cancers

Review
Optimal Use of Novel Immunotherapeutics in B-Cell
Precursor ALL
Federico Lussana 1,2, * , Gianluca Cavallaro 1,2 , Pantaleo De Simone 1,2 and Alessandro Rambaldi 1,2

1 Department of Oncology and Hematology, University of Milan, 20122 Milan, Italy

2 Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, 24127 Bergamo, Italy
* Correspondence: [email protected]; Tel.: +39-03-5267-3639

Simple Summary: In this review, we discussed the impact of novel immune therapies on the
treatment of child and adult acute lymphoblastic leukemia (ALL). Based on the promising results
achieved in the relapsed/refractory (R/R) setting, several trials are currently evaluating the clinical
benefit of incorporating these new drugs into frontline treatments. The results emerging from the
most recent studies are opening new avenues to further the significant improvements in the clinical
outcome of this disease.

Abstract: Novel immune therapies are currently being used for patients with R/R ALL based on
their ability to induce not only hematologic but also molecular remission. Despite promising results,
specific clinical conditions, such as high tumor burden or extra medullary relapse, are still associated
with a remarkably poor clinical outcome. Therefore, how to optimize the choice and the timing of such
new treatments within different clinical settings remains a matter of debate. In addition, with the aim
of increasing the rate and depth of molecular remission, clinical studies are currently evaluating the
combination of these immunotherapies with chemotherapy in the contest of frontline treatment. The
preliminary data suggest that this approach may increase the cure rate and perhaps reduce the use of
allogeneic stem cell transplantation (alloHSCT) in first remission. In Ph-positive ALL, reproducible
results are showing that frontline treatment programs, based on the combination of tyrosine kinase
inhibitors and immunotherapy, can achieve unprecedented rates of hematologic and molecular
remission as well as a long-term cure, even in the absence of chemotherapy and alloHSCT. The results
Citation: Lussana, F.; Cavallaro, G.; from these studies have led to the development of potentially curative treatment modalities, even for
De Simone, P.; Rambaldi, A. Optimal
older ALL patients who cannot be treated with conventional intensive chemotherapy. The present
Use of Novel Immunotherapeutics in
review examined the evidence for an appropriate use of the new immunotherapies in ALL patients
B-Cell Precursor ALL. Cancers 2023,
and provided some appraisal of the current and future possible uses of these drugs for achieving
15, 1349. https://doi.org/10.3390/
further therapeutic improvement in the treatment of this disease.
cancers15041349

Academic Editors: Elisabetta Keywords: B-cell precursor acute lymphoblastic leukemia; relapsed/refractory; blinatumomab;
Abruzzese and William Arcese inotuzumab; CAR-T cells
Received: 6 January 2023
Revised: 5 February 2023
Accepted: 15 February 2023
Published: 20 February 2023 1. Introduction
Acute lymphoblastic leukemia (ALL) is a hematologic malignancy that originates
from the proliferation of a single B- or T-lymphocyte progenitor, which proliferates and
accumulates in the blood; the bone marrow; and, possibly, the extramedullary sites. This
Copyright: © 2023 by the authors.
disease represents the most frequent cancer in the pediatric population, especially in infants
Licensee MDPI, Basel, Switzerland.
aged 1–4 years, with a subsequent decrease in the overall incidence which reaches its lowest
This article is an open access article
point in young adults aged 25–45 years. The majority of ALL is of B-cell origin. Remarkable
distributed under the terms and
advances have been achieved in the treatment of ALL thanks to the optimal use of intensive
conditions of the Creative Commons
chemotherapy, the implementation of minimal residual disease monitoring, and better sup-
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
portive care, allowing most pediatric and adult patients to achieve a complete hematologic
4.0/).
remission (CR). Despite these significant advances in treatment, less than 50% of adult

Cancers 2023, 15, 1349. https://doi.org/10.3390/cancers15041349 https://www.mdpi.com/journal/cancers

Cancers 2023, 15, 1349 2 of 23

patients survive 5 years or more, and about 15–20% of children suffer a relapse. Relapsed
or refractory (R/R) ALL is still a major concern, with a dismal prognosis in both children
and adults and with an overall survival of 3 to 12 months depending on the duration of
the first remission and the number of lines of salvage therapy. In the last few years, the
advent of novel targeted immunotherapies, specifically blinatumomab, inotuzumab, and
chimeric antigen receptor T cells (CAR-T cells), has changed the therapeutic landscape of
R/R B-cell precursor ALL (BCP-ALL). These novel immunotherapies showed impressive
results in terms of response rates and a greater likelihood of proceeding to allogeneic stem
cell transplantation (alloHSCT) consolidation, thus significantly changing the perspectives
of this very unfavorable condition. Based on the promising results in the R/R setting,
immunotherapies are currently under investigation in the early phases of the disease. More-
over, these targeted immunotherapies are usually well tolerated, so the associated clinical
benefit is not limited to younger and fit patients but also to older and frail patients. The
main objectives of this review were to focus on how to choose among these novel agents
according to specific conditions and to highlight the important challenges to be addressed
in the coming years.

2. Immunotherapies
2.1. Blinatumomab
Bispecific T-cell engagers (BiTE) are a class of bispecific antibodies designed for cancer
immunotherapy [1]. Among these, blinatumomab is a bispecific T-cell engager with a
dual affinity for CD3 and CD19 that brings effector T lymphocytes and target B cells into
close proximity, allowing T cells to recognize and exert their cytotoxic activity against
CD19-positive B cells. Blinatumomab has been approved by the US Food and Drug Ad-
ministration for the treatment of pediatric and adult patients with R/R BCP-ALL as well
as for the treatment of those with measurable residual disease (MRD). Based on the re-
sults of the phase II studies on R/R adult ALL [2,3], the current treatment schedule is
based on a ramp-up with 9 mcg daily over the first week followed by 28 mcg daily for
28 days in each subsequent cycle. The efficacy and safety of blinatumomab for the treat-
ment of adult patients with R/R Ph-negative B-cell precursor ALL have been demonstrated
through the pivotal, phase III TOWER study (ClinicalTrials.gov identifier: NCT02013167)
that randomized (2:1 ratio) 405 patients to either blinatumomab monotherapy or multia-
gent chemotherapy [4]. Compared with the chemotherapy arm, the blinatumomab-treated
patients achieved a significantly better complete remission (CR, 34% versus 16%), event-
free survival (EFS, 31% versus 12%), and median overall survival (OS, 7.7 months versus
4.4 months), no matter their age, prior to salvage treatment or prior to alloHSCT. Among
the patients who achieved CR, 76% were MRD negative in the blinatumomab treatment
group versus 48% in the chemotherapy group. These results have been confirmed in
the pediatric setting. In a phase I/II dose-escalation/dose-expansion trial conducted on
Ph-negative ALL patients with a refractory disease or a disease relapsed after at least two
lines of therapy or alloHSCT, blinatumomab showed a complete remission rate of 39%,
with MRD negativity in 52% of the responders. The median OS was 7.5 months [5]. In a
phase III randomized study AALL1331 trial (ClinicalTrials.gov identifier: NCT02101853),
the effect of postreinduction/consolidation with blinatumomab vs. chemotherapy was
evaluated in patients < 30 years. Blinatumomab proved superior compared to chemother-
apy, with a 2-year OS rate of 71% vs. 58%. The rate of MRD negativity was 75% for the
blinatumomab group vs. 32% for the chemotherapy group (p < 0.001). Given the better
response, the likelihood of proceeding alloHSCT was greater for the blinatumomab group
(70% vs. 43%) [6]. In a parallel study, blinatumomab, compared with intensive multidrug
chemotherapy, before alloHSCT was confirmed to have a clinical benefit, with an improved
EFS (66% vs. 27%) and OS (85% vs. 70%) [7].
The efficacy of blinatumomab has also been tested as a single agent in patients with
R/R Ph+ ALL (ALCANTARA; ClinicalTrials.gov identifier: NCT02000427), showing a
response rate of 36% associated with a rate of complete MRD response of 88%, which was
Cancers 2023, 15, 1349 3 of 23

