Stimulation of Anti-Tumor Immunity Using Dendritic Cell/Tumor Fusions and Anti-CD3/CD28.

Abstract Dendritic Cells (DCs) are potent antigen presenting cells that prominently express costimulatory molecules and are uniquely capable of stimulating primary immune responses. We have developed a promising tumor vaccine involving the fusion of patient derived tumor cells and autologous DCs. However, vaccine efficacy is limited by effector cell dysfunction and increased presence of regulatory T cells characteristic of cancer patients. Ligation of CD3/CD28 has been shown to deliver a powerful antigen-independent stimulus to resting T cell populations. We postulated that the combined exposure to antiCD3/CD28 and DC/tumor fusions would result in the expansion of activated T cells targeting tumor antigens. We have examined the phenotypic and functional characteristics of T cells that have undergone in vitro stimulation with DC/renal carcinoma (RCC) fusion cells in conjunction with expansion using antiCD3/CD28. DCs were generated from adherent mononuclear cells cultured with rhIL-4 and GM-CSF for five days, and matured by 48 hour exposure to TNFa. DCs were fused with RCC by coculture in 50% solution of polyethylene glycol. CD3/CD28 mediated activation was accomplished by culturing cells on plates coated with antiCD3/CD28 antibody for 48 hours. Exposure to fusion cells, antiCD3/CD28 alone, or antiCD3/CD28 followed by DC/tumor fusions resulted in no significant evidence of T cell expansion with a stimulation index (SI) of 0.9, 1.0, and 1.0, respectively. In contrast, a marked synergistic effect on proliferation was observed when T cells underwent stimulation with DC/tumor fusion cells followed by expansion using antiCD3/CD28 (SI 13.2) (p= 0.02, p=0.01, and p= 0.03 compared to anti-CD3/CD28 alone, fusions alone, and antiCD3/CD28 followed by fusion cells, respectively). We assessed the phenotypic characteristics of T cells stimulated by antiCD3/CD28, fusion cells, or their combination. In 10 experiments, stimulation with DC/RCC fusions followed by exposure to antiCD3/CD28 resulted in a nearly 8 fold expansion of CD4+/CD25+ cells (p=0.001 compared to unstimulated T cells). A 16 fold increase in CD4/CD25/CD69+ cells was observed consistent with the dramatic expansion of activated T cells. In contrast, exposure to antiCD3/CD28 alone or antiCD3/CD28 followed by stimulation with fusion cells resulted in a 3 fold expansion of CD4/CD25+ T cells and a modest expansion of CD4/CD25/CD69+ cells. In concert with these findings, IFNγ production by CD4+ T cells was most pronounced (7-fold expansion) following stimulation with DC/tumor fusion vaccine and expansion with anti-CD3/CD28 (p<0.01). We also examined the effect of stimulation with DC/RCC fusions followed by antiCD3/CD28 on the expansion of regulatory T cells. In 9 experiments, stimulation with DC/RCC fusions followed by expansion with antiCD3/CD28 also resulted in a 5-fold and 4.6 fold expansion of CD4/CD25/Foxp3+ and IL-10 expressing T cells, respectively. Stimulation with fusions and antiCD3/CD28 resulted in an increase in CD45RO+ memory effector T cells (2-fold increase, p= 0.08) and a decrease in CD45RA+, naïve T cells. In conclusion, we have shown that stimulation of T cells by DC/RCC fusions followed by exposure to CD3/CD28 antibodies results in the expansion of tumor reactive T cells that predominantly express markers of activation. We are developing a clinical trial in which patients will receive fusion/CD3/CD28 expanded T cells following in vivo depletion of regulatory T cells.