CN106432494B

CN106432494B - Novel anti-PD-1 antibodies

Info

Publication number: CN106432494B
Application number: CN201610656318.4A
Authority: CN
Inventors: 郑勇; 李竞; 陈智胜
Original assignee: Guangzhou Yuheng Biotechnology Co ltd
Current assignee: Guangzhou Yuheng Biotechnology Co., Ltd.
Priority date: 2015-08-11
Filing date: 2016-08-11
Publication date: 2021-07-16
Anticipated expiration: 2036-08-11
Also published as: CN106432494B9; CN106432494A

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a novel anti-PD-1 antibody, which comprises a heavy chain CDR sequence selected from the following group: 1, 3, 5, 13, 15, 21, 23, 25, 33, 35 and 37, which are capable of blocking the binding of PD-1 ligand to programmed death molecule 1(PD-1), thus blocking the inhibitory effect of PD-1 ligand on T cells expressing PD-1. The antibodies of the present invention provide very effective agents for the treatment of various cancers by modulating human immune function.

Description

Novel anti-PD-1 antibodies

Technical Field

The present invention relates to novel anti-PD-1 antibodies.

Background

Evidence of increasing preclinical and clinical outcomes suggests that targeting immune checkpoints is becoming the most promising approach to treating cancer patients. Programmed cell death molecule 1 is one of the immune checkpoint proteins that plays a major role in limiting T cell activity, which provides the major immune resistance mechanism by which tumor cells can evade immune surveillance. The interaction of PD-1 expressed on activated T cells with PD-L1 expressed on tumor cells down-regulates the immune response and attenuates anti-tumor immunity. The expression of PD-L1 on tumors is associated with a decreased survival rate in esophageal, pancreatic and other types of cancer, highlighting the potential of this pathway as a new promising target for tumor immunotherapy. Pharmaceutical companies have developed a variety of drugs for the PD-1 pathway, such as BMSs, merck, roche, and Glatiramer Smith (GSK). Data from clinical trials show early evidence of persistent clinical activity and good safety in patients with various tumor types. Nivolumab is a PD-1 drug developed by BMS that is being put into the central phase of the next generation field. Currently in 6 late stage studies, treatment promoted tumor shrinkage in 3 of the 5 cancer groups studied, including 18% in 72 patients with lung cancer, nearly one-third in 98 patients with melanoma, and 27% in 33 patients with renal cancer. Lambrolizumab, developed by merck corporation, is a fully human monoclonal IgG4 antibody that acts on PD-1, seizing new breakthrough indicators of the FDA after impressive IB data obtained for skin cancer. Results from the phase IB study showed an objective anti-tumor response in 51% of 85 cancer patients and a complete response in 9% of patients. Experimental MPDL3280A from roche demonstrated shrinkage of tumors in 29 (21%) of 140 advanced cancer patients with tumors of various sizes.

However, existing therapeutic approaches are not entirely satisfactory, and there is therefore still a need for new anti-PD-1 antibodies.

Brief description of the invention

The present application provides novel anti-PD-1 monoclonal antibodies (particularly fully human antibodies), polynucleotides encoding the same, and methods of using the same.

In one aspect, the present application provides an isolated monoclonal antibodyA body or antigen binding fragment thereof, which can be present at no more than 10^-8M (e.g. ≦ 9x10^-9M、≤8x10^-9M、≤7x10^-9M、≤6x10^-9M、≤5x10^-9M、≤4x10^-9M、≤3x10^-9M、≤2x10^-9M、or≤10^-9M) specifically binds to human PD-1, said Kd value being determined by plasmon resonance binding.

In certain embodiments, the antibody or antigen-binding fragment thereof has an EC of no more than 100nM or no more than 10nM (e.g., no more than 50nM, 40nM, 30nM, 20nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or 1nM)₅₀Binds to monkey PD-1. In certain embodiments, the antibody or antigen-binding fragment thereof does not bind to mouse PD-1, but binds to monkey PD-1 with a similar binding affinity as human PD-1. In some embodiments, the antibody or antigen-binding fragment thereof has an IC of no more than 100nM (e.g., no more than 50nM, 40nM, 30nM, 20nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, 0.02nM or 0.01nM)₅₀Effectively inhibit the binding of human or monkey PD-1 with its ligand (such as PD-L1, PD-L2). In certain embodiments, the EC is₅₀Or IC₅₀The assay was performed by Fluorescence Activated Cell Sorting (FACS) analysis.

In certain embodiments, the antibody or antigen-binding fragment thereof has substantially reduced effector function. In certain embodiments, the antibody or antigen-binding fragment thereof does not mediate ADCC or CDC or both.

In certain embodiments, an antibody or antigen-binding fragment thereof described herein comprises a heavy chain CDR sequence selected from the group consisting of: 1, 3, 5, 13, 15, 21, 23, 25, 33, 35 and 37. .

In certain embodiments, an antibody or antigen-binding fragment thereof described herein comprises a light chain CDR sequence selected from the group consisting of: 7, 9, 11, 17, 19, 27, 29, 31, 39, 41, 43 and 65.

In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise a heavy chain variable region selected from the group consisting of

SEQ ID NOs

1, 3, 5, 7, 9, and 11; or selected from SEQ ID NO 13, 15, 5, 7, 17 and 11; or selected from SEQ ID NO 1, 15, 5, 7, 17 and 19; or selected from SEQ ID NO 1, 15, 5, 7, 17 and 65; or selected from SEQ ID NO 1, 15, 5, 7, 17 and 19; or selected from SEQ ID NOs 21, 23, 25, 27, 29 and 31; or at least 1, 2, 3, 4,5 or 6 CDRs selected from SEQ ID NOs:33, 35, 37, 39, 41 and 43.

In certain embodiments, the antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable region selected from the group consisting of:

a) a heavy chain variable region comprising SEQ ID NO 1, SEQ ID NO 3 and/or SEQ ID NO 5;

b) a heavy chain variable region comprising SEQ ID NO 13, SEQ ID NO 15 and/or SEQ ID NO 5;

c) a heavy chain variable region comprising SEQ ID NO 1, SEQ ID NO 15 and/or SEQ ID NO 5;

d) a heavy chain variable region comprising SEQ ID NO 21, SEQ ID NO 23 and/or SEQ ID NO 25; and

e) a heavy chain variable region comprising SEQ ID NO 33, SEQ ID NO 35 and/or SEQ ID NO 37.

In certain embodiments, the antibody or antigen-binding fragment thereof described herein comprises a light chain variable region selected from the group consisting of:

a) a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 9 and/or SEQ ID NO 11;

b) a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 17 and/or SEQ ID NO 11;

c) a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 17 and/or SEQ ID NO 19;

d) a light chain variable region comprising SEQ ID NO 27, SEQ ID NO 29, and/or SEQ ID NO 31;

e) a light chain variable region comprising SEQ ID NO 39, SEQ ID NO 41 and/or SEQ ID NO 43; and

f) a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 17 and/or SEQ ID NO 65.

In certain embodiments, an antibody or antigen-binding fragment thereof described herein comprises:

a) a heavy chain variable region comprising SEQ ID NO 1, SEQ ID NO 3 and/or SEQ ID NO 5; and a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 9 and/or SEQ ID NO 11;

b) a heavy chain variable region comprising SEQ ID NO 13, SEQ ID NO 15 and/or SEQ ID NO 5; and a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 17 and/or SEQ ID NO 11;

c) a heavy chain variable region comprising SEQ ID NO 1, SEQ ID NO 15 and/or SEQ ID NO 5; and a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 17 and/or SEQ ID NO 19;

d) a heavy chain variable region comprising SEQ ID NO 21, SEQ ID NO 23 and/or SEQ ID NO 25; and a light chain variable region comprising SEQ ID NO 27, SEQ ID NO 29, and/or SEQ ID NO 31;

e) a heavy chain variable region comprising SEQ ID NO 33, SEQ ID NO 35 and/or SEQ ID NO 37; and a light chain variable region comprising SEQ ID NO 39, SEQ ID NO 41 and/or SEQ ID NO 43; and

f) a heavy chain variable region comprising SEQ ID NO 1, SEQ ID NO 15 and/or SEQ ID NO 5; and a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 17 and/or SEQ ID NO 65.

In certain embodiments, the antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable region selected from the group consisting of SEQ ID NO 45, SEQ ID NO 49, SEQ ID NO 53, SEQ ID NO 57, and SEQ ID NO 61.

In certain embodiments, the antibody or antigen-binding fragment thereof described herein comprises a light chain variable region selected from the group consisting of SEQ ID NO 47, SEQ ID NO 51, SEQ ID NO 55, SEQ ID NO 59, SEQ ID NO 63, and SEQ ID NO 67.

a) a heavy chain variable region comprising SEQ ID NO 45; and a light chain variable region comprising SEQ ID NO 47;

b) a heavy chain variable region comprising SEQ ID NO 49; and a light chain variable region comprising SEQ ID NO 51;

c) a heavy chain variable region comprising SEQ ID NO 53; and a light chain variable region comprising SEQ ID NO: 55;

d) a heavy chain variable region comprising SEQ ID NO 57; and a light chain variable region comprising SEQ ID NO 59;

e) a heavy chain variable region comprising SEQ ID NO 61; and a light chain variable region comprising SEQ ID NO 63; or

f) A heavy chain variable region comprising SEQ ID NO 53; and a light chain variable region comprising SEQ ID NO 67.

In certain embodiments, an antibody or antigen-binding fragment thereof described herein includes, e.g., 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2hAb, 1.139.15hAb, and 1.153.7 hAb.

In certain embodiments, the antibody or antigen binding fragment thereof described herein competes for the same epitope as antibody 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2hAb, 1.139.15hAb, or 1.153.7 hAb. In certain embodiments, the epitope to which the antibody or antigen-binding fragment thereof described herein binds comprises at least one of the following PD-L1 amino acid residues: v64, P83, D85, L128, A129, P130, K131, A132 and Q133.

In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are capable of blocking binding of human PD-1 to its ligand, and thus provide at least one of the following activities:

a) in CD4⁺Inducing IL-2 production in T cells;

b) in CD4⁺Inducing production of IFN γ in T cells;

c) induction of CD4⁺Proliferation of T cells; and

d) reversing the T reg inhibitory function.

In certain embodiments, the antibody described herein is a monoclonal antibody, a fully human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a diabody, a labeled antibody, a diabody, or an anti-idiotypic antibody. In certain embodiments, the antibody or antigen-binding fragment thereof is a fully human monoclonal antibody, optionally produced by a transgenic rat, e.g., a transgenic rat in which endogenous rat immunoglobulin gene expression is inactivated and which carries a recombinant human immunoglobulin locus having a J locus deletion and a C-kappa mutation.

In certain embodiments, an antigen-binding fragment provided herein is a camelized single domain antibody (camelized single domain antibody), a diabody, a scFv dimer, a BsFv, a dsFv, (dsFv)2, dsFv-dsFv ', a Fv fragment, a Fab ', a F (ab ')2, a ds diabody, a nanobody, a domain antibody, or a diabody.

In certain embodiments, the antibodies or antigen-binding fragments thereof described herein further comprise an immunoglobulin constant region.

In certain embodiments, the antibodies or antigen-binding fragments thereof described herein further comprise a conjugate.

In certain embodiments, the conjugate can be a detectable label, a pharmacokinetic modifying moiety, or a purifying moiety.

In another aspect, the present application provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof as described herein. In certain embodiments, the polynucleotides provided herein encode the amino acid sequence of an antibody or antigen-binding fragment thereof as described herein. In certain embodiments, the present application provides vectors comprising these polynucleotides. In certain embodiments, the present application provides methods of expressing one or more antibodies or antigen-binding fragments described herein by culturing a host cell under conditions in which the antibody or antigen-binding fragment encoded by the polynucleotide is expressed in a vector. In certain embodiments, the polynucleotides provided herein are operably linked to a promoter, such as the SV40 promoter, in a vector. In certain embodiments, the host cell comprising the vector provided herein is a chinese hamster ovary cell, or 293F cell.

In another aspect, the present application provides a kit comprising an antibody or antigen-binding fragment thereof described herein.

In another aspect, the PD-1 antibodies of the present application, e.g., 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2hAb, 1.139.15hAb, and 1.153.7hAb, have good tolerance and high in vivo anti-tumor activity in animals. In certain embodiments, an animal having a tumor administered a PD-1 antibody described herein has at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least an 80%, at least a 90%, or at least a 95% reduction in tumor volume as compared to a control animal having a similar baseline tumor volume administered solvent alone.

In another aspect, the present application provides a method of treating a condition associated with PD-1 in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein. In certain embodiments, the individual is identified as having a disorder or condition that is likely to respond to a PD-1 antagonist. In certain embodiments, the individual is identified as being positive for PD-L1 or up-regulated in PD-L1 levels in a test biological sample from the individual.

In another aspect, the present application provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein and one or more pharmaceutically acceptable carriers. In certain embodiments, the pharmaceutical carrier may be, for example, a diluent, an antioxidant, an adjuvant, an excipient, or a non-toxic auxiliary substance.

In another aspect, the present application provides a method of treating a condition in a subject that would benefit from an upregulated immune response, comprising administering to the subject an effective amount of an antibody, or antigen-binding fragment thereof, described herein. In certain embodiments, the subject has up-regulated PD-L1 expression, or is identified as positive for PD-L1 expression.

In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof as described herein in the manufacture of a medicament for the treatment of a condition that would benefit from an upregulated immune response. In certain embodiments, the condition is cancer or a chronic viral infection.

Brief Description of Drawings

FIG. 1 shows FACS analysis-determined binding of fully human anti-PD-1 antibodies to CHO cells expressing PD-1.

FIG. 2 shows FACS analysis of fully human PD-1 antibody with CHO cells expressing PD-1 at an EC of about 2nM₅₀And (4) combining.

FIG. 3 shows FACS analysis-determined binding of fully human anti-PD-1 antibody to activated CD4+ T cells expressing PD-1).

FIG. 4 shows the IC of fully human anti-PD-1 antibodies at about 3-8nM as determined by FACS analysis₅₀Blocking the binding of PD-L1 to PD-1 transfected CHO cells.

Figure 5 shows that fully human anti-PD-1 antibodies specifically bind to PD-1, but not to family members CD28 and CTLA4 as determined by FACS analysis.

