Engineered T Cells for Leukemia: A Review of Current Approaches and Applications

Sungyub Lew, MD

Pallawi Torka, MD

Department of Medicine,

Roswell Park Cancer Institute

Buffalo, New York

Elizabeth A. Griffiths, MD

Elizabeth A. Griffiths, MD

Department of Medicine

Roswell Park Cancer Institute

Buffalo, New York

Abstract

Chimeric antigen receptor T cells (CAR T cells), engineered from individual patients to specifically target tumor-associated antigens, have shown extraordinary promise in the management of B-cell malignancies. Moreover, CAR T cells are being developed for a myriad of other hematologic malignancies. First-generation CAR T cells demonstrated only modest efficacy because of inadequate activation, persistence, and proliferation, but the addition of one (second-generation) or more (third-generation) costimulatory molecules has produced a marked improvement in efficacy. Clinical trials are recruiting patients with leukemia, and CARs are being tested for patients with a large variety of both solid and liquid tumors. It seems likely that CAR T-cell therapy will become an established option in the near future for patients with B-cell malignancies. Significant questions remain about optimal timing for administration, product preparation, and CAR design to optimize response across malignancy types. This review addresses the basics of CAR T-cell design and reviews data from published clinical studies in leukemia. It also contains commentary on the prospects of this promising new therapeutic strategy.

Introduction

The most familiar immunotherapeutic approach for treatment of leukemia is allogeneic hematopoietic stem cell transplant (allo-HSCT). Manipulation of graft-vs-leukemia (GVL) effects can be further accomplished through the use of donor lymphocyte infusions (DLIs) and donor natural killer (NK) cell infusions. These conventional approaches are unfortunately characterized by unpredictable donor T-cell-mediated graft-versus-host disease (GVHD), and in some cases, by a failure to achieve antileukemic activity. More specific immune-based approaches have long been the topic of significant research, but until recently had been unsuccessful.

Hematopoietic malignancies express a variety of antigens that could be potential targets for immunotherapy, but overlap between leukemic and normal cell antigen expression has been a significant barrier. Leukemias have been shown to evade host immunosurveillance through a variety of different mechanisms, including downregulation of human leukocyte antigens and expression of programmed cell death ligand-1 (PD-L1 [among others]). Furthermore, leukemia-associated self-antigens are not targeted endogenously because most T cells directed against these self-antigens are deleted during thymic education, and the residual population is held in check by regulatory T cells. Ex vivo gene modification of T cells through the introduction of recombinant receptors directed against these self-antigens can overcome host immune tolerance. Chimeric antigen receptor T-cell (CAR T) therapy based on this concept is a form of “living” immunotherapy that has shown unprecedented responses in leukemia, specifically B-cell acute lymphoblastic leukemia (BALL). Currently, CAR T therapy is under simultaneous development at many centers, and although the most well-developed CAR T therapies are directed at CD-19, a pan B-cell marker present on both normal and malignant B cells, numerous other targets are also promising. Despite the intense enthusiasm for CAR T therapy, many questions and uncertainties remain about its optimal design, long-term safety, and optimal timing.

Figure 1 . Chimeric antigen receptors (CARs): (a) anatomy and evolution of CAR design, and (b) CAR T cells recognize specific antigens on cancer cell surface and trigger T-cell—mediated killing.

Adapted from Brentjens et al, 2012, and Kochenderfer et al, 2013.

What Are CAR T Cells?

In its simplest form, a CAR consists of an antigen recognition domain derived from an antibody that is fused to a T-cell receptor (TCR) signaling domain. Key steps in the manufacturing process include the selection and activation of isolated T cells, transduction of T cells to express CARs, ex vivo expansion of the modified T cells, and cryopreservation in infusible media.¹Once the patient has received lymphodepleting chemotherapy, the CAR T cells are reinfused. In this way, CARs graft the specificity of a monoclonal antibody onto the dynamic and persisting characteristics of an effector T cell.

The advantage of an engineered CAR T cell is that, unlike native TCRs, CAR T cells can recognize a variety of cell surface antigens without the constraint of major histocompatibility complex (MHC) human leucocyte antigen (HLA) class restriction. This HLA independence overcomes the tumor’s ability to escape immunodetection by downregulating HLA molecules on its surface. CARs can be engineered against any tumor antigen that is expressed on the cell surface for which a monoclonal antibody can be generated, including proteins, carbohydrates, and glycolipids, thus opening a world of opportunities for cancer treatment. Although the generation of a large quantity of CAR T cells is labor intensive and requires significant infrastructure and expertise, the turnaround time is just a few days, making this strategy feasible for use in the clinical setting. Because the patient’s own cells are reprogrammed to express tumor-specific antigens, the risk of GVHD is limited, a distinct advantage over all HSCT-based immunotherapy. Furthermore, because of long-term persistence of the CAR T cells, a single infusion may be sufficient to produce a sustained response, thus offering the potential for “living” immunotherapy.

The Evolving Anatomy of a CAR T Cell (FIGURE 1)

Gross et al are credited with introducing a T-cell hybridoma in 1989.²A CAR consists of a polypeptide that is the single-chain variable fragment (scFv) from an antibody, linked to the signaling machinery of a T-cell receptor, typically the ζ chain.³The scFv binds to a defined target antigen and triggers effector cell activation upon target engagement.

