), and is a protein encoded by the CD274 gene (in humans). PD-L1 can bind to its receptor, such as programmed death 1 (PD-1). The complexation of PD-L1 and PD-1 exerts an immunosuppressive effect by inhibiting T cell proliferation and producing cytokines IL-2 and IFN-γ. The term "PD-L1" encompasses any natural PD-L1 or modified PD-1 of any vertebrate origin, including mammals such as primates (e.g., humans or monkeys) and rodents (e.g., mice or rats). The term encompasses "full length", unprocessed PD-L1, and any form of PD-L1 produced by processing in cells. PD-L1 can exist as a transmembrane protein or as a soluble protein. The term also encompasses naturally occurring variants of PD-L1, such as splice variants or allelic variants. The basic structure of PD-L1 includes four domains: an extracellular Ig-like V-type domain and an Ig-like C2-type domain, a transmembrane domain, and a cytoplasmic domain. The PD-L1 sequence is known in the art. For example, information about the human PD-L1 gene (including genomic DNA sequence) can be found under NCBI Gene ID No. 29126. The amino acid sequence of an exemplary full-length human PD-L1 protein can be found under NCBI Accession No. NP_054862 or UniProt Accession No. Q9NZQ7.

[0047] In this application, the term "PD-L2" generally refers to programmed death ligand 2 protein, which may also be referred to as "programmed death 2", "CD273", "cluster of differentiation 273", "B7-DC" or "PDCD1LG2". The term "PD-L2" covers any natural PD-L2 or modified PD-L2 of any vertebrate origin, including mammals, such as primates (e.g., humans or monkeys) and rodents (e.g., mice or rats). The term covers "full-length", unprocessed PD-1 and any form of PD-L2 produced by processing in cells. PD-L2 can exist as a transmembrane protein or as a soluble protein. "PD-L2" includes complete PD-L2 and fragments thereof, as well as functional variants, isoforms, species homologs, derivatives, analogs of PD-L2, and analogs having at least one common epitope with PD-L2. The PD-L2 sequence is known in the art. For example, an exemplary full-length human PD-L2 protein sequence can be found under NCBI Accession No. NP_079515.2.

[0048] In this application, the term "VEGFR" generally refers to the vascular endothelial growth factor receptor, including its naturally occurring allelic forms and processed forms. VEGFR can include VEGFR1, CEGFR2, and VEGFR3, as well as other alternatively spliced ​​variants. VEGFR can be membrane-bound or soluble. The term "VEGFR" can also refer to VEGFR from non-human species such as mouse, rat, or primate. In this application, VEGFR from a particular species can be represented as follows: hVEGFR represents human VEGFR, and mVEGFR represents murine VEGFR. Generally, VEGFR can refer to human VEGFR. The term "VEGFR" is also used to refer to truncated forms or fragments of intact VEGFR, and also includes functional variants, isoforms, species homologs, derivatives, analogs of VEGFR, and analogs that share at least one epitope with VEGFR. VEGFR sequences are known in the art. For example, an exemplary full-length human VEGFR2 protein sequence can be found under NCBI Accession No. NP_002244.

[0049] In this application, when referring to a protein, polypeptide and / or nucleic acid molecule, homologs of the protein, polypeptide and / or nucleic acid molecule are also included. In this application, the term "homolog" generally refers to an amino acid sequence or nucleotide sequence that has a certain homology to a wild-type amino acid sequence and a wild-type nucleotide sequence. The term "homology" can be equivalent to sequence "identity". Homologous sequences can include amino acid sequences that are at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the subject sequence. Typically, a homolog will contain the same active site, etc. as the subject amino acid sequence. Homology can be considered in terms of similarity (i.e., amino acid residues with similar chemical properties / functions), or it can be expressed in terms of sequence identity. In the present application, a sequence having a percentage identity to any one of the SEQ ID NOs of an amino acid sequence or a nucleotide sequence refers to a sequence having the said percentage identity over the entire length of the mentioned SEQ ID NO.

[0050] In this application, the term "antigen binding protein" generally refers to a protein comprising a portion that binds to an antigen, and optionally a scaffold or backbone portion that allows the portion that binds to the antigen to adopt a conformation that promotes the binding of the antigen binding protein to the antigen. Antigen binding proteins can typically comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both, and functional fragments thereof. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. Examples of antigen binding proteins include, but are not limited to, antibodies, antigen binding fragments, immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, as long as they exhibit the desired antigen binding activity.

[0051] In this application, the term "antibody" generally refers to an immunoglobulin that is reactive to a specified protein or peptide or a fragment thereof. The antibody can be an antibody from any class, including but not limited to IgG, IgA, IgM, IgD and IgE, and an antibody from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). The antibody can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody can also have a light chain selected from, for example, kappa (κ) or lambda (λ). The antibodies of the present application can be derived from any species.

[0052] In this application, the term "antigen-binding fragment" generally refers to a portion of an antibody molecule that comprises amino acid residues that interact with the antigen and impart specificity and affinity to the antibody for the antigen. Examples of antigen-binding fragments may include, but are not limited to, Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, and / or dAb. In this application, the term "Fab" generally refers to a fragment containing a heavy chain variable domain and a light chain variable domain, and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain; the term "Fab'" generally refers to a fragment that differs from Fab by the addition of a small number of residues (including one or more cysteines from the antibody hinge region) to the carboxyl terminus of the heavy chain CH1 domain; the term "F(ab')2" generally refers to a dimer of Fab', an antibody fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region. The term "Fv" generally refers to the smallest antibody fragment that contains a complete antigen recognition and binding site. In some cases, the fragment may be composed of a dimer of a heavy chain variable region and a light chain variable region in tight non-covalent association; the term "dsFv" generally refers to a disulfide-stabilized Fv fragment in which the bond between a single light chain variable region and a single heavy chain variable region is a disulfide bond. The term "dAb fragment" generally refers to an antibody fragment consisting of a VH domain. In this application, the term "scFv" generally refers to a monovalent molecule formed by a pair of heavy chain variable domains and a light chain variable domain of an antibody covalently linked by a flexible peptide linker; such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. In this application, the term "VHH" generally refers to an antibody comprising the variable antigen binding domain of a heavy chain antibody. VHH may also be referred to as a nanobody.

[0053] In this application, the term "variable region" or "variable domain" generally refers to the domain of the antibody heavy chain or light chain involved in the binding of the antibody to the antigen. In this application, the term "variable" generally refers to that some parts of the sequence of the variable domain of the antibody vary strongly, forming the binding and specificity of various specific antibodies to their specific antigens. Variability is not evenly distributed in the entire variable region of the antibody. It is concentrated in three segments in the light chain variable region and the heavy chain variable region, known as the complementary determining region (CDR) or hypervariable region (HVR), namely LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3. The more highly conserved parts in the variable domain are called framework regions (FR). The variable domains of natural heavy and light chains each include four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), most of which adopt a β-pleated sheet configuration and are connected by three CDR structure loop regions. The CDRs in each chain are held together by the FR regions and, together with the CDRs from the other chain, form the antibody antigen-binding site.

[0054] In the art, a variety of methods can be used to encode the variable region of an antibody or divide the CDR of an antibody, such as the Kabat numbering scheme and definition rules based on sequence variability, the Chothia numbering scheme and definition rules based on the position of the structural loop region, the IMGT numbering scheme and definition rules based on the amino acid sequence alignment of germline V genes by efranc et al., Honneger's numbering scheme (AHo's), Martin numbering scheme, Gelfand numbering scheme, etc.

[0055] As used herein, the term "monoclonal antibody" generally refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.

[0056] In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parent antibody"), and the constant region is derived from a human antibody, so that the resulting chimeric antibody is less likely to induce an adverse immune response in a human individual than the parent (e.g., mouse-derived) antibody.

[0057] In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., a mouse antibody) are replaced by corresponding amino acids derived from human immunoglobulins. In the CDR region, the addition, deletion, insertion, replacement or modification of amino acids may also be permitted, as long as they still retain the ability of the antibody to bind to a specific antigen. Humanized antibodies may optionally comprise at least a portion of a human immunoglobulin constant region. "Humanized antibodies" retain antigenic specificity similar to that of the original antibody. The "humanized" form of a non-human (e.g., mouse) antibody may minimally comprise a chimeric antibody derived from a sequence of a non-human immunoglobulin. In some cases, the CDR region residues in a human immunoglobulin (recipient antibody) may be replaced with CDR region residues of a non-human species (donor antibody) (such as mouse, rat, rabbit or non-human primate) with desired properties, affinity and / or ability. In some cases, the FR region residues of a human immunoglobulin may be replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain amino acid modifications that are not found in the recipient antibody or in the donor antibody.

[0058] In this application, the term "fully human antibody" generally refers to an antibody expressed by a genetically engineered animal by transferring a human antibody-encoding gene into an animal lacking the antibody gene. All parts of the antibody (including the variable and constant regions) are encoded by genes of human origin. Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, ribosome display technology, and RNA-peptide technology.

