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T cells genetically engineered to recognize and eliminate tumor cells through synthetic chimeric antigen receptors (CARs) have demonstrated remarkable clinical efficacy against B cell leukemia over the past decade. This therapy is a form of highly personalized medicine that involves genetically modifying a patient's T cells to recognize and kill cancer cells. With the FDA approval of 5 CAR T cell products, this approach has been validated as a powerful new drug in the therapeutic armamentarium against cancer. Researchers are now studying how to expand this technology beyond its use in conventional polyclonal αβ T cells to address limitations to the current therapy in cancer and applications beyond it. Considering the specific characteristics of immune cell from diverse lineages, several preclinical and clinical studies are under way to assess the advantages of CAR-redirected function in these cells and apply the lessons learned from CAR T cell therapy in cancer to other diseases.

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Sequences of CAR T cells with mCD8 signal peptide annotated in orange, scFv annotated in red, myc tag annotated in highlighted gray, mCD28 transmembrane and intracellular annotated in purple, mZeta annotated in green, IRES annotated in pink, mIL12 annotated in black, and m4-1BB annotated in yellow.

T-cell receptor (TCR)–modified T-cell gene therapy can target a variety of extracellular and intracellular tumor-associated antigens, yet has had little clinical success. A potential explanation for limited antitumor efficacy is a lack of T-cell activation in vivo. We postulated that expression of proinflammatory cytokines in TCR-modified T cells would activate T cells and enhance antitumor efficacy. We demonstrate that expression of interleukin 18 (IL18) in tumor-directed TCR-modified T cells provides a superior proinflammatory signal than expression of interleukin 12 (IL12). Tumor-targeted T cells secreting IL18 promote persistent and functional effector T cells and a proinflammatory tumor microenvironment. Together, these effects augmented overall survival of mice in the pmel-1 syngeneic tumor model. When combined with sublethal irradiation, IL18-secreting pmel-1 T cells were able to eradicate tumors, whereas IL12-secreting pmel-1 T cells caused toxicity in mice through excessive cytokine secretion. In another xenograft tumor model, IL18 secretion enhanced the persistence and antitumor efficacy of NY-ESO-1–reactive TCR-modified human T cells as well as overall survival of tumor-bearing mice. These results demonstrate a rationale for optimizing the efficacy of TCR-modified T-cell cancer therapy through expression of IL18.See related commentary by Wijewarnasuriya et al., p. 732

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We present a case of a patient with multiply relapsed, refractory myeloma whose clinical course showed evidence of a synergistic abscopal-like response to chimeric antigen receptor (CAR) T-cell therapy and localized radiotherapy (XRT). Shortly after receiving B-cell maturation antigen (BCMA)–targeted CAR T-cell therapy, the patient required urgent high-dose steroids and XRT for spinal cord compression. Despite the steroids, the patient had a durable systemic response that could not be attributed to XRT alone. Post-XRT findings included a second wave of fever and increased CRP and IL6, beginning 21 days after CAR T cells, which is late for cytokine-release syndrome from CAR T-cell therapy alone on this trial. Given this response, which resembled cytokine-release syndrome, immediately following XRT, we investigated changes in the patient's T-cell receptor (TCR) repertoire over 10 serial time points. Comparing T-cell diversity via Morisita's overlap indices (CD), we discovered that, although the diversity was initially stable after CAR T-cell therapy compared with baseline (CD = 0.89–0.97, baseline vs. 4 time points after CAR T cells), T-cell diversity changed after the conclusion of XRT, with >30% newly expanded TCRs (CD = 0.56–0.69, baseline vs. 4 time points after XRT). These findings suggest potential synergy between radiation and CAR T-cell therapies resulting in an abscopal-like response.

