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CN116284406A - PD-1 binding protein and application thereof - Google Patents

PD-1 binding protein and application thereof Download PDF

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Publication number
CN116284406A
CN116284406A CN202310322301.5A CN202310322301A CN116284406A CN 116284406 A CN116284406 A CN 116284406A CN 202310322301 A CN202310322301 A CN 202310322301A CN 116284406 A CN116284406 A CN 116284406A
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seq
amino acid
antigen binding
acid sequence
binding protein
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刘雅容
李海豹
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Zhuhai Gravel Biotechnology Co ltd
Shanghai Grit Biotechnology Co Ltd
Suzhou Grit Biotechnology Co Ltd
Zhuhai Tuoyu Biotechnology Co Ltd
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Zhuhai Gravel Biotechnology Co ltd
Shanghai Grit Biotechnology Co Ltd
Suzhou Grit Biotechnology Co Ltd
Zhuhai Tuoyu Biotechnology Co Ltd
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Abstract

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

Description

PD-1 binding protein and application thereof
Technical Field
The application relates to the field of biological medicine, in particular to a PD-1 binding protein and application thereof.
Background
Programmed Death receptor 1 (pd-1) is a type I membrane protein with 288 amino acids that is expressed predominantly 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, weaken the secretion of cytokines and play an immunosuppressive role. The PD-1/PD-L1 channel inhibitor can block the combination of PD-1 and PD-L1, block negative regulation signals, enable T cells to restore activity, play a role in killing tumor cells, and further inhibit tumor growth. Therefore, the immunomodulation targeting PD-1/PD-L1 is of great significance for tumor suppression. Currently, the blocking type 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 there is still a need to continuously develop more effective anti-PD-1 antibodies and humanized antibodies thereof.
Disclosure of Invention
The present application provides an antigen binding protein that binds PD-1 that exhibits one or more desired functional properties, such as high affinity binding to PD-L1, the ability to inhibit PD-1 binding to PD-1, the ability to enhance T cell activation including proliferation, IFN- γ and/or IL-2 secretion, the ability to stimulate an antibody response, and/or the ability to reverse the inhibitory function of an immunosuppressive cell, such as a T regulatory cell. In one embodiment, the present application also provides an antibody of the scFv-huIgG1 Fc type, conveniently for use in an autocrine format in combination with a cytostatic agent. The present application also provides nucleic acid molecules encoding the isolated antigen binding proteins, expression vectors, host cells, and methods for preparing the isolated antigen binding proteins. The PD-1-binding antigen binding proteins disclosed herein can be used (alone or in combination with other active agents or therapeutic forms) to treat, prevent and/or diagnose 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 a light chain variable region comprising HCDR1, HCDR2 and HCDR3, said HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 2; the HCDR3 comprises an amino acid sequence shown in SEQ ID NO. 3; the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises an amino acid sequence shown in SEQ ID NO. 4; the LCDR2 comprises an amino acid sequence shown in SEQ ID NO. 5 (YAS); the LCDR3 comprises an amino acid sequence shown in SEQ ID NO. 6; and the heavy chain variable region of the antigen binding protein comprises at least one FR in an antibody heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID No. 67.
In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises at least one FR in an antibody heavy chain variable region VH comprising the amino acid sequence set forth in any one of SEQ ID NOs 54 to 56.
In another preferred embodiment, the light chain variable region of the antigen binding protein comprises at least one FR of an antibody light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO. 68.
In another preferred embodiment, the light chain variable region of the antigen binding protein comprises at least one FR of an antibody light chain variable region VL comprising the amino acid sequence set forth in any one of SEQ ID NOs 57 to 59.
In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR4 comprising the amino acid sequence shown in SEQ ID NO. 45.
In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR3 comprising the amino acid sequence shown in SEQ ID NO. 63.
In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR3 comprising the amino acid sequence shown in any one of SEQ ID NOs 42 to 44.
In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR2 comprising the amino acid sequence shown in SEQ ID NO. 41.
In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1 comprising the amino acid sequence shown in SEQ ID NO. 62.
In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1 comprising the amino acid sequence shown in any one of SEQ ID NOs 39 to 40.
In another preferred embodiment, the light chain variable region of the antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR4 comprising the amino acid sequence shown in SEQ ID NO. 53.
In another preferred embodiment, the light chain variable region of the antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR3 comprising the amino acid sequence shown in SEQ ID NO. 66.
In another preferred embodiment, the light chain variable region of the antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR3 comprising the amino acid sequence shown in any one of SEQ ID NOs 51 to 52.
In another preferred embodiment, the light chain variable region of the antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR2 comprising the amino acid sequence shown in SEQ ID NO. 65.
In another preferred embodiment, the light chain variable region of the antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR2 comprising the amino acid sequence shown in any one of SEQ ID NOs 49 to 50.
In another preferred embodiment, the light chain variable region of the antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR1 comprising the amino acid sequence shown in SEQ ID NO. 64.
In another preferred embodiment, the light chain variable region of the antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR1 comprising the amino acid sequence shown in any one of SEQ ID NOs 46 to 48.
In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO. 67.
In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NOs 54 to 56.
In another preferred embodiment, the light chain variable region of the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO. 68.
In another preferred embodiment, the light chain variable region of the antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NOs 57 to 59.
In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO. 67 and the light chain variable region of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO. 68.
In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO. 54 and the light chain variable region of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO. 57.
In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO. 55 and the light chain variable region of the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO. 58.
In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO. 56 and the light chain variable region of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO. 59.
In another preferred embodiment, the antigen binding protein further comprises an antibody heavy chain constant region.
In another preferred embodiment, the antigen binding protein comprises an antibody heavy chain constant region derived from a human IgG constant region.
In another preferred embodiment, the antibody heavy chain constant region of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO. 60.
In another preferred embodiment, the antigen binding protein comprises an antibody light chain constant region.
In another preferred embodiment, the antibody light chain constant region of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO. 61.
In another preferred embodiment, the antigen binding protein comprises an antibody or antigen binding fragment thereof.
In another preferred embodiment, the antibody comprises a Fab, fab ', fv fragment, F (ab') 2, scFv, di-scFv and/or dAb.
In another preferred embodiment, the antigen binding fragment is selected from the group consisting of: humanized antibodies, chimeric antibodies, and fully human antibodies.
In another preferred embodiment, the antigen binding protein has one or more of the properties selected from the group consisting of: can bind to human PD-1, can block PD-1 and PD-L1 binding, can block PD-1 and PD-L2 binding, can stimulate secretion of IL-2, TNF-alpha and/or IFN-gamma in immune cells, can inhibit tumor growth and/or tumor cell proliferation, and can increase killing capacity of immune cells.
In another aspect, the present application provides a polypeptide comprising the isolated antigen binding protein, and optionally a tag sequence that facilitates expression and/or purification.
In another aspect, the present application provides an isolated nucleic acid molecule or molecules encoding the isolated antigen binding protein and/or the polypeptide.
In another aspect, the present application provides vectors comprising the nucleic acid molecules.
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, an immunoconjugate is provided comprising said isolated antigen binding protein, and a conjugated moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
In another aspect, the application provides the use of the isolated antigen binding protein, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition and/or the immunoconjugate in the manufacture of a medicament for treating a PD-1 mediated disease or disorder.
In certain embodiments, the PD-1 mediated disease or disorder comprises a cancer or tumor.
In another aspect, the application provides the use of the isolated antigen binding protein and/or the immunoconjugate in the manufacture of a detection reagent, detection plate or detection kit for detecting PD-1 molecules in a sample.
In another aspect, the present application provides a method of detecting PD-1 protein in a sample (including non-diagnostic and diagnostic purposes), the method comprising the steps of:
(1) Contacting the sample with said isolated antigen binding protein and/or said immunoconjugate;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of PD-1 protein in the sample.
In another aspect, the present application provides a method of affecting cytokine production by a target cell for non-therapeutic and/or diagnostic purposes, the method comprising administering the antigen binding protein, the polypeptide, the nucleic acid, the vector, and/or a cell expressing the antigen binding protein, the polypeptide, or comprising the nucleic acid and/or the vector.
In another preferred embodiment, the target cells comprise immune cells, such as tumor-infiltrating lymphocytes (TILs), peripheral Blood Mononuclear Cells (PBMCs).
In another preferred embodiment, the cytokine comprises IL-2, TNF- α and/or IFN- γ.
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, alleviating or treating a PD-1 mediated disease or condition 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 a cancer or tumor.
In another preferred embodiment, the cancer or tumor comprises a solid tumor or a hematological tumor.
Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the invention as described herein. Accordingly, the drawings and descriptions herein are to be regarded as illustrative in nature and not as restrictive.
Drawings
The specific features of the invention related to this application are set forth in the appended claims. The features and advantages of the invention that are related to the present application will be better understood by reference to the exemplary embodiments and the drawings that are described in detail below. The drawings are briefly described as follows:
FIG. 1 shows the detection curves of the affinity of tetravalent human PD-L1 extracellular fragment mutants described in the present application to PD-1-huIgG1 Fc (based on BLI method);
FIG. 2 shows the inhibition curves of binding of PD-1 and PD-L1 by huIgG1 antibodies to 6H6 and pembrolizumab;
FIG. 3 shows the binding curves of huIgG1 antibodies to PD-1 for 6H6 and pembrolizumab.
FIG. 4 shows a graph of the measured CD107a cell fraction following addition of PD-1 antibodies to tumor-infiltrating lymphocyte (TIL) cell culture media derived from donor A and donor B.
FIG. 5 shows graphs of cytokine secretion results measured after addition of PD-1 antibodies to tumor-infiltrating lymphocytes (TIL) cell culture media derived from donor A and donor B. Wherein A-C shows that tumor-infiltrating lymphocytes derived from donor A secrete cytokines; D-F shows that tumor-infiltrating lymphocytes derived from donor B secrete cytokines.
