Binding molecules for BCMA and CD3

A technology that combines molecules and CDR-L3, which is applied in the direction of peptides containing affinity tags, drug combinations, fusion peptides, etc., and can solve the problems of non-use and rarely achieve complete remission

Inactive Publication Date: 2014-11-26
AMGEN RES (MUNICH) GMBH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Stem cell transplantation may not be an option for most patients because of advanced age, the presence of other serious medical conditions, or other physical limitations
Chemotherapy only partially controls multiple myeloma and rarely achieves complete remission

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

[0590] Example A1 Generation of CHO cells expressing chimeric BCMA

[0591] 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 the mouse sequence. Build the following molecules:

[0592] ●Human BCMA ECD / E1 mouse (SEQ ID NO: 1009)

[0593] 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 replaced by the corresponding murine cluster (SEQ ID NO: 1004 or 1008 Amino acid residues 1-4) replacement

[0594] → Deletion of amino acid residues 1-3 and G6Q mutation in SEQ ID NO: 1002 or 1007

[0595] ●Human BCMA ECD / E2 mouse (SEQ ID NO: 1010)

[0596] 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 replaced by the corresponding murine cluster (SEQ ID NO...

Embodiment A2

[0618] 2.1 Transient expression in HEK293 cells

[0619] Clones of expression plasmids with sequence-verified nucleotide sequences were used for transfection and protein expression in the FreeStyle293 expression system (Invitrogen GmbH, Karlsruhe, Germany) according to the manufacturer's instructions. A supernatant containing expressed protein was obtained, cells were removed by centrifugation and the supernatant was stored at -20°C.

[0620] 2.2 Stable expression in CHO cells

[0621] Clones of expression plasmids with nucleotide sequences of verified sequences were transfected into DHFR-deficient CHO cells for eukaryotic expression constructs. Eukaryotic protein expression in DHFR-deficient CHO cells was performed as described in Kaufman R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the constructs was induced by increasing concentrations of methotrexate (MTX) up to a final concentration of 2OnM MTX. After two passages of static culture, cells were grown...

Embodiment A3

[0628] Example A3 Epitope clustering of murine scFv-fragments

[0629] Cells transfected with human or murine BCMA, or with chimeric BCMA molecules, were stained with native, undiluted periplasmic extracts containing scFv bound to human / cynomolgus BCMA. 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 incubated with PBS / 2% FCS instead of periplasmic extract. 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 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

Background technique [0001] 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, i.e., as an intracellular protein (Gras et al. (1995) International Immunol 7(7): 1093-1105), which showed that BCMA appears to be involved in B-cell development and play an important role in homeostasis. The findings of Gras et al. may be related to the fact that the BCMA protein described by Gras et al. is a fusion protein between BCMA and IL-2 due to a chromosomal translocation. Simultaneously, BCMA is also set to be critical for B-cell development and B-cell marker essential for homeostasis (Schliemann et al. (2001) Science 293(5537):2111-2114). [0002] BCMA expression is restricted to the B-cell lineage and is mainly present on plasma cells and plasmablasts, and to some extent on memory B-cells, but virtually absen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P35/02A61P37/00
CPCC07K2317/34C07K2317/622C07K2319/30A61K2039/505C07K2319/43C07K2319/00C07K2317/56C07K2317/94C07K16/468C07K2319/21C07K2317/565C07K2317/31C07K2317/73C07K2319/31C07K16/2809C07K16/2878C07K14/70578C07K2317/33C07K2317/92A61P17/00A61P19/00A61P3/00A61P35/00A61P35/02A61P35/04A61P37/00A61P37/02A61P37/06A61P43/00A61P7/00A61K39/0005A61K2039/575C07K16/2875
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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