VEGFR2 as a target for CAR T cell therapy of Ewing sarcoma.

BACKGROUND: T cells engineered to express chimeric antigen receptors (CARs) are a novel modality to treat refractory cancers. The development of CAR T cells against Ewing sarcoma (EwS) is limited by a lack of targetable surface antigens. We investigated vascular endothelial growth factor receptor 2 (VEGFR2) expressed on tumor-associated blood vessels as potential CAR target in this cancer. METHODS: Expression of VEGFR2 was studied by immunohistochemistry in human EwS biopsies and in murine xenografts and by flow cytometry in EwS cell lines. CARs with short, medium, and long hinge domains against either human or murine VEGFR2 were generated and expressed in human T cells by retroviral gene transfer. The capacity of the individual CARs to activate T cells in response to VEGFR2-expressing cells was compared in vitro. RESULTS: Tumor-associated endothelial cells in human EwS biopsies and in xenografts expressed VEGFR2. Tumor cells in the majority of EwS biopsies were also VEGFR2-positive. Following modification with anti-mouse or anti-human VEGFR2-specific CAR genes, T cells specifically lysed VEGFR2-expressing target cells of the respective species. CAR T cells with short-length or medium-length hinge domains were functionally superior over those with the long hinge region by in vitro parameters, including antigen-specific degranulation responses, lysis of tumor spheroids, tumor necrosis factor α secretion, sequential killing, and proliferation. CONCLUSIONS: VEGFR2 is consistently expressed on endothelial cells of the tumor stroma in EwS and thus is a candidate target for CAR T cells in this cancer. Among various VEGFR2-specific CARs, a construct with a short hinge domain was chosen to be further developed toward clinical translation.

EZH2 Inhibition in Ewing Sarcoma Upregulates GD2 Expression for Targeting with Gene-Modified T Cells.

Chimeric antigen receptor (CAR) engineering of T cells allows one to specifically target tumor cells via cell surface antigens. A candidate target in Ewing sarcoma is the ganglioside GD2, but heterogeneic expression limits its value. Here we report that pharmacological inhibition of Enhancer of Zeste Homolog 2 (EZH2) at doses reducing H3K27 trimethylation, but not cell viability, selectively and reversibly induces GD2 surface expression in Ewing sarcoma cells. EZH2 in Ewing sarcoma cells directly binds to the promoter regions of genes encoding for two key enzymes of GD2 biosynthesis, and EZH2 inhibition enhances expression of these genes. GD2 surface expression in Ewing sarcoma cells is not associated with distinct in vitro proliferation, colony formation, chemosensitivity, or in vivo tumorigenicity. Moreover, disruption of GD2 synthesis by gene editing does not affect its in vitro behavior. EZH2 inhibitor treatment sensitizes Ewing sarcoma cells to effective cytolysis by GD2-specific CAR gene-modified T cells. In conclusion, we report a clinically applicable pharmacological approach for enhancing efficacy of adoptively transferred GD2-redirected T cells against Ewing sarcoma, by enabling recognition of tumor cells with low or negative target expression.

Programmed cell death ligand 1 (PD-L1) expression is not a predominant feature in Ewing sarcomas.

BACKGROUND: Programmed cell death 1 (PD-1) receptor engagement on T cells by its ligand programmed cell death ligand 1 (PD-L1) is a key mechanism of immune escape, and antibody blockade of the interaction has emerged as an effective immunotherapeutic strategy in some cancers. The role and relevance of the PD-1 checkpoint in Ewing sarcoma (EwS) is not yet understood. PROCEDURE: Here, we investigated expression of PD-L1 and PD-1 in EwS by immunohistochemistry analysis of pretherapeutic tumor biopsies and in tumor xenografts following treatment with human T cells engineered to express a chimeric antigen receptor (CAR) against the tumor-associated antigen GD2 . PD-L1 surface expression in EwS cell lines was assessed by flow cytometry. RESULTS: PD-L1 expression was not detectable on tumor cells in any of the 60 EwS biopsies. Infiltrating PD-L1 positive T cells were found in one tumor, and four biopsies contained PD-1-positive T cells. Of 13 EwS cell lines, none constitutively expressed PD-L1 on the cell surface. Interferon-γ cytokine stimulation induced upregulation of the ligand on all cell lines. Adoptive therapy with CAR gene-modified T cells in a mouse model did not induce PD-L1 expression in EwS xenografts despite tumor infiltration with PD-1+ CD3+ T cells. CONCLUSIONS: EwS cells can upregulate PD-L1 under inflammatory conditions, but do not express the ligand in the pretherapeutic tumor microenvironment or postexposure to CAR T cells. PD-1 checkpoint blockade alone is thus unlikely to evoke potent immune responses against EwS. Identification of the relevant immune evasion strategies in EwS will be vital for the development of effective immune targeting strategies.

