Stealth transgenes enable CAR-T cells to evade host immune responses.

BACKGROUND: Adoptive cell therapy, such as chimeric antigen receptor (CAR)-T cell therapy, has improved patient outcomes for hematological malignancies. Currently, four of the six FDA-approved CAR-T cell products use the FMC63-based αCD19 single-chain variable fragment, derived from a murine monoclonal antibody, as the extracellular binding domain. Clinical studies demonstrate that patients develop humoral and cellular immune responses to the non-self CAR components of autologous CAR-T cells or donor-specific antigens of allogeneic CAR-T cells, which is thought to potentially limit CAR-T cell persistence and the success of repeated dosing. METHODS: In this study, we implemented a one-shot approach to prevent rejection of engineered T cells by simultaneously reducing antigen presentation and the surface expression of both Classes of the major histocompatibility complex (MHC) via expression of the viral inhibitors of transporter associated with antigen processing (TAPi) in combination with a transgene coding for shRNA targeting class II MHC transactivator (CIITA). The optimal combination was screened in vitro by flow cytometric analysis and mixed lymphocyte reaction assays and was validated in vivo in mouse models of leukemia and lymphoma. Functionality was assessed in an autologous setting using patient samples and in an allogeneic setting using an allogeneic mouse model. RESULTS: The combination of the Epstein-Barr virus TAPi and an shRNA targeting CIITA was efficient and effective at reducing cell surface MHC classes I and II in αCD19 'stealth' CAR-T cells while retaining in vitro and in vivo antitumor functionality. Mixed lymphocyte reaction assays and IFNγ ELISpot assays performed with T cells from patients previously treated with autologous αCD19 CAR-T cells confirm that CAR T cells expressing the stealth transgenes evade allogeneic and autologous anti-CAR responses, which was further validated in vivo. Importantly, we noted anti-CAR-T cell responses in patients who had received multiple CAR-T cell infusions, and this response was reduced on in vitro restimulation with autologous CARs containing the stealth transgenes. CONCLUSIONS: Together, these data suggest that the proposed stealth transgenes may reduce the immunogenicity of autologous and allogeneic cellular therapeutics. Moreover, patient data indicate that repeated doses of autologous FMC63-based αCD19 CAR-T cells significantly increased the anti-CAR T cell responses in these patients.

Mesothelin CAR T Cells Secreting Anti-FAP/Anti-CD3 Molecules Efficiently Target Pancreatic Adenocarcinoma and its Stroma.

PURPOSE: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells). EXPERIMENTAL DESIGN: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids. RESULTS: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors. CONCLUSIONS: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.

Non-cleavable hinge enhances avidity and expansion of CAR-T cells for acute myeloid leukemia.

Chimeric antigen receptor (CAR) T cell therapy is effective in lymphoid malignancies, but there has been limited data in myeloid cancers. Here, we start with a CD27-based CAR to target CD70 ("native") in acute myeloid leukemia (AML), and we find modest efficacy in vivo, consistent with prior reports. We then use orthogonal approaches to increase binding on both the tumor and CAR-T cell sides of the immune synapse: a pharmacologic approach (azacitidine) to increase antigen density of CD70 in myeloid tumors, and an engineering approach to stabilize binding of the CAR to CD70. To accomplish the latter, we design a panel of hinge-modified regions to mitigate cleavage of the extracellular portion of CD27. Our CD8 hinge and transmembrane-modified CD70 CAR-T cells are less prone to cleavage, have enhanced binding avidity, and increased expansion, leading to more potent in vivo activity. This enhanced CD70-targeted CAR is a promising candidate for further clinical development.

CAR T cell killing requires the IFNγR pathway in solid but not liquid tumours.

Chimeric antigen receptor (CAR) therapy has had a transformative effect on the treatment of haematologic malignancies1-6, but it has shown limited efficacy against solid tumours. Solid tumours may have cell-intrinsic resistance mechanisms to CAR T cell cytotoxicity. Here, to systematically identify potential resistance pathways in an unbiased manner, we conducted a genome-wide CRISPR knockout screen in glioblastoma, a disease in which CAR T cells have had limited efficacy7,8. We found that the loss of genes in the interferon-γ receptor (IFNγR) signalling pathway (IFNGR1, JAK1 or JAK2) rendered glioblastoma and other solid tumours more resistant to killing by CAR T cells both in vitro and in vivo. However, loss of this pathway did not render leukaemia or lymphoma cell lines insensitive to CAR T cells. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell-adhesion pathways after exposure to CAR T cells. We found that loss of IFNγR1 in glioblastoma cells reduced overall CAR T cell binding duration and avidity. The critical role of IFNγR signalling in susceptibility of solid tumours to CAR T cells is surprising, given that CAR T cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumours, IFNγR signalling was required for sufficient adhesion of CAR T cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumours differ in their interactions with CAR T cells and suggests that enhancing binding interactions between T cells and tumour cells may yield improved responses in solid tumours.

Tumor Interferon Signaling Is Regulated by a lncRNA INCR1 Transcribed from the PD-L1 Locus.

