WO2026008009A1

WO2026008009A1 - Pd-1 targeting antibodies and uses thereof

Info

Publication number: WO2026008009A1
Application number: PCT/CN2025/106810
Authority: WO
Inventors: Dandan Chen; Xuan HE
Original assignee: Xiamen Jiuheyipin Biopharmaceutical Co Ltd
Current assignee: Xiamen Jiuheyipin Biopharmaceutical Co Ltd
Priority date: 2024-07-04
Filing date: 2025-07-03
Publication date: 2026-01-08
Anticipated expiration: 2027-01-04

Abstract

Provided are novel anti-PD-1 agonist antibodies and antigen-binding fragments, as well as fusion proteins comprising such antibodies and a CTLA-4 peptide. Polynucleotides encoding the antibodies and fusion proteins, pharmaceutical compositions comprising the antibodies and fusion proteins, methods of manufacturing and medical uses of these anti-PD-1 antibodies and fusion proteins described herein are also provided.

Description

PD-1 TARGETING ANTIBODIES AND USES THEREOF

1. Reference to Sequence Listing Submitted Electronically

This application incorporates by reference a Sequence Listing entitled 091A001WO02_SL. XML created on June 20, 2025, and having a size of 94, 115 bytes.
2. Related Application

This application claims priority to PCT Patent Application No. PCT/CN2024/103524, filed July 4, 2024, which is incorporated herein by reference in its entirety.

3. Field

The present invention relates to molecular biology, cell biology, and immunology. Provided herein include anti-PD-1 antibodies and uses thereof in treating autoimmune or inflammatory diseases.

4. Background

Programmed cell death 1 (PD-1) is a key regulator in the induction and maintenance of immunological tolerance. The protein functions as an “immune checkpoint” inhibitor, that is, it acts to modulate the activity of cells in the immune system to regulate and limit autoimmune diseases. It is well understood that many cancers can protect themselves from the immune system by modifying “immune checkpoint” inhibitors and thus avoid detection. PD-1 inhibitors, a new class of drugs that block PD-1, activate the immune system to attack tumors and are used to treat certain types of cancer. In contrast, defective PD-1 inhibitory functions have also been associated with pathophysiology of immune-mediated diseases, and dysregulated expression of PD-1 or its ligands can contribute to certain autoimmune diseases. Induction of PD-1 activation represents an alternative approach to suppress the immune response and provide treatments for various immune and inflammatory disorders.

Despite the therapeutic potential of PD-1 agonists in treating autoimmunity and inflammatory diseases, such agents are currently lacking. The compositions and methods provided herein address this need and provide related advantages.

5. Summary

Provided herein are antibodies or antigen-binding fragments thereof that specifically binds human PD-1, comprising: (a) a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence of SEQ ID NO: 18; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or (b) a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 from a VH having the amino acid sequence of SEQ ID NO: 27; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, the antibodies or antigen-binding fragments provided herein comprise (a) a VL comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or (b) a VH comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 11, 12 and 13, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, the antibodies or antigen-binding fragments provided herein comprise VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 having the amino acid sequences of SEQ ID NOs: 8, 9, 10, 11, 12 and 13, respectively. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 having the amino acid sequences of SEQ ID NOs: 8, 9, 10, 11, 14 and 13, respectively.

In some embodiments, the antibodies or antigen-binding fragments provided herein are chimeric antibodies or antigen-binding fragments, humanized antibodies or antigen-binding fragments, or human antibodies or antigen-binding fragments.

In some embodiments, the antibodies or antigen-binding fragments provided herein are humanized antibodies or antigen-binding fragments.

In some embodiments, the antibodies or antigen-binding fragments provided herein comprise (a) a VL has at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44; and/or (b) a VH has at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54.

In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VL and a VH having the amino acid sequences of (1) SEQ ID NOs: 18 and 27, respectively; (2) SEQ ID NOs: 19 and 28, respectively; (3) SEQ ID NOs: 19 and 29, respectively; (4) SEQ ID NOs: 20 and 28, respectively; (5) SEQ ID NOs: 20 and 29, respectively; (6) SEQ ID NOs: 21 and 28, respectively; (7) SEQ ID NOs: 21 and 29, respectively; (8) SEQ ID NOs: 22 and 30, respectively; (9) SEQ ID NOs: 22 and 31, respectively; (10) SEQ ID NOs: 22 and 32, respectively; (11) SEQ ID NOs: 24 and 30, respectively; (12) SEQ ID NOs: 24 and 31, respectively; (13) SEQ ID NOs: 24 and 32, respectively; (14) SEQ ID NOs: 22 and 33, respectively; (15) SEQ ID NOs: 24 and 33, respectively; (16) SEQ ID NOs: 22 and 34, respectively; (17) SEQ ID NOs: 24 and 35, respectively; (18) SEQ ID NOs: 40 and 50, respectively; (19) SEQ ID NOs: 38 and 48, respectively; (20) SEQ ID NOs: 38 and 49, respectively; (21) SEQ ID NOs: 39 and 48, respectively; (22) SEQ ID NOs: 39 and 49, respectively; (23) SEQ ID NOs: 41 and 51, respectively; (24) SEQ ID NOs: 41 and 52, respectively; (25) SEQ ID NOs: 42 and 51, respectively; (26) SEQ ID NOs: 42 and 52, respectively; (27) SEQ ID NOs: 39 and 51, respectively; (28) SEQ ID NOs: 39 and 52, respectively; (29) SEQ ID NOs: 43 and 53, respectively; (30) SEQ ID NOs: 43 and 54, respectively; (31) SEQ ID NOs: 44 and 53, respectively; or (32) SEQ ID NOs: 44 and 54, respectively.

In some embodiments, the antibodies or antigen-binding fragments provided herein are selected from the group consisting of a Fab, a Fab’, a F (ab’) 2, a Fv, a scFv, a (scFv) 2, a single domain antibody (sdAb) , and a heavy chain antibody (HCAb) . In some embodiments, the antibodies or antigen-binding fragments provided herein are IgG1 antibodies, an IgG2 antibodies, an IgG3 antibodies, or an IgG4 antibodies.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that compete with the antibody or antigen-binding fragment described herein for binding to human PD-1.

In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain constant region (CL) that is kappa CL (Cκ; SEQ ID NO: 58) . In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain constant region (CL) that is lambda CL (Cλ; SEQ ID NO: 59) .

In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain constant region (CH) having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-63. In some embodiments, the heavy chain constant regions (CH) provided herein comprise at least one amino acid mutation to enhance its antibody-dependent cellular cytotoxicity (ADCC) .

In some embodiments, the antibodies or antigen-binding fragments provided herein are IgG1 antibodies.

In some embodiments, the heavy chain constant regions (CH) of the IgG1 antibodies provided herein comprise at least one amino acid substitution selected from the group consisting of L234A, L235E, G236A, S239D, F243L, D265A, S298A, A330L and I332E, according to EU numbering. In some embodiments, the CH regions of the IgG1 antibodies provided herein have S239D/A330L/I332E substitutions, according to EU numbering. In some embodiments, the CH region lacks core fucose on the Fc N-glycan on N297, according to EU numbering.

In some embodiments, the antibodies or antigen-binding fragments provided herein activate PD-1 signaling. In some embodiments, the antibodies or antigen-binding fragments provided herein inhibit T cell activity. In some embodiments, the antibodies or antigen-binding fragments provided herein inhibit T cell proliferation. In some embodiments, the antibodies or antigen-binding fragments provided herein inhibit T cell activity and proliferation. In some embodiments, the antibodies or antigen-binding fragments provided herein suppress T cell reconstitution.

In some embodiments, the antibodies or antigen-binding fragments provided herein are monospecific antibodies, bispecific antibodies or multispecific antibodies.

In some embodiments, the antibodies or antigen-binding fragments provided herein are monoclonal antibodies or antigen-binding fragments.

In some embodiments, provided herein are polynucleotides that encode or a plurality of polynucleotides that collectively encode the polypeptide chain (s) of the antibodies or antigen-binding fragments disclosed herein. In some embodiments, provided herein are vectors comprising a polynucleotide or plurality of polynucleotides disclosed herein. In some embodiments, provided herein are host cells comprising a polynucleotide or plurality of polynucleotides disclosed herein, or vectors disclosed herein.

In some embodiments, provided herein are methods of making an antibody or antigen-binding fragment thereof that specifically binds human PD-1, comprising culturing the cell disclosed herein under conditions that allow expression of the antibody or antigen-binding fragment. In some embodiments, the methods provided herein comprise isolating the antibody or antigen-binding fragment from the culture.

In some embodiments, provided herein are pharmaceutical compositions comprising a therapeutically effective amount of the antibody or antigen-binding fragment disclosed herein, and a pharmaceutically acceptable carrier.

In some embodiments, provided herein are methods of activating PD-1 signaling in an immune effector cell, comprising contacting the immune effector cell with the antibody or antigen-binding fragment disclosed herein. In some embodiments, provided herein are methods of inhibiting activity of an immune effector cell, comprising contacting the immune effector cell with the antibody or antigen-binding fragment disclosed herein. In some embodiments, provided herein are methods of reducing autoimmunity or inflammation in a subject in need thereof, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment disclosed herein. In some embodiments, provided herein are methods of treating an autoimmune or inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment disclosed herein. In some embodiments, the methods provided herein further comprise administering an additional therapy to the subject.

In some embodiments, the subject is a human.

In some embodiments, provided herein are uses of the antibody or antigen-binding fragment disclosed herein in reducing autoimmunity or inflammation. In some embodiments, provided herein are uses of the antibody or antigen-binding fragment disclosed herein for the preparation of a medicament for reducing autoimmunity or inflammation. In some embodiments, provided herein are uses of the antibody or antigen-binding fragment disclosed herein in treating an autoimmune or inflammatory disease. In some embodiments, provided herein are uses of the antibody or antigen-binding fragment disclosed herein for the preparation of a medicament for treating an autoimmune or inflammatory disease.

In some embodiments, provided herein are fusion proteins thereof comprising the anti-PD-1 antibody or antigen-binding fragment disclosed herein and a CTLA-4 peptide. In some embodiments, the CTLA-4 peptide provided herein comprises human CTLA-4 extracellular domain. In some embodiments, the CTLA-4 peptide provided herein has amino acid sequence of SEQ ID NO: 4.

In some embodiments, provided herein are fusion proteins wherein the CTLA-4 peptide and the anti-PD-1 antibody or antigen-binding fragment are connected by a linker. In some embodiments, the linker provided herein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 65-68.

In some embodiments, provided herein are fusion proteins wherein the CTLA-4 peptide is fused to N-terminus of the antibody or antigen-binding fragment. In some embodiments, provided herein are fusion proteins wherein the CTLA-4 peptide is fused to C-terminus of the antibody or antigen-binding fragment.

In some embodiments, the fusion proteins provided herein comprise a heavy chain and a light chain, wherein the heavy chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-78, and wherein the light chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 70.

In some embodiments, provided herein are polynucleotides that encode or a plurality of polynucleotides that collectively encode polypeptide chain (s) of the fusion proteins disclosed herein. In some embodiments, provided herein are vectors comprising the polynucleotides or plurality of polynucleotides disclosed herein. In some embodiments, provided herein are host cells comprising the polynucleotides or plurality of polynucleotides disclosed herein, or the vectors disclosed herein.

In some embodiments, provided herein are methods of making the fusion proteins disclosed herein, comprising culturing the cells disclosed herein under conditions that allow expression of the fusion protein. In some embodiments, the methods provided herein comprise isolating the antibody or antigen-binding fragment from the culture.

In some embodiments, provided herein are pharmaceutical compositions comprising a therapeutically effective amount of the fusion proteins disclosed herein and a pharmaceutically acceptable carrier.

In some embodiments, provided herein are methods of activating PD-1 signaling in an immune effector cell, comprising contacting the immune effector cell with the fusion proteins disclosed herein. In some embodiments, provided herein are methods of inhibiting activity of an immune effector cell, comprising contacting the immune effector cell with the fusion proteins disclosed herein. In some embodiments, provided herein are methods of reducing autoimmunity or inflammation in a subject in need thereof, comprising administering to the subject an effective amount of the fusion proteins disclosed herein. In some embodiments, provided herein are methods of treating an autoimmune or inflammatory disease associated in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the fusion proteins disclosed herein. In some embodiments, the methods provided herein further comprise administering an additional therapy to the subject.

In some embodiments, the subject provided herein is a human.

In some embodiments, provided herein are uses of the fusion proteins disclosed herein in reducing autoimmunity or inflammation. In some embodiments, provided herein are uses of the fusion proteins disclosed herein for the preparation of a medicament for reducing autoimmunity or inflammation. In some embodiments, provided herein are uses of the fusion proteins disclosed herein in treating an autoimmune or inflammatory disease. In some embodiments, provided herein are uses of the fusion proteins disclosed herein for the preparation of a medicament for treating an autoimmune or inflammatory disease.

6. Brief Description of Drawings

FIGs. 1A-1C provide representative results of T cell activation assay showing that candidate anti-PD-1 antibodies inhibited T cell activation. Results for candidate antibodies 00F8XM002, 00F8XM004, 00F8XM006, 00F8XM008, 00F8XM012, and 00F8XM013 are provided in FIG. 1A. Results for candidate antibodies CT-122, CT-123, CT-124, CT-125, CT-126, and 00F8XM013 are provided in FIG. 1B. Results for candidate antibodies CT-150, CT-151, CT-152, 00F8XM012, CT-143, CT-144, CT-145, CT-146, CT-147, CT-148, CT-149, as well as reference antibody peresolimab are provided in FIG. 1C.

FIGs. 2A-2D provide representative results of human primary CD4+ T cell proliferation activity showing that candidate anti-PD-1 antibodies inhibited proliferation of primary human CD4+T cells. Results for candidate antibodies 00F8XM008, CT-110, CT-112, CT-114, and CT-116 are provided in FIG. 2A. Results for candidate antibodies 00FFQY003, CT-111, CT-113, CT-115, and CT-117 are provided in FIG. 2B. Results for candidate antibodies CT-143, CT-145, and CT-146 are provided in FIG. 2C. Results for candidate antibodies CT-143, CT-145, CT-146, 00F8XM008, 00FFQY003, and CT-122 are provide in FIG. 2D.

FIG. 3 provides representative in vivo results in PBMC-humanized NCG-IL15 transgenic mice showing that candidate anti-PD-1 antibody CT-147 significantly suppressed T cell reconstitution.

7. Detailed Description

The present disclosure provides novel antibodies, including antigen-binding fragments that specifically bind PD-1 (e.g., human PD-1) . Pharmaceutical compositions comprising a therapeutically effective amount of such antibodies or antigen-binding fragments are also disclosed herein. Also disclosed herein are uses of such pharmaceutical compositions for treating autoimmune or inflammatory diseases.

Programmed cell death protein 1 (PD-1) , also known as CD279 cluster of differentiation 279, is a protein encoded in humans by the PDCD1 gene. PD-1 is an immune checkpoint and guards against autoimmunity through two mechanisms. It promotes apoptosisof antigen-specific T-cells in lymph nodes and reduces apoptosis in regulatory T cells. PD-1 is a type I membrane protein of 288 amino acids and is a member of the extended CD28/CTLA-4 family of T cell regulators (SEQ ID NO: 1) . The protein’s structure includes an extracellular IgV domain followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located in an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif, which suggests that PD-1 negatively regulates T-cell receptor TCR signals.

The sequence of human PD-1 is provided below:

PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 protein is upregulated on macrophages and dendritic cells (DC) in response to LPS and GM-CSF treatment, and on T cells and B cells upon TCR and B cell receptor signaling, whereas in resting mice, PD-L1 mRNA can be detected in the heart, lung, thymus, spleen, and kidney. PD-L1 is expressed on almost all murine tumor cell lines, including PA1 myeloma, P815 mastocytoma, and B16 melanoma upon treatment with IFN-γ. PD-L2 expression is more restricted and is expressed mainly by DCs and a few tumor lines.

PD-L1-mediated activation of PD-1 requires accumulation of the ligand at the cell-cell interface, promoting the colocalization of PD-1 and the T cell receptor (TCR) complex at the immune synapse. Ligation of PD-1 enables the recruitment and activation of the SHP2 phosphatase, which in turn dephosphorylates TCR complex-signaling molecules, including ZAP-70, SLP-76, and PLCγ, resulting in T cell inhibition.

The PD-1 pathway is a key immune checkpoint that plays an important role in maintaining peripheral T cell tolerance and regulating adaptive immune responses. PD-1 pathway impairment has been associated with pathogenesis of autoimmune diseases. PD-1, PD-L1, and PD-L2 gene polymorphisms are associated with several autoimmune diseases, and perturbance of PD-1 pathway components has been reported in some indications. For example, abnormally low PD-L1 expression was noted in samples from type 1 diabetes and Crohn’s disease patients. Additionally, PD-1 expression on effector T cells is elevated in a number of autoimmune diseases, and administration of PD-1 pathway antagonists can cause autoimmune-like symptoms in cancer patients, including aggravation of preexisting autoimmunity.

An inhibitor of both adaptive and innate immune responses, PD-1 is expressed on activated T, natural killer (NK) and B lymphocytes, macrophages, dendritic cells (DCs) and monocytes. Han et al., Am J Cancer Res. 2020; 10 (3) : 727-742. One promising approach to generate efficacious PD-1 agonists is to mimic natural PD-L1-PD-1 engagement with immune effector cells (e.g., T cells) . Provided herein are novel anti-PD-1 antibodies that activate the PD-1 signaling and thereby inhibit T cell activation, proliferation, and/or reconstitution.

Before the present disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments set forth herein, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments, and is not intended to be limiting.
7.1 Definitions

Unless otherwise defined herein, scientific and technical terms used in the present disclosures shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

The term “a” or “an” entity refers to one or more of that entity; for example, “an antibody, ” is understood to represent one or more antibodies.

The term “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B, ” “A or B, ” “A” (alone) , and “B” (alone) . Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone) ; B (alone) ; and C (alone) .

As used herein, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. The term “about” encompasses the exact number recited. In some embodiments, “about” means within plus or minus 10%of a given value or range. In certain embodiments, “about” means that the variation is ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1%of the value to which “about” refers. In some embodiments, “about” means that the variation is ±1%, ±0.5%, ±0.2%, or ±0.1%of the value to which “about” refers.

