Chimeric antigen receptors (CARs) are engineered molecules that can be introduced into T cells to enable them to target specific tumor antigens. CAR T cells targeting CD19 have shown promise in patients with relapsed and refractory B-cell neoplasms, including those with acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphomas. Notably, durable responses have been observed in patients who had not undergone consolidative stem cell transplant, a finding that correlates with reports of T-cell persistence and B-cell aplasia in studies of anti-CD19 treatment in vivo. Cytokine release syndrome, correlating with activation and expansion of T cells, and neurologic toxicity are the most significant treatment-related adverse effects. Efforts are underway to extend the benefits of immunotherapy with anti-CD19 CAR T cells to other targets and tumor types.
Introduction
Cancer immunotherapy aims to harness the antitumor potential of the immune system and translate it into effective therapies for patients. One such approach, chimeric antigen receptor (CAR) T-cell therapy, has yielded promising initial clinical results. CAR T-cell therapy employs gene transfer techniques to reprogram endogenous T cells to target a specific tumor antigen. Most of the collective experience with this treatment has involved the use of CAR T cells to target CD19, a pan–B-cell antigen expressed in most B-cell malignancies. Genetically modified T cells expressing an anti-CD19 CAR have shown promise in several clinical trials for patients with acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and B-cell non-Hodgkin lymphoma (NHL).[1-7] However, CAR T-cell therapy is associated with toxicities including cytokine release syndrome (CRS), a novel inflammatory process correlating with the in vivo activation and expansion of CD19 CAR T cells; neurologic effects; and B-cell aplasia.
The promise of CAR-modified T-cell therapy derives from its combined immunologic benefits; these include the specificity of a targeted antibody, the ability to expand the T-cell population in vivo for an amplified antitumor response, and the potential for long-term persistence to facilitate ongoing tumor surveillance. Given the successes of early-phase trials with anti-CD19 CAR T cells, larger studies are underway to assess the feasibility of evaluating this treatment modality across multiple centers and in larger patient cohorts. In addition, modification of CARs to target new antigens and a variety of tumor types, including solid tumors, is the focus of ongoing investigations. In this review, we will describe the mechanism of action of CAR T cells, discuss outcomes of current clinical trials, and highlight emerging directions for this exciting approach to cancer treatment.
CAR T Cells
CARs are engineered to combine an extracellular single-chain antigen-recognition domain (usually the variable regions of a specific monoclonal antibody) with one or more intracellular T-cell signaling domains. With the use of gene transfer techniques, CARs can then be introduced into normal T cells, redirecting them to target new antigens (such as CD19) in a manner independent of major histocompatibility complexes.[8,9] Transduction of T cells with CARs encoded in lentiviral or retroviral vectors results in permanent modification of the genome and thus the potential for ongoing expression of the CAR protein for the life of the T cell. The components of the CAR signaling domain are critical for maximal activation, expansion, and persistence of CAR T cells, and therefore are a key target for manipulation. The so-called “first-generation” CARs, which included only the antigen-recognition domain with an intracellular CD3ζ signaling domain, had limited clinical activity[10-12] (Figure 1). They were unable to adequately activate T cells in vivo, especially when tumor cells lacked expression of T-cell costimulatory molecules.[8,9,11,13]
Several groups have worked to improve treatment efficacy by developing “second-generation” CARs, which include a costimulatory domain typically derived from CD28 or CD137 (also known as 4-1BB), in addition to the CD3ζ domain. The strong indication from preclinical trials that second-generation CARs would have greater antitumor potency[14,15] has now been proven by the clinically significant treatment outcomes seen in patients with relapsed and refractory (R/R) CD19-positive malignancies (see Figure 1).[1-4,6] Although the optimal costimulatory domain for second-generation CARs is not known, preclinical studies suggest that 4-1BB CARs may improve in vivo expansion and persistence. In clinical studies, 4-1BB CARs have exhibited prolonged persistence when used in the treatment of patients with CLL and ALL, which may correlate with sustained remissions.[4,6,15] Investigation of third-generation CARs (which have multiple costimulatory domains) is underway to assess whether the efficacy of CAR T-cell therapy can be further improved (see Figure 1).[16]
