EA042479B1 - MONOCLONAL ANTIBODY AGAINST PD1, ITS PHARMACEUTICAL COMPOSITION AND THEIR USE - Google Patents

Description

The field of technology to which the invention belongs

The present invention belongs to the field of tumor therapy and molecular immunology and relates to an anti-PD1 antibody, a pharmaceutical composition and methods of use. In particular, the present invention relates to a monoclonal antibody against PD1.

State of the art

The PD1 transmembrane receptor (programmed cell death protein 1, also known as PD-1) is a member of the CD28 gene family expressed in activated T cells, B cells, and myeloid cells. Both PD1 ligands (ie PDL1 and PDL2) belong to the B7 superfamily; moreover, PDL1 is widely expressed in a variety of cells, including T cells, B cells, endothelial cells and epithelial cells, while PDL2 is expressed only in antigen presenting cells, such as dendritic cells and macrophages.

T cells play a very important role in the elimination of viral infections, and the antiviral response of T cells is usually associated with immunopathogenesis. PD1 plays a vital role in the down regulation of T cell activation. Although PD1-mediated downregulation on T cells can reduce tissue damage caused by infection, blocking or inhibiting the negative regulatory effect of PD1 can lead to autoimmune diseases, for example, viral infection of the pancreas can be more effectively eliminated in PD1 knockout mice, but may lead to more severe liver damage (Isai et al., 2003, J. Exp. Med., 198:39-50). In addition, tumors with high expression of PD1 often develop in cancers that are difficult to detect (Hamanishi et al., 2007, Proc. Natl. Acad. Sci., USA, 104:3360-5). A common way to regulate PD1 expression is to introduce antibodies into the body.

Due to the broad antitumor prospects and the amazing efficacy of antibodies to PD1, it is generally believed that antibodies against PD1 metabolic pathways will lead to breakthroughs in the treatment of various tumors: non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma (Homet M.B., Parisi G. et al., Anti-PDl Therapy in Melanoma Semin Oncol., 2015 Jun, 42(3):466-473), Held S.A., Heine A. et al., Advances in immunotherapy of chronic myeloid leukemia CML , Curr. Cancer Drug Targets, 2013 Sep, 13(7):768-74).

Since the disclosure of unprecedented clinical performance data at the annual meetings of the American Association for Cancer Research (AACR) and the American Society of Clinical Oncology (ASCO) in 2012 and 2013. Anti-PD1 antibodies have become the most sought-after new drugs in research and development in the global pharmaceutical industry.

At present, there is still a need to develop new anti-PD1 antibodies with better binding efficiency in order to effectively block the binding of PD1 to PDL1.

The essence of the invention

Through in-depth research and creative work by immunizing mice with recombinant PD1 expressed in the mammalian cell expression system as an antigen, the inventors obtained hybridoma cells by fusing mouse splenocytes and myeloma cells. By screening a large number of samples, the inventors obtained the LT003 hybridoma cell line (deposit accession number CCTCC: C2015105).

The inventors surprisingly found that the LT003 hybridoma cell line is able to secrete a specific monoclonal antibody (named 14C12) that binds specifically to PD1, and this monoclonal antibody can effectively block the fusion of PD1 to PDL1.

In addition, the inventors created a humanized anti-PD1 antibody (named 14C12H1L1) in a novel way.

Even more surprisingly, the inventors found that the 14C12 and 14C12H1L1 antibodies herein can effectively bind to human T cells, activate T cells, and induce secretion of IFN-γ and IL-2 from human lymphocytes. Antibodies 14C12 and 14C12H1L1 herein have the potential to be drugs for the prevention and treatment of malignant neoplasms, including lung cancer, melanoma, kidney cancer, ovarian cancer and leukemia, as well as anemia.

The following is provided by the present invention.

The present invention relates to a monoclonal antibody or antigennegative fragment, in which the variable region of the heavy chain (VH) of the specified monoclonal antibody contains a CDR with the amino acid sequences of SEQ ID NO: 9-11; and/or the light chain variable region (VL) of said monoclonal antibody contains CDRs with the amino acid sequences of SEQ ID NOs: 12-14.

In some examples of the invention, said monoclonal antibody or antigen-binding fragments thereof are disclosed, wherein the VH amino acid sequence of the monoclonal antibody is selected from SEQ ID NO: 2 and SEQ ID NO: 6; and/or the V _L amino acid sequence of the monoclonal antibody is selected from SEQ ID NO: 4 and

- 1 042479

SEQ ID NO: 8.

In an example of the present invention, said monoclonal antibody or antigen-binding fragments thereof is disclosed, wherein the monoclonal antibody comprises (1) VH represented by SEQ ID NO: 2 and VL represented by SEQ ID NO: 4;

(2) VH represented by SEQ ID NO: 6 and VL represented by SEQ ID NO: 8.

The variable regions in the heavy and light chains of an antibody regulate binding activity. Each chain contains three hypervariable regions, namely the complementarity determining region (CDR) (HCDR1, HCDR2 and HCDR3 in the heavy (h) chain and LCDR1, LCDR2 and LCDR3 in the light (L) chain) as defined by Kabat et al. (Sequences of Proteins of Immunological Interest, Fifth Edition (1991), volume 1-3, NIH Publication 91-3242, Bethesda MD).

Through techniques well known to one of skill in the art described herein, such as analyzing the amino acid sequences in the CDRs of monoclonal antibodies in (1) and (2) above through the VBASE2 database, the 14C12 and 14C12H1L1 antibodies herein contain the same CDRs, with the amino acid sequences of the 3 V _H CDR regions are as follows:

HCDRl: GFAFSSYD (SEQ ID NO:9)

HCDR2: ISGGGRYT (SEQ ID NO:10)

HCDR3: ANRYGEAWFAY (SEQ ID NO:11);

wherein the amino acid sequences of the 3 CDR V _L regions are as follows:

LCDRl: QDINTY (SEQ ID NO:12),

LCDR2: RAN (SEQ ID NO :13)

LCDR3: LQYDEFPLT (SEQ ID NO:14).

In a specific example, said monoclonal antibody or antigen-binding fragment thereof is disclosed, wherein said monoclonal antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, CDR, single chain antibodies (e.g., scFv), humanized antibodies, chimeric antibodies or bispecific antibodies.

In certain embodiments, said monoclonal antibody or antigen-binding fragment thereof is disclosed, wherein said monoclonal antibody binds to a PD1 protein with a K _D of less than about 10 ^-5 M, such as less than about 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 M or less; preferably measured with Fortebio molecular interaction equipment.

In certain embodiments, said monoclonal antibody or antigen-binding fragment thereof is disclosed, wherein said monoclonal antibody binds to a PD1 protein with an EC ₅₀ of less than about 100 nM, such as less than about 10, 1, 0.9, 0.8, 0, 7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 nM, or less. In particular, said EC ₅₀ is determined by indirect ELISA.

