Kwong-Yok Tsang

Sublethal Irradiation of Human Tumor Cells Modulates Phenotype Resulting in Enhanced Killing by Cytotoxic T Lymphocytes

Local radiation of tumor masses is an established modality for the therapy of a range of human tumors. It has recently been recognized that doses of radiation, lower than or equal to those that cause direct cytolysis, may alter the phenotype of target tissue by up-regulating gene products that may make tumor cells more susceptible to T-cell–mediated immune attack. Previously, we demonstrated that radiation increased Fas (CD95) gene expression in carcinoembryonic antigen (CEA)-expressing murine tumor cells, which consequently enhanced their susceptibility to CEA-specific CTL-mediated killing. The present study was designed to determine whether these phenomena also occur with human tumor cells. Here, 23 human carcinoma cell lines (12 colon, 7 lung, and 4 prostate) were examined for their response to nonlytic doses of radiation (10 or 20 Gy). Seventy-two hours postirradiation, changes in surface expression of Fas (CD95), as well as expression of other surface molecules involved in T-cell–mediated immune attack such as intercellular adhesion molecule 1, mucin-1, CEA, and MHC class I, were examined. Twenty-one of the 23 (91%) cell lines up-regulated one or more of these surface molecules postirradiation. Furthermore, five of five irradiated CEA+/A2+ colon tumor cells lines demonstrated significantly enhanced killing by CEA-specific HLA-A2–restricted CD8+ CTLs compared with nonirradiated counterparts. We then used microarray analysis to broaden the scope of observed changes in gene expression after radiation and found that many additional genes had been modulated. These up-regulated gene products may additionally enhance the tumor cells’ susceptibility to T-cell–mediated immune attack or serve as additional targets for immunotherapy. Overall, the results of this study suggest that nonlethal doses of radiation can be used to make human tumors more amenable to immune system recognition and attack and form the rational basis for the combinatorial use of cancer vaccines and local tumor irradiation.

Combining a Recombinant Cancer Vaccine with Standard Definitive Radiotherapy in Patients with Localized Prostate Cancer

Purpose: Many patients with clinically localized prostate cancer develop biochemical failure despite excellent local therapy perhaps due to occult metastatic disease. One potential solution is the utilization of a well-tolerated systemic therapy (e.g., vaccine) in concert with local therapy. Experimental Design: We present a randomized phase II clinical trial designed to determine if a poxviral vaccine encoding prostate-specific antigen (PSA) can induce a PSA-specific T-cell response when combined with radiotherapy in patients with clinically localized prostate cancer. Thirty patients were randomized in a 2:1 ratio into vaccine plus radiotherapy or radiotherapy-only arms. Those patients in the combination arm received a “priming” vaccine with recombinant vaccinia (rV) PSA plus r V containing the T-cell costimulatory molecule B7.1 (rV-B7.1) followed by monthly booster vaccines with recombinant fowlpox PSA. The vaccines were given with local granulocyte-macrophage colony-stimulating factor and low-dose systemic interleukin-2. Standard external beam radiation therapy was given between the fourth and the sixth vaccinations. Results: Seventeen of 19 patients in the combination arm completed all eight vaccinations and 13 of these 17 patients had increases in PSA-specific T cells of at least 3-fold versus no detectable increases in the radiotherapy-only arm (P < 0.0005). There was also evidence of de novo generation of T cells to well-described prostate-associated antigens not found in the vaccine, providing indirect evidence of immune-mediated tumor killing. The vaccine was well tolerated. Conclusion: This vaccine regimen can be safely given in patients undergoing radiation therapy for localized prostate cancer, with the majority of patients generating a PSA-specific cellular immune response to vaccine.

