TGF beta1 antigen binding molecule and application thereof

A technology of antigen-binding molecules and structural domains, which is applied in the fields of application, anti-growth factor immunoglobulin, and medical preparations of non-active ingredients, etc., can solve the problems of high toxicity and side effects, low specific affinity of antibodies, and poor heterosexuality, and achieve The effect of high binding ability

Active Publication Date: 2022-04-12
SANYOU BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the defects in the prior art that the antibodies targeting TGFβ1 have low specific affinity, poor heterosexuality, relatively high toxicity and side effects, and poor curative effect, the present invention provides a TGFβ1 antigen-binding molecule and its application

Method used

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  • TGF beta1 antigen binding molecule and application thereof
  • TGF beta1 antigen binding molecule and application thereof
  • TGF beta1 antigen binding molecule and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] The preparation of embodiment 1 raw material

[0061] 1.1 Preparation of fusion protein huTGFβR2-Fc

[0062] According to the sequence provided by the UniProt database, the human TGFβR2 extracellular region was synthesized (UniProt numbering P37173 23-166), and the C-terminus of the gene sequence encoding the human TGFβR2 extracellular region was linked to the human IgG1 Fc segment (such as SEQ ID NO: 1), and then constructed into the eukaryotic expression vector pcDNA3.4 (Invitrogen). The obtained expression vector was expressed using the ExpiCHO transient expression system (Gibco, A29133), and the resulting supernatant was filtered through 0.22 μm and then purified using the Protein A / G affinity purification method, and then eluted with 100 mM glycine salt (pH 3.0) The qualified huTGFβR2-Fc fusion protein was obtained.

[0063] 1.2 Positive antibody preparation

[0064] The positive control antibodies GC1008 (see US20170342144 A1 for antibody sequence) and M7824 (M...

Embodiment 2

[0070] Example 2 Animal immunity and serum immune titer detection

[0071] Recombinant human TGFβ1 (purchased from Sino Biological, product number 10804-H08H1) was used as the antigen to immunize one alpaca (Nanchang Dajia Technology), once every 2 weeks, and immunized 4 times in total, each alpaca was immunized with 500 μg antigen each time , supplemented with CFA (complete Freund's adjuvant). After the alpaca immunization, the alpaca serum was taken for immune titer detection. The determination of immune titer is to measure the binding ability of immune serum to recombinant human TGFβ1 by ELISA method, and judge the immune effect according to the antibody titer of binding antigen.

[0072] The specific method is as follows: one day before the immunopotency determination, the recombinant human TGFβ1 was diluted with PBS to a final concentration of 2 μg / mL to obtain a dilution. Take 30 μL of the diluted solution and add it to the ELISA plate, and coat overnight at 4°C. On t...

Embodiment 4

[0085] Example 4 Production and Expression of Candidate Chimeric Antibodies

[0086] The VHH obtained by screening in Example 3 was fused with the human IgG1 Fc segment (as shown in SEQ ID NO: 1), wherein the C-terminal of the VHH gene sequence was connected to the N-terminal of the human IgG1 Fc segment gene sequence to construct the VHH- The expression vector of Fc chimeric antibody pcDNA3.4 (Invitrogen). Expressed by the ExpiCHO transient expression system (Gibco, A29133), the cell culture supernatant expressing the target protein was centrifuged at 15,000 g for 10 min at high speed, and the obtained supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), and then purified with 100 mM acetic acid The target protein was eluted with sodium (pH 3.0), then neutralized with 1M Tris-HCl, and finally the resulting protein was replaced into PBS buffer through an ultrafiltration concentrator tube (Millipore, UFC901096) to obtain a candidate chimeric antibody. After...

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PUM

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Abstract

The invention discloses a TGF (Transforming Growth Factor) beta1 antigen binding molecule and application thereof. The invention relates to a TGF [beta] 1 antigen binding molecule comprising a single variable domain of an immunoglobulin, the single variable domain comprising CDR1, CDR2 and CDR3, the CDR1 comprising the amino acid sequence as shown in SEQ ID NO: 2, 5, 8, 11, 14, 17, 28 or 32-33, the CDR2 comprising the amino acid sequence as shown in SEQ ID NO: 3, 6, 9, 12, 15 or 18, and the CDR3 comprising the amino acid sequence as shown in SEQ ID NO: 4, 7, 10, 13, 16, 19, 30, 31, 34 or 35. The TGF [beta] 1 antigen binding molecule has no binding ability to TGF [beta] subtypes except human TGF [beta] 1, and has strong affinity to TGF [beta] 1.

Description

technical field [0001] The invention belongs to the field of antibody medicines, and in particular relates to a TGFβ1 antigen-binding molecule, a multispecific antigen-binding molecule, a pharmaceutical composition and its application in the preparation of medicines for diagnosis, prevention and / or treatment of cancer, and immunodetection or determination thereof The method for TGFβ1, the present invention also relates to the isolated nucleic acid, recombinant expression vector, transformant and preparation method related to the TGFβ1 antigen-binding molecule and the multispecific antigen-binding molecule, as well as the TGFβ1 antigen-binding molecule comprising the TGFβ1 antigen-binding molecule, the Kits and kits of the multispecific antigen-binding molecules and / or the pharmaceutical compositions. Background technique [0002] TGFβ (transforming growth factor beta, transforming growth factor β) is a pleiotropic, pleiotropic cytokine that regulates cell proliferation, diff...

Claims

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Application Information

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IPC IPC(8): C07K16/22C12N15/13C12N15/85C12N5/10G01N33/68A61K47/68A61P35/00
Inventor 郎国竣孔超谭永聪刘亚茹胡宇豪闫鑫甜
Owner SANYOU BIOPHARMACEUTICALS CO LTD
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