WO2020211804A1 - 抗pd-1抗体在制备治疗实体瘤的药物中的用途 - Google Patents

抗pd-1抗体在制备治疗实体瘤的药物中的用途 Download PDF

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WO2020211804A1
WO2020211804A1 PCT/CN2020/085046 CN2020085046W WO2020211804A1 WO 2020211804 A1 WO2020211804 A1 WO 2020211804A1 CN 2020085046 W CN2020085046 W CN 2020085046W WO 2020211804 A1 WO2020211804 A1 WO 2020211804A1
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antibody
cancer
antigen
tumor
binding fragment
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PCT/CN2020/085046
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English (en)
French (fr)
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冯辉
武海
姚盛
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上海君实生物医药科技股份有限公司
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Priority to JP2021561915A priority Critical patent/JP2022529969A/ja
Priority to US17/604,058 priority patent/US20220154296A1/en
Priority to CN202080003181.7A priority patent/CN112292151A/zh
Priority to BR112021019524A priority patent/BR112021019524A2/pt
Priority to EP20791286.6A priority patent/EP3957326A4/en
Publication of WO2020211804A1 publication Critical patent/WO2020211804A1/zh
Priority to ZA2021/07993A priority patent/ZA202107993B/en

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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the use of anti-PD-1 antibodies in the treatment of tumors.
  • the present invention relates to the use of anti-PD-1 antibodies in the treatment of solid tumors, especially esophageal cancer, more preferably the use of esophageal squamous cell carcinoma (ESCC); and the use of anti-PD-1 antibodies in the preparation of therapeutic entities Tumors, especially esophageal cancer, acral cutaneous melanoma, more preferably esophageal squamous cell carcinoma (ESCC) in medicine; and the use of biomarkers to predict the use of anti-PD-1 antibodies in the treatment of solid tumors, Especially the use in esophageal cancer, more preferably the therapeutic method in esophageal squamous cell carcinoma (ESCC); and the use of the combination therapy or composition containing the anti-PD-1 antibody in the treatment of solid tumors.
  • ESCC esophageal cancer
  • ESCC esophageal squam
  • Immune escape is one of the characteristics of cancer.
  • Ahmadzadeh, M. et al., Blood, 114:1537-44 disclosed that tumor-specific T lymphocytes often exist in the tumor microenvironment, draining lymph nodes and peripheral blood, but due to the immunosuppressive mechanism network existing in the tumor microenvironment, its It is usually impossible to control the progression of the tumor.
  • CD8 + tumor-infiltrating T lymphocytes (TIL) usually express activation-induced inhibitory receptors, including CTLA-4 and PD-1, while tumor cells often express immunosuppressive ligands, including PD-1 ligand 1 (PD-L1, Also called B7-H1 or CD274), this ligand inhibits T cell activation and effector functions.
  • PD-1 and its ligands have become an important way for tumor cells to inhibit T cells activated in the tumor microenvironment.
  • PD-1 Programmed death receptor 1 plays an important role in immune regulation and peripheral tolerance maintenance.
  • PD-1 is mainly expressed in activated T cells and B cells, and its function is to inhibit the activation of lymphocytes. This is a normal peripheral tissue tolerance mechanism of the immune system to prevent and treat immune overload.
  • activated T cells that infiltrate the tumor microenvironment highly express PD-1 molecules, and the inflammatory factors secreted by activated white blood cells will induce tumor cells to highly express PD-1 ligands PD-L1 and PD-L2, resulting in tumor microenvironment
  • the PD-1 pathway of activated T cells is continuously activated, and the function of T cells is inhibited and cannot kill tumor cells.
  • Therapeutic PD-1 antibody can block this pathway, partially restore the function of T cells, so that activated T cells can continue to kill tumor cells.
  • PD-1/PD-L1 pathway blockade has been proven to be an effective way to induce durable anti-tumor responses in various cancer indications.
  • Monoclonal antibodies (mAbs) that block the PD/PD-L1 pathway can enhance the activation and effector functions of tumor-specific T cells, reduce tumor burden, and improve survival.
  • Esophageal cancer is one of the most common malignant tumors in humans. In the past few decades, the incidence and mortality of the disease have continued to rise. At present, 400,000 people worldwide die from this disease every year. Among them, esophageal squamous cell carcinoma (ESCC) is the most common histological subtype of esophageal cancer in developing countries. It is the most important histological subtype of esophageal cancer in the population of South America and East Asia. The tumor is still worldwide. Unmet need for treatment. In China, EC is the third common cancer and the fourth cause of cancer-related death. However, ESCC accounts for more than 90% of all esophageal cancers in China, and is often treated with chemotherapy and radiotherapy.
  • ESCC esophageal squamous cell carcinoma
  • the first aspect of the present invention provides a use of an anti-PD-1 antibody or an antigen-binding fragment thereof alone or in combination with other anti-cancer agents in the preparation of drugs for the treatment of cancer.
  • the other anticancer agents described in the present invention are small molecule targeted anticancer agents.
  • the other anticancer agents of the present invention are selected from CDK4/6 inhibitors, FGF/FGFR inhibitors.
  • the drug contains an anti-PD-1 antibody or antigen-binding fragment thereof and a CDK4/6 inhibitor. In one or more embodiments, the drug contains an anti-PD-1 antibody or antigen-binding fragment thereof and an FGF/FGFR inhibitor.
  • the present invention provides the use of an anti-PD-1 antibody or an antigen-binding fragment thereof in the preparation of a medicament for the treatment of cancer patients in combination with a CDK4/6 inhibitor and FGF/FGFR inhibitor.
  • the present invention provides the use of an anti-PD-1 antibody or an antigen-binding fragment thereof in combination with a CDK4/6 inhibitor and an FGF/FGFR inhibitor to prepare a medicine for treating cancer patients.
  • the cancer of the present invention is a solid tumor.
  • the cancer includes, but is not limited to, gastric cancer, esophageal cancer, nasopharyngeal cancer, head and neck squamous cell carcinoma, breast cancer, bladder cancer, and colon cancer.
  • the cancer is preferably esophageal cancer. In one or more preferred embodiments, the cancer is esophageal squamous cell carcinoma. In one or more preferred embodiments, the cancer is advanced ESCC. In one or more preferred embodiments, the cancer is ESCC refractory to chemotherapy. In other embodiments, the esophageal squamous cell carcinoma is advanced, metastatic and/or refractory esophageal squamous cell carcinoma.
  • the solid tumor does not have gene amplification of the 11q13 region of chromosome.
  • the present invention provides an anti-PD-1 antibody (preferably triprolizumab) and a CDK4/6 inhibitor (preferably reboxinil or pabocinil, more preferably pabocinil Use of the combination of) in the preparation of a medicament for the treatment of esophageal cancer or colon cancer;
  • the present invention provides the use of an anti-PD-1 antibody (preferably teriprizumab) in the preparation of a medicament for the treatment of esophageal cancer .
  • the individual has been pre-treated.
  • the pre-treatment includes but is not limited to chemotherapy or radiotherapy.
  • the individual has been treated systemically.
  • the pre-treatment or systemic treatment is at least 2 times.
  • the individual has no history of autoimmune disease or autoimmune disease.
  • the individual does not have any concomitant diseases that require long-term immunosuppressive drug treatment.
  • the individual has not previously received any immune check site blocker treatment.
  • the anti-PD-1 antibody or antigen-binding fragment thereof is administered alone or in combination with other anti-cancer agents to induce a durable clinical response in the individual.
  • the anti-PD-1 antibody or antigen-binding fragment thereof is administered alone or in combination with other anti-cancer agents.
  • the therapeutically effective dose of the anti-PD-1 antibody or antigen-binding fragment thereof ranges from About 0.1 to about 10.0 mg/kg body weight, intravenous infusion about once every 2 weeks.
  • the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a fixed dose of about 1 mg/kg body weight, 3 mg/kg body weight or 10 mg/kg body weight or 240 g/kg, once every 2 weeks.
  • the single administration dose of the anti-PD-1 antibody or antigen-binding fragment thereof is 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. kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 5mg/kg, or 10mg/kg body weight, or selected from about 120mg to about 480mg fixed dose, such as 120mg, 240mg, 360mg or 480mg fixed dose .
  • the dosing cycle frequency of the anti-PD-1 antibody or antigen-binding fragment thereof is 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.
  • the single administration dose of the anti-PD-1 antibody or antigen-binding fragment thereof is 1 mg/kg individual body weight, 3 mg/kg individual body weight, 10 mg/kg individual body weight, or 240 mg fixed dose per Apply once every two weeks.
  • the anti-PD-1 antibody or antigen-binding fragment thereof is administered in a liquid dosage form, such as an injection, via a parenteral route, such as by intravenous infusion.
  • the administration period 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, half a year or longer, optionally, the time of each dosing cycle can be the same or different, and the interval between each dosing cycle can be the same or different.
  • the second aspect of the present invention provides a kit for treating an individual suffering from cancer, the kit comprising: (a) a monoclonal antibody or an antigen-binding fragment thereof that specifically binds and inhibits PD-1, and optionally a Anti-cancer agents other than PD-1 antibody or its antigen-binding fragment; and (b) administration of anti-PD-1 antibody or its antigen-binding fragment alone, or combined administration of monoclonal antibodies or their antigens that specifically bind and inhibit PD-1 Instructions for combining fragments and other anticancer agents to treat individual cancers.
  • the cancer is a solid tumor.
  • the cancer includes but is not limited to gastric cancer, esophageal cancer, nasopharyngeal cancer, head and neck squamous cell carcinoma, breast cancer, bladder cancer and colon cancer.
  • the cancer is preferably esophageal cancer.
  • the cancer is esophageal squamous cell carcinoma.
  • the cancer is ESCC.
  • the solid tumor does not have gene amplification of the 11q13 region of chromosome.
  • the other anticancer agents described in the present invention are small molecule targeted anticancer agents.
  • the other anticancer agents of the present invention are selected from one or more of CDK4/6 inhibitors and FGF/FGFR inhibitors.
  • the kit contains an anti-PD-1 antibody or antigen-binding fragment thereof and a CDK4/6 inhibitor. In one or more embodiments, the kit contains an anti-PD-1 antibody or antigen-binding fragment thereof and an FGF/FGFR inhibitor.
  • the third aspect of the present invention provides a use of a CDK4/6 inhibitor or FGF/FGFR inhibitor in the preparation of a medicine for treating cancer.
  • the fourth aspect of the present invention provides a method for the treatment of cancer, which includes the step of sequencing an individual before treatment; wherein the anti-PD-1 antibody or its antigen is administered alone to an individual who does not have the amplification of the 11q13 region of chromosome Combining fragments, optionally in combination with a CDK4/6 inhibitor, and/or one or a combination of FGF/FGFR inhibitors; administering a CDK4/6 inhibitor alone to an individual with amplification of the 11q13 region of chromosome, and / Or a combination of one or more of the FGF/FGFR inhibitors, optionally combined administration of anti-PD-1 antibodies or antigen-binding fragments thereof.
  • the individual is a human.
  • the individual described in the present invention is a human and his cancer is a solid tumor.
  • the individual of the present invention is a human and his cancer is selected from the group including but not limited to gastric cancer, esophageal cancer, nasopharyngeal cancer, head and neck squamous cell carcinoma, breast cancer, bladder Cancer and colon cancer.
  • the individual described in the present invention is a human and his cancer is esophageal cancer.
  • the individual described in the present invention is a human and his cancer is ESCC.
  • the individual of the present invention has esophageal cancer and has not previously received immunotherapy.
  • the fifth aspect of the present invention provides a method for predicting the therapeutic effect of an anti-PD-1 antibody in a tumor patient, which includes detecting whether the patient has gene amplification in the 11q13 region of chromosome, wherein the absence of gene amplification in the 11q13 region of chromosome indicates that The tumor patients are suitable for treatment with anti-PD-1 antibodies.
  • the anti-PD-1 antibody is a monoclonal antibody or an antigen-binding fragment thereof.
  • the anti-PD-1 antibody specifically binds to PD-1 and blocks the binding of PD-L1 or PD-L2 to PD-1.
  • the anti-PD-1 antibody specifically binds to PD-L1 or/and PD-L2, and blocks the binding of PD-L1 and/or PD-L2 to PD-1.
  • the anti-PD-1 antibody is an antibody comprising a complementarity determining region (CDR), and the amino acid sequence of the light chain complementarity determining region (LCDR) is shown in SEQ ID NO: 1, 2 and 3.
  • the amino acid sequence of the chain complementarity determining region (HCDR) is shown in SEQ ID NO: 4, 5 and 6.
  • the anti-PD-1 antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), where VL is shown in SEQ ID NO: 7 and VH is shown in SEQ ID NO :8 shown.
  • VL light chain variable region
  • VH heavy chain variable region
  • the anti-PD-1 antibody is an anti-PD-1 antibody comprising a light chain and a heavy chain, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 9, and the heavy chain comprises SEQ ID NO: The amino acid sequence shown in 10.
  • the length of the bar graph indicates the maximum reduction or minimum increase of the target lesion.
  • the color of the bar graph reflects the results of previous system treatments. Blue: ⁇ 3L; Orange: 2L; Green: 1L.
  • the target lesions had the best change from baseline, with a decrease of >30%, but the response could not be confirmed or new lesions occurred.
  • the target lesions changed the best from baseline, with an increase of less than 20%, but due to the occurrence of new lesions or the progression of non-target lesions, they were characterized by progressive disease (PD).
  • PD progressive disease
  • FIG. 1 Clinical response related to tumor PD-L1 expression or tumor mutation burden (TMB).
  • the PD-L1 positive state is defined as the presence of tumor cells or immune cells with membrane staining intensity ⁇ 1% including IHC staining by SP142.
  • TMB status is determined by whole-exome sequencing of tumor biopsy and paired PBMCs.
  • the WES genome map shows: TP53 (p53) (76%), RYR2 (22%), NOTCH1 (20%), LRP1B (17%) and TRIO (17%) missense mutations or truncations are the most common in ESCC tumor biopsies. 5 common mutant genes.
  • Figure 4 11q13 gene locus amplification and RNA expression analysis of selected ESCC patients.
  • Genome profiling was performed by next-generation sequencing of FFPE tumors and paired peripheral blood samples from 51 available patients. 11q13 amplified 24 cases. At the top is the chromosome 11q13 region and coding gene map. Three algorithms are used to determine the amplification event. Every individual has amplified genes. Mutations or deletions of amplified genes are also marked.
  • Figure 5 The clinical response is related to the gene amplification status of the 11q13 region of chromosome.
  • Tumor 11q13 status was determined by whole exome sequencing of tumor biopsy and paired PBMCs.
  • Figure 6 The clinical trial progress of NCT02915432 study evaluating the effects of teriprizumab in patients with advanced GC, ESCC, NPC and HNSCC.
  • Figure 7 Research results of the inhibitory effect of anti-PD-1 antibody combined with CDK4/6 inhibitor on tumor growth in mice.
  • the present invention relates to tumor treatment methods.
  • the method of the present invention includes administering an anti-PD-1 antibody or antigen-binding fragment thereof alone to a patient in need; or administering an anti-PD-1 antibody or an antigen-binding fragment thereof in combination with other anticancer agents to a patient in need.
  • the present invention also relates to a method for using biomarkers to predict the efficacy of anti-PD-1 antibodies in the treatment of cancer, especially in patients with esophageal cancer.
  • administering refers to introducing a composition containing a therapeutic agent into a subject using any of various methods or delivery systems known to those skilled in the art.
  • the administration route of anti-PD-1 antibody includes intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral administration routes, such as injection or infusion.
  • Parenteral administration refers to administration methods usually by injection other than enteral or local administration, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, and intrasaccular , Intraframe, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspine, intradural and intrasternal injections and infusions, and intracorporeal electroporation.
  • AE adverse reaction
  • Medical treatments can have one or more related AEs, and each AE can have the same or different severity levels.
  • subject includes any organism, preferably animals, more preferably mammals (e.g., rats, mice, dogs, cats, rabbits, etc.), and most preferably humans.
  • mammals e.g., rats, mice, dogs, cats, rabbits, etc.
  • subject and “individual” are used interchangeably 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 single domain antibodies. It specifically binds to an antigen and includes at least two heavy (H) and two light (L) chains interconnected by disulfide bonds, or antigen-binding fragments thereof. Each heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region, and the heavy chain constant region includes three constant domains CH1, CH2 and CH3.
  • Each light chain contains a light chain variable region (VL) and a light chain constant region, and the light chain constant region contains a constant domain CL.
  • VL light chain variable region
  • CL constant domain
  • the VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDR), which are interspersed in more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • both the light chain and heavy chain variable domains include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • Amino acids are usually assigned to each domain according to the following definition: Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th edition; NIH publication number 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.
  • the carboxy terminal part of the heavy chain can define the constant region that is mainly responsible for effector functions.
  • human light chains are divided into ⁇ chain and ⁇ chain.
