Fludarabine | Others signal

Fludarabine and neurotoxicity in engineered T-cell therapy

Kate L. Lowe1 ● Crystal L. Mackall2 ● Elliot Norry3 ● Rafael Amado3 ● Bent K. Jakobsen3 ● Gwendolyn Binder3

Received: 20 October 2017 / Revised: 25 January 2018 / Accepted: 9 March 2018
© The Author(s) 2018. This article is published with open access

Abstract
Adoptive T-cell therapy, incorporating engineered T cell receptors (TCRs) or chimeric antigen receptors (CARs), target tumor antigens with high afﬁnity and speciﬁcity. To increase the potency of adoptively transferred T cells, patients are conditioned with lymphodepleting chemotherapy regimens prior to adoptive T-cell transfer (ACT), and data suggest that ﬂudarabine is an important component of an effective regimen. In a recent clinical trial using CAR-T cells engineered to target the CD19 B-cell antigen to treat acute lymphoblastic leukemia, JCAR-015 (NCT02535364), two patient deaths due to cerebral edema led to trial suspension. The lymphodepleting agent ﬂudarabine was suggested as the causative agent, in part due to its known association with neurotoxicity and its ability to induce greater potency. In a similar CAR-T study also incorporating ﬂudarabine in the preconditioning regimen, ZUMA-1 (NCT02348216), one patient died of cerebral edema. However, subsequent deaths in the JCAR-015 study after removal of ﬂudarabine and improved understanding behind the mechanisms of CAR-T-related encephalopathy syndrome (CRES) indicate that ﬂudarabine is not the primary causative agent of cerebral edema and that it can be safely incorporated into the preconditioning regimen for ACT. Since entering clinical use in the late 1980s as a chemotherapy agent, ﬂudarabine and similar analogs have been associated with lethal neurological toxicity, yet the manifestation and timing of symptoms are distinct to those observed recently in ACT. Herein, we review the history of ﬂudarabine development as a chemotherapeutic agent, and discuss the safety of its continued use in preconditioning regimens for ACT.

Introduction

The term “adoptive immunotherapy” was ﬁrst used to explain the graft-vs.-tumor effect observed following allo- geneic stem cell transplants for leukemia [1]. In these early studies, chemotherapy-induced immunosuppression was found to be necessary to enable successful stem cell grafts. Later studies demonstrated the importance of the T-cell component of the stem cell graft in preventing relapse [2]. Allogenic stem cell transplantation (ASCT) signiﬁed the ﬁrst potentially curative approach to improve cancer therapy

Electronic supplementary material The online version of this article (https://doi.org/10.1038/s41434-018-0019-6) contains supplementary material, which is available to authorized users.

* Gwendolyn Binder [email protected]

1 Immunocore, Oxford, UK
2 Department of Pediatrics, Hematology and Oncology, Stanford Cancer Institute, Stanford, CA, USA
3 Adaptimmune, Oxford, UK, Philadelphia, PA, USA

for patients with hematological malignancies, and it was also a ﬁrst example of cell-based immune oncology.
Lymphodepleting agents were selected based on clinical experience from single agent and combination che- motherapies and include the alkylating agents chlorambucil and cyclophosphamide, which were among the ﬁrst identiﬁed chemotherapeutics. The family of nucleoside analogs shortly followed, encompassing the pyrimidine nucleoside cytarabine and the purine nucleosides cladribine, pentostatin and ﬂudarabine that demonstrate potent cytotoxic activity [3, 4]. Purine nucleosides were developed speciﬁcally to improve on the activity of cytarabine, and ﬂudarabine’s relative resistance to adeno- sine deaminase improved its bioavailability. While not used in the original transplant regimens, ﬂudarabine became an important part of pre-transplantation condition- ing regimens due to its improved tolerability and outcomes [5–7]. Fludarabine induces cellular cytotoxicity via multiple pathways that ultimately lead to an inhibition of DNA synthesis. A rate-limiting step in this process is the activity of deoxycytidine kinase, which is abundant in lymphocytes, making them susceptible to accumulation of F-ara-ATP, the active metabolite of ﬂudarabine, and hence

Table 1 Details of neurotoxicity observed in clinical trials using ﬂudarabine chemotherapy

Reference Year No. of patients Disease Fludarabine Treatment regimen Onset of symptoms Reported symptoms Recovered Autopsy
(mg/m2) (days) (Y/N) (Y/N)
Spriggs et al. [22]
1986 2 AML 60 x d1−5; 15 d; 2 c 70 Dementia, incontinence, blindness and quadriparesis N Y
Spriggs et al. [22]
1986 1 Acute leukemia 100 x d1−5; 15 d; 1 c 35 Dementia, incontinence, blindness, quadriparesis and coma N Y
Warrell et al. [21]
1986 5 Acute leukemia 150 x d1−5; 7 d; 1 c 21−43 Dementia, incontinence, blindness, seizure and coma 1Y 4N 1Y 3N
Chun et al. [23]
1986 13 Acute leukemia 96 x d1−5; x d; x c NS Dementia, hallucination, blindness and quadriparesis N N
Chun et al. [23]
1986 1 Acute leukemia 125 Single bolus NS Dementia, blindness, quadriparesis N N
Merkel et al. [24]
1986 1 T-ALL 77 x d1−5; 28 d; x c 60 Dementia, myoclonus and coma N N
Weiss et al. [25]
1986 1 NSLCC 22 x d1−5; 28 d; x c 41 Dementia, cerebral atrophy and coma N N
Rainey et al. [26]
1988 1 SLCC 1 x d1−5; 28 d; x c NS Ataxia and dizziness NS NS
Hochster et al. [27]
1992 6 NHL 18 x d1−5; 28 d; 1−25 c NS Vision loss, hearing loss and confusion Y N
Kornblau et al. [28]
1993 2 Acute leukemia 30 x d1−5; x d; x c NS Severe, progressive cerebral dysfunction N N
Cohen et al. [29]
1993 1 CLL 20−25 x d1−5; 28 d; 6 c >170 Blurred vision and gait disturbance Y N
Cohen et al [29]
1993 1 Mycosis fungoides 20−25 x d1−5; 28 d; 8 c >220 Seizure, loss of consciousness and hemiparesis Y N
Cheson et al.a [30]
1994 3 CLL 25 x d1−5; 28 d; x c >120 Dementia, vision loss, seizure and paralysis N N
Cheson et al.a [30]
1994 1 FSCL 18.25 x d1−5; 28 d; x c >220 Ataxia and dysphasia N N
Cheson et al.a [30]
1994 5 CLL 25−40 x d1−5; 28 d; 3−10 c >90 Dementia, dizziness, seizures and quadriparesis Y N
Johnson et al. [31]
1994 5 CLL 25 x d1−5; 28 d; x c 20-Jan Headaches, hemiparesis and quadriparesis 4 Y 1N Y
Zabernigg et al. [32]
1994 1 CLL 20−25 x d1−5; 28 d; 6 c 360 Dementia, hemiparesis and coma N Y
Johnson et al. [33]
1996 3 CLL 25 x d1−5; x d; 6 c NS NS N N
Gonzalez et al. [34]
1999 3 CLL 25 x d1−5; x d; x c 120−250 Paralysis, quadriparesis and dementia N 1Y 2N
Sorensen et al. [35]
1997 1 CLL 25 x d1−5; 21−28 d; 1−12 c 180 Visual and cerebral disturbances Y N
Cid et al.C [36]
2000 1 CLL 18−25 x d1−5; 21−28 d; x c >220 Hemiparesis NS NS
Leonard et al.C [37]
2002 1 CLL NS NS 150 Aphasia and right hemiparesis NS NS
Saumoy et al.C [38]
2002 1 CLL 25 x d1−5; 21−28 d; x c NS Progressive neurological syndrome N N

giving ﬂudarabine particularly potent lymphodepleting properties [8, 9].
In the setting of adoptive T-cell transfer (ACT)-based immune oncology, use of lymphodepleting chemotherapy enhances potency, in addition to making “room” for the infused cells to engraft [10, 11]. Lymphodepletion was applied to the adoptive transfer of tumor-inﬁltrating lym- phocytes (TILs) for the treatment of metastatic melanoma and led to enhanced TIL homing and anti-tumor efﬁcacy [12]. Use of the non-myeloablative transplant regimen containing ﬂudarabine and cyclophosphamide improved overall response rates compared to patients who did not receive preconditioning, and this regimen therefore was taken forward in a majority of subsequent engineered ACT studies [13]. ACT engineered with chimeric antigen receptors (CARs) and high afﬁnity T-cell receptors (TCRs) targeted against tumor-speciﬁc antigens have shown pro- mise in the clinic [14–16].
Recently there have been reports of lethal neurotoxicity due to cerebral edema in six patients treated with CD19 CAR-T cells, across two different CD19 CAR-T studies [17–19]. After the ﬁrst two patients were reported, links between this neurotoxicity and ﬂudarabine were suggested, as these patients had received ﬂudarabine in addition to cyclophosphamide as part of their preconditioning regimen, and neurotoxicity is known to be associated with this drug. However, the neurological symptoms associated with ﬂu- darabine as a chemotherapeutic agent are distinct from CD19 CAR-T neurotoxicity in both symptoms and timing. Speciﬁcally, ﬂudarabine-associated neurotoxicity has a later onset, and cerebral edema is not reported. Since initial reports, signiﬁcant neurotoxicity, including cerebral edema, occurred in the absence of ﬂudarabine-containing pre- conditioning regimens.
Although it is now broadly accepted that ﬂudarabine is not a primary driver of neurotoxicity at the doses used in ACT studies, it is important to understand its role in the efﬁcacy and toxicity of adoptive T-cell therapy, in order to develop products with the optimal balance of risk and beneﬁt for patients. Here, we summarize the use of ﬂudar- abine from a historical perspective to the present day, reporting efﬁcacy and toxicity of ﬂudarabine compared to alternative agents and in different therapeutic regimens, and the rationale for its use in combination with ACT.

