Eric R. Lutz

Mesothelin-specific CD8+ T Cell Responses Provide Evidence of In Vivo Cross-Priming by Antigen-Presenting Cells in Vaccinated Pancreatic Cancer Patients

Tumor-specific CD8+ T cells can potentially be activated by two distinct mechanisms of major histocompatibility complex class I–restricted antigen presentation as follows: direct presentation by tumor cells themselves or indirect presentation by professional antigen-presenting cells (APCs). However, controversy still exists as to whether indirect presentation (the cross-priming mechanism) can contribute to effective in vivo priming of tumor-specific CD8+ T cells that are capable of eradicating cancer in patients. A clinical trial of vaccination with granulocyte macrophage–colony stimulating factor–transduced pancreatic cancer lines was designed to test whether cross-presentation by locally recruited APCs can activate pancreatic tumor-specific CD8+ T cells. Previously, we reported postvaccination delayed-type hypersensitivity (DTH) responses to autologous tumor in 3 out of 14 treated patients. Mesothelin is an antigen demonstrated previously by gene expression profiling to be up-regulated in most pancreatic cancers. We report here the consistent induction of CD8+ T cell responses to multiple HLA-A2, A3, and A24-restricted mesothelin epitopes exclusively in the three patients with vaccine-induced DTH responses. Importantly, neither of the vaccinating pancreatic cancer cell lines expressed HLA-A2, A3, or A24. These results provide the first direct evidence that CD8 T cell responses can be generated via cross-presentation by an immunotherapy approach designed to recruit APCs to the vaccination site.

Evaluation of Ipilimumab in combination with allogeneic pancreatic tumor cells transfected with a GM-CSF gene in previously treated pancreatic cancer

Preclinical reports support the concept of synergy between cancer vaccines and immune checkpoint blockade in nonimmunogenic tumors. In particular, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) antibodies have been successfully combined with GM-CSF cell-based vaccines (GVAX). Ipilimumab (anti-CTLA-4) has been tested as a single agent in patients with pancreatic ductal adenocarcinoma (PDA) resulting in a delayed response at a dose of 3 mg/kg. Our study evaluated ipilimumab 10 mg/kg (arm 1) and ipilimumab 10 mg/kg+GVAX (arm 2). A total of 30 patients with previously treated advanced PDA were randomized (1:1). Induction doses were administered every 3 weeks for a total of 4 doses followed by maintenance dosing every 12 weeks. Two patients in arm 1 showed evidence of stable disease (7 and 22 wk) but none demonstrated CA19-9 biochemical responses. In contrast, 3 patients in arm 2 had evidence of prolonged disease stabilization (31, 71, and 81 wk) and 7 patients experienced CA19-9 declines. In 2 of these patients, disease stabilization occurred after an initial period of progression. The median overall survival (OS) (3.6 vs. 5.7 mo, hazards ratio: 0.51, P=0.072) and 1 year OS (7 vs. 27%) favored arm 2. Similar to prior ipilimumab studies, 20% of patients in each arm had grade 3/4 immune-related adverse events. Among patients with OS>4.3 months, there was an increase in the peak mesothelin-specific T cells (P=0.014) and enhancement of the T-cell repertoire (P=0.031). In conclusion, checkpoint blockade in combination with GVAX has the potential for clinical benefit and should be evaluated in a larger study.

Safety and Survival With GVAX Pancreas Prime and Listeria Monocytogenes–Expressing Mesothelin (CRS-207) Boost Vaccines for Metastatic Pancreatic Cancer