The first generation of CARs consisted of:

1. An extracellular target-binding or antigen-recognition domain (ectodomain). The target-binding domain was derived from a scFv of an antibody against the target antigen, composed of light and heavy chain portions linked in series via a polypeptide sequence. Antibody-ligand interactions are not MHC restricted, in contrast to TCR-ligand interactions, which need antigen processing and presentation.
2. A hinge region, usually derived from CD8 or immunoglobulin G4 (IgG4) molecules.⁴The length of the hinge region affects the flexibility of the scFv and hence its interaction with the ligand.⁵The optimal length is unknown and may vary with type of ligand and CAR.
3. A transmembrane domain that anchors the CAR to the cell membrane (eg, from CD28).
4. An intracellular-signaling domain (endodomain), usually the CD3 ζ T-cell coreceptor signaling domain.

In clinical trials involving patients with various cancers, including lymphoma and ovarian cancer, the first generation of CAR-T cells triggered T-cell killing but showed only modest efficacy because of inadequate activation, persistence, and proliferation of the modified T cells, all of which were attributed to a lack of costimulatory molecules.^6-9CD28 was the first costimulatory molecule used in the CAR construct and dramatically enhanced antigen-specific cytokine production (eg, interleukin 2 [IL-2]), cytotoxicity, and proliferation.^10-12These second-generation CARs were also relatively resistant to suppression by regulatory T cells.¹³Since then, many other costimulatory molecules have been introduced, including CD27,¹⁴CD134 (OX40, TNFRSF4), CD137 (4-1BB, TNFRSF9),^15,16CD244,¹⁷and inducible T-cell costimulator (ICOS).¹⁸

CAR constructs with a single costimulatory molecule are known as second generation, and those with two or more costimulatory molecules are referred to as third generation. A variety of studies have proved the superiority of second- over first-generation CARs in terms of persistence and cytotoxic activity,⁸but the superiority of third-generation CARs over second-generation CARs remains unproven. Preclinical data comparing CD19 CARs containing either CD28 or 4-1BB costimulatory domains suggested that while cytotoxicity is similar with both modifications, the CAR containing 4-1BB may be associated with improved proliferation,¹⁵although this observation may be restricted to CD19 CARs. An ongoing clinical trial is designed to identify the best costimulatory domain for CD19 CARs (NCT00466531).

The Manufacturing Process: Nuts and Bolts (FIGURE 2)T-Cell Collection

Currently, various CAR constructs that differ in scFv, hinge, transmembrane, stimulatory, and costimulatory domains are being studied in clinical trials at different centers. The optimal combination is yet to be identified and may differ according to the clinical indication.Peripheral blood mononuclear cells (PBMCs) are collected from the patient by apheresis, typically before the administration of lymphodepleting chemotherapy.¹An absolute T-lymphocyte count below 200 to 300/μL may result in poor T-cell collection and, therefore, chemotherapy immediately before harvest is not ideal.¹If the patient is stable post allo-HSCT, then donor PBMCs can be engineered. To date, no significant GVHD has been observed after infusion of allogeneic donor-type CAR T cells in patients with B-cell ALL who relapsed after allo-HSCT. Leukapheresis is typically performed without granulocyte-colony stimulating factor (G-CSF) support, since some reports suggest that G-CSF mobilization may skew T cells toward a Th2 phenotype and impair signaling through CD28.¹⁹The potency of T cells may vary, depending on the mobilization regimen, patient treatment history, method of T-cell isolation, and stimulation. The apheresed PBMC product is then transduced and expanded before reinfusion into the patient.

Figure 2 . Overview of CAR T-cell therapy in clinic.

CAR indicates chimeric antigen receptor; PBMC, peripheral blood mononuclear cell.

Gene Transfer Into T cells

Stable gene transfer is a prerequisite for sustained CAR-T cell expansion and is required for the persis- tence of effector cells in patients following reinfusion. The genetic material encoding the CAR can be introduced into T cells by several methods, each of which has its own pros and cons in terms of complexity, stability of transgene expression, safety, regulations, and cost. The commonly used techniques are:

1. Viral techniques
   • Gamma retroviral vector
   • Lentiviral vector
2. Nonviral techniques
   • Transposons/transposase systems
   • RNA electroporation

Viral vectors can integrate into the host genome, leading to permanent transgene expression. These vectors are highly efficient in transducing T cells after in vitro activation with various cytokines. The advantage of gamma retroviral and lentiviral vectors compared with adenoviral and adeno-associated viral vectors is low immunogenicity. In addition, lentiviruses can integrate in nondividing quiescent cells, are less susceptible to silencing by host restriction factors, and can deliver larger DNA sequences.⁴The disadvantages of viral vectors are the high cost, need for highly specialized staff and facilities required for production, strict regulatory requirements, and risk of mutagenesis.²⁰

