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Adoptive immunotherapy with T cells genetically modified to express chimeric antigen receptors (CARs) is a promising approach to improve outcomes for cancer patients. While CAR T cell therapy is effective for hematological malignancies, there is a need to improve the efficacy of this therapeutic approach for patients with solid tumors and brain tumors. At present, several approaches are being pursued to improve the antitumor activity of CAR T cells including i) targeting multiple antigens, ii) improving T cell expansion/persistence, iii) enhancing homing to tumor sites, and iv) rendering CAR T cells resistant to the immunosuppressive tumor microenvironment (TME). Augmenting signal 3 of T cell activation by transgenic expression of cytokines or engineered cytokine receptors has emerged as a promising strategy since it not only improves CAR T cell expansion/persistence but also their ability to function in the immunosuppressive TME. In this review, we will provide an overview of cytokine biology and highlight genetic approaches that are actively being pursued to augment cytokine signaling in CAR T cells.

Adoptive cellular immunotherapy using immune cells expressing chimeric antigen receptors (CARs) has shown promise, particularly for the treatment of hematological malignancies. To date, the majority of clinically evaluated CAR cell products have been derived from autologous immune cells. While this strategy can be effective it also imposes several constraints regarding logistics. This includes i) availability of center to perform leukapheresis, ii) necessity for shipment to and from processing centers, and iii) time requirements for product manufacture and clinical release testing. In addition, previous cytotoxic therapies can negatively impact the effector function of autologous immune cells, which may then affect efficacy and/or durability of resultant CAR products. The use of allogeneic CAR cell products generated using cells from healthy donors has the potential to overcome many of these limitations, including through generation of “off the shelf” products. However, allogeneic CAR cell products come with their own challenges, including potential to induce graft-versus-host-disease, as well as risk of immune-mediated rejection by the host. Here we will review promises and challenges of allogeneic CAR immunotherapies, including those being investigated in preclinical models and/or early phase clinical studies.

Immunotherapy with chimeric antigen receptor (CAR) T cells offers a promising method to improve cure rates and decrease morbidities for patients with cancer. In this regard, CD19-specific CAR T cell therapies have achieved dramatic objective responses for a high percent of patients with CD19-positive leukemia or lymphoma. Most patients with solid tumors however, have experienced transient or no benefit from CAR T cell therapies. Novel strategies are therefore needed to improve CAR T cell function for patients with solid tumors. One obstacle for the field is limited CAR T cell persistence after infusion into patients. In this review we highlight genetic engineering strategies to improve CAR T cell persistence for enhancing antitumor activity for patients with solid tumors.

Oncolytic vaccinia virus (VV) therapy has shown promise in preclinical models and in clinical studies. However, complete responses have rarely been observed. This lack of efficacy is most likely due to suboptimal virus spread through the tumor resulting in limited tumor cell destruction. We reasoned that redirecting T cells to the tumor has the potential to improve the antitumor activity of oncolytic VVs. We, therefore, constructed a VV encoding a secretory bispecific T-cell engager consisting of two single- chain variable fragments specific for CD3 and the tumor cell surface antigen EphA2 (EphA2-T-cell engager-armed VV (EphA2-TEA-VV)). In vitro, EphA2-TEA-VV's ability to replicate and induce oncolysis was similar to that of unmodified virus. However, only tumor cells infected with EphA2-TEA-VV induced T-cell activation as judged by the secretion of interferon-γ and interleukin-2. In coculture assays, EphA2-TEA-VV not only killed infected tumor cells, but in the presence of T cells, it also induced bystander killing of noninfected tumor cells. In vivo, EphA2-TEA-VV plus T cells had potent antitumor activity in comparison with control VV plus T cells in a lung cancer xenograft model. Thus, arming oncolytic VVs with T-cell engagers may represent a promising approach to improve oncolytic virus therapy.

Refractory metastatic rhabdomyosarcoma is largely incurable. Here we analyze the response of a child with refractory bone marrow metastatic rhabdomyosarcoma to autologous HER2 CAR T cells. Three cycles of HER2 CAR T cells given after lymphodepleting chemotherapy induces remission which is consolidated with four more CAR T-cell infusions without lymphodepletion. Longitudinal immune-monitoring reveals remodeling of the T-cell receptor repertoire with immunodominant clones and serum autoantibodies reactive to oncogenic signaling pathway proteins. The disease relapses in the bone marrow at six months off-therapy. A second remission is achieved after one cycle of lymphodepletion and HER2 CAR T cells. Response consolidation with additional CAR T-cell infusions includes pembrolizumab to improve their efficacy. The patient described here is a participant in an ongoing phase I trial (NCT00902044; active, not recruiting), and is 20 months off T-cell infusions with no detectable disease at the time of this report. Recurrent metastatic rhabdomyosarcoma remains largely incurable. Here, the authors describe a child with metastatic rhabdomyosarcoma who has durable response to HER2-specific CAR T cells and shows endogenous immune reactivity.

