Formation Of Inhibitory Antibodies Research Articles

First modern Russian plasma-derived coagulation factor VIII concentrate (Eytoplasm): Results of clinical study of pharmacokinetics, efficacy and safety

Introduction. Replacement therapy with coagulation factor VIII concentrates remains the standard of care for patients with hemophilia A. In 2023, the drug Eytoplasm, the first modern plasma-derived coagulation factor VIII concentrate developed in the Russian Federation, was authorized for medical use in the Russian Federation.Aim: to study the efficacy, safety, immunogenicity, and pharmacokinetic properties of Eytoplasm.Methods. A multicenter, prospective, open-label clinical trial was conducted in 55 patients over 12 years of age with severe hemophilia A who had previously received treatment with coagulation factor VIII concentrates (at least 150 exposure days). All patients received the drug for prophylactic treatment 2–3 times a week; the treatment duration was 6 months (at least 50 exposure days). In addition, the drug was used to treat bleeding. Ten patients underwent 10 surgical interventions (2 major and 8 minor). Pharmacokinetic parameters were determined after the first administration of the drug to patients and after 6 months of therapy.Results. Eytoplasm pharmacokinetics properties are comparable with other plasma-derived coagulation factor VIII concentrates. No bleeding was recorded in 75.9% of patients. In 92.3 % of patients, a single administration of the drug was sufficient to stop an episode of bleeding. In all participants who completed the study, the residual activity of coagulation factor VIII 48–72 hours after drug administration was at least 1 %. Positive dynamics of APTT was showed during the study. Serious adverse events, allergic reactions, thrombotic and thromboembolic complications were absent in patients. In 3 patients, 4 adverse events associated with the use of the drug were registered: 2 cases of increased serum concentration of direct bilirubin and 2 cases of headache. An inhibitor to coagulation factor VIII was not detected in any patient. In none of the patients who did not have antibodies to parvovirus B19 before the first dose of Eytoplasm, antibodies were detected after 6 months of therapy.Conclusion. Eytoplasm is an effective option for the prevention and treatment of bleeding, and during surgical interventions, including major ones, in patients with hemophilia A. The drug has a favorable safety profi le; its use was not associated with the formation of inhibitory antibodies, allergic reactions, thrombotic and thromboembolic complications.

Cryo-EM Structure of Coagulation Factor VIII Bound to a Patient-Derived Anti-A2 Domain Antibody Inhibitor

Coagulation factor VIII (FVIII) promotes hemostasis by binding to activated factor IX on activated platelet surfaces to form the intrinsic tenase complex. The formation of inhibitory antibodies against FVIII occurs in approximately 30% of congenital hemophilia A patients receiving FVIII replacement therapy, significantly complicating treatments. By identifying the FVIII amino acids which bind to pathogenic inhibitory antibodies, FVIII replacement therapies can be engineered with reduced immunogenicity. Here, we aimed to structurally characterize FVIII bound to NB11B2, a patient-derived antibody inhibitor which binds to the A2 domain and disrupts tenase complex activity, using cryogenic electron microscopy (cryo-EM). The FVIII:NB11B2 structure was solved to a global resolution of 4.0 Å, representing the first structure of FVIII bound to an anti-A2 domain antibody inhibitor. Structural analysis revealed that NB11B2 binds to a discontinuous epitope encompassing FVIII residues S470-P472, R490-K493, and P502-E507. Furthermore, our structure identified a 20° swivel-like movement adopted by the C2 domain while the C1 domain remains immobile, consistent with a previous X-ray crystal structure of FVIII bound to an anti-C1 domain inhibitor. These results provide the structural basis for inhibition of FVIII by NB11B2 and elucidate a novel role for portions of the FVIII A2 domain in regulating tenase complex assembly and/or activity.

Generation of FVIII-Specific Tregs Expressing a Chimeric Antigen Receptor (CAR) to Suppress Inhibitor Development in Hemophilia a Mice

