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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.

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.

Donor T cells have been shown to be reactive against and effective in adoptive immunotherapy of Epstein-Barr virus (EBV) lymphomas which develop in some leukemia patients post marrow transplantation. These T cells may be genetically modified by incorporation of a replication-incompetent viral vector (NIT) encoding both an inactive mutant nerve growth factor receptor (LNGFR), as an immunoselectable surface marker, and a herpes simplex virus thymidine kinase (HSV-TK), rendering the cells sensitive to ganciclovir. The current studies are based on the selective HSV-TK-catalyzed trapping (phosphorylation) of the thymidine analog [131I]-2′-fluoro-2′-deoxy-1-β-D-arabinofuransyl-5-iodo-uracil (FIAU) as a means of stably labeling such T cells for in vivo trafficking (including tumor targeting) studies. Because of the radiosensitivity of lymphocytes and the potentially high absorbed dose to the nucleus from intracellular 131I (even at tracer levels), the nucleus absorbed dose (D n ) and dose-dependent immune functionality were evaluated for NIT+ T cells labeled ex vivo in [131I]FIAU-containing medium. Based on in vitro kinetic studies of [131I]FIAU uptake by NIT+ T cells, D n was calculated using an adaptation of the MIRD formalism and the recently published MIRD cellular S factors. Immune cytotoxicity of [131I]FIAU-labeled cells was assayed against 51Cr-labeled target cells [B-lymphoblastoid cells (BLCLs)] in a standard 4-h release assay. At median nuclear absorbed doses up to 830 cGy, a 51Cr-release assay against BLCLs showed no loss of immune cytotoxicity, thus demonstrating the functional integrity of genetically transduced, tumor-reactive T cells labeled at this dose level for in vivo cell trafficking and tumor targeting studies.

Infusions of donor peripheral blood T cells can induce durable remissions of Epstein-Barr virus (EBV) lymphomas complicating marrow grafts, but they contain alloreactive T cells capable of inducing graft-versus-host disease. EBV-specific T-cell lines or clones avoid this problem but require 30 to 40 days of culture to establish. To accelerate the generation of EBV-specific T cells, we tested whether retroviral vectors, which only integrate in dividing cells, could be used to transduce and select antigen-reactive T cells early after sensitization to autologous EBV-transformed B cells. T cells were transduced with a dicistronic retroviral vector, NIT, which encodes low-affinity nerve growth factor receptor as an immunoselectable marker and herpes simplex virus thymidine kinase as a suicide gene, at different time points after sensitization. EBV-specific cytotoxic T lymphocyte precursor (CTLp) frequencies in purified NIT+T-cell fractions transduced on day 8 of culture were comparable to those of EBV-specific T-cell lines cultured for 30 days or more. Alloreactive CTLp frequencies were markedly reduced in the NIT+ fraction relative to the untransduced T-cell population. NIT+ fractions transduced on day 8 possessed more CD4+ T cells than the cell lines at day 30 and exhibited the same selective pattern of reactivity against immunodominant antigens presented by specific HLA alleles. In contrast, T cells transduced with NIT 5 days after stimulation with mitogen and interleukin-2 were relatively depleted of T cells specific for autologous EBV-transformed cells. Thus, retroviral vectors may be used for rapid selection of viral antigen-reactive T cells depleted of alloreactive T cells.

Recombinant retroviruses are efficient vectors for introducing genes into many mammalian cell types. They are useful in the context of clinical as well as experimental applications, owing to the ability to generate high-titer and helper-free viral stocks. Retroviral vectors are especially appropriate for the transduction of primary lymphocytes, because gene transfer is stable and mediated by nonimmunogenic vectors. Stable integration in chromosomes of cells undergoing clonal expansion ensures that the foreign genetic material will be faithfully transmitted to the cells' progeny. However, oncoretroviral vectors derived from murine leukemia viruses (MLV) require target cell division to integrate. Here we review factors that determine retroviral-mediated gene transfer efficiency in primary T-lymphocytes, in particular, T-cell activation status, viral receptor expression, and culture conditions.

