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WO2019076277A1 - Utilisations d'un anticorps anti-pd-1 et d'un anticorps anti-lag-3 conjointement dans la préparation d'un médicament pour le traitement d'une tumeur - Google Patents

Utilisations d'un anticorps anti-pd-1 et d'un anticorps anti-lag-3 conjointement dans la préparation d'un médicament pour le traitement d'une tumeur Download PDF

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WO2019076277A1
WO2019076277A1 PCT/CN2018/110324 CN2018110324W WO2019076277A1 WO 2019076277 A1 WO2019076277 A1 WO 2019076277A1 CN 2018110324 W CN2018110324 W CN 2018110324W WO 2019076277 A1 WO2019076277 A1 WO 2019076277A1
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Prior art keywords
seq
variable region
chain variable
antibody
heavy chain
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PCT/CN2018/110324
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English (en)
Chinese (zh)
Inventor
黄晓辉
曹国庆
杨昌永
张连山
Original Assignee
江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
苏州盛迪亚生物医药有限公司
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Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司, 苏州盛迪亚生物医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to CN201880059078.7A priority Critical patent/CN111094339B/zh
Publication of WO2019076277A1 publication Critical patent/WO2019076277A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the use of an anti-PD-1 antibody or antigen-binding fragment thereof and a LAG-3 antibody or antigen-binding fragment thereof in combination for the preparation of a medicament for treating tumors and/or enhancing T cell activity.
  • LAG-3 lymphocyte activation gene-3
  • CD223 is a membrane protein of members of the immunoglobulin superfamily. It has been shown to be associated with various diseases such as immune diseases, tumors, Plasmodium infections, and hypersensitivity reactions. Related ([J]. Advances in Modern Biomedicine, 2014, 15: 047); and PD-1 (programmed death receptor-1) antibodies can specifically recognize and bind to PD-1 on the surface of lymphocytes, blocking PD-1 /PD-L1 signaling pathway, which in turn activates the immune killing effect of T cells on tumors, and modulates the immune system of the body to eliminate tumor cells in vivo. In some diseases, LAG-3 expression is elevated and corresponding immunosuppression occurs.
  • Patent application WO2015042246, WO2015048312, and WO2016196560 disclose an anti-LAG antibody and an anti-PD-1 or anti-PD-L1 antibody in combination for treating malignant tumors such as melanoma, non-small cell lung cancer, hematological tumor, etc., and administration methods, and the like;
  • WO2016110593 discloses A composition comprising a LAG-3 protein selected from BMS-936559, MED14736, and the like, and a PD-1 pathway inhibitor, for treating a plurality of tumors;
  • WO2017025498 discloses a bispecific fusion polypeptide of LGA-3 and PD-1 capable of Co-stimulation of T cell responses.
  • WO2015085847 and WO2017054646 disclose a novel PD-1 antibody and its preparation.
  • the anti-PD-1 antibody provided by the invention is in the clinical phase I of China, and has good safety.
  • the clinical research results reported have shown that it has certain anti-tumor effects ([J].Journal of Clinical Oncology 35(2017): E15572-e15572)
  • the present invention provides the use of an anti-PD-1 antibody or antigen-binding fragment thereof and an anti-LAG-3 antibody or antigen-binding fragment thereof in combination for the preparation of a medicament for treating tumors and/or enhancing T cell activity, or
  • the heavy chain variable region and the light chain variable region of the antigen-binding fragment thereof comprise the sequence of the CDR regions of (i) or (ii) below:
  • each of the aforementioned CDR sequences is as follows:
  • the enhanced T cell activity includes, but is not limited to, restoring T cell function or stimulating T cell proliferation.
  • the LAG-3 antibody or antigen-binding fragment thereof according to the present invention wherein the antibody or antigen-binding fragment thereof is a LAG-3 humanized antibody or antigen-binding thereof, is provided Fragment.
  • the LAG-3 antibody or antigen-binding fragment thereof according to the present invention, wherein the heavy chain FR region sequence on the heavy chain variable region of the humanized antibody is derived Human germline heavy chain, combination sequence of IGHV7-4-1*02 and hjh6.1 or its mutant sequence; it comprises FR1, FR2, FR3 region and hjh6.1 of human germline heavy chain IGHV7-4-1*02 FR4 region or a mutated sequence thereof; preferably, wherein the humanized antibody heavy chain FR region sequence has a back mutation of 0-10 amino acids, more preferably one or more selected from the group consisting of E46K, R38K, V93T and Y95F Amino acid back mutation.
  • the humanized LAG-3 antibody or antigen-binding fragment thereof according to the present invention, wherein the humanized antibody heavy chain variable region sequence is SEQ ID NO: 21
  • the humanized LAG-3 antibody or antigen-binding fragment thereof according to the present invention wherein the humanized antibody heavy chain variable region comprises SEQ ID NO: 21, The sequence set forth in SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25, or an amino acid sequence having at least 85% (preferably 95%) sequence identity thereto.
  • the humanized LAG-3 antibody or antigen-binding fragment thereof according to the present invention, wherein the heavy chain FR region on the heavy chain variable region of the humanized antibody is provided
  • the sequence is derived from the combined sequence of human germline heavy chain IGHV1-3*01 and hjh6.1 and its mutated sequence; it comprises FR1, FR2, FR3 region of human germline heavy chain IGHV1-3*01 and FR4 of hjh6.1 a region or a mutated sequence thereof; wherein the humanized antibody heavy chain FR region sequence has a back mutation of 0-10 amino acids, more preferably one or more selected from the group consisting of F29L, A97T, M48I, V68A, I70L, R72V and Amino acid back mutation of T74K.
  • the humanized LAG-3 antibody or antigen-binding fragment thereof according to the present invention, wherein the humanized antibody heavy chain variable region sequence is SEQ ID NO: 29
  • the humanized LAG-3 antibody or antigen-binding fragment thereof according to the present invention wherein the humanized antibody heavy chain variable region sequence is selected from the group consisting of SEQ ID NO: 29.
  • SEQ ID NO: 29 A sequence as set forth in SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33, or an amino acid sequence having at least 85% (preferably 95%) sequence identity thereto.
  • the humanized LAG-3 antibody or antigen-binding fragment thereof according to the present invention wherein the light chain FR region on the light chain variable region of the humanized antibody is provided
  • the sequence is derived from the combined sequence of human germline light chain template IGKV1-39*01 and hjk4.1 and its mutant sequence; it comprises FR1, FR2, FR3 region and hjk4.1 of human germline light chain IGKV1-39*01 FR4 region and its mutant sequence.
  • the humanized LAG-3 antibody or antigen-binding fragment thereof according to the present invention, wherein the humanized antibody variable region light chain sequence is SEQ ID NO: 22
  • a humanized LAG-3 antibody or antigen-binding fragment thereof according to the present invention, wherein the humanized antibody light chain FR region sequence has 0-10 amino acids
  • the back mutation is preferably one or more amino acid back mutations selected from the group consisting of D70Q, F71Y, I48V and A43S.
  • the humanized LAG-3 antibody or antigen-binding fragment thereof according to the present invention wherein the humanized antibody light chain variable region sequence is selected from the group consisting of SEQ ID NO: 22.
