Prospective Analysis of Adoptive TIL Therapy in Patients with Metastatic Melanoma: Response, Impact of Anti-CTLA4, and Biomarkers to Predict Clinical Outcome.

Purpose: Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) has consistently demonstrated clinical efficacy in metastatic melanoma. Recent widespread use of checkpoint blockade has shifted the treatment landscape, raising questions regarding impact of these therapies on response to TIL and appropriate immunotherapy sequence.Patients and Methods: Seventy-four metastatic melanoma patients were treated with autologous TIL and evaluated for clinical response according to irRC, overall survival, and progression-free survival. Immunologic factors associated with response were also evaluated.Results: Best overall response for the entire cohort was 42%; 47% in 43 checkpoint-naïve patients, 38% when patients were exposed to anti-CTLA4 alone (21 patients) and 33% if also exposed to anti-PD1 (9 patients) prior to TIL ACT. Median overall survival was 17.3 months; 24.6 months in CTLA4-naïve patients and 8.6 months in patients with prior CTLA4 blockade. The latter patients were infused with fewer TIL and experienced a shorter duration of response. Infusion of higher numbers of TIL with CD8 predominance and expression of BTLA correlated with improved response in anti-CTLA4 naïve patients, but not in anti-CTLA4 refractory patients. Baseline serum levels of IL9 predicted response to TIL ACT, while TIL persistence, tumor recognition, and mutation burden did not correlate with outcome.Conclusions: This study demonstrates the deleterious effects of prior exposure to anti-CTLA4 on TIL ACT response and shows that baseline IL9 levels can potentially serve as a predictive tool to select the appropriate sequence of immunotherapies. Clin Cancer Res; 24(18); 4416-28. ©2018 AACR.

Utilizing T-cell Activation Signals 1, 2, and 3 for Tumor-infiltrating Lymphocytes (TIL) Expansion: The Advantage Over the Sole Use of Interleukin-2 in Cutaneous and Uveal Melanoma.

In this study, we address one of the major critiques for tumor-infiltrating lymphocyte (TIL) therapy-the time needed for proper expansion of a suitable product. We postulated that T-cell receptor activation in the first phase of expansion combined with an agonistic stimulation of CD137/4-1BB and interleukin-2 would favor preferential expansion of CD8 TIL. Indeed, this novel 3-signal approach for optimal T-cell activation resulted in faster and more consistent expansion of CD8CD3 TIL. This new method allowed for successful expansion of TIL from cutaneous and uveal melanoma tumors in 100% of the cultures in <3 weeks. Finally, providing the 3 signals attributed to optimal T-cell activation led to expansion of TIL capable of recognizing their tumor counterpart in cutaneous and uveal melanoma. This new methodology for the initial phase of TIL expansion brings a new opportunity for translation of TIL therapy in challenging malignancies such as uveal melanoma.

A Novel Method to Generate and Expand Clinical-Grade, Genetically Modified, Tumor-Infiltrating Lymphocytes.

Following the clinical success achieved with the first generation of adoptive cell therapy (ACT) utilizing in vitro expanded tumor-infiltrating lymphocytes (TILs), the second and third generations of TIL ACT are evolving toward the use of genetically modified TIL. TIL therapy generally involves the transfer of a high number of TIL, ranging from 109 to 1011 cells. One of the technical difficulties in genetically modifying TIL, using a retroviral vector, is the ability to achieve large expansion of transduced TIL, while keeping the technique suitable to a Good Manufacturing Practices (GMP) environment. Consequently, we developed and optimized a novel method for the efficient production of large numbers of GMP-grade, gene-modified TIL for the treatment of patients with ACT. The chemokine receptor CXCR2 was used as the gene of interest for methodology development. The optimized procedure is currently used in the production of gene-modified TIL for two clinical trials for the treatment of metastatic melanoma at MD Anderson Cancer Center.

Intrathecal Administration of Tumor-Infiltrating Lymphocytes Is Well Tolerated in a Patient with Leptomeningeal Disease from Metastatic Melanoma: A Case Report.

UNLABELLED: Patients with leptomeningeal disease (LMD) from melanoma have very poor outcomes and few treatment options. We present a case of intrathecal (i.t.) administration of autologous tumor-infiltrating lymphocytes (TIL) in a patient with LMD from metastatic melanoma. The patient developed LMD after previous treatments with surgery, high-dose bolus interleukin-2 (HD IL2), and systemic TIL infusion and experienced radiographic progression after intrathecal IL2 (i.t. IL2) therapy. The patient received weekly treatment with increasing numbers of i.t. TIL followed by twice-weekly i.t. IL2. The patient received three i.t. TIL infusions and did not experience any toxicities beyond those expected with i.t. IL2 therapy. Analysis of cerebrospinal fluid demonstrated increased inflammatory cytokines following the i.t. TREATMENTS: Subsequent imaging demonstrated disease stabilization, and neurological deficits also remained stable. The patient expired 5 months after the initiation of i.t. TIL therapy with disease progression in the brain, liver, lung, and peritoneal and retroperitoneal lymph nodes, but without LMD progression. These results demonstrate the safety of i.t. administration of TIL in melanoma patients with LMD and support the feasibility of conducting a prospective clinical trial to determine this therapy's clinical benefit among these patients.

