Adoptive T cell therapy remains a promising form of treatment for metastatic melanoma. As highlighted above, T cell activation used to generate tumor-reactive T cells results in a phenotype that is likely suboptimal for therapy. In addition, TIL, TCR-modified T cells, and CAR T cells all typically undergo in vitro expansion to obtain a sufficient number of cells for clinical response . Continued stimulation during expansion leads to further T cell differentiation, shortening of telomeres, and activation induced cell death (AICD) . As cytokines have been shown to render T cells susceptible to lentiviral transduction in the absence of CD3 stimulation, there have been efforts to generate melanoma-reactive T cells in the absence of activation . One group has previously demonstrated successful transfer of a TCR reactive to the melanoma antigen, MART-1, without prior stimulation of CD3 . However, TCR-modified or CAR T cells generated in the absence of activation have yet to be evaluated in vivo. This is likely due to the low absolute yield and the inability to select for transduced cells. In these studies, we demonstrate the efficient transfer of TIL1383I TCR and a CD34 marker allowing for magnetic-based sorting without CD3 activation. In addition, we evaluate TCR-modified T cells generated in the absence of activation using an in vivo system for the first time.
IL-2, IL-7, and IL-15 have been demonstrated to enhance lentiviral transduction of T cells without CD3 stimulation . However, IL-2 has been known to result in terminal differentiation and enhance AICD while both IL-2 and IL-15 induce T cell proliferation [28, 29]. In contrast, IL-7 is a T cell homeostatic regulator that promotes T cell survival without stimulating cell division . Cell cycle analysis in our studies confirmed that IL-7 culture transitions lymphocytes from G0 to G1 without commitment to S phase and therefore proliferation (Fig. 1). Thus, we utilized IL-7 in our studies to facilitate gene transfer in attempt to preserve maximal immune-competence.
Using our lentiviral construct, we were able to efficiently generate TIL1383I TCR-modified T cells following IL-7 culture (Fig. 2b). In the donors we tested, cells transduced following IL-7 culture maintained a relatively balanced CD4+/CD8+ ratio compared to the low CD4+/CD8+ ratio we observed in activated cells. Prior studies from another group found a similarly high proportion of CD8+ cells when transducing stimulated T cells . Infusion products used in different CD19 CAR T cell trials also frequently skewed toward an either predominantly CD4+ or CD8+ population [31–33]. Although the causes are not completely clear, differences in transduction and in vitro expansion protocols, including methods of T cell stimulation, cytokines, feeder cells, viral vectors, and length of expansion, all may account for inconsistent CD4+/CD8+ ratios. Patient sex, age, ethnicities, or prior exposures may also play a role. As variability in the infusion product likely accounts for the unpredictable clinical responses to adoptive cell therapy, there have been efforts in generating a more consistent product. Studies have shown that an infusion product formulated with defined CD4+ and CD8+ subsets in a 1:1 ratio enhances in vivo anti-tumor activity [25, 34]. This has led to a CAR T trial in which CD4+ and specific CD8+ subsets were purified and transduced separately before formulated in a 1:1 ratio [35–37]. Although the trial demonstrated significant clinical responses, the manufacturing process remains laborious. Generating TCR-modified T cells following IL-7 culture appears to be a straightforward strategy to achieve a similar CD4+/CD8+ composition without the potential consequences of T cell activation.
As evidence suggests that less-differentiated T cells have higher therapeutic potential, there are ongoing efforts to preserve less-differentiated phenotypes of TCR-modified or CAR T cells. The group that formulated an infusion product with a defined CD4+/CD8+ ratio used a purified CD8+ TCM starting population for transduction [35–37]. In addition, the use of IL-7 and IL-15 during in vitro expansion has been used to maintain less-differentiated T cell populations [16, 38]. However, both of these strategies continue to rely on CD3 stimulation for efficient gene transfer. Even when cell products were generated from a less-differentiated CD8+ TCM population, the patients received a product that consisted of CD8+ cells that were mostly TEM [35, 37]. This is consistent with our studies that demonstrate significant T cell differentiation following activation (Fig. 3b and c). In addition, a recent study evaluating TCR-modified T cells specific to the cancer-testis antigen, NY-ESO-1, following expansion with IL-7 and IL-15 demonstrated a limited enrichment of less-differentiated cells and no absolute benefit . By transducing T cells in the complete absence of activation, we obtain TIL1383I TCR-modified T cells with preserved TN and TSCM populations and retained telomere lengths (Figs. 3 and 4).
We confirmed the tyrosinase-specific reactivity of TIL1383I TCR-modified T cells generated following IL-7 culture using both in vitro and in vivo systems. Intracellular cytokine assays demonstrated cytokine production when TIL1383I TCR-modified cells generated in the absence of activation were incubated with tyrosinase-loaded targets. However, there appears to be a lower percentage of cytokine producing cells compared to activated TIL1383I TCR-modified T cells (Fig. 5). This is likely due to the nature of TN and TSCM subpopulations, which require more time to react in vitro, but have been shown to be a superior therapy in vivo . The results following overnight incubation in our experiments are likely not reflective of the overall therapeutic potential of TIL1383I TCR-modified T cells generated in the absence of activation. Indeed, we found a superior anti-melanoma response when mice were treated with TIL1383I TCR-modified T cells generated following IL-7 culture compared to mice treated with activated cells (Fig. 6).
Even with efficient gene-transfer of TIL1383I TCR without CD3 stimulation, the percentage of transduced cells was lower than a typical infusion product. However, our CD34t transduction marker permits clinical-grade magnetic selection, resulting in a highly pure transduced product with enhanced tumor-reactivity . In vitro expansion is commonly used to increase the percentage of transduced cells and obtain the required number of cells for infusion, but this comes at the expense of telomere shortening and reduced proliferative potential . It would be difficult to obtain comparable absolute numbers of gene-modified cells typical for infusion without in vitro expansion. However, TIL1383I TCR-modified T cells generated in the absence of activation have desirable qualities for therapy, so the number of cells required to see a clinical response may not be as high. Furthermore, generating a potent therapy without in vitro expansion would shorten production time and permit more timely treatment.
In conclusion, we demonstrate efficient generation of melanoma-reactive TIL1383I TCR-modified T cells in the absence of activation by utilizing IL-7 treatment and a lentiviral vector. TIL1383I TCR-modified T cells generated following IL-7 culture have a balanced CD4+/CD8+ ratio, less-differentiated phenotype, and increased proliferative potential. These TIL1383I TCR-modified T cells generated in the absence of activation have anti-melanoma reactivity both in vitro and in vivo. A growing body of data suggests that less-differentiated phenotypes and longer telomeres correlate with clinical response. Thus, generating TIL1383I TCR-modified T cells in the complete absence of activation is a feasible strategy to improve therapeutic potential. These findings may be applied to other tumor reactive TCRs and CARs and improve adoptive T cell therapy for melanoma and other malignancies.