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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 emergence of the novel SARS-CoV-2 virus is the most important public-health issue of our time. Understanding the diverse clinical presentations of the ensuing disease, COVID-19, remains a critical unmet need. Here we present a comprehensive listing of the diverse clinical indications associated with COVID-19. We explore the theory that anti-SARS-CoV-2 antibodies could cross-react with endogenous human proteins driving some of the pathologies associated with COVID-19. We describe a novel computational approach to estimate structural homology between SARS-CoV-2 proteins and human proteins. Antibodies are more likely to interrogate 3D-structural epitopes than continuous linear epitopes. This computational workflow identified 346 human proteins containing a domain with high structural homology to a SARS-CoV-2 Wuhan strain protein. Of these, 102 proteins exhibit functions that could contribute to COVID-19 clinical pathologies. We present a testable hypothesis to delineate unexplained clinical observations vis-à-vis COVID-19 and a tool to evaluate the safety-risk profile of potential COVID-19 therapies.

Reliable and reproducible monitoring of the conformational state of therapeutic protein products remains an unmet technological need. This need is amplified by the increasing number of biosimilars entering the drug development pipeline as many branded biologics are reaching the end of their market exclusivity period. Availability of methods to better characterize protein conformation may improve detection of counterfit and unlicensed therapeutic proteins. In this study, we report the use of a set of modified DNA aptamers with enhanced chemical diversity to probe the conformational state of 12 recombinant human erythropoietin (rHuEPO) therapeutic protein products; one FDA-licensed rHuEPO originator biological product, three rHuEPO products that are approved for marketing in the US or EU as biosimilars, and eight rHuEPO products that are not approved for marketing in the US or EU. We show that several of these modified aptamers are able to distinguish rHuEPO reference products or approved biosimilars from non-licensed rHuEPO products on the basis of differences in binding kinetics and equilibrium affinity constants. These reagents exhibit sensitivity to the conformational integrity of various forms of rHuEPO and as such represent powerful, simple-to-use analytical tools to monitor the conformational integrity of therapeutic-proteins during manufacture and to screen for and identify both substandard and counterfeit products.

Background: Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. The aim of our longitudinal study was to describe ageing‐related cognitive changes in men with FXS. Method: A neuropsychologist determined the raw scores (RSs) of 19 men with FXS twice with the Leiter International Performance Scale at an average interval of 22 years. The ages of the participants at baseline ranged from 16 to 50 (mean 27) years. Results: At follow‐up, the RSs improved in two men, remained the same in two men and declined in 15 men. Overall, the RS of the study group deteriorated by an average 4 points in RSs (p < 0.001). Conclusion: Cognitive ageing in men with FXS started earlier than that in men in the general population; in many cases, cognitive ageing in men with FXS began before middle age, usually without any medical or other underlying cause.

Hemophilia B is a blood clotting disorder caused by deficient activity of coagulation factor IX (FIX). Multiple recombinant FIX proteins are currently approved to treat hemophilia B, and several gene therapy products are currently being developed. Codon optimization is a frequently used technique in the pharmaceutical industry to improve recombinant protein expression by recoding a coding sequence using multiple synonymous codon substitutions. The underlying assumption of this gene recoding is that synonymous substitutions do not alter protein characteristics because the primary sequence of the protein remains unchanged. However, a critical body of evidence shows that synonymous variants can affect cotranslational folding and protein function. Gene recoding could potentially alter the structure, function, and in vivo immunogenicity of recoded therapeutic proteins. Here, we evaluated multiple recoded variants of F9 designed to further explore the effects of codon usage bias on protein properties. The detailed evaluation of these constructs showed altered conformations, and assessment of translation kinetics by ribosome profiling revealed differences in local translation kinetics. Assessment of wild-type and recoded constructs using a major histocompatibility complex (MHC)-associated peptide proteomics assay showed distinct presentation of FIX-derived peptides bound to MHC class II molecules, suggesting that despite identical amino acid sequence, recoded proteins could exhibit different immunogenicity risks. Posttranslational modification analysis indicated that overexpression from gene recoding results in suboptimal posttranslational processing. Overall, our results highlight potential functional and immunogenicity concerns associated with gene-recoded F9 products. These findings have general applicability and implications for other gene-recoded recombinant proteins.