  • Human heavy chains are usually classified into ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotype of the antibody is defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the IgG subclass is well known to those skilled in the art and includes but is not limited to IgG1, IgG2, IgG and IgG4.
  • 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.
  • antibody fragment refers to an antigen-binding fragment of an antibody, that is, an antibody fragment that retains the ability of a full-length antibody to specifically bind to an antigen, such as retaining one or Fragments of multiple CDR regions.
  • antibody 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.
  • Chimeric antibody refers to an antibody and its fragments in which a part of the heavy chain and/or light chain is the same or homologous to the corresponding sequence in an antibody derived from a specific species (such as human) or belonging to a specific antibody class or subclass , And the rest of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species (such as mouse) or belonging to another antibody class or subclass, as long as it exhibits the desired biological activity.
  • Human antibody refers to an antibody that contains 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, “mouse antibody” or “rat antibody” refers to antibodies that only contain mouse or rat immunoglobulin sequences, respectively.
  • Humanized antibody refers to a form of antibody that contains sequences derived from non-human (such as murine) antibodies as well as human antibodies. Such antibodies contain the smallest sequence derived from one side of a non-human immunoglobulin. Generally, a humanized antibody will contain substantially all of at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all The FR region is the FR region of human immunoglobulin. The humanized antibody optionally also includes at least a portion of an immunoglobulin constant region (Fc) (usually a human immunoglobulin constant region).
  • Fc immunoglobulin constant region
  • cancer refers to a wide range of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division, growth division, and growth lead to the formation of malignant tumors, which invade adjacent tissues and can also metastasize to remote parts of the body through the lymphatic system or bloodstream. Examples of cancer include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma, and sarcoma.
  • cancer More specific examples include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myelogenous leukemia, multiple myeloma , Gastrointestinal (tract) 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, hepatocytoma, breast cancer, colon cancer and head and neck cancer.
  • cancers of the present invention include those cancers characterized by a sequenced individual having gene amplification in the 11q13 region of chromosome.
  • the cancers of the present invention include those cancers that are characterized in that the sequenced individual does not have gene amplification of the 11q13 region of chromosome.
  • EC esophageal cancer
  • EC esophageal cancer
  • ESCC esophageal squamous cell carcinoma
  • immunotherapy refers to the treatment of a subject suffering from a disease or at risk of infection or suffering from recurrence of the disease by methods including inducing, enhancing, suppressing or otherwise modifying the immune response.
  • Treatment or “therapy” of a subject refers to any type of intervention or process performed on the subject, or administration of an active agent to the subject, with the purpose of reversing, alleviating, improving, alleviating or preventing symptoms and complications Or the onset, progression, severity or recurrence of the disease, or biochemical indicators related to the disease.
  • PD-1 Programmed death receptor-1
  • PD-1 is mainly expressed on previously activated T cells in the body and binds two ligands, PD-L1 and PD-L2.
  • the term "PD-1” as used herein includes human PD-1 (hPD-1), variants, isotypes and species homologs of hPD-1, and analogs that have at least one epitope in common with hPD-1 .
  • a “therapeutically effective amount” or “therapeutically effective dose” of a drug or therapeutic agent is any amount of a drug that protects the subject from the onset of disease or promotes the regression of the disease when used alone or in combination with another therapeutic agent.
  • the resolution of the disease is evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or the prevention of injury or disability caused by the pain of the disease.
  • the ability of therapeutic agents 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 that predict human efficacy, or by in vitro assays The activity of the agent is determined in the.
  • a therapeutically effective amount of a drug includes a "prophylactically effective amount", that is, any amount that inhibits the development or recurrence of cancer when administered to a subject at risk of developing cancer or a subject suffering from recurrence of cancer, alone or in combination with an antitumor agent medicine.
  • Biotherapeutics refers to biomolecules, such as antibodies or fusion proteins, that block ligand/receptor signaling in any biological pathway that supports tumor maintenance and/or growth or inhibits anti-tumor immune responses.
  • CDR as used herein means that the immunoglobulin variable region is a complementarity determining region defined using the Kabat numbering system.
  • Anticancer agent refers to any therapeutic agent that can be used to treat cancer.
  • the categories of anticancer agents include but are not limited to: alkylating agents, antimetabolites, kinase inhibitors, spindle poisons, plant alkaloids, cytotoxic/antitumor antibiotics, photosensitizers, antiestrogens and selective estrogen receptor modulation
  • Antisense oligonucleotides antiprogesterone agents, aromatase inhibitors, CDK4/6 inhibitors, FGF/FGFR inhibitors, etc., and antisense oligonucleotides that inhibit the expression of genes involved in abnormal cell proliferation or tumor growth.
  • the term "about” refers to a value or composition within an acceptable error range of a specific value or composition as determined by a person of ordinary skill in the art, which partly depends on how the value or composition is measured or determined, that is, the limitation of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation according to practice in the art. Alternatively, “about” can refer to a range of up to 10% or 20% (ie, ⁇ 10% or ⁇ 20%). For example, about 3 mg/kg may include any number between 2.7 mg/kg and 3.3 mg/kg (relative to 10%), and between 2.4 mg/kg and 3.6 mg/kg (relative to 20%). In this article, when a specific value or composition is provided, unless expressly stated otherwise, the meaning of "about” should be assumed to be within the acceptable error range of the specific value or composition.
  • Therapeutic anti-PD-1 monoclonal antibody refers to an antibody that specifically binds to a mature form of specific PD-1 expressed on the surface of certain mammalian cells. Mature PD-1 has no presecretory leader sequence, or leader peptide.
  • the terms “PD-1” and “mature PD-1” are used interchangeably herein, and unless clearly defined otherwise, or clearly seen from the context, they should be understood as the same molecule.
  • a therapeutic anti-human PD-1 antibody or anti-hPD-1 antibody refers to a monoclonal antibody that specifically binds to mature human PD-1.
  • framework region or "FR” refers to immunoglobulin variable regions that do not include CDR regions.
  • isolated antibody or antigen-binding fragment thereof refers to a purified state and in this case the designated molecule does not substantially contain other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates or other materials (such as cell debris or growth Medium).
  • Patient refers to any single subject that requires medical treatment or participates in clinical trials, epidemiological studies or used as a control, including humans and mammals, such as horses, cows, and dogs Or cat.
  • PD-1 antibody refers to binding to the PD-1 receptor, blocking the binding of PD-L1 expressed on cancer cells with PD-1 expressed on immune cells (T, B, NK cells) and preferably can also block Any chemical compound or biological molecule that binds PD-L2 expressed on cancer cells to PD-1 expressed on immune cells.
  • Alternative nouns or synonyms for PD-1 and its ligands include: for PD-1, PDCD1, PD1, CD279, and SLEB2; for PD-L1, PDCD1L1, PDL1, B7-H1, B7H1, B7-4, CD274 and B7-H; and for PD-L2, there are PDCD1L2, PDL2, B7-DC and CD273.
  • the PD-1 antibody blocks the binding of human PD-L1 and human PD-1, and preferably blocks both human PD-L1 and PD-L2 and human PD1 binding.
  • the amino acid sequence of human PD-1 can be found in NCBI locus number: NP_005009.
  • the amino acid sequences of human PD-L1 and PD-L2 can be found in NCBI locus numbers: NP_054862 and NP_079515, respectively.
  • Anti-PD-1 antibodies that can be used for any of the uses, therapies, drugs and kits described in the present invention include monoclonal antibodies (mAb) or antigen-binding fragments thereof, which specifically bind to PD-1, and preferably specifically bind 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.
  • the constant region is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 constant regions, and in a preferred embodiment, the constant region is a human IgG4 constant region.
  • the PD-1 antibody is a monoclonal antibody or an antigen-binding fragment thereof, which comprises: (a) the light chain CDR is SEQ ID NO:1
  • the amino acids shown in, 2 and 3 and the heavy chain CDR are the amino acids shown in SEQ ID NO: 4, 5 and 6.
  • the PD-1 antibody specifically binds to human PD-1 and includes: (a) a light chain comprising SEQ ID NO: 7 Variable region, and (b) a monoclonal antibody comprising the heavy chain variable region of SEQ ID NO: 8.
  • the PD-1 antibody specifically binds to human PD-1 and includes: (a) a light chain comprising SEQ ID NO: 9, And (b) a monoclonal antibody comprising the heavy chain of SEQ ID NO: 10.
  • the PD-1 antibody is a monoclonal antibody or an antigen-binding fragment thereof.
  • Table A below provides the use and therapy of the present invention List of amino acid sequences of exemplary anti-PD-1 antibody mAbs in drugs and kits:
  • anti-PD-1 antibodies that bind to human PD-1 and can be used in the uses, therapies, drugs, and kits of the present invention are described in WO2014206107.
  • the human PD-1 mAb that can be used as an anti-PD-1 antibody in the uses, therapies, drugs, and kits of the present invention includes any of the anti-PD-1 antibodies described in WO2014206107, including: teriprizumab ( Teriplizumab) (a kind of structure described in WHO Drug Information (Volume 32, Issue 2, Pages 372-373 (2018)) and containing the sequence SEQ ID NO: 9 and 10 Humanized IgG4 mAb of light chain and heavy chain amino acid sequence).
  • the anti-PD-1 antibodies that can be used for the uses, therapies, drugs, and kits of the present invention also include nivolumab and pamuzumab that have been approved by the FDA, and NMPA approved letters Dilimumab, as well as SHR-1210 and Tislelizumab which are still in the clinical stage, and the preclinical and clinical stages that can block PD-L1 expressed on cancer cells and immune cells (T, B, NK cells)
  • the PD-1 on) binds and preferably can also block any chemical compound or biological molecule that can also block the binding of PD-L2 expressed on cancer cells to PD-1 expressed on immune cells.
  • the anti-PD-1 antibodies that can be used in the uses, therapies, drugs, and kits of the present invention also include anti-PD that specifically binds to PD-L1 to block the binding of PD-L1 to PD-1.
  • -L1 monoclonal antibody such as atezolizumab, avelumab, durvalumab, or any chemical compound or biological molecule that can specifically bind to PD-L1 to block the binding of PD-L1 to PD-1.
  • PD-L1 or PD-L2 refers to any detectable expression level of a specific PD-L protein on the surface of a cell or a specific PD-L mRNA in a cell or tissue.
  • PD-L protein expression can be detected by diagnostic PD-L antibody in IHC analysis of tumor tissue sections or by flow cytometry.
  • the PD-L protein expression of 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).
  • One method uses a simple binary endpoint where PD-L1 expression is positive or negative, where a positive result of PD-L1 expression is defined by the percentage of tumor cells showing histological evidence of cell surface membrane staining. Counting tumor tissue sections as at least 1% of the total tumor cells is defined as positive for PD-L1 expression.
  • PD-L1 expression in tumor tissue sections is quantified in tumor cells and in infiltrating immune cells.
  • the percentages of tumor cells and infiltrating immune cells exhibiting membrane staining were individually quantified as ⁇ 1%, 1% to 50%, and subsequent 50% to 100%. For tumor cells, if the score is ⁇ 1%, the PD-L1 expression count is negative, and if the score is ⁇ 1%, it is positive.
  • the expression of PD-L1 in the tumor tissue section of the subject is ⁇ 1%. Compared with the expression of PD-L1 in the tumor tissue section of the subject ⁇ 1%, no comparison is achieved. Good benefit.
  • the cancer is a solid tumor. In some specific embodiments, the cancer is esophageal cancer. As a preferred embodiment, the cancer is ESCC.
  • the "RECIST 1.1 efficacy standard" as described herein refers to the definition of Eisenhauver et al., E.A. et al., Eur. J Cancer 45:228-247 (2009) for target damage or non-target damage based on the background of the measured response.
  • ECOG scoring standard is an indicator of a patient's physical strength to understand his general health and tolerance to treatment.
  • ECOG physical fitness scoring standard score 0 points, 1 point, 2 points, 3 points, 4 points and 5 points.
  • a score of 0 means that the activity ability is completely normal, and there is no difference in the activity ability before the onset.
  • a score of 1 refers to the ability to walk around freely and engage in light physical activities, including general housework or office work, but cannot engage in heavier physical activities.
  • sustained response refers to the sustained therapeutic effect after cessation of the therapeutic agent or combination therapy described herein.
  • the sustained response has a duration at least the same as the duration of the treatment or at least 1.5, 2.0, 2.5, or 3 times the duration of the treatment.
  • tissue section refers to a single part or piece of a tissue sample, such as a tissue slice cut from a sample of normal tissue or tumor.
  • Treatment of cancer as described herein refers to subjects suffering from cancer or having been diagnosed with cancer using the treatment regimens described herein (such as administration of anti-PD-1 antibodies, or administration of anti-PD-1 antibodies and CDK4/6 inhibition Or FGF/FGFR inhibitor combination therapy) to achieve at least one positive therapeutic effect (e.g., decrease in the number of cancer cells, decrease in tumor volume, decrease in the rate of cancer cell infiltration into surrounding organs, or decrease in the rate of tumor metastasis or tumor growth ).
  • the positive treatment effect in cancer can be measured in various ways (see W.A. Weber, J. Nucl. Med., 50:1S-10S (2009)).
  • T/C ⁇ 42% is the minimum level of anti-tumor activity.
  • T/C(%) median value of treated tumor volume/median value of control tumor volume ⁇ 100.
  • 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 increase in life expectancy compared to the initial or untreated individual or patient.
  • the response to the combination of the invention is any of PR, CR, PFS, DFS, OR, or OS, which is assessed using the RECIST 1.1 efficacy standard.
  • the treatment plan of the combination of the present invention that is effective in treating cancer patients may vary according to various factors such as the patient's disease state, age, weight, and the ability of the therapy to stimulate the subject's anti-cancer response.
  • the embodiment 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.
  • mode of administration and “dosage regimen” are used interchangeably, and refer to the dosage and time of each therapeutic agent in the combination of the present invention.
  • Tumor when applied to subjects diagnosed with or suspected of having cancer refers to malignant or potentially malignant neoplasms or tissue masses of any size, and includes primary tumors and secondary neoplasms. Solid tumors usually do not contain abnormal growths or clumps of tissue in cysts or fluid areas. Different types of solid tumors are named for the cell types that form them. Examples of solid tumors are sarcoma, carcinoma, and lymphoma. Blood cancer usually does not form a solid tumor.
  • Tumor burden refers to the total amount of tumor material distributed throughout the body. Tumor burden refers to the total number of cancer cells in the entire body or the total size of the tumor. Tumor burden can be measured by a variety of methods known in the art, such as using calipers after the tumor is removed from the subject, or using imaging techniques (such as ultrasound, bone scanning, computed tomography when in vivo) (CT) or magnetic resonance imaging (MRI) scan) to measure its size.
  • imaging techniques such as ultrasound, bone scanning, computed tomography when in vivo) (CT) or magnetic resonance imaging (MRI) scan
  • tumor size refers to the total size of the tumor, which can be measured as the length and width of the tumor.
  • the size of the tumor can be measured by various methods known in the prior art, such as using a caliper after the tumor is removed from the subject, or using imaging techniques (such as bone scan, ultrasound, CT or MRI scan) while in the body. size.
  • tumor mutation burden refers to the total number of somatic gene coding errors, base substitutions, gene insertion or deletion errors detected per million bases.
  • tumor mutation burden is estimated by analyzing somatic mutations (including coding base substitutions and macrobase insertions of the studied panel sequence).
  • the subject’s tumor mutation burden (TMB) is greater than or equal to 12 mutations/Mb, which is not better than the subject’s tumor mutation burden (TMB) ⁇ 12 mutations/Mb.
  • the cancer is a solid tumor.
  • the cancer is esophageal cancer.
  • the cancer is ESCC.
  • gene amplification refers to a process in which the copy number of a gene encoding a specific protein is selectively increased while other genes are not increased proportionally. Under natural conditions, gene amplification is achieved by removing the repetitive sequence of the gene from the chromosome and then performing extrachromosomal replication in a plasmid or by transcribing all the repetitive sequences of ribosomal RNA to generate RNA transcripts and then transcribing them to generate additional copies of the original DNA molecule. of.
  • gene sequencing analysis is disclosed in some embodiments.
  • the subject of the invention has certain unique gene amplifications.
  • the subject has gene amplification of the 11q13 region of chromosome.
  • the subject has CDK4/6 gene amplification; in some further preferred embodiments, the subject has FGF3/4/19 gene amplification.
  • the esophageal cancer subject has CDK4/6 gene amplification.
  • the esophageal cancer subject has FGF3/4/19 gene amplification.
  • the tumor patient has gene amplification in the 11q13 region of chromosome, which indicates that it can be used (a) to administer CDK4/6 inhibitors and/or FGF/FGFR inhibitors alone, or (b) to administer anti-inflammatory drugs in combination.
  • the combination of PD-1 antibody or its antigen-binding fragment and one or more of CDK4/6 inhibitor and/or FGF/FGFR inhibitor has better therapeutic effect.
