Edwin B. Walker

OX40 Is a Potent Immune-Stimulating Target in Late-Stage Cancer Patients

OX40 is a potent costimulatory receptor that can potentiate T-cell receptor signaling on the surface of T lymphocytes, leading to their activation by a specifically recognized antigen. In particular, OX40 engagement by ligands present on dendritic cells dramatically increases the proliferation, effector function, and survival of T cells. Preclinical studies have shown that OX40 agonists increase antitumor immunity and improve tumor-free survival. In this study, we performed a phase I clinical trial using a mouse monoclonal antibody (mAb) that agonizes human OX40 signaling in patients with advanced cancer. Patients treated with one course of the anti-OX40 mAb showed an acceptable toxicity profile and regression of at least one metastatic lesion in 12 of 30 patients. Mechanistically, this treatment increased T and B cell responses to reporter antigen immunizations, led to preferential upregulation of OX40 on CD4(+) FoxP3(+) regulatory T cells in tumor-infiltrating lymphocytes, and increased the antitumor reactivity of T and B cells in patients with melanoma. Our findings clinically validate OX40 as a potent immune-stimulating target for treatment in patients with cancer, providing a generalizable tool to favorably influence the antitumor properties of circulating T cells, B cells, and intratumoral regulatory T cells.

Phase 1 study of stereotactic body radiotherapy and interleukin-2--tumor and immunological responses.

Stereotactic body radiation therapy enhances tumor response rate to high-dose interleukin-2 in a phase 1 study. Let’s Work Together Despite decades of research into causes and potential therapies for cancer, cancers still account for almost 13% of all deaths every year. It is becoming increasingly clear that monotherapies are not the answer, and combining drugs to improve efficacy and prevent resistance is becoming the norm. However, care must be taken when combining even drugs already in the clinic—two treatments may not necessarily be better than one and may even cause harm. Thus, there is a need for rationally designed combination therapy. Here, Seung et al. conduct a phase 1 trial on one such rationally combined therapy—interleukin-2 (IL-2) and stereotactic body radiation therapy (SBRT). IL-2, an immune activator, has been long used in the clinic either as a single-agent immunotherapy or in combination with various drugs for melanoma and renal cell carcinoma, with limited success. Here, the authors combine high-dose IL-2 with targeted radiation therapy based on clinical observation of enhanced efficacy in patients as well as the still to be proven hypothesis that radiation damage induces tumor antigen release and microenvironment changes that should enhance the immune-activating effects of IL-2. They found that the combination therapy was safe, and, albeit in a small number of patients, appeared to have improved efficacy over IL-2 alone. Intriguingly, they found a greater frequency of proliferating early effector memory T cells in the peripheral blood of these patients. Although studies with more patients and more detailed mechanistic follow-up must be performed, this study suggests that the rational combination of SBRT and IL-2 may improve upon current therapies for metastatic melanoma and renal cell carcinoma. Preclinical models suggest that focal high-dose radiation can make tumors more immunogenic. We performed a pilot study of stereotactic body radiation therapy (SBRT) followed by high-dose interleukin-2 (IL-2) to assess safety and tumor response rate and perform exploratory immune monitoring studies. Patients with metastatic melanoma or renal cell carcinoma (RCC) who had received no previous medical therapy for metastatic disease were eligible. Patients received one, two, or three doses of SBRT (20 Gy per fraction) with the last dose administered 3 days before starting IL-2. IL-2 (600,000 IU per kilogram by means of intravenous bolus infusion) was given every 8 hours for a maximum of 14 doses with a second cycle after a 2-week rest. Patients with regressing disease received up to six IL-2 cycles. Twelve patients were included in the intent-to-treat analysis, and 11 completed treatment per the study design. Response Evaluation Criteria in Solid Tumors criteria were used to assess overall response in nonirradiated target lesions. Eight of 12 patients (66.6%) achieved a complete (CR) or partial response (PR) (1 CR and 7 PR). Six of the patients with PR on computed tomography had a CR by positron emission tomography imaging. Five of seven (71.4%) patients with melanoma had a PR or CR, and three of five (60%) with RCC had a PR. Immune monitoring showed a statistically significantly greater frequency of proliferating CD4+ T cells with an early activated effector memory phenotype (CD3+CD4+Ki67+CD25+FoxP3−CCR7−CD45RA−CD27+CD28+/−) in the peripheral blood of responding patients. SBRT and IL-2 can be administered safely. Because the response rate in patients with melanoma was significantly higher than expected on the basis of historical data, we believe that the combination and investigation of CD4+ effector memory T cells as a predictor of response warrant further study.

