WO2024032662A1

WO2024032662A1 - Antibody targeting pd-1 and vegf, and use thereof

Info

Publication number: WO2024032662A1
Application number: PCT/CN2023/112024
Authority: WO
Inventors: 王宗达; 顾春银; 邓俗俊
Original assignee: 上海济煜医药科技有限公司
Priority date: 2022-08-09
Filing date: 2023-08-09
Publication date: 2024-02-15
Also published as: TW202413427A

Abstract

Provided are an antibody targeting PD-1 and VEGF, and a use thereof. Also provided is an antigen-binding protein, comprising a first targeting moiety having a function of specifically binding to PD-1, and a second targeting moiety having a function of specifically binding to VEGF.

Description

An antibody targeting PD-1 and VEGF and its application

Technical field

This application relates to the field of biomedicine, specifically to an antibody targeting PD-1 and VEGF and its application.

Background technique

Malignant tumors are diseases that seriously threaten human health worldwide and are the main type of disease causing human death. With the aging of the domestic population, the incidence of tumors continues to increase, and effective therapeutic drugs are urgently needed to be developed.

The development of immune checkpoint drugs has become a research hotspot in recent years. Programmed death 1 (programmed death 1), referred to as PD-1, is widely expressed in immune cells and is an important immunosuppressive molecule. The main ligand of PD-1 is PD-L1, and PD-L1 is mainly expressed on the surface of tumor cells. After the ligand PD-L1 binds to the receptor PD-1, it inhibits T cell activation in the tumor microenvironment, causing the immune system such as T cells to be unable 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 to prevent the recognition of PD-1 and PD-L1 and restore the normal function of T cells, thereby making T cells effective. Kill tumor cells. A number of therapeutic antibodies targeting this signaling pathway have been developed, such as Pembrolizumab and Nivolumab. However, the response rate is generally low during the treatment process and drug resistance is easy to develop. sexual phenomena.

In addition, epidermal growth factor VEGF can specifically stimulate the proliferation of endothelial cells during tumor growth and plays a key role in various types of tumor angiogenesis. After VEGF binds to the epidermal growth factor receptor VEGFR, it can mediate the transcription and expression of intracellular related protein genes through downstream signaling pathways and promote the proliferation of vascular endothelial cells. A number of humanized monoclonal antibodies targeting human VEGF, such as Bevacizumab, have been developed. However, the response rate is still low during treatment and drug resistance is easy to develop.

At the same time, most of the therapeutic antibody drugs currently on sale are monoclonal antibodies, which are specific for a single target. During the treatment process, they are more likely to have low response rates and easy development of drug resistance. Therefore, there is an urgent need in this field for a multispecific antibody targeting PD-1 and VEGF that is structurally stable, has good efficacy, and is suitable for large-scale industrial production.

Contents of the invention

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

In one aspect, the present application provides an antigen-binding protein comprising a first targeting moiety and a second targeting moiety, the first targeting moiety comprising an antibody that specifically binds PD-1 or an antigen-binding fragment thereof, so The second targeting moiety contains specific Antibodies or antigen-binding fragments thereof that bind VEGF;

Wherein, the first targeting portion includes HCDR1, HCDR2 and HCDR3 of the antibody or its antigen-binding fragment, the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5 or 11, and the HCDR2 includes SEQ ID NO: 6 or The amino acid sequence shown in 12, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 7 or 13;

Wherein, the first targeting portion includes LCDR1, LCDR2 and LCDR3 of the antibody or its antigen-binding fragment, and the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 8 or 14, and the LCDR2 includes SEQ ID NO: 9 Or the amino acid sequence shown in 15, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 10 or 16;

Wherein, the second targeting portion includes HCDR1, HCDR2 and HCDR3 of the Nanobody, the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 26, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 27, and The HCDR3 includes the amino acid sequence shown in SEQ ID NO: 28.

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

In one aspect, the application provides a vector comprising the nucleic acid of the application.

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

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

In one aspect, the present application provides a pharmaceutical composition, which includes 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 cells of the present application, and any Choose a pharmaceutically acceptable carrier.

On the one hand, the application provides a kit, which contains the antigen-binding protein of the application, the nucleic acid of the application, the vector of the application, the immunoconjugate of the application, the cell of the application, and/or the application of Pharmaceutical compositions.

In one aspect, the present application provides a method for preparing the antigen-binding protein of the present application, which includes culturing the cells of the present application under conditions that enable the expression of the antigen-binding protein.

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 the present application. Use of the applied kit in the preparation of medicaments for preventing, alleviating and/or treating tumors.

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 the present application. Requested kit for treatment.

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 the present application. The requested kit is for the prevention, mitigation and/or treatment of tumors.

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 The immunoconjugate, the cell of the present application, the pharmaceutical composition of the present application, and/or the kit of the present application.

On the one hand, 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 of the present application Binding protein, nucleic acid of the present application, vector of the present application, immunoconjugate of the present application, cell of the present application, pharmaceutical composition of the present application, and/or kit of the present application.

A method for regulating immune response, 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 cells of the present application, the pharmaceutical composition of the present application, and/or the kit of the present application.

The molecular structure of the bispecific antibody of this application can be IgG-VHH. Using this form of bispecific antibody against PD-1 or PD-L1 and VEGF targets can effectively avoid mismatching of antibody light and heavy chains, and the molecular stability is better High, 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 effect.

Those skilled in the art will readily appreciate other aspects and advantages of the present application from the detailed description below. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will realize, the contents of this application enable those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention covered by this application. Accordingly, the drawings and descriptions of the present application are illustrative only and not restrictive.

Description of drawings

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

Figure 1 shows that the antigen-binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described in this application bind to human PD-1 protein on the surface of CHOK1 cells.

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

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

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

Figure 5 shows the results that both humanized antibodies 1910h3hzL3H4 and 41D2HzL4H3 can stimulate lymphocytes to secrete IL2.

Figure 6 shows the construction scheme of single domain antibodies displayed on the yeast cell wall surface.

Figure 7 shows the results of single domain antibodies blocking the binding of VEGF165 and human VEGFR2.

Figure 8 shows the blocking results of Ab1910VE18 and Ab1910VE21 antibodies.

Figure 9 shows the binding results of bispecific antibodies Kab5 and Kab6 to cell surface antigen PD-1.

Figure 10 shows the results of bispecific antibodies blocking the binding of cell surface antigens PD-1 and PD-L1.

Figure 11 shows the results of bispecific antibodies blocking the binding of cell surface antigens PD-1 and PD-L2.

Figure 12 shows the results of the activity of bispecific antibodies competing with VEGFR2 for binding to the antigen VEGF.

Figure 13 shows the results of bispecific antibodies stimulating mixed lymphocytes to secrete the cytokine IL2.

Detailed ways

The implementation of the invention of the present application will be described below with specific examples. Those skilled in the art can easily understand other advantages and effects of the invention of the present application from the contents disclosed in this specification.

In the present 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.

In this application, the term "linker" generally refers to a moiety that joins or connects two or more discrete separate monomeric domains. The linker may be a peptide linker, which contains a polypeptide of 2 or more amino acid residues linked by peptide bonds, and is used to link one or more antigen-binding moieties. 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 may be ADIEGRMD.

In this application, the term "VEGF" generally refers to vascular endothelial cell growth factor, including its naturally occurring allelic and processed forms. The VEGF may include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and/or PlGF. In this application, the VEGF may be VEGF-A, which may also be called 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 binding to VEGFR2, VEGF165 mediates the transcription and expression of intracellular related protein genes through the downstream PLC-γ-PKC-Raf-MEK-MAPK signaling pathway, promoting the proliferation of vascular endothelial cells. The term "VEGF" may also refer to proteins from non-human species Such as mouse, rat or primate VEGF. In this application, VEGF from a particular species may be represented as follows, hVEGF represents human VEGF and mVEGF represents murine 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 those that have at least one epitope in common with VEGF analogues. 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.

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". PD-1 proteins usually include an extracellular IgV domain, a transmembrane region and an intracellular tail. PD-1 is commonly expressed on T cells, B cells, natural killer T cells, activated monocytes, and dendritic cells (DC). PD-1 can bind to its ligands PD-L1 and PD-L2. The term "PD-1" encompasses any native PD-1 or modified PD-1 from any vertebrate source, including mammals, such as primates (e.g., humans or monkeys) and rodents ( e.g. mouse or rat). The term encompasses "full-length", unprocessed PD-1 as well as any form of PD-1 resulting from processing in the cell. PD-1 can exist as a transmembrane protein or as a soluble protein. "PD-1" includes complete PD-1 and its fragments, and also includes functional variants, isoforms, species homologs, derivatives, analogs of PD-1, and those that have at least one property in common with PD-1 Analogues of epitopes. 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 BOLAJ3 turn up.

