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CN-121991232-A - Anti-LOXL 2 nano antibody or antigen binding fragment and application

CN121991232ACN 121991232 ACN121991232 ACN 121991232ACN-121991232-A

Abstract

The invention belongs to the field of medical biology, and relates to an anti-LOXL 2 nano antibody or antigen binding fragment and application. The anti-LOXL 2 nanobody or antigen binding fragment comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequence of the CDR1 is shown as one of SEQ ID NO: 1-SEQ ID NO:3, the amino acid sequence of the CDR2 is shown as one of SEQ ID NO: 5-SEQ ID NO:8, and the amino acid sequence of the CDR3 is shown as one of SEQ ID NO: 10-SEQ ID NO: 13. The anti-LOXL 2 nano antibody has the advantages of small molecular weight, high affinity and better specificity. Has good prospect for diagnosing fibrosis.

Inventors

  • YANG XING
  • Wei Zhuxin
  • ZHANG NING
  • WAN WUZHOU

Assignees

  • 北京大学人民医院
  • 云南白药集团股份有限公司
  • 云核医药(天津)有限公司

Dates

Publication Date
20260508
Application Date
20250521

Claims (10)

  1. 1. An anti-LOXL 2 nanobody or antigen binding fragment comprising three complementarity determining regions CDR1, CDR2 and CDR3 wherein, The amino acid sequence of the CDR1 is shown as one of SEQ ID NO 1-SEQ ID NO 3; the amino acid sequence of the CDR2 is shown as one of SEQ ID NO 5-SEQ ID NO 8; The amino acid sequence of the CDR3 is shown as one of SEQ ID NO 10-SEQ ID NO 13.
  2. 2. The anti-LOXL 2 nanobody or antigen-binding fragment of claim 1, further comprising four framework regions FR1, FR2, FR3 and FR4 alternating with three complementarity determining regions, wherein, The amino acid sequence of FR1 is shown as one of SEQ ID NO. 15-SEQ ID NO. 19; the amino acid sequence of FR2 is shown as one of SEQ ID NO. 20-SEQ ID NO. 24; the amino acid sequence of FR3 is shown as one of SEQ ID NO. 25-SEQ ID NO. 29; the amino acid sequence of FR4 is shown as one of SEQ ID NO. 30-SEQ ID NO. 31.
  3. 3. The anti-LOXL 2 nanobody or antigen-binding fragment of claim 1, wherein the nanobody or antigen-binding fragment comprises one of the following sequences: (i) An amino acid sequence shown as one of SEQ ID NO 32-SEQ ID NO 35; (ii) Amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity and identical function to the amino acid sequences shown in one of SEQ ID NO's 32-SEQ ID NO. 35; (iii) And adding, substituting, deleting or inserting an amino acid sequence with 1 or more amino acid residues into the amino acid sequence shown as one of SEQ ID NO. 32-SEQ ID NO. 35, and retaining the function of the amino acid sequence shown as one of SEQ ID NO. 32-SEQ ID NO. 35.
  4. 4. A nucleic acid molecule encoding an anti-LOXL 2 nanobody or an antigen-binding fragment according to any one of claims 1-3.
  5. 5. A vector comprising the nucleic acid molecule of claim 4.
  6. 6. A host cell comprising the vector of claim 5 or the nucleic acid molecule of claim 4, preferably a bacterial cell, a fungal cell, an animal cell, a plant cell or a progeny cell of these cells.
  7. 7. A method of producing anti-LOXL 2 nanobodies comprising the steps of: (a) Culturing the host cell of claim 6 under conditions suitable for nanobody production, thereby obtaining a culture comprising said anti-LOXL 2 nanobody; (b) Isolating and/or recovering the anti-LOXL 2 nanobody from the culture, and optionally (C) Purifying and/or modifying the anti-LOXL 2 nanobody obtained in step (b).
  8. 8. An antibody-conjugated drug comprising the anti-LOXL 2 nanobody or antigen-binding fragment of any one of claims 1-3, a linker, and an effector, preferably wherein the effector comprises at least one of a nuclide, a cytotoxic agent, a fluorophore, an enzyme that catalyzes the development of a substrate, a chemiluminescent agent, and a nanoparticle-based label; The nuclide is a diagnostic radionuclide or a therapeutic radionuclide, the diagnostic radionuclide is preferably at least one of 18 F、 32 P、 33 P、 45 Ti、 47 Sc、 52 Fe、 59 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 75 Sc、 77 As、 86 Y、 90 Y、 89 Sr、 89 Zr、 94 Tc、 94 Tc、 99m Tc、 99 Mo、 105 Pd、 105 Rh、 111 Ag、 111 ln、 123 I、 124 I、 125 I、 131 I、 142 Pr、 143 Pr、 149 Pm、 153 Sm、 154"1581 Gd、 161 Tb、 166 Dy、 166 Ho、 169 Er、 175 Lu、 177 Lu、 186 Re、 188 Re、 189 Re、 194 lr、 198 Au、 199 Au、 211 At、 211 Pb、 212 Bi、 212 Pb、 213 Bi、 223 Ra and 225 Ac, and the therapeutic radionuclide is preferably at least one of 32 P、 47 Sc、 57 Co、 89 Sr、 90 Y、 103 Pd、 106 Ru、 124 I、 125 I、 131 I、 131 Cs、 137 Cs、 177 Lu、 192 Ir、 212 Bi and 225 Ac.
  9. 9. A pharmaceutical composition comprising an anti-LOXL 2 nanobody or antigen-binding fragment according to any one of claims 1-3, or an antibody-conjugated drug according to claim 8.
  10. 10. The use of the anti-LOXL 2 nanobody or antigen-binding fragment of any one of claims 1-3, or the antibody-conjugated drug of claim 8, for: (i) Use of a reagent for detecting a LOXL 2-mediated disease, preferably a kit for detecting a LOXL 2-mediated disease, or (Ii) Use in the manufacture of a medicament for the treatment of LOXL2 mediated diseases; The LOXL2 mediated disease is preferably cancer, connective tissue disease or cardiovascular disease; The cancer is preferably at least one of head and neck squamous cell carcinoma, breast cancer, lung cancer, colorectal cancer, gastric cancer, cervical cancer, liver cancer, esophageal cancer, pancreatic cancer, oral squamous cell carcinoma, and renal cell carcinoma; the connective tissue disease is preferably at least one of skin relaxation and fibrosis; the cardiovascular disease is preferably at least one of atherosclerosis and myocardial ischemia.

