Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes for metastatic melanoma: current status and future outlook

Abstract

Immunotherapy using autologous T cells has emerged to be a powerful treatment option for patients with metastatic melanoma. These include the adoptive transfer of autologous tumor-infiltrating lymphocytes (TILs), T cells transduced with high-affinity T cell receptors against major tumor antigens, and T cells transduced with chimeric antigen receptors composed of hybrid immunoglobulin light chains with endodomains of T-cell signaling molecules. Among these and other options for T-cell therapy, TILs together with high-dose interleukin 2 have had the longest clinical history with multiple clinical trials in centers across the world consistently demonstrating durable clinical response rates near 50% or more. A distinct advantage of TIL therapy making it still the T-cell therapy of choice is the broad nature of the T-cell recognition against both defined and undefined tumors antigens against all possible major histocompatibility complex, rather than the single specificity and limited major histocompatibility complex coverage of the newer T cell receptors and chimeric antigen receptor transduction technologies. In the past decade, significant inroads have been made in defining the phenotypes of T cells in TIL-mediating tumor regression. CD8+ T cells are emerging to be critical, although the exact subset of CD8+ T cells exhibiting the highest clinical activity in terms of memory and effector markers is still controversial. We present a model in which both effector-memory and more differentiated effector T cells ultimately may need to cooperate to mediate long-term tumor control in responding patients. Although TIL therapy has shown great potential to treat metastatic melanoma, a number of issues have emerged that need to be addressed to bring it more into the mainstream of melanoma care. First, we have a reached the point where a pivotal phase II or phase III trial is needed in an attempt to gain regulatory approval of TILs as standard of care. Second, improvements in how we expand TILs for therapy are needed that minimize the time the T cells are in culture and improve the memory and effector characteristics of the T cells for longer persistence and enhanced anti-tumor activity in vivo. Third, there is a critical need to identify surrogate and predictive biomarkers to better select suitable patients for TIL therapy to improve response rate and duration. Overall, the outlook for TIL therapy for melanoma is very bright. We predict that TILs will indeed emerge to become an approved treatment in the upcoming years through pivotal clinical trials. Moreover, new approaches combining TILs with targeted signaling pathway drugs, such as mutant B-RAF inhibitors, and synergistic immunomodulatory interventions enhancing T-cell costimulation and preventing negative regulation should further increase therapeutic efficacy and durable complete response rates.

Figure 1. Schematic representation of the process of TIL expansion and TIL therapy for metastatic melanoma starting from tumor fragments

Suitable tumors from eligible stage IIIc-IV patients are resected and taken to the lab under sterile conditions where they are cut up into small 3–5 mm² fragments and placed in culture plates or small culture flasks with growth medium and high-dose (HD) IL-2. The TIL are initially expanded for 3–5 weeks during this “pre-rapid expansion protocol” (pre-REP) phase to at least 50 × 10⁶ cells. The cells are then subjected to a rapid expansion protocol (REP) over two weeks by stimulating the T cells using anti-CD3 in the presence of PBMC feeders cells and IL-2. The expanded TIL (now billions of cells) are washed, pooled, and infused into the patient followed by one or two cycles of HD IL-2 therapy. Before TIL transfer, the patient is treated with a preparative regimen using cyclophosphamide (Cy) and fludaribine (Flu) that transiently depletes host lymphocytes “making room” for the infused TIL and removing cytokine sinks and regulatory T cells in order to facilitate TIL persistence.

Figure 2. Stages of CD8⁺ T-cell differentiation found in melanoma CD8⁺ T-cell in TIL and phenotypic markers assessed by flow cytometry used to delineate these stages

CD8⁺ T cells in TIL can be found having markers of central memory (T_CM), effector-memory (T_EM), effector (T_EFF) or terminally-differentiated effector (T_TDE) stages. These stages and markers are based on previous work on anti-viral CD8⁺ T-cell response and the states of CTL differentiation associated with the control of both acute and chronic viral infections in humans. These markers are only a guide to identify these functional memory or effector stages and may not always fit into these defined categories. In addition, the expression of these subset markers may be transiently down-modulated, permanently lost, or re-expressed under different cytokine or in different tissue niches in vivo. Nevertheless, understanding the changes in these effector and memory markers in TIL is very useful in biomarker studies to identify clinically active T-cell subsets. Melanoma TIL expanded for therapy are a mixture of mostly T_EM, T_EFF, T_TDE (that have lost CD28 expression). Relatively very few TCM are found (<5%). Initially, NK cells are found in initial TIL expansions (pre-REP), but these are lost after the REP which selectively activates the T cells. Abbreviations: GB, granzyme B; Perf, perforin.

Figure 3. Model for how CD8⁺ T-cells at different stages of CTL differentiation can synergize or cooperate in controlling tumors following TIL adoptive transfer into patients

The model reconciles a number of contrasting results on phenotypic analysis on TIL subsets and their correlation with clinical response from a number of groups. It proposes that both T_EM and T_EFF and T_TDE may all ultimately play a critical role in adoptive cell therapy at different times to control tumor growth. The TIL need to become cytolytic effector cells to kill tumor cells requiring differentiation into T_EFF and T_TDE cells. However, T_EM cell are long-lived, but have little cytolytic potential. The T_EFF and T_TDE (more oligoclonal) may rapidly home into tumors and orchestrate an early wave of tumor killing. These cells are relatively short-lived and can be replaced by T_EM (more polyclonal) that have a higher intrinsic proliferative capacity and can differentiate later into T_EFF and T_TDE and mediate later waves of tumor killing. In addition to T_EM and T_EFF/T_TDE cells, endogenous T cells re-emerging in the patients after recovery from the transient lymphodepletion may also play a role at these later times by recognizing tumor antigens initially released from earlier waves of tumor killing. Overall, this model explains data on TCR clonotype analysis during TIL therapy where different clonotypes that predominate in the infused decrease and other less frequent clonotypes increase, while other clonotypes are lost altogether to be replaced by TCR clonotypes later on that were not detected in the infused TIL (Radvanyi et al., unpublished data).