Cindy L. Zuleger

A Phase I Study of Immunization Using Particle-Mediated Epidermal Delivery of Genes for gp100 and GM-CSF into Uninvolved Skin of Melanoma Patients

Purpose: We examined in vivo particle-mediated epidermal delivery (PMED) of cDNAs for gp100 and granulocyte macrophage colony-stimulating factor (GM-CSF) into uninvolved skin of melanoma patients. The aims of this phase I study were to assess the safety and immunologic effects of PMED of these genes in melanoma patients. Experimental Design: Two treatment groups of six patients each were evaluated. Group I received PMED with cDNA for gp100, and group II received PMED with cDNA for GM-CSF followed by PMED for gp100 at the same site. One vaccine site per treatment cycle was biopsied and divided for protein extraction and sectioning to assess transgene expression, gold-bead penetration, and dendritic cell infiltration. Exploratory immunologic monitoring of HLA-A2+ patients included flow cytometric analyses of peripheral blood lymphocytes and evaluation of delayed-type hypersensitivity to gp100 peptide. Results: Local toxicity in both groups was mild and resolved within 2 weeks. No systemic toxicity could be attributed to the vaccines. Monitoring for autoimmunity showed no induction of pathologic autoantibodies. GM-CSF transgene expression in vaccinated skin sites was detected. GM-CSF and gp100 PMED yielded a greater infiltration of dendritic cells into vaccine sites than did gp100 PMED only. Exploratory immunologic monitoring suggested modest activation of an antimelanoma response. Conclusions: PMED with cDNAs for gp100 alone or in combination with GM-CSF is well tolerated by patients with melanoma. Moreover, pathologic autoimmunity was not shown. This technique yields biologically active transgene expression in normal human skin. Although modest immune responses were observed, additional investigation is needed to determine how to best utilize PMED to induce antimelanoma immune responses.

MAGE-C2 Promotes Growth and Tumorigenicity of Melanoma Cells, Phosphorylation of KAP1, and DNA Damage Repair

Melanoma-associated antigen-encoding (MAGE) genes are expressed in melanoma and other cancers but not in normal somatic cells. MAGE expression is associated with aggressive tumor growth, poor clinical outcome, and resistance to chemotherapy, but the mechanisms have not been completely elucidated. In this study, we show that downregulation of MAGE-C2 in A375 melanoma cells and low-passage cultures from human metastatic melanomas (MRA cells) results in increased apoptosis and decreased growth of tumor xenografts in athymic nude mice. Previously, we showed that MAGE-C2 binds KAP1, a scaffolding protein that regulates DNA repair. Phosphorylation of KAP1-Serine 824 (Ser824) by ataxia-telangiectasia-mutated (ATM) kinase is necessary for repair of DNA double-strand breaks (DSBs); now we show that MAGE-C2 knockdown reduces, whereas MAGE-C2 overexpression increases, ATM kinase-dependent phosphorylation of KAP1-Ser824. We demonstrate that MAGE-C2 increases co-precipitation of KAP1 with ATM and that binding of MAGE-C2 to KAP1 is necessary for increased KAP1-Ser824 phosphorylation. Furthermore, ectopic expression of MAGE-C2 enhances repair of I-SceI endonuclease-induced DSBs in U-2OS cells. As phosphorylation of KAP1-Ser824 facilitates relaxation of heterochromatin, which is necessary for DNA repair and cellular proliferation, our results suggest that MAGE-C2 can promote tumor growth by phosphorylation of KAP1-Ser824 and by enhancement of DNA damage repair.

OMIP-008: measurement of Th1 and Th2 cytokine polyfunctionality of human T cells.

Purpose and Appropriate Sample Types This panel was optimized to assess CD4+ and CD8+ T cell responses to various tumor antigens from melanoma patients. The panel was tested on single-cell derived T cell isolates (SCD-T) and T cell lines derived from peripheral blood mononuclear cells (PBMC) from melanoma patients, T cell lines from the tumor environment of melanoma patients, and fresh and cryopreserved PBMC (healthy donors). Staining can be performed in 96-well plates for high-throughput.

