Cancer Immunotherapy with Chimeric Antigen Receptor (CAR) T Cells

Chimeric antigen receptor (CAR) T-cell therapy is a rapidly growing treatment modality. In approved products, a patient’s own T lymphocytes are collected by apheresis and transduced with a gene that encodes for a CAR to direct the T cells against cancer cells. The  genetically modified autologous T cells are expanded in vitro at a production facility and then reinfused into the patient. ^[1]

Several types of adoptive cell transfer are under investigation, but CAR T-cell therapy is the first to enter clinical practice. Manufacture of CAR T cells is most commonly done using retroviral vectors. Considerable research continues to enhance current efforts as well as extend this therapy to many tumor types. Currently approved CAR T-cell threapies are listed in Table 1, below. 

Table 1. CAR T-cell therapies approved by the US Food and Drug Administration (Open Table in a new window)

 

Chimeric antigen receptors (CARs) are synthetic proteins expressed on the surface of T cells. These receptors have both extracellular and intracellular components. The extracellular component is an antigen-recognizing domain composed of fragments of monoclonal antibodies; it recognizes a specific protein on the surface of malignant cells (eg, CD19 on B-cells). The intracellular domains ensure intracellular signaling necessary to activate the effector functions of the CAR T cell. First-generation CAR T cells utilized an intracellular domain from the CD3 ζ-chain of the T-cell receptor (TCR), which induced cytotoxicity against targeted malignant cells but failed to support CAR T cell expansion in vivo after reinfusion.

In contrast, second- and third-generation CAR T cells have an additional costimulatory intracellular domain (eg, CD28, 41BB, OX40) that enhances the CAR T cells’ ability to proliferate, expand, and persist in vivo. Establishing a favorable cytokine environment within the patient through lymphocyte depletion (commonly accomplished with fludarabine and cyclophosphamide) further enhances the ability of CAR T cells to expand in vivo. ^{[2, 3]}  The modified T cells are typically infused 2-14 days after lymphocyte depletion. Once infused, the cells continue to expand in number and bind to cancer cells via the engineered receptor, resulting in immunologic cancer cell death. ^[4]  Persistence of CAR T cells for as long as 3 years has been reported. ^[5]

Currently, CAR T cells are approved by the US Food and Drug Administration (FDA) for the following indications:

• Acute lymphoblastic leukemia (ALL) that is refractory or in second or later relapse, in patients younger than 25 years of age
• Relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL), after failure of at least 2 prior lines of therapy
• R/R mantle cell lymphoma, after failure of therapy with a Bruton's tyrosine kinase (BTK) inhibitor

The first report of CAR T-cell therapy was in 2010, in a patient with advanced follicular lymphoma. Kochenderfer et al described dramatic regression of the lymphoma after infusion of CAR T cells engineered to target CD19. ^[6] Since then, several trials have been launched; key studies will be reviewed in this article.

Acute lymphoblastic leukemia

ALL is the most common pediatric cancer, with approximately 3100 cases diagnosed in children and adolescents younger than 20 years each year in the United States. Eighty to 85% of pediatric ALL cases originate in B-cells. ^[7]

Approximately 98% of children with ALL attain complete remission (CR) with standard treatment, and 85% of patients aged 1 to 18 years with newly diagnosed ALL treated with current regimens have long-term event-free survival. ^[7]  However, relapsed/refractory (R/R) ALL carries a poor prognosis: less than 25% of these patients achieve a CR with standard salvage chemotherapy, and responses typically last only 4-9 weeks. ^[5]

CD19 is uniformly expressed on B-cell precursor ALL cells, making it an attractive target for anti-CD19 CAR T cells. In 2011, researchers at Memorial Sloan Kettering Cancer Center first reported a case in which an adult patient with R/R ALL received anti-CD19 CAR T, and subsequently underwent allogeneic stem cell transplantation (ASCT) after being in remission for 8 weeks following the CAR T infusion. ^[3]  The same group later published their experience in 53 patients with heavily pre-treated R/R ALL who received autologous T cells modified to express 19-28z, a second-generation CAR specific to CD19. CR was achieved in 83% of patients, with overall survival of 13 months after a median follow-up of 29 months. ^[8]