FIG. 6 shows that fully human anti-PD-1 antibodies against PD-1 bind to cynomolgus monkey PD-1 but not to murine PD-1.

FIG. 7 shows the complete kinetics of the binding affinity of the PD-1 antibody to human PD-1 as determined by plasmon resonance methods at from 3.76E-9 to 1.76E-10 mol/L.

FIG. 8 shows the effect of fully human anti-PD-1 antibodies on IL-2 production in a Mixed Lymphocyte Reaction (MLR).

FIG. 9 shows the effect of fully human anti-PD-1 antibodies on IFN γ production in MLR.

FIG. 10 shows that fully human anti-PD-1 antibody promotes T cell proliferation in MLR.

Figure 11 shows that fully human anti-PD-1 antibody promotes T cell proliferation in a specific T cell response.

Figure 12 shows that anti-PD-1 antibodies reversed Treg suppression function.

Figure 13 shows that anti-PD-1 antibodies lack ADCC in activated T cells.

Figure 14 shows that anti-PD-1 antibodies lack CDC in activated T cells.

FIG. 15 shows that 1.103.11-v2hAb binds with similar affinity to the human PD-1 extracellular domain in different buffers as determined by ELISA. "1.103.11-v 2hAb in buffer" means that the antibody is in formulation buffer, and "1.103.11-v 2hAb in PBS" means that the antibody is in 1xPBS (pH 7.4).

FIG. 16 shows FACS-determined similarity of binding of 1.103.11-v2hAb to PD-1 expressing CHO cells in different buffers. "1.103.11-v 2hAb in buffer" means that the antibody is in formulation buffer, and "1.103.11-v 2hAb in PBS" means that the antibody is in 1xPBS (pH 7.4).

Figure 17 shows hot-spot residues (shaded regions) on the crystal structure of antibody-bound human PD-L1. A shows the common hotspot residue; b to D show the hot spot residues of 1.103.11hAb, Keytruda and 11.148.10hAb, respectively.

Detailed Description

The following description of the present application is intended to be illustrative of various embodiments of the present application. Therefore, the specific modifications discussed herein should not be construed as limitations on the scope of the application. Numerous equivalents, changes, and modifications will readily occur to those skilled in the art without departing from the scope of the present application, and it is intended that such equivalents be included within the scope of the present invention. All documents, including publications, patents, and patent applications, cited in this application are incorporated by reference in their entirety.

Definition of

The term "antibody" as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody that binds a particular antigen. A natural intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second and third constant regions; each light chain consists of a variable region and a constant region. Mammalian heavy chains can be classified as α, δ, ε, γ, and μ, and mammalian light chains as λ or κ. The antibody is "Y" shaped, the neck of the "Y" structure consisting of the second and third constant regions of the two heavy chains, which are bound by disulfide bonds. Each arm of the "Y" structure includes the variable and first constant regions of one of the heavy chains, which are associated with the variable and constant regions of one of the light chains. The variable regions of the light and heavy chains determine the binding of the antigen. The variable region of each chain contains three hypervariable regions, called Complementarity Determining Regions (CDRs) (CDRs for the light chain (L) comprise LCDR1, LCDR2, LCDR3 and CDRs for the heavy chain (H) comprise HCDR1, HCDR2, HCDR 3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be named or identified by the Kabat, Chothia or Al-Lazikani nomenclature. (Al-Lazikani, B., Chothia, C., Lesk, A.M., J.mol.biol.,273(4),927 (1997)), Chothia, C., et Al, J Mol biol.Dec 5; 186(3):651-63 (1985); Chothia, C.and Lesk, A.M., J.mol.biol.,196,901 (1987); Chothia, C. et Al, Nature.Dec 21-28; 342(6252):877-83 (1989); Kabat E.A. et Al, National Institutes of Health, Bethesda, Md. (1991)). Where three CDRs are separated by flanking continuous portions called Framework Regions (FRs), which are more highly conserved than CDRs and form a scaffold-supported hypervariable loop. The constant regions of the heavy and light chains are not involved in antigen binding, but have multiple effector functions. Antibodies can be classified into several classes depending on the amino acid sequence of the heavy chain constant region. Depending on whether it contains alpha, delta, epsilon, gamma and mu heavy chains, antibodies can be classified into five major classes or isoforms, respectively: IgA, IgD, IgE, IgG and IgM. Several major antibody classes can also be divided into subclasses, such as IgG1(γ 1 heavy chain), IgG2(γ 2 heavy chain), IgG3(γ 3 heavy chain), IgG4(γ 4 heavy chain), IgA1(α 1 heavy chain), or IgA2(α 2 heavy chain), among others.

The term "antigen-binding fragment" as used herein refers to an antibody fragment formed from an antibody portion containing one or more CDRs or any other antibody fragment that binds an antigen but does not have an intact antibody structure. Examples of antigen binding fragments include, but are not limited to, antibodies such as bifunctional antibodies (diabodies), Fab ', F (ab')₂Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)₂Bispecific dsFv (dsFv-dsFv'), disulfide stabilized bifunctional antibodies (ds diabody), single chain antibody molecules (scFv), scFv dimers (diabodies), bivalent single chain antibodies (BsFv), multispecific antibodies, camelized single domain antibodies (camelized single domain antibodies), nanobodies, domain antibodies, and bivalent domain antibodies. The antigen-binding fragment may bind to the same antigen as the maternal antibody. In certain embodiments, an antigen-binding fragment can compriseFrom a specific human antibody of one or more CDR, grafted to from one or more different human antibody framework region.

An "Fab" fragment of an antibody refers to the portion of the antibody molecule that is disulfide bonded to the variable and constant regions of a light chain (which includes both the variable and constant regions) and a heavy chain.

By "Fab'" fragment is meant a Fab fragment comprising part of the hinge region.

“F(ab')₂"refers to a dimer of Fab.

The "Fc" of an antibody refers to that portion of the antibody which consists of the second and third constant regions of the heavy chain bound via disulfide bonds. The Fc portion of antibodies is responsible for a variety of different effector functions such as ADCC and CDC, but is not involved in antigen binding.

The "Fv" segment of an antibody refers to the smallest antibody fragment that contains the entire antigen-binding site. The Fv fragment consists of the variable region of one light chain and the variable region of one heavy chain.

"Single chain Fv antibody" or "scFv" refers to an engineered antibody having a light chain variable region directly linked to a heavy chain variable region or linked via a peptide chain (Huston JS et al, Proc Natl Acad Sci USA, 85:5879 (1988)).

"Single chain antibody Fv-Fc" or "scFv-Fc" refers to an engineered antibody consisting of an scFv linked to an Fc portion of an antibody.

"Camelidized single domain antibodies", "Heavy chain antibodies" or "HCAb (Heavy-chain-only antibodies)" all refer to antibodies containing two V-chains_HAntibodies that do not contain a light chain in the domain (Riechmann L. and Muydermans S., J Immunol methods. Dec 10; 231(1-2):25-38 (1999); Muydermans S., J Biotechnol. Jun; 74(4):277-302 (2001); WO 94/04678; WO 94/25591; U.S. patent No.6,005,079). Heavy chain antibodies were originally derived from camelidae (camels, dromedary and llamas). Despite the absence of light chains, camelized antibodies (camelized antibodies) have all the functions of antigen binding confirmed (Hamers-Casterman C. et al, Nature. Jun 3; 363(6428):446-8 (1993); Nguyen VK. et al, "Heavy-chain antibodies in camelids; a case of evolution innovation," immunogenetics. Apr(ii) a 54(1) 39-47 (2002); nguyen VK. et al, immunology. May; 109(1):93-101(2003)). The variable region of the heavy chain antibody (VHH domain) is the smallest known antigen-binding unit produced by adaptive immunity (Koch-Nolte F. et al, FASEB J. Nov; 21(13):3490-8.Epub 2007Jun 15 (2007)).

By "nanobody" is meant an antibody fragment consisting of one VHH domain from a heavy chain antibody and two constant regions CH2 and CH 3.

"bifunctional antibodies" (diabodies) include small antibody fragments with two antigen-binding sites, wherein the fragments comprise V's connected on the same polypeptide chain_HField and V_LDomain (V)_H-V_LOr V_H-V_L) (see, Holliger P. et al, Proc Natl Acad Sci U S.Jul 15; 90(14) 6444-8 (1993); EP 404097; WO 93/11161). The linker between the two domains is so short that the two domains on the same chain do not pair with each other, thereby forcing the two domains to pair with the complementary domains of the other chain, forming two antibody binding sites. The two antibody binding sites may be targeted to bind to the same or different antigens (or epitopes).

"Domain antibody" refers to an antibody fragment containing only one heavy chain variable region or one light chain variable region. In some cases, two or more V_HThe domains are covalently bound by a polypeptide linker and form bivalent domain antibodies. Two V of bivalent domain antibody_HThe domains may be targeted to the same or different antigens.

In certain embodiments, "(dsFv)₂"contains three peptide chains: two V_HGroups are linked by a polypeptide linker and linked to two V groups by disulfide bonds_LThe groups are combined.

In certain embodiments, a "bispecific ds bifunctional antibody" comprises V_L1-V_H2(connected by a polypeptide linker) and V_H1-V_L2(again, linked by a polypeptide linker) both at V_H1And V_L1The two are bonded through disulfide bonds.

A "bispecific dsFv" or "dsFv-dsFv" comprises three polypeptide chains: v_H1-V_H2Group (a) in whichThe heavy chains of both are linked by polypeptide linkers (e.g., long elastic linkers) and are linked to V via disulfide bonds_L1And V_L2Groups are combined, each pair of heavy and light chains paired by a disulfide bond having different antigen specificity.

In certain embodiments, an "scFv dimer" is a diabody or a diabody (BsFv), comprising two V s that dimerize_H-V_L(attached by a polypeptide linker) group, V of one of the groups_HWith V of another group_LThe two binding sites cooperate to form two binding sites that can be targeted to bind to the same antigen (or epitope) or to different antigens (or epitopes). In other embodiments, an "scFv dimer" is a bispecific diabody comprising interconnected V_L1-V_H2(ligated by polypeptide linker) and V_H1-V_L2(ligated by polypeptide linkers) wherein V_H1And V_L1Collaboration, V_H2And V_L2In cooperation, and each cooperative pair has a different antigen specificity.

The term "fully human", when used in this application, means that the antibody or antigen-binding fragment has or consists of an amino acid sequence corresponding to that of an antibody produced by a human or human immune cell or derived from a non-human source, e.g., a transgenic non-human animal utilizing a human antibody repertoire, or other sequence encoding a human antibody. In certain embodiments, a fully human antibody does not comprise amino acid residues (particularly antigen binding residues) derived from a non-human antibody.

The term "humanized" as used herein, when applied to an antibody or antigen-binding fragment, refers to an antibody or antigen-binding fragment that includes CDRs derived from a non-human animal, FR regions derived from a human, and constant regions derived from a human, where applicable. Because the humanized antibody or antigen binding fragment has reduced immunogenicity, it may be used in certain embodiments as a therapeutic agent in humans. In some embodiments, the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In some embodiments, the humanized antibody or antigen-binding fragment consists essentially entirely of human-derived sequences, except for CDR sequences that are non-human. In some embodiments, the human-derived FR region may include the same amino acid sequence as the human-derived antibody from which it is derived, or it may include some amino acid changes, e.g., no more than 10, 9, 8, 7, 6,5, 4,3, 2, or 1 amino acid change. In some embodiments, the amino acid change can be present in only the heavy chain FR region, only the light chain FR region, or both chains. In some preferred embodiments, the humanized antibody comprises human FR1-3 and human JH and jk.

The term "chimeric" as used herein refers to an antibody or antigen-binding fragment having a portion of a heavy and/or light chain derived from one species, and the remainder of the heavy and/or light chain derived from a different species. In an illustrative example, a chimeric antibody can include constant regions derived from a human and variable regions derived from a non-human animal, such as a mouse.

As used herein, "PD-1" refers to a programmed cell death protein that belongs to the immunoglobulin superfamily and functions as a cosuppression antibody for negative regulation of the immune system. PD-1 is a member of the CD28/CTLA-4 family, with two inhibitory ligands, including PD-L1 and PD-L2. A representative amino acid sequence of human PD-1 is disclosed by NCBI accession No. NP _005009.2, and a representative nucleic acid sequence encoding said human PD-1 is disclosed by NCBI accession No. NP _ 005018.2.

"PD-L1" as used in this application refers to programmed cell death ligand 1(PD-L1, see, e.g., Freeman et al (2000) J.Exp.Med.192: 1027). The amino acid sequence of representative humanized PD-L1 is NCBI accession No. NP-054862.1, and the nucleic acid sequence of representative humanized PD-L1 is NCBI accession No. NM-014143.3. PD-L1 is expressed in placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found on many tumor or cancer cells. PD-L1 binds to the receptor PD-1 or B7-1 expressed on activated T cells, B cells and bone marrow cells. Binding of PD-L1 to its receptor triggers signal transduction to inhibit TCR-mediated activation of cytokine production and T cell proliferation. Thus, PD-L1 plays a major role in suppressing the immune system in specific events, such as pregnancy, autoimmune diseases, tissue transplantation, and it is thought to allow tumor or cancer cells to circumvent immune checkpoints and evade immune responses.

An "anti-PD-1 antibody" as used herein refers to an antibody that is capable of specifically binding to PD-1 (e.g., human or monkey PD-1) with sufficient affinity to provide diagnostic and/or therapeutic use.

"specific binding" or "specific binding" in this application refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen. In certain embodiments, the antibodies or antigen binding fragments thereof of the present application specifically bind to human and/or monkey PD-1, and their binding affinity (K)_D)≤10^-6M (e.g.. ltoreq.5 x10^-7M，≤2x10^-7M，≤10^-7M，≤5x10^-8M，≤2x10^-8M，≤10^-8M，≤5x10^-9M，≤2x10^-9M，≤10^-9M，≤10^-10M). KD in the present application refers to the ratio of dissociation rate to association rate (koff/kon), and can be determined by surface plasmon resonance methods, for example using instruments such as Biacore.