Insulin-like growth factor-1 receptor (IGF-1R) inhibition promotes expansion of human NK cells which maintain their potent antitumor activity against Ewing sarcoma cells.

BACKGROUND: Patients with primary metastatic or relapsed Ewing sarcomas (EwS) have a poor prognosis. While inhibitory insulin-like growth factor 1 receptor (IGF-1R)-specific antibodies have shown single agent activity in some patients with refractory disease, effective therapeutic targeting will rely on optimal combinations with conventional or innovative therapies. Specifically, combination of inhibitory IGF-1R antibodies with adoptive transfer of activated natural killer (NK) cells may have therapeutic benefit in EwS without adding toxicity. PROCEDURE: We investigated the in vitro effects of IGF-1R targeting on the immunological profile of EwS cells and on the survival and tumor targeting capacity of K-562-activated NK cells. RESULTS: IGF-1R inhibition reliably reduced EwS cell viability without affecting expression of immune-modulatory and MHC molecules. In NK cells, we observed a significant superior expansion following in vitro activation in the presence of IGF-1R-specific antibodies, while expression of differentiation markers and activating receptors remained unaffected. Activated NK cells coincubated with EwS cells showed potent degranulation responses unaffected by IGF-1R inhibition. These findings were reproducible in a stimulator cell-free NK cell expansion system, suggesting that direct effects of IGF-R1 antibodies on the IGF-R1 pathway in NK cells induce their activation and expansion. CONCLUSIONS: Activated human NK cells respond to IGF-1R inhibition with superior expansion kinetics while maintaining potent antitumor responses against EwS. Combination of adoptive NK cell transfer with IGF-1R targeting may be an efficient means to eliminate minimal residual disease after conventional therapy and thereby rescue patients at the highest risk of relapse.

Common Ewing sarcoma-associated antigens fail to induce natural T cell responses in both patients and healthy individuals.

Disseminated or relapsed Ewing sarcoma (EwS) has remained fatal in the majority of patients. A promising approach to preventing relapse after conventional therapy is to establish tumor antigen-specific immune control. Efficient and specific T cell memory against the tumor depends on the expansion of rare T cells with native specificity against target antigens overexpressed by the tumor. Candidate antigens in EwS include six-transmembrane epithelial antigen of the prostate-1 (STEAP1), and the human cancer/testis antigens X-antigen family member 1 (XAGE1) and preferentially expressed antigen in melanoma (PRAME). Here, we screened normal donors and EwS patients for the presence of circulating T cells reactive with overlapping peptide libraries of these antigens by IFN-γ Elispot analysis. The majority of 22 healthy donors lacked detectable memory T cell responses against STEAP1, XAGE1 and PRAME. Moreover, ex vivo detection of T cells specific for these antigens in both blood and bone marrow were limited to a minority of EwS patients and required nonspecific T cell prestimulation. Cytotoxic T cells specific for the tumor-associated antigens were efficiently and reliably generated by in vitro priming using professional antigen-presenting cells and optimized cytokine stimulation; however, these T cells failed to interact with native antigen processed by target cells and with EwS cells expressing the antigen. We conclude that EwS-associated antigens fail to induce efficient T cell receptor (TCR)-mediated antitumor immune responses even under optimized conditions. Strategies based on TCR engineering could provide a more effective means to manipulating T cell immunity toward targeted elimination of tumor cells.

High proportions of CD4⁺ T cells among residual bone marrow T cells in childhood acute lymphoblastic leukemia are associated with favorable early responses.