History of ﬂudarabine safety and efﬁcacy as a chemotherapeutic agent

Initial phase I clinical trials testing ﬂudarabine for the treatment of solid tumors started intravenous (i.v.) dosing at 260 mg/m2 for 5 days, which was soon reduced by 60% due to severe neutropenia [20]. Anti-tumor activity was most

potent against hematological malignancies resulting in dose escalating Phase II trials for treatment of acute leukemia (≥100 mg/m2) (Supplementary Tables 1 and 2). Although efﬁcacious, high incidence of delayed, progressive central nervous system (CNS) toxicity was reported (described in more detail in the following section). This toxicity was associated with high dose of ﬂudarabine and was addressed by reducing doses to 25–40 mg/m2 for 5 days i.v. in monthly cycles [21–24]. Generally, 25–40 mg/m2 dose regimens were well tolerated, although neurotoxic events continued to be reported [25–44] (Table 1, Supplementary Table 2). By the late 1990s, ﬂudarabine-based chemother- apy regimens became the standard ﬁrst- and second-line therapies for the most common adult leukemia, chronic lymphocytic leukemia (CLL), and a common therapeutic for many other leukemias and lymphomas.
Compared to alternative chemotherapy regimens, ﬂu- darabine as a monotherapy produced superior or statistically similar response rates to the chlorambucil, cyclophosphamide-doxorubicin hydrochloride-vincristine (CHOP) regimen, cyclophosphamide-adrianmycin-cisplatin (CAP) regimen and ﬂudarabine-prednisone, with no sig- niﬁcant increase in toxic effects. However, no difference in survival was observed (Supplementary Table 3). Combining ﬂudarabine and cyclophosphamide (FC) gave rise to both enhanced response rates and extended survival with no increase in toxicity (Supplementary Table 3). The addition of targeted monoclonal antibodies, such as the anti-CD20 antibody rituximab (FCR) or the anti-CD52 antibody ale- mutuzumab, further enhanced response and survival in CLL and B-cell non-Hodgkin’s lymphoma (B-NHL), although increasing potency also led to increased incidence of toxi- city in more vulnerable patients, such as the elderly (Sup- plementary Table 3). Lowering the dose of FC (FCR-Lite regimens) or replacing FC with alternate agents such as bendamustine often showed more favorable toxicity pro- ﬁles, making them more suitable for vulnerable patient populations (Supplementary Table 3).

Fludarabine-induced neurotoxicity

Fludarabine is rapidly converted in plasma to F-araA, which accumulates in cells where it is phosphorylated to its active metabolite F-araATP by deoxycytidine kinase [9]. This metabolite has a half-life of approximately 20 h in vivo, and its clearance is dependent upon adequate renal function. The main dose-limiting toxicities associated with ﬂudarabine that are common among chemotherapy agents were mye- losuppression and risk of infection, which accounted for the majority of fatalities and failed outcomes. Neurological abnormalities continued to cause concern and were initially confounded by an absence of a mechanism to explain their

onset and occurrence. Somnolence and peripheral neuro- pathy during and immediately following ﬂudarabine infu- sion were frequently observed but usually reversible, and these symptoms had been described previously with other anti-metabolite drugs [22] (Supplementary Table 2). Of greater concern were the late-onset neurological symptoms (20–250 days) that manifested in a pattern of progressive visual disturbances, peripheral neuropathy, dementia, ataxia, hemiparesis, quadriparesis and blindness, sometimes leading to coma and death (Table 1). Cheson et al. [30] described these late onset symptoms to be associated spe- ciﬁcally with purine analogs. While the mechanisms of purine analog-mediated neurotoxicity are still not entirely clear, there is evidence to support the hypothesis that neu- rotoxicity results from disruption of normal synaptic func- tion. Purine analogs are known to cross the blood brain barrier (BBB), and reduced uptake into the CNS protects against neurotoxicity [45–47]. Adenosine is a major reg- ulator of neuromodulation, and purine analogs have been shown to bind to adenosine receptors [48]. Speciﬁcally, ﬂudarabine is an A1 receptor agonist [49]. The A1 receptor is expressed primarily on neurons, and agonism causes somnolence and may induce coma, while antagonism can induce seizures. The concentration at which ﬂudarabine is predicted to agonize the A1 receptor is pharmacologically relevant for patients receiving higher doses of ﬂudarabine [50].
Where autopsies or MRI could be performed, neurolo- gical abnormalities were characterized by demyelination of white matter, variable extents of necrosis, areas of enlarged astrocytes and oligodendrocytes, and multiple lesions in white matter identiﬁed by high signal by MRI [21–23, 29, 30, 34, 36, 38, 42, 43] (Table 1). Evidence of JC virus in cerebrospinal ﬂuid or brain biopsies supported the diagnosis of the leukoencephalopathy (LE) or progressive multifocal leukoencephalopathy (PML) [32, 34, 36–39, 44] as a contributing factor to purine analog neurotoxicity. JC virus infects over 70% of the population and only becomes pathological in immunosuppressive environments, again increasing susceptibility to the elderly and patients with advanced disease. The virus infects oligodendrocytes and astrocytes leading to irreparable degeneration [34]. In the case of CLL, 90% of PML diagnoses since 1990 occurred in patients treated with purine analogs, such as ﬂudarabine [51].
In summary, ﬂudarabine demonstrated enhanced potency against hematological malignancies compared to alternative agents but was associated with a distinct set of late onset, progressive neurological symptoms in isolated cases. Neu- rological abnormalities were more prominent at high doses and in more vulnerable patients, such as those with advanced disease or elderly patients that often harbor renal insufﬁciency, thereby heightening ﬂudarabine exposure.

Dose reduction of ﬂudarabine is associated with a reduction of these toxicities in patients.

Development of lymphodepleting pre- conditioning regimens for ASCT

Despite increasing response rates with new combination- based chemotherapy regimens, patients with advanced hematological malignancies gained little survival advantage until the emergence of ASCT as the ﬁrst potentially curative treatment strategy [1, 52, 53]. Successful engraftment and durability of cells relied heavily on conditioning the host immune system to “create room” by increasing the levels of homeostatic cytokines including IL-7 and IL-15 and diminishing regulatory mechanisms [54, 55].
Myeloablative pre-conditioning regimens were asso- ciated with high risk of toxicity and were considered unsuitable in vulnerable patients [56]. While non- myeloablative regimens reduced toxicity, durability of remission was compromised, leading to the development of reduced intensity conditioning (RIC) regimens that char- acteristically included a nucleoside analog, such as ﬂudar- abine, in combination with an alkylating agent, such as melphalan or busulfan [57–59] (Table 2).
In clinical trials, ﬂudarabine-melphalan (Flu-M) RIC proved superior to FC in increasing engraftment of ASCT and prolonging disease-free survival [53, 59–62] (Table 2). However, non-relapse mortality (NRM) rates that largely resulted from acute graft-vs.-host disease (GVHD) remained high, often correlating with starting disease bur- den [58, 59, 61]. Efforts to enhance cytotoxic activity of pre-conditioning regimens led to trials replacing ﬂudarabine for a more active nucleoside analog, clofarabine, increasing durability [63–68]. However, RIC regimens consisting of Flu-M, Flu-busulfan, clofarabine-M or clofarabine-busulfan resulted in similar progression-free survival (PFS) and NRM rates across malignancies, making it difﬁcult to dis- sect optimal treatment strategies [58, 61, 65–77]. Irrespec- tive of individual combinatorial approaches, pre- conditioning regimens containing nucleoside analogs were associated with increased engraftment and durability of transferred cells.

Fludarabine-associated neurotoxicity in ASCT

A retrospective analysis of patients receiving ﬂudarabine- containing pre-conditioning for hematopoietic cell trans- plantation showed a 2−3% incidence of grade 3 or higher neurologic events, manifesting in a similar pattern of symptoms to those observed previously from ﬂudarabine

alone, from somnolence during infusion, headaches, blurred vision to blindness, seizure, dementia, cognitive decline and paralysis [78]. These chemotherapy-induced LEs can be characterized by MRI as being posterior reversible ence- phalopathy syndrome (PRES), acute toxic leukoencepha- lopathy (ATL), or other leukoencephalophathy (OLE), which is similar to ATL but with lesser white matter changes. PRES is associated with better survival outcomes than ATL. The timing of events tended to plateau at one- month post treatment. A low incidence of neurological events were also recorded in studies reviewed here using either ﬂudarabine or clofarabine preconditioning [43, 58, 63, 64, 71, 74, 78, 79] (Table 2). In a speciﬁc case study, neurological symptoms were described to be consistent with LE [43]. Beitinjaneh et al. [78] performed a comprehensive analysis of CNS toxicity associated with ﬂudarabine pre- conditioning for ASCT. Distinct stages of CNS disease were categorized by MRI and correlated with different forms and severities of LE that inﬂuenced survival. Notably, cerebral edema was not described in the imaging ﬁndings. The exact occurrence of neurological toxicity following ASCT is likely masked by short follow-up times resulting from disease progression, incidence of GVHD or transition to other treatment regimens.