  • the humanized LAG-3 antibody or antigen-binding fragment thereof according to the present invention, wherein the humanized antibody variable region light chain sequence is SEQ ID NO: 30
  • a humanized LAG-3 antibody or antigen-binding fragment thereof according to the present invention, wherein the humanized antibody light chain FR region sequence has 0-10 amino acids
  • the back mutation is preferably one or more amino acid back mutations selected from the group consisting of L46R, G66R, S60K, P44F, Y36L, K42G, I21L and T85D.
  • the humanized LAG-3 antibody or antigen-binding fragment thereof according to the present invention wherein the humanized antibody light chain variable region sequence is selected from the group consisting of SEQ ID NO: 30.
  • the humanized LAG-3 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region sequence,
  • the heavy chain variable region sequence has at least 85% (preferably 95%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25;
  • a light chain variable region sequence having at least 85 of the amino acid sequence set forth in SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28 % sequence identity.
  • the LAG-3 antibody or antigen-binding fragment thereof of the present invention wherein the humanized antibody comprises (a) a heavy chain variable region sequence, the heavy chain variable a region sequence having at least 85% (preferably 95%) sequence identity to the amino acid sequence set forth in SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33; and (b) light a chain variable region sequence having the amino acid sequence set forth in SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37 At least 85% (preferably 95%) sequence identity.
  • a LAG-3 antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody comprises a heavy chain variable region and a light chain variable region selected from the group consisting of combination:
  • a LAG-3 antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody comprises a heavy chain variable region and a light chain variable region selected from the group consisting of combination:
  • the chimeric or humanized LAG-3 antibody or antigen-binding fragment thereof according to the present invention, wherein the chimeric antibody or humanized antibody heavy chain further comprises a human The heavy chain constant region of the source IgG1, IgG2, IgG3 or IgG4 or variant thereof, preferably comprising a heavy chain constant region of human IgG4 or a variant thereof, most preferably a heavy chain constant region as set forth in SEQ ID NO:38;
  • the chimeric or humanized antibody light chain further comprises a light chain constant region of a human kappa, lambda chain or variant thereof, most preferably a light chain constant region as set forth in SEQ ID NO:39.
  • the light chain variable region of the anti-PD-1 antibody or antigen-binding fragment thereof comprises SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, respectively.
  • the heavy chain variable region of the PD-1 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42 respectively HCDR3;
  • the PD-1 antibody is a humanized antibody.
  • a preferred humanized antibody light chain sequence is the sequence set forth in SEQ ID NO: 47 or a variant thereof; said variant preferably has a 0-10 amino acid change in the light chain variable region; more preferably A43S Amino acid changes.
  • the humanized antibody heavy chain sequence is the sequence set forth in SEQ ID NO: 46 or a variant thereof; the variant preferably has an amino acid change of 0-10 in the heavy chain variable region; more preferably an amino acid of G44R Variety.
  • the humanized antibody light chain sequence is the sequence set forth in SEQ ID NO: 47 and the heavy chain sequence is the sequence set forth in SEQ ID NO:46.
  • sequences of the aforementioned humanized antibody heavy and light chains are as follows:
  • the anti-PD-1 antibody or antigen-binding fragment thereof may also be selected from the group consisting of Pidilizumab, MEDI-0680, AMP-224, PF-06801591, TSR-042, JS-001, GLS- 010, PDR-001, Genolimzumab, Camrelizumab, BGB-A317, IBI-308, REGN-2810, Pembrolizumab, Nivolumab, and combinations thereof.
  • the tumor is selected from the group consisting of a malignant tumor, a benign tumor; the malignant tumor is selected from the group consisting of a malignant epithelial tumor, a sarcoma, a myeloma, a leukemia, a lymphoma, a melanoma, a head and neck tumor, and a brain.
  • Tumor peritoneal cancer, mixed tumor, childhood malignant tumor
  • the malignant epithelial tumor is selected from the group consisting of lung cancer, breast cancer, liver cancer, pancreatic cancer, colorectal cancer, stomach cancer, gastroesophageal adenocarcinoma, esophageal cancer, small intestine cancer, cardiac cancer , endometrial cancer, ovarian cancer, fallopian tube cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer, kidney cancer, bladder cancer, anal cancer, gallbladder cancer, cholangiocarcinoma, teratoma, cardiac tumor;
  • the tumor is selected from the group consisting of nasopharyngeal carcinoma, laryngeal cancer, thyroid cancer, tongue cancer, and oral cancer;
  • the sarcoma is selected from the group consisting of Askin tumor, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannomas
  • the myeloma is selected from the group consisting of isolated myeloma, multimodal myeloma, diffuse myeloma, leukemia myeloma, extramedullary myeloma;
  • the leukemia is selected from acute lymphoid white Disease, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, hairy cell leukemia, T cell lymphocytic leukemia, large granular lymphocytic leukemia, adult T cell leukemia;
  • said lymphoma is selected from non-Hodgkin Lymphoma, Hodgkin's lymphoma
  • the brain tumor is selected from the group consisting of a neuroepithelial neoplasm, a cranial nerve and a spinal nerve tumor, a meningeal tumor
  • the child malignant tumor is selected from the group consisting of a nephroblastoma, a neuroblastom
  • the lung cancer is selected from the group consisting of the lung cancer selected from the group consisting of non-small cell lung cancer and small cell lung cancer; and the breast cancer is selected from the group consisting of the hormone receptor (HR) positive.
  • Breast cancer human epidermal growth factor receptor-2 (HER2)-positive breast cancer, triple-negative breast cancer;
  • the renal cancer is selected from the group consisting of transparent renal cell carcinoma, papillary renal cell carcinoma, chromophobe renal cell carcinoma, and collecting duct
  • the cancer of the neuroepithelial tissue is selected from the group consisting of: preferably astrocytoma, anaplastic astrocytoma, glioblastoma;
  • the liver cancer is selected from the group consisting of primary liver cancer, secondary liver cancer, and primary liver cancer. It is selected from the group consisting of hepatocellular carcinoma, cholangiocarcinoma, and mixed liver cancer; and the colorectal cancer is selected from the group consisting of colon cancer and rectal cancer.
  • the tumor is selected from the group consisting of a microsatellite unstable high MSI-H or a mismatch repaired solid tumor, Hodgkin's lymphoma, non-Hodgkin's lymphoma, prostate cancer, Pancreatic cancer, lung cancer, esophageal cancer, liver cancer, cholangiocarcinoma, breast cancer, colorectal cancer, gastric cancer, kidney cancer, acute myeloid lymphocytic leukemia, myelodysplastic syndrome, glioma, basal cell carcinoma, squamous cell carcinoma Human papillomavirus-associated tumors, preferably microsatellite unstable high MSI-H or mismatch repaired solid tumors.
  • a microsatellite unstable high MSI-H or mismatch repaired solid tumor preferably microsatellite unstable high MSI-H or mismatch repaired solid tumors.
  • the tumor is mediated and/or expresses PD-L1 by PD-1.
  • the tumor further expresses LAG-3.
  • the tumor is selected from the group consisting of a RAS mutant tumor, a RAF mutant tumor.
  • the tumor is selected from a tumor that is not associated with a RAS mutant and is not associated with a RAF mutant tumor.
  • the RAS mutant is selected from the group consisting of a HRas mutant, a KRas mutant, and an NRas mutant; and the RAF mutant is selected from the group consisting of an A-RAF mutant and a B-RAF mutant.