close to that reported in R/R Ph-negative ALL. Among the responders, 44% proceeded to
alloHSCT. After a median follow-up of 16.1 months, the median relapse-free survival (RFS)
was 6.8 months, while the median OS was 9.0 months in the whole cohort and 19.8 months
in the blinatumomab responders [8]. A propensity score analysis was also performed to
compare the outcomes of the patients treated with blinatumomab in the AZCANTARA
study with those of an external historical cohort of patients receiving standard chemother-
apy. A higher rate of CR/CRh (36% vs. 25%) and a better OS with a hazard ratio of 0.81
(95%CI, 0.57 to 1.14) was seen after treatment with blinatumomab [9].
Based on the significant results obtained in the R/R setting, blinatumomab was
also evaluated in patients with molecular evidence of MRD in their first or later CR. In
a multicenter phase II study, which was conducted in 116 adult patients with ALL in
hematologic CR but with a high MRD level (>10−3 ), blinatumomab induced an excellent
78% molecular response rate after the first cycle. The median OS was 36.5 months, and a
prolonged survival was occasionally achieved without alloHSCT [10]. Interestingly, for the
patients in first CR achieving MRD negativity, there was no evidence that the OS improved
in patients who did or did not undergo transplantation. On the contrary, the outcomes
of the patients receiving blinatumomab who were in their second or later CR and who
did not proceed to HCT were inferior [10]. On the basis of these results, the US Food
and Drug Administration (FDA) and the European Medicines Agency (EMA) granted the
first approval for the treatment of MRD with blinatumomab. In a large real-world study,
blinatumomab confirmed strong efficacy outcomes in adults with MRD+ B-cell precursor
ALL, with 91% of Ph− and 59% of Ph+ patients achieving an MRD response within two
cycles of blinatumomab. The overall MRD response did not differ by the CR state (CR1 or
CR2) in both the Ph− and the Ph+ subgroups [11].
Finally, an escalation phase Ib study (NCT04521231) is investigating the safety and
efficacy of subcutaneous blinatumomab for the treatment of adults with R/R BCP-ALL.
Blinatumomab given subcutaneously does not only simplify its administration but also
maximizes the pharmacokinetics and dynamics while decreasing the patients’ discomfort.
Preliminary results are in keeping with this hypothesis [12].
To sum up, despite the high rate of responses leading to the approval of blinatumomab
as a single agent in R/R ALL, the cure rate remains rather limited without subsequent
consolidation with alloHSCT [13]. Based on these observations and on the optimal results
of blinatumomab in the treatment of MRD positive patients, an anticipated use of this drug
in frontline therapy seems to be the best way to optimize its clinical benefit as we discuss
later in this review.

2.2. Inotuzumab Ozogamicin

Inotuzumab ozogamicin is an anti-CD22 antibody conjugated to calicheamicin. Its
regular approval by the FDA and the EMA for R/R B-cell precursor ALL came from a phase
III trial, the INOVATE trial (ClinicalTrials.gov identifier: NCT 01564784) [13]. Compared
with the conventional chemotherapy arm, patients treated with inotuzumab achieved a
significantly higher rate of CR and CRi (81% versus 29%) and had a significantly better
OS (median of 7.7 versus 6.7 months) and PFS (5 versus 1.8 months). Among the patients
who achieved CR, 78% were found to be MRD negative through flow cytometry in the
inotuzumab treatment group versus 28% in the chemotherapy group. The duration of
remission was in favor of inotuzumab as confirmed by a long-term follow-up reporting
a 3-year rate of 20.3% versus 6.5% in the control group [14]. Interestingly, inotuzumab
remained efficacious in patients with a high baseline disease burden, and this did not
seem to negatively impact the safety profile of this drug [15]. The main inotuzumab
toxicity causing concern was hepatoxicity, including a more frequent incidence of veno-
occlusive disease (VOD) (13% vs. 1%). About 3% of cases occurred in patients receiving
inotuzumab therapy alone, while most cases occurred after alloHSCT and with the use of a
dual-alkylator conditioning regimen.
Cancers 2023, 15, 1349 4 of 23

Nonetheless, the survival with the inotuzumab single agent remained suboptimal. For
this reason, inotuzumab was combined with low-intensity chemotherapy based on 8 cycles
of mini-hyper-CVD with or without blinatumomab in 96 patients with BCP-ALL in their
first relapse (29 with blinatumomab and 67 without). The majority of the patients achieved
CR or CRi (80% for the entire cohort and 91% for those in their first relapse). Among
75 evaluable patients, for MRD assessment through MFC, 62 patients (83%) achieved nega-
tive MRD, with higher rates of MRD negativity in those in their first relapse (89%). Forty-
four patients (46%) received alloHSCT. The 3-year OS rate was 33% for the entire cohort
and 42% for those in their first relapse, which was superior to that observed with either ino-
tuzumab or blinatumomab single agents when using a propensity score analysis [16–19]. In
the SWOG1312 phase I trial, inotuzumab was combined with reduced-intensity chemother-
apy with encouraging results. The last reported data showed a composite rate of remission
of 61% among 23 heavily pretreated patients. The treatment was well tolerated, with three
patients experiencing hepatic VOD after their second transplantation or when receiving
the higher dose of inotuzumab [20]. Recently, the single-arm phase II trial AALL1621
(ClinicalTrials.gov identifier: NCT02981628), conducted by the Children’s Oncology Group,
showed that inotuzumab is effective and well tolerated in heavily pretreated children and
adolescents (age of 1–21 years) with R/R CD22-positive BCP-ALL. VOD after hematopoietic
stem-cell transplantation and prolonged cytopenias were notable toxicities, while partial
CD22 expression and lower CD22 site density were associated with a lower likelihood of
response [21].
In summary, inotuzumab is approved as a single agent for relapsed/refractory B-cell
precursor ALL due to the high rate of response in terms of CR and MRD negativity, but
its efficacy remains limited by a short duration of response. As mentioned later in the
review, to improve the clinical benefit of inotuzumab, ongoing studies are exploring its
use in frontline therapies or as a bridge to allogeneic transplants or CAR-T cell therapy.
Finally, VOD is still a concern, but the hematologic community is progressively learning to
prevent this serious complication by avoiding a dual-alkylator conditioning regimen and
by increasing the time between the last dose of inotuzumab and the conditioning regimen.

2.3. Chimeric Antigen Receptor T Cells

Cellular immunotherapy with chimeric antigen receptor T-cell (CAR-T cell) anti-CD19
represents an attractive therapeutic option in the R/R setting. Different generations of
CAR-T cells have been developed with different costimulatory domains (commonly 4–1 BB
or CD28), which are relevant due to their expansion and persistence [22]. Tisagenlecleucel
(Tisa-cel) is the first CAR-T cell medication approved for R/R ALL for those up to 25 years
of age. In the first phase I/IIa study, in 30 patients, the CR rate was 90%, 88% of whom
achieved MRD negativity [23]. The responses were quite durable from 2 to 24 months,
with 68% CAR-T cell persistence and B-cell aplasia at 6 months. A subsequent larger study
including 79 children and young adults showed a CR rate and concomitant MRD negativity
of 82%. In the long-term follow-up, the 5-year EFS and OS rates were 42% (95%CI, 29–54)
and 55% (95%CI, 43–66), respectively [24,25]. In a pediatric real-life analysis, Tisa-cel was
confirmed to be an effective salvage treatment. It is worth remembering that a high disease
burden, defined as bone marrow blasts ≥ 5%, was associated with a significantly worse OS
(58% vs. 85%) [26].
Based on the excellent remission rates and the significant clinical benefit observed
particularly in the responding patients who achieved MRD negativity, the COG cooperative
group launched the first trial of the earlier use of CAR-T cell therapy in the setting of patients
with MRD positivity. The AALL1721/Cassiopeia study (Clinical Trials.gov identifier:
NCT03876769) was a phase II single-arm trial of tisagenlecleucel in children and young
adults in CR1 with high-risk criteria and persistent MRD positivity (>0.01% through flow
cytometry) at the end of chemotherapy. The primary end point of this study was the 5-year
DFS, and the secondary end points included the overall survival, safety, the percentage of
patients in remission without alloHSCT at 1 year, and the time to reach B-cell recovery.
Cancers 2023, 15, 1349 5 of 23