The ability to "block binding" or "compete for the same epitope" in this application refers to the ability of an antibody or antigen-binding fragment thereof to inhibit the interaction of two intermolecular bindings (e.g., human PD-1 and anti-PD-1 antibodies) to any detectable degree. In certain embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules can inhibit the interaction of binding between two molecules by at least 50%. In certain embodiments, such inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

As used herein, "epitope" refers to the portion of an amino acid or group of atoms of an antigenic molecule that binds to an antibody. If two antibodies exhibit competitive binding to the antigen, it is possible to bind to the same epitope on the antigen. For example, if an antibody or antigen-binding fragment thereof provided herein blocks the binding of an exemplified antibody, e.g., 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2hAb, 1.139.15hAb, and 1.153.7hAb, to human PD-1, then the antibody or antigen-binding fragment thereof can be considered to bind to the same epitope as those exemplified antibodies.

Mutations of specific amino acid residues in the epitope, e.g., by alanine scanning mutagenesis (alanine scanning mutagenesis), identify mutations that reduce or prevent protein binding. "alanine scanning mutation" is a method that can be used to identify certain residues or regions of a protein that affect the interaction of an epitope with other compounds or proteins to which it binds. A residue or group of target residues in a protein is substituted with a neutral or negatively charged amino acid (most preferably alanine or polyalanine, or a conservative amino acid substitution). Any mutation that reduces the binding of the protein or the codon encoding it to an extent that exceeds a threshold or that maximizes the reduction of binding of the protein compared to other mutations is likely to be in an epitope bound by the protein. In certain embodiments of the present application, the epitope important for the PD-1 antibody comprises at least one of the following amino acid residues: v64, P83, D85, L128, A129, P130, K131, A132 and Q133.

The term "1.7.3 hAb" as used herein refers to a peptide having the sequence as shown in SEQ ID NO:45, as shown in SEQ ID NO:47 and a human IgG4 isotype constant region.

As used herein, "1.49.9 hAb" refers to a fully human monoclonal antibody having the heavy chain variable region shown in SEQ ID NO:49, the light chain variable region shown in SEQ ID NO:51, and a human IgG4 isotype constant region.

As used herein, "1.103.11 hAb" refers to a fully human monoclonal antibody having the heavy chain variable region shown in SEQ ID NO:53, the light chain variable region shown in SEQ ID NO:55, and a human IgG4 isotype constant region.

As used herein, "1.103.11 _ v2 hAb" refers to a fully human monoclonal antibody having the heavy chain variable region shown in SEQ ID NO:53, the light chain variable region shown in SEQ ID NO:67, and a human IgG4 isotype constant region. As used herein, "1.139.15 hAb" refers to a fully human monoclonal antibody having the heavy chain variable region shown in SEQ ID NO:57, the light chain variable region shown in SEQ ID NO:59, and a human IgG4 isotype constant region.

As used herein, "1.153.7 hAb" refers to a fully human monoclonal antibody having the heavy chain variable region shown in SEQ ID NO:61, the light chain variable region shown in SEQ ID NO:63, and a human IgG4 isotype constant region.

In the present application, "conservative substitution" when used in reference to an amino acid sequence means the substitution of one amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions may be made between hydrophobic side chain amino acid residues (e.g., Met, Ala, Val, Leu, and Ile), between neutral hydrophilic side chain residues (e.g., Cys, Ser, Thr, Asn, and Gln), between acidic side chain residues (e.g., Asp, Glu), between basic side chain amino acids (e.g., His, Lys, and Arg), or between directional side chain residues (e.g., Trp, Tyr, and Phe). It is known in the art that conservative substitutions do not generally result in significant changes in the conformational structure of a protein, and therefore the biological activity of the protein can be retained.

"percent sequence identity," when used with respect to an amino acid sequence (or nucleic acid sequence), refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to a reference sequence to the amino acid (or nucleic acid) residues in the candidate sequence after alignment and, if necessary, introduction of a spacer to maximize the number of identical amino acids (or nucleic acids). Conservative substitutions of the amino acid residues may or may not be considered identical residues. Sequences can be aligned to determine percent sequence identity of amino acid (or Nucleic acid) sequences by tools disclosed in the art, such as BLASTN, BLASTp (national center for Biotechnology information website (NCBI), see also, Altschul S.F. et al, J.mol.biol., 215: 403-. One skilled in the art can use default parameters for the tool or adjust the parameters appropriately as needed for the alignment, for example by choosing an appropriate algorithm.

"T cells" as used herein includes CD4⁺T cell, CD8⁺T cells, T helper type 1T cells, T helper type 2T cells, T helper type 17T cells, and suppressor T cells.

"effector function" as used herein refers to the biological activity of the Fc region of an antibody to bind its effectors such as the C1 complex and Fc receptor. Exemplary effector functions include Complement Dependent Cytotoxicity (CDC) induced by interaction of the antibody with C1q on the C1 complex, antibody dependent cell mediated cytotoxicity (ADCC) induced by binding of the Fc region of the antibody to Fc receptors on effector cells, and phagocytosis.

"cancer" or "cancerous condition" in the present application refers to any medical condition which is mediated by the growth, proliferation or metastasis of neoplastic or malignant cells and which causes solid and non-solid tumors, such as leukemia. The term "tumor" as used herein refers to a solid substance of a tumor and/or malignant cells.

"treating" or "treatment" of a condition includes preventing or alleviating the condition, reducing the rate at which a condition develops or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination thereof. For cancer, "treatment" or "therapy" may refer to inhibiting or slowing the growth, reproduction, or metastasis of a tumor or malignant cell, or some combination of the above. In the context of a tumor, "treating" or "therapy" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination thereof.

The "isolated" material has been artificially altered from its natural state. If a "separated" substance or component occurs in nature, it has been altered or removed from its original state, or both. For example, a polynucleotide or polypeptide naturally present in a living animal is not isolated, but is considered to be "isolated" if the polynucleotide or polypeptide is sufficiently isolated from the materials with which it naturally coexists in its natural state and is present in a sufficiently pure state. In certain embodiments, the antibodies and antigen-binding fragments are at least 90%, 93%, 95%, 96%, 97%, 98%, 99% pure, as determined by electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatographic methods (e.g., ion exchange chromatography or reverse phase HPLC).

A "vector" in the present invention refers to a vehicle into which a polynucleotide encoding a protein is operably inserted and the protein is expressed. The vector may be used to transform, transduce or transfect a host cell so that the genetic material elements it carries are expressed in the host cell. By way of example, the carrier includes: plasmids, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses, among others. Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma vacuolium viruses (e.g., SV 40). The vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site. The vector may also include components that facilitate its entry into the cell, including, but not limited to, viral particles, liposomes, or protein coats.

The term "host cell" as used herein refers to a cell into which an exogenous polynucleotide and/or vector has been introduced.

The term "PD-1 associated or related disease" as used herein refers to any condition that is caused, exacerbated or otherwise associated by an increase or decrease in the expression or activity of PD-1 (e.g., human PD-1).

As used herein, a "therapeutically effective amount" or "effective dose" refers to a dose or concentration of a drug effective to treat a disease or condition associated with human PD-1. For example, for use of the antibodies or antigen-binding fragments thereof disclosed in the present invention, a therapeutically effective amount is at that dose or concentration, and the antibody or antigen-conjugate can eliminate all or a portion of the tumor, inhibit or slow the growth of the tumor, inhibit the growth or proliferation of cells that mediate the cancerous condition, inhibit the metastasis of tumor cells, alleviate any symptoms or markers associated with the tumor or cancerous condition, prevent or delay the progression of the tumor or cancerous condition, or some combination thereof.

By "pharmaceutically acceptable" is meant a carrier, vehicle, diluent, adjuvant and/or salt that is, in general, chemically and/or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

anti-PD-1 antibodies

In one aspect, the invention provides anti-PD-1 antibodies and antigen-binding fragments thereof. PD-1, also known as CD279, is a key immune checkpoint receptor known to be expressed by activated T cells, which modulates immunosuppression. PD-1 ligand 1(PD-L1) is a 40kDa transmembrane protein expressed on a variety of tumor cells, stromal cells, or both, that binds to PD-1. Inhibition of the interaction between PD-1 and PD-L1 can enhance T cell responses thereby mediating anticancer activity.

In certain embodiments, the present application provides exemplary fully human monoclonal antibodies 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2hAb, 1.139.15hAb, and 1.153.7hAb, the CDR sequences of which are set forth in Table 1, and the heavy or light chain variable region sequences are also set forth below.

TABLE 1

1.7.3hAb-VH (23466-VH) (SEQ ID NO:45 is an amino acid, SEQ ID NO:46 is a nucleic acid) heavy chain CDR1-3 SEQ ID NO:1, 3, 5 is an amino acid sequence and SEQ ID NO:2, 4,6 is a nucleic acid sequence.

Section V: IGHV 4-39X 01

And (D) section: IGHD 1-26X 01

Segment J: IGHJ4 × 02

1.7.3hAb-VL (23195-VL) (SEQ ID NO:47 is an amino acid and SEQ ID NO:48 is a nucleic acid) light chain CDR1-3: 7, 9 and 11 are amino acid sequences and 8, 10 and 12 are nucleic acid sequences:

section V: IGLV2-14 x 01

Segment J: IGLJ3 × 02

1.49.9hAb-VH (20951-VH) (SEQ ID NO:49 amino acid, SEQ ID NO:50 nucleic acid) heavy chain CDR1-3: 13, 15 and 5 are amino acid sequences and 14, 16 and 6 are nucleic acid sequences:

section V: IGHV 4-39X 01

And (D) section: IGHD 1-26X 01

Segment J: IGHJ4 × 02

1.49.9hAb-VL (21526-VL) (SEQ ID NO:51 is an amino acid and SEQ ID NO:52 is a nucleic acid) light chain CDR1-3: 7, 17 and 11 are amino acid sequences and 8, 18 and 12 are nucleic acid sequences:

section V: IGLV2-14 x 01

Segment J: IGLJ3 × 02

1.103.11hAb-VH (20975-VH) (SEQ ID NO:53 is amino acid, SEQ ID NO:54 is nucleic acid) heavy chain CDR1-3: 1, 15 and 5 are amino acid sequences and 2, 16 and 6 are nucleic acid sequences:

section V: IGHV 4-39X 01

And (D) section: IGHD 1-26X 01

Segment J: IGHJ4 × 02

1.103.11hAb-VL (21038-VL) (SEQ ID NO:55 is an amino acid and SEQ ID NO:56 is a nucleic acid) light chain CDR1-3: 7, 17 and 19 are amino acid sequences and 8, 18 and 20 are nucleic acid sequences:

section V: IGLV2-14 x 01

Segment J: IGLJ3 × 02

1.139.15hAb-VH (23521-VH): (SEQ ID NO:57 is an amino acid and SEQ ID NO:58 is a nucleic acid) heavy chain CDR1-3: 21, 23, 25 are amino acid sequences and 22, 24, 26 are nucleic acid sequences:

section V: IGHV 4-39X 01

And (D) section: IGHD6-13 x 01

A J segment; IGHJ4 × 02

1.139.15hAb-VL (22895-VL) (SEQ ID NO:59 is an amino acid and SEQ ID NO:60 is a nucleic acid) light chain CDR1-3: 27, 29, 31 are amino acid sequences and 28, 30, 32 are nucleic acid sequences:

section V: IGLV2-18 x 02

Segment J: IGLJ3 × 02

1.153.7hAb-VH (20942-VH) (SEQ ID NO:61 is amino acid, SEQ ID NO:62 is nucleic acid) heavy chain CDR1-3: 33, 35, 37 are amino acid sequences and 34, 36, 38 are nucleic acid sequences:

section V: IGHV 3-23X 01

And (D) section: IGHD 7-27X 01

Segment J: IGHJ4 × 02

1.153.7hAb-VL (21110-VL) (SEQ ID NO:63 is an amino acid and SEQ ID NO:64 is a nucleic acid) light chain CDR1-3: 39, 41, 43 and 40, 42, 44 and the nucleotide sequences of SEQ ID NOs:

section V: IGLV3-9 x 01

Segment J: IGLJ3 × 02

1.103.11-v2hAb-VH (20975-VH) (SEQ ID NO:53 is amino acid, SEQ ID NO:54 is nucleic acid) light chain CDR1-3: 1, 15 and 5 are amino acid sequences and 2, 16 and 6 are nucleic acid sequences:

section V: IGHV 4-39X 01

And (D) section: IGHD 1-26X 01

Segment J: IGHJ4 × 02

1.103.11-v2hAb-VL (21038-2-VL) (SEQ ID NO:67 is an amino acid and SEQ ID NO:68 is a nucleic acid) light chain CDR1-3: 7, 17 and 65 are amino acid sequences and 8, 18 and 66 are nucleic acid sequences:

section V: IGLV2-14 x 01

Segment J: IGLJ3 × 02

In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof comprise heavy chain CDR sequences selected from the group consisting of: 1, 3, 5, 13, 15, 21, 23, 25, 33, 35 and 37. In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof comprise light chain CDR sequences selected from the group consisting of: 7, 9, 11, 17, 19, 27, 29, 31, 39, 41, 43 and 65. In some embodiments, one or more of the CDR sequences described herein may be modified or altered such that the resulting antibody has improved one or more properties (e.g., improved antigen binding, improved glycosylation pattern, reduced risk of glycosylation at the CDR residues, increased pharmacokinetic half-life, pH sensitivity, and compatibility with respect to conjugation) relative to the original antibody, or is comparable to the original antibody (i.e., an antibody having the same CDR sequences in addition to the modifications and alterations described above), or at least substantially retains the antigen binding properties of the original antibody.

In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof comprise a heavy chain variable region selected from the group consisting of seq id nos: a heavy chain variable region comprising SEQ ID NO 1, SEQ ID NO 3, and/or SEQ ID NO 5; a heavy chain variable region comprising SEQ ID NO 13, SEQ ID NO 15, and/or SEQ ID NO 5; a heavy chain variable region comprising SEQ ID NO 1, SEQ ID NO 15, and/or SEQ ID NO 5; a heavy chain variable region comprising SEQ ID NO 21, SEQ ID NO 23, and/or SEQ ID NO 25; and a heavy chain variable region comprising SEQ ID NO 33, SEQ ID NO 35, and/or SEQ ID NO 37.

In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof comprise a light chain variable region, wherein the light chain variable region is selected from the group consisting of: a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 9 and/or SEQ ID NO 11; a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 17 and/or SEQ ID NO 11; a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 17 and/or SEQ ID NO 19; a light chain variable region comprising SEQ ID NO 27, SEQ ID NO 29, and/or SEQ ID NO 31; a light chain variable region comprising SEQ ID NO 39, SEQ ID NO 41 and/or SEQ ID NO 43; and a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 17 and/or SEQ ID NO 65.

In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof comprise: a) a heavy chain variable region comprising SEQ ID NO 1, SEQ ID NO 3, and/or SEQ ID NO 5; and a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 9 and/or SEQ ID NO 11; b) a heavy chain variable region comprising SEQ ID NO 13, SEQ ID NO 15, and/or SEQ ID NO 5; and a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 17 and/or SEQ ID NO 11; c) a heavy chain variable region comprising SEQ ID NO 1, SEQ ID NO 15, and/or SEQ ID NO 5; and a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 17 and/or SEQ ID NO 19; d) a heavy chain variable region comprising SEQ ID NO 21, SEQ ID NO 23, and/or SEQ ID NO 25; and a light chain variable region comprising SEQ ID NO 27, SEQ ID NO 29, and/or SEQ ID NO 31; e) a heavy chain variable region comprising SEQ ID NO 33, SEQ ID NO 35, and/or SEQ ID NO 37; and a light chain variable region comprising SEQ ID NO 39, SEQ ID NO 41 and/or SEQ ID NO 43; or f) a heavy chain variable region comprising SEQ ID NO 1, SEQ ID NO 15, and/or SEQ ID NO 5; and a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 17 and/or SEQ ID NO 65.

It will be appreciated by those skilled in the art that the CDR sequences provided in table 1 may be modified to include substitutions of one or more amino acids, thereby resulting in improved biological activity, such as improved binding affinity to human PD-1. For example, libraries of antibody variants (e.g., Fab or FcFv variants) can be produced and expressed using phage display technology, and subsequently screened for antibodies with affinity for human PD-1. In another example, computer software can be used to simulate the binding of the antibody to human PD-1 and to identify the amino acid residues on the antibody that form the binding interface. Substitutions of these residues may be avoided to prevent a decrease in binding affinity, or may be targeted for substitution to form stronger binding. In certain embodiments, at least one (or all) substitution in a CDR sequence is a conservative substitution.

In certain embodiments, the antibodies and antigen-binding fragments comprise one or more CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the sequences listed in table 1, while retaining similar or even higher binding affinity to human PD-1 than its parent antibody, which has substantially identical sequences, but whose corresponding CDR sequences have 100% sequence identity to the sequences listed in table 1.

In certain embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof are fully human. The fully human antibodies do not have problems in humans such as immunogenicity or reduced binding affinity as often observed in humanized antibodies.

In some embodiments, the fully human anti-PD-1 antibodies and antigen-binding fragments thereof comprise a heavy chain variable region selected from the group consisting of SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:61, and homologous sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity thereto; and/or a light chain variable region, wherein the light chain variable region is selected from the group consisting of SEQ ID NO 47, SEQ ID NO 51, SEQ ID NO 55, SEQ ID NO 59, SEQ ID NO 63, SEQ ID NO 67, and homologous sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity thereto. These fully human antibodies retain binding affinity to human PD-1, preferably with the exemplified antibodies: levels of 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2hAb, 1.139.15hAb and 1.153.7hAb were similar.

In some embodiments, the fully human anti-PD-1 antibodies and antigen-binding fragments thereof comprise a) a heavy chain variable region comprising SEQ ID NO 45; and a light chain variable region comprising SEQ ID NO 47; b) a heavy chain variable region comprising SEQ ID NO 49; and a light chain variable region comprising SEQ ID NO 51; c) a heavy chain variable region comprising SEQ ID NO 53; and a light chain variable region comprising SEQ ID NO: 55; d) a heavy chain variable region comprising SEQ ID NO 57; and a light chain variable region comprising SEQ ID NO 59; e) a heavy chain variable region comprising SEQ ID NO 61; and a light chain variable region comprising SEQ ID NO 63; and f) a heavy chain variable region comprising SEQ ID NO 53; and a light chain variable region comprising SEQ ID NO 67.

The application also comprisesAn anti-PD-1 antibody and an antigen-binding fragment thereof are claimed to compete for antibodies and antigen-binding fragments thereof of the same epitope. In certain embodiments, the antibody is administered at less than 10^-6M, less than 10^-7M, less than 10^-7.5M, less than 10^-8M, less than 10^-8.5M or less than 10^-9M or less than 10^-10IC of M₅₀Values (i.e., half inhibitory concentrations) blocked binding of 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2hAb, 1.139.15hAb, or 1.153.7hAb to human or monkey PD-1. IC (integrated circuit)₅₀Values are determined by competitive assays such as ELISA assays, radioligand competition binding assays, and FACS analysis.

In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof described herein can have a value of ≦ 10^-6M(e.g.,≤5x10^-7M,≤2x10^-7M,≤10^-7M,≤5x10^-8M,≤2x10^-8M,≤10^-8M,≤5x10^-9M,≤2x10^-9M,≤10^-9M,10^-10M) binds specifically to human PD-1, as measured by plasmon resonance binding. Binding affinity can be defined by K_DValues are expressed as calculated by the ratio of off-rate to on-rate (koff/kon) when the binding of antigen and antigen binding molecule reaches equilibrium. The antigen binding affinity (e.g. K)_D) May suitably be determined by suitable methods known in the art, including the use of instruments such as the plasmon resonance binding method of Biacore (see, for example, Murphy, M.et al, Current protocols in protein science, Chapter 19, Unit 19.14,2006).

In certain embodiments, the antibodies and antigen-binding fragments thereof described herein have an EC of 0.1nM to 100nM (e.g., 0.1nM to 50nM, 0.1nM to 30nM, 0.1nM to 20nM, or 0.1nM to 10nM, or 0.1nM to 1nM) with human PD-1₅₀(i.e., half the binding concentration) binding. Binding of the antibody to human PD-1 can be determined by methods known in the art such as sandwich assays such as ELISA, Western blotting, FACS or other binding assays. In an illustrative example, the antibody to be tested (i.e., the primary antibody) is bound to immobilized human PD-1 or cells expressing human PD-1, followed by washing away unbound antibody, introducing a labeled secondary antibody,it is capable of binding to a primary antibody and thus capable of detecting the bound primary antibody. The detection can be performed on a microplate reader plate when immobilized PD-1 is used, or can be performed using FACS analysis when cells expressing human PD-1 are used. In certain embodiments, the antibodies and antigen-binding fragments thereof described herein bind to human PD-1 with EC50 (i.e., an effective concentration of 50%) at 1nM to 10nM or 1nM to 5nM (as determined using FACS analysis).

In certain embodiments, the antibodies and antigen-binding fragments thereof described herein have an IC of 0.2nM to 100nM (e.g., 0.2nM to 50nM, 0.2nM to 30nM, 0.2nM to 20nM, 0.2nM to 10nM, or 1nM to 10nM)₅₀Inhibits the binding of human PD-1 to its ligand, as measured by a competitive assay.

In certain embodiments, the antibodies and antigen-binding fragments thereof described herein inhibit the binding of human PD-1 to its ligand, and thus provide methods that include, for example, inducing the production of cytokines by activated T cells (e.g., CD 4)⁺T cells and CD8⁺T cells), inducing proliferation of activated T cells (e.g., CD 4)⁺T cells and CD8⁺T cells) and biological activity to reverse the suppressive function of regulatory tregs. Exemplary cytokines include IL-2 and IFN gamma. The term "IL-2" refers to interleukin 2, a cytokine signaling molecule that modulates the activity of white blood cells (e.g., leukocytes) in the immune system. The term "interferon gamma (IFN γ)" is derived from Natural Killer (NK) cells, NK T cells, CD4⁺And CD8⁺T cells produce cytokines, which are important activators of macrophages and inducers of Major Histocompatibility Complex (MHC) molecule expression. Cytokine production can be determined by methods known in the art, such as ELISA. These methods can also be used to detect T cell proliferation, comprising³H]Thymidine incorporation assay.

The anti-PD-1 antibodies and antigen-binding fragments thereof are PD-1 specific. In certain embodiments, the antibodies and antigen-binding fragments thereof do not bind to CD28 and/or CTLA-4. For example, the binding affinity to CD28 and/or CTLA-4 is less than 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the binding affinity of PD-1.

In certain embodiments, the antibodies and antigen-binding fragments thereof are administered with an EC of no greater than 100nM, e.g., no greater than or about 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, 0.02nM, or 0.01nM₅₀Binding to monkey PD-1 (determined by ELISA). In certain embodiments, the antibodies and antigen-binding fragments thereof bind monkey PD-1 with an EC50 of about 1nM to 10 nM.

In certain embodiments, the antibodies and antigen-binding fragments thereof do not bind to murine PD-1, but bind to monkey PD-1 with similar binding affinity as human PD-1. For example, binding of exemplary antibodies 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2hAb, 1.139.15hAb, and 1.153.7hAb to murine PD-1 was not detectable by commonly used binding assays such as ELISA or FACS analysis, which detected affinity or EC of these antibodies to monkey PD-1 similar to human PD-1₅₀The values are combined.

In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof have reduced or eliminated effector function. In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof have a constant region of the IgG4 isotype with reduced or depleted effector function. Equivalent functions such as ADCC and CDC can result in cytotoxicity to cells expressing PD-1. Many cells, such as T cells, are capable of normally expressing PD-1. To avoid potential undesirable toxicity to these normal cells, certain embodiments of the antibodies and antigen-binding fragments thereof described herein have reduced or even eliminated effector function. A number of assays are known to assess ADCC or CDC activity, such as Fc receptor binding assays, complement C1q binding assays and cell lysis methods, and can be readily selected by one skilled in the art. Without wishing to be bound by theory, it is believed that antibodies with reduced or eliminated effector functions such as ADCC and CDC do not cause or minimize cytotoxicity to PD-1 expressing cells (e.g., those T cells), thus avoiding undesirable side effects. At the same time, blocking PD-1 would trigger immune system therapies such as cancer or chronic infectious conditions.

In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof described herein have reduced side effects. For example, the anti-PD-1 antibodies and antigen-binding fragments thereof described herein have fully human IgG sequences and are therefore less immunogenic than humanized antibodies. As another example, the anti-PD-1 antibodies and antigen-binding fragments thereof described herein can be in the form of IgG4 to abrogate ADCC and CDC.

In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof described herein are advantageous in that they can be used in combination with immunogenic agents, such as tumor cells, purified tumor antigens, and cells transfected with an antigen encoding an immunostimulatory factor, tumor vaccines. Furthermore, the anti-PD-1 antibodies and antigen-binding fragments thereof can be included in combination therapies, including standard chemotherapy and radiotherapy, target-based small molecule therapy, other emerging immune checkpoint modulator therapies. In some embodiments, the antibodies and antigen-binding fragments thereof can be used as the base molecule for antibody-drug conjugates, bispecific or multivalent antibodies.

The anti-PD-1 antibodies and antigen-binding fragments thereof described herein can be monoclonal antibodies, polyclonal antibodies, fully human antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, bispecific antibodies, labeled antibodies, bivalent antibodies, or anti-idiotypic antibodies. Recombinant antibodies are antibodies that are produced in vitro using recombinant methods rather than animals. Bispecific antibodies or diabodies are artificial antibodies having fragments of two different monoclonal antibodies, which are capable of binding two different antigens. A "bivalent" antibody and antigen-binding fragment thereof includes two antigen-binding sites. The two antigen binding sites may bind to the same antigen, or may each bind to a different antigen, in which case the antibody or antigen binding fragment is "bispecific".

In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof described herein are fully human antibodies. In some embodiments, the fully human antibody is produced using recombinant methods. For example, a transgenic animal such as a mouse can be prepared to carry a transgene or transchromosome of a human immunoglobulin gene and thus be capable of producing fully human antibodies upon immunization with a suitable antigen such as human PD-1. Fully human antibodies can be isolated from such transgenic animals, or alternatively, can be prepared by hybridoma technology, the spleen cells of which are fused to an immortal cell line to produce hybridoma cells that secrete the fully human antibodies. Exemplary transgenic animals include, but are not limited to, Omni rats, in which the expression of endogenous rat immunoglobulin genes is inactivated and simultaneously genetically engineered to contain a functional recombinant human immunoglobulin locus; omni mice, whose endogenous mouse immunoglobulin gene expression is inactivated and at the same time genetically engineered to contain a recombinant human immunoglobulin locus with J-locus deletions and C-kappa mutations. OmniFilc, a transgenic rat, whose expression of endogenous rat immunoglobulin genes was inactivated and simultaneously genetically engineered to contain a recombinant human immunoglobulin locus with a single common, recombinant VkJk light chain and functional heavy chain. For specific information please see further: et al, Journal of Immunology,2013,190: 1481-90; et al, Journal of Immunological Methods 400-401(2013) 78-86; geurts A.et al, Science,2009,325: 433; us patent 8,907,157; european patent 2152880B 1; european patent 2336329B1, each of which is incorporated by reference herein in its entirety. Other suitable transgenic animals may also be used, for example, HuMab mice (see in particular Lonberg, N.et al. Nature 368(6474): 856859 (1994)), Xeno-mice (Mendez et al. Nat Genet.,1997,15: 146-.

In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof described herein are camelized single domain antibodies (camelized single chain domain antibodies), diabodies (diabodies), scfvs, scFv dimers, BsFv, dsFv, (dsFv)2, dsFv-dsFv ', Fv fragments, Fab ', F (ab ')2, ds diabodies (ds diabodies), nanobodies, domain antibodies, or diabodies.

In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof described herein further comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain and/or light chain constant region. The heavy chain constant region includes the CH1, CH1-CH2, or CH1-CH3 regions. In some embodiments, the immunoglobulin constant region may further comprise one or more modifications to achieve a desired property. For example, the constant region may be modified to reduce or deplete one or more effector functions to enhance FcRn receptor binding or to introduce one or more cysteine residues.