Background Phelan–McDermid syndrome (PMD) is usually not only caused by 22q13.3 deletion but also pathogenic variants (mutations) of SHANK3 gene. PMD is characterized by global intellectual disability, severely delayed or absent speech, and features of autism spectrum disorder and susceptibility to psychotic behavior. Here, we describe a neurocognitive follow‐up and genetic etiology for two siblings with PMD. Method Comparative genomic hybridization (CGH) array test was normal and no 22q13.3 deletion was observed. For this reason, whole exome sequencing (WES) analyzed the siblings’ and the parents’ DNA sample. Results The results of the siblings strongly suggest that the SHANK3 gene variant c.2313+1G>A is pathogenic and PMD can be inherited from a mosaic father for this gene variant. Both siblings learned new skills until puberty but experienced a neuropsychiatric disaster after the age of 14 years experienced neurocognitive decline and it was sharp for one of the siblings. Conclusion The long‐term observations are sparse in PMD and SHANK3 mutations. This is the neurocognitive follow‐up from childhood to middle ages, where a sharp neurocognitive decline was observed. We conclude that progressive neuropsychiatric symptoms in adolescence are a universal clinical clue for PMD diagnosis and an underlying SHANK3 splicing site mutation., Here, we describe two siblings with a novel SHANK3 gene variant of c.2313+1G>A which is inherited from the healthy mosaic father. The results of the siblings point that the variant is pathogenic. The long‐term observations are sparse in PMD and SHANK3 mutations. This is the neurocognitive follow‐up from childhood to middle ages, where a sharp neurocognitive decline was observed.

Synonymous codons occur with different frequencies in different organisms, a phenomenon termed codon usage bias. Codon optimization, a common term for a variety of approaches used widely by the biopharmaceutical industry, involves synonymous substitutions to increase protein expression. It had long been presumed that synonymous variants, which, by definition, do not alter the primary amino acid sequence, have no effect on protein structure and function. However, a critical mass of reports suggests that synonymous codon variations may impact protein conformation. To investigate the impact of synonymous codons usage on protein expression and function, we designed an optimized coagulation factor IX (FIX) variant and used multiple methods to compare its properties to the wild-type FIX upon expression in HEK293T cells. We found that the two variants differ in their conformation, even when controlling for the difference in expression levels. Using ribosome profiling, we identified robust changes in the translational kinetics of the two variants and were able to identify a region in the gene that may have a role in altering the conformation of the protein. Our data have direct implications for codon optimization strategies, for production of recombinant proteins and gene therapies.

The liver is the primary source of a large number of plasma proteins and plays a critical role in multiple biological processes. Inadequate oxygen supply characterizing various clinical settings such as liver transplantation exposes the liver to hypoxic conditions. Studies assessing hypoxia-induced global translational changes in liver are lacking. Here, we employed a recently developed ribosome-profiling technique to assess global translational responses of human primary hepatocytes exposed to acute hypoxic stress (1% O2) for the short term. In parallel, transcriptome profiling was performed to assess mRNA expression changes. We found that translational responses appeared earlier and were predominant over transcriptional responses. A significant decrease in translational efficiency of several ribosome genes indicated translational inhibition of new ribosome protein synthesis in hypoxia. Pathway enrichment analysis highlighted altered translational regulation of MAPK signaling, drug metabolism, oxidative phosphorylation, and nonalcoholic fatty liver disease pathways. Gene Ontology enrichment analysis revealed terms related to translation, metabolism, angiogenesis, apoptosis, and response to stress. Transcriptional induction of genes encoding heat shock proteins was observed within 30 min of hypoxia. Induction of genes encoding stress response mediators, metabolism regulators, and proangiogenic proteins was observed at 240 min. Despite the liver being the primary source of coagulation proteins and the implicated role of hypoxia in thrombosis, limited differences were observed in genes encoding coagulation-associated proteins. Overall, our study demonstrates the predominance of translational regulation over transcription and highlights differentially regulated pathways or biological processes in short-term hypoxic stress responses of human primary hepatocytes.NEW & NOTEWORTHY The novelty of this study lies in applying parallel ribosome- and transcriptome-profiling analyses to human primary hepatocytes in hypoxia. To our knowledge, this is the first study to assess global translational responses using ribosome profiling in hypoxic hepatocytes. Our results demonstrate the predominance of translational responses over transcriptional responses in early hepatic hypoxic stress responses. Furthermore, our study reveals multiple pathways and specific genes showing altered regulation in hypoxic hepatocytes.