FIG. 6 shows a graph of the results of Mixed Lymphocyte Reaction (MLR) experiments to detect stimulation of PD-1 antibodies against T lymphocytes.
FIG. 7 is a graph showing the result of enhancing the killing ability of TCR-T cells by the addition of PD-1 antibody.
FIG. 8 shows the results of antigen binding affinity of the 6H6 antibody.
FIG. 9 shows the results of antigen binding affinity of the 6H6-5 antibody.
FIG. 10 shows the results of antigen binding affinity of the 6H6-25 antibody.
FIG. 11 shows the results of antigen binding affinity of the 6H6-29 antibody.
FIG. 12 shows half maximal inhibitory concentration IC of 6H6 and humanized 6H6-5, 6H6-25, 6H6-29 huIgG1 antibodies on PD-1 and PD-L1 binding 50 。
Detailed Description
Further advantages and effects of the invention of the present application will become apparent to those skilled in the art from the disclosure of the present application, from the following description of specific embodiments.
Definition of terms
In the present application, the term "PD-1" generally refers to programmed cell death 1, also referred to as "programmed death 1", "CD279", "cluster 279", "PD1", "PDCD1". 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 can bind to two ligands, PD-L1 and PD-L2. The term "PD-1" includes any natural 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, and any form of PD-1 produced by cellular processing. PD-1 may be present as a transmembrane protein or as a soluble protein. "PD-1" includes intact PD-1 and fragments thereof, and also functional variants, isoforms, species homologs, derivatives, analogs of PD-1, and analogs having at least one epitope in common 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" generally refers to the programmed cell death 1 ligand 1, which may also be referred to as B7 homolog 1, B7-H1, cluster of differentiation 274, (3) 274 or CD274, which down-regulates T-cell activation and cytokine secretion upon binding to PD-1. "PD-L1" includes any natural PD-L1 of any vertebrate origin, including mammals, such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). The term encompasses "full length", unprocessed PD-L1, as well as any form of PD-L1 produced by cellular processing. PD-L1 may be present as a transmembrane protein or as a soluble protein. "PD-L1" includes intact PD-L1 and fragments thereof, and also functional variants, isoforms, species homologs, derivatives, analogs of PD-L1, and analogs having at least one epitope in common with PD-L1. The basic structure of PD-L1 comprises 4 domains: extracellular Ig-like V-type domains and Ig-like C2-type domains, transmembrane domains, and cytoplasmic domains. Complete hPD-L1 sequences can be found under GenBank accession number Q9 NZQ.
In this application, the term "isolated" or "purified" generally refers to a molecule (e.g., antibody, nucleic acid, etc.) that is at least partially separated from other molecules to which it is normally bound in its natural 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 in its natural state is typically flanked by polynucleotides. Thus, polynucleotides fused to regulatory or coding sequences to which they normally do not bind are considered isolated in this application, for example, due to recombinant techniques. Such molecules are considered isolated even when present, for example, in the chromosome of a host cell or in a nucleic acid solution. In general, the terms "isolated" and "purified" are not intended to refer to the complete absence of such materials or the absence of water, buffers or salts unless they are present in amounts that interfere in the experimental or therapeutic use of the molecule in large amounts. The antigen binding proteins of the present application and nucleic acids encoding the antigen binding proteins of the present application are isolated/purified.
In the present application, the term "antigen binding protein" is used in its broad sense and means a protein comprising a portion that binds to an antigen or target and optionally 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 the antigen. Examples of antigen binding proteins include human antibodies, humanized anti-antibodiesA body; a chimeric antibody; a recombinant antibody; a single chain antibody; single domain antibodies (or referred to as nanobodies); a bifunctional antibody; a trifunctional antibody; a four-functional antibody; fab fragments; f (ab') 2 Fragments; igD antibodies; igE antibodies; igM antibodies; 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, for example, to 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 mimics ("PAMs") may be used, as well as frameworks based on antibody mimics that utilize fibronectin components as frameworks.
In this application, the term "antibody" is used in its 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), or antigen-binding fragments thereof, that are interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins, i.e., igM, igD, igG, igA and IgE, with their respective heavy chains being the μ, δ, γ, α, epsilon chains, respectively. The Ig of the same class can be further classified into different subclasses according to the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chain, for example, igG can be classified into IgG1, igG2, igG3 and IgG4. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. Each class Ig of the five classes of Igs may have either a kappa chain or a lambda chain.
In certain naturally occurring IgG, igD, and IgA antibodies, the heavy chain constant region comprises three domains, CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region comprises one domain, CL. VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), alternating with regions of greater conservation termed Framework Regions (FR). Each VH and VL comprises three CDRs and four Framework Regions (FR), arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable domains of the natural heavy and light chains each comprise four FR regions (HFR 1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR 4), mostly in a β -sheet configuration, connected by three CDRs, forming a loop connection, and in some cases forming part of a β -sheet structure. The CDRs in each chain are in close proximity by the FR region and form together with the CDRs from the other chain an antigen binding site of the antibody. The constant region of an antibody may mediate the binding of an 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 parts of the sequence of the variable domain of an antibody vary strongly, which results in the binding and specificity of various specific antibodies for their specific antigens. However, variability is not evenly distributed throughout the variable regions of antibodies. It concentrates in three segments in the light and heavy chain variable regions, known as Complementarity Determining Regions (CDRs) or hypervariable regions (HVRs). The more highly conserved parts in the variable domain are called Frameworks (FR). The variable domains of the natural heavy and light chains each comprise four FR regions, mostly in a β -sheet configuration, connected by three CDRs, forming a loop connection, and in some cases forming part of a β -sheet structure. The CDRs in each chain are in close proximity by the FR regions and together with the CDRs from the other chain form the antigen binding site of the antibody, and the constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity. In the art, CDRs of antibodies can be defined by a variety of methods, for example, kabat definition rules based on sequence variability (see, kabat et al, immunological protein sequences, fifth edition, national institutes of health, bescens da, maryland (1991). In this application, amino acid residues in variable domain sequences and full length antibody sequences are determined using rules comprising Kabat definition (see table 1).
TABLE 1 CDR definitions of antibodies of the present application based on 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 may refer to the amino acid sequence from aa to bb starting from the N-terminus of the antibody light chain; haa-Hbb may refer to the amino acid sequence from aa to bb starting from the N-terminus of the heavy chain of the antibody. For example, L24-L34 may refer to the amino acid sequence from position 24 to position 34 starting from the N-terminus of the light chain of the antibody; 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 this application, the term "Fab" refers to an antigen-binding fragment of an antibody. As described above, papain can be used to digest intact antibodies. The antibodies, after digestion with papain, produce two identical antigen-binding fragments, a "Fab" fragment, and a residual "Fc" fragment (i.e., fc region, supra). Fab fragment consists of a complete L chain and a heavy chain variable region and the H chain (V H ) Is a constant region (C) H 1) Composition is prepared.
In this application, the term "Fab' fragment" refers to a monovalent antigen binding fragment of a human monoclonal antibody that 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 a heavy chain. For example, a Fab' fragment can 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 held together by disulfide bonds and a partial hinge region. F (ab') 2 fragments have divalent antigen binding activity and are capable of cross-linking 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 and light chain variable regions, and lacking the heavy and light chain constant regions. 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 variable region antibody fragment comprising a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light chain 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 specifically stated otherwise, as used herein, an scFv may have the VL and VH variable regions described in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), an scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.
In this application, the term "dAb" generally refers to an antigen binding fragment having a VH domain, a VL domain or having a VH domain or a VL domain, reference is made, for example, to Ward et al (Nature, 1989Oct 12;341 (6242): 544-6), reference is made to Holt et al, trends Biotechnol.,2003,21 (11): 484-490; and other published patent applications such as WO 06/030220, WO 06/003388 and Domantis 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 the small number of natural mutations that may be present. Monoclonal antibodies are generally 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 without contamination by other immunoglobulins. The modifier "monoclonal" refers to the characteristics of the antibody as 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 as used herein may be prepared in hybridoma cells or may be prepared by recombinant DNA methods.
In this application, the term "chimeric antibody" generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species. Typically, the variable 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 the 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, provided that 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. A "humanized" form of a non-human (e.g., murine) antibody may minimally comprise chimeric antibodies derived from sequences of non-human immunoglobulins. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) may be replaced with CDR region residues of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired properties, affinity and/or capability. In some cases, the FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, the humanized antibody may comprise amino acid modifications that are not 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 this application, the term "reference antibody" generally refers to any antibody that can bind to an antigen (e.g., PD-1). In certain instances, the antigen binding proteins described herein can compete with a reference antibody for binding to an antigen (e.g., PD-1).
In this application, the term "isolated nucleic acid molecule" or "isolated polynucleotide" generally refers to DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, that is not associated with all or a portion of a polynucleotide found in nature, or that 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 mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having a plurality of functions as described above. The vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell comprising the vector.
In the present application, the term "cell" generally refers to an individual cell, cell line or cell culture that may or has contained a plasmid or vector comprising a nucleic acid molecule as described herein, or that is capable of expressing an antibody or antigen binding fragment thereof as described herein. The cell may comprise progeny of a single host cell. The daughter cells may not necessarily be identical in morphology or in genome to the original parent cells due to natural, unexpected or deliberate mutation, but are capable of expressing the antibodies or antigen-binding fragments thereof described herein. The cells may be obtained by transfecting the cells in vitro using the vectors described herein. The cells may be prokaryotic cells (e.g., E.coli) or eukaryotic cells (e.g., yeast cells, e.g., COS cells, chinese Hamster Ovary (CHO) cells, heLa cells, HEK293 cells, COS-1 cells, NS0 cells, or myeloma cells). 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 is introduced. The recombinant host cell includes not only a particular cell but also the progeny of such a cell.
In this 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, chelators, sugar alcohols, salt-forming counter ions, such as sodium; and/or nonionic surfactants.