The anti-CD19 CAR T cells used in various clinical trials may differ in regard to the design of the CAR molecule, the method of CAR transfer into T cells, and other aspects of the CAR T-cell culture environment. The basic steps involved in the manufacture of anti-CD19 CAR T cells at the University of Pennsylvania (Penn) and in most other clinical trial programs are summarized in Figure 2. Patients undergo an apheresis procedure to collect autologous T cells. Once collected, T cells are genetically modified with the CAR construct and expanded ex vivo for clinical use. While lentiviral and retroviral vectors are the methods of gene transfer in the represented studies, other methods such as electroporation and RNA-based methods can be employed.[12,17] The type of cell culture manufacturing system used may impact the final phenotypic composition of the CAR T-cell product, yielding different clinical outcomes.[18-20] As an example, the manufacturing process at our institution uses an anti-CD3/CD28 antibody-coated magnetic bead system that results in a final product capable of memory function and persistence.[19] Prior to anti-CD19 CAR T-cell infusion, patients typically receive chemotherapy; the goal is to induce lymphodepletion and thereby enhance CAR T-cell expansion and persistence in vivo.[21] Lymphodepletion may have the additional benefit of tumor cytoreduction, which can potentially improve CAR T-cell treatment efficacy and minimize toxicity. Notably, however, some patients have responded to CAR T-cell therapy in the absence of prior lymphodepletion.[4] Besides variations in the investigational product, other differences across CAR T-cell treatment protocols include the type and intensity of lymphodepletion, the timing and dose of anti-CD19 CAR T-cell infusions, and of course the target patient population and malignancy.
Clinical Outcomes of Anti-CD19 CAR T-Cell Therapy
The greatest clinical experience with CAR T cells is with targeting CD19, which is a pan–B-cell antigen expressed in the majority of B-cell malignancies, including ALL, CLL, and NHL. This ubiquitous expression of CD19 on malignant B cells, plus the limitation of off-tumor expression on healthy B cells, make CD19 an attractive target associated with limited “on-target/off-tumor” cytotoxicities.
B-cell ALL
The prognosis for adult patients with R/R B-cell ALL is poor, and almost all patients will die from their disease.[22-29] The only possible curative approach in this setting is the use of a salvage strategy, followed by allogeneic stem cell transplantation (SCT) in patients who achieve complete remission. In a retrospective review of 547 adult patients with first relapse of Philadelphia chromosome–negative ALL, no patient who had not undergone SCT was alive after 1 year, compared with 38% of patients who received SCT after initial salvage therapy.[24] Even poorer outcomes are seen in adult patients who relapse after SCT, most of whom have chemotherapy-resistant disease with low response rates to donor leukocyte infusion.[30] While the majority of children diagnosed with ALL respond well to traditional treatment approaches, those with primary refractory disease have a poor prognosis, with an overall survival rate of about 30% at 10 years.[31] Children with relapsed disease are traditionally more responsive to initial attempts at salvage therapy than their adult counterparts, but relapsed ALL remains a leading cause of cancer deaths in children.[32,33] The dismal prognosis and lack of conventional treatment options for patients with R/R ALL have driven the pursuit of therapies with alternative mechanisms of action. Immunotherapeutic approaches that target ALL antigens (such as CD19, CD20, and CD22) are proving to be remarkably effective but transient in the R/R ALL population.[1,3,4,29,34-36] Outcomes using CAR T cells directed at anti-CD19 highlight the potential of an immunologic approach.
