RU2788616C2 - Antibodies against pd-1 and their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: antibodies or their antigen-binding fragments, which bind to PD-1, and their use for the production of a bispecific antibody are presented. Nucleic acid, expression vectors, a pair of expression vectors, cells for expression of an anti-PD-1 antibody or its antigen-binding fragment, a method for the production of an anti-PD-1 antibody or its antigen-binding fragment, an antibody-drug conjugate for increasing an immune response, a method for the treatment of an individual having malignant neoplasm, a method for reduction in a tumor growth rate, a method for destruction of a tumor cell, and pharmaceutical compositions for increasing an immune response are also disclosed.

EFFECT: invention allows for obtainment of new antibodies or their antigen-binding fragments, which have high affinity to PD-1.

68 cl, 6 tbl, 20 dwg, 8 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to antibodies against PD-1 (programmed cell death protein 1) and their use.

BACKGROUND OF THE INVENTION

Currently, malignant neoplasms are one of the types of diseases with the highest mortality among humans. According to statistics from the World Health Organization, in 2012 the number of cases of malignant neoplasms and deaths worldwide reached 14 million and 8.2 million, respectively. In China, the number of cases of newly diagnosed malignancies is 3.07 million and the death toll is 2.2 million.

The recent clinical and commercial success of antitumor antibodies has attracted considerable interest in antibody therapeutics. There is a need to develop anti-tumor antibodies for use in various antibody-based therapeutics for the treatment of cancer.

SUMMARY OF THE INVENTION

The present invention relates to antibodies against PD-1, their antigen-binding fragments and their use.

In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that binds to PD-1 (programmed cell death protein 1) comprising: a heavy chain variable region (VH) containing complementarity determining regions (CDRs) 1, 2 and 3, where the CDR1 VH region contains an amino acid sequence that is at least 80% identical to the selected amino acid sequence of the CDR1 VH, the CDR2 VH region contains an amino acid sequence that is at least 80% identical to the selected amino acid sequence of the CDR2 VH, and the CDR3 VH region contains the amino acid sequence , which is at least 80% identical to the selected amino acid sequence of CDR3 VH; and a light chain variable region (VL) containing CDRs 1, 2 and 3, where the CDR1 VL region contains an amino acid sequence that is at least 80% identical to the selected amino acid sequence of the CDR1 VL, the CDR2 VL region contains an amino acid sequence that is at least 80% identical to the selected amino acid sequence of VL CDR2, and the CDR3 VL region contains an amino acid sequence that is at least 80% identical to the selected amino acid sequence of CDR3 VL, where the selected amino acid sequences of CDR 1, 2 and 3 VH and the selected amino acid sequences of CDR 1, 2 and 3 VL are one of the following:

(1) selected amino acid sequences of VH CDRs 1, 2, 3 are shown in SEQ ID NOs: 1, 2, 3, respectively, and selected amino acid sequences of VL CDRs 1, 2, 3 are shown in SEQ ID NOs: 4, 5, 6, respectively;

(2) selected amino acid sequences of CDR 1, 2, 3 VH are shown in SEQ ID NOS: 7, 8, 9, respectively, and selected amino acid sequences of CDR 1, 2, 3 VL are shown in SEQ ID NOS: 10, 11, 12, respectively;

(3) selected amino acid sequences of CDR 1, 2, 3 VH are shown in SEQ ID NOS: 7, 13, 14, respectively, and selected amino acid sequences of CDR 1, 2, 3 VL are shown in SEQ ID NOS: 10, 11, 15, respectively.

In some embodiments, the VH contains CDRs 1, 2, 3 with the amino acid sequences shown in SEQ ID NO: 1, 2 and 3, respectively, and the VL contains CDR 1, 2, 3 with the amino acid sequences shown in SEQ ID NO: 4, 5 and 6, respectively.

In some embodiments, the VH contains CDRs 1, 2, 3 with the amino acid sequences shown in SEQ ID NO:7, 8 and 9, respectively, and the VL contains CDRs 1, 2, 3 with the amino acid sequences shown in SEQ ID NO:10 , 11 and 12, respectively.

In some embodiments, the VH contains CDRs 1, 2, 3 with the amino acid sequences shown in SEQ ID NO: 7, 13 and 14, respectively, and the VL contains CDR 1, 2, 3 with the amino acid sequences shown in SEQ ID NO: 10 , 11 and 15, respectively.

In some embodiments, the antibody or antigen-binding fragment specifically binds to human PD-1. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a single chain variable fragment (scFV).

In another aspect, the present invention relates to a nucleic acid containing a polynucleotide encoding a polypeptide containing:

(1) an immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively, and where VH, when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO:29, 30, 31 or 40, binds to PD-1;

(2) an immunoglobulin light chain or fragment thereof containing a VL containing CDRs 1, 2 and 3 containing the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively, and where VL when paired with a VH containing the amino acid sequence given in SEQ ID NO:26, 27, 28 or 39 binds to PD-1;

(3) an immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2 and 3 containing the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9, respectively, and where VH when paired with the variable region of the light chain (VL), containing the amino acid sequence shown in SEQ ID NO:36, 37, 38 or 42, associated with PD-1;

(4) an immunoglobulin light chain or fragment thereof containing a VL containing CDRs 1, 2 and 3 containing the amino acid sequences shown in SEQ ID NOs: 10, 11 and 12, respectively, and where VL when paired with a VH containing the amino acid sequence given in SEQ ID NO:32, 33, 34, 35 or 41 binds to PD-1;

(5) an immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2 and 3 containing the amino acid sequences shown in SEQ ID NOs: 7, 13 and 14, respectively, and where VH when paired with the variable region of the light chain (VL), containing the amino acid sequence shown in SEQ ID NO:44, associated with PD-1;

(6) an immunoglobulin light chain or fragment thereof containing a VL containing CDRs 1, 2 and 3 containing the amino acid sequences shown in SEQ ID NOs: 10, 11 and 15, respectively, and where VL when paired with a VH containing the amino acid sequence shown in SEQ ID NO:43 binds to PD-1.

In some embodiments, the nucleic acid comprises a polynucleotide encoding an immunoglobulin heavy chain-containing polypeptide or fragment thereof, containing a VH containing CDRs 1, 2, and 3, containing the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively.

In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide containing an immunoglobulin light chain or a fragment thereof, containing a VL containing CDRs 1, 2 and 3, containing the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively.

In some embodiments, the nucleic acid comprises a polynucleotide encoding an immunoglobulin heavy chain-containing polypeptide or fragment thereof, containing a VH containing CDRs 1, 2, and 3, containing the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, respectively.

In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide containing an immunoglobulin light chain or a fragment thereof containing a VL containing CDRs 1, 2 and 3 containing the amino acid sequences shown in SEQ ID NOs: 10, 11 and 12, respectively.

In some embodiments, the nucleic acid comprises a polynucleotide encoding an immunoglobulin heavy chain-containing polypeptide or fragment thereof, containing a VH containing CDRs 1, 2, and 3, containing the amino acid sequences shown in SEQ ID NOs: 7, 13, and 14, respectively.

In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide containing an immunoglobulin light chain or a fragment thereof containing a VL containing CDRs 1, 2 and 3 containing the amino acid sequences shown in SEQ ID NOs: 10, 11 and 15, respectively.

In some embodiments, the VH, when paired with VL, specifically binds to human PD-1, or the VL, when paired with VH, specifically binds to human PD-1.

In some embodiments, the immunoglobulin heavy chain, or fragment thereof, is a humanized immunoglobulin heavy chain, or fragment thereof, and the immunoglobulin light chain, or fragment thereof, is a humanized immunoglobulin light chain, or fragment thereof.

In some embodiments, the nucleic acid encodes a single chain variable fragment (scFv). In some embodiments, the nucleic acid is cDNA.

In one aspect, the present invention relates to a vector containing one or more of the nucleic acids presented in the present description. In some embodiments, the vector encodes a VL region and a VH region that together bind to PD-1.

In one aspect, the present invention relates to a pair of vectors, where each vector contains one of the nucleic acids presented in the present description, where the pair of vectors jointly encodes a VL region and a VH region that together bind to PD-1.

In another aspect, the present invention relates to a cell containing the vector presented in the present description, or a pair of vectors presented in the present description. In some embodiments, the cell is a CHO cell.

In one aspect, the present invention also relates to a cell containing one or more of the nucleic acids presented in the present description.

In another aspect, the present invention relates to a cell containing two of the nucleic acids presented in the present description. In some embodiments, the two nucleic acids co-code for a VL region and a VH region that together bind to PD-1.

In another aspect, the present invention relates to methods for producing an antibody or antigen-binding fragment. The methods include the steps

(a) culturing a cell as described herein under conditions sufficient for the cell to produce an antibody or antigen-binding fragment; and

(b) collecting the antibody or antigen-binding fragment produced by the cell.

In one aspect, the present invention relates to an antibody or antigen-binding fragment thereof that binds to PD-1, containing a heavy chain variable region (VH) containing an amino acid sequence that is at least 90% identical to the selected VH sequence, and a light chain variable region (VL) containing an amino acid sequence that is at least 90% identical to the selected VL sequence, where the selected VH sequence and the selected VL sequence are one of the following:

(1) the selected VH sequence is SEQ ID NO: 26, 27, 28, or 39, and the selected VL sequence is SEQ ID NO: 29, 30, 31, or 40;

(2) the selected VH sequence is SEQ ID NO:32, 33, 34, 35, or 41 and the selected VL sequence is SEQ ID NO:36, 37, 38, or 42;

(3) the selected VH sequence is SEQ ID NO:43 and the selected VL sequence is SEQ ID NO:44.

In some embodiments, the VH contains the sequence of SEQ ID NO:26, and the VL contains the sequence of SEQ ID NO:31.

In some embodiments, the VH contains the sequence of SEQ ID NO:27, and the VL contains the sequence of SEQ ID NO:31.

In some embodiments, the antibody or antigen-binding fragment specifically binds to human PD-1.

In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a single chain variable fragment (scFV).

In one aspect, the present invention provides an antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof as described herein covalently linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

In another aspect, the present invention provides methods for treating a subject having cancer. The methods include the steps of administering to an individual a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof as provided herein or an antibody-drug conjugate as provided herein.

In some embodiments, the individual has a solid tumor. In some embodiments, the cancer is an inoperable melanoma or a metastatic melanoma. In some embodiments, the malignancy is non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), head and neck cancer, renal cell carcinoma (RCC), melanoma, bladder cancer, gastric cancer, urothelial cancer, Merkel cell carcinoma , triple negative breast cancer (TNBC), or colorectal carcinoma.

In one aspect, the present invention relates to methods for reducing the rate of tumor growth. The methods include the steps of bringing the tumor cell into contact with an effective amount of a composition comprising an antibody or antigen-binding fragment provided herein, or an antibody-drug conjugate provided herein.

In another aspect, the present invention relates to methods for killing tumor cells. The methods include the steps of bringing the tumor cells into contact with an effective amount of a composition comprising an antibody or antigen-binding fragment provided herein or an antibody-drug conjugate provided herein.

In one aspect, the present invention relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment provided in the present description, and a pharmaceutically acceptable carrier.

In another aspect, the present invention also provides a pharmaceutical composition comprising the antibody-drug conjugate described herein and a pharmaceutically acceptable carrier.

Within the scope of the invention, the term "malignant neoplasm" refers to cells capable of autonomous growth. Examples of such cells include cells having an abnormal condition characterized by the growth of rapidly proliferating cells. The term is intended to include malignant growth, such as tumors; oncogenic processes, metastatic tissues and malignantly transformed cells, tissues or organs, regardless of the histopathological type or stage of invasiveness. The term also includes malignant tumors of various organ systems such as the respiratory, cardiovascular, renal, reproductive, hematological, nervous, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas, including malignant tumors such as most colon cancers, renal cell carcinoma, prostate and/or testicular cancer, non-small cell lung carcinoma, and small intestine cancer. Malignancies that are "naturally occurring" include any malignancy that is not experimentally induced by implantation of malignant cells into an individual, and includes, for example, spontaneously occurring malignancy, malignancy resulting from exposure of the patient to carcinogens, malignancy resulting from insertions of a transgenic oncogene or tumor suppressor gene knockout; and cancer caused by infections, such as viral infections. The term "carcinoma" is known in the art and refers to malignant tumors of epithelial or endocrine tissues. The term also includes carcinosarcomas, including malignant tumors consisting of carcinomatous and sarcomatous tissues. The term "adenocarcinoma" refers to a carcinoma derived from glandular tissue, or in which tumor cells form recognizable glandular structures. The term "sarcoma" is known in the art and refers to malignant tumors of mesenchymal origin. The term "hematopoietic neoplastic disorders" includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin. A hematopoietic neoplastic disorder may arise from myeloid, lymphoid, or erythroid lineages or their progenitor cells.

As used herein, the term "antibody" refers to any antigen-binding molecule containing at least one (e.g., one, two, three, four, five, or six) complementarity-determining region (CDR) (e.g., any of the three CDRs of an immunoglobulin light chain or any of the three immunoglobulin heavy chain CDRs) and capable of specifically binding to an epitope. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), single chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, the antibody may comprise the Fc region of a human antibody. The term "antibody" also includes derivatives, such as bispecific antibodies, single chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody, where the portion of the antibody can specifically bind to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (eg, a heavy chain variable domain or a light chain variable domain). Non-limiting examples of antibody fragments include, for example, Fab-, Fab'-, F(ab') ₂ - and Fv fragments.

As used herein, the term "human antibody" refers to an antibody encoded by an endogenous nucleic acid (eg, a rearranged human immunoglobulin heavy or light chain locus) present in a human. In some embodiments, the human antibody is derived from a human or obtained in culture of human cells (eg, human hybridoma cells). In some embodiments, the human antibody is produced in a non-human cell (eg, a mouse or hamster cell line). In some embodiments, the human antibody is produced in a bacterial or yeast cell. In some embodiments, a human antibody is produced in a transgenic, non-human animal (eg, a bovine) containing an unrearranged or rearranged human immunoglobulin locus (eg, a human immunoglobulin heavy or light chain locus).

As used herein, the term "chimeric antibody" refers to an antibody containing a sequence present in at least two different antibodies (eg, antibodies from two different mammalian species, such as a human and a mouse antibody). A non-limiting example of a chimeric antibody is an antibody comprising the variable domain sequences (eg, all or part of the light chain and/or heavy chain variable domain sequence) of a non-human (eg, murine) antibody and human antibody constant domains. Additional examples of chimeric antibodies are provided herein and are known in the art.

