Intratumoral Activation of 41BB Costimulatory Signals Enhances CD8 T Cell Expansion and Modulates Tumor-Infiltrating Myeloid Cells.

The activation of 41BB costimulatory signals by agonistic Abs enhances the expansion and function of tumor-infiltrating lymphocytes (TILs) for treating cancer patients with adoptive cell therapy. However, the impact of 41BB agonism is not limited to enhancing the activity of T cells, and the mechanism by which additional activation of this costimulatory axis in tumor-associated myeloid cells is poorly understood. In this study, we describe that the intratumoral administration of 41BB agonistic Abs led to increases in CD8 T cell infiltration followed by tumor regression in murine models. We found that granulocytes and monocytes rapidly replaced macrophages and dendritic cells in tumors following administration of anti-41BB Abs. Overall, myeloid cells from anti-41BB-treated tumors had an improved capacity to stimulate T cells in comparison with control-treated tumors. In human coculture systems, we demonstrated that the agonism of the 41BB-41BBL axis enhanced costimulatory signals and effector functions among APC and autologous TILs. Overall, these findings suggest that the effect of 41BB agonistic Abs are supported by additional costimulatory signals from tumor-associated myeloid cells,v leading to enhanced TIL expansion and function.

Reactive Myelopoiesis Triggered by Lymphodepleting Chemotherapy Limits the Efficacy of Adoptive T Cell Therapy.

Adoptive T cell therapy (ACT) in combination with lymphodepleting chemotherapy is an effective strategy to induce the eradication of tumors, providing long-term regression in cancer patients. Despite that lymphodepleting regimens condition the host for optimal engraftment and expansion of adoptively transferred T cells, lymphodepletion concomitantly promotes immunosuppression during the course of endogenous immune recovery. In this study, we have identified that lymphodepleting chemotherapy initiates the mobilization of hematopoietic progenitor cells that differentiate to immunosuppressive myeloid cells, leading to a dramatic increase of peripheral myeloid-derived suppressor cells (MDSCs). In melanoma and lung cancer patients, MDSCs rapidly expanded in the periphery within 1 week after completion of a lymphodepleting regimen and infusion of autologous tumor-infiltrating lymphocytes (TILs). This expansion was associated with disease progression, poor survival, and reduced TIL persistence in melanoma patients. We demonstrated that the interleukin 6 (IL-6)-driven differentiation of mobilized hematopoietic progenitor cells promoted the survival and immunosuppressive capacity of post-lymphodepletion MDSCs. Furthermore, the genetic abrogation or therapeutic inhibition of IL-6 in mouse models enhanced host survival and reduced tumor growth in mice that received ACT. Thus, the expansion of MDSCs in response to lymphodepleting chemotherapy may contribute to ACT failure, and targeting myeloid-mediated immunosuppression may support anti-tumor immune responses.

Phase Ib trial of IRX-2 plus durvalumab in patients with recurrent and/or metastatic head and neck squamous cell carcinoma.

OBJECTIVES: IRX-2 is a multi-cytokine immune-activating agent with anti-tumor activity in non-metastatic head and neck squamous cell carcinoma (HNSCC). Here, we evaluated combined IRX-2 and durvalumab in patients with recurrent and/or metastatic HNSCC. MATERIALS AND METHODS: This was a phase Ib trial consisting of dose escalation and expansion. Primary endpoints were safety and biomarkers to assess the immune response in the tumor microenvironment including significant increases in PD-L1 expression and CD8 + tumor infiltrating lymphocytes (TIL) comparing pre- and on-treatment tumor biopsies. Secondary endpoints were objective response rates (ORR) and survival outcomes. RESULTS: Sixteen patients were evaluable for response, and nine patients were evaluable for biomarkers. Thirteen patients (68 %) had exposure to prior anti-PD-1 therapy. No dose-limiting or grade ≥ 3 treatment-related adverse events were observed. On-treatment biopsies showed significantly increased PD-L1 (p = 0.005), CD3+ (p = 0.020), CD4+ (p = 0.022), and CD8 + T cells (p = 0.017) compared to pre-treatment. Median overall survival and progression-free survival (PFS) were 6.18 months (95 % CI, 2.66-8.61) and 2.53 months (95 % CI, 1.81-4.04), respectively. One patient had an objective response (ORR, 5.3 %) with an ongoing PFS of > 25 months. Disease control rate was 42 %. The responder harbored an ARID1A variant of unknown significance (VUS) that was predicted to bind her HLA-I alleles with a higher affinity than the reference peptide. CONCLUSIONS: IRX-2 and durvalumab were safe and elicited the evidence of immune activation in the tumor microenvironment determined by increased PD-L1 expression and CD8+ TILs. CLINICAL TRIAL REGISTRATION NUMBER: NCT03381183.