Introduction: The development of inhibitory antibodies (inhibitors) against coagulation factor VIII (FVIII) is a critical complication in the treatment of hemophilia A, as hemostasis can no longer be re-established by FVIII replacement therapy. Cellular Treg therapy is an attractive candidate for targeted treatment of B and T cells involved in inhibitor formation, as it can suppress both cell types through direct and indirect mechanisms. Here, we generated antigen specific Tregs by engineering and retrovirally transducing a chimeric antigen receptor (CAR) molecule with specificity to human FVIII. Antigen recognition via the single chain antibody variable regions triggers signaling via the CD28 and CD3z co-stimulatory domains, which induces CAR-Treg activation and proliferation independent of MHC recognition. We show that human FVIII specific CAR-Tregs with second generation mouse signaling domains are activated and proliferate in response to antigen recognition, which can cause contact dependent or cytokine dependent suppression of inhibitor producing cells. Methods:A single chain variable fragment specific for human FVIII was cloned (Creative Biolabs, NY) out of an EBV transformed B cell from a hemophilia A patient (BO2C11), which produces IgG4 directed against residues 2125-2332 of huFVIII, corresponding to the carboxy-terminal end of C1 and the complete highly immunogenic C2 domain. This was fused to a 2nd generation CAR construct expressing CD28 and CD3ζ signaling molecules and packaged in a retroviral system (pMys-IRES-eGFP). CD3/CD28 activated polyclonal Tregs were retrovirally transduced to generate FVIII CAR-Tregs. Binding to Fc-FVIII was tested using an A647 conjugated Fc antibody. Upregulation of activation and exhaustion markers, specifically CD69, CD44, LAP, GARP and PD1 was tested 24 h after stimulation with either Fc-FVIII or B domain deleted (BDD) FVIII. Proliferation of CellTrace Violet labeled and FVIII stimulated CAR-Tregs was tested both in vitro and in vivo upon stimulation with BDD FVIII. 1x106 GFP+ FVIII CAR-Tregsorted cells were adoptively transferred into F8 e16−/− hemophilia A mice, and recipients were challenged, starting 1 day later, with weekly IV injections of 1 IU BDD-FVIII for 4 weeks. Plasma was tested for inhibitor formation at 4 and 8 weeks using the Bethesda assay and IgG1 ELISA. Results:FVIII CAR-Tregs bound Fc-FVIII (Eloctate) in a specific manner, with no binding observed to control Fc-FIX (Alprolix). Stimulation for 24 hours with either Fc-FVIII or BDD-FVIII resulted in upregulation of activation and exhaustion markers such as CD69, CD44, GARP and PD-1. No non-specific activation with the unrelated Fc-FIX antigen was observed. CellTrace Violet labeled CAR-Treg and CAR transduced conventional T cells underwent antigen-specific proliferation upon 72 hours of in vitro stimulation with FVIII. In vivo, persistence of adoptively transferred CAR-Tregs was observed only when antigen was concurrently administered. We are currently testing for the suppression of inhibitor development in BALB/c F8 e16−/− hemophilia A mice following adoptive immunotherapy with FVIII CAR-Tregs and continued weekly administrations of BDD-FVIII. Conclusions: Here, we demonstrate that CAR-Tregs directed to human FVIII show antigen-specific binding, activation and proliferation. We found persistence of adoptively transferred FVIII CAR-Tregs in response to antigen stimulation in mice. The suppressive activity of FVIII CAR-Treg on inhibitory antibody formation is now being evaluated in hemophilia A mice that are given factor replacement therapy with FVIII. CAR-Tregs represent an effective way to generate a large pool of antigen-specific cells, with no requirement for MHC restriction. DisclosuresBiswas:Bayer Hemophilia Awards Program: Research Funding. Herzog:Novo Nordisk: Research Funding; Applied Genetic Technologies Corporation: Consultancy; Spark Therapeutics: Patents & Royalties.

