PD-1 agonistic antibody and use thereof

Abstract

Provided in the present invention are a PD-1 agonistic antibody and the use thereof. Specifically provided in the present invention are an agonistic antibody against PD-1, a pharmaceutical composition containing the PD-1 agonistic antibody, the use of the anti-human-PD-1 agonistic antibody in the preparation of a drug for treating autoimmune diseases, and a method for treating autoimmune diseases using the anti-human-PD-1 agonistic antibody.

Description

PD-1 agonistic antibodies and uses thereof

[0001] Cross-reference to related applications

[0002] This application claims the benefit of Chinese Patent Application No. CN202411165494.9, filed August 23, 2024; the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application belongs to the technical field of medicine, and specifically relates to PD-1 agonistic antibodies and uses thereof. BACKGROUND

[0004] Excessive activation of the immune system, and lack of effective negative regulation, leads to autoimmune diseases. When the body's own tissues are subject to an adverse immune response, the immune system is equipped with mechanisms to suppress immune cells to reduce the immune response in order to prevent falling into such a pathological state. Immune checkpoint molecules, such as CTLA-4, PD-1, can initiate immune suppression regulatory mechanisms during the activation of immune cells, and down-regulate the intensity of the immune response. In fact, blocking the function of immune checkpoints CTLA-4 or PD-1 can reverse the suppression of anti-tumor immunity, and the resulting immune activation brings significant anti-tumor effects. At the same time, in cancer patients receiving PD-1 inhibitor immunotherapy, a certain proportion of adverse reactions caused by excessive immune response were observed. This fact shows that the immune checkpoint-mediated immune suppression mechanism plays a very significant role and can become an important target for the treatment of autoimmune diseases caused by excessive activation of the immune response.

[0005] PD-1 (Programmed Death 1) is a receptor expressed on activated immune cells (such as T cells, B cells, NK, NKT cells, etc.), and its expression is strictly regulated. PD-1 belongs to the Ig superfamily, containing an extracellular IgV domain, a transmembrane domain and an intracellular domain. PD-L1 and PD-L2, which are ligands of PD-1, are expressed in a variety of cell types, including antigen-presenting cells, cells of the lung, heart, thymus, spleen and kidney. Once PD-L1 or PD-L2 binds to PD-1, the immunoreactivity of T cells is inhibited through a signal transduction pathway, such as reduced cell proliferation, reduced cell survival, and inhibition of inflammatory cytokines (e.g., IFNγ, TNFα and IL-2).

[0006] Upon stimulation, PD-1 is phosphorylated at the immunoreceptor switch motif (ITSM) of its cytoplasmic portion, which then binds to the SH2-containing phosphatase 2 (SHP2), initiating T cell inactivation. In addition, in PD-1-mediated immune suppression, CD28 is dephosphorylated, further inhibiting T cell function.

[0007] There is a strong correlation between autoimmune disease conditions and the PD-1 pathway. Mice lacking PD-1 develop spontaneous lupus-like autoimmune disease. In humans, single nucleotide polymorphisms in the PD-1 gene are associated with higher incidence of systemic lupus erythematosus, type 1 diabetes, multiple sclerosis, rheumatoid arthritis progression. In addition, patients with SLE in the flare stage often have lower PD-L1 expression.

[0008] In the treatment of autoimmune diseases, the immune response is inhibited by the action of stimulating the immunosuppression pathway, which is to fully utilize the endogenous immune checkpoint pathway in the body to regulate the autoimmune response. The result can lead to significant disease improvement and response persistence, which is an effective supplement to current immunomodulatory therapy. SUMMARY

[0009] To achieve the above technical purpose, the present application provides an agonistic antibody against PD-1 (also known as CD279), a composition comprising such an anti-human PD-1 agonistic antibody, and a method for treating autoimmune diseases using such a PD-1 agonistic antibody, and the specific technical solutions are as follows:

[0010] In a first aspect, the present application provides an antibody or an antigen-binding portion thereof that specifically binds to PD-1 or a functional fragment thereof, the antibody or the antigen-binding portion thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL);

[0011] The VH includes:

[0012] I. a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3;

[0013] II. a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, wherein SEQ ID NO: 23: YIDPYYGNTRYX1QKFX2G; and / or

[0014] III. a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5; and

[0015] The VL includes:

[0016] I) a light chain complementarity determining region 1 (LCDR1) having at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6;

[0017] II) a light chain complementarity determining region 2 (LCDR2) having at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; and

[0018] III) a light chain complementarity determining region 3 (LCDR3) having at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24, wherein SEQ ID NO: 24: QHYYX3SPX4T;

[0019] wherein the X1is selected from A, N, or S;

[0020] the X2is selected from Q or K;

[0021] the X3is selected from N or S;

[0022] the X4is selected from P or V.

[0023] In some embodiments, the CDRs are defined based on the Kabat numbering system. However, one skilled in the art would appreciate that the CDRs are defined herein by way of example only, and other numbering systems such as IMGT, Chothia, Martin, Gelfand, AbM, Contact, and the like are also suitable for defining the CDRs of the antibodies or antigen-binding portions thereof provided herein.

[0024] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL);

[0025] wherein the VHcomprises:

[0026] i. a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence as set forth in SEQ ID NO: 3; or a heavy chain complementarity determining region 1 (HCDR1) having at least one amino acid difference compared to the amino acid sequence set forth in SEQ ID NO: 3;

[0027] ii. a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence as set forth in SEQ ID NO: 23; or a heavy chain complementarity determining region 2 (HCDR2) having at least one amino acid difference compared to the amino acid sequence set forth in SEQ ID NO: 23, wherein SEQ ID NO: 23: YIDPYYGNTRYX1QKFX2G; and

[0028] iii. a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence as set forth in SEQ ID NO: 5; or a heavy chain complementarity determining region 3 (HCDR3) having at least one amino acid difference compared to the amino acid sequence as set forth in SEQ ID NO: 5; and

[0029] The VL comprises:

[0030] i) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence as set forth in SEQ ID NO: 6; or a light chain complementarity determining region 1 (LCDR1) having at least one amino acid difference compared to the amino acid sequence as set forth in SEQ ID NO: 6;

[0031] ii) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence as set forth in SEQ ID NO: 7; or a light chain complementarity determining region 2 (LCDR2) having at least one amino acid difference compared to the amino acid sequence as set forth in SEQ ID NO: 7;

[0032] iii) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence as set forth in SEQ ID NO: 24; or a light chain complementarity determining region 3 (LCDR3) having at least one amino acid difference compared to the amino acid sequence as set forth in SEQ ID NO: 24, wherein SEQ ID NO: 24: QHYYX3SPX4T;

[0033] wherein the X1is selected from A, N, or S;

[0034] the X2is selected from Q or K;

[0035] the X3is selected from N or S;

[0036] the X4is selected from P or V;

[0037] In some embodiments, the amino acid difference is 1-3, e.g., 1, 2, 3.

[0038] In some embodiments, the amino acid difference is an amino acid substitution.

[0039] In some preferred embodiments, the amino acid difference is a substitution of a conservative amino acid.

[0040] In some embodiments, the antibody or antigen binding portion thereof comprises:

[0041] A1 : the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 3, 4, and 5, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively; or

[0042] A2: the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 3, 19, and 5, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively; or

[0043] A3: the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 3, 4, and 5, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 20, respectively; or

[0044] A4: the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 3, 21, and 5, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively; or

[0045] A5: the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 3, 21, and 5, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 22, respectively.

[0046] In some embodiments, the VH has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of:

[0047] SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17.

[0048] In some embodiments, the VL has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of:

[0049] SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18.

[0050] In some embodiments, the VH and VL are selected from the group consisting of:

[0051] S1) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 1; and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 2; or

[0052] S2) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 9; and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 10; or

[0053] S3) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 11; and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 12; or

[0054] S4) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 13; and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 14; or

[0055] S5) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 15; and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 16; or

[0056] S6) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 17; and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 18.

[0057] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL);

[0058] wherein,

[0059] the VH comprises HCDR1, HCDR2, and HCDR3 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17:

[0060] the VL comprises LCDR1, LCDR2, and LCDR3 contained in an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18;

[0061] wherein the CDRs can be defined based on Kabat numbering, or other numbering systems such as IMGT, Chothia, Martin, Gelfand, AbM, Contact, etc.

[0062] In some embodiments, the antibody or antigen-binding portion thereof is a murinized, chimerized, or humanized antibody or antigen-binding portion thereof.

[0063] In some preferred embodiments, the antibody or antigen-binding portion thereof is a humanized antibody or antigen-binding portion thereof.

[0064] In some embodiments, the antibody or antigen-binding portion thereof comprises an immunoglobulin Fc region or a functional variant thereof.

[0065] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a human immunoglobulin Fc region or a functional variant thereof.

[0066] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises an Fc region of human IgG1, IgG2, IgG3, or IgG4, or a functional variant thereof.

[0067] In some preferred embodiments, the antibody or antigen-binding portion thereof has a human immunoglobulin Fc region or a functional variant thereof with V12 mutations, which include any one of the following amino acid mutations or a combination of multiple amino acid mutations:

[0068] E233D, G237D, P238D, H268D, P271G, A330R.

[0069] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a human immunoglobulin Fc region or a functional variant thereof with amino acid mutations E233D / G237D / P238D / H268D / P271G / A330R.

[0070] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises an Fc region of human IgG1, or a functional variant thereof.

[0071] In some embodiments, the antibody or antigen-binding portion thereof is directly linked to the Fc region or linked through a linker.

[0072] In some preferred embodiments, the linker is a polypeptide.

[0073] In some preferred embodiments, the linker is a glycine (G) and serine (S) rich polypeptide, such as GGGGS, (GGGGS) n (GGGGGS) n and the like.

[0074] In some embodiments, the antibody is a monoclonal antibody.

[0075] In some embodiments, the antigen binding moiety is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, dAb, nanobody, Fd, and Fd'.

[0076] In a second aspect, the present application provides a fusion protein comprising any of the aforesaid antibodies or antigen binding moieties thereof.

