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CRISPR–Cas9 mediated genome editing offers unprecedented opportunities for treating human diseases. There are several reports that demonstrate pre-existing immune responses to Cas9 which may have implications for clinical development of CRISPR-Cas9 mediated gene therapy. Here we use 209 overlapping peptides that span the entire sequence of Staphylococcus aureus Cas9 (SaCas9) and human peripheral blood mononuclear cells (PBMCs) from a cohort of donors with a distribution of Major Histocompatibility Complex (MHC) alleles comparable to that in the North American (NA) population to identify the immunodominant regions of the SaCas9 protein. We also use an MHC Associated Peptide Proteomics (MAPPs) assay to identify SaCas9 peptides presented by MHC Class II (MHC-II) proteins on dendritic cells. Using these two data sets we identify 22 SaCas9 peptides that are both presented by MHC-II proteins and stimulate CD4+ T-cells., There have been reports of immune responses against Cas9 which may impair clinical use. Here the authors scan a cohort comparable to the North American population vis-à-vis distribution of MHC-II variants to identify Cas9 peptides presented by MHC-II proteins and can stimulate CD4 + T-cells.

The most challenging complication associated with Factor VIII (FVIII) replacement therapy is the development of neutralizing anti-drug antibodies, or inhibitors, which occur in 23-35% of severe (FVIII level via neonatal Fc receptor (FcRn) binding, other Fc-mediated interactions, including engagement with Fc gamma receptors (FcγR), may have immunological consequences. Several case reports and retrospective analyses suggest that rFVIIIFc offers superior outcomes with respect to ITI compared to other FVIII products. Previously we and others demonstrated rFVIIIFc interactions with activating FcγRIIIA/CD16. Here, we investigated if rFVIIIFc activates natural killer (NK) cells via CD16. We demonstrated rFVIIIFc signaling via CD16 independent of Von Willebrand Factor (VWF):FVIII complex formation. We established that rFVIIIFc potently activated NK cells in a CD16-dependent fashion resulting in IFNγ secretion and cytolytic perforin and granzyme B release. We also demonstrated an association between rFVIIIFc-mediated NK cell IFNγ secretion levels and the high-affinity (158V) CD16 genotype. Furthermore, we show that rFVIIIFc-activated CD16+ NK cells were able to lyse a B-cell clone (BO2C11) bearing an anti-FVIII B-cell receptor in an antibody-dependent cellular cytotoxicity (ADCC) assay. These in vitro findings provide an underlying molecular mechanism that may help explain clinical case reports and retrospective studies suggesting rFVIIIFc may be more effective in tolerizing HA patients with anti-FVIII inhibitors compared to FVIII not linked to Fc. Our in vitro findings suggest a potential use of Fc-fusion proteins acting via NK cells to target antigen-specific B-cells, in the management of unwanted immune responses directed against immunogenic self-antigens or therapeutic protein products.

Therapeutic proteins provide interventions for some of the most complex and intractable diseases and are an essential part of modern medicine. Immunogenicity is the development of immune responses, usually measured by antibodies, to therapeutic proteins. These responses can adversely affect the safety and efficacy of the therapeutic agent and may have the following consequences: neutralization of a life-saving biotherapeutic, crossreactivity to non-redundant endogenous proteins, and hypersensitivity responses. These concerns have been underscored by the discontinuation of development of several drugs in recent years owing to immunogenicity issues. We review here recent progress in technological approaches that are useful for the clinical and non-clinical risk assessment of immunogenicity as well as mitigation strategies including deimmunizing protein molecules and inducing immune tolerance to the therapeutic protein.

The repurposing of the CRISPR/Cas microbial adaptive immune system for gene editing has resulted in an exponential rise in new technologies and promising approaches for treating numerous human diseases. While some of the approaches being currently developed involve ex vivo editing by CRISPR/Cas9, many more potential applications will require in vivo editing. The in vivo use of this technology comes with challenges, one of which is the immune response to Cas9, a protein of microbial origin. Thus, the prevalence of pre-existing antibodies to Cas9 could also be a relevant parameter. There are many avenues for how CRISPR/Cas9 technologies will be applied in vivo, including the mode of delivery. These may be expected to invoke different immunological pathways. Nonetheless, as with all protein therapeutics, it may be desirable to monitor for anti-Cas9 antibodies during clinical development. This will require the development of robust and reliable assays. Here, we describe ELISA-based assays that are capable of detecting antibodies to Cas9 from Staphylococcus aureus (SaCas9) and Streptococcs pyogenes (SpCas9) in human sera. Furthermore, using these assays to screen for pre-existing antibodies in 200 human serum samples, we found the prevalence of anti-SaCas9 and anti-SpCas9 antibodies to be 10% and 2.5%, respectively. Keywords: CRISPR/Cas9, immunogenicity, anti-drug antibodies, pre-existing antibodies, gene editing, drug development

Background Plasma-derived (pd) or recombinant (r) therapeutic factor VIII proteins (FVIIIs) are infused to arrest/prevent bleeding in patients with hemophilia A (PWHA). However, FVIIIs are neutralized if anti-FVIII-antibodies (inhibitors) develop. Accumulating evidence suggests that pdFVIIIs with von Willebrand factor (VWF) are less immunogenic than rFVIIIs and that distinct rFVIIIs are differentially immunogenic. Since inhibitor development is T-helper-cell-dependent, human leukocyte antigen (HLA)-class-II (HLAcII) molecules constitute an important early determinant. Objectives Use dendritic cell (DC)-protein processing/presentation assays with mass-spectrometric and peptide-proteomic analyses to quantify the DP-bound, DQ-bound, and DR-bound FVIII-derived peptides in individual HLAcII repertoires and compare the immunogenic potential of six distinct FVIIIs based on their measured peptide counts. Patients/methods Monocyte-derived DCs from normal donors and/or PWHA were cultured with either: Mix-rFVIII, a VWF-free equimolar mixture of a full-length (FL)-rFVIII [Advate® (Takeda)] and four distinct B-domain-deleted (BDD)-rFVIIIs [Xyntha® (Pfizer), NovoEight® (Novo-Nordisk), Nuwiq® (Octapharma), and Afstyla® (CSL Behring GmBH)]; a pdFVIII + pdVWF [Beriate® (CSL Behring GmBH)]; Advate ± pdVWF; Afstyla ± pdVWF; and Xyntha + pdVWF. Results We showed that (i) Beriate had a significantly lower immunogenic potential than Advate ± pdVWF, Afstyla - pdVWF, and Mix-rFVIII; (ii) distinct FVIIIs differed significantly in their immunogenic potential in that, in addition to (i), Afstyla + pdVWF had a significantly lower immunogenic potential than Beriate, while the immunogenic potential of Beriate was not significantly different from that of Xyntha + pdVWF; and (iii) rFVIIIs with pdVWF had significantly lower immunogenic potentials than the same rFVIIIs without pdVWF. Conclusions Our results provide HLAcII peptidomic level explanations for several important clinical observations/issues including the differential immunogenicity of distinct FVIIIs and the role of HLAcII genetics in inhibitor development.

