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Abstract Background Relapse remains a challenge after transplantation in pediatric patients with hematological malignancies. Myeloablative regimens used for disease control are associated with acute and long-term adverse effects. We used a CD45RA-depleted haploidentical graft for adoptive transfer of memory T cells combined with NK-cell addback and hypothesized that maximizing the graft-versus-leukemia (GVL) effect might allow for reduction in intensity of conditioning regimen. Methods In this phase II clinical trial (NCT01807611), 72 patients with hematological malignancies (complete remission (CR)1: 25, ≥ CR2: 28, refractory disease: 19) received haploidentical CD34 + enriched and CD45RA-depleted hematopoietic progenitor cell grafts followed by NK-cell infusion. Conditioning included fludarabine, thiotepa, melphalan, cyclophosphamide, total lymphoid irradiation, and graft-versus-host disease (GVHD) prophylaxis consisted of a short-course sirolimus or mycophenolate mofetil without serotherapy. Results The 3-year overall survival (OS) and event-free-survival (EFS) for patients in CR1 were 92% (95% CI:72–98) and 88% (95% CI: 67–96); ≥ CR2 were 81% (95% CI: 61–92) and 68% (95% CI: 47–82) and refractory disease were 32% (95% CI: 11–54) and 20% (95% CI: 6–40). The 3-year EFS for all patients in morphological CR was 77% (95% CI: 64–87) with no difference amongst recipients with or without minimal residual disease (P = 0.2992). Immune reconstitution was rapid, with mean CD3 and CD4 T-cell counts of 410/μL and 140/μL at day + 30. Cumulative incidence of acute GVHD and chronic GVHD was 36% and 26% but most patients with acute GVHD recovered rapidly with therapy. Lower rates of grade III-IV acute GVHD were observed with NK-cell alloreactive donors (P = 0.004), and higher rates of moderate/severe chronic GVHD occurred with maternal donors (P = 0.035). Conclusion The combination of a CD45RA-depleted graft and NK-cell addback led to robust immune reconstitution maximizing the GVL effect and allowed for use of a submyeloablative, TBI-free conditioning regimen that was associated with excellent EFS resulting in promising long-term outcomes in this high-risk population. The trial is registered at ClinicalTrials.gov (NCT01807611).

Abstract 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.

Abstract Virus-specific T cells (VST) from partially-HLA matched donors have been effective for treatment of refractory viral infections in immunocompromised patients in prior studies with a good safety profile, but rare adverse events have been described. Here we describe a unique and severe adverse event of VST therapy in an infant with severe combined immunodeficiency, who receives, as part of a clinical trial (NCT03475212), third party VSTs for treating cytomegalovirus viremia following bone marrow transplantation. At one-month post-VST infusion, rejection of graft and reversal of chimerism is observed, as is an expansion of T cells exclusively from the VST donor. Single-cell gene expression and T cell receptor profiling demonstrate a narrow repertoire of predominantly activated CD4+ T cells in the recipient at the time of rejection, with the repertoire overlapping more with that of peripheral blood from VST donor than the infused VST product. This case thus demonstrates a rare but serious side effect of VST therapy.

Despite intensive therapies, pediatric patients with relapsed or refractory solid tumors have poor outcomes and need novel treatments. Immune therapies offer an alternative to conventional treatment options but require the identification of differentially expressed antigens to direct antitumor activity to sites of disease. B7-H3 (CD276) is an immune regulatory protein that is expressed in a range of malignancies and has limited expression in normal tissues. B7-H3 is highly expressed in pediatric solid tumors including osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, Wilms tumor, neuroblastoma, and many rare tumors. In this article we review B7-H3-targeted chimeric antigen receptor (B7-H3-CAR) T cell therapies for pediatric solid tumors, reporting preclinical development strategies and outlining the landscape of active pediatric clinical trials. We identify challenges to the success of CAR T cell therapy for solid tumors including localizing to and penetrating solid tumor sites, evading the hostile tumor microenvironment, supporting T cell expansion and persistence, and avoiding intrinsic tumor resistance. We highlight strategies to overcome these challenges and enhance the effect of B7-H3-CAR T cells, including advanced CAR T cell design and incorporation of combination therapies.