Residual nonmalignant T cells in the bone marrow of patients with acute leukemias may be involved in active immune responses to leukemic cells. Here, we investigated the phenotypic signature of T cells present at diagnosis in 39 pediatric patients with acute lymphoblastic leukemia (ALL) treated within standardized ALL-BFM study protocols. Previously described age associations of lymphocyte subpopulations in the peripheral blood of healthy children were reproduced in leukemic bone marrow. Analysis of individual lymphocyte parameters and risk-associated variables using univariate linear regression models revealed a correlation of higher CD4/CD8 ratios at diagnosis with a favorable bone marrow response on day 15. Separate analysis of CD4⁺ cells with the CD4⁺CD25(hi)FoxP3⁺ T(reg) cell phenotype showed that the association was caused by non-T(reg) CD4⁺ cells. The association of higher CD4/CD8 ratios with a favorable bone marrow response on day 15 of treatment persisted in a cohort extended to 69 patients. We conclude that CD4⁺ non-T(reg) cells in leukemic bone marrow at diagnosis may have a role in early response to treatment. Prospective analysis of the CD4/CD8 ratio in a large cohort of pediatric patients is now needed. Moreover, future experiments will establish the functional role of the individual T cell subsets in immune control in pediatric ALL.

Preclinical Development of CAR T Cells with Antigen-Inducible IL18 Enforcement to Treat GD2-Positive Solid Cancers.

PURPOSE: Cytokine-engineering of chimeric antigen receptor-redirected T cells (CAR T cells) is a promising principle to overcome the limited activity of canonical CAR T cells against solid cancers. EXPERIMENTAL DESIGN: We developed an investigational medicinal product, GD2IL18CART, consisting of CAR T cells directed against ganglioside GD2 with CAR-inducible IL18 to enhance their activation response and cytolytic effector functions in the tumor microenvironment. To allow stratification of patients according to tumor GD2 expression, we established and validated immunofluorescence detection of GD2 on paraffin-embedded tumor tissues. RESULTS: Lentiviral all-in-one vector engineering of human T cells with the GD2-specific CAR with and without inducible IL18 resulted in cell products with comparable proportions of CAR-expressing central memory T cells. Production of IL18 strictly depends on GD2 antigen engagement. GD2IL18CART respond to interaction with GD2-positive tumor cells with higher IFNγ and TNFα cytokine release and more effective target cytolysis compared with CAR T cells without inducible IL18. GD2IL18CART further have superior in vivo antitumor activity, with eradication of GD2-positive tumor xenografts. Finally, we established GMP-compliant manufacturing of GD2IL18CART and found it to be feasible and efficient at clinical scale. CONCLUSIONS: These results pave the way for clinical investigation of GD2IL18CART in pediatric and adult patients with neuroblastoma and other GD2-positive cancers (EU CT 2022-501725-21-00).

Ganglioside SSEA-4 in Ewing sarcoma marks a tumor cell population with aggressive features and is a potential cell-surface immune target.

Carbohydrate markers of immature cells during prenatal human development can be aberrantly expressed in cancers and deserve evaluation as immune targets. A candidate target in Ewing sarcoma is the globo-series ganglioside stage-specific embryonic antigen-4 (SSEA-4). We detected SSEA-4 expression on the cell surface of all of 14 EwS cell lines and in 21 of 31 (68%) primary EwS tumor biopsies. Among paired subpopulations of tumor cells with low versus high SSEA-4 expression, SSEA-4high expression was significantly and consistently associated with functional characteristics of tumor aggressiveness, including higher cell proliferation, colony formation, chemoresistance and propensity to migrate. SSEA-4low versus SSEA-4high expression was not related to expression levels of the EWSR1-FLI1 fusion transcript or markers of epithelial/mesenchymal plasticity. SSEA-4low cells selected from bulk populations regained higher SSEA-4 expression in vitro and during in vivo tumor growth in a murine xenograft model. T cells engineered to express SSEA-4-specific chimeric antigen receptors (CARs) specifically interacted with SSEA-4 positive EwS cells and exerted effective antigen-specific tumor cell lysis in vitro. In conclusion, with its stable expression and functional significance in EwS, SSEA-4 is an attractive therapeutic immune target in this cancer that deserves further evaluation for clinical translation.

CAR T cells as micropharmacies against solid cancers: Combining effector T-cell mediated cell death with vascular targeting in a one-step engineering process.