Unprecedented remission rates of 67% to 90% have been observed in adult and pediatric patients with R/R ALL treated with second-generation anti-CD19 CAR T cells (see the Table).[1,3,4,7] In our program, we reported a 90% complete response (CR) rate in 25 pediatric and 5 adult patients treated with CTL019 cells, which are anti-CD19 CAR T cells incorporating the 4-1BB costimulatory domain.[4] Flow cytometry to assess for minimal residual disease (MRD) was negative in 22 patients, positive in 3 patients (with 0.1%, 0.09%, and 0.22% levels of MRD), and not performed in 2 patients who achieved a CR. Of interest, responders included 2 patients who were refractory to blinatumomab, an agent that uses a bispecific antibody to redirect cytotoxic T cells to eliminate B cells with its anti-CD3 and anti-CD19 arms.[36] Eighteen of the 30 patients treated had relapsed after a prior allogeneic SCT; T cells were successfully collected and manufactured from these recipients, and no post-infusion graft-vs-host disease (GVHD) was observed. Of the 27 responding patients, 7 relapsed (6 weeks to 8.5 months after infusion) and 1 developed myelodysplastic syndrome. Three of these relapses occurred in patients with CD19-negative leukemia. Importantly, sustained remissions lasting from 2 months to more than 24 months were noted in the remaining 19 patients, 15 of whom did not receive further treatment (3 were consolidated in remission with SCT). The durable remissions observed in patients not bridged to allogeneic SCT correlated with CAR T-cell persistence and the biological correlate of ongoing CAR T-cell activity, B-cell aplasia.[4]
Similarly high response rates have also been observed in an ALL treatment study by researchers from Memorial Sloan Kettering Cancer Center (MSKCC), using anti-CD19–directed CAR T cells containing a CD28 costimulatory domain (as opposed to the 4-1BB domain used in Penn/Children’s Hospital of Philadelphia [CHOP] protocols).[1] Of 16 adults with R/R ALL who were treated with the regimen, 14 (88%) achieved a CR. Four patients had relapsed after allogeneic SCT, and no patient developed GVHD. Of the 16 patients treated, 7 underwent allogeneic SCT, with 2 deaths related to SCT complications and the remaining 5 patients in ongoing remission (with follow-up ranging from 2 to 24 months). The duration of CAR T-cell persistence in this study was limited to 1 to 3 months after infusion.
In a National Cancer Institute (NCI) study (ClinicalTrials.gov identifier: NCT01593696), a CR rate of 67% was observed in a phase I intent-to-treat analysis of 20 children and young adults with B-cell ALL treated with anti-CD19–directed CAR T cells containing a CD28 domain.[3] Ten of the 14 responding patients subsequently underwent SCT. Two patients who were not consolidated with SCT developed CD19-negative relapse, similar to the Penn/CHOP reports. Three patients who did not respond to initial treatment were reinfused with CAR T cells, but they did not respond to therapy. CAR T cells did not persist beyond 68 days. Similar to the MSKCC experience described previously, no patient treated with CAR T cells after allogeneic SCT (n = 8) developed GVHD.[3]
Recently, investigators from the Fred Hutchinson Cancer Research Center reported similar outcomes in 29 adult patients with ALL in a trial with several unique features.[7] First, CD4-positive and CD8-positive central memory T cells were enriched. CAR T cells were manufactured separately from the CD4-positive and CD8-positive T-cell subsets; this was achieved by using an anti-CD19 CAR incorporating the CD3ζ signaling and 4-1BB costimulatory domains transduced via a retrovirus that also contained a truncated epidermal growth factor receptor (EGFR). Inclusion of this truncated EGFR allowed identification and selection of virally transduced cells. Cells were then reconstituted in a 1:1 ratio of CD4:CD8 cells for infusion. The authors reported a CR rate of 93% (86% of whom were MRD-negative). Ten of 12 patients who received cyclophosphamide or etoposide alone achieved a CR, but 7 of them subsequently relapsed. The next 17 patients received fludarabine and cyclophosphamide. They had higher peak expansion and persistence through day 28. While 94% of these patients achieved a CR, the follow-up duration was short; however, disease-free survival seemed to be improved compared with that of patients treated with cyclophosphamide alone. This suggests that, indeed, the intensity of the lymphodepletion may impact overall outcomes.[7] This interesting approach is the first attempt at using a defined T-cell population for CAR therapy; it is hoped that future studies will identify the ideal ratio of CAR-modified T-cell subtypes, and evaluate whether or not this approach can improve outcomes, compared with trials that do not preselect T-cell populations.
Anti-CD19 CAR T-cell therapy can be used successfully as a bridge to curative allogeneic SCT. In patients who do not undergo consolidation with SCT, long-term disease control may be dependent in large part on the persistence of anti-CD19 CAR T cells, although the optimal length of persistence (months to lifelong) is unknown. An important problem (even in patients with CAR T-cell persistence) is CD19-negative relapse. Anti-CD19 CAR T cells are effective in the post-SCT setting, where it is feasible to collect and manufacture donor-derived T cells from the recipient and safely infuse anti-CD19–directed CAR T cells into the patient without triggering GVHD.[1,3,4] Multicenter studies using anti-CD19–directed CAR T cells in adult and pediatric ALL are underway or in development.
