CN116284406A - A kind of PD-1 binding protein and its application - Google Patents

Abstract

A PD-1 binding protein and its use are provided, as are uses of the antigen binding protein.

Description

A kind of PD-1 binding protein and its application

technical field

This application relates to the field of biomedicine, in particular to a PD-1 binding protein and its application.

Background technique

Programmed Death-1 (PD-1) is a type I membrane protein with 288 amino acids, mainly expressed on the surface of activated T cells. PD-1 has two ligands, namely programmed death ligand-1 (Programmed Death Ligand-1, PD-L1) and PD-L2. The interaction of PD-1 with PD-L1 and PD-L2 will down-regulate the activity of T cells, weaken the secretion of cytokines, and play an immunosuppressive role. PD-1/PD-L1 pathway inhibitors can block the combination of PD-1 and PD-L1, block negative regulatory signals, restore the activity of T cells, play a role in killing tumor cells, and then inhibit tumor growth. Therefore, immune regulation targeting PD-1/PD-L1 is of great significance for tumor suppression. At present, antibody drugs that block the PD-1/PD-L1 pathway still face many challenges in clinical practice, such as low effectiveness, drug resistance, and side effects. It is still necessary to continue to develop more effective anti-PD-1 antibodies and its humanized antibodies.

Contents of the invention

The present application provides an antigen-binding protein that binds to PD-1, which exhibits one or more desired functional properties, such as high-affinity binding to PD-L1, the ability to inhibit the binding of PD-1 to PD-1, Enhanced T cell activation includes the ability to proliferate, secrete IFN-γ and/or IL-2, stimulate antibody responses, and/or reverse the suppressive function of immunosuppressive cells such as T regulatory cells. In one embodiment, the present application also provides a scFv-huIgG1 Fc-type antibody, which can be conveniently used in combination with cellular drugs in an autocrine form. The present application also provides nucleic acid molecules encoding the isolated antigen-binding protein, expression vectors, host cells and methods for preparing the isolated antigen-binding protein. Antigen binding proteins that bind PD-1 disclosed herein can be used (alone or in combination with other active agents or treatment modalities) to treat, prevent and/or diagnose diseases, such as cancer diseases (eg, solid and soft tissue tumors).

In one aspect, the application provides an isolated antigen binding protein, said antigen binding protein comprising a light chain variable region and a heavy chain variable region, said heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, said HCDR1 Comprising the amino acid sequence shown in SEQ ID NO: 1; the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 2; the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 3; the light chain variable region contains LCDR1 , LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5 (YAS); the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6 sequence; and the heavy chain variable region of the antigen binding protein comprises at least one FR in the antibody heavy chain variable region VH, the VH comprising the amino acid sequence shown in SEQ ID NO:67.

In another preference, the heavy chain variable region of the antigen-binding protein comprises at least one FR in the antibody heavy chain variable region VH, and the VH comprises any one of SEQ ID NO:54 to 56. amino acid sequence.

In another preferred example, the light chain variable region of the antigen-binding protein comprises at least one FR in the antibody light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:68.

In another preference, the light chain variable region of the antigen-binding protein comprises at least one FR in the VL of the light chain variable region of an antibody, and the VL comprises any one of SEQ ID NO:57 to 59. amino acid sequence.

In another preferred example, the heavy chain variable region of the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR4 comprises the amino acid shown in SEQ ID NO:45 sequence.

In another preferred example, the heavy chain variable region of the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR3 comprises the amino acid shown in SEQ ID NO:63 sequence.

In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR3 comprises any of SEQ ID NO:42 to 44 Amino acid sequence shown in one item.

In another preference, the heavy chain variable region of the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR2 comprises the amino acid shown in SEQ ID NO:41 sequence.

In another preference, the heavy chain variable region of the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises the amino acid shown in SEQ ID NO:62 sequence.

In another preferred example, the heavy chain variable region of the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises any of SEQ ID NO:39 to 40 Amino acid sequence shown in one item.

In another preference, the light chain variable region of the antigen-binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR4 comprises the amino acid shown in SEQ ID NO:53 sequence.

In another preference, the light chain variable region of the antigen-binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR3 comprises the amino acid shown in SEQ ID NO:66 sequence.

In another preference, the light chain variable region of the antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR3 comprises any of SEQ ID NO:51 to 52 Amino acid sequence shown in one item.

In another preference, the light chain variable region of the antigen-binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR2 comprises the amino acid shown in SEQ ID NO:65 sequence.

In another preferred embodiment, the light chain variable region of the antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR2 comprises any of SEQ ID NO:49 to 50 Amino acid sequence shown in one item.

In another preference, the light chain variable region of the antigen-binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1 comprises amino acids shown in SEQ ID NO:64 sequence.

In another preferred embodiment, the light chain variable region of the antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1 comprises any of SEQ ID NO:46 to 48 Amino acid sequence shown in one item.

In another preferred example, the heavy chain variable region of the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:67.

In another preferred example, the heavy chain variable region of the antigen-binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:54 to 56.

In another preferred example, the light chain variable region of the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:68.

In another preferred example, the light chain variable region of the antigen-binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:57-59.

In another preferred example, the heavy chain variable region of the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:67, and the light chain variable region of the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:68 amino acid sequence.

In another preferred example, the heavy chain variable region of the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:54, and the light chain variable region of the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:57 amino acid sequence.

In another preference, the heavy chain variable region of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:55, and the light chain variable region of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:58 amino acid sequence.

In another preferred example, the heavy chain variable region of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:56, and the light chain variable region of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:59 amino acid sequence.

In another preferred example, the antigen-binding protein further includes an antibody heavy chain constant region.

In another preferred example, the antigen-binding protein includes an antibody heavy chain constant region derived from a human IgG constant region.

In another preferred example, the antibody heavy chain constant region of the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:60.

In another preferred example, the antigen-binding protein includes an antibody light chain constant region.

In another preferred example, the antibody light chain constant region of the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:61.

In another preferred example, the antigen-binding protein includes an antibody or an antigen-binding fragment thereof.

In another preferred embodiment, the antibody includes Fab, Fab', Fv fragment, F(ab')2, scFv, di-scFv and/or dAb.

In another preferred example, the antigen-binding fragment is selected from the group consisting of humanized antibodies, chimeric antibodies and fully human antibodies.

In another preferred example, the antigen-binding protein has one or more properties selected from the following groups: capable of binding to human PD-1, capable of blocking the binding of PD-1 and PD-L1, capable of blocking The combination of PD-1 and PD-L2 can stimulate the secretion of IL-2, TNF-α and/or IFN-γ in immune cells, can inhibit tumor growth and/or tumor cell proliferation, and can improve the killing ability of immune cells.

In another aspect, the present application provides a polypeptide comprising the isolated antigen-binding protein, and optionally a tag sequence that facilitates expression and/or purification.

In another aspect, the application provides one or more isolated nucleic acid molecules encoding said isolated antigen binding protein and/or said polypeptide.

In another aspect, the present application provides a vector comprising the nucleic acid molecule.

In another aspect, the present application provides a cell comprising the nucleic acid molecule or the vector.

In another aspect, the present application provides a method for preparing the isolated antigen-binding protein, the method comprising culturing the cell under conditions that allow the expression of the isolated antigen-binding protein.

In another aspect, the present application provides a pharmaceutical composition, which comprises the isolated antigen-binding protein, the nucleic acid molecule, the carrier and/or the cell, and optionally a pharmaceutically acceptable adjuvant.

In another aspect, there is provided an immunoconjugate comprising said isolated antigen binding protein, and a conjugation moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme .

In another aspect, the application provides the isolated antigen-binding protein, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition and/or the immunoconjugate in Use in the preparation of medicaments for treating PD-1-mediated diseases or conditions.

In certain embodiments, the PD-1-mediated disease or condition includes cancer or tumor.

In another aspect, the application provides the use of the isolated antigen-binding protein and/or the immunoconjugate in the preparation of a detection reagent, detection plate or detection kit for detecting PD-1 molecules in a sample .

In another aspect, the application provides a method for detecting PD-1 protein in a sample (including non-diagnostic purposes and diagnostic purposes), the method comprising the steps of:

(1) contacting the sample with the isolated antigen-binding protein and/or the immunoconjugate;

(2) Detect whether the antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of PD-1 protein in the sample.

In another aspect, the present application provides a method for influencing target cells to produce cytokines for non-therapeutic and/or diagnostic purposes, said method comprising administering said antigen-binding protein, said polypeptide, said nucleic acid, said said vector, and/or express said antigen-binding protein, said polypeptide, or a cell comprising said nucleic acid and/or said vector.

In another preferred example, the target cells include immune cells, such as tumor infiltrating lymphocytes (TIL), peripheral blood mononuclear cells (PBMC).

In another preferred example, the cytokines include IL-2, TNF-α and/or IFN-γ.

In another aspect, the present application provides a method for inhibiting the combination of PD-1 and PD-L1, which includes administering an effective amount of the antigen-binding protein, the nucleic acid molecule, and the carrier to a subject in need. , the cell and/or the pharmaceutical composition.

In another aspect, the present application provides a method for inhibiting the combination of PD-1 and PD-L2, which includes administering an effective amount of the antigen-binding protein, the nucleic acid molecule, and the carrier to a subject in need. , the cell and/or the pharmaceutical composition.

In another aspect, the present application provides a method for preventing, alleviating or treating a PD-1-mediated disease or disorder, which includes administering an effective amount of the antigen-binding protein and the nucleic acid molecule to a subject in need. , the carrier, the cell and/or the pharmaceutical composition.

In certain embodiments, the PD-1-mediated disease or condition includes cancer or tumor.

In another preferred example, the cancer or tumor includes solid tumors and blood tumors.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the specific embodiments which are disclosed without departing from the spirit and scope of the invention to which this application relates. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary rather than restrictive.

Description of drawings

The particular 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 with reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the accompanying drawings is as follows:

Figure 1 shows the detection curve of the affinity (based on BLI method) between the tetravalent human PD-L1 extracellular segment mutant described in the present application and PD-1-huIgG1 Fc;

Figure 2 shows the inhibition curves of 6H6 and pembrolizumab's huIgG1 antibody binding to PD-1 and PD-L1;

Figure 3 shows the binding curves of huIgG1 antibodies of 6H6 and pembrolizumab to PD-1.

Figure 4 shows the results of the proportion of CD107a cells measured after adding PD-1 antibody to the tumor infiltrating lymphocyte (TIL) cell culture medium from donor A and donor B.

Figure 5 shows the results of cytokine secretion measured after adding PD-1 antibody to the tumor infiltrating lymphocyte (TIL) cell culture medium from donor A and donor B. Among them, A-C show the secretion of cytokines by tumor infiltrating lymphocytes from donor A; D-F show the secretion of cytokines by tumor infiltrating lymphocytes from donor B.