Adjuvant Immunization of HLA-A2-Positive Melanoma Patients With a Modified gp100 Peptide Induces Peptide-Specific CD8+ T-Cell Responses

PURPOSE To measure the CD8+ T-cell response to a melanoma peptide vaccine and to compare an every-2-weeks with an every-3-weeks vaccination schedule. PATIENTS AND METHODS Thirty HLA-A2-positive patients with resected stage I to III melanoma were randomly assigned to receive vaccinations every 2 weeks (13 vaccines) or every 3 weeks (nine vaccines) for 6 months. The synthetic, modified gp100 peptide, g209-2M, and a control peptide, HPV16 E7, were mixed in incomplete Freunds adjuvant and injected subcutaneously. Peripheral blood mononuclear cells obtained before and after vaccination by leukapheresis were analyzed using a fluorescence-based HLA/peptide-tetramer binding assay and cytokine flow cytometry. RESULTS Vaccination induced an increase in peptide-specific T cells in 28 of 29 patients. The median frequency of CD8+ T cells specific for the g209-2M peptide increased markedly from 0.02% before to 0.34% after vaccination (P <.0001). Eight patients (28%) exhibited peptide-specific CD8+ T-cell frequencies greater than 1%, including two patients with frequencies of 4.96% and 8.86%, respectively. Interferon alfa-2b-treated patients also had significant increases in tetramer-binding cells (P <.0001). No difference was observed between the every-2-weeks and the every-3-weeks vaccination schedules (P =.59). CONCLUSION Flow cytometric analysis of HLA/peptide-tetramer binding cells was a reliable means of quantifying the CD8+ T-cell response to peptide immunization. This assay may be suitable for use in future trials to optimize different vaccination strategies. Concurrent interferon treatment did not inhibit the development of a peptide-specific immune response and vaccination every 2 weeks, and every 3 weeks produced similar results.

Signaling Through OX40 Enhances Antitumor Immunity

The existence of tumor-specific T cells, as well as their ability to be primed in cancer patients, confirms that the immune response can be deployed to combat cancer. However, there are obstacles that must be overcome to convert the ineffective immune response commonly found in the tumor environment to one that leads to sustained destruction of tumor. Members of the tumor necrosis factor (TNF) superfamily direct diverse immune functions. OX40 and its ligand, OX40L, are key TNF members that augment T-cell expansion, cytokine production, and survival. OX40 signaling also controls regulatory T-cell differentiation and suppressive function. Studies over the past decade have demonstrated that OX40 agonists enhance antitumor immunity in preclinical models using immunogenic tumors; however, treatment of poorly immunogenic tumors has been less successful. Combining strategies that prime tumor-specific T cells together with OX40 signaling could generate and maintain a therapeutic antitumor immune response.

Vaccination of Women with Metastatic Breast Cancer, Using a Costimulatory Gene (CD80)-Modified, HLA-A2-Matched, Allogeneic, Breast Cancer Cell Line: Clinical and Immunological Results

MDA-MB-231, an HLA-A2(+), HER2/neu(+) allogeneic breast cancer cell line genetically modified to express the costimulatory molecule CD80 (B7-1), was used to vaccinate 30 women with previously treated stage IV breast cancer. Expression of CD80 conferred the ability to deliver a costimulatory signal and thereby improved the antigen presentation capability of the tumor cells to patient T cells in vitro. Patients were vaccinated with 10(7) or 10(8) irradiated gene-modified tumor cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) or BCG, three times at 2-week intervals and then monthly until progressive disease developed. GM-CSF-related flulike symptoms and minor injection site reactions were observed frequently. Prolonged disease stabilization was observed in four patients but no objective tumor regressions were seen. Immune responses were measured in matched peripheral blood samples collected before and after treatment from 9 of 15 patients treated at the 10(8) tumor cell dose. Four patients exhibited MHC class I-restricted cytokine production in response to the parental breast cancer cell line. One patient maintained an increased number of circulating tumor-specific, interferon gamma-secreting CD8(+) T cells for 24 months after the last vaccination. One patient exhibited a tumor-specific interleukin 5 response to an autologous tumor cell line. This immunization strategy proved to be safe and feasible, and induced tumor-specific immune responses in a minority of patients; however, no objective tumor regressions were observed.

