Neil Berinstein

Combination Chemotherapy and ALVAC-CEA/B7.1 Vaccine in Patients with Metastatic Colorectal Cancer

Purpose: The combination of vaccines and chemotherapy holds promise for cancer therapy, but the effect of cytotoxic chemotherapy on vaccine-induced antitumor immunity is unknown. This study was conducted to assess the effects of systemic chemotherapy on ALVAC-CEA/B7.1–induced T-cell immunity in patients with metastatic colorectal cancer. Experimental Design: Patients with metastatic colorectal cancer were treated with fluorouracil, leucovorin, and irinotecan and were also given ALVAC-CEA/B7.1 vaccine with or without tetanus toxoid adjuvant. Eligible patients were randomized to ALVAC followed by chemotherapy and booster vaccination (group 1), ALVAC and tetanus toxoid followed by chemotherapy (group 2), or chemotherapy alone followed by ALVAC in patients without disease progression (group 3). Humoral immune responses were measured by standard ELISA assay, and carcinoembryonic antigen (CEA)-specific T-cell responses were measured by IFN-γ enzyme-linked immunospot assay. Results: One hundred eighteen patients were randomized to receive either ALVAC before and concomitantly with chemotherapy (n = 39), ALVAC with tetanus adjuvant before and concomitantly with chemotherapy (n = 40), or chemotherapy followed by ALVAC (n = 39). Serious adverse events were largely gastrointestinal (n = 30) and hematologic (n = 24). Overall, 42 patients (40.4%) showed objective clinical responses. All patients developed antibody responses against ALVAC, but increased anti-CEA antibody titers were detected in only three patients. Increases in CEA-specific T cells were detected in 50%, 37%, and 30% of patients in groups 1, 2, and 3, respectively. There were no differences in clinical or immune responses between the treatment groups. Conclusion: The combination of ALVAC-CEA/B7.1 vaccine and systemic chemotherapy has an acceptable safety profile in patients with metastatic colorectal cancer. Systemic chemotherapy did not affect the generation of CEA-specific T-cell responses following vaccination.

Therapeutic vaccines against melanoma and colorectal cancer.

Our overall strategy is to develop multivalent recombinant vaccines capable of eliciting broad immune responses in patients with malignant melanoma or colorectal cancer. We report herein results from initial studies conducted in cancer patients to evaluate the effect of intratumoral administration of recombinant canarypox viruses carrying cytokine genes. Our current focus is on the induction of tumor-specific T-cell responses using a prime/boost immunization schedule with a unique vector system derived from the canary pox virus called ALVAC, in which we incorporate genes encoding Tumor Associated Antigens (TAAs) of interest. Clinical studies in colorectal cancer evaluating an ALVAC CEA candidate vaccine have shown that this approach is safe and can induce tumor-specific T cell responses. Additional clinical studies evaluating candidate vaccines against melanoma and colorectal cancer, targeting either the gp100, Mage 1, Mage 3 or p53 molecules are ongoing.

Overview of therapeutic vaccination approaches for cancer.

Recently, immunotherapy has been widely investigated for the treatment of cancer. While much of the success in this area lies with passive immunotherapy, the focus of this review is active vaccination strategies, which are showing promise. Cancer cells express a wide profile of different proteins, some of which are related to oncogenic transformation and are specific to cancer cells only. However, in cancer, most protein targets presented to the immune system from tumor cells are self-antigens. The immune system is highly tolerant of, and therefore does not react to, these self-antigens. Active immunotherapy aims to reverse this immune tolerance so the immune system can respond appropriately to self-antigens. To generate a successful antitumor response, several features should be considered: a target antigen on tumor cells to direct the immune response, a platform to present the vaccine-derived antigen to the immune system, an adjuvant to enhance immune stimulation, and appropriate monitoring techniques. New tools are becoming available for monitoring, although the clinical relevance for these assays needs to be established. Optimism for cancer vaccine approaches are increasing. Recent studies have shown that it is possible to break immune tolerance to self-antigens. Promising therapeutic clinical activity has been demonstrated, and other studies have shown that cancer vaccines are most beneficial for minimal residual disease states. Further optimization of cancer vaccines and larger clinical studies are required.

Clinical phase I intratumoral administration of two recombinant ALVAC canarypox viruses expressing human granulocyte-macrophage colony-stimulating factor or interleukin-2: the transgene determines the composition of the inflammatory infiltrate.

Immunotherapy employs cytokines for modifying local inflammatory reactions. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to activate dendritic cells, macrophages, and granulocytes leading to clinical trials using GM-CSF-based cancer vaccine approaches. Interleukin-2 (IL-2) is an important T cell stimulatory cytokine approved as exogenous antitumor agent. The ALVAC viral vector system uses a recombinant canarypox virus for local gene expression. We report a phase I clinical trial using intratumoral administration of ALVAC GM-CSF or ALVAC IL-2 in skin metastases of melanoma or leiomyosarcoma. ALVAC GM-CSF and ALVAC IL-2 were injected at 107.12 and 106.92, 50% cell culture infectious dose in eight metastases with acceptable tolerability. Local and systemic inflammatory reactions were observed. The transgene determined the local infiltrate: GM-CSF induced monocyte and macrophage enrichment of the peritumoral inflammatory infiltrate, whereas IL-2 increased local T lymphocytes. Stable disease of injected lesions was seen after ALVAC GM-CSF application, whereas ALVAC IL-2 treatment led to partial regression in three out of eight injected tumors, accompanied by decreased expression of melanocytic antigens. Local GM-CSF expression could be induced. In summary, ALVAC GM-CSF and ALVAC IL-2 injections are safe and can mediate local biologic and immunologic effects.

