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.

The development of neutralizing antibodies-termed "inhibitors"-to infused therapeutic (t) factor VIII proteins (tFVIIIs) is the most serious obstacle to effective treatment of bleeding in Hemophilia A (HA) patients. As clinically significant FVIII immune responses are only initiated if dendritic cell (DC) cII-HLAs can present foreign tFVIII-derived peptides to naïve FVIII-specific T cells, we posit the "Gate Keeper" hypothesis in which the limiting determinant of inhibitor formation are patients' cII-HLA repertoires with the majority being individually distinct and each contributing slightly to the vast population level diversity of cII-HLAs. While cII-HLAs are critical at the cellular level for initiating immune responses, conflicting results from population studies have led some to describe their encoding HLA-II structural genes as weak determinants of inhibitor causation. Our main objective here is to test a hypothesis that gets at the heart of this disconnect between molecular-based expectations and population-level data by analyzing cII-HLA peptidomic data from DC-protein processing and presentation assays (PPPAs). The chief variable of DC-PPPA data is the peptide count, which we assume to be directly proportional to immunogenic potential (IP). Our working model is that inhibitor formation requires at minimum, in its initial stages, a complex between cII-HLAs and specific tFVIII-derived peptides. A testable null hypothesis under this thinking posits that a given cII-HLA allotype will have the same IP when exposed to several tFVIIIs. To test this hypothesis, we first performed model selection to determine the best set of predictor allotypes. To analyze the data, we employed a log-linear model where the peptide count is the dependent variable and allotype is a categorical independent variable consisting of 29 levels for 29 allotypes (8 DP, 10 DQ, and 11 DR allotypes). We used elastic net regression (ENR) to select the best set of allotype levels thus giving the best overall model consisting now of only four DR allotypes (Table 1). We then performed interaction analysis under the best-selected allotypes model in which we introduced as additional predictor variables, a tFVIII categorical variable consisting of five levels for five different tFVIIIs, namely full length (FL)-recombinant (r) FVIII (FL-rFVIII) ± von Willebrand Factor (VWF), B domain truncated (BDT)-rFVIII ± VWF, and plasma derived (pd) FVIII (pdFVIII) + VWF, and 12 interaction terms for the (4 - 1) × (5 - 1) possible interactions between the cII-HLA allotype and tFVIII variables. We found significant cII-HLA allotype × tFVIII interactions (Table 2). To get at the specific null hypothesis of interest, we examined within-allotype risk ratios (RRs) and their appropriately adjusted confidence intervals (CIs).1-4 It can be shown that an 84% CI is sufficient to achieve a significance level of α = 0.05 for the CI difference.2-4 Although there are 12 total interaction terms, per allotype there are only three possible CI comparisons on using the interaction term with the highest RR as a fixed reference. On constructing the adjusted CIs and correcting for multiple hypothesis testing,2 we found that two comparisons in Table 2 corresponded to significantly different RRs. We determined statistical power to detect a CI difference.1,3 As seen in Table 2, our study was extremely underpowered, which may explain why only two significant differences were found. Thus, at least for the two comparisons showing significant difference, we have refuted the null hypothesis of no difference across tFVIIIs for a given allotype, and have affirmed our working model that specific combinations of cII-HLAs and tFVIII-derived peptides are the triggering factor in inhibitor development.Schenker N, Gentleman J. On judging the significance of differences by examining the overlap between confidence intervals. Am Statistician. 2001; 55(3): 182-6.Julious S. Using confidence intervals around individual means to assess statistical significance between two means. Pharmaceut Statist. 2004; 3: 217-22.Maghsoodloo S, Huang C-Y. Comparing the overlapping of two independent confidence intervals with a single confidence interval for two normal population parameters. J Statist Plan & Infer. 2010; 140: 3295-305.Knol M, Pestman W, Grobbee D. The (mis)use of overlap of confidence intervals to assess effect modification. Eur J Epidemiol. 2011; 26(4): 253-4. Disclosures Hofmann: CSL Behring: Employment. Dinh:Haplomics Biotechnology Corporation: Employment, Equity Ownership. Escobar:Pfizer: Research Funding; Bayer, CSL Behring, Genentech, Hemabiologics, Kedrion, Novo Nordisk, Octapharma, Pfizer and Shire: Consultancy. Maraskovsky:CSL Behring: Employment. Howard:CSL Behring: Research Funding; Haplomics Biotechnology Corporation: Equity Ownership, Other: Chief Scientific Officer, Patents & Royalties: Patent applications and provisional patent applications .

