The majority of lung cancers are nonsmall cell lung cancers (NSCLCs), which are associated with several gene mutations. Targeted therapies have shown great promise compared with cytotoxic chemotherapy in patients with NSCLC whose tumor cells harbor a gene mutation. The echinoderm microtubule associated protein like 4 (

) gene was found to be fused with the anaplastic lymphoma kinase (

) gene, resulting in a mutant fusion gene product,

, which drives tumorigenesis in a subset of patients with NSCLCs. In less than a decade since this discovery, several drugs have been designed to target this mutant gene. Crizotinib was the first drug approved for use in patients with ALK-rearranged NSCLC; however, resistance patterns have begun to emerge within a year of use of this drug. Newer, more potent ALK inhibitors include ceritinib and alectinib, which have been approved for use in patients who have progressed on crizotinib in the United States and in Japan, respectively. This review article focuses on these next-generation ALK inhibitors, specifically with respect to their pharmacokinetics, spectrum of activity, and ongoing clinical trials. This article also reviews the available literature on other next- generation ALK inhibitors, which are currently in various phases of clinical development.

Lung cancer remains the leading cause of cancer-related mortality, accounting for 26.8% of all cancer deaths. An estimated 158,040 Americans will die from lung cancer in 2015.

This is higher than the mortality attributed to breast (6.8%), colorectal (8.4%), and prostate cancers (4.7%) combined. Therefore, tremendous efforts are being made to discover improved treatment options for patients diagnosed with lung cancer. More than 80% of lung cancers are nonsmall cell lung cancers (NSCLCs). In these tumors, molecular profiling has revealed molecular aberrations that can be specifically targeted to yield significantly improved outcomes.

The success of targeting the BCR-ABL gene in patients with chronic myelogenous leukemia (CML) led to a search for similarly targetable mutations in NSCLCs. Although gefitinib was approved by the US Food and Drug Administration (FDA) as a third-line therapy in patients with NSCLC, the responses to treatment have not been consistent. Retrospective studies of phase II trials demonstrated that responses typically were observed mostly in patients who were female, never-smokers, and in those who had bronchoalveolar carcinoma or adenocarcinoma with bronchoalveolar features.

However, even among these patients, the response was not consistent.

revealed for the first time the specific activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR), which correlated with exquisite sensitivity to gefitinib. As a result of this study, patients could now be screened for the presence or absence of these mutations, and a better response to therapy could be predicted. It has been established that in an unselected population, the response rate to EGFR mutation-targeted therapy is roughly 9%,

while among patients with NSCLC who are known to have EGFR mutations this response rate is about 67%.

Figure 1 . EML4-ALK Rearrangement and ALK Signaling Pathway

) gene was originally identified in 1994 in anaplastic large-cell lymphoma where the mutation was a translocation from chromosome 2 on to chromosome 5, resulting in a fusion protein to nucleophosmin (NPM).

discovered that in lung adenocarcinomas the ALK gene was rearranged and fused to the echinoderm microtubule associated protein like 4 (EML4) promoter. Both the

genes are located on the short arm of chromosome 2 and are transcribed in opposite directions (

). However, in a subset of individuals with NSCLC, there is an inversion mutation resulting in the fusion of the N-terminal domain of EML4 to the intracellular kinase domain of ALK.

fusion gene, which, when transcribed, results in a constitutively active ALK tyrosine kinase.

) usually involve the Ras/Raf/MEK/ERK (resulting in uncontrolled cell proliferation) or the JAK-STAT3 or phosphoinositide 3 (PI3)-kinase signaling mechanisms (resulting in cell survival and cytoskeletal changes).

This chromosomal rearrangement acting as an oncogene was among the first to be discovered in solid tumors and for that reason was considered a fascinating target, dispelling the widely held notion that these rearrangements were restricted to hematologic malignancies. Soda et al

established transgenic mice models that expressed

in the lung alveolar epithelial cells and showed that these mice developed several adenocarcinoma nodules in both lungs. Subsequently, the study researchers also showed that injection of NIH 3T3 fibroblasts expressing the

gene product into athymic mice resulted in tumorigenesis and fatal respiratory failure, which could be prevented by treating these mice with ALK inhibitors. These experiments confirmed that the fusion kinase could serve as a driver mutation when present.