Clonal expansions of 6-thioguanine resistant T lymphocytes in the blood and tumor of melanoma patients.

The identification of specific lymphocyte populations that mediate tumor immune responses is required for elucidating the mechanisms underlying these responses and facilitating therapeutic interventions in humans with cancer. To this end, mutant hypoxanthine‐guanine phosphoribosyltransferase (HPRT) deficient (HPRT‐) T‐cells were used as probes to detect T‐cell clonal amplifications and trafficking in vivo in patients with advanced melanoma. Mutant T‐cells from peripheral blood were obtained as clonal isolates or in mass cultures in the presence of 6‐thioguanine (TG) selection and from tumor‐bearing lymph nodes (LNs) or metastatic melanoma tissues by TG‐selected mass cultures. Nonmutant (wild‐type) cells were obtained from all sites by analogous means, but without TG selection. cDNA sequences of the T‐cell receptor (TCR) beta chains (TCR‐β), determined directly (clonal isolates) or following insertion into plasmids (mass cultures), were used as unambiguous biomarkers of in vivo clonality of mature T‐cell clones. Clonal amplifications, identified as repetitive TCR‐β V‐region, complementarity determining region 3 (CDR3), and J‐region gene sequences, were demonstrated at all sites studied, that is, peripheral blood, LNs, and metastatic tumors. Amplifications were significantly enriched among the mutant compared with the wild‐type T‐cell fractions. Importantly, T‐cell trafficking was manifested by identical TCR‐β cDNA sequences, including the hypervariable CDR3 motifs, being found in both blood and tissues in individual patients. The findings described herein indicate that the mutant T‐cell fractions from melanoma patients are enriched for proliferating T‐cells that infiltrate the tumor, making them candidates for investigations of potentially protective immunological responses. Environ. Mol. Mutagen., 2008. Published 2008 Wiley‐Liss, Inc.

In vivo 6-thioguanine-resistant T cells from melanoma patients have public TCR and share TCR beta amino acid sequences with melanoma-reactive T cells

In vivo hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient T cells (MT) from melanoma patients are enriched for T cells with in vivo clonal amplifications that traffic between blood and tumor tissues. Melanoma is thus a model cancer to test the hypothesis that in vivo MT from cancer patients can be used as immunological probes for immunogenic tumor antigens. MT were obtained by 6-thioguanine (TG) selection of lymphocytes from peripheral blood and tumor tissues, and wild-type T cells (WT) were obtained analogously without TG selection. cDNA sequences of the T cell receptor beta chains (TRB) were used as unambiguous biomarkers of in vivo clonality and as indicators of T cell specificity. Public TRB were identified in MT from the blood and tumor of different melanoma patients. Such public TRB were not found in normal control MT or WT. As an indicator of T cell specificity for melanoma, the >2600 MT and WT TRB, including the public TRB from melanoma patients, were compared to a literature-derived empirical database of >1270 TRB from melanoma-reactive T cells. Various degrees of similarity, ranging from 100% conservation to 3-amino acid motifs (3-mer), were found between both melanoma patient MT and WT TRBs and the empirical database. The frequency of 3-mer and 4-mer TRB matching to the empirical database was significantly higher in MT compared with WT in the tumor (p=0.0285 and p=0.006, respectively). In summary, in vivo MT from melanoma patients contain public TRB as well as T cells with specificity for characterized melanoma antigens. We conclude that in vivo MT merit study as novel probes for uncharacterized immunogenic antigens in melanoma and other malignancies.