The immunogenicity of protein therapeutics is an important safety and efficacy concern during drug development and regulation. Strategies to identify individuals and subpopulations at risk for an undesirable immune response represent an important unmet need. The major histocompatibility complex (MHC)–associated peptide proteomics (MAPPs) assay directly identifies the presence of peptides derived from a specific protein therapeutic on a donor’s MHC class II (MHC-II) proteins. We applied this technique to address several questions related to the use of factor VIII (FVIII) replacement therapy in the treatment of hemophilia A (HA). Although >12 FVIII therapeutics are marketed, most fall into 3 categories: (i) human plasma-derived FVIII (pdFVIII), (ii) full-length (FL)–recombinant FVIII (rFVIII; FL-rFVIII), and (iii) B-domain–deleted rFVIII. Here, we investigated whether there are differences between the FVIII peptides found on the MHC-II proteins of the same individual when incubated with these 3 classes. Based on several observational studies and a prospective, randomized, clinical trial showing that the originally approved rFVIII products may be more immunogenic than the pdFVIII products containing von Willebrand factor (VWF) in molar excess, it has been hypothesized that the pdFVIII molecules yield/present fewer peptides (ie, potential T-cell epitopes). We have experimentally tested this hypothesis and found that dendritic cells from HA patients and healthy donors present fewer FVIII peptides when administered pdFVIII vs FL-rFVIII, despite both containing the same molar VWF excess. Our results support the hypothesis that synthesis of pdFVIII under physiological conditions could result in reduced heterogeneity and/or subtle differences in structure/conformation which, in turn, may result in reduced FVIII proteolytic processing relative to FL-rFVIII.

High-Throughput (HT) SELEX combines SELEX (Systematic Evolution of Ligands by EXponential Enrichment), a method for aptamer discovery, with massively parallel sequencing technologies. This emerging technology provides data for a global analysis of the selection process and for simultaneous discovery of a large number of candidates but currently lacks dedicated computational approaches for their analysis. To close this gap, we developed novel in-silico methods to analyze HT-SELEX data and utilized them to study the emergence of polymerase errors during HT-SELEX. Rather than considering these errors as a nuisance, we demonstrated their utility for guiding aptamer discovery. Our approach builds on two main advancements in aptamer analysis: AptaMut-a novel technique allowing for the identification of polymerase errors conferring an improved binding affinity relative to the 'parent' sequence and AptaCluster-an aptamer clustering algorithm which is to our best knowledge, the only currently available tool capable of efficiently clustering entire aptamer pools. We applied these methods to an HT-SELEX experiment developing aptamers against Interleukin 10 receptor alpha chain (IL-10RA) and experimentally confirmed our predictions thus validating our computational methods.

Immunogenicity is an important consideration in the licensure of a therapeutic protein because the development of neutralizing anti-drug antibodies (ADAs) can affect both safety and efficacy. Neoantigens introduced by bioengineering of a protein drug are a particular cause for concern. The development of a bioengineered recombinant factor VIIa (rFVIIa) analog was discontinued after phase 3 trials because of the development of ADAs. The unmodified parent molecule (rFVIIa), on the other hand, has been successfully used as a drug for more than two decades with no reports of immunogenicity in congenital hemophilia patients with inhibitors. We used computational and experimental methods to demonstrate that the observed ADAs could have been elicited by neoepitopes in the engineered protein. The human leukocyte antigen type of the patients who developed ADAs is consistent with this hypothesis of a neoepitope-driven immune response, a finding that might have implications for the preclinical screening of therapeutic protein analogs.

Aim T cells specific for tFVIIIs in HA patients can induce neutralizing FVIII antibodies called inhibitors. DCs that present tFVIII derived peptides in their HLAcII repertoire may activate T cells. We employed DC protein processing and presentation assays (DCAs) to quantify tFVIII peptides in HLAcII peptidomes and used these as measures of the immunogenic potential (IP) of tFVIIIs as a function of their associated HLAcII/tFVIII peptide density. Methods We used DCs from 28 healthy donors and 5 HA patients [2 inhibitor (Inh) positive and 3 Inh negative] in 3 DCAs. In DCA 1, DCs from healthy donors 1–12 were given an equimolar mix of 5 recombinant (r) FVIIIs. In DCA 2, DCs from healthy donors 13–24 were given a plasma derived (pd) FVIII + vWF or 1 of 2 rFVIIIs (BDT or FL) ± vWF. In DCA 3, DCs from healthy donors 25–28 and HA patients 1–5 were incubated with pdFVIII or FL, BDD, or BDT rFVIII, all + vWF (Fig. 1A). After DC lysis and DR, DQ and DP isolation, we identified tFVIII peptides by LC-MS/MS. We performed a log linear analysis of the DR bound tFVIII peptides to identify determinants of Inh risk (Fig. 1B-E) with random effects to control for potential donor- and DCA-clustering. Space limits prevent description of the DQ and DP peptides. Results The IP of tFVIIIs was greater in HA patients than in healthy donors and in Inh positive than in Inh negative patients (Fig. 1B-E). We found that BDT + vWF and FL ± vWF had lesser and greater IP, respectively, than pdFVIII + vWF; the IP of BDD + vWF was comparable to that of pdFVIII + vWF. The rFVIII mix had the greatest IP. The higher IP in HA patients versus healthy donors and in Inh positive vs Inh negative patients reflect significant correlations of 0.91 and 0.52 with the presence/absence of the FVIII Inh risk allele DRB1*15:01. Download : Download high-res image (400KB) Download : Download full-size image Conclusions DC protein processing and presentation is affected by HLAcII repertoires but not by HA patient or FVIII Inh status after accounting for correlation with DRB1*15:01. Relative to pdFVIII, rFVIIIs have different IP with BDT and FL being significantly less and greater, respectively. T.E. Howard: 4. Scientific/Medical Advisor; Company/Organization; Haplomics Biotechnology. M. Hofmann: 5. Employee; Company/Organization; CSL Behring. L.V. Dinh: 5. Employee; Company/Organization; Haplomics Biotechnology. J. Powell: 5. Employee; Company/Organization; CSL Behring. E. Maraskovsky: 5. Employee; Company/Organization; CSL Limited.