[0077] In some embodiments, the fusion protein further comprises a protein or polypeptide that increases the stability and / or delays the half-life of the antibody or antigen binding moiety thereof.

[0078] In some embodiments, the fusion protein further comprises a protease cleavage site.

[0079] In a third aspect, the present application provides a multispecific antibody comprising any of the aforesaid antibodies or antigen binding moieties thereof or fusion proteins, and one or more additional antigen binding regions, wherein the one or more additional antigen binding regions bind to a different antigen or to a different epitope of the same antigen.

[0080] In a fourth aspect, the present application provides an isolated nucleic acid molecule encoding any of the aforesaid antibodies or antigen binding moieties thereof, fusion proteins, or multispecific antibodies.

[0081] In a fifth aspect, the present application provides an expression vector comprising any of the aforesaid nucleic acid molecules.

[0082] In some embodiments, the nucleic acid molecule is operably linked to an expression control element.

[0083] In a sixth aspect, the present application provides a recombinant cell comprising any of the aforesaid nucleic acid molecules or expression vectors.

[0084] In a seventh aspect, the present application provides a composition comprising a group consisting of:

[0085] any of the aforesaid antibodies or antigen binding moieties thereof, fusion proteins, multispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells.

[0086] In some embodiments, the composition further comprises at least one pharmaceutically acceptable carrier.

[0087] In some preferred embodiments, the pharmaceutically acceptable carrier comprises, but is not limited to, a solvent, a diluent, a disintegrant, a precipitation inhibitor, a surfactant, a glidant, a binder, a lubricant, a dispersant, a suspending agent, an isotonic agent, a thickening agent, an emulsifying agent, a preservative, a stabilizer, a hydrating agent, an emulsion accelerator, a buffer, an absorbent, a coloring agent, a flavoring agent, a sweetening agent, an ion exchange agent, a release agent, a coating agent, a flavoring agent, or an antioxidant.

[0088] In an eighth aspect, the present application provides a method for preparing any of the foregoing antibodies or antigen-binding portions thereof, fusion proteins, or multispecific antibodies, the method comprising culturing the foregoing recombinant cell under conditions that allow expression of the antibodies or antigen-binding portions thereof, fusion proteins, or multispecific antibodies, and isolating and / or purifying the antibodies or antigen-binding portions thereof, fusion proteins, or multispecific antibodies from the culture of the recombinant cell.

[0089] In a ninth aspect, the present application provides use of any of the foregoing antibodies or antigen-binding portions thereof, fusion proteins, multispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells, and / or compositions in the manufacture of a medicament for preventing and / or treating a PD-1 related disease.

[0090] In a tenth aspect, the present application provides any of the foregoing antibodies or antigen-binding portions thereof, fusion proteins, multispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells, and / or compositions for use in preventing and / or treating a PD-1 related disease.

[0091] In some embodiments, the PD-1 related disease comprises an autoimmune disease.

[0092] In some embodiments, the autoimmune disease comprises, but is not limited to, graft-versus-host disease, solid organ transplant rejection, vasculitis, systemic lupus erythematosus, type I diabetes, multiple sclerosis, giant cell arteritis, psoriasis, rheumatoid arthritis, inflammatory bowel disease (IBD), non-specific ulcerative colitis, autoimmune hepatitis.

[0093] In an eleventh aspect, the present application provides use of any of the foregoing antibodies or antigen-binding portions thereof, fusion proteins, multispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells, and / or compositions for treating a PD-1 related disease in a subject in need thereof.

[0094] In a twelfth aspect, the present application provides a method for preventing and / or treating a PD-1 related disease, comprising administering to a subject in need thereof a therapeutically effective amount of any of the foregoing antibodies or antigen-binding portions thereof, fusion proteins, multispecific antibodies, or compositions.

[0095] In some embodiments, the subject is a mammal.

[0096] In some preferred embodiments, the subject is a human.

[0097] In some embodiments, the PD-1 related disease comprises an autoimmune disease.

[0098] In some preferred embodiments, the autoimmune disease comprises graft versus host disease, solid organ transplant rejection, vasculitis, systemic lupus erythematosus, type I diabetes, multiple sclerosis, giant cell arteritis, psoriasis, rheumatoid arthritis, IBD, non-specific ulcerative colitis, autoimmune hepatitis.

[0099] In some embodiments, the method comprises administering to a subject in need thereof the antibody or antigen-binding portion thereof, the fusion protein, or the multispecific antibody in combination with another therapeutic agent.

[0100] In some embodiments, the therapeutic agent is a therapeutic antibody.

[0101] In some preferred embodiments, the therapeutic agent is a therapeutic antibody against an immune checkpoint.

[0102] In some preferred embodiments, the immune checkpoint comprises, but is not limited to, CTLA-4, PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, 4-1BB, OX40, etc.

[0103] The antibody provided by the present disclosure has the following advantages:

[0104] (1) High affinity, can specifically recognize human PD-1 protein, and the binding activity is close to that of the positive control Peresolimab;

[0105] (2) The antibody is humanized, avoiding the immunogenicity of murine antibodies;

[0106] (3) Strong agonistic activity, similar to the agonistic activity of the positive control Peresolimab;

[0107] (4) Significantly slows down the weight loss of GvHD mice, prolongs the survival of mice, and shows potential in the treatment of autoimmune diseases. BRIEF DESCRIPTION OF DRAWINGS

[0108] Figure 1 shows the binding of antibody 5A3 of the present application to recombinant human PD-1 protein, with ANB030, Peresolimab as controls.

[0109] Figure 2 shows the binding of antibody 5A3 of the present application to recombinant cynomolgus monkey PD-1 protein, with ANB030, Peresolimab as controls.

[0110] Figure 3 shows the binding of antibody 5A3-hu01, 5A3-hu02, 5A3-hu03, 5A3-hu-R2, 5A3 of the present application to Jurkat-NFAT-luc-PD-1 cells, with ANB030, Peresolimab as controls.

[0111] Figure 4 and Figure 5 show the agonistic activity of antibody 5A3-hu01, 5A3-hu02, 5A3-hu03, 5A3-hu-R2, 5A3 of the present application in reporter cell lines, with ANB030, Peresolimab as controls.

[0112] Figure 6 and Figure 7 show the agonistic activity of antibody 5A3-hu02 in primary cell lines, with Peresolimab as control.

[0113] Figure 8 shows the binding of antibody 5A3-AM17 after affinity maturation to Jurkat-NFAT-luc-PD-1 cells, with ANB030, Peresolimab as controls.

[0114] Figure 9 shows the ADCC activity of antibody 5A3-AM17 and 5A3-AM17-V12 after affinity maturation, with Peresolimab, Peresolimab-V12 as controls.

[0115] Figure 10 shows the agonistic activity of antibody 5A3-AM17 and 5A3-AM17-V12 after affinity maturation in reporter cell lines, with Peresolimab, Peresolimab-V12 as controls.

[0116] Figure 11 shows the agonistic activity of antibody 5A3-AM17 and 5A3-AM17-V12 after affinity maturation in primary T cells, with Peresolimab, Peresolimab-V12 as controls.

[0117] FIG. 12 and FIG. 13 show the therapeutic effects of antibody 5A3-AM17 after affinity maturation on GVHD mice, Peresolimab and Rosnilimab as controls. FIG. 12 shows the effects of each antibody on the weight change of mice, and FIG. 13 shows the effects of each antibody on the survival rate of mice. DETAILED DESCRIPTION

[0118] In order to make the objects, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions of the embodiments of the present disclosure will be described clearly and completely below with reference to the drawings of the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of protection of the present disclosure.

[0119] The present disclosure can be implemented in other specific forms without departing from the essential attributes of the present disclosure. It should be understood that any and all embodiments of the present disclosure can be combined with the technical features in any other embodiment or multiple other embodiments to obtain additional embodiments, without conflict. The present disclosure includes the additional embodiments obtained by such combination.

[0120] All publications and patents mentioned in the present disclosure are hereby incorporated by reference in their entirety. If the use or the term used in any publication and patent incorporated by reference herein conflicts with the use or the term used in the present disclosure, the use and the term in the present disclosure shall prevail.

[0121] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0122] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following definitions are applied to the descriptions and claims herein, and apply equally when used in the singular or in the plural.

[0123] Unless the context clearly indicates otherwise, as used herein, the construction "a" or "an" is taken to include plural referents unless the context clearly indicates otherwise. For example, reference to "a cell" includes a plurality of such cells as well as equivalents thereof known to those skilled in the art, and so forth.

[0124] As shown herein, the term "about" denotes a range of ±20% of the numerical value that follows. In some embodiments, the term "about" denotes a range of ±10% of the numerical value that follows. In some embodiments, the term "about" denotes a range of ±5% of the numerical value that follows.

[0125] Numerical ranges as used herein are intended to include all numbers and combinations of numbers within the range. For example, a range of 1 to 20 is intended to include any number from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0126] As used in the present disclosure, the terms "comprises", "comprising", "includes", "including" or "comprising" means "including, but not limited to". The terms are intended to be open-ended to designate the presence of any stated features, elements, integers, steps, or components, but do not preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" encompasses the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" as used throughout the disclosure, and in the claims, can be replaced with the term "consisting of".

[0127] As used in the present disclosure, the terms "optional", "any", "any of", or "any one of" mean that the subsequently described event or circumstance can or can not occur, and the description includes instances where the event or circumstance occurs and instances where it does not. As used in the present disclosure, "one" and "an" are used to refer to one or more than one of the grammatical object of the article.

[0128] As used in the present disclosure, "and / or" is to be taken as specific

[0129] As shown herein, the term "antibody" refers to an immunoglobulin molecule that is generally composed of two pairs of polypeptide chains (each pair having one light (LC) and one heavy (HC) chain). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define a different class of antibodies as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids. The heavy chains also contain a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant domains are not involved directly in binding of an antibody to an antigen, but exhibit various effector functions, such as mediating the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy chain / light chain pair form the antigen binding site.

[0130] As used herein, the term "complementarity determining region" or "CDR" refers to amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these regions can be defined according to various numbering systems known in the art, e.g., as defined by the Rabat numbering system (Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system, or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one of skill in the art will readily be able to identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well understood by those skilled in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). In this document, the assignment of amino acids to various regions or domains can be defined according to the Rabat numbering system, or according to the IMGT, Chothia, or Contact numbering system.