As used herein, the term "humanized antibody" refers to a non-human antibody containing a minimal sequence derived from a non-human (eg, mouse) immunoglobulin and containing sequences derived from a human immunoglobulin. In non-limiting examples, humanized antibodies are human antibodies (recipient antibody) in which hypervariable region (e.g., CDR) residues of the recipient antibody are replaced with hypervariable region (e.g., CDR) residues from a non-human antibody (e.g., donor antibody), e.g., mouse antibody , rat or rabbit having the desired specificity, affinity and capacity. In some embodiments, Fv framework residues of a human immunoglobulin are replaced with corresponding non-human (eg, murine) immunoglobulin residues. In some embodiments, humanized antibodies may contain residues not found in the recipient antibody or donor antibody. These modifications can be made to further improve the properties of the antibody. In some embodiments, the humanized antibody contains substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops (CDRs) correspond to loops from a non-human (e.g., murine) immunoglobulin, and all or substantially all of the framework regions are human immunoglobulin framework regions. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin. Humanized antibodies can be produced by molecular biology techniques known in the art. The present description provides non-limiting examples of methods for producing humanized antibodies.

As used herein, the term "single chain antibody" refers to a single polypeptide containing at least two immunoglobulin variable domains (eg, a mammalian immunoglobulin heavy chain or light chain variable domain) capable of specifically binding to an antigen. The present description provides non-limiting examples of single chain antibodies.

As used herein, the term "multimeric antibody" refers to an antibody containing four or more (eg, six, eight, or ten) immunoglobulin variable domains. In some embodiments, the multimeric antibody can cross-link one target molecule (eg, PD-1) with at least one second target molecule (eg, CTLA-4) on the surface of a mammalian cell (eg, human T cells).

Within the scope of the invention, the terms "individual" and "patient" are used interchangeably throughout the present description, they mean an animal, human or non-human animal, which is subjected to treatment with the methods of the present invention. The present invention provides for veterinary and non-veterinary use. Human patients may be adults or adolescents (eg, people less than 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g. marmosets, chimpanzees, gorillas, etc.), rodents (e.g. rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g. common pigs, miniature pigs), horses, dogs, cats, cows and other domestic, farm and zoo animals.

In the context of the invention, in relation to an antibody, the phrases "specifically binding" and "specifically binding" mean that the antibody preferentially interacts with the target molecule (eg, PD-1) relative to other molecules, t. the interaction depends on the presence of a specific structure (ie, antigenic determinant or epitope) on the target molecule; in other words, the reagent recognizes and binds molecules that include a specific structure, and not all molecules in general. An antibody that specifically binds to a target molecule may be referred to as a target-specific antibody. For example, an antibody that specifically binds to a PD-1 molecule may be referred to as a PD-1 specific antibody or an anti-PD-1 antibody.

Within the scope of the invention, the terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.

Within the scope of the invention, the terms "polynucleotide", "nucleic acid molecule", and "nucleic acid sequence" are used interchangeably herein to refer to polymers of nucleotides of any length, at least two nucleotides, and include, but are not limited to, DNA, RNA , DNA/RNA hybrids and their modifications.

Unless otherwise indicated, all technical and scientific terms used in the present description have the meaning generally understood by a person skilled in the art to which the invention belongs. The present description provides methods and materials for use in the present invention, but other suitable methods and materials known in this field can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries and other references mentioned in the present description are incorporated herein by reference in their entirety. In case of conflict, the present description, including definitions, shall take precedence.

Other features and advantages of the invention will be apparent from the following detailed description and figures, as well as the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a diagram showing the first part of an exemplary method for producing anti-hPD-1 antibodies.

Fig. 2 is a diagram showing the second part of an exemplary method for producing anti-hPD-1 antibodies.

Fig. 3 is a set of flow cytometry diagrams showing that anti-hPD-1 antibodies block binding of hPD-1 to hPD-L1.

Fig. 4 is a set of graphs showing flow cytometry results of anti-hPD-1 antibody cross-reactivity with monkey PD-1 (rmPD-1), mouse PD-1 (mPD-1), and human-mouse chimeric PD-1 ( chiPD-1). "NC" means negative control.

Fig. 5 is a graph showing the results of surface plasmon resonance (SPR) using a chimeric anti-hPD-1 antibody 1A7-mHvKv-IgG4-S228P and human PD-1.

Fig. 6 is a graph showing the results of surface plasmon resonance (SPR) using the chimeric anti-hPD-1 antibody 3F1-mHvKv-IgG4-S228P and human PD-1.

Fig. 7 is a graph showing body weight over time in humanized PD-1 (B-hPD-1) mice with MC-38 tumor cells treated with mouse anti-hPD-1 antibodies and Keytruda®. "PS" stands for physiological saline.

Fig. 8 is a graph showing the percentage change in body weight over time in humanized PD-1 (B-hPD-1) mice with MC-38 tumor cells treated with mouse anti-hPD-1 and Keytruda® antibodies. "PS" stands for physiological saline.

Fig. 9 is a graph showing tumor size over time in humanized PD-1 (B-hPD-1) mice with MC-38 tumor cells treated with mouse anti-hPD-1 and Keytrud® antibodies. "PS" stands for physiological saline.

Fig. 10 is a graph showing body weight over time in humanized PD-1 (B-hPD-1) mice with MC-38 tumor cells treated with chimeric anti-hPD-1 antibodies and Keytruda®. "PS" stands for physiological saline.

Fig. 11 is a graph showing the percentage change in body weight over time in humanized PD-1 (B-hPD-1) mice with MC-38 tumor cells treated with chimeric anti-hPD-1 and Keytrud® antibodies. "PS" stands for physiological saline.

Fig. 12 is a graph showing tumor size over time in humanized PD-1 (B-hPD-1) mice with MC-38 tumor cells treated with chimeric anti-hPD-1 antibodies and Keytruda®. "PS" stands for physiological saline.

Fig. 13 is a graph showing body weight over time in humanized PD-1 (B-hPD-1) mice with MC-38 tumor cells treated with humanized anti-hPD-1 antibodies and Keytruda®. "PS" stands for physiological saline.

Fig. 14 is a graph showing the percent change in body weight over time in humanized PD-1 (B-hPD-1) mice with MC-38 tumor cells treated with humanized anti-hPD-1 and Keytrud® antibodies. "PS" stands for physiological saline.

Fig. 15 is a graph showing tumor size over time in humanized PD-1 (B-hPD-1) mice with MC-38 tumor cells treated with humanized anti-hPD-1 antibodies and Keytruda®. "PS" stands for physiological saline.

In FIG. 16 shows the CDR sequences of mouse anti-hPD-1 antibodies (25-1A7, 18-3F1, 3-6G1) and the CDR sequences of humanized anti-hPD-1 antibodies determined using Kabat numbering.

In FIG. 17 shows the CDR sequences of mouse anti-hPD-1 antibodies (25-1A7, 18-3F1, 3-6G1) and the CDR sequences of humanized anti-hPD-1 antibodies determined using Chothia numbering.

In FIG. 18 shows the amino acid sequences of human PD-1 (hPD-1), mouse PD-1 (mPD-1), monkey PD-1 (rmPD-1), and chimeric PD-1 (chiPD-1).

In FIG. 19 shows the amino acid sequences of the heavy chain variable regions and the light chain variable regions of humanized anti-hPD-1 antibodies.

In FIG. 20 shows the amino acid sequence of the heavy chain variable regions and the light chain variable regions of mouse antibodies against hPD-125-1A7, 18-3F1 and 3-6G1.

DETAILED DESCRIPTION

The present invention relates to exemplary antibodies and antigen-binding fragments thereof that bind to PD-1 (programmed cell death protein 1; also known as CD279).

PD-1 and malignancies

The immune system can distinguish between normal body cells and cells that it recognizes as "foreign", which allows the immune system to attack foreign cells while leaving normal cells intact. Sometimes this mechanism involves proteins called immune checkpoints. Immune checkpoints are molecules in the immune system that turn on (costimulatory molecules) or turn off a signal.

Checkpoint inhibitors can prevent the immune system from attacking normal tissue and thus prevent autoimmune diseases. Many tumor cells also express checkpoint inhibitors. These tumor cells elude immune surveillance by engaging some of the immune checkpoint pathways, particularly in T cells that are specific for tumor antigens (Creelan, Benjamin C. "Update on immune checkpoint inhibitors in lung cancer." Cancer Control 21, 1 (2014): 80-89). Because many immune checkpoints are initiated by ligand-receptor interactions and can be easily blocked by antibodies against the ligands and/or their receptors.

PD-1 (programmed cell death protein-1) is an immune checkpoint and protects against autoimmunity through a dual mechanism of stimulating apoptosis (programmed cell death) in antigen-specific T cells in the lymph nodes while reducing apoptosis in regulatory T cells (anti-inflammatory). , suppressor T cells).

PD-1 is mainly expressed on the surface of T cells and primary B cells; two PD-1 ligands (PD-L1 and PD-L2) are widely expressed on antigen presenting cells (APCs). The interaction of PD-1 with its ligands plays an important role in the negative regulation of the immune response. Inhibition of binding between PD-1 and its ligand can make tumor cells susceptible to the cytolytic effect of the immune system, and thus can achieve the effect of killing tumor tissues and treating malignant neoplasms.

PD-L1 is expressed on neoplastic cells from many different cancers. As a result of PD-1 binding on T cells leading to their inhibition, PD-L1 expression is the primary mechanism by which tumor cells can elude immune surveillance. Overexpression of PD-L1, in essence, can be the result of 2 mechanisms, natural and adaptive. Natural expression of PD-L1 on malignant cells is associated with cellular/genetic aberrations in these neoplastic cells. Activation of cellular signaling, including the AKT and STAT pathways, results in increased expression of PD-L1. In primary mediastinal B-cell lymphoma, MHC class II transactivator (CIITA) genes are fused to either PD-L1 or PD-L2, resulting in overexpression of these proteins. Amplification of chromosome 9p23-24, on which PD-L1 and PD-L2 are localized, leads to increased expression of both proteins in classical Hodgkin's lymphoma. The adaptive mechanism is associated with the induction of PD-L1 expression in the tumor microenvironment. PD-L1 can be induced on neoplastic cells in response to interferon γ. In colon cancer with microsatellite instability, PD-L1 is mainly expressed on myeloid cells in tumors, which then suppress the function of cytotoxic T cells.

The use of PD-1 blockade to enhance antitumor immunity originates from an observation in models of chronic infections where preventing PD-1 interactions reverses T-cell depletion. Similarly, blockade of PD-1 prevents PD-1 T cells/PD-L1 tumor cells or PD-1 T cells/PD-L2 tumor cells from interacting, resulting in restoration of T-cell mediated antitumor immunity.

A detailed description of PD-1 and the use of anti-PD-1 antibodies for the treatment of malignancies is given, for example, in Topalian, Suzanne L., et al. "Safety, activity, and immune correlates of anti-PD-1 antibody in cancer." New England Journal of Medicine 366.26 (2012): 2443-2454; Hirano, Fumiya, et al. "Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity." Cancer research 65.3 (2005): 1089-1096; Raedler, Lisa A. "Keytruda (pembrolizumab): first PD-1 inhibitor approved for previously treated unresectable or metastatic melanoma." American health & drug benefits 8.Spec Feature (2015): 96; Kwok, Gerry, et al. "Pembrolizumab (Keytruda)." (2016): 2777-2789; US Patent Application No. 20170247454, US Patent No. 9834606 B and US Patent No. 8728474; each of which is incorporated herein by reference in its entirety.

The present invention relates to several anti-PD-1 antibodies, antigen-binding fragments thereof, and methods of using these anti-PD-1 antibodies and antigen-binding fragments to inhibit tumor growth and treat malignancies.

Antibodies and antigen-binding fragments

The present invention relates to antibodies against PD-1 and their antigennegative fragments. Basically, antibodies (also called immunoglobulins) consist of two classes of polypeptide chains - light chains and heavy chains. A non-limiting antibody of the present invention may be an intact antibody of four immunoglobulin chains, including two heavy chains and two light chains. The heavy chain of an antibody may be of any isotype, including IgM, IgG, IgE, IgA, or IgD, or subisotype, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain may be a kappa light chain or a lambda light chain. The antibody may contain two identical copies of the light chain and two identical copies of the heavy chain. The heavy chains, each containing one variable domain (or variable region, V _H ) and a plurality of constant domains (or constant regions), are linked to each other via a disulfide bond between their constant domains to form an antibody stem. Light chains, each of which contains one variable domain (or variable region, V _L ) and one constant domain (or constant region), each of which is associated with one heavy chain through a disulfide bond. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is linked. The variable regions of light chains and heavy chains contain three hypervariable regions located between the more conserved framework regions (FRs).

These hypervariable regions, known as complementarity determining regions (CDRs), form loops containing the main antigen-binding surface of an antibody. The four framework regions primarily adopt the beta sheet conformation and the CDRs form loops connecting the beta sheet structure and in some cases forming part of it. The CDRs in each strand are held in close proximity by the framework regions and, together with the CDRs of the other strand, participate in the formation of the antigen-binding region.

Methods for identifying CDR regions of an antibody by analyzing the amino acid sequence of an antibody are well known, and a number of CDR definitions are widely used. The Kabat definition is based on sequence variability and the Chothia definition is based on the localization of structural loop regions. These methods and definitions are described, for example, in Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan, et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular immunology 45,14 (2008): 3832-3839; Wu, T.T. and Kabat, E.A. (1970) J. Exp. Med. 132:211-250; Martin et al., Methods Enzymol. 203:121-53 (1991); Morea et al., Biophys Chem. 68(1-3):9-16 (Oct. 1997); Morea et al., J Mol Biol. 275(2):269-94 (Jan. 1998); Chothia et al., Nature 342(6252):877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each of which is incorporated herein by reference in its entirety. Unless specifically stated otherwise in the present description, Kabat numbering is used by default in the present invention.

CDRs are important for antigen epitope recognition. As used herein, an "epitope" is the smallest portion of a target molecule capable of being specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope may be approximately three, four, five, six, or seven amino acids, but these amino acids may not be in the sequential linear sequence of the antigen's primary structure, as the epitope may depend on the three-dimensional configuration of the antigen, taking into account its secondary and tertiary structure.

In some embodiments, the antibody is an intact immunoglobulin molecule (eg, IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3 and IgG4) are highly conserved, distinguished by their constant region, in particular hinge regions and upper CH2 domains. The sequences and distinctions of IgG subclasses are known in the art and are described, for example, in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions." Frontiers in immunology 5 (2014); Irani, et al. "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases." Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

The antibody may also be an immunoglobulin molecule derived from any species (eg, human, rodent, mouse, camelid). The antibodies provided herein also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific antibodies, and chimeric antibodies comprising an immunoglobulin binding domain fused to another polypeptide. The term "antigen-binding domain" or "antigen-binding fragment" means the portion of an antibody that retains the specific binding activity of an intact antibody, i.e. any portion of an antibody capable of specifically binding to an epitope on an intact antibody target molecule. It includes, for example, Fab, Fab', F(ab')2 and variants of these fragments. Thus, in some embodiments, an antibody or antigen-binding fragment thereof may be, for example, a scFv, Fv, Fd, dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a single chain antibody molecule, a multispecific antibody formed from antibody fragments, and any a polypeptide comprising a binding domain that is an antibody binding domain or is homologous to an antibody binding domain. Non-limiting examples of antigen binding domains include, for example, heavy chain and/or light chain CDRs of an intact antibody, heavy and/or light chain variable regions of an intact antibody, full-length heavy or light chains of an intact antibody, or a single CDR from an intact antibody heavy chain or light chain.

