CN113372445B - anti-PD-1 monoclonal antibody - Google Patents

Abstract

The invention belongs to the field of biological medicine, provides an antibody or a functional fragment combined with human PD-1 with high affinity, and also provides an amino acid sequence for encoding the antibody or the functional fragment, comprising a light chain and a heavy chain of the antibody and an amino acid sequence of a complementarity determining region, comprising an expression vector and a host cell for expressing the antibody or the functional fragment thereof, and a production method of the antibody or the functional fragment thereof; the antibody provided by the invention has high affinity, blocks the PD-1/PD-L1 signal path, and plays an important role in human tumor treatment.

Description

anti-PD-1 monoclonal antibody

Technical Field

The invention relates to the field of biological medicine, in particular to an anti-PD-1 monoclonal antibody.

Background

Programmed Death receptor-1 (pd-1) is an important class of immunonegative regulatory molecules (also known as "immune checkpoint molecules") belonging to the CD28 family members, structurally consisting of 288 amino acid class I transmembrane glycoproteins, consisting of an extracellular region, a transmembrane region and an intracellular region. Wherein the extracellular region of the PD-1 molecule is an immunoglobulin-like variable region (IgV) domain, and the intracellular region comprises an immunoreceptor tyrosine-inhibiting motif (immunoreceptor tyrosine based inhibitory motif, ITIM) and an immunoreceptor tyrosine-converting motif (immunoreceptor tyrosine based switch motif, ITSM), and ITSM is a key motif for intracellular signaling of the PD-1 molecule. PD-1 is expressed predominantly on the surfaces of CD4+ T cells, CD8+ T cells, NK-T cells, B cells and activated monocytes, PD-1 being an immunosuppressive receptor expressed on the surfaces of activated T cells and B cells (Okazaki et al Current optics in immunology.2002,14:779-782;Bennett et al.J Immunol.2003,170:711-718), predominantly induced by T Cell Receptor (TCR) or B Cell Receptor (BCR) signals; play an important role in regulating activation and inhibition signals in the immune system (Okazaki, taku et al International immunology 2007,19 (7): 813-824).

PD-1 has two ligands PD-L1 and PD-L2, PD-L1 is a transmembrane protein consisting of 290 amino acids, and the extracellular domain has the constant region IgV of two immunoglobulins and an IgC-like domain. It is expressed mainly on mature macrophages, B cells, CD4-T cells, dendritic cells and is highly expressed in tumor cells. PD-L2 is a transmembrane protein consisting of 270 amino acids, but the PD-L2 expression region is extremely limited, being present only in macrophages and dendritic cells, and is thought to play a major role in immune presentation; it is 34% homologous to PD-L1, which belongs to the B7 ligand family, and both share a common structural basis similar to the other members of this family. PD-1 is capable of interacting with its ligands (PD-L1 and PD-L2), significantly inhibiting CD3 and CD28 mediated T cell activation and cytokine production through intracellular signaling pathways, and is therefore an important immune whistle card regulating T cell responses. Under normal conditions, the PD-1/PD-Ls signaling pathway can induce and maintain immune tolerance of peripheral tissues, and has positive effects on preventing excessive inflammatory reaction of tissues and occurrence of autoimmune diseases; when tumors and viruses are infected, the expression of the cells PD-L1 and PD-L2 is up-regulated, and the cells PD-L1 and PD-L2 interact with the surface of the T cells, so that the activation and proliferation of the T cells and the killing of the tumors can be inhibited, and the T cell function is disturbed; in the occurrence of tumors, after PD-L1 expressed by tumor cells is combined with PD-1, the tumor cells can inhibit the generation and cell proliferation of growth factors, the secretion of T cell immunostimulating cytokines such as IFN-gamma, IL-2 and TNF-alpha and the expression of survivin are regulated, the secretion of immunosuppressive cytokine IL-10 is promoted, and the immune escape of tumor cells is promoted. The PD-1/PD-L1 signaling pathway has a close relationship with tumor progression, and in tumor patients, high expression of PD-L1 can enhance the metastatic capacity of tumors, leading to increased mortality in patients and associated with poor prognosis in patients. It was found that highly expressed PD-L1 protein was detected in human tumor tissues such as lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer. Blocking PD-1/PD-L1 signals with blocking anti-PD-1 monoclonal antibodies can be achieved by up-regulating IFN-gamma, IL-2 and IL-10 secretion, effectively reversing proliferation inhibition of CD4+ and CD8+ T cells, and remarkably enhancing activation degree and killing capacity of the T cells.

Because of the broad spectrum of tumor prospects and the surprising efficacy of PD-1 antibodies, it is widely recognized by the industry that antibodies directed against the PD-1 pathway would bring breakthrough progress in the treatment of a variety of tumors. Existing PD-1/PD-L1 antibodies are FDA approved in the united states for the treatment of nearly ten tumors, including: melanoma, non-small cell lung cancer, bladder cancer, renal cancer, head and neck cancer, gastric cancer, liver cancer, hodgkin lymphoma, solid tumors of MSI-H, and the like. There are 6 anti-PD-1/PD-L1 mab drugs currently marketed by the FDA in the united states, of which anti-PD-1 mab has Pembrolizumab, nivolumab, pidilizumab; the anti-PD-L1 monoclonal antibody has Atezolizumab, durvalumab, avelumab, wherein Pembrolizumab is more a first-mentioned broad-spectrum anticancer drug. At present, 4 domestic anti-PD-1 antibodies are marketed in batches, namely Xindi monoclonal antibody of Xinda organisms, terlipressin Li Shan antibody of Junzhen organisms, tirelizumab of Baiji China and Cari Li Zhushan antibody of Hengrui.

However, there is still a need to develop new anti-PD-1 antibodies with better binding efficiency to effectively block PD-1/PD-L1 signaling pathway, exerting anti-tumor effect.

Disclosure of Invention

The present invention is based on the mechanism of action of PD-1/PD-L1 in the development of tumorigenesis, thereby providing an anti-PD-1 antibody that binds to programmed death factor 1 (PD-1) and exhibits many advantageous properties, including, for example, binding to human PD-1 with high affinity, having good biological activity, being able to block PD-1/PD-L1 signaling pathways, inhibiting the growth of tumor cells in vivo, and thus exerting an anti-tumor effect.

In a first aspect, the invention relates to an anti-PD-1 antibody or antigen-binding fragment, which antibody preferably exhibits one or more of the following characteristics:

(a) Inhibit PD-1 binding to PD-L1;

(b) At 1.0X10 ^-10 M or higher affinity binds human PD-1;

(c) Blocking the PD-1/PD-L1 signal path;

(d) Has stronger biological activity;

(e) Inhibit the growth of tumor cells in vivo.

Preferably, the antibodies of the invention are preferably humanized antibodies, and in alternative embodiments the anti-PD-1 antibodies may also be mouse or chimeric or human antibodies.

Preferably, the antibody is present in a 2.5X10 form ^-12 KD of M or lower binds human PD-1;

more preferably, the antibody is present at 1.82×10 ^-12 KD of M or lower binds human PD-1.

In a second aspect of the invention there is provided a monoclonal antibody or antigen-binding fragment that specifically binds PD-1, the antibody or antigen-binding fragment of the invention having a light chain variable region and a heavy chain variable region.

In some embodiments, the antibody or antigen binding fragment thereof comprises: a light chain variable region comprising CDR1, CDR2 and CDR3 sequences; and a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein

Preferably, the antibody variable region comprises:

(1) VL domain comprising LCDR1, LCDR2, LCDR3 as set forth in SEQ ID NO: 17. 18, 19; and

(2) VH domain comprising HCDR1, HCDR2, HCDR3 as set forth in SEQ ID NO: 20. 21, 22; or (b)

(3) Amino acid sequences having more than one amino acid conservative modification compared to the sequences of (1) - (2), or sequences having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identity or homology to the sequences.

More preferably, the antibody variable region comprises:

(1) The amino acid sequence is shown in SEQ ID NO:9, or a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identity or homology to said sequence, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions); and

(2) The amino acid sequence is shown in SEQ ID NO:10, or a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identity or homology to said sequence, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions).

(3) The amino acid sequence is shown in SEQ ID NO:13, or a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identity or homology to said sequence, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions).

(4) The amino acid sequence is shown in SEQ ID NO:14, or a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identity or homology to said sequence, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions).

The third aspect of the invention relates to an isolated nucleotide molecule comprising a nucleic acid sequence capable of encoding the variable region of an antibody of the invention, or a complement of nucleotides encoding the variable region of an antibody of the invention.

In a preferred embodiment, the nucleotide sequence encoding the antibody variable region comprises a sequence selected from the group consisting of:

a. comprises a murine antibody light chain variable region nucleotide sequence shown in SEQ ID NO. 11 or a complementary sequence thereof; and, a step of, in the first embodiment,

b. comprises a murine antibody heavy chain variable region nucleotide sequence shown in SEQ ID NO. 12 or a complementary sequence thereof; and/or

c. Comprising a nucleotide sequence selected from the group consisting of the humanized antibody light chain variable region nucleotide sequence set forth in SEQ ID NO. 15 or its complement; and, a step of, in the first embodiment,

d. comprising a nucleotide sequence selected from the group consisting of the heavy chain variable region nucleotide sequences of the humanized antibodies shown in SEQ ID NO. 16 or a complement thereof.

In a fourth aspect of the invention, there is provided an antigen, which is PD-1-mFc, which is used as an immunogen for the preparation of antibodies.

