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Anti-CD47 antibody and production method and application thereof

An antibody and affinity technology, applied in the field of biomedicine, can solve problems such as unreported anemia, overestimation of curative effect, and difference in curative effect

Pending Publication Date: 2020-06-09
SHANGHAI PHARMAEXPLORER +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

All 16 patients using Hu5F9-G4 developed varying degrees of anemia, and some developed hyperbilirubinemia. However, low platelets were not reported. On the contrary, in the TTI-621 test group, 5 patients Thrombocytopenia of G3 and G4 grades occurred in 4 patients, but all 11 patients had stable hemoglobin, and no anemia was reported
In addition, preclinical models of anti-CD47 therapy have factors that overestimate the efficacy and the efficacy varies greatly between different tumors

Method used

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  • Anti-CD47 antibody and production method and application thereof
  • Anti-CD47 antibody and production method and application thereof
  • Anti-CD47 antibody and production method and application thereof

Examples

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preparation example Construction

[0307] The preparation method of the nucleic acid is a conventional preparation method in the art. Preferably, it includes the following steps: obtaining the nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining the nucleic acid molecule encoding the above-mentioned protein by artificial full-sequence synthesis .

[0308] Those skilled in the art know that substitutions, deletions, alterations, insertions or additions can be appropriately introduced into the base sequence encoding the amino acid sequence of the above protein to provide a polynucleotide homologue. The homologue of the polynucleotide in the present invention can be prepared by replacing, deleting or adding one or more bases in the gene encoding the protein sequence within the scope of maintaining antibody activity.

[0309] carrier

[0310] The invention also provides a recombinant expression vector comprising the nucleic acid.

[0311] The recombinant expression v...

Embodiment 1

[0383] Example 1 Preparation of CD47 Antibody

[0384] (1), preparation of immunogen A

[0385] The amino acid sequence 19-141 (Gln19-Glu141) of the extracellular region encoding the human CD47 protein (wherein, the protein sequence number of the human CD47 protein in the UniProtKB database is Q08722) was cloned into the pCPC vector (purchased from Invitrogen, V044-50) And prepare according to established standard molecular biology method (Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press) Recombinant plasmid. HEK293 cells (purchased from Invitrogen) were transiently transfected with the prepared recombinant plasmid [polyetherimide (PEI) was purchased from Polysciences] and expanded for cultivation. After 4 days, the cell culture fluid was collected and centrifuged to remove cell components. Obtain the culture supernatant that contains CD47 albumen.After adding...

Embodiment 2

[0407] Example 2 Production and Purification of Murine Antibody

[0408] The antibody concentration produced by hybridoma cells is relatively low, only about 1-10 μg / ml, and the concentration varies greatly. Moreover, various proteins produced by cell culture in the culture medium and fetal bovine serum components contained in the culture medium have varying degrees of interference with many biological activity analysis methods, so small-scale (1-5 mg) antibody production and purification are required.

[0409] The hybridoma cells obtained in Example 1 were inoculated into T-75 cell culture flasks and acclimatized and passaged for 3 generations with a production medium (Hybridomaserum free medium, purchased from Invitrogen). When the growth state is good, inoculate the cell culture spinner bottle. Add 500 ml of production medium to each 2-liter culture spinner bottle, and inoculate the cell density at 1.0×10 5 / ml. Close the bottle cap tightly, and place the spinner bottle ...

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Abstract

The invention discloses a CD47-targted antibody and a production method and application thereof, particularly discloses a novel CD47-targeted mouse antibody or a novel CD47-targeted human-mouse chimeric antibody, and discloses a method for producing the monoclonal antibody. The monoclonal antibody can bind to a CD47 antigen highly specifically, has very high affinity, and has the activity of significant anti-tumor activity and the like.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a CD47 antibody and its preparation method and application. Background technique [0002] CD47, also known as integrin-associated protein (IAP), ovarian cancer antigen OA3, Rh-associated antigen, and MER6, is a transmembrane glycoprotein widely expressed on the cell surface and belongs to the immunoglobulin superfamily. The molecular weight of CD47 is between 47-55kD, and it contains an extracellular Ig-like variable domain, five highly hydrophobic extended transmembrane segments, and a segment-selectively spliced ​​carboxy-terminal cytoplasmic tail. [0003] SIRPα (Signal Regulatory Protein α) is also a transmembrane protein, mainly expressed on the surface of macrophages, dendritic cells and nerve cells, and its extracellular region contains three immunoglobulin superfamily-like regions, of which the N-terminal The region of CD47 mediates the binding to CD47, and regulat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N5/10A61K39/395A61P35/00A61P35/02
CPCC07K16/2803A61P35/00A61P35/02C07K2317/24C07K2317/565C07K2317/567C07K2317/73C07K2317/92A61K2039/505C07K2317/76A61K35/17
Inventor 刘礼乐杨达志S·可汗段清高婧姚婧妘许志强孟亚丽王亮潘明明周文明夏珂郭翠翠童国珍王璐赵加洵王冬旭戴朝晖
Owner SHANGHAI PHARMAEXPLORER
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