CAR-T Cells and Oncolytic Viruses: Joining Forces to Overcome the Solid Tumor Challenge

Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells has resulted in unprecedented rates of long-lasting complete responses in patients with leukemia and lymphoma. However, despite the impressive results in patients with hematologic malignancies, CAR-T cells have showed limited effect against solid cancers. New approaches will need to simultaneously overcome the multiple challenges that CAR-T cells encounter in solid tumors, including the immunosuppressive tumor microenvironment and heterogeneity of antigen expression. Oncolytic viruses are lytic and immunogenic anti-cancer agents with the potential to synergize with CAR-T cells for the treatment of solid tumors. In addition, viruses can be further modified to deliver therapeutic transgenes selectively to the tumor microenvironment, which could enhance the effector functions of tumor-specific T cells. This review summarizes the major limitations of CAR-T cells in solid tumors and discusses the potential role for oncolytic viruses as partners for CAR-T cells in the fight against cancer

mRNAs encoding IL-12 and a decoy-resistant variant of IL-18 synergize to engineer T cells for efficacious intratumoral adoptive immunotherapy

Interleukin-12 (IL-12) gene transfer enhances the therapeutic potency of adoptive T cell therapies. We previously reported that transient engineering of tumor-specific CD8 T cells with IL-12 mRNA enhanced their systemic therapeutic efficacy when delivered intratumorally. Here, we mix T cells engineered with mRNAs to express either single-chain IL-12 (scIL-12) or an IL-18 decoy-resistant variant (DRIL18) that is not functionally hampered by IL-18 binding protein (IL-18BP). These mRNA-engineered T cell mixtures are repeatedly injected into mouse tumors. Pmel-1 T cell receptor (TCR)-transgenic T cells electroporated with scIL-12 or DRIL18 mRNAs exert powerful therapeutic effects in local and distant melanoma lesions. These effects are associated with T cell metabolic fitness, enhanced miR-155 control on immunosuppressive target genes, enhanced expression of various cytokines, and changes in the glycosylation profile of surface proteins, enabling adhesiveness to E-selectin. Efficacy of this intratumoral immunotherapeutic strategy is recapitulated in cultures of tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR) T cells on IL-12 and DRIL18 mRNA electroporation.Fil: Olivera, Irene. Universidad de Navarra; España. Instituto de Investigación Sanitaria de Navarra; EspañaFil: Bolaños, Elixabet. Universidad de Navarra; España. Instituto de Investigación Sanitaria de Navarra; EspañaFil: Gonzalez Gomariz, Jose. Universidad de Navarra; España. Instituto de Investigación Sanitaria de Navarra; EspañaFil: Hervas Stubbs, Sandra. Universidad de Navarra; España. Instituto de Investigación Sanitaria de Navarra; EspañaFil: Mariño, Karina Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Luri Rey, Carlos. Universidad de Navarra; España. Instituto de Investigación Sanitaria de Navarra; EspañaFil: Etxeberria, Iñaki. Universidad de Navarra; España. Instituto de Investigación Sanitaria de Navarra; EspañaFil: Cirella, Assunta. Universidad de Navarra; España. Instituto de Investigación Sanitaria de Navarra; EspañaFil: Egea, Josune. Universidad de Navarra; España. Instituto de Investigación Sanitaria de Navarra; EspañaFil: Glez Vaz, Javier. Universidad de Navarra; España. Instituto de Investigación Sanitaria de Navarra; EspañaFil: Garasa, Saray. Universidad de Navarra; España. Instituto de Investigación Sanitaria de Navarra; España. Centro de Investigación Biomédica en Red de Cáncer ; EspañaFil: Alvarez, Maite. Universidad de Navarra; España. Instituto de Investigación Sanitaria de Navarra; España. Centro de Investigación Biomédica en Red de Cáncer ; EspañaFil: Eguren Santamaria, Iñaki. Universidad de Navarra; España. Instituto de Investigación Sanitaria de Navarra; EspañaFil: Guedan, Sonia. Institut D'investigacions Biomèdiques August Pi Isunyer; EspañaFil: Sanmamed, Miguel F.. Universidad de Navarra; España. Instituto de Investigación Sanitaria de Navarra; España. Centro de Investigación Biomédica en Red de Cáncer ; EspañaFil: Berraondo, Pedro. Universidad de Navarra; España. Instituto de Investigación Sanitaria de Navarra; España. Centro de Investigación Biomédica en Red de Cáncer ; EspañaFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Teijeira, Alvaro. Universidad de Navarra; España. Instituto de Investigación Sanitaria de Navarra; EspañaFil: Melero, Ignacio. Universidad de Navarra; España. Instituto de Investigación Sanitaria de Navarra; España. Centro de Investigación Biomédica en Red de Cáncer ; España. Clínica Universidad de Navarra; Españ

