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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 Fc γRIII (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. [ABSTRACT FROM AUTHOR]

Summary: A subset of individuals with 'primary' or 'idiopathic' immune thrombocytopenia (ITP) who fail to respond to conventional first‐ and second‐line agents or who lose responsiveness are considered to have 'refractory' disease (rITP), placing them at increased risk of bleeding and complications of intensive treatment. However, the criteria used to define the refractory state vary among studies, which complicates research and clinical investigation. Moreover, it is unclear whether rITP is simply 'more severe' ITP, or if there are specific pathogenic pathways that are more likely to result in refractory disease, and whether the presence or development of rITP can be established or anticipated based on these differences. This paper reviews potential biological features that may be associated with rITP, including genetic and epigenetic risk factors, dysregulation of T cells and cytokine networks, antibody affinity and specificity, activation of complement, impaired platelet production and alterations in platelet viability and clearance. These findings indicate the need for longitudinal studies using novel clinically available methodologies to identify and monitor pathogenic T cells, platelet antibodies and other clues to the development of refractory disease. [ABSTRACT FROM AUTHOR]