To enhance the potency of chimeric antigen receptor (CAR) engineered T cells in solid cancers, we designed a novel cell-based combination strategy with an additional therapeutic mode of action. CAR T cells are used as micropharmacies to produce a targeted pro-coagulatory fusion protein, truncated tissue factor (tTF)-NGR, which exerts pro-coagulatory activity and hypoxia upon relocalization to the vascular endothelial cells that invade tumor tissues. Delivery by CAR T cells aimed to induce locoregional tumor vascular infarction for combined immune-mediated and hypoxic tumor cell death. Human T cells that were one-vector gene-modified to express a GD2-specific CAR along with CAR-inducible tTF-NGR exerted potent GD2-specific effector functions while secreting tTF-NGR that activates the extrinsic coagulation pathway in a strictly GD2-dependent manner. In murine models, the CAR T cells infiltrated GD2-positive tumor xenografts, secreted tTF-NGR into the tumor microenvironment and showed a trend towards superior therapeutic activity compared with control cells producing functionally inactive tTF-NGR. In vitro evidence supports a mechanism of hypoxia-mediated enhancement of T cell cytolytic activity. We conclude that combined CAR T cell targeting with an additional mechanism of antitumor action in a one-vector engineering strategy is a promising approach to be further developed for targeted treatment of solid cancers.

Activated human γδ T cells induce peptide-specific CD8+ T-cell responses to tumor-associated self-antigens.

Specific cellular immunotherapy of cancer requires efficient generation and expansion of cytotoxic T lymphocytes (CTLs) that recognize tumor-associated self-antigens. Here, we investigated the capacity of human γδ T cells to induce expansion of CD8+ T cells specific for peptides derived from the weakly immunogenic tumor-associated self-antigens PRAME and STEAP1. Coincubation of aminobisphosphonate-stimulated human peripheral blood-derived γδ T cells (Vγ9+Vδ2+), loaded with HLA-A*02-restricted epitopes of PRAME, with autologous peripheral blood CD8+ T cells stimulated the expansion of peptide-specific cytolytic effector memory T cells. Moreover, peptide-loaded γδ T cells efficiently primed antigen-naive CD45RA+ CD8+ T cells against PRAME peptides. Direct comparisons with mature DCs revealed equal potency of γδ T cells and DCs in inducing primary T-cell responses and peptide-specific T-cell activation and expansion. Antigen presentation by γδ T-APCs was not able to overcome the limited capacity of peptide-specific T cells to interact with targets expressing full-length antigen. Importantly, T cells with regulatory phenotype (CD4+ CD25hiFoxP3+) were lower in cocultures with γδ T cells compared to DCs. In summary, bisphosphonate-activated γδ T cells permit generation of CTLs specific for weakly immunogenic tumor-associated epitopes. Exploiting this strategy for effective immunotherapy of cancer requires strategies that enhance the avidity of CTL responses to allow for efficient targeting of cancer.

The Cellular Tumor Immune Microenvironment of Childhood Solid Cancers: Informing More Effective Immunotherapies.

Common pediatric solid cancers fail to respond to standard immuno-oncology agents relying on preexisting adaptive antitumor immune responses. The adoptive transfer of tumor-antigen specific T cells, such as CAR-gene modified T cells, is an attractive strategy, but its efficacy has been limited. Evidence is accumulating that local barriers in the tumor microenvironment prevent the infiltration of T cells and impede therapeutic immune responses. A thorough understanding of the components of the functional compartment of the tumor microenvironment and their interaction could inform effective combination therapies and novel engineered therapeutics, driving immunotherapy towards its full potential in pediatric patients. This review summarizes current knowledge on the cellular composition and significance of the tumor microenvironment in common extracranial solid cancers of childhood and adolescence, such as embryonal tumors and bone and soft tissue sarcomas, with a focus on myeloid cell populations that are often present in abundance in these tumors. Strategies to (co)target immunosuppressive myeloid cell populations with pharmacological anticancer agents and with selective antagonists are presented, as well as novel concepts aiming to employ myeloid cells to cooperate with antitumor T cell responses.

Relevance of Abnormal KCNN1 Expression and Osmotic Hypersensitivity in Ewing Sarcoma.