Figure 6 shows the results of the mixed lymphocyte reaction (MLR) test to detect the stimulation of T lymphocytes by the PD-1 antibody.

Figure 7 shows the result of adding PD-1 antibody to enhance the killing ability of TCR-T cells.

Figure 8 shows the antigen binding affinity results of the 6H6 antibody.

Figure 9 shows the antigen binding affinity results of the 6H6-5 antibody.

Figure 10 shows the antigen binding affinity results of the 6H6-25 antibody.

Figure 11 shows the antigen binding affinity results of the 6H6-29 antibody.

Figure 12 shows the half inhibitory concentration IC ₅₀ of 6H6 and humanized 6H6-5, 6H6-25, 6H6-29 huIgG1 antibodies on the binding of PD-1 and PD-L1.

Detailed ways

The implementation of the invention of the present application will be described in the following specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

In this application, the term "PD-1" generally refers to programmed cell death 1, also known as "programmed death 1", "CD279", "cluster of differentiation 279", "PD1", "PDCD1". PD-1 is normally expressed on T cells, B cells, natural killer T cells, activated monocytes and dendritic cells (DCs), and is involved in apoptosis. PD-1 generally consists of an extracellular IgV domain, a transmembrane region and an intracellular domain. PD-1 can bind two ligands, PD-L1 and PD-L2. The "PD-1" includes any native PD-1 of any vertebrate origin, including mammals, such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). The term encompasses "full length", unprocessed PD-1 as well as any form of PD-1 produced by cellular processing. 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 homologues, derivatives, analogs of PD-1, and PD-1 with at least one Epitope analogs. The amino acid sequence of human PD1 is shown in UniProt (www.uniprot.org), accession number Q15116.

In this application, the term "PD-L1" generally refers to programmed cell death 1 ligand 1, also known as B7 homolog 1, B7-H1, cluster of differentiation 274, (3)274 or CD274, which is related to PD-1 binding downregulates T cell activation and cytokine secretion. "PD-L1" includes any native PD-L1 of any vertebrate origin, including mammals, such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats) ). The term encompasses "full length", unprocessed PD-L1 as well as any form of PD-L1 produced by cellular processing. PD-L1 can exist as a transmembrane protein or as a soluble protein. "PD-L1" includes intact PD-L1 and its fragments, and also includes functional variants, isoforms, species homologues, derivatives, analogs of PD-L1, and PD-L1 with at least one Epitope analogs. 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. The complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ7.

In this application, the terms "isolated" or "purified" generally refer to a molecule (eg, antibody, nucleic acid, etc.) that is at least partially separated from other molecules with which it is normally associated in its native state. An "isolated or purified polypeptide" is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, cell debris, and growth media. An "isolated or purified nucleic acid" is at least partially separated from nucleic acids that, in their native state, normally flank the polynucleotide. Thus, polynucleotides fused to regulatory or coding sequences with which they are not normally associated are considered isolated in this application, eg, as a result of recombinant techniques. Such molecules are considered isolated even when present, for example, in the chromosome of a host cell or in a nucleic acid solution. In general, the terms "isolated" and "purified" are not intended to refer to the complete absence of such substances or the absence of water, buffers or salts, unless they are present in amounts which substantially interfere with the experimental or therapeutic use of the molecule. The antigen binding proteins of the present application and nucleic acids encoding the antigen binding proteins of the present application are isolated/purified.

In this application, the term "antigen binding protein" is used in its broadest sense and means comprising a moiety that binds to an antigen or target and optionally comprises a framework or framework that allows the antigen binding moiety to adopt a configuration that facilitates binding of the antigen binding protein to the antigen. Framework part of the protein. Examples of antigen-binding proteins include human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; single-domain antibodies (or nanobodies); diabodies; an F(ab') ₂ fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgGl antibody; an IgG2 antibody; an IgG3 antibody; or an IgG4 antibody and fragments thereof. Antigen binding proteins may comprise, for example, alternative protein frameworks or artificial frameworks with grafted CDRs or CDR derivatives. Such frameworks include, but are not limited to: antibody-derived frameworks comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antigen binding protein; and fully synthetic frameworks comprising, eg, biocompatible polymers. See, eg, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). In addition, peptibody antibody mimics ("PAMs") may be used, as may frameworks based on antibody mimics that utilize fibronectin components as frameworks.

In this application, the term "antibody" is used in the broadest sense and specifically encompasses, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies comprising two light chains and two heavy chains), polyclonal antibodies , multispecific antibodies (eg, bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, heavy chain antibodies, and camelized single domain antibodies (eg, heavy chain variable domain antibodies). Antibodies generally have the structure of an immunoglobulin, a protein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely, IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are respectively μ chains, delta chains, and gamma chains , α chain, ε chain. The same class of Ig can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa chains or lambda chains by difference in the constant region. Each of the five Ig classes can have either a kappa chain or a lambda chain.

In certain naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region comprises three domains, CH1, CH2 and CH3. In certain naturally occurring antibodies, each light chain comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs). Each VH and VL comprises three CDRs and four framework regions (FRs), arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable domains of native heavy and light chains each comprise four FR regions (HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4), mostly in a β-sheet configuration, connected by three CDRs, Loop links are formed and in some cases form part of a β-sheet structure. The CDRs in each chain are in close proximity by the FR regions and, together with the CDRs from the other chain, form the antigen-binding site of the antibody. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

In this application, the term "variable" generally refers to the fact that certain parts of the sequence of the variable domains of antibodies vary strongly, which contributes to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments in the light and heavy chain variable regions, called complementarity determining regions (CDRs) or hypervariable regions (HVR). The more highly conserved portions of variable domains are called the frameworks (FRs). The variable domains of native heavy and light chains each comprise four FR regions, mostly in a β-sheet configuration, connected by three CDRs, forming loops, and in some cases forming part of the β-sheet structure. The CDRs in each chain are in close proximity through the FR region and together with the CDRs from the other chain form the antigen-binding site of the antibody, the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit distinct effector functions , eg involved in antibody-dependent cytotoxicity of antibodies. In the art, the CDRs of antibodies can be defined by various methods, such as the Kabat definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, 5th edition, National Institutes of Health, Besse Star, Md. (1991). In this application, amino acid residues in variable domain sequences and in full-length antibody sequences were determined using rules that included Kabat definitions (see Table 1).

Table 1 The CDR definition of the antibody of this application based on the Kabat definition rule

Kabat Residues LCDR1 L24-L34 LCDR2 L50-L56 LCDR3 L89-L97 HCDR1 H31-H35 HCDR2 H50-H66 HCDR3 H99-H107

Among them, Laa-Lbb can refer to the amino acid sequence from the N-terminal of the antibody light chain, from the aa to the bb position; Haa-Hbb can refer to the amino acid sequence from the aa to the bb position from the N-terminal of the antibody heavy chain sequence. For example, L24-L34 can refer to the amino acid sequence from position 24 to position 34 starting from the N-terminal of the antibody light chain; H231-H35 can refer to the amino acid sequence from position 31 to position 35 starting from the N-terminus of the antibody heavy chain.

In this application, the term "Fab" refers to an antigen-binding fragment of an antibody. Intact antibodies can be digested using papain as described above. Papain digestion of antibodies yields two identical antigen-binding fragments, the "Fab" fragment, and a residual "Fc" fragment (ie, the Fc region, supra). The Fab fragment consists of a complete L chain with the variable region of a heavy chain and the first constant region ( _CH 1 ) of the H chain ( _VH ).

In this application, the term "Fab' fragment" refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which fragment is slightly larger than a Fab fragment. For example, a Fab' fragment includes all of the light chain, all of the variable domains of the heavy chain and all or part of the first and second constant domains of the heavy chain. For example, a Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.

In this application, the term "F(ab')2" refers to antibody fragments produced by pepsin digestion of intact antibodies. The F(ab')2 fragment contains two Fab fragments and part of the hinge region held together by disulfide bonds. F(ab')2 fragments have bivalent antigen binding activity and are capable of cross-linking antigen.

In this application, the term "Fv fragment" refers to a monovalent antigen-binding fragment of a human monoclonal antibody comprising all or part of the heavy and light chain variable regions and lacking the heavy and light chain constant regions. The heavy and light chain variable regions include, for example, CDRs. For example, an Fv fragment includes all or part of the approximately 110 amino acid amino-terminal variable regions of the heavy and light chains.

In this application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chains can be The variable regions are contiguous (eg via a synthetic linker such as a short flexible polypeptide linker) and can be expressed as a single chain polypeptide wherein the scFv retains the specificity of the intact antibody from which it was derived. Unless otherwise specified, as used in this application, a scFv can have the VL and VH variable regions described in any order (eg, relative to the N-terminal and C-terminal of the polypeptide), and the scFv can include a VL-linker-VH Or VH-linker-VL can be included.

In the present application, the term "dAb" generally refers to an antigen-binding fragment having a VH domain, a VL domain, or having a VH domain or a VL domain, see e.g. Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6) , with reference to Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and to other published patent applications such as WO 06/030220, WO 06/003388 and Domantis Ltd.

In this application, the term "monoclonal antibody" generally refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies in the population are identical except for minor natural mutations that may be present. Monoclonal antibodies are usually highly specific against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma cultures without contamination from other immunoglobulins. The modifier "monoclonal" denote the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies used herein can be produced in hybridoma cells, or can be produced by recombinant DNA methods.

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 of a laboratory animal, such as a rodent ("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 parental (e.g., 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 other than the CDR region of a non-human antibody (such as a mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulins. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids may also be permissible so long as they still retain the ability of the antibody to bind a particular antigen. A humanized antibody optionally will comprise at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains antigen specificity similar to the original antibody. "Humanized" forms of non-human (eg, murine) antibodies may contain, at a minimum, chimeric antibodies of sequence derived from non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) can be replaced with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or capabilities. , rabbit or non-human primate) residue substitution in the CDR region. In certain instances, FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, humanized antibodies can comprise amino acid modifications that are absent in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody properties, such as binding affinity.

In this application, the term "reference antibody" generally refers to any antibody that can bind to an antigen (eg, PD-1). In certain instances, an antigen binding protein described herein can compete with a reference antibody for binding to an antigen (eg, PD-1).

In this application, the term "isolated nucleic acid molecule" or "isolated polynucleotide" generally refers to DNA or RNA of genomic, mRNA, cDNA or synthetic origin, or some combination thereof, which is not identical to the polynucleoside molecule found in nature. All or a portion of the acid is associated with, or linked to, a polynucleotide to which it is not attached in nature.

