Method for generating multispecific antibodies from monospecific antibodies

A multimer, domain technology used in the field of generating multispecific antibodies from monospecific antibodies

Pending Publication Date: 2020-06-05
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Prior art methods for converting monospecific antibodies or antibody derivatives into bsAbs have disadvantages such as, for example, limitations related to the process of post-assembly bsAb preparations and their composition

Method used

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  • Method for generating multispecific antibodies from monospecific antibodies
  • Method for generating multispecific antibodies from monospecific antibodies
  • Method for generating multispecific antibodies from monospecific antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[1489] Design and module composition of 2 / 3-IgG

[1490] overall evaluation

[1491] figure 1 The design and modular composition of the 2 / 3-IgG used in the method of the invention is shown. These 2 / 3-IgGs consist of three separate chains: a light chain (typically, a full-length light chain comprising the light chain variable domain and a light chain constant domain), a heavy chain (typically, Full-length heavy chain with heavy chain variable domain and all heavy chain constant domains (including hinge region), and one heavy chain Fc region polypeptide (typically, heavy chain Fc region fragment including hinge-CH2-CH3) . The light and heavy chain variable domains form a functional binding site. The heavy chain (typically, it is derived from the human IgG1 subclass) contains a knob-cys mutation or a hole-cys mutation in CH3 that enables dimerization of the knob-into-hole Fc region (in the CH3 domain of the antibody heavy chain The mutations T366W and S354C are cal...

Embodiment 2

[1497] Expression and purification of 2 / 3-IgG of the present invention

[1498] 2 / 3-IgG is achieved by co-transfection of plasmids encoding the light chain, heavy chain (with a knob mutation or a hole mutation) and matching MHC FcRP (hole or knob) into mammalian cells (e.g. HEK293) using state-of-the-art techniques Express.

[1499] In more detail, e.g., for the production of 2 / 3-IgG by transient transfection (e.g. in HEK293 cells), cDNA preparation based on the CMV-intron A promoter or a genome based on the CMV promoter was applied. Prepared expression plasmids.

[1500] In addition to the antibody expression cassette, this plasmid contains:

[1501] - origin of replication, which allows the plasmid to replicate in E. coli,

[1502] - the β-lactamase gene that confers ampicillin resistance in E. coli, and

[1503] - the dihydrofolate reductase gene from Mus musculus (Mus musculus) as a selectable marker in eukaryotic cells.

[1504] The transcription unit...

Embodiment 3

[1526] Generation of bispecific antibodies (bsAbs) via a 2 / 3-IgG-exchange reaction

[1527] 2 / 3-IgG containing one light chain, one heavy chain and MHCFcRP has been produced as two types of KiH heterodimers: full-length heavy chain-knot::MHCFcRP-hole and full-length heavy chain-hole::MHCFcRP -Knot. Both types of 2 / 3-IgG are more or less 'flawed' because MHCFcRP lacks the additional CH3 cysteine ​​necessary to form an interchain disulfide bond with the heavy chain, and MHCFcRP contains a mismatched full-length heavy chain The charge mutation of the counterpart. However, the modules that make up those defective heterodimers can rearrange into Figure 4 Matchingly charged bispecific heterodimers shown in . The full-length heavy chain (knob-cys) from 2 / 3-IgG A and the full-length heavy chain from 2 / 3-IgG B (hole-cys) form matching heterodimers. Compatible heterodimers are also formed when MHCFcRP (hole charge) interacts with MHCFcRP (knot charge). Thus, an exchange reacti...

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Abstract

Herein is reported a method for the generation of multispecific antibodies by a half- antibody exchange reaction between two 2 / 3-IgGs destabilized in one half by asymmetric perturbing mutations fostering the generation of correctly assemble full length bispecific antibodies. The method can be performed in the absence of reducing agents and does not require hinge region disulfide bonds in the starting 2 / 3-IgGs.

Description

[0001] Here we report an easy-to-use and scalable method for generating bispecific and multispecific antibodies using a novel half-antibody exchange method. Background technique [0002] Prior art methods for the biochemical transformation of monospecific antibody derivatives to assemble bispecific antibodies employ (i) half-antibody complementation reactions and (ii) IgG-IgG exchange reactions. [0003] These techniques are disclosed, for example, in WO 2015 / 046467, Rispens et al., J. Biol. Chem. 289 (2014) 6098-6109, US 9,409,989, WO 2013 / 060867, WO 2011 / 131746, WO 2011 / 133886, WO 2011 / 143545, WO 2010 / 151792, Gunasekaran et al., J. Biol. Chem. 285 (2010) 19637–19646, WO 2009 / 041613, WO 2009 / 089004, WO 2008 / 119353, WO 2007 / 114325, US, 412, 76 US 8,642,745, WO 2006 / 047340, WO 2006 / 106905, WO 2005 / 042582, WO 2005 / 062916, WO 2005 / 000898, US 7,183,076, US 7,951,917, Segal, D.M. et al., Curr. 558-562, WO 98 / 50431, WO 98 / 04592, Merchant, A.M. et al., Nat. Biotechnol. 16 (1998) 677...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395C07K16/46
CPCC07K16/468C07K2317/10C07K2317/31C07K2317/35C07K2317/50C07K2317/515C07K2317/526C07K2317/56C07K2317/64C07K2317/66C07K1/22C07K16/42C07K2317/522C07K2317/524C07K2317/53C07K2317/622
Inventor U·布林克曼S·登格尔G·乔治斯E·霍夫曼K·迈尔F·鲍曼
Owner F HOFFMANN LA ROCHE & CO AG
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