Randomized phase II trial of lymphodepletion plus adoptive cell transfer of tumor-infiltrating lymphocytes, with or without dendritic cell vaccination, in patients with metastatic melanoma.

BACKGROUND: The adoptive transfer of tumor-infiltrating lymphocytes (TIL) has demonstrated robust efficacy in metastatic melanoma patients. Tumor antigen-loaded dendritic cells (DCs) are believed to optimally activate antigen-specific T lymphocytes. We hypothesized that the combined transfer of TIL, containing a melanoma antigen recognized by T cells 1 (MART-1) specific population, with MART-1-pulsed DC will result in enhanced proliferation and prolonged survival of transferred MART-1 specific T cells in vivo ultimately leading to improved clinical responses. DESIGN: We tested the combination of TIL and DC in a phase II clinical trial of patients with advanced stage IV melanoma. HLA-A0201 patients whose early TIL cultures demonstrated reactivity to MART-1 peptide were randomly assigned to receive TIL alone or TIL +DC pulsed with MART-1 peptide. The primary endpoint was to evaluate the persistence of MART-1 TIL in the two arms. Secondary endpoints were to evaluate clinical response and survival. RESULTS: Ten patients were given TIL alone while eight patients received TIL+DC vaccine. Infused MART-1 reactive CD8+ TIL were tracked in the blood over time by flow cytometry and results show good persistence in both arms, with no difference in the persistence of MART-1 between the two arms. The objective response rate was 30% (3/10) in the TIL arm and 50% (4/8) in the TIL+DC arm. All treatments were well tolerated. CONCLUSIONS: The combination of TIL +DC showed no difference in the persistence of MART-1 TIL compared with TIL therapy alone. Although more patients showed a clinical response to TIL+DC therapy, this study was not powered to resolve differences between groups. TRIAL REGISTRATION NUMBER: NCT00338377.

COVID-19: a pandemic experience that illuminates potential reforms to health research.

COVID-19 has halted research around the globe and forced researchers out of their laboratories. Non-emergency medical appointments were canceled. Ongoing clinical trials were challenged to create new modes of operation while public pressure mounted to find therapeutic options against COVID-19. Yet, the inability to conduct research during COVID-19 was overcome with cooperation, resource sharing, and compassion, which provides important lessons on how to improve health related research as we enter a new normal.

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.

Single-cell profiling of dynamic cytokine secretion and the phenotype of immune cells.

Natural killer (NK) cells are a highly heterogeneous population of innate lymphocytes that constitute our first line of defense against several types of tumors and microbial infections. Understanding the heterogeneity of these lymphocytes requires the ability to integrate their underlying phenotype with dynamic functional behaviors. We have developed and validated a single-cell methodology that integrates cellular phenotyping and dynamic cytokine secretion based on nanowell arrays and bead-based molecular biosensors. We demonstrate the robust passivation of the polydimethylsiloxane (PDMS)-based nanowells arrays with polyethylene glycol (PEG) and validated our assay by comparison to enzyme-linked immunospot (ELISPOT) assays. We used numerical simulations to optimize the molecular density of antibodies on the surface of the beads as a function of the capture efficiency of cytokines within an open-well system. Analysis of hundreds of individual human peripheral blood NK cells profiled ex vivo revealed that CD56dimCD16+ NK cells are immediate secretors of interferon gamma (IFN-γ) upon activation by phorbol 12-myristate 13-acetate (PMA) and ionomycin (< 3 h), and that there was no evidence of cooperation between NK cells leading to either synergistic activation or faster IFN-γ secretion. Furthermore, we observed that both the amount and rate of IFN-γ secretion from individual NK cells were donor-dependent. Collectively, these results establish our methodology as an investigational tool for combining phenotyping and real-time protein secretion of individual cells in a high-throughput manner.

Multifaceted Role of BTLA in the Control of CD8+ T-cell Fate after Antigen Encounter.