Neoantigen-specific CD4+ tumor-infiltrating lymphocytes are potent effectors identified within adoptive cell therapy products for metastatic melanoma patients.

BACKGROUND: Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) is a promising immunotherapeutic approach for patients with advanced solid tumors. While numerous advances have been made, the contribution of neoantigen-specific CD4+T cells within TIL infusion products remains underexplored and therefore offers a significant opportunity for progress. METHODS: We analyzed infused TIL products from metastatic melanoma patients previously treated with ACT for the presence of neoantigen-specific T cells. TILs were enriched on reactivity to neoantigen peptides derived and prioritized from patient sample-directed mutanome analysis. Enriched TILs were further investigated to establish the clonal neoantigen response with respect to function, transcriptomics, and persistence following ACT. RESULTS: We discovered that neoantigen-specific TIL clones were predominantly CD4+ T cells and were present in both therapeutic responders and non-responders. CD4+ TIL demonstrated an effector T cell response with cytotoxicity toward autologous tumor in a major histocompatibility complex class II-dependent manner. These results were validated by paired TCR and single cell RNA sequencing, which elucidated transcriptomic profiles distinct to neoantigen-specific CD4+ TIL. CONCLUSIONS: Despite methods which often focus on CD8+T cells, our study supports the importance of prospective identification of neoantigen-specific CD4+ T cells within TIL products as they are a potent source of tumor-specific effectors. We further advocate for the inclusion of neoantigen-specific CD4+ TIL in future ACT protocols as a strategy to improve antitumor immunity.

Branched-chain keto acids promote an immune-suppressive and neurodegenerative microenvironment in leptomeningeal disease.

Leptomeningeal disease (LMD) occurs when tumors seed into the leptomeningeal space and cerebrospinal fluid (CSF), leading to severe neurological deterioration and poor survival outcomes. We utilized comprehensive multi-omics analyses of CSF from patients with lymphoma LMD to demonstrate an immunosuppressive cellular microenvironment and identified dysregulations in proteins and lipids indicating neurodegenerative processes. Strikingly, we found a significant accumulation of toxic branched-chain keto acids (BCKA) in the CSF of patients with LMD. The BCKA accumulation was found to be a pan-cancer occurrence, evident in lymphoma, breast cancer, and melanoma LMD patients. Functionally, BCKA disrupted the viability and function of endogenous T lymphocytes, chimeric antigen receptor (CAR) T cells, neurons, and meningeal cells. Treatment of LMD mice with BCKA-reducing sodium phenylbutyrate significantly improved neurological function, survival outcomes, and efficacy of anti-CD19 CAR T cell therapy. This is the first report of BCKA accumulation in LMD and provides preclinical evidence that targeting these toxic metabolites improves outcomes.

Biomarkers on melanoma patient T cells associated with ipilimumab treatment.

