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Anti-hla-dq2.5 antibody

An antibody, antibody pair technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, recombinant DNA technology, DNA/RNA fragments, etc., can solve problems such as difficulty in completely eliminating gluten exposure

Pending Publication Date: 2020-06-02
CHUGAI PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In practice, however, it is difficult to completely eliminate gluten exposure even with GFD

Method used

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  • Anti-hla-dq2.5 antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0315] Expression and purification of recombinant proteins

[0316] 1.1. Recombinant HLA-DQ2.5 / 33-mer gliadin peptide complex, HLA-DQ8 / gliadin peptide complex, HLA-DQ5.1 / DBY peptide complex, HLA-DQ2.2 / CLIP Expression and purification of peptide complexes and HLA-DQ7.5 / CLIP peptide complexes

[0317] Expression and purification of recombinant HLA-DQ2.5 / 33-mer gliadin peptide complex

[0318] The sequences used for expression and purification are: HLA-DQA1*0501 (Protein Database Accession No. 4OZG) and HLA-DQB1*0201 (Protein Database Accession No. 4OZG), both of which have the CAMPATH-1H signal sequence: MGWSCIILFLVATATGVHS (SEQ ID NO :99). HLA-DQA1*0501 has a C47S mutation, a GGGG linker (SEQ IDNO: 100) and a c-fos leucine zipper sequence (PNAS, September 29, 1998; 95(20): 11828-33) and in HLA-DQA1 *Flag-Tag at the C-terminus of 0501. HLA-DQB1*0201 has a 33-mer gliadin peptide sequence: LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO: 101), and a Factor X cleavage linker (Acta...

Embodiment 2

[0334] 2.1 Establishment of J.RT3-T3.5 cell line expressing D2 TCR

[0335] The D2 TCRα chain cDNA (SEQ ID NO: 110) was inserted into the expression vector pCXND3 (WO2008 / 156083). The D2 TCRβ chain cDNA (SEQ ID NO: 111) was inserted into the expression vector pCXZD1 (US2009 / 0324589). Linearized D2 TCRα chain-pCXND3 and D2 TCRβ chain-pCXZD1 (1500 ng each) were simultaneously introduced into the J.RT-T3.5 cell line by electroporation (LONZA, 4D-Nucleofector X). The transfected cells were then cultured in a medium containing geneticin and bleomycin (Zeocin), and then sorted using AriaIII (Becton Dickinson) to obtain a high-expressing cell population. Single cell cloning is then performed to obtain cells that highly express the desired D2 TCR molecule.

[0336] 2.2 Expression of HLA-DQ2.5, HLA-DQ2.5 / gliadin peptide, HLA-DQ2.5 / CLIP peptide, HLA-DQ2.2, HLA-DQ7.5, HLA-DQ8, HLA-DQ5.1 Establishment of Ba / F3 cell lines of HLA-DQ6.3, HLA-DQ7.3, HLA-DR and HLA-DP

[0337] HLA-DQA1*050...

Embodiment 3

[0341] Generation of anti-DQ2.5 antibodies

[0342] Anti-DQ2.5 antibodies were prepared, selected and assayed as follows:

[0343] NZW rabbits were immunized intradermally with HLA-DQ2.5 / 33-mer gliadin peptide complex. Four repeated doses were given over a 2-month period, followed by blood and spleen collection. For B cell selection, biotinylated HLA-DQ5.1 / DBY peptide complexes, biotinylated HLA-DQ8 / gliadin peptide complexes, and Alexa Fluor 488-tagged HLA-DQ2.5 / 33 were prepared Polymeric gliadin peptide complex. B cells capable of binding HLA-DQ2.5 but not HLA-DQ5.1 or HLA-DQ8 were stained with the above-mentioned labeled proteins, sorted using a cell sorter, and then plated and cultured according to the steps described in WO2016098356A1. After incubation, B cell culture supernatants were collected for further analysis and B cell pellets were cryopreserved.

[0344] Specific binding to the HLA-DQ2.5 / 33-mer gliadin peptide complex was evaluated and confirmed binding to the...

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Abstract

The antibodies of the present invention have specific binding activity to HLA-DQ2.5 and may have binding activity to HLA-DQ2.2 and / or HLA-DQ7.5, but substantially no binding activity to HLA-DQ8, HLA-DQ5.1, HLA-DQ6.3, HLA-DQ7.3, HLA-DR, HLA-DP, or a complex of the invariant chain (CD74) and HLA-DQ2.5. The antibodies bind to HLA-DQ2.5 in the presence of a gluten peptide such as gliadin, i.e., bind to HLA-DQ2.5 forming a complex with the gluten peptide. The antibodies have neutralizing activity against the binding between HLA-DQ2.5 and TCR, and thus block the interaction between HLA-DQ2.5 and anHLA-DQ2.5-restricted CD4+ T cell. The antibodies do not undergo rapid internalization mediated by the invariant chain.

Description

technical field [0001] The present invention relates to anti-HLA-DQ2.5 antibodies Background technique [0002] Celiac disease is an autoimmune disorder in which ingestion of gluten causes damage to the small intestine in genetically susceptible patients (NPL 1 to 5). About 1% of the Western population, or 8 million people in the United States and the European Union, is thought to have celiac disease; however, no significant therapeutic advances have been made since the disease was recognized in the 1940s. [0003] Human leukemia antigens (HLA) belonging to class II of the major histocompatibility complex (MHC) include HLA-DR, HLA-DP, and HLA-DQ molecules, such as the HLA-DQ2.5 isotype (hereinafter referred to as "HLA- DQ2.5"), which form heterodimers consisting of α and β chains on the cell surface. The majority (>90%) of celiac disease patients have the HLA-DQ2.5 haplotype allele (NPL 6). This isoform is thought to have a stronger affinity for gluten peptides. Like ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/11
CPCC07K16/2833C07K2317/33C07K2317/76C07K2317/92A61P37/00
Inventor 大仓有生高桥德行津岛崇祖尔卡尔奈恩·哈尔富丁
Owner CHUGAI PHARMA CO LTD
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