CAR'TCR-T cells co-expressing CD33-CAR and dNPM1-TCR as superior dual-targeting approach for AML treatment.

Acute myeloid leukemia (AML), a fast-progressing hematological malignancy affecting myeloid cells, is typically treated with chemotherapy or hematopoietic stem cell transplantation. However, approximately half of the patients face relapses and 5-year survival rates are poor. With the goal to facilitate dual-specificity, boosting anti-tumor activity, and minimizing the risk for antigen escape, this study focused on combining chimeric antigen receptor (CAR) and T cell receptor (TCR) technologies. CAR'TCR-T cells, co-expressing a CD33-CAR and a transgenic dNPM1-TCR, revealed increased and prolonged anti-tumor activity in vitro, particularly in case of low target antigen expression. The distinct transcriptomic profile suggested enhanced formation of immunological synapses, activation, and signaling. Complete elimination of AML xenografts in vivo was only achieved with a cell product containing CAR'TCR-T, CAR-T, and TCR-T cells, representing the outcome of co-transduction with two lentiviral vectors encoding either CAR or TCR. A mixture of CAR-T and TCR-T cells, without CAR'TCR-T cells, did not prevent progressive tumor outgrowth and was comparable to treatment with CAR-T and TCR-T cells individually. Overall, our data underscore the efficacy of co-expressing CAR and transgenic TCR in one T cell, and might open a novel therapeutic avenue not only for AML but also other malignancies.

Adoptive cell therapy in paediatric extracranial solid tumours: current approaches and future challenges.

Immunotherapy has ignited hope to cure paediatric solid tumours that resist traditional therapies. Among the most promising methods is adoptive cell therapy (ACT). Particularly, ACT using T cells equipped with chimeric antigen receptors (CARs) has moved into the spotlight in clinical studies. However, the efficacy of ACT is challenged by ACT-intrinsic factors, like lack of activation or T cell exhaustion, as well as immune evasion strategies of paediatric solid tumours, such as their highly immunosuppressive microenvironment. Novel strategies, including ACT using innate-like lymphocytes, innovative cell engineering techniques, and ACT combination therapies, are being developed and will be crucial to overcome these challenges. Here, we discuss the main classes of ACT for the treatment of paediatric extracranial solid tumours, reflect on the available preclinical and clinical evidence supporting promising strategies, and address the challenges that ACT is still facing. Ultimately, we highlight state-of-the-art developments and opportunities for new therapeutic options, which hold great potential for improving outcomes in this challenging patient population.

Joining Forces for Cancer Treatment: From "TCR versus CAR" to "TCR and CAR".

T cell-based immunotherapy has demonstrated great therapeutic potential in recent decades, on the one hand, by using tumor-infiltrating lymphocytes (TILs) and, on the other hand, by engineering T cells to obtain anti-tumor specificities through the introduction of either engineered T cell receptors (TCRs) or chimeric antigen receptors (CARs). Given the distinct design of both receptors and the type of antigen that is encountered, the requirements for proper antigen engagement and downstream signal transduction by TCRs and CARs differ. Synapse formation and signal transduction of CAR T cells, despite further refinement of CAR T cell designs, still do not fully recapitulate that of TCR T cells and might limit CAR T cell persistence and functionality. Thus, deep knowledge about the molecular differences in CAR and TCR T cell signaling would greatly advance the further optimization of CAR designs and elucidate under which circumstances a combination of both receptors would improve the functionality of T cells for cancer treatment. Herein, we provide a comprehensive review about similarities and differences by directly comparing the architecture, synapse formation and signaling of TCRs and CARs, highlighting the knowns and unknowns. In the second part of the review, we discuss the current status of combining CAR and TCR technologies, encouraging a change in perspective from "TCR versus CAR" to "TCR and CAR".

Combination of GD2-directed bispecific trifunctional antibody therapy with Pd-1 immune checkpoint blockade induces anti-neuroblastoma immunity in a syngeneic mouse model.