In some embodiments, the antigen-binding fragment may form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptors are single chain variable fragment (scFv) fusion proteins as described herein, fused to the transmembrane domain and the CD3 zeta endodomain. In some embodiments, the chimeric antigen receptor also contains intracellular signaling domains from various co-stimulatory protein receptors (eg, CD28, 41BB, ICOS). In some embodiments, the implementation of the chimeric antigen receptor contains multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to increase activity. Thus, in one aspect, the present invention further provides cells (eg, T cells) expressing the chimeric antigen receptors described herein.

In some embodiments, the scFv comprises one heavy chain variable domain and one light chain variable domain.

Anti-PD-1 antibodies and antigen-binding fragments

The present invention relates to antibodies and their antigen-binding fragments that specifically bind to PD-1. The antibodies and antigen-binding fragments provided herein can bind to PD-1 and can stimulate the PD-1 signaling pathway, thereby enhancing the immune response. The present invention relates, for example, to mouse anti-PD-1 antibodies 25-1A7 ("1A7"), 18-3F1 ("3F1") and 3-6G1 ("6G1"), their chimeric antibodies, and their humanized antibodies (e.g., antibodies presented in table 2).

The sequences of the CDRs of 1A7 and of antibodies derived from 1A7 (e.g., humanized antibodies) include the heavy chain variable domain CDRs, SEQ ID NOs: 1-3, and the light chain variable domain CDRs, SEQ ID NOs: 4-6, determined using Kabat numbering. . CDR can also be determined by the Chothia system. When numbered according to Chothia, the CDR sequences of the heavy chain variable domain are given in SEQ ID NOs: 16, 17, 3 and the CDR sequences of the light chain variable domain are given in SEQ ID NOs: 4-6.

Similarly, the sequences of the CDRs of 3F1 and 3F1-derived antibodies include the heavy chain variable domain CDR, SEQ ID NO:7-9, and the light chain variable domain CDR, SEQ ID NO:10-12, determined using Kabat numbering. When numbered according to Chothia, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 18, 19, 9, and the light chain variable domain CDRs are shown in SEQ ID NOs: 10-12.

The sequences of the CDRs of 6G1 and 6G1 derived antibodies include the heavy chain variable CDRs, SEQ ID NOs: 7, 13, 14, and the light chain variable CDRs, SEQ ID NOs: 10, 11, 15, determined using Kabat numbering. When numbered according to Chothia, the sequences of the CDRs of the heavy chain variable domain are given in SEQ ID NOS: 20, 21, 14, and the CDRs of the light chain variable domain are given in SEQ ID NOS: 10, 11, 15.

The present invention also relates to the amino acid sequences of the heavy chain variable regions and the light chain variable regions of humanized antibodies. Because there are many ways to humanize a mouse antibody (eg, the sequence can be modified with different amino acid substitutions), the heavy chain and light chain of an antibody can have more than one version of the humanized sequences. The amino acid sequences of the heavy chain variable regions of the humanized 1A7 antibody are shown in SEQ ID NOS:26-28. The amino acid sequences of the light chain variable regions of the humanized 1A7 antibody are shown in SEQ ID NOS:29-31. Any of these heavy chain variable region sequences (SEQ ID NO:26-28) can be paired with any of these light chain variable region sequences (SEQ ID NO:29-31).

Similarly, the amino acid sequences of the heavy chain variable region of the humanized 3F1 antibody are shown in SEQ ID NOS:32-35. The amino acid sequences of the light chain variable region of the humanized 3F1 antibody are shown in SEQ ID NOS:36-38. Any of these heavy chain variable region sequences (SEQ ID NOs:32-35) can be paired with any of these light chain variable region sequences (SEQ ID NOs:36-38).

As shown in FIG. 19, the term "percent humanization" means the percentage sequence identity of a heavy chain or light chain variable region compared to human antibody sequences in the International Immunogenetics Information System (IMGT) database. The term "best match" means that the sequence of the heavy chain or light chain variable region is closer to a particular species than to another. For example, the term "best match" to a human means that the sequence is closer to a human than to another species. The best match to human and Macaca fascicularis means that the sequence has the same percent identity with respect to human and Macaca fascicularis sequences, and these percent identity is the highest relative to sequences of other species. In some embodiments, the percentage of humanization is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%. A detailed description regarding the determination of the percentage of humanization and the determination of the best matches is known in this field and is presented, for example, in Jones, Tim D., et al. "The INNs and outs of antibody nonproprietary names." MAbs. Vol. 8. No. 1. Taylor & Francis, 2016, incorporated herein by reference in its entirety. Often, a high percentage of humanization has various advantages, for example, the antibody is safer and more effective in humans, more likely to be well tolerated in humans, and/or less likely to have side effects.

In addition, in some embodiments, the antibodies or antigen-binding fragments provided herein may also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 1-3, SEQ ID NOs: 7-9 , SEQ ID NO: 7, 13, 14, SEQ ID NO: 16, 17, 3, SEQ ID NO: 18, 19, 9, and SEQ ID NO: 20, 21, 14; and/or one, two or three light chain variable region CDRs selected from the group of SEQ ID NOS: 4-6, SEQ ID NOS: 10-12 and SEQ ID NOS: 10, 11, 15.

In some embodiments, the antibodies may comprise a heavy chain variable region (VH) containing complementarity determining regions (CDRs) 1, 2, 3, where the CDR1 region contains or consists of an amino acid sequence that is at least 80%, 85%, 90 % or 95% identical with respect to the selected amino acid sequence of the CDR1 VH, the CDR2 region contains or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical with respect to the selected amino acid sequence of the CDR2 VH, and the CDR3 region contains or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical with respect to the selected amino acid sequence to a VH CDR3 and a light chain variable region (VL) containing CDRs 1, 2, 3, where the CDR1 region contains or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical with respect to the selected amino acid CDR1 VL foreign acid sequence, the CDR2 region contains or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical with respect to the selected amino acid sequence of the VL CDR2, and the CDR3 region contains or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical with respect to the selected amino acid sequence of the VL CDR3. Selected amino acid sequences of VH CDRs 1, 2, 3 and selected amino acid sequences of VL CDRs 1, 2, 3 are shown in FIG. 16 (CDR by Kabat) and FIG. 17 (CDR by Chothia).

In some embodiments, an antibody or antigen-binding fragment provided herein may comprise a heavy chain variable domain comprising one, two, or three CDRs of SEQ ID NO:1 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:2 with zero, one or two insertions, deletions or amino acid substitutions; SEQ ID NO:3 with zero, one or two insertions, deletions or amino acid substitutions.

In some embodiments, an antibody or antigen-binding fragment provided herein may comprise a heavy chain variable domain comprising one, two, or three CDRs of SEQ ID NO:7 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:8 with zero, one or two insertions, deletions or amino acid substitutions; SEQ ID NO:9 with zero, one or two insertions, deletions or amino acid substitutions.

In some embodiments, an antibody or antigen-binding fragment provided herein may comprise a heavy chain variable domain comprising one, two, or three CDRs of SEQ ID NO:7 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:13 with zero, one or two insertions, deletions or amino acid substitutions; SEQ ID NO:14 with zero, one or two amino acid insertions, deletions or substitutions.

In some embodiments, an antibody or antigen-binding fragment provided herein may comprise a heavy chain variable domain comprising one, two, or three CDRs of SEQ ID NO:16 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:17 with zero, one or two insertions, deletions or amino acid substitutions; SEQ ID NO:3 with zero, one or two insertions, deletions or amino acid substitutions.

In some embodiments, an antibody or antigen-binding fragment provided herein may comprise a heavy chain variable domain comprising one, two, or three CDRs of SEQ ID NO:18 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:19 with zero, one or two insertions, deletions or amino acid substitutions; SEQ ID NO:9 with zero, one or two insertions, deletions or amino acid substitutions.

In some embodiments, an antibody or antigen-binding fragment provided herein may comprise a heavy chain variable domain comprising one, two, or three CDRs of SEQ ID NO:20 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:21 with zero, one or two insertions, deletions or amino acid substitutions; SEQ ID NO:14 with zero, one or two amino acid insertions, deletions or substitutions.

In some embodiments, an antibody or antigen-binding fragment provided herein may comprise a light chain variable domain comprising one, two, or three CDRs of SEQ ID NO:4 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:5 with zero, one or two insertions, deletions or amino acid substitutions; SEQ ID NO:6 with zero, one or two amino acid insertions, deletions or substitutions.

In some embodiments, an antibody or antigen-binding fragment provided herein may comprise a light chain variable domain comprising one, two, or three CDRs of SEQ ID NO:10 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:11 with zero, one or two insertions, deletions or amino acid substitutions; SEQ ID NO:12 with zero, one or two amino acid insertions, deletions or substitutions.

In some embodiments, an antibody or antigen-binding fragment provided herein may comprise a light chain variable domain comprising one, two, or three CDRs of SEQ ID NO:10 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:11 with zero, one or two insertions, deletions or amino acid substitutions; SEQ ID NO:15 with zero, one or two amino acid insertions, deletions or substitutions.

Insertions, deletions and substitutions may be within the CDR sequence or at one or both ends of the CDR sequence.

The present invention also relates to antibodies, or antigen-binding fragments thereof, that bind to PD-1. Antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) containing or consisting of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical with respect to the selected VH sequence, and a light chain variable region ( VL) containing or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical with respect to the selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO:26, 27, 28, or 39, and the selected VL sequence is SEQ ID NO:29, 30, 31, or 40. In some embodiments, the selected VH sequence is SEQ ID NO: 32, 33, 34, 35, or 41, and the selected VL sequence is SEQ ID NO: 36, 37, 38, or 42. In some embodiments, the selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is is SEQ ID NO:44.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, sequences are aligned for optimal comparison (e.g., gaps in one or both of the first and second amino acid or nucleic acid sequences can be included for optimal alignment, and non-homologous sequences can be neglected for comparison purposes). ). The length of the reference sequence fragment aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100%. The amino acid residues or nucleotides at the respective amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that must be included to optimally align the two sequences. For purposes of the present invention, sequence comparisons and determination of percent identity between two sequences can be performed using a Blossum 62 weight matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

The present invention also relates to a nucleic acid containing a polynucleotide encoding a polypeptide containing an immunoglobulin heavy chain or an immunoglobulin light chain. An immunoglobulin heavy chain or immunoglobulin light chain contains the CDRs shown in FIG. 16 or FIG. 17 or have the sequences shown in FIG. 19 or FIG. 20. When polypeptides are paired with a corresponding polypeptide (eg, corresponding heavy chain variable region or corresponding light chain variable region), the paired polypeptides bind to PD-1 (eg, human PD-1).

Anti-PD-1 antibodies and antigen binding fragments can also be variants (including derivatives and conjugates) of antibodies or antibody fragments and multispecific (eg, bispecific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multispecific antibodies (multimeric, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single chain antibodies, intracellularly produced antibodies (i.e., intrabodies ) and their antigen-binding fragments. Antibodies or antigen-binding fragments thereof may be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.

Antibody fragments are suitable for use in the methods of the present invention provided that they retain the desired affinity and specificity of the full length antibody. Thus, an antibody fragment that binds to PD-1 will retain the ability to bind to PD-1. An Fv fragment is an antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain closely linked, eg covalently, eg in scFv. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs, or a subset thereof, confer antigen-binding specificity on the antibody. However, even a single variable domain (or half of the Fv containing only three CDRs specific for an antigen) may have the ability to recognize and bind an antigen, although typically at a lower affinity than the entire binding site.

Single chain Fv antibody fragments (or scFv) contain the VH and VL domains (or regions) of the antibody, where these domains are in the same polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains to allow the scFv to form the desired structure for antigen binding.

The Fab fragment contains the variable and constant domain of the light chain and the variable domain and the first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments contain a pair of Fab fragments, typically covalently linked near their carboxy ends with hinged cysteines in between. Other chemistries of antibody fragments are also known in the art.

Diabodies are small antibody fragments with two antigen binding sites, where the fragments contain a VH connected to a VL in the same polypeptide chain (VH and VL). Using a linker that is too short to allow pairing of two domains on the same strand, the domains are forced to pair with complementary domains on the other strand and two antigen-binding sites are obtained.

Linear antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1) which together with complementary light chain polypeptides form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific.

The antibodies and antibody fragments of the present invention can be modified in the Fc region to achieve desired effector functions or serum half-life.

Multimerization of antibodies can be achieved using natural antibody aggregation or chemical or recombinant coupling methods known in the art. For example, a certain percentage of purified antibody preparations (eg, purified IgG ₁ molecules) spontaneously form protein aggregates containing antibody homodimers and other higher order antibody multimers.

Alternatively, antibody homodimers can be prepared by chemical coupling methods known in the art. For example, heterobifunctional cross-linkers can be used to prepare antibody multimers, including, but not limited to, SMCC (succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate) and SATA (N-succinimidyl-S-acetylthio-acetate). An example of a method for producing antibody homodimers is described in Ghetie et al. ( Proc. Natl. Acad. Sci. USA 94: 7509-7514, 1997). Antibody homodimers can be converted to Fab' ₂ homodimers by pepsin digestion. Another way to obtain antibody homodimers is to use the autophilic T15 peptide described by Zhao et al. ( J. Immunol. 25:396-404, 2002).

In some embodiments, the multispecific antibody is a bispecific antibody. Bispecific antibodies can be generated by designing a contact region between a pair of antibody molecules to maximize the percentage of heterodimers recovered from a recombinant cell culture. For example, the contact region may contain at least a portion of the CH3 domain of the constant domain of the antibody. In this method, one or more amino acids with small side chains from the interface of the first antibody molecule are replaced with amino acids with larger side chains (eg, tyrosine or tryptophan). At the site of contact of the second antibody molecule, compensatory "cavities" of identical or similar size for large side chains are created by replacing amino acids with large side chains with amino acids with smaller side chains (eg, alanine or threonine). This is a mechanism to increase the yield of the heterodimer relative to other undesirable end products such as homodimers. This method is described, for example, in WO 96/27011, incorporated herein by reference in its entirety.

Bispecific antibodies include cross-linked or "heteroconjugated" antibodies. For example, one of the antibodies in the heteroconjugate can be bound to avidin and the other to biotin. Heteroconjugated antibodies can also be made by any convenient cross-linking methods. Suitable cross-linking agents and methods for cross-linking are well known in the art and are described in US Pat. No. 4,676,980, incorporated herein by reference in its entirety.

Methods for producing bispecific antibodies from antibody fragments are also known in the art. For example, bispecific antibodies can be generated using a chemical compound. Brennan et al. ( Science 229:81, 1985) describe a method in which intact antibodies are proteolytically cleaved to obtain F(ab') ₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize the vicinal dithiols and prevent the formation of intermolecular disulfide bonds. The resulting Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the TNB-Fab' derivatives is then converted back to a thiol-Fab' by reduction with mercaptoethylamine and mixed with an equimolar amount of another TNB-Fab' derivative to form a bispecific antibody.