It is essential for the improvement of adoptive cell therapies to generate efficiently large populations of human primary T cells that reliably express a suicide gene conferring drug sensitivity, such as herpes simplex virus thymidine kinase (HSVtk). We show here that an optimized dicistronic vector containing the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) is functional in human primary. T lymphocytes that bear on average one integrated vector copy per cell. We demonstrate reliable coexpression of the marker NTP, an inactive mutant of the human low-affinity nerve growth factor receptor and HSVtk. In the dicistronic vector NIT, NTP is expressed as a cap-dependent marker and HSVtk as a nonselectable IRES-dependent gene. Cell-surface expression of NTP is sufficient to allow for the efficient and rapid enrichment of the transduced cells to high purity. Of these purified lymphocytes, 97 +/- 4% and 92 +/- 6% are selectively eliminated when cultured in the presence of 1.0 or 0.1 microM ganciclovic respectively, establishing that the EMCV IRES ensures efficient and sufficient expression of two genes in human primary T cells.

Dendritic cells are attractive candidates for vaccine-based immunotherapy because of their potential to function as natural adjuvants for poorly immunogenic proteins derived from tumors or microbes. In this study, we evaluated the feasibility and consequences of introducing foreign genetic material by retroviral vectors into dendritic cell progenitors. Proliferating human bone marrow and cord blood CD34+ cells were infected by retroviral vectors encoding the murine CD2 surface antigen. Mean transduction efficiency in dendritic cells was 11.5% from bone marrow and 21.2% from cord blood progenitors. Transduced or untransduced dendritic cell progeny expressed comparable levels of HLA-DR, CD83, CD1a, CD80, CD86, S100, and p55 antigens. Granulocytes, macrophages, and dendritic cells were equally represented among the transduced and mock-transduced cells, thus showing no apparent alteration in the differentiation of transduced CD34+ precursors. The T-cell stimulatory capacity of retrovirally modified and purified mCD2-positive allogeneic or nominal antigen-pulsed autologous dendritic cells was comparable with that of untransduced dendritic cells. Human CD34+ dendritic cell progenitors can therefore be efficiently transduced using retroviral vectors and can differentiate into potent immunostimulatory dendritic cells without compromising their T-cell stimulatory capacity or the expression of critical costimulatory molecules and phenotypic markers. These results support ongoing efforts to develop genetically modified dendritic cells for immunotherapy.

It is essential for the study of T-cell function and the improvement of adoptive cell therapies to efficiently generate large populations of human primary T cells that reliably express foreign genes. This goal is achieved by using recombinant retroviruses pseudotyped with either the gibbon ape leukemia virus (GaLV) envelope or the vesicular stomatitis virus G (VSV-G) glycoprotein. We show here that both retroviral particles mediate stable gene transfer in CD4+ and in CD8+ peripheral blood lymphocytes cultured under optimized conditions. However, VSV-G–pseudotyped virions may cause transduction artifacts that must be carefully excluded. The VSV-G virions require 10- to 100-fold higher concentrations of infectious particles to achieve levels of gene transfer comparable to GaLV-virions. Nonetheless, the physical stability of VSV-G–coated particles enables the concentration of viral stocks to 109 infectious particles per milliliter or more.

RNA decoys are oligonucleotides corresponding to the TAR and RRE sequences of HIV which inhibit the HIV-encoded regulatory proteins Tat and Rev, respectively. Adeno-associated viral vectors encoding RNA decoys stably transduced into the human T-cell line CEM-SS expressed transactivating region (TAR) and Rev-responsive element (RRE) RNA decoys from tRNA pol III promoters at high levels, without any apparent deleterious effects on cell growth or expression of CD4. DNA blot analysis indicated that RNA decoy-encoding vectors were not rearranged and were integrated into the genomic DNA of selected cell lines. Vector DNA with the appropriate TAR and RRE sequences was isolated from transduced cell lines after prolonged growth in culture, further confirming that the vector DNA was present in a stable form through multiple cell cycles. Cell lines expressing TAR and RRE decoys transiently inhibited HIV gene expression and replication by 70–99% as determined by measurement of intracellular and extracellular HIV p24 production. Adeno-associated vectors encoding RNA decoys may be useful for gene therapy of HIV infection.

