Combination Therapy of Humanized Chimeric-Anti-Human VLA4 Monoclonal Antibodies and Anti-Cancer Drugs for AML.

Bone marrow (BM) minimal residual disease causes relapse after chemotherapy in AML. We have previously reported that VLA4-positive leukemic cells acquired resistance to drug-induced apoptosis through the PI-3K/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA4 and fibronectin on BM stromal cells. This resistance was negated by mouse-anti-human VLA4Ab (mouse VLA4Ab). In human leukemia SCID mouse model, we demonstrated a 100% survival rate with combination of mouse VLA4 Ab and AraC, while with AraC alone, only slight prolongation of survival was attained. In clinical study, overall survival at 5 years was 90% for 10 VLA4⁻ patients and 25% for 15 VLA4⁺ patients (Matsunaga T et al, Nature Med 2003, 9, 1158–1165). In the present study, to perform the translational research, we first examined the myelosuppressive effect of the combination of rat-anti-mouse VLA4 Ab and AraC in C57/BL6 mice, and found that CBC data were almost the same as those of the mice treated with AraC alone. We next produced humanized chimeric-anti-human VLA4 Ab (chimeric-VLA4Ab), and examined its efficacy in combination with anti-cancer drugs in vitro and in vivo (human leukemia SCID mouse model). Chimeric-VLA4Abs were produced as follows: (i) total RNA of mouse VLA4 Abs were extracted from two hybridomas (SG/17 and SG/73), (ii) cDNA were synthesized by reverse transcriptase, (iii) variable region gene of mouse VLA4 Abs were amplified by 5′RACE method, (iv) TA cloning of amplified gene was performed, (v) sequence of mouse VLA4Abs gene was determined, (v) cloned variable region gene of mouse VLA4 Abs and constant region gene of human IgG1 were inserted into expression vector, and the expression vector was transfected into 293T cells, (vi) supernatant of the 293T cells was collected and purified to obtain chimeric-VLA4 Abs. The effects of chimeric-VLA4 Abs thus obtained in in vitro and in vivo (human leukemia SCID mouse model) were comparable to those of mouse VLA4 Abs. To perform the clinical study, we are presently producing the GMP-graded chimeric-VLA4 Abs.