Ewing sarcoma (EwS) is a rare and highly malignant bone tumor occurring mainly in childhood and adolescence. Physiologically, the bone is a central hub for Ca2+ homeostasis, which is severely disturbed by osteolytic processes in EwS. Therefore, we aimed to investigate how ion transport proteins involved in Ca2+ homeostasis affect EwS pathophysiology. We characterized the expression of 22 candidate genes of Ca2+-permeable or Ca2+-regulated ion channels in three EwS cell lines and found the Ca2+-activated K+ channel KCa2.1 (KCNN1) to be exceptionally highly expressed. We revealed that KCNN1 expression is directly regulated by the disease-driving oncoprotein EWSR1-FL1. Due to its consistent overexpression in EwS, KCNN1 mRNA could be a prognostic marker in EwS. In a large cohort of EwS patients, however, KCNN1 mRNA quantity does not correlate with clinical parameters. Several functional studies including patch clamp electrophysiology revealed no evidence for KCa2.1 function in EwS cells. Thus, elevated KCNN1 expression is not translated to KCa2.1 channel activity in EwS cells. However, we found that the low K+ conductance of EwS cells renders them susceptible to hypoosmotic solutions. The absence of a relevant K+ conductance in EwS thereby provides an opportunity for hypoosmotic therapy that can be exploited during tumor surgery.

GMP-Compliant Manufacturing of TRUCKs: CAR T Cells targeting GD2 and Releasing Inducible IL-18.

Chimeric antigen receptor (CAR)-engineered T cells can be highly effective in the treatment of hematological malignancies, but mostly fail in the treatment of solid tumors. Thus, approaches using 4th advanced CAR T cells secreting immunomodulatory cytokines upon CAR signaling, known as TRUCKs ("T cells redirected for universal cytokine-mediated killing"), are currently under investigation. Based on our previous development and validation of automated and closed processing for GMP-compliant manufacturing of CAR T cells, we here present the proof of feasibility for translation of this method to TRUCKs. We generated IL-18-secreting TRUCKs targeting the tumor antigen GD2 using the CliniMACS Prodigy® system using a recently described "all-in-one" lentiviral vector combining constitutive anti-GD2 CAR expression and inducible IL-18. Starting with 0.84 x 108 and 0.91 x 108 T cells after enrichment of CD4+ and CD8+ we reached 68.3-fold and 71.4-fold T cell expansion rates, respectively, in two independent runs. Transduction efficiencies of 77.7% and 55.1% was obtained, and yields of 4.5 x 109 and 3.6 x 109 engineered T cells from the two donors, respectively, within 12 days. Preclinical characterization demonstrated antigen-specific GD2-CAR mediated activation after co-cultivation with GD2-expressing target cells. The functional capacities of the clinical-scale manufactured TRUCKs were similar to TRUCKs generated in laboratory-scale and were not impeded by cryopreservation. IL-18 TRUCKs were activated in an antigen-specific manner by co-cultivation with GD2-expressing target cells indicated by an increased expression of activation markers (e.g. CD25, CD69) on both CD4+ and CD8+ T cells and an enhanced release of pro-inflammatory cytokines and cytolytic mediators (e.g. IL-2, granzyme B, IFN-γ, perforin, TNF-α). Manufactured TRUCKs showed a specific cytotoxicity towards GD2-expressing target cells indicated by lactate dehydrogenase (LDH) release, a decrease of target cell numbers, microscopic detection of cytotoxic clusters and detachment of target cells in real-time impedance measurements (xCELLigence). Following antigen-specific CAR activation of TRUCKs, CAR-triggered release IL-18 was induced, and the cytokine was biologically active, as demonstrated in migration assays revealing specific attraction of monocytes and NK cells by supernatants of TRUCKs co-cultured with GD2-expressing target cells. In conclusion, GMP-compliant manufacturing of TRUCKs is feasible and delivers high quality T cell products.

Using stroma-anchoring cytokines to augment ADCC: a phase 1 trial of F16IL2 and BI 836858 for posttransplant AML relapse.

