Use of anti-PD-1 antibodies in treating tumors

Abstract

The present invention discloses the use of anti-PD-1 antibodies in the treatment of melanoma. The present invention also discloses the use of reagents for detecting BRAF, NRAS, and CDK4 / CCND1 gene mutations in a test kit for predicting the efficacy of melanoma patients in response to treatment with anti-PD-1 antibodies and / or antigen-binding fragments thereof alone.

Description

Technical Field

[0001] The present invention relates to the use of anti-PD-1 antibodies in treating tumors. Specifically, the present invention relates to the use of anti-PD-1 antibodies in treating melanoma; the use of anti-PD-1 antibodies in preparing a medicament for treating melanoma; and a method for predicting the efficacy of anti-PD-1 antibodies alone in treating melanoma using biomarkers. Background Art

[0002] Immune escape is one of the characteristics of cancer. Ahmadzadeh, M. et al., Blood, 114:1537-44, disclosed that tumor-specific T lymphocytes are often present in the tumor microenvironment, draining lymph nodes and peripheral blood, but due to the network of immunosuppressive mechanisms in the tumor microenvironment, they are usually unable to control the progression of the tumor. + Tumor-infiltrating T lymphocytes (TILs) typically express activation-induced inhibitory receptors, including CTLA-4 and PD-1, while tumor cells frequently express immunosuppressive ligands, including PD-1 ligand 1 (PD-L1, also known as B7-H1 or CD274), which inhibits T cell activation and effector function. Among these inhibitory mechanisms, PD-1 and its ligands have emerged as important pathways that tumor cells exploit to suppress activated T cells in the tumor microenvironment.

[0003] Programmed death receptor 1 (PD-1) plays an important role in immune regulation and maintaining peripheral tolerance. PD-1 is primarily expressed in activated T and B cells and functions to inhibit lymphocyte activation, a normal peripheral tissue tolerance mechanism of the immune system that prevents immune hyperactivity. However, activated T cells infiltrating the tumor microenvironment highly express the PD-1 molecule. Inflammatory factors secreted by activated leukocytes induce tumor cells to highly express PD-1 ligands PD-L1 and PD-L2, leading to persistent activation of the PD-1 pathway on activated T cells in the tumor microenvironment. This suppresses T cell function and prevents them from killing tumor cells. Therapeutic PD-1 antibodies can block this pathway, partially restoring T cell function and enabling activated T cells to continue killing tumor cells.

[0004] Over the past decade, PD-1 / PD-L1 pathway blockade has been shown to be an effective approach for inducing durable anti-tumor responses in various cancer indications. Monoclonal antibodies (mAbs) that block the PD-1 / PD-L1 pathway can enhance the activation and effector function of tumor-specific T cells, reduce tumor burden, and improve survival. Between 2014 and 2017, the FDA approved two anti-PD1 monoclonal antibodies (nivolumab and pembrolizumab) and three anti-PD-L1 monoclonal antibodies (atezolizumab, avelumab, and durvalumab) for the treatment of human tumors. Melanoma was the first indication for nivolumab and pembrolizumab approved in 2014.

[0005] Melanoma has long been considered a highly immunogenic cancer due to the frequent observation of lymphocyte infiltration into tumors and clinical responses to high-dose IL-2 immunotherapy. Mechanistically, chronic UV radiation exposure, associated with the induction of DNA damage, is the primary cause of melanoma in Western populations. Chronic UV-induced sunburn (CSD) melanomas account for 95% of cutaneous melanomas in the United States and other Western countries. In contrast, acral lentiginous melanoma (ALM) (~50%) and mucosal melanoma (MM) (~20%) are the two most common melanoma subgroups in Asian populations, a finding published by Chi, Z. et al., BMC Cancer (2011), 11:85. Furney, S.J. et al., Pigment Cell Melanoma Res (2014), 27:835-836, reported that both ALM and MM are not associated with chronic UV exposure and carry fewer DNA mutations. A retrospective study by Cho, J. et al., Invest New Drugs (2016), 34:677-84, showed that immunotherapy is less effective in treating ALM and MM than CSD melanoma. Therefore, there is uncertainty about the efficacy of immunotherapy, such as anti-PD-1 antibodies, in treating melanoma, especially in treating mucosal and acral melanomas, which are common in Asian populations. How to further improve its therapeutic effect is a technical problem that urgently needs to be solved in this field. In addition, although immune checkpoint inhibitor therapy has made significant improvements in the treatment of metastatic melanoma in the past decade, most of the studies are still on several types of skin-type (also called non-acral) melanoma in Caucasians. Research on Asian and African races is very limited, resulting in a large gap in the treatment of non-acral and primary melanoma in these two races, which needs to be addressed urgently. Summary of the Invention

[0006] In one aspect, the present invention provides a method for treating a melanoma patient, comprising administering to the patient a therapeutically effective amount of an anti-PD1-1 antibody.

[0007] A second aspect of the present invention provides a use of an anti-PD-1 antibody in the preparation of a medicament for treating melanoma patients.

[0008] In one or more embodiments, the melanoma is advanced or metastatic melanoma.

[0009] In one or more embodiments, the melanoma patient has a BRAF mutation.

[0010] In one or more embodiments, the melanoma patient is selected from the group consisting of acral melanoma, mucosal melanoma, non-acral cutaneous melanoma, and melanoma of uncertain primary lesion.

[0011] In one or more embodiments, the melanoma patient is a non-acral melanoma patient.

[0012] In one or more embodiments, the melanoma patient is a melanoma patient with uncertain primary lesion.

[0013] As one or more embodiments, the PD-L1 expression in the cancer tissue or section of the melanoma patient is tested positive. In one or more embodiments, the tumor mutation rate (TMB) of the biopsy sample or paired peripheral blood sample of the melanoma patient's cancer is ≥3.6Muts / Mb. In one or more embodiments, the PD-L1 expression in the cancer tissue or section of the melanoma patient's cancer is tested positive and the tumor mutation rate (TMB) of the biopsy sample or paired peripheral blood sample of the cancer is ≥3.6Muts / Mb.

[0014] A third aspect of the present invention provides a method for treating a melanoma patient, comprising administering to the patient a therapeutically effective amount of a CDK4 / 6 inhibitor alone, or administering a therapeutically effective amount of a CDK4 / 6 inhibitor in combination with an anti-PD-1 antibody.

[0015] In one or more embodiments, the melanoma patient has CDK4 or CCDN1 gene amplification.

[0016] In a fourth aspect, the present invention provides a kit for predicting the effect of anti-PD-1 antibody treatment on an individual with a tumor, the kit comprising: (a) a reagent for detecting BRAF mutations in an individual's tumor tissue or peripheral blood; and (b) instructions for using the reagent described in (a) to detect the effect of anti-PD-1 antibody treatment on an individual.

[0017] In a fifth aspect, the present invention provides a kit for predicting the effect of anti-PD-1 antibody treatment on an individual suffering from a tumor, the kit comprising: (a) a reagent for detecting CDK4 / CCND1 gene mutation or amplification in an individual's tumor tissue or peripheral blood; and (b) instructions for using the reagent described in (a) to detect the effect of CDK4 / 6 inhibitors alone or in combination with anti-PD-1 antibodies on the individual.

[0018] In a sixth aspect, the present invention provides a kit for predicting the effect of anti-PD-1 antibody treatment on an individual suffering from a tumor, the kit comprising: (a) a reagent for detecting NRAS gene mutations in an individual's tumor tissue or peripheral blood; and (b) instructions for using the reagent described in (a) to detect the effect of anti-PD-1 antibody treatment on an individual.

[0019] In the uses, methods, and kits described herein, the anti-PD-1 antibody is a monoclonal antibody or an antigen-binding fragment thereof. In certain embodiments, the anti-PD-1 antibody specifically binds to PD-1, blocking the binding of PD-L1 or PD-L2 to PD-1. In certain embodiments, the anti-PD-1 antibody specifically binds to PD-L1 and / or PD-L2, blocking the binding of PD-L1 and / or PD-L2 to PD-1.

[0020] In one or more embodiments, the anti-PD-1 antibody is an antibody comprising at least one complementarity determining region (CDR), the amino acid sequence of which complementarity determining region (CDR) is selected from the following: SEQ ID NO: 1, 2, 3, 4, 5 or 6.

[0021] In one or more embodiments, the anti-PD-1 antibody is an antibody comprising a complementarity determining region (CDR), wherein the amino acid sequence of the light chain complementarity determining region (LCDR) is shown in SEQ ID NOs: 1, 2, and 3, and the amino acid sequence of the heavy chain complementarity determining region (HCDR) is shown in SEQ ID NOs: 4, 5, and 6.

[0022] In one or more embodiments, the anti-PD-1 antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the amino acid sequence of VL is shown in SEQ ID NO: 7, and the amino acid sequence of VH is shown in SEQ ID NO: 8.

[0023] In one or more embodiments, the anti-PD-1 antibody is an anti-PD-1 antibody comprising a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence shown in SEQ ID NO: 9, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 10.

[0024] In one or more embodiments, the anti-PD-1 antibody is selected from one or more of nivolumab, pembrolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, and cemiplimab.

[0025] In one or more embodiments, the single administration dose of the anti-PD-1 antibody or antigen-binding fragment thereof is from about 0.1 mg / kg to about 10.0 mg / kg of the individual's body weight, for example, about 0.1 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 5 mg / kg or 10 mg / kg of the individual's body weight, or is selected from a fixed dose of about 120 mg to about 480 mg, for example, a fixed dose of about 120 mg, 240 mg, 360 mg or 480 mg.