[0059] In this application, the terms "binding", "specific binding" or "specific for" generally refer to a measurable and reproducible interaction, such as binding between an antigen and an antibody, that can determine the presence of a target in the presence of a heterogeneous population of molecules (including biological molecules). For example, an antibody binds to an epitope through its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope. For example, an antibody that specifically binds to a target (which may be an epitope) is one that binds to that target with greater affinity, avidity, more readily, and / or for a greater duration than it binds to other targets. An antibody is said to "specifically bind" to an antigen when it binds to an epitope more readily through its antigen binding domain than it would to a random, unrelated epitope.

[0060] In this application, the terms "KD", "K D" are used interchangeably and generally refer to the equilibrium dissociation constant, and "KD" is the ratio of the dissociation rate constant (kdis, also known as "off-rate (koff)" or "kd") to the association rate constant (kon, also known as "binding rate (kon)" or "ka"). The association rate constant (kon), dissociation rate constant (kdis) and equilibrium dissociation constant (KD) can be used to express the binding affinity of an antigen-binding protein (e.g., an antibody) for an antigen. Methods for determining association and dissociation rate constants are well known in the art and include, but are not limited to, biomembrane interferometry (BLI), radioimmunoassay (RIA), equilibrium dialysis, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), co-immunoprecipitation (Co-IP), and protein chip technology. The affinity of a particular protein-protein interaction measured may be different if measured under different conditions (e.g., salt concentration, pH).

[0061] In this application, the term "primate" generally refers to monkey and ape species, and includes monkey species such as monkeys from the genus Macaca (e.g., crab-eating monkeys (Macaca fascicularis) and or rhesus monkeys (Macaca mulatta)) and baboons (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri), and tamarins (species from the genus Saguinus), and ape species such as chimpanzees (Pan troglodytes), and also includes Homo sapiens.

[0062] In this application, the terms "polypeptide" or "protein" are used interchangeably and generally refer to polymers of amino acid residues. The term also applies to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. The term may also include modified amino acid polymers, for example, by adding sugar residues to form glycoproteins or modified by phosphorylation. Polypeptides and proteins can be produced by naturally occurring and non-recombinant cells or by genetically engineered or recombinant cells, and may include molecules having the amino acid sequence of a native protein, or molecules having one or more amino acids deleted, added and / or substituted from the native sequence. The terms "polypeptide" and "protein" particularly include sequences of one or more amino acids deleted, added and / or substituted from the antigen-binding proteins described herein.

[0063] In this application, the term "isolated" generally refers to a biological material (e.g., a virus, nucleic acid, or protein) that is substantially free of components that are typically associated with or interact with the biological material in its naturally occurring environment. The isolated biological material optionally includes additional materials that are not found in the biological material in its natural environment (e.g., nucleic acid or protein). In this application, when referring to proteins, "isolated" generally refers to the separation and separation of the molecule from the entire organism in which the molecule is found naturally occurring, or the substantial absence of other biological macromolecules of the same type. When referring to nucleic acid molecules, it is completely or partially separated from the sequence to which it is naturally bound, or the nucleic acid has a heterologous sequence to which it is bound, or the nucleus is separated from the chromosome.

[0064] In this application, the term "immunoconjugate" generally refers to a substance formed by linking an antigen binding protein with other active agents, which can be small molecule active agents such as chemotherapeutic agents, toxins, immunotherapeutic agents, imaging probes or spectroscopic probes.

[0065] In this application, the term "nucleic acid" molecule generally refers to isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or their analogs, isolated from their natural environment or artificially synthesized.

[0066] In this application, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector primarily used for inserting DNA or RNA into a cell, a vector primarily used for replicating DNA or RNA, and a vector primarily used for expression of the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above-mentioned functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell containing the vector.

[0067] In this application, the term "cell" generally refers to an individual cell, cell line or cell culture that may contain or have contained a plasmid or vector comprising a nucleic acid molecule as described herein, or that can express an antigen-binding protein as described herein. The cell may include progeny of a single host cell. Due to natural, accidental or deliberate mutations, the progeny cells may not necessarily be identical in morphology or genome to the original parent cell, but may be able to express the antibody or antigen-binding fragment thereof as described herein. The cell may be obtained by in vitro transfection of cells using the vectors described herein. The cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, e.g., a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NSO cell or a myeloma cell). In some cases, the cell may be a mammalian cell. For example, the mammalian cell may be a CHO-K1 cell.

[0068] As used herein, the term "pharmaceutical composition" generally refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0069] In this application, the term "treatment" generally refers to the desire to change the natural course of the disease in the individual being treated, and can be achieved by preventing or treating the disease or by clinical intervention during the course of a clinical disease. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some cases, antigen binding proteins (e.g., anti-VEGF antibodies) can be used to delay disease development or slow disease progression.

[0070] In this application, the term "administering" generally refers to a method of administering a dose of a compound (e.g., an anticancer therapeutic agent) or a pharmaceutical composition (e.g., a pharmaceutical composition comprising an anticancer therapeutic agent) to a subject (e.g., a patient). Administration can be performed in any suitable manner, including parenteral, intrapulmonary, and intranasal, and (if desired for local treatment) intralesional administration. Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0071] In this application, the term "tumor" generally refers to all neoplastic cell growth and proliferation (whether malignant or benign) and all precancerous and cancerous cells and tissues. In this application, the tumor can be a tumor with high expression of VEGF or VEGFR in cells and tissues. Tumors can include solid tumors and / or non-solid tumors (e.g., hematologic tumors, lymphomas).

[0072] In this application, the term "between" generally means that the C-terminus of a certain amino acid segment is directly or indirectly linked to the N-terminus of a first amino acid segment, and its N-terminus is directly or indirectly linked to the C-terminus of a second amino acid segment. In the light chain, for example, the N-terminus of L-FR2 is directly or indirectly linked to the C-terminus of LCDR1, and the C-terminus of L-FR2 is directly or indirectly linked to the N-terminus of LCDR2. For another example, the N-terminus of L-FR3 is directly or indirectly linked to the C-terminus of LCDR2, and the C-terminus of L-FR3 is directly or indirectly linked to the N-terminus of LCDR3. In the heavy chain, for example, the N-terminus of H-FR2 is directly or indirectly linked to the C-terminus of HCDR1, and the C-terminus of H-FR2 is directly or indirectly linked to the N-terminus of HCDR2. For another example, the N-terminus of H-FR3 is directly or indirectly linked to the C-terminus of HCDR2, and the C-terminus of H-FR3 is directly or indirectly linked to the N-terminus of HCDR3. In the present application, the "first amino acid fragment" and the "second amino acid fragment" can be any identical or different amino acid fragments.

[0073] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0074] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0075] Detailed Description of the Invention

[0076] In one aspect, the present application provides an antigen-binding protein comprising a first targeting moiety and a second targeting moiety, wherein the first targeting moiety comprises an antibody or an antigen-binding fragment thereof that specifically binds to PD-1, and the second targeting moiety comprises an antibody or an antigen-binding fragment thereof that specifically binds to VEGF;

[0077] wherein the first targeting moiety comprises HCDR1, HCDR2, and HCDR3 of an antibody or antigen-binding fragment thereof, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 5 or 11, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 6 or 12, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7 or 13;

[0078] wherein the first targeting moiety comprises LCDR1, LCDR2, and LCDR3 of an antibody or antigen-binding fragment thereof, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8 or 14, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9 or 15, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10 or 16;

[0079] Wherein, the second targeting portion comprises HCDR1, HCDR2 and HCDR3 of an antibody or an antigen-binding fragment thereof, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 26, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 27, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 28.

[0080] For example, the first targeting portion of the present application may comprise HCDR1, HCDR2 and HCDR3 of an antibody or an antigen-binding fragment thereof, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 6, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 7.

[0081] For example, the first targeting portion of the present application may comprise LCDR1, LCDR2 and LCDR3 of an antibody or an antigen-binding fragment thereof, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10.

[0082] For example, the first targeting portion of the present application may comprise HCDR1, HCDR2 and HCDR3 of an antibody or an antigen-binding fragment thereof, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 12, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 13.

[0083] For example, the first targeting portion of the present application may comprise LCDR1, LCDR2 and LCDR3 of an antibody or an antigen-binding fragment thereof, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16.

[0084] For example, the first targeting moiety of the present application may comprise HCDR3, and the HCDR3 may comprise the HCDR3 of a VH having an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 3, 22, and 24. For example, the first targeting moiety of the present application may comprise HCDR2, and the HCDR2 may comprise the HCDR2 of a VH having an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 3, 22, and 24. For example, the first targeting moiety of the present application may comprise HCDR1, and the HCDR1 may comprise the HCDR1 of a VH having an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 3, 22, and 24. For example, the first targeting moiety of the present application may comprise HCDR3, HCDR2, and HCDR1, and the HCDR3, HCDR2, and HCDR1 may comprise the HCDR3, HCDR2, and HCDR1 of a VH having an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 3, 22, and 24. For example, the first targeting moiety of the present application may comprise HFR1, HFR2, HFR3, and / or HFR4, wherein the HFR1, HFR2, HFR3, and HFR4 may respectively comprise the HFR1, HFR2, HFR3, and HFR4 of the VH having an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3, 22, and 24. For example, the classification may be based on the Chothia rule.