BACKGROUND: Ipilimumab induces long-lasting clinical responses in a minority of patients with metastatic melanoma. To better understand the mechanism(s) of action and to identify novel biomarkers associated with the clinical benefit and toxicity of ipilimumab, baseline characteristics and changes in CD4(+) and CD8(+) T cells from melanoma patients receiving ipilimumab were characterized by gene profiling and flow cytometry. METHODS: Microarray analysis of flow-cytometry purified CD4(+) and CD8(+) T cells was employed to assess gene profiling changes induced by ipilimumab. Selected molecules were further investigated by flow cytometry on pre, 3-month and 6-month post-treatment specimens. RESULTS: Ipilimumab up-regulated Ki67 and ICOS on CD4(+) and CD8(+) cells at both 3- and 6-month post ipilimumab (p ≤ 0.001), decreased CCR7 and CD25 on CD8(+) at 3-month post ipilimumab (p ≤ 0.02), and increased Gata3 in CD4(+) and CD8(+) cells at 6-month post ipilimumab (p ≤ 0.001). Increased EOMES(+)CD8(+), GranzymeB(+)EOMES(+)CD8(+) and decreased Ki67(+)EOMES(+)CD4(+) T cells at 6 months were significantly associated with relapse (all p ≤ 0.03). Decreased Ki67(+)CD8(+) T cells were significantly associated with the development of irAE (p = 0.02). At baseline, low Ki67(+)EOMES(+)CD8(+) T cells were associated with relapse (p ≤ 0.001), and low Ki67(+)EOMES(+)CD4(+) T cells were associated with irAE (p ≤ 0.008). CONCLUSIONS: Up-regulation of proliferation and activation signals in CD4(+) and CD8(+) T cells were pharmacodynamic markers for ipilimumab. Ki67(+)EOMES(+)CD8(+) and Ki67(+)EOMES(+)CD4(+)T cells at baseline merit further testing as biomarkers associated with outcome and irAEs, respectively.

Combination Nivolumab, CD137 Agonism, and Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes for Patients with Metastatic Melanoma.

PURPOSE: Metastatic melanoma is a tumor amenable to immunotherapy in part due to the presence of antigen-specific tumor-infiltrating lymphocytes (TIL). These T cells can be activated and expanded for adoptive cell transfer (ACT), which has resulted in relatively high rates of clinical responses. Similarly, immune checkpoint inhibitors, specifically programmed cell death protein 1 (PD-1) blocking antibodies, augment antitumor immunity and increase the influx of T cells into tumors. Thus, we hypothesized that addition of PD-1 inhibition may improve the outcomes for patients undergoing ACT with TILs. PATIENTS AND METHODS: Patients with stage III/IV metastatic melanoma with unresectable disease who were anti-PD-1 treatment-naïve were enrolled. TILs were generated in the presence of anti-4-1BB antibody in vitro and expanded for ACT. Patients in cohort 1 received TIL infusion followed by nivolumab. Patients in cohort 2 also received nivolumab prior to surgical harvest and during TIL production. RESULTS: A total of 11 patients were enrolled, all of whom were evaluated for response, and nine completed ACT. Predominantly CD8+ TILs were successfully expanded from all ACT-treated patients and were tumor reactive in vitro. The trial met its safety endpoint, as there were no protocol-defined dose-limiting toxicity events. The objective response rate was 36%, and median progression-free survival was 5 months. Two nonresponders who developed new metastatic lesions were analyzed to determine potential mechanisms of therapeutic resistance, which included clonal divergence and intrinsic TIL dysfunction. CONCLUSIONS: Combination therapy with TILs and nivolumab was safe and feasible for patients with metastatic melanoma and provides important insights for future therapeutic developments in ACT with TILs.

Expansion of tumor-infiltrating lymphocytes (TIL) from penile cancer patients.

Penile cancer is a rare but highly lethal cancer, and therapeutic options for patients presenting with lymph nodal disease are very limited. Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) was shown to provide durable objective response in patients with metastatic melanoma and TIL have been expanded from solid tumors at rates between 70 and 90% depending on the specific diagnosis. We evaluated whether TIL could be expanded from surgical specimens of patients with penile cancer. Tumor samples from metastatic lymph nodes obtained at the time of inguinal lymph node dissection were collected, minced into fragments, placed in individual wells of a 24-well plate, and propagated in high dose IL-2 for four weeks. The phenotype of expanded TILs was assessed by flow cytometry and their anti-tumor reactivity was assessed by IFN-γ ELISA. TIL were expanded from 11 out of 12 (91.6%) samples of metastatic lymph nodes. Expanded TIL were predominantly CD3+ (mean 67.5%, SD 19.4%) with a mean of 46.8% CD8+ T cells (SD 21.1%). Five out of 11 samples (45.4%) from expanded TIL secreted IFN-γ in response to autologous tumor. TIL expansion and phenotype of expanded T cell lymphocytes were independent of previous HPV infection and treatment with neoadjuvant chemotherapy. This is the first report demonstrating successful expansion of tumor-reactive TIL from penile cancer patients, which support development of ACT strategies using TIL for the treatment of advanced and recurrent penile cancer.

