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. An in vitro method of enriching or selecting human T-cells comprising a genetically modified Epidermal Growth Factor Receptor (EGFR) gene, the gene comprising a nucleotide sequence encoding a truncated non-immunogenic endogenous cell surface molecule, the cell surface molecule comprising an EGFR Domain III and an EGFR Domain IV; but lacking 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 nonimmunogenic 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, the method comprising:
contacting a population of cells comprising the human T cells comprising a genetically modified EGFR gene with an antibody that binds to the truncated non-immunogenic endogenous cell surface molecule.
2. The in vitro method of claim 1 , wherein the genetically modified EGFR gene comprises nucleotides 67-1071 of SEQ ID NO:2.
3. The in vitro method of claim 1 , wherein the genetically modified EGFR gene encodes an amino acid sequence comprising residues 23-357 of SEQ ID NO:3.
4. The in vitro method of claim 1 , wherein the genetically modified EGFR gene further comprises a GMCSFR alpha chain signal sequence.
5. The in vitro method of claim 4 , wherein the genetically modified EGFR gene comprises SEQ ID NO:2.
6. The in vitro method of claim 4 , wherein the genetically modified EGFR gene encodes an amino acid sequence comprising at least 90% identical to SEQ ID NO:3.
7. The in vitro method of claim 4 , wherein the genetically modified EGFR gene encodes an amino acid sequence comprising SEQ ID NO:3.
8. The in vitro method of claim 1 , wherein the genetically modified EGFR gene is inserted into a vector.
9. The in vitro method 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, wherein the tumor associated antigen is selected from the group consisting of CD19, CD20 and CD22.
10. The in vitro method of claim 1 , wherein the modified EGFR is coupled to a codon-optimized anti-CD 19 costimulatory chimeric antigen receptor (CD 19CAR) and a C-terminal 2A cleavable linker.
11. An in vitro method of enriching or selecting human T-cells comprising a genetically modified Epidermal Growth Factor Receptor (EGFR) gene that is coupled to a CD19CAR and a C-terminal 2A cleavable linker, wherein the gene encodes an amino acid sequence comprising SEQ ID NO:6.
12. The in vitro method of claim 11 , wherein the genetically modified EGFR gene is inserted into a vector to transfect the population of human T-cells.
13. An in vivo of method of depleting T cells comprising a genetically modified Epidermal Growth Factor Receptor (EGFR) gene, the gene comprising a nucleotide sequence encoding a truncated non-immunogenic endogenous cell surface molecule, the cell surface molecule comprising an EGFR Domain III and an EGFR Domain IV; but lacking 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 nonimmunogenic 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, the method comprising:
administering, to a patient that has been previously administered a population of cells comprising the human T cells comprising a genetically modified EGFR gene with, an antibody that binds to the truncated non-immunogenic endogenous cell surface molecule.