Although clinical responses with CD19-targeting chimeric antigen receptor (CAR) T-cell treatment have been observed in patients with certain hematologic malignancies, high rates of disease relapse highlight the necessity to understand and improve mechanisms of CAR T-cell failure. Because T-cell dysfunction is thought to contribute to CAR T-cell treatment failure, understanding what mechanisms drive T cells into this dysfunctional state may aid optimal design of efficacious CAR T cells. Dysfunctional CAR T cells have been characterized as having upregulated inhibitory receptors and decreased cytolytic capabilities. Previous studies have identified a role for sustained CAR CD3ζ signaling in CAR T-cell dysfunction. Here, we demonstrate a mechanism that drives dysfunction in CAR T cells through excessive costimulation. Fully activated CD19-targeted CAR T cells were rendered dysfunctional upon stimulation with both endogenous CD28 stimulation and CAR-mediated CD28 costimulation. Costimulation-driven dysfunction of CAR T cells was demonstrated in a syngeneic immunocompetent mouse model, in which CAR T cells were activated with signals 1 (CD3ζ), 2 (CD28), and 3 (IL12). Thus, we show that CAR T-cell dysfunction can be driven through excessive CD28 and 4-1BB costimulation.See related article by Drakes et al., p. 743

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Table S1: Detailed descriptions of all grade neurotoxicity; Table S2: Correlation between CAR T cell phenotype and neurotoxicity; Table S3: Correlation between CSF cytokines and CSF protein concentrations; Table S4: Definitions of CRS

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Malignant pleural diseases, comprising metastatic lung and breast cancers and malignant pleural mesothelioma (MPM), are aggressive solid tumors with poor therapeutic response. We developed and conducted a first-in-human, phase I study of regionally delivered, autologous, mesothelin-targeted chimeric antigen receptor (CAR) T-cell therapy. Intrapleural administration of 0.3M to 60M CAR T cells/kg in 27 patients (25 with MPM) was safe and well tolerated. CAR T cells were detected in peripheral blood for >100 days in 39% of patients. Following our demonstration that PD-1 blockade enhances CAR T-cell function in mice, 18 patients with MPM also received pembrolizumab safely. Among those patients, median overall survival from CAR T-cell infusion was 23.9 months (1-year overall survival, 83%). Stable disease was sustained for ≥6 months in 8 patients; 2 exhibited complete metabolic response on PET scan. Combination immunotherapy with CAR T cells and PD-1 blockade agents should be further evaluated in patients with solid tumors.Significance:Regional delivery of mesothelin-targeted CAR T-cell therapy followed by pembrolizumab administration is feasible, safe, and demonstrates evidence of antitumor efficacy in patients with malignant pleural diseases. Our data support the investigation of combination immunotherapy with CAR T cells and PD-1 blockade agents in solid tumors.See related commentary by Aldea et al., p. 2674.This article is highlighted in the In This Issue feature, p. 2659

Figure S1: Detailed time course of neurotoxicity for each patient; Figure S2: Serum cytokines in relation to tocilizumab and corticosteroid administration; Figure S3: Hematopoietic toxicity and coagulopathy in severe neurotoxicity; Figure S4: Angiopoietin 1 and 2 alterations in severe neurotoxicity

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Supplemental Material

Purpose: Most patients diagnosed with ovarian cancer will ultimately die from their disease. For this reason, novel approaches to the treatment of this malignancy are needed. Adoptive transfer of a patient's own T cells, genetically modified ex vivo through the introduction of a gene encoding a chimeric antigen receptor (CAR) targeted to a tumor-associated antigen, is a novel approach to the treatment of ovarian cancer.Experimental Design: We have generated several CARs targeted to the retained extracellular domain of MUC16, termed MUC-CD, an antigen expressed on most ovarian carcinomas. We investigate the in vitro biology of human T cells retrovirally transduced to express these CARs by coculture assays on artificial antigen-presenting cells as well as by cytotoxicity and cytokine release assays using the human MUC-CD+ ovarian tumor cell lines and primary patient tumor cells. Further, we assess the in vivo antitumor efficacy of MUC-CD–targeted T cells in SCID-Beige mice bearing peritoneal human MUC-CD+ tumor cell lines.Results: CAR-modified, MUC-CD–targeted T cells exhibited efficient MUC-CD–specific cytolytic activity against both human ovarian cell and primary ovarian carcinoma cells in vitro. Furthermore, expanded MUC-CD–targeted T cells infused through either i.p. injection or i.v. infusion into SCID-Beige mice bearing orthotopic human MUC-CD+ ovarian carcinoma tumors either delayed progression or fully eradicated disease.Conclusion: These promising preclinical studies justify further investigation of MUC-CD–targeted T cells as a potential therapeutic approach for patients with high-risk MUC16+ ovarian carcinomas. Clin Cancer Res; 16(14); 3594–606. ©2010 AACR.