Natural killer (NK) cells are key effectors in cancer immunosurveillance and posttransplant immunity, but deficiency of environmental signals and insufficient tumor recognition may limit their activity. We hypothesized that the antibody-mediated anchoring of interleukin-2 (IL-2) to a spliced isoform of the extracellular matrix (ECM) glycoprotein tenascin-C would potentiate NK-cell-mediated antibody-dependent cellular cytotoxicity against leukemic blasts. In this novel-novel combination, dose-escalation, phase 1 trial, we enrolled patients with posttransplant acute myeloid leukemia (AML) relapse to evaluate the safety, pharmacokinetics, pharmacodynamics, and preliminary activity of the antibody-cytokine fusion F16IL2 (10 × 106 to 20 × 106 IU IV; days 1, 8, 15, and 22 of each 28-day cycle) in combination with the anti-CD33 antibody BI 836858 (10-40 mg IV, 2 days after each F16IL2 infusion). Among the 15 patients (median [range] age, 50 [20-68] years) treated across 4 dose levels (DLs), 6 (40%) had received 2 or 3 prior transplantations. The most frequent adverse events were pyrexia, chills, and infusion-related reactions, which were manageable, transient and of grade ≤2. One dose-limiting toxicity occurred at each of DLs 3 (pulmonary edema) and 4 (graft-versus-host disease). Three objective responses were observed among 7 patients treated at the 2 higher DLs, whereas no responses occurred at the 2 starting DLs. Combination therapy stimulated the expansion and activation of NK cells, including those expressing the FcγRIIIA/CD16 receptor. ECM-targeted IL-2 combined with anti-CD33 immunotherapy represents an innovative approach associated with acceptable safety and encouraging biologic and clinical activity in posttransplant AML relapse. This trial was registered at EudraCT as 2015-004763-37.

Surface expression of the immunotherapeutic target GD2 in osteosarcoma depends on cell confluency.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy of pediatric sarcomas is challenged by the paucity of targetable cell surface antigens. A candidate target in osteosarcoma (OS) is the ganglioside GD2 , but heterogeneous expression of GD2 limits its value. AIM: We aimed to identify mechanisms that upregulate GD2 target expression in OS. METHODS AND RESULTS: GD2 surface expression in OS cells, studied by flow cytometry, was found to vary both among and within individual OS cell lines. Pharmacological approaches, including inhibition of the histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2) and modulation of the protein kinase C, failed to increase GD2 expression. Instead, cell confluency was found to be associated with higher GD2 expression levels both in monolayer cultures and in tumor spheroids. The sensitivity of OS cells to targeting by GD2 -specific CAR T cells was compared in an in vitro cytotoxicity assay. Higher cell confluencies enhanced the sensitivity of OS cells to GD2 -antigen specific, CAR T-cell-mediated in vitro cytolysis. Mechanistic studies revealed that confluency-dependent upregulation of GD2 expression in OS cells is mediated by increased de novo biosynthesis, through a yet unknown mechanism. CONCLUSION: Expression of GD2 in OS cell lines is highly variable and associated with increasing cell confluency in vitro. Strategies for selective upregulation of GD2 are needed to enable effective therapeutic targeting of this antigen in OS.

HLA-G and HLA-E Immune Checkpoints Are Widely Expressed in Ewing Sarcoma but Have Limited Functional Impact on the Effector Functions of Antigen-Specific CAR T Cells.

Immune-inhibitory barriers in the tumor microenvironment of solid cancers counteract effective T cell therapies. Based on our finding that Ewing sarcomas (EwS) respond to chimeric antigen receptor (CAR) gene-modified effector cells through upregulation of human leukocyte antigen G (HLA-G), we hypothesized that nonclassical HLA molecules, HLA-G and HLA-E, contribute to immune escape of EwS. Here, we demonstrate that HLA-G isotype G1 expression on EwS cells does not directly impair cytolysis by GD2-specific CAR T cells (CART), whereas HLA-G1 on myeloid bystander cells reduces CART degranulation responses against EwS cells. HLA-E was induced in EwS cells by IFN-γ stimulation in vitro and by GD2-specific CART treatment in vivo and was detected on tumor cells or infiltrating myeloid cells in a majority of human EwS biopsies. Interaction of HLA-E-positive EwS cells with GD2-specific CART induced upregulation of HLA-E receptor NKG2A. However, HLA-E expressed by EwS tumor cells or by myeloid bystander cells both failed to reduce antitumor effector functions of CART. We conclude that non-classical HLA molecules are expressed in EwS under inflammatory conditions, but have limited functional impact on antigen-specific T cells, arguing against a relevant therapeutic benefit from combining CART therapy with HLA-G or HLA-E checkpoint blockade in this cancer.