T-Cell Expansion

Nonviral approaches are cheaper and potentially safer; however, the CAR T-cell constructs are usually not durable. Transposon/transposase systems such as Sleeping Beauty introduce plasmid DNA by elec- troporation along with DNA or RNA that codes for a transposase.²¹They can deliver a large payload and have been used by MD Anderson Cancer Center in Houston, Texas. Specific terminal repeats flanking the CAR expression cassette are recognized by the transposase and the cassette is inserted into the T-cell genome. RNA electroporation is the process of introducing synthetic mRNA into T cells without integration into the host genome.²²It leads to generation of short-lived CAR-T cells, with limited antitumor activity, that last 3 to 5 days. Their short lifespan translates into limited toxicity, and as a result they have been used in early experiments to study the safety and efficacy of new CAR T-cell constructs.T-cell expansion ex vivo requires activating signals, appropriate culture media, and expertise. The optimal duration of ex vivo expansion is unknown, but longer expansion periods may result in more terminally differentiated T cells that may have impaired antitumor efficacy.²³Most reported clinical studies have involved transfer of bulk-transduced T cells with wide variation in the CD4:CD8 ratio, proportion of regulatory T cells (Tregs), and memory T-cell subsets. Graft engineering consists of selection and expansion of a particular cohort of T cells from the apheresed product and is being developed at some centers (eg, Fred Hutchinson Cancer Research Center in Seattle). Preclinical studies suggest that naïve T cells, central memory T cells, and Th17 or memory stem cells—rather than terminally differentiated effector cells—possess the optimal characteristics for adoptive cellular therapy because they have a high proliferative potential, persist long term, and have the ability to repopulate both memory compartments.^24,25Immunomagnetic separation of distinct T-cell subsets before CAR engineering may make the infused product more homogeneous but adds significantly to the complexity of their generation.

Currently, cell culture is done on an individual basis at a few large academic centers. The challenge for the future is streamlining this resource-intensive process into a more automated, assembly line—like culture system. Before infusion into the patient, the CAR T-cell product must be tested to ensure safety, sterility, purity, potency, and identity.¹

Figure 3. Summary of the Key Studies of CD19 CAR T Cells in ALL

allo-HSCT indicates allogeneic hematopoietic stem cell transplant; CR, complete response; MRD, minimal residual disease; NR, not reported.

Preparing the Patient

Preclinical and clinical studies have shown that T- cell homeostatic proliferation and persistence are increased by lymphodepletion, probably by eliminating Tregs²⁶and by reducing competition for avail- able cytokines (IL-2 and IL-7).²⁷Functional immune reconstitution is superior when T-cell infusion occurs on day 2 after high-dose conditioning compared with later timepoints. The optimal conditioning regimen is not defined, but regimens that include cyclophosphamide and fludarabine have been used in almost all current CAR studies.^28-31

The level of engraftment and expansion does not correlate strongly with the infused T-cell dose; hence, the optimal dosing strategy for T-cell infusion is unknown. After infusion, an initial peak in CAR T-cell number is noted in the peripheral blood, followed by a reduction within the first few days from redistribution into tissues, and subsequent resurgence in the peripheral blood from proliferation.⁶Strategies to augment T-cell persistence after transfer include exogenous cytokine administration, engineered autocrine production of cytokines (IL-12), and overexpression of prosurvival signals or reversal of antisurvival signals. In the first human studies, IL-2 was administered pre-infusion, but it was eliminated later because of lack of evidence of benefit.³²

Clinical Application

CD19 is a transmembrane glycoprotein expressed on a multitude of B-cell malignancies, including >95% of all B-cell ALLs, B-cell non-Hodgkin lymphomas, and chronic lymphocytic leukemia (CLL). It is also expressed on normal B cells from the early pro-B to mature B-cell stages but not on any other cell types. The restricted expression of CD19 to B-lineage cells means that anti-CD19 CAR T cells have on-target/off- tumor effects resulting from B-cell depletion. Loss of B-cells produces hypogammaglobulinemia, which can be easily managed with periodic intravenous immunoglobulin (IVIG) infusions, but is associated with limited other toxicity. As a result, most of the clinical trials to date have been done with CD19 CAR T cells as described in the subsequent paragraphs. A sum- mary of these studies is presented in TABLE 1. Other target antigens under investigation for hema- tologic malignancies include CD20,³³ROR1,³⁴immunoglobulin kappa light chain,³⁵CD33,^36,37IL-3— receptor α chain/CD123, and Lewis Y antigen.³⁸

ALL

The first studies in ALL were published in the spring of 2013 by investigators from the University of Pennsylvania²⁹in Philadelphia and the Memorial Sloan Kettering Cancer Center (MSKCC)³⁹in New York City. Updated reports with expanded patient cohorts have been published since then. Maude et al reported on 30 patients with relapsed/refractory CD19+ ALL who received lentivirally transduced second-generation CD19 CARs using the FMC63 scFv and 4-1BB costimulatory domain.³¹This study enrolled 25 children and five adults (26-60 years of age); three patients had primary refractory ALL; the remainder had relapsed disease. One patient had CD19-expressing T-cell ALL; 18/30 patients had relapsed following allo-HSCT; three were refractory to blinatumumab, the CD3/CD19 bispecific antibody; and two had asymptomatic central nervous system (CNS) involvement. At first assessment after 1 month, 27/30 patients had morphologic complete remission (CR) and 22/30 patients were negative for minimal residual disease (MRD) by multiparametric flow cytometry, implying a CR rate of >90%. After a median follow-up of 7 months, 19 patients remained in CR, and 15 did not require any further therapy. Event-free survival (EFS) was 67% (95% CI, 51-88) and overall survival (OS) was 78% (95% CI, 65-95) at 6 months. No differences in OS and EFS were seen between patients who had received allo-HSCT and those who had not. Two patients with CNS disease showed clearance of CNS blasts. There was no discernable effect of CD19 density or cell dose on efficacy or toxicity; however, durable responses appeared to correlate with higher peak levels of circulating CAR T-cells and duration of B-cell aplasia. Of the seven patients who relapsed after CAR T-cell therapy, three occurred early from loss of CAR T cells, one had refractory disease, and three relapsed because of loss of CD19 expression on the leukemia cells in the face of persistent CAR T-cell circulation. CAR T cells were detectable up to 11 months, and the probability of persistence at 6 months was 68% (50%-92%).