As a result of these findings, the search for ALK inhibitors was initiated with the hope of improving outcomes for patients with

Table 1. Clinical Trials Evaluating the Efficacy of Crizotinib

AE indicates adverse events; ALT, alanine transaminase; AST, aspartate aminotransferase; ORR, objective response rate; OS, overall survival; PFS, progression-free survival

Crizotinib, or PF-02341066, is an orally bioavailable, small-molecule tyrosine kinase inhibitor (TKI) that competes for the adenosine 5&rsquo;-triphosphate (ATP)-binding sites on tyrosine kinases, which was found to have potent in vitro and in vivo inhibitory activity against c-MET and ALK.

It has a half maximal inhibitory concentration (IC50) of about 0.003 uM.

In a comprehensive kinase assay including more than 120 kinases, crizotinib was found to have a nearly 20-fold higher selectivity for ALK and MET compared with the other kinases.

additionally showed that ROS1 autophosphorylation was also inhibited by crizotinib with a IC50 of 40 nM to 60 nM.

The first phase I study (PROFILE 1001) from August 2008 was conducted by Camidge et al

; 143 patients with ALK-positive advanced NSCLC were assessed and about 60.8% were found to have an objective re- sponse. Median progression-free survival (PFS) was found to be 9.7 months, with the overall survival (OS) estimated at 87.9% at 6 and 12 months. Another phase I trial to study the toxicity profile and efficacy of crizotinib in patients with ALK-rearranged lung cancers was initiated in February 2010.

This study also found an impressive response rate, with about 57% of the patients demonstrating a partial response (PR) or complete response (CR) and 33% patients demonstrating stable disease, with a favorable adverse event (AE) profile.

PROFILE 1005, a phase II trial, enrolled 261 patients with ALK-positive disease who had been treated with at least one line of chemotherapy before enrollment. Even patients with stable brain metastases were deemed eligible for participation in the study. Updated results so far show that treatment with crizotinib leads to a response rate of 60% and a median PFS of 8 months.

A phase III study (PROFILE 1007) was conducted to compare the responses seen in patients with ALK-rearranged NSCLC treated with crizotinib versus systemic chemotherapy using either pemetrexed or docetaxel.

The results were unequivocally in favor of targeted therapy, with a response rate of about 65% with crizotinib versus 20% with chemotherapy. Moreover, the median PFS was 7.7 months in the crizotinib arm versus 3.0 months in patients receiving chemotherapy. Although no significant improvement was found in OS, the conclusion was that crizotinib was clearly associated with improved response and tolerability compared with standard chemotherapy in patients with ALK-positive NSCLC.

another multicenter trial, is ongoing to determine the efficacy and safety of crizotinib versus pemetrexed-cisplatin or pemetrexed-carboplatin in patients with previously untreated ALK-positive advanced lung cancers. This phase III study has so far demonstrated significant improvement in PFS (10.9 months vs 7.0 months) and an overall objective response rate (ORR) of 74% in the groups treated with crizotinib versus 45% in those treated with systemic chemotherapy. Median OS had not been reached in either group. An updated report by Mok et al

had shown improvement in the rate of intracranial events with respect to time to intracranial progression in patients who receive crizotinib versus systemic chemotherapy. However, there have been other isolated case reports of patients on crizotinib

who have a systemic response but are found to have central nervous system (CNS) disease that either develops anew or progresses.

These findings suggest that crizotinib may decrease the CNS failure rates compared with systemic chemo- therapy, but patients may also develop CNS progression. Therefore, further studies may be warranted to better identify the differences between the two groups. The initial enthusiasm caused by the dramatic responses to crizotinib therapy has since been tempered by the rising incidence of acquired resistance patterns. Studies have shown that the average time of relapse is 8.9 months to 10.5 months.

The known mechanisms of resistance may be divided into two categoriesALK dominant and ALK nondominant.