Recurrent and/or refractory (R/R) pediatric acute myeloid leukemia (AML) remains a recalcitrant disease with poor outcomes. Cell therapy with genetically modified immune effector cells holds the promise to improve outcomes for R/R AML since it relies on cytotoxic mechanisms that are distinct from chemotherapeutic agents. While T cells expressing chimeric antigen receptors (CAR T cells) showed significant anti-AML activity in preclinical models, early phase clinical studies have demonstrated limited activity, irrespective of the targeted AML antigen. Lack of efficacy is most likely multifactorial, including: (i) a limited array of AML-specific targets and target antigen heterogeneity; (ii) the aggressive nature of R/R AML and heavy pretreatment of patients; (iii) T-cell product manufacturing, and (iv) limited expansion and persistence of the CAR T cells, which is in part driven by the immunosuppressive AML microenvironment. Here we review the results of early phase clinical studies with AML-specific CAR T cells, and avenues investigators are exploring to improve their effector function.

CRISPR-Cas9-based therapeutic genome editing approaches hold promise to cure a variety of human diseases. Recent findings demonstrate pre-existing immunity for the commonly used Cas orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans, which threatens the success of this powerful tool in clinical use. Thus, a comprehensive investigation and potential risk assessment are required to exploit the full potential of the system. Here, we investigated existence of immunity to SpCas9 and SaCas9 in control rhesus macaques (Macaca mulatta) alongside monkeys transplanted with either lentiviral transduced or CRISPR-SpCas9 ribonucleoprotein (RNP)-edited cells. We observed significant levels of Cas9 antibodies in the peripheral blood of all transplanted and non-transplanted control animals. Transplantation of ex vivo transduced or SpCas9-mediated BCL11A enhancer-edited cells did not alter the levels of Cas9 antibodies in rhesus monkeys. Following stimulation of peripheral blood cells with SpCas9 or SaCas9, neither Cas9-specific T cells nor cytokine induction were detected. Robust and durable editing frequencies and expression of high levels of fetal hemoglobin in BCL11A enhancer-edited rhesus monkeys with no evidence of an immune response (>3 years) provide an optimistic outlook for the use of ex vivo CRISPR-SpCas9 (RNP)-edited cells.

Chimeric antigen receptor (CAR) cell-based therapies have demonstrated limited success in solid tumors, including glioblastoma (GBM). GBMs exhibit high heterogeneity and create an immunosuppressive tumor microenvironment (TME). In addition, other challenges exist for CAR therapy, including trafficking and infiltration into the tumor site, proliferation, persistence of CARs once in the tumor, and reduced functionality, such as suboptimal cytokine production. Cytokine modification is of interest, as one can enhance therapy efficacy and minimize off-target toxicity by directly combining CAR therapy with cytokines, antibodies, or oncolytic viruses that alter cytokine response pathways. Alternatively, one can genetically modify CAR T-cells or CAR NK-cells to secrete cytokines or express cytokines or cytokine receptors. Finally, CARs can be genetically altered to augment or suppress intracellular cytokine signaling pathways for a more direct approach. Codelivery of cytokines with CARs is the most straightforward method, but it has associated toxicity. Alternatively, combining CAR therapy with antibodies (e.g., anti-IL-6, anti-PD1, and anti-VEGF) or oncolytic viruses has enhanced CAR cell infiltration into GBM tumors and provided proinflammatory signals to the TME. CAR T- or NK-cells secreting cytokines (e.g., IL-12, IL-15, and IL-18) have shown improved efficacy within multiple GBM subtypes. Likewise, expressing cytokine-modulating receptors in CAR cells that promote or inhibit cytokine signaling has enhanced their activity. Finally, gene editing approaches are actively being pursued to directly influence immune signaling pathways in CAR cells. In this review, we summarize these cytokine modification methods and highlight any existing gaps in the hope of catalyzing an improved generation of CAR-based therapies for glioblastoma.

Background CD47 is an attractive immunotherapeutic target because it is highly expressed on multiple solid tumors. However, CD47 is also expressed on T cells. Limited studies have evaluated CD47-chimeric antigen receptor (CAR) T cells, and the role of CD47 in CAR T-cell function remains largely unknown.Methods Here, we describe the development of CD47-CAR T cells derived from a high affinity signal regulatory protein α variant CV1, which binds CD47. CV1-CAR T cells were generated from human peripheral blood mononuclear cells and evaluated in vitro and in vivo. The role of CD47 in CAR T-cell function was examined by knocking out CD47 in T cells followed by downstream functional analyses.Results While CV1-CAR T cells are specific and exhibit potent activity in vitro they lacked antitumor activity in xenograft models. Mechanistic studies revealed CV1-CAR T cells downregulate CD47 to overcome fratricide, but CD47 loss resulted in their failure to expand and persist in vivo. This effect was not limited to CV1-CAR T cells, since CD47 knockout CAR T cells targeting another solid tumor antigen exhibited the same in vivo fate. Further, CD47 knockout T cells were sensitive to macrophage-mediated phagocytosis.Conclusions These findings highlight that CD47 expression is critical for CAR T-cell survival in vivo and is a ‘sine qua non’ for successful adoptive T-cell therapy.