Cytokine release syndrome (CRS) occurred in all patients, with 8/30 (27%) requiring care in the intensive care unit (ICU). Severe CRS (sCRS) was noted to present early (after 1 day compared with 4 days in patients with mild-moderate CRS and was associated with higher baseline disease burden and higher levels of IL-6, C-reactive protein (CRP), ferritin, interferon-γ, and soluble IL-2 receptor. Because of high IL-6 levels, tocilizumab was incorporated into the management of early, sCRS and resulted in significant abrogation of symptoms. Neurologic toxicity was noted in 13 patients and was self-limiting.

Davila et al treated 16 adult patients with re-lapsed/refractory B-ALL with autologous T-cells expressing a CD19-specific CD28/CD3ζ second- generation CAR.²⁸Fourteen patients had detectable disease at the time of CAR T-cell infusion, despite salvage chemotherapy and cyclophosphamide conditioning. The overall CR rate was 88%, with 86% of patients from the CR group further classified (12/14) as MRD negative. Seven of 11 eligible patients were able to undergo allo-HSCT; no relapses were noted post-transplant. Of the remaining patients, three had suboptimal response to CAR T-cell therapy, two declined transplant despite achieving molecular CR, and three had medical comorbidities that ruled out a transplant. This precluded assessment of durability of responses in these patients. In four of eight non-transplanted patients, morphologic remission was noted at up to 24 months. The persistence of 19-28z CAR T cells was noted to be about 3 months, in contrast to the University of Pennsylvania study, where patients exhibited CAR T-cell persistence for several months. One possible explanation for this difference is that the 19-28z CAR T-cell expansion and subsequent contraction are CD19 antigen dependent, resulting in T-cell clearance on elimination of normal and malignant B cells, as seen in a normal T-cell immune response to antigen.³⁹The persistence of CD19-targeted CARs incorporating a 4-1BB moiety rather than the CD28 costimulatory molecule may therefore result from antigen-independent signaling through the 4-1BB CAR, as previously demonstrated in preclinical studies.¹⁵

Toxicities were similar across the two studies. Stringent criteria were applied for diagnosis of sCRS, which included a triad of persistent fever for ≥3 days, selected cytokine elevation (75-fold in two cytokines or 250-fold in one cytokine), and evidence of clinical toxicity (hypotension requiring at least one pressor, or hypoxia, or neurologic manifesta- tions). Patients with sCRS required a prolonged hospital stay (56.7 days vs 15.1 days).

The National Institutes of Health (NIH) recently published results of a phase I intention-to-treat dose-escalation study assessing feasibility, toxicity, maximum tolerated dose (MTD), response rate, and biological correlates of response in children and young adults with relapsed or refractory ALL treated with 19-28z CAR T cells.³⁰In all, 21 patients were enrolled and all received fludarabine and cyclophosphamide preconditioning. The MTD was 1 × 106 CD19-CAR T-cells per kg; all but two patients received the anticipated T-cell dose. Efficacy was similar to that reported in earlier studies, with 67% of patients achieving a CR and 60% achieving MRD negativity. Ten patients who were MRD negative and underwent allo-HSCT remained disease-free at last follow-up. Two patients who were MRD negative and ineligible for transplant relapsed with CD19-negative ALL at 3 and 5 months. With a median follow-up of 10 months, OS was 51.6%. Eleven of 18 evaluable patients demonstrated the presence of CAR T cells in the cerebrospinal fluid (CSF), and CNS disease was cleared in two patients with asymptomatic CNS involvement. Grade 4 CRS was noted in 3/21 (14%) patients, and all toxicities were fully reversible. Neurotoxicity was noted in 6/21 patients; the concentration of CAR T-cells in the CSF was higher in patients who developed neurotoxicity than in those who did not. In evaluable patients, CAR T-cell persistence was lost after 68 days.

