Monoclonal antibodies: new chance in the management of B-cell acute lymphoblastic leukemia

ABSTRACT Objectives This review aims to see the progress of several clinically-used monoclonal antibodies in treating ALL patients and how they improved patients' outcomes. Methods We searched Web of Science, Elsevier and PubMed for relevant published studies, and summarized eligible evidence on the management of newly-diagnosed and relapsed or refractory ALL with monoclonal antibodies. Ongoing trials were identified from ClinicalTrials.gov. Results Rituximab, an anti-CD20 monoclonal antibody, prolonged patients' complete remission duration and overall survival when combined with hyper-CVAD regimen. Another anti-CD20 monoclonal antibody, Ofatumumab, was reported to have similar benefits. Blinatumomab allows endogenous CD3-positive cytotoxic T cells to target and eliminate CD19-positive blasts. FDA has approved its efficacy in patients with R/R B-ALL and eliminating minimal residual disease (MRD). It serves as a bridge to eradicate MRD before transplantation, and may also be a new choice for patients unable to undergo transplantation. An anti-CD22 monoclonal antibody named Inotuzumab Ozogamicin showed great improvement in patients' outcome, but its toxicity to liver is also worthy of our attention. Conclusion Monoclonal antibodies are proven to be a promising immunotherapeutic strategy to improve ALL patients' outcome in the long term. There's still a need for individualized treatment with effective and well-tolerated medicines. Trial registration: ClinicalTrials.gov identifier: NCT01363128. Trial registration: ClinicalTrials.gov identifier: NCT01466179. Trial registration: ClinicalTrials.gov identifier: NCT02013167. Trial registration: ClinicalTrials.gov identifier: NCT02000427. Trial registration: ClinicalTrials.gov identifier: NCT01564784. Trial registration: ClinicalTrials.gov identifier: NCT03677596. Trial registration: ClinicalTrials.gov identifier: NCT01363297. Trial registration: ClinicalTrials.gov identifier: NCT02981628. Trial registration: ClinicalTrials.gov identifier: NCT03094611. Trial registration: ClinicalTrials.gov identifier: NCT01371630. Trial registration: ClinicalTrials.gov identifier: NCT04224571. Trial registration: ClinicalTrials.gov identifier: NCT02458014. Trial registration: ClinicalTrials.gov identifier: NCT04546399. Trial registration: ClinicalTrials.gov identifier: NCT02879695. Trial registration: ClinicalTrials.gov identifier: NCT03913559. Trial registration: ClinicalTrials.gov identifier: NCT03441061. Trial registration: ClinicalTrials.gov identifier: NCT03739814. Trial registration: ClinicalTrials.gov identifier: NCT02877303. Trial registration: ClinicalTrials.gov identifier: NCT03698552. Trial registration: ClinicalTrials.gov identifier: NCT04601584. Trial registration: ClinicalTrials.gov identifier: NCT04684147. Trial registration: ClinicalTrials.gov identifier: NCT04681105.


Introduction
Acute lymphoblastic leukemia (ALL) is a malignant hematological disease derived from the abnormal proliferation and accumulation of immature lymphocytes in the bone marrow, peripheral blood and extramedullary organs. It occurs most frequently in childhood, and reaches a second peak in patients over 60 years [1]. Conventional treatment is based on multi-agent chemotherapy, typically consisting of an induction phase to reduce initial leukemic burden and obtain remission, a consolidation phase to eradicate residual blasts, a late intensification phase and long-term maintenance. Routine central nervous system (CNS) prophylaxis is recommended to start early, reducing the chance of relapse in CNS. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is recognized as a curative approach for suitable high-risk patients in certain subgroups.
Currently, the proportion of pediatric patients with long-term survival reaches up to nearly 90% [2,3]. While the majority of adult patients could achieve complete remission (CR) following upfront chemotherapy, their outcome in the long term is far from satisfying, owing to factors such as advanced age, poor tolerance to chemotherapy, higher rate of recurrence and higherrisk quality of the disease itself [4][5][6]. Only about half of adult patients treated with conventional chemotherapy could survive for more than 5 years [7]. The application of pediatric-inspired therapy and its further exploration of tolerable upper age limit has improved the rate of 5year overall survival in first adolescents and young adults (AYAs), and then patients younger than 55 years old, to nearly 60%, and even to 80% in some cases [8][9][10][11]. Meanwhile, the presence of minimal residual disease (MRD) also challenges the treating strategy with a higher rate of relapse.