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).

Intracellular immunization is an anti-viral gene therapy strategy based on the introduction of DNA templates into cells to stably express genetic elements which inhibit viral gene expression and replication. We have recently developed an intracellular immunization strategy for human immunodeficiency virus (HIV) infection that uses RNA decoys. RNA decoys are short RNA oligonucleotides corresponding to the HIV trans activation response element (TAR) or Rev response element (RRE) sequences, which function by inhibiting the binding of the HIV regulatory proteins Tat and Rev to the authentic HIV RNA TAR and RRE regions, respectively. In this report we describe the characterization of potent RRE decoys containing the minimal 13-nucleotide primary Rev binding domain of the RRE. Using an improved tRNA cassette to express high levels of RRE transcripts in CEM cells, we found that this new generation of minimal RRE decoys were more potent inhibitors of HIV in isolated cell lines than previously described TAR or RRE decoys. CEM cells expressing RRE decoys exhibited diminished Rev function in cotransfection assays, confirming the specificity of inhibition of HIV by RRE decoys and indicating that the 13-nucleotide minimal Rev binding domain defined by using in vitro binding studies also binds Rev in vivo. Significant differences in the degree of HIV inhibition between individual CEM cell lines transduced with RRE decoy vectors which were not due to sequence alterations in the tRNA-RRE DNA template, differences in RRE decoy expression level, or endogenous variations in the resistance of CEM clonal cell lines to HIV were observed. In order to evaluate the efficacy of RRE decoys in a more realistic fashion than by comparison of individual clonal cell lines, polyclonal populations of transduced CEM cells were infected with HIV. By using a novel flow cytometric method for quantitating intracellular p24 expression, one version of the RRE decoys tested in this study was found to be capable of durably protecting polyclonal populations of CEM cells from HIV.

Identifying the spectrum of genetic alterations that cooperate with critical oncogenes to promote transformation provides a foundation for understanding the diversity of clinical phenotypes observed in human cancers. Here, we performed integrated analyses to identify genomic alterations that co-occur with oncogenic BRAF in melanoma and abrogate cellular dependence upon this oncogene. We identified concurrent mutational inactivation of the PTEN and RB1 tumor suppressors as a mechanism for loss of BRAF/MEK dependence in melanomas harboring (V600E)BRAF mutations. RB1 alterations were mutually exclusive with loss of p16(INK4A), suggesting that whereas p16(INK4A) and RB1 may have overlapping roles in preventing tumor formation, tumors with loss of RB1 exhibit diminished dependence upon BRAF signaling for cell proliferation. These findings provide a genetic basis for the heterogeneity of clinical outcomes in patients treated with targeted inhibitors of the mitogen-activated protein kinase pathway. Our results also suggest a need for comprehensive screening for RB1 and PTEN inactivation in patients treated with RAF and MEK-selective inhibitors to determine whether these alterations are associated with diminished clinical benefit in patients whose cancers harbor mutant BRAF.

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.

Objective To determine the relationship between cerebrospinal fluid (CSF) beta 2-microglobulin (beta 2M) and severity of AIDS dementia complex (ADC), and between CSF beta 2M and response of ADC to zidovudine. Design A prospective study. Setting Tertiary referral hospital. Patients, participants Seventy-eight patients with varying stages of ADC were selected from a subgroup of a cohort of HIV-seropositive patients who are being studied prospectively for the neurological complications of HIV-1 infection. To enter our study, patients had to have an ADC stage of at least 0.5 (equivocal symptoms or abnormal neurological signs in the absence of functional impairment). A control group of 11 HIV-1-seropositive, neurologically normal patients was chosen randomly from the patients followed in the Multicenter AIDS Cohort Study. Interventions Patients were assessed neurologically and neuropsychologically and computed tomography of the brain and CSF studies were performed. Main outcome measures Patients were staged according to severity of ADC on clinical criteria. Neuropsychological test scores were converted to an impairment score. CSF beta 2M was quantified in both serum and CSF of all patients and in 10 patients with pre- and post-zidovudine assessments. Results There was a high correlation between CSF beta 2M concentration and severity of ADC (P less than 0.0001); treatment with zidovudine significantly reduced these concentrations (P = 0.013). CSF beta 2M concentration was independent of CSF white-cell count and blood-brain barrier impairment. Other CSF changes in the same patients (including blood-brain barrier permeability to albumin, intrathecal synthesis of immunoglobulin G and HIV-1-p24-antigen levels) were less useful as objective correlates of ADC severity and response to zidovudine therapy. Conclusions CSF beta 2M may be a valuable marker of ADC severity and response to antiviral therapy.