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

In the present application, the term "cell" generally refers to individual cells, cell lines or cell culture. The cells may include progeny of a single host cell. Due to natural, accidental or deliberate mutations, the progeny cells may not necessarily be completely identical in shape or genome to the original parent cells, but it is sufficient to 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 with the vectors described in this application. The cells can be prokaryotic cells (such as Escherichia coli) or eukaryotic cells (such as yeast cells, such as 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 can be 293T cells. In this application, the term "recombinant cell" generally refers to a cell into which a recombinant expression vector has been introduced. The recombinant host cells include not only certain specific cells, but also the progeny of these cells.

In this application, the term "pharmaceutically acceptable adjuvant" generally includes a pharmaceutically acceptable carrier, excipient or stabilizer which is incompatible with the cells or mammals to which it is exposed at the dosage and concentration employed. poisonous. Typically, the physiologically acceptable carrier is a pH buffered aqueous solution. Examples of physiologically acceptable carriers may include buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions such as sodium; and/or nonionic surfactants.

In this application, the protein, polypeptide and/or amino acid sequence involved should also be understood to include at least the following scope: variants or homologues having the same or similar functions as the protein or polypeptide.

In the present application, the variant may be, in the amino acid sequence of the protein and/or the polypeptide (for example, an antibody or fragment thereof that specifically binds to the PD-1 protein) with substitution, deletion or addition of one or A protein or polypeptide of multiple amino acids. For example, the functional variant may comprise at least 1, such as 1-30, 1-20 or 1-10, further such as 1, 2, 3, 4 or 5 amino acid substitutions , proteins or polypeptides with amino acid changes by deletion and/or insertion. Said functional variant may substantially retain the biological properties of said protein or said polypeptide prior to alteration (eg, substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the protein or polypeptide prior to the alteration. For example, the substitutions may be conservative substitutions.

In the present application, the homologue may be at least about 85% (for example, 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, the homology generally refers to the similarity, similarity or association between two or more sequences. "Percentage of sequence homology" can be calculated in the following manner: compare the two sequences to be aligned in the comparison window, and determine that there are identical nucleic acid bases (for example, A, T, C, G, I) in the two sequences ) or the same amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) Number of positions To obtain the number of matching positions, the number of matching positions was divided by the total number of positions in the comparison window (ie, window size), and the result was multiplied by 100 to yield the percent sequence identity. Alignment for purposes of determining percent sequence homology can be accomplished in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared or over a region of sequence of interest. The homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in "An Improved Tool for Biological Sequence Comparison" by W.R.Pearson and D.J. Lipman, Proc. Natl. Acad. Sci., 85:2444-2448, 1988; and D.J. Lipman and W.R. Pearson "Fast and sensitive protein similarity search", Science, 227:1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers, and D. Lipman, "A Basic Local Alignment Search Tool", Journal of Molecular Biology, 215: 403-410 , 1990.

In this application, the term "optional" or "optionally" means that the subsequently described event or circumstance can but does not have to occur.

In this application, the term "comprises" generally refers to the meanings comprising, encompassing, comprising or encompassing. In some cases, it also means "for" and "consisting of".

In this application, the terms "about" and "approximately" shall generally mean an acceptable degree of error for a measured quantity given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10 percent, and more typically within 5 percent of a stated value or range of values.

In the present application, the term "therapeutically effective amount" refers to the amount of antibody which, when administered to a human or animal, elicits a response sufficient to produce a therapeutic effect in said human or animal. The effective amount can be easily determined by those of ordinary skill in the art according to routine methods.

Detailed description of the invention

antigen binding protein

In one aspect, the present application provides an antigen-binding protein, and the antigen-binding protein may comprise at least one CDR in the variable region VH of an antibody heavy chain, and the VH comprises the amino acid sequence shown in SEQ ID NO:7 or 9.

Antigen-binding proteins described herein include antibodies or antigen-binding fragments thereof. The antibodies described herein may be monoclonal antibodies, chimeric antibodies, humanized antibodies and/or fully human antibodies. Antigen-binding fragments of the antibodies described herein may be Fab, Fab', Fv fragments, F(ab') ₂ , scFv, di-scFv and/or dAb.

The antigen binding proteins described herein can compete with the reference antibody for binding to PD-1. The reference antibody may comprise a light chain variable region and a heavy chain variable region. For example, the light chain variable region of the reference antibody comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5 (YAS). Amino acid sequence; the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6; and the heavy chain variable region of the reference antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid shown in SEQ ID NO:1 Sequence; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.

The antigen binding proteins described herein may comprise heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3.

In the present application, the HCDR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:3.

In the present application, the HCDR2 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:2.

In the present application, the HCDR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:1.

In the present application, HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in sequence.

The antigen binding proteins described herein may also comprise heavy chain framework regions HFR1, HFR2, HFR3 and HFR4.

In the present application, the HFR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 19, and the C-terminus of the HFR1 is directly or indirectly connected to the N-terminus of the HCDR1.

In the present application, the HFR2 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 20, and the HFR2 is located between the HCDR1 and the HCDR2.

In the present application, the HFR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 21, and the HFR3 is located between the HCDR2 and the HCDR3.

In the present application, the HFR4 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 22, and the N-terminal of the HFR4 is connected to the C-terminal of the HCDR3.

In the present application, the antigen-binding protein may comprise an antibody heavy chain variable region (VH), and the VH may comprise the amino acid sequence shown in SEQ ID NO:7 or 9.

In some embodiments, HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein may respectively comprise the amino acids shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14 in sequence sequence.

For example, the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the HFR1, HFR2 of the antigen-binding protein , HFR3 and HFR4 may respectively comprise the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

In some embodiments, HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein may respectively comprise the amino acids shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22 in sequence sequence.

For example, the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the HFR1, HFR2 of the antigen-binding protein , HFR3 and HFR4 may respectively comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22.

The antigen binding proteins described herein may comprise a heavy chain constant region.

In the present application, the heavy chain constant region may comprise a human IgG constant region. In certain instances, the heavy chain constant region may comprise a human IgG4 constant region and/or a human IgGl heavy chain constant region.

In some embodiments, the heavy chain constant region may comprise the amino acid sequence set forth in any one of SEQ ID NO:27 or SEQ ID NO:28.

For example, HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the heavy chain of the antigen-binding protein is constant The region may comprise the amino acid sequence set forth in either of SEQ ID NO:27 or SEQ ID NO:28.

For example, the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the HFR1, HFR2 of the antigen-binding protein , HFR3 and HFR4 can respectively comprise the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, and the heavy chain constant region of the antigen-binding protein can comprise The amino acid sequence shown in any one of SEQ ID NO:27 or SEQ ID NO:28.

For example, the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the HFR1, HFR2 of the antigen-binding protein , HFR3 and HFR4 can respectively comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, and the heavy chain constant region of the antigen-binding protein can comprise The amino acid sequence shown in any one of SEQ ID NO:27 or SEQ ID NO:28.

The antigen binding protein described in the present application may comprise at least one CDR in the variable region VL of the antibody light chain, and the VL comprises the amino acid sequence shown in SEQ ID NO:8 or 10.

In the present application, HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in sequence.

In some embodiments, HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the antigen-binding protein At least one CDR in the VL of an antibody light chain variable region comprising the amino acid sequence shown in SEQ ID NO:8 may be included.

In some embodiments, HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the antigen-binding protein At least one CDR in the VL of an antibody light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 16 may be included.

The antigen binding proteins described herein may comprise light chain complementarity determining regions LCDR1, LCDR2 and LCDR3.

In the present application, the LCDR1 of the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:4.

In the present application, the LCDR2 of the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO: 5 (YAS).

In the present application, the LCDR3 of the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:6.

In the present application, the LCDR1, LCDR2 and LCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 (YAS) and SEQ ID NO: 6 in sequence.

In some embodiments, the antigen binding protein can comprise HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3, wherein, the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 can respectively comprise SEQ ID NO: 1, SEQ ID NO: 1, and LCDR3 respectively. Amino acid sequences shown in ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5(YAS) and SEQ ID NO:6.

In some embodiments, the LCDR1, LCDR2 and LCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 (YAS) and SEQ ID NO: 6, and the The VH of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:7.

In some embodiments, the LCDR1, LCDR2 and LCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 (YAS) and SEQ ID NO: 6, and the The VH of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:9.

In some embodiments, the LCDR1, LCDR2 and LCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 (YAS) and SEQ ID NO: 6, and the The VH of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 7, and the antigen binding protein may comprise a heavy chain constant region, and the heavy chain constant region may comprise SEQ ID NO: 27 or SEQ ID NO: The amino acid sequence shown in any one of 28.

In some embodiments, the LCDR1, LCDR2 and LCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 (YAS) and SEQ ID NO: 6, and the The VH of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 9, and the antigen binding protein may comprise a heavy chain constant region, and the heavy chain constant region may comprise SEQ ID NO: 27 or SEQ ID NO: The amino acid sequence shown in any one of 28.

The antigen binding proteins described herein may comprise the light chain framework regions LFR1, LFR2, LFR3 and LFR4.

In the present application, the LFR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 23, and the C-terminus of the LFR1 is directly or indirectly connected to the N-terminus of the LCDR1.

In the present application, the LFR2 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 24, and the LFR2 is located between the LCDR1 and the LCDR2.

In the present application, the LFR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 25, and the LFR3 is located between the LCDR2 and the LCDR3.

In the present application, the LFR4 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 18 or SEQ ID NO: 26, and the N-terminal of the LFR4 is connected to the C-terminal of the LCDR3.

In the present application, the antigen-binding protein may comprise VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:8 or 10.

In some embodiments, LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 in sequence.

For example, the LCDR1, LCDR2 and LCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 (YAS) and SEQ ID NO: 6, and the antigen-binding protein LFR1, LFR2, LFR3 and LFR4 may comprise the amino acid sequences shown in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18 in sequence.

For example, the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 (YAS) and SEQ ID NO:6, and the LFR1 of the antigen binding protein, LFR2, LFR3 and LFR4 can comprise SEQ ID NO: Amino acid sequences shown in ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

In some embodiments, LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26 in sequence.

For example, the LCDR1, LCDR2 and LCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 (YAS) and SEQ ID NO: 6, and the antigen-binding protein LFR1, LFR2, LFR3 and LFR4 may comprise the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26 in sequence.

For example, the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 (YAS) and SEQ ID NO:6, and the LFR1 of the antigen binding protein, LFR2, LFR3 and LFR4 can comprise SEQ ID NO: Amino acid sequences shown in ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26.

In the present application, the antigen binding protein may comprise VH and VL, the VH may comprise the amino acid sequence shown in SEQ ID NO: 7 or 15, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 8 or 10. amino acid sequence.

In some embodiments, the VH may comprise the amino acid sequence set forth in SEQ ID NO:7, and the VL may comprise the amino acid sequence set forth in SEQ ID NO:8.

For example, the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 (YAS) and SEQ ID NO:6, and the HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein can respectively comprise sequentially The amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, and LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein may comprise SEQ ID NO: 15. The amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

In some embodiments, the VH can comprise the amino acid sequence set forth in SEQ ID NO:9, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:10.

