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Humanized anti-CD47 monoclonal antibody and application thereof

A monoclonal antibody, humanized technology, applied in the application, antibody, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problem of not completely eliminating NHL and so on

Active Publication Date: 2020-03-10
CHANGCHUN GENESCIENCE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

CHAO et al.’s research on acute lymphoblastic leukemia found that anti-CD47 monoclonal antibody combined with rituximab can not only eliminate the tumor at the original transplant site, but also eliminate the tumor in the blood circulation and spread to the liver, spleen, lymph nodes and other parts, reaching Long-term survival and inhibition of tumor recurrence, while anti-CD47 monoclonal antibody or anti-CD20 monoclonal antibody can only inhibit the growth rate of NHL but cannot completely eliminate NHL

Method used

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  • Humanized anti-CD47 monoclonal antibody and application thereof
  • Humanized anti-CD47 monoclonal antibody and application thereof
  • Humanized anti-CD47 monoclonal antibody and application thereof

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preparation example Construction

[0137] The preparation method of the humanized anti-CD47 monoclonal antibody provided by the invention comprises:

[0138] Step 1: Antibody humanization, through sequence alignment on NCBI tools, humanization transformation is completed, and the transformation antibody is screened;

[0139] Step 2: Affinity maturation is performed on the screened humanized antibodies by phage display method to prepare humanized anti-CD47 monoclonal antibodies.

[0140] Specifically, the method includes:

[0141] The preparation method of the CD47 humanized antibody is as follows: using the murine antibody 059-1.43.1 prepared in the patent CN106084052A as a template, PCR amplification obtains the antibody heavy chain variable region gene VH and light chain variable region gene VL, Translate it into an amino acid sequence, then compare the amino acid sequence with the human antibody sequences in the NCBI database, and select the 5 human antibody sequences with the highest similarity to the vari...

Embodiment 1

[0143] The preparation of embodiment 1 antigenic protein, chimeric antibody and positive control antibody

[0144] 1. Antigen gene synthesis and expression vector construction

[0145] The amino acid sequence of the extracellular region of the human CD47 protein is fused with the amino acid sequence of the connecting peptide-hIgG1Fc or the connecting peptide-7his, and the amino acid sequence design is as follows:

[0146] The amino acid sequence of human CD47-hIgGFc is as follows (SEQ ID NO:47):

[0147] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPSGGGGSASEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0148] The amino acid sequence of human CD47-7His is as follows (SEQ ID NO: 48):

[0149] MWPLVAA...

Embodiment 2

[0174] Embodiment 2 Humanization

[0175] The murine antibody 059-1.43.1 in the patent CN106084052A was selected for humanization. The humanization process mainly includes human template search and transplantation.

[0176] The main target of humanization transformation is the FR sequence in the variable region. Using the amino acids of the murine antibody 059-1.43.1 VH and VL in patent CN106084052A as templates, sequence alignment was performed on the NCBI website, and 5 humanized reference sequences were found, which were used as reference templates for humanized transformation of antibody FR regions Design humanized transformation sequences.

[0177] The specific sequence of the CDR region is shown in Table 1, the specific sequence of Blastp is shown in Table 2, and the sequence of the humanized antibody after transplantation with the retained FR region is shown in Table 3.

[0178] Table 1. The specific sequence of the CDR region of the 059-1.43.1 antibody

[0179]

...

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Abstract

The invention relates to the technical field of antibody drugs, in particular to a humanized anti-CD47 monoclonal antibody and an application thereof. The humanized anti-CD47 monoclonal antibody provided by the invention can effectively inhibit tumor growth, dose-dependently block the binding of human SIRP alpha and human CD47, promote the phagocytosis effect of macrophages on tumor cells, preventthe tumor cells from evading a defense system of tumor immunity, and play an anti-tumor role; the monoclonal antibody can block the binding of the CD47 on the surfaces of the tumor cells and the SIRPalpha on the surfaces of the macrophages, block a ''do not eat me'' signal of the tumor cells, promote the recognition and uptake of the tumor cells by the macrophages, and promote the progress of phagocytizing the tumor cells; and the binding of the CD47 on the surfaces of the tumor cells and the SIRP alpha on the surfaces of the macrophages is a universal ''do not eat me''signal, and the CD47 can be used as a very promising target in the tumor immune system, and can play a powerful and effective role in human tumor treatment.

Description

technical field [0001] The invention relates to the technical field of antibody medicines, in particular to humanized anti-CD47 monoclonal antibodies and applications thereof. Background technique [0002] CD47 [0003] CD47, also known as integrin-associated protein (IAP), was originally known from the co-purification of human placenta with integrin aVβ3 and from the co-immunoprecipitation of platelets with β3 integrin, and is a transmembrane protein widely expressed on the cell surface. Glycoproteins that belong to the immunoglobulin superfamily. [0004] CD47 is a crucial marker on the cell surface, with a molecular weight between 47-55kDa, and its structure includes an amino-terminal extracellular variable region, a transmembrane region composed of 3-5 highly hydrophobic transmembrane segments and A hydrophilic carboxy-terminal cytoplasmic tail. It interacts with various ligands such as: integrins, SIRPα (signal regulatory protein alpha), SIRPγ and thrombospondin. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13G01N33/68A61K39/395A61P35/00A61P35/02
CPCA61K2039/505A61P35/00A61P35/02C07K16/2803C07K2317/24C07K2317/565C07K2317/567C07K2317/76C07K2317/92
Inventor 冯晓王涛金磊郝倩秦锁富孙大为梁阳秋闫宏博车翀肖亮
Owner CHANGCHUN GENESCIENCE PHARM CO LTD
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