Lymphodepletion in adoptive T-cell therapy

TILs

Following the clinical success of using preconditioning regimens to support engraftment of ASCT for the treatment of both hematological and solid malignancies, the same principle was applied to ACT of TILs for the treatment of metastatic melanoma [80, 81]. Dudley et al. [13, 82] applied FC non-myeloablative conditioning to ACT of TILs cul- tured from patients with metastatic melanoma and demon- strated enhancement of anti-tumor activity (Table 3). A series of studies expanded on these observations, including combining FC with total body irradiation (supplemented with hematopoietic stem cells) to induce more potent lym- phodepletion, resulting in higher objective responses [83– 87]. The rationale for chemotherapy preconditioning and the mechanisms by which it enhances anti-tumor activity are multi-pronged, including the reduction of immunosup- pressive cell populations (e.g. regulatory T cells and myeloid-derived suppressor cells), an increase in tumor antigen presentation by inducing cell death, a reduction of sinks for the T-cell proliferative cytokines IL-7 and IL-15 and an improvement in the reactivity of the adoptively transferred cells [10, 88, 89].
One major caveat of ACT using TILs is that many cancers are unsuitable for TIL extraction, and the process is

Table 2 Results from clinical trials using reduced intensity conditioning regimens prior to ASCT

References Year No. of patients Disease Pre-conditioning regimen Engraftment success (%) PFS (%);
Years (Yr) NRM (%); Years (Yr)/Days (d)
Giralt et al. [59]
2001 78 HMs Flu-M 80−100 53; 1Yr 37; 100d
van Besien et al. [53]
2003 31 HMs Flu-M 100 33; 1Yr 23; NS
Schetelig et al. [77]
2003 30 CLL Flu-Busulfan 93 67; 2Yr 15; 2Yr
Morris et al. [58]
2004 88 NHL Flu-M- Alemtuzumab 97 50; 3Yr 29; 100d
Anderlini et al. [60]
2005 14 NHL FC 69 21; 1.5Yr 5; 1.5Yr
26 NHL Flu-M 100 37; 1.5Yr 22; 1.5Yr
Brown et al. [75]
2006 46 CLL Flu-Busulfan >75 34; 2Yr 15; 2Yr
Delgado et al. [76]
2006 24 CLL Flu-M- Alemtuzumab 85 45; 2Yr 26; 2Yr
Oran et al. [61]
2007 112 AML & MDS Flu-M 82 15; 2Yr 54;>2Yr
Shimoni et al. [74]
2007 72 HMs Flu-Busulfan 97 72; 2Yr 16; 2Yr
79 HMs Flu-M 99 36; 2Yr 40; 2Yr
Anderlini et al. [62]
2008 58 NHL Flu-M NS 32; 2Yr 15; 2Yr
Valcarcel et al. [69]
2008 93 AML & MDS Flu-Busulfan NS 43; 4yr 16; 1Yr
Lee et al.C [43] 2010 2 AML FC − − 2a

Beitinjaneh et al. [78]
2011 1596 HMs Flu (+various agents) NS NS NS
Santarone et al. [70]
2011 44 ALL Flu-Busulfan 100 63; 2Yr 18; 2Yr
Kebriaei et al. [71]
2012 51 ALL Clo-Busulfan 94 54; 1Yr 6; 100d
van Besien et al. [64]
2012 72 HMs Clo-M NS 45; 1Yr 26; 1Yr
Kirschbaum et al. [63]
2012 14 HMs Clo-M 100 61;>1Yr 21;<1Yr
Baron et al. [73]
2015 218 AML Flu-Busulfan 99 53; 2Yr 18; 2Yr
176 Flu-M 100 60; 2Yr 20; 2Yr
Annalaro et al.C [79] 2015 1 Myeloﬁbrosis Flu CR − 1a

Rambaldi et al. [72]
2015 125 AML Cyc-Busulfan NS NS 17.2; 1Yr
127 AML Flu-Busulfan NS NS 7.9; 1Yr
El-Jawahri et al. [66]
2016 33 AML, ALL and MDS Clo-Busulfan 100 50; 2Yr 24; 1Yr
Alatrash et al. [67]
2016 70 AML, CML & MDS Clo-Flu-Busulfan 100 0.9Yrb 4; 100d
Chevallier et al. [65]
2016 316 AML & MDS Flu-Busulfan NS 51.1; 2Yr 17.3; 2Yr
39 AML & MDS Clo-Busulfan NS 61.5; 2Yr 10.3; 2Yr
Kebriaei et al. [68]
2017 107 ALL Clo-Busulfan NS 44; 2Yr 34; 2Yr
Incidence of NRM were primarily related to opportunistic infections and GVHD-related morbidities, including mucosal, hepatic, pulmonary, renal, cardiac, skin and gastrointestinal
PFS progression-free survival, NRM non-relapse mortality, NS not stated, C case study, HMs hematological malignancies, CLL chronic lymphocytic leukemia, NHL non-Hodgkin’s lymphoma, AML acute myeloid leukemia, MDS myelodysplastic syndromes, ALL acute lymphoblastic leukemia, CML chronic myeloid leukemia, Flu ﬂudarabine, M melphalan, FC ﬂudarabine-cyclophosphamide, Clo clofarabine
aIndividual instances of NRM
bMedian PFS

both cost and labor intensive [90]. As a result, a search for a more widely applicable ACT methodology has continued, which led to the emergence of genetically modiﬁed lym- phocytes expressing tumor-targeting receptors in the form of TCRs or CARs.

Engineered TCR-transduced T cells

TCR engineered T cells are attractive because the TCR recognizes the HLA-peptide complex, and the presented

peptides are derived from both membrane and intracellular proteins. This enables TCRs to recognize virtually any protein in the cell, which is useful when engineering spe- ciﬁcity for tumor recognition. Afﬁnity optimization of tumor antigen-speciﬁc TCR-transduced T cells offers sig- niﬁcant advantages over natural afﬁnity TCRs, since the majority of tumor antigens are self-antigens, thus rendering the naturally occurring repertoire of TCRs to be low afﬁnity due to thymic selection mechanisms [91]. Afﬁnity optimi- zation has been shown to improve the potency and

Table 3 Results from clinical trials using ACT of TILs with or without pre-conditioning regimens

References Year No. of patients Disease Therapeutic regimen CR/ORR (%) PFS >1
year Neurotoxicity
≤Grade 3 Neurotoxicity
≥Grade 4
Rosenberg et al. [80]
1988 20 MM IL-2 −; 55 − − −
Rosenberg et al. [81]
1994 29 MM IL-2 −; 31 − − −
Rosenberg et al. [81]
1994 57 MM FC + IL-2 −; 35 − − −
Dudley et al. [13]
2002 13 MM FC + IL-2 −; 46 − − −
Dudley et al. [82]
2005 35 MM FC + IL-2 9; 51 6% − 1
Dudley et al. [83]
2008 43 MM FC + IL-2 9.3; 48 9% − −
Dudley et al. [83]
2008 25 MM FC + TBI + IL-2 16; 72 12% 4 −
Rosenberg et al. [84]
2011 43 MM FC + IL-2 12; 49 12% − −
Rosenberg et al. [84]
2011 25 MM FC + TBI + IL-2 40; 72 40% − −
Pilon-Thomas et al. [86]
2012 13 MM FC + IL-2 15; 26 NS − −
Besser et al. [87]
2013 54 MM FC + IL-2 9; 40 9% 4 -
Goff et al. [85]
2016 50 MM FC + IL-2 24; 45 24% − −
Goff et al. [85]
2016 51 MM FC + TBI + IL-2 24; 54 24% − −
CR complete response, ORR overall response rate, PFS progression-free survival, MM metastatic melanoma, TIL tumor-inﬁltrating lymphocyte,
IL-2 interleukin-2, FC ﬂudarabine-cyclophosphamide, TBI today body irradiation

functionality of TCR engineered T cells, which is necessary to recognize immune-selected tumors [92]. TCR-transduced T cells utilize the natural T-cell signaling infrastructure and therefore maintain the inherent capability to induce phy- siologically appropriate levels of T-cell activation, costi- mulation, expansion, memory cell formation and APC interactions [93].
Several TCR engineered T-cell therapy studies have been carried out, including both natural and afﬁnity-optimized TCRs [94]. The antigens MART-1 and gp100 were identi- ﬁed as melanoma-speciﬁc antigens during the course of ACT clinical trials with TILs and became the ﬁrst targets for TCR-transduced T-cell therapy [95–97]. In the majority of these studies, an FC regimen was used for preconditioning. In the ﬁrst application of ACT using TCR-transduced T cells for the treatment of metastatic melanoma, response rates were low despite preparative lymphodepletion [95] (Table 4). Efforts to enhance the afﬁnity of TCRs to their target antigen, MART-1, reﬂected positively in response rates but simultaneously raised issues of enhanced on target, off tumor toxicity, with patients experiencing non-lethal adverse reactions in the skin, eyes and ears due to Mart-1 expression in the cells in those tissues [97]. Subsequent studies expanded into non-melanoma tumors, targeting the NY-ESO-1 cancer testis antigen, with promising results in melanoma and synovial sarcoma [15, 16, 98]. In a TCR- transduced T-cell therapy targeting the cancer testis antigen MAGE-A3, two patients experienced lethal neurological abnormalities said to result from cross-reactivity to other members of the MAGE family that were expressed in brain [99]. A different MAGE-A3 TCR also showed off tumor
−off target toxicity in recognition of the cardiomyocyte

protein titin, resulting in two patient deaths [100]. In no cases were these adverse events related to use of ﬂudarabine in the preconditioning regimen. Dose reduction of ﬂudar- abine is under investigation in a pilot study in synovial sarcoma, as this may reduce other ﬂudarabine-related toxi- cities such as prolonged neutropenia and bone marrow failure [16]. In this same study, removal of ﬂudarabine appeared to have a negative effect on efﬁcacy.
In summary, the incidence of neurotoxicity appears to be lower with TCR engineered ACT studies, and ﬂudarabine appears to play role in enhancing ACT potency.