, the former category accounts for about one-third of the cases with acquired resistance to crizotinib. This category includes secondary gene mutations in the

kinase domain, ALK or copy number gain or escape of the tumor cells to so-called sanctuary sites such as the CNS where crizotinib has poor penetration. The most common and well characterized among the gene mutations is the substitution of methionine for leucine, or the

of ALK is located at the bottom of the ATP-binding pocket and considered the gatekeeper position. The presence of an amino acid with a bulky side chain at this crucial position, it is postulated, may render the hydrophobic pocket inaccessible, thereby interfering with the binding of crizotinib.

Several other mutations have been characterized, including

The ALK-independent mechanisms involve activation of other signaling pathways such as the EGFR, heat shock protein 90 (HSP 90), the PI3K/ AKT/mTOR pathways

or transformation to sarco- matoid carcinoma.

In the remaining third of patients, the mechanism by which tumor cells acquire resistance to crizotinib is unknown.

Figure 2 . Mechanisms of crizotinib resistance.

Figure 3 . Generation of LDK378 from TAE684

The poor prognostic significance of ALK, the logical ease with which it can be targeted, and the impressive results seen with crizotinib have all contributed to the frenzied search for other inhibitors with an increased spectrum of activity in terms of potency, bioavailability, and durability of response.

Ceritinib (LDK378) is a selective small-molecule ALK inhibitor developed by Zykadia. Similar to crizotinib, ceritinib also competes for the ATP-binding site but has a much higher potency, with an IC50 of 0.00015 uM.

Crizotinib owes its existence to Marsilje et al at Novartis,

whose previous work had shown the high potency of a compound TAE684 in mediating ALK inhibition.

However, TAE684 itself could not be applied to clinical practice because of the toxic metabolites it generates. Hence, as shown in

, it was modified to yield LDK378 or compound 6, as it was then known. In Ba/F3 cells transfected with various kinases, this compound was found to inhibit ALK activation with an IC50 value of 40.7 nM but had IC50 values >100 nM against all other kinases.

The time taken to reach maximal concentration after an orally ingested dose was roughly 6 hours. The mean terminal half-life was estimated at 40 hours, suggesting a drug-accumulation effect with increasing doses. The drug was found to be metabolized by cytochrome p450 3A (CYP3A), with potential drug interactions with other drugs that are similarly metabolized.

The drug is primarily eliminated by the liver but requires dose reduction only in moderate or severe hepatic impairment.

mutations revealed that ceritinib was able to suppress ALK phosphorylation and its downstream signaling pathways at much lower doses than crizotinib. The

-mutant cell line MGH045 was implanted in mice, who were treated with vehicle, crizotinib, and ceritinib at 25 mg/kg/day. The group treated with ceritinib demonstrated significantly lower tumor volumes, underlining the efficacy of ceritinib in controlling tumor growth. Ceritinib was also found to have activity against cells expressing other ALK kinase domain mutations, such as

. However, it lacked the capacity to inhibit ALK containing the

ASCEND 1 was the initial phase I trial conducted by Shaw et al.

Of the 130 patients with ALK-rearrangementpositive disease enrolled by October 2012, 122 had advanced NSCLC and of these, 83 patients had received crizotinib before enrollment in the trial. The remaining eight were non-lung cancer patients, including those with breast cancer, alveolar rhabdomyosarcoma, rectal adenocarcinoma, anaplastic large-cell lymphoma, and inflammatory myofibroblastic tumor. These patients received LDK378 at doses starting from 50 mg/day to 750 mg/day. The primary objective was to determine the maximal tolerated dose (MTD), with secondary objectives of determining the safety and side-effect profile, pharmacokinetic profile, and antitumor activity.

Patients who received a dose of at least 400 mg/day were found to have an increase in alanine aminotransferase (ALT) that was dose limiting. Patients receiving doses of 600 mg daily or more developed diarrhea and dehydration, while those receiving 750 mg/ day developed vomiting and nausea more frequently. Overall, the most common AEs were gastrointestinal complaints (82% with nausea, 75% with diarrhea, and 65% with vomiting). Fatigue was listed as a frequent side effect, seen in about 47% of the study population, and an increase in ALT was seen in 35%. Four cases of interstitial lung disease were attributed to the drug, as was one case of asymptomatic QT prolongation. More than half the patients required a dose reduction during the course of treatment.