In this application, the term "PD-L1" generally refers to the programmed death ligand 1 protein. PD-L1 is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), and is the protein encoded by the CD274 gene (in humans). PD-L1 can bind to its receptors, 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 the cytokines IL-2 and IFN-γ. The term "PD-L1" encompasses any native PD-L1 or modified PD-1 from any vertebrate source, including mammals, such as primates (e.g., humans or monkeys) and rodents ( e.g. mouse or rat). The term encompasses "full-length", unprocessed PD-L1 as well as any form of PD-L1 resulting from processing in the cell. 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: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain. PD-L1 sequences are known in the art. Information on the human PD-L1 gene, including the genomic DNA sequence, can be found, for example, 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 number NP_054862 or UniProt accession number Q9NZQ7.

In this application, the term "PD-L2" generally refers to the 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" encompasses any native PD-L2 or modified PD-L2 from any vertebrate source, including mammals, such as primates (e.g., humans or monkeys) and rodents ( e.g. mouse or rat). The term encompasses "full-length," unprocessed PD-1 as well as any form of PD-L2 resulting from processing in the cell. PD-L2 can exist as a transmembrane protein or as a soluble protein. "PD-L2" includes complete PD-L2 and its fragments, as well as functional variants, isoforms, species homologues, derivatives, analogs of PD-L2, and those that have at least one property in common with PD-L2 Analogues of epitopes. PD-L2 sequences are known in the art. For example, an exemplary full-length human PD-L2 protein sequence can be found under NCBI accession number NP_079515.2.

In this application, the term "VEGFR" generally refers to the vascular endothelial growth factor receptor, including its naturally occurring allelic and processed forms. VEGFRs may include VEGFR1, CEGFR2, and VEGFR3, as well as other alternatively spliced variants. VEGFR can be membrane-bound or soluble. The term "VEGFR" may also refer to VEGFR from non-human species such as mouse, rat or primate. In this application, VEGFR from a particular species may be represented as follows, hVEGFR represents human VEGFR and mVEGFR represents murine VEGFR. Generally, VEGFR may 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 homologues, derivatives, analogs of VEGFR, and those that have at least one epitope in common with VEGFR analogues. VEGFR sequences are known in the art. For example, an exemplary full-length human VEGFR2 protein sequence can be found under NCBI accession number NP_002244.

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 "homologue" generally refers to an amino acid sequence or a nucleotide sequence that has certain homology to a wild-type amino acid sequence and a wild-type nucleotide sequence. The term "homology" may be equated with sequence "identity." Homologous sequences may include amino acid sequences that may be 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, homologs will contain active sites, etc. that are identical to the subject amino acid sequence. Homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), or homology can be expressed in terms of sequence identity. In this application, reference to a sequence having a percent identity with any one of the SEQ ID NOs of an amino acid sequence or a nucleotide sequence means that the sequence has said percent identity over the entire length of the SEQ ID NO mentioned. the sequence of.

As used herein, the term "antigen-binding protein" generally refers to a protein comprising an antigen-binding portion, and optionally a scaffold or backbone portion that allows the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding protein to the antigen. Antigen binding proteins may 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 binding domains that interact with the antigen. Examples of antigen binding proteins include but Not limited to antibodies, antigen-binding fragments, immunoconjugates, multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, etc., as long as they display the required antigen-binding activity That’s it.

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

As used herein, the term "antigen-binding fragment" generally refers to a portion of an antibody molecule that contains the amino acid residues that interact with the antigen and confer 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 fragment, F(ab') ₂ , scFv, di-scFv and/or dAb. In this application, the term "Fab" generally refers to a fragment containing the variable domain of the heavy chain and the variable domain of the light chain, and also containing 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 is different from Fab by adding 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 " usually refers to a dimer of Fab', an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region. The term "Fv" generally refers to the smallest antibody fragment containing intact antigen recognition and binding sites. In some cases, the fragment may consist of a heavy chain variable domain and a light chain variable domain as a dimer in tight non-covalent association; the term "dsFv" generally refers to a disulfide-stabilized Fv fragment, The bond between its single light chain variable domain and its single heavy chain variable domain 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 covalently connecting a heavy chain variable domain and a light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules may have general Structure: NH ₂ -VL-linker-VH-COOH or NH ₂ -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 can also be called Nanobodies.

In this application, the term "variable region" or "variable domain" generally refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. In this application, the term "variable" generally means that certain portions of the sequence of the variable domain of an antibody vary strongly, resulting in the binding and specificity of various specific antibodies for their specific antigens. Variability is not evenly distributed throughout the variable regions of an antibody. It is concentrated in three segments in the light chain variable region and heavy chain variable region, known as the complementarity determining region (CDR) or hypervariable region (HVR), which are LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3. The more highly conserved portions of the variable domains are called framework regions (FR). The variable domains of the native heavy and light chains each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4) , most of them adopt β-fold Stacked configuration, connected by three CDR structural loop regions. The CDRs in each chain are held closely together by the FR region and, together with the CDRs from the other chain, form the antigen-binding site of the antibody.

In the art, various methods can be used to encode the variable region of an antibody or divide the CDRs 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, efranc et al.'s IMGT numbering scheme and definition rules based on amino acid sequence alignment of germline V genes, as well as Honneger's numbering scheme (AHo's), Martin numbering scheme, Gelfand numbering scheme, etc.

In this application, the term "monoclonal antibody" generally refers to an antibody obtained from a population of antibodies that are essentially homogeneous, i.e., the individual antibodies making up the population are identical except for possible naturally occurring mutations that may be present in minimal amounts and/or In addition to post-translational modifications (such as isomerization, amidation). Monoclonal antibodies are highly specific and target a single antigenic site.

In this application, the term "chimeric antibody" generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species. Typically, the variable regions are derived from an antibody from a laboratory animal, such as a rodent (a "parent antibody"), and the constant regions are derived from a human antibody, such that the resulting chimeric antibody is more effective in a human individual than the parent (eg, mouse-derived) antibody. Less likely to trigger an adverse immune response.

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 (eg, a mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulins. Additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions may also be allowed as long as they retain the ability of the antibody to bind a specific antigen. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. "Humanized antibodies" retain antigen specificity similar to the original antibody. "Humanized" forms of non-human (eg, murine) antibodies may minimally comprise chimeric antibodies derived from sequences derived from non-human immunoglobulins. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) can be used with a non-human species (donor antibody) having the desired properties, affinity, and/or ability (such as mouse, rat , rabbit or non-human primate) CDR region residue substitution. In some cases, FR region residues of a human immunoglobulin can be replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody.

In this application, the term "fully human antibody" generally refers to an antibody in which the human gene encoding the antibody is transferred into a genetically engineered antibody gene-deficient animal to express it in the animal. All parts of the antibody (including the variable and constant regions of the antibody) are encoded by genes of human origin. Methods for obtaining fully human antibodies in this field include phage display technology, transgenic mouse technology, ribosome display technology, and RNA-polypeptide technology.

In this application, the terms "binding,""specificbinding," or "specific for" generally refer to a measurable and reproducible interaction, such as binding between an antigen and an antibody, which can be determined in the presence of a molecule heterogeneity (including biological molecules) Presence of target in a group situation. 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 a target (which may be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and/or for a greater duration than it binds other targets. An antibody is said to "specifically bind" an antigen when it binds to an epitope more readily through its antigen-binding domain than it would to a random, unrelated epitope.

In this application, the terms "KD" and " _KD " are used interchangeably and generally refer to the equilibrium dissociation constant. "KD" is the dissociation rate constant (kdis, also known as "off-rate". )(koff)" or "kd") to the binding rate constant (kon, also known as "binding rate (kon)" or "ka"). The binding affinity of an antigen-binding protein (eg, an antibody) for an antigen can be expressed using the association rate constant (kon), the dissociation rate constant (kdis), and the equilibrium dissociation constant (KD). Methods for determining association and dissociation rate constants are well known in the art, including but not limited to biofilm interference technique (BLI), radioimmunoassay (RIA), equilibrium dialysis, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET) , co-immunoprecipitation (Co-IP) and protein chip technology. The measured affinity for a particular protein-protein interaction can differ if measured under different conditions (eg, salt concentration, pH).

