Recombinant antibody-like T cell antigen receptor, T cell antigen receptor conjugate, bi-specific molecule and application

Abstract

The invention discloses a recombinant antibody-like T cell antigen receptor, a T cell antigen receptor conjugate, a bi-specific molecule and application. By designing various IgG1 constant region and1G4113 fusion expression forms, the fusion protein form that soluble expression can be conducted in mammalian cells is obtained through screening, and the high-purity recombinant antibody-like T cellantigen receptor can be obtained simply by one-step purification. The recombinant antibody-like T cell antigen receptor is coupled with MMAE micromolecules to obtain the T cell antigen receptor conjugate, and the T cell antigen receptor conjugate can be used for being prepared into an anti-tumor drug. The recombinant antibody-like T cell antigen receptor is coupled with fluorescent dye molecules so as to be used for in-vivo positioning of the drug or tumor imaging. The recombinant antibody-like T cell antigen receptor is coupled with an anti-CD3 antibody to obtain the soluble expression bi-specific molecule, and the bi-specific molecule can be used for being prepared into the anti-tumor drug.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a recombinant antibody-like T cell antigen receptor, a T cell antigen receptor coupled drug, a bispecific molecule and its application. Background technique [0002] Cancer has become an important disease that endangers human health. In recent years, tumor immunotherapy represented by tumor immune checkpoint inhibitors, adoptive immune cell therapy and individualized tumor vaccines has shown unprecedented anti-tumor therapeutic effects, and has become a hot spot in cancer research and clinical treatment. The selection of tumor antigen targets is a key factor for the success of tumor immunotherapy. Tumor neoantigens have attracted extensive attention from researchers due to their tumor specificity, and have become potential ideal targets for tumor immunotherapy. [0003] Somatic mutation is an important driving factor for the development of tumors. There are a large number of...

AI Technical Summary

Problems solved by technology

Although the antibody affinity (generally 10-100nM) obtained by screening is already at a relatively high level, it is still difficult to reach the level of antibody drugs (generally <10nM), and further modification and maturation are needed.

Among the more than 50 published TCRm, there are only 2 cases of antibodies targeting tumor neoantigens, respectively targeting the G12V mutation of KRAS and HLA-A*0201, the L858R mutation of EGFR and the pMHC formed by HLA-A*0301 , and the specificity of the latter is not ideal

There are only a small number of amino acid residue changes in the pMHC formed between the mutant protein and the normal protein, and the amino acid residues of the antigenic peptide are easily buried by MHC, which makes the screening of TCRm antibodies targeting tumor neoantigens extremely difficult and workload-intensive. Disadvantages such as poor specificity

The recognition of TCR to pMHC is highly specific, and the specific TCR that can recognize tumor neoantigens is expected to become a tumor-specific TCR targeting tumor cells, but the affinity of TCR is not satisfactory (generally 1-100 μM), which is also a limitation of TCR. As a bottleneck for the widespread application of drugs

[0005] On the other hand, TCR is a membrane protein, which is difficult to express solublely, which also limits the application of TCR as a protein drug and increases the difficulty of evaluating TCR activity in vitro

Jonathan (BOULTER J M, GLICK M, TODOROV P T, et al. Stable, soluble T-cell receptor molecules for crystallization and therapeutics [J]. Protein Eng, 2003, 16 (9): 707-711.) etc. T48C and TRBC of TRAC S57C was mutated and expressed in Escherichia coli, and a variety of soluble TCRs were successfully obtained through renaturation. Although this method is suitable for the preparation of a variety of soluble TCRs, there are the following problems: 1. The renaturation efficiency is low (≈40 %); 2. Escherichia coli has endotoxin and other toxic substances that are difficult to remove and are harmful to the human body; 3. The method includes expression and purification of E. coli inclusion bodies, renaturation and purification by ion exchange columns and molecular sieves after renaturation , the process is very cumbersome, and the technical requirements are high; 4. The purity of the obtained product is about 95%, which still needs to be further improved to meet the follow-up medicine, etc.

Therefore, the application of TCR as a drug molecule is currently limited

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