The early results from this phase I study also revealed an ORR of about 58% for patients who received at least 400 mg/day. The ORR was estimated at 59% for those who received 750 mg/day. The median duration of response (DOR) was 8.2 months and median PFS was estimated at 7.0 months. Among patients who had previously been treated with crizotinib, the ORR was 56% at both dose levels, while the ORR was about 62% for patients who were crizotinib-naiÌˆvereceiving the 400-mg day dosage. The median PFS in these patients was about 6.9 and 10.2 months, respectively.

was later carried out to evaluate the differences in tolerability and efficacy of ceritinib among the Asian patient population versus the Caucasian group. Interestingly, it was found that fewer Asians developed grade 3 or 4 side effects (55% vs 72%, respectively). Moreover, the Asian subgroup also had a higher ORR than the Caucasian group (69% vs 57%, respectively) and a higher me- dian DOR (10.1 months vs 6.9 months, respectively).

Table 2 . Clinical trials evaluating the efficacy of ceritinib

Based on these early results, the FDA granted accelerated approval in April 2014, for the use of ceritinib in patients with ALK-rearranged metastatic NSCLC who had progressed through treatment with crizotinib. This was noteworthy because it is one of the few times a drug has received FDA approval based on phase I results alone.

However, this achievement entails some potential pitfalls. The MTD was set at 750 mg/day; however, more than 50% of the patients enrolled in the trial required dose reductions at lower doses of 400 mg and 600 mg. Hence, this suggested that a lower dose that is therapeutically effective and better tolerated may need to be determined. The terminal half-life of 40 hours also argues in favor of dose adjustments, with possibly considering an initial loading dose followed by a maintenance dose that may improve the tolerability profile. Although the study did include patients with stable asymptomatic intracranial metastases, the pharmacokinetic profile did not include any information about the level of the drug in the cerebrospinal fluid (CSF). Also, it remains unknown if the bioavailability of the drug is affected by the intake of food. Finally, because the delayed AE/ toxicity profile remains to be uncovered, more studies examining these factors must be conducted before the drug can be used more widely.

Results from the expansion phase of the ASCEND-I trial continue to be updated. At the 2014 ASCO Annual Meeting, recent findings were presented based on 255 patients,

246 of whom had ALK-rearranged NSCLC (including both the group of patients previously treated with an ALK inhibitor, such as crizotinib, and the group of patients who were ALK inhibitor-naiÌˆve). The median ORR for all ALK+ NSCLC was estimated at 61.8%,

the median DOR was 9.7 months, and the median PFS was 9 months. In general, the ALK inhibitor-naiÌˆve group fared significantly better than the group that was previously treated with ALK inhibition (ORR = 72.3% vs 56.4%, respectively; DOR = 17 months vs 8.3 months, respectively; and median PFS of 18.4 months vs 6.9 months, respectively).

Of the 124 patients with stable brain metastases in this cohort,

26 patients were ALK-inhibitor-naiÌˆve, while the remaining 98 had received an ALK inhibitor previously. The ORR in these two groups was 69.2% and 50%, respectively. The median DOR was not estimable in the former group, while it was 6.93 months in the latter group. The median PFS in these two groups was 8.3 months and 6.7 months, respectively, while the intracranial response rates were again significantly higher in the group that was ALK inhibitor-naiÌˆve (75%) versus the group that had received prior ALK inhibition (40%).

Thus, the overall results were highly promising for therapeutic efficacy with continued ALK inhibition in the setting of crizotinib resistance, unlike the case of other TKIs such as EGFR inhibitors where the response rate was around 10% once resistance developed to the first-line TKI.

Several trials are evaluating the use of ceritinib in various clinical contexts.