Recombinant therapeutic proteins are a broad class of biological products used to replace dysfunctional human proteins in individuals with genetic defects (e.g., factor VIII for hemophilia) or, in the case of monoclonal antibodies, bind to disease targets involved in cancers, autoimmune disorders, or other conditions. Unfortunately, immunogenicity (immune response to the drug) remains a key impediment, potentially affecting the safety and efficacy of these therapeutics. Immunogenicity risk is routinely evaluated during the licensure of therapeutic proteins. However, despite eliciting anti-drug immune responses in at least some patients, most protein drugs are nevertheless licensed as they address unmet medical needs. The pre-licensure immunogenicity assessments of therapeutic proteins are the subject of numerous reviews and white papers. However, observation and clinical management of the immunogenicity of approved therapeutic proteins face additional challenges. We survey the immunogenicity of approved therapeutic proteins, discuss the clinical management of immunogenicity, and identify the challenges to establishing clinically relevant immunogenicity assays for use in routine clinical practice.

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

Hemophilia A (HA) is associated with defects in the F8 gene, encoding coagulation factor VIII (FVIII). Our previous studies show that F8-targeting micro RNAs (miRNAs), a group of small RNAs involved in gene regulation, can downregulate F8 expression causing HA in individuals with normal F8-genotypes and increased HA severity in patients with mutations in F8. Understanding the mechanistic underpinnings of human genetic diseases caused or modulated by miRNAs require a small animal model, such as a mouse model. Here, we report a foundational study to develop such a model system. We identified the mouse 3&prime, untranslated region (3&prime, UTR) on murine F8-mRNA (muF8-mRNA) that can bind to murine miRNAs. We then selected three miRNAs for evaluation: miR-208a, miR-351 and miR-125a. We first demonstrate that these three miRNAs directly target the 3&prime, UTR of muF8-mRNA and reduce the expression of a reporter gene (luciferase) mRNA fused to the muF8-3&prime, UTR in mammalian cells. Furthermore, in mouse cells that endogenously express the F8 gene and produce FVIII protein, the ectopic expression of these miRNAs downregulated F8-mRNA and FVIII protein. These results provide proof-of-concept and reagents as a foundation for using a normal F8-containing mouse as a model for the miRNA regulation of normal F8 in causing or aggravating the genetic disease HA.

MicroRNAs (miRNA) play an important role in gene expression at the posttranscriptional level by targeting the untranslated regions of messenger RNA (mRNAs). These small RNAs have been shown to control cellular physiological processes including cell differentiation and proliferation. Dysregulation of miRNAs have been associated with numerous diseases. In the past few years miRNAs have emerged as potential biopharmaceuticals and the first miRNA-based therapies have entered clinical trials. Our recent studies suggest that miRNAs may also play an important role in the pathology of genetic diseases that are currently considered to be solely due to mutations in the coding sequence. For instance, among hemophilia A patients there exist a small subset, with normal wildtype genes; i.e., lacking in mutations in the coding and non-coding regions of the F8 gene. Similarly, in many patients with missense mutations in the F8 gene, the genetic defect does not fully explain the severity of the disease. Dysregulation of miRNAs that target mRNAs encoding coagulation factors have been shown to disturb gene expression. Alterations in protein levels involved in the coagulation cascade mediated by miRNAs could lead to bleeding disorders or thrombosis. This review summarizes current knowledge on the role of miRNAs in hemophilia and thrombosis. Recognizing and understanding the functions of miRNAs by identifying their targets is important in identifying their roles in health and diseases. Successful basic research may result in the development and improvement of tools for diagnosis, risk evaluation or even new treatment strategies.

