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MAGE-A4 T cell receptors and methods of use thereof

A MAGE-A4, cell receptor technology, applied in chemical instruments and methods, DNA/RNA fragments, receptors/cell surface antigens/cell surface determinants, etc.

Pending Publication Date: 2022-06-03
REGENERON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] There remains an unmet need in the art for new targeting agents based on T cell receptors that specifically bind the MAGE-A4 antigen and methods for the production and use of such targeting agents in therapeutic and diagnostic settings

Method used

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  • MAGE-A4 T cell receptors and methods of use thereof
  • MAGE-A4 T cell receptors and methods of use thereof
  • MAGE-A4 T cell receptors and methods of use thereof

Examples

Experimental program
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Effect test

example 1

[0228] Example 1 MAGE-A4-specific T cell receptor recognition

[0229] MAGE-A4(286-294) peptide (KVLEHVVRV, SEQ ID NO: 609) (specifically presented by human HLA-A2, diluted in PBS and mixed with adjuvant (eg, with complete Freund's adjuvant (CFA); Chondrex)) to humanize the cellular immune system components in mice Mice (see PCT Publication No. WO 2016 / 164492, the entire contents of which are hereby incorporated by reference and made a part hereof) were immunized. Spleen suspensions of immunized mice were obtained and dissociated. Red blood cells were lysed in ACK Lysis Buffer (Life Technologies), and splenocytes were suspended in RPMI complete medium. Isolated splenocytes were sorted and single T cells bound to MAGE-A4(286-294) peptide in the context of MHC were isolated by fluorescence activated cell sorting (FACS). Isolated T cells were seeded in a single well and mixed with TCR alpha and beta chain variable region specific PCR primers. The cDNA of each single T cell w...

example 2M

[0266] Example 2MAGE-A4-specific T cell receptor recognition

[0267] MAGE-A4(230-239) peptide (GVYDGREHTV, SEQ ID NO: 612) (specifically presented by human HLA-A2, diluted in PBS and mixed with adjuvant (eg, with complete Freund's adjuvant (CFA); Chondrex)) to humanize the cellular immune system components in mice Mice (see PCT Publication No. WO 2016 / 164492, the entire contents of which are hereby incorporated by reference and made a part hereof) were immunized. Spleen suspensions of immunized mice were obtained and dissociated. Red blood cells were lysed in ACK Lysis Buffer (Life Technologies), and splenocytes were suspended in RPMI complete medium. Isolated splenocytes were sorted and single T cells bound to MAGE-A4(230-239) peptide in the context of MHC were isolated by fluorescence activated cell sorting (FACS). Isolated T cells were seeded in a single well and mixed with TCR alpha and beta chain variable region specific PCR primers. The cDNA of each single T cell w...

example 3

[0296] Example 3 Cytotoxicity of MAGE-A4 TCR on A375 melanoma cells

[0297] This example illustrates the selective killing ability of certain MAGE-A4 TCRs of the present invention to A375 melanoma cells.

[0298] method

[0299] T cell manipulation

[0300] Whole T cells were purified from human peripheral blood mononuclear cells (PBMCs) by negative selection using the EasySep Human T Cell Isolation Kit (Stem Cell Technology, 17951), and then cryopreserved. On day 0 of the experiment, T cells were thawed and cultured in cells containing 200 U / ml human IL-2 and anti-CD3 / anti-CD28 activated beads (Life Technologies, 111.32D) (bead:cell ratio 1:1) The medium (CTS OpTmizer medium [Life Technologies, A1048501], supplemented with 10 mg / ml gentamicin, 4 mM L-glutamine) was inoculated with 1 × 10 6 cells / ml. On day 3, the activated microbeads were removed and used Nucleofector IIb nucleofection instrument and human T cell nucleofection kit (Lonza, VVPA-1002) and 20 mg Cas9 protei...

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Abstract

The present invention provides isolated T cell receptors (TCRs) that specifically bind to the HLA presenting cancer-testicular antigen melanoma associated antigen A4 (MAGE-A4) peptide, and therapeutic and diagnostic methods of using these isolated TCRs. The present invention provides a T cell receptor (TCR) produced for an anti-MAGE-A4 peptide antigen in the case of MHC (HLA-A2). In reporter gene detection, the determined unique TCR sequence can be specifically combined with the small peptide MAGE-A4 in the HLA molecular groove, and T cells are activated.

Description

[0001] Related patent applications [0002] This application claims priority to and the benefit of US Provisional Application No. 62 / 862,726, filed June 18, 2019, and US Provisional Patent Application No. 62 / 871,793, filed July 9, 2019. The entire contents of the above-mentioned applications are incorporated herein by reference and form part of the present invention. [0003] sequence listing [0004] This application contains a Sequence Listing, which is submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy was created on June 15, 2020 with the name 118003_00320_SL.txt and a size of 344832 kilobytes. Background technique [0005] T cell receptors (TCRs) are membrane-bound heterodimers containing an alpha and beta chain similar to the variable (V) and constant (C) regions of immunoglobulins. The TCR alpha chain comprises a V-alpha chain covalently linked to a C-alpha chain, and the beta chain comprises a V-beta cha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/30C12N15/62C07K19/00
CPCC07K14/7051A61P35/00C07K14/4748A61K39/4611A61K39/4632C12N2740/15041A61K39/461A61K39/464486A61K39/464488A61K39/4644A61K2121/00A61K2300/00A61K9/0019A61K9/0053A61K35/00A61K47/00
Inventor N·鲍尔曼C·古雷尔J·汉森
Owner REGENERON PHARM INC
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