Subsequently, in 2014, investigators from the University of Pennsylvania published early results with CTL019 (a CD-19–directed CAR T with a 41BB costimulatory molecule). A total of 30 pediatric and young adult patients (25 years old or younger) with R/R ALL received CTL019 and 90% of them achieved CR. Nineteen patients had sustained remissions beyond 2 to 3 months, suggesting continued function of the infused cells. ^[5]  Three patients in CR subsequently underwent ASCT. In the patients who did not undergo transplantation, event-free survival at median follow-up of 6 months was 67%. In patients with relapse who received salvage therapy, overall survival at 6 months was 78%. ^[5]  

Those excellent results led to an open-label, multi-center, global phase II trial (ELIANA), which enrolled pediatric and young adult patients with R/R ALL for treatment with tisagenlecleucel. Among 75 patients who received tisagenlecleucel, the overall response rate was 81% at 3 months, with 60% of patients achieving CR (all patients achieving CR were negative for minimum residual disease [MRD]) and event-free survival of 50% at 12 months. ^[9]  Median overall survival was not reached, and overall survival at 18 months was 70% in the updated results. ^[10]  On the basis of this study, in August 2017, the FDA approved tisagenlecleucel (Kymriah) for the treatment of patients up to 25 years of age with B-cell precursor ALL that is refractory or in second or later relapse. ^[11]

Currently, there is an unmet clinical need in patients with R/R ALL who are older than 25 years of age. CAR T clinical trials are ongoing in this age group. In the Zuma-3 phase I study, which evaluated KTE-X19 in adult patients with R/R ALL, the CR rate was 68% and all patients were MRD negative. The phase II portion of the study is ongoing. ^[12]  KTE-X19 is an anti-CD19 CAR T cell that has the same construct of axicabtagene ciloleucel, with a CD28 costimulatory domain. However, it is manufactured differently, by removing circulating CD19-expressing malignant cells from the initial pheresis product prior to the manufacturing process. The removal of these cells reduces manufacturing failures and possible activation and exhaustion of anti-CD19 CAR T cells during the ex vivo manufacturing process.

Diffuse large B-cell lymphoma

CAR T-cell therapies approved for treatment of DLBCL include axicabtagene ciloleucel and tisagenlecleucel. A third CAR T-cell therapy, lisocabtagene maraleucel, is awaiting approval for this indication.

Axicabtagene ciloleucel

The second CAR T-cell therapy to enter clinical practice, axicabtagene ciloleucel (Yescarta), was approved by the FDA in October 2017. Axicabtagene ciloleucel is CD19-directed with a CD28 co-stimulatory molecule and is indicated for use in adults with DLBCL who have not responded to or who have relapsed after at least two other lines of therapy. Approved uses include DLBCL, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, and DLBCL arising from follicular lymphoma. Axicabtagene ciloleucel is not indicated for the treatment of primary central nervous system lymphoma. ^[13]

The ZUMA-1 trial in refractory aggressive non-Hodgkin lymphoma (DLBCL, primary mediastinal B-cell lymphoma, or transformed follicular lymphoma) enrolled 111 patients, 101 of whom received axicabtagene ciloleucel. The objective response rate (ORR)—consistent across disease subtypes and other key covariates—was 82%. ^[14] At a median follow up of 27.1 months, 83% of patients had a response and 58% had a complete response. The median duration of response was 11.1 months and the median overall survival was not reached. ^[15]

Since the approval of axicabtagene ciloleucel, data in patients treated outside of clinical trials have become available. These data show that patients treated in the “real world” have outcomes similar to those reported in the ZUMA-1 trial, with approximately 40-50% of patients remaining in remission at 12 months. Patients with a baseline high lactate dehydrogenase level or poor performance status were found to have the highest risk of progression or death after CAR T-cell therapy. ^{[16, 17]}

