Manufacture of recombinant polyclonal antibodies.

Polyclonal antibody therapy in the form of hyper-immune serum has for more than a century been used for treatment of many infectious diseases. However, with the emergence of first antibiotics and later recombinant monoclonal antibody therapy, the use of hyper-immune serum has declined. The main reason for this is that methods for consistent manufacturing of safe hyper immune immunoglobulin products have been lacking. In contrast, manufacturing processes of recombinant monoclonal antibodies follow a well established schedule and it appears obvious to use similar methods to produce recombinant polyclonal products. However, the methods for monoclonal antibody manufacturing are, for several reasons, not directly applicable to generation and manufacture of polyclonal recombinant antibodies. A new production strategy based on recombinant mammalian producer cells has recently been developed to support consistent generation of recombinant polyclonal antibodies for therapeutic use. This review describes aspects of this novel technology with emphasis on the generation, production and characterization procedures employed, and provides comparison with alternative polyclonal and monoclonal antibody manufacturing strategies.

Functional screening of a retroviral peptide library for MHC class I presentation.

We have used retroviral vector technology to develop a method for functional screening of combinatorial peptide libraries expressed inside mammalian cells with the ultimate goal of identifying new drug targets. The method was validated in a library screening experiment based on antigen presentation of small peptides. A library encoding SIXNXEKX-peptides, where X designates randomised positions corresponding to major histocompatibility (MHC) class I anchor residues, was generated in a retroviral vector. The library was transduced into a population of antigen presenting cells (APCs) known to mediate MHC class I restricted presentation of the SIINFEKL peptide. The cellular library was screened by using an antigen presentation assay in which a T cell hybridoma recognising the MHC class I/SIINFEKL peptide complex was employed. Using this experimental model, we identified two positive cellular clones both encoding SIINFEKL peptides with identical codon usage. This number corresponded well to the expected frequency of SIINFEKL in the library. The lack of identification of other peptides capable of activating the T-hybridoma supports previous findings of a high degree of specificity at the level of peptide-loading of MHC-molecules. The result further demonstrates the potential of using combinatorial libraries for functional screening and selection of effector peptides stably expressed in mammalian cells.

Transcriptional regulation of the interferon-gamma-inducible tryptophanyl-tRNA synthetase includes alternative splicing.

We have investigated the transcriptional control elements of the human interferon (IFN)-gamma-induced tryptophanyl-tRNA synthetase (hWRS) gene and characterized the transcripts. Transcription leads to a series of mRNAs with different combinations of the first exons. The full-length mRNA codes for a 55-kDa protein (hWRS), but a mRNA lacking exon II is present in almost as high amounts as the full-length transcript. This alternatively spliced mRNA is probably translated into a 48-kDa protein starting from Met48 in exon III. The predicted 48-kDa protein corresponds exactly to an IFN-gamma-inducible protein previously detected by two-dimensional gel electrophoresis. By isolation of genomic clones and construction of plasmids containing hWRS promoter fragments fused to the secreted alkaline phosphatase reporter gene we have mapped a promoter region essential for IFN-mediated gene activation. This region contains IFN-stimulated response elements (ISRE) as well as a Y-box and a gamma-activated sequence (GAS) element. IFN-gamma inducibility of hWRS depends on ongoing protein synthesis, suggesting that so far undescribed transcription factors apart from the latent GAS-binding protein p91 contribute to gene activation. This could be interferon-regulatory factor-1, which binds ISRE elements.

Differential regulation of the human, interferon inducible tryptophanyl-tRNA synthetase by various cytokines in cell lines.

Cytokines regulate the expression of specific sets of proteins which mediate their biological effects. We have comprehensively delineated the regulation of the human tryptophanyl-tRNA synthetase (hWRS) by eight different cytokines (including IFNs) and poly(I).poly(C) in several cell lines. Six non-lymphoid cell lines were tested, and all of these produced human, IFN inducible hWRS (gamma 2) mRNA upon stimulation with IFN-gamma. In all these cell lines the level of gamma 2 mRNA increased 2-4 h after induction reaching a stable plateau after 8-12 h. The IFN-gamma induction of gamma 2 mRNA could be blocked by cycloheximide in human amniotic (AMA) cells, epithelial HeLa cells and HT1080 fibroblasts, but not in T98G glioblastoma cells. IFN-alpha and poly(I).poly(C) elicited small, transient gamma 2 responses in a few of the non-lymphoid cell lines, whereas none of the other six cytokines tested elicited a response. The six lymphoid cell lines tested did not show the same induction pattern. In the monocytic cells, THP-1, gamma 2 mRNA was highly induced by IFN-gamma, whereas in the B-cell line, Daudi, gamma 2 mRNA was transiently induced by IFN-alpha and poly(I).poly(C), and not by IFN-gamma. Altered mRNA turnover rate as a consequence of IFN-gamma treatment did not appear to play a significant role in the accumulation of gamma 2 transcript, since the stability essentially was the same in induced versus non-induced cells. We conclude that the hWRS gene is induced preferentially by IFN-gamma, and that the induction pattern resembles the one reported for the IFN induced enzyme, indoleamine 2,3-dioxygenase (IDO).