Any of the antibodies or antigen-binding fragments provided herein can be conjugated to a stabilizing molecule (eg, a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in the subject's body or in solution). Non-limiting examples of stabilizing molecules include: polymer (eg, polyethylene glycol) or protein (eg, serum albumin, such as human serum albumin). Conjugation of the stabilizing molecule may increase the half-life or prolong the biological activity of the antibody or antigen-binding fragment in vitro (eg, in tissue culture or when stored as a pharmaceutical composition) or in vivo (eg, in the human body).

In some embodiments, the antibodies or antigen-binding fragments provided herein can be conjugated to a therapeutic agent. An antibody-drug conjugate containing an antibody, or an antigen-binding fragment thereof, can be covalently or non-covalently linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine, maytansinoids such as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranololoo, puromycin, epirubicin, cyclophosphamide and their analogues).

Characteristics of antibodies

The antibodies or antigen-binding fragments thereof provided herein can block binding between PD-1 and PD-L1 and/or binding between PD-1 and PD-L2.

In some embodiments, by binding to PD-1, the antibody can inhibit the PD-1 signaling pathway and positively regulate the immune response. Thus, in some embodiments, the antibodies or antigen-binding fragments provided herein are PD-1 antagonists. In some embodiments, the antibodies, or antigen-binding fragments thereof, are PD-1 agonists.

In some embodiments, the antibodies or antigen-binding fragments provided herein can increase the immune response, activity, or number of T cells (e.g., CD8 ⁺ and/or CD4 ⁺ cells) by at least 10%, 20%, 30%. , 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 3 times, 5 times, 10 times or 20 times. In some embodiments, the antibodies or antigen-binding fragments provided herein can reduce the activity or number of T cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90%, 100%, 2 times, 3 times, 5 times, 10 times or 20 times.

In some embodiments, the antibody (or antigen-binding fragments thereof) specifically binds to PD-1 (e.g., human PD-1, monkey PD-1, mouse PD-1, and/or chimeric PD-1) with a dissociation rate (k _off ) of less than 0.1 s ^-1 , less than 0.01 s ^-1 , less than 0.001 s ^-1 , less than 0.0001 s ^-1 , or less than 0.00001 s ^-1 . In some embodiments, the dissociation rate (k _off ) is greater than 0.01 s ^-1 , greater than 0.001 s ^-1 , greater than 0.0001 s ^-1 , greater than 0.00001 s ^-1 , or greater than 0.000001 s ^-1 .

In some embodiments, the association rate (k _on ) is greater than 1x10 ² /Ms, greater than 1x10 ³ /Ms, greater than 1x10 ⁴ /Ms, greater than 1x10 ⁵ /Ms, or greater than 1x10 ⁶ /Ms . In some embodiments, the association rate (k _on ) is less than 1x10 ⁵ /Ms, less than 1x10 ⁶ /Ms, or less than 1x10 ⁷ /Ms.

Affinities can be derived from the ratio of kinetic rate constants (KD=k _off /k _on ). In some embodiments, the KD is less than 1×10 ^-6 M, less than 1×10 ^-7 M, less than 1×10 ^-8 M, less than 1×10 ^-9 M, or less than 1×10 ^-10 M. In some embodiments, the implementation KD is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is greater than ^{1x10 -7} M, greater than ^{1x10 -8} M, greater than ^{1x10 -9} M, greater than ^{1x10 -10} M, greater than ^{1x10 -11} M, or greater than 1x10 ^-12 M. In some embodiments, the antibody binds to human PD-1 with a KD of about 3 nM or less.

Common methods for measuring the affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the antibody binds to human PD-1 (SEQ ID NO:22), monkey PD-1 (e.g., rhesus monkey PD-1, SEQ ID NO:24), chimeric PD-1 (SEQ ID NO:25 ) and/or mouse PD-1 (SEQ ID NO:23). In some embodiments, the antibody does not bind to human PD-1 (SEQ ID NO:22), monkey PD-1 (e.g., rhesus monkey PD-1, SEQ ID NO:24; cynomolgus monkey PD-1), chimeric PD- 1 (SEQ ID NO:25) and/or mouse PD-1 (SEQ ID NO:23).

In some embodiments, thermal stability is determined. The antibodies or antigen-binding fragments provided herein may have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95°C.

Because IgG can be described as a multidomain protein, the melting curve sometimes shows two transitions with a first denaturation temperature Tm D1 and a second denaturation temperature Tm D2. The presence of these two peaks often indicates denaturation of the Fc domains (Tm D1) and Fab domains (Tm D2), respectively. If there are two peaks, the term "Tm" usually refers to Tm D2. Thus, in some embodiments, the antibodies or antigen-binding fragments provided herein have a Tm D1 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 , 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95°C. In some embodiments, the antibodies or antigen-binding fragments provided herein have a Tm D2 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95°C.

In some embodiments, Tm, Tm D1, Tm D2 are less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 , 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95°C.

In some embodiments, the antibody has a percent tumor growth inhibition (TGI%) greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120% , 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200%. In some embodiments, the antibody has a percent tumor growth inhibition of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200%. TGI% can be determined, for example, through 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the start of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. Within the scope of the invention, the percentage of tumor growth inhibition (TGI%) is calculated by the following formula:

TGI (%) = [1-(Ti-T0)/(Vi-V0)]×100

Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day zero. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day zero.

In some embodiments, the antibodies or antigen-binding fragments provided herein are PD-1 antagonists. In some embodiments, the antibodies or antigen-binding fragments reduce PD-1 signaling in a target cell expressing PD-1.

In some embodiments, the antibodies or antigen-binding fragments increase the function of CD4 ⁺ effector T cells, for example, by increasing the proliferation of CD4 ⁺ effector T cells and/or increasing the production of interferon-gamma by CD4 ⁺ effector T cells (for example, compared to proliferation and /or the production of cytokines before the introduction of antibodies or antigen-binding fragments). In some embodiments, the implementation of the cytokine is interferon-gamma. In some embodiments, antibodies or antigen-binding fragments increase the number of intratumoral (infiltrating) CD4 ⁺ effector T cells (e.g., total number of CD4 ⁺ effector T cells, or, e.g., percentage of CD4 ⁺ cells among CD45 ⁺ cells), e.g., compared to the number of intratumoral (infiltrating) CD4 ⁺ T cells before the introduction of antibodies or antigen-binding fragments. In some embodiments, the antibodies or antigen-binding fragments increase the number of intratumoral (infiltrating) CD4 ⁺ effector T cells expressing interferon-gamma (e.g., all CD4 ⁺ interferon-gamma expressing cells, or, for example, the percentage of interferon-gamma-expressing CD4 ⁺ cells among all CD4 ⁺ cells), for example, compared with the number of intratumoral (infiltrating) CD4 ⁺ T cells expressing interferon-gamma before administration.

In some embodiments, antibodies or antigen-binding fragments increase the number of intratumoral (infiltrating) CD8 ⁺ effector T cells (e.g., total number of CD8 ⁺ effector T cells or, e.g., percentage of CD8 ⁺ cells among CD45 ⁺ cells), e.g., compared to the number of intratumoral (infiltrating) CD8 ⁺ effector T cells before administration. In some embodiments, antibodies or antigen-binding fragments increase the number of intratumoral (infiltrating) CD8 ⁺ effector T cells expressing interferon-gamma (e.g., the percentage of CD8 ⁺ cells expressing interferon-gamma among all CD8 ⁺ cells), for example, compared to the number of intratumoral (infiltrating) CD8 ⁺ T cells expressing interferon-gamma, before the introduction of antibodies against human PD-1.

In some embodiments, the antibodies or antigen-binding fragments increase memory T cell function, for example, by increasing the proliferation of memory T cells and/or increasing the production of cytokines (eg, interferon-gamma) by memory cells.

In some embodiments, the antibodies or antigen-binding fragments inhibit T _reg function, for example, by reducing T _reg suppression of effector T cell function (eg, effector T cell proliferation and/or effector T cell secretion of cytokines). In some embodiments, the effector T cell is a CD4 ⁺ effector T cell. In some embodiments, the antibodies or antigen-binding fragments reduce the number of intratumoral (infiltrating) T _regs (eg, the total number of T _regs or, for example, the percentage of Fox3p ⁺ cells among CD4 ⁺ cells).

In some embodiments, the antibodies or antigen-binding fragments are an anti-hPD-1 depleting antibody (eg, it depletes cells expressing human PD-1). In some embodiments, the antibodies or antigen-binding fragments deplete human PD-1 expressing cells in vitro . In some embodiments, the human PD-1 expressing cells are CD4 ⁺ effector T cells or T _reg cells. In some embodiments, depletion occurs via ADCC and/or phagocytosis.

In some embodiments, the antibodies or antigen-binding fragments have a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis. In some embodiments, the Fc region is from human IgG1, human IgG2, human IgG3, or human IgG4.

In some embodiments, the antibodies or antigen-binding fragments do not induce apoptosis of PD-1 expressing cells (eg, T _reg ).

In some embodiments, the antibodies or antigen-binding fragments do not contain a functional Fc region. For example, antibodies or antigen binding fragments are Fab, Fab', F(ab')2 and Fv fragments.

Methods for obtaining antibodies against PD-1

An isolated fragment of human PD-1 can be used as an immunogen to generate antibodies by standard methods for producing polyclonal and monoclonal antibodies. Polyclonal antibodies can be induced in animals by multiple injections (eg, subcutaneous or intraperitoneal injections) of the antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic to the species to be immunized. Animals can be injected with the antigenic peptide or protein more than once (eg, two, three or four times).

As immunogens, you can use a full-length polypeptide or protein, or, alternatively, their antigenic peptide fragments. An antigenic peptide of a protein contains at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues from the PD-1 amino acid sequence and includes an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein . As described above, the full length sequence of human PD-1 is known in the art (SEQ ID NO:22).

An immunogen is typically used to generate antibodies by immunizing a suitable individual (eg, a human or a transgenic animal expressing at least one human immunoglobulin locus). A suitable immunogenic preparation may comprise, for example, a recombinantly expressed or chemically synthesized polypeptide (eg, a fragment of human PD-1). The formulation may further include an adjuvant such as complete or incomplete Freund's adjuvant, or a similar immunostimulatory agent.

Polyclonal antibodies can be obtained as described above by immunizing a suitable individual with a PD-1 polypeptide or antigenic peptide (eg, part of PD-1) as an immunogen. The antibody titer of an immunized individual can be monitored over time by standard methods such as enzyme-linked immunosorbent assay (ELISA) using an immobilized polypeptide or PD-1 peptide. If desired, antibody molecules can be isolated from a mammal (eg, blood) and further purified by well-known methods such as protein A or protein G chromatography to obtain an IgG fraction. At an appropriate time after immunization, for example when specific antibody titers are highest, antibody-producing cells can be obtained from the individual and used to generate monoclonal antibodies by standard methods such as the hybridoma method originally described by Kohler et al. ( Nature 256:495-497, 1975), human B cell hybridoma method (Kozbor et al., Immunol. Today 4:72, 1983), EBV hybridoma method (Cole et al., Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, Inc., pp. 77-96, 1985) or trioma methods. The technology for producing hybridomas is well known (see, in general, Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY). Monoclonal antibody-producing hybridoma cells are determined by screening hybridoma culture supernatants for antibodies binding to the polypeptide or epitope of interest, for example, using a standard ELISA assay.

Variants of the antibodies or antigen-binding fragments provided herein can be obtained by making appropriate nucleotide changes to the DNA encoding a human antibody, humanized antibody, or chimeric antibody or antigen-binding fragment thereof provided herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues in the amino acid sequences constituting the antigen-binding site of an antibody or antigen-binding domain. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein, such as PD-1. Any combination of deletions, insertions and/or combinations can be used to produce an antibody or antigen-binding fragment thereof having increased binding affinity for the target. Amino acid changes made to an antibody or antigen binding fragment may also change or introduce new post-translational modifications to the antibody or antigen binding fragment, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence in such a way that enzymes present in the cell will attach another sugar) or insert new glycosylation sites.

The antibodies provided herein can be obtained from any animal species, including mammals. Non-limiting examples of native antibodies include those derived from humans, primates, e.g., marmosets and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters). and rabbits), including transgenic rodents genetically engineered to produce human antibodies.

Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as derived from) human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations inserted by in vitro random or site-directed mutagenesis or by in vivo somatic mutation), for example, in CDRs.

A humanized antibody typically has a human framework (FR) onto which non-human CDRs are grafted. Thus, a humanized antibody contains one or more amino acid sequences inserted into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, typically taken from an "import" variable domain. Humanization can be accomplished essentially, for example, by replacing the rodent CDR sequences with corresponding sequences from a human antibody. These methods are described, for example, in Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al. Science 239:1534-1536 (1988); each of which is incorporated herein by reference in its entirety. Thus, "humanized" antibodies are chimeric antibodies where the substantially smaller than intact human V domain is replaced with the corresponding non-human species sequence. In practice, humanized antibodies are typically mouse antibodies in which some CDR residues and some FR residues are replaced with residues from analogous regions in human antibodies.

The choice of human VH and VL domains to use in making humanized antibodies is very important to reduce immunogenicity. In the so-called "best match" method, the V domain sequence of a mouse antibody is screened against an entire library of known human domain sequences. The human sequence closest to the mouse sequence is then adopted as the human FR for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987)).

It is also important to humanize antibodies while maintaining high specificity and affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be generated by analyzing parental sequences and various conceptual humanized products using 3D models of parental and humanized sequences. Three-dimensional immunoglobulin models are publicly available and well known to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows one to analyze the possible role of the residues in the functioning of the candidate immunoglobulin sequence, i.e. analyze residues that affect the ability of a candidate immunoglobulin to bind to its antigen. Thus, FR residues from recipient and import sequences can be selected and combined to achieve the desired antibody characteristic, such as increased affinity for target antigens.

Typically, amino acid sequence variants of a human, humanized, or chimeric anti-PD-1 antibody will comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity in relation to the sequence present in the light or heavy chain of the parent antibody.

The term "identity" or "homology" with respect to a parent sequence generally means the percentage of amino acid residues present in a candidate sequence that are identical to the sequence present in a human, humanized, or chimeric anti-PD-1 antibody, or fragment thereof, after sequence alignment. and including gaps, if necessary, to achieve the maximum percent sequence identity without considering any conservative substitutions as part of the sequence identity.

Additional modifications can be made to the anti-PD-1 antibodies or antigen-binding fragments. For example, one can insert cysteine residues into the Fc region, thereby allowing an interchain disulfide bond to form in this region. The homodimeric antibody thus obtained may have an increased in vitro and/or in vivo half-life. Homodimeric antibodies with increased in vitro and/or in vivo half-life can also be made using heterobifunctional cross-linkers, as described, for example, in Wolff et al. ( Cancer Res. 53:2560-2565, 1993). Alternatively, an antibody can be engineered to have dual Fc regions (see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

In some embodiments, a covalent modification can be made to an anti-PD-1 antibody or antigen-binding fragment thereof. These covalent modifications can be carried out by chemical or enzymatic synthesis or by enzymatic or chemical cleavage. Other types of covalent modifications to an antibody or antibody fragment are introduced into the molecule by reacting the target amino acid residues in the antibody or fragment with an organic derivatization agent capable of reacting with selected side chains or N- or C-terminal residues.