CLL

Acute Myeloid Leukemia

Overall, CAR T-cells have shown unprecedented response rates of up to 90% in patients with relapsed/ refractory ALL (TABLE 1). Response rates appear to be higher in ALL than in CLL, likely from defects in T-cell function in CLL⁴⁰and from preferential homing of CD19 CAR T cells to the bone marrow over the lymph nodes. Efficacy appears to depend on the degree of expansion of CAR T-cells rather than on the dose infused. CAR T-cell therapy demonstrates considerable promise as a bridge to transplantation, even in chemorefractory disease, but the potential to replace transplant with engineered T-cells remains a tantalizing possibility. Important prerequisites for response appear to include host lymphodepletion, incorporation of a costimulatory domain, short-term culture, and persistence of the infused product. Longer follow-up is required to assess the optimal duration of persistence for response durability.The initial report of activity of CARs in CLL came from the University of Pennsylvania, where three patients with refractory CLL were treated with CD19 CARs with a 4-1BB costimulatory domain.⁴¹Among the treated patients were two prolonged CRs and one partial response (PR). Investigators at MSKCC treated eight patients with relapsed, heavily pretreated, purine-analogue refractory CLL with CD28 costimulatory domain-based CD19 CARs.⁴²They found that T cells from patients with CLL expanded poorly when compared with T cells derived from patients with ALL, but overall gene transfer was more effective. The median age of the eight treated patients was 68 years; all patients had bulky adenopathy. Of four evaluable patients from this cohort, one patient had a PR and two had stable disease (SD) without concomitant development of B-cell aplasia. An inverse correlation was noted between preexisting peripheral blood tumor burden and persistence of CAR T cells, suggesting that T cells are more rapidly cleared from the circulation in the presence of greater target cell numbers. The investigators concluded that clinical benefit was more likely to occur in the context of MRD. A third study was reported by investigators from the National Cancer Institute (NCI), who reported results from four patients with advanced, heavily-treated CLL responding to CD19 CAR T-cell therapy. Three of four patients attained CR, which was durable for up to 23 months.⁴³Investigators at Baylor College of Medicine in Texas had one patient with CLL in their treatment cohort. This patient demonstrated SD for 10 months after T-cell infusion without prior lymphodepleting chemotherapy.⁸All four trial investigators used second-generation CARs with a CD28 costimulatory domain and gammaretroviral vector transduction, with the exception of the University of Pennsylvania investigators, who used 4-1BB as their costimulatory molecule and a lentiviral transduction approach. The best response in these studies, a CR with long-term B-cell aplasia, was observed in a patient with low estimated disease burden, suggesting that maximal cytoreduction before CAR T-cell infusion may be necessary for success in patients with CLL.Unlike lymphoid malignancies, where abrogation of CD19 results in B-cell aplasia but no other on-target/ off-tumor effects, the challenge in AML is identification of an appropriate target that is not present on normal hematopoietic cells. The lack of AML-specific surface markers has been a significant barrier to the development of CAR-based approaches. The IL-3 receptor α chain/CD123 is expressed on most AML cells, and human CD123-redirected T cells have resulted in AML remissions in mice.⁴⁴However, because CD123 is also present on marrow precursors, anti-CD123 CAR T cells have the potential to eradicate myelopoiesis, which would require allo-HSCT as a rescue strategy. Similarly, CD33 is expressed on immature myeloid cells, including AML; anti-CD33 CAR T cells have been shown to have equal anti-AML efficacy but more severe myeloablation compared with anti-CD123 CAR T cells.⁴⁵Despite the potentially chilling side effect profile of such strategies, clinical trials targeting CD123 and CD33 have commenced at the City of Hope (NCT02159495) and in China (NCT01864902).

Recently, researchers at the University of Pennsylvania have published preclinical data on 4-1BB- costimulated anti-CD33 CAR T cells in a humanized mouse model bearing primary AML xeongrafts.⁴⁶These authors confirmed the antileukemic activity of CD33-targeted CARs at the expense of marked toxicity toward the myeloid compartment. They further developed a “biodegradable” RNA-based anti-CD33 CAR T-cell construct that persisted for a limited period.⁴⁶This construct demonstrated less robust antileukemic activity, but limited myeloid toxicity, potentially opening the door to a CAR T-cell-based bridging approach to enable allogeneic bone marrow transplantation in patients with chemotherapy-refractory AML.

Adverse EffectsCytokine release syndrome (CRS)

CD44v6 is another potential target present on AML cells. This molecule is an adhesion protein expressed on some AML blasts and myeloma cells, but its presence on keratinocytes has the potential for lethal epithelial toxicity, a phenomenon seen previously in one patient treated with the anti-CD44v6 monoclonal antibody, bivatuzumab.⁴⁷Lewis Y antigen has also been targeted by a CD28-costimulated retrovirally tranduced CAR T-cell construct at the University of Melbourne. So far, four patients have been treated with minimal toxicity.^38,48Another study evaluating the safety of CAR T cells directed against NKG2D-ligands is recruiting patients with AML and myelodysplastic syndrome (MDS) with refractory anemia with excess blasts (RAEB) (NCT02203825).CRS is the most common adverse effect (AE) of CAR T-cell therapy and can be potentially life-threatening. The symptoms range from mild ‘flu-like’ illness to multiorgan/respiratory failure requiring intubation and blood pressure support. Some degree of CRS is seen in most patients receiving anti-CD19 CAR T cells, with sCRS reported in about 30% of patients. CRS symptoms appeared earlier with CD28-containing CD19-CAR T cells, compared with 4-1BB-containing CD19-CAR T cells, likely because of a more rapid T-cell expansion in the former group. Severe CRS is associated with elevations in serum IL-6, IL-10, and interferon-γ concentrations. In addition, retrospective analysis of serum samples in the MSKCC study²⁸has shown a significant difference between the C-reactive protein (CRP) levels of patients with sCRS compared with patients demonstrating mild or no CRS; CRP levels ≥20 mg/dL were associated with sCRS. CRP might therefore have use as a predictive biomarker of sCRS. Tocilizumab, the anti-IL-6 antibody, is the preferred first-line therapy for sCRS, with the use of corticosteroids restricted only to the most severe cases because administration of high-dose steroids has the potential to ablate the CAR T cells.^28,29,39Suppression of CAR T-cell expansion would be presumed to have a negative impact on leukemic control, especially if required in the early period after CAR T-cell infusion.

Neurotoxicity

Reversible neurologic complications, including delirium and seizure-like activity, have been noted in all CD19 CAR T-cell studies in ALL; similar symp- toms have been noted in patients receiving blina- tumomab, the bispecific CD19-directed CD3 T-cell engager. Patients may develop a gradual progression of confusion, word-finding difficulty, and aphasia, and ultimately become obtunded. Imaging, electroencephalograms (EEGs), and lumbar punctures are typically unrevealing, but a higher concentration of CAR T cells has been noted in the CSF in these patients.³⁰The possible mechanisms responsible for these symptoms include either passage of systemic cytokines across the blood-brain barrier or cross reactivity of CAR T cells with targets in the CNS. The symptoms are typically transient and management is supportive.