In the present application, reference to proteins, polypeptides and/or amino acid sequences is also to be understood as comprising at least the following ranges: variants or homologues having the same or similar function as the protein or polypeptide.
In the present application, the variant may be a protein or polypeptide in which one or more amino acids have been substituted, deleted or added in 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). For example, the functional variant may comprise a protein or polypeptide that has been altered in amino acids by at least 1, such as 1-30, 1-20, or 1-10, and yet another such as 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 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 a PD-1 protein).
In this application, homology generally refers to similarity, similarity or association between two or more sequences. "percent 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 where 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, gln, cys and Met) is present is determined to give the number of matched positions, the number of matched positions is divided by the total number of positions in the comparison window (i.e., window size), and the result is multiplied by 100 to produce the percent 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 needed to achieve maximum alignment over the full length sequence being compared or over the region of the target sequence. The homology can also be determined by the following method: FASTA and BLAST. For a description of FASTA algorithm, see w.r.pearson and d.j.lipman, "improved tools for biological sequence comparison", proc.Natl. Acad.Sci., U.S. Proc., 85:2444-2448, 1988; "quick sensitive protein similarity search" by d.j.lipman and w.r.pearson, science,227:1435-1441, 1989. For a description of the BLAST algorithm, see "a basic local contrast (alignment) search tool", journal of molecular biology, 215:403-410, 1990.
In this application, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur.
In this application, the terms "comprises," "comprising," and "includes" are used in their plain, inclusive, and open-ended meaning. In some cases, the meaning of "as", "consisting of … …" is also indicated.
In this 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 this application, the term "therapeutically effective amount" refers to an amount of antibody that, when administered to a human or animal, elicits a response sufficient to produce a therapeutic effect in the human or animal. The effective amount is readily determined by one of ordinary skill in the art following routine methods.
Detailed Description
Antigen binding proteins
In one aspect, the present application provides an antigen binding protein that may comprise at least one CDR in an antibody heavy chain variable region VH that comprises the 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 may be monoclonal, chimeric, humanized and/or fully human. The antigen binding fragments of the antibodies described herein may be Fab, fab ', fv fragments, F (ab') 2 scFv, di-scFv and/or dAb.
The antigen binding proteins described herein can compete with the reference antibody for binding to PD-1. The reference antibody may comprise a light chain variable region and a heavy chain variable region. For example, the light chain variable region of the reference antibody comprises LCDR1, LCDR2 and LCDR3, said LCDR1 comprising the amino acid sequence of SEQ ID No. 4 and said LCDR2 comprising the amino acid sequence of SEQ ID No. 5 (YAS); the LCDR3 comprises an amino acid sequence shown in SEQ ID NO. 6; and the heavy chain variable region of the reference antibody comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence of SEQ ID No. 1; the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 2, and the HCDR3 comprises an amino acid sequence shown in SEQ ID NO. 3.
The antigen binding proteins described herein may comprise heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3.
In this application, the HCDR3 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO. 3.
In this application, the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 2.
In the present application, the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 1.
In the present application, 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 sequence.
The antigen binding proteins described herein may further comprise heavy chain framework regions HFR1, HFR2, HFR3 and HFR4.
In the present application, the HFR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 11 or SEQ ID NO. 19, and the C-terminus of the HFR1 is directly or indirectly linked to the N-terminus of the HCDR 1.
In this application, HFR2 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 12 or SEQ ID NO. 20, and the HFR2 is located between the HCDR1 and the HCDR 2.
In this application, HFR3 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 13 or SEQ ID NO. 21, and the HFR3 is located between the HCDR2 and the HCDR 3.
In this application, HFR4 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 14 or SEQ ID NO. 22, and the N-terminus of HFR4 is linked to the C-terminus of HCDR 3.
In this 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 include the amino acid sequences shown in SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, respectively.
For example, 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, and 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 that order.
In some embodiments, HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein, respectively, can include the amino acid sequences shown in SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21 and SEQ ID NO. 22, respectively.
For example, 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, and 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 that order.
The antigen binding proteins described herein may comprise a heavy chain constant region.
In this application, the heavy chain constant region may comprise a constant region of human IgG. In certain instances, the heavy chain constant region can comprise a human IgG4 constant region and/or a heavy chain constant region of human IgG 1.
In some embodiments, the heavy chain constant region may comprise the amino acid sequence set forth in any one of SEQ ID NO. 27 or SEQ ID NO. 28.
For example, HCDR1, HCDR2, and HCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, respectively, 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, 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, and 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 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, 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, and 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 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.
The antigen binding proteins described herein may comprise at least one CDR in the antibody light chain variable region VL 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 sequence.
In some embodiments, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein can comprise the amino acid sequences of SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively, and the antigen binding protein can comprise at least one CDR in an antibody light chain variable region VL comprising the amino acid sequence of SEQ ID No. 8.
In some embodiments, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein can comprise the amino acid sequences of SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively, and the antigen binding protein can comprise at least one CDR in an antibody light chain variable region VL comprising the amino acid sequence of SEQ ID No. 16.
The antigen binding proteins described herein may comprise light chain complementarity determining regions LCDR1, LCDR2 and LCDR3.
In this application, LCDR1 of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO. 4.
In this application, LCDR2 of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO. 5 (YAS).
In this application, 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 (YAS) and SEQ ID NO. 6, respectively, in sequence.
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 shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 (YAS) and SEQ ID NO:6, respectively.
In some embodiments, 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 (YAS), and SEQ ID NO. 6, respectively, and the VH of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 7.
In some embodiments, 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 (YAS), and SEQ ID NO. 6, respectively, and the VH of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 9.
In some embodiments, LCDR1, LCDR2, and LCDR3 of the antigen binding protein can each comprise the amino acid sequence of SEQ ID No. 4, SEQ ID No. 5 (YAS), and SEQ ID No. 6, respectively, and the VH of the antigen binding protein can comprise the amino acid sequence of 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 of any one of SEQ ID No. 27 or SEQ ID No. 28.
In some embodiments, LCDR1, LCDR2, and LCDR3 of the antigen binding protein can each comprise the amino acid sequence of SEQ ID No. 4, SEQ ID No. 5 (YAS), and SEQ ID No. 6, respectively, and the VH of the antigen binding protein can comprise the amino acid sequence of 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 of any one of SEQ ID No. 27 or SEQ ID No. 28.
The antigen binding proteins described herein may comprise light chain framework regions LFR1, LFR2, LFR3 and LFR4.
In this application, LFR1 of said antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 15 or SEQ ID NO. 23, and the C-terminus of said LFR1 is directly or indirectly linked to the N-terminus of said LCDR 1.
In this application, LFR2 of said antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 16 or SEQ ID NO. 24, and said LFR2 is located between said LCDR1 and said LCDR 2.
In this application, LFR3 of said antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 17 or SEQ ID NO. 25, and said LFR3 is located between said LCDR2 and said LCDR 3.
In this application, LFR4 of said antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 18 or SEQ ID NO. 26, and the N-terminus of said LFR4 is linked to the C-terminus of said LCDR 3.
In the present application, the antigen binding protein may comprise VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO. 8 or 10.
In some embodiments, LFR1, LFR2, LFR3 and LFR4 of said 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, 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 (YAS), and SEQ ID NO. 6, respectively, 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, respectively.
For example, 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, and 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 (YAS), and SEQ ID NO. 6, respectively, 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, respectively.
In some embodiments, LFR1, LFR2, LFR3 and LFR4 of said 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, 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 (YAS), and SEQ ID NO. 6, respectively, 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, respectively.
For example, 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, and 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 (YAS), and SEQ ID NO. 6, respectively, 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, respectively.
In this application, the antigen binding protein may comprise a VH and a VL, the VH may comprise the amino acid sequence shown in SEQ ID No. 7 or 15, and the VL may comprise the amino acid sequence shown in SEQ ID No. 8 or 10.
In some embodiments, the VH may comprise the amino acid sequence shown in SEQ ID NO. 7, and the VL may comprise the amino acid sequence shown in SEQ ID NO. 8.
For example, 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, and 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 (YAS), and SEQ ID NO:6, respectively, and 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 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.
In some embodiments, the VH may comprise the amino acid sequence shown in SEQ ID NO. 9, and the VL may comprise the amino acid sequence shown in SEQ ID NO. 10.
For example, 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, and 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 (YAS), and SEQ ID NO. 6, respectively, and 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. LFR1, LFR2, LFR3 and LFR4 of said antigen binding protein may comprise in sequence the amino acid sequences shown as 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 the amino acid sequence shown in any one of SEQ ID NO. 27 or SEQ ID NO. 28.
In some embodiments, the VH may comprise the amino acid sequence shown in SEQ ID NO. 7 and the VL may comprise the amino acid sequence shown in SEQ ID NO. 8, and the heavy chain constant region of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO. 27 or SEQ ID NO. 28.
For example, HCDR1, HCDR2, and HCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and 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 (YAS), and SEQ ID NO:6, respectively, and 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 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 VH may comprise the amino acid sequence shown in SEQ ID NO:7, and the antigen binding protein may comprise the constant amino acid sequence shown in SEQ ID NO:8, and the antigen binding protein may comprise either the amino acid sequence shown in VL chain of SEQ ID NO: 28.
In some embodiments, the VH may comprise the amino acid sequence shown in SEQ ID NO. 9, and the VL may comprise the amino acid sequence shown in SEQ ID NO. 10.
For example, 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, and 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 (YAS), and SEQ ID NO:6, respectively, and 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 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, and the VH may comprise the amino acid sequence shown in SEQ ID NO:15, and the constant chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:16, and either the amino acid sequence shown in SEQ ID NO: 28.
