Anti-cxcr4 antibodies

An antibody and CDR2L technology, applied in the direction of antibodies, anti-tumor drugs, radioactive carriers, etc.

Pending Publication Date: 2019-09-13
エムエスエムプロテインテクノロジーズインコーポレイテッド
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Despite the existence of several agents targeting CXCR4 available or und

Method used

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  • Anti-cxcr4 antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1: Preparation of Fab phage library and screening of phage antibodies

[0088] The antibodies of the invention were originally derived from a Fab library of size 10^11 comprising a pSF1 phagemid carrying a Fab E. coli codon-optimized synthetic gene encoding a human Fab heavy chain with randomized CDRs and human Fab light chain. For the heavy chain the framework DP47 was used, for the light chain the framework DPK22 was used.

[0089] Procedures as described in Knappik et al. (2000) J.Mol.Biol.296:57-86; Lee et al. (2004) J.Mol.Biol.340:1073-93; Hoet et al. (2005) 23:344-8 were used and CDR randomization schemes to generate phage libraries.

[0090] More specifically, in the Fab library, heavy chain CDR1, CDR2 and CDR3 and light chain CDR3 were randomized. For randomization of heavy chain CDR3, trinucleotide-based oligonucleotides were employed as described in Knappik et al., while for the other CDRs standard nucleotide mixtures were used to generate CDR oli...

Embodiment 2

[0097] Example 2: Affinity maturation

[0098] Initial candidates were then subjected to affinity maturation as described above. Two affinity maturation libraries were generated by randomization by CDR2H or CDR3L, respectively. Each of these libraries was subjected to two rounds of high stringency selection. Selected Fabs are expressed individually and clones with improved binding properties are retained. The best clones were reconstituted into IgG characterized by the best CXCR4 binding affinity and selectivity and the best ability to prevent ligand-induced Ca-flux. As a final step, the most promising heavy and light chains were recombined and the resulting IgG recharacterized as previously described.

[0099] In addition, CDR3H is matured by introducing point mutations. The resulting mutated Fab fragments were characterized to identify optimal CXCR4 binders.

[0100] The following mature antibody candidates were further obtained:

[0101]

[0102]

[0103] *R10...

Embodiment 3

[0105] Example 3: Synthesis of IgG Antibody

[0106] The CHO / pTT transient transfection system from the National Research Council Biotechnology Research Institute of Canada (NRC-BRI) was used according to the protocol provided by NRC-BRI. See International Patent Application Publication WO2009 / 137911 Al. More specifically, each IgG of interest was produced in CHO-3E7 cells co-transfected with pTT vectors expressing the light and heavy chains of that IgG, respectively, using polyethyleneimine (PEI) as the transfection reagent.

[0107]IgG containing cell culture medium was collected and IgG was purified on protein A plus agarose (Pierce) using standard methodology. Use sterile, endotoxin-free solutions for all purification procedures.

[0108] In the following examples, the Fab fragment of interest or the IgG antibody of interest is referred to as "test Fab", "test antibody" or "test IgG", as appropriate.

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Abstract

The present invention relates to monospecific antibodies against CXCR4 or binding fragments thereof, to the use of such anti-CXCR4 antibodies or binding fragments in treating diseases whose pathogenesis is related to activation of CXCR4, as well as to pharmaceutical compositions comprising such anti-CXCR4 antibodies or binding fragments.

Description

[0001] field of invention [0002] The present invention relates to monospecific anti-CXCR4 antibodies and binding fragments, to the use of such anti-CXCR4 antibodies and binding fragments in the treatment of diseases whose mechanism of the invention involves the activation of CXCR4, and to pharmaceutical combinations comprising such anti-CXCR4 antibodies and binding fragments Materials and kits. Background technique [0003] G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors, form a superfamily of proteins that often play important roles in a variety of biological and pathological processes. Representing a subfamily of GPCRs, chemokine receptors are named for the ability of their ligands (ie, chemokines) to induce directional chemotaxis in nearby responding cells. Among these chemokine receptors, CXCR4 (also known in the art, for example, LESTR, fusin or CD 184) plays an important role in immune and inflammatory responses by mediating dir...

Claims

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Application Information

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IPC IPC(8): A61K39/395A61K51/10C07K16/28C12N15/13
CPCA61K51/1033G01N33/574C07K16/2866A61K2039/505C07K2317/21C07K2317/55C07K2317/565C07K2317/732C07K2317/76C07K2317/92G01N33/57426A61P35/00A61P35/04A61K39/395G01N33/57415
Inventor A·麦卡利斯特C·穆隆C·斯陶佛-凯莱赫S·阿巴索娃V·瓦西里耶娃O·里姆科维奇A·乌利廷R·米哈伊洛夫T·米尔扎别科夫D·克莱默E·金姆
Owner エムエスエムプロテインテクノロジーズインコーポレイテッド
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