Characterization of genetically modified T-cell receptors that recognize the CEA:691-699 peptide in the context of HLA-A2.1 on human colorectal cancer cells.
OBJECTIVE
Carcinoembryonic antigen (CEA) is a tumor-associated protein expressed on a variety of adenocarcinomas. To develop an immunotherapy for patients with cancers that overexpress CEA, we isolated and genetically modified a T-cell receptors (TCRs) that specifically bound a CEA peptide on human cancer cells.


METHODS
HLA-A2.1 transgenic mice were immunized with CEA:691-699. A CEA-reactive TCR was isolated from splenocytes of these mice and was genetically introduced into human peripheral blood lymphocytes via RNA electroporation or retroviral transduction. Amino acid substitutions were introduced throughout the complementarity determining regions (CDR1, CDR2, and CDR3) of both TCR alpha and beta chains to improve recognition of CEA.


RESULTS
Murine lymphocytes bearing the CEA-reactive TCR specifically recognized peptide-loaded T2 cells and HLA-A2.1(+) CEA(+) human colon cancer cells. Both CD8(+) and CD4(+) human lymphocytes expressing the murine TCR specifically recognized peptide-loaded T2 cells. However, only gene-modified CD8(+) lymphocytes specifically recognized HLA-A2.1(+) CEA(+) colon cancer cell lines, and tumor cell recognition was weak and variable. We identified two substitutions in the CDR3 of the alpha chain that significantly influenced tumor cell recognition by human peripheral blood lymphocytes. One substitution, T for S at position 112 (S112T), enhanced tumor cell recognition by CD8(+) lymphocytes, and a second dually substituted receptor (S112T L110F) enhanced tumor cell recognition by CD4(+) T cells.


CONCLUSIONS
The modified CEA-reactive TCRs are good candidates for future gene therapy clinical trials and show the power of selected amino acid substitutions in the antigen-binding regions of the TCR to enhance desired reactivities.
Parkhurst
Maria R
MR
Surgery Branch, National Cancer Institute/NIH, Bethesda, Maryland 20892, USA. Maria_Parkhurst@nih.gov
Joo
Jayne
J
Riley
John P
JP
Yu
Zhiya
Z
Li
Yong
Y
Robbins
Paul F
PF
Rosenberg
Steven A
SA
D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002272:Carcinoembryonic Antigen / Q000737:chemistry / Q000276:immunology*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000276:immunology*; D018274:Electroporation; D015789:HLA-A2 Antigen; D006801:Humans; D008213:Lymphocyte Activation; D051379:Mice; D008822:Mice, Transgenic; D010455:Peptides; D011948:Receptors, Antigen, T-Cell / Q000235:genetics* / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
D002272:Carcinoembryonic Antigen; D015789:HLA-A2 Antigen; D010455:Peptides; D011948:Receptors, Antigen, T-Cell