Phase I Study of Sequential Vaccinations With Fowlpox-CEA(6D)-TRICOM Alone and Sequentially With Vaccinia-CEA(6D)-TRICOM, With and Without Granulocyte-Macrophage Colony-Stimulating Factor, in Patients With Carcinoembryonic Antigen–Expressing Carcinomas

PURPOSE Our previous clinical experience with vaccinia and replication-defective avipox recombinant carcinoembryonic antigen (CEA) vaccines has demonstrated safety and clinical activity with a correlation between CEA-specific immune response and survival. Preclinical evidence demonstrated that the addition of the transgenes for three T-cell costimulatory molecules (B7-1, ICAM-1, LFA-3, designated TRICOM) results in a significant improvement in antigen-specific T-cell responses and antitumor activity. We describe here the first trial in humans of the CEA-TRICOM vaccines (also including an enhancer agonist epitope within the CEA gene). PATIENTS AND METHODS Fifty-eight patients with advanced CEA-expressing cancers were accrued to eight cohorts that involved vaccinations with the following: replication-defective fowlpox recombinant (rF)-CEA(6D)-TRICOM; primary vaccination with recombinant vaccinia (rV)-CEA(6D)-TRICOM plus rF-CEA(6D)-TRICOM booster vaccinations; and rV-CEA(6D)-TRICOM and then rF-CEA(6D)-TRICOM, plus granulocyte-macrophage colony-stimulating factor (GM-CSF) with vaccines, or with divided doses of vaccine with GM-CSF. Vaccines were administered every 28 days for six doses and then once every 3 months. Reverting to treatments every 28 days was allowed if patients progressed on the 3-month schedule. RESULTS In this phase I study, no significant toxicity was observed. Twenty-three patients (40%) had stable disease for at least 4 months, with 14 of these patients having prolonged stable disease (> 6 months). Eleven patients had decreasing or stable serum CEA, and one patient had a pathologic complete response. Enhanced CEA-specific T-cell responses were observed in the majority of patients tested. CONCLUSION We demonstrated that the CEA-TRICOM vaccines are safe and can generate significant CEA-specific immune responses, and they seem to have clinical benefit in some patients with advanced cancer.

Pilot Study of Vaccination with Recombinant CEA-MUC-1-TRICOM Poxviral-Based Vaccines in Patients with Metastatic Carcinoma

Purpose: Poxviral vectors have a proven safety record and can be used to incorporate multiple transgenes. Prior clinical trials with poxviral vaccines have shown that immunologic tolerance to self-antigens can be broken. Carcinoembryonic antigen (CEA) and MUC-1 are overexpressed in a substantial proportion of common solid carcinomas. The primary end point of this study was vaccine safety, with immunologic and clinical responses as secondary end points. Experimental Design: We report here a pilot study of 25 patients treated with a poxviral vaccine regimen consisting of the genes for CEA and MUC-1, along with a triad of costimulatory molecules (TRICOM; composed of B7.1, intercellular adhesion molecule 1, and lymphocyte function–associated antigen 3) engineered into vaccinia (PANVAC-V) as a prime vaccination and into fowlpox (PANVAC-F) as a booster vaccination. Results: The vaccine was well tolerated. Apart from injection-site reaction, no grade ≥2 toxicity was seen in more than 2% of the cycles. Immune responses to MUC-1 and/or CEA were seen following vaccination in 9 of 16 patients tested. A patient with clear cell ovarian cancer and symptomatic ascites had a durable (18-month) clinical response radiographically and biochemically, and one breast cancer patient had a confirmed decrease of >20% in the size of large liver metastasis. Conclusions: This vaccine strategy seems to be safe, is associated with both CD8 and CD4 immune responses, and has shown evidence of clinical activity. Further trials with this agent, either alone or in combination with immunopotentiating and other therapeutic agents, are warranted.

Safety and immunologic response of a viral vaccine to prostate-specific antigen in combination with radiation therapy when metronomic-dose interleukin 2 is used as an adjuvant.