  • the tumor patient does not have gene amplification of the 11q13 region of chromosome, indicating that it can be used (a) to administer the anti-PD-1 antibody or its antigen-binding fragment alone, or (b) to administer the anti-PD- 1
  • a combination of antibodies or antigen-binding fragments thereof and one or more of CDK4/6 inhibitors and/or FGF/FGFR inhibitors has a better therapeutic effect.
  • CDK refers to cyclin-dependent kinases, which is a group of serine/threonine protein kinases. CDK drives the cell cycle through the phosphorylation of serine/threonine protein through the synergistic effect of cyclin. It is an important factor in cell cycle regulation.
  • CDK 1-8 There are 8 types of CDK family, including CDK 1-8. Each CDK binds to different types of cyclin to form a complex, which regulates the process of cells transitioning from G1 phase to S phase or G2 phase to M phase and exiting M phase.
  • the FDA has approved the listing of CDK4/6 inhibitors, mainly Rebocinil and Pabocinil, and there are dozens of CDK4/6 inhibitors in the clinical research stage.
  • FGF refers to the fibroblast growth factor (Fibroblast growth factor) protein family, which has 23 members, FGF1-23. According to their different mechanisms of action, FGFs can be divided into three categories: endocrine (FGF15/19/21/23), paracrine (FGF1-10, FGF16-18, FGF20, FGF22) and exocytosis FGF11/12/13/14 .
  • the process of paracrine FGFs to regulate biological activity is to use HS as a cofactor to specifically bind to the FGF receptor (Fibroblast Growth Factor Receptor, FGFRs) on the cell surface.
  • FGFRs mainly include 4 kinds: FGFR1, FGFR2, FGFR3, FGFR4.
  • IHC Immunohistochemistry
  • luciferin enzymes, metal ions, isotopes
  • Polypeptides and proteins and methods for localization, qualitative and relative quantitative research.
  • the tumor tissue samples of the subject are tested for PD-L1 expression, and the detection uses Roche's anti-human PD-L1 antibody SP142 (Cat No: M4422) Perform staining experiments.
  • membrane staining intensity of tumor cells ⁇ 1% is defined as PD-L1 positive.
  • the therapeutic agent of the present invention can constitute a pharmaceutical composition, such as a pharmaceutical composition containing the anti-PD-1 antibody described herein or/and other anti-cancer agents other than the anti-PD-1 antibody and other pharmaceutically acceptable carriers .
  • pharmaceutically acceptable carrier includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the carrier suitable for the composition containing the anti-PD-1 antibody is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration, such as by injection or infusion, for containing other anticancer agents
  • the carrier of the composition is suitable for parenteral administration, such as oral administration.
  • the pharmaceutical composition 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, emulsifying agents and dispersing agents.
  • the dosage regimen is adjusted to provide the best desired response, such as maximum therapeutic response and/or minimum adverse effects.
  • the dosage range may be about 0.01 to about 20 mg/kg, about 0.1 to about 10 mg/kg of the individual’s body weight, or 120 mg, 240 mg, 360 mg, 480 mg fixed dose.
  • the dosage may be about 0.1, about 0.3, about 1, about 2, about 3, about 5, or about 10 mg/kg of the individual's body weight.
  • Dosing regimens are usually designed to achieve such exposures that result in sustained receptor occupancy (RO) based on the typical pharmacokinetic properties of Ab.
  • RO sustained receptor occupancy
  • a representative dosing 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 more once.
  • the anti-PD-1 antibody is administered to the individual about once every two weeks.
  • the administration schedule of the CDK4/6 inhibitor or FGF/FGFR inhibitor varies for different subtypes.
  • the CDK4/6 inhibitor or FGF/FGFR inhibitor is administered at its approved or recommended dose, and continue Treatment until clinical effects are observed or until unacceptable toxicity or disease progression occurs.
  • the present invention relates to a method for treating cancer patients, comprising (a) administering a therapeutically effective amount of anti-PD-1 antibody or its antigen-binding fragment alone, or (b) administering a therapeutically effective amount of anti-PD-1 antibody or its One or a combination of antigen-binding fragments and optionally other anticancer agents other than anti-PD-1 antibodies and antigen-binding fragments thereof.
  • the present invention is suitable for the present invention to (a) administer a therapeutically effective amount of anti-PD-1 antibody or its antigen-binding fragment alone, or (b) administer a therapeutically effective amount of anti-PD-1 antibody or
  • a cancer patient treated with a combination of one or more of its antigen-binding fragments and optionally other anticancer agents other than the anti-PD-1 antibody and its antigen-binding fragments is preferably a cancer for which gene amplification of the 11q13 region of chromosome has not been detected patient. Therefore, in some embodiments, the cancer treatment method further includes the step of sequencing cancer patients.
  • the present invention relates to a method of treating cancer patients, comprising (a) administering a therapeutically effective amount of another anticancer agent alone, or (b) administering a therapeutically effective amount of anti-PD-1 antibody in combination, or One or a combination of its antigen-binding fragments and optionally other anti-cancer agents other than anti-PD-1 antibodies.
  • the present invention is suitable for the present invention to (a) administer a therapeutically effective amount of other anticancer agents alone, or (b) administer a therapeutically effective amount of anti-PD-1 antibodies or antigen-binding fragments thereof and any
  • the selected cancer patients who are treated with one or more combinations of anti-cancer agents other than anti-PD-1 antibodies preferably have gene amplification in the 11q13 region of chromosome, especially those with CDK4/6 gene amplification or FGF3/ 4/19 cancer patients with gene amplification. Therefore, in some embodiments, the cancer treatment method further includes the step of sequencing cancer patients.
  • the other anticancer agent is a therapeutically effective amount of a CDK4/6 inhibitor and/or FGF/FGFR inhibitor.
  • the present invention provides methods of treating individuals/patients with esophageal cancer.
  • the method includes administering to the individual a therapeutically effective dose of the following combination: (a) as an Ab or antigen-binding fragment thereof that specifically binds to the PD-1 receptor and inhibits PD-1 activity; and (b) Another anti-cancer therapy.
  • the method includes administering to the individual an effective amount of a combination of (i) the standard therapy for esophageal cancer disclosed elsewhere herein, or (ii) other anticancer agents.
  • the other anticancer agent is selected from CDK4/6 inhibitors and/or FGF/FGFR inhibitors.
  • the esophageal cancer is esophageal squamous cell carcinoma (ESCC) .
  • the method further includes the step of sequencing the patient before administering the therapeutic drug; according to the sequencing results, to individuals without amplification of the 11q13 region of chromosome, administering anti-PD-1 antibody or Its antigen-binding fragments are optionally combined with CDK4/6 inhibitors and/or one or a combination of FGF/FGFR inhibitors; for the amplification of the 11q13 region of chromosome, especially the CDK4/6 gene amplification
  • the CDK4/6 inhibitor and/or FGF/FGFR inhibitor are administered alone or a combination of one or more, optionally in combination with anti-PD-1 antibodies or Its antigen-binding fragment.
  • Anti-PD-1 antibody suitable for use in the method of the present invention
  • the antibody PD-1 antibody suitable for the method of the present invention binds PD-1 with high specificity and affinity, blocks the binding of PD-L1/2 and PD-1, and inhibits the immunity achieved by PD-1 signal transduction Inhibitory effect.
  • the anti-PD-1 antibody includes an antigen-binding portion or fragment that binds to the PD-1 receptor and exhibits functional properties similar to the intact Ab in inhibiting ligand binding and upregulating the immune system.
  • the anti-PD-1 antibody or antigen-binding fragment thereof is an anti-PD-1 antibody or antigen-binding fragment thereof that cross-competes with teriprizumab for binding to human PD-1.
  • the anti-PD-1 antibody or antigen-binding fragment thereof is a chimeric, humanized or human Ab or antigen-binding fragment thereof.
  • the Ab is a humanized Ab.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain constant region of human IgG1 or IgG4 isotype.
  • the sequence of the IgG4 heavy chain constant region of the anti-PD-1 antibody or antigen-binding fragment thereof contains the S228P mutation, which replaces the hinge region with a proline residue that is usually present at the corresponding position of an antibody of the IgG1 isotype Serine residues in.
  • the anti-PD-1 antibody is teriprizumab.
  • the anti-PD-1 antibody is selected from the humanized antibodies 38, 39, 41 and 48 described in WO2014206107.
  • the anti-PD-1 antibody is as described in the "anti-PD-1 antibody" section above.
  • CDK4/6 inhibitors suitable for use in the method of the present invention
  • CDK4/6 inhibitors are used in combination with anti-PD-1 antibodies, or the anti-cancer agents used alone.
  • the FDA has approved the listing of CDK4/6 inhibitors, mainly Eli Lilly’s Verzenio (abemaciclib), which is mainly used to treat hormone receptor (HR) positive and human epidermal growth factor receptor 2 (HER2) after receiving endocrine therapy.
  • HR hormone receptor
  • HER2 human epidermal growth factor receptor 2
  • FGF/FGFR inhibitor suitable for the method of the present invention
  • other anti-cancer agents that is, in addition to anti-PD-1 antibodies, are used in combination with anti-PD-1 antibodies, or the anti-cancer agents used alone are FGF/FGFR inhibitors.
  • FGF/FGFR inhibitors There are no FGF/FGFR inhibitors currently on the market. Boehringer Ingeham’s VEGFR/PDGFR/FGFR inhibitor nintedanib was used to treat liver cancer, non-small cell lung cancer, and idiopathic fibrosis. It obtained the FDA’s breakthrough drug qualification in 2014. In the country, there is currently deritinib in the clinical stage.
  • a method for treating cancer in an individual comprising administering to the individual one of an anti-PD-1 antibody or an antigen-binding fragment thereof, a CDK4/6 inhibitor, and an FGF/FGFR inhibitor One or more combination therapy.
  • Combination therapy may also include one or more additional therapeutic agents.
  • the additional therapeutic agent may be a chemotherapeutic agent or a biotherapeutic agent other than CDK4/6 inhibitor and FGF/FGFR inhibitor.
  • each therapeutic agent in the combination therapy of the present invention can be administered alone or in a pharmaceutical composition comprising the therapeutic agent and one or more pharmaceutically acceptable carriers and excipients.
  • Agent and diluent are pharmaceutically acceptable carriers and excipients.
  • Each of the therapeutic agents in the combination therapy of the present invention can be administered simultaneously, concurrently, or sequentially in any order.
  • the therapeutic agents in 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 administered at different administration times, such as chemotherapeutic agents are administered at least daily and
  • the biotherapeutics are administered infrequently, such as once every week, or every two weeks or every three weeks.
  • the CDK4/6 inhibitor, FGF/FGFR inhibitor is administered prior to the administration of the anti-PD-1 antibody, and in other embodiments, the CDK4/6 inhibitor, FGF/FGFR inhibitor is administered Anti-PD-1 antibody is administered afterwards.
  • At least one of the therapeutic agents in the combination therapy is administered using the same dosing schedule (dose, frequency, treatment duration).
  • Each small molecule therapeutic agent in the combination therapy of the present invention can be administered orally or parenterally (such as intravenous, intramuscular, intraperitoneal, subcutaneous, rectal, topical or transdermal administration routes).
  • the combination therapy of the present invention can be administered before or after surgery, and can be administered before, during or after radiotherapy.
  • the combination therapies described in the present invention are administered to patients who have not previously been treated with biotherapeutics or chemotherapeutics. In other embodiments, the combination therapy is administered to patients who cannot achieve a sustained response after treatment with biotherapeutics or chemotherapeutics.
  • the combination therapy of the present invention can be used to treat tumors discovered by palpation or by imaging techniques known in the prior art, such as MRI, ultrasound or CAT scan.
  • the choice of the dosing regimen of the combination therapy of the present invention depends on several factors, including but not limited to serum or tissue conversion rate, degree of symptoms, immunogenicity, and accessibility of target cells, tissues, and organs of the individual to be treated.
  • the dosage regimen should match the acceptable degree of side effects to deliver the maximum amount of various therapeutic agents to the patient. Therefore, the dosage and frequency of administration of each biotherapeutic agent and chemotherapeutic agent in the combination therapy depend on the specific therapeutic agent, the severity of the cancer being treated, and the characteristics of the patient.
  • One or more of the anti-PD-1 antibody or its antigen-binding fragment thereof, CDK4/6 inhibitor and FGF/FGFR inhibitor of the present invention can be used as a kit comprising a first container, a second container and a package insert provide.
  • the first container contains at least one dose of the drug containing anti-PD-1 antibodies or antigen-binding fragments thereof
  • the second container contains at least one dose of one or more of CDK4/6 inhibitors and FGF/FGFR inhibitors Medicine
  • the package insert or label contains instructions for using the medicine to treat cancer.
  • the kit may further include other materials that can be used to administer drugs, such as diluents, filter paper, IV bags and threads, needles and syringes.
  • Anti-PD-1 antibody predicts the effect of cancer treatment
  • the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention includes sequencing the individual before treatment.
  • the cancer is esophageal cancer; in other preferred embodiments, the cancer is ESCC.
  • the prediction method of the present invention is to test the individual's genes before treatment to evaluate whether the gene in the 11q13 region of chromosome has amplification.
  • the gene of the 11q13 region of the chromosome of the individual is not amplified.
  • the gene in the 11q13 region of the chromosome of the individual has amplification.
  • the individual has CCND1 gene and/or FGF/FGFR gene amplification.
  • the individual has CDK4/6 gene and/or FGF3/4/19 gene amplification.
  • the present invention also includes a method for predicting the therapeutic effect of anti-PD-1 antibodies or antigen-binding fragments thereof administered alone or in combination in tumor patients by using genes in the 11q13 region of chromosome.
  • the absence of gene amplification in the 11q13 region of chromosome indicates that the tumor patient is suitable for treatment with anti-PD-1 antibody or antigen-binding fragment thereof alone or in combination.
  • the present invention also provides the use of biomarker detection reagents in preparing a kit for predicting the effect of anti-PD-1 antibody in treating cancer.
  • reagents include, for example, reagents for detecting the presence or absence of gene amplification of the 11q13 region of chromosome in individual genes.
  • reagents include, for example, reagents for detecting the presence of CCND1 gene and/or FGF/FGFR gene amplification in individual genes.
  • such reagents include, for example, reagents for detecting the presence of CDK4/6 gene and/or FGF3/4/19 gene amplification in individual genes.
  • the present invention also includes the use of reagents for detecting genes in the 11q13 region of chromosome in preparing a kit for predicting the effect of anti-PD-1 antibodies in treating cancer.
  • reagents include, but are not limited to, reagents commonly used in tests, including but not limited to primers, probes, and reagents required for PCR.
  • Eligible subjects must (1) be between 18 and 75 years old, (2) histological and/or cytologically proven to have advanced and/or metastatic ESCC, (3) patients with advanced ESCC , Must have received or received at least one treatment and still have progress (including but not limited to chemotherapy or radiotherapy), (4) ECOG score is 0 or 1.
  • Subjects must have evaluable lesions according to RECIST v 1.1 standards, no other past or concurrent malignant tumors, no active autoimmune diseases or history of autoimmune diseases, and long-term immunosuppression Drug treatment of concomitant diseases, no previous anti-CTLA4, anti-PD-1, anti-PD-L1 antibody treatment, no active hepatitis B or hepatitis C virus infection, no pregnancy or breast-feeding, no enrollment Have received anti-tumor therapy, radiotherapy or any surgical treatment in the previous 4 weeks.
  • the demographic statistics of the enrolled subjects are shown in Table 1.
  • Table 1 Demographic statistics of the enrolled subjects
  • tested drug anti-PD-1 antibody teriprizumab (teriprizumab) (WO2014206107).
  • the dose group of anti-PD-1 antibody in this experiment is: this study first conducts a 3mg/kg dose group study. After enrollment, the subjects will receive treatment every 2 weeks (Q2W), and every 4 weeks is a cycle until the disease progresses, unbearable toxicity occurs, the subject withdraws the consent form, and the investigator judges that there is no further benefit , Or death.
  • the content of the present invention mainly evaluates the results of cohort 2.
  • the planned dose is 3mg/kg, Q2W.
  • TRAE Treatment-related adverse events
  • TRAEs include weight loss (18.6%), anemia (18.6%), loss of appetite (18.6%), fever (16.9%), cough (16.9%), leukopenia (15.3%), and elevated AST (13.6%) , Hypothyroidism (13.6%), elevated ALT (11.9%), fatigue (11.9%), nausea (11.9%), increased total bilirubin (10.2%), low hemoglobin count (10.2%) and constipation ( 10.2%) (Table 2).
  • the 6 deaths related to treatment may not be related to treatment, including 1 case of cachexia, 2 cases of pneumonia and 3 cases of unknown etiology.
  • the median treatment time is 3.5 months (between 0.1-19.1 months).
  • CR patients have previously received first-line chemotherapy and have not received radiotherapy.
  • 10 PR patients had received radiotherapy in the past, of which 4 had also received 2 previous systemic chemotherapy, and the other 6 had received 3 or 4 previous chemotherapy.
