A kind of anti-lap monoclonal antibody, its antigen-binding fragment and application thereof
A monoclonal antibody and binding fragment technology, applied in the direction of antibodies, applications, antibody medical components, etc., can solve serious and limited clinical benefits and other problems, and achieve the effect of improving tumor immune response
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Embodiment 1
[0052] Example 1 of the present invention provides an anti-LAP monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, and the amino acid sequence of HCDR1 in the heavy chain variable region is selected from SEQ ID NO : 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 24 or SEQ ID NO: 30; the amino acid sequence of the HCDR2 of the heavy chain variable region is selected from SEQ ID NO: 2. SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 25 or SEQ ID NO: 31; the amino acid sequence of HCDR3 of the heavy chain variable region is selected from SEQ ID NO: 3 , SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 26 or SEQ ID NO: 32; the amino acid sequence of the LCDR1 of the light chain variable region is selected from SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 27 or SEQ ID NO: 33; the amino acid sequence of the LCDR2 of the light chain variable region is se...
Embodiment 2
[0054] In Example 2 of the present invention, an anti-LAP monoclonal antibody was obtained by immunizing mice with LAP antigen, optimizing the immunization method, and creating a phage display library. The anti-LAP monoclonal antibody or its antigen-binding fragment obtained by screening was selected from any of the following kind:
[0055]
Embodiment 3
[0057] Example 3 of the present invention specifically provides a screening method for anti-LAP single-chain antibody on the basis of Example 2 as follows: Step 1: LAP antigen immunization of mice
[0058] 1. Experimental animals:
[0059] Species and strains: BALB / c, female, mouse; body weight: 18-20g;
[0060] Laboratory animal provider: Beijing Huafukang Biotechnology Co., Ltd.
[0061] 2. Immunization: the mice were immunized, and the immune antigen was human LAP (a gene synthesized by Nanjing KingScript Biotechnology Co., Ltd., the company constructed a vector and expressed and purified it).
[0062] Step 2: Construction of phage antibody library
[0063]Take mouse splenocytes with higher titer, use Trizol reagent (purchased from Ambion, article number: 15596026), extract total RNA in mouse splenocytes, obtain cDNA by RT-PCR, use cDNA as a template, and use degenerate primers ( The degenerate primer reference used: Journal of Immunological Methods 233 (2000) 167-177) f...
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