Engineered antibodies currently represent over 40% of biopharmaceuticals in clinical practice, with widespread use for both diagnosis and therapy, especially for oncology and inflammatory diseases. Antibodies are immunoglobulins and circulate in the blood as stable, soluble, mammalian proteins. Antibodies provide unique, specific targeting of disease biomarkers. They are widely used for blood and tissue diagnosis (immunoassay) and as injectable therapeutics can deliver a variety of payloads such as diagnostic radioisotopes or cytotoxic drugs. With many successful therapies approved by the US Food and Drug Administration and European Medicines Agency, the biotechnology industry is expanding to new medical opportunities. Recombinant antibodies have been reduced in size, rebuilt into multivalent molecules and fused with many molecules including radionuclides, toxins, enzymes, nanoparticles, and viruses. The emergence of recombinant technologies has revolutionized the selection, humanization, and production of antibodies, thereby superseding hybridoma technology and allowing the design of antibody-based reagents with any specificity and for a wide variety of disease applications. This article describes the latest developments in the design, production, and clinical use of recombinant antibodies.

Meiotic behavior based on observations of the first and second divisions was studied in males of four taxa of the Australian tiger beetle genus Pseudotetracha of the tribe Megacephalini (Coleoptera). Pseudotetracha blackburni clade 1 shows 10 pairs of autosomes plus a trivalent that is hypothesized to be the result of either a translocation or a fusion in which the original heterosomes (very likely XY) and an autosomal pair are involved, giving rise to a recently established neo-X1X2Y sex chromosome system of chiasmatic nature. The origin of this karyotype has been determined to have taken place 2.30–3.72 million years in the past using a molecular clock based on the 16S rRNA substitution rate. Pseudotetracha blackburni clade 2 shows a meioformula of the type n = 11 + XY, the same as that found in the related taxon P. australis. Previous data for P. whelani, with 12 pairs of autosomes and an XY sex chromosome system, are confirmed in this survey. The multiple chiasmatic sex chromosome system of P. blackburni clade 1 is considered to be of recent origin and with an evolutionarily short-life confined to this species, where close relatives exhibit simple genetic systems, in contrast to the long evolutionary life of the multiple achiasmatic sex chromosome system broadly found in the tribes Cicindelini and Collyrini. The implications of this chromosomal rearrangement in terms of recombination and speciation are discussed. The results of this work, together with the available cytogenetic data for other Megacephalini species, are interpreted in the light of recent molecular phylogenies of the tribe, showing evidence of a possible process of karyotypic orthoselection with recurrent cycles of incorporation of autosomes to the heterosome pair and subsequent loss of the Y chromosome in Tetracha and Pseudotetracha.

OBJECTIVES:To determine the costs of severe hypoglycaemia (SH) in a population of patients with type 1 diabetes mellitus in the Spanish healthcare system and the cost-effectiveness of insulin lispro over regular insulin in preventing SH episodes. METHODS:A retrospective study of 100 patients in three Spanish health centres was performed. Resource utilisation data were collected only for interventions specifically relating to the hypoglycaemic episode. The direct medical costs determined in the analyses were: costs of hospitalisation, diagnostic tests carried out, costs of treatment administered and other associated costs such as visits to the endocrinologist and re-training in glucose control, transportation and assistance of a care-giver. In addition, indirect costs such as days of lost productivity were measured. The incidence rates of SH for insulin lispro and regular insulin were obtained from the literature. The incremental cost-effectiveness of insulin lispro over regular insulin was calculated. RESULTS:The overall mean cost per episode of SH was 366 euro, comprised of 65.4% direct costs and 35.6% indirect costs. The largest cost was for hospitalisation at 183 euro per episode. The SH episodes incidence rates for 100 patients per year were 33 and 73 for insulin lispro and 48 (p < 0.05) and 117 (p < 0.01) for regular insulin, in the two clinical trials found in the literature. The additional cost to prevent one episode of SH with insulin lispro over regular insulin ranged from 277 euro to insulin lispro dominance. CONCLUSIONS:Severe hypoglycaemia has a significant impact on the total cost of diabetes. The use of insulin lispro is associated with reductions in annual costs because of SH and, possibly, the overall effect may be cost neutral or cost saving when total costs are considered. The cost of SH should be included in the analysis of total socio-economic burden of diabetes.

