Harald Kropshofer

The safety and side effects of monoclonal antibodies.

Monoclonal antibodies (mAbs) are now established as targeted therapies for malignancies, transplant rejection, autoimmune and infectious diseases, as well as a range of new indications. However, administration of mAbs carries the risk of immune reactions such as acute anaphylaxis, serum sickness and the generation of antibodies. In addition, there are numerous adverse effects of mAbs that are related to their specific targets, including infections and cancer, autoimmune disease, and organ-specific adverse events such as cardiotoxicity. In March 2006, a life-threatening cytokine release syndrome occurred during a first-in-human study with TGN1412 (a CD28-specific superagonist mAb), resulting in a range of recommendations to improve the safety of initial human clinical studies with mAbs. Here, we review some of the adverse effects encountered with mAb therapies, and discuss advances in preclinical testing and antibody technology aimed at minimizing the risk of these events.

High-sensitivity miniaturized immunoassays for tumor necrosis factor α using microfluidic systems

We use microfluidic chips to detect the biologically important cytokine tumor necrosis factor alpha (TNF- alpha) with picomolar sensitivity using sub-microliter volumes of samples and reagents. The chips comprise a number of independent capillary systems (CSs), each of which is composed of a filling port, an appended microchannel, and a capillary pump. Each CS fills spontaneously by capillary forces and includes a self-regulating mechanism that prevents adventitious drainage of the microchannels. Thus, interactive control of the flow in each CS is easily achieved via collective control of the evaporation in all CSs by means of two Peltier elements that can independently heat and cool. Long incubation times are crucial for high sensitivity assays and can be conveniently obtained by adjusting the evaporation rate to have low flow rates of approximately 30 nL min(-1). The assay is a sandwich fluorescence immunoassay and takes place on the surface of a poly(dimethylsiloxane)(PDMS) slab placed across the microchannels. We precoat PDMS with capture antibodies (Abs), localize the capture of analyte molecules using a chip, then bind the captured analyte molecules with fluorescently-tagged detection Abs using a second chip. The assay results in a mosaic of fluorescence signals on the PDMS surface which are measured using a fluorescence scanner. We show that PDMS is a compatible material for high sensitivity fluorescence assays, provided that detection antibodies with long excitation wavelength fluorophores ( > or =580 nm) are employed. The chip design, long incubation times, proper choice of fluorophores, and optimization of the detection Ab concentration all combine to achieve high-sensitivity assays. This is exemplified by an experiment with 170 assay sites, occupying an area of approximately 0.6 mm(2) on PDMS to detect TNF-alpha in 600 nL of a dendritic cell (DC) culture medium with a sensitivity of approximately 20 pg mL(-1)(1.14 pM).

Clustering of MHC–peptide complexes prior to their engagement in the immunological synapse: lipid raft and tetraspan microdomains

Summary: Protein reorganization at the interface of a Tu2003cell and an antigen‐presenting cell (APC) plays an important role in Tu2003cell activation. Imaging techniques reveal that reorganization of particular receptor–ligand pairs gives rise to an intercellular junction, termed the immunological synapse. In this synapse antigenic peptides associated with major histocompatibility complex (MHC) molecules form multimolecular arrays on the APC side, engaging an equivalent number of clustered Tu2003cell receptors (TCRs) on the Tu2003cell. The accumulation of MHC molecules carrying cognate peptide in the APC–Tu2003cell interface was thought to depend on the specificity and presence of TCRs. Recent evidence, however, suggests that the APC is equipped to preorganize MHC–peptide complexes in the absence of Tu2003cells. To this end, MHC molecules become incorporated into two types of membrane microdomains: (i) cholesterol‐ and glycosphingolipid‐enriched domains, denoted lipid rafts, that preconcentrate MHC classu2003II molecules; and (ii) microdomains made up of tetraspan proteins, such as CD9, CD63, CD81 or CD82, that mediate enrichment of MHC classu2003II molecules loaded with a select set of peptides. It follows that the integrity, composition and dynamics of these microdomains are candidate determinants favoring activation or silencing of Tu2003cells.

