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Evaluation of myeloid-derived suppressor cells (MDSC), a cell type implicated in T-cell suppression, may inform immune status. However, a uniform methodology is necessary for prospective testing as a biomarker. We report the use of a computational algorithm-driven analysis of whole blood and cryopreserved samples for monocytic MDSC (m-MDSC) quantity that removes variables related to blood processing and user definitions. Applying these methods to samples from patients with melanoma identifies differing frequency distribution of m-MDSC relative to that in healthy donors. Patients with a pretreatment m-MDSC frequency outside a preliminary definition of healthy donor range (

The abscopal effect is a phenomenon in which local radiotherapy is associated with the regression of metastatic cancer at a distance from the irradiated site. The abscopal effect may be mediated by activation of the immune system. Ipilimumab is a mono‑ clonal antibody that inhibits an immunologic checkpoint on T cells, cytotoxic T‑lymphocyte–associated antigen 4 (CTLA ‑ 4). We report a case of the abscopal effect in a patient with melanoma treated with ipilimumab and radiotherapy. Temporal associations were noted: tumor shrinkage with antibody responses to the cancer– testis antigen NY‑ ESO‑ 1, changes in peripheral‑ blood immune cells, and increases in antibody responses to other antigens after radiotherapy. (Funded by the National Institutes of Health and others.)

Ipilimumab, a monoclonal antibody against cytotoxic T lymphocyte antigen 4 (CTLA-4), has been shown to improve survival in patients with advanced metastatic melanoma. It also enhances immunity to NY-ESO-1, a cancer/testis antigen expressed in a subset of patients with melanoma. To characterize the association between immune response and clinical outcome, we first analyzed NY-ESO-1 serum antibody by ELISA in 144 ipilimumab-treated patients with melanoma and found 22 of 140 (16%) seropositive at baseline and 31 of 144 (22%) seropositive following treatment. These NY-ESO-1–seropositive patients had a greater likelihood of experiencing clinical benefit 24 wk after ipilimumab treatment than NY-ESO-1–seronegative patients ( P = 0.02, relative risk = 1.8, two-tailed Fisher test). To understand why some patients with NY-ESO-1 antibody failed to experience clinical benefit, we analyzed NY-ESO-1–specific CD4 + and CD8 + T-cell responses by intracellular multicytokine staining in 20 NY-ESO-1–seropositive patients and found a surprising dissociation between NY-ESO-1 antibody and CD8 responses in some patients. NY-ESO-1–seropositive patients with associated CD8 + T cells experienced more frequent clinical benefit (10 of 13; 77%) than those with undetectable CD8 + T-cell response (one of seven; 14%; P = 0.02; relative risk = 5.4, two-tailed Fisher test), as well as a significant survival advantage ( P = 0.01; hazard ratio = 0.2, time-dependent Cox model). Together, our data suggest that integrated NY-ESO-1 immune responses may have predictive value for ipilimumab treatment and argue for prospective studies in patients with established NY-ESO-1 immunity. The current findings provide a strong rationale for the clinical use of modulators of immunosuppression with concurrent approaches to favor tumor antigen-specific immune responses, such as vaccines or adoptive transfer, in patients with cancer.