The ongoing phase II trial NCT01685138 is a single-arm, multicenter, open-label trial evaluating the efficacy of ceritinib in patients who were either treatment-naiÌˆve (crizotinib or chemotherapy) or had been previously treated with cytotoxic chemotherapy. The initial results from this study were presented at ASCO 2015, and showed an impressive ORR in patients with and without brain metastases (67.6% and 58%, respectively). The median DOR and PFS have been estimated at 10.8 and 11.1, respectively, for patients without brain metastases, while these values are 9.1 months and 10.8 months, respectively, for patients with brain metastases.

Another multicenter, single-arm phase II trial (NCT01685060) seeks to determine the ORR with ceritinib in patients who have previously received chemotherapy and crizotinib. The updated results were presented at ASCO 2015 and showed impressive intracranial disease control rates (IDCR) in patients that matched or surpassed the whole body (WB) response. The most common AEs seen were nausea, diarrhea, and vomiting. The researchers conclude that ceritinib can provide sustained responses in patients treated with crizotinib with or without intracranial metastases.

Table 3 . Other ALK Inhibitors in Development/Trials

ALT indicats alanine aminotranferase; AST, aspartate aminotransferase; CNS, central nervous system; NSCLC, nonsmall cell lung cancer; TRAE, treatment-related AE.

Two phase III trials are evaluating the efficacy of ceritinib in terms of PFS compared with standard chemotherapy agents. Trial NCT01828099 includes patients who are treatment-naiÌˆve and compares ceritinib (arm 1) against combinations of pemetrexed and cisplatin (arm 2) versus pemetrexed and carboplatin (arm 3). This is an interesting study because it may provide information about the utility of ceritinib as a first-line agent in ALK-rearranged NSCLC. The other phase III trial (NCT01828112) includes patients who were previously treated (chemotherapy with the platinum doublet or crizotinib) and has three groups: group 1 receives ceritinib, group 2 receives single-agent pemetrexed, and group 3 receives docetaxel.

11 patients developed disease relapse after initially responding to ceritinib. These relapsed tumors were biopsied and 5 of the 11 showed new mutations in the ALK kinase domain at either G1202 or F1174 loci. There remains a paucity of data identifying other resistance mechanisms in these relapsed cases.

Alectinib, CH5424802, manufactured by Chugai Pharmaceuticals/Roche Pharmaceuticals, is another orally available, small-molecule inhibitor of ALK with an IC50 of 0.0019 uM.

) was found to be about 8.6 hours and its oral bioavailability in mice was estimated at 70.8%.

ALK-rearranged lung cancer cells that have the following mutations conferring resistance to crizotinib are still sensitive to alectinib:

. In fact, kinase activity assays by Sakomoto et al

showed that this drug mediated substantially potent inhibition of both native ALK and the

mutant ALK (Ki = 0.83 nM and 1.56 nM, respectively). In contrast, crizotinib had a 10-fold higher Ki value for the mutant ALK compared with wild-type ALK.

mutation has been reported in a case of acquired resistance to alectinib.

Interestingly, this mutation also confers resistance to crizotinib and ceritinib.

A phase I/II study (AF-001JP) was conducted in Japan

that included patients with ALK-rearranged NSCLC from across 13 institutions. The phase I portion of this study included 24 patients, and the primary objective was to determine the dose-limiting toxicities and ascertain the MTD for the phase II portion of the study. Patients on this study received doses ranging from 20 mg twice daily to 300 mg twice daily. No dose-limiting toxicities were noted at the maximal dose; hence, 300 mg twice daily was set as the recommended dose for the subsequent part of the study. The phase II portion of the study recruited 46 patients of whom 2 had CR and 41 had PR. The ORR was an impressive 93.5%. Twelve of the 46 patients had grade 3 treatment-related AEs (neutropenia and increased creatine phosphokinase [CPK] levels) while 5 of the 46 developed serious AEs. However, no grade 4 AEs or deaths were noted. The drug was approved for use in Japan in 2014 for patients with ALK-positive NSCLC that is advanced, recurrent, or unresectable.

