CRISPR/Cas9-mediated TGFßRII disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells in vitro.

BACKGROUND: CAR T-cell therapy has been recently unveiled as one of the most promising cancer therapies in hematological malignancies. However, solid tumors mount a profound line of defense to escape immunosurveillance by CAR T-cells. Among them, cytokines with an inhibitory impact on the immune system such as IL-10 and TGFß are of great importance: TGFß is a pleiotropic cytokine, which potently suppresses the immune system and is secreted by a couple of TME resident and tumor cells. METHODS: In this study, we hypothesized that knocking out the TGFß receptor II gene, could improve CAR T-cell functions in vitro and in vivo. Hereby, we used the CRISPR/Cas9 system, to knockout the TGFßRII gene in T-cells and could monitor the efficient gene knock out by genome analysis techniques. Next, Mesothelin or Claudin 6 specific CAR constructs were overexpressed via IVT-RNA electroporation or retroviral transduction and the poly-functionality of these TGFßRII KO CAR T-cells in terms of proliferation, cytokine secretion and cytotoxicity were assessed and compared with parental CAR T-cells. RESULTS: Our experiments demonstrated that TGFßRII KO CAR T-cells fully retained their capabilities in killing tumor antigen positive target cells and more intriguingly, could resist the anti-proliferative effect of exogenous TGFß in vitro outperforming wild type CAR T-cells. Noteworthy, no antigen or growth factor-independent proliferation of these TGFßRII KO CAR T-cells has been recorded. TGFßRII KO CAR T-cells also resisted the suppressive effect of induced regulatory T-cells in vitro to a larger extent. Repetitive antigen stimulation demonstrated that these TGFßRII KO CAR T-cells will experience less activation induced exhaustion in comparison to the WT counterpart. CONCLUSION: The TGFßRII KO approach may become an indispensable tool in immunotherapy of solid tumors, as it may surmount one of the key negative regulatory signaling pathways in T-cells.

A TCR-like CAR Promotes Sensitive Antigen Recognition and Controlled T-cell Expansion Upon mRNA Vaccination.

Chimeric antigen receptor (CAR) T cells are efficacious in patients with B-cell malignancies, while their activity is limited in patients with solid tumors. We developed a novel heterodimeric TCR-like CAR (TCAR) designed to achieve optimal chain pairing and integration into the T-cell CD3 signaling complex. The TCAR mediated high antigen sensitivity and potent antigen-specific T-cell effector functions in short-term in vitro assays. Both persistence and functionality of TCAR T cells were augmented by provision of costimulatory signals, which improved proliferation in vitro and in vivo. Combination with a nanoparticulate RNA vaccine, developed for in vivo expansion of CAR T cells, promoted tightly controlled expansion, survival, and antitumor efficacy of TCAR T cells in vivo. Significance: A novel TCAR is tightly controlled by RNA vaccine-mediated costimulation and may provide an alternative to second-generation CARs for the treatment of solid tumors.

An RNA vaccine drives expansion and efficacy of claudin-CAR-T cells against solid tumors.

Chimeric antigen receptor (CAR)-T cells have shown efficacy in patients with B cell malignancies. Yet, their application for solid tumors has challenges that include limited cancer-specific targets and nonpersistence of adoptively transferred CAR-T cells. Here, we introduce the developmentally regulated tight junction protein claudin 6 (CLDN6) as a CAR target in solid tumors and a strategy to overcome inefficient CAR-T cell stimulation in vivo. We demonstrate that a nanoparticulate RNA vaccine, designed for body-wide delivery of the CAR antigen into lymphoid compartments, stimulates adoptively transferred CAR-T cells. Presentation of the natively folded target on resident antigen-presenting cells promotes cognate and selective expansion of CAR-T cells. Improved engraftment of CAR-T cells and regression of large tumors in difficult-to-treat mouse models was achieved at subtherapeutic CAR-T cell doses.

CIMT 2018: Pushing frontiers in cancer immunotherapy - Report on the 16th Annual Meeting of the Association for Cancer Immunotherapy.

The 16th Annual Meeting of the Association for Cancer Immunotherapy (CIMT), Europe's largest meeting series of its kind, took place in Mainz, Germany from 15-17 May, 2018. Cutting-edge advancements in cancer immunotherapy were discussed among more than 700 scientists under the motto "Pushing Frontiers in Cancer Immunotherapy". This meeting report is a summary of some of the CIMT 2018 highlights.

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.

Sulfur and nitrogen mustards induce characteristic poly(ADP-ribosyl)ation responses in HaCaT keratinocytes with distinctive cellular consequences.

Mustard agents are potent DNA alkylating agents with mutagenic, cytotoxic and vesicant properties. They include bi-functional agents, such as sulfur mustard (SM) or nitrogen mustard (mustine, HN2), as well as mono-functional agents, such as "half mustard" (CEES). Whereas SM has been used as a chemical warfare agent, several nitrogen mustard derivatives, such as chlorambucil and cyclophosphamide, are being used as established chemotherapeutics. Upon induction of specific forms of genotoxic stimuli, several poly(ADP-ribose) polymerases (PARPs) synthesize the nucleic acid-like biopolymer poly(ADP-ribose) (PAR) by using NAD(+) as a substrate. Previously, it was shown that SM triggers cellular poly(ADP-ribosyl) ation (PARylation), but so far this phenomenon is poorly characterized. In view of the protective effects of PARP inhibitors, the latter have been proposed as a treatment option of SM-exposed victims. In an accompanying article (Debiak et al., 2016), we have provided an optimized protocol for the analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to further analyze mustard-induced PARylation and its functional consequences, in general. Thus, in the present study, we performed a comprehensive characterization of the PARylation response in HaCaT cells after treatment with four different mustard agents, i.e., SM, CEES, HN2, and chlorambucil, on a qualitative, quantitative and functional level. In particular, we recorded substance-specific as well as dose- and time-dependent PARylation responses using independent bioanalytical methods based on single-cell immuno-fluorescence microscopy and quantitative isotope dilution mass spectrometry. Furthermore, we analyzed if and how PARylation contributes to mustard-induced toxicity by treating HaCaT cells with CEES, SM, and HN2 in combination with the clinically relevant PARP inhibitor ABT888. As evaluated by a novel immunofluorescence-based protocol for the detection of N7-ETE-guanine DNA adducts, the excision rate of CEES-induced DNA adducts was not affected by PARP inhibition. Furthermore, while CEES induced moderate changes in cellular NAD(+) levels, annexin V/PI flow cytometry analysis revealed that these changes did not affect CEES-induced short-term cytotoxicity 24h after treatment. In contrast, PARP inhibition impaired cell proliferation and clonogenic survival, and potentiated micronuclei formation of HaCaT cells upon CEES treatment. Similarly, PARP inhibition affected clonogenic survival of cells treated with bi-functional mustards such as SM and HN2. In conclusion, we demonstrate that PARylation plays a functional role in mustard-induced cellular stress response with substance-specific differences. Since PARP inhibitors exhibit therapeutic potential to treat SM-related pathologies and to sensitize cancer cells for mustard-based chemotherapy, potential long-term effects of PARP inhibition on genomic stability and carcinogenesis should be carefully considered when pursuing such a strategy.