CAR-T cells

CAR-T cells are engineered with synthetic receptors com- prised of an antibody single chain variable fragment (scFv) speciﬁc for a cell surface protein, a transmembrane domain, and intracellular signaling domains [101]. Second- generation CARs incorporate various T-cell costimulatory activation domains such as CD137 (4-1BB) and CD28 to enhance expansion and persistence of the CAR-T cells [102, 103]. Signaling mechanisms are likely to be different with CARs than with TCRs, which harness the natural immu- nologic synapse, and whether this may lead to differences in the toxicity proﬁles of the two modalities is an area of active investigation.
As with TIL and TCR-transduced T-cell therapy, preparative lymphodepleting regimens using mainly cyclophosphamide and ﬂudarabine were used to increase efﬁcacy and durability of adoptively transferred CAR-T cells [104–124] (Table 5). In mouse tumor models and human studies, lymphodepletion by cyclophosphamide was

critical for CD19 CAR-T-cell engraftment and persistence [106, 125]. Fludarabine enhanced in vivo persistence of adoptively transferred T cells 2.9 fold, in part by reducing starting tumor burden prior to ACT [106, 126]. Considered here are clinical trials using second-generation CAR-T cells engineered to target B-cell-related hematological malig- nancies through the CD19 or CD20 surface B-cell antigens. Overall, response rates were much higher than conventional chemotherapy, other ACT methods, and the bispeciﬁc anti- CD19 BiTE blinatumomab [127] (Table 5).
Few studies have directly compared pre-conditioning regimens; however, trials that did not use pre-conditioning gave rise to substantially lower response rates and PFS [106, 107, 110] (Table 5). Turtle et al. [120] used both cyclophosphamide and FC pre-conditioning regimens and showed that FC improved CAR–T-cell persistence and disease-free survival compared to cyclophosphamide alone. Clear differences in toxicities between lymphodepleting regimens were not described. While enhanced lymphode- pletion correlated with increased efﬁcacy and durability of CAR-T cells, deeper conditioning can promote enhanced initial expansion of CAR-T, which along with cell dose and tumor burden, is known to increase the frequency of severe cytokine release syndrome (CRS).
CRS is a systemic inﬂammatory response caused by high levels of inﬂammatory cytokines that potentiate T-cell activation and proliferation, and its occurrence in the con- text of engineered T-cell therapy has a unique algorithm for accurate diagnosis, classiﬁcation and treatment [128, 129]. CRS can be associated with a broad range of symptoms, often mimicking infection or sepsis. Typical presenting symptoms include fever, anorexia, nausea, fatigue and myalgia/arthralgia, and symptoms can progress to more serious life-threatening complications, including hypoten- sion, capillary leak, and hypoxia. More severe cases can be associated with organ toxicity such as cardiac dysfunction, adult respiratory distress syndrome, and neurologic invol- vement [118]. In most cases, symptoms of CRS can be effectively managed with supportive care and by blocking the trans IL-6 signaling mechanism through the IL-6 receptor blocking antibody tocilizumab or the IL-6 block- ing antibody situximab, and earlier administration provides more effective management of symptoms. Tociluzimab is generally considered not to reduce initial anti-tumor activ- ity, although more research is needed to determine what effect blocking IL-6 activity has on CAR-T cell durability [122]. CRS observed in TCR studies has been less frequent and less severe than in CAR-T studies, which may be due to differences in T-cell signaling, tumor target density, and tumor burden and accessibility [16].
We described earlier a distinct set of late-onset neuro- logical symptoms associated with ﬂudarabine therapy and other purine nucleoside analogs. Neurological events

Table 5 Results from clinical trials using CAR-T-cell therapy with or without pre-conditioning regimens

References Year No. of patients Disease CAR Pre-conditioning regimen ORR (%) PFS >1
year Neurotoxicity
≤Grade 3 Neurotoxicity
≥Grade 4
Till et al. [124]
2008 9 FL or MCL CD20 Various 42 14 − −
Kochenderfer et al.c [104]
2010 1 FL CD19:CD28+IL-2 FC PR Y − −
Porter et al.c [105]
2011 1 CLL CD19:CD137 Pentostatin-cyclophosphamide CR N − -
Brentjens et al. [106]
2011 3 CLL or B-ALL CD19:CD28 − − − − −
Brentjens et al. [106]
2011 4 CLL or B-ALL CD19:CD28 Cyclophosphamide − − − −
Savoldo et al. [107]
2011 6 NHL CD19:CD28 − − − NS NS
Kalos et al. [108]
2011 3 CLL CD19:CD137 Bendamustine, bendamustine & rituximab, pentostatin-cyclophosphamide 100 − − −
Kochenderfer et al. [109]
2012 8 B-cell malignancies CD19:CD28+IL-2 FC 88 38 − −
Cruz et al. [110]
2013 8 B-cell malignancies CD19:CD28 (VST) − 25 − NS NS
Brentjens et al. [111]
2013 5 B-ALL CD19:CD28 Cyclophosphamide 100 NS − −
Grupp et al. [112]
2013 2 B-ALL CD19:CD137 Cyclophosphamide 100 50 1 −
Davila et al. [113]
2014 16 B-ALL CD19:CD28 Cyclophosphamide 88 NS 3 3
Maude et al. [114]
2014 30 B-ALL CD19:CD137 FC 90 67 13 8
Park et al. [115]
2015 43 B-ALL CD19:CD28 Cyclophosphamide 84 16 NS NS
Kochenderfer et al. [116]
2015 15 B-cell malignancies CD19:CD28 FC 80 46 3 1
Lee et al. [117]
2015 19 B-cell malignancies CD19:CD28 FC 68 NS 7 −
Porter et al. [118]
2015 14 CLL CD19:CD137 FC, cyclophosphamide-pentostatin or bendamustine 57 29 6 1
Dai et al. [119]
2015 9 B-ALL CD19:CD137 (autologous) C-MOAD/- 44 − 1 1
Turtle et al. [120] 2016 12 B-ALL CD19:CD137 Cyclophosphamide 83 8 15a 6a
Turtle et al. [120]
2016 17 B-ALL CD19:CD137 FC 95 65
Turtle et al. [17]
2017 24 CLL CD19:CD28:4-1BB:CD3ζ Cyclophosphamide, ﬂudarabine, or FC 71 − 7 1
Gardner et al.A [121]
2016 43 B-ALL (Pediatric) CD19:CD28:4-1BB Cyclophosphamide-FC 93 51 21 10
Brudno et al. [122]
2016 20 B-cell malignancies CD19:CD28 − 40 NS − −
Maude et al.A [123]
2016 30 B-ALL CD19:CD137 FC 87 NS 9 9
All patients had undergone previous treatment. All studies report varying degrees of infection and symptoms of cytokine release syndrome, including hepatic, pulmonary, renal, cardiac and skin abnormalities as well as fever, vascular leak, hypotension and hypoxia
c case study, A abstract, CR complete response, ORR overall response rate, PFS progression-free survival, IL-2 interleukin-2, NHL non-Hodgkin’s lymphoma, CLL chronic lymphocytic leukemia, HL Hodgkin’s lymphoma, FL follicular lymphoma, MCL mantle cell lymphoma, FC ﬂudarabine-cyclophosphamide, VST virus-speciﬁc T cell, C-MOAD methotrexate, rituximab, vincristine, pegylated L-asparaginase and dexamethasone
aNot detailed whether patients treated with cyclophosphamide or FC

reported following CD19-targeted CAR-T-cell therapy, recently named CAR-T-cell-related encephalopathy syn- drome (CRES), manifest in a pattern distinct from that previously described with ﬂudarabine, occurring within the ﬁrst week of therapy. Several groups reported diagnoses of encephalopathy associated with symptoms of delirium, confusion and hallucinations that occurred soon after CAR- T cell transfer. Symptoms in many cases were severe but reversible [113, 114, 116–118, 120] (Table 5). Factors speculated to contribute to the onset of neurological events include circulating CAR-T-cell concentration and the onset of CRS [117]. Studies identiﬁed a correlation between the severity of CRS, the intensity of lymphodepletion and starting tumor burden [111, 113]. In one study, a high incidence of grade 3 or above neurotoxicity (50%) was reported using CAR-T cells for treatment of B-ALL. Peak levels of IL-6, IFN-γ, ferritin, and CRP were signiﬁcantly higher in patients who developed neurotoxicity. Further- more, six of six patients with high tumor burden and also treated with higher cell doses developed severe neurotoxi- city [120]. In an analysis of the incidence and grade of neurotoxicity, CRS, and correlative biomarkers in blood and CSF in adult patients with relapsed or refractory B- ALL, severe neurotoxicity correlated with pre-infusion disease burden and peak CAR-T-cell expansion in the blood. Neurotoxicity grade correlated with elevations of CSF protein level as well as elevated cytokines IL6, IL8, IL10, IFNγ and G-CSF in CSF over serum at the time of neurotoxicity [130]. These factors suggest a causal role of CRS in the development of neurotoxicity.
CRES is often biphasic, with an initial phase occurring during CRS, and a second phase occurring days later. This second phase may be why some neurological events do not correlate with cytokine levels or CRS. Treatment algorithms for CRES include blockade of IL-6 signaling as well as systemic steroids; however, neurotoxicity has not been consistently responsive to these therapies, making it difﬁ- cult to elucidate whether early identiﬁcation and treatment of CRS will be effective in treating neurological symptoms [116, 120, 123, 129]. The presentation, time course, pro- posed monitoring, and treatment algorithm for CRES was recently reported by Neelapu et al. [129].
In a recent clinical trial, JCAR-015 (NCT02535364), investigating the application of CD19 CAR-T cells for the treatment of B-ALL, two patient deaths due to cerebral edema resulted in the trial being suspended, pending investigation. This ﬁnding was not previously reported in CAR-T studies. The cerebral edema was initially thought to be associated with the addition of ﬂudarabine to the treat- ment cohort. Fludarabine was subsequently removed from the preconditioning regimen in the trial; however, two fur- ther patient deaths reported shortly after the trial was re- initiated indicated that ﬂudarabine was not the causal agent