In the adult patients, although the impressive rate of CR was confirmed, the long-term
outcomes achieved with academic CAR-T cells were initially less significant, with a median
EFS and OS of 6.1 months and 12.9 months [27]. The largest set of safety and efficacy data
on Tisa-cel in the real-world setting included 255 children and young adult ALL patients.
With a median follow-up of 13.4 months and a CR rate of 85.5%, the 12-month duration of
response (DOR), EFS, and OS rates were 60.9%, 52.4%, and 77.2%, respectively [28].
More recently, the single-arm ZUMA-3 trial evaluated KTE-X19 CAR-T cell therapy in
the largest population of adults with R/R B-cell precursor ALL to date. KTE-X19 allowed
39 patients (71%) to achieve CR or CRi, and 97% of these responders obtained MRD
negativity. After a median follow-up of 16 months, the median overall survival was
18 months across all the treated patients. The safety profile of KTE-X19 was manageable
and was without deaths due to cytokine release syndrome. These survival outcomes com-
pare favorably with those achieved by blinatumomab and inotuzumab, leading the FDA
to approve KTE-X19 as the first CAR-T cell product for adult ALL patients. [29]. Overall,
however, a very recent systematic review of 16 studies involving 489 adults confirmed
a very high early remission rate; however, the duration of response remains challeng-
ing [30], and the need for a subsequent allogeneic transplantation is often considered to be
necessary [31–34].
A novel second-generation CD19 CAR-T cell therapy with a fast binding off rate for
CD19 (AUTO1), designed with the aim to reduce toxicity and to improve persistence,
was tested in a phase I study of 20 adult patients with R/R ALL. Interestingly, the study
design planned a double infusion of CAR-T cells at different dosages according to the blast
percentage. The preliminary results of this study were encouraging, with a high remission
rate (85% had a MRD negative response) and an EFS and OS at 12 months of 48% and
64%, respectively. No patients experienced ≥ grade III CRS, and 15% experienced grade III
neurotoxicity that resolved within 72 h with steroids [35]. To improve the clinical results
and to shorten the time from cell collection to infusion into the patient, new laboratory
approaches have been developed by our Chinese colleagues, such as the so-called FasT
CAR-T (F-CAR-T) cell next-day manufacturing platform. The underlying idea is to freeze
the cells a few hours after the viral transduction and to allow CAR-T cell expansion in vivo
after the cells are thawed and infused back into the patient. Reducing the duration of
the ex vivo culture should limit the differentiation of T cells, favoring the preservation
of naïve and stem cell memory T cells in the final product. This is expected to be useful
for obtaining a longer CAR-T cell persistence and, consequently, higher response rates
and a longer duration of response. The preclinical studies and the first human clinical
studies confirmed a superior in vivo CAR-T cell proliferation and expansion compared to
conventional CAR-T cells, which showed a younger phenotype [36]. A similar clinical trial
is also currently ongoing in the US and in Europe with a novel autologous CD19-targeting
CAR-T cell manufactured using the T-Charge™ Platform (YTB323) for the treatment of
R/R CD19-positive lymphoid neoplasms. The T-Charge™ Platform minimizes the ex
vivo culture time and reduces the manufacturing process time to < 2 days. This novel
platform also preserves naïve/TSCM cells in the final product, leading to a potentially
higher potency and persistence [37] (Clinical Trials.gov identifier: NCT03960840).
New approaches to overcoming the main obstacles of CAR-T cell therapy, such as
the high costs and logistical complexity of the transduction viral process, and to allowing
lymphodepleted patients to access CAR-T cell treatment are underway. In addition, the
time of production may be clinically relevant, as some patients may face disease progression
during this time, precluding the treatment itself. In this regard, to replace patient-derived
CAR-T cells with ready-to-go cellular products, such as off-the-shelf allogeneic CAR-T
cells, represents an ambitious goal of the ongoing research. However, the possibility of life-
threatening graft-versus-host disease (GvHD) with an allogenic cell therapy product raises
major concerns. Therefore, clinical research with immune cells other than “conventional” T
cells is underway (Figure 1A,B).
Cancers 2023, 15, 1349 6 of 25

CAR-T cells, represents an ambitious goal of the ongoing research. However, the possibil-
ity of life-threatening graft-versus-host disease (GvHD) with an allogenic cell therapy
Cancers 2023, 15, 1349 6 of 23
product raises major concerns. Therefore, clinical research with immune cells other than
“conventional” T cells is underway (Figure 1A,B).

Figure 1. Novel approaches for exploration of immunotherapies in relapse/refractory BCP-ALL.

Figure 1. Novel approaches for exploration of immunotherapies in relapse/refractory BCP-ALL.
Panel (A) new approaches with different adoptive cell therapies in relapse/refractory BCP-ALL
Panel
Panel (A)
(B) new approaches with
key characteristics of different adoptive cell therapies
the new manufacturing in relapse/refractory
platforms BCP-ALL
for the fast production Panel
of CAR-T
(B) key characteristics of the new manufacturing platforms for the fast production of CAR-T cells.
cells.

ALL
ALL represents
representsacute
acutelymphoblastic
lymphoblasticleukemia,
leukemia,CAR
CAR represents chimeric
represents antigen
chimeric recep-
antigen re-
tor, CIK represents cytokine-induced killer cells, CRS represents cytokine release syndrome,
ceptor, CIK represents cytokine-induced killer cells, CRS represents cytokine release syn-
ICANS represents immune-effector-cell-associated neurotoxicity syndrome, NK represents
drome, ICANS represents immune-effector-cell-associated neurotoxicity syndrome, NK
natural killer, GVHD represents graft-versus-host disease, PD represents programmed
represents natural killer, GVHD represents graft-versus-host disease, PD represents pro-
death, T-reg represents regulatory T cells, and TRAC represents T-cell receptor alpha
grammed death, T-reg represents regulatory T cells, and TRAC represents T-cell receptor
constant gene.
alpha constant gene.
The figure was created with BioRender.com.
The figure was created with BioRender.com.
In a recent phase I and II clinical trial (NCT03389035), we examined the treatment
feasibility of BCP-ALL patients who relapsed after alloHSCT with donor-derived anti-
CD19 CAR-T cells (CARCIK-CD19) engineered with the sleeping beauty (SB) transposon.
A complete response was achieved in 18 out of 27 patients (66.7%) and in 16 out of the
21 patients treated with the 2 highest doses (76.2%). A total of 14 (77.8%) of the overall
Cancers 2023, 15, 1349 7 of 23

responders and 13 of the responders at the highest doses (81.3%) were MRD negative. With
a median follow up of 2.8 years, a significantly better EFS was observed in the patients
treated with the highest doses (p = 0.0265). For the 21 patients treated at the 2 highest
dose levels, the median EFS and OS were 4 and 12 months, respectively. The median DOR
of the 16 patients in CR was 9.5 months, with a 6-month DOR of 54.4% (SE = 13.8). CRS
occurred in nine patients (four patients with grade I CRS and five with grade II CRS), and
immune-effector-cell-associated neurotoxicity (ICAN) (grade III) occurred in two patients
at the two highest doses. Although 10 out of 27 had experienced GvHD after the previous
alloHSCT, GvHD never occurred after treatment with CARCIK-CD19 [38,39]. In keeping
with our results, a very recent phase I study of 25 patients treated with allogeneic CAR-
T cells (UCART19), manufactured from healthy donor peripheral blood, confirmed the
safety profile of allogeneic CAR-T cells, with a low risk of inducing GvHD (only 2 patients
developed grade I acute cutaneous GvHD) [40].
Due to their ability to recognize tumor cells in a non-HLA (human leukocyte antigen)-
restricted manner, other “nonconventional” allogeneic cells used in CAR-T cell technology
are natural killer (NK) cells. Preliminary promising results have been reported in different
tumors, such as multiple myeloma or neuroblastoma [41,42], but the observed short-term
responses remain as the main concern for the use of this cell therapy. Liu and colleagues
published a small study including 11 patients with CD19-positive lymphoid tumors. Eight
patients (73%) achieved a hematologic response, and seven (four with lymphoma and
three with CLL) had complete remission. The responses were rapid and were seen within
30 days after infusion at all dose levels. However, most patients had short-term responses,
and five of eight patients needed post-remission therapy [43].
In conclusion, CAR-T cell therapy represents a potential curative treatment for R/R
ALL, particularly when used for a low burden relapse or MRD positive disease. Given
the complexity of this therapy, its possible early placing in ALL therapy remains a major
open spot. Moreover, a subsequent allogeneic transplant is a matter of debate, and some
biological parameters, such as B-cell aplasia and CAR-T cell persistence, could be helpful
parameters in this difficult choice. A longer follow-up on the pivotal clinical trial will
probably address some of these questions. Finally, there are many new platforms developed
with the aim to make manufacturing easier and faster as well as to improve cell therapy
efficacy. In this regard, it is worth noting that the time of production may be clinically
relevant, as some patients may undergo clinical disease progression during this time,
precluding the treatment itself.
A summary of the evidence leading to the regulatory approval of the immunotherapies
(blinatumomab, inotuzumab, tisagenlecleucel, and brexucabtagene autoleucel) for R/R
ALL is presented in Table 1.
Cancers 2023, 15, 1349 8 of 23

Table 1. Summary of the main clinical studies on the immunotherapies approved for treating R/R BCP-ALL (blinatumomab, inotuzumab, tisagenlecleucel, and
brexucabtagene autoleucel).

