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Bispecifc antibodies against HER2

A bispecific antibody and antibody technology, applied in the direction of antibodies, specific peptides, anti-tumor drugs, etc., can solve problems such as HER2 damage

Active Publication Date: 2014-05-14
健玛保
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, internalization of HER2 is impaired when compared to EGFR

Method used

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  • Bispecifc antibodies against HER2
  • Bispecifc antibodies against HER2
  • Bispecifc antibodies against HER2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0920] Example 1 - Expression constructs for HER2 and HER2 variants

[0921] A fully codon-optimized construct was generated for expression of full-length HER2 (1255 aa, Swissprot P04626), the extracellular domain (ECD) of HER2 (HER2-ECDHis, aa1-653 with a C-terminal His6 tag), the naturally occurring A splice variant of HER2 (HER2-delex16, derived from exon 16 deletion and lacking aa633-648), and a truncated form of the HER2 receptor (HER2-stumpy, aa648-1256). The construct contained appropriate restriction sites and an optimized Kozak sequence for cloning (Kozak, M., Gene 1999; 234(2): 187-208). The construct was cloned into the mammalian expression vector pEE13.4 (Lonza Biologics; Bebbington, C.R., et al., Biotechnology (N Y) 1992; 10(2):169-75) and fully sequenced to confirm the construct correctness.

Embodiment 2

[0922] Example 2 - Expression Constructs for Pertuzumab, Trastuzumab, C1, and F5

[0923] Complete codon-optimized constructs for the expression of the heavy chain (HC) and light chain (LC) of the IgG1 antibodies pertuzumab, C1 and F5 in HEK cells were generated. The variable regions encoded by these constructs are identical to those described for the Pertuzumab heavy and light chains in U.S. Patent No. 6,949,245 and for the C1 and F5 heavy and light chains in U.S. Patent No. 7,244,826 Those are the same. For C1 and F5, mammalian vectors p33G1f and p33K or p33L (pcDNA3.3 (Invitrogen)). For pertuzumab, mammalian expression vectors pG1f (pEE12.4 (Lonza Biologics) and pKappa (pEE6.4 (Lonza Biologics).

[0924] Trastuzumab It can be produced in the same manner, using the heavy and light chain sequences described in, eg, US Pat. No. 7,632,924.

[0925] The sequence disclosures of US Patent Nos. 6,949,245; 7,244,826; and 7,632,924 are hereby incorporated by reference in their...

Embodiment 3

[0926] Example 3 - Transient expression of HER2 and HER2 variants in HEK-293 or CHO cells

[0927] Freestyle TM 293-F (a subclone of HEK-293 adapted to suspension growth and chemically defined Freestyle medium, (HEK-293F)) cells were obtained from Invitrogen and 293fectin (Invitrogen) was used according to the manufacturer's instructions with the appropriate plasmid DNA transfection. In the case of antibody expression, appropriate heavy and light chain expression vectors are co-expressed.

[0928] pEE13.4HER2, pEE13.4HER2-delex16, and pEE13.4HER2-stumpy were transfected into Freestyle MAX transfection reagent (Invitrogen) TM in CHO-S (Invitrogen) cell line. Expression of HER2 and HER2-delex16 was tested by FACS analysis as described below.

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PUM

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Abstract

Bispecific antibodies which comprise antigen-binding regions binding to two different epitopes of human epidermal growth factor receptor 2 (HER2), and related antibody-based compositions and molecules, are disclosed. Pharmaceutical compositions comprising the antibodies and methods of preparing and using the antibodies are also disclosed.

Description

field of invention [0001] The present invention relates to bispecific antibodies against human epidermal growth factor receptor 2 (HER2), and uses of such antibodies, especially in the treatment of cancer. Background of the invention [0002] HER2 is a 185 kDa cell surface receptor tyrosine kinase and member of the epidermal growth factor receptor (EGFR) family, which includes four distinct receptors: EGFR / ErbB-1, HER2 / ErbB-2, HER3 / ErbB-3, and HER4 / ErbB-4. The four members of the EGFR family form homodimers and heterodimers, with HER2 being the preferred and strongest dimeric partner of the other ErbB receptors (Graus-Porta et al., Embo J 1997; 16: 1647-1655; Tao et al., J Cell Sci 2008; 121:3207-3217). HER2 can be activated by overexpression or by heterodimerization with other ErbBs that can be activated by ligand binding (Riese and Stern, Bioessays 1998; 20:41-48). For HER2, no ligand has been identified. HER2 activation leads to receptor phosphorylation, which trigger...

Claims

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Application Information

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IPC IPC(8): A61K39/395A61P35/00C07K16/32C07K16/10C07K16/28C07K16/46
CPCC07K16/1063C07K16/2809C07K16/2863C07K16/2887C07K16/468C07K2317/21C07K2317/24C07K2317/31C07K2317/41C07K2317/526C07K2317/53C07K2317/55C07K2317/73C07K2317/732C07K2317/734C07K2317/77C07K2317/94C07K16/32A61P35/00A61K47/6855A61K47/6829A61K47/6803A61K47/6849A61K47/6879A61K39/395A61K45/06A61K2039/505C07K2317/30C07K2317/33C07K2317/92
Inventor B.德戈伊杰P.范伯克尔K.斯特鲁马尼A.F.拉布里杰恩J.J.尼杰森J.I.米斯特斯P.帕伦J.舒尔曼
Owner 健玛保
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