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.

CD20 Abs induce clinical responses in lymphoma patients, but there are considerable differences between individual patients. In 51Cr release assays with whole blood as effector source, RAJI cells were effectively killed by a mouse/human chimeric IgG1 construct of CD20 Ab 1F5, whereas ARH-77 proved resistant to killing by this Ab. When whole blood was fractionated into plasma, mononuclear cells, or granulocytic effector cells, RAJI cells were effectively killed in the presence of complement-containing plasma, whereas the mature B cell line ARH-77 proved complement resistant. However, with a bispecific Ab (BsAb) against the myeloid receptor for IgA (CD89; FcαRI) and CD20, a broad range of B cell lines were effectively killed. FcαRI is expressed on monocytes/macrophages, neutrophils, and eosinophils. As the numbers of these effector cells and their functional activity can be enhanced by application of G-CSF or GM-CSF, lysis via (FcαRI × CD20) BsAb was significantly enhanced in blood from patients during therapy with these myeloid growth factors. Interestingly, the major effector cell population for this BsAb were polymorphonuclear neutrophils, which proved ineffective in killing malignant B cells with murine, chimeric IgG1, or FcγRI- or FcγRIII-directed BsAbs against CD20. Experiments with blood from human FcαRI/FcγRI double-transgenic mice showed corresponding results, allowing the establishment of relevant syngenic animal models in these mice. In conclusion, the combination of myeloid growth factors and an (FcαRI × CD20) BsAb may represent a promising approach to improve effector cell recruitment for CD20-directed lymphoma therapy.

IntroductionOver the last 5 years, monoclonal antibodies have broadened the therapeutic armentarium in oncology. 1,2 Hematologic malignancies are recognized as particularly promising targets, 3 which is also reflected by today's list of approved antibodies. 4 Among those investigated in early clinical trials against B-cell lymphomas are antibodies against human leukocyte antigen (HLA) class II, such as Lym-1 5 and Hu1D10. 6 Preclinical studies demonstrated that HLA class II antibodies were particularly effective in killing Blymphoma cells. For example, they inhibited B-cell proliferation, triggered apoptosis and complement-dependent cytotoxicity (CDC), and mediated antibody-dependent cellular cytotoxicity (ADCC) by mononuclear and polymorphonuclear neutrophil (PMN) effector cells. 7-10 Importantly, studies against xenogenic tumors in severe combined immunodeficient (SCID) mice confirmed the potential of HLA class II antibodies in vivo, 11,12 although HLA class II antibodies were less efficient than anti-idiotype antibodies in syngenic lymphoma models. 13 However, HLA class II is constitutively expressed on normal B cells, monocytes/macrophages, and dendritic cells, and exposure to interferon (IFN)-␥ may induce HLA class II expression on many other cell types, including activated T cells and endothelial cells. Correspondingly, HLA class II antibodies demonstrated considerable toxicity in primate models, 14 and side effects in phase 1 clinical trials were more pronounced than those usually observed with rituximab. Importantly, however, some durable responses were documented in this phase 1 trial, even at low antibody doses. 15 Our understanding of what effector mechanisms operate with monoclonal antibodies in vivo is comparatively limited. 16 Novel studies demonstrated that some antibodies act by directly stimulating immune effector cells [17][18][19][20] or by blocking inhibitory immune interactions. 21 Traditionally, however, effector mechanisms were divided into direct mechanisms, which are mediated by the antibody's variable region-including growth inhibition, induction of apoptosis, and blockade of growth factors-and indirect mechanisms, which are triggered by the constant regions of antibodies. The latter include complement-dependent tumor cell lysis and effector cell-mediated killing by ADCC or phagocytosis. Effector cell-mediated mechanisms typically require interactions between antibodies' constant Fc region and their cellular receptors. For IgG, 3 classes of leukocyte receptors are distinguished: Fc␥RI (CD64), Fc␥RII (CD32), and Fc␥RIII (CD16), which associate with the common FcR␥ chain for signaling. 22 Recent studies in mice, in which expression and function of activating Fc receptors were disrupted by "knockout" of this FcR␥ chain, have demonstrated that Fc␥ receptors are critical for the therapeutic efficacy of rituximab and herceptin, 23 currently the most widely used antibodies in oncology. Like rituximab and herceptin, most of the tumor-directed unconjugated antibodies employed in clinical trials ...