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Novel bispecific antibody binding to human TLR2 and human TLR4

A bispecific antibody and antibody technology, applied in the direction of antibodies, antibody medical components, antibody mimics/scaffolds, etc., can solve the problems of bispecific antibodies that have not been reported

Active Publication Date: 2016-10-26
ASTELLAS PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, bispecific antibodies that bind to human TLR2 and human TLR4 have not been reported so far

Method used

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  • Novel bispecific antibody binding to human TLR2 and human TLR4
  • Novel bispecific antibody binding to human TLR2 and human TLR4
  • Novel bispecific antibody binding to human TLR2 and human TLR4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0264]Anti-human TLR2 antibodies were produced using human monoclonal antibody development technology "VelocImmune" (VelocImmune antibody technology; Regeneron Corporation (US Patent No. 6596541)) mice. VelocImmune mice were immunized with human TLR2 protein (R&D systems, 2616-TR-050) together with an adjuvant to induce an immune response. Lymphocytes were collected from the spleen or lymph nodes of the immunized mice according to a conventional method, and were fused with mouse myeloma cells SP2 / 0 (ATCC: CRL-1581) to produce hybridomas. Monoclonalization was carried out, and culture was carried out in CD hybridoma medium (Life Technologies Co., Ltd.), which is a serum-free medium. Antibodies were purified from the obtained culture supernatant using a Protein G column (GE ヘルスケア). In the VelocImmune technology, transgenic mice in which the variable regions of the endogenous immunoglobulin heavy and light chains are replaced by the corresponding human variable regions are used....

Embodiment 2

[0265] (Example 2: Evaluation of neutralizing activity of anti-human TLR2 antibody)

[0266] In order to evaluate the neutralizing activity of the anti-human TLR2 antibodies determined in Example 1, the human monocytic THP1-xBlue cells endogenously expressing human TLR2 were used to detect the induction of alkaline phosphatase (AP) against the TLR2 / 6 agonist Pam2CSK4. ) produced inhibition.

[0267] As a result, it was found that the anti-human TLR2 antibody (chimeric antibody) named 31-5F5-2F3 inhibited AP production and had neutralizing activity against human TLR2.

Embodiment 3

[0268] (Example 3: Sequence determination of anti-human TLR2 antibody and production of mutants)

[0269] The sequences of the genes encoding the heavy and light chains of the antibody were analyzed from the hybridoma producing the anti-human TLR2 antibody 31-5F5-2F3, and sequence determination was performed.

[0270] After the sequence of the antibody was determined, in order to improve the physical properties and stability of the antibody, the FRs of the heavy chain and light chain of 31-5F5-2F3 were replaced with FRs of other human antibodies to produce the anti-human TLR2 antibody 31-5F5-2F3.m1.

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Abstract

The invention provides a bispecific antibody for the prevention or treatment of immune inflammatory diseases by acting on both human TLR2 and human TLR4, and inhibiting a human TLR2- and human TLR4-mediated immune inflammatory effect. Provided is a bispecific antibody for human TLR2 and human TLR4, said antibody including: a heavy chain variable region of an anti-human TLR2 antibody comprising the amino acid sequence from amino acid number 1 to 120 of SEQ ID NO: 4; a light chain variable region of an anti-human TLR2 antibody comprising the amino acid sequence from amino acid number 1 to 113 of SEQ ID NO: 6; a heavy chain variable region of an anti-human TLR4 antibody comprising the amino acid sequence from amino acid number 491 to 609 of SEQ ID NO: 4; and a light chain variable region of an anti-human TLR4 antibody comprising the amino acid sequence from amino acid number 625 to 732 of SEQ ID NO: 4.

Description

technical field [0001] The invention relates to a novel bispecific antibody combined with human TLR2 and human TLR4. Background technique [0002] Toll-like receptor 2 (Toll-Like Receptor 2; TLR2) and Toll-like receptor 4 (Toll-Like Receptor 4; TLR4) are immunocompetent cells including macrophages and neutrophils, vascular endothelial cells, renal tubules A single-transmembrane protein expressed in a wide range of renal intrinsic cells such as epithelial cells and tissues (Folia Biol (Praha)., 2005, Vol.51, No.6, p.188-197). TLR2 forms a complex with TLR1 or TLR6, and reacts with bacterial components such as peptidoglycan and lipoprotein. On the other hand, TLR4 forms a complex with myeloid differentiation factor 2 (MD2) protein, CD14 protein, etc., and reacts with bacterial components such as lipopolysaccharide (LPS) represented by lipopolysaccharide (LPS). In addition, these receptors are activated by binding to damage-associated molecular patterns (DAMPs), which are end...

Claims

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Application Information

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IPC IPC(8): C12N15/09C07K16/28C07K16/46C12N1/15C12N1/19C12N1/21C12N5/10C12P21/08A61K39/395A61P31/04
CPCA61P1/18A61P29/00A61P31/04A61P37/02C07K16/2896C07K2317/31C07K2317/622C07K2317/64C07K2317/76C07K2317/92C07K2319/00A61K39/39533A61K2039/505C07K16/28C07K2317/40C07K2317/515C07K2317/52C07K2317/56C07K2317/565C12N5/12C12N15/63C12N15/64
Inventor 竹内聪佐藤雅人铃木丈太郎曽我真司高崎淳青山康二奥山千枝
Owner ASTELLAS PHARMA INC
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