[0085] For example, the first targeting moiety of the present application may comprise LCDR3, and the LCDR3 may comprise LCDR3 of VL having an amino acid sequence as shown in any one of SEQ ID NOs: 2, 4, 21, and 23. For example, the first targeting moiety of the present application may comprise LCDR2, and the LCDR2 may comprise LCDR2 of VL having an amino acid sequence as shown in any one of SEQ ID NOs: 2, 4, 21, and 23. For example, the first targeting moiety of the present application may comprise LCDR1, and the LCDR1 may comprise LCDR1 of VL having an amino acid sequence as shown in any one of SEQ ID NOs: 2, 4, 21, and 23. For example, the first targeting moiety of the present application may comprise LCDR3, LCDR2, and LCDR1, and the LCDR3, LCDR2, and LCDR1 may comprise LCDR3, LCDR2, and LCDR1 of VL having an amino acid sequence as shown in any one of SEQ ID NOs: 2, 4, 21, and 23. For example, the first targeting moiety of the present application may comprise LFR1, LFR2, LFR3, and / or LFR4, wherein the LFR1, LFR2, LFR3, and LFR4 may respectively comprise the LFR1, LFR2, LFR3, and LFR4 of the VL of any one of the amino acid sequences as shown in SEQ ID NOs: 2, 4, 21, and 23. For example, the classification may be based on the Chothia rule.

[0086] In one embodiment, the present application provides an antigen binding protein comprising a first targeting moiety and a second targeting moiety, wherein the first targeting moiety comprises an antibody or an antigen-binding fragment thereof that specifically binds to PD-1, and the second targeting moiety comprises an antibody or an antigen-binding fragment thereof that specifically binds to VEGF:

[0087] Wherein, the antibody or antigen-binding fragment thereof that specifically binds to PD-1 comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 5, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 6, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 7; LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; or

[0088] wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13; LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16,

[0089] The antibody or antigen-binding fragment thereof that specifically binds to VEGF comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 26, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 27, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 28.

[0090] In a specific embodiment, the second targeting moiety is linked directly or via a linker to the C-terminus of the antibody heavy chain of the first targeting moiety.

[0091] For example, the first targeting moiety comprises an antibody heavy chain variable region VH, and the VH comprises an amino acid sequence selected from any one of SEQ ID NOs: 1, 3, 22 and 24.

[0092] For example, the first targeting moiety comprises an antibody light chain variable region VL, and the VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 2, 4, 21 and 23.

[0093] For example, wherein the first targeting moiety comprises an antibody heavy chain variable region VH and an antibody light chain variable region VL, the VH comprises the amino acid sequence set forth in SEQ ID NO: 1, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 2. For example, the VH comprises the amino acid sequence set forth in SEQ ID NO: 3, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 4. For example, the VH comprises the amino acid sequence set forth in SEQ ID NO: 22, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 21. For example, the VH comprises the amino acid sequence set forth in SEQ ID NO: 24, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0094] For example, the first targeting moiety further comprises an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from an IgG constant region. For example, it is derived from an IgG4 constant region. For example, the first targeting moiety can comprise the amino acid sequence set forth in SEQ ID NO: 32. For example, the first targeting moiety further comprises an antibody light chain constant region. For example, the first targeting moiety can comprise the amino acid sequence set forth in SEQ ID NO: 31.

[0095] According to the antigen-binding protein of the present application, the second targeting moiety comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 25. According to the antigen-binding protein of the present application, the second targeting moiety comprises an antibody heavy chain constant region. For example, derived from an IgG1 constant region. For example, the second targeting moiety can comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the second targeting moiety is a nanobody.

[0096] In one embodiment, the present application provides an antigen-binding protein comprising a first targeting moiety and a second targeting moiety, wherein the first targeting moiety comprises an antibody or an antigen-binding fragment thereof that specifically binds to PD-1, and the second targeting moiety comprises an antibody or an antigen-binding fragment thereof that specifically binds to VEGF, wherein the antigen-binding protein is a bispecific antibody Kab5 or Kab6.

[0097] In one embodiment, the bifunctional antibody Kab5 comprises a heavy chain or a light chain, wherein the heavy chain comprises the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 29 and SEQ ID NO: 25 from N-terminus to C-terminus; and the light chain comprises the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 31 from N-terminus to C-terminus.

[0098] In one embodiment, the bifunctional antibody Kab6 comprises a heavy chain or a light chain, wherein the heavy chain comprises the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 32 and SEQ ID NO: 25 from N-terminus to C-terminus; and the light chain comprises the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 31 from N-terminus to C-terminus.

[0099] In the present application, a portion of each heavy chain or light chain amino acid sequence of the antigen-binding protein is homologous to the corresponding amino acid sequence in an antibody from a specific species, or belongs to a specific class. For example, the variable region and constant portion of the light chain and heavy chain are both from the variable region and constant region of an antibody from an animal species (such as a human). In the present application, the homolog can be a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the protein and / or the polypeptide.

[0100] In the present application, described homology generally refers to the similarity, similarity or association between two or more sequences.In order to determine the comparison of sequence homology percentage, can realize by multiple ways known in the art, for example, use publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the alignment sequence, comprise for realizing in the full-length sequence range that is being compared or in the target sequence zone maximum to required any algorithm.Described homology also can be measured by following method: FASTA and BLAST.

[0101] According to the antigen-binding protein of the present application, the antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, and a fully human antibody. For example, the antigen-binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and / or dAb. For example, the antigen-binding protein comprises a bispecific antibody. For example, the antigen-binding protein comprises an IgG-VHH type bispecific antibody.

[0102] For example, the second targeting moiety is located at the C-terminus and / or N-terminus of the first targeting moiety.For example, the first targeting moiety comprises an antibody heavy chain and an antibody light chain, and the second targeting moiety is located at the C-terminus of the antibody heavy chain and / or the antibody light chain of the first targeting moiety.

[0103] For example, the second targeting moiety is directly or indirectly linked to the first targeting moiety. For example, the second targeting moiety is linked to the first targeting moiety via a linker. For example, a linker connecting two polypeptides at both ends is known in the art to link the second targeting moiety to the first targeting moiety. For example, the linker comprises the amino acid sequence set forth in SEQ ID NO: 29.

[0104] According to the antigen-binding protein of the present application, the antigen-binding protein comprises the antibody heavy chain or antigen-binding fragment thereof of the first targeting moiety, an optional linker and the antibody or antigen-binding fragment thereof of the second targeting moiety, and the second polypeptide chain comprises the antibody light chain or antigen-binding fragment thereof of the first targeting moiety. For example, the first targeting moiety and the second targeting moiety in the first polypeptide chain in the present application can be directly connected, such as the linker is absent or connected by a chemical bond. For example, the first targeting moiety and the second targeting moiety in the first polypeptide chain in the present application can be indirectly connected, such as through a linker. For example, the linker structure can be a connecting peptide known in the present application. For example, the first polypeptide chain can comprise: the heavy chain variable region of the first targeting moiety-the heavy chain constant region of the first targeting moiety-X-the variable region of the second targeting moiety-the constant region of the second targeting moiety, and the second polypeptide chain can comprise: the light chain variable region of the first targeting moiety-the light chain constant region of the first targeting moiety, wherein the X is absent or comprises the linker.

[0105] For example, the first polypeptide chain comprises, from N-terminus to C-terminus, an antibody heavy chain or an antigen-binding fragment thereof of a first targeting moiety and an antibody or an antigen-binding fragment thereof of a second targeting moiety. For example, the first polypeptide chain comprises, from N-terminus to C-terminus, an antibody heavy chain or an antigen-binding fragment thereof of a first targeting moiety, a linker, and an antibody or an antigen-binding fragment thereof of a second targeting moiety.

[0106] In the present application, the isolated antigen binding protein can be expressed as 1×10 -8 M or lower K D The K D The value can be approximately 1×10 -8 M or smaller, approximately 9×10 -9 M or smaller, about 8×10 -9 M or smaller, about 7×10 -9 M or smaller, about 6×10 -9 M or smaller, about 5×10 -9 M or smaller, about 4×10 -9 M or smaller, about 3×10 -9 M or smaller, about 2×10-9 M or less, about 1×10 -9 A value of M or less binds to human-derived PD-1, for example, as detected using the FortieBio Octet Molecular Interaction Analyzer.