In some embodiments, the present invention relates to antibody variants having a carbohydrate structure lacking a fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the Asn297 sugar chain relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 /077546. The term "Asn297" refers to an asparagine residue located approximately at position 297 in the Fc region (with Eu numbering of Fc region residues; or at position 314 with Kabat numbering); however, Asn297 can also be approximately ±3 amino acids upstream or downstream of position 297, ie. between positions 294 and 300, due to minor sequence variants in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of an antibody can be further engineered to replace the asparagine at position 297 with alanine (N297A).

In some embodiments, to improve production efficiency by eliminating Fab exchange, the antibody Fc region is further engineered by replacing the serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description regarding the S228 mutation is provided, for example, in Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preperation." Journal of Biological Chemistry 290.9 (2015): 5462-5469, incorporated herein by reference in its entirety.

Recombinant vectors

The present invention also relates to recombinant vectors (e.g., expression vectors) comprising an isolated polynucleotide as provided herein (e.g., a polynucleotide encoding a polypeptide as described herein), host cells into which the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or the vector containing the polynucleotide), and the production of recombinant antibody polypeptides or fragments thereof by recombinant methods.

As used herein, the term "vector" means any construct that can deliver one or more polynucleotides of interest to a host cell when the vector is inserted into the host cell. An "expression vector" can deliver and express one or more polynucleotides of interest as an encoded polypeptide in a host cell into which the expression vector is inserted. Thus, in an expression vector, a polynucleotide of interest is positioned for expression in the vector by operably linking it to regulatory elements such as a promoter, enhancer and/or poly-A tail in the vector or host cell genome at or near the insertion site of the polynucleotide of interest. it, or flanking it such that the polynucleotide of interest will be translated in the host cell into which the expression vector is inserted.

The vector can be inserted into the host cell by methods known in the art, for example, by electroporation, chemical transfection (eg, DEAE-dextran), transformation, transfection and infection, and/or transduction (eg, using a recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant virus), deproteinized DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

In some embodiments, a polynucleotide provided herein (e.g., a polynucleotide encoding a polypeptide provided herein) is inserted using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may include the use of a non-pathogenic (defective) ), a replication-competent virus, or it may use a replication-defective virus. In the case of the latter, virus replication will generally only occur in virus-matched packaging cells. Suitable systems are described, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. sci. USA 86:317-321; Flexner et al., 1989, Ann. N.Y. Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine 8:17-21; U.S. Patent Nos. 4,603,112, 4,769,330 and 5,017,487; WO 89/01973; US Pat. No. 4,777,127; GB 2200651; EP 0345242; WO 91/02805; Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. sci. USA 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. sci. USA 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res. 73:1202-1207. Methods for inserting DNA into such expression systems are well known to those skilled in the art. The DNA can also be "deproteinized" as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692. Uptake of deproteinized DNA can be enhanced by coating DNA on biodegradable particles that are efficiently transported by cells.

For expression, a DNA insert comprising an antibody-encoding or polypeptide-encoding polynucleotide provided herein may be operably linked to an appropriate promoter (e.g., a heterologous promoter), such as, but not limited to, the lambda phage PL promoter, promoters lac, trp and tacE. coli, SV40 early and late promoters, and retroviral LTR promoters. Other suitable promoters are known to those skilled in the art. Expression constructs may further comprise transcription initiation sites, transcription termination sites, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs may include a translation initiation site at the beginning of the polypeptide and a termination codon (UAA, UGA or UAG) appropriately located at the end of the polypeptide to be translated.

As indicated, expression vectors may include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance gene for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria. Typical non-limiting examples of suitable host cells include bacterial cells such as E. coli , Streptomyces , and Salmonella typhimurium cells; fungal cells such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma and HK 293 cells; and plant cells. Suitable culture media and conditions for the host cells provided herein are known in the art.

Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9 available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be apparent to those skilled in the art.

Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the SV40 early and late promoters, retroviral LTR promoters such as Rous sarcoma virus (RSV) promoters, and metallothionein promoters such as the mouse metallothionein-I promoter.

in yeastSaccharomyces cerevisiae you can use a number of vectors containing constitutive or inducible promoters, such as promoters of factor alpha, alcohol oxidase and PGH. For an overview, see Ausubelet al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y, and Grantet al., Methods Enzymol., 153:516-544 (1997).

Insertion of the construct into the host cell can be accomplished by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals such as Davis et al ., Basic Methods In Molecular Biology (1986), incorporated herein by reference in its entirety.

Transcription of DNA encoding an antibody of the present invention by higher eukaryotes can be increased by inserting an enhancer sequence into a vector. Enhancers are cis-acting DNA elements, typically about 10 to 300 bp, that increase the transcriptional activity of a promoter in a specified host cell type. Examples of enhancers include the SV40 enhancer located at the late origin of replication at base pair position 100-270, the cytomegalovirus early promoter-enhancer, the polyoma enhancer at the late origin of replication, and adenovirus enhancers.

Appropriate secretion signals can be inserted into the expressed polypeptide to secrete the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment. The signals may be endogenous to the polypeptide or may be heterologous signals.

The polypeptide (eg, antibody) may be expressed in a modified form, such as a fusion protein (eg, GST fusion protein), or with a histidine tag, and may include not only secretion signals, but also additional heterologous functional regions. For example, a region of additional amino acids, in particular charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell during purification or subsequent transport and storage. In addition, peptide residues can be added to the polypeptide to facilitate purification. Such regions may be removed prior to final production of the polypeptide. The addition of peptide residues to polypeptides for secretion or excretion, to improve stability, and to facilitate purification, among other things, are known and routine methods in this field.

Methods of treatment

The antibodies, or antibody, or antigen-binding fragments thereof of the present invention can be used for various therapeutic purposes. In one aspect, the present invention relates to methods for treating cancer in an individual, methods for reducing the rate of tumor expansion in an individual over time, methods for reducing the risk of metastasis, or methods for reducing the risk of an individual developing additional metastases. In some embodiments, the treatment can stop, slow, delay, or inhibit the progression of a cancer. In some embodiments, treatment may result in a reduction in the number, severity, and/or duration of one or more symptoms of cancer in an individual.

In one aspect, the present invention relates to methods comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment provided herein to an individual in need thereof (e.g., an individual having or is determined or diagnosed to have a malignant neoplasm) having, for example, breast cancer (eg, triple-negative breast cancer), carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, cancer pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, stomach cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hemoblastosis. In some embodiments, the cancer is inoperable melanoma or metastatic melanoma, non-small cell lung carcinoma (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the individual has a solid tumor. In some embodiments, the cancer is head and neck squamous cell carcinoma (SCCHN), renal cell carcinoma (RCC), triple negative breast cancer (TNBC), or colorectal carcinoma. In some embodiments, the individual has Hodgkin's lymphoma. In some embodiments, the individual has triple negative breast cancer (TNBC), gastric cancer, urothelial cancer, Merkel cell carcinoma, or head and neck cancer.

In some embodiments, the compositions and methods provided herein can be used to treat patients at risk of developing cancer. Cancer patients can be identified by various methods known in the art.

As used herein, the term "effective amount" means an amount or dose sufficient to produce beneficial or desired results, including stopping, slowing, halting or inhibiting the progression of a disease, such as cancer. The effective amount will vary depending on, for example, the age and body weight of the individual to whom the antibody is administered, the antigen-binding fragment encoding the antibody polynucleotide, the vector containing the polynucleotide, and/or their composition, the severity of the symptoms and the route of administration, and thus the administration can be determined individually.

An effective amount can be administered in one or more administrations. By way of example, an effective amount of an antibody or antigen-binding fragment is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay the progression of cancer in a patient, or an amount sufficient to improve, stop, stabilize, reverse, slow and/or delaying the proliferation of cells (eg, cells in a biopsy, any of the cancer cells provided herein, or a cell line (eg, a cancer cell line)) in vitro . As is known in the art, the effective amount of an antibody or antigen-binding fragment may vary, among other things, depending on the history of the patient, as well as other factors such as the type (and/or dose) of the antibody used.

Effective amounts and regimens for administering antibodies, antibody-encoding polynucleotides and/or compositions provided herein can be determined empirically, as is known to those skilled in the art. Those skilled in the art will appreciate that the dose to be administered will vary depending on, for example, the mammal to which the antibodies, antibody-encoding polynucleotides and/or compositions provided herein will be administered, the route of administration, the particular type of antibody used, antibody-encoding polynucleotides, antigen-binding fragments and/or compositions provided herein and other drugs administered to a mammal. Guidance on the selection of appropriate doses of antibody or antigen binding fragment can be found in the literature on the therapeutic use of antibodies and antigen binding fragments, for example, Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., 1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp. 365-389.

A typical daily dose of an effective amount of antibody is from 0.01 mg/kg to 100 mg/kg. In some embodiments, the dose may be less than 100 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg /kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg or 0.1 mg/kg. In some embodiments, the dosage may be greater than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg /kg, 1 mg/kg, 0.5 mg/kg, 0.1 mg/kg, 0.05 mg/kg or 0.01 mg/kg. In some embodiments, the dose is approximately 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg. kg, 1 mg/kg, 0.9 mg/kg, 0.8 mg/kg, 0.7 mg/kg, 0.6 mg/kg, 0.5 mg/kg, 0.4 mg/kg, 0 .3 mg/kg, 0.2 mg/kg or 0.1 mg/kg.

In any of the methods provided herein, at least one antibody, an antigen-binding fragment thereof, or a pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions provided herein) and optionally at least one additional therapeutic the agent can be administered to the individual at least once a week (eg, once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and/or antigen-binding fragments are administered in a single composition (eg, liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in a single composition (eg, liquid composition). In some embodiments, at least one antibody or antigen binding fragment and at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, at least one additional therapeutic agent is administered in a sustained release oral formulation.

In some embodiments, one or more additional therapeutic agents can be administered to the individual before or after administration of at least one antibody, antibody antigen-binding fragment, or pharmaceutical composition (e.g., any of the antibodies, antibody antigen-binding fragments, or pharmaceutical compositions described herein). In some embodiments, one or more additional therapeutics and at least one antibody, antibody antigen-binding fragment, or pharmaceutical composition (e.g., any of the antibodies, antibody antigen-binding fragments, or pharmaceutical compositions provided herein) is administered to an individual such that overlapping the bioactive period of one or more additional therapeutic agents and at least one antibody or antigennegative fragment (eg, any of the antibodies or antigennegative fragments provided herein) in an individual.

In some embodiments, an individual may be administered at least one antibody, antibody antigen-binding fragment, or pharmaceutical composition (e.g., any of the antibodies, antibody antigen-binding fragments, or pharmaceutical compositions described herein) over an extended period of time (e.g., for at least least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years or 5 years). One of skill in the medical field can determine the duration of the treatment period by any of the methods provided herein to diagnose or monitor the effectiveness of treatment (eg, observation of at least one symptom of malignancy). As described herein, one of skill in the medical field can also change the type and amount (e.g., increase or decrease) of antibodies or antibody antigen-binding fragments (and/or one or more additional therapeutic agents) administered to an individual and can also adjust (e.g., increase or decrease) the dose or frequency of administration of at least one antibody or antigen-binding fragment of an antibody (and/or one or more additional therapeutic agents) to an individual, taking into account the evaluation of the effectiveness of treatment (for example, by any methods presented in the present description and known in this field) .

In some embodiments, one or more additional therapeutic agents may be administered to the individual. The additional therapeutic agent may contain one or more inhibitors selected from the group consisting of B-Raf inhibitor, EGFR inhibitor, MEK inhibitor, ERK inhibitor, K-Ras inhibitor, c-Met inhibitor, anaplastic lymphoma kinase (ALK) inhibitor, phosphatidylinositol inhibitor -3-kinase (PI3K), an Akt inhibitor, an mTOR inhibitor, a dual PI3K/mTOR inhibitor, a Bruton's tyrosine kinase (BTK) inhibitor, and an inhibitor of isocitrate dehydrogenase 1 (IDH1) and/or isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an indolamine-2,3-dioxygenase-1 (IDO1) inhibitor (eg, epacadostat).

In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of a HER3 inhibitor, an LSD1 inhibitor, an MDM2 inhibitor, a BCL2 inhibitor, a CHK1 inhibitor, an inhibitor of an activated hedgehog signaling pathway, and an estrogen receptor selective degrader.

In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, reolizin, alimta, zycadia, sutenta , temsirolimus, axitinib, everolimus, sorafenib, votrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitor, Ad-GM-CSF, temazolomide, IL-2, IFNα, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomide, amrubicin, carfilzomib, pralatrexate, and enzastaurine.

In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of adjuvant, TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, IL-10 antagonist, IL-4 antagonist, IL-13 antagonist, IL-17 antagonist, HVEM antagonist, ICOS agonist, CX3CL1-targeted treatment, CXCL9-targeted treatment, CXCL10-targeted treatment, CCL5-targeted treatment, LFA-1 agonist, ICAM1 agonist, and selectin agonist .

In some embodiments, the individual is administered carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody.

Methods for modifying and using anti-PD-1 antibodies are described, for example, in US Patent Application Nos. US20170247454, US20170081409A1, US20170044259A1 and US20160159905; each of which is incorporated herein by reference in its entirety.

Pharmaceutical compositions and routes of administration

The present invention also relates to pharmaceutical compositions containing at least one (for example, one, two, three or four) of the antibodies or antigennegative fragments presented in the present description. Two or more (eg, two, three, or four) of any of the antibodies or antigen-binding fragments provided herein may be present in a pharmaceutical composition in any combination. Pharmaceutical compositions can be formulated by any method known in this field.

Pharmaceutical compositions are formulated to be compatible with the intended route of administration (eg, intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). Compositions may include a sterile diluent (eg, sterile water or saline), fatty oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, antibacterial or antifungal agents such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thiomersal, etc. .p., antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates or phosphates and isotonic agents such as sugars (eg dextrose), polyalcohols (eg mannitol or sorbitol), salts (eg sodium chloride), or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, for example, US Pat. No. 4,522,811). The formulations of the compositions can be formulated and enclosed in ampoules, disposable syringes or multi-dose vials. If necessary (eg, as in the case of injectable formulations), proper fluidity can be maintained, for example, using a coating such as lecithin or a surfactant. The absorption of an antibody or antigen-binding fragment thereof can be prolonged by the inclusion of an absorption delaying agent (eg, aluminum monostearate and gelatin). Alternatively, controlled release can be achieved with implants and microencapsulated delivery systems that may include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

Compositions containing one or more of any of the antibodies or antigen-binding fragments provided herein can be formulated for parenteral (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in unit dosage form (i.e., physically discrete units containing a predetermined amount of active compound for ease of administration and dose uniformity).