In another aspect, the present application provides an isolated antigen binding protein comprising a light chain variable region comprising HCDR1, HCDR2 and HCDR3, said HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 2; the HCDR3 comprises an amino acid sequence shown in SEQ ID NO. 3; the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises an amino acid sequence shown in SEQ ID NO. 4; the LCDR2 comprises an amino acid sequence shown in SEQ ID NO. 5 (YAS); the LCDR3 comprises an amino acid sequence shown in SEQ ID NO. 6.
For example, the heavy chain variable region of the antigen binding protein of the present application comprises at least one FR in an antibody heavy chain variable region VH comprising the amino acid sequence set forth in any one of SEQ ID NOs 54 to 56.
For example, the antigen binding proteins of the present application comprise at least one FR in the antibody heavy chain variable region VH shown in SEQ ID NO. 67. For example, the antigen binding protein of the present application comprises at least one FR in the antibody heavy chain variable region VH as set forth in any one of SEQ ID NOs 54 to 56. For example, the antigen binding proteins of the present application comprise 1, 2, 3 or 4 FR in the VH described above. For example, the antigen binding proteins of the present application comprise 4H-FRs in the VH described above. For example, the antigen binding proteins of the present application comprise H-FRs 1 to 4 of the same or different VHs described above.
Further, the light chain variable region of the antigen binding protein of the present application comprises at least one FR in the antibody light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO. 68. For example, the light chain variable region of the antigen binding protein of the present application comprises at least one FR in an antibody light chain variable region VL comprising the amino acid sequence set forth in any one of SEQ ID NOs 57 to 59.
For example, the antigen binding proteins of the present application comprise at least one FR in the variable region VL of the light chain of the antibody shown in SEQ ID NO. 68. For example, the antigen binding proteins of the present application comprise at least one FR of the antibody light chain variable region VL as set forth in any one of SEQ ID NOS: 57 to 59. For example, the antigen binding proteins of the present application comprise 1, 2, 3 or 4 FR of the VL described above. For example, the antigen binding proteins of the present application comprise the 4L-FRs of VL described above. For example, the antigen binding proteins of the present application comprise L-FRs 1 through 4 of the same or different VL as described above.
For example, the antigen binding proteins of the present application comprise at least one FR in the antibody heavy chain variable region VH shown in SEQ ID NO. 67 and at least one FR in the antibody light chain variable region VL shown in SEQ ID NO. 68. For example, the antigen binding protein of the present application comprises at least one FR of the antibody heavy chain variable region VH as set forth in any one of SEQ ID NOS: 54 to 56 and comprises at least one FR of the antibody light chain variable region VL as set forth in any one of SEQ ID NOS: 57 to 59.
For example, the heavy chain variable region of the antigen binding proteins of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 45.
For example, H-FR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO. 63.
For example, H-FR3 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOS: 42 to 44.
For example, H-FR2 of the present application may comprise the amino acid sequence shown in SEQ ID NO. 41.
For example, H-FR1 of the present application may comprise the amino acid sequence shown as SEQ ID NO. 62.
For example, H-FR1 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOS: 39 to 40.
For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 45, said H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 63, said H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 41 and said H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 62.
For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 45, said H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO. 42 to 44, said H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 41, and said H-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO. 39 to 40.
For example, the heavy chain variable region of the antigen binding proteins of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 45, said H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 42, said H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 41, and said H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 39.
For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 45, said H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 43, said H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 41 and said H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 40.
For example, the heavy chain variable region of the antigen binding proteins of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 45, said H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 44, said H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 41 and said H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 40.
For example, the light chain variable region of the antigen binding proteins of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, and said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 53.
For example, L-FR3 of the present application may comprise the amino acid sequence shown as SEQ ID NO. 66.
For example, L-FR3 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOs 51 to 52.
For example, L-FR2 of the present application may comprise the amino acid sequence shown as SEQ ID NO. 65.
For example, L-FR2 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOs 49 to 50.
For example, L-FR1 of the present application may comprise the amino acid sequence shown as SEQ ID NO. 64.
For example, L-FR1 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOS: 46 to 48.
For example, the light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 53, said L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 66, said L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 65, and said L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 64.
For example, the light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 53, said L-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO. 51 to 52, said L-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NO. 49 to 50, and said L-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO. 46 to 48.
For example, the light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 53, said L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 51, said L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 49, and said L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 46.
The light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:53, said L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:52, said L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:50, and said L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 47.
The light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 53, said L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 52, said L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 40, and said L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 48.
For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 45, said H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 63, said H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 41 and said H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 62; and the light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 53, said L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 66, said L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 65, and said L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 64.
For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 45, said H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO. 42 to 44, said H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 41, and said H-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO. 39 to 40; and the light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 53, said L-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO. 51 to 52, said L-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NO. 49 to 50, and said L-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO. 46 to 48.
For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 45, said H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 42, said H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 41, and said H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 39; and the light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 53, said L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 51, said L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 49, and said L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 46.
For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 45, said H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 43, said H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 41 and said H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 40; and the light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:53, L-FR3 of the present application may comprise the amino acid sequence shown in SEQ ID NO:52, said L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:50, and said L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 47.
For example, the heavy chain variable region of the antigen binding protein of the present application may comprise H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 45, said H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 44, said H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 41 and said H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 40; and the light chain variable region of the antigen binding protein of the present application may comprise L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 53, said L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 52, said L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 40, and said L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 48.
For example, the heavy chain variable region of the antigen binding proteins of the present application comprises the amino acid sequence shown as SEQ ID NO. 67. For example, the heavy chain variable region of the antigen binding proteins of the present application comprises the amino acid sequence set forth in any one of SEQ ID NOs 54 to 56.
For example, the light chain variable region of the antigen binding proteins of the present application comprises the amino acid sequence shown as SEQ ID NO. 68. For example, the light chain variable region of the antigen binding proteins of the present application comprises the amino acid sequence set forth in any one of SEQ ID NOs 57 to 59.
For example, the heavy chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO. 67, and the light chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO. 68. For example, the heavy chain variable region of the antigen binding protein of the present application comprises the amino acid sequence set forth in any one of SEQ ID NOS: 54 to 56, and the light chain variable region of the antigen binding protein of the present application comprises the amino acid sequence set forth in any one of SEQ ID NOS: 57 to 59.
For example, the heavy chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO. 54, and the light chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO. 57.
For example, the heavy chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO. 55, and the light chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO. 58.
For example, the heavy chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO. 56, and the light chain variable region of the antigen binding protein of the present application comprises the amino acid sequence shown in SEQ ID NO. 59.
Still further, the antigen binding proteins of the present application include an antibody heavy chain constant region. For example, the antigen binding proteins of the present application include antibody heavy chain constant regions derived from human IgG constant regions. For example, the antibody heavy chain constant region of the antigen binding proteins of the present application comprises the amino acid sequence shown as SEQ ID NO. 60.
Still further, the antigen binding proteins of the present application include antibody light chain constant regions. For example, the antibody light chain constant region of the antigen binding proteins of the present application comprises the amino acid sequence shown as SEQ ID NO. 61.
For example, the antigen binding proteins of the present application include antibodies or antigen binding fragments thereof. For example, antibodies of the present application include Fab, fab ', fv fragments, F (ab') 2, scFv, di-scFv and/or dAb. For example, the antigen binding fragments of the present application are selected from the group consisting of: humanized antibodies and fully human antibodies.
The physical/chemical properties and/or biological activity 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 blot), 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 (e.g., a PD-1 antibody) that "specifically binds" to a PD-1 antigen may typically bind to PD-1, but not to other proteins lacking the PD-1 sequence. The antigen binding proteins described herein (e.g., PD-1 antibodies) are capable of specifically binding to PD-1 antigen or a labeled form thereof (e.g., a fluorescently labeled PD-1 antigen), but do not bind 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 biological membrane interference (BLI).
The antigen binding proteins described herein may bind to human PD-1 protein. In certain instances, the antigen binding proteins described herein can also cross-react with PD-1 of a monkey (e.g., cynomolgus monkey). For example, by flow assay techniques and enzyme-linked immunosorbent assay. 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 the antigen binding proteins described herein to PD-1 can be detected using an enzyme-linked immunoassay. For example, in ELISA, the human PD-1 antigen protein is used, and the EC50 value of the PD-1 antigen binding protein and PD-1 may be between about 0.0001nM and about 100nM, for example, may be between about 0.001nM and about 10nM, may be between about 0.001nM and about 5nM, may be between about 0.001nM and about 1nM, or may 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 and or PD-L2. In some cases, the antigen binding protein blocks the binding enzyme-linked immunosorbent ELISA assay of PD-1 with PD-L1 and/or PD-L2. For example, PD-L1 and/or PD-L2 antigen proteins are first coated on a plate, and a reduced amount of unlabeled said antigen binding protein and biotin-labeled PD-1 protein are mixed and incubated together. The cells were then analyzed using ELISA to confirm that the antigen binding proteins could block the binding of PD-1 and PD-L1 and or PD-L2.
The antigen binding proteins described herein are capable of stimulating the proportion of immune cells expressing CD07a cells. The antigen binding proteins described herein are capable of stimulating 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., CD3 antibody, CD28 antibody, or nanomaterial TransAct comprising CD3 antibody and CD28 antibody TM ) On the basis of activation, the effect on immune cell activation 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 cells may comprise artificially modified immune cells. For example, the immune cells may comprise TCR-T cells. Secretion of cytokines in immune cells can be determined by any method known to those skilled in the art, for example, by quantitative measurement of immune cell (e.g., T cell) proliferation or cytokine production by immune cells (e.g., IFN- γ or IL-2 produced by T cells) by enzyme-linked immunosorbent assay (ELISA). For example, the stimulation of T lymphocytes by PD-1 antibodies can be detected by Mixed Lymphocyte Reaction (MLR) experiments.
