Edmund C. Lattime

A Phase I Trial of Pox PSA vaccines (PROSTVAC®-VF) with B7-1, ICAM-1, and LFA-3 co-stimulatory molecules (TRICOM™) in Patients with Prostate Cancer

PurposeBased on previous studies that demonstrated the safety profile and preliminary clinical activity of prostate specific antigen (PSA) targeted therapeutic vaccines, as well as recent laboratory data supporting the value of the addition of co-stimulatory molecules B7-1, ICAM-1, and LFA-3 (designated TRICOM™) to these vaccines, we conducted a Phase I study to evaluate the safety and immunogenicity of a novel vaccinia and fowlpox vaccine incorporating the PSA gene sequence and TRICOM.MethodsIn this study, ten patients with androgen independent prostate cancer with or without metastatic disease were enrolled. Patients were treated with 2 × l08 pfu of a recombinant vaccinia virus vaccine (PROSTVAC-V) followed by 1 × 109 pfu of the booster recombinant fowlpox virus (PROSTVAC-F) both with gene sequences for PSA and TRICOM. The mean age of patients enrolled in the study was 70 (range 63 to 79). The mean PSA at baseline was 434 (range 9 – 1424).ResultsThere were no deaths, and no Grade 3 or 4 adverse events. The most commonly reported adverse events, regardless of causality, were injection site reactions and fatigue. One serious adverse event (SAE) occurred that was unrelated to vaccine; this patient developed progressive disease with a new sphenoid metastasis. PSA was measured at week 4 and week 8. Four patients had stable disease (with less than 25% increase in PSA) through the week 8 study period. Anti-PSA antibodies were not induced with therapy: however, anti-vaccinia titers increased in all patients.ConclusionThis study demonstrated that vaccination with PROSTVAC-V and PROSTVAC-F combined with TRICOM is well-tolerated and generated an immune response to vaccinia. Therefore, PROSTVAC-VF/TRICOM represents a feasible therapeutic approach for further phase II and III study in patients with prostate cancer.

Cellular autophagy machinery is not required for vaccinia virus replication and maturation.

The origin of the primary membrane of vaccinia virus, the double-membrane structure that surrounds the immature virions (IV), is not fully understood. Here we investigated whether the primary membrane is of autophagy origin. Morphologic studies by electron microscopy showed no apparent difference in viral maturation in the autophagy deficient cell lines, the Atg5-/- mouse embryonic fibroblast (MEF) and the Beclin 1 -/- embryonic stem (ES) cells, compared to their isogenic wild-type counterparts. Moreover, quantitative assays indicated that the viruses replicate and maturate in the autophagy deficient cell lines as efficiently as they do in the corresponding isogenic wild type cells. This study indicates that the cellular autophagy machinery is not required for the life-cycle of vaccinia virus, suggesting that the primary vaccinia viral membrane likely does not originate from the autophagic membrane.

A National Multicenter Phase 2 Study of Prostate-specific Antigen (PSA) Pox Virus Vaccine with Sequential Androgen Ablation Therapy in Patients with PSA Progression: ECOG 9802

BACKGROUND E9802 was a phase 2 multi-institution study conducted to evaluate the safety and effectiveness of vaccinia and fowlpox prostate-specific antigen (PSA) vaccine (step 1) followed by combination with androgen ablation therapy (step 2) in patients with PSA progression without visible metastasis. OBJECTIVE To test the hypothesis that vaccine therapy in this early disease setting will be safe and have a biochemical effect that would support future studies of immunotherapy in patients with minimal disease burden. DESIGN, SETTING, AND PARTICIPANTS Patients who had PSA progression following local therapy were treated with PROSTVAC-V (vaccinia)/TRICOM on cycle 1 followed by PROSTVAC-F (fowlpox)/TRICOM for subsequent cycles in combination with granulocyte-macrophage colony-stimulating factor (step 1). Androgen ablation was added on progression (step 2). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Step 1 primary end points included progression at 6 mo and characterization of change in PSA velocity pretreatment to post-treatment. Step 2 end points included PSA response with combined vaccine and androgen ablation. RESULTS AND LIMITATIONS In step 1, 25 of 40 eligible patients (63%) were progression free at 6 mo after registration (90% confidence interval [CI], 48-75). The median pretreatment PSA velocity was 0.13 log(PSA)/mo, in contrast to median postregistration velocity of 0.09 log(PSA)/mo (p=0.02), which is an increase in median PSA doubling time from 5.3 mo to 7.7 mo. No grade ≥4 treatment-related toxicity was observed. In the 27 patients eligible and treated for step 2, 20 patients achieved a complete response (CR) at 7 mo (CR rate: 74%; 90% CI, 57-87). Although supportive of larger studies in the cooperative group setting, this study is limited by the small number of patients and the absence of a control group as in a phase 3 study. CONCLUSIONS A viral PSA vaccine can be administered safely in the multi-institutional cooperative group setting to patients with minimal disease volume alone and combined with androgen ablation, supporting the feasibility of future phase 3 studies in this population. PATIENT SUMMARY These data support consideration of vaccine therapy earlier in the course of prostate cancer progression with minimal disease burden in future studies of vaccine approaches in earlier stages of disease.