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With the US Food and Drug Administration (FDA) approval of four CD19- and one BCMA-targeted chimeric antigen receptor (CAR) therapy for B cell malignancies, CAR T cell therapy has finally reached the status of a medicinal product. The successful manufacturing of autologous CAR T cell products is a key requirement for this promising treatment modality. By analyzing the composition of 214 apheresis products from 210 subjects across eight disease indications, we found that high CD14+ cell content poses a challenge for manufacturing CAR T cells, especially in patients with non-Hodgkin's lymphoma and multiple myeloma caused by the non-specific phagocytosis of the magnetic beads used to activate CD3+ T cells. We demonstrated that monocyte depletion via rapid plastic surface adhesion significantly reduces the CD14+ monocyte content in the apheresis products and simultaneously boosts the CD3+ content. We established a 40% CD14+ threshold for the stratification of apheresis products across nine clinical trials and demonstrated the effectiveness of this procedure by comparing manufacturing runs in two phase 1 clinical trials. Our study suggests that CD14+ content should be monitored in apheresis products, and that the manufacturing of CAR T cells should incorporate a step that lessens the CD14+ cell content in apheresis products containing more than 40% to maximize the production success.

Malignant pleural diseases, comprising metastatic lung and breast cancers and malignant pleural mesothelioma (MPM), are aggressive solid tumors with poor therapeutic response. We developed and conducted a first-in-human, phase I study of regionally delivered, autologous, mesothelin-targeted chimeric antigen receptor (CAR) T-cell therapy. Intrapleural administration of 0.3M to 60M CAR T cells/kg in 27 patients (25 with MPM) was safe and well tolerated. CAR T cells were detected in peripheral blood for >100 days in 39% of patients. Following our demonstration that PD-1 blockade enhances CAR T-cell function in mice, 18 patients with MPM also received pembrolizumab safely. Among those patients, median overall survival from CAR T-cell infusion was 23.9 months (1-year overall survival, 83%). Stable disease was sustained for ≥6 months in 8 patients; 2 exhibited complete metabolic response on PET scan. Combination immunotherapy with CAR T cells and PD-1 blockade agents should be further evaluated in patients with solid tumors. Significance: Regional delivery of mesothelin-targeted CAR T-cell therapy followed by pembrolizumab administration is feasible, safe, and demonstrates evidence of antitumor efficacy in patients with malignant pleural diseases. Our data support the investigation of combination immunotherapy with CAR T cells and PD-1 blockade agents in solid tumors. See related commentary by Aldea et al., p. 2674. This article is highlighted in the In This Issue feature, p. 2659

CD19-targeted chimeric antigen receptor (CAR) T-cell therapy has become a breakthrough treatment of patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, despite the high initial response rate, the majority of adult patients with B-ALL progress after CD19 CAR T-cell therapy. Data on the natural history, management, and outcome of adult B-ALL progressing after CD19 CAR T cells have not been described in detail. Herein, we report comprehensive data of 38 adult patients with B-ALL who progressed after CD19 CAR T therapy at our institution. The median time to progression after CAR T-cell therapy was 5.5 months. Median survival after post–CAR T progression was 7.5 months. A high disease burden at the time of CAR T-cell infusion was significantly associated with risk of post–CAR T progression. Thirty patients (79%) received salvage treatment of post–CAR T disease progression, and 13 patients (43%) achieved complete remission (CR), but remission duration was short. Notably, 7 (58.3%) of 12 patients achieved CR after blinatumomab and/or inotuzumab administered following post–CAR T failure. Multivariate analysis revealed that a longer remission duration from CAR T cells was associated with superior survival after progression following CAR T-cell therapy. In summary, overall prognosis of adult B-ALL patients progressing after CD19 CAR T cells was poor, although a subset of patients achieved sustained remissions to salvage treatments, including blinatumomab, inotuzumab, and reinfusion of CAR T cells. Novel therapeutic strategies are needed to reduce risk of progression after CAR T-cell therapy and improve outcomes of these patients.

