Integration of ζ-deficient CARs into the CD3ζ gene conveys potent cytotoxicity in T and NK cells.

ABSTRACT: Chimeric antigen receptor (CAR)-redirected immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in nonphysiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Random gene transfer modalities pose a risk of malignant transformation by insertional mutagenesis. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR expression and redirection of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. In T cells, CD3ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3ζ-CD19-CAR-T cells exhibited comparable leukemia control to TCRα chain constant (TRAC)-replaced and lentivirus-transduced CAR-T cells in vivo. Tuning of CD3ζ-CAR-expression levels significantly improved the in vivo efficacy. Notably, CD3ζ gene editing enabled redirection of NK cells without impairing their canonical functions. Thus, CD3ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes.

CD171- and GD2-specific CAR-T cells potently target retinoblastoma cells in preclinical in vitro testing.

BACKGROUND: Chimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects. METHODS: CD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3+) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs. RESULTS: All retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing. CONCLUSION: Both CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.

Integration of ζ-deficient CARs into the CD3-zeta gene conveys potent cytotoxicity in T and NK cells.

Chimeric antigen receptor (CAR)-reprogrammed immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3 ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3 ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and reprogramming of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. In T cells, CD3 ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3 ζ-CD19-CAR-T cells exhibited comparable leukemia control to T cell receptor alpha constant ( TRAC )-replaced and lentivirus-transduced CAR-T cells in vivo . Tuning of CD3 ζ-CAR-expression levels significantly improved the in vivo efficacy. Compared to TRAC -edited CAR-T cells, integration of a Her2-CAR into CD3 ζ conveyed similar in vitro tumor lysis but reduced susceptibility to activation-induced cell death and differentiation, presumably due to lower CAR-expression levels. Notably, CD3 ζ gene editing enabled reprogramming of NK cells without impairing their canonical functions. Thus, CD3 ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes. Key points: Integration of ζ-deficient CARs into CD3 ζ gene allows generation of functional TCR-ablated CAR-T cells for allogeneic off-the-shelf use CD3 ζ-editing platform allows CAR reprogramming of NK cells without affecting their canonical functions.

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.

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.

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.

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.