Expanded Tumor-infiltrating Lymphocytes From Soft Tissue Sarcoma Have Tumor-specific Function.

Adoptive cell transfer (ACT) with tumor-infiltrating lymphocytes (TILs) can generate durable clinical responses in patients with metastatic melanoma and ongoing trials are evaluating efficacy in other advanced solid tumors. The aim of this study was to develop methods for the expansion of tumor-reactive TIL from resected soft tissue sarcoma to a degree required for the ACT. From 2015 to 2018, 70 patients were consented to an institutional review board-approved protocol, and fresh surgical specimens were taken directly from the operating room to the laboratory. Fragments of the tumor (1 mm3) or fresh tumor digest were placed in culture for a period of 4 weeks. Successfully propagated TIL from these cultures were collected and analyzed by flow cytometry. TIL were cocultured with autologous tumor and function was assessed by measurement of interferon-γ in the supernatant by enzyme-linked immunosorbent assay. Initial TIL cultures were further expanded using a rapid expansion protocol. Nearly all specimens generated an initial TIL culture (91% fragment method, 100% digest method). The phenotype of the TIL indicated a predominant CD3+ population after culture (43% fragment, 52% digest) and TIL were responsive to the autologous tumor (56% fragment, 40% digest). The cultured TIL expanded to a degree required for clinical use following rapid expansion protocol (median: 490-fold fragment, 403-fold digest). The data demonstrate the feasibility of TIL culture from fresh soft tissue sarcoma. The derived TIL have tumor-specific reactivity and can be expanded to clinically relevant numbers. An active ACT clinical trial using the methods described in this report is now approved for patients with metastatic soft tissue sarcoma.

Combination of Ipilimumab and Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes for Patients with Metastatic Melanoma.

PURPOSE: Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) for metastatic melanoma can be highly effective, but attrition due to progression before TIL administration (32% in prior institutional experience) remains a limitation. We hypothesized that combining ACT with cytotoxic T lymphocyte-associated antigen 4 blockade would decrease attrition and allow more patients to receive TIL. EXPERIMENTAL DESIGN: Thirteen patients with metastatic melanoma were enrolled. Patients received four doses of ipilimumab (3 mg/kg) beginning 2 weeks prior to tumor resection for TIL generation, then 1 week after resection, and 2 and 5 weeks after preconditioning chemotherapy and TIL infusion followed by interleukin-2. The primary endpoint was safety and feasibility. Secondary endpoints included of clinical response at 12 weeks and at 1 year after TIL transfer, progression free survival (PFS), and overall survival (OS). RESULTS: All patients received at least two doses of ipilimumab, and 12 of the 13 (92%) received TIL. A median of 6.5 × 1010 (2.3 × 1010 to 1.0 × 1011) TIL were infused. At 12 weeks following infusion, there were five patients who experienced objective response (38.5%), four of whom continued in objective response at 1 year and one of which became a complete response at 52 months. Median progression-free survival was 7.3 months (95% CI 6.1-29.9 months). Grade ≥ 3 immune-related adverse events included hypothyroidism (3), hepatitis (2), uveitis (1), and colitis (1). CONCLUSION: Ipilimumab plus ACT for metastatic melanoma is feasible, well tolerated, and associated with a low rate of attrition due to progression during cell expansion. This combination approach serves as a model for future efforts to improve the efficacy of ACT.

Expansion of tumor infiltrating lymphocytes (TIL) from bladder cancer.