CLL and NHL
Despite a wide array of available treatment options, CLL remains incurable without an allogeneic SCT, and the prognosis is quite poor for patients with refractory or multiply relapsed disease and short progression-free intervals between treatments.[37] Anti-CD19 CAR T cells have been used by several centers to treat patients with R/R CLL.[2,6,38] We recently reported outcomes of long-term follow-up of the first 14 patients with CLL treated at Penn using CTL019 cells.[6] The overall response rate (ORR) in this heavily pretreated cohort was 57%, with 4 CRs and 4 partial responses (PRs). Similar to our experience with CAR T-cell immunotherapy for ALL, durable remissions have been observed that correlate with anti-CD19 CAR T-cell expansion and persistence. Remarkably, 2 of these patients with CLL completed CAR T-cell infusion more than 5 years ago, and both are in molecular remission with detectable levels of functional anti-CD19 CAR T cells.[6] In the Penn program, we have not yet been able to identify baseline disease- or patient-related characteristics that differentiate CLL responders from nonresponders.[6]
In an NCI-led study, researchers used anti-CD19 CAR T cells with a CD28 costimulatory domain to treat 15 patients with advanced CD19-positive B-cell malignancies. All 4 patients with CLL in the study responded to treatment, with 3 having sustained CRs lasting longer than 1 year.[2] However, the initial experiences of MSKCC investigators using anti-CD19 CAR T cells with a CD28 costimulatory domain in patients with chemotherapy-refractory or relapsed B-cell lymphomas were more disappointing, with 0 of 7 evaluable CLL patients responding. The reasons for this lack of response are not clear but may be related to differences in the protocol (a phase I design with 3 of 7 CLL patients not undergoing lymphodepletion) or in patient selection criteria, compared with the NCI and Penn studies.[38]
Data on the use of anti-CD19 CAR T cells as treatment for NHL are less mature. Nine patients with diffuse large B-cell lymphoma (DLBCL) or primary mediastinal B-cell lymphoma received this therapy at the NCI, with 4 achieving a CR, 2 a PR, 1 stable disease, and 2 not evaluable for response.[2] Preliminary results describing outcomes using anti-CD19 CAR T cells in CD19-positive NHL from Penn and the University of Washington, Seattle, were presented at the 2015 American Society of Hematology Annual Meeting.[39,40] Researchers from Penn reported an ORR of 68% following treatment with CTL019 in 22 patients with heavily pretreated NHL (13 with DLBCL, 7 with follicular lymphoma, and 2 with mantle cell lymphoma). At a median follow-up of 11.7 months, the rate of progression-free survival was 62%.[40] The group from Seattle reported their experience using an anti-CD19 CAR containing the 4-1BB costimulatory domain in 28 patients with NHL (18 with DLBCL, 6 with follicular lymphoma, and 4 with mantle cell lymphoma). A differential response was noted in 12 patients treated with cyclophosphamide as lymphodepletion, compared with 12 (of 16 evaluable) patients who received fludarabine plus cyclophosphamide as lymphodepletion. Response rates were improved in patients pretreated with fludarabine (ORR, 67% vs 50%), and correlated with higher peak levels of CAR T cells in vivo and improved CAR T-cell persistence[39]; similar to results observed in patients with ALL, these findings suggest that the process of lymphodepletion may have an important impact on overall treatment outcomes for patients with B-cell malignancies.
Toxicity
CRS
CRS is a systemic inflammatory response that correlates with the in vivo activation and proliferation of CAR T cells. The first clinical sign of CRS is fever, which is often low initially but can escalate to levels as high as 105°F (40.5°C). In the vast majority of patients, CRS occurs within 1 to 14 days of anti-CD19 CAR T-cell infusion. Unfortunately, this syndrome can progress beyond fevers and malaise to life-threatening vasodilatory shock. Depending on its severity, CRS can either be self-limited (requiring only supportive care with antipyretics and intravenous fluids) or may require intervention with anti-cytokine–directed therapy. The duration of CRS is variable and dependent on intervention, with resolution typically occurring within 2 to 3 weeks after anti-CD19 CAR T-cell infusion.
