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Multispecific binding molecules having specificity to dystroglycan and laminin-2

A muscular dystrophy, multispecific technology, applied in the field of multispecific binding molecules, which can solve problems such as impracticality

Pending Publication Date: 2020-04-10
SANOFI SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, systemic delivery of recombinant α-dystroglycan indicated that this protein failed to reach the interstitial space for incorporation into the sarcolemma (Han, R. et al., (2009) PNAS 106(31) ,12573-12579)
Therefore, the use of recombinant α-dystroglycan as a protein replacement therapy for α-dystroglycan diseases is considered technically impractical

Method used

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  • Multispecific binding molecules having specificity to dystroglycan and laminin-2
  • Multispecific binding molecules having specificity to dystroglycan and laminin-2
  • Multispecific binding molecules having specificity to dystroglycan and laminin-2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0494] Example 1: Identification of anti-β-DG ECD, anti-LG-5 and anti-LG-4 / 5 antibodies.

[0495] method

[0496] protein expression

[0497] For expression of the murine β-DG extracellular domain (mβ-DG ECD), a construct containing an E. coli codon-optimized cassette encoding the N-terminal maltose-binding protein, TEV cleavage site, mβ-DG ( UniProt Q62165, amino acids 652-746) and a C-terminal HPC4 tag, with pET22b as the parental vector backbone. The constructs were transformed into chemically competent Origami B(DE3)pLysS cells (Novagen). Expression was performed at 37°C and IPTG induction was performed at OD = 0.6. Cells were pelleted and resuspended in lysis buffer containing EDTA-free protease inhibitors (Roche) and lysed by sonication. mβ-DG-HPC4 was purified from clarified cell lysates by treating the cell lysates on an amylose resin column (NewEngland Biolabs), cleaving the maltose-binding protein with Turbo TEV protease (EtonBiosciences), The digest was proce...

Embodiment 2

[0508] Example 2: Generation of hybridomas, monoclonal and chimeric antibodies targeting β-DG, LG-5 and LG-4 / 5.

[0509] method

[0510] Cell line production

[0511] By codon-optimizing constructs containing the extracellular and endogenous transmembrane domains of the N-terminal myc tag and β-DG (mouse UniProtQ62165, amino acids 652-893; human UniProt Q14118, amino acids 654-895), the Stable cell lines expressing human or murine β-DG on the surface. Adherent human embryonic kidney cells (HEK) and adherent Chinese hamster ovary cells (CHO-K1 ) were transfected using lipofectamine (ThermoFisher), and cells were selected with Geneticin (Gibco). Surviving cells were serially diluted to achieve uniclonal capacity and surface expression of β-DG was confirmed by anti-myc flow cytometry.

[0512] Construction of Tfr1 transmembrane domain containing N-terminal myc tag, Gly / Ser linker, LG-5 (mouse UniProt Q60675, amino acids 2932-3118; human UniProt P24043, amino acids 2936-3122)...

Embodiment 3

[0546] Example 3: Generation of bispecific antibodies recognizing β-DG and the LG-4 / 5 domain of the laminin-2α subunit

[0547] method

[0548] Generation of tetravalent bispecific tandem Ig (TBTI) antibodies

[0549] The VH and VL sequences obtained from the resulting hybridoma cells were codon optimized and synthesized for mammalian expression (Genscript). To generate a construct expressing the light chain, a VL sequence specific for β-DG, (G4S) 2 The linker, a VL sequence specific to LG-4 / 5, and human κ chain (Genbank Q502W4) or mouse κ chain (Genbank BAB33404) were fused together and cloned into the transient episomal expression vector pXL, which was derived from An analogue of the pTT vector described by Durocher et al. (Nucl. Acids Res. 2002, 30(2), E9). To generate a construct expressing the heavy chain, a VH sequence specific for β-DG, (G4S) 2 A linker, a VH sequence specific for LG-4 / 5, and human IgG1 (Genbank Q569F4) or mouse IgG1 (GenBank AAA75163.1) fused toget...

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Abstract

Provided herein multispecific (e.g., bispecific) binding molecules comprising a first binding domain that binds an extracellular portion of dystroglycan and a second binding domain that binds laminin-2. Further provided herein are methods for making such binding molecules and uses of such binding molecules for treating and / or preventing alpha-dystroglycanopathies.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 460,663, filed February 17, 2017, and European Application No. EP18305168.9, filed February 16, 2018, each of which is incorporated by reference in its entirety and into this article. [0003] Submit a sequence listing as an ASCII text file [0004] The contents of the following submitted ASCII text file are hereby incorporated by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (File Name: 183952028140SEQLIST.txt, Date of Record: February 16, 2018, Size: 315KB ). technical field [0005] The present disclosure relates to multispecific (e.g. multispecific and trivalent, or bispecific and bivalent or tetravalent) binding molecules comprising a first binding structure that binds an extracellular portion of dystrophin domain and binds the second binding domain of laminin-2. The present disclosure also relates to m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C07K16/28A61K39/395A61P21/00
CPCA61K2039/505A61P21/00C07K16/18C07K16/28C07K2317/31
Inventor L·塞维尼C·贝尔W·H·布朗迪克陈扬德S·H·程T·D·康纳斯C·德沃D·霍夫曼C·兰格M·马格纳T·马格纳C·普拉德斯E·雷奥R·魏赵紅梅朱云祥
Owner SANOFI SA
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