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Polypeptide variants and uses thereof

An antibody, selected technology, applied in the direction of peptide, specific peptide, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problem of undesired activation

Pending Publication Date: 2019-11-15
GENMAB BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Enhanced Fc-Fc interactions between antibodies can be used to amplify the binding of an antibody to its target on a cell surface, but where the target cell is an effector cell such as a T cell, NK cell or the mechanism of action involves binding to an effector cell (e.g. In the case of other effector cells such as in bispecific antibodies), then interaction with C1q or Fc-γR and / or activation of Fc effector functions such as CDC and / or ADCC may be undesirable

Method used

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  • Polypeptide variants and uses thereof
  • Polypeptide variants and uses thereof
  • Polypeptide variants and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0565] Antibody generation, production and purification

[0566] Antibody expression constructs

[0567] For antibody expression, variable heavy (VH) and variable light (VL) chain sequences were prepared by gene synthesis (GeneArt Gene Synthesis; ThermoFisher Scientific, Germany) and cloned into IgG1 containing heavy chain (HC) and light chain (LC) The pcDNA3.3 expression vector (ThermoFisher Scientific, US) of the constant region. Desired mutations are introduced by gene synthesis or site-directed mutagenesis. Antibodies mentioned in this application have been derived from previously described CD38 antibody HuMAB 005 (WO2006 / 099875), DR5 antibody hDR5-01, hDR5-05 (WO2014 / 009358), CD52 antibody IgG1-Campath (alemtuzumab ( alemtuzumab), Crowe et al., Clin Exp Immunol. 1992, 87(1):105-110) and the VH and VL sequences of CD20 antibodies IgG1-7D8 and IgG1-11B8 (WO2004 / 035607). In some embodiments, human IgGl antibody b12 (gp120-specific antibody) was used as a negative control ...

Embodiment 2

[0573] Analysis of the effect of mutations previously shown to inhibit C1q binding and CDC in wild-type antibodies on the in vitro CDC efficacy of IgG-005 variants with enhanced Fc-Fc interactions

[0574] The C1q binding center in the CH2 domain of human IgG1 was mapped to residues D270, K322, P329 and P331 by alanine substitution (Idusogie et al., 2000 J. Immunol.). Mutants D270A, K322A and P329A were able to significantly reduce C1q binding and complement activation of rituximab in a complement concentration-dependent manner (Idusogie et al., 2000 J. Immunol).

[0575] IgG hexamerization upon target binding on the cell surface has been shown to support efficient binding of the hexameric structure of C1q, resulting in strong C1q binding (Diebolder et al., Science 2014). IgG hexamerization on the cell surface is mediated through intermolecular noncovalent Fc-Fc interactions and can be enhanced by point mutations in the CH2 domain, including E345R and E430G (Diebolder et al., ...

Embodiment 3

[0581] Analysis of the effect of selection of mutations at positions D270, K322 and P329 of the C1q-binding core on the in vitro CDC efficacy of IgG1-005 variants with enhanced Fc-Fc interactions

[0582] Mutations at positions D270, K322, and P329 of the human IgG1 C1q binding site were designed to interfere with the protein-protein interaction established upon C1q binding to IgG1. Thus, the WT amino acid was replaced by a charged amino acid with a new or opposite charge: D270R, K322E, P329D and P329R. The impact of these additional mutants on the CDC efficacy of the E430G mutated IgG1-005 variant with enhanced Fc-Fc interaction was tested. A concentration series of purified antibodies (0.001-10.0 μg / mL final antibody concentration in 3-fold dilutions) was tested in an in vitro CDC assay on Daudi cells with 20% NHS as described in Example 2.

[0583] mutation amino acid charge D270A -→Neutral (non-polar) D270R -→+ K322A +→neutral (non-polar) ...

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Abstract

Described herein are polypeptides and antibodies comprising a variant Fc region. The variant Fc region provides for stabilized Fc-Fc interactions when the polypeptide(s), antibody or antibodies are bound to its target, antigen or antigens on the surface of a cell, while at the same time also having decreased complement-dependent cytotoxicity (CDC) and may also have decreased activation of other effector functions resulting from one or more amino acid modifications in the Fc region.

Description

[0001] field of invention [0002] The present invention relates to polypeptides containing an Fc region, such as antibodies, which have reduced Fc effector function, such as reduced binding to C1q, reduced complement-dependent cytotoxicity (CDC), and may also have reduced activation of the Fc region. One or more amino acid modifications cause activation of other effector functions. [0003] Background of the invention [0004] Fc-mediated effector functions of mAbs such as complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) contribute to Therapeutic window defined by efficacy and toxicity. CDC is initiated by the binding of C1q to the Fc region of an antibody. C1q is a multimeric protein consisting of six globular binding heads attached to a stem. Each globular binding head has low affinity for IgG; and CIq must acquire affinity by binding multiple IgG1 molecules on the cell s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C07K16/28
CPCC07K16/00C07K16/2878C07K16/2887C07K16/2893C07K16/2896C07K2317/524C07K2317/526C07K2317/71C07K2317/73C07K2317/75C07K2317/94A61P29/00A61P31/00A61P31/04A61P31/10A61P31/12A61P35/00A61P37/06A61P43/00C07K2317/522C07K2317/528C07K2317/72C07K2317/732C07K2317/734C07K2317/92
Inventor R.德容F.博尔斯肯斯M.奥弗迪克K.斯特鲁马内J.舒尔曼P.帕伦
Owner GENMAB BV