Purpose: Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes (TIL) has shown an overall clinical response rate 40%-50% in metastatic melanoma patients. BTLA (B-and-T lymphocyte associated) expression on transferred CD8+ TILs was associated with better clinical outcome. The suppressive function of the ITIM and ITSM motifs of BTLA is well described. Here, we sought to determine the functional characteristics of the CD8+BTLA+TIL subset and define the contribution of the Grb2 motif of BTLA in T-cell costimulation.Experimental Design: We determined the functional role and downstream signal of BTLA in both human CD8+ TILs and mouse CD8+ T cells. Functional assays were used including single-cell analysis, reverse-phase protein array (RPPA), antigen-specific vaccination models with adoptively transferred TCR-transgenic T cells as well as patient-derived xenograft (PDX) model using immunodeficient NOD-scid IL2Rgammanull (NSG) tumor-bearing mice treated with autologous TILs.Results: CD8+BTLA- TILs could not control tumor growth in vivo as well as their BTLA+ counterpart and antigen-specific CD8+BTLA- T cells had impaired recall response to a vaccine. However, CD8+BTLA+ TILs displayed improved survival following the killing of a tumor target and heightened "serial killing" capacity. Using mutants of BTLA signaling motifs, we uncovered a costimulatory function mediated by Grb2 through enhancing the secretion of IL-2 and the activation of Src after TCR stimulation.Conclusions: Our data portrays BTLA as a molecule with the singular ability to provide both costimulatory and coinhibitory signals to activated CD8+ T cells, resulting in extended survival, improved tumor control, and the development of a functional recall response. Clin Cancer Res; 23(20); 6151-64. ©2017 AACR.

Inhibition of the B7-H3 immune checkpoint limits tumor growth by enhancing cytotoxic lymphocyte function.

The interaction between tumor and the immune system is still poorly understood. Significant clinical responses have been achieved in cancer patients treated with antibodies against the CTLA4 and PD-1/PD-L1 checkpoints; however, only a small portion of patients responded to the therapies, indicating a need to explore additional co-inhibitory molecules for cancer treatment. B7-H3, a member of the B7 superfamily, was previously shown by us to inhibit T-cell activation and autoimmunity. In this study, we have analyzed the function of B7-H3 in tumor immunity. Expression of B7-H3 was found in multiple tumor lines, tumor-infiltrating dendritic cells, and macrophages. B7-H3-deficient mice or mice treated with an antagonistic antibody to B7-H3 showed reduced growth of multiple tumors, which depended on NK and CD8+ T cells. With a putative receptor expressed by cytotoxic lymphocytes, B7-H3 inhibited their activation, and its deficiency resulted in increased cytotoxic lymphocyte function in tumor-bearing mice. Combining blockades of B7-H3 and PD-1 resulted in further enhanced therapeutic control of late-stage tumors. Taken together, our results indicate that the B7-H3 checkpoint may serve as a novel target for immunotherapy against cancer.

4-1BB-Enhanced Expansion of CD8+ TIL from Triple-Negative Breast Cancer Unveils Mutation-Specific CD8+ T Cells.

Triple-negative breast cancer (TNBC) highly infiltrated with CD8+ tumor-infiltrating lymphocytes (TIL) has been associated with improved prognosis. This observation led us to hypothesize that CD8+ TIL could be utilized in autologous adoptive cell therapy for TNBC, although this concept has proven to be challenging, given the difficulty in expanding CD8+ TILs in solid cancers other than in melanoma. To overcome this obstacle, we used an agonistic antibody (urelumab) to a TNFR family member, 4-1BB/CD137, which is expressed by recently activated CD8+ T cells. This approach was first utilized in melanoma and, in this study, led to advantageous growth of TILs for the majority of TNBC tumors tested. The agonistic antibody was only added in the initial setting of the culture and yet favored the propagation of CD8+ TILs from TNBC tumors. These expanded CD8+ TILs were capable of cytotoxic functions and were successfully utilized to demonstrate the presence of immunogenic mutations in autologous TNBC tumor tissue without recognition of the wild-type counterpart. Our findings open the way for a successful adoptive immunotherapy for TNBC. Cancer Immunol Res; 5(6); 439-45. ©2017 AACR.

Novel algorithmic approach predicts tumor mutation load and correlates with immunotherapy clinical outcomes using a defined gene mutation set.

