# US 2019/0315864 A1 · Single domain antibody and derivative proteins thereof against ctla4 > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2019/0315864 A1 - Document ID: US20190315864A1 - Status: Published application - Kind: A1 - Application number: 16/302,583 - Filed: 2017-05-19 - Published: 2019-10-17 - Earliest claimed: 2016-05-19 - Assignees: Suzhou Alphamab Co., Ltd.; Xitian Zhang; Xin Zhang - Applicants: Suzhou Alphamab Co., Ltd.; Xitian Zhang; Xin Zhang - Inventors: Ting Xu; Xiaoxiao Wang; Jie Li; Haiyan Wu; Li Gao; Qian Chu; Yu Bai - Web page: https://patentgrep.com/patent/US20190315864A1 ## CPC - C07K 16/2818: {against CD28 or CD152} - A61K 39/395: Antibodies (agglutinins A61K38/36 {; as drug carriers A61K47/50}); Immunoglobulins; Immune serum, e.g. antilymphocytic serum - A61K 39/3955: {against proteinaceous materials, e.g. enzymes, hormones, lymphokines} - A61K 39/39558: {against tumor tissues, cells, antigens} - A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants - A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics - A61P 7/00: Drugs for disorders of the blood or the extracellular fluid - A61P 11/00: Drugs for disorders of the respiratory system - A61P 11/02: Nasal agents, e.g. decongestants - A61P 13/10: of the bladder - A61P 13/12: of the kidneys - A61P 15/00: Drugs for genital or sexual disorders (for disorders of sex hormones A61P5/24); Contraceptives - A61P 17/00: Drugs for dermatological disorders - A61P 19/08: for bone diseases, e.g. rachitism, Paget's disease - A61P 25/00: Drugs for disorders of the nervous system - A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics - A61P 31/04: Antibacterial agents - A61P 31/12: Antivirals - A61P 31/16: for influenza or rhinoviruses - A61P 31/18: for HIV - A61P 31/22: for herpes viruses - A61P 33/02: Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis - A61P 33/06: Antimalarials - A61P 35/00: Antineoplastic agents - A61P 35/02: specific for leukemia - C07K 16/22: against growth factors {; against growth regulators} - C07K 16/28: against receptors, cell surface antigens or cell surface determinants - C07K 16/2863: {against receptors for growth factors, growth regulators} - C07K 16/32: against translation products of oncogenes - C12N 15/70: Vectors or expression systems specially adapted for E. coli - A61K 2039/505: {comprising antibodies} - C07K 2317/22: from camelids, e.g. camel, llama or dromedary - C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered - C07K 2317/52: Constant or Fc region; Isotype - C07K 2317/565: Complementarity determining region [CDR] - C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody® - C07K 2317/60: characterized by non-natural combinations of immunoglobulin fragments - Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE ## Abstract The present invention relates to the field of medical biology, and discloses a single domain antibody and derivative proteins thereof against CTLA4. In particular, the present invention discloses a CTLA4 binding protein and the use thereof, especially the use for treating and/or preventing CTLA4 relevant diseases such as tumor. ## Claims ### Claim 1 A CTLA4-binding protein, which can specifically bind to CTLA4 and comprises at least one immunoglobulin single variable domain, said immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 selected from: (1) CDR1 set forth in SEQ ID NO:1, CDR2 set forth in SEQ ID NO:2, CDR3 set forth in SEQ ID NO:3; (2) CDR1 set forth in SEQ ID NO:4, CDR2 set forth in SEQ ID NO:5, CDR3 set forth in SEQ ID NO:6; (3) CDR1 set forth in SEQ ID NO:7, CDR2 set forth in SEQ ID NO:8, CDR3 set forth in SEQ ID NO:9; (4) CDR1 set forth in SEQ ID NO:10, CDR2 set forth in SEQ ID NO:11, CDR3 set forth in SEQ ID NO:12; (5) CDR1 set forth in SEQ ID NO:13, CDR2 set forth in SEQ ID NO:14, CDR3 