[0026] In one or more embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a frequency of about once a week, once every two weeks, once every three weeks, once every four weeks, or once a month, preferably once every two weeks.

[0027] In one or more embodiments, the single administration dose of the anti-PD-1 antibody or antigen-binding fragment thereof is 1 mg / kg body weight, 3 mg / kg body weight, 10 mg / kg body weight, or a fixed dose of 240 mg, 480 mg, administered once every two weeks.

[0028] In one or more embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered in a liquid dosage form, such as an injection, parenterally, such as by intravenous infusion.

[0029] In one or more embodiments, the administration cycle of the anti-PD-1 antibody or antigen-binding fragment thereof can be one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months or longer. Optionally, the duration of each administration cycle can be the same or different, and the interval between each administration cycle can be the same or different.

[0030] In one or more embodiments, the CDK4 / 6 inhibitor is selected from palbociclib, ribociclib, and pomacicillin.

[0031] In the uses, methods and kits of the present invention, the individual is a human.

[0032] In one or more embodiments of the uses, methods, and kits of the present invention, the subject has melanoma and has not previously received anti-PD-1 or anti-PD-L1 immunotherapy. In a preferred embodiment, the cancer is advanced melanoma. In a preferred embodiment, the cancer is metastatic melanoma.

[0033] A seventh aspect of the present invention provides a method for predicting the therapeutic effect of anti-PD-1 antibodies on tumor patients, comprising detecting a biomarker in the patient's tumor tissue or peripheral blood before treatment, wherein the biomarker is a BRAF gene mutation, and wherein the presence of the BRAF gene mutation indicates that the tumor patient is suitable for treatment with anti-PD-1 antibodies.

[0034] An eighth aspect of the present invention provides a method for predicting the efficacy of anti-PD-1 antibody treatment in a cancer patient, comprising detecting a biomarker in the patient's tumor tissue, wherein the biomarker is CDK4 or CCND1 gene amplification. In one or more embodiments, the presence of CDK4 or CCND1 gene amplification indicates that the cancer patient is suitable for treatment with a combination of an anti-PD-1 antibody and a CDK4 / 6 inhibitor. In another or more embodiments, the presence of CDK4 or CCND1 gene amplification indicates that the cancer patient is suitable for treatment with a CDK4 / 6 inhibitor.

[0035] A ninth aspect of the present invention provides a method for predicting the efficacy of anti-PD-1 antibody treatment in a cancer patient, comprising detecting a biomarker in the patient's tumor tissue or peripheral blood before treatment, wherein the biomarker is a NRAS gene mutation. In one or more embodiments, the presence of the NRAS gene mutation indicates that treatment with the anti-PD-1 antibody alone is ineffective or unsuitable.

[0036] In the uses, methods and kits of the present invention, the tumor is a solid tumor. In one or more embodiments, the tumor is a melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : Diagram of a Phase II clinical study of toripalimab in patients with locally advanced or metastatic melanoma versus standard of care.

[0038] Figure 2Clinical response was assessed according to RECIST v1.1. A: Change in maximum tumor size from baseline in patients with at least one post-treatment imaging assessment (n = 119). The length of the bar represents the maximum reduction or minimum increase in target lesions. B: Change in individual tumor burden from baseline over time (n = 119). C: Exposure and duration of response in confirmed responders (n = 22).

[0039] Figure 3 : Progression-free survival (PFS) (A) and overall survival (OS) (B) of melanoma patients in this study.

[0040] Figure 4 : Overall survival (OS) of the responder subgroup (A) and the melanoma subgroup (B) in this study.

[0041] Figure 5 : Progression-free survival (PFS) of melanoma subtypes in this study.

[0042] Figure 6 : Relationship between clinical response and tumor PD-L1 expression and TMB. A: PD-L1 positivity was defined as membrane staining of tumor cells or immune cells with any intensity ≥1% using SP142 IHC staining; TMB was calculated by whole-exome sequencing of somatic mutations within coding regions; 3.6 muts / Mb was used as the cutoff value; B: PD-L1 + Or the percentage of TMB ≥ 3.6 Muts / Mb in the melanoma subgroup; C: PD-L1 + and PD-L1 - Progression-free survival (PFS) of patients with D: PD-L1 + and PD-L1 - E: progression-free survival (PFS) of patients with TMB ≥ 3.6 Muts / Mb and TMB < 3.6 Muts / Mb; F: overall survival (OS) of patients with TMB ≥ 3.6 Muts / Mb and TMB < 3.6 Muts / Mb.

[0043] Figure 7 : Gene variants and frequencies in 98 patients by whole exome sequencing (WES).

[0044] Figure 8 : Relationship between clinical response and signature scores of IFN-γ-related gene set, inflammation-related gene set, and angiogenesis-related gene set. DETAILED DESCRIPTION

[0045] The present invention relates to methods for treating tumors. These methods comprise administering an anti-PD-1 antibody or an antigen-binding fragment thereof alone to a patient in need thereof; or administering an anti-PD-1 antibody in combination with another anticancer agent to a patient in need thereof. The present invention also relates to methods for using biomarkers to predict the efficacy of anti-PD-1 antibodies in treating cancer, particularly melanoma.

[0046] the term

[0047] In order to make the present invention easier to understand, certain technical terms are specifically defined below. Unless otherwise explicitly stated elsewhere in this document, the technical terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0048] "Administering," "giving," and "treating" refer to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those skilled in the art. Routes of administration of anti-PD-1 antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal, or other parenteral routes of administration, such as injection or infusion. "Parenteral administration" refers to administration other than enteral or topical administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intracortical, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intradural, and intrasternal injection and infusion, as well as in vivo electroporation.

[0049] An "adverse effect" (AE) as described herein is any unfavorable and generally unintended or undesirable sign, symptom, or disease associated with the use of a medical treatment. For example, an adverse effect may be associated with activation of the immune system or expansion of immune system cells in response to the treatment. A medical treatment may have one or more associated AEs, and each AE may have the same or different levels of severity.

[0050] "Tumor burden" refers to the total amount of tumor material distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of a tumor throughout the body. Tumor burden can be determined by a variety of methods known in the art, such as measuring the size of a tumor after it has been removed from a subject using calipers, or while in vivo using imaging techniques such as ultrasound, bone scans, computed tomography (CT), or magnetic resonance imaging (MRI) scans.

[0051] The term "tumor size" refers to the overall size of a tumor, which can be measured as the length and width of the tumor. Tumor size can be determined by a variety of methods known in the art, such as measuring the size of the tumor using calipers after removal from the subject, or using imaging techniques (such as bone scans, ultrasound, CT or MRI scans) while in vivo.

[0052] The terms "subject," "individual," and "object" include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. The terms "subject" and "patient" are used interchangeably herein.

[0053] As used herein, "antibody" refers to any form of antibody that can achieve the desired biological activity or binding activity. Therefore, it is used in the broadest sense, but is not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies, humanized full-length human antibodies, chimeric antibodies, and camelid-derived single-domain antibodies. An "antibody" specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region, the heavy chain constant region comprising three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (VL) and a light chain constant region, the light chain constant region comprising one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, known as complementarity determining regions (CDRs), which are interspersed with more conserved regions known as framework regions (FRs). Generally speaking, from N-terminus to C-terminus, both the light and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Amino acids are typically assigned to each domain according to the following definitions: Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th Edition; NIH Publication No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat et al., (1977) J. Biol. Chem. 252: 6609-6616; Chothia et al., (1987) J Mol. Biol. 196: 901-917 or Chothia et al., (1989) Nature 341: 878-883.

[0054] The carboxyl-terminal portion of the heavy chain defines the constant region primarily responsible for effector function. Human light chains are typically classified as kappa and lambda chains. Human heavy chains are typically classified as μ, δ, γ, α, or ε, and define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG subclasses are well known to those skilled in the art and include, but are not limited to, IgG1, IgG2, IgG, and IgG4.

[0055] The term "antibody" includes: naturally occurring and non-naturally occurring Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or non-human Abs; fully synthetic Abs; and single-chain Abs. Non-human Abs can be humanized by recombinant methods to reduce their immunogenicity in humans.

[0056] Unless otherwise expressly indicated, "antibody fragments" or "antigen-binding fragments" as used herein refer to antigen-binding fragments of antibodies, i.e., antibody fragments that retain the ability of a full-length antibody to specifically bind to an antigen, such as fragments that retain one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; nanobodies; and multispecific antibodies formed from antibody fragments.

[0057] "Chimeric antibodies" refer to antibodies and fragments thereof in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species (such as human) or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species (such as mouse) or belonging to another antibody class or subclass, as long as it exhibits the desired biological activity.

[0058] A "human antibody" refers to an antibody that comprises only human immunoglobulin sequences. If the human antibody is produced in a mouse, mouse cell, or hybridoma derived from a mouse cell, it may contain murine carbohydrate chains. Similarly, a "mouse antibody" or "rat antibody" refers to an antibody that comprises only mouse or rat immunoglobulin sequences, respectively.

[0059] "Humanized antibody" refers to an antibody form containing sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins. Typically, a humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin. The humanized antibody optionally further comprises at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region.

