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CN-121975015-A - Anti-TROP 2 nano-antibody, nano-antibody coupled drug and application thereof

CN121975015ACN 121975015 ACN121975015 ACN 121975015ACN-121975015-A

Abstract

The invention provides an anti-TROP 2 nano-antibody, a nano-antibody coupled drug and application thereof. The amino acid sequence of a heavy chain variable region CDR1 of the anti-TROP 2 nano antibody is shown as SEQ ID NO. 1, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 3. The nano antibody with small molecular weight and high affinity is obtained by screening, the antibody coupling medicine developed based on the nano antibody can specifically identify and bind the human TROP2 protein on the cell surface, has specific cytotoxicity, and has important application prospect in the development of tumor treatment medicines.

Inventors

  • Nan Shufeng
  • Gao Junbi

Assignees

  • 广州医科大学附属第一医院（广州呼吸中心）

Dates

Publication Date
20260505
Application Date
20260202

Claims (10)

  1. 1. The anti-TROP 2 nano antibody is characterized in that the amino acid sequence of a heavy chain variable region CDR1 of the anti-TROP 2 nano antibody is shown as SEQ ID NO. 1, the amino acid sequence of the CDR2 is shown as SEQ ID NO.2, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 3.
  2. 2. The anti-TROP 2 nanobody of claim 1, wherein the amino acid sequence of the heavy chain variable region of said anti-TROP 2 nanobody is shown as SEQ ID No. 4 or SEQ ID No. 6.
  3. 3. A nucleotide molecule encoding the anti-TROP 2 nanobody of claim 1 or 2; Preferably, the sequence of the nucleotide molecule is selected from the group shown in SEQ ID NO. 5 or SEQ ID NO. 7.
  4. 4. An expression vector, characterized in that, the expression vector contains the nucleotide molecule of claim 3.
  5. 5. A method for preparing an anti-TROP 2 nanobody, comprising the steps of: (S1) preparing an expression vector containing a nucleotide molecule encoding an anti-TROP 2 nanobody; (S2) transfecting a eukaryotic or prokaryotic host cell with the expression vector of step (S1); (S3) culturing the eukaryotic or prokaryotic host cell transfected in step (S2); And (S4) separating and purifying to obtain the anti-TROP 2 nano antibody.
  6. 6. A nanobody-conjugated drug, characterized in that it contains the anti-TROP 2 nanobody according to claim 1 or 2.
  7. 7. A lentiviral expression vector comprising a nucleotide molecule encoding a human TROP2 molecular membrane protein.
  8. 8. A method for constructing a cell line expressing a human TROP2 molecular membrane protein, comprising the steps of: (S1) preparing a lentiviral expression vector containing a nucleotide molecule encoding a human TROP2 molecular membrane protein; (S2) co-transfecting 293T/FT cells with the lentiviral expression vector and the lentiviral packaging plasmid of step (S1); (S3) culturing the 293T/FT cells transfected in step (S2); (S4) centrifugally collecting culture solution to obtain slow virus solution; (S5) infecting the cell line with the lentiviral solution obtained in the step (S4) to obtain the cell line expressing the human TROP2 molecular membrane protein.
  9. 9. A chimeric antigen receptor cell, characterized in that the chimeric antigen receptor cell is a CAR-T cell, a CAR-NK cell or a CAR-M cell, and the antigen binding domain of the chimeric antigen receptor is the anti-TROP 2 nanobody of claim 1 or 2.
  10. 10. Use of an anti-TROP 2 nanobody according to claim 1 or 2, a nanobody coupled drug according to claim 6 or a chimeric antigen receptor cell according to claim 9 for the preparation of a medicament for the treatment of tumors.