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.

Overexpression of trans-acting response element (TAR)-containing sequences (TAR decoys) in CEM SS cells renders cells resistant to human immunodeficiency type 1 (HIV-1) replication. Mutagenesis of TAR was used to investigate the molecular mechanism underlying the observed inhibition. A nucleotide change which disrupts the stem structure of TAR or sequence alterations in the loop abolish the ability of the corresponding TAR decoy RNAs to inhibit HIV replication. A compensatory mutation which restores the stem structure also restores TAR decoy RNA function. Synthesis of viral RNA is drastically reduced in cells expressing a functional TAR decoy RNA, but it is unaffected in cells expressing a mutant form of TAR decoy RNA. It is therefore concluded that overexpression of TAR-containing sequences in CEM SS cells interferes with the process of Tat-mediated transactivation of viral gene expression. However, the phenotype of several mutations suggests that TAR decoy RNA does not inhibit HIV-1 gene expression by simply sequestering Tat but rather does so by sequestering a transactivation protein complex, implying that transactivation requires the cooperative binding of both Tat and a loop-binding cellular factor(s) to TAR. Expression of wild-type or mutant forms of TAR had no discernible effects on cell viability, thus reducing concerns about using TAR decoy RNAs as part of an intracellular immunization protocol for the treatment of AIDS.

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.

Cancer-testis or germ cell antigens (GCAs) are a category of tumor antigens expressed by male germ cells and by cancers of diverse histological origin, but not usually by normal adult somatic tissue. These antigens include products encoded by the MAGE, BAGE, GAGE, SSX, and LAGE/NY-ESO-1 families that encode antigenic peptides recognized by T lymphocytes. In this study, we exploit oligonucleotide technology to identify genes in melanoma and soft tissue sarcoma (STS) that display a cancer-testis/GCA expression profile. We identified 59 such genes, including GCAs we knew to be recognized by T lymphocytes. Among our findings are the expression of PRAME in monophasic synovial sarcoma, PRAME and NY-ESO-1 in myxoid/round cell liposarcoma, and SSX2 and members of the GAGE family in malignant fibrous histiocytoma. Furthermore, the proto-oncogene DBL/MCF2 was identified as encoding a novel candidate GCA expressed by clear cell sarcoma/melanoma of soft parts (MSP). DBL/MCF2 peptides that are bound to HLA-A*0201 were identified and recognized by T lymphocytes. These results show the utility of high-throughput expression analysis in the efficient screening and identification of GCA candidates in cancer, and its application to the discovery of candidate targets for T cell immunity against GCAs expressed by cancer.