[0060] The term "melanoma" refers to a type of malignant tumor originating from melanocytes, which is commonly found in the skin and also in the mucosa, choroid and other parts of the eye. Melanoma is the most malignant type of skin tumor and is prone to distant metastasis. Melanoma is divided into four subtypes, including acral, mucosal, cutaneous and indeterminate primary lesions. The cutaneous type is further divided into long-term sun-exposed cutaneous type (CSD) and non-long-term positive exposure damage type (non-CSD). CSD and non-CSD are collectively referred to as solar type. McLaughlin et al., Cancer, 2005, Mar 1, 103(5): 1000-1007; Chi Z. et al., BMC Cancer, 2011; 11: 85 disclosed that acral and mucosal melanomas are the most common subtypes in Asians, and their carcinogenesis is not caused by DNA mutations caused by ultraviolet radiation. Compared with solar melanoma, acral and mucosal melanomas contain only a small number of DNA mutations. In the United States, 95% of melanomas are solar melanomas, while in Asia, particularly China, over 70% are acral and mucosal melanomas, with mucosal melanoma accounting for over 50%. Invest New Drugs, 34:677-684; Cho, J. et al., reported that immunotherapy is less effective in treating acral and mucosal melanomas than in treating solar melanomas.

[0061] The term "immunotherapy" refers to the treatment of a subject who has a disease or is at risk of contracting or suffering from a recurrence of a disease by methods that include inducing, enhancing, suppressing, or otherwise modifying an immune response. "Treatment" or "therapy" of a subject refers to any type of intervention or procedure performed on a subject, or the administration of an active agent to a subject, with the intent to reverse, alleviate, ameliorate, slow, or prevent the onset, progression, severity, or recurrence of symptoms, complications, or conditions, or biochemical markers associated with a disease.

[0062] "Programmed death receptor-1 (PD-1)" refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 is primarily expressed on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. The term "PD-1" as used herein includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs that share at least one common epitope with hPD-1.

[0063] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes disease regression, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of impairment or disability resulting from the affliction of the disease. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predictive of human efficacy, or by measuring the activity of the agent in in vitro assays.

[0064] A therapeutically effective amount of a drug includes a "prophylactically effective amount," ie, any amount of a drug that, when administered alone or in combination with an anti-tumor agent to a subject at risk of developing cancer or to a subject who has had a recurrence of cancer, inhibits the development or recurrence of cancer.

[0065] "Biotherapeutic agent" refers to a biological molecule, such as an antibody or fusion protein, that blocks ligand / receptor signaling in any biological pathway that supports tumor maintenance and / or growth or suppresses anti-tumor immune responses.

[0066] Unless expressly indicated otherwise, "CDR" as used herein refers to the complementarity determining regions of immunoglobulin variable regions as defined using the Kabat numbering system.

[0067] "Therapeutic anti-PD-1 monoclonal antibodies" refer to antibodies that specifically bind to a specific mature form of PD-1 expressed on the surface of certain mammalian cells. Mature PD-1 lacks a pre-secretory leader sequence, or leader peptide. The terms "PD-1" and "mature PD-1" are used interchangeably herein and, unless otherwise explicitly defined or clearly evident from the context, should be understood to refer to the same molecule.

[0068] As described herein, therapeutic anti-human PD-1 antibodies or anti-hPD-1 antibodies refer to monoclonal antibodies that specifically bind to mature human PD-1.

[0069] As used herein, "framework region" or "FR" refers to an immunoglobulin variable region excluding the CDR regions.

[0070] An "isolated antibody or antigen-binding fragment thereof" refers to a purified state and in which case the designated molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials (such as cell debris or growth medium).

[0071] "Patient," "patient," or "subject" refers to any single human, typically a mammal, including humans and other mammals such as horses, cows, dogs, or cats, who is in need of medical treatment or is participating in a clinical trial, epidemiological study, or serving as a control.

[0072] The "RECIST 1.1 efficacy criteria" described herein refer to the definitions of target damage or non-target damage described in Eisenhauver et al., EA et al., Eur. J Cancer 45: 228-247 (2009) based on the context of the measured response. Before immunotherapy, it was the most commonly used standard for evaluating the efficacy of solid tumors. However, with the advent of the immune era, many problems that had not previously appeared in tumor evaluation have emerged. Therefore, based on the emerging phenomena caused by immunotherapy itself, in 2016, the RECIST Working Group revised the existing "RECIST v.1.1" and proposed a new judgment standard, namely the "irRECIST standard" described herein, which aims to better evaluate the efficacy of immunotherapy drugs.

[0073] The Eastern Cooperative Oncology Group (ECOG) performance status score (ECOG) is an indicator of a patient's general health and ability to tolerate treatment based on their physical strength. The ECOG performance status score is 0, 1, 2, 3, 4, or 5. A score of 0 indicates completely normal activity, no difference from pre-onset activity. A score of 1 indicates the patient can move freely and engage in light physical activity, including general housework or office work, but cannot engage in heavy physical activity.

[0074] "Sustained response" refers to a sustained therapeutic effect after cessation of treatment with a therapeutic agent or combination therapy described herein. In some embodiments, the sustained response has a duration that is at least the same as the duration of treatment or at least 1.5, 2.0, 2.5, or 3 times the duration of treatment.

[0075] "Tissue section" refers to a single portion or piece of a tissue sample, such as a thin slice of tissue cut from a sample of normal tissue or a tumor.

[0076] "Treatment" of cancer as described herein refers to the use of the treatment regimen described herein (such as the administration of an anti-PD-1 antibody or a combination therapy of an anti-PD-1 antibody and a CDK4 / 6 inhibitor) in a subject suffering from or diagnosed with cancer to achieve at least one positive therapeutic effect (e.g., a decrease in the number of cancer cells, a decrease in tumor volume, a decrease in the rate of cancer cell infiltration into peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth). Positive therapeutic effects in cancer can be measured in a variety of ways (see WA Weber, J. Nucl. Med., 50: 1S-10S (2009)). For example, with respect to tumor growth inhibition, according to NCI standards, T / C≦42% is the minimum level of anti-tumor activity. It is considered that T / C (%) = median volume of treated tumors / median volume of control tumors × 100. In some embodiments, the therapeutic effect achieved by the combination of the present invention is any one of PR, CR, OR, PFS, DFS and OS. PFS (also called "time to tumor progression") refers to the length of time that cancer does not grow during and after treatment, and includes the amount of time the patient experiences CR or PR and the amount of time the patient experiences SD. DFS refers to the length of time the patient remains disease-free during and after treatment. OS refers to the extension of life expectancy compared to an initial or untreated individual or patient. In some embodiments, the response to the combination of the present invention is any one of PR, CR, PFS, DFS, OR, or OS, which is assessed using RECIST 1.1 efficacy criteria. The treatment regimen of the combination of the present invention for effectively treating cancer patients can vary according to a variety of factors (such as the patient's disease state, age, weight, and the ability of the therapy to stimulate the subject's anti-cancer response). Although embodiments of the present invention may not achieve an effective positive therapeutic effect in every subject, it should be effective and achieve a positive therapeutic effect in a statistically significant number of subjects.

[0077] The terms "administration method" and "dosage regimen" are used interchangeably to refer to the dosage and timing of each therapeutic agent in the combination of the present invention.

[0078] The term "immunohistochemistry (IHC)" refers to a method for determining the location, qualitative, and relative quantitative analysis of antigens (peptides and proteins) within tissue cells by using the principle of specific binding between antigens and antibodies to develop color with a colorant (fluorescein, enzyme, metal ion, isotope) labeled with the antibody through a chemical reaction. In some embodiments of the present invention, prior to treatment with an anti-PD-1 antibody, a PD-L1 test is performed on a tumor tissue sample from a subject using Roche's anti-human PD-L1 antibody SP142 (Cat No: M4422) for staining. In some embodiments, a membrane staining intensity of ≥1% of tumor cells is defined as PD-L1 positive.

[0079] In the following paragraphs, various aspects of the invention are described in further detail.

[0080] Anti-PD-1 antibodies

[0081] As used herein, "PD-1 antibody" refers to any chemical compound or biological molecule that binds to the PD-1 receptor, blocks the binding of PD-L1 expressed on cancer cells to PD-1 expressed on immune cells (T, B, NK cells), and preferably also blocks the binding of PD-L2 expressed on cancer cells to PD-1 expressed on immune cells. Alternative terms or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279, and SLEB2 for PD-1; PDCD1L1, PDL1, B7-H1, B7H1, B7-4, CD274, and B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC, and CD273 for PD-L2. In any of the methods, medicaments, and uses of the present invention for treating human subjects, the PD-1 antibody blocks the binding of human PD-L1 to human PD-1, and preferably blocks the binding of both human PD-L1 and PD-L2 to human PD1. The amino acid sequence of human PD-1 can be found at NCBI Locus Accession No. NP_005009. The amino acid sequences of human PD-L1 and PD-L2 can be found at NCBI Locus Accession No. NP_054862 and NP_079515, respectively.

[0082] Herein, when referring to an "anti-PD-1 antibody," the term includes antigen-binding fragments thereof unless otherwise indicated or described.