Immune responses to therapeutic proteins and peptides can adversely affect their safety and efficacy; consequently, immunogenicity risk-assessments are part of the development, licensure and clinical use of these products. In most cases the development of anti-drug antibodies is mediated by T cells which requires antigen presentation by Major Histocompatibility Complex Class II (MHCII) molecules (also called Human Leucocyte Antigen, HLA in humans). Immune responses to many protein therapeutics are thus HLA-restricted and it is important that the distribution of HLA variants used in the immunogenicity assessments provides adequate coverage of the target population. Due to biases inherent to the collection of samples in a blood bank or donor pool, simple random sampling will not achieve a truly representative sample of the population of interest. To help select a donor cohort we introduce SampPick, an implementation of simulated annealing which optimizes cohort selection to closely match the frequency distribution of a target population or subpopulation. With inputs of a target background frequency distribution for a population and a set of available, HLA-typed donors, the algorithm will iteratively create a cohort of donors of a user selected size that will closely match the target population rather than a random sample. In addition to optimizing the HLA types of donor cohorts, the software presented can be used to optimize donor cohorts for any other biallelic or monoallelic trait.

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.

Background: Williams syndrome (WBS) is a genetic multisystem disorder. The main symptom is borderline (intelligence quotient, IQ 70–79) or abnormally low intelligence (IQ < 70). According to earlier studies young individuals with WBS demonstrate generally a slightly higher verbal IQ (VIQ) compared to performance/nonverbal IQ (PIQ). WBS was recognized as a distinct entity already about 60 years ago, but still cognition in adults with WBS is poorly known. Methods: We followed 25 adults (age at baseline 19–68, median 38) with genetically confirmed WBS for about 20 years. The study subjects underwent medical and neuropsychological assessments at the baseline and at the end of follow‐up. Results: The mean VIQ remained quite stable from early adulthood up to 40 years of age after which it declined. The mean PIQ kept on improving from early adulthood until 50 years of age after which it gradually declined. At the end of the study, all study subjects had at least two longstanding health problems out of which hypertension, psychiatric disorder, and scoliosis or kyphosis occurred most frequently. At end of the study, two patients suffered from vascular dementia. Seven patients died during the follow‐up. Conclusions: In adults with WBS, the course of cognition is uneven across the cognitive profile. Their verbal functions both develop and deteriorate earlier than performance/nonverbal functions. Frequent somatic co‐morbidities may increase risk to shortened life span.

Background Hemophilia A (HA) is associated with mutations in the F8 gene that expresses factor VIII (FVIII). Unexpectedly, HA also manifests in a small subset of individuals with no mutations (exonic or intronic) in their F8 gene. MicroRNAs (miRNAs) cause translational interference, affecting protein quality and stoichiometry. Here, by analyzing miRNAs of two patients from this subset, we evaluated miRNA-based FVIII suppression as a testable hypothesis to explain FVIII deficiency in patients with HA with no F8 gene mutations. Study design and methods To test the hypothesis, miRNA sequencing from two patients with mild and moderate HA with no mutations in their F8 gene, followed by experimental verification, was used to identify a group of upregulated miRNAs in patients with HA compared to normal controls; with binding sites in the 3' untranslated region (UTR) of F8 messenger RNA (mRNA), a prerequisite for miRNA-based gene regulation. From this pool, miR-374b-5p and miR-30c-5p, known to be expressed in human liver, where FVIII is expressed, were subjected to extensive characterization. Results In two cell lines that constitutively express FVIII, we demonstrated that overexpression of miR-374b or miR-30c decreased FVIII expression, while an miR-30c inhibitor partially restored FVIII expression. Conclusion These data support a role for microRNAs in fine-tuning F8 gene regulation. Based on our findings, our current model suggests that in HA cases where the F8 gene is normal and is predicted to express normal levels of FVIII, F8 mRNA 3' UTR targeting miRNAs may be responsible for a FVIII-deficiency phenotype clinically manifesting as HA.

