Tom E. Howard

The critical role of tissue angiotensin-converting enzyme as revealed by gene targeting in mice.

Angiotensin-converting enzyme (ACE) generates the vasoconstrictor angiotensin II, which plays a critical role in maintenance of blood pressure in mammals. Although significant ACE activity is found in plasma, the majority of the enzyme is bound to tissues such as the vascular endothelium. We used targeted homologous recombination to create mice expressing a form of ACE that lacks the COOH-terminal half of the molecule. This modified ACE protein is catalytically active but entirely secreted from cells. Mice that express only this modified ACE have significant plasma ACE activity but no tissue-bound enzyme. These animals have low blood pressure, renal vascular thickening, and a urine concentrating defect. The phenotype is very similar to that of completely ACE-deficient mice previously reported, except that the renal pathology is less severe. These studies strongly support the concept that the tissue-bound ACE is essential to the control of blood pressure and the structure and function of the kidney.

Sperm-specific expression of angiotensin-converting enzyme (ACE) is mediated by a 91-base-pair promoter containing a CRE-like element.

The gene encoding the testis isozyme of angiotensin-converting enzyme (testis ACE) is one example of the many genes expressed uniquely during spermatogenesis. This protein is expressed by developing germ cells late in their development and results from the activation of a sperm-specific promoter that is located within intron 12 of the gene encoding the somatic isozyme of ACE. In vitro transcription, DNase footprinting, gel shift assays, and transgenic mouse studies have been used to define the minimal testes ACE promoter and to characterize DNA-protein interactions mediating germ cell-specific expression. These studies show that proper cell- and stage-specific expression of testis ACE requires only a small portion of the immediate upstream sequence extending to -91. A critical motif within this core promoter is a cyclic AMP-responsive element sequence that interacts with a testis-specific transactivating factor. Since this putative cyclic AMP-responsive element has been conserved within the testis ACE promoters of different species and is found at the same site in other genes that are expressed specifically in the testis, it may provide a common mechanism for the recognition of sperm-specific promoters.

