Method for improving VEGF-receptor blocking selectivity of an anti-VEGF antibody

A technology of VEGF-R2 and VEGF-A121, applied in the field of antibodies, can solve problems such as insignificant inhibition, and achieve the effect of reducing side effects and improving safety characteristics

Pending Publication Date: 2020-07-31
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Some anti-VEGF antibodies that preferentially inhibit VEGF binding to VEGF-R2 but not significantly inhibit VEGF binding to VEGF-R1 have been reported, but have not been clinically successful (WO200064946 describes an antibody called "2C3", WO2009060198 An antibody called "r84" is described, and WO2012089176 describes an antibody called "L3H6", and EP3006465 describes an antibody called "HF2-1", "HF2-5", "HF2-9" and "HF2-11" antibody)

Method used

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  • Method for improving VEGF-receptor blocking selectivity of an anti-VEGF antibody
  • Method for improving VEGF-receptor blocking selectivity of an anti-VEGF antibody
  • Method for improving VEGF-receptor blocking selectivity of an anti-VEGF antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Production of Human Anti-VEGF Antibody (Antibody VEGF-0089)

[0086] figure 1 Superposition of the crystal structure of the human VEGF dimer in complex with VEGF-R1 domain 2 and VEGF-R3 domains 2 and 3 is depicted. This overlay illustrates that both VEGF receptors bind to highly similar regions on the VEGF dimer, so antibodies that bind VEGF in the same manner without inhibiting VEGF binding to both receptors VEGF-R1 and VEGF-R2 appear to be highly challenging. Consistent with this, among the various anti-VEGF antibodies known in the art, only a few antibodies have been reported to selectively block the binding of VEGF to VEGF-R2 but not the binding of VEGF to VEGF-R1.

[0087] The antibody VEGF-0089 described herein was derived from Roche's proprietary transgenic rabbits expressing a humanized antibody repertoire following immunization with VEGF-derived antigens. Transgenic rabbits comprising human immunoglobulin loci are reported in WO2000 / 46251, WO2002 / 12437, WO20...

Embodiment 2

[0100] Characterization of the generated human anti-VEGF antibody (antibody VEGF-0089)

[0101] VEGF binding of antibody VEGF-0089 Fab fragment was assessed by surface plasmon resonance (SPR) as described below.

[0102] Determination of Antibody Binding Affinity by Surface Plasmon Resonance (SPR)

[0103] An anti-His capture antibody (GE Healthcare 28995056) was immobilized to a Series S sensor chip C1 (GE Healthcare 29104990) using standard amine coupling chemistry, resulting in a surface density of 500-1000 resonance units (RU). As running and dilution buffers, HBS-P+ (10 mM HEPES, 150 mg / mL NaCl, pH 7.4, 0.05% surfactant P20) was used, and the measurement temperatures were set at 25°C and 37°C, respectively. hVEGF-A121 was captured to the surface resulting in capture levels of 5-35RU. A dilution series (0.37-30 nM) of anti-VEGF antibody was injected for 120 s and dissociation was monitored for at least 600 s at a flow rate of 30 μl / min. The surface was regenerated by in...

Embodiment 3

[0111] X-ray crystallography and epitope determination of antibody VEGF-0089 in complex with VEGF-dimer

[0112] The crystal structure of the VEGF-0089 Fab fragment described above was analyzed according to standard methods known in the art.

[0113] X-ray crystallography of VEGF-0089 Fab fragment complexed with VEGF-A121 was performed as follows:

[0114] Complex formation and purification of the dimeric complex VEGF-A121-VEGF-0089 Fab. For complex formation, VEGF-0089 Fab fragment and human VEGF-A121 (Peprotech) were mixed in a molar ratio of 1.1:1. After overnight incubation at 4°C for 16 hours, the complex was purified by gel filtration chromatography on a Superdex200 (16 / 600) column in 20 mM MES, 150 mM NaCl, pH 6.5. Fractions containing the dimer complex were pooled and concentrated to 1.44 mg / ml.

[0115] Crystallization of the dimeric VEGF-A121-VEGF-0089 Fab complex. Initial crystallization experiments were performed at 21°C in a sitting drop vapor diffusion appara...

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Abstract

The present invention relates to methods for improving anti-VEGF antibodies in order to provide or improve antibodies that preferentially inhibit binding to VEGF to VEGF-R2 rather than VEGF binding toVEGF-R1; and antibodies provided by said methods.

Description

[0001] field of invention [0002] The present invention relates to methods for improving anti-VEGF antibodies to provide or improve antibodies that preferentially inhibit the binding of VEGF to VEGF-R2 rather than VEGF to VEGF-R1, and antibodies provided by said methods. Background technique [0003] Anti-VEGF antibodies approved for clinical use, such as with Inhibits VEGF binding to two receptors, VEGF-R1 (FLT-1, fms-like tyrosine kinase) and VEGF-R2 (KDR / FLK-1, fetal liver kinase). VEGF-R1 and VEGF-R2 are closely related receptor tyrosine kinases (RTKs). Although VEGF-R2 is assumed to be primarily responsible for VEGF-mediated angiogenesis (Holash, J. et al., Proc Natl Acad Sci U S A. 2002 Aug 20;99(17):11393-8), VEGF-R1 is known to have a role in angiogenesis Unrelated to other important biological roles, eg in osteoclast differentiation (Aldridge, S.E. et al., Biochem Biophys Res Commun. 2005 Sep30;335(3):793-8). [0004] Some anti-VEGF antibodies that preferential...

Claims

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Application Information

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IPC IPC(8): A61K39/395C07K16/22
CPCA61K39/39591C07K16/22C07K2317/24C07K2317/34C07K2317/55C07K2317/565C07K2317/567C07K2317/76C07K2317/92C07K2317/94A61K39/3955C07K2317/21G16B15/00G16C20/50
Inventor 约尔格·本茨斯特凡·登格尔塞巴斯蒂安·芬恩约尔格·默勒肯安德烈亚斯·埃勒
Owner F HOFFMANN LA ROCHE & CO AG
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