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CD20/CD47 bispecific antibody and application thereof

A bispecific antibody, CD20 technology, applied in the field of biomedicine, can solve the problems of maximizing the activation of immune functions, such as obstacles and declines

Inactive Publication Date: 2020-07-17
BETA PHARM SUZHOU LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with CD20 monoclonal antibody, the CDC effect on tumor cells has decreased. It may be that this structure has a certain steric hindrance to the simultaneous targeting of CD20 / CD47, and there is a certain obstacle to the maximum activation of immune function.
In addition, the N80A mutation has no significant effect on the blocking of SIRPa and CD47, and the patent CN201811187563.0 has made an experimental demonstration

Method used

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  • CD20/CD47 bispecific antibody and application thereof
  • CD20/CD47 bispecific antibody and application thereof
  • CD20/CD47 bispecific antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0076] Example Construction of bispecific antibody (double antibody) expression vector and preparation of protein expression

[0077] 1. Construction of expression vector

[0078] Human SIRPα has two pairs of wild-type V1 or V2, the amino acid sequence 27-504 constitutes the mature V1 (NP_542970.1NCBI), V2 differs from V1 by 13 amino acids, and the 30-504 amino acid constitutes the mature V2 (CAA71403. 1GeneBank), the present invention selects wild type 2, ie V2, the amino acid sequence 31-148 of SIRPαD1 (SEQ ID NO: 1).

[0079] Link SIRPαD1 with the N-terminal of the heavy chain or the N-terminal of the light chain or the C-terminal of the light chain of the CD20 antibody through a Linker to construct gene expression vectors 9020-1RP, 9020-2RP, and 9020-6RP. SIRPaD1 mutant CD002 was constructed as a bispecific antibody corresponding to 9020-1RP-1C1, 9020-1RP replaced by Linker corresponding to 9020-1RP-1C2, and the Fc mutation of 9020-1RP corresponding to 9020-T1RP.

[0080...

experiment example 1

[0093] Experimental example 1 Bispecific antibody CD47 / CD20 binding experiment

[0094] 1. The combination of double antibody and CD47 is detected by ELISA:

[0095] Coat hCD47-his (Cat#CD7-H5227, Lot#C56P1-737F1-FA) at 1 μg / ml, choose PBS (HyCloneLot: AC13298279) as the coating buffer, 100 μl / well, and coat at room temperature (25°C) 16-18h, wash the plate twice with TBST, block with PBS+3%BSA, 200μl / well, block at room temperature (25°C) for 16-18h, wash the plate once with TBST, tap dry, and dry at 37°C for 2 hours.

[0096] Serial dilution of bispecific antibodies: prepare 9020-1RP, 9020-2RP, 9020-6RP, 9020-1RP-1C1, 9020-1RP-1C2, 9020-T1RP, CD20mAb-SD1 and antibody Rituximab according to 330 μl 10 μg / ml, recombinant protein For SIRPaD1 and CD002, 100 μg / ml is the first gradient, and a 3-fold serial dilution is performed. For example, the second gradient is to add 80 μl of the first gradient to 160 μl of PBS, and so on, a total of 8 gradient concentrations.

[0097] Incub...

experiment example 2

[0103] Experimental example 2 CD47 / CD20 expression level on cell surface

[0104] In target-specific detection, in order to be able to better select a reasonable cell line as the test object. We detected the expression levels of CD47 and CD20 on the cell surface of multiple tumor cell lines.

[0105] The CD20 / CD47 expression levels of Raji, Daudi, Ramous, HL60, MDA-MB231, SHP-77 and other blood tumor and solid tumor cell lines and RBC were measured by flow cytometry (BDFACSCelestaCellAnalyzer). The reagents and instruments used in this experiment are listed in Table 1.

[0106] Table 1. The reagent and instrument list that experimental example 2 of the present invention adopts

[0107]

[0108] The specific implementation is as follows:

[0109] 1. First observe each cell under a microscope, collect the cells when the cells are round, transparent and normal, count, and centrifuge at 1500rpm for 5min.

[0110] 2. Discard the supernatant, resuspend each cell with PBS (Hyc...

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Abstract

The invention discloses a CD20 / CD47 bispecific antibody and an application thereof. The bispecific antibody can simultaneously target CD20 and CD47 and includes an antibody against CD20 and a first functional region (SIRPalphaD1) of a human SIRPalpha membrane extracellular segment, the SIRPalphaD1 is connected with N-terminus of a heavy chain of a CD20 antibody by a connecting peptide, and the Fcpart adopts partial point mutation to enhance the ADCC / CDC; for the CD20 part, the bispecific antibody shows a stronger effect on ADCC / CDC of tumor cells than that of CD20 monoclonal antibody activated ADCC / CDC; and for the CD47 part, the bispecific antibody can effectively block the binding of CD47-positive tumor cells to SIRPalpha, and activate the phagocytic activity (ADCP) of macrophages on the tumor cells, and the phagocytic activity is stronger than that of recombinant protein SIRPalphaD1-Fc and CD47 monoclonal antibody activated ADCP. The bispecific antibody shows more specific targetedbinding and killing effects on CD20<+> / CD47<+> double-positive tumor cells, shows no binding or weak binding to single-positive cells, especially CD47<+> single-positive cells (such as erythrocyte RBC), shows no binding or weak binding to human peripheral blood monouclear cells (PBMC), has no ADCC / CDC immune killing side effects on normal cells such as RBC and PBMC, and has no agglutination sideeffects on red blood cells.

Description

technical field [0001] The present invention relates to the field of biomedicine, specifically to the field of antibody engineering technology and the field of immunity, and more specifically to a CD20 / CD47 bispecific antibody and its application. Background technique [0002] CD20 is expressed on the surface of B cells at all stages of development and differentiation except plasma cells (B cells that secrete immunoglobulin). It acts directly on B cells by regulating the flow of transmembrane calcium ions, and plays an important role in B cell proliferation and differentiation. the regulating effect. CD20 antigen is a B cell differentiation antigen, which is only located in pre-B cells and mature B cells. It is expressed in more than 95% of B-cell lymphomas, but not in hematopoietic stem cells, plasma cells and other normal tissues. Aiming at the relative specificity of CD20 antigen expression in B-cell lymphoma, multiple CD20 antibody drugs have been approved for marketing...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K39/395A61K38/17A61P35/00A61P35/02
CPCA61K38/177A61K39/3955A61P35/00A61P35/02C07K14/70596C07K16/2887C07K2317/31
Inventor 王雷尹建河董丽莎张崇骞赵永浩汤春张晓霞J·彭D·张
Owner BETA PHARM SUZHOU LTD
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