Daniel Haley

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.

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.

Tumor-Derived Autophagosome Vaccine: Mechanism of Cross-Presentation and Therapeutic Efficacy

Purpose: We previously reported that autophagy in tumor cells plays a critical role in cross-presentation of tumor antigens and that autophagosomes are efficient antigen carriers for cross-priming of tumor-reactive CD8+ T cells. Here, we sought to characterize further the autophagosome-enriched vaccine named DRibble (DRiPs-containing blebs), which is derived from tumor cells after inhibition of protein degradation, and to provide insights into the mechanisms responsible for their efficacy as a novel cancer immunotherapy. Experimental Design: DRibbles were characterized by Western blot and light or transmission electron microscopy. The efficiency of cross-presentation mediated by DRibbles was first compared with that of whole-tumor cells and pure proteins. The mechanisms of antigen cross-presentation by DRibbles were analyzed, and the antitumor efficacy of the DRibble vaccine was tested in 3LL Lewis lung tumors and B16F10 melanoma. Results: The DRibbles sequester both long-lived and short-lived proteins, including defective ribosomal products (DRiP), and damage-associated molecular pattern molecules exemplified by HSP90, HSP94, calreticulin, and HMGB1. DRibbles express ligands for CLEC9A, a newly described C-type lectin receptor expressed by a subset of conventional and plasmacytoid dendritic cells (DC), and cross-presentation was partially CLEC9A dependent. Furthermore, this autophagy-assisted antigen cross-presentation pathway involved both caveolae- and clathrin-mediated endocytosis and endoplasmic reticulum–associated degradation machinery. It depends on proteasome and TAP1, but not lysosome functions of antigen-presenting cells. Importantly, DCs loaded with autophagosome-enriched DRibbles can eradicate 3LL Lewis lung tumors and significantly delay the growth of B16F10 melanoma. Conclusions: These data documented the unique characteristics and potent antitumor efficacy of the autophagosome-based DRibble vaccine. The efficacy of DRibble cancer vaccine will be further tested in clinical trials. Clin Cancer Res; 17(22); 7047–57. ©2011 AACR.

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.

Anti-OX40 stimulation in vivo enhances CD8+ memory T cell survival and significantly increases recall responses.

There is growing evidence that engagement of OX40 (CD134), a member of the TNF receptor superfamily, can directly stimulate antigen‐specific CD8+ T cells. It has been shown that CD8+ T cells express OX40 following activation, but the response of antigen‐specific CD8+ T cells to OX40 stimulation has not been fully characterized. We utilized an antigen‐specific transgenic CD8+ T cell model (OT‐I) to determine if OX40 engagement can boost the generation of antigen‐specific CD8+ T cell memory. Our results demonstrate that enhanced OX40 costimulation, via an agonist anti‐OX40 antibody, increases CD25 and phospho‐Akt expression on the antigen‐specific CD8+ T cells and significantly increases the generation of long‐lived antigen‐specific CD8+ memory T cells. The increased numbers of memory CD8+ T cells generated via anti‐OX40 treatment still required the presence of CD4+ T cells for their long‐term maintenance in vivo. In addition, anti‐OX40 costimulation greatly enhanced antigen‐specific CD8+ T cell recall responses. These data show that OX40 engagement in vivo increases the number of antigen‐specific CD8+ memory T cells surviving after antigen challenge and has implications for the development of more potent vaccines against pathogens and cancer.

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.

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.

Disruption of TGF-β Signaling Prevents the Generation of Tumor-Sensitized Regulatory T Cells and Facilitates Therapeutic Antitumor Immunity

