Novel immunotherapies for adult patients with B-lineage acute lymphoblastic leukemia
Wei et al. Journal of Hematology & Oncology
Novel immunotherapies for adult patients with B-lineage acute lymphoblastic leukemia
Guoqing Wei 0
Jiasheng Wang 0
He Huang 0
Yanmin Zhao 0
0 Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University , Hangzhou 310000 , China
The past decade witnessed the rapid development of adult B-lineage acute lymphoblastic leukemia (ALL) treatment. Beyond the development of chemotherapy regimens, immunotherapy is starting a new era with unprecedented complete remission (CR) rate. Targeting B-lineage-specific surface markers such as CD19, CD20, CD22, or CD52, immunotherapy has been demonstrating promising clinical results. Among the immunotherapeutic methods, naked monoclonal antibodies (mAbs), antibody-drug conjugate (ADC), bispecific T cell engager (BiTE), and chimeric antigen receptor (CAR) T cells are the main types. In this review, we will examine the emerging preclinical and clinical development on (1) anti-CD20 naked mAbs rituximab, ofatumumab, and obinutuzumab; (2) anti-CD19 ADCs SAR3419 and SGN-CD19A and anti-CD19 BiTE blinatumomab; (3) anti-CD22 naked mAb epratuzumab and anti-CD22 ADC inotuzumab ozogamicin; (4) anti-CD52 naked mAb alemtuzumab; and (5) anti-CD19 CAR T cells. We will discuss their efficacy, adverse effects, as well as future development.
For children with B cell acute lymphoblastic leukemia (B
ALL), current chemotherapy regimens can achieve
longterm overall survival (OS) of 80–90%. However, similar
results have not been seen in adults. Despite a high
initial complete response (CR) rate of 80–90%, most of
the adults will eventually relapse with
chemotherapyresistant disease. Long-term OS in adults with B ALL
remains in the range of 30–50%; the prognosis of
relapsed or refractory (R/R) ALL is even more dismal
with a 5-year OS of only 10% [
]. For R/R ALL
patients, the only option to achieve long-term survival is
allogeneic hematopoietic stem cell transplantation
(allo-HSCT), which requires reinduction chemotherapy
prior to the transplantation. The chemotherapy in the
context, however, is generally poorly tolerated with
unsatisfied outcomes, as only 5 to 10% patients can be
bridged to allo-HSCT . Although a few new cytotoxic
drugs have been approved over the last decade such as
clofarabine and liposomal vincristine, the low single-agent
response rates (17% with clofarabine monotherapy, 20%
with liposomal vincristine monotherapy) still emphasize
an urgent need for different alternative treatment
strategies in R/R adult ALL [
Altogether, four types of immunotherapies have been
developed to date, including naked monoclonal antibodies
(mAbs) (such as rituximab, epratuzumab, and
alemtuzumab), conjugated monoclonal antibodies (such as
inotuzumab ozogamicin, SAR3419, and SGN-CD19A),
bispecific T cell engager (BiTE) (such as blinatumomab),
and chimeric antigen receptor (CAR) T cell therapy
(Fig. 1). Naked monoclonal antibodies exert their
cytotoxic effects through mechanisms such as
antibodydependent cytotoxicity, complement-dependent
cytotoxicity, and direct induction of apoptosis; moreover,
direct blocking of leukemic cell receptors can lead to
cell death if the signalings through the receptors are
crucial for leukemic cell to survive. If a surface marker
is known to internalize upon binding (such as CD19
and CD22), potent cytotoxins can be conjugated to the
monoclonal antibody, resulting in an additional
cytotoxic mechanism. BiTE conjugates two monoclonal
antibodies recognizing leukemic cell and cytotoxic T
cells (CTLs) and exerts its effects by specifically
bridging CTLs and leukemic cells. CAR T cells utilizes
engineered T cells by introducing leukemic
celltargeting single-chain variant fragment (scFv)
chimerized with intracellular T cell activation domains. Both
BiTE and CAR T cells lead to leukemic cell killing in
mechanisms similar to cancer-specific CTLs, including
releasing of cytotoxic granules, activation of
deathrelated receptors, and releasing of cytokines. Compared
with CAR T cell therapy, naked/conjugated mAbs and
BiTE are more readily available and easier to
manufacture; however, CAR T cell therapy as a “living drug” is
more durable and repeat infusions are usually not
needed. Based on clinical data, BiTE and CAR T cell
therapy are more potent and generate better outcomes
than naked/conjugated mAbs; however, these two
modalities are associated with more severe side effects,
such as cytokine release syndrome (CRS), and adverse
neurologic events. In this review, we will discuss
clinical and pre-clinical results of these different modalities
in treating B ALL, focusing on the efficacy (Table 1)
and the side effects (Table 2).
CD20-targeting agents—rituximab, ofatumumab, and
CD20 is a surface marker of B-lineage lymphocytes with
robust expression after the mid-stage of development. It
is presented in 25% of pre-B ALL and nearly all mature
ALL. The GRAALL (a Group for Research in Adult
Acute Lymphoblastic Leukemia) study demonstrated a
higher relapse rate (39 vs 20%, P = 0.04) and a worse
event-free survival (EFS) exclusively in pre-B ALL
patients with positive CD20 expression and a high white
blood count, indicating worse outcomes are associated
with positive CD20 expression [
Rituximab is a humanized murine mAb targeting CD20.