Chimeric antigen receptor (CAR) T-cell therapy for multiple myeloma targeting B-cell maturation antigen (TNFRSF17; BCMA) induces high overall response rates; however, relapse occurs commonly. A reservoir of multiple myeloma cells lacking sufficient BCMA surface expression (antigen escape) may be implicated in relapse. We demonstrate that simultaneous targeting of an additional antigen—here, G protein-coupled receptor class-C group-5 member-D (GPRC5D)—can prevent BCMA escape–mediated relapse in a model of multiple myeloma. To identify an optimal approach, we compare subtherapeutic doses of different forms of dual-targeted cellular therapy. These include; (i) parallel-produced and pooled mono-targeted CAR T cells, (ii) bicistronic constructs expressing distinct CARs from a single vector, and (iii) a dual-scFv “single-stalk” CAR design. When targeting BCMA-negative disease, bicistronic and pooled approaches had the highest efficacy, whereas for dual-antigen–expressing disease, the bicistronic approach was more efficacious than the pooled approach. Mechanistically, expressing two CARs on a single cell enhanced the strength of CAR T-cell/target cell interactions. Significance: Myeloma frequently relapses post-CAR T-cell therapy; antigen escape–mediated relapse can be mitigated with upfront dual-targeting (BCMA/GPRC5D). A bicistronic vector encoding two CARs avoids the challenge of parallel manufacturing separate CAR T-cell products, while providing superior efficacy; this dual-targeted approach may enhance the durability of responses to cellular therapy for myeloma. See related commentary by Simon and Riddell, p. 130. This article is highlighted in the In This Issue feature, p. 127

Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable responses in B-cell malignancies. However, many patients suffer from limited response and tumor relapse due to lack of persisting CAR T cells and immune escape. These clinical challenges have compromised the long-term efficacy of CAR T-cell therapy and call for the development of novel CAR designs. We demonstrated that CAR T cells secreting a cytokine interleukin-36γ (IL-36γ) showed significantly improved CAR T-cell expansion and persistence, and resulted in superior tumor eradication compared with conventional CAR T cells. The enhanced cellular function by IL-36γ was mediated through an autocrine manner. In addition, activation of endogenous antigen-presenting cells (APCs) and T cells by IL-36γ aided the formation of a secondary antitumor response, which delayed the progression of antigen-negative tumor challenge. Together, our data provide preclinical evidence to support the translation of this design for an improved CAR T-cell-mediated antitumor response.

BackgroundWaldenström macroglobulinemia (WM) is an incurable disease and, while treatable, can develop resistance to available therapies and be fatal. Chimeric antigen receptor (CAR) T cell therapy directed against the CD19 antigen has demonstrated efficacy in relapsed or refractory B lymphoid malignancies, and is now approved for B cell acute lymphoblastic leukemia and certain B cell lymphomas. However, CAR T therapy has not been evaluated for use in WM.Methods and resultsWe performed preclinical studies demonstrating CAR T cell activity against WM cells in vitro, and developed an in vivo murine model of WM which demonstrated prolonged survival with use of CAR T therapy. We then report the first three patients with multiply relapsed and refractory WM treated for their disease with CD19-directed CAR T cells on clinical trials. Treatment was well tolerated, and observed toxicities were consistent with those seen in CAR T treatment for other diseases, and no grade 3 or higher cytokine release syndrome or neurotoxicity events occurred. All three patients attained at least a clinical response to treatment, including one minimal residual disease-negative complete response, though all three eventually developed recurrent disease between 3 and 26 months after initial treatment.ConclusionsThis report summarizes preclinical and clinical activity of CD19-directed CAR T therapy in WM, demonstrating early tolerability and efficacy in patients with WM, and representing a possible treatment option in patients with heavily pretreated and relapsed or refractory WM. Larger studies evaluating CAR T therapy in WM are warranted, along with further evaluation into mechanisms of resistance to CAR T therapy.