In a fifth aspect of the invention, there is provided an expression vector comprising the polynucleotide encoding the anti-PD-1 antibody or antigen-binding fragment.

In a sixth aspect of the invention there is provided a host cell transformed with an expression vector as described above, said host cell being capable of producing an antibody according to the invention, preferably said host cell is a prokaryotic cell or a yeast or a mammalian cell, more preferably said host cell is a mammalian cell, even more preferably said mammalian cell is a CHO cell.

In a seventh aspect of the invention, there is provided a hybridoma cell line. The hybridoma cell strain of the Anti-PD-1 monoclonal antibody obtained after mice are immunized by taking PD-1-mFc as an antigen is named as Anti-PD-1-15C6D10, and is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.19199, and the preservation address is North Chen West Lu No.1 of Beijing Chaoyang area, the preservation date is 2019, 12 months 11, and the hybridoma cell strain is classified and named as the mouse hybridoma cell strain.

In an eighth aspect of the present invention, there is provided a method for preparing an anti-PD-1 monoclonal antibody, comprising the steps of:

a. Constructing eukaryotic expression vectors, and transfecting host cells to obtain antigens;

b. after the antigen provided by the invention is used for immunizing a mouse, spleen cells of the mouse are obtained;

c. preparing hybridoma cell strain by adopting cell fusion technology, and screening hybridoma cell strain capable of producing high-titer monoclonal antibody.

In a preferred embodiment, the eukaryotic expression vector of step a is pCHO1.0-PD-1-mFc, and the preparation method mainly comprises the following steps: connecting PD-1 with mFc to synthesize PD-1-mFc, and optimally synthesizing a PUC-57-PD-1-mFc plasmid; then a series of operations such as enzyme digestion, enzyme ligation, transformation, cloning, plasmid extraction, identification and the like are carried out to obtain the pCHO1.0-PD-1-mFc plasmid.

The following details a preparation method of pCHO1.0-PD-1-mFc plasmid, comprising the following steps:

(1) the 24-170 amino acids of PD-1 protein are connected with mouse mFc protein through GG (GGGGS) 3linker, and sequence optimization and synthesis of PD-1-mFc gene are carried out by Kirschner Biotechnology Co.

(2) And (3) carrying out double digestion on the optimized synthesized PUC-57-PD-1-mFc plasmid and the pCHO1.0 plasmid by using an AVR II and BSTZ 17I, recovering a target fragment by using an agarose gel recovery kit, connecting the target fragment with T4 DNA ligase at 16 ℃ overnight, transforming DH5 alpha vector, picking clone, inoculating bacteria, extracting the plasmid, and carrying out double digestion identification to obtain the pCHO1.0-PD-1-mFc plasmid.

In a preferred embodiment, step b is specifically the obtaining of spleen cells from the mice after 4 immunizations of the mice.

In a preferred embodiment, step c specifically comprises fusing spleen cells of the mouse with syngeneic myeloma cells to obtain a hybridoma cell line.

In a preferred embodiment, step c specifically comprises screening hybridoma cell lines producing anti-PD-1 monoclonal antibodies by ELISA and FACS methods.

The following details a preparation method of the anti-PD-1 monoclonal antibody, comprising the following steps:

a. expressing PD-1-mFc Protein, selecting 24-170 amino acids of PD-1 Protein, connecting with mouse mFc Protein through GG (GGGGS) 3linker, performing sequence optimization and DNA synthesis by Kirschner Biotechnology, constructing eukaryotic expression vector pCHO1.0-PD-1-mFc with target gene, transfecting CHO-S cell, pressure screening and expanding culture, collecting cell culture supernatant, purifying PD-1-mFc Protein by Protein G affinity chromatography to obtain antigen with SEQ ID NO: 3.

b. Female Balb/c mice are selected for subcutaneous and intraperitoneal immunization, and spleen is boosted after three times of immunization to obtain immune spleen cells.

c. Preparing hybridoma cell lines by adopting a cell fusion technology, and screening the hybridoma cell lines capable of producing the anti-PD-1 antibody:

Prepared syngeneic myeloma cells SP2/0 and immunized mice spleen cells were prepared according to spleen cells: SP 2/0=2: 1, two cells were mixed in the ratio of 1. Cells were washed three times with 20mL of cell fusion solution added under centrifugation at 1000rpm for 5 min. Cell pellet at 1.0X10 ⁷ The density of individual/mL was suspended in the electrofusion solution and 9mL of the cell suspension was added to the fusion pool.

d. After electrofusion, the fused cells were gently transferred to a pre-warmed conditioned medium at 37℃and left at room temperature for a further 1h. The cells were packed at 1.0X10X 10 ⁴ The cells/wells were inoculated into 96-well plates for culturing.

e. Cell culture supernatants were collected, the resulting antibodies were further purified and humanized, and affinity of the purified antibodies, blocking PD-1/PD-L1 efficacy and anti-tumor effects were determined.

In a preferred embodiment, the method for determining antibody affinity in step e is the ForteBio method, which comprises the following specific steps: and (3) fixing the purified Anti-PD-1-15C6D10, 23E7D9, 35D9F4, anti-PD-1-15C6D10-H, opdivo and Keystuda by using a protein A sensor, combining and dissociating the diluted PD-1 protein with the protein A sensor of the solidified PD-1 antibody to obtain a combination constant and a dissociation constant respectively, and finally obtaining the affinity constant of the Anti-PD-1 antibody.

In a preferred embodiment, the method for determining the competitive blocking of Anti-PD-1-15C6D10-H with Opdivo and Keystuda for PD-1/PD-L1 signal pathway in step e is ELISA, comprising the following specific steps: PD-1 protein is taken as a coating antigen, anti-PD-1-15C6D10-H, OPdivo and Keystuda antibodies are respectively diluted and added, simultaneously biotin-labeled PD-L1 protein is added, goat Anti-human IgG (H+L) -HRP is taken as a secondary antibody, and finally OD450 is measured. The result shows that the antibody can strongly block the combination of PD-1 and PD-L1 and block the PD-1/PD-L1 signal path, and the effect is equivalent to that of the Opdivo and Keystuda under the experimental condition.

In a ninth aspect of the invention, there is provided a method of obtaining a humanized Anti-PD-1 monoclonal antibody Anti-PD-1-15C6D10-H, the antibody being an antibody in which murine CDR sequences are grafted onto human framework sequences, and further framework region modifications may be made within the human framework sequences. In a preferred embodiment, murine CDRs are grafted onto framework regions of human antibodies to make "humanized antibodies". Mainly comprises the following operation steps:

(1) the invention expresses PD-1-mFc protein by a method of constructing a stable cell bank, immunizes a mouse, obtains mouse hybridoma cells by a method of fusing with SP2/0 cells, screens by ELISA and FACS methods, obtains cells with better affinity, and obtains variable region genes of the hybridoma cells by an RT-PCR method;

(2) Humanization of the antibodies was performed using molecular biology software to obtain humanized antibodies.

In a preferred embodiment, the step (2) specifically includes:

a. the light and heavy chain CDR gene sequence of the murine monoclonal antibody is obtained by utilizing a genetic engineering technology (the sequence is shown as SEQ ID NO: 17-22);

b. selection of human templates (CDR grafting receptors);

c. the connection of the mouse source CDR and the human source antibody FR;

d. molecular construction and expression of humanized antibodies.

In a tenth aspect of the present invention, there is provided a method for obtaining a variable region sequence of an anti-PD-1 antibody comprising: primers were synthesized based on the constant region sequence of the antibody gene, total RNA of the hybridoma cell line was extracted, and reverse transcribed into cDNA. The heavy and light chain variable regions of the monoclonal antibodies were amplified by primer PCR. And (3) carrying out electrophoresis on the PCR product, and recovering the target fragment by gel. The target fragment is connected to a T vector pMD19-T, E.coli DH5 alpha is transformed, positive clones are picked up by plating, plasmids are extracted, and sequencing is carried out.

In an eleventh aspect of the invention, there is provided a pharmaceutical composition comprising an effective amount of the anti-PD-1 antibody that binds to PD-1 and a pharmaceutically acceptable carrier.

In a twelfth aspect of the invention, there is provided the use of the anti-PD-1 antibody that binds to PD-1 in a medicament for the treatment or alleviation of cancer, infectious diseases, wherein the cancer includes, but is not limited to lymphoma, melanoma, lung cancer, liver cancer, stomach cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, bladder cancer, glioma. Wherein the infectious disease includes, but is not limited to, a flavivirus, dengue virus, parvovirus, mumps virus.

The invention has the advantages that:

the anti-PD-1 monoclonal antibody disclosed by the invention has the advantages of high affinity, good stability, high affinity of the humanized anti-PD-1 antibody developed by the anti-PD-1 monoclonal antibody, better biological activity, stronger effect of blocking PD-1/PD-L1 signal paths and obvious effect in inhibiting tumor development.

In order to make the invention easier to understand, certain terms are first defined, but these terms are not meant to define or limit the scope of the invention.

Abbreviations and definitions

PD-1 programmed death receptor-1

PD-L1 PD-1 ligand 1

PD-L2 PD-1 ligand 2

Complementarity determining regions in immunoglobulin variable regions defined by the CDR using the Kabat numbering system

EC ₅₀ Concentration yielding 50% efficacy or binding

ELISA enzyme-linked immunosorbent assay

FR antibody framework region: immunoglobulin variable region excluding CDR regions

HRP horseradish peroxidase

IgG immunoglobulin G

mAb monoclonal antibodies

PCR polymerase chain reaction

The V region is an IgG chain segment of variable sequence between different antibodies.

