CN114761434A

CN114761434A - PD-1 antibody, preparation method and application thereof

Info

Publication number: CN114761434A
Application number: CN202180007011.0A
Authority: CN
Inventors: 刘雅容; 种孟阳
Original assignee: Zhuhai Gravel Biotechnology Co ltd; Shanghai Grit Biotechnology Co Ltd; Suzhou Grit Biotechnology Co Ltd; Zhuhai Tuoyu Biotechnology Co Ltd
Current assignee: Shenzhen Shali Biotechnology Co ltd; Shanghai Grit Biotechnology Co Ltd; Suzhou Grit Biotechnology Co Ltd; Zhuhai Tuoyu Biotechnology Co Ltd
Priority date: 2020-09-30
Filing date: 2021-09-29
Publication date: 2022-07-15
Anticipated expiration: 2041-09-29
Also published as: CN114761434B; WO2022068891A1

Abstract

An antigen binding protein is provided that is capable of binding to PD-1. Also provides a preparation method and application of the antigen binding protein.

Description

PD-1 antibody, preparation method and application thereof

Technical Field

The application relates to the field of biomedicine, in particular to an antigen binding protein combined with PD-1.

Background

Programmed Death receptor 1 (PD-1) is a type I membrane protein with 288 amino acids, which is mainly expressed on the surface of activated T cells. PD-1 has two ligands, Programmed Death Ligand-1 (Programmed Death Ligand-1, PD-L1) and PD-L2. The interaction of PD-1, PD-L1 and PD-L2 can reduce the activity of T cells, reduce the secretion of cytokines and play a role in immunosuppression. The PD-1/PD-L1 pathway inhibitor can block the combination of PD-1 and PD-L1, block negative regulation signals, restore the activity of T cells, play a role in killing tumor cells and further inhibit the growth of tumors. Therefore, the immunoregulation taking PD-1/PD-L1 as a target has important significance for tumor inhibition. Currently, the blocking antibody drugs of the PD-1/PD-L1 pathway still face a plurality of challenges in clinic, such as low effectiveness, drug resistance, side effects and the like, and the development of more effective anti-PD-1 antibodies is still needed.

Disclosure of Invention

The present application provides an antigen binding protein that binds PD-1 and 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, the ability to enhance T cell activation including proliferative capacity, IFN- γ and/or IL-2 secretion, the ability to stimulate an antibody response, and/or the ability to reverse the inhibitory function of immunosuppressive cells, such as T regulatory cells. In one embodiment, the present application also provides an antibody of the scFv-huIgG1 Fc type for use conveniently in an autocrine format in combination with a cellular drug. Nucleic acid molecules encoding the isolated antigen binding proteins, expression vectors, host cells, and methods for making the isolated antigen binding proteins are also provided. The antigen binding proteins disclosed herein that bind to PD-1 can be used (alone or in combination with other active agents or therapeutic modalities) for the treatment, prevention, and/or diagnosis of diseases, such as cancer diseases (e.g., solid and soft tissue tumors).

In one aspect, the present application provides an isolated antigen binding protein comprising at least one CDR in a VH of a heavy chain variable region of an antibody, said VH comprising an amino acid sequence shown in SEQ ID NO 7 or 9.

In certain embodiments, the isolated antigen binding protein comprises an antibody or antigen binding fragment thereof.

In certain embodiments, the antigen binding fragment comprises a Fab, Fab ', Fv fragment, F (ab') 2, scFv, di-scFv, and/or dAb.

In certain embodiments, the antibody is selected from the group consisting of: monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

In certain embodiments, the isolated antigen binding protein competes for binding to PD-1 with a reference antibody, wherein the reference antibody comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID No. 4; the LCDR2 comprises an amino acid sequence shown as SEQ ID NO. 5; the LCDR3 comprises an amino acid sequence shown in SEQ ID NO. 6, the heavy chain variable region of the reference antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises an amino acid sequence shown in SEQ ID NO. 1; the HCDR2 comprises an amino acid sequence shown as SEQ ID NO. 2; the HCDR3 comprises an amino acid sequence shown in SEQ ID NO. 3.

In certain embodiments, the VH of the isolated antigen binding protein comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in SEQ ID No. 3.

In certain embodiments, the HCDR2 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID No. 2.

In certain embodiments, the HCDR1 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID No. 1.

In certain embodiments, said VH of said isolated antigen binding protein comprises FR1, FR2 and FR3, FR4, wherein said FR1 comprises the amino acid sequence shown in SEQ ID No. 11 or SEQ ID No. 19.

In certain embodiments, the FR2 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO. 12 or SEQ ID NO. 20.

In certain embodiments, the FR3 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO 13 or SEQ ID NO 21.

In certain embodiments, the FR4 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO. 14 or SEQ ID NO. 22.

In certain embodiments, the isolated antigen binding protein comprises a VH, and the VH comprises the amino acid sequence shown in SEQ ID No. 7 or 9.

In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region.

In certain embodiments, the heavy chain constant region of the isolated antigen binding protein is derived from a human IgG constant region.

In certain embodiments, the heavy chain constant region of the isolated antigen binding protein is derived from a human IgG4 constant region and/or a human IgG1 constant region.

In certain embodiments, the isolated antigen binding protein comprises at least one CDR in the variable region VL of an antibody comprising the amino acid sequence set forth in SEQ ID No. 8 or 10.

In certain embodiments, the VL of the isolated antigen binding protein comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID No. 4.

In certain embodiments, the LCDR2 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID No. 5.

In certain embodiments, the LCDR3 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID No. 6.

In certain embodiments, said VL of said isolated antigen binding protein comprises FR1, FR2 and FR3, FR4, wherein said FR1 comprises the amino acid sequence set forth in SEQ ID No. 15 or SEQ ID No. 23.

In certain embodiments, the FR2 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO 16 or SEQ ID NO 24.

In certain embodiments, the FR3 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:17 or SEQ ID NO: 25.

In certain embodiments, the FR4 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO. 18 or SEQ ID NO. 26.

In certain embodiments, the isolated antigen binding protein comprises a VL, and the VL comprises an amino acid sequence set forth in SEQ ID NO 8 or 10.

In certain embodiments, the isolated antigen binding protein has one or more of the following properties:

a) (ii) is capable of binding human PD-1;

b) can block the combination of PD-1 and PD-L1;

c) can block the combination of PD-1 and PD-L2;

d) capable of stimulating the secretion of IL-2, TNF-alpha and/or IFN-gamma in immune cells;

e) capable of inhibiting tumor growth and/or tumor cell proliferation;

f) can improve the killing ability of immune cells.

a) (ii) is capable of binding to human PD-1;

b) can block the combination of PD-1 and PD-L1;

c) can block the combination of PD-1 and PD-L2;

d) capable of stimulating the secretion of IL-2 and/or IFN-gamma in immune cells;

e) Can inhibit tumor growth and/or tumor cell proliferation.

In another aspect, the present application provides an isolated nucleic acid molecule or molecules encoding the isolated antigen binding protein.

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

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

In another aspect, the present application provides a method of making the isolated antigen binding protein, the method comprising culturing the cell under conditions such that the isolated antigen binding protein is expressed.

In another aspect, the present application provides a pharmaceutical composition comprising said isolated antigen binding protein, said nucleic acid molecule, said vector and/or said cell, and optionally a pharmaceutically acceptable adjuvant.

In another aspect, the present application provides the use of the isolated antigen binding protein, the nucleic acid molecule, the vector, the cell, and/or the pharmaceutical composition in the preparation of a medicament for treating a PD-1 mediated disease or disorder.

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

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

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

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

In certain embodiments, the PD-1 mediated disease or disorder comprises cancer.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

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

FIG. 1 shows the detection curve of the affinity (based on BLI method) of extracellular domain mutant of tetravalent human PD-L1 to PD-1-huIgG1 Fc;

FIG. 2 shows the inhibition curves of the huIgG1 antibodies against PD-1 and PD-L1 for 6H6 and pembrolizumab;

FIG. 3 shows the binding curves of huIgG1 antibody to PD-1 for 6H6 and pembrolizumab.

FIGS. 4A-4B are graphs showing the results of a proportion of CD107a cells in Tumor Infiltrating Lymphocyte (TIL) cell culture media derived from donor A and donor B to which PD-1 antibody was added.

FIGS. 5A-5F are graphs showing cytokine secretion by the addition of PD-1 antibody to Tumor Infiltrating Lymphocyte (TIL) cell culture media derived from donor A and donor B.

FIGS. 6A-6B are graphs showing the results of Mixed Lymphocyte Reaction (MLR) experiments testing the stimulation of PD-1 antibody to T lymphocytes.