Therefore, exploring effective and well-tolerated treatment strategies has been on going over the past decade. The development of monoclonal antibodies has been one of the most promising options due to their activity to enhance the efficacy of conventional chemotherapy without adding therapy-related toxicity by specifically targeting certain leukemia-associated antigens. Studies using monoclonal antibodies in the management of acute lymphoblastic leukemia are shown in Table 1. In this review, we focus on recent progress in how monoclonal antibodies being used in treating patients with ALL and improving their outcomes.

Rituximab
CD20 is expressed during nearly all stages of differentiation on both normal and malignant B lymphocytes, but only 30-50% of precursor B-ALL blasts and increases the occurrence of relapse and death [12][13][14][15]. Rituximab is a chimeric monoclonal antibody against CD20 and can trigger cytotoxic effects and induce apoptosis of leukemic cells. At present, adding Rituximab to upfront chemotherapy has shown considerable improvement in prognosis of patients with diffuse large-B-cell lymphoma, aggressive mantle-cell lymphoma, chronic lymphocytic leukemia (CLL) and Burkitt and Burkitt-type lymphoma/leukemia [16][17][18][19][20][21][22][23].
In 2012, a pilot study reported 9 adult patients with CD20 + relapsed/refractory (R/R) B-ALL treated by the combination of Rituximab and chemotherapy, suggesting it may be a feasible approach to achieve CR and reduce MRD in this group of patients, but required larger series [24]. The MD Anderson Cancer Center (MDACC) trial recruited 282 adolescents and adults with de novo Ph (Philadelphia chromosome) negative precursor B-cell lymphoblastic leukemia (BCP-ALL) and evaluated the efficacy of additional Rituximab into the standard treatment for patients with more than 20% CD20 expression. Based on the hyper-CVAD (hyper-fractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone) regimen, an effective first-line therapy in this subset, the incorporation of Rituximab demonstrated its superiority with better 3-year rates of CR duration (70%: 38%; P < 0.001) and 3-year overall survival (OS, time from treatment initiation to death or date of last follow-up) (75%: 47%; P = 0.003) in patients younger than 60 years old. But for the older patient group, prognosis was poorer regardless of the usage of Rituximab [25].
In the meantime, the Group for Research on Adult Acute Lymphoblastic Leukemia 2005 (GRAALL-2005) trial enrolled 787 patients aged 18-59 with newly diagnosed Ph negative ALL between 2006 and 2014 from multiple centers in France and Switzerland. Among them, 220 patients with CD20 expression (assessed

Ofatumumab
Ofatumumab, a human anti-CD20 monoclonal antibody, targets a distinct extracellular epitope from Rituximab. Due to its closer binding to the cell membrane, Ofatumumab was proved to promote a more potent complement-dependent cytotoxicity in vitro than Rituximab [27,28]. Ofatumumab has been demonstrated to be safe and effective in treating CLL and has gained approval by the US Food and Drug Administration (FDA) in 2014 [29,30]. Therefore, promising results may also exist in patients with ALL by introducing Ofatumumab to intensive chemotherapy. Previous evidence suggests an increase in CD20 expression after induction. Relevant mechanisms are as follows. The instability of leukemic phenotypes makes it susceptible to external influences in cellular processes, and medical intervention especially glucocorticoid may trigger the up-regulation of CD20 expression during induction chemotherapy [31,32]. Based on these results, researchers hypothesized that patients whose CD20 expression level was less than 20% at diagnosis might also benefit from the treatment of anti-CD20 monoclonal antibodies.