Purpose: We have previously reported on the safety and immunologic response of a poxvirus-based vaccine encoding prostate-specific antigen (PSA) used in combination with radiation therapy in patients with localized prostate cancer. We hypothesized that a “metronomic” dose of interleukin 2 (IL-2) as a biological adjuvant would cause less toxicity while maintaining immunologic response. Experimental Design: Eighteen patients with localized prostate cancer were treated in a single-arm trial using previously established doses of vaccine and radiation therapy. The vaccine used was a recombinant vaccinia virus engineered to encode PSA admixed with a recombinant vaccinia encoding the costimulatory molecule B7.1, followed by booster vaccinations with a recombinant fowlpox vector expressing PSA. Patients received a total of eight planned vaccination cycles, once every 4 weeks, with granulocyte-macrophage colony-stimulating factor given on days 1 to 4 and interleukin 2 (IL-2) at a dose of 0.6 MIU/M2 given from days 8 to 21 after each vaccination. Definitive external beam radiation therapy was initiated after the third vaccination cycle. Patients were evaluated for safety and immunologic response. Toxicity and immunologic activity were compared with the previously reported regimen containing a higher dose of IL-2. Results: Seventeen of 18 patients received all eight cycles of vaccine with IL-2. Five of eight HLA-A2+ patients evaluated had an increase in PSA-specific T cells of ≥3-fold. Toxicities were generally mild, with only seven vaccination cycles of 140 given resulting in grade 3 toxicities possibly attributable to IL-2. Conclusions: Metronomic-dose IL-2 in combination with vaccine and radiation therapy is safe, can induce prostate-specific immune responses, and has immunologic activity similar to low-dose IL-2, with markedly reduced toxicities.

A Pilot Study of MUC-1/CEA/TRICOM Poxviral-Based Vaccine in Patients with Metastatic Breast and Ovarian Cancer

Purpose: PANVAC is a recombinant poxviral vaccine that contains transgenes for MUC-1, CEA, and 3 T-cell costimulatory molecules. This study was conducted to obtain preliminary evidence of clinical response in metastatic breast and ovarian cancer patients. Experimental design: Twenty-six patients were enrolled and given monthly vaccinations. Clinical and immune outcomes were evaluated. Results: These patients were heavily pretreated, with 21 of 26 patients having 3 or more prior chemotherapy regimens. Side effects were largely limited to mild injection-site reactions. For the 12 breast cancer patients enrolled, median time to progression was 2.5 months (1–37+) and median overall survival was 13.7 months. Four patients had stable disease. One patient had a complete response by RECIST and remained on study for 37 months or more, with a significant drop in serum interleukin (IL)-6 and IL-8 by day 71. Another patient with metastatic disease confined to the mediastinum had a 17% reduction in mediastinal mass and was on study for 10 months. Patients with stable or responding disease had fewer prior therapies and lower tumor marker levels than patients with no evidence of response. For the ovarian cancer patients (n = 14), the median time to progression was 2 months (1–6) and median overall survival was 15.0 months. Updated data are presented here for one patient treated with this vaccine in a previous trial, with a time to progression of 38 months. Conclusions: Some patients who had limited tumor burden with minimal prior chemotherapy seemed to benefit from the vaccine. Further studies to confirm these results are warranted. Clin Cancer Res; 17(22); 7164–73. ©2011 AACR.

Combination Chemotherapy and Radiation of Human Squamous Cell Carcinoma of the Head and Neck Augments CTL-Mediated Lysis