Introduction The immunosuppressive tumor microenvironment (TME) is a major barrier to the efficacy of chimeric antigen receptor T cells (CAR-T cells) in glioblastoma (GBM). Transgenic expression of IL15 is one attractive strategy to modulate the TME. However, at present, it is unclear if IL15 could be used to directly target myeloid-derived suppressor cells (MDSCs), a major cellular component of the GBM TME. Here, we explored if MDSC express IL15Rα and the feasibility of exploiting its expression as an immunotherapeutic target.Methods RNA-seq, RT-qPCR, and flow cytometry were used to determine IL15Rα expression in paired peripheral and tumor-infiltrating immune cells of GBM patients and two syngeneic murine GBM models. We generated murine T cells expressing IL13Rα2-CARs and secretory IL15 (CAR.IL15s) or IL13Rα2-CARs in which IL15 was fused to the CAR to serve as an IL15Rα-targeting moiety (CAR.IL15f), and characterized their effector function in vitro and in syngeneic IL13Rα2+glioma models.Results IL15Rα was preferentially expressed in myeloid, B, and dendritic cells in patients’ and syngeneic GBMs. In vitro, CAR.IL15s and CAR.IL15f T cells depleted MDSC and decreased their secretion of immunosuppressive molecules with CAR.IL15f T cells being more efficacious. Similarly, CAR.IL15f T cells significantly improved the survival of mice in two GBM models. TME analysis showed that treatment with CAR.IL15f T cells resulted in higher frequencies of CD8+T cells, NK, and B cells, but a decrease in CD11b+cells in tumors compared with therapy with CAR T cells.Conclusions We demonstrate that MDSC of the glioma TME express IL15Ra and that these cells can be targeted with secretory IL15 or an IL15Rα-targeting moiety incorporated into the CAR. Thus, IL15-modified CAR T cells act as a dual targeting agent against tumor cells and MDSC in GBM, warranting their future evaluation in early-phase clinical studies.

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.

The outcome of patients with acute myeloid leukemia remains poor, and immunotherapy has the potential to improve this. T cells expressing chimeric antigen receptors or bispecific T-cell engagers targeting CD123 are actively being explored in preclinical and/or early phase clinical studies. We have shown that T cells expressing CD123-specific bispecific T-cell engagers (CD123.ENG T cells) have anti-acute myeloid leukemia activity. However, like chimeric antigen receptor T cells, their effector function diminishes rapidly once they are repeatedly exposed to antigen-positive target cells. Here we sought to improve the effector function of CD123.ENG T cells by expressing inducible co-stimulatory molecules consisting of MyD88 and CD40 (iMC), MyD88 (iM), or CD40 (iC), which are activated by a chemical inducer of dimerization. CD123.ENG T cells expressing iMC, iM, or iC maintained their antigen specificity in the presence of a chemical inducer of dimerization, as judged by cytokine production (interferon-γ, interleukin-2) and their cytolytic activity. In repeat stimulation assays, activating iMC and iM, in contrast to iC, enabled CD123.ENG T cells to secrete cytokines, expand, and kill CD123-positive target cells repeatedly. Activating iMC in CD123.ENG T cells consistently improved antitumor activity in an acute myeloid leukemia xenograft model. This translated into a significant survival advantage in comparison to that of mice that received CD123.ENG or CD123.ENG.iC T cells. In contrast, activation of only iM in CD123.ENG T cells resulted in donor-dependent antitumor activity. Our work highlights the need for both toll-like receptor pathway activation via MyD88 and provision of co-stimulation via CD40 to consistently enhance the antitumor activity of CD123.ENG T cells.

