Multispecific antibodies comprising full length antibodies and single chain fab fragments

A multi-specific antibody and full-length antibody technology, applied in the direction of antibodies, antibody mimics/scaffolds, anti-receptors/cell surface antigens/cell surface determinant immunoglobulins, etc., can solve problems such as low expression yield

Active Publication Date: 2012-03-07
ROCHE GLYCART AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular, genetically engineered bispecific tetravalent antibodies reported in WO 1995 / 009917 and Müller, D. et al., Handbook of Therapeutic antibodies, Part III, Chapter 2, (2008) 345-378 showed only very low expression yield

Method used

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  • Multispecific antibodies comprising full length antibodies and single chain fab fragments
  • Multispecific antibodies comprising full length antibodies and single chain fab fragments
  • Multispecific antibodies comprising full length antibodies and single chain fab fragments

Examples

Experimental program
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Effect test

Embodiment 1

[0248] Design of Multispecific Antibody Molecules According to the Present Invention Recognizing Human IGF1 Receptor and Human EGF Receptor

[0249] In the following as one embodiment of the invention, a tetravalent bispecific antibody comprising a full-length antibody binding to a first antigen (IGF-1R or EGFR) and two single-chain Fab fragments, the two Single-chain Fab fragments are linked to full-length antibodies (both single-chain Fab fragments are at both C-terminal ends of the heavy chain or at both C-terminal ends of the light chain) via a peptide linker and linked to a second, different antigen (IGF-1R or Another) binding of EGFR. The antibody domains and linker have the following order in N-terminal to C-terminal direction in the single chain Fab fragment: VL-CL-Linker-VH-CH1.

[0250] as used for For the heavy chain variable region VH of the antigen binding site, SEQ ID NO:15 was used. as used for For the light chain variable region VL of the antigen bind...

Embodiment 2

[0259] bispecific Expression & purification of antibody scFabXGFR1 molecule

[0260] The light and heavy chains of the corresponding bispecific antibodies are constructed in expression vectors carrying prokaryotic and eukaryotic selectable markers. These plasmids were amplified in E. coli, purified and subsequently transfected into HEK293F cells for transient expression of recombinant proteins (using Invitrogen's freesyle system). After 7 days, HEK 293 cell supernatants were obtained and purified by protein A and size exclusion chromatography. The homogeneity of all bispecific antibody constructs was confirmed by SDS-PAGE under non-reducing and reducing conditions. Under reducing conditions ( Figure 8 ), polypeptide chains carrying C- and N-terminal scFab fusions showed surface molecular sizes based on SDS-PAGE similar to the calculated molecular weights. The expression levels of all constructs were analyzed by protein A HPLC and were similar to or in some cases slig...

Embodiment 3

[0269] bispecific Stability and aggregation tendency of antibody scFab-XGFR molecules

[0270] HP size exclusion chromatography was performed to determine the amount of aggregates present in the prepared recombinant antibody derivatives. For this, bispecific antibody samples were analyzed by high performance SEC on an UltiMate 3000 HPLC system (Dionex) using a Superdex 200 analytical size exclusion column (GE Healthcare, Sweden). Figure 9 shows examples of these analyses. Aggregates appear as separate peaks or shoulders preceding the fractions containing monomeric antibody derivatives. For this work, we defined a desired 'monomer' molecule consisting of two heterodimers comprising a heavy chain and a light chain with a scFab attached to either of them. After removal of N-glycans by enzymatic treatment with Peptide-N-Glycosidase F (Roche Molecular Biochemicals), the integrity of the reduced bispecific antibody light and heavy chains and the amino acid backbone of the fus...

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Abstract

The present invention relates to multispecific, especially bispecific antibodies comprising full length antibodies and single chain Fab fragments, methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

Description

[0001] The present invention relates to multispecific antibodies comprising full-length antibodies and single-chain Fab fragments, in particular bispecific antibodies, methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof. Background of the invention [0002] Recently, a variety of multispecific recombinant antibody formats have been developed, e.g., by fusing, e.g., an IgG antibody format with a single chain region of a tetravalent bispecific antibody (see e.g., Coloma, M.J., et al., Nature Biotech 15 (1997) 159-163; WO 2001 / 077342; and Morrison, S.L., Nature Biotech 25 (2007) 1233-1234). [0003] Several other novel formats capable of binding two or more antigens have also been developed, such as bi-, triple- or quadruple-chain antibodies, minibodies, several single-chain antibodies (scFv, Bis-scFv), in which no antibody remains Core structure (IgA, IgD, IgE, IgG or IgM) (Holliger, P., et al., Nature Biotech 23 (2005) 1126-113...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C07K16/28
CPCA61K2039/505C07K16/00C07K16/468C07K2317/21C07K2317/55C07K2317/56C07K2317/565C07K2317/626C07K2317/64C07K2317/73C07K2319/00C07K16/2863C07K16/46C07K16/28C12N15/62A61K39/395
Inventor U·布林克曼P·布鲁恩克尔R·克罗斯代尔C·克莱因E·科佩茨基E·默斯纳J·T·雷古拉J·M·尚策J·O·施特拉克P·乌马纳
Owner ROCHE GLYCART AG
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