ALEX-1, a phase I/II trial, was conducted in the United States by Gadgeel et al

to ascertain the MTD of alectinib because the Japanese study showed no AEs at the maximal studied dose of 300 mg twice daily. In the US study, 47 patients received increasing doses of alectinib, from 300 mg twice daily to 900 mg twice daily. The most common AEs were fatigue, myalgias, and peripheral edema. Dose-limiting toxicities were seen in two patients receiving the 900-mg twice-daily dosing and included grade 3 headache and grade 3 neutropenia. At the data cutoff point at a median of 126 days follow-up, about 44 patients were evaluable and the ORR was 55%, which was dramatically lower than the response seen with the Japanese study. More impressively, however, 21 of the 47 patients were diagnosed with stable CNS metastases at baseline, and of these, 6 had a CR following treatment with alectinib, 5 had a PR, and 8 had stable disease. The ratio of alectinib concentration in the CSF to serum was 0.75, suggesting a very high level of CNS penetration. Based on this study, the dose of alectinib for the phase II portion (ongoing) was recommended at 600 mg twice daily. At the 2015 ASCO Annual Meeting, data presented from the phase II portion of the study showed a particularly impressive CNS response to alectinib.

Subsequently, some reports have described the use of alectinib on a compassionate basis for leptomeningeal disease that developed in patients treated with crizotinib and ceritinib.

Of the four patients who were treated with 600 mg twice daily of alectinib, three developed significant clinical and radiologic improvement, while the fourth patient had stable disease for 4 months prior to progressing.

However, patients treated with these next-generation ALK inhibitors also develop resistance to the drugs. With alectinib, Katayama et al

mutation.Interestingly, these muta- tions were associated with continued sensitivity to ceritinib and other next-generation ALK inhibitors (

) but were resistant to crizotinib. Studies have also shown that activating the MET signal pathway predisposes to resistance to alectinib. Kogita et al demonstrated that the MET ligand hepatocyte growth factor (HGF) enables the H3122 and H2228 ALK-positive NSCLC cell lines to develop resistance to alectinib; however, the cells still retain their sensitivity to crizotinib.

Over the past decade, significant progress has been made in targeted therapy in NSCLC, especially with the use of ALK inhibitors for ALK-rearranged NSCLC. While these have followed the patterns seen with the use of other TKIs, such as those that inhibit

, the dramatic initial response has been followed by emergence of resistance patterns within a year. There have been heartening differences with significant responses seen with the next-generation ALK inhibitors, in contrast to the second-line of therapy in

and other mutant pathways. However, this also raises interesting questions about the most optimal sequence in which to employ this arsenal of inhibitor drugs.

Multiple treatment algorithms have been proposed to answer these questions, some of which are being attempted in clinical trials. One school of thought suggests using crizotinib up front for ALK- rearranged NSCLC. On disease relapse, rebiopsy of the tumor specimen and use of ceritinib (if sensitive) is suggested. Given the PFS with crizotinib of 8.2 months and median PFS of crizotinib-exposed patients on ceritinib at 7.8 months, the combination is expected to yield a median PFS of 17.4 months and a median OS of 49.4 months.

Alternatively, if the rebiopsy reveals a mutant-cell population that would be resistant to ceritinib (containing the

mutation, for example) or if CNS relapse was the more notable feature, alectinib may be a more reasonable second-line therapy. In case of a third case of crizotinib resistance, activation of alternate signaling pathways such as EGFR-, insulin-like growth factor 1 receptor (IGF-1R), or KRAS is noted. In these cases, instead of using another ALK-TKI, it may be more prudent to use an anti-EGFR/ anti-IGF-1R drug, or systemic chemotherapy. Other non-TKI drugs are also being investigated, including HSP 90 inhibitors and immunotherapeutic agents.

It is important to determine what the consequences are of using these TKIs in any given order. It has been reported that the incidence of leptomeningeal metastases is increasing among patients with ALK-rearranged NSCLC. Given the rising use of crizotinib and even ceritinib in these patients, could there be a link between these events? If so, how would this influence our choice of initial and subsequent lines of therapy for ALK-positive tumors?

