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Anti-SIRP alpha monoclonal antibody

A monoclonal antibody and antigen technology, applied in the direction of antibodies, anti-tumor drugs, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problems of reducing immune response and adverse effects

Pending Publication Date: 2022-07-01
LUNAN PHARMA GROUP CORPORATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Due to the high sequence similarity between SIRPα and sirpg (especially in the region interacting with CD47), existing published anti-SIRPα antibodies also bind sirpg and have adverse effects in humans, such as inhibiting T cell proliferation and reducing immunity answer

Method used

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  • Anti-SIRP alpha monoclonal antibody
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  • Anti-SIRP alpha monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0185] Example 1 Acquisition of anti-SIRPα antibody

[0186] (1) Prepare antigen, the concrete steps are as follows:

[0187] a. Obtain the target gene

[0188] In this example, the protein sequence of SIRPα (Uniprot: P78324) was searched from Uniprot, and amino acids 31-373 were selected, and the SIRPα extracellular region was connected with the mouse heavy chain constant region through three linking peptides (GGGGS). Sequence optimization and DNA synthesis were performed by GenScript Biotechnology Co., Ltd. The optimally synthesized PUC-57-SIRPa-mFC plasmid and PGS plasmid were digested with HindIII and EcoRI, and the target fragment was recovered by agarose gel recovery kit, and ligated with T4 DNA ligase at 16°C overnight.

[0189] b. Construction of recombinant eukaryotic expression vector

[0190] Both the sequence and the vector PGS were digested with restriction enzymes HindIII and EcoRI, and the target fragment was recovered and ligated with T4 ligase to construct ...

Embodiment 2

[0226] Example 2 Detection of Antibody Molecular Weight by SDS-PAGE

[0227] SDS-PAGE electrophoresis was carried out according to the method in the third appendix of Chinese Pharmacopoeia to identify its molecular weight and expression. The SDS-PAGE pattern of the purified antibody is as follows figure 1 As shown, the molecular weights are about 50kd and 25kd respectively, which are in line with the molecular weights of the antibody heavy chain and light chain, and the purity is above 95%.

Embodiment 3

[0228] Example 3 Variable region amplification of anti-SIRPα murine monoclonal antibody

[0229] The candidate hybridoma cells 36D6, 36G4, 39D8, 40F3, 52B3, 55G4, and 57B10 were cultured to logarithmic growth phase, and the cells were collected by centrifugation at 1000 rpm for 10 min. box prime script TM The first-strand cDNA was synthesized by RT-PCR, and the DNA sequence of the antibody variable region corresponding to the hybridoma cells was amplified by using the first-strand cDNA as the subsequent template. According to the subtype identification results, the heavy chain and light chain constant region sequences of the antibody subtype are obtained, involving specific nested PCR primers. The primer sequences used in the amplification reaction are the same as the antibody variable region first framework region and antibody variable region. Complementary constant regions.

[0230] (1) Cloning of heavy chain variable regions of murine antibodies 36D6, 36G4, 39D8, 40F3, 52...

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Abstract

The invention belongs to the technical field of biology, particularly relates to a monoclonal antibody or an antigen binding fragment combined with human sirp alpha with high affinity, and further provides an amino acid sequence and a nucleotide sequence for coding the antibody or the antigen binding fragment. The antibody provided by the invention can block the interaction between sirpalpha and CD47 without influencing the interaction between the CD47 and sirpg.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an anti-SIRPα monoclonal antibody, a preparation method and an application thereof. Background technique [0002] Signal regulatory protein alpha or SIRPα (also known as SIRPα / CD172a or SHPS-1) is a cell surface glycoprotein, a transmembrane protein belonging to the immunoglobulin superfamily, which is predominantly expressed in myeloid cell lineages (including MΦ, DC, It is expressed in granulocytes, etc.), and is characterized in that the extracellular region contains two proximal membrane IgC domains and one distal IgV domain; in the intracellular tail, there are two immunoreceptor tyrosine inhibitory motifs (ITIM). Once receptors are cross-linked, tyrosine phosphorylated ITIM sites recruit and activate SHP phosphatases to negatively regulate cellular functions, such as phagocytosis or inflammatory cytokine release. The cloning and expression of human SIRPα is described in US6541...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00
CPCC07K16/2896A61P35/00C07K2317/565C07K2317/56C07K2317/92A61K2039/505
Inventor 张贵民赵丽丽刘忠曹宇李振宇朱中松张娜
Owner LUNAN PHARMA GROUP CORPORATION
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