set forth in SEQ ID NO:15; (6) CDR1 set forth in SEQ ID NO:16, CDR2 set forth in SEQ ID NO:17, CDR3 set forth in SEQ ID NO:18; (7) CDR1 set forth in SEQ ID NO:19, CDR2 set forth in SEQ ID NO:20, CDR3 set forth in SEQ ID NO:21; (8) CDR1 set forth in SEQ ID NO:22, CDR2 set forth in SEQ ID NO:23, CDR3 set forth in SEQ ID NO:24; (9) CDR1 set forth in SEQ ID NO:25, CDR2 set forth in SEQ ID NO:26, CDR3 set forth in SEQ ID NO:27; (10) CDR1 set forth in SEQ ID NO:28, CDR2 set forth in SEQ ID NO:29, CDR3 set forth in SEQ ID NO:30; (11) CDR1 set forth in SEQ ID NO:31, CDR2 set forth in SEQ ID NO:32, CDR3 set forth in SEQ ID NO:33; (12) CDR1 set forth in SEQ ID NO:34, CDR2 set forth in SEQ ID NO:35, CDR3 set forth in SEQ ID NO:36; (13) CDR1 set forth in SEQ ID NO:37, CDR2 set forth in SEQ ID NO:38, CDR3 set forth in SEQ ID NO:39; (14) CDR1 set forth in SEQ ID NO:40, CDR2 set forth in SEQ ID NO:41, CDR3 set forth in SEQ ID NO:42; (15) CDR1 set forth in SEQ ID NO:43, CDR2 set forth in SEQ ID NO:44, CDR3 set forth in SEQ ID NO:45; (16) CDR1 set forth in SEQ ID NO:46, CDR2 set forth in SEQ ID NO:47, CDR3 set forth in SEQ ID NO:48; (17) CDR1 set forth in SEQ ID NO:49, CDR2 set forth in SEQ ID NO:50, CDR3 set forth in SEQ ID NO:51; (18) CDR1 set forth in SEQ ID NO:52, CDR2 set forth in SEQ ID NO:53, CDR3 set forth in SEQ ID NO:54; (19) CDR1 set forth in SEQ ID NO:55, CDR2 set forth in SEQ ID NO:56, CDR3 set forth in SEQ ID NO:57; (20) CDR1 set forth in SEQ ID NO:58, CDR2 set forth in SEQ ID NO:59, CDR3 set forth in SEQ ID NO:60; (21) CDR1 set forth in SEQ ID NO:61, CDR2 set forth in SEQ ID NO:62, CDR3 set forth in SEQ ID NO:63; (22) CDR1 set forth in SEQ ID NO:64, CDR2 set forth in SEQ ID NO:65, CDR3 set forth in SEQ ID NO:66; (23) CDR1 set forth in SEQ ID NO:67, CDR2 set forth in SEQ ID NO:68, CDR3 set forth in SEQ ID NO:69; (24) CDR1 set forth in SEQ ID NO:70, CDR2 set forth in SEQ ID NO:71, CDR3 set forth in SEQ ID NO:72; and (25) CDR1 set forth in SEQ ID NO:73, CDR2 set forth in SEQ ID NO:74, CDR3 set forth in SEQ ID NO:75. ### Claim 2 (depends on 1) The CTLA4-binding protein of claim 1, wherein said immunoglobulin single variable domain is a VHH. ### Claim 3 (depends on 2) The CTLA4-binding protein of claim 2, wherein the VHH is a humanized VHH. ### Claim 4 (depends on 2) The CTLA4-binding protein of claim 2, wherein said VHH comprises an amino acid sequence of any one of SEQ ID NOs:76-100. ### Claim 5 (depends on 3) The CTLA4-binding protein of claim 3, wherein said VHH comprises an amino acid sequence having at least 80%, or at least 90%, or at least 95%, or at least 99% sequence identity to any one of SEQ ID NOs:76-100. ### Claim 6 (depends on 3) The CTLA4-binding protein of claim 3, wherein said humanized VHH comprises an amino acid sequence of any one of SEQ ID NOs:101-109. ### Claim 7 (depends on 1, 5) A CTLA4-binding protein, which is obtained by affinity maturation of the CTLA4-binding protein of claim 1 or 5. ### Claim 8 (depends on 1, 5) The CTLA4-binding protein of claim 1 or 5, which comprises two said immunoglobulin single variable domain. ### Claim 9 (depends on 1, 5) The CTLA4-binding protein of claim 1 or 5, which further comprises an immunoglobulin Fc region. ### Claim 10 (depends on 9) The CTLA4-binding protein of claim 9, wherein the immunoglobulin Fc region is an Fc region of human immunoglobulin, preferably an Fc region of human IgG1. ### Claim 11 (depends on 10) The CTLA4-binding protein of claim 10, wherein the amino acid sequence of the immunoglobulin Fc region is set forth in SEQ ID NO:132. ### Claim 12 (depends on 9) The CTLA4-binding protein of claim 9, which comprises an amino acid sequence selected from SEQ ID NOs:114-128. ### Claim 13 (depends on 1, 5) The CTLA4-binding protein of claim 1 or 5, which has at least one of the following