[0083] The anti-PD-1 antibodies suitable for any of the uses, therapies, drugs and kits described herein bind to PD-1 with high specificity and affinity, block the binding of PD-L1 / 2 to PD-1, and inhibit PD-1 signal transduction, thereby achieving an immunosuppressive effect. In any of the uses, therapies, drugs and kits disclosed herein, the anti-PD-1 antibodies include the full-length antibodies themselves, as well as antigen-binding portions or fragments that bind to the PD-1 receptor and exhibit functional properties similar to those of the complete Ab in inhibiting ligand binding and upregulating the immune system. In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment is an anti-PD-1 antibody or its antigen-binding fragment that cross-competes with toripalimab for binding to human PD-1. In other embodiments, the anti-PD-1 antibody or its antigen-binding fragment is a chimeric, humanized or human Ab or its antigen-binding fragment. In certain embodiments for treating human individuals, the Ab is a humanized Ab.

[0084] In some embodiments, the anti-PD-1 antibody for any of the uses, therapies, medicaments, and kits described herein comprises a monoclonal antibody (mAb) or an antigen-binding fragment thereof that specifically binds to PD-1, and preferably specifically binds to human PD-1. The mAb can be a human antibody, a humanized antibody, or a chimeric antibody, and can include a human constant region. In some embodiments, the constant region is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 constant regions; preferably, the anti-PD-1 antibody or antigen-binding fragment thereof for any of the uses, therapies, medicaments, and kits described herein comprises a heavy chain constant region of the human IgG1 or IgG4 isotype, more preferably a human IgG4 constant region. In some embodiments, the sequence of the IgG4 heavy chain constant region of the anti-PD-1 antibody or antigen-binding fragment thereof comprises an S228P mutation, which replaces a serine residue in the hinge region with a proline residue that is typically present at the corresponding position in antibodies of the IgG1 isotype.

[0085] Preferably, in any embodiment of the use, therapy, drug and kit described in the present invention, the PD-1 antibody is a monoclonal antibody or an antigen-binding fragment thereof, and its light chain CDRs are the amino acids shown in SEQ ID NOs: 1, 2 and 3, and the heavy chain CDRs are the amino acids shown in SEQ ID NOs: 4, 5 and 6.

[0086] More preferably, in any embodiment of the use, therapy, medicine and kit described in the present invention, the PD-1 antibody is a monoclonal antibody that specifically binds to human PD-1 and comprises: (a) a light chain variable region represented by SEQ ID NO: 7, and (b) a heavy chain variable region represented by SEQ ID NO: 8.

[0087] Further preferably, in any embodiment of the use, therapy, medicine and kit described in the present invention, the PD-1 antibody is a monoclonal antibody that specifically binds to human PD-1 and comprises: (a) a light chain represented by SEQ ID NO: 9, and (b) a heavy chain represented by SEQ ID NO: 10.

[0088] Table A below provides the amino acid sequence numbers of the light chain CDRs and heavy chain CDRs of exemplary anti-PD-1 antibody mAbs for use in the uses, therapies, medicaments, and kits described herein:

[0089] Table A: Light and heavy chain CDRs of exemplary anti-human PD-1 antibodies (Kabat)

[0090] LCDR1 SEQ ID NO: 1 LCDR2 SEQ ID NO: 2 LCDR3 SEQ ID NO: 3 HCDR1 SEQ ID NO: 4 HCDR2 SEQ ID NO: 5 HCDR3 SEQ ID NO: 6

[0091] Examples of anti-PD-1 antibodies that bind to human PD-1 and can be used in the uses, therapies, medicaments, and kits described herein are described in WO2014206107. Human PD-1 mAbs that can be used as anti-PD-1 antibodies in the uses, therapies, medicaments, and kits described herein include any of the anti-PD-1 antibodies described in WO2014206107, including Toripalimab (a humanized IgG4 mAb having a structure described in WHO Drug Information (Vol. 32, No. 2, pp. 372-373 (2018)) and comprising the light and heavy chain amino acid sequences set forth in SEQ ID NOs: 9 and 10). In a preferred embodiment, the anti-PD-1 antibody that can be used in any of the uses, therapies, medicaments, and kits described herein is selected from humanized antibodies 38, 39, 41, and 48 described in WO2014206107. In a particularly preferred embodiment, the anti-PD-1 antibody that can be used in any of the uses, therapies, medicaments, and kits described herein is Toripalimab.

[0092] Anti-PD-1 antibodies that can be used in any of the uses, therapies, drugs, and kits described in the present invention also include Nivolumab and Pembrolizumab approved by the FDA.

[0093] In certain embodiments, the anti-PD-1 antibodies that can be used in any of the uses, therapies, drugs, and kits described herein also include anti-PD-L1 monoclonal antibodies that specifically bind to PD-L1 to block the binding of PD-L1 to PD-1, such as nivolumab, pembrolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, and cemiplimab.

[0094] As used herein, "PD-L1" expression or "PD-L2" expression refers to any detectable expression level of a specific PD-L protein on the surface of a cell or a specific PD-L mRNA within a cell or tissue. PD-L protein expression can be detected using diagnostic PD-L antibodies in IHC analysis of tumor tissue sections or by flow cytometry. Alternatively, PD-L protein expression by tumor cells can be detected by PET imaging using a binding agent that specifically binds to the desired PD-L target (such as PD-L1 or PD-L2).

[0095] For methods for quantifying PD-L1 protein expression in IHC analysis of tumor tissue sections, see, but are not limited to, Thompson, RH et al., PNAS 101(49): 17174-17179 (2004); Taube, JM et al., Sci Transl Med 4, 127ra37 (2012); and Toplian, SL et al., New Eng. J. Med. 366(26): 2443-2454 (2012).

[0096] One approach uses a simple binary endpoint of positive or negative PD-L1 expression, where a positive result is defined as the percentage of tumor cells showing histological evidence of cell surface membrane staining. Positive PD-L1 expression is defined as a count of at least 1% of total tumor cells in a tumor tissue section.

[0097] In another approach, PD-L1 expression in tumor tissue sections is quantified in tumor cells and in infiltrating immune cells. The percentage of tumor cells and infiltrating immune cells that exhibit membrane staining is quantified separately as <1%, 1% to 50%, and then 50% to 100%. For tumor cells, PD-L1 expression is counted as negative if the score is <1% and as positive if the score is ≥1%.

[0098] In some embodiments, the level of PD-L1 expression by malignant cells and / or by infiltrating immune cells within a tumor is determined to be "overexpressed" or "elevated" based on comparison to the level of PD-L1 expression by an appropriate control. For example, the control PD-L1 protein or mRNA expression level can be the level quantified in non-malignant cells of the same type or in sections from matched normal tissue.

[0099] CDK4 / 6 inhibitors

[0100] As used herein, the term "CDK" stands for cyclin-dependent kinases, a group of serine / threonine protein kinases. CDKs drive the cell cycle by phosphorylating serine / threonine proteins in concert with cyclins, making them crucial factors in cell cycle regulation. The CDK family comprises eight members, CDK 1-8, each of which binds to a different type of cyclin to form a complex that regulates the transition from G1 to S phase, or from G2 to M phase, and the process of exiting M phase.

[0101] Currently, the FDA-approved CDK4 / 6 inhibitors include ribociclib, palbociclib, and abemaciclib. Ribociclib, in combination with an aromatase inhibitor, can be used as a first-line treatment for postmenopausal women with advanced metastatic breast cancer that is HR-positive and HER2-negative. Palbociclib, in combination with letrozole, can be used to treat postmenopausal women with estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer. Abemaciclib is primarily used to treat adult patients with hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer whose disease has progressed after endocrine therapy.

[0102] There are dozens of CDK4 / 6 inhibitors in the clinical research stage, such as Milciclib, Trilaciclib, Lerociclib and Voruciclib.

[0103] In any use, therapy, drug and kit described in the present invention, CDK4 / 6 inhibitors include but are not limited to palbociclib.

[0104] In any of the uses, therapies, drugs and kits described in the present invention, the combination includes but is not limited to Toripalimab and Palbociclib.

[0105] Diseases and their treatment

[0106] The present invention relates to the treatment of cancer. The present invention comprises administering an anti-PD-1 antibody or an antigen-binding fragment thereof alone to a patient in need thereof; or comprises administering an anti-PD-1 antibody in combination with other anticancer agents to a patient in need thereof.

[0107] As used herein, the term "cancer" refers to a broad range of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division, proliferation, and growth lead to the formation of malignant tumors, which invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. Examples of cancers suitable for treatment or prevention using the methods, medicaments, and kits of the present invention include, but are not limited to, carcinomas, lymphomas, leukemias, blastomas, and sarcomas. More specific examples of cancer include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer.

[0108] Preferably, cancer patients suitable for the present invention are those with positive PD-L1 expression, or those with a tumor mutation burden (TMB) ≥ 3.6 Muts / Mb in peripheral blood or tumor tissue, or those with positive PD-L1 expression and a tumor mutation burden (TMB) ≥ 3.6 Muts / Mb in peripheral blood or tumor tissue. In certain embodiments, cancer patients suitable for treatment by the methods of the present invention are preferably those with BRAF gene mutations detected in tumor tissue.

[0109] As used herein, the term "tumor mutation burden (TMB)" refers to the total number of somatic gene coding errors, base substitutions, gene insertions, or deletions detected per million bases. In some embodiments of the present invention, tumor mutation burden (TMB) is estimated by analyzing somatic mutations (including coding base substitutions and megabase insertions of the panel sequences studied). In the present invention, when the tumor mutation burden (TMB) of a subject is greater than or equal to 3.6 Muts / Mb, it indicates that administering anti-PD-1 antibodies alone to such subjects, or administering anti-PD-1 antibodies in combination with other anticancer agents, can achieve better therapeutic effects than TMB < 3.6 Muts / Mb.

