Sipuleucel-T immune parameters correlate with survival: an analysis of the randomized phase 3 clinical trials in men with castration-resistant prostate cancer.

PURPOSE: Sipuleucel-T, the first FDA-approved autologous cellular immunotherapy for treatment of advanced prostate cancer, is manufactured by activating peripheral blood mononuclear cells, including antigen presenting cells (APCs), with a fusion protein containing prostatic acid phosphatase. Analysis of data from three phase 3 trials was performed to immunologically characterize this therapy during the course of the three doses, and to relate the immunological responses to overall survival (OS). METHODS: Sipuleucel-T product characteristics [APC numbers, APC activation (CD54 upregulation), and total nucleated cell (TNC) numbers] were assessed in three randomized, controlled phase 3 studies (N = 737). Antigen-specific cellular and humoral responses were assessed in a subset of subjects. The relationships between these parameters and OS were assessed. RESULTS: APC activation occurred in the first dose preparation [6.2-fold, (4.65, 7.70); median (25th, 75th percentile)] and increased in the second [10.6-fold (7.83, 13.65)] and third [10.5-fold (7.89, 13.65)] dose preparations. Cytokines and chemokines associated with activated APCs were produced during the manufacture of each dose; T-cell activation-associated cytokines were detected in the second and third dose preparations. Antigen-specific T cells were detectable after administration of the first sipuleucel-T dose. Cumulative APC activation, APC number, and TNC number correlated with OS (P < 0.05). Antigen-specific immune responses were observed in 78.8 % of monitored subjects and their presence correlated with OS (P = 0.003). CONCLUSION: Sipuleucel-T broadly engages the immune system by activating APCs ex vivo and inducing long-lived immune responses in vivo. These data indicate antigen-specific immune activation as a mechanism by which sipuleucel-T prolongs OS.

Concurrent trastuzumab and HER2/neu-specific vaccination in patients with metastatic breast cancer.

PURPOSE: The primary objectives of this phase I/II study were to evaluate the safety and immunogenicity of combination therapy consisting of concurrent trastuzumab and human epidermal growth factor receptor 2 (HER2)/neu-specific vaccination in patients with HER2/neu-overexpressing metastatic breast cancer. PATIENTS AND METHODS: Twenty-two patients with stage IV HER2/neu-positive breast cancer receiving trastuzumab therapy were vaccinated with an HER2/neu T-helper peptide-based vaccine. Toxicity was graded according to National Cancer Institute criteria, and antigen specific T-cell immunity was assessed by interferon gamma enzyme-linked immunosorbent spot assay. Data on progression-free and overall survival were collected. RESULTS: Concurrent trastuzumab and HER2/neu vaccinations were well tolerated, with 15% of patients experiencing an asymptomatic decline in left ventricular ejection fraction below the normal range during combination therapy. Although many patients had pre-existing immunity specific for HER2/neu and other breast cancer antigens while treated with trastuzumab alone, that immunity could be significantly boosted and maintained with vaccination. Epitope spreading within HER2/neu and to additional tumor-related proteins was stimulated by immunization, and the magnitude of the T-cell response generated was significantly inversely correlated with serum transforming growth factor beta levels. At a median follow-up of 36 months from the first vaccine, the median overall survival in the study population has not been reached. CONCLUSION: Combination therapy with trastuzumab and a HER2/neu vaccine is associated with minimal toxicity and results in prolonged, robust, antigen-specific immune responses in treated patients.

Insulin-like growth factor-binding protein-2 is a target for the immunomodulation of breast cancer.

Breast cancer is immunogenic and well suited to treatment via immunomodulation. The disease is often treated to remission and time to relapse is generally measured in years in many cases. Immune-based therapeutics, such as cancer vaccines, may be able to affect the clinical progression of micrometastatic disease. Immune targets must be identified that have the potential to inhibit tumor growth. Insulin-like growth factor-binding protein-2 (IGFBP-2) has direct effects on breast cancer proliferation via stimulation of critical signaling pathways. We questioned whether IGFBP-2 was an immune target in breast cancer. IGFBP-2-specific IgG antibody immunity was preferentially detected in breast cancer patients compared with controls (P = 0.0008). To evaluate for the presence of T-cell immunity, we identified potential pan-HLA-DR binding epitopes derived from IGFBP-2 and tested the peptides for immunogenicity. The majority of epitopes elicited peptide-specific T cells in both patients and controls and had high sequence homology to bacterial pathogens. IGFBP-2 peptide-specific T cells could respond to naturally processed and presented IGFBP-2 protein, indicating that these peptides were native epitopes of IGFBP-2. Finally, both immunization with IGFBP-2 peptides as well as adoptive transfer of IGFBP-2-competent T cells mediated an antitumor effect in a transgenic mouse model of breast cancer. This is the first report of IGFBP-2 as a human tumor antigen that may be a functional therapeutic target in breast cancer.

Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses.

BACKGROUND: Standardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of two commonly used cell-based assays, tritiated thymidine incorporation (proliferation) and IFN-gamma ELISPOT, to predict T cell responses to HER-2/neu, tetanus toxoid (tt), and cytomegalovirus (CMV) antigens. These antigens were determined to be low (HER-2/neu), moderate (tt), and robustly (CMV) immunogenic proteins. Samples from 27 Stage II, III, and IV HER-2/neu positive breast cancer patients, vaccinated against the HER-2/neu protein and tt, were analyzed by tritiated thymidine incorporation and IFN-gamma ELISPOT for T cell response. RESULTS: Linear regression analysis indicates that both stimulation index (SI) (p = 0.011) and IFN-gamma secreting precursor frequency (p < 0.001) are significant indicators of antigen specific immunity. ROC curves plotted to assess the performance of tritiated thymidine incorporation and the ELISPOT assay indicate that SI is a significant indicator of low T cell response to the HER-2/neu vaccine (p = 0.05), and of moderate and robust responses to tt (p = 0.01) and CMV (p = 0.016), respectively. IFN-gamma precursor frequency is a significant indicator of a robust T cell response to CMV (p = 0.03), but not of moderate tt (p = 0.09), or low HER-2/neu (p = 0.09) T cell responses. CONCLUSION: These data underscore the importance of taking into consideration the performance characteristics of assays used to measure T cell immunity. This consideration is particularly necessary when determining which method to utilize for assessing responses to immunotherapeutic manipulations in cancer patients.

Functional T cell responses to tumor antigens in breast cancer patients have a distinct phenotype and cytokine signature.

The overall prevalence with which endogenous tumor Ags induce host T cell responses is unclear. Even when such responses are detected, they do not usually result in spontaneous remission of the cancer. We hypothesized that this might be associated with a predominant phenotype and/or cytokine profile of tumor-specific responses that is different from protective T cell responses to other chronic Ags, such as CMV. We detected significant T cell responses to CEA, HER-2/neu, and/or MAGE-A3 in 17 of 21 breast cancer patients naive to immunotherapy. The pattern of T cell cytokines produced in response to tumor-associated Ags (TAAs) in breast cancer patients was significantly different from that produced in response to CMV or influenza in the same patients. Specifically, there was a higher proportion of IL-2-producing CD8(+) T cells, and a lower proportion of IFN-gamma-producing CD4(+) and/or CD8(+) T cells responding to TAAs compared with CMV or influenza Ags. Finally, the phenotype of TAA-responsive CD8(+) T cells in breast cancer patients was almost completely CD28(+)CD45RA(-) (memory phenotype). CMV-responsive CD8(+) T cells in the same patients were broadly distributed among phenotypes, and contained a high proportion of terminal effector cells (CD27(-)CD28(-)CD45RA(+)) that were absent in the TAA responses. Taken together, these results suggest that TAA-responsive T cells are induced in breast cancer patients, but those T cells are phenotypically and functionally different from CMV- or influenza-responsive T cells. Immunotherapies directed against TAAs may need to alter these T cell signatures to be effective.

Phenotype and in vitro function of mature MDDC generated from cryopreserved PBMC of cancer patients are equivalent to those from healthy donors.

