CTLA-4 targeting antibody and preparation method and application thereof
A CTLA-4, antibody technology, applied in the field of biomedicine, can solve problems such as lack of activity and safety
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[0156] Antibody preparation
[0157] The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences for the light and heavy chains can be fused together to form single-chain antibodies.
[0158] Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
[0159] In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.
[0160] At present, the DNA sequence encoding the antibody of the ...
Embodiment 1
[0191] The preparation of embodiment 1CTLA-4 antibody
[0192] (1) Preparation of Immunogen A
[0193] The amino acid sequence Lys36-Asp161 of the extracellular region of the human CTLA-4 protein was cloned into the pCpC vector (purchased from Invitrogen, V044-50) with a human IgG Fc fragment (hFc), and the plasmid was prepared according to established standard molecular biology methods, For specific methods, see Sambrook, J., Fritsch, E.F., and Maniatis T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). HEK293 cells (purchased from Invitrogen) were transiently transfected (PEI, Polysciences) and used FreeStyle TM 293 (Invitrogen) was expanded at 37°C. After 4 days, the cell culture fluid was collected, and the cell components were removed by centrifugation to obtain the culture supernatant containing the extracellular region of CTLA-4 protein. The culture supernatant was loaded onto a protein A aff...
Embodiment 2
[0213] Example 2 Identification of Chimeric Antibody
[0214] (1) Flow cytometry (FACS) detection of antibody binding to CTLA-4 expressing cells
[0215] Transfect the 293F cell line with the pIRES plasmid containing the full-length nucleotide sequence encoding human CTLA-4 described in step (2) of Example 1 to obtain a 293F stably transfected cell line containing human CTLA-4 (herein referred to as HEK293 -hCTLA-4 stable cell line), the pIRES plasmid with the full-length gene of monkey-derived CTLA-4, wherein the database accession number of the monkey-derived CTLA-4 nucleotide sequence is XM_005574014.1, was transfected into the HEK293 cell line HEK293 stably transfected cell line containing monkey CTLA-4 (referred to here as HEK293-cCTLA-4 stable cell line). The HEK293-hCTLA-4 stable cell line and the HEK293-cCTLA-4 stable cell line were expanded to 90% confluence in T-75 cell culture flasks, the medium was aspirated, and HBSS buffer (Hanks Balanced SaltSolution, purchased...
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Abstract
Description
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