Alternative Approaches to Oral Tolerance Induction to Factor FVIII

A serious complication in the treatment X-linked bleeding disorder hemophilia A is the formation of inhibitory antibodies against factor VIII (FVIII), which compromise traditional replacement therapy. We previously developed an Oral immunotherapy (OTI) based on repeated uptake of a mixture of lettuce plant cells transgenic for heavy chain (HC) or C2 domain of human FVIII fused to cholera toxin B (CTB) subunit [Blood 124:1659; Plant Biotechnol J. 16:1148]. Fusion proteins were transgenically expressed in the chloroplasts. Repeated oral uptake of a mixture of freeze-dried powder of lettuce cells accomplished antigen delivery to the immune system of the small intestine by targeting of the GM1 receptor that is highly expressed on the surface of the gut epithelium, resulting in induction of regulatory T cells (Treg) that suppress inhibitor formation upon subsequent intravenous (iv) FVIII replacement therapy. An alternative to oral antigen delivery is the oral delivery of immune modulatory antibodies. Here, we compared the plant cell-based method with oral delivery of anti-CD3, which has been successful in murine models of autoimmune disease and is currently evaluated in clinical trials. Unlike in iv administration, oral anti-CD3 does not systemically deplete T cells. Hemophilia A BALB/c mice (F8 e16 gene deletion) received oral gavage of a mixture of CTB-FVIII-HC/-C2 (1.5 µg/antigen) expressing lettuce leaf cells 2x/week for 9 weeks. Starting at 4 weeks into the experiment, 1 IU/mouse of BDD-FVIII (Xyntha) was given iv, once per week for 5 weeks. Alternatively, following a published protocol that was successful in other models, 5 µg of hamster anti-murine CD3 was given by oral gavage daily for 5 straight days, followed by 5 weekly iv injections of BDD-FVIII. Control animals (no OTI) developed inhibitors with an average titer of 18 ± 3 BU/ml (n=16). Of these, 88% were high-titer (i.e >5 BU/ml, up to 43 BU/ml). Inhibitor formation was significantly reduced in plant cell-treated mice (10 ± 2.5 BU/ml, n=17), with 47% showing no or low-titer inhibitors (<5 BU/ml). Interestingly, inhibitor formation in anti-CD3 treated mice showed a unique pattern, with two distinct outcomes. One subset of animals developed inhibitors of 0-15 BU/ml (on average 2.5-fold lower than control group), while the second subset showed increased titers of 41-89 BU/ml. There were no animals with intermediate titers. In general, FVIII-specific IgG titers followed the same patterns as inhibitor responses. When the 2 approaches (plant cells and anti-CD3) were combined, 50% of mice developed <5 BU/ml (as compared to 36% for anti-CD3 only). However, this did not represent an improvement over giving lettuce cells only. Finally, we tested anti-CD3 Fab fragment in place of FL anti-CD3. While a daily dose of 5 µg anti-CD3 Fab/mouse was unsuccessful in suppressing inhibitor formation, a dose of 0.5 µg marginally decrease titers. In summary, oral anti-CD3 delivery was not as effective in reducing inhibitor formation as plant cells expressing CTB-FVIII antigen fusions. While FL anti-CD3 showed promise in a subset of animals, it also posed a risk of an increased response in others, which complicates development of this approach. Distinct from all other treatment groups, lettuce-fed mice showed elevated frequencies in Foxp3+ Treg, a reduction in Teff cells, and the highest increase in LAP+ Treg. Further studies on induced CD4+LAP+FoxP3- Treg cells from lettuce-fed mice showed high expression of ICOS, CD69 and Ki-67, but not CTLA-4. Among the different subsets of Treg, LAP+ T cell contained the highest proportion of IL-10 expressing cells, and oral antigen delivery induced IL-10+LAP+ Treg, which are likely key to the suppression of the immune response against FVIII. Disclosures Daniell: Takeda Pharmaceuticals: Patents & Royalties. Herzog:Takeda Pharmaceuticals: Patents & Royalties.

Role of Small Intestine and Gut Microbiome in Plant-Based Oral Tolerance for Hemophilia.

Fusion proteins, which consist of factor VIII or factor IX and the transmucosal carrier cholera toxin subunit B, expressed in chloroplasts and bioencapsulated within plant cells, initiate tolerogenic immune responses in the intestine when administered orally. This approach induces regulatory T cells (Treg), which suppress inhibitory antibody formation directed at hemophilia proteins induced by intravenous replacement therapy in hemophilia A and B mice. Further analyses of Treg CD4+ lymphocyte sub-populations in hemophilia B mice reveal a marked increase in the frequency of CD4+CD25−FoxP3−LAP+ T cells (but not of CD4+CD25+FoxP3+ T cells) in the lamina propria of the small but not large intestine. The adoptive transfer of very small numbers of CD4+CD25−LAP+ Treg isolated from the spleen of tolerized mice was superior in suppression of antibodies directed against FIX when compared to CD4+CD25+ T cells. Thus, tolerance induction by oral delivery of antigens bioencapsulated in plant cells occurs via the unique immune system of the small intestine, and suppression of antibody formation is primarily carried out by induced latency-associated peptide (LAP) expressing Treg that likely migrate to the spleen. Tolerogenic antigen presentation in the small intestine requires partial enzymatic degradation of plant cell wall by commensal bacteria in order to release the antigen. Microbiome analysis of hemophilia B mice showed marked differences between small and large intestine. Remarkably, bacterial species known to produce a broad spectrum of enzymes involved in degradation of plant cell wall components were found in the small intestine, in particular in the duodenum. These were highly distinct from populations of cell wall degrading bacteria found in the large intestine. Therefore, FIX antigen presentation and Treg induction by the immune system of the small intestine relies on activity of a distinct microbiome that can potentially be augmented to further enhance this approach.

Reprogrammed CD4+ T Cells That Express FoxP3+ Control Inhibitory Antibody Formation in Hemophilia A Mice.

