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Antibody targeting Sirp alpha or antigen binding fragment of antibody as well as preparation and application of antibody or antigen binding fragment

A technology for combining fragments and antibodies, applied in the field of biomedicine, which can solve the problems of low expression, the inability of antibodies to be combined at the same time, and the reduction of side effects.

Pending Publication Date: 2020-09-08
L&L BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is to overcome the fact that antibodies in the prior art cannot simultaneously bind to two forms of human Sirpα, Sirpα-V1 and Sirpα-V2, so as to target more patient populations, and cannot bind to human Sirpα while not binding to human Sirpβ and Sirpγ so as to reduce side effects and low expression levels after humanization, the present invention provides an antibody targeting Sirpα or its antigen-binding fragment, bispecific antibody and its preparation method and application

Method used

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  • Antibody targeting Sirp alpha or antigen binding fragment of antibody as well as preparation and application of antibody or antigen binding fragment
  • Antibody targeting Sirp alpha or antigen binding fragment of antibody as well as preparation and application of antibody or antigen binding fragment
  • Antibody targeting Sirp alpha or antigen binding fragment of antibody as well as preparation and application of antibody or antigen binding fragment

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Experimental program
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Embodiment 1

[0130] Cloning, expression and purification of embodiment 1 antigen and antibody

[0131] Antigens used in the present invention may be purchased from different companies as follows.

[0132] Beijing Yiqiao Shenzhou Technology Co., Ltd.: Human Sirpα-V1-his (Cat. No.: 11612-H08H), Human Sirpα-V1-mFc (Cat. No.: 11612-H38H), Mouse Sirpα-his (Cat. No.: 50956-M08H), Human Sirpγ (Cat. No.: 11828-H08H); or Beijing Baipusaisi Biotechnology Co., Ltd.: human Sirpβ-hFc (Cat. No.: SIA-H5257), human Sirpγ-hFc (Cat. his (product number: B2048) or obtained by expression and purification of the present invention.

[0133] The expressed human Sirpα-V1 protein (his, or Fc Tag) sequence is NCBI Reference Sequence: NP_001035111, with a total length of 504 amino acids, of which the 1-30th is the signal peptide; the extracellular domain (ECD) is the 31-373rd amino acid. The 31st-137th amino acid of ECD is the Ig-like-V-type region, the 148th-247th amino acid is the Ig-like C1-type1 region, and t...

Embodiment 2

[0163] Embodiment 2 High expression cell line construction and cell viability (ELISA) detection

[0164] The high-expression cell lines used in the present invention are all constructed by the inventors through our company's stable cell line construction platform. The following takes the construction of human Sirpα high-expression cell lines as an example to illustrate the construction process. Specific steps are as follows:

[0165] On the first day of the experiment, 293T cells (Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, Cat#: GNHu17) were seeded in two 6cm culture dishes, and the number of cells in each culture dish reached 7.5×10 5 . On the second day, add 4 μg of the encapsulating plasmid (pGag-pol, pVSV-G and other BioVector plasmid vector strains and cell gene collection centers) and the plasmid pBabe-hSirpα for cloning the human Sirpα gene into OPTI-MEM (Thermofisher Scientific, Cat#: 31985070) , to make the final volume 20...

Embodiment 3

[0169] Example 3 Anti-Sirpα antibody and antigen binding assay (ELISA)

[0170] The human Sirpα-V1-hFc described in Example 1, Sirpα-V1-his, Sirpα-V2-his, Sirpβ-his, Sirpγ-his, monkey Sirpα-his (cynoSirpα-his, His) or different antigens (recombinant proteins) such as NOD-mSirpα-his were diluted to 1 μg / ml, 2 μg / ml, or 5 μg / ml, and added to a 96-well microtiter plate (Corning, CLS3590-100EA) at a volume of 50 μl / well. placed in a 37°C incubator for 2 hours. After discarding the liquid, add 230 μl / well of 5% skimmed milk (bright skimmed milk powder) blocking solution diluted with PBS, incubate at 37°C for 3 hours or place at 4°C overnight (16-18 hours) for blocking. Discard the blocking solution and wash the plate 5 times with PBST buffer (PH7.4PBS containing 0.05% tweeen-20), then add 50μl / well supernatant (containing detection antibody) or 10μg / ml initial, 5-fold serial dilution of the antibody to be tested , incubate at 37°C for 1 hour, wash the plate 5 times with PBST, add...

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Abstract

The invention discloses an antibody targeting Sirp alpha or an antigen binding fragment of the antibody. The antibody comprises a light chain variable region and / or a heavy chain variable region, theantibody or the antigen binding fragment thereof combines human Sirp alpha-V1 and human Sirp alpha-V2, but weakly combines or does not combine human Sirp beta and Sirp gamma, does not combine human Tcells, and has a function of blocking combination of Sirp alpha and CD47. The invention also discloses a bispecific antibody containing the antibody, and a preparation method and application of the antibody or the antigen binding fragment thereof. Due to the unique characteristics of the antibody or the antigen binding fragment thereof, the antibody or the antigen binding fragment thereof is moresuitable for drug development of the antibody or the antigen-binding fragment thereof aiming at the human Sirp alpha target, can be independently or jointly administrated as a candidate drug, and particularly provides a new or even better choice for combined immunotherapy of tumors.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to a Sirpα-targeting antibody or an antigen-binding fragment thereof and a preparation method and application thereof. The present invention also relates to a bispecific antibody comprising the Sirpα-targeting antibody or an antigen-binding fragment thereof Sexual antibodies. Background technique [0002] The SIRP family is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. According to the structure of the transmembrane region and the intracellular region, it is generally divided into Sirpα, Sirpβ, and Sirpγ. The structures of the three extracellular domains are highly homologous, and they all consist of three Ig-like domains. The intracellular regions of Sirpβ and Sirpγ are very short, only six and four amino acids, respectively, and neither has a signal motif interacting with phosphatase. The four tyrosine residues in the cytoplasmic region of Sirpα (Si...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K16/46C12N15/13C12N1/21C12N5/10A61K39/395A61P35/00
CPCC07K16/2896C07K16/2818C07K16/2827A61P35/00C07K2317/24C07K2317/31C07K2317/92C07K2317/76C07K2317/56C07K2317/565C07K2317/51C07K2317/515A61K2039/505A61K39/00C07K2317/33C07K16/2803C07K2317/622C07K2317/90A61K39/3955A61K2039/507C07K2317/20
Inventor 刘佳建
Owner L&L BIOPHARMA CO LTD
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