N. Rebecca Haley

Enhancement of anti‐tumor immunity through local modulation of CTLA‐4 and GITR by dendritic cells

Cancer vaccines have now demonstrated clinical efficacy, but immune modulatory mechanisms that prevent autoimmunity limit their effectiveness. Systemic administration of mAbs targeting the immune modulatory receptors CTLA‐4 and glucocorticoid‐induced TNFR‐related protein (GITR) on Treg and effector T cells augments anti‐tumor immunity both experimentally and clinically, but can induce life‐threatening autoimmunity. We hypothesized that local delivery of anti‐CTLA‐4 and anti‐GITR mAbs to the sites where T cells and tumor antigen‐loaded DC vaccines interact would enhance the induction of anti‐tumor immunity while avoiding autoimmunity. To achieve this goal, DCs transfected with mRNA encoding the H and L chains of anti‐mouse CTLA‐4 and GITR mAbs were co‐administered with tumor antigen mRNA‐transfected DCs. We observed enhanced induction of anti‐tumor immunity and significantly improved survival in melanoma‐bearing mice, without signs of autoimmunity. Using in vitro assays with human DCs, we demonstrated that DCs transfected with mRNA encoding a humanized anti‐CTLA‐4 mAb and mRNA encoding a soluble human GITR‐L fusion protein enhance the induction of anti‐tumor CTLs in response to DCs transfected with mRNAs encoding either melanoma or breast cancer antigens. Based on these results, this approach of using local delivery of immune modulators to enhance vaccine‐induced immunity is currently being evaluated in a phase I clinical cancer immunotherapy trial.

A prospective, double‐blind, randomized clinical feasibility trial of controlling the storage age of red blood cells for transfusion in cardiac surgical patients

BACKGROUND: Recent evidence demonstrates an association between duration of storage of red blood cells (RBC) and morbidity and mortality after cardiac surgery. We studied the feasibility of two different schemes for categorizing and randomizing age of RBC units transfused in cardiac surgical patients.

Melanoma immunotherapy using mature DCs expressing the constitutive proteasome

BACKGROUND Many cancers, including melanoma, exclusively express constitutive proteasomes (cPs) and are unable to express immunoproteasomes (iPs). In contrast, mature DCs used for immunotherapy exclusively express iPs. Since proteasomes generate peptides presented by HLA class I molecules, we hypothesized that mature melanoma antigen-loaded DCs engineered to process antigens through cPs would be superior inducers of antimelanoma immunity in vivo. METHODS Subjects with metastatic melanoma were vaccinated with mature DCs transfected with RNAs encoding melanoma antigens MART1, MAGE-3, gp100, and tyrosinase. These DCs were derived from monocytes that were untransfected (Arm A; n = 4), transfected with control siRNA (Arm B; n = 3), or transfected with siRNAs targeting the 3 inducible iP subunits (Arm C; n = 5). RESULTS Vaccination stimulated antigen-specific T cell responses in all subjects, which peaked after 3-4 vaccinations, but remained elevated in Arm C subjects. Also in Arm C, circulating melanoma cell levels (as detected by quantitative PCR) fell, and T cell lytic activity against autologous melanoma was induced. In HLA-A2⁺ subjects, CD8⁺ T cells that bound tetramers loaded with cP-derived melanoma antigenic peptides were found in the peripheral blood only in Arm C subjects. Of 2 subjects with active disease (both in Arm C), one had a partial clinical response, while the other, who exhibited diffuse dermal and soft tissue metastases, had a complete response. CONCLUSION These results suggest that the efficacy of melanoma DC-based immunotherapy is enhanced when tumor antigen-loaded DCs used for vaccination express cPs. TRIAL REGISTRATION Clinicaltrials.gov NCT00672542. FUNDING Duke Clinical Research Institute/Duke Translational Medicine Institute, Duke Melanoma Consortium, and Duke University Department of Surgery.

Increased yield of endothelial cells from peripheral blood for cell therapies and tissue engineering.

AIM Peripheral blood-derived endothelial cells (pBD-ECs) are an attractive tool for cell therapies and tissue engineering, but have been limited by their low isolation yield. We increase pBD-EC yield via administration of the chemokine receptor type 4 antagonist AMD3100, as well as via a diluted whole blood incubation (DWBI). MATERIALS & METHODS Porcine pBD-ECs were isolated using AMD3100 and DWBI and tested for EC markers, acetylated LDL uptake, growth kinetics, metabolic activity, flow-mediated nitric oxide production and seeded onto titanium tubes implanted into vessels of pigs. RESULTS DWBI increased the yield of porcine pBD-ECs 6.6-fold, and AMD3100 increased the yield 4.5-fold. AMD3100-mobilized ECs were phenotypically indistinguishable from nonmobilized ECs. In porcine implants, the cells expressed endothelial nitric oxide synthase, reduced thrombin-antithrombin complex systemically and prevented thrombosis. CONCLUSION Administration of AMD3100 and the DWBI method both increase pBD-EC yield.