Purpose: To develop a genome-based classification scheme for clear-cell sarcoma (CCS), also known as melanoma of soft parts (MSP), which would have implications for diagnosis and treatment. This tumor displays characteristic features of soft tissue sarcoma (STS), including deep soft tissue primary location and a characteristic translocation, t(12;22)(q13;q12), involving EWS and ATF1 genes. CCS/MSP also has typical melanoma features, including immunoreactivity for S100 and HMB45, pigmentation, MITF-M expression, and a propensity for regional lymph node metastases. Materials and Methods: RNA samples from 21 cell lines and 60 pathologically confirmed cases of STS, melanoma, and CCS/MSP were examined using the U95A GeneChip (Affymetrix, Santa Clara, CA). Hierarchical cluster analysis, principal component analysis, and support vector machine (SVM) analysis exploited genomic correlations within the data to classify CCS/MSP. Results: Unsupervised analyses demonstrated a clear distinction between STS and melanoma and, furthermore, showed that CCS/MSP cluster with the melanomas as a distinct group. A supervised SVM learning approach further validated this finding and provided a user-independent approach to diagnosis. Genes of interest that discriminate CCS/MSP included those encoding melanocyte differentiation antigens, MITF, SOX10, ERBB3, and FGFR1. Conclusion: Gene expression profiles support the classification of CCS/MSP as a distinct genomic subtype of melanoma. Analysis of these gene profiles using the SVM may be an important diagnostic tool. Genomic analysis identified potential targets for the development of therapeutic strategies in the treatment of this disease.

New technologies are needed to characterize the migration, survival, and function of antigen-specific T cells in vivo. Here, we demonstrate that Epstein-Barr virus (EBV)--specific T cells transduced with vectors encoding herpes simplex virus-1 thymidine kinase (HSV-TK) selectively accumulate radiolabeled 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU). After adoptive transfer, HSV-TK+ T cells labeled in vitro or in vivo with [131I]FIAU or [124I]FIAU can be noninvasively tracked in SCID mice bearing human tumor xenografts by serial images obtained by scintigraphy or positron emission tomography (PET), respectively. These T cells selectively accumulate in EBV+ tumors expressing the T cells' restricting HLA allele but not in EBV- or HLA-mismatched tumors. The concentrations of transduced T cells detected in tumors and tissues are closely correlated with the concentrations of label retained at each site. Radiolabeled transduced T cells retain their capacity to eliminate targeted tumors selectively. This technique for imaging the migration of ex vivo-transduced antigen-specific T cells in vivo is informative, nontoxic, and potentially applicable to humans.

Retroviral infection is restricted by the expression of a viral receptor on the surface of the target cell. Retrovirus-mediated gene transfer is therefore not possible in cells that fail to express sufficient levels of the appropriate receptor, representing one major obstacle to the use of recombinant retroviruses in experimental and clinical applications. In this study, we utilized an adenoviral vector to express transiently the receptor for the ecotropic murine leukemia virus in a panel of human cell lines. Following adenoviral infection, the susceptibility to ecotropic retroviral particles of A549, HeLa, RC39 and Meso 33 cells, derived from human lung epithelium, cervical epithelium, kidney and mesothelium, respectively, was measured on a single-cell basis by the detection of a cell surface marker encoded by the recombinant retrovirus. The marker, termed NTP, was found in 10-30%, 25-50% and 50-90% of cells infected at 5, 50 and 250 adenovirus multiplicity of infection, respectively. Southern blot analysis demonstrated the integration of intact retroviral DNA. The integrated vector copy number increased with the adenoviral multiplicity of infection, suggesting that retrovirus infection is proportional to receptor expression by the target cell, albeit not in a linear fashion. Susceptibility to ecotropic retroviral infection was maintained undiminished for at least 3 days, indicating the persistent expression of ecotropic receptor by the adenovirus-transduced cells in that time period and the lack of a major cellular defense triggered by adenovirus infection against the subsequent retroviral infection. Thus, the infection of human cells of various tissues with a recombinant adenovirus expressing the ecotropic murine leukemia virus receptor generates a window of susceptibility where a high retroviral infection rate can be achieved. Increased efficiency of retroviral infection obtained in this fashion is amenable to specific regulation via the controlled expression of the adenovirus-encoded retroviral receptor.