In administering anti-CD19 CAR T-cell therapy to our first 30 adult and pediatric patients with ALL, we found that all experienced some degree of CRS. This was defined as severe in 8 patients who required care in an intensive care unit, including vasopressor support and supplemental oxygen.[4] Other centers have reported a similar incidence, duration, and severity of CRS in their patients treated with CAR T-cell therapy, as summarized in the Table. Given the life-threatening nature of CRS, it is useful to attempt to identify patient-, disease-, and therapy-related factors that may predict for the severity of CRS. Disease type (ALL) and disease burden (in ALL) correlate strongly with the severity of CRS.[1,4,17] Unlike with more traditional agents, there is not an obvious dose-toxicity relationship for CD19-directed CAR T-cell therapy, in which the infusion dose is grossly underpredictive of the final expanded active dose; also, dose is only one of many factors that may correlate with peak in vivo expansion. However, there is some indication that the infusion dose of CAR T cells may be correlated with the severity of CRS. For example, in the phase I portion of the NCI-led ALL study, the authors found that an increasing severity of CRS was associated with escalating CAR T-cell dose levels.[3]
The clinical features of CRS are associated with high levels of inflammatory markers and cytokines, including C-reactive protein, ferritin, interferon-γ, and interleukin-6 (IL-6).[1-4,6,41] Effector cytokines such as interferon-γ and soluble interleukin-2 receptor α are elevated, but so are cytokines traditionally associated with macrophage activation, such as IL-6 and interleukin-10. It is interesting that many of the clinical manifestations of CRS overlap with those of macrophage activation syndrome/hemophagocytic lymphohistiocytosis.[4]
Given the very high levels of IL-6 observed in patients with CRS-as well as the initial observation that tocilizumab, an antibody against the IL-6 receptor, rapidly reversed fevers, hypotension, and life-threatening hypoxia in a child who developed CRS after treatment with CTL019[41]-the use of tocilizumab to manage CRS has become standard practice. Tocilizumab remains an attractive treatment option for CRS, since it continues to be effective for most patients and has limited inherent toxicity. In addition, it may have a less negative impact on the antitumor effect of anti-CD19 CAR T cells when compared with steroids. A study from MSKCC has shown differential survival of CD19 CAR T cells in the bone marrow at day 28 that was dependent on whether patients’ severe CRS was managed with steroid- or tocilizumab-based approaches.[1] It remains to be seen whether the administration of tocilizumab earlier in the clinical course of CRS will have a negative impact on response rates.
Neurologic toxicity
As summarized in the Table, most studies across different disease types have reported neurologic side effects within the first few weeks of anti-CD19 CAR T-cell therapy. Although observed adverse effects of this treatment are usually self-limited and include encephalopathy, delirium, focal deficits, and seizures, the neurologic toxicities can result in death.[7] The mechanisms involved in mediating these different neurologic events are unclear, and indeed may vary. It is established that anti-CD19 CAR T cells readily cross the blood-brain barrier and are detectable in cerebrospinal fluid, but their presence does not correlate with neurologic abnormalities. In our initial Penn/CHOP ALL cohort, CAR T cells were identified in 17 of 19 evaluable cerebrospinal fluid samples at day 28. Only a fraction of patients in this subset did not have central nervous system toxicity.[4] In our experience, the administration of tocilizumab does not improve symptoms. Further studies are needed to determine the mechanism of action, associated risk factors, and optimal management of neurologic toxicity after CAR T-cell therapy.
