An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells.

Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/ß T-cell receptors (TCRα/ß). However, potential mispairing of introduced TCRα/ß-chains with endogenous ß/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vß-fragment to the TCR Cß-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/ß-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vß-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vß. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/ß-positive T-cells.

The chemotherapeutic agent topotecan differentially modulates the phenotype and function of dendritic cells.

The camptothecin analogue topotecan (TPT) induces tumor cell apoptosis due to interference with topoisomerase I and is clinically used as a second-line chemotherapeutic in the treatment for metastasizing ovarian and small cell lung carcinoma. Based on the more recent finding of TPT-mediated inhibition of the transcription factor hypoxia-induced factor-1α, a hallmark of solid tumors, TPT, is currently tested in clinical trials for its suitability as a first-line chemotherapeutic for the treatment for various types of tumors. Due to the gained clinical interest in TPT and in light of its modulatory effect on signaling pathways, which are also of importance for immune cell functions, we asked for potential effects of TPT on dendritic cells (DCs), the main antigen-presenting cell population of the immune system. Here, we show that TPT at a therapeutically relevant dose partially activated monocyte-derived DCs as reflected by enhanced migratory activity, elevated expression of HLA-DR and costimulatory/maturation markers, and accordingly an increased allogenic CD4(+) T cell stimulation. In marked contrast, TPT prevented full maturation of DCs stimulated with a cocktail of proinflammatory mediators, accompanied by somewhat lower upregulation of NF-κB factors p65 and RelB.

Cytotoxicity of tumor antigen specific human T cells is unimpaired by arginine depletion.

Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8(+) T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i) CD8(+) T cells with specificity against the MART-1aa26-35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii) clonal CMV pp65aa495-503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495-503 specific T cell receptor were analyzed. Our data demonstrate that human CD8(+) T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency.