Selection of Gene-Marked Tumor Infiltrating Lymphocytes from Post-Treatment Biopsies: A Case Study

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Abstract
Patients with malignant melanoma have been treated with interleukin-2 (IL-2) and tumor-infiltrating lymphocytes (TIL) marked by retroviral gene transduction. The retroviral vector contained a gene coding for the bacterial enzyme neomycin phosphotransferase, such that transduced TIL expressing the enzyme could survive otherwise toxic concentrations of the neomycin analogue G418. For 1 patient, who exhibited a complete regression of cancer after treatment with TIL, lymphocytes from post-treatment blood and tumor biopsies were cultured in IL-2, and transduced TIL were recovered by G418 selection. Analysis of T-cell receptor heterogeneity indicated that the transduced TIL recovered from the tumor biopsy were different from TIL that were kept strictly in vitro and selected in G418. The selection process required weeks in culture, during which time control cultures changed radically in subset composition, so there was also a simultaneous selection for long-term in vitro growth advantage. It cannot be certain that the TIL subsets preferentially recovered from the tumor biopsy corresponded to those that mediated complete elimination of tumor in this patient. The first human gene transfer clinical protocol involved the insertion of a marker gene into tumor infiltrating lymphocytes (TIL) to study how these immune cells traffic in the body. The marker was NeoR, a bacterial gene that provides resistance to the neomycin-like antibiotic G418. Aebersold and his colleagues demonstrate that the TIL that were marked with NeoR were resistant to G418 at the time that they were given to the patient, and that it was possible, though difficult, to isolate G418-resistant TIL back from patient blood and tumor tissue.