Abstract Purpose: Dendritic cells (DC) initiate adaptive immune responses through the uptake and presentation of antigenic material. In preclinical studies, intratumorally injected activated DCs (aDCs; DCVax-Direct) were superior to immature DCs in rejecting tumors from mice. Experimental Design: This single-arm, open-label phase I clinical trial evaluated the safety and efficacy of aDCs, administered intratumorally, in patients with solid tumors. Three dose levels (2 million, 6 million, and 15 million aDCs per injection) were tested using a standard 3 + 3 dose-escalation trial design. Feasibility, immunogenicity, changes to the tumor microenvironment after direct injection, and survival were evaluated. We also investigated cytokine production of aDCs prior to injection. Results: In total, 39 of the 40 enrolled patients were evaluable. The injections of aDCs were well tolerated with no dose-limiting toxicities. Increased lymphocyte infiltration was observed in 54% of assessed patients. Stable disease (SD; best response) at week 8 was associated with increased overall survival. Increased secretion of interleukin (IL)-8 and IL12p40 by aDCs was significantly associated with survival (P = 0.023 and 0.024, respectively). Increased TNFα levels correlated positively with SD at week 8 (P < 0.01). Conclusions: Intratumoral aDC injections were feasible and safe. Increased production of specific cytokines was correlated with SD and prolonged survival, demonstrating a link between the functional profile of aDCs prior to injection and patient outcomes. Clin Cancer Res; 24(16); 3845–56. ©2018 AACR.

Abstract Background: Activated, autologous dendritic cells (aaDC) can be used to induce anti-tumor immune responses. A unique method of applying aaDC is through intratumoral injection, where the tumor cells serve as the source of antigen required for an adaptive anti-tumor response. A local effect may also occur as a result of cytokine production by the injected DC which makes the tumor more susceptible to a pre-existing or an induced immune attack. Methods: Forty patients with locally advanced or metastatic solid tissue cancers were treated in a dose escalation trial in which aaDC were injected percutaneously under image guidance into a single tumor. Subjects had a median of 3 tumors (range 1 - 5) and had received an average of 3.1 prior treatments. To generate the aaDC, autologous monocytes were converted ex vivo into DC which were then activated. All batches of DC were released based on pre-specified criteria which included immunophenotyping and a T cell-stimulation assay, as well as sterility and endotoxin levels. Cytokine levels produced by the activated DC during manufacturing were measured and patient outcomes were correlated to these expression levels. Results: All three doses levels were well tolerated. The main adverse events related to treatment were grade 1 and 2 fevers. Twenty-one patients achieved stable disease (SD) 8 weeks after initiating treatment, and this was found to correlate with survival (p = 0.01). Levels of certain cytokines, such as such IL-8 and IL-12 p40, and TNFα were substantially elevated in vitro and IL-8 and IL-12 p40 production were predictive of survival (p = 0.001 and p = 0.008 resp.). TNFα levels also correlated with SD at week 8 (p = 0.01). More than 70% of patients tested were found to have significant T cell responses, and/or de novo or significantly enhanced PD-L1 expression in the tumor post treatment, with a trend towards improved survival (p = 0.1). Conclusions: Study outcomes such as stabilization of disease and survival correlated with high DC cytokine levels, in the absence of meaningful toxicity. The DCVax treatment may be mediated through direct cytotoxic effects, as well as modulation of the tumor microenvironment to increase tumor infiltration by T cells, and attraction of inflammatory cells to the tumor. The development of PD-L1 expression likely reflects an induced immune response. Citation Format: Vivek Subbiah, Ravi Murthi, Robert Prins, Kyle Hendricks, Chitra Hosing, Lori Noffsinger, Mary McGuire, Robert Brown, Aung Naing, David Hong, Siqing Fu, Anthony Conley, Indreshpal Kaur, Sarah Campion, Marnix Bosch. Cytokine production by intratumorally administered activated dendritic cells correlates with survival in a Phase I clinical trial in diverse cancers [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B005.

Meeting abstracts Dendritic cells (DC) are proficient in initiating adaptive immune responses, through the uptake and subsequent presentation to the immune system of antigenic compounds. In preclinical studies, activated DC (aDC; DCVax®-Direct) were shown to be superior to immature DC in clearing

Rheumatoid arthritis (RA) is a systemic autoimmune disease with unknown etiology where tumor necrosis factor-α (TNFα) plays a critical role. Etanercept, a recombinant fusion protein of human soluble tumor necrosis factor receptor II (hsTNFR) linked to the Fc portion of human IgG1, is used to treat RA based on the rationale that sTNFR binds TNFα and blocks TNFα-mediated inflammation. We compared hsTNFR protein delivery from genetically engineered human mesenchymal stem cells (hMSCs) with etanercept. Blocking TNFα-dependent intercellular adhesion molecule-1 expression on transduced hMSCs and inhibition of nitric oxide production from TNFα-treated bovine chondrocytes by conditioned culture media from transduced hMSCs demonstrated the functionality of the hsTNFR construction. Implanted hsTNFR-transduced mesenchymal stem cells (MSCs) reduced mouse serum circulating TNFα generated from either implanted TNFα-expressing cells or lipopolysaccharide induction more effectively than etanercept (TNFα, 100%; interleukin [IL]-1α, 90%; and IL-6, 60% within 6 hours), suggesting faster clearance of the soluble tumor necrosis factor receptor (sTNFR)-TNFα complex from the animals. In vivo efficacy of sTNFR-transduced MSCs was illustrated in two (immune-deficient and immune-competent) arthritic rodent models. In the antibody-induced arthritis BalbC/SCID mouse model, intramuscular injection of hsTNFR-transduced hMSCs reduced joint inflammation by 90% compared with untransduced hMSCs; in the collagen-induced arthritis Fischer rat model, both sTNFR-transduced rat MSCs and etanercept inhibited joint inflammation by 30%. In vitro chondrogenesis assays showed the ability of TNFα and IL1α, but not interferon γ, to inhibit hMSC differentiation to chondrocytes, illustrating an additional negative role for inflammatory cytokines in joint repair. The data support the utility of hMSCs as therapeutic gene delivery vehicles and their potential to be used in alleviating inflammation within the arthritic joint.

As an alternative approach to recombinant protein therapy, we tested and compared recombinant protein to cell-based delivery of a therapeutic gene. We chose human erythropoietin (hEPO) as the therapeutic gene and human adult mesenchymal stem cells (hMSCs) as the delivery vehicle. In vivo experiments were performed in NOD/SCID mice, with hMSCs bound to ceramic cubes, using both ELISA and hematocrit analysis to measure systemic hEPO expression and function. Hematocrit (normally 40% in NOD/SCID mice) was elevated to between 60 and 80% for 92 days with a single hEPO-transduced hMSC implantation, comparable to the subcutaneous injection of 100 IU of recombinant hEPO (rhEPO) three times per week for a total of 40 injections. As few as 0.5x10(6) cells caused an increase in hematocrit; both hematocrit and hEPO protein level correlated with cell numbers, suggesting that cell dose can be used to regulate therapeutic protein serum levels. An injection of a lethal dose of phenylhydrazine resulted in survival of only the animals receiving hEPO-transduced hMSCs; all rhEPO injected mice died. In vivo efficacy of hEPO-transduced hMSCs was further demonstrated by their ability to elevate hematocrit in anemic animals. These results illustrate the ability of hMSCs to express genes of therapeutic value and demonstrate their potential clinical utility in diseases that would benefit from cell-based gene delivery.