T-cell dependent immunogenicity of protein therapeutics: Preclinical assessment and mitigation

Protein therapeutics hold a prominent and rapidly expanding place among medicinal products. Purified blood products, recombinant cytokines, growth factors, enzyme replacement factors, monoclonal antibodies, fusion proteins, and chimeric fusion proteins are all examples of therapeutic proteins that have been developed in the past few decades and approved for use in the treatment of human disease. Despite early belief that the fully human nature of these proteins would represent a significant advantage, adverse effects associated with immune responses to some biologic therapies have become a topic of some concern. As a result, drug developers are devising strategies to assess immune responses to protein therapeutics during both the preclinical and the clinical phases of development. While there are many factors that contribute to protein immunogenicity, T cell- (thymus-) dependent (Td) responses appear to play a critical role in the development of antibody responses to biologic therapeutics. A range of methodologies to predict and measure Td immune responses to protein drugs has been developed. This review will focus on the Td contribution to immunogenicity, summarizing current approaches for the prediction and measurement of T cell-dependent immune responses to protein biologics, discussing the advantages and limitations of these technologies, and suggesting a practical approach for assessing and mitigating Td immunogenicity.

Functional HLA-DM on the surface of B cells and immature dendritic cells

HLA‐DM (DM) plays a critical role in antigen presentation through major histocompatibility complex (MHC) class II molecules. DM functions as a molecular chaperone by keeping class II molecules competent for antigenic peptide loading and serves as an editor by favoring presentation of high‐stability peptides. Until now, DM has been thought to exert these activities only in late endosomal/lysosomal compartments of antigen‐presenting cells. Here we show that a subset of DM resides at the cell surface of B cells and immature dendritic cells. Surface DM engages in complexes with putatively empty class II molecules and controls presentation of those antigens that rely on loading on the cell surface or in early endosomal recycling compartments. For example, epitopes derived from myelin basic protein that are implicated in the autoimmune disease multiple sclerosis are down‐modulated by DM, but are presented in the absence of DM. Thus, this novel concept of functional DM on the surface may be relevant to both protective immune responses and autoimmunity.

Evaluation of the Immuno-Stimulatory Potential of Stopper Extractables and Leachables by Using Dendritic Cells as Readout

Recombinant protein pharmaceuticals may bear some risks and undesirable side effects, such as the appearance of immunogenic reactions. The increased incidence of antibody-mediated pure red cell aplasia (PRCA) outside the United States after administration of a human serum albumin (HSA)-free EPREX (recombinant human erythropoietin alpha) formulation was explained with the generation of rubber stopper related leachables, possibly acting as immunogenic adjuvants. In our study, we have investigated the potential of extractable and leachable preparations of three different pharmaceutical relevant stoppers to generate a danger signal in a dendritic cell assay. Furthermore, the investigated extractable and leachable preparations were characterized by NMR and a micelle-based polysorbate quantification method. In summary, we could demonstrate that stopper extractables, either generated by extraction or by leaching conditions, were not acting as danger signals for dendritic cells. Instead we identified degradation products of polysorbate 80, oleic acid and follow-up products, occur only under very accelerated conditions (100 degrees C for 4 days) as a potential stimulator for these immune cells. As this degradation did not occur at real-time, the authors however do not consider their finding to be linked to any direct safety implications of polysorbate-containing formulations in clinical practice.

DM Loss in k Haplotype Mice Reveals Isotype-Specific Chaperone Requirements

DM actions as a class II chaperone promote capture of diverse peptides inside the endocytic compartment(s). DM mutant cells studied to date express class II bound by class II-associated invariant chain-derived peptide (CLIP), a short proteolytic fragment of the invariant chain, and exhibit defective peptide-loading abilities. To evaluate DM functional contributions in k haplotype mice, we engineered a novel mutation at the DMa locus via embryonic stem cell technology. The present experiments demonstrate short-lived Ak/CLIP complexes, decreased Ak surface expression, and enhanced Ak peptide binding activities. Thus, we conclude that DM loss in k haplotype mice creates a substantial pool of empty or loosely occupied Ak conformers. On the other hand, the mutation hardly affects Ek activities. The appearance of mature compact Ek dimers, near normal surface expression, and efficient Ag presentation capabilities strengthen the evidence for isotype-specific DM requirements. In contrast to DM mutants described previously, partial occupancy by wild-type ligands is sufficient to eliminate antiself reactivity. Mass spectrometry profiles reveal Ak/CLIP and a heterogeneous collection of relatively short peptides bound to Ek molecules. These experiments demonstrate that DM has distinct roles depending on its specific class II partners.