Trial/Study Setting Study Design Total Patients Immunotherapeutic Treatment Main Results
[Reference]
CR: blinatumomab 34% vs. ChT 16%.
Blinatumomab 9 µg/day first Molecular response: blinatumomab 76% vs.
Tower 2017 [13] R/R Ph-negative adults Phase III 271 blinatumomab vs. 134 ChT
randomized trial week then 28 µg/day c.i. ChT 48%. Median RFS: blinatumomab 7.3
vs. ChT 4.6 months. OS: blinatumomab 7.7
vs. ChT 4.0 months.
Molecular response: blinatumomab 75% vs.
AALL1331 [6]
R/R Ph-negative children and
Phase III randomized trial 107 blinatumomab vs. 109 ChT Blinatumomab 15 µg/m2 day c.i.
young adults (1–30 years) 32% ChT. RFS: blinatumomab 54% vs. ChT
for 28 days 39%. OS: blinatumomab 71% vs. ChT 58%.
54 blinatumomab vs. 54 ChT for Minimal residual disease remission:
Locatelli et al. [7]
High risk first relapse Ph-negative
Phase III randomized trial the 3rd consolidation Blinatumomab 15 µg/m2 day c.i. blinatumomab 90% vs. ChT 54%. EFS:
children before alloHSCT for 28 days blinatumomab 66% vs. ChT 27%. OS:
blinatumomab 85% vs. ChT 70%.
Blinatumomab 9 µg/day first CR: 36%. Molecular response: 88%. Median
ALCANTARA [8] R/R Ph-positive adults Phase II trial 45
week then 28 µg/day c.i. RFS: 6.7 months. OS: 7.1 months.
Blast [10] MRD positive after induction Phase II trial 113 Blinatumomab 15 µg/m2 / Molecular response: 78%. Medina RFS: 18.9
day c.i. months. OS: 36.5 months.
CR: inotuzumab 81% vs. ChT 29%.
Molecular response: inotuzumab 78% vs.
Inotuzumab 0.8 mg/m2 on day 1 ChT 28%. Median RFS: inotuzumab 5
Phase III
INO-VATE ALL [14] R/R Ph-negative or -positive adults randomized trial 109 inotuzumab vs. 109 ChT of each cycle and 0.5 mg/m2 on months vs. ChT 1.8 months. OS:
days 8 and 15 inotuzumab 7.7 vs. ChT 6.7 months. VOD:
inotuzumab 13% vs. ChT 1% (most cases
occurred after alloHSCT).
CR: 58%. MRD response: 67%. Minimal
residual disease measured by flow
Inotuzumab dosing was 0.8 cytometry: 18 (66.7%) had minimal residual
R/R Ph- and Ph+ mg/m2 intravenously on day 1 disease < 0.01%. VOD: 29% among 21
AALL1621 [21] children and adolescents Phase II trial 48
(age of 1–21 years) and 0.5 mg/m2 on days 8 and 15 patients undergoing alloHSCT. Partial
of a 28-day cycle CD22 expression and lower CD22 site
density were associated with a lower
likelihood of response to inotuzumab.
R/R Ph- and Ph+ Single infusion of
children and young adults 79 CR rate: 82%. MRD negativity: 100%.
Eliana study [23–25] Phase II trial 5-year RFS: 49%. 5y OS: 55%.
(age of 3–21) tisagenlecleucel

Tisagenlecleucel R/R Ph- and Ph+ Single infusion of commercial

children and young adults Real-world setting 255 CR: 86%. DOR: 61%. EFS: 52%. OS: 77%.
real-world study [28] (age of 1–26) tisagenlecleucel

Single infusion of CR: 71%. MRD response: 97%. Median

ZUMA-3 trial [29] R/R Ph- and Ph+ adults Phase II trial 55 DOR: 13 months. Median RFS: 12 months.
brexucabtagene autoleucel Median OS: 18 months.
R/R represents relapsed/refractory, Ph represents Philadelphia, ChT represents chemotherapy, c.i. represents continuous infusion, CR represents complete remission, RFS represents
relapse-free survival, OS represents overall survival, EFS represents event-free survival, MRD represents measurable residual disease, VOD represents veno-occlusive disease, and
alloHSCT represents allogeneic stem cell transplantation.
Cancers 2023, 15, 1349 9 of 23

3. Mechanisms of Resistance to Immunotherapies

Different mechanisms of tumor escape after treatment with novel immunotherapies
have been described, and, in general, either the capacity of the leukemic clone to escape
from the therapy-related immune selection or the inability of the immune system to generate
an adequate response against leukemia can be referred to [44]. Some of these mechanisms
of relapse are shared among the different therapies; others are treatment-specific and rely
on the peculiar mechanism of action of each treatment.
The efficacy of blinatumomab depends on cytotoxic CD3+ T-cell engagement against
CD19-expressing cells. The emergence of CD19-negative leukemic clones has been observed
in about one-third of patients who relapsed after blinatumomab [45]. Multiple mechanisms
can contribute to CD19 loss, such as alternative splicing, truncated CD19 variants, or a
disrupted CD19 membrane export in the postendoplasmic reticulum compartment [46,47].
A myeloid lineage switch in patients with KMT2A(MLL) or ZNF384 rearrangements is also
possible [48]. In addition, the possible mechanisms of resistance include the inhibition of
blinatumomab-induced T-cell expansion due to exhausted T cells or the presence of an
excessive number of regulatory T cells (T-regs) [49,50]. In a recent study using bulk tumor
and single-cell sequencing on 44 R/R BCP-ALL patients, blinatumomab nonresponders
displayed a T-cell repertoire with clonal expansion that was enriched in mucosal-associated
invariant T cells (MAIT), unconventional T cells involved in nonpeptidic antigen recog-
nition [47]. A threshold of T-regs of >10% in the peripheral blood has been proposed to
identify patients with the highest likelihood to be resistant to blinatumomab [50]. The
overexpression of PD1/PD-L1 can also be important [51]. Besides the quality of the T cell re-
sponse, the tumor burden also affects the treatment outcomes. Patients with a bone marrow
blast infiltration of >50% have a lower probability to achieve CR [4]. Thus, the optimal ratio
between the CD3+ T cells and the target CD19+ cells is probably crucial. For such patients,
a pretreatment phase is suggested to reduce the load of the disease, with the ultimate goal
of also reducing adverse reactions, including cytokine release syndrome [52,53].
Similar to blinatumomab, downregulation or antigen loss is a common mechanism of
tumor escape after anti-CD19 CAR-T cell therapy, with observed CD19-negative relapse
rates of 10–25% in several clinical trials [23,24,27,54]. In a recently published paper, three
main patterns of relapse were found. Among 420 CAR-T cell treated children and young
patients, 51% were CD19-positive, 41% were CD19-negative, and 7% showed a lineage
switch relapse. Notably, in a multivariable model, high preinfusion disease burden, prior
blinatumomab nonresponse, older age, and a 4–1BB CAR-T cell construct were associated
with an increased risk of CD19-negative relapses [55].
Among the different modalities of CD19 modulation, genetic and posttranscriptional
aberrations have been well documented: frameshift code insertions; deletions in exons 2–5;
and mRNA splice variants, in particular exon 2, are associated with the loss of either the
transmembrane domain, with the consequent absence of surface antigen expression [56],
or the extracellular epitope recognized by CAR-T cells [57]. Interestingly, the presence
of CD19-negative subclones [58] or leukemic cells harboring exon 2 splicing variants [59]
have also been observed in samples of patients with BCP-ALL at diagnosis, suggesting
that the possible mechanisms of escape could be acquired even before exposure to the
immunotherapies. Moreover, antigen masking due to accidental transfection and the
consequential aberrant expression of the anti-CD19 CAR-T cells by BCP-ALL cells [60]; the
transfer of CD19 from blast to T cells via a phenomenon known as trogocytosis [61]; or a
phenomena of leukemia plasticity with phenotype conversion, in particular, in patients
harboring KMT2A gene rearrangements [62–64] are other modalities by which leukemic
cells can modulate antigen expression. Usually observed early after the infusion, relapses of
BCP-ALL that maintain the expression of CD19 are associated with a failure of the CAR-T
cells to expand and persists in vivo. In these cases, the mechanisms of resistance are related
to some current limitations of CAR-T cell technology, in particular, the quality of the T
cells selected for transfection (long-lasting memory vs. effector T cells), the ratio of the
Cancers 2023, 15, 1349 10 of 23