Coagulation Factor VIII (FVIII) replacement therapy in hemophilia A patients is complicated by the development of inhibitory antibodies, which often render the treatment ineffective. Previous studies demonstrated a strong correlation between induction of regulatory T cells (Treg) and tolerance to the therapeutic protein. We, therefore, set out to evaluate whether the adoptive transfer of FVIII-specific CD4+ Treg cells prevents inhibitor response to FVIII protein therapy. To this end, we first retrovirally transduced FoxP3+ into FVIII-specific CD4+ cells, which resulted in cells that stably express FoxP3, are phenotypically similar to peripherally induced Tregs and are antigen specific suppressors, as judged by in vitro assays. Upon transfer of the FVIII-specific CD4+ FoxP3+ cells into hemophilia A mice, development of inhibitory antibodies in response to administering FVIII protein was completely suppressed. Suppression was extended for 2 months, even after transferred cells were no longer detectable in the secondary lymphoid organs of recipient animals. Upon co-transfer of FoxP3+-transduced cells with the B cell depleting anti-CD20 into mice with pre-existing inhibitory antibodies to FVIII, the escalation of inhibitory antibody titers in response to subsequent FVIII protein therapy was dramatically reduced. We conclude that reprogramed FoxP3 expressing cells are capable of inducing the in vivo conversion of endogenous FVIII peripheral Tregs, which results in sustained suppression of FVIII inhibitors caused by replacement therapy in recipient hemophilia A animals.

FVIII-specific human chimeric antigen receptor T-regulatory cells suppress T- and B-cell responses to FVIII

AbstractReplacement therapy with factor VIII (FVIII) is used in patients with hemophilia A for treatment of bleeding episodes or for prophylaxis. A common and serious problem with this therapy is the patient's immune response to FVIII, because of a lack of tolerance, leading to the formation of inhibitory antibodies. Development of tolerogenic therapies, other than standard immune tolerance induction (ITI), is an unmet goal. We previously generated engineered antigen-specific regulatory T cells (Tregs), created by transduction of a recombinant T-cell receptor (TCR) isolated from a hemophilia A subject's T-cell clone. The resulting engineered T cells suppressed both T- and B-cell effector responses to FVIII. In this study, we have engineered an FVIII-specific chimeric antigen receptor (ANS8 CAR) using a FVIII-specific scFv derived from a synthetic phage display library. Transduced ANS8 CAR T cells specific for the A2 domain proliferated in response to FVIII and ANS8 CAR Tregs were able to suppress the proliferation of FVIII-specific T-effector cells with specificity for a different FVIII domain in vitro. These data suggest that engineered cells are able to promote bystander suppression. Importantly, ANS8 CAR-transduced Tregs also were able to suppress the recall antibody response of murine splenocytes from FVIII knockout mice to FVIII in vitro and in vivo. In conclusion, CAR-transduced Tregs are a promising approach for future tolerogenic treatment of hemophilia A patients with inhibitors.

FVIII tolerance induction in hemophilia A mice utilizing low dose IL2 treatment

Abstract Hemophilia A patients lack or possess low levels of functional FVIII protein, which results in an inability for the blood to clot when injury occurs. The current treatment for hemophilia A patients is FVIII protein replacement, but this treatment is expensive and often results in anti-FVIII immune responses, neutralizing the clotting effect. Regulatory T (Treg) cells help balance T effector (Teff) cells through their suppressive function during an immune response and keep autoimmunity in check. Treg cells possess a high affinity epitope of the IL2 receptor. With a low dose of IL2, the Treg population outcompetes the Teff cells, leading to an increase in activation and number. The increased suppressive activity may induce tolerance in hemophilia A mice treated with FVIII. Hemophilia A mice were hydrodynamically injected with IL2 and FVIII plasmids. Cell staining data showed a marked increase in Treg cell population and activation. In addition, the Treg/Teff ratio was significantly increased in the first 3 weeks and maintained at increased levels over several weeks afterwards. On Day 28-post FVIII injection, control mice had started to develop inhibitors associated with significant decrease of FVIII expression, while mice treated with IL2 showed no inhibitors with persistent FVIII expression. The treated mice are monitored to determine the potential long term tolerance induced by low dose IL2. Combined gene transfer of FVIII and IL2 plasmids can produce therapeutic FVIII and simultaneously prevent inhibitory antibody formation, thus providing a potentially effective treatment of hemophilia A.