Introduction: Despite advances in treating high-risk neuroblastoma, 50-60% of patients still suffer relapse, necessitating new treatment options. Bispecific trifunctional antibodies (trAbs) are a promising new class of immunotherapy. TrAbs are heterodimeric IgG-like molecules that bind CD3 and a tumor-associated antigen simultaneously, whereby inducing a TCR-independent anti-cancer T cell response. Moreover, via their functional Fc region they recruit and activate cells of the innate immune system like antigen-presenting cells potentially enhancing induction of adaptive tumor-specific immune responses. Methods: We used the SUREK trAb, which is bispecific for GD2 and murine Cd3. Tumor-blind trAb and the monoclonal ch14.18 antibody were used as controls. A co-culture model of murine dendritic cells (DCs), T cells and a neuroblastoma cell line was established to evaluate the cytotoxic effect and the T cell effector function in vitro. Expression of immune checkpoint molecules on tumor-infiltrating T cells and the induction of an anti-neuroblastoma immune response using a combination of whole cell vaccination and trAb therapy was investigated in a syngeneic immunocompetent neuroblastoma mouse model (NXS2 in A/J background). Finally, vaccinated mice were assessed for the presence of neuroblastoma-directed antibodies. We show that SUREK trAb-mediated effective killing of NXS2 cells in vitro was strictly dependent on the combined presence of DCs and T cells. Results: Using a syngeneic neuroblastoma mouse model, we showed that vaccination with irradiated tumor cells combined with SUREK trAb treatment significantly prolonged survival of tumor challenged mice and partially prevent tumor outgrowth compared to tumor vaccination alone. Treatment led to upregulation of programmed cell death protein 1 (Pd-1) on tumor infiltrating T cells and combination with anti-Pd-1 checkpoint inhibition enhanced the NXS2-directed humoral immune response. Conclusion: Here, we provide first preclinical evidence that a tumor vaccination combined with SUREK trAb therapy induces an endogenous anti-neuroblastoma immune response reducing tumor recurrence. Furthermore, a combination with anti-Pd-1 immune checkpoint blockade might even further improve this promising immunotherapeutic concept in order to prevent relapse in high-risk neuroblastoma patients.

Inhibiting Lysine Demethylase 1A Improves L1CAM-Specific CAR T Cell Therapy by Unleashing Antigen-Independent Killing via the FAS-FASL Axis.

Chimeric antigen receptor (CAR) T cell therapy has emerged as a promising treatment strategy, however, therapeutic success against solid tumors such as neuroblastoma remains modest. Recurrence of antigen-poor tumor variants often ultimately results in treatment failure. Using antigen-independent killing mechanisms such as the FAS receptor (FAS)-FAS ligand (FASL) axis through epigenetic manipulation may be a way to counteract the escape achieved by antigen downregulation. Analysis of public RNA-sequencing data from primary neuroblastomas revealed that a particular epigenetic modifier, the histone lysine demethylase 1A (KDM1A), correlated negatively with FAS expression. KDM1A is known to interact with TP53 to repress TP53-mediated transcriptional activation of genes, including FAS. We showed that pharmacologically blocking KDM1A activity in neuroblastoma cells with the small molecule inhibitor, SP-2509, increased FAS cell-surface expression in a strictly TP53-dependent manner. FAS upregulation sensitized neuroblastoma cells to FAS-FASL-dependent killing and augmented L1CAM-directed CAR T cell therapy against antigen-poor or even antigen-negative tumor cells in vitro. The improved therapeutic response was abrogated when the FAS-FASL interaction was abolished with an antagonistic FAS antibody. Our results show that KDM1A inhibition unleashes an antigen-independent killing mechanism via the FAS-FASL axis to make tumor cell variants that partially or totally suppress antigen expression susceptible to CAR T cell therapy.

GD2-directed bispecific trifunctional antibody outperforms dinutuximab beta in a murine model for aggressive metastasized neuroblastoma.