The term “antibody” as used herein refer to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of any of the foregoing, through at least one antigen-binding site wherein the antigen-binding site is usually within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single-domain antibodies (sdAbs; e.g., camelid antibodies, alpaca antibodies) , single-chain Fv (scFv) antibodies, heavy chain antibodies (HCAbs) , light chain antibodies (LCAbs) , multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, and any other modified immunoglobulin molecule comprising an antigen-binding site (e.g., dual variable domain immunoglobulin molecules) as long as the antibodies exhibit the desired biological activity. Antibodies also include, but are not limited to, mouse antibodies, camel antibodies, chimeric antibodies, humanized antibodies, and human antibodies. An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) , based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. Unless expressly indicated otherwise, the term “antibody” as used herein includes “antigen-binding fragment” of intact antibodies. The term “antigen-binding fragment” as used herein refers to a portion or fragment of an intact antibody that is the antigenic determining variable region of an intact antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F (ab’) 2, Fv, linear antibodies, single chain antibody molecules (e.g., scFv) , heavy chain antibodies (HCAbs) , light chain antibodies (LCAbs) , disulfide-linked scFv (dsscFv) , diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD) , single variable domain antibodies (sdAbs; e.g., camelid antibodies, alpaca antibodies) , and single variable domain of heavy chain antibodies (VHH) , and bispecific or multispecific antibodies formed from antibody fragments. A “bispecific” antibody is an artificial hybrid antibody having two different antigen binding sites, which recognize and specifically bind two different targets. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab'fragments. See, e.g., Songsivilai &Lachmann, Clin. Exp. Immunol. 79: 315-321 (1990) ; Kostelny et al., J. Immunol. 148, 1547-1553 (1992) .

The term “humanized antibody” as used herein refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulin. In some instances, the Fv framework region residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species. In some instances, residues of the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, hamster, camel) that have the desired specificity, affinity, and/or binding capability. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or binding capability. The term “human antibody” as used herein refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any of the techniques known in the art.

The term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids and a carboxy-terminal portion that includes a constant region. The constant region can be one of five distinct types, referred to as alpha (α) , delta (δ) , epsilon (ε) , gamma (γ) and mu (μ) , based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: α, δ and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes of antibodies, IgA, IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, namely IgGl, IgG2, IgG3 and IgG4. A heavy chain can be a human heavy chain.

The term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids and a carboxy-terminal portion that includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as kappa (κ) and lambda (λ) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. A light chain can be a human light chain.

The term “variable domain” or “variable region” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chains and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and is used in the binding and specificity of each particular antibody for its particular antigen. The variable domain of the light chain is referred to as the “VL” ; and the variable domain of the heavy chain is referred to as the “VH. ” The variable domains differ extensively in sequence between different antibodies. The variability in sequence is concentrated in the CDRs while the less variable portions in the variable domain are referred to as framework regions (FR) . The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen. The numbering of amino acid positions used herein is according to the EU Index, as in Kabat et al. (1991) Sequences of proteins of immunological interest. (U.S. Department of Health and Human Services, Washington, D.C. ) 5^th ed. A variable region can be a human variable region.

A CDR refers to one of three hypervariable regions (H1, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH β-sheet framework, or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the antibody VL β-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by a variety of methods/systems. These systems and/or definitions have been developed and refined over years and include Kabat, Chothia, IMGT, AbM, and Contact. For example, Kabat defines the regions of most hypervariability within the antibody variable (V) domains (Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977) ; Kabat, Adv. Prot. Chem. 32: 1-75 (1978) ) . The Chothia definition is based on the location of the structural loop regions, which defines CDR region sequences as those residues that are not part of the conserved β-sheet framework, and thus are able to adapt different conformations (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) ) . Both terminologies are well recognized in the art. Additionally, the IMGT system is based on sequence variability and location within the structure of the variable regions. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on analyses of the available antibody crystal structures. Software programs (e.g., abYsis) are available and known to those of skill in the art for analysis of antibody sequence and determination of CDRs. The positions of CDRs within a canonical antibody variable domain have been determined by comparison of numerous structures (Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997) ; Morea et al., Methods 20: 267-279 (2000) ) . Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al., supra (1997) ) . Such nomenclature is similarly well known to those skilled in the art.

For example, CDRs defined according to either the Kabat (hypervariable) or Chothia (structural) designations, are set forth in the table below.

¹Residue numbering follows the nomenclature of Kabat et al., supra
²Residue numbering follows the nomenclature of Chothia et al., supra

One or more CDRs also can be incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin. An immunoadhesin can incorporate the CDR (s) as part of a larger polypeptide chain, can covalently link the CDR (s) to another polypeptide chain, or can incorporate the CDR (s) noncovalently. The CDRs permit the immunoadhesin to bind to a particular antigen of interest. The CDR regions can be analyzed by, for example, abysis website (http: //abysis. org/) .

The terms “epitope” and “antigenic determinant” as used interchangeably herein refer to the site on the surface of a target molecule to which an antibody or antigen-binding fragment binds, such as a localized region on the surface of an antigen. The target molecule can comprise, a protein, a peptide, a nucleic acid, a carbohydrate, or a lipid. An epitope having immunogenic activity is a portion of a target molecule that elicits an immune response in an animal. An epitope of a target molecule having antigenic activity is a portion of the target molecule to which an antibody binds, as determined by any method well known in the art, including, for example, by an immunoassay. Antigenic epitopes need not necessarily be immunogenic. Epitopes often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. The term, “epitope” includes linear epitopes and conformational epitopes. A region of a target molecule (e.g., a polypeptide) contributing to an epitope can be contiguous amino acids of the polypeptide or the epitope can come together from two or more non-contiguous regions of the target molecule. The epitope may or may not be a three-dimensional surface feature of the target molecule. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.

The term “specifically bind, ” and its grammatical equivalents, as used herein mean that a polypeptide or molecule interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including related and unrelated proteins. A binding moiety (e.g., antibody) that specifically binds a target molecule (e.g., antigen) can be identified, for example, by immunoassays, ELISAs, Bio-Layer Interferometry ( “BLI” ) , SPR (e.g., Biacore) , or other techniques known to those of skill in the art. Typically, a specific reaction will be at least twice background signal or noise and can be more than 10 times background. See, e.g., Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336 for a discussion regarding antibody specificity. A binding moiety that specifically binds a target molecule can bind the target molecule at a higher affinity than its affinity for a different molecule. In some embodiments, a binding moiety that specifically binds a target molecule can bind the target molecule with an affinity that is at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, or at least 100 times greater, than its affinity for a different molecule. In some embodiments, a binding moiety that specifically binds a particular target molecule binds a different molecule at such a low affinity that binding cannot be detected using an assay described herein or otherwise known in the art. In some embodiments, “specifically binds” means, for instance, that a binding moiety binds a molecule target with a K_D of about 0.1 mM or less. In some embodiments, “specifically binds” means that a polypeptide or molecule binds a target with a K_D of about 10 μM or less or about 1 μM or less. In some embodiments, “specifically binds” means that a polypeptide or molecule binds a target with a K_D of about 0.1 μM or less, about 0.01 μM or less, or about 1 nM or less. Because of the sequence identity between homologous proteins in different species, specific binding can include a polypeptide or molecule that recognizes a protein or target in more than one species. Likewise, because of homology within certain regions of polypeptide sequences of different proteins, specific binding can include a polypeptide or molecule that recognizes more than one protein or target. It is understood that, in some embodiments, a binding moiety (e.g., antibody) that specifically binds a first target may or may not specifically bind a second target. As such, “specific binding” does not necessarily require (although it can include) exclusive binding, i.e., binding to a single target. Thus, a binding moiety (e.g., antibody) can, in some embodiments, specifically bind more than one target. For example, an antibody can, in certain instances, comprise two identical antigen-binding sites, each of which specifically binds the same epitope on two or more proteins. In certain alternative embodiments, an antibody can be bispecific and comprise at least two antigen-binding sites with differing specificities.

The term “binding affinity” as used herein generally refers to the strength of the sum total of noncovalent interactions between a binding moiety (e.g., antibody) and a target molecule (e.g., antigen) . The binding of a binding moiety and a target molecule is a reversible process, and the affinity of the binding is typically reported as an equilibrium dissociation constant (K_D) . K_D is the ratio of a dissociation rate (k_off or k_d) to the association rate (k_on or k_a) . The lower the K_D of a binding pair, the higher the affinity. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific illustrative embodiments include the following. In some embodiments, the “K_D” or “K_D value” can be measured by assays known in the art, for example by a binding assay. The K_D may be measured in a radiolabeled antigen binding assay (RIA) (Chen, et al., (1999) J. Mol. Biol. 293: 865-881) . The K_D or K_D value can also be measured by using biolayer interferometry (BLI) using, for example, the Gator system (Probe Life) , or the Octet-96 system (Sartorius AG) . The K_D or K_D value can also be measured by using surface plasmon resonance assays (SPR) by Biacore, using, for example, a BIAcoreTM-2000 or a BIAcoreTM-3000 BIAcore, Inc., Piscataway, NJ) . The binding affinity can also be quantified with EC₅₀, which is the concentration of ligand at which half of the target is present in the bound state in a binding assay.

The terms “activate, ” “stimulate, ” and “agonize, ” and their grammatical equivalents as used interchangeably herein in relation to a molecule or a signaling pathway refer to initiate, turn on, trigger, strengthen or increase the function or the activity of the molecule or the signaling pathway. In some embodiments, it is a process by which the targeted molecule or signaling is brought into an active state to exert its physiological functions.

The terms “inhibit, ” “block, ” and “antagonize” and their grammatical equivalents used interchangeably herein in relation to a molecule or a signaling pathway refer to stop, eliminate, decrease, or reduce the function or the activity of the molecule or the signaling pathway. Total abolition is not required; partial inhibition, which corresponds to a reduction but not complete abolition of the biological effect -is also contemplated.

The terms “polypeptide, ” “peptide, ” and “protein, ” as used interchangeably herein refer to polymers of amino acids of any length, which can be linear or branched. It can include unnatural or modified amino acids or be interrupted by non-amino acids. A polypeptide, peptide, or protein can also be modified with, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.

The term “immune effector cell” as used herein means a type of cell within the immune system that directly acts to carry out an immune response. These cells are responsible for performing specific functions to combat pathogens, infected cells, or abnormal cells. They include various types such as cytotoxic T cells that can directly kill target cells, natural killer cells with innate cytotoxic abilities, B cells that produce antibodies, and macrophages that phagocytose and destroy foreign substances or cells. Immune effector cells play a crucial role in defending the body against invaders, maintaining immune homeostasis, and mounting an appropriate immune reaction to clear threats or restore normal physiological conditions.

The term “contact” and its grammatical equivalents as used herein in relation to a molecule (e.g., an antibody) and a cell or a second molecule refers to the act of bringing the molecule into physical proximity or interaction with the cell or the second molecule. The contact enables a direct encounter or engagement between the molecule and the surface of the cell or the second molecule. The contact can be in vivo or in vitro.

The term “variant” as used herein in relation to a protein or a polypeptide with particular sequence features (the “reference protein” or “reference polypeptide” ) refers to a different protein or polypeptide having one or more (such as, for example, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and/or additions as compared to the reference protein or reference polypeptide. The changes to an amino acid sequence can be amino acid substitutions. The changes to an amino acid sequence can be conservative amino acid substitutions. The changes to an amino acid sequence can be amino acid deletions. A variant can be a fragment of the reference protein or polypeptide. A functional variant of a protein or polypeptide maintains the basic structural and functional properties of the reference protein or polypeptide. For example, a functional variant or a functional fragment of human CTLA-4 maintains its binding to CD80 and CD86 and its function to deliver the inhibitory signal to T cells.

The terms “polynucleotide, ” “plurality of polynucleotides, ” and “nucleic acid, ” as used interchangeably herein refer to a polymer or oligomer of nucleotides of any length. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases (such as methylated, hydroxymethylated, or glycosylated) , non-natural nucleotides, non-nucleotide building blocks that exhibit similar structure and/or function as natural nucleotides (i.e., “nucleotide analogs” ) , and/or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. The nucleic acids or polynucleotides can be heterogenous or homogenous in composition, can be isolated from naturally occurring sources, or can be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and can exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. Nucleic acid structures also include, for instance, a DNA/RNA helix, peptide nucleic acid (PNA) , morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 4 (14) : 4503-4510 (2002) and U.S. Patent 5,034,506) , locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000) ) , cyclohexenyl nucleic acid (see Wang, Am. Chem. Soc., 122: 8595-8602 (2000) ) , and/or a ribozyme.

The terms “identical, ” percent “identity, ” and their grammatical equivalents as used herein in the context of two or more polynucleotides or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two polynucleotides or polypeptides provided herein are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 bases, such as at least about 80-100 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.

The term “vector” as used herein refer to a vehicle that is used to carry genetic material (e.g., a polynucleotide sequence) , which can be introduced into a host cell, where it can be replicated and/or expressed. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more polynucleotides are to be co-expressed, both polynucleotides can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding polynucleotides can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of polynucleotides into a host cell can be confirmed using methods well known in the art. It is understood by those skilled in the art that the polynucleotides are expressed in a sufficient amount to produce a desired product (e.g., an anti-PD-1 antibody or antigen-binding fragment as described herein) , and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

As used herein, the term “encode” and its grammatical equivalents refer to the inherent property of specific sequences of nucleotides in a polynucleotide or a nucleic acid, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA can include introns.

A polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

The term “treat” and its grammatical equivalents as used herein in connection with a disease or a condition, or a subject having a disease or a condition refer to an action that suppresses, eliminates, reduces, and/or ameliorates a symptom, the severity of the symptom, and/or the frequency of the symptom associated with the disease or disorder being treated.

The term “administer” and its grammatical equivalents as used herein refer to the act of delivering, or causing to be delivered, a therapeutic or a pharmaceutical composition to the body of a subject by a method described herein or otherwise known in the art. The therapeutic can be a compound, a polypeptide, an antibody, a cell, or a population of cells. Administering a therapeutic or a pharmaceutical composition includes prescribing a therapeutic or a pharmaceutical composition to be delivered into the body of a subject. Exemplary forms of administration include oral dosage forms, such as tablets, capsules, syrups, suspensions; injectable dosage forms, such as intravenous (IV) , intramuscular (IM) , or intraperitoneal (IP) ; transdermal dosage forms, including creams, jellies, powders, or patches; buccal dosage forms; inhalation powders, sprays, suspensions, and rectal suppositories.

The terms “effective amount, ” “therapeutically effective amount, ” as used herein refer to the administration of an agent to a subject, either alone or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to the subject. The therapeutically effective amount can be ascertained by measuring relevant physiological effects. The exact amount required varies from subject to subject, depending on the age, weight, and general condition of the subject, the severity of the condition being treated, the judgment of the clinician, and the like. An appropriate “effective amount” in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” refers to a material that is suitable for drug administration to an individual along with an active agent without causing undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition.

The term “subject” as used herein refers to any animal (e.g., a mammal) , including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. A subject can be a human. A subject can have a particular disease or condition.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Exemplary genes and polypeptides are described herein with reference to GenBank numbers, GI numbers and/or SEQ ID NOs. It is understood that one skilled in the art can readily identify homologous sequences by reference to sequence sources, including but not limited to GenBank (ncbi. nlm. nih. gov/genbank/) and EMBL (embl. org/) .
7.2 Anti-PD-1 antibodies and antigen-binding fragments

Provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 (e.g., human PD-1) . In some embodiments, provided herein are anti-PD-1 antibodies. In some embodiments, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgA antibody. In some embodiments, the antibody is an IgD antibody. In some embodiments, the antibody is an IgE antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibodies provided herein can be an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody.

In some embodiments, provided herein are antigen-binding fragments of an anti-PD-1 antibody. In some embodiments, antigen-binding fragments provided herein can be a single domain antibody (sdAb) , a heavy chain antibody (HCAb) , a Fab, a Fab’, a F (ab’) ₂, a Fv, a single-chain variable fragment (scFv) , or a (scFv) ₂. In some embodiments, the antigen-binding fragment of an anti-PD-1 antibody is a single domain antibody (sdAb) . In some embodiments, the antigen-binding fragment of an anti-PD-1 antibody is a heavy chain antibody (HCAb) . In some embodiments, the antigen-binding fragment of an anti-PD-1 antibody is a Fab. In some embodiments, the antigen-binding fragment of an anti-PD-1 antibody is a Fab’. In some embodiments, the antigen-binding fragment of an anti-PD-1 antibody is a F (ab’) ₂. In some embodiments, the antigen-binding fragment of an anti-PD-1 antibody is a Fv. In some embodiments, the antigen-binding fragment of an anti-PD-1 antibody is a scFv. In some embodiments, the antigen-binding fragment of an anti-PD-1 antibody is a disulfide-linked scFv [ (scFv) ₂] . In some embodiments, the antigen-binding fragment of an anti-PD-1 antibody is a diabody (dAb) .

In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments provided herein comprise recombinant antibodies or antigen-binding fragments. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments provided herein comprise monoclonal antibodies or antigen-binding fragments. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments provided herein comprise polyclonal antibodies or antigen-binding fragments. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments provided herein comprise camelid (e.g., camels, dromedary and llamas) antibodies or antigen-binding fragments. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments provided herein comprise chimeric antibodies or antigen-binding fragments. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments provided herein comprise humanized antibodies or antigen-binding fragments. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments provided herein comprise human antibodies or antigen-binding fragments. In some embodiments, provided herein are anti-PD-1 human scFvs.

In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments provided herein are isolated. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments provided herein are substantially pure.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment provided herein comprises a multispecific antibody or antigen-binding fragment. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment provided herein comprises a bispecific antibody or antigen-binding fragment. In some embodiments, the bispecific antibody or antigen-binding fragment comprises an anti-PD-1 antibody or antigen-binding fragment provided herein. In some embodiments, the bispecific antibody or antigen-binding fragment comprises an anti-PD-1 scFv provided herein.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment provided herein comprises a monovalent antigen-binding site. In some embodiments, an anti-PD-1 antibody or antigen-binding fragment comprises a monospecific binding site. In some embodiments, an anti-PD-1 antibody or antigen-binding fragment comprises a bivalent binding site.

In some embodiments, an anti-PD-1 antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment. Monoclonal antibodies can be prepared by any method known to those of skill in the art. In some embodiments, the monoclonal antibodies are prepared using hybridoma technology. and Milstein (1975) Nature, 256 (5517) , 495-497; Harlow and Lane (1988) , ANTIBODIES: A LABORATORY MANUAL. Cold Spring Harbor Laboratory Press.

In some embodiments, a monoclonal antibody is modified by using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light chain and heavy chain of a mouse monoclonal antibody are replaced with the constant regions of a human antibody to generate a chimeric antibody. In some embodiments, the constant regions are truncated or removed to generate a desired antibody fragment of a monoclonal antibody. In some embodiments, site-directed or high-density mutagenesis of the variable region (s) is used to optimize specificity and/or affinity of a monoclonal antibody.