For example, the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 (YAS) and SEQ ID NO:6, and the HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein can respectively comprise sequentially Amino acid sequences shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22. LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26 in sequence.

In the present application, the antigen-binding protein may comprise a heavy chain constant region, and the heavy chain constant region may comprise the amino acid sequence shown in any one of SEQ ID NO:27 or SEQ ID NO:28.

In some embodiments, the VH may comprise the amino acid sequence shown in SEQ ID NO: 7, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 8, and the heavy chain constant region of the antigen binding protein The amino acid sequence shown in any one of SEQ ID NO:27 or SEQ ID NO:28 may be included.

For example, the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 (YAS) and SEQ ID NO:6, and the HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein can respectively comprise sequentially The amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, and LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein may comprise SEQ ID NO: 15. The amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and the VH may include the amino acid sequence shown in SEQ ID NO: 7, and the VL may include SEQ ID NO 8, and the heavy chain constant region of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 27 or SEQ ID NO: 28.

In some embodiments, the VH can comprise the amino acid sequence set forth in SEQ ID NO:9, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:10.

For example, the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 (YAS) and SEQ ID NO:6, and the HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein can respectively comprise sequentially The amino acid sequences shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, and LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein may comprise SEQ ID NO: 23. The amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, and the VH may include the amino acid sequence shown in SEQ ID NO: 15, and the VL may include SEQ ID NO 16, and the heavy chain constant region of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 27 or SEQ ID NO: 28.

In another aspect, the application provides an isolated antigen binding protein, the antigen binding protein comprising a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the HCDR1 Comprising the amino acid sequence shown in SEQ ID NO: 1; the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 2; the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 3; the light chain variable region contains LCDR1 , LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5 (YAS); the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6 sequence.

For example, the heavy chain variable region of the antigen binding protein of the present application comprises at least one FR in the antibody heavy chain variable region VH, said VH comprising the amino acid sequence shown in any one of SEQ ID NO:54 to 56.

For example, the antigen binding protein of the present application comprises at least one FR in the antibody heavy chain variable region VH shown in SEQ ID NO:67. For example, the antigen binding protein of the present application comprises at least one FR in the antibody heavy chain variable region VH shown in any one of SEQ ID NO:54 to 56. For example, the antigen-binding protein of the present application comprises 1, 2, 3 or 4 FRs in the above-mentioned VH. For example, the antigen-binding protein of the present application comprises 4 H-FRs in the above-mentioned VH. For example, the antigen-binding protein of the present application comprises H-FR1 to 4 of the above-mentioned same or different VH.

Further, the light chain variable region of the antigen-binding protein of the present application comprises at least one FR in the antibody light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:68. For example, the light chain variable region of the antigen binding protein of the present application comprises at least one FR in the VL of the antibody light chain variable region, said VL comprising the amino acid sequence shown in any one of SEQ ID NO:57 to 59.

For example, the antigen binding protein of the present application comprises at least one FR in the antibody light chain variable region VL shown in SEQ ID NO:68. For example, the antigen binding protein of the present application comprises at least one FR in the antibody light chain variable region VL shown in any one of SEQ ID NO:57 to 59. For example, the antigen-binding protein of the present application comprises 1, 2, 3 or 4 FRs of the above-mentioned VL. For example, the antigen-binding protein of the present application comprises the four L-FRs of the above-mentioned VL. For example, the antigen-binding protein of the present application comprises L-FR1 to 4 of the above-mentioned same or different VL.

For example, the antigen binding protein of the present application comprises at least one FR in the antibody heavy chain variable region VH shown in SEQ ID NO:67, and includes at least one FR in the antibody light chain variable region VL shown in SEQ ID NO:68 a fr. For example, the antigen binding protein of the present application comprises at least one FR in the antibody heavy chain variable region VH shown in any one of SEQ ID NO:54 to 56, and comprises any one of SEQ ID NO:57 to 59. At least one FR in the variable region VL of the antibody light chain shown.

For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:45.

For example, the H-FR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO:63.

For example, the H-FR3 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO:42 to 44.

For example, the H-FR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO:41.

For example, H-FR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO:62.

For example, H-FR1 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO:39 to 40.

For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:45, The H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:63, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:41, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:62 The amino acid sequence shown.

For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:45, The H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:42 to 44, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:41, and the H-FR1 may comprise The amino acid sequence shown in any one of SEQ ID NO:39 to 40.

For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:45, The H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:42, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:41, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:39 The amino acid sequence shown.

For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:45, The H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:43, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:41, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:40 The amino acid sequence shown.

For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:45, The H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:44, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:41, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:40 The amino acid sequence shown.

For example, the light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, and said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:53.

For example, L-FR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO:66.

For example, L-FR3 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO:51 to 52.

For example, L-FR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO:65.

For example, L-FR2 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO:49 to 50.

For example, L-FR1 of the present application may comprise the amino acid sequence shown in SEQ ID NO:64.

For example, L-FR1 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO:46 to 48.

For example, the light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, and said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:53, The L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:66, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:65, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:64 The amino acid sequence shown.

For example, the light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, and said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:53, The L-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:51 to 52, the L-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NO:49 to 50, and the The L-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:46 to 48.

For example, the light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, and said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:53, The L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:51, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:49, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:46 The amino acid sequence shown.

The light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, and said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:53, said L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:52, said L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:50, and said L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:47 amino acid sequence.

The light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, and said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:53, said L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:52, said L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:40, and said L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:48 amino acid sequence.

For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:45, The H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:63, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:41, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:62 and the light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, and said L-FR4 may comprise SEQ ID NO:53 The amino acid sequence of the L-FR3 can comprise the amino acid sequence shown in SEQ ID NO:66, the L-FR2 can comprise the amino acid sequence shown in SEQ ID NO:65, and the L-FR1 can comprise the amino acid sequence shown in SEQ ID NO:65 The amino acid sequence shown in NO:64.

For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:45, The H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:42 to 44, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:41, and the H-FR1 may comprise The amino acid sequence shown in any one of SEQ ID NO:39 to 40; And the light chain variable region of the antigen binding protein of the present application can comprise L-FR1, L-FR2, L-FR3 and L-FR4, described L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:53, said L-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:51 to 52, said L-FR2 may comprise SEQ ID NO: 49-50, and the L-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:46-48.

For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:45, The H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:42, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:41, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:39 and the light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, and said L-FR4 may comprise SEQ ID NO:53 The amino acid sequence of the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:51, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:49, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:49 The amino acid sequence shown in NO:46.

For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:45, The H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:43, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:41, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:40 and the light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, and said L-FR4 may comprise SEQ ID NO:53 Amino acid sequence, the L-FR3 of the present application can comprise the amino acid sequence shown in SEQ ID NO:52, described L-FR2 can comprise the amino acid sequence shown in SEQ ID NO:50, and described L-FR1 can comprise SEQ ID NO: Amino acid sequence shown in ID NO:47.

For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:45, The H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:44, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:41, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:40 and the light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, and said L-FR4 may comprise SEQ ID NO:53 The amino acid sequence of the L-FR3 can comprise the amino acid sequence shown in SEQ ID NO:52, the L-FR2 can comprise the amino acid sequence shown in SEQ ID NO:40, and the L-FR1 can comprise the amino acid sequence shown in SEQ ID NO:40 The amino acid sequence shown in NO:48.

For example, the heavy chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO:67. For example, the heavy chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in any one of SEQ ID NO:54 to 56.

For example, the light chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO:68. For example, the light chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in any one of SEQ ID NO:57-59.

For example, the heavy chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO:67, and the light chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO:68 . For example, the heavy chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in any one of SEQ ID NO:54 to 56, and the light chain variable region of the antigen binding protein of the present application comprises SEQ ID NO: The amino acid sequence shown in any one of 57 to 59.

For example, the heavy chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO:54, and the light chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO:57 .

For example, the heavy chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO:55, and the light chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO:58 .

For example, the heavy chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO:56, and the light chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO:59 .

Furthermore, the antigen-binding protein of the present application includes an antibody heavy chain constant region. For example, the antigen binding proteins of the present application include antibody heavy chain constant regions derived from human IgG constant regions. For example, the antibody heavy chain constant region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO:60.

Furthermore, the antigen-binding protein of the present application includes an antibody light chain constant region. For example, the antibody light chain constant region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO:61.

For example, the antigen binding proteins of the present application include antibodies or antigen binding fragments thereof. For example, antibodies of the present application include Fab, Fab', Fv fragments, F(ab')2, scFv, di-scFv and/or dAb. For example, the antigen-binding fragment of the present application is selected from the group consisting of humanized antibodies and fully human antibodies.

The physical/chemical properties and/or biological activities of the PD-1 antigen binding proteins described herein can be identified, screened or characterized by various assays known in the art.

In one aspect, the present application can be tested, for example, by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blot), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, etc. Antigen binding activity of the antigen binding protein or fusion protein. Antigen binding proteins (eg, PD-1 antibodies) described herein are capable of specifically binding PD-1 antigens. An antigen binding protein (eg, a PD-1 antibody) that "specifically binds" a PD-1 antigen can generally bind PD-1, but not other proteins that lack PD-1 sequences. The antigen binding proteins described herein (e.g., PD-1 antibodies) are capable of specifically binding PD-1 antigen or its labeled form (e.g., fluorescently labeled PD-1 antigen), but not PD-1 epitopes lacking of other proteins. Whether an antigen binding protein (eg, antibody) binds a PD-1 antigen can be determined using any assay known in the art. Examples of assays known in the art to determine binding affinity include biofilm interferometry (BLI).

The antigen binding proteins described herein can bind to human PD-1 protein. In certain instances, the antigen binding proteins described herein may also cross-react with monkey (eg, cynomolgus) PD-1. For example, detected by flow cytometry and ELISA. In this application, "cross-reactivity" refers to the ability of an antibody to react with homologous proteins from other species.

In some cases, the PD-1 binding activity of the antigen binding proteins described herein can be detected using an enzyme-linked immunoassay assay. For example, in ELISA, using human PD-1 antigen protein, the EC50 value of the PD-1 antigen binding protein and PD-1 can be between about 0.0001 nM to about 100 nM, for example, can be between about 0.001 nM to about 10 nM Between, can be between about 0.001 nM to about 5 nM, can be between about 0.001 nM to about 1 nM or can be between about 0.01 nM to about 1 nM.

In another aspect, the antigen binding proteins described herein can block the binding of PD-1 to PD-L1 and or PD-L2. In certain instances, the antigen binding protein blocks the binding of PD-1 to PD-L1 and or PD-L2 in an ELISA assay. For example, first coat the PD-L1 and or PD-L2 antigen protein on the plate, mix decreasing amounts of the unlabeled antigen-binding protein and the biotin-labeled PD-1 protein, and incubate together. Then, the cells are analyzed using ELISA to confirm that the antigen binding protein can block the binding of PD-1 and PD-L1 and or PD-L2.