  • the confirmed ORR is 15.3% (95% CI 7.2 to 27.0).
  • the average response time is 1.8 months.
  • the median duration of response was 11.2 months.
  • an arbitrary size reduction of the target lesion relative to baseline was observed ( Figures 1A and 1B).
  • the median PFS was 2.1 months, and the median OS was 6.9 months ( Figures 1C and 1D).
  • Example 2 Correspondence between PD-L1 expression and anti-PD-1 antibody therapeutic effect on tumor
  • PD-L1 positive status is defined as the presence of membrane staining intensity ⁇ 1% of tumor cells or the presence of PD-L1 staining intensity of tumor infiltrating immune cells covering ⁇ 1% of tumor cells occupied by tumor cells, related within the tumor, continuous pericarcinoma stromal tumors area.
  • PD-L1 expression was measured on tumor biopsy samples taken from 57 subjects in Example 1. 19 (33.3%) PD-L1 expression positive and 38 (66.7%) PD-L1 expression negative samples were identified ( Figure 2A).
  • PD-L1 positive is defined as SP142IHC staining with ⁇ 1% tumor cell (TC) or immune cell (IC) positive.
  • TAB tumor mutation burden
  • TMB whole-exome sequencing
  • ESCC patients generally had lower TMB.
  • No biopsy sample has more than 20 mutations per million base pair, and only 13 biopsy samples have 10-20 mutations per million base pair.
  • Example 4 Mutation panorama of anti-PD-1 therapy and its corresponding relationship with clinical efficacy
  • the tumor tissue and matching PBMC samples were collected.
  • the 4 ⁇ m part of hematoxylin and the eosin-stained FFPE samples were subjected to pathological examination to ensure that each sample has more than 80% nucleocytosis and the tumor content is not less than 20%.
  • the library was prepared by sonicating the double-stranded DNA to 250bp. Subsequently, SureSelect XT library preparation kit or KAPA Hyper preparation kit (KAPA Biosystems) was used to construct a library with end repair, dA addition and adaptor ligation, and then PCR amplification was performed and quantified by Qubit evaluation.
  • PCR amplification was performed and quantified by Qubit evaluation.
  • Sequence reading is generated using Illumina NovaSeq 6000 (Illumina Inc., San Diego, CA). Check and control data quality, and establish a set of customized bioinformatics pipelines for the discovery of SNV, long and short insertions and deletions, CNA, gene rearrangements, TMB and MSI. Finally, all detected mutations are compared to our internal annotation database for clinical treatment. Based on the annotated mutations, a concise report based on clinical trials and literature will be generated.
  • the Burrows-Wheeler sequencer was used to compare the original read sequence with the human genome reference sequence (hg19), and then the Picard marker replication algorithm was used to remove PCR repetitive sequences.
  • the original calls of SNV and S-indel are called by internal algorithms. At least 5 reads are required to support optional calls. Remove the variation with read depth less than 50x, chain offset less than or greater than 90%, and VAF ⁇ 1%.
  • SNPs are defined as frequencies that exceed 1.5% in the dbSNP database (version 147) or Exome Sequencing Project 6500 (ESP6500) or more than 1.5% in 1000 genome projects are also excluded for further consideration. Products from sequencing errors or populations are also excluded.
  • the aligned reads in each exon region were normalized, and then CNVKIT (version 0.9.1) was used to further correct the results for GC content and reference genome mappability.
  • the cyclic binary division algorithm is used to divide the obtained copy rate.
  • the allele frequency of more than 4000 sequenced SNPs single nucleotide polymorphisms
  • the copy rate of tumor tissue and matched normal blood sample is calculated. If log2 (copy rate) exceeds 0.8 or does not exceed -1, the fragment is considered to be enlarged or deleted, respectively.
  • Example 5 Relationship between 11q13 gene amplification and resistance to anti-PD-1 therapy
  • RNA sequence Use miRNeasy kit (cat#217504, Qiagen) to extract RNA from unstained FFPE, and then use The rRNA depletion kit (cat#E6310L, New England Biolabs) was further processed to delete rRNA.
  • M-MLV RT RNase (H-) (cat#M3683, Promega) and NEB second-strand mRNA synthesis kit (cat#E6111L, New England Biolabs) were used to synthesize first-strand and second-strand cDNA, respectively.
  • the cDNA product was sonicated to produce a ⁇ 200bp fragment (Covaris E220).
  • the adapter-linked library was created by cDNA using the KAPA Hyper preparation kit (cat#07962363001, Roche) and sequenced on the NovaSeq 5000/6000 platform. The relative abundance of each annotated transcript is expressed in transcripts per million (TPM) and log2 conversion before analysis.
  • Example 4 The WES analysis of Example 4 showed that 24 of the 50 biopsies (48.0%) contained the amplification of the 11q13 region of chromosome ( Figure 4A).
  • the messenger RNA expression analysis further confirmed the genomic DNA amplification event.
  • the mRNA level is related to the amplification status of the corresponding genes in this region, including CCND1 (Cyclin D1) and FGF family members (FGF3/4/19) ( Figure 4B).
  • Example 6 Other biomarkers and subgroup analysis
  • biomarkers or subgroup analyses related to clinical effects include: age, gender, ECOG score, pre-treatment, the presence of liver metastases at baseline, tumor volume, and baseline serum LDH levels. Among them, patients with an ECOG score of 0, low baseline tumor volume and baseline LDH levels above the upper limit of normal have numerically better ORR. However, these differences were not statistically significant (Table 4).
  • Example 7 Study on the inhibitory effect of anti-PD-1 antibody combined with CDK4/6 inhibitor on tumor growth in mice
  • CT-26 cells were inoculated subcutaneously on the right side of hPD-1 humanized female mice at a concentration of 2.5 ⁇ 10 5 cells/0.1 mL, and when the tumor grew to about 93 mm 3 , they were randomly divided into groups according to the tumor volume, each with 7 cells. There are 4 groups in total, namely:
  • G1 Anti-KLH hIgG4 (1mg/kg) negative control group
  • G2 toripalimab (teriprolizumab) (1mg/kg) group;
  • G3 Pabocinil (100mg/kg);
  • G4 toripalimab (1mg/kg) combined with Pabocinil (100mg/kg) group.
  • Anti-KLH hIgG4 and toripalimab are administered by intraperitoneal injection, twice a week, three consecutive times, and pabocinil is administered orally, once a day.
  • the tumor volume and body weight were measured twice a week, and the mouse body weight and tumor volume were recorded.
  • the animals were euthanized and the tumor volume inhibition rate (TGI Tv %) was calculated.
  • test drug Pabocinil has a certain inhibitory effect on tumor growth; compared with toripalimab (1mg/kg) and Pabocinib (100mg/kg) single drugs, toripalimab (1mg/kg) combined with Pabocinib ( 100mg/kg) administration has a more significant inhibitory effect on tumor growth.
  • Table 5 Effect of toripalimab combined with RT391 on tumor volume of B-hPD-1 mice transplanted with CT-26 cells
  • a mean ⁇ standard error
  • b the tumor volume of the treatment group and the tumor volume of the Anti-KLH hIgG4 control group are statistically compared 11 days after the treatment, t-test.

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Abstract

本发明公开了抗PD-1抗体在治疗肿瘤中的用途，以及用于检测染色体11q13区基因扩增的试剂在制备预测肿瘤病人对抗PD-1抗体和/或抗原结合片段治疗的效果的检测试剂盒中的用途。

Description

抗PD-1抗体在制备治疗实体瘤的药物中的用途 技术领域