FIG. 7 is a graph showing the results of enhancing the killing ability of TCR-T cells by adding PD-1 antibody.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

Definition of terms

In this application, the term "PD-1" generally refers to programmed cell death 1, also known as "programmed death 1"),

"CD 279", "cluster of differentiation 279", "PD 1", "PDCD 1". PD-1 is typically expressed on T cells, B cells, natural killer T cells, activated monocytes, and Dendritic Cells (DCs) and is involved in apoptosis. PD-1 typically comprises an extracellular IgV domain, a transmembrane region and an intracellular domain. PD-1 may 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 that results from cellular processing. PD-1 may exist as a transmembrane protein or as a soluble protein. "PD-1" includes intact PD-1 and fragments thereof, as well as functional variants, isoforms, species homologs, derivatives, analogs of PD-1, and analogs having at least one common epitope with PD-1. The amino acid sequence of human PD1 is shown in UniProt (www.uniprot.org), accession number Q15116.

In the present application, the term "PD-L1" refers generally to programmed cell death 1 ligand 1, also known as B7 homolog 1, B7-H1, cluster of differentiation 274, (3)274 or CD274, which upon binding to PD-1 down regulates 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 that results from cellular processing. PD-L1 may be present as a transmembrane protein or as a soluble protein. "PD-L1" includes intact PD-L1 and fragments thereof, and also includes functional variants, isoforms, species homologs, derivatives, analogs of PD-L1, and analogs having at least one common epitope with PD-L1. The basic structure of PD-L1 includes 4 domains: an extracellular Ig-like V-type domain and an Ig-like C2-type domain, a transmembrane domain, and a cytoplasmic domain. The complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ 7.

In the present application, the term "isolated" or "purified" generally refers to a molecule (e.g., an 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 biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, cell debris, and growth media. An "isolated or purified nucleic acid" is at least partially isolated from a nucleic acid that normally flanks a polynucleotide in its natural state. Thus, it is considered in the present application that polynucleotides fused to regulatory or coding sequences to which they are not normally bound are isolated, e.g., due to recombinant techniques. Such molecules are considered isolated even when present, for example, in the chromosome of the 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 that interfere with the experimental or therapeutic use of the molecule in large quantities. The antigen binding proteins of the present application and the 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 is meant to encompass a moiety that binds to an antigen or target and optionallyAnd optionally a protein comprising a framework or framework portion that allows the antigen binding portion to adopt a configuration that facilitates binding of the antigen binding protein to an antigen. Examples of antigen binding proteins include human antibodies, humanized antibodies; a chimeric antibody; a recombinant antibody; a single chain antibody; a bifunctional antibody; a trifunctional antibody; a tetrafunctional antibody; a Fab fragment; f (ab')₂A fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgG1 antibody; an IgG2 antibody; an IgG3 antibody; or IgG4 antibodies and fragments thereof. Antigen binding proteins may include, for example, alternative protein frameworks or artificial frameworks with grafted CDRs or CDR derivatives. Such frameworks include, but are not limited to: an antibody-derived framework comprising mutations introduced to, for example, stabilize the three-dimensional structure of an antigen-binding protein; and fully synthetic frameworks comprising, for example, biocompatible polymers. See, e.g., 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, peptide antibody mimetics ("PAM") can be used, as can frameworks based on antibody mimetics that utilize a fibronectin component as a framework.

In the present application, the term "antibody" is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies comprising two light chains and two heavy chains), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, heavy chain antibodies, and camelized single domain antibodies (e.g., heavy chain variable domain antibodies). Antibodies generally have the structure of an immunoglobulin and may comprise proteins of at least two Heavy Chains (HC) and two Light Chains (LC) linked to each other by disulfide bonds, or antigen-binding fragments thereof. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus in their antigenicity. Accordingly, immunoglobulins can be classified into five classes, or isotypes called immunoglobulins, i.e., IgM, IgD, IgG, IgA and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, IgG can be classified into IgG1, IgG2, IgG3 and IgG 4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. In the five classes of igs, the second class of igs can have either kappa chains or lambda chains.

In certain naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region comprises three domains, CH1, CH2 and CH 3. 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 comprises one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), which alternate with more conserved regions termed 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 FR 4. The variable domains of native heavy and light chains each comprise four FR regions (HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4), largely adopting a β -sheet configuration, connected by three CDRs, forming a loop linkage, and in some cases forming part of a β -sheet structure. The CDRs in each chain are held in close proximity by the FR region and form, together with the CDRs from the other chain, the antigen-binding site of the antibody. The constant region of the antibody can mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

In the present application, the term "variable" generally refers to the fact that certain portions of the sequence of the variable domains of antibodies vary strongly, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the light and heavy chain variable regions, called Complementarity Determining Regions (CDRs) or hypervariable regions (HVRs). The more highly conserved portions of the variable domains are called the Framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely in a β -sheet configuration, connected by three CDRs, forming a loop junction, and in some cases forming part of a β -sheet structure. The CDRs in each chain are held in close proximity by the FR region and form together with the CDRs from the other chain the antigen binding site of the antibody, and the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, for example, involved in antibody-dependent cytotoxicity of the antibody. In the art, CDRs of an antibody can be defined by a variety of methods, such as Kabat definition rules based on sequence variability (see Kabat et al, immunological protein sequences, fifth edition, national institutes of health, bessel, maryland (1991). in this application, amino acid residues in variable domain sequences and full-length antibody sequences are determined using the Kabat definition rules (see table 1).

TABLE 1 CDR definition of the antibodies of the present application based on the Kabat definition rules

Kabat	Residues
LCDR1	L24-L34

LCDR2	L50-L56
LCDR3	L89-L97
HCDR1	H31-H35
HCDR2	H50-H66
HCDR3	H99-H107

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

In the present application, the term "Fab" refers to an antigen-binding fragment of an antibody. As described above, the intact antibody can be digested with papain. Papain digestion of antibodies produces two identical antigen-binding fragments, a "Fab" fragment, and a residual "Fc" fragment (i.e., the Fc region, supra). The Fab fragment consists of one complete L chain with the variable region of one heavy chain and the H chain (V)_H) Of (a) a first constant region (C)_H1) And (4) forming.

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

In the present application, the term "F (ab') 2" refers to an antibody fragment produced by pepsin digestion of an intact antibody. The F (ab')2 fragment contains two Fab fragments and a partial hinge region held together by disulfide bonds. F (ab')2 fragments have divalent antigen binding activity and are capable of crosslinking antigens.

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 chain variable region and the light chain variable region, and lacking the heavy chain constant region and the light chain constant region. The heavy chain variable region and the light chain variable region include, for example, CDRs. For example, Fv fragments comprise all or part of the amino-terminal variable region of about 110 amino acids of the heavy and light chains.

In the present application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment comprising the variable region of a light chain and at least one antibody fragment comprising the variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker) and are capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, a scFv may have the VL and VH variable regions described in any order (e.g., relative to the N-and C-termini of the polypeptide), may comprise a VL-linker-VH or may comprise a VH-linker-VL.

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

In the present application, the term "monoclonal antibody" generally refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies in the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are typically highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which typically have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful herein can be prepared in hybridoma cells, or can be prepared by recombinant DNA methods.

In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parent antibody") and the constant region is derived from a human antibody, such that the resulting chimeric antibody has a reduced likelihood of eliciting an adverse immune response in a human individual as compared to the parent (e.g., mouse-derived) antibody.

In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR regions of a non-human antibody (e.g., a mouse antibody) are replaced with corresponding amino acids derived from a human immunoglobulin. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions may also be permissible as long as they still retain the ability of the antibody to bind to a particular antigen. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. "humanized antibodies" retain antigen specificity similar to the original antibody. "humanized" forms of non-human (e.g., murine) antibodies may be chimeric antibodies that minimally comprise sequences derived from non-human immunoglobulins. In certain instances, CDR region residues in a human immunoglobulin (recipient antibody) can be replaced with CDR region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired properties, affinities and/or capabilities. In some cases, residues from the FR region of a human immunoglobulin may be replaced with corresponding non-human residues. In addition, humanized antibodies may comprise amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve the properties of the antibody, such as binding affinity.

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

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

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

In the present application, the term "cell" generally refers to an individual cell, cell line or cell culture that may or may already contain a plasmid or vector comprising a nucleic acid molecule described herein, or that is capable of expressing an antibody or antigen-binding fragment thereof described herein. The cell may comprise progeny of a single host cell. Due to natural, accidental, or deliberate mutation, the progeny cells may not necessarily be identical in morphology or in genome to the original parent cell, but may be capable of expressing an antibody or antigen-binding fragment thereof as described herein. The cells can be obtained by transfecting cells in vitro with the vectors described herein. The cell may be a prokaryotic cell (e.g., E.coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese Hamster Ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, an NS0 cell, or a myeloma cell). In some cases, the cell may be a mammalian cell. For example, the mammalian cell may be a 293T cell. In the present application, the term "recombinant cell" generally refers to a cell into which a recombinant expression vector has been introduced. The recombinant host cell includes not only a specific cell but also a progeny of such a cell.