Vatreptacog alfa (VA), a recombinant activated human factor VII (rFVIIa) variant with 3 amino acid substitutions, was developed to provide increased procoagulant activity in hemophilia patients with inhibitors to factor VIII or factor IX. In phase 3 clinical trials, changes introduced during the bioengineering of VA resulted in the development of undesired anti-drug antibodies in some patients, leading to the termination of a potentially promising therapeutic protein product. Here, we use preclinical biomarkers associated with clinical immunogenicity to validate our deimmunization strategy applied to this bioengineered rFVIIa analog. The reengineered rFVIIa analog variants retained increased intrinsic thrombin generation activity but did not elicit T-cell responses in peripheral blood mononuclear cells isolated from 50 HLA typed subjects representing the human population. Our algorithm, rational immunogenicity determination, offers a broadly applicable deimmunizing strategy for bioengineered proteins.

Fusing the human immunoglobulin G1 (IgG1) constant region (Fc-domain) to therapeutic proteins or peptides increases their circulating plasma half-life via neonatal Fc receptor (FcRn) binding and recycling. However, Fc-mediated interactions with other molecules including complement C1q and Fc gamma receptors (FcγRs) can have immunological consequences and the potential to modulate the immunogenicity of Fc-fusion therapeutics. In a comparative study, we carried out a comprehensive assessment of Fc-mediated interactions for five FDA-approved Fc-fusion therapeutics. C1q binding and complement activation were measured by ELISA, while FcγR binding and signaling were evaluated using BW5147:FcγR-ζ reporter cell lines. We demonstrate that FIX-Fc and FVIII-Fc bound C1q as well as activating and inhibitory FcγRs (I, IIA, IIB, IIIA). These coagulation factor Fc-fusions also signaled via FcγRIIIA, and to a lesser extent via FcγRI and FcγRIIB. TNFR-Fc and CTLA4-Fc bound FcγRI, while TNFR-Fc also bound FcγRIIIA, but these interactions did not result in FcγR signaling. Our comprehensive assessment demonstrates that (i) different Fc-fusion drugs have distinct C1q/FcγR binding and signaling properties, (ii) FcγR binding does not predict signaling, and (iii) the fusion partner (effector molecule) can influence Fc-mediated interactions.

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.

Background Intellectual disability (ID) and dementia reflect disturbed cortical function during and after developmental age, respectively. Due to the wide heterogeneity of ID population the decline in cognitive and adaptive skills may be different in distinct genetic subgroups. Methods Using the British Present Psychiatric State–learning Disabilities assessment (PPS‐LD) questionnaire the dementia signs were screened in 62, 22 and 44 individuals (> 35 year of age) with Down (DS, OMIM number 190685), Williams (WS, OMIM number, 194050), and Fragile X syndrome (FXS, OMIM number 309550), respectively. The median age of those with FXS (59 years) was higher than of those with DS (50 years) and WS (53 years). Results Most study participants with DS (80%) and FXS (89%) were or had been moderately or severely intellectually disabled while most participants with WS (73%) were or had been mildly or moderately disabled at adolescent age. The adolescent (premorbid) level of ID did not correlate with the dementia score. The median scores were 11/27, 1/27, and 0/27 in DS, WS, and FXS subgroups, respectively. Dementia that was confirmed by brain imaging, manifested as Alzheimer disease and as moya‐moya disease associated vascular dementia in DS and as vascular dementia in WS. Conclusions This survey suggests that the risk of dementia varies depending on the cause of ID and that the severity of ID in adolescence does not predict the development of dementia at a later age. Consequently, the ID and dementia should be understood as separate clinical entities that need to be taken into account in the health management of intellectually disabled people. This is important for the arrangement of appropriate and timely interventions, which can be expected to delay the need for institutionalization.

Objective Here we provide a critical review of the state of the art with respect to non-clinical assessments of immunogenicity for therapeutic proteins. Key findings The number of studies on immunogenicity published annually has more than doubled in the last 5 years. The science and technology, which have reached a critical mass, provide multiple of non-clinical approaches (computational, in vitro, ex vivo and animal models) to first predict and then to modify or eliminate T-cell or B-cell epitopes via de-immunization strategies. We discuss how these may be used in the context of drug development in assigning the immunogenicity risk of new and marketed therapeutic proteins. Summary Protein therapeutics represents a large share of the pharma market and provide medical interventions for some of the most complex and intractable diseases. Immunogenicity (the development of antibodies to therapeutic proteins) is an important concern for both the safety and efficacy of protein therapeutics as immune responses may neutralize the activity of life-saving and highly effective protein therapeutics and induce hypersensitivity responses including anaphylaxis. The non-clinical computational tools and experimental technologies that offer a comprehensive and increasingly accurate estimation of immunogenic potential are surveyed here. This critical review also discusses technologies which are promising but are not as yet ready for routine use.

