Therese Seldon

A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies

Recombinant monoclonal antibodies currently dominate the protein biologics marketplace. The path from target antigen discovery and screening, to a recombinant therapeutic antibody can be time-consuming and laborious. We describe a set of expression vectors, termed mAbXpress, that enable rapid and sequence-independent insertion of antibody variable regions into human constant region backbones. This method takes advantage of the In Fusion cloning system from Clontech, which allows ligation-free, high-efficiency insertion of the variable region cassette without the addition of extraneous amino acids. These modular vectors simplify the antibody reformatting process during the preliminary evaluation of therapeutic or diagnostic candidates. The resulting constructs can be used directly for transient or amplifiable, stable expression in mammalian cells. The effectiveness of this method was demonstrated by the creation of a functional, fully human anti-human CD83 monoclonal antibody.

Immunosuppressive human anti-CD83 monoclonal antibody depletion of activated dendritic cells in transplantation

Current immunosuppressive/anti-inflammatory agents target the responding effector arm of the immune response and their nonspecific action increases the risk of infection and malignancy. These effects impact on their use in allogeneic haematopoietic cell transplantation and other forms of transplantation. Interventions that target activated dendritic cells (DCs) have the potential to suppress the induction of undesired immune responses (for example, graft versus host disease (GVHD) or transplant rejection) and to leave protective T-cell immune responses intact (for example, cytomegalovirus (CMV) immunity). We developed a human IgG1 monoclonal antibody (mAb), 3C12, specific for CD83, which is expressed on activated but not resting DC. The 3C12 mAb and an affinity improved version, 3C12C, depleted CD83+ cells by CD16+ NK cell-mediated antibody-dependent cellular cytotoxicity, and inhibited allogeneic T-cell proliferation in vitro. A single dose of 3C12C prevented human peripheral blood mononuclear cell-induced acute GVHD in SCID mouse recipients. The mAb 3C12C depleted CMRF-44+CD83bright activated DC but spared CD83dim/- DC in vivo. It reduced human T-cell activation in vivo and maintained the proportion of CD4+ FoxP3+ CD25+ Treg cells and also viral-specific CD8+ T cells. The anti-CD83 mAb, 3C12C, merits further evaluation as a new immunosuppressive agent in transplantation.

Beyond patents: Effective intellectual property strategy in biotechnology

Abstract Dispelling the linear view of intellectual property protection in biotechnology, intellectual property (IP) and patents are almost synonymous in the biotechnology industry. In this innovation-intensive industry, it is unsurprising that patents are the foremost means of protection, since they provide (at least) 20 years exclusivity. However, a recent seven-year international study challenged the preconception that patenting leads to heightened innovation (International Expert Group on Biotechnology 2008). The following analysis uses case studies to demonstrate that leading companies take a holistic approach to IP management.

CD83 Antibody Inhibits Human B Cell Responses to Antigen as well as Dendritic Cell–Mediated CD4 T Cell Responses

Anti-CD83 Ab capable of Ab-dependent cellular cytotoxicity can deplete activated CD83+ human dendritic cells, thereby inhibiting CD4 T cell–mediated acute graft-versus-host disease. As CD83 is also expressed on the surface of activated B lymphocytes, we hypothesized that anti-CD83 would also inhibit B cell responses to stimulation. We found that anti-CD83 inhibited total IgM and IgG production in vitro by allostimulated human PBMC. Also, Ag-specific Ab responses to immunization of SCID mice xenografted with human PBMC were inhibited by anti-CD83 treatment. This inhibition occurred without depletion of all human B cells because anti-CD83 lysed activated CD83+ B cells by Ab-dependent cellular cytotoxicity and spared resting (CD83−) B cells. In cultured human PBMC, anti-CD83 inhibited tetanus toxoid–stimulated B cell proliferation and concomitant dendritic cell–mediated CD4 T cell proliferation and expression of IFN-γ and IL-17A, with minimal losses of B cells (<20%). In contrast, the anti-CD20 mAb rituximab depleted >80% of B cells but had no effect on CD4 T cell proliferation and cytokine expression. By virtue of the ability of anti-CD83 to selectively deplete activated, but not resting, B cells and dendritic cells, with the latter reducing CD4 T cell responses, anti-CD83 may be clinically useful in autoimmunity and transplantation. Advantages might include inhibited expansion of autoantigen- or alloantigen-specific B cells and CD4 T cells, thus preventing further production of pathogenic Abs and inflammatory cytokines while preserving protective memory and regulatory cells.