Purpose: The combination of systemic multiagent chemotherapy (5-fluorouracil + cisplatin) and tumor irradiation is standard of care for head and neck squamous cell carcinoma (HNSCC). Furthermore, it has been shown that sublethal doses of radiation or chemotherapeutic drugs in diverse cancer types may alter the phenotype or biology of neoplastic cells, making them more susceptible to CTL-mediated cytotoxicity. However, little is known about the potential synergistic effect of drug plus radiation on CTL killing. Here, we examined whether the combination of two chemotherapeutics and ionizing radiation enhanced CTL-mediated destruction of HNSCC more so than either modality separately, as well as the basis for the enhanced tumor cell lysis. Experimental Design: Several HNSCC cell lines with distinct biological features were treated with sublethal doses of cisplatin and 5-fluorouracil for 24 hours and with 10-Gy irradiation. Seventy-two hours postirradiation, tumor cells were exposed to an antigen-specific CD8+ CTL directed against carcinoembryonic antigen or MUC-1. Results: In three of three tumor cell lines tested, enhanced CTL activity was observed when the two modalities (chemotherapy and radiation) were combined as compared with target cells exposed to either modality separately. CTL-mediated lysis was MHC restricted and antigen specific and occurred almost entirely via the perforin pathway. Moreover, the combination treatment regimen led to a 50% reduction in Bcl-2 expression whereas single modality treatment had little bearing on the expression of this antiapoptotic gene. Conclusions: Overall, these results reveal that (a) CTL killing can be enhanced by combining multiagent chemotherapy and radiation and (b) combination treatment enhanced or sensitized HNSCC to the perforin pathway, perhaps by down-regulating Bcl-2 expression. These studies thus form the rational basis for clinical trials of immunotherapy concomitant with the current standard of care of HNSCC.

A human cytotoxic t-lymphocyte epitope and its agonist epitope from the non-variable number of tandem repeat sequence of muc-1

Purpose: MUC-1/DF-3 remains an attractive target for vaccine therapy. It is overexpressed in the majority of human carcinomas and multiple myeloma. Clinical trials using MUC-1-based vaccines have demonstrated safety, clinical responses, and the induction of T-cell responses directed against MUC-1. Previous studies in experimental models and in clinical trials have demonstrated that altering the amino acid sequence of a “self” epitope can lead to the generation of an enhancer agonist epitope capable of eliciting stronger T-cell responses than the native epitope can. Experimental Design and Results: We describe here the identification of six novel class I HLA-A2 epitopes of MUC-1 that reside outside of the variable number of tandem repeat region. Each is shown to have the ability to activate human T cells as measured by IFN-γ production. One epitope (ATWGQDVTSV, at amino acid position 92–101 and designated P-92), which demonstrated the highest level of binding to HLA-A2 and which induced the highest level of IFN-γ in human T cells, was further studied for the generation of potential enhancer agonist epitopes. Of four potential agonists identified, one epitope (ALWGQDVTSV, designated P-93L) was identified as an enhancer agonist. Compared with the native P-92 peptide, the P-93L agonist (a) bound HLA-A2 at lower peptide concentrations, (b) demonstrated a higher avidity for HLA-A2 in dissociation assays, (c) when used with antigen-presenting cells, induced the production of more IFN-γ by T cells than with the use of the native peptide, and (d) was capable of more efficiently generating MUC-1-specific human T-cell lines from normal volunteers and pancreatic cancer patients. Most importantly, the T-cell lines generated using the agonist epitope were more efficient than those generated with the native epitope in the lysis of targets pulsed with the native epitope and in the lysis of HLA-A2 human tumor cells expressing MUC-1. Conclusions: In addition to the identification of novel MUC-1 epitopes outside the variable number of tandem repeat region, the studies reported here describe the first agonist epitope of MUC-1. The employment of this agonist epitope in peptide-, protein-, and vector-based vaccines may well aid in the development of effective vaccines for a range of human cancers.

The use of a rapid ELISPOT assay to analyze peptide-specific immune responses in carcinoma patients to peptide vs. recombinant poxvirus vaccines.

