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T cell receptors

A cell receptor, surface plasmon technology, applied in animal cells, vertebrate cells, genetically modified cells, etc., can solve problems such as unknown functions

Active Publication Date: 2019-03-15
ADAPTIMMUNE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Function is unknown although it is thought to possibly play a role in embryonic development

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1 - Cloning of reference MAGE-A4 TCR α and β chain variable region sequences into pGMT7-based expression plasmids

[0099] The parental MAGE-A4 TCR variable alpha and TCR variable beta domains of SEQ ID NO: 3 and SEQ ID NO: 4 were synthesized by standard methods described in (Molecular Cloning a Laboratory Manual 3rd Edition, Sambrook and Russell) Cloning into pGMT7-based expression plasmids containing Cα or Cβ. Plasmids were sequenced using an Applied Biosystems 3730xl DNA Analyzer. In the same manner, the reference MAGE-A4 TCR variable alpha and TCR variable beta domains of SEQ ID NO: 4 and SEQ ID NO: 5 were cloned.

[0100] The DNA sequence encoding the variable region of the TCRα chain was ligated into pEX956 cut with restriction enzymes. The DNA sequence encoding the variable region of the TCR beta chain was ligated into pEXb21, which was also cut with restriction enzymes.

[0101] The ligated plasmids were transformed into competent E. coli strain XL1-b...

Embodiment 2

[0102] Example 2 - Expression, refolding and purification of a soluble reference MAGE-A4 TCR

[0103] The expression plasmids containing the reference TCR α-chain and β-chain prepared in Example 1 were respectively transformed into Escherichia coli strain BL21pLysS, and a single ampicillin-resistant clone was incubated at 37°C in TYP (ampicillin 100 μg / ml ) medium grown to OD 600 was about 0.6-0.8, and then 0.5 mM IPTG was used to induce protein expression. Cells were harvested 3 hours after induction by centrifugation at 4000 rpm for 30 minutes in a Beckman J-6B. in MgCl 2 The cell pellet was lysed with 25 ml BugBuster (NovaGen) in the presence of DNaseI. Inclusion body pellets were recovered by centrifugation in a Beckman J2-21 centrifuge at 13000 rpm for 30 minutes. Three detergent washes were then performed to remove cellular debris and membrane components. Each inclusion body pellet was homogenised in Triton buffer (50 mM Tris-HCl pH 8.0, 0.5% Triton-X100, 200 mM NaC...

Embodiment 3

[0106] Example 3 - Binding Characterization

[0107] BIAcore analysis

[0108] Surface plasmon resonance biosensor (BIAcore 3000 TM ) can be used to analyze the binding of soluble TCRs to their peptide-MHC ligands. This is facilitated by the generation of soluble biotinylated peptide-HLA ("pHLA") complexes that can be immobilized to streptavidin-coated binding surfaces (sensor chips). The sensor chip contains four individual flow cells that are capable of simultaneously measuring the binding of T cell receptors to four different pHLA complexes. Manual injection of pHLA complexes allows easy manipulation of precise levels of immobilized class I molecules.

[0109] In vitro refolding of biotinylated class I HLA-A*0201 molecules from bacterially expressed inclusion bodies containing constitutive subunit proteins and synthetic peptides, followed by purification and in vitro enzymatic biotinylation (O'Callaghan et al. (1999) Anal. Biochem. 266:9-15). The HLA-A*0201-heavy chain...

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Abstract

The present invention relates to T cell receptors (TCRs) which bind the HLA-A*0201 restricted peptide GVYDGEEHSV (SEQ ID NO: 1) derived from the MAGE-B2 protein. The TCRs of the invention demonstrateexcellent specificity profiles for this MAGE epitope. Also provided are nucleic acids encoding the TCRs, cells engineered to present the TCRs, cells harbouring expression vectors encoding the TCRs andpharmaceutical compositions comprising the TCRs, nucleic acids or cells of the invention.

Description

[0001] sequence listing [0002] The present invention relates to a T cell receptor (TCR) that binds the HLA-A*0201-restricted decapeptide GVYDGEEHSV (SEQ ID NO: 2) derived from the melanoma-associated antigen (MAGE) B2 protein (amino acids 230-239) , and binds the HLA-A*0201-restricted decapeptide GVYDGREHTV (SEQ ID NO: 1 ) derived from the melanoma-associated antigen (MAGE) A4 protein. The TCRs of the present invention show an excellent specificity profile for these MAGE epitopes. Background technique [0003] Cancer-testis antigens (CTAs) are a subclass of tumor-associated antigens (TAAs) encoded by approximately 140 genes. Expression of these antigens is restricted to immune privileged sites such as the testis, placenta, and fetal ovary; they are usually not expressed in other tissues. Expression of these genes has been observed in malignant tumors. The immunogenicity of CTAs has led to the widespread development of cancer vaccines targeting these antigens in many solid...

Claims

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Application Information

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IPC IPC(8): C07K14/725C07K14/47
CPCC07K14/7051C12N5/0636A61K39/001111A61P35/00C12N2510/00A61K39/464486A61K39/4611A61K39/4632C07K14/4748A61K38/00A61K35/17
Inventor N·特里布尔W·劳伦斯E·巴格
Owner ADAPTIMMUNE
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