PURPOSE GVAX pancreas, granulocyte-macrophage colony-stimulating factor-secreting allogeneic pancreatic tumor cells, induces T-cell immunity to cancer antigens, including mesothelin. GVAX is administered with low-dose cyclophosphamide (Cy) to inhibit regulatory T cells. CRS-207, live-attenuated Listeria monocytogenes-expressing mesothelin, induces innate and adaptive immunity. On the basis of preclinical synergy, we tested prime/boost vaccination with GVAX and CRS-207 in pancreatic adenocarcinoma. PATIENTS AND METHODS Previously treated patients with metastatic pancreatic adenocarcinoma were randomly assigned at a ratio of 2:1 to two doses of Cy/GVAX followed by four doses of CRS-207 (arm A) or six doses of Cy/GVAX (arm B) every 3 weeks. Stable patients were offered additional courses. The primary end point was overall survival (OS) between arms. Secondary end points were safety and clinical response. RESULTS A total of 90 patients were treated (arm A, n = 61; arm B, n = 29); 97% had received prior chemotherapy; 51% had received ≥ two regimens for metastatic disease. Mean number of doses (± standard deviation) administered in arms A and B were 5.5 ± 4.5 and 3.7 ± 2.2, respectively. The most frequent grade 3 to 4 related toxicities were transient fevers, lymphopenia, elevated liver enzymes, and fatigue. OS was 6.1 months in arm A versus 3.9 months in arm B (hazard ratio [HR], 0.59; P = .02). In a prespecified per-protocol analysis of patients who received at least three doses (two doses of Cy/GVAX plus one of CRS-207 or three of Cy/GVAX), OS was 9.7 versus 4.6 months (arm A v B; HR, 0.53; P = .02). Enhanced mesothelin-specific CD8 T-cell responses were associated with longer OS, regardless of treatment arm. CONCLUSION Heterologous prime/boost with Cy/GVAX and CRS-207 extended survival for patients with pancreatic cancer, with minimal toxicity.

Allogeneic Granulocyte Macrophage Colony-Stimulating Factor–Secreting Tumor Immunotherapy Alone or in Sequence with Cyclophosphamide for Metastatic Pancreatic Cancer: A Pilot Study of Safety, Feasibility, and Immune Activation

Purpose: The combination of chemotherapy and immunotherapy has not been examined in patients with advanced pancreatic cancer. We conducted a study of two granulocyte macrophage colony-stimulating factor–secreting pancreatic cancer cell lines (CG8020/CG2505) as immunotherapy administered alone or in sequence with cyclophosphamide in patients with advanced pancreatic cancer. Experimental Design: This was an open-label study with two cohorts: cohort A, 30 patients administered a maximum of six doses of CG8020/CG2505 at 21-day intervals; and cohort B, 20 patients administered 250 mg/m2 of cyclophosphamide i.v. 1 day before the same immunotherapy given as in cohort A. The primary objective was to evaluate safety and duration of immunity. Secondary objectives included time to disease progression and median overall survival. Results: The administration of CG8020/CG2505 alone or in sequence with cyclophosphamide showed minimal treatment-related toxicity. Median survival values in cohort A and cohort B were 2.3 and 4.3 months, respectively. CD8+ T-cell responses to HLA class I–restricted mesothelin epitopes were identified predominantly in patients treated with cyclophosphamide + CG8020/CG2505 immunotherapy. Conclusion: Granulocyte macrophage colony-stimulating factor–secreting pancreatic cancer cell lines CG8020/CG2505 alone or in sequence with cyclophosphamide showed minimal treatment-related toxicity in patients with advanced pancreatic cancer. Also, mesothelin-specific T-cell responses were detected/enhanced in some patients treated with CG8020/CG2505 immunotherapy. In addition, cyclophosphamide-modulated immunotherapy resulted in median survival in a gemcitabine-resistant population similar to chemotherapy alone. These findings support additional investigation of cyclophosphamide with CG8020/CG2505 immunotherapy in patients with advanced pancreatic cancer.

A Live-Attenuated Listeria Vaccine (ANZ-100) and a Live-Attenuated Listeria Vaccine Expressing Mesothelin (CRS-207) for Advanced Cancers: Phase I Studies of Safety and Immune Induction

Purpose: Listeria monocytogenes (Lm)-based vaccines stimulate both innate and adaptive immunity. ANZ-100 is a live-attenuated Lm strain (Lm ΔactA/ΔinlB). Uptake by phagocytes in the liver results in local inflammatory responses and activation and recruitment of natural killer (NK) and T cells, in association with increased survival of mice bearing hepatic metastases. The Lm ΔactA/ΔinlB strain, engineered to express human mesothelin (CRS-207), a tumor-associated antigen expressed by a variety of tumors, induces mesothelin-specific T-cell responses against mesothelin-expressing murine tumors. These two phase I studies test ANZ-100 and CRS-207 in subjects with liver metastases and mesothelin-expressing cancers, respectively. Experimental Design: A single intravenous injection of ANZ-100 was evaluated in a dose escalation study in subjects with liver metastases. Nine subjects received 1 × 106, 3 × 107, or 3 × 108 colony-forming units (cfu). CRS-207 was evaluated in a dose-escalation study in subjects with mesothelioma, lung, pancreatic, or ovarian cancers. Seventeen subjects received up to 4 doses of 1 × 108, 3 × 108, 1 × 109, or 1 × 1010 cfu. Results: A single infusion of ANZ-100 was well tolerated to the maximum planned dose. Adverse events included transient laboratory abnormalities and symptoms associated with cytokine release. Multiple infusions of CRS-207 were well tolerated up to 1 × 109 cfu, the determined maximum tolerated dose. Immune activation was observed for both ANZ-100 and CRS-207 as measured by serum cytokine/chemokine levels and NK cell activation. In the CRS-207 study, listeriolysin O and mesothelin-specific T-cell responses were detected and 37% of subjects lived ≥15 months. Conclusions: ANZ-100 and CRS-207 administration was safe and resulted in immune activation. Clin Cancer Res; 18(3); 858–68. ©2011 AACR.

