beta-thalassemias are the most common single gene disorders and are potentially amenable to gene therapy. However, retroviral vectors carrying the human beta-globin cassette have been notoriously unstable. Recently, considerable progress has been made using lentiviral vectors, which stably transmit the beta-globin expression cassette. Thus far, mouse studies have shown correction of the beta-thalassemia intermedia phenotype and a partial, variable correction of beta-thalassemia major phenotype. We tested a lentiviral vector carrying the human beta-globin expression cassette flanked by a chromatin insulator in transfusion-dependent human thalassemia major, where it would be ultimately relevant. We demonstrated that the vector expressed normal amounts of human beta-globin in erythroid cells produced in in vitro cultures for unilineage erythroid differentiation. There was restoration of effective erythropoiesis and reversal of the abnormally elevated apoptosis that characterizes beta-thalassemia. The gene-corrected human beta-thalassemia progenitor cells were transplanted into immune-deficient mice, where they underwent normal erythroid differentiation, expressed normal levels of human beta-globin, and displayed normal effective erythropoiesis 3 to 4 months after xenotransplantation. Variability of beta-globin expression in erythroid colonies derived in vitro or from xenograft bone marrow was similar to that seen in normal controls. Our results show genetic modification of primitive progenitor cells with correction of the human thalassemia major phenotype.

Only 10% of metastatic melanoma patients survive 5 years, even though many can achieve substantial tumor reduction by surgical resection and/or radiation therapy and/or systemic therapy. An effective, nontoxic, consolidation immunotherapy could benefit such patients. We initiated a randomized trial to compare 2 promising patient-specific immunotherapy cell products. Patients had to have a diagnosis of metastatic melanoma and availability of an autologous melanoma cell line. Patients were stratified by whether their most advanced stage had been regional or distant metastases, and by whether they had measurable disease at the time of treatment, then they were randomized to receive irradiated autologous proliferating tumor cells or autologous dendritic cells (DC) loaded with antigens from such cells. Both products were injected subcutaneously in 500 µg of granulocyte-macrophage colony stimulating factor, weekly for 3 weeks and then monthly for 5 months. Patients in the 2 arms did not differ in baseline characteristics. All patients received prescribed therapy. Treatment was well tolerated. At the time of initial analysis, with no patients lost to follow-up, 50% of patients deceased, and all surviving patients followed for at least 6 months after randomization, survival is superior in the DC arm (hazard ratio, 0.27; 95% confidence interval, 0.098-0.729) with median survival not reached versus 15.9 months, and 2-year survival rates of 72% versus 31% (P=0.007). This trial provides evidence that a DC vaccine is associated with longer survival compared with a tumor cell vaccine, and is consistent with previous data suggesting a survival benefit from this patient-specific immunotherapy.

Personalized vaccine, recognized after the failure of allogenic melanoma whole cell and lysate vaccine phase III trials, involves culturing cells from a patient's 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 (> or =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.

Significant 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.

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 5 34) were immunized with dendritic cells cocultured with irradiated, IFNc-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 1 SD of normal and healthy volunteers on weeks 0, 4 and 24, patients were categorized into cohorts-I (LLL, n 5 16), II (HHL/HLL, n 5 4), III (LLH/LHH/LHL, n 5 7) and IV (HHH/HLH, n 5 7). The cohorts were regrouped as sTG-downregulators (sTG-DR; n 5 20) and upregulators (sTG-UR; n 5 14). These two cohorts differed significantly in their overall (p < 0.012) and progression-free (p 5 0.0001) survival posttreatment. 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 antigangliosideIgM 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. ' 2007 Wiley-Liss, Inc.