Pharmacogenetics and the immunogenicity of protein therapeutics

volume 29 number 10 october 2011 nature biotechnology To the Editor: The development of anti-drug antibodies (ADAs) to therapeutic proteins can lead to adverse events and also make a biologic less effective for its intended use. Immunogenicity assessments are now critical for the development, regulatory licensure and use of biologics and it has been argued that it is highly unlikely that regulatory approval would be granted for a biologic without an assessment of its immunogenicity1. The development of ADAs does not necessarily affect the safety and/or efficacy of a protein therapeutic and thus risk-based approaches are generally advocated to evaluate the clinical consequences2. For example, although therapies that involve replacement of proteins that are lacking or nonfunctional in patients have had spectacular results in the clinical management of many chronic diseases, such therapies are particularly prone to have adverse consequences as a result of so-called neutralizing ADAs3. Several reviews have previously catalogued the product and patient-related risk factors for immunogenicity (refs. 4,5 and references therein and Supplementary Fig. 1). The past decade has seen a steady shift in the use of recombinant human proteins, substantial improvements in quality and less heterogeneity in therapeutic protein products owing to the adoption of strategies such as quality by design6. Even so, improvements in product quality cannot control for the genetic variability of the patient population due to which some individuals, racial and/or ethnic groups or other subpopulations develop inhibitory antibodies at a higher frequency than others. In this article, using the example of factor VIII in the treatment of hemophilia A, we propose that a pharmacogenetic approach may be an important factor in the accurate prediction of immunogenicity of some categories of therapeutic proteins. Recombinant human protein drugs are mostly recognized by the body as ‘self ’; tolerance of recombinant proteins can be affected, however, by two key differences from the corresponding endogenous protein: (i) mutations in the endogenous protein that render it defective and (ii) the occurrence of nonsynonymous (ns) single-nucleotide polymorphisms (SNPs). Genotyping the endogenous (albeit nonfunctional) protein in a patient can identify regions of the infused protein that are ‘foreign’ to that individual (Fig. 1 and Supplementary Fig. 2a). Thus, in the case of factor VIII, consistent with the precept that synthesis of the factor VIII polypeptide chain is necessary for inducing central tolerance, the nature of the mutation in the patient’s factor VIII gene (F8) should be a good predictor of the frequency of factor VIII inhibitor formation. Hemophilia A patients with missense mutations in F8 develop inhibitors with a frequency of ~5%, whereas the rate of inhibitor development for patients with large gene deletions has been reported to be as high as 88%7. Nonetheless, even within each class of patients, there is variability in the immune response, that is, some patients with a missense mutation develop inhibitory antibodies, whereas a fraction of patients with large deletions do not develop inhibitors. SNPs are the most common source of genetic variation in the human population8 and a recent report has investigated the effects of ns-SNPs on factor VIII immunogenicity9. This study demonstrates the presence of several ns-SNPs in F8 that result in primary amino acid sequence mismatches between the infused factor VIII and the endogenous factor VIII protein of some patients with hemophilia A. Large differences in the frequency of inhibitor development between patients of white-European and black-African descent may be traced to distinct populationspecific distributions of these ns-SNPs10. Concomitantly, there is both indirect and direct evidence that the CD4+ T-cell response is essential for the development of inhibitory antibodies, and recent studies have been successful in identifying T-cell epitopes on the factor VIII protein11,12. On the basis of the above findings, we suggest that a sequence mismatch between the endogenous (tolerizing) peptides and those derived from the infused protein-drug may be exploited as a basis for understanding the pharmacogenetics of immunogenicity. Additionally, a critical determinant for T cell–dependent alloimmunization in an infused protein is the strength at which any foreign (non-self) peptide(s) derived from it (potential T-cell epitopes) binds to one or more of the distinct major histocompatibility complex (MHC)-II molecules on the surface of an individual patient’s antigen-presenting cells13. MHC-II proteins are extremely polymorphic and their distributions also exhibit clear racial and ethnic differences14 (Supplementary Fig. 1c). Thus, even identical non-self peptides will interact differently with the MHC-II repertoire of different patients, for example, binding with high affinity to an MHC-II protein in one individual while not binding at all in another. Several studies have endeavored to associate the nature and location of hemophilia A– causing mutations to immunogenicity, and whether particular human leukocyte antigen (HLA) alleles occur more frequently in individuals who develop inhibitory ADAs. In the past, however, these parameters have been considered independently. Here, we propose that all three parameters, mutations (as well as SNPs) in factor VIII, HLA type and sequence of factor VIII infusions be determined in individual patients. The decision making is hierarchical (Fig. 1) based, first, on determining the regions of sequence mismatch between the endogenous and infused proteins; next, on whether the peptides that represent the sequence variation bind to that patient’s MHC class II proteins (http://tools.immuneepitope.org/analyze/ html/mhc_II_binding.html); and, finally, whether the peptide-MHC interaction elicits T-cell responses. Such a strategy unfortunately cannot be fully validated using clinical data that are currently available. For example, the Hemophilia A Mutation Database (Hemophilia ADB15; http://hadb. org.uk), which is the most comprehensive database of F8 mutations and repository Pharmacogenetics and the immunogenicity of protein therapeutics CHMp/EWp/433478/2010. <http://www.ema.europa. eu/ema/index.jsp?curl=search.jsp&q=22+July+2010. +EMA%2Fchmp%2Fewp%2F433478%2F2010&mu rl=menus%2Fregulations%2Fregulations.jsp&mid=> 12. Finke, L.H. et al. Vaccine 25, B97–B109 (2007). 13. Hoos, A. et al. Semin. Oncol. 37, 533–546 (2010). 14. Marshall, M., Ribas, A. & Huang, B. Evaluation of baseline serum C-reactive protein and benefit from tremelimumab compared to chemotherapy in firstline melanoma. Abstract no. 2609, presented at the 2010 annual meeting of the American Society Clinical Oncology, Chicago, IL, June 4–8, 2010. 15. Robert, C. et al. N. Engl. J. Med. 364, 2517–2526 (2011). 16. Taylor, C.F. et al. Nat. Biotechnol. 26, 889–896 (2008). 17. Finn, O.J. N. Engl. J. Med. 358, 2704–2715 (2008). 18. Schreiber, R., Old, L.J. & Smyth, M.J. Science 331, 1565–1570 (2011). 19. Zitvogel, L., Kepp, O. & Kroemer, G. Nat. Rev. Clin. Oncol. 8, 151–160 (2011). CORRESpONdENCE

Polymorphisms in the F8 Gene and MHC-II Variants as Risk Factors for the Development of Inhibitory Anti-Factor VIII Antibodies during the Treatment of Hemophilia A: A Computational Assessment

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.