In some embodiments, provided herein are anti-PD-1 antibodies with the sequence features are described below. The specific CDR sequences defined herein are generally based on Kabat definition. However, it is understood that a general reference to a heavy chain CDR or CDRs and/or a light chain CDR or CDRs of a specific antibody encompasses all CDR definitions as known to those of skill in the art. In some embodiments, provided herein are anti-PD-1 antibodies having the VL CDRs and/or VH CDRs of antibody clone antibody disclosed herein, wherein the CDRs are defined by Kabat, Chothia, IMGT, AbM, or Contact. In some embodiments, the CDRs are defined by Kabat (as exemplified in detail below) . In some embodiments, the CDRs are defined by Chothia. In some embodiments, the CDRs are defined by IMGT. In some embodiments, the CDRs are defined by AbM. In some embodiments, the CDRs are defined by Contact.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof provided herein has a VL that comprises VL CDRs 1, 2, and 3 from the VL having the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof provided herein has a VH that comprises VH CDRs 1, 2, and 3 from the VH having the amino acid sequence of SEQ ID NO: 27. The anti-PD-1 antibody or antigen-binding fragment thereof provided herein can have a VL comprising VL CDRs 1, 2, and 3 and a VH comprising VH CDRs 1, 2, and 3 from the VL and VH having the amino acid sequences of SEQ ID NOs: 18 and 27, respectively. The CDRs can be defined by any system known in the art. In some embodiments, the CDRs are defined by Kabat, Chothia, IMGT, AbM, or Contact. In some embodiments, the CDRs are defined by Kabat. In some embodiments, the CDRs are defined by Kabat. In some embodiments, the CDRs are defined by IMGT. In some embodiments, the CDRs are defined by AbM. In some embodiments, the CDRs are defined by Contact.

In some embodiments, anti-PD-1 antibodies or antigen-binding fragments provided herein comprise one, two, three, four, five, and/or six CDRs of any one of the antibodies described herein. In some embodiments, anti-PD-1 antibodies or antigen-binding fragments provided herein comprise a light chain variable region (VL) comprising one, two, and/or three, light chain CDRs (VL CDRs) from Table 1. In some embodiments, anti-PD-1 antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region (VH) comprising one, two, and/or three heavy chain CDRs (VH CDRs) from Table 2. In some embodiments, anti-PD-1 antibodies or antigen-binding fragments provided herein comprise one, two, and/or three VL CDRs from Table 1 and one, two, and/or three VH CDRs from Table 2.

Table 1 Amino acid sequences of light chain variable region CDRs (VL CDRs) of anti-PD-1 antibodies or antigen-binding fragments

Table 2 Amino acid sequences of heavy chain variable region CDRs (VH CDRs) of anti-PD-1 antibodies or antigen-binding fragments

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 (e.g., human PD-1) , comprising a VL comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 8; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 9; and/or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 10; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 11; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 12; and/or (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 13; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1, comprising a VL comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 8; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 9; or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 10; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VL CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDR. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1, comprising a VL comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 8; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 9; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 10; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VL CDRs. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 having a VL, wherein the VL comprises VL CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively, as defined by Kabat; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VL CDRs. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 having a VL, wherein the VL comprises VL CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively, as defined by Kabat; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VL CDRs. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 11; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 12; or (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 13; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VH CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDR. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 11; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 12; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 13; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VH CDRs. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs. In some embodiments, the VH CDR2 has an amino acid substitution at the G7 of SEQ ID NO: 12. In some embodiments, the substitution is G7S. In some embodiments, the VH CDR2 has the amino acid sequence of SEQ ID NO: 14.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 having a VH, wherein the VH comprises VH CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 11, 12 and 13, respectively, as defined by Kabat; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VH CDRs. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs. In some embodiments, the VH comprises VH CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 11, 14 and 13, respectively, as defined by Kabat.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1, comprising, as defined by Kabat, (a) a VL comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or (b) a VH comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 11, 12 and 13, respectively; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VH CDRs. In some embodiments, the VH CDR2 has an amino acid substitution at the G7 of SEQ ID NO: 12. In some embodiments, the substitution is G7S. In some embodiments, the VH CDR2 has the amino acid sequence of SEQ ID NO: 14.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3, having the amino acid sequences of SEQ ID NOs: 8, 9, 10, 11, 12 and 13, respectively, as defined by Kabat, or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the CDRs. In some embodiments, the VH CDR2 has an amino acid substitution at the G7 of SEQ ID NO: 12. In some embodiments, the substitution is G7S. In some embodiments, the antibodies or antigen-binding fragments thereof comprise VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3, having the amino acid sequences of SEQ ID NOs: 8, 9, 10, 11, 14 and 13, respectively, as defined by Kabat.

Table 3 Amino acid sequences of VLs and VHs.

Table 4A Humanized Antibodies (HuAb) with specific VLs and VHs.

Table 4B Humanized Antibodies (HuAb) with specific humanized VLs and VHs.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof provided herein comprises a VL having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof provided herein comprises a VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof provided herein comprises a VL having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44 and a VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof provided herein is a variant of an antibody provided herein. The variant VL can have up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44. The variant VL can have up to about 5 amino acid substitutions, additions, and/or deletions in an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44. The variant VH can have up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54. The variant VH can have up to about 5 amino acid substitutions, additions, and/or deletions in an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54. In some embodiments, the variant of an antibody has up to about 5 conservative amino acid substitutions.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising: (a) a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44; and/or (b) a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100%sequence identity to SEQ ID NO: 18. In some embodiments, the VL can have the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VH that is any VH disclosed herein. In some embodiments, the VH can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 19. In some embodiments, the VL can have the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VH that is any VH disclosed herein. In some embodiments, the VH can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 20. In some embodiments, the VL can have the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VH that is any VH disclosed herein. In some embodiments, the VH can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 21. In some embodiments, the VL can have the amino acid sequence of SEQ ID NO: 21. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VH that is any VH disclosed herein. In some embodiments, the VH can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 22. In some embodiments, the VL can have the amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VH that is any VH disclosed herein. In some embodiments, the VH can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 24. In some embodiments, the VL can have the amino acid sequence of SEQ ID NO: 24. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VH that is any VH disclosed herein. In some embodiments, the VH can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 38. In some embodiments, the VL can have the amino acid sequence of SEQ ID NO: 38. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VH that is any VH disclosed herein. In some embodiments, the VH can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 39. In some embodiments, the VL can have the amino acid sequence of SEQ ID NO: 39. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VH that is any VH disclosed herein. In some embodiments, the VH can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 40. In some embodiments, the VL can have the amino acid sequence of SEQ ID NO: 40. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VH that is any VH disclosed herein. In some embodiments, the VH can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 41. In some embodiments, the VL can have the amino acid sequence of SEQ ID NO: 41. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VH that is any VH disclosed herein. In some embodiments, the VH can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 42. In some embodiments, the VL can have the amino acid sequence of SEQ ID NO: 42. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VH that is any VH disclosed herein. In some embodiments, the VH can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 43. In some embodiments, the VL can have the amino acid sequence of SEQ ID NO: 43. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VH that is any VH disclosed herein. In some embodiments, the VH can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 44. In some embodiments, the VL can have the amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VH that is any VH disclosed herein. In some embodiments, the VH can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 27. In some embodiments, the VH can have the amino acid sequence of SEQ ID NO: 27. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VL that is any VL disclosed herein. In some embodiments, the VL can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 28. In some embodiments, the VH can have the amino acid sequence of SEQ ID NO: 28. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VL that is any VL disclosed herein. In some embodiments, the VL can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 29. In some embodiments, the VH can have the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VL that is any VL disclosed herein. In some embodiments, the VL can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 30. In some embodiments, the VH can have the amino acid sequence of SEQ ID NO: 30. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VL that is any VL disclosed herein. In some embodiments, the VL can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 31. In some embodiments, the VH can have the amino acid sequence of SEQ ID NO: 31. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VL that is any VL disclosed herein. In some embodiments, the VL can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 32. In some embodiments, the VH can have the amino acid sequence of SEQ ID NO: 32. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VL that is any VL disclosed herein. In some embodiments, the VL can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 33. In some embodiments, the VH can have the amino acid sequence of SEQ ID NO: 33. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VL that is any VL disclosed herein. In some embodiments, the VL can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 34. In some embodiments, the VH can have the amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VL that is any VL disclosed herein. In some embodiments, the VL can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 35. In some embodiments, the VH can have the amino acid sequence of SEQ ID NO: 35. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VL that is any VL disclosed herein. In some embodiments, the VL can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 48. In some embodiments, the VH can have the amino acid sequence of SEQ ID NO: 48. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VL that is any VL disclosed herein. In some embodiments, the VL can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 49. In some embodiments, the VH can have the amino acid sequence of SEQ ID NO: 49. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VL that is any VL disclosed herein. In some embodiments, the VL can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 50. In some embodiments, the VH can have the amino acid sequence of SEQ ID NO: 50. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VL that is any VL disclosed herein. In some embodiments, the VL can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 51. In some embodiments, the VH can have the amino acid sequence of SEQ ID NO: 51. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VL that is any VL disclosed herein. In some embodiments, the VL can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 52. In some embodiments, the VH can have the amino acid sequence of SEQ ID NO: 52. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VL that is any VL disclosed herein. In some embodiments, the VL can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 53. In some embodiments, the VH can have the amino acid sequence of SEQ ID NO: 53. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VL that is any VL disclosed herein. In some embodiments, the VL can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 54. In some embodiments, the VH can have the amino acid sequence of SEQ ID NO: 54. In some embodiments, the antibodies or antigen-binding fragments can further comprise have a VL that is any VL disclosed herein. In some embodiments, the VL can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44.

Expressly contemplated herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising any combination of any VL and any VH disclosed herein. For exemplary purposes, in some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising a VL and a VH, wherein the VL and VH can have the amino acid sequences of SEQ ID NOs: 18 and 27, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 19 and 28, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 19 and 29, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 20 and 28, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 20 and 29, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 21 and 28, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 21 and 29, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 22 and 30, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 22 and 31, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 22 and 32, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 24 and 30, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 24 and 31, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 24 and 32, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 22 and 33, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 24 and 33, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 22 and 34, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 24 and 35, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 40 and 50, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 38 and 48, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 38 and 49, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 39 and 48, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 39 and 49, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 41 and 51, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 41 and 52, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 42 and 51, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 42 and 52, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 39 and 51, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 39 and 52, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 43 and 53, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 43 and 54, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 44 and 53, respectively. In some embodiments, the VL and VH can have the amino acid sequences of SEQ ID NOs: 44 and 54, respectively.

In some embodiments, provided herein are anti-PD-1 antibodies or antigen-binding fragments thereof that comprise VL CDRs from a VL described herein (SEQ ID NO: 18, 19, 20, 21, 22, 24, 38, 39, 40, 41, 42, 43 or 44) , and/or VH CDRs from a VH described herein (SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 35, 48, 49, 50, 51, 52, 53 or 54) . Methods to identify CDRs are well known in the art. For example, software programs (e.g., abYsis) on publicly available websites are known to those of skill in the art for analysis of antibody sequence and determination of CDRs. In some embodiments, the CDRs are defined by Kabat, Chothia, IMGT, AbM, or Contact. In some embodiments, the CDRs are defined by Kabat (as exemplified in detail below) . In some embodiments, the CDRs are defined by Chothia. In some embodiments, the CDRs are defined by IMGT. In some embodiments, the CDRs are defined by AbM. In some embodiments, the CDRs are defined by Contact.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 18; and/or (b) a VH comprising VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 27. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind PD-1 comprising (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24, 38-44; and/or (b) a VH comprising VH CDRs 1, 2, and 3 from a VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35, 48-54.

The anti-PD-1 antibodies or antigen-binding fragments thereof can comprise a combination of any VL disclosed herein and any VH disclosed herein. In some embodiments, the VL and VH are connected by a linker. The linker can be a flexible linker or a rigid linker. In some embodiments, the linker has the amino acid sequence of (GGGGS) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 65) . In some embodiments, the linker has the amino acid sequence of (EAAAK) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 66) . In some embodiments, the linker has the amino acid sequence of (PA) nPAP, n=0, 1, 2, 3, or 4 (SEQ ID NO: 67) . In some embodiments, the linker has the amino acid sequence of GGGGSGGGS (SEQ ID NO: 68) .

In some embodiments, anti-PD-1 antibodies provided herein are IgA, IgD, IgE, IgG, or IgM antibodies. In some embodiments, the antibody is an IgA antibody. In some embodiments, the antibody is an IgD antibody. In some embodiments, the antibody is an IgE antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibodies provided herein can be an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody.

In some embodiments, anti-PD-1 antibodies provided herein comprise a light chain and a heavy chain. The light chain can comprise a light chain constant domain (CL) and a light chain variable domain (VL) . The heavy chain can comprise a heavy chain variable domain (VH) and a heavy chain constant domain (CH) . The VL/VH can be any VL/VH disclosed herein. In some embodiments, the light chain constant region (CL) is kappa CL (Cκ; SEQ ID NO: 58) . In some embodiments, the light chain constant region (CL) is lambda CL (Cλ; SEQ ID NO: 59) . In some embodiments, the heavy chain can comprise a heavy chain constant domain (CH) from human IgA. In some embodiments, the heavy chain can comprise a heavy chain constant domain (CH) from human IgD. In some embodiments, the heavy chain can comprise a heavy chain constant domain (CH) from human IgE. In some embodiments, the heavy chain can comprise a heavy chain constant domain (CH) from human IgG. In some embodiments, the heavy chain can comprise a heavy chain constant domain (CH) from human IgM. In some embodiments, the heavy chain can comprise a heavy chain constant domain (CH) from human IgG1 (e.g., SEQ ID NO: 60) . In some embodiments, the heavy chain can comprise a heavy chain constant domain (CH) from human IgG2 (e.g., SEQ ID NO: 61) . In some embodiments, the heavy chain can comprise a heavy chain constant domain (CH) from human IgG3 (e.g., SEQ ID NO: 62) . In some embodiments, the heavy chain can comprise a heavy chain constant domain (CH) from human IgG4 (e.g., SEQ ID NO: 63) . The CH can further include a C-terminal lysine (K) . Expressly contemplated here are any and all combinations of the VL/VH pairs disclosed herein that specifically bind PD-1 (e.g., human PD-1) and the CL/CH disclosed herein or otherwise known in the art.

Table 5 Sequences of constant regions

Epitope mapping is a method of identifying the binding site, region, or epitope on a target protein where an antibody binds. A variety of methods are known in the art for mapping epitopes on target proteins. These methods include mutagenesis, including but not limited to, shotgun mutagenesis, site-directed mutagenesis, and alanine scanning; domain or fragment scanning; peptide scanning (e.g., Pepscan technology) ; display methods (e.g., phage display, microbial display, and ribosome/mRNA display) ; methods involving proteolysis and mass spectroscopy; and structural determination (e.g., X-ray crystallography and NMR) . In some embodiments, anti-PD-1 antibodies or antigen-binding fragments described herein are characterized by assays including, but not limited to, N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, ion exchange chromatography, and papain digestion.

The anti-PD-1 antibodies or antigen-binding fragments of the present disclosure can be analyzed for their physical, chemical and/or biological properties by various methods known in the art. In some embodiments, an anti-PD-1 antibody is tested for its ability to bind PD-1 (e.g., human PD-1) . Binding assays include, but are not limited to, BLI, SPR (e.g., Biacore) , ELISA, and FACS. In addition, antibodies can be evaluated for solubility, stability, thermostability, viscosity, expression levels, expression quality, and/or purification efficiency.

In some embodiments, anti-PD-1 antibodies or antigen-binding fragments described herein bind to human PD-1 with high affinity, for example, with a K_D of 10^-7 M or less, 5×10^-8 M or less, 10^-8 M or less, 5×10^-9 M or less, 10^-9 M or less, 5×10^-10 M or less, or 10^-10 M or less. In some embodiments, anti-PD-1 antibodies or antigen-binding fragments described herein bind to human PD-1 with a K_D of 5×10^-8 M or less. In some embodiments, anti-PD-1 antibodies or antigen-binding fragments described herein bind to human PD-1 with high affinity, for example, with a K_D of about 10^-7 M, about 5×10^-8 M, about 10^-8 M, about 5×10^-9 M, about 10^-9 M, about 5×10^-10 M, or about 10^- ¹⁰ M. In some embodiments, anti-PD-1 antibodies or antigen-binding fragments described herein bind to human PD-1 with a K_D of about 5×10^-9 M. In some embodiments, anti-PD-1 antibodies or antigen-binding fragments described herein bind to human PD-1 with a K_D ranging from 10^-10 M to 10^-7 M, from 10^-9 M to 10^-7 M, from 10^-8 M to 10^-7 M, from 10^-10 M to 5×10^-8 M, from 10^-9 M to 5×10^-8 M, from 10^-8 M to 5×10^-8 M, from 10^-10 M to 10^-8 M, from 10^-9 M to 10^-8 M, from 10^-10 M to 5×10^-9 M, from 10^-9 M to 5×10^-9 M, or from 10^-10 M to 10^-9 M. In some embodiments, anti-PD-1 antibodies or antigen-binding fragments described herein bind to human PD-1 with high affinity, for example, with a K_D from 10^-9 M to 5×10^-8 M. In some embodiments, the K_D is determined by BLI. In some embodiments, the K_D is determined by SPR. In some embodiments, anti-PD-1 antibodies or antigen-binding fragments described herein bind to human PD-1 with high affinity, for example, with a K_D of 10^-7 M or less, 5×10^-8 M or less, 10^-8 M or less, 5×10^-9 M or less, 10^-9 M or less, 5×10^-10 M or less, or 10^-10 M or less; or about 10^-7 M, about 5×10^-8 M, about 10^-8 M, about 5×10^-9 M, about 10^-9 M, about 5×10^-10 M, or about 10^-10 M; or ranging from 10^-10 M to 10^-7 M, from 10^-9 M to 10^-7 M, from 10^-8 M to 10^-7 M, from 10^-10 M to 5×10^-8 M, from 10^-9 M to 5×10^-8 M, from 10^-8 M to 5×10^-8 M, from 10^-10 M to 10^-8 M, from 10^-9 M to 10^-8 M, from 10^-10 M to 5×10^-9 M, from 10^-9 M to 5×10^-9 M, or from 10^-10 M to 10^-9 M, as measured by SPR.

In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments described herein specifically bind to human PD-1. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments described herein bind to both human PD-1 and cynomolgus PD-1.