for the fatal cerebral edema seen in the trial. The trial has since been suspended [131]. Another study, ZUMA-1 (NCT02348216), reported a similar incident, where a patient with advanced refractory NHL experienced cerebral edema following CD19 CAR-T-cell infusion after FC pre- conditioning, resulting in lethality [132]. This event was an isolated incident, and the trial continued (now complete). However, the CAR constructs engineered for both of these studies share the same CD28 signaling domain, which has been reported to result in greater T-cell expansion [133]. This may contribute to the CRS toxicity and thus neuro- toxicity. Investigations into the pathophysiology of these events have recently been reported and include an early endothelial activation event mediated by the CAR-T cells. This initiates a cascade of coagulopathy concomitant with increased BBB permeability. Cytokines produced during CRS, as well as the CAR-T cells, then trafﬁc into the CNS, inducing neurotoxicity [134].
The contribution of the target antigen to neurological abnormalities should be considered. We previously noted the incidence of neurological toxicity within days following adoptive transfer of T cells transduced with high afﬁnity TCRs against the MAGE-A3 cancer testis antigen, which also cross-reacted with the MAGE-A9 and MAGE-A12 antigens that can be expressed in the CNS. Two patients exhibited lethal global encephalopathy characterized by white matter abnormalities, vacuolation and edema by MRI [99]. Although the cause of toxicity was attributed to cross- reactivity with another MAGE family member, the impact of lymphodepletion on the exposure of these potent mole- cules was not assessed. The contribution of a speciﬁc antigen is unique in each case and relates to the level of antigen expression, highlighting the need for thorough research into antigen expression proﬁles of antigen and related protein sequences.
The spotlight on CD19 CAR-T toxicity is driven by the success and potency of these molecules in the clinic. Interestingly, early results in a primate model suggest that similar neurotoxicity occurs following treatment with CD20 CAR-T cells [135]. In a small number of non-human pri- mates, CRS and neurotoxicity were recapitulated following infusion of CD20 CAR-T cells in healthy animals following preconditioning with cyclophosphamide alone. The autopsy of the animals showed extensive inﬁltration into the brain of CD20+ CAR-T cells [135]. This increased CNS inﬁltration may be due to the CAR-T cells reacting to naturally traf- ﬁcking B cells in the brain parenchyma [136]. Furthermore, CD19 CAR-T cells have been identiﬁed in the CSF of some subjects with neurotoxicity and the extent of inﬁltration correlates with severity in some but not all studies [117, 119, 130]. Neurotoxicity with encephalopathic symptoms has also been described with the bispeciﬁc anti-CD19 BiTE blinatumomab. Despite the recently described

pathophysiology of CD19 CAR-T-mediated neurotoxicity described earlier, the signiﬁcance of CD19 or other antigens on the mechanism of neurological toxicity cannot be excluded and will only be further understood with con- tinuing evaluation of affected subjects and as increasing numbers of CAR-T cells targeting different antigens are tested for a range of malignancies.

Conclusions

The early onset neurological symptoms observed following CD19 CAR-T-cell therapy that include encephalopathy and more rarely cerebral edema appear to be driven by a distinct set of mechanisms from those that drive late-onset neuro- logical toxicity associated directly with ﬂudarabine. To our knowledge, late-onset neurotoxicity associated with ﬂudar- abine has not been reported in ACT studies. There should be careful monitoring for this potential toxicity, particularly in patients with increased sensitivity to ﬂudarabine, such as heavily pre-treated patients or patients with renal insufﬁciency.
The mechanisms that induce early onset cerebral edema are just beginning to be elucidated. CRES severity is highly correlated with CRS severity, which is known to be related to expansion of CAR-T cells in vivo. As suspected then, risk factors for CRES include tumor burden and the depth of preconditioning, as both of these factors affect the expansion of infused T cells. This may explain why the ﬁrst cerebral edema events on the JCAR015 study were seen once ﬂu- darabine was added to the preconditioning regimen, as it leads to a more profound lymphopenia. Presence of the target in the CNS (e.g. on B cells circulating through the CNS) may also contribute to CRES. CRES is a common neurotoxic side effect in CAR-T-cell studies, and similar side effects appear to be less frequent and less severe in TCR and TIL studies. Although encephalopathy can be severe, complete recovery is typical. A second neurological toxicity, cerebral edema, has only rarely been observed in CAR-T- cell studies, has not been observed to date in TCR T cell studies, and is acutely life threatening. It now appears that cerebral edema is an extreme manifestation of CRES. The recent elucidation of the mechanism behind this toxicity and components of the etiology with other described medical events, such as thrombotic thrombocytopenic purpura and malaria-associated neurologic dysfunction, suggest approa- ches for therapeutic intervention [137]. Early identiﬁcation of certain biomarkers and careful management of CRS may help reduce side effects of neurotoxicity [129]
Fludarabine is an important component of the pre- conditioning regimen for effective ACT. Consideration of the intensity of ﬂudarabine preconditioning should include reduction of dose for patients with known risk factors for

ﬂudarabine toxicity, as described in the product label. Next generation engineered T-cell therapies that include approa- ches to circumvent the requirement for preconditioning may lead to ACT regimens with improved tolerability. Until then, ﬂudarabine continues to be an important component of the preconditioning regimen for supporting the potency of ACT. At doses used in this context, the evidence to date does not support a direct role for ﬂudarabine in initiation or exacerbation of CRES.

Compliance with ethical standards
Conﬂict of interest KLL is an employee of Immunocore; EN, RA, BKJ and GB are employees of Adaptimmune. CLM is a member of the Parker Institute for Cancer Immunotherapy, which supports the Stan- ford University Cancer Immunotherapy Program.

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References

1. Mathe G, Amiel JL, Schwarzenberg L, Cattan A, Schneider M, Devries MJ, et al. Successful Allogenic Bone Marrow Trans- plantation in Man: Chimerism, Induced Speciﬁc Tolerance and Possible Anti-Leukemic Effects. Blood. 1965;25:179–96.
2. Horowitz MM, Gale RP, Sondel PM, Goldman JM, Kersey J, Kolb HJ, et al. Graft-versus-leukemia reactions after bone mar- row transplantation. Blood. 1990;75:555–62.
3. DeVita VT Jr., Chu E. A history of cancer chemotherapy. Cancer Res. 2008;68:8643–53.
4. Chun HG, Leyland-Jones B, Cheson BD. Fludarabine phosph/ ate: a synthetic purine antimetabolite with signiﬁcant activity against lymphoid malignancies. J Clin Oncol. 1991;9:175–88.
5. Srinivasan R, Takahashi Y, McCoy JP, Espinoza-Delgado I, Dorrance C, Igarashi T, et al. Overcoming graft rejection in heavily transfused and allo-immunised patients with bone mar- row failure syndromes using ﬂudarabine-based haematopoietic cell transplantation. Br J Haematol. 2006;133:305–14.
6. George B, Mathews V, Shaji RV, Srivastava V, Srivastava A, Chandy M. Fludarabine-based conditioning for allogeneic stem cell transplantation for multiply transfused patients with Fanco- ni’s anemia. Bone Marrow Transplant. 2005;35:341–3.
7. Dhere V, Edelman S, Waller EK, Langston A, Graiser M, Connolly EC, et al. Myeloablative busulfan/cytoxan condition- ing versus reduced-intensity ﬂudarabine/melphalan conditioning for allogeneic hematopoietic stem cell transplant in patients with acute myelogenous leukemia. Leuk Lymphoma 2017;59:1–7.
8. Fidias P, Chabner BA, Grossbard ML. Purine Analogs for the Treatment of Low-Grade Lymphoproliferative Disorders. Oncologist. 1996;1:125–39.

9. Gandhi V, Plunkett W. Cellular and clinical pharmacology of
ﬂudarabine. Clin Pharmacokinet. 2002;41:93–103.
10. Gattinoni L, Finkelstein SE, Klebanoff CA, Antony PA, Palmer DC, Spiess PJ, et al. Removal of homeostatic cytokine sinks by lymphodepletion enhances the efﬁcacy of adoptively transferred tumor-speciﬁc CD8 + T cells. J Exp Med. 2005;202:907–12.
11. Klebanoff CA, Khong HT, Antony PA, Palmer DC, Restifo NP. Sinks, suppressors and antigen presenters: how lymphodepletion enhances T cell-mediated tumor immunotherapy. Trends Immunol. 2005;26:111–7.
12. Grifﬁth KD, Read EJ, Carrasquillo JA, Carter CS, Yang JC, Fisher B, et al. In vivo distribution of adoptively transferred indium-111-labeled tumor inﬁltrating lymphocytes and periph- eral blood lymphocytes in patients with metastatic melanoma. J Natl Cancer Inst. 1989;81:1709–17.
13. Dudley ME, Wunderlich JR, Yang JC, Hwu P, Schwartzentruber DJ, Topalian SL, et al. A phase I study of nonmyeloablative chemotherapy and adoptive transfer of autologous tumor antigen-speciﬁc T lymphocytes in patients with metastatic mel- anoma. J Immunother. 2002;25:243–51.
14. Riaz IB, Zahid U, Kamal MU, Husnain M, McBride A, Hua A, et al. Anti-CD 19 and anti-CD 20 CAR-modiﬁed T cells for B- cell malignancies: a systematic review and meta-analysis. Immunotherapy. 2017;9:979–93.
15. Rapoport AP, Stadtmauer EA, Binder-Scholl GK, Goloubeva O, Vogl DT, Lacey SF, et al. NY-ESO-1-speciﬁc TCR-engineered T cells mediate sustained antigen-speciﬁc antitumor effects in myeloma. Nat Med. 2015;21:914–21.
16. Mackall C, Tap WD, Glod J, Druta M, Chow WA, Araujo DM, Grupp SA, Van Tine BA, Chagin K, Van Winkle E, Kari G, Trivedi T, Norry E, Holdich T, Bartlett-Pandite AN, Amado RG, D’Angelo SP. Open label, non-randomized, multi-cohort pilot study of genetically engineered NY-ESO-1c259T in HLA-A2+ patients with synovial sarcoma (NCT01343043). J Clin Oncol. 2017;35(15 suppl):p3000.
17. Turtle CJ, Hay KA, Hanaﬁ LA, Li D, Cherian S, Chen X, et al. Durable molecular remissions in chronic lymphocytic leukemia treated with CD19-speciﬁc chimeric antigen receptor-modiﬁed T cells after failure of ibrutinib. J Clin Oncol: Off J Am Soc Clin Oncol. 2017;35:3010–20.
18. Juno Therapeutics places JCAR015 phase II ROCKET trial on clinical hold [press release]. 11/23/16. 2016.
19. Locke FL, Neelapu SS, Bartlett NL,Siddiqi T, Chavez JC,Hosing CM, et al. Phase 1 clinical results of the ZUMA-1 (KTE-C19- 101) study: a phase 1-2 multi-center study evaluating the safety and efﬁcacy of anti-CD19 CAR T cells (KTE-C19) in subjects with refractory aggressive non-Hodgkin lymphoma (NHL). Blood. 2015;126:3991.
20. Malspeis L, Grever MR, Staubus AE, Young D. Pharmacoki- netics of 2-F-ara-A (9-beta-D-arabinofuranosyl-2-ﬂuoroadenine) in cancer patients during the phase I clinical investigation of ﬂudarabine phosphate. Semin Oncol. 1990;17(5Suppl 8): 18–32.
21. Warrell RP Jr., Berman E. Phase I and II study of ﬂudarabine phosphate in leukemia: therapeutic efﬁcacy with delayed central nervous system toxicity. J Clin Oncol. 1986;4:74–9.
22. Spriggs DR, Stopa E, Mayer RJ, Schoene W, Kufe DW. Flu- darabine phosphate (NSC 312878) infusions for the treatment of acute leukemia: phase I and neuropathological study. Cancer Res. 1986;46:5953–8.
23. Chun HG, Leyland-Jones BR, Caryk SM, Hoth DF. Central nervous system toxicity of ﬂudarabine phosphate. Cancer Treat Rep. 1986;70:1225–8.
24. Merkel DE, Grifﬁn NL, Kagan-Hallet K, Von Hoff DD. Central nervous system toxicity with ﬂudarabine. Cancer Treat Rep. 1986;70:1449–50.