Tisagenlecleucel

After its approval for ALL, in May 2018 tisagenlecleucel gained approval for adults with R/R DLBCL, including DLBCL not otherwise specified, high-grade B-cell lymphoma, and DLBCL arising from follicular lymphoma, after 2 or more lines of systemic therapy. ^[18]

Approval was based on the single-arm, open-label, multicenter, phase II JULIET trial in adults with R/R DLBCL and DLBCL after transformation from follicular lymphoma. Eligible patients must have received at least 2 prior lines of therapy, including an anthracycline and rituximab, or relapsed following ASCT. Patients received a single infusion of tisagenlecleucel following completion of lymphodepleting chemotherapy. ^[19]

The ORR for the 99 evaluable patients was 54% (95% CI:43%-64%) with a CR rate of 40%. Responses were consistent across all subgroups, including prior autologous stem cell transplantation and double/triple hit lymphoma. With a median follow-up time of 19.3 months, the median duration of response (DOR) was not reached. ^[20]

Lisocabtagene maraleucel 

Lisocabtagene maraleucel (Breyanzi) is a CD19-directed CAR T-cell therapy for adults with R/R large B-cell lymphoma (LBCL), including DLBCL not otherwise specified (including DLBCL arising from indolent lymphoma), high-grade B-cell lymphoma, primary mediastinal large B-cell lymphoma, and follicular lymphoma grade 3B, after previous treatment with two or more lines of systemic therapy. It is not indicated for primary CNS lymphoma.

Safety and efficacy were evaluated in the TRANSCEND trial, an open-label, multicenter, single-arm trial in 268 patients with R/R LBCL. After a single infusion of lisocabtagene maraleucel, following completion of lymphodepleting chemotherapy, 54% of patients achieved CR (95% CI: 47%-61%) and 19% achieved PR (95% CI:14%-26%). Median duration of response for all responders was 16.7 months (CR was not reached; PR: 1.4 months [95% CI: 1.1-2.2 months). Remission persisted for at least 6 months in 65% of responders and for at least 9 months in 62%. Most common adverse reactions were fatigue, CRS, musculoskeletal pain, nausea, headache, encephalopathy, infections (pathogen unspecified), decreased appetite, diarrhea, hypotension, tachycardia, dizziness, cough, constipation, abdominal pain, vomiting, and edema. ^[50]

Mantle cell lymphoma

The FDA granted accelerated approval to KTE-X19 (Tecartus) in 2020 for patients with R/R mantle cell lymphoma after failure of therapy with a BTK inhibitor. Approval was based on the Zuma-2 trial, which showed an impressive 93% ORR (95% confidence interval [CI], 84-98%) and 67% CR (95% CI, 53-78%) in the primary efficacy analysis of 60 patients. After a median follow up of 12.3 months, 57% of those patients were still in remission. Subgroup analysis showed that KTE-X19 was effective in patients with high-risk mantle cell lymphoma (eg, TP53 mutation, blastoid morphologic features, Ki-67 proliferation index of 50% or higher). ^[22]

Follicular lymphoma

Currently, no FDA-approved CAR T products are available for patients with follicular lymphoma. Earlier studies included small numbers of follicular lymphoma patients who underwent CAR T with CTL019. ^[23]  In the 14 follicular lymphoma patients who received CTL019, the ORR was 89%, with 71% of patients achieving a CR.