Purpose: Prior studies show that i.m. injection of xenogeneic orthologues of melanosomal antigens (tyrosinase, gp100) induces CD8+ T-cell responses to the syngeneic protein. To further define the optimal vaccination strategy, we conducted a pilot clinical trial comparing i.m. injection with particle-mediated epidermal delivery (PMED).Experimental Design: Human leukocyte antigen (HLA)-A*0201+ disease–free melanoma patients were randomized to the PMED or i.m. arm, receiving eight vaccinations over 4 months. Patients received 4 μg or 2,000 μg per injection, respectively, of mouse gp100 DNA. Peripheral blood mononuclear cells were collected, cultured with gp100 peptides, and analyzed by tetramer and intracellular cytokine staining for responses to HLA-A*0201–restricted gp100 epitopes [gp100209-217 (ITDQVPFSV) and gp100280-288 (YLEPGPVTA)].Results: Twenty-seven patients with stage IIB-IV melanoma were analyzable for immune response. The only common toxicity was grade 1 injection site reaction in nine patients with no intergroup difference, and one dose-limiting toxicity of acute hypersensitivity occurred in a PMED patient with undiagnosed gold allergy. Four of 27 patients produced gp100 tetramer+CD8+ T cells, all carrying the CCR7loCD45RAlo effector-memory phenotype. Five of 27 patients generated IFN-γ+CD8+ T cells, one who was also tetramer-positive. Overall, vaccination induced a response in 30% of patients, which was not significantly associated with study arm or clinical outcome. However, the PMED group showed a trend toward increased IFN-γ+CD8+ T-cell generation (P = 0.07).Conclusion: A comparable efficacy and safety profile was shown between the i.m. and PMED arms, despite a significantly decreased dose of DNA used for PMED injection. Clin Cancer Res; 16(15); 4057–65. ©2010 AACR.

5553Background: WT1 is highly expressed in serous ovarian cancer. GPS is WT1-targeting heteroclitic peptide tetravalent non-HLA-restricted vaccine, and has shown promising phase 2 activity in AML, ...

Blockade of inhibitory signals mediated by cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) has been shown to enhance T cell responses and induce durable clinical responses in patients with metastatic melanoma. The functional impact of anti-CTLA-4 therapy on human immune responses is still unclear. To explore this, we analyzed immune-related adverse events and immune responses in metastatic melanoma patients treated with ipilimumab, a fully human anti-CTLA-4 monoclonal antibody. Fifteen patients were selected on the basis of availability of suitable specimens for immunologic monitoring, and eight of these showed evidence of clinical benefit. Five of the eight patients with evidence of clinical benefit had NY-ESO-1 antibody, whereas none of seven clinical non-responders was seropositive for NY-ESO-1. All five NY-ESO-1 seropositive patients had clearly detectable CD4+and CD8+T cells against NY-ESO-1 following treatment with ipilimumab. One NY-ESO-1 seronegative clinical responder also had a NY-ESO-1 CD4+and CD8+T cell response, possibly related to prior vaccination with NY-ESO-1. Among five clinical non-responders analyzed, only one had a NY-ESO-1 CD4+T cell response and this patient did not have detectable anti-NY-ESO-1 antibody. Overall, NY-ESO-1-specific T cell responses increased in frequency and functionality during anti-CTLA-4 treatment, revealing a polyfunctional response pattern of IFN-γ, MIP-1β and TNF-α. We therefore suggest that CTLA-4 blockade enhanced NY-ESO-1 antigen-specific B cell and T cell immune responses in patients with durable objective clinical responses and stable disease. These data provide an immunologic rationale for the efficacy of anti-CTLA-4 therapy and call for immunotherapeutic designs that combine NY-ESO-1 vaccination with CTLA-4 blockade.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances immune responses by inducing proliferation, maturation, and migration of dendritic cells (DCs) as well as expansion and differentiation of B and T lymphocytes. The potency of DNA vaccines can be enhanced by the addition of DNA encoding cytokines, acting as molecular adjuvants. We conducted a phase I/II trial of human GM-CSF DNA in conjunction with a multipeptide vaccine (gp100 and tyrosinase) in stage III/IV melanoma patients. Nineteen human leukocyte antigen (HLA)-A*0201+ patients were treated. Three dose levels were studied: 100, 400, and 800 microg DNA/injection, administered subcutaneously every month with 500 microg of each peptide. In the dose-ranging study, three patients were treated at each dose level. The remaining patients were then treated at the highest dose. Most toxicities were grade 1 injection-site reactions. Eight patients (42%) developed CD8+ T-cell responses, defined by aor =3 SD increase in baseline reactivity to tyrosinase or gp100 peptide in tetramer or intracellular cytokine staining (ICS) assays. There was no relationship between dose and T-cell response. Responding T cells had an effector memory cell phenotype. Polyfunctional T cells were also demonstrated. At a median of 31 months follow-up, median survival has not been reached. Human GM-CSF DNA was found to be a safe adjuvant.