Articles

Clinical Articles

The majority of lung cancers are nonâ€“small cell lung cancers (NSCLCs), which are associated with several gene mutations.

Second-Generation ALK inhibitors for Therapy of ALK-Rearranged Non-Small Cell Lung Cancer

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.

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 &ldquo;living&rdquo; 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.

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.

Once 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&rsquo;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&rsquo;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 &ldquo;living&rdquo; immunotherapy.

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

Gross et al are credited with introducing a T-cell hybridoma in 1989.

A 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 &zeta; chain.

The scFv binds to a defined target antigen and triggers effector cell activation upon target engagement.

The first generation of CARs consisted of:

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.

A hinge region, usually derived from CD8 or immunoglobulin G4 (IgG4) molecules.

The length of the hinge region affects the flexibility of the scFv and hence its interaction with the ligand.

The optimal length is unknown and may vary with type of ligand and CAR.

A transmembrane domain that anchors the CAR to the cell membrane (eg, from CD28).

An intracellular-signaling domain (endodomain), usually the CD3 &zeta; 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.

CD28 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.

These second-generation CARs were also relatively resistant to suppression by regulatory T cells.

Since then, many other costimulatory molecules have been introduced, including CD27,

CD134 (OX40, TNFRSF4), CD137 (4-1BB, TNFRSF9),

and inducible T-cell costimulator (ICOS).

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,

but 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,

although 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)

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.

An absolute T-lymphocyte count below 200 to 300/&mu;L may result in poor T-cell collection and, therefore, chemotherapy immediately before harvest is not ideal.

If 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.

The 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.

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:

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.

The 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.

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.

They 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.

It 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.

Most 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 naiÌˆve T cells, central memory T cells, and Th17 or memory stem cellsrather than terminally differentiated effector cells&mdash;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.

Immunomagnetic 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.

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.

Preclinical and clinical studies have shown that T- cell homeostatic proliferation and persistence are increased by lymphodepletion, probably by eliminating Tregs

and by reducing competition for avail- able cytokines (IL-2 and IL-7).

Functional 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.

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.

Strategies 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.

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,

IL-3 receptor &alpha; chain/CD123, and Lewis Y antigen.

The first studies in ALL were published in the spring of 2013 by investigators from the University of Pennsylvania

in Philadelphia and the Memorial Sloan Kettering Cancer Center (MSKCC)

in 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.

This 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-&gamma;, 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/CD3&zeta; second- generation CAR.

Fourteen 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.

The 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.

Toxicities were similar across the two studies. Stringent criteria were applied for diagnosis of sCRS, which included a triad of persistent fever for &ge;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.

In all, 21 patients were enrolled and all received fludarabine and cyclophosphamide preconditioning. The MTD was 1 &times; 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.

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 CLL

and 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.

Among 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.

They 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.

Investigators 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.

All 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 &alpha; chain/CD123 is expressed on most AML cells, and human CD123-redirected T cells have resulted in AML remissions in mice.

However, 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.

Despite 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.

These authors confirmed the antileukemic activity of CD33-targeted CARs at the expense of marked toxicity toward the myeloid compartment. They further developed a &ldquo;biodegradable&rdquo; RNA-based anti-CD33 CAR T-cell construct that persisted for a limited period.

This 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.

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.

Lewis 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.

Another 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 &lsquo;flu-like&rsquo; 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-&gamma; concentrations. In addition, retrospective analysis of serum samples in the MSKCC study

has 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 &ge;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.

Suppression 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.

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.

The 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.

This review addresses the basics of CAR T-cell design and reviews data from published clinical studies in leukemia.

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

Saint Barnabas Medical Center, Livingston, New Jersey

Saint George&rsquo;s University School of Medicine

Chairman and Surgeon-in-Chief, Department of Surgery

Saint Barnabas Medical Center, Professor of Surgery, New Jersey Medical School

Sunitinib treatment is associated with a significantly increased risk of all-grade and high-grade hypertension.

June 2015 | The Journal of Targeted Therapies in Cancer

Second-Generation ALK inhibitors for Therapy of ALK-Rearranged Non-Small Cell Lung Cancer

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

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