Before its introduction to ALL treatment, rituximab has
shown prolonged survival for patients with CD20+
nonHodgkin’s lymphoma and Burkitt’s lymphoma/leukemia
]. Emerging results have confirmed its efficacy in
MD Anderson Cancer Center recruited patients with
Ph-negative B ALL and treated with the modified
hyperCVAD (cyclophosphamide, vincristine, doxorubicin and
dexamethasone) regimens with or without rituximab.
For the younger (age < 60 years) subgroup, complete
remission duration (CRD) and OS were better with the
combination of hyper-CVAD and rituximab than with
hyper-CVAD alone (69 vs 38%; P < .001 and 71 vs 47%,
P = .003). However, no benefit from the addition of
rituximab was noted in elderly patients (age ≥ 60 years), in
part because of deaths in CR from infections. The results
suggest a role for rituximab use in patients with CD20+
ALL under 60 years old [
]. Similar outcomes were
observed in a multicenter randomized trial comparing
the pediatric-inspired GRAALL protocol to the same
regimen plus rituximab [
]. Two hundred twenty
patients aged 18–59 years old with newly diagnosed
CD20positive Ph-negative B cell precursor (BCP)-ALL were
enrolled from 2005 to 2014. Rituximab (375 mg/m2) was
given from induction to the first year of maintenance for
a total of 16 to 18 infusions. After induction ± salvage
reinduction, the CR rate was 92 and 91% in rituximab
and control arm, respectively. Patients treated in the
rituximab arm had a lower cumulative incidence of
relapse (CIR) (2-year CIR, 18 vs 30.5% in the control
arm; p = 0.02) and longer EFS (2-year EFS, 65 vs 52%
220 pts aged 18–59 years Rituximab (375 mg/m2) was added After induction ± salvage reinduction, Phase
old with newly diagnosed to pediatric-inspired GRAALL CR rate was 92 and 91% in the rituximab III 
CD20-positive Ph-negative B cell protocol from induction to the first and control arm. Pts treated in the
precursor (BCP) ALL year of maintenance for a total of 16 rituximab arm had a lower CIR (2-year
to 18 infusions CIR, 18 vs 30.5% in the control arm;
p = 0.02) and longer EFS (2-year EFS, 65
vs 52% in the control arm; p = 0.038),
but not longer OS (2-year OS, 71 vs 64%
in the control arm; p = 0.095)
INO group: INO (0.8–0.5 mg/m2, CR rate was higher with INO than with Phase
weekly, 3 times per cycle; cycle standard therapy (80.7 vs. 29.4% p < 0.001) III [
length, 21–28 days; total number and a higher percentage of pts in the INO
of cycles, 6); standard intensive group achieved < 0.01% MRD (78.4 vs.
chemotherapy: FLAG for up to 28.1%, P < 0.001). Both PFS and OS were
4 cycles, cytarabine plus mitoxantrone longer with INO (median PFS, 5.0 vs.
for up to 4 cycles, or high-dose 1.8 months, P < 0.001; median OS, 7.7 vs.
cytarabine for up to 1 cycle 6.7 months, P = 0.04)
Mini-hyper-CVD regimen plus INO The ORR was 71%: 31 (53%) CR, 13 (23%) Phase
administered on day 3 of each of CRp, and 1 (2%) CRi. II [
the first 4 cycles, rituximab (in pts 27 (47%) pts proceeded to receive
whose cells were CD20-positive) and allo-HSCT. Pts who were treated with
intrathecal chemotherapy were mini-hyper-CVD plus INO had a higher
given for the first 4 courses PFS rate and improved OS compared to a
historical cohort with single-agent INO in
R/R ALL (2-year PFS; 52 vs 36%; p = 0.20:
2-year OS; 44 vs. 25%; p = 0.01)
Pts patients, CIR cumulative incidence of relapse, OS overall survival, CRD complete remission duration, CR complete remission, MRD minimal residual disease,
RFS relapse-free survival, DFS disease-free survival, R/R refractory/relapsed, CRp complete remission in the absence of total platelet recovery, CRi complete
remission with incomplete hematologic recovery, ORR overall response rate
in the control arm; p = 0.038), but not longer OS
(2year OS, 71 vs 64% in the control arm; p = 0.095).
When censoring patients who received allogeneic
HSCT in first CR at transplant time, EFS and OS were
longer in the rituximab arm (2-year EFS, 66 vs 53%,
and 2-year OS, 74 vs 63%; p = 0.021 and 0.018,
respectively). These data indicated adding rituximab to the ALL
chemotherapy protocol could improve the outcome for
patients with CD20-positive, Ph-negative ALL, especially
in younger adults.
Rituximab is among the most studied immunotherapies,
and its safety has been well addressed. Due to its low
toxicity, rituximab may be particularly useful in elder patients
and patients who are unfit for more aggressive treatment
]. However, several limitations were discovered: its
efficacy is limited when administered alone; moreover, CNS
relapse is common when rituximab is used as
monotherapy because mAbs cannot cross the blood-brain barrier.