Developing CAR T cells for acute myeloid leukemia (AML) has been hampered by a paucity of targets that are expressed on AML blasts and not on hematopoietic progenitor cells (HPCs). Here we demonstrate that GRP78 is expressed on the cell surface of primary AML blasts but not HPCs. To target GRP78, we generate T cell expressing a GRP78-specific peptide-based CAR, which show evidence of minimal fratricide post activation/transduction and antigen-dependent T cell differentiation. GRP78-CAR T cells recognize and kill GRP78-positive AML cells without toxicity to HPCs. In vivo, GRP78-CAR T cells have significant anti-AML activity. To prevent antigen-dependent T cell differentiation, we block CAR signaling and GRP78 cell surface expression post activation by using dasatinib during GRP78-CAR T cell manufacturing. This significantly improves their effector function in vitro and in vivo. Thus, targeting cell surface GRP78-positive AML with CAR T cells is feasible, and warrants further active exploration. There is an unmet need to discover suitable targets for CAR-T therapy in patients with acute myeloid leukemia (AML). Here the authors show that GRP78, a key regulator of the unfolded protein response, is highly expressed on the surface of primary AML blasts, but not on normal lymphocytes and hematopoietic progenitor cells, and that GRP78-CAR T have anti-AML activity in preclinical models.

Glioblastoma is the most aggressive primary brain tumor in humans and is virtually incurable with conventional therapies. Chimeric antigen receptor (CAR) T cell therapy targeting the glioblastoma antigen EphA2 is an attractive approach to improve outcomes because EphA2 is expressed highly in glioblastoma but only at low levels in normal brain tissue. Building upon our previous findings in this area, we generated and evaluated a panel of EphA2-specific CARs. We demonstrate here that T cells expressing CD28.ζ and 41BB.ζ CARs with short spacers had similar effector function, resulting in potent antitumor activity. In addition, incorporating the 41BB signaling domain into CD28.ζ CARs did not improve CAR T cell function. While we could not determine functional differences between CD28.ζ, 41BB.ζ, and CD28.41BB.ζ CAR T cells, we selected CD28.ζ CAR T cells for further clinical development based on safety consideration.

ABSTRACT Purpose Natural killer (NK) cell cytotoxicity correlates with the ligation of activating receptors (e.g., NKG2D) by their ligands (e.g., MHC class I–related chains [MIC] A and B) on target cells. Histone deacetylase inhibitors (HDACi) at high concentrations inhibit tumor growth and can increase NKG2D ligand expression on tumor targets, but are widely regarded as toxic to NK cells. Methods We investigated the mechanism of entinostat, a benzamide-derivative narrow-spectrum HDACi, in augmenting the cytotoxicity of NK cells against human colon carcinoma and sarcoma by assessing gene and protein expression, histone acetylation, and cytotoxicity in in vitro and murine models. Results We observed that entinostat dose- and time-dependent increase in MIC expression in tumor targets and NKG2D in primary human NK cells, both correlating with increased acetylated histone 3 (AcH3) binding to associated promoters. Entinostat pretreatment of colon carcinoma and sarcoma cells, NK cells, or both led to enhanced overall cytotoxicity in vitro , which was reversed by NKG2D blockade, and inhibited growth of tumor xenografts. Lastly, we showed decreased expression of MICA and ULBP2 transcription in primary human osteosarcoma. Conclusions Entinostat enhances NK cell killing of cancer cells through upregulation of both NKG2D and its ligands, suggesting an attractive approach for augmenting NK cell immunotherapy of solid tumors such as colon carcinoma and sarcomas.

Adoptive transfer of virus-specific T cells can prevent and treat serious infections with Epstein-Barr virus (EBV), cytomegalovirus (CMV), and adenovirus (Adv) after allogeneic hematopoietic stem cell transplant. It has, however, proved difficult to make this approach widely available since infectious virus and viral vectors are required for T cell activation, followed by an intensive and prolonged culture period extending over several months. We now show that T cells targeting a range of viral antigens derived from EBV, CMV, and Adv can be reproducibly generated in a single culture over a 2–3-week period, using methods that exclude all viral components and employ a much-simplified culture technology. When administered to recipients of haploidentical (n = 5), matched unrelated (n = 3), mismatched unrelated (n = 1) or matched related (n = 1) transplants with active CMV (n = 3), Adv (n = 1), EBV (n = 2), EBV+Adv (n = 2) or CMV+Adv (n = 2) infections, the cells produced complete virological responses in 80%, including all patients with dual infections. In each case, a decrease in viral load correlated with an increase in the frequency of T cells directed against the infecting virus(es); both immediate and delayed toxicities were absent. This approach should increase both the feasibility and applicability of T cell therapy. The trial was registered at www.clinicaltrials.gov as NCT01070797.

Immunotherapy with chimeric antigen receptor T cells for pediatric solid and brain tumors is constrained by available targetable antigens. Cancer-specific exons present a promising reservoir of targets; however, these have not been explored and validated systematically in a pan-cancer fashion. To identify cancer specific exon targets, here we analyze 1532 RNA-seq datasets from 16 types of pediatric solid and brain tumors for comparison with normal tissues using a newly developed workflow. We find 2933 exons in 157 genes encoding proteins of the surfaceome or matrisome with high cancer specificity either at the gene ( n  = 148) or the alternatively spliced isoform ( n  = 9) level. Expression of selected alternatively spliced targets, including the EDB domain of fibronectin 1, and gene targets, such as COL11A1, are validated in pediatric patient derived xenograft tumors. We generate T cells expressing chimeric antigen receptors specific for the EDB domain or COL11A1 and demonstrate that these have antitumor activity. The full target list, explorable via an interactive web portal ( https://cseminer.stjude.org/ ), provides a rich resource for developing immunotherapy of pediatric solid and brain tumors using gene or AS targets with high expression specificity in cancer. CAR T cell immunotherapy for paediatric solid and brain tumours is constrained by the availability of targetable antigens. Here, the authors investigate the landscape of cancer-specific exons as potential targets by analysing 1,532 RNAseq datasets from 16 types of paediatric solid and brain tumours.