Date of Graduation
5-2015
Document Type
Dissertation (PhD)
Program Affiliation
Immunology
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Laurence J. N. Cooper
Committee Member
Samir Hanash
Committee Member
Dean Lee
Committee Member
Willem Overwijk
Committee Member
Michelle Barton
Abstract
Patients with metastatic melanoma have a poor prognosis due to resistance to conventional therapies. Thus, new targeted treatment strategies are required to improve therapeutic outcome. One prospective approach is to infuse T cells that are rendered specific for tumor-associated antigens (TAAs) preferentially expressed on melanoma cells. Recognition of cell-surface TAAs independent of major histocompatibility complex can be achieved by introducing a TAA-specific chimeric antigen receptor (CAR) on T cells using gene therapy. This approach is being used in clinical trials to adoptively transfer CD19-specific CAR+ T cells in patients with B-lineage malignancies. To generate T-cell therapy for melanoma we targeted a TAA derived from human endogenous retroviruses (HERV), whose genome stably integrated into humans millions of years ago. During oncogenesis, biologically active variants of HERV, such as the envelope (env) protein of HERV-K, are expressed on the surface of melanoma, but not normal cells. To target HERV-K, T cells were engineered to express a CAR specific for this env protein, by replacing the antigen-binding exodomain of CD19-specific CAR with the single chain antibody (scFv) sequence of an anti-HERV-K env specific monoclonal antibody. This new CAR was cloned as a transposon into our Sleeping Beauty (SB) system that we have adapted for human application. DNA plasmids coding for the HERV-K env-specific CAR and SB transposase were electro-transferred into primary human T cells, and genetically modified CAR+ T cells were selectively propagated on irradiated artificial activating and propagating cells (AaPC) expressing HERV-K env and the desired T-cell co-stimulatory molecules. After co-culture on g-irradiated AaPC, 95% of CD3+ T cells expressed the CAR and these HERV-K env-specific CAR+ T cells were able to specifically kill HERV-K env+, but not HERV-K env-, melanoma targets in vitro in contrast to control (no DNA) T cells. Specificity of these CAR+ T cells was proved by over- expressing HERV-K env protein in antigen negative EL4 mouse cells that were preferentially killed compared to HERV-K env- EL4 parental cells. HERV-K env knockdown by shRNA on A888 cells resulted in reduced killing compared to parental A888 melanoma cells. A novel observation was that the antigen is not a type 1 transmembrane protein and that it is shed from its surface which is recognized by HERV-K env-specific CAR+ T cells. The CAR+ T cells were also successful in reducing tumor growth and metastasis of A375-super metastatic (SM) tumor cells from lungs to liver in vivo. The tumor-bearing mice receiving the CAR+T cells lived longer and appeared healthier than the control tumor-bearing mice who received no CAR+ T cells. In aggregate, these data demonstrate for the first time that T cells targeting an active ancient retrovirus can be used as an immunotherapy for melanoma, using an approach that has translational appeal for clinical trials.
Keywords
Immunotherapy, Human endogenous retrovirus, melanoma