Ofatumumab (HuMax-CD20) is a second-generation
anti-CD20 mAb that binds to a site different from
rituximab. Ofatumumab targets a membrane proximal
small-loop epitope on the CD20 molecule and is more
potent than rituximab in inducing antibody-dependent
cell-mediated cytotoxicity (ADCC) and
complementdependent cytotoxicity (CDC) [
]. As a single agent,
ofatumumab’s adverse effects are mild, consisting
primarily of grade 1 or 2 infusion reactions or infections.
Only 9% of patients have grade 3 or 4 infections. In a
phase II study of hyper-CVAD/MTX-Ara-C in
combination with ofatumumab for adults with CD20-positive
ALL, promising results have been achieved. Altogether,
55 patients with de novo ALL and 4 patients in CR with
previous treatment received a median of 8 cycles (range,
1–8) of ofatumumab therapy. All but one patient (98%)
achieved a CR after cycle 1, and 53 patients (93%)
achieved minimal residual disease (MRD) negativity. The
3-year CRD and OS rates were 78 and 68%, respectively.
The 3-year OS in patients with CD20 < 20 and ≥ 20%
were 82 and 64%, respectively (p = 0.96). This study
proved safety and high effectiveness in patients with
CD20-positive ALL [
]. As ofatumumab binds to a
different epitope than rituximab, the medication may
also be used to overcome rituximab-resistant disease.
Nonetheless, longer follow-up and randomized studies
are warranted in the future.
Obinutuzumab, a type II glycoengineered humanized
anti-CD20 mAb, has shown potent activity in CLL and
has been FDA-approved for the upfront treatment of
CLL. The post-translational glycoengineering
modification of obinutuzumab can enhance its binding affinity
to the FccRIII receptors on immune effector cells that
would promote ADCC, while at the same time decrease
CDC. Obinutuzumab is potentially more potent than
other anti-CD20 mAbs due to its ability to directly
induce cell death [
Encouraging preclinical results have been reported in
ALL cell lines and xenografts [
]. However, no clinical
studies in ALL patients have been performed yet. Thus,
future studies are awaited as it may offer another option
for the treatment of ALL.
CD19-targeting agents—SAR3419, SGN19a, and
CD19 is present in 90% of pre-B and mature ALL
leukemic cells. Its high expression rate makes it an ideal
target for immunotherapy. However, the antigen is
known to internalize upon binding to antibody, making
it an unsuitable target for naked mAbs. Nonetheless, the
internalization property makes it an attractive target for
SAR3419 is an antibody-drug conjugate (ADC) with a
humanized anti-CD19 antibody conjugated to
maytansin, a potent antimitotic agent. After binding to
CD19positive lymphoblast, SAR3419 is internalized and
processed to release the active maytansin metabolites
that induce both cell cycle arrest and apoptosis [
In phase I studies conducted in patients with
nonHodgkin’s lymphoma, the major dose-limiting toxicity is
reversible severe blurred vision, which was associated
with epithelial corneal changes. Given the adverse effect,
the maximally tolerated dose was 160 mg/m2
intravenously every 3 weeks [
]. In studies using CD19-positive
pre-B cell ALL and mixed lineage leukemia (MLL)
xenografts, the administration of SAR3419 delayed disease
progression, even in chemotherapy-resistant xenograft
]. Unfortunately, a phase II multicenter,
single-arm clinical trial (NCT01440179) was prematurely
terminated due to modest activity of SAR3419 compared
with its competitors. In this study, patients received
SAR3419 induction monotherapy (55, 70, and 90 mg/
m2, ≤ 8 weekly dosed); the responding patients were
eligible for maintenance therapy (biweekly for ≤24 weeks).
Of the 17 evaluable patients, only 4 had a disease
response (estimated overall response rate (ORR), 25.5%;
80% CI, 14.2–39.6%). The duration of response (DOR)
was only 1.9 months (range, 1–5.6 months) [
indicating SAR3419 monotherapy is unpromising in the
treatment of R/R ALL. Thus, the unfavorable efficacy
and dose-limiting toxicity may hinder further
development of SAR3419.
SGN-CD19A (denintuzumab) is a novel ADC composed
of a humanized anti-CD19 mAb linked to a
microtubuledisrupting agent monomethyla-uristatin F (MMAF).
The ADC binds to CD19, internalizes, and releases
MMAF, which ultimately results in G2-M phase growth
arrest and induction of apoptosis. In a phase I
doseescalation study (NCT 01786096), 49 patients with R/R
ALL (n = 40) or lymphoma (n = 9) were treated with
weekly IV SGN-CD19A (0.3–4.5 mg/kg) or every
3 weeks (0.5–6 mg/kg). Among the 33 evaluable
patients, objective responses were observed in 30% (10 of
33), including 6 of 25 patients on the weekly schedule
and 4 of 8 patients on the every 3-week schedule.
SGNCD19A was generally well tolerated with superficial
microcystic keratopathy being the most common
toxicity, which might require routine steroid eye drop
]. After all, promising results in heavily
pretreated R/R patients and safety profile suggest
opportunities for combination with other conventional
anti-leukemic therapies in lymphoblastic malignancies.