Immunotherapy converts nonimmunogenic pancreatic tumors into immunogenic foci of immune regulation.

Lutz and colleagues show that an allogeneic vaccine given with or without low-dose cyclophosphamide converted pancreatic cancer into an immunogenic cancer with infiltrating effector lymphocytes and formation of tertiary lymphoid aggregates that are regulatory structures of adaptive immunity. Pancreatic ductal adenocarcinoma (PDAC) is considered a “nonimmunogenic” neoplasm. Single-agent immunotherapies have failed to demonstrate significant clinical activity in PDAC and other “nonimmunogenic” tumors, in part due to a complex tumor microenvironment (TME) that provides a formidable barrier to immune infiltration and function. We designed a neoadjuvant and adjuvant clinical trial comparing an irradiated, granulocyte-macrophage colony-stimulating factor (GM-CSF)–secreting, allogeneic PDAC vaccine (GVAX) given as a single agent or in combination with low-dose cyclophosphamide to deplete regulatory T cells (Treg) as a means to study how the TME is altered by immunotherapy. Examination of resected PDACs revealed the formation of vaccine-induced intratumoral tertiary lymphoid aggregates in 33 of 39 patients 2 weeks after vaccine treatment. Immunohistochemical analysis showed these aggregates to be regulatory structures of adaptive immunity. Microarray analysis of microdissected aggregates identified gene-expression signatures in five signaling pathways involved in regulating immune-cell activation and trafficking that were associated with improved postvaccination responses. A suppressed Treg pathway and an enhanced Th17 pathway within these aggregates were associated with improved survival, enhanced postvaccination mesothelin-specific T-cell responses, and increased intratumoral Teff:Treg ratios. This study provides the first example of immune-based therapy converting a “nonimmunogenic” neoplasm into an “immunogenic” neoplasm by inducing infiltration of T cells and development of tertiary lymphoid structures in the TME. Post-GVAX T-cell infiltration and aggregate formation resulted in the upregulation of immunosuppressive regulatory mechanisms, including the PD-1–PD-L1 pathway, suggesting that patients with vaccine-primed PDAC may be better candidates than vaccine-naïve patients for immune checkpoint and other immunomodulatory therapies. Cancer Immunol Res; 2(7); 616–31. ©2014 AACR.

PD-1/PD-L1 blockade together with vaccine therapy facilitates effector T-cell infiltration into pancreatic tumors

Pancreatic ductal adenocarcinoma (PDA) has a poor prognosis due to late detection and resistance to conventional therapies. Published studies show that the PDA tumor microenvironment is predominantly infiltrated with immune suppressive cells and signals that if altered, would allow effective immunotherapy. However, single-agent checkpoint inhibitors including agents that alter immune suppressive signals in other human cancers such as cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed death 1 (PD-1), and its ligand PD-L1, have failed to demonstrate objective responses when given as single agents to PDA patients. We recently reported that inhibition of the CTLA-4 pathway when given together with a T cell inducing vaccine gives objective responses in metastatic PDA patients. In this study, we evaluated blockade of the PD-1/PD-L1 pathway. We found that PD-L1 is weakly expressed at a low frequency in untreated human and murine PDAs but treatment with a granulocyte macrophage colony-stimulating factor secreting PDA vaccine (GVAX) significantly upregulates PD-L1 membranous expression after treatment of tumor-bearing mice. In addition, combination therapy with vaccine and PD-1 antibody blockade improved murine survival compared with PD-1 antibody monotherapy or GVAX therapy alone. Furthermore, PD-1 blockade increased effector CD8+ T lymphocytes and tumor-specific interferon-&ggr; production of CD8+ T cells in the tumor microenvironment. Immunosuppressive pathways, including regulatory T cells and CTLA-4 expression on T cells were overcome by the addition of vaccine and low-dose cyclophosphamide to PD-1 blockade. Collectively, our study supports combining PD-1 or PD-L1 antibody therapy with a T cell inducing agent for PDA treatment.