BACKGROUND: While clinical outcomes following immunotherapy have shown an association with tumor mutation load using whole exome sequencing (WES), its clinical applicability is currently limited by cost and bioinformatics requirements. METHODS: We developed a method to accurately derive the predicted total mutation load (PTML) within individual tumors from a small set of genes that can be used in clinical next generation sequencing (NGS) panels. PTML was derived from the actual total mutation load (ATML) of 575 distinct melanoma and lung cancer samples and validated using independent melanoma (n = 312) and lung cancer (n = 217) cohorts. The correlation of PTML status with clinical outcome, following distinct immunotherapies, was assessed using the Kaplan-Meier method. RESULTS: PTML (derived from 170 genes) was highly correlated with ATML in cutaneous melanoma and lung adenocarcinoma validation cohorts (R2 = 0.73 and R2 = 0.82, respectively). PTML was strongly associated with clinical outcome to ipilimumab (anti-CTLA-4, three cohorts) and adoptive T-cell therapy (1 cohort) clinical outcome in melanoma. Clinical benefit from pembrolizumab (anti-PD-1) in lung cancer was also shown to significantly correlate with PTML status (log rank P value < 0.05 in all cohorts). CONCLUSIONS: The approach of using small NGS gene panels, already applied to guide employment of targeted therapies, may have utility in the personalized use of immunotherapy in cancer.

Defective STAT1 activation associated with impaired IFN-γ production in NK and T lymphocytes from metastatic melanoma patients treated with IL-2.

High dose (HD) IL-2 therapy has been used for almost two decades as an immunotherapy for metastatic melanoma. IL-2 promotes the proliferation and effector function of T and NK cells through the tyrosine phosphorylation and activation of signal transducer and activator of transcription factors (STAT), especially STAT5. However, whether any defects in STAT activation exist in T and NK lymphocytes from melanoma patients are under debate. Here, we measured the extent of HD IL-2-induced phosphorylation of STAT5 and STAT1 in lymphocyte subsets from metastatic melanoma patients and healthy controls at a single cell level using flow cytometry. We found no defects in IL-2-induced STAT5 phosphorylation and induction of proliferation in T and NK cell subsets in vitro. This was confirmed by measuring ex vivo STAT5 activation in whole blood collected from patients during their first bolus HD IL-2 infusion. IL-2 also induced STAT1 phosphorylation via IFN-γ receptors in T and NK cell subsets through the release of IFN-γ by CD56hi and CD56lo NK cells. Further analysis revealed that melanoma patients had a sub-optimal STAT1 activation response linked to lower IL-2-induced IFN-γ secretion in both CD56hi and CD56low NK cell subsets. STAT1 activation in response to IL-2 also showed an age-related decline in melanoma patients not linked to tumor burden indicating a premature loss of NK cell function. Taken together, these findings indicate that, although STAT5 activation is normal in metastatic melanoma patients in response to IL-2, indirect STAT1 activation is defective owing to deficiencies in the NK cell response to IL-2.

The beneficial effects of a gas-permeable flask for expansion of Tumor-Infiltrating lymphocytes as reflected in their mitochondrial function and respiration capacity.

Adoptive transfer of autologous ex vivo expanded tumor-infiltrating lymphocytes (TIL) is a highly successful cell therapy approach in the treatment of late-stage melanoma. Notwithstanding the success of this therapy, only very few centers worldwide can provide it. To make this therapy broadly available, one of the major obstacles to overcome is the complexity of culturing the TIL. Recently, major efforts have been deployed to resolve this issue. The use of the Gas-permeable flask (G-Rex) during the REP has been one application that has facilitated this process. Here we show that the use of this new device is able to rescue poor TIL growth and maintain clonal diversity while supporting an improved mitochondrial function.

Tumor-Infiltrating Lymphocyte Therapy: Addressing Prevailing Questions.

