Rituximab and Alemtuzumab in Combination, but Not Alone, Induce Complete Remissions in a Preclinical Animal Model of Primary Human ALL: Rationale for Combination Treatment.
Authors: Nijmeijer Bart , van Schie Marianke L.J. , Willemze Roel , Falkenburg J.H. Frederik 
Published: October 2019

Abstract
Acute lymphoblastic leukemia (ALL) in adults has a poor prognosis, despite intensive chemotherapy or allogeneic stem cell transplantation. Further treatment intensification is limited by treatment-related toxicity. Monoclonal antibodies display a relatively favorable toxicity profile. Rituximab recognizing CD20, and alemtuzumab recognizing CD52, have shown clinical activity in hematological malignancies. Precursor-B ALL (preB-ALL) may express CD20 and/or CD52. However, little is known on the in vivo activity of these antibodies in preB-ALL. We evaluated the activity of rituximab and alemtuzumab on preB-ALL in a preclinical model. We first evaluated the expression of CD20 and CD52 on primary preB-ALL cells. For this, 18 primary samples were randomly selected, stained with rituximab or alemtuzumab, and secondary anti-human-Ig antibodies. Mean fluorescence intensity (MFI) was analyzed by flow cytometry. Ten samples expressed CD20 (56%, median MFI 102, range 97 to 951), eight samples did not (44%, median MFI: 6.9, range 6.3 to 8.3). Twelve samples expressed CD52 (67%, median MFI: 324, range 111–633), six samples did not (33%, median MFI: 8.1, range 6.5–8.8). All CD20 positive samples expressed CD52. Subsequently, we evaluated the in vivo activity of rituximab and alemtuzumab, alone and in combination.We selected 2 primary ALL (coded COA and VBK) that expressed CD20 and CD52. NOD/scid mice were engrafted with COA or VBK cells. Three weeks after inoculation, treatment was started by daily injection of either 250 ug rituximab or 250 ug alemtuzumab, or 250 ug of both antibodies. Throughout the treatment period, therapeutic concentrations of antibody were measured in serum of treated animals (rituximab: median 399 ug/mL, range 200–739 ug/mL, alemtuzumab: median 152ug/mL, range 51–199 ug/mL). After four weeks of treatment, animals were sacrificed. Peripheral blood (PB), spleen (SPL) and bone marrow (BM) were isolated and analyzed by flow cytometry. In animals treated with single antibody, leukemic cells persisted though at lower levels when compared to control animals, suggesting limited activity. After engraftment of COA leukemia, in rituximab treated animals 1, 6 and 59%, in alemtuzumab treated animals 1, 8 and 19%, and in control treated animals 37, 80 and 70% leukemic cells were present in PB, SPL and BM, respectively. After engraftment of VKB leukemia in rituximab treated animals 42, 45 and 75%, in alemtuzumab treated animals 2, 2 and 24%, and control treated animals 50, 59 and 86% leukemic cells were found in PB, SPL and BM, respectively. In contrast, combination treatment with rituximab and alemtuzumab induced complete remissions in all of 7 COA-engrafted, and in 2 of 3 VBK-engrafted animals. The VBK-engrafted animal that did not reach a remission showed less than 1% leukemic cells in BM only. Cells that persisted during single antibody treatment were analyzed by flow cytometry. In all animals treated with rituximab or alemtuzumab alone, the persisting leukemic cells did not have therapeutic antibody present on their surface, nor could the targeted antigen be detected. These observations suggested that persistence of leukemic cells was due to loss of the targeted antigen. In the control animals no change of the expression of CD20 or CD52 during leukemic outgrowth was observed. These results suggest that combined treatment with rituximab and alemtuzumab may be far more effective than treatment with either agent alone in a significant number of preB-ALL patients.