B-cell aplasia

Clinical Pearls

• Second-generation and third-generation CAR T cells combine the specificity of a monoclonal antibody with one or more secondary activation signaling molecules to activate T-cell killing in the absence of major histocompatability complex (MHC) restriction.
• Engineered, or chimeric antigen receptor, T cells have been successfully translated to the clinic for B-cell malignancies and show significant promise as a bridge to transplantation in this context.
• Successful CAR T cells must expand and persist within the patient, avoid suppression by the immunosuppressive tumor milieu, and attack a target or targets whose expression is sustained on the surface of the tumor.

CAR-Expressing NK Cells

Future Directions

This is an on-target/off-tumor effect that serves as a surrogate for persistence and activity of the CAR T cells. The duration of aplasia depends on the CAR construct and subjects patients to increased risk of infection. Patients are typically given prophylactic monthly IVIG infusions, in a manner analogous to that used for the management of patients with X- linked agammaglobulinemia.NK cells are of great clinical interest because they contribute to the graft-versus-leukemia effect, but unlike T cells, play no role in GVHD. Feasibility and safety of NK-cell therapies have been shown for leukemia in both the pretransplant and posttransplant settings.⁴⁹In a fashion analogous to that previously described for CAR T cells, CARs have been introduced into NK cells by either viral-based transduction or by electroporation with varying degrees of success(<10%-93%). In preclinical studies, CAR-NK cells directed against CD19 and CD20 have shown promise against B-cell malignancies.^50-53Two clinical studies using CAR-NK cells are ongoing and both are assessing the safety of anti-CD19-BB-&zeta; CAR-NK cells in patients with B-lineage ALL in the relapsed/ refractory setting (NCT00995137, NCT01974479).CAR T-cell therapy is a valuable addition to the battle against leukemia, but it is still in the early stages of development and much work needs to be done to maximize its efficacy and limit its toxicity. To enhance safety, the next advance in construction of CARs will be the introduction of a suicide gene so that CAR T cells can be depleted in cases of severe, unacceptable toxicity. Various approaches are being studied, including cotransduction of the herpes simplex virus-derived thymidine kinase gene (killed by ganciclovir),⁵⁴expression of an inducible caspase or Fas that can induce apoptosis when exposed to a synthetic dimerization agent,⁵⁵and incorporation of extracellular portions of the EGF receptor, CD34 or CD20,^56,57which are susceptible to existing therapeutic agents (eg, rituximab). The introduction of two separate CARs into the same T cell could also be used to prevent off-tumor/on-target side effects and enhance specificity. In such a construct, one CAR would provide a low-affinity activating CD3&zeta; receptor specific for a first tumor-associated antigen, while another CAR would contain a receptor harboring a costimulatory domain fused to an antigen-binding domain of a second antigen. In this scenario, only interactions that trigger both CAR components would be sufficient to activate T-cell cytolysis.

Multispecific CAR T cells could combat relapse from antigen loss escape by targeting multiple disease-specific B-cell antigens essential for survival/proliferation. This can be achieved by several methods: designing multiple recognition sites on the same CAR, expressing a number of CARs on the same cell using multicistronic vectors, or transducing independent T-cell populations with CARs of different specificities.⁵⁸Developing chimeric receptors that provide activating signals on engagement of inhibitory ligands in the tumor microenvironment, such as PD-L1, may help overcome the immunosuppressive molecules secreted by the cancer cells.⁵⁹Furthermore, CAR T cells that express proliferative T-cell costimulatory ligands (4-1BBL) or proinflammatory cytokines (IL-12), called armored fourth-generation CAR T cells, are already under construction.^60,61

Conclusion

Acknowledgments

Currently, CAR T-cell therapy is personalized, with the raw T cells being derived from the patient. Clearly, there are technical and financial challenges in manufacturing single-patient product lots. To make CAR T-cell therapy more commercially feasible, universal off-the-shelf products need to be developed.¹Preclinical work is ongoing in this regard.CAR T-cell therapy holds great promise to revolutionize treatment for hematologic malignancy and has already shown impressive results in ALL, although in small clinical studies. However, significant variables in the CAR constructs, generation, manufacturing process, and clinical trial design make direct comparisons between these studies difficult. At least 30 clinical trials targeting CD19 are recruiting patients in North America, Europe, and Asia.⁴Ideally, CAR therapy should be experimentally optimized for each target antigen and application, and this can only materialize through collaborative efforts of the key players. A clinical trial is planned at the University of Pennsylvania Children&rsquo;s Hospital and at MSKCC funded by an NIH Special Translational Research Acceleration Project award to address the issue of optimal costimulatory domain choice. In this study, patients treated with the MSKCC 19-28z CAR will be compared head to head with those treated with the University of Pennsylvania&rsquo;s 19-BBz CAR for cytotoxicity, expansion, and persistence. It is a forgone conclusion that engineered T cells will transform the way we treat B-cell ALL, and it is likely they will significantly affect the management of other B-cell disorders. What remains to be seen is whether or not the potential of engineered immunity will fulfill its promise in the management of myeloid malignancy, and even more provokingly, provide a significant shift for the management of all cancer types.The authors would like to thank Michael J. Nemeth, PhD, for critical review of the manuscript prior to submission. PT and EAG wrote the review and reviewed the literature. PT has no disclosures. EAG would like to disclose honoraria from Celgene, Inc and Alexion Pharmaceuticals and grant funding from Astex Pharmaceuticals.