[0107] In another embodiment, the binding activity of the PD-1 antigen-binding protein described herein to PD-1 can be detected using flow cytometry or enzyme-linked immunosorbent assay. For example, in a FACS assay, using host cells stably expressing human PD-1 (such as CHOK1 cells), the PD-1 antigen-binding protein binds to PD-1 with an EC50 value of between about 0.0001 nM and about 100 nM, for example, between about 0.001 nM and about 10 nM, between about 0.001 nM and about 5 nM, between about 0.001 nM and about 1 nM, between about 0.01 nM and about 1 nM, between about 0.01 nM and about 1.2 nM, or between about 0.1 nM and about 1.5 nM.

[0108] For example, in a FACS assay, using host cells stably expressing monkey PD-1 (such as CHOK1 cells), the PD-1 antigen binding protein binds to PD-1 with an EC50 value of between about 0.0001 nM and about 100 nM, for example, between about 0.001 nM and about 10 nM, between about 0.001 nM and about 5 nM, between about 0.01 nM and about 1 nM, between about 0.02 nM and about 1.0 nM, or between about 0.1 nM and about 1.0 nM.

[0109] In another aspect, the antigen binding proteins described herein are capable of blocking the binding of PD-1 to PD-L1. In certain instances, the blocking of the binding of PD-1 to PD-L1 by the antigen binding proteins can be determined by flow cytometry (FACS) or enzyme-linked immunosorbent assay (ELISA).

[0110] For example, host cells stably expressing human PD-1 (such as HEK293 cells) are first incubated with decreasing amounts of the unlabeled antigen binding protein, followed by incubation with biotin-labeled PD-L1 protein. The cells are then analyzed using FACS to confirm that the antigen binding protein blocks the binding of PD-1 to PD-L1. For example, the IC50 value is between about 0.001 nM and about 10 nM, between about 0.001 nM and about 5 nM, between about 0.01 nM and about 5 nM, between about 0.1 nM and about 1.0 nM, and between about 0.5 nM and about 1.5 nM.

[0111] For example, host cells stably expressing human PD-1 (e.g., CHOK1 cells) are first incubated with decreasing amounts of the unlabeled antigen-binding protein, followed by incubation with biotin-labeled PD-L2 protein. The cells are then analyzed using FACS to confirm that the antigen-binding protein blocks the binding of PD-1 to PD-L2. For example, the concentration range is between about 0.001 nM and about 10 nM, between about 0.001 nM and about 5 nM, between about 0.1 nM and about 2.5 nM, or between about 0.1 nM and about 3 nM.

[0112] In the present application, the isolated antigen binding protein can be expressed as 1×10 -7 M or lower KD value to bind VEGF (e.g., VEGF165). For example, the KD value can be about 5×10 -8 M or smaller, about 4×10 -8 M or smaller, about 3×10 -8 M or smaller, about 2×10 -8 M or less, about 1×10 -8 M or smaller, approximately 9×10 -9 M or smaller, about 8×10 -9 M or smaller, about 7×10 -9 M or smaller, about 6×10 -9 M or smaller, about 5×10 -9 M or smaller, about 4×10 -9 M or smaller, about 3×10 -9 M or smaller, about 2×10 -9 M or less, about 1×10 -9 M or smaller, approximately 9×10 -10 M or smaller, about 8×10 -10 M or smaller, about 7×10 -10 Values ​​of M or less bind to human-derived VEGF, for example, as detected using the FortieBio Octet Molecular Interaction Analyzer.

[0113] The antigen binding proteins described herein can block the combination of VEGF (e.g., VEGF165) and VEGFR (e.g., VEGFR2). In some cases, the combination of VEGF and VEGFR blocked by the antigen binding proteins can be measured by flow cytometry (FACS) or enzyme-linked immunosorbent assay (ELISA).

[0114] For example, first human VEGFR (e.g., VEGFR2) is incubated with decreasing amounts of unlabeled antigen-binding proteins, followed by incubation with labeled VEGF protein. The OD450 is then measured to confirm that the antigen-binding proteins block VEGF (e.g., VEGF165) from binding to VEGFR (e.g., VEGFR2). For example, the IC50 for blocking activity is between about 0.001 μg / mL and about 10 μg / mL, between about 0.001 μg / mL and about 5 μg / mL, between about 0.01 μg / mL and about 1 μg / mL, between about 0.02 μg / mL and about 0.5 μg / mL, between about 0.2 μg / mL and about 15 μg / mL, between about 0.2 μg / mL and about 12 μg / mL, between about 0.2 μg / mL and about 10 μg / mL, between about 0.3 μg / mL and about 8 μg / mL, between about 0.3 μg / mL and about 6 μg / mL, between about 0.5 μg / mL and about 5 μg / mL, between about 0.1 μg / mL and about 2 μg / mL, or between about 0.5 μg / mL and about 1.5 μg / mL.

[0115] In one aspect, the present application provides a nucleic acid encoding an antigen-binding protein of the present application. The one or more nucleic acid molecules may encode the antigen-binding protein described herein. For example, each of the one or more nucleic acid molecules may encode a complete polypeptide chain of the antigen-binding protein. The nucleic acid molecules described herein may be isolated.

[0116] On the one hand, the application provides a kind of vector, it comprises the nucleic acid of the application.The nucleic acid molecule described in the application can be isolated.For example, it can be produced or synthesized by the following method: (i) amplified in vitro, for example, produced by polymerase chain reaction (PCR) amplification, (ii) produced by cloning and recombination, (iii) purified, for example, by enzyme cutting and gel electrophoresis fractionation, or (iv) synthesized, for example, by chemical synthesis.In some embodiments, the nucleic acid of the isolation is the nucleic acid molecule prepared by recombinant DNA technology.

[0117] In one aspect, the present application provides an immunoconjugate comprising an antigen-binding protein of the present application. For example, the immunoconjugate of the present application may further comprise a compound having cell-killing ability. For example, the immunoconjugate of the present application may further comprise a transmembrane domain, an intracellular stimulatory domain, and / or an intracellular signaling domain.

[0118] In one aspect, the present application provides a cell comprising the antigen-binding protein, nucleic acid, vector, and / or immunoconjugate of the present application. For example, the present application provides a host cell, which may comprise one or more nucleic acid molecules and / or one or more vectors described herein. In certain embodiments, each or each host cell may comprise one or more nucleic acid molecules or vectors described herein. For example, the cells of the present application do not have the potential to develop into a complete organism.

[0119] On the one hand, the present application provides a pharmaceutical composition comprising the antigen-binding protein of the present application, the nucleic acid of the present application, the vector of the present application, the immunoconjugate of the present application, and / or the cell of the present application, and optionally a pharmaceutically acceptable carrier. The pharmaceutically acceptable adjuvant is non-toxic to the recipient at the dosage and concentration adopted. The pharmaceutical composition of the present application may also contain more than one active compound, generally those active compounds with complementary activities that do not adversely affect each other.

[0120] In one aspect, the present application provides a kit comprising the antigen-binding protein of the present application, the nucleic acid of the present application, the vector of the present application, the immunoconjugate of the present application, the cell of the present application, and / or the pharmaceutical composition of the present application.

[0121] In one aspect, the present application provides a method for preparing the antigen-binding protein of the present application, comprising culturing the cell of the present application under conditions that allow the antigen-binding protein to be expressed.

[0122] On the one hand, the present application provides an antigen-binding protein of the present application, a nucleic acid of the present application, a vector of the present application, an immunoconjugate of the present application, a cell of the present application, a pharmaceutical composition of the present application, and / or a kit of the present application for the preparation of a drug, wherein the drug is used to prevent, alleviate and / or treat tumors. For example, wherein the tumor includes a tumor with high expression of PD-1, PD-L1 and / or VEGF. According to the use of the present application, wherein the tumor includes solid tumors and / or non-solid tumors. According to the use of the present application, wherein the tumor includes colorectal cancer, breast cancer, blastoma, cervical cancer, ovarian cancer, melanoma, lung cancer, kidney cancer, esophageal cancer, head and neck cancer, lymphoma, liver cancer and / or gastric cancer.

[0123] On the one hand, the present application provides an antigen-binding protein of the present application, a nucleic acid of the present application, a vector of the present application, an immunoconjugate of the present application, a cell of the present application, a pharmaceutical composition of the present application, and / or a kit of the present application, which is used to prevent, alleviate and / or treat tumors. For example, wherein the tumor includes a tumor with high expression of PD-1, PD-L1 and / or VEGF. According to the use of the present application, wherein the tumor includes solid tumors and / or non-solid tumors. According to the use of the present application, wherein the tumor includes colorectal cancer, breast cancer, blastoma, cervical cancer, ovarian cancer, melanoma, lung cancer, kidney cancer, esophageal cancer, head and neck cancer, lymphoma, liver cancer and / or gastric cancer.

[0124] On the one hand, the present application provides a method for preventing, alleviating and / or treating tumors, comprising administering the antigen-binding proteins of the present application, the nucleic acids of the present application, the vectors of the present application, the immunoconjugates of the present application, the cells of the present application, the pharmaceutical compositions of the present application, and / or the kits of the present application. For example, wherein the tumor comprises a tumor with high expression of PD-1, PD-L1 and / or VEGF. According to the use of the present application, wherein the tumor comprises a solid tumor and / or a non-solid tumor. According to the use of the present application, wherein the tumor comprises colorectal cancer, breast cancer, blastoma, cervical cancer, ovarian cancer, melanoma, lung cancer, kidney cancer, esophageal cancer, head and neck cancer, lymphoma, liver cancer and / or gastric cancer.