25. Weiss GR, Crowley J, Von Hoff DD, Taylor SA, Belt RJ, Coltman CA Jr., et al. Phase II study of ﬂudarabine phosphate for the treatment of advanced non-small cell carcinoma of the lung: a Southwest Oncology Group Study. Cancer Treat Rep. 1986;70:1123–4.
26. Rainey JM, Hill JB, Crowley J. Evaluation of ﬂudarabine phosphate in small cell carcinoma. A Southwest Oncology Group Study. Invest New Drugs. 1988;6:45–6.
27. Hochster HS, Kim KM, Green MD, Mann RB, Neiman RS, Oken MM, et al. Activity of ﬂudarabine in previously treated non-Hodgkin’s low-grade lymphoma: results of an Eastern Cooperative Oncology Group study. J Clin Oncol. 1992;10:28–32.
28. Kornblau SM, Cortes-Franco J, Estey E. Neurotoxicity asso- ciated with ﬂudarabine and cytosine arabinoside chemotherapy for acute leukemia and myelodysplasia. Leukemia. 1993;7:378–83.
29. Cohen RB, Abdallah JM, Gray JR, Foss F. Reversible neurologic toxicity in patients treated with standard-dose ﬂudarabine phos- phate for mycosis fungoides and chronic lymphocytic leukemia. Ann Intern Med. 1993;118:114–6.
30. Cheson BD, Vena DA, Foss FM, Sorensen JM. Neurotoxicity of purine analogs: a review. J Clin Oncol. 1994;12:2216–28.
31. Johnson PW, Fearnley J, Domizio P, Goldin J, Nagendran K, Gawler J, et al. Neurological illness following treatment with ﬂudarabine. Br J Cancer. 1994;70:966–8.
32. Zabernigg A, Maier H, Thaler J, Gattringer C. Late-onset fatal neurological toxicity of ﬂudarabine. Lancet. 1994;344:1780.
33. Johnson S, Smith AG, Lofﬂer H, Osby E, Juliusson G, Emmerich B, et al. Multicentre prospective randomised trial of ﬂudarabine versus cyclophosphamide, doxorubicin, and pre- dnisone (CAP) for treatment of advanced-stage chronic lym- phocytic leukaemia. The French Cooperative Group on CLL. Lancet. 1996;347:1432–8.
34. Gonzalez H, Bolgert F, Camporo P, Leblond V. Progressive multifocal leukoencephalitis (PML) in three patients treated with standard-dose ﬂudarabine (FAMP). Hematol Cell Ther. 1999;41:183–6.
35. Sorensen JM, Vena DA, Fallavollita A, Chun HG, Cheson BD. Treatment of refractory chronic lymphocytic leukemia with ﬂu- darabine phosphate via the group C protocol mechanism of the National Cancer Institute: ﬁve-year follow-up report. J Clin Oncol. 1997;15:458–65.
36. Cid J, Revilla M, Cervera A, Cervantes F, Munoz E, Ferrer I, et al. Progressive multifocal leukoencephalopathy following oral ﬂudarabine treatment of chronic lymphocytic leukemia. Ann Hematol. 2000;79:392–5.
37. Leonard S, Hulin C, Anxionnat R, Grignon Y, Taillandier L, Vespignani H. [Multifocal progressive leukoencephalitis in a patient given ﬂudarabine for chronic lymphoid leukemia]. Rev Neurol (Paris). 2002;158:1121–3.
38. Saumoy M, Castells G, Escoda L, Mares R, Richart C, Ugarriza
A. Progressive multifocal leukoencephalopathy in chronic lym- phocytic leukemia after treatment with ﬂudarabine. Leuk Lym- phoma. 2002;43:433–6.
39. Kiewe P, Seyfert S, Korper S, Rieger K, Thiel E, Knauf W. Progressive multifocal leukoencephalopathy with detection of JC virus in a patient with chronic lymphocytic leukemia parallel to onset of ﬂudarabine therapy. Leuk Lymphoma. 2003;44:1815–8.
40. Rossi JF, van Hoof A, de Boeck K, Johnson SA, Bron D, Foussard C, et al. Efﬁcacy and safety of oral ﬂudarabine phos- phate in previously untreated patients with chronic lymphocytic leukemia. J Clin Oncol. 2004;22:1260–7.
41. Hagenbeek A, Eghbali H, Monfardini S, Vitolo U, Hoskin PJ, de Wolf-Peeters C, et al. Phase III intergroup study of ﬂudarabine phosphate compared with cyclophosphamide, vincristine, and

prednisone chemotherapy in newly diagnosed patients with stage III and IV low-grade malignant Non-Hodgkin’s lymphoma. J Clin Oncol. 2006;24:1590–6.
42. Kalita J, Patel NS, Misra UK. Magnetic resonance imaging may simulate progressive multifocal leucoencephalopathy in a patient with chronic lymphocytic leukemia after ﬂudarabine therapy. Ann Indian Acad Neurol. 2008;11:114–5.
43. Lee MS, McKinney AM, Brace JR, Santacruz K. Clinical and imaging features of ﬂudarabine neurotoxicity. J neuro- Ophthalmol. 2010;30:37–41.
44. Lejniece S, Murovska M, Chapenko S, Breiksa B, Jaunmuktane Z, Feldmane L, et al. Progressive multifocal leukoencephalo- pathy following ﬂudarabine treatment in a chronic lymphocytic leukemia patient. Exp Oncol. 2011;33:239–41.
45. Adjei AA, Dagnino L, Wong MM, Paterson AR. Protection against ﬂudarabine neurotoxicity in leukemic mice by the nucleoside transport inhibitor nitrobenzylthioinosine. Cancer Chemother Pharmacol. 1992;31:71–5.
46. Major PP, Agarwal RP, Kufe DW. Deoxycoformycin: neurolo- gical toxicity. Cancer Chemother Pharmacol. 1981;5:193–6.
47. Grever MR, Siaw MF, Jacob WF, Neidhart JA, Miser JS, Coleman MS, et al. The biochemical and clinical consequences of 2’-deoxycoformycin in refractory lymphoproliferative malig- nancy. Blood. 1981;57:406–17.
48. Ribeiro JA, Sebastiao AM, de Mendonca A. Adenosine receptors in the nervous system: pathophysiological implications. Prog Neurobiol. 2002;68:377–92.
49. Jensen K, Johnson LA, Jacobson PA, Kachler S, Kirstein MN, Lamba J, et al. Cytotoxic purine nucleoside analogues bind to A1, A2A, and A3 adenosine receptors. Naunyn-Schmiedeberg’s Arch Pharmacol. 2012;385:519–25.
50. Long-Boyle JR, Green KG, Brunstein CG, Cao Q, Rogosheske J, Weisdorf DJ, et al. High ﬂudarabine exposure and relationship with treatment-related mortality after nonmyeloablative hema- topoietic cell transplantation. Bone Marrow Transplant. 2011;46:20–6.
51. Isidoro L, Pires P, Rito L, Cordeiro G. Progressive multifocal leukoencephalopathy in a patient with chronic lymphocytic leukaemia treated with alemtuzumab. BMJ Case Reports. 2014;2014. pii: bcr2013201781.
52. Dickinson AM, Norden J, Li S, Hromadnikova I, Schmid C, Schmetzer H, et al. Graft-versus-leukemia effect following hematopoietic stem cell transplantation for leukemia. Front Immunol. 2017;8:496.
53. Van Besien K, Devine S, Wickrema A, Jessop E, Amin K, Yassine M, et al. Regimen-related toxicity after ﬂudarabine- melphalan conditioning: a prospective study of 31 patients with hematologic malignancies. Bone Marrow Transplant. 2003;32:471–6.
54. Sandau MM, Winstead CJ, Jameson SC. IL-15 is required for sustained lymphopenia-driven proliferation and accumulation of CD8 T cells. J Immunol. 2007;179:120–5.
55. Fry TJ, Connick E, Falloon J, Lederman MM, Liewehr DJ, Spritzler J, et al. A potential role for interleukin-7 in T-cell homeostasis. Blood. 2001;97:2983–90.
56. Ricci F, Tedeschi A, Morra E, Montillo M. Fludarabine in the treatment of chronic lymphocytic leukemia: a review. Ther Clin Risk Manag. 2009;5:187–207.
57. Bethge WA, Hegenbart U, Stuart MJ, Storer BE, Maris MB, Flowers ME, et al. Adoptive immunotherapy with donor lym- phocyte infusions after allogeneic hematopoietic cell transplan- tation following nonmyeloablative conditioning. Blood. 2004;103:790–5.
58. Morris E, Thomson K, Craddock C, Mahendra P, Milligan D, Cook G, et al. Outcomes after alemtuzumab-containing reduced- intensity allogeneic transplantation regimen for relapsed and