Genetic determinants of normal variation in coagulation factor (F) IX levels: Genome-wide scan and examination of the FIX structural gene

Summary.  Background: High‐normal and elevated plasma FIX activity (FIX:C) levels are associated with increased risk for venous‐ and possibly arterial‐thrombosis. Objective: Because the broad normal range for FIX:C involves a substantial unknown genetic component, we sought to identify quantitative‐trait loci (QTLs) for this medically important hemostasis trait. Methods: We performed a genome‐wide screen and a resequencing‐based variation scan of the known functional regions of every distinct FIX gene (F9) in the genetic analysis of idiopathic thrombophilia project (GAIT), a collection of 398 Spanish‐Caucasians from 21 pedigrees. Results: We found no evidence for linkage (LOD scores <1.5) despite genotyping more than 540 uniformly‐spaced microsatellites. We identified 27 candidate F9 polymorphisms, including three in cis‐elements responsible for the increase in FIX:C that occurs with aging, but found no significant genotype‐specific differences in mean FIX:C levels (P‐values ≥ 0.11) despite evaluating every polymorphism in GAIT by marginal multicovariate measured‐genotype association analysis. Conclusions: The heritable component of interindividual FIX:C variability likely involves a collection of QTLs with modest effects that may reside in genes other than F9. Nevertheless, because the alleles of these 27 polymorphisms exhibited a low overall degree of linkage disequilibrium, we are currently defining their haplotypes to interrogate several highly‐conserved non‐exonic sequences and other F9 segments not examined here.

The 57 kb deletion in cystinosis patients extends into TRPV1 causing dysregulation of transcription in peripheral blood mononuclear cells

Background Cystinosis is an autosomal recessive disease characterised by the abnormal accumulation of lysosomal cystine. Mutations in the cystinosin gene (CTNS) represent known causes for the disease. The major cystinosis mutation is a 57 kb deletion on human chromosome 17p13 that removes the majority of CTNS and the entire adjacent gene, CARKL/SHPK. Objectives In order to identify other genes that may influence the cystinosis pathobiological pathway, peripheral blood mononuclear cells (PBMC) were collected from cystinosis family members, and DNA and RNA extracted. Results Using whole genome transcriptional profiling, transient receptor potential vanilloid 1 (TRPV1) was found to be differentially expressed in association with cystinosis. This was verified using TaqMan qRT-PCR. There was a 72% reduction in PBMC TRPV1 mRNA levels in cystinosis individuals homozygous for the 57 kb deletion (n=6) compared to unaffected individuals without the deletion (n=6) (p=0.002). TRPV1 is a sensory receptor located on chromosome 17p13, adjacent to CARKL/SHPK. It was ascertained that the 57 kb deletion extends from exon 10 of CTNS, upstream through CARKL/SHPK, to intron 2 of TRPV1, thus deleting the first two non-coding exons. Conclusion This is the first study to report that the 57 kb deletion extends into the TRPV1 gene causing dysregulation of transcription in PBMC isolated from cystinosis patients.

Haemophilia management: time to get personal?

Summary.  The possibility of alloimmunization in patients receiving protein replacement therapy depends on (at least) three risk factors, which are necessary concomitantly but insufficient alone. The first is the degree of structural difference between the therapeutic protein and the patient’s own endogenous protein, if expressed. Such differences depend on the nature of the disease mutation and the pre‐mutation endogenous protein structure as well as on post‐translational changes and sequence‐engineered alterations in the therapeutic protein. Genetic variations in the recipients’ immune systems comprise the second set of risk determinants for deleterious immune responses. For example, the limited repertoire of MHC class II isomers encoded by a given person’s collection of HLA genes may or may not be able to present a ‘foreign’ peptide(s) produced from the therapeutic protein – following its internalization and proteolytic processing – on the surface of their antigen‐presenting cells (APCs). The third (and least characterized) variable is the presence or absence of immunologic ‘danger signals’ during the display of foreign‐peptide/MHC‐complexes on APCs. A choice between existing therapeutic products or the manufacture of new proteins, which may be less immunogenic in some patients or patient populations, may require prior definition of the first two of these variables. This leads then to the possibility of developing personalized therapies for disorders due to genetic deficiencies in endogenous proteins, such as haemophilia A and B. [Correction made after online publication 11 July 2011: several critical corrections have been made to the abstract].

Observations regarding the immunogenicity of BDD-rFVIII derived from a mechanistic personalized medicine perspective

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

Detection of intracellular Factor VIII protein in peripheral blood mononuclear cells by flow cytometry.

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 methods 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.