Engineering and Design of Chimeric Antigen Receptors

T cells engineered with chimeric antigen receptors (CARs) have emerged as a potent new class of therapeutics for cancer, based on their remarkable potency in blood cancers. Since the first clinical reports of their efficacy emerged 7 years ago, investigators have focused on the mechanisms and properties that make CARs effective or toxic, and their effects on T cell biology. Novel CAR designs coupled with improvements in gene transfer technology, incorporating advances in gene editing, have the potential to increase access to engineered cell therapies, as well as improve their potency in solid tumors. Keywords: CAR T cells, immunotherapy, chimeric antigen receptors, cance

Genome Editing in Engineered T Cells for Cancer Immunotherapy

Advanced gene transfer technologies and profound immunological insights have enabled substantial increases in the efficacy of anticancer adoptive cellular therapy (ACT). In recent years, the U.S. Food and Drug Administration and European Medicines Agency have approved six engineered T cell therapeutic products, all chimeric antigen receptor-engineered T cells directed against B cell malignancies. Despite encouraging clinical results, engineered T cell therapy is still constrained by challenges, which could be addressed by genome editing. As RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats technology passes its 10-year anniversary, we review emerging applications of genome editing approaches designed to (1) overcome resistance to therapy, including cancer immune evasion mechanisms; (2) avoid unwanted immune reactions related to allogeneic T cell products; (3) increase fitness, expansion capacity, persistence, and potency of engineered T cells, while preserving their safety profile; and (4) improve the ability of therapeutic cells to resist immunosuppressive signals active in the tumor microenvironment. Overall, these innovative approaches should widen the safe and effective use of ACT to larger number of patients affected by cancer

Minimal RB-responsive E1A Promoter Modification to Attain Potency, Selectivity, and Transgene-arming Capacity in Oncolytic Adenoviruses

Oncolytic adenoviruses are promising anticancer agents due to their ability to self-amplify at the tumor mass. However, tumor stroma imposes barriers difficult to overcome by these agents. Transgene expression is a valuable strategy to counteract these limitations and to enhance antitumor activity. For this purpose, the genetic backbone in which the transgene is inserted should be optimized to render transgene expression compatible with the adenovirus replication cycle and to keep genome size within the encapsidation size limit. In order to design a potent and selective oncolytic adenovirus that keeps intact all the viral functions with minimal increase in genome size, we inserted palindromic E2F-binding sites into the endogenous E1A promoter. The insertion of these sites controlling E1A-Δ24 results in a low systemic toxicity profile in mice. Importantly, the E2F-binding sites also increased the cytotoxicity and the systemic antitumor activity relative to wild-type adenovirus in all cancer models tested. The low toxicity and the increased potency results in improved antitumor efficacy after systemic injection and increased survival of mice carrying tumors. Furthermore, the constrained genome size of this backbone allows an efficient and potent expression of transgenes, indicating that this virus holds promise for overcoming the limitations of oncolytic adenoviral therapy

Single residue in CD28-costimulated CAR-T cells limits long-term persistence and antitumor durability