Immunotherapy with T-cells expressing bispecific T-cell engagers (ENG T-cells) is a promising approach to improve the outcomes for patients with recurrent/refractory acute myeloid leukemia (AML). However, similar to T-cells expressing chimeric antigen receptors (CARs), their antitumor activity is limited in the setting of chronic antigen stimulation. We therefore set out to explore whether transgenic expression of IL15 improves the effector function of ENG T-cells targeting CD123-positive AML. T-cells expressing CD123-specific ENG (CD123-ENG) ± IL15 were generated by retroviral transduction from peripheral blood T cells from healthy donors or patients with AML. In this study, we characterized in detail the phenotype and effector functions of ENG T-cell populations in vitro and in vivo. IL15-expressing CD123-ENG (CD123-ENG.IL15) T-cells retained their antigen-specificity and effector function in the setting of chronic antigen exposure for more 30 days of coculture with AML blasts in contrast to CD123-ENG T-cells, whose effector function rapidly eroded. Furthermore, CD123-ENG.IL15 T-cells remained in a less differentiated state as judged by a high frequency of naïve/memory stem T-cell-like cells (CD45RA+CCR7+/CD45RO−CD62L+ cells) without evidence of T-cell exhaustion. Single cell cytokine profiling using IsoPlexis revealed enhanced T-cell polyfunctionality of CD123-ENG.IL15 T-cells as judged by effector cytokine production, including, granzyme B, IFN-γ, MIP-1α, perforin, TNF-α, and TNF-β. In vivo, CD123-ENG.IL15 T-cells exhibited superior antigen-specific anti-AML activity and T-cell persistence in both peripheral blood and tissues (BM, spleens, and livers), resulting in a significant survival advantage in one AML xenograft model and two autologous AML PDX models. In conclusion, we demonstrate here that the expansion, persistence, and anti-AML activity of CD123-ENG T-cells can be significantly improved by transgenic expression of IL15, which promotes a naïve/TSCM-like phenotype. However, we also highlight that targeting a single tumor antigen (CD123) can lead to immune escape, reinforcing the need to develop approaches to target multiple antigens. Likewise, our study demonstrates that it is feasible to evaluate autologous T cells in AML PDX models, which will be critical for future preclinical evaluations of next generation AML-redirected T-cell therapies.

CD19-specific chimeric antigen receptor (CAR) T cell therapy has changed the treatment paradigm for pediatric, adolescent and young adult (AYA) patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, data on the associated infectious disease challenges in this patient population are scarce. Knowledge of infections presenting during treatment, and associated risk factors, is critical for pediatric cellular therapy and infectious disease specialists as we seek to formulate effective anti-infective prophylaxis, infection monitoring schemas, and empiric therapy regimens. In this work we describe our institutional experience in a cohort of 38 pediatric and AYA patients with CD19-positive malignancy treated with lymphodepleting chemotherapy (fludarabine/cyclophosphamide) followed by a single infusion of CD19-CAR T cells (total infusions, n=39), including tisagenlecleucel (n=19; CD19/4-1BB) or on an institutional clinical trial (n=20; CD19/4-1BB; NCT03573700). We demonstrate that infections were common in the 90 days post CAR T cells, with 19 (50%) patients experiencing a total of 35 infections. Most of these (73.7%) occurred early post infusion (day 0 to 28; infection density of 2.36 per 100 patient days-at-risk) compared to late post infusion (day 29 to 90; infection density 0.98 per 100 patient days-at-risk), respectively. Bacterial infections were more frequent early after CAR T cell therapy, with a predominance of bacterial blood stream infections. Viral infections occurred throughout the post infusion period and included primarily systemic reactivations and gastrointestinal pathogens. Fungal infections were rare. Pre-infusion disease burden, intensity of bridging chemotherapy, lymphopenia post lymphodepleting chemotherapy/CAR T cell infusion and development of CAR-associated hemophagocytic lymphohistiocytosis (carHLH) were all significantly associated with either infection density or time to first infection post CAR T cell infusion. A subset of patients (n=6) had subsequent CAR T cell reinfusion and did not appear to have increased risk of infectious complications. Our experience highlights the risk of infections after CD19-CAR T cell therapy, and the need for continued investigation of infectious outcomes as we seek to improve surveillance, prophylaxis and treatment algorithms.