refractory non-Hodgkin lymphoma. Blood. 2004;104: 3865–71.
59. Giralt S, Thall PF, Khouri I, Wang X, Braunschweig I, Ippolitti C, et al. Melphalan and purine analog-containing preparative regimens: reduced-intensity conditioning for patients with hematologic malignancies undergoing allogeneic progenitor cell transplantation. Blood. 2001;97:631–7.
60. Anderlini P, Saliba R, Acholonu S, Okoroji GJ, Donato M, Giralt S, et al. Reduced-intensity allogeneic stem cell transplantation in relapsed and refractory Hodgkin’s disease: low transplant-related mortality and impact of intensity of conditioning regimen. Bone Marrow Transplant. 2005;35:943–51.
61. Oran B, Giralt S, Saliba R, Hosing C, Popat U, Khouri I, et al. Allogeneic hematopoietic stem cell transplantation for the treat- ment of high-risk acute myelogenous leukemia and myelodys- plastic syndrome using reduced-intensity conditioning with ﬂudarabine and melphalan. Biol Blood Marrow Transplant. 2007;13:454–62.
62. Anderlini P, Saliba R, Acholonu S, Giralt SA, Andersson B, Ueno NT, et al. Fludarabine-melphalan as a preparative regimen for reduced-intensity conditioning allogeneic stem cell trans- plantation in relapsed and refractory Hodgkin’s lymphoma: the updated M.D. Anderson Cancer Center experience. Haematolo- gica. 2008;93:257–64.
63. Kirschbaum MH, Stein AS, Popplewell L, Delioukina M, Chen R, Nakamura R, et al. A phase I study in adults of clofarabine combined with high-dose melphalan as reduced-intensity con- ditioning for allogeneic transplantation. Biol Blood Marrow Transplant. 2012;18:432–40.
64. van Besien K, Stock W, Rich E, Odenike O, Godley LA, O’Donnell PH, et al. Phase I-II study of clofarabine-melphalan- alemtuzumab conditioning for allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant. 2012; 18:913–21.
65. Chevallier P, Labopin M, de La Tour RP, Lioure B, Bulabois CE, Huynh A, et al. Clofarabine versus ﬂudarabine-based reduced-intensity conditioning regimen prior to allogeneic transplantation in adults with AML/MDS. Cancer Med. 2016;5:3068–76.
66. El-Jawahri A, Li S, Ballen KK, Cutler C, Dey BR, Driscoll J, et al. Phase II trial of reduced-intensity busulfan/clofarabine conditioning with allogeneic hematopoietic stem cell transplan- tation for patients with acute meloid leukemia, myelodysplastic syndromes, and acute lymphoid leukemia. Biol Blood Marrow Transplant. 2016;22:80–5.
67. Alatrash G, Thall PF, Valdez BC, Fox PS, Ning J, Garber HR, et al. Long-term outcomes after treatment with clofarabine+/− ﬂudarabine with once-daily intravenous busulfan as pretransplant conditioning therapy for advanced myeloid leukemia and mye- lodysplastic syndrome. Biol Blood Marrow Transplant. 2016;22:1792–800.
68. Kebriaei P, Bassett R, Lyons G, Valdez B, Ledesma C, Rondon G, et al. Clofarabine plus busulfan is an effective conditioning regimen for allogeneic hematopoietic stem cell transplantation in patients with acute lymphoblastic leukemia: long-term study results. Biol Blood Marrow Transplant. 2017;23:285–92.
69. Valcarcel D, Martino R, Caballero D, Martin J, Ferra C, Nieto JB, et al. Sustained remissions of high-risk acute myeloid leu- kemia and myelodysplastic syndrome after reduced-intensity conditioning allogeneic hematopoietic transplantation: chronic graft-versus-host disease is the strongest factor improving sur- vival. J Clin Oncol. 2008;26:577–84.
70. Santarone S, Pidala J, Di Nicola M, Field T, Alsina M, Ayala E, et al. Fludarabine and pharmacokinetic-targeted busulfan before allografting for adults with acute lymphoid leukemia. Biol Blood Marrow Transplant. 2011;17:1505–11.

71. Kebriaei P, Basset R, Ledesma C, Ciurea S, Parmar S, Shpall EJ, et al. Clofarabine combined with busulfan provides excellent disease control in adult patients with acute lymphoblastic leu- kemia undergoing allogeneic hematopoietic stem cell transplan- tation. Biol Blood Marrow Transplant. 2012;18:1819–26.
72. Rambaldi A, Grassi A, Masciulli A, Boschini C, Mico MC, Busca A, et al. Busulfan plus cyclophosphamide versus busulfan plus ﬂudarabine as a preparative regimen for allogeneic hae- mopoietic stem-cell transplantation in patients with acute mye- loid leukaemia: an open-label, multicentre, randomised, phase 3 trial. Lancet Oncol. 2015;16:1525–36.
73. Baron F, Labopin M, Peniket A, Jindra P, Afanasyev B, Sanz MA, et al. Reduced-intensity conditioning with ﬂudarabine and busulfan versus ﬂudarabine and melphalan for patients with acute myeloid leukemia: a report from the Acute Leukemia Working Party of the European Group for Blood and Marrow Transplantation. Cancer . 2015;121:1048–55.
74. Shimoni A, Hardan I, Shem-Tov N, Rand A, Herscovici C, Yerushalmi R, et al. Comparison between two ﬂudarabine-based reduced-intensity conditioning regimens before allogeneic hematopoietic stem-cell transplantation: ﬂudarabine/melphalan is associated with higher incidence of acute graft-versus-host dis- ease and non-relapse mortality and lower incidence of relapse than ﬂudarabine/busulfan. Leukemia. 2007;21:2109–16.
75. Brown JR, Kim HT, Li S, Stephans K, Fisher DC, Cutler C, et al. Predictors of improved progression-free survival after non- myeloablative allogeneic stem cell transplantation for advanced chronic lymphocytic leukemia. Biol Blood Marrow Transplant: J Am Soc Blood Marrow Transplant. 2006;12:1056–64.
76. Delgado J, Thomson K, Russell N, Ewing J, Stewart W, Cook G, et al. Results of alemtuzumab-based reduced-intensity allogeneic transplantation for chronic lymphocytic leukemia: a British Society of Blood and Marrow Transplantation Study. Blood. 2006;107:1724–30.
77. Schetelig J, Thiede C, Bornhauser M, Schwerdtfeger R, Kiehl M, Beyer J, et al. Evidence of a graft-versus-leukemia effect in chronic lymphocytic leukemia after reduced-intensity con- ditioning and allogeneic stem-cell transplantation: the Coopera- tive German Transplant Study Group. J Clin Oncol. 2003;21:2747–53.
78. Beitinjaneh A, McKinney AM, Cao Q, Weisdorf DJ. Toxic leukoencephalopathy following ﬂudarabine-associated hemato- poietic cell transplantation. Biol Blood Marrow Transplant. 2011;17:300–8.
79. Annaloro C, Costa A, Fracchiolla NS, Mometto G, Artuso S, Saporiti G, et al. Severe ﬂudarabine neurotoxicity after reduced intensity conditioning regimen to allogeneic hematopoietic stem cell transplantation: a case report. Clin Case Rep. 2015;3:650–5.
80. Rosenberg SA, Packard BS, Aebersold PM, Solomon D, Topa- lian SL, Toy ST, et al. Use of tumor-inﬁltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. A preliminary report. N Eng J Med. 1988;319:1676–80.
81. Rosenberg SA, Yannelli JR, Yang JC, Topalian SL, Schwart- zentruber DJ, Weber JS, et al. Treatment of patients with meta- static melanoma with autologous tumor-inﬁltrating lymphocytes and interleukin 2. J Natl Cancer Inst. 1994;86:1159–66.
82. Dudley ME, Wunderlich JR, Yang JC, Sherry RM, Topalian SL, Restifo NP, et al. Adoptive cell transfer therapy following non- myeloablative but lymphodepleting chemotherapy for the treat- ment of patients with refractory metastatic melanoma. J Clin Oncol. 2005;23:2346–57.
83. Dudley ME, Yang JC, Sherry R, Hughes MS, Royal R, Kam- mula U, et al. Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens. J Clin Oncol. 2008;26:5233–9.

