Zehra Elgundi

The state-of-play and future of antibody therapeutics ☆

Abstract It has been over four decades since the development of monoclonal antibodies (mAbs) using a hybridoma cell line was first reported. Since then more than thirty therapeutic antibodies have been marketed, mostly as oncology, autoimmune and inflammatory therapeutics. While antibodies are very efficient, their cost‐effectiveness has always been discussed owing to their high costs, accumulating to more than one billion dollars from preclinical development through to market approval. Because of this, therapeutic antibodies are inaccessible to some patients in both developed and developing countries. The growing interest in biosimilar antibodies as affordable versions of therapeutic antibodies may provide alternative treatment options as well potentially decreasing costs. As certain markets begin to capitalize on this opportunity, regulatory authorities continue to refine the requirements for demonstrating quality, efficacy and safety of biosimilar compared to originator products. In addition to biosimilars, innovations in antibody engineering are providing the opportunity to design biobetter antibodies with improved properties to maximize efficacy. Enhancing effector function, antibody drug conjugates (ADC) or targeting multiple disease pathways via multi‐specific antibodies are being explored. The manufacturing process of antibodies is also moving forward with advancements relating to host cell production and purification processes. Studies into the physical and chemical degradation pathways of antibodies are contributing to the design of more stable proteins guided by computational tools. Moreover, the delivery and pharmacokinetics of antibody‐based therapeutics are improving as optimized formulations are pursued through the implementation of recent innovations in the field. Graphical abstract Figure. No Caption available.

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.

The Analysis of CD83 Expression on Human Immune Cells Identifies a Unique CD83+-Activated T Cell Population

CD83 is a member of the Ig gene superfamily, first identified in activated lymphocytes. Since then, CD83 has become an important marker for defining activated human dendritic cells (DC). Several potential CD83 mRNA isoforms have been described, including a soluble form detected in human serum, which may have an immunosuppressive function. To further understand the biology of CD83, we examined its expression in different human immune cell types before and after activation using a panel of mouse and human anti-human CD83 mAb. The mouse anti-human CD83 mAbs, HB15a and HB15e, and the human anti-human CD83 mAb, 3C12C, were selected to examine cytoplasmic and surface CD83 expression, based on their different binding characteristics. Glycosylation of CD83, the CD83 mRNA isoforms, and soluble CD83 released differed among blood DC, monocytes, and monocyte-derived DC, and other immune cell types. A small T cell population expressing surface CD83 was identified upon T cell stimulation and during allogeneic MLR. This subpopulation appeared specifically during viral Ag challenge. We did not observe human CD83 on unstimulated human natural regulatory T cells (Treg), in contrast to reports describing expression of CD83 on mouse Treg. CD83 expression was increased on CD4+, CD8+ T, and Treg cells in association with clinical acute graft-versus-host disease in allogeneic hematopoietic cell transplant recipients. The differential expression and function of CD83 on human immune cells reveal potential new roles for this molecule as a target of therapeutic manipulation in transplantation, inflammation, and autoimmune diseases.

New insights into the phenotype of human dendritic cell populations

HLDA10 is the Tenth Human Leukocyte Differentiation Antigen (HLDA) Workshop. The HLDA Workshops provide a mechanism to allocate cluster of differentiation (CD) nomenclature by engaging in interlaboratory studies. As the host laboratory, we invited researchers from national and international academic and commercial institutions to submit monoclonal antibodies (mAbs) to human leukocyte surface membrane molecules, particularly those that recognised molecules on human myeloid cell populations and dendritic cells (DCs). These mAbs were tested for activity and then distributed as a blinded panel to 15 international laboratories to test on different leukocyte populations. These populations included blood DCs, skin‐derived DCs, tonsil leukocytes, monocyte‐derived DCs, CD34‐derived DCs, macrophage populations and diagnostic acute myeloid leukaemia and lymphoma samples. Each laboratory was provided with enough mAb to perform five repeat experiments. Here, we summarise the reactivity of different mAb to 68 different cell‐surface molecules expressed by human myeloid and DC populations. Submitted mAbs to some of the molecules were further validated to collate data required to designate a formal CD number. This collaborative process provides the broader scientific community with an invaluable data set validating mAbs to leukocyte‐surface molecules.

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

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