A recombinant bispecific single‐chain Fv antibody against HLA class II and FcγRIII (CD16) triggers effective lysis of lymphoma cells

Abstract

Summary Bispecific antibodies offer the possibility of improving effector‐cell recruitment for antibody therapy. For this purpose, a recombinant bispecific single‐chain Fv antibody (bsscFv), directed against FcγRIII (CD16) and human leucocyte antigen (HLA) class II, was constructed and tested in functional assays. RNA from the hybridomas 3G8 and F3.3, reacting with CD16 and HLA class II, respectively, was used to generate phage display libraries. From these libraries, reactive phages were isolated and the bsscFv was constructed by connecting both single‐chain Fv components through a 20 amino acid flexible linker. After expression in SF21 insect cells and chromatographic purification, the bsscFv bound specifically and simultaneously to both antigens. The affinities of the anti‐CD16 and the anti‐HLA class II scFv components of the bsscFv were 8·6 × 10−8 mol/l and 13·7 × 10−8 mol/l, respectively, which was approximately sevenfold lower than the F(ab) fragments of the parental antibodies. In antibody‐dependent cellular cytotoxicity experiments with human mononuclear cells as effectors, the bsscFv‐mediated specific lysis of both HLA class II‐positive, malignant human B‐lymphoid cell lines and primary cells from patients with chronic B‐cell lymphocytic leukaemia. Optimal lysis was obtained at bsscFv concentrations of approximately 400 ng/ml, similar to the concentration required for maximum lysis by the corresponding chemically linked bispecific antibody. Thus, this recombinant bsscFv‐antibody is an efficient molecule for effector‐cell mediated lysis of malignant human B‐lymphoid cells.

Mimicking an Induced Self Phenotype by Coating Lymphomas with the NKp30 Ligand B7-H6 Promotes NK Cell Cytotoxicity

“…To analyze binding of B7-H6:7D8 and B7-H6:4D5, Ramos or MDA-MB-361 cells were incubated with either protein on ice for 45 min, then washed with 2 ml PBA buffer and subsequently stained with a secondary Alexa Fluor 488-coupled anti-penta His Ab (Qiagen) on ice for 30 min. To determine the affinity of B7-H6:7D8 for CD20, binding curves were recorded and equilibrium constants (K D values) were calculated as described (31). To demonstrate simultaneous binding, Ramos cells were preincubated with B7-H6:7D8 (ULBP2:7D8 as negative control) at 50 mg/ml followed by the fusion protein NKp30-Fc ko at 100 mg/ml.…”

“…Single clones were isolated by limiting dilution. The Histagged proteins were purified by affinity chromatography with Ni-NTA agarose beads (Qiagen, Hilden, Germany) as described (31). Fc containing fusion proteins were purified as described (28).…”

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

“…Thereby the vector pSecTag2HygroC-STREP-His-CD123 · dsCD16 · CD33 was produced. In these constructs, the index ds designates the disulfide-stabilized variants (Bruenke et al, 2004). To generate the vector pSecTag2HygroC-STREP-His-CD123 · dsCD16 · CD123, the sequence coding for the CD123-specific scFv was amplified by polymerase chain reaction (PCR) from the vector pAK400-CD123scFv and ligated into pSecTag2-HygroC-STREP-His-CD123 · dsCD16 · CD33, using XhoI/ EcoRV restriction sites, and replacing the coding sequence for the C-terminal CD33-specific scFv.…”

Generation and selection of immunized Fab phage display library against human B cell lymphoma

“…As for lymphoma, so far only two studies have reported the generation of specific phage display library. Bruenke et al [22] generated bispecific single-chain Fv antibody (bsscFv) from hybridomas 3G8 and F3.3 using the phage display library, which could mediate specific lysis of malignant human B-lymphoid cell lines. In another report, Dong et al [23] isolated two Fab clones against the viral capsid antigen (VCA) of Epstein-Barr virus (EBV) from a patient with marginal zone B cell lymphoma.…”