  • the B-RAF mutant is preferably selected from the B-RAF V600E mutant, the B-RAF V600K mutant, the B-RAF V600D mutant, and the B-RAF V600R mutant.
  • the above mutation is a positive mutation.
  • the above tumor expresses PD-L1 and/or LAG-3 as overexpression or normal expression.
  • the tumor is selected from the group consisting of a middle-stage tumor, a relapsed and refractory tumor, a chemotherapy-treated drug failure and/or a recurrent tumor, a radiotherapy failure and/or a recurrent tumor, and a targeted drug treatment. Failure and/or recurrence of the tumor, failure of immunotherapy, and/or recurrence of the tumor.
  • the tumor is either resistant or resistant to immunotherapeutics or immunotherapy
  • the immunotherapeutic agent is PD-1 and/or PD-L1 or CTLA- 4 (cytotoxic T lymphocyte-associated protein 4) as a target
  • the immunotherapy is selected from the group consisting of immunological checkpoint block (ICB) therapy, chimeric antigen receptor T cell immunotherapy (CAR-T therapy), autologous cellular immunity Therapy (CIK therapy).
  • IB immunological checkpoint block
  • CAR-T therapy chimeric antigen receptor T cell immunotherapy
  • CIK therapy autologous cellular immunity Therapy
  • the immunotherapeutic agent is selected from the group consisting of a PD-1 antibody, a PD-L1 antibody, and a CTLA-4 antibody, and the PD-1 antibody includes, but is not limited to, Pidilizumab, MEDI-0680.
  • PD-L1 Antibodies include, but are not limited to, MSB-0011359-C, CA-170, LY-3300054, BMS-936559, Durvalumab, Avelumab, Atezolizumab;
  • CTLA-4 antibodies include, but are not limited to, ipilimumab, AK-104, JHL-1155, ATOR -1015, AGEN-1884, PRS-010, tremelimumab, IBI-310, MK-1308, BMS-986218, SN-CA21, FPT-155, KN-044, CG-0161, ONC-392, AGEN-2041, PBI -5D3H5.
  • the dose of the anti-LAG-3 antibody or antigen-binding fragment thereof is selected from 0.01 to 1000 mg, preferably from 0.1 mg, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 2 mg, 3 mg, 4mg, 5mg, 6mg, 7mg, 8mg, 9mg, 10mg, 11mg, 12mg, 12.5mg, 15mg, 17.5mg, 20mg, 22.5mg, 25mg, 30mg, 40mg, 45mg, 50mg, 60mg, 70mg, 75mg, 80mg, 90mg , 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 1000 mg, more preferably 50 mg, 60 mg 70, 75, 75, 80
  • the anti-LAG-3 antibody or antigen-binding fragment thereof is selected from the group consisting of 1-20 mg/kg, preferably from 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg. , 5mg/kg, 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 12mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 18mg/kg, 20mg/kg, more Preferred are 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, and 10 mg/kg.
  • the anti-PD-1 antibody or antigen-binding fragment thereof is selected from the group consisting of 50-600 mg, preferably from 50 mg, 60 mg, 70 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 600 mg, more preferably from 100 mg, 200 mg, 400 mg.
  • the anti-PD-1 antibody or antigen-binding fragment thereof is selected from the group consisting of 1-10 mg/kg, preferably from 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg. 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, more preferably 3 mg/kg, 4 mg/kg, 5 mg/kg.
  • an anti-PD-1 antibody or antigen-binding fragment thereof is used in combination with an anti-LAG-3 antibody or antigen-binding fragment thereof for tumor therapy, and the order of administration is either an anti-LAG-3 antibody or an antigen-binding fragment thereof.
  • the PD-1 antibody or antigen-binding fragment thereof is administered prior to administration, or both, or the anti-LAG-3 antibody or antigen-binding fragment thereof is administered after administration of the PD-1 antibody or antigen-binding fragment thereof.
  • an anti-PD-1 antibody or antigen-binding fragment thereof is used in combination with an anti-LAG-3 antibody or an antigen-binding fragment thereof for tumor treatment, and both may be administered in the same administration cycle or in different administrations. Drug administration.
  • the treatment period may be 1 day, 3 days, 1 week, 2 weeks, 3 weeks (21 days), 3-4 weeks (21-28 days), 4 weeks (28 days), preferably 3 Week or 3-4 weeks or 4 weeks.
  • the treatment cycle includes, but is not limited to, a chemotherapy cycle or a radiation therapy cycle or other related targeted drug therapy cycle or immunotherapy cycle.
  • the anti-LAG-3 antibody or antigen-binding fragment thereof and the anti-PD-1 antibody or antigen-binding fragment thereof can be used in combination for treating tumors in the same or different treatment cycles, and in the process of treating tumors,
  • the LAG-3 antibody or antigen-binding fragment thereof may be administered in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof, or may be combined with a preferred chemotherapy regimen or a radiotherapy treatment scheme or a targeted small molecule drug according to different tumor types.
  • the treatment regimen or immunotherapy regimen treats tumors, including but not limited to cellular immunotherapy (eg, CAR-T therapy, tumor vaccine, CIK therapy, etc.); in addition, anti-LAG-3 antibody or antigen-binding fragment thereof and anti-PD
  • cellular immunotherapy eg, CAR-T therapy, tumor vaccine, CIK therapy, etc.
  • anti-LAG-3 antibody or antigen-binding fragment thereof eg., anti-PD
  • the combined administration of the -1 antibody or antigen-binding fragment thereof may also be carried out separately in combination with other therapeutic regimens.
  • the anti-LAG-3 antibody or antigen-binding fragment thereof and the anti-PD-1 antibody or antigen-binding fragment thereof may be used in combination with, or before or after, various tumor diagnosis norms or guidelines.
  • the treatment protocols specified in different pathological types and stages of tumor progression include, but are not limited to, NCCN (National Comprehensive Cancer Network publishes guidelines for clinical practice of various malignancies) or malignancy promulgated by the Chinese Ministry of Health. Tumor diagnosis and treatment specifications.
  • an anti-LAG-3 antibody or antigen-binding fragment thereof is used in combination with an anti-PD-1 antibody or antigen-binding fragment thereof in the same treatment cycle (eg, 28 days for one treatment cycle or 21 days for one treatment cycle).
  • the anti-LAG-3 antibody or antigen-binding fragment thereof is administered simultaneously with the anti-PD-1 antibody or antigen-binding fragment thereof or before the anti-PD-1 antibody or antigen-binding fragment thereof or in the anti-PD-1 antibody or
  • the antigen-binding fragment is administered; the anti-LAG-3 antibody or antigen-binding fragment thereof is administered on the first day of the treatment cycle or on the first and the 15th day of a treatment cycle during the same administration period.
  • the treatment cycle may be 21 days or 28 days; the anti-PD-1 antibody or antigen-binding fragment thereof is in the same dosing period as the anti-LAG-3 antibody or antigen-binding fragment thereof (eg, 28 days for a treatment cycle or When administered for 21 days for a treatment cycle, the anti-PD-1 antibody or antigen-binding fragment thereof can be administered at a frequency of 2 weeks/time or 3 weeks/time or 4 weeks/time, or at the first of a treatment cycle. Day and day 15 dosing.