[0131] As used herein, the term "antigen binding portion" is used interchangeably with "antigen binding fragment," and refers to a polypeptide fragment of an antibody, e.g., a polypeptide fragment of a full length antibody, that retains the ability to specifically bind to the same antigen bound by the full length antibody, and / or competes with the full length antibody for specific binding to the antigen, which is also known as an "antigen binding portion." Antigen binding fragments of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (e.g., technology from Ablynx), domain antibodies (e.g., technology from Domantis), and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136. Preferably, an antigen binding portion retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parent antibody to the target.

[0132] As used herein, the term "full length antibody" means an antibody composed of two "full length heavy chains" and two "full length light chains". Among them, the "full length heavy chain" refers to a polypeptide chain composed of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain in the direction from N-terminus to C-terminus; and, when the full length antibody is of IgE isotype, optionally further comprising a heavy chain constant region CH4 domain. Preferably, the "full length heavy chain" is a polypeptide chain composed of VH, CH1, HR, CH2 and CH3 in the direction from N-terminus to C-terminus. The "full length light chain" is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the direction from N-terminus to C-terminus. The two pairs of full length antibody chains are connected together by a disulfide bond between CL and CH1 and a disulfide bond between the hinge regions of the two full length heavy chains. The full length antibody of the present disclosure can be from a single species, for example, human; can also be a chimeric antibody or a humanized antibody, preferably a humanized antibody. The full length antibody of the present disclosure comprises two antigen binding sites formed by a pair of VH and VL, respectively, which specifically recognize / bind the same antigen.

[0133] The term "murine antibody" in the present disclosure is a monoclonal antibody derived from mice prepared according to the knowledge and skills in the art. When preparing, the test subject is injected with an antigen, and then a hybridoma expressing an antibody with the desired sequence or functional characteristics is isolated. When the injected test subject is a mouse, the resulting antibody is a murine antibody.

[0134] The term "chimeric antibody" is an antibody in which the variable region of a murine antibody is fused with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, a hybridoma secreting a murine-specific monoclonal antibody is first established, and then the variable region gene is cloned from the murine hybridoma cells, and the constant region gene of the human antibody is cloned as needed. The murine variable region gene is linked to the human constant region gene to form a chimeric gene, which is then inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system. The antibody light chain of the chimeric antibody further comprises the light chain constant region of the human κ, λ chain or its variant. The antibody heavy chain of the chimeric antibody further comprises the heavy chain constant region of the human IgG1, IgG2, IgG3, IgG4 or its variant, preferably the heavy chain constant region of the human IgG1, IgG2 or IgG4, or the IgG1, IgG2 or IgG4 heavy chain constant region variant using amino acid mutations (such as YTE mutation or back mutation, L234A and / or L235A mutation, or S228P mutation).

[0135] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting a mouse CDR sequence into a human antibody variable region framework, i.e., a human germline antibody framework sequence of different types. This overcomes the heterologous response induced by chimeric antibodies carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available at www.mrccpe.com.ac.uk / vbase). To avoid a decrease in activity along with a decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse or reversion mutations to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies further demonstrated by yeast with affinity maturation mutations of the CDR.

[0136] The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids arranged side-by-side through the ternary folding of a protein. Epitopes formed from adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost after treatment with denaturing solvents. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitopes bind to a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of epitopes include techniques in the art, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0137] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K0) of the interaction. D () indicates. In this invention, the term "K" is used. D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. In some embodiments, an antibody that specifically binds to a certain antigen (or an antibody that is specific to a certain antigen) means that the antibody binds at a dissociation equilibrium constant of less than about 10-1. -9 M, for example, less than about 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M or lower affinity (K) D ) binds to the antigen. In some implementations, when KD ≤ 10 x 10 -8 M (preferably K D ≤ 5 x 10 -9 M) the antibody of the application or antigen-binding fragment thereof is considered to specifically bind PD-1. The specific binding property between two molecules can be determined using methods well known in the art. One method involves measuring the rate of antigen binding site / antigen complex formation and dissociation. The "association rate constant" (k a or k on ) and the "dissociation rate constant" (k dis or k off ) can both be calculated from the concentration and the actual rates of association and dissociation. The ratio of k dis / k on equals the dissociation constant K D . K D , k on and k dis values can be measured using any effective method. In certain embodiments, the dissociation constant can be measured using bioluminescence interferometry (e.g., ForteBio Octet method). In addition to this, the dissociation constant can be measured using surface plasmon resonance technology (e.g., Biacore) or Kinexa.

[0138] As used herein, the terms "conservative substitution," "conservative amino acid substitution," and "conservative replacement" are used interchangeably and mean an amino acid substitution that does not adversely affect or alter the intended properties of a protein / polypeptide comprising the amino acid sequence. Conservative replacements can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis, for example. Conservative amino acid substitutions include substitutions of an amino acid residue for an amino acid residue with similar side chains, e.g., substitutions that take place within a family of amino acid residues that have similar side chains e.g., residues that are physically or functionally similar (e.g., have similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a preferred substitution is one in which the replacement amino acid residue is from the same side chain family as the corresponding amino acid residue. Methods of identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); and Burks et al. Proc. Natl Acad Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0139] As shown in the present disclosure, "identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent identity between two sequences is a function of the number of matching or homologous positions shared by the sequences divided by the number of positions compared, multiplied by 100% to yield the percentage. For example, if 6 of 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous over the 10 positions. Generally, the comparison is made over the length of the sequence claimed, when the two sequences are optimally aligned. According to some embodiments of the present disclosure, "identity" refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence or a nucleotide sequence can be determined using homology search sites on the Internet, such as the BLAST page of the NCBI home page website. For example, the identity of a pair of amino acid sequences can be calculated in Advanced BLAST 2.1 by using blastp as the program, setting Expect value to 10, setting all Filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, and performing the search, and then the value of identity (%) is obtained.

[0140] As used herein, the terms "monoclonal antibody", "monoclonal", "mAb" have the same meaning and are used interchangeably to refer to an antibody or a fragment of an antibody from a population of highly homogenous antibody molecules, i.e., a population of antibody molecules that are identical except for naturally occurring mutations that can arise during production. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are in contrast to monoclonal antibodies and generally comprise at least 2 or more different antibodies that generally recognize different epitopes on an antigen. Furthermore, the term "monoclonal" indicates only that the antibody is characterized as being obtained from a population of highly homogenous antibodies and does not indicate that the antibody must be prepared by any particular method.

[0141] The writing of the twenty conventional amino acids referred to in the present disclosure follows the conventional usage. In the present disclosure, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present disclosure, amino acids are generally represented by the single and three letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0142] As used herein, the term "isolated" refers to a state that is obtained by artificial means from a natural state. If a certain "isolated" substance or component exists naturally, it can be because its natural environment has changed, or the substance is separated from the natural environment, or both. For example, a certain non-isolated polynucleotide or polypeptide exists naturally in a certain living organism, and a high-purity same polynucleotide or polypeptide separated from the natural state is referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixed artificial or synthetic substances, nor does it exclude other impurities that do not affect the activity of the isolated substance. For example, an isolated antibody can be substantially free of other cell materials and / or chemicals.

[0143] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer of at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) that are typically linked together by phosphodiester bonds. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded, and can be cDNA.

[0144] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a polynucleotide inserted into it, the vector is referred to as an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by the vector are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 -derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papova viruses (such as SV40). A vector can contain various elements to control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain a replication initiation site.

[0145] As used herein, the terms "recombinant cell" and "host cell" are used interchangeably and refer to a cell that can be used to introduce a vector, including but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0146] As used in the present disclosure, the term "pharmaceutically acceptable carrier / excipient" refers to a carrier / excipient that is compatible, in pharmacology and / or physiology, with the subject and the active ingredient, which is well known in the art, and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents to delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thiomersal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art, which are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), and the like.

[0147] As used in the present disclosure, the term "prevention" refers to a method implemented in order to stop or delay the occurrence of a disease or disorder or a symptom (e.g., a tumor, an infection or an autoimmune disease) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present disclosure, a beneficial or desired clinical result includes, but is not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of the state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Moreover, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0148] As used in the present disclosure, the term "subject" refers to a mammal, such as a primate, e.g., a human. In certain embodiments, the subject (e.g., human) has an autoimmune disease, or is at risk of having an autoimmune disease. In certain embodiments, the subject (e.g., human) has an autoimmune disease, or is at risk of having an autoimmune disease.

[0149] As used in the present disclosure, the term "effective amount" means an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, a prophylactically effective amount refers to an amount that is sufficient to prevent, hinder or delay the onset of a disease; a therapeutically effective amount refers to an amount that is sufficient to cure, or at least partially arrest the disease and its complications in a patient already having the disease. Determining such an effective amount is well within the capabilities of those skilled in the art, e.g., an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight, and gender, the mode of administration of a drug, and other therapies being administered concurrently, etc.

[0150] As used herein, the term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a form of cytotoxicity in which Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, or macrophages), causing these cytotoxic effector cells to bind specifically to target cells that are coated with antigen and subsequently kill the target cells through secretion of cytotoxins. Methods to test the ADCC activity of antibodies are known in the art, e.g., by measuring the binding activity between the antibody to be tested and Fc receptors (e.g., CD16a).

[0151] As shown in the present disclosure, "EC 50 " also referred to as "half maximal effective concentration", refers to the concentration of a drug, antibody or poison that induces a response halfway between the baseline and maximum after a specified exposure time. EC 50 and affinity are inversely related, the lower the EC 50 value, the higher the affinity of the antibody.