Nucleic acid molecules, vectors and cells
In another aspect, the present application provides one or more nucleic acid molecules that may encode an isolated antigen binding protein described herein. The nucleic acid molecules described herein may be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, e.g. by Polymerase Chain Reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified, e.g. fractionated by cleavage and gel electrophoresis, 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 that may comprise a nucleic acid molecule as 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 also contain expression control elements that allow for 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 which regulate gene transcription or mRNA translation, and the like. 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, which may comprise a nucleic acid molecule as described herein or a vector as described herein. In certain embodiments, each or each host cell may comprise one or more 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 may be introduced into such host cells, e.g., eukaryotic cells, such as cells from plants, fungal or yeast cells, 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 that may comprise an antigen binding protein described herein and/or the nucleic acid molecule, the vector, the host cell, and optionally a pharmaceutically acceptable adjuvant. The pharmaceutically acceptable adjuvants are non-toxic to the recipient 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 active compounds having complementary activities that do not adversely affect each other. The type and effective amount of such drugs depends, for example, on the amount and type of antagonist present in the formulation, as well as on 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 a dose required to be able to prevent and/or treat (at least partially treat) a disease or disorder and/or any complications 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, such methods are known to those of ordinary skill in the art by using an appropriate medium, an appropriate temperature, an appropriate incubation time, and the like.
Any method suitable for producing monoclonal antibodies may be used to produce the antigen binding proteins of the present application. For example, animals may be immunized with a linked or naturally occurring PD-1 protein or fragment thereof. Suitable immunization methods may be used, 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 generating non-human antibodies specific for PD-1, and screening the antibodies for biological activity. The priming immunogen may be full length mature human PD-1, including natural homodimers, or a peptide containing single/multiple epitopes. The immunogens may be used alone or in combination with one or more immunogenicity enhancing agents known in the art.
The humanized antibody may be selected from any class of immunoglobulins, including IgM, igD, igG, igA and IgE. In this application, the antibody is an IgG antibody, and an IgG1 subtype is used. Optimization of the necessary constant domain sequences to produce the desired biological activity can be achieved by screening antibodies using the biological assays described in the examples below. Also, any type of light chain may 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 may be obtained by conventional techniques, such as by PCR amplification or genomic library screening. In addition, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very 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 a variety of existing DNA molecules (or vectors, for example) and cells known in the art.
The present application also relates to vectors comprising the above-described suitable nucleic acid molecules 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 recombinant DNA may be performed using techniques well known in the art. The resulting transformants may be cultured by conventional methods, and the transformants express the polypeptide encoded by the nucleic acid molecules of the present application. Depending on the host cell used, it is cultivated in conventional medium under suitable conditions. Typically, the resulting host cells are cultured under conditions suitable for expression of the antigen binding proteins of the present application. The antigen binding proteins of the present application are 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 using immunoprecipitation or in vitro binding assays, such as flow cell sorting (FACS) or enzyme-linked immunosorbent assay (ELISA).
Method and use
In another aspect, the present application provides the use of the antigen binding protein in the manufacture of a medicament. The antigen binding proteins 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 conditions, e.g., 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 PD-1 mediated diseases or conditions. The prevention or treatment of a disease or disorder may refer to inhibiting or slowing the progression or progression of the disease or disorder. For example, can be used to inhibit the development or progression of a tumor. For example, tumor growth or tumor cell proliferation can be inhibited.
In another aspect, the present application provides said antigen binding proteins for use in the prevention or treatment of PD-1 mediated diseases or conditions.
In another aspect, the present application provides a method of inhibiting the binding of PD-1 to PD-L1 and/or PD-L2 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 and/or PD-L2 described herein under conditions that allow the antigen binding protein and/or PD-1 to bind to PD-L1 and/or PD-L2, detecting whether a complex is formed between the antigen binding protein and PD-1, and detecting whether a complex is formed between PD-1 and PD-L1 and/or PD-L2.
In another aspect, the present application provides a method of preventing, alleviating or treating a PD-1 mediated disease or condition comprising administering an antibody or antigen binding fragment thereof, the molecular nucleic acid, the vector, the host cell and/or the pharmaceutical composition described herein to a subject in need thereof. For example, can be used to inhibit the development or progression of a tumor. For example, tumor growth or tumor cell proliferation can be inhibited.
Without intending to be limited by any theory, the following examples are meant to illustrate the proteins, methods of preparation, uses, etc. of the present application and are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Examples
EXAMPLE 1 construction of expression vector for recombinant fusion proteins of amino acid sequences 25 to 167 of human apoptosis receptor 1 (PD-1) with human IgG1 Fc region (PD-1-huIgG 1 Fc) and eukaryotic expression
Synthesis of Gene sequence of the region between amino acids 25 and 167 of PD-1 and construction of expression vector of PD-1-huIgG1 Fc fusion protein
The gene sequence (SEQ ID NO: 29) encoding the amino acid sequence shown in SEQ ID NO:30 was synthesized by chemical synthesis from isoleucine 25 to glutamine 167 of programmed death receptor 1 (PD-1) (NCBI access No. NP-005009.2). The gene sequence from proline at position 100 to lysine amino acid at position 330 of the constant region of the heavy chain of the human IgG1 is synthesized by a chemical synthesis mode. The upstream primer containing the mouse Igkv3-10 signal peptide gene sequence is synthesized chemically and used for constructing expression vector. The PD-1 gene fragment was spliced with the human IgG1 Fc gene fragment by molecular cloning. The splice products were cloned into pcdna3.1 (Thermo) using TaKaRa seamless cloning kit.
2. Expression and purification of recombinant PD-1-huIgG1 Fc fusion proteins
After transfection of 293T cells (ATCC) with this expression vector for 5 days, the culture supernatant was collected and the recombinant PD-1-huIgG1 Fc fusion protein was purified using AKTAExplorer 100 (GE). Due to glycosylation modification and the like, the recombinant PD-1-huIgG1 Fc fusion protein is subjected to reducing SDS-PAGE electrophoresis and then is stained by Coomassie brilliant blue to show that the recombinant PD-1-huIgG1 Fc fusion protein has a size of about 60 kDalton.
Example 2 construction of expression vector for recombinant human apoptosis receptor 1 (PD-1) amino acid sequence 25 to 167 and polyhistidine tag fusion protein (PD-1-his) and eukaryotic expression
Synthesis of Gene sequence of the region between amino acids 25 and 167 of PD-1 and construction of expression vector of Polyhistidine tag fusion protein
The gene sequence of the isoleucine 25 to 167 glutamine regions of programmed death receptor 1 (PD-1) (NCBI access No. NP-005009.2) was synthesized by chemical synthesis. The upstream primer containing the mouse Igkv3-10 signal peptide gene sequence and the downstream primer containing the polyhistidine tag (having the amino acid sequence shown in SEQ ID NO: 32) gene sequence (SEQ ID NO: 31) were chemically synthesized for constructing the pCDNA3.1 vector expressing PD-1-his.
2. Expression and purification of recombinant PD-1-his proteins
After transfection of 293T cells (ATCC) with this expression vector for 5 days, the culture supernatant was collected and the recombinant PD-1-his protein was purified by AKTAExplorer 100 (GE). Recombinant PD-1-his proteins were visualized by Coomassie blue staining after reducing SDS-PAGE for glycosylation modification and the like to a size of about 40 kDalton.
Example 3: construction of expression vector for anti-human PD-1 antibody (Nivolumab) and eukaryotic expression
Acquisition of Nivolumab antibody variable region Gene and construction of expression vector
The light and heavy chain variable region genes of the Nivolumab antibody are respectively SEQ ID NO. 33 and SEQ ID NO. 34, the light chain variable region of the Nivolumab antibody has the amino acid sequence shown as SEQ ID NO. 35, and the heavy chain variable region of the Nivolumab antibody has the amino acid sequence shown as SEQ ID NO. 36. The heavy chain variable region gene is used as a template, the heavy chain variable region fragment is amplified by PCR, and the amplified product is cloned into pFDEs-CHIg-hG 1 (invivo) containing the human IL-2 signal peptide gene and the human IgG1 heavy chain constant region gene by using a TaKaRa seamless cloning kit. The light chain variable region gene is used as a template, the light chain variable region fragment is amplified by PCR, and the amplified product is cloned into pFUSE2ss-CLIg-hK (invivogen) containing the human IL-2 signal peptide gene and the human kappa light chain constant region gene by using a TaKaRa seamless cloning kit.
Expression and purification of Nivolumab antibodies
After 5 days of co-transfection of 293T cells (ATCC) at this double plasmid 1:1 ratio, the culture supernatants were collected and purified by AKTAExplorer100 (GE) for Nivolumab antibody. The Nivolumab antibody was visualized by Coomassie blue staining after non-reducing SDS-PAGE electrophoresis to show its size to be about 150 kDalton.
Example 4: ELISA detection of binding of recombinant human PD-1 (PD-1-huIgG 1 Fc or PD-1-his) to Nivolumab antibody
Recombinant human PD-1 (PD-1-huIgG 1Fc or PD-1-his) was detected for binding to Nivolumab antibodies using an enzyme-linked immunosorbent assay (ELISA) which was performed as follows: the PD-1-huIgG 1Fc or PD-1-his fusion protein prepared above was added to the microplate at 100 ng/well and coated overnight at 4 ℃. The cells were washed three times with PBS, 1% BSA/PBS,200 uL/well, and blocked at 37℃for 1 hour. 100ul PBS was added to the plate after washing, and 100 ng/well of Nivolumab chimeric antibody was bound for 1 hour at 37 ℃. PBST was washed three times and 100ul of HRP-goat anti-human IgG (Fab specific) diluted 1:5000 was added and bound for 1 hour at 37 ℃. PBST was washed three times, 100 uL/well TMB developing solution was added, developed at 37℃for 10 minutes, 100 uL/well ELISA stop solution was added, and the OD450 value was read by an ELISA reader. OD450 values may reflect binding of the Nivolumab antibody to recombinant human PD-1.
