Development of novel protein scaffolds as alternatives to whole antibodies for imaging and therapy: status on discovery research and clinical validation.

Recent advances in combinatorial protein engineering have made it possible to develop antibody-based and non-Ig protein scaffolds that can potentially substitute for most whole antibody-associated properties. In theory, many different natural human protein backbones are suitable to be used as recombinant templates for engineering : antibody-derived scaffolds, carrier proteins that display a single binding interface, backbones that provide a rigid core structure suitable for grafting loops or protein scaffolds allowing the incorporation of variable loops in a favorable 3D configuration. In practice however, only a few have yielded the necessary properties to be translated into 'druggable Biologicals'. Amongst these properties, potential broad specificities towards any kind of target, ease of production, small size, good tolerability and low immunogenicity are essential and will be discussed in this review. Intellectual property is another key issue for the development of these protein scaffolds; although circumventing antibody-associated patents is often a major if not primary goal, clear advantages compared to whole antibodies must be presented to translate scaffold discovery into successful therapeutic drug candidates. In this review, a particular emphasis will be given to the most validated scaffolds that have reached the clinical development phase. Although the question of their immunogenicity is still open, preliminary clinical data do not point to any particular adverse immunogenic reactions although these are highly dependent on dosage, administration route and therapeutic indication. Finally, some of the emerging Biotechs developing protein scaffolds have been associated during the last two years with successful acquisitions by Big Pharmas and we will speak on the perspective positions of these proteins within the global Biologicals market.

Cellular distribution of transforming growth factor betas 1, 2, and 3 and their types I and II receptors during postnatal development and spermatogenesis in the boar testis.

Transforming growth factor betas (TGF betas) 1, 2, and 3 and their types I and II receptors (TGF betas RI and RII) were immunolocalized 1) during testicular development from the perinatal to the adult period and 2) in maturing germ cell populations at successive seminiferous epithelium stages. In the perinatal testis, TGF beta isoforms and receptors were both preponderant in Leydig cells and in spermatogonia. At prepuberty, their expression appeared in Sertoli cells, while germ cells showed specific TGF beta1 and TGF betaRI staining in the spermatocytes. In the adult testis, TGF beta ligands exhibited a preferential tubular distribution. TGF beta1 was mainly detected in young spermatocytes, TGF beta2 in Sertoli cells, and TGF beta3 in Sertoli and premeiotic germ cells. Although the two receptors were systematically observed together in various cells, our data indicate a predominance of one in comparison with the other depending on the cell type. TGF betaRI was predominant in meiotic and differentiated germ cells and TGF betaRII in somatic cells. Finally, in the adult testis, TGF betas 1, 3, and RI showed a germ-cell pattern that depended upon the stage of the seminiferous epithelium cycle. Specifically, staining for the ligands was predominant before meiosis, and TGF betaRI was present particularly during meiosis and spermiogenesis. Together, the temporal and spatial distribution of the TGF beta system components suggests that these signaling molecules may play a crucial role during specific steps of testicular development and during different waves of seminiferous epithelium maturation leading to spermatogenesis.

Cellular distribution of EGF, TGFalpha and their receptor during postnatal development and spermatogenesis of the boar testis.

The epidermal growth factor (EGF), the transforming growth factor alpha (TGFalpha) and the epidermal growth factor receptor (EGFr) have been immunolocalized, (i) during the testicular postnatal development (i.e. at the perinatal, prepubertal and adult periods), and (ii) during the seminiferous epithelium cycle in the different germ cell types. While TGFalpha was essentially observed in somatic cells, specifically in perinatal Leydig cells and in mature Sertoli cells, EGF was localized both in germ cells and in somatic cells with a preferential tubular expression. Furthermore, identification of EGFr in different testicular cell types indicates that during postnatal development and spermatogenesis, testicular cells are potentially responsive to EGF in that they express EGFr. Indeed, in the course of the gonadal development, the EGFr distribution was evidenced both in somatic and germ cells with a specific germ cell pattern depending upon the seminiferous epithelium cycle. A predominant EGFr staining was evidenced during the meiotic process and the spermiogenesis. Together, the present data are in favor of the involvement of the TGFalpha/EGF system in the local control of testicular cells during development and particularly of its potential direct implication in crucial steps of spermatogenesis such as meiosis and spermiogenesis.

Tumor necrosis factor alpha receptor p55 is under hormonal (follicle-stimulating hormone) control in testicular Sertoli cells.

Identification of TNF alpha receptors and their regulation by the endocrine system were examined in testicular Sertoli cells. By using cross-linking experiments as well as northern blotting analysis, we detected the p55 receptor type (MW: 55 kDa, mRNA: 2.3 kb), but not the p75 TNF alpha receptor type. Furthermore, we report that Sertoli cell TNF alpha receptors are under the control of FSH, but not of other hormones which affect Sertoli cell activity. FSH enhances (ED50: 2 ng/ml) TNF alpha binding to Sertoli cells. This stimulating effect of FSH occurs at the level of p55 TNF alpha receptor, as indicated by the increase in p55 mRNA after FSH treatment. Together, these observations show that the action of TNF alpha (which originates in the testis from germ cells and/or interstitial macrophages) on Sertoli cells remains under the control of the endocrine system as shown by the regulatory effect of FSH on TNF alpha p55 receptors.