Most recently, the interim analysis from the Zuma-5 study were presented at ASCO 2020 for patients with R/R indolent lymphomas (80 follicular lymphoma, 14 marginal zone lymphoma) in whom at least 2 prior lines of therapy had failed. All patients received a single infusion of axicabtagene ciloleucel. Of the 87 evaluable patients, the ORR was 94%, with 79% achieving CR. ^[24] After a median follow-up of 15.3 months, the estimated median progression-free survival (PFS) was 23.5 months (95% CI, 22.8%–not estimable) in all patients, and 68% of patients with follicular lymphoma had an ongoing response. This compares very favorably with the 1-year PFS currently achieved with PI3K inhibitors. ^[25]

Multiple myeloma

Several myeloma target antigens are being investigated in clinical trials of patients with R/R multiple myeloma, including B-cell maturation antigen (BCMA), CD38, CD138, SLAMF7, and natural killer group 2D. Idecabtagene vicleucel (ide-cel; bb2121) is a BCMA-targeted CAR T cell that showed excellent activity in a phase I study; 33 patients with high risk R/R myeloma were enrolled, and 85% had a response (45% CR). All 16 patients who had a response and who could be evaluated for MRD had MRD-negative status. ^[26]  

These excellent results were confirmed in a larger phase II study (KarMMa); the initial results showed an ORR of 73% with a median PFS of 8.6 months in heavily pre-treated patients with R/R multiple myeloma. Moreover, the rates of severe grade 3 or higher toxicity were relatively low (cytokine release syndrome, 5%; neurotoxicity: 3%). ^[27]  Based on this study, Ide-cel was granted breakthrough therapy designation by the FDA and a Biologics License Application (BLA) is currently under review.

Another promising CAR T agent, developed in China, is LCAR-B38, which targets 2 distinct BCMA epitopes with theoretical high avidity and distinguishes LCAR-B38M from other BCMA-targeting CAR T constructs. The phase I (LEGEND-2) study enrolled 57 patients with R/R multiple myeloma and 88% of patients achieved a response (74% CR). Moreover, the toxicity profile was favorable, with a grade 3 cytokine release syndrome rate of 7% and only one patient experiencing grade 1 neurotoxicity. After a median follow-up of 19 months, the median OS was not reached. ^[28]  

The same construct is currently undergoing a phase I/II study in the United States and Japan (CARTITUDE-1) under the name JNJ-4528. In the phase Ib part of the study in 29 patients, all patients achieved a response; 22 (76%) achieved a stringent CR. Neurotoxicity was reported in 4 patients (grade I-2 in three patients, grade 3 in one patients). However, there was one death from cytokine release syndrome, so further results from the phase II part of the study are needed to clarify the safety profile of the construct. ^[29]

Solid tumors

Preclinical data on CAR T therapy in solid tumors are exciting but clinical data is still in its infancy, involving mostly phase I studies. Several challenges stand in the way of using CAR T cells in solid tumors, such as identification of a truly tumor-specific antigen that is not expressed on normal tissues, as well as a hostile tumor micro-environment. Serious off-tumor toxicities have been observed in solid tumor CAR T trials, including liver, cardiac, central nervous system, and lung toxicity. ^{[30, 31, 32]} There are currently many ongoing clinical trials using CAR T cells in solid tumors, including common malignancies such as colorectal, prostate, breast, and pancreatic cancer.

CAR T cell therapy has a unique set of acute toxicities that include cytokine release syndrome (CRS), neurologic toxicity, cytopenias, and B-cell aplasia. These adverse events can be fatal and require special management. For this reason, the FDA mandates inclusion of a risk evaluation and mitigation strategy (REMS) that requires special certification for hospitals and clinics as well as additional training for physicians and support staff. ^[11]

Patients often require hospitalization to receive CAR T therapy, although this depends on the CAR T product, the bulk of the disease, and the proximity of the patient to the CAR T center. Patients receiving CAR T constructs with accelerated expansion kinetics (eg, CD28-based constructs) often require hospitalization for at least 7 days after the CAR T infusion.

Reporting and grading of toxicities is challenging. Comparison across clinical trials is difficult, as different grading criteria were often used. However, in 2019 the American Society for Transplantation and Cellular Therapy (ASTCT) published a consensus grading system to simplify the grading and reporting of CRS and neurologic toxicities (immune effector cell–mediated neurotoxicity syndrome [ICANS]). The ASTCT scoring system is easy to calculate clinically at the bedside, and is gaining rapid adoption in clinical practice. ^[33]  

Other challenges of CAR T cell therapy include its extremely high cost and the need for complicated logistical planning. This restricts the administration of this therapy to large tertiary referral centers, which can limit the ability of patients to receive it.

