Polyethylene glycol 20 kDa-induced vacuolation does not impair phagocytic function of human monocyte-derived macrophages

Conjugation to polyethylene glycol (PEG) is commonly used to enhance drug delivery and efficacy by extending the half-life of the drug molecule. This has important implications for reducing treatment burden in diseases that require chronic prophylaxis, such as hemophilia. Clearance of PEG molecules with high molecular weights (≥ 40 kDa) has been reported to cause cellular vacuolation in mammals. Rurioctocog alfa pegol (PEGylated recombinant coagulation factor VIII) contains a 20-kDa PEG. This study investigated the effects of exposure to 20-kDa PEG (10 μg/ml to 10 mg/ml) on the morphology and function of human monocyte-derived macrophages (MDMs) in vitro. Exposure to PEG for 24 hours was associated with significant vacuolation only at concentrations of 1 mg/ml or more, which far exceed the levels associated with clinically relevant doses of rurioctocog alfa pegol. Immunofluorescence staining of PEG was detected in the cytoplasm of MDMs, indicating uptake into the cells. No impairment of MDM phagocytic activity (ability to ingest fluorescently labeled Escherichia coli) was observed with 24-hour exposure to PEG, even at concentrations associated with significant vacuolation. Furthermore, PEG exposure did not have significant effects on cytokine secretion in resting or lipopolysaccharide-stimulated MDMs, or on the expression of cell surface markers in stimulated MDMs. Cell viability was not affected by 24-hour exposure to PEG. In conclusion, vacuolation of human MDMs after exposure to 20-kDa PEG only occurred with PEG concentrations far in excess of those equivalent to clinically relevant doses of rurioctocog alfa pegol and did not affect MDM viability or functionality. Together, these results support the concept that PEG-mediated vacuolation is an adaptive cellular response rather than a toxic effect

Qualification of Hemophilia Treatment Centers to Enable Multi-Center Studies of Gene Expression Signatures in Blood Cells from Pediatric Patients

Hemophilia A is a rare congenital bleeding disorder caused by a deficiency of functionally active coagulation factor VIII (FVIII). Most patients with the severe form of the disease require FVIII replacement therapies, which are often associated with the development of neutralizing antibodies against FVIII. Why some patients develop neutralizing antibodies while others do not is not fully understood. Previously, we could demonstrate that the analysis of FVIII-induced gene expression signatures in peripheral blood mononuclear cells (PBMC) obtained from patients exposed to FVIII replacement therapies provides novel insights into underlying immune mechanisms regulating the development of different populations of FVIII-specific antibodies. The aim of the study described in this manuscript was the development of training and qualification test procedures to enable local operators in different European and US clinical Hemophilia Treatment Centers (HTC) to produce reliable and valid data for antigen-induced gene expression signatures in PBMC obtained from small blood volumes. For this purpose, we used the model antigen Cytomegalovirus (CMV) phosphoprotein (pp) 65. We trained and qualified 39 local HTC operators from 15 clinical sites in Europe and the US, of whom 31 operators passed the qualification at first attempt, and eight operators passed at the second attempt

Prospective Hemophilia Inhibitor PUP Study Reveals Distinct Antibody Signatures during FVIII Inhibitor Eradication