Description

Anti-TROP 2 nano-antibody, nano-antibody coupled drug and application thereof Technical Field The invention belongs to the biomedical field, and in particular relates to an anti-TROP 2 nano antibody, a nano antibody coupling drug and application thereof. Background TROP2 is a cell surface glycoprotein encoded by the TACSTD (tumor associated calcium signaling 2) gene in the P32 region of chromosome 1, and was first identified as a highly expressed protein on feeder cells in 1981. The TROP2 protein consists of 323 amino acids, has a molecular size of 36 kD and is a single transmembrane surface glycoprotein. It comprises mainly the following domains, hydrophobic leader peptide (1-26), extracellular domain (27-274), transmembrane domain (275-297) and hydrophobic cytoplasmic tail (298-323). The protein also contains 4 predicted N-linked glycosylation sites at positions 33, 120, 168 and 208, respectively. TROP2 can bind to various proteins such as IGF-1, claudin-7, beta-catenin and the like to act, and then the intracellular tail serine residue (S303) of TROP2 protein is phosphorylated through the action of Protein Kinase C (PKC), so that the downstream calcium ion signal channel and cyclin expression are regulated, the adhesion of fibronectin is reduced, and the growth, proliferation and transfer of cells are further promoted. TROP2 is expressed mainly in epithelial cells under normal physiological conditions and has a key role in embryonic development. The expression of TROP2 can be detected by normal tissues such as skin, cornea, salivary gland, respiratory tract, lung and the like, but the expression level is low, however, the high expression condition exists in various human malignant tumors such as breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, ovarian cancer and the like, and the expression level of TROP2 is related to the malignant degree of the disease. The cause of TROP2 overexpression in various tumor cells is not completely understood, and some studies now show that a series of transcription factor networks, including TP63/TP53, ERG, GRHL1/Get-1, HNF1A/TCF-1, etc., can regulate TROP2 expression, thereby causing deregulation of transcription and post-transcriptional levels. It is also because of the expression characteristics and action mechanism of TROP2, which makes it an excellent natural tumor therapeutic target, and drug development for this target is also being continuously performed. An Antibody conjugated drug (anti-body-Drug Conjugates, ADC) is formed by coupling a monoclonal Antibody (anti-body) targeting tumor specific antigen or tumor related antigen with different numbers of small molecule cytotoxins (Payload) through a Linker (Linker). The monoclonal antibody has the dual advantages of high targeting property of the monoclonal antibody and high activity of the cytotoxic drug in tumor tissues, and is one of the drug categories which are most rapidly developed in the tumor targeted treatment field in recent years. ADC drugs against TROP2 targets have been successful in various tumor treatments, for example, golian Sha Tuozhu mab (Tuodawei, trodelvy) has been batched for the treatment of advanced Triple Negative Breast Cancer (TNBC) and advanced HR +/HER2- breast cancer. In addition, ADC drugs such as Dato-Dxd, SKB264 and the like also show good curative effects in other solid tumors such as non-small cell lung cancer and the like. However, to date, nanobody ADC drugs against TROP2 have not been published on the market. Whereas nanobodies (Nb), i.e. Heavy Chain single domain antibodies VHH (Variable domain of HEAVY CHAIN of Heavy-Chain antibodies), are derived from a Heavy Chain Antibody (HCAb) that lacks a light Chain naturally occurring in camelids. The single domain antibody consisting of only one heavy chain variable region obtained by cloning the variable region is the minimum unit which has complete functions, is stable and can be combined with antigen. The nano antibody has the characteristics of high stability, good water solubility, simple humanization, high targeting property, strong penetrability, convenient transformation and the like, and plays a great role in immune experiments, diagnosis and treatment as an emerging force in the diagnosis and treatment of a new generation of antibodies. Therefore, there is a need in the art to develop an anti-TROP 2 nanobody, in particular an anti-TROP 2 nanobody with good TROP2 antigen binding capacity and stronger functional activity, and to construct an ADC medicament for anti-tumor application based on the same. Disclosure of Invention Aiming at the defects existing in the prior art, the invention aims to provide an anti-TROP 2 nano-antibody, a nano-antibody coupling drug and application thereof. The invention provides a research foundation for the research based on TROP2 targets, including antibody coupling drugs, antigen chimeric antibody cell drug