[0125] In one aspect, the present application provides a method for inhibiting the binding of PD-1 protein to PD-L1 protein and / or PD-L2 protein, and / or inhibiting the binding of VEGF protein to VEGFR protein, the method comprising administering the antigen-binding protein, nucleic acid, vector, immunoconjugate, cell, pharmaceutical composition, and / or kit of the present application. For example, the method may be an in vitro or in vitro method. For example, the method may be a method for non-therapeutic purposes. In certain cases, the method may comprise contacting a biological sample with an antigen-binding protein and / or PD-1 described herein under conditions that allow the antigen-binding protein and / or PD-1 to bind to PD-L1, and detecting whether a complex is formed between PD-1 and PD-L1. In certain cases, the method may comprise contacting a biological sample with an antigen-binding protein and / or VEGF described herein under conditions that allow the antigen-binding protein and / or VEGF to bind to VEGFR, and detecting whether a complex is formed between VEGFR and VEGFR.

[0126] A method for regulating an immune response, the method comprising administering the antigen-binding protein, nucleic acid, vector, immunoconjugate, cell, pharmaceutical composition, and / or kit of the present invention. For example, the regulating an immune response comprises stimulating immune cells to secrete cytokines. For example, the regulating an immune response comprises stimulating immune cells to secrete IL-2.

[0127] Without intending to be bound by any theory, the following embodiments are merely intended to illustrate various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0128] Example

[0129] Example 1 Preparation and detection of anti-PD-1 antibodies

[0130] 1.1 Hybridoma Antibody Screening and Preparation

[0131] Five Balb / c and five SJL mice were immunized with human PD-1, mouse IgG2a Fc Tag fusion protein (Acrobiosystem, PD1-H5255a) and pcDNA3.4-hPD-1 plasmid DNA, 2 to 3 weeks apart. After three booster immunizations, five mice were given one to three times with human PD-1, mouse IgG2a Fc Tag fusion protein or CHOK1-hPD-1 stably transfected cell lines (GenScript, M00529). Blood was collected for titer determination. Flow cytometry was used to measure the binding of serum to CHOK1 cells or 293T cells overexpressing human PD-1. Splenocytes from mice with high binding titers were then fused with myeloma (Sp2 / 0) cells.

[0132] Flow cytometry was used to screen clones from the fusion plate that specifically bound to CHOK1-hPD-1 cells and simultaneously blocked the binding of ligands PD-L1 and PD-L2 to CHOK1-hPD1 cells. Subcloning was performed using limiting dilution, ultimately resulting in the identification of monoclonal clones that secreted specific antibodies. Small-scale production and purification of the monoclonal antibodies in serum-free medium were performed for subsequent characterization, including testing for antibody binding to CHOK1-hPD1, blocking the binding of PD-L1 and PD-L2 to CHOK1-hPD-1, and mixed lymphocyte assays. Finally, candidate anti-human PD-1 monoclonal antibodies 41D2-2C3D7 and 46H3A8 were identified. Monoclonal antibody sequencing revealed the variable region sequences of the murine hybridoma clones 41D2-2C3D7 and 46H3A8 as follows:

[0133] >41D2-2C3D7 heavy chain variable region sequence (SEQ ID NO: 1)

[0134] >41D2-2C3D7 light chain variable region sequence (SEQ ID NO: 2)

[0135] >46H3A8 heavy chain variable region sequence (SEQ ID NO: 3)

[0136] >46H3A8 light chain variable region sequence (SEQ ID NO: 4)

[0137] Table 1 Heavy and light chain CDR sequences of murine hybridoma clone 41D2-2C3D7

[0138] Table 2 Heavy and light chain CDR sequences of murine hybridoma clone 46H3A8

[0139] 1.2 Humanization of mouse hybridoma 41D2-2C3D7 and 46H3A8 monoclonal antibodies

[0140] The most homologous human germline antibody (e.g., from IMGT) was selected through sequence alignment as the humanization design framework.

[0141] The light chain framework was IGKV1-9*01 (SEQ ID NO: 17) and IGKJ4*01 (SEQ ID NO: 18), while the heavy chain framework was IGHV3-21*01 (SEQ ID NO: 19) and IGHJ5*01 (SEQ ID NO: 20). Based on sequence alignment and variable region structure information, the light and heavy chain variable regions of 41D2-2C3D7 and 46H3A8 underwent one to two rounds of humanization mutagenesis. After mutagenesis, the humanized light and heavy chain variable regions 41D2HzL4 (SEQ ID NO: 21) and 41D2HzH3 (SEQ ID NO: 22) were obtained for 41D2-2C3D7, and the humanized light and heavy chain variable regions 1910h3hzL3 (SEQ ID NO: 23) and 1910h3hzH4 (SEQ ID NO: 24) were obtained for 46H3A8. The humanized antibody light and heavy chain variable regions can be numbered using Chothia to define the antibody light and heavy chain CDR regions. The 41D2HzL4 and 1910h3hzL3 sequences were combined with the human kappa chain constant region CL (amino acid sequence SEQ ID NO: 31) to form the antibody light chain, and the 41D2HzH3 and 1910h3hzH4 sequences were combined with the human IgG4 constant region CH (amino acid sequence SEQ ID NO: 32) to form the antibody heavy chain. After these combinations, the humanized antibodies 41D2HzL4H3 (also referred to as 1910D2HzL4H3) and 1910h3hzL3H4 were paired.

[0142] >41D2HzL4 light chain variable region sequence (SEQ ID NO: 21)

[0143] >41D2HzH3 heavy chain variable region sequence (SEQ ID NO: 22)

[0144] >1910h3hzL3 light chain variable region sequence (SEQ ID NO: 23)

[0145] >1910h3hzH4 heavy chain variable region sequence (SEQ ID NO: 24)

[0146] 1.3 Determination of humanized antibody kinetic parameters

[0147] (1) Octet RED96e (Fortebio) was used to determine the affinity of humanized antibodies 1910h3hzL3H4 and 41D2HzL4H3 to human PD-1 (Sino Biological, Catalog No.: 10377-H08H). Antigen and antibody were diluted with 1xPBST (1xPBS: Biochemical, B548117-0500; 0.02% Tween 20: Sigma-Aldrich, P1379). The antigen concentration was 100 nM and the antibody concentration was 50 nM.

[0148] (2) Sample detection (Octet Data Acquisition 11.1.0.11): First, add the sample to a 96-well plate (Greiner bio-one, 655209) with a system of 200 μL / well. Then set the software parameters, set the plate temperature to 30°C, and collect the standard kinetic signal at a frequency of 5.0 Hz. Next, pre-wet the AHC sensor (Fortébio, catalog number: 18-0015) with 1xPBST for 10 minutes and then detect it on the machine. Each cycle includes the following steps: 1) immersion in buffer for 60 seconds; 2) detection of non-specific binding of antigen to sensor; 3) regeneration with 10 mM glycine solution at pH 1.7; 4) immersion in buffer for 60 seconds; 5) antibody immobilization on the sensor for 20 seconds; 6) sensor immersion in buffer for 180 seconds; 7) antigen binding to antibody for 180 seconds; 8) antigen-antibody dissociation for 10 minutes; 9) sensor regeneration.

[0149] (3) Data Analysis

[0150] The association rate (Ka) and dissociation rate (Kd) of the antigen-antibody binding were measured using Fortebio's Data Analysis 11.0 software in a 1:1 ratio, and the equilibrium dissociation constant (Kd) of the antibody was calculated. D The results are shown in Table 3:

[0151] Table 3 Affinity of humanized antibodies to human PD-1

[0152] The results showed that the affinity of 1910h3hzL3H4 for human PD-1 was comparable to that of pembrolizumab, while the affinity of 41D2HzL4H3 for human PD-1 was approximately 4.2 times higher than that of pembrolizumab, indicating that the humanized antibody has a higher binding affinity for PD-1.

[0153] 1.4 Binding activity of candidate antibodies to human or cynomolgus monkey PD-1 on cell surfaces

[0154] CHOK-hPD-1 (GenScript, M00529) or CHOK1-cynoPD-1 (GenScript, M00572) cells were harvested, washed once with FACS buffer (PBS containing 2% FBS), and resuspended. The cells were aliquoted into a 96-well plate (Corning 3799) at a ratio of 1E5 / 50 μl / well. Antibodies were added to the 96-well plate at a ratio of 50 μl / well, mixed, and incubated at 4°C for 1 hour. The cells were centrifuged at 2000 rpm for 5 minutes, the supernatant removed, and the cells were washed once with 200 μl of FACS buffer per well. Alexa Fluor 488 secondary antibody (Invitrogen) at a 1:1000 dilution was added to each well at a ratio of 100 μl / well, mixed, and incubated at 4°C for 1 hour. The cells were centrifuged at 2000 rpm for 5 minutes, the supernatant removed, and the cells were washed once with 200 μl of FACS buffer per well. The cells were resuspended in 30 μl of FACS buffer and analyzed.