Advanced bladder cancer patients have limited therapeutic options resulting in a median overall survival (OS) between 12 and 15 months. Adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) has been used successfully in treating patients with metastatic melanoma, resulting in a median OS of 52 months. In this study, we investigated the feasibility of expanding TIL from the tumors of bladder cancer patients. Primary bladder tumors and lymph node (LN) metastases were collected. Tumor specimens were minced into fragments, placed in individual wells of a 24-well plate, and propagated in high dose IL-2 for four weeks. Expanded TIL were phenotyped by flow cytometry and anti-tumor reactivity was assessed after co-culture with autologous tumor digest and IFN-gamma ELISA. Of the 28 transitional cell bladder or LN tumors collected, 14/20 (70%) primary tumors and all of the LN metastases demonstrated TIL expansion. Expanded TIL were predominantly CD3+ (median 63%, range 10-87%) with a median of 30% CD8 + T cells (range 5-70%). TIL secreted IFN-gamma in response to autologous tumor. Addition of agonisitic 4-1BB antibody improved TIL expansion from primary bladder tumors regardless of pre-treatment with chemotherapy. This study establishes the practical first step towards an autologous TIL therapy process for therapeutic testing in patients with bladder cancer.

Expansion of tumor-infiltrating lymphocytes (TIL) from human pancreatic tumors.

BACKGROUND: We evaluated whether tumor infiltrating lymphocytes (TIL) could be expanded from surgically resected tumors from pancreatic cancer patients. METHODS: Tumors were resected from pancreatic cancer patients. Tumors were minced into fragments and cultured in media containing high dose interleukin-2 (IL-2) for up to 6 weeks. T cell phenotype, activation markers, and reactivity were measured. RESULTS: TIL expansion was measured in 19 patient samples. The majority of these TIL were CD4+ T cells and were highly activated. Purified CD8+ T cells produced IFN-γ in response to HLA-matched pancreatic tumor targets. PD-1 blockade and 4-1BB stimulation were demonstrated as effective strategies to improve effective TIL yield, including the production of tumor-reactive pancreatic TIL. CONCLUSIONS: TIL expanded from pancreatic tumors are functional and able to respond to pancreatic tumor associated antigens. PD-1 blockade, 41BB stimulation, and CD8+ T cell enrichment are effective strategies to improve TIL yield and tumor reactivity. These results support the development of adoptive cell therapy strategies using TIL for the treatment of pancreatic cancer.

Extended dose ipilimumab with a peptide vaccine: immune correlates associated with clinical benefit in patients with resected high-risk stage IIIc/IV melanoma.

PURPOSE: To determine safety and feasibility of adjuvant ipilimumab following resection of high-risk melanoma and to identify surrogate markers for benefit. EXPERIMENTAL DESIGN: In this phase II trial, 75 patients with resected stage IIIc/IV melanoma received the CTLA-4 antibody ipilimumab every 6 to 8 weeks for 1 year. Eligible patients received further maintenance treatments. The first 25 patients received 3 mg/kg of ipilimumab, and an additional 50 patients received 10 mg/kg. HLA-A*0201+ patients received multipeptide immunizations in combination with ipilimumab. Leukapheresis was performed prior to and 6 months after initiation of treatment. RESULTS: Median overall and relapse-free survivals were not reached after a median follow-up of 29.5 months. Significant immune-related adverse events were observed in 28 of 75 patients and were positively associated with longer relapse-free survival. Antigen-specific T cell responses to vaccine were variable, and vaccine combination was not associated with additional benefit. No effects on T regulatory cells were observed. Higher changes in Th-17 inducible frequency were a surrogate marker of freedom from relapse (P = 0.047), and higher baseline C-reactive protein (CRP) levels were associated with freedom from relapse (P = 0.035). CONCLUSIONS: Adjuvant ipilimumab following resection of melanoma at high risk for relapse appeared to be associated with improved outcome compared to historical reports. Significant immune-related adverse events were generally reversible and appeared to be associated with improved relapse-free survival. Although vaccination failed to induce a consistent in vitro measurable response, a higher change in Th-17 inducible cells and higher baseline CRP levels were positively associated with freedom from relapse.