[0152] I. Anti-PD-1 antibodies

[0153] In some embodiments, the present disclosure provides antibodies, e.g., murinized antibodies, humanized antibodies, preferably humanized antibodies, with desired functions or properties. The present disclosure provides anti-PD-1 antibodies with desirable properties as therapeutic agents that function to agonize immune suppression pathways. These properties include one or more of the following: the ability to bind to human PD-1 with high affinity, high agonistic activity to human PD-1, and enhanced ADCC activity. The antibodies provided by the present disclosure are suitable for use in PD-1 related diseases, especially autoimmune diseases caused by PD-1 related defects, preferably at least one of graft versus host disease, solid organ transplant rejection, vasculitis, systemic lupus erythematosus, type I diabetes, multiple sclerosis, giant cell arteritis, psoriasis, rheumatoid arthritis, IBD, non-specific ulcerative colitis, autoimmune hepatitis.

[0154] Anti-PD-1 antibodies with specific functional properties

[0155] The present disclosure provides antibodies that specifically bind to human PD-1 and can bind to PD-1 from certain other species, e.g., monkey (cynomolgus monkey, etc.) PD-1, murine (mouse, etc.) PD-1. Preferably, the antibodies of the present disclosure bind to human PD-1 with high affinity.

[0156] The antibodies are characterized by particular functional features or properties. For example, the antibodies specifically bind to human PD-1 with high affinity. Binding to human PD-1 can be assessed using one or more conventional techniques in the art. For example, in some embodiments, the antibodies can be tested by flow cytometry analysis, in which the antibodies are reacted with a cell line that expresses human PD-1 (e.g., Jurkat cells, CHO cells, 293 cells, etc., whose cell surface stably expresses human PD-1). In some embodiments, Bio-Layer Interferometry (BLI), ELISA assays can also be used for binding analysis.

[0157] In some embodiments, the antibodies or antigen binding portions thereof of the present disclosure bind to human PD-1 protein with a Kd of 5.0 x 10 -8 M or less. D In some embodiments, the antibodies or antigen binding portions thereof of the present disclosure bind to human PD-1 protein with a Kd of 4.0 x 10 -8 M or less. D In some embodiments, the antibodies or antigen binding portions thereof of the present disclosure bind to human PD-1 protein with a Kd of 3.0 x 10 -8 M or less. D In some embodiments, the antibodies or antigen binding portions thereof of the present disclosure bind to human PD-1 protein with a Kd of 2.0 x 10 -8 M or less. D In some embodiments, the antibodies or antigen binding portions thereof of the present disclosure bind to human PD-1 protein with a Kd of 1.0 x 10 -8 M or less. D In some embodiments, the antibodies or antigen binding portions thereof of the present disclosure bind to human PD-1 protein with a Kd of 9.0 x 10 -9 M or less. D In some embodiments, the antibodies or antigen binding portions thereof of the present disclosure bind to human PD-1 protein with a Kd of 8.0 x 10 -9 M or less. D In some embodiments, the antibodies or antigen binding portions thereof of the present disclosure bind to human PD-1 protein with a Kd of 7.0 x 10 -9 M or less. D In some embodiments, the antibodies or antigen binding portions thereof of the present disclosure bind to human PD-1 protein with a Kd of 6.0 x 10 -9 M or less. D In some embodiments, the antibodies or antigen binding portions thereof of the present disclosure bind to human PD-1 protein with a Kd of 6.0 x 10

[0158] In some preferred embodiments, the antibodies or antigen binding portions thereof of the present disclosure are humanized monoclonal antibodies. In the present disclosure, the antibodies or antigen binding portions thereof can also be referred to using the following designations instead:

[0159] 5A3, 5A3-hu01, 5A3-hu02, 5A3-hu03, 5A3-hu-R2, 5A3-AM17.

[0160] In one aspect, the present disclosure provides an antibody or an antigen-binding portion thereof that specifically binds to PD-1 or a functional fragment thereof, the antibody or the antigen-binding portion thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL);

[0161] wherein the VH comprises:

[0162] I. a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3;

[0163] II. a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, wherein SEQ ID NO: 23: YIDPYYGNTRYX1QKFX2G; and

[0164] III. a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5; and

[0165] the VL comprises:

[0166] I) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6;

[0167] II) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; and

[0168] III) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24, wherein SEQ ID NO: 24: QHYYX3SPX4T;

[0169] wherein the X1 is selected from A, N, or S;

[0170] the X2 is selected from Q or K;

[0171] the X3 is selected from N or S;

[0172] the X4 is selected from P or V.

[0173] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL);

[0174] wherein the VHcomprises:

[0175] i. a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence as set forth in SEQ ID NO: 3; or a heavy chain complementarity determining region 1 (HCDR1) having at least one amino acid difference compared to the amino acid sequence as set forth in SEQ ID NO: 3;

[0176] ii. a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence as set forth in SEQ ID NO: 23; or a heavy chain complementarity determining region 2 (HCDR2) having at least one amino acid difference compared to the amino acid sequence as set forth in SEQ ID NO: 23, wherein SEQ ID NO: 23: YIDPYYGNTRYX1QKFX2G; and

[0177] iii. a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence as set forth in SEQ ID NO: 5; or a heavy chain complementarity determining region 3 (HCDR3) having at least one amino acid difference compared to the amino acid sequence as set forth in SEQ ID NO: 5; and

[0178] the VLcomprises:

[0179] i) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence as set forth in SEQ ID NO: 6; or a light chain complementarity determining region 1 (LCDR1) having at least one amino acid difference compared to the amino acid sequence as set forth in SEQ ID NO: 6;

[0180] ii) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence as set forth in SEQ ID NO: 7; or a light chain complementarity determining region 2 (LCDR2) having at least one amino acid difference compared to the amino acid sequence as set forth in SEQ ID NO: 7;

[0181] iii) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence as set forth in SEQ ID NO: 24; or a light chain complementarity determining region 3 (LCDR3) having at least one amino acid difference compared to the amino acid sequence as set forth in SEQ ID NO: 24, wherein SEQ ID NO: 24: QHYYX3SPX4T;

[0182] wherein the X1is selected from A, N, or S;

[0183] the X2is selected from Q or K;

[0184] X3 is selected from N or S;

[0185] X4 is selected from P or V;

[0186] In some embodiments, the amino acid difference is 1-3, e.g., 1, 2, 3, preferably 2 or 3.

[0187] In some embodiments, the amino acid difference is an amino acid substitution, preferably a substitution of a conservative amino acid.

[0188] Examples of conservative amino acid substitutions in the present disclosure are as follows:

[0189] Such conservative substitutions are preferably of one amino acid in the following groups (a) to (e) to another amino acid residue in the same group: (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gin; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, non-polar residues: Met, Leu, He, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0190] In particular, conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gin or to His; Asp to Glu; Cys to Ser; Gin to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gin; He to Leu or to Val; Leu to He or to Val; Lys to Arg, to Gin, or to Glu; Met to Leu, to Tyr, or to He; Phe to Met, to Leu, or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to He, or to Leu.

[0191] In some embodiments, the antibody or antigen-binding portion thereof comprises:

[0192] A1 : the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 3, 4, and 5, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively; or

[0193] A2: the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 3, 19, and 5, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively; or

[0194] A3: the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 3, 4, and 5, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 20, respectively; or

[0195] A4: the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 3, 21, and 5, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively; or

[0196] A5: the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 3, 21, and 5, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 22, respectively.

[0197] In some embodiments, the VH has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of:

[0198] SEQ ID NO: 19, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17.

[0199] In some embodiments, the VL has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of:

[0200] SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18.

[0201] In some embodiments, the VH and VL are selected from the group consisting of:

[0202] S1) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 1; and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 2; or

[0203] S2) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 9; and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 10; or

[0204] S3) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 11; and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 12; or

[0205] S4) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 13; and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 14; or

[0206] S5) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 15; and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 16; or

[0207] S6) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 17; and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 18.

[0208] In some embodiments, the antibody or antigen-binding portion thereof is a murinized, chimerized, or humanized antibody or antigen-binding portion thereof.

[0209] In some preferred embodiments, the antibody or antigen-binding portion thereof is a humanized antibody or antigen-binding portion thereof.

[0210] In some embodiments, the antibody or antigen-binding portion thereof comprises an immunoglobulin Fc region or functional variant thereof.

[0211] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a human immunoglobulin Fc region or functional variant thereof.

[0212] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises an Fc region of human IgGl, IgG2, IgG3, or IgG4 or functional variant thereof. In some preferred embodiments, the antibody or antigen-binding portion thereof has a human immunoglobulin Fc region or functional variant thereof with a V12 mutation comprising any one of the following amino acid mutations or a combination of multiple amino acid mutations: E233D, G237D, P238D, H268D, P271G, A330R.

[0213] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a human immunoglobulin Fc region or functional variant thereof comprising amino acid mutations E233D / G237D / P238D / H268D / P271G / A330R.

[0214] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises an Fc region of human IgGl or functional variant thereof.

[0215] In some embodiments, the antibody or antigen-binding portion thereof is directly linked to the Fc region or linked via a linker.

[0216] In some preferred embodiments, the linker is a polypeptide.

[0217] In some preferred embodiments, the linker is a glycine (G) and serine (S) rich polypeptide, such as GGGGS, (GGGGS) n (GGGGGS) n and the like, wherein n can be 1-100, preferably 1-50, more preferably 1-10, such as 4.

[0218] In some embodiments, the antigen-binding portion is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, dAb, nanobody, Fd, and Fd'.

[0219] III. Fusion Proteins

[0220] The present disclosure provides fusion proteins comprising any of the aforementioned antibodies or antigen-binding portions thereof.

[0221] The fusion proteins are constructed for the purpose of enhancing the stability and / or delaying the half-life of the antibody, or facilitating the purification production.

[0222] In some embodiments, the fusion protein further comprises a biologically active protein or functional variant thereof, such as: immunoglobulin Fc domain, serum albumin, albumin-binding polypeptide, prealbumin, carboxy-terminal peptide, elastin-like polypeptide, His tag, GST tag, MBP tag, FLAG tag, and SUMO tag.

[0223] In some embodiments, the fusion protein comprises one or more linker peptides.

[0224] In some embodiments, the fusion protein comprises one or more protease cleavage sites.

[0225] III. Multispecific Molecules

[0226] In some embodiments, the antibodies described herein can be multispecific antibodies (e.g., bispecific or trispecific antibodies). The multispecific antibodies comprise two or more antigen binding sites, at least one of which is a PD-1 antigen binding site.