Quality Control in Cord Blood Banking

The hematopoietic progenitor cells present in the umbilical cord (HPC, Cord Blood) have the potential to engraft and differentiate in the bone marrow with subsequent trilineage hematopoiesis. The collection, processing and storage of HPC, Cord Blood should preserve the cell number and characteristics to protect their hematopoietic potential. Federal regulations establish guidelines for HPC, Cord Blood manufacturing and testing to ensure the safety, purity, potency and identity of the product. Accrediting organizations for cord blood banks, including the AABB and the Foundation for Accreditation of Cellular Therapy (FACT), provide a series of guidelines for developing safe processes and procedures in cellular therapy activities. Additionally, clinical data based on outcome analysis provide evidence to help in the process of unit selection for a given patient, based on compatibility, cell dose and potency of the product.

Abstract 5373: Altering the composition of the proteasome of melanoma antigen RNA-transfected dendritic cells enhances vaccination-induced anti-melanoma immunity: results of a Phase I trial

Background: Dendritic cells (DC) must be matured to maximally stimulate antigen-specific immune responses and avoid the induction of tolerance. During maturation, the proteasome, the multi-subunit complex that generates peptides presented in the context of HLA class I, is altered to exclusively consist of immunoproteasomes (iPs). Compared to the constitutive proteasome (cP), the iP has altered protease activities and generates a different repertoire of peptide epitopes. Vaccination with mature tumor-antigen loaded DC will thus stimulate immunity against iP-generated peptides. Since melanomas, and other cancers, are frequently defective in inducible iP expression, this anti-tumor immune response may be miss-targeted and not recognize melanomas presenting cP-derived peptides. We hypothesized that mature melanoma antigen-loaded DC expressing the cP, rather than the iP, would be superior inducers of anti-melanoma immunity in subjects with metastatic melanoma. Methods: 12 subjects with metastatic melanoma were immunized with 6 weekly intradermal injections of 10 million mature autologous monocyte-derived DC transfected with RNA encoding full-length MART-1, MAGE-3, gp100 and tyrosinase. The monocytes from which the DC were generated were either untransfected (Study Arm A, n=4), transfected with control siRNA (Study Arm B, n=3), or transfected with siRNAs targeting the 3 inducible iP subunits LMP-2, LMP-7 and MECL-1 (Study Arm C, n=5). Results: 10 subjects received a full course of vaccination, while only enough DC were available for 4 and 5 total vaccine doses in 2 subjects. All 12 subjects tolerated vaccination without toxicity. Vaccination stimulated melanoma antigen-specific CD4+ and CD8+ T cell responses detected in peripheral blood by IFN-γ ELISPOT in all subjects, which peaked after 3-4 vaccinations. These T cell responses decreased thereafter in subjects in Study Arms A and B, but remained elevated in subjects in Study Arm C. Melanoma cell levels in peripheral blood, as detected by qPCR, fell after vaccination in Study Arm C. Using autologous melanoma cells (and autologous normal skin fibroblasts as controls) derived from each subject, we found that vaccination in Study Arm C, but not A or B, induced peripheral T cells with lytic activity against autologous melanoma. Two subjects had active disease, both in Study Arm C. The one subject with diffuse extremity in-transit disease had a partial clinical response with transient liquefaction of dermal lesions. The other subject with diffuse dermal and soft tissue metastatic lesions had complete resolution of detectable metastasis by PET scanning. Conclusion: The efficacy of melanoma immunotherapy using antigen RNA-transfected mature DC is enhanced when DC express the cP rather than the iP. We feel that this novel approach warrants further clinical investigation. 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 5373. doi:1538-7445.AM2012-5373

Conference Scene: Cell and gene therapies: clinical and near-clinical focus

The Phacilitate Cell and Gene Therapy Forum was a showcase of the steps required for translational medicine. The major focus was on requirements from early phase discovery through to the regulatory and business processes to bring treatments to patients. Talks on discovery and new methodologies and technologies were presented in light of adaptability to treatment regimens. Venture capitalists and bank representatives, external evaluators of these new treatments, gave valuable financial process insight. US FDA officials offered explanation and advice on successful approaches to the regulatory challenges to this new and emerging field. Completing the picture were talks from contract suppliers and manufacturers of necessary materials and equipment.