The Monoclonal Antibody 3C12C, Targeting CD83 Is a T Cell Sparing, Potential New Immunosuppressive Agent

A42 Immunosuppressive Capacities of Human Renal Tubular Epithelial Cells; a Role for Indoleamine 2,3-Dioxygenase? M. Demmers, C. Baan, M. Roemeling-van Rhijn, T. van den Bosch, M. Hoogduijn, M. Betjes, W. Weimar, A. Rowshani. Internal Medicine, Section Nephrology and Transplantation, Erasmus MC University Medical Center, Rotterdam, Netherlands. Introduction Renal tubular epithelial cells (TECs) are one of the main targets of T cell attack during acute cellular rejection. We hypothesize that TECs modulate the outcome of allo-immunity in a bi-directional way executing immunosuppressive effects and dampening the local infl ammation. Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme inhibiting T-cell proliferation. TECs express cytoplasmic IDO during acute rejection. We studied whether TECs possess immunosuppressive capacities and if IDO might play a role suppressing T-cell alloactivity. Materials and Methods Anti CD3/CD28 activated peripheral blood mononuclear cells were cocultured with IFN-γ/TNF-α activated TECs for 3 days. We analysed CD4+ T-cell and CD8+ T-cell proliferation response in the absence or presence of IDO inhibitor 1-L-MT. Next we analysed early and late apoptosis as increased IDO acitivity is associated with increased apoptosis. Further we examined whether inhibition of T cell proliferation was cell-cell contact dependent using transwell membrane experiments. Results We found that TECs dose-dependently inhibited CD4+ T-cell and CD8+ T-cell proliferation. TEC mRNA analysis and supernatant L-kynurenine showed that activated TECs express IDO mRNA expression and signifi cantly upregulated L-kynureninen, which was signifi cantly downregulated using 1-L-MT. Transwell experiments showed that TEC-mediated immunosuppression is cell-cell contact dependent. Downregulated CD4+ T-cell proliferation was partly recovered after addition of 1-L-MT, while CD8+ T-cell proliferation was not affected by 1-L-MT. Activated TECs increased early and late apoptosis of proliferating CD4+ T-cells, 1-L-MT abrogated both early and late CD4+ T-cell apoptosis. Discussion Our data show that TECs possess immunosuppressive capacities and inhibit the allo-reactive T cell proliferation that can partly be explained by indoleamine 2,3-dioxygenase immune regulation. Abstract# A43 Introduction of a New Cell Model of Biopsy-Derived Human Proximal Tubule Cells to Study the Role of Pharmacogenetics in CNIAssociated Nephrotoxicity. N. Knops,1,2 D. Kuypers,3 R. Masereeuw,4 E. Levtchenko,1,2 L. Van den Heuvel.2 1Pediatric Nephrology and Solid Organ Transplantation, University Hospital Leuven, Leuven, Belgium; 2Labarotory for Pediatrics, Dept of Development & Regeneration, KU Leuven, Leuven, Belgium; 3Nephrology, University Hospital Leuven, Leuven, Belgium; 4Pharmacology and Toxicology, Radboud University, Nijmegen, Netherlands. Background: Calcineurin inhibitors (CNI) constitute the basis of immunosuppressive regimes in transplantation, but are associated with the development of histological lesions leading to kidney failure. CNI’s are metabolized by CYP3A and excreted by Pgp (ABCB1) in the gut and liver but also in proximal tubular cells (PTC). Clinical studies demonstrated a relation between common variants of CYP3A5/ ABCB1 genes and CNI-associated nephrotoxicity (CNIT). The mechanism is unknown. We established a model of human PTC that can be used to study the pathogenesis of CNIT. Methods: A technique was developed to culture cells from a protocol biopsy in renal allograft recipients. Primary cells were transfected with SV40T and hTERT virus for conditional immortalization and differentiation. Subclones were selected based upon specifi c PTC markers (AQP1 and CD13) using Western Blot (WB) and FACS. Light and scanning electron microscopy were performed to detect PTC morphology. PCR and sequencing was used to assess genotype. Quantative RT-PCR, WB and immunohistochemistry was performed for CYP3A5 an ABCB1 expression. CYP3A5 activity was assessed by differential midazolam(MDZ) hydroxylation using LC-MS and Pgp activity by calcein effl ux. Results: From 27 out of 38 biopsies cell lines were generated. Based upon genotype 11 subclones with PTC biomarkers were selected. In vitro PTC morphology with brush border microvilli was observed. We confi rmed CYP3A5 and Pgp mRNA and protein expression. CYP3A5*1 carriers had increased 1OH/4OH MDZ formation vs.*3/*3 (1,44 vs 0,7; p<0,05). Pgp activity was confirmed by 39% calcein accumulation(95% CI:33-44), but not related ABCB1 3435CT genotype. Tacrolimus disappearance was 49 times higher in CYP3A5*1 vs.