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

In this application, the terms "polypeptide" or "protein" are used interchangeably and generally refer to a polymer 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 the addition of sugar residues to form glycoproteins or by phosphorylation. Polypeptides and proteins may be produced from naturally occurring and non-recombinant cells or from genetically engineered or recombinant cells, and may comprise molecules having the amino acid sequence of the native protein, or having the deletion, addition, or deletion of one or more amino acids of the native sequence. and/or substituted molecules. The terms "polypeptide" and "protein" particularly include sequences in which one or more amino acids of the antigen-binding proteins described herein are deleted, added and/or substituted.

In this application, the term "isolated" generally refers to biological material (eg, viruses, nucleic acids, or proteins) that is substantially free of components that normally accompany or interact with it in its naturally occurring environment. The isolated biological material optionally contains additional materials that the biological material is not found to have in its natural environment (eg, nucleic acids or proteins). In this application, when referring to a protein, "isolated" generally refers to the isolation and separation of the molecule from the entire organism in which it is found naturally occurring. separate, or essentially non-existent, other biological macromolecules of the same type. When it comes to a nucleic acid molecule, it is completely or partially separated from the sequence to which it is naturally associated, or the nucleic acid has heterologous sequences to which it is associated, or the nucleic acid is separated from the chromosome.

In this application, the term "immunoconjugate" generally refers to a substance formed by connecting an antigen-binding protein to other active agents. The other active agents can be small molecule active agents, such as chemotherapeutic agents, toxins, immunotherapeutic agents, and imaging probes. or spectroscopic probes.

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

In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells. The vectors may include vectors primarily used for insertion of DNA or RNA into cells, vectors primarily used for replication of DNA or RNA, and vectors primarily used for expression of transcription and/or translation of DNA or RNA. The vectors also include vectors having a variety of the above-mentioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce the desired expression product by culturing a suitable host cell containing the vector.

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

In this application, the term "pharmaceutical composition" generally refers to a preparation that is in a form that is effective to allow the biological activity of the active ingredients and does not contain unacceptable toxicity to the subject to whom the composition is to be administered. additional ingredients.

In this application, the term "treatment" generally refers to a clinical intervention intended to alter the natural course of the disease in the individual being treated, and may be to achieve prevention or treatment during the clinical course of the disease. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of disease, preventing metastasis, reducing the rate of disease progression, ameliorating or relieving disease status, and alleviating or improving prognosis. In some cases, antigen-binding proteins (eg, anti-VEGF antibodies) can be used to delay disease progression or slow disease progression.

In this application, the term "administering" generally refers to administering to a subject (e.g., a patient) a dose of a compound (e.g. (e.g., an anti-cancer therapeutic agent) or a pharmaceutical composition (e.g., a pharmaceutical composition comprising an anti-cancer therapeutic agent). Administration may be by any suitable means, including parenteral, intrapulmonary and intranasal, and, if required for local treatment, intralesional administration. Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

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 may be a tumor with high expression of VEGF or VEGFR in cells and tissues. Tumors may include solid tumors and/or non-solid tumors (eg, hematological tumors, lymphomas).

In this application, the term "between" usually means that the C-terminus of a certain amino acid fragment is directly or indirectly connected to the N-terminus of the first amino acid fragment, and its N-terminus is directly or indirectly connected to the C-terminus of the second amino acid fragment. indirect connection. In the light chain, for example, the N-terminus of the L-FR2 is directly or indirectly connected to the C-terminus of the LCDR1, and the C-terminus of the L-FR2 is directly or indirectly connected to the N-terminus of the LCDR2. For another example, the N terminus of the L-FR3 is directly or indirectly connected to the C terminus of the LCDR2, and the C terminus of the L-FR3 is directly or indirectly connected to the N terminus of the LCDR3. In the heavy chain, for example, the N-terminus of the H-FR2 is directly or indirectly connected to the C-terminus of the HCDR1, and the C-terminus of the H-FR2 is directly or indirectly connected to the N-terminus of the HCDR2. For another example, the N terminus of the H-FR3 is directly or indirectly connected to the C terminus of the HCDR2, and the C terminus of the H-FR3 is directly or indirectly connected to the N terminus of the HCDR3. In this application, the "first amino acid fragment" and the "second amino acid fragment" can be any amino acid fragments that are the same or different.

In this application, the term "comprises" generally means including, encompassing, containing or encompassing. In some cases, it also means "for" or "composed of".

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

Detailed description of the invention

In one aspect, the present application provides an antigen-binding protein comprising a first targeting moiety and a second targeting moiety, the first targeting moiety comprising an antibody that specifically binds PD-1 or an antigen-binding fragment thereof, so The second targeting portion includes an antibody that specifically binds VEGF or an antigen-binding fragment thereof;

wherein the first targeting portion comprises LCDR1, LCDR2 and LCDR3 of the antibody or antigen-binding fragment thereof, and The LCDR1 includes the amino acid sequence shown in SEQ ID NO: 8 or 14, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 9 or 15, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 10 or 16. sequence;

Wherein, the second targeting portion includes HCDR1, HCDR2 and HCDR3 of the antibody or its antigen-binding fragment, the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 26, and the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 27 Amino acid sequence, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 28.

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, the HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 6 The amino acid sequence of HCDR3 includes the amino acid sequence shown in SEQ ID NO: 7.

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

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, the HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 11, and the HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 12 The amino acid sequence of HCDR3 includes the amino acid sequence shown in SEQ ID NO: 13.

For example, the first targeting part of the present application may include LCDR1, LCDR2 and LCDR3 of the antibody or its antigen-binding fragment, and the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 14, and the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 15 The amino acid sequence shown is, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 16.

For example, the first targeting portion of the present application may comprise an HCDR3, which may comprise an HCDR3 of a VH having an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3, 22, and 24. For example, the first targeting portion of the present application may comprise an HCDR2, which may comprise an HCDR2 of a VH with an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3, 22, and 24. For example, the first targeting portion of the present application may comprise an HCDR1, which may comprise an HCDR1 of a VH with an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3, 22, and 24. For example, the first targeting portion of the present application can include HCDR3, HCDR2, and HCDR1, and the HCDR3, HCDR2, and HCDR1 can include the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, 22, and 24. HCDR3, HCDR2, and HCDR1 of VH. For example, the first targeting portion of the present application may include HFR1, HFR2, HFR3, and/or HFR4, and the HFR1, HFR2, HFR3, and HFR4 may respectively include amino acid sequences such as SEQ ID NO: 1, 3, 22, and 24. HFR1, HFR2, HFR3, and HFR4 of the VH represented by any one of them. For example, it can be divided according to Chothia rules.

For example, the first targeting moiety of the present application may comprise LCDR3, which may comprise the LCDR3 of a VL with 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 include LCDR2, The LCDR2 may comprise the LCDR2 of VL with an amino acid sequence as shown in any one of SEQ ID NO: 2, 4, 21 and 23. For example, the first targeting moiety of the present application may comprise LCDR1, which may comprise the LCDR1 of a VL with an amino acid sequence as shown in any one of SEQ ID NOs: 2, 4, 21 and 23. For example, the first targeting portion of the present application may include LCDR3, LCDR2, and LCDR1, and the LCDR3, LCDR2, and LCDR1 may include a VL with an amino acid sequence as shown in any one of SEQ ID NO: 2, 4, 21, and 23 LCDR3, LCDR2, and LCDR1. For example, the first targeting portion of the present application may include LFR1, LFR2, LFR3, and/or LFR4, and the LFR1, LFR2, LFR3, and LFR4 may respectively include amino acid sequences such as SEQ ID NO: 2, 4, 21, and 23. LFR1, LFR2, LFR3, and LFR4 of the VL represented by any one of them. For example, it can be divided according to Chothia rules.

In one embodiment, the application provides an antigen-binding protein comprising a first targeting moiety and a second targeting moiety, the first targeting moiety comprising an antibody that specifically binds PD-1 or an antigen-binding protein thereof. Fragment, the second targeting portion comprises an antibody that specifically binds VEGF or an antigen-binding fragment thereof:

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

Among them, HCDR1 contains the amino acid sequence shown in SEQ ID NO: 11, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 12, HCDR3 contains the amino acid sequence shown in SEQ ID NO: 13; LCDR1 contains the amino acid sequence shown in SEQ ID NO: 14 Amino acid sequence, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 15, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16,

Wherein the antibody specifically binding to VEGF or its antigen-binding fragment includes HCDR1, HCDR2 and HCDR3, the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 26, and the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 27 , and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 28.