In certain embodiments, said monoclonal antibody or antigen-binding fragment thereof is disclosed, wherein said monoclonal antibody binds to a PD1 protein with a KD of less than about 10 ^-5 M, such as less than about 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 M or less.

In some embodiments, said monoclonal antibody or antigen-binding fragment thereof is disclosed, wherein said monoclonal antibody contains regions other than CDRs derived from a non-mouse species, such as a human.

In certain embodiments, said monoclonal antibody or antigen-binding fragments thereof are disclosed, wherein said monoclonal antibody is produced by the LT003 hybridoma cell line and said LT003 hybridoma cell line is deposited with the China Type Culture Collection Center (CCTCC) with CCCCC Deposit Accession Number: C2015105.

The present invention relates to an isolated nucleic acid molecule containing a nucleotide sequence capable of encoding the VH of an antibody, the VH of said antibody having a CDR with the amino acid sequences of SEQ ID NOs: 9-11.

In particular, the heavy chain of said antibody has the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 6.

More specifically, said nucleic acid molecule has the nucleotide sequences of SEQ ID NO: 1 or SEQ ID NO: 5.

The present invention relates to an isolated nucleic acid molecule containing a nucleotide sequence capable of encoding the VL of an antibody, the VL of said antibody having a CDR with the amino acid sequences of SEQ ID NOs: 12-14.

In particular, the V _L of said antibody has the amino acid sequences of SEQ ID NO: 4 or SEQ ID NO: 8.

More specifically, said nucleic acid molecule has nucleotide sequences

- 2 042479 SEQ ID NO: 3 or SEQ ID NO: 7.

The present invention relates to a vector containing the isolated nucleic acid molecule described in this invention.

The present invention relates to a host cell containing an isolated nucleic acid molecule described in this invention, or a vector described in this invention.

The present invention relates to a method for producing a monoclonal antibody or antigen-binding fragments thereof described in this invention by culturing a host cell in this invention under appropriate conditions and isolating said monoclonal antibody or antigen-binding fragments thereof from cell culture.

The present invention relates to the LT003 hybridoma cell line, which is deposited with the China Type Culture Collection Center (CTCCC) with CCCCC Deposit Accession Number: C2015105.

The present invention relates to a conjugate that consists of a monoclonal antibody or antigen-binding fragments thereof and a conjugate portion, wherein said monoclonal antibody is a monoclonal antibody or antigen-binding fragments thereof described in this invention, and said conjugated portion is a detectable marker. In particular, said conjugated part is radioactive isotopes, fluorescein, luminescent substances, dyes or enzymes.

The present invention relates to kits of reagents, consisting of a monoclonal antibody or its antigen-binding fragments or their conjugates described in the invention.

In particular, reagent kits may contain a secondary antibody that specifically recognizes said monoclonal antibody or antigen-binding fragments thereof; optionally said secondary antibody may contain detectable markers such as radioactive isotopes, fluorescein, luminescent substances, dyes or enzymes.

The present invention relates to the use of said monoclonal antibody, or antigen-binding fragments thereof, or conjugates thereof, as described herein, to create kits of reagents, said kits of reagents being used to detect the presence or level of PD1 in samples.

The present invention relates to a pharmaceutical composition containing the specified monoclonal antibody, or its antigen-binding fragments, or their conjugates described in the invention. Optionally, it may also contain a pharmaceutically acceptable carrier or excipient.

The present invention relates to the use of said monoclonal antibody, or its antigen-binding fragments, or their conjugates described in the invention, in the manufacture of drugs for the prevention and/or treatment and/or adjuvant treatment and/or diagnosis of tumors or anemia; in particular, said tumors may be melanoma, kidney cancer, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, non-small cell lung cancer, ovarian cancer and leukemia.

The present inventors have found in animal experiments that 14C12H1L1 can effectively inhibit the growth of MC38 tumor cells inoculated subcutaneously in the right flank of PD-1 HuGEMM mice, in which 14C12H1L1 antibody drug can significantly inhibit tumor growth in PD tumor-bearing mice -1 HuGEMM, with equivalent potency to the marketed monoclonal antibody nivolumab, which is an approved drug that targets the same target.

The present invention relates to the use of a monoclonal antibody, or its antigen-binding fragments, or their conjugates described in this invention, for the manufacture of drugs for the following purposes:

blocking PD1 binding to a PD1 ligand, regulation (eg, decrease) of PD1 activity or level, attenuation of PD1 immunosuppression, or increased expression of IFN-γ and/or IL-2 in T lymphocytes;

specifically, said PD1 ligand is PDL1 or PDL2, preferably PDL1.

The present invention relates to an in vivo or in vitro method of administering to cells or subjects in need thereof an effective dose of a monoclonal antibody, or antigen-binding fragments thereof, or conjugates thereof, described herein, said method being selected from the following:

methods for blocking PD1 binding to a PD1 ligand, methods for regulating (eg, lowering) PD1 activity or levels, methods for attenuating PD1 immunosuppression, or methods for increasing expression of IFN-γ and/or IL-2 in T lymphocytes;

specifically, said PD1 ligand is PDL1 or PDL2, preferably PDL1.

- 3 042479

In a specific example of the invention, said in vitro method is for non-therapeutic or non-diagnostic purposes.

Interferon y (IFNy) is mainly produced by natural killer (NK) cells and natural killer T cells (NKT) or produced by effector T cells consisting of CD4 Th1 cells and CD8 cytotoxic T lymphocytes after stimulation with specific antigens. As an important cytokine of innate and adaptive immunity, IFN-y plays an important role in counteracting or inhibiting viral, some bacterial and protozoan infections. Meanwhile, IFN-y can activate macrophages and induce major histocompatibility complex (MCHC) type 2 expression to activate immune responses to control tumor progression (Schoenborn J.R., Wilson S.V., Regulation of Interferon-γ During Innate and Adaptive Immune Responses, Advances in Immunology, 2007, 96:41-101). In an in vitro study of the present invention, an anti-PD1 antibody can induce the secretion of IFN-a to activate immune responses.

Interleukin 2 (IL-2), produced by T cells, is a growth factor that regulates T cell subpopulations and is a critical factor that regulates immune responses, promotes the proliferation of activated B cells, and is involved in antibody responses, hematopoiesis, and cancer surveillance. Recombinant human IL-2 has been approved by the US FDA (Food and Drug Administration) for the treatment of malignant tumors (including melanoma, kidney tumor, etc.) during clinical trials for the treatment of chronic viral infections (Chavez, A.R. et al., Pharmacologic administration of interleukin-2, Ann. N. Y. Acad. Sci., 2009, 1182, p.14-27). In in vitro studies, the anti-PD1 antibody of the present invention can specifically attenuate PD1 immunosuppression, activate T cells, and induce IL-2 production, showing promise for widespread use in gene therapy for tumor and parasitic diseases.

