A Recombinant Bispecific Single-Chain Fragment Variable Specific for HLA Class II and FcαRI (CD89) Recruits Polymorphonuclear Neutrophils for Efficient Lysis of Malignant B Lymphoid Cells

Abstract

Bispecific Abs offer new perspectives for cancer immunotherapy. In this study, we describe a recombinant bispecific single-chain fragment variable (bsscFv) directed against FcαRI (CD89) on polymorphonuclear neutrophils (PMNs) or monocytes/macrophages and HLA class II on lymphoma target cells. FcαRI and HLA class II-directed single-chain fragment variable (scFv) fragments were isolated from phage display libraries, established from the hybridomas A77 and F3.3, respectively. The two scFv molecules were connected with a 20 aa flexible linker sequence. After expression in SF21 insect cells and chromatographic purification, the bispecific molecule showed specific binding to both Ags at KD values of 148 ± 42 nM and 113 ± 25 nM for the anti-FcαRI and anti-HLA class II scFv components in the bsscFv, respectively. In Ab-dependent cytotoxicity assays with PMNs as effectors and a series of lymphoma-derived cell lines (ARH-77, RAJI, REH, NALM-6, RS4;11), the bsscFv was significantly more cytotoxic than the parental murine IgG1 and its chimeric IgG1 derivative. When targeting primary tumor cell isolates from six patients with B cell malignancies, the killing capacity of the (FcαRI × HLA class II) bsscFv compared favorably to conventional HLA class II mAb. Importantly, the cell lines NALM-6 and RS411, as well as two primary tumor cell isolates, were exclusively lysed by the bsscFv. To our knowledge, this is the first report of an FcαRI-directed bsscFv effectively recruiting PMNs for redirected cytotoxicity against human B cell malignancies. Our data show that an (FcαRI × HLA class II) bsscFv is an interesting candidate for further engineering of small, modular immunopharmaceuticals.

The Multifaceted Roles Neutrophils Play in the Tumor Microenvironment

“…GM-CSF enhanced the anti-tumor neutrophil-dependent ADCC through enhanced expression of CD11/CD18 molecules [336]. Neutrophils were also shown to contribute to the anti-tumor ADCC in Non-Hodgkin's Lymphoma using antibodies to CD20 (Rituximab) [341], in breast cancer using a Tn antigen-specific chimeric mAb [342], and in B-cell lymphoma using a bispecific singlechain fragment variable-specific for HLA class II and FcαRI (CD89) [343]. The bispecific antibody against the myeloid receptor for IgA (FcαR1; CD89) and the B-cell surface marker CD20 induced neutrophil-dependent ADCC toward broad range of B cell lines [344].…”

Natural Killer (NK)- and T-Cell Engaging Antibody-Derived Therapeutics

“…The first are F(ab') 2 conjugates targeting the tumor antigens HER2/neu or CD30, respectively. The second is a bispecific ab targeting the EGFR, and the third a tandem bispecific single chain Fv (bsscFv) targeting HLA class II [39,[45][46][47]. All of these agents mediated ADCC in vitro by recruiting CD89-positive effector cells, i.e., mainly neutrophilic granulocytes and monocytes.…”

Fc Receptors in Mucosal Immunology

“…Moreover, it has now been demonstrated that FcαRI is the most potent neutrophil Fc receptor compared to Fcγ receptors for the induction of antibody-dependent killing of tumor cells (Deo et al, 1998;Huls et al, 1999aHuls et al, , 1999bvan Egmond et al, 1999a;Keler et al, 2000;Stockmeyer et al, 2000;Dechant and Valerius, 2001;Stockmeyer et al, 2001;Dechant et al, 2002;Stadick et al, 2002;Stockmeyer et al, 2003;Dechant et al, 2007;Zhao et al, 2008;Guettinger et al, 2010;Lohse et al, 2010). Furthermore, cross-linking of neutrophil FcαRI by (dimeric) IgA leads to the release of LTB4 and neutrophil recruitment, resulting in amplification of neutrophil migration (van der Steen et al, 2009).…”