Chimeric antigen receptor T cell therapies for lymphoma

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1 Chimeric antigen receptor T cell therapies for lymphoma Jennifer N. Brudno and James N. Kochenderfer Abstract New therapies are needed for patients with Hodgkin or non-hodgkin lymphomas that are resistant to standard therapies. Indeed, unresponsiveness to standard chemotherapy and relapse after autologous stem-cell transplantation are indicators of an especially poor prognosis. Chimeric antigen receptor (CAR) T cells are emerging as a novel treatment modality for these patients. Clinical trial data have demonstrated the potent activity of anti CD19 CAR T cells against multiple subtypes of B cell lymphoma, including diffuse large B cell lymphoma (DLBCL), follicular lymphoma, mantle cell lymphoma, and marginal zone lymphoma. Importantly, anti CD19 CAR T cells have impressive activity against chemotherapy-refractory lymphoma, inducing durable complete remissions lasting >2 years in some patients with refractory DLBCL. CAR T cell therapies are, however, associated with potentially fatal toxicities, including cytokine-release syndrome and neurological toxicities. CAR T cells with novel target antigens, including CD20, CD22, and κ light chain for B cell lymphomas, and CD30 for Hodgkin and T cell lymphomas, are currently being investigated in clinical trials. Centrally manufactured CAR T cells are also being tested in industry-sponsored multicentre clinical trials, and will probably soon become a standard therapy. Herein, we review the clinical efficacy and toxicity of CAR T cell therapies for lymphoma, and discuss their limitations and future directions with regard to toxicity management, CAR designs and CAR T cell phenotypes, conditioning regimens, and combination therapies. REVIEWS Experimental Transplantation and Immunology Branch, National Cancer Institute, NIH Building 10 room , Bethesda, Maryland 20892, USA. Correspondence to J.N.K. kochendj@mail.nih.gov doi: /nrclinonc Published online 31 Aug 2017 Non-Hodgkin lymphoma (NHL) is a heterogeneous disease entity comprising diverse B cell and T cell lymphoma subtypes that collectively account for approximately 4% of all cancers in the USA 1. In 2012, an estimated 385,700 patients worldwide were diagnosed with NHL, and an estimated 199,700 patients died as a result of the disease 2. Diffuse large B cell lymphoma (DLBCL) is the most-common form of B cell NHL, with an estimated 27,650 new cases in the USA in 2016, accounting for approximately 26% of all mature- B cell NHL neoplasms diagnosed 3. By comparison, follicular lymphoma (FL), marginal-zone lymphoma (MZL), and mantle-cell lymphoma (MCL) accounted for an estimated 13%, 7%, and 3%, respectively, of new mature B cell NHL diagnoses in the USA in 2016 (REF. 3). Hodgkin lymphoma (HL) and peripheral T cell lymphomas (PTCL) are less common, with 8,500 new cases of HL and 7,190 new cases of mature T cell or natural killer (NK) cell neoplasms estimated in the USA in 2016 (REF. 3). In many of the most-common forms of NHL, cancer cells originate from the B cell lineage and, therefore, often express B lymphocyte differentiation antigens, including CD20 and CD19. The development of the anti CD20 monoclonal antibody rituximab and addition of this agent to conventional cytotoxic chemo therapy regimens has improved patient outcomes across multiple B cell NHL subtypes 4 6 ; however, the prog noses of most patients with chemotherapy- refractory or multiply- relapsed B cell NHL remain poor DLBCL is curable with first-line anthracycline-based immunochemotherapy regimens, typically R CHOP (comprising rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), but a substantial number of patients have disease relapse, or primary-refractory lymphoma 8,11 ; the standard second-line approach is salvage chemotherapy followed by autologous haematopoietic stem-cell transplantation (auto HSCT) 12,13. Primaryrefractory DLBCL, which is defined by a response of less than partial remission (PR) to first-line immunochemotherapy, is associated with especially poor outcomes: only 23 29% of patients with primary-refractory DLBCL respond to second-line treatment 7,14, with a median progression-free survival (PFS) of 3 months 14. Patients with DLBCL refractory to second-line therapy have a median overall survival of only 4 months 7, with an estimated response rate to third-line chemotherapy of only 14% 15. The prognosis of patients with DLBCL NATURE REVIEWS CLINICAL ONCOLOGY ADVANCE ONLINE PUBLICATION MacmilanPublishersLimited,partofSpringerNature.Alrightsreserved.