[0155] Figure 1 shows the binding of the antigen-binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described herein to human PD-1 protein on the surface of CHOK1 cells. Figure 2 shows the binding of the antigen-binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described herein to cynomolgus monkey PD-1 protein on the surface of CHOK1 cells. The results demonstrate that the humanized antibodies exhibit comparable binding activity to human and cynomolgus monkey PD-1 on cell surfaces as pembrolizumab.

[0156] 1.5 Candidate antibodies block the binding activity of human PD-1 to PD-L1 or PD-L2 on the cell surface

[0157] CHOK1-hPD-1 cells were harvested, washed once with FACS buffer, and resuspended. The cells were then aliquoted into a 96-well U-shaped plate at a concentration of 1E5 / 50 μl / well. Antibodies were diluted and added to the plate at a concentration of 25 μl / well. Biotinylated PD-L1 (Sinobiological, Catalog No. 10084-H02H-B) (or PD-L2, Acrobiosystem, Catalog No. PD2-H82F6) was diluted and added to the plate at a concentration of 25 μl / well. The cells were mixed and incubated at 4°C for 1 hour. The cells were centrifuged at 2000 rpm for 5 minutes, the supernatant removed, and the cells were washed once with 200 μl of FACS buffer per well. SA-Alexa Fluor 488 (Invitrogen, Catalog No. S32354) at a dilution of 1:1000 was added to each well at a concentration of 100 μl / well. The cells were mixed and incubated at 4°C for 30 minutes. Centrifuge at 2000 rpm for 5 minutes, remove the supernatant, add 200 μl FACS buffer to each well to wash once, resuspend in 30 μl FACS buffer and test on the instrument.

[0158] Figure 3 shows that the antigen-binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described herein blocked the binding of human PD-L1 to the human PD-1 protein on the surface of CHOK1 cells. Figure 4 shows that the antigen-binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described herein blocked the binding of human PD-L2 to the human PD-1 protein on the surface of CHOK1 cells. The results demonstrate that the humanized antibodies have good activity in blocking the binding of human PD-1 to PD-L1 or PD-L2 on the cell surface.

[0159] 1.6 Mixed lymphocyte reaction induced by candidate antibodies

[0160] Human dendritic cells (DCs) were revived using cell culture medium (1640 + 2% FBS) and the DC cell density was adjusted to 1*10^5-1*10^7 cells / mL. Mitomycin C was then added to a final concentration of 50μg / mL. After incubation at 37°C in the dark for 30 minutes, 10ml of culture medium was added to terminate the incubation. The cells were centrifuged at 400g for 10 minutes and then washed once with 10ml of culture medium. Anti-PD-1 antibody was serially diluted: the highest final concentration of the antibody was 2.5μg / mL (prepared at 10μg / mL). A 10-fold serial dilution (5 concentration points + 1 zero concentration) was performed. 50μL of the prepared anti-PD-1 antibody was then added to the corresponding cell culture plate (Corning, Cat. No. 3599). Human peripheral blood lymphocytes (PBMCs) and mitomycin C-treated dendritic cells (DCs) were collected and the DC density was adjusted to 2 x 10^5 cells / mL. 50 μL of cells were then added to each well of the culture plate, resulting in a DC cell count of 1 x 10^4 cells / well. PBMC density was adjusted to 2 x 10^6 cells / mL and 100 μL of cells were then added to each well of the culture plate, resulting in a PBMC cell count of 2 x 10^5 cells / well. The cell culture plates were incubated at 37°C, 5% CO2 in a cell culture incubator for 5 days. After 5 days, the cells were centrifuged at 300 g for 5 minutes, and the supernatant was collected and assayed for IL-2 content using a Human IL-2 ELISA kit (BD, Cat. No. 550611). The assay was performed strictly according to the kit instructions, and the data were processed using GraphPad Prism software.

[0161] The results are shown in Figure 5 , which show that both humanized antibodies 1910h3hzL3H4 and 41D2HzL4H3 can stimulate lymphocytes to secrete IL2, with activity similar to that of Pembrolizumab, indicating that the antigen-binding proteins of the present application have a good ability to stimulate immune cells to secrete cytokines.

[0162] Example 2 Preparation and Detection of Anti-VEGF Single Domain Antibodies

[0163] 2.1 Construction and panning of phage single-domain antibody library

[0164] Blood samples of 150 ml were collected from three alpacas that had not been immunized with the target antigen. Lymphocytes were isolated from the blood samples and total RNA was extracted to construct a phage display nanoantibody library with a library capacity of 3*10 9 Take 6 ml of transformed antibody library bacteria to prepare phage for specific panning, and the total amount of bacteria should be 50 times greater than the library capacity.

[0165] 50ml of Streptavidin Magnetic Beads (Thermo Fisher, Catalog No. 88817) were pre-bound to 1ml of phage and incubated at room temperature for 30 minutes to remove nonspecific binding. After background removal, the nanobody library phage were added with 10ug of Biotinylated Human VEGF165 (Biopsy, Catalog No. VE5-H82Q0) and 150ul of Streptavidin Magnetic Beads and incubated at room temperature for 15 minutes. Unbound phage were then washed 14 times with PBST (PBS containing 0.05% Tween-20) to remove the antigen. Antigen-specific phage were eluted with 450ul of 100mM hydrochloric acid, neutralized with 50ul of 1M Tris-HCl (pH 11), and infected with Escherichia coli SS320 cells in the logarithmic growth phase to generate and purify phage for the next round of screening. The screening method was the same as the first round, except that the amount of antigen was reduced to 4ug. The enriched phages after two rounds of screening were identified by enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 4. The results showed that the phages were significantly enriched after two rounds of panning.

[0166] Table 4 ELISA identification after the second round of enrichment

[0167] 2.2 Yeast display library construction and screening

[0168] As shown in Figure 6, the construction scheme of single-domain antibodies displayed on the yeast cell wall surface was as follows: using the plasmid after two rounds of panning of the phage antibody library as a template, primers were designed to perform polymerase chain reaction (PCR) amplification of the nanobody gene (V H H); PCR amplified V H After the H gene fragment is recovered, it is co-transformed with the yeast display plasmid into the Saccharomyces cerevisiae strain EBY100 (purchased from ATCC). Through homologous recombination of Saccharomyces cerevisiae, the VHH gene is inserted into the yeast display plasmid, thereby realizing the display of single-domain antibodies on the surface of the yeast cell wall.

[0169] The yeast display library was sorted twice using a flow cytometer. The yeast obtained from the sorting was spread on a nutrient-deficient plate culture medium. 46 monoclonal clones were selected for sequencing, and finally 5 single-domain heavy chain antibodies with unique sequences were obtained. The corresponding yeast monoclonal colonies were subjected to flow cytometric analysis, and 1×10 6 The cells were stained according to the various protocols shown in Table 5.

[0170] Table 5 Flow cytometry identification protocol for monoclonal yeast colonies

[0171] The strength of binding to human VEGF165-Bio in protocol 1 was measured by the mean fluorescence intensity (MFI) of PE. Similarly, protocols 2 and 3 were used to assess mouse VEGF-Bio and nonspecific binding. The competition signal in protocol 4 was measured by the mean fluorescence intensity (MFI) of APC. The results are shown in Table 6. By eliminating clones that did not bind to human or mouse VEGF, the anti-human VEGF165 single-domain heavy chain monoclonal antibody, Y20A6, was obtained. Clone Y20A6 exhibited both satisfactory binding and competitive abilities.

[0172] Table 6 Flow cytometry analysis results of yeast monoclonal colonies after the second round of screening of yeast display library

[0173] 2.3 Humanized Single-Domain Antibody Design and Expression

[0174] IMGT / Domain Gap Align was performed on the sequence of the monoclonal antibody Y20A6, and the human germline with the highest homology was found to be IGHV3-23*04. Antibody sequences were numbered according to the Chothia convention. The mutations shown in Table 7 below were performed on the monoclonal antibody Y20A6 to generate Hz20A6.2 and Hz20A6.3.

[0175] Table 7 Humanized design of single domain antibody sequences

[0176] Y20A6 was fused with the heavy chain variable regions of humanized clones Hz20A6.1 and Hz20A6.3 to IgG1 Fc N297A (SEQ ID NO: 30) to form the antibody heavy chain, forming the heavy chain single-domain antibodies Ab1910VE6 (corresponding to Y20A6), Ab1910VE8 (corresponding to Hz20A6.2), and Ab1910VE9 (corresponding to Hz20A6.3). Codon optimization was performed, and the genes were synthesized and inserted into the expression vector pcDNA3.4 (Life Technologies). After expression plasmid amplification and plasmid extraction, the plasmid was transformed into ExpiCHO cells (ThermoFisher Scientific, A29133), and the antibody was transiently expressed using the supplier's ExpiCHO expression system protocol. Purified antibody expression and purification data are shown in Table 8.