VH immunoglobulin heavy chain variable regions

VL immunoglobulin light chain variable regions

KD equilibrium dissociation constant

Kon binding rate constant

Kd dissociation rate constant

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. The antibodies or fragments thereof used in the present invention may be prepared by conventional techniques known in the art, such as genetic recombination or other modification methods, alone or in combination, and methods for introducing such modifications in their DNA sequences based on the amino acid sequence of an antibody are well known to those skilled in the art; see, e.g., sambrook, molecular cloning: a laboratory manual, cold Spring Harbor Laboratory (1989) n.y. The modification referred to is preferably carried out at the nucleic acid level. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

The term "PD-1" is Programmed Death receptor-1 (PD-1) which is a member of the CD28 family, and is an immunosuppressive receptor expressed on the surface of activated T cells and B cells. PD-1 is predominantly expressed in CD4 ⁺ T cells, CD8 ⁺ T cells, NK-T cells, B cells and activated monocyte surfaces are mainly expressed by induction of T Cell Receptor (TCR) or B Cell Receptor (BCR) signaling. The term also includes any variant, isoform, species identity or analogue of PD-1 which comprises at least one common epitope with PD-1, which is expressed naturally by cells including tumor cells, or by cells transfected with the PD-1 gene or cDNA. Also, the terms are used interchangeably with programmed death 1, "programmed cell death 1," "protein PD-1," "PD1," "PDCD1," "hPD-1," "hPD1," and complete PD-1 sequences can be found in UNIPAT.

The terms "PD-L1", "PD-L2" refer to two ligands of PD-1. PD-1 is capable of interacting with PD-L1 and PD-L2, significantly inhibiting CD3 and CD28 mediated T cell activation and cytokine production through intracellular signaling pathways, and is therefore an important immune whistle card regulating T cell responses. Under normal conditions, the PD-1/PD-Ls signaling pathway can induce and maintain immune tolerance in peripheral tissues, with positive effects on preventing excessive inflammatory responses in tissues and the occurrence of autoimmune diseases. In pathological conditions, PD-1 interacts with PD-L1, PD-L2, down-regulates secretion of T-cell immunostimulatory cytokines such as IFN-gamma, IL-2 and TNF-alpha and expression of survivin, and promotes secretion of the immunosuppressive cytokine IL-10, thereby suppressing T-cell immune responses.

The PD-1/PD-L1 signaling pathway has a close relationship with tumor progression, and in tumor patients, high expression of PD-L1 can enhance the metastatic capacity of tumors, leading to increased mortality in patients and associated with poor prognosis in patients. Blocking PD-1/PD-L1 signaling with blocking anti-PD-1 mab can be accomplished by up-regulating IFN-gamma, IL-2, IL-10 secretion and effectively reversing CD4 ⁺ And CD8 ⁺ Proliferation inhibition of T cells, and at the same time, significantly enhances the activation degree and killing ability of T cells. Indications for PD-1 targets also include other related diseases or conditions found in the prior art (e.g., in lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer, etc.), as well as those not found in the future.

The term "antibody" refers herein to an immunoglobulin or antigen binding fragment, and includes any polypeptide comprising an antigen binding site that can be produced in vivo or in vitro. The term includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, nonspecific antibodies, humanized antibodies, single chain antibodies, chimeric antibodies, recombinant antibodies. As used herein, the term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of a single molecular composition. The term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., an "antigen-binding portion") or single chain thereof. An "antibody" refers to a glycoprotein or antigen binding portion thereof comprising at least two "light chains" (abbreviated LC) and two "heavy chains" (abbreviated HC) linked together by disulfide bonds. The light and heavy chains of such antibodies are polypeptides composed of several domains, each comprising a variable region, a constant region and a framework region. An antibody "variable region" refers to an antibody light chain variable region (abbreviated herein as VL) or an antibody heavy chain variable region (abbreviated herein as VH), either alone or in combination, each of which is further divided into a "hypervariable region" or "complementarity determining region" (abbreviated herein as CDR), as well as a conserved region-framework region (abbreviated herein as FR), in an antigen-binding site that facilitates the formation of the antibody. If variants of the variable regions of the antibody are desired, amino acid residue conservative substitutions in the framework regions may be made, and the antibody compared to the variable regions of other antibodies that contain the same CDR1 and CDR2 sequences as the variable regions of the antibody (Chothia and Lesk, J Mol Biol 196 (4): 901-917, 1987). The term "hypervariable region" or "CDR region" or "complementarity determining region" refers to the amino acid residues of an antibody responsible for antigen binding, non-contiguous amino acid sequences, which are distributed over a conserved region-Framework Region (FR). Each LCDR and HCDR region is composed of three CDRs and four FRs, with the order from N-terminus to C-terminus being: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are abbreviated herein as LCDR1, LCDR2, LCDR3; the three CDR regions of the heavy chain are abbreviated as HCDR1, HCDR2, HCDR3. The CDR regions comprise residues that bind to the antigen. CDR region sequences may be defined by IMGT, kabat, chothia and AbM methods or amino acid residues within the variable region identified by any CDR region sequence determination method known in the art. The Chothia CDR definitions (Chothia et al, "Canonical structures for the hypervariable regions of immunoglobulins", journal of Molecular Biology,1987,196 (4): 901-917; al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", journal ofMolecular Biology,1997,273 (4): 927-948.) are based on the three-dimensional structure of the antibody and topology of the CDR loops except for HCDR1 and HCDR2, the Chothia CDR definitions are the same as the Kabat CDR definitions, the North CDR definitions (North et al, "ANew Clustering ofAntibody CDR Loop Conformations", journal ofMolecular Biology,2011,406 (2): 228-256) are based on neighbor-propagation clusters (affinitypropagation clustering) using a large number of crystal structures.

Antibody "constant regions" refer to antibody light chain constant regions (abbreviated CL) or antibody heavy chain constant regions (abbreviated CH), individually or in combination, the light chain constant regions comprising a single constant domain, the heavy chain constant regions comprising heavy chain constant domains CH1, CH2 and CH3 (IgA, igD and IgG antibodies) and CH4 (IgE and IgM antibodies), located between IgG antibody CH1 and CH2 domains is a "hinge" domain ("H"). The structure of the light chain of an IgG molecule can be represented as N-VL-CL-C; the structure of an IgG heavy chain can be represented as N-VH-CH1-H-CH2-CH3-C (where H is the hinge domain and N and C represent the N-terminal and C-terminal ends of the polypeptide, respectively).

Antibodies can be divided into five classes, depending on the heavy chain constant region of the antibody: igG, igA, igD, igE, or IgM; the heavy chain constant regions corresponding to the different types of immunoglobulins are: gamma, alpha, delta, epsilon, and mu. Different immunoglobulins include different subtypes, such as IgG1, igG2, igG3, igG4, igA1, and IgA2. The subunit structure and spatial configuration of different types of immunoglobulins are well known in the art. There are only two types of antibody light chains: kappa and lambda. The constant regions of the heavy and light chains of antibodies are not directly involved in binding of the antibody to the target, but exhibit various effector functions.

The term "framework" residue or "FR" residue is a variable domain residue other than a hypervariable region residue as defined herein. The FR residues are relatively conserved in a given species and they can provide a scaffold for CDRs, and when non-human antibodies are prepared against a particular antigen, "humanized" variable domains can be obtained by grafting CDRs from a non-human antibody into FR regions in a human antibody.

As used herein, the term "antibody" or "antigen binding fragment" also refers to one or more fragments of an antibody comprising at least the portion of the antibody required to specifically bind to antigen PD-1, or to retain the antigen (e.g., PD-1) specific binding capacity of a parent antibody molecule, including fragments provided by any known technique (e.g., enzymatic cleavage, peptide synthesis, and recombinant techniques). For example, an antigen binding fragment may comprise at least one variable region (heavy or light chain variable region) or one or more CDRs of an antibody known to bind a particular antigen. Examples of suitable antigen binding fragments include, but are not limited to, bispecific antibodies and single chain molecules and Fab, F (ab') 2, fc, fabc, and Fv molecules, single chain (Sc) antibodies, individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains or CDRs and other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy chain and one light chain, dimers consisting of VL, VH, CL, and CH1 domains, fd fragments consisting of VH and CH1 domains; fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody, all antibody isotypes can be used to generate antigen-binding fragments. In addition, antigen binding fragments may include a non-antibody protein framework that can successfully incorporate polypeptide fragments into an orientation that confers affinity to a given antigen (e.g., a protein scaffold). Antigen binding fragments may be recombinantly produced or produced by enzymatic or chemical cleavage of intact antibodies. The terms "antigen binding fragment" and "antigen binding domain", "binding fragment" are used interchangeably. Although antigen binding fragments typically include an antibody light chain variable region and an antibody heavy chain variable region, both need not necessarily be included. For example, the Fd antibody fragments described above consist only of VH and CH1 domains.

As used herein, the term "Fab fragment" is composed of CH1 and variable regions of one light and one heavy chain.

The "Fc fragment" contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody.

"Fab ' fragments" contain portions of one light chain and one heavy chain comprising the VH domain and the CH1 domain, and the region between the CH1 and CH2 domains, and the formation of an interchain disulfide bond between the two heavy chains of two Fab ' fragments can form a F (ab ') 2 molecule.

"F (ab') 2 fragments" contain two light chains and two heavy chains comprising a constant region portion between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, a F (ab ') 2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

The "Fv region" comprises variable regions from both the heavy and light chains, but lacks constant regions.