The antigen-binding protein described in the present application can stimulate the proportion of CD07a-expressing cells among immune cells. The antigen binding proteins described herein can stimulate the secretion of IFN-γ, TNF-α and/or IL2 in immune cells. For example, the stimulating ability of the antigen-binding protein described in the present application can be based on the activation of known activators in the art (such as CD3 antibody, CD28 antibody, or nanomaterial TransAct ^™ comprising CD3 antibody and CD28 antibody), increasing the The effect of immune cell activation. The immune cells may include lymphocytes such as B cells, T cells, natural killer cells, myeloid cells such as monocytes, macrophages, mast cells, basophils and granulocytes. For example, the immune cells may comprise tumor infiltrating lymphocytes (TILs). For example, the immune cells may comprise artificially modified immune cells. For example, the immune cells may comprise TCR-T cells. The secretion of cytokines in immune cells can be measured by any method known to those skilled in the art, for example, by enzyme-linked immunoassay (ELISA) to quantitatively measure the proliferation of immune cells (such as T cells) or the cytokines produced by immune cells (such as IFN-γ or IL-2 produced by T cells). For example, the stimulation result of PD-1 antibody on T lymphocytes can be detected by mixed lymphocyte reaction (MLR) assay.

Nucleic acid molecules, vectors and cells

In another aspect, the application provides one or more nucleic acid molecules that encode the isolated antigen binding proteins described herein. The nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified (iv) synthetic, for example by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA techniques.

In another aspect, the present application provides a vector, which may comprise the nucleic acid molecule described in the present application. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector may also contain expression control elements that permit proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like. Such vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector.

In another aspect, the present application provides a cell, which may comprise the nucleic acid molecule or the vector described in the present application. In certain embodiments, each or each host cell may comprise one or more of the nucleic acid molecules or vectors described herein. In certain embodiments, each or each host cell may comprise a plurality (eg, 2 or more) or a plurality (eg, 2 or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein can be introduced into the host cells, such as eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like. The vectors described in this application can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection and the like.

pharmaceutical composition

On the other hand, the application provides a pharmaceutical composition, which may comprise the antigen binding protein described in the application and/or the nucleic acid molecule, the carrier, the host cell, and optionally a pharmaceutical acceptable adjuvants. Such pharmaceutically acceptable adjuvants are nontoxic to recipients at the doses and concentrations employed, and may include buffers, antioxidants, preservatives, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic Aqueous polymers, amino acids, carbohydrates, salt-forming counterions, metal complexes, and/or nonionic surfactants. The pharmaceutical compositions herein may also contain more than one active compound, generally those active compounds with complementary activities that do not adversely affect each other. The type and effective amount of such drug depends, for example, on the amount and type of antagonist present in the formulation, as well as the clinical parameters of the subject.

The pharmaceutical compositions described herein may comprise a prophylactically and/or therapeutically effective amount of the antigen binding protein. The prophylactically and/or therapeutically effective amount is the dose required to be able to prevent and/or treat (at least partially treat) the disease or condition and/or any complications thereof in a subject suffering from or at risk of developing.

Preparation

In another aspect, the present application provides a method for preparing the antigen-binding protein. The method may comprise culturing the host cell described herein under conditions such that the antigen binding protein is expressed. For example, by using appropriate medium, appropriate temperature and incubation time, etc., these methods are understood by those of ordinary skill in the art.

Any method suitable for producing monoclonal antibodies can be used to produce the antigen binding proteins of the present application. For example, animals can be immunized with linked or naturally occurring PD-1 protein or fragments thereof. Suitable immunization methods may be used, including adjuvants, immunostimulants, repeated booster immunizations, one or more routes may be used.

Any suitable form of PD-1 can be used as an immunogen (antigen) to generate non-human antibodies specific to PD-1, and to screen the biological activity of the antibodies. The stimulating immunogen can be full-length mature human PD-1, including natural homodimers, or peptides containing single/multiple epitopes. Immunogens can be used alone or in combination with one or more immunogenicity enhancers known in the art.

Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA and IgE. In this application, the antibody is an IgG antibody, and the IgG1 subtype is used. Optimization of the necessary constant domain sequences to produce the desired biological activity can be achieved by screening antibodies using the biological assays described in the Examples below. Likewise, either type of light chain can be used in the compounds and methods of the present application. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the present application.

The sequence of the DNA molecule of the antigen-binding protein of the present application or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences for the light and heavy chains can be fused together to form single-chain antibodies.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods. In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them. The nucleic acid molecule can then be introduced into various existing DNA molecules (or, eg, vectors) and cells known in the art.

The present application also relates to vectors comprising appropriate nucleic acid molecules as described above together with appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein. The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. For example, animal cells can include, but are not limited to: 293T cells.

The step of transforming host cells with recombinant DNA described in this application can be performed by techniques well known in the art. The obtained transformant can be cultured by conventional methods, and the transformant expresses the polypeptide encoded by the nucleic acid molecule of the present application. Depending on the host cell used, it is cultured under appropriate conditions using a conventional medium. Typically, the transformed host cells are cultured under conditions suitable for expression of the antigen-binding protein of the present application. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc. The antigen-binding protein of the present application was purified by conventional separation and purification methods well known to personnel.

The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as flow cytometry (FACS) or enzyme-linked immunosorbent assay (ELISA).

method and use

In another aspect, the present application provides a use of the antigen-binding protein in the preparation of medicaments. The antigen binding protein may be administered alone or in combination with one or more additional therapies, such as chemotherapy radiation therapy, immunotherapy, surgical intervention, or any combination of these. The medicament can be used to treat PD-1-mediated diseases or conditions, for example, to treat cancer, inhibit tumor growth and/or inhibit tumor cell proliferation.

On the other hand, the antigen-binding protein provided in this application can be used to prevent or treat PD-1-mediated diseases or disorders. The preventing or treating a disease or condition may refer to inhibiting or delaying the development or progression of the disease or condition. For example, it can be used to inhibit the development or progression of tumors. For example, tumor growth or tumor cell proliferation can be inhibited.

In another aspect, the present application provides the antigen-binding protein for preventing or treating PD-1-mediated diseases or disorders.

In another aspect, the present application provides a method for inhibiting the combination of PD-1 and PD-L1 and or PD-L2, comprising administering the antigen-binding protein described in the present application. The method can be an ex vivo or in vitro method. In certain instances, the method may comprise subjecting a biological sample to an antigen binding protein and/or PD-L1 and or PD-L2 described herein in a manner that allows the antigen binding protein and/or PD-1 to bind to PD- Contacting under the conditions of L1 and or PD-L2, detecting whether a complex is formed between the antigen-binding protein and PD-1, and detecting whether a complex is formed between PD-1 and PD-L1 or PD-L2.

In another aspect, the present application provides a method for preventing, alleviating or treating a PD-1-mediated disease or disorder, which includes administering the antibody or antigen-binding fragment thereof described herein, the Molecular nucleic acid, the carrier, the host cell and/or the pharmaceutical composition. For example, it can be used to inhibit the development or progression of tumors. For example, tumor growth or tumor cell proliferation can be inhibited.

Not intending to be limited by any theory, the following examples are only for explaining the protein, preparation method and application of the present application, and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are usually followed. Percentages and parts are by weight unless otherwise indicated.

Example

Example 1: Expression vector construction and eukaryotic expression of the fusion protein (PD-1-huIgG1 Fc) of recombinant human programmed death receptor 1 (PD-1) amino acid sequence 25 to 167 and human IgG1 Fc region

1. Synthesis of the gene sequence between the 25th and 167th amino acids of PD-1 and the construction of the expression vector of the PD-1-huIgG1 Fc fusion protein

The gene sequence between the 25th isoleucine and the 167th glutamine of programmed death receptor 1 (PD-1) (NCBI accession No.NP_005009.2) was synthesized by chemical synthesis (SEQ ID NO: 29), the gene sequence encodes the amino acid sequence shown in SEQ ID NO:30. The gene sequence between the 100th proline and the 330th lysine of the human IgG1 heavy chain constant region was synthesized by chemical synthesis. The upstream primer containing mouse Igkv3-10 signal peptide gene sequence was chemically synthesized and used for expression vector construction. Through molecular cloning, the PD-1 gene fragment was spliced with the human IgG1 Fc gene fragment. The spliced product was cloned into pCDNA3.1 (Thermo) using TaKaRa seamless cloning kit.

2. Expression and purification of recombinant PD-1-huIgG1 Fc fusion protein

After transfecting 293T cells (ATCC) with this expression vector for 5 days, the culture supernatant was collected, and the recombinant PD-1-huIgG1 Fc fusion protein was purified with AKTAexplorer 100 (GE). Due to glycosylation modification and other reasons, the size of the recombinant PD-1-huIgG1 Fc fusion protein was about 60k Daltons after reducing SDS-PAGE electrophoresis and Coomassie brilliant blue staining.

Example 2: Expression vector construction and eukaryotic expression of recombinant human programmed death receptor 1 (PD-1) 25-167 amino acid sequence and polyhistidine tag fusion protein (PD-1-his)

1. Synthesis of the gene sequence between the 25th and 167th amino acids of PD-1 and the construction of the expression vector of the polyhistidine tag fusion protein

The gene sequence from the 25th isoleucine to the 167th glutamine of programmed death receptor 1 (PD-1) (NCBI accession No.NP_005009.2) was synthesized by chemical synthesis. Chemically synthesized upstream primer containing mouse Igkv3-10 signal peptide gene sequence and downstream primer containing polyhistidine tag (with the amino acid sequence shown in SEQ ID NO:32) gene sequence (SEQ ID NO:31) for The pCDNA3.1 vector expressing PD-1-his was constructed.

2. Expression and purification of recombinant PD-1-his protein

After transfecting 293T cells (ATCC) with this expression vector for 5 days, the culture supernatant was collected, and the recombinant PD-1-his protein was purified with AKTAexplorer 100 (GE). Due to glycosylation modification and other reasons, the size of the recombinant PD-1-his protein was about 40k Daltons after reducing SDS-PAGE electrophoresis and Coomassie brilliant blue staining.