In the present application, the term "pharmaceutically acceptable adjuvant" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers which are non-toxic to the cells or mammals to which they are exposed at the dosages and concentrations employed. Typically, the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers can 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 a nonionic surfactant.

In the present application, reference to protein, polypeptide and/or amino acid sequences is also to be understood as including at least the following ranges: variants or homologues having the same or similar function as said protein or polypeptide.

In the present application, the variant may be a protein or polypeptide having substitution, deletion or addition of one or more amino acids in the amino acid sequence of the protein and/or the polypeptide (e.g., an antibody or a fragment thereof that specifically binds to PD-1 protein). For example, the functional variant may comprise a protein or polypeptide that has been altered by at least 1, such as 1-30, 1-20 or 1-10, yet for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions and/or insertions. The functional variant may substantially retain the biological properties of the protein or the polypeptide prior to the alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding capacity) of the protein or the polypeptide prior to alteration. For example, the substitution may be a conservative substitution.

In the present application, the homolog may be a protein or polypeptide having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the protein and/or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to PD-1 protein).

In the present application, homology refers generally to similarity, similarity or relatedness between two or more sequences. The "percentage of sequence homology" can be calculated by: the two sequences to be aligned are compared in a comparison window, the number of positions in the two sequences at which the same nucleobase (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, gin, Cys, and Met) is determined to yield the number of matched positions, the number of matched positions is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to yield the percentage of sequence homology. Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared or over a region of the target sequence. The homology can also be determined by the following method: FASTA and BLAST. The FASTA algorithm is described in "improved tools for biological sequence comparison" by w.r.pearson and d.j.lipman, proceedings of the national academy of sciences of the united states (proc.natl.acad.sci.), 85: 2444 2448, 1988; and "rapid and sensitive protein similarity search" by d.j.lipman and w.r.pearson, Science, 227: 1435-1441, 1989. BLAST algorithms are described in "a basic local contrast (alignment) search tool" by s.altschul, w.gish, w.miller, e.w.myers and d.lipman, journal of molecular biology, 215: 403-410, 1990.

In this application, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may, but need not, occur.

In this application, the term "comprising" is used in a generic sense to mean including, summarizing, containing or encompassing. In some cases, the meaning of "is", "consisting of … …" is also indicated.

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

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

Detailed Description

Antigen binding proteins

In one aspect, the present application provides an antigen binding protein comprising at least one CDR in a VH of a heavy chain variable region of an antibody, said VH comprising an amino acid sequence shown in SEQ ID NO 7 or 9.

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

The antigen binding proteins described herein can compete with a reference antibody for binding to PD-1. The reference antibody can 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 set forth in SEQ ID No. 4, and the LCDR2 comprises the amino acid sequence set forth in SEQ ID No. 5; the LCDR3 comprises an amino acid sequence shown as 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 sequence shown in SEQ ID NO. 1; the HCDR2 comprises an amino acid sequence shown as SEQ ID NO. 2, and the HCDR3 comprises an amino acid sequence shown as SEQ ID NO. 3.

The antigen binding proteins described herein may comprise the heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR 3.

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

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

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

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

The antigen binding proteins described herein may also comprise the heavy chain framework regions HFR1, HFR2, HFR3 and HFR 4.

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 linked directly or indirectly to the N-terminus of the HCDR 1.

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 HCDR 2.

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 HCDR 3.

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-terminus of the HFR4 is linked to the C-terminus of the HCDR 3.

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 comprise the amino acid sequences set forth in SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 14, respectively, in that order.

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

In some embodiments, the HFR1, HFR2, HFR3, and HFR4 of the antigen binding protein may comprise the amino acid sequences set forth in SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, and SEQ ID NO 22, respectively, in that order.

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

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

In the present application, the heavy chain constant region may comprise the constant region of a human IgG. In certain instances, the heavy chain constant region can include a human IgG4 constant region and/or a human IgG1 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, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, respectively, in that order, 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.

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

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

The antigen binding proteins described herein may comprise at least one CDR in the variable region VL of an antibody comprising the amino acid sequence shown in SEQ ID NO. 8 or 10.

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

In some embodiments, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein can comprise the amino acid sequences set forth in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively, in order, and the antigen binding protein can comprise at least one CDR in an antibody light chain variable region VL comprising the amino acid sequence set forth in SEQ ID No. 8.

In some embodiments, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein may comprise the amino acid sequences set forth in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, respectively, in that order, and the antigen binding protein may comprise at least one CDR in the antibody light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO. 16.

The antigen binding proteins described herein may comprise the light chain complementarity determining regions LCDR1, LCDR2 and LCDR 3.

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.

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, LCDR1, LCDR2 and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively, in the order named.

In some embodiments, the antigen binding protein may comprise HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 may comprise the amino acid sequences set forth in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, respectively, in that order.

In some embodiments, the LCDR1, LCDR2, and LCDR3 of the antigen binding protein may comprise the amino acid sequences set forth in SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6, respectively, in order, and the VH of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID No. 7.

In some embodiments, the LCDR1, LCDR2, and LCDR3 of the antigen binding protein may comprise the amino acid sequences set forth in SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6, respectively, in order, and the VH of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID No. 9.

In some embodiments, the LCDR1, LCDR2, and LCDR3 of the antigen binding protein can comprise the amino acid sequences set forth in SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6, respectively, in order, and the VH of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID No. 7, and the antigen binding protein can comprise a heavy chain constant region, and the heavy chain constant region can comprise the amino acid sequence set forth in any one of SEQ ID No. 27 or SEQ ID No. 28.

In some embodiments, the LCDR1, LCDR2, and LCDR3 of the antigen binding protein can comprise the amino acid sequences set forth in SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6, respectively, in order, and the VH of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID No. 9, and the antigen binding protein can comprise a heavy chain constant region, and the heavy chain constant region can comprise the amino acid sequence set forth in any one of SEQ ID No. 27 or SEQ ID No. 28.

The antigen binding proteins described herein may comprise the light chain framework regions LFR1, LFR2, LFR3, and LFR 4.

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 linked directly or indirectly to the N-terminus of the LCDR 1.

In the present application, LFR2 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO. 16 or SEQ ID NO. 24, and the LFR2 is located between the LCDR1 and the LCDR 2.

In the present application, LFR3 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:17 or SEQ ID NO:25, and the LFR3 is located between the LCDR2 and the LCDR 3.

In the present application, 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-terminus of the LFR4 is linked to the C-terminus of the LCDR 3.

In the present application, the antigen binding protein may comprise a VL, and the VL may comprise an 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 set forth in SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, and SEQ ID NO 18, in that order.

For example, LCDR1, LCDR2, and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively, in that order, and 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 that order.

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

For example, LCDR1, LCDR2, and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively, in that order, and 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 that order.

For example, the HCDR1, HCDR2 and HCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively, in order, and the LCDR1, LCDR2 and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively, in order, and the 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, respectively.

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

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 comprise, in order, the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, respectively, and the LCDR1, LCDR2, and LCDR3 of the antigen binding protein may comprise, in order, the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively, and the HFR1, HFR2, HFR3, and HFR4 of the antigen binding protein may comprise, in order, the amino acid sequences shown in SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 14, respectively, and the LFR1, LFR2, LFR3, and LFR4 of the antigen binding protein may comprise, in order, the amino acid sequences shown in SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, and SEQ ID NO. 18.

In some embodiments, the VH may comprise the amino acid sequence set forth in SEQ ID NO 9 and the VL may 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 sequentially comprise the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively, and the LCDR1, LCDR2 and LCDR3 of the antigen binding protein may sequentially comprise the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively, and the HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein may sequentially comprise the amino acid sequences shown in SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21 and SEQ ID NO. 22, respectively. LFR1, LFR2, LFR3, and LFR4 of the antigen binding protein may comprise, in order, the amino acid sequences shown in SEQ 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 a heavy chain constant region, and the heavy chain constant region may comprise an amino acid sequence set forth 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 7, and the VL can comprise the amino acid sequence set forth in SEQ ID NO 8, and the heavy chain constant region of the antigen-binding protein can comprise the amino acid sequence set forth 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 comprise the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively, in order, and the LCDR1, LCDR2 and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively, in order, and the HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, respectively, and the 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, respectively, and the LFR 7 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 7, and the VL may comprise the amino acid sequence set forth in SEQ ID NO 8 and the heavy chain constant region of the antigen binding protein may comprise the amino acid sequence set forth 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 comprise the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively, in order, and the LCDR1, LCDR2 and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively, in order, and the HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21 and SEQ ID NO. 22, respectively, and the 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, respectively, in order, and the amino acid sequence shown in SEQ ID NO. 15 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 15, and the VL may comprise the amino acid sequence set forth in SEQ ID NO 16 and the heavy chain constant region of the antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO 27 or SEQ ID NO 28.