[0227] In some embodiments, the antibodies or antigen binding portions thereof comprise a bispecific antibody of at least one first binding specificity for PD-1 and a second binding specificity for a second target antigenic determinant. Further, in certain embodiments the multispecific antibody has a third binding specificity.

[0228] Although fully human monoclonal antibodies are preferred, the multispecific antibodies described herein can employ murine, chimeric, or humanized monoclonal antibodies.

[0229] When the other binding specificity moiety is an antibody, it can be conjugated via the sulfhydryl groups of the C-terminal hinge regions of the two heavy chains. Alternatively, both binding specificities can be encoded by the same vector and expressed and assembled in the same host cell. This approach is particularly suitable for bispecific antibodies having combinations of binding specificities such as (mAb x mAb), (mAb x Fab), (Fab x F(ab')2), or (ligand x Fab) fusion proteins. The bispecific antibodies described herein can be single chain molecules comprising one single chain antibody and a binding determinant or single chain bispecific molecules comprising two binding determinants. Bispecific molecules can comprise at least two single chain molecules.

[0230] IV. Isolated nucleic acid molecules / vectors / recombinant cells

[0231] The present application also provides nucleic acid molecules encoding the antibodies or antigen binding portions thereof, fusion proteins, and / or multispecific antibodies. The nucleic acid molecules can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when it is separated from other cellular material or other contaminants, such as other nucleic acids or proteins, of the cell of origin, when purified by standard well known techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. The nucleic acid molecules of the present disclosure can be, for example, DNA or RNA, and can or can not contain intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0232] The nucleic acids of the present disclosure can also be in the form of, can be present in, and / or can be part of a vector, such as a plasmid, cosmid, or YAC. The vector can be, inter alia, an expression vector, i.e., a vector that can provide for the expression of the antibodies or antigen-binding portions thereof of the present disclosure in vitro and / or in vivo, i.e., in suitable host cells, host organisms, and / or expression systems. Such expression vectors typically comprise at least one nucleic acid of the present disclosure operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). Selection of such elements and their sequences for expression in a particular host is within the skill of those in the art. Particular examples of control elements and other elements useful or necessary for the expression of the antibodies or antigen-binding portions thereof of the present disclosure, such as promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

[0233] The nucleic acids of the present disclosure can be prepared or obtained by known means, e.g., by de novo DNA synthesis and / or recombinant DNA technology, based on information regarding the amino acid sequences of the polypeptides of the present disclosure given herein, and / or can be isolated from suitable natural sources.

[0234] In another aspect, the present disclosure provides recombinant cells capable of expressing the antibodies or antigen-binding portions thereof, fusion proteins, or multispecific antibodies of the present disclosure, and / or containing the nucleic acid molecules or expression vectors of the present disclosure. In some embodiments, the recombinant cells are bacterial cells, fungal cells, or mammalian cells.

[0235] Suitable bacterial cells include cells of Gram-negative bacterial strains, such as Escherichia coli strains, Proteus strains, and Pseudomonas strains, and Gram-positive bacterial strains, such as Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains.

[0236] Suitable fungal cells include cells of species of Trichoderma, Neurospora, and Aspergilus; or of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0237] Suitable mammalian cells include, e.g., HEK293 cells, Jurkat cells, CHO cells, BHK cells, HeLa cells, or COS cells, etc.

[0238] The antibodies or antigen-binding portions thereof, fusion proteins, or multispecific antibodies of the present disclosure can be produced intracellularly (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies) in the cells as described above, then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g., in the culture medium of the host cells), then isolated from the culture medium and optionally further purified.

[0239] Methods and reagents for recombinantly producing polypeptides, e.g., particular suitable expression vectors, transformation or transfection methods, selection markers, methods of inducing protein expression, culture conditions, etc., are known in the art. Similarly, protein isolation and purification techniques suitable for use in manufacturing the antibodies or antigen-binding portions thereof, fusion proteins, or multispecific antibodies of the present disclosure in the methods are well known to those skilled in the art.

[0240] V. Preparation or production of antibodies

[0241] The monoclonal antibodies of the present disclosure can be made by a variety of techniques, such as the hybridoma technique, recombinant DNA technology, or phage antibody library techniques.

[0242] For example, the following procedure can be used to produce a monoclonal antibody. A mouse or other suitable host animal is first immunized with an immunogen, if necessary, with an adjuvant. After immunization, the animal will produce lymphocytes that secrete antibodies specific for the immunogen. Alternatively, lymphocytes can be obtained from an in vitro immunization. The desired lymphocytes are collected and fused with myeloma cells using a suitable fusing agent, such as PEG, to form hybrid cells (hybridomas). The hybridomas so formed are seeded and grown in a suitable culture medium that preferably contains an inhibitor of lymphocyte growth to prevent the growth of un-fused cells. The resulting cells are plated and grown in selective media until only the cells that have fused grow and secrete antibodies of the desired specificity. The culture medium in which the hybridoma cells are growing is then removed and the supernatant tested for production of monoclonal antibodies directed against the specific antigen. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined, for example, by immunoprecipitation or an in vitro binding assay, such as a radioimmunoassay (RIA), an enzyme-linked immunosorbent assay (ELISA), or a fluorescent immunoassay. The affinity of the monoclonal antibodies can be determined, for example, using the Scatchard analysis. After the specificity, affinity, and reactivity of the antibodies produced by the hybridoma are determined, the desired cell line can be subcloned by a standard limited dilution procedure. Suitable culture media for bringing lymphocytes to proliferation can be DMEM or RPMI-1640, etc. Alternatively, the hybridoma cells can be grown in vivo in the animal from which they were derived. The monoclonal antibodies secreted by the subclones are separated from the culture medium or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0243] Monoclonal antibodies can also be produced by recombinant DNA methods. DNA molecules encoding the heavy and light chains of the monoclonal antibodies can be isolated from the hybridoma cells by PCR amplification using nucleic acid primers specific for the heavy and light chain genes. The resulting DNA molecules are inserted into expression vectors, which are then transfected into host cells (such as E. coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins) and cultured under suitable conditions to obtain recombinant expression of the desired antibodies.

[0244] Antibodies can be purified by known techniques, such as affinity chromatography using protein A or protein G. Subsequently or alternatively, the specific antigen (the target molecule recognized by the antibody) or an antigenic epitope thereof can be immobilized on a column and the immunospecific antibody purified by immunoaffinity chromatography.

[0245] VI. Compositions / Pharmaceutical Compositions

[0246] In another aspect, the present application further provides a composition comprising a group consisting of:

[0247] any of the foregoing antibodies or antigen-binding portions thereof, fusion proteins, multispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells.

[0248] In some embodiments, the composition further comprises at least one pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutically acceptable carrier or excipient includes, but is not limited to, a carrier, a surface active agent, a thickening or emulsifying agent, a solid binder, a dispersing or suspending agent, a solubilizer, a colorant, a flavoring agent, a coating agent, a disintegrant, a lubricant, a sweetening agent, a preservative, an isotonic agent, and combinations thereof.

[0249] Preferably, the composition includes a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0250] Preferably, examples of suitable aqueous and nonaqueous carriers that can be employed in the composition include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0251] Preferably, these compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms can be ensured through sterilization procedures and through the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid and the like. It can also be desirable to include isotonic agents, such as sugars, sodium chloride and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

[0252] The use of these media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions described herein is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0253] A composition is typically required to be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like), and suitable mixtures thereof.

[0254] Preferably, the composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or topical administration (e.g., injection or infusion). Depending on the route of administration, the active compound can be coated with a material to protect it from acid and other natural conditions which can cause it to denature. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinally, epidural and intrasternal injection and infusion. Alternatively, the antibodies of the present disclosure can be administered via a non-parenteral route, such as a topical, transdermal or mucosal route, e.g., intranasal, oral, vaginal, rectal, sublingual or topical administration.

[0255] The compositions described in the present disclosure can also be used or administered as a combination therapy (i.e., the anti-PD-1 antibody in combination with other agents). For example, the combination therapy can include the anti-PD-1 antibody of the present disclosure or antigen binding portion thereof in combination with at least one other agent that treats an autoimmune disease and / or a PD-1 agonist.

[0256] In some embodiments, the compositions described in the present disclosure can include other agents that treat an autoimmune disease, preferably a PD-1 agonist. In some embodiments, the composition comprises a first antibody and a second antibody, wherein the first antibody or the second antibody is an antibody specific for PD-1. In some embodiments, the first antibody and the second antibody are present in the composition at a fixed dose (i.e., a fixed ratio). This fixed dose is between at least about 1 :200 to at least about 200: 1, at least about 1 : 150 to at least about 150: 1, at least about 1 : 100 to at least about 100: 1, at least about 1 : 75 to at least about 75: 1, at least about 1 : 50 to at least about 50: 1, at least about 1 : 25 to at least about 25: 1, at least about 1 : 10 to at least about 10: 1.

[0257] In some examples, the second antibody is an antibody against an immune checkpoint, preferably one or more of an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-TIGIT antibody, an anti-OX40 (also known as CD134, TNFRSF4, ACT35, and / or TXGP1L) antibody, an anti-LAG-3 antibody, an anti-CD73 antibody, an anti-CD137 antibody, an anti-CD27 antibody, or an anti-CSF-1R antibody.

[0258] VII. Uses / Methods of Treatment

[0259] The antibodies or antigen-binding portions thereof, fusion proteins, bispecific antibodies, and / or compositions provided herein have a variety of uses in vitro and in vivo, such as detecting the presence and / or amount of PD-1 and / or agonizing PD-1 to inhibit an immune response. In a preferred embodiment, the antibodies of the present disclosure are humanized antibodies that can be administered to cells cultured in vitro or ex vivo, or to a human subject (which can be in vivo administration) to achieve the intended therapeutic effect.

[0260] In one aspect, the present disclosure provides a method of altering an immune response in a subject, comprising administering to a subject (preferably a human) in need thereof a therapeutically effective amount of an antibody or antigen-binding portion thereof, fusion protein, bispecific antibody, and / or composition of the present disclosure. In some embodiments, the subject has an immune response disorder in vivo, preferably the subject has an immune response that is enhanced, upregulated, and / or over-activated.

