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.

SummaryA recombinant bispecific single-chain fragment variable antibody (bsscFv), directed against the B-cell antigen CD19 and the low affinity Fc-receptor FccRIII (CD16), was designed for use in the treatment of patients with leukaemias and lymphomas. The Fc-portions of whole antibodies were deliberately eliminated in this construct to avoid undesired effector functions. A stabilised bsscFv, ds[CD19 · CD16], was generated, in which disulphide bonds bridging the respective variable light (VL) and variable heavy (VH) chains were introduced into both component single-chain (sc)Fvs. After production in 293T cells and chromatographic purification, ds[CD19 · CD16] specifically and simultaneously bound both antigens. The serum stability of ds[CD19 · CD16] was increased more than threefold when compared with the unstabilised counterpart, while other biological properties were not affected by these mutations. In antibody-dependent cellular cytotoxicity experiments, ds[CD19 · CD16] mediated specific lysis of both CD19-positive malignant human B-lymphoid cell lines and primary tumour cells from patients with B-cell chronic lymphocytic leukaemia or B-cell acute lymphoblastic leukaemia. Natural killer cells, mononuclear cells (MNCs) from healthy donors and, in some instances, MNCs isolated from patients after allogeneic stem cell transplantation, were used as effectors. Thus, ds[CD19 · CD16] holds promise for the treatment of CD19 + B-lineage malignancies.

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.

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New antimicrobial materials will be more and more in the focus for hygienic and clinical disease control. Antimicrobial materials have to be distinguished in leaching and nonleaching materials. For many applications of antimicrobial materials on implants the use of nonleaching materials is essential. Therefore, the antimicrobial efficiency of leaching and nonleaching polymers has been investigated quantitatively in vitro in direct comparison on a highly relevant implant of central venous catheters (CVCs) using a well‐established called Certika test. This test is especially designed to test antimicrobial properties of leachable and nonleachable materials. This contribution demonstrates that newly developed nonleaching antimicrobial CVCs are equivalent to conventional leaching CVC systems in their antimicrobial performance against gram‐positive and gram‐negative bacteria, as well as Candida species. The use of new nonleaching antimicrobial polymers as shown here for CVCs represents a different mode of action with the aim to prevent infections also with antibiotic‐resistant strains and reduced side effects.