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 antigen binding activity of an antigen binding protein or fusion protein of the present application can be tested, for example, by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., western blotting), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, and the like. The antigen binding proteins described herein (e.g., PD-1 antibodies) are capable of specifically binding to PD-1 antigen. An antigen binding protein that "specifically binds" to a PD-1 antigen (e.g., a PD-1 antibody) can generally bind to PD-1, but not to other proteins that lack the PD-1 sequence. An antigen binding protein (e.g., a PD-1 antibody) described herein is capable of specifically binding to a PD-1 antigen or a labeled form thereof (e.g., a fluorescently labeled PD-1 antigen), but does not bind to other proteins lacking a PD-1 epitope. Whether an antigen binding protein (e.g., an antibody) binds to 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 interference technique (BLI).

The antigen binding proteins described herein can bind to human PD-1 protein. In certain instances, the antigen binding proteins described herein can also cross-react with monkey (e.g., cynomolgus monkey) PD-1. For example, by flow assay techniques and enzyme-linked immunoassays. In this application, "cross-reactive" refers to the ability of an antibody to react with homologous proteins from other species.

In certain instances, the binding activity of an antigen binding protein described herein to PD-1 can be detected using an enzyme linked immunosorbent assay. For example, in an ELISA using a human PD-1 antigen protein, the EC50 value for PD-1 antigen binding protein to PD-1 can be between about 0.0001nM and about 100nM, e.g., can be between about 0.001nM and about 10nM, can be between about 0.001nM and about 5nM, can be between about 0.001nM and about 1nM, or can be between about 0.01nM and about 1 nM.

In another aspect, the antigen binding proteins described herein are capable of blocking the binding of PD-1 to PD-L1. In some cases, the antigen binding protein blocks binding of PD-1 to PD-L1 in an enzyme linked immunosorbent ELISA assay. For example, the PD-L1 antigen protein is first coated on a plate, and the antigen binding protein and biotin-labeled PD-1 protein are mixed in reduced amounts and incubated together. The cells were then analyzed using ELISA to confirm that the antigen binding protein can block the binding of PD-1 and PD-L1.

The antigen binding proteins described herein are capable of stimulating the proportion of cells expressing CD07a in immune cells. The antigen binding proteins described herein are capable of stimulating the secretion of IFN-. gamma., TNF-. alpha.and/or IL2 in immune cells. For example, the stimulatory capacity of the antigen binding proteins described herein may be an activator known in the art (e.g., a CD3 antibody, a CD28 antibody, or a nanomaterial TransAct comprising a CD3 antibody and a CD28 antibody) ^TM) On the basis of the activation, the effect on the activation of immune cells is increased. 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 cell may comprise an artificially modified immune cell. For example, the immune cells can comprise TCR-T cells. Cytokine secretion in immune cells can be measured by any method known to those skilled in the art, for example, by quantifying immune cell (e.g., T cell) proliferation or cytokine production by immune cells (e.g., IFN-. gamma.or IL-2 production by T cells) by enzyme-linked immunosorbent assay (ELISA). For example, the stimulation of T lymphocytes by PD-1 antibodies can be detected by a Mixed Lymphocyte Reaction (MLR) assay.

Nucleic acid molecules, vectors and cells

In another aspect, the present application provides one or more nucleic acid molecules that can encode an isolated antigen binding protein described herein. The nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) in vitro amplified, e.g., by Polymerase Chain Reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, e.g., by enzymatic cleavage and gel electrophoresis fractionation, or (iv) synthesized, e.g., 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 can comprise a nucleic acid molecule described herein. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may contain expression control elements that allow for the 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, among others. The vector may include, for example, a plasmid, cosmid, virus, phage, or other vector commonly used in, for example, genetic engineering. For example, the vector is an expression vector.

In another aspect, the present application provides a cell that can comprise a nucleic acid molecule described herein or a vector described herein. 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 (e.g., 2 or more) or a plurality (e.g., 2 or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein can be introduced into the host cell, e.g., a eukaryotic cell, such as a plant-derived cell, a fungal or yeast cell, and the like. The vectors described herein can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamine transfection, and the like.

Pharmaceutical composition

In another aspect, the present application provides a pharmaceutical composition comprising an antigen binding protein and/or a nucleic acid molecule as described herein, a vector as described herein, a host cell as described herein, and optionally a pharmaceutically acceptable adjuvant. The pharmaceutically acceptable adjuvants are non-toxic to recipients at the dosages and concentrations employed, and may include buffers, antioxidants, preservatives, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic 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, typically those with complementary activities that do not adversely affect each other. The type and effective amount of such drugs will depend, 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) a disease or disorder and/or any complication thereof in a subject suffering from or at risk of developing the disease or disorder.

Preparation method

In another aspect, the present application provides methods of making the antigen binding proteins. The method may comprise culturing the host cell described herein under conditions such that the antigen binding protein is expressed. For example, these methods can be performed by using an appropriate medium, an appropriate temperature, an appropriate incubation time, and the like, which are known to 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, an animal can be immunized with a linked or naturally occurring PD-1 protein or fragment thereof. Suitable immunization methods, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used.

Any suitable form of PD-1 may be used as an immunogen (antigen) for the production of non-human antibodies specific for PD-1, which antibodies are screened for biological activity. The challenge immunogen may be full-length mature human PD-1, including a native homodimer, or a peptide containing a single/multiple epitope. The immunogen may be used alone or in combination with one or more immunogenicity enhancing agents known in the art.

Humanized antibodies may be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE. In the present application, the antibody is an IgG antibody, and an IgG1 subtype is used. Optimization of the sequence of the essential constant domains to produce the desired biological activity can be achieved by screening antibodies using the biological assays described in the examples below. Likewise, any 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 or fragment thereof of the present application can be obtained by conventional techniques, such as by PCR amplification or genomic library screening. Alternatively, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into cells, and isolating the relevant sequence from the propagated host cells by conventional methods. In addition, the sequence of interest can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The nucleic acid molecule can then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.

The application also relates to vectors comprising suitable nucleic acid molecules as described above and suitable promoters or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. For example, animal cells may include (but are not limited to): 293T cells.

The steps described herein for transforming a host cell with a recombinant DNA may be performed using techniques well known in the art. The transformants obtained can be cultured by conventional methods and express the polypeptides encoded by the nucleic acid molecules of the present application. Depending on the host cell used, it is cultivated in a conventional medium under suitable conditions. Typically, the transformed host cell is cultured under conditions suitable for expression of the antigen binding protein of the present application. The antigen binding protein of the present application is then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.

The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays, such as flow cytometric sorting (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 manufacture of a medicament. The antigen binding protein may be administered alone or in combination with one or more additional therapies, such as chemotherapeutic radiation therapy, immunotherapy, surgical intervention, or any combination of these. The medicaments are useful for treating PD-1 mediated diseases or disorders, for example, for treating cancer, inhibiting tumor growth, and/or inhibiting tumor cell proliferation.

In another aspect, the antigen binding proteins provided herein are useful for preventing or treating a PD-1 mediated disease or condition. The prevention or treatment of a disease or disorder may refer to inhibiting or delaying the development or progression of a disease or disorder. 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 said antigen binding protein for use in the prevention or treatment of a PD-1 mediated disease or disorder.

In another aspect, the present application provides a method of inhibiting the binding of PD-1 to PD-L1, comprising administering an antigen binding protein described herein. The method may be an ex vivo or in vitro method. In certain instances, the methods can include contacting a biological sample with an antigen binding protein and/or PD-L1 described herein under conditions that allow the antigen binding protein and/or PD-1 to bind to PD-L1, 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.

In another aspect, the present application provides a method of preventing, ameliorating, or treating a PD-1 mediated disease or disorder, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof, a molecular nucleic acid, a vector, a host cell, and/or a pharmaceutical composition described herein. 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.

Without intending to be bound by any theory, the following examples are intended only to illustrate the fusion proteins, preparation methods, uses, etc. of the present application, and are not intended to limit the scope of the present invention.

