Psoriasis is a human skin condition characterized by epidermal hyperproliferation and infiltration of multiple leukocyte populations. In characterizing a novel insulin growth factor (IGF)-like (IGFL) gene in mice (mIGFL), we found transcripts of this gene to be most highly expressed in skin with enhanced expression in models of skin wounding and psoriatic-like inflammation. A possible functional ortholog in humans, IGFL1, was uniquely and significantly induced in psoriatic skin samples. In vitro IGFL1 expression was up-regulated in cultured primary keratinocytes stimulated with tumor necrosis factor α but not by other psoriasis-associated cytokines. Finally, using a secreted and transmembrane protein library, we discovered high affinity interactions between human IGFL1 and mIGFL and the TMEM149 ectodomain. TMEM149 (renamed here as IGFLR1) is an uncharacterized gene with structural similarity to the tumor necrosis factor receptor family. Our studies demonstrate that IGFLR1 is expressed primarily on the surface of mouse T cells. The connection between mIGFL and IGFLR1 receptor suggests mIGFL may influence T cell biology within inflammatory skin conditions.

The selection of high-producing mammalian cell lines is a crucial step in process development for the production of biopharmaceuticals. Previously, cloning by limiting dilution method was used to isolate monoclonal NS0 cells secreting high levels of humanized-C2 monoclonal antibodies. However limiting dilution method is time consuming, has low probability of monoclonality and is significantly limited by the number of clones that can be feasibly screened. In order to minimize the duration and to increase the probability of obtaining high-producing clones with high monoclonality, an automated colony picker, Clone Pix FL system was used to replace limiting dilution method. We were able to screen 1 x 105 clones secreting humanized monoclonal antibodies and high producer clones were selected in just 7 days. Briefly, semi-solid media was used to immobilize single cells separately and allow them to proliferate into discrete clones. The high viscosity nature of the semi-solid media retains the secreted products in the vicinity of the associated clones. Using Clone Pix FL system, all clones were screened and the producer clones with different exterior fluorescent intensities were automatically isolated. We were able to isolate rare high-producers (> 3000 FU) with frequency of as low as 0.003% of the population. A quantitative ELISA was also performed to evaluate the correlation between the fluorescence intensity of clones with its corresponding antibody productivity. Clones with fluorescence intensity of <1000 FU showed relatively low antibody productivity compared with those greater than 1000 FU; however above this there was no correlation of production with the increase in fluorescence intensity. Hence, although the high-throughput, rapid and automated nature of Clone Pix FL system allows the screening of large number of cells in a short period of time with also an increased in the probability of obtaining rare and precious high-producing clones, downstream analysis are still vital to determine the ‘actual' and stable high producer clones.

Abstract
The recent remarkable rise in biomedical applications of antibodies and their recombinant constructs has shifted the interest in determination of antigenic epitopes in target proteins from the areas of protein science and molecular immunology to the vast fields of modern biotechnology. In this article, we demonstrated that measuring binding induced changes in two-dimensional NMR spectra enables rapid determination of antibody binding footprints on target protein antigens. Such epitopes recognized by six high-affinity monoclonal murine antibodies (mAbs) against human neutrophil gelatinase-associated lipocalin (NGAL) were determined by measuring chemical shifts or broadening of peaks in (1)H-(15)N-TROSY HSQC and (1)H-(13)C HSQC spectra of isotope-labeled NGAL occurring upon its binding to the antibodies. Locations of the epitopes defined by the NMR studies are in good agreement with the results of antibody binding pairing observed by dual-color fluorescence cross-correlation spectroscopy. In all six cases, the antibodies recognize conformational epitopes in regions of relatively rigid structure on the protein. None of the antibodies interact with the more flexible funnel-like opening of the NGAL calyx. All determined epitope areas in NGAL reflect the dimensions of respective antibody binding surface (paratopes) and contain amino acid residues that provide strong interactions. This NMR-based approach offers comprehensive information on antigenic epitopes and can be applied to numerous protein targets of diagnostic or therapeutic interest.

Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.

Abstract
The AttSite Recombinase Technology from Intrexon, Blacksburg, VA, utilizes specific DNA sequences and proprietary recombinase enzymes to catalyze the insertion of a gene of interest at a specific location in the host cell genome. Using this technology, we have developed Chinese Hamster Ovary (CHO) cell lines that have incorporated attB recombination sites at highly transcriptionally active loci or 'hot spots' within the cell genome. Subsequently, these attB site containing host cell lines could then be used for the expression of future Centocor products. Candidate production cell lines would be generated by a simple recombination event. Since the therapeutic gene of interest would preferentially integrate into the pre-selected high-expressing attB site, candidate cell lines would consistently express high levels of the gene of interest. We have been able to demonstrate that the AttSite Recombinase Technology could be a valid approach for the development of high-expressing production cell lines.

Production of antisera able to recognize individual heterotrimeric G protein subunits resulted in rapid expansion of information on their distribution and function. However, no antibodies that specifically recognize the active state have been available. Four-way primary screening of 763 hybridomas generated from mice immunized with guanosine 5'-O-(3-thio)triphosphate-loaded Gi1 and isolated using an automated robotic colony picker identified three antibodies that interacted with the constitutively active, Q204L, mutant but neither the constitutively inactive, G203A, mutant nor wild-type Gi1. This profile extended to other closely related Gi family G proteins but not to the less closely related Gs and Gq/G11 families. Each antibody was, however, also able to identify wild-type, GDP-bound Gi family G proteins in the presence of fluoroaluminate, which mimics the presence of the terminal phosphate of GTP and hence generates an active/transition state conformation. Stimulation of cells coexpressing a wild-type Gi subunit and the dopamine D2 receptor with the agonist ligand nor-apomorphine also allowed these conformationally selective antibodies to bind the G protein. Such reagents allow the specific identification of activated G proteins in a native environment and may allow the development of label-free screening assays for G protein-coupled receptor-mediated activation of Gi family G proteins.—Lane, J. R., Henderson, D., Powney, B., Wise, A., Rees, S., Daniels, D., Plumpton, C., Kinghorn, I., Milligan, G. Antibodies that identify only the active conformation of Gi family G protein subunits.

Production of IgG antibodies is usually done in mamalian cells. Predominantly, chinese hamster ovary (CHO) cells are used for generating stably transfected cell lines and for manufacturing. In addition to large scale manufacturing, stably transfected CHO cells are also well suited for repeated laboratory scale prodution of antibodies. Once generated, such cell lines can be expanded for production according to the antibody amounts needed. In this chapter, a general introduction to CHO cell line deveopment for antibody expression and a more detailed example protocol for establishing a basic CHO cell antibody expression system are given. All high performance stable mammalian expression systems are result of intensive optimisation efforts and strongly depend on the vector, selection system, host cell line and media that are used. The basic protocol described here is based on generally available materials and should be only seen as a starting point for laboratory scale production of antibodies in CHO cells.