BACKGROUND: Neuroblastoma is the most common extracranial solid tumor of childhood. Patients with high-risk disease undergo extremely aggressive therapy and nonetheless have cure rates below 50%. Treatment with the ch14.18 monoclonal antibody (dinutuximab beta), directed against the GD2 disialoganglioside, improved 5-year event-free survival in high-risk patients when administered in postconsolidation therapy and was recently implemented in standard therapy. Relapse still occurred in 57% of these patients, necessitating new therapeutic options. Bispecific trifunctional antibodies (trAbs) are IgG-like molecules directed against T cells and cancer surface antigens, redirecting T cells (via their CD3 specificity) and accessory immune cells (via their functioning Fc-fragment) toward tumor cells. We sought proof-of-concept for GD2/CD3-directed trAb efficacy against neuroblastoma. METHODS: We used two GD2-specific trAbs differing only in their CD3-binding specificity: EKTOMUN (GD2/human CD3) and SUREK (GD2/mouse Cd3). This allowed trAb evaluation in human and murine experimental settings. Tumor-blind trAb and the ch14.18 antibody were used as controls. A coculture model of human peripheral blood mononuclear cells (PBMCs) and neuroblastoma cell lines was established to evaluate trAb antitumor efficacy by assessing expression of T-cell surface markers for activation, proinflammatory cytokine release and cytotoxicity assays. Characteristics of tumor-infiltrating T cells and response of neuroblastoma metastases to SUREK treatment were investigated in a syngeneic immunocompetent neuroblastoma mouse model mimicking minimal residual disease. RESULTS: We show that EKTOMUN treatment caused effector cell activation and release of proinflammatory cytokines in coculture with neuroblastoma cell lines. Furthermore, EKTOMUN mediated GD2-dependent cytotoxic effects in human neuroblastoma cell lines in coculture with PBMCs, irrespective of the level of target antigen expression. This effect was dependent on the presence of accessory immune cells. Treatment with SUREK reduced the intratumor Cd4/Cd8 ratio and activated tumor infiltrating T cells in vivo. In a minimal residual disease model for neuroblastoma, we demonstrated that single-agent treatment with SUREK strongly reduced or eliminated neuroblastoma metastases in vivo. SUREK as well as EKTOMUN demonstrated superior tumor control compared with the anti-GD2 antibody, ch14.18. CONCLUSIONS: Here we provide proof-of-concept for EKTOMUN preclinical efficacy against neuroblastoma, presenting this bispecific trAb as a promising new agent to fight neuroblastoma.

A Reproducible Bioprinted 3D Tumor Model Serves as a Preselection Tool for CAR T Cell Therapy Optimization.

Chimeric antigen receptor (CAR) T cell performance against solid tumors in mouse models and clinical trials is often less effective than predicted by CAR construct selection in two-dimensional (2D) cocultures. Three-dimensional (3D) solid tumor architecture is likely to be crucial for CAR T cell efficacy. We used a three-dimensional (3D) bioprinting approach for large-scale generation of highly reproducible 3D human tumor models for the test case, neuroblastoma, and compared these to 2D cocultures for evaluation of CAR T cells targeting the L1 cell adhesion molecule, L1CAM. CAR T cells infiltrated the model, and both CAR T and tumor cells were viable for long-term experiments and could be isolated as single-cell suspensions for whole-cell assays quantifying CAR T cell activation, effector function and tumor cell cytotoxicity. L1CAM-specific CAR T cell activation by neuroblastoma cells was stronger in the 3D model than in 2D cocultures, but neuroblastoma cell lysis was lower. The bioprinted 3D neuroblastoma model is highly reproducible and allows detection and quantification of CAR T cell tumor infiltration, representing a superior in vitro analysis tool for preclinical CAR T cell characterization likely to better select CAR T cells for in vivo performance than 2D cocultures.

CD28 Co-Stimulus Achieves Superior CAR T Cell Effector Function against Solid Tumors Than 4-1BB Co-Stimulus.