Examples

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

Synthesis of gene sequence of PD-125-167 amino acid interval and construction of expression vector of PD-1-huIgG1 Fc fusion protein

A gene sequence (SEQ ID NO:29) in the region from isoleucine at position 25 to glutamine at position 167 of programmed death receptor 1(PD-1) (NCBI access No. NP-005009.2) encoding the amino acid sequence shown in SEQ ID NO:30 was synthesized by chemical synthesis. The gene sequence of the interval from proline at position 100 to lysine at position 330 of the constant region of the heavy chain of human IgG1 was synthesized by chemical synthesis. Upstream primers containing mouse Igkv3-10 signal peptide gene sequences are chemically synthesized and used for constructing expression vectors. The PD-1 gene fragment is spliced with a human IgG1 Fc gene fragment through molecular cloning. 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 the expression vector for 5 days, culture supernatants were collected, and the recombinant PD-1-huIgG1 Fc fusion protein was purified using AKTA explorer 100 (GE). Due to glycosylation modification and the like, the size of the recombinant PD-1-huIgG1 Fc fusion protein is shown to be about 60k daltons by Coomassie blue staining after reduction SDS-PAGE electrophoresis.

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

Synthesis of gene sequence of PD-125-167 amino acid interval and construction of expression vector of polyhistidine tag fusion protein

The gene sequence of the region from isoleucine 25 to glutamine 167 of programmed death receptor 1(PD-1) (NCBI access No. np _005009.2) was synthesized by chemical synthesis. An upstream primer containing a mouse Igkv3-10 signal peptide gene sequence and a downstream primer containing a polyhistidine tag (having an amino acid sequence shown in SEQ ID NO: 32) gene sequence (SEQ ID NO:31) are chemically synthesized to construct a pCDNA3.1 vector for expressing PD-1-his.

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

After transfecting 293T cells (ATCC) with the expression vector for 5 days, culture supernatants were collected and the recombinant PD-1-his protein was purified using AKTA explorer 100 (GE). Due to glycosylation modification and the like, the size of the recombinant PD-1-his protein is about 40 kDalton as shown by Coomassie blue staining after electrophoresis by reduced SDS-PAGE.

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

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

The genes of the Nivolumab antibody light chain variable region synthesized by a chemical synthesis mode are respectively SEQ ID NO. 33 and SEQ ID NO. 34, the Nivolumab antibody light chain variable region has an amino acid sequence shown by SEQ ID NO. 35, and the Nivolumab antibody heavy chain variable region has an amino acid sequence shown by SEQ ID NO. 36. Taking the heavy chain variable region gene as a template, carrying out PCR amplification on the heavy chain variable region fragment, and cloning the amplified product into pFUSs-CHIg-hG 1(invivogen) containing a human IL-2 signal peptide gene and a human IgG1 heavy chain constant region gene by using a TaKaRa seamless cloning kit. The light chain variable region fragment was PCR-amplified using the light chain variable region gene as a template, and the amplified product was cloned into pFUSE2ss-CLIg-hK (invivogen) containing human IL-2 signal peptide gene and human kappa light chain constant region gene using TaKaRa seamless cloning kit.

Expression and purification of Nivolumab antibody

After 293T cells (ATCC) were co-transfected with the two plasmid 1:1 ratio for 5 days, culture supernatants were collected and the Nivolumab antibody was purified using AKTA explorer 100 (GE). The Nivolumab antibody was visualized by Coomassie blue staining after electrophoresis on non-reducing SDS-PAGE to a size of about 150 kDalton.

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

The enzyme-linked immunosorbent assay (ELISA) is used for detecting the combination of the recombinant human PD-1(PD-1-huIgG1 Fc or PD-1-his) and the Nivolumab antibody, and the ELISA experiment is specifically operated as follows: the plate was coated overnight at 4 ℃ with 100 ng/well of PD-1-huIgG1 Fc or PD-1-his fusion protein prepared above. PBS wash three times, add 1% BSA/PBS, 200 uL/well, 37 degrees C blocking for 1 hours. After washing the plate with 100ul PBS, 100 ng/well of the Nivolumab chimeric antibody was added and bound at 37 ℃ for 1 hour. PBST was washed three times and 100ul of HRP-goat anti-human IgG (Fab specific) diluted 1:5000 was added for binding at 37 ℃ for 1 hour. PBST is washed for three times, 100 uL/hole TMB developing solution is added, color development is carried out for 10 minutes at 37 ℃, 100 uL/hole ELISA stopping solution is added, and an enzyme-linked immunosorbent assay (OD 450) value is read by an enzyme-linked immunosorbent assay (ELISA) instrument. The OD450 value reflects the binding of the Nivolumab antibody to recombinant human PD-1.

Example 5: expression of tetravalent human cell programmed death-ligand 1(PD-L1) extracellular domain mutant and determination of affinity with human PD-1 molecule

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

The gene sequence (SEQ ID NO:37) of two human PD-L1 extracellular segment mutants connected by a flexible peptide is synthesized by a chemical synthesis mode, and the gene sequence codes the amino acid sequence shown in SEQ ID NO: 38. The gene sequence of the region from proline at position 100 to lysine at position 330 of the constant region of the heavy chain of human IgG1 (UniProtKB/Swiss-Process No. P01857.1) was synthesized by chemical synthesis. Upstream primers containing mouse Igkv3-10 signal peptide gene sequences are chemically synthesized and used for constructing expression vectors. The gene fragment of the PD-L1 extracellular mutant repeat structure is spliced with the human IgG1 Fc gene fragment through molecular cloning. The spliced product was cloned into pCDNA3.1(Thermo) using TaKaRa seamless cloning kit.

2. Expression and purification of tetravalent human PD-L1 extracellular domain mutant

After 293T cells (ATCC) were transfected with the expression vector for 5 days, culture supernatants were collected and the tetravalent human PD-L1 extracellular domain mutant protein was purified using AKTA explorer 100 (GE). Due to glycosylation modification and the like, the tetravalent human PD-L1 extracellular domain mutant protein is subjected to reduction SDS-PAGE electrophoresis and then is stained by Coomassie brilliant blue to show that the size of the protein is about 85k daltons.

Biotinylation of PD-1-huIgG1 Fc fusion protein

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

4. Determination of the affinity (avidity) of the tetravalent human PD-L1 extracellular domain mutant for PD-1-huIgG1 Fc

Affinity of the tetravalent human PD-L1 extracellular domain mutant to PD-1-huIgG1 Fc was determined using an Octet K2(ForteBio) molecular interaction analyzer. 100nM of biotinylated PD-1-huIgG1 Fc fusion protein was prepared and immobilized on an SA probe (ForteBio) at a height of 1nM, following the standard protocol for Octet K2. Experiments were carried out with the tetravalent human PD-L1 extracellular domain mutant diluted in a two-fold gradient starting from a concentration of 50nM as analyte, and the affinity of the tetravalent human PD-L1 extracellular domain mutant to PD-1-huIgG1 Fc was determined to be 9.4nM, and the results of the affinity determination are shown in FIG. 1. Wild-type PD-1 and PD-L1 are known to bind with an 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 murine antibody, and murine anti-humanization

1. Immunising an animal

2mg/mL of PD-1-huIgG1 Fc fusion protein was emulsified as an antigen by mixing with an equal volume of complete Freund's adjuvant (Sigma-Aldrich), and 10 6-week-old female Balb/c mice (immunized subcutaneously, 100ug of each antigen) were boosted every ten days after the initial immunization for a total of four subcutaneous immunizations, and the spleen was directly shocked with PD-1-huIgG1 Fc fusion protein at the fifth immunization.

2. Serum titer detection

50uL of blood was taken from the tail vein before each booster immunization, and the cells were removed by centrifugation, and the serum was retained. ELISA plates were coated overnight at 4 ℃ with 50ng PD-1-his (ACRO biosystems). PBS wash three times, add 1% BSA/PBS, 200 uL/well, 37 degrees C blocking for 1 hours. A gradient of diluted mouse serum was added and allowed to bind for 1 hour at 37 ℃. PBST was washed three times and 100ul of a 1:5000 dilution of HRP-goat anti-mouse IgG (Shanghai assist in peptide) was added and combined for 1 hour at 37 ℃. PBST is washed for three times, 100 uL/hole TMB developing solution is added, color development is carried out for 10 minutes at 37 ℃, 100 uL/hole ELISA stopping solution is added, and an enzyme-linked immunosorbent assay (OD 450) value is read by an enzyme-linked immunosorbent assay (ELISA) instrument.

3. Construction of an immune library

3.1 Total cDNA acquisition of mouse splenocytes

Four days after the four-day shock immunization with the PD-1-huIgG1 Fc fusion protein by direct intraperitoneal injection, the spleen was removed. Spleen cells were obtained by grinding the whole spleen with a 70 μm cell mesh (BD). After washing twice with PBS, spleen cells were obtained by centrifugation at 1000g for 10 minutes. Total RNA was extracted using Trizol RNA extraction kit.

The RNA was used as a template, and SuperScript was used^TMIV First-Strand Synthesis System kit for the First Strand cDNA Synthesis.