[0110] The method of treating cancer of the present invention comprises administering to a subject in need thereof a therapeutically effective amount of an anti-PD-1 antibody, or an anti-PD-1 antibody and a CDK4 / 6 inhibitor. The anti-PD-1 antibody may be as described in any embodiment herein, more preferably an antibody having a light chain CDR with the amino acids set forth in SEQ ID NOs: 1, 2, and 3, and a heavy chain CDR with the amino acids set forth in SEQ ID NOs: 4, 5, and 6, more preferably a monoclonal antibody comprising a light chain variable region set forth in SEQ ID NO: 7 and a heavy chain variable region set forth in SEQ ID NO: 8, more preferably a monoclonal antibody comprising a light chain set forth in SEQ ID NO: 9 and a heavy chain set forth in SEQ ID NO: 10, more preferably humanized antibodies 38, 39, 41, and 48 described in WO2014206107, and most preferably toripalimab. The CDK4 / 6 inhibitor is selected from the group consisting of Milciclib, Trilaciclib, Lerociclib, and Voruciclib, preferably Palbociclib. In a preferred embodiment, the present invention uses toripalimab and Palbociclib in combination to treat cancer.

[0111] In a particularly preferred embodiment, the present invention provides a method for treating melanoma, comprising administering a therapeutically effective amount of toripalimab to a melanoma patient; preferably, the patient is positive for PD-L1 expression, or has a tumor burden (TMB) ≥ 3.6Muts / Mb in peripheral blood or tumor tissue, or is positive for PD-L1 expression and has a tumor burden (TMB) ≥ 3.6Muts / Mb in peripheral blood or tumor tissue. In certain embodiments, the melanoma patient is preferably a melanoma patient in whom a BRAF gene mutation is detected in tumor tissue, more preferably a non-acral melanoma or a primary melanoma. In some embodiments, the melanoma is an acral or / and mucosal melanoma. In another particularly preferred embodiment, the present invention provides a method for treating melanoma, comprising administering a therapeutically effective amount of toripalimab and palbociclib to a melanoma patient; preferably, CDK4 or CCND1 gene amplification is present in the patient's peripheral blood or tumor tissue.

[0112] According to standard pharmaceutical practice, when two or more therapeutic agents are administered (i.e., "combination therapy"), each therapeutic agent can be administered alone or in a pharmaceutical composition comprising the therapeutic agent and one or more pharmaceutically acceptable carriers, excipients, and diluents.

[0113] Each therapeutic agent in the combination therapy of the present invention can be administered simultaneously, concurrently, or sequentially in any order. The therapeutic agents in the combination therapy are administered in different dosage forms, such as one drug is a tablet or capsule and the other drug is a sterile liquid, and / or are administered at different dosing times, such as a chemotherapeutic agent is administered at least daily and a biotherapeutic agent is administered less frequently, such as once every week, or every two weeks, or every three weeks.

[0114] In some embodiments, the CDK4 / 6 inhibitor is administered prior to administration of the anti-PD-1 antibody, while in other embodiments, the CDK4 / 6 inhibitor is administered after administration of the anti-PD-1 antibody.

[0115] In some embodiments, at least one of the therapeutic agents in the combination therapy is administered using the same dosing regimen (dose, frequency, duration of treatment) as when the drugs are used as monotherapy to treat the same cancer.

[0116] Each small molecule therapeutic agent in the combination therapy described herein can be administered orally or parenterally (eg, intravenous, intramuscular, intraperitoneal, subcutaneous, rectal, topical, or transdermal routes of administration).

[0117] The combination therapies described herein can be administered before or after surgery and can be administered before, during, or after radiation therapy.

[0118] In some embodiments, the combination therapy described herein is administered to patients who have not previously been treated with a biotherapeutic agent (typically an anti-PD-1 antibody) or a chemotherapeutic agent (typically a CDK4 / 6 inhibitor). In other embodiments, the combination therapy is administered to patients who have not achieved a sustained response after treatment with a biotherapeutic agent or a chemotherapeutic agent.

[0119] The combination therapies of the present invention may be used to treat tumors that are detected by palpation or by imaging techniques known in the art, such as MRI, ultrasound, or CAT scans.

[0120] The combination therapy of the present invention is preferentially administered to cancer patients whose PD-L1 expression is positive or whose TMB is ≥ 3.6 Muts / Mb.

[0121] The choice of dosing regimen for the combination therapies of the present invention depends on several factors, including but not limited to seroconversion rate or tissue conversion rate, severity of symptoms, immunogenicity, and accessibility of target cells, tissues, and organs in the individual being treated. Preferably, the dosing regimen is designed to deliver the maximum amount of each therapeutic agent to the patient while maintaining an acceptable level of side effects. Therefore, the dosage and frequency of each biotherapeutic and chemotherapeutic agent in the combination therapy will depend on the specific therapeutic agent, the severity of the cancer being treated, and the characteristics of the patient.

[0122] The anti-PD-1 antibody and CDK4 / 6 inhibitor of the present invention can be provided as a kit comprising a first container, a second container, and a package insert.

[0123] The first container contains at least one dose of a drug comprising an anti-PD-1 antibody, the second container contains at least one dose of a drug comprising a CDK4 / 6 inhibitor, and the package insert or label contains instructions for using the drug to treat cancer. The kit may further contain other materials useful for administering the drug, such as diluents, filter paper, IV bags and lines, needles, and syringes. As a preferred embodiment, the instructions may state that the drug is intended for use in treating cancer patients whose PD-L1 expression is positive as tested by immunohistochemistry (IHC) analysis.

[0124] The therapeutic agents described in the present invention may constitute pharmaceutical compositions, such as pharmaceutical compositions containing the anti-PD-1 antibodies described herein and / or other anticancer agents other than the anti-PD-1 antibodies, and other pharmaceutically acceptable carriers. As described herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. Preferably, carriers suitable for compositions containing anti-PD-1 antibodies are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration, such as by injection or infusion, while carriers for compositions containing other anticancer agents are suitable for parenteral administration, such as oral administration. The pharmaceutical compositions of the present invention may contain one or more pharmaceutically acceptable salts, antioxidants, water, non-aqueous carriers, and / or adjuvants such as preservatives, wetting agents, emulsifiers and dispersants.

[0125] The dosage regimen is adjusted to provide the best desired response, such as maximum therapeutic response and / or minimum adverse effect. For anti-PD-1 antibodies, including administration in combination with another anticancer agent, the dosage range may be about 0.01 to about 20 mg / kg, about 0.1 to about 10 mg / kg of individual body weight, or a fixed dose of 120 mg, 240 mg, 360 mg, or 480 mg. For example, the dosage may be about 0.1, about 0.3, about 1, about 2, about 3, about 5, or about 10 mg / kg of individual body weight. Dosage regimens are typically designed to achieve such exposure that results in sustained receptor occupancy (RO) based on the typical pharmacokinetic properties of Ab. A representative dosage regimen may be about once a week, about once every two weeks, about once every three weeks, about once every four weeks, about once a month, or longer. In some embodiments, anti-PD-1 antibodies are administered to an individual about once every two weeks.

[0126] The dosing schedule of other anticancer agents varies for different drugs. In some embodiments of the present invention, the dosing schedule of the CDK inhibitor varies for different subtypes.

[0127] use

[0128] The present invention also includes an anti-PD-1 antibody or a combination thereof with a CDK4 / 6 inhibitor for treating cancer patients, and the use of an anti-PD-1 antibody or a combination thereof with a CDK4 / 6 inhibitor in the preparation of a medicament for treating cancer patients. The cancer patient or the cancer he or she is suffering from may be as described in any of the foregoing embodiments; preferably, the cancer patient is a cancer patient with positive PD-L1 expression, or a tumor mutation burden (TMB) ≥ 3.6 Muts / Mb in peripheral blood or tumor tissue, or a PD-L1 expression-positive cancer patient with a tumor mutation burden (TMB) ≥ 3.6 Muts / Mb in peripheral blood or tumor tissue. In certain embodiments, the cancer patient is a cancer patient with a BRAF gene mutation detected in tumor tissue. More preferably, the melanoma patient is a patient with positive PD-L1 expression, or a tumor burden (TMB) ≥ 3.6 Muts / Mb in peripheral blood or tumor tissue, or a PD-L1 expression-positive cancer patient with a tumor burden (TMB) ≥ 3.6 Muts / Mb in peripheral blood or tumor tissue. In certain embodiments, the melanoma patient is more preferably a non-acral melanoma or a melanoma of uncertain primary focus. In some embodiments, the melanoma patient is an acral melanoma patient or a mucosal melanoma patient.

[0129] Preferred anti-PD-1 antibodies for treating cancer patients can be as described in any embodiment herein, more preferably antibodies having light chain CDRs with amino acids set forth in SEQ ID NOs: 1, 2, and 3, and heavy chain CDRs with amino acids set forth in SEQ ID NOs: 4, 5, and 6, more preferably monoclonal antibodies comprising a light chain variable region set forth in SEQ ID NO: 7 and a heavy chain variable region set forth in SEQ ID NO: 8, more preferably monoclonal antibodies comprising a light chain set forth in SEQ ID NO: 9 and a heavy chain set forth in SEQ ID NO: 10, more preferably humanized antibodies 38, 39, 41, and 48 described in WO2014206107, and most preferably toripalimab. The CDK4 / 6 inhibitor is preferably palbociclib.