Most immune responses to biotherapeutic proteins involve the development of anti-drug antibodies (ADAs). New drugs must undergo immunogenicity assessments to identify potential risks at early stages in the drug development process. This immune response is T cell-dependent. Ex vivo assays that monitor T cell proliferation often are used to assess immunogenicity risk. Such assays can be expensive and time-consuming to carry out. Furthermore, T cell proliferation requires presentation of the immunogenic epitope by major histocompatibility complex class II (MHCII) proteins on antigen-presenting cells. The MHC proteins are the most diverse in the human genome. Thus, obtaining cells from subjects that reflect the distribution of the different MHCII proteins in the human population can be challenging. The allelic frequencies of MHCII proteins differ among subpopulations, and understanding the potential immunogenicity risks would thus require generation of datasets for specific subpopulations involving complex subject recruitment. We developed TCPro, a computational tool that predicts the temporal dynamics of T cell counts in common ex vivo assays for drug immunogenicity. Using TCPro, we can test virtual pools of subjects based on MHCII frequencies and estimate immunogenicity risks for different populations. It also provides rapid and inexpensive initial screens for new biotherapeutics and can be used to determine the potential immunogenicity risk of new sequences introduced while bioengineering proteins. We validated TCPro using an experimental immunogenicity dataset, making predictions on the population-based immunogenicity risk of 15 protein-based biotherapeutics. Immunogenicity rankings generated using TCPro are consistent with the reported clinical experience with these therapeutics.

Essentials Fc-fusion increases a therapeutic's half-life, but FcγR interactions may impact immunogenicity. Species-specific Fc-FcγR interactions allow for mechanistic in vivo studies using mouse models. Fc fusion modulates the immune response to factor IX in hemophilia B mice by eliciting Th1 bias. This model could inform future studies of IgE-associated anaphylaxis in hemophilia B patients. SummaryBackground Fc fusion is a platform technology used to increase the circulating half-life of protein and peptide therapeutics. However, there are potential immunological consequences with this approach, such as changes in the molecule's immunogenicity as well as possible interactions with a repertoire of Fc receptors (FcR) that can modulate immune responses. Objectives/Methods Using a mouse hemophilia B (HB) model, we compared the immune responses to infusions of recombinant human factor IX (hFIX) and hFIX fused to mouse IgG2a-Fc (hFIX-mFc). The mFc was employed to allow species-specific Fc–FcγR interactions. Results Although treatment with hFIX-mFc altered the early development of anti-FIX IgG, no significant differences in anti-FIX antibody titers were observed at the end of the treatment regimen (5 weeks) or upon anamnestic response (5 months). However, treatment with hFIX-mFc elicited higher FIX-neutralizing antibody levels and resulted in reduced IgE titers compared with the hFIX-treated group. Additionally, differences in plasma cytokine levels and in vitro CD4+ T-cell responses suggest that whereas hFIX treatment triggered a Th2-biased immune response, hFIX-mFc treatment induced Th1-biased CD4+ T cells. We also show that hFIX-mFc bound to soluble FcγRs and engaged with FcγRs on different cell types, which may impact antigen presentation. Conclusions These studies provide a model system to study how Fc-fusion proteins may affect immune mechanisms. We used this model to demonstrate a plausible mechanism by which Fc fusion may modulate the IgE response to hFIX. This model may be appropriate for investigating the rare but severe IgE-mediated anaphylaxis reaction to hFIX infusions in HB patients.

ADAMTS13 is a secreted metalloprotease that cleaves von Willebrand Factor multimers in order to maintain proper homeostasis. A severe deficiency in ADAMTS13 triggers a disorder known as thrombotic thrombocytopenic purpura. At present, ADAMTS13 expression levels are determined by immunoblotting. We established a flow cytometry methodology to detect intracellular ADAMTS13 in liver and kidney cells using a polyclonal antibody, BL154G, and several monoclonal antibodies previously used to detect ADAMTS13 by immunoblotting. The results were validated using confocal microscopy, immunoblotting, and an activity assay (FRETS-VWF73). We show that labeling ADAMTS13 with specific antibodies and detection by flow cytometry yields results that are comparable with previously established methods for ADAMTS13 detection. Specifically, we compared the endogenous expression levels of ADAMTS13 in various liver cell lines using flow cytometry and obtained results that parallel immunoblot analysis. Knockdown of ADAMTS13 expression via targeted siRNA resulted in significantly reduced median signal, displaying the sensitivity of this detection method. A further analysis of reliability and specificity was achieved through plasmid DNA and transfection reagent dose response studies. The flow cytometry method described here is useful in determining the expression of both endogenous and recombinant forms of intracellular ADAMTS13. Flow cytometry is a convenient, efficient, and cost-effective way to measure the expression levels of ADAMTS13. Published 2009 Wiley-Liss, Inc.

Synonymous single-nucleotide polymorphisms (SNPs) do not produce altered coding sequences, and therefore they are not expected to change the function of the protein in which they occur. We report that a synonymous SNP in the Multidrug Resistance 1 ( MDR 1) gene, part of a haplotype previously linked to altered function of the MDR 1 gene product P-glycoprotein (P-gp), nonetheless results in P-gp with altered drug and inhibitor interactions. Similar mRNA and protein levels, but altered conformations, were found for wild-type and polymorphic P-gp. We hypothesize that the presence of a rare codon, marked by the synonymous polymorphism, affects the timing of cotranslational folding and insertion of P-gp into the membrane, thereby altering the structure of substrate and inhibitor interaction sites.

The efflux of drugs by the multidrug transporter P-glycoprotein (Pgp; ABCB1) is one of the principal means by which cancer cells evade chemotherapy and exhibit multidrug resistance. Mechanistic studies of Pgp-mediated transport, however, transcend the importance of this protein per se as they help us understand the transport pathway of the ATP-binding cassette proteins in general. The ATP-binding cassette proteins comprise one of the largest protein families, are central to cellular physiology, and constitute important drug targets. The functional unit of Pgp consists of two nucleotide-binding domains (NBD) and two transmembrane domains that are involved in the transport of drug substrates. Early studies postulated that conformational changes as a result of ATP hydrolysis were transmitted to the transmembrane domains bringing about drug transport. More recent structural and biochemical studies on the other hand suggested that ATP binds at the interface of the two NBDs and induces the formation of a closed dimer, and it has been hypothesized that this dimerization and subsequent ATP hydrolysis powers transport. Based on the mutational and biochemical work on Pgp and structural studies with isolated NBDs, we review proposed schemes for the catalytic cycle of ATP hydrolysis and the transport pathway. [Mol Cancer Ther 2007;6(1):13–23]