Blinatumomab is the first-in-class BiTE construct, which
binds to both CD3 on CTLs and CD19 on B cells. The
construct can facilitate CTL activation and expansion,
which will result in effective lysis of CD19-positive cells,
through release of cytokines and the pore-forming
perforin system [
]. (Fig. 1).
Pharmacokinetic analyses from a phase II study in
ALL patients found rapid and sustained depletion of B
cells, with the level becoming undetectable at a mean of
2.18 days (range, 0.03 to 13.94 days) [
]. On the
other hand, circulating T cells were initially depleted
from the peripheral blood within hours of
blinatumomab infusion but recovered to baseline or higher within
a few days, followed by a polyclonal expansion of T cells
expressing the activation marker CD69 . Accordingly,
many patients experienced a CRS 1–2 days after the
infusion, which was mediated by transient release of
inflammatory cytokines interleukin (IL)-2, IL-6,
IL10, tumor necrosis factor and interferon-γ from
blinatumomab-engaged T cell effectors. Cytokine levels
declined after day 2, and these spikes generally did not
recur with future cycles [
In a phase II study of 21 patients with persistent MRD,
the single-agent blinatumomab was continuously infused
at 15 μg/m2/day for a 4-week period followed by a
2week treatment-free interval before starting the next
cycle. The study achieved a molecular response rate of
80% and long-term relapse-free survival (RFS) of 61%,
after a median follow-up of 33 months [
]. In the
BLAST study, 88 of the 106 ALL patients (76%) achieved
MRD after the first cycle of blinatumomab, including
high-risk subgroups such as patients in second-line
treatment, patients with high MRD burden, and older
patients. With median follow-up of 29.5 months, median
OS and RFS were 18.9 and 36.5 months, respectively.
MRD complete response achieved in the first cycle
was associated with longer OS (median OS 40.4 vs
12.0 months, P = .001) and RFS (35.2 vs 7.1 months,
P = 0.002), compared with not achieving an MRD
complete response after the first cycle [
Based on the positive experience in adult patients with
MRD-positive B cell lineage ALL, a phase II study of
blinatumomab enrolled 36 Ph-negative pre-B ALL
adults with primary refractory disease or relapsed after
chemotherapy or HSCT. It led to 69% CR or CR with
incomplete count recovery (CRi), and 88% of the
responders also obtaining a molecular response (MRD
level below 10− 4 by PCR) within the first two cycles of
drug administration. The median OS was 9.8 months
(95% CI, 8.5 to 14.9 months), and the median RFS was
7.6 months (95% CI, 4.5 to 9.5 months) [
study of 189 patients with high-burden R/R B ALL
showed a CR/CRi rate of 43% and a median OS rate of
6.1 months [
]. Safety profile appears favorable in
these studies, with CRS and neurologic events being
the most severe toxicities.
Despite the promising initial results, some patients do
not respond to blinatumomab or experience disease
progression after an initial response. The causes of primary
resistance remain unknown. However, several
mechanisms have been proposed. One possible mechanism is
the selection of CD19-negative subclones, as leukemic
cells can still maintain proliferation without CD19
]. Further, increased regulatory T (Treg)
cells in combination with high lactic dehydrogenase level
predicted resistance to blinatumomab [
Tregs secrete IL-10 suppressing T cell proliferation,
leading to treatment failure. Upregulation of programmed
death-ligand 1 (PD-1) on leukemia cells after
blinatumomab treatment is also a potential immune escape
]. Further studies are warranted to analyze the
significance of the PD-1/PD-L1 interplay as a resistance
mechanism to blinatumomab.
Besides blinatumomab, other BiTEs are also under active
developing. Recently, a disulfide-stabilized form of BiTE
was tested which showed increased efficient [
CD22-targeting agents—epratuzumab and inotuzumab
CD22 is expressed on leukemic blasts in 90% of
preB ALL and mature ALL. Like CD19, CD22 is rapidly
internalized upon antibody binding. Possible
mechanisms of action of anti-CD22 antibodies include
ADC, modulation of B cell signaling, and inhibition
of proliferation [
Epratuzumab is a naked, humanized anti-CD22
immunoglobulin G1 (IgG1) that is internalized after binding to
CD22. Engagement of CD22 with epratuzumab leads to
direct phosphorylation of key upstream inhibitory
receptors of BCR signaling [
]. Epratuzumab has been used
as a single agent (4 doses of intravenous epratuzumab
360 mg/m2 twice a week) followed by standard
Children’s Oncology Group (COG) reinduction
chemotherapy regimen in 15 pediatric patients with relapsed pre-B
ALL. Although this strategy did not improve CR in
comparison with historical controls (65 vs. 66%), more
patients achieved negative MRD status (42 vs. 25%,
p = 0.001). The more favorable rate of MRD negativity
after addition of epratuzumab suggests that this
antibody may enhance the response to cytotoxic
]. However, further follow-up is needed to
determine whether the deeper remission level translates
into improved DFS or OS. In adult ALL, the Southwest
Oncology Group (SWOG) evaluated epratuzumab
combined with clofarabine plus cytarabine in 31 patients
experiencing first or later relapsed disease. Overall, the
response rate was 52%, significantly higher than that of
SWOG’s previous trial with clofarabine/cytarabine alone
]. These data provided the groundwork for a
randomized phase III trial in children with relapsed ALL
evaluating chemotherapy with or without epratuzumab
(NCT01802814). Recently, in a phase II prospective
study, epratuzumab was combined with hyper-CVAD in
younger patients (18–59 years old) with R/R CD22+ B
ALL. Among the 30 patients ultimately considered for
analyses, the ORR was only 50%, with 45% of CR/CRi
patients achieved negative MRD. All patients in CR/CRi
and 1 patient in PR received a consolidation
chemotherapy. However, at the time of analysis, all patients died
except for the recently enrolled responders still in CR
]. The short-lived improvement and overall
disappointing outcome could be explained by an insufficient
disease load decrease and/or by escape of the blast cells
to epratuzumab. The results suggested that epratuzumab
should be tested within first-line chemotherapies as it
may participate to decrease the MRD level. Because
CD22 is internalized upon binding, we expect
conjugating cytotoxic agents with epratuzumab would result in
more favorable outcomes.