Antineoplastic Effects of Partially HLA-Matched Irradiated Blood Mononuclear Cells in Patients With Renal Cell Carcinoma

PURPOSE Vaccines, cytokines, and other biologic-based therapies are being developed as antineoplastic agents. Many of these agents are designed to induce an autologous immune response directed against the malignancy. In contrast, hematopoietic stem-cell transplantation is being developed as a form of allogeneic immunotherapy. This study tests the tolerance and antineoplastic activity of sequential infusions of partially HLA-matched allogeneic blood mononuclear cells (obtained from relatives) when administered outside of the context of a hematopoietic stem-cell transplantation. The cells are irradiated to prevent graft-versus-host disease. PATIENTS AND METHODS Fifteen patients with relapsed or refractory malignancies for which no standard therapy was available were enrolled onto a clinical trial designed to assess the tolerability and antineoplastic effects of irradiated partially HLA-matched blood mononuclear cells obtained from relatives. RESULTS There was disease regression in three patients with metastatic renal cell carcinoma during treatment. There was disease progression in six patients with metastatic renal cell carcinoma and two patients with metastatic melanoma during treatment. There was no change in disease state in several other patients. CONCLUSION Irradiated allogeneic blood mononuclear cells administered outside the context of hematopoietic stem-cell transplantation may induce disease responses in patients with relapsed or refractory malignancies. Transfusion of irradiated allogeneic blood mononuclear cells should be developed further as a novel therapeutic antineoplastic approach.

Vaccinia virus leads to ATG12–ATG3 conjugation and deficiency in autophagosome formation

The interactions between viruses and cellular autophagy have been widely reported. On the one hand, autophagy is an important innate immune response against viral infection. On the other hand, some viruses exploit the autophagy pathway for their survival and proliferation in host cells. Vaccinia virus is a member of the family of Poxviridae which includes the smallpox virus. The biogenesis of vaccinia envelopes, including the core envelope of the immature virus (IV), is not fully understood. In this study we investigated the possible interaction between vaccinia virus and the autophagy membrane biogenesis machinery. Massive LC3 lipidation was observed in mouse fibroblast cells upon vaccinia virus infection. Surprisingly, the vaccinia virus induced LC3 lipidation was shown to be independent of ATG5 and ATG7, as the atg5 and atg7 null mouse embryonic fibroblasts (MEFs) exhibited the same high levels of LC3 lipidation as compared with the wild-type MEFs. Mass spectrometry and immunoblotting analyses revealed that the viral infection led to the direct conjugation of ATG3, which is the E2-like enzyme required for LC3-phosphoethanonamine conjugation, to ATG12, which is a component of the E3-like ATG12–ATG5-ATG16 complex for LC3 lipidation. Consistently, ATG3 was shown to be required for the vaccinia virus induced LC3 lipidation. Strikingly, despite the high levels of LC3 lipidation, subsequent electron microscopy showed that vaccinia virus-infected cells were devoid of autophagosomes, either in normal growth medium or upon serum and amino acid deprivation. In addition, no autophagy flux was observed in virus-infected cells. We further demonstrated that neither ATG3 nor LC3 lipidation is crucial for viral membrane biogenesis or viral proliferation and infection. Together, these results indicated that vaccinia virus does not exploit the cellular autophagic membrane biogenesis machinery for their viral membrane production. Moreover, this study demonstrated that vaccinia virus instead actively disrupts the cellular autophagy through a novel molecular mechanism that is associated with aberrant LC3 lipidation and a direct conjugation between ATG12 and ATG3.