Example 5: expression of tetravalent human apoptosis-ligand 1 (PD-L1) extracellular segment mutants and determination of affinity with human PD-1 molecules
1. Construction of expression vector of tetravalent human PD-L1 extracellular segment mutant
The gene sequence (SEQ ID NO: 37) of the two human PD-L1 extracellular-segment mutants linked by a flexible peptide, which encodes the amino acid sequence shown in SEQ ID NO:38, was synthesized by chemical synthesis. The gene sequence of the human IgG1 heavy chain constant region (UniProtKB/Swiss-Prot access No. P01857.1) was synthesized by chemical synthesis from proline at position 100 to lysine amino acid at position 330. The upstream primer containing the mouse Igkv3-10 signal peptide gene sequence is synthesized chemically and used for constructing expression vector. And splicing the gene fragment of the PD-L1 extracellular segment mutant repetitive structure with the human IgG1Fc gene fragment by molecular cloning. The splice products were cloned into pcdna3.1 (Thermo) using TaKaRa seamless cloning kit.
2. Expression and purification of tetravalent human PD-L1 extracellular segment mutant
After transfection of 293T cells (ATCC) with this expression vector for 5 days, the culture supernatant was collected and purified using AKTAExplorer 100 (GE) as a tetravalent human PD-L1 extracellular domain mutant protein. Due to glycosylation modification and other reasons, tetravalent human PD-L1 extracellular mutant proteins are subjected to reducing SDS-PAGE electrophoresis and then are stained by Coomassie brilliant blue to show that the size of the tetravalent human PD-L1 extracellular mutant proteins is about 85 kDalton.
Biotinylation of PD-1-huIgG1Fc fusion proteins
PD-1-huIgG1Fc fusion proteins were randomly biotinylated using standard procedures provided by EZ-Link Sulfo-NHS-LC-Biotin (Thermo). ELISA was used to verify the binding activity of biotinylated PD-1-huIgG1Fc fusion protein to Nivolumab antibody.
4. Determination of affinity (avidity) of tetravalent human PD-L1 extracellular fragment mutants with PD-1-huIgG1Fc
The affinity of tetravalent human PD-L1 extracellular domain mutants to PD-1-huIgG1Fc was determined using an Octet K2 (forteBio) molecular interaction analyzer. 100nM of biotinylated PD-1-huIgG1Fc fusion protein was prepared and immobilized on SA probe (ForteBio) to a height of 1nM according to the standard protocol of Octet K2. Experiments were performed with tetravalent human PD-L1 extracellular domain mutants diluted in a two-fold gradient starting from a concentration of 50nM as analyte, and the affinity of tetravalent human PD-L1 extracellular domain mutants to PD-1-huIgG1Fc was measured to be 9.4nM, and the results of the affinity measurements are shown in fig. 1. Wild-type PD-1 and PD-L1 binding affinities of 8.2uM are known (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. Immunization of animals
2mg/mL of the PD-1-huIgG1 Fc fusion protein prepared in example 1 was mixed and emulsified as antigen with an equal volume of complete Freund's adjuvant (Sigma-Aldrich), 10 6-week old female Balb/c mice (immunized subcutaneously with 100ug of antigen each) were given, four subcutaneous immunizations were performed every ten days after the initial immunization, and spleen impact immunization was performed directly with the PD-1-huIgG1 Fc fusion protein at the fifth immunization.
2. Serum titer detection
Blood was collected from the anterior-caudal vein after each boost for 50uL, and the cells were removed by centrifugation, leaving serum. PD-1-his (ACRO Biosystems) ng/well was added to ELISA microwells and coated overnight at 4 ℃. The cells were washed three times with PBS, 1% BSA/PBS was added thereto, 200 uL/well, and the cells were blocked at 37℃for 1 hour. The mouse serum was added in gradient dilution and combined at 37℃for 1 hour. PBST was washed three times, and 100ul of HRP-goat anti-mouse IgG (Shanghai substrate) diluted 1:5000 was added and bound for 1 hour at 37 ℃. PBST was washed three times, 100 uL/well TMB developing solution was added, developed at 37℃for 10 minutes, 100 uL/well ELISA stop solution was added, and the OD450 value was read by an ELISA reader.
3. Construction of an immune library
3.1 total cDNA acquisition of mouse spleen cells
Mice were sacrificed four days after impact immunization with PD-1-huIgG1 Fc fusion protein directly by intraperitoneal injection, and spleens were taken. The whole spleen was ground with a 70 μm cell screen (BD) to obtain spleen cells. After washing twice with PBS, 1000g was centrifuged for 10 minutes to obtain spleen cells. Total RNA was extracted using Trizol RNA extraction kit.
Using the RNA as a template and SuperScript TM The First strand cDNA was synthesized using the IV First-Strand Synthesis System kit.
3.2 antibody Gene amplification and light-heavy chain splicing
The cDNA was used as a template, and Antibody amplification primers described in the reference (Schaefer J.V., honegger A., pluckthun A. (2010) Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR assembly.In: kontermann R., dubel S. (eds)) were used to PCR amplify heavy chain variable domain genes using heavy chain variable domain upstream and downstream primers, and kappa chain variable domain genes using light chain variable domain upstream and downstream primers. In a 50uL reaction system, 25. 25uL phusion master mix (Thermo), 2.5uL of the upstream primer (25 pmol), 2.5uL of the downstream primer (25 pmol), 1.5uL DMSO,0.5uL cDNA and 18uL of ddH2O were added, respectively. The PCR reaction was performed according to the following procedure: after 1 minute of pre-denaturation at 98 ℃, the mixture enters a temperature cycle, denaturation at 98 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, extension at 72 ℃ for 1 minute, 30 times of cycle and final extension at 72 ℃ for 10 minutes.
The amplified VH gene and VL gene were recovered using a DNA gel recovery kit. Equal amounts of VH gene and VL gene were mixed and used as templates, and scFv genes were amplified by overlap PCR using the upstream primer scFv-F and the downstream primer scFv-R. In a 50uL reaction system, 25. 25uL phusion master mix, 2.5uL (25 pmol) of the upstream primer, 2.5uL (25 pmol) of the downstream primer, 1.5uL DMSO,0.5uL cDNA and 18uL of ddH2O were added, respectively. The PCR reaction was performed according to the following procedure: after 1 minute of pre-denaturation at 98 ℃, the mixture enters a temperature cycle, denaturation at 98 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, extension at 72 ℃ for 1 minute, 30 times of cycle and final extension at 72 ℃ for 10 minutes.
And (3) recovering the amplified scFv gene fragment by using a DNA gel recovery kit.
3.3 construction of immune library
The scFv gene fragment and pcomb3XTT vector (Scipps research, USA) were digested with SfiI DNA endonucleases, respectively. Into the 50uL reaction system, sfiI 2uL,10 Xbuffer 5uL, DNA3ug and ddH2O were added to 50uL, respectively. After thoroughly mixing, incubation was carried out at 50℃for 3 hours.
And (3) recovering the digested scFv gene fragment and the pcomb3XTT vector by using a DNA gel recovery kit. The digested scFv gene fragment and 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 were added to 50uL, respectively. After thoroughly mixing, incubation was carried out at 4℃for 16 hours. A small amount of the product was taken and the ligation efficiency was verified by agarose gel electrophoresis.
10uL of the above ligation cyclized product was added to the homemade TG1 electrotransformation competence, and then subjected to electric shock transformation by an electrotransformation apparatus. 10ul of the electrotransformed bacteria were removed, counted and the phage antibody library size counted by reasonable dilution and streaking on ampicillin-containing plates. The remaining electrotransformed bacteria were added to 2XYT medium containing 100ug/mL ampicillin and 2% glucose and incubated in a heated incubator. After the completion of the culture, the culture was centrifuged at 4000G for 10 minutes at 4℃to supplement the precipitant with an appropriate amount of glycerol and stored at-80℃as an antibody strain library. scFv immune libraries were obtained with a capacity exceeding 3E9 pool by multiple electrotransformation accumulation.
4. Screening and identification of murine immune antibody phage library
Biotinylation of the 4.1PD-1-huIgG1 Fc fusion protein
PD-1-huIgG1 Fc fusion proteins were randomly biotinylated using standard protocols provided by EZ-Link Sulfo-NHS-LC-Biotin. ELISA was used to verify the binding activity of biotinylated PD-1-huIgG1 Fc fusion protein to Nivolumab antibody.
4.2 biopanning
The PD-1-huIgG1 Fc fusion protein is taken as a target protein, biopanning is performed on the murine immune antibody library to obtain the antibody combined with the PD-1-huIgG1 Fc fusion protein (especially the PD-1 extracellular domain). 100OD bacteria were recovered from the antibody pool at an initial od600=0.1 density and grown to log phase, and the antibody library was rescued using M13KO7 helper phage, centrifuged, resuspended in 2xYT medium containing ampicillin and kanamycin and amplified overnight at 30 ℃. PEG/NaCl precipitation phage, using glycerol/PBST dissolved phage precipitation to obtain immune library phage suspension. Casein blocked phages were placed 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 casein blocked biotinylated PD-1-huIgG1 Fc fusion protein and casein blocked Dynabeads M-270 streptavidin co-incubation system, and the beads were washed with PBST to remove phages that could not bind to the PD-1-huIgG1 Fc fusion protein. Under proper elution conditions, the self-made tetravalent PD-L1 extracellular segment mutant is used for carrying out competitive elution on phage. 10ul of eluted phage solution was left for determination of total phage output, the remaining phage solution was used to infect log-growing TG1, and after overnight amplification was considered as antibody library for the next round of panning. Biopanning was performed three times in total and the elutriation experimental parameters are detailed in table 2.
Table 2 experimental parameters of biopanning of antibody library
Figure BDA0004152237690000331
4.3 Primary screening clones that specifically bind to the extracellular Domain of PD-1 and have PD-1-tetravalent PD-L1 blocking action
The antibody library obtained after the completion of the third biopanning was diluted and plated on ampicillin-containing plates to obtain a monoclonal, and the monoclonal was selected and cultured overnight in a deep well plate. The deep-well plate was subjected to repeated three times of freeze thawing the next day, and the supernatant was used for the subsequent two types of ELISA reactions.