gp100 209 -2M Peptide Immunization of Human Lymphocyte Antigen- A2 Stage I-III Melanoma Patients Induces Significant Increase in Antigen-Specific Effector and Long-Term Memory CD8 T Cells

Thirty-five HLA-A2+ patients with completely resected stage I-III melanoma were vaccinated multiple times over 6 months with a modified melanoma peptide, gp100209–2M, emulsified in Montanide adjuvant. Direct ex vivo gp100209–2M tetramer analysis of pre- and postvaccine peripheral blood mononuclear cells (PBMCs) demonstrated significant increases in the frequency of tetramer+ CD8+ T cells after immunization for 33 of 35 evaluable patients (median, 0.36%; range, 0.05–8.9%). Ex vivo IFN-γ cytokine flow cytometry analysis of postvaccine PBMCs after brief gp100209–2M in vitro activation showed that for all of the patients studied tetramer+ CD8+ T cells produced IFN-γ; however, some patients had significant numbers of tetramer+ IFN-γ− CD8+T cells suggesting functional anergy. Additionally, 8 day gp100209–2M in vitro stimulation (IVS) of pre- and postvaccine PBMCs resulted in significant expansion of tetramer+ CD8+ T cells from postvaccine cells for 34 patients, and these IVS tetramer+ CD8+ T cells were functionally responsive by IFN-γ cytokine flow cytometry analysis after restimulation with either native or modified gp100 peptide. However, correlated functional and phenotype analysis of IVS-expanded postvaccine CD8+ T cells demonstrated the proliferation of functionally anergic gp100209–2M- tetramer+ CD8+ T cells in several patients and also indicated interpatient variability of gp100209–2M stimulated T-cell proliferation. Flow cytometry analysis of cryopreserved postvaccine PBMCs from representative patients showed that the majority of tetramer+ CD8+ T cells (78.1 ± 4.2%) had either an “effector” (CD45 RA+/CCR7−) or an “effector-memory” phenotype (CD45RA−/CCR7−). Notably, analysis of PBMCs collected 12–24 months after vaccine therapy demonstrated the durable presence of gp100209–2M-specific memory CD8+ T cells with high proliferation potential. Overall, this report demonstrates that after vaccination with a MHC class I-restricted melanoma peptide, resected nonmetastatic melanoma patients can mount a significant antigen-specific CD8+ T-cell immune response with a functionally intact memory component. The data further support the combined use of tetramer binding and functional assays in correlated ex vivo and IVS settings as a standard for immunomonitoring of cancer vaccine patients.

Combined IL-21 and Low-Dose IL-2 therapy induces anti-tumor immunity and long-term curative effects in a murine melanoma tumor model