Cancers 2023, 15, 1349 11 of 25

CD4+ and CD8+ T cells to be transfected, the choice of the costimulatory domain (4–1 BB
vs. CD28), the affinity for CD19, and the antigenicity of the scFv [65].
The mechanisms of resistance to InO are less defined and are quite dissimilar from
the CD4+ and CD8+ T cells to be transfected, the choice of the costimulatory domain (4–1
those
BB vs.described
CD28), the previously, giving
affinity for CD19, andathe
different targeted
antigenicity of theantigen
scFv [65]. and distinctive mode of
action.The mechanisms of resistance to InO are less defined and are quite relapse
In particular, a loss of CD22 expression by the leukemia at dissimilaris from
not frequent
and
thosehas been described
described previously,only givingin athe case reports
different targetedofantigen
both pediatric
and distinctiveand adult
mode of BCP-ALL
patients
action. In[66,67].
particular,When
a lossanalyzing the patients
of CD22 expression treated
by the leukemia in the INOVATE
at relapse is not trial,
frequentKantarjian
and coll.been
and has observed
described a only
reduction in CD22
in the case reportsexpression from and
of both pediatric baseline in the subjects
adult BCP-ALL pa- who
tients [66,67].
relapsed afterWhen analyzing the
InO treatment, butpatients treated in the
no CD22-negative INOVATE
clones trial, Kantarjian
were detected and
[68]. Furthermore,
coll.same
the observed
group a reduction
analyzed in the
CD22 expression
bone marrow from baselinefrom
samples in thethe
subjects
patientswhobefore
relapsed treatment
after InO treatment, but no CD22-negative clones were detected
with InO and reported a reduction in CR and the DOR in those with lower blast CD22 [68]. Furthermore, the
same group compared
expression analyzed the bonethe
with marrow samples
patients withfrom
CD22 thepositivity
patients before≥ 90% treatment
measured withthrough
InO and reported a reduction in CR and the DOR in those with lower blast CD22 expres-
flow cytometry [69]. Intriguingly, the patients with leukemic cells harboring KMT2A
sion compared with the patients with CD22 positivity ≥ 90% measured through flow cy-
rearrangements were characterized at baseline by a lower expression of CD22 and, both
tometry [69]. Intriguingly, the patients with leukemic cells harboring KMT2A rearrange-
in the subgroup analysis of the phase III trial and in the follow-up studies, these patients
ments were characterized at baseline by a lower expression of CD22 and, both in the sub-
did
groupnotanalysis
benefitoffrom InO inIIIterms
the phase of response
trial and and survival
in the follow-up studies,rates
thesein comparison
patients did notwith the
standard of care [13,69]. These data suggest that the efficacy of InO
benefit from InO in terms of response and survival rates in comparison with the standard and the consequent risk
of
of relapse are associated
care [13,69]. These data withsuggestthethat
level
theof antigen
efficacy of expression
InO and the and densityrisk
consequent on ofthere-leukemic
blast, which
lapse are influences
associated thelevel
with the amount of the
of antigen drug that
expression andisdensity
delivered on theinto the cells.
leukemic blast,Finally, a
recent
which work on pediatric
influences the amount patients
of the with
drug BCP-ALL showed
that is delivered intothat
the the specific
cells. Finally,aberrant
a recent splicing
work on of
variants pediatric
the CD22 patients
genewithwere BCP-ALL showed
associated withthat the specific
antigen aberrant splicing
downregulation and var-
an acquired
iants of thetoCD22
resistance gene were associated with antigen downregulation and an acquired
InO [70].
resistance to InO [70].
The possible mechanisms of tumor escape after exposure to the novel immunothera-
The possible
pies are schematized mechanisms
in Figure of tumor
2. escape after exposure to the novel immunothera-
pies are schematized in Figure 2.

Figure 2. Mechanisms of relapse after exposure to different immunotherapies. Panel (A) T-cell
Figure 2. Mechanisms of relapse after exposure to different immunotherapies. Panel (A) T-cell
activation and expansion can be impaired by the leukemia-derived mechanisms of immune es-
activation and expansion
cape, primarily antigen losscan be impaired
or phenomena by the
derived leukemia-derived
from mechanisms
T-cell proliferation of antigen
under chronic immune escape,
primarily antigen
stimulation, such asloss orexhaustion.
T-cell phenomena derived
Panel from T-cell
(B) relapses proliferation
after CAR-T under
cell infusion chronic
could antigen stim-
be related
to antigen loss (CD19-relapses) or to impaired CAR-T cell function (CD19+ relapses). Panel
ulation, such as T-cell exhaustion. Panel (B) relapses after CAR-T cell infusion could be related C: re-
sistance to inotuzumab ozogamicin is mainly related to reduced CD22 expression and impaired
to antigen loss (CD19-relapses) or to impaired CAR-T cell function (CD19+ relapses). Panel C: re-
drug internalization.
sistance to inotuzumab ozogamicin is mainly related to reduced CD22 expression and impaired
drug internalization.
Cancers 2023, 15, 1349 11 of 23

Ag represents antigen, ALL represents acute lymphoblastic leukemia, CAR represents

chimeric antigen receptor, PD represents programmed death, TCR represents T-cell receptor.
The figure was created with BioRender.com.

4. Optimal Use of the Different Immunotherapies According to Specific Clinical

Conditions
4.1. Extramedullary Relapse
According to the available literature, among pediatric and adult patients, nearly 40%
of relapses occur in the extramedullary sites (EMD, or extramedullary disease) either iso-
lated or along with a hematologic relapse. In the absence of head-to-head comparisons,
the previously described immunotherapies seem to perform differently in this setting. In
a subanalysis of the TOWER trial, blinatumomab proved to be less effective in patients
with EMD [4]. Aldoss and coauthors [71] reported a lower probability of obtaining CR
in patients with active EMD at the time of initiating blinatumomab or with a history of
prior EMD. The same authors in a following casuistry of 132 patients confirmed similar
findings: among the patients who were refractory to blinatumomab, 43% of the patients
had EMD, 40% of whom had CNS involvement. In addition, for the patients who primarily
responded to blinatumomab but had a history of EMD, the risk for relapse/progression
at the extramedullary sites (particularly CNS) was significantly higher and not abrogated
through consolidation with alloHSCT [72]. However, in a European real-life analysis on the
use of blinatumomab in adult ALL patients, 10 out of 20 patients with EMD achieved com-
plete hematological remission with 5 of them also achieving an MRD response, suggesting
that the exact role of blinatumomab in this setting is not fully established [11].
In a post hoc study on the pivotal trial INOVATE, De Angelo and colleagues analyzed
the efficacy of inotuzumab among 18 patients with EMD or lymphoblastic lymphoma. The
CR/CRi rate was 66.7%, with 58% of the responders achieving MRD negativity. Among
the patients with baseline EMD, 71% achieved CR/CRi with a durable resolution of the
EMD [15].
In a real-world analysis, the use of CAR-T cells in patients with non-CNS-EMD proved
effective even though the bone marrow response was higher and quicker compared to that
of the extramedullary sites [73]. Interestingly, tisagenlecleucel was safe and efficacious in
a small study of 12 relapsed patients with primary CNS lymphoma. A total of 7 of the
12 patients (58.3%) obtained a response, and it was complete in 6 patients. CAR-T cells
were also noted to expand and traffic to the CNS in the absence of a systemic disease [74].
Very recently, a study including 48 patients with R/R BCP-ALL with CNS involvement
confirmed that CAR-T cells could induce similar high response rates in both BM and CNS
diseases [75]. In keeping with these results, Shah NN et al., in a phase I study, demonstrated
the efficacy of CD19-CAR-T cells in the treatment of active CNS leukemia among 13
patients with CNS involvement at infusion of which one had extensive leptomeningeal
involvement [76]. In our experience with CARCIK-CD19 cells, we confirmed that these
cells can move into the cerebrospinal fluid and exert antileukemic activity with manageable
toxicity if the leukemic burden in this site is not overwhelming (Figure 3).
Based on the limited experience in the previously quoted studies, at our institution we
considered inotuzumab to be the first option for R/R ALL patients with extramedullary
disease. For patients with limited SNC involvement, CAR-T cells may represent a valuable
option.
Cancers 2023, 15,
Cancers 2023, 15, 1349
1349 12 of
13 of 23
25