The monoclonal antibody, or antigen-binding fragments thereof, or conjugates thereof described in this invention are used for the prevention and/or treatment and/or adjuvant treatment and/or diagnosis of tumors or anemia; in particular, said tumors may be melanoma, kidney cancer, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, non-small cell lung cancer, ovarian cancer or leukemia.

The monoclonal antibody, or antigen-binding fragments thereof, or conjugates thereof, described in this invention are used to block the binding of PD1 to a PD1 ligand, regulate (e.g., decrease) the activity or level of PD1, attenuate PD1 immunosuppression, or increase the expression of IFN-γ and/or IL-2 in T-lymphocytes;

specifically, said PD1 ligand is PDL1 or PDL2, preferably PDL1.

In a specific example of this invention, the monoclonal antibody, or its antigen-binding fragments, or their conjugates, described in this invention, block only the binding of PD1 to PDL1.

The present invention relates to a method for the prevention and/or treatment and/or adjuvant treatment and/or diagnosis of tumors or anemia, comprising the procedure of applying to subjects an effective dose of a monoclonal antibody, or its antigen-binding fragments, or their conjugates described in this invention; in particular, said tumors may be melanoma, kidney cancer, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, non-small cell lung cancer, ovarian cancer or leukemia.

Unless otherwise indicated herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. In addition, the laboratory methods of cell and tissue culture, molecular genetics, oligo- or polynucleotide chemistry, and immunology described herein are well known and widely used in the art. Meanwhile, in order to better understand the present invention, it should be understood that the following terms, unless otherwise indicated, have the following meanings.

As used herein, the term PD1 protein amino acid sequence (Programmed Cell Death Protein 1, NCBI GenBank: NP_005009.2) encompasses full-length PD1 protein or PD1ECD (PD1 extracellular segment) or PD1ECD-containing fragments; and also includes a PD1ECD fusion protein, eg fused to mouse or human IgG Fc protein fragments (mFc or hFc). In addition, those skilled in the art will recognize that the amino acid sequence of the PD1 protein may be naturally or artificially mutated (including, without limitation, substitutions, deletions, and/or insertions) without affecting its biological functions. Therefore, in this invention, the term PD1 protein should include all such sequences and their natural or artificial variants. In addition, when describing fragments of the PD1 protein sequence,

- 4 042479 specified sequence fragments include both sequence fragments and corresponding sequence fragments in its natural or artificial variants.

As used herein, the term PDL1 protein amino acid sequence (programmed cell death ligand 1, NCBI Genebank ID: NP_054862.1) encompasses the full-length PDL1 protein or PDL1ECD (extracellular segment of PDL1) or PDL1ECD-containing fragments; and also includes a PDL1ECD fusion protein, eg fused to mouse or human IgG Fc protein fragments (mFc or hFc). In addition, those skilled in the art will appreciate that the amino acid sequence of the PDL1 protein may be naturally or artificially mutated (including, without limitation, substitutions, deletions, and/or insertions) without affecting its biological functions. Therefore, in this invention, the term PDL1 protein should include all such sequences and their natural or artificial variants. In addition, when describing sequence fragments of the PDL1 protein, said sequence fragments include both sequence fragments and corresponding sequence fragments in natural or artificial variants.

Used in this invention, the term EC ₅₀ refers to the concentration to provide 50% of the maximum effect.

As used herein, the term antibody refers to immunoglobulin proteins, which typically consist of two pairs of polypeptide chains (each pair having a light (L) chain and a heavy (H) chain). Light chains are classified as k and λ light chains. Heavy chains are classified as μ, δ, γ, α and ε and define the antibody isotype as IgM, IgD, IgG, IgA and IgE respectively. In light chains and heavy chains, the variable regions and constant regions are linked by a J region of about 12 or more amino acids. The heavy chain also contains a D-region with about 3 or more amino acids. Each heavy chain contains a variable region (VH) and a constant region ( _CH ), which consists of 3 domains ( _CH 1, CH2 and _CH 3). Each light chain contains a variable region (VL) and a constant region (CL), which consists of a single CL domain. The constant region can mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the complement component 1q (C1q) of the classical complement system. VH and VL can also be subdivided into regions of high variability (called the complementarity determining region (CDR)), which are separated by relatively conserved regions, called framework regions (FR). From the amino end to the carboxyl end, each V _H and V _L consists of 3 CDRs and 4 FRs in the order FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions (VH and VL) of the heavy chain and light chain form the binding site of the antibody. The distribution of amino acids by region or domain follows Kabat's definitions in Sequences of Proteins of Immunological Interest (National Institutes of Health Bethesda, MD (1987 and 1991)) or Chothia & Lesk (1987), Mol. Biol., 196:901-917; or Chothia et al. (1989), Nature, 342:878-883. The term antibody is not limited to any particular method for their preparation. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, for example IgG antibodies (such as IgGl, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM.

As used herein, the term antigen-binding fragments refers to a polypeptide containing fragments of a full-length antibody that retains the ability to specifically bind to the same antigen and/or compete with the full-length antibody for binding to the antigen, also referred to as the antigen-binding portion. See Fundamental Immunology, Ch. 7 (Paul, W., ed. 2, Raven Press, NY (1989)), including the entire article and references in this invention for all purposes. Antigen-binding fragments can be generated by recombinant DNA techniques or by digestion of intact antibodies with proteolytic enzymes or chemicals. In some instances, antigen-binding fragments include Fab, Fab', F(ab') ₂ , Fd, Fv, dAb, and CDR fragments, single chain antibodies (eg, scFv), chimeric antibodies, diabodies, and polypeptides that contain at least a portion of an antibody, sufficient to confer specific antigen-binding ability to the polypeptides.

In some cases, the antigen-binding fragment is a diabody, namely a dimeric antibody fragment whose V _H and V _L domains are expressed on the same polypeptide chain, however, due to the use of a linker too short to allow pairing between two domains on the same chain, the domains are forced to pair. with complementary domains in the other strand to form two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci., USA, 90:6444-6448 (1993); and Poljak RJ et al. , Structure 2: 1121-1123 (1994)).

Using conventional techniques known to those skilled in the art (such as recombinant DNA technology or enzymatic/chemical digestion), an antigen-binding fragment (such as the antibody fragments described above) can be obtained from a given antibody (e.g., monoclonal antibodies 14C12, 14C12H1L1 provided in of this invention) and screened for specificity in the same manner as for a full-length antibody.

- 5 042479

In this invention, unless otherwise indicated, the term antibody refers not only to an intact antibody, but also to antigen-binding fragments of an antibody.