"Single chain Fv antibody (scFv antibody)" refers to an antibody fragment comprising the VH and VL domains of an antibody, which domains are present in a single polypeptide chain. Generally, fv polypeptides additionally comprise a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding.

The term "antibody" includes, for example, murine antibodies, human antibodies, chimeric antibodies, humanized antibodies, and genetically engineered antibodies (variant or mutant antibodies) as long as their characteristic properties remain unchanged. Particularly preferred are human or humanized antibodies, in particular recombinant human or humanized antibodies.

The term "chimeric antibody" refers to an antibody comprising variable region sequences derived from an antibody of one species and constant regions derived from an antibody of another species, e.g., an antibody in which the variable region sequences are derived from a murine antibody and the constant regions are derived from human antibody constant regions.

The term "human antibody" is an antibody that has an amino acid sequence corresponding to an antibody produced by a human and/or has been prepared using techniques for preparing human antibodies. The definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding fragments.

The term "humanized antibody" generally refers to chimeric molecules prepared using recombinant techniques that have the basic sequence of the antigen-binding portion of an immunoglobulin from a non-human species and the remainder of the immunoglobulin structure of a molecule based on the structure and/or sequence of a human immunoglobulin. Humanized monoclonal antibodies as used herein, "humanized antibodies" refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences, and additional framework region modifications may be made within the human framework sequences, wherein the murine antibody CDRs have the desired specificity, affinity, and other advantageous characteristics. The "humanized" form of a murine antibody is a chimeric antibody that contains minimal sequence derived from a mouse immunoglobulin, with the majority of the humanized antibody being human immunoglobulin. In a preferred embodiment, murine CDRs are grafted onto framework regions of human antibodies to make "humanized antibodies". See, e.g., riechmann, L. et al, nature,1988,332:323-327; and Neuberger, M.S. et al, nature,1985,314:268-270. As used herein, the term "humanized antibody" refers in one embodiment to antibodies specifically of murine origin with the heavy and light chain CDRs modified with human antibody framework regions, and to other forms of antibodies according to the prior art.

Thus, if the antibody moiety is comprised in an antibody according to the invention, the antibody (or antibody moiety) may in one embodiment be an antigen binding fragment in the circumstances described above.

The term "Kon" as used herein refers to the equilibrium binding rate of a particular antibody-antigen interaction, while the term "Koff" as used herein refers to the equilibrium dissociation rate of a particular antibody-antigen interaction. The term "KD" as used herein refers to the equilibrium dissociation constant, which is obtained from the ratio of Koff to Kon, i.e. Koff/Kon, the KD value of an antibody can be determined by methods established in the art. KD binding affinity constants can be determined, for example, by enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (BIAcore tm) or biological layer interferometry (e.g., using ProteOn ^ТМ XPR36SPR (Berle Life medicine) or Octet ^ТМ System) to measure.

High affinity is a precondition for successful immune experiments and is also a basic index for measuring high-quality monoclonal antibodies. Non-specific interactions of antibody drugs can cause side effects, so high affinity is important for the preparation of some high quality antibody drugs. For IgG antibodies, the term "high affinity" refers to a target antigen of 10 ^-8 M or less, more preferably 10 ^-9 M or less, even more preferably 10 ^-10 Antibodies to KD of M or lower. However, for other antibody isotypes, "high affinity" binding may be different. For example, "high affinity" binding for IgM isotype refers to having 10 ^-7 M or less, more preferably 10 ^-8 M or less, even more preferably 10 ^-9 Antibodies to KD of M or lower. By "specifically binds to PD-1 or to PD-1" is meant that the antibody is capable of binding to the target PD-1 with sufficient affinity such that the antibody is useful as a therapeutic agent targeting PD-1. In one embodiment, the antibody that binds to PD-1 has 10 ^-8 M or less, preferably 10 ^-12 Dissociation constant (Kon) of M.

The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence" or "polynucleotide sequence" and "polynucleotide" are used interchangeably. They refer to any length of nucleotide in polymeric form, including deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides comprise single-stranded and double-stranded forms. The nucleic acid may be present in whole cells, in cell lysates, or in partially purified or substantially pure form. Other cellular components or contaminants may be purified by standard techniques, including column chromatography, agarose gel electrophoresis, and other techniques well known in the art, and standard molecular biology techniques may be used to obtain the nucleic acids of the invention. For antibodies expressed from hybridomas, cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques.

The term "vector", as used herein, refers to a construct capable of delivering and preferably expressing one or more genes or sequences in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid vectors, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

The following will describe various aspects of the invention in further detail.

anti-PD-1 antibodies

The antibodies of the invention are characterized by specific functional features or properties of the antibodies, e.g., the specific antibodies specifically bind PD-1. Preferably, the antibodies of the invention bind PD-1 with high affinity, e.g.at 1.0X10 ^-10 M or higher affinity binds PD-1. The anti-PD-1 antibodies of the invention preferably exhibit one or more of the following characteristics:

(a) Inhibit PD-1 binding to PD-L1;

(b) At 1.0X10 ^-10 M or higher affinity binds human PD-1;

(c) Competitive blocking of the PD-1/PD-L1 signaling pathway with marketed anti-PD-1 antibodies;

(d) Has stronger biological activity;

(e) Inhibit the growth of tumor cells in vivo.

In general, common techniques for humanizing murine antibodies typically produce such humanized antibodies: has reduced antigen binding affinity compared to the original murine antibody (Almagro et al front in bioscience.2008,13:1619-1633;Foote et al.Jorunal of Molecular Biology.1992,224 (2): 487-499;Hwang et al.Methods.2005,36 (1): 35-42). Surprisingly, the humanized antibodies of the invention show affinities very close to those of murine antibodies.

Preferably, the antibody is present in a 2.5X10 form ^-12 M or higher affinity binds human PD-1; more preferably, the antibody is present at 1.82×10 ^-12 M or higher affinity binds human PD-1.

Antibodies of the invention may exhibit any combination of the above features, such as two, three or more of the above features.

Standard assays for assessing the binding capacity of the antibodies to PD-1 are known in the art and include ELISA, westem block and RIA. Suitable assays for assessing any of the above features are described in detail in the examples.

Preferred antibodies of the invention are monoclonal antibodies Anti-PD-1-15C6D10 and humanized monoclonal antibodies Anti-PD-1-15C6D10-H, in some embodiments humanized antibodies Anti-PD-1-15C6D10-H retain all CDR sequences of murine antibodies Anti-PD-1-15C6D10, in other embodiments humanized antibodies may have one or two, three, four, five or six CDRs whose sequences differ from the original antibody sequences, the isolation and structural characterization of which are described in the examples. In one aspect, the invention provides heavy chain CDR1, CDR2, CDR3 and light chain CDR1, CDR2 and CDR3 of an anti-PD-1 antibody. The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 17. The amino acid sequence of the light chain CDR2 is shown as SEQ ID NO. 18; the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 19; the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO:20; the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO. 21; the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 22. CDR regions were identified using the Kabat system (Kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest, fifth Edition, U.S. device ofHealth and Human Services, NIH Publication No. 91-3242).

In another aspect, the invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds PD-1, preferably human PD-1.

Preferred heavy and light chain combinations of anti-PD-1 antibodies or antigen-binding fragments thereof comprise:

(1) Murine antibody Anti-PD-1-15C6D10 heavy and light chain variable region amino acid sequences:

a. a light chain variable region comprising SEQ ID NO: 9; and

b. a heavy chain variable region comprising SEQ ID NO:10, an amino acid sequence of seq id no; and/or

(2) Humanized antibody Anti-PD-1-15C6D10-H heavy and light chain variable region amino acid sequences:

c. a light chain variable region comprising SEQ ID NO:13, an amino acid sequence of seq id no; and

d. a heavy chain variable region comprising SEQ ID NO:14, and a sequence of amino acids.

In other embodiments, one of skill in the art may substitute (e.g., conservatively substitute), add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to the CDR regions or light or heavy chain variable region sequences of the invention, to obtain variants of said antibody or antigen binding fragment sequences, resulting in modified antibody sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to the source antibody sequence, without materially affecting the biological activity of the antibody of the invention. Antibodies in which VH and/or VL have higher homology to VH and VL of the above sequences can also be obtained by mutagenesis or conservative sequence substitution of the nucleic acid molecule encoding SEQ ID nos:9, 10, 13, 14, followed by testing the encoded altered antibodies for retained function (i.e., the functions described in (a) to (e) above) using the functional assays described herein. They are all considered to be included within the scope of the present invention.

The amino acid sequence encoding the light chain of the murine antibody (SEQ ID NO: 9) is shown below, with the amino acid sequence encoding the CDR shown bolded:

the heavy chain amino acid sequence (SEQ ID NO: 10) encoding the murine antibody is shown below, with the amino acid sequence encoding the CDR shown bolded:

the amino acid sequence encoding the humanized antibody light chain (SEQ ID NO: 13) is shown below, with the amino acid sequence encoding the CDR shown bolded:

the amino acid sequence encoding the heavy chain of the humanized antibody (SEQ ID NO: 14) is shown below, with the amino acid sequence encoding the CDR shown bolded:

by "conservative sequence modifications" is meant amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications may be introduced to the antibodies of the invention by standard techniques well known to those skilled in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). It is known in the art that conservative substitutions typically do not cause a significant change in the conformational structure of the protein, and thus are capable of preserving the biological activity of the protein.