[0130] In a preferred embodiment, the present invention relates to a combination of toripalimab or toripalimab and palbociclib for treating cancer patients. More specifically, the present invention relates to a combination of toripalimab or toripalimab and palbociclib for treating melanoma patients; more preferably, the melanoma patient is positive for PD-L1 expression, or the tumor burden (TMB) in peripheral blood or tumor tissue is ≥3.6Muts / Mb, or PD-L1 expression is positive, while the tumor burden (TMB) in peripheral blood or tumor tissue is ≥3.6Muts / Mb, or the melanoma patient in whom BRAF gene mutation is detected in tumor tissue, more preferably non-acral melanoma or primary melanoma. In some embodiments, the melanoma patient is an acral melanoma patient or a mucosal melanoma patient.

[0131] Method for predicting the effect of anti-PD-1 antibodies on cancer treatment

[0132] This article can predict the efficacy of anti-tumor treatment with anti-PD-1 antibodies, CDK4 / 6 inhibitors, or a combination of both by performing whole-exome sequencing (such as using second-generation sequencing technology) on tumor biopsy samples or corresponding peripheral blood samples from patients to identify whether certain genes are mutated or amplified.

[0133] The "gene mutation" or "gene change" described herein includes gene truncation, gene rearrangement / fusion, gene amplification, gene deletion and gene substitution / insertion, etc.

[0134] As used herein, the term "gene amplification" refers to a process in which the copy number of a gene encoding a specific protein is selectively increased while the number of copies of other genes is not proportionally increased. Under natural conditions, gene amplification occurs by excising repetitive sequences of a gene from chromosomes and then replicating them extrachromosomally in a plasmid, or by generating RNA transcripts from the entire repetitive sequence of ribosomal RNA, which are then transcribed to generate additional copies of the original DNA molecule. Some embodiments of the present invention disclose gene sequencing analysis.

[0135] In some embodiments of the present invention, the subjects described herein have certain unique gene mutations, such as some subjects with CDK4 or CDDN 1 gene amplification; some subjects with BRAF gene mutations, particularly those with non-acral melanoma or melanoma of uncertain primary; some subjects with mucosal melanoma have NF1 gene mutations; and some subjects (including patients with acral, mucosal, non-acral (cutaneous), and unknown primary melanoma) have NRAS gene mutations. In some embodiments, the BRAF gene mutation is selected from gene rearrangement / fusion and gene substitution / insertion. In some embodiments, the NRAS gene mutation is a gene substitution / insertion.

[0136] In some embodiments of the present invention, a BRAF gene mutation generally indicates that the patient will have a better therapeutic effect when treated with the anti-PD-1 antibody of the present invention. In some embodiments of the present invention, a CDK4 or CCND1 gene amplification indicates that the patient will have a better therapeutic effect when treated with the anti-PD-1 antibody of the present invention in combination with a CDK4 / 6 inhibitor or with a CDK4 / 6 inhibitor alone. In some embodiments of the present invention, a NRAS gene mutation indicates that the patient will have an unsatisfactory therapeutic effect when treated with the anti-PD-1 antibody of the present invention alone.

[0137] Therefore, the present invention provides a method for predicting the efficacy of treating cancer in an individual using the anti-PD-1 antibodies of the present invention, particularly toripalimab, comprising detecting biomarkers in the patient's peripheral blood before treatment, wherein the biomarkers are selected from, but not limited to, BRAF, NRAS, CDK4, or CCND1. The presence of a BRAF gene mutation indicates that the individual is suitable for treatment with an anti-PD-1 antibody, the presence of an NRAS gene mutation indicates that the individual is not suitable for treatment with an anti-PD-1 antibody, and the presence of a CDK4 or CCND1 gene amplification indicates that the individual is suitable for treatment with an anti-PD-1 antibody and a CDK4 / 6 inhibitor or a CDK4 / 6 inhibitor alone. Preferably, the cancer is melanoma.

[0138] The present invention also includes a method for predicting the efficacy of anti-PD-1 antibody treatment in a tumor patient using the BRAF gene or the NRAS gene. The presence of a BRAF gene mutation indicates that the tumor patient is suitable for treatment with an anti-PD-1 antibody. The presence of an NRAS gene mutation indicates that the tumor patient is not suitable for treatment with an anti-PD-1 antibody.

[0139] In certain embodiments, the present invention also provides the use of detection reagents for biomarkers (especially BRAF gene, and / or NRAS gene, and / or CDK4 gene, and / or CCND1 gene) in the preparation of a kit for predicting the effect of anti-PD-1 antibodies and / or CDK4 / 6 inhibitors in treating cancer. Such reagents include, but are not limited to, reagents routinely used in testing, including, but not limited to, primers, probes, reagents required for PCR, etc. The cancer is preferably melanoma. Preferably, the prediction includes: the presence of a BRAF gene mutation indicates that the individual is suitable for treatment with an anti-PD-1 antibody, the presence of an NRAS gene mutation indicates that the individual is not suitable for treatment with an anti-PD-1 antibody, and the presence of a CDK4 or CCND1 gene amplification indicates that the individual is suitable for treatment with an anti-PD-1 antibody and a CDK4 / 6 inhibitor or suitable for treatment with a CDK4 / 6 inhibitor alone.

[0140] Abbreviations

[0141] Throughout the description and examples of the present invention, the following abbreviations are used:

[0142] One dose BID, 2 times a day

[0143] CDR complementarity determining regions

[0144] DFS disease-free survival

[0145] FR framework region

[0146] IgG

[0147] IHC immunohistochemistry

[0148] OR Overall Response

[0149] ORR objective response rate

[0150] OS (overall survival)

[0151] PD disease progression

[0152] PFS progression-free survival

[0153] PR partial response

[0154] CR complete response

[0155] SD stable disease

[0156] DLT dose-limiting toxicity

[0157] MTD Maximum tolerated dose

[0158] AE adverse events

[0159] One dose every two weeks

[0160] One dose per day

[0161] CSD long-term sunshine type

[0162] non-CSD non-long-term sunshine type

[0163] IRC Independent Review Committee

[0164] TRAEs and treatment-related adverse events

[0165] SAE serious adverse reactions

[0166] MM melanoma

[0167] The present invention is further illustrated by the following examples, which should not be construed as limiting the present invention.The contents of all references cited throughout this application are expressly incorporated herein by reference.

[0168] Example

[0169] Example 1: Clinical study of anti-PD-1 antibody monotherapy for the treatment of melanoma

[0170] Eligible subjects must be (1) at least 18 years old, (2) have locally advanced or metastatic melanoma, (3) be refractory to standard systemic therapy, (4) have an ECOG performance status of 0 or 1, (5) have no history of autoimmune disease or persistent infection, and (6) have not received any previous anti-PD-1 / or anti-PD-L1 immunotherapy.

[0171] Subjects must have evaluable lesions according to RECIST v 1.1 criteria, and are not allowed to use anti-tumor drugs concurrently, systemic steroids, or have not used anti-CTLA4, anti-PD-1, or anti-PD-L1 antibody treatment.

[0172] Test drug: anti-PD-1 antibody toripalimab (WO2014206107).

[0173] The dose of anti-PD-1 antibody used in this trial was 3 mg / kg, administered intravenously once every two weeks (Q2W).

[0174] Clinical design:

[0175] This is a single-arm, Phase II, open-label clinical trial to evaluate the safety and anti-tumor activity of anti-PD-1 antibodies in the treatment of patients with advanced melanoma.

[0176] From December 26, 2016, to September 15, 2017, a total of 161 melanoma patients who were refractory to standard treatment were screened at 6 centers, and a total of 128 patients (eg, Figure 1 The demographic data of the enrolled subjects are shown in Table 1. The mean age was 52.5 years, with 57 males (45.5%) and 71 females (55.5%). One patient was subsequently diagnosed with non-recurrent cancer and was excluded from the efficacy analysis.

[0177] Among melanoma subtypes, 50 (39.4%) had acral melanoma, 22 (17.3%) had mucosal melanoma, 29 (22.9%) had non-acral cutaneous melanoma, and 26 (20.5%) had undetermined primary tumors. BRAF mutations were documented in 34 (26.6%) of the patients enrolled. Most patients had received multiple lines of prior therapy, with 88 (68.7%) receiving at least two prior lines of systemic therapy. Ninety-nine (78.0%) patients had received prior systemic chemotherapy, and nine (7.0%) had received ipilimumab.

[0178] Table 1: Demographic data of the enrolled subjects

[0179]

[0180]

[0181] Note: PD-L1 positivity is defined as PD-L1 expression of ≥1% of tumor cells using SP142 IHC staining.