Immunity to self antigens on cancer is constrained by tolerance/ignorance. DNA vaccines encoding xenogeneic differentiation antigens, such as tyrosinase (TYR), mediate tumor protection and regression in implantable mouse models, and dogs with spontaneous melanoma. We conducted a trial of mouse and human TYR DNA vaccines in stage III/IV melanoma patients. Eighteen human leukocyte antigen (HLA)-A*0201(+) melanoma patients were randomized as follows: one group received three mouse TYR DNA injections followed by three human TYR DNA injections; the other group received the same vaccines in opposite sequence. The study was conducted at three dose levels: 100, 500, and 1,500 microg DNA/injection, administered intramuscularly (IM) every 3 weeks. Most toxicities were grade 1 injection site reactions. Seven patients developed CD8(+) T-cell responses, defined by a3 SD increase in baseline reactivity to TYR peptide in tetramer or intracellular cytokine staining (ICS) assays. There was found to be no relationship between dose, assigned schedule, and T-cell response. At a median of 42 months follow-up, median survival has not been reached. Mouse and human TYR DNA vaccines were found safe and induced CD8(+) T-cell responses in 7 of 18 patients. T cells recognizing a native TYR peptide had a phenotype consistent with that of effector memory cells.

Delays in immune recovery after allogeneic hematopoietic stem cell transplantation (allo-HSCT) are associated with increased risks of infection and relapse. IL-7 has a central role in T-cell development and survival and enhances immune recovery in murine models of allo-HSCT. We performed a phase 1 trial of r-hIL-7 (CYT107) in recipients of T-cell depleted allo-HSCTs. Twelve patients were treated with escalating doses of r-hIL-7 administered weekly for 3 weeks. The study drug was well tolerated with only one patient developing acute skin GVHD. At baseline, patients were profoundly lymphopenic. CYT107 induced a doubling in CD4+ and CD8+ T cells. The main effect of IL-7 was an expansion of effector memory T cells, the predominant subset identified in our patients. There was no significant effect on CD4+CD25+FoxP3+ T cells, NK, or B cells. Importantly, we not only saw quantitative increases in T cells after a short course of IL-7 but also demonstrated an increase in functional T cells, including viral-specific T cells that recognize CMV. Enhanced TCR diversity was also observed after treatment. Our results indicate that r-hIL-7 can enhance immune recovery after a T cell–depleted allo-HSCT without causing significant GVHD or other serious toxicity (www.clinicaltrials.gov; NCT00684008).

Anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibodies, such as ipilimumab, have generated measurable immune responses to Melan-A, NY-ESO-1, and gp100 antigens in metastatic melanoma. Vaccination against such targets has potential for immunogenicity and may produce an effector-memory T-cell response.To determine the effect of CTLA-4 blockade on antigen-specific responses following vaccination, in-depth immune monitoring was performed on three ipilimumab-treated patients prevaccinated with gp100 DNA (IMF-24), gp100(209-217) and tyrosinase peptides plus GM-CSF DNA (IMF-32), or NY-ESO-1 protein plus imiquimod (IMF-11); peripheral blood mononuclear cells were analyzed by tetramer and/or intracellular cytokine staining following 10-day culture with HLA-A*0201-restricted gp100(209-217) (ITDQVPFSV), tyrosinase(369-377) (YMDGTMSQV), or 20-mer NY-ESO-1 overlapping peptides, respectively. Tumors from IMF-32 were analyzed by immunohistochemistry to help elucidate mechanism(s) underlying tumor escape.Following vaccination, patients generated weak to no CD4(+) or CD8(+) T-cell response specific to the vaccine antigen but demonstrated increases in effector-memory (CCR7(lo)CD45RA(lo)) tetramer(+)CD8(+) T cells. After ipilimumab induction, patients experienced a robust, although sometimes transient, antigen-specific response for gp100 (IMF-32 and IMF-24) or NY-ESO-1 (IMF-11) and produced polyfunctional intracellular cytokines. Primary and metastatic tumors expressed tyrosinase but not gp100 or class I/II MHC molecules.Vaccination induced a measurable antigen-specific T-cell response that increased following CTLA-4 blockade, potentially "boosting" the vaccine-primed response. Tumor escape may be related to antigen loss or lack of MHC expression necessary for immune activity. These results in a limited number of patients support the need for further research into combining vaccination with ipilimumab and provide insight into mechanisms underlying tumor escape.