No abstract

Eltrapuldencel-T (CLBS20) consists of autologous dendritic cells loaded with antigens from irradiated, self-renewing, autologous tumor cells that potentially present the entire repertoire of unique patient-specific tumor-associated antigens resulting from nonsynonymous mutations in each patient's melanoma tumor cell line. In a single-arm phase 2 trial, metastatic melanoma patients treated with s.c. injections of CLBS20 had a 2-year overall survival (OS) of 73% (NCT00948480). In a randomized phase 2 trial (NCT00436930), 2-year OS was 72% compared to 31% for a tumor cell vaccine (TCV) consisting of irradiated cancer cells from an autologous tumor cell line. Mild local injection site reactions was the most common toxicity. This report focuses on 5-year follow up data from the randomized trial. During October 2007 to February 2011 42 patients were randomized 1:1 to receive CLBS20 or TCV. After resection of a metastatic tumor, if/when a cell line was established, patients were eligible for randomization if/when they were referred by their managing physician. Both products were mixed in 500 micrograms GM-CSF and injected s.c. weekly for three weeks, then monthly for up to 5 months. Cell lines were successfully established rapidly enough for possible clinical use for 78/183 (43%); 42/78 (54%) patients were referred for randomization. The median time from tumor submission to cell line success was 3.0 months, then 2.5 weeks for safety testing and documentation, then another 3.7 months to randomization. At the time of tumor harvest 24 patients were stage 4 and 18 were recurrent stage 3; at randomization 33 were stage 4 and 9 stage 3. Trends toward imbalances of baseline characteristics were biased against the CLBS20 arm (elevated LDH, detectable disease, brain metastases). There were no differences in tumor resection site, days in cell culture, or days to randomization. Because of leukapheresis and dendritic cell production for CLBS20, the median time from randomization to first dose was 43 days for CLBS20 vs 8 days for TCV. All patients were treated as randomized; survival was calculated from randomization date. At this analysis, 33 (79%) were dead and the 9 survivors had been followed 5 years. OS was higher in the CLBS20 arm: median 42.2 vs 19.9 months for all and 40.4 vs 16.9 for stage 4 patients, and in subsets defined by measurable disease, and serum LDH. The only variable associated with 3-year OS was randomization to CLBS20 (p = 0.018). A patient with refractory progressive measurable disease experienced a delayed complete response that was ongoing at 5 years. In an era before widespread use of anti-BRAF/anti-MEK and anti-checkpoint agents, CLBS20 monotherapy was associated with encouraging 5-year OS. Because of its unique mechanism of action, absence of toxicity, and apparent benefit regardless of tumor burden, CLBS20 is worthy of further evaluation as a monotherapy or in combination with other anti-melanoma therapies. Citation Format: Robert O. Dillman, Edward F. McClay, Thomas T. Amatruda, Carol DePriest, Denysha J. Carbonell, Andrew N. Cornforth. Randomized trial of dendritic vs tumor cell patient-specific vaccines: 5-year analysis. [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 CT105.

The use of whole cell tumor vaccines and various means of loading antigen onto dendritic cells have been under investigation for over a decade. Induction of apoptosis and the exposure of immune-stimulating proteins are thought to be beneficial for the use in immunotherapy protocols, but conclusive evidence in the clinical setting has been lacking. Incubation of melanoma cell lines with interferon-gamma (IFN-γ) increased phosphatidylserine and calreticulin exposure, but not in the IFN-γ-resistant cell line Lu-1205. Short-term autologous melanoma cell lines used for loading dendritic cells for immunotherapy showed differential response to the pro-apoptotic effects of IFN-γ. These IFN-γ-treated tumor cells (TCs) were irradiated and used for loading antigen for dendritic cell therapy. A log-rank comparison of survival for patients whose TCs were found to be either sensitive (upregulated phosphatidylserine and calreticulin) or insensitive to IFN-γ revealed a strongly significant correlation to progression-free (p = 0.003) and overall survival (p = 0.002) favorably in those patients whose cell lines were resistant to the proapoptotic effect of IFN-γ. These results suggest that the use of IFN-γ in anti-melanoma dendritic cell-based immunotherapy may only be beneficial when the cells do not undergo apoptosis in response to IFN-γ and support the contention that the use of some apoptotic cells in vaccines may be detrimental.