Characterization of Antigen-Specific Immune Responses Induced by Canarypox Virus Vaccines

Avipoxvirus-based vectors, such as recombinant canarypox virus ALVAC, are studied extensively as delivery vehicles for vaccines against cancer and infectious diseases. Effective use of such vaccines is expected to benefit from proper understanding of the interaction between these viral vectors and the host immune system. We performed preclinical vaccination experiments in a murine tumor model to analyze the immunogenic properties of an ALVAC-based vaccine against carcinoembryonic Ag (ALVAC-CEA), a tumor-associated autoantigen commonly overexpressed in colorectal cancers. The protective CEA-specific immunity induced by this vaccine consisted of CD4+ T cell responses with a mixed Th1/Th2 cytokine profile that were accompanied by potent humoral responses, but not by CEA-specific CD8+ CTL immunity. In contrast, protective immunity induced by a CEA-specific DNA vaccine (DNA-CEA) consisted of Th1 and CTL responses. Modification of the ALVAC-CEA vaccine through coinjection of DNA-CEA, admixture with CpG oligodeoxynucleotides, or supplementation with additional transgenes encoding a triad of costimulatory molecules (TRICOM) did not result in induction of CEA-specific CTL responses. Even though these results suggested that ALVAC does not elicit Ag-specific CTLs, immunization with ALVAC vaccines against other Ags efficiently induced CTL responses. Our data show that the capacity of ALVAC vaccines to elicit CTL immunity against transgene-encoded Ags critically depends on the presence of highly immunogenic CTL epitopes in these Ags. This consideration needs to be taken into account with respect to the design and evaluation of vaccination strategies that use ALVAC-based vaccine.

Quantitative Immunohistochemical Analysis and Prognostic Significance of TRPS-1, a New GATA Transcription Factor Family Member, in Breast Cancer

The trichorhinophalangeal syndrome 1 (TRPS-1) gene is a novel GATA transcription factor family member. Previously, using a gene expression profiling and immunohistochemistry (IHC) screen, we identified TRPS-1 as a highly prevalent gene in breast cancer (BC), expressed in >90% of estrogen receptor alpha (ERα)+ and ERα− BC subtypes. TRPS-1 was also shown to be expressed in prostate cancer where it was shown to play a proapoptotic function during androgen withdrawal possibly through regulating antioxidant metabolism. The role of TRPS-1 and its prognostic significance in hormone-dependent and hormone-independent BC however is not known. In this study, we developed a new quantitative IHC (qIHC) method to further study TRPS-1 as a marker and possible prognostic indicator in BC. By using this method, a quantitative parameter for TRPS-1 expression called a quick score (QS) was derived from the measured labeling index and mean optical density after IHC and applied to a set of 152 stage II/III BC patients from 1993 to 2006 who did not receive preoperative chemotherapy. Associations between QS and tumor characteristics were evaluated using the Kruskal–Wallis test. A wide range of TRPS-1 QS was found among the sample set with higher TRPS-1 QS significantly associated with tumor ERα (pu2009=u20090.023 for QS and pu2009=u20090.028 for Allred score), progesterone receptor (pu2009=u20090.009), and GATA-3 (pu2009<u20090.0001). TRPS-1 QS was also positively associated with HER2 status (pu2009=u20090.026). Further analysis of different ductal structures in ten BC cases revealed that TRPS-1 expression was expressed at low levels in the remaining normal ducts and in areas of usual ductal hyperplasia but showed marked increase in expression in ductal carcinoma in situ and invasive carcinoma lesions in the tissue. An analysis of TRPS-1 expression in association with overall survival in the 152 stage II/III sample set also revealed that TRPS-1 QS (≥4.0) was significantly associated with improved survival (pu2009=u20090.0165). Patients with TRPS-1 QS <4 had a hazard ratio of 2 (pu2009=u20090.019) after univariate Cox proportional hazards analysis. In summary, this new qIHC approach was found to reveal critical differences in TRPS-1 expression in primary BC samples and found that it is a promising prognostic marker that should be further evaluated as a possible tumor suppressor gene facilitating improved survival in different subtypes of BC.

Critical issues in the clinical testing of cancer vaccines: summary of a panel discussion at a symposium entitled “cancer vaccines: are they here yet?” conducted in Toronto, Canada, December 2002

It is important to define the best and most appropriate population in which to test the vaccine. We need to take into account established therapies for this patient population, and develop a strategy to combine this therapy with the vaccine therapy. In early phase I clinical trials, we need to establish the best surrogate endpoints to optimize vaccine activity; immunology endpoints are mechanistically relevant. These early trials should have small patient groups and should be designed to optimize combination therapies using these surrogate endpoints. In these trials, it will be important to randomize early to address questions about the best treatment schedules and the best antigens. Statistical methods can be used to assess these immunologic endpoints. Finally, we should use the best arm to carry to the next phase of clinical testing. For cancer vaccines to be considered therapeutic, we need to demonstrate clear clinical endpoints. We need to show increased survival rates, tumor eradication or tumor control. To achieve these endpoints, a strong commitment to carrying out randomized clinical trials needs to be made. The difficulty with making decisions to proceed to advanced clinical testing of cancer vaccines is the fact that we can always improve the vaccine by adding a new antigen adjuvant or improving the dosing schedule. However, there is no reason why the vaccine refinement work cannot continue while we move forward to test clinical activity of promising vaccines in these large randomized clinical trials. There has been a tremendous amount of work completed to date in terms of evaluating immune responses as surrogate endpoints. But these are only surrogate endpoints. We need to move the work from the laboratory into the clinical setting to evaluate the clinical benefits of these vaccines.