[0182] 1.1 Safety Study:

[0183] As of August 15, 2019, 23 months after the last patient was enrolled, patients had received a median of 10 doses of toripalimab (range, 1 to 73 doses). 116 of 128 patients (97.7%) experienced treatment-related adverse events (STAs). Most were grade 1 or 2. Common (>5%) treatment-related adverse events are shown in Table 2. Treatment-related SAEs occurred in 10 (7.8%) patients. 25 (19.5%) patients experienced TRAEs of grade 3 or higher, including 13 (10.2%) grade 3 and 12 (9.4%) grade 4 side effects; no patient experienced a grade 5 adverse event. Fifteen (11.7%) patients discontinued treatment due to TRAEs; 8 (6.3%) patients had dose delays due to TRAEs. TRAEs of special interest occurred in fewer than 5% of patients and included 5 patients (3.9%) with vitiligo, 3 patients (2.3%) with liver injury (2 grade 4), 2 patients (1.6%) with acute pancreatitis, 2 patients (1.6%) with interstitial lung disease (1 grade 3), 2 patients (1.6%) with adrenal insufficiency, 2 patients (1.6%) with hypopituitarism, and 1 patient (0.8%) with uveitis (grade 3).

[0184] Table 2: Common (≥20%) adverse events associated with toripalimab treatment in all subjects (n=36)

[0185] N(％) all Level 1 Level 2 Level 3 Level 4 Level 5 All adverse reactions 125(97.7 42(32.8) 42(32.8) 29(22.7) 12(9.4) 0 TSH rises 42(32.8 34(26.6) 8(6.3) 0 0 0 ALT rise 41(32.0) 34(26.6) 5(3.9) 2(1.6) 0 0 Hyperglycemia 41(32.0) 35(27.3) 4(3.1) 1(0.8) 1(0.8) 0 proteinuria 35(27.3) 33(25.8) 1(0.8) 1(0.8) 0 0 Hypothyroidism 35(27.3) 19(14.8) 16(12.5) 0 0 0 Increased creatine kinase 33(25.8) 26(20.3) 3(2.3) 2(1.6) 2(1.6) 0 rash 30(23.4) 27(21.1) 3(2.3) 0 0 0 AST rise 30(23.4) 28(21.9) 1(0.8) 1(0.8) 0 0 Skin discoloration 30(23.4) 28(21.9) 2(1.6) 0 0 0 Leukocyturia 29(22.7) 27(21.1) 1(0.8) 1(0.8) 0 0 hematuria 29(22.7) 29(22.7) 0 0 0 0 Leukopenia 28(21.9) 22(17.2) 6(4.7) 0 0 0 anemia 27(21.1) 9(7.0) 15(11.7) 3(2.3) 0 0

[0186] Increased amylase 25(19.5) 15(11.7) 5(3.9) 2(1.6) 3(2.3) 0 Neutropenia 24(18.8) 18(14.1) 6(4.7) 0 0 0 itching 24(18.8) 20(15.6) 3(2.3) 1(0.8) 0 0 Hyperthyroidism 23(18.0) 18(14.1) 5(3.9) 0 0 0 Elevated DBIL 22(17.2) 15(11.7) 7(5.5) 0 0 0 Elevated TBIL 22(17.2) 22(17.2) 0 0 0 0 TSH decrease 19(14.8) 16(12.5) 3(2.3) 0 0 0 fatigue 19(14.8) 19(14.8) 0 0 0 0 decreased appetite 19(14.8) 17(13.3) 2(1.6) 0 0 0 fever 15(11.7) 15(11.7) 0 0 0 0 cough 15(11.7) 12(9.4) 3(2.3) 0 0 0 elevated blood lipids 14(10.9) 2(1.6) 5(3.9) 6(4.7) 1(0.8) 0

[0187] 1.2 Anti-tumor activity study:

[0188] As of August 15, 2019, in the intention-to-treat (ITT) population (n=127), 61 patients (48.0%) had died, 46 patients (36.2%) had discontinued treatment, and 20 patients (15.7%) remained on study. The median duration of treatment was 4.6 months (range, 0.2 to 33.6 months). Among the 127 patients assessed by IRC / RECIST v1.1, 22 patients achieved a confirmed objective response (1 complete response and 21 partial responses). The ORR per RECIST v1.1 was 17.3% (95% CI 11.2-25.0) and the ORR per irRECIST was 18.1% (95% CI 11.8-25.9). The specific results are shown in Table 3.

[0189] Table 3: Clinical efficacy assessed according to RECIST v1.1 or irRECIST criteria

[0190]

[0191] Note: ORR = (CR + PR) / total number × 100%; DCR = (CR + PR + uPR + SD) / total number × 100%.

[0192] CR: complete response; PR: partial response; uPR: unconfirmed PR; SD: stable disease; PD: progressive disease; NE: not evaluable; ORR: objective response rate; DCR: disease control rate; CI: confidence interval.

[0193] Target lesion size decreased relative to baseline in 49 subjects (38.6%), as shown in Figure 2 (A) Three subjects with SD initially had partial responses, but these responses could not be confirmed. The median duration of response was 3.5 months (95% CI 1.7-3.6), the median duration of response (DOR) was not reached, and only 9 of the 22 patients experienced disease progression after the initial response, as shown in the table below. Figure 2 (B) and Figure 2 (C). The DCR per RECISTv1.1 was 57.5% (95% CI 48.4-66.2), and the DCR per irRECIST was 59.8% (95% CI 50.8-68.4). The median PFS per RECISTv1.1 was 3.6 months (95% CI 3.7-5.5), and the median PFS per irRECIST was 3.7 months (95% CI 3.3-9.1), see Figure 3 (A).

[0194] The median overall OS in the ITT population (n=127) was estimated to be 22.2 months (95% CI 15.3 to not reached). Figure 3 (B).

[0195] The median OS of subjects who experienced an objective response (n=22) and stable disease (n=51) was not reached. Only 2 of the 22 PR / CR patients died, and 17 of the 51 SD patients died. The median overall survival (mOS) of the 54 patients with progressive disease was 9.7 months. Figure 4 (A).

[0196] In the four melanoma subtypes of acral, mucosal, nonacral cutaneous, and indeterminate primary, the ORRs per RECIST v1.1 as assessed by the independent review committee (IRC) were 14.0%, 0%, 31.0%, and 23.1%, respectively. The median PFS per RECIST v1.1 was 3.2, 1.9, 5.5, and 7.3 months, respectively. Specific results are shown in Tables 4 and Figure 5 The median OS for acral and mucosal subtypes was 16.9 months and 10.3 months, respectively, whereas the median OS for melanoma of uncertain primary site and non-acral cutaneous melanoma was not reached by the cutoff date. Figure 4 (B).

[0197] Table 4: Clinical efficacy and survival evaluation of RECIST v1.1 in melanoma subgroups

[0198]

[0199]

[0200] NE: Unpredictable.

[0201] 1.3 Pharmacokinetics and Immunogenicity

[0202] The steady-state mean trough plasma concentration of toripalimab was 39.8 μg / mL (range 4.9-92.4 μg / mL), which is much higher than the full PD-1 blockade concentration of 1.5 ug / ml (10 nmol / L). Anti-drug antibodies (ADA) were tested in 128 patients. 16 patients (12.5%) were ADA positive, of which 3 were positive before treatment and 13 (10.2%) were ADA positive after treatment. However, none of the ADA-positive individuals had consecutive positive samples. Only one of the 16 patients had a reduced trough concentration of toripalimab and ADA was detected at the same time, indicating the presence of neutralizing activity. There was no significant difference between ADA-positive and ADA-negative patients in terms of AEs, SAEs, the incidence of grade 3 or higher AEs, discontinuation or dose delays, and clinical efficacy.

[0203] Example 2: Study on the correlation between biomarkers and clinical efficacy

[0204] 2.1 PD-L1 expression in tumors

[0205] Tumor biopsy specimens from 127 patients were analyzed to analyze the correlation between tumor histology and the clinical efficacy of anti-PD-1 antibodies. Roche's rabbit anti-human PD-L1 antibody SP142 was used for detection. PD-L1 positivity was defined as the presence of membrane staining intensity ≥1% of tumor cells.

[0206] like Figure 6 As shown in (A), 26 patients (20.5%) were PD-L1 positive, 84 patients (66.1%) were PD-L1 negative, and 17 patients (13.4%) had unknown PD-L1 expression. Figure 6 (B) As shown, PD-L1 is expressed in four melanoma subtypes. + The proportion in acral melanoma (6.8%) and mucosal melanoma (10.5%) was significantly lower than that in non-acral melanoma (37.5%) and melanoma of uncertain primary site (52.2%).

[0207] like Figure 6 As shown in (C) and 6(D), PD-L1 + Patients with PD-L1 - Patients treated with toripalimab responded better in terms of ORR (38.5% vs. 11.9%, p=0.0065) and DCR (80.8% vs. 48.8%, p=0.006). + Patients with PD-L1 expression also showed a better progression-free survival (PFS) and overall survival (OS). - Patients had a significant survival advantage, with median PFS of 7.7 months and 2.7 months, respectively, HR=0.53 (95% CI 0.32-0.88), p=0.013; median OS was not reached versus 14.4 months, HR=0.35 (95% CI 0.19-0.63), p=0.0005.

[0208] 2.2 Tumor mutation burden (TMB) determination

[0209] In the experiment of Example 1, whole exome sequencing was performed on tumor biopsy specimens and paired peripheral blood samples from patients using second-generation sequencing technology. Tumor mutation burden (TMB) is determined by analyzing somatic mutations within the coding regions of the human genome. Figure 6 As shown in (A), 98 patients achieved valid results. TMB was generally low in this study, with a median TMB of 1.5 mutations per million base pairs (Muts / Mb). No patients with MSI-high were identified. Only 6 patients had a TMB exceeding 10 Muts / Mb, of which 3 exceeded 20 Muts / Mb.

