The ability of human gd T cells from healthy donors to kill pancreatic ductal adenocarcinoma (PDAC) in vitro and in vivo in immunocompromised mice requires the addition of gd T-cell-stimulating antigens. In this study, we demonstrate that gd T cells isolated from patients with PDAC tumor infiltrates lyse pancreatic tumor cells after selective stimulation with phosphorylated antigens. We determined the absolute numbers of gd T-cell subsets in patient whole blood and applied a real-time cell analyzer to measure their cytotoxic effector function over prolonged time periods. Because phosphorylated antigens did not optimally enhance gd T-cell cytotoxicity, we designed bispecific antibodies that bind CD3 or Vg9 on gd T cells and Her2/neu (ERBB2) expressed by pancreatic tumor cells. Both antibodies enhanced gd T-cell cytotoxicity with the Her2/Vg9 antibody also selectively enhancing release of granzyme B and perforin. Supporting these observations, adoptive transfer of gd T cells with the Her2/Vg9 antibody reduced growth of pancreatic tumors grafted into SCID-Beige immunocompromised mice. Taken together, our results show how bispecific antibodies that selectively recruit gd T cells to tumor antigens expressed by cancer cells illustrate the tractable use of endogenous gd T cells for immunotherapy.

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.

In the last two decades, monoclonal antibodies have revolutionized the therapy of cancer patients. Although antibody therapy has continuously been improved, still a significant number of patients do not benefit from antibody therapy. Therefore, rational optimization of the antibody molecule by Fc engineering represents a major area of translational research to further improve this potent therapeutic option. Monoclonal antibodies are able to trigger a variety of effector mechanisms. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement- dependent cytotoxicity (CDC) are considered important in antibody therapy of cancer. Novel mechanistic insights into the action of monoclonal antibodies allowed the development of various Fc engineering approaches to modulate antibodies' effector functions. Strategies in modifying the Fc glycosylation profile (Fc glyco-engineering) or approaches in engineering the protein backbone (Fc protein engineering) have been intensively evaluated. In the current review, Fc engineering strategies resulting in improved ADCC, ADCP and CDC activity are summarized and discussed.

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