CN-121591903-B - Nanometer antibody targeting CDH6 and application thereof

Abstract

The invention relates to the technical field of immunity, in particular to a nano antibody targeting CDH6 and application thereof. According to the invention, the human CDH6 nano antibody sequence is obtained through screening a natural phage library, affinity verification and CAR structure design are carried out on the human CDH6 nano antibody sequence, the human CDH6 nano antibody sequence is constructed on CAR-T, the anti-tumor function of the human CDH6 nano antibody to ovarian cancer is researched through in vitro experiments, the humanized transformation is carried out on the sequence with good anti-tumor effect, the anti-tumor function in vivo and in vitro of animals is further verified, the optimal sequence is finally obtained, and the human CDH6 nano antibody sequence can be applied to development of antibody medicines, ADC medicines and the like.

Inventors

• YAN ZHONGHUI
• GU SONGHAI
• He Yurou
• Deng Weiyan
• CAI ZHENZHEN
• ZHANG YILI

Assignees

• 赛德特生物制药有限公司

Dates

Publication Date

20260508

Application Date

20260128

Claims (10)

1. 1. The CDH6 targeting nanobody is characterized by comprising a CDR1, a CDR2 and a CDR3, wherein the sequence of the CDR1 is SEQ ID NO. 4, the sequence of the CDR2 is SEQ ID NO. 5, and the sequence of the CDR3 is SEQ ID NO. 6.
2. 2. The nanobody of claim 1, wherein the sequence is SEQ ID No.1, SEQ ID No. 2 or SEQ ID No. 3.
3. 3. A nucleic acid molecule comprising a gene encoding the nanobody of claim 1 or 2.
4. 4. An expression vector comprising the nucleic acid molecule of claim 3.
5. 5. A host cell comprising: (i) A nucleic acid molecule according to claim 3, or (Ii) The expression vector of claim 4.
6. 6. Use of any of the following in the preparation of a CDH 6-targeted chimeric antigen receptor or a CDH 6-targeted chimeric antigen receptor T cell: (a) The nanobody of claim 1 or 2; (b) The nucleic acid molecule of claim 3; (c) The expression vector of claim 4; (d) The host cell of claim 5.
7. 7. A chimeric antigen receptor targeting CDH6 comprising a signal peptide, a hinge, a transmembrane domain, a co-stimulatory domain, a signal domain, and a nanobody according to claim 1 or 2 linked to the antibody as antigen recognition domain; the costimulatory domain is 4-1BB; the signal domain is cd3ζ.
8. 8. A CDH 6-targeting chimeric antigen receptor T cell having the chimeric antigen receptor of claim 7.
9. 9. Use of any of the following in the preparation of a CDH 6-targeted drug or a CDH 6-targeted detection reagent: (a) The nanobody of claim 1 or 2; (b) The nucleic acid molecule of claim 3; (c) The expression vector of claim 4; (d) The host cell of claim 5; (e) The chimeric antigen receptor of claim 7; (f) The chimeric antigen receptor T cell of claim 8; The medicine is an ovarian cancer medicine with high CDH6 expression resistance.
10. 10. A product comprising an acceptable adjuvant or adjuvant and the chimeric antigen receptor T cell of claim 8; the product does not include food products.

Description

Nanometer antibody targeting CDH6 and application thereof Technical Field The invention relates to the technical field of immunity, in particular to a nano antibody screening targeting CDH6 and application thereof. Background Cadherin-6 (CDH 6) is a member of the Cadherin family of proteins, a single membrane glycoprotein consisting of 790 amino acids, belonging to type II cadherins, consisting of three distinct domains, an extracellular domain (ECD) containing five Cadherin sequences, a transmembrane region and an intracellular tail. CDH6 is highly expressed in malignant tumors such as human renal cell carcinoma, ovarian cancer, thyroid cancer, cholangiocarcinoma, small cell lung cancer and the like, particularly ovarian cancer (OVC) and Renal Cell Carcinoma (RCC), and CDH6 expression can be observed in about 65% -85% of OVC patients. Taking ovarian cancer as an example, about 70% -80% of patients with advanced ovarian cancer still have recurrent disease after receiving standard platinum-containing chemotherapy regimen. There is currently an unmet clinical need for such patients and a need for new therapeutic approaches. Chimeric antigen receptor-modified T cell (CHIMERIC ANTIGEN receptor modified T cells, CAR-T cells) therapy is one of the most rapidly developing adoptive cellular immunotherapy. The therapy is to transduce patient T cells with genetically engineered synthetic receptors to target cancer cell surface antigens to mediate antitumor effects. The CAR-T cell therapy mainly utilizes the immune system of a patient to attack cancer cells, and has the advantages of strong pertinence, lasting treatment effect, small side effect and the like. The prior art has the following defects that (1) the antibody is large in steric hindrance and high in immunogenicity of the traditional single-chain antibody, and (2) the target point is a CAR-T product which is lack of CDH6 targets at home and abroad at present. Aiming at solid tumors which are difficult to cure, such as ovarian cancer, renal cancer, cholangiocarcinoma and the like, which express CDH6 in a high way, a huge unmet clinical demand exists. Disclosure of Invention Therefore, the human CDH6 nanometer antibody sequence is obtained through screening a natural phage library, affinity verification and CAR structure design are carried out on the human CDH6 nanometer antibody sequence, the human CDH6 nanometer antibody sequence is constructed on CAR-T, anti-tumor functions of ovarian cancer, renal cancer and the like are researched through in vitro experiments, humanized transformation is carried out on the sequence with good anti-tumor effects, the anti-tumor functions in and out of animals are further verified, and finally a preferred sequence is obtained, and the human CDH6 nanometer antibody sequence can be applied to development of antibody medicines, ADC and the like. Compared with the traditional single-chain antibody, the alpaca-derived nano antibody has the advantages of small steric hindrance, low immunogenicity and the like. In order to achieve the above object, the present invention provides the following technical solutions: the invention provides a protein fragment, which has sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6. In some embodiments of the invention, the protein fragment may be an antibody fragment. The invention also provides a nano antibody, the sequence of the CDR1 is SEQ ID NO. 4, the sequence of the CDR2 is SEQ ID NO. 5, and the sequence of the CDR3 is SEQ ID NO. 6. In some embodiments of the invention, the nanobody described above has: (1) An amino acid sequence shown as SEQ ID NO. 1, SEQ ID NO.2 or SEQ ID NO. 3, or (2) An amino acid sequence obtained by substituting, deleting or adding one or more residues in the amino acid sequence shown in (1) and having the same or similar functions as those of (1), or (3) An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence as set forth in (1) or (2); the plurality is 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The invention also provides a nucleic acid molecule comprising a gene encoding the nanobody described above. In some embodiments of the invention, the nucleic acid molecules described above further include acceptable genetic elements, including but not limited to promoters, signal peptides, coding elements for hinges, coding elements for transmembrane domains, coding elements for co-stimulatory domains, coding elements for signal domains. In some embodiments of the invention, the morphology of the nucleic acid molecules described above may be linear or circular. The invention also provides an expression vector comprising the nucleic acid molecule. In some embodiments of the invention, the expression vectors described above include, but are not limited to, lentiviral vectors. The invention also provides a host cell comprising the nucleic