B-cell aplasia
When considering any targeted therapy, expression of the target antigens on healthy tissue can help in the prediction of on-target/off-tumor toxicity. For example, on-target/off-tumor toxicity from anti-CD19 CAR T cells has been limited to healthy B cells. In our patients with persistence of CTL019, B-cell aplasia and associated hypogammaglobulinemia have been observed. Indeed, B-cell aplasia may be a biological marker for ongoing CAR T-cell activity, or a measure of functional persistence. To date, with follow-up beyond 5 years, B-cell aplasia has been successfully managed with intravenous immunoglobulin replacement therapy.[4,6]
Future Directions: Fulfilling the Promise of Anti-CD19 CAR T-Cell Therapy
TO PUT THAT INTO CONTEXT
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David G. Maloney, MD, PhD
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Cameron J. Turtle, MBBS, PhDFred Hutchinson Cancer Research Center
Seattle, WashingtonHow Have CAR T Cells Advanced Cancer Treatment? Redirection of T-cell specificity by genetic modification of T cells with chimeric antigen receptors (CARs) has shown activity in CD19-positive malignancies, including ALL, CLL, and NHL. Especially promising are the initial rates of remission induced by anti-CD19 CAR T-cell therapy in ALL, where up to 90% of refractory patients may achieve an MRD-negative remission. It is unknown to what extent the treatment outcome is affected by the design or delivery of the CAR T-cell product, including the dose and T-cell subsets infused, the costimulatory domains included in the CAR, and the intensity of lymphodepletion. Remission duration is linked to CAR T-cell expansion and persistence, and may be shortened in patients who develop an immune response that causes elimination of the CAR T cells.What Questions and Considerations Should Be Addressed in Future Clinical Trials?In the Seattle series, intense lymphodepletion with cyclophosphamide plus fludarabine was associated with better expansion, persistence, and more durable remissions. Future use of constructs with humanized or human CD19 binding single-chain variable fragment domains may decrease immune rejection. A useful surrogate for functional CAR T-cell persistence is ongoing depletion of CD19-positive normal B cells, a process that can be measured easily using established clinical tests. Toxicity appears to be correlated with CAR T-cell proliferation; it differs between patients with ALL, CLL, and NHL, possibly due in part to the ease of access of the CAR T cells to CD19-positive cells in most patients with ALL. Lower rates of complete remission have been observed in patients with NHL and CLL, which may be due to factors in the tumor microenvironment of these diseases. Trials incorporating antibodies to programmed death 1 and programmed death ligand 1 have been initiated to assess their impact on treatment outcomes. A continuing challenge in the field of CAR T-cell–based immunotherapy is the detection and treatment of neurotoxicity, which appears to be related to cytokine release syndrome rather than direct CAR T-cell toxicity in the central nervous system, since it has been observed with other B cell–targeted CAR T-cell treatments, including CD22. Trials assessing CAR T-cell therapy in solid cancers are underway. The potential extension of this treatment to patients with solid tumors, while hopeful, will likely present additional clinical challenges.Financial Disclosure:Dr. Maloney and Dr. Turtle receive research funding from Juno Therapeutics.
While responses to anti-CD19 CAR T-cell therapy are dramatic, there are opportunities to improve outcomes and mitigate toxicities.
Improving access and feasibility
Until recently, anti-CD19 CAR T-cell therapy was available at only a handful of specialized centers. While CAR T-cell therapy is still only available through participation in a clinical trial, several groups have now launched multicenter trials for ALL and NHL, enabling broader access.
A major limitation to the use of CAR-modified T cells is the need to collect adequate numbers of functional T cells from patients with an active malignancy that has often been heavily pretreated with lymphotoxic agents. Preselecting specific cell populations for manufacturing adds another level of complexity and manipulation. In patients who relapse after allogeneic SCT, this potential limitation can be circumvented by collecting T cells for manipulation from the original donor.[42] After apheresis, completion of the manufacturing process may be delayed up to several weeks, which may be impractical in the treatment of patients with rapidly progressive disease. Importantly, the NCI-led group, when describing their study of autologous anti-CD19 CAR T cells as treatment for pediatric ALL, used an intent-to-treat analysis and reported that 19 of 21 enrolled patients successfully received the intended dose of cells, while 2 patients received lower than the intended dose.[3] Similarly, to adequately assess the potential role of CAR T cells for a general population, and in relation to other available treatment options for patients with R/R ALL, randomized studies should incorporate an intent-to-treat design, and larger phase II studies should report outcomes of enrolled patients who do not receive the intended dose of cells.
Inherent in the previously described approaches is the fact that one product is manufactured to treat only one patient. An exciting area of active research is the development of universal CAR T cells, wherein T cells are collected from a third party and manufactured with the intent to treat several patients. This approach requires further engineering of the T cells to mitigate risks of GVHD and T-cell rejection by the host.[43] While the availability of “off-the-shelf” CARs would dramatically improve access to CAR T-cell therapy, it is not known whether the efficacy and safety of this approach would be comparable to the autologous approach.
Improving long-term outcomes in ALL
In ALL, initial remission rates for patients treated with anti-CD19 CAR T cells are impressive. In our experience, the persistence of CTL019 cells correlates with sustained remissions.[4] Strategies to augment chances of persistence through CAR T-cell modifications or immunomodulatory interventions are important areas of investigation.
