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CN-121975016-A - ScFV fragment and application thereof in preparation of medicines for treating neuroblastoma

CN121975016ACN 121975016 ACN121975016 ACN 121975016ACN-121975016-A

Abstract

The invention belongs to the technical field of biological medicines, and discloses an scFV fragment and application thereof in preparing medicines for treating neuroblastoma. The invention discloses an anti-ERBB 4 scFV antibody. According to the invention, by designing the scFV fragment specifically combined with the extracellular section of ERBB4 and simultaneously designing ERBB4.CAR T, experimental verification shows that compared with the traditional CD3 + T cell, the CART and NB cell co-culture of the extracellular section scFV of the target ERBB4 can effectively activate T cells, has stronger tumor killing effect, prompts the feasibility of using ERBB4.CAR T for preventing NB from progressing and recurrence, has better clinical transformation value, and has important significance for treating neuroblastoma.

Inventors

  • MIAO LEI
  • YANG TIANYOU
  • SONG YE
  • HE XINHENG
  • WANG HAIYUN

Assignees

  • 广州医科大学附属妇女儿童医疗中心

Dates

Publication Date
20260505
Application Date
20260130

Claims (10)

  1. 1. An anti-ERBB 4 scFV antibody, which is formed by connecting a heavy chain variable region and a light chain variable region through a linker, wherein the heavy chain variable region comprises an antigen complementarity determining regions CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, the light chain variable region comprises an antigen complementarity determining regions CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, or The heavy chain variable region comprises the complementarity determining regions CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 15, and the light chain variable region comprises the complementarity determining regions CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 17.
  2. 2. The scFV antibody of claim 1, wherein the heavy chain variable region has the amino acid sequence: A1 SEQ ID NO:10 or SEQ ID NO. 16; or (b) A2 Amino acid sequence with the same or similar functions after the amino acid sequence shown in SEQ ID NO. 10 or SEQ ID NO. 16 is modified by substitution, deletion or addition of one or more amino acids; preferably, the amino acid sequence of the light chain variable region is: B1 SEQ ID NO:14 or SEQ ID NO. 18; or (b) B2 Amino acid sequence 14 or 18, and functionally identical or similar thereto, modified by substitution, deletion or addition of one or more amino acids.
  3. 3. The scFV antibody of claim 1 or 2, wherein the linker has an amino acid sequence of (GGCAGCGCCAGC) n, (GGCGGCGGCAGC) n, (GGCGGCGGCGGCAGC) n, YAPVDV, (GGGS) n, (GGSG) n, (GGGGS) n, wherein 1≤n≤10, and n is an integer.
  4. 4. A scFV antibody according to claim 3 wherein the amino acid sequence of the scFV antibody is shown in SEQ ID No. 2 or SEQ ID No. 3.
  5. 5. A chimeric antigen receptor comprising the scFV antibody of any one of claims 1-4.
  6. 6. The chimeric antigen receptor according to claim 5, further comprising a spacer domain, a transmembrane domain, and an intracellular signaling domain; preferably, the transmembrane domain comprises a transmembrane region selected from at least one of the following proteins, polypeptide CD28、NKp30、CDS、DAP10、4-1BB、DAP12、CD3C、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、KIRDS2、OX40、CD2、CD27、LFA-1、CD278、4-1BB、GITR、CD40、BAFFR、HVEM、SLAMF7、NKp80、CD160、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、CD226、CD244、2B4、CD84、CD96、CEACAM1、CRTAM、CD229、CD160、PSGL1、CD100、SLAMF6、SLAM、BLAME、CD162、LTBR、PAG/Cbp; Preferably, the intracellular signaling domain comprises a primary signaling domain and/or a co-stimulatory signaling domain; preferably, the spacer domain comprises CH2 and CH3 regions selected from hinge domains and/or immunoglobulins; preferably, the nucleotide sequence of the chimeric antigen receptor is shown as SEQ ID NO. 6.
  7. 7. A biological material associated with the scFV antibody of any one of claims 1-4 or the chimeric antigen receptor of claim 5 or 6, wherein the biological material is any one of a 1) -a 12): a1 A nucleic acid molecule encoding the scFV antibody of any one of claims 1-4 or the chimeric antigen receptor of claim 5 or 6; a2 An expression cassette comprising a 1) said nucleic acid molecule; a3 A recombinant vector comprising the nucleic acid molecule of a 1); a4 A recombinant vector comprising the expression cassette of a 2); a5 A) a recombinant microorganism comprising a nucleic acid molecule according to a 1); a6 A) a recombinant microorganism comprising the expression cassette of a 2); a7 A) a recombinant microorganism comprising the recombinant vector of a 3); a8 A) a recombinant microorganism comprising the recombinant vector of a 4); a9 A transgenic cell line comprising a 1) said nucleic acid molecule; a10 A transgenic cell line comprising the expression cassette of a 2); a11 A transgenic cell line comprising a 3) the recombinant vector; a12 A transgenic cell line comprising the recombinant vector of a 4).
  8. 8. A chimeric antigen receptor immune cell expressing the chimeric antigen receptor of claim 5 or 6; Preferably, the chimeric antigen receptor-related biological material of claim 5 or 6 is integrated into the genome of the chimeric antigen receptor immune cell; preferably, the immune cells include any one of T cells, immune cells generated by differentiation of stem cells, monocytes, macrophages or NK cells.
  9. 9. Use of the scFV antibody of any one of claims 1-4, the chimeric antigen receptor of claim 5 or 6, the biomaterial of claim 7 or the chimeric antigen receptor immune cell of claim 8 for the manufacture of a medicament for the prevention and/or treatment of cancer.
  10. 10. A medicament comprising the scFV antibody of any one of claims 1-4, the chimeric antigen receptor of claim 5 or 6, the biological material of claim 7 or the chimeric antigen receptor immune cell of claim 8.