Example 3: Anti-human PD-1 antibody (Nivolumab) expression vector construction and eukaryotic expression

1. Acquisition of variable region gene of Nivolumab antibody and construction of expression vector

The Nivolumab antibody light and heavy chain variable region genes synthesized by chemical synthesis are SEQ ID NO: 33 and SEQ ID NO: 34, the Nivolumab antibody light chain variable region has the amino acid sequence shown in SEQ ID NO: 35, and the Nivolumab antibody heavy chain variable region The chain variable region has the amino acid sequence shown in SEQ ID NO:36. Using the heavy chain variable region gene as a template, the heavy chain variable region fragment was amplified by PCR, and the amplified product was cloned into pFUSEss containing the human IL-2 signal peptide gene and the human IgG1 heavy chain constant region gene using the TaKaRa seamless cloning kit - CHIg-hG1 (invivogen). Using the light chain variable region gene as a template, the light chain variable region fragment was amplified by PCR, and the amplified product was cloned into a human IL-2 signal peptide gene and human kappa light chain constant region gene using the TaKaRa seamless cloning kit. In pFUSE2ss-CLIg-hK (invivogen).

2. Expression and purification of Nivolumab antibody

After 5 days of co-transfection of 293T cells (ATCC) with this double plasmid at a ratio of 1:1, the culture supernatant was collected, and Nivolumab antibody was purified with AKTAexplorer100 (GE). The size of the Nivolumab antibody is about 150k Daltons by Coomassie Brilliant Blue staining after non-reducing SDS-PAGE electrophoresis.

Example 4: ELISA detection of recombinant human PD-1 (PD-1-huIgG1 Fc or PD-1-his) binding to Nivolumab antibody

Use enzyme-linked immunosorbent assay (ELISA) to detect the combination of recombinant human PD-1 (PD-1-huIgG1 Fc or PD-1-his) and Nivolumab antibody. The specific operation of the ELISA experiment is as follows: add the prepared above to the microwell plate PD-1-huIgG1Fc or PD-1-his fusion protein 100ng/well, coated overnight at 4°C. Wash three times with PBS, add 1% BSA/PBS, 200uL/well, block at 37°C for 1 hour. After washing the plate with 100ul PBS, add 100ng/well Nivolumab chimeric antibody, and bind at 37°C for 1 hour. Wash three times with PBST, add 100ul 1:5000 diluted HRP-goat anti-human IgG (Fab-specific) and combine for 1 hour at 37°C. Wash with PBST three times, add 100uL/well TMB color development solution, develop color at 37°C for 10 minutes, add 100uL/well ELISA stop solution, and read the OD450 value with a microplate reader. The OD450 value can reflect the binding of Nivolumab antibody to recombinant human PD-1.

Example 5: Expression of tetravalent human programmed death-ligand 1 (PD-L1) extracellular segment mutants and determination of affinity with human PD-1 molecules

1. Construction of the expression vector of the tetravalent human PD-L1 extracellular segment mutant

The gene sequence (SEQ ID NO:37) of two human PD-L1 extracellular segment mutants connected by a flexible peptide was synthesized by chemical synthesis, and the gene sequence encoded the amino acid sequence shown in SEQ ID NO:38. Synthesize the gene sequence from the 100th proline to the 330th lysine of the human IgG1 heavy chain constant region (UniProtKB/Swiss-Prot accession No.P01857.1) by chemical synthesis. The upstream primer containing mouse Igkv3-10 signal peptide gene sequence was chemically synthesized and used for expression vector construction. Through molecular cloning, the gene fragment of the repeat structure of the PD-L1 extracellular segment mutant was spliced with the human IgG1 Fc gene fragment. The spliced product was cloned into pCDNA3.1 (Thermo) using TaKaRa seamless cloning kit.

2. Expression and purification of tetravalent human PD-L1 extracellular segment mutants

After transfecting 293T cells (ATCC) with this expression vector for 5 days, the culture supernatant was collected, and the tetravalent human PD-L1 extracellular segment mutant protein was purified with AKTAexplorer 100 (GE). Due to glycosylation modification and other reasons, the tetravalent human PD-L1 extracellular segment mutant protein was electrophoresed by reducing SDS-PAGE and stained with Coomassie brilliant blue to show that its size was about 85k Daltons.

3. Biotinylation of PD-1-huIgG1 Fc fusion protein

The PD-1-huIgG1 Fc fusion protein was randomly biotinylated using the standard operating procedure provided by EZ-Link Sulfo-NHS-LC-Biotin (Thermo). The binding activity of biotinylated PD-1-huIgG1 Fc fusion protein to Nivolumab antibody was verified by ELISA method.

4. Determination of the avidity of tetravalent human PD-L1 extracellular segment mutants and PD-1-huIgG1 Fc

The affinity between tetravalent human PD-L1 extracellular segment mutants and PD-1-huIgG1Fc was determined by Octet K2 (ForteBio) molecular interaction analyzer. According to the standard operating procedure of Octet K2, 100 nM of biotinylated PD-1-huIgG1Fc fusion protein was prepared and immobilized on the SA probe (ForteBio), and the immobilization height was 1 nM. The quadrivalent human PD-L1 extracellular segment mutant was used as an analyte in two-fold serial dilution starting from 50nM concentration, and the affinity between the tetravalent human PD-L1 extracellular segment mutant and PD-1-huIgG1 Fc was measured. The resultant force is 9.4nM, and the results of affinity determination are shown in Fig. 1 . It is known that wild-type PD-1 and PD-L1 have a binding affinity of 8.2uM (see Cheng, X. et al., J. Biol. Chem., 288(17): 11771-85, 2013).

Example 6: Preparation of anti-human PD-1 mouse antibody, mouse anti-humanization

1. Immunization of animals

Mix and emulsify the PD-1-huIgG1 Fc fusion protein prepared in Example 1 at 2 mg/mL as an antigen with an equal volume of complete Freund's adjuvant (Sigma-Aldrich), and take 10 6-week-old female Balb/c mice (Subcutaneous immunization, each with 100ug of antigen. After the initial immunization, a booster immunization was performed every ten days, and a total of four subcutaneous immunizations were performed. In the fifth immunization, PD-1-huIgG1 Fc fusion protein was directly used for spleen shock immunization .

2. Serum titer detection

Before each booster immunization, 50uL of blood was collected from the tail vein, and the cells were removed by centrifugation, and the serum was retained. Add PD-1-his (ACRO Biosystems) 50ng/well to the ELISA microwell plate, and coat overnight at 4°C. Wash three times with PBS, add 1% BSA/PBS, 200uL/well, block at 37°C for 1 hour. Add serially diluted mouse serum and combine for 1 hour at 37°C. Wash three times with PBST, add 100ul 1:5000 diluted HRP-goat anti-mouse IgG (Shanghai Yisheng) and combine for 1 hour at 37°C. Wash with PBST three times, add 100uL/well TMB color development solution, develop color at 37°C for 10 minutes, add 100uL/well ELISA stop solution, and read the OD450 value with a microplate reader.

3. Construction of immune library

3.1 Total cDNA acquisition of mouse splenocytes

The mice were sacrificed four days after the PD-1-huIgG1 Fc fusion protein was directly injected intraperitoneally for pulse immunization, and the spleen was taken. Splenocytes were harvested by grinding whole spleens with a 70 micron cell mesh (BD). After washing twice with PBS, centrifuge at 1000g for 10 minutes to obtain spleen cells. Total RNA was extracted using Trizol RNA extraction kit.

Using the RNA as a template, the first-strand cDNA was synthesized using the SuperScript ^™ IV First-Strand Synthesis System kit.

3.2 Antibody gene amplification and light and heavy chain splicing

Using the cDNA as a template, refer to the antibody amplification primers described in the literature (Schaefer J.V., Honegger A., Plückthun A. (2010) Construction of scFv Fragments from Hybridoma or SpleenCells by PCR Assembly. In: Kontermann R., Dübel S. (eds) AntibodyEngineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg), PCR amplification of the heavy chain variable domain gene using the heavy chain variable domain upstream and downstream primers, and PCR using the light chain variable domain upstream and downstream primers Amplification of kappa chain variable domain genes. In the 50uL reaction system, 25uL phusion mastermix (Thermo), 2.5uL (25pmol) of upstream primer, 2.5uL (25pmol) of downstream primer, 1.5uL DMSO, 0.5uL cDNA and 18uL ddH2O were added respectively. The PCR reaction was performed according to the following program: 98°C pre-denaturation for 1 minute, followed by temperature cycling, 98°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 1 minute, 30 cycles, and 72°C final extension for 10 minutes.

The amplified VH gene and VL gene were recovered using a DNA gel recovery kit. The equal amount of VH gene and VL gene was mixed as a template, and the scFv gene was amplified by overlapping PCR using the upstream primer scFv-F and the downstream primer scFv-R. In the 50uL reaction system, add 25uL phusion master mix, 2.5uL (25pmol) of upstream primer, 2.5uL (25pmol) of downstream primer, 1.5uL DMSO, 0.5uL cDNA and 18uL ddH2O. The PCR reaction was performed according to the following program: 98°C pre-denaturation for 1 minute, followed by temperature cycling, 98°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 1 minute, 30 cycles, and 72°C final extension for 10 minutes.

The amplified scFv gene fragment was recovered using a DNA gel recovery kit.

3.3 Construction of immune library

The scFv gene fragment and the pcomb3XTT vector (Scripps Research Institute, USA) were digested with SfiI DNA endonuclease, respectively. In the 50uL reaction system, add 2uL of SfiI, 5uL of 10x buffer, 3ug of DNA, and add ddH2O to 50uL. After mixing well, incubate at 50°C for 3 hours.

Use a DNA gel recovery kit to recover the digested scFv gene fragment and pcomb3XTT vector. Use T4 ligase to cyclize the digested scFv gene fragment and digested pcomb3XTT vector. In the 50uL reaction system, add T4 ligase 1uL, 10x buffer 5uL, scFv gene 100ng, pComb3XTT vector 500ng, add ddH2O to 50uL. After mixing well, incubate at 4°C for 16 hours. A small amount of product was taken to verify the ligation efficiency by agarose gel electrophoresis.

Add 10uL of the above ligated cyclization product into the self-made TG1 electrotransformation competent body, and then perform electroporation transformation by electroporator. Take out 10ul of the electrotransformed bacteria and pass a reasonable dilution and streak on the plate containing ampicillin, so as to count and count the size of the phage antibody library. The remaining electrotransformed bacteria were added to 2xYT medium containing 100 ug/mL ampicillin and 2% glucose, and placed in a heating incubator for culture. After the cultivation, centrifuge at 4000G for 10 minutes at 4°C, add an appropriate amount of glycerol to the precipitated bacteria and store at -80°C as the antibody strain library. The scFv immune library with more than 3E9 storage capacity was accumulated through multiple electroporations.

4. Screening and identification of murine immune antibody phage library

4.1 Biotinylation of PD-1-huIgG1 Fc fusion protein

Random biotinylation of PD-1-huIgG1 Fc fusion protein was performed using the standard operating procedure provided by EZ-Link Sulfo-NHS-LC-Biotin. The binding activity of biotinylated PD-1-huIgG1 Fc fusion protein to Nivolumab antibody was verified by ELISA method.