[0177] Table 8 Single domain antibody expression and purification data

[0178] 2.4 Determination of the binding activity of single-domain antibodies to human VEGF165

[0179] Referring to the method in Example 1.3, the affinity of the single-domain antibodies to human VEGF165 before and after humanization was determined. The results are shown in Table 9 below. AB1910VE8 and AB1910VE9 have good affinity for human VEGF, and AB1910VE8 and AB1910VE9 antibodies were selected for the blocking function experiment.

[0180] Table 9 Affinity of single domain antibodies to human VEGF165

[0181] 2.5 Determination of the activity of single-domain antibodies in blocking the binding of human VEGF165 to human VEGFR2

[0182] The single-domain antibody's ability to block the binding of VEGF165 to human VEGFR2 was determined using a competitive ELISA. Human VEGFR2 (Acro, Catalog No. KDR-H5227) was diluted to 1 μg / mL in PBS (Gibco, Catalog No. 10010-023) and added to a 96-well plate at 100 μL per well. The plates were sealed with film and incubated overnight at 4°C. The next day, the plates were washed three times with a wash buffer (PBST with 0.05% TWEEN-20; Sigma-Aldrich, P1379). A blocking buffer (PBST + 2% BSA; BSA: VWR, 0332-1KG) was prepared using the wash buffer. 300 μL of the blocking buffer was added to each well and the plates were blocked at 37°C for 1 hour. Anti-VEGF and Biotin-hVEGF (Acro, Catalog No. VE5-H82Q0) were prepared in blocking buffer. The starting concentration of Anti-VEGF was 200 μg / mL. A 5-fold serial dilution (7 concentration points + 1 zero concentration point) was performed to obtain a Biotin-hVEGF concentration of 90 ng / mL. The blocked ELISA plate was then removed and washed three times with washing buffer. 50 μL of the diluted Anti-VEGF was added to a 96-well plate, followed by 50 μL of Biotin-hVEGF. The plates were then incubated at 37°C for 1 hour. The plates were then washed three times with washing buffer. Secondary antibody SA-HRP (Sigma, S2438) was diluted 1:5000 in blocking buffer and added at 100 μL / well to the plate. The plates were incubated at 37°C for 1 hour. Wash the plate three times with washing solution, add 100 μL TMB colorimetric solution (Biopanda, Catalog No.: TMB-S-003) to each well, develop color at room temperature in the dark, then add 50 μL stop solution (Solarbio, Catalog No.: C1058) to each well to terminate the reaction, and measure the absorbance at a wavelength of 450 nm.

[0183] The results of blocking the binding of VEGF165 and human VEGFR2 by single domain antibodies are shown in FIG7 . The results show that Ab1910VE8, Ab1910VE9, and Ab1910VE6 all have blocking function, and AB1910VE9 was selected for affinity maturation.

[0184] 2.6 Construction of single domain antibody affinity maturation library

[0185] The Ab1910VE9 sequence was affinity matured to improve its affinity for human VEGF. The CDR regions of Ab1910VE9 were randomly mutated at the HCDR1-3 and LCDR1-3 positions according to the Chothia definition to construct a mutation library. NNK mutation primers were designed to perform polymerase chain reaction (PCR) amplification of each CDR mutation library gene fragment. Each CDR mutation library gene fragment and a yeast display plasmid were separately transferred into the Saccharomyces cerevisiae strain EBY100 (purchased from ATCC), and each CDR mutation library was displayed on the yeast surface. The parental sequence of Ab1910VE9 was also displayed on the yeast surface as a control.

[0186] After culturing and induction, the library was sorted using Biotin-Human VEGF165 for three rounds, starting at 3 nM and followed by a 10-fold dilution series. Cell populations with high expression levels and strong antigen binding capacity were collected. After sorting, the cells were plated on SD-Trp solid medium and incubated at 30°C for 3 days.

[0187] Single clones were selected for sequencing, and the resulting clones with unique sequences were identified by flow cytometry. The clones were then incubated with 1 nM Biotin-Human VEGF165 and stained. The ratio of the displayed mean fluorescence signal intensity to the antigen-binding mean fluorescence signal intensity of the different clones was compared, reflecting the binding ability of individual molecules to the antigen. Based on the binding affinity values, the expressed antibody Ab1910VE21 (heavy chain variable region amino acid sequence of SEQ ID NO: 25) was selected. The antibody expression number and single clone identification results are shown in Table 10 below. The Chothia-numbered CDR region amino acid sequence of Ab1910VE21 is shown in Table 11 below.

[0188] Table 10 Monoclonal identification results and antibody expression numbers

[0189] >1910VE21 single domain antibody heavy chain variable region amino acid sequence (SEQ ID NO: 25)

[0190] Table 11 1910VE21 single domain antibody CDR region sequences

[0191] 2.7 Affinity Maturation Single Domain Antibody Expression and SEC-HPLC Purity Analysis

[0192] The single domain antibodies in Table 10 were expressed and purified. The expressed antibodies were subjected to SEC-HPLC purity analysis as follows:

[0193] (1) Dilute the sample to 1 mg / mL, mix thoroughly, centrifuge at 12,000 rpm for 5 min, transfer the supernatant to a sample bottle, and place it on the HPLC sample plate. Set the chromatographic conditions as follows:

[0194] (2) After the chromatographic column was equilibrated with the mobile phase (200 mM phosphate buffer, pH 6.8), the sample was injected and analyzed. The data were analyzed using chromatography software, and the peak area percentage of each peak was calculated using the peak area normalization method.

[0195] The expression and purification data of affinity matured single domain antibodies are shown in Table 12 below.

[0196] Table 12 Affinity matured single domain antibody expression and purification data

[0197] 2.8 Determination of Kinetic Parameters of Affinity Matured Single Domain Antibodies

[0198] The affinity of the humanized antibody to human VEGF165 was determined by referring to the method in Example 1.3. The results are shown in Table 13. The affinity of the antibody molecule AB1910VE21 after affinity maturation was increased several times.

[0199] Table 13 Affinity of affinity matured antibodies to human VEGF165

[0200] 2.9 Functional experiment of affinity-matured VEGF antibody blocking the binding of human VEGF165 and human VEGFR2

[0201] Referring to the method of Example 2.5, an experiment was conducted to determine the ability of affinity matured VEGF antibodies to block the binding function of human VEGF165 and human VEGFR2.

[0202] The experimental results are shown in FIG8 , which show that the blocking effects of Ab1910VE18 and Ab1910VE21 antibodies are comparable to those of Bevacizumab.

[0203] Example 3 Design and Detection of Anti-PD-1 / VEGF Bispecific Antibodies Kab5 and Kab6

[0204] 3.1 Bispecific Antibody Design and Preparation

[0205] The bispecific antibodies Kab5 and Kab6 were designed as shown in the table below, i.e., a nanobody fragment was connected to the C-terminus of the two heavy chains of an IgG antibody.

[0206] Table 14 Design of bispecific antibodies Kab5 and Kab6

[0207] Linker amino acid sequence is (SEQ ID NO: 29): ADIEGRMD

[0208] Kab5 and Kab6 genes were synthesized and inserted into the expression vector pcDNA3.4 (Life Technologies). After amplification and extraction, the expression plasmids were transformed into ExpiCHO cells (ThermoFisher Scientific, A29133). Antibodies were transiently expressed using the ExpiCHO expression system protocol provided by the supplier, and purified for subsequent characterization.

[0209] 3.2 Affinity determination of bispecific antibodies Kab5 and Kab6 for human VEGF165

[0210] The affinity of the biotin human VEGF165 (ACRO, Catalog No. VE5-H82Q0) and the bispecific antibodies Kab5 and Kab6 was determined using the method described in Example 1.3. The results are shown in Table 15 below. The affinity of Kab5 and Kab6 for human VEGF165 was slightly lower than that of bevacizumab.

[0211] Table 15 Affinity of bispecific antibodies Kab5, Kab6 for human VEGF165

[0212] 3.3 Affinity determination of bispecific antibodies Kab5 and Kab6 for human PD-1

[0213] The affinity of bispecific antibodies Kab5 and Kab6 for PD-1 (Sino Biological, Catalog No. 10377-H08H) was determined using the method described in Example 1.3. The results are shown in Table 16 below. The results show that Kab5 and Kab6 have similar affinities for human PD-1, both being superior to pembrolizumab.

