The discovery and clinical application of immune-checkpoint inhibitors has dramatically improved the treatments, outcomes and therapeutic concepts in multiple tumor settings. This breakthrough was mainly based on monoclonal antibodies blocking the inhibitory molecule CTLA-4 and or the PD-1/PD-L1 axis, with the aim of counteracting major tumor immune evasion mechanisms. Even acknowledging these important successes, not all the patients benefit from these treatments. Translational and clinical research efforts are ongoing to explore the potentialities of a new generation of immune-modulatory molecules to extend current clinical applications and contrast the unsolved issues of resistance and disease relapse that still affects a considerable rate of patients. New immune-checkpoints, with either stimulatory or inhibitory functions are emerging with key roles in regulating T cell response but also affecting other crucial effectors belonging to the innate immune response (e.g., natural killer). Their therapeutic exploitation, either alone or in strategical combinations, is providing important preclinical results, holding promises currently explored in initial clinical trials. The first results point toward favorable safety profiles with selective hints of activity in challenging settings. Important issues regarding the dose, schedule and rational combinations remain open and data from the clinical studies are needed. Here we provide an overview of the main emerging stimulatory or inhibitory immune-checkpoints exploitable in cancer treatment, briefly reporting their biological function, preclinical activity and preliminary clinical data.

Chimeric antigen receptor (CAR)-engineered T lymphocytes (CAR Ts) produced impressive clinical results against selected hematological malignancies, but the extension of CAR T cell therapy to the challenging field of solid tumors has not, so far, replicated similar clinical outcomes. Many efforts are currently dedicated to improve the efficacy and safety of CAR-based adoptive immunotherapies, including application against solid tumors. A promising approach is CAR engineering of immune effectors different from αβT lymphocytes. Herein we reviewed biological features, therapeutic potential, and safety of alternative effectors to conventional CAR T cells: γδT, natural killer (NK), NKT, or cytokine-induced killer (CIK) cells. The intrinsic CAR-independent antitumor activities, safety profile, and ex vivo expansibility of these alternative immune effectors may favorably contribute to the clinical development of CAR strategies. The proper biological features of innate immune response effectors may represent an added value in tumor settings with heterogeneous CAR target expression, limiting the risk of tumor clonal escape. All these properties bring out CAR engineering of alternative immune effectors as a promising integrative option to be explored in future clinical studies.

Purpose of our study was to explore a new immunotherapy for high grade soft tissue sarcomas (STS) based on cytokine-induced killer cells (CIK) redirected with a chimeric antigen receptor (CAR) against the tumor-promoting antigen CD44v6. We aimed at generating bipotential killers, combining the CAR specificity with the intrinsic tumor-killing ability of CIK cells (CAR⁺.CIK). We set a patient-derived experimental platform. CAR⁺.CIK were generated by transduction of CIK precursors with a lentiviral vector encoding for anti-CD44v6-CAR. CAR⁺.CIK were characterized and assessed in vitro against multiple histotypes of patient-derived STS. The anti-sarcoma activity of CAR⁺.CIK was confirmed in a STS xenograft model. CD44v6 was expressed by 40% (11/27) of patient-derived STS. CAR⁺.CIK were efficiently expanded from patients (n = 12) and killed multiple histotypes of STS (including autologous targets, n = 4). The killing activity was significantly higher compared with unmodified CIK, especially at low effector/target (E/T) ratios: 98% vs 82% (E/T = 10:1) and 68% vs 26% (1:4), (p<0.0001). Specificity of tumor killing was confirmed by blocking with anti-CD44v6 antibody. CAR⁺.CIK produced higher amounts of IL6 and IFN-γ compared to control CIK. CAR⁺.CIK were highly active in mice bearing subcutaneous STS xenografts, with significant delay of tumor growth (p<0.0001) without toxicities. We report first evidence of CAR⁺.CIK's activity against high grade STS and propose CD44v6 as an innovative target in this setting. CIK are a valuable platform for the translation of CAR-based strategies to challenging field of solid tumors. Our findings support the exploration of CAR⁺.CIK in clinical trials against high grade STS.