In certain embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof further comprise a conjugate. It is contemplated that the antibodies or antigen-binding fragments thereof of the present invention may be linked to a variety of conjugates (see, e.g., "Conjugate Vaccines", constraints to Microbiology and Immunology, j.m.cruse and r.e.lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugates may be attached to the antibody or antigen conjugate by covalent binding, affinity binding, intercalation, coordinate binding, complexation, binding, mixing or addition, among other means. In certain embodiments, the antibodies and antigen-binding fragments disclosed herein can be engineered to contain specific sites other than the epitope-binding portion that can be used to bind to one or more conjugates. For example, such sites may comprise one or more reactive amino acid residues, such as cysteine and histidine residues, for facilitating covalent attachment to the conjugate. In certain embodiments, the antibody may be linked indirectly to the conjugate, or via another conjugate. For example, the antibody or antigen-binding fragment thereof can bind to biotin and then indirectly bind to a second conjugate, which is linked to avidin. The conjugate can be a detectable label, a pharmacokinetic modifying moiety, a purifying moiety, or a cytotoxic moiety. Examples of detectable labels may include fluorescent labels (e.g., fluorescence)A biotin-substrate marker (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, carbohydrate oxidase, or beta-D-galactosidase), a radioisotope (e.g., rhodamine, dansyl, phycoerythrin, or Texas Red), a fluorescent dye,¹²³I、¹²⁴I、¹²⁵I、¹³¹I、³⁵S、³H、¹¹¹In、¹¹²In、¹⁴C、⁶⁴Cu、⁶⁷Cu、⁸⁶Y、⁸⁸Y、⁹⁰Y、¹⁷⁷Lu、²¹¹At、¹⁸⁶Re、¹⁸⁸Re、¹⁵³Sm、²¹²Bi、and ³²p, other lanthanides, luminescent labels), chromophore moieties, digoxin, biotin/avidin, DNA molecules, or gold for detection. In certain embodiments, the conjugate can be a pharmacokinetic modifying moiety such as PEG, which helps to extend the half-life of the antibody. Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, ethylene glycol/propylene glycol copolymers, and the like. In certain embodiments, the conjugate can be a purification moiety such as a magnetic bead. A "cytotoxic moiety" may be any agent that is harmful to or may damage or kill a cell. Examples of cytotoxic moieties include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazide), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU), and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, doxycycline, and pharmaceutically acceptable salts thereof, Mitomycin C and cis-dichlorodiammineplatinum (II) (DDP) cisplatin), anthracycline antibiotics (e.g., daunorubicin (formerly daunorubicin) and doxorubicin), antibiotics (e.g., doxorubicinDactinomycin (formerly known as actinomycin), bleomycin, mithramycin and anthranilic Acid (AMC)) and antimitotic agents such as vincristine and vinblastine.

Polynucleotides and recombinant methods

The present application provides isolated polynucleotides encoding anti-PD-1 antibodies and antigen-binding fragments thereof. In certain embodiments, the isolated polynucleotide comprises one or more nucleotide sequences as in table 1 that encode a CDR sequence as in table 1.

In some embodiments, the isolated polynucleotide encodes a heavy chain variable region and comprises a sequence selected from the group consisting of seq id no: SEQ ID NO 46, SEQ ID NO 50, SEQ ID NO 54, SEQ ID NO 58, SEQ ID NO 62, and homologous sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity thereto. In some embodiments, the isolated polynucleotide encodes a light chain variable region and comprises a sequence selected from the group consisting of seq id no: SEQ ID NO 48, SEQ ID NO 52, SEQ ID NO 56, SEQ ID NO 60, SEQ ID NO 64, SEQ ID NO 68, and homologous sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity thereto. In certain embodiments, the percentage of identity is derived from the degeneracy of the genetic code, while the encoded protein sequence remains unchanged.

Vectors comprising polynucleotides encoding the anti-PD-1 antibodies and antigen-binding fragments thereof (e.g., comprising the sequences shown in table 1) can be introduced into host cells for cloning (amplification of DNA) or gene expression using recombinant techniques well known in the art. In another embodiment, the antibody can be made by methods of homologous recombination as are well known in the art. The DNA encoding the monoclonal antibody may be isolated and sequenced by conventional methods (e.g., oligonucleotide probes may be used which specifically bind to the genes encoding the heavy and light chains of the antibody). Various carriers can be selected. Carrier components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer sequence, a promoter (e.g., SV40, CMV, EF-1. alpha.) and a transcription termination sequence.

In some embodiments, the vector system comprises a mammalian, bacterial, yeast system, etc., and will include plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2, etc., other vectors available from the laboratory or commercially available. Suitable vectors may include plasmids or viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses).

Vectors comprising polynucleotides encoding the antibodies and antigen-binding fragments thereof may be introduced into host cells for cloning or gene expression. The host cells suitable for cloning or expressing the DNA in the vector of the present invention are prokaryotic cells, yeast or the above-mentioned higher eukaryotic cells. Prokaryotic cells suitable for use in the present invention include eubacteria such as, for example, gram-negative or gram-positive bacteria, e.g., Enterobacteriaceae, e.g., E.coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescens, and Shigella, and bacilli, e.g., Bacillus subtilis and Bacillus licheniformis, Pseudomonas, e.g., Pseudomonas aeruginosa, and Streptomyces.

In addition to prokaryotic cells, eukaryotic microorganisms such as filamentous fungi or yeast may also be used as host cells for cloning or expressing vectors encoding anti-PD-1 antibodies. Saccharomyces cerevisiae, or Saccharomyces cerevisiae, is the most commonly used lower eukaryotic host microorganism. However, many other genera, species and strains are more commonly used and are suitable for use in the present invention, such as Schizosaccharomyces pombe; kluyveromyces hosts such as Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces williamsii (ATCC 24,178), Kluyveromyces lactis (ATCC 56,500), Kluyveromyces drosophilus (ATCC 36,906), Kluyveromyces thermotolerans, and Kluyveromyces marxianus; yarrowia lipolytica (EP 402,226); pichia pastoris (EP 183,070); candida species; trichoderma reesei (EP 244,234); performing Neurospora; schwann yeast in western countries, such as: schwann yeast western; and filamentous fungi, such as: neurospora, Penicillium, Tolypocladium and Aspergillus, such as: aspergillus nidulans and Aspergillus niger.

The host cells provided herein that are suitable for expressing glycosylated antibodies or antigen binding fragments thereof are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Various baculovirus strains (bacterial strains) and variants thereof, as well as corresponding permissive insect host cells (permissive insect host cells), have been found to be derived from hosts such as: spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. A variety of viral strains for transfection are publicly available, such as Autographa californica nuclear polyhedrosis virus and Bm-5 variants of Bombyx mori nuclear polyhedrosis virus, all of which can be used in the present invention, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be used as hosts.

However, the most interesting are the vertebral cells, and the culture of the vertebral cells (tissue culture) has become a routine procedure. As examples of mammalian host cells which may be used, there are SV40 transformed monkey kidney cell CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 cell subclones in suspension culture, Graham et al, j.gen virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamster ovary cells/-DHFR (CHO, Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse testicular support cells (TM4, Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV1ATCC CCL 70); vero cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL 51); TRI cells (Mather et al, Annals N.Y.Acad.Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human liver cancer cell line (Hep G2). In certain preferred embodiments, the host cell is a 293F cell.

Host cells are transformed with the above-described expression or cloning vectors that produce anti-PD-1 antibodies and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformed cells, or amplifying genes encoding the sequences of interest.

The host cells of the invention used to produce the antibodies or antigen-binding fragments thereof can be cultured in a variety of media. Commercially available culture media such as Ham's F10(Sigma), minimal essential Medium (MEM, (Sigma)), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma may be used to culture the host cells. Additionally, any of the methods described in Ham et al, meth.enz.58:44(1979), Barnes et al, anal. biochem.102:255(1980), U.S. patent No. 4,767,704; 4,657,866, respectively; 4,927,762, respectively; 4,560,655, respectively; or 5,122,469; WO 90/03430; WO 87/00195; or the medium described in U.S. patent application Re.30,985, can be used as the medium for the host cells. These media may be supplemented with the necessary hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium chloride, magnesium chloride and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds, usually in the micromolar range, at final concentrations), and glucose or an equivalent energy source. The medium may also contain any other necessary additives at appropriate concentrations known in the art. The conditions of the medium, such as temperature, pH, and the like, which have been previously used to select host cells for expression, are well known to those of ordinary skill.

When using recombinant techniques, the antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, the particulate debris of the host cells or lysed fragments is first removed, for example, by centrifugation or sonication. Carter et al, Bio/Technology 10:163-167(1992) describes a method for isolating antibodies secreted into the membrane space of E.coli walls. Briefly, the cell paste (cell paste) was opened in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethanesulfonyl fluoride (PMSF) for about 30 minutes or more. Cell debris was removed by centrifugation. If the antibody is secreted into the culture medium, the supernatant of the expression system is usually first concentrated using commercially available protein concentration filters, such as Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be added in any of the foregoing steps to inhibit proteolytic degradation, as well as antibiotics to prevent the growth of adventitious contaminants.

The antibody produced from the cells can be purified by purification methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being a preferred purification technique. The class of the antibody and the presence of the Fc domain of any immunoglobulin in the antibody determines whether protein a is suitable as an affinity ligand. Protein a can be used to purify antibodies based on human gamma 1, gamma 2 or gamma 4 heavy chains (Lindmark et al, j. immunol. meth.62:1-13 (1983)). Protein G is applicable to all murine isoforms and human gamma 3(Guss et al, EMBO J.5: 15671575 (1986)). Agarose is the most commonly used affinity ligand attachment matrix, but other matrices may be used. Mechanically stable matrices such as controlled pore glass or poly (styrene) benzene can achieve faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, it can be purified using Bakerbond ABX. TM. resin (J.T.Baker, Phillipsburg, N.J.). Other techniques for protein purification may also be determined depending on the antibody to be obtained, such as fractionation in ion exchange columns, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin sepharose chromatography based on anion or cation exchange resins (e.g.polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

After any preliminary purification steps, the mixture containing the antibody of interest and impurities can be treated by low pH hydrophobic interaction chromatography, with a wash buffer at a pH of about 2.5-4.5, preferably at low salt concentrations (e.g., from about 0 to 0.25M salt concentration).

Reagent kit

Kits comprising the anti-PD-1 antibodies and antigen-binding fragments thereof are provided. In some embodiments, the kit is used to detect the presence or level of PD-1 in a biological sample. The biological sample may comprise a cell or tissue.

In some embodiments, the kit comprises an anti-PD-1 antibody and antigen-binding fragments thereof conjugated to a detectable label. In some embodiments, the kit comprises an unlabeled anti-PD-1 antibody and antigen-binding fragments thereof, and further comprises a secondary antibody that is capable of binding to the label of the unlabeled anti-PD-1 antibody and antigen-binding fragments thereof. The kit may further include instructions for use and packaging separating each component in the kit.

In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof are linked to a substrate or instrument for use in a sandwich assay such as an ELISA or immunochromatographic assay. Suitable substrates or instruments may be, for example, microplates and test strips.

Pharmaceutical compositions and methods of treatment

The present application further provides pharmaceutical compositions comprising the anti-PD-1 antibodies and antigen-binding fragments thereof and one or more pharmaceutically acceptable carriers.

Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein can include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous media, non-aqueous media, antimicrobial substances, isotonic substances, buffers, antioxidants, anesthetics, suspending/dispersing agents, chelating agents, diluents, adjuvants or nontoxic auxiliary substances, other components well known in the art, or various combinations thereof.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, mercaptosorbitol, butyl methyl anisole, butylated hydroxytoluene, and/or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in a composition comprising an antibody or antigen-binding fragment thereof disclosed herein will reduce oxidation of the antibody or antigen-binding fragment thereof. The reduction in oxidation prevents or reduces the reduction in binding affinity, thereby improving antibody stability and extending shelf life. Thus, in certain embodiments, the invention provides compositions comprising one or more of the antibodies or antigen-binding fragments thereof and one or more antioxidants, such as methionine. The present invention further provides methods of preventing oxidation, extending shelf life and/or increasing activity of an antibody or antigen-binding fragment thereof provided herein by admixing the antibody or antigen-binding fragment thereof with one or more antioxidants, such as methionine.

Further, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, ringer's solution injection, isotonic glucose injection, sterile water injection, or dextrose and lactate injection, non-aqueous media such as: fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil, antibacterial substances at bacteriostatic or fungistatic concentrations, isotonic agents such as: sodium chloride or glucose, buffers such as: phosphate or citrate buffers, antioxidants such as: sodium bisulfate, local anesthetics such as: procaine hydrochloride, suspending and dispersing agents such as: sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone, emulsifiers such as: polysorbate 80 (tween-80), chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antibacterial agents as carriers may be added to the pharmaceutical composition in multi-dose containers, including phenolics or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride, and benzalkonium chloride. Suitable excipients may include, for example, water, salt, glucose, glycerol or ethanol. Suitable non-toxic auxiliary substances may include, for example, emulsifiers, pH buffers, stabilizers, solubilizers, or substances such as sodium acetate, sorbitan laurate, triethanolamine oleate or cyclodextrins.

The pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharine, cellulose, magnesium carbonate, and the like.

In certain embodiments, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions may be prepared in any conventional form, for example, liquid solvents, suspending agents, emulsifying agents, or solid forms suitable for the production of liquid solvents, suspending agents or emulsifying agents. Injectable preparations may include ready-to-use sterile and/or pyrogen-free solutions, sterile dried solubles, e.g., lyophilized powders, combined with solvents prior to use, including subcutaneous tablets, sterile suspensions ready for injection, sterile dried insoluble products, combined with vehicles prior to use, and sterile and/or pyrogen-free emulsions. The solvent may be aqueous or non-aqueous.

In certain embodiments, a unit dose of an injectable formulation is packaged in an ampoule, a manifold, or a syringe with a needle. It is well known in the art that all formulations for injection administration should be sterile pyrogen free.

In certain embodiments, sterile lyophilized powders can be prepared by dissolving an antibody or antigen-binding fragment thereof disclosed herein in an appropriate solvent. The solvent may contain a compound that enhances the stability of the powder or reconstituted solution prepared from the powder, or improves the pharmacological properties of the powder or reconstituted solution. Suitable excipients include, but are not limited to, water, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable materials. The solvent may contain a buffer, such as citric acid buffer, sodium or potassium phosphate buffer or other buffers known to those skilled in the art, and in one embodiment, the pH of the buffer is neutral. The dissolution is followed by sterile filtration under standard conditions well known in the art and then lyophilized to produce the desired formulation. In one embodiment, the resulting solvent is dispensed into vials for lyophilization. Each tubule may contain a single dose or multiple doses of the anti-PD-1 antibody or antigen binding fragment thereof or composition thereof. The loading per vial may be slightly higher than that required for each dose or for multiple doses (e.g., 10% excess), thereby ensuring accurate sampling and accurate dosing. The lyophilized powder may be stored under appropriate conditions, such as in the range of about 4 ℃ to room temperature.