Chimeric antigen receptor (CAR) T cells targeting CD123, an acute myeloid leukemia (AML) antigen, hold the promise of improving outcomes for patients with refractory/recurrent disease. We generated five lentiviral vectors encoding CD20, which may serve as a target for CAR T cell depletion, and 2nd or 3rd generation CD123-CARs since the benefit of two costimulatory domains is model dependent. Four CARs were based on the CD123-specific single-chain variable fragment (scFv) 26292 (292) and one CAR on the CD123-specific scFv 26716 (716), respectively. We designed CARs with different hinge/transmembrane (H/TM) domains and costimulatory domains, in combination with the zeta (z) signaling domain: 292.CD8aH/TM.41BBz (8.41BBz), 292.CD8aH/TM.CD28z (8.28z), 716.CD8aH/TM.CD28z (716.8.28z), 292.CD28H/TM. CD28z (28.28z), and 292.CD28H/TM.CD28.41BBz (28.28.41BBz). Transduction efficiency, expansion, phenotype, and target cell recognition of the generated CD123-CAR T cells did not significantly differ. CAR constructs were eliminated for the following reasons: (1) 8.41BBz CARs induced significant baseline signaling, (2) 716.8.28z CAR T cells had decreased anti-AML activity, and (3) CD28.41BBz CAR T cells had no improved effector function in comparison to CD28z CAR T cells. We selected the 28.28z CAR since CAR expression on the cell surface of transduced T cells was higher in comparison to 8.28z CARs. The clinical study (NCT04318678) evaluating 28.28z CAR T cells is now open for patient accrual.

B7-H3 is actively being explored as an immunotherapy target for pediatric patients with solid tumors using monoclonal antibodies or T cells expressing chimeric antigen receptors (CARs). B7-H3-CARs containing a 41BB costimulatory domain are currently favored by several groups based on preclinical studies. In this study, we initially performed a detailed analysis of T cells expressing B7-H3-CARs with different hinge/transmembrane (CD8α versus CD28) and CD28 or 41BB costimulatory domains (CD8α/CD28, CD8α/41BB, CD28/CD28, CD28/41BB). Only subtle differences in effector function were observed between CAR T cell populations in vitro. However, CD8α/CD28-CAR T cells consistently outperformed other CAR T cell populations in three animal models, resulting in a significant survival advantage. We next explored whether adding 41BB signaling to CD8α/CD28-CAR T cells would further enhance effector function. Surprisingly, incorporating 41BB signaling into the CAR endodomain had detrimental effects, while expressing 41BBL on the surface of CD8α/CD28-CAR T cells enhanced their ability to kill tumor cells in repeat stimulation assays. Furthermore, 41BBL expression enhanced CD8α/CD28-CAR T cell expansion in vivo and improved antitumor activity in one of four evaluated models. Thus, our study highlights the intricate interplay between CAR hinge/transmembrane and costimulatory domains. Based on our study, we selected CD8α/CD28-CAR T cells expressing 41BBL for early phase clinical testing.

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.

Cell-based immunotherapies have had remarkable success in the clinic, specifically in the treatment of hematologic malignancies. However, these strategies have had limited efficacy in patients with solid tumors. To better understand the challenges involved, the National Cancer Institute (NCI) convened an initial workshop with immuno-oncology thought leaders in December 2018 and a follow-up workshop in December 2020. The goals of the NCI workshops on cell-based immunotherapy for solid tumors were to discuss the current state of the field of cell-based immunotherapy, obtain insights into critical knowledge gaps, and identify ways in which NCI could facilitate progress. At both meetings, subjects emphasized four main types of challenges in further developing cell-based immunotherapy for patients with solid tumors: scientific, technical, clinical, and regulatory. The scientific barriers include selecting appropriate targets, ensuring adequate trafficking of cell therapy products to tumor sites, overcoming the immunosuppressive tumor microenvironment, and identifying appropriate models for these investigations. While mouse models may provide some useful data, the majority of those that are commonly used are immunodeficient and unable to fully recapitulate the immune response in patients. There is therefore a need for enhanced support of small early-phase human clinical studies, preferably with adaptive trial designs, to provide proof of concept for novel cell therapy approaches. Furthermore, the requirements for manufacturing, shipping, and distributing cell-based therapies present technical challenges and regulatory questions, which many research institutions are not equipped to address. Overall, workshop subjects identified key areas where NCI support might help the research community in driving forward innovation and clinical utility: 1) provide focused research support on topics such as tumor target selection, immune cell fitness and persistence, cell trafficking, and the immunosuppressive tumor microenvironment; 2) support the rapid translation of preclinical findings into proof of concept clinical testing, harmonize clinical trial regimens, and facilitate early trial data sharing (including negative results); 3) expand manufacturing support for cell therapies, including vectors and reagents, and provide training programs for technical staff; and 4) develop and share standard operating procedures for cell handling and analytical assays, and work with the Food and Drug Administration to harmonize product characterization specifications.

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.