4.2 Biopanning

Biopanning was applied to the PD-1-huIgG1 Fc fusion protein as the target protein, and the above mouse immune antibody library was biopanned to obtain antibodies that bind to the PD-1-huIgG1 Fc fusion protein (especially the extracellular domain of PD-1) . Take 100OD bacteria from the antibody strain library to recover at initial OD600=0.1 density and grow to the logarithmic phase, then use M13KO7 helper phage to rescue the antibody library, centrifuge, resuspend in 2xYT medium containing ampicillin and kanamycin and Amplify overnight at 30°C. Phage was precipitated with PEG/NaCl, and the precipitated phage was dissolved with glycerol/PBST to obtain the phage suspension of the immune library. The casein-blocked phages were put into the co-incubation system of casein-blocked biotinylated huIgG1 Fc (ACRO Biosystems) fusion protein and casein-blocked Dynabeads M-270 streptavidin, and the supernatant phage suspension was collected. Further, put the collected phage suspension into the co-incubation system of casein-blocked biotinylated PD-1-huIgG1 Fc fusion protein and casein-blocked Dynabeads M-270 streptavidin, and wash the magnetic beads with PBST Phage unable to bind to the PD-1-huIgG1Fc fusion protein were removed. Under appropriate elution conditions, the phages were eluted competitively with a self-made tetravalent PD-L1 extracellular segment mutant. 10ul of the eluted phage solution was used to determine the total amount of output phage, and the remaining phage solution was used to infect TG1 with logarithmic growth. After overnight amplification, it was regarded as the antibody library for the next round of panning. A total of three rounds of biopanning were performed, and the parameters of the panning experiment are shown in Table 2.

Table 2 Antibody library biopanning experimental parameters

4.3 Preliminary screening of clones that specifically bind to the extracellular domain of PD-1 and have PD-1-tetravalent PD-L1 blocking effect

The antibody library obtained after the third round of biopanning was diluted and coated on a plate containing ampicillin to obtain a single clone, and the single clone was selected and cultured overnight in a deep well plate. The next day, the deep-well plate was repeatedly frozen and thawed three times, and the centrifuged supernatant was used for the subsequent two types of ELISA reactions.

ELISA reaction for detection of binding activity: coated with 50ng PD-1-his overnight, washed three times with PBS, added 1% BSA/PBS, 200uL/well, blocked at 37°C for 1 hour. After washing three times with PBS, 100ul of centrifuged supernatant was added and incubated at 37°C for 1 hour. After washing with PBST three times, 100ul of HRP-coupled goat anti-mouse IgG (Fab-specific) (Thermo) diluted at 1:5000 was added. Wash with PBST three times, add 100uL/well TMB color development solution, develop color at 37°C for 10 minutes, add 100uL/well ELISA stop solution, and read the OD450 value with a microplate reader. This screening step was repeated twice independently to ensure the accuracy of the data, and clones with an average OD450 greater than 0.15 were selected for subsequent analysis.

ELISA reaction for detection of blocking activity: coated with PD-1-his overnight, washed three times with PBS, added 1% BSA/PBS, 200uL/well, blocked at 37°C for 1 hour. After washing with PBS three times, add 100ul centrifuged supernatant and 20ng tetravalent PD-L1 mixture and incubate at 37°C for 1 hour, after washing with PBST three times, add 100ul 1:5000 diluted HRP-coupled goat anti-mouse IgG (Fab-specific) (Thermo). Wash with PBST three times, add 100uL/well TMB color development solution, develop color at 37°C for 10 minutes, add 100uL/well ELISA stop solution, and read the OD450 value with a microplate reader. This screening step was repeated twice for independent experiments to ensure the accuracy of the data, and clones with an average OD450 number less than 1 were selected for subsequent analysis.

Further, the candidate clones meeting the above two conditions are sequenced to obtain the antibody light chain variable domain and heavy chain variable domain sequences.

4.4 Rescreening clones that specifically bind to the extracellular domain of PD-1 and have PD-1-tetravalent PD-L1 blocking effect

The candidate clones obtained from the primary screening were expanded and cultivated with a 50ml shaker tube (thermo), and the periplasmic cavity extract was obtained by using lysozyme (Shanghai Sangong) combined with three freeze-thaw methods, and the binding and binding of the clones were again identified by two ELISAs blocking activity. Coat with 50ng PD-1-his overnight, wash three times with PBS, add 1% BSA/PBS, 200uL/well, block at 37°C for 1 hour. After washing three times with PBS, 100ul of centrifuged supernatant was added and incubated at 37°C for 1 hour. After washing with PBST three times, 100ul of HRP-coupled goat anti-mouse IgG (Fab-specific) (Thermo) diluted at 1:5000 was added. Wash with PBST three times, add 100uL/well TMB color development solution, develop color at 37°C for 10 minutes, add 100uL/well ELISA stop solution, and read OD450 value with microplate reader to verify binding activity. Coat overnight with PD-1-his, wash three times with PBS, add 1% BSA/PBS, 200uL/well, block at 37°C for 1 hour. After washing three times with PBS, add 100ul centrifuged supernatant and different quality tetravalent PD-L1 mixtures and incubate at 37°C for 1 hour. After washing three times with PBST, add 100ul 1:5000 diluted HRP-coupled goat anti-mouse IgG (Fab specific of) (Thermo). Wash with PBST three times, add 100uL/well TMB color development solution, develop color at 37°C for 10 minutes, add 100uL/well ELISA stop solution, and read the OD450 value with a microplate reader to verify the blocking activity. Select the clone with low ELISA reading value (that is, strong blocking effect) for sequencing to obtain the sequence of the antibody, and select 6H6 with the strongest blocking effect for subsequent analysis. The amino acid sequences of the antibody heavy chain variable region and light chain variable region of 6H6 They are the sequences SEQ ID NO.7 and SEQ ID NO.8, respectively.

4.5 Expression of 6H6 Cloned hIgG1 Antibody

The heavy chain variable region sequence of 6H6 was cloned into pFUSEss-CHIg-hG1 respectively. The 6H6 light chain variable region sequences were cloned into pFUSE2ss-CLIg-hK, respectively. After 5 days of co-transfection of 293T cells (ATCC) with the two plasmids at a ratio of 1:1, the culture supernatant was collected, and the 6H6 antibody was purified with AKTAexplorer 100 (GE). The size of the 6H6 antibody was about 150k Daltons by Coomassie brilliant blue staining after non-reducing SDS-PAGE electrophoresis.

4.6 Detection of the blocking effect of each hIgG1 antibody (6H6 and Nivolumab) at the molecular level

6H6, Nivolumab and hIgG1 isotype control (R&D Systems, USA) were used together for competition ELISA experiments. The brief steps are: coat each well with 100ng of PD-1-huIgG1 Fc fusion protein overnight, then add 50ul of 2ng/ul of different hIgG1 antibodies (6H6, Nivolumab and isotype control antibody) and 50ul of different concentrations of PD-L1- his (ACROBiosystems) mixture, 100ul 1:5000 mouse anti-his tag-HRP (Thermo) for color development to detect the blocking effect of each antibody molecule level. This detection step contains three duplicate wells, and the average value of the ELISA results is shown in Table 3. The blocking effect of 6H6 molecular level is better than that of Nivolumab.

Table 3 Molecular level blocking effect of 6H6, Nivolumab and hIgG1 isotype control antibody

5. Humanization of anti-human PD-1 mouse antibody 6H6

In view of the superior molecular level blocking activity of 6H6 molecule, humanization was carried out for 6H6. First, carry out antibody humanization based on complementarity-determining region transplantation, and select IGKV6-21*02 and IGKJ4*01, IGHV3-23*04 and IGHJ4*01 human germline genes to replace the mouse light chain and heavy chain corresponding to 6H6, respectively. chain of germline genes. Secondly, predict the key amino acids that contribute to the affinity of the antibody and complete the back mutation: the first type of amino acid residues are located on the VL and VH binding interface residues, which play a key role in the packing of the two domains, and the second type of amino acid residues The base is close to the CDR region and is buried inside the protein. The third type of amino acid residues is the residues that directly interact with the CDR region, including hydrophobic interactions/hydrogen bonds/salt bridges, etc. Three candidate molecules were constructed based on the above rules, and the Hu_6H6 molecule was selected as the humanized 6H6 antibody version through in vitro affinity measurement. The amino acid sequences of the antibody heavy chain variable region and light chain variable region of Hu_6H6 are the sequence SEQID NO.9 and SEQ ID NO.10.

Example 7: Preliminary in vitro evaluation of anti-PD-1 mouse antibody

1. In vitro neutralization test of huIgG1 PD-1 antibody

Use ELISA to conduct PD-1 antibody neutralization test in vitro to verify the ability of 6H6 and pembrolizumab huIgG1 antibodies to block the binding of PD-L1 to PD-1: coat PD-L1-huIgG1 Fc (ACRO Biosystems) 1ug/ ml100ul per well, wash the plate and pat dry, then incubate with 1% BSA in PBS 200ul/well at 37°C for 1 hour to block. After washing the plate with 0.1% PBST 3 times and patting dry, add the mixture of scFv-huIgG1 Fc antibody and Biotinylated PD-1-huIgG1Fc (Kactus Biosystems) and incubate at room temperature for 1 hour (2ug/ml 50ul of PD-1-huIgG1 Fc with 160ug/ ml initial two-fold serial dilution of 50ul antibody mix, a total of eight gradients). Wash the plate 3 times with 0.1% PBST, add Streptavidin-HRP (R&D Systems) 100ul per well (1:200 dilution) and incubate at room temperature for 1 hour. After washing the plate 6 times with 0.1% PBST, add 100ul of TMB chromogenic solution to each well and incubate at room temperature for 10 minutes, add 100ul of stop solution to each well and use a microplate reader to read OD450. The data were analyzed and processed, and the IC50 of the half-maximum inhibitory concentration of the huIgG1 antibody of 6H6h and pembrolizumab binding to PD-1 and PD-L1 were calculated to be 3.605ug/ml and 6.662ug/ml, respectively, as shown in Figure 2.

2. In vitro binding test of huIgG1 PD-1 antibody

Use ELISA to measure the affinity of PD-1 antibody to verify the affinity of huIgG1 antibody of 6H6 and pembrolizumab: coat PD-1 Protein, Human, Recombinant (His Tag) (Beijing Yiqiao Shenzhou) 0.5ug/ml100ul per well four times overnight, wash After the plate was patted dry, it was blocked with 1% BSA in PBS 200ul/well and incubated at 37°C for 1 hour. Wash the plate 3 times with 0.1% PBST and pat dry, then incubate with scFv-huIgG1 Fc type antibody for 1 hour at room temperature (starting from 10ug/ml two-fold serial dilution, a total of eight gradients). Wash the plate 3 times with 0.1% PBST, add Goat anti-Mouse IgG F(ab')2Secondary Antibody, HRP (Thermo)/Goat Anti-Human IgG Secondary Antibody (HRP) (Beijing Sino Biological) 100ul per well (1:10000 diluted) and incubated at room temperature for 1 hour. After washing the plate 6 times with 0.1% PBST, add 100ul of TMB chromogenic solution to each well and incubate at room temperature for 10 minutes, add 100ul of stop solution to each well and use a microplate reader to read OD450. The data were analyzed and processed, and the calculated EC50 of 6H6 and pembrolizumab huIgG1 antibody binding to PD-1 were 0.01723ug/ml and 0.01106ug/ml, respectively, as shown in Figure 3.