[0261] In another aspect, the present disclosure provides a method of agonizing an immune suppressive pathway in a subject, comprising administering to a subject (preferably a human) in need thereof a therapeutically effective amount of an antibody or antigen-binding portion thereof, fusion protein, bispecific antibody, and / or composition of the present disclosure. In some embodiments, the subject has an immune response disorder in vivo, preferably the subject has an immune response that is enhanced, upregulated, and / or over-activated.

[0262] In yet another aspect, the present disclosure provides a method of preventing and / or treating a PD-1 related disease, comprising administering to a subject (preferably a human) in need thereof a therapeutically effective amount of an antibody or antigen-binding portion thereof, fusion protein, bispecific antibody, and / or composition of the present disclosure. In some embodiments, the subject has a PD-1 mediated immune response disorder in vivo, preferably the subject has an immune response that is enhanced, upregulated, and / or over-activated.

[0263] In yet another aspect, the present disclosure provides a method of preventing and / or treating an autoimmune disease, comprising administering to a subject (preferably a human) in need thereof a therapeutically effective amount of an antibody or antigen-binding portion thereof, fusion protein, bispecific antibody, and / or composition of the present disclosure. In some embodiments, the autoimmune disease is caused or contributed to by a PD-1 mediated immune response disorder. In some preferred embodiments, the autoimmune disease includes, but is not limited to, graft versus host disease, solid organ transplant rejection, vasculitis, systemic lupus erythematosus, type I diabetes, multiple sclerosis, giant cell arteritis, psoriasis, rheumatoid arthritis, inflammatory bowel disease (IBD), non-specific ulcerative colitis, autoimmune hepatitis, and the like.

[0264] Those skilled in the art will appreciate that an appropriate dosage can vary from patient to patient. Determining the optimal dosage will generally involve balancing the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to the activity of the particular compound, the route of administration, the time of administration, the rate of clearance of the compound, the duration of the treatment, other drugs, compounds, and / or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient. But generally the dosage will be chosen to achieve local concentrations at the site of desired action which achieve the desired effects without causing substantial harmful or deleterious side effects.

[0265] In certain preferred embodiments, the course of treatment involving the antibodies or antigen binding portions thereof of the present disclosure will comprise multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the antibodies or antigen binding portions thereof of the present disclosure can be administered daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it is understood that the dosage or interval can be altered based on patient response and clinical practice.

[0266] To enhance the therapeutic effect of the antibodies or antigen binding portions thereof, fusion proteins, multispecific antibodies, and / or compositions provided by the present disclosure, they can be administered in combination with at least one other therapeutic agent (e.g., a cytotoxic agent, a radiotoxic agent, an antitumor agent, an antiangiogenic agent, or an immunosuppressive agent).

[0267] In the context of administering a treatment, the term "combination" or "co-administration" as used herein refers to the use of more than one treatment or therapeutic agent. The use of the term "combination" does not restrict the order in which treatments or therapeutic agents are administered to a subject. A treatment or therapeutic agent can be administered prior to, simultaneously with, or following the administration of a second treatment or therapeutic agent to a patient. Preferably, the treatments or therapeutic agents are administered to a subject in an order, amount, and / or at an interval such that they can act together. In a particular embodiment, the treatments or therapeutic agents are administered to a subject in an order, amount, and / or at an interval such that they provide an increased benefit than if administered otherwise, particularly independently of one another. Preferably, the increased benefit is a synergistic effect.

[0268] To make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in details below with embodiments. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturers. All reagents or instruments not mentioned by the manufacturers are the conventional products which can be purchased in the market. In order to better illustrate the present application, numerous specific details are given in the following specific embodiments. The specific embodiments described herein are only used to explain the present application and do not constitute any limitation to the present application. In addition, in the following description, the description of the well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present application. Such structures and technologies are also described in the publication of many publications.

[0269] Example 1: Animal immunization and antibody screening

[0270] BALB / c mice were immunized with human PD-1 antigen protein (ACRO Biosystems, PD1-H5221) for 4 times in 42 days cycle. The first immunization used 50 μg antigen protein mixed with equal volume of Freund's complete adjuvant (Sigma, F5881) for multiple subcutaneous injection. The following boost immunization used Freund's incomplete adjuvant (Sigma, F5506). 25 μg antigen was used for subcutaneous multiple injection at day 14, 25 μg antigen was used for subcutaneous multiple injection at day 28, and 25 μg antigen was used for intraperitoneal injection at day 42. The mouse spleen was dissected out 4 days after the last immunization, and mouse B cells were enriched with mouse Pan-B cell enrichment kit (Stemcell, Cat#19844). The B cells were resuspended with PBS+2% FBS and stained with the following antibodies: anti-mouse IgM, anti-mouse IgD, anti-mouse IgG1, anti-mouse B220 (all antibodies purchased from BD Bioscience). The PD-1 antigen was labeled with AF647 and added to the cells for staining at 4°C in the dark for 30 minutes. After staining, the cells were washed with PBS for 3 times, resuspended with PBS+2% FBS and loaded to the flow cytometry for sorting. The sorting was performed with Sony MA900 flow cytometry. The B220+IgG+IgM-IgD-B cells were gated according to the fluorescence signals, and the single cell mode was used for sorting the single B cells into 96-well PCR plate. The DNA sequences of the light and heavy chains of the antibodies were obtained by the method of classical PCR and Sanger sequencing. The amino acid sequences of the light and heavy chain variable regions of the representative clone 5A3 are listed in Table 1, and the CDR sequences are listed in Table 2 (defined based on the kabat numbering).

[0271] Table 1

[0272] Table 2

[0273] Example 2: Antibody humanization

[0274] The murine antibody 5A3 screened in Example 1 was subjected to humanization engineering by CDR grafting. The germline genes with the highest homology to the original murine antibody variable regions were obtained by IgBlast and IMGT / DomainGapAlign as the templates for humanization. The germline heavy chain V gene IGHV1-46*01, IGHV1-3*01 and the germline heavy chain J gene IGHJ4*01 were selected as the human germline gene templates for the heavy chain variable region of 5A3, and the germline light chain V gene IGKV4-1*01 and the germline light chain J gene IGKJ1*01 were selected as the human germline gene templates for the light chain variable region of 5A3. The 6 CDRs of the murine parent antibody were grafted onto the corresponding human germline gene templates. The key amino acids in the framework regions of the murine antibody variable regions (e.g., the residues in the Vernier zone, the interchain interface residues of VH-VL) were determined by structure modeling algorithm, and then selective back mutations were performed at the corresponding positions in the grafted sequences to complete the design of the humanized antibody variable region sequences. The light and heavy chain variable region sequences are shown in Table 3, and the CDR sequences (defined based on the kabat numbering) are shown in Table 4. The designed variable regions were spliced with the constant region of wild-type hlgG1 to form the complete monoclonal antibody. The detailed method can be referred to Lo, B. K. C. (2004). Antibody Humanization by CDR Grafting. In: Lo, B. K. C. (eds) Antibody Engineering. Methods in Molecular Biology TM , vol 248. Humana Press.

[0275] The antibody affinity maturation was performed according to the reported phage display method. Finally, the recombinantly expressed mutant 5A3-AM17 was obtained.

[0276] Table 3

[0277] Table 4

[0278] Example 3: Determination of antibody affinity by BLI

[0279] The binding kinetics of antibodies 5A3-hu01, 5A3-hu02, 5A3-hu03 and 5A3-hu-R2 to human PD-1 (ACRO Biosystems, PD1-H5221) protein were detected by the method of biofilm interference (BLI).

[0280] The antibody to be tested was immobilized onto the AHC2.0 biosensor, and the antigen protein PD-1 was diluted to 100 nM, 50 nM, and 25 nM, respectively, using 0.02% PBST20, and then injected. Kinetic analysis was performed using a binding model (Octet RH96 data analysis software) using a 1:1 binding model to calculate the association rate (K on ), the dissociation rate (K off ), and the equilibrium dissociation constant (K D ) as the ratio of K off / K on . The affinity of the antibody is shown in Table 5.

[0281] Table 5

[0282] Example 4: ELISA binding experiment

[0283] The binding of antibody 5A3 to human PD-1 protein and cynomolgus monkey PD-1 protein was detected by ELISA. Human PD-1 protein (ACRO Biosystems, PD1-H5221) or cynomolgus monkey PD-1 protein (ACRO Biosystems, PD1-C52H5) was diluted with PBS to 1 pg / mL, 100 pL per well, added to the ELISA plate. The plate was covered with a plate sealing film and incubated at 2-8 °C overnight. The liquid was aspirated, and each well was washed with 300 pL of washing buffer (0.05% Tween 20 + PBS) for 3 times. 200 pL of blocking solution (0.05% Tween 20 + 2% BSA + PBS) was added to each well and incubated at room temperature for 1 hour. The antibody was diluted with the blocking solution. The diluted antibody was added to the ELISA plate at 100 pL per well. Incubate at room temperature for 1 hour with gentle shaking. Repeat the washing step 3 times. Dilute the secondary antibody (goat anti-human IgG-HRP, Jackson ImmunoResearch) with the blocking buffer. Add 100 pL of diluted secondary antibody to each well. Incubate at room temperature for 30 minutes with gentle shaking. Repeat the washing step 3 times. Add 100 pL of TMB substrate solution (catalog TMB-S-001, Innoreagents) to each well. Incubate the plate at room temperature for 10 minutes in the dark. Add 50 pL of stop solution (catalog C1058, Solarbio) to each well. Within 30 minutes after adding the stop solution, measure the absorbance at 450 nm using a microplate reader (ENVISION, PerkinElmer). The detection results are shown in FIG. 1-FIG. 2 and Table 6-Table 7. The experimental results show that the binding activity of 5A3 to human PD-1 protein is better than ANB030 (WO2020247648A2) and similar to Peresolimab (WO2019168745A1). 5A3 can bind to cynomolgus monkey PD-1 protein, and the binding activity is similar to human PD-1 protein.