Nucleic acid molecules encoding antibodies of the invention:

another aspect of the invention relates to nucleic acid molecules encoding the antibodies of the invention. The nucleic acid molecules include nucleotide sequences encoding the light chain variable regions of the antibodies of the invention, nucleotide sequences encoding the heavy chain variable regions of the antibodies of the invention, nucleotide sequences encoding the light chains of the antibodies of the invention, and/or nucleotide sequences encoding the heavy chains of the antibodies of the invention.

Preferred nucleic acid molecules of the invention are nucleic acid molecules which encode VL and VH sequences of Anti-PD-1-15C6D10 and Anti-PD-1-15C6D 10-H. DNA sequences encoding the VH sequences of Anti-PD-1-15C6D10 and Anti-PD-1-15C6D10-H are shown in SEQ ID NO:12 and 16, respectively. DNA sequences encoding VL sequences of Anti-PD-1-15C6D10 and Anti-PD-1-15C6D10-H are shown in SEQ ID NO:11 and 15, respectively.

An exemplary polynucleotide encoding Anti-PD-1-15C6D10 VL (SEQ ID NO: 11) is shown below:

an exemplary polynucleotide encoding an Anti-PD-1-15C6D10 VH (SEQ ID NO: 12) is shown below:

an exemplary polynucleotide encoding Anti-PD-1-15C6D10-H VL (SEQ ID NO: 15) is shown below:

an exemplary polynucleotide encoding an Anti-PD-1-15C6D10-H VH (SEQ ID NO: 16) is shown below:

the polynucleotide sequences of the variable domains of antibodies may be used to express anti-PD-1 antibodies in large amounts, or may be used to produce such derivatives and/or to improve the affinity or other characteristics of such antibodies. Methods for preparing antibody derivatives, e.g., humanized antibodies, chimeric antibodies, and other types of antibodies, are well known in the art. In the alternative, antibodies may also be recombinantly produced by phage display technology.

In some embodiments, the nucleotides encoding the antibodies CH and/or CL may be converted to full length antibody heavy and/or light chain genes. For example, the DNA fragments encoding VH and VL are obtained first, and further manipulation of these DNA fragments by standard recombinant DNA techniques can convert the genes of the antibody variable region into full-length antibody chain genes, fab fragment genes or (Fab') 2 fragment genes or scFv genes. The above DNA fragment encoding VL or VH may be linked to another protein, such as an antibody constant region. Ligating the DNA encoding the VH to another DNA molecule encoding a heavy chain constant region may convert the isolated DNA encoding the VH into a full length heavy chain gene. Ligating the DNA encoding the VL to another DNA molecule encoding a light chain constant region may convert the isolated DNA encoding the VH to a full length heavy chain gene.

The invention also relates to expression vectors comprising the above nucleotide sequences, which can be used to transform suitable host cells for expression of the antibodies, a means for allowing expression of the antibodies. Expression vectors include, but are not limited to, plasmids, phages, lentiviruses.

As used herein, "host cell" refers to a cell harboring a recombinant expression vector, and the invention provides host cells comprising a nucleotide sequence encoding an antibody or heavy chain or antigen-binding fragment thereof of the invention, and/or a nucleotide sequence encoding an antibody or light chain or antigen-binding fragment thereof of the invention, and/or both. The host cell may be a prokaryotic cell, a eukaryotic cell. Prokaryotic cells include, but are not limited to, bacterial cells; eukaryotic cells include, but are not limited to, yeast cells, E.coli, etc., wherein host cells-mammalian cells for expression are well known to those skilled in the art, e.g., CHO cells, NSO cells, or HEK-293 cells. Preferred cell lines are determined by high expression levels, and in one embodiment of the invention, CHO cells expressing anti-PD-1 antibodies are preferred.

The recombinant DNA may be transformed/transfected into a suitable host cell using conventional techniques well known to those skilled in the art. When the expression vector of the gene encoding the antibody is introduced into a host cell, the antibody expressed by the host cell may be secreted to the outside of the cell by selecting a conventional medium according to the host cell used, and the antibody may be recovered and purified by methods well known to those skilled in the art. Separation and purification methods include, but are not limited to, salting out, analytical sieve chromatography, ion exchange chromatography, high performance liquid chromatography, or a combination of methods.

Preparation of monoclonal antibodies of the invention:

the monoclonal antibodies of the invention can be prepared by a variety of techniques, including conventional monoclonal antibody preparation methods, such as classical hybridoma techniques (the hybridoma method described by Kohler & Milstein, nature.1975, 256:495-497), although in principle, other methods can be used to prepare monoclonal antibodies. The present invention employs classical hybridoma technology to produce monoclonal antibodies, and immunization protocols and techniques, as well as cell fusion techniques, are well known in the art.

The antibodies of the invention can be readily produced in mammalian cells (e.g., CHO, NSO, COS cells), and techniques for culturing host cells are well known in the art.

The humanized antibody of the present invention can be prepared according to the sequence of the above-prepared murine monoclonal antibody, and DNA encoding heavy and light chain immunoglobulins can be obtained from the target murine hybridoma and engineered to contain human immunoglobulin sequences using standard molecular biology techniques. To create humanized antibodies, murine CDR regions can be inserted into human framework regions using methods known in the art (see U.S. Pat. No. 5,372 to Winter and U.S. Pat. No. 5,30101 to Queen et al; U.S. Pat. No. 62,42; U.S. Pat. No. 5,83 and U.S. Pat. No. 62,62).

Characterization of antibodies binding to antigen:

antibodies of the invention can be assayed for binding to PD-1 by methods such as standard ELSIA. In other embodiments of the invention PD-1 is coated in an ELISA plate and then blocked with BSA. Blood from immunized mice was added to each well and incubated at 37℃for 2h. Washing with PBST, then adding enzyme-labeled antibody-secondary antibody, and incubating at 37 ℃ for 1h. The wash was washed with PBST, developed, and OD450 values were determined. Preferably, mice producing high titers of antibodies are used for subsequent fusion experiments. The ELSIA method described above can also be used to screen hybridoma cell lines that react positively with PD-1 and subcloning them, from which high titer hybridoma cell lines are selected for culture for purification of antibodies.

To purify the anti-PD-1 antibodies, the cell culture supernatant may be filtered and concentrated, followed by affinity chromatography with protein a. And simultaneously, carrying out identification of the purity of the antibody by SDS-PAGE. And (5) sub-packaging the obtained monoclonal antibody and preserving at low temperature.

To determine the binding capacity of an antibody to an antigen, fortebio may be used to determine the affinity of the protein of interest. And (3) solidifying the purified PD-1 antibody by using the protein A sensor, diluting the PD-1 protein by 6 times, combining with the protein A sensor of the solidified PD-1 antibody, dissociating to obtain a combination constant and a dissociation constant respectively, and finally obtaining the affinity constant of the PD-1 monoclonal antibody.

The application of the invention:

the antibodies and methods of the invention have numerous in vitro and in vivo uses, including, for example, detection of PD-1 or enhancement of immune responses by blocking PD-1/PD-L1 signaling pathways. For example, antibodies can be administered to cells in vitro or cultured ex vivo or to animals, e.g., in vivo, and can enhance immunity in a variety of situations. In a specific embodiment, the method is particularly suitable for inhibiting the development of cancer cells in vivo, and in a preferred embodiment, the antibody of the invention is a humanized antibody, or the antibody may be a chimeric antibody or a human anti-PD-1 antibody.

Preferred cancers that may be inhibited using the antibodies of the invention should include cancers that are generally responsive to immunotherapy. Examples of preferred cancers for treatment may include, but are not limited to, melanoma, prostate, breast, lung, bone, pancreas, uterus, ovary, colorectal cancer, for example.

PD-1 blockade can also be combined with standard cancer therapies. PD-1 blockade may be effective in combination with chemotherapeutic regimens; PD-1 blocking antibodies may also be used in combination with bispecific antibodies that target effector cells expressing fcα receptors to tumor cells (see, e.g., US5922845 and US 5837243).

The antibodies of the invention may be used for in vitro diagnostic purposes, e.g., the antibodies of the invention may be used to detect the expression level of PD-1 in a sample (e.g., blood sample, urine sample, etc.) from a patient; methods that can be used for detection include immunological methods such as flow cytometry, ELISA, immunohistochemistry, and also PD-1 antibody diagnostic kits.

Drawings

FIG. 1 determination of PD-1 expressing positive CHO-K1 cells and negative CHO-K1 cells

FIG. 2 SDS-PAGE identification of purified antibodies

FIG. 3 determination of Anti-PD-1-15C6D10-H, opdivo and Keystuda blocking PD-1/PD-L1 Signal pathway

FIG. 4 determination of biological Activity of anti-PD-1 antibodies

FIG. 5 measurement results of inhibition of tumor growth by anti-PD-1 antibodies

Detailed Description

The present invention is described in further detail below by way of examples, which are set forth to illustrate, but not limit, the invention. The contents of all figures and references, patents and published patent applications cited throughout the application are hereby expressly incorporated by reference.

In the following examples, materials used may be purchased or prepared by reference to the techniques disclosed herein; both source and gauge are not indicated as commercially available; various processes and methods not described in detail are conventional methods well known in the art.

Example 1 preparation of immunogens

(1) Expression of PD-1-mFc proteins

(1) The amino acid sequence of PD-1 is from Uniprot Q15116, and the sequence is shown in SEQ ID NO:1, selecting 24-170 amino acids of PD-1 protein, then connecting with mouse mFc protein (mouse IgG Fc (3 HKF-A) 2-214 bit sequence is shown as SEQ ID NO: 2) through GG (GGGGS) 3, and performing sequence optimization and PD-1-mFc DNA synthesis by Kirschner Biotechnology Co.