Targeting neoantigens to augment antitumour immunity

The past decade of cancer research has been marked by a growing appreciation of the role of immunity in cancer. Mutations in the tumour genome can cause tumours to express mutant proteins that are tumour specific and not expressed on normal cells (neoantigens). These neoantigens are an attractive immune target because their selective expression on tumours may minimize immune tolerance as well as the risk of autoimmunity. In this Review we discuss the emerging evidence that neoantigens are recognized by the immune system and can be targeted to increase antitumour immunity. We also provide a framework for personalized cancer immunotherapy through the identification and selective targeting of individual tumour neoantigens, and present the potential benefits and obstacles to this approach of targeted immunotherapy.

HER-2/neu-Specific Monoclonal Antibodies Collaborate with HER-2/neu-Targeted Granulocyte Macrophage Colony-Stimulating Factor Secreting Whole Cell Vaccination to Augment CD8+ T Cell Effector Function and Tumor-Free Survival in Her-2/neu-Transgenic Mice

HER-2/neu is overexpressed in several cancers including 30% of breast carcinomas, and correlates with a poor outcome. HER-2/neu-transgenic (neu-N) mice that overexpress the non-transforming rat neu develop spontaneous mammary carcinomas and demonstrate immunotolerance to the neu protein similar to that observed in patients with neu-expressing cancers. In neu-N mice, neu-targeted vaccination induces weak T cell and negligible Ab responses sufficient to delay but not eradicate transplanted neu-expressing tumor. Here we demonstrate that passive infusion of neu-specific mAbs in sequence with whole cell vaccination significantly improves tumor-free survival over either modality alone. Importantly, treatment of neu-N mice with vaccine in combination with two distinct neu-specific Abs is particularly efficacious, preventing tumor in 70% and eradicating established tumor in 30% of neu-N mice. In vivo lymphocyte subpopulation depletion experiments demonstrate that the efficacy of Ab, alone or combined with vaccine, is dependent on both CD4+ and CD8+ T cells. Furthermore, the in vivo antitumor effects of vaccine and Ab are associated with a significant increase in the number and function of neu-specific CD8+ T cells. Collectively, these observations suggest that similarly increased efficacy could be obtained by combining neu-targeted vaccination and neu-specific Abs such as trastuzumab (Herceptin) in patients with neu-expressing cancers.

OX40 Costimulation Synergizes with GM-CSF Whole-Cell Vaccination to Overcome Established CD8+ T Cell Tolerance to an Endogenous Tumor Antigen

T cell costimulation via OX40 is known to increase CD4+ T cell expansion and effector function and enhances the development of T cell memory. OX40 costimulation can also prevent, and even reverse, CD4+ T cell anergy. However, the role of OX40 in CD8+ T cell function is less well defined, particularly in the setting of immune tolerance. To determine the effects of OX40 costimulation on the induction of the host CD8+ T cell repertoire to an endogenous tumor Ag, we examined the fate of CD8+ T cells specific for the immunodominant rat HER-2/neu epitope, RNEU420–429, in FVB MMTV-neu (neu-N) mice, which express rat HER-2/neu protein in a predominantly mammary-restricted fashion. We show that the RNEU420–429-specific T cell repertoire in neu-N mice expands transiently after vaccination with a neu-targeted GM-CSF-secreting whole-cell vaccine, but quickly declines to an undetectable level. However, OX40 costimulation, when combined with GM-CSF-secreting tumor-targeted vaccination, can break established CD8+ T cell tolerance in vivo by enhancing the expansion, and prolonging the survival and effector function of CD8+ T cells specific for RNEU420–429. Moreover, we demonstrate that OX40 expression is up-regulated on both CD4+ and CD8+ T cells shortly after administration of a GM-CSF expressing vaccine. These studies highlight the increased efficacy of OX40 costimulation when combined with a GM-CSF-secreting vaccine, and define a new role for OX40 costimulation of CD8+ T cells in overcoming tolerance and boosting antitumor immunity.