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Binding molecules for bcma and cd3

A technology for binding molecules, CDR-H3, applied in the field of producing binding molecules of the present invention

Inactive Publication Date: 2016-09-28
AMGEN RES (MUNICH) GMBH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] A large number of biopharmaceuticals, such as antibodies and / or other physiologically active proteins, have been produced in the past decade, but their efficient production remains a challenging task

Method used

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  • Binding molecules for bcma and cd3
  • Binding molecules for bcma and cd3
  • Binding molecules for bcma and cd3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment A1

[1008] Example A1 Generation of CHO cells expressing chimeric BCMA

[1009] In order to construct a chimeric epitope mapping molecule, the amino acid sequence or single amino acid residue of the corresponding epitope domain of human BCMA was replaced with a murine sequence. Construct the following molecules:

[1010] ●Human BCMA ECD / E1 mouse (SEQ ID NO: 1009)

[1011] Chimeric extracellular BCMA domain: human extracellular BCMA domain, in which epitope cluster 1 (amino acid residues 1-7 of SEQ ID NO: 1002 or 1007) is divided by the corresponding murine cluster (SEQ ID NO: 1004 or 1008) Amino acid residues 1-4) replacement

[1012] →SEQ ID NO: 1002 or 1007 deletion of amino acid residues 1-3 and G6Q mutation

[1013] ●Human BCMA ECD / E2 mouse (SEQ ID NO: 1010)

[1014] Chimeric extracellular BCMA domain: human extracellular BCMA domain, in which epitope cluster 2 (amino acid residues 8-21 of SEQ ID NO: 1002 or 1007) is divided by the corresponding murine cluster (SEQ ID NO: 1004 or 1008...

Embodiment A2

[1036] 2.1 Transient expression in HEK293 cells

[1037] The clone of the expression plasmid with the nucleotide sequence of the verified sequence was used for transfection and protein expression was performed in the FreeStyle 293 expression system (Invitrogen GmbH, Karlsruhe, Germany) according to the manufacturer's instructions. The supernatant containing the expressed protein is obtained, the cells are removed by centrifugation and the supernatant is stored at -20°C.

[1038] 2.2 Stable expression in CHO cells

[1039] The clone of the expression plasmid with the nucleotide sequence of the verified sequence was transfected into DHFR-deficient CHO cells for eukaryotic expression construct. The eukaryotic protein expression in DHFR-deficient CHO cells is performed as described in Kaufman R.J. (1990) Methods Enzymol. 185, 537-566. The gene amplification of the construct was induced by increasing concentrations of methotrexate (MTX) to a final concentration of 20 nM MTX. After two ...

Embodiment A3

[1047] Example A3 Epitope clustering of mouse scFv-fragments

[1048] Cells transfected with human or murine BCMA, or with chimeric BCMA molecules were stained with natural, undiluted periplasmic extract containing scFv bound to human / cyno BCMA. The bound scFv was detected using 1 μg / ml anti-FLAG antibody (Sigma F1804) and R-PE-labeled anti-mouse Fcγ-specific antibody (1:100; Dianova#115-116-071). All antibodies were diluted in PBS with 2% FCS. As a negative control, cells were cultured with PBS / 2% FCS instead of periplasmic extract. The samples were measured by flow cytometry on a FACSCanto II instrument (Becton Dickinson) and analyzed with FlowJo software (version 7.6).

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Abstract

The invention discloses binding molecules for BCMA and CD3. The present invention relates to a binding molecule comprising a first and a second binding domain, wherein the first binding domain is capable of binding to epitope clusters of BCMA, and the second binding domain is capable of binding to the T cell CD3 receptor complex. Moreover, the invention provides a nucleic acid sequence encoding the binding molecule, a vector comprising said nucleic acid sequence and a host cell transformed or transfected with said vector. Furthermore, the invention provides a process for the production of the binding molecule of the invention, a medical use of said binding molecule and a kit comprising said binding molecule.

Description

Technical field [0001] The present invention relates to at least bispecific binding molecules comprising first and second binding domains, wherein the first binding domain is capable of binding to the epitope cluster of BCMA and the second binding domain is capable of binding to the T cell CD3 receptor Complex. In addition, the present invention provides a nucleic acid sequence encoding the binding molecule, a vector containing the nucleic acid sequence, and a host cell transformed or transfected with the vector. Furthermore, the present invention provides a method for producing the binding molecule of the present invention, the medical use of the binding molecule and a kit containing the binding molecule. Background technique [0002] BCMA (B-cell maturation antigen, TNFRSF17, CD269) is a transmembrane protein belonging to the TNF receptor superfamily. BCMA was originally reported as an integral membrane protein in the Golgi body of human mature B lymphocytes, that is, as an i...

Claims

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Application Information

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IPC IPC(8): C07K16/28
CPCC07K16/2809A61K2039/505C07K2317/31C07K2317/33C07K2317/34C07K2317/622C07K16/2878A61P17/00A61P3/00A61P35/00A61P35/02A61P37/00A61P37/02A61P7/00C07K2317/92C07K2317/94A61K39/3955C07K16/2896C07K2319/20C07K16/468C07K2319/21C07K2319/74C07K2317/565
Inventor 彼得·库菲托拜厄斯·劳姆帕特里克·霍夫曼罗曼·基谢尔拉尔夫·鲁特布瑟桃瑞丝·芳保罗·亚当E·伯格斯B·赫比斯苏珊娜·希普
Owner AMGEN RES (MUNICH) GMBH
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