84. Rosenberg SA, Yang JC, Sherry RM, Kammula US, Hughes MS, Phan GQ, et al. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin Cancer Res. 2011;17:4550–7.
85. Goff SL, Dudley ME, Citrin DE, Somerville RP, Wunderlich JR, Danforth DN, et al. Randomized, prospective evaluation com- paring intensity of lymphodepletion before adoptive transfer of tumor-inﬁltrating lymphocytes for patients with metastatic mel- anoma. J Clin Oncol. 2016;34:2389–97.
86. Pilon-Thomas S, Kuhn L, Ellwanger S, Janssen W, Royster E, Marzban S, et al. Efﬁcacy of adoptive cell transfer of tumor- inﬁltrating lymphocytes after lymphopenia induction for meta- static melanoma. J Immunother. 2012;35:615–20.
87. Besser MJ, Shapira-Frommer R, Itzhaki O, Treves AJ, Zippel DB, Levy D, et al. Adoptive transfer of tumor-inﬁltrating lym- phocytes in patients with metastatic melanoma: intent-to-treat analysis and efﬁcacy after failure to prior immunotherapies. Clin Cancer Res. 2013;19:4792–800.
88. Wrzesinski C, Restifo NP. Less is more: lymphodepletion fol- lowed by hematopoietic stem cell transplant augments adoptive T-cell-based anti-tumor immunotherapy. Curr Opin Immunol. 2005;17:195–201.
89. Muranski P, Boni A, Wrzesinski C, Citrin DE, Rosenberg SA, Childs R, et al. Increased intensity lymphodepletion and adoptive immunotherapy—how far can we go? Nat Clin Pract Oncol. 2006;3:668–81.
90. Rosenberg SA, Restifo NP. Adoptive cell transfer as persona- lized immunotherapy for human cancer. Science. 2015; 348:62–8.
91. Aleksic M, Liddy N, Molloy PE, Pumphrey N, Vuidepot A, Chang KM, et al. Different afﬁnity windows for virus and cancer-speciﬁc T-cell receptors: implications for therapeutic strategies. Eur J Immunol. 2012;42:3174–9.
92. Tan MP, Gerry AB, Brewer JE, Melchiori L, Bridgeman JS, Bennett AD, et al. T cell receptor binding afﬁnity governs the functional proﬁle of cancer-speciﬁc CD8+ T cells. Clin Exp Immunol. 2015;180:255–70.
93. Perica K, Varela JC, Oelke M, Schneck J. Adoptive T cell immunotherapy for cancer. Rambam Maimonides Med J. 2015;6:e0004.
94. Rapoport APYJ. T-cell receptor (TCR)-gene modiﬁed T-cells for cancer: methods, data and challenges. In: Perales M-A, Abutalib SA, Bollard C, editors. Advances & controversies in hemato- poietic cell transplants and cell therapy. New York: Springer; 2018.
95. Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science. 2006;314:126–9.
96. Burns WR, Zheng Z, Rosenberg SA, Morgan RA. Lack of speciﬁc gamma-retroviral vector long terminal repeat promoter silencing in patients receiving genetically engineered lympho- cytes and activation upon lymphocyte restimulation. Blood. 2009;114:2888–99.
97. Johnson LA, Morgan RA, Dudley ME, Cassard L, Yang JC, Hughes MS, et al. Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood. 2009;114:535–46.
98. Robbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME, et al. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engi- neered lymphocytes reactive with NY-ESO-1. J Clin Oncol: Off J Am Soc Clin Oncol. 2011;29:917–24.
99. Morgan RA, Chinnasamy N, Abate-Daga D, Gros A, Robbins PF, Zheng Z, et al. Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy. J Immunother. 2013;36:133–51.

100. Linette GP, Stadtmauer EA, Maus MV, Rapoport AP, Levine BL, Emery L, et al. Cardiovascular toxicity and titin cross- reactivity of afﬁnity-enhanced T cells in myeloma and mela- noma. Blood. 2013;122:863–71.
101. Park JH, Brentjens RJ. Adoptive immunotherapy for B-cell malignancies with autologous chimeric antigen receptor mod- iﬁed tumor targeted T cells. Discov Med. 2010;9:277–88.
102. Curran KJ, Pegram HJ, Brentjens RJ. Chimeric antigen receptors for T cell immunotherapy: current understanding and future directions. J Gene Med. 2012;14:405–15.
103. Maus MV, June CH. Making better chimeric antigen receptors for adoptive T-cell therapy. Clin Cancer Res. 2016;22:1875–84.
104. Kochenderfer JN, Wilson WH, Janik JE, Dudley ME, Stetler- Stevenson M, Feldman SA, et al. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19. Blood. 2010;116:4099–102.
105. Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modiﬁed T cells in chronic lymphoid leukemia. N Eng J Med. 2011;365:725–33.
106. Brentjens RJ, Riviere I, Park JH, Davila ML, Wang X, Stefanski J, et al. Safety and persistence of adoptively transferred auto- logous CD19-targeted T cells in patients with relapsed or che- motherapy refractory B-cell leukemias. Blood. 2011; 118:4817–28.
107. Savoldo B, Ramos CA, Liu E, Mims MP, Keating MJ, Carrum G, et al. CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modiﬁed T cells in lymphoma patients. J Clin Invest. 2011;121:1822–6.
108. Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A, et al. T cells with chimeric antigen receptors have potent anti- tumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med. 2011;3:95ra73.
109. Kochenderfer JN, Dudley ME, Feldman SA, Wilson WH, Spaner DE, Maric I, et al. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti- CD19 chimeric-antigen-receptor-transduced T cells. Blood. 2012;119:2709–20.
110. Cruz CR, Micklethwaite KP, Savoldo B, Ramos CA, Lam S, Ku S, et al. Infusion of donor-derived CD19-redirected virus-speciﬁc T cells for B-cell malignancies relapsed after allogeneic stem cell transplant: a phase 1 study. Blood. 2013;122:2965–73.
111. Brentjens RJ, Davila ML, Riviere I, Park J, Wang X, Cowell LG, et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med. 2013;5:177ra38.
112. Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, et al. Chimeric antigen receptor-modiﬁed T cells for acute lymphoid leukemia. N Eng J Med. 2013;368:1509–18.
113. Davila ML, Riviere I, Wang X, Bartido S, Park J, Curran K, et al. Efﬁcacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med. 2014;6:224ra25.
114. Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Eng J Med. 2014;371:1507–17.
115. Park JH, Brentjens RJ. Are all chimeric antigen receptors created equal? J Clin Oncol. 2015;33:651–3.
116. Kochenderfer JN, Dudley ME, Kassim SH, Somerville RP, Carpenter RO, Stetler-Stevenson M, et al. Chemotherapy- refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Oncol. 2015;33:540–9.
117. Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Del- brook C, Feldman SA, et al. T cells expressing CD19 chimeric

antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet. 2015;385:517–28.
118. Porter DL, Hwang WT, Frey NV, Lacey SF, Shaw PA, Loren AW, et al. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med. 2015;7:303ra139.
119. Dai H, Zhang W, Li X, Han Q, Guo Y, Zhang Y, et al. Tolerance and efﬁcacy of autologous or donor-derived T cells expressing CD19 chimeric antigen receptors in adult B-ALL with extra- medullary leukemia. Oncoimmunology. 2015;4:e1027469.
120. Turtle CJ, Hanaﬁ LA, Berger C, Gooley TA, Cherian S, Hudecek M, et al. CD19 CAR-T cells of deﬁned CD4+:CD8+ compo- sition in adult B cell ALL patients. J Clin Invest. 2016; 126:2123–38.
121. Gardner R, Wu D, Cherian S, Fang M, Hanaﬁ LA, Finney O, et al. Acquisition of a CD19-negative myeloid phenotype allows immune escape of MLL-rearranged B-ALL from CD19 CAR-T- cell therapy. Blood. 2016;127:2406–10.
122. Brudno JN, Kochenderfer JN. Toxicities of chimeric antigen receptor T cells: recognition and management. Blood. 2016;127:3321–30.
123. Maude S, Barrett DM. Current status of chimeric antigen receptor therapy for haematological malignancies. Br J Haema- tol. 2016;172:11–22.
124. Till BG, Jensen MC, Wang J, Chen EY, Wood BL, Greisman HA, et al. Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically modiﬁed autologous CD20-speciﬁc T cells. Blood. 2008;112: 2261–71.
125. Cheadle EJ, Hawkins RE, Batha H, Rothwell DG, Ashton G, Gilham DE. Eradication of established B-cell lymphoma by CD19-speciﬁc murine T cells is dependent on host lymphopenic environment and can be mediated by CD4+ and CD8+ T cells. J Immunother. 2009;32:207–18.
126. Wallen H, Thompson JA, Reilly JZ, Rodmyre RM, Cao J, Yee
C. Fludarabine modulates immune response and extends in vivo survival of adoptively transferred CD8 T cells in patients with metastatic melanoma. PLoS ONE. 2009;4:e4749.
127. Topp MS, Gokbuget N, Stein AS, Zugmaier G, O’Brien S, Bargou RC, et al. Safety and activity of blinatumomab for adult patients with relapsed or refractory B-precursor acute lympho- blastic leukaemia: a multicentre, single-arm, phase 2 study. Lancet Oncol. 2015;16:57–66.
128. Lee DW, Gardner R, Porter DL, Louis CU, Ahmed N, Jensen M, et al. Current concepts in the diagnosis and management of cytokine release syndrome. Blood. 2014;124:188–95.
129. Neelapu SS, Tummala S, Kebriaei P, Wierda W, Gutierrez C, Locke FL, et al. Chimeric antigen receptor T-cell therapy— assessment and management of toxicities. Nat Rev Clin Oncol. 2018;15:47–62.
130. Santomasso B,Park JH,Riviere I,Mead E,Halton E,Diamonte C, et al. Biomarkers associated with neurotoxicity in adult patients with relapsed or refractory B-ALL (R/R B-ALL) treated with CD19 CAR T cells. J Clin Oncol. 2017;35(15_suppl): 3019.
131. De Angelo DJG, Park A, Dinner JH, Mannis SN, Lunning GN, Khalde MA, et al. Clinical outcomes for the phase 2, single-arm, multicenter trial of JCAR015 in adult B-ALL (ROCKET Study). J Immunother Cancer. 2017;5(Suppl 2):P217.
132. Harris J OncLive [Internet] 2017. http://www.onclive.com/web- exclusives/kite-reports-cerebral-edema-death-in-zuma1-car-tcell- trial. Accessed 17 Jan 2018.
133. van der Stegen SJ, Hamieh M, Sadelain M. The pharmacology of second-generation chimeric antigen receptors. Nat Rev Drug Discov. 2015;14:499–509.

134. Gust J, Hay KA, Hanaﬁ LA, Li D, Myerson D, Gonzalez-Cuyar LF, et al. Endothelial activation and blood−brain barrier disruption in neurotoxicity after adoptive immunotherapy with CD19 CAR-T cells. Cancer Discov. 2017;7: 1404–19.
135. Taraseviciute A, Kean L, Jensen MC. Creation of the ﬁrst non- human primate model that faithfully recapitulates chimeric antigen receptor (CAR) T cell-mediated cytokine release

syndrome (CRS) and neurologic toxicity following B cell- directed CAR-T cell therapy. Blood. 2016;128:651.
136. Alter A, Duddy M, Hebert S, Biernacki K, Prat A, Antel JP, et al. Determinants of human B cell migration across brain endothelial cells. J Immunol. 2003;170:4497–505.
137. Mackall CLM, Miklos DB. Endothelial cell activation emerges as a driver of CAR-T cell associated neurotoxicity. Cancer Discov. 2017;7:1371–3.