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Cross-species-specific psmaxcd3 bispecific single chain antibody

A single-chain antibody and bispecific technology, applied in the direction of antibody, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, specific peptide, etc., can solve the problems of increasing the risk of prostate cancer and achieve maximum safety , good safety effect

Inactive Publication Date: 2013-04-03
AMGEN RES (MUNICH) GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other studies have shown that genetic variations in specific regions of chromosome 8 can increase a person's risk of developing prostate cancer

Method used

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  • Cross-species-specific psmaxcd3 bispecific single chain antibody
  • Cross-species-specific psmaxcd3 bispecific single chain antibody
  • Cross-species-specific psmaxcd3 bispecific single chain antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0297] Example 1 Generation and characterization of PSMA and CD3 cross-species specific bispecific single chain antibody molecules

[0298] 1.1 Cloning and expression of human PSMA antigen on CHO cells

[0299] The sequence of the human PSMA antigen ('AY101595', Homo sapiens prostate specific membrane antigen mRNA, complete cds, was synthesized by gene synthesis according to standard operating procedures, National Center for Biotechnology Information, http: / / www.ncbi.nlm.nih .gov / entrez) for obtaining synthetic molecules. Gene synthesis fragments were also designed to contain Kozak sites for eukaryotic expression of the construct and restriction sites at the beginning and end of the DNA. The introduced restriction sites (XbaI at the 5' end and SalI at the 3' end) were used during the cloning step in Mack et al. (Mack M et al., ProcNatl Acad Sci USA 1995;92:7021-5 and Raum et al. Human Cancer Immunol Immunother (2001) 50 (3)) described in the expression plasmid called pEFDH...

Embodiment 2

[0314] 2.1 Flow cytometry binding analysis of PSMA and CD3 cross-species specific bispecific antibody

[0315] To test the functionality of the cross-species specific bispecific antibody constructs with respect to binding ability to human and macaque PSMA and to human and macaque CD3, FACS analysis was performed. For this, CHO cells transfected with human PSMA and the human CD3-positive T-cell leukemia cell line HPB-ALL (DSMZ, Braunschweig, ACC483) were used to examine binding to human antigens. Generated by using macaque PSMA transfectants and macaque T cell line 4119LnPx (kindly provided by Prof. Fickenscher (Hygiene Institute, Virology, Erlangen-Nuernbe); published in Knappe A et al., and Fickenscher H., Blood 2000, 95, 3256 -61] to test the binding reactivity to macaque antigens.

[0316] Flow cytometry analysis was performed as follows:

[0317] Incubation of 200.000 cells of different cell lines with 50 μl of purified protein (2 μg / ml) of the cross-species specific bis...

Embodiment 3

[0326] Example 3 Binding assays of scFv against various cell lines

[0327] 3.1. Expression of single-chain antibody constructs in E. coli

[0328]The scFv molecules EpCAM 4-7 (WO 99 / 25818), PM74-G3, PM52-H3, PM52-C3, PM75-A10 and PM91-B6 are expressed by using the plasmid pComb3H5BFlag / His, where the expression construct (eg scFv) includes Flag -tag (DYKDDDDK) and His6-tag. Plasmid DNA of each scFv molecule was transformed into 100 [mu]l of heat shock competent E. coli TG1 and plated on carbenicillin LB-agar. E. coli transformed with pComb3H5BFlag / His containing VL- and VH-segments produced sufficient quantities of soluble scFv after induction with 1 mM IPTG. Due to an appropriate signal sequence, the scFv-chain is exported into the periplasm where it folds into a functional conformation.

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Abstract

The present invention relates to a bispecific single chain antibody molecule comprising a first binding domain capable of binding to an epitope of human and non-chimpanzee primate CD3 epsilon chain, wherein the epitope is part of an amino acid sequence comprised in the group consisting of SEQ ID NOs. 2, 4, 6, and 8, and a second binding domain capable of binding to prostate-specific membrane antigen (PSMA). The invention also provides nucleic acids encoding said bispecific single chain antibody molecule as well as vectors and host cells and a process for its production. The invention further relates to pharmaceutical compositions comprising said bispecific single chain antibody molecule and medical uses of said bispecific single chain antibody molecule.

Description

technical field [0001] The present invention relates to a bispecific single chain antibody molecule comprising a first binding domain capable of binding to an epitope of the human and non-chimpanzee primate CD3ε chain, wherein the epitope is contained in SEQ ID NO.2, SEQ ID NO .4. A part of the amino acid sequence in SEQ ID NO.6 and SEQ ID NO.8, said bispecific single chain antibody molecule further comprising a second binding domain capable of binding to prostate-specific membrane antigen (PSMA). The present invention also provides nucleic acid encoding the bispecific single chain antibody molecule, as well as vectors and host cells and methods for their production. The invention further relates to pharmaceutical compositions comprising said bispecific single chain antibody molecule and to the medical use of said bispecific single chain antibody molecule. Background technique [0002] T cell recognition is mediated by clonally distributed αβ and γδ T cell receptors (TcRs) ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K16/30C07K16/40A61K39/395
CPCC07K2317/31C07K2317/33C07K2317/622C07K2317/565C07K16/468C07K16/40C07K16/2809C07K16/30C07K16/3069A61P13/10A61P35/00A61P37/00A61K38/17A61K39/395A61K49/16C07K14/435C07K14/475C07K16/18C07K16/22C07K16/24C07K16/28C07K16/46
Inventor P·库褔尔T·劳姆R·基谢尔R·鲁特布瑟P·霍夫曼D·劳S·曼戈尔德M·克林格尔E·沙勒S·豪斯曼P·弗卢尔C·斯泰格尔
Owner AMGEN RES (MUNICH) GMBH
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