Spacer or co-stimulatory components in chimeric antigen receptor (CAR) design influence CAR T cell effector function. Few preclinical mouse models optimally support CAR candidate pre-selection for clinical development. Here we use a model in which murine CAR T cells can be exploited with human tumor xenografts. This mouse-in-mouse approach avoids limitations caused by species-specific factors crucial for CAR T cell survival, trafficking and function. We compared trafficking, expansion and tumor control for T cells expressing different CAR construct designs targeting two antigens (L1CAM or HER2), structurally identical except for spacer (long or short) or co-stimulatory (4-1BB or CD28) domains to be evaluated. Using monoclonal, murine-derived L1CAM-specific CAR T cells in Rag-/- mice harboring established xenografted tumors from a human neuroblastoma cell line revealed a clear superiority in CAR T cell trafficking using CD28 co-stimulation. L1CAM-targeting short spacer-CD28/ζ CAR T cells expanded the most at the tumor site and induced initial tumor regression. Treating patient-derived neuroblastoma xenografts with human L1CAM-targeting CAR T cells confirmed the superiority of CD28 co-stimulus. CD28 superiority was also demonstrated with HER2-specific CAR T cells (targeting ovarian carcinoma xenografts). Our findings encourage incorporating CD28 signaling into CAR design for adoptive T cell treatment of solid tumors.

The immune system prevents recurrence of transplanted but not autochthonous antigenic tumors after oncogene inactivation therapy.

Targeted oncogene inactivation by small molecule inhibitors can be very effective but tumor recurrence is a frequent problem in the clinic. Therapy by inactivation of the cancer-driving oncogene in transplanted tumors was shown to be augmented in the presence of T cells. However, these experiments did not take into account the long-term, usually tolerogenic, interaction of de novo malignancies with the immune system. Here, we employed mice, in which SV40 large T (Tag) and firefly luciferase (Luc) as fusion protein (TagLuc) could be regulated with the Tet-on system and upon activation resulted in tumors after a long latency. TagLuc inactivation induced profound tumor regression, demonstrating sustained oncogene addiction. While tumor relapse after TagLuc inactivation was prevented in immunocompetent mice bearing transplanted tumors, autochthonous tumors relapsed or recurred after therapy discontinuation indicating that the immune system that coevolved with the malignancy over an extended period of time lost the potency to mount an efficient anti-tumor immune response. By contrast, adoptively transferred CD8+ T cells targeting the cancer-driving oncogene eradicated recurrent autochthonous tumors, highlighting a suitable therapy option in a clinically relevant model.

Preventing tumor escape by targeting a post-proteasomal trimming independent epitope.

Adoptive T cell therapy (ATT) can achieve regression of large tumors in mice and humans; however, tumors frequently recur. High target peptide-major histocompatibility complex-I (pMHC) affinity and T cell receptor (TCR)-pMHC affinity are thought to be critical to preventing relapse. Here, we show that targeting two epitopes of the same antigen in the same cancer cells via monospecific T cells, which have similar pMHC and pMHC-TCR affinity, results in eradication of large, established tumors when targeting the apparently subdominant but not the dominant epitope. Only the escape but not the rejection epitope required postproteasomal trimming, which was regulated by IFN-γ, allowing IFN-γ-unresponsive cancer variants to evade. The data describe a novel immune escape mechanism and better define suitable target epitopes for ATT.

Fas expression by tumor stroma is required for cancer eradication.

The contribution of molecules such as perforin, IFN-γ (IFNγ), and particularly Fas ligand (FasL) by transferred CD8(+) effector T (T(E)) cells to rejection of large, established tumors is incompletely understood. Efficient attack against large tumors carrying a surrogate tumor antigen (mimicking a "passenger" mutation) by T(E) cells requires action of IFNγ on tumor stroma cells to avoid selection of antigen-loss variants. Because "cancer-driving" antigens (CDAs) are rarely counterselected, IFNγ may be expected to be dispensable in elimination of cancers by targeting a CDA. Here, initial regression of large, established tumors required neither IFNγ, FasL, nor perforin by transferred CD8(+) T(E) cells targeting Simian Virus (SV) 40 large T as CDA. However, cytotoxic T(E) cells lacking IFNγ or FasL could not prevent relapse despite retention of the rejection antigen by the cancer cells. Complete tumor rejection required IFNγ-regulated Fas by the tumor stroma. Therefore, T(E) cells lacking IFNγ or FasL cannot prevent progression of antigenic cancer because the tumor stroma escapes destruction if its Fas expression is down-regulated.

Molecular pathways: comparing the effects of drugs and T cells to effectively target oncogenes.

