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. The resulting fusion protein was transiently expressed by use of viral vectors in Nicotiana benthamiana((Nb)) leaves and purified. Competitive ELISA and cell cytotoxicity assays revealed that the dimerized anti-(Nb)TNF-V(H)H(Ckappa) proteins blocked TNFalpha-activity more effectively than either the monomeric Escherichia coli((Ec)) produced anti-(Ec)TNF-V(H)H or the monomeric anti-(Nb)TNF-V(H)H(Ckappa). We suggest that enhanced inhibition shown by dimeric anti-(Nb)TNF-V(H)H(Ckappa) proteins is achieved by an increase in avidity.

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. We investigated the possibility to mitigate the parasitic infection by feeding poultry with antibody expressing transgenic crop seeds. RESULTS: Using the phage display antibody library, we generated a panel of anti-Eimeria scFv antibody fragments with high sporozoite-neutralizing activity. These antibodies were expressed either transiently in agrobacteria-infiltrated tobacco leaves or stably in seeds of transgenic pea plants. Comparison of the scFv antibodies purified either from tobacco leaves or from the pea seeds demonstrated no difference in their antigen-binding activity and molecular form compositions. Force-feeding experiments demonstrated that oral delivery of flour prepared from the transgenic pea seeds had higher parasite neutralizing activity in vivo than the purified antibody fragments isolated from tobacco. The pea seed content was found to protect antibodies against degradation by gastrointestinal proteases (>100-fold gain in stability). Ad libitum feeding of chickens demonstrated that the transgenic seeds were well consumed and not shunned. Furthermore, feeding poultry with shred prepared from the antibody expressing pea seeds led to significant mitigation of infection caused both by high and low challenge doses of Eimeria oocysts. CONCLUSION: The results suggest that our strategy offers a general approach to control parasitic infections in production animals using cost-effective antibody expression in crop seeds affordable for the animal health market.

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." With careful control, this can generate scFv-based proteins able to bind two very distinct targets, "bispecific diabodies." Further manipulation enables the assembly of higher-order complexes.

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.

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. tenella sporozoites has been identified as a target of neutralizing monoclonal antibody 2H10E3. To clarify the mechanism of invasion inhibition caused by the EtSAG1-specific antibodies, a structural model of EtSAG1 was generated. It appears that "EtSAG fold" does not bear an evolutionary relationship to any known protein structure. The intra- and interchain disulfide bonds could be assigned to certain pairs of six conserved cysteines found in members of the EtSAG protein family. The outward-facing surface of the antigen was found to comprise an expanded positively charged patch, thus suggesting that the parasite invasion process may be initiated by sporozoite attachment to negatively charged sulfated proteoglycans on the surface of the host cell.

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. To overcome this variability, we developed a novel bispecific diabody that simultaneously and agonistically targeted CD40 on AML-DC and CD28 on naïve T cells. Beside optimization of CD28-mediated signalling, the resulting cellular cross-linking was also hypothesized to increase the strength and duration of T cell/AML-DC interactions, thus increasing T-cell responsiveness to AML antigens. The alphaCD40/alphaCD28-bispecific diabody was found to bind to its target antigens and provoked increased T-cell-DC cluster formation. The alphaCD40/alphaCD28 diabody is capable of increasing T-cell proliferation induced by AML-DC as well as the induction of DC maturation. Importantly, priming efficacy of tumour-specific cytotoxic T cells can also be improved by cross-linking AML-DC and T cells with the alphaCD40/alphaCD28 diabody. We propose that the alphaCD40/alphaCD28-bispecific diabody can serve as a potent therapeutic tool to effectively augment anti-tumour T-cell responses elicited by AML-DC.

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. A second generation of bispecific molecules, bispecific single-chain antibodies and hetero-oligomeric-engineered antibodies, has now been produced by using DNA recombinant technology; these may theoretically improve tumor targeting and minimize side effects, eventually replacing the full-length bispecific antibodies. Over the next few years, several recombinant bispecific antibodies are expected to enter clinical trials and ultimately emerge as new pharmaceutical products. This review briefly summarizes major trends in the development of bispecific antibodies for tumor therapy, and describes a rationale for the generation of novel recombinant molecules.

