Purpose: Tumor antigen-loaded dendritic cells (DC) are believed to activate antitumor immunity by stimulating T cells, and CTL-associated antigen 4 (CTLA4)-blocking antibodies should release a key negative regulatory pathway on T cells. The combination was tested in a phase I clinical trial in patients with advanced melanoma. Experimental Design: Autologous DC were pulsed with MART-1 26-35 peptide and administered with a dose escalation of the CTLA4-blocking antibody tremelimumab. Sixteen patients were accrued to five dose levels. Primary end points were safety and immune effects; clinical efficacy was a secondary end point. Results: Dose-limiting toxicities of grade 3 diarrhea and grade 2 hypophysitis developed in two of three patients receiving tremelimumab at 10 mg/kg monthly. Four patients had an objective tumor response, two partial responses and two complete responses, all melanoma free between 2 and 4 years after study initiation. There was no difference in immune monitoring results between patients with an objective tumor response and those without a response. Exploratory gene expression analysis suggested that immune-related gene signatures, in particular for B-cell function, may be important in predicting response. The CTL-associated antigen 4 (CTLA4) is a main negative regulator of the immune system, which inhibits costimulatory signaling provided by dendritic cells (DC) to activate T lymphocytes. Antibodies to CTLA4 block this negative signaling, which allows a dominant positive signaling provided by costimulatory molecules on DCs recognized by CD28 on T cells (1). In animal models, administration of CTLA4-blocking antibodies alone induced rejection of established tumors, provided that these were immunogenic tumor models (1, 2). However, in poorly immunogenic tumor models, which may more closely resemble human disease, prior immunization with tumor vaccines, including DC-based vaccines, was required for CTLA4 blockade to exert robust antitumor effects (1).Tremelimumab 206) is a fully human CTLA4-blocking monoclonal antibody being developed for the treatment of cancer (3). In phase I testing, plasma levels of antibody of 30 μg/mL, which corresponds to levels predicted to result in continuous CTLA4 blockade in vitro, were achieved for at least 1 month at doses beyond 6 mg/kg (4). Cumulative clinical data suggests that single agent tremelimumab has antitumor activity in ∼10% of patients with advanced melanoma, and these responses tend to be long

Background CTLA4 blocking monoclonal antibodies provide durable clinical benefit in a subset of patients with advanced melanoma mediated by intratumoral lymphocytic infiltrates. A key question is defining if the intratumoral infiltration is a differentiating factor between patients with and without tumor responses. Methods Paired baseline and post-dosing tumor biopsies from 19 subjects, including three patients with an objective tumor response, were prospectively collected from patients with metastatic melanoma receiving the anti-CTLA4 antibody tremelimumab within a clinical trial with primary endpoint of quantitating CD8+ cytotoxic T lymphocyte (CTL) infiltration in tumors. Samples were analyzed for cell density using automated imaging capture, and further characterized for functional lymphocyte properties by assessing the cell activation markers HLA-DR and CD45RO, the cell proliferation marker Ki67 and the T regulatory cell marker FOXP3. Results There was a highly significant increase in intratumoral infiltration by CD8+ cells in biopsies taken after tremelimumab treatment. This included increases between 1-fold and 100-fold changes in 14 out of 18 evaluable cases regardless of clinical tumor response or progression. There was no difference between the absolute number, location or cell density of infiltrating cells between clinical responders and patients with non-responding lesions that showed acquired intratumoral infiltrates. There were similar levels of expression of T cell activation markers (CD45RO, HLA-DR) in both groups, and no difference in markers for cell replication (Ki67) or the suppressor cell marker FOXP3. Conclusion CTLA4 blockade induces frequent increases in intratumoral T cell infiltration despite which only a minority of patients have objective tumor responses.