3.2 antibody Gene amplification and light-heavy chain splicing

Using the cDNA as a template, Antibody amplification primers described in the literature (Schaefer j.v., honeyger a., pluckthun a. (2010) Construction of scFv Fragments from hybrid or spring Cells by PCR assembly.in: Kontermann r., D ü bel S. (eds) Antibody engineering. In a 50uL reaction system, 25uL of phusion master mix (Thermo), 2.5uL of the forward primer (25pmol), 2.5uL of the reverse primer (25pmol), 1.5uL of DMSO, 0.5uL of cDNA, and 18uL of ddH2O were added, respectively. The PCR reaction was performed according to the following procedure: pre-denaturation at 98 ℃ for 1 min, then temperature cycling, denaturation at 98 ℃ for 30 sec, annealing at 58 ℃ for 30 sec, extension at 72 ℃ for 1 min, cycling for 30 times, and final extension at 72 ℃ for 10 min.

The amplified VH gene and VL gene were recovered using DNA gel recovery kit. The same amount of VH gene and VL gene were mixed as a template, and scFv gene was amplified by overlap PCR using upstream primer scFv-F and downstream primer scFv-R. In a 50uL reaction system, 25uL phusion master mix, 2.5uL (25pmol) upstream primer, 2.5uL (25pmol) downstream primer, 1.5uL DMSO, 0.5uL cDNA and 18uL ddH2O were added, respectively. The PCR reaction was performed as follows: pre-denaturation at 98 ℃ for 1 min, then temperature cycling, denaturation at 98 ℃ for 30 sec, annealing at 58 ℃ for 30 sec, extension at 72 ℃ for 1 min, cycling for 30 times, and final extension at 72 ℃ for 10 min.

And recovering the scFv gene fragment obtained by amplification by using a DNA gel recovery kit.

3.3 construction of immune libraries

The scFv gene fragment and pcomb3XTT vector (Scripps research, USA) were digested with SfiI endonuclease DNA, respectively. In a 50uL reaction system, SfiI 2uL, 10 Xbuffer 5uL, DNA 3ug, ddH2O to 50uL were added. After mixing well, incubation was carried out at 50 ℃ for 3 hours.

And recovering the scFv gene fragment and the pcomb3XTT vector after enzyme digestion by using a DNA gel recovery kit. The digested scFv gene fragment and the digested pcomb3XTT vector were circularized using T4 ligase. In a 50uL reaction system, 1uL of T4 ligase, 5uL of 10 Xbuffer, 100ng of scFv gene, 500ng of pComb3XTT vector, and ddH2O to 50uL were added. After mixing well, incubation was carried out at 4 ℃ for 16 hours. A small amount of the product was run through an agarose gel to verify ligation efficiency.

10uL of the above ligation-cyclization product was added to the electroporation competent TG1 in-house, followed by electroporation using an electrotransfer apparatus. 10ul of the electro-transformed bacteria were removed and counted for phage antibody library size by appropriate dilution and streaking on ampicillin containing plates. The remaining electrotransformed bacteria were added to 2XYT medium containing 100ug/mL ampicillin and 2% glucose and placed in a heated incubator for culture. After the completion of the culture, the cells were centrifuged at 4000G for 10 minutes at 4 ℃ and the precipitated cells were supplemented with an appropriate amount of glycerol and stored at-80 ℃ as an antibody cell bank. scFv immune libraries with over 3E9 library capacity were obtained by multiple accumulation of electrotransformations.

4. Screening and identification of murine immune antibody phage libraries

Biotinylation of PD-1-huIgG1 Fc fusion protein

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

4.2 biopanning

And (2) performing biological panning on the murine immune antibody library by using the PD-1-huIgG1 Fc fusion protein as a target protein to obtain an antibody combined with the PD-1-huIgG1 Fc fusion protein (especially PD-1 extracellular domain). After recovering 100OD bacteria from the antibody seed library at the starting OD600 ═ 0.1 density and growing to logarithmic phase, the antibody library was rescued using M13KO7 helper phage, resuspended after centrifugation in 2XYT medium containing ampicillin and kanamycin and amplified overnight at 30 ℃. PEG/NaCl precipitation of phage, with glycerol/PBST dissolved phage precipitation to obtain immune library phage suspension. Casein-blocked phage were dropped into a casein-blocked biotinylated huIgG1 Fc (ACRO biosystems) fusion protein and casein-blocked Dynabeads M-270 streptavidin co-incubation system, and the supernatant phage suspension was collected. Further, the collected phage suspension was put into a co-incubation system of biotinylated PD-1-huIgG1 Fc fusion protein blocked by casein and Dynabeads M-270 streptavidin blocked by casein, and PBST was used to wash the magnetic beads to remove phage that could not bind to the PD-1-huIgG1 Fc fusion protein. Under appropriate elution conditions, the phage were competitively eluted using the home-made tetravalent PD-L1 extracellular domain mutant. 10ul of the eluted phage solution was retained for determination of total output phage amount, and the remaining phage solution was used to infect log-increased TG1, which was used as the antibody library for the next round of panning after overnight amplification. Three rounds of biopanning were performed, with the parameters of the panning experiments detailed in table 2.

TABLE 2 antibody library biopanning Experimental parameters

4.3 Primary screening of clones binding specifically to the extracellular domain of PD-1 and having a blocking action against PD-1-tetravalent PD-L1

The antibody library obtained after the end of the third round of biopanning was diluted and plated on ampicillin-containing plates to obtain single clones, which were selected for overnight culture in deep well plates. The deep-well plate was freeze-thawed three times the next day repeatedly, and the supernatant was centrifuged for two subsequent ELISA reactions.

ELISA reaction to detect binding activity: the coating was applied overnight with 50ng PD-1-his, washed three times with PBS, and blocked by the addition of 1% BSA/PBS at 200 uL/well for 1 hour at 37 ℃. PBS wash three times followed by 100ul centrifugation of supernatant incubated for 1 hour at 37 deg.C, PBST wash three times followed by 100ul 1:5000 dilution of HRP-conjugated goat anti-mouse IgG (Fab-specific) (Thermo). PBST is washed for three times, 100 uL/hole TMB developing solution is added, color development is carried out for 10 minutes at 37 ℃, 100 uL/hole ELISA stopping solution is added, and an enzyme-linked immunosorbent assay (OD 450) value is read by an enzyme-linked immunosorbent assay (ELISA) instrument. The screening step is repeated twice independent experiments to ensure accurate data, and clones with the OD450 number average value larger than 0.15 are selected for subsequent analysis.

ELISA reaction to detect blocking activity: the coating was done overnight with PD-1-his, washed three times with PBS, added 1% BSA/PBS at 200 uL/well and blocked for 1 hour at 37 ℃. PBS was washed three times, 100ul of the centrifuged supernatant and 20ng of tetravalent PD-L1 mixture were added and incubated for 1 hour at 37 deg.C, and PBST was washed three times, 100ul of a 1:5000 dilution of HRP-conjugated goat anti-mouse IgG (Fab-specific) (Thermo). PBST is washed for three times, 100 uL/hole TMB developing solution is added, color development is carried out for 10 minutes at 37 ℃, 100 uL/hole ELISA stopping solution is added, and an enzyme-linked immunosorbent assay (OD 450) value is read by an enzyme-linked immunosorbent assay (ELISA) instrument. The screening step is repeated twice independent experiments to ensure accurate data, and clones with the OD450 number average value smaller than 1 are selected for subsequent analysis.

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

4.4 rescreening clones with specific binding of PD-1 extracellular domain and blocking of PD-1-tetravalent PD-L1

The candidate clones obtained from the primary screening were expanded in 50ml shake tubes (thermo), periplasmic cavity extracts were obtained by lysozyme (Shanghai worker) combined with three freeze-thaw cycles, and the binding and blocking activities of the clones were again identified by two ELISAs. The coating was applied overnight with 50ng PD-1-his, washed three times with PBS, and blocked by the addition of 1% BSA/PBS at 200 uL/well for 1 hour at 37 ℃. After three washes with PBS 100ul of the centrifuged supernatant was added and incubated for 1 hour at 37 deg.C, after three washes with PBST 100ul of a 1:5000 dilution of HRP conjugated goat anti-mouse IgG (Fab specific) (Thermo) was added. PBST was washed three times, 100 uL/well TMB developing solution was added, color development was carried out at 37 ℃ for 10 minutes, 100 uL/well ELISA stop solution was added, and OD450 value was read with an microplate reader to verify binding activity. The coating was done overnight with PD-1-his, washed three times with PBS, added with 1% BSA/PBS at 200 uL/well and blocked for 1 hour at 37 ℃. PBS was washed three times and then incubated for 1 hour at 37 ℃ with 100ul of the centrifugation supernatant and different masses of tetravalent PD-L1 mixture, PBST was washed three times and then 100ul of a 1:5000 dilution of HRP-conjugated goat anti-mouse IgG (Fab-specific) (Thermo) was added. PBST was washed three times, 100 uL/well TMB developing solution was added, development was carried out at 37 ℃ for 10 minutes, 100 uL/well ELISA stop solution was added, and OD450 value was read by an microplate reader to verify blocking activity. Clones with low ELISA reading values (namely, strong blocking effect) are selected for sequencing to obtain antibody sequences, 6H6 with the strongest blocking effect is selected from the antibody sequences for subsequent analysis, and the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody of 6H6 are respectively shown as sequences No.7 and No. 8.