Single‐nucleotide variations defining previously unreported ADAMTS13 haplotypes are associated with differential expression and activity of the VWF‐cleaving protease in a Salvadoran congenital thrombotic thrombocytopenic purpura family

Although autosomal recessive haematological disorders, such as congenital thrombotic thrombocytopenic purpura (cTTP), are individually rare and difficult to ascertain, studies involving one or more homozygous affected children and their unaffected heterozygous parents have led to expanded understanding of known and discovery of previously unknown molecular-genetic characteristics. We present an in-depth examination of ADAMTS13 haplotypes, mRNA levels and protein expression, activity, and enzyme kinetics in this case study of two Salvadoran children with cTTP – the first reported cases of this disease in individuals originally from Central America – and their parents. The propositi – a son and a daughter born to asymptomatic, non-consanguineous (but possibly distantly related) parents from the same town in El Salvador – were diagnosed with cTTP at ages 6 and 2 years, respectively. Although they developed haemolytic anaemia and thrombocytopenia 2 d (son) and 17 months (daughter) after birth, their cTTP diagnosis was not rendered until both were hospitalized simultaneously with fever, respiratory symptoms, haemolytic anaemia and thrombocytopenia. Peripheral blood smears for both demonstrated schistocytes, and their von Willebrand factor-cleaving protease (VWF-CPase) activities were <1% without ADAMTS13 IgG antibodies. Fresh frozen plasma (FFP) infusion induced rapid (within 48 h) normalization of their blood counts and resolution of the microangiopathic changes. Both children have been treated with prophylactic infusions of FFP (10 ml/kg every 2 5 weeks) without long-term neurological or renal sequelae. Following approval by institutional review boards and receipt of informed consent, blood samples were collected from the children, prior to FFP administration, and from both parents. Genomic DNA and total RNA were extracted from peripheral blood leukocytes using QIAamp DNA Blood Maxi and PAXgene Blood RNA kits, respectively (Qiagen; Germantown, MD, USA). All ADAMTS13 exons, at least 50 base pairs (bp) of each flanking intron junction, ~500 bp 5’ of the promoter, and ~200 bp of 3’-flanking genomic DNA were amplified using polymerase chain reaction (PCR), cleaned using Exo-SAP-IT (Affymetrix; Santa Clara, CA, USA), and subjected to direct bi-directional Sanger sequencing followed by capillary electrophoresis and analysis on ABI-3730 instruments. Reverse transcription was performed (Shomron et al, 2010), and plasma VWF-CPase antigen was measured using the Technozym ADAMTS13 enzyme-linked immunosorbent assay (Technoclone; Vienna, Austria) and the Victor X3 multilabel plate reader (PerkinElmer; Waltham, MA, USA). Fluorogenic FRETS-VWF73 (Peptides International; Louisville, KY, USA) was prepared and assayed (Sauna et al, 2009). The kinetic characteristics of ADAMTS13 were obtained using GraphPad Prism software. ADAMTS13 sequencing revealed both children to be homozygous and their parents to be heterozygous for the previously described, cTTP-causing, single-base-substitution mutation 20506C > T (Table IA) (Levy et al, 2001; Snider et al, 2004; Hing et al, 2013). The children’s VWF-CPase antigen and activity levels were undetectable, although steady-state levels of the ADAMTS13 mRNA were >2 5-fold higher in the daughter than in the son. The re-sequenced regions of the ADAMTS13 loci segregating within this family contained 26 additional single-nucleotide variations (SNVs), including two previously unreported non-synonymous (ns)-SNVs: 27852C > T (c.3362C > T ⇒ 972Arg > Trp) and 33325G > A (c.3733G > A ⇒ 1096Arg > His) (Table IA). The parents’ genotypes differed at nine positions, including three ns-SNVs, creating two distinct, non-mutant haplotypes (designated I and III) at the gene, mRNA, and protein levels (Table IA). The quantitative realtime-PCR assay revealed >4-fold higher steady-state mRNA levels in the father compared to the mother (P < 0 001; Table IB). Plasma VWF-CPase activity and antigen levels were ~2-fold greater in the father than in the mother (P = 0 00164 and P = 0 0633, respectively), but the specific activities of these structurally distinct VWF-CPase proteins were notably almost identical (253 5 vs. 256 2 U/lg). Initial velocity kinetic analysis using the Michelis-Menten equation demonstrated that the Vmax of the father’s VWFCPase was twice that of the mother’s (1 4 vs. 0 7; P < 0 0001) while its affinity for substrate (FRETS-VWF73) was one-third that of her VWF-CPase (Km = 0 3 vs. 0 1; P = 0 0585).