As used herein, the terms mAb and monoclonal antibody refer to an antibody or antibody fragment derived from a group of highly homologous antibodies, i. from a group of identical antibody molecules, with the exception of mutations that may occur spontaneously. A monoclonal antibody is highly specific for one epitope on an antigen. Polyclonal antibodies are distinct from monoclonal antibodies containing at least 2 or more different antibodies, which typically recognize different epitopes on an antigen. Monoclonal antibodies can be obtained using hybridoma technology, which was originally reported by Kohler et al. (Nature, 256:495 (1975)), as well as recombinant DNA technology (see US patent 4816567).

As used herein, the term humanized antibody refers to an antibody or fragments thereof derived from a human immunoglobulin (receptor antibody) whose CDRs or part of the CDRs have been replaced by CDR regions of a non-human derived antibody (donor antibody), wherein the donor antibody may be an antibody non-human (eg, mouse, rat, or rabbit) antibody with predictable specificity, binding affinity, or reactivity. In addition, some framework region (FR) amino acid residues of a receptor antibody can also be replaced with corresponding non-human amino acid residues or amino acid residues of other antibodies to further improve or optimize antibody performance. For more information on humanized antibodies, see, for example, Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op. Struct. Biol. 2:593-596 (1992); and Clark, Immunol. Today, 21:397-402 (2000).

As used in this invention, the term isolate or isolated means artificially obtained in a natural state. If a certain kind of isolated substance or component exists in nature, it may be produced due to a change in its natural environment, or isolated from the natural environment, or both. For example, a polynucleotide or polypeptide in its natural state in a living animal will be said to be isolated if it has been isolated with a high degree of purity in the same natural state. The term isolated or isolated does not exclude the presence of an artificial or synthetic substance or other impurities that do not interfere with activity.

As used herein, the term vector refers to a nucleic acid delivery vehicle that can be introduced with a polynucleotide. A vector that can contain a protein that is encoded by the introduced polynucleotide to be expressed is called an expression vector. Vectors can be introduced into a host cell by transformation, transduction, or transfection so that the genetic substances carried by the vector can be expressed in the host cell. Vectors are well known to those of skill in the art, including, without limitation, the plasmid; phasmid; cosmid; an artificial chromosome such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a P1-derived artificial chromosome (PAC); phage such as λ phage or M13 phage, as well as animal viruses, etc. Animal viruses can include, without limitation, reverse transcriptase virus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (eg, herpes simplex virus), varicella-zoster virus, baculovirus, papillomavirus, and papovavirus (such as SV40). The vector may contain a variety of components that control expression, including, but not limited to, a promoter, a transcription initiation factor, an enhancer, a selection element, and a reporter gene. In addition, the vector may also contain an origin of replication.

As used herein, the term host cell refers to cells that can import vectors, including, but not limited to, prokaryotic cells such as E. coli and Bacillus subtilis, fungal cells such as yeast and Aspergillus, insect cells such as Drosophila S2 cells, and Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

As used herein, the term specific binding refers to non-random binding between two molecules, such as an interaction between an antibody and its target antigen. In some embodiments, specific binding of an antibody to an antigen means an affinity (KD), e.g., less than about 10 ^-5 M, such as less than 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 M, or less.

As used herein, the term KD refers to the dissociation equilibrium constant of a specific interaction between an antibody and an antigen to describe the binding affinity between antibodies and antigens. The smaller the equilibrium dissociation constant, the stronger the antibody binds to the antigen, the higher the affinity between the antibody and antigen. Typically, antibodies (eg, monoclonal antibodies 14C12, 14C12H1L1 in this invention) bind to antigens (eg, PD1 protein) with a KD of less than about 10 ^-5 M, for example, less than 10 ^-6 , 10 ^-7 , 10 ^-8 . 10 ^-9 or 10 ^-10 M, or even less. KD can be measured by any method well known to those of skill in the art, such as using the Fortebio Octet system. Used in this invention, the terms monoclonal antibody and mAb have the same meaning and are used interchangeably; the terms polyclonal antibody and PcAb have the same meaning and are used interchangeably; the terms polypeptide and protein have the same meaning and are used interchangeably. Also in this invention, amino acids are usually represented by one-letter or three-letter abbreviations known in the art. For example, alanine can be represented as A or Ala.

As used herein, the terms hybridoma and hybridoma cell line may be used interchangeably and, when referred to herein, include subclone and hybridoma progeny cells. For example, when referred to herein, the LT003 hybridoma cell line also includes the LT003 subclone and progeny cells.

As used herein, the term pharmaceutically acceptable carrier or excipient refers to a carrier and/or excipient that is pharmaceutically and/or physiologically compatible with the subjects and active ingredients and is widely recognized in the art (Remington's Pharmaceutical Sciences, Edited by Gennaro AR, 19th ed., Pennsylvania: Mack Publishing Company, 1995), including, without limitation, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, without limitation, phosphate buffer; surfactants include, without limitation, cationic, anionic, or nonionic surfactants such as Tween-80; ionic strength enhancers include, without limitation, sodium chloride.

Used in this invention, the term effective dose is defined as the amount of therapeutic agent sufficient to achieve or at least partially achieve the desired effect. For example, an effective prophylactic dose (eg, for cancer) is an amount to prevent, stop, or delay the onset of diseases (eg, cancer); an effective dose of treatment is an amount that cures or at least partially stops the disease and its complications in patients with the disease. Determination of such an effective dose is entirely within the ability of technical personnel in the art. For example, the effective dose of treatment will depend on the severity of the disease, the general immune system of the patient, the general condition of the patients such as age, weight and sex, the route of administration of said drug and other concomitant treatments, and the like.

effects of the invention.

The monoclonal antibodies of this invention, especially 14C12H1L1, are able to specifically bind to PD1, effectively blocking the binding of PD1 to PDL1 and attenuating PD1 immunosuppression to activate T-lymphocytes. At the same time, the anti-PD1 antibody 14C12H1L1 can induce the secretion of IFN-γ and IL-2 much better than the control antibody 5C4 (5C4: PD1 antibody from Medarex Inc.: Alan J. Korman, et al., Human monoclonal antibodies to programmed death 1 ( PD1) and methods for treating cancer using anti-PDl antibodies alone or in combination with other immunotherapeutics, US Patent No. US 8008449 B2). The monoclonal antibodies of this invention, especially 14C12H1L1, have an antitumor effect equivalent to the approved nivolumab for the same target. The antibodies of this invention have the potential to be or be used in medicaments for the prevention and/or treatment of non-small cell lung cancer, renal cell cancer, ovarian cancer, melanoma, leukemia, or anemia.

Description of drawings

In FIG. 1 shows the results of SDS polyacrylamide gel electrophoresis (SDS-PAGE) of the humanized monoclonal antibody 14C12H1L1. Left to right: 1 - 1 μg antibody in unreconstituted loading buffer; 2 - 1 μg of antibody in reconstituted loading buffer; 5 µl marker; 3 - 1 mcg of BSA.