Description

ScFV fragment and application thereof in preparation of medicines for treating neuroblastoma Technical Field The invention belongs to the technical field of biological medicines, and particularly relates to a scFV segment and application thereof in preparing medicines for treating neuroblastoma. Background Neuroblastoma (Neuroblastoma, NB) is the most common childhood extracranial solid tumor, NB is frequently found in the adrenal gland and retroperitoneal space, and primary NB affected parts are accompanied by metastases in bone marrow, liver, skin, etc. In addition to the risk stratification system of the international neuroblastoma staging system (International Neuroblastoma STAGING SYSTEM, INSS), the international neuroblastoma risk group (International Neuroblastoma Risk Group, INRG) also provides non-INSS and targeted pre-treatment risk assessment, classifying localized tumors into L1 and L2, and metastatic tumors into M and MS, while further defining risk factors based on clinical criteria in combination with PET-CT. The high heterogeneity of NB is mainly reflected in genetic, gene expression and tumor microenvironment, low-risk and medium-risk NB infants are good in prognosis, high in cure rate and high in five-year survival rate, the survival rate is over 90%, and the total survival rate of the high-risk group, especially the infants with MYCN gene amplification, age over 18 months and specific or non-specific metastasis is not more than 50% after being treated by a first-line treatment scheme for 5 years. The current NB infants subjected to surgery combined radiotherapy and chemotherapy obviously improve the overall survival rate after receiving targeted immunotherapy such as GD2 antibody, cytokine and the like, but still have relapse and metastasis phenomena (most of the NB infants are evaluated as M-phase high risk). While current NB-targeted immunotherapy is mainly focused on antibody therapy of immune checkpoint molecules and adoptive immune cell feedback. Among them, the antibody therapy targeting GD2 and CAR-T cell feedback therapy show a certain potential, dinutuximab is a monoclonal antibody developed against GD2 on NB cell membrane, and FDA approval has been obtained for the comprehensive treatment of high risk neuroblastoma, and the optimized antibodies Dinutuximab beta, hu3F8 (naxitamab, danyelza) and CAR-T cell feedback therapy against GD2 have all achieved good effects in clinical trials of recurrent or refractory NB treatment. However, as the research is in depth, NB cells cannot fully and effectively activate the anti-tumor immune effect of effector T cell groups under the immune checkpoint inhibitors such as PD-1/PDL-1 and the immunotherapy such as CAR-T cells due to tumor mutation load and low expression level of HLA molecules, PD-1 and the like, and immune cells are exhausted to influence the effective treatment rate, so that the risk of relapse still exists. Therefore, how to further optimize the anti-GD 2 therapeutic effect and the combination method are one of the major problems to be solved. Disclosure of Invention The present invention aims to solve at least one of the technical problems in the prior art described above. Provides a scFV segment and application thereof in preparing medicaments for treating neuroblastoma. An object of the first aspect of the present invention is to provide an scFV antibody against ERBB 4. The object of the second aspect of the present invention is to provide a chimeric antigen receptor. In a third aspect, the present invention provides a biological material related to the scFV antibody of the first aspect or the chimeric antigen receptor of the second aspect. The object of the fourth aspect of the present invention is to provide a chimeric antigen receptor immune cell. An object of a fifth aspect of the present invention is to provide the use of an scFV antibody of the first aspect of the present invention, a chimeric antigen receptor of the second aspect of the present invention, a biomaterial of the third aspect of the present invention or a chimeric antigen receptor immune cell of the fourth aspect of the present invention for the preparation of a medicament for the prevention and/or treatment of cancer. The sixth aspect of the present invention is directed to a medicament. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: In a first aspect of the present invention, there is provided an anti-ERBB 4 scFV antibody comprising a heavy chain variable region comprising the antigen complementarity determining regions CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO:7 (GFDFSSYG), SEQ ID NO:8 (ISTGGSA) and SEQ ID NO:9 (ARNAGFSLYLDI) and a light chain variable region comprising the antigen complementarity determining regions CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO:11 (QASQSISSYLS), SEQ ID NO:12 (AASNLAS) and SEQ ID NO:13 (CLGTADCRFVRCNA) joined by a linker,