The MD Anderson Cancer Center conducted a single-arm, phase 2 trial (NCT01363128) to recruit untreated Ph negative B-cell ALL patients with at least 1% of CD20 expression [33]. All patients received hyper-CVAD therapy on course 1,3,5,7, and MTX-Ara-C therapy (high-dose methotrexate and cytarabine) on course 2,4,6,8. Ofatumumab was given for 2 separate days on course 1,2,3,4 with a total of 8 doses. During the following 30 courses of POMP therapy (6-mercaptopurine, vincristine, methotrexate, and prednisone) for maintenance, another 4 doses of Ofatumumab were given on course 6,7,18,19 as intensification on top of high-dose methotrexate and pegylated asparaginase. After a median follow-up of 44 months (26-53 months), two thirds of the patients were still alive, of which more than half (54%) remained in their first CR duration. For adolescents and young adults aging 18-39, 4-year EFS and OS were 69% and 74%, respectively, comparable to those treated with pediatric or pediatric-inspired regimens as previously reported [34][35][36][37][38][39][40]. Of note, although patients with CD20 expression less than 20% were not eligible in previous studies focusing on Rituximab, this patient group could gain different levels of clinical benefits, and therefore, may also be indicated for the use of CD20 monoclonal antibodies. Infection was the most common grade 3 or 4 adverse event (AE), and none of the deaths were considered to be directly Ofatumumab-related.

Blinatumomab
CD19 is a B-lineage surface antigen essential for the development and survival of B cells, and is expressed on virtually all stages of B-lineage ALL. Its frequent expression on these blasts makes it an ideal target for the utilization of Blinatumomab, a bispecific T-cell engager (BiTE) antibody that enables endogenous CD3-positive cytotoxic T cells to recognize and eliminate CD19-positive B cells [41,42].
In order to further verity the efficacy and safety of single-agent immunotherapy with Blinatumomab for adults with Ph negative R/R BCP-ALL, a multi-institutional phase 3 clinical trial (NCT02013167), also known as the TOWER trial, was conducted. Patients were assigned randomly to receive either Blinatumomab or standard chemotherapy in a 2:1 ratio. Obvious clinical benefits were reported in the Blinatumomab group with a significantly higher CR (34%: 16%; P < 0.001) within 12 weeks after treatment, as well as a 3.7 months' extension of median OS (7.7 months: 4.0 months; HR: 0.71; P = 0.01), and a higher rate of 6-month EFS (31%:12%; HR: 0.55; 95%CI: 0.43-0.71; P < 0.001). AEs were common in both groups, and the Blinatumomab group had an overall lower rate of serious AEs and was also associated with a lower incidence of myelosuppression and related complications than the chemotherapy group [45].
After 2 cycles of Blinatumomab induction, patients who achieved a bone marrow response (≤5% bone marrow blasts) or CR/CRh/CRi (platelets >100,000/μL, or absolute neutrophil count >1000/μL) could continue to use Blinatumomab for another 3 cycles of consolidation and long-term maintenance therapy. Thirty six of 271 patients finally entered maintenance therapy, of whom the median OS was not reached at the end of the study, and for those who discontinued Blinatumomab early, median OS was 15.5 months, with a relative odds ratio of 0.37 (95%CI: 0.16-0.88). The median RFS was 14.5 months (95%CI: 7.1-21.9) and 9.8 months (95%CI: 8.5-11.1) respectively for patients with or without maintenance therapy, with a relative odds ratio of 0.48 (95%CI: 0.22-1.03). The incidence of adverse events decreased from 97.2% during induction, to 86.1% during consolidation, to 72.2% during maintenance. Although the follow-up result was limited by its insufficient sample size, we could still consider Blinatumomab as a potential option for long-term treatment in R/R BCP-ALL [46].