  • the term "combination" is a mode of administration, which means administering at least one dose of an anti-PD-1 antibody or antigen-binding fragment thereof and at least one dose of an anti-LAG-3 antibody over a certain period of time. Or an antigen-binding fragment thereof, wherein both substances exhibit a pharmacological effect.
  • the time period may be within one administration period, preferably within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, or within 24 hours, within 12 hours.
  • the PD-1 antibody or antigen-binding fragment thereof and the anti-LAG-3 antibody or antigen-binding fragment thereof can be administered simultaneously or sequentially.
  • Such a term includes a treatment in which a PD-1 antibody or antigen-binding fragment thereof and an anti-LAG-3 antibody or antigen-binding fragment thereof are administered by the same administration route or different administration routes.
  • the combined modes of administration of the present invention are selected from the group consisting of simultaneous administration, independent formulation and co-administration or independently formulated and administered sequentially.
  • the invention further relates to the use of the medicament, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is administered once a day, twice a day, three times a day, once a week, two times Once a week, once every three weeks, once a month; the frequency of administration of the anti-LAG-3 antibody or antigen-binding fragment thereof is once a day, twice a day, three times a day, once a week, once every two weeks, three weeks Once, once a month.
  • the anti-LAG-3 antibody or antigen-binding fragment thereof is administered by injection, for example subcutaneously or intravenously, and the anti-LAG-3 antibody or antigen-binding fragment thereof is prepared before injection.
  • the anti-PD-1 antibody or antigen-binding fragment thereof is administered by injection, for example subcutaneously or intravenously, and the anti-PD-1 antibody or antigen-binding fragment thereof is formulated for injection before injection. form.
  • Particularly preferred injectable forms of the anti-PD-1 antibody or antigen-binding fragment thereof are injection or lyophilized powders comprising an anti-PD-1 antibody or antigen-binding fragment thereof, a buffer, a stabilizer, optionally further comprising Surfactant.
  • the buffering agent may be selected from one or more of the group consisting of acetate, citrate, succinate, and phosphate.
  • the stabilizer may be selected from sugars or amino acids, preferably disaccharides such as sucrose, lactose, trehalose, maltose.
  • the surfactant is selected from the group consisting of polyoxyethylene hydrogenated castor oil, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, preferably the polyoxyethylene sorbitan fatty acid ester is polysorbate 20, 40, 60 or 80. Most preferred is polysorbate 20. Injectable forms of the most preferred anti-PD-1 antibodies or antigen-binding fragments thereof comprise an anti-PD-1 antibody or antigen-binding fragment thereof, acetate buffer, trehalose and polysorbate 20.
  • the combined modes of administration of the present invention are selected from the group consisting of simultaneous administration, independent formulation and co-administration or independently formulated and administered sequentially.
  • the combined administration routes of the present invention are selected from the group consisting of oral administration, parenteral administration, and transdermal administration, and include, but are not limited to, intravenous injection, subcutaneous injection, and intramuscular injection.
  • the present invention provides the above anti-PD-1 antibody or antigen-binding fragment thereof in combination with the above anti-LAG-3 antibody or antigen-binding fragment thereof as a medicament for treating tumors and/or enhancing T cell activity.
  • a method of treating tumors and/or enhancing T cell activity comprising administering to a patient an anti-PD-1 antibody or antigen-binding fragment thereof as described above in combination with the above anti-LAG-3 antibody or antigen-binding fragment thereof.
  • the present invention also provides a pharmaceutical kit, or a pharmaceutical kit comprising the above anti-PD-1 antibody or antigen-binding fragment thereof in combination with the above anti-LAG-3 antibody or antigen-binding fragment thereof.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof in combination with the above anti-LAG-3 antibody or antigen-binding fragment thereof, and one or more pharmaceutically acceptable An excipient, diluent or carrier.
  • Figure 3 Effect of Antibody A and Antibody B on human malignant glioma U-87MG mouse xenografts, where *p ⁇ 0.05, **p ⁇ 0.01 vs FC control-6mpk; *p ⁇ 0.05 vs antibody B-3mpk By student T test;
  • the term “LAG-3” refers to lymphocyte activating gene 3.
  • the term “LAG-3” encompasses variants, isoforms, homologs, orthologs, and paralogs.
  • the term “human LAG-3” refers to the human sequence LAG-3, for example the complete amino acid sequence of human LAG-3 with Uniprot number: P18627. LAG-3, such as CD223, is also known in the art.
  • the human LAG-3 sequence differs from the human LAG-3 of Uniprot No.: P18627 in that it has, for example, a conservative mutation or a mutation in a non-conserved region, and the human LAG-3 of LAG-3 and Uniprot No.: P18627 has substantial The same biological function.
  • the biological function of human LAG-3 is to have an epitope in the extracellular domain of LAG-3 that is specifically bound by the antibodies of the present disclosure, or the biological function of human LAG-3 is to bind to MHC class II molecules.
  • the specific human LAG-3 sequence is typically at least 90% identical in amino acid sequence to human LAG-3 of Uniprot No.: P18627 and contains a human amino acid sequence when compared to the LAG-3 amino acid sequence of other species (eg, murine). Amino acid residues.
  • human LAG-3 may be at least 85% or even at least 95%, 96%, 97%, 98% or 99% identical to the LAG-3 of Uniprot number: P18627 in the amino acid sequence.
  • the human LAG-3 sequence shows no more than 10 amino acid differences from the LAG-3 sequence of Uniprot No.: P18627.
  • human LAG-3 may exhibit no more than 5 or even no more than 4, 3, 2 or 1 amino acid differences than the LAG-3 sequence of Uniprot No.: P18627. Percent identity can be determined as set forth herein.
  • Sequence identity refers to the degree of identity between two nucleic acids or two amino acid sequences when optimally aligned and compared in the presence of a suitable substitution, insertion or deletion.
  • sequence identity between the sequences described in the present invention and their identical sequences may be at least 85%, 90% or 95%, preferably at least 95%.
  • Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 100%.
  • the "antibody” as used in the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds.
  • immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain, respectively. , ⁇ chain, and ⁇ chain.
  • the same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either a kappa chain or a lambda chain by the constant region. Each class Ig of the five classes of Ig may have a kappa chain or a lambda chain.
  • the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or a variant thereof.
  • the antibody heavy chain of the present invention may further comprise a heavy chain constant region comprising IgG1, IgG2, IgG3, IgG4 or a variant thereof of human or murine origin.
  • variable region The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, being the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • CDR complementarity determining region
  • Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment of the present invention conform to the known Kabat numbering rules (LCDR1-3, HCDE2-3) in number and position, or conform to the numbering rules of kabat and chothia. (HCDR1).
  • the antibody of the present invention includes a murine antibody, a chimeric antibody, a humanized antibody, preferably a humanized antibody.
  • murine antibody is in the present invention a monoclonal antibody against human LAG-3 prepared according to the knowledge and skill in the art.
  • the test subject is injected with the LAG-3 antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional properties is isolated.
  • the murine LAG-3 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprising a murine IgG1 , heavy chain constant region of IgG2, IgG3 or variants thereof.
  • chimeric antibody is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody.
  • a hybridoma that secretes a murine-specific monoclonal antibody is first established, and then the variable region gene is cloned from the murine hybridoma cell, and the variable region gene of the human antibody is cloned as needed, and the murine variable region gene is cloned.
  • the human constant region gene is ligated into a chimeric gene, inserted into an expression vector, and finally expressed in a eukaryotic or prokaryotic system.