Generation of an NFκB-Driven Alpharetroviral "All-in-One" Vector Construct as a Potent Tool for CAR NK Cell Therapy.

Immune cell therapeutics are increasingly applied in oncology. Especially chimeric antigen receptor (CAR) T cells are successfully used to treat several B cell malignancies. Efforts to engineer CAR T cells for improved activity against solid tumors include co-delivery of pro-inflammatory cytokines in addition to CARs, via either constitutive cytokine expression or inducible cytokine expression triggered by CAR recognition of its target antigen-so-called "T cells redirected for universal cytokine-mediated killing" (TRUCKs) or fourth-generation CARs. Here, we tested the hypothesis that TRUCK principles could be expanded to improve anticancer functions of NK cells. A comparison of the functionality of inducible promoters responsive to NFAT or NFκB in NK cells showed that, in contrast to T cells, the inclusion of NFκB-responsive elements within the inducible promoter construct was essential for CAR-inducible expression of the transgene. We demonstrated that GD2CAR-specific activation induced a tight NFκB-promoter-driven cytokine release in NK-92 and primary NK cells together with an enhanced cytotoxic capacity against GD2+ target cells, also shown by increased secretion of cytolytic cytokines. The data demonstrate biologically relevant differences between T and NK cells that are important when clinically translating the TRUCK concept to NK cells for the treatment of solid malignancies.

Overcoming Heterogeneity of Antigen Expression for Effective CAR T Cell Targeting of Cancers.

Chimeric antigen receptor (CAR) gene-modified T cells (CAR T cells) can eradicate B cell malignancies via recognition of surface-expressed B lineage antigens. Antigen escape remains a major mechanism of relapse and is a key barrier for expanding the use of CAR T cells towards solid cancers with their more diverse surface antigen repertoires. In this review we discuss strategies by which cancers become amenable to effective CAR T cell therapy despite heterogeneous phenotypes. Pharmaceutical approaches have been reported that selectively upregulate individual target antigens on the cancer cell surface to sensitize antigen-negative subclones for recognition by CARs. In addition, advanced T cell engineering strategies now enable CAR T cells to interact with more than a single antigen simultaneously. Still, the choice of adequate targets reliably and selectively expressed on the cell surface of tumor cells but not normal cells, ideally by driving tumor growth, is limited, and even dual or triple antigen targeting is unlikely to cure most solid tumors. Innovative receptor designs and combination strategies now aim to recruit bystander cells and alternative cytolytic mechanisms that broaden the activity of CAR-engineered T cells beyond CAR antigen-dependent tumor cell recognition.

Design and Characterization of an "All-in-One" Lentiviral Vector System Combining Constitutive Anti-GD2 CAR Expression and Inducible Cytokines.

Genetically modified T cells expressing chimeric antigen receptors (CARs) so far have mostly failed in the treatment of solid tumors owing to a number of limitations, including an immunosuppressive tumor microenvironment and insufficient CAR T cell activation and persistence. Next-generation approaches using CAR T cells that secrete transgenic immunomodulatory cytokines upon CAR signaling, known as TRUCKs ("T cells redirected for universal cytokine-mediated killing"), are currently being explored. As TRUCKs were engineered by the transduction of T cells with two separate vectors, we developed a lentiviral modular "all-in-one" vector system that combines constitutive CAR expression and inducible nuclear factor of activated T cells (NFAT)-driven transgene expression for more efficient production of TRUCKs. Activation of the GD2-specific CAR via GD2+ target cells induced NFAT promoter-driven cytokine release in primary human T cells, and indicated a tight linkage of CAR-specific activation and transgene expression that was further improved by a modified NFATsyn promoter. As proof-of-concept, we showed that T cells containing the "all-in-one" vector system secrete the immunomodulatory cytokines interleukin (IL)12 or IL18 upon co-cultivation with primary human GD2+ tumor cells, resulting in enhanced effector cell properties and increased monocyte recruitment. This highlights the potential of our system to simplify application of TRUCK-modified T cells in solid tumor therapy.