Denysha Carbonell

Establishment of stable cell lines for personalized melanoma cell vaccine.

Personalized vaccine, recognized after the failure of allogenic melanoma whole cell and lysate vaccine phase III trials, involves culturing cells from a patients own tumor within a short duration and with less passages but with optimized expression of tumor-associated antigens (TAAs). Its feasibility is established by comparing pure cell lines generated from fresh and cryopreserved tissues (n=164) of patients with lymph node (LN) and distant metastases. Stable cell lines (from 67% of specimens) are subcultured after cryopreserving them. Pure cell lines established after eliminating fibroblasts (from 96% of the cell lines) include those from LN (69%), soft tissues including cutaneous (60%), liver (64%), lung (75%), bone (80%), brain (75%), and other sites (73%). Within 3.5 months, stable cell lines (≥50 million cells) are established from initiating the cell culture. For LN metastases, the duration differs significantly (P2<0.05) between fresh (1.4–3.4 months) and cryopreserved (2.4–4.7 months) tissues. The expression of TAAs varies as follows: Tyrosinase (81%) >Melan-A (80%) >HMB45/gp-100 (75%) >Mel-5/TRP-1 (65%) >MAGE-1 (47%) > S-100 (28%). The number of TAAs per cell line differs between early (<7) and late (>7) passages. Among late passage cell lines, lesser percentage of cell lines express three to six antigens pointing out that early passage (<7) cell lines may be needed for antigen-targeted immunotherapy. This study provides a protocol for establishing cell lines within 2–5 months for personalized vaccine therapy for nodal and organ metastatic melanoma patients.

Monitoring response to treatment in melanoma patients: Potential of a serum glycomic marker

A mechanistic marker correlating with tumor progression and clinical response is useful for assessing therapeutic response and determining the course of therapy. Since serum‐total‐ganglioside (sTG) and antiganglioside‐IgM antibody levels reflected tumor progression, the feasibility of utilizing sTG for assessing the response to immunotherapy of metastatic‐melanoma was tested. Patients (n = 34) were immunized with dendritic cells cocultured with irradiated, IFNγ‐treated autologous tumor cells admixed with GM‐CSF. Levels of sTG and antiganglioside‐IgM antibody titers were measured in sera of vaccine recipients at 0, 4 and 24 weeks of treatment. Based on sTG‐level, whether lower (L) or higher (H) than the mean + 1 SD of normal and healthy volunteers on weeks 0, 4 and 24, patients were categorized into cohorts‐I (LLL, n = 16), II (HHL/HLL, n = 4), III (LLH/LHH/LHL, n = 7) and IV (HHH/HLH, n = 7). The cohorts were regrouped as sTG‐ downregulators (sTG‐DR; n = 20) and upregulators (sTG‐UR; n = 14). These two cohorts differed significantly in their overall (p < 0.012) and progression‐free (p = 0.0001) survival post‐treatment. 43% sTG‐UR died within 39 months, with a median survival of 39 months, whereas 61% of the sTG‐DR survived for 48 months. Both endogenous and vaccine‐induced antiganglioside‐IgM antibodies appeared to regulate sTG levels. Nonresponders had increased sTG with no or low IgM antibody response. The sTG level is regulated within 24 weeks post‐treatment and therefore, may serve as an ideal biomarker for assessing therapeutic responses in patients. Clinical correlations of sTG indicate that sTG‐downregulating therapy may be an effective treatment strategy for melanoma.

Increases in Serum TARC/CCL17 Levels Are Associated with Progression-Free Survival in Advanced Melanoma Patients in Response to Dendritic Cell-Based Immunotherapy