*3/*3 carriers, but again not related to ABCB1 3435CT genotype. Conclusion: PTC cell lines can be generated from a kidney biopsy and demonstrate functional expression of genes involved in CNI metabolism after immortalization. Differences in protein function were detected for CYP3A5 genotype. This in vitro model can be used to study the role of pharmacogenetic variation in CNIT. DISCLOSURES: Knops, N.: Grant/Research Support, Astellas. Kuypers, D.: Grant/ Research Support, Astellas. Levtchenko, E.: Grant/Research Support, Astellas. A43 Introduction of a New Cell Model of Biopsy-Derived Human Proximal Tubule Cells to Study the Role of Pharmacogenetics in CNIAssociated Nephrotoxicity. N. Knops,1,2 D. Kuypers,3 R. Masereeuw,4 E. Levtchenko,1,2 L. Van den Heuvel.2 1Pediatric Nephrology and Solid Organ Transplantation, University Hospital Leuven, Leuven, Belgium; 2Labarotory for Pediatrics, Dept of Development & Regeneration, KU Leuven, Leuven, Belgium; 3Nephrology, University Hospital Leuven, Leuven, Belgium; 4Pharmacology and Toxicology, Radboud University, Nijmegen, Netherlands. Background: Calcineurin inhibitors (CNI) constitute the basis of immunosuppressive regimes in transplantation, but are associated with the development of histological lesions leading to kidney failure. CNI’s are metabolized by CYP3A and excreted by Pgp (ABCB1) in the gut and liver but also in proximal tubular cells (PTC). Clinical studies demonstrated a relation between common variants of CYP3A5/ ABCB1 genes and CNI-associated nephrotoxicity (CNIT). The mechanism is unknown. We established a model of human PTC that can be used to study the pathogenesis of CNIT. Methods: A technique was developed to culture cells from a protocol biopsy in renal allograft recipients. Primary cells were transfected with SV40T and hTERT virus for conditional immortalization and differentiation. Subclones were selected based upon specifi c PTC markers (AQP1 and CD13) using Western Blot (WB) and FACS. Light and scanning electron microscopy were performed to detect PTC morphology. PCR and sequencing was used to assess genotype. Quantative RT-PCR, WB and immunohistochemistry was performed for CYP3A5 an ABCB1 expression. CYP3A5 activity was assessed by differential midazolam(MDZ) hydroxylation using LC-MS and Pgp activity by calcein effl ux. Results: From 27 out of 38 biopsies cell lines were generated. Based upon genotype 11 subclones with PTC biomarkers were selected. In vitro PTC morphology with brush border microvilli was observed. We confi rmed CYP3A5 and Pgp mRNA and protein expression. CYP3A5*1 carriers had increased 1OH/4OH MDZ formation vs.*3/*3 (1,44 vs 0,7; p<0,05). Pgp activity was confirmed by 39% calcein accumulation(95% CI:33-44), but not related ABCB1 3435CT genotype. Tacrolimus disappearance was 49 times higher in CYP3A5*1 vs.*3/*3 carriers, but again not related to ABCB1 3435CT genotype. Conclusion: PTC cell lines can be generated from a kidney biopsy and demonstrate functional expression of genes involved in CNI metabolism after immortalization. Differences in protein function were detected for CYP3A5 genotype. This in vitro model can be used to study the role of pharmacogenetic variation in CNIT. DISCLOSURES: Knops, N.: Grant/Research Support, Astellas. Kuypers, D.: Grant/ Research Support, Astellas. Levtchenko, E.: Grant/Research Support, Astellas. Abstract# A44 Angiogenin Promotes Cell Survival During Cyclosporine-Induced Endoplasmic Reticulum Stress. I. Mami,1 N. Bovier,1 S. Pezet,1 P. Beaune,1,2 N. Pallet,1,2 E. Thervet.1,3 1INSERM U-775, INSERM, Paris, France; 2Service de Biochimie, Hopital Europeen Georges Pompidou, Paris, France; 3Service de Nephrologie, Hopital Europeen Georges Pompidou, Paris, France. Background Calcineurin inhibitors nephrotoxicity promotes chronic kidney injury, and contributes to chronic allograft nephropathy. We have demonstrated previously that cyclosporine is an ER stress inducer, ER stress mediates its nephrotoxicity. ER stress contributes to kidney disease, and constitutes a progression factor. Recent studies suggest that Angiogenin (ANG), a stress-activated and secreted ribonuclease, cleaves tRNA to generate fragments called tiRNA. These tiRNA contribute to stress-induced translational repression, indicating that ANG and tiRNA help to reprogram protein translation during stress, and are previously unappreciated components of the stress response. The implication of tiRNA in the ER stress-induced translational repression is unknown. Objectives Our hypothesis is that cyclosporine regulates the production and activation of ANG during the Unfolded Protein Response (UPR), the adaptive program activated in response to ER stress, in the kidney epithelium. That ANG promotes cellular adaptation during stress, mediated by tiRNA integrated in the UPR-induced translational repression. The purpose of this study is to characterize the mechanisms of ANG synthesis, cellular localization and biological functions, during ER stress activated by cyclosporine. Results In a model of human epithelial cells, we have demonstrated that ANG expression is induced during ER stress, that ANG production depends on IRE1a, and that ANG expression is regulated by the transcription factor sXBP1 and NF-kB. ER stress promotes a nucleo-cytoplasmic transfert of ANG which localizes in part in stress granules. ANG inhibits ER stress-indu