BACKGROUND: Monocyte-derived-dendritic-cells (MDDC) are the major DC type used in vaccine-based clinical studies for a variety of cancers. In order to assess whether in vitro differentiated MDDC from cryopreserved PBMC of cancer patients are functionally distinct from those of healthy donors, we compared these cells for their expression of co-stimulatory and functional markers. In addition, the effect of cryopreservation of PBMC precursors on the quality of MDDC was also evaluated using samples from healthy donors. METHODS: Using flow cytometry, we compared normal donors and cancer patients MDDC grown in the presence of GM-CSF+IL-4 (immature MDDC), and GM-CSF+IL-4+TNFalpha+IL-1beta+IL-6+PGE-2 (mature MDDC) for (a) surface phenotype such as CD209, CD83 and CD86, (b) intracellular functional markers such as IL-12 and cyclooxygenase-2 (COX-2), (c) ability to secrete IL-8 and IL-12, and (d) ability to stimulate allogeneic and antigen-specific autologous T cells. RESULTS: Cryopreservation of precursors did affect MDDC marker expression, however, only two markers, CD86 and COX-2, were significantly affected. Mature MDDC from healthy donors and cancer patients up-regulated the expression of CD83, CD86, frequencies of IL-12+ and COX-2+ cells, and secretion of IL-8; and down-regulated CD209 expression relative to their immature counterparts. Compared to healthy donors, mature MDDC generated from cancer patients were equivalent in the expression of nearly all the markers studied and importantly, were equivalent in their ability to stimulate allogeneic and antigen-specific T cells in vitro. CONCLUSION: Our data show that cryopreservation of DC precursors does not significantly affect the majority of the MDDC markers, although the trends are towards reduced expression of co-stimulatory makers and cytokines. In addition, monocytes from cryopreserved PBMC of cancer patients can be fully differentiated into mature DC with phenotype and function equivalent to those derived from healthy donors.

Tumor antigen-specific T-cell expansion is greatly facilitated by in vivo priming.

PURPOSE: Adoptive T-cell therapy is a promising strategy for the treatment of patients with established tumors but is often limited to specific cancers where tumor-infiltrating lymphocytes, the source of T cells for ex vivo culture, can be obtained. In this study, we evaluated the feasibility of expanding HER-2/neu-specific T cells derived from peripheral blood ex vivo following in vivo priming with a HER-2/neu peptide vaccine. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells from cytomegalovirus (CMV)-seronegative and CMV-seropositive donors as well as HER-2/neu-positive cancer patients who had or had not been vaccinated with a HER-2/neu peptide-based vaccine was used as a source of T lymphocytes. Antigen-specific T-cell lines were generated by in vitro stimulation with antigen followed by nonspecific expansion on CD3/CD28 beads. The ability to expand antigen-specific T cells was assessed using IFN-gamma and granzyme B enzyme-linked immunosorbent spot. The phenotype of the resultant T-cell lines was evaluated by flow cytometry, including the presence of FOXP3-expressing CD4(+) T cells. RESULTS: The frequencies of CMV-specific T cells generated from CMV(+) donors were >11-fold higher than the frequencies from CMV(-) donors (P = 0.001), with 22-fold increase of total number of CD3(+) T cells. The frequencies of HER-2/neu-specific T cells generated from the primed patients were >25-fold higher than the frequencies from unvaccinated patients (P = 0.006), with an average of a 19-fold increase of total number of CD3(+) T cells. Using peripheral blood as the source of T cells did not result in concurrent expansion of FOXP3(+)CD4(+) regulatory T cells despite the use of interleukin-2 in in vitro culture. Both CD4(+) and CD8(+) HER-2/neu-specific T cells could be expanded. The extent of ex vivo expansion correlated with the magnitude of immunity achieved during immunization (P = 0.008). CONCLUSION: Tumor-specific T cells can be efficiently expanded from the peripheral blood ex vivo following in vivo priming with a vaccine. This approach provides an effective method to generate tumor-specific polyclonal T cells for therapeutic use that could be applied to cancer patients with any tumor type.

Laboratory analysis of T-cell immunity.

Immune-based strategies for treating and preventing cancer are increasingly being tested and include cancer vaccination, adoptive T cell therapy, and cytokine therapy. An important component of testing and development of immune-based strategies is monitoring the immunologic response. The ability to monitor T cell immunity has been suboptimal. The measurement of tumor-specific immunity will aid in defining which strategies should be moved forward in clinical trials and which should be eliminated or evaluated further in the preclinical realm. Immunologic monitoring is necessary for determining if an approach has immunologic efficacy and ultimately whether immunologic responses correlate with a clinical response. This article discusses several important elements of measuring T cell immunity such as validation principles, laboratory issues, current approaches, and new paradigms and concepts for future testing. Informative immunologic monitoring of T cells will be one of the driving forces in advancing the field of tumor immunology.

Maximizing the retention of antigen specific lymphocyte function after cryopreservation.