Monitoring cellular immune responses is one prerequisite for the rational development of cancer vaccines.We describe an extensive effort to optimize and validate quantitatively an in vitro T-cell culture method by determining the phenotype and function of both CD4(+) and CD8(+) T cells, including measurement of the phenotype markers CCR7, CD45RA, CD28 and CD27 and the functional markers interferon (IFN)-gamma, interleukin (IL)-2, macrophage inflammatory protein (MIP)-1beta, tumor necrosis factor (TNF)-alpha and CD107a.Autologous peripheral blood mononuclear cells (PBMC) were potent stimulators that expanded antigen (Ag)-specific CD8(+) T cells during short-term culture with the addition of IL-2 and IL-15 cytokines. Polyfunctional Ag-specific CD4(+) and CD8(+) T cells were detectable using this method.Our culture system represents a robust human T-cell culture protocol that permits phenotypic, quantitative and qualitative evaluation of vaccine-induced CD4(+) and CD8(+) T-cell responses.

A differentiation antigen commonly expressed on melanoma cells, gp100 is the target of infiltrating T cells. We conducted a phase I randomized cross-over trial of melanoma patients with either xenogeneic (mouse) or human gp100 plasmid DNA injected intramuscularly at three dosages (100, 500 or 1,500 microg) every three weeks for three doses. After the first three injections, patients were then immunized three times with gp100 from the other species. Peripheral blood samples were analyzed at various time points following 10-day culture with gp100 peptides using multi-parametric flow cytometry. A total of 19 patients were enrolled, with 18 assessable for immune function and survival. 14 (74%) were male, with a median age of 56 years (range, 20-82). All patients had no evidence of disease; 10 (53%) had stage III disease, 3 each (16%) had stage IIB and IV disease, 2 (11%) had choroidal and 1 (5%) had anal mucosal involvement. With a median follow-up of 30 months, median progression-free survival (PFS) is 44 months. Median survival is not reached. There was no grade 3/4 toxicity; the most common grade 1/2 toxicity was an injection site reaction in 12 patients (63%, all grade 1). Five patients developed CD8+ cells binding gp100(280-288) HLA-A2-restricted tetramer. One patient had an increase in CD8+ IFN-gamma+ cells. This xenogeneic immunization strategy was safe and associated with minimal toxicity. There was also evidence of immune response.