Purpose: No effective therapy is available for unresectable soft-tissue sarcomas (STS). This unmet clinical need prompted us to test whether chondroitin sulfate proteoglycan 4 (CSPG4)-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CAR.CIK) are effective in eliminating tumor cells derived from multiple STS histotypes in vitro and in immunodeficient mice. Experimental Design: The experimental platform included patient-derived CAR.CIK and cell lines established from multiple STS histotypes. CAR.CIK were transduced with a retroviral vector encoding second-generation CSPG4-specific CAR (CSPG4-CAR) with 4-1BB costimulation. The functional activity of CSPG4-CAR.CIK was explored in vitro, in two- and three-dimensional STS cultures, and in three in vivo STS xenograft models. Results: CSPG4-CAR.CIK were efficiently generated from patients with STS. CSPG4 was highly expressed in multiple STS histotypes by in silico analysis and on all 16 STS cell lines tested by flow cytometry. CSPG4-CAR.CIK displayed superior in vitro cytolytic activity against multiple STS histotypes as compared with paired unmodified control CIK. CSPG4-CAR.CIK also showed strong antitumor activity against STS spheroids; this effect was associated with tumor recruitment, infiltration, and matrix penetration. CSPG4-CAR.CIK significantly delayed or reversed tumor growth in vivo in three STS xenograft models (leiomyosarcoma, undifferentiated pleomorphic sarcoma, and fibrosarcoma). Tumor growth inhibition persisted for up to 2 weeks following the last administration of CSPG4-CAR.CIK. Conclusions: This study has shown that CSPG4-CAR.CIK effectively targets multiple STS histotypes in vitro and in immunodeficient mice. These results provide a strong rationale to translate the novel strategy we have developed into a clinical setting.

Purpose: BRAF and MEK inhibitors (BRAF/MEKi) favor melanoma-infiltrating lymphocytes, providing the rationale for current combinatorial trials with anti–PD-1 antibody. A portion of melanoma cells may express PD-1, and anti–PD-1 antibody could have a direct antitumor effect. Here, we explore whether BRAF/MEKi modulate rates of PD-1+ melanoma cells, supporting an additional—lymphocyte-independent—basis for their therapeutic combination with anti–PD-1 antibody. Experimental Design: With data mining and flow cytometry, we assessed PD-1, PD-L1/2 expression on melanoma cell lines (CCLE, N = 61; validation cell lines, N = 7) and melanoma tumors (TCGA, N = 214). We explored in vitro how BRAF/MEKi affect rates of PD-1+, PD-L1/2+ melanoma cells, and characterized the proliferative and putative stemness features of PD-1+ melanoma cells. We tested the functional lymphocyte-independent effect of anti–PD-1 antibody alone and in combination with BRAF/MEKi in vitro and in an in vivo immunodeficient murine model. Results: PD-1 is consistently expressed on a small subset of melanoma cells, but PD-1+ cells increase to relevant rates during BRAF/MEKi treatment [7.3% (5.6–14.2) vs. 1.5% (0.7–3.2), P = 0.0156; N = 7], together with PD-L2+ melanoma cells [8.5% (0.0–63.0) vs. 1.5% (0.2–43.3), P = 0.0312; N = 7]. PD-1+ cells proliferate less than PD-1− cells (avg. 65% less; t = 7 days) and are preferentially endowed with stemness features. In vivo, the direct anti-melanoma activity of PD-1 blockage as monotherapy was negligible, but its association with BRAF/MEKi significantly delayed the development of drug resistance and tumor relapse. Conclusions: BRAF/MEKi increase the rates of PD-1+ melanoma cells that may sustain tumor relapse, providing a lymphocyte-independent rationale to explore combinatory strategies with anti–PD-1 antibody. Clin Cancer Res; 24(14); 3377–85. ©2018 AACR.

The term “cancer stem cells” (CSCs) commonly refers to a subset of tumor cells endowed with stemness features, potentially involved in chemo-resistance and disease relapses. CSCs may present peculiar immunogenic features influencing their homeostasis within the tumor microenvironment. The susceptibility of CSCs to recognition and targeting by the immune system is a relevant issue and matter of investigation, especially considering the multiple emerging immunotherapy strategies. Adoptive cellular immunotherapies, especially those strategies encompassing the genetic redirection with chimeric antigen receptors (CAR), hold relevant promise in several tumor settings and might in theory provide opportunities for selective elimination of CSC subsets. Initial dedicated preclinical studies are supporting the potential targeting of CSCs by cellular immunotherapies, indirect evidence from clinical studies may be derived and new studies are ongoing. Here we review the main issues related to the putative immunogenicity of CSCs, focusing on and highlighting the existing evidence and opportunities for cellular immunotherapy approaches with T and non-T antitumor lymphocytes.