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Antibodies against the ectodomain of ErbB3 and uses thereof

An extracellular domain and structural domain technology, applied in the field of antibodies against the extracellular domain of ErbB3 and its application, can solve the problems that ErbB3 has not been taken seriously

Inactive Publication Date: 2013-03-27
MERRIMACK PHARMACEUTICALS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although the role of ErbB3 in cancer has been studied (see, for example, Horst et al. (2005) 115, 519-527; Xue et al. (2006) Cancer Res. 66, 1418-1426), ErbB3 Still underappreciated as a target for clinical intervention

Method used

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  • Antibodies against the ectodomain of ErbB3 and uses thereof
  • Antibodies against the ectodomain of ErbB3 and uses thereof
  • Antibodies against the ectodomain of ErbB3 and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0300] Example 1: Preparation of Antibodies Using Phage Display

[0301] To obtain human anti-ErbB3 antibodies referred to herein as Ab #6, Ab #3, Ab #14, Ab #17, and Ab #19, human Fab phage containing a unique combination of immunoglobulin sequences from a human donor library (Hoet et al., supra) for initial screening of ErbB3 binders.

[0302] Using purified ErbB3 and a Chinese Hamster Ovary (CHO) cell line expressing cell surface ErbB3, 73 unique Fab sequences (obtained using the method described above or slight variations thereof) were identified from the library. These 73 clones were then reformatted into phage-free Fabs only. Using high-throughput methods, these Fabs were expressed on a small scale and tested for binding using ELISA and FLEXCHIP methods, which are a high-throughput surface plasmon resonance (SPR) technique. Phage-free 73 Fabs were spotted on the chip surface and binding kinetics and epitope blocking to ErbB3-his fusion target protein or ErbB3-Fc prot...

Embodiment 2

[0304] Example 2: Optimization of Anti-ErbB3 Fab

[0305] Following the identification of Fabs that block the binding of the ErbB3 ligand (neuretonin) to ErbB3, the following codon optimization was performed on the VH and VL sequences of the Fabs.

[0306] VH and VL regions were rearranged using expression constructs expressed as IgGl or IgG2 isotypes. This construct includes a SELEXIS backbone with expression cassettes for replacement of the appropriate heavy and light chain sequences. The SELEXIS vector contains the CMV promoter and matching poly-A signal.

[0307] The VH and VL nucleic acid sequences of codon-optimized Ab #6 (obtained using the method described above or slight variations thereof) are shown in SEQ ID NO: 25 and 26, respectively, and those sequences of Ab #3 are shown in SEQ ID NO: 27 and 28, as shown in Figure 22.

Embodiment 3

[0308] Example 3: Binding affinity of ErbB3

[0309] The dissociation constants of anti-ErbB3 antibodies were measured using two independent techniques, surface plasmon resonance analysis and cell binding assay using MALME-3M cells.

[0310] Surface Plasmon Resonance Analysis

[0311] Surface plasmon resonance analysis (e.g. FLEXCHIP analysis) was generally performed on a BIACORE 3000 instrument, etc., as described in Wassaf et al. ). Based on formula K D =K d / K a to calculate K D value.

[0312] K of Ab #6 and Ab #3 measured using the method described above or a slight variation thereof using surface plasmon resonance analysis D The values ​​are shown in Figure 2A and 2B middle. K of Ab #6 D Values ​​are shown as approximately 4nM, K for Ab #3 DValues ​​are shown to be approximately 8nM. Furthermore, surface plasmon resonance indicated that Ab #6 competes with HRG for binding to ErbB3.

[0313] Cell Binding Assay

[0314] Perform cell binding assay to d...

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Abstract

The present invention provides a novel class of antibodies and antigen binding fragments thereof that bind the extracellular domain of ErbB3 receptor and inhibit various ErbB3 functions. For example, the antibodies and antigen binding fragments described herein are capable of binding to the receptor designated ErbB3 and inhibiting EGF-like ligand mediated phosphorylation of the receptor. Such antibodies and antigen binding fragments thereof have the useful characteristic of inhibiting the proliferation of cancer cells expressing ErbB3.

Description

Background technique [0001] The ErbB / HER subfamily of polypeptide growth factor receptors includes epidermal growth factor (EGF) receptors (EGFR, ErbB1 / HER1), neu oncogene products (ErbB2 / HER2), and the recently identified ErbB3 / HER3 and ErbB4 / HER4 receptors Proteins (see, eg, Hynes et al. (1994) Biochim. Biophys. Acta. Rev. Cancer 1198, 165-184). Each of these receptors is speculated to consist of an extracellular domain (extracellular ligand-binding domain), a transmembrane domain, a cytoplasmic protein tyrosine kinase (PTK) domain, and a C-terminal phosphorylation domain Composition (see, eg Kim et al. (1998) Biochem. J. 334, 189-195). The extracellular domain of the ErbB receptor is also characterized by being divided into four domains (I-IV). Domains I and III of the ErbB extracellular domain are involved in ligand binding (see, eg, Hynes et al. (2005) Nature Rev. Cancer 5, 341-354). Ligands for these receptors include neuregulin (HRG) and betacellulin (BTC). [0002]...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395
CPCC07K2317/34C07K2317/622C07K2317/92C07K2317/21C07K16/32A61K2039/505C07K2317/55C07K2317/73A61P35/00A61P43/00
Inventor B·舍贝尔U·尼尔森M·菲尔德豪斯
Owner MERRIMACK PHARMACEUTICALS INC
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