The outcome for metastatic pediatric osteosarcoma (OS) remains poor. Thus, there is an urgent need to develop novel therapies, and immunotherapy with CAR T cells has the potential to meet this challenge. However, there is a lack of preclinical models that mimic salient features of human disease including reliable development of metastatic disease post orthotopic OS cell injection. To overcome this roadblock, and also enable real-time imaging of metastatic disease, we took advantage of LM7 OS cells expressing firefly luciferase (LM7.ffLuc). LM7.ffLuc were implanted in a collagen mesh into the tibia of mice, and mice reliably developed orthotopic tumors and lung metastases as judged by bioluminescence imaging and histopathological analysis. Intratibial implantation also enabled surgical removal by lower leg amputation and monitoring for metastases development post-surgery. We then used this model to evaluate the antitumor activity of CAR T cells targeting B7-H3, an antigen that is expressed in a broad range of solid tumors including OS. B7-H3-CAR T cells had potent antitumor activity in a dose-dependent manner and inhibited the development of pulmonary metastases resulting in a significant survival advantage. In contrast T cells expressing an inactive B7-H3-CAR had no antitumor activity. Using unmodified LM7 cells also enabled us to demonstrate that B7-H3-CAR T cells traffic to orthotopic tumor sites. Hence, we have developed an orthotopic, spontaneously metastasizing OS model. This model may improve our ability not only to predict the safety and efficacy of current and next generation CAR T cell therapies but also other treatment modalities for metastatic OS.

Allogeneic hematopoietic stem cell transplant (HSCT) for relapsed pediatric non-Hodgkin lymphoma (NHL) is often reserved for patients with certain NHL subtypes or high-risk disease whereas the remainder receive autologous HSCT. Given the aggressive nature of pediatric NHL, we performed allogeneic HSCTs for all patients regardless of disease risk. We report overall survival (OS) and prognostic variables in 36 pediatric patients who underwent allogeneic HSCT between 1998 and 2016. OS at 3 years was 67%. The 3-year OS varied based on NHL subtype: 100% for anaplastic large cell lymphoma (n = 14), 63% for diffuse large B-cell lymphoma (n = 8), 17% for lymphoblastic lymphoma (LL; n = 9) and 80% for other subtypes combined (n = 5). Disease status influenced outcome with 3-year OS of 100% for patients in complete remission (n = 15), 59% with partial remission (PR; n = 17), and 0% with progressive/stable disease (n = 3) (P = .004). Of the 17 patients in PR, all 6 with LL died of relapsed disease, whereas the other 11 attained remission after HSCT and remained disease-free. The cumulative incidence of relapse after HSCT for LL was 78% compared with 15% for all other NHL subtypes combined (P < .0001). Cumulative incidence of nonrelapse mortality (NRM) was low in our cohort at 6%. Hence, allogeneic HSCT is a well-tolerated and useful therapeutic option with low rates of NRM and relapse for all NHL subtypes except LL with active disease at HSCT.

Abstract: Cytomegalovirus (CMV) infections remain a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT), and standard antiviral therapies are associated with significant side effects and development of drug-resistant mutants. Adoptively transferred donor-derived CMV-specific T cells (CMVSTs) can provide an alternative treatment modality with few side effects but are not widely available due to their patient-specific nature. Here we report the establishment and use of a bank of CMVSTs derived from just 8 CMV-seropositive donors, with HLA types representing the diverse US population, as an “off-the-shelf” therapy to treat drug-refractory infections. To date, we have screened 29 patients for study participation and identified a suitable line, with ≥2 of 8 shared HLA antigens, for 28 (96.6%) patients with a median of 4 shared HLA antigens. Of these, 10 patients with persistent/refractory CMV infections or disease were eligible for treatment; a single infusion of cells produced 3 partial responses and 7 complete responses, for a cumulative response rate of 100% (95% confidence interval, 69.2-100) with no graft-versus-host disease, graft failure, or cytokine release syndrome. Potential wider use of the tested CMVSTs across transplant centers is made more feasible by our ability to produce sufficient material to generate cells for >2000 infusions from a single donor collection. Our data indicate that a “mini” bank of CMVSTs prepared from just 8 well-chosen third-party donors can supply the majority of patients with an appropriately matched line that produces safe and effective anti-CMV activity post-HSCT.

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.