Description

Anti-LOXL 2 nano antibody or antigen binding fragment and application The application is a divisional application of an application patent application with the application date of 2025, 5, 21, the application number of 202510658983.6 and the name of 'an anti-LOXL 2 nano antibody or antigen binding fragment and application'. Technical Field The invention belongs to the field of medical biology, and particularly relates to an anti-LOXL 2 nano antibody or antigen binding fragment and application. Background Early fibrosis is reversible and curable, so early diagnosis of fibrosis and judgment of the extent thereof are of great importance. Aiming at the molecular level change in the fibrosis process, a fibrosis related biological marker is searched, and the method has important clinical value for realizing early diagnosis of fibrosis. Furthermore, although there is currently much evidence for the pathological and molecular biological basis of fibrosis, there is still a need to develop more intensive studies in terms of defining anti-fibrosis targets. Related intervention targets currently have a novel molecular target mechanism such as a TGF-beta classical pathway, a peroxisome proliferator-activated receptor (PPAR), an angiotensin receptor, an endothelin receptor, a farnesyl X receptor, a cannabinoid receptor and the like. Numerous antibody drugs targeting different targets are now approved for clinical use but the efficacy needs to be further verified, and there is an urgent need to discover new therapeutic targets and develop new safer and more effective therapies. LOXL2 is a member of the LOX family, having a conserved catalytic domain that promotes oxidative deamination of lysine residues, thereby cross-linking collagen and elastin, affecting collagen fiber stiffness, involved in remodeling of the extracellular matrix of tumors and cardiovascular-related diseases, whose expression and activity changes can lead to a variety of diseases, and is considered a biomarker of progression of various diseases such as liver, lung and heart fibrosis, scleroderma and cancer. LOXL2 is therefore a potential target for diagnosis and treatment of tumor and fibrosis-related diseases. Currently, LOXL2 has been studied as a variety of targeted inhibition drugs, including GB2064, which has entered phase II clinical trials as a selective, mechanism-based small molecule inhibitor. Nanobodies are the smallest fragments known to date that can bind antigen, only 1/10 the size of monoclonal antibodies, with structural stability and binding activity comparable to the original heavy chain antibody. Compared with the traditional antibody, the nano antibody has various unique advantages, such as good tissue penetrating capacity, quick removal, easy production and modification, high stability, lower immunogenicity and the like, and is a novel antibody molecule with very good application prospect. The nano antibody fuses the advantages of the small molecular polypeptide and the traditional antibody, has wide application prospect and clinical value in the aspects of targeted therapy and accurate diagnosis, but the antibodies entering clinical experiments at present are few. The traditional monoclonal antibody has the disadvantages of complex preparation process, high production cost, high molecular weight and poor tissue penetrating capacity. Nanobodies against LOXL2 targets have not been reported and clinically used, so there is an urgent need in the art to develop new specific nanobodies that are effective against LOXL2 targets. Disclosure of Invention Definition of the definition Unless otherwise indicated or defined, all terms used have the usual meaning in the art, which will be understood by those skilled in the art. Moreover, unless otherwise indicated, all methods, steps, techniques and operations not specifically detailed may be, and have been, performed in a manner known per se, which will be appreciated by those skilled in the art. The terms "antibody" or "immunoglobulin" are used interchangeably herein to refer to either heavy chain antibodies or conventional four chain antibodies, unless otherwise indicated, as general terms to include full length antibodies, individual chains thereof, and all portions, domains, or fragments thereof (including but not limited to antigen binding domains or fragments, e.g., VHH domains or VH/VL domains, respectively). Furthermore, the term "sequence" (e.g. in terms of "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence", etc.) as used herein is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding said amino acid sequence, unless the context requires a more defined interpretation. The invention aims to provide an anti-human/mouse nanobody capable of blocking LOXL2 fiber crosslinking, and simultaneously provides a coding sequence of the nanobody, a preparation method and applicatio