8583 Background: Ipilimumab (ipi), a monoclonal antibody blocking CTLA-4, demonstrated survival benefit in metastatic melanoma in two randomized controlled trials. With its approval by the FDA, the use of this immunotherapy is increasing. The surgical safety of ipi has not been reported. Methods: From our prospective database, we identified patients undergoing surgery within 30 days of receiving a dose of ipi or on maintenance ipi between October 2007 and August 2011. Surgical toxicity was graded 1-5 by the Clavien classification. In 10 patients matched blood and tumor infiltrating lymphocytes were harvested. Phenotype of T cell subsets were analyzed by flow cytometry. Results: 23 patients were identified who underwent 34 operations a median of 27 weeks after initiation of ipi (1-123 weeks). Indications for surgery were symptomatic, progressive, or isolated persistent disease in 52%, 39% and 9% respectively. Subcutaneous resections were the most frequent, followed by intra-abdominal and nodal procedures (Table). Grade 1 wound complications were seen in 17% of patients. One patient had a Grade 2 wound complication. No Grade 3-5 complications were seen. With a median follow up from starting ipi of 86 weeks, 3 patients have no evidence of disease, 10 are alive with disease and 10 have died of disease. Analysis of the T cell infiltrate and peripheral blood from 10 patients with progressive or symptomatic disease shows an elevated % of CD4+FOXP3+ T regulatory cells and a 2.8 fold reduction in CD8+/ CD4+FOXP3+ T regulatory ratio in the tumor compared to blood (p=0.02). Conclusions: Results from this single-center experience suggest that surgery is safe in patients receiving ipi. Immune modulation caused by CTLA-4 blockade does not appear to impact wound healing, even in the bowel. In carefully selected patients metastectomy may be appropriate for breakthrough metastases. The high percentage of T regulatory cells and low T effector cells in the progressive tumors suggests a mechanism of immune escape. [Table: see text]

We prospectively evaluated 94 patients with AIDS-dementia complex (ADC) and a smaller group of 27 patients with other HIV-1 related neurological conditions to determine the usefulness of cerebrospinal fluid (CSF) p24 antigen and HIV-1 culture in the diagnosis of ADC. The presence of ADC correlated with detectable CSF p24, but not with a positive culture. However, only 54% of the patients with severe or end stage (stages 3 and 4) ADC had detectable CSF p24 and only 25% had a positive culture. Among those with detectable CSF p24, there was no discernible relationship between the severity of ADC and the amount of CSF p24. The diagnostic sensitivity of CSF p24 in ADC was 21% whereas the specificity was 98%. CSF HIV-1 culture had a sensitivity of 30% and a specificity of 80%. To address the possibility of binding of p24 in immune complexes, thereby escaping detection, an acid hydrolysis procedure was performed on the CSF and serum samples. This did not, however, make an appreciable difference in the detection rate of p24. To delineate whether the finding of cell free virus in the CSF was associated with ADC, CSF culture for HIV-1 was performed on both cell depleted and cell associated fractions. It was uncommon for CSF to be culture positive in only the cell free fraction and there was no relationship to the presence or severity of ADC.

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.

e13053 Background: Prior studies have shown intramuscular (IM) injection of xenogeneic orthologues of melanosomal antigens (tyrosinase, gp100) result in CD8+ T-cell responses to the syngeneic prote...