(A) (PB) (B) (CSF)

Figure 3.
Figure 3. Detection
Detection of
of CARCIK-CD19 cells in
CARCIK-CD19 cells in the
the peripheral blood (Panel
peripheral blood (Panel A,
A, PB)
PB) and
and cerebral
cerebral spinal
spinal
fluid (Panel B, CSF) in an adult ALL patient. At day 28 from infusion, CARCIK-CD19 cells were
fluid (Panel B, CSF) in an adult ALL patient. At day 28 from infusion, CARCIK-CD19 cells were
simultaneously in PB (11/mcl) and CSF (1/mcl).
simultaneously in PB (11/mcl) and CSF (1/mcl).

Based on theDisease
4.2. High-Burden limited
at experience
Relapse in the previously quoted studies, at our institution
we considered
In both theinotuzumab toassessing
pivotal trials be the first option
the valuefor
of R/R ALL patients
blinatumomab with
and extramedullary
inotuzumab, high-
disease. For patients with limited SNC involvement, CAR-T cells may represent
burden disease was defined by a marrow blast count greater than 50%. The use a valuable
of blina-
option. in this setting was clearly associated with a reduced probability of achieving
tumomab
CR [4,77]. Even in the setting of MRD, the disease burden matters when blinatumomab
4.2.
is High-Burden
employed. Disease
In the at Relapse
recent retrospective analysis of the GRAAL group, the 3-year OS of
In both
patients withthe pivotal trials
molecular MRD assessing
< 0.1%, the valueor
0.1–1%, of >1%
blinatumomab
was 86%, and 58%,inotuzumab,
and 33%, respec- high-
burden disease was defined by a marrow blast count greater than
tively [78]. These results suggest that an optimal ratio between the CD3+ T cells and the 50%. The use of blina-
tumomab
target CD19+in this setting
cells was clearly
is probably a crucialassociated
parameter.with For
a reduced probability
this reason, of achieving
pre-blinatumomab
CR [4,77].
mild Even in the
cytoreduction settingbeofconsidered
should MRD, the diseasewhenever burden matters
possible when blinatumomab
to improve leukemic con- is
employed.
trol and to In the recent
reduce retrospective
the risk of cytokine analysis
releaseofsyndrome
the GRAAL group,
(CRS) andthe 3-year OS
neurologic of pa-
toxicity
(ICANS)
tients with [52,53,79].
molecular MRD < 0.1%, 0.1–1%, or >1% was 86%, 58%, and 33%, respectively
[78]. A postresults
These hoc analysis
suggestofthat
the anINOVATE
optimal study [15] divided
ratio between patients
the CD3+ intoand
T cells three
thegroups
target
according to their baseline blast counts: low (< 50%), moderate (50–90%),
CD19+ cells is probably a crucial parameter. For this reason, pre-blinatumomab mild cy- and high (>90%).
The authors should
toreduction found be thatconsidered
inotuzumab’s activity
whenever is independent
possible to improvefrom the disease
leukemic controlburden;
and to
moreover,
reduce thethe rateofofcytokine
risk MRD negativity
release among
syndrome the responders
(CRS) and did not seemtoxicity
neurologic to be affected
(ICANS) by
the baseline disease burden. In contrast to blinatumomab, a significant clinical benefit was
[52,53,79].
also observed
A post hoc in analysis
the patients
of thewith a high disease
INOVATE studyburden.
[15] divided patients into three groups
Similar to blinatumomab, the impact
according to their baseline blast counts: low (< 50%), of the disease
moderateburden in CAR-T
(50–90%), and highcell therapy
(> 90%).
seems to be unfavorable. In a study from MSKCC [27], patients
The authors found that inotuzumab’s activity is independent from the disease burden; with a higher disease
burden (BM > 5% blasts) showed a lower CR rate. A recent
moreover, the rate of MRD negativity among the responders did not seem to be affectedwork on a large pediatric
population
by the baselineanalyzing
diseasethe impactInofcontrast
burden. blinatumomab and subsequent
to blinatumomab, anti-CD19
a significant CAR-T
clinical cell
benefit
therapy
was alsoshowed
observedthat a nonresponse
in the patients withtoa blinatumomab
high disease burden.and a high burden disease (>5%
blastsSimilar
in the bone marrow) werethe
to blinatumomab, associated
impact ofwith the the worstburden
disease EFS and in RFS
CAR-T independent
cell therapyof
other prognostic factors [80].
seems to be unfavorable. In a study from MSKCC [27], patients with a higher disease bur-
In summary,
den (BM > 5% blasts)these data suggest
showed a loweraCR preference for inotuzumab
rate. A recent work on a largefor patients
pediatric with high-
popula-
burden disease. In such cases, a proper tumor lysis syndrome
tion analyzing the impact of blinatumomab and subsequent anti-CD19 CAR-T cell therapyprophylaxis and, possibly,
inpatient
showed that management
a nonresponse for theto first cycle couldand
blinatumomab be considered.
a high burden Thedisease
use of blinatumomab
(> 5% blasts in
likely remains preferable in patients lacking a robust expression
the bone marrow) were associated with the worst EFS and RFS independent of other of CD22, those whoprog-
were
previously
nostic factors treated
[80]. with inotuzumab, or those with a higher risk of hepatotoxicity. In this
respect, no more than two cycles of inotuzumab should be advised in patients who are
In summary, these data suggest a preference for inotuzumab for patients with high-
candidates for subsequent consolidation with allogeneic transplantation. Inotuzumab may
burden disease. In such cases, a proper tumor lysis syndrome prophylaxis and, possibly,
also represent an ideal bridging therapy for CAR-T cells due to the possibility of providing
inpatient management for the first cycle could be considered. The use of blinatumomab
a sequential target therapy on two different antigens.
likely remains preferable in patients lacking a robust expression of CD22, those who were
Cancers 2023, 15, 1349 13 of 23