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.

Synonymous codon changes, which do not alter protein sequence, were previously thought to have no functional consequence. Although this concept has been overturned in recent years, there is no unique mechanism by which these changes exert biological effects. A large repertoire of both experimental and bioinformatic methods has been developed to understand the effects of synonymous variants. Results from this body of work have provided global insights into how biological systems exploit the degeneracy of the genetic code to control gene expression, protein folding efficiency, and the coordinated expression of functionally related gene families. Although it is now clear that synonymous variants are important in a variety of contexts, from human disease to the safety and efficacy of therapeutic proteins, there is no clear consensus on the approaches to identify and validate these changes. Here, we review the diverse methods to understand the effects of synonymous mutations.

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.

Haemophilia B is an X-linked recessive disorder caused by deficiency of functional coagulation factor IX, which results almost exclusively from mutations in the F9 gene. We sought to determine features, which could distinguish between mutations that cause severe disease symptoms from those that cause non-severe disease symptoms. Towards this objective, we have performed a statistical analysis of reported point mutations in F9. These include: potential local changes in mRNA free energy, codon usage, charge and type of mutated amino acid, location of the mutation with regard to protein secondary structure and functional domain and amino acids' evolutionary conservation scores. Wilcoxon signed-rank tests showed highly significant differences between severe and non-severe disease causing mutations in their effect on free energy of small mRNA fragments and evolutionarily conserved amino acids. Our results suggest that information at the mRNA level as well as conservation of the amino acid correlate well with disease severity. This study demonstrates that computational tools may be used to characterize the severity of haemophilia B associated with point mutations and suggests their utility in predicting the outcome of sequence changes in recombinant proteins.

The infusion of plasma-derived or recombinant factors to treat bleeding disorders such as hemophila A and B is a success story in the management of a chronic disease. The effectiveness of this approach is however limited by challenges with adverse effects of treatment. The most notable of these are the development of inhibitory antibodies that target the protein therapeutic. The current standard of care for management of hemophiliacs is prophylactic treatment that includes frequent infusions of a Factor VIII product. Failure to comply with the prophylactic regimen is a major hurdle in the management of these patients. We discuss here more recent findings that argue for a pharmacogenetic approach to understanding (and eventually circumventing) immunogenicity. We also review strategies used to bioengineer coagulation factors to extend the half-lives of coagulation proteins. The rapid progress in the last few years to bioengineer coagulation factors in different ways to attain this goal is described. Finally, novel technologies and potential products are emerging that utilize synthetic molecules in lieu of replacement proteins obviating the limitations associated with replacement therapies.

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.

How can we understand a case in which a given amino acid sequence folds into structurally and functionally distinct molecules? Synonymous single-nucleotide polymorphisms in the MDR1 (multidrug resistance 1 or ABCB1) gene involving frequent-to-rare codon substitutions lead to identical protein sequences. Remarkably, these alternative sequences give a protein product with similar but different structures and functions. Here, we propose that long-enough ribosomal pause time scales may lead to alternate folding pathways and distinct minima on the folding free energy surface. While the conformational and functional differences between the native and alternate states may be minor, the MDR1 case illustrates that the barriers may nevertheless constitute sufficiently high hurdles in physiological time scales, leading to kinetically trapped states with altered structures and functions. Different folding pathways leading to conformationally similar trapped states may be due to swapping of (fairly symmetric) segments. Domain swapping is more likely in the no-pause case in which the chain elongates and folds simultaneously; on the other hand, sufficiently long pause times between such segments may be expected to lessen the chances of swapping events. Here, we review the literature in this light.

