Characterization of CD33/CD3 Tetravalent Bispecific Tandem Diabodies (TandAbs) for the Treatment of Acute Myeloid Leukemia.

Purpose: Randomized studies with gemtuzumab ozogamicin have validated CD33 as a target for antigen-specific immunotherapy of acute myeloid leukemia (AML). Here, we investigated the potential of CD33/CD3-directed tandem diabodies (TandAbs) as novel treatment approach for AML. These tetravalent bispecific antibodies provide two binding sites for each antigen to maintain the avidity of a bivalent antibody and have a molecular weight exceeding the renal clearance threshold, thus offering a longer half-life compared to smaller antibody constructs. Experimental design: We constructed a series of TandAbs comprised of anti-CD33 and anti-CD3 variable domains of diverse binding affinities and profiled their functional properties in CD33+ human leukemia cell lines, xenograft models, and AML patient samples. Results: Our studies demonstrated that several CD33/CD3 TandAbs could induce potent, dose-dependent cytolysis of CD33+ AML cell lines. This effect was modulated by the effector-to-target cell ratio and strictly required the presence of T-cells. Activation and proliferation of T-cells and maximal AML cell cytolysis correlated with high avidity to both CD33 and CD3. High-avidity TandAbs were broadly active in primary specimens from patients with newly diagnosed or relapsed/refractory AML in vitro, with cytotoxic properties independent of CD33 receptor density and cytogenetic risk. Tumor growth delay and inhibition were observed in both prophylactic and established HL-60 xenograft models in immunodeficient mice. Conclusion: Our data show high efficacy of CD33/CD3 TandAbs in various preclinical models of human AML. Together, these findings support further study of CD33/CD3 TandAbs as novel immunotherapeutics for patients with AML.