Chimeric antigen receptor (CAR)-T-cell therapy for solid tumors is limited due to heterogeneous target antigen expression and outgrowth of tumors lacking the antigen targeted by CAR-T cells directed ...against single antigens. Here, we developed a bicistronic construct to drive expression of a CAR specific for EGFRvIII, a glioblastoma-specific tumor antigen, and a bispecific T-cell engager (BiTE) against EGFR, an antigen frequently overexpressed in glioblastoma but also expressed in normal tissues. CART.BiTE cells secreted EGFR-specific BiTEs that redirect CAR-T cells and recruit untransduced bystander T cells against wild-type EGFR. EGFRvIII-specific CAR-T cells were unable to completely treat tumors with heterogenous EGFRvIII expression, leading to outgrowth of EGFRvIII-negative, EGFR-positive glioblastoma. However, CART.BiTE cells eliminated heterogenous tumors in mouse models of glioblastoma. BiTE-EGFR was locally effective but was not detected systemically after intracranial delivery of CART.BiTE cells. Unlike EGFR-specific CAR-T cells, CART.BiTE cells did not result in toxicity against human skin grafts in vivo.
Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases
has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)
. To address this limitation, we engineered an enhanced ...Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants
to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing.
Cell-based therapies are emerging as effective agents against cancer and other diseases. As autonomous "living drugs," these therapies lack precise control. Chimeric antigen receptor (CAR) T cells ...effectively target hematologic malignancies but can proliferate rapidly and cause toxicity. We developed ON and OFF switches for CAR T cells using the clinically approved drug lenalidomide, which mediates the proteasomal degradation of several target proteins by inducing interactions between the CRL4
E3 ubiquitin ligase and a C2H2 zinc finger degron motif. We performed a systematic screen to identify "super-degron" tags with enhanced sensitivity to lenalidomide-induced degradation and used these degradable tags to generate OFF-switch degradable CARs. To create an ON switch, we engineered a lenalidomide-inducible dimerization system and developed split CARs that required both lenalidomide and target antigen for activation. Subtherapeutic lenalidomide concentrations controlled the effector functions of ON- and OFF-switch CAR T cells. In vivo, ON-switch split CARs demonstrated lenalidomide-dependent antitumor activity, and OFF-switch degradable CARs were depleted by drug treatment to limit inflammatory cytokine production while retaining antitumor efficacy. Together, the data showed that these lenalidomide-gated switches are rapid, reversible, and clinically suitable systems to control transgene function in diverse gene- and cell-based therapies.
Chimeric antigen receptor (CAR) T-cell therapy represents a revolutionary treatment for haematological malignancies (i.e. B-ALL). However, the success of this type of treatment has not yet been ...achieved in solid tumors. One hypothesis is that the immunosuppressive nature of the tumor microenvironment (TME) influences and affects the efficacy of adoptive immunotherapy. Understanding the role of the TME and its interaction with CAR T-cells is crucial to improve the potency of adoptive immunotherapy. In this review, we discuss the strategies and potential combinatorial approaches recently developed in mouse models to enhance the efficacy of CAR T-cells, with particular emphasis on the translational potential of these approaches.
CAR T cells with different costimulation domains have proven clinical efficacy in leukemia and lymphoma but have different kinetics of activation, antigen sensitivity, and susceptibility to ...exhaustion. Two recent studies identified that these functions are shaped by a balance among opposing signaling complexes and transcription factors competing for binding motifs.
CAR T cells with different costimulation domains have proven clinical efficacy in leukemia and lymphoma but have different kinetics of activation, antigen sensitivity, and susceptibility to exhaustion. Two recent studies identified that these functions are shaped by a balance among opposing signaling complexes and transcription factors competing for binding motifs.
Despite remarkable success in the treatment of hematological malignancies, CAR T-cell therapies for solid tumors have floundered, in large part due to local immune suppression and the effects of ...prolonged stimulation leading to T-cell dysfunction and exhaustion. One mechanism by which gliomas and other cancers can hamper CAR T cells is through surface expression of inhibitory ligands such as programmed cell death ligand 1 (PD-L1). Using the CRIPSR-Cas9 system, we created universal CAR T cells resistant to PD-1 inhibition through multiplexed gene disruption of endogenous T-cell receptor (TRAC), beta-2 microglobulin (B2M) and PD-1 (PDCD1). Triple gene-edited CAR T cells demonstrated enhanced activity in preclinical glioma models. Prolonged survival in mice bearing intracranial tumors was achieved after intracerebral, but not intravenous administration. CRISPR-Cas9 gene-editing not only provides a potential source of allogeneic, universal donor cells, but also enables simultaneous disruption of checkpoint signaling that otherwise impedes maximal antitumor functionality.
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.
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•Azacitidine increases CD70 antigen density and potentiates CD70 CAR function•CD8H&TM-modified CAR-T cells have improved avidity and in vivo performance•In vitro avidity correlates with in vivo potency
Leick et al. identify an immunotherapy strategy using CAR-T cells for leukemia by increasing binding avidity on both sides of the synapse through pharmacologic enhancement of tumor antigen density with azacitidine and via hinge modification on the T cell.
Anaplastic large-cell lymphoma (ALCL) is a clinical and biological heterogeneous disease that includes systemic anaplastic lymphoma kinase (ALK)-positive and ALK-negative entities. To discover ...biomarkers and/or genes involved in ALK-negative ALCL pathogenesis, we applied the cancer outlier profile analysis algorithm to a gene expression profiling data set including 249 cases of T-cell non-Hodgkin lymphoma and normal T cells. Ectopic coexpression of ERBB4 and COL29A1 genes was detected in 24% of ALK-negative ALCL patients. RNA sequencing and 5′ RNA ligase-mediated rapid amplification of complementary DNA ends identified 2 novel ERBB4-truncated transcripts displaying intronic transcription start sites. By luciferase assays, we defined that the expression of ERBB4-aberrant transcripts is promoted by endogenous intronic long terminal repeats. ERBB4 expression was confirmed at the protein level by western blot analysis and immunohistochemistry. Lastly, we demonstrated that ERBB4-truncated forms show oncogenic potentials and that ERBB4 pharmacologic inhibition partially controls ALCL cell growth and disease progression in an ERBB4-positive patient-derived tumorgraft model. In conclusion, we identified a new subclass of ALK-negative ALCL characterized by aberrant expression of ERBB4-truncated transcripts carrying intronic 5′ untranslated regions.
•Endogenous intronic long terminal repeats promote the ectopic expression of truncated ERBB4 transcripts in 24% of ALK-negative ALCL.•The expression of ERBB4-aberrant transcripts defines a new subclass of ALK-negative ALCL and may contribute to ALCL transformation.
Cutaneous T cell lymphomas (CTCL) are a heterogeneous group of malignancies characterized by the expansion of a malignant T cell clone. Chimeric Antigen Receptor (CAR) T cell therapy has shown ...impressive results for the treatment of B-cell tumors, but several challenges have prevented this approach in the context of T cell lymphoma. These challenges include the possibilities of fratricide due to shared T-cell antigens, T cell immunodeficiency, and CAR transduction of malignant cells if CAR T are manufactured in the autologous setting. In this review, we discuss these and other challenges in detail and summarize the approaches currently in development to overcome these challenges and offer cellular targeting of T cell lymphomas.
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