Chimeric Antigen Receptor (CAR)-T cells represent a major breakthrough in cancer therapy, wherein a patient’s own T cells are engineered to recognize a tumor antigen, resulting in activation of a local cytotoxic immune response. However, CAR-T cell therapies are currently limited to the treatment of B-cell cancers and their effectiveness is hindered by resistance from antigen-negative tumor cells, immunosuppression in the tumor microenvironment, eventual exhaustion of T-cell immunologic functions, and frequent severe toxicities. To overcome these problems, we have developed a novel class of CAR-T cells engineered to express an enzyme that activates a systemically-administered small-molecule prodrug in situ at a tumor site. We show that these Synthetic Enzyme-Armed KillER (SEAKER) cells exhibit enhanced anticancer activity with small-molecule prodrugs, both in vitro and in vivo in mouse tumor models. This modular platform enables combined targeting of cellular and small-molecule therapies to treat cancers and potentially a variety of other diseases.

In the New England Journal of Medicine, Wang et al. report favorable ZUMA-2 trial results of CAR T cells for patients with relapsed and refractory mantle cell lymphoma following prior failed Bruton's tyrosine kinase inhibitor therapy, with an overall response rate of 93% and an expected safety profile.

Radiotherapy is potentially an important salvage strategy post-chimeric antigen receptor T cell therapy (CART), but limited data exist. We reviewed 14 patients treated with salvage radiation post-CART progression (SRT). Most received SRT for first post-CART relapse (71%) to sites previously PET-avid pre-CART (79%). Median overall survival (OS) post-SRT was 10 months. Post-SRT, six localized relapses achieved 100% response (3 = complete, 3 = partial), with improved freedom from subsequent relapse (P = 0·001) and OS (P = 0·004) compared to advanced stage relapses. Three were bridged to allogeneic transplantation; at analysis, all were alive/NED. SRT has diverse utility and can integrate with novel agents or transplantation to attempt durable remissions.

Chimeric antigen receptor (CAR) T cells have demonstrated clinical benefit in patients with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). We undertook a multicenter clinical trial to determine toxicity, feasibility, and response for this therapy. A total of 25 pediatric/young adult patients (age, 1-22.5 years) with R/R B-ALL were treated with 19-28z CAR T cells. Conditioning chemotherapy included high-dose (3 g/m2) cyclophosphamide (HD-Cy) for 17 patients and low-dose (≤1.5 g/m2) cyclophosphamide (LD-Cy) for 8 patients. Fifteen patients had pretreatment minimal residual disease (MRD

T cells genetically engineered to express chimeric antigen receptors (CARs) have proven — and impressive — therapeutic activity in patients with certain subtypes of B cell leukaemia or lymphoma, with promising efficacy also demonstrated in patients with multiple myeloma. Nevertheless, various barriers restrict the efficacy and/or prevent the widespread use of CAR T cell therapies in these patients as well as in those with other cancers, particularly solid tumours. Key challenges relating to CAR T cells include severe toxicities, restricted trafficking to, infiltration into and activation within tumours, suboptimal persistence in vivo, antigen escape and heterogeneity, and manufacturing issues. The evolution of CAR designs beyond the conventional structures will be necessary to address these limitations and to expand the use of CAR T cells to a wider range of malignancies. Investigators are addressing the current obstacles with a wide range of engineering strategies in order to improve the safety, efficacy and applicability of this therapeutic modality. In this Review, we discuss the innovative designs of novel CAR T cell products that are being developed to increase and expand the clinical benefits of these treatments in patients with diverse cancers., Chimeric antigen receptor (CAR) T cell therapy, the first approved therapeutic approach with a genetic engineering component, holds substantial promise in the treatment of a range of cancers but is nevertheless limited by various challenges, including toxicities, intrinsic and acquired resistance mechanisms, and manufacturing issues. In this Review, the authors describe the innovative approaches to the engineering of CAR T cell products that are providing solutions to these challenges and therefore have the potential to considerably improve the safety and effectiveness of treatment., Key points Chimeric antigen receptor (CAR) T cells have induced remarkable responses in patients with certain haematological malignancies, yet various barriers restrict the efficacy and/or prevent the widespread use of this treatment.Investigators are addressing these challenges with engineering strategies designed to improve the safety, efficacy and applicability of CAR T cell therapy.CARs have modular components, and therefore the optimal molecular design of the CAR can be achieved through many variations of the constituent protein domains.Toxicities currently associated with CAR T cell therapy can be mitigated using engineering strategies to make CAR T cells safer and that potentially broaden the range of tumour-associated antigens that can be targeted by overcoming on-target, off-tumour toxicities.CAR T cell efficacy can be enhanced by using engineering strategies to address the various challenges relating to the unique biology of diverse haematological and solid malignancies.Strategies to address the manufacturing challenges can lead to an improved CAR T cell product for all patients.