4.3. Blinatumomab, Inotuzumab, and CAR-T Cells: Do They Stand Alone or Bridge Treatments to
Allogeneic Transplants?
At present, the need for subsequent alloHSCT in patients successfully treated with
blinatumomab, inotuzumab, or CAR-T cells remains a matter of debate. This uncertainty is
partly due to the fact that none of the published studies were designed to assess the role
of alloHSCT in posttreatment consolidation, and the decision for alloHSCT was left to the
treating physician. With these limitations, a clear clinical benefit for patients proceeding to
alloHSCT after blinatumomab has not yet been demonstrated [3,81] but has been repeatedly
suggested [11,82]. In particular, blinatumomab maintenance therapy (≥ six cycles) was
associated with an increased OS and RFS compared with patients not receiving maintenance
independently from consolidation with alloHSCT [83]. In the MRD setting, the OS was
not different in patients who did or did not undergo transplantation, whereas, for those
treated during their second or later CR, the outcome of patients who did not undergo
transplantation was inferior [10]. Nonetheless, in a long-term analysis of a phase II study
conducted in the MRD setting, younger patients (≤ 35 years) who underwent alloHSCT
had a better 3-year survival compared with those who did not (62% vs. 22%) [82]. Finally, a
single-center experience confirmed alloHSCT after blinatumomab salvage therapy to be an
effective postremission treatment, with a 2-year RFS of 40% [84].
A post hoc analysis of the INOVATE trial revealed better outcomes in the patients
achieving MRD negativity and proceeding to alloHSCT compared to the nontransplanted
subjects [85,86]. In a pooled analysis of the phase I/II and phase III studies, the best
outcome was observed in the patients who underwent alloHSCT directly upon first re-
mission [87]. The median OS was not reached with a 2-year survival probability of 51%.
As expected, the MRD negative patients had a better median posttransplant OS and PFS
compared to the MRD positive patients (17.8 months vs. 5 months and 10.8 months vs.
3.4 months, respectively).
When considering CAR-T cells, different studies showed high rates of response and
a prolonged survival with a different proportion of patients subsequently treated with
alloHSCT after treatment with CAR-T cells. The pivotal phase II trial ELIANA showed
a rate of RFS up to 60% at 1 year and 49% at 5 years, with only a minority of patients
receiving alloHSCT [24,25]. Using a different CAR-T construct, Lee et al. demonstrated
a role of transplantation in consolidation in reducing the relapse rate in young patients
with R/R BCP-ALL [88]. Similarly, in a phase I trial using CD19.28 CAR-T cells in R/R
children and AYAs, the patients with a molecular response who received a transplant
showed a sustained response and survival at long-term follow-up. All the MRD negative
patients not proceeding to alloHSCT relapsed at a median time of 5 months post-CAR-T
cell infusion [76]. In the adult setting, conflicting results have been reported on the role of
consolidative alloHSCT in adult patients achieving MRD negative remission after CAR-T
cell therapy. Park and colleagues showed no differences in the survival outcomes between
the patients receiving alloHSCT after achieving MRD negativity compared to those who
did not [27]. Similarly, when analyzing the recent results of the phase II ZUMA-3 trial, Shah
and colleagues showed no effect of alloHSCT on the duration of response nor on the RFS.
However, the proportion of the patients receiving transplantation was significantly low
(18%) [29]. In contrast, three other studies showed that alloHSCT in adult patients achieving
MRD negativity after CD19 CAR-T cell therapy was associated with an improvement in the
EFS. However, this benefit did not translate into a clear benefit in terms of the OS [31–33].
All in all, with the novel immunotherapies, a prolonged clinical benefit can be achieved
in a fair proportion of the responding patients who also achieved an MRD negative status.
In this context, a subsequent alloHSCT should be taken into consideration as a possible
effective strategy at least for younger and fit patients in order to limit the risk of an early
relapse after immunotherapy.
Table 2 summarizes our suggested approach according to different clinical conditions.
Cancers 2023, 15, 1349 14 of 23

Table 2. Summary of suggested strategies according to different clinical conditions.

Condition Our Approach

• Inotuzumab or CAR-T cells as the first option.

• In patients lacking CD22 surface expression or
those previously treated with inotuzumab,
Extramedullary relapse blinatumomab is a valid alternative.
• For patients with SNC involvement (if not
rapidly progressive), CAR-T cells may represent
a valuable option.

• Inotuzumab should be preferred if bone marrow

blasts are >50%.
• For patients with CD22 negativity, those
High-burden disease at relapse previously treated with inotuzumab, or those at
high risk of hepatotoxicity, blinatumomab is a
valid option. In this case, mild cytoreductive
therapy before starting blinatumomab is advised.

• In younger and fit patients achieving an MRD

negative CR after inotuzumab or blinatumomab,
we usually advise further consolidation with
their first alloHSCT. For those relapsed after a
previous transplant, a second alloHSCT should
be discussed with the patient. MRD negative
responders after CAR-T cell therapy may proceed
to their subsequent first alloHSCT, and this
option should be discussed with the patient.
• In younger and fit patients with an MRD positive
Consolidation treatment after CR
hematologic remission, alloHSCT is
achievement with or without alloHSCT
always advised.
• In older or frail patients not eligible to receive
alloHSCT, additional continuation/maintenance
therapy with blinatumomab or inotuzumab
should be considered.
• Sequential treatments with inotuzumab,
blinatumomab and/or CAR-T cells are
potentially valid and should be considered in the
setting of clinical studies (some are
already underway).

5. Future Perspectives
5.1. Anti-CD19 and Anti-CD22 Immunotherapies as Frontline Treatments
Both anti-CD19 and anti-CD22 immunotherapies have been explored in recent clinical
trials as frontline treatments in association or sequentially to chemotherapy. In the phase II
study GIMEMA LAL2317 (NCT03367299), blinatumomab was added to a pediatric-like
risk-oriented regimen in a sequential manner following the early and late consolidation
phases based on high-dose methotrexate and ara-C. Upfront alloHSCT was limited to
patients with a very high clinical or cytogenetic risk profile at diagnosis and patients re-
maining MRD positive (i.e., ≥ 10−4 ) after early consolidation. A total of 149 BCP-ALL
Ph-negative patients were enrolled (median age of 41 and range of 18–65) of whom 90%
obtained a complete hematological response. No matter the MRD status after consolida-
tion, blinatumomab was given to all the patients. MRD negativity improved from 72%
achieved after consolidation chemotherapy up to 95% following one blinatumomab cycle
(p = 0.018) [89]. The use of blinatumomab in first-line treatment was also recently explored
in a phase II single-arm monocentric study by the MD Anderson researchers using a se-
quential approach of four cycles (instead of eight) of high-dose induction chemotherapy
Cancers 2023, 15, 1349 15 of 23

based on the hyper-CVAD/MTX-ara-C scheme followed by four cycles of blinatumomab.

The immunotherapy was also incorporated in the maintenance scheme and was alternated
with POMP chemotherapy. MRD was detected through flow cytometry (MFC) and through
next-generation sequencing. In the updated results of this trial, with a median follow-up of
37 months, all 32 of the untreated patients obtained a complete response at any time during
treatment, with 97% of the subjects achieving MRD negativity through MFC overall. Of
note, 12% of the patients that received blinatumomab converted their MRD positivity after
the first cycle of treatment. Treatment was well tolerated, with less than half of the patients
experiencing a neurological toxicity of any grade, and there was only one discontinuation
due to treatment-related neurotoxicity [90]. Although with the limit of a short follow-up,
the improved MRD responses in the above-mentioned studies were also associated with
superior survival outcomes compared to historic controls. With a median follow up of
10 months, the GIMEMA trial reported a 1-year OS and DFS of 84% and 72%, respec-
tively [89]. Likewise, the patients treated in the MD Anderson study experienced an
estimated 3-year PFS and OS of 73% and 81%, respectively [90]. Other cooperative groups
are favorably applying these immunotherapies as frontline therapies in ongoing clini-
cal studies. The Eastern Cooperative Oncology Group (ECOG) recently completed and
presented at the 64th American Society of Hematology Annual Meeting a phase III trial
comparing standard chemotherapy with or without blinatumomab (NCT02003222). In
this study, the patients with MRD negativity at the end of induction were randomized to
receive a standard course of consolidation chemotherapy with or without blinatumomab.
The results showed that patients receiving blinatumomab had an OS of 83% compared with
65% in the control arm [91]. Furthermore, the Dutch–Belgian phase II HOVON146ALL
trial is now evaluating the ability of blinatumomab in addition to prephase treatment
and consolidation therapy to induce a deeper molecular response and to possibly obtain
durable remission (NCT03541083).
Taking into consideration the good safety profile shown by inotuzumab in combi-
nation with chemotherapy in the relapse setting, this association is now being explored
in frontline treatments. In particular, a phase III trial comparing inotuzumab in addition
to standard induction treatment in newly diagnosed young adult BCP-ALL patients is
ongoing (NCT03150693). This study could further elucidate the feasibility of inotuzumab
as a first-line treatment with intense chemotherapy in terms of the response rate, sur-
vival advantage, and expected toxicity. A summary of the main studies exploring the
immunotherapies is available in the Supplementary Materials (Table S1).