2 Key points New treatments are needed for patients with chemotherapy-refractory or multiply-relapsed lymphoma Chimeric antigen receptor (CAR) T cells targeting CD19 have demonstrated efficacy in multiple subtypes of B cell lymphoma, with activity seen in patients with chemotherapy-refractory lymphoma; durable remissions are possible Multicentre clinical trials have demonstrated that centralized CAR T cell processing is feasible, and response rates in early studies of centrally manufactured CAR T cell therapies are similar to those reported in single-centre studies CARs targeting novel antigens, such as CD20, CD22, CD30 and κ light chains, are in development and will extend the applicability of CAR T cell therapy to patients with Hodgkin lymphoma, T cell lymphoma, or CD19 negative B cell lymphoma Cytokine-release syndrome and neurological toxicity are severe adverse events commonly associated with CAR T cell therapies for lymphoma, and reducing the risk of such toxicities is a major avenue for improving CAR T cell therapies CAR T cell therapy is likely to become safer and more effective, and will probably become a standard treatment option for patients with relapsed and primary-chemotherapy-refractory lymphoma in the near future that has relapsed after an initial response to auto HSCT is also dismal, with a median overall survival of 10 months 9,16. Most patients with FL respond to first-line rituximab-based chemoimmunotherapy regimens 17 19, although these conventional treatments are not curative. A subgroup of approximately 20% of patients with FL develop progressive lymphoma within 24 months of initial treatment and have poor long-term outcomes, with a median overall survival duration of <5 years 10. MCL has traditionally been managed using intensive immunochemotherapy regimens, followed by auto HSCT, an approach that is associated with considerable toxicities and that lacks curative potential 20,21. The introduction of novel targeted therapies for MCL, such as bortezomib 22, lenalidomide 23, and ibrutinib 24, has yielded responses that are usually not durable beyond 2 years 25. Standard first-line therapy for PTCL consists of anthracycline-based chemotherapy regimens, such as CHOP 26,27. Unfortunately, the responses to therapy of patients with T cell lymphomas are generally not as robust as those of patients with B cell NHLs, with median overall survival durations in the range of months Only 25 29% of patients with relapsed or refractory PTCL respond to second-line therapy Patients with HL are often cured with first-line anthracycline-based chemotherapy regimens, typically doxo rubicin, bleomycin, vinblastine, and dacarbazine (ABVD), with or without radiation therapy ; however, approximately half of the subgroup of patients with primary- refractory or relapsed disease do not obtain durable remission with standard salvage chemo therapy followed by auto HSCT 37,38. Despite typically originating from the B lymphocyte lineage, the malignant Reed Sternberg cells that underlie HL do not ubiquitously express CD20. Thus, unlike in B cell NHLs, rituximab has limited clinical utility in patients with HL 39. Nevertheless, in the past 5 years, novel targeted therapies have provided alternative treatment options for these patients. CD30, a characteristic marker of Reed Sternberg cells, can be targeted using brentuximab vedotin; monotherapy with this antibody drug conjugate after failure of auto HSCT has been associated with an overall response rate (ORR) of 75% 40, and a 5 year PFS of 22% 41. In addition, amplification of the 9p24.1 locus containing the genes encoding the inhibitory immune-checkpoint proteins PD L1 and PD L2 is a defining feature of classical HL. Correspondingly, inhibition of the PD 1 PD L1/PD L2 immune checkpoint using nivolumab 42,43 or pembrolizumab 44 is associated with high ORRs (65 87%) in patients with relapsed and/or refractory HL; however, complete remissions (CRs) are in the minority (9 17%), and the median duration of response to nivolumab is just 13.1 months 45. Together, these findings indicate that further improvement of lymphoma therapy is possible; indeed, new treatments with greater efficacy are urgently needed, particularly for patients with relapsed and refractory lymphomas. Although allogeneic HSCT (allo HSCT) offers the potential to cure patients with various subtypes of lymphoma, transplant-related mortality remains high, and long-term sequelae, including chronic graftversus-host disease (GVHD), can have a substantial negative effect on quality of life A key goal of the chimeric antigen receptor (CAR)-T cell field is to develop T cell-based therapies that are safer and more effective than allo-hsct, by genetically engineering T cells to recognize specific tumour-associated antigens including the validated targets, CD19, CD20, and CD30. This cellular immunotherapy approach has now been tested in a number of clinical trials involving patients with lymphoma, with many more studies ongoing. The results reported to date are encouraging, although toxicity remains a concern. Herein, we review the current clinical data on CAR T cell therapies for lymphoma. We also discuss the limitations of the current CAR T cell products, and highlight avenues for ongoing research aimed at improving their effectiveness and safety. Chimeric antigen receptors CARs are fusion proteins that include both antigenrecognition moieties and T cell signalling s (FIG. 1a). The antigen-recognition is usually a single- chain variable fragment (scfv) derived from a monoclonal antibody targeting a tumour-associated antigen, predominantly CD19 in the context of lymphoma 50 52, In the first-generation of CARs, the T cell signalling comprised an intracellular portion of the T cell receptor (TCR) CD3ζ subunit (FIG. 1b). By contrast, the second and later generations of CARs incorporate two types of T cell signalling s: co stimulatory s, derived from co stimulatory receptors, such as CD28 and/or 4 1BB; and a T cell-activation derived from CD3ζ 54, The gene encoding the CAR construct is transferred into the genomes of T cells using a gene-therapy vector, which is usually a replicationincompetent γ retrovirus 62 67, or a lentivirus 68 70, although transposon systems have also been used 71. Most CAR T cell infusions are preceded by a conditioning chemotherapy regimen, with extensive evidence from mouse studies indicating that lymphocyte depletion in the recipient enhances the activity of the adoptively transferred T cells by causing increased serum levels of 2 ADVANCE ONLINE PUBLICATION