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. human B non-Hodgkin's lymphoma in severe immune deficient mice using a recombinant bispecific CD19 x CD16 diabody (targeting natural killer cells to CD19 cells) and the angiogenesis inhibitor thalidomide. Monotherapy with either thalidomide or diabody caused an approximate 50% reduction in tumor growth rate. The combined treatment showed evidence for a synergistic effect resulting in a 74% reduction in median tumor size. In the combined treatment group, two of five animals had complete remissions of their s.c. tumor. These results suggest that a combination treatment with recombinant diabodies and angiogenesis inhibition represents a useful approach in cancer therapy.

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. Use of tetravalent tanDbs caused almost quantitative elimination of malignant B cells from the blood samples of 19 patients and some cytotoxic activity in 3 of 23 analyzed cases. In contrast, the structurally similar but bivalent diabody and single-chain diabody demonstrated nearly no antitumor activity in an autologous system. tanDb-induced activation and proliferation of T cells occurred only in the presence of CD19+ target cells. Expression of the B7-1 (CD80) and B7-2 (CD86) molecules on the surface of leukemia cells made unnecessary the additional CD28-costimulation of T cells. When only a few tanDb molecules were present, the effect of CD28 costimulation on T-cell activation was more pronounced. Depending on the patient sample, we observed a 10- to 1,000-fold decrease of the half-maximal concentrations of tanDb for cell lysis. Upon CD28 crosslinking by agonistic MAb, specific tumor cell lysis was found at tanDb concentrations as low as 0.5 pM. These data demonstrate that the tetravalent CD19xCD3 tanDb might be a promising tool for the immunotherapy of human B-cell leukemias and lymphomas.

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. We generated a number of single-chain constructs using four V(H) and V(L) domains specific either for human CD3, a component of T-cell receptor (TCR) complex, or for CD19, a human B-cell antigen, separated by different rationally designed peptide linkers of 6-27 amino acid residues. The generated bispecific constructs were expressed in bacterial periplasm and their molecular forms, antigen-binding properties, stability, and T-cell proliferative and anti-tumor activities were compared. Using peripheral blood mononuclear cell cultures from patients suffering from B-cell chronic lymphocytic leukemia, we demonstrated that the tandab-mediated activation of autologous T cells and depletion of malignant cells correlates with the stability of the recombinant molecule and with the distance between the CD19 and CD3 binding sites.

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. Single-chain Fv (scFv) monomers, dimers and trimers were generated by changing the linker length between the V(H) and V(L) domains. The linkers used were the natural extensions of the V(H) into the C(H)1 domain. The dimeric molecules (diabodies) demonstrated the best CD3-binding activity. The diabody with the six amino acid linker was produced in bacteria with a tenfold higher yield than other scFvs and possessed CD3-binding affinity approaching that of the parental mAb. In contrast to OKT3 mAb, the anti-CD3 diabody and scFv monomer did not cause any T-cell activation and cytokine release in vitro, while demonstrating CD3 modulation. In mixed lymphocyte cultures, both diabody and scFv, but not the monoclonal antibody OKT3, were able to suppress T-cell activation and secretion of IL-2 and IFN-gamma in a dose-dependent manner. The anti-CD3 diabody may provide a potent immunosuppressive drug with low toxicity and immunogenicity.

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. When expressed in bacteria, all (scFv)(2) variants appeared to be only half-functional, binding to CD19 and demonstrating no CD3-binding activity. Only the diabody-like scBsDb could bind both antigens. Comparison of the scBsDb with a structurally similar non-covalent dimer (diabody) demonstrated a stabilizing effect of the linker in the middle of the scBsDb molecule. We demonstrated that the mechanism of inactivation of CD19xCD3 diabody under physiological conditions is initiated by a dissociation of the weaker (anti-CD3) VH/VL interface followed by domain swapping with the formation of non-active homodimers. The instability of one homodimer makes the process of diabody dissociation/reassociation irreversible, thus gradually decreasing the fraction of active molecules. The structural parameters influencing the formation of functional bispecific single-chain antibodies are indicated and ways of making relatively stable bispecific molecules are proposed.

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. The combination of the CD19 x CD16 BsDb with a previously described CD19 x CD3 BsDb and CD28 costimulation significantly increased the lytic potential of human PBLs. Treatment of SCID mice bearing an established Burkitt's lymphoma (5 mm in diameter) with human PBLs, CD19 x CD16 BsDb, CD19 x CD3 BsDb, and anti-CD28 mAb resulted in the complete elimination of tumors in 80% of animals. In contrast, mice receiving human PBLs in combination with either diabody alone showed only partial tumor regression. These data clearly demonstrate the synergistic effect of small recombinant bispecific molecules recruiting different populations of human effector cells to the same tumor target.