Development of a practical and sensitive assay for evaluating immune responses against cancer Ag has been a challenge for immune monitoring of patients. We have established a reproducible method using peptide-pulsed K562-A*0201 cells as APC to expand Ag-specific T cells in vitro. This method may be applied for monitoring T-cell responses in cancer immunotherapy clinical trials.Autologous PBMC from HLA-A*0201+ healthy donors and patients with melanoma were stimulated with peptide-pulsed K562-A*0201 cells under varying conditions. We investigated (1) different culture conditions, including the requirements for serum and cytokines for expansion of CD8+ T lymphocytes; (2) a range of peptide concentrations for Ag loading; (3) phenotypic characterization of responding T cells; and (4) APC:responder ratios and their effects on T-cell expansion. We validated these conditions by ELISPOT and intracellular cytokine staining (ICS) assays using peptides from influenza, Epslein-Barr Virus (EBV) and tyrosinase.Conditions for optimal T-cell expansion using K562-A*0201 APC included input of 2 x 10(6) PBMC, a 10 microg/mL peptide concentration to pulse K562-A*0201 cells, a 1:30 APC:responder T-cell ratio and culture in 10% autologous plasma supplemented with IL-2 and IL-15. In these conditions, Ag-specific T cells expanded100-fold over a 10-day culture period (peak at day 12).This bulk culture method is simple and reliable for expanding human Ag-specific T cells using peptide-pulsed K562-A*0201 cells. This HLA-matched APC line can be adapted to other HLA haplotypes, and has advantages for monitoring clinical trials of immunotherapy with limited availability of autologous APC and PBMC from patients.

Abstract 674 Immune recovery is an important determinant in multiple outcomes following allogeneic hematopoietic stem cell transplant (allo-HSCT). Delays in B and T cell reconstitution are associated with an increased risk of infection, relapse and secondary malignancy. Strategies to enhance post-transplant T-cell reconstitution could therefore improve morbidity and mortality after allo-HSCT. The cytokine Interleukin-7 (IL-7) is a unique therapeutic candidate to promote immune reconstitution because it has a central role in T cell development and survival. Murine models of allo-HSCT have demonstrated that IL-7 can enhance thymopoiesis as well as promote peripheral T cell survival and expansion. Initial clinical trials performed with recombinant human IL-7 (rhIL-7) have demonstrated a dose-dependent expansion of CD4+ and CD8+ T cells with an acceptable toxicity profile in patients with solid tumors or HIV infection. Hence we are conducting a phase I trial of post-transplant administration of rhIL-7 (CYT107, Cytheris Inc) in recipients of a T cell depleted (TCD) allo-HSCT to determine the safety, toxicity and biological activity on T cell reconstitution. To date, 9 patients (AML=7, MDS=2), with a median age of 59.3 years (range 27–67 years) have been treated with escalating doses of rhIL-7 (3 at 10 mcg/kg, 6 at 20 mcg/kg) administered subcutaneously weekly for 3 weeks following TCD allo-HSCT from an HLA compatible donor. Accrual is ongoing in the final cohort (30 mcg/kg). Recombinant hIL-7 was started at a median of 96 days post allo-HSCT (range 61–244 days). Most patients experienced transient minor injection site reactions. One patient (20 mcg/kg) developed a biopsy proven hypersensitivity drug rash a week after the first injection and was removed from the study (evaluable for toxicity but not immune recovery endpoints). No other significant injection-related toxicities have occurred, and no patients have developed GVHD. No anti-IL-7 antibodies or neutralizing antibodies have developed following rhIL-7 injection. Two of 9 patients with high-risk AML have relapsed (4 and 9 months post rhIL-7), an incidence consistent with published data in patients undergoing allo-HSCT for AML in CR, irrespective of T-cell depletion. Eight patients remain alive with a median follow-up of 14.5 months post rhIL-7 administration. At baseline, the median T cell counts were 91/mm3 (range 5 – 219 /mm3), 43/mm3 (range 9 – 299 /mm3) and 0 (range 0 – 17 /mm3) for CD4+, CD8+ and CD45RA+ T cells, respectively. Preliminary assessment of the immunological effects of rhIL-7 in 8 evaluable patients has demonstrated an increase in CD4+ T cells exhibiting a naïve or central memory phenotype (69% median increase over baseline at day 21 – range 8% to 35-fold increase), and CD8+ T cells exhibiting a naïve or effector memory phenotype (94% median increase over baseline at day 28 – range 0 to 11-fold increase). There was no observed effect on the frequency of CD4+CD25+FoxP3+ T cells or CD19+ B cells. TCR excision circles (TREC) analysis performed on CD4+ and CD8+ subsets in the first 6 patients, using absolute quantification real-time PCR, demonstrated increases in TRECs in 5/6 patients indicating enhanced T cell production. Finally, all 3 CMV-seropositive patients developed CD8+ T cell CMV-specific responses detected by intracellular IFNγ production to overlapping CMV-pp65 pentadecapeptides peptide pools after administration of rhIL-7. In one patient, we also analyzed CMV-specific T-cell frequency using HLA-A*0201 restricted MHC-tetramers. The highest CMV-specific response levels were noted in this patient with a history of CMV viremia and low-level CMV-specific CD8+ T cells prior to rhIL-7 (5.3-fold increase to the A0201-restricted immunodominant NLV peptide by tetramer assay after rhIL-7). Our pre-clinical data and early clinical results suggest that administration of rhIL-7 in recipients of a TCD allo-HSCT has minimal toxicity and can enhance post-transplant immune recovery without causing GVHD. Disclosures: Perales: Cytheris: Research Funding. Croughs:Cytheris: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Morre:Cytheris: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. van den Brink:Cytheris: Research Funding.