  • the antibody light chain of the LAG-3 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof.
  • the antibody heavy chain of the LAG-3 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising a human IgG1, IgG2 or IgG4 heavy chain constant region, or An IgGl, IgG2 or IgG4 variant of an amino acid mutation (such as a YTE mutation).
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of murine CDR sequences into human antibody variable region frameworks, ie different types of human germline antibodies An antibody produced in a framework sequence. It is possible to overcome the heterologous reaction induced by the chimeric antibody by carrying a large amount of the mouse protein component.
  • framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), as well as in Kabat, EA, etc.
  • humanized antibodies of the invention also include humanized antibodies that are further affinity matured by phage display.
  • the CDR sequence of the mouse in the LAG-3 humanized antibody is selected from the group consisting of SEQ ID NOs: 9-20; the human antibody variable region framework is designed and selected, wherein the antibody a heavy chain FR region sequence on the variable region of the heavy chain, derived from the combined sequence of the human germline heavy chain IGKV1-39*01 and hjk4.1; wherein the light chain FR region sequence on the variable region of the antibody light chain, A combined sequence derived from the human germline heavy chain IGHV3-23*04 and hjh6.1.
  • the human antibody variable region can be subjected to minimal reverse mutation to maintain activity.
  • the CDR graft can attenuate the affinity of the LAG-3 antibody or antigen-binding fragment thereof to the antigen due to the framework residues that are in contact with the antigen. Such interactions can be the result of high mutations in somatic cells. Therefore, it may still be necessary to graft such donor framework amino acids to the framework of humanized antibodies. Amino acid residues involved in antigen binding from a non-human LAG-3 antibody or antigen-binding fragment thereof can be identified by examining the murine monoclonal antibody variable region sequences and structures. Each residue in the CDR donor framework that differs from the germline can be considered to be related.
  • the sequence can be compared to a subtype consensus sequence or a consensus sequence of a murine sequence with a high percent similarity.
  • Rare framework residues are thought to be the result of high somatic mutations and thus play an important role in binding.
  • antibody fragment refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, LAG-3). It has been shown that fragments of full length antibodies can be utilized for antigen binding function of antibodies.
  • binding fragment contained in the term "antigen-binding fragment" of an antibody examples include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) a F(ab') 2 fragment, including a divalent fragment of two Fab fragments joined by a disulfide bridge on the hinge region, (iii) an Fd fragment consisting of a VH and CH1 domain; (iv) an Fv fragment consisting of a single arm VH and VL domain of the antibody (v) a single domain or dAb fragment (Ward et al, (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) A combination of two or more separate CDRs, optionally joined by a synthetic linker.
  • CDR complementarity determining region
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined by a synthetic linker using a recombinant method such that they are capable of producing a single protein in which the VL and VH regions are paired to form a monovalent molecule.
  • Chains referred to as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883).
  • Such single chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody.
  • the antigen binding portion can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the intact immunoglobulin.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • single-chain antibody single-chain Fv
  • scFv single-chain Fv
  • scFv antibody heavy chain variable domain
  • VL antibody light chain variable domain
  • scFv molecules can have the general structure: NH 2 -VL- linker -VH-COOH or NH 2 -VH- linker -VL-COOH.
  • Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or variant thereof, for example using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) .
  • linkers useful in the present invention are by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996). , Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that contribute primarily to antigen binding.
  • One of the most commonly used definitions of the six CDRs is provided by Kabat E. A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).
  • the Kabat definition of a CDR applies only to the CDR1, CDR2 and CDR3 (LCDR1, LCDR2, LCDR3 or L1, L2, L3) of the light chain variable domain, as well as the CDR2 and CDR3 of the heavy chain variable domain. (HCDR2, HCDR3 or H2, H3).
  • antibody framework refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain that does not have a CDR.
  • epitopes refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (eg, a specific site on a LAG-3 molecule).
  • Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).
  • the terms “specifically binds”, “selectively binds”, “selectively binds” and “specifically binds” refers to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of less than about 10 -7 M, such as less than about 10 -8 M, 10 -9 M, or 10 -10 M or less.
  • KD affinity
  • competitive binding refers to an antibody that recognizes the same epitope (also referred to as an antigenic determinant) or a portion of the same epitope on the extracellular region of human LAG-3 and binds to the antigen with the monoclonal antibody of the present invention.
  • An antibody that binds to the same epitope as the monoclonal antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of human LAG-3 recognized by the monoclonal antibody of the present invention.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction.
  • an antibody of the invention binds to LAG-3 with a dissociation equilibrium constant (KD) of less than about 10-7 M, such as less than about 10 -8 M, 10 -9 M, or 10-10 M or less, for example, as used Surface plasmon resonance (SPR) techniques were measured in a BIACORE instrument.
  • SPR Surface plasmon resonance
  • nucleic acid molecule refers to a DNA molecule and an RNA molecule.
  • the nucleic acid molecule may be single stranded or double stranded, but is preferably a double stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to the coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in a host cell into which they have been introduced (for example, a bacterial vector having an origin of replication of bacteria and an episomal mammalian vector) or can be integrated into the genome of the host cell after introduction into the host cell, thereby The host genome is replicated together (eg, a non-episomal mammalian vector).
  • a mouse can be immunized with human LAG-3 or a fragment thereof, and the obtained antibody can be renatured, purified, and subjected to amino acid sequencing by a conventional method.
  • the antigen-binding fragment can also be prepared by a conventional method.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in a non-human CDR region.
  • the human FR germline sequence can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Immunoglobulin Journal, 2001 ISBN 014441351. obtain.
  • IMGT ImMunoGeneTics
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include bacterial, microbial, plant or animal cells.
  • Bacteria susceptible to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.
  • the engineered antibodies or antigen-binding fragments of the invention can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human LAG-3. Positive clones were expanded in a serum-free medium in a bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified by a conventional technique.
  • purification is carried out using an A or G Sepharose FF column containing an adjusted buffer.
  • the non-specifically bound components are washed away.
  • the bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected.
  • the antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange.
  • the resulting product needs to be frozen immediately, such as -70 ° C, or lyophilized.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic agent, diagnostic agent or composition and animal, human, subject Contact of the test subject, cell, tissue, organ or biological fluid.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells.
  • administeristering and “treating” also means treating, for example, cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • Treatment when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering to a patient a therapeutic agent for internal or external use, for example a composition comprising any of the binding compounds of the present invention, the patient having one or more symptoms of the disease, and the therapeutic agent is known to have Therapeutic effect.
  • a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases to induce such symptoms to degenerate or to inhibit the progression of such symptoms to any degree of clinical right measurement.
  • the amount of therapeutic agent also referred to as "therapeutically effective amount" effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect in the patient.
  • Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition. While embodiments of the invention (e.g., methods of treatment or preparations) may be ineffective in ameliorating the symptoms of each target disease, any statistical test methods known in the art such as Student's t-test, chi-square test, according to Mann and Whitney U-test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.
  • any statistical test methods known in the art such as Student's t-test, chi-square test, according to Mann and Whitney U-test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.
  • Constantly modified refers to amino acids in other amino acid substitution proteins having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that Changes are made without altering the biological activity of the protein. It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate the diagnosis.