Recombinant Poxvirus and the Tumor Microenvironment: Oncolysis, Immune Regulation and Immunization

Oncolytic viruses (OVs) are being extensively studied for their potential roles in the development of cancer therapy regimens. In addition to their direct lytic effects, OVs can initiate and drive systemic antitumor immunity indirectly via release of tumor antigen, as well as by encoding and delivering immunostimulatory molecules. This combination makes them an effective platform for the development of immunotherapeutic strategies beyond their primary lytic function. Engineering the viruses to also express tumor-associated antigens (TAAs) allows them to simultaneously serve as therapeutic vaccines, targeting and amplifying an immune response to TAAs. Our group and others have shown that vaccinating intratumorally with a poxvirus that encodes TAAs, in addition to immune stimulatory molecules, can modulate the tumor microenvironment, overcome immune inhibitory pathways, and drive both local and systemic tumor specific immune responses.

Oncolytic viruses: focusing on the tumor microenvironment

Recent developments in cancer immunotherapy have generated renewed interest in oncolytic viruses as platforms for the development of anti-tumor immunotherapy regimens. Several reviews have detailed exciting new approaches in this area, as well as emphasized the potential success of such agents as part of combinatorial treatment strategies. Indeed, we agree that this treatment modality will most likely achieve its greatest potential as part of combination therapeutic anticancer regimens. However, we would emphasize that the most successful oncolytic virusbased treatments, as well as any combinatorial strategies into which they are incorporated, will likely be those that take advantage of modifying the tumor microenvironment. We and others have used recombinant poxvirus and herpesvirus vectors in both preclinical and clinical studies to demonstrate that oncolytic viruses can be used to modify the cytokine and antigen milieu of the tumor microenvironment, ultimately promoting systemic tumor-specific immune responses that overcome immune escape mechanisms. A central hypothesis of our studies has been that in situ treatment with oncolytic viruses can be utilized to release an autologous tumor antigen into the tumor microenvironment, thus inducing a systemic tumor-specific immune response against both primary and metastatic tumor. This strategy effectively relies on autologous tumor as a source of multiple antigens, allowing development of a multivalent tumor-specific immune response. Support for this approach can be found in studies that demonstrate an average of 90 mutations per tumor in human breast and colorectal cancers. Furthermore, it is believed that the immunogenic cell death in the tumor microenvironment that occurs because of oncolytic virus infection triggers release of damage-associated molecular pattern and pathogen-associated molecular pattern molecules, as well as inflammatory cytokines, that aid in the activation and recruitment of antigen presenting cells, ultimately yielding a Th1 immune response. An essential factor in such immune therapeutic strategies is the idea that the tumor microenvironment acts as the center for tumor immune escape and regulation of anti-tumor immune responses (Figure 1a). Multiple cytokines produced by tumor, particularly IL-10, can have pivotal roles in suppressing the immune response. Studies by our group demonstrated that tumor-induced IL-10 can prevent the induction of both tumor-specific and poxvirus-encoded antigen-specific type 1 immune responses at the tumor site. Furthermore, at least part of this effect was due to suppression of dendritic cell maturation, thus preventing antigen presentation. Immune suppressive cytokines, such as IL-10 and TGF-β, also attenuate cytotoxic T-cell (CTL) function and the Th1 response. The tumor microenvironment promotes the development of suppressive cell populations such as regulatory T cells and myeloid-derived suppressor cells. Both cell populations have inhibitory effects on T-cell function and infiltration into the tumor microenvironment. Given these and other mechanisms of immune escape, we hypothesize that the immune population of the tumor microenvironment can be advantageously adapted by using modified poxviruses to deliver immune stimulatory cytokines, while still taking advantage of the aforementioned direct oncolytic effects of poxviruses. As reviewed by Lichty et al., oncolytic viruses have been modified to encode numerous immune enhancing cytokines, including IL-2, IL-12 and interferon gamma. We focused our initial efforts on granulocyte macrophage colony-stimulating factor (GM-CSF), based on this cytokine’s important role in recruitment and activation of antigen presenting cells. We carried out a phase I trial of intralesional vaccinia-GM-CSF in patients with refractory melanoma. This led to rejection of injected lesions with infiltration of CD4+ and CD8+ T lymphocytes. Moreover, four of seven patients exhibited regression of untreated tumors, suggesting development of a tumor-specific immune response. This agent, studied as JX-594, has been further demonstrated to preferentially replicate in the tumor microenvironment following intravenous administration in both preclinical and clinical studies. Further evidence supporting this approach was found in subsequent phases I and II clinical trials evaluating an oncolytic herpes simplex virus expressing GM-CSF, which demonstrated that intratumoral injection with this oncolytic virus in melanoma patients resulted in generation of systemic tumor antigen-specific T-lymphocyte responses, as well as decreases in regulatory T cells, suppressor CD8+ T cells and myeloid-derived suppressor cells in patients that developed clinical responses. A complimentary approach which we and others are actively exploring is to utilize oncolytic viruses as vectors for expression of tumor-associated antigen (TAA). Further supporting our central hypothesis that oncolytic viruses can be used as tools to modify the immunological components of the tumor microenvironment, we found in the MB49 murine model of bladder cancer that delivery of recombinant vaccinia virus expressing the immunodominant major histocompatibility complex class I epitope of the TAA overcame systemic anergy and resulted in a systemic CTL response. The Schlom group independently arrived at similar conclusions in a transgenic colon cancer model using intratumoral subcutaneous priming and intratumoral boosting with recombinant vaccinia virus expressing the carcinembryonic antigen (CEA). Most recently using our NBT1 orthotopic breast cancer model we found that TAA addition resulted in reduction of myeloid-derived suppressor cell in the tumor and periphery, induction of a systemic T-cell response and tumor regression. It is possible that the effectiveness of intratumoral oncolytic virus expressing TAA involves multiple mechanisms, including tumor oncolysis, improved TAA presentation by antigen presenting cells leading to a systemic TAA-specific immune response, and increased antigen expression by infected tumor cells enabling improved CTL-mediated killing (Figure 1b). Lee et al. found that addition of cisplatin to intratumoral vaccinia expressing TAA resulted in increased CD11c+ dendritic cells in the tumor and increased antigen-specific CD8+ T cells systemically, further supporting the critical role that antigen presentation in the tumor microenvironment likely plays in generation of tumor-specific T-cell responses from intratumoral vaccination with oncolytic vaccines expressing TAA. The finding of increased inflammatory and T-cell infiltration in both local and distant lesions after intratumoral treatment with Newcastle disease virus, and subsequent systemic response to CTLA-4 blockade, shows the critical role that the inflammatory mileu induced by oncolytic virus itself likely plays in attracting anti-tumor T lymphocytes. Clinical studies support the advantages of intratumoral poxviruses expressing TAA, including ongoing clinical trials of intraprostatic Cancer Gene Therapy (2015) 22, 169–171