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

For example, wherein the first targeting portion comprises an antibody heavy chain variable region VH, and the VH comprises an amino acid sequence selected from any one of SEQ ID NO: 1, 3, 22 and 24.

For example, wherein 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 NO: 2, 4, 21 and 23.

For example, wherein the first targeting portion comprises an antibody heavy chain variable region VH and an antibody light chain variable region VL, the VH comprises the amino acid sequence shown in SEQ ID NO: 1, and the VL comprises SEQ ID NO : The amino acid sequence shown in 2. For example, the VH includes the amino acid sequence shown in SEQ ID NO: 3, and the VL includes the amino acid sequence shown in SEQ ID NO: 4. For example, the VH includes the amino acid sequence shown in SEQ ID NO: 22, and the VL includes the amino acid sequence shown in SEQ ID NO: 21. For example, the VH includes the amino acid sequence shown in SEQ ID NO: 24, and the VL includes the amino acid sequence shown in SEQ ID NO: 23.

For example, wherein the first targeting moiety further comprises an antibody heavy chain constant region derived from an IgG constant region. For example, derived from the IgG4 constant region. For example, the first targeting moiety may comprise the amino acid sequence shown in SEQ ID NO: 32. For example, wherein the first targeting moiety further comprises an antibody light chain constant region. For example, the first targeting moiety may comprise the amino acid sequence shown in SEQ ID NO: 31.

According to the antigen-binding protein of the present application, the second targeting portion includes the antibody heavy chain variable region VH, and the VH includes the amino acid sequence shown in SEQ ID NO: 25. According to the antigen-binding protein of the present application, the second targeting portion comprises an antibody heavy chain constant region. For example, derived from the IgG1 constant region. For example, the second targeting moiety may comprise the amino acid sequence shown in SEQ ID NO: 30. For example, wherein the second targeting moiety is a Nanobody.

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

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

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

In this application, a part of each heavy chain or light chain amino acid sequence of the antigen-binding protein is homologous to the corresponding amino acid sequence in the antibody from a specific species, or belongs to a specific class. For example, the variable and constant portions of the light and heavy chains are derived from the variable and constant regions of an antibody from one animal species (eg, human). In this application, the homolog may be at least about 85% identical to the amino acid sequence of the protein and/or the polypeptide (e.g., having 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 higher) Proteins or polypeptides with sequence homology.

In this application, homology generally refers to similarity, similarity or association between two or more sequences. Alignment for the purpose of determining percent sequence homology can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximal alignment within the full-length sequences being compared or within the sequence region of interest. The homology can also be determined by the following methods: FASTA and BLAST.

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

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 portion includes an antibody heavy chain and an antibody light chain, and the second targeting portion is located at the C-terminus of the antibody heavy chain and/or the antibody light chain of the first targeting portion.

For example, the second targeting moiety is directly or indirectly linked to the first targeting moiety. For example, the second targeting moiety is connected to the first targeting moiety through a linker. For example, in the art, a linker connecting polypeptides at both ends can be used to connect the second targeting moiety and the first targeting moiety. For example, the linker includes the amino acid sequence shown in SEQ ID NO: 29.

According to the antigen-binding protein of the present application, the antigen-binding protein includes the first polypeptide chain, an antibody heavy chain or an antigen-binding fragment thereof including the first targeting portion, an optionally present or absent linker, and the The second targeting moiety is an antibody or an antigen-binding fragment thereof, and the second polypeptide chain comprises an antibody light chain of the first targeting moiety or an antigen-binding fragment thereof. For example, the first targeting moiety and the second targeting moiety in the first polypeptide chain in the present application can be directly connected, for example, without a linker or connected through chemical bonds. For example, the first targeting moiety and the second targeting moiety in the first polypeptide chain of the present application can be connected indirectly, for example, through a linker. For example, the linker structure may be a linker peptide known in the present application. For example, the first polypeptide chain may 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 may comprise: the light chain variable region of the first targeting moiety - the light chain constant region of the first targeting moiety, wherein said X means that it does not exist or contains the linker.

For example, the first polypeptide chain sequentially includes an antibody heavy chain or an antigen-binding fragment thereof of the first targeting moiety from the N-terminus to the C-terminus. segment and the second targeting portion of the antibody or antigen-binding fragment thereof. For example, the first polypeptide chain sequentially includes the antibody heavy chain of the first targeting portion or its antigen-binding fragment, a linker and the second targeting portion of the antibody or its antigen-binding fragment from the N-terminus to the C-terminus.

In the present application, the isolated antigen-binding protein is capable of binding to primate-derived PD-1 with a K _D value of 1×10 ^-8 M or lower. For example, the value of K _D may be about 1 × 10 ^-8 M or less, about 9 × 10 ^-9 M or less, about 8 × 10 ^-9 M or less, about 7 × 10 ^-9 M or less, About 6×10 ^-9 M or less, about 5×10 ^-9 M or less, about 4×10 ^-9 M or less, about 3×10 ^-9 M or less, about 2×10 ^-9 M or less, Values of about 1×10 ⁻⁹ M or less bind human-derived PD-1, for example, as detected using the FortieBio Octet Molecular Interaction Analyzer.

In another case, the binding activity of the PD-1 antigen-binding protein described in the present application to PD-1 can be detected using flow cytometry or enzyme-linked immunoassay assay. For example, in FACS detection, using host cells that stably express human PD-1 (such as CHOK1 cells), the EC50 value of the PD-1 antigen-binding protein binding to PD-1 is 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, about 0.1 nM to about 1.5nM.

For example, in FACS detection, using host cells (such as CHOK1 cells) that stably express monkey PD-1, the EC50 value of the PD-1 antigen-binding protein binding to PD-1 is 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, between about 0.1 nM and about 1.0 nM.

In another aspect, the antigen-binding protein described in the present application can block the binding of PD-1 to PD-L1. In some cases, the blocking of the binding of PD-1 and PD-L1 by the antigen-binding protein can be measured by flow cytometry FACS or enzyme-linked immunoassay ELISA.

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

For example, host cells stably expressing human PD-1 (such as CHOK1 cells) are first incubated with decreasing amounts of unlabeled said antigen-binding protein, and subsequently incubated with biotin-labeled PD-L2 protein. The cells were then analyzed using FACS to confirm that the antigen-binding protein blocked the binding of PD-1 to PD-L2. For example, between about 0.001nM and about 10nM, about 0.001nM to about 5nM, between about 0.1nM to about 2.5nM, and between about 0.1nM to about 3nM.

In this application, the isolated antigen-binding protein is capable of binding to VEGF (eg, VEGF165) with a KD value of 1×10 ^-7 M or lower. For example, the value of the KD may be about 5×10 ^-8 M or less, about 4×10 ^-8 M or less, about 3×10 ^-8 M or less, about 2×10 ^-8 M or less, about 1×10 ^-8 M or less, about 9×10 ^-9 M or less, about 8×10 ^-9 M or less, about 7×10 -9 M or less, about 6× ^{10 -9} ^M or less, about 5×10 ^-9 M or less, about 4×10 ^-9 M or less, about 3×10 ^-9 M or less, about 2×10 ^-9 M or less, about 1×10 ^-9 M or less, about Values of 9 × 10 ^-10 M or less, about 8 × 10 ^-10 M or less, about 7 × 10 ^-10 M or less bind human-derived VEGF, e.g., as detected using the FortieBio Octet Molecular Interaction Analyzer .

The antigen-binding proteins described herein are capable of blocking the binding of VEGF (eg, VEGF165) to VEGFR (eg, VEGFR2). In some cases, the antigen-binding protein blocking the binding of VEGF and VEGFR can be determined by flow cytometry FACS or enzyme-linked immunoassay ELISA.

For example, human VEGFR (eg VEGFR2) is first incubated with decreasing amounts of unlabeled said antigen binding protein and subsequently with labeled VEGF protein. Then, OD450 is measured to confirm that the antigen-binding protein blocks the binding of VEGF (eg, VEGF165) to VEGFR (eg, 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, and about 0.02 μg /mL to about 0.5μg/mL, between about 0.2μg/mL to about 15μg/mL, between about 0.2μg/mL to about 12μg/mL, between about 0.2μg/mL to about 10μg/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. between, or between about 0.5 μg/mL and about 1.5 μg/mL.