Transport of Streptococcus pneumoniae Capsular Polysaccharide in MHC Class II Tubules

Bacterial capsular polysaccharides are virulence factors and are considered T cell–independent antigens. However, the capsular polysaccharide Sp1 from Streptococcus pneumoniae serotype 1 has been shown to activate CD4+ T cells in a major histocompatibility complex (MHC) class II–dependent manner. The mechanism of carbohydrate presentation to CD4+ T cells is unknown. We show in live murine dendritic cells (DCs) that Sp1 translocates from lysosomal compartments to the plasma membrane in MHCII-positive tubules. Sp1 cell surface presentation results in reduction of self-peptide presentation without alteration of the MHCII self peptide repertoire. In DM-deficient mice, retrograde transport of Sp1/MHCII complexes resulting in T cell–dependent immune responses to the polysaccharide in vitro and in vivo is significantly reduced. The results demonstrate the capacity of a bacterial capsular polysaccharide antigen to use DC tubules as a vehicle for its transport as an MHCII/saccharide complex to the cell surface for the induction of T cell activation. Furthermore, retrograde transport requires the functional role of DM in self peptide–carbohydrate exchange. These observations open new opportunities for the design of vaccines against microbial encapsulated pathogens.

Overview of Cell-Based Tools for Pre-Clinical Assessment of Immunogenicity of Biotherapeutics

With the increasing number of biotherapeutic drugs entering clinical trials, drug-induced immunogenicity becomes more and more a topic in immunotoxicology of drug development. Immunogenicity relies on the induction or presence of antibodies recognizing a biotherapeutic protein after its administration. Anti-drug antibodies (ADA) that may either bind to a protein drug and/ or neutralize its potency may modulate the pharmacokinetics of therapeutic proteins or evoke adverse events, ranging from hypersensitivity to autoimmune reactions. Screening for binding and neutralizing ADA is integral part of the monitoring program of biotherapeutics in clinical studies. In light of the availability of powerful in silico and in vitro tools for immunogenicity risk assessment, de-risking possibilities during pre-clinical development have become worth being considered. This review, which summarizes a presentation from the Conference on Immunogenicity for Biologics, gives an overview on novel cell-based approaches in immunogenicity risk assessment in the lead optimization phase of biotherapeutics. In particular, a strategy combining a human dendritic cell and a mass spectrometry analysis is compared with in silico algorithms as to its suitability to identify T-cell epitopes conferring immunogenicity. Moreover, the possibility of utilizing T-cell activation assays to rank lead candidates according to their immunogenicity potential is discussed. Finally, a strategy is outlined as to how the results of cell-based risk assessment tools can be exploited to reduce the immunogenicity of biotherapeutic proteins in the future.

A simple whole blood bioassay detects cytokine responses to anti-CD28SA and anti-CD52 antibodies

INTRODUCTIONnIn 2006 the anti-CD28 superagonistic IgG4 TGN1412, having passed pre-clinical safety screens, caused a severe cytokine storm in 6 healthy volunteers. Others have shown that for TGN1412 to induce an inflammatory signal in human peripheral blood mononuclear cells (PBMCs) or in human diluted blood, endothelial cells or bound monoclonal antibody (mAb) is required as part of a bioassay complex. These types of protocols rely on different donor cells and therefore have limitations as bioassays for pre-clinical testing.nnnMETHODSnWe performed studies using human PBMC/endothelial cell co-cultures, whole blood/endothelial cell co-cultures and human whole blood alone. We bracketed responses of a CD28 superagonist antibody with mAbs against CD52 (alemtuzumab, MabCampath-1H) or epidermal growth factor receptor (cetuximab, Erbitux) and with the immunostimulant lipopolysaccharide. We detected cytokine responses at the level of protein release (using ELISAs and Luminex assays) and gene induction (using real-time PCR arrays).nnnRESULTSnHere we confirm that IL-8 release was induced in a mixed endothelial cell-PBMC system by the anti-CD28 mAb. We go on to show that an alemtuzumab and an anti-CD28 mAb, but not cetuximab induced the release of a range of cytokines including IL-8, IL-6, IFNγ, IL-2 and IL10 after 24h and induced cytokine gene induction after 1h. Co-cultures of whole blood and HUVECS showed larger variability but no superiority over whole blood alone at a range of time points (0.5-48h).nnnDISCUSSIONnWe suggest that, whilst limitations exist, human blood-based in vitro assays may prove useful in assessing the potential of mAbs and other biotherapeutics to cause release of cytokines in humans.