Identifying a Clinically Applicable Mutational Burden Threshold as a Potential Biomarker of Response to Immune Checkpoint Therapy in Solid Tumors

Purpose An association between mutational burden and response to immune checkpoint therapy has been documented in several cancer types. The potential for such a mutational burden threshold to predict response to immune checkpoint therapy was evaluated in several clinical datasets, where mutational burden was measured either by whole-exome sequencing (WXS) or using commercially available sequencing panels. Methods WXS and RNA-seq data of 33 solid cancer types from TCGA were analyzed to determine whether a robust immune checkpoint activating mutation (iCAM) burden threshold associated with evidence of immune checkpoint activation exists in these cancers that may serve as a biomarker for response to immune checkpoint blockade therapy. Results We find that a robust iCAM threshold, associated with signatures of immune checkpoint activation, exists in 8 of 33 solid cancers: melanoma, lung adenocarcinoma, colon adenocarcinoma, endometrial cancer, stomach adenocarcinoma, cervical cancer, ER+HER2- breast cancer, and bladder-urothelial cancer. Tumors with mutational burden higher than the threshold (iCAM+) also had clear histologic evidence of lymphocytic infiltration. In published datasets of melanoma, lung adenocarcinoma and colon cancer, patients with iCAM+ tumors had significantly better response to immune checkpoint therapy compared to those with iCAM- tumors. ROC analysis using TCGA predictions as gold standard showed that iCAM+ tumors are accurately identifiable using clinical sequencing assays, such as FoundationOne or StrandAdvantage. Using the FoundationOne derived threshold, analysis of 113 melanoma tumors, showed that iCAM+ patients have significantly better response to immune checkpoint therapy. iCAM+ and iCAM- tumors have distinct mutation patterns and different immune microenvironments. Conclusion In 8 solid cancers, a mutational burden threshold exists that may predict response to immune checkpoint blockade. This threshold is identifiable using available clinical sequencing assays.

Poxvirus-Based Strategies for Combined Vaccine and Tumor Microenvironment Manipulation