Study in MRD+ patients
MRD is the major cause of relapse, it can be detected by techniques such as flow cytometry and polymerase chain reaction (PCR). We normally define more than 10 −4 detectable blasts as MRD positive. Treatment for patients with persistent or relapsed positive MRD is one of the trickiest issues in ALL therapy, considering the widely-accepted fact that MRD is always linked to a poor prognosis [47][48][49]. In such condition, allo-HSCT was highly recommended, but the benefit was less satisfying due to the long waiting period of a suitable donor or a relatively higher chance of getting relapsed after transplantation [50][51][52][53]. Early application of Blinatumomab maybe another feasible solution in this setting. In the phase 2 BLAST trial, Nicola et al. analyzed 116 B-ALL patients in hematological CR with MRD (≥10 −3 ), the majority of which (78%) were able to obtain complete MRD response after receiving Blinatumomab. It was reported that complete MRD responders were associated with a significantly longer RFS (23.6 months: 5.7 months; P = 0.002) and OS (38.9 months: 12.5 months; P = 0.002) than MRD non-responders, and the treatment begun after first CR was more beneficial [46]. The efficacy of Blinatumomab to eliminate MRD makes it the first FDA-approved therapy for patients with BCP-ALL in morphologic remission with minimal residual disease [54].
In the final follow-up report of the BLAST trial, when every patient completed the 5-year assessment, median survival was reported to be not reached (29.5-NR) for those who had complete MRD response and 14.4 months (3.8-32.3) for those who were not (p = 0.002). Among those who underwent HSCT, median OS was NR and 16.5 months (p = 0.065) respectively for patients with or without MRD response, and for those who did not undergo HSCT, median OS was 56.4 and 6.2 months (p = 0.043) respectively [55], suggesting that Blinatumomab may serve as an effective bridging therapy to eradicate MRD before HSCT, and for those who were not conditioned for HSCT, Blinatumomab could also be a promising option to achieve long-term survival. Similar result was reported in the subsequent study of the TOWER trial, where no significant benefit of HSCT was seen for patients who achieved complete remission with full, partial, or incomplete hematologic recovery with Blinatumomab (p = 0.69), but those with an MRD response in the first salvage therapy, regardless of their HSCT status had the best outcome. In addition, no unusual toxicities or increased risk and mortality were observed post-HSCT compared with the standard chemotherapy group [56].
In a recent retrospective study, Blinatumomab also showed remarkable potential to reduce MRD in infant patients. After receiving 1 or 2 cycles of Blinatumomab, 2 of 11 (81.8%) patients achieved a complete MRD response prior to HSCT. Three-year EFS and OS were 47% and 81%, respectively, although limited by the small sample size, improvement was obvious compared with the historical cohort [57][58][59], making it possible to reset the age limit for this therapy.

Study in Ph+ patients
The presence of Ph chromosome is one of the most common chromosomal abnormalities in ALL patients with an incidence of approximately 15-30%. It used to be associated with an inferior prognosis, however, since the introduction of tyrosine kinase inhibitors (TKIs), patients' outcomes have been greatly improved with the 5-year OS increasing from 10% to 40%-50% [60][61][62][63][64][65]. But for patients who get relapsed after the treatment of TKI-based therapy, or are refractory or intolerant to TKIs, options are limited to achieve a long-term survival.
A single-arm, multicenter, phase 2 study (NCT02000427) was conducted to explore the possibility and efficacy of single-agent Blinatumomab in this patient population. 45 patients were included, and Blinatumomab was administered in 28-day cycles. Anti-leukemia activity was revealed with a 36% of CR/ CRh rate after the first two cycles, of which 88% also achieved a complete MRD response. Median RFS and OS were 6.7 and 7.1 months, respectively. Frequent AEs involved pyrexia (58%), febrile neutropenia (40%), and headache (31%), consistent with those observed in previous Ph negative ALL studies [66]. In the subsequent propensity score analysis (PSA), an external cohort was established by patients with similar baseline characteristics and receiving standard of care (SOC) chemotherapy instead of Blinatumomab (n = 55) from the existing databases. The rate of CR/CRh was 36% in the Blinatumomab cohort versus 25% in the SOC cohort, leading to an odds ratio of 1.54 (95%CI: 0.61-3.89) or 1.70 (95% credible interval [CrI]: 0. 94-2.94) with Bayesian data augmentation. The Bayesian-augmented (80% power) hazard ratio estimate for OS was 0.77 (95%CrI: 0.61-0.96), suggesting a 23% reduction in the risk of death in the Blinatumomab group compared with the SOC group [67].