3 cytokines, such as IL 15, and possibly by depleting regulatory T (T reg )-cell numbers The first preclinical studies of CAR T cells in the treatment of cancer were conducted in the early 1990s; these studies involved CARs targeting ovarian cancer cells 76. The first preclinical studies that demonstrated in vitro and in vivo activity of anti CD19 CAR T cells were published in 2003 (REFS 77,78). Early clinical results in B cell NHLs The first published report of the anticancer activity of anti CD19 CAR T cells in humans was published in 2010, and described a patient with multiply-relapsed FL who was treated at the US National Cancer Institute (NCI) 79. The patient received a conditioning chemotherapy regimen comprising cyclophosphamide and fludarabine, followed by an infusion of autologous T cells transduced to express an anti CD19 CAR containing a CD28 co stimulatory 79 this CAR has been designated FMC63 28Z 54. The CAR T cell infusion was followed by a course of high-dose IL 2 (REF. 79). The patient achieved a PR lasting 7 months, was retreated in an identical manner upon progression, achieving a second PR 80, and remains progression-free 7 years later, despite receiving no further lymphoma therapy since this second CAR T cell infusion in March 2010 (Kochenderfer, J. N., unpublished data). The patient also remains free of any chronic toxicities 80 (Kochenderfer, J. N., unpublished data), although he did experience protracted B cell aplasia owing to the CAR T cell-mediated eradication of all cells carrying the CD19 antigen 79. This same group reported the successful treatment of four other patients with advanced-stage B cell lymphomas that were refractory to all standard therapies, excluding allo HSCT, using the same therapeutic approach (TABLE 1); an additional patient with FL died of influenza during therapy and was not evaluable for response 80. These findings confirmed that CAR T cells can eradicate lymphoma masses, as well as malignancy in the bone marrow 80. Moreover, a correlation between increasing serum levels of interferon γ (IFNγ) and tumour necrosis factor α (TNFα) with greater degrees of overall toxicity was reported 80. This toxicity syndrome associated with elevated serum cytokine levels after CAR T cell infusions has since been termed cytokine-release syndrome (CRS; BOX 1), and reflects an intense systemic inflammatory response resulting from CAR T cell activation 63,64,81. In a separate early study, two patients with FL treated with T cells transduced with a first-generation anti CD19 CAR construct incorporating a CD3ζ T cell activation, without a co stimulatory, did not achieve objective anti-lymphoma responses 82 (TABLE 1). In another trial, five patients with relapsed and/or refractory B cell NHL simultaneously received two different CAR T cell products: T cells transduced with a CAR construct containing an anti CD19 scfv and a CD3ζ T cell-activation, and T cells expressing CARs that were identical except for addition of a a Hinge region ScFv Transmembrane region Co-stimulatory region T-cell-activation Figure 1 Chimeric antigen receptor (CAR) structures. a The diagram shows the schematic structure of a second-generation CAR: a single-chain variable region (scfv) of an antibody, which provides target specificity; hinge and transmembrane regions; a co-stimulatory ; and a T cell-activation. b The evolution of CAR designs. In contrast to second-generation CARs, first-generation CARs lack a co-stimulatory. In all of the second-generation CARs tested in lymphoma clinical trials to date, the co stimulatory has been derived from either CD28 or 4 1BB, which are co stimulatory receptors expressed on the surface of T cells. Third-generation CARs incorporate two co stimulatory s, derived from different co stimulatory proteins, such as CD28 and 4 1BB. b T-cellactivation CD28 co-stimulatory T-cell-activation 4-1BB co-stimulatory T-cell-activation CD28 co-stimulatory 4-1BB co-stimulatory T-cell-activation First-generation CAR Second-generation CARs Third-generation CAR NATURE REVIEWS CLINICAL ONCOLOGY ADVANCE ONLINE PUBLICATION 3

4 Table 1 Selected published trials of autologous anti CD19 CAR T cell therapy for patients with B cell NHL Study Lymphoma subtypes (n) Co stimulatory Conditioning chemotherapy Jensen et al. FL (2) None Flu 25 mg/m 2 daily for 5 days (2010) 82 starting after the first of multiple CAR T cell infusions Kochenderfer FL (1) CD28 60 mg/kg Cy daily for 2 days, et al. (2010) 79 followed by Flu 25 mg/m 2 daily for 5 days Savoldo et al. DLBCL (2); TFL (2); (2011) 65 PCNSL with systemic relapse (1) Two CAR T cell products: one without and one with a CD28 co stimulatory None Kochenderfer FL (4); SMZL (1) CD28 60 mg/kg Cy daily for 2 days, et al. (2012) 80 followed by Flu 25 mg/m 2 daily for 5 days Kochenderfer DLBCL NOS (4); et al. (2015) 62 PMBL (4); DLBCL transformed from CLL (1); indolent NHL NOS (1); SMZL (1) CD28 Wang et al. (2016) 89 DLBCL (11); MCL (5) One trial (NHL1) using a first-generation CAR lacking a co stimulatory, and one trial (NHL2) using a second-generation CAR with a CD28 co-stimulatory Turtle et al. Aggressive B cell (2016) 85 NHL (11); TFL (11); FL (6); MCL (4) 4 1BB Cy at a total dose of either 120 mg/kg or 60 mg/kg, followed by Flu 25 mg/m 2 daily for 5 days Auto-HSCT conditioning One of four regimens: Cy 2 4 g/m 2 on day 1 Cy 2 4 g/m 2 on day 1; etoposide mg/m 2 daily on days 1 3 Cy 60 mg/kg on day 1; Flu 25 mg/m 2 daily on days 2 4 Cy 60 mg/kg on day 1; Flu 25 mg/m 2 daily on days 2 6 Locke et al. DLBCL (7) CD28 Cy 500 mg/m 2 IV and Flu (2017) mg/m 2 daily for 3 days Kochenderfer DLBCL (13); TFL (4); et al. (2017) 67 FL (2); PMBL (2); MCL (1) CD28 Cy 300 mg/m 2 or 500 mg/m 2 IV daily for 3 days, and Flu 30 mg/m 2 daily for 3 days Cell dose ORR and CRR 4 5 escalating doses of ORR: 0/2 (0%) cells/m 2 CRR: 0/2 (0%) CAR T cells, followed by CAR T cells the following day 1 2 simultaneous infusions of the two CAR T cell products at escalating doses of cells/m 2 ORR: 1/1 (100%) CRR: 0/1 (0%) ORR: 0/5 (0%) CRR: 0/5 (0%) CAR T cells/kg ORR: 4/4 (100%)* CRR: 0/4 (0%) CAR T cells/kg ORR: 8/9 (89%) NHL1: 25, 50, or total CAR T cells per infusion NHL2: 50 or CAR T cells per infusion , , or CAR T cells/kg CRR: 5/9 (56%) NHL1 ORR: 7/8 (88%) NHL1 CRR: 5/8 (63%) NHL2 ORR: 8/8 (100%) NHL2 CRR: 8/8 (100%) ORR: 19/30 (63%) # CRR: 10/30 (33%) # CAR T cells/kg ORR: 5/7 (71%) CRR: 4/7 (57%) 1, 2 or CAR T cells/kg ORR: 