Mutant cancer-driving oncogenes are the best therapeutic targets, both with drugs like small-molecule inhibitors (SMI) and adoptive T-cell therapy (ATT), the most effective form of immunotherapy. Cancer cell survival often depends on oncogenes, which implies that they are homogeneously expressed by all cancer cells and are difficult to select against. Mutant oncogene-directed therapy is relatively selective, as it targets preferentially the oncogene-expressing cancer cells. Both SMI and ATT can be highly effective in relevant preclinical models as well as selected clinical situations, and both share the risk of therapy resistance, facilitated by the frequent genetic instability of cancer cells. Recently, both therapies were compared in the same experimental model targeting the same oncogene. It showed that the oncogene-inactivating drug selected resistant clones, leading eventually to tumor relapse, whereas ATT eradicated large established tumors completely. The mode of tumor destruction likely explained the different outcome with only ATT destroying the tumor vasculature. Elucidating the cellular and molecular mechanisms responsible for tumor regression and relapse will define optimal conditions for the clinic. We argue that the ideal conditions of ATT in the experimental cancer model can be translated to individuals with cancer.

Depot formation of doxycycline impairs Tet-regulated gene expression in vivo.

The tetracycline (Tet) system is widely used for regulation of gene expression in vitro and in vivo. We constructed C57BL/6 transgenic mice (rtTA-CM2) with strong and ubiquitous reverse transactivator (rtTA2(S)-M2) gene expression. rtTA-CM2 mice were crossed to Tet-responsive reporter mice (LC-1) conditionally expressing the firefly luciferase (FLuc) gene under control of a Tet-responsive element, which allowed sensitive quantification of the transactivator activity by bioluminescent imaging. Following doxycycline (dox) application, up to 10(5)-fold increase in BL signal was measured. rtTA activity was inducible in most analyzed organs. After dox withdrawal the BL signal decreased significantly but did not disappear completely, most likely due to a dox depot formation in vivo. The residual dox was sufficient to partly down-regulate a Tet-off controlled oncogene in a tumor transplantation experiment, resulting in reduced tumor growth. rtTA-CM2 mice may be a useful tool to analyze the function of genes in various organs but also reveal that down-regulation of gene expression is not complete.

Oncogene-targeting T cells reject large tumors while oncogene inactivation selects escape variants in mouse models of cancer.

The genetic instability of cancer cells frequently causes drug resistance. We established mouse cancer models, which allowed targeting of an oncogene by drug-mediated inactivation or monospecific CD8(+) effector T (T(E)) cells. Drug treatment of genetically unstable large tumors was effective but selected resistant clones in the long term. In contrast, T(E) cells completely rejected large tumors (≥500 mm(3)), if the target antigen was cancer-driving and expressed in sufficient amounts. Although drug-mediated oncogene inactivation selectively killed the cancer cells and left the tumor vasculature intact, which likely facilitated survival and growth of resistant clones, T(E) cell treatment led to blood vessel destruction and probably "bystander" elimination of escape variants, which did not require antigen cross-presentation by stromal cells.

In vivo imaging of an inducible oncogenic tumor antigen visualizes tumor progression and predicts CTL tolerance.

Visualizing oncogene/tumor Ag expression by noninvasive imaging is of great interest for understanding processes of tumor development and therapy. We established transgenic (Tg) mice conditionally expressing a fusion protein of the SV40 large T Ag and luciferase (TagLuc) that allows monitoring of oncogene/tumor Ag expression by bioluminescent imaging upon Cre recombinase-mediated activation. Independent of Cre-mediated recombination, the TagLuc gene was expressed at low levels in different tissues, probably due to the leakiness of the stop cassette. The level of spontaneous TagLuc expression, detected by bioluminescent imaging, varied between the different Tg lines, depended on the nature of the Tg expression cassette, and correlated with Tag-specific CTL tolerance. Following liver-specific Cre-loxP site-mediated excision of the stop cassette that separated the promoter from the TagLuc fusion gene, hepatocellular carcinoma development was visualized. The ubiquitous low level TagLuc expression caused the failure of transferred effector T cells to reject Tag-expressing tumors rather than causing graft-versus-host disease. This model may be useful to study different levels of tolerance, monitor tumor development at an early stage, and rapidly visualize the efficacy of therapeutic intervention versus potential side effects of low-level Ag expression in normal tissues.