(2) And (3) carrying out double digestion on the optimized synthesized PUC-57-PD-1-mFc plasmid and the pCHO1.0 plasmid by using an AVR II and BSTZ 17I, recovering a target fragment by using an agarose gel recovery kit, connecting the target fragment with T4 DNA ligase at 16 ℃ overnight, transforming DH5 alpha vector, picking clone, inoculating bacteria, extracting the plasmid, and carrying out double digestion identification.

(3) Identification of the correct plasmid was sent to Shanghai Bioengineering Co.Ltd for sequencing and identification, and the amino acid sequence was shown as SEQ ID NO:3, the gene sequence is shown as SEQ ID NO: 4.

The linearized plasmid pCHO 1.0-PD-1-mFc was transfected into CHO-S cells using the liposome method (Freestyle MAX, invitrogen) to detect PD-1-mFc fusion protein expression. The specific operation is as follows:

(1) plasmid transfection was followed by Freestyle ^TM Instructions for use of MAX transfection reagent the day before transfection of CHO-S cells was followed by 0.5X10 ⁶ passaging/mL, counting the day of transfection, and adjusting the cell density to 1.0X10 ⁶ /mL，Culturing at 37 ℃.

(2) Preparation of transfection solution 50. Mu.g of linearized plasmid pCHO 1.0-PD-1-mFc was added to OptiPRO ^TM SFM medium to 1.5mL, 50. Mu. LFRESTYLE ^TM MAX transfection reagent 1.45mL OptiPRO was added ^TM SFM culture medium, respectively mixing, slowly adding the transfection reagent mixed solution into the DNA mixed solution, mixing, standing at room temperature for 10min, and slowly dripping the transfection mixed solution into 30mL of CHO-S cells. 8% CO ₂ Culturing at 37℃and 130 rpm.

(3) After 48h transfection, puromycin and MTX with different concentrations are pressurized, and after 2 rounds of pressurization, the cell activity is recovered to 90%, and 4 branches are frozen; and taking a part of the cells to 0.3X10 ⁶ Inoculating 30 mL/mL, feeding and culturing, and collecting supernatant.

(2) Purification of PD-1-mFc proteins

Cell culture supernatants were collected and antibody purification was performed using Protein G affinity chromatography.

After the column was first equilibrated with 20mmol/LPBS (pH 7.4), the cell culture supernatant centrifuged and filtered through a 0.4 μm filter was passed through the column. Then, the sample was washed with 20mmol/LPBS (pH 7.4) to reach an OD value to a base line, eluted with 20mmol/L citric acid (pH 3.2) solution, the eluate in the peak region was collected, the pH was adjusted, and the protein concentration was measured by the BCA method after dialysis.

EXAMPLE 2 acquisition of CHO-K1-PD-1 positive cells

(1) Synthesizing a target gene for encoding PD-1 by using a gene synthesis technology, wherein the nucleotide sequence of the target gene is shown as SEQ ID NO: 23.

(2) The optimized synthesized PUC-57-PD-1 plasmid and pCHO 1.0 plasmid are digested with AvrII and BstZ 17I, target fragments are recovered by agarose gel recovery kit, the target fragments are connected with T4 DNA ligase at 16 ℃ overnight, E.coli DH5 alpha is transformed, and positive clones are picked up for small plasmid extraction after overnight culture.

(3) The restriction enzymes avrII and BstZ 17I are subjected to double digestion and then agarose gel electrophoresis identification, the plasmid with correct identification is sent to Shanghai bioengineering limited company for sequencing identification, and clones with correct sequencing are selected for large quantity of plasmid extraction.

(4) Linearizing with restriction enzyme NruI, recovering plasmid by ethanol precipitation, sterilizing by heating at 95deg.C for 15min, cooling at room temperature, and preserving at-20deg.C.

(5) The linearized plasmid pCHO1.0-PD-1 was transfected into CHO-K1 cells using the liposome method (Freestyle MAX, invitrogen) and protein expression was examined in a flow-through manner. Plasmid transfection was followed by Freestyle ^TM MAX transfection reagent was prepared according to the instructions for use, and the day before transfection of CHO-K1 cells was performed according to 0.5X10 ⁶ passaging/mL, counting the day of transfection, and adjusting the cell density to 1.0X10 ⁶ Culture at 37 ℃. Then, a transfection solution was prepared and 50. Mu.g of linearized plasmid pCHO1.0-PD-1 was added to OptiPRO ^TM SFM medium to 1.5mL, 50. Mu.L Freestyle ^TM MAX transfection reagent 1.45mL OptiPRO was added ^TM SFM culture medium, respectively mixing, slowly adding the transfection reagent mixed solution into the DNA mixed solution, mixing, standing at room temperature for 10min, and slowly dripping the transfection mixed solution into 30mL of CHO-K1 cells. 8% CO ₂ Culturing at 37℃and 130 rpm.

(6) After 48h of transfection, puromycin and MTX with different concentrations are pressurized, and the cell viability is recovered to 90% after 2 rounds of pressurization; and taking a part of the cells to 0.3X10 ⁶ 30mL was inoculated per mL for flow-through detection of cell monoclonalization.

(7) Cells in logarithmic growth phase were diluted to 2cells/mL, 200. Mu.L of cells per well was inoculated into 96-well plates, and after 7 days, the monoclonal was observed under a microscope and labeled, and after the cells were grown, the cells were subjected to expansion culture for flow assay for the expression of cell surface PD-1. And taking untransfected CHO-K1 cells as negative cells, taking transfected cells as cells to be tested, adding FITC-labeled PD-1 antibodies for incubation after PBS washing, detecting by a flow cytometer after washing, and screening to obtain the CHO-K1 cells with high PD-1 expression. The measurement results are shown in FIG. 1.

EXAMPLE 3 preparation and identification of monoclonal antibodies

(1) Immunization of mice

Mixing the obtained PD-1-mFc protein with Freund's complete adjuvant or Freund's incomplete adjuvant, stirring for 1.5 hr, emulsifying to obtain water-in-oil emulsion, performing subcutaneous and intraperitoneal immunization, wherein the primary immunization dose is 100 μg/dose, the other immunization doses are halved, the immunization interval is 14 days, spleen is strengthened after three times of immunization, and cell fusion is performed after three days.

(2) Cell fusion

(1) Preparation of spleen cells

The immunized mice were sacrificed by cervical removal and soaked in 75% alcohol for 3-4min. The spleen was removed, placed in a petri dish with a cell strainer, a small amount of 1640 medium without serum was added, crushed with a syringe plunger until no tissue clumps and cells were uniform, and the cell suspension was transferred to a 50mL centrifuge tube. 1000rpm, centrifuging for 5min, discarding supernatant, adding 20mL of erythrocyte lysate, and standing at room temperature for 3min. The cell suspension was filtered into a 50mL centrifuge tube with a cell strainer. 1000rpm, centrifuging for 5min, discarding the supernatant, suspending in 20mL of 1640 medium without serum, and counting for later use.

(2) Preparation of myeloma cell SP2/0

The cultured SP2/0 cells were blown evenly with a sterile pipette, transferred to a 50mL centrifuge tube, centrifuged at 1000rmp for 5min, and the supernatant was aspirated. SP2/0 cells were suspended in 20mL of 1640 medium without serum, 1000rmp, centrifuged for 5min, and the supernatant was discarded. Suspended in 20mL of 1640 medium without serum. Counting for standby.

(3) Electrofusion operation

According to spleen cells: SP 2/0=2: 1, two cells were mixed in the ratio of 1. Cells were washed three times with 20mL of cell fusion solution added under centrifugation at 1000rpm for 5 min. Cell pellet at 1.0X10 ⁷ The density of individual/mL was suspended in the electrofusion. 9mL of the cell suspension was added to the fusion cell, and electrofusion was performed for 30 seconds as follows.

AC 40-60V	30s
		DC 800V-1900V	40μs，3×
POSTAC 4-6V	3s

After electrofusion, the fused cells were gently transferred to a pre-warmed conditioned medium at 37℃and left at room temperature for a further 1h. The cells were packed at 1.0X10X 10 ⁴ The individual/wells were seeded into 96-well plates.

(3) Selection of hybridoma cells

(1) PD-1-His protein (Beijing Yiqiao Shenzhou) coated ELISA plate, 40 ng/well, 4 ℃ coating overnight.

(2) Skim milk with mass fraction of 5% was blocked at 37℃for 2h, serial diluted mouse antisera were added and incubated at 37℃for 2h.

(3) Wash 3 times with PBST, add horseradish peroxidase labeled goat anti-mouse secondary antibody (1:8000) and incubate for 1h.

(4) Washed 5 times with PBST and developed for 10min in the dark with TMB.

(5) The coloration was stopped by 2mol/L sulfuric acid.

(6) And (3) measuring an OD value at 450nm by using an enzyme-labeled instrument, and screening positive clone cells.

(4) FACS-based cell-level positive clone selection

To confirm that anti-PD-1 antibodies are capable of binding naturally to conformational PD-1 molecules expressed on the cell membrane, the present invention utilizes FACS for further screening on transfected CHO-K1 cell lines expressing human PD-1 molecules.

(1) Taking positive cells CHO-K1/PD-1 and negative cells CHO-K1 in exponential culture period, centrifuging to collect cells, re-suspending cells with PBS, and adjusting cell concentration to make cell number per well 2.5X10 ⁵ And each.