Inotuzumab ozogamicin (INO)
Inotuzumab ozogamicin (INO) is an ADC that consists of
calicheamicin (a potent DNA-binding cytotoxic agent)
attached to an engineered humanized monoclonal
immunoglobulin G4 (IgG4) antibody targeting CD22. INO binds
CD22 with sub-nanomolar affinity and is rapidly
internalized, delivering the conjugated calicheamicin intracellularly.
Calicheamicin binds to the minor DNA grove, causing
double-strand DNA cleavage and cell apoptosis.
In a single-institution phase II study, 49 patients with
R/R ALL were treated with 1.8 mg/m2 INO every 3–
4 weeks as a single agent. It resulted in an ORR of 57%
(18% CR and 39% CRi) and median OS of 5.1 and
7.9 months in all patients and 28 responders,
respectively. A total of 27 patients who achieved complete
morphological response were assessed for MRD, and 63%
(17 of 27) of patients was found to have MRD-negative
status. Abnormal liver function tests were the most
significant adverse events observed and were graded as
severe in 31% of patients [
]. To optimize the benefit-risk
ratio of INO, a weekly dosing regimen was evaluated in
41 patients based on preclinical studies [
drug administration at a lower dose (0.8 mg/m2 on day
1, 0.5 mg/m2 on days 8 and 15) was associated with
similar efficacy (59% ORR). However, liver toxicity was
seen in only 11 of 41 (27%) patients with weekly INO
compared with 28 of 49 (57%) with single-dose INO.
This study indicates that more frequent but lower dose
of INO may reduce toxicities while maintain the efficacy.
Recently, a global, open-label, randomized phase III
study confirmed the notion. Patients were randomly
assigned to receive either INO (0.8–0.5 mg/m2, weekly,
three times per cycle, cycle length of 21–28 days, 6 total
cycles) or standard intensive chemotherapy. The CR rate
was higher with INO than with standard chemotherapy
(80.7 vs. 29.4% p < 0.001). Moreover, a higher percentage
of patients in the INO group achieved MRD-negative
status (78.4 vs. 28.1%, P < 0.001). Both PFS and OS were
longer with INO (median PFS, 5.0 vs. 1.8 months,
P < 0.001; median OS, 7.7 vs. 6.7 months, P = 0.04) [
Because HSCT is considered to be the only curative
treatment option, the capacity of INO to increase
the number of patients who can proceed to HSCT is
The promising results of INO as a single agent led to
the initiation of a clinical trial combining INO with
non-myelosuppressive chemotherapy. A total of 57
patients with R/R CD22+ B ALL received INO, in
combination with mini-hyper-CVD regimen, rituximab,
and intrathecal chemotherapy. The ORR was 71%, with
47% patients proceeding to allo-HSCT. Compared with
historical cohort, patients treated with
mini-hyperCVD plus INO had a higher PFS rates and improved
OS (2-year PFS; 52 vs. 36%; p = 0.20: 2-year OS; 44 vs.
25%; p = 0.01) [
]. These encouraging results
provided an option of combining INO with low-intensity
chemotherapy in patients with R/R ALL.
The same regimen of mini-hyper-CVD combined with
INO was also evaluated in elderly patients ≥ 60 years
with newly diagnosed B ALL [
]. Of the 42 patients
evaluable for response, 40 (95%) achieved CR/CRp. Of
the 44 patients assessed for MRD, 41 (93%) achieved
negative MRD. The results appear superior to historical
data with hyper-CVAD alone in a similar patients’
population (3-year overall survival (OS) rates of 52 and
36%, respectively, p = 0.05) [
CD52, an antigen involved in T cell activation, is
expressed in 70% of T ALL and pre-B ALL.