The toxicity and therapeutic efficacy of the compositions can be determined by standard pharmaceutical methods in cell cultures or experimental animals (eg, monkeys). For example, LD50 (50% lethal dose in a population) and ED50 (therapeutically effective dose in 50% of a population) can be determined, with the therapeutic index being the ratio of LD50:ED50. Agents having a high therapeutic index are preferred. If an agent exhibits an undesirable side effect, steps should be taken to minimize possible harm (i.e., reduce unwanted side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical methods.

Data obtained from cell culture assays and animal studies can be used in formulating appropriate dosages of any of the agents indicated for use in an individual (eg, human). A therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments provided herein) will be an amount that treats a disease (e.g., destroys malignant cells) in an individual (e.g., a person identified as having cancer) or an individual identified as being at risk of developing a disease (e.g., an individual who has previously developed cancer but has been cured) reduce the severity, frequency, and/or duration one or more symptoms of a disease in an individual (eg, human). One of skill in the medical or veterinary arts can determine the potency and dosage of any of the antibodies or antigen-binding fragments provided herein by methods known in the art, as well as by observing one or more symptoms of a disease in an individual (e.g., human). Several factors may affect the dose and timing required to effectively treat an individual (eg, severity of disease or disorder, prior treatment, general health and/or age of the individual, and presence of other medical conditions).

Exemplary dosages include milligram or microgram amounts of any of the antibodies or antigen-binding fragments provided herein per kilogram of body weight of an individual (e.g., about 1 µg/kg to about 500 mg/kg; about 100 µg/kg to about 500 mg/kg). kg; from about 100 µg/kg to about 50 mg/kg; from about 10 µg/kg to about 5 mg/kg; from about 10 µg/kg to about 0.5 mg/kg, or from about 1 µg/kg to approximately 50 µg/kg). While these dosages cover a wide range, one of skill in the art will appreciate that therapeutic agents, including antibodies and antigen-binding fragments thereof, vary in potency and effective amounts can be determined by methods known in the art. Typically, relatively low amounts are administered initially, and then the medical or veterinarian (in the case of therapeutic use) or the researcher (in the development stage) can gradually increase the dose until an appropriate response is achieved. In addition, it should be understood that the specific dosage level for any particular individual will depend on various factors, including the activity of the particular compound used, age, body weight, general health, sex and nutrition of the individual, time of administration, route of administration, rate of excretion, and time half-life of an antibody or antibody fragment in vivo .

Pharmaceutical compositions may be included in a container, pack or dispenser along with instructions for administration. The present invention also relates to methods for the production of antibodies or antigen-binding fragments for various applications, as described in the present description.

EXAMPLES

Further, the present invention is described using the following non-limiting examples of the invention described in the claims.

Example 1 Obtaining Mouse Anti-hPD-1 Antibodies

To generate mouse antibodies against human PD-1 (hPD-1; SEQ ID NO:22), 6-8 week old female BALB/c mice were immunized with human PD-1. Anti-hPD-1 antibodies were collected by the methods described below and shown in FIG. 1 and FIG. 2.

Immunization of mice

Female BALB/c mice 6-8 weeks old were immunized with His-tagged human PD-1 proteins at 20 μg/mouse at a concentration of 100 μg/ml. His-tagged human PD-1 proteins were emulsified with adjuvant and injected at four positions on the back of the mouse. For the first subcutaneous (s.c.) injection, the diluted antigen was emulsified with an equal volume of Freund's complete adjuvant (CFA). On subsequent subcutaneous injections, the protein was emulsified with an equal volume of Incomplete Freund's Adjuvant (IFA). Three days after the third injection or booster immunization, blood (serum) was collected and analyzed for antibody titer using ELISA.

In another experiment, 6-8 week old female BALB/c mice were immunized by injection into mice of an expression plasmid encoding human PD-1. Plasmids encoding the antigen were injected into the tibialis anterior muscle (intramuscular injection; i.m. injection) of mice using a gene gun at a concentration of 1000 μg/μl in an amount of 60 μg per mouse. At least four injections were performed with at least 14 days between two injections. Blood (serum) was collected seven days after the last immunization and the serum was tested for antibody titer by ELISA.

Methods were also performed to boost immunization at least fourteen days after prior immunization (via plasmid injection or protein injection). CHO cells expressing the PD-1 antigen on their surface were injected intravenously into mice via the tail veins. Four days after injection, spleens were obtained.

Fusion of SP2/0 cells and spleen cells

The spleen tissues were crushed. Spleen cells were first selected with CD3ε microparticles and anti-mouse IgM microparticles and then fused with SP2/0 cells. The cells were then seeded in 96-well plates with hypoxanthine-aminopterin-thymidine medium (HAT).

Primary screening for hybridomas

Primary screening of hybridoma supernatant in 96-well plates was performed using fluorescence-activated cell sorting (FACS) by standard methods. Before screening, Chinese hamster ovary (CHO) cells were added to 96-well plates (2×10 ⁴ cells per well). 50 µl of supernatant was used. The antibodies used in the experiments were as follows

(1) AffiniPure goat anti-mouse IgG fluorescein (FITC)-conjugated F(ab) ₂ fragment, Fcγ fragment-specific, and

(2) Alexa Fluor® 647-conjugated AffiniPure goat anti-human IgG F(ab) ₂ fragment, Fcγ fragment-specific.

subcloning

Subcloning was carried out using ClonePix2. Briefly, the contents of the positive wells identified during the primary screen were transferred to semi-solid medium and IgG-positive clones were identified and tested. An FITC-conjugated anti-mouse IgG Fc antibody was used.

Ascitic fluid antibodies

1x10 ⁶ positive hybridoma cells were intraperitoneally injected into B- ^NDG® mice (Beijing Biocytogen, Beijing, China). Monoclonal antibodies were obtained by growing hybridoma cells in the abdominal cavity of the mouse. Hybridoma cells divided and produced ascitic fluid in the abdominal cavity of mice. The liquid contained a high concentration of antibody, which can be collected for later use.

Purification of antibodies

Antibodies in ascitic fluid were purified using protein chromatography with GE AKTA (GE Healthcare, Chicago, Illinois, United States). 25-1A7 ("1A7"), 18-3F1 ("3F1") and 3-6G1 ("6G1") were among the mouse antibodies obtained by the methods described above.

The VH, VL and CDR regions of the antibodies were determined. The amino acid sequences of heavy chain CDR1, CDR2, CDR3 and light chain CDR1, CDR2 and CDR3 of 1A7 are shown in SEQ ID NOs: 1-6 (Kabat numbering) or SEQ ID NOs: 16, 17, 3, 4, 5, 6 (numbering according to Chothia).

The amino acid sequences of CDR1, CDR2, CDR3 of the heavy chain and CDR1, CDR2 and CDR3 of the light chain of 3F1 are given in SEQ ID NO:7-12 (Kabat numbering) or SEQ ID NO:18, 19, 9, 10, 11, 12 (numbering according to Chothia).

The amino acid sequences of heavy chain CDR1, CDR2, CDR3 and light chain CDR1, CDR2 and CDR3 of 6G1 are shown in SEQ ID NOs: 7, 13, 14, 10, 11, 15 (Kabat numbering) or SEQ ID NOs: 20, 21, 14 , 10, 11, 15 (numbered according to Chothia).

Example 2 Humanization of Mouse Antibodies

The starting point for humanization was mouse antibodies (eg 1A7 and 3F1). The amino acid sequences of the heavy chain variable region and the light chain variable region of these mouse antibodies were determined.

Three humanized variants of the heavy chain variable region (SEQ ID NO:26-28) and three humanized variants of the light chain variable region (SEQ ID NO:29-31) of 1A7 were constructed, containing various modifications or substitutions.

Four humanized variants of the heavy chain variable region (SEQ ID NO:32-35) and three humanized variants of the light chain variable region (SEQ ID NO:36-38) of 3F1 were constructed, containing various modifications or substitutions.

These humanized heavy chain variable region variants can be combined with any light chain variable region variants based on the same mouse antibody. For example, 1A7-H1 (SEQ ID NO:26) can be combined with any humanized light chain variable region variant based on the same mouse 1A7 antibody (e.g., 1A7-K3 (SEQ ID NO:31)), and the antibody is labeled as such (for example, 1A7-H1K3).

These humanized antibodies were then generated using BioLuminate 1.0 (Schrodinger, Shanghai, China).

Example 3: Testing in vitro mouse antibodies against hPD-1: blocking the binding of human PD-1 (hPD-1) and human PD-L1 (hPD-L1)

Blocking assays were performed to determine if anti-hPD-1 antibodies could block the binding of hPD-1 to its hPD-L1 ligand.

Anti-hPD-1 antibodies were collected from mouse ascitic fluid and purified by chromatography. 25 μl of CHO cells transiently transfected with human PD-1 was added to each well of the plate. Purified antibodies were titrated to final concentrations of 50, 5, 0.5, 0.05, 0.005 µg/ml. Titratable antibodies were added to each well at 25 μl per well at 4° C. and incubated for 30 minutes.

50 μl of biotin-hPD-L1 (with a final concentration of 2 μg/ml in each well) was added to each well. Cells with biotin-hPD-L1 and antibodies were incubated at 4°C for 15 minutes.

After washing twice with phosphate-buffered saline (PBS), 50 µl of FITC-labeled anti-mouse Fc IgG (anti-mIgG-FITC Fc) antibody at a 1:100 dilution and PE-labeled streptavidin (streptavidin-PE) at a 1 dilution were added to each well: 100 and incubated for 30 minutes at 4°C followed by washing with PBS. FITC and PE signals were determined by flow cytometry.

As shown in FIG. 3, when the concentration of mouse anti-PD-1 antibodies 25-1A7, 18-3F1 and 03-6G1 increased, the signal of cells binding to PD-L1 decreased (y-axis), while the signal of cells binding to mouse anti-PD-1 antibodies PD-1 increased, suggesting that binding between human PD-1 and PD-L1 was blocked by anti-hPD-1 antibodies.

Example 4 Anti-hPD-1 Antibody Cross-Reactivity Against Monkey PD-1, Mouse PD-1 and Human-Mouse Chimeric PD-1

In each experiment, CHO cells were transfected with mouse PD-1 (mPD-1, SEQ ID NO:23), monkey (rhesus monkey) PD-1 (rmPD-1, SEQ ID NO:24), or chimeric PD-1 ( mouse and human) (chiPD-1, SEQ ID NO:25).

25 μl of CHO cells were added to each well. 25 μl of purified anti-hPD-1 antibody (1 μg/ml) (1A7, 3F1 or 6G1) was added to each well and incubated at 4°C for 30 minutes.

After washing twice with PBS (1200 rpm, 5 min), 50 µl of FITC-labeled anti-mouse Fc IgG (anti-mIgG-FITC Fc) antibody at a 1:500 dilution was added to each well, followed by incubation at 4°C for 30 minutes and rinsing with PBS (1200 rpm, 5 min). FITC signals were determined by flow cytometry.

As shown in FIG. 4, 1A7, 3F1 and 6G1 did not cross-react with mouse PD-1 but had strong cross-reactivity with rmPD-1 and some cross-reactivity with chimeric PD-1. In FIG. 4 "NC" means negative control.

Example 5 Anti-hPD-1 Antibodies Binding Affinity

The binding affinity of antibodies against hPD-1 was measured using surface plasmon resonance (SPR) using a Biacore T200 biosensor (Biacore, INC, Piscataway N.J.) equipped with pre-immobilized protein A sensor chips.

Anti-hPD-1 antibody 1A7-mHvKv-IgG4-S228P (1 μg/ml) was injected onto the Biacore T200 biosensor at 10 μl/min for 30 seconds to achieve the desired protein density (approximately 67 response units (RU)). Then histidine-labeled human PD-1 proteins (hPD-1-His) at a concentration of 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 nM were injected at 30 µl/min for 100 seconds . Dissociation was monitored for 400 seconds. The chip was regenerated with glycine (pH 2.0, 30 μl/min for 12 seconds) after the last injection of each titer. Results for 1A7-mHvKv-IgG4-S228P are shown in FIG. five.

Kinetic rates of association (k _on ) and rates of dissociation (k _off ) were obtained simultaneously by a global fit to the data using the 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6 99-110) using Biacore T200 Evaluation Software 3.0. Affinities can be derived from the ratio of kinetic rate constants (KD=k _off /k _on ).

As will be appreciated by one of skill in the art, the same method, with appropriate parameter adjustments (eg, antibody concentrations), was performed for each antibody tested. For example, in FIG. 6 is a graph showing the results for 3F1-mHvKv-IgG4-S228P. The results for the tested antibodies are summarized in Table 1 below.

Table 1

Anti-hPD-1 antibodies Association rate (k _on ) (1/Ms) Dissociation rate (k _off ) (1/s) Affinity (KD) (M) 1A7-mHvKv-IgG4-S228P 4.07E+05 9.56E-04 2.35E-09 1A7-H1K1-IgG4-S228P 5.68E+04 2.72E-03 4.79E-08 1A7-H1K2-IgG4-S228P 8.26E+04 2.65E-03 3.21E-08 1A7-H1K3-IgG4-S228P 1.18E+05 1.19E-03 1.01E-08 1A7-H2K1-IgG4-S228P 4.89E+04 2.80E-03 5.73E-08 1A7-H2K2-IgG4-S228P 4.88E+04 2.82E-03 5.78E-08 1A7-H2K3-IgG4-S228P 1.15E+05 1.36E-03 1.19E-08 1A7-H3K1-IgG4-S228P 6.62E+04 2.64E-03 3.99E-08 1A7-H3K2-IgG4-S228P 4.81E+04 2.80E-03 5.82E-08 1A7-H3K3-IgG4-S228P 1.02E+05 1.32E-03 1.29E-08 3F1-mHvKv-IgG4-S228P 3.70E+03 1.28E-03 3.46E-07 3F1-H1K1-IgG4-S228P 4.81E+03 1.81E-03 3.76E-07 3F1-H1K2-IgG4-S228P 5.27E+03 1.55E-03 2.94E-07 3F1-H1K3-IgG4-S228P 4.79E+03 1.32E-03 2.75E-07 3F1-H2K1-IgG4-S228P 4.55E+03 1.92E-03 4.22E-07 3F1-H2K2-IgG4-S228P 4.81E+03 1.70E-03 3.54E-07 3F1-H2K3-IgG4-S228P 4.41E+03 1.45E-03 3.29E-07 3F1-H3K1-IgG4-S228P 4.53E+03 1.79E-03 3.95E-07 3F1-H3K2-IgG4-S228P 5.06E+03 1.50E-03 2.96E-07 3F1-H3K3-IgG4-S228P 4.38E+03 1.39E-03 3.18E-07 3F1-H4K1-IgG4-S228P 4.42E+03 2.04E-03 4.60E-07 3F1-H4K2-IgG4-S228P 4.80E+03 1.77E-03 3.69E-07 3F1-H4K3-IgG4-S228P 4.06E+03 1.61E-03 3.97E-07

Among these antibodies tested, 1A7 and 3F1 are the mouse anti-hPD-1 antibodies described in Example 1. Based on 1A7 and 3F1, chimeric anti-hPD-1 antibodies were prepared, including 1A7-mHvKv-IgG4-S228P and 3F1-mHvKv-IgG4-S228P . The chimeric antibodies comprise a heavy chain variable domain and a light chain variable domain from the corresponding mouse anti-hPD-1 antibodies with constant domains from a human IgG4 antibody (including, for example, the CL, CH1, CH2, and CH3 domains).