Chimeric antigen receptor (CAR) T-cell therapy combines antigen-specific properties of monoclonal antibodies with the lytic capacity of T cells. An effective and safe CAR-T cell therapy strategy relies on identifying an antigen that has high expression and is tumor specific. This strategy has been successfully used to treat patients with CD19+ B-cell acute lymphoblastic leukemia (B-ALL). Finding a suitable target antigen for other cancers such as acute myeloid leukemia (AML) has proven challenging, as the majority of currently targeted AML antigens are also expressed on hematopoietic progenitor cells (HPCs) or mature myeloid cells. Herein, we developed a computational method to perform a data transformation to enable the comparison of publicly available gene expression data across different datasets or assay platforms. The resulting transformed expression values (TEVs) were used in our antigen prediction algorithm to assess suitable tumor-associated antigens (TAAs) that could be targeted with CAR-T cells. We validated this method by identifying B-ALL antigens with known clinical effectiveness, such as CD19 and CD22. Our algorithm predicted TAAs being currently explored preclinically and in clinical CAR-T AML therapy trials, as well as novel TAAs in pediatric megakaryoblastic AML. Thus, this analytical approach presents a promising new strategy to mine diverse datasets for identifying TAAs suitable for immunotherapy.

Chimeric antigen receptor (CAR) T cell therapy for solid tumors has shown limited efficacy in early-phase clinical studies. The majority of CARs encode CD28 and/or 41BB costimulatory endodomains, and we explored whether MyD88 and CD40 (MC) costimulatory endodomains in CARs could improve their antitumor activity. We generated CD28-, 41BB-, and MC-CAR T cells and demonstrated that MC-CAR T cells have greater proliferative capacity and antitumor activity in repeat stimulation assays and in tumor models in vivo. Transcriptomic analysis revealed that MC-CAR T cells expressed higher levels of MYB and FOXM1, key cell cycle regulators, and were activated at baseline. After stimulation, MC-CAR T cells remained in a less differentiated state than CD28- and 41BB-CAR T cells as judged by low levels of transcription factor TBET and B lymphocyte induced maturation protein 1 expression and lower cytolytic activity in comparison with CD28- and 41BB-CAR T cells. Thus, including MyD88 and CD40 signaling domains in CARs may improve current CAR T cell therapy approaches for solid tumors.

Chimeric antigen receptor (CAR) T cells targeting CD19 have been successful treating patients with relapsed/refractory B cell acute lymphoblastic leukemia (ALL) and B cell lymphomas. However, relapse after CAR T cell therapy is still a challenge. In addition, preclinical and early clinical studies targeting acute myeloid leukemia (AML) have not been as successful. This can be attributed in part to the presence of an AML microenvironment that has a dampening effect on the antitumor activity of CAR T cells. The AML microenvironment includes cellular interactions, soluble environmental factors, and structural components. Suppressive immune cells including myeloid derived suppressor cells and regulatory T cells are known to inhibit T cell function. Environmental factors contributing to T cell exhaustion, including immune checkpoints, anti-inflammatory cytokines, chemokines, and metabolic alterations, impact T cell activity, persistence, and localization. Lastly, structural factors of the bone marrow niche, secondary lymphoid organs, and extramedullary sites provide opportunities for CAR T cell evasion by AML blasts, contributing to treatment resistance and relapse. In this review we discuss the effect of the AML microenvironment on CAR T cell function. We highlight opportunities to enhance CAR T cell efficacy for AML through manipulating, targeting, and evading the anti-inflammatory leukemic microenvironment.

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. Keywords: GBM, CAR T cells, EphA2, brain tumor

Current therapies against hepatitis B virus (HBV) do not reliably cure chronic infection, necessitating new therapeutic approaches. The T cell response can clear HBV during acute infection, and the adoptive transfer of antiviral T cells during bone marrow transplantation can cure patients of chronic HBV infection. To redirect T cells to HBV-infected hepatocytes, we delivered plasmids encoding bispecific antibodies directed against the viral surface antigen (HBsAg) and CD3, expressed on almost all T cells, directly into the liver using hydrodynamic tail vein injection. We found a significant reduction in HBV-driven reporter gene expression (184-fold) in a mouse model of acute infection, which was 30-fold lower than an antibody only recognizing HBsAg. While bispecific antibodies triggered, in part, antigen-independent T cell activation, antibody production within hepatocytes was non-cytotoxic. We next tested the bispecific antibodies in a different HBV mouse model, which closely mimics the transcriptional template for HBV, covalently closed circular DNA (cccDNA). We found that the antiviral effect was noncytopathic, mediating a 495-fold reduction in HBsAg levels at day 4. At day 33, bispecific antibody-treated mice exhibited 35-fold higher host HBsAg immunoglobulin G (IgG) antibody production versus untreated groups. Thus, gene therapy with HBsAg/CD3-bispecific antibodies represents a promising therapeutic strategy for patients with HBV.

