International intellectual property strategies for therapeutic antibodies

Therapeutic antibodies need international patent protection as their markets expand to include industrialized and emerging countries. Because international intellectual property strategies are frequently complex and costly, applicants require sound information as a basis for decisions regarding the countries in which to pursue patents. While the most important factor is the size of a given market, other factors should also be considered.Key words: antibody, patent, international, PCT, filing strategy     

Expression and purification of recombinant hemoglobin in Escherichia coli

Background

Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species.

Methodology/Principal Findings

As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus), a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications.

Conclusion/Significance

Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis.     

Evolution of the globin gene family in deuterostomes: lineage-specific patterns of diversification and attrition

In the Metazoa, globin proteins display an underlying unity in tertiary structure that belies an extraordinary diversity in primary structures, biochemical properties, and physiological functions. Phylogenetic reconstructions can reveal which of these functions represent novel, lineage-specific innovations, and which represent ancestral functions that are shared with homologous globin proteins in other eukaryotes and even prokaryotes. To date, our understanding of globin diversity in deuterostomes has been hindered by a dearth of genomic sequence data from the Ambulacraria (echinoderms + hemichordates), the sister group of chordates, and the phylum Xenacoelomorpha, which includes xenoturbellids, acoelomorphs, and nemertodermatids. Here, we report the results of a phylogenetic and comparative genomic analysis of the globin gene repertoire of deuterostomes. We first characterized the globin genes of the acorn worm, Saccoglossus kowalevskii, a representative of the phylum Hemichordata. We then integrated genomic sequence data from the acorn worm into a comprehensive analysis of conserved synteny and phylogenetic relationships among globin genes from representatives of the eight lineages that comprise the superphylum Deuterostomia. The primary aims were 1) to unravel the evolutionary history of the globin gene superfamily in deuterostomes and 2) to use the estimated phylogeny to gain insights into the functional evolution of deuterostome globins. Results of our analyses indicate that the deuterostome common ancestor possessed a repertoire of at least four distinct globin paralogs and that different subsets of these ancestral genes have been retained in each of the descendant organismal lineages. In each major deuterostome group, a different subset of ancestral precursor genes underwent lineage-specific expansions of functional diversity through repeated rounds of gene duplication and divergence. By integrating results of the phylogenetic analysis with available functional data, we discovered that circulating oxygen-transport hemoglobins evolved independently in several deuterostome lineages and that intracellular nerve globins evolved independently in chordates and acoelomorph worms.     

Copy number polymorphism in the α-globin gene cluster of European rabbit (Oryctolagus cuniculus)

Comparative genomic studies have revealed that mammals typically possess two or more tandemly duplicated copies of the α-globin (HBA) gene. The domestic rabbit represents an exception to this general rule, as this species was found to possess a single HBA gene. Previous electrophoretic surveys of HBA polymorphism in natural populations of the European rabbit (Oryctolagus cuniculus) revealed extensive geographic variation in the frequencies of three main electromorphs. The variation in frequency of two electromorphs is mainly partitioned between two distinct subspecies of European rabbit, and a third is restricted to the hybrid zone between the two rabbit subspecies in Iberia. Here we report the results of a survey of nucleotide polymorphism, which revealed HBA copy number polymorphism in Iberian populations of the European rabbit. By characterizing patterns of HBA polymorphism in populations from the native range of the European rabbit, we were able to identify the specific amino-acid substitutions that distinguish the previously characterized electromorphs. Within the hybrid zone, we observed the existence of a second HBA gene duplicate, named HBA2, that mostly represents a novel sequence haplotype, which occurs in higher frequency within the hybrid zone, and thus appears to have arisen in hybrids of the two distinct subspecies. Although this novel gene is also present in other wild Iberian populations, it is almost absent from French populations, which suggest a recent ancestry, associated with the establishment of the post-Pleistocene contact zone between the two European rabbit subspecies.     