IntroductionChanges in the levels of serum cytokines and growth factors are associated with response to therapy. We examined cytokine, chemokine, and growth factor levels in serum collected from normal volunteers or metastatic melanoma patients receiving dendritic cell-based immunotherapy.Materials and MethodsUsing an array for 42 cytokines, chemokines, or growth factors, sera from normal controls and metastatic melanoma patients at baseline and week 4 were analyzed for qualitative changes. Quantitative determination of the levels of the chemokine thymus and activation-regulated chemokine (TARC/CCL17) was determined by enzyme-linked immunosorbent assay (ELISA).ResultsSignificant qualitative differences were noted in serum cytokine, chemokine, and growth factor levels of metastatic melanoma patients versus the normal controls at baseline. The results also demonstrated a significant decrease in the level of angiogenin (P = 0.026) and a significant increase in TARC/CCLl7 (P = 0.008) from week 0 to week 4 which was associated with improved overall survival (P = 0.059). Higher TARC/CCL17 levels were observed by ELISA at week 4 and a log-rank comparison revealed a significant association between high serum TARC/CCL17 levels at week 4 and progression-free survival (P = 0.005). Receiver–operator characteristic analysis revealed that week 4 serum TARC/CCL17 levels were predictive of progression-free and overall survival, indicating that serum TARC/CCL17 might be of predictive value of response to dendritic cell-based anti-melanoma immunotherapy.

Characterization of interferon-γ-treated melanoma tumor cells for use in dendritic cell-based immunotherapy.

Efficient delivery of tumor-associated antigens to professional antigen-presenting cells is important for inducing a response in patients receiving cancer immunotherapy. Interferon-gamma (IFN-γ) is used by the immune system to combat viral and fungal infections by restricting cell proliferation and, in some cases, inducing apoptosis. Using IFN-γ to activate target tumor cells prior to antigen loading of dendritic cells (DCs) may enhance the beneficial qualities of whole-cell tumor vaccines. The incubation of melanoma cell cultures with IFN-γ resulted in an increase in the expression of major histocompatibility complex molecules and ICAM-1 but generally decreased the expression of melanoma-associated tumor antigens. Additionally, important immune-stimulating molecules (heat-shock proteins, high-mobility group box-1 protein, and calreticulin) were also present but differentially regulated by IFN-γ. Loading of DCs with IFN-γ-treated tumor cells resulted in a small but significant increase in the expression of CD83-positive DCs, indicating the initiation of DC maturation (p=0.019). IFN-γ treatment of melanoma cell lines prior to antigen loading of DCs may aid in antigen processing and presentation.

Superiority of dendritic cell vaccine vs tumor cell vaccine: survival by stratification subsets in MACVAC randomized Phase II trial of patient-specific vaccines utilizing antigens from autologous melanoma tumor cell lines

In a randomized Phase II trial conducted in patients with metastatic melanoma, superior overall survival (p=0.007) was observed for 18 patients treated with vaccines that consisted of autologous dendritic cells loaded with antigens from irradiated autologous melanoma stem cells, (DC-TC, aka eltrapuldencel-T, NBS20 and CBLS20) compared to 24 patients treated with vaccines consisting of autologous irradiated melanoma stem cells (TC) [ClinicalTrials.gov NCT00436930].[1] Both vaccines were admixed with GM-CSF as an adjuvant. Tumor cell lines that served as the source of patient-specific tumor-associated antigens were derived from metastases resected from patients with stage IV or recurrent stage III melanoma. The treatment schedule consisted of weekly subcutaneous injections for 3 weeks, and then monthly for 5 months. The current analysis was undertaken to determine the treatment effects of DC-TC vs TC in each of the subsets defined by the pre-randomization stratifications that were based on whether patients had measurable or non-measurable disease as defined by RECIST, and whether their most advanced stage of disease at the time of randomization had been stage IV or recurrent stage III disease. At the time of this analysis 5 DC-TC and 3 TC patients had been followed for 5 years; 5 patients (3 TC and 2 DC-TC) were alive but followed less than 5 years (minimum 3.5 years); 29 were deceased. No patients were lost to follow up. The survival results are summarized in Table ​Table1.1. Although the numbers are small, DC-TC immunotherapy was associated with superior survival in each of the four different subsets defined by the stratification variables. Eltrapuldencel-T has moved forward into a pivotal Phase III trial sponsored by Caladrius BioSciences, Inc. Table 1