Protein microarrays combine aspects of DNA microarrays and ELISA for the parallel interrogation of a biological sample using a multiplex of protein biomarkers. Here we report the development of a protein microarray consisting of a subset of CD antibodies and CRP. Several preparations (culture supernatant, ascites fluid and purified Ig) of each antibody were used in a forward phase protein microarray. Microarrays were fabricated using a non-contact printer delivering 300 pL (±30 pL) to specific locations on polyacrylamide gel-based substrates. Following production, microarrays were blocked for non-specific binding and incubated with sera conjugated directly with Cy3. Using CRP as a control biomarker, 12 clinical samples (inflammatory conditions and controls) were interrogated using the protein microarray format and results compared to CRP measured by conventional immunoassay. The data obtained from the microarray correlated with CRP assessed by immunoassay. Subsequently CRP ‘positive’ samples were interrogated for CD antigen expression; which revealed CD25 and CD45RO expression in all samples. Whilst this study focussed on a subset of CD antibodies, it is anticipated that this array could be expanded to include a larger number of CD antibodies and allow screening of sera from multiple conditions in order to identify disease markers.

A recombinant Fab antibody, designated 1E8-4b, which reacts with the Alzheimer's disease (AD)-related Abeta peptides, Abeta[1-40], Abeta[1-42] and Abeta[1-43] has been developed. The 1E8-4b Fab was constructed by cloning the V(H)C(H1) and V(L)C(L) domains from the parent hybridoma 1E8 antibody, reported previously to recognize these Abeta peptides. Briefly, a C-terminal Flag tag sequence was incorporated into this construct, which was ligated into the vector pHFA2 and expressed in Escherichia coli. Following purification on an M2 anti-Flag affinity column, the 1E8-4b recombinant Fab antibody was shown to bind plaques within sections of brain tissue from CERAD-defined AD patients by immunohistochemistry. ELISA, epitope mapping and immunoblotting confirmed the recognition of the Abeta1-40/42/43] peptides by the 1E8-4b Fab. The 1E8-4b Fab did not recognize APP695 or APP770 which contain the Abeta sequence. The Abeta specificity of the recombinant 1E8-4b Fab antibody was identical to the parent 1E8 monoclonal antibody.

The T lymphocyte membrane glycoprotein CD8 enhances antigen recognition by class I-restricted T cells. There are two naturally occurring forms of CD8, an αβ heterodimer expressed by the majority of CD8+ T cells, and a less abundant αα homodimer found on specialised T cell subsets. An expression strategy was developed for production of soluble CD8αα and CD8αβ extracellular domains for use in ligand binding studies. Mouse CD8α was expressed autonomously as a homodimer at 10 mg/l in mammalian fibroblasts, but CD8β was not expressed at significant levels in the absence of CD8α. Co-expression with CD8α led to significant enhancement in the level of CD8β expression, which was secreted as a non-covalent heterodimer at 3 mg/l with CD8α. Despite the marked increase of CD8β expression in the presence of CD8α, an excess of soluble CD8αα homodimer was also present in the supernatant of co-expressing cell clones. In order to resolve the CD8αα homodimer from the CD8αβ heterodimer, affinity chromatographic techniques specific for the CD8β subunit were employed. Purification procedures requiring elution from affinity matrices at low pH led to substantial losses in the total antigenic activity and partial subunit dissociation of the soluble CD8αβ heterodimer. The inclusion of a hexahistidine tag at the C-terminus of CD8β enabled affinity purification of soluble CD8αβ (and sCD8αα) under neutral conditions, yielding recombinant protein with the correct stoichiometry and full antigenic activity. This method may prove useful for production of other soluble recombinant heterodimeric receptor proteins whose antigenicity is affected by denaturation during immunoaffinity purification.

MHC class I molecules (MHC-I) display peptides from the intracellular pool at the cell surface for recognition by T lymphocytes bearing alphabeta TCR. Although the activation of T cells is controlled by the interaction of the TCR with MHC/peptide complexes, the degree and extent of the activation is influenced by the binding in parallel of the CD8 coreceptor with MHC-I. In the course of quantitative evaluation of the binding of purified MHC-I to engineered CD8, we observed that peptide-deficient H-2Ld (MHC-I) molecules bound with moderate affinity (Kd = 7.96 x 10(-7) M), but in the presence of H-2Ld-binding peptides, no interaction was observed. Examination of the amino terminal sequences of CD8alpha and beta chains suggested that H-2Ld might bind these protein termini via its peptide binding cleft. Using both competition and real-time direct assays based on surface plasmon resonance, we detected binding of empty H-2Ld to synthetic peptides representing these termini. These results suggest that some MHC molecules are capable of binding the amino termini of intact cell surface proteins through their binding groove and provide alternative explanations for the observed binding of MHC molecules to a variety of cell surface receptors and coreceptors.