e16149 Background: Ipilimumab (Ipi) is a human IgG1 monoclonal antibody that blocks the inhibitory action of the checkpoint molecule CTLA-4. Recent data [Proc Amer Soc Clin Onc, Abstr#5004, 2008] from castrate metastatic PC pts suggested that Ipi was active but was associated with grade 3 autoimmune adverse events (AEs), such as colitis, hepatitis, hypophysitis or rash, which required high dose steroids. We studied pts who were treated on a clinical trial with Ipi 10 mg/kg alone or with radiation to a single site in bone to determine whether there was any correlation between serum cytokine levels and autoimmune AEs. Methods: Thirteen pts were treated, of whom 10 had sufficient specimens for analysis. Of the 10 pts, 8 had ≥1 AE during treatment. Pts were stratified by ascertainment of clinical benefit (CB) vs. no clinical benefit (NCB) based on time-to-PSA-baseline relapse (TTBR) and also by toxicity (tox) using standard NCI tox criteria. TTBR was defined as time from study entry until the time when a PSA measurement reached or exceeded the baseline value. Pts with a TTBR greater than the median value were judged to have CB; those with a TTBR less than the median value had NCB. Pts with grade (gd) 0/1/2 tox were termed “low tox” while those with gd 3/4 tox were “high tox”. Sera at serial time points were analyzed for interferon-gamma (IFN-γ), tumor necrosis factor α and interleukins (IL)-1b, 2, 4, 8, 10, 12, 13 with the Meso Scale Discovery Multiplex Assay (MSD, Gaithersburg, MD). Results: IFN-γ was the predominant cytokine produced in 4 of 5 pts with CB and AEs; it increased at week 4 or 7 after two doses of Ipi, then declined. Four of 5 pts with NCB had minimal IFN-γ production. There was no difference in IL-10 production between those with CB vs. NCB or high vs. low tox. However, there was a suggestion that the IFN-γ /IL-10 ratio may be increased in pts who had high tox and/or CB vs. pts with low tox and NCB. This awaits confirmation. Conclusions: While this experience with Ipi in PC is small, there may be differences between pts with CB vs. NCB and pts with low vs. high tox. Investigation of the immunophenotype of pts who remain in follow- up and continue to be enrolled is ongoing as well as the impact of prior chemotherapy on development of toxicities. [Table: see text]

Overexpression of TAR-containing sequences (TAR decoys) was used to render cells resistant to HIV replication. A chimeric tRNA(meti)-TAR transcription unit contained in a double copy murine retroviral vector was used to express high levels of HIV-1 TAR-containing transcripts in CEM SS cells. Replication of HIV-1 was inhibited over 99% in cells expressing chimeric tRNA-TAR transcripts, but an amphotropic murine retrovirus replicated normally in these cells. Expression of TAR sequences in CEM SS cells had no adverse effects on cell viability, indicating that essential cellular factors are not being sequestered in these cells. TAR decoy RNA-mediated HIV inhibition may also be effective against natural HIV isolates in spite of their hypervariable nature, as suggested by the fact that replication of SIVmac was also inhibited in cells expressing HIV-1 TAR decoys.

We evaluated cerebrospinal fluid (CSF) concentrations of neopterin, a putative marker of activated macrophages, in 97 subjects infected with human immunodeficiency virus type 1 who had a spectrum of neurological complications. The highest CSF neopterin concentrations occurred in those with neurological opportunistic infections, primary central nervous systems lymphoma, and acquired immunodeficiency syndrome (AIDS) dementia complex. In general, the CSF concentration of neopterin was independent of CSF cell count and blood-brain barrier disruption to albumin. In the patients with AIDS dementia complex, CSF neopterin concentrations correlated with severity of disease and decreased in conjunction with clinical improvement following treatment with zidovudine. These results suggest that CSF neopterin, although not disease-specific, may be useful as a surrogate marker for the presence of AIDS dementia complex and its response to antiviral therapy.

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.

Many mutations of the housekeeping gene encoding glucose-6-phosphate dehydrogenase (G6PD) cause G6PD deficiency in humans. Some underlie severe forms of chronic nonspherocytic hemolytic anemia (CNSHA) for which there is no definitive treatment. By using retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein that harbor the human G6PD (hG6PD) complementary DNA, stable and lifelong expression of hG6PD was obtained in all the hematopoietic tissues of 16 primary bone marrow transplant (BMT) recipient mice and 14 secondary BMT recipients. These findings demonstrate the integration of a functional gene in totipotent stem cells. The average total G6PD in peripheral blood cells of these transplanted mice, measured as enzyme activity, was twice that of untransplanted control mice. This allowed the inference that the amount of G6PD produced by the transduced gene must be therapeutically effective. With the same vectors both the cloning efficiency and the ability to form embryoid bodies were restored in embryonic stem cells, in which the G6PD gene had been inactivated by targeted homologous recombination, thus effectively rescuing their defective phenotype. Finally, expression of normal human G6PD in hG6PD-deficient primary hematopoietic cells and in human hematopoietic cells engrafted in nonobese diabetic/severe combined immunodeficient mice was obtained. This approach could cure severe CNSHA caused by G6PD deficiency.

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)