本发明涉及抗PD-1抗体在治疗肿瘤中的用途。具体而言，本发明涉及抗PD-1抗体在治疗实体瘤，尤其是食管癌中的用途，更优选是食管鳞状细胞癌(ESCC)中的用途；以及抗PD-1抗体在制备治疗实体瘤，尤其是食管癌，肢端型皮肤黑色素瘤中的用途，更优选是食管鳞状细胞癌(ESCC)的药物中的用途；以及利用生物标记物预测抗PD-1抗体在治疗实体瘤，尤其是食管癌中的用途，更优选是食管鳞状细胞癌(ESCC)中的疗效的方法；以及包含该抗PD-1抗体的组合疗法或组合物在治疗实体瘤中的用途。
背景技术
免疫逃逸是癌症的特征之一。Ahmadzadeh,M.等，Blood,114:1537-44中公开了肿瘤特异性T淋巴细胞常存在于肿瘤微环境、引流淋巴结和外周血中，但由于肿瘤微环境中存在的免疫抑制机制网络，其通常无法控制肿瘤的进展。CD8 +肿瘤浸润T淋巴细胞(TIL)通常表达活化诱导的抑制受体，包括CTLA-4和PD-1，而肿瘤细胞经常表达免疫抑制配体，包括PD-1配体1(PD-L1，也叫B7-H1或CD274)，该配体抑制T细胞激活和效应功能。在抑制机制中，PD-1及其配体已成为肿瘤细胞利用其抑制肿瘤微环境中激活的T细胞的重要途径。
程序性死亡受体1(PD-1)在免疫调节及周边耐受性维持中起重要作用。PD-1主要在激活的T细胞和B细胞中表达，功能是抑制淋巴细胞的激活，这是免疫系统的一种正常的防治免疫过激的外周组织耐受机制。但是，在肿瘤微环境中浸润的活化T细胞高表达PD-1分子，活化白细胞分泌的炎症因子会诱导肿瘤细胞高表达PD-1的配体PD-L1和PD-L2，导致肿瘤微环境中活化T细胞PD-1通路持续激活，T细胞功能被抑制，无法杀伤肿瘤细胞。治疗型PD-1抗体可以阻断这一通路，部分恢复T细胞的功能，使活化T细胞能够继续杀伤肿瘤细胞。
近十年来，PD-1/PD-L1通路阻断已被证明是在各种癌症适应症中诱导持久抗肿瘤应答的有效途径。阻断PD/PD-L1通路的单克隆抗体(mAbs)可以增强肿瘤特异性T细胞的活化和效应功能，减轻肿瘤负担，提高生存率。
食管癌(EC)是人类最常见的恶性肿瘤之一，在过去的几十年中，该病的发病率和死亡率持续上升，目前全球每年有400,000人死于此病。其中，食管鳞状细胞癌(ESCC) 是发展中国家中最常见的食管癌组织学亚型，其是在南美洲和东亚人群中最主要的食管癌组织学亚型，该肿瘤在世界范围内仍未满足治疗需求。而在中国，EC是第三种常见的癌症，且排在癌症相关死亡原因的第四位。而ESCC在中国的整个食管癌中占比高于90％，且常用化疗和放疗方法来治疗。目前转移性ESCC最常用的化疗药物有顺铂、5-氟尿嘧啶和紫杉烷类。并且ESCC病人的5-年生存率很低，仅有15％-20％。目前对于化疗难治性EC患者没有标准疗法。最近，Clin Cancer Res 2018；24(6):1296-304公开了抗PD-1抗体SHR-1210治疗中国的化疗难治性ESCC患者具有可控的安全性及有效的抗肿瘤活性，并发现PD-L1阳性肿瘤比PD-L1阴性肿瘤客观有效率更高，且发现突变负荷(TMB)以及潜在的突变相关新抗原数量与更好的治疗反应相关。而在J Clin Oncol 2019；37,2019(suppl 4；abstr 2)中公开了经一线治疗后疾病进展的晚期ESCC患者，利用帕姆单抗治疗在PD-L1结合阳性得分(CPS)≥10人群中相比化疗具有显著的整体生存率。因此，虽然前期研究已经证实PD-1靶向疗法在转移性ESCC子群的病人中具有有效性，但抗PD-1抗体免疫疗法的有效的预测生物标记物仍不明确。
发明内容
本发明第一方面提供一种单独施用抗PD-1抗体或其抗原结合片段，或与其他抗癌剂组合施用在制备用于治疗癌症的药物中的用途。
在一个或多个实施方式中，本发明所述的其他抗癌剂为小分子靶向抗癌剂。在一个实施方式中，本发明所述的其他抗癌剂选自CDK4/6抑制剂，FGF/FGFR抑制剂。
在一个或多个实施方案中，所述药物含有抗PD-1抗体或其抗原结合片段和CDK4/6抑制剂。在一个或多个实施方案中，所述药物含有抗PD-1抗体或其抗原结合片段和FGF/FGFR抑制剂。
在一个或多个实施方式中，本发明提供抗PD-1抗体或其抗原结合片段在用于制备与CDK4/6抑制剂，FGF/FGFR抑制剂组合用于治疗癌症患者的药物中的用途。
在一个或多个实施方式中，本发明提供抗PD-1抗体或其抗原结合片段与CDK4/6抑制剂，FGF/FGFR抑制剂组合的用途，其用于制备治疗癌症患者的药物。
在一个或多个实施方案中，本发明所述癌症为实体瘤。
在一个或多个实施方案中，所述癌症包含但不局限于胃癌、食管癌、鼻咽癌、头颈部鳞状细胞癌、乳腺癌、膀胱癌和结肠癌。
在一个或多个实施方案中，所述癌症优选是食管癌。在一个或多个优选实施方式中，所述的癌症是食管鳞状细胞癌。在一个或多个优选的实施方式中，所述的癌症是晚期ESCC。在一个或多个优选的实施方式中，所述的癌症是化疗难治性ESCC。在其他实施 方式中，所述的食管鳞状细胞癌是晚期、转移性和/或难治性食管鳞状细胞癌。
在一个或多个实施方案中，所述实体瘤不具有染色体11q13区的基因扩增。
在一个或多个实施方案中，本发明提供抗PD-1抗体(优选特瑞普利单抗)与CDK4/6抑制剂(优选瑞博西尼或帕博西尼，更优选帕博西尼)的组合在制备治疗食管癌或结肠癌的药物中的用途；在一些实施方案中，本发明提供抗PD-1抗体(优选特瑞普利单抗)在制备治疗食管癌的药物中的用途。
在某些实施方式中，所述个体已经进行了预先治疗。在某些实施方式中，所述预先治疗包括但不限于化疗或放疗。在某些实施方式中，所述个体已经进行了系统性治疗。在一个或多个优选的实施方式中，所述预先治疗或系统性治疗至少为2次。在某些实施方式中，所述个体无自身免疫性疾病史或自身免疫性疾病史。在某些实施方式中，所述个体无任何需要长期免疫抑制药物治疗的伴随性疾病。在某些实施方式中，所述个体既往未接受过任何免疫检查位点阻断剂治疗。
在一些实施方式中，单独施用抗PD-1抗体或其抗原结合片段，或与其他抗癌剂组合施用，以诱导所述个体中持久的临床反应。
在一个或多个实施方案中，单独施用抗PD-1抗体或其抗原结合片段，或与其他抗癌剂组合施用的治疗中，抗PD-1抗体或其抗原结合片段治疗有效剂量的范围为约0.1至约10.0mg/kg体重，约每2周静脉输注一次。在一些实施方式中，抗PD-1抗体或其抗原结合片段以约1mg/kg体重，3mg/kg体重或10mg/kg体重或240g/kg固定剂量施用，每2周一次。
在一个或多个实施方案中，抗PD-1抗体或其抗原结合片段的单次施用剂量为约0.1mg/kg至约10.0mg/kg个体体重，例如约0.1mg/kg，约0.3mg/kg，约1mg/kg、约2mg/kg、约3mg/kg、约5mg/kg，或10mg/kg个体体重，或选自约120mg至约480mg固定剂量，例如120mg、240mg、360mg或480mg固定剂量。
在一个或多个实施方案中，抗PD-1抗体或其抗原结合片段的给药周期频率为约每一周一次，每两周一次、每三周一次、每四周一次或一个月一次，优选为每两周一次。
在一个或多个实施方案中，抗PD-1抗体或其抗原结合片段的单次施用剂量为1mg/kg个体体重、3mg/kg个体体重、10mg/kg个体体重、或240mg固定剂量，以每两周一次施用。
在一个或多个实施方案中，抗PD-1抗体或其抗原结合片段以液体剂型例如注射剂，经胃肠外途径例如经静脉输注施用。
在一个或多个实施方案中，抗PD-1抗体或其抗原结合片段的给药周期可以为一周、二周、三周、一个月、两个月、三个月、四个月、五个月、半年或更长时间，任选地，每个给药周期的时间可以相同或不同，且每个给药周期之间的间隔可以相同或不同。
本发明第二方面提供用于治疗患有癌症的个体的试剂盒，所述试剂盒包括：(a)特异性结合并抑制PD-1的单抗或其抗原结合片段，和任选的除抗PD-1抗体或其抗原结合片段以外的其他抗癌剂；和(b)单独施用抗PD-1抗体或其抗原结合片段，或组合施用特异性结合并抑制PD-1的单抗或其抗原结合片段和其他抗癌剂以治疗个体癌症的说明书。所述癌症为实体瘤。作为一种优选的实施方式，所述癌症包含但不局限于胃癌、食管癌、鼻咽癌、头颈部鳞状细胞癌、乳腺癌、膀胱癌和结肠癌。作为一种优选的实施方式，所述癌症优选是食管癌。作为一种优选的实施方式，所述的癌症是食管鳞状细胞癌。作为更进一步的优选方式，所述的癌症是ESCC。作为一种优选的实施方式，所述实体瘤不具有染色体11q13区的基因扩增。
在一个或多个实施方式中，本发明所述的其他抗癌剂为小分子靶向抗癌剂。在一个实施方式中，本发明所述的其他抗癌剂选自CDK4/6抑制剂和FGF/FGFR抑制剂中的一种或多种。
在一个或多个实施方案中，所述试剂盒含有抗PD-1抗体或其抗原结合片段和CDK4/6抑制剂。在一个或多个实施方案中，所述试剂盒含有抗PD-1抗体或其抗原结合片段和FGF/FGFR抑制剂。
本发明第三方面提供一种CDK4/6抑制剂或FGF/FGFR抑制剂在制备用于治疗癌症的药物中的用途。
本发明第四方面提供一种用于治疗癌症的方法，其包括在治疗前对个体进行测序的步骤；其中，对不具有染色体11q13区的扩增的个体单独施用抗PD-1抗体或其抗原结合片段，任选地组合施用CDK4/6抑制剂、和/或FGF/FGFR抑制剂的一种或几种的组合；对具有染色体11q13区的扩增的个体单独施用CDK4/6抑制剂、和/或FGF/FGFR抑制剂的一种或几种的组合，任选地组合施用抗PD-1抗体或其抗原结合片段。
本发明所述的用途、方法、药物及试剂盒中，个体是人。
在一个或多个优选的实施方式中，本发明所述的个体是人且其癌症是实体瘤。在一个或多个优选的实施方式中，本发明所述的个体是人且其癌症选自包含但不局限于胃癌、食管癌、鼻咽癌、头颈部鳞状细胞癌、乳腺癌、膀胱癌和结肠癌。在一个优选的实施方式中，本发明所述的个体是人且其癌症是食管癌。在一个优选的实施方式中，本发明所述的个体是人且其癌症是ESCC。在一个或多个实施方式中，本发明所述个体患有食管癌且既往未接受过免疫治疗。
本发明第五方面提供一种预测肿瘤病人对抗PD-1抗体治疗效果的方法，其包括检测患者是否具有染色体11q13区的基因扩增，其中，不存在所述染色体11q13区的基因扩增表明所述肿瘤病人适于用抗PD-1抗体进行治疗。
本发明所述的用途、方法、药物及试剂盒中，所述抗PD-1抗体是单克隆抗体或其抗原结合片段。在某些实施方案中，所述抗PD-1抗体特异性结合PD-1，阻断PD-L1或PD-L2与PD-1结合。在某些实施方案中，所述抗PD-1抗体特异性结合PD-L1或/和PD-L2，阻断PD-L1和/或PD-L2与PD-1的结合。
在一个或多个实施方式中，抗PD-1抗体是包含互补决定区(CDR)的抗体，其轻链互补决定区(LCDR)氨基酸序列如SEQ ID NO：1、2和3所示，重链互补决定区(HCDR)氨基酸序列如SEQ ID NO：4、5和6所示。
在一个或多个实施方式中，抗PD-1抗体包含轻链可变区(VL)和重链可变区(VH)，其中，VL如SEQ ID NO：7所示，VH如SEQ ID NO：8所示。
在一个或多个实施方式中，抗PD-1抗体是包含轻链和重链的抗PD-1抗体，且轻链包含SEQ ID NO:9所示的氨基酸序列，重链包含SEQ ID NO：10所示的氨基酸序列。
附图说明
图1：(A)研究者根据RECIST v1.1评估的肿瘤大小与基线相比的最大变化(n＝46，受试者具有基线和至少一种治疗后影像学评价)。条形图的长度表示目标病变的最大减少或最小增加。条形图的颜色反映了先前系统治疗的结果。蓝色：≥3L；橙色:2L；绿色:1L。5例患者(标记为#)，目标病灶较基线变化最好，>下降30％，但反应不能证实或有新的病灶发生。5例患者(标记为+)，靶病灶较基线变化最好，增幅小于20％的增加，但由于新病灶的发生或非靶病灶的进展，以进展性疾病(PD)为特征。
(B)研究者评估了每RECIST v1.1(n＝46，受试者基线及至少1次治疗后x线片评价)的个体肿瘤负担随时间的变化。*更改百分比被截断至100％。
(C)研究中所有患者无进展生存期。
(D)研究中所有患者的总生存率。在指定的时间点的存活患者的百分比显示。被审查的病人在图中标有“┃”。在指定时间点有危险的病人人数显示在X-轴下面。
图2：与肿瘤PD-L1表达或肿瘤突变负担(TMB)相关的临床反应。
(A)PD-L1阳性状态被定义为存在膜染色的强度≥1％的肿瘤细胞或免疫细胞通过SP142包含IHC染色。
(B)TMB状态通过肿瘤活检的全外显子组测序和配对的PBMCs来确定。
(C)PD-L1阳性与PD-L1阴性患者的PFS比较。
(D)PD-L1阳性与PD-L1阴性患者的OS比较。
(E)TMB高和TMB低的病人的PFS比较。
(F)TMB高(≥12)和TMB低(＜12)的病人的OS比较。生存患者的百分比显示在指定的时间点。被审查病人在图种标有“┃”。x轴下方显示了在指定时间点处于危险中的患者数量。
图3：晚期EC患者的突变情况。
WES的基因组图谱显示：TP53(p53)(76％)、RYR2(22％)、NOTCH1(20％)、LRP1B(17％)和TRIO(17％)的错义突变或截断是ESCC肿瘤活检中最常见的5个突变基因。
图4：入选ESCC患者11q13基因位点扩增及RNA表达分析。
(A)通过对51例可用患者的FFPE肿瘤和配对外周血样本进行下一代测序进行基因组图谱分析。11q13扩增24例。顶部为染色体11q13区域和编码基因图。采用三种算法确定放大事件。每个个体都有扩增的基因。扩增基因的突变或缺失也被标记。
(B)扩增11q13的信使RNA表达分析。对现有患者进行mRNA测序和表达谱分析。在每个个体中mRNA表达水平与基因扩增相关。
图5：临床反应与染色体11q13区的基因扩增状态有关。
(A)肿瘤11q13状态通过肿瘤活检的全外显子组测序和配对的PBMCs来确定。
(B)11q13野生型与11q13扩增型患者无进展生存。
(C)11q13野生型与11q13扩增型患者的总生存率。生存患者的百分比显示在指定的时间点。被审查病人在图种标有“┃”。x轴下方显示了在指定时间点处于危险中的患者数量。
图6：NCT02915432研究评价特瑞普利单抗在晚期GC，ESCC，NPC及HNSCC患者中的作用的临床试验进程。
图7：抗PD-1抗体联合CDK4/6抑制剂对小鼠肿瘤生长的抑制作用研究结果。
具体实施方式
本发明涉及肿瘤治疗方法。本发明的方法包括向有需要的患者单独施用抗PD-1抗体或其抗原结合片段；或包括向有需要的患者施用与其他抗癌剂组合的抗PD-1抗体或其抗原结合片段。本发明还涉及利用生物标记物预测抗PD-1抗体在治疗癌症，尤其是食管癌患者中的疗效的方法。
术语
为了更易于理解本发明，下文具体定义某些科技术语。除非本文中别处另有明确说明， 否则本文所用的科技术语皆具有本发明所属技术领域的普通技术人员通常所了解的含义。
“施用”、“给与”及“处理”是指采用本领域技术人员已知的各种方法或递送系统中的任意一种将包含治疗剂的组合物引入受试者。抗PD-1抗体的给药途径包括静脉内、肌内、皮下、腹膜、脊髓或其他胃肠外给药途径，比如注射或输注。“胃肠外给药”是指除了肠内或局部给药以外的通常通过注射的给药方式，包括但不限于静脉内、肌内、动脉内、鞘内、淋巴内、损伤内、囊内、框内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜内和胸骨内注射和输注以及经体内电穿孔。
本文所述的“不良反应”(AE)是与使用医学治疗相关的任何不利的和通常无意的或不期望的迹象、症状或疾病。例如，不良反应可能与在响应治疗时免疫系统的激活或免疫系统细胞的扩增相关。医学治疗可以具有一种或多种相关的AE，并且每种AE可以具有相同或不同的严重性水平。
术语“受试者”包括任何生物体，优选动物，更优选哺乳动物(例如大鼠、小鼠、狗、猫、兔等)，且最优选的是人。术语“受试者”、“患者”和“个体”在本文中可以互换使用。
本文所述的“抗体”是指能达到期望的生物活性或结合活性的任何形式的抗体。因此，它以最广泛含义使用，但不限于单克隆抗体、多克隆抗体、多特异性抗体、人源化全长人抗体、嵌合抗体及骆驼源单一结构域抗体。其特异性结合抗原并包含通过二硫键互连的至少两条重(H)和两条轻(L)链，或其抗原结合片段。每条重链包含重链可变区(VH)和重链恒定区，重链恒定区包含三个恒定结构域CH1、CH2和CH3。每条轻链包含轻链可变区(VL)和轻链恒定区，轻链恒定区包含一个恒定结构域CL。VH和VL区可进一步细分为称为互补决定区(CDR)的高变区，其散布于更为保守的称为框架区(FR)的区域。一般而言，自N末端至C末端，轻链及重链可变结构域二者皆包含FR1、CDR1、FR2、CDR2、FR3、CDR3及FR4。通常是根据如下的定义将氨基酸分配至每一个结构域的：Sequences of Proteins of Immunological Interest,Kabat等人；National Institutes of Health,Bethesda,Md.；第5版；NIH出版号91-3242(1991)：Kabat(1978)Adv.Prot.Chem.32:1-75；Kabat等人，(1977)J.Biol.Chem.252:6609-6616；Chothia等人，(1987)J Mol.Biol.196:901-917或Chothia等人，(1989)Nature 341:878-883。
重链的羧基末端部分可定义主要负责效应子功能的恒定区。通常，人轻链分为κ链及λ链。人重链通常分为μ、δ、γ、α或ε，且将抗体的同种型分别定义为IgM、IgD、IgG、IgA及IgE。IgG亚类是本领域技术人员熟知的，包括但不限于IgG1、IgG2、IgG和IgG4。