Chimeric antigen receptor (CAR) T-cell therapy has dramatically shifted the landscape of treatment for lymphoid malignancies, especially diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). However, there continue to be significant limitations of this therapy, such as incomplete or nonsustained responses and severe toxicities in a subset of patients. Furthermore, expanding the role of CAR T-cell therapy to new disease types is an important next step. In this review, we will highlight landmark trials for anti-CD19 CAR T cells and first-in-human trials of novel CARs, as well as discuss promising innovative CAR designs that are still undergoing preclinical development. Lastly, we will discuss toxicity and mechanisms of CAR T-cell resistance and failure, as well as potential future treatment approaches to these common issues.

CD8(+) T cells are critical mediators of adaptive immunity but may also exhibit innate-like properties such as surface expression of NKG2C, an activating receptor typically associated with natural killer (NK) cells. We demonstrate that, similar to NK cells, NKG2C(+)TCRαβ(+)CD8(+) T cells are associated with prior human cytomegalovirus (HCMV) exposure. In addition to expressing several NK cell markers such as CD56 and KIR, NKG2C(+)CD8(+) T cells are oligoclonal and do not upregulate PD-1 even in response to persistent activation. Furthermore, we found that NKG2C(+)CD8(+) T cells from some individuals exhibited strong effector function against leukemia cells and HCMV-infected fibroblasts, which was dictated by both NKG2C and TCR specificity. Transcriptomic analysis revealed that the transcription factor BCL11B, a regulator of T cell developmental fate, is significantly downregulated in NKG2C(+)CD8(+) T cells when compared to conventional NKG2C(−)CD8(+) T cells. BCL11B deletion in conventional CD8(+) T cells resulted in the emergence of a similar innate-like CD56(+)CD94(+)DAP12(+)NKG2C(+)CD45RA(+)CCR7(−)PD-1(−/low) T cell population with activity against HLA-E(+) targets. Based on their intrinsic capacity to recognize diseased cells coupled with lack of PD-1 induction, NKG2C(+)CD8(+) T cells represent a lymphocyte population that resides at the boundary between innate and adaptive immunity, presenting an attractive alternative for cellular therapy, including CAR T-based therapies.

CD8+ T cells not only are critical mediators of adaptive immunity but also may exhibit innate-like properties such as surface expression of NKG2C, an activating receptor typically associated with n...

Anti-CD19 chimeric antigen receptor (CAR) T cell therapy has led to unprecedented responses in patients with high-risk hematologic malignancies. However, up to 60% of patients still experience disease relapse and up to 80% of patients experience CAR-mediated toxicities, such as cytokine release syndrome or immune effector cell-associated neurotoxicity syndrome. We investigated the role of the intestinal microbiome on these outcomes in a multicenter study of patients with B cell lymphoma and leukemia. We found in a retrospective cohort (n = 228) that exposure to antibiotics, in particular piperacillin/tazobactam, meropenem and imipenem/cilastatin (P-I-M), in the 4 weeks before therapy was associated with worse survival and increased neurotoxicity. In stool samples from a prospective cohort of CAR T cell recipients (n = 48), the fecal microbiome was altered at baseline compared to healthy controls. Stool sample profiling by 16S ribosomal RNA and metagenomic shotgun sequencing revealed that clinical outcomes were associated with differences in specific bacterial taxa and metabolic pathways. Through both untargeted and hypothesis-driven analysis of 16S sequencing data, we identified species within the class Clostridia that were associated with day 100 complete response. We concluded that changes in the intestinal microbiome are associated with clinical outcomes after anti-CD19 CAR T cell therapy in patients with B cell malignancies.