Alemtuzumab is a fully humanized monoclonal antibody against
CD52. Alemtuzumab has been evaluated in the CALGB
phase I trial of 24 patients with de novo CD52+ ALL
after the successful achievement of CR with induction
]. The drug was given three times
weekly for a target dose of 30 mg subcutaneously. After
51 months of follow-up, median OS was 55 months
and DFS was 53 months. Notably, alemtuzumab was
associated with increased risk of neutropenia and CMV
viremia, supporting a role for stimulating factor
support. Recently, a phase II study assessed the efficacy
of alemtuzumab combined with granulocyte
colonystimulating factor (G-CSF) in 12 patients with R/R
ALL. G-CSF was administered during alemtuzumab
administration. Although 4 of the 12 patients achieved
CR, all patients progressed within a few months and all
but 1 died [
]. The current studies only demonstrate
modest activity of alemtuzumab against T ALL or B
ALL, yet significant adverse effects have been reported
extensively. These results would potentially limit further
development of alemtuzumab.
Chimeric antigen receptor T cell therapy
Chimeric antigen receptors (CARs) consist of an
extracellular binding domain (scFv), hinge domain,
transmembrane domain, and intracellular signaling domains.
When expressed in autologous T cells (or donor T
cells in the post-transplant setting), CARs redirect the
CTLs toward antigen-expressing tumor cells in an
HLA-independent manner. DNA constructs encoding
such CARs could be stably incorporated into human T
cells via lentiviral or γ-retroviral transductions,
electroporation, or “Sleeping Beauty” transposon.
The engineering of CAR T cells evolves over time. The
first-generation CAR T cells are only incorporated with
T cell receptor CD3ζ signaling domain. This construct
revealed weak proliferation ability, poor anti-tumor
effect, and short survival of T cells [
]. Second- and
third-generation CAR T cells are incorporated with
costimulatory domains, such as CD137 (4-1BB), CD28,
CD27, or CD134 (OX40), which significantly enhanced
the expansion, persistence, and potency of CAR T cells.
Among them, CD28 and 4-1BB are currently the most
widely used costimulatory domains. Studies showed
that CD28 endued CAR T cells with stronger killing
ability, while 4-1BB granted longer persistence in vivo.
Expansion of CAR T cells is essential. The CAR T
cells are first expanded ex vivo to a goal cell count
around 3 × 106 cells/kg [
]. Following infusion,
circulating CAR T cells expand in vivo by 1000 folds
within 7–14 days. CAR T cells have the potential to
enter cerebrospinal fluid, exerting anti-leukemic activity in
this sanctuary site, and persist after infusion .
However, the persistence of CAR T cells within patients varied
across the studies. Stephan et al. report persistence of
CD19-CAR T cells for 3–39 months after infusion in
patients with ongoing responses, whereas Davila et al. found
the levels undetectable by 3 months [
Clinical outcome of CD19-targeted CAR T cells in R/R ALL
CAR T cell therapy has achieved promising results in
multiple clinical trials (Table 3). The majority of the
trials are led by three research institutes—the University
of Pennsylvania (UPenn), the National Cancer
Institute (NCI), and the Memorial Sloan Kettering Cancer
1 or 2 × 106
CAR T cells/kg
1–17.4 × 106
0.2–4 × 106
4-1BB, Lentivirus Children and young adults Investigator’s
with R/R ALL, n = 53 choice
R/R ALL aged ≥ 18
years (ZUMA-3) or 2–21
years (ZUMA-4) with ≥ 25%
Ph+ ALL and low-burden
central nervous system
disease are eligible
4-1BB, Lentivirus Adult with R/R ALL,
n = 30
Cy ± etoposide
2 × 105 or 2 × 106 CR: 27/29 (93%)
or 2 × 107 cells/kg
4-1BB, Lentivirus Children and young adults Flu/cy
with R/R ALL, 29 pts
reaching D28 prior to the
MSKCC Memorial Sloan Kettering Cancer Center, NCI National Cancer Institute, FHCRC Fred Hutchinson Cancer Research Center, UPenn University of Pennsylvania,
CHOP Children’s Hospital of Philadelphia, Cy cyclophosphamide, Flu fludarabine, FLAG fludarabine, high-dose cytarabine, and G-CSF
A recent study from MSKCC reported the efficacy of
CAR T cell therapy in 44 adults with R/R B ALL. Of the
43 patients evaluable for response, 36 patients (84%)
achieved CR after 19-28z CAR T cell (JCAR015, Juno
Therapeutics, Seattle, WA) infusion. Among them, 29
(83%) achieved MRD negativity (MRD-CR). The median
OS for all patients and MRD-CR patients was 8.5 and
10.8 months, respectively. Posttreatment MRD status
emerged as a strong predictive marker of OS. The OS at
6 months was 76% (95% CI 51–89) in the MRD-CR
cohort vs. 14% (95% CI 8–45) in the MRD+ CR cohort.