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

In one aspect, the application provides a vector comprising the nucleic acid of the application. The nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) amplification in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) production by clonal recombination, (iii) purification , for example by enzymatic digestion and gel electrophoresis fractionation, or (iv) synthetic, for example by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

In one aspect, the present application provides an immunoconjugate comprising an antigen-binding protein of the present application. For example, the immunoconjugates of the present application may also include compounds with cell-killing capabilities. For example, the immunoconjugate of the present application can also include Contains a transmembrane domain, an intracellular stimulation domain, and/or an intracellular signaling domain.

In one aspect, the present application provides a cell comprising an antigen-binding protein of the present application, a nucleic acid of the present application, a vector of the present application, and/or an immunoconjugate of the present application. For example, the present application provides host cells that may comprise one or more nucleic acid molecules described herein 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.

In one aspect, the present application provides a pharmaceutical composition, which includes 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 cells of the present application, and any Choose a pharmaceutically acceptable carrier. The pharmaceutically acceptable adjuvant is nontoxic to the recipient at the dose and concentration used. The pharmaceutical composition in the present application may also contain more than one active compound, usually one that does not adversely affect each other. Those active compounds that have complementary activities.

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 the present application. Use of the applied kit in the preparation of medicaments for preventing, alleviating and/or treating tumors. For example, the tumor includes a tumor with high expression of PD-1, PD-L1 and/or VEGF. For use according to the present application, wherein the tumor includes solid tumors and/or non-solid tumors. Use according to 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 .

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 the present application. Application kit for preventing, alleviating and/or treating tumors. For example, the tumor includes a tumor with high expression of PD-1, PD-L1 and/or VEGF. For use according to the present application, wherein the tumor includes solid tumors and/or non-solid tumors. Use according to 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 .

On the one hand, the present application provides a method for preventing, alleviating and/or treating tumors, 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 cells, the drug of the present application compositions, and/or kits of the present application. For example, the tumor includes a tumor with high expression of PD-1, PD-L1 and/or VEGF. For use according to the present application, wherein the tumor includes solid tumors and/or non-solid tumors. Use according to 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 .

On the one hand, 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 of the present application Binding protein, nucleic acid of the present application, vector of the present application, immunoconjugate of the present application, cell of the present application, pharmaceutical composition of the present application, and/or kit of the present application. For example, the method may be an ex vivo or in vitro method. For example, the method may be a non-therapeutic method. In some cases, the method may include 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 PD-L1. , to detect whether a complex is formed between PD-1 and PD-L1. In some cases, the method may include 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 the relationship between VEGFR and VEGFR. whether a complex is formed between them.

A method for regulating immune response, 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 cells of the present application, the pharmaceutical composition of the present application, and/or the kit of the present application. For example, the regulating immune response includes stimulating immune cells to secrete cytokines. For example, the modulating immune response includes stimulating immune cells to secrete IL-2.

Without intending to be limited by any theory, the following examples are only for illustrating various technical solutions of the invention of the present application, and are not intended to limit the scope of the invention of the present application.

Example

Example 1 Preparation and detection of anti-PD-1 antibodies

1.1 Hybridoma antibody screening and preparation

Human PD-1, Mouse IgG2a Fc Tag fusion protein (Acrobiosystem, PD1-H5255a) and pcDNA3.4-hPD-1 plasmid DNA were used to immunize 5 Balb/c and SJL mice each time at an interval of 2 to 3 weeks, 3 After several times, booster immunization was carried out 1-3 times with Human PD-1, Mouse IgG2a Fc Tag fusion protein or CHOK1-hPD-1 stably transformed cell line (Genscript, M00529), and blood was collected to measure the titer. The titer test uses flow cytometry to measure the binding of serum to CHOK1 or 293T cells that overexpress human PD-1 protein. Mouse splenocytes with high binding titers are fused with myeloma (Sp2/0) cells.

Use flow cytometry to screen out ligands from the fusion plate that specifically bind to CHOK1-hPD-1 cells and can simultaneously block them. Clones that bind to PD-L1 and PD-L2 and cellular CHOK1-hPD1 are subcloned using the limiting dilution method, and monoclonal clones that can secrete specific antibodies are finally screened. Use serum-free medium for small-scale production and purification to obtain monoclonal antibodies for subsequent identification, including detection of the binding ability of the antibody to CHOK1-hPD1, blocking function of the binding of PD-L1, PD-L2 and CHOK1-hPD-1, and mixing Lymphocyte experiments finally yielded candidate anti-human PD-1 monoclonal antibodies 41D2-2C3D7 and 46H3A8. The variable region sequences of mouse hybridoma clones 41D2-2C3D7 and 46H3A8 were obtained through monoclonal antibody sequencing as follows:

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

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

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

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

Table 1 Sequences of heavy chain and light chain CDR regions of mouse hybridoma clone 41D2-2C3D7

Table 2 Sequences of heavy chain and light chain CDR regions of mouse hybridoma clone 46H3A8

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

Select the most homologous human Germline antibody (for example, derived from IMGT) through sequence comparison as the humanization design framework.

The light chain is based on IGKV1-9*01 (SEQ ID NO: 17), IGKJ4*01 (SEQ ID NO: 18), and the heavy chain is based on IGHV3-21*01 (SEQ ID NO: 19), IGHJ5*01 (SEQ ID NO: 20) was used as a framework, and 1-2 rounds of humanized mutations were carried out on the amino acids in the variable regions of 41D2-2C3D7 and 46H3A8 light and heavy chains based on sequence alignment and variable region structure information. After mutation, 41D2-2C3D7 obtained the humanized light and heavy chain variable regions 41D2HzL4 (SEQ ID NO:21) and 41D2HzH3 (SEQ ID NO:22); 46H3A8 obtained the humanized light and heavy chain variable region 1910h3hzL3 (SEQ ID NO:23 ) and 1910h3hzH4 (SEQ ID NO:24). Chothia numbering can be performed on the light and heavy chain variable regions of humanized antibodies to define the antibody light chain 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 an 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 an antibody. The weight chains are combined and paired to form humanized antibodies 41D2HzL4H3 (also known as 1910D2HzL4H3) and 1910h3hzL3H4.

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

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

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

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

1.3 Determination of kinetic parameters of humanized antibodies

(1) Octet RED96e (Fortebio) was used to determine the relationship between humanized antibodies 1910h3hzL3H4 and 41D2HzL4H3 and Affinity of human PD-1 (Sino Biological, Cat. No.: 10377-H08H), both antigen and antibody were diluted with 1xPBST (1xPBS: Sangon, B548117-0500; 0.02% Tween 20: sigma-alorich, P1379), and the concentration of antigen used is 100nM, and the antibody concentration used is 50nM.

(2) Sample on-machine detection (Octet Data Acquisition 11.1.0.11): First, add the sample to a 96-well plate (Greiner bio-one, 655209), and the system is 200 μL/well. Then set the software parameters, the plate temperature is set to 30°C, and the frequency of collecting standard kinetic signals is 5.0Hz. Next, pre-wet the AHC sensor (Fortébio, Cat. No.: 18-0015) with 1xPBST for 10 minutes, and then put it on the machine for detection. Each cycle includes the following steps: 1) Immerse in buffer for 60 seconds; 2) Detect whether the antigen binds non-specifically to the sensor; 3) Regenerate with 10mM glycine solution at pH 1.7; 4) Immerse in buffer for 60 seconds; 5) Antibody solidification On the sensor, the time is 20 seconds; 6) The sensor is immersed in the buffer for 180 seconds; 7) The antigen and antibody are combined, the time is 180 seconds; 8) The dissociation of the antigen and antibody, the time is 10 minutes; 9) The sensor is regenerated.

(3)Data analysis

Fortebio's Data Analysis 11.0 software was used to measure the binding rate (Ka) and dissociation rate (Kd) of the antigen-antibody in a 1:1 binding manner to calculate the equilibrium dissociation constant (K _D ) of the antibody. The results are shown in Table 3:

Table 3 Affinity of humanized antibodies to human PD-1

The results show that compared with Pembrolizumab, the affinity of the antibody to human PD-1 is equivalent to that of 1910h3hzL3H4; the affinity of 41D2HzL4H3 to human PD-1 is approximately 4.2 times higher than that of Pembrolizumab. This shows that the humanized antibody has higher binding affinity to PD-1.