And re-dissolving the freeze-dried powder with water for injection to obtain the preparation for injection administration. In one embodiment, the lyophilized powder can be reconstituted by addition to sterile pyrogen-free water or other suitable liquid carrier. The precise amount is determined by the selected therapy and can be determined empirically.

Also provided are methods of treatment comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein to a subject in need thereof, thereby treating or preventing a condition or disorder associated with PD-1. In another aspect, there is also provided a method of treating a condition in a subject that would benefit from an upregulated immune response, comprising administering to the subject in need thereof a therapeutically effective amount of an antibody, or antigen-binding fragment thereof, as described herein.

The therapeutically effective dose of the antibody or antigen-binding fragment thereof provided herein depends on a variety of factors well known in the art, such as body weight, age, past medical history, current therapy, the health status of the subject and the potential for cross-infection, allergies, hypersensitivity and side effects, as well as the route of administration and the extent of tumor development. One skilled in the art (e.g., a physician or veterinarian) can proportionately lower or raise the dosage based on these or other conditions or requirements.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein can be administered at a therapeutically effective dose of between about 0.01mg/kg and about 100mg/kg (e.g., about 0.01mg/kg, about 0.5mg/kg, about 1mg/kg, about 2mg/kg, about 5mg/kg, about 10mg/kg, about 15mg/kg, about 20mg/kg, about 25mg/kg, about 30mg/kg, about 35mg/kg, about 40mg/kg, about 45mg/kg, about 50mg/kg, about 55mg/kg, about 60mg/kg, about 65mg/kg, about 70mg/kg, about 75mg/kg, about 80mg/kg, about 85mg/kg, about 90mg/kg, about 95mg/kg, or about 100 mg/kg). In certain embodiments, the antibody or antigen-binding fragment thereof is administered at a dose of about 50mg/kg or less, and in certain embodiments, 10mg/kg or less, 5mg/kg or less, 1mg/kg or less, 0.5mg/kg or less, or 0.1mg/kg or less. A particular dose can be administered at multiple intervals, such as once daily, twice or more monthly, once weekly, once every two weeks, once every three weeks, once monthly, or once every two or more months. In certain embodiments, the dosage administered may vary over the course of treatment. For example, in certain embodiments, the initial administered dose may be higher than the subsequent administered dose. In certain embodiments, the dosage administered is adjusted during the course of treatment according to the response of the subject to whom it is administered.

The dosage regimen may be adjusted to achieve an optimal response (e.g., therapeutic response). For example, administration can be carried out as a single dose or in multiple divided doses over a period of time.

The antibodies and antigen-binding fragments disclosed in the present invention can be administered by administration means well known in the art, such as injection (e.g., subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous drip, intramuscular injection, or intradermal injection) or non-injection (e.g., oral, nasal, sublingual, rectal, or topical administration).

The PD-1 associated condition or disorder can be an immune-related disease or disorder. In certain embodiments, the conditions and disorders associated with PD-1 include tumors and cancers, such as non-small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoides (mycoses fungoides), meckel cell carcinoma and other hematologic malignancies, such as Classical Hodgkin Lymphoma (CHL), primary mediastinal large B cell lymphoma, B-rich lymphoma of T cells/histiocytes, EBV positive and negative PTLD and EBV-associated Diffuse Large B Cell Lymphoma (DLBCL), plasmacytic lymphoma, extranodal NK/T cell lymphoma, nasopharyngeal cancer and HHV 8-associated primary effusion lymphoma, Hodgkin's lymphoma, Central Nervous System (CNS) tumors, e.g. primary CNS lymphoma, spinal axis tumors, brain stem glioma. In certain embodiments, the tumors and cancers are metastatic, particularly metastatic tumors that express PD-L1. In certain embodiments, the conditions and disorders associated with PD-1 include autoimmune diseases, such as Systemic Lupus Erythematosus (SLE), psoriasis, systemic scleroderma, autoimmune diabetes. In certain embodiments, the conditions and disorders associated with PD-1 include chronic viral infections, such as hepatitis b, hepatitis c, herpes virus, Epstein-Barr virus, aids virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus 2, human papilloma virus, viral infections of adenovirus, kaposi's sarcoma-associated herpes virus epidemics, pseudocyclopia virus (Torquetenovirus), JC virus or BK virus, and the like.

Application method

The application further provides methods of using the anti-PD-1 antibodies or antigen-binding fragments thereof.

In some embodiments, the present application provides a method of treating a condition or disorder associated with PD-1 in an individual comprising administering a therapeutically effective amount of a PD-1 antibody or antigen-binding fragment thereof described herein. In some embodiments, the individual is identified as having a disorder or condition that is likely to respond to a PD-1 antagonist.

The presence and level of PD-L1 in a target biological tissue can indicate whether the individual from which the biological sample is derived is likely to respond to a PD-1 antagonist. Various methods may be used to determine the presence or level of PD-L1 in a test biological sample from the individual. For example, the biological sample to be tested may be exposed to an anti-PD-L1 antibody or antigen-binding fragment thereof, which binds to the expressed PD-L1 protein and detects the expressed PD-L1 protein. Alternatively, PD-L1 can be detected at the nucleic acid expression level using, e.g., qPCR, reverse transcription PCR, microarray, SAGE, FISH, and the like. In some embodiments, the test sample is derived from a cancer cell or tissue, or an immune cell that enters a tumor. In some embodiments, the presence or level of PD-L1 in the biological sample to be tested is upregulated indicating the likelihood of a response. The term "upregulation" as used herein means a total increase in the level of PD-L1 protein detected in a test sample of no less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or more, using an antibody or antigen binding fragment thereof as described herein, as compared to the level of PD-L1 protein in a reference sample detected using the same antibody. The reference sample may be a control sample obtained from a healthy or disease-free individual, or a healthy or disease-free sample obtained from an individual from which the test sample is derived. For example, the reference sample can be a disease-free sample that is near or adjacent to the test sample (e.g., tumor).

The antibodies and antigen-binding fragments disclosed herein can be administered alone or in combination with one or more other therapeutic means or substances. For example, the antibodies and antigen-binding fragments disclosed herein may be used in combination with chemotherapy, radiation therapy, cancer treatment surgery (e.g., tumor resection), treatment of one or more anti-emetic drugs or other chemotherapy-induced complications, or any other therapeutic substance for cancer or any other condition mediated by PD-1. In certain such embodiments, the antibodies and antigen-binding fragments disclosed herein, when used in combination with one or more therapeutic agents, may be administered concurrently with the one or more therapeutic agents, and in certain such embodiments, the antibodies and antigen-binding fragments may be administered concurrently as part of the same pharmaceutical composition. However, the antibody and antigen conjugate "in combination" with other therapeutic substances need not be administered simultaneously or in the same composition as the therapeutic substance. The meaning of "in combination" in the present invention also includes that an antibody and an antigen-binding agent administered before or after another therapeutic substance are also considered to be "in combination" with the therapeutic substance, even if the antibody or antigen-binding fragment thereof and the second substance are administered by different administration means. Where possible, other therapeutic agents to be used in combination with the antibodies or antigen-binding fragments thereof disclosed herein may be administered by Reference to the methods of the product specification for the other therapeutic agent, or by Reference to surgeon's docket No. 2003(Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (11 months 2002)), or by Reference to other methods known in the art.

In certain embodiments, the therapeutic substance is capable of inducing or enhancing an immune response against the cancer. For example, tumor vaccines can be used to induce an immune response to certain tumors or cancers. Cytokine therapy can be used to enhance the presentation of tumor antigens to the immune system. Examples of cytokine therapy include, but are not limited to, interferons such as interferon alpha, beta and gamma, colony stimulating factors such as macrophage CSF, granulocyte macrophage CSF and granulocyte-CSF, interleukins such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12, tumor necrosis factors such as TNF-alpha and TNF-beta. Agents that inactivate immunosuppressive targets, such as TGF- β inhibitors, IL-10 inhibitors, and Fas ligand inhibitors, may also be used. Another group of agents include those that activate an immune response against a tumor or cancer cell, for example, those that increase T cell activation (e.g., T cell co-stimulatory molecule agonists such as CTLA-4, ICOS, and OX-40), and those that increase dendritic cell function and antigen presentation.

The following examples are intended to better illustrate the invention and should not be construed as limiting the scope of the invention. All of the specific compositions, materials and methods described below, in whole or in part, are within the scope of the invention. These specific compositions, materials and methods are not intended to limit the invention, but are merely illustrative of specific embodiments within the scope of the invention. Those skilled in the art may develop equivalent compositions, materials, and methods without adding inventive step and without departing from the scope of the invention. It will be appreciated that various modifications to the method of the invention may still be included within the scope of the invention. The inventors intend such variations to be included within the scope of the present invention.

Example 1: generation of antibody hybridomas

1.1 Generation of immunogen: the full length DNA encoding the ECD of PD-1 and PD-L1 or both was synthesized and inserted into the expression vector pcDNA3.3. Plasmid DNA was prepared in large quantities and the inserted DNA sequence was verified by sequencing. Fusion proteins of PD-1ECD and PD-L1ECD, which contain a variety of tags, including human Fc, mouse Fc and His tags, were prepared by transfection of the human PD-1ECD gene into CHO-S or HEK293 cells. After 5 days, the supernatant harvested from the transiently transfected cell culture was used for protein purification. The fusion protein was purified and quantified for immunization and screening.

1.2 establishment of stable cell lines. To obtain a tool for antibody screening and validation, we established PD-1 and PD-L1 transfected cell lines. Briefly, the pCND3.3 expression vectors containing full-length PD-1 or PD-L1 were transfected into CHO-K1, 293F or Ba/F3 cells using the Lipofectamine 2000 transfection kit according to the manufacturer's instructions. 48-72 hours after transfection, the transfected cells were cultured in medium containing Blasticidin (Blasticidin) or G418 for selection. After a period of time, cells stably incorporating the PD-1 or PD-L1 gene in the genomic DNA will be selected. Meanwhile, whether the cells have the expression of the target genes PD-1 and PD-L1 is verified. Once the expression is verified, individual clones of interest are picked by limited dilution and scaled up to large capacity. The established monoclonal cell lines were then maintained in medium containing low doses of the antibiotics Blasticidin (Blasticidin) or G418.

1.3 establishment of antibody hybridomas.

1.3.1 immunization and cell fusion: primary challenge immunizations were performed using 8-10 week old OMT-rats (available from Open Monoclonal Technology, inc., Palo Alto, US) on the footpad with 10 μ g of human PD-1ECD protein in TiterMax, repeated every 3 days with PD-1ECD formulated with aluminum. Sera were collected from rats every 2 weeks and antibody titers were determined by ELISA or FACS assays. When the antibody titer reached a sufficiently high value, rats were given a final challenge without adjuvant (100. mu.l of 1XPBS was added instead) and cell fusion was performed as follows: lymph node B isolated from immunized OMT-ratThe lymphocytes were cell fused with myeloma cells (in a 1:1 ratio). The cell mixture was washed and suspended with 5-10ml of ECF solution. The ECF solution was added to adjust the concentration to 2x10⁶Cells/ml. Immediately after electrofusion of the cells, the cell suspension in the fusion chamber was transferred into a sterile tube containing a larger volume of medium. After incubation at 37 ℃ for more than 24 hours, the cell suspensions were mixed and pipetted into 96-well plates (0.5 × 10)⁶Cells/plate). At 37 deg.C, 5% CO₂The cells are cultured under conditions. When the clones were large enough, 100 μ Ι of supernatant was transferred from the 96-well plate for antibody screening test.

1.3.2 first round and confirmation screening of hybridoma supernatants: an ELISA test was used as a first round of screening to test hybridoma supernatants for binding to PD-1 protein. Briefly, plates were coated with 1. mu.g/ml of soluble protein of the extracellular domain of human PD-1 overnight at 4 ℃. After blocking and washing, the hybridoma supernatants were transferred to the coated plates and incubated at room temperature for 1 hour. The plates were then washed and then incubated for 45 minutes with goat anti-rat IgG1HRP (Bethyl) and goat anti-rat IgG2b HRP (Bethyl) secondary antibody. After washing, TMB substrate was added and the reaction was stopped with 2M HCl. The absorbance at 450nm was read using a microplate reader (Molecular Device). To confirm the natural binding of the PD-1 antibody to the conformational PD-1 molecule expressed on the cell membrane, FACS analysis was performed on PD-1 transfected CHO-S cell lines. At 1x10⁶Concentration of cells/ml CHO-S cells expressing PD-1 were transferred to a 96-well U-bottom plate (BD). The hybridoma supernatants were then transferred to the plates and incubated at 4 ℃ for 1 hour. After washing with 1 XPBS/1% BSA, goat anti-rat FITC (Jackson Immunoresearch Lab) secondary antibody was added and the cells were incubated at 4 ℃ for 1 hour in the dark. The cells were then washed and resuspended in 1 XPBS/1% BSA or fixed in 4% formalin and analyzed by flow cytometry (BD). Binding of the antibody to the parental CHO-S cell line was performed using the same method. FIG. 1 shows the binding of anti-human PD-1 antibody to CHO cells expressing PD-1. CHO cells transfected with full-length human PD-1 were conjugated with anti-human PD-1 antibody from rat hybridoma, followed by FITC-conjugated goat anti-rat-IgG FcSecondary antibody staining was performed and analyzed by FACS. The data show that the antibody specifically binds to PD-1 expressed on CHO cells.

To test the binding affinity of the antibodies to native PD-1 expressed on human CD4+ T cells, human CD4+ T cells were obtained from PBMC cultures cultured for 3 days in IL-2 and OKT3 and bound with anti-human PD-1 antibodies. Binding of the antibody to PD-1 on the T cells was analyzed by FACS. As shown in fig. 3, FACS analysis showed that the antibody specifically binds to native PD-1 expressed on CD4+ T cells.