4.5 expression of 6H6 clone hIgG1 form antibody

The heavy chain variable region sequence of 6H6 was cloned into pFUSEs-CHIg-hG 1, respectively. The 6H6 light chain variable region sequence was cloned into pFUSE2ss-CLIg-hK, respectively. After 293T cells (ATCC) were co-transfected at the two-plasmid 1:1 ratio for 5 days, culture supernatants were collected and the 6H6 antibody was purified by AKTA explorer 100 (GE). The 6H6 antibody was visualized by Coomassie blue staining after electrophoresis on a non-reducing SDS-PAGE to a size of about 150 kDalton.

4.6 detection of the blocking Effect of the hIgG1 antibodies (6H6 and Nivolumab) at the molecular level

6H6, Nivolumab, and hIgG1 isotype control (U.S. R & D Systems) were used together for competition ELISA experiments. The brief steps are as follows: 100ng of PD-1-huIgG1 Fc fusion protein per well was used for overnight coating, 50ul of 2ng/ul of different hIgG1 antibodies (6H6, Nivolumab and isotype control antibody) and 50ul of PD-L1-his (ACRO biosystems) mixtures at different concentrations were added in sequence, and 100ul 1:5000 mouse anti-his tag-HRP (thermo) was developed to detect blocking effect at each antibody molecule level. The detection steps all contained three duplicate wells and the average values of the ELISA results are detailed in Table 3. The blocking effect of 6H6 molecular level is better than that of Nivolumab.

TABLE 36 molecular level blocking effects of H6, Nivolumab and hIgG1 isotype control antibodies

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

In view of the superior molecular-level blocking activity of the 6H6 molecule, humanization was made against 6H 6. Humanization of antibodies based on CDR grafting was first performed by selecting human germline genes for IGKV6-21 x 02 and IGKJ4 x 01, IGHV3-23 x 04 and IGHJ4 x 01, respectively, in place of germline genes for the murine light and heavy chains, respectively, corresponding to 6H 6. Second we predicted key amino acids contributing to antibody affinity and completed back mutations: the first class of amino acid residues, residues located at the VL and VH binding interfaces, play a key role in Packing of the two domains, the second class of amino acid residues are residues near the CDR regions and embedded inside the protein, and the third class of amino acid residues are residues that have direct interactions with the CDR regions, including hydrophobic interactions/hydrogen bonds/salt bridges, etc. Three candidate molecules are constructed based on the above rules, and Hu _6H6 molecule is preferably selected as humanized 6H6 antibody version by in vitro affinity determination, the amino acid sequences of heavy chain variable region and light chain variable region of Hu _6H6 are sequence No.15 and No.16 respectively.

Example 7: preliminary in vitro evaluation of anti-PD-1 murine antibodies

In vitro neutralization assay for huIgG1 type PD-1 antibody

The ability of antibodies huIgG1 from 6H6 and pembrolizumab to block the binding of PD-L1 to PD-1 was verified by performing a PD-1 antibody in vitro neutralization assay using ELISA: four degrees overnight coated with PD-L1-huIgG1 Fc (ACRO biosystems)1ug/ml 100ul per well, washed plate after patting dry using 1% BSA in PBS 200ul/well 37 ℃ incubation for 1 hour for blocking. The plates were washed 3 beats dry with 0.1% PBST and incubated for 1 hour at room temperature with the addition of a mixture of scFv-huIgG1 Fc type antibody and Biotinylated PD-1-huIgG1 Fc (Kactus biosystems) (2ug/ml of 50ul PD-1-huIgG1 Fc mixed with 50ul antibody diluted in a two-fold gradient starting from 160ug/ml for eight gradients). Plates were washed 3 times with 0.1% PBST and incubated with Streptavidin-HRP (R & D Systems)100ul per well (1:200 dilution) for 1 hour at room temperature. After washing the plate for 6 times by 0.1% PBST, adding 100ul of TMB color development solution into each well for incubation for 10 minutes, adding 100ul of stop solution into each well, and reading OD450 by using an enzyme-labeling instrument. The data were analyzed and calculated to give half maximal inhibitory concentrations IC50 of 6H6H and pembrolizumab's huIgG1 antibody to PD-1 and PD-L1 binding of 3.605ug/ml and 6.662ug/ml, respectively, as shown in FIG. 2.

In vitro binding assay for huIgG1 type PD-1 antibody

PD-1 antibody affinity assay was performed using ELISA to verify the affinity of 6H6 and the humgg 1 antibody to pembrolizumab: PD-1 Protein, Human, Recombinant (His tag) (Beijing Yiqian, Chi) was coated 0.5ug/ml 100ul per well four times overnight, and blocked by incubation with 1% BSA in PBS 200ul/well for 1 hour at 37 ℃ after washing and patting dry. The 0.1% PBST plates were washed 3 beats dry and incubated with the addition of scFv-huIgG1 Fc type antibody for 1 hour at room temperature (two-fold gradient dilution starting from 10ug/ml for eight gradients). The plates were washed 3 times with 0.1% PBST, and incubated with Goat Anti-Mouse IgG F (ab')2Secondary Antibody, HRP (thermo)/Goat Anti-Human IgG Secondary Antibody (HRP) (Beijing Yi Qian Shen) 100ul per well (1:10000 dilution) for 1 hour at room temperature. After washing the plate for 6 times by 0.1% PBST, adding 100ul of TMB color development solution into each well, incubating for 10 minutes at room temperature, adding 100ul of stop solution into each well, and reading OD450 by using an enzyme-labeling instrument. The data were analyzed and calculated to give an EC50 of 0.01723ug/ml and 0.01106ug/ml for binding of the huIgG1 antibody to PD-1, 6H6 and pembrolizumab, respectively, as shown in FIG. 3.

Example 8: stimulation of Tumor Infiltrating Lymphocytes (TIL) by PD-1 antibody

The stimulation of Tumor Infiltrating Lymphocytes (TILs) by the antibodies of the present application was confirmed by the addition of PD-1 antibody to the cell culture medium of TILs. Two batches of TIL cells (donor A and donor B) were revived, containedThe culture was carried out in the presence of IL-2 for 48 hours. After the culture was completed, IL-2 was washed off, 1E 5/well cells were added to a 96-well plate, and T Cell TransAct was added to the test group (Transact + PD-1) at a ratio of 1:1000 according to the instructions^TM(Miltenyi Biotec) and 6H6 PD-1 antibody at 1. mu.g/ml, control group was stimulated with T Cell TransAct alone^TMThe results of the measurement of the proportion of cells expressing CD107a and the secretion of cytokines after overnight incubation.

FIG. 4A is a graph showing the results of a proportion of CD107a cells added with PD-1 antibody in Tumor Infiltrating Lymphocyte (TIL) cell culture medium derived from donor A. FIG. 4B shows the results of the proportion of CD107a cells added with PD-1 antibody in Tumor Infiltrating Lymphocyte (TIL) cell culture medium derived from donor B. The results show that the PD-1 antibodies of the present application can increase the proportion of cells expressing CD107 a.

FIGS. 5A-5F are graphs showing the results of cytokine IL-2, TNF- α and/or IFN- γ secretion by the addition of PD-1 antibody to Tumor Infiltrating Lymphocyte (TIL) cell culture media derived from donor A and donor B. The results show that the PD-1 antibodies of the present application can increase the secretion of IL-2, TNF-alpha and/or IFN-gamma; the PD-1 antibodies of the present application have the ability to activate immune cells.

Example 9: mixed Lymphocyte Reaction (MLR) assay to detect stimulation of T lymphocytes by PD-1 antibodies

The Mixed Lymphocyte Reaction (MLR) experiment is used for detecting that the PD-1 antibody can stimulate the T lymphocyte to secrete IL-2 and IFN-gamma. Human PBMC were adjusted to a cell density of 2E7 cells/ml and monocytes were sorted using CD14 MicroBeads, human (Miltenyi Biotec). The cells were cultured for 5 days with the addition of 50ng/ml GM-CSF (ACRO Biosystems) and 50ng/ml IL-4(ACRO Biosystems). Supplementation with 10ng/ml LPS (Sigma) and 20ng/ml TNF-. alpha. (ACRO Biosystems) induced DC cell maturation. CD4+ T cells were isolated from PBMCs of different human origin using Naive CD4+ T Cell Isolation Kit II, human (miltenyi biotec). In 96-well plates, 250. mu.1 culture medium per well contained 1.0E5 isolated T cells, 2E4 maturation-inducing DC cells, and a series of concentration gradients of pembrolizumab or PD-1 antibody 6H6 of the present application. A Human IgG1 Isotype control (Isotype control) was used as a negative control. Mixed lymphocytes at 37 ℃ with 5% CO ₂After 3 days in the cell culture chamber, 100ul of culture supernatant was removed from each well of the 96-well plate for IL-2 and IFN- γ assays.