Regulatory T (Treg) cells represent a major roadblock to the induction of antitumor immunity through vaccine approaches. TGF-β is a cytokine implicated in the generation and maintenance of Treg cells, as well as in their suppressive function. These experiments examined whether the generation of tumor-sensitized Treg cells was TGF-β dependent and evaluated whether TGF-β produced by Treg cells blocked the priming of tumor-specific T cells in vaccinated reconstituted lymphopenic mice. We show that tumor-sensitized Treg cells (CD25+/FoxP3+) obtained from tumor-bearing mice block the generation of tumor-specific T cells in reconstituted lymphopenic mice. Strikingly, this suppression is absent if tumor-sensitized Treg cells are acquired from tumor-bearing mice expressing the dominant-negative TGFβRII in T cells. This loss of suppression was a result of the crucial role of TGF-β in generating tumor-sensitized Treg cells, and not due to the insensitivity of naive or tumor-primed effector T cells to the direct suppressive influence of TGF-β. We conclude that blocking TGF-β in a tumor-bearing host can inhibit the induction of highly suppressive tumor-sensitized Treg cells. These data suggest that an integrative strategy combining “up-front” Treg cell ablation followed by vaccination and TGF-β blockade may limit generation of new tumor-sensitized Treg cells and improve the generation of therapeutic immune responses in patients with cancer.

Phenotype and Functional Characterization of Long-term gp100-Specific Memory CD8+ T Cells in Disease-Free Melanoma Patients Before and After Boosting Immunization

Purpose: Effective cancer vaccines must both drive a strong CTL response and sustain long-term memory T cells capable of rapid recall responses to tumor antigens. We sought to characterize the phenotype and function of gp100 peptide-specific memory CD8+ T cells in melanoma patients after primary gp100209-2M immunization and assess the anamnestic response to boosting immunization. Experimental Design: Eight-color flow cytometry analysis of gp100-specific CD8+ T cells was done on peripheral blood mononuclear cells collected shortly after the primary vaccine regimen, 12 to 24 months after primary vaccination, and after boosting immunization. The anamnestic response was assessed by comparing the frequency of circulating gp100-specific T cells before and after boosting. Gp100 peptide-induced in vitro functional avidity and proliferation responses and melanoma-stimulated T-cell CD107 mobilization were compared for cells from all three time points for multiple patients. Results: The frequency of circulating gp100-specific memory CD8+ T cells was comparable with cytomegalovirus-specific and FLU-specific T cells in the same patients, and the cells exhibited anamnestic proliferation after boosting. Their phenotypes were not unique, and individual patients exhibited one of two distinct phenotype signatures that were homologous to either cytomegalovirus-specific or FLU-specific memory T cells. Gp100-specific memory T cells showed some properties of competent memory T cells, such as heightened in vitro peptide-stimulated proliferation and increase in central memory (TCM) differentiation when compared with T-cell responses measured after the primary vaccine regimen. However, they did not acquire enhanced functional avidity usually associated with competent memory T-cell maturation. Conclusions: Although vaccination with class I–restricted melanoma peptides alone can break tolerance to self-tumor antigens, it did not induce fully competent memory CD8+ T cells—even in disease-free patients. Data presented suggest other vaccine strategies will be required to induce functionally robust long-term memory T cells.

Depletion of tumor-induced Treg prior to reconstitution rescues enhanced priming of tumor-specific, therapeutic effector T cells in lymphopenic hosts.

We reported previously that vaccination of reconstituted, lymphopenic mice resulted in a higher frequency of tumor‐specific effector T cells with therapeutic activity than vaccination of normal mice. Here, we show that lymphopenic mice reconstituted with spleen cells from tumor‐bearing mice (TBM), a situation that resembles the clinical condition, failed to generate tumor‐specific T cells with therapeutic efficacy. However, depletion of CD25+ Treg from the spleen cells of TBM restored tumor‐specific priming and therapeutic efficacy. Adding back TBM CD25+ Treg to CD25− naïve and TBM donor T cells prior to reconstitution confirmed their suppressive role. CD25+ Treg from TBM prevented priming of tumor‐specific T cells since subsequent depletion of CD4+ T cells did not restore therapeutic efficacy. This effect may not be antigen‐specific as three histologically distinct tumors generated CD25+ Treg that could suppress the T‐cell immune response to a melanoma vaccine. Importantly, since ex vivo depletion of CD25+ Treg from TBM spleen cells prior to reconstitution and vaccination fully restored the generation of therapeutic effector T cells, even in animals with established tumor burden, we have initiated a translational clinical trial of this strategy in patients with metastatic melanoma.