Quantitative Comparison of the Antimicrobial Efficiency of Leaching versus Nonleaching Polymer Materials.

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.

Heterodimeric bispecific antibody-derivatives against CD19 and CD16 induce effective antibody-dependent cellular cytotoxicity against B-lymphoid tumor cells.

Bispecific scFv antibody-derivatives (bsscFvs) recruiting natural killer (NK) cells for the lysis of malignant cells have therapeutic potential. However, a bsscFv specific for the B-lymphoid tumor antigen CD19 and the trigger molecule CD16 on NK cells had similar affinities for both antigens (42 and 58nM, respectively) and was not optimal for cytotoxicity. Therefore, a bispecific tribody (bsTb) was constructed with two binding sites for CD19 and one for CD16. This bsTb contained a CD19-specific Fab fragment carrying a CD16-specific scFv fused to its light chain and a CD19-specific scFv fused to its heavy chain. The bsTb was compared with a bispecific bibody (bsBb) lacking the CD19-specific scFv. The bsTb had 3-fold greater avidity for CD19 than the bsBb (8 and 24nM, respectively), while both had equal affinity for CD16 (56nM). Both molecules mediated antibody-dependent cellular cytotoxicity (ADCC) of leukemia-derived SEM cells and primary cells from leukemia patients. The bsTb showed half-maximum effective concentrations (EC(50)) of 55pM and promoted equal lysis as the bsBb and the bsscFv at 6- and 12-fold lower concentrations, respectively. Among these three molecules the bsTb showed the most promising in vitro properties which are anticipated to be displayed also in vivo.

A recombinant bispecific single-chain fragment variable specific for HLA class II and Fc alpha RI (CD89) recruits polymorphonuclear neutrophils for efficient lysis of malignant B lymphoid cells.

Bispecific Abs offer new perspectives for cancer immunotherapy. In this study, we describe a recombinant bispecific single-chain fragment variable (bsscFv) directed against Fc alpha RI (CD89) on polymorphonuclear neutrophils (PMNs) or monocytes/macrophages and HLA class II on lymphoma target cells. Fc alpha RI 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 K(D) values of 148 +/- 42 nM and 113 +/- 25 nM for the anti-Fc alpha RI 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 (Fc alphaRI x 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 Fc alpha RI-directed bsscFv effectively recruiting PMNs for redirected cytotoxicity against human B cell malignancies. Our data show that an (Fc alpha RI x HLA class II) bsscFv is an interesting candidate for further engineering of small, modular immunopharmaceuticals.

A novel CD19-directed recombinant bispecific antibody derivative with enhanced immune effector functions for human leukemic cells.

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.

Effective lysis of lymphoma cells with a stabilised bispecific single-chain Fv antibody against CD19 and FcgammaRIII (CD16).

A recombinant bispecific single-chain fragment variable antibody (bsscFv), directed against the B-cell antigen CD19 and the low affinity Fc-receptor FcgammaRIII (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 x 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 x CD16] specifically and simultaneously bound both antigens. The serum stability of ds[CD19 x 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 x 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 x CD16] holds promise for the treatment of CD19(+) B-lineage malignancies.

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

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 FcgammaRIII (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 x 10(-8) mol/l and 13.7 x 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.

Efficient eukaryotic expression of fluorescent scFv fusion proteins directed against CD antigens for FACS applications.

Two sets of expression vectors were constructed that permitted the efficient expression of single-chain Fv fragments (scFvs) fused N-terminally to an enhanced mutant of the green fluorescent protein GFP+ or the red fluorescent protein DsRed in insect and mammalian cells. The vectors allowed rapid cloning of scFv fragments and secretion of the fusion proteins in a native conformation. Fluorescent scFv fusion proteins directed against a series of cluster of differentiation (CD) antigens were efficiently secreted by transiently transfected mammalian cells and insect cells infected with baculoviral expression constructs. Yields of the secreted proteins varied from 100 microg/l to 3 mg/l. The purified proteins were functionally active in flow cytometry, immunofluorescent microscopy, and competition binding experiments performed to delineate the epitopes recognized by different monoclonal antibodies against the same polypeptide. The use of two different scFv fragments fused with red and green fluorescent proteins and reacting with T- and B-cell lineage markers (CD7 and CD19), respectively, allowed a simplified quantitation of both subsets in two-color flow cytometry experiments with mixed populations of T- and B-lymphoid cells. Due to the lack of Fc domains in the scFv proteins, the fluorescent fusion proteins showed more than 20-fold reduced background fluorescence compared with whole antibodies of the same specificity in experiments with effector cells expressing the high affinity FcgammaRI receptor CD64. Thus, for a number of analytical applications, fluorescent scFv fusion proteins offer advantages over the use of complete primary antibodies and chemically labeled fluorescent secondary antibodies.

HLA class II antibodies in the treatment of hematologic malignancies.

Promising results have suggested human leukocyte antigen (HLA) class II as potential target for antibody-based lymphoma therapy. Thus, antibodies against HLA class II induced apoptosis in vitro, as well as complement-dependent cytotoxicity (CDC) and effector cell-mediated cytotoxicity (ADCC). Furthermore, animal models demonstrated strong antitumor effects in vivo. Importantly, early clinical studies with antibodies Lym-1 or Hu1D10, both against HLA-DR variants, suggested therapeutic potential. However, the increased risk of serious toxicity may require an improved understanding of the clinically relevant mechanisms of action of HLA class II-directed antibodies, which may then allow generation of optimized antibody constructs to achieve optimal therapeutic efficacy.