Truncated epiderimal growth factor receptor (EGFRt) for transduced T cell selection
A non-immunogenic selection epitope may be generated by removing certain amino acid sequences of the protein. For example, a gene encoding a truncated human epidermal growth factor receptor polypeptide (EGFRt) that lacks the membrane distal EGF-binding domain and the cytoplasmic signaling tail, but retains an extracellular epitope recognized by an anti-EGFR antibody is provided. Cells may be genetically modified to express EGFRt and then purified without the immunoactivity that would accompany the use of full-length EGFR immunoactivity. Through flow cytometric analysis, EGFRt was successfully utilized as an in vivo tracking marker for genetically modified human T cell engraftment in mice. Furthermore, EGFRt was demonstrated to have cellular depletion potential through cetuximab mediated antibody dependent cellular cytotoxicity (ADCC) pathways. Thus, EGFRt may be used as a non-immunogenic selection tool, tracking marker, a depletion tool or a suicide gene for genetically modified cells having therapeutic potential.
1. A genetically modified Epidermal Growth Factor Receptor (EGFR) gene, comprising a nucleotide sequence encoding a truncated non-immunogenic endogenous cell surface molecule, said cell surface molecule comprising an EGFR Domain III and an EGFR Domain IV; but lacking nucleotides all of the domains consisting of an EGFR Domain I, an EGFR Domain II, an EGFR Juxtamembrane Domain, and an EGFR Tyrosine Kinase Domain; wherein the truncated non-immunogenic endogenous cell surface molecule (i) does not have endogenous signaling or trafficking function; (ii) binds a therapeutic anti-EGFR antibody; (iii) does not bind an endogenous EGFR ligand; and (iv) acts as a marker.
2. The gene of claim 1 , further comprising a GMCSFR alpha chain signal sequence.
3. The gene of claim 2 comprising SEQ ID NO:2.
4. The gene of claim 2 , wherein the gene encodes an amino acid sequence comprising at least 90% identical to SEQ ID NO:3.
5. The gene of claim 2 , wherein the gene encodes an amino acid sequence comprising SEQ ID NO:3.
6. The gene of claim 1 , wherein the gene is part of a construct which comprises the modified EGFR coupled via a C-terminal 2A cleavable linker to a chimeric antigen receptor specific for a tumor associated antigen selected from CD19, a codon-optimized anti-CD19 costimulatory chimeric antigen receptor (CD19CAR), CD20 or CD22.
7. The gene of claim 6 , wherein the modified EGFR is coupled to a CD19CAR and a C-terminal 2A cleavable linker.
8. The gene of claim 1 , comprising nucleotides 67-1071 of SEQ ID NO:2.
9. The gene of claim 1 , comprising nucleotides 67-1071 of SEQ ID NO:1.
10. The gene of claim 2 comprising SEQ ID NO:1.
11. The gene of claim 1 , wherein the gene encodes an amino acid sequence comprising residues 23-357 of SEQ ID NO:2.
12. A modified EGFR gene coupled to a CD19CAR and a C-terminal 2A cleavable linker, wherein the gene encodes an amino acid sequence comprising SEQ ID NO:6.
13. The modified EGFR gene of claim 1 , wherein the marker is used to enrich cells.
14. The modified EGFR gene of claim 1 , wherein the marker is used to select cells.
15. The modified EGFR gene of claim 1 , wherein the marker is used to induce cell suicide in cells expressing the truncated non-immunogenic endogenous cell surface molecule.
16. The modified EGFR gene of claim 1 , wherein the endogenous EGFR ligand is EGF.
17. The modified EGFR gene of claim 1 , wherein the therapeutic anti-EGFR antibody is cetuximab.
18. A genetically modified Epidermal Growth Factor Receptor (EGFR) gene, comprising a nucleotide sequence encoding a truncated non-immunogenic endogenous cell surface molecule, said cell surface molecule comprising an EGFR Domain III and an EGFR Domain IV; but lacking nucleotides all of the domains consisting of an EGFR Domain I, an EGFR Domain II, an EGFR Juxtamembrane Domain, and an EGFR Tyrosine Kinase Domain; wherein the truncated non-immunogenic endogenous cell surface molecule (i) does not contain endogenous signaling or trafficking function; (ii) binds a therapeutic anti-EGFR antibody; (iii) does not bind an endogenous EGFR ligand; and (iv) acts as a marker used to enrich cells, select cells, or induce cell suicide in cells expressing said cell surface molecule.
19. The modified EGFR gene of claim 18 , wherein the endogenous EGFR ligand is EGF.
20. The modified EGFR gene of claim 18 , wherein the therapeutic anti-EGFR antibody is cetuximab.