  • the effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the methodological route and dosage of the administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Exogenous refers to a substance that is produced outside of a living being, cell or human, depending on the situation.
  • Endogenous refers to a substance produced in a cell, organism or human body, depending on the circumstances.
  • “Homology” refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both comparison sequences are occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecule is homologous at that position .
  • the percent homology between the two sequences is a function of the number of matches or homology positions shared by the two sequences divided by the number of positions compared x 100.
  • the expression "cell”, “cell line” and “cell culture” are used interchangeably and all such names include progeny.
  • the words “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. In the case of a different name, it is clearly visible from the context.
  • PCR polymerase chain reaction
  • oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strand of the template to be amplified.
  • the 5' terminal nucleotides of the two primers may coincide with the ends of the material to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See generally, Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.).
  • PCR used herein is considered as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which comprises using a known nucleic acid and a nucleic acid polymerase as a primer to amplify or Produce a specific portion of the nucleic acid.
  • “Pharmaceutical composition” means a mixture comprising one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, such as physiological/pharmaceutically acceptable Carrier and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • Immunotherapy refers to the use of the immune system to treat diseases. In the present invention, it mainly refers to stimulating and enhancing the body's anti-tumor immune response by increasing the immunogenicity of tumor cells and sensitivity to effector cell killing, and applying immunity. The cells and effector molecules are infused into the host to cooperate with the body's immune system to kill tumors and inhibit tumor growth.
  • Enhanced T cell activity in the present invention means not only enhancing the activity of existing T cells, but also restoring the function of T cells, reactivation of inactivated T cells or stimulation of T cell proliferation, and production of antitumor activity.
  • the invention is further described in the following examples, which are not intended to limit the scope of the invention.
  • the experimental methods in the examples of the present invention which do not specify the specific conditions are usually in accordance with conventional conditions, such as the cold spring harbor antibody technology experiment manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer.
  • Reagents without specific source are routine reagents purchased from the market.
  • the UniProt Lymphocyte activation gene 3 protein (human LAG-3, Uniprot No.: P18627) was used as a template for the LAG-3 of the present invention, and the amino acid sequences of the antigen and the detection protein of the present invention were designed, and the LAG-3 protein was selected.
  • Different tags were ligated into pHr vector (self-produced) or pTT5 vector (Biovector, Cat#: 102762) or pTargeT vector (promega, A1410), transiently expressed in 293 cells or purified by CHO-S.
  • An antigen encoding the antigen of the present invention and a protein for detection are obtained.
  • the following LAG-3 antigens are not specifically described as human LAG-3
  • LAG-3 extracellular domain with Flag tag LAG-3-Flag, used to immunize mice
  • the horizontal line is the signal peptide and the italic part is the Flag-tag label.
  • Full-length LAG-3 used to construct LAG-3 overexpressing cell lines, immunized mice and tested
  • the cross-hatched portion is the signal peptide
  • the double-lined portion is the linker
  • the italicized portion is the Fc.
  • Fusion protein of LAG-3 extracellular domain and mIgG2a Fc LAG-3-mFc, for detection
  • the cross-hatched portion is the signal peptide
  • the double-lined portion is the linker
  • the italicized portion is the mFc.
  • the sample was centrifuged at high speed to remove impurities and concentrated to an appropriate volume.
  • the flag affinity column was equilibrated with 0.5 x PBS and washed 2-5 column volumes.
  • the supernatant cells were subjected to supernatant analysis and the supernatant samples were applied to the column.
  • the column was rinsed with 0.5 x PBS until the A280 reading dropped to baseline. Rinse the column with PBS, rinse the heteroprotein, and collect.
  • the protein of interest was eluted with 100 mM glycine, pH 3.0. and collected for subsequent in vitro activation and further purification.
  • the cell expression supernatant samples were centrifuged at high speed to remove impurities, and the hybridoma expression supernatant was purified by Protein G column, and the recombinant antibody and Fc fusion protein expression supernatant were purified using a Protein A column. Rinse the column with PBS until the A280 reading drops to baseline.
  • the protein of interest was eluted with 100 mM acetic acid pH 3.0 and neutralized with 1 M Tris-HCl, pH 8.0.
  • the eluted sample was appropriately concentrated and further purified by PBS-balanced gel chromatography Superdex 200 (GE). The peak of the depolymerized product was collected and used.
  • Anti-human LAG-3 monoclonal antibodies are produced by immunizing mice.
  • Experimental SJL white mice female, 6 weeks old (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001).
  • Feeding environment SPF level. After the mice were purchased, the laboratory environment was kept for 1 week, 12/12 hours light/dark cycle adjustment, temperature 20-25 ° C; humidity 40-60%. Mice that have adapted to the environment are immunized according to the following protocol.
  • the immunizing antigen is the Flag tagged human LAG-3 extracellular domain (SEQ ID NO: 1).
  • Immunization program A use Gold Adjuvant (Sigma Cat No. T2684) and Thermo Alum (Thermo Cat No. 77161) was cross-immunized.
  • the antigen was emulsified and inoculated for 0, 7, 14, 21, 28, 35, and 42 days. On day 0, subcutaneous (SC) multiple injections of 50 ⁇ g/only of the emulsified antigen. On day 7, 25 ⁇ g/head was injected intraperitoneally (IP).
  • SC subcutaneous
  • IP intraperitoneally
  • the back or intraperitoneal injection of antigen was selected according to the agglomeration of the back and the swelling of the abdomen.
  • Blood was taken on days 21, 35, and 49, and antibody titers in the serum of the mice were determined by ELISA.
  • spleen cell fusion was performed in mice with high antibody titers in serum and titers that tended to plate.
  • the immunization was boosted 3 days before the splenocyte fusion, and an antigen solution prepared by intraperitoneal (IP) injection of 50 ⁇ g/vial of physiological saline was administered.
  • IP intraperitoneal
  • Immunization Protocol B Mice were immunized with QuickAntibody-Mouse 5W (KX0210041). The ratio of antigen to adjuvant was 1:1, 25 ⁇ g/mouse/time (first exempt/enhanced immunization). The antigen and the adjuvant were quickly and thoroughly mixed and inoculated for 0, 21, and 35 days. On day 0, the mouse calf muscle (IM) was injected with 25 ⁇ g/antigen. On the 21st and 35th day, 25 ⁇ g/mouse was injected in the same manner (depending on the titer, whether or not the third exemption was performed). Blood was taken on the 28th and 42nd day, and the antibody titer in the serum of the mouse was determined by ELISA.
  • IM mouse calf muscle
  • Splenocyte fusion was performed in mice with high antibody titers in serum and titers that tended to plate. The immunization was boosted 3 days before the splenocyte fusion, and an antigen solution prepared by intraperitoneal (IP) injection of 50 ⁇ g/vial of physiological saline was administered.
  • IP intraperitoneal
  • Spleen lymphocytes and myeloma cell Sp2/0 cells were optimized using an optimized PEG-mediated fusion step ( CRL-8287 (TM ) was fused to obtain hybridoma cells.
  • the fused hybridoma cells were resuspended in complete medium (DMEM medium containing 20% FBS, 1 ⁇ HAT, 1 ⁇ OPI) at a density of 0.5-1 ⁇ 10 6 /ml, and 100 ⁇ l/well in 96-well plates. After incubating for 3-4 days at 37 ° C, 5% CO 2 , HAT complete medium was supplemented with 100 ⁇ l/well, and culture was continued for 3-4 days until a needle-like clone was formed.
