Development of Anti-CD99scFvs for the Treatment of Acute Myeloid Leukemia

CD99 has gained much attention in recent years as a novel therapeutic target in hematological malignancies, due to its upregulation in acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). We have recently shown that targeting CD99 with a knockdown approach or with commercial antibodies results in antileukemia activity in AML cells. We have also developed anti-CD99 nanoparticles and demonstrated excellent in vitro and in vivo antileukemia activity. Here we report the development of human single chain variable fragment targeting CD99 (anti-CD99 scFv) and the preclinical activity in AML cells and in AML xenograft mouse model. The anti-CD99 scFv was developed by inserting CD99scFv DNA sequence into the pFUSE vector, encoding pATL103-CD99 scFvs. To produce anti-CD99 scFv, the plasmids were transfected in Expi293 (HEK293T) cells. Then the medium was harvested and underwent two cycles of dialysis. To determine product purity, proteins were analyzed on SDS-PAGE gel stained with Coomassie blue. For each 240ml cell culture media with recombinant CD99 plasmids, we obtained about 1-2mg anti-CD99 scFv. The binding affinity of CD99 scFv to CD99 surface protein was assessed in 293T cells (CD99 null cells) which exhibited no binding and in MOLM-13 cells and MV4-11 (CD99 positive AML cells) which demonstrated strong binding. Treatment with 5uM of anti-CD99 scFv significantly reduced cell viability in both leukemic cell line MOLM-13 and MV4-11 (MOLM-13: 35.14%, P= 0.008; MV4-11: 30.17%, P=0.002) and primary AML patient blasts (29.37%, P=0.048) compared with control cells. Colony forming assay showed that anti-CD99 scFv treated AML blasts exhibited less number of colonies compared with control cells (plating efficiency (PE): 0.035% vs 0.12%). We also established the in vivo antileukemia activity of anti-CD99 scFv using MOLM-13 cells (FLT3-ITD positive AML cells) NOD scid gamma (NSG) xenograft mouse model. MOLM-13 (2.5x10^6) cells were engrafted into NSG mice via IV tail injection (N=4 mice per group). Mice were treated with PBS (group 1) or 4mg/kg of anti-CD99 scFV on days 10, 14, 18 and 22 post cell engraftment. Mice were euthanized on day 24 and levels of leukemia engraftment were assessed by flow cytometry measurements of huCD45 staining of cells collected from the bone marrow and the peripheral blood and compared between the two groups. Mice treated with four doses of 4mg/kg CD99scFv demonstrated significant reduction in leukemia engraftment in the bone marrow assessed by flow cytometry measurements of huCD45 staining compared with the PBS mice group (huCD45%: 39.7 vs 56, P = 0.0017). In conclusion, we report the development of anti-CD99 single chain variable fragments for the treatment of AML. Our study demonstrates good binding affinity and specificity and a promising preclinical antileukemia activity both in AML cells and in xenograft mouse model.