[0284] Table 6. Antibody binding to recombinant human PD-1 protein

[0285] Table 7. Antibody binding to recombinant cynomolgus monkey PD-1 protein

[0286] Example 5: FACS flow binding experiment

[0287] To test the binding ability of the antibodies at the cellular level, human PD-1 gene was transferred into Jurkat-NFAT-luc cells (Novigen, DD1302-01) to obtain Jurkat-NFAT-luc-PD-1 cells. Jurkat-NFAT-luc-PD-1 cells were added to a 96-well V-bottom plate at a density of 100,000 cells per well, and a gradient dilution (concentrations were 100, 20, 4, 0.8, 0.16, 0.032, 0.064, 0.0128 nM, respectively) of antibodies 5A3, 5A3-hu01, 5A3-hu02, 5A3-hu03 and 5A3-hu-R2 was added to the plate, and incubated at 4°C for 1 hour. After washing with PBS for 3 times, a fluorescent secondary antibody AF647-labeled anti-human IgG antibody (Jackson, 109-605-098) was added and incubated at 4°C for 1 hour. After washing with PBS for 3 times, an Attune NxT (Thermo) flow cytometer was used for analysis, and the MFI (mean fluorescence intensity) value was detected. The EC50value was calculated by fitting with Graphpad software. The results are shown in Figure 3 and Table 8. 50

[0288] Table 8

[0289] Example 6: Agonistic activity of PD-1 agonists

[0290] 6.1: Establishment of reporter-related cell lines

[0291] ​Gene human PD-1 (Uniprot, Q15116) and OKT3 (SCFV)-CD28 (sequence: QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTVTVSAGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGDPPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS, SEQ ID NO: 26) were cloned into pLVX-IRES-Puro vector (Clontech, KLV3501) to obtain lentiviral vectors. Lentivirus production, target cell transfection were performed according to the manufacturer's instructions: lentiviral vectors were mixed with pH1 and pH2 (Clontech, KLV3501) and transfected into 293T cell line (ATCC, CRL-3216) using Lipofectamine 3000 reagent (Thermo, L3000015) to produce lentivirus.

[0292] Jurkat-NFAT-Luc (Novoprotein, DD1302-01) and CHOK1-hCD32B cells (Kang Yuanbochuang, KC-1251) were transfected with PD-1 and OKT3 (SCFV)-CD28 lentivirus respectively, and the transfected cells were amplified and screened using medium containing 1 mg / mL G418 and 700 μg / mL G418 (Gibco, 11811031) respectively, to obtain stable cells Jurkat-NFAT-PD-1-Luc and CHOK1-hCD32B-OKT3.

[0293] CHOK1-hCD32B-OKT3 was used as crosslinking cells and Jurkat-NFAT-PD-1-Luc cells as effector cells to detect the agonistic activity of antibodies.

[0294] In this assay, Jurkat-NFAT-PD-1-Luc is a Jurkat-NFAT-luc cell stably expressing PD-1 (Novigen, DD1302-01), expressing firefly luciferase driven by NFAT response element. CHOK1-hCD32B-OKT3 cell is a cell line stably expressing OKT3. When Jurkat-NFAT-PD-1-Luc cell is co-cultured with CHOK1-hCD32B-OKT3 cell, Jurkat-NFAT-PD-1-Luc gets the first signal stimulation by OKT3, and the antibody binds to PD-1 on Jurkat-NFAT-PD-1-Luc cell, and under the crosslinking action of hCD32B, the firefly luciferase expressed by the NFAT response element is activated. The biological activity in this mechanism is quantitatively determined by the luciferase produced by the activation of the NFAT pathway, and the luciferase activity in the effector cell is quantitatively determined by the bioluminescence reading. The larger the luminescence reading, the more luciferase and the stronger the activity, and the stronger the agonistic effect of the antibody.

[0295] The procedure of the reporter gene detection experiment is as follows:

[0296] CHOK1-hCD32B-OKT3 cells (0.2 x 10^5 cells / well) and Jurkat-NFAT-PD-1-Luc cells (1 x 10^5 cells / well) were inoculated in a 96-well plate containing the antibody to be tested (100 μL / well) and co-cultured at 37°C. Anti-HEL human IgG1 antibody (Cat. No. B422203, Biointron) (hereinafter referred to as IgG1-Isotype) was used as an isotype control. After 6 hours of incubation, the fluorescent enzyme substrate (Bright-Glo Luciferase Substrate, Cat. No. DD1204-03, Vazyme) was added to the corresponding wells. The luminescence value was read by a detector (Envision, model 2105). The luminescence reading was expressed as RLU (relative light unit), and the EC TM 50 .

[0297] The results are shown in Figures 4-5 and Table 9. On the reporter gene cell line, 5A3, 5A3-hu01, 5A3-hu02, 5A3-hu03, and 5A3-hu-R2 have similar agonistic activity to the positive control Peresolimab.

[0298] Table 9

[0299] ​Example 7: Agonistic activity of antibodies on PBMC (peripheral blood mononuclear cell)

[0300] Antibody function was tested using CHOK1-hCD32B-OKT3 (constructed in the same way as described in Example 6) and PBMC cells. Specifically as follows:

[0301] Pre-coat CD3 Monoclonal Antibody (OKT3) (Thermo, 16-0037-85) in T75 cell culture flask (Corning, 430641-FQ2), discard the suspended OKT3 the next day, wash twice with PBS (Solarbio, P1010) and then add PBMC pre-activated for 48h, inoculate CHOK1-hCD32B-OKT3 cells (0.2x10^5 cells / well) in a 96-well flat-bottom plate (Corning, 3599), the next day add PBMC cells (1x10^5 cells / well) and the antibody to be tested (total volume 100 μL / well) to the 96-well plate containing CHOK1-hCD32B-OKT3 cells, and incubate at 37°C. After incubation, collect the supernatant and use the IFN gamma Human Uncoated ELISA Kit (Thermo, 88-7316-77) to detect the amount of IFNγ released in the supernatant. The agonistic activity of the PD-1 agonist is calculated by the amount of IFNγ released.

[0302] The results are shown in Figures 6-7 and Table 10. 5A3-hu02 has similar agonistic activity to the positive control Peresolimab on primary cell lines.

[0303] Table 10

[0304] Example 8: Construction of PD-1 agonist antibodies with Fc variants

[0305] Studies have shown that enhancing the binding to FcγRIIb can further enhance the activity of PD-1 agonists. By mutating the Fc region of the antibody, the affinity to FcγRIIb receptor can be increased, while the affinity to FcγRIIIa (CD16a) is reduced, thereby avoiding the activity of antibody-induced ADCC. The Fc mutant V12 (E233D / G237D / P238D / H268D / P271G / A330R) can increase the affinity to FcγRIIb receptor by 62 times. In the present application, the Fc mutant V12 is used to construct new PD-1 agonist antibodies. The sequence of the constant region is as follows:

[0306] >V12_hIgG1 constant region

[0307] Example 9: FACS flow binding experiment

[0308] According to the method in Example 5, the binding of 5A3-AM17 on Jurkat-NFAT-Luc-PD1 cell line was determined, as shown in Figure 8: 5A-AM17 has strong binding activity to Jurkat-NFAT-luc-PD-1 cells, which is much better than ANB030 molecule, and has similar binding activity to Peresolimab.

[0309] Example 10: PD-1 agonistic antibody with Fc variant eliminates ADCC

[0310] ADCC reporter effector cells (ADCC FcyRIIIa (V158) Jurkat Effector Cell line) and target cells (Jurkat-NFAT-luc-hPD-1) were mixed according to the effector-target ratio of 5:1. Then gradient-diluted antibodies were added, mixed, and then placed in a 37-degree incubator for 5h. Then one-Lite luciferase detection substrate (Vazyme, DD1203-03) was added to detect the ADCC activity of the antibody. As shown in Figure 9: compared with PD-1 agonistic antibody peresolimab and 5A3-AM17 with wt human IgG1 Fc, the corresponding Fc variants Peresolimab-V12 and 5A3-AM17-V12 completely lost the ADCC activity.

[0311] Example 11: PD-1 agonistic antibody with Fc variant still retains agonistic activity of PD-1

[0312] In the reporter gene system (experimental method same as Example 6), PD-1 agonistic antibody Peresolimab-V12 and 5A3-AM17-V12 containing Fc variant (V12) still have the function of inhibiting T cell activation, but the agonistic activity is weaker than that of PD-1 agonistic antibody with wt human IgG1 Fc (Figure 10-11). The maximum inhibition rate decreases from 60% to about 30%, indicating that although Fc V12 variant is reported to promote the affinity to FcyRIIb (CD32b), it has a negative effect on the activity of PD-1 agonist. Further primary T cell activation experiment results confirm that the agonistic activity of PD-1 agonist containing V12 Fc variant will be reduced in vitro.

[0313] Example 12: Efficacy of antibodies in the graft-versus-host disease (GvHD) model induced by human PBMC transplantation into B-NDG mice

[0314] B-NDG mice (purchased from B&K Universal) were injected with 1 x 10 6 cells of human PBMC cells. Antibody treatment was initiated one day after injection of PBMC, at a dose of 5 mpk, administered via the tail vein. The administration frequency was twice a week, and the treatment lasted for 4 weeks. Body weight and GvHD scoring were measured during the treatment. GvHD scoring criteria: body weight reduction, changes in the activity state, posture, hair, skin, jaundice, etc. of the mice, and the maximum score was given if the mice died. The survival period scoring criteria was that the mice had a 20% or more reduction in body weight for 3 consecutive days.

[0315] The experimental results showed that, compared with the isotype control antibody, 5A3-AM17 could significantly slow down the weight loss of the mice and improve the symptoms of GvHD (Figure 12). The survival period of the mice after drug treatment was significantly prolonged (Figure 13).