术语“抗体”包括：天然存在的和非天然存在的Ab；单克隆和多克隆Ab；嵌合和人源化Ab；人或非人Ab；全合成Ab；和单链Ab。非人Ab可以通过重组方法人源化以降低其在人中的免疫原性。
除非另有明确表示，否则本文所述的“抗体片段”或“抗原结合片段”是指抗体的抗原结合片段，即保留了全长抗体的特异性结合至抗原能力的抗体片段，例如保留一个或多个CDR区的片段。抗体结合片段的实例包括但不限于Fab、Fab’、F(ab’)2及Fv片段；双链抗体；线形抗体；单链抗体分子；纳米抗体及由抗体片段形成的多特异性抗体。
“嵌合抗体”是指如下的抗体以及其片段：其中重链和/或轻链的一部分与源自特定物种(如人)或属于特定抗体种类或亚类的抗体中相应序列相同或同源，而链的其余部分与源自另一物种(如小鼠)或属于另一抗体种类或亚类的抗体中相应序列相同或同源，只要其表现出期望的生物活性即可。
“人抗体”是指仅包含人免疫球蛋白序列的抗体。若人抗体是在小鼠、小鼠细胞或源自小鼠细胞的杂交瘤中产生，则其可含有鼠类碳水化合物链。类似的，“小鼠抗体”或“大鼠抗体”是指仅分别包含小鼠或大鼠免疫球蛋白序列的抗体。
“人源化抗体”是指含有来自非人(如鼠类)抗体以及人抗体的序列的抗体形式。此类抗体含有源自非人免疫球单边的最小序列。通常，人源化抗体将包含实质上全部的至少一个且通常两个可变结构域，其中全部或实质上全部超变环对应于非人免疫球蛋白的超变环，且全部或实质上全部FR区为人免疫球蛋白的FR区。人源化抗体任选还包括免疫球蛋白恒定区(Fc)(通常为人免疫球蛋白恒定区)的至少一部分。
本文中，术语“癌症”是指以身体中异常细胞不受控制的生长为特征的广泛的各种疾病。不受调节的细胞分裂、生长分裂和生长导致恶性肿瘤的形成，其侵入邻近组织并还可以通过淋巴系统或血流转移至身体的远端部分。癌症的实例包括但不限于癌、淋巴瘤、白血病、母细胞瘤及肉瘤。癌症的更特定的实例包括鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌、胶质瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、急性骨髓性白血病、多发性骨髓瘤、胃肠(道)癌、肾癌、卵巢癌、肝癌、淋巴母细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、肾癌、前列腺癌、甲状腺癌、黑色素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性神经胶质母细胞瘤、子宫颈癌、脑癌、胃癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌及头颈癌。癌症的另一个实施方式包括食管癌。癌症的另一特定实施例包括ESCC。癌症的另一特定实施例包括化疗难治性ESCC。癌症的另一特定实施例包括晚期ESCC。在某些实施方案中，本发明所述的癌症包括那些特征在于测序的个体具有染色体11q13区基因扩增的癌症。本发明所述的癌症包括那些特征在于测序的个体不具有染色体11q13区基因扩增的癌症。
术语“食管癌”或“EC”是人类最常见的恶性肿瘤之一，其是常见的消化道肿瘤，按照组织学及病理分型，其可分为鳞状细胞癌、腺癌和小细胞癌三种。其中，“食管鳞状细胞癌”或“ESCC”是发展中国家最常见的EC类型，该种癌症的治疗预后很差，5年总生存率 仅为20-30％。
术语“免疫治疗”是指通过包括诱导、增强、抑制或以其他方式修饰免疫反应的方法治疗患有疾病或具有感染或遭受疾病复发风险的受试者。受试者的“治疗”或“疗法”是指对受试者进行的任何类型的干预或过程，或给与受试者活性剂，目的在于逆转、缓解、改善、减缓或预防症状、并发症或病症的发作、进展、严重性或复发，或与疾病相关的生化指标。
“程序性死亡受体-1(PD-1)”是指属于CD28家族的免疫抑制性受体。PD-1主要在体内先前活化的T细胞上表达，并且结合两种配体PD-L1和PD-L2。本文使用的术语“PD-1”包括人PD-1(hPD-1)、hPD-1的变体、同种型和物种同源物，以及与hPD-1具有至少一个共同表位的类似物。
药物或治疗剂的“治疗有效量”或“治疗有效剂量”是当单独使用或与另一种治疗剂组合使用时保护受试者免于疾病发作或促进疾病消退的任何量的药物，所述疾病消退通过疾病症状的严重性的降低，疾病无症状期的频率和持续时间的增加，或由疾病痛苦引起的损伤或失能的预防来证明。治疗剂促进疾病消退的能力可以使用本领域技术人员已知的多种方法来评价，比如在临床试验期间的人受试者中，在预测人类功效的动物模型系统中，或通过在体外测定法中测定所述药剂的活性。
药物治疗有效量包括“预防有效量”，即当单独或与抗肿瘤剂组合给与处于发展癌症风险的受试者或患有癌症复发的受试者时，抑制癌症的发展或复发的任何量的药物。
“生物治疗剂”是指在支持肿瘤维持和/或生长或抑制抗肿瘤免疫应答的任何生物途径中阻断配体/受体信号传导的生物分子，例如抗体或融合蛋白。
除非另有明确表示，否则本文所用的“CDR”是指免疫球蛋白可变区是使用Kabat编号系统定义的互补决定区。
“抗癌剂”是指可用于治疗癌症的任何治疗剂。抗癌剂的类别包括但不限于：烷化剂、抗代谢药、激酶抑制剂、纺锤体毒剂植物碱、细胞毒性/抗肿瘤抗生素、光敏化剂、抗雌激素及选择性雌激素受体调节剂、抗孕酮剂、芳香酶抑制剂、CDK4/6抑制剂、FGF/FGFR抑制剂等，及抑制参与异常细胞增殖或肿瘤生长的基因表达的反义寡核苷酸。
术语“约”是指如本领域普通技术人员所确定的特定值或组成的可接受误差范围内的值或组成，其部分取决于如何测量或确定所述值或组成，即测量系统的限制。比如，“约”可以是指根据本领域中的实践在1个或大于1个标准偏差内。或者，“约”可以是指多达10％或20％的范围(即，±10％或±20％)。例如，约3mg/kg可包括2.7mg/kg和3.3mg/kg之间(相对于10％)，和2.4mg/kg和3.6mg/kg之间(相对于20％)的任何数目。在本文中，提供特定值或组成时，除非另有明确说明，“约”的含义应假定设为该特定值或组成的可接受误差范围内。
“治疗性抗PD-1单克隆抗体”是指特异性结合至在某些哺乳动物细胞表面上表达的特定PD-1的成熟形式的抗体。成熟的PD-1无前分泌前导序列，或叫前导肽。术语“PD-1”及“成熟的PD-1”在本文中可互换使用，且除非另有明确定义，或明确能从上下文看出，否则应理解为相同分子。
如本文所述，治疗性抗人PD-1抗体或抗hPD-1抗体是指特异性结合至成熟人PD-1的单克隆抗体。
本文所述的“框架区”或“FR”是指不包括CDR区的免疫球蛋白可变区。
“分离的抗体或其抗原结合片段”是指纯化状态且在该情况下所指定的分子实质上不含有其他生物分子，诸如核酸、蛋白质、脂质、碳水化合物或其他材料(诸如细胞碎片或生长培养基)。
“患者”、“病人”或“受试者”是指需要医疗方法或参与临床试验、流行病学研究或用作对照的任意单一受试者，包括人及哺乳动物，比如马、牛、狗或猫。
在以下段落中，进一步详细描述本发明的各个方面。
抗PD-1抗体
“PD-1抗体”是指结合PD-1受体，阻断表达于癌细胞上的PD-L1与表达于免疫细胞(T、B、NK细胞)上的PD-1结合且优选也能阻断表达于癌细胞上的PD-L2与表达于免疫细胞上的PD-1结合的任何化学化合物或生物分子。PD-1及其配体的替代名词或同义词包括：对于PD-1而言有PDCD1、PD1、CD279及SLEB2；对于PD-L1而言有PDCD1L1、PDL1、B7-H1、B7H1、B7-4、CD274及B7-H；且对于PD-L2而言有PDCD1L2、PDL2、B7-DC及CD273。在治疗人个体的任何本发明治疗方法、药物及用途中，PD-1抗体阻断人PD-L1与人PD-1的结合，且优选阻断人PD-L1和PD-L2二者与人PD1结合。人PD-1氨基酸序列可见于NCBI基因座编号：NP_005009。人PD-L1及PD-L2氨基酸序列可分别见于NCBI基因座编号：NP_054862及NP_079515。
可用于本发明所述的任何用途、疗法、药物及试剂盒的抗PD-1抗体包括单克隆抗体(mAb)或其抗原结合片段，其特异性结合至PD-1，且优选特异性结合至人PD-1。mAb可以为人抗体、人源化抗体或嵌合抗体，且可包括人恒定区。在一些实施方式中，恒定区是选自人IgG1、IgG2、IgG3及IgG4恒定区组成的组，且在优选实施方式中，恒定区是人IgG4恒定区。
在本发明所述的用途、疗法、药物及试剂盒的任意一个实施方式中，PD-1抗体是单克隆抗体或其抗原结合片段，其包含：(a)轻链CDR为SEQ ID NO：1、2和3所示的氨基酸，及重链CDR为SEQ ID NO：4、5和6所示的氨基酸。
在本发明所述的用途、疗法、药物及试剂盒的任意一个实施方式中，PD-1抗体是特异性结合至人PD-1且包含：(a)包含SEQ ID NO：7的轻链可变区，及(b)包含SEQ ID NO：8的重链可变区的单克隆抗体。
在本发明所述的用途、疗法、药物及试剂盒的任意一个实施方式中，PD-1抗体是特异性结合至人PD-1且包含：(a)包含SEQ ID NO：9的轻链，及(b)包含SEQ ID NO：10的重链的单克隆抗体。
在本发明所述的用途、疗法、药物及试剂盒的任意一个实施方式中，PD-1抗体是单克隆抗体或其抗原结合片段，下表A提供了用于本发明所述的用途、疗法、药物及试剂盒中的示例性抗PD-1抗体mAb的氨基酸序列的列表：
表A：示例性抗人PD-1抗体的轻重链CDR
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
结合至人PD-1且可用于本发明所述的用途、疗法、药物及试剂盒的抗PD-1抗体的实施例阐述于WO2014206107中。在本发明所述的用途、疗法、药物及试剂盒中可用作抗PD-1抗体的人PD-1mAb包括WO2014206107中描述的任意一个抗PD-1抗体，包括：特瑞普利单抗(特瑞普利单抗)(一种具有WHO Drug Information(第32卷，第2期，第372-373页(2018))中所述的结构且包含序列SEQ ID NO：9和10所示的轻链及重链氨基酸序列的人源化IgG4mAb)。在某些实施方案中，可用于本发明所述的用途、疗法、药物及试剂盒的抗PD-1抗体还包括FDA已经批准的纳武单抗和帕姆单抗，以及NMPA已经批准的信迪利单抗，以及尚在临床阶段的SHR-1210和Tislelizumab，以及尚在临床前及临床阶段的能够阻断表达于癌细胞上的PD-L1与表达于免疫细胞(T、B、NK细胞)上的PD-1结合且优选也能阻断表达于癌细胞上的PD-L2与表达于免疫细胞上的PD-1结合的任何化学化合物或生物分子。
在某些实施方案中，可用于本发明所述的用途、疗法、药物及试剂盒的抗PD-1抗体也包括特异性结合PD-L1以阻断PD-L1与PD-1结合的抗PD-L1单克隆抗体，如atezolizumab、avelumab、durvalumab、或任何能特异性结合PD-L1以阻断PD-L1与PD-1 结合的化学化合物或生物分子。
如本文所述的“PD-L1”表达或“PD-L2”表达是指细胞表面上的特定PD-L蛋白质或细胞或组织内的特定PD-L mRNA的任何可检测的表达水平。PD-L蛋白质表达可利用诊断性PD-L抗体在肿瘤组织切片的IHC分析中或通过流式细胞术检测。或者，肿瘤细胞的PD-L蛋白质表达可通过PET成像使用特异性结合至期望PD-L靶标(比如PD-L1或PD-L2)的结合剂检测。
用于在肿瘤组织切片的IHC分析中定量PD-L1蛋白质表达的方法，可参见以下但不限于Thompson,R.H.等人，PNAS 101(49)：17174-17179(2004)；Taube,J.M.等人，Sci Transl Med 4，127ra37(2012)；及Toplian,S.L.等人，New Eng.J.Med.366(26):2443-2454(2012)等。
一种方法采用PD-L1表达呈阳性或阴性的简单二元终点，其中PD-L1表达阳性结果用显示细胞表面膜染色的组织学证据的肿瘤细胞百分比来定义。将肿瘤组织切片计数为总肿瘤细胞的至少1％定义为PD-L1表达呈阳性。
在另一方法中，在肿瘤细胞中以及在浸润免疫细胞中定量肿瘤组织切片中的PD-L1表达。将展现膜染色的肿瘤细胞及浸润免疫细胞的百分比单独的定量为<1％、1％至50％，及随后的50％直至100％。对于肿瘤细胞，若评分<1％，则将PD-L1表达计数为阴性，若评分≥1％则为阳性。
在本发明的一些实施方式中，受试者的肿瘤组织切片中的PD-L1表达≥1％，与受试者的肿瘤组织切片中的PD-L1表达<1％相比，并未取得较好的获益。在一些优选的实施方式中，所述的癌症为实体瘤。在一些具体的实施方式中，所述的癌症为食管癌。作为一种优选的实施方式，所述的癌症为ESCC。
如本文所述的“RECIST 1.1疗效标准”是指Eisenhauver等人、E.A.等人，Eur.J Cancer45:228-247(2009)基于所测量反应的背景针对靶标损伤或非靶标损伤所述的定义。
术语“ECOG”评分标准，是从患者的体力来了解其一般健康状况和对治疗耐受能力的指标。ECOG体力状况评分标准记分：0分、1分、2分、3分、4分和5分。评分为0是指活动能力完全正常，与起病前活动能力无任何差异。评分为1是指能自由走动及从事轻体力活动，包括一般家务或办公室工作，但不能从事较重的体力活动。
“持续应答”是指在停止用本文所述治疗剂或组合疗法后的持续治疗效应。在一些实施方式中，持续应答具有至少与治疗持续时间相同或为治疗持续时间的至少1.5,2.0,2.5或3倍的持续时间。
“组织切片”是指组织样品的单一部分或片，比如从正常组织或肿瘤的样品切割的组织薄片。
本文所述的“治疗”癌症是指向患有癌症或经诊断患有癌症的受试者采用本文所述治疗方案(如施用抗PD-1抗体，或施用抗PD-1抗体与CDK4/6抑制剂或FGF/FGFR抑制剂的组合疗法)以达到至少一种阳性治疗效果(比如，癌症细胞数目减少、肿瘤体积减小、癌细胞浸润至周边器官的速率降低或肿瘤转移或肿瘤生长的速率降低)。癌症中的阳性治疗效果可以多种方式测量(参见W.A.Weber,J.Nucl.Med.,50:1S-10S(2009))。比如，关于肿瘤生长抑制，根据NCI标准，T/C≦42％是抗肿瘤活性的最小水平。认为T/C(％)＝经治疗肿瘤体积中值/对照肿瘤体积中值×100。在一些实施方式中，通过本发明的组合达到的治疗效果是PR、CR、OR、PFS、DFS及OS中的任一个。PFS(也叫“至肿瘤进展的时间”)是指治疗期间及之后癌症不生长的时间长度，且包括患者经历CR或PR的时间量以及患者经历SD的时间量。DFS是指治疗期间及之后患者仍无疾病的时间长度。OS是指与初始或未经治疗的个体或患者相比预期寿命的延长。在一些实施方式中，对本发明组合的应答是PR、CR、PFS、DFS、OR或OS中的任一个，其使用RECIST 1.1疗效标准评定。有效治疗癌症患者的本发明组合的治疗方案可根据多种因素(比如患者的疾病状态、年龄、体重及疗法激发受试者的抗癌反应的能力)而变。尽管本发明的实施方式可不在每个受试者中达到有效的阳性治疗效果，但在统计学上显著数目的受试者中应有效并达到了阳性治疗效果。
术语“给药方式”、“给药方案”可互换使用，是指本发明组合中每一治疗剂的使用剂量及时间。
“肿瘤”在应用于经诊断患有或怀疑患有癌症的受试者时是指任何大小的恶性或潜在恶性的赘生物或组织块，且包括原发性肿瘤及继发性赘生物。实体瘤通常不含有囊肿或液体区域的组织的异常生长或块。不同类型的实体瘤针对形成其的细胞类型来命名。实体瘤的例子有肉瘤、癌及淋巴瘤。血液癌通常不会形成实体瘤。
“肿瘤负荷”是指分布于整个体内的肿瘤物质的总量。肿瘤负荷是指整个体内的癌细胞的总数目或肿瘤的总大小。肿瘤负荷可通过现有技术中已知的多种方法测定，比如在肿瘤自受试者移除后使用卡尺、或在体内时使用成像技术(比如超声、骨扫描、计算层析X射线照相术(CT)或磁共振成像(MRI)扫描)测量其尺寸。
术语“肿瘤大小”是指肿瘤的总大小，其可测量为肿瘤的长度及宽度。肿瘤大小可通过现有技术中已知的多种方法测定，例如在肿瘤自受试者移除后使用卡尺、或在体内时使用成像技术(比如骨扫描、超声、CT或MRI扫描)测量其尺寸。
术语“肿瘤突变负荷(TMB)”是指每百万碱基中被检测出的，体细胞基因编码错误、碱基替换、基因插入或缺失错误的总数。在本发明的一些实施方式中，肿瘤突变负荷(TMB)是通过分析体细胞突变(包括编码基置换和研究的面板序列的巨碱基插入)来估计的。在本 发明的一些实施方式中，受试者的肿瘤突变负荷(TMB)大于等于12个突变/Mb，与受试者肿瘤突变负荷TMB<12个突变/Mb相比，并未取得较好的获益。在一些优选的实施方式中，所述的癌症为实体瘤。在一些具体的实施方式中，所述的癌症为食管癌。作为一种优选的实施方式，所述的癌症为ESCC。
术语“基因扩增”是指某特异蛋白质编码的基因的拷贝数选择性地增加而其他基因并未按比例增加的过程。在自然条件下，基因扩增是通过从染色体切除基因的重复序列再在质粒中进行染色体外复制或通过将核糖体RNA的全部重复序列生成RNA转录物再转录生成原来DNA分子的额外拷贝而实现的。本发明中，一些实施例中公开了基因测序分析。本发明的一些实施方式中，本发明所述的受试者中具有某些独特的基因扩增。在一些优选实施方式中，所述受试者具有染色体11q13区的基因扩增。在更进一步的一些优选实施方式中，所述受试者具有CDK4/6基因扩增；在更进一步的一些优选实施方式中，所述受试者具有FGF3/4/19基因扩增。作为一种优选的实施方式，所述食管癌受试者中具有CDK4/6基因扩增。作为一种优选的实施方式，所述食管癌受试者中具有FGF3/4/19基因扩增。在一些实施方式中，所述的肿瘤患者具有染色体11q13区的基因扩增，预示着其可用(a)单独施用CDK4/6抑制剂和/或FGF/FGFR抑制剂，或(b)组合施用抗PD-1抗体或其抗原结合片段与CDK4/6抑制剂和/或FGF/FGFR抑制剂中的一种或几种的组合进行治疗的疗效更好。在一些实施方式中，所述的肿瘤患者不具有染色体11q13区的基因扩增，预示着其可用(a)单独施用抗PD-1抗体或其抗原结合片段，或(b)组合施用抗PD-1抗体或其抗原结合片段与CDK4/6抑制剂和/或FGF/FGFR抑制剂中的一种或几种的组合进行治疗的疗效更好。