Introduction: Factor (F)VIII immunogenicity is the main obstacle to both successfully treating hemophilia and receiving FDA approval for new therapeutics. Since immune responses to allogeneically distinct proteins require T-cell activation and proliferation, at least one foreign peptide must be liberated from FVIII that can bind with high affinity to one or more of a patient's HLA-class-II (HLA-II) isomers. Due to the complex pathogenesis of inhibitor development, which involves numerous genetic variations and both treatment and product related variables, our ability to predict which patients will become alloimmunized to FVIII remains suboptimal. The role of FVIII glycosylation in inhibitor development appears to be an important, yet not well characterized, modifying influence; defining the mechanism has been complicated by the fact that some FVIII products are highly glycosylated with thousands of potential glycoforms. Here we describe studies of the spectrum of FVIII peptides eluted from HLA-II complexes and the impact of glycosylation on the proteolysis and/or binding of these peptides to a patient's individual HLA-II isomers, i.e. to further incorporate personalized medicine in hemophilia care. Methods: Immature monocyte-derived dendritic cells (DCs) from 12 unrelated donors were generated in vitro and matured in the presence of an equimolar pool of a full length (FL) and 4 B-domain deleted (BDD) rFVIII proteins free of VWF and other proteins. Following harvest and lysis, HLA-DP, -DQ, and -DR molecules were recovered using a specific immunoaffinity step. Peptides were then eluted from these complexes and sequenced by high resolution mass spectrometry (LC/MS/MS). The bound peptides were mapped to the FVIII reference sequence. The estimated binding affinity of FVIII peptides to the HLA-II alleles found in these donors were obtained from NetMHCIIpan and compared to the peptides observed to be bound. Results: The 12 DC donors express 29 distinct HLA-II isomers (6 DP, 11 DQ, 12 DR) from an overall HLA-II gene repertoire having the following alleles: DP: 01:03/02:01, 01:03/03:01, 01:03/04:01, 01:03/04:02, 02:01/01:01, 02:02/05:01 DQ: 01:01/05:03, 01:02/06:02, 01:02/06:09, 01:03/05:01, 01:03/06:03, 01:03/06:04, 02:01/02:01, 02:01/02:02, 03:01/03:02, 03:02/03:03, 05:01/03:01 DR: 01:01, 03:01, 04:01, 04:04, 07:01, 09:01, 11:01, 11:04, 13:01, 13:02, 14:01, 15:01 These 29 HLA-II isomers were found to have 77 bound peptides that covered 1,202 of the 2,332 total amino acid residues in the NCBI reference FL-FVIII protein, whose domain structure and consensus N-linked glycosylation (NLG) sites are shown in panel A of the Figure. The HLA-II bound peptides were mapped to the FVIII regions from which they derive (panel B). One of the 20 NLG sites known to be glycosylated and 3 of the 4 known non-glycosylated NLG sites were found to be in a bound peptide. A NetMHCIIpan analysis predicted that no FVIII nonamer containing a consensus NLG site binds strongly to any of the 12 DR isomers; but, it also clearly showed that more peptides bind with weak or strong affinity as peptide length increases (data not shown). Conclusion: Despite finding 3 of FVIII's 4 non-glycosylated consensus NLG sites in the tightly bound HLA-II/peptide complexes, only 1 of the 20 glycosylated NLG sites was found among these bound peptides. The near complete absence of N-linked glycans in the presented HLA-II bound peptides is compelling, but incomplete, evidence that glycosylation influences the immunogenicity of FVIII. Potential mechanisms may include effects on internalization, proteolysis and/or HLA-II binding. Further direct studies are required to determine if these post-translational modifications affect proteolysis and/or HLA-II binding; core peptides that can be proteolyzed and/or bound in the absence of glycosylation or with alternative glycan conformations may provide more evidence that glycosylation can modulate immunogenicity. We hypothesize that HLA-II bound and unbound FVIII peptides constitute a novel immune-response-related biomarker that will improve the accuracy of inhibitor risk prediction by allowing pertinent patient-specific pharmacogenomic inputs to be analyzed in a more biologically relevant manner. References: Karosiene et al. NetMHCIIpan-3.0, a pan-specific MHC-II prediction method including all 3 human MHC-II isotypes: HLA-DR, -DP and -DQ. Immunogenetics, 2013. Disclosures Hofmann: CSL Behring: Employment. Zollner:CSL Behring: Employment. Powell:CSL Behring: Employment. Maraskovsky:CSL Behring: Employment. Howard:Baxter: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Haplomics, Inc.: Patents & Royalties; CSL Behring: Consultancy, Honoraria, Research Funding.

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