Antibodies tested also include humanized antibodies, e.g. 3F1-H1K1-IgG4-S228P, 3F1-H1K2-IgG4-S228P, 3F1 3F1-H2K1-IgG4-S228P -S228P, 3F1-H4K1-IgG4-S228P, 3F1-H4K2-IgG4-S228P and 3F1-H4K3-IgG4-S228P. Humanized antibodies comprise human IgG4 antibody constant domains (including, for example, CL, CH1, CH2, and CH3 domains). Humanized heavy chain variable domains are numbered as H1, H2, H3, etc.; and humanized light chain variable domains are numbered as K1, K2, K3, etc. The sequences of the humanized variable domains are shown in FIG. 19. For example, 1A7-H1K1-IgG4-S228P is based on mouse antibody 1A7 and contains a humanized H1 heavy chain variable domain (SEQ ID NO:26) and a humanized K1 light chain variable domain (SEQ ID NO:29). Similarly, 3F1-H1K1-IgG4-S228P is based on the mouse antibody 3F1 and contains a humanized H1 heavy chain variable domain (SEQ ID NO:32) and a humanized 9H3 K1 light chain variable domain (SEQ ID NO:36).

The names and sequences of chimeric anti-PD-1 antibodies and humanized anti-PD-1 antibodies are shown in Table 2 below. To improve production efficiency by eliminating Fab exchange, the antibody Fc region was further constructed by replacing serine at position 228 (EU numbering) with proline (S228P).

table 2

Type Name of the antibody VH SEQ ID NO: VL SEQ ID NO: Constant Regions Chimeric antibody based on 1A7 1A7-mHvKv-IgG4-S228P 39 40 Human IgG4 with S228P mutation Humanized antibodies based on 1A7 1A7-H1K1-IgG4-S228P 26 29 Human IgG4 with S228P mutation 1A7-H1K2-IgG4-S228P 26 thirty Human IgG4 with S228P mutation 1A7-H1K3-IgG4-S228P 26 31 Human IgG4 with S228P mutation 1A7-H2K1-IgG4-S228P 27 29 Human IgG4 with S228P mutation 1A7-H2K2-IgG4-S228P 27 thirty Human IgG4 with S228P mutation 1A7-H2K3-IgG4-S228P 27 31 Human IgG4 with S228P mutation 1A7-H3K1-IgG4-S228P 28 29 Human IgG4 with S228P mutation 1A7-H3K2-IgG4-S228P 28 thirty Human IgG4 with S228P mutation 1A7-H3K3-IgG4-S228P 28 31 Human IgG4 with S228P mutation Chimeric antibody based on 3F1 3F1-mHvKv-IgG4-S228P 41 42 Human IgG4 with S228P mutation Humanized antibodies based on 3F1 3F1-H1K1-IgG4-S228P 32 36 Human IgG4 with S228P mutation 3F1-H1K2-IgG4-S228P 32 37 Human IgG4 with S228P mutation 3F1-H1K3-IgG4-S228P 32 38 Human IgG4 with S228P mutation 3F1-H2K1-IgG4-S228P 33 36 Human IgG4 with S228P mutation 3F1-H2K2-IgG4-S228P 33 37 Human IgG4 with S228P mutation 3F1-H2K3-IgG4-S228P 33 38 Human IgG4 with S228P mutation 3F1-H3K1-IgG4-S228P 34 36 Human IgG4 with S228P mutation 3F1-H3K2-IgG4-S228P 34 37 Human IgG4 with S228P mutation 3F1-H3K3-IgG4-S228P 34 38 Human IgG4 with S228P mutation 3F1-H4K1-IgG4-S228P 35 36 Human IgG4 with S228P mutation 3F1-H4K2-IgG4-S228P 35 37 Human IgG4 with S228P mutation 3F1-H4K3-IgG4-S228P 35 38 Human IgG4 with S228P mutation

Example 6 Thermal Stability of Anti-hPD-1 Antibodies

Thermofluor assays were performed using the Protein Thermal Shift™ dye kit (Thermo Fisher Scientific) and QuantStudio™ 5 real-time PCR systems (Thermo Fisher Scientific). This assay measures thermal stability using a fluorescent dye that binds to hydrophobic regions exposed when the protein unfolds.

The experiment was carried out according to the manufacturer's instructions. 2 µl of antibody, 10.5 µl of water, 5 µl of Protein Thermal Shift buffer and 2.5 µl of diluted Protein Thermal Shift dye were mixed. The samples were heated to 25° C. at a rate of 1.6° C./second and then heated to 99° C. at a rate of 0.05° C./second.

The table below shows the Tm of four humanized antibodies against hPD-1.

Table 3

Antibody Variable domains Type (constant domains) Thermal stability (Tm, °C) 1A7-H1K3-IgG4-S228P 1A7 H1K3 Human IgG4 with S228P mutation 75.94 1A7-H2K3-IgG4-S228P 1A7 H2K3 Human IgG4 with S228P mutation 76.09 3F1-H1K2-IgG4-S228P 3F1 H1K2 Human IgG4 with S228P mutation 82.82 3F1-H1K3-IgG4-S228P 3F1 H1K3 Human IgG4 with S228P mutation 82.37

Example 7 Testing in vivo mouse antibodies and anti-hPD-1 chimeric antibodies

To test antibodies against hPD-1 in vivo and predict their effects on humans, a humanized PD-1 mouse model was generated. A humanized mouse PD-1 model was constructed to express the chimeric PD-1 protein (SEQ ID NO:25), where a portion of the extracellular region of the mouse PD-1 protein was replaced with the corresponding extracellular region of human PD-1. Amino acid residues 31-141 of mouse PD-1 (SEQ ID NO:23) were replaced with amino acid residues 31-141 of human PD-1 (SEQ ID NO:22). The humanized mouse model (B-hPD-1) is a novel tool for testing new therapeutic treatments in the clinical setting by significantly reducing the difference between clinical outcome in humans and normal mice expressing mouse PD-1. A detailed description of the humanized PD-1 mouse model can be found in International Application No. PCT/CN2017/090320, incorporated herein by reference in its entirety.

Anti-hPD-1 antibodies were tested for their effect on tumor growth in vivo in a colonic carcinoma model. MC-38 tumor cells (colon adenocarcinoma cells) were subcutaneously injected into B-hPD-1 mice. When tumors in mice reached a volume of 150 ± 50 mm ³ , mice were randomly assigned to different groups based on tumor volume (five mice per group).

Mice were then injected with saline (PS) and anti-hPD-1 antibodies via intraperitoneal injection. The antibody was administered on the first day and fourth day of each week for 3 weeks (6 injections in total).

The injection volume was calculated based on the mouse weight as 1 mg/kg. The tumor was measured along the long axis and short axis, and the tumor volume was calculated as 0.5×(long axis)×(short axis) ² . The pre-injection mouse weight was also measured when the mice were divided into different groups (before the first antibody injection), twice a week during the antibody injection period, and before sacrifice.

The percentage of tumor growth inhibition (TGI%) was calculated by the following formula: TGI (%) = [1-(Ti-T0)/(Vi-V0)]×100. Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day zero. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day zero.

For statistical analysis, Student's t-test was used. TGI% above 60% indicates significant suppression of tumor growth. P<0.05 is the threshold for statistical significance.

In vivo results for mouse anti-hPD-1 antibodies 25-1A7 and 18-3F1

In each of the four groups (G1, G2, G3, G4), B-hPD-1 mice were injected with saline (PS) as controls (G1), Keytruda® (G2), mouse anti-hPD-125-1A7 (G3) or mouse anti-hPD-1 antibody 18-3F1 (G4). The weight of mice was monitored throughout the treatment period. The mass of mice in all groups increased (Fig. 7 and Fig. 8). No significant differences in weight were observed among the four groups. The results indicate that 1A7 and 3F1 were well tolerated and were not toxic to mice.

Tumor size in the Keytruda®, 1A7 or 3F1 treated groups increased to a lesser extent than in the control group (FIG . 9). In particular, the size of the tumor in G3 is smaller than in G2; and tumor size in G4 is smaller than in G2.

Also calculated TGI% on day 24 (24 days after distribution to groups), as shown in the table below.

Table 4

Group Antibodies Average tumor size by day 24 TGI% P value (relative to control) G1 PS (1 mg/kg; i.p. administration; twice a week, 6 injections total) 1078 - - G2 Keytruda® (1 mg/kg) (1 mg/kg; i.p. administration; twice a week, 6 injections total) 842 25.3% 0.691 G3 25-1A7 (1 mg/kg) (1 mg/kg; i.p. administration; twice a week, 6 injections total) 258 87.9% 0.153 G4 18-3F1 (1 mg/kg) (1 mg/kg; i.p. administration; twice a week, 6 injections total) 646 46.3% 0.439

In vivo results for anti-hPD-1 chimeric antibodies

B-hPD-1 mice (humanized PD-1 mice) were injected with chimeric anti-hPD-1 antibodies 03-6G1-mHvKv-IgG4-S228P (Group 2, G2), 18-3F1-mHvKv-IgG4-S228P (Group 3, G3), 25-1A7-mHvKv-IgG4-S228P (group 4, G4) and Keytruda® (group 5) via intraperitoneal administration. Saline was injected as a control (Group 1, G1). The injected amount of antibodies was calculated taking into account the weight of the mouse as 1 mg/kg. Antibodies were administered on the first day and fourth day of each week (6 injections in total).

Carried out monitoring of the weight of mice during the entire period of treatment. In all groups, the weight of mice increased (Fig. 10 and Fig. 11). No significant differences in weight were observed between groups. The results indicate that the anti-hPD-1 antibodies were well tolerated and were not toxic to mice.

Significant differences in tumor size were observed in the groups treated with the chimeric antibodies compared to the control group (FIG . 12).

TGI% on day 20 (20 days after grouping) for each treatment group was calculated as shown in the table below.

Table 5

Group Antibodies Average tumor size per day 20 TGI% P value G1 PS 1427 - - G2 03-6G1-mHvKv-IgG4-S228P (1 mg/kg; i.p. administration; twice a week, 6 injections total) 564 66.0% 0.032 G3 18-3F1-mHvKv-IgG4-S228P (1 mg/kg; i.p. administration; twice a week, 6 injections total) 700 55.6% 0.056 G4 25-1A7-mHvKv-IgG4-S228P (1 mg/kg; i.p. administration; twice a week, 6 injections total) 356 81.9% 0.011 G5 Keytruda® (1 mg/kg; i.p. administration; twice a week, 6 injections total) 200 93.8% 0.005

The results indicate that the 03-6G1-mHvKv-IgG4-S228P and 25-1A7-mHvKv-IgG4-S228P chimeric antibodies significantly inhibited tumor growth. Among the three chimeric antibodies, 25-1A7-mHvKv-IgG4-S228P had the highest TGI%.

Example 8 Testing in vivo humanized antibodies against hPD-1

Humanized anti-hPD-1 antibodies were tested in humanized PD-1 (B-hPD-1) mice to demonstrate their effect on tumor growth in vivo .

B-hPD-1 mice were injected subcutaneously with MC-38 tumor cells (colon adenocarcinoma cells). When tumors in mice reached a volume of 150 ± 50 mm ³ , mice were randomly assigned to different groups based on tumor volume (five mice in each group).

Mice were then injected with saline as a control (G1), humanized anti-PD-1 antibody 1A7-H1K3-IgG4-S228P (G2), humanized anti-PD-1 antibody 1A7-H2K3-IgG4-S228P (G3), or Keytruda® (G4 ). Antibodies were administered on the second day and fifth day of each week by intraperitoneal injection at 1 mg/kg for 3 weeks (6 injections in total).

Carried out monitoring of the weight of mice during the entire period of treatment. In all groups, the weight of mice increased (Fig. 13 and Fig. 14). No significant differences in weight were observed between groups. The results indicate that the anti-hPD-1 antibodies were well tolerated and were not toxic to mice.

Significant differences in tumor size were observed in the groups treated with anti-hPD-1 antibodies (FIG. 15). In particular, the size of the tumor in G2 is smaller than in G4; and tumor size in G3 is smaller than in G4.

Also calculated TGI% on day 21 (21 after grouping) in each treatment group, as shown in the table below.

Table 6

Group Antibodies Average tumor size per day 18 TGI% P value G1 PS 1986 - - G2 1A7-H1K3-IgG4 (1 mg/kg) 233 94.5% 0.001 G3 1A7-H2K3-IgG4 (1 mg/kg) 150 99.0% 0.001 G4 Keytruda® (1 mg/kg) 422 84.4% 0.006

The results described above indicate that all anti-hPD-1 antibodies can inhibit tumor growth. Among them, 1A7-H2K3-IgG4 (1mg/kg) had the highest percentage of tumor growth inhibition (TGI%).

OTHER IMPLEMENTATION OPTIONS

It should be understood that while the present invention has been presented in conjunction with the detailed description, the foregoing description is intended to be illustrative and not to limit the scope of the invention as defined by the scope of the claims. Other aspects, advantages and modifications are within the scope of the claims.