Abstract An enzyme-linked immunosorbent spot (ELISPOT) assay for interferon γ production has been used to analyze specific T cell responses to a Flu 9-mer peptide, and a 9-mer peptide of carcinoembryonic antigen (CEA). Assays were performed on peripheral blood mononuclear cells (PBMC) of HLA-A2-positive patients with CEA-expressing carcinomas, both before and after vaccination with CEA-based vaccines, and from HLA-A2-positive healthy blood donors. The ELISPOT assay utilized aliquots of frozen PBMC, and assays were performed after 24 h in culture with peptide to rule out any artifacts due to long-term in vitro stimulation cycles. An internal standard was used for each assay to define reproducibility of the assay, and all samples from a given patient (pre- and post-vaccination, with both the Flu and CEA peptides) were analyzed simultaneously. The results indicated a trend towards healthy blood donors having higher levels of Flu-specific T cell precursors than do colon carcinoma patients, but these results were not statistically significant (P = 0.06). On the other hand, slightly higher CEA-specific T cell responses were observed in cancer patients with CEA-expressing carcinomas than in healthy blood donors. PBMC from two CEA-based vaccine clinical trials were analyzed for T cell responses to the same CEA peptide and to the Flu control peptide. The first trial consisted of three monthly vaccinations of CEA peptide (designated PPP) in adjuvant. The second trial consisted of cohorts receiving three monthly vaccinations of avipox-CEA recombinant (designated AAA) or cohorts receiving a primary vaccination with recombinant vaccinia-CEA followed by two monthly vaccinations with avipox-CEA (designated VAA). Few, if any, CEA-specific T cell responses were seen in the PPP vaccinations, while the majority of patients receiving the poxvirus CEA recombinants demonstrated increases in CEA-specific T cell responses and no increases in Flu-specific responses. CEA-specific IgG responses were also demonstrated in patients following recombinant CEA poxvirus vaccinations. Statistical analyses of the T cell responses to the same CEA peptide demonstrated a P value of 0.028 for the recombinant poxvirus vaccines, as compared with the peptide vaccine. There were no differences seen (P = 0.37) in Flu-specific responses after these two types of CEA vaccination. These results thus provide the first evidence that poxvirus recombinant-based vaccines are more potent in the initiation of tumor-antigen-specific T cell responses than vaccines employing peptide in adjuvant, when assays are conducted in an identical manner, and in defining responses to the same peptide. These results also demonstrate for the first time that an ELISPOT assay, performed over a 24-h period and without in vitro sensitization, can be successfully used to monitor immune responses to a tumor-associated antigen in cancer patients.

A Viral Vaccine Encoding Prostate-Specific Antigen Induces Antigen Spreading to a Common Set of Self-Proteins in Prostate Cancer Patients

Purpose: We previously reported a randomized phase II clinical trial combining a poxvirus-based vaccine encoding prostate-specific antigen (PSA) with radiotherapy in patients with localized prostate cancer. Here, we investigate whether vaccination against PSA induced immune responses to additional tumor-associated antigens and how this influenced clinical outcome. Experimental Design: Pretreatment and posttreatment serum samples from patients treated with vaccine + external beam radiation therapy (EBRT) versus EBRT alone were evaluated by Western blot and serologic screening of a prostate cancer cDNA expression library (SEREX) to assess the development of treatment-associated autoantibody responses. Results: Western blotting revealed treatment-associated autoantibody responses in 15 of 33 (45.5%) patients treated with vaccine + EBRT versus 1 of 8 (12.5%) treated with EBRT alone. SEREX screening identified 18 antigens, which were assembled on an antigen array with 16 previously identified antigens. Antigen array screening revealed that 7 of 33 patients (21.2%) treated with vaccine + EBRT showed a vaccine-associated autoantibody response to four ubiquitously expressed self-antigens: DIRC2, NDUFS1, MRFAP1, and MATN2. These responses were not seen in patients treated with EBRT alone, or other control groups. Patients with autoantibody responses to this panel of antigens had a trend toward decreased biochemical-free survival. Conclusions: Vaccine + EBRT induced antigen spreading in a large proportion of patients. A subset of patients developed autoantibodies to a panel of four self-antigens and showed a trend toward inferior outcomes. Thus, cancer vaccines directed against tumor-specific antigens can trigger autoantibody responses to self-proteins, which may influence the efficacy of vaccination. Clin Cancer Res; 16(15); 4046–56. ©2010 AACR.