Bispecific antibodies targeting EGFR and HER2

A bispecific antibody, antibody technology, applied in the direction of antibody, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, specific peptide, etc., can solve the problem that the full efficacy of EGFR and HER2 inhibition has not yet been achieved.

Active Publication Date: 2019-04-02
BIOMUNEX PHARMA +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, binding to both antigens is monovalent rather than divalent as in natural antibodies, so the full potency of EGFR and HER2 inhibition may not have been achieved yet

Method used

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  • Bispecific antibodies targeting EGFR and HER2
  • Bispecific antibodies targeting EGFR and HER2
  • Bispecific antibodies targeting EGFR and HER2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0511] Example 1: Preparation of bispecific antibodies BiXAb-3486, BiXAb-3489 and BiXAb-3732SS of the present invention

[0512] gene synthesis

[0513] After codon optimization for mammalian expression, anti-HER2 (trastuzumab, clone humAb4D5-8) (Carter P., Presta L., Gorman C.M., Ridgway J.B., Henner D., Wong W.L., Rowland A.M., Kotts C., Carver M.E., Shepard H.M. (1992) Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci U SA.15, 4285-4289) and anti-EGFR (cetuximab) The amino acid sequence of (Humblet Y. (2004). Cetuximab: an IgG1 monoclonal antibody for the treatment of epidermal growth factor receptor expressing tumors. Expert Opin Pharmacother 5: 1621-1633) was used to design the DNA sequence. For the heavy chain, DNA encoding the signal peptide, the variable region of Fab1 and the constant CH1 domain followed by a pseudo-hinge junction and the variable region and constant CH1 domain of Fab2 were synthesized by GeneScript with flankin...

Embodiment 2

[0531] Example 2: Preparation of the bispecific antibody BiXAb-E06528 of the present invention

[0532] gene synthesis

[0533] After codon optimization for mammalian expression, the amino acid sequences of anti-EGFR (cetuximab) and anti-HER2 (trastuzumab) were used to design the DNA sequence using the GeneScript program. These antibodies are referred to as "parental" anti-EGFR and "parental" anti-HER2 mAbs.

[0534] The DNA construct of the heavy chain was designed in the following way: signal peptide, followed by a sequence consisting of the variable region, followed by the constant CH1 domain of Fab1 (anti-HER2), followed by the AP linker, followed by the variable region , followed by the constant CH1 domain of Fab2 (anti-EGFR), where a Thr to Glu mutation was introduced at Kabat position 192; flanking sequences for restriction enzyme digestion were introduced at both ends of the heavy chain DNA construct. The DNA construct for the light chain was designed in the follow...

Embodiment 3

[0546] Example 3: Characterization by Differential Scanning Calorimetry

[0547] The thermal stability of BiXAb-3489, parental anti-HER2 mAb and parental anti-EGFR mAb was compared using differential scanning calorimetry (DSC). Differential scanning calorimetry experiments were performed using a Microcal™ VP-capillary DSC system (Malvern Instruments).

[0548] All samples were centrifuged (20,000 x g, 5 min, 4°C) and their protein content was quantified using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific) with IgG analysis program before DSC analysis. For assays, all samples were diluted in PBS to a final concentration of 1 mg / mL.

[0549] The pre-equilibration time was 3 min, and the resulting thermograms were collected between 20 and 110°C, at a scan rate of 60°C / h, with a filtration period of 25 seconds and media feedback. Prior to sample analysis, 5 buffer / buffer scans were measured to stabilize the instrument, with buffer / buffer scans performed between each pr...

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Abstract

The present disclosure relates to bispecific antibodies targeting EGFR and HER2, and methods for the production of these antibodies. The bispecific antibodies consist of one complete antibody on whichtwo VH-VL chains are attached via a linker to each NH terminal region of both VH chains of the antibody. The bispecific antibodies constructed use the amino acid sequences of the heavy chain (VH) andthe light chain (VL) variable regions of two monoclonal antibodies targeting EGFR and HER2, namely cetuximab and trastuzumab, respectively

Description

[0001] The present invention relates to bispecific antibodies targeting EGFR and HER2, methods for producing the antibodies, compositions and uses thereof. Background of the invention [0002] The HER family, which includes four tyrosine kinase receptors (EGFR / HER1, HER2, HER3 and HER4), activates multiple partially redundantly interconnected downstream signaling cascades (eg, MAPK and PI3K / AKT pathways) involved in cell proliferation. HER aberrant signaling has been observed in a number of solid tumors (lung, colorectal, pancreatic, etc.). EGFR and HER2 are cell surface receptor tyrosine kinases (TKs) that transduce growth signals through homodimerization and heterodimerization with HER family receptors. Heterodimerization of EGFR with HER2 induces more potent activation of TK signaling than EGFR or HER2 homodimerization. When tumor cells overexpress both EGFR and HER2, they exhibit aggressive tumor cell growth due to increased potential for EGFR / HER2 heterodimerization and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K16/46
CPCC07K16/468C07K2317/515C07K2317/73C07K16/2863A61P35/00A61K38/00C07K16/32C07K2317/31C07K2317/51C07K2317/53C07K2317/732C07K2317/92A61K2039/505
Inventor E.朱科夫斯基O.莱杰P-E.杰拉德A.佩雷格林C.拉宝勒特
Owner BIOMUNEX PHARMA
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