Couturier, MA et al retrospectively analyzed 26 patients with R/R Ph+ ALL, the conjunction of Blinatumomab and Ponatinib also showed promising results with 25 of 26 patients achieving complete morphologic remission, and 23 achieving a complete molecular response. The median OS and EFS were 20 and 15.3 months, respectively, with a median follow-up of 34.4 months. No significant difference of OS or EFS was found in patients proceeding to allo-HSCT or not [68]. Another retrospective study examined 11 Ph+ ALL patients treated with concurrent Blinatumomab and TKIs. After a median treatment of one cycle, 8 of 9 patients in positive MRD status achieved complete molecular response (CMR), 2 remaining patients without detectable MRD maintained CMR, suggesting that the combination of Blinatumomab and TKIs may be used as a consolidation therapy to obtain MRD negativity and sustain CMR [69].
The global phase 3 INO-VATE trial (NCT01564784) compared the efficacy of single-agent Inotuzumab Ozogamicin against standard intensive chemotherapy in patients with CD22-positive relapsed or refractory B-ALL. In each cycle, Inotuzumab Ozogamicin was administrated 0.8-0.5 mg/m 2 on day 1 and 0.5 mg/ m 2 on day 8 and day 15 for up to 6 cycles. Those treated by Inotuzumab Ozogamicin were associated with a significantly higher rate of CR (80.7%: 29.4%; p < 0.001) and a longer CR duration (4.6 months: 3.1 months; P = 0.03). Among patients who achieved CR, 78.4% were MRD negative in the Inotuzumab Ozogamicin group versus 28.1% in the standard-therapy group (P < 0.001). Survival analysis also documented the superiority of Inotuzumab Ozogamicin with median progression-free survival (PFS) improved from 1.8 months to 5.0 months (HR: 0.45; p < 0.001) and median OS improved from 6.7 months to 7.7 months (HR: 0.77; P = 0.04) [76]. Results were further confirmed in the long-term survival follow-up with a higher 2-year OS rate in the Inotuzumab Ozogamicin group (22.8%: 10.0%; HR: 0.75; P = 0.0105) [77]. In the subset analysis by age cohort, although patients < 55 years old were associated with a longer median OS (8.6: 5.6 months; HR:0.610) compared to those ≥55 years old, no significant difference of CR/CRi rates (75%: 70%; P = 0.24), MRD-negativity rate (76%: 79%; P = 0.64), duration of remission (DOR) (5.4 months: 4.7 months; HR = 0.748; P = 0.0934), PFS (5.0 months: 4.9 months, P = 0.1010) or incidences of any-grade treatment-emergent adverse events (TEAEs) (99%: 100%) was seen between the two age groups, suggesting Inotuzumab Ozogamicin could be well tolerated by the elderly [78]. It was noteworthy that liver damage of any degree occurred in about half of the patients (51%) in Inotuzumab Ozogamicin group, compared to 34% in standard care group. Sinusoidal obstruction syndrome (SOS) was more frequently observed in the Inotuzumab Ozogamicin group (13%: <1%) as well, thus liver-related adverse events should not be neglected during and after the treatment [79]. Trials on whether a lower dose of Inotuzumab Ozogamicin can have a similar effect while reducing its treatment-emergent hepatotoxicity are still ongoing. (e.g. NCT03677596) A pooled analysis investigated the outcome of 101 patients who proceeded to allo-HSCT after attaining CR with Inotuzumab Ozogamicin from the INO-VATE trial and another earlier phase 1/2 trial (NCT01363297) [80]. The most favorable 2-year survival laid in patients without previous HSCT or extra salvage treatment as a ratio of 51% [81].
In a retrospective study, pediatric patients with relapsed or refractory ALL were likewise well responded to Inotuzumab Ozogamicin, with 67% of CR/CRi rates. Among those in remission, 71% were MRD negative. Although SOS only occurred in the post-HSCT population, the incidence of which was much higher than that of in the INO-VATE trial (52%: 22%) [82]. Prospective studies are still ongoing (eg. NCT02981628, NCT03094611) to further assess the use of Inotuzumab Ozogamicin in children.