In FIG. 2 shows the binding kinetics of the 14C12 antibody.

In FIG. 3 - kinetics of binding of antibody 14C12H1L1.

In FIG. 4 - 5C4 antibody binding kinetics.

In FIG. 5 shows the results of ELISA for the binding of 14C12H1L1 and 5C4 to PD1.

In FIG. 6 - results of competitive ELISA for binding of 14C12H1L1 and 5C4 to PD1 in comparison with PDL1.

In FIG. 7 shows EC ₅₀ of 14C12H1L1 binding to PD1 on the surface of 293T-PD1 cells.

In FIG. 8 - 14C12H1L1 binding activity to PD1 surface antigen of T cells.

In FIG. 9 shows the effect of 14C12H1L1 on the secretion of IFN-γ mixed lymphocytes.

In FIG. 10 - the effect of 14C12H1L1 on the secretion of IL-2 mixed lymphocytes.

In FIG. 11 - Effect of 14C12H1L1 on IL-2 cytokine secretion induced by admixture of PBMC, MDA-MB-2 31 and Raji cells.

In FIG. 12 - Effect of 14C12H1L1 on tumor growth in the MC38 tumor model in PD-1 HuGEMM mice.

- 7 042479

The LT003 hybridoma cell line was deposited with the China Type Culture Collection Center (CCTCC) at Wuhan University, Wuhan, China, 430072 on June 16, 2015, with CCCCC Deposit Accession Number: C2015105.

Detailed description of the invention

The invention will now be described in detail. As will be understood by a person skilled in the art, the following examples are used only to describe the invention and should not be construed as limiting the scope of the invention. Cases without specific description of methods or conditions were performed according to the literature in the art (e.g., Guide to Molecular Cloning, by J. Sambrook et al., translated by Peitang Huang et al., third Edition, Science Press) or according to with instructions for using the product. All reagents or instruments without a manufacturer's name were common commercially available products.

In the examples below, the T cells used were obtained from Akeso Biopharma, Inc.; BALB/C mice were purchased from Guangdong Animal Medical Center. The PD-1 HuGEMM mice used were obtained from Nanjing Galaxy Biopharma Co., Ltd.; MC38 cells were obtained from Shanghai Fudan IBS Cell Center; a commercial drug against the same target, used nivolumab (Opdivo®), was obtained from the Bristol-Myers Squibb Company.

Example 1 Preparation of LT003 hybridoma cell line and production of monoclonal antibody 14C12.

1. Creation of the LT003 hybridoma cell line.

Used PD1-mFc (PD1: programmed cell death protein 1, NCBI GenBank ID: NP_005009.2) fusion protein as antigen and fused splenocytes from immunized BALB/C mice (purchased from Guangdong Animal Medical Center) and mouse myeloma cells into hybridoma cells using a currently accepted method (e.g., Stewart, S.J., Monoclonal Antibody Production, in Basic Methods in antibody Production and Characterization, Eds.G.C. Howard and D.R. Bethell, Boca Raton: CRC Press, 2000).

The microplate was coated with PD1-mFc as antigen and hybridoma cells were screened with mediated ELISA to obtain hybridoma cells that secrete new antibodies that specifically bind to PD1.

In screening, hybridoma cell lines capable of secreting PD1-binding monoclonal antibodies were obtained by competitive ELISA against PDL1-hFc ligand fusion protein (PDL1: programmed cell death protein ligand 1, NCBI Genebank ID: NP 054862.1), and stable hybridoma cell lines were obtained by the limited dilution method, and then stable LT003 cell lines were obtained by the limited dilution method (the monoclonal antibody secreted from LT003 is called 14C12).

The LT003 hybridoma cell line (PD1-14C12) was deposited with the China Type Culture Collection Center (CCTCC) at Wuhan University, Wuhan, China, 430072 on June 16, 2015, with CCCCC Deposit Accession Number: C2015105.

2. Obtaining a monoclonal antibody 14C12.

The LT003 cell line of the present invention was cultured using IMDM medium containing 10% low IgG fetal bovine serum for 7 days, and then the cell culture supernatant was harvested and purified to obtain 14C12 antibody.

Example 2 Preparation of light chain and heavy chain sequences for monoclonal antibody 14C12.

Extracted mRNA from the hybridoma cell line LT003, obtained in example 1 above, in accordance with the manual for the kit of reagents for isolation of total cellular/bacterial RNA (Tiangen, product no. DP430).

cDNA was synthesized according to the manual for the Invitrogen Superscript® III First Strand Synthesis System for RT-PCR Kit (Reverse Transcriptase PCR) and PCR amplification was performed.

TA cloning was performed directly using PCR amplification products according to the instructions of the pEASY-T1 cloning kit (Transgen CT101).

The TA cloning products were sequenced and the following results were obtained.

VH DNA sequencing results: (354 bp).

GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGTCACTGAAACT

GAGCTGCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGACATGTCATGGGTGAGGCAGACCCCT

GAGAAGCGCCTGGAATGGGTCGCTACTATCAGCGGAGGCGGGCGATACACCTACTATCCTGACT

C T G T CAAAG G GAGAT T CACAAT TAG T C G G GATAAC G C CAGAAAATAC TCTGTATCTGCAGATGTC

TAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGTGCAAACCGGTACGGCGAAGCATGGTTT

GCCTATTGGGGACAGGGCACCCTGGTGACAGTCTCTGCC(SEQ ID NO: 1)

- 8 042479

Encoded protein sequence: (118 a.k.).

EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISGGGRYTY

YPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA (SEQ ID NO: 2)

VL DNA sequencing results: (318 bp).

GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGCCTGGGCGAGCGAGTGAC

CTTCACATGCAAAGCATCCCAGGACATCAACACATACCTGTCTTGGTTTCAGCAGAAGCCAGGC

AAAAGCCCCAAGACCCTGATCTACCGGGCCAATAGACTGGTGGACGGGGTCCCCAGCAGATTCT

CCGGATCTGGCAGTGGGCAGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACATGGG

CATCTACTATTGCCCTGCAGTATGATGAGTTCCCTCTGACCTTTGGAGCAGGCACAAAACTGGAA

CTG (SEQ ID NO: 3)

Encoded protein sequence: (106 a.k.).

DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIYRANRLVDGVP

SRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLEL (SEQ ID NO: 4)

2. Obtaining a recombinant monoclonal antibody 14C12 (Re).

The heavy chain cDNA sequence (variable region sequence SEQ ID NO: 1) and the light chain cDNA sequence (variable region sequence SEQ ID NO: 3) were cloned separately from 14C12 (Re) into the pUC57simple vector (provided by GenScript Biotech Corp.) (enzymatic cleavage sites : XbaI and BamHI) and obtained plasmids pUC57simple-14C12H and pUC57 simple-14C12L, respectively.