[0316] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

An antibody or an antigen-binding portion thereof that specifically binds to PD-1 or a functional fragment thereof, characterized in that, The antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises: I. a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence that has at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; II. a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence that has at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, wherein SEQ ID NO: 23: YIDPYYGNTRYX1QKFX2G; and III. a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence that has at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5; and the VL comprises: I) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence that has at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6; II) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence that has at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; and III) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence that has at least about 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24, wherein SEQ ID NO: 24: QHYYX3SPX4T; wherein the X1 is selected from A, N, or S; the X2 is selected from Q or K; the X3 is selected from N or S; the X4 is selected from P or V. The antibody or antigen binding portion thereof of claim 1, wherein The antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises: i. a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence as set forth in SEQ ID NO: 3; or a heavy chain complementarity determining region 1 (HCDR1) comprising at least one, preferably 1-3, amino acid difference(s) compared to the amino acid sequence set forth in SEQ ID NO: 3; ii. a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence as set forth in SEQ ID NO: 23; or a heavy chain complementarity determining region 2 (HCDR2) comprising at least one, preferably 1-3, amino acid difference(s) compared to the amino acid sequence set forth in SEQ ID NO: 23, wherein SEQ ID NO: 23: YIDPYYGNTRYX1QKFX2G; and iii. a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence as set forth in SEQ ID NO: 5; or a heavy chain complementarity determining region 3 (HCDR3) comprising at least one, preferably 1-3, amino acid difference(s) compared to the amino acid sequence set forth in SEQ ID NO: 5; and the VL comprises: i) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence as set forth in SEQ ID NO: 6; or a light chain complementarity determining region 1 (LCDR1) having at least one, preferably 1-3, amino acid difference when compared to the amino acid sequence as set forth in SEQ ID NO: 6; ii) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence as set forth in SEQ ID NO: 7; or a light chain complementarity determining region 2 (LCDR2) having at least one, preferably 1-3, amino acid difference when compared to the amino acid sequence as set forth in SEQ ID NO: 7; iii) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence as set forth in SEQ ID NO: 24; or a light chain complementarity determining region 3 (LCDR3) having at least one, preferably 1-3, amino acid difference when compared to the amino acid sequence as set forth in SEQ ID NO: 24, wherein SEQ ID NO: 24: QHYYX3SPX4T; wherein said X1 is selected from A, N or S; said X2 is selected from Q or K; said X3 is selected from N or S; said X4 is selected from P or V; Preferably, the amino acid difference is an amino acid substitution; more preferably, the amino acid difference is a substitution of a conservative amino acid. The antibody or antigen binding portion thereof of claim 2, wherein The antibody or antigen binding portion thereof comprises: A1 : the VH comprises HCDR1, HCDR2 and HCDR3 of an amino acid sequence as set forth in SEQ ID NOs: 3, 4 and 5, respectively; and the VL comprises LCDR1, LCDR2 and LCDR3 of an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8, respectively; or A2: the VH comprises HCDR1, HCDR2 and HCDR3 of an amino acid sequence as set forth in SEQ ID NOs: 3, 19 and 5, respectively; and the VL comprises LCDR1, LCDR2 and LCDR3 of an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8, respectively; or A3: the VH comprises HCDR1, HCDR2 and HCDR3 of an amino acid sequence as set forth in SEQ ID NOs: 3, 4 and 5, respectively; and the VL comprises LCDR1, LCDR2 and LCDR3 of an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 20, respectively; or A4: the VH comprises HCDR1, HCDR2 and HCDR3 of an amino acid sequence as set forth in SEQ ID NOs: 3, 21 and 5, respectively; and the VL comprises LCDR1, LCDR2 and LCDR3 of an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 8, respectively; or A5: the VH comprises HCDR1, HCDR2 and HCDR3 of an amino acid sequence as set forth in SEQ ID NOs: 3, 21 and 5, respectively; and the VL comprises LCDR1, LCDR2 and LCDR3 of an amino acid sequence as set forth in SEQ ID NOs: 6, 7 and 22, respectively. The antibody or antigen binding portion thereof of any one of claims 1-3, wherein the VH has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17; and / or the VL has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO:

18. The antibody or antigen binding portion thereof of any one of claims 1-4, wherein the VH and VL are selected from the group consisting of: S1) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 1; and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 2; or S2) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 9; and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 10; or S3) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 11; and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 12; or S4) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 13; and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 14; or S5) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 15; and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 16; or S6) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 17; and a VL comprising an amino acid sequence as set forth in SEQ ID NO:

18. The antibody or antigen binding portion thereof of any one of claims 1-5, wherein The antibody or antigen-binding portion thereof is a murinized, chimerized, or humanized antibody or antigen-binding portion thereof, preferably a humanized antibody or antigen-binding portion thereof. The antibody or antigen binding portion thereof of claim 6, wherein The antibody or antigen-binding portion thereof comprises an immunoglobulin Fc region or functional variant thereof, preferably a human immunoglobulin Fc region or functional variant thereof, more preferably an Fc region of human IgGl, IgG2, IgG3, or IgG4 or functional variant thereof, most preferably a human immunoglobulin Fc region or functional variant thereof having a V12 mutation comprising any one of the following amino acid mutations or a combination of multiple amino acid mutations: E233D, G237D, P238D, H268D, P271G, A330R, particularly preferably a human immunoglobulin Fc region or functional variant thereof comprising the amino acid mutations E233D / G237D / P238D / H268D / P271G / A330R. The antibody or antigen binding portion thereof of claim 7, wherein The antibody or antigen-binding portion thereof is directly linked to the Fc region or linked via a linker. The antibody or antigen binding portion thereof of any one of claims 1-8, wherein The antibody is a monoclonal antibody; and / or the antigen binding moiety is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, dAb, nanobody, Fd and Fd'. A fusion protein characterized in that, comprising the antibody or antigen binding moiety of any one of claims 1-9. A multispecific antibody, characterized in that comprising the antibody or antigen binding moiety of any one of claims 1-9 or the fusion protein of claim 10, and a further antigen binding region or regions, wherein the further antigen binding region or regions bind to a different antigen or to a different epitope of the same antigen than the antibody or antigen binding moiety. An isolated nucleic acid molecule characterized in that, encoding the antibody or antigen binding moiety of any one of claims 1-9, the fusion protein of claim 10 or the multispecific antibody of claim 11. An expression vector, characterized in that, comprising the nucleic acid molecule of claim 12; optionally the nucleic acid molecule is operably linked to an expression control element. A recombinant cell, characterized in that, comprising the nucleic acid molecule of claim 12 or the expression vector of claim 13. Composition, characterized in that comprising the group consisting of: the antibody or antigen binding moiety of any one of claims 1-9, the fusion protein of claim 10, the multispecific antibody of claim 11, the nucleic acid molecule of claim 12, the expression vector of claim 13, the recombinant cell of claim 14; optionally the composition further comprises at least one pharmaceutically acceptable carrier. the antibody or antigen binding moiety of any one of claims 1-9, the fusion protein of claim 10, the multispecific antibody of claim 11, the nucleic acid molecule of claim 12, the expression vector of claim 13, the recombinant cell of claim 14 and / or the composition of claim 15 for use in the prevention and / or treatment of a PD-1 associated disease; preferably the PD-1 associated disease comprises an autoimmune disease; more preferably the autoimmune disease comprises graft versus host disease, solid organ transplant rejection, vasculitis, systemic lupus erythematosus, type I diabetes, multiple sclerosis, giant cell arteritis, psoriasis, rheumatoid arthritis, inflammatory bowel disease, non-specific ulcerative colitis, autoimmune hepatitis. the antibody or antigen binding moiety of any one of claims 1-9, the fusion protein of claim 10, the multispecific antibody of claim 11, the nucleic acid molecule of claim 12, the expression vector of claim 13, the recombinant cell of claim 14 and / or the composition of claim 15 for use in the prevention and / or treatment of a PD-1 associated disease; preferably the PD-1 associated disease comprises an autoimmune disease; more preferably the autoimmune disease comprises graft versus host disease, solid organ transplant rejection, vasculitis, systemic lupus erythematosus, type I diabetes, multiple sclerosis, giant cell arteritis, psoriasis, rheumatoid arthritis, inflammatory bowel disease, non-specific ulcerative colitis, autoimmune hepatitis. Use of the antibody or antigen-binding portion thereof according to any one of claims 1-9, the fusion protein according to claim 10, the multispecific antibody according to claim 11, the nucleic acid molecule according to claim 12, the expression vector according to claim 13, the recombinant cell according to claim 14 and / or the composition according to claim 15 for the treatment of a PD-1 related disease in a subject in need thereof; Preferably, the subject is a mammal; more preferably, the subject is a human; Preferably, the PD-1 related disease comprises an autoimmune disease; more preferably, the autoimmune disease comprises graft versus host disease, solid organ transplant rejection, vasculitis, systemic lupus erythematosus, type I diabetes, multiple sclerosis, giant cell arteritis, psoriasis, rheumatoid arthritis, inflammatory bowel disease, non-specific ulcerative colitis, autoimmune hepatitis. A method of preventing and / or treating a PD-1 related disease, characterized by, It comprises administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding portion thereof according to any one of claims 1-9, the fusion protein according to claim 10, the multispecific antibody according to claim 11 and / or the composition according to claim 15; Preferably, the subject is a mammal; more preferably, the subject is a human; Preferably, the PD-1 related disease comprises an autoimmune disease; more preferably, the autoimmune disease comprises graft versus host disease, solid organ transplant rejection, vasculitis, systemic lupus erythematosus, type I diabetes, multiple sclerosis, giant cell arteritis, psoriasis, rheumatoid arthritis, inflammatory bowel disease, non-specific ulcerative colitis, autoimmune hepatitis. The method of claim 19, wherein It comprises administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding portion thereof according to any one of claims 1-9, the fusion protein according to claim 10, the multispecific antibody according to claim 11 and / or the composition according to claim 15; Preferably, the subject is a mammal; more preferably, the subject is a human; Preferably, the PD-1 related disease comprises an autoimmune disease; more preferably, the autoimmune disease comprises graft versus host disease, solid organ transplant rejection, vasculitis, systemic lupus erythematosus, type I diabetes, multiple sclerosis, giant cell arteritis, psoriasis, rheumatoid arthritis, inflammatory bowel disease, non-specific ulcerative colitis, autoimmune hepatitis. It comprises administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding portion thereof according to any one of claims 1-9, the fusion protein according to claim 10, the multispecific antibody according to claim 11 and / or the composition according to claim 15;

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