Although vaccine strategies for the generation of tumor-specific immunity in patients continue to have great promise, to date they have been less than overwhelming in their antitumor efficacy. Studies from our laboratory and other laboratories have examined the lack of apparent immune recognition of tumor and limited effectiveness of tumor antigen-based vaccines. Such studies have resulted in the definition of a series of “immune escape mechanisms” whereby the tumor microenvironment and draining lymph nodes manifest a balance between effector and regulatory pathways resulting in a systemic lack of responsiveness to tumor and, in some cases, vaccine therapy. Our studies, reviewed in this chapter, have focused on the use of recombinant poxviral vectors to infect/transfect tumor and stromal components in the tumor microenvironment aimed at overcoming “immune escape” and resulting in an effective systemic antitumor immunity. Studies outlined here demonstrate that poxviral vectors are effective in infecting/transfecting tumor in vitro and in vivo, leading to the production of immune-active cytokines at the tumor site. In a series of phase I trials in melanoma and bladder cancer patients, we demonstrated that infection/transfection of the tumor using poxvirus encoding the immune active cytokine granulocyte–macrophage colony-stimulating factor is effective in inducing local immune cell infiltration into the tumor microenvironment. Parallel preclinical studies have found that inclusion of genes encoding tumor antigen into the recombinant poxvirus with or without additional pharmacologic agents modulating immune escape mechanisms is effective in inducing systemic antitumor immunity and manifesting positive antitumor effects. Finally, we describe the translation of these latter findings to an ongoing phase I study of antigen-encoding poxvirus given intratumorally in patients with locally advanced pancreatic cancer.

Abstract B100: Panvac-F and Panvac-V: Phase I study of intratumoral and systemic vaccination

Patients with adenocarcinoma of the pancreas were treated using a combination of EUS-guided intrapancreatic tumor injection and systemic boost using PANVAC-F and PANVAC-V in this first-in-man Phase I trial. The study represents the translation of our unique demonstration in murine models of bladder and orthotopic mammary tumors that anergy to systemic immunization using antigen encoding vaccinia recombinants could be overcome by immunization into the tumor microenvironment. Consented patients with locally advanced or minimally metastatic pancreatic cancer received EUS-guided intrapancreatic injections (IT) of recombinant Panvac-F (Fowlpox encoding MUC-1, CEA, TRICOM), systemic subcutaneous (SC) Panvac-V (vaccinia) and SC Panvac-F boosts. Systemic SC vaccines were accompanied by subcutaneous rH-GM-CSF, 100 mcg X 4 days. Patients received 2 intrapancreatic injections of Panvac-F (2 weeks apart) with systemic Panvac-V and Panvac-F boosts given with GMCSF extending to day 71 (total of 2 IT Panvac-F, 1 SC Panvac-V, 4 SC Panvac-F). Patients were allowed to transition to standard care at day 31. Patients were evaluated for toxicity and tumor progression. Follow-up care was provided by referring oncologists. In this dose escalation study, the first cohort of 6 pts received IT Panvac-F (10 8 PFU), SC Panvac-V (2 X 10 8 PFU), and SC Panvac-F (10 9 PFU). The second dose cohort of 8 pts received IT Panvac-F (10 9 PFU), SC Panvac-V (2 X 10 8 PFU), and SC Panvac-F (10 9 PFU ). At dose level 1, two of six patients were removed from study after approximately two weeks due to rapid disease progression and died one and six months after trial initiation. At dose level 2, one patient was removed due to rapidly progressive disease and died at 1 mo and a second patient withdrew following 1 IT inoculation and died at month 16. Of the remaining 10 pts, 3 presented with distant metastatic disease (Median Survival 7 mos, range 1-25) and 7 presented without distant metastases (Median Survival 16 mos, range 3-35). Of note, none of the 7 patients presenting without metastatic disease developed distant visceral metastases, by imaging available to us but died of sequellae associated with progressive local disease. Of the above 10 pts, all but one transitioned initially to treatment with gemcitabine-based therapy. (the remaining pt did not begin systemic treatment). Initial RNAseq transcriptome analysis of Fine Needle Aspirate (FNA) comparing Day 1 and Day 14 samples demonstrated a significant increase in a series of chemokines associated with the induction of an immune response in the tumor microenvironment. A planned series of immunologic and genetic analyses are underway. Results demonstrate that the “first in man” intrapancreatic administration of recombinant poxvirus was well tolerated with the complete regimen suggesting an encouraging period of stable disease. The finding that none of the patients who presented without distant visceral metastases developed such would be consistent with the generation of a systemic immune response with effects on seeded metastatic cells. Analysis of local and systemic immune responses is currently proceeding and may provide further insights. This study is supported by the NCI Cancer Therapeutics Evaluation Program (CTEP) and by NCI U01-CA07031 and P30-CA72720. Citation Format: Elizabeth A. Poplin, David A. August, Rebecca A. Moss, Tamir Ben-Menachem, Hazar Michael, Aparna Repaka, Renee Artymyshyn, Chang Chan, James L. Gulley, Robert S. DiPaola, Edmund C. Lattime. Panvac-F and Panvac-V: Phase I study of intratumoral and systemic vaccination. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B100.