Plasmids pUC57simple-14C12H and pUC57simple14C12L, respectively, were enzymatically digested (HindIII and EcoRI), and then the heavy and light chains extracted after electrophoresis were subcloned into the pcDNA3.1 vector, both recombinant plasmids were extracted, and 293F cells were co-transfected.

After 7 days of cell culture, the cell culture supernatant was centrifuged by high-speed centrifugation and filtered by vacuum filtration through a microporous membrane and loaded into a HiTrap MabSelectSuRe column, and then the antibody was eluted with elution buffer in one step, and then passed through a HiTrap desalting column and washed in phosphate-buffered saline (PBS), and then a recombinant 14C12 (Re) antibody was obtained after further purification.

As confirmed by ELISA for binding activity, the recombinant 14C12 (Re) antibody had a binding activity equivalent to the 14C12 antibody, and therefore can be further used in the subsequent construction of antibody humanization.

Example 3 Construction of the Light Chain and Heavy Chain Sequences of the Humanized 14C12H1L1 Antibody.

1. Construction of the light chain and heavy chain sequences of the humanized 14C12H1L1 antibody.

According to the three-dimensional crystal structure of the PD1 protein (Shinohara, T. et al., Structure and chromosomal localization of the human PD1 gene (PDCD1), Genomics, 1995, 23(3):704-6)) and the sequences of the 14C12 antibody obtained in example 2, using computer simulation of antibodies, amino acid mutations were designed according to the model and the sequences of the variable region of the antibody 14C12H1L1 were obtained (heavy chain constant region is the C region of the Ig gamma-1 chain, ACCESSION: P01857; light chain constant region is C kappa chain Ig region, ACCESSION:P01834) below.

Heavy chain variable region DNA sequence: (354 bp).

GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACT

GAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCA

GGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATCCTGACA

G C G T CAAG GGCCGGTT CACAAT CTC TAGAGATAACAG TAAGAACAAT CTGTATCTGCAGAT GAA

CAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTT

GCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGT (SEQ ID NO: 5)

Encoded protein sequence: (118 a.k.).

EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTY

YPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS (SEQ ID NO: 6)

- 9 042479

Light chain variable region DNA sequence: (321 bp).

GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCAC

CTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGG

(SEQ ID NO: 7)

Encoded protein sequence: (107 a.k.).

DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVP

SRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 8)

Example 4 Preparation and SDS-PAGE electrophoresis of the humanized monoclonal antibody 14C12H1L1.

Heavy chain cDNA (variable region sequence is SEQ ID NO: 5, heavy chain constant region is Ig gamma-1 chain C region, ACCESSION: P01857) and light chain cDNA (variable region sequence is SEQ ID NO 7) were cloned separately. : light chain constant region is the C region of kappa chain Ig, ACCESSION: P01834) 14C12H1L1 into the pUC57simple vector (provided by GenScript Biotech Corp.) (enzymatic cleavage sites: XbaI and BamHI) to generate plasmid pUC57simple-14C12H1 and pUC57simple-14C12Ll, respectively . These plasmids were subcloned into the pcDNA3.1 vector respectively (enzymatic cleavage sites: XbaI and BamHI), both recombinant plasmids were extracted and co-transfected with 293F cells.

After 7 days of cell culture, the cell culture was centrifuged by high-speed centrifugation and filtered by vacuum filtration through a microporous membrane and loaded into a HiTrap MabSelectSuRe column, and then the antibody was eluted with elution buffer in one step, and then passed through a HiTrap desalting column and washed in buffer PBS, and further purified to obtain recombinant antibody 14C12H1L1, which was analyzed by SDS-PAGE electrophoresis.

The results are shown in FIG. 1, the reduced target protein appeared at approximately 24.5 and 49 kDa, and the unreduced target protein appeared at approximately 147 kDa.

Example 5 Kinetic Measurements of Antibodies.

The binding kinetics of the 14C12 antibody and the humanized 14C12H1L1 antibody to the PD1 antigen was measured using the Fortebio Octet system.

1. Digest PD1-mFc protein with TEV protease and obtain PD1 antigen by column purification.

2. PD1 antigen (concentration 1 μg/ml) labeled with biotin was immobilized on the surface of the SA sensor and equilibrated in PBST buffer, and then antibodies 14C12, 14C12H1L1 and 5C4 were bound, and the antibodies were diluted in each dilution three times the previous concentration starting with 200 nM. Dissociation of antigen and antibody was also performed in PBST.

The kinetics of binding of antibodies 14C12, 14C12H1L1 and 5C4 is shown in Table. 1 and in FIG. 2, 3 and 4, respectively. The results showed that both 14C12 and 14C12H1L1 have good affinity for PD1 with higher affinity than 5C4.

Table 1

Dynamic parameters of the 14C12 antibody.

Name of the antibody K _D (M) cop (1/ms) Error cop Kdis (1/s) Error kdis 14С12 1.81Е-11 3.38E+05 8.23E+03 6.12E-06 1.04Е-05 14C12H1L1 2.42E-11 3.17E+05 5.90E+03 7.66E-06 8.70E-06 5С4 6.46Е-10 5.63E+05 1.38E+04 3.63E-04 9.77Е-06

K _D : dissociation constant;

Ko _n : antigen-antibody binding rate;

Kd _is : dissociation rate of antigen and antibody;

KD=Kdis/Con.

Example 6 Antibody-PD1 antigen binding activity measured by mediated ELISA.

The binding activities of antibodies 14C12H1L1 and 5C4 with PD1 were measured separately using mediated ELISA as follows.

After incubation with PD1-mFc at 4°C overnight, the microplate was blocked with 1% BSA at 37°C for 2 h, and then antibodies were added separately and incubated at 37°C for 30 min,

- 10 042479 HRP (horseradish peroxidase) labeled secondary antibody (goat anti-human IgG (H+L)) (Jackson, 109-035-088) was then added and then TMB (Neogen, 308177) was added to run the reaction for 5 min and measured the absorbance at a wavelength of 450 nm in a microplate reader.

The results of binding of antibodies 14C12H1L1 and 5C4 to the PD1 antigen are shown in FIG. 5. As shown in FIG. 5, both 14C12H1L1 and 5C4 antibodies can efficiently bind to the PD1 protein in a dose dependent manner. Absorption rates at different doses are shown in Table. 2. By modeling the curve using the results of quantitative analysis in the form of optical density values, it was determined that the EC50 binding of 14C12H1L1 and 5C4 to PD1 was determined to be 0.032 and 0.043 nM, respectively.

table 2

Uptake rates for 14C12H1L1 and 5C4 binding to PD1

Antigen coating: PDl-mFc (0.5 µg/ml) Antibody concentration (µg/ml) 14C12H1L1 5С4 1 2,970 2,954 2.959 2,991 0.3 2.886 2,961 2.978 3.079 0.1 2.864 2.868 2.838 2,926 0.03 2,674 2,669 2.617 2,659 0.01 2.222 2.201 1.981 2.221 0.003 1.383 1.464 1.169 1.222 0.001 0.676 0.736 0.527 0.548 0 0.062 0.062 0.065 0.073 secondary antibody HRP labeled secondary antibody (goat anti-human IgG)

Example 7 Antibody binding activities against PD1 antigen against PDL1 determined by competitive ELISA.