However, this study did not show significant survival
superiority in patients who underwent allo-HSCT after
CAR T cell infusion than those who did not (OS at
6 months was 70% in patients who underwent allo-HSCT
vs. 64% in patients who did not receive allo-HSCT) [
In contrast, researchers from NCI showed that long-term
outcomes were superior among patients with post-CAR T
HSCT. In their study, 31 (60.8%) of 51 R/R ALL patients
achieved a CR with 28/31 (90%) of responders negative
for MRD. Relapse was significantly more common in
patients who did not have a HSCT after CAR T therapy
(6/7; 85.7%) compared to those who did (2/21; 9.5%)
(p = 0.0001). Even when counting in the transplant-related
mortality, the median leukemia-free survival (LFS) in
the HSCT group was significantly longer (HR = 16.9,
p = 0.0006) [
]. Based on the aforementioned excellent
outcomes, a multicenter, phase 1/2 study called ZUMA-3
and ZUMA-4 that enroll adult (aged ≥ 18 years) and
pediatric (aged 2–21 years) R/R ALL patients were started
]. Researchers from the UPenn also reported their
experience with CTL019 (Novartis, Basel, Switzerland) in
patients with R/R ALL. In a recent study of largely
pediatric patients, 94% (50/53) achieved CR, with MRD
negativity in 47 responding patients. RFS was 72% at
6 months (95% CI, 59–87%) and 44% at 12 months (95%
CI, 30–65%), and OS was 78% at 12 months (95% CI, 67–
]. This single-center trial of CTL019 for R/R ALL
showed prolonged CAR T cell persistence along with
long-term CR without further therapy in the majority of
patients. However, the high efficacy and a similar safety
profile of CTL019 have yet to be reproduced in
multicenter trials, such as the first US multicenter trial (ENSIGN)
and the first global trial (ELIANA) [
Although the above studies differed in CAR designs, T
cell manufacturing, conditioning regimens, patients’ age,
leukemia burdens, and T cell dosages, each trial was
comparably effective in treating R/R ALL, reaching 90%
even in heavily pretreated patients. As a result, these
preliminary clinical trials have paved the way for more
In addition to autologous CD19-CAR T cells, the
potential of donor-derived CD19-CAR T cells to treat
relapse after allo-HSCT was also intriguing [
preliminary results have been reported [
et al. conducted a clinical trial using allogeneic
donorderived CAR T cells in B cell malignancy patients who
relapsed after allo-HSCT . Eight of the 20 treated
patients obtained remission. None of the patients
developed new onset graft-versus-host disease (GVHD) after
the infusion. The result was similar with a previous
study from Baylor College of Medicine using
donorderived CD19-redirected virus-specific T cells, in which
no evidence of GVHD was reported [
]. However, in
another study, Dai et al. reported for the first time of
GVHD occurrence in 2 of the 3 patients that received
donor-derived CAR T cells [
]. The GVHD might be
related to a higher dose of infused CAR T as well as
mixed chimerism in recipients. In addition, different
CAR designs may also play a role. Using a mouse
alloHSCT model, Smith et al. found that adoptive transfer
of murine donor m1928z CAR T cells (CAR with CD28
costimulatory domain) caused significantly less GVHD
compared to m19delta T cells (CAR with no
costimulation) and m19BBz T cells (CAR with 4-1BB
costimulatory domain) [
]. This result is in accordance with the
clinical observations, as the 4-1BB domain was
incorporated in the donor-derived CAR T cells that caused
GVHD in Dai’s case [
Even more interestingly, allogeneic CD19 CAR T cells
could be applied to patients without prior HSCT. The
infusion of allogeneic CAR T cells could potentially augment
the killing effect through additional alloreactive-attacking
capability. Cai et al. reported a case where the patient
received co-infusion of haplo-identical allogeneic CAR T
cells and mobilized peripheral blood stem cells (PBSCs)
following induction therapy. The patients achieved
MRDCR and full donor engraftment, with only mild toxicity
]. Although the result seems intriguing, potential
complications such as GVHD need further study.
Toxicities of CD19-targeted CAR T cells
Toxicities associated with CAR T cell therapy are reported
in nearly all clinical trials. Most toxicities were mild and
reversible, including fever, chills, hypotension, dyspnea,
headaches, and fatigue. The three main concerning
toxicities are on-tumor off-target toxicity, CRS, and
neurotoxicity. On-tumor off-target toxicity is related to the inability
of CAR T to distinguish between tumor and normal cells,
since the CD19 antigen is homogeneously expressed by
both normal and malignant B lymphocytes. The toxicity
can lead to B cell aplasia, which might cause
agammaglobulinemia. Low immunoglobulin level will increase the
risk of opportunistic infections. But it is preventable with
immunoglobulin infusions and/or administration of
antibiotics. CRS is caused by significant production of
inflammatory cytokines secreted by activated CAR T cells
]. The onset of CRS is variable, ranging from
24 h to 3 weeks following CAR T infusion [
While mild CRS is reported in almost all patients,
severe CRS occurs in about 30% of patients. Patients with
high leukemia burden are at increased risk of more
severe CRS, presenting as vascular leak and hypotension
leading to multi-organ system dysfunction. These severe
complications will often need aggressive medical
management, including hemodynamic support, mechanical
ventilation, and application of tocilizumab (an IL-6 inhibitor)
and, in life-threatening conditions, corticosteroids. It is
worth noting, however, that administration of