Pilot study of safety and feasibility of DNA microseeding for treatment of spontaneous canine melanoma

Abstract Spontaneous canine malignant melanoma provides an excellent pre‐clinical model to study DNA vaccines for melanoma immunotherapy. A USDA‐approved xenogeneic human tyrosinase (huTYR) plasmid DNA vaccine delivered intramuscularly induces detectable immune responses and has clinical activity in some dogs with melanoma. The objective of this pilot study was to evaluate the feasibility, safety and immunogenicity of huTYR plasmid DNA administered to the skin via microseeding in dogs with spontaneous melanoma. DNA microseeding utilizes a modified tattooing device as an alternate and potentially more potent delivery method for DNA immunization. DNA was delivered to shaved inner thigh skin of six companion dogs with melanoma approximately every 14 days for a planned total of four vaccination time points. An anti‐huTYR ELISA was used to test pre‐ and post‐treatment sera. Biopsies of treated skin were obtained for detection of huTYR transgene expression. DNA microseeding was well tolerated with no significant toxicity detected beyond local site irritation, and there were no signs of autoimmunity. huTYR‐expressing cells were observed in biopsies of huTYR DNA microseeding sites. Increased humoral anti‐huTYR antibodies were seen in two of five evaluable dogs following microseeding compared to baseline. DNA microseeding is well tolerated in companion dogs with melanoma. Further investigation is needed to determine if combining DNA microseeding with other immunotherapy regimens potentiates this delivery platform for cancer immunotherapy.

Abstract 2366: Pilot study of DNA microseeding to activate immune rejection of canine melanoma

Background: Canine malignant melanoma provides a model to study DNA vaccine delivery systems. A xenogeneic human tyrosinase (huTYR) DNA vaccine delivered by Biojector2000 received United States Department of Agriculture licensure when it appeared to prolong survival of dogs with melanoma compared to historical, stage-matched controls and to stimulate immune responses in some dogs. The current study evaluates toxicity, transgene expression, and antibody responses to huTYR in companion dogs with spontaneously developing melanoma following delivery of huTYR DNA to the skin via a modified tattoo device, a method termed DNA microseeding. Methods: Five companion dogs with melanoma were scheduled to receive huTYR DNA at two sites (Site A and Site B) on the inner thigh by DNA microseeding every 2 weeks for 4 administrations at a range of huTYR DNA doses; 2 dogs (50 μg [Site A] and 100 μg [Site B]); 2 dogs (200 μg [Site A] and 400 μg [Site B]); and 1 dog (83 μg [Site A] and 83 μg commercial huTYR plasmid [Site B]). Vaccine site biopsies were obtained to determine transgene expression 24 hours after the 1st and 3rd vaccination time-points, and 48 hours after the 2nd and 4th vaccination time-points. Blood samples were obtained at baseline and 2 weeks after the 2nd, 3rd, and 4th vaccinations to quantify TYR-specific antibodies via indirect ELISA. Results: No toxicity, beyond local site irritation, related to the vaccine administration was observed. The 3 dogs with known disease at study entry received 3, 1 and 4 treatments before discontinuation of treatment due to progressive disease. The 2 dogs without evidence of disease at study entry received all 4 planned treatments and remained without evidence for recurrence after treatment for the duration of the study (6 weeks). Only rare huTYR+ cells with macrophage-like morphology were observed in some vaccine site biopsies. A significant increase in anti-huTYR IgG was detected at Day 57 compared to pre-treatment in 2 of the 4 evaluable dogs (p = 0.03 Wilcoxon Mann Whitney), and these were the 2 dogs without evidence of disease at study entry. Baseline anti-huTYR IgG levels were also greater compared to IgG levels against an irrelevant control antigen. Conclusions: While microseeding of huTYR plasmid DNA resulted in only rare transgene expression at DNA doses up to 400 μg, humoral responses against huTYR were substantially boosted in 2 of 4 evaluable dogs. Additional testing is needed to determine if DNA microseeding enhances huTYR DNA vaccine immunogenicity compared to Biojector delivery. Citation Format: Cindy L. Zuleger, Chulhi Kang, Erik A. Ranheim, Ilene Kurzman, Michael D. Macklin, Michael A. Newton, David M. Vail, Jedd D. Wolchok, Elof Eriksson, Mark R. Albertini. Pilot study of DNA microseeding to activate immune rejection of canine melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2366.