The ability to cryopreserve lymphocytes in peripheral blood mononuclear cells (PBMC) to retain their function after thawing is critical to the analysis of cancer immunotherapy studies. We evaluated a variety of cryopreservation strategies with the aim of developing an optimized protocol for freezing and thawing PBMC to retain viability and function. We determined several factors which do not affect cell viability after cryopreservation such as shipping frozen samples on dry ice, the length of time and speed at which samples are washed and centrifuged after thawing, and the number of cells frozen per container. Different media additives, however, did impact the viability of the cells after thawing. There was a significant reduction in the viability of the cells after freezing when using human AB serum compared to all other additives tested (p<0.000). A second critical parameter was the temperature of the media used to wash the cells after removal from the cryotubes. When the media was cooled to 4 degrees C prior to washing, the mean viability was 69.7+/-12.5%, at 25 degrees C 92.55+/-3.1%, and at 37 degrees C 95.11+/-2.5%. Finally, we used an optimized cryopreservation protocol with different media additives to determine if functional T cell responses to tetanus toxoid could be preserved. There was a statistically significant correlation between the tetanus specific stimulation index (S.I.) of the non-cryopreserved PBMC and SI obtained from cells frozen with media containing human serum albumin as compared to other additives such as dextran or fetal bovine serum.

Effect of dose on immune response in patients vaccinated with an her-2/neu intracellular domain protein--based vaccine.

PURPOSE: To evaluate the safety of an HER-2/neu intracellular domain (ICD) protein vaccine and to estimate whether vaccine dose impacts immunogenicity. PATIENTS AND METHODS: Twenty-nine patients with HER-2/neu-overexpressing breast or ovarian cancer and with no evidence of disease after standard therapy received a low- (25 microg), intermediate- (150 microg), or high-dose (900 microg) HER-2/neu ICD protein vaccine. The vaccine was administered intradermally, monthly for 6 months, with granulocyte-macrophage colony-stimulating factor as an adjuvant. Toxicity and both cellular and humoral HER-2/neu-specific immunity was evaluated. RESULTS: The vaccine was well tolerated. The majority of patients (89%) developed HER-2/neu ICD-specific T-cell immunity. The dose of vaccine did not predict the magnitude of the T-cell response. The majority of patients (82%) also developed HER-2/neu-specific immunoglobulin G antibody immunity. Vaccine dose did not predict magnitude or avidity of the HER-2/neu-specific humoral immune response. Time to development of detectable HER-2/neu-specific immunity, however, was significantly earlier for the high- versus low-dose vaccine group (P =.003). Over half the patients retained HER-2/neu-specific T-cell immunity 9 to 12 months after immunizations had ended. CONCLUSION: The HER-2/neu ICD protein vaccine was well tolerated and effective in eliciting HER-2/neu-specific T-cell and antibody immunity in the majority of breast and ovarian cancer patients who completed the vaccine regimen. Although the dose of vaccine did not impact the magnitude of T-cell or antibody immunity elicited, patients receiving the highest dose developed HER-2/neu-specific immunity more rapidly than those who received the lowest dose.

Flt3 ligand as a vaccine adjuvant in association with HER-2/neu peptide-based vaccines in patients with HER-2/neu-overexpressing cancers.

Dendritic cells (DCs) are potent antigen-presenting cells and have shown promise to function as "natural" vaccine adjuvants. Currently, most cancer vaccine trials using DCs generate autologous DCs ex vivo for each patient. Systemic treatment with Flt3 ligand (FL) results in a marked increase of DCs in tissues such as spleen and lymph nodes in mice and in the peripheral blood and skin of humans. In light of these observations, we questioned whether FL could be used systemically as a vaccine adjuvant to stimulate DC mobilization in vivo, circumventing the need to generate DCs ex vivo. Ten patients with HER-2/neu-overexpressing cancer were enrolled in a phase 1 study to receive a HER-2/neu peptide-based vaccine targeting the intracellular domain of the HER-2/neu protein. All patients received 20 microg/kg FL per day subcutaneously for 14 days. Five patients received the HER-2/neu peptide-based vaccine alone on day 7 of the 14-day cycle, and 5 patients received the vaccine admixed with 150 microg granulocyte macrophage-colony-stimulating factor (GM-CSF) on day 7 of the FL cycle. T-cell proliferative responses to HER-2/neu peptides and intracellular domain protein suggest that vaccine regimens including FL as an adjuvant were not effective in eliciting a significant HER-2/neu protein-specific T-cell proliferative response. However, including FL as a vaccine adjuvant was effective in boosting the precursor frequency of interferon-gamma-secreting HER-2/neu-specific T cells. The small sample size of each group, however, did not allow a statistically significant comparison of immune responses between the FL alone and FL with GM-CSF arms. Finally, vaccine regimens including FL as a vaccine adjuvant were associated with the development of apparent autoimmune phenomena in some patients.