Example 8: PD-1 antibody stimulates tumor infiltrating lymphocytes (TIL)

The stimulating effect of the antibody of the present application on TIL cells was verified by adding PD-1 antibody to the cell culture medium of tumor infiltrating lymphocytes (TIL). Two batches of TIL cells (donor A and donor B) were revived and cultured in IL-2-containing medium for 48 hours. After the culture was completed, IL-2 was washed and removed, and 1E5/well cells were added to a 96-well plate. The test group (Transact+PD-1) was added T Cell TransAct ^TM (Miltenyi Biotec) and 1 μg /ml of 6H6 PD-1 antibody stimulation, the control group was only added T Cell TransAct ^TM , after incubation overnight, the results of the proportion of cells expressing CD107a and the secretion of cytokines were detected.

A in Figure 4 shows the results of the ratio of CD107a cells with PD-1 antibody added to the tumor infiltrating lymphocyte (TIL) cell culture medium derived from donor A. B in Figure 4 shows the results of the proportion of CD107a cells with PD-1 antibody added to the tumor infiltrating lymphocyte (TIL) cell culture medium from donor B. The results showed that the PD-1 antibody of the present application can increase the proportion of cells expressing CD107a.

Figure 5 shows the results of cytokine IL-2, TNF-α and/or IFN-γ secretion in tumor infiltrating lymphocyte (TIL) cell culture medium from donor A and donor B with PD-1 antibody picture. The results show that the PD-1 antibody of the present application can increase the secretion of IL-2, TNF-α and/or IFN-γ; the PD-1 antibody of the present application has the ability to activate immune cells.

Example 9: Detection of PD-1 antibody stimulation of T lymphocytes by mixed lymphocyte reaction (MLR) assay

Mixed lymphocyte reaction (MLR) assay was used to detect that PD-1 antibody could stimulate T lymphocytes to secrete IL-2 and IFN-γ. The human PBMCs were adjusted to a cell density of 2E7/ml, and CD14 MicroBeads, human (MiltenyiBiotec) were used to sort monocytes. Add 50ng/ml GM-CSF (ACRO Biosystems) and 50ng/ml IL-4 (ACRO Biosystems) and culture for 5 days. Adding 10 ng/ml of LPS (Sigma) and 20 ng/ml of TNF-α (ACRO Biosystems) induced DC cell maturation. CD4+T cells were isolated from PBMCs from different human sources using Naive CD4+T Cell Isolation Kit II, human (Miltenyi Biotec). In a 96-well plate, 250 μl of culture medium per well contains 1.0E5 isolated T cells, 2E4 induced mature DC cells, and a series of concentration gradients of pembrolizumab or the PD-1 antibody 6H6 of the application. Human IgG1 isotype control (Isotype control) was used as a negative control. The mixed lymphocytes were cultured in a 37°C, 5% CO ₂ cell incubator for 3 days, and then 100ul culture supernatant was taken from each well of the 96-well plate for IL-2 and IFN-γ determination.

Figure 6 shows the results of the mixed lymphocyte reaction (MLR) test to detect the stimulation of PD-1 antibody on the secretion of IL-2 (A) and IFN-γ (B) by T lymphocytes. The results show that the PD-1 antibody of the present application has the ability to activate immune cells.

Example 10: PD-1 antibody enhances the killing ability of immune cells

By adding PD-1 antibody to the killing of A375 cells by NYESO-1 TCR-T cells, it was verified whether the PD-1 antibody enhanced the killing ability of TCR-T. CD3 positive T cells were sorted from freshly isolated human PBMC cells by CD3 MicroBeads, human (MiltenyiBiotec130-050-101). TCR-T cells can be obtained by any transduction method, for example, the lentivirus carrying NY-ESO-1 TCR (Kite Pharma) is used to transduce the sorted CD3 positive T cells, and the transduction efficiency can be 40% according to flow cytometry . A375 cells were plated into 96-well plates with 2E4 cells per well, and 2E4/well transduced NYESO-1TCR cells were added. At the same time, 10 μg/ml of 6H6 antibody and control antibody pembrolizumab were added as the test group, and the same amount of PBMC cells were added to A375 cells. As a negative control, only A375 cells were used as a blank control, and fluorescent dye SuperView ^TM 488 Caspase-3 was added to each well of all groups at the same time. Transfer the 96-well plate to IncuCyte S3 and automatically take pictures every 3 hours and then statistically analyze the killing results after 33 hours.

Figure 7 shows the result of adding PD-1 antibody to enhance the killing ability of TCR-T cells. The results show that the PD-1 antibody of the present application can enhance the killing ability of immune cells.

Example 11: Humanization of anti-human PD-1 murine antibody

(1) Humanized design of antibodies

Humanized against 6H6. Firstly, antibody humanization based on complementarity-determining region transplantation was carried out, and the 6H6 mouse antigene was compared with the human germline gene. This application obtained key amino acids that contribute to antibody affinity and completed back mutation: the first type of amino acid residues The residues located on the binding interface of VL and VH play a key role in the packing of the two domains. The second type of amino acid residues is close to the CDR region and embedded in the protein. The third type of amino acid residues is related to There are directly interacting residues in the CDR region, including hydrophobic interactions/hydrogen bonds/salt bridges, etc. Construct candidate molecules based on the above rules, synthesize sequences and construct expression vectors, transfect into 293F cells to express purified antibodies in suspension, and select 6H6-5, 6H6-25, 6H6-29 antibody molecules as humanized by in vitro affinity measurement 6H6 antibody version. The heavy chain constant region of the antibody may be the sequence shown in SEQ ID NO:60; the light chain constant region of the antibody may be the sequence shown in SEQ ID NO:61.

(2) Binding affinity test

The binding affinity of the huIgG1 type PD-1 antibody was measured by surface plasmon resonance (SPR) with a Biacore T200 biosensor equipped with a pre-immobilized protein A sensor chip. The humanized antibody (1ug/ml) was injected into the Biacore T200 biosensor at 10ul/min for about 24-33 seconds, and the desired protein density (about 44-57 response units) had been reached. Then the human PD1 protein (PD -1Protein, Human, Recombinant His Tag) was injected for 300 seconds at a concentration of 50ug/ml, 25ug/ml, 12.5ug/ml, 6.25ug/ml, 3.125ug/ml, and 1.5625ug/ml at a concentration of 30ul/min, and the dissociation was detected at 250 S. Figures 8, 9, 10 and 11 show the antigen binding affinity results of 6H6, 6H6-5, 6H6-25 and 6H6-29 antibodies, respectively.

Antibody 6H6 6H6-5 6H6-25 6H6-29 Affinity (KD) 4.50E-08 1.09E-08 8.07E-09 1.78E-08

(3) Antibody neutralization test in vitro

In vitro neutralization test of humanized 6H6 antibody was carried out by ELISA to verify the ability of humanized 6H6 huIgG1 antibody to block the combination of PD-L1 and PD-1: coat PD-L1-huIgG1 Fc (Kactus Biosystems ) 1ug/ml 100ul per well, wash the plate and pat dry, then incubate with 1% BSA in PBS 200ul/well at 37°C for 1 hour to block. After washing the plate with 0.1% PBST 3 times and patting dry, add scFv-huIgG1 Fc antibody and Biotinylated PD-1-huIgG1Fc (Kactus Biosystems) mixture and incubate at room temperature for 1 hour (2ug/ml 50ul of PD-1-huIgG1 Fc with 160ug/ml ml initial two-fold serial dilution of 50ul antibody mix, a total of 15 gradients). Wash the plate 3 times with 0.1% PBST, add Streptavidin-HRP (R&D Systems) 100ul per well (1:200 dilution) and incubate at room temperature for 1 hour. After washing the plate 6 times with 0.1% PBST, add 100ul of TMB chromogenic solution to each well and incubate at room temperature for 10 minutes, add 100ul of stop solution to each well and use a microplate reader to read OD450. The data were analyzed and processed, and the half inhibitory concentration IC ₅₀ of 6H6 and humanized 6H6-5, 6H6-25, 6H6-29 huIgG1 antibodies to PD-1 and PD-L1 binding was calculated, as shown in Figure 12.

Antibody 6H6 6H6-5 6H6-25 6H6-29 _IC50 (ug/ml) 2.6477 2.9335 0.8004 1.9073

The foregoing detailed description has been offered by way of explanation and example, not to limit the scope of the appended claims. Variations on the presently recited embodiments of this application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims (10)

1. An antigen binding protein that binds PD-1, said antigen binding protein comprising a light chain variable region and a heavy chain variable region, said heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, said HCDR1 having an amino acid sequence of SEQ ID No. 1; the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 2; the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3; the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4; the sequence of the LCDR2 is shown as SEQ ID NO. 5 (YAS); the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; and, the heavy chain variable region of the antigen binding protein comprises FRs 1 to 4 in an antibody heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO. 67; and the light chain variable region of the antigen binding protein comprises FRs 1 to 4 in an antibody light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO. 68.

2. The antigen binding protein of claim 1, comprising the heavy chain variable region of SEQ ID No. 67.

3. The antigen binding protein of claim 1, comprising a heavy chain variable region as set forth in any one of SEQ ID NOs 54 to 56.

4. The antigen binding protein of claim 1, comprising the light chain variable region of SEQ ID No. 68.

5. The antigen binding protein of claim 1, comprising a light chain variable region as set forth in any one of SEQ ID NOs 57 to 59.

6. The antigen binding protein of any one of claims 1-5, comprising an antibody or antigen binding fragment thereof.

7. A polypeptide comprising the antigen binding protein of any one of claims 1-6.

8. A nucleic acid encoding the antigen binding protein of any one of claims 1-6 and/or the polypeptide of claim 7.

9. A cell expressing the antigen binding protein of any one of claims 1-6, the polypeptide of claim 7, and/or comprising the nucleic acid of claim 8.

10. A method of affecting cytokine production by a target cell for non-therapeutic and/or diagnostic purposes, the method comprising administering the antigen binding protein of any one of claims 1-6, the polypeptide of claim 7, the nucleic acid of claim 8, a vector comprising the nucleic acid, and/or a cell expressing the antigen binding protein, the polypeptide, or comprising the nucleic acid and/or the vector.

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