BackgroundIn vivo studies have recently demonstrated that interleukin 21 (IL-21) enhances the anti-tumor function of T-cells and NK cells in murine tumor models, and the combined use of IL-21 and IL-15 has resulted in prolonged tumor regression and survival in mice with previously established tumors. However, the combined anti-tumor effects of IL-21 and low dose IL-2 have not been studied even though IL-2 has been approved for human use, and, at low dose administration, stimulates the proliferation of memory T cells, and does not significantly increase antigen-induced apoptosis or regulatory T cell (Treg) expansion. This study examined whether recombinant IL-21 alone or in combination with low-dose IL-2 could improve the in vivo anti-tumor function of naïve, tumor-antigen specific CD8+ T cells in a gp10025–33 T cell receptor transgenic pmel murine melanoma model.MethodsCongenic C57BL/6 (Ly5.2) mice bearing subcutaneous B16F10 melanoma tumors were sublethally irradiated to induce lymphopenia. After irradiation naive pmel splenocytes were adoptively transferred, and mice were immunized with bone marrow-derived dendritic cells pulsed with human gp10025–33 (hgp10025–33). Seven days after vaccination groups of mice received 5 consecutive days of intraperitoneal administration of IL-2 alone (20 × 103 IU), IL-21 alone (20 μg) or IL-21 and IL-2. Control animals received no cytokine therapy.ResultsIL-21 alone and IL-2 alone both delayed tumor progression, but only IL-21 significantly augmented long-term survival (20%) compared to the control group. However, combination therapy with IL-21 and IL-2 resulted in the highest long-term (>150 days) tumor-free survival frequency of 46%. Animals that were tumor-free for > 150 days demonstrated tumor-specific protection after rechallenge with B16F10 melanoma cells. At peak expansion (21 days post vaccination), the combination of IL-21 plus IL-2 resulted in a 2- to 3-fold higher absolute number of circulating tumor antigen-specific pmel CD8+ T cells than was stimulated by IL-2 or IL-21 alone. Pmel CD8+ T cells were predominantly partitioned into central memory (CD62L+/CD127+) or effector-memory (CD62L-/CD127+) phenotypes by day 28-post vaccination in IL-21 + IL-2 treated mice.ConclusionThese observations support the potential use of IL-21 and low-dose IL-2 therapy in combination with a tumor-antigen vaccine and lymphopenic conditioning in future cancer clinical trials to maintain high numbers of anti-tumor memory CD8+ T cells with the potential to sustain long term tumor regression and survival.

Short-term dietary administration of celecoxib enhances the efficacy of tumor lysate-pulsed dendritic cell vaccines in treating murine breast cancer.

Cyclooxygenase‐2 (COX‐2) is a rate‐limiting enzyme in the synthesis of prostaglandins. It is over‐expressed in multiple cancers and has been associated with diminished tumor immunity. Dendritic cells (DCs) are considered candidates for cancer immunotherapy due to their ability to process and present antigens to T cells and stimulate immune responses. However, DC‐based vaccines have exhibited minimal effectiveness against established tumors. In this study, we evaluated the effect of short‐term administration of the selective COX‐2 inhibitor celecoxib on the efficacy of DC‐based vaccines in preventing and treating established 4T1 murine mammary tumors. We show that dietary celecoxib alone significantly suppresses the growth of primary tumors and the incidence of lung metastases in the prophylactic setting but is less effective against pre‐established tumors. However, we demonstrate that celecoxib administered after primary tumor establishment synergizes with tumor lysate‐pulsed DC and the adjuvant, GM‐CSF, to improve the antitumor immune response by suppressing primary tumor growth and markedly reducing the occurrence of lung metastases. This triple combination therapy elicits a tumor‐specific immune response evidenced by elevated IFN‐γ and IL‐4 secretion by CD4+ T cells and results in increased infiltration of CD4+ and CD8+ T cells to the tumor site. In addition, dietary celecoxib inhibits angiogenesis evidenced by decreased vascular proliferation within the tumor and serum vascular endothelial growth factor levels. These studies suggest that short‐term celecoxib therapy in combination with DC vaccines may be safely used for treating metastatic breast cancer.

Polychromatic flow cytometry: A rapid method for the reduction and analysis of complex multiparameter data

Recent advances in flow cytometry have resulted in the development of reliable techniques for performing polychromatic (5–17 color) flow cytometry analysis. However, the data reduction and analysis involved in the resolution of hundreds of possible cellular subphenotypes identified, using a single polychromatic flow cytometry staining panel, presents a major obstacle to the successful application of this technology.

MONITORING IMMUNE RESPONSES IN CANCER PATIENTS RECEIVING TUMOR VACCINES

Clinical evaluation of therapeutic tumor vaccines has resulted in examination and comparison of the types of immune function assays required to monitor tumor antigen-stimulated T cell effector function in immunized patients. Three of the most commonly used assays include ELISPOT, tetramer assay, and cytokine flow cytometry (CFC). Discussed are the method and principles for each assay and an assessment of important methodological, reagent, and data acquisition issues that are relevant for the accurate and effective use of the assays. The sensitivity and utility of the assays and present arguments advocating their integrated use in future immunomonitoring studies are also discussed.