1.4 Binding activity of candidate antibodies to cell surface human or cynomolgus PD-1

Collect CHOK-hPD-1 (GenScript, M00529) cells or CHOK1-cynoPD-1 (GenScript, M00572) cells, wash them once with FACS buffer (PBS containing 2% FBS) and resuspend them at 1E5/50ul /well, dispense into a 96-well plate (Corning3799). Add the antibody to the above 96-well U-shaped plate, 50ul/well, mix well and incubate at 4°C for 1 hour. Centrifuge at 2000rpm for 5 minutes to remove the supernatant, add 200ul FACS buffer to each well and wash once. Add 1:1000 Alexa Fluor 488 secondary antibody (Invitrogen) to each well, 100ul/well, mix well and incubate at 4°C for 1 hour. Centrifuge at 2000rpm for 5 minutes to remove the supernatant, add 200ul FACS buffer to each well and wash once, resuspend in 30ul FACS buffer and run on the machine for detection.

Figure 1 shows that the antigen-binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described in the present application bind to CHOK1 cells. Human PD-1 protein on the cell surface. Figure 2 shows that the antigen-binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described in this application bind to the cynomolgus monkey PD-1 protein on the surface of CHOK1 cells. The results showed that the binding activity of the humanized antibody to human or cynomolgus monkey PD-1 on the cell surface was comparable to that of Pembrolizumab.

1.5 Candidate antibodies block the binding activity of human PD-1 and PD-L1 or PD-L2 on the cell surface

Collect CHOK1-hPD-1 cells, wash them once with FACS buffer and resuspend them. Distribute the cells into a 96-well U-shaped plate at 1E5/50ul/well. The antibody was diluted and added to the above 96-well plate, 25ul/well. Biotinylated PD-L1 (Sinobiological, Cat. No. 10084-H02H-B) (or PD-L2, Acrobiosystem, Cat. No. PD2-H82F6) was diluted and added to the above. In a 96-well plate, 25ul/well, mix well and incubate at 4°C for 1 hour. Centrifuge at 2000rpm for 5 minutes to remove the supernatant, add 200ul FACS buffer to each well and wash once. Add 1:1000 SA-Alexa Fluor 488 (Invitrogen cat: S32354) to each well, 100ul/well, mix well and incubate at 4°C for 30 minutes. Centrifuge at 2000rpm for 5 minutes to remove the supernatant, add 200ul FACS buffer to each well and wash once, resuspend in 30ul FACS buffer and run on the machine for detection.

Figure 3 shows that the antigen-binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described in this application block the binding of human PD-L1 to the human PD-1 protein on the surface of CHOK1. Figure 4 shows that the antigen-binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described in this application block human PD-L2 and human PD-1 protein on the surface of CHOK1 cells. The results show that humanized antibodies have better blocking activity on the binding of human PD-1 to PD-L1 or PD-L2 on the cell surface.

1.6 Mixed lymphocyte reaction induced by candidate antibodies

Use cell culture medium (1640+2% FBS) to resuscitate human dendritic DC cells, adjust the DC cell density to 1*10^5-1*10^7cells/mL, and then add mitomycin at a final concentration of 50μg/ml C. Treat at 37 degrees in the dark for 30 minutes, add 10 ml of culture medium to terminate, centrifuge at 400 g for 10 minutes, and then wash once with 10 mL of culture medium. Gradient dilution of anti-PD-1 antibody: The highest final concentration of the antibody is 2.5 μg/mL (prepared concentration is 10 μg/mL), 10-fold gradient dilution (5 concentration points + 1 0 concentration), and then the corresponding cell culture plate (Corning , Cat. No.: 3599), add 50 μL of prepared anti-PD-1 antibody. Collect DC cells treated with human peripheral blood lymphocytes PBMC and mitomycin C, adjust the DC cell density to 2*10^5 cells/mL, and then add the cells to the culture plate, 50 μL per well, that is, the number of DC cells per well is 1*10^4 cells/well; adjust the PBMC cell density to 2*10^6 cells/mL, and then add cells to the culture plate, 100 μL per well, that is, the number of PBMC cells per well is 2*10^5 cells/well. Place the cell culture plate in a 37°C, 5% carbon dioxide cell culture incubator for 5 days. After 5 days, centrifuge at 300g for 5 minutes. Collect the supernatant and use the Human IL-2 ELISA kit (BD, Cat. No.: 550611) to detect the IL-2 content. The detection method was carried out in strict accordance with the kit instructions, and the data was processed with GraphPad Prism software.

The results are shown in Figure 5. The results show that both humanized antibodies 1910h3hzL3H4 and 41D2HzL4H3 can stimulate lymphocytes to secrete IL2, and their activity is similar to that of Pembrolizumab. Explain that the antigen-binding protein of the present application stimulates immune cells to secrete cells Factor ability is better.

Example 2 Preparation and detection of anti-VEGF single domain antibody

2.1 Construction and panning of phage single domain antibody library

Take blood samples from three alpacas that have not been immunized with the target antigen, and take 150ml from each. Lymphocytes were isolated from blood samples and total RNA was extracted to construct a phage display nanobody library with a library size of 3*10 ⁹ cfu. Take 6 ml of transformed antibody library bacteria to prepare phage for specific panning. The total number of bacteria is 50 times greater than the library capacity.

Pre-bind 50ml Streptavidin Magnetic Beads (Thermo fisher, Cat. No.: 88817) with 1ml phage and incubate it at room temperature for 30 minutes to remove non-specific binding. After removing the background, add 10ug Biotinylated Human VEGF165 (Cat. No.: VE5-H82Q0), 150ul Streptavidin Magnetic Beads to the nanobody library phage, incubate at room temperature for 15 minutes, and wash 14 times with PBST (PBS containing 0.05% Tween-20). Unbound phages are washed away. The antigen-specific binding phage was eluted with 450ul100mM hydrochloric acid, and 50ul of 1M Tris-HCl at pH 11 was added to neutralize and infect E. coli SS320 in the logarithmic growth phase, and the phage were generated and purified 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 phages enriched after two rounds of screening were used to identify the enrichment status using enzyme-linked immunoassay (ELISA). The results are shown in Table 4. The results showed that the phages were significantly enriched after two rounds of panning.

Table 4 Enzyme-linked immunoassay (ELISA) identification after the second round of enrichment

2.2 Yeast display library construction and screening

As shown in Figure 6, the construction scheme of single domain antibody displayed on the surface of yeast cell wall uses the plasmid after two rounds of panning of the phage antibody library as a template to design primers for polymerase chain reaction (PCR) to amplify the nanobody gene (V _H H ); the PCR-amplified _VHH gene fragment is recovered and co-transfected with the yeast display plasmid into Saccharomyces cerevisiae strain EBY100 (purchased from ATCC). The VHH gene is inserted into the yeast display plasmid through homologous recombination of Saccharomyces cerevisiae, thereby achieving Yeast cell wall surface displays single domain antibodies.

Use a flow sorter to conduct two rounds of sorting of the yeast display library. The sorted yeasts are spread on auxotrophic plate culture medium, and 46 single clones are selected for sequencing. Finally, 5 single domains with unique sequences are obtained. Heavy chain antibodies. Perform flow cytometry staining analysis on the corresponding yeast monoclonal colonies, and take 1×10 ⁶ cells for staining according to each protocol shown in Table 5.

Table 5 Scheme for identification of monoclonal yeast colonies by flow cytometry

The strength of the cell population that binds to human VEGF165-Bio in Scheme 1 is reflected by the PE mean fluorescence signal intensity (MFI). Similarly, Scheme 2 and Scheme 3 can evaluate the Mouse VEGF-Bio and non-specific binding levels. The competition signal in Scheme 4 is determined by APC. Reflected by mean fluorescence signal intensity (MFI). The results are shown in Table 6. After excluding clones that did not bind to human VEGF and mouse VEGF, monoclonal Y20A6, an anti-human VEGF165 single domain heavy chain antibody, was finally obtained. The binding and competition abilities of clone Y20A6 were both ideal.

Table 6 Flow cytometric staining analysis results of yeast single clone colonies after the second round of screening of yeast display library

2.3 Humanized design and expression of single domain antibodies

Perform IMGT/Domain Gap Align on the sequence of monoclonal antibody Y20A6, and find the human germline with the highest homology to it, which is IGHV3-23*04. The antibody sequence is numbered according to Chothia rules. The monoclonal Y20A6 was mutated as shown in Table 7 below to obtain Hz20A6.2 and Hz20A6.3.