Extending body space in immersive virtual reality: a very long arm illusion

Recent studies have shown that a fake body part can be incorporated into human body representation through synchronous multisensory stimulation on the fake and corresponding real body part - the most famous example being the Rubber Hand Illusion. However, the extent to which gross asymmetries in the fake body can be assimilated remains unknown. Participants experienced, through a head-tracked stereo head-mounted display a virtual body coincident with their real body. There were 5 conditions in a between-groups experiment, with 10 participants per condition. In all conditions there was visuo-motor congruence between the real and virtual dominant arm. In an Incongruent condition (I), where the virtual arm length was equal to the real length, there was visuo-tactile incongruence. In four Congruent conditions there was visuo-tactile congruence, but the virtual arm lengths were either equal to (C1), double (C2), triple (C3) or quadruple (C4) the real ones. Questionnaire scores and defensive withdrawal movements in response to a threat showed that the overall level of ownership was high in both C1 and I, and there was no significant difference between these conditions. Additionally, participants experienced ownership over the virtual arm up to three times the length of the real one, and less strongly at four times the length. The illusion did decline, however, with the length of the virtual arm. In the C2-C4 conditions although a measure of proprioceptive drift positively correlated with virtual arm length, there was no correlation between the drift and ownership of the virtual arm, suggesting different underlying mechanisms between ownership and drift. Overall, these findings extend and enrich previous results that multisensory and sensorimotor information can reconstruct our perception of the body shape, size and symmetry even when this is not consistent with normal body proportions.     

Levels of angiopoietins 1 and 2 in induced sputum supernatant in patients with COPD

Pathological features of chronic obstructive pulmonary disease (COPD) include lung vascular remodeling and angiogenesis. Angiopoietin-1 (Ang-1), is an essential mediator of angiogenesis by establishing vascular integrity, whereas angiopoietin-2 (Ang-2) acts as its natural inhibitor. We determined the levels of angiopoietins in sputum supernatants of patients with COPD and investigated their possible association with mediators and cells involved in the inflammatory and remodeling process. Fifty-nine patients with COPD, 25 healthy smokers and 20 healthy non-smokers were studied. All subjects underwent lung function tests, sputum induction for cell count identification and Ang-1, Ang-2, VEGF, TGF-β1, MMP-2, LTB4, IL-8, albumin measurement in sputum supernatants. Airway vascular permeability (AVP) index was also assessed. Ang-2 levels were significantly higher in patients with COPD compared to healthy smokers and healthy non-smokers [median, interquartile ranges pg/ml, 267 (147-367) vs. 112 (67-171) and 98 (95-107), respectively; p<0.001]. Regression analysis showed a significant association between Ang-2 levels and AVP index, VEGF, IL-8 and MMP-2 levels in COPD, the strongest being with VEGF. Our results indicate that induced sputum Ang-2 levels are higher in COPD compared to healthy smokers and healthy non-smokers. Moreover, Ang-2 is associated with AVP, IL-8, MMP-2, and VEGF, indicating a possible role for Ang-2 in the pathogenesis of the disease.     

Repression of small toxic protein synthesis by the Sib and OhsC small RNAs

The sequences encoding the QUAD1 RNAs were initially identified as four repeats in Escherichia coli. These repeats, herein renamed SIB, are conserved in closely related bacteria, although the number of repeats varies. All five Sib RNAs in E. coli MG1655 are expressed, and no phenotype was observed for a five-sib deletion strain. However, a phenotype reminiscent of plasmid addiction was observed for overexpression of the Sib RNAs, and further examination of the SIB repeat sequences revealed conserved open reading frames encoding highly hydrophobic 18- to 19-amino-acid proteins (Ibs) opposite each sib gene. The Ibs proteins were found to be toxic when overexpressed and this toxicity could be prevented by coexpression of the corresponding Sib RNA. Two other RNAs encoded divergently in the yfhL-acpS intergenic region were similarly found to encode a small hydrophobic protein (ShoB) and an antisense RNA regulator (OhsC). Overexpression of both IbsC and ShoB led to immediate changes in membrane potential suggesting both proteins affect the cell envelope. Whole genome expression analysis showed that overexpression of IbsC and ShoB, as well as the small hydrophobic LdrD and TisB proteins, has both overlapping and unique consequences for the cell.