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Innovative Therapeutic Strategies for Mixed Lineage Leukemia-rearranged B-cell Acute Lymphoblastic Leukemia

Periodic Reporting for period 1 - InTheMLLrBALL (Innovative Therapeutic Strategies for Mixed Lineage Leukemia-rearranged B-cell Acute Lymphoblastic Leukemia)

Reporting period: 2018-04-01 to 2020-03-31

B-cell acute lymphoblastic leukemia (B-ALL) is the commonest cancer in children and many B-ALL patients are refractory to chemotherapy and eventually relapse.
Adoptive transfer of T-cells engineered to express an artificial chimeric antigen (Ag) receptor (CAR) targeting a tumor cell surface-specific Ag is a promising approach for Cancer Immunotherapy. In B-ALL, CAR T-cells targeting CD19 have generated unprecedented results, and ~50% of treated patients are disease-free after 1 year. However, relapse after treatment is common. Moreover, the expression of CD22 has been demonstrated in B-ALL cells as well as in patients who relapse as CD19- after CD19-CAR T-cell therapy. Consequently, a CD22-CAR T-cell approach has been recently developed, and preliminary clinical results in refractory/relapse (R/R) B-ALL are promising; however, relapse also occurred. A strategy to offset Ag-loss relapse is to modify T-cells with one CAR molecule containing two different binding domains in tandem. Therefore, tandem CAR T-cells targeting both CD19 and CD22 may overcome Ag-loss escape and reduce the risk of relapse in B-ALL.
Immuno checkpoints (IC) receptors such as PD-1, CTLA-4, LAG-3, and TIM-3 regulate immune function homeostasis, but many cancers overexpress their ligands (Ls) as immune-regulatory mechanism to achieve immune escape. Moreover, the involvement of tumor microenvironment in the loss of CAR T-cell function by PD-1/PD-L1 has also been suggested. To date, there is a paucity of clinical data showing that blockade of IC can enhance CAR-T cell function. In the setting of B-ALL, recent studies suggest that B-ALL cells seem to regulate T-cell function through PD-1/PD-L1 axis, thus impairing the therapeutic efficacy of T-cells against B-ALL cells. Overall, these studies underscore the need to investigate the role of IC molecules in the B-ALL cell response treatment.
B-ALL originates in the bone marrow (BM), and the BM microenvironment (BMM) is particularly important in B-ALL given the high dependence of leukemic cells on external factors for proliferation and survival. The BM is also the primary site where residual leukemic cells survive during standard chemotherapy, and is the most frequent location of B-ALL relapse. Mesenchymal stromal cells (MSC) are vital constituents of the BMM and are involved in drug resistance of B-ALL cells during chemotherapy. The successful function/persistence of CAR T-cells within the host depend on complex in vivo processes and, in recent years, it has been extensively demonstrated that MSC regulate T-cells by modifying their activation, proliferation, and effector functions. However, the impact of BM-MSC on CD19-CAR T-cells, that are likely to experience the same loss of function/persistence as T-cells is understudied.
The rationale for launching this project was to develop a new CAR T-cell immunotherapy for B-ALL patients, who otherwise have no current alternative treatment. In the time of this Marie Skłodowska-Curie Actions Individual Fellowship, by using both in vitro and in vivo approaches, I generated a novel tandem CD22/CD19-CAR for the treatment of R/R B-ALL. In parallel, I contributed to characterized IC axes in B-ALL to decide which of them has the major implication in B-ALL to develop future immunotherapies. Finally, I studied the influence of BM-MSC from B-ALL patients in the activity of CD19-CAR T-cells to understand better the interplay between T-cells, B-ALL cells and BM-MSC to improve the efficiency of this immunotherapy. In general, the findings of this project not only generate frontier knowledge on the disease, they are also very important for society. In particular, for patients with B-ALL who do not have current alternative treatment because the findings of this project provide key information for the clinical translation of a CD22/CD19-CAR for B-ALL.
A tandem CD22/CD19-CAR is not only useful to treat MLL-r B-ALL but also to treat a broader spectrum of B-ALL subtypes given the expression of CD22 in almost B-ALL patients. We have generated and functionally validated in vitro and in vivo a tandem CD22/CD19-CAR, using in-house developed CD19 and CD22 monoclonal antibodies, and compared it with CD19-CAR. CD19-CAR- and CD22/CD19-CAR-expressing T cells showed similar expansion in vitro and activity in vitro and in vivo using B-ALL cell lines, patient samples and patient-derived xenografts. Collectively, our tandem CD22/CD19-CAR warrants a clinical opportunity to test whether targeting both CD19 and CD22 may overcome Ag-loss escape and reduce the risk of relapse in B-ALL.
Besides, we characterized the immunomodulatory effects of B-ALL BM-MSC on human T-cells and CD19-CAR T-cells. While B-ALL BM-MSC were less proliferative than equivalent cells from healthy donors (HD) in vitro, the morphology, immunophenotype, differentiation potential and chemoprotection was very similar in both populations. Likewise, both HD and B-ALL BM-MSC were equally immunosuppressive in vitro and anti-inflammatory in an in vivo model of severe colitis. Interestingly, B-ALL BM-MSC failed to impair CD19-CAR T-cell proliferation, cytotoxicity or cytokine production in vitro using B-ALL cell lines and patient-matched primary B-ALL cells. Finally, in vivo the growth of NALM6 cells was controlled by CD19-CAR T-cells irrespective of the presence/absence of B-ALL or HD BM-MSC.
This proposal focuses on Cancer, one of the major challenges to European Health Research Area and one priority in H2020 and in the 3rd EU Health Programme. Cancer is one of the “Major Chronic Diseases” and is a key public health concern across Member States and a central priority of EU health policy. Health is one of the aspects addressed by H2020, whose three key priorities are those of Europe 2020, Health 2020 and Innovation Union, together designed to boost Europe’s economy. Thus, this project therefore addresses two of these three key aims that are Excellent Science as well as Health as a Societal Challenge.
Further innovative research in CAR-T cells is essential, especially in the EU where CAR clinical trials remain underdeveloped. In line with this, the findings during this fellowship demonstrate that a CD22/CD19-CAR warrants a clinical opportunity for B-ALL. Regarding the findings related to IC molecules demonstrate that targeting IC axes in combination with CD19-CAR T-cell therapy could be a way to further improve the efficiency of the current immunotherapy for B-ALL. These findings contribute to strengthen European excellence and competitiveness in Cancer Immunotherapy and to improve the clinical treatments and finally increase the quality of life of the patients affected by this illness.
Besides my research, as Marie Skolodowska-Curie Ambassador I participated twice at IGTP in Badalona, to explain to PhD students and researchers how to prepare a winning MSCA proposal. Moreover, I shared our findings with science and non-scientific communities. I participated in every other week seminars organized by the Hematology and Immunology Departments of the Hospital Clinic of Barcelona where I also presented data obtained from my project once per semester, and as well the results have been presented in several scientific conferences. Regarding transfer of knowledge to non-scientific community, in 2019 I participated in the UniStem Day at the School of Medicine (University of Barcelona), and in the European Researcher Night at the French Lyceum of Barcelona.
Woman In Science Day 2020, IJC, Spain.
Round Table: MSCA IF 2018 at IGTP, Badalona, Spain.