The antibodies were tested for blocking activity as confirmation screens to select potential antibodies of interest. Selected antibodies were tested for their ability to block the binding of ligand PD-L1 to PD-1 transfected CHO-S cells by FACS analysis. At 1x10⁶Concentration of cells/ml CHO-S cells expressing human PD-1 were transferred to a 96-well U-bottom plate (BD). The antibodies were serially diluted in wash buffer (1 XPBS/1% BSA) and the cells were incubated for 1 hour at 4 ℃. After washing, human Fc fusion-human PD-L1 protein was added and incubated at 4 ℃ for 1 hour. A secondary antibody of goat anti-human IgG Fc FITC antibody (no cross-reactivity with rat IgG Fc, Jackson Immunoresearch Lab) was incubated with the cells at 4 ℃ for 1 hour in the absence of light. The cells were then washed and resuspended in 1 XPBS/1% BSA or fixed in 4% formalin and analyzed by flow cytometry (BD).

1.3.3 hybridoma subcloning: once specific binding and blocking is verified by the first round and confirmation screening, the positive hybridoma cell line can be used for subcloning. Briefly, for each hybridoma cell line, cells were counted and diluted in cloning media to 5 cells/well, 1 cell/well, and 0.5 cells/well. 200 μ l/well were plated into 96-well plates, one plate at 5 cells/well, one plate at 1 cell/well and four plates at 0.5 cells/well. All plates were incubated at 37 ℃ with 5% CO₂. Incubate until all cell lines can be checked by ELISA assay.

Example 2: antibody hybridoma cell sequencing and fully human antibody characterization

2.1 antibody hybridoma cell sequencing: RNA was isolated from monoclonal hybridoma cells using Trizol reagent. VH and VL of PD-1 antibody were amplified using the following protocol: briefly, RNA is first reverse transcribed into cDNA using reverse transcriptase as described in the present application, reaction system (20 μ l):

reaction conditions

The resulting cDNA was used as a template for subsequent PCR amplification using specific primers for the gene of interest. The PCR reaction was performed by the following steps:

reaction conditions are as follows:

mu.l of the PCR reaction product was taken for ligation with pMD18-T vector. Top10 competent cells were transformed with 10. mu.l of the ligation product and the mixture was transferred to 2-YT + Cab plates pre-warmed according to standard protocols and incubated overnight. Positive clones were checked by PCR using M13-48 and M13-47 primers and subsequently sequenced.

2.2 construction of fully human antibody molecules: VH and VL of PD-1 antibody were amplified as described above. The PCR reaction product was purified by PCR clean-up kit and VL and pCI vectors were digested with restriction enzymes Pme I and BssH II at 37 ℃ for 2 hours. The reaction products were electrophoresed in a 1% agarose gel and gel extracted according to the manufacturer's instructions. The digested VL and pCI vectors were ligated using the following steps:

the mixture was incubated at 16 ℃ for 30 minutes. Transformation and clonal propagation were performed with 10. mu.l of reaction product. Plasmid pCI-VL DNA extraction was performed using the confirmed clones. The pCI-VL vector and VH fragment were then digested with Xbal and Sal I and the purified digested VH and vector were ligated with T4DNA ligase at 16 ℃ for 30 min. Once the inserted VL and VH sequences were verified by sequencing, transient transfection and establishment of stable cell lines were performed using an expression vector containing whole IgG of fully human PD-1 antibody.

Example 3: characterization of fully human antibodies

3.1 full kinetic binding affinity of Surface Plasmon Resonance (SPR) assay: the affinity and binding kinetics of the antibody to PD-1 were characterized by SPR using ProteOn XPR36 (Bio-Rad). Protein A protein (Sigma) was immobilized on a GLM sensor chip by amine coupling (Bio-Rad). The purified antibody was flowed over the sensor chip and captured by protein a. The chip was rotated 90 ℃ and washed with running buffer (1 XPBS/0.01% Tween20, Bio-Rad) until the baseline stabilized. 5 concentrations of human PD-1 and running buffer were flowed through the antibody flow cell at a flow rate of 100. mu.L/min, first for 240s for the bound phase and then 600s for the dissociated phase. After each run with H pH 1.7₃PO₄Regenerating the chip. Binding and dissociation curves were fitted to a 1:1 Langmiur binding model using ProteOn software.

As shown in FIG. 7, the affinity of the anti-PD-1 antibody detected by using surface plasmon resonance for recombinant human-derived PD-1 was from 3.76E-9 to 1.76E-10 mol/L.

3.2 binding affinity of PD-1 antibodies to cell surface PD-1 molecules as determined by flow cytometry (FACS): the binding affinity of the antibodies to cell surface PD-1 was tested by FACS analysis. At 5x10⁵Concentration of cells/ml CHO-S cells expressing PD-1 were transferred to96-well U-shaped bottom plate (BD). The test antibody was diluted 1:2 serially with wash buffer (1 XPBS/1% BSA) and incubated at 4 ℃ for 1 hour. Secondary goat anti-human IgG Fc FITC (3.0 moles FITC per mole IgG, (Jackson Immunoresearch Lab)) was added and incubated at 4 ℃ for 1 hour protected from light. The cells were then washed once and resuspended in 1 XPBS/1% BSA and analyzed using flow cytometry (BD). Based on the quantified beads (Quantum. MESF Kit (Bangs Laboratories, Inc.), fluorescence intensity was converted to bound molecules per cell. K was calculated using Graphpad Prism5_D. FIG. 2 shows the binding of fully human PD-1 antibodies (i.e., 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15hAb, and 1.153.7hAb) to CHO cells expressing PD-1. Binding of PD-1 transfected CHO cells with fully human antibodies against human PD-1 and FACS analysis showed that fully human PD-1 antibodies had an EC of 2nmol/L₅₀Specifically binds to PD-1.

CHO cells expressing human PD-1 were incubated with different concentrations of anti-PD-1 antibody, and mouse Fc-labeled human PD-L1 was subsequently added to the cells. Binding of human PD-L1 to PD-1 expressing cells was detected using FITC-conjugated goat anti-mouse IgG, followed by FACS analysis. As shown in FIG. 4, anti-PD-1 antibody blocked the binding of PD-L1 to PD-1 transfected CHO cells.

3.3 homologous Gene (across species) and homolog (across family) screens:

3.3.1 Cross-reactivity with cynomolgus monkey PD-1 and murine PD-1: cross-reactivity was determined by ELISA. Plates (Nunc) were coated with 1. mu.g/ml cynomolgus monkey PD-1(Sino Biological) and murine PD-1(Sino Biological) overnight at 4 ℃. After blocking and washing, 1. mu.g/ml antibody was added to the plate and incubated for 1 hour at room temperature. The plates were then washed and then incubated with goat anti-rat IgG1HRP (Bethyyl) and goat anti-rat IgG2bHRP (Bethyyl) for 45 minutes. After washing, TMB substrate was added and the reaction was stopped with 2M HCl. The absorbance at 450nm was read using a microplate reader (Molecular Device).

The results of the cross-species experiments showed that the anti-PD-1 antibody bound to cynomolgus monkey PD-1 but not murine PD-1 (fig. 6).

3.3.2 Cross-reactivity with PD-1 family members CD28, CTLA4 and ICOS: to detect the cross-family binding activity of the fully human antibody, cell lines expressing PD-1, CD28, CTLA4, or ICOS were stained with antibody, followed by secondary antibody binding to goat anti-human-IgG Fc conjugated FITC. PD-1 expressing cells served as positive controls. The corresponding parental cell line served as a negative control. The bound cells were analyzed using BD Biosciences FACSCAnto II and FlowJo version software.

FIG. 5 shows PD-1, or CD28 transfected CHO cells, and CTLA4 transfected 293F cells stained with anti-PD-1 antibodies and analyzed by FACS. The results show that the PD-1 antibody specifically binds to PD-1, but not to CD28 and CTLA4 of the PD-1 family.

3.4 epitope Classification (Binning) test

3.4.1 binding epitopes of PD-1 antibodies sorting for reference antibodies A and B was performed by SPR assay using ProteOn XPR36 (Bio-Rad). The reference antibodies A and B were immobilized on a GLC sensor chip (Bio-Rad) by amine-based coupling. The human PD-1 solution was flowed through the path of the immobilized antibody and captured with the reference antibody. The chip was then rotated 90 ° and washed with running buffer until the baseline stabilized. Flowing the selected antibody through the sensor chip.

3.4.2 binding epitopes of the PD-1 antibody were classified by FACS against the reference antibodies A and B. CHO cells expressing human PD-1 at the cell surface were incubated with reference antibodies A and B at 10. mu.g/ml for 1 hour. The cells were washed and incubated for 1 hour with the addition of PD-1 antibody of the present application. A secondary antibody against rat IgG-FITC was added and incubated at 4 ℃ for 1 hour. The cells were then washed 1 time and resuspended in 1 XPBS/1% BSA and analyzed by flow cytometry (BD).

The SPR test and FACS results of the classification test indicate that the binding epitope of the fully human PD-1 antibody (i.e., 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15hAb, and 1.153.7hAb) on human PD-1 is different from that of the known PD-1 antibodies (i.e., reference antibodies a and B).

3.5 cell-based assays to determine in vitro function of PD-1 antibodies

3.5.1 Effect of human PD-1 antibody on T cell proliferation. The effect of PD-1 antibodies on T lymphocyte proliferation was tested using an allogeneic response. In 96-well U-bottomed tissue culture plates containing 10% FCS and anti-mousePrimary Dendritic Cell (DC) -stimulated MLR was performed in 200. mu.l of RPMI 1640 of biotin. Mixing DC with 1X10⁵Allogeneic total CD4⁺T cells were mixed at a ratio of DC to T cells of 1:10 and 1: 100. Culturing in the presence or absence of neutralizing mAb: the human PD-1 antibody and the reference antibodies A and B were used at a concentration of 10. mu.g/ml. Incubation for 5 days, 1 uCi/well of [ 2 ] was added during the last 16 hours³H]Thymidine. Measurement by scintillation counting³H]Incorporation of thymidine using a triple-well mean [ 2 ]³H]Thymidine incorporation (counts per minute) indicates a proliferative response. The DC-only counting is conventionally<1000 cpm. The results shown are representative examples of a minimum of 5 trials.

Human Dendritic Cells (DCs) and CD4 used in the above allogeneic MLR⁺T、CD8⁺T and total cells were generated from PBMCs as follows: human monocytes were purified from PBMCs by negative selection using a human monocyte concentration kit (human monocyte enrichment cocktail kit) according to the instructions of the manufacturer (StemCell Meylan). Briefly, PBMCs were isolated from healthy donor blood using a Ficoll-Paque gradient. Cells were washed twice with PBS, followed by 1X10 in isolation buffer⁸Cells/ml were resuspended and the Ab mix was concentrated with monocytes and incubated at 4 ℃ for 30 min. Unlabeled monocytes passed through the MACS column were collected. To generate iDCs, monocytes were cultured with GM-CSF (PeproTech, Rocky Hill, N.J.; 800U/ml) and IL-4 (PeproTech; 500U/ml) in RPMI 1640 medium containing 10% FCS and antibiotics at a cell concentration of 2X10⁶Cells/ml. Half of the medium was replaced daily with medium containing GM-CSF and IL-4. iDCs were stimulated with LPS (026: B6; Sigma-Aldrich, St. Louis, MO; 1. mu.g/ml) for an additional 24 hours on day 5 to generate mature DCs. PBMC were prepared by contacting human CD4 with PBMC according to the manufacturer's instructions (Stemsep)⁺T、CD8⁺Purification of CD4 by negative selection of concentrated mixtures of T and Total T cells incubated with magnetic colloids⁺T、CD8⁺T and total T cells.

Stimulation of human CD4 with allogeneic DCs in the presence or absence of PD-1 antibodies 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15hAb and 1.153.7hAb⁺T cells. Warp beam[³H]Evaluation of Thymidine incorporation CD4⁺Proliferation of T cells. FIG. 10 shows that 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15hAb, and 1.153.7hAb increase concentration-dependent T cell proliferation.

3.5.2 effects of in vitro humanized PD-1 antibodies on cytokine IFN γ secretion: to evaluate the blocking effect of human PD-1 antibody on the production of cytokine IFN γ, we performed an experiment of IFN γ production in allogeneic-MLR. Briefly, CD4 was used according to the manufacturer's instructions⁺T cell concentration kit (CD 4)⁺T cell enrichment cocktail kit) human CD4 was negative screened⁺T cells were purified from PBMC. Immature DCs were generated from monocytes cultured in GM-CSF and IL-4 for 5 days and differentiated into mature DCs by overnight stimulation with LPS at 1. mu.g/ml. Will CD4⁺T cells and iDC/mDC were mixed at T: DC ratios of 10:1 and 100: 1. The culture is performed in the presence or absence of a human PD-1 antibody and a standard antibody. After 5 days, supernatants from each culture were collected for determination of the cytokine IFN γ. IFN γ levels in the supernatant were determined by ELISA assay. Briefly, Maxisorp plates (0.75. mu.g/ml; i.e., 1/1360 diluted) were coated with anti-human IFN γ mAb diluted in coating buffer, 50. mu.l/well (i.e., 3.7. mu.l of antibody was added to a full 96-well plate in 5ml of coating buffer) and incubated overnight at 4 ℃. Add 200. mu.l/well of blocking buffer for 2 hours to block excess protein binding capacity. Dilutions of recombinant IFN γ were prepared as standards and were diluted two-fold with complete medium from 8000pg/ml to 125pg/ml, plus only complete medium. Plates were washed, standard and test supernatants (100. mu.l/well) were added and incubated for 2-4 hours. Biotinylated anti-IFN γ mAb (1/1333) in blocking buffer was added followed by additional avidin peroxidase. The reaction was performed by adding TMB substrate and quenched with 2M HCl. Absorbance was measured at 450 nm.

FIG. 9 shows the stimulation of human CD4 with allogeneic DCs in the presence or absence of the 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15hAb, and 1.153.7hAb antibodies⁺T cells. IFN γ levels were determined by ELISA. The results show that the fully human PD-1 antibody increases IFN γ in a dose-dependent mannerAnd (4) secreting.

3.5.3 Effect of human PD-1 on Interleukin 2(IL-2) production in vitro: will CD4⁺T cells and iDC/mDC were mixed at T: DC ratios of 10:1 and 100: 1. The culture is performed in the presence or absence of a human PD-1 antibody and a standard antibody. After 5 days, supernatants from each culture were collected for cytokine determination. IL-2 levels in the supernatants were determined by ELISA assays.