Table 7 Humanized design of single domain antibody sequences

Y20A6 was fused with the heavy chain variable region of humanized clones Hz20A6.1 and Hz20A6.3 and IgG1 Fc N297A (SEQ ID NO: 30) to form an antibody heavy chain, forming a heavy chain single domain antibody Ab1910VE6 (corresponding to Y20A6) , Ab1910VE8 (corresponding to Hz20A6.2) and Ab1910VE9 (corresponding to Hz20A6.3). Codon optimization was performed, and the gene was synthesized and loaded into the expression vector pcDNA3.4 (Life Technologies). After expression plasmid amplification and plasmid extraction, the plasmid is transferred into ExpiCHO cells. (ThermoFisher Scientific, A29133), the antibody was transiently expressed according to the supplier's ExpiCHO expression system method, and the purified antibody expression and purification data are shown in Table 8.

Table 8 Single domain antibody expression and purification data

2.4 Determination of binding activity of single domain antibody to human VEGF165

Referring to the method in Example 1.3, the affinity of single domain antibodies to human VEGF165 before and after humanization was measured. The results are shown in Table 9 below. AB1910VE8 and AB1910VE9 have better affinity with human VEGF. AB1910VE8 and AB1910VE9 antibodies were selected for blocking function experiments.

Table 9 Affinity of single domain antibodies to human VEGF165

2.5 Determination of the activity of single domain antibodies in blocking the binding of human VEGF165 to human VEGFR2

The competitive ELISA method was used to determine the binding activity of single domain antibodies to block VEGF165 and human VEGFR2. Specifically, human VEGFR2 (Acro, Cat. No.: KDR-H5227) was diluted to 1 μg/mL with PBS (Gibco, Cat. No.: 10010-023), and then added Pour 100 μL into each well of a 96-well plate, cover with sealing film, and incubate at 4°C overnight. The next day, wash the plate three times with washing solution PBST (0.05% TWEEN-20: sigma-alorich, P1379), then use the washing solution to prepare blocking solution PBST+2% BSA (BSA: VWR, 0332-1KG), and add it to each well 300 μL blocking solution, block at 37°C for 1 hour. Use blocking solution to prepare Anti-VEGF and Biotin-hVEGF (Acro, Cat. No.: VE5-H82Q0). The starting concentration of Anti-VEGF is 200 μg/mL, 5-fold gradient dilution (7 concentration points + 1 0 concentration point) , the preparation concentration of Biotin-hVEGF is 90ng/mL. Then take out the blocked enzyme plate, wash the plate three times with washing solution, add 50 μL of diluted Anti-VEGF to the 96-well plate, then add 50 μL of Biotin-hVEGF to the 96-well plate, and incubate at 37°C. 1 hour. Wash the plate three times with washing solution, dilute the secondary antibody SA-HRP (sigma, S2438) in blocking solution 1:5000, add 100 μL/well to the enzyme plate, and incubate at 37°C for 1 Hour. Wash the plate three times with washing solution, add 100 μL of TMB chromogenic solution (Biopanda, Cat. No.: TMB-S-003) to each well, and develop the color at room temperature in the dark. Then add 50 μL of stop solution (Solarbio, Cat. No.: C1058) to each well to terminate the reaction. , measure the absorbance value at a wavelength of 450nm.

The results of single domain antibodies blocking the binding of VEGF165 and human VEGFR2 are shown in Figure 7. The results show that Ab1910VE8, Ab1910VE9 and Ab1910VE6 all have blocking functions, and AB1910VE9 was selected for affinity maturation.

2.6 Construction of single domain antibody affinity maturation library

The Ab1910VE9 sequence was affinity matured to improve its affinity with human VEGF. The CDR region of Ab1910VE9 was defined according to Chothia, and the amino acids at the HCDR1-3 and LCDR1-3 sites were randomly mutated to construct a mutation library. Design NNK mutation primers for polymerase chain reaction (PCR) amplification of each CDR mutation library gene fragment. The gene fragments of each CDR mutation library and the yeast display plasmid were transferred into Saccharomyces cerevisiae strain EBY100 (purchased from ATCC), so that each CDR mutation library was displayed on the yeast surface. At the same time, the parental sequence of Ab1910VE9 was displayed on the yeast surface and used as a control.

After the library was cultured and induced, Biotin-Human VEGF165 was used for three rounds of sorting, with a starting concentration of 3nM and 10-fold gradient dilution. Collect cell populations with high display levels and strong antigen-binding ability; after sorting, the cells are spread on SD-Trp solid medium and cultured statically at 30°C for 3 days.

Single clones were selected for sequencing, and the single clones with unique sequences obtained were identified by flow cytometry, incubated and stained with 1nM Biotin-Human VEGF165, and the ratio of the average fluorescence signal intensity displayed by different clones to the average fluorescence signal intensity of antigen binding was compared. Reflects the ability of a single molecule to bind to an antigen. Based on the binding force value, the expressed antibody Ab1910VE21 (heavy chain variable region amino acid sequence is SEQ ID NO: 25) was finally selected. The antibody expression number and monoclonal identification results are shown in Table 10 below. The Chothia numbered CDR region amino acid sequence of Ab1910VE21 is as shown in Table 11.

Table 10 Monoclonal identification results and antibody expression numbers

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

Table 11 1910VE21 single domain antibody CDR region sequence

2.7 Affinity matured single domain antibody expression and SEC-HPLC purity analysis

Express and purify the single domain antibodies in Table 10. Perform SEC-HPLC purity analysis of expressed antibodies as follows:

(1) Dilute the sample to 1mg/mL, mix well, centrifuge at 12000rpm for 5 minutes, transfer the supernatant to a sample bottle, and put it into the HPLC sample tray. Set the chromatographic conditions as follows:

(2) After equilibrating the chromatographic column with the mobile phase (200mM phosphate buffer, pH 6.8), inject samples for analysis, use chromatography software for data analysis, and calculate the peak area percentage of each peak using the peak area normalization method.

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

Table 12 Affinity matured single domain antibody expression and purification data

2.8 Determination of kinetic parameters of affinity matured single domain antibodies

Referring to the method in Example 1.3, the affinity of the humanized antibody and human VEGF165 was measured. The results are shown in Table 13. The affinity of the antibody molecule AB1910VE21 after affinity maturation increased several times.

Table 13 Affinity of affinity matured antibodies to human VEGF165

2.9 Functional experiment of affinity mature VEGF antibody blocking the binding of human VEGF165 and human VEGFR2

Referring to the method of Example 2.5, the affinity matured VEGF antibody was tested for blocking the binding function of human VEGF165 and human VEGFR2.

The experimental results are shown in Figure 8. The results show that the blocking effect of Ab1910VE18 and Ab1910VE21 antibodies is equivalent to that of Bevacizumab.

Example 3 Design and detection of anti-PD-1/VEGF bispecific antibodies Kab5 and Kab6

3.1 Bispecific antibody design and preparation

The bispecific antibodies Kab5 and Kab6 are designed as shown in the table below, that is, a nanobody fragment is connected to the C-termini of two heavy chains of an IgG antibody.

Table 14 Design of bispecific antibodies Kab5 and Kab6

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

Kab5 and Kab6 genes were synthesized and loaded into the expression vector pcDNA3.4 (Life Technologies). After expression plasmid amplification and plasmid extraction, the plasmid was transferred into ExpiCHO cells (ThermoFisher Scientific, A29133), and the antibody was transiently expressed according to the supplier's ExpiCHO expression system method. The antibody was purified for subsequent property determination.

3.2 Affinity determination of bispecific antibodies Kab5, Kab6 and human VEGF165

Referring to the method in Example 1.3, the affinity of biotin human VEGF165 (ACRO, product number: VE5-H82Q0) and bispecific antibodies Kab5 and Kab6 was determined. The results are shown in Table 15 below. The difference in affinity between Kab5 and Kab6 and human VEGF165 is small, slightly lower than that of Bevacizumab.

Table 15 Affinity of bispecific antibodies Kab5 and Kab6 with human VEGF165

3.3 Affinity determination of bispecific antibodies Kab5 and Kab6 with human PD-1

Referring to the method in Example 1.3, determine the affinity between PD-1 (Sino Biological, Cat. No.: 10377-H08H) and bispecific antibodies Kab5 and Kab6. The results are shown in Table 16 below. The results show that the affinity difference between Kab5 and Kab6 with human PD-1 is small, and both are better than Pembrolizumab.

Table 16 Affinity of bispecific antibodies Kab5 and Kab6 with human PD-1

3.4 Binding activity of bispecific antibodies Kab5 and Kab6 to cell surface antigen PD-1

Referring to the method in Example 1.4, the binding activity of the bispecific antibodies Kab5 and Kab6 to the cell surface antigen PD-1 was determined. The binding results of bispecific antibodies Kab5 and Kab6 to cell surface antigen PD-1 are shown in Figure 9. The results showed that the binding activity of bispecific antibodies Kab5 and Kab6 to cell surface antigen PD-1 was similar to that of humanized antibody 41D2HzL4H3.