Unlike pediatric patients, adults and the elderly were characterized by poor tolerance to chemotherapy according to past experience. In order to improve this situation, a single-arm, phase 2 study (NCT01371630) was performed to explore the effect of Inotuzumab Ozogamicin plus mini-hyper-CVD. Mini-hyper-CVD, a modified version of standard hyper-CVAD with lower dose, referred to cyclophosphamide and dexamethasone reduced to 50%, methotrexate reduced to 25% and no anthracycline. In 59 R/R ALL patients aged 18-87 years, 78% responded, of whom 82% were negative for MRD. The 1-year RFS and OS rates were 40% and 46%, respectively [83]. Encouraging results were also reported in patients with newly-diagnosed Ph-ALL aged 60 years and over. All but one patient (98%) achieved responses, and estimated 3-year EFS and OS were 49%  and 56% , respectively [84].

Conclusion and discussion
The last decade has witnessed exciting survival improvements in ALL patients due to the progress made in monoclonal antibodies. Common targeted antigens include CD20、CD19 and CD22. Rituximab is a chimeric monoclonal antibody against CD20. For patients expressing at least 20% of CD20 and younger than 60 years old, combining Rituximab with hyper-CVAD regimen prolonged their CR duration and OS. Based on the positive response in treating adult patients, now a CCCG Relapsed ALL 2017 Study assesses the remission rate and MRD response in pediatric group treated with Rituximab and Bortezomib, a proteasome inhibitor [85]. Because the course of induction often goes with an increased level of CD20, the use of another anti-CD20 monoclonal antibody, Ofatumumab, which makes AYAs' 4-year EFS and OS comparable to those treated with pediatric-inspired regimens may expand the indication to patients with at least 1% of CD20 expression. Blinatumomab, a bispecific T-cell binding antibody, has been approved by FDA for patients with R/R B-ALL, as well as patients with morphologic remission but MRD+ ALL. We R/R, relapsed or refractory; CR, complete remission; CRi, CR with incomplete marrow recovery; ORR, overall response rate; OS, overall survival; PFS, progression-free survival; RFS, relapse-free survival; EFS, event-free survival; MRD, minimal residual disease; DOR, duration of remission; HCT, hematopoietic cell transplantation; CRh, CR with incomplete hematologic recovery.
suggest it to be an effective bridging approach to eradicate MRD before transplantation, and serves as a promising choice to achieve long-term survival for patients ineligible to undergo transplantation.
Ongoing clinical trials include how well Blinatumomab works in treating patients with MRD [86] and how effective Blinatumomab combines with Nivolumab (PD-1 checkpoint inhibitor) or Ipilimumab (CTLA-4 checkpoint inhibitor) in patients with R/R ALL [87,88]. Inotuzumab Ozogamicin is an anti-CD22 monoclonal antibody conjugated to a cytotoxic agent called calicheamicin. Among patients with CD22+ R/R B-ALL, encouraging results have been seen in all ages, so single-agent Inotuzumab Ozogamicin is recommended for children, adults and the elderly. Inotuzumab Ozogamicin plus mini-hyper-CVD chemotherapy is recommended for patients aged 60 years and over with newly-diagnosed Ph− ALL, but hepatotoxicity of any degree should be worthy of our attention at any time. Phase II trials evaluate Inotuzumab Ozogamicin in both pediatric and adult patients with detectable MRD are underway [89,90]. The combination of two different monoclonal antibodies, such as Blinatumomab and Inotuzumab Ozogamicin [91,92], and the development of new antibodies include antibody to CD20, CD19, CD123, and bispecific antibody to CD19 / CD3 are on progress [93][94][95][96]. Ongoing clinical trials mentioned above are listed in Table 2. In conclusion, the rise of monoclonal antibodies provides doctors with more options, but also brings new challenges in how to choose the most suitable therapy for each patient and achieve Individualized precision treatment.

Disclosure statement
No potential conflict of interest was reported by the author (s).