The binding activities of the humanized 14C12H1L1 and 5C4 antibodies to the anti-PDL1 PD1 antigen were measured by competitive ELISA as follows.

After overnight incubation with PD1-hFc at 4°C overnight, the microplate was blocked with 1% BSA for 2 h, 14C12H1L1 and 5C4 antibodies at various concentrations (see Table 3 for diluted concentrations) were mixed with PDL1-mFc for 10 min, and the mixtures were incubated at 37°C for 30 min, and then the respective anti-human and anti-mouse enzyme-labeled secondary antibodies, respectively, were added and incubated at 37°C for 30 min. Absorbance at 450 nm was read in a microplate reader.

The results of binding of antibodies 14C12H1L1 and 5C4 to the PD1 antigen are shown in FIG. 6. As shown in FIG. 6, the 14C12H1L1 antibody can compete with PDL1 and bind efficiently to the PD1 protein in a dose dependent manner. The intensity of absorption at different doses are shown in table. 3. By curve modeling using absorbance quantification results, the EC50s of 14C12H1L1 and 5C4 binding activities were determined to be 1.322 and 1.199 nM, respectively.

Table 3

Competitive binding of 14C12H1L1 and 5C4 to PD1 against PDL1

Antigenic coating PDl-mFc 0.5 µg/ml Antibody Concentration/Dilution Gradient 14C12H1L1 5С4 1.5 µg/ml 0.062 0.064 0.070 0.075 13 0.069 0.064 0.081 0.086 19 0.363 0.305 0.372 0.269 1:27 1.727 1.543 1.429 1,604 1:81 1,892 1.752 1.766 1,881

- 11 042479

1:243 1,984 2,029 2,045 2.005 1:729 1,937 1,978 1,934 1.954 0 1,870 1,977 1,933 1.977 ligand PDLl-mFc 0.3 µg/ml secondary antibody HRP labeled goat anti-mouse secondary antibody

Example 8 Antibody binding activity to cell surface antigen PD1 by flow cytometry.

293T host cells expressing the PD1 antigen were constructed and labeled with the humanized 14C12H1L1 antibody prepared according to the invention (see Example 4). The ability of the 14C12H1L1 antibody to specifically bind to the cell surface antigen in its native conformation was analyzed and verified by flow cytometry.

1. Construction of a 293T host cell expressing the PD1 antigen.

The specific steps were as follows.

Construction of a 293T host cell expressing the PD1 antigen.

293T cells were transfected with the PD1-containing pLenti6.3-PD1 vector (pLenti6.3 vector purchased from Invitrogen Corporation) according to the LipofectaminTransfection Kit manual (purchased from Invitrogen Corporation) to obtain a stable pool of PD1-expressing 293T-PD1 by screening.

2. Binding of antibodies to the cell surface antigen 293T-PD1.

Antibody labeling and flow cytometry.

The 293T-PD1 obtained in the above step was digested with trypsin and distributed into tubes, each containing 2x10 ⁵ cells. PD1 antibodies were diluted using PBS buffer (1% BSA) at concentrations of 50, 10, 5, 1, 0.1, and 0.01 nM and added to tubes and incubated on ice with PD1-expressing 293T cells for 2 h. the tube was added with 100 µl of FITC-labeled secondary goat anti-human antibody (1:500) and incubated on ice for 1 h. FITC channel.

The results of binding of the humanized 14C12H1L1 antibody to 293T-PD1 cells are shown in FIG. 7. As shown in FIG. 7, the 14C12H1L1 antibody can efficiently bind the target PD1 protein expressed on the surface of 293T-PD1 host cells in a dose dependent manner. Fluorescence intensities at different doses are shown in Table 1. 4. Through curve modeling using quantification results as fluorescence intensity values, the EC50 of 14C12H1L1 binding to PD1 was determined to be 1.89 nM.

Table 4

Fluorescence intensities of 14C12H1L1 binding to 293T-PD1 surface antigen detected by flow cytometry

Concentration (nM) 0.01 0.1 1 5 10 50 Fluorescence intensity 8.32 20.31 174.62 579.41 686.49 669.54

Example 9 Antibody Binding Activity to T Cell Surface Antigen PD1 by Flow Cytometry.

PBMCs were isolated using Ficoll-Paque Plus (GE Healthcare lot #171440-02), then CD4+ cells were isolated and the cells were stimulated with PHA (phytohemagglutinin) (Shanghai Shenqi Biotech Co., Ltd, 50 μl/ml) for three days, and then washed once with PBS and antibodies at various concentrations were added and incubated on ice for 1.5 h. Cells were then washed with PBS once after incubation, FITC labeled goat anti-human secondary antibody (Jackson immunoresearch lot. 102155) was added, incubated on ice in the dark for 1 h and after a single wash with PBS, fluorescence signals were measured on a flow cytometer.

The results of binding of the humanized 14C12H1L1 antibody to T cells are shown in FIG. 8. It appears that the 14C12H1L1 antibody can effectively bind the PD1 protein on the surface of T cells in a dose dependent manner.

Example 10. The reaction of mixed lymphocytes.

Cytokine secretion IFN-γ, IL-2 PBMCs were isolated using Ficoll-Paque Plus (GE Healthcare lot #171440-02), additional isolated PBMCs induced IL-4 (Peprotech K2513, 1000 U/mL) and GM-CSF ( Peprotech H1513, 1000 U/ml) for 6 days, and then TNF-α (Peprotech G1513, 200 U/ml) was added for induction for 3 days to obtain DC cells (dendritic cells).