术语“CDK”即周期蛋白依赖性激酶(cyclin-dependent kinases)，是一组丝氨酸/苏氨酸蛋白激酶，CDK通过和周期蛋白cyclin协同作用对丝氨酸/苏氨酸蛋白的磷酸化驱动细胞周期，是细胞周期调控中的重要因子。CDK家族有CDK 1-8等8种，每种CDK结合不同类型的cyclin形成复合物，调节细胞从G1期过渡到S期或G2期过渡到M期以及退出M期的进程。目前FDA已经批准上市的CDK4/6抑制剂主要有瑞博西尼、帕博西尼，而处于临床研究阶段的CDK4/6抑制剂有几十种。
术语“FGF”即成纤维细胞生长因子(Fibroblast growth factor)蛋白类家族，该家族成员共有23个，FGF1-23。根据FGFs的不同作用机制可将其分为三类：内分泌(FGF15/19/21/23)、旁分泌(FGF1-10、FGF16-18、FGF20、FGF22)和胞分泌FGF11/12/13/14。旁分泌FGFs调节生物活性的过程是利用HS作为辅助因子，特异性结合细胞表面的FGF受体(Fibroblast Growth Factor Receptor，FGFRs)。而FGFRs主要包括4种：FGFR1、FGFR2、FGFR3、FGFR4。目前尚未有FGF/FGFR抑制剂上市，勃林格英格汉的 VEGFR/PDGFR/FGFR抑制剂nintedanib用于治疗肝癌、非小细胞肺癌、特发性纤维化于2014年获得了FDA的突破性药物资格，而在国内目前有德立替尼处于临床阶段。
术语“免疫组化(IHC)”是指利用抗原与抗体特异性结合的原理，通过化学反应使标记抗体的显色剂(荧光素、酶、金属离子、同位素)显色来确定组织细胞内抗原(多肽和蛋白质)，并对其进行定位、定性及相对定量的研究的方法。本发明的一些实施方式中，在利用抗PD-1抗体治疗之前，对受试者的肿瘤组织样品进行PD-L1表达检测，所述检测使用罗氏的抗人PD-L1抗体SP142(Cat No：M4422)进行染色实验。在一些实施方式中，肿瘤细胞的膜染色强度≥1％被定义为PD-L1阳性。
药物组合物及剂量
本发明所述的治疗剂可以构成药物组合物，比如含有本文所述抗PD-1抗体或/和该抗PD-1抗体以外的其他抗癌剂以及其他药学上可接受的载体的药物组合物。如本发明所述，“药学上可接受的载体”包括生理上相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗和吸收延迟剂等。优选的，适用于含有抗PD-1抗体的组合物的载体适合于静脉内、肌内、皮下、胃肠外、脊椎或表皮施用，比如通过注射或输注，而用于含有其他抗癌剂的组合物的载体适合于胃肠外施用，比如口服。本发明的药物组合物可以含有一种或多种药学上可接受的盐、抗氧化剂、水、非水载体、和/或佐剂比如防腐剂、润湿剂、乳化剂和分散剂。
调整给药方案以提供最佳所需的反应，比如最大治疗反应和/或最小不良效果。对于抗PD-1抗体，包括与另一抗癌剂组合时的施用，剂量范围可为约0.01至约20mg/kg，约0.1至约10mg/kg个体体重，或120mg、240mg、360mg、480mg固定剂量。例如，剂量可以是约0.1，约0.3，约1，约2，约3，约5，或约10mg/kg个体体重。通常设计给药方案以实现这样的暴露，其导致基于Ab的典型药代动力学特性的持续受体占用(RO)。代表性的给药方案可能为约每周一次，约每两周一次，约每三周一次，约每四周一次，约一个月一次，或更长一次施用。在一些实施方式中，约每两周一次向个体施用抗PD-1抗体。
其他抗癌剂的给药时间表针对不同的药物而有所不同。
本发明的一些具体实施方式中，所述的CDK4/6抑制剂或FGF/FGFR抑制剂的给药时间表针对不同的亚型而变化。对于抗PD-1抗体与CDK4/6抑制剂或FGF/FGFR抑制剂的组合治疗，在一些实施方式中，将CDK4/6抑制剂或FGF/FGFR抑制剂以其批准或推荐的剂量施用，继续治疗，直到观察到临床效果或直到不可接受的毒性或疾病进展发生。
本发明的方法
本发明涉及治疗癌症患者的方法，包括向癌症患者(a)单独施用治疗有效量的抗PD-1抗体或其抗原结合片段，或(b)组合施用治疗有效量的抗PD-1抗体或其抗原结合片段和任选的除抗PD-1抗体和其抗原结合片段外的其他抗癌剂的一种或几种的组合。
在某些实施方案中，适用于本发明所述的(a)单独施用治疗有效量的抗PD-1抗体或其抗原结合片段，或(b)组合施用治疗有效量的抗PD-1抗体或其抗原结合片段和任选的除抗PD-1抗体和其抗原结合片段外的其他抗癌剂的一种或几种的组合治疗的癌症患者优选是未测出染色体11q13区基因扩增的癌症患者。因此，一些实施方案中，所述癌症治疗方法还包括对癌症患者进行测序的步骤。
在某些实施方案中，本发明涉及治疗癌症患者的方法，包括向癌症患者(a)单独施用治疗有效量的其他抗癌剂，或(b)组合施用治疗有效量的抗PD-1抗体或其抗原结合片段和任选的除抗PD-1抗体外的其他抗癌剂的一种或几种的组合。
在某些实施方案中，适用于本发明所述的(a)单独施用治疗有效量的其他抗癌剂，或(b)组合施用治疗有效量的抗PD-1抗体或其抗原结合片段和任选的除抗PD-1抗体外的其他抗癌剂的一种或几种的组合治疗的癌症患者优选是测出染色体11q13区基因扩增、尤其是具有CDK4/6基因扩增或具有FGF3/4/19基因扩增的癌症患者。因此，一些实施方案中，所述癌症治疗方法还包括对癌症患者进行测序的步骤。
在某些实施方案中，所述的其他抗癌剂为治疗有效量的CDK4/6抑制剂和/或FGF/FGFR抑制剂。
在某些实施方案中，本发明提供治疗患有食管癌的个体/患者的方法。在一些实施方式中，所述方法包括向个体施用治疗有效剂量的以下组合：(a)作为特异性结合PD-1受体并抑制PD-1活性的Ab或其抗原结合片段；和(b)另一抗癌疗法。在一个实施方式中，所述的方法包括向个体施用以下有效量的组合：(i)本文其他部分公开的治疗食管癌的标准疗法，或(ii)其他抗癌剂。在一些实施方式中，所述的其他抗癌剂选自CDK4/6抑制剂和/或FGF/FGFR抑制剂。由于在发展中国家食管癌患者中最常见的食管癌组织学亚型是食管鳞状细胞癌(ESCC)，因此，在一些实施方式中，所述的食管癌为食管鳞状细胞癌(ESCC)。在某些实施方案中，所述方法还包括，在给予治疗药物之前，对患者进行测序的步骤；根据测序结果，对不具有染色体11q13区的扩增的个体，单独施用抗PD-1抗体或其抗原结合片段，任选地组合施用CDK4/6抑制剂、和/或FGF/FGFR抑制剂的一种或几种的组合；对具有染色体11q13区的扩增、尤其是具有CDK4/6基因扩增或具有FGF3/4/19基因扩增的个体，单独施用CDK4/6抑制剂、和/或FGF/FGFR抑制剂的一种或几种的组合，任选地组合施用抗PD-1抗体或其抗原结合片段。
适用于本发明的方法中的抗PD-1抗体
适用于本发明的方法中的抗体PD-1抗体是以高特异性和亲和力结合PD-1，阻断PD-L1/2与PD-1的结合，并抑制PD-1信号转导达到的免疫抑制效果。在任何本文公开的治疗方法中，抗PD-1抗体包括结合PD-1受体并在抑制配体结合和上调免疫系统方面表现出类似完整Ab的功能特性的抗原结合部分或片段。在一些实施方式中，抗PD-1抗体或其抗原结合片段为与特瑞普利单抗交叉竞争结合人PD-1的抗PD-1抗体或其抗原结合片段。在其他的实施方式中，抗PD-1抗体或其抗原结合片段是嵌合、人源化或人Ab或其抗原结合片段。在用于治疗人个体的某些实施方式中，所述的Ab为人源化Ab。
在一些实施方式中，抗PD-1抗体或其抗原结合片段包含人IgG1或IgG4同种型的重链恒定区。在一些实施方式中，抗PD-1抗体或其抗原结合片段的IgG4重链恒定区的序列包含S228P突变，其用IgG1同种型抗体的相应位置处通常存在的脯氨酸残基替代铰链区中的丝氨酸残基。在包括施用抗PD-1抗体的本文描述的任何治疗方法的某些实施方式中，所述抗PD-1抗体是特瑞普利单抗。在一些实施方式中，所述的抗PD-1抗体选自WO2014206107中描述的人源化抗体38,39,41和48。在一些实施方案中，所述抗PD-1抗体如前文“抗PD-1抗体”部分所述。
适用于本发明的方法中的CDK4/6抑制剂
本发明用于治疗肿瘤的一些实施方式中，其他的抗癌剂，即除了抗PD-1抗体以外与抗PD-1抗体组合使用，或单独使用的抗癌剂是CDK4/6抑制剂。目前FDA已经批准上市的CDK4/6抑制剂主要有礼来公司的Verzenio(abemaciclib)，主要用于治疗接受内分泌疗法后疾病进展的激素受体(HR)阳性以及人类表皮生长因子受体2(HER2)阴性的晚期或转移性乳腺癌成年患者；以及诺华公司的Kisqali(Ribociclib，瑞博西尼)，其与芳香酶抑制剂联用可作为一线用药治疗HR阳性以及HER2阴性的绝经后晚期转移性乳腺癌女性患者；第三个是辉瑞公司的Ibrance(Palbociclib，帕博西尼)，其与来曲唑联用治疗雌激素受体(ER)阳性、人表皮生长因子受体2(HER2)阴性的绝经后妇女转移性乳腺癌。
适用于本发明的方法中的FGF/FGFR抑制剂
本发明用于治疗肿瘤的一些实施方式中，其他的抗癌剂，即除了抗PD-1抗体以外与抗PD-1抗体组合使用，或单独使用的抗癌剂是FGF/FGFR抑制剂。目前尚未有FGF/FGFR抑制剂上市，勃林格英格汉的VEGFR/PDGFR/FGFR抑制剂nintedanib用于治疗肝癌、非 小细胞肺癌、特发性纤维化于2014年获得了FDA的突破性药物资格，而在国内目前有德立替尼处于临床阶段。
用途、疗法、药物及试剂盒
本发明一方面，提供了用于治疗个体中癌症的方法，该方法包含向所述个体施用包含抗PD-1抗体或其抗原结合片段及CDK4/6抑制剂、FGF/FGFR抑制剂中的一种或多种的组合疗法。
组合疗法也可以包含一种或多种额外治疗剂。所述的额外治疗剂可以是除CDK4/6抑制剂、FGF/FGFR抑制剂以外的化学治疗剂或生物治疗剂。
根据标准医药实践，本发明的组合疗法中的每一个治疗剂均可单独施用或在药物组合物中施用，该药物组合物包含治疗剂及一种或多种药学上可接受的载体、赋形剂及稀释剂。
本发明的组合疗法中的每一个治疗剂可同时施用、并行施用或以任何次序依次施用。在组合疗法中的治疗剂以不同剂型施用，比如一种药物是片剂或胶囊且另一种药物是无菌液体，和/或以不同给药时间施用，比如化学治疗剂至少每日施用且生物治疗剂不频繁施用，比如每一周、或每两周或每三周一次。
在一些实施方式中，CDK4/6抑制剂、FGF/FGFR抑制剂是在施用抗PD-1抗体之前施用，而在另一些实施方式中，CDK4/6抑制剂、FGF/FGFR抑制剂是在施用抗PD-1抗体之后施用。
在一些实施方式中，在药物作为单一疗法用于治疗相同癌症时，组合疗法中的治疗剂中至少一种使用相同的给药方案(剂量、频次、治疗持续时间)施用。
本发明所述的组合疗法中的每一个小分子治疗剂可经口或胃肠外(比如静脉内、肌内、腹膜内、皮下、经直肠、局部或透皮施用途径)施用。
本发明所述的组合疗法可在手术之前或之后施用，且可在放疗之前、期间或之后施用。
在一些实施方式中，将本发明所述的组合疗法施用于先前未经生物治疗剂或化学治疗剂治疗的患者中。在另一些实施方式中，将组合疗法施用于用生物治疗剂或化学治疗剂治疗之后而不能达到持续响应的患者中。
本发明所述的组合疗法可用于治疗通过触诊或通过现有技术中已知的成像技术发现的肿瘤中，比如MRI、超声或CAT扫描。
本发明的组合疗法的给药方案的选择取决于若干的因素，包括但不限于血清或组织转化率、症状的程度、免疫原性及所治疗个体的靶标细胞、组织、器官的可达性。优选的，给药方案要配合可接受的副作用程度来给患者递送最大量的各种治疗剂。因此，组合疗法中的每一个生物治疗剂和化学治疗剂的剂量及给药频率取决于特定的治疗剂、所治疗的癌 症的严重程度及患者的特性。
本发明所述的抗PD-1抗体或其抗原结合片段和CDK4/6抑制剂、FGF/FGFR抑制剂中的一种或几种可作为包含第一容器及第二容器及包装插页的试剂盒提供。
第一容器含有至少一个剂量的包含抗PD-1抗体或其抗原结合片段的药物，第二容器含有至少一个剂量的包含CDK4/6抑制剂、FGF/FGFR抑制剂中的一种或几种的药物，且包装插页或标签包含使用药物治疗癌症的说明。试剂盒可进一步包含可用于施用药物的其他材料，比如稀释剂、滤纸、IV袋及线、针及注射器。
抗PD-1抗体对治疗癌症效果的预测方法
本发明所述的抗PD-1抗体或其抗原结合片段，尤其是特瑞普利单抗单独施用或组合施用对治疗个体中癌症的效果的预测方法，包括在治疗前对个体进行测序。在一些实施方式中，所述的癌症为食管癌；在另一些优选的实施方式中，所述的癌症是ESCC。
在一个实施方式中，本发明所述的预测方法为在治疗前对个体的基因检测，评价染色体11q13区的基因是否存在扩增。在一个实施方式中，所述的个体的染色体11q13区的基因无扩增。在另一个实施方式中，所述的个体的染色体11q13区的基因具有扩增。在一个实施方式中，所述的个体具有CCND1基因和/或FGF/FGFR基因扩增。在一个实施方式中，所述的个体具有CDK4/6基因和/或FGF3/4/19基因扩增。
本发明还包括利用染色体11q13区的基因预测肿瘤患者对抗PD-1抗体或其抗原结合片段单独或组合施用的治疗效果的方法。不存在染色体11q13区的基因扩增表明所述肿瘤患者适于用抗PD-1抗体或其抗原结合片段单独或组合施用进行治疗。
在某些实施方案中，本发明还提供生物标记物的检测试剂在制备预测抗PD-1抗体治疗癌症的效果用的试剂盒中的用途。这类试剂包括例如对个体基因中是否存在染色体11q13区的基因扩增进行检测的试剂。优选地，这类试剂包括例如对个体基因中是否存在CCND1基因和/或FGF/FGFR基因扩增进行检测的试剂。更优选地，这类试剂包括例如对个体基因中是否存在CDK4/6基因和/或FGF3/4/19基因扩增行检测的试剂。
本发明还包括检测染色体11q13区的基因的试剂在制备预测抗PD-1抗体治疗癌症的效果的试剂盒中的用途。这类试剂包括但不限于测试中常规使用到的试剂，包括但不限于引物、探针、PCR所需试剂等。
缩略语
贯穿于本发明的说明书及实施例中，使用以下缩略语：
BID 一个剂量，每日2次
CDR 互补决定区
FR 框架区
IgG 免疫球蛋白G
IHC 免疫组织化学
WES 全外显子组测序
PBMC 外周血单个核细胞
OR 总体应答
ORR 客观缓解率
OS 总生存
PD 疾病进展
PFS 无进展生存
DFS 无疾病生存
DCR 疾病控制率
PR 部分应答
CR 完全应答
SD 疾病稳定
DLT 剂量限制性毒性
MTD 最大耐受剂量
AE 不良事件
Q2W 每两周一个剂量
QD 每天一个剂量
TRAE 与治疗相关的不良反应
SAE 严重不良反应
TMB 肿瘤突变负荷
EC 食管癌
ESCC 食管鳞状细胞癌
LDH 乳酸脱氢酶
ECOG 东部肿瘤协作组
本发明通过以下实施例进一步阐述，但所述实施例不应被解释为限制本发明。整个申请中引用的所有参考文献的内容通过引用的方式明确并入本文。
实施例
实施例1：抗PD-1抗体单用治疗肿瘤的临床研究
入组标准：符合条件的受试者必须(1)年龄在18至75岁之间，(2)组织学和/或细胞学证实患有晚期和/或转移性ESCC，(3)晚期ESCC患者，必须已经接受过或至少接受过一种治疗仍有进展(包括但不限于化疗或放疗)，(4)ECOG评分为0或1。
受试者必须是按RECIST v 1.1标准有可评估病灶，不允许具有其他既往或并发恶性肿瘤，不允许具有任何活跃的自身免疫性疾病或自身免疫性疾病史，不允许具有需要长期的免疫抑制药物治疗的伴随性疾病，不允许既往接受过抗-CTLA4、抗PD-1、抗PD-L1抗体治疗，不允许具有活性乙肝或丙肝病毒感染，不允许怀孕或正在哺乳，不允许在入组前4周进行过抗肿瘤治疗、放疗或任何手术治疗。入组受试者的人口学统计数据如表1所示。
表1：入组受试者人口学统计数据
Figure PCTCN2020085046-appb-000001
说明：*阳性定义为用SP142IHC染色≥1％肿瘤细胞或免疫细胞；缩写词：ECOG，东部肿瘤协作组；LDH，乳酸脱氢酶。
受试药物：抗PD-1抗体特瑞普利单抗(特瑞普利单抗)(WO2014206107)。
本试验抗PD-1抗体剂量组为：本研究先进行3mg/kg剂量组研究。入组后，受试者将每2周(Q2W)接受一次治疗，每4周为一个周期，直至病情进展、发生无法忍受的毒性、受试者撤回同意书、研究者判断无进一步的获益，或死亡。
临床设计：
这是一个开放式、多中心临床Ib/II期试验，分为8个独立队列，本发明内容主要评估队列2的结果。用以评价抗PD-1抗体在治疗化疗难治性晚期ESCC中的安全性、耐受性、和抗肿瘤活性。
从2017.3.9至2017.8.24，共计纳入60名晚期ESCC受试者，其中59位受试者用特瑞普利单抗治疗，1位受试者在治疗前撤回同意书。
计划剂量为3mg/kg，Q2W。
1.1安全性研究：