Claims (146)

1. An antibody or antigen-binding fragment thereof that binds to PD-1 (programmed cell death protein 1), containing: a heavy chain variable region (VH) containing complementarity determining regions (CDRs) 1, 2 and 3, where the CDR1 VH region contains an amino acid sequence that is identical to the selected amino acid sequence of the CDR1 VH, the CDR2 VH region contains an amino acid sequence that is identical to the selected amino acid sequence of the CDR2 VH, and the CDR3 VH region contains an amino acid sequence that is identical to the selected amino acid sequence of the CDR3 VH; and a light chain variable region (VL) containing CDRs 1, 2 and 3, where the CDR1 VL region contains an amino acid sequence that is identical to the selected CDR1 VL amino acid sequence, the CDR2 VL region contains an amino acid sequence that is identical to the selected CDR2 VL amino acid sequence, and the CDR3 VL region contains an amino acid sequence that is identical to the selected CDR3 VL amino acid sequence, where the selected amino acid sequences of VH CDRs 1, 2 and 3 and the selected amino acid sequences of VL CDRs 1, 2 and 3 are one of the following: (1) the selected amino acid sequences of CDR 1, 2, 3 VH are shown in SEQ ID NOs: 1, 2, 3, respectively, and selected amino acid sequences of CDR 1, 2, 3 VL are shown in SEQ ID NO: 4, 5, 6, respectively; (2) selected amino acid sequences of VH CDRs 1, 2, 3 are shown in SEQ ID NOs: 16, 17, 3, respectively, and selected amino acid sequences of CDR 1, 2, 3 VL are shown in SEQ ID NO: 4, 5, 6, respectively; (3) selected amino acid sequences of VH CDRs 1, 2, 3 are shown in SEQ ID NOs: 7, 8, 9, respectively, and selected amino acid sequences of CDR 1, 2, 3 VL are given in SEQ ID NO: 10, 11, 12, respectively; (4) selected amino acid sequences of VH CDRs 1, 2, 3 are shown in SEQ ID NOs: 18, 19, 9, respectively, and selected amino acid sequences of CDR 1, 2, 3 VL are given in SEQ ID NO: 10, 11, 12, respectively; (5) selected amino acid sequences of VH CDRs 1, 2, 3 are shown in SEQ ID NOs: 7, 13, 14, respectively, and selected amino acid sequences of CDR 1, 2, 3 VL are shown in SEQ ID NO: 10, 11, 15, respectively; and (6) selected amino acid sequences of VH CDRs 1, 2, 3 are shown in SEQ ID NOs: 20, 21, 14, respectively, and selected amino acid sequences of VL CDRs 1, 2, 3 are shown in SEQ ID NOs: 10, 11, 15, respectively. 2. An antibody or antigen-binding fragment according to claim 1, where VH contains CDRs 1, 2, 3 with the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively, and VL contains CDRs 1, 2, 3 with amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively, where CDR 1, 2, 3 VH and CDR 1, 2, 3 VL are defined according to Kabat numbering. 3. The antibody or antigen-binding fragment according to claim 1, where VH contains CDRs 1, 2, 3 with the amino acid sequences of SEQ ID NOs: 16, 17 and 3, respectively, and VL contains CDRs 1, 2, 3 with amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively, where CDR 1, 2, 3 VH and CDR 1, 2, 3 VL are defined according to Chothia numbering. 4. The antibody or antigen-binding fragment according to claim 1, where VH contains CDRs 1, 2, 3 with the amino acid sequences of SEQ ID NOs: 7, 8 and 9, respectively, and VL contains CDRs 1, 2, 3 with the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, where CDR 1, 2, 3 VH and CDR 1, 2, 3 VL are defined according to Kabat numbering. 5. The antibody or antigen-binding fragment according to claim 1, where VH contains CDRs 1, 2, 3 with the amino acid sequences of SEQ ID NOs: 18, 19 and 9, respectively, and VL contains CDRs 1, 2, 3 with the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, where CDR 1, 2, 3 VH and CDR 1, 2, 3 VL are defined according to Chothia numbering. 6. The antibody or antigen-binding fragment according to claim 1, where VH contains CDRs 1, 2, 3 with the amino acid sequences of SEQ ID NOs: 7, 13 and 14, respectively, and VL contains CDRs 1, 2, 3 with amino acid sequences of SEQ ID NOs: 10, 11 and 15, respectively, where CDR 1, 2, 3 VH and CDR 1, 2, 3 VL are defined according to Kabat numbering. 7. The antibody or antigen-binding fragment according to claim 1, where VH contains CDRs 1, 2, 3 with the amino acid sequences of SEQ ID NOs: 20, 21 and 14, respectively, and VL contains CDRs 1, 2, 3 with amino acid sequences of SEQ ID NOs: 10, 11 and 15, respectively, where CDR 1, 2, 3 VH and CDR 1, 2, 3 VL are defined according to Chothia numbering. 8. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment specifically binds to human PD-1. 9. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof. 10. An antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein the antigen-binding fragment is a single chain variable fragment (scFV). 11. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 to obtain a bispecific antibody. 12. Nucleic acid for producing an antibody or its antigen-binding fragment, where the nucleic acid encodes: (1) an immunoglobulin heavy chain or fragment thereof containing a VH containing CDRs 1, 2 and 3 containing the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively, and where VH, when paired with a light chain variable region (VL) containing the amino acid sequences of SEQ ID NO: 29, 30, 31 or 40, forms an antibody or antigen-binding fragment containing VH and VL, where the antibody or antigennegative fragment binds to PD-1; (2) an immunoglobulin light chain or fragment thereof, comprising a VL containing CDRs 1, 2, and 3, comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, and where VL, when paired with a VH containing the amino acid sequences of SEQ ID NO: 26, 27, 28 or 39, forms an antibody or antigen-binding fragment containing VH and VL, where the antibody or antigennegative fragment binds to PD-1; (3) an immunoglobulin heavy chain or fragment thereof containing a VH containing CDRs 1, 2 and 3 containing the amino acid sequences of SEQ ID NOs: 7, 8 and 9, respectively, and where VH, when paired with a light chain variable region (VL) containing the amino acid sequences of SEQ ID NO: 36, 37, 38 or 42, forms an antibody or antigen-binding fragment containing VH and VL, where the antibody or antigennegative fragment binds to PD-1; (4) an immunoglobulin light chain or fragment thereof containing VL containing an amino acid sequence that is identical to SEQ ID NO: 36, 37 or 38, and where VL, when paired with a VH containing the amino acid sequences of SEQ ID NO: 32, 33, 34, 35 or 41, forms an antibody or antigen-binding fragment containing VH and VL, where the antibody or antigennegative fragment binds to PD-1; (5) an immunoglobulin heavy chain, or fragment thereof, containing VH containing CDRs 1, 2 and 3 containing the amino acid sequences of SEQ ID NOs: 7, 13 and 14, respectively, and where VH, when paired with a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 44, forms an antibody or antigen-binding fragment containing VH and VL, where the antibody or antigennegative fragment binds to PD-1; (6) an immunoglobulin light chain or fragment thereof containing VL containing CDR 1, 2 and 3 containing the amino acid sequences of SEQ ID NO: 10, 11 and 15, respectively, and where VL, when paired with a VH containing the amino acid sequence of SEQ ID NO: 43, forms an antibody or antigen-binding fragment containing VH and VL, where the antibody or antigennegative fragment binds to PD-1. 13. The nucleic acid according to claim 12, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide containing an immunoglobulin heavy chain or a fragment thereof containing a VH containing CDRs 1, 2 and 3 containing the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively . 14. Nucleic acid according to claim 12, where the nucleic acid contains a polynucleotide encoding a polypeptide containing an immunoglobulin light chain or a fragment thereof, containing a VL containing CDRs 1, 2 and 3, containing the amino acid sequences of SEQ ID NO: 4, 5 and 6, respectively . 15. The nucleic acid according to claim 12, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide containing an immunoglobulin heavy chain or a fragment thereof containing a VH containing CDRs 1, 2 and 3 containing the amino acid sequences of SEQ ID NOs: 7, 8 and 9, respectively . 16. Nucleic acid according to claim 12, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide containing an immunoglobulin heavy chain or a fragment thereof containing a VH containing CDRs 1, 2 and 3 containing the amino acid sequences of SEQ ID NOs: 7, 13 and 14, respectively . 17. The nucleic acid according to claim 12, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide containing an immunoglobulin light chain or a fragment thereof containing a VL containing CDRs 1, 2 and 3 containing the amino acid sequences of SEQ ID NOs: 10, 11 and 15, respectively . 18. Nucleic acid according to any one of claims 12-17, where VH, when paired with VL, forms an antibody or antigen-binding fragment containing VH and VL, wherein the antibody, or antigen-binding fragment thereof, can specifically bind to human PD-1, or VL, when paired with VH, forms an antibody or its antigen-binding fragment containing VH and VL, wherein the antibody, or antigen-binding fragment thereof, can specifically bind to human PD-1. 19. The nucleic acid of any one of claims 12-18, wherein the immunoglobulin heavy chain or fragment thereof is a humanized immunoglobulin heavy chain or fragment thereof and the immunoglobulin light chain or fragment thereof is a humanized immunoglobulin light chain or fragment thereof. 20. A nucleic acid according to any one of claims 12-19, wherein the nucleic acid encodes a single chain variable fragment (scFv). 21. A nucleic acid according to any one of claims 12-20, wherein the nucleic acid is cDNA. 22. An expression vector containing one or more of the nucleic acids according to any one of claims 12-21. 23. An expression vector containing two nucleic acids according to any one of claims 12-21, where two nucleic acids encode a VL region and a VH region, forming an antibody or antigen-binding fragment containing VH and VL, where the antibody or antigennegative fragment can contact PD-1. 24. A pair of expression vectors containing a first expression vector and a second expression vector, where each expression vector contains one nucleic acid according to any one of claims 12-21, where the first expression vector encodes a VH region and the second expression vector encodes a VL region, forming an antibody or antigen-binding fragment containing a VH region and a VL region, where the antibody or antigennegative fragment can contact PD-1. 25. A cell for expressing an anti-PD-1 antibody or antigen-binding fragment thereof, comprising an expression vector according to claim 22 or 23 or a pair of expression vectors according to claim 24. 26. The cell of claim 25, wherein the cell is a CHO cell. 27. A cell for expressing an anti-PD-1 antibody or antigen-binding fragment thereof, comprising one or more nucleic acids according to any one of claims 12-21. 28. A cell for expressing an anti-PD-1 antibody or antigen-binding fragment thereof, comprising two nucleic acids according to any one of claims 12-21. 29. The cell according to claim 28, where two nucleic acids together encode a VL region and a VH region, forming an antibody or antigen-binding fragment containing VH or VL, where the antibody or antigennegative fragment can contact PD-1. 30. A method for producing an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the method comprises (a) culturing the cell according to any one of claims 25 to 29 under conditions sufficient for the cell to produce an antibody or antigen-binding fragment; and (b) collecting the antibody or antigen-binding fragment produced by the cell. 31. Antibody or its antigen-binding fragment that binds to PD-1, containing a heavy chain variable region (VH) containing an amino acid sequence that is identical to the selected VH sequence, and a light chain variable region (VL) containing an amino acid sequence that is identical to the selected VL sequence, where the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 26, 27, 28, or 39, and the selected VL sequence is SEQ ID NO: 29, 30, 31 or 40; (2) the selected VH sequence is SEQ ID NO: 32, 33, 34, 35, or 41, and the selected VL sequence is SEQ ID NO: 36, 37, 38 or 42; and (3) the selected VH sequence is SEQ ID NO: 43 and the selected VL sequence is SEQ ID NO: 44. 32. The antibody or antigen-binding fragment thereof according to claim 31, where VH contains the sequence of SEQ ID NO: 26 and VL contains the sequence of SEQ ID NO: 31. 33. The antibody or antigen-binding fragment thereof according to claim 31, where VH contains the sequence of SEQ ID NO: 27 and VL contains the sequence of SEQ ID NO: 31. 34. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 26 and the selected VL sequence is SEQ ID NO: 29. 35. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 26 and the selected VL sequence is SEQ ID NO: 30. 36. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 27 and the selected VL sequence is SEQ ID NO: 29. 37. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 27 and the selected VL sequence is SEQ ID NO: 30. 38. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 28 and the selected VL sequence is SEQ ID NO: 29. 39. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 28 and the selected VL sequence is SEQ ID NO: 30. 40. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 28 and the selected VL sequence is SEQ ID NO: 31. 41. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 32 and the selected VL sequence is SEQ ID NO: 36. 42. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 32 and the selected VL sequence is SEQ ID NO: 37. 43. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 32 and the selected VL sequence is SEQ ID NO: 38. 44. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 33 and the selected VL sequence is SEQ ID NO: 36. 45. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 33 and the selected VL sequence is SEQ ID NO: 37. 46. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 33 and the selected VL sequence is SEQ ID NO: 38. 47. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 34 and the selected VL sequence is SEQ ID NO: 36. 48. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 34 and the selected VL sequence is SEQ ID NO: 37. 49. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 34 and the selected VL sequence is SEQ ID NO: 38. 50. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 35 and the selected VL sequence is SEQ ID NO: 36. 51. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 35 and the selected VL sequence is SEQ ID NO: 37. 52. An antibody or antigen-binding fragment thereof according to claim 31, wherein the selected VH sequence is SEQ ID NO: 35 and the selected VL sequence is SEQ ID NO: 38. 53. An antibody or antigen-binding fragment thereof according to any one of claims 31-52, wherein the antibody or antigen-binding fragment specifically binds to human PD-1. 54. An antibody or antigen-binding fragment thereof according to any one of claims 31-53, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof. 55. An antibody or antigen-binding fragment thereof according to any one of claims 31-54, wherein the antigen-binding fragment is a single chain variable fragment (scFV). 56. The use of an antibody or antigen-binding fragment thereof according to any one of claims 31-55 to obtain a bispecific antibody. 57. An antibody-drug conjugate for enhancing an immune response, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-11 and 31-55 covalently linked to a therapeutic agent. 58. The antibody-drug conjugate of claim 57, wherein the therapeutic agent is a cytotoxic or cytostatic agent. 59. A method of treating a subject having a cancer, wherein the method comprises administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-11 and 31-55 or an antibody-drug conjugate according to claim 57 or 58 . 60. The method of claim 59 wherein the subject has a solid tumor, inoperable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), head and neck cancer, renal cell carcinoma (RCC), melanoma, cancer bladder cancer, gastric cancer, urothelial cancer, Merkel cell carcinoma, triple negative breast cancer (TNBC), colorectal carcinoma, breast cancer, cervical cancer, endometrial cancer, glioma, liver cancer, lung cancer, lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, testicular cancer, thyroid cancer, urethral cancer, hemablastosis, small cell lung cancer (SCLC), metastatic hormone-refractory prostate cancer, or Hodgkin's lymphoma. 61. A method of reducing a tumor growth rate, wherein the method comprises contacting a tumor cell with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-11 and 31-55 or an antibody-drug conjugate according to claim 57, or 58. 62. A method of killing a tumor cell, wherein the method comprises contacting the tumor cell with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-11 and 31-55 or an antibody-drug conjugate according to claim 57 or 58 . 63. A pharmaceutical composition for enhancing an immune response, comprising an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-11 and 31-55 and a pharmaceutically acceptable carrier. 64. A pharmaceutical composition for enhancing an immune response, comprising an effective amount of the antibody drug conjugate of claim 57 or 58 and a pharmaceutically acceptable carrier. 65. An antibody or antigen-binding fragment thereof that binds to PD-1 containing a VH containing CDRs 1, 2, 3 of a selected VH sequence and a VL containing CDRs 1, 2, 3 of a selected VL sequence, where (1) the selected VH sequence is SEQ ID NO: 26, 27, 28, or 39 and the selected VL sequence is SEQ ID NO: 29, 30, 31, or 40; (2) the selected VH sequence is SEQ ID NO: 32, 33, 34, 35, or 41 and the selected VL sequence is SEQ ID NO: 36, 37, 38, or 42; or (3) the selected VH sequence is SEQ ID NO: 43 and the selected VL sequence is SEQ ID NO: 44. 66. An antibody or antigen-binding fragment thereof according to claim 65, wherein the selected VH sequence is SEQ ID NO: 26 and the selected VL sequence is SEQ ID NO: 29. 67. An antibody or antigen-binding fragment thereof according to claim 65, wherein the selected VH sequence is SEQ ID NO: 32 and the selected VL sequence is SEQ ID NO: 36. 68. An antibody or antigen-binding fragment thereof according to claim 65, wherein the selected VH sequence is SEQ ID NO: 43 and the selected VL sequence is SEQ ID NO: 44.

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