Multidrug resistance protein 4 (MRP4/ABCC4), transports cyclic nucleoside monophosphates, nucleoside analog drugs, chemotherapeutic agents, and prostaglandins. In this study we characterize ATP hydrolysis by human MRP4 expressed in insect cells. MRP4 hydrolyzes ATP (Km, 0.62 mm), which is inhibited by orthovanadate and beryllium fluoride. However, unlike ATPase activity of P-glycoprotein, which is equally sensitive to both inhibitors, MRP4-ATPase is more sensitive to beryllium fluoride than to orthovanadate. 8-Azido[alpha-32P]ATP binds to MRP4 (concentration for half-maximal binding approximately 3 microm) and is displaced by ATP or by its non-hydrolyzable analog AMPPNP (concentrations for half-maximal inhibition of 13.3 and 308 microm). MRP4 substrates, the prostaglandins E1 and E2, stimulate ATP hydrolysis 2- to 3-fold but do not affect the Km for ATP. Several other substrates, azidothymidine, 9-(2-phosphonylmethoxyethyl)adenine, and methotrexate do not stimulate ATP hydrolysis but inhibit prostaglandin E2-stimulated ATP hydrolysis. Although both post-hydrolysis transition states MRP4.8-azido[alpha-32P]ADP.Vi and MRP4.8-azido[alpha-32P]ADP.beryllium fluoride can be generated, nucleotide trapping is approximately 4-fold higher with beryllium fluoride. The divalent cations Mg2+ and Mn2+ support comparable levels of nucleotide binding, hydrolysis, and trapping. However, Co2+ increases 8-azido[alpha-32P]ATP binding and beryllium fluoride-induced 8-azido[alpha-32P]ADP trapping but does not support steady-state ATP hydrolysis. ADP inhibits basal and prostaglandin E2-stimulated ATP hydrolysis (concentrations for half-maximal inhibition 0.19 and 0.25 mm, respectively) and beryllium fluoride-induced 8-azido[alpha-32P]ADP trapping, whereas Pi has no effect up to 20 mm. In aggregate, our results demonstrate that MRP4 exhibits substrate-stimulated ATP hydrolysis, and we propose a kinetic scheme suggesting that ADP release from the post-hydrolysis transition state may be the rate-limiting step during the catalytic cycle.

ABCG2 (BCRP/MXR/ABCP) is a half-transporter associated with multidrug resistance that presumably homodimerizes for function. It has a conserved GXXXG motif in its first transmembrane segment, a motif that has been linked with dimerization in other proteins, e.g., glycophorin A. We substituted either or both glycines of this GXXXG motif with leucines to evaluate the impact on drug transport, ATP hydrolysis, cross-linking, and susceptibility to degradation. All mutants also carried the R482G gain-of-function mutation, and all migrated to the cell surface. The mutations resulted in lost transport for rhodamine 123 and impaired mitoxantrone, pheophorbide a, and BODIPY-prazosin transport, particularly in the double leucine mutant (G406L/G410L). Basal ATPase activity of the G406L/G410L mutant was comparable to the empty vector transfected cells with no substrate induction. Despite impaired function, the mutants retained susceptibility to cross-linking using either disuccinimidyl suberate (DSS) or the reducible dithiobis(succinimidyl propionate) (DSP) and demonstrated a high molecular weight complex under nonreducing conditions. Mutations to alanine at the same positions yielded fully functional transporters. Finally, we exposed cells to mitoxantrone to promote folding and processing of the mutant proteins, which in the leucine mutants resulted in increased amounts detected on immunoblot and by immunofluorescence. These studies support a hypothesis that the GXXXG motif promotes proper packing of the transmembrane segments in the functional ABCG2 homodimer, although it does not solely arbitrate dimerization.

Intron-22-inversion patients express the entire Factor VIII (FVIII)-amino-acid sequence intracellularly as 2 non-secreted polypeptides and have a positive “intracellular (I)-FVIII-CRM” status. Mutations conferring a positive I-FVIII-CRM status are associated with low inhibitor risk and are pharmacogenetically relevant because inhibitor risk may be affected by the nature of the therapeutic FVIII-protein (tFVIII), the affinity of any tFVIII-derived foreign peptide (tFVIII-fp) for any HLA class-II isomer (HLA-II) comprising individual major histocompatibility complex (MHC) repertoires, and the stability of any tFVIII-fp/HLA-II complex. We hypothesize that mutations conferring a completely or substantially negative I-FVIII-CRM status are pharmacogenetically irrelevant because inhibitor risk is high with any tFVIII and individual MHC repertoire.

The platform technology of fragment crystallizable (Fc) fusion, in which the Fc region of an antibody is genetically linked to an active protein drug, is among the most successful of a new generation of bioengineering strategies. Immunogenicity is a critical safety concern in the development of any protein therapeutic. While the therapeutic goal of generating Fc-fusion proteins has been to extend half-life, there is a critical mass of literature from immunology indicating that appropriate design of the Fc component has the potential to engage the immune system for product-specific outcomes. In the context of Fc-fusion therapeutics, a review of progress in understanding Fc biology suggests the prospect of engineering products that have an extended half-life and are able to modulate the immune system.

P-glycoprotein (Pgp), the ATP-binding cassette multidrug transporter, exhibits a drug (substrate)-stimulatable ATPase activity, and vanadate (Vi) inhibits this activity by stably trapping the nucleoside diphosphate in the Pgp.ADP.Vi conformation. We recently demonstrated that Vi-induced 8-azido-[alpha-(32)P]ADP trapping into Pgp in the absence of substrate occurs both in the presence of 8-azido-[alpha-(32)P]ATP (following 8-azido-ATP hydrolysis) or 8-azido-[alpha-(32)P]ADP (without hydrolysis) and, the transition state intermediates generated under either condition are functionally indistinguishable. In this study, we compare the effect of substrates on Vi-induced 8-azido-[alpha-(32)P]ADP trapping into Pgp under both non-hydrolysis and hydrolysis conditions. We demonstrate that whereas substrates stimulate the Vi-induced trapping of 8-azido-[alpha-(32)P]ADP under hydrolysis conditions, they strongly inhibit Vi-induced trapping under non-hydrolysis conditions. This inhibition is concentration-dependent, follows first order kinetics, and is effected by drastically decreasing the affinity of nucleoside diphosphate for Pgp during trapping. However, substrates do not affect the binding of nucleoside diphosphate in the absence of Vi, indicating that the substrate-induced conformation exerts its effect at a step distinct from nucleoside diphosphate-binding. Our results demonstrate that during the catalytic cycle of Pgp, although the transition state, Pgp x ADP x P(i) (Vi), can be generated both via the hydrolysis of ATP or by directly providing ADP to the system, in the presence of substrate the reaction is driven in the forward direction, i.e. hydrolysis of ATP. These data suggest that substrate-stimulated ATP hydrolysis by Pgp is a vectorial process.

Structural perturbations in biopolymers with hydrophobic interiors i.e. specific proteins and dimyristoylphosphatidylcholine (DMPC) vesicles were investigated as a function of solute concentrations in the medium. 1,6-diphenyl-1,3,5-hexatriene (DPH) was used as fluorescent probe. Response of DPH was comparable to that of intrinsic tryptophan in BSA in terms of steady state and time resolved fluorescence. The solutes induced a decrease in steady state anisotropy as well as rotational correlation time (computed from lifetime measurements) for DPH in both proteins and membranes. Enhanced access of the quencher potassium iodide to tryptophan in bovine serum albumin (BSA) and ovalbumin, and enhanced terbium leakage in DMPC vesicles induced by various solutes concomitant with decreased anisotropy/correlation time were consistent with structural perturbations of the nature of defects or voids in these polymers.

A series of five homodimeric polyethylene glycol (PEG) linked homodimers based on the multidrug resistance reversal agent (−)-stipiamide were made and tested for their ability to interact with P-glycoprotein, the protein responsible for multidrug resistance, using ATPase and photoaffinity displacement assays. Key reactions include a new alkoxide-mesylate displacement for the assembly of the PEG linkers and a double Sonogashira coupling reaction.