References

1. Levine BL. Performance-enhancing drugs: design and production of redirected chimeric antigen receptor (CAR) T cells. Cancer Gene Ther. 2015;22(2):79-84.
2. Gross G, Gorochov G, Waks T, Eshhar Z. Generation of effector T cells expressing chimeric T cell receptor with antibody type-specificity. Transplant Proc. 1989;21(1 pt 1):127-130.
3. Eshhar Z, Waks T, Gross G, Schindler DG. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors. Proc Natl Acad Sci U S A. 1993;90(2):720-724.
4. Gill S, June CH. Going viral: chimeric antigen receptor T-cell therapy for hematological malignancies. Immunol Rev. 2015;263(1):68-89.
5. James SE, Greenberg PD, Jensen MC, et al. Antigen sensitivity of CD22- specific chimeric TCR is modulated by target epitope distance from the cell membrane. J Immunol. 2008;180(10):7028-7038.
6. Kershaw MH, Westwood JA, Parker LL, et al. A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer. Clin Cancer Res. 2006;12(20 pt 1):6106-6115.
7. Lamers CH, Sleijfer S, Vulto AG, et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. J Clin Oncol. 2006;24(13):e20-e22.
8. Savoldo B, Ramos CA, Liu E, et al. CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients. J Clin Invest.2011;121(5):1822-1826.
9. Till BG, Jensen MC, Wang J, et al. Adoptive immunotherapy for indolent non- Hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-specific T cells. Blood. 2008;112(6):2261-2271.
10. Brentjens RJ,Santos E,Nikhamin Y,et al.Genetically targeted T cells eradicate systemic acute lymphoblastic leukemia xenografts. Clin Cancer Res. 2007;13(18 pt 1):5426-5435.
11. Kowolik CM,Topp MS,Gonzalez S,et al.CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells. Cancer Res. 2006;66(22):10995-10004.
12. Maher J,Brentjens RJ,Gunset G,Riviere I,Sadelain M. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta / CD28 receptor. Nat Biotechnol. 2002;20(1):70-75.
13. Loskog A,Giandomenico V,Rossig C,Pule M,Dotti G,Brenner MK. Addition of the CD28 signaling domain to chimeric T-cell receptors enhances chimeric T-cell resistance to T regulatory cells. Leukemia. 2006;20(10):1819-1828.
14. Song DG,Powell DJ.Pro-survival signaling via CD27 costimulation drives effective CAR T-cell therapy. Oncoimmunology. 2012;1(4):547-549.
15. Milone MC,Fish JD,Carpenito C,et al.Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol Ther. 2009;17(8):1453- 1464.
16. Song DG,Ye Q,Carpenito C,et al.In vivo persistence,tumor localization, and antitumor activity of CAR-engineered T cells is enhanced by costimulatory signaling through CD137 (4-1BB). Cancer Res. 2011;71(13):4617-4627.
17. Altvater B,Landmeier S,Pscherer S,et al.2B4(CD244)signaling by recombinant antigen-specific chimeric receptors costimulates natural killer cell activation to leukemia and neuroblastoma cells. Clin Cancer Res. 2009;15(15):4857-4866.
18. Guedan S,Chen X,Madar A,et al.ICOS-based chimeric antigen receptors program bipolar TH17/TH1 cells. Blood. 2014;124(7):1070-1080.
19. Tanaka J,Mielcarek M,Torok-Storb B.Impaired induction of the CD28-responsive complex in granulocyte colony-stimulating factor mobilized CD4 T cells. Blood. 1998;91(1):347-352.
20. Hacein-Bey-AbinaS,GarrigueA,WangGP,et al.Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J Clin Invest. 2008;118(9):3132-3142.
21. Hackett PB,Largaespada DA,Cooper LJ.A transposon and transposase system for human application.Mol Ther. 2010;18(4):674-683.
22. Zhao Y,Moon E,Carpenito C,et al.Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor. Cancer Res. 2010;70(22):9053-9061.
23. Gattinoni L,Klebanoff CA,Palmer DC,et al.Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+T cells. J Clin Invest.2005;115(6):1616-1626.
24. Burger JA,Gribben JG.Th emicroenvironment in chronic lymphocytic leukemia (CLL) and other B cell malignancies: insight into disease biology and new targeted therapies. Semin Cancer Biol. 2014;24:71-81.
25. Hinrichs CS,Borman ZA,Gattinoni L,et al.Human effector CD8+T cells derived from naive rather than memory subsets possess superior traits for adoptive immunotherapy. Blood. 2011;117(3):808-814.
26. Antony PA,Piccirillo CA,Akpinarli A,et al.CD8+T cell immunity against a tumor/self-antigen is augmented by CD4+ T helper cells and hindered by naturally occurring T regulatory cells. J Immunol. 2005;174(5):2591-2601.
27. Gattinoni L,Finkelstein SE,Klebanoff CA,et al.Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells. J Exp Med. 2005;202(7):907-912.
28. Davila ML,Riviere I,Wang X,et al.Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med. 2014;6(224):224ra25.
29. Grupp SA,Kalos M,Barrett D,et al.Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med. 2013;368(16):1509-1518.
30. Lee DW,Kochenderfer JN,Stetler-Stevenson M,et al.T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet. 2015;385(9967):517-528.
31. Maude SL,Frey N,Shaw PA,et al.Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med. 2014;371(16):1507-1517.
32. Kochenderfer JN, Rosenberg SA. Treating B-cell cancer with T cells expressing anti-CD19 chimeric antigen receptors. Nat Rev Clin Oncol.2013;10(5):267-276.