-

Claims (25)

T cells were isolated from PBMC and mixed with DC cells obtained above at a ratio of 10:1 for co-culturing with 14C12H1L1, 5C4 and hIgG antibodies (hIgG as isotype control) in various ratios for 5-6 days. IFN-γ and IL-2 secretion values were measured using appropriate ELISA kits (both purchased from Dakewe), respectively. The secretions of IFN-γ and IL-2 after mixing the culture of DC and T cells were shown in FIG. 9 and 10. The 14C12H1L1 antibody can effectively induce IFN-γ and IL-2 secretion in a dose dependent manner. The 14C12H1L1 antibody can induce higher secretion of both IFN-γ and IL-2 than the control 5C4 antibody. Example 11. Induced secretion of IL-2. Isolated PBMCs (same method as in Example 10) were stimulated with PHA (Shanghai Shenqi Biotech Co., Ltd, 50 μl/ml) for 3 days, and then mature PBMCs (5×10 ⁴ cells/well) were mixed with Raji cells (Chinese Academy of Sciences Shanghai Branch) (5 x 10 ⁴ cells/well) and MDA-MB-231 (ATCC) cells (1 x 10 ⁴ cells/well) in a 96-well plate. Added 100 nm 14C12H1L1 or control antibody 5C4 or hIgG (hIgG as isotype control), mixed and cultured together for 3 days. IL-2 secretion was determined using an ELISA kit (purchased from Dakewe) according to kit instructions. The results of IL-2 secretion after mixed cell culture incubation are shown in FIG. 11. As shown in FIG. 11, the antibody can effectively induce PBMC to secrete IL-2, and the secretion of IL-2 induced by 14C12H1L1 is significantly higher than that for the control 5C4 antibody. Example 12 Effect of 14C12H1L1 antibody on tumor growth in the MC38 tumor model in PD-1 HuGEMM mice. MC38 tumor cells (1×10 ⁶ cells/mouse) were inoculated subcutaneously into the right flank of HuGEMM PD-1 mice (human PD-1 transgenic mice). When the average tumor volume reached approximately 118 mm ³ , mice were randomly divided into 4 experimental groups of 8 mice in each group. Antibodies were given by administration through the abdominal cavity. Specific groups and dosages were as follows: isotype control group (dose: 8 mg/kg), 14C12H1L1 high dose group (dose: 8 mg/kg), 14C12H1L1 low dose group (dose: 0.8 mg/kg), nivolumab group (dose: 8 mg/kg). The above 4 groups were given antibodies twice a week for a total of 5 doses. After injection, tumor sizes were measured twice a week. The results are shown in FIG. 12. The results indicate that, statistically, tumor sizes in the nivolumab group, the high-dose 14C12H1L1 group, and the low-dose 14C12H1L1 group were significantly smaller than those in the isotype control group (P<0.01, P<0.01, P<0, 05 respectively). The high-dose 14C12H1L1 (8 mg/kg) group demonstrated a statistically significant antitumor effect in the MC38 tumor model in PD-1 HuGEMM mice and was equivalent to the approved drug for the same target, nivolumab (8 mg/kg). Although specific embodiments of the present invention have been described in detail, as will be understood by a person skilled in the art, these details may entail various modifications and substitutions in accordance with all of the principles disclosed by us. All these changes are covered by the scope of this invention. The entire scope of this invention is covered by the appended claims and any equivalents. CLAIM 1. Anti-PD-I monoclonal antibody or antigennegative fragment, where the variable region of the heavy chain (VH) of the monoclonal antibody contains a CDR with the amino acid sequences of SEQ ID NO: 9-11; and the light chain variable region (V _L ) of the monoclonal antibody contains CDRs with the amino acid sequences of SEQ ID NOs: 12-14. 2. Monoclonal antibody or antigennegative fragment according to claim 1, where the amino acid sequence VH of the monoclonal antibody is selected from SEQ ID NO: 2 and SEQ ID NO: 6; and the VL amino acid sequence of the monoclonal antibody is selected from SEQ ID NO: 4 and SEQ ID NO: 8. 3. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein the monoclonal antibody or antigen-binding fragment thereof comprises VH with the amino acid sequence of SEQ ID NO: 2 and V _L with the amino acid sequence of SEQ ID NO: 4. 4. A monoclonal antibody or antigen-binding fragment according to claim 2, where the monoclonal - 13 042479 antibody or its antigen-binding fragment contains VH with the amino acid sequence of SEQ ID NO: 6 and V _L with the amino acid sequence of SEQ ID NO: 8. 5. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antigen-binding fragment is a Fab, Fab', F(ab')2, Fd, Fv, dAb, a CDR fragment, or a single chain antibody (scFv). 6. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the binding affinity (KD) for PD1 of the monoclonal antibody or antigen-binding fragment thereof is less than 10 ^-5 M. 7. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the binding affinity (KD) for PD1 of the monoclonal antibody or antigen-binding fragment thereof is 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 or 10 ^{- 10} M or less. 8. The monoclonal antibody or antigen-binding fragment thereof according to claim 6 or 7, wherein the KD is measured with a Fortebio molecular interaction analyzer. 9. The monoclonal antibody or antigen-binding fragment thereof according to claims 1 to 4, wherein the monoclonal antibody or antigen-binding fragment thereof contains regions other than CDRs derived from a species other than a mouse. 10. The monoclonal antibody or antigen-binding fragment thereof according to claim 9, wherein the monoclonal antibody or antigen-binding fragment thereof contains regions other than human-derived CDRs. 11. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the monoclonal antibody or antigen-binding fragment thereof is produced by the LT003 hybridoma cell line and the LT003 hybridoma cell line is deposited with the China Type Culture Collection Center (CCTCC) with the deposit accession number CTCCC: C2015105. 12. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain variable region (VH) of a monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the VH contains a CDR with the amino acid sequences of SEQ ID NOs: 9-11. 13. The isolated nucleic acid molecule according to claim 12, wherein the V _H contains the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6. 14. The isolated nucleic acid molecule according to claim 13, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 5. 15. An expression construct containing an isolated nucleic acid molecule according to any one of claims 12-14. 16. The expression construct according to claim 15, wherein the expression construct further comprises an isolated nucleic acid molecule or an expression construct containing a nucleotide sequence encoding the light chain variable region (VL) of a monoclonal antibody or antigen-binding fragment thereof according to claim 1, where V _L contains a CDR with the amino acid sequences of SEQ ID NO: 12-14. 17. Expression construct according to claim 16, where V _L contains the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 8. 18. The expression construct according to claim 17, wherein the nucleotide sequence encoding the light chain variable region (V _L ) of the monoclonal antibody or its antigen-binding fragment according to claim 1 contains the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 7. 19. An expression cell line containing an isolated nucleic acid molecule according to any one of claims 12-14 or an expression construct according to any one of claims 15-18. 20. The LT003 hybridoma cell line deposited with the China Type Culture Collection Center (CCTCC) with CCCCC Accession Number: C2015105 for the production of an anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11. 21. A conjugate comprising a monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-11 and a conjugation partner as a detectable marker. 22. The conjugate of claim 21 wherein the conjugation partner is a radioactive isotope, fluorescein, luminescent substance, dyes or enzymes. 23. Pharmaceutical composition containing a monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 11 or a conjugate according to claim 21 or 22 and a pharmaceutically acceptable carrier or excipient. 24. The use of a monoclonal antibody or an antigen-binding fragment thereof according to claims 1 to 11, or a conjugate according to claim 21 or 22, or a pharmaceutical composition according to claim 23 for the treatment of tumors. 25. Use according to claim 24, wherein the tumors are melanoma, kidney cancer, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, non-small cell lung cancer, ovarian cancer and leukemia. -