16/22 (73%) CRR: 12/22 (55%) The data shown relate only to the patients with NHL and not patients with other B cell malignancies treated in each trial. Auto-HSCT, autologous haematopoietic stem-cell transplantation; CAR, chimeric antigen receptor; CLL, chronic lymphocytic leukaemia; Cy, cyclophosphamide; CRR, complete remission rate; DLBCL: diffuse large B cell lymphoma; FL, follicular lymphoma; Flu, fludarabine; IV, intravenously; MCL, mantle cell lymphoma; n, number of patients; NHL, non-hodgkin lymphoma; NOS, not otherwise specified; ORR, objective response rate; PMBL, primary mediastinal B cell lymphoma; PCNSL, primary central nervous system lymphoma; SMZL, splenic marginal-zone lymphoma; TFL, transformed follicular lymphoma. *One patient with FL was reported in the earlier study by these authors 79, but after disease progression, underwent re treatment with CAR T cells in this trial and had reinduction of a PR; the response of one other patient with FL was not evaluable owing to death while on study. The responses of two patients were not evaluable, one owing to death while on study and one owing to noncompliance with the follow up protocol. All patients received bis-chloroethylnitrosourea, etoposide, cytarabine, and melphalan high-dose chemotherapy as conditioning, and received CAR T cell infusions following auto-hsct. Before auto HSCT, three patients were in CR and two patients were in PR; responses represent combined effect of auto-hsct followed by CAR T cell infusions. Before auto HSCT, six patients were in CR and two patients were in PR; responses represent combined effect of auto-hsct followed by CAR T cell infusions. # Two patients were unevaluable for response owing to death on study. CD28 co stimulatory 65. Stable disease was the best anti-lymphoma response observed among these patients (TABLE 1); however, superior in vivo expansion and persistence of the T cells with CARs incorporating a co stimulatory was demonstrated 65. Single-centre trials in B cell NHLs Another trial conducted at the NCI was the first to demonstrate the efficacy of anti CD19 CAR T cells against DLBCL 62. In this study, 11 patients with various B cell NHLs received lymphocyte-depleting cyclo phosphamide and fludarabine chemotherapy followed by a single infusion of FMC63 28Z CAR T cells (TABLE 1). Infiltration of malignant lymph nodes by CAR T cells was demonstrated 62. Notably, CRs were achieved in two of four patients with chemotherapy-refractory DLBCL not otherwise specified, and in two of four patients with primary mediastinal B cell lymphoma (PMBL) 62 populations of patients with poor prognoses and few standard treatment options available 7,14,83,84. Of note, the persistent presence 4 ADVANCE ONLINE PUBLICATION

5 Box 1 Summary of common chimeric antigen receptor (CAR)-T cell toxicities Toxicities that occur after infusion of CAR T cells generally belong to one of two groups: cytokine-release syndrome (CRS) and neurological toxicities. CRS is associated with elevated serum levels of a wide range of cytokines. The aetiology of neurological toxicity is not clear; however, similar to CRS, neurological toxicity is associated with elevated levels of cytokines and other substances secreted by CAR T cells. Common manifestations of CRS and neurological toxicities that can occur after infusions of CAR T cells are listed below. CRS Fevers Tachycardia Hypotension Hypoxia Decreased cardiac ejection fraction Renal insufficiency Increased serum levels of hepatic enzymes Prolongation of prothrombin time and partial thromboplastin time Neurological toxicities Delirium Dysphasia Tremors Imbalance and gait instability Obtundation Seizures of high numbers of CAR T cells in the blood was not necessary for sustained remission. B cell aplasia was common, but recovery of polyclonal B cell populations was demonstrated in the absence of lymphoma recurrence 62. In a trial by Turtle et al patients with B cell NHL received infusions of T cells transduced with an anti CD19 CAR containing a 4 1BB co stimulatory. The CAR T cell products were produced in a defined 1:1 ratio of CD4 + :CD8 + T cells, with the intent of administering a more-homogenous and possibly more-efficacious cell product, an approach that had been associated with favourable antitumour activity in mouse models 86. The researchers found that high CAR T cell doses posed an added risk of CRS and neurotoxicity 85 (BOX 1): two deaths, one owing to pontine haemorrhage and the other to gastrointenstinal haemorrhage, occurred in patients treated with CAR T cells/kg, leading the investigators to administer lower doses to all subsequent patients 85. Nevertheless, responses were observed across all lymphoma subtypes, including DLBCL, transformed FL (TFL), FL, and MCL, with an ORR of 63% and a CR rate of 33% 85 (TABLE 1). Among 18 response-evaluable patients who received fludarabine as part of their conditioning chemotherapy, the ORR was 72% and the CR rate was 50%, compared with 50% and 8%, respectively, among the 12 patients who received conditioning regimens lacking fludarabine 85. Indeed, the patients who received fludarabine had higher serum levels of IL 15 and IL 7 on the day of cell infusion, and a greater degree of subsequent CAR T cell expansion than those who did not receive fludarabine 85. In some of the patients who did not receive fludarabine conditioning, poor CAR T cell proliferation was associated with the development of a cytotoxic T cell response directed at the CAR construct 85. Similar immune responses to CARs have been reported in other clinical trials involving patients with NHL 82 and acute lymphoblastic leukaemia (ALL) 70,87. In June 2017, results from the NCI demonstrated that a low-dose cyclophosphamide plus fludarabine conditioning regimen administered before infusion of anti CD19 CAR T cells was sufficient to deplete lymphocyte numbers and increase levels of serum cytokines, including IL 15 and IL 7, compared with baseline values 67. In 22 patients with various B cell NHLs, this conditioning regimen was followed by an infusion of 1, 2, or CAR T cells/kg, resulting in a ORR of 73%, with a CR rate of 55% 67 (TABLE 1). Among 19 patients with one of the various subtypes of DLBCL, the ORR was 68%, and 47% had a CR 67. The low-dose chemotherapy regimen used in this study would not be expected to have substantial anticancer activity against chemotherapy-refractory DLBCL; therefore, the responses observed are almost certainly attributable to the CAR T cell therapy. Importantly, treatment