FIG. 8 shows stimulation of human CD4 with allogeneic DCs in the presence or absence of an antibody of the present application or a control antibody⁺T cells. IL-2 levels were determined by ELISA. The results show that fully human PD-1 antibody increases IFN γ secretion in a dose-dependent manner. The results show that anti-PD-1 antibodies increase IL-2 secretion in a dose-dependent manner.

3.5.4 Effect of human PD-1 antibody on cell proliferation and cytokine production by autoantigen specific immune response: in this assay, T cells and DC cells are from the same donor. Briefly, CD4 was purified from PBMCs⁺T cells were cultured in CMV pp65 peptide and low dose IL2(20U/ml) while DCs were generated from monocytes cultured in PBMCs of the same donor in GM-CSF and IL-4. After 5 days, CD4 treated with CMV pp65 peptide⁺T cells were co-cultured with DCs to which pp65 peptide was pulsed in the presence or absence of human PD-1 antibody and a reference antibody (as a control).

On day 5, 100. mu.l of supernatant from each culture was used for the determination of the cytokines IFN. gamma. and IL-2. The level of production of IFN γ and IL-2 was detected by ELISA assay. A specific T cell proliferation targeting DC pulsed with CMV pp65 peptide³H]Thymidine incorporation assay.

As shown in FIG. 11, PD-1 antibodies increased the concentration-dependent CMV stimulated by autologous CMV pp65 peptide-loaded DCs⁺-CD4⁺Proliferation of T cells.

3.5.5 Effect of human PD-1 antibodies on regulatory T cell (Tregs) suppressive function: tregs are a subset of T cells, which are key immune regulators and play a key role in maintaining self-tolerance.

CD4⁺CD25⁺Regulatory T cells are associated with tumors because of the increased number of Tregs found in patients with a variety of cancers and are associated with a poorer prognosis. To directly assess the effect of human PD-1 antibodies on the immunosuppressive response, we performed Tregs experiments. Separation of CD4 Using specific anti-CD 25 Microbeads (Miltenyi Biotec, Auburn, Calif.) and Positive or negative selection, respectively⁺CD25⁺And CD4⁺CD25^-T cells. Initially, human CD4 was used according to the manufacturer's instructions (Stemsep)⁺T cell concentration mixture and magnetic colloid incubation of PBMC, negative selection purification of CD4⁺T cells. Then resuspend CD4 in MACS buffer⁺T cells, on ice with CD25⁺The beads were incubated for 30 minutes, washed and packed. Collecting non-column bound CD4 from the effluent solution⁺CD25^-T cells, and washed prior to use. CD4 was subsequently recovered from the column⁺CD25⁺T cells and washed prior to use. Tregs were combined with CD4 in the presence or absence of human PD-1 antibody at a concentration of 10. mu.g/ml⁺CD25^-T cells and DCs (Treg: Teff ratio 1:1) were cultured. No antibody or isotype antibody was used as a negative control. On day 5, the supernatant of the culture was taken for detection of cytokines by ELISA, by adding at a concentration of 1 uCi/well³H]Thymidine and further cultured for 18 hours, and cell proliferation was detected. [³H]Thymidine incorporation was counted by scintillation. As shown in figure 12, the PD-1 antibody abolished Treg inhibitory function and restored responsive T cell proliferation and IFN γ secretion.

3.6ADCC/CDC assay: for healthy PD-1⁺The undesired cytotoxicity was minimized and the selected anti-PD-1 fully human antibodies were confirmed to be free of ADCC and CDC functions.

3.6.1 ADCC: activated T cells expressing high levels of cell surface PD-1 were used as target cells and pre-incubated with varying concentrations of fully human antibody in 96-well plates for 30 minutes, followed by the addition of IL-2 activated PBMC (used as a source of Natural Killer (NK) cells, i.e., effector cells) at an effector/target ratio of 50: 1. [ 5% CO at 37 ℃%₂Incubate the plate in the incubator for 6 hours. By cytotoxicity testAssay kit (Roche) measures target cell lysis. The optical density was determined by Molecular Devices SpectraMax M5e microplate reader. The results show that the fully human anti-PD-1 antibodies tested do not mediate ADCC (figure 13).

3.6.2 CDC: target cells (activated T cells), diluted human serum complement (Quidel-a112), and various concentrations of fully human PD-1 antibody were mixed in 96-well plates. At 37 deg.C, 5% CO₂Incubate the plate in the incubator for 4 hours. Target cell lysis was measured by CellTiter glo (Promega-G7573). Rituxan (Roche) and human B lymphocyte cell fine Raji (CD20 positive) were used as positive controls. The data show that CDC was not mediated by PD-1 antibodies (fig. 14).

Example 4: epitope identification of fully human antibodies

To determine the epitope difference between the antibodies 1.103.11hAb and Keytruda (the current hPD-1 antibody) described herein, alanine scanning mutagenesis experiments (alanine scanning experiment) and antibody binding efficacy evaluations against hPD-1 were performed using 1.103.11hAb, Keytruda, and 11.148.10 (which are control hPD-1 antibodies that bind epitopes that do not overlap with the epitope of 1.103.11hAb or Keytruda).

The alanine residue in hPD-1 was mutated to a glycine codon and all other residues were mutated to alanine codons. Point substitutions of amino acids were made for each residue of the extracellular domain (ECD) of hPD-1 using two-step sequential PCR. The plasmid pcDNA3.3-hPD-1_ ECD.His, which encodes the ECD and C-terminal His-tag of human PD-1, was used as a template, a set of mutagenic primers was used as a first step PCR, and a QuikChangeLighting multipoint site-directed mutagenesis kit (Agilent technologies, Palo Alto, Calif.) was used. The parent template was digested after the mutation combination reaction using the Dpn I endonuclease. In the second PCR, a linear DNA expression cassette containing the CMV promoter, PD-1 extracellular domain (ECD), His-tag, and polyadenylation of herpes simplex virus Thymidine Kinase (TK) was amplified and transiently expressed in HEK293F cells (Life Technologies, Gaithersburg, MD).

Monoclonal antibodies 1.103.11hAb, keytruda, and 11.148.10hAb were coated on plates for ELISA binding assays. HRP-conjugated anti-His antibody (Rockland, Cat # 200-. The absorbance was normalized according to the mean value of the control mutations. After additional thresholding of the binding fold changes (<0.55), the finally determined epitope residues are identified.

The hPD-1 mutation of the first 30 point substitutions that significantly reduced antibody binding is listed in table 2. Checking the position of all these residues on the hPD-1 crystal structure (PDB codes 3RRQ and 4ZQK) shows that some amino acids (e.g. val144, Leu142, Val110, Met108, Cys123, etc.) are completely buried in the protein and are unlikely to come into direct contact with any antibody. The observed reduction in binding is most likely due to instability or even structural collapse of the hPD-1 structure following alanine substitution. To avoid misinterpreting these data as epitopic hotspots, control antibody 11.148.10hAb (which binds to a completely different location on the antigen, but is expected to respond to the collapse of the hPD-1 structure) was used. Mutations affecting both antibodies were considered as false hot spots and removed from the list. After additional thresholding of the fold change of binding (<0.55), the finally determined epitope residues are listed in table 3. They were 9 positions for 1.103.11hAb and 5 positions for Keytruda, and 10 residues for the control antibody 11.148.10 hAb.

TABLE 2 Effect of PD-1 point mutations on antibody binding

Bold: amino acids overlapping with control 11.148.10hAb for structural maintenance were excluded from the hot spot list.

^aFold changes in binding are relative to binding of several silent alanine substitutions.

TABLE 3 potential epitopes recognized

Critical value fold change <0.55

^*C, C ', F, G, A' represent a peptide chain having a crystal structure of hPD-1 in FIG. 17. The C "chain observed in mPD-1 is absent in the hPD-1 structure. The beta-sheet is replaced with an unstructured loop at hPD-1. For ease of comparison with mPD-1 only, this region is still marked with C ".

Comparison of the epitope residues of 1.103.11hAb and Keytruda in Table 3 shows two overlapping hot spot residues. The other residues appeared to be quite diverse, indicating that the two antibodies may have very different mechanisms in binding hPD-1 and blocking hPD-L1. Reading the residue ID in table 3 does not provide an intuitive interpretation of the mechanism. Therefore, all the data in Table 3, as well as the hPD-L1 binding site, were plotted on the crystal structure of hPD-1 for better visualization and comparison (FIG. 17).

As shown in FIG. 17, the hot spot residues responsible for hPD-L1 binding were all centered between C, F and the G chain (FIG. 17A). The two antibodies 1.103.11hAb and Keytruda studied, while functional in both binding hPD-1 and blocking hPD-L1, apparently have different epitopes (1.103.11 hAb in FIG. 17B and Keytruda in FIG. 17C). The Keytruda epitope is mainly contributed by residues on the C' D loop (corresponding to the C "chain of mPD-1), which do not cleave the PD-L1 binding site at all. This suggests that the function of the Keytruda block hPD-L1 is more dependent on the steric hindrance effect provided by the size of the antibody. In contrast, the epitope of antibody 1.103.11hAb of the present application consists of hot spots distributed at multiple locations and directly overlaps with the hPD-L1 binding site (fig. 17A and 17B). The 1.103.11hAb of the present application blocked hPD-L1 by having a higher competition than hPD-L1 with the common binding site of both. Therefore, 1.103.11hAb is expected to be more functional in downstream development.

However, antibody 11.148.10hAb bound a completely different position compared to the two functional antibodies (fig. 17D), which demonstrates itself as a good control antibody for monitoring the function of hPD-1 in making alanine substitutions.

While the disclosure has been particularly shown and described with reference to specific embodiments, some of which are preferred, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. An isolated antibody or antigen-binding fragment thereof, comprising:

a) a heavy chain variable region comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 3 and CDR3 of SEQ ID NO. 5; and a light chain variable region comprising CDR1 of SEQ ID NO. 7, CDR2 of SEQ ID NO. 9 and CDR3 of SEQ ID NO. 11;

b) a heavy chain variable region comprising CDR1 of SEQ ID NO 13, CDR2 of SEQ ID NO 15 and CDR3 of SEQ ID NO 5; and a light chain variable region comprising CDR1 of SEQ ID NO. 7, CDR2 of SEQ ID NO. 17 and CDR3 of SEQ ID NO. 11;

c) a heavy chain variable region comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 15 and CDR3 of SEQ ID NO. 5; and a light chain variable region comprising CDR1 of SEQ ID NO. 7, CDR2 of SEQ ID NO. 17 and CDR3 of SEQ ID NO. 19;

d) a heavy chain variable region comprising CDR1 of SEQ ID NO:21, CDR2 of SEQ ID NO:23 and CDR3 of SEQ ID NO: 25; and a light chain variable region comprising CDR1 of SEQ ID NO:27, CDR2 of SEQ ID NO:29 and CDR3 of SEQ ID NO: 31; or

e) A heavy chain variable region comprising CDR1 of SEQ ID NO:33, CDR2 of SEQ ID NO:35 and CDR3 of SEQ ID NO: 37; and a light chain variable region comprising CDR1 of SEQ ID NO:39, CDR2 of SEQ ID NO:41 and CDR3 of SEQ ID NO: 43.

2. The antibody or antigen-binding fragment thereof of claim 1, comprising:

d) a heavy chain variable region comprising SEQ ID NO 57; and a light chain variable region comprising SEQ ID NO 59; or

e) A heavy chain variable region comprising SEQ ID NO 61; and a light chain variable region comprising SEQ ID NO 63.

3. The antibody or antigen binding fragment thereof of claim 1 or 2, which is present in an amount of no more than 10^-8The Kd value of M, which is determined by plasmon resonance binding, specifically binds to human PD-1.

4. The antibody or antigen-binding fragment thereof of claim 1 or 2, with an EC of no more than 100nM, or no more than 10nM₅₀Binding to monkey PD-1, and/or no binding to mouse PD-1.

5. The antibody or antigen-binding fragment thereof of claim 1 or 2, which blocks binding of human or monkey PD-1 to its ligand with an IC50 of no more than 100 nM.

6. The antibody or antigen binding fragment thereof of claim 1 or 2, which does not bind to CD28 or CTLA 4.

7. The antibody or antigen-binding fragment thereof of claim 1 or 2, which does not mediate ADCC or CDC or both.

8. The antibody or antigen-binding fragment thereof of claim 1 or 2, which is a fully human monoclonal antibody.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein said fully human monoclonal antibody is produced by a transgenic rat.

10. The antibody or antigen binding fragment thereof of claim 1 or 2, which blocks binding of human PD-1 to its ligand and thus provides at least one of the following activities:

a) in CD4⁺Inducing IL-2 production in T cells;

b) in CD4⁺Inducing production of IFN γ in T cells;

c) induction of CD4⁺Proliferation of T cells; and

d) reversing the T reg inhibitory function.

11. The antibody or antigen-binding fragment thereof of claim 1 or 2, which is a bifunctional antibody (diabody), scFv dimer, dsFv, (dsFv)2, dsFv-dsFv ', Fv fragment, Fab ', or F (ab ') 2.

12. The antibody or antigen-binding fragment thereof of claim 11, wherein the diabody is BsFv or ds diabody (ds diabody).

13. The antibody or antigen-binding fragment thereof of claim 1 or 2, further comprising an immunoglobulin constant region.

14. The antibody or antigen-binding fragment thereof of claim 1 or 2, further comprising a conjugate.

15. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-14.

16. A vector comprising the isolated polynucleotide of claim 15.

17. A host cell comprising the vector of claim 16.

18. A method of expressing the antibody or antigen-binding fragment thereof of any one of claims 1-14, comprising culturing the host cell of claim 17 under conditions in which the isolated polynucleotide of claim 15 is expressed.

19. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-14.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-14 in the manufacture of a medicament for treating a PD-1-associated condition in an individual, said PD-1-associated condition being a tumor, cancer, autoimmune disease, or chronic viral infection associated with immunosuppression by PD-1.

21. The use of claim 20, wherein the individual is identified as having a disorder or condition responsive to a PD-1 antagonist.

22. The use of claim 21, wherein the individual is identified as being positive for PD-L1 or up-regulated in PD-L1 levels in a test biological sample from the individual.

23. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-14 and one or more pharmaceutically acceptable carriers.

24. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-14 in the manufacture of a medicament for treating immunosuppression caused by PD-1.

25. The use of claim 24, wherein the medicament is for treating cancer or chronic viral infection associated with immunosuppression by PD-1.