Tumor interferon (IFN) signaling promotes PD-L1 expression to suppress T cell-mediated immunosurveillance. We identify the IFN-stimulated non-coding RNA 1 (INCR1) as a long noncoding RNA (lncRNA) transcribed from the PD-L1 locus and show that INCR1 controls IFNγ signaling in multiple tumor types. Silencing INCR1 decreases the expression of PD-L1, JAK2, and several other IFNγ-stimulated genes. INCR1 knockdown sensitizes tumor cells to cytotoxic T cell-mediated killing, improving CAR T cell therapy. We discover that PD-L1 and JAK2 transcripts are negatively regulated by binding to HNRNPH1, a nuclear ribonucleoprotein. The primary transcript of INCR1 binds HNRNPH1 to block its inhibitory effects on the neighboring genes PD-L1 and JAK2, enabling their expression. These findings introduce a mechanism of tumor IFNγ signaling regulation mediated by the lncRNA INCR1 and suggest a therapeutic target for cancer immunotherapy.

Glioblastoma infiltration of both tumor- and virus-antigen specific cytotoxic T cells correlates with experimental virotherapy responses.

The mode of action for oncolytic viruses (OVs) in cancer treatment is thought to depend on a direct initial cytotoxic effect against infected tumor cells and subsequent activation of immune cell responses directed against the neoplasm. To study both of these effects in a mouse model of glioblastoma (GBM), we employed murine GBM cells engineered to constitutively express the type I Herpes Simplex Virus (HSV1) HSV-1 receptor, nectin-1, to allow for more efficient infection and replication by oncolytic HSV (oHSV). These cells were further engineered with a surrogate tumor antigen to facilitate assays of T cell activity. We utilized MRI-based volumetrics to measure GBM responses after injection with the oHSV and bioluminescent imaging (BLI) to determine oHSV replicative kinetics in the injected tumor mass. We found increased infiltration of both surrogate tumor antigen- and oHSV antigen-specific CD8+ T cells within 7 days after oHSV injection. There was no increase in tumor infiltrating CD8+ T cells expressing "exhaustion" markers, yet oHSV infection led to a reduction in PD-1+ CD8+ T cells in injected GBMs and an increase in IFNγ+ CD8+ T cells. There was a significant direct correlation between oHSV-mediated reduction in GBM volume and increased infiltration of both viral and tumor antigen-specific CD8+ T cells, as well as oHSV intratumoral gene activity. These findings imply that CD8+ T cell cytotoxicity against both tumor and viral antigens as well as intratumoral oHSV gene expression are important in oHSV-mediated GBM therapy.

Cytomegalovirus promotes murine glioblastoma growth via pericyte recruitment and angiogenesis.

Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM); however, a mechanistic connection in vivo has not been established. The purpose of this study is to characterize the effects of murine CMV (MCMV) on GBM growth in murine models. Syngeneic GBM models were established in mice perinatally infected with MCMV. We found that tumor growth was markedly enhanced in MCMV+ mice, with a significant reduction in overall survival compared with that of controls (P < 0.001). We observed increased angiogenesis and tumor blood flow in MCMV+ mice. MCMV reactivation was observed in intratumoral perivascular pericytes and tumor cells in mouse and human GBM specimens, and pericyte coverage of tumor vasculature was strikingly augmented in MCMV+ mice. We identified PDGF-D as a CMV-induced factor essential for pericyte recruitment, angiogenesis, and tumor growth. The antiviral drug cidofovir improved survival in MCMV+ mice, inhibiting MCMV reactivation, PDGF-D expression, pericyte recruitment, and tumor angiogenesis. These data show that MCMV potentiates GBM growth in vivo by increased pericyte recruitment and angiogenesis due to alterations in the secretome of CMV-infected cells. Our model provides evidence for a role of CMV in GBM growth and supports the application of antiviral approaches for GBM therapy.

Arming an Oncolytic Herpes Simplex Virus Type 1 with a Single-chain Fragment Variable Antibody against PD-1 for Experimental Glioblastoma Therapy.

PURPOSE: Glioblastoma (GBM) is resistant to standard of care. Immune checkpoints inhibitors (such as anti-PD-1 mAbs) efficiently restore antitumor T-cell activity. We engineered a new oncolytic herpes simplex virus (oHSV) expressing a single-chain antibody against PD-1 (scFvPD-1) to evaluate its efficacy in mouse models of GBM. EXPERIMENTAL DESIGN: NG34scFvPD-1 expresses the human GADD34 gene transcriptionally controlled by the Nestin promoter to allow replication in GBM cells and a scFvPD-1 cDNA transcriptionally controlled by the CMV promoter. ELISA assays were performed to detect binding of scFvPD-1 to mouse and human PD-1. In vitro cytotoxicity and replication assays were performed to measure NG34scFvPD-1 oncolysis, and scFvPD-1 expression and secretion were determined. In vivo survival studies using orthotopic mouse GBM models were performed to evaluate the therapeutic potency of NG34scFvPD-1. RESULTS: NG34scFvPD-1-infected GBM cells express and secrete scFvPD-1 that binds mouse PD-1. The introduction of the scFvPD-1 sequence in the viral backbone does not alter the oncolytic properties of NG34scFvPD-1. In situ NG34scFvPD-1 treatment improved the survival with a tail of durable survivorship in 2 syngeneic immunocompetent mouse models of GBM. Mice that survived the first GBM challenge rejected the second challenge of GBM when implanted in the contralateral hemisphere. However, this was not true when athymic mice were employed as the recipients of the second challenge, consistent with the need for an intact immune system to obtain a memory response. CONCLUSIONS: NG34scFvPD-1 treatment induces a durable antitumor response in 2 preclinical mouse models of GBM with evidence for antitumor memory.