FIGS. 6A-6B are graphs showing the results of Mixed Lymphocyte Reaction (MLR) experiments to detect the stimulation of PD-1 antibody to IL-2 and IFN-gamma secretion by T lymphocytes. The results show that the PD-1 antibodies of the present application have the ability to activate immune cells.

Example 10: PD-1 antibodies enhance the killing ability of immune cells

Whether the PD-1 antibody enhances TCR-T killing ability was verified by adding the PD-1 antibody to the NYESO-1TCR-T cell killing of A375 cells. CD3 positive T cells were sorted from freshly isolated human PBMC cells by CD3MicroBeads, human (Miltenyi Biotec 130-050-. TCR-T cells can be obtained by any transduction method, for example, a lentivirus carrying NY-ESO-1TCR (Kite Pharma) is used to transduce selected CD3 positive T cells, and the transduction efficiency in flow assay can be 40%. A375 cells per well 2E4 cells were plated on 96-well plates, 2E 4/well transduced NYESO-1TCR cells were added, 10. mu.g/ml 6H6 antibody and control antibody pembrolizumab were added simultaneously as test groups, the same amount of PBMC cells as negative control were added to A375 cells, only A375 cells as blank control, and fluorescent dye SuperView was added simultaneously to each well of all groups^TM488 Caspase-3. The killing results were statistically analyzed after transferring the 96-well plate to IncuCyte S3 for 33 hours of automatic photographing every 3 hours.

FIG. 7 is a graph showing the results of enhancing the killing ability of TCR-T cells by adding PD-1 antibody. The results show that the PD-1 antibody of the present application can enhance the killing ability of immune cells.

The foregoing detailed description is provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications of the presently recited embodiments will be apparent to those of ordinary skill in the art and are intended to be within the scope of the appended claims and their equivalents.

Claims

An isolated antigen binding protein comprising at least one CDR in the variable region VH of an antibody heavy chain, said VH comprising the amino acid sequence shown in SEQ ID NO 7 or 9.
The isolated antigen binding protein of claim 1, comprising an antibody or antigen binding fragment thereof.
The isolated antigen binding protein of any of claims 1-2, wherein said antigen binding fragment comprises a Fab, Fab ', Fv fragment, F (ab')₂scFv, di-scFv and/or dAb.
The isolated antigen binding protein of any one of claims 2-3, wherein the antibody is selected from the group consisting of: monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
The isolated antigen binding protein of any one of claims 1-4, which competes for binding to PD-1 with a reference antibody, wherein the reference antibody comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3, and a heavy chain variable region, the LCDR1 comprising the amino acid sequence set forth in SEQ ID NO 4; the LCDR2 comprises an amino acid sequence shown as SEQ ID NO. 5; the LCDR3 comprises an amino acid sequence shown in SEQ ID NO. 6, the heavy chain variable region of the reference antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises an amino acid sequence shown in SEQ ID NO. 1; the HCDR2 comprises an amino acid sequence shown as SEQ ID NO. 2; the HCDR3 comprises an amino acid sequence shown in SEQ ID NO. 3.
The isolated antigen-binding protein of any of claims 1-5, said VH comprising HCDR1, HCDR2 and HCDR3, wherein said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO 3.
The isolated antigen binding protein of claim 6, wherein the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 2.
The isolated antigen binding protein of any of claims 6-7, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO 1.
The isolated antigen binding protein of any of claims 1-8, said VH comprising HFR1, HFR2, HFR3, and HFR4, wherein said HFR1 comprises the amino acid sequence set forth in SEQ ID No. 11 or SEQ ID No. 19.
The isolated antigen binding protein of any of claim 9, wherein said HFR2 comprises the amino acid sequence set forth in SEQ ID No. 12 or SEQ ID No. 20.
The isolated antigen binding protein of any of claims 9-10, wherein said HFR3 comprises the amino acid sequence set forth in SEQ ID No. 13 or SEQ ID No. 21.
The isolated antigen binding protein of any of claims 9-11, wherein said HFR4 comprises the amino acid sequence set forth in SEQ ID No. 14 or SEQ ID No. 22.
The isolated antigen binding protein of any of claims 1-12, comprising a VH, and the VH comprises the amino acid sequence set forth in SEQ ID No. 7 or 9.
The isolated antigen binding protein of any one of claims 1-13, comprising an antibody heavy chain constant region.
The isolated antigen binding protein of claim 14, wherein the heavy chain constant region is derived from a human IgG constant region.
The isolated antigen binding protein of any one of claims 14-15, wherein the heavy chain constant region is derived from a human IgG4 constant region and/or a human IgG1 constant region.
The isolated antigen binding protein of any of claims 1-16, comprising at least one CDR in an antibody light chain variable region VL comprising the amino acid sequence set forth in SEQ ID No. 8 or 10.
The isolated antigen binding protein of claim 17, wherein the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID No. 4.
The isolated antigen binding protein of claim 18, wherein said LCDR2 comprises the amino acid sequence set forth in SEQ ID No. 5.
The isolated antigen binding protein of any of claims 18-19, wherein the LCDR3 comprises the amino acid sequence set forth in SEQ ID No. 6.
The isolated antigen binding protein of any of claims 17-20, said VL comprises LFR1, LFR2, LFR3, and LFR4, wherein said LFR1 comprises the amino acid sequence set forth in SEQ ID No. 15 or SEQ ID No. 23.
The isolated antigen binding protein of any of claim 21, wherein said LFR2 comprises the amino acid sequence set forth in SEQ ID No. 16 or SEQ ID No. 24.
The isolated antigen binding protein of any of claims 21-22, wherein said LFR3 comprises the amino acid sequence set forth in SEQ ID No. 17 or SEQ ID No. 25.
The isolated antigen binding protein of any of claims 21-23, wherein said LFR4 comprises the amino acid sequence set forth in SEQ ID No. 18 or SEQ ID No. 26.
The isolated antigen binding protein of any of claims 1-24, comprising a VL, and the VL comprises an amino acid sequence set forth in SEQ ID No. 8 or 10.
The isolated antigen binding protein of any one of claims 1-25, having one or more of the following properties:

a) (ii) is capable of binding to human PD-1;

b) can block the combination of PD-1 and PD-L1;

c) can block the combination of PD-1 and PD-L2;

d) capable of stimulating the secretion of IL-2, TNF-alpha and/or IFN-gamma in immune cells;

e) Capable of inhibiting tumor growth and/or tumor cell proliferation;

f) can improve the killing ability of immune cells.
An isolated one or more nucleic acid molecules encoding the isolated antigen binding protein of any one of claims 1-26.
A vector comprising the nucleic acid molecule of claim 27.
A cell comprising the nucleic acid molecule of claim 27 or the vector of claim 28.
A method of making the isolated antigen binding protein of any one of claims 1-26, the method comprising culturing the cell of claim 29 under conditions such that the isolated antigen binding protein of any one of claims 1-26 is expressed.
A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-26, the nucleic acid molecule of claim 27, the vector of claim 28, and/or the cell of claim 29, and optionally a pharmaceutically acceptable adjuvant.
Use of the isolated antigen binding protein of any one of claims 1-26, the nucleic acid molecule of claim 27, the vector of claim 28, the cell of claim 29, and/or the pharmaceutical composition of claim 31 in the preparation of a medicament for treating a PD-1 mediated disease or disorder.
The use of claim 32, wherein the PD-1 mediated disease or condition comprises cancer.
A method of inhibiting PD-1 binding to PD-L1, comprising administering to a subject in need thereof an effective amount of the antigen binding protein of any one of claims 1-26, the nucleic acid molecule of claim 27, the vector of claim 28, the cell of claim 29, and/or the pharmaceutical composition of claim 31.
A method of inhibiting PD-1 binding to PD-L2, comprising administering to a subject in need thereof an effective amount of the antigen binding protein of any one of claims 1-26, the nucleic acid molecule of claim 27, the vector of claim 28, the cell of claim 29, and/or the pharmaceutical composition of claim 31.
A method of preventing, ameliorating, or treating a PD-1 mediated disease or disorder comprising administering to a subject in need thereof an effective amount of the antigen binding protein of any one of claims 1-26, the nucleic acid molecule of claim 27, the vector of claim 28, the cell of claim 29, and/or the pharmaceutical composition of claim 31.
The method of claim 36, wherein the PD-1 mediated disease or disorder comprises cancer.