Abstract 4845: Microarray analysis of melanoma autologous tumor cell lines used as the source of tumor associated antigens in patient-specific dendritic cell immunotherapy phase II trial in patients with metastatic melanoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Melanoma cells that are proliferating and self-renewing in short-term cell culture, have the phenotypic and functional characteristics of tumor stem cells, and they express unique patient-specific neoantigens, as well as numerous common melanoma associated antigens. Patient-specific therapeutic vaccines, produced by Incubating autologous dendritic cells with autologous tumor cells from such cell lines (DC-TC), have yielded promising results in phase II trials in metastatic melanoma. In this study we examined the expression of genes on the tumor cell lines which had served as the sources of tumor associated antigens loaded onto autologous dendritic cells as part of a phase II trial in which there was an observed 5-year survival rate of 50%.[Dillman 2009] There was sufficient melanoma tumor cells to analyze for 50 of the 54 patients enrolled in the phase II trial. Microarray analysis was performed by Response Genetics, Inc. (Los Angeles) for 54,000 genes. The Excel data file was entered into an Affymetrix data processing algorithm using the R programming language. Gene ranking was performed using prediction analysis of microarrays.[Tibshirani 2002] Correlation with overall survival was done for 26 patients who survived 5 years or longer, and 24 patients who survived less than 5 years. Hierarchical clustering for over and under expression yielded 9 genes, VEPH1, ZNF280B, FGF13, ST6GALNAC3, QPCT, ZNF280B, BST, PBRM1 and C21orf91 which identified 11/11 patients who had a survival greater than 5 years, and 17/18 patients who died in less than 2 years. Using the top six genes, overall survival greater than 5 years was correctly predicted for 80.3% of patients, and survival less than 5 years was correctly predicted for 74.2% of patients. Gene array data obtained from self-renewing, proliferating, autologous tumor cells was prognostic for survival. It is unclear whether these genes are predictive of an effective immune response after repeated injections of a therapeutic dendritic cell-tumor cell vaccine. Citation Format: Andrew N. Cornforth, Gary Fogel, Denysha Carbonell, Robert O. Dillman. Microarray analysis of melanoma autologous tumor cell lines used as the source of tumor associated antigens in patient-specific dendritic cell immunotherapy phase II trial in patients with metastatic melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4845. doi:10.1158/1538-7445.AM2015-4845

Abstract 3703: Randomized trial of autologous dendritic cell vs tumor cell vaccines in patients with metastatic melanoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Autologous proliferating tumor cells may represent tumor stem cells and/or early progenitor cells that are capable of establishing new sites of metastatic cancer, and may provide excellent antigenic targets for patient-specific vaccines. In successive trials in metastatic melanoma, 74 patients treated with irradiated proliferating autologous tumor cell vaccines (TC) had a median survival of 20.5 months (29% 5-yr survival), and 54 patients treated with autologous dendritic cell vaccines (DC), in which dendritic cells were loaded with antigen from autologous proliferating tumor cells, had a 50% 5-yr survival (Cancer Biother Radiopharm 2007, 2009). A randomized trial was initiated to determine whether there was a difference in the two approaches. Key eligibility criteria included a diagnosis of metastatic melanoma, successful establishment of a short-term cell line from resected metastatic tumor with expansion to 200 million proliferating tumor cells by the Hoag Cell Biology Laboratory, and the ability of patients to travel to Newport Beach, CA. There were no eligibility restrictions for types or numbers of prior therapies. Patients were stratified by whether they had measurable disease and by whether their most advanced stage had been regional or distant metastases, then randomized to receive either TC or DC injected s.c. with 500 micrograms of GM-CSF, weekly for 3 weeks and then monthly for five months. This was designed as a randomized phase III trial with 90% power to detect a 50% difference in survival by intent-to-treat analysis. In April 2011 Hoag Hospital advised that the Cell Biology Laboratory was to be closed for strategic and economic reasons, forcing discontinuation of the trial. 42 of a planned 200 patients were randomized, and all 42 received the assigned treatment. Because of the premature closure, an unplanned interim analysis was performed. There were no differences between the two arms in patient characteristics for 17 different variables including age, gender, measurable disease, location of metastases, LDH, and prior therapies. One patient in the DC arm experienced complete regression of multiple measurable soft-tissue metastases. With 21 patients deceased, survival is superior in the DC arm (HR=0.27 95% CI 0.098-0.729) with median survival not reached vs 15.9 months and 2-year survival rates 72% vs 30% (p=.007 Mantle-Cox log rank test, and p=.011 Breslow generalized Wilcoxon). This study establishes the importance of denditic cells in active specific immunotherapy, confirms an earlier study showing a high rate of long-term survival in patients with metastatic melanoma treated with such therapy, and shows that a patient-specific DC product is superior to a patient-specific TC product in death reduction. ([NCT00436930][1]) Supported by the Hoag Hospital Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3703. doi:1538-7445.AM2012-3703 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00436930&atom=%2Fcanres%2F72%2F8_Supplement%2F3703.atom