features: (a) binding to human CTLA4 with a KD of less than 1×10-7 M; (b) blocking the interaction of CTLA4 with CD80 and/or CD86; (c) enhancing activation of PBMCs and/or T cells; (d) inhibiting tumor growth. ### Claim 14 (depends on 1, 5) The CTLA4-binding protein of claim 1 or 5, which binds to CTLA4 with a KD of less than 1×10-7 M, preferably less than 1×10-8 M, more preferably less than 1×10-9 M, more preferably less than 1×10-10 M. ### Claim 15 (depends on 1, 5) A nucleic acid molecule encoding the CTLA4-binding protein of claim 1 or 5. ### Claim 16 (depends on 15) An expression vector, which comprises the nucleic acid molecule of claim 15 operably linked to an expression regulation element. ### Claim 17 (depends on 15) A recombination cell, which comprises the nucleic acid molecule of claim 15 or is transformed with an expression vector which comprises the nucleic acid molecule of claim 15 operably linked to an expression regulation element, and is capable of expressing said CTLA4-binding protein. ### Claim 18 (depends on 1, 5) A method for producing the CTLA4-binding protein of claim 1 or 5, which comprises: a) culturing a recombination cell which comprises a nucleic acid molecule encoding the CTLA4-binding protein of claim 1 or 5 or is transformed with an expression vector which comprises a nucleic acid molecule encoding the CTLA4-binding protein of claim 1 or 5 operably linked to an expression regulation element and is capable of expressing said CTLA4-binding protein under conditions that allow expression of the CTLA4-binding protein; b) recovering the CTLA4-binding protein expressed by the host cell from the culture of step a); and c) optionally further purifying and/or modifying the CTLA4-binding protein obtained from step b). ### Claim 19 (depends on 1, 5) A pharmaceutical composition comprising the CTLA4-binding protein of claim 1 or 5, and a pharmaceutically acceptable carrier. ### Claim 20 (depends on 1, 5) A method for preventing and/or treating cancer in a subject, comprising administering an effective amount of the CTLA4-binding protein of claim 1 or 5 or a pharmaceutical composition comprising the CTLA4-binding protein of claim 1 or 5, and a pharmaceutically acceptable carrier to the subject. ### Claim 21 (depends on 20) The method of claim 20, further comprising administering additional anti-tumor therapeutic means to said subject. ### Claim 22 (depends on 21) The method of claim 21, wherein said additional anti-tumor therapeutic means comprises chemotherapy, radiotherapy, antibody therapy targeting tumor-specific antigen, other tumor immunotherapy or tumor-targeting small molecule drugs. ### Claim 23 (depends on 21) The method of claim 21, wherein said additional anti-tumor therapeutic means comprises antibody therapy using anti-EGFR antibody, anti-EGFR variant antibody, anti-VEGFa antibody, anti-HER2 antibody, or anti-CMET antibody or combinations thereof. ### Claim 24 (depends on 20) The method of claim 20, wherein said cancer is selected from the group consisting of lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, renal cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, corpus carcinoma, osteosarcoma. ### Claim 25 (depends on 1, 5) A method for preventing and/or treating infectious disease in a subject, comprising administering an effective amount of the CTLA4-binding protein of claim 1 or 5 or a pharmaceutical composition comprising the CTLA4-binding protein of claim 1 or 5, and a pharmaceutically acceptable carrier to the subject. ### Claim 26 (depends on 25) The method of claim 25, wherein said infectious disease is caused by a pathogen selected from the group consisting of HIV, hepatitis virus, influenza virus, herpes virus, Giardia, Plasmodium, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa. ### Claim 27 (canceled)