The anti-PD-1 antibodies or antigen-binding fragments provided herein can have one or more of the functional properties described above. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments provided herein (1) bind to human PD-1 with high affinity; (2) activate PD-1 signaling; (3) inhibit T cell activity, (4) inhibit T cell proliferation, or (5) suppress T cell reconstitution, or any combination of (1) - (5) . In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments provided herein bind to human PD-1 with high affinity. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments provided herein activate PD-1 signaling. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments provided herein inhibit T cell activity. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments provided herein inhibit T cell proliferation. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments provided herein suppress T cell reconstitution.

In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments provided herein can lessen autoimmune activity in vivo. For example, in some embodiment, the anti-PD-1 antibodies or antigen-binding fragments provided herein can reduce disease scores. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments provided herein can reduce inflammatory cytokines, such as IL-2, IFNγ, etc.

In some embodiments, provided herein are also antibodies or antigen-binding fragments that compete with the antibody or antigen-binding fragment provided above for binding to PD-1 (e.g., human PD-1) . Antibodies that “compete with another antibody for binding to a target” refer to antibodies that inhibit (partially or completely) the binding of the other antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to a target, can be determined using known competition experiments, e.g., surface plasmon resonance (SPR) analysis. In some embodiments, an anti-PD-1 antibody or antigen-binding fragment competes with, and inhibits binding of another antibody or antigen-binding fragment to PD-1 by at least 50%, 60%, 70%, 80%, 90%or 100%. Competition assays can be conducted as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: l0. H0l/pdb. prot4277 or in Chapter 11 of “Using Antibodies” by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999.

The present disclosure further contemplates additional variants and equivalents that are substantially homologous to the recombinant, monoclonal, chimeric, humanized, and human antibodies, or antibody fragments thereof, described herein. In some embodiments, it is desirable to improve the binding affinity of the antibody. In some embodiments, it is desirable to modulate biological properties of the antibody, including but not limited to, specificity, thermostability, expression level, effector function (s) , glycosylation, immunogenicity, and/or solubility. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of an antibody, such as changing the number or position of glycosylation sites or altering membrane anchoring characteristics.

Antibodies comprising functional variants of the heavy chain, light chains, VL regions, VH regions, or one or more CDRs of the antibodies of the examples as also provided herein. A functional variant of a heavy chain, a light chain, VL, VH, or CDRs used in the context of an antibody still allows the antibody to retain at least a substantial proportion (at least about 90%, 95%or more) of functional features of the “reference” and/or “parent” antibody, including affinity and/or the specificity/selectivity, Fc inertness and PK parameters such as half-life, Tmax, Cmax. Such functional variants typically retain significant sequence identity to the parent antibody and/or have substantially similar length of heavy and light chains. Exemplary variants include those which differ from heavy and/or light chains, VH and/or VL, and/or CDR regions of the parent antibody sequences mainly by conservative substitutions, e.g., 10, such as 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant cam be conservative amino acid residue replacements.

Variations can be a substitution, deletion, or insertion of one or more nucleotides encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native antibody or polypeptide sequence. In some embodiments, amino acid substitutions are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements. Insertions or deletions can be in the range of about 1 to 5 amino acids. In some embodiments, the substitution, deletion, or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the parent molecule. In some embodiments, variations in the amino acid sequence that are biologically useful and/or relevant can be determined by systematically making insertions, deletions, or substitutions in the sequence and testing the resulting variant proteins for activity as compared to the parent protein.

In some embodiments, provided herein are variants of anti-PD-1 antibodies or antigen-binding fragments described herein. In some embodiments, provided herein are variants of anti-PD-1 antibody clone antibody, chimeric antibody or humanized antibody. In some embodiments, a variant comprises one to 30 amino acid substitutions, additions, and/or deletions in the parent antibody or antigen-binding fragment. In some embodiments, a variant comprises one to 25 amino acid substitutions, additions, and/or deletions in the parent antibody or antigen-binding fragment. In some embodiments, a variant comprises one to 20 substitutions, additions, and/or deletions in the parent antibody or antigen-binding fragment. In some embodiments, a variant comprises one to 15 substitutions, additions, and/or deletions in the parent antibody or antigen-binding fragment. In some embodiments, a variant comprises one to 10 substitutions, additions, and/or deletions in the parent antibody or antigen-binding fragment. In some embodiments, a variant comprises one to five amino acid substitutions, additions, and/or deletions in the parent antibody or antigen-binding fragment. In some embodiments, a variant comprises one to three amino acid substitutions, additions, and/or deletions in the parent antibody or antigen-binding fragment. In some embodiments, the amino acid substitution (s) is in a CDR of the antibody or antigen-binding fragment. In some embodiments, the amino acid substitution (s) is not in a CDR of the antibody or antigen-binding fragment. In some embodiments, the amino acid substitution (s) is in a framework region of the antibody or antigen-binding fragment. In some embodiments, the amino acid substitutions, additions, and/or deletions are conservative amino acid substitutions.

The variant antibodies or antigen-binding fragments described herein can be generated using methods known in the art, including but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983) TECHNIQUES IN MOLECULAR BIOLOGY (MacMillan Publishing Company, New York) ; Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-492 (1985) ; Kunkel et al., Methods Enzymol. 54: 367-382 (1987) ; Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor, N. Y. ) ; U.S. Pat. No. 4,873,192; and the references cited therein; herein incorporated by reference. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the polypeptide of interest can be found in the model of Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C. ) , pp. 345-352, herein incorporated by reference in its entirety. The model of Dayhoff et al. uses the Point Accepted Mutation (PAM) amino acid similarity matrix (PAM 250 matrix) to determine suitable conservative amino acid substitutions. Conservative substitutions, such as exchanging one amino acid with another having similar properties, can be beneficial. Examples of conservative amino acid substitutions as taught by the PAM 250 matrix of the Dayhoff et al. model include those identified in the table below.

It is known in the art that the constant region (s) of an antibody mediates several effector functions and these effector functions can vary depending on the isotype of the antibody. For example, binding of the C1 component of complement to the Fc region of IgG or IgM antibodies (bound to antigen) activates the complement system. Activation of complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and can be involved in autoimmune hypersensitivity. In addition, the Fc region of an antibody can bind a cell expressing a Fc receptor (FcR) . There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors) , IgE (epsilon receptors) , IgA (alpha receptors) and IgM (mu receptors) . Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell cytotoxicity or ADCC) , release of inflammatory mediators, placental transfer, and control of immunoglobulin production.

In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments described herein comprise a constant region of a human IgA antibody. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments described herein comprise a constant region of a human IgD antibody. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments described herein comprise a constant region of a human IgE antibody. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments described herein comprise a constant region of a human IgG antibody. In some embodiments, anti the anti-PD-1 antibodies or antigen-binding fragments described herein comprise a constant region of a human IgM antibody. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments described herein comprise a constant region of a human IgG1 antibody. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments described herein comprise a constant region of a human IgG2 antibody. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments described herein comprise a constant region of a human IgG3 antibody. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments described herein comprise a constant region of a human IgG4 antibody.

In some embodiments, at least one or more of the constant regions has been modified or deleted in the anti-PD-1 antibody or antigen-binding fragment described herein. In some embodiments, the antibodies comprise modifications to one or more of the three heavy chain constant regions (CH1, CH2 or CH3) and/or to the light chain constant region (CL) .

In some embodiments, the heavy chain constant region of the modified antibodies comprises at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibodies comprises more than one human constant region. In some embodiments, modifications to the constant region comprise additions, deletions, or substitutions of one or more amino acids in one or more regions. In some embodiments, one or more regions are partially or entirely deleted from the constant regions of the modified antibodies. In some embodiments, the entire CH2 domain has been removed from an antibody (ΔCH2 constructs) . In some embodiments, a deleted constant region is replaced by a short amino acid spacer that provides some of the molecular flexibility typically imparted by the absent constant region. In some embodiments, a modified antibody comprises a CH3 domain directly fused to the hinge region of the antibody. In some embodiments, a modified antibody comprises a peptide spacer inserted between the hinge region and modified CH2 and/or CH3 domains.

In some embodiments, an anti-PD-1 antibody or antigen-binding fragment comprises a Fc region. In some embodiments, the Fc region is fused via a hinge. The hinge can be an IgG1 hinge, an IgG2 hinge, or an IgG3 hinge. The amino acid sequences of the Fc region of human IgG1, IgG2, IgG3, and IgG4 are known to those of ordinary skill in the art. In some cases, Fc regions with amino acid variations have been identified in native antibodies. In some embodiments, the modified antibodies (e.g., modified Fc region) provide for altered effector functions that, in turn, affect the biological profile of the antibody. For example, in some embodiments, the deletion or inactivation (through point mutations or other means) of a constant region reduces Fc receptor binding of the modified antibody as it circulates. In some embodiments, the constant region modifications reduce the immunogenicity of the antibody. In some embodiments, the constant region modifications increase the serum half-life of the antibody. In some embodiments, the constant region modifications reduce the serum half-life of the antibody. In some embodiments, the constant region modifications affect ADCC and/or CDC of the antibody.

The in vivo half-life of an antibody can impact its effector functions. The half-life of an antibody can be increased or decreased to modify its therapeutic activities. FcRn is a receptor that is structurally similar to MHC Class I antigen that non-covalently associates with b2-microglobulin. FcRn regulates the catabolism of IgGs and their transcytosis across tissues (Ghetie and Ward, 2000, Annu. Rev. Immunol. 18: 739-766; Ghetie and Ward, 2002, Immunol. Res. 25: 97-113) . The IgG-FcRn interaction takes place at pH 6.0 (pH of intracellular vesicles) but not at pH 7.4 (pH of blood) ; this interaction enables IgGs to be recycled back to the circulation. The region on human IgG1 involved in FcRn binding has been mapped (Shields et al., 2001, J. Biol. Chem. 276: 6591-604) . Alanine substitutions at positions Pro238, Thr256, Thr307, Gln311, Asp312, Glu380, Glu382, or Asn434 of human IgG1 enhance FcRn binding. IgG1 molecules harboring these substitutions have longer serum half-lives. Consequently, these modified IgG1 molecules can be able to carry out their effector functions, and hence exert their therapeutic efficacies, over a longer period of time compared to unmodified IgG1. Other exemplary substitutions for increasing binding to FcRn include a Gln at position 250 and/or a Leu at position 428. EU numbering is used for all positions in the constant region.

Other Fc mutations are incorporated to improve serum half-life. Exemplary mutations include, for example, M252Y/S254T/T256E (YTE) substitutions, M428L/N434S (LS) substitutions, T307A/E380A/N434A (TM) substitutions, and H433K/N434F (HS) substations. These mutations are specifically designed to enhance the interaction of the antibody with the FcRn receptor, thereby reducing lysosomal degradation and prolonging the antibody's circulation time in the bloodstream.

Oligosaccharides covalently attached to the conserved Asn297 are involved in the ability of the Fc region of an IgG to bind FcγR (Lund et al., 1996, J. Immunol. 157: 4963-69; Wright and Morrison, 1997, Trends Biotechnol. 15: 26-32) . Engineering of this glycoform on IgG can significantly improve IgG-mediated ADCC. Addition of bisecting N-acetylglucosamine modifications (Umana et al., 1999, Nat. Biotechnol. 17: 176-180; Davies et al., 2001, Biotech. Bioeng. 74: 288-94) to this glycoform or removal of fucose (Shields et al., 2002, J. Biol. Chem. 277: 26733-40; Shmkawa et al., 2003, J. Biol. Chem. 278: 6591-604; Niwa et al., 2004, Cancer Res. 64: 2127-33) from this glycoform are two examples of IgG Fc engineering that improves the binding between IgG Fc and FcγR, thereby enhancing Ig-mediated ADCC activity. In some embodiments, an anti-PD-1 antibody described herein has a glycan attached to the conserved Asn297 residue of the constant region, wherein the numbering of amino acid residues in the constant region is according to the EU-index. In some embodiments, the glycan is biantennary. In some embodiments, the glycan is core fucosylated. In some embodiments, the glycan has zero terminal galactose residues. In some embodiments, the glycan is biantennary and core fucosylated. In some embodiments, the glycan is biantennary and has zero terminal galactose residues. In some embodiments, the glycan is core fucosylated and has zero terminal galactose residues. In some embodiments, the glycan is biantennary, core fucosylated and has zero galactose residues. In some embodiments, in a population of anti-PD-1 antibodies described herein the conserved Asn297 residues of the constant regions, wherein the numbering of amino acid residues in the constant region is according to the EU-index are predominantly occupied by biantennary, core fucosylated glycans with zero terminal galactose residues. Additionally, an afucosylated IgG1 which lacks core fucose on Fc N-glycans on the N297 position has been shown to have increased binding affinity to FcγRIIIa on NK cells and enhanced ADCC activity (Pereira NA et al., MAbs . 2018 Jul; 10 (5) : 693-711. ) . In some embodiments, the anti-PD-1 antibodies described herein are afucosylated on N297.

A systemic substitution of solvent-exposed amino acids of human IgG1 Fc region has generated IgG variants with altered ADCC/CDC activities and/or FcγR binding affinities. For example, when compared to parental IgG1, a subset of these variants involving substitutions at T256/S298, S298/E333, S298/K334, or S298/E333 K334 to Ala demonstrate increased in both binding affinity toward FcγR and ADCC activity (Shields et al., 2001, J. Biol. Chem. 276: 6591-604; Okazaki et al., 2004, J. Mol. Biol. 336: 1239-49) . In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments described herein comprise a variant of human IgG1 heavy chain constant region that comprises one or more amino acid substitutions selected from the group consisting of L234, L235, G236, S239, F243, H268, D270, R292, S298, Y300, V305, K326, A330, I332, E333, K334 and P396. In some embodiments, an anti-PD-1 antibody or antigen-binding fragment described herein comprises an IgG1 heavy chain constant region that comprises at least one amino acid substitution. The IgG1 heavy chain constant region can comprise a L234 substitution. The L234 substitution can be, e.g., L234Y. The IgG1 heavy chain constant region can comprise a L235 substitution. The L235 substitution can be, e.g., L235Q or L235V. The IgG1 heavy chain constant region can comprise a G236 substitution. The G236 substitution can be, e.g., G236A or G236W. The IgG1 heavy chain constant region can comprise an S239 substitution. The S239 substitution can be, e.g., S239D or S239M. The IgG1 heavy chain constant region can comprise an F243 substitution. The F243 substitution can be, e.g., F243L. The IgG1 heavy chain constant region can comprise an H268 substitution. The H268 substitution can be, e.g., H268D. The IgG1 heavy chain constant region can comprise a D270 substitution. The D270 substitution can be, e.g., D270E. The IgG1 heavy chain constant region can comprise an R292 substitution. The R292 substitution can be, e.g., R292P. The IgG1 heavy chain constant region can comprise an S298 substitution. The S298 substitution can be, e.g., S298A. The IgG1 heavy chain constant region can comprise a Y300 substitution. The Y300 substitution can be, e.g., Y300L. The IgG1 heavy chain constant region can comprise a V305 substitution. The V305 substitution can be, e.g., V305I. The IgG1 heavy chain constant region can comprise a K326 substitution. The K326 substitution can be, e.g., K326D. The IgG1 heavy chain constant region can comprise an A330 substitution. The A330 substitution can be, e.g., A330M or A330L. The IgG1 heavy chain constant region can comprise an I332 substitution. The I332 substitution can be, e.g., I332E. The IgG1 heavy chain constant region can comprise an E333 substitution. The E333 substitution can be, e.g., E333A. The IgG1 heavy chain constant region can comprise a K334 substitution. The K334 substitution can be, e.g., K334A or K334E. The IgG1 heavy chain constant region can comprise a P396 substitution. The P396 substitution can be, e.g., P396L. All are numbered according to the EU Index.

In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments described herein comprise a variant of human IgG1 heavy chain constant region that comprises one or more amino acid substitutions selected from the group consisting of L234Y, L235Q, L235V, G236A, G236W, S239D, S239M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330M, A330L, I332E, E333A, K334A, K334E, and P396L. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, A330S, P331S, D356E, and L358M. In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments described herein comprise a variant of human IgG1 heavy chain constant region modified by amino acid substitutions S298A, E333A, and K334A. In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments described herein comprise a variant of human IgG1 heavy chain constant region modified by amino acid substitutions S239D and I332E. In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments described herein comprise a variant of human IgG1 heavy chain constant region modified by amino acid substitutions S239D, A330L, and I332E (e.g., SEQ ID NO: 64) . In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments described herein comprise a variant of human IgG1 heavy chain constant region modified by amino acid substitution G236A. In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments described herein comprise a variant of human IgG1 heavy chain constant region modified by amino acid substitutions G236A, S239D, and I332E. In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments described herein comprise a variant of human IgG1 heavy chain constant region modified by amino acid substitutions G236A, A330L, and I332E. In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments described herein comprise a variant of human IgG1 heavy chain constant region modified by amino acid substitutions G236A, S239D, A330L, and I332E. In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments described herein comprise a variant of human IgG1 heavy chain constant region modified by amino acid substitutions F243L, R292P, Y300L, V305I, and P396L. In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments described herein comprise a variant of human IgG1 heavy chain constant region modified by amino acid substitutions L235V, F243L, R292P, Y300L, and P396L. In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments described herein comprise a variant of human IgG1 heavy chain constant region modified by amino acid substitutions L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A. In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments described herein comprise a variant of human IgG1 heavy chain constant region modified by amino acid substitutions D270E, K326D, A330M, and K334E. In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments described herein comprise a variant of human IgG1 heavy chain constant region modified by well-known amino acid substitutions specifically increase FcγRIIB binding affinities. In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments described herein comprise a variant of human IgG1 heavy chain constant region modified by amino acid substitutions S267E/L328F which specifically increase FcγRIIB binding affinities. In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments described herein comprise a variant of human IgG1 heavy chain constant region that lacks core fucose on Fc N-glycans on N297. All are numbered according to the EU Index.

In some embodiments, variants can include addition of amino acid residues at the amino-and/or carboxyl-terminal end of the antibody or polypeptide. The length of additional amino acids residues can range from one residue to a hundred or more residues. In some embodiments, a variant comprises an N-terminal methionyl residue. In some embodiments, the variant comprises an additional polypeptide/protein (e.g., Fc region) to create a fusion protein. In some embodiments, a variant is engineered to be detectable and can comprise a detectable label and/or protein (e.g., a fluorescent tag or an enzyme) .

In constructing variants of an anti-PD-1 binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, modifications are made such that variants continue to possess the desired properties, e.g., being capable of specifically binding to an PD-1, and in certain embodiments being able to activate PD-1 signaling. Obviously, any mutations made in the DNA encoding the variant polypeptide must not place the sequence out of reading frame. In some embodiments, mutations made in the DNA do not create complementary regions that could produce secondary mRNA structure.