5.2. Should Less Intensive Chemotherapy or a Chemo-Free Approach Be Used to Treat Old and
Frail Patients?
The treatment landscape of older patients is rapidly changing, and their results are
improving thanks to the addition of innovative immunotherapies. The dismal outcome of
older patients with an expected 5-year overall survival of less than 20% [92] will rapidly
improve thanks to these innovative treatments. The MD Anderson group proposed a
reduced-intensity chemotherapy based on eight cycles of mini-hyper-CVD associated
with fractionated inotuzumab for the first four cycles for newly diagnosed patients aged
≥ 60 years. The study protocol was further amended to incorporate four cycles of blina-
tumomab as a consolidation treatment, reducing the chemotherapy to four cycles and
lowering the dose of fractionated inotuzumab. Blinatumomab was continued as a mainte-
nance treatment alternated with POMP. Among 64 patients evaluable for a morphologic
response with a median age of 68 years (range of 60–81), 96% achieved an MRD negativity
overall. The induction mortality was very low (< 5%), and the incidence of VOD was
< 10% [93]. Interestingly, a recent propensity score analysis on 58 old patients treated with
mini-HCVD and inotuzumab revealed improved complete responses and outcomes when
compared to historic controls [19]. The German cooperative group GMALL evaluated the
efficacy of inotuzumab as an induction treatment followed by a conventional high-dose con-
solidation chemotherapy. Patients aged ≥ 55 years were enrolled in this study. Complete
Cancers 2023, 15, 1349 16 of 23

remission was achieved by all the 27 patients who completed the induction phase, with
74% of the patients available for the MRD study reaching a molecular response. Reported
severe adverse events were mainly hematological, while approximately one-third of the
patients experienced a grade ≥ III transaminitis. No VOD cases were reported [94].
The role of blinatumomab as the first-line treatment for older patients with BCP-ALL
was investigated in the SWOG 1318 trial. In this chemo-free protocol, the bispecific antibody
was given as an induction treatment for two cycles followed by an additional three consoli-
dation cycles and POMP maintenance. Treatment was well tolerated, with no early deaths
reported during the induction phase. Of the 29 evaluable patients (median age of 75 years),
two-thirds obtained a complete response, with an estimated 3-year DFS and OS of 37% and
37%, respectively [95]. In line with previous studies, two phase II clinical trials performed
by the European working group on adult ALL (EWALL) are evaluating the combination of
either inotuzumab (NCT03249870) or blinatumomab (NCT03480438) with mild-intensity
chemotherapy in patients older than 55 years (see the Supplementary Table S1).

5.3. The Use of Immunotherapies in Ph-Positive ALL

In the Ph-positive setting, the introduction of tyrosine kinase inhibitors (TKIs) radically
changed the natural history of this otherwise poor-prognosis subset. Given the high
prevalence of older patients in this subset of BCP-ALL patients, several studies investigated
the association of different TKIs with less-intensive chemotherapy, showing a high rate of
induction responses and a lower induction toxic mortality, although the best consolidation
strategy is still a matter of debate [92]. In this regard, several clinical trials are evaluating
the best consolidation strategy with a particular interest in chemo-free schemes based on
the association of TKIs and the novel immunotherapies.
Following pioneering studies showing a high remission rate with TKIs only [96–98],
the GIMEMA phase II trial D-ALBA investigated a chemo-free program based on the
sequential administration of dasatinib and blinatumomab. Of the 63 enrolled patients (me-
dian age of 54 years), all but 1 obtained a complete response after the induction treatment.
Interestingly, the rate of the molecular response obtained after the first two cycles of consoli-
dation therapy further improved with subsequent blinatumomab infusions, with more than
80% of patients being MRD negative after four cycles of blinatumomab consolidation [99].
Based on the excellent results of D-ALBA, the GIMEMA group is now heading a phase III
randomized clinical trial comparing the combination of third-generation TKI ponatinib and
blinatumomab with the association of imatinib and chemotherapy (GIMEMA ALL2820 trial,
NCT04722848). The MD Anderson group very recently published the results of a phase II
study based on the combination of ponatinib and blinatumomab in newly diagnosed or
R/R Ph-positive BCP-ALL patients. The bispecific antibody was given up to five cycles,
while TKI monotherapy was continued as a maintenance treatment for at least five years.
Among the patients in the first-line treatment (40 patients, median age of 57 years), they all
achieved a complete response of whom 33 of 38 evaluable patients (87%) had a molecular
response. At 16 months of follow-up, the 1-year EFS and OS were both 95%; one patient
died in CR due to hypovolemic shock following cardiac catheterization, and one patient
received alloHSCT due to a persistently detectable BCR-ABL1 transcript [100].
The recent and ongoing clinical trials on frontline immunotherapy in Ph+ ALL are
available in the Supplementary Table S1.

5.4. Combination with Checkpoint Inhibitors

Among patients who relapsed after the immunotherapies, different mechanisms of
escape have been described, and new studies are now investigating the efficacy of new
combinations of treatments that target those pathways. In the patients who relapsed
after blinatumomab, there is evidence that leukemic cells and the tumor microenviron-
ment can lead to T-cell suppression. In particular, PD-L1 overexpression on BCP-ALL
blasts and the upregulation of T-reg cells have been described as possible phenomena of
resistance in relapsed patients [51]. Thus, the possible options of treatment are the combi-
Cancers 2023, 15, 1349 17 of 23

nation of blinatumomab with checkpoint inhibitors in CD19-positive relapses [49,101,102];

the use of CAR-T cells with a different affinity for CD19 [103]; the use of other im-
munotherapies, such as inotuzumab, in CD19-negative cases, or the use of bicistronic
CAR-T cells targeting CD19 and CD22 [104,105]. Many clinical trials combining blinatu-
momab and checkpoint inhibitors or new CAR-T cell constructs are currently ongoing
(Table S2 in the Supplementary Materials).

6. Conclusions
Based on the results obtained in the R/R setting showing the ability of immunothera-
pies to induce not only hematologic but also molecular remission, several trials are currently
evaluating their use as part of frontline treatments. All these trials may lead to the de-
velopment of an innovative combination of chemoimmunotherapies for patients with
ALL with less toxicity and to the establishment of their best positioning in future upfront
therapeutic regimens with the goal of increasing the rate and quality of deep molecular
responses in ALL (Figure 1A,B and Table S1 in the Supplementary Materials). In our opin-
ion, blinatumomab or inotuzumab could be better incorporated into the earlier phases of
treatment based on some characteristics, such as their availability off the shelf and no need
of lymphodepleting chemotherapy, leaving a wider therapeutic role of CAR-T cells in an
earlier R/R setting. The encouraging preliminary results coming from the ongoing studies
suggest that immunotherapies really have the potential to reduce the need to use intensive
chemotherapy or to reduce the indication of alloHSCT as a consolidation treatment. In
this regard, the combination of blinatumomab and the new generation of TKIs in Ph+
ALL represents the first evidence that a frontline chemotherapy-free regimen can be very
effective, also avoiding the need for alloHSCT in most patients. Thus, the landscape of
treatment is now really changing, and, in the coming years, a wider use of these drugs in
frontline treatments is likely. The clinical benefit of incorporating these drugs will not be
limited to younger and fit patients but will also apply to older and more frail patients when
considering that the immunotherapies are better tolerated than conventional chemotherapy.
At the same time, these immunotherapies could improve transplantation outcomes when
given either as a prophylaxis or as a postrelapse treatment [106].

Supplementary Materials: The following supporting information can be downloaded at https:

//www.mdpi.com/article/10.3390/cancers15041349/s1, Table S1: Recent and ongoing studies ex-
ploring immunotherapies in the frontline treatment of BCP-ALL patients and Table S2: Novel
approaches in relapse/refractory BCP-ALL patients.
Author Contributions: Conceptualization, F.L. and A.R.; writing—original draft preparation, F.L.,
G.C., P.D.S., and A.R.; supervision, A.R. All authors have read and agreed to the published version of
the manuscript.
Funding: The review was supported by the Associazione Italiana per la Ricerca sul Cancro-AIRC
5 × 1000 Program: “Metastatic disease: the key unmet need in oncology”-ISM-Code:21147 and the
Associazione Italiana Lotta alla Leucemia, Linfoma e Mieloma (AIL) Sezione Bergamo.
Conflicts of Interest: Federico Lussana received payment or honoraria for lectures, presentations,
educational events, or consulting fees from AbbVie, Amgen, Bristol Myers Squibb, Clinigen, Janssen,
Incyte, and Pfizer. Alessandro Rambaldi received payment or honoraria for lectures, presentations,
educational events, or consulting fees from Amgen, Pfizer, Novartis, Kite–Gilead, Astellas, Jazz,
AbbVie, Incyte, Janssen, and Omeros. The funders had no role in the design of the study or in the
writing of the manuscript.

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