(2) 100 mu L of hybridoma clone supernatant to be detected and 50 mu L of prepared cell suspension are added into a 96-well plate, the mixture is incubated for 50min by a shaking table at 4 ℃, 150 mu L of cold PBS is added into the 96-well plate after the first-antibody incubation is finished, the mixture is blown and evenly mixed by a gun head, the 96-well plate is placed into a plate centrifuge for balancing, the supernatant is carefully sucked by a gun after centrifugation at 2000rpm for 3min, and the operation is repeated for 1 time.

(3) Adding a gold anti-mouse IgG (H+L) iFluor 647 fluorescent secondary antibody, 80 mu L per well, incubating for 40min at 4 ℃ by a shaker, adding 150 mu L of cold PBS into a 96-well plate after the secondary antibody incubation is finished, blowing and uniformly mixing by a gun head, putting the 96-well plate into a plate-type centrifuge for balancing, centrifuging at 2000rpm for 3min, carefully sucking the supernatant by a gun, and repeating the operation for 1 time.

(4) Positive clones were detected by flow cytometry, and hybridoma cell lines numbered Anti-PD-1-15C6D10, 23E7D9 and 35D9F4, respectively, were finally obtained by screening.

(5) Mass production and purification of antibodies

The Anti-PD-1-15C6D10, 23E7D9 and 35D9F4 obtained in example 2 were resuscitated, passaged and expanded, and cell culture supernatants were collected. Antibodies were purified using protein a affinity chromatography. The preparation of ProteinA affinity column, with PBS balance column, centrifugation and 0.4 μm filter membrane filtration of the cell culture supernatant column, then with PBS to the OD value close to zero, 50mmol/LpH3.2 glycine-hydrochloric acid buffer elution, collecting peak area eluent, dialysis for use.

(6) Identification of antibodies

SDS-PAGE reduction electrophoresis is used for detecting the size and purity of the target protein, electrophoresis is carried out according to the method of the fourth part of the 2015 edition of Chinese pharmacopoeia, and the gray level scanning of an electrophoresis chart is carried out to identify the molecular weight and the expression quantity of the monoclonal antibody. As shown in FIG. 2, the purity of the protein is greater than 95% for light chain of about 55KD and heavy chain of about 25 KD.

Example 4 affinity assay of antibodies

The target protein affinity was measured using a biological layer interferometry (Fortebio) assay. And (3) solidifying the purified PD-1 antibody by using the protein A sensor, diluting the PD-1-his protein by 6 concentrations, combining with the protein A sensor of the solidified PD-1 antibody, dissociating to obtain a combination constant and a dissociation constant respectively, and finally obtaining the affinity constant of the PD-1 monoclonal antibody. The results of the affinity measurements for the antibodies are shown in Table 1.

TABLE 1 affinity assay results for murine anti-PD-1 monoclonal antibodies

Antibodies to	KD(M)	Kon(1/Ms)	Koff(1/s)
				Anti-PD-1-15C6D10	1.82E-12	8.34E+06	1.52E-5
23E7D9	1.41E-08	1.53E+05	2.17E-03
				35D9F4	4.72E-08	1.04E+05	4.91E-03

EXAMPLE 5 humanization of monoclonal antibodies

Selecting Anti-PD-1-15C6D10 with high antibody affinity for antibody sequencing and subsequent humanization of the antibody, and mainly comprises the following operation steps:

(1) the monoclonal antibody variable region was sequenced and the following primers were synthesized based on the constant region sequence of the antibody gene:

total RNA of the hybridoma cell line was extracted with TAKARA's RNA extraction kit, and the RNA was reverse transcribed into cDNA. Primers were used to amplify the light and heavy chain complementarity determining regions of the monoclonal antibody, and PCR conditions were: 94 ℃ for 5min;94 ℃ for 30s,58 ℃ for 30s and 72 ℃ for 1min; and at 72℃for 10min. And (3) carrying out agarose gel electrophoresis on the PCR product, and recovering the target fragment by gel. The fragment of interest was ligated to T vector pMD19-T, E.coli DH 5. Alpha. Transformed, plated on ampicillin-resistant LB plates, positive clones were picked, miniplasmids were sequenced, and the results were shown in tables 2 and 3.

(2) Selecting a monoclonal antibody Anti-PD-1-15C6D10 with high affinity for humanized transformation: obtaining a light and heavy chain CDR gene sequence of the murine monoclonal antibody; selecting a human template (CDR grafting receptor); the molecular construction of humanized antibodies is accomplished by linking murine CDRs with human antibody FRs and designing back mutations for some amino acids that may affect the structure of the antibody.

(3) Then ligating to expression vector to transform E.coli DH 5. Alpha. Competent cells.

(4) The humanized antibody Anti-PD-1-15C6D10-H is expressed by the transformed CHO cells.

TABLE 2 variable region amino acid sequence and nucleotide sequence of anti-PD-1 monoclonal antibodies

TABLE 3 amino acid sequences of Anti-PD-1-15C6D10 heavy chain complementarity determining regions

EXAMPLE 6 humanized monoclonal antibody affinity assay

(1) Diluting Anti-PD-1-15C6D10-H, keytruda, opdivo to 10 μg/mL with a sample diluent (PBST dilution kinetic Buffer); PD-1 protein was also diluted with sample dilutions to 100nM, 50nM, 25nM, 12.5nM, 6.25nM.

(2) Placing the sample, the analyte, the buffer solution and the sensor into a corresponding 96-well plate and placing into an instrument; setting the attribute of each hole in the instrument according to the adding sequence of the samples; the experimental steps are set: baseline 60s, loading 120s (immobilized antibody set up to jump to the next step when the signal reaches 2 nm), baseline2180 s, association s (antigen binding), association 600s, sensor regeneration.

(3) After the setting is finished, running a test; after the test run was completed, the results were analyzed using ForteBio DateAnalysis 10.0-kinetic. The results of the antibody affinity measurements are shown in Table 4. The KD value for Anti-PD-1-15C6D10-H is 2.50E-12, which is significantly higher than the KD value of Keytruda, opdivo, indicating that Anti-PD-1-15C6D10-H binds PD-1 with higher affinity.

TABLE 4 humanized monoclonal antibody affinity assay results

Antibodies to	KD(M)	Kon(1/Ms)	Koff(1/s)
				Anti-PD-1-15C6D10-H	2.5E-12	7.50E+6	1.89E-5
Anti-PD-1-15C6D10	1.82E-12	8.34E+6	1.52E-5
				Keytruda	1.01E-9	2.83E+5	2.86E-4
Opdivo	1.63E-9	1.20E+5	1.95E-4

Example 7 Anti-PD-1-15C6D10-H, keytruda, opdivo Competition blocking of PD-1/PD-L1 Signal pathway

The humanized antibody Anti-PD-1-15C6D10-H can be detected to block the PD-1/PD-L1 signal path, and the specific implementation method is as follows:

(1) human PD-1 protein was diluted with PBS and 100. Mu.L per well was plated in 96-well plates, membrane-sealed with a sealing plate, allowed to stand at 4℃overnight.

(2) Discarding the supernatant; preparing 2% skimmed milk powder-PBST sealing solution, adding 350 mu L of the sealing solution into a 96-well plate, and sealing and incubating for 2h at room temperature; anti-PD-1-15C6D10-H, keytruda, opdivo was diluted with 2% nonfat dry milk-PBST.

(3) After PBST washing the plate for 4-5 times, 50 mu L of diluted antibody to be detected is added into each hole, and 50 mu L of biotin-labeled PD-L1 protein is added at the same time, and the plate is kept stand for incubation for 1.5h at room temperature.

(4) After washing the plate with PBST for 4-5 times, 100. Mu.L of Goat anti-human IgG (H+L) -HRP was added to each well, and the plate was allowed to stand at room temperature for incubation for 1.5 hours.

(5) After PBST washing the plate for 4-5 times, adding TMB substrate into 100 mu L of each hole, and incubating at room temperature for color development for 10min; the color development was stopped by adding 100. Mu.L of 1M HCl per well and the absorbance was read at 450nm using a microplate reader.

(6) The graph Padprism5 software is used for analyzing data by taking the logarithm of concentration as an abscissa and the absorbance value as an ordinate, the results are shown in fig. 1 and table 5, and experimental results show that Anti-PD-1-15C6D10-H can strongly block the combination of PD-1 and PD-L1 and block the PD-1/PD-L1 signal path, and the experimental results are equivalent to the effects of Opdivo and Keystuda under the experimental conditions, and the experimental results are shown in fig. 3 and table 5:

TABLE 5 determination of competitive blocking of PD-1/PD-L1 Signal pathway by antibodies

Antibodies to	EC50(nM)
		Anti-PD-1-15C6D10-H	172.3
Keytruda	110.1
		Opdivo	175.4

Example 8 PD-1 determination of antibody biological Activity

The pharmacodynamic activity of the recombinant humanized anti-PD-1 monoclonal antibody is based on high affinity with human PD-1 protein and blocking PD-1/PD-L1 signal path. GS-C2/PD-L1 cells are tool cells for stably overexpressing PD-L1 (GS-C2 is a CHO cell for gold), GS-J2/PD-1 cells are tool cells for stably overexpressing PD-1 and contain a reporter gene system in the cells (GS-J2 is a Jurkat cell for gold). The recombinant humanized anti-PD-1 monoclonal antibody can be combined with the PD-1 protein on the surface of a GS-J2/PD-1 cell, the combination of PD-1 and PD-L1 is blocked, a reporter gene system in the GS-J2/PD-1 cell is activated, the expression quantity of Luciferase in the cell is increased, and qualitative and quantitative analysis can be carried out on the Luciferase by using a Promega Bio-Glo Luciferase kit, so that the activity of the recombinant humanized anti-PD-1 monoclonal antibody is evaluated.