Abstract 768: Lymphokine activated killer cell conditioned media induces monocytes to differentiate into a dendritic cell-like/activated monocyte phenotype with cytotoxic potential against glioblastoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Despite advances in surgery and multimodal treatment regimens, Glioblastoma (GBM) continues to have poor overall survival outcomes and associated treatment morbidity. Cell-based immunotherapy is being explored as an option in the treatment of GBM. Treatment of GBM with lymphokine activated killer cells (LAK) generated by interleukin-2 (IL-2) stimulation has been associated with encouraging overall survival rates (Dillman RO et al. J Immunotherapy 2009 32:914-919). Since it has been reported that IL-2-activated lymphocytes can produce cytokines that may influence the differentiation and activation of monocytes (Kirsch M et al. J Neurooncol 1994 20:35-45), we explored the generation of activated monocytes and/or dendritic cells (DC) in LAK cell therapy products. Methods: Magnetic cell isolation of major histocompatibility complex II positive cells (HLA-DR) from LAK cell preparations revealed a small subpopulation of cells expressing CD80 and CD86 which also retained some CD14 expression. We then explored the possibility of generating activated monocytes and/or DC by incubating elutriated monocytes in the conditioned media of LAK cell preparations (LAK-CM) and examining their characteristics in comparison to standard GM-CSF/IL-4 DC or LAK cells. Results: The resulting LAK-CM incubated monocytes showed high levels of expression of CD80 (65.3%), CD86 (72.3%), HLA-DR (83.6%), comparable to the standard GM-CSF/IL-4 DC. The LAK-CM treated monocytes also retained their CD14 expression at (58.2% vs. 58.4%) and had similar levels of phagocytic ability compared to DC (14.7% vs. 12.7%) and demonstrated higher cytotoxicity levels against a model GBM cell line, U251, compared to either LAK cells themselves or the standard GM-CSF/IL-4 generated DC (85.6% vs. 55.9% vs. 60.9%, respectively). Conclusions: Combining lymphocytes and monocytes from elutriated lymphocyte fractions under LAK cell generation conditions (1000 IU/mL IL-2 in AIMV) also yielded similar dendritic cell/activated monocyte phenotypes suggesting that new combinations of cell therapy could be produced using this method with higher tumor cytotoxicity potentials as well as antigen cross presentation capabilities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 768. doi:10.1158/1538-7445.AM2011-768

Dynamic Expression of Human Leukocyte and Tumor-Associated Antigens in Primary Melanoma Cell Lines: Differential Modulation by IFN-γ

Senthamil Selvan, Abner Fowler, Annette Arora, Ashwin Gowda, Denysha Carbonell, Andrea Beatty, Karen Spencer, Robert Dillman. Cell Biology, Hoag Cancer Center, Newport Beach, CA. IFN-g has been recognized to have role in endogenous tumor surveillance mechanisms with its pleiotropic functions in the processes of antigen presentation, T cell and macrophage activation, and cellular cytotoxicity. However, the role of IFN-g in the expression of tumor-associated antigens by tumor cells is poorly understood. In the present study, we determined IFN-ginduced differential expression of various tumor-associated antigens (TAA), and HLA I and II in a number of primary melanoma cell lines. Since January 2000, pure primary tumor cell lines were established from fresh or cryopreserved tumor tissues in 80/108 melanoma (success rate of 74%). Melanoma lines were incubated in 10 U/ml IFN-g containing RPMI-1640 for 72 hrs and were analyzed by immunocytochemistry and Flow Cytometry. The following table shows the pattern of expression of TAAs and HLAs before and after IFN-g treatment. Data includes change in both cell number and intensity after incubation with IFN-g. Conclusion: Primary melanoma cell line generation is feasible in a high percentage of melanoma tissues. Although IFN-g up-regulates the expression of HLA I and II in most cell lines, a number of tumor-associated antigens expressions were either unaltered or mostly down-regulated upon exposure to IFN-g. Implication of this to an effective inducibility of tumor-immune response with IFN-g-treated tumor cell vaccine needs to be further determined.