  • complete medium DMEM medium containing 20% FBS, 1 ⁇ HAT, 1 ⁇ OPI
  • HT complete medium RPMI-1640 medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI
  • RPMI-1640 medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI was added, and cultured at 37 ° C, 5% CO 2 for 3 days, and then subjected to ELISA.
  • the hybridoma culture supernatant was detected by a binding ELISA method according to the growth density of the hybridoma cells (see Test Example 1).
  • the positive well cell supernatant detected by ELISA was subjected to a cell blocking experiment (see Test Example 3).
  • the well cells that were positive for both binding and blocking were expanded in a timely manner for cryopreservation and two to three subclones until a single cell clone was obtained.
  • the sequence of cloning from a positive hybridoma is as follows. Hybridoma cells in logarithmic growth phase were collected, using Trizol (Invitrogen, Cat No.15596-018) according to kit instructions an RNA extraction step, with PrimeScript TM Reverse Transcriptase reverse transcription kit (Takara, Cat No.2680A). The cDNA obtained by reverse transcription was subjected to PCR amplification using a mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503), and sent to a sequencing company for sequencing. The amino acid sequences corresponding to the DNA sequences of the heavy and light chains of the obtained hybridoma clone mAb229 and mAb303 are represented by SEQ ID NO: 5, 6 and SEQ ID NO: 7, 8.
  • the obtained positive clones were subjected to an ELISA assay in which human LAG-3 was combined (test example 1, results show that the protein level binding activity EC50 value in Table 2), and ELISA assay in which human LAG-3 overexpresses CHO-s cells (test example 2)
  • the results are shown in Table 2 for cell-level binding activity EC50 values) and blocking LAG-3 antigen binding to Daudi cells (Test Example 3, results are shown in Table 2 for blocking active EC50 values), and tested for human LAG-3
  • the affinity of the protein see test example 4, the results are shown in Table 3).
  • the data in Table 2 shows that the LAG-3 antibodies mAb229 and mAb303 have good binding activity to human LAG-3 protein.
  • the LAG-3 antibodies mAb229 and mAb303 have good binding activity to CHO-S cells overexpressing the human LAG-3 full-length protein. Both the LAG-3 antibodies mAb229 and mAb303 significantly blocked the binding of human LAG-3 antigen to Daudi cells.
  • the data in Table 3 indicates that the LAG-3 antibodies mAb229 and mAb303 of the present invention have strong binding activity and affinity to human LAG-3 protein.
  • the heavy and light chain variable region germline genes with high homology to mAb229 were selected as templates, and the CDRs of the murine antibody were respectively determined.
  • the amino acid residues are determined and annotated by the Kabat numbering system.
  • the humanized light chain template of the murine antibody mAb229 is IGKV1-39*01 and hjk4.1, and the humanized heavy chain template is IGHV7-4-1*02 and hjh6.1.
  • the sequence of the humanized variable region is as follows:
  • the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • I48V indicates that 48-bit I is mutated back to V according to the Kabat numbering system.
  • Grafted represents the murine antibody CDRs implanted into the human germline FR region sequence.
  • Hu229-005 indicates that there are two mutations in the light chain HumAb229_VL.1A and heavy chain HumAb229_VH.1 on the humanized mouse antibody Hu229-005. Other analogies.
  • Hu229VH.1 (same as Hu229VH-CDR graft)
  • Hu229VL.1 (same as Hu229VL-CDR graft)
  • the heavy and light chain variable region germline genes with high homology to mAb303 were selected as templates, and the CDRs of the murine antibody were respectively determined.
  • the amino acid residues are determined and annotated by the Kabat numbering system.
  • the humanized light chain templates of the murine antibody mAb303 are IGKV1-39*01 and hjk4.1, and the humanized heavy chain templates are IGHV1-3*01 and hjh6.1.
  • the humanized variable region sequences are as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • L46R indicates that 46-bit L is mutated back to R according to the Kabat numbering system. Grafted represents the murine antibody CDRs implanted into the human germline FR region sequence.
  • Hu303-005 indicates that there are two mutations in the light chain HumAb303_VL.1A and heavy chain HumAb303_VH.1 on the humanized mouse antibody Hu303-005. Other analogies.
  • Hu303_VH.1 (same as Hu303VH-CDR graft)
  • Hu303_VL.1 (same as Hu303VL-CDR graft)
  • the antibody uses the constant region of the human heavy chain IgG4/light chain kappa in combination with each variable region, and the S228P mutation is made in the Fc segment to increase the stability of the IgG4 antibody, and other mutations known in the art can be used to increase its performance.
  • the positive antibody molecule obtained by hybridoma screening is sequenced to obtain a variable region coding gene sequence.
  • the primers were designed to sequence the primers, and the sequencing gene was used as a template.
  • the VH/VK gene fragment of each antibody was constructed by PCR, and then the expression vector pHr (with signal peptide and hIgG4/hkappa constant region gene (CH1-FC/CL) fragment).
  • the homologous recombination was carried out to construct a recombinant antibody full-length expression plasmid VH-CH1-FC-pHr/VL-CL-pHr.
  • the antibody sequence after human design was codon-optimized to generate the coding sequence of the human codon-preferred gene.
  • the primers were designed to construct the VH/VK gene fragment of each antibody, and then the expression vector pHr (with signal peptide and hIgG4/hkappa constant region).
  • the gene (CH1-FC/CL) fragment was subjected to homologous recombination to construct a humanized antibody full-length expression plasmid VH-CH1-FC-pHr/VL-CL-pHr.
  • the plasmid expressing the light heavy chain of the antibody was transfected into HEK293E cells at a ratio of 1:1.2. After 6 days, the expression supernatant was collected, and the impurities were removed by high-speed centrifugation and purified by a Protein A column. Rinse the column with PBS until the A280 reading drops to baseline. The protein of interest was eluted with an acidic eluent of pH 3.0 - pH 3.5 and neutralized with 1 M Tris-HCl, pH 8.0-9.0. The eluted sample was appropriately concentrated, and further purified by PBS-balanced gel chromatography Superdex 200 (GE) to remove the aggregate, collect the monomer peak, and equilibrate the device.
  • PBS-balanced gel chromatography Superdex 200 GE
  • LAG-3 antibody (Hu303-005 in Table 7 of the above Example 4, is the heavy chain variable region shown in SEQ ID NO: 29 and the light chain variable region shown in SEQ ID NO: 34 and the corresponding heavy
  • the LAG3 antibody was dissolved in PBS buffer at a storage concentration of more than 2 mg/ml.
  • the PD-1 antibody (the heavy and light chain sequences are SEQ ID NO: 46 and SEQ ID NO: 47, respectively referred to as antibody B), and the PD-1 antibody was dissolved in PBS buffer at a storage concentration of 40 mg/ml.
  • Superantigen Staphylococcus aureus enterotoxin B SEB lyophilized powder is dissolved in deionized water, completely dissolved, filtered and sterilized by 0.22 ⁇ M filter, stored in storage at a concentration of 500 ⁇ g/ml; when working concentration, PBS is used. Buffer dilution.
  • PBMCs human peripheral blood mononuclear cells
  • the PBMCs used in this experiment were extracted from fresh blood of 2 volunteers.