In some embodiments, a variant of an anti-PD-1 antibody or antigen-binding fragment disclosed herein can retain the ability to bind PD-1 to a similar extent, the same extent, or to a higher extent, as the parent antibody or antigen-binding fragment. In some embodiments, the variant can be at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%or more identical in amino acid sequence to the parent antibody or antigen-binding fragment. In certain embodiments, a variant of an anti-PD-1 antibody or antigen-binding fragment comprises the amino acid sequence of the parent anti-PD-1 antibody or antigen-binding fragment with one or more conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties.

In some embodiments, a variant of an anti-PD-1 antibody or antigen-binding fragment comprises the amino acid sequence of the parent antibody or antigen-binding fragment with one or more non-conservative amino acid substitutions. In some embodiments, a variant of an anti-PD-1 antibody or antigen-binding fragment comprises the amino acid sequence of the parent binding antibody or antigen-binding fragment with one or more non-conservative amino acid substitution, wherein the one or more non-conservative amino acid substitutions do not interfere with or inhibit one or more biological activities of the variant (e.g., PD-1 binding) . In certain embodiments, the one or more conservative amino acid substitutions and/or the one or more non-conservative amino acid substitutions can enhance a biological activity of the variant, such that the biological activity of the functional variant is increased as compared to the parent antibody or antigen-binding fragment.

In some embodiments, the variant can have 1, 2, 3, 4, or 5 amino acid substitutions in the CDRs (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3) of the binding moiety.

In some embodiments, anti-PD-1 antibodies or antigen-binding fragments described herein are chemically modified naturally or by intervention. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments have been chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and/or linkage to a cellular ligand or other protein. Any of numerous chemical modifications can be carried out by known techniques. The anti-PD-1 antibodies or antigen-binding fragments can comprise one or more analogs of an amino acid (including, for example, unnatural amino acids) , as well as other modifications known in the art.

In some embodiments, anti-PD-1 antibodies or antigen-binding fragments disclosed herein can be linked to at least one agent to form an antibody conjugate. The conjugate can be, for example, an antibody conjugated to another protein, carbohydrate, lipid, steroids, immunosuppressors, or mixed moiety molecule (s) . Such antibody conjugates include, but are not limited to, modifications that include linking the antibody to one or more polymers. For example, an antibody or antigen-binding fragment can be linked to one or more water-soluble polymers. Linkage to a water-soluble polymer reduces the likelihood that the antibody or antigen-binding fragment precipitate in an aqueous environment, such as a physiological environment. One skilled in the art can select a suitable water-soluble polymer based on considerations including, but not limited to, whether the polymer/antibody conjugate will be used in the treatment of a patient and, if so, the pharmacological profile of the antibody (e.g., half-life, dosage, activity, antigenicity, and/or other factors) .

In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety can be, but is not limited to, at least one effector or reporter molecule. Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, an enzyme (e.g., that catalyzes a colorimetric or fluorometric or bioluminescent reaction) , a substrate, a solid matrix, such as biotin. An antibody can comprise one, two, or more of any of these labels.

Antibody conjugates are also used as diagnostic agents. In some embodiments, an anti-PD-1 antibody or antigen-binding fragment described herein is conjugated to a detectable substance or molecule that allows the agent to be used for diagnosis and/or detection. A detectable substance can include, but is not limited to, enzymes; prosthetic groups (e.g., biotin and flavine (s) ) ; fluorescent materials; bioluminescent materials, such as luciferase; radioactive materials; positron emitting metals; and magnetic metal ions positron emitting metals; and magnetic metal ions.

An anti-PD-1 antibody or antigen-binding fragment described herein can be attached to a solid support. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, an immobilized anti-PD-1 antibody or antigen-binding fragment is used in an immunoassay. In some embodiments, an immobilized anti-PD-1 antibody or antigen-binding fragment is used in purification of the target antigen (e.g., human PD-1) .

Provided herein are anti-PD-1 antibodies and antigen-binding fragments thereof that include but are not limited to monoclonal antibodies, polyclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, and antigen-binding fragments thereof. The anti-PD-1 antibodies or antigen-binding fragments described herein can be produced by any method known in the art, including chemical synthesis and recombinant expression techniques. The practice of the invention employs, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art.

In some embodiments, monoclonal antibodies are made using recombinant DNA techniques as known to one skilled in the art. Provided herein are also methods of producing an antibody or antigen-binding fragment thereof that specifically binds human PD-1, comprising culturing the cell disclosed herein under conditions suitable for expression of the antibody or antigen-binding fragment. In some embodiments, the methods further comprise isolating the antibody or antigen-binding fragment from the culture. Polynucleotides of the antibodies or antigen-binding fragments provided herein can be prepared, manipulated, and/or expressed using any of the well-established techniques known and available in the art. In some embodiments, polynucleotides of the antibodies or antigen-binding fragments provided herein can be prepared recombinantly. Many vectors can be used. Exemplary vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of categories of animal viruses useful as vectors include, without limitation, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40) . Examples of expression vectors are pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DEST^TM, pLenti6/V5-DEST^TM, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.

In some embodiments, a recombinant expression vector is used to express a polynucleotide encoding a polypeptide described herein. For example, a recombinant expression vector can be a replicable DNA construct that includes synthetic or cDNA-derived DNA fragments encoding a polypeptide operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes. In some embodiments, coding sequences of polypeptides disclosed herein can be ligated into such expression vectors for their expression in mammalian cells. In some embodiments, a viral vector is used. DNA regions are “operatively linked” when they are functionally related to each other. For example, a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. In some embodiments, structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. In some embodiments, in situations where recombinant protein is expressed without a leader or transport sequence, a polypeptide can include an N-terminal methionine residue.

A wide variety of expression host/vector combinations can be employed. Suitable host cells for expression include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts, as well as methods of protein production, including antibody production are well-known in the art. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCR1, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and other filamentous single-stranded DNA phages.

Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney-derived) , L-929 (murine fibroblast-derived) , C127 (murine mammary tumor-derived) , 3T3 (murine fibroblast-derived) , CHO (Chinese hamster ovary-derived) , HeLa (human cervical cancer-derived) , BHK (hamster kidney fibroblast-derived) , HEK-293 (human embryonic kidney-derived) cell lines and variants thereof. Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5’ or 3’ flanking non-transcribed sequences, and 5’ or 3’ non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences. Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also offers a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.

Peptides can also be synthesized, in whole or in part, using chemical methods (see, e.g., Caruthers (1980) . Nucleic Acids Res. Symp. Ser. 215; Horn (1980) ; and Banga, A.K., THERAPEUTIC PEPTIDES AND PROTEINS, FORMULATION, PROCESSING AND DELIVERY SYSTEMS (1995) Technomic Publishing Co., Lancaster, PA) . Peptide synthesis can be performed using various solid phase techniques (see, e.g., Roberge, Science 269: 202 (1995) ; Merrifield, Methods. Enzymol. 289: 3 (1997) ) and automated synthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer (Perkin Elmer) in accordance with the manufacturer’s instructions. Peptides can also be synthesized using combinatorial methodologies. Synthetic residues and polypeptides can be synthesized using a variety of procedures and methodologies known in the art (see, e.g., ORGANIC SYNTHESES COLLECTIVE VOLUMES, Gilman, et al. (Eds) John Wiley &Sons, Inc., NY) . Modified peptides can be produced by chemical modification methods (see, for example, Belousov, Nucleic Acids Res. 25: 3440 (1997) ; Frenkel, Free Radic. Biol. Med. 19: 373 (1995) ; and Blommers, Biochemistry 33: 7886 (1994) ) . Peptide sequence variations, derivatives, substitutions and modifications can also be made using methods such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR based mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13: 4331 (1986) ; Zoller et al., Nucl. Acids Res. 10: 6487 (1987) ) , cassette mutagenesis (Wells et al., Gene 34: 315 (1985) ) , restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA 317: 415 (1986) ) and other techniques can be performed on cloned DNA to produce invention peptide sequences, variants, fusions and chimeras, and variations, derivatives, substitutions and modifications thereof.

For in vivo use of antibodies in humans, it may be preferable to use human or humanized antibodies. Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences, including improvements to these techniques. See, also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety. A human antibody can also be an antibody wherein the heavy and light chains are encoded by a nucleotide sequence derived from one or more sources of human DNA. In some embodiments, an anti-PD-1 antibody or antigen-binding fragment is a human antibody or antigen-binding fragment. Human antibodies can be prepared using various techniques known in the art.

Alternatively, in some embodiments, a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human. In some embodiments, the antigen binding domain portion is humanized. Various methods for generating humanized antibodies are known in the art, including but not limited to, CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein in its entirety by reference) , veneering or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28 (4/5) : 489-498; Studnicka et al., 1994, Protein Engineering, 7 (6) : 805-814; and Roguska et al., 1994, PNAS, 91: 969-973, each of which is incorporated herein by its entirety by reference) , chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein in its entirety by reference) , and techniques disclosed in, e.g., U.S. Patent Application Publication No. US2005/0042664, U.S. Patent Application Publication No. US2005/0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 93/17105, Tan et al., J. Immunol., 169: 1119-25 (2002) , Caldas et al., Protein Eng., 13 (5) : 353-60 (2000) , Morea et al., Methods, 20 (3) : 267-79 (2000) , Baca et al., J. Biol. Chem., 272 (16) : 10678-84 (1997) , Roguska et al., Protein Eng., 9 (10) : 895-904 (1996) , Couto et al., Cancer Res., 55 (23 Supp) : 5973s-5977s (1995) , Couto et al., Cancer Res., 55 (8) : 1717-22 (1995) , Sandhu J S, Gene, 150 (2) : 409-10 (1994) , and Pedersen et al., J. Mol. Biol., 235 (3) : 959-73 (1994) , each of which is incorporated herein in its entirety by reference. Often, framework residues in the framework regions can be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332: 323, which are incorporated herein by reference in their entireties. )

A humanized antibody has one or more amino acid residues introduced into it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Thus, humanized antibodies comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions from human. Humanization of antibodies is well-known in the art and can essentially be performed following the method of Winter and co-workers (Jones et al., Nature, 321: 522-525 (1986) ; Riechmann et al., Nature, 332: 323-327 (1988) ; Verhoeyen et al., Science, 239: 1534-1536 (1988) ) , by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR-grafting (EP 239, 400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference herein in their entirety) . In such humanized chimeric antibodies, substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanization of antibodies can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28 (4/5) : 489-498; Studnicka et al., Protein Engineering, 7 (6) : 805-814 (1994) ; and Roguska et al., PNAS, 91: 969-973 (1994) ) or chain shuffling (U.S. Pat. No. 5,565,332) , the contents of which are incorporated herein by reference herein in their entirety.

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity. According to the so-called “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993) ; Chothia et al., J. Mol. Biol., 196: 901 (1987) , the contents of which are incorporated herein by reference herein in their entirety) . Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992) ; Presta et al., J. Immunol., 151: 2623 (1993) , the contents of which are incorporated herein by reference herein in their entirety) .

Antibodies can be humanized with retention of high affinity for the target antigen and other favorable biological properties. For example, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind the target antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

A humanized antibody retains a similar antigenic specificity as the original antibody, for example, the ability to bind human PD-1 antigen. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody for a particular antigen can be increased using methods of “directed evolution, ” as described by Wu et al., J. Mol. Biol., 294: 151 (1999) , the contents of which are incorporated herein by reference herein in their entirety.

A variety of methods are known in the art to purify anti-PD-1 antibodies, such as affinity chromatography. Eluted IgG can be checked by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer solution can be exchanged, and the concentration can be determined. The monoclonal antibodies can be aliquoted and stored.

Methods for analyzing binding affinity, cross-reactivity, and binding kinetics of various anti-PD-1 antibodies include standard assays known in the art, for example, Western Blot, ELISA, and flow cytometry. Further methods available in the art include biolayer interferometry (BLI) using, for example, Gator system (Probe Life) or the Octet-96 system (Sartorius AG) , or BIACORE^TMsurface plasmon resonance (SPR) analysis using a BIACORE^TM 2000 SPR instrument (Biacore AB, Uppsala, Sweden) .
7.3 Fusion proteins

CTLA-4 (Cytotoxic T-Lymphocyte-Associated Protein 4) is a critical regulatory protein in the immune system, classified as a member of the immunoglobulin superfamily. It is a type I transmembrane protein, having an extracellular domain, a transmembrane domain, and a cytoplasmic domain. The extracellular portion of CTLA-4 comprises a single V-set immunoglobulin (Ig) domain, which is responsible for binding to its ligands, CD80 (B7-1) and CD86 (B7-2) , on antigen-presenting cells. This domain exhibits a typical Ig-like fold, with a compact structure featuring a β-sheet scaffold flanked by loops and short α-helices. This hydrophobic transmembrane domain anchors CTLA-4 in the cell membrane, playing a crucial role in the stability and localization of the protein within T-cells. The cytoplasmic tail of CTLA-4, although short and lacking intrinsic enzymatic activity, contains motifs essential for signal transduction. These motifs, including a tyrosine-based motif, facilitate interactions with intracellular phosphatases such as SHP-2 and PP2A, which are integral to the inhibitory signaling pathway.

CTLA-4 typically functions as a dimer, which enhances its binding avidity to CD80 and CD86, outcompeting the costimulatory receptor CD28 and delivering an inhibitory signal to the T-cell.

CTLA-4 is a key negative regulator of T-cell activation and immune response. It is primarily expressed on activated T-cells and regulatory T-cells (Tregs) . Upon binding to CD80 or CD86 on antigen-presenting cells, CTLA-4 transmits an inhibitory signal that decreases T-cell proliferation, cytokine production, and cellular immune responses. This mechanism is vital for maintaining self-tolerance and preventing autoimmunity.

The inhibitory action of CTLA-4 is crucial for the fine-tuning of immune responses, ensuring that activation of T-cells is appropriately controlled to avoid excessive inflammation or autoimmunity.

Human CTLA-4 exists in five (5) isoforms (UniProt: P16410) . The canonical isoform has a total of 223 amino acids (SEQ ID NO: 3) , of which amino acids 36-161 constitute the extracellular domain, amino acids 162-182 constitute the transmembrane domain, and amino acids 183-223 constitute the cytoplasmic domain. More information about human CTLA-4 can be found on public databases with the following IDs: HGNC: 2505; neXtProt: NX P16410; MIM: 109100; UniProt: P16410.

The term “CTLA-4 peptide” as used herein includes wildtype full length CTLA-4, as well its functional variants (e.g., splice variants or allelic variants) and functional fragments (e.g., a functional domain or a form resulted from processing in the cell) . A CTLA-4 peptide can be of natural origin or synthetically made. A CTLA-4 peptide can be of any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (cynos) ) and rodents (e.g., mice and rats) , unless otherwise indicated. A CTLA-4 peptide can also include a signal peptide. A CTLA-4 peptide is typically secretory, meaning that it is produced and released into the extracellular environment by cells. In some embodiments, the CTLA-4 peptides provided herein comprise human CTLA-4 (SEQ ID NO: 3) or its functional variant or fragment. In some embodiments, the CTLA-4 peptides provided herein comprise the extracellular domain of human CTLA-4. In some embodiments, the CTLA-4 peptides provided herein comprise the wildtype extracellular domain of human CTLA-4 (SEQ ID NO: 4) . In some embodiments, the CTLA-4 peptides provided herein comprise a functional variant or fragment of the wildtype extracellular domain of human CTLA-4.

In some embodiments, provided herein are fusion proteins comprising the anti-PD-1 antibodies or antigen binding fragments disclosed herein. In some embodiments, provided herein are fusion proteins comprising the anti-PD-1 antibodies or antigen binding fragments disclosed herein and a CTLA-4 peptide. In some embodiments, the fusion protein can be useful in treating autoimmune and inflammatory diseases.

The fusion proteins can comprise any anti-PD-1 antibodies or antigen-binding fragments disclosed herein. For exemplary purpose, in some embodiments, the fusion proteins disclosed herein comprise 00F8XM013, 00F8XM001, 00F8XM003, 00F8XM005, 00F8XM002, 00F8XM004, 00F8XM006, 00F8XM007, 00F8XM010, 00F8XM008, 00F8XM011, 00F8XM009, 00F8XM012, 00FFQY001, 00FFQY002, 00FFQY003, 00FFQY004, CT-122, CT-123, CT-125, CT-124, CT-126, CT-147, CT-143, CT-145, CT-148, CT-144, CT-146, CT-149, CT-151, CT-150, or CT-152.

The fusion proteins disclosed herein comprise a CTLA-4 peptide. In some embodiments, the CTLA-4 peptide is a human CTLA-4 peptide. In some embodiments, the CTLA-4 peptide is full-length wildtype human CTLA-4. In some embodiments, the CTLA-4 peptide has the amino acid sequence of SEQ ID NO: 3. In some embodiments, the CTLA-4 peptide is a functional variant of full-length wildtype human CTLA-4. In some embodiments, the CTLA-4 peptide has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 3. In some embodiments, the CTLA-4 peptide comprises a functional fragment of full-length wildtype human CTLA-4. In some embodiments, the CTLA-4 peptide comprises the extracellular domain of a human CTLA-4. In some embodiments, the CTLA-4 peptide has the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CTLA-4 peptide has at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to SEQ ID NO: 4.

In some embodiments of the fusion proteins disclosed herein, the CTLA-4 peptide and the anti-PD-1 antibody or antigen-binding fragment are connected by a linker, such as a peptide linker. The linker can be e.g., a glycine linker, a glycine-rich linker, or a glycine-serine linker. The linker can be, for example, 1 to 20 amino acids in length. A person of ordinary skill in the art would understand that the fusion proteins disclosed herein are not limited by the specific linkers exemplified herein. Any peptide linker with the appropriate length and flexibility that allows both the anti-PD-1 antibody and the CTLA-4 peptide to properly function can be used.

In some embodiments, the linker has an amino acid sequence selected from the group consisting of SEQ ID NOs: 65-68. The linker can be a flexible linker or a rigid linker. In some embodiments, the linker has the amino acid sequence of (GGGGS) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 65) . In some embodiments, the linker has the amino acid sequence of (EAAAK) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 66) . In some embodiments, the linker has the amino acid sequence of (PA) nPAP, n=0, 1, 2, 3, or 4 (SEQ ID NO: 67) . In some embodiments, the linker has the amino acid sequence of GGGGSGGGS (SEQ ID NO: 68) .