Autologous adoptive T-cell therapies have made tremendous strides over the last few years with excitement currently being generated by technologies that can reprogram T-cell specificities toward any desired antigen including chimeric antigen receptors and recombinant T-cell receptors. Time will tell whether these new genetically engineered T-cell technologies will be effective as advertised, especially in solid tumors, considering the limited availability of specific antigens and the difficulty in managing the unpredictable on-target, off-tissue toxicities. However, a form of T-cell therapy that has been utilized in patients more than any other and has left a lasting mark in the field is tumor-infiltrating lymphocytes (TILs). Tumor-infiltrating lymphocyte therapy has consistently yielded durable clinical responses in selected patients with metastatic melanoma and is now being increasingly applied to treat other solid tumors, including head and neck squamous cell carcinoma, cervical cancer, breast cancer, and lung cancer. Despite its long history in the clinic and key developments over the last few decades that have augmented response rates and have made TIL manufacturing more streamlined, a number of key outstanding conceptual questions remain to be answered in the TIL therapy field. In this review, we address critical questions, including the mechanism of action of TILs and active T-cell subsets, the current need for lymphoablative preconditioning, predictive biomarkers, the role of combination therapy such as checkpoint blockade, new excitement over the recognition of mutated antigens (the "mutanome") by TILs, and issues in developing TILs for nonmelanoma indications. In each case, we will critically discuss the main issues and concerns and how they can affect the eventual positioning of TIL therapy in the mainstream of cancer care.

BTLA marks a less-differentiated tumor-infiltrating lymphocyte subset in melanoma with enhanced survival properties.

In a recent adoptive cell therapy (ACT) clinical trial using autologous tumor-infiltrating lymphocytes (TILs) in patients with metastatic melanoma, we found an association between CD8+ T cells expressing the inhibitory receptor B- and T-lymphocyte attenuator (BTLA) and clinical response. Here, we further characterized this CD8+BTLA+ TIL subset and their CD8+BTLA- counterparts. We found that the CD8+ BTLA+ TILs had an increased response to IL-2, were less-differentiated effector-memory (TEM) cells, and persisted longer in vivo after infusion. In contrast, CD8+BTLA- TILs failed to proliferate and expressed genes associated with T-cell deletion/tolerance. Paradoxically, activation of BTLA signaling by its ligand, herpes virus entry mediator (HVEM), inhibited T-cell division and cytokine production, but also activated the Akt/PKB pathway thus protecting CD8+BTLA+ TILs from apoptosis. Our results point to a new role of BTLA as a useful T-cell differentiation marker in ACT and a dual signaling molecule that curtails T-cell activation while also conferring a survival advantage for CD8+ T cells. These attributes may explain our previous observation that BTLA expression on CD8+ TILs correlates with clinical response to adoptive T-cell therapy in metastatic melanoma.

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.

BRAFV600E Co-opts a Conserved MHC Class I Internalization Pathway to Diminish Antigen Presentation and CD8+ T-cell Recognition of Melanoma.

Oncogene activation in tumor cells induces broad and complex cellular changes that contribute significantly to disease initiation and progression. In melanoma, oncogenic BRAF(V600E) has been shown to drive the transcription of a specific gene signature that can promote multiple mechanisms of immune suppression within the tumor microenvironment. We show here that BRAF(V600E) also induces rapid internalization of MHC class I (MHC-I) from the melanoma cell surface and its intracellular sequestration within endolysosomal compartments. Importantly, MAPK inhibitor treatment quickly restored MHC-I surface expression in tumor cells, thereby enhancing melanoma antigen-specific T-cell recognition and effector function. MAPK pathway-driven relocalization of HLA-A*0201 required a highly conserved cytoplasmic serine phosphorylation site previously implicated in rapid MHC-I internalization and recycling by activated immune cells. Collectively, these data suggest that oncogenic activation of BRAF allows tumor cells to co-opt an evolutionarily conserved MHC-I trafficking pathway as a strategy to facilitate immune evasion. This link between MAPK pathway activation and the MHC-I cytoplasmic tail has direct implications for immunologic recognition of tumor cells and provides further evidence to support testing therapeutic strategies combining MAPK pathway inhibition with immunotherapies in the clinical setting.

Peptide-based vaccination and induction of CD8+ T-cell responses against tumor antigens in breast cancer.