corticosteroids may also minimize or eliminate the CAR T cell
activity. Neurological toxicities are also accompanied by CAR
T infusion, including delirium, dysphasia, akinetic mutism,
and seizures. In November 2016, two patients died from
cerebral edema in a phase II ROCKET study testing the
investigational anti-CD19 CAR T cell JCAR015 (Juno
Therapeutics, Seattle, WA). Considering in the previous
three deaths in July 2016, the trial was halted due to
concern of safety [
]. Juno highlighted the lower levels
of toxicity in both ALL and other B cell malignancies
using their 4-1BB CAR T products with defined
CD4:CD8 composition (JCAR017 and JCAR014). Thus,
they speculated the flu/cy conditioning regimen or the
CD28 costimulatory domain used in JCAR015 that may
trigger more rapid expansion of T cells and disruption of
the blood-brain barrier might be the culprits. However,
other groups (such as Kite/NCI) using CD28 CARs
appeared to have no significant neurotoxicity, even though
they also used flu/cy for conditioning. Thus, the etiology
Causes and management of relapse after CAR T therapy
If CAR T could be durably detected in the recipients,
the chances of relapse would decrease [
]. In one study,
loss of functional persistence of CAR T was associated
with CD19+ relapse with a hazard ratio (HR) of 34
(p = 0.013) [
]. Some studies sought to prolong CAR T
persistence by reinfusion of CAR T in patients with
evidence of poor persistence. However, investigators from
Fred Hutchinson Cancer Research Center (FHCRC)
detected a cytotoxic CD8+ T cell response to CAR in some
patients who failed to achieve engraftment of CAR T
cells after second infusions; epitope mapping in 1 patient
identified immunogenic epitopes within the murine
FMC63 scFv [
]. Thus, repeated infusion would be
ineffective in this subset of patients with rapid CAR cell
loss mediated by immune rejection. In this condition,
retreatment with CD19-directed CAR T expressing a
humanized scFv may induce second remission of ALL [
UPenn has published the results of a phase I study of
humanized CTL119 at the 2016 ASH meeting. Thirty
children and young adults with R/R B ALL with or
without prior exposure to CAR T cells were enrolled. They
found CTL119-induced remissions in 64% (7/11) of
patients previously treated with murine CD19-directed
CAR T cells and 100% of CAR-naïve patients [
Inadequate lymphodepletion may be another reason for
limited CAR T persistence. Indeed, addition of fludarabine
to the lymphodepletion regimen was showed to enhance
CAR T persistence and prevent transgene rejection [
In addition, combining CAR T therapy with other
medications such as PD-1 inhibitor [
] or BTK inhibitor
] was also showed to improve CAR T cell persistence.
Loss of surface CD19 expression is another cause of
]. Interestingly, Lacey et al. reported a case
where the relapsed CD19-negative leukemia was
originated from a single leukemic cell accidentally transduced
with CAR19 that survived the manufacturing process.
This leukemic clone evaded CTL019 detection via
downregulation of the target antigen in a cell
autonomous fashion [
]. Recently, some promising studies
investigated the use of CAR T targeting CD22 to treat
CD19-negative relapses [
]. Targeting additional B
cell antigens is an active area of investigation.
With the rapid development of naked monoclonal
antibodies, antibody-drug conjugates, bispecific T cell
engagers, and adoptive T cell therapies, the era of R/R ALL
treatment is fast forwarding. CD19, CD20, CD22, and
CD52 are the main markers presented in the majority of
B ALL patients and thus are targeted in
immunotherapies. With CAR T therapy moving to the market,
achieving higher CR rates than frontline chemotherapies
or even chemotherapies combined with mAbs, the roles
of mAbs need to be newly defined. Monoclonal antibodies
may still play important roles in induction therapy of ALL
due to their availability and relatively safe profile. In the
future, immunotherapies may substitute or allow a lower
dose of chemotherapy to achieve long-term remission in
ALL patients, even in those with poor performance status
who are otherwise ineligible for traditional chemotherapy.
Despite high remission rate that could be achieved by the
aforementioned therapies, relapse still remains as the
major problem. As the choices of targeted and immune
therapies increase, we must look beyond high remission
rates and search for the strategies to prevent relapse and
ADC: Antibody-drug conjugate; ADCC: Antibody-dependent cell-mediated
cytotoxicity; ALL: Acute lymphoblastic leukemia; BiTE: Bispecific T cell
engager; CAR: Chimeric antigen receptor; CDC: Complement-dependent
cytotoxicity; CR: Complete remission; CRi: Complete remission with
incomplete blood cell count recovery; CRp: Complete remission with
incomplete platelet recovery; CRS: Cytokine release syndrome; EFS: Event-free
survival; HR: Hazard ratio; HSCT: Hematopoietic stem cell transplantation;
INO: Inotuzumab ozogamicin; mAb: Monoclonal antibody; MRD: Minimal
residual disease; ORR: Overall response rate; OS: Overall survival; R/R:
Refractory/relapsed; RFS: Relapse-free survival
This work was jointly supported by the Zhejiang Province Natural Science
Funds (LY13H080002) and National Natural Science Funds of China (81470307).
Availability of data and materials
Data sharing is not applicable to this article as no datasets were generated
or analyzed during the current study.
GW, JW, and YZ drafted the manuscript. HH helped to review the manuscript.
All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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