While CD19-positive relapses correlate with loss of CAR T-cell persistence, patients receiving anti-CD19 CAR or antibody therapy are at risk for CD19-negative relapses.[3,4] This finding is not particularly surprising, considering that the success of chemotherapy-based approaches in ALL is dependent on the use of a multidrug regimen. It has been shown that CD19-negative escape clones retain expression of CD22.[3,44] This suggests the potential feasibility of a combined CAR T-cell approach (targeting CD19 and CD22) either sequentially or concurrently, which may decrease the risk of relapse. Single-agent studies exploring the use of anti-CD22 CAR T cells for ALL are underway.
CLL: Improving response rates
While relapse is the major obstacle to successful CAR T-cell therapy for ALL, in CLL treated with CAR T cells, most remissions are sustained without the need for additional therapy. It has been hypothesized that the decreased treatment response in CLL may be due to T-cell functional defects or the negative impact of the CLL tumor microenvironment on antigen engagement by T cells.[6,16] Several mechanisms that are known to block immune-mediated antitumor responses have been observed in CLL, including overexpression of immunosuppressive cytokines and immune checkpoint inhibitors.[45-47] These findings suggest that combining checkpoint inhibitors with anti-CD19 T-cell therapy may improve treatment efficacy, an approach that will be explored in clinical trials. It has recently been shown that T cells collected from CLL patients undergoing treatment with the Bruton tyrosine kinase inhibitor ibrutinib have improved ex vivo expansion corresponding with decreased expression of immune inhibitor receptors such as programmed death 1. In mouse models of ALL and CLL, adding ibrutinib was shown to improve the efficacy of anti-CD19 CAR T cells.[48] These findings led to a study now ongoing at Penn using anti-CD19 CARs in conjunction with ibrutinib for patients with R/R CLL. Given the hypothesized dysfunction of T cells in CLL patients, another approach is to treat this population with universal CARs from healthy donors, as described previously in this article.
Beyond CD19: Developing CAR T-cell regimens for the treatment of solid tumors and acute myeloid leukemia
While there are many hurdles in the development of CAR therapy for solid tumors, significant progress has been made in the past few years.[49-51] Promising results have been reported in studies of CAR T cells targeting the human tumor antigen NY-ESO-1 in patients with synovial cell sarcoma and melanoma, and clinical trials are underway to target mesothelin in lung and pancreatic cancer and the EGFR variant III tumor antigen in glioblastoma.[51-53] The first challenge is to identify an appropriate target antigen. CD19 is an ideal target due to its ubiquitous expression on malignant B cells and the limitation of off-tumor expression to healthy B cells. Candidate targets on solid tumors are often also expressed on healthy tissues, predicting for significant off-tumor toxicities. Despite the limited toxicity seen with antibody therapy targeting human epidermal growth factor receptor 2 (HER2), its low level of expression on healthy lung tissue resulted in significant treatment-related toxicity after infusion of anti-HER2 CAR T cells.[54] Similar problems arise in patients with acute myeloid leukemia, with candidate antigens often being expressed on hematopoietic stem cells and posing a risk of aplasia. In addition to the ongoing pursuit of more specific tumor antigens, other strategies to address this issue are being developed. One approach is to design a CAR T cell that requires two tumor antigens for optimal activation, thereby improving the specificity of the therapy.[55] Another approach is to engineer CAR T cells whose activity can be controlled, allowing the practitioner to turn the T cells on (through an induction system) or off (with a suicide switch).[56,57]
Another limitation in the treatment of solid tumors is the decreased ability of CAR T cells to infiltrate tumors and exert an effective antitumor response. Treatment success is limited in large part by the immunosuppressive nature of the tumor microenvironment. Different genetic modifications to CAR T cells are being explored in the preclinical setting to equip the CAR T cells with additional molecular armor to combat this inhospitable environment.[49,58] Other strategies combining CAR T cells with other agents such as checkpoint inhibitors are particularly attractive in the design of immunotherapies for the treatment of solid tumors.[49]
Conclusion
The success of second-generation anti-CD19 CAR T cells has revolutionized the field of cancer immunotherapy and provided hope for patients with previously limited options. Advances in the field continue to develop rapidly, with ongoing studies aimed at further improving outcomes for patients with CD19-positive diseases and moving forward to achieve success in the use of CAR T-cell therapy for management of other malignancies. Without question, we are just beginning to see the promise of CAR T cells realized in the treatment of cancer.
Financial Disclosure:Dr. Frey receives research support from Novartis. Dr. Porter receives research support and royalty payments, and holds intellectual property rights (managed according to policies of the University of Pennsylvania) from Novartis; and his spouse is employed by Genentech.
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