with anti CD19 CAR T cells produced durable remissions in this study: 11 of the 12 CR are ongoing, with durations of 7 24 months at the time of publication; the 12 month PFS for all participants was 63%. The findings of this study also demonstrated that CAR T cells can induce CRs in patients with so called double-hit, MYC translocated and BCL2 translocated DLBCL 67 a form of the disease that is associated with a poor prognosis. Peak IL 15 levels were positively correlated with peak blood CAR T cell numbers, both of which were in turn associated with the likelihood of obtaining a PR or CR 67. Toxicities related to CRS and neurological effects were manageable with supportive care, and intensive care in some cases; three patients requied vasopressor drugs, and two patients required mechanical ventilation 67. Transfusion-dependent thrombocytopenia occurred in only two of 22 patients (9%), and in most patients, neutrophil counts did not drop below 500 cells/μl 67. The most-prominent toxicities were neurological, with 55% of patients experiencing grade 3 or 4 neurological toxicities. In one patient, neurological toxicity was treated successfully with dexamethasone, and in another patient, resolved spontaneously after unsuccessful treatment with the IL 6 receptor antagonist tocilizumab; all other cases of neurological toxicity resolved spontaneously without treatment 67. Recovery of normal polyclonal B cell populations was demonstrated in the patients with a CR, with an absence of lymphoma recurrence 67. Data from the University of Pennsylvania have confirmed that anti CD19 CAR T cells containing a 4 1BB co stimulatory (CTL019) have significant anticancer efficacy in various NHL subtypes, including DLBCL, FL, and MCL 88. The results of this study, presented at the 2015 American Society of Hematology meeting 88, demonstrated an ORR of 68%, and PFS of 62% at 12 months. Results published in 2016 have demonstrated the safety and feasibility of administration of anti CD19 CAR T cells following auto-hsct conditioning for patients with DLBCL, TFL, and MCL 89. These results included data derived from two trials of anti CD19 CAR central-memory-enriched T cells, one using a NATURE REVIEWS CLINICAL ONCOLOGY ADVANCE ONLINE PUBLICATION 5

6 Table 2 Open clinical trials of anti CD19 CAR T cell therapies for lymphoma ClinicalTrials.gov identifier Trial title/description Lymphoma subtype Centre; location NCT CD19 + CAR T cells for lymphoid malignancies CD19 + B cell NHL MD Anderson Cancer Center, Houston, Texas, USA NCT NCT NCT NCT Phase IIa study of redirected autologous T cells engineered to contain anti CD19 attached to TCRζ and 4 1BB signalling s in patients with chemotherapy relapsed or refractory CD19 + lymphomas Laboratory-treated T cells in the treatment of patients with relapsed or refractory CLL, NHL, or ALL Autologous CD19 CAR T cells in relapsed or refractory B cell lymphoma A phase I study evaluating the safety and efficacy of C CAR011 treatment in patients with DLBCL (C-CAR011) DLBCL; FL; TFL; MCL CD19 + B cell NHL CD19 + B cell NHL DLBCL University of Pennsylvania, Philadelphia, USA University of Washington Cancer Consortium, Seattle, USA Peking University Cancer Hospital, Beijing, China People s Hospital of Jiangsu Province, Nanjing City, China NCT Anti CD19 CAR-T-cell immunotherapy for relapsed or refractory MCL MCL Chinese PLA General Hospital, Beijing, China NCT NCT NCT NCT NCT NCT NCT NCT NCT NCT Study evaluating the efficacy and safety with CAR T for recurrent or refractory DLBCL T cells expressing a fully-human anti CD19 CAR T cells for the treatment of B cell malignancies Pilot study of redirected autologous T cells engineered to contain humanized anti CD19 in patients with relapsed or refractory CD19 + leukaemia and lymphoma previously treated with cell therapy Administration of anti CD19 CAR-transduced T cells from the original transplant donor to patients with recurrent or persistent B cell malignancies after allogeneic stem-cell transplantation In vitro expanded allogeneic EBV-specific cytotoxic T lymphocytes genetically targeted to the CD19 antigen in B cell malignancies Memory-enriched CAR T cell immunotherapy for B cell lymphoma (MeCAR) Activated T cells expressing 2nd or 3rd generation CD19 specific CAR in advanced-stage B cell NHL, ALL, and CLL (SAGAN) Evaluation of 4th generation safety-designed CAR T cells targeting high-risk and refractory B cell lymphomas (4SCAR19273) Pilot study of non-viral, RNA-redirected autologous T cells in patients with refractory or relapsed hodgkin lymphoma Evaluation of CAR19 T cells as an optimal bridge to allogeneic stem-cell transplantation (COBALT) NCT Activated T lymphocytes expressing CARs for relapsed CD19 + malignancies post allogeneic stem-cell transplantation (CARPASCIO) NCT NCT NCT NCT NCT NCT NCT Phase I/II study of pembrolizumab in patients failing to respond to or relapsing after anti CD19-CAR-modified T cell therapy for relapsed or refractory CD19 + lymphomas* JCAR014 and durvalumab in the treatment of patients with relapsed or refractory B cell NHL A phase I/II multicentre study evaluating KTE C19 in patients with refractory aggressive NHL (ZUMA 1) A phase II multicentre study evaluating patients with relapsed/ refractory MCL (ZUMA 2) A study evaluating KTE C19 in combination with atezolizumab in patients with refractory DLBCL (ZUMA 6) Study of efficacy and safety of CTL019 in adult patients with DLBCL (JULIET) Study evaluating the safety and pharmacokinetics of JCAR017 in B cell NHL DLBCL CD19 + B cell malignancies DLBCL B cell malignancy CD19 + B cell NHL CD19 + B cell NHL CD19 + B cell NHL CD19 + B cell NHL HL CD19 + DLBCL CD19 + NHL FL; DLBCL; MCL DLBCL; PMBL; tdlbcl DLBCL; PMBL; TFL MCL DLBCL DLBCL DLBCL; tdlbcl; FL (grade 3B); MCL; PMBL First Affiliated Hospital of Anhui Medical University, Hefei, China National Cancer Institute, Bethesda, Maryland, USA Children s Hospital of Philadelphia, Pennsylvania, USA National Cancer Institute, Bethesda, Maryland, USA Memorial Sloan Kettering Cancer Center, New York, USA Xinqiao Hospital, Chongqing, China Houston Methodist Hospital and Texas Children s Hospital, USA Peking University Cancer Hospital, Beijing, China Children s Hospital of Philadelphia, Pennsylvania, USA University College London Hospital, UK Houston Methodist Hospital and Texas Children s Hospital, USA University of Pennsylvania, Philadelphia, USA University of Washington Cancer Consortium, Seattle, USA International multicentre study US multicentre study US multicentre study International multicentre study US multicentre study Information derived from database on 23rd March 2017; all trials identified are phase I and/or II studies. ALL, acute lymphoblastic leukaemia; CAR, chimeric antigen receptor; CLL, chronic lymphoblastic leukaemia; DLBCL, diffuse large B cell lymphoma; EBV, Epstein Barr virus; FL, follicular lymphoma; MCL, mantle-cell lymphoma; NHL, non-hodgkin lymphoma; PMBL, Primary mediastinal B-cell lymphoma; TCRζ, T-cell receptor CD3ζ chain; tdlbcl, DLBCL transformed from an indolent histology; TFL, transformed FL. *Eligible patients must have received CTL019/CTL119 CAR T cells at the University of Pennsylvania. 6 ADVANCE ONLINE PUBLICATION

