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.

A novel bispecific antibody-derived recombinant protein targeting leukemias and lymphomas was designed, a single-chain Fv triple body (sctb) consisting of 1 polypeptide chain with 3 scFvs connected in tandem. The distal scFvs were specific for the tumor antigen CD19, and the central scFv for the trigger molecule CD16 (FcgammaRIII) on natural killer (NK) cells and macrophages. We had previously built a disulphide stabilized (ds) bsscFv [19 x 16] with monovalent binding for CD19 from ds components. The sctb ds[19 x 16 x 19] also used ds components and displayed 3-fold greater avidity for CD19 than the bsscFv (KD = 13 vs. 42 nM), whereas both had equal affinity for CD16 (KD = 58 nM). Plasma half-lives in mice were 4 and 2 hours for the sctb and the bsscFv, respectively. In antibody-dependent cellular cytotoxicity reactions with human mononuclear cells as effectors, the sctb promoted equal lysis of leukemic cell lines and primary cells from leukemia and lymphoma patients at 10-fold to 40-fold lower concentrations than the bsscFv. This new format may also be applicable to a variety of other tumor antigens and effector molecules. With half-maximum effective concentrations (EC50) in the low picomolar range, the sctb ds[19 x 16 x 19] is an attractive candidate for further preclinical evaluation.

D-2-hydroxyglutarate (D-2HG) is released by various types of malignant cells including acute myeloid leukemia (AML) blasts carrying isocitrate dehydrogenase (IDH) gain-of-function mutations. D-2HG acting as an oncometabolite promotes proliferation, anoikis, and differentiation block of hematopoietic cells in an autocrine fashion. However, prognostic impact of IDH mutations and high D-2HG levels remains controversial and might depend on the overall mutational context. An increasing number of studies focus on the permissive environment created by AML blasts to promote immune evasion. Impact of D-2HG on immune cells remains incompletely understood. Here, we sought out to investigate the effects of D-2HG on T-cells as key mediators of anti-AML immunity. D-2HG was efficiently taken up by T-cells in vitro, which is in line with high 2-HG levels measured in T-cells isolated from AML patients carrying IDH mutations. T-cell activation was slightly impacted by D-2HG. However, D-2HG triggered HIF-1a protein destabilization resulting in metabolic skewing towards oxidative phosphorylation, increased regulatory T-cell (Treg) frequency, and reduced T helper 17 (Th17) polarization. Our data suggest for the first time that D-2HG might contribute to fine tuning of immune responses.

Single-chain Fv triplebodies (sctb), consisting of a single polypeptide chain with 3 single-chain antibody variable fragments connected in tandem, were generated as antileukemic agents. A CD19-specific sctb of this format has previously been shown to be superior to a bispecific single-chain Fv antibody fragment (bsscFv) for the elimination of leukemic B-lineage cells, but corresponding targeted agents for the treatment of acute myeloid leukemia are still lacking. For this purpose, both a bsscFv and a sctb specific for CD33 and the trigger molecule CD16 (FcgammaRIII) were produced. The sctb displayed 3.5-fold greater avidity for CD33 than the bsscFv 33xds16, whereas both had close to equal affinity for CD16. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, both the bsscFv 33xds16 and the sctb induced lysis of tumor cells with half maximum effective concentrations (EC50) in the low picomolar range. It is interesting to note that the sctb promoted equal lysis of human leukemia-derived cell lines at 10 to 200-fold lower concentrations than the bsscFv. Both molecules mediated ADCC of primary patient cells. In conclusion, both the bsscFv 33xds16 and the sctb 33xds16x33 eliminated acute myeloid leukemia cells in ADCC reactions, but the novel sctb format showed significantly greater specific activity.

SummaryFour new single-chain Fv antibody fragments (scFvs) specific for the human leucocyte surface antigen CD123 (interleukin-3 receptor a) were generated to achieve preferential targeting of leukaemia stem cells (LSCs) in acute myeloid leukaemia (AML). The scFvs were isolated from a phage display library generated with spleen RNA from mice, immunized with a fusion protein consisting of the extracellular domain of CD123 and the Fc domain of a human immunoglobulin G1. The scFvs displayed CD123-specific binding on tumour cells (binding constants (K D ) 4AE5-101 nmol/l). The scFv with the highest affinity was used to design two cell death-inducing molecules. First, an immunotoxin, a fusion protein with truncated Pseudomonas Exotoxin A, induced potent apoptosis of AML-derived MOLM-13 and SKNO-1 cells at nanomolar concentrations. Second, the fusion to another scFv, specific for the low affinity