Multivalent FZD and WNT binding molecules and uses thereof

A technology for binding molecules and binding sites, applied in chemical instruments and methods, anti-receptor/cell surface antigen/cell surface determinant immunoglobulins, peptides, etc., and can solve problems such as inability to use

Pending Publication Date: 2022-04-29
ANTLERA THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The hydrophobicity and sensitive tertiary structure of Wnt proteins make their biochemical purification challenging and their use in vivo and in vitro

Method used

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  • Multivalent FZD and WNT binding molecules and uses thereof
  • Multivalent FZD and WNT binding molecules and uses thereof
  • Multivalent FZD and WNT binding molecules and uses thereof

Examples

Experimental program
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preparation example Construction

[0138] One embodiment of the present invention is a method for preparing a multivalent binding molecule that activates the Wnt signaling pathway, comprising:

[0139] a) selecting an Fc domain with a C-terminus and an N-terminus, such as that of an immunoglobulin comprising a CH3 domain, such as IgG, such as IgG1,

[0140] b) recognize antibodies with binding specificity for more than one FZD receptor and

[0141] c) recognizes an antibody with binding specificity to a Wnt co-receptor;

[0142] d) producing a nucleic acid molecule comprising

[0143] (i) a nucleotide sequence encoding the selected Fc domain,

[0144] (ii) a nucleotide sequence encoding the VL and / or VH of the antibody derived from step b, and

[0145] (iii) a nucleotide sequence encoding the VL and / or VH of the antibody derived from step c,

[0146] d) expressing the nucleic acid molecule of (d) to produce a polypeptide that forms a multivalent binding molecule through dimerization of the Fc domain, the mu...

Embodiment I

[0179] 1. Development of multivalent FZD agonists

[0180] To prepare multivalent binding molecules with a first binding domain comprising a FZD diabody and a second binding domain comprising a co-receptor diabody, we selected the enriched half of the FZD receptor by using conventional phage display technology. Those bound by the cystine domain (CRD), FZD-specific antibodies were identified from a synthetic Fab phage library (Library F; see US Pub. No. 2016 / 0194394, inventor Sidhu et al.). Affinity or specific maturation is performed as desired. For example, pan-FZD binding antibody #5019 (recognizing FZD1, 2, 4, 5, 7 and 8) was matured from an FZD7-derived antibody using the FZD4 CRD as antigen. Our previous work also identified several antibodies fully specific to FZD4 (5038, 5044, 5048, 5062, 5063, 5080, 5081) or to FZD5 (2928) (see, for example, US20160194394, inventor Sidhu et al. and WO2017127933A1 , inventor Pan et al).

[0181] These FZD antibodies are used to prepa...

Embodiment II

[0220] Example II - Synthetic Antibodies Targeting FZD and LRP6

[0221] We previously applied phage display to generate hundreds of synthetic antibodies using nine recombinant FZD CRDs as antigens (the FZD3 CRD could not be purified) (Steinhart et al. Nat. Med. 23, 60 (2016); Pavlovic et al. MAbs (2018), doi: 10.1080 / 19420862.2018.1515565). System characterization revealed a continuous spectrum of specificity, with some Abs exhibiting broad specificity, such as the pan-FZD Ab (FP) recognizing FZD1 / 2 / 4 / 5 / 7 / 8 ( Figure 11A ), other Abs showed more restricted specificity, and some were monospecific ( Figure 11B ). Functional characterization revealed that some antibodies competed with Wnt and inhibited β-catenin signaling, while others were noncompetitive and did not interfere with Wnt signaling ( Figure 11B ). Altogether, we fully characterized 161 anti-FZD antibodies, including 47 Wnt signaling inhibitors. Unexpectedly, as discussed herein, regardless of whether they co...

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Abstract

Described herein are methods of affecting binding of a multivalent binding molecule to a FZD receptor and a Wnt co-receptor on a cell, wherein binding of the multivalent binding molecule to both the FZD receptor and the co-receptor on the cell activates the Wnt signaling pathway. Also described herein are multivalent binding molecules that activate the Wnt signaling pathway, comprising an FZD receptor binding domain and a Wnt co-receptor binding domain on either end of the Fc domain, and methods of using the same.

Description

Background technique [0001] This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application No. 62 / 860,161, filed June 11, 2019, the entire contents of which are incorporated herein by reference. [0002] This application contains a Sequence Listing, which has been electronically filed in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy was created on June 10, 2020, named 115773_PC424WO_SL.txt, and was 279,203 bytes in size. [0003] The Wnt signaling pathway is critical for embryonic development and tissue homeostasis in adults. Wnt ligands are secreted growth factors that regulate various cellular processes such as proliferation, differentiation, survival and migration. Wnt ligands are universally important for controlling tissue stem cell self-renewal and regulating many progenitor cell populations. The hydrophobicity and sensitive tertiary structure of Wnt proteins make their biochemical purification challe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395C12N5/071
CPCC07K16/28C07K2317/33C07K2317/92C07K2317/31C07K16/2863C07K2317/76C07K2317/622C07K2317/64C07K2317/35C07K2317/73C07K2317/526C07K2317/21C07K2317/55C07K2317/626C07K2317/75C07K16/18A61K2039/505
Inventor S·安格斯S·西杜陶余勇
Owner ANTLERA THERAPEUTICS INC
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