7 first-generation CAR construct and the other using a CAR containing a CD28 co stimulatory (TABLE 1). In these trials, patients received auto-hsct shortly before CAR T cell infusion; therefore, the antilymphoma activity of the CAR T cells was impossible to interpret. Of note, the persistence of the central memory T cells was not demonstrably longer than that of other CAR T cell products infused to patient involved in other clinical trials 89. Numerous clinical trials testing anti CD19 CAR T cells are currently recruiting patients with lymphoma, including studies in different treatment settings, of novel CAR designs, and/or of combination therapies (TABLE 2). Multicentre trials of CAR T cells A FMC63 28Z expressing CAR T cell product 54,62,67 designated axicabtagene ciloleucel (and formerly KTE C19) is currently in commercial development. In a phase I multicentre trial of axicabtagene ciloleucel 90, the feasibility of treatment involving central manufacturing of CAR T cell was clearly demonstrated. Treatment with this product, preceded by a conditioning regimen comprising 500 mg/m 2 of cyclophosphamide and 30 mg/m 2 of fludarabine given daily on the same 3 days, was associated with a high ORR (71%) in seven patients with DLBCL who were either refractory to the last course of chemotherapy or had a disease relapse within 12 months of auto-hsct 90. Anticancer activity was observed in both the germinal centre B cell (GCB) and non-gcb subtypes of DLBCL 90. CRS and neurotoxicities were common and sometimes severe, but could be managed effectively across centres 90. In a subsequent phase II multicentre study 91,92, with preliminary results presented in 2017, 101 patients with refractory aggressive NHL received axicabtagene ciloleucel; the ORR was 82% and the CR rate was 54%, demonstrating that CAR T cell therapy can be delivered successfully to large numbers of patients. At a median follow up duration of 8.7 months, the ongoing ORR was 44%, with 39% of the patients remaining in CR 92. Findings of a multicentre trial of the JCAR017 anti CD19 CAR T cell product containing a 4 1BB co stimulatory, preceded by a fludarabine and cyclophosphamide conditioning chemotherapy regimen, showed an ORR of 80% and a 60% CR rate in 20 patients who were evaluable for lymphoma response 93. A total of 28 patients were evaluable for safety outcomes (25 with DLBCL, two with MCL, and one with FL), with CRS and neurological toxicities reported to be manageable; 14% of patients had at least one grade 3 or 4 neurological toxicity 93. CTL019, consisting of T cells expressing an anti CD19 CAR containing a 4 1BB co stimulatory, is now being evaluated in an international multicentre study involving patients with DLBCL 94. CTL019 administration is being preceded by a chemotherapy conditioning regimen of fludarabine 25 mg/m 2 for 3 days and bendamustine 90 mg/m 2 for 2 days 94. Interim results for 51 patients with 3 months of follow up data demonstrate an ORR of 59% and CR rate of 43%, with a median duration of response that has not been reached; CRS was reported in 57% of patients, and 13% had grade 3 or 4 neurological toxicities 94. CAR T cell therapies after allo HSCT Patients with progressive B cell malignancies after allo HSCT have extremely limited treatment options 95,96, and are often treated with donor lymphocyte infusion (DLI) administration of unmanipulated allogeneic lymphocytes from the original haematopoietic stemcell donor 97,98. DLIs have inconsistent efficacy and often cause GVHD, owing to an immune response of the donor lympho cytes to nonmaligant tissues in the recipient 99. Thus, transduction of allogeneic T cells to express anti CD19 CARs might provide an advantage over standard DLI, by specifically targeting the immune response to malignant cells. Administration of allogeneic anti CD19 CAR T cells from original allo-hsct stem-cell donors to recipients with B cell malignancies has been demonstrated to be feasible and safe in two clinical trials In one trial 101, eight patients with ALL or chronic lymphoblastic leukaemia (CLL) who had undergone prior allo-hsct received virus-specific cytotoxic T cells transduced with an anti CD19 CAR incorporating a CD28 co stimulatory. Two of six evaluable patients had objective anti-leukaemic responses 101. Toxicities were mild, and, interestingly, the allogeneic CAR T cells did not induce GVHD 101. In the other trial 100,102, 20 patients with various B cell malignancies, including ALL, CLL, DLBCL, and MCL, were treated with allogeneic T cells expressing the aforementioned anti CD19 FMC63 28Z CAR 54. Unlike in most CAR T cell clinical trials, conditioning chemo therapy was not administered. Nevertheless, responses were observed in all disease subgroups 102, clearly demonstrating the anticancer activity of the CAR T cells alone 100,102. Importantly, no patient developed acute GVHD, and only one patient had progression of previously-existing chronic GVHD 102. The pronounced lack of GVHD seen in these trials, in contrast with the high rates of GVHD after standard DLIs, is compelling and not fully explained. Persistence of the allogeneic FMC63 28Z CAR T cells was shown to be limited to 1 2 months 102, which is the most likely explanation for the absence of GVHD. Remissions occurred within 2 weeks in some patients on this trial 102 ; therefore, we hypothesize that the CAR T cells can cause remissions and are then eliminated before the development of GVHD. Subsequent data from mouse models of lymphoma support this theory by showing that CD28 co stimulated CAR T cells undergo increased activation, compared with bulk donor T cells, followed by progressive exhaustion and deletion, seemingly owing to dual signalling through the CAR and endogenous TCRs 103. In the same mouse model, first-generation CARs and CARs with a 4 1BB co stimulatory did not protect against GVHD, compared with bulk DLI 103. Other investigators are using gene editing techniques, such as transcription activator-like effector nuclease (TALEN)-based DNA cleavage, to produce CAR T cells that lack TCR genes, with the goal of generating CAR T cell products from unrelated donors who are not HLA matched with the recipient 104. In addition to these two clinical trials, anti CD19 CAR T cells generated using a transposon system have been used as adjuvant treatments after auto-hsct or NATURE REVIEWS CLINICAL ONCOLOGY ADVANCE ONLINE PUBLICATION 7