3.5 Bispecific antibodies block the binding activity of cell surface antigen PD-1 to PD-L1 and PD-L2

Referring to the method in Example 1.5, the binding activity of the bispecific antibody in blocking the cell surface antigen PD-1 and PD-L1 and PD-L2 was determined. The results of bispecific antibodies blocking the binding of cell surface antigens PD-1 and PD-L1 are shown in Figure 10. The results of bispecific antibodies blocking the binding of cell surface antigens PD-1 and PD-L2 are shown in Figure 11. The results showed that the binding activity of the bispecific antibody to block cell surface antigens PD-1, PD-L1, and PD-L2 was similar to that of the humanized antibody 41D2HzL4H3.

3.6 Determination of the activity of bispecific antibodies competing with VEGFR2 for binding to VEGF

The competitive ELISA method was used to measure the activity of the bispecific antibody competing with VEGFR2 for binding to the antigen VEGF. Referring to the measurement method in Example 2.5, the activity of the bifunctional antibody competing with VEGFR2 for binding to the antigen VEGF was measured. The activity results of bispecific antibodies competing with VEGFR2 for binding to the antigen VEGF are shown in Figure 12. The results show that Kab5 and Kab6 blocking effects are equivalent and both are better than Bevacizumab.

3.7 Bispecific antibodies induce mixed lymphocyte reactions

Referring to the experimental method of Example 1.6, the reaction of the bispecific antibody in inducing mixed lymphocytes to secrete the cytokine IL2 was measured. The results of the bispecific antibody stimulating the mixed lymphocytes to secrete the cytokine IL2 are shown in Figure 13. The results show that Kab5 and Kab6 bispecific antibodies have similar functions to pembrolizumab.

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 modifications to the embodiments described herein will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims

An antigen-binding protein comprising a first targeting portion and a second targeting portion, the first targeting portion comprising an antibody that specifically binds PD-1 or an antigen-binding fragment thereof, the second targeting portion comprising Antibodies or antigen-binding fragments thereof that specifically bind to VEGF;

Wherein, the first targeting portion includes HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 of an antibody that specifically binds to PD-1 or an antigen-binding fragment thereof, wherein the HCDR1 includes the amino acid shown in SEQ ID NO: 5 Sequence, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 6, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 7, the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 8, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 9, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 10; or;

Among them, HCDR1 contains the amino acid sequence shown in SEQ ID NO: 11, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 12, HCDR3 contains the amino acid sequence shown in SEQ ID NO: 13; LCDR1 contains the amino acid sequence shown in SEQ ID NO: 14 The amino acid sequence of LCDR2 includes the amino acid sequence shown in SEQ ID NO: 15, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 16;

Wherein, the second targeting portion includes HCDR1, HCDR2 and HCDR3 of the antibody or its antigen-binding fragment, the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 26, and the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 27 Amino acid sequence, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 28.
The antigen-binding protein of 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 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 1, and the VL includes an amino acid sequence selected from SEQ ID NO: 2;

(ii) the VH includes the amino acid sequence shown in SEQ ID NO: 3, and the VL includes the amino acid sequence shown in SEQ ID NO: 4;

(iii) The VH includes the amino acid sequence shown in SEQ ID NO: 22, and the VL includes the amino acid sequence shown in SEQ ID NO: 21; or

(iv) The VH includes the amino acid sequence shown in SEQ ID NO: 24, and the VL includes the amino acid sequence shown in SEQ ID NO: 23.
The antigen-binding protein of any one of claims 1-2, wherein the first targeting portion further comprises an antibody heavy chain constant region derived from an IgG constant region, for example comprising SEQ ID The amino acid sequence shown in NO:32.
The antigen-binding protein according to any one of claims 1-3, wherein the first targeting portion further comprises an antibody light chain constant region, for example, comprising the amino acid sequence shown in SEQ ID NO: 31.
The antigen-binding protein according to any one of claims 1-4, wherein the second targeting portion comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 25.
The antigen-binding protein of any one of claims 1-5, wherein the second targeting portion comprises an antibody heavy chain constant region derived from an IgG constant region, for example comprising SEQ ID NO. : The amino acid sequence shown in 30.
The antigen-binding protein according to any one of claims 1-6, wherein the antibody is selected from the group consisting of murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies.
The antigen-binding protein according to any one of claims 1-7, wherein the antigen-binding fragment includes Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
The antigen-binding protein according to any one of claims 1-8, the antigen-binding protein comprising a bispecific antibody, for example, the antigen-binding protein comprising an IgG-VHH type bispecific antibody, such as a bispecific antibody Kab5, Kab6.
The antigen-binding protein according to any one of claims 1 to 9, the first targeting portion includes an antibody heavy chain and an antibody light chain, and the second targeting portion is located at the anti-antibody portion of the first targeting portion. The C-terminus of the heavy chain, wherein the second targeting moiety is directly connected to the first targeting moiety or indirectly connected through a linker.
The antigen-binding protein according to claim 10, the linker comprises the amino acid sequence shown in SEQ ID NO: 29.
The antigen-binding protein according to any one of claims 1-11, said antigen-binding protein comprising a first polypeptide chain and a second polypeptide chain, said first polypeptide chain comprising said first targeting moiety an antibody heavy chain or an antigen-binding fragment thereof, an optionally present or absent linker and the second targeting portion of the antibody or an antigen-binding fragment thereof, and the second polypeptide chain comprises the first targeting portion Part of an antibody light chain or antigen-binding fragment thereof.
The antigen-binding protein according to claim 12, 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 - Variable region of two targeting moieties - the second targeting moiety the constant region, and the second polypeptide chain comprising: 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 includes the linkage son.
According to the antigen-binding protein according to any one of claims 12-13, the first polypeptide chain sequentially includes an antibody heavy chain or an antigen-binding fragment thereof of the first targeting portion from the N-terminus to the C-terminus and the third An antibody with two targeting portions or an antigen-binding fragment thereof, or the first polypeptide chain sequentially includes an antibody heavy chain of the first targeting portion or an antigen-binding fragment thereof, a linker and the third targeting portion from the N-terminus to the C-terminus. Two targeting portions of antibodies or antigen-binding fragments thereof.
A nucleic acid encoding the antigen-binding protein of any one of claims 1-14.
A vector comprising the nucleic acid of claim 15.
An immunoconjugate comprising the antigen-binding protein of any one of claims 1-14.
A cell comprising the antigen-binding protein of any one of claims 1-14, the nucleic acid of claim 15, the vector of claim 16, and/or the immunoconjugation of claim 17 things.
A pharmaceutical composition comprising the antigen-binding protein of any one of claims 1-14, the nucleic acid of claim 15, the carrier of claim 16, and the immunoconjugate of claim 17 , and/or the cell of claim 18, and optionally a pharmaceutically acceptable carrier.
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, and the immunoconjugate of claim 17, The cell according to claim 18, and/or the pharmaceutical composition according to claim 19.
A method of preparing the antigen-binding protein of any one of claims 1-14, which includes culturing the cells of claim 18 under conditions that enable expression of the antigen-binding protein.
An antigen-binding protein according to any one of claims 1 to 14, the nucleic acid according to claim 15, the vector according to claim 16, the immunoconjugate according to claim 17, or the immunoconjugate according to claim 18 The use of cells, the pharmaceutical composition of claim 19, and/or the kit of claim 20 in the preparation of medicines for preventing, alleviating, and/or treating tumors.
The use according to claim 22, wherein the tumor comprises a tumor with high expression of PD-1, PD-L1 and/or VEGF.
The use according to any one of claims 22-23, wherein the tumor comprises a solid tumor and/or a non-solid tumor.
The use according to any one of claims 22 to 24, wherein the tumor includes colorectal cancer, breast cancer, blastoma, cervical cancer, ovarian cancer, melanoma, lung cancer, renal cancer, esophageal cancer, head and neck cancer , lymphoma, liver and/or gastric cancer cancer.
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 any one of claims 1-14 The antigen-binding protein, 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 according to claim 20.
A method of regulating 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, or the vector of claim 17 The immunoconjugate, the cell of claim 18, the pharmaceutical composition of claim 19, and/or the kit of claim 20.
According to the method of claim 27, the regulating immune response comprises stimulating immune cells to secrete cytokines.