Cytokine release syndrome 

CRS is a potentially life-threatening complication of CAR T cell therapy. It is a systemic inflammatory response that results when the activated CAR T cells rapidly release large amounts of cytokines, including interleukin-6 (IL-6) and interferon γ, into the bloodstream. Clinically, CRS presents as flulike illness, with fever and systemic symptoms (eg, nausea, fatigue, headache, myalgias, malaise). With higher grades of CRS, patients can develop hypotension and multi-organ toxicity (eg, acute respiratory distress syndrome, renal failure, liver failure, cardiac dysfunction, disseminated intravascular coagulation, and encephalopathy). ^{[4, 34]}

ASTCT CRS grading is based on the presence and severity of fever, hypotension, and hypoxia (see Table 1, below). Organ toxicities associated with CRS may be graded according to Common Terminology Criteria for Adverse Events, version 5.0 (CTCAE v5.0), but they do not influence CRS grading. ^[33]

Table 2. American Society for Transplantation and Cellular Therapy Consensus Grading of Cytokine Release Syndrome (CRS) (Open Table in a new window)

The onset of CRS is typically within 14 days after CAR T-cell infusion and coincides with maximal expansion of CAR T cells. The incidence and severity of CRS appear to be greater in patients with a large tumor burden and in those with an active infection at the time of CAR T infusion, presumably because those patients experience higher levels of T-cell activation. ^[34]

CRS is a diagnosis of exclusion. Therapy begins with symptomatic measures for mild manifestations (fluids, fever management, antibiotics). As CRS severity progresses, patients may need supplemental oxygen, mechanical ventilation, or vasopressors. ^{[34, 33]}

Tocilizumab, an antibody targeting the IL-6 receptor, has become standard therapy for CRS and is the first-line treatment for progressive CRS. Fever and hypotension often resolve within a few hours after administration of tocilizumab, allowing patients to be weaned off vasopressors and other supportive care measures. In patients whose condition does not improve or stabilize after they receive tocilizumab, an additional 1 or 2 doses of tocilizumab can be administered.

Second-line treatment options for CRS include other immunosuppressant medications such as glucocorticosteroids or siltuximab. ^{[34, 35]} Theoretically, steroids may inhibit CAR T efficacy, but judicious use is necessary to dampen the severity of CRS and to minimize toxicity.

Neurologic toxicities: immune effector cell–mediated neurotoxicity syndrome 

Neurologic toxicity is common after CAR T cell therapy and can occur with or without CRS. Symptoms can be mild to moderate (eg, headache, confusion, dysmetria, ataxia, dysphasia, tremors), but can also be severe and life-threatening (eg, seizures, coma requiring intubation and mechanical ventilation for airway protection).

ICANS typically occurs within 4-5 days after CAR T infusion, lasts 5-10 days, and is typically reversible. The incidence of neurologic toxicity in published reports varies from 0% to 50% and differs among the different CAR T constructs. ^{[5, 36, 37]}  

The mechanism of neurologic toxicity after CAR T cell therapy is poorly understood, but might be related to increased trafficking of cytokines into the central nervous system, or related to the CAR T cells that cross the blood-brain barrier. ^[38]  Several groups have found anti-CD19 CAR T cells with elevated IL-6 levels in the cerebrospinal fluid in patients who developed neurotoxicity. ^{[5, 37, 39]} However, neurotoxicity is not exclusive to CAR T cell therapy and has also been reported with other immunotherapies, such as bispecific antibodies. ^[40]  

ICANS grading includes the Immune Effector Cell–Associated Encephalopathy (ICE) score. The assessments and points are as follows:

• Orientation (to year, month, city, hospital): 4 points
• Naming (ability to name 3 objects [eg, point to clock, pen, button]): 3 points
• Following commands (ability to follow simple commands [eg, “Show me 2 fingers” or “Close your eyes and stick out your tongue”]): 1 point
• Writing (ability to write a standard sentence [eg, “Our national bird is the bald eagle”]: 1 point
• Attention (ability to count backwards from 100 by 10): 1 point

ICE scoring is as follows:

• 10 points: no impairment
• 7-9 points: grade 1 ICANS
• 3-6 points: grade 2 ICANS
• 0-2 points: grade 3 ICANS
• 0 points (patient unable to be assessed because unarousable): grade 4 ICANS

Incorporation of ICE scoring into ASTCT ICANS grading is shown in Table 2, below.

Table 3. American Society for Transplantation and Cellular Therapy Immune Effector Cell–mediated Neurotoxicity Syndrome (ICANS) Consensus Grading for Adults (Open Table in a new window)

Treatment of ICANS is mainly with glucocorticosteroids and supportive care. Treatment is mostly effective at reversing the signs and symptoms.

B-cell aplasia and hypogammaglobulinemia

B-cell aplasia occurs when anti-CD19 CAR T cells inadvertently damage normal B-lymphocytes that express CD19. Patients are typically at high risk of developing infections because of their hypogammaglobulinemia. However, this can be treated with intravenous immunoglobulin (IVIG) replacement therapy. ^[41] While B-cell aplasia can cause long-term hypogammaglobulinemia, some patients will reconstitute their B-cells and restore their immunoglobulin levels without requiring life-long IVIG. ^[42]

Cytopenias

Low blood counts, including neutropenia and thrombocytopenia, are common after CAR T therapy and can last from weeks to several months. Prolonged neutropenia can place patients at increased risk of infection. The etiology of cytopenias post–CAR T therapy is poorly understood, and while early cytopenias can be attributed to lymphodepleting therapy, prolonged cytopenias can occur in patients who have not received lymphodepleting chemotherapy. ^[43]  Interestingly, a prolonged CD4 T cell deficit has been described. ^{[44, 45]}  While the optimal strategy is not known, many centers provide re-vaccination and prophylaxis against Pneumocystis jirovecii pneumonia and varicella-zoster virus infection.

 

The field of CAR T-cell therapy continues to grow, with new data and indications, and more approvals are expected in the near future. The field is moving beyond lymphoid malignancies, and many trials in other hematological malignancies and solid tumors are ongoing. At the time of this writing, 288 clinical trials that are currently recruiting patients are listed on clinicaltrials.gov. Some of these address limitations of anti-CD19 therapy, while others expand CAR T-cell therapy to other indications.

Novel CAR T constructs targeting more than one antigen are now in late clinical stages, with clinical data generated. For example, dual CD19- and CD22-targeting CAR T-cell constructs are showing early promise in ALL and DLBCL, including cases in which a CD19-targeting CAR T-cell product had previously failed. ^{[46, 4, 47]}  Other similar concepts include anti-myeloma CAR T constructs targeting more than one antigen on myeloma cells. In addition, initial research suggests that use of CAR T cells that target both CD19 and CD123, another antigen commonly found on leukemia cells, may prevent antigen loss. ^[48]

Allogeneic CAR T constructs are also in clinical trials and would theoretically offer an ‘off-the-shelf” product that would simplify the process of delivering CAR T therapy, particularly in patients with rapidly progressive disease who cannot wait several weeks for autologous CAR T manufacturing. Early phase I data in DLBCL and follicular lymphoma have been presented, but more data are needed to determine how efficacious these products will be compared with autologous CAR T constructs. ^[49]

In summary, current CAR T-cell therapies represent a major advance in the treatment of B-cell malignancies as well as a new paradigm for future efforts directed at other currently incurable tumor types. CAR T cell use can be associated with severe and even life-threatening side effects, but these toxicities must be viewed in the context of the risk-benefit ratio. Fortunately, with experience, these toxicities can be ameliorated. One may reasonably hope that over the next few years, new and effective applications of this exciting treatment modality will expand to more cancers.