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

Abstract

Purpose: Randomized studies with gemtuzumab ozogamicin have validated CD33 as a target for antigen-specific immunotherapy of acute myelogenous 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 halflife compared to smaller antibody constructs.Experimental Design: We constructed a series of TandAbs composed of anti-CD33 and anti-CD3 variable domains of diverse binding affinities and profiled their functional properties in CD33 þ 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 CD33þ 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. Conclusions: 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. Clin Cancer Res; 22(23); 5829-38. Ó2016 AACR.

In Vitro and In Vivo Efficacy of a Novel CD33-Targeted Thorium-227 Conjugate for the Treatment of Acute Myeloid Leukemia

“…Similarly, IMGN779 (9) makes use of a new class of DNA alkylating agents called indolinobenzodiazepine pseudodimers (termed IGN) and has recently entered clinical phase I. Another strategy being pursued includes the development of bispecific T-cell engaging molecules including BiTE AMG330 (10-13) and a recently developed tetravalent TandAb CD33/CD3 molecule (41). The development of BiTEs for the treatment of hematopoietic disorders might be promising as exemplified by blinatumomab, a CD19-CD3 BiTE, approved for the treatment of acute lymphoblastic leukemia (42).…”

Highly Specific and Effective Targeting of EGFRvIII-Positive Tumors with TandAb Antibodies

“…The standard domain order and linker setting for TandAbs was as follows: ${\text{V}}_{\text{L}}^{\text{CD3}}-{\left(\text{GGS}\right)}_{\text{2}}-{\text{V}}_{\text{H}}^{\text{EGFRvIII}}-{\left(\text{GGS}\right)}_{\text{2}}-{\text{V}}_{\text{L}}^{\text{EGFRvIII}}-{\left(\text{GGS}\right)}_{\text{2}}-{\text{V}}_{\text{H}}^{\text{CD3}}.$ Some additional TandAb variants containing (GGS) 3 - or (GGS) 4 -linkers between V L and V H segments were constructed. Anti-CD3 sequence clone LcHC21ktay (26) or humanized anti-human and cynomolgus CD3 (CD3 x ) variants differing in their affinities for CD3 (28) were used in TandAbs for T-cell engagement. Gene sequences of published EGFRvIII-targeting comparator antibodies MR1-1 × OKT3 tandem single-chain bispecific antibody (31–33) and ch806 (34) were synthesized (Geneart).…”

“…The biodistribution of TandAbs, the ability to extravasate and also reach abluminal tumors is in agreement with predictions based on pharmacokinetic modeling and also confirmed by others (25). The TandAb platform is clinically validated and has shown potent cytotoxicity against target cells in vitro and in vivo (26–28). …”

abYsis: Integrated Antibody Sequence and Structure—Management, Analysis, and Prediction

“…Third, antibodies can be used as a delivery mechanism, either through antibody-drug conjugates with cytotoxic moieties or through 'antibody-directed enzyme prodrug therapy' (ADEPT), which aims to restrict the action of a cytotoxic drug to cancer sites [3]. Fourth, more recent developments have led to bispecific antibodies capable of binding two antigens, either for convenience of having a single agent, or deliberately cross-linking different molecules [4,5,6].…”