3005 Background: T-cell and antibody responses to self antigens on cancer are usually constrained by immunologic tolerance and ignorance. We found that DNA vaccines encoding xenogeneic differentiation antigens, such as tyrosinase (TYR), can mediate tumor protection and regression in implantable mouse models and dogs with spontaneously arising melanoma. Based on this, we conducted a trial of DNA vaccines encoding mouse and human TYR in patients with AJCC stage III/IV melanoma. Methods: HLA-A*0201+ melanoma patients were randomized to 2 different schedules: one group received 3 injections of mouse TYR DNA followed by 3 injections of human TYR DNA while the other group received 3 injections of human TYR DNA followed by 3 injections with the mouse gene. The study was conducted a three different dose levels: 100, 500 and 1,500 mcg DNA/injection, administered IM every 3 weeks. A total of 18 patients were treated, 6 at each dose level being randomized to one of the two schedules. Anti-TYR antibodies and CD8+ T cells recognizing the native human tyrosinase369-377 (YMDGTMSQV) peptide were measured at fixed time points. T-cell responses were monitored with MHC tetramer and intracytoplasmic IFN-γ staining assays using 10-day in vitro stimulation. Multiparametric flow cytometry was performed to further define the phenotype of responding cells. Results: Most toxicities were transient grade I injection site reactions. Seven patients had CD8+ T cell responses, defined as a >3 standard deviation increase in baseline reactivity to the TYR peptide in either the tetramer or intracellular IFN-γ assay. There was no relationship between dose level or assigned schedule and occurrence of T-cell response. Phenotypic characterization of responding T cells showed that most were consistent with an effector memory phenotype including the expression of granzyme B and surface expression of CD107a. No antibody responses were observed. At a median of 42 months of follow-up, median survival has not been reached and 6/18 patients have died from melanoma (1 in the group of patients who had a T cell response and 5 in the non-responders). Conclusions: Mouse and human TYR DNA vaccines were safe and induced CD8+ T cell responses in 7/18 patients. T cells recognizing a native TYR peptide had a phenotype consistent with that of effector memory cells. No significant financial relationships to disclose.

Printed by RECOMBINANT HUMAN INTERLEUKIN-7 (CYT107) PROMOTES T CELL RECOVERY FOLLOWING T-CELL DEPLETED ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTION M-A Perales1, JD Goldberg1 , L Lechner 1 , G Koehne1, J Yuan1, EB Papadopoulos1 , JW Young1, AA Jakubowski1, B Zaidi1, H Gallardo1, R Kendle1, C Liu1, T Rasalan1 , Y Xu1, JD Wolchok1, T Croughs2, M Morre2, M Maloy1, G Heller1, MRM van den Brink1 1Memorial Sloan-Kettering Cancer Center (New York, US); 2Cytheris, Inc (Issy Les Moulineaux, FR)