Tumor-associated antigens (TAAs) have been identified in many malignant tumors. Within these TAAs are peptide sequences that bind major histocompatibility complex (MHC) class I and class II molecules recognized by T cells triggering antigen-specific CD8+ cytotoxic T-cell and CD4+ T-helper cell responses. Efforts to develop vaccines for breast cancer have been underway for more than 20 years, including peptide and whole inactivated tumor cell vaccines as well as antigen-loaded dendritic cell vaccines. The majority of vaccine trials have used peptides, including single-peptide and multiple-peptide formulations using either MHC class I and class II epitopes in oil-based emulsions alone or in combination with an adjuvant, such as granulocyte-macrophage colony-stimulating factor, and Toll-like receptor agonists. Preclinical research in vitro and in animal models has been aimed at improving vaccine efficacy by identifying more immunogenic peptides and combinations of peptides and adjuvants and cytokine adjuvants that induce stronger immune responses and prolong T-cell memory. Clinical studies investigating the therapeutic potential of active immunization using peptide vaccines has found no serious side effects. In this review, we examine TAA peptide-based vaccination regimens showing promise in breast cancer patients that are also being investigated in clinical trials of safety and efficacy. We also discuss the current limitations in the peptide vaccination field and areas for future development.

Activation and propagation of tumor-infiltrating lymphocytes on clinical-grade designer artificial antigen-presenting cells for adoptive immunotherapy of melanoma.

PURPOSE: Adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL) is a therapy for metastatic melanoma with response rates of up to 50%. However, the generation of the TIL transfer product is challenging, requiring pooled allogeneic normal donor peripheral blood mononuclear cells (PBMC) used in vitro as "feeders" to support a rapid-expansion protocol. Here, we optimized a platform to propagate TIL to a clinical scale using K562 cells genetically modified to express costimulatory molecules such as CD86, CD137-ligand, and membrane-bound IL-15 to function as artificial antigen-presenting cells (aAPC) as an alternative to using PBMC feeders. EXPERIMENTAL DESIGN: We used aAPC or γ-irradiated PBMC feeders to propagate TIL and measured rates of expansion. The activation and differentiation state was evaluated by flow cytometry and differential gene expression analyses. Clonal diversity was assessed on the basis of the pattern of T-cell receptor usage. T-cell effector function was measured by evaluation of cytotoxic granule content and killing of target cells. RESULTS: The aAPC propagated TIL at numbers equivalent to that found with PBMC feeders, whereas increasing the frequency of CD8 T-cell expansion with a comparable effector-memory phenotype. mRNA profiling revealed an upregulation of genes in the Wnt and stem-cell pathways with the aAPC. The aAPC platform did not skew clonal diversity, and CD8 T cells showed comparable antitumor function as those expanded with PBMC feeders. CONCLUSIONS: TIL can be rapidly expanded with aAPC to clinical scale generating T cells with similar phenotypic and effector profiles as with PBMC feeders. These data support the clinical application of aAPC to manufacture TIL for the treatment of melanoma.

An Antibody-based Blood Test Utilizing a Panel of Biomarkers as a New Method for Improved Breast Cancer Diagnosis.

In order to develop a new tool for diagnosis of breast cancer based on autoantibodies against a panel of biomarkers, a clinical trial including blood samples from 507 subjects was conducted. All subjects showed a breast abnormality on exam or breast imaging and final biopsy pathology of either breast cancer patients or healthy controls. Using an enzyme-linked immunosorbent assay, the samples were tested for autoantibodies against a predetermined number of biomarkers in various models that were used to determine a diagnosis, which was compared to the clinical status. Our new assay achieved a sensitivity of 95.2% [CI = 92.8-96.8%] at a fixed specificity of 49.5%. Receiver-operator characteristic curve analysis showed an area under the curve of 80.1% [CI = 72.6-87.6%]. These results suggest that a blood test which is based on models comprising several autoantibodies to specific biomarkers may be a new and novel tool for improving the diagnostic evaluation of breast cancer.

Continuous 4-1BB co-stimulatory signals for the optimal expansion of tumor-infiltrating lymphocytes for adoptive T-cell therapy.

Co-stimulation through members of the tumor necrosis factor receptor (TNFR) family appears to be critical for the generation of T cells with optimal effector-memory properties for adoptive cell therapy. Our work suggests that continuous 4-1BB/CD137 co-stimulation is required for the expansion of T cells with an optimal therapeutic profile and that the administration of 4-1BB agonists upon adoptive cell transfer further improves antitumor T-cell functions.