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Anti-human CD19 and CD206 bispecific antibody as well as preparation method and application thereof

A bispecific antibody and specific technology, applied in the field of molecular biology, can solve problems such as lack of

Active Publication Date: 2022-04-12
BEIJING CANCER HOSPITAL PEKING UNIV CANCER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently targeting CD163 + M2 macrophages and CD20 + The mechanism of direct interaction between B lymphocytes is still lacking, but CD86 + The occurrence of M1 cell polarization response requires the induction of IFN-γ and TNF-α

Method used

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  • Anti-human CD19 and CD206 bispecific antibody as well as preparation method and application thereof
  • Anti-human CD19 and CD206 bispecific antibody as well as preparation method and application thereof
  • Anti-human CD19 and CD206 bispecific antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1, Structural design of IgG1 type anti-CD19 / CD206 bispecific antibody

[0034] First, the design of Anti-CD19-scFv and Fc region

[0035] (1) Anti-CD19 signal peptide design

[0036] Amino acid sequence (SEQ ID NO.1):

[0037] METGLRWLLLVAVLKGVQC

[0038] Corresponding nucleic acid sequence (SEQ ID NO.2):

[0039] ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGT

[0040] (2) Anti-CD19-scFv (VHs) design

[0041] Amino acid sequence (SEQ ID NO.3):

[0042] EVKLQSGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

[0043] Corresponding nucleic acid sequence (SEQ ID NO.4):

[0044] GAGGTGAAACTGCAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTAT...

Embodiment 2

[0101] Example 2. Construction of anti-CD19 / CD206 bispecific antibody eukaryotic expression system

[0102] According to the antibody structure design in Example 1, a vector that can be expressed in 293F cells was constructed using the "self-cleaving 2A peptide" system. The vector construction steps are as follows:

[0103] 1) Splicing the nucleic acid sequences corresponding to the Anti-CD19 signal peptide, Anti-CD19-scFv (VH), Anti-CD19-scFv (VL) Anti-CD19-CH2, Anti-CD19-CH3 domains according to the above sequence , and obtain the corresponding nucleic acid fragment sequence by artificial synthesis. Among them, a Linker amino acid sequence (SEQ ID NO.27: GGGGSGGGGSGGGGS) was added between the Anti-CD19-scFv (VH) and Anti-CD19-scFv (VL) structural domains, and named as Sequence fragment 1.

[0104] 2) The nucleic acid sequences corresponding to the Anti-CD206 heavy chain signal peptide, Anti-CD206-VH, Anti-CD206-CH1, Anti-CD206-CH2, and Anti-CD206-CH3 domains were spliced ​​...

Embodiment 3

[0111] Example 3, Purification of anti-CD19 / CD206 bispecific antibody

[0112] The 293F expressing cells and culture supernatants expressed for 48 hours were harvested, and the anti-CD19 / CD206 bispecific antibody molecule was purified by Protein A affinity chromatography, and the expression purity and the structure of the heavy chain and light chain of the bispecific antibody molecule were analyzed by SDS-PAGE reasonableness is checked. The results show that the anti-CD19 / CD206 molecule designed using the "self-cleaving 2A peptide" system in Example 2 of the present invention can be correctly expressed and cleaved in 293F cells, and the heavy chains of anti-CD19 and anti-CD206 Some, the molecular weight is around 49-50KD. The light chain part of Anti-CD206 has a molecular weight of about 19-25KD ( image 3 ).

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Abstract

The invention discloses an anti-human CD19 and CD206 bispecific antibody as well as a preparation method and application thereof. The antibody comprises a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein the first polypeptide chain is specifically combined with an epitope of CD19, and the second polypeptide chain and the third polypeptide chain are specifically combined with an epitope of CD206; the second polypeptide chain is covalently bound with the first polypeptide chain and the third polypeptide chain respectively. The antibody can target CD19 molecules on the surface of human CD20 + B lymphocytes and CD206 molecules on the surface of CD163 + M2 macrophages in vivo and in vitro at the same time, so that the polar distance between the CD20 + B cells and the CD163 + M2 macrophages in a tumor microenvironment is adjusted, co-localization interaction of the CD20 + B cells and the CD163 + M2 macrophages is induced, the CD163 + M2 macrophages in the tumor microenvironment are promoted, and the tumor microenvironment is improved. Under the induction of TNF (Tumor Necrosis Factor) or IFN-gamma secreted by CD20 + B lymphocytes, the macrophages are subjected to polar differentiation into CD86 + M1 macrophages to mediate tumor inhibition and killing.

Description

technical field [0001] The present application relates to the technical field of molecular biology, in particular, to a humanized IgG1 bispecific antibody against the extracellular domain of CD19 and CD206 receptors and its preparation method and application. Background technique [0002] In the current research on tumor-associated macrophages (TAMs), the consensus has been reached that TAMs are usually divided into two functional subtypes, classically activated CD86 + M1 macrophages and selectively activated CD163 + M2 macrophages. The former usually exerts anti-tumor functions, including directly mediating cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) to kill tumor cells; the latter can promote the occurrence and metastasis of tumor cells and inhibit CD8 + T cell-mediated anti-tumor immune responses promote tumor angiogenesis, leading to tumor progression. CD86 + M1 and CD163 + M2 macrophages are highly plastic and thus can transform into each ...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/85C12N5/10A61K39/395A61P35/00
Inventor 沈琳赵兴旺
Owner BEIJING CANCER HOSPITAL PEKING UNIV CANCER HOSPITAL
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