[0214] Table 16 Affinity of bispecific antibodies Kab5, Kab6 to human PD-1

[0215] 3.4 Binding activity of bispecific antibodies Kab5 and Kab6 with the cell surface antigen PD-1

[0216] The binding activity of bispecific antibodies Kab5 and Kab6 to the cell surface antigen PD-1 was determined using the method described in Example 1.4. The binding results of bispecific antibodies Kab5 and Kab6 to the cell surface antigen PD-1 are shown in Figure 9 . The results demonstrate that the binding activity of bispecific antibodies Kab5 and Kab6 to the cell surface antigen PD-1 is similar to that of the humanized antibody 41D2HzL4H3.

[0217] 3.5 Bispecific antibodies block the binding activity of cell surface antigens PD-1 with PD-L1 and PD-L2

[0218] Referring to the method in Example 1.5, the bispecific antibody's binding activity against the cell surface antigens PD-1, PD-L1, and PD-L2 was determined. The results of the bispecific antibody blocking the binding of the cell surface antigens PD-1 and PD-L1 are shown in Figure 10 , and the results of the bispecific antibody blocking the binding of the cell surface antigens PD-1 and PD-L2 are shown in Figure 11 . These results demonstrate that the bispecific antibody's binding activity against the cell surface antigens PD-1, PD-L1, and PD-L2 is similar to that of the humanized antibody 41D2HzL4H3.

[0219] 3.6 Determination of the activity of bispecific antibodies competing with VEGFR2 for VEGF binding

[0220] The competitive ELISA method was used to determine the activity of the bispecific antibody in competing with VEGFR2 for binding to the antigen VEGF. The competitive binding activity of the bispecific antibody against VEGFR2 for binding to the antigen VEGF was determined using the same assay method as described in Example 2.5. The results of the competitive binding activity of the bispecific antibody against VEGFR2 for binding to the antigen VEGF are shown in Figure 12 . The results demonstrate that Kab5 and Kab6 have comparable blocking effects and both are superior to bevacizumab.

[0221] 3.7 Bispecific Antibodies Induce Mixed Lymphocyte Reactions

[0222] Referring to the experimental method of Example 1.6, the bispecific antibody-induced secretion of cytokine IL2 by mixed lymphocytes was measured. The results of bispecific antibody-stimulated secretion of cytokine IL2 by mixed lymphocytes are shown in Figure 13 , indicating that the Kab5 and Kab6 bispecific antibodies have similar functions to Pembrolizumab.

[0223] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments listed in the present application are obvious to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents.

Claims

1. An antigen binding protein comprising a first targeting moiety and a second targeting moiety, wherein the first targeting moiety comprises an antibody or antigen-binding fragment thereof that specifically binds to PD-1, and the second targeting moiety comprises an antibody or antigen-binding fragment thereof that specifically binds to VEGF; in, The first targeting moiety comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 of an antibody or antigen-binding fragment thereof that specifically binds to PD-1, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 6, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10; or wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13; LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16; Wherein, the second targeting portion comprises HCDR1, HCDR2 and HCDR3 of an antibody or an antigen-binding fragment thereof, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 26, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 27, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

28.

2. The antigen-binding protein according to claim 1, wherein the first targeting moiety comprises an antibody heavy chain variable region VH and an antibody light chain variable region VL, wherein: (i) the VH comprises an amino acid sequence selected from SEQ ID NO: 1, and the VL comprises an amino acid sequence selected from SEQ ID NO: 2; (ii) the VH comprises the amino acid sequence set forth in SEQ ID NO: 3, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 4; (iii) the VH comprises the amino acid sequence set forth in SEQ ID NO: 22, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 21; or (iv) the VH comprises the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises the amino acid sequence shown in SEQ ID NO:

23.

3. The antigen binding protein according to any one of claims 1-2, wherein the first targeting moiety further comprises an antibody heavy chain constant region, the antibody heavy chain constant region is derived from an IgG constant region, for example, comprises the amino acid sequence shown in SEQ ID NO:

32.

4. The antigen binding protein according to any one of claims 1 to 3, wherein the first targeting moiety further comprises an antibody light chain constant region, for example comprising the amino acid sequence shown in SEQ ID NO:

31.

5. The antigen binding protein according to any one of claims 1 to 4, wherein the second targeting moiety comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:

25.

6. The antigen binding protein according to any one of claims 1 to 5, wherein the second targeting moiety comprises an antibody heavy chain constant region, which is derived from an IgG constant region, for example, comprises the amino acid sequence shown in SEQ ID NO:

30.

7. The antigen binding protein according to any one of claims 1 to 6, wherein the antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

8. The antigen binding protein according to any one of claims 1 to 7, wherein the antigen binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

9. The antigen-binding protein according to any one of claims 1 to 8, wherein the antigen-binding protein comprises a bispecific antibody, for example, the antigen-binding protein comprises an IgG-VHH type bispecific antibody, for example, bispecific antibodies Kab5 and Kab6.

10. The antigen binding protein according to any one of claims 1 to 9, wherein the first targeting moiety comprises an antibody heavy chain and an antibody light chain, and the second targeting moiety is located at the C-terminus of the antibody heavy chain of the first targeting moiety, wherein The second targeting moiety is directly linked to the first targeting moiety or indirectly linked via a linker. The antigen-binding protein according to claim 10 , wherein the linker comprises the amino acid sequence shown in SEQ ID NO:

29.

12. An antigen-binding protein according to any one of claims 1-11, wherein the antigen-binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the antibody heavy chain or antigen-binding fragment thereof of the first targeting portion, an optionally present or absent linker, and the antibody or antigen-binding fragment thereof of the second targeting portion, and the second polypeptide chain comprises the antibody light chain or antigen-binding fragment thereof of the first targeting portion.

13. The antigen binding protein of claim 12, wherein the first polypeptide chain comprises: the heavy chain variable region of the first targeting moiety-the heavy chain constant region of the first targeting moiety-X-the variable region of the second targeting moiety-the constant region of the second targeting moiety, and the second polypeptide chain comprises: the light chain variable region of the first targeting moiety-the light chain constant region of the first targeting moiety, wherein X is absent or comprises the linker.

14. The antigen-binding protein according to any one of claims 12-13, wherein the first polypeptide chain comprises, from N-terminus to C-terminus, the antibody heavy chain or its antigen-binding fragment of the first targeting portion and the antibody or its antigen-binding fragment of the second targeting portion, or the first polypeptide chain comprises, from N-terminus to C-terminus, the antibody heavy chain or its antigen-binding fragment of the first targeting portion, a linker and the antibody or its antigen-binding fragment of the second targeting portion.

15. A nucleic acid encoding the antigen binding protein of any one of claims 1-14. A vector comprising the nucleic acid according to claim 15 .

17. An immunoconjugate comprising the antigen binding protein of any one of claims 1-14.

18. A cell comprising the antigen binding protein of any one of claims 1 to 14, the nucleic acid of claim 15, the vector of claim 16, and / or the immunoconjugate of claim 17.

19. A pharmaceutical composition comprising the antigen binding protein of any one of claims 1 to 14, the nucleic acid of claim 15, the vector of claim 16, the immunoconjugate of claim 17, and / or the cell of claim 18, and optionally a pharmaceutically acceptable carrier.

20. A kit comprising the antigen-binding protein of any one of claims 1-14, the nucleic acid of claim 15, the vector of claim 16, the immunoconjugate of claim 17, the cell of claim 18, and / or the pharmaceutical composition of claim 19.

21. A method for preparing the antigen binding protein of any one of claims 1 to 14, comprising culturing the cell of claim 18 under conditions allowing expression of the antigen binding protein.

22. Use of the antigen-binding protein of any one of claims 1 to 14, the nucleic acid of claim 15, the vector of claim 16, the immunoconjugate of claim 17, the cell of claim 18, the pharmaceutical composition of claim 19, and / or the kit of claim 20 in the preparation of a medicament for preventing, alleviating and / or treating tumors.

23. The use according to claim 22, wherein the tumor comprises a tumor with high expression of PD-1, PD-L1 and / or VEGF.

24. The use according to any one of claims 22-23, wherein the tumor comprises a solid tumor and / or a non-solid tumor.

25. The use according to any one of claims 22-24, wherein the tumor comprises colorectal cancer, breast cancer, blastoma, cervical cancer, ovarian cancer, melanoma, lung cancer, kidney cancer, esophageal cancer, head and neck cancer, lymphoma, liver cancer and / or gastric cancer.

26. A method for inhibiting the binding of PD-1 protein to PD-L1 protein and / or PD-L2 protein, and / or inhibiting the binding of VEGF protein to VEGFR protein, the method comprising administering the antigen binding protein of any one of claims 1 to 14, the nucleic acid of claim 15, the vector of claim 16, the immunoconjugate of claim 17, the cell of claim 18, the pharmaceutical composition of claim 19, and / or the kit of claim 20.

27. A method of modulating an immune response, the method comprising administering the antigen binding protein of any one of claims 1-14, the nucleic acid of claim 15, the vector of claim 16, the immunoconjugate of claim 17, the cell of claim 18, the pharmaceutical composition of claim 19, and / or the kit of claim 20. The method according to claim 27 , wherein regulating the immune response comprises stimulating immune cells to secrete cytokines.

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