Chimeric antigen receptors and uses thereof
a technology of chimeric antigen and receptors, applied in the field of chimeric antigen receptors, can solve the problems of inability to isolate and expand t cell populations, distinctly heterogeneous, unpredictable and variable efficacy of this method, and achieve the effect of facilitating cell auto-stimulation
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example 1
Protocol for PEP2-2-3 binding peptide Chimeric Antigen Receptor (CAR) Design and Expression
[0207]An exemplified protocol detailing the process of designing and expressing an anti-non-functional (nf) P2X7 receptor CAR according to an embodiment of the present invention is detailed as follows.
Design of PEP2-2-3 (anti-nf P2x7) Chimeric Antigen Receptor
[0208]An anti-nfP2x7 chimeric antigen receptor (CAR) was designed according to the schematic illustrated in FIG. 1.
[0209]An antigen-recognition domain 1 of the CAR was generated which included the amino acid sequence of the PEP2-2-3 binding peptide (amino acid sequence set forth in SEQ ID NO: 10 and nucleotide sequence set forth in SEQ ID NO: 11). The PEP2-2-3 sequence was shown to have specific affinity for the dysfunctional P2X7 receptor expressed on cancer cells, such as prostate LNCap cells, without significant affinity for monocytes or lymphocytes.
[0210]A CD8a signalling peptide 2 (having the amino acid sequence set forth in SEQ ID N...
example 2
Design of Alternative Anti-nfP2X7 Chimeric Antigen Receptor
[0249]A further exemplified protocol detailing the process of designing, and expressing on a T cell, an anti-non-functional (nf) P2X7 receptor CAR, according to an embodiment of the present invention, is detailed below.
[0250]Anti-nfP2X7 CARs were designed utilising three anti-non-functioning P2X7 binding peptides. Specifically, CARs were designed to include antigen recognition domains with sequence homology to peptides PEP2-2-1-1, PEP2-472-2 or PEP2-2-12 (having the amino acid sequences set forth in SEQ ID NOs: 32, 33 and 34 respectively). These binding peptides have been shown to bind to the non-functional P2X7 receptor (Barden, J. A., Sluyter, R., Gu, B. J. & Wiley, J. S. 2003. Specific detection of non-functional human P2X(7) receptors in HEK293 cells and B-lymphocytes. FEBS Lett 538, 159-162).
[0251]The alignment of the above binding peptides to the heavy chain variable regions of antibodies that recognise non-functional ...
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