P-glycoprotein (Pgp) is a plasma membrane protein whose overexpression confers multidrug resistance to tumor cells by extruding amphipathic natural product cytotoxic drugs using the energy of ATP. An elucidation of the catalytic cycle of Pgp would help design rational strategies to combat multidrug resistance and to further our understanding of the mechanism of ATP-binding cassette transporters. We have recently reported (Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515–2520) that there are two independent ATP hydrolysis events in a single catalytic cycle of Pgp. In this study we exploit the vanadate (Vi)-induced transition state conformation of Pgp (Pgp·ADP·Vi) to address the question of what are the effects of ATP hydrolysis on the nucleotide-binding site. We find that at the end of the first hydrolysis event there is a drastic decrease in the affinity of nucleotide for Pgp coincident with decreased substrate binding. Release of occluded dinucleotide is adequate for the next hydrolysis event to occur but is not sufficient for the recovery of substrate binding. Whereas the two hydrolysis events have different functional outcomesvis a vis the substrate, they show comparablet for both incorporation and release of nucleotide, and the affinities for [α-32P]8-azido-ATP during Vi-induced trapping are identical. In addition, the incorporation of [α-32P]8-azido-ADP in two ATP sites during both hydrolysis events is also similar. These data demonstrate that during individual hydrolysis events, the ATP sites are recruited in a random manner, and only one site is utilized at any given time because of the conformational change in the catalytic site that drastically reduces the affinity of the second ATP site for nucleotide binding. In aggregate, these findings provide an explanation for the alternate catalysis of ATP hydrolysis and offer a mechanistic framework to elucidate events at both the substrate- and nucleotide-binding sites in the catalytic cycle of Pgp.

P-glycoprotein (Pgp) is a transmembrane protein conferring multidrug resistance to cells by extruding a variety of amphipathic cytotoxic agents using energy from ATP hydrolysis. The objective of this study was to understand how substrates affect the catalytic cycle of ATP hydrolysis by Pgp. The ATPase activity of purified and reconstituted recombinant human Pgp was measured using a continuous cycling assay. Pgp hydrolyzes ATP in the absence of drug at a basal rate of 0.5 μmol·min·mg−1 with aK m for ATP of 0.33 mm. This basal rate can be either increased or decreased depending on the Pgp substrate used, without an effect on the K m for ATP or 8-azidoATP and K i for ADP, suggesting that substrates do not affect nucleotide binding to Pgp. Although inhibitors of Pgp activity, cyclosporin A, its analog PSC833, and rapamycin decrease the rate of ATP hydrolysis with respect to the basal rate, they do not completely inhibit the activity. Therefore, these drugs can be classified as substrates. Vanadate (Vi)-induced trapping of [α-32P]8-azidoADP was used to probe the effect of substrates on the transition state of the ATP hydrolysis reaction. TheK m for [α-32P]8-azidoATP (20 μm) is decreased in the presence of Vi; however, it is not changed by drugs such as verapamil or cyclosporin A. Strikingly, the extent of Vi-induced [α-32P]8-azidoADP trapping correlates directly with the fold stimulation of ATPase activity at steady state. Furthermore, Pi exhibits very low affinity for Pgp (K i∼30 mm for Vi-induced 8-azidoADP trapping). In aggregate, these data demonstrate that the release of Vi trapped [α-32P]8-azidoADP from Pgp is the rate-limiting step in the steady-state reaction. We suggest that substrates modulate the rate of ATPase activity of Pgp by controlling the rate of dissociation of ADP following ATP hydrolysis and that ADP release is the rate-limiting step in the normal catalytic cycle of Pgp.

P-glycoprotein (Pgp), the ATP-binding cassette (ABC) transporter, confers multidrug resistance to cancer cells by extruding cytotoxic natural product amphipathic drugs using the energy of ATP hydrolysis. Our studies are directed toward understanding the mechanism of action of Pgp and recent work deals with the assessment of interaction between substrate and ATP sites and elucidation of the catalytic cycle of ATP hydrolysis. The kinetic analyses of ATP hydrolysis by reconstituted purified Pgp suggest that ADP release is the rate-limiting step in the catalytic cycle and the substrates exert their effect by modulating ADP release. In addition, we provide evidence for two distinct roles for ATP hydrolysis in a single turnover of Pgp, one in the transport of drug and the other in effecting conformational changes so as to reset the transporter for the next catalytic cycle. Detailed kinetic measurements determined that both nucleotide-binding domains behave symmetrically and during individual hydrolysis events the ATP sites are recruited in a random manner. Furthermore, only one nucleotide site hydrolyzes ATP at any given time, causing (in this site) a conformational change that drastically decreases (>30-fold) the affinity of the second site for ATP-binding. Thus, the blocking of ATP-binding to the second site while the first one is in catalytic conformation appears to be the basis for the alternate catalytic cycle of ATP hydrolysis by Pgp, and this may be applicable as well to other ABC transporters linked with the development of multidrug resistance.

A combinatorial library of polyenes, based on (-)-stipiamide, has been constructed and evaluated for the discovery of new multidrug resistance reversal agents. A palladium coupling was used to react each individual vinyl iodide with a mixture of the seven acetylenes at near 1:1 stoichiometry. The coupling was also used to react each individual acetylene with the mixture of six vinyl iodides to create 13 pools indexed in two dimensions for a total of 42 compounds. Individual compounds were detected at equimolar concentration. The vinyl iodides, made initially using a crotylborane addition to generate the anti1,2-hydroxylmethyl products, were now made using a more efficient norephedrine propionate boron enolate aldol reaction. The indexed approach, ideally suited for cellular assays that involve membrane-bound targets, allowed for the rapid identification of reversal agents using assays with drug-resistant human breast cancer MCF7-adrR cells. Intersections of potent pools identified new compounds with promising activity. Aryl dimension pools showed R = ph and naphthyl as the most potent. The acetylene dimension had R' = phenylalaninol and alaninol as the most potent. Isolated individual compounds, both active and nonpotent, were assayed to confirm the library results. The most potent new compound was 4ek (R = naphthyl, R' = phenylaninol) at 1.45 microM. Other nonnatural individual naphthyl-amide compounds showed potent MDR reversal including the morpholino-amide 4ej (1.69 microM). Synergistic activities attributed to the two ends of the molecule were also identified. Direct interaction with Pgp was established by ATPase and photoaffinity displacement assays. The results indicate that both ends of the polyene reversal agent are involved in Pgp interaction and can be further modified for increased potency.

Intercellular transfer of proteins is a mode of communication between cells that is crucial for certain physiological processes. Chemotherapy is the treatment of choice for approximately 50% of all cancers. However, multidrug resistance mediated by drug-efflux pumps such as P-glycoprotein (Pgp) minimizes the effectiveness of such therapy in a large number of patients. A new study demonstrates the functional intercellular transfer of Pgp. Non-genetic transfer of the multidrug resistance phenotype raises fascinating questions about the mechanism and regulation of cell-surface membrane-protein-mediated spread of traits.

Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs). An indirect intracellular staining (ICS) method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI) values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels.

Neutralizing antibodies (inhibitors) to replacement factor VIII (FVIII, either plasma derived or recombinant) impair the effective management of hemophilia A. Individuals with hemophilia A due to major deletions of the FVIII gene (F8) lack antigenically cross-reactive material in their plasma ("CRM-negative"), and the prevalence of inhibitors in these individuals may be as high as 90%. Conversely, individuals with hemophilia A caused by F8 missense mutations are CRM-positive, and their overall prevalence of inhibitors is

The protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder thrombotic thrombocytopenic purpura (TTP). It has been observed that cyclosporin A (CsA) treatment, particularly in transplant patients, may sometimes be linked to the development of TTP. Until now, the reason for such a link was unclear. Here we provide evidence demonstrating that cyclophilin B (CypB) activity plays an important role in the secretion of active ADAMTS13. We found that CsA, an inhibitor of CypB, reduces the secretion of ADAMTS13 and leads to conformational changes in the protein resulting in diminished ADAMTS13 proteolytic activity. A direct, functional interaction between CypB (which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone functions) and ADAMTS13 is demonstrated using immunoprecipitation and siRNA knockdown of CypB. Finally, CypB knock-out mice were found to have reduced ADAMTS13 levels. Taken together, our findings indicate that cyclophilin-mediated activity is an important factor affecting secretion and activity of ADAMTS13. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization in the proper folding of this protein. These results altogether provide a novel mechanistic explanation for CsA-induced TTP in transplant patients.

The development of effective clinical interventions against multidrug resistance (MDR) in cancer remains a significant challenge. Single nucleotide polymorphisms (SNPs) contribute to wide variations in how individuals respond to medications and there are several SNPs in human P-glycoprotein (P-gp) that may influence the interactions of drug-substrates with the transporter. Interestingly, even some of the synonymous SNPs have functional consequences for P-gp. It is also becoming increasingly evident that an understanding of the transport pathway of P-gp may be necessary to design effective modulators. In this review we discuss: (1) The potential importance of SNPs (both synonymous and non-synonymous) in MDR and (2) How new concepts that have emerged from structural studies with isolated nucleotide binding domains of bacterial ABC transporters have prompted biochemical studies on P-gp, leading to a better understanding of the mechanism of P-gp mediated transport. Our results suggest that the power-stroke is provided only after formation of the pre-hydrolysis transition-like (E·S) state during ATP hydrolysis.

Structural and biochemical studies of ATP-binding cassette (ABC) transporters suggest that an ATP-driven dimerization of the nucleotide-binding domains (NBDs) is an important reaction intermediate of the transport cycle. Moreover, an asymmetric occlusion of ATP at one of the two ATP sites of P-glycoprotein (Pgp) may follow the formation of the symmetric dimer. It has also been postulated that ADP drives the dissociation of the dimer. In this study, we show that the E.S conformation of Pgp (previously demonstrated in the E556Q/E1201Q mutant Pgp) can be obtained with the wild-type protein by use of the nonhydrolyzable ATP analogue ATP-gamma-S. ATP-gamma-S is occluded into the Pgp NBDs at 34 degrees C but not at 4 degrees C, whereas ATP is not occluded at either temperature. Using purified Pgp incorporated into proteoliposomes and ATP-gamma-35S, we demonstrate that the occlusion of ATP-gamma-35S has an Eact of 60 kJ/mol and the stoichiometry of ATP-gamma-35S:Pgp is 1:1 (mol/mol). Additionally, in the conserved Walker B mutant (E556Q/E1201Q) of Pgp, we find occlusion of the nucleoside triphosphate but not the nucleoside diphosphate. Furthermore, Pgp in the occluded nucleotide conformation has reduced affinity for transport substrates. These data provide evidence for the ATP-driven dimerization and ADP-driven dissociation of the NBDs, and although two ATP molecules may initiate dimerization, only one is driven to an occluded pre-hydrolysis intermediate state. Thus, in a full-length ABC transporter like Pgp, it is unlikely that there is complete association and disassociation of NBDs and the occluded nucleotide conformation at one of the NBDs provides the power-stroke at the transport-substrate site.

Each nucleotide-binding domain (NBD) of mammalian P-glycoproteins (Pgps) and human ATP-binding cassette (ABC) B subfamily members contains a tyrosine residue approximately 25 residues upstream of the Walker A domain. To assess the role of the conserved Y401 and Y1044 residues of human Pgp, we substituted these residues with F, W, C, or A either singly or together. The mutant proteins were expressed in a Vaccinia virus-based transient expression system as well as in baculovirus-infected HighFive insect cells. The Y401F, Y401W, Y1044F, Y1044W, or Y401F/Y1004F mutants transported fluorescent substrates similar to the wild-type protein. On the other hand, Y401L and Y401C exhibited partial (30-50%) function, and transport was completely abolished in Y401A, Y1044A, and Y401A/Y1044A mutant Pgps. Similarly, in Y401A, Y1044A, and Y401A/Y1044A mutants, TNP-ATP binding, vanadate-induced trapping of nucleotide, and ATP hydrolysis were completely abolished. Thus, an aromatic residue upstream of the Walker A motif in ABC transporters is critical for binding of ATP. Additionally, the crystal structures of several NBDs in the nucleotide-bound form, data mining, and alignment of 18,514 ABC domains with the consensus conserved sequence in a database of all nonredundant proteins indicate that an aromatic residue is highly conserved in approximately 85% of ABC proteins. Although the role of this aromatic residue has previously been studied in a few ABC proteins, we provide evidence for a near-universal structural and functional role for this residue and recognize its presence as a conserved subdomain approximately 25 amino acids upstream of the Walker A motif that is critical for ATP binding. We named this subdomain the "A-loop" (aromatic residue interacting with the adenine ring of ATP).

Members of the superfamily of ATP-binding cassette (ABC) transporters mediate the movement of a variety of substrates including simple ions, complex lipids and xenobiotics. At least 18 ABC transport proteins are associated with disease conditions. P-glycoprotein (Pgp, ABCB1) is the archetypical mammalian ABC transport protein and its mechanism of action has received considerable attention. There is strong biochemical evidence that Pgp moves molecular cargo against a concentration gradient using the energy of ATP hydrolysis. However, the molecular details of how the energy of ATP hydrolysis is coupled to transport remain in dispute and it has not been possible to reconcile the data from various laboratories into a single model. The functional unit of Pgp consists of two nucleotide binding domains (NBDs) and two trans-membrane domains which are involved in the transport of drug substrates. Considerable progress has been made in recent years in characterizing these functionally and spatially distinct domains of Pgp. In addition, our understanding of the domains has been augmented by the resolution of structures of several non-mammalian ABC proteins. This review considers: (i) the role of specific conserved amino acids in ATP hydrolysis mediated by Pgp; (ii) emerging insights into the dimensions of the drug binding pocket and the interactions between Pgp and the transport substrates and (iii) our current understanding of the mechanisms of coupling between energy derived from ATP binding and/or hydrolysis and efflux of drug substrates.