8 allo-hsct in patients with advanced-stage B cell NHL or ALL 71. In the seven patients who received CAR T cells after auto-hsct, 30 month PFS and overall survival were 83% and 100%, respectively; in the 19 patients treated in the allo-hsct setting, these rates were 53% and 63%, respectively, at 12 months 71. This non comparative study provides limited insights into anticancer activity of CAR T cell therapy in these settings; however, toxicities were limited and treatment did not exacerbate the severity of GVHD 71, thus illustrating the safety and feasibility of this approach. Targeting antigens other than CD19 Two main reasons exist for targeting antigens other than CD19 for the treatment of lymphoma. First, many types of lymphoma, including Hodgkin lymphoma, T cell lymphomas, and even some B cell NHLs, do not express CD19 (REF. 105). Second, treatment of CD19 + lymphomas with anti CD19 CAR T cells might fail if CD19 expression is not expressed on all lymphoma cells before treatment, or is lost from lymphoma cells during treatment, owing to persistence and outgrowth of resistant clones. For these reasons, investigators are developing CARs targeting alternative lymphoma-associated antigens 66,82, The success of the anti CD20 monoclonal antibody rituximab in the treatment of B cell NHL underscores CD20 as an attractive target of CAR T cell therapy. Of note, CD20 negative relapse of B cell malignancies following rituximab-based therapy has been reported, at rates of up to 60% 110,111, which might present a challenge to anti CD20 CAR T cell therapy. Nevertheless, early clinical studies of anti CD20 CAR T cells have been conducted, in which patients were screened to ensure CD20 expression by lymphoma cells. In one study 82, two patients with DLBCL received adjuvant therapy using T cells transduced with a first-generation anti CD20 CAR immediately following auto-hsct, making the response to the CAR T cell infusion difficult to evaluate. One of these patients had an ongoing CR for 9 years at the time of publication, an outcome that is not uncommon with auto HSCT alone, and the other patient achieved a CR lasting 19 months 82. Another clinical trial was conducted using T cells transduced through plasmid electroporation to express a third-generation anti CD20 CAR with both CD28 and 4 1BB co- stimulatory s 106. In this pilot study 106, patients with MCL or FL received conditioning chemotherapy with cyclophosphamide and three subsequent infusions of CAR T cells, followed by 14 days of subcutaneous IL 2. Two patients with no evaluable lymphoma before cell infusion remained in CR, and one patient had an objective PR 106 (TABLE 3). The CAR T cells were shown to persist in the blood for 9 12 months 106. A third study was conducted with T cells transduced with a lenti viral vector to express an anti CD20 CAR with a 4 1BB co stimulatory 107. This trial included seven patients with heavily pretreated DLBCL, who received variable conditioning chemotherapy regimens before administration of escalating doses of CAR T cells over 3 4 days; five out of six evaluable patients had an objective response, mostly PRs 107 (TABLE 3). However, one patient who entered the study in PR achieved an ongoing CR, lasting 14 months at the time of publication 107. This patient received no conditioning chemotherapy and had detectable CAR T cells in the blood >6 months after cell infusion, which provides clear evidence of the anti cancer activity of the CAR T cells themselves. CRS-related toxicities were observed in four patients, with notably delayed occurrence 3 8 weeks after cell infusion. Other notably adverse events included delayed tumour-lysis syndrome and gastrointestinal haemorrhage in two patients with gastrointestinal involvement of lymphoma, one resulting in death 107. Results of a phase II study of the same anti CD20 CAR in 11 patients with DLBCL or indolent NHL demonstrated an ORR of 82% and a CR rate of 55% 108 (TABLE 3). CRs occurred in the one patient with FL and the one patient with MCL, as well as in four of eight patients with DLBCL (including those with GCB or activated-b like-cell subtype disease) 108. Thus, the available clinical results are preliminary, but together indicate that anti CD20 CAR T cells have encouraging therapeutic potential in B cell NHLs. The immunoglobulin kappa (κ) light chain antigen is expressed on κ restricted B cell lymphomas, and not on all nonmalignant B cells; therefore, the κ light chain is an attractive target for CAR T cell therapy because complete B cell aplasia will be avoided. In a phase I trial, seven patients with various NHL subtypes that expressed κ light chains, including DLBCL, TFL, MCL and lymphoplasmacytic lymphoma (LPL), have been treated with anti κ light chain CAR T cells 66 (TABLE 3). Cyclophosphamide conditioning was administered before CAR T cell infusions in the patients who were not already lymphopenic; lymphopenic patients received no conditioning 66. Overall, three of nine patients (33%) had an objective response: one of two patients with LPL achieved a PR and both patients with TFL achieved a CR. One CR was ongoing at 32 months after CAR T cell infusion. Of note, no adverse events were attributable to CRS 66. The B cell marker CD22 is another potential therapeutic target in patients with B cell malignancies. Anti CD22 CAR T cells have been evaluated in vitro and in mouse models; in mouse models of CD22 + leukaemia, anti CD22 CAR T cells had anticancer activity that was similar to that of anti CD19 CAR T cells 112,113. Anti CD22 CAR T cells are currently being tested in early clinical trials involving patients with CD22 + ALL or NHL (TABLE 4). Targeting CD30 in HL and PTCL CD30 is a transmembrane receptor and a member of the TNF receptor superfamily 114, and is highly expressed on malignant Reed Sternberg cells in HL 115. The efficacy of targeting CD30 to treat HL 40, and PTCL has been demonstrated in multiple clinical trials of the antibody-drug conjugate brentuximab vedotin 123. Nevertheless, only a minority (~20%) of patients with HL who receive brentuximab monotherapy remain progression-free at 5 years 41, suggesting that CD30 targeted treatments could be improved to increase the durability of remissions. 8 ADVANCE ONLINE PUBLICATION