In some embodiments of the fusion proteins disclosed herein, the CTLA-4 peptide is fused to N-terminus of the anti-PD-1 antibody or antigen-binding fragment. In some embodiments of the fusion proteins disclosed herein, the CTLA-4 peptide is fused to C-terminus of the antibody or antigen-binding fragment.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment has two or more peptide chains, and the CTLA-4 peptide is linked to one of peptide chains. In some embodiments, the CTLA-4 peptide is linked to two or more of peptide chains. In some embodiments, fusion proteins provided herein comprise an anti-PD-1 IgG disclosed herein and an CTLA-4 peptide. In some embodiments, the CTLA-4 peptide is linked to the heavy chain of the anti-PD-1 IgG disclosed herein. In some embodiments, the CTLA-4 peptide is linked to N-terminus of the heavy chain of the anti-PD-1 IgG disclosed herein. In some embodiments, the CTLA-4 peptide is linked to the C-terminus of the heavy chain of the anti-PD-1 IgG disclosed herein. In some embodiments, the CTLA-4 peptide is linked to the light chain of the anti-PD-1 IgG disclosed herein. In some embodiments, the CTLA-4 peptide is linked to the N-terminus of the light chain of the anti-PD-1 IgG disclosed herein. In some embodiments, the CTLA-4 peptide is linked to the C-terminus of light chain of the anti-PD-1 IgG disclosed herein.

In some embodiments, fusion proteins provided herein comprise a heavy chain and a light chain, wherein the heavy chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-78, and wherein the light chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 70. In some embodiments, the heavy chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 71. In some embodiments, the heavy chain has the amino acid sequence of SEQ ID NO: 71. In some embodiments, the heavy chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the heavy chain has the amino acid sequence of SEQ ID NO: 72. In some embodiments, the heavy chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the heavy chain has the amino acid sequence of SEQ ID NO: 73. In some embodiments, the heavy chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the heavy chain has the amino acid sequence of SEQ ID NO: 74. In some embodiments, the heavy chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the heavy chain has the amino acid sequence of SEQ ID NO: 75. In some embodiments, the heavy chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 76. In some embodiments, the heavy chain has the amino acid sequence of SEQ ID NO: 76. In some embodiments, the heavy chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the heavy chain has the amino acid sequence of SEQ ID NO: 77. In some embodiments, the heavy chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the heavy chain has the amino acid sequence of SEQ ID NO: 78. In some embodiments, the light chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 70. In some embodiments, the light chain has the amino acid sequence of SEQ ID NO: 70.

In some embodiments, fusion proteins provided herein comprise a heavy chain and a light chain, wherein the heavy chain and light chain have the amino acid sequences of SEQ ID NOs: 71 and 70, respectively. In some embodiments, the heavy chain and light chain have the amino acid sequences of SEQ ID NOs: 72 and 70, respectively. In some embodiments, the heavy chain and light chain have the amino acid sequences of SEQ ID NOs: 73 and 70, respectively. In some embodiments, the heavy chain and light chain have the amino acid sequences of SEQ ID NOs: 74 and 70, respectively. In some embodiments, the heavy chain and light chain have the amino acid sequences of SEQ ID NOs: 75 and 70, respectively. In some embodiments, the heavy chain and light chain have the amino acid sequences of SEQ ID NOs: 76 and 70, respectively. In some embodiments, the heavy chain and light chain have the amino acid sequences of SEQ ID NOs: 77 and 70, respectively. In some embodiments, the heavy chain and light chain have the amino acid sequences of SEQ ID NOs: 78 and 70, respectively.

Table 6: Sequences of peptide chains of exemplary fusion proteins

7.4 Polynucleotides, Vectors, and Cells

Also provided herein are polynucleotides that encode a polypeptide of an anti-PD-1 antibody or antigen-binding fragment, or a fusion protein described herein. In some embodiments, the anti-PD-1 antibody or fusion protein provided herein comprise one or more polypeptide chain (s) . In some embodiments, provided herein is a polynucleotide that encodes the polypeptide chain (s) of the antibody. In some embodiments, provided herein are a plurality of polynucleotides that encode the polypeptide chain (s) of the antibody. In some embodiments, provided herein is a polynucleotide that encodes the polypeptide chain (s) of the fusion protein. In some embodiments, provided herein are a plurality of polynucleotides that encode the polypeptide chain (s) of the fusion protein.

The term “polynucleotide that encodes a polypeptide” encompasses a polynucleotide which includes only coding sequences for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences. The polynucleotides of the disclosure can be in the form of RNA or in the form of DNA. DNA can be cDNA, genomic DNA, or synthetic DNA, and can be double-stranded or single-stranded. Single stranded DNA can be the coding strand or non-coding (anti-sense) strand. The polynucleotides of the disclosure can be mRNA.

Expressly contemplated herein are polynucleotides encode any anti-PD-1 antibody or antigen-binding fragment disclosed herein. For illustrative purposes, in some embodiments, the polynucleotides provided herein encode an anti-PD-1 antibody or antigen-binding fragment comprising: as defined by Kabat, (a) a light chain variable region (VL) comprising VL CDR1, VL CDR2, VL CDR3 having the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or (b) a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, VH CDR3 having the amino acid sequences of SEQ ID NOs: 11, 12 and 13, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, the polynucleotides provided herein encode an anti-PD-1 antibody or antigen-binding fragment comprising: as defined by Kabat, (a) a light chain variable region (VL) comprising VL CDR1, VL CDR2, VL CDR3 having the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or (b) a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, VH CDR3 having the amino acid sequences of SEQ ID NOs: 11, 14 and 13, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, the polynucleotides provided herein encode an anti-PD-1 antibody or antigen-binding fragment comprising (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 18; and/or (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 27. The polynucleotides provided herein can be in the form of DNA. The polynucleotides can be in the form of mRNA.

In some embodiments, the polynucleotides provided herein encode an anti-PD-1 antibody or antigen-binding fragment disclosed herein comprising a VL and a VH, wherein the VL comprises VL CDR1, CDR2 and CDR3 and the VH comprises VH CDR1, CDR2 and CDR3, and wherein the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 have the amino acid sequences of (1) SEQ ID NOs: 8, 9, 10, 11, 12 and 13, respectively; or (2) SEQ ID NOs: 8, 9, 10, 11, 14, and 13, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the CDRs. The polynucleotides can be in the form of DNA. The polynucleotides can be in the form of mRNA.

In some embodiments, the polynucleotides provided herein encode an anti-PD-1 antibody or antigen-binding fragment disclosed herein comprising a VL and a VH, wherein the VL and VH have the amino acid sequences of (1) SEQ ID NOs: 18 and 27, respectively; (2) SEQ ID NOs: 19 and 28, respectively; (3) SEQ ID NOs: 19 and 29, respectively; (4) SEQ ID NOs: 20 and 28, respectively; (5) SEQ ID NOs: 20 and 29, (6) SEQ ID NOs: 21 and 28, respectively; (7) SEQ ID NOs: 21 and 29, respectively; (8) SEQ ID NOs: 22 and 30, respectively; (9) SEQ ID NOs: 22 and 31, respectively; (10) SEQ ID NOs: 22 and 32, respectively; (11) SEQ ID NOs: 24 and 30, respectively; (12) SEQ ID NOs: 24 and 31, respectively; (13) SEQ ID NOs: 24 and 32, respectively; (14) SEQ ID NOs: 22 and 33, respectively; (15) SEQ ID NOs: 24 and 33, respectively; (16) SEQ ID NOs: 22 and 34, respectively; (17) SEQ ID NOs: 24 and 35, respectively; (18) SEQ ID NOs: 40 and 50, respectively; (19) SEQ ID NOs: 38 and 48, respectively; (20) SEQ ID NOs: 38 and 49, respectively; (21) SEQ ID NOs: 39 and 48, respectively; (22) SEQ ID NOs: 39 and 49, respectively; (23) SEQ ID NOs: 41 and 51, respectively; (24) SEQ ID NOs: 41 and 52, respectively; (25) SEQ ID NOs: 42 and 51, respectively; (26) SEQ ID NOs: 42 and 52, respectively; (27) SEQ ID NOs: 39 and 51, respectively; (28) SEQ ID NOs: 39 and 52, respectively; (29) SEQ ID NOs: 43 and 53, respectively; (30) SEQ ID NOs: 43 and 54, respectively; (31) SEQ ID NOs: 44 and 53, respectively; (32) SEQ ID NOs: 44 and 54, respectively. The polynucleotides can be in the form of DNA. The polynucleotides can be in the form of mRNA.

In some embodiments, the VL and VH are connected by a linker. The linker can be a flexible linker or a rigid linker. In some embodiments, the linker has the amino acid sequence of (GGGGS) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 65) . In some embodiments, the linker has the amino acid sequence of (EAAAK) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 66) . In some embodiments, the linker has the amino acid sequence of (PA) nPAP, n=0, 1, 2, 3, or 4 (SEQ ID NO: 67) . In some embodiments, the linker has the amino acid sequence of GGGGSGGGS (SEQ ID NO: 68) .

Additionally, in some embodiments, the polynucleotide (s) or plurality of polynucleotides provided herein encode or collectively encode fusion proteins comprising a heavy chain and a light chain, wherein the heavy chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-78, and wherein the light chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 70.

The present disclosure also provides variants of the polynucleotides described herein, wherein the variants encode, for example, fragments, analogs, and/or derivatives of an anti-PD-1 antibody or fusion protein disclosed herein. In some embodiments, the present disclosure provides a polynucleotide having a nucleotide sequence at least about 80%identical, at least about 85%identical, at least about 90%identical, at least about 95%identical, at least about 96%identical, at least about 97%identical, at least about 98%identical, or at least about 99%identical to a polynucleotide sequence encoding an anti-PD-1 antibody or fusion protein described herein.

As used herein, the phrase “a polynucleotide having a nucleotide sequence at least about 95%identical to a polynucleotide sequence” means that the nucleotide sequence of the polynucleotide is identical to a reference sequence except that the polynucleotide sequence can include up to five-point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence, up to 5%of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5’ or 3’ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

The polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments, a polynucleotide variant contains alterations which produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide. In some embodiments, a polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code) . Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (e.g., change codons in the human mRNA to those preferred by a bacterial host such as E. coli) . In some embodiments, a polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence.

In some embodiments, a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.

In some embodiments, a polynucleotide comprises the coding sequence for a polypeptide (e.g., an antibody or fusion proteins) fused in the same reading frame to a polynucleotide which aids in expression and secretion of a polypeptide from a host cell (e.g., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide) . The polypeptide can have the leader sequence cleaved by the host cell to form a “mature” form of the polypeptide. In some embodiments, a leader sequence could have an amino acid sequence of SEQ ID NO: 79.

In some embodiments, a polynucleotide comprises the coding sequence for a polypeptide (e.g., an antibody or fusion protein) fused in the same reading frame to a marker or tag sequence. For example, in some embodiments, a marker sequence is a hexa-histidine tag (HIS-tag) that allows for efficient purification of the polypeptide fused to the marker. In some embodiments, a marker sequence is a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used. In some embodiments, the marker sequence is a FLAG^TM tag. In some embodiments, a marker can be used in conjunction with other markers or tags.

In some embodiments, a polynucleotide is isolated. In some embodiments, a polynucleotide is substantially pure.

Vectors and cells comprising the polynucleotides described herein are also provided. In some embodiments, provided herein are vectors comprising a polynucleotide provided herein. The vectors can be expression vectors. In some embodiments, vectors provided herein comprise a polynucleotide encoding one peptide chain of the anti-PD-1 antibody or fusion protein described herein. In some embodiments, vectors provided herein comprise a polynucleotide encoding all polypeptide that is part of an anti-PD-1 antibody or fusion protein described herein.

In some embodiments, provided herein are recombinant expression vectors, which can be used to amplify and express a polynucleotide encoding an anti-PD-1 antibody or fusion protein described herein. For example, a recombinant expression vector can be a replicable DNA construct that includes synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of an anti-PD-1 antibody or fusion protein, operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes. In some embodiments, a viral vector is used. DNA regions are “operatively linked” when they are functionally related to each other. For example, a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. In some embodiments, structural elements intended for use in certain expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. In some embodiments, in situations where recombinant protein is expressed without a leader or transport sequence, a polypeptide can include an N-terminal methionine residue.

A wide variety of expression host/vector combinations can be employed. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCR1, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and other filamentous single-stranded DNA phages. In some embodiments, an anti-PD-1 antibody or fusion protein described herein is expressed from one or more vectors.

Provided herein are suitable host cells comprising vectors described herein. In some embodiments, the host cells can be used for recombination expression of the anti-PD-1 antibodies or fusion proteins described herein. The host cells can include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts, as well as methods of protein production, including antibody production are well-known in the art.

Examples of suitable mammalian host cells include, but are not limited to, COS-7 (monkey kidney-derived) , L-929 (murine fibroblast-derived) , C127 (murine mammary tumor-derived) , 3T3 (murine fibroblast-derived) , CHO (Chinese hamster ovary-derived) , HeLa (human cervical cancer-derived) , BHK (hamster kidney fibroblast-derived) , HEK-293 (human embryonic kidney-derived) cell lines and variants thereof. Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5’ or 3’ flanking non-transcribed sequences, and 5’ or 3’ non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences. Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also offers a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.

The present disclosure also provides host cells comprising the polypeptides described herein, polynucleotides encoding polypeptides described herein, or vectors comprising such polynucleotides. In some embodiments, provided herein are host cells comprising a vector comprising a polynucleotide disclosed herein. In some embodiments, host cells provided herein comprise a vector comprising a polynucleotide encoding an anti-PD-1 antibody or fusion protein described herein. In some embodiments, host cells provided herein comprise a vector comprising a polynucleotide encoding a polypeptide that is part of an anti-PD-1 antibody or fusion protein described herein. In some embodiments, host cells provided herein comprise a polynucleotide encoding the polypeptide (s) of an anti-PD-1 antibody or fusion protein described herein. In some embodiments, host cells provided herein comprise a plurality of polynucleotides encoding the polypeptides of an anti-PD-1 antibody or fusion protein described herein. In some embodiments, the cells produce the polypeptide (s) of anti-PD-1 antibody or fusion protein described herein.
7.5 Pharmaceutical Compositions

Provided herein are pharmaceutical compositions comprising the anti-PD-1 antibodies or antigen-binding fragments disclosed herein. Provided herein are also pharmaceutical compositions comprising the fusion proteins disclosed herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the anti-PD-1 antibodies or antigen-binding fragments disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the fusion proteins disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions are useful in suppressing autoimmunity or inflammation. In some embodiments, the pharmaceutical compositions are useful in treating an autoimmune disease or an inflammatory disease.

Provided herein are also kits for preparation of pharmaceutical compositions having the anti-PD-1 antibodies or antigen-binding fragments disclosed herein. In some embodiments, the kit comprises the anti-PD-1 antibodies or antigen-binding fragments disclosed herein and a pharmaceutically acceptable carrier in one or more containers. In another embodiment, the kits can comprise anti-PD-1 antibodies or antigen-binding fragments disclosed herein for administration to a subject. In specific embodiments, the kits comprise instructions regarding the preparation and/or administration of the anti-PD-1 antibodies or antigen-binding fragments.

Provided herein are also kits for preparation of pharmaceutical compositions having the fusion proteins disclosed herein. In some embodiments, the kit comprises the fusion proteins disclosed herein and a pharmaceutically acceptable carrier in one or more containers. In another embodiment, the kits can comprise fusion proteins disclosed herein for administration to a subject. In specific embodiments, the kits comprise instructions regarding the preparation and/or administration of the fusion proteins.

In some embodiments, provided herein is a pharmaceutical composition suitable for local administration. In some embodiments, provided herein is a pharmaceutical composition suitable for systemic administration.

Pharmaceutically acceptable carriers that can be used in compositions provided herein include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion) . Depending on the route of administration, the active ingredient (i.e., anti-PD-1 antibodies or fusion proteins) can be coated in a material to protect the active ingredient from the action of acids and other natural conditions that can inactivate the active ingredient.

Provided herein are also pharmaceutical compositions or formulations that improve the stability of the anti-PD-1 antibodies or fusion proteins to allow for their long-term storage. In some embodiments, the pharmaceutical composition or formulation disclosed herein comprises: (a) anti-PD-1 antibodies or fusion proteins disclosed herein; (b) a buffering agent; (c) a stabilizing agent; (d) a salt; (e) a bulking agent; and/or (f) a surfactant. In some embodiments, the pharmaceutical composition or formulation is stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years or more. In some embodiments, the pharmaceutical composition or formulation is stable when stored at 4℃, 25℃, or 40℃.

Buffering agents useful in the pharmaceutical compositions or formulations disclosed herein can be a weak acid or base used to maintain the acidity (pH) of a solution near a chosen value after the addition of another acid or base. Suitable buffering agents can maximize the stability of the pharmaceutical formulations by maintaining pH control of the formulation. Suitable buffering agents can also ensure physiological compatibility or optimize solubility. Rheology, viscosity and other properties can also depend on the pH of the formulation. Common buffering agents include, but are not limited to, histidine, citrate, succinate, acetate and phosphate. In some embodiments, a buffering agent comprises histidine (e.g., L-histidine) with isotonicity agents and potentially pH adjustment with an acid or a base known in the art. In certain embodiments, the buffering agent is L-histidine. In certain embodiments, the pH of the formulation is maintained between about 2 and about 10, or between about 4 and about 8.

Stabilizing agents are added to a pharmaceutical product to stabilize that product. Such agents can stabilize proteins in different ways. Common stabilizing agents include, but are not limited to, amino acids such as glycine, alanine, lysine, arginine, or threonine, carbohydrates such as glucose, sucrose, trehalose, rafftnose, or maltose, polyols such as glycerol, mannitol, sorbitol, cyclodextrins or dextran of any kind and molecular weight, or PEG. In some embodiments, the stabilizing agent is chosen to maximize the stability of FIX polypeptide in lyophilized preparations. In certain embodiments, the stabilizing agent is sucrose and/or arginine.

Bulking agents can be added to a pharmaceutical composition or formulation to add volume and mass to the product, thereby facilitating precise metering and handling thereof. Common bulking agents include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

Surfactants are amphipathic substances with lyophilic and lyophobic groups. A surfactant can be anionic, cationic, zwitterionic, or nonionic. Examples of nonionic surfactants include, but are not limited to, alkyl ethoxylate, nonylphenol ethoxylate, amine ethoxylate, polyethylene oxide, polypropylene oxide, fatty alcohols such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbates, or dodecyl dimethylamine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

The pharmaceutical compositions disclosed herein can further comprise one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer and/or a surfactant, as well as various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers and surfactants in pharmaceutical compositions is well-known to the skilled person. Reference may be made to Remington: The Science and Practice of Pharmacy, 19^th edition, 1995.

In some embodiments, the pharmaceutical composition is an aqueous formulation. Such a formulation is typically a solution or a suspension, but can also include colloids, dispersions, emulsions, and multi-phase materials. The term “aqueous formulation” is defined as a formulation comprising at least 50%w/w water. Likewise, the term “aqueous solution” is defined as a solution comprising at least 50 %w/w water, and the term “aqueous suspension” is defined as a suspension comprising at least 50 %w/w water.

