Prophylactic and/or therapeutic agent for inflammatory lung disease

An inflammatory and therapeutic agent technology, applied in the field of preventive and/or therapeutic agents for inflammatory lung diseases, can solve the problems that cannot be said to be fundamental treatment, cannot expect fibrosis inhibition, improvement, etc., to achieve inhibition of proliferation, inhibition of differentiation effect

Pending Publication Date: 2022-05-27
UNIV OKAYAMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a drug for the treatment of idiopathic pulmonary fibrosis, two molecular targeted therapy drugs (pirfenidone (Pirfenidone) and nintedanib (Nintedanib)) are known (non-patent literature 2, 3), but the actual situation It is all symptomatic therapy, and the suppression and improvement of fibrosis cannot be expected, and it cannot be said to be a fundamental treatment.

Method used

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  • Prophylactic and/or therapeutic agent for inflammatory lung disease
  • Prophylactic and/or therapeutic agent for inflammatory lung disease
  • Prophylactic and/or therapeutic agent for inflammatory lung disease

Examples

Experimental program
Comparison scheme
Effect test

reference example 1

[0114] (Reference Example 1) Preparation of S100A8 / A9 Heterodimer for Anti-S100A8 / A9 Antibody Production

[0115] In this reference example, the preparation of the S100A8 / A9 heterodimer, which is an antigen used for the preparation of the anti-S100A8 / A9 antibody shown in the following examples, will be described. An expression vector obtained by incorporating full-length S100A8 and full-length S100A9 into pET21 (see figure 1 ) S100A8 / A9 heterodimer was produced by E. coli and purified (see Futami J. et al., Biochem Biophys Rep., 19; 6: 94-100, (2016)). As a comparative example, full-length S100A8 or full-length S100A9 was incorporated into pET21, produced in E. coli by the same method as above, and purified (see Futami J. et al. (2016)).

[0116] The purified S100A8 / A9 heterodimer and the S100A8 monomer and S100A9 monomer as comparative examples were subjected to SDS-PAGE and CBB staining, and the results are shown in figure 2 . For the S100A8 / A9 heterodimer, it was confir...

Embodiment 1

[0118] (Example 1) Production of anti-S100A8 / A9 monoclonal antibody

[0119] In this example, the preparation of the anti-S100A8 / A9 monoclonal antibody used in the following examples and experimental examples will be described. The anti-S100A8 / A9 monoclonal antibody of this example was prepared using the S100A8 / A9 heterodimer prepared in the above (Reference Example 1) as an antigen.

[0120] (1) Preparation of hybridomas

[0121] The anti-S100A8 / A9 monoclonal antibody of this example was produced using the S100A8 / A9 heterodimer prepared in the above (Reference Example 1) as an antigen, using a monoclonal antibody customization service, Genostaff (Genetics, Japan). Mice (Balb / c) were used as immunized animals, and Titer-MAX was used as an adjuvant in the immunization of the antigen. According to the conventional method, the spleen of the immunized animal was fused with mouse myeloma cells (P3U1) using polyethylene glycol (PEG1500) to prepare hybridomas, and 160 clones were o...

Embodiment 2

[0128] (Example 2) Screening of neutralizing antibodies

[0129] In this Example, the monoclonal antibodies produced by the 160 cloned hybridomas produced and selected in Example 1 were examined for their influence on the production of S100A8 / A9-induced inflammatory cytokines. Using human keratinocytes in which inflammatory cytokines are strongly induced by S100A8 / A9, the inhibitory effect of each antibody on S100A8 / A9 signaling was evaluated using the mRNA expression levels of inflammatory cytokines as an index. Specifically, 30 ng / mL of purified S100A8 / A9 and each anti-S100A8 / A9 monoclonal antibody obtained by purification from 1 mL of 160 cloned hybridoma culture supernatants by a Protein G column were added to keratinocytes. (Normal Human Keratinocytes: NHK), the cells cultured at 37°C for 3 hours were collected, and the mRNA expression levels of TNF-α, IL-6, and IL-8 were analyzed by real-time quantitative PCR (qPCR).

[0130] Real-time quantitative PCR (qPCR) analysis w...

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Abstract

The present invention provides an inflammatory lung disease agent capable of effectively preventing and / or treating inflammatory lung diseases, and more specifically, to a prophylactic and / or therapeutic agent for inflammatory lung diseases, comprising, as an active ingredient, an antibody or an antibody fragment having antigen-binding activity with respect to S100A8 / A9 heterodimers. By blocking the interaction between S100A8 / A9 and its receptor group, inflammatory lung diseases can be effectively prevented and / or treated. Specifically, by blocking the interaction between S100A8 / A9 and RAGE, which is a receptor thereof, the expression of NF-[kappa] B, which induces the expression of various inflammatory cytokines as a transcription factor downstream of RAGE, is inhibited, and the proliferation of activated fibroblasts is inhibited, and further, by inhibiting the differentiation of activated fibroblasts into myofibroblasts, the proliferation of myofibroblasts is inhibited. The inflammatory lung diseases can be effectively prevented and / or treated. In addition, the prophylactic and / or therapeutic agent for inflammatory lung disease according to the present invention can be suitably used as a prophylactic and / or therapeutic agent for COVID-19.

Description

technical field [0001] The present invention relates to the prevention of inflammatory lung diseases using, as an active ingredient, an antibody or antibody fragment having an antigen-binding activity to a heterodimer of S100A8 protein (also referred to as "S100A8") and a heterodimer of S100A9 protein (also referred to as "S100A9"). and / or therapeutic agents. [0002] This application claims the priority of Japanese Patent Application No. 2019-197222 incorporated herein by reference. Background technique [0003] S100 proteins are cell-type-specifically expressed calcium-binding proteins with two EF-hands, and 20 subfamilies have been identified so far. S100A8 (MRP8, calgranulin A, calgranulin A) is a member of the S100 family of calcium binding proteins, and is usually co-expressed with S100A9 (MRP14, calgranulin B, calgranulin B). S100A8 / A9 (Calprotectin), which is a heterodimer of S100A8 and S100A9, accumulates in body fluids during inflammation and is thought to be inv...

Claims

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Application Information

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IPC IPC(8): A61K39/395A61P11/00A61P29/00A61P43/00C07K16/18
CPCC07K16/18A61P11/00A61P29/00A61P43/00A61K2039/505C07K16/24C07K2317/76C07K2317/24A61P31/14C07K2317/33C07K2317/565
Inventor 阪口政清丰冈伸一木下理惠荒木恒太
Owner UNIV OKAYAMA
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