Factor VIII (FVIII) inhibitor formation is a major clinical concern during replacement therapy in patients with hemophilia A. Immune tolerance induction (ITI) is the only therapeutic approach to attempt inhibitor eradication and establishment of long-term immune tolerance to FVIII. Hemophilia Inhibitor Previously Untreated Patient (PUP) Study (HIPS) was a prospective clinical trial to investigate changes in the immune system of PUPs with severe hemophilia A. Five patients who developed persistent FVIII inhibitors during HIPS entered an ITI extension arm (HIPS-ITI). During HIPS-ITI, inhibitor patients received ITI with the same FVIII product (a single source of recombinant, human full-length FVIII) used in HIPS until successful tolerance, declared failure, or a maximum of 2 years after HIPS-ITI enrollment, whichever came first. Blood samples and clinical data were collected monthly. Longitudinal FVIII-binding antibody signatures, associated binding specificities, and apparent affinities were determined for each patient at each sampling time point. ITI was successful or partially successful in 2 patients and failed in 3. Both groups presented with distinct FVIII-specific antibody signatures. ITI success required the disappearance of FVIII inhibitors, which was associated with the eradication or sustained titer minimization of high-affinity FVIII-specific antibodies, particularly of the immunoglobulin G1 (IgG1) and IgG4 subclasses. In contrast, ITI failure, as reflected by FVIII inhibitor persistence, was associated with persistent high-affinity FVIII-specific antibodies. Interestingly, 1 patient with partial ITI success and 1 patient with ITI failure developed apparent oligoreactive FVIII-binding antibodies during ITI. The explanation of the true nature of these antibodies requires more comprehensive follow-ups in future studies. This trial was registered at www.clinicaltrials.gov as #NCT01652027

The interleukin-33 receptor (ST2) is a novel therapeutic target to attenuate the progression of hemophilic arthropathy

Hemophilia A is an X-linked bleeding disorder caused by a blood clotting protein factor VIII deficiency. Patients with hemophilia develop recurrent bleeding episodes. When bleeding occurs in the joints, hemophilic arthropathy (HA) may develop, resulting in hemarthroses and joint deformation. A novel congenic mouse model of severe hemophilia A was generated using CRISPR/CRISPR-associated protein 9 targeting of exon 1 of the F8 gene (F8em1-/-) to explore changes in the bleeding and inflammation during HA. F8em1-/- mice have a high penetrance of spontaneous bleeding, with joint bleeds progressing to arthropathy. F8em1-/- mice were subjected to needle-induced damage to the knee to assess synchronized joint bleeding, and the development of HA and synovial inflammation was assessed. The synovium of injured joints of F8em1-/- mice had differential and temporal expression of inflammatory genes after injury. Pathway analysis identified upregulation of the interleukin-1 (IL-1) family cytokines, IL-1β and IL-33; and respective receptors IL-1 receptor accessory protein and T1/ST2 (ST2) in the synovium of mice after needle-induced HA. Soluble ST2 and IL-33 levels were elevated in the plasma of F8em1-/- mice in acute stages after needle injury to the joints. Dual ST2-deficient F8em1-/- mice were generated, with ST2-deficient hemophilic mice developing significantly reduced joint damage after needle injury relative to F8em1-/- mice. Using a therapeutic intervention, blocking ST2 after joint injury significantly ameliorated joint damage during HA in hemophilic mice. These studies in a new mouse model of HA identify a crucial role of ST2 in HA pathogenesis and highlight its potential as a novel therapeutic target.</p

BAX 335 hemophilia B gene therapy clinical trial results: potential impact of CpG sequences on gene expression

Gene therapy has the potential to maintain therapeutic blood clotting factor IX (FIX) levels in patients with hemophilia B by delivering a functional human F9 gene into liver cells. This phase 1/2, open-label dose-escalation study investigated BAX 335 (AskBio009, AAV8.sc-TTR-FIXR338Lopt), an adeno-associated virus serotype 8 (AAV8)-based FIX Padua gene therapy, in patients with hemophilia B. This report focuses on 12-month interim analyses of safety, pharmacokinetic variables, effects on FIX activity, and immune responses for dosed participants. Eight adult male participants (aged 20-69 years; range FIX activity, 0.5% to 2.0%) received 1 of 3 BAX 335 IV doses: 2.0 × 1011; 1.0 × 1012; or 3.0 × 1012 vector genomes/kg. Three (37.5%) participants had 4 serious adverse events, all considered unrelated to BAX 335. No serious adverse event led to death. No clinical thrombosis, inhibitors, or other FIX Padua-directed immunity was reported. FIX expression was measurable in 7 of 8 participants; peak FIX activity displayed dose dependence (32.0% to 58.5% in cohort 3). One participant achieved sustained therapeutic FIX activity of ∼20%, without bleeding or replacement therapy, for 4 years; in others, FIX activity was not sustained beyond 5 to 11 weeks. In contrast to some previous studies, corticosteroid treatment did not stabilize FIX activity loss. We hypothesize that the loss of transgene expression could have been caused by stimulation of innate immune responses, including CpG oligodeoxynucleotides introduced into the BAX 335 coding sequence by codon optimization. This trial was registered at www.clinicaltrials.gov as #NCT01687608

Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency

One important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an in vitro cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized F8 nucleotide sequences were employed. A comprehensive immunogenicity assessment using in vitro stimulation assays and various in vivo preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A

Risky business of inhibitors: HLA haplotypes, gene polymorphisms, and immune responses

The development of neutralizing antibodies against factor VIII (FVIII inhibitors) and factor IX (FIX inhibitors) is the major complication in hemophilia care today. The antibodies neutralize the biological activity of FVIII and FIX and render replacement therapies ineffective. Antibodies are generated as a result of a cascade of tightly regulated interactions between different cells of the innate and the adaptive immune system located in distinct compartments. Any event that modulates the repertoire of specific B or T cells, the activation state of the innate and adaptive immune system, or the migration pattern of immune cells will therefore potentially influence the risk for patients to develop inhibitors. This chapter reviews our current understanding of different pathways of antibody development that result in different qualities of antibodies. Potential differences in differentiation pathways leading to high-affinity neutralizing or low-affinity non-neutralizing antibodies and the potential influence of gene polymorphisms such as HLA haplotype, FVIII haplotype, and polymorphisms of immunoregulatory genes are discussed.</jats:p

Qualification of Hemophilia Treatment Centers to Enable Multi-Center Studies of Gene Expression Signatures in Blood Cells from Pediatric Patients

Hemophilia A is a rare congenital bleeding disorder caused by a deficiency of functionally active coagulation factor VIII (FVIII). Most patients with the severe form of the disease require FVIII replacement therapies, which are often associated with the development of neutralizing antibodies against FVIII. Why some patients develop neutralizing antibodies while others do not is not fully understood. Previously, we could demonstrate that the analysis of FVIII-induced gene expression signatures in peripheral blood mononuclear cells (PBMC) obtained from patients exposed to FVIII replacement therapies provides novel insights into underlying immune mechanisms regulating the development of different populations of FVIII-specific antibodies. The aim of the study described in this manuscript was the development of training and qualification test procedures to enable local operators in different European and US clinical Hemophilia Treatment Centers (HTC) to produce reliable and valid data for antigen-induced gene expression signatures in PBMC obtained from small blood volumes. For this purpose, we used the model antigen Cytomegalovirus (CMV) phosphoprotein (pp) 65. We trained and qualified 39 local HTC operators from 15 clinical sites in Europe and the US, of whom 31 operators passed the qualification at first attempt, and eight operators passed at the second attempt.&nbsp

Risky business of inhibitors: HLA haplotypes, gene polymorphisms, and immune responses

Abstract The development of neutralizing antibodies against factor VIII (FVIII inhibitors) and factor IX (FIX inhibitors) is the major complication in hemophilia care today. The antibodies neutralize the biological activity of FVIII and FIX and render replacement therapies ineffective. Antibodies are generated as a result of a cascade of tightly regulated interactions between different cells of the innate and the adaptive immune system located in distinct compartments. Any event that modulates the repertoire of specific B or T cells, the activation state of the innate and adaptive immune system, or the migration pattern of immune cells will therefore potentially influence the risk for patients to develop inhibitors. This chapter reviews our current understanding of different pathways of antibody development that result in different qualities of antibodies. Potential differences in differentiation pathways leading to high-affinity neutralizing or low-affinity non-neutralizing antibodies and the potential influence of gene polymorphisms such as HLA haplotype, FVIII haplotype, and polymorphisms of immunoregulatory genes are discussed.</jats:p