Covalent dimerization of camelidae anti-human TNF-alpha single domain antibodies by the constant kappa light chain domain improves neutralizing activity.

The tumor necrosis factor-alpha (TNFalpha) plays an important role in a number of chronic inflammatory disorders. Monoclonal camelidae variable heavy chain domain-only antibodies (V(H)H) have been developed to antagonize the action of human TNFalpha (anti-TNF-V(H)H). Here we describe a strategy to obtain functional dimeric anti-TNF-V(H)H molecules, based on the C-terminal fusion of a kappa light chain domain to the anti-TNF-V(H)H.

Antibody expressing pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens.

Background: Coccidiosis caused by protozoans of genus Eimeria is a chicken parasitic disease of great economical importance. Conventional disease control strategies depend on vaccination and prophylactic use of anticoccidial drugs. Alternative solution to prevent and treat coccidiosis could be provided by passive immunization using orally delivered neutralizing antibodies.

High-level periplasmic expression and purification of scFvs.

The isolation of recombinant antibodies by phage display naturally leads to experiments to evaluate their biological and immunological properties. Although crude preparations may have their value in initial studies, the need often exists for highly purified protein that can be tested in vivo. This chapter describes methods to generate high yields of scFv from bacterial cultures and to purify protein to the degree of homogeneity required for the most exacting analysis.

Generation of bispecific and tandem diabodies.

Conventionally, antibody phage display has been used to isolate recombinant antibodies that are monovalent in their interaction with target antigens. These antibodies can be reengineered for expression in mammalian cell culture as full-length, monospecific immunoglobulins. An emerging branch of research has sought to generate bivalent recombinant antibodies by manipulating the length of the linker separating heavy- and light-chain variable domains in single-chain Fv proteins, thereby promoting inter-scFv interaction and the formation of "diabodies.

Model structure of the immunodominant surface antigen of Eimeria tenella identified as a target for sporozoite-neutralizing monoclonal antibody.

Eimeria tenella is a coccidian parasite of great economical importance for poultry industry. The surface of Eimeria invasive agents, sporozoites and merozoites, is coated with a family of developmentally regulated glycosylphosphatidylinositol (GPI)-linked surface antigens (SAGs), some of them involved in the initiation of the infection process. Using 2D gel electrophoresis followed by mass spectrometry, an antigenic surface protein EtSAG1 (TA4) of E.

The novel bispecific diabody alphaCD40/alphaCD28 strengthens leukaemic dendritic cell-induced T-cell reactivity.

Dendritic cell (DC)-based immunotherapy faces new challenges because the efficacy of DC vaccines in clinical trials has been inconsistent. Strategies to improve immune responses induced by DC are currently being explored. We have recently shown the feasibility of generating fully functional DC from acute myeloid leukaemic (AML) blasts, but with varying expression levels of the important costimulatory molecule CD86.

Recent advances in the generation of bispecific antibodies for tumor immunotherapy.

Bispecific antibodies are currently in clinical and preclinical development for the treatment of various cancers. Designed to direct and enhance the body's immune response to specific tumors, bispecific antibodies consist of a targeting domain (typically an antibody fragment that binds to a tumor antigen) linked to a triggering arm that is specific for a cell-surface molecule capable of mediating a phagocytic or lytic response by macrophages, natural killer cells, T-cells, or other effector cells. Recent animal studies have confirmed the therapeutic potential of bispecific antibodies; however, results from clinical trials so far have been less promising and have revealed clear limitations of these molecules, such as immunogenicity and severe side effects caused by mass release of inflammatory cytokines.

Combined effect of recombinant CD19 x CD16 diabody and thalidomide in a preclinical model of human B cell lymphoma.

Combining different treatment strategies offers the possibility of improving treatment results for cancer patients. The aim of our study was therefore to investigate the combination of treatment of established s.c.

Effect of tetravalent bispecific CD19xCD3 recombinant antibody construct and CD28 costimulation on lysis of malignant B cells from patients with chronic lymphocytic leukemia by autologous T cells.

To develop an effective antitumor immunotherapy for B-lineage non-Hodgkin's lymphoma, we constructed a tetravalent tandem diabody (tanDb) specific for both human CD19 (B-cell marker) and CD3 (T-cell antigen). Here, we report the effective killing of malignant primary B cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) by autologous T cells induced by tanDb at very low E:T ratios. Mononuclear cells from patients with B-CLL were cultured with bispecific antibody fragments in either the presence or absence of monospecific anti-CD28 antibody.

Effect of linker sequences between the antibody variable domains on the formation, stability and biological activity of a bispecific tandem diabody.

Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (V(H) and V(L)) domains of two different specificities connected by three linkers. When assembled in the order V(H)(A)-linker(1)-V(L)(B)-linker(2)-V(H)(B)-linker(3)-V(L)(A), the single-chain molecule either folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody, or forms a homodimer that is twice as large, a so-called tandem diabody. The formation of the tandem diabody is determined by the association of complementary V(H) and V(L) domains located on different polypeptide chains, and depends on the length and probably the amino acid composition of the three linkers joining the variable domains.

Immunosuppressive properties of anti-CD3 single-chain Fv and diabody.

The mouse anti-human CD3 monoclonal antibody OKT3 is a potent immunosuppressive agent used in clinical transplantation. However, OKT3 therapy is associated with unpleasant and often serious side effects which appear to result from cytokine release, complement activation and a human anti-mouse antibody (HAMA) response. To decrease these adverse side effects, we constructed antibody fragments comprising OKT3 variable domains without any constant domains.

Generation and production of engineered antibodies.

Various forms of recombinant monoclonal antibodies are being used increasingly, mainly for therapeutic purposes. This review specifically focuses on what is now called antibody engineering, and discusses the generation of chimeric, humanized, and fully human recombinant antibodies, immunoglobulin fragments, and artificial antigen-binding molecules. Since the production of recombinant antibodies is a limiting factor in their availability, and a shortage is expected in the future, different expression systems for recombinant antibodies and transgenic organisms as bioreactors are also discussed, along with their advantages and drawbacks.

Effect of domain order on the activity of bacterially produced bispecific single-chain Fv antibodies.

Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (VH and VL) domains of two different specificities. Depending on the order of the VH and VL domains and on the length of peptides separating them, the single-chain molecule either forms two single-chain Fv (scFv) modules from the adjacent domains of the same specificity, a so-called scFv-scFv tandem [(scFv)(2)], or folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody (scBsDb). We generated a number of four-domain constructs composed of the same VH and VL domains specific either for human CD19 or CD3, but arranged in different orders.

Synergistic antitumor effect of bispecific CD19 x CD3 and CD19 x CD16 diabodies in a preclinical model of non-Hodgkin's lymphoma.

To target NK cells against non-Hodgkin's lymphoma, we constructed a bispecific diabody (BsDb) with reactivity against both human CD19 and FcgammaRIII (CD16). Bacterially produced CD19 x CD16 BsDb specifically interacted with both CD19(+) and CD16(+) cells and exhibited significantly higher apparent affinity and slower dissociation from the tumor cells than from effector cells. It was able to induce specific lysis of tumor cells in the presence of isolated human NK cells or nonfractionated PBLs.