Resistance to chemically different natural product anti-cancer drugs (multidrug resistance, or MDR) results from decreased drug accumulation, resulting from expression of one or more ATP-dependent efflux pumps. The first of these to be identified was P-glycoprotein (P-gp), the product of the human MDR1 gene, localized to chromosome 7q21. P-gp is a member of the large ATP-binding cassette (ABC) family of proteins. Although its crystallographic 3-D structure is yet to be determined, sequence analysis and comparison to other ABC family members suggest a structure consisting of two transmembrane (TM) domains, each with six TM segments, and two nucleotide-binding domains. In the epithelial cells of the gastrointestinal tract, liver, and kidney, and capillaries of the brain, testes, and ovaries, P-gp acts as a barrier to the uptake of xenobiotics, and promotes their excretion in the bile and urine. Polymorphisms in the MDR1 gene may affect the pharmacokinetics of many commonly used drugs, including anticancer drugs. Substrate recognition of many different drugs occurs within the TM domains in multiple-overlapping binding sites. We have proposed a model for how ATP energizes transfer of substrates from these binding sites on P-gp to the outside of the cell, which accounts for the apparent stoichiometry of two ATPs hydrolysed per molecule of drug transported. Understanding of the biology, genetics, and biochemistry of P-gp promises to improve the treatment of cancer and explain the pharmacokinetics of many commonly used drugs.

Rae1p and Mex67p/Tap are conserved mRNA export factors. We have used synthetic lethal genetic screens in Schizosaccharomyces pombe to identify mutations in genes that are functionally linked to rae1 and mex67 in mRNA export. From these screens, we have isolated mutations in a putative S. pombe homologue of the Candida albicans elf1 gene. The elf1 of S. pombe is not an essential gene. When elf1 mutations are combined with rae1-167 mutation, growth and mRNA export is inhibited in the double mutants. This inhibition can be suppressed by the multicopy expression of mex67 suggesting that Mex67p can substitute for the loss of Elf1p function. Elf1p is a non-membrane member of the ATP-binding cassette (ABC) class of ATPase and the GFP-Elf1p fusion localizes to the cytoplasm. Elf1p, expressed and purified from Escherichia coli, binds and hydrolyzes ATP. A mutant Elf1p that carries a glycine to aspartic acid (G731D) mutation within the Walker A domain of the second ATP site retains the ATP binding but loses its ATPase activity in vitro. This mutant protein no longer functions in mRNA export. Taken together, our results show that Elf1p functions as a mRNA export factor along with Rae1p and Mex67p in S. pombe.

A series of five homodimeric polyethylene glycol (PEG) linked homodimers based on the multidrug resistance reversal agent (−)-stipiamide were made and tested for their ability to interact with P-glycoprotein, the protein responsible for multidrug resistance, using ATPase and photoaffinity displacement assays. Key reactions include a new alkoxide-mesylate displacement for the assembly of the PEG linkers and a double Sonogashira coupling reaction.

P-glycoprotein (Pgp) is an ATP-dependent drug efflux pump whose overexpression confers multidrug resistance to cancer cells. Pgp exhibits a robust drug substrate-stimulable ATPase activity, and vanadate (Vi) blocks this activity effectively by trapping Pgp nucleotide in a non-covalent stable transition state conformation. In this study we compare Vi-induced [alpha-(32)P]8-azido-ADP trapping into Pgp in the presence of [alpha-(32)P]8-azido-ATP (with ATP hydrolysis) or [alpha-(32)P]8-azido-ADP (without ATP hydrolysis). Vi mimics P(i) to trap the nucleotide tenaciously in the Pgp.[alpha-(32)P]8-azido-ADP.Vi conformation in either condition. Thus, by using [alpha-(32)P]8-azido-ADP we show that the Vi-induced transition state of Pgp can be generated even in the absence of ATP hydrolysis. Furthermore, half-maximal trapping of nucleotide into Pgp in the presence of Vi occurs at similar concentrations of [alpha-(32)P]8-azido-ATP or [alpha-(32)P]8-azido-ADP. The trapped [alpha-(32)P]8-azido-ADP is almost equally distributed between the N- and the C-terminal ATP sites of Pgp in both conditions. Additionally, point mutations in the Walker B domain of either the N- (D555N) or C (D1200N)-terminal ATP sites that arrest ATP hydrolysis and Vi-induced trapping also show abrogation of [alpha-(32)P]8-azido-ADP trapping into Pgp in the absence of hydrolysis. These data suggest that both ATP sites are dependent on each other for function and that each site exhibits similar affinity for 8-azido-ATP (ATP) or 8-azido-ADP (ADP). Similarly, Pgp in the transition state conformation generated with either ADP or ATP exhibits drastically reduced affinity for the binding of analogues of drug substrate ([(125)I]iodoarylazidoprazosin) as well as nucleotide (2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate). Analyses of Arrhenius plots show that trapping of Pgp with [alpha-(32)P]8-azido-ADP (in the absence of hydrolysis) displays an approximately 2.5-fold higher energy of activation (152 kJ/mol) compared with that observed when the transition state intermediate is generated through hydrolysis of [alpha-(32)P]8-azido-ATP (62 kJ/mol). In aggregate, these results demonstrate that the Pgp.[alpha-(32)P]8-azido-ADP (or ADP).Vi transition state complexes generated either in the absence of or accompanying [alpha-(32)P]8-azido-ATP hydrolysis are functionally indistinguishable.

P-glycoprotein (Pgp) is an ATP-dependent hydrophobic natural product anticancer drug efflux pump whose overexpression confers multidrug resistance to tumor cells. The work reported here deals with the elucidation of the energy requirement for substrate interaction with Pgp during the catalytic cycle. We show that the K d (412 nM) of the substrate analogue [ 125 I]iodoarylazidoprazoin for Pgp is not altered by the presence of the nonhydrolyzable nucleotide 5′-adenylylimididiphosphate and vanadate ( K d = 403 nM). Though binding of nucleotide per se does not affect interactions with the substrate, ATP hydrolysis results in a dramatic conformational change where the affinity of [ 125 I]iodoarylazidoprazoin for Pgp trapped in transition-state conformation (Pgp⋅ADP⋅vanadate) is reduced >30-fold. To transform Pgp from this intermediate state of low affinity for substrate to the next catalytic cycle, i.e., a conformation that binds substrate with high affinity, requires conditions that permit ATP hydrolysis. Additionally, there is an inverse correlation ( R 2 = 0.96) between 8AzidoADP (or ADP) release and the recovery of substrate binding. These results suggest that the release of nucleotide is necessary for reactivation but not sufficient. The hydrolysis of additional molecule(s) of ATP (or 8AzidoATP) is obligatory for the catalytic cycle to advance to completion. These data are consistent with the observed stoichiometry of two ATP molecules hydrolyzed for the transport of every substrate molecule. Our data demonstrate two distinct roles for ATP hydrolysis in a single turnover of the catalytic cycle of Pgp, one in the transport of substrate and the other in effecting conformational changes to reset the pump for the next catalytic cycle.

