Endogenous factor VIII synthesis from the intron 22-inverted F8 locus may modulate the immunogenicity of replacement therapy for hemophilia A.

Pandey GSYanover CMiller-Jenkins LMGarfield SCole SACurran JEMoses EKRydz NSimhadri VKimchi-Sarfaty CLillicrap DViel KRPrzytycka TM,Pierce GFHoward TESauna ZEPATH (Personalized Alternative Therapies for Hemophilia) Study InvestigatorsLusher JChitlur MAmeri ANatarajan KIyer RVThompson AAWatts RGKempton CLKessler CBarrett JCMartin EJKey NKruse-Jarres RLessinger CPratt KPJosephson NMcRedmond K, Withycombe JWalsh CMatthews DMahlangu JKrause ASchwyzer RThejpal RRapiti NGoga YCoetzee MStones DMann KButenas SAlmasy L, Blangero JCarless MRaja RReed E.

Source: Laboratory of Hemostasis, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA.

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 <10% (ref. 2). Individuals with the F8 intron 22 inversion (found in ∼50% of individuals with severe hemophilia A) have been grouped with the former on the basis of their genetic defect and CRM-negative status. However, only ∼20% of these individuals develop inhibitors. Here we demonstrate that the levels of F8 mRNA and intracellular FVIII protein in B lymphoblastoid cells and liver biopsies from individuals with the intron 22 inversion are comparable to those in healthy controls. These results support the hypothesis that most individuals with the intron 22 inversion are tolerized to FVIII and thus do not develop inhibitors. Furthermore, we developed a new pharmacogenetic algorithm that permits the stratification of inhibitor risk for individuals and subpopulations by predicting the immunogenicity of replacement FVIII using, as input, the number of putative T cell epitopes in the infused protein and the competence of major histocompatibility complex class II molecules to present such epitopes. This algorithm showed statistically significant accuracy in predicting the presence of inhibitors in 25 unrelated individuals with the intron 22 inversion.

Nature Medicine 2013 Oct;19(10):1318-24

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.

Pandey GSYanover CHoward TESauna ZE.

Source: Laboratory of Hemostasis, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA.


The 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.

PLoS Comput Biol. 2013 May;9(5).


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

Pandey GSTseng SCHoward TESauna ZE.

Source: Laboratory of Hemostasis, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, 29 Lincoln Drive, Bethesda, MD 20892, USA.


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.

Biomed Res Int. 2013; 2013:793502

Pharmacogenetics and the immunogenicity of protein therapeutics

Chen Yanover Ph.D, Nisha Jain, Ph.D. (FDA) Glenn Pierce Ph.D. MD (Biogen-IDEC), Tom E Howard Ph.D. MD (Los Angeles VA) & Zuben E Sauna, Ph.D. (FDA)

nature biotechnology volume 29 number 10 October 2011