Bispecific bivalent scfv-Fc molecules

A technology of identity and linker, applied in the field of protein engineering

Inactive Publication Date: 2016-01-06
AMGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Production of bispecific antibodies in the standard IgG format can be challenging as they contain four different polypeptide chains

Method used

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  • Bispecific bivalent scfv-Fc molecules
  • Bispecific bivalent scfv-Fc molecules
  • Bispecific bivalent scfv-Fc molecules

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] Example 1: Construction of anti-HER2CD3ε and anti-FOLR1 / CD3ε Bi-Fc molecules and single-chain bispecific molecules

[0130] Bi-Fc molecules were generated using the method essentially described above. et al. (2000), Blood95(6):2098-2103. In more detail, the construct encoding heterodimer anti-HER2 / CD3εBi-Fc was prepared as follows. DNA fragments encoding the VH region (SEQ ID NO: 5) and the VL region (SEQ ID NO: 6) of the anti-HER2 IgG antibody and the VH region (SEQ ID NO: 7) and the VL region (SEQ ID NO: 8) of the anti-human CD3ε IgG antibody were used in the forward direction and reverse primers were amplified by PCR and spliced ​​together by a flexible linker. The resulting DNA fragment encoding a linear fusion DNA encoding two scFvs joined by a linker is referred to herein as a single-chain anti-HER2 / CD3ε (SEQ ID NO: 9). This construct was subcloned into a mammalian expression vector for antibody production.

[0131] A heterodimeric anti-HER2 / CD3ε Bi-Fc (SEQ I...

Embodiment 2

[0133] Example 2: Determination of Binding of BiTE:Fc Molecules to Target Cells and Immune Effector Cells

[0134] Binding of heterodimeric anti-HER2 / CD3ε Bi-Fc and single chain anti-HER2 / CD3ε to CD3 expressing T cells and HER2 expressing JIMT-1 cells was assessed as follows. Human pan-T cells (purified using Human PanT Cell Isolation Kit II, MiltenyiBiotec, Auburn, CA) or purified JIMT-1 cells in the absence or presence of 10 μg / mL heterodimer anti-HER2 / CD3εBi- In the case of Fc or single chain anti-HER2 / CD3ε, they were incubated at 4°C for 16 hours. Cellular binding of the heterodimeric anti-HER2 / CD3ε Bi-Fc was detected using an allophycocyanin (APC)-labeled anti-human Fc secondary antibody. using mouse anti- The antibody was then detected with an APC-labeled mouse Ig-specific antibody containing Labeled single chain anti-HER2 / CD3ε.

[0135] shown in figure 2 In the fluorescence-activated cell sorting (FACS) histogram of , unfilled curves represent data from cells in...

Embodiment 3

[0136] Example 3: Lysis of Tumor Cell Lines in the Presence of Bi-Fc and T Cells

[0137] Detection of the aforementioned heterodimeric anti-HER2 / CD3ε and anti-FOLR1 / CD3ε Bi-Fc and single-chain anti-HER2 / CD3ε and anti-FOLR1 / CD3ε molecules to determine their function in tumor cells expressing HER2 or FOLR1 as Activity of target cells in a T-cell-dependent cell lysis (TDCC) assay. Briefly, panT cells were isolated from healthy human donors using the Human PanT Cell Isolation Kit II (MiltenyiBiotec, Auburn, CA). The T cells were cultured with CFSE-labeled tumor target cells at a ratio of 10:1, with or without the presence of image 3 with Figure 4 Heterodimer anti-HER2 / CD3ε or anti-FOLR1 / CD3ε Bi-Fc or single chain anti-HER2 / CD3ε or anti-FOLR1 / CD3ε described in Example 1 at varying concentrations indicated in . As controls, some samples contained T cells and tumor target cells, but no Bi-Fc or single chain molecules.

[0138] Target cells for anti-FOLR1 / CD3ε heterodimeric Bi-...

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Abstract

Described herein is a bispecific molecule containing an Fc polypeptide chain and immunoglobulin variable regions. Also provided are pharmaceutical formulations comprising such molecules, nucleic acids encoding such molecules, host cells containing such nucleic acids, methods of making such molecules, and methods of using such molecules.

Description

[0001] Cross references to related patent applications [0002] This patent application claims the benefit of US Provisional Patent Application 61 / 791,424, filed March 15, 2013, which is hereby incorporated by reference in its entirety. technical field [0003] The invention belongs to the field of protein engineering. Background technique [0004] Bispecific antibodies hold promise as therapeutic agents for a variety of indications. Production of bispecific antibodies in the standard IgG format can be challenging because they contain four different polypeptide chains. The efficacy of a smaller and easier-to-manufacture bispecific molecule has been clinically demonstrated in non-Hodgkin's lymphoma. See, eg, Bargou et al. (2008), Science 321(5891):974-977. Due to the short in vivo half-life of this small single-chain molecule, prolonged administration by continuous intravenous infusion was used to achieve the above results. Id. Therefore, there is a need in the art for b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K16/32C07K16/46
CPCC07K16/28C07K16/2803C07K16/2809C07K16/32A61K2039/505C07K2317/31C07K2317/52C07K2317/60C07K2317/622C07K2317/71C07K2317/73C07K2317/92C07K2319/00C07K16/00C07K2317/64C07K16/468A61P1/16A61P11/00A61P11/08A61P13/12A61P19/04A61P29/00A61P31/04A61P31/12A61P33/00A61P33/02A61P35/00A61P35/02A61P37/06A61P9/10C07K16/2863C07K16/40C07K2317/56C07K2317/565
Inventor L·G·伯格斯P·A·博伊尔勒炜阎M·L·麦考斯
Owner AMGEN INC
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