Abstract 2538: Immune responses to common melanoma-associated antigens following intratumoral injection of alpha-gal glycolipids in patients with advanced melanoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: One mechanism of immune escape in the tumor microenvironment is the inhibition of processes involved in tumor antigen uptake by antigen presenting cells (APC). Effective uptake of tumor cells by APC is achieved pre-clinically by labeling them with α-gal glycolipids (α-gal) and exploiting the natural anti-Gal antibody response that constitutes approximately 1% of immunoglobulins in humans. The current study involves intratumoral injection of glycolipids expressing α-gal epitopes in patients (pts) with advanced melanoma and evaluates, in HLA-A2+ treated pts, the development of immune responses to common melanoma-associated antigens (MAA). Methods: Eligibility criteria included unresectable metastatic melanoma (recurrent stage III or stage IV); at least one readily resectable cutaneous, subcutaneous, or readily palpable lymph node metastasis; and a serum anti-Gal titer above 1:50. Pts received 2 injections of alpha-gal glycolipids four weeks apart at 0.1 mg/injection (n=3; dose level 1), 1.0 mg/injection (n=3; dose level 2), or 10 mg/injection (n=3; dose level 3). Ex vivo pre-treatment and Day 57 post-treatment peripheral blood mononuclear cells (PBMC) from pts who were HLA-A2+ (8 of 9 treated pts) were stained with pooled MAA pentamers (gp100209-217, gp100154-162, Melan-A/MART-126-35, NY-ESO-1157-165, and Tyrosinase368-376) and evaluated by flow cytometry. Cells stained with cytomegalovirus (CMV) pp65495-504 or HTLV-1 Tax11-19 served as positive or negative controls, respectively. Results: MAA pentamer+ T cells were detected in 7 of 8 HLA-A2+ pts, both pre- and post-treatment. The frequency of MAA pentamer+ T cells post- versus pre-treatment increased approximately 2-fold for 2 pts [one from dose level 2 and one from dose level 3 (0.16% vs. 0.082%, and 0.033% vs. 0.017%, respectively (background subtracted))]. Similar increases were not observed for the other 5 pts with detectable MAA pentamer+ cells. Furthermore, the median fluorescence intensity (MFI) of MAA pentamer+ T cells, an indirect measure of T cell activation potential, was markedly higher post-treatment, as compared to pre-treatment, for 2 of 3 pts in dose level 1 (11227 vs. 3027, and 4045 vs. 1581). Substantial changes in MFI were not observed for the other pts.. Conclusions: Intratumoral injection of α-gal glycolipids can increase the ex vivo frequency of circulating MAA reactive T cells in some pts with advanced melanoma. Based on this finding, we are pursuing functional characterization of these pre- and post-treatment PBMC. Citation Format: Cindy L. Zuleger, Paul M. Sondel, Jacquelyn A. Hank, Erik A. Ranheim, Thomas A. McFarland, Jennifer Collins, Erin Clements, Giles Whalen, Uri Galili, Mark R. Albertini. Immune responses to common melanoma-associated antigens following intratumoral injection of alpha-gal glycolipids in patients with advanced melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2538. doi:10.1158/1538-7445.AM2014-2538

A conditional predictive p-value to compare a multinomial with an overdispersed multinomial in the analysis of T-cell populations

Immunological experiments that record primary molecular sequences of T-cell receptors produce moderate to high-dimensional categorical data, some of which may be subject to extra-multinomial variation caused by technical constraints of cell-based assays. Motivated by such experiments in melanoma research, we develop a statistical procedure for testing the equality of two discrete populations, where one population delivers multinomial data and the other is subject to a specific form of overdispersion. The procedure computes a conditional-predictive p-value by splitting the data set into two, obtaining a predictive distribution for one piece given the other, and using the observed predictive ordinate to generate a p-value. The procedure has a simple interpretation, requires fewer modeling assumptions than would be required of a fully Bayesian analysis, and has reasonable operating characteristics as evidenced empirically and by asymptotic analysis.