Chimeric antigen receptor–T (CAR-T) cell therapies can eliminate relapsed and refractory tumors, but the durability of antitumor activity requires in vivo persistence. Differential signaling through the CAR costimulatory domain can alter the T cell metabolism, memory differentiation, and influence long-term persistence. CAR-T cells costimulated with 4-1BB or ICOS persist in xenograft models but those constructed with CD28 exhibit rapid clearance. Here, we show that a single amino acid residue in CD28 drove T cell exhaustion and hindered the persistence of CD28-based CAR-T cells and changing this asparagine to phenylalanine (CD28-YMFM) promoted durable antitumor control. In addition, CD28-YMFM CAR-T cells exhibited reduced T cell differentiation and exhaustion as well as increased skewing toward Th17 cells. Reciprocal modification of ICOS-containing CAR-T cells abolished in vivo persistence and antitumor activity. This finding suggests modifications to the costimulatory domains of CAR-T cells can enable longer persistence and thereby improve antitumor response

Pancreatic cancer therapy with combined mesothelin-redirected chimeric antigen receptor T cells and cytokine-armed oncolytic adenoviruses

Pancreatic ductal adenocarcinoma (PDA) is characterized by its highly immunosuppressive tumor microenvironment (TME) that limits T cell infiltration and induces T cell hypofunction. Mesothelinredirected chimeric antigen receptor T cell (meso-CAR T cell) therapy has shown some efficacy in clinical trials but antitumor efficacy remains modest. We hypothesized that combined meso-CAR T cells with an oncolytic adenovirus expressing TNF-α and IL-2 (Ad5/3-E2F-D24-TNFa-IRES-IL2, or OAd-TNFa-IL2) would improve efficacy. OAd-TNFa-IL2 enhanced the antitumor efficacy of meso-CAR T cells in human-PDA-xenograft immunodeficient mice and efficacy was associated with robustly increased tumor-infiltrating lymphocytes (TILs), enhanced and prolonged T cell function. Mice treated with parental OAd combined with meso-CAR T developed tumor metastasis to the lungs even if primary tumors were controlled. However, no mice treated with combined OAd-TNFa-IL2 and meso-CAR T died of tumor metastasis. We also evaluated this approach in a syngeneic mouse tumor model by combining adenovirus expressing murine TNF-α and IL-2 (Ad-mTNFa-mIL2) and mouse CAR T cells. This approach induced significant tumor regression in mice engrafted with highly aggressive and immunosuppressive PDA tumors. Ad-mTNFa-mIL2 increased both CAR T cell and host T cell infiltration to the tumor and altered host tumor immune status with M1 polarization of macrophages and increased dendritic cell maturation. These findings indicate that combining cytokine-armed oncolytic adenovirus to enhance the efficacy of CAR T cell therapy is a promising approach to overcome the immunosuppressive TME for the treatment of PDA

CAR affinity modulates the sensitivity of CAR-T cells to PD-1/PD-L1-mediated inhibition

Abstract Chimeric antigen receptor (CAR)-T cell therapy for solid tumors faces significant hurdles, including T-cell inhibition mediated by the PD-1/PD-L1 axis. The effects of disrupting this pathway on T-cells are being actively explored and controversial outcomes have been reported. Here, we hypothesize that CAR-antigen affinity may be a key factor modulating T-cell susceptibility towards the PD-1/PD-L1 axis. We systematically interrogate CAR-T cells targeting HER2 with either low (LA) or high affinity (HA) in various preclinical models. Our results reveal an increased sensitivity of LA CAR-T cells to PD-L1-mediated inhibition when compared to their HA counterparts by using in vitro models of tumor cell lines and supported lipid bilayers modified to display varying PD-L1 densities. CRISPR/Cas9-mediated knockout (KO) of PD-1 enhances LA CAR-T cell cytokine secretion and polyfunctionality in vitro and antitumor effect in vivo and results in the downregulation of gene signatures related to T-cell exhaustion. By contrast, HA CAR-T cell features remain unaffected following PD-1 KO. This behavior holds true for CD28 and ICOS but not 4-1BB co-stimulated CAR-T cells, which are less sensitive to PD-L1 inhibition albeit targeting the antigen with LA. Our findings may inform CAR-T therapies involving disruption of PD-1/PD-L1 pathway tailored in particular for effective treatment of solid tumors