In some embodiments, the pharmaceutical compositions disclosed herein are freeze-dried, to which the physician or the patient adds solvents and/or diluents prior to use.

Pharmaceutical compositions disclosed herein can also include 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.

Examples of suitable aqueous and nonaqueous carriers that can be employed in the pharmaceutical compositions or formulations described herein 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 dispersions, and by the use of surfactants.

These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms can be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, 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.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such 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, use thereof in the pharmaceutical compositions described herein is contemplated. A pharmaceutical composition or formulation can comprise a preservative or can be devoid of a preservative. Supplementary active compounds can be incorporated into the compositions.

Pharmaceutical compositions or formulations typically must 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. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, the compositions can include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, some methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carrier material in the pharmaceutical compositions or formulations disclosed herein can vary. In some embodiments, the amount of active ingredient which can be combined with a carrier material is the amount that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.

The pharmaceutical compositions disclosed herein can be prepared with carriers that protect the active ingredient against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and poly lactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See. e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
7.6 Methods and Uses

The anti-PD-1 antibodies or antigen-binding fragments, fusion proteins, compositions and methods described herein have numerous in vitro and in vivo utilities. The present disclosure also provides methods of uses of the anti-PD-1 antibodies or fusion proteins, polynucleotides encoding such anti-PD-1 antibodies or fusion proteins, vectors comprising such polynucleotides, or pharmaceutical compositions having such antibodies or fusion proteins disclosed herein in activating PD-1 signaling, or inhibiting the activation or proliferation of immune effector cells (e.g., T cells) , or suppressing immune effector cells (e.g., T cells) reconstitution, reducing autoimmunity or inflammation, or treating a disease or disorder associated with PD-1 signaling, such as autoimmune and inflammatory disease.

In some embodiments, provided herein are methods of activating PD-1 signaling in an immune effector cell, comprising contacting the immune effector cell with the anti-PD-1 antibodies or fusion proteins disclosed herein. In some embodiments, provided herein are methods of inhibiting activity of an immune effector cell, comprising contacting the immune effector cell with the anti-PD-1 antibodies or fusion proteins disclosed herein. Immune effector cells include T cells, B cell, natural killer (NK) cells, NKT cells, macrophages, granulocytes, neutrophils, eosinophils, mast cells, and basophils. In some embodiments, the immune effector cells are PD-1 expressing immune effector cells. In some embodiments, the immune effector cells are T cells, NK cells, NKT cells, B cells, macrophages, DCs, monocytes, or any combination thereof. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is a NK cell. In some embodiments, the immune effector cell is a macrophage. The immune effector cells can also be genetically engineered. For example, the immune effector cell can be T cells expressing a chimeric antigen receptor (CAR) , or CAR T cells.

In some embodiments, provided herein are methods of activating PD-1 signaling in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the anti-PD-1 antibodies or fusion proteins disclosed herein. In some embodiments, provided herein are uses of the anti-PD-1 antibodies or fusion proteins disclosed herein for activating PD-1 signaling. In some embodiments, provided herein are uses of the anti-PD-1 antibodies or fusion proteins provided herein for the preparation of a medicament for activating PD-1 signaling. In some embodiments, provided herein are methods of activating PD-1 signaling in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the pharmaceutical compositions disclosed herein. In some embodiments, provided herein are uses of the pharmaceutical compositions disclosed herein for activating PD-1 signaling. In some embodiments, provided herein are uses of the pharmaceutical compositions provided herein for the preparation of a medicament for activating PD-1 signaling. In some embodiments, the methods provided herein inhibit the activity of immune effector cells (e.g., T cells) . In some embodiments, the methods provided herein inhibit the proliferation of immune effector cells (e.g., T cells) . In some embodiments, the methods provided herein suppress the reconstitution of immune effector cells (e.g., T cells) .

In some embodiments, provided herein are methods of reducing autoimmunity or inflammation in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the anti-PD-1 antibodies or fusion proteins disclosed herein. In some embodiments, provided herein are uses of the anti-PD-1 antibodies or fusion proteins disclosed herein for reducing autoimmunity or inflammation. In some embodiments, provided herein are uses of the anti-PD-1 antibodies or fusion proteins provided herein for the preparation of a medicament for reducing autoimmunity or inflammation. In some embodiments, provided herein are methods of reducing autoimmunity or inflammation in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the pharmaceutical compositions disclosed herein. In some embodiments, provided herein are uses of the pharmaceutical compositions disclosed herein for reducing autoimmunity or inflammation. In some embodiments, provided herein are uses of the pharmaceutical compositions provided herein for the preparation of a medicament for reducing autoimmunity or inflammation.

In some embodiments, provided herein are uses of the anti-PD-1 antibodies or fusion proteins disclosed herein for treating an autoimmune or inflammatory disease. In some embodiments, provided herein are uses of the anti-PD-1 antibodies or fusion proteins provided herein for the preparation of a medicament for treating an autoimmune or inflammatory disease. In some embodiments, provided herein are methods of treating an autoimmune or inflammatory disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the pharmaceutical compositions disclosed herein. In some embodiments, provided herein are uses of the pharmaceutical compositions disclosed herein for treating an autoimmune or inflammatory disease. In some embodiments, provided herein are uses of the pharmaceutical compositions provided herein for the preparation of a medicament for treating an autoimmune or inflammatory disease. In some embodiments, the disease to be treated with methods disclosed herein can be an autoimmune or inflammatory disease associated with insufficient PD-1 signaling.

The subject to be treated by methods disclosed herein is typically a human. The subject can also be a mammal, such as a mouse, rat or primate (e.g., a marmoset or monkey) . The subject can be a non-human animal. The antibodies, fusion proteins, and pharmaceutical compositions disclosed herein can also have veterinary use. The subject to be treated can be a farm animal for example, a cow or bull, sheep, pig, ox, goat or horse or can be a domestic animal such as a dog or cat. The animal can be any age, or a mature adult animal. In some embodiments, treatment can be therapeutic, prophylactic or preventative. The subject can be one who is in need thereof. Those in need of treatment can include individuals already suffering from a medical disease in addition to those who are at risk of developing the disease in the future. In some embodiments, the subject to be treated with methods disclosed herein have deficient PD-1 signaling. In some embodiments, the subject to be treated with methods disclosed herein has excess autoimmunity or inflammation. In some embodiments, the subject has been diagnosed with an autoimmune or inflammatory disease associated with PD-1 signaling. In some embodiments, the subject is at risk of developing an autoimmune or inflammatory disease associated with PD-1 signaling.

In some embodiments, methods provided herein can promote a beneficial therapeutic response with respect to an autoimmune or inflammatory response. In some embodiments, methods provided herein result in an improvement of symptoms associated with the disease.

Actual dosage levels of the active ingredients (i.e., the anti-PD-1 antibodies or fusion proteins) in the pharmaceutical compositions described herein can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions described herein, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

The anti-PD-1 antibodies or fusion proteins can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the anti-PD-1 antibodies or fusion proteins in the patient. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and until the patient shows partial or complete amelioration of symptoms of disease.

The anti-PD-1 antibodies or fusion proteins or pharmaceutical compositions provided herein can be administered to a subject by any methods known in the art, including, but not limited to, pleural administration, intravenous administration, subcutaneous administration, intranodal administration, intramuscular administration, intradermal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, intracranial administration, spinal or other parenteral routes of administration, for example by injection or infusion, or direct administration to the thymus. 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, intraspinal, epidural and intrastemal injection and infusion. In some embodiments, subcutaneous administration is adopted. In some embodiments, intravenous administration is adopted. In some embodiments, oral administration is adopted. In one embodiment, the antibodies or fusion proteins provided herein can be delivered locally. In another embodiment, the antibodies or fusion proteins provided herein can be administered systemically.

In the methods disclosed herein, a therapeutically effective amount of the anti-PD-1 antibodies or fusion proteins or pharmaceutical compositions disclosed herein is administered to a subject that can benefit from activation of PD-1 signaling. The subject can have deficient or insufficient, or complete lack of activation of PD-1 signaling pathway. The subject can be a mammal. In some embodiments, the subject is a human.

Anti-PD-1 antibodies or fusion proteins or pharmaceutical compositions provided herein can be administered with medical devices known in the art. For example, in some embodiments, a needleless hypodermic injection device can be used, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules for use described herein include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

In some embodiments, the anti-PD-1 antibodies or fusion proteins or pharmaceutical compositions provided herein can be administered with an additional therapy. The additional therapy can be administered prior to, concurrently with, or subsequent to administration of the anti-PD-1 antibodies or fusion proteins, cells, or pharmaceutical compositions described herein. Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously. A person skilled in the art can readily determine appropriate regimens for administering a pharmaceutical composition described herein and an additional therapy in combination, including the timing and dosing of an additional agent to be used in a combination therapy, based on the needs of the subject being treated.

The antibodies or fusion proteins provided herein can also be used in detection of PD-1. Also encompassed are methods for detecting the presence of human PD-1 antigen in a sample, or measuring the amount of human PD-1 antigen, comprising contacting the sample, and a control sample, with a monoclonal antibody, e.g., a humanized monoclonal antibody, or an antigen-binding fragment thereof, which specifically binds to human PD-1, under conditions that allow for formation of a complex between the antibody or antigen-binding fragment and human PD-1. The formation of a complex is then detected, wherein a difference complex formation between the sample compared to the control sample is indicative the presence of human PD-1 antigen in the sample. Moreover, the anti-PD-1 antibodies or fusion proteins described herein can be used to purify human PD-1 via immunoaffinity purification. In some embodiments, the anti-PD-1 antibodies or fusion proteins described herein are used for detecting PD-1-expressing cells. In some embodiments, the anti-PD-1 antibodies or fusion proteins described herein are used for quantifying PD-1 antigen, or PD-1-expressing cells.
7.7 Exemplified embodiments

Embodiment 1: An antibody or antigen-binding fragment thereof that specifically binds human PD-1, comprising: (a) a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence of SEQ ID NO: 18; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or (b) a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 from a VH having the amino acid sequence of SEQ ID NO: 27; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

Embodiment 2: The antibody or antigen-binding fragment of Embodiment 1, wherein (a) the VL comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or (b) the VH comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 11, 12 and 13, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

Embodiment 3: The antibody or antigen-binding fragment of Embodiment 1, comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 having the amino acid sequences of SEQ ID NOs: 8, 9, 10, 11, 12 and 13, respectively.

Embodiment 4: The antibody or antigen-binding fragment of Embodiment 1, comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 having the amino acid sequences of SEQ ID NOs: 8, 9, 10, 11, 14 and 13, respectively.

Embodiment 5: The antibody or antigen-binding fragment of any one of Embodiments 1 to 4 that is a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a human antibody or antigen-binding fragment.

Embodiment 6: The antibody or antigen-binding fragment of Embodiment 5 that is a humanized antibody or antigen-binding fragment.

Embodiment 7: The antibody or antigen-binding fragment of any one of Embodiments 1 to 6, wherein: (a) the VL has at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44; and/or (b) the VH has at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54.

Embodiment 8: The antibody or antigen-binding fragment of Embodiment 7 wherein the VL and VH have the amino acid sequences of (1) SEQ ID NOs: 18 and 27, respectively; (2) SEQ ID NOs: 19 and 28, respectively; (3) SEQ ID NOs: 19 and 29, respectively; (4) SEQ ID NOs: 20 and 28, respectively; (5) SEQ ID NOs: 20 and 29, respectively; (6) SEQ ID NOs: 21 and 28, respectively; (7) SEQ ID NOs: 21 and 29, respectively; (8) SEQ ID NOs: 22 and 30, respectively; (9) SEQ ID NOs: 22 and 31, respectively; (10) SEQ ID NOs: 22 and 32, respectively; (11) SEQ ID NOs: 24 and 30, respectively; (12) SEQ ID NOs: 24 and 31, respectively; (13) SEQ ID NOs: 24 and 32, respectively; (14) SEQ ID NOs: 22 and 33, respectively; (15) SEQ ID NOs: 24 and 33, respectively; (16) SEQ ID NOs: 22 and 34, respectively; (17) SEQ ID NOs: 24 and 35, respectively; (18) SEQ ID NOs: 40 and 50, respectively; (19) SEQ ID NOs: 38 and 48, respectively; (20) SEQ ID NOs: 38 and 49, respectively; (21) SEQ ID NOs: 39 and 48, respectively; (22) SEQ ID NOs: 39 and 49, respectively; (23) SEQ ID NOs: 41 and 51, respectively; (24) SEQ ID NOs: 41 and 52, respectively; (25) SEQ ID NOs: 42 and 51, respectively; (26) SEQ ID NOs: 42 and 52, respectively; (27) SEQ ID NOs: 39 and 51, respectively; (28) SEQ ID NOs: 39 and 52, respectively; (29) SEQ ID NOs: 43 and 53, respectively; (30) SEQ ID NOs: 43 and 54, respectively; (31) SEQ ID NOs: 44 and 53, respectively; or (32) SEQ ID NOs: 44 and 54, respectively.

Embodiment 9: An antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of any one of Embodiments 1 to 8 for binding to human PD-1.

Embodiment 10: The antibody or antigen-binding fragment of any one of Embodiments 1 to 9 that is selected from the group consisting of a Fab, a Fab’, a F (ab’) 2, a Fv, a scFv, a (scFv) 2, a single domain antibody (sdAb) , and a heavy chain antibody (HCAb) .

Embodiment 11: The antibody or antigen-binding fragment of any one of Embodiments 1 to 9 that is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

Embodiment 12: The antibody of Embodiment 11 comprising a light chain constant region (CL) that is kappa CL (Cκ; SEQ ID NO: 58) .

Embodiment 13: The antibody of Embodiment 11 or 12 comprising a heavy chain constant region (CH) having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-63.

Embodiment 14: The antibody of Embodiment 13, wherein the CH region comprises at least one amino acid mutation to enhance its antibody-dependent cellular cytotoxicity (ADCC) .

Embodiment 15: The antibody of any one of Embodiments 11 to 14 that is an IgG1 antibody.

Embodiment 16: The antibody of Embodiment 15, wherein the CH region of the IgG1 antibody has at least one amino acid substitution selected from the group consisting of L234A, L235E, G236A, S239D, F243L, D265A, S298A, A330L and I332E, according to EU numbering.

Embodiment 17: The antibody of Embodiment 16, wherein the CH region of the IgG1 antibody has S239D/A330L/I332E substitutions, according to EU numbering.

Embodiment 18: The antibody of Embodiment 16, wherein the CH region lacks core fucose on the Fc N-glycan on N297, according to EU numbering.

Embodiment 19: The antibody or antigen-binding fragment of any one of Embodiments 1 to 18 that activates PD-1 signaling.

Embodiment 20: The antibody or antigen-binding fragment of any one of Embodiments 1 to 19 that inhibits T cell activity, proliferation, reconstitution, or any combination thereof.

Embodiment 21: The antibody or antigen-binding fragment of any one of Embodiments 1 to 20 that is a monospecific antibody, bispecific antibody or a multispecific antibody.

Embodiment 22: The antibody or antigen-binding fragment of any one of Embodiments 1 to 21 that is a monoclonal antibody or antigen-binding fragment.

Embodiment 23: A polynucleotide that encodes or a plurality of polynucleotides that collectively encode the polypeptide chain (s) of the antibody or antigen-binding fragment of any one of the Embodiments 1 to 22.

Embodiment 24: A vector comprising the polynucleotide or plurality of polynucleotides of Embodiment 23.

Embodiment 25: A host cell comprising the polynucleotide or plurality of polynucleotides of Embodiment 23, or the vector of Embodiment 24.

Embodiment 26: A method of making an antibody or antigen-binding fragment thereof that specifically binds human PD-1, comprising culturing the cell of Embodiment 25 under conditions that allow expression of the antibody or antigen-binding fragment.

Embodiment 27: The method of Embodiment 26 that comprises isolating the antibody or antigen-binding fragment from the culture.

Embodiment 28: A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment of any one of Embodiments 1 to 22, and a pharmaceutically acceptable carrier.

Embodiment 29: A method of activating PD-1 signaling in an immune effector cell, comprising contacting the immune effector cell with the antibody or antigen-binding fragment of any one of Embodiments 1 to 22.

Embodiment 30: A method of inhibiting activity of an immune effector cell, comprising contacting the immune effector cell with the antibody or antigen-binding fragment of any one of Embodiments 1 to 22.

Embodiment 31: A method of reducing autoimmunity or inflammation in a subject in need thereof, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of Embodiments 1 to 22.

Embodiment 32: A method of treating an autoimmune or inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment of any one of Embodiments 1 to 22.

Embodiment 33: The method of Embodiment 31 or 32, further comprising administering an additional therapy to the subject.

Embodiment 34: The method of any one of Embodiments 31 to 33, wherein the subject is a human.

Embodiment 35: Use of the antibody or antigen-binding fragment of any one of Embodiments 1 to 22 in reducing autoimmunity or inflammation.

Embodiment 36: Use of the antibody or antigen-binding fragment of any one of Embodiments 1 to 22 for the preparation of a medicament for reducing autoimmunity or inflammation.

Embodiment 37: Use of the antibody or antigen-binding fragment of any one of Embodiments 1 to 22 in treating an autoimmune or inflammatory disease.

Embodiment 38: Use of the antibody or antigen-binding fragment of any one of Embodiments 1 to 22 for the preparation of a medicament for treating an autoimmune or inflammatory disease.

Embodiment 39: A fusion protein thereof comprising the anti-PD-1 antibody or antigen-binding fragment of any one of Embodiments 1 to 22 and a CTLA-4 peptide.

Embodiment 40: The fusion protein of Embodiment 39, wherein the CTLA-4 peptide comprises human CTLA-4 extracellular domain.

Embodiment 41: The fusion protein of Embodiment 39, wherein the CTLA-4 peptide has the amino acid sequence of SEQ ID NO: 4.

Embodiment 42: The fusion protein of any one of Embodiments 39 to 41, wherein the CTLA-4 peptide and the anti-PD-1 antibody or antigen-binding fragment are connected by a linker.

Embodiment 43: The fusion protein of Embodiment 42, wherein the linker has an amino acid sequence selected from the group consisting of SEQ ID NOs: 65-68.

Embodiment 44: The fusion protein of any one of Embodiments 39 to 43, wherein the CTLA-4 peptide is fused to N-terminus of the antibody or antigen-binding fragment.

Embodiment 45: The fusion protein of any one of Embodiments 39 to 43, wherein the CTLA-4 peptide is fused to C-terminus of the antibody or antigen-binding fragment.

Embodiment 46: A fusion protein comprising a heavy chain and a light chain, wherein the heavy chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-78, and wherein the light chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 70.