ELISA reaction to detect binding Activity: the cells were coated overnight with 50ng of PD-1-his, washed three times with PBS, added 1% BSA/PBS,200 uL/well, and blocked at 37℃for 1 hour. 100ul of supernatant 37℃was added after three PBS washes and incubated for 1 hour, 100ul of HRP-conjugated goat anti-mouse IgG (Fab-specific) (Thermo) was added after three PBST washes with 1:5000 dilution. PBST was washed three times, 100 uL/well TMB developing solution was added, developed at 37℃for 10 minutes, 100 uL/well ELISA stop solution was added, and the OD450 value was read by an ELISA reader. The screening step was repeated twice to ensure accurate data, and clones with OD450 number average values greater than 0.15 were selected for subsequent analysis.
ELISA reaction to detect blocking Activity: the cells were coated overnight with PD-1-his, washed three times with PBS, added with 1% BSA/PBS,200 uL/well, and blocked at 37℃for 1 hour. 100ul of supernatant and 20ng of tetravalent PD-L1 cocktail were added after three PBS washes and incubated for 1 hour at 37℃and 100ul of HRP-conjugated goat anti-mouse IgG (Fab-specific) (Thermo) diluted 1:5000 was added after three PBST washes. PBST was washed three times, 100 uL/well TMB developing solution was added, developed at 37℃for 10 minutes, 100 uL/well ELISA stop solution was added, and the OD450 value was read by an ELISA reader. The screening step is repeated twice to ensure accurate data, and clones with OD450 number average value less than 1 are selected for subsequent analysis.
Further, candidate clones meeting both conditions are sequenced to obtain antibody light chain variable domain and heavy chain variable domain sequences.
4.4 rescreening clones specifically binding to the extracellular domain of PD-1 and having a PD-1-tetravalent PD-L1 blocking action
The candidate clones obtained by the primary screening were subjected to expansion culture with 50ml shaking tube (thermo), periplasmic cavity extract was obtained by the method of lysozyme (Shanghai) combined with three freeze thawing, and the binding and blocking activities of the clones were identified again by two ELISA. The cells were coated overnight with 50ng of PD-1-his, washed three times with PBS, added 1% BSA/PBS,200 uL/well, and blocked at 37℃for 1 hour. 100ul of supernatant 37℃was added after three PBS washes and incubated for 1 hour, 100ul of HRP-conjugated goat anti-mouse IgG (Fab-specific) (Thermo) was added after three PBST washes with 1:5000 dilution. PBST was washed three times, 100 uL/well TMB developing solution was added, developing was performed at 37℃for 10 minutes, 100 uL/well ELISA stop solution was added, and the OD450 value was read by an ELISA reader, thereby verifying the binding activity. The cells were coated overnight with PD-1-his, washed three times with PBS, added with 1% BSA/PBS,200 uL/well, and blocked at 37℃for 1 hour. 100ul of supernatant and different quality tetravalent PD-L1 cocktail were added after three PBS washes and incubated for 1 hour at 37℃and 100ul of HRP-conjugated goat anti-mouse IgG (Fab specific) (Thermo) diluted 1:5000 was added after three PBST washes. PBST was washed three times, 100 uL/well TMB developing solution was added, developed at 37℃for 10 minutes, 100 uL/well ELISA terminator was added, and the OD450 value was read by an ELISA reader, thereby verifying blocking activity. Clones with low ELISA reading value (namely strong blocking effect) are selected for sequencing to obtain the sequence of the antibody, and 6H6 with the strongest blocking effect is selected from the sequences for subsequent analysis, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the 6H6 antibody are respectively SEQ ID NO.7 and SEQ ID NO.8.
4.5 expression of 6H6 clone hIgG 1-form antibodies
The heavy chain variable region sequence of 6H6 was cloned into pFUSSs-CHIg-hG 1, respectively. The 6H6 light chain variable region sequences were cloned into pFUSE2ss-CLIg-hK, respectively. After 5 days of co-transfection of 293T cells (ATCC) at this two plasmid 1:1 ratio, the culture supernatants were collected and the 6H6 antibody was purified using AKTAExplorer 100 (GE). The 6H6 antibody was visualized by Coomassie blue staining after non-reducing SDS-PAGE electrophoresis to a size of about 150 kDalton.
4.6 detection of the blocking Effect of the molecular level of each hIgG1 antibody (6H 6 and Nivolumab)
Competition ELISA experiments were performed with 6H6, nivolumab and hIgG1 isotype control (U.S. R & D Systems). The method comprises the following steps: 100ng of PD-1-huIgG1 Fc fusion protein per well was used for overnight coating, and 50ul of different hIgG1 antibodies (6H 6, nivolumab and isotype control antibodies) and 50ul of a mixture of PD-L1-his (ACRO Biosystems) at different concentrations were added sequentially and 100ul of 1:5000 mouse anti-his tag-HRP (Thermo) was developed to detect the blocking effect of each antibody molecule level. The assay procedure contained three duplicate wells and the average ELISA results are shown in Table 3. The blocking effect of the 6H6 molecular level is better than that of Nivolumab.
TABLE 3 blocking effects of levels of 6H6, nivolumab and hIgG1 isotype control antibody molecules
Figure BDA0004152237690000351
5. Humanization of anti-human PD-1 murine antibody 6H6
Humanization was performed for 6H6 in view of the superior molecular level blocking activity of 6H6 molecules. First, humanized antibody based on complementarity determining region transplantation is carried out, IGKV6-21 x 02 and IGKJ4 x 01 are selected respectively, and the human germline genes IGHV3-23 x 04 and IGHJ4 x 01 replace the germline genes of murine light chain and heavy chain corresponding to 6H6 respectively. Next, key amino acids contributing to antibody affinity were predicted and back mutations were completed: the residues of the first type of amino acid residues located at the VL and VH binding interface play a key role for Packing of the two domains, the residues of the second type of amino acid residues near the CDR regions and embedded within the protein, the residues of the third type of amino acid residues being residues with direct interactions with the CDR regions, including hydrophobic interactions/hydrogen bonds/salt bridges, etc. Three candidate molecules are constructed based on the above rule, and Hu_6H2 molecules are preferably used as humanized 6H6 antibody versions through in vitro affinity measurement, and the amino acid sequences of the heavy chain variable region and the light chain variable region of the Hu_6H2 are respectively SEQ ID NO.9 and SEQ ID NO.10.
Example 7: preliminary in vitro evaluation of anti-PD-1 murine antibodies
In vitro neutralization assay for huIgG1 type PD-1 antibodies
In vitro neutralization assays of PD-1 antibodies were performed using ELISA to verify the ability of huIgG1 type antibodies of 6H6 and pembrolizumab to block PD-L1 binding to PD-1: PD-L1-huIgG1 Fc (ACRO Biosystems) ug/ml100 ul/well was coated overnight at four degrees, plate washed and blotted dry and incubated with 1% BSA in PBS 200ul/well for 1 hour at 37℃for blocking. After 3 beats of 0.1% PBST wash plates, the plates were incubated with scFv-huIgG1 Fc type antibody and Biotinylated PD-1-huIgG1 Fc (Kactus Biosystems) mixture at room temperature for 1 hour (2 ug/ml 50ul of PD-1-huIgG1 Fc mixed with 50ul antibody diluted with a total of eight gradients starting with a two-fold gradient of 160 ug/ml). Plates were washed 3 times with 0.1% PBST and incubated with 100ul of strepavidin-HRP (R & D Systems) per well (1:200 dilution) for 1 hour at room temperature. After 6 times washing the plates with 0.1% PBST, 100ul of TMB chromogenic solution was added and incubated at room temperature for 10 minutes per well, 100ul of stop solution was added and OD450 readings were performed using an ELISA reader. The data were analyzed and calculated to give half inhibition concentrations IC50 of the huIgG1 antibodies of 6H6H and pembrolizumab against 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 antibodies
PD-1 antibody affinity assay was performed using ELISA to verify the affinity of the huIgG1 antibodies of 6H6 and pembrolizumab: four-degree overnight coating of PD-1protein, human, recombint (His Tag) (Beijing Yiqiao Shenzhou) 0.5ug/ml100ul per well, plate washing and drying, and incubation at 1%BSA in PBS 200ul/well 37℃for 1 hour for blocking. Plates were washed 3 times with 0.1% PBST and incubated with scFv-huIgG1 Fc type antibody at room temperature for 1 hour (initial two-fold gradient dilution from 10ug/ml, total eight gradients). Plates were washed 3 times with 0.1% PBST, and incubated with gold Anti-Mouse IgG F (ab') 2Secondary Antibody,HRP (Thermo)/gold Anti-Human IgG Secondary Antibody (HRP) (Beijing Yiqiao Shenzhou) 100ul per well (1:10000 dilution) for 1 hour at room temperature. After 6 times washing the plates with 0.1% PBST, 100ul of TMB chromogenic solution was added and incubated at room temperature for 10 minutes per well, 100ul of stop solution was added and OD450 readings were performed using an ELISA reader. The data were analyzed and calculated to have EC50 s of 0.01723ug/ml and 0.01106ug/ml, respectively, for binding of the huIgG1 antibody to PD-1 of 6H6 and pembrolizumab, as shown in FIG. 3.
Example 8: PD-1 antibody stimulates Tumor Infiltrating Lymphocytes (TIL)
The stimulatory effect of the antibodies of the present application on Tumor Infiltrating Lymphocytes (TIL) was verified by adding the PD-1 antibody to the cell culture medium of TIL cells. Two batches of TIL cells (donor A and donor B) were resuscitated and cultured in medium containing IL-2 for 48 hours. IL-2 was removed by washing after the completion of the culture, 1E 5/well cells were added to a 96-well plate, and the test group (transact+PD-1) was prepared according to the instructionsAdding T Cell TransAct at a ratio of 1:1000 TM (Miltenyi Biotec) and 1. Mu.g/ml of 6H6 PD-1 antibody stimulation, control group plus T Cell TransAct only TM The results of the cell proportion expressing CD107a and cytokine secretion were examined after overnight incubation.