截至2018.12.31，最后一位患者入组已经16个月，59例患者接受至少一剂特瑞普利单抗治疗，并纳入安全性分析。59例患者中有56例(94.9％)发生治疗相关不良事件(TRAE)，但大多数TRAE为1级或2级(表2)。常见的TRAEs包括体重减轻(18.6％)、贫血(18.6％)、食欲减少(18.6％)、发烧(16.9％)、咳嗽(16.9％)、白细胞减少(15.3％)、AST升高(13.6％)、甲状腺功能减退(13.6％)、ALT升高(11.9％)、疲劳(11.9％)、恶心(11.9％)、总胆红素增加(10.2％),、低血红蛋白计数(10.2％)和便秘(10.2％)(表2)。3级和更高的TRAEs在19位(32.2％)患者上发生。其中11位(18.6％)患者出现3级TRAEs，包括2位贫血、2位低钠血症、1例高血压、1例厌食症、1例吞咽困难、1例食管狭窄、1例疲劳、1例心包积水、1例高钙血症、1例白细胞减少、1例上呼吸道感染和1例颈部感染。2位(3.4％)患者出现4级TRAEs，包括1例高尿酸症，1例高位截瘫。与治疗相关的6例死亡均可能与治疗不相关，包括1例恶病质、2例肺炎和3例病因不明。14例(23.7％)患者因TRAE而永久停药；6例(10.2％)患者因TRAE而出现剂量中断。可见，特瑞普利单抗在ESCC患者中具有可控的安全性。
表2：队列中常见的治疗相关不良反应(>10％)
Figure PCTCN2020085046-appb-000002
Figure PCTCN2020085046-appb-000003
1.2抗肿瘤活性研究：
至2018.12.31，受试者中67.8％(40/59)的患者已经死亡。5.1％(3/59)随访中断，11.9％(7/59)撤回同意，74.5％(44/59)停止治疗，8.5％(5/59)仍在研究中。
中位治疗时间是3.5个月(0.1-19.1个月之间)。
至数据截止日期，研究者采用RECIST v1.1评估，59例患者中共有1例CR，10例PR(包括2例未确诊PR)和17例SD。最佳应答率为18.6％(95％CI 9.7 to 30.9)，DCR为47.5％(95％CI 34.3 to 60.9)。CR患者既往接受过1线化疗，未接受过放疗。10位PR患者既往接受过放疗，其中4位还接受过2种先前全身化疗，其他6位接受过3种或4种先前化疗。
无进展生存率和总体活率
确认的ORR是15.3％(95％CI 7.2 to 27.0)。平均响应时间是1.8个月。中位响应持续时间为11.2个月。在25例(42.4％)受试者中观察到相对于基线的靶病灶任意大小的减小(图1A和1B)。中位PFS是2.1个月，中位OS是6.9个月(图1C和1D)。
实施例2：PD-L1表达与抗PD-1抗体治疗肿瘤疗效对应关系研究
在实施例1所述的临床试验的特瑞普利单抗治疗之前，对受试者进行了存档或新鲜的肿瘤活检。采用免疫组化(IHC)方法检测福尔马林固定石蜡包埋(FFPE)肿瘤组织样本中的PD-L1，并在临床中心实验室(Q2实验室，北京)的VENTANA Benchmark Ultra系统上进行验证。采用Spring Bioscience(Roche)兔抗人PD-L1单克隆抗体(克隆SP142,Cat No: M4422)进行PD-L1免疫组化检测。由认证病理学家利用染色的肿瘤组织评估肿瘤细胞(TC)和肿瘤浸润免疫细胞(IC)上PD-L1的表达。PD-L1阳性状态被定义为存在膜染色的强度≥1％的肿瘤细胞或存在PD-L1染色强度的肿瘤浸润免疫细胞覆盖≥1％的被肿瘤细胞占据,肿瘤内相关,连续癌周基质肿瘤区域。
对取自实施例1中的57位受试者的肿瘤活检样本进行PD-L1表达测定。鉴定出19个(33.3％)PD-L1表达阳性和38个(66.7％)PD-L1表达阴性样本(图2A)。
PD-L1阳性被定义为采用SP142IHC染色，具有≥1％的肿瘤细胞(TC)或免疫细胞(IC)阳性。
在本研究中，PD-L1阳性患者与PD-L1阴性受试者在ORR(15.8％vs.18.4％)或OS(6.7月vs.6.9月)中并无显著差异(图2A、2C、2D)。而PD-L1阳性受试者的中位PFS在数值上优于PD-L1阴性受试者(3.4个月vs.2.0个月)，但该差异并无统计学意义(p＝0.85)(图2C)。
本研究的肿瘤组织活检中，仅有5位(8.8％)受试者具有超过5％的PD-L1阳性表达(图2A)。PD-L1表达超过5％的受试者比PD-L1表达低或无表达的患者的临床响应更好，PD-L1阳性受试者的40％ORR和100％DCR相对阴性受试者的15.4％ORR和42.3％DCR。ORR差异无统计学意义(p＝0.21)，DCR差异有统计学意义(p＝0.019)。
实施例3：肿瘤突变负担(TMB)与抗PD-1抗体治疗肿瘤疗效对应关系研究
在实施例1所述的临床试验过程中，对取自50位受试者的肿瘤活检样本进行全外显子组测序(WES)。TMB是通过分析人类基因组编码区域内的体细胞突变来确定的。47例患者中获得了有效TMB结果(表3)。
本研究中，ESCC患者的TMB普遍较低。没有一个活检样本具有每百万碱基对突变超过20个，并且只有13个活检样本具有每百万碱基对突变10-20个。
将每个Mb具有12个突变作为截止值，TMB大于等于12个突变/Mb(n＝11)的患者与TMB小于12个突变/Mb(n＝36)的患者具有相似的ORR(18.2％比19.4％)(图2B)。TMB高(≥12)的组比TMB低(＜12)的组在数值上具有更好的PFS(4.0个月vs.1.9个月)和OS(11.5个月vs.6.7个月)，尽管这种区别并无统计学意义(图2E和2F)。
表3：TMB与抗PD-1抗体治疗肿瘤疗效对应关系
Figure PCTCN2020085046-appb-000004
Figure PCTCN2020085046-appb-000005
Figure PCTCN2020085046-appb-000006
实施例4：抗PD-1治疗的突变全景及其与临床疗效对应关系
4.1全外显子测序(WES)和数据分析
收集了肿瘤组织和与之匹配的PBMC样本，苏木精的4μm部分和曙红-染色的FFPE样本进行了病理学检查，确保每个样本均具有核细胞性大于80％，肿瘤含量不少于20％。使用足够的未染色FFPE切片提取不少于200ng的DNA。大约从与通常生成1～5μg DNA匹配的200μl PBMC匹配作为参照。
文库制备采用超声破碎双链DNA至250bp。随后采用SureSelect XT库准备工具包，或KAPA Hyper预备包(KAPA生物系统公司)进行末端修复、dA添加和适配器连接的文库构建，然后进行PCR扩增，并通过Qubit评估进行量化。按照协议杂交捕获目标区域并制备库。序列读取是使用Illumina NovaSeq 6000(Illumina Inc.，San Diego,CA)生成的。对数据质量进行检查和控制，并建立一套定制的生物信息学管道，用于发现SNV、长和 短插入和缺失、CNA、基因重排、TMB和MSI。最后，所有检测到的突变都对照我们的内部注释数据库进行临床治疗。根据注释的突变，将生成一份基于临床试验和文献的简明报告.
4.2体细胞信噪比、短缩进和拷贝数改变的识别(SCNAs)
利用Burrows-Wheeler测序仪对原始读序列与人类基因组参考序列(hg19)进行比对，然后使用Picard标记复制算法去除PCR重复序列。SNV和S-indel的原始调用由内部算法调用。支持可选调用至少需要5次读取。去除读取深度小于50x、链偏置小于或大于90％、VAF<1％的变异。一般SNPs定义为来自dbSNP数据库(147版)或Exome测序项目6500(ESP6500)中超过1.5％的频率或1000个基因组项目中超过1.5％的频率也被排除在外，以供进一步考虑。来自测序错误或群体的产品也被排除在外。
为了定义SCNA事件，对每个外显子区域内的对齐读取进行归一化，然后使用CNVKIT(version 0.9.1)对结果进行GC含量和参考基因组可映射性的进一步校正。利用循环二值分割算法对得到的复制率进行分割。按照ASCAT的方法，用超过4000个测序SNPs(单核苷酸多态性)的等位基因频率估计畸变肿瘤细胞的比例。经深度归一化、GC、可映射性校正，计算肿瘤组织与匹配的正常血样的拷贝率。如果log2(拷贝率)超过0.8或不超过-1，则分别认为片段被放大或删除。
WES的基因组图谱显示：本研究中，TP53(p53)(76％)、RYR2(22％)、NOTCH1(20％)、LRP1B(17％)和TRIO(17％)的错义突变或截断是ESCC肿瘤活检的前五大突变基因(图3)。但这五个基因的错义突变或截断或其他功能突变的丧失均与临床响应无关。
实施例5：11q13基因扩增与抗PD-1治疗耐药的关系
采用以下方法进行全转录组测序(RNA序列)。使用miRNeasy试剂盒(cat#217504,Qiagen公司)从未染色的FFPE部分提取RNA，然后使用
Figure PCTCN2020085046-appb-000007
rRNA消耗试剂盒(cat#E6310L,新英格兰生物实验室)进一步处理以删除rRNA。分别使用M-MLV RT RNase(H-)(cat#M3683,Promega公司)和NEB第二链mRNA合成试剂盒(cat#E6111L,新英格兰生物实验室)合成第一链和第二链cDNA。对cDNA产物进行超声处理产生～200bp的片段(Covaris E220)。适配器连接的库是由cDNA使用KAPA Hyper制备试剂盒(cat#07962363001,罗氏)创建，在NovaSeq 5000/6000平台上测序。每个注释转录本的相对丰度以百万分之转录量(TPM)和分析前的log2转化表示。
实施例4的WES分析显示：50例活检中有24例(48.0％)包含了染色体11q13区的扩增(图4A)。信使RNA表达分析进一步证实了基因组DNA扩增事件，mRNA水平与该区域相应基因的扩增状态相关，包括CCND1(Cyclin D1)和FGF家族成员(FGF3/4/19)(图 4B)。重要的是，无11q13扩增的患者(n＝26)比11q13扩增的患者(n＝24)具有统计学意义的更好的客观响应率(30.8％相比4.2％，p＝0.024)(图5A)和更长的PFS(3.7个月相比1.0个月；HR＝0.47[95％CI 0.24到0.91]，p＝0.025)。无11q13扩增的患者也具有更长的中位OS(11.5个月相比5.6个月；HR＝0.60[95％CI 0.30到1.20])(图5C)。
实施例6：其他的生物标记物和亚组分析
其他的与临床效果相关的生物标记物或亚组分析包括：年龄、性别、ECOG得分、前期治疗、基线时肝转移的存在、肿瘤体积、基线血清LDH水平。其中，ECOG得分为0的、低基线肿瘤体积和基线LDH水平高于正常值的上限的病人具有数字上更好的ORR。但是这些区别均无统计学意义(表4)。
表4：其他的标记物及亚组与临床疗效的相关性
Figure PCTCN2020085046-appb-000008
实施例7：抗PD-1抗体联合CDK4/6抑制剂对小鼠肿瘤生长的抑制作用研究
将CT-26细胞以2.5×10 5个/0.1mL浓度接种于hPD-1人源化雌性小鼠的右侧皮下，待肿瘤生长到约93mm 3时按肿瘤体积随机分组，每组7只，共4组，分别为：
G1：Anti-KLH hIgG4(1mg/kg)阴性对照组；
G2：toripalimab(特瑞普利单抗)(1mg/kg)组；
G3：帕博西尼(100mg/kg)；
G4：toripalimab(1mg/kg)联合帕博西尼(100mg/kg)组。
Anti-KLH hIgG4和toripalimab给药途径均为腹腔注射，每周给药2次，连续给药3次，帕博西尼为口服给药，每天给药1次。首次给药后11天结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。实验结束时，动物安乐死，计算肿瘤体积抑制率(TGI Tv％)。
结果如表5、图7所示。表明受试药物帕博西尼对肿瘤生长有一定抑制作用；与toripalimab(1mg/kg)、帕博西尼(100mg/kg)单药相比，toripalimab(1mg/kg)联合帕博西尼(100mg/kg)给药具有更显著抑制肿瘤生长的作用。
表5：toripalimab联合RT391对CT-26细胞移植B-hPD-1小鼠肿瘤体积的影响
Figure PCTCN2020085046-appb-000009
注：a：平均数±标准误；b：给药组肿瘤体积与Anti-KLH hIgG4对照组肿瘤体积在给药11天后统计学比较，t-test。

Claims (21)

  1. 抗PD-1抗体和/或其抗原结合片段单独，或抗PD-1抗体和/或其抗原结合片段与除抗PD-1抗体以外的抗癌剂组合在制备治疗实体瘤的药物中的用途，所述实体瘤不具有染色体11q13区的基因扩增。
  2. 如权利要求1所述的用途，其特征在于，所述抗癌剂为小分子靶向抗癌剂。
  3. 如权利要求1所述的用途，其特征在于，所述抗癌剂选自CDK4/6抑制剂、FGF/FGFR抑制剂中的一种或多种；优选地，所述CDK4/6抑制剂选自瑞博西尼和帕博西尼。
  4. CDK4/6抑制剂、FGF/FGFR抑制剂中的一种或多种单独，或与抗PD-1抗体和/或其抗原结合片段组合在制备治疗实体瘤的药物中的用途，所述实体瘤具有染色体11q13区的基因扩增；优选地，所述CDK4/6抑制剂选自瑞博西尼和帕博西尼。
  5. 如权利要求1-4任一项所述的用途，其特征在于，所述抗PD-1抗体是单克隆抗体或其抗原结合片段，其特异性结合PD-1，且阻断PD-L1与PD-1结合。
  6. 如权利要求5所述的用途，其特征在于，所述抗PD-1抗体的轻链互补决定区氨基酸序列如SEQ ID NO：1、2和3所示，重链互补决定区氨基酸序列如SEQ ID NO：4、5和6所示。
  7. 如权利要求5所述的用途，其特征在于，所述抗PD-1抗体的轻链可变区氨基酸序列如SEQ ID NO：7所示，重链可变区氨基酸序列如SEQ ID NO：8所示。
  8. 如权利要求5所述的用途，其特征在于，所述抗PD-1抗体的轻链包含SEQ ID NO:9所示的氨基酸序列、重链包含SEQ ID NO：10所示的氨基酸序列。
  9. 如权利要求5所述的用途，其特征在于，所述抗PD-1抗体选自纳武单抗、帕姆单抗、特瑞普利单抗、信迪利单抗、卡瑞利珠单抗和替雷利珠单抗或其组合。
  10. 如权利要求1-9任一项所述的用途，其特征在于，所述抗PD-1抗体或其抗原结合片段的施用剂量为约0.1mg/kg至约10.0mg/kg个体体重，例如约0.1mg/kg，约0.3mg/kg，约1mg/kg、约2mg/kg、约3mg/kg、约5mg/kg，或10mg/kg个体体重，或选自约120mg至约480mg固定剂量，例如120mg、240mg、360mg或480mg固定剂量。
  11. 如权利要求10所述的用途，其特征在于，所述抗PD-1抗体或其抗原结合片段的给药频率为约每一周一次、每两周一次、每三周一次、每四周一次或一个月一次，优选为每两周一次。
  12. 如权利要求10所述的用途，其特征在于，所述抗PD-1抗体或其抗原结合片段的施用剂量为1mg/kg个体体重、3mg/kg个体体重、10mg/kg个体体重、或240mg固定剂量、480mg固定剂量，每两周一次施用。
  13. 如权利要求10所述的用途，其特征在于，所述抗PD-1抗体或其抗原结合片段以液体剂型例如注射剂，经胃肠外途径例如经静脉输注施用。
  14. 如权利要求10所述的用途，其特征在于，所述抗PD-1抗体或其抗原结合片段的给药周期为一周、二周、三周、一个月、两个月、三个月、四个月、五个月、半年或更长时间，任选地，每个给药周期的时间相同或不同，且每个给药周期之间的间隔相同或不同。
  15. 一种用于实体瘤治疗的方法，其包括在治疗前对个体进行测序的步骤，其特征在于，对不具有染色体11q13区扩增的个体单独施用抗PD-1抗体或其抗原结合片段，任选地组合施用CDK4/6抑制剂、和/或FGF/FGFR抑制剂的一种或几种的组合；对具有染色体11q13区扩增的个体单独施用CDK4/6抑制剂、和/或FGF/FGFR抑制剂的一种或几种的组合，任选地组合施用抗PD-1抗体或其抗原结合片段；优选地，所述CDK4/6抑制剂选自瑞博西尼和帕博西尼。
  16. 如权利要求1-14任一项所述的用途或权利要求15所述的方法，其特征在于，所述实体瘤为食管癌或结肠癌。
  17. 如权利要求16所述的用途或方法，其特征在于，所述食管癌为食管鳞状细胞癌。
  18. 用于检测染色体11q13区基因扩增的试剂在制备预测肿瘤病人对抗PD-1抗体和/或其抗原结合片段治疗的效果的检测试剂盒中的用途。
  19. 用于检测CDK4/6基因扩增的试剂在制备预测肿瘤病人对抗PD-1抗体和/或其抗原结合片段治疗的效果的检测试剂盒中的用途。
  20. 用于检测FGF/FGFR基因扩增的试剂在制备预测肿瘤病人对抗PD-1抗体和/或其抗原结合片段治疗的效果的检测试剂盒中的用途。
  21. 一种用于治疗患有癌症的个体的试剂盒，所述试剂盒包括：(a)抗PD-1的抗体或其抗原结合片段，和任选的除抗PD-1抗体或其抗原结合片段以外的其他抗癌剂；和(b)单独施用抗PD-1抗体或其抗原结合片段，或组合施用除抗PD-1抗体或其抗原结合片段以外的其他抗癌剂以治疗个体癌症的说明书；所述癌症为实体瘤；作为一种优选的实施方式，所述癌症包含但不局限于胃癌、食管癌、鼻咽癌、头颈部鳞状细胞癌、乳腺癌、膀胱癌和结肠癌；优选地，所述其他抗癌剂为CDK4/6抑制剂，更优选为瑞博西尼和/或帕博西尼。
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