  • the extraction methods are as follows:
  • the PBMCs were freshly isolated and inoculated into 96-well cell culture plates at a cell density of approximately 1 ⁇ 10 ⁇ 5/well. Stimulated with 80 ng/mL SEB superantigen; PD1 antibody and LAG-3 antibody samples were separately diluted and added to each concentration. The corresponding cell culture wells were spotted; the background control wells were only the SEB stimulation wells. After incubating for 72 hours at 37 ° C in a 5% CO 2 incubator, the cell culture supernatant was collected. The IL-2 secretion level in the cell culture supernatant was measured by ELISA (BD, CAT# 550611). Refer to the reagent instructions for specific operations. As a result, as shown in Figure 1, the LAG-3 antibody sample in combination with the PD1 antibody enhanced the secretion of the cytokine IL-2 by activated T lymphocytes, and had a drug concentration dose effect.
  • Example 7 LAG-3 antibody (antibody A) combined with PD-1 antibody against human malignant glioma U-87MG
  • NOD/SCID female mice 4-6 weeks old, weighing about 19g, 5/cage rearing, 12/12 hour light/dark cycle adjustment, temperature 23 ⁇ 1°C constant temperature, humidity 40-60%, free access to water.
  • FC control antibody concentration: 13.3 mg / ml, batch number: 20151126, from Shanghai Hengrui Pharmaceutical Co., Ltd.
  • the drug was diluted with PBS under sterile conditions to a concentration of the drug, and stored at 4 ° C.
  • LAG-3 antibody (Hu303-005 in Table 7 of the above Example 4, is the heavy chain variable region shown in SEQ ID NO: 29 and the light chain variable region shown in SEQ ID NO: 34 and the corresponding heavy
  • the sequence consisting of the strand constant region SEQ ID NO: 38 and the light chain constant region SEQ ID NO: 39, hereinafter referred to as antibody A) is prepared according to the above Examples 1-5; concentration: 1.9 mg/ml, before administration Dilute to a dosing concentration under sterile conditions in PBS and store at 4 °C.
  • PD-1 antibody (the heavy and light chain sequences are SEQ ID NO: 46 and SEQ ID NO: 47, respectively referred to as antibody B), prepared according to the method described in patent application WO201508584; concentration: 50 mg/ml, before administration Dilute to a dosing concentration under sterile conditions in PBS and store at 4 °C.
  • CD3 mAb diluted in PBS 40 ng / ml was added to a 6-well cell culture plate, 1 ml / well, coated at 37 ° C for one hour;
  • PBMCs (1640 medium culture) of 2 volunteers were added separately: about 2 ⁇ 10 6 Cells per well, 2 ml/well;
  • PBMCs were injected twice in the whole test period, and PBMCs of two volunteers stimulated by CD3 antibody for 4 days in Day0 were mixed in a ratio of 1:1, and injected into tumor tissues of tumor-bearing mice at 5 ⁇ 10 5 cells/mouse. The remaining PBMCs were stopped and cultured, and intraperitoneally injected into the tumor-bearing mice at 5 ⁇ 10 6 cells/mouse on Day7. The administration was performed by intraperitoneal injection of (IP) antibody B and antibody A from Day 0 three times a week (TIW) for a total of 6 times (Table 1). Tumor volume, animal weight, and data were recorded twice a week.
  • IP intraperitoneal injection
  • V 1/2 ⁇ a ⁇ b 2 where a and b represent length and width, respectively.
  • T/C(%) (TT 0 )/(CC 0 ) ⁇ 100
  • T and C are the tumor volume of the treatment group and the control group at the end of the experiment
  • T 0 and C 0 are the tumors at the beginning of the experiment. volume.
  • TGI (%) 1 - T / C (%).
  • the tumor-bearing mice were well tolerated by either single or combined use of antibody A and antibody B.
  • the body weight increased steadily throughout the administration, and no obvious drug-induced weight loss occurred (Fig. 4).
  • the combination of antibody A and antibody B (6mpk+3mpk, IP, TIW ⁇ 6) can significantly inhibit the growth of subcutaneous xenografts in human malignant glioma U-87MG mice, and has certain advantages over the single use group. . Tumor-bearing mice are well tolerated by the above drugs.
  • Example 8 Inhibition of U-87MG subcutaneous xenografts by LAG-3 antibody (antibody A, antibody C) alone and in combination with PD-1 antibody
  • FC control antibody concentration: 13.3 mg / ml, batch number: 20151126, from Shanghai Hengrui Pharmaceutical Co., Ltd.
  • the drug was diluted with PBS under sterile conditions to a concentration of the drug, and stored at 4 ° C.
  • LAG-3 antibody (Hu303-005 in Table 7 of the above Example 4, is the heavy chain variable region shown in SEQ ID NO: 29 and the light chain variable region shown in SEQ ID NO: 34 and the corresponding heavy
  • Hu229-013 in Table 5 of the above Example 3 is the heavy chain shown in SEQ ID NO:
  • the preparation protocol was carried out with reference to the above Examples 1-5; concentration: 1.9 mg/ml, diluted to a dosing concentration with PBS under aseptic conditions before administration, and stored at 4 ° C.
  • PD-1 antibody (the heavy and light chain sequences are SEQ ID NO: 46 and SEQ ID NO: 47, respectively referred to as antibody B), prepared according to the method described in patent application WO201508584; concentration: 50 mg/ml, before administration Dilute to a dosing concentration under sterile conditions in PBS and store at 4 °C.
  • PBMCs were injected twice in the whole test period, and PBMCs of two volunteers stimulated by CD3 antibody for 4 days in Day0 were mixed in a ratio of 1:1, and injected into tumor tissues of tumor-bearing mice at 5 ⁇ 10 5 cells/mouse. The remaining PBMCs were stopped and cultured, and intraperitoneally injected into the tumor-bearing mice at 5 ⁇ 10 6 cells/mouse on Day7.
  • the administration was performed by intraperitoneal injection (IP) of antibody B, antibody A, and antibody C from Day 0 three times a week (TIW) for a total of 6 times (Table 1). Tumor volume, animal weight, and data were recorded twice a week.
  • IP intraperitoneal injection
  • the results of the experiment are shown in Table 9 and Figure 5.
  • the LAG-3 antibody ie, antibody C-6mpk and antibody A-6mpk
  • the tumor inhibition rate was 27.25% (p ⁇ 0.05).
  • the antibody B-3mpk alone was significantly different (p ⁇ 0.001 vs FC control, p ⁇ 0.01 vs antibody B-3mpk).

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Abstract

L'invention concerne des utilisations d'un anticorps anti-PD-1 et d'un anticorps anti-LAG-3 conjointement dans la préparation d'un médicament pour le traitement d'une tumeur. L'invention concerne plus particulièrement, des utilisations de l'anticorps anti-PD-1 ou d'un fragment de liaison à l'antigène de celui-ci et de l'anticorps anti-LAG-3 ou d'un fragment de liaison à l'antigène de celui-ci conjointement dans la préparation d'un médicament pour le traitement d'une tumeur et/ou l'amélioration des activités de lymphocytes T.
PCT/CN2018/110324 2017-10-17 2018-10-16 Utilisations d'un anticorps anti-pd-1 et d'un anticorps anti-lag-3 conjointement dans la préparation d'un médicament pour le traitement d'une tumeur WO2019076277A1 (fr)

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