Down syndrome (DS) is characterized by intellectual disability and development of dementia that are attributed to similar neuropathological features as observed in Alzheimer's disease (AD). The aim of this study was to investigate whether DS patients have similar impairment of preattentive auditory processing as observed in AD. Sinusoidal tones were presented to DS patients and healthy controls, and evoked auditory evoked fields (AEF) were measured with a whole-head magnetoencephalography (MEG) system. Patients with DS had significantly delayed and attenuated N100m, and delayed but not attenuated P50m responses over both hemispheres. Present results indicate that preattentive auditory processing underlying stimulus detection is impaired in DS. Given that anticholinergic drugs modulate AEFs, degeneration of cholinergic system in DS could contribute to the damaged auditory processing.

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]

The transport cycle of ABC transporters in general and P-glycoprotein in particular has been extensively studied, but the molecular mechanism remains controversial. We identify stable reaction intermediates in the progression of the P-glycoprotein-mediated ATPase reaction equivalent to the enzyme-substrate (E.S, P-glycoprotein.ATP) and enzyme-product (E.P, P-glycoprotein.ADP.P(i)) reaction intermediates. These have been characterized using the photoaffinity analog 8-azido-[alpha-32P]ATP as well as under equilibrium conditions using [alpha-32P]ATP, in which a cross-linking step is not involved. Similar results were obtained when 8-azido-[alpha-32P]ATP or [alpha-32P]ATP was used. The reaction intermediates were characterized based on their kinetic properties and the nature (triphosphate/diphosphate) of the trapped nucleotide. Using this defined framework and the Walker B E556Q/E1201Q mutant that traps nucleotide in the absence of vanadate or beryllium fluoride, the high to low affinity switch in the transport substrate binding site can be attributed to the formation of the E.S reaction intermediate of the ATPase reaction. Importantly, the posthydrolysis E.P state continues to have low affinity for substrate, suggesting that conformational changes that form the E.S complex are coupled to the conformational change at the transport substrate site to do mechanical work. Thus, the formation of E.S reaction intermediate during a single turnover of the catalytic cycle appears to provide the initial power stroke for movement of drug substrate from inner leaflet to outer leaflet of lipid bilayer. This novel approach applies transition state theory to elucidate the mechanism of P-glycoprotein and other ABC transporters and has wider applications in testing cause-effect hypotheses in coupled systems.

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.

The human MDR1 (ABCB1) gene product, P-glycoprotein (Pgp), functions as an ATP-dependent efflux pump for a variety of chemotherapeutic drugs. In this study, we assessed the role of conserved glutamate residues in the Walker B domain of the two ATP sites (E556 and E1201, respectively) during the catalytic cycle of human Pgp. The mutant Pgps (E556Q, E556A, E1201Q, E1201A, E556/1201Q, and E556/1201A) were characterized using a vaccinia virus based expression system. Although steady-state ATP hydrolysis and drug transport activities were abrogated in both E556Q and E1201Q mutant Pgps, [alpha-(32)P]-8-azidoADP was trapped in the presence of vanadate (Vi), and the release of trapped [alpha-(32)P]-8-azidoADP occurred to a similar extent as in wild-type Pgp. This indicates that these mutations do not affect either the first hydrolysis event or the ADP release step. Similar results were also obtained when Glu residues were replaced with Ala (E556A and E1201A). Following the first hydrolysis event and release of [alpha-(32)P]-8-azidoADP, both E556Q and E1201Q mutant Pgps failed to undergo another cycle of Vi-induced [alpha-(32)P]-8-azidoADP trapping. Interestingly, the double mutants E556/1201Q and E556/1201A trapped [alpha-(32)P]-8-azidoADP even in the absence of Vi, and the occluded nucleotide was not released after incubation at 37 degrees C for an extended period. In addition, the properties of transition state conformation of the double mutants generated in the absence of Vi were found to be similar to that of the wild-type protein trapped in the presence of Vi (Pgp x [alpha-(32)P]-8-azidoADP xVi). Thus, in contrast to the single mutants, the double mutants appear to be defective in the ADP release step. In aggregate, these data suggest that E556 and E1201 residues in the Walker B domains may not be critical as catalytic carboxylates for the cleavage of the bond between the gamma-P and the beta-P of ATP during hydrolysis but are essential for the second ATP hydrolysis step and completion of the catalytic cycle.

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.