Embodiment 47: A polynucleotide that encodes or a plurality of polynucleotides that collectively encode polypeptide chain (s) of the fusion protein of any one of Embodiments 39 to 46.

Embodiment 48: A vector comprising the polynucleotide or plurality of polynucleotides of Embodiment 47.

Embodiment 49: A host cell comprising the polynucleotide or plurality of polynucleotides of Embodiment 47, or the vector of Embodiment 48.

Embodiment 50: A method of making the fusion protein of any one of Embodiments 39 to 46, comprising culturing the cell of Embodiment 49 under conditions that allow expression of the fusion protein.

Embodiment 51: The method of Embodiment 50 that comprises isolating the antibody or antigen-binding fragment from the culture.

Embodiment 52: A pharmaceutical composition comprising a therapeutically effective amount of the fusion protein of any one of Embodiments 39 to 46 and a pharmaceutically acceptable carrier.

Embodiment 53: A method of activating PD-1 signaling in an immune effector cell, comprising contacting the immune effector cell with the fusion protein of any one of Embodiments 39 to 46.

Embodiment 54: A method of inhibiting activity of an immune effector cell, comprising contacting the immune effector cell with the fusion protein of any one of Embodiments 39 to 46.

Embodiment 55: A method of reducing autoimmunity or inflammation in a subject in need thereof, comprising administering to the subject an effective amount of the fusion protein of any one of Embodiments 39 to 46.

Embodiment 56: A method of treating an autoimmune or inflammatory disease associated in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the fusion protein of any one of Embodiments 39 to 46.

Embodiment 57: The method of Embodiment 55 or 56, further comprising administering an additional therapy to the subject.

Embodiment 58: The method of any one of Embodiments 55 to 57, wherein the subject is a human.

Embodiment 59: Use of the fusion protein of any one of Embodiments 39 to 46 in reducing autoimmunity or inflammation.

Embodiment 60: Use of the fusion protein of any one of Embodiments 39 to 46 for the preparation of a medicament for reducing autoimmunity or inflammation.

Embodiment 61: Use of the fusion protein of any one of Embodiments 39 to 46 in treating an autoimmune or inflammatory disease.

Embodiment 62: Use of the fusion protein of any one of Embodiments 39 to 46 for the preparation of a medicament for treating an autoimmune or inflammatory disease.

The practice of the invention employs, unless otherwise indicated, conventional techniques in molecular biology, cell biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described in the references cited herein and are fully explained in the literature. See, e.g., Maniatis et al. (1982) MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989) , MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons (1987 and annual updates) ; CURRENT PROTOCOLS IN IMMUNOLOGY, John Wiley &Sons (1987 and annual updates) ; Gait (ed. ) (1984) OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH, IRL Press; Eckstein (ed. ) (1991) OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, IRL Press; Birren et al. (eds. ) (1999) GENOME ANALYSIS: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; Borrebaeck (ed. ) (1995) ANTIBODY ENGINEERING, Second Edition, Oxford University Press; Lo (ed. ) (2006) ANTIBODY ENGINEERING: METHODS AND PROTOCOLS (METHODS IN MOLECULAR BIOLOGY) ; Vol. 248, Humana Press, Inc; Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) ; Hammerling et al., in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS 563 681 (Elsevier, N.Y., 1981) ; each of which is incorporated herein by reference in its entirety.
7.8 Experimental

The examples provided below are for purposes of illustration only, which are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.

Briefly, studies described below included the generation and characterization of candidate anti-PD-1 agonist antibodies. In particular, candidate humanized antibodies were found to bind to human PD-1 with high affinity and to activate PD-1 signaling with strong potency. Indeed, the candidate anti-PD-1 agonist antibodies also effectively inhibited T cell activation and proliferation, and significantly suppressed T cell reconstitution in vivo.
7.8.1 Example 1: Candidate antibodies bound to human PD-1 with high affinity

Methods: The binding kinetics of the candidate antibodies to Human PD-1/PDCD1 Protein, His Tag (Acro: PD-1-H5221) were measured by surface plasmon resonance (SPR) at 25℃ using a 8K instrument. The samples were dissolved in HBS-EP+ running buffer and the monoclonal antibody was captured using a Protein A-coupled CM5 Series S sensor chip (GE Healthcare CAS #2913913 l-AA) , and each cycle was performed at 25℃ at a flow rate of 10 μl/min for antibody capture and 30 μl/min for ligand binding and dissociation. Each cycle included the following steps: injection of 2 μg/mL monoclonal antibody, with a contact time of 60 seconds, with a target capture level of approximately 220RU on flow cell 2; injection of Human PD-1 /PDCD1 protein (concentrations ranging from 100 nM to 0.625 nM by 2-fold serial dilutions) for 100 seconds, followed by a 120-second dissociation phase with 10mM pH1.5 Gly-HCL and regeneration with a contact time of 30 seconds. The association (Kon) and dissociation rates (Koff) of each cycle were determined using standard double referencing and fitting to the “affinity kinetics” model in the parallel kinetics batch mode in Biacore 8K evaluation. Affinity (KD) was calculated from the binding kinetics according to the relationship KD = Koff /Kon.

Results and conclusions: As shown in Table 1, all candidate humanized antibodies bound to human PD-1 with high affinity.

Table 1 The affinity binding kinetics of humanized antibodies to human PD-1

7.8.2 Example 2: Candidate antibodies effectively inhibited T cell activation

Methods: Jurkat T cells (BPS) transfected with NFAT luciferase reporter and human PD-1 were activated with human CD3 antibody and THP-1 cells (ATCC) . THP-1 cells (40,000 cells/well) were plated in 100 μl of culture medium in a white 96-well plate (Corning, #3917) . Human anti-CD3 antibody (OKT3) (eBioscience, #16-0037) at a final concentration of 2 μg/mL and treatment antibodies (candidate anti-PD-1 antibody or isotype control IgG1) were added in a five-fold 8-point antibody gradient dilution to THP-1 cells. The cell and antibody mixture were incubated at 37℃, 5%CO₂ for 30 minutes. NFAT-luciferase Jurkat reporter cells expressing PD-1 (50,000 cells/well) were added to the THP-1 cells, CD3 antibody and treatment antibody mixture on the 96-well plate and incubated at 37℃, 5%CO₂ for 5 hours. After the incubation, the plate was placed at room temperature for 10 minutes. 80 μl of BioGlo reagent (Promega, #G7940) was added to the wells and incubated at room temperature for 10 minutes. The luminescent signals were read using SpectraMax i3x (Molecular Devices) .

Results and conclusions: As shown in FIGs. 1A-1C, all candidate anti-PD-1 antibodies inhibited T cell activation in a dose-dependent manner, comparable to peresolimab.
7.8.3 Example 3: Candidate antibodies effectively inhibited proliferation of
human primary T cells

Methods: PBMCs were washed three times in PBS. CFSE labeling was performed in PBS at a concentration of 1 mM CFSE and incubated for 20 minutes at 37℃, 5%CO₂. CFSE was quenched with 4℃ cold FBS followed by a wash step. CFSE-labeled PBMCs (150,000 cells/well) were added to each 96-well round-bottom well containing the candidate antibodies or isotype control IgG1. Starting with a concentration of 100 nM, the antibodies were subjected to a five-fold 8-point serial dilution into the PBMC cells. Cells were stimulated with 4 ng/mL of superantigen (Staphylococcal enterotoxin, SEB) (Toxin Technologies BT202) and incubated for 72 hours at 37℃, 5%CO₂. After incubation and washing with PBS buffer, live/dead cell staining was performed using the Zombie Yellow^TM Fixable Viability Kit (Biolegend, #423103) according to the manufacturer's protocol. The plate was washed with FACS buffer (PBS + 1%FBS) and then stained with labeled anti-CD4 (Biolegend, #317415) antibody for 30 minutes on ice. The cells were analyzed on a Fortessa flow cytometer, and the flow cytometry data were processed and analyzed using FlowJo software. The CD4+ cell proliferation rate was calculated using CFSE staining: treatment group CFSE low%/Mean IgG1 CFSE low%.

Results and conclusions: As shown in FIGs2A-2D, the candidate antibodies effectively inhibited the proliferation of human primary CD4+ T cells.
7.8.4 Example 4: Candidate antibodies significantly suppressed T cell
reconstitution in vivo

Methods: Human PBMCs were injected intravenously into NCG-IL15 transgenic mice on Day 0 to establish a humanized immune system. Beginning on Day 4 the mice received twice-weekly (BIW) intraperitoneal doses of either vehicle, the reference antibody peresolimab (5 mg/kg) , or candidate anti-PD1 antibody CT-147 (5 mg/kg) . Interim blood samples were taken on the days indicated in the timeline (Day 6, 15, 22, 29, 36 and 43) for flow cytometric analysis of circulating lymphocyte subsets.

Results and conclusions: As shown in FIG. 3, vehicle-and peresolimab-treated mice showed robust T-cell outgrowth, reaching ～40–60 %of total lymphocytes by Day 29–36. In contrast, the candidate anti-PD-1 antibody kept circulating T-cell levels below ～10 %throughout the study, demonstrating a sustained suppression of T-cell reconstitution in this PBMC-humanized model.
7.9 Sequences

***

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. Unless the context indicates otherwise, it is specifically intended that the various features described herein can be used in any combination. 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. Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Claims

An antibody or antigen-binding fragment thereof that specifically binds human PD-1, comprising:

(a) a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence of SEQ ID NO: 18; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or

(b) a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 from a VH having the amino acid sequence of SEQ ID NO: 27; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.
The antibody or antigen-binding fragment of claim 1, wherein

(a) the VL comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or

(b) the VH comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 11, 12 and 13, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.
The antibody or antigen-binding fragment of claim 1, comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 having the amino acid sequences of SEQ ID NOs: 8, 9, 10, 11, 12 and 13, respectively.
The antibody or antigen-binding fragment of claim 1, comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 having the amino acid sequences of SEQ ID NOs: 8, 9, 10, 11, 14 and 13, respectively.
The antibody or antigen-binding fragment of any one of claims 1 to 4 that is a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a human antibody or antigen-binding fragment.
The antibody or antigen-binding fragment of claim 5 that is a humanized antibody or antigen-binding fragment.
The antibody or antigen-binding fragment of any one of claims 1 to 6, wherein:

(a) the VL has at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 18-22, 24 and 38-44; and/or

(b) the VH has at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 27-35 and 48-54.
The antibody or antigen-binding fragment of claim 7 wherein the VL and VH have the amino acid sequences of (1) SEQ ID NOs: 18 and 27, respectively; (2) SEQ ID NOs: 19 and 28, respectively; (3) SEQ ID NOs: 19 and 29, respectively; (4) SEQ ID NOs: 20 and 28, respectively; (5) SEQ ID NOs: 20 and 29, respectively; (6) SEQ ID NOs: 21 and 28, respectively; (7) SEQ ID NOs: 21 and 29, respectively; (8) SEQ ID NOs: 22 and 30, respectively; (9) SEQ ID NOs: 22 and 31, respectively; (10) SEQ ID NOs: 22 and 32, respectively; (11) SEQ ID NOs: 24 and 30, respectively; (12) SEQ ID NOs: 24 and 31, respectively; (13) SEQ ID NOs: 24 and 32, respectively; (14) SEQ ID NOs: 22 and 33, respectively; (15) SEQ ID NOs: 24 and 33, respectively; (16) SEQ ID NOs: 22 and 34, respectively; (17) SEQ ID NOs: 24 and 35, respectively; (18) SEQ ID NOs: 40 and 50, respectively; (19) SEQ ID NOs: 38 and 48, respectively; (20) SEQ ID NOs: 38 and 49, respectively; (21) SEQ ID NOs: 39 and 48, respectively; (22) SEQ ID NOs: 39 and 49, respectively; (23) SEQ ID NOs: 41 and 51, respectively; (24) SEQ ID NOs: 41 and 52, respectively; (25) SEQ ID NOs: 42 and 51, respectively; (26) SEQ ID NOs: 42 and 52, respectively; (27) SEQ ID NOs: 39 and 51, respectively; (28) SEQ ID NOs: 39 and 52, respectively; (29) SEQ ID NOs: 43 and 53, respectively; (30) SEQ ID NOs: 43 and 54, respectively; (31) SEQ ID NOs: 44 and 53, respectively; or (32) SEQ ID NOs: 44 and 54, respectively.
An antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of any one of claims 1 to 8 for binding to human PD-1.
The antibody or antigen-binding fragment of any one of claims 1 to 9 that is selected from the group consisting of a Fab, a Fab’, a F (ab’) ₂, a Fv, a scFv, a (scFv) ₂, a single domain antibody (sdAb) , and a heavy chain antibody (HCAb) .
The antibody or antigen-binding fragment of any one of claims 1 to 9 that is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.
The antibody of claim 11 comprising a light chain constant region (CL) that is kappa CL (Cκ; SEQ ID NO: 58) .
The antibody of claim 11 or 12 comprising a heavy chain constant region (CH) having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-63.
The antibody of claim 13, wherein the CH region comprises at least one amino acid mutation to enhance its antibody-dependent cellular cytotoxicity (ADCC) .
The antibody of any one of claims 11 to 14 that is an IgG1 antibody.
The antibody of claim 15, wherein the CH region comprises at least one amino acid substitution selected from the group consisting of L234A, L235E, G236A, S239D, F243L, D265A, S298A, A330L and I332E, according to EU numbering.
The antibody of claim 16, wherein the CH region of the IgG1 antibody has S239D/A330L/I332E substitutions, according to EU numbering.
The antibody of claim 16, wherein the CH region lacks core fucose on the Fc N-glycan on N297, according to EU numbering.
The antibody or antigen-binding fragment of any one of claims 1 to 18 that activates PD-1 signaling.
The antibody or antigen-binding fragment of any one of claims 1 to 19 that inhibits T cell activity, proliferation, reconstitution, or any combination thereof.
The antibody or antigen-binding fragment of any one of claims 1 to 20 that is a bispecific antibody or a multispecific antibody.
The antibody or antigen-binding fragment of any one of claims 1 to 21 that is a monoclonal antibody or antigen-binding fragment.
A polynucleotide that encodes or a plurality of polynucleotides that collectively encode the polypeptide chain (s) of the antibody or antigen-binding fragment of any one of the claims 1 to 22.
A vector comprising the polynucleotide or plurality of polynucleotides of claim 23.
A host cell comprising the polynucleotide or plurality of polynucleotides of claim 23, or the vector of claim 24.
A method of making an antibody or antigen-binding fragment thereof that specifically binds human PD-1, comprising culturing the cell of claim 25 under conditions that allow expression of the antibody or antigen-binding fragment.
The method of claim 26 that comprises isolating the antibody or antigen-binding fragment from the culture.
A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 22, and a pharmaceutically acceptable carrier.
A method of activating PD-1 signaling in an immune effector cell, comprising contacting the immune effector cell with the antibody or antigen-binding fragment of any one of claims 1 to 22.
A method of inhibiting activity of an immune effector cell, comprising contacting the immune effector cell with the antibody or antigen-binding fragment of any one of claims 1 to 22.
A method of reducing autoimmunity or inflammation in a subject in need thereof, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 22.
A method of treating an autoimmune or inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 22.
The method of claim 31 or 32, further comprising administering an additional therapy to the subject.
The method of any one of claims 31 to 33, wherein the subject is a human.
Use of the antibody or antigen-binding fragment of any one of claims 1 to 22 in reducing autoimmunity or inflammation.
Use of the antibody or antigen-binding fragment of any one of claims 1 to 22 for the preparation of a medicament for reducing autoimmunity or inflammation.
Use of the antibody or antigen-binding fragment of any one of claims 1 to 22 in treating an autoimmune or inflammatory disease.
Use of the antibody or antigen-binding fragment of any one of claims 1 to 22 for the preparation of a medicament for treating an autoimmune or inflammatory disease.
A fusion protein thereof comprising the anti-PD-1 antibody or antigen-binding fragment of any one of claims 1 to 22 and a CTLA-4 peptide.
The fusion protein of claim 39, wherein the CTLA-4 peptide comprises human CTLA-4 extracellular domain.
The fusion protein of claim 39, wherein the CTLA-4 peptide has the amino acid sequence of SEQ ID NO: 4.
The fusion protein of any one of claims 39 to 41, wherein the CTLA-4 peptide and the anti-PD-1 antibody or antigen-binding fragment are connected by a linker.
The fusion protein of claim 42, wherein the linker has an amino acid sequence selected from the group consisting of SEQ ID NOs: 65-68.
The fusion protein of any one of claims 39 to 43, wherein the CTLA-4 peptide is fused to N-terminus of the antibody or antigen-binding fragment.
The fusion protein of any one of claims 39 to 43, wherein the CTLA-4 peptide is fused to C-terminus of the antibody or antigen-binding fragment.
A fusion protein comprising a heavy chain and a light chain, wherein the heavy chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-78, and wherein the light chain has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 70.
A polynucleotide that encodes or a plurality of polynucleotides that collectively encode polypeptide chain (s) of the fusion protein of any one of claims 39 to 46.
A vector comprising the polynucleotide or plurality of polynucleotides of claim 47.
A host cell comprising the polynucleotide or plurality of polynucleotides of claim 47, or the vector of claim 48.
A method of making the fusion protein of any one of claims 39 to 46, comprising culturing the cell of claim 49 under conditions that allow expression of the fusion protein.
The method of claim 50 that comprises isolating the antibody or antigen-binding fragment from the culture.
A pharmaceutical composition comprising a therapeutically effective amount of the fusion protein of any one of claims 39 to 46 and a pharmaceutically acceptable carrier.
A method of activating PD-1 signaling in an immune effector cell, comprising contacting the immune effector cell with the fusion protein of any one of claims 39 to 46.
A method of inhibiting activity of an immune effector cell, comprising contacting the immune effector cell with the fusion protein of any one of claims 39 to 46.
A method of reducing autoimmunity or inflammation in a subject in need thereof, comprising administering to the subject an effective amount of the fusion protein of any one of claims 39 to 46.
A method of treating an autoimmune or inflammatory disease associated in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the fusion protein of any one of claims 39 to 46.
The method of claim 55 or 56, further comprising administering an additional therapy to the subject.
The method of any one of claims 55 to 57, wherein the subject is a human.
Use of the fusion protein of any one of claims 39 to 46 in reducing autoimmunity or inflammation.
Use of the fusion protein of any one of claims 39 to 46 for the preparation of a medicament for reducing autoimmunity or inflammation.
Use of the fusion protein of any one of claims 39 to 46 in treating an autoimmune or inflammatory disease.
Use of the fusion protein of any one of claims 39 to 46 for the preparation of a medicament for treating an autoimmune or inflammatory disease.