FIG. 4A shows a graph of the CD107a cell fraction of PD-1 antibody added to tumor-infiltrating lymphocytes (TIL) cell culture medium derived from donor A. FIG. 4B shows the CD107a cell fraction results of PD-1 antibodies added to tumor-infiltrating lymphocytes (TIL) cell culture medium from donor B. The results show that the PD-1 antibodies of the present application can increase the proportion of cells expressing CD107 a.
FIG. 5 shows graphs of the secretion results of cytokines IL-2, TNF- α and/or IFN- γ from PD-1 antibodies in tumor-infiltrating lymphocytes (TIL) cell culture medium derived from donor A and donor B. The results show that the PD-1 antibodies of the present application can increase secretion of IL-2, TNF- α and/or IFN- γ; the PD-1 antibodies of the present application have the ability to activate immune cells.
Example 9: mixed Lymphocyte Reaction (MLR) assay to detect PD-1 antibody stimulated T lymphocytes
Detection of PD-1 antibodies using Mixed Lymphocyte Reaction (MLR) experiments was able to stimulate IL-2 secretion and IFN-gamma secretion by T lymphocytes. Human PBMCs were adjusted to cell densities of 2E 7/ml and monocytes were sorted using CD14 microblades, human (Miltenyi Biotec). 50ng/ml GM-CSF (ACRO Biosystems) and 50ng/ml IL-4 (ACRO Biosystems) were added and incubated for 5 days. DC cell maturation was induced by addition of 10ng/ml LPS (Sigma) and 20ng/ml TNF- α (ACRO Biosystems). CD4+ T cells were isolated from PBMC from different human sources using Naive CD4+ T Cell Isolation Kit II, human (Miltenyi Biotec). In 96-well plates, 250 μ1 culture broth per well contained 1.0E5 isolated T cells, 2E4 mature-inducing DC cells, and a series of concentration gradients of pembrolizumab or PD-1 antibody 6H6 of the present application. As negative control, a Human IgG1 Isotype control (Isotype control) was used. Mixed lymphocytes were incubated at 37℃with 5% CO 2 The cells were cultured in a cell incubator for 3 days, and 100ul of culture supernatant was removed from each well of the 96-well plate for IL-2 and IFN-gamma measurement.
FIG. 6 shows the results of Mixed Lymphocyte Reaction (MLR) experiments to detect the stimulation of PD-1 antibodies to T lymphocytes secreting IL-2 (A) and IFN-gamma (B). 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 capacity of immune cells
Whether the PD-1 antibody enhances TCR-T killing was verified by adding the PD-1 antibody to the killing of A375 cells by NYESO-1TCR-T cells. CD3 positive T cells were isolated from freshly isolated human PBMC cells by CD3 microblades, human (Miltenyi Biotec 130-050-101). TCR-T cells can be obtained by any transduction method, for example, using a slow virus carrying NY-ESO-1TCR (Kite Pharma) for transduction of selected CD3 positive T cells, the transduction efficiency of the flow assay can be 40%. 2E4 cells per well of A375 cells are plated into 96-well plates, NYESO-1TCR cells after 2E 4/well transduction are added, simultaneously 10 mug/ml of 6H6 antibody and control antibody pembrolizumab are added as test groups, equivalent PBMC cells are added into A375 cells as negative control, only A375 cells are used as blank control, and fluorescent dye SuperView is added simultaneously per well of all groups TM 488 Caspase-3. The killing results were statistically analyzed after automatic photographing for 33 hours every 3 hours by transferring the 96-well plate into the intucyte S3.
FIG. 7 is a graph showing the result of enhancing the killing ability of TCR-T cells by the addition of PD-1 antibody. The results show that the PD-1 antibodies of the present application can enhance the killing ability of immune cells.
Example 11: humanization of anti-human PD-1 murine antibodies
(1) Humanized design of antibodies
Humanization was performed for 6H 6. Firstly, carrying out antibody humanization based on complementarity determining region transplantation, comparing a 6H6 mouse anti-gene with a human germline gene, obtaining key amino acids contributing to antibody affinity and finishing back mutation: the residues of the first type of amino acid residues located at the VL and VH binding interface play a key role for Packing of the two domains, the residues of the second type of amino acid residues near the CDR regions and embedded within the protein, the residues of the third type of amino acid residues being residues with direct interactions with the CDR regions, including hydrophobic interactions/hydrogen bonds/salt bridges, etc. Candidate molecules are constructed based on the rules, sequences are synthesized, expression vectors are constructed, the expression purified antibodies are transfected into 293F cells in a suspension mode, and 6H6-5, 6H6-25 and 6H6-29 antibody molecules are selected as humanized 6H6 antibody versions through in vitro affinity determination. Wherein the heavy chain constant region of the antibody may be SEQ ID NO:60, a sequence shown in seq id no; wherein the light chain constant region of the antibody may be SEQ ID NO: 61.
Figure BDA0004152237690000381
(2) Binding affinity assay
The binding affinity of huIgG1 type PD-1 antibodies was measured by Surface Plasmon Resonance (SPR) with a Biacore T200 biosensor equipped with a pre-immobilized protein a sensor chip. Humanized antibody (1 ug/ml) was injected into Biacore T200 biosensing at 10ul/min for about 24-33 seconds to reach the desired protein density (about 44-57 response units. Human PD 1protein (PD-1Protein,Human,Recombinant His Tag) was then injected at 50ug/ml, 25ug/ml, 12.5ug/ml, 6.25ug/ml, 3.125ug/ml, 1.5625ug/ml for 30ul/min for 300 seconds and dissociation was detected for 250 seconds. FIGS. 8, 9, 10 and 11 show the antigen binding affinity results for the 6H6, 6H6-5, 6H6-25, 6H6-29 antibodies, respectively.
Antibodies to 6H6 6H6-5 6H6-25 6H6-29
Affinity (KD) 4.50E-08 1.09E-08 8.07E-09 1.78E-08
(3) In vitro neutralization assay for antibodies
In vitro neutralization test of humanized 6H6 antibody by ELISA, and the capacity of humanized 6H6 huIgG1 type antibody to block the combination of PD-L1 and PD-1 is verified: PD-L1-huIgG1 Fc (Kactus Biosystems) ug/ml 100 ul/well was coated overnight at four degrees Celsius, plate washed and flash dried, and then blocked by incubation at 1%BSA in PBS 200ul/well 37℃for 1 hour. After 3 beats of 0.1% PBST wash plates, the plates were incubated with scFv-huIgG1 Fc type antibody and Biotinylated PD-1-huIgG1 Fc (Kactus Biosystems) mixture at room temperature for 1 hour (2 ug/ml 50ul of PD-1-huIgG1 Fc mixed with 50ul antibody diluted with a total of 15 gradients starting at 160ug/ml in a twofold gradient). Plates were washed 3 times with 0.1% PBST and added with strepavidin-HRP (R&D Systems) 100ul per well (1:200 dilution) were incubated for 1 hour at room temperature. After 6 times washing the plates with 0.1% PBST, 100ul of TMB chromogenic solution was added and incubated at room temperature for 10 minutes per well, 100ul of stop solution was added and OD450 readings were performed using an ELISA reader. Analyzing and processing the data to calculate the half inhibition concentration IC of the 6H6 and the humanized 6H6-5, 6H6-25 and 6H6-29 huIgG1 antibodies to the combination of PD-1 and PD-L1 50 As shown in fig. 12.
Antibodies to 6H6 6H6-5 6H6-25 6H6-29
IC 50 (ug/ml) 2.6477 2.9335 0.8004 1.9073
The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Numerous variations of the presently exemplified embodiments of the present application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and equivalents thereof.

Claims (10)

1. An antigen binding protein that binds PD-1, said antigen binding protein comprising a light chain variable region and a heavy chain variable region, said heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, said HCDR1 having an amino acid sequence of SEQ ID No. 1; the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 2; the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3; the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4; the sequence of the LCDR2 is shown as SEQ ID NO. 5 (YAS); the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; and, the heavy chain variable region of the antigen binding protein comprises FRs 1 to 4 in an antibody heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO. 67; and the light chain variable region of the antigen binding protein comprises FRs 1 to 4 in an antibody light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO. 68.
2. The antigen binding protein of claim 1, comprising the heavy chain variable region of SEQ ID No. 67.
3. The antigen binding protein of claim 1, comprising a heavy chain variable region as set forth in any one of SEQ ID NOs 54 to 56.
4. The antigen binding protein of claim 1, comprising the light chain variable region of SEQ ID No. 68.
5. The antigen binding protein of claim 1, comprising a light chain variable region as set forth in any one of SEQ ID NOs 57 to 59.
6. The antigen binding protein of any one of claims 1-5, comprising an antibody or antigen binding fragment thereof.
7. A polypeptide comprising the antigen binding protein of any one of claims 1-6.
8. A nucleic acid encoding the antigen binding protein of any one of claims 1-6 and/or the polypeptide of claim 7.
9. A cell expressing the antigen binding protein of any one of claims 1-6, the polypeptide of claim 7, and/or comprising the nucleic acid of claim 8.
10. A method of affecting cytokine production by a target cell for non-therapeutic and/or diagnostic purposes, the method comprising administering the antigen binding protein of any one of claims 1-6, the polypeptide of claim 7, the nucleic acid of claim 8, a vector comprising the nucleic acid, and/or a cell expressing the antigen binding protein, the polypeptide, or comprising the nucleic acid and/or the vector.
CN202310322301.5A 2022-03-30 2023-03-29 PD-1 binding protein and application thereof Pending CN116284406A (en)

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