9 Table 3 Selected published trials of CAR T cells targeting antigens other than CD19 in patients with lymphoma Study CAR target antigen Lymphoma subtypes (n) Co stimulatory Conditioning chemotherapy Jensen et al. CD20 DLBCL (2) None Patients underwent (2010) 82 auto-hsct days before CAR T cell infusion Till et al. CD20 MCL (2); FL (1) CD28 and 4 1BB Cy 1,000 mg/m 2 on day -2 (2012) 106 before CAR T cell infusion Wang et al. CD20 DLBCL (7) 4 1BB One of four regimens: (2014) 107 COED; COD; CHODE; or ESHAP Zhang et al. CD20 DLBCL (8); (2016) 108 FL (1); MCL (1); PCMZL (1) Ramos et al. (2016) 66 Kappa (κ) light chain DLBCL (2); TFL (2); LPL (2); MCL (1) Wang et al. CD30 Hodgkin (2017) 109 lymphoma (17); C ALCL (1) 4 1BB CD28 4 1BB No conditioning, or one of six regimens: CHOP; MACH; FC; EOCH; CHOD; or CHODE Cy 12.5 mg/kg 4 days before cell infusion, or no conditioning No conditioning, or one of seven regimens: FC; PC; GEMC; EAMC; GMC; MAMC; or GMMC Cell dose Three escalating doses of CAR T cells/m 2 Three cell infusions 2 5 days apart at incremental doses of , , and cells/m escalating daily doses ( CAR T cells/kg) 3 5 escalating daily doses ( CAR T cells/kg) ORR and CRR ORR: 2/2 (100%)* CRR: 2/2 (100%)* ORR: 3/3 (100%) CRR: 2/3 (67%) ORR: 5/6 (83%) CRR: 1/6 (17%) ORR: 9/11 (82%) CRR: 6/11 (55%) ORR: 3/7 (43%) CAR T cells/m 2 CRR: 2/7 (29%) CAR T cells/kg ORR: 7/18 (39%) CRR: 0/18 (0%) The data shown relate only to the patients with lymphoma and not patients with other B cell malignancies treated in each trial. Auto-HSCT, autologous haematopoietic stem-cell transplantation; C ALCL, cutaneous anaplastic large cell lymphoma; CAR, chimeric antigen receptor; CHOD, cyclophosphamide, doxorubicin, vincristine, and dexamethasone; CHODE, cyclophosphamide, doxorubicin, vincristine, dexamethasone, and etoposide; CHOP, cyclophosphamide, doxorubicin, vincristine, and prednisone; COD, cyclophosphamide, vincristine, and dexamethasone; COED, cyclophosphamide, vincristine, etoposide, and dexamethasone; Cy, cyclophosphamide; CRR, complete remission rate; DLBCL, diffuse large B cell lymphoma; EAMC, etoposide, cytarabine, mustargen, and cyclophosphamide; EOCH, etoposide, vincristine, cyclophosphamide, and doxorubicin; ESHAP, methylprednisolone, etoposide, carboplatin, and high-dose cytosine arabinoside; FC, fludarabine and cyclophosphamide; FL, follicular lymphoma; GEMC, gemcitabine, epirubicin, mustargen, and cyclophosphamide; GMC, gemcitabine, mustargen, and cyclophosphamide; GMMC, gemcitabine, mitoxantrone, mustargen, and cyclophosphamide; LPL, lymphoplasmacytic lymphoma; MACH, mitoxantrone, cytarabine, cyclophosphamide, and doxorubicin; MAMC, mitoxantrone, cytarabine, mustargen, and cyclophosphamide; MCL, mantle-cell lymphoma; n, number of patients; ORR, objective response rate; PC, nab-paclitaxel and cyclophosphamide; PCMZL, primary cutaneous marginal-zone lymphoma; TFL, transformed FL. *Responses represent combined effect of auto-hsct followed by CAR T cell infusions; one patient remained in CR for 9 years at time of publication, and the other patient achieved a CR lasting 19 months. Two patients with no evaluable disease before cell infusion remained in CR until the time of reporting, one for 12 months and the other for 24 months. One patient not evaluable owing to death on study. First-generation anti CD30 CAR T cells were developed in the 1990s, and preclinical studies demonstrated the ability of these cells to lyse CD30 expressing HL cell lines in vitro 124,125. Notably, the presence of soluble CD30 did not attenuate cytolysis, suggesting that CD30 shed from HL cells into the blood would not inhibit the efficacy of anti CD30 CAR T cells in vivo 124. Indeed, Epstein Barr-virus-specific cytotoxic T cells transduced with an anti CD30 CAR have been shown to have activity against CD30 + cancer cell lines in vitro, as well as in vivo, in a mouse xenograft model 126,127. This research has led to a phase I clinical trial of anti CD30 CAR T cells with a CD28 co stimulatory in seven patients with HL and two with anaplastic large cell lymphoma (ALCL; a T cell NHL subtype) 128,129. Of note, eight of these patients had brentuximab-refractory disease 128,129. Preliminary results of this study demonstrated one CR and one PR 128,129. No patients received conditioning chemotherapy 129, therefore, these responses were clearly attributable to anti CD30 CAR T cells. Moveover, no evidence of CRS was reported 129. In another phase I trial, 18 patients (17 with HL and one with cutaneous ALCL) were treated with T cells expressing an anti CD30 CAR containing a 4 1BB co stimulatory 109. Patients received a variety of chemotherapy conditioning regimens, and 28% of the patients had received brentuximab 109 (TABLE 3). Seven patients had a PR, with a median PFS of 6 months 109. CRS toxicities similar to those seen in clinical trials of anti CD19 CAR T cells occurred. Of note, CAR T cell persistence was <8 weeks in this study, but tumour biopsies showed efficient trafficking of T cells to lymphoma sites 109. A number of other clinical trials of anti CD30 CAR T cell therapy are ongoing (TABLE 4), and will provide more information on the efficacy of this approach. Summary of CAR T cell toxicities Arguably, toxicity is currently the biggest barrier to more effective CAR T cell therapies for lymphoma. Toxicities hamper efforts to increase anticancer efficacy of CAR T cells because approaches such as administering higher cell doses or enhancing T cell activity might lead to more severe toxicities. Most of the toxicities associated with CAR T cell therapies resolve within 2 weeks of CAR T cell infusion without any treatment other than supportive care, but these short-lived toxicities can be severe, and in rare cases fatal 62,85,130. Early CAR studies provided evidence of a syndrome with prominent signs of fever, tachycardia, and hypotension, and other toxicities occurring after CAR T cell infusions; this syndrome was designated CRS 63,80,81,131 (BOX 1). CRS also includes many other abnormalities including, but not NATURE REVIEWS CLINICAL ONCOLOGY ADVANCE ONLINE PUBLICATION 9