Hemophilia A (HA) is a bleeding disorder caused by deficiency of functional plasma clotting factor VIII (FVIII). Genetic mutations in the gene encoding FVIII (F8) have been extensively studied. Over a thousand different mutations have been reported in the F8 gene. These span a diverse range of mutation types, namely, missense, splice-site, deletions of single and multiple exons, inversions, etc. There is nonetheless evidence that other molecular mechanisms, in addition to mutations in the gene encoding the FVIII protein, may be involved in the pathobiology of HA. In this study, global small ncRNA expression profiling analysis of whole blood from HA patients, and controls, was performed using high-throughput ncRNA microarrays. Patients were further sub-divided into those that developed neutralizing-anti-FVIII antibodies (inhibitors) and those that did not. Selected differentially expressed ncRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. We identified several ncRNAs, and among them hsa-miR-1246 was significantly up-regulated in HA patients. In addition, miR-1246 showed a six-fold higher expression in HA patients without inhibitors. We have identified an miR-1246 target site in the noncoding region of F8 mRNA and were able to confirm the suppressory role of hsa-miR-1246 on F8 expression in a stable lymphoblastoid cell line expressing FVIII. These findings suggest several testable hypotheses vis-a-vis the role of nc-RNAs in the regulation of F8 expression. These hypotheses have not been exhaustively tested in this study as they require carefully curated clinical samples.

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.

Summary Inhibitors are an impediment to the effective management of haemophilia B (HB), but there is limited understanding of the underlying genetic risk factors. In this study we aim to understand the role of F9 gene mutations on inhibitor development in patients with HB. Mutations in the F9 gene were identified and HLA typing performed for five boys with severe HB. Data from the CDC Haemophilia B Mutation Project (CHBMP) database were used to assess association between F9 gene mutation type and inhibitor development. Analysis of the CHBMP database showed that larger disruptions in the F9 gene are associated with a higher life-time prevalence of inhibitors. We detected the following mutations in the five subjects, including four novel mutations: Nonsense in three patients (c.223 C>T; p.Arg75* in two siblings, c.553 C>T; p.Glu185*); Splice site in two patients (c.723 + 1 G>A, c.278-27 A>G); Missense in one patient (c.580 A>G, p.Thr194Ala; c.723 G>T; p.Gln241His). Of the two siblings only one responded to immune tolerance induction (ITI). These siblings have identical F9 gene mutations but differ with respect to the HLA alleles. Interestingly, an analysis of peptide-MHC binding affinities shows a significantly higher (one-sided unpaired t-test, P = 0.0018) median affinity for FIX-derived peptides in the sibling that responded to ITI. We conclude that the nature of the F9 gene mutation may be an important risk factor for the development of inhibitors. In addition, the HLA alleles of the individual patients, in conjunction with the mutation type, could be a predictor for the development of inhibitors as well as the response to ITI.

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.

A benzophenone photoaffinity label 9 based on the polyene natural product (-)-stipiamide has been constructed using a diaminoethane spacer and the radioactive agent [3H]-BZDC (N-succinimidyl p-benzoyl-(2,3-3H)-dehydrocinnamate). Photoaffinity experiments show specific binding to human P-glycoprotein (Pgp) in the presence of cis-flupentixol but not with cyclosporin A.

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.

Most native proteins do not make optimal drugs and thus a second- and third-generation of therapeutic proteins, which have been engineered to improve product attributes or to enhance process characteristics, are rapidly becoming the norm. There has been unprecedented progress, during the past decade, in the development of platform technologies that further these ends. Although the advantages of engineered therapeutic proteins are considerable, the alterations can affect the safety and efficacy of the drugs. We discuss both the key technological innovations with respect to engineered therapeutic proteins and advancements in the underlying basic science. The latter would permit the design of science-based criteria for the prediction and assessment of potential risks and the development of appropriate risk management plans. This in turn holds promise for more predictable criteria for the licensure of a class of products that are extremely challenging to develop but represent an increasingly important component of modern medical practice.

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.

Author(s): Sauna, ZE; Ameri, A; Kim, B; Yanover, C; Viel, KR; Rajalingam, R; Cole, SA; Howard, TE