Detection of Anti-PD-1-15C6D10-H organisms by reporter gene methodThe biological activity is compared with that of the marketed drugs Keystuda and Opdivo. The specific implementation method is as follows: GS-C2/PD-L1 cells were collected and the cell density was adjusted, inoculated into a 96-well plate at 100. Mu.L/well, and left to stand at 37℃with 5% CO ₂ After overnight incubation in a cell incubator, the supernatant was discarded. Diluting antibodies to be tested with different concentration gradients by using buffer solution, spreading the antibodies to be tested into the 96-well plate according to 50 mu L/well, collecting GS-J2/PD-1, adjusting cell density, inoculating the antibodies to be tested into the 96-well plate according to 50 mu L/well, fully mixing, standing at 37 ℃ and 5% CO ₂ The cells were incubated for 6h. After the incubation, bio-Glo Luciferase detection reagent was added, and after thoroughly mixing, the mixture was incubated at room temperature for 5min in a dark place. And (3) detecting the RLU value by using a multifunctional enzyme-labeled instrument, taking the logarithmic value of the antibody concentration as the X axis and the activation percentage as the Y axis, and adopting data processing software to perform four-parameter curve fitting. EC50 s of Anti-PD-1-15C6D10-H, keytruda and Opdivo were determined and compared for biological activity. The results are shown in FIG. 4 and Table 6.

TABLE 6 determination of biological Activity of antibodies

Antibodies to	EC50(nM)
		Anti-PD-1-15C6D10-H	0.07411
Keytruda	0.1421
		Opdivo	0.6336

Experimental results show that the Anti-PD-1-15C6D10-H has stronger activation activity for exciting T cells, and compared with Keytruda, opdivo under the same experimental conditions, the Anti-PD-1-15C6D10-H has stronger activation activity.

Example 9 evaluation of anti-tumor Effect of PD-1 antibody

The specific implementation method is as follows:

(1) mouse colorectal cancer MC38 cells were purchased from the short time Hirudin Biotechnology Co., ltd, and cultured at 37℃in 5% CO ₂ The culture medium composition was DMEM medium containing 10% inactivated fetal bovine serum. Cells were sub-bottled for 1 passage every 3 to 4 days.

(2) PBS-resuspended MC38 tumor cells were grown at 3-6X10 ⁵ A total of 32 mice were inoculated subcutaneously on the right flank of B-hPD-1 humanized mice (purchased from Baioser's plot) at a concentration of 0.1mL and 0.1 mL.

(3) When the average tumor volume reached about 100mm ³ When the mice with moderate tumor volume of the individual are selected to be put into groups, animals are randomly distributed into experimental groups according to the tumor volume, 8 animals in each group are firstly dosed on the same day, two days are separated, the dose of the 6 dosing groups is 20mg/Kg, and the control group is given with PBS buffer solution with the same volume. The tumor volume was measured 2 times per week using a vernier caliper, and the long and short diameters of the tumor were measured, and the volume calculation formula was: tumor volume = 0.5 x long diameter x short diameter ² The measurement result is shown in fig. 5, and the result shows that the Anti-PD-1-15C6D10-H tumor inhibition effect is better than that of Kettruda and Opdivo.

Sequence listing

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Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

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Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

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Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

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Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

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Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

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Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

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Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

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Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

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Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly

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Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys

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Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro

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Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly

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Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro

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Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly

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Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

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Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

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Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser

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Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly

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Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly

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Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys

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Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln

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Thr Leu Val Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

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Gly Gly Gly Ser Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro

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Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala

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Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu

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Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys

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Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn Tyr Asn Trp Pro Gln

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

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Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Phe Gln Gly Arg Val Thr Ile Thr Leu Asp Thr Ser Thr Asp Ile Ala

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Thr Leu Val Thr Val Ser Ser

115

<210> 15

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<210> 16

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<212> DNA

<213> Artificial sequence (Artificial Sequence)

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gaagtccagc tcgtccaatc tggtgcagaa gtcaaaaaac ccggagcaac tgtgaaaata 60

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ccaggcaaag gtcttgaatg gatggcaagg atagacccat ggaataatgg tcacacaaag 180

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<210> 17

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

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Arg Ala Ser Gln Asn Ile Asp Asn Tyr Ile Ala

1 5 10

<210> 18

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

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Ser Ala Ser Asn Arg Pro Ser

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<210> 19

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<213> Artificial sequence (Artificial Sequence)

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Gln Gln Asn Tyr Asn Trp Pro Gln Thr

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<212> PRT

<213> Artificial sequence (Artificial Sequence)

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Asp Tyr Tyr Met His

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<210> 21

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<212> PRT

<213> Artificial sequence (Artificial Sequence)

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Arg Ile Asp Pro Trp Asn Asn Gly His Thr Lys Tyr Asp Pro Lys Phe

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Gln Gly

<210> 22

<211> 9

<212> PRT

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<400> 22

Gln Gln Ser Arg Asn Trp Pro Arg Thr

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<210> 23

<211> 864

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

atgcagattc cacaggcacc ctggccagtc gtttgggcag tcctccagct tggttggaga 60

ccaggttggt ttctcgacag tccagaccgc ccatggaatc ctcctacctt ctcacctgca 120

ctcctcgtgg taacagaagg agataatgct accttcactt gctccttctc caatacatca 180

gagtccttcg tgctcaattg gtatagaatg tcaccttcta atcaaacaga taaactcgca 240

gcattccctg aagatcggag ccaaccaggc caagattgta ggtttcgagt aacccaactt 300

cctaatgggc gggatttcca tatgagcgtt gtccgcgccc ggagaaatga ctctggaact 360

tacttgtgtg gcgctatctc tctcgcccca aaagcacaga ttaaagagtc tcttcgagct 420

gaactgcgcg tcactgaacg gagagccgaa gtacccacag cacacccctc accctcaccc 480

cgccccgcag gccaatttca aactttggta gtcggagttg tgggcggact tctcggatct 540

ttggtcctgc tcgtgtgggt gctcgccgtc atctgcagta gggcagccag gggaacaatc 600

ggagcacgga gaacaggaca acccttgaag gaagaccctt ctgccgtccc cgttttcagt 660

gtcgactatg gagaactcga tttccagtgg agagagaaga cccctgaacc accagtaccc 720

tgcgttcccg aacaaactga atacgctaca atagttttcc ccagtggtat gggtacaagc 780

tcacccgccc gaagaggctc cgccgacggc ccccggagtg ctcagccatt gcgcccagag 840

gacggccatt gttcatggcc tctc 864

Claims (14)

1. An anti-PD-1 monoclonal antibody, or an antigen-binding fragment thereof, comprising:

(1) VL domain comprising LCDR1, LCDR2, LCDR3 as set forth in SEQ ID NO: 17. 18, 19; and

(2) VH domain comprising HCDR1, HCDR2, HCDR3 as set forth in SEQ ID NO: 20. 21, 22.

2. An anti-PD-1 monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises:

(1) The amino acid sequence is shown in SEQ ID NO:13, and

(2) The amino acid sequence is shown in SEQ ID NO: 14.

3. An anti-PD-1 monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a murine, chimeric or humanized antibody.

4. A murine anti-PD-1 monoclonal antibody, or an antigen-binding fragment thereof, comprising:

(1) The amino acid sequence is shown in SEQ ID NO:9, and

(2) The amino acid sequence is shown in SEQ ID NO: 10.

5. A hybridoma cell strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.19199, and has a preservation date of 2019, 12 months and 11 days, and is classified and named as a mouse hybridoma cell strain.

6. A polynucleotide encoding the anti-PD-1 antibody or antigen-binding fragment thereof of any one of claims 1-4, or a complement of a polynucleotide encoding the anti-PD-1 antibody or antigen-binding fragment thereof of claim 1 or 4.

7. A polynucleotide according to claim 6, wherein said polynucleotide has a polynucleotide sequence shown in one of the following:

(1) A polynucleotide sequence shown in SEQ ID NO. 11 or a complement thereof;

(2) A polynucleotide sequence shown in SEQ ID NO. 12 or a complement thereof;

(3) A polynucleotide sequence shown in SEQ ID NO. 15 or a complement thereof; and

(4) The polynucleotide sequence shown in SEQ ID NO. 16 or the complementary sequence thereof.

8. An expression vector comprising the polynucleotide sequence of claim 6 or 7.

9. A host cell transformed with the expression vector of claim 8.

10. A host cell according to claim 9, wherein said host cell is a mammalian cell.

11. The host cell of claim 10, wherein the mammalian cell is a CHO cell.

12. A pharmaceutical composition comprising the anti-PD-1 antibody or antigen-binding fragment thereof according to claim 1 that binds PD-1 and a pharmaceutically acceptable carrier.

13. Use of an anti-PD-1 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4 for the manufacture of a medicament that blocks the PD-1/PD-L1 signaling pathway for the treatment or alleviation of cancer or an infectious disease.

14. The use according to claim 13, wherein the cancer includes, but is not limited to, lymphoma, melanoma, lung cancer, liver cancer, stomach cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, bladder cancer, glioma; the infectious diseases include, but are not limited to, flaviviruses, dengue viruses, parvoviruses, mumps viruses.