A recombinant trispecific single‐chain Fv derivative directed against CD123 and CD33 mediates effective elimination of acute myeloid leukaemia cells by dual targeting

Abstract

Two trivalent constructs consisting of single-chain Fv antibody fragments (scFvs) specific for the interleukin-3 receptor a chain (CD123), CD33 and the Fcc-receptor III (CD16) were designed and characterized for the elimination of acute myeloid leukaemia (AML) cells. The dual targeting single-chain Fv triplebody (sctb) [123 · ds16 · 33] and the mono targeting sctb [123 · ds16 · 123] both specifically bound their respective target antigens and were stable in human serum at 37°C for at least 5 d. Both constructs induced potent antibody-dependent cellular cytotoxicity (ADCC) of two different AML-derived CD33-and CD123 double-positive cell lines in the low picomolar range using isolated mononuclear cells (MNCs) as effector cells. In these experiments the dual targeting molecule produced significantly stronger lysis than the mono targeting agent. In addition, the sctbs showed a high potency in mediating ADCC of primary leukaemia cells isolated from peripheral blood or bone marrow of seven AML patients. Hence, these novel molecules displayed potent anti-leukaemic effects against AML cells in vitro and represent attractive candidates for further preclinical development.

Dual-targeting triplebody 33-16-123 (SPM-2) mediates effective redirected lysis of primary blasts from patients with a broad range of AML subtypes in combination with natural killer cells

“…59 The original scFv domains were of murine origin 59 and were humanized, disulfide-stabilized and stability-engineered for incorporation into SPM-2. A final clinical candidate named SPM-2 was identified, and a production process in stably transfected human Freestyle TM 293F-cells, as well as a downstream purification process following industry standard procedures was developed.…”

“…We also urgently wish to determine, whether the dual-targeting triplebody mediates more potent lysis at lower EC50 doses in RDL experiments than the mono-targeting related proteins CD33-16 and CD123–16, either individually or in combination, similar to the studies performed earlier with the triplebodies 123-16-33, HLAII-16–19 and 33–3-19. 59,68–70 …”

“…A number of therapeutic agents with similar molecular formats as those described for CD33 have been developed, including immunoglobulins, 47–49 a radio-immunoconjugate, 50 ADCs and a fusion protein between the cytokine IL-3 and a fragment of diphtheria toxin, 51–54 bi-specific T cell-recruiting agents (BiTEs) and Dual-Antigen Recruiting T-cell engagers (DARTs), 55–58 dual-targeting triplebodies, 59,60 and CAR-transfected T cells. 61–64 Expression of CD123 is largely restricted to hematopoietic cells, but expression on endothelial cells has been reported.…”

“…8 Larger studies in patients with many different subtypes of the disease are needed to investigate, whether new dual-targeting agents simultaneously addressing both antigens on the same cell 59,60 or combinations of corresponding mono-targeting agents are promising for clinical applications for a broad group of AML patients. In a first large study pursuing this goal, the expression of CD33 and CD123 alone and in combination was investigated on cellular samples from 319 AML patients.…”

Mono- and dual-targeting triplebodies activate natural killer cells and have anti-tumor activityin vitroandin vivoagainst chronic lymphocytic leukemia

“…Several trispecific immunoconstructs targeting the FcgRIIIa receptor on NK cells have been developed and compared with their bispecific counterparts. [26][27][28] An additional tumor-specific arm in such trispecific construct increased its avidity, leading to enhanced target cell killing. [26][27][28] Moreover, increase in the construct size beyond the kidney exclusion limit of 65 kDa was an additional clinical advantage.…”