Specific lymphocyte subsets predict response to adoptive cell therapy using expanded autologous tumor-infiltrating lymphocytes in metastatic melanoma patients.

PURPOSE: Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) is a promising treatment for metastatic melanoma unresponsive to conventional therapies. We report here on the results of an ongoing phase II clinical trial testing the efficacy of ACT using TIL in patients with metastatic melanoma and the association of specific patient clinical characteristics and the phenotypic attributes of the infused TIL with clinical response. EXPERIMENTAL DESIGN: Altogether, 31 transiently lymphodepleted patients were treated with their expanded TIL, followed by two cycles of high-dose interleukin (IL)-2 therapy. The effects of patient clinical features and the phenotypes of the T cells infused on the clinical response were determined. RESULTS: Overall, 15 of 31 (48.4%) patients had an objective clinical response using immune-related response criteria (irRC) with 2 patients (6.5%) having a complete response. Progression-free survival of more than 12 months was observed for 9 of 15 (60%) of the responding patients. Factors significantly associated with the objective tumor regression included a higher number of TIL infused, a higher proportion of CD8(+) T cells in the infusion product, a more differentiated effector phenotype of the CD8(+) population, and a higher frequency of CD8(+) T cells coexpressing the negative costimulation molecule "B- and T-lymphocyte attenuator" (BTLA). No significant difference in the telomere lengths of TIL between responders and nonresponders was identified. CONCLUSION: These results indicate that the immunotherapy with expanded autologous TIL is capable of achieving durable clinical responses in patients with metastatic melanoma and that CD8(+) T cells in the infused TIL, particularly differentiated effectors cells and cells expressing BTLA, are associated with tumor regression.

Oncogenic BRAF(V600E) promotes stromal cell-mediated immunosuppression via induction of interleukin-1 in melanoma.

PURPOSE: In this study, we assessed the specific role of BRAF(V600E) signaling in modulating the expression of immune regulatory genes in melanoma, in addition to analyzing downstream induction of immune suppression by primary human melanoma tumor-associated fibroblasts (TAF). EXPERIMENTAL DESIGN: Primary human melanocytes and melanoma cell lines were transduced to express WT or V600E forms of BRAF, followed by gene expression analysis. The BRAF(V600E) inhibitor vemurafenib was used to confirm targets in BRAF(V600E)-positive melanoma cell lines and in tumors from melanoma patients undergoing inhibitor treatment. TAF lines generated from melanoma patient biopsies were tested for their ability to inhibit the function of tumor antigen-specific T cells, before and following treatment with BRAF(V600E)-upregulated immune modulators. Transcriptional analysis of treated TAFs was conducted to identify potential mediators of T-cell suppression. RESULTS: Expression of BRAF(V600E) induced transcription of interleukin 1 alpha (IL-1α) and IL-1ß in melanocytes and melanoma cell lines. Further, vemurafenib reduced the expression of IL-1 protein in melanoma cell lines and most notably in human tumor biopsies from 11 of 12 melanoma patients undergoing inhibitor treatment. Treatment of melanoma-patient-derived TAFs with IL-1α/ß significantly enhanced their ability to suppress the proliferation and function of melanoma-specific cytotoxic T cells, and this inhibition was partially attributable to upregulation by IL-1 of COX-2 and the PD-1 ligands PD-L1 and PD-L2 in TAFs. CONCLUSIONS: This study reveals a novel mechanism of immune suppression sensitive to BRAF(V600E) inhibition, and indicates that clinical blockade of IL-1 may benefit patients with BRAF wild-type tumors and potentially synergize with immunotherapeutic interventions.

Intranasal immunization with synthetic peptides corresponding to the E6 and E7 oncoproteins of human papillomavirus type 16 induces systemic and mucosal cellular immune responses and tumor protection.

The E6 and E7 oncoproteins of the high-risk HPV type16 represent ideal targets for HPV vaccine development, they being consistently expressed in cervical cancer lesions. Since HPV-16 is primarily transmitted through genital mucosal route, mucosal immune responses constitute an essential feature for vaccination strategies against HPV-associated lesions. We present here evidence showing that mucosal immunization of mice by the intranasal route with a mixture of peptides E7(44-62) and E6(43-57) from the E7 and E6 oncoproteins of HPV-16, respectively, using a mutant cholera toxin adjuvant (CT-2*), primed strong antigen-specific cellular immune responses in systemic and mucosal tissues. Significant levels of IFN-gamma production by both CD4 and CD8 cells were observed along with CTL responses that were effective against both peptide-pulsed targets as well as syngeneic tumor cells (TC-1) expressing the cognate E6 and E7 proteins. Furthermore, mice immunized with the peptide mixture and CT-2* effectively resisted TC-1 tumor challenge. These results together with our earlier observations that T cell responses to these peptides correlate with recurrence-free survival in women after ablative treatment for HPV-associated cervical intraepithelial neoplasia, support the potential of these E6 and E7 peptides for inclusion in vaccine formulations.