33. Till BG, Jensen MC, Wang J, et al. CD20-specific adoptive immunotherapy for lymphoma using a chimeric antigen receptor with both CD28 and 4-1BB domains: pilot clinical trial results. Blood. 2012;119(17):3940-3950.
34. Hudecek M, Schmitt TM, Baskar S, et al. The B-cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor. Blood. 2010;116(22):4532-4541.
35. Vera J, Savoldo B, Vigouroux S, et al. T lymphocytes redirected against the kappa light chain of human immunoglobulin efficiently kill mature B lymphocyte-derived malignant cells. Blood. 2006;108(12):3890-3897.
36. Dutour A, Marin V, Pizzitola I, et al. In vitro and in vivo antitumor effect of anti-CD33 chimeric receptor-expressing EBV-CTL against CD33 acute myeloid leukemia. Adv Hematol. 2012;2012:683065.
37. Schirrmann T, Pecher G. Specific targeting of CD33(+) leukemia cells by a natural killer cell line modified with a chimeric receptor. Leuk Res.2005;29(3):301-306.
38. Peinert S, Prince HM, Guru PM, et al. Gene-modified T cells as immunotherapy for multiple myeloma and acute myeloid leukemia expressing the Lewis Y antigen. Gene Ther. 2010;17(5):678-686.
39. Brentjens RJ, Davila ML, Riviere I, et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med. 2013;5(177):177ra38.
40. Christopoulos P, Pfeifer D, Bartholome K, et al. Definition and characterization of the systemic T-cell dysregulation in untreated indolent B-cell lymphoma and very early CLL. Blood. 2011;117(14):3836- 3846.
41. Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 2011;365(8):725-733.
42. Brentjens RJ, Riviere I, Park JH, et al. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood. 2011;118(18):4817-4828.
43. Kochenderfer JN, Dudley ME, Kassim SH, et al. Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Oncol. 2015;33(6):540-549.
44. Gill S, Tasian SK, Ruella M, et al. Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells. Blood. 2014;123(15):2343-2354.
45. Pizzitola I, Anjos-Afonso F, Rouault-Pierre K, et al. Chimeric antigen receptors against CD33/CD123 antigens efficiently target primary acute myeloid leukemia cells in vivo. Leukemia. 2014;28(8):1596-1605.
46. Kenderian SS, Ruella M, Shestova O, et al. CD33 specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia [published online February 27, 2015]. Leukemia. doi:10.1038/leu.2015.52.
47. Tijink BM,Buter J,de Bree R,et al.A phase I dose escalation study with anti-CD44v6 bivatuzumab mertansine in patients with incurable squamous cell carcinoma of the head and neck or esophagus. Clin Cancer Res. 2006;12(20 pt 1):6064-6072.
48. Ritchie DS, Neeson PJ, Khot A, et al. Persistence and efficacy of second generation CAR T cell against the LeY antigen in acute myeloid leukemia. Mol Ther. 2013;21(11):2122-2129.
49. Glienke W, Esser R, Priesner C, et al. Advantages and applications of CAR-expressing natural killer cells. Front Pharmacol. 2015;6:21.
50. Imai C, Iwamoto S, Campana D. Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood. 2005;106(1):376-383.
51. Muller T, Uherek C, Maki G, et al. Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells. Cancer Immunol Immunother. 2008;57(3):411-423.
52. Boissel L, Betancur M, Wels WS, Tuncer H, Klingemann H. Transfection with mRNA for CD19 specific chimeric antigen receptor restores NK cell mediated killing of CLL cells. Leuk Res. 2009;33(9):1255-1259.
53. Shimasaki N, Fujisaki H, Cho D, et al. A clinically adaptable method to enhance the cytotoxicity of natural killer cells against B-cell malignancies. Cytotherapy. 2012;14(7):830-840.
54. Marktel S, Magnani Z, Ciceri F, et al. Immunologic potential of donor lymphocytes expressing a suicide gene for early immune reconstitution after hematopoietic T-cell-depleted stem cell transplantation. Blood. 2003;101(4):1290-1298.
55. Zhou X, Di Stasi A, Tey SK, et al. Long-term outcome after haploidentical stem cell transplant and infusion of T cells expressing the inducible caspase 9 safety transgene. Blood. 2014;123(25):3895-3905.
56. Griffioen M, van Egmond EH, Kester MG, et al. Retroviral transfer of human CD20 as a suicide gene for adoptive T-cell therapy. Haematologica. 2009;94(9):1316-1320.
57. Philip B, Kokalaki E, Mekkaoui L, et al. A highly compact epitope- based marker/suicide gene for easier and safer T-cell therapy. Blood. 2014;124(8):1277-1287.
58. Ghorashian S, Pule M, Amrolia P. CD19 chimeric antigen receptor T cell therapy for haematological malignancies. Br J Haematol. 2015;169(4):463-478.
59. Prosser ME, Brown CE, Shami AF, Forman SJ, Jensen MC. Tumor PD-L1 co-stimulates primary human CD8(+) cytotoxic T cells modified to express a PD1:CD28 chimeric receptor. Mol Immunol. 2012;51(3-4):263-272.
60. Stephan MT, Ponomarev V, Brentjens RJ, et al. T cell-encoded CD80 and 4-1BBL induce auto- and transcostimulation, resulting in potent tumor rejection. Nat Med. 2007;13(12):1440-1449.
61. Pegram HJ, Lee JC, Hayman EG, et al. Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning. Blood. 2012;119(18):4133-4141.