Anticancer activity of osmium(VI) nitrido complexes in patient-derived glioblastoma initiating cells and in vivo mouse models.

Glioblastoma is the most prevalent and lethal primary intrinsic brain tumor with a median patient survival of less than two years, even with the optimal standard of care, namely, surgical resection followed by radiotherapy with adjuvant temozolomide chemotherapy. Long-term survival is extremely rare and there is a tremendous need for novel GBM therapies. Following our prior reports on the anticancer activity of osmium(VI) nitrido compounds and their effectiveness against cancer initiating cells, we investigated the efficacy of Os(VI) on GBM initiating cells in vitro and in vivo. Conventional MTT and 3D cytotoxicity assays revealed that patient-derived GBM models were sensitive to cisplatin, TMZ, and two Os(IV) derivatives. Rapid cell death occurred at low micromolar concentrations of the Os(IV) compounds. Cell cycle analysis, Os uptake studies, and cellular distribution experiments provided further insight into the anticancer properties of these compounds, indicating differential uptake for both compounds and a modest G2/M arrest after treatment. Moreover, in vivo experiments showed a significant increase in survival after a single intracranial chemotherapeutic injection, results that warrant further studies using this approach.

Role of NK cells in immunotherapy and virotherapy of solid tumors.

Although natural killer (NK) cells are endowed with powerful cytolytic activity against cancer cells, their role in different therapies against solid tumors has not yet been fully elucidated. Their interactions with various elements of the tumor microenvironment as well as their possible effects in contributing to and/or limiting oncolytic virotherapy render this potential immunotherapeutic tool still difficult to exploit at the bedside. Here, we will review the current literature with the aim of providing new hints to manage this powerful cell type in future innovative therapies, such as the use of NK cells in combination with new cytokines, specific mAbs (inducing ADCC), Tyr-Kinase inhibitors, immunomodulatory drugs and/or the design of oncolytic viruses aimed at optimizing the effect of NK cells in virotherapy.

Modulation of CD112 by the alphaherpesvirus gD protein suppresses DNAM-1-dependent NK cell-mediated lysis of infected cells.

Natural killer (NK) cells are key players in the innate response to viruses, including herpesviruses. In particular, the variety of viral strategies to modulate the recognition of certain herpesviruses witnesses the importance of NK cells in the control of this group of viruses. Still, NK evasion strategies have remained largely elusive for the largest herpesvirus subfamily, the alphaherpesviruses. Here, we report that the gD glycoprotein of the alphaherpesviruses pseudorabies virus (PRV) and herpes simplex virus 2 (HSV-2) displays previously uncharacterized immune evasion properties toward NK cells. Expression of gD during infection or transfection led to degradation and consequent down-regulation of CD112, a ligand for the activating NK receptor DNAX accessory molecule 1 (DNAM-1). CD112 downregulation resulted in a reduced ability of DNAM-1 to bind to the surface of both virus-infected and gD-transfected cells. Consequently, expression of gD suppressed NK cell degranulation and NK cell-mediated lysis of PRV- or HSV-2-infected cells. These data identify an alphaherpesvirus evasion strategy from NK cells and point out that interactions between viral envelope proteins and host cell receptors can have biological consequences that stretch beyond virus entry.

Natural killer cells: frequency, phenotype and function in healthy cats.

Natural killer (NK) cells play a central role in innate immunity and have been shown to influence adaptive immune responses as well. This study aimed to provide a general NK cell quantification and phenotyping in several compartments of healthy cats and assess their functional properties. The results indicated that NK numbers, both absolute and relative, and phenotype mostly correspond with those found in bovine, ovine, human and murine immunology. However, there were also distinct differences, especially with regard to the expression of the integrin CD11b and the selectin CD62L (between 10 and 30% of feline NK cells stain positive for these markers) and the relative frequencies in lymph nodes (6.7%), which stand central in NK cell development. Caution should be taken when extrapolating findings on NK cell properties over species, notwithstanding the generally accepted evolutionary conservation of NK cells and their subtypes. It was also shown that K562 cells, the 'golden' target cell line for NK functionality tests did not work for feline cells. The feline kidney cell line CRFK proved to be very responsive to NK- and NKT-mediated lysis and therefore, represents an ideal alternative target. This study is a good reference for NK cell numbers, both absolute and relative, phenotype and function in several anatomical compartments of healthy cats and for cat-specific cytotoxic assays involving both NK and NKT cells.