Correlative studies of clinical studies for hematological malignancies have implicated that less differentiated, CD8+-dominant CAR T cell products have greater antitumor activity. Here, we have investigated whether the differentiation status of CAR T cell products affects their antitumor activity in preclinical models of solid tumors. We explored if different activation/expansion protocols, as well as different co-stimulatory domains in the CAR construct, influence the short- and long-term efficacy of CAR T cells against HER2-positive tumors. We generated T cell products that range from the most differentiated (CD28.z; OKT3-antiCD28/RPMI expansion) to the least differentiated (41BB.z; OKT3-RetroNectin/LymphoONE expansion), as judged by cell surface expression of the differentiation markers CCR7 and CD45RA. While the effect of differentiation status was variable with regard to antigen-specific cytokine production, the most differentiated CD28.z CAR T cell products, which were enriched in effector memory T cells, had the greatest target-specific cytolytic activity in vitro. These products also had a greater proliferative capacity and maintained CD4+ T cells upon repeated stimulation in vitro. In vivo, differentiated CD28.z CAR T cells also had the greatest antitumor activity, resulting in complete response. Our results highlight that it is critical to optimize CAR T cell production and that optimal product characteristics might depend on the targeted antigen and/or cancer.

The outcome for advanced stage hepatocellular carcinoma (HCC) remains poor, highlighting the need for novel therapies. Genetically modified mesenchymal stem cells (MSCs) are actively being explored as cancer therapeutics due to their inherent ability to migrate to tumor sites. We reasoned that MSCs can be genetically modified to redirect T cells to Glypican-3 (GPC3)+ HCC, and genetically modified these with viral vectors encoding a GPC3/CD3 bispecific T cell engager (GPC3-ENG), a bispecifc T cell engager specific for an irrelevant antigen (EGFRvIII), and/or costimulatory molecules (CD80 and 41BBL). Coculture of GPC3+ cells, GPC3-ENG MSCs, and T cells resulted in T cell activation, as judged by interferon γ (IFNγ) production and killing of tumor cells by T cells. Modification of GPC3-ENG MSCs with CD80 and 41BBL was required for antigen-dependent interleukin-2 (IL-2) production by T cells and resulted in faster tumor cell killing by redirected T cells. In vivo, GPC3-ENG MSCs ± costimulatory molecules had antitumor activity in the HUH7 HCC xenograft model, resulting in a survival advantage. In conclusion, MSCs genetically modified to express GPC3-ENG ± costimulatory molecules redirect T cells to GPC3+ tumor cells and have potent antitumor activity. Thus, further preclinical exploration of our modified approach to GPC3-targeted immunotherapy for HCC is warranted.

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.

The efficacy of T cells expressing chimeric antigen receptors (CARs) for solid tumors has been limited by insufficient CAR T cell expansion and persistence. The use of virus-specific T cells (VSTs) as carriers for CARs may overcome this limitation since CAR-VSTs can be boosted by viral vaccines or oncolytic viruses. However, there is limited understanding of the optimal combination of endodomains and their influence on the native T cell receptor (TCR) in VSTs. We therefore compared the function of GD2.CARs expressing the TCR zeta chain (ζ) alone or combined with endodomains from CD28 and 4-1BB in varicella zoster virus-specific (VZV) T cells. VZVSTs expressing GD2-CARs recognized VZV-derived peptides and killed GD2-expressing tumor cells. However, after repeated stimulation through their native TCR, the expansion of GD2-CAR.CD28ζ-VZVSTs was 3.3-fold greater (p < 0.001) than non-transduced VZVSTs, whereas GD2-CARζ- and GD2-CAR.41BBζ inhibited VZVST expansion (p < 0.01). Compared to control VZVSTs, GD2-CAR.ζ VZVSTs showed a greater frequency of apoptotic (p < 0.01) T cells, whereas prolonged downregulation of the native αβ TCR was observed in GD2-CAR.41BBζ VZVSTs (p < 0.001). We confirmed that CD28ζ can best maintain TCR function by expressing GD2.CARs in Epstein-Barr virus-specific T cells and CD19-CARs in VZVSTs. In response to CAR stimulation VSTs with CD28ζ endodomains also showed the greatest expansion (6 fold > GD2-CAR.41BBζ VZVSTs (p < 0.001), however anti-tumor efficacy was superior in GD2-CAR.41BBζ-VZVSTs. These findings demonstrate that CAR signaling domains can enhance or diminish the function of the native TCR and indicate that only CD28ζ may preserve the function of the native TCR in tonically signaling CAR-VSTs.