The development of neutralizing anti-drug-antibodies to the Factor VIII protein-therapeutic is currently the most significant impediment to the effective management of hemophilia A. Common non-synonymous single nucleotide polymorphisms (ns-SNPs) in the F8 gene occur as six haplotypes in the human population (denoted H1 to H6) of which H3 and H4 have been associated with an increased risk of developing anti-drug antibodies. There is evidence that CD4+ T-cell response is essential for the development of anti-drug antibodies and such a response requires the presentation of the peptides by the MHC-class-II (MHC-II) molecules of the patient. We measured the binding and half-life of peptide-MHC-II complexes using synthetic peptides from regions of the Factor VIII protein where ns-SNPs occur and showed that these wild type peptides form stable complexes with six common MHC-II alleles, representing 46.5% of the North American population. Next, we compared the affinities computed by NetMHCIIpan, a neural network-based algorithm for MHC-II peptide binding prediction, to the experimentally measured values and concluded that these are in good agreement (area under the ROC-curve of 0.778 to 0.972 for the six MHC-II variants). Using a computational binding predictor, we were able to expand our analysis to (a) include all wild type peptides spanning each polymorphic position; and (b) consider more MHC-II variants, thus allowing for a better estimation of the risk for clinical manifestation of anti-drug antibodies in the entire population (or a specific sub-population). Analysis of these computational data confirmed that peptides which have the wild type sequence at positions where the polymorphisms associated with haplotypes H3, H4 and H5 occur bind MHC-II proteins significantly more than a negative control. Taken together, the experimental and computational results suggest that wild type peptides from polymorphic regions of FVIII constitute potential T-cell epitopes and thus could explain the increased incidence of anti-drug antibodies in hemophilia A patients with haplotypes H3 and H4., Author Summary The development of anti-drug antibodies to therapeutic proteins is a significant impediment to development and licensure of therapeutic proteins and limits their clinical utility. The development of such antibodies requires CD4+ T-cell activation, which is mediated by the recognition of epitopes presented by MHC class-II (MHC-II) molecules. Here, we use experimental measurements and computational predictions of peptide-MHC-II affinities to study the clinical observation that African-American hemophilia A patients have a higher incidence of anti-drug antibodies to Factor VIII than Caucasian patients. Specifically, we used the experimental data to select and validate a computational prediction method which, in turn, allowed us to expand our analysis to a larger repertoire of peptide-MHC-II complexes. We showed that wild type peptides spanning haplotype polymorphisms common in the African American population bind MHC-II proteins significantly more than a negative control, thus providing a mechanistic explanation of the phenomenon in this population.

Abstract 3349 Introduction: The protease ADAMTS13 (A Disintegrin and Metalloprotease with Thrombo Spondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder Thrombotic Thrombocytopenic Purpura (TTP). TTP has been in particular observed in transplant patients following treatment with Cyclosporin A (CsA), a drug which is frequently administered in patients to suppress allogeneic immune response. However, a mechanistic explanation for such a link has remained unclear. Here, we report that cyclophilin-B mediated peptidyl-prolyl cis–trans isomerase (PPIase) activity, which is also targeted by CsA, plays an important role in the production of active ADAMTS13. Methods and Results: An in vitro transient expression system was used to study the effects of CsA on the synthesis and activity of ADAMTS13. We found that exposing HEK293 cells to CsA results in a dose-dependent reduction in the level of secreted ADAMTS13, but not the ubiquitously-expressed housekeeping protein Hsp70. Moreover, treatment with other immunosuppressive drugs, such as rapamycin or tacrolimus/FK506 did not impact the secretion of ADAMTS13. CsA is a known inhibitor of several cellular substrates, including cyclophilin B (CypB), which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone activities. We show a direct, functional interaction between CypB and ADAMTS13 using co-immunoprecipitation analysis. Specifically, ADAMTS13 and CypB could be detected in association together in untreated cells, but not in those treated with CsA, where only ADAMTS13 could be detected. ADAMTS13 levels are also reduced in the setting of siRNA-mediated CypB knockdown, suggesting a direct link between CypB expression/activity and ADAMTS13 maturation. We have found that CsA leads to intracellular conformational changes in the ADAMTS13 protein, evidenced by a unique trypsin digestion pattern of ADAMTS13 from CsA-treated cells relative to untreated cells. Additionally we found that treatment of cells with N-acetyl-Leu-Leu-norleucinal (ALLN), a proteasome inhibitor, resulted in about a 2.5 fold rescue in ADAMTS13 expression in the setting of CsA treatment. This observation suggests that CsA treatment leads to an enhanced proteasomal degradation of ADAMTS13, possibly due altered conformation. Although CsA did not influence the overall level of intracellular ADAMTS13, it resulted in diminished specific proteolytic activity, further suggesting improper ADAMTS13 folding. Finally, we show that plasma samples from CypB knockout mice have reduced levels of circulating ADAMTS13. Conclusions: Our findings indicate that cyclophilin-mediated activity is an important factor in ADAMTS13 secretion and activity. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization process mediated by CypB in its proper folding and maturation. We suggest that CsA treatment disrupts the interaction between ADAMTS13 and CypB. Although this may not be the sole mechanism by which CsA ultimately lowers levels of secreted ADAMTS13, this work altogether provides a novel mechanistic explanation for CsA-induced TTP in transplant patients. Disclosures: No relevant conflicts of interest to declare.

The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB pump that catalyzes extrusion of the metalloids As(III) and Sb(III), conferring metalloid resistance. The catalytic subunit, ArsA, is an ATPase with two homologous halves, A1 and A2, connected by a short linker. Each half contains a nucleotide binding domain. The overall rate of ATP hydrolysis is slow in the absence of metalloid and is accelerated by metalloid binding. The results of photolabeling of ArsA with the ATP analogue 8-azidoadenosine 5'-[alpha-(32)P]-triphosphate at 4 degrees C indicate that metalloid stimulation correlates with a10-fold increase in affinity for nucleotide. To investigate the relative contributions of the two nucleotide binding domains to catalysis, a thrombin site was introduced in the linker. This allowed discrimination between incorporation of labeled nucleotides into the two halves of ArsA. The results indicate that both the A1 and A2 nucleotide binding domains bind and hydrolyze trinucleotide, even in the absence of metalloid. Sb(III) increases the affinity of the A1 nucleotide binding domain to a greater extent than the A2 nucleotide binding domain. The ATP analogue labeled with (32)P at the gamma position was used to measure hydrolysis of trinucleotide at 37 degrees C. Under these catalytic conditions, both nucleotide binding domains hydrolyze ATP, but hydrolysis in A1 is stimulated to a greater degree by Sb(III) than A2. These results suggest that the two homologous halves of the ArsA may be functionally nonequivalent.