[0210] like Figure 6 As shown in (B), among the four melanoma subtypes, mucosal melanoma had the lowest TMB, with a median TMB of 1.6 Muts / Mb, and only one patient (6.7%) had a TMB exceeding 3.6 Muts / Mb. Based on the recommendations of Robert M. Samstein et al., this study selected a cutoff value (3.6 Muts / Mb) in the top 20% of TMB values ​​to define the TMB-high population. Using 3.6 Muts / Mb as the cutoff, patients with TMB ≥ 3.6 Muts / Mb (n = 20) had a better response than patients with TMB < 3.6 Muts / Mb (n = 78) (ORR 30.0% vs. 12.8%), but the difference was not statistically significant (p = 0.088). Figure 6 (A) and Table 5. The PFS and OS values ​​of the TMB ≥ 3.6 Muts / Mb group were also better, but the difference was not statistically significant ( Figure 6 , E and F).

[0211] In this study, the TMB≥3.6Muts / Mb (n=20) and PD-L1+ (n=26) groups were mainly two independent groups. Among the 26 PD-L1-positive patients, only 7 had TMB≥3.6Muts / Mb ( Figure 6 , A).

[0212] Table 5: Top 20% TMB values ​​in each subtype as cutoff values ​​for efficacy analysis

[0213]

[0214] Example 3: Gene sequencing analysis

[0215] In the experiment of Example 1, whole exome sequencing was performed on tumor biopsies and paired peripheral blood samples from patients using second-generation sequencing technology, and 19,278 gene mutations were identified from 98 patients, including 7,964 missense mutations, 509 gene deletions, 482 rearrangements, 288 alternative splicing sites, 129 frameshift truncations, and 8,157 gene amplifications. After excluding genes that frequently mutated in the public exome, the most frequently altered genes (≥10%) were BRAF (33%), TERT (32%), CDKN2A (12%), NRAS (16%), CDK4 (12%), APOB (11%), CCND1 (11%), AGAP2 (11%), NF1 (10%), LRP1B (10%), MDM2 (10%), and KIT (10%). For details, see Figure 7 .

[0216] As shown in Table 6, sequencing results from 98 patients revealed distinct patterns of genomic alterations across melanoma subtypes. BRAF mutations were more frequent in non-acral cutaneous melanomas (11 / 23, 48%) and melanomas of unknown primary (14 / 21, 67%), but less frequent in acral melanomas (7 / 39, 18%) and mucosal melanomas (0 / 15, 0%). In contrast, NF1 mutations were more enriched in mucosal melanomas (4 / 15, 27%) than in the other three subtypes (8%, 4%, and 10%). Furthermore, CDK4 or CCND1 (Cyclin D1) amplification was observed in 33% (13 / 39) of acral melanomas and 20% (3 / 15) of mucosal melanomas, but only in 9% (2 / 23) of non-acral cutaneous melanomas and 10% (2 / 21) of unknown primary melanomas. This study found that 70% (7 / 10) of CCND1 copy number variations were observed in the acral subtype as part of 11q13 gene amplification. Subjects with CCND1 amplification (n=10) had a low response to toripalimab treatment (ORR 0%). This study shows that for patients with CCND1 amplification, CDK4 / 6 targeted therapy or combination therapy with CDK4 / 6 inhibitors and anti-PD-1 antibodies has good potential.

[0217] This study also found that subjects with NRAS mutations (n=16), including 7 acral type, 3 mucosal type, 3 non-acral type (cutaneous type), and 3 primary unknown type, had an ORR of 6.3% (1 / 16) for toripalimab treatment. This study showed that NRAS mutations can be used as a predictor of poor prognosis for immunotherapy.

[0218] Table 6: Whole-exome sequencing of gene mutations from 98 patients

[0219]

[0220] Example 4: Messenger RNA Expression Profiling in Tumor Biopsies

[0221] In the experiment of Example 1, RNA sequencing and expression profiling were performed on mRNA extracted from tumor biopsies. Valid results were obtained from 46 patients. The gene expression signatures of the IFN-γ-related gene set (IDO1, CXCL10, CXCL9, HLA-DRA, STAT1, IFNG), the inflammation-related gene set (IL-6, CXCL1, CXCL2, CXCL3, CXCL8, PTGS2), and the angiogenesis-related gene set (VEGFA, KDR, ESM1, PECAM1, ANGPTL4, CD34) were evaluated. It was found that there was no significant difference in the signature scores of the angiogenesis-related gene set, the IFN-related gene set, and the inflammation-related gene set between patients with clinical benefit (CR+PR+SD) and patients with progressive disease ( Figure 8 ). Sequence Listing <110> Shanghai Junshi Biopharmaceuticals Co., Ltd. <120> Use of anti-PD-1 antibodies in treating tumors <130> 200154 <150> CN 202010090829.0 <151> 2020-02-13 <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 1 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 2 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 3 Phe Gln Gly Ser His Val Pro Leu Thr 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 4 Asp Tyr Glu Met His 1 5 <210> 5 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 5 Val Ile Glu Ser Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 6 Glu Gly Ile Thr Thr Val Ala Thr Thr Tyr Tyr Trp Tyr Phe Asp Val 1 5 10 15 <210> 7 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region <400> 7 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 8 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region <400> 8 Gln Gly Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Ile His Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Glu Ser Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Ile Thr Thr Val Ala Thr Thr Tyr Tyr Trp Tyr Phe 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 9 <211> 219 [[ID=4​​ <220> <223> Light chain <400> 9 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 10 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 10 Gln Gly Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Ile His Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Glu Ser Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Ile Thr Thr Val Ala Thr Thr Tyr Tyr Trp Tyr Phe 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Leu Gly Lys 450

Claims

1. Use of an anti-PD-1 antibody or an antigen-binding fragment thereof in the preparation of a medicament for treating cancer patients, characterized in that: The cancer patient is a cancer patient with positive PD-L1 expression, or a cancer patient with a tumor mutation burden ≥3.6Muts / Mb in peripheral blood or tumor tissue; Or cancer patients with positive PD-L1 expression and tumor mutation burden (TMB) ≥ 3.6 Muts / Mb in peripheral blood or tumor tissue; or BRAF A cancer patient with a gene mutation; wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the anti-PD-1 antibody are shown as SEQ ID NOs: 1, 2 and 3, respectively, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown as SEQ ID NOs: 4, 5 and 6, respectively; and wherein the cancer patient is a patient with non-acral melanoma or melanoma of uncertain primary lesion.

2. The use according to claim 1, characterized in that The non-acral melanoma or melanoma of uncertain primary focus is advanced or metastatic melanoma.

3. The use according to claim 1, characterized in that The anti-PD-1 antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the amino acid sequence of VL is shown in SEQ ID NO: 7, and the amino acid sequence of VH is shown in SEQ ID NO:

8.

4. The use according to claim 3, characterized in that The anti-PD-1 antibody is an anti-PD-1 antibody comprising a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence shown in SEQ ID NO: 9, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:

10.

5. The use according to claim 1, characterized in that The anti-PD-1 antibody is toripalimab.

6. The use according to claim 1, wherein The anti-PD-1 antibody is a monoclonal antibody or an antigen-binding fragment thereof.

7. The use according to any one of claims 1 to 6, characterized in that The single administration dose of the anti-PD-1 antibody or antigen-binding fragment thereof is 0.1 mg / kg to 10.0 mg / kg of individual body weight, or a fixed dose selected from 120 mg to 480 mg.

8. The use according to claim 7, characterized in that The single administration dose of the anti-PD-1 antibody or antigen-binding fragment thereof is 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg or 10 mg / kg of individual body weight, or a fixed dose selected from 120 mg, 240 mg, 360 mg or 480 mg.

9. The use according to claim 7, characterized in that The anti-PD-1 antibody or antigen-binding fragment thereof is administered once a week, once every two weeks, once every three weeks, once every four weeks, or once a month.

10. The use according to claim 9, characterized in that The anti-PD-1 antibody or antigen-binding fragment thereof is administered once every two weeks.

11. The use according to claim 7, wherein: The single administration dose of the anti-PD-1 antibody or antigen-binding fragment thereof is 1 mg / kg body weight, 3 mg / kg body weight, 10 mg / kg body weight, or a fixed dose of 240 mg or 480 mg, administered once every two weeks.

12. The use according to claim 7, characterized in that The anti-PD-1 antibody or antigen-binding fragment thereof is in liquid dosage form and administered parenterally.

13. The use according to claim 12, characterized in that The liquid dosage form is an injection.

14. The use according to claim 12, characterized in that The parenteral administration is administration via intravenous infusion.

15. The use according to claim 7, wherein: The administration cycle of the anti-PD-1 antibody or antigen-binding fragment thereof is one week, two weeks, three weeks, one month, two months, three months, four months, five months or six months.

16. The use according to claim 15, characterized in that The duration of each dosing cycle is the same or different, and the interval between each dosing cycle is the same or different.

17. The use according to any one of claims 1 to 6, characterized in that The cancer patient is detected in tumor tissue BRAF For patients with melanoma with a gene mutation, the anti-PD-1 antibody is toripalimab.

18. The use according to any one of claims 1 to 6, characterized in that The application is the combination of an anti-PD-1 antibody or an antigen-binding fragment thereof and a CDK4 / 6 inhibitor, wherein the anti-PD-1 antibody is toripalimab, and the CDK4 / 6 inhibitor includes palbociclib, ribociclib or pomacicillin.

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