441. IL2 Plasmid Treated Hemophilia A Mice Show an Increase in Regulatory T Cell Population and Initial Tolerance to FVIII

Hemophilia A is an inherited X chromosomal linked recessive disease. Hemophilia A patients lack or possess low levels of functional FVIII protein, which results in an inability for the blood to clot when injury occurs. This inability to clot can result in major blood loss and potential death. The current treatment for patients with hemophilia A is FVIII protein replacement, but this treatment is expensive and often results in anti-FVIII immune responses, neutralizing the clotting effect. In order to overcome this potential immune response patients undergo a high dose regimen of FVIII to induce tolerance. However, a third of patients still develop an immune response.Regulatory T (Treg) cells help balance T effector (Teff) cells through their suppressive function during an immune response and keep autoimmunity in check. Both of these cell types bind to interleukin 2 (IL2), a cytokine which promotes the differentiation, expansion and activation of these cell populations. Treg cells possess a high affinity epitope of the IL2 receptor. With a low dose of IL2, the Treg population outcompetes the Teff cells, leading to an increase in activation and number. The increased suppressive activity may induce tolerance in hemophilia A mice treated with FVIII plasmid.Hemophilia A mice were hydrodynamically injected with either 2 µg or 5 µg IL2 plasmid and 50 µg FVIII plasmid, either sequentially (1 week apart) or simultaneously. Both plasmids were driven by a liver-specific promoter (hAAT-HCR). Cell staining data showed a marked increase in Treg cell population and activation, demonstrating that a small amount of IL2 plasmid incorporated into liver cells is enough to dramatically increase the Treg population. A larger increase of Teff cells were observed in mice treated with 5 µg IL2 plasmid than in mice treated with 2 µg IL2 plasmid. Importantly, Treg/Teff ratio was significantly increased in the 2 µg IL2 plasmid group in the first 3 weeks and maintained at increased levels over several weeks afterwards. ELISA showed an initial higher level of IL2 production; IL2 expression dropped and maintained at low levels after one week. On Day 28-post FVIII plasmid injection, control mice had started to develop inhibitors associated with significant decrease of FVIII expression, while mice treated with IL2 plasmid showed no inhibitor development with persistent FVIII gene expression. The treated mice are monitored to determine the potential long term tolerance effects induced by low dose IL2 plasmid. Using gene therapy to slightly increase the amount of a cytokine in patients could provide a better treatment option than existing drug regimens. FVIII protein is also extremely expensive, especially in the amounts needed for treating hemophilia A patients. Combined gene transfer of FVIII and IL2 plasmids has the potential to produce therapeutic FVIII and simultaneously prevent inhibitory antibody formation, therefore reducing the amount of expensive reagents needed, the number of doctor visits required, and the overall cost, potential morbidity and stress to the patient.

Characterization of a genetically engineered mouse model of hemophilia A with complete deletion of the F8 gene

ESSENTIALS: Anti-factor VIII (FVIII) inhibitory antibody formation is a severe complication in hemophilia A therapy. We genetically engineered and characterized a mouse model with complete deletion of the F8 coding region. F8(TKO) mice exhibit severe hemophilia, express no detectable F8 mRNA, and produce FVIII inhibitors. The defined background and lack of FVIII in F8(TKO) mice will aid in studying FVIII inhibitor formation. The most important complication in hemophilia A treatment is the development of inhibitory anti-Factor VIII (FVIII) antibodies in patients after FVIII therapy. Patients with severe hemophilia who express no endogenous FVIII (i.e. cross-reacting material, CRM) have the greatest incidence of inhibitor formation. However, current mouse models of severe hemophilia A produce low levels of truncated FVIII. The lack of a corresponding mouse model hampers the study of inhibitor formation in the complete absence of FVIII protein. We aimed to generate and characterize a novel mouse model of severe hemophilia A (designated the F8(TKO) strain) lacking the complete coding sequence of F8 and any FVIII CRM. Mice were created on a C57BL/6 background using Cre-Lox recombination and characterized using in vivo bleeding assays, measurement of FVIII activity by coagulation and chromogenic assays, and anti-FVIII antibody production using ELISA. All F8 exonic coding regions were deleted from the genome and no F8 mRNA was detected in F8(TKO) mice. The bleeding phenotype of F8(TKO) mice was comparable to E16 mice by measurements of factor activity and tail snip assay. Similar levels of anti-FVIII antibody titers after recombinant FVIII injections were observed between F8(TKO) and E16 mice. We describe a new C57BL/6 mouse model for severe hemophilia A patients lacking CRM. These mice can be directly bred to the many C57BL/6 strains of genetically engineered mice, which is valuable for studying the impact of a wide variety of genes on FVIII inhibitor formation on a defined genetic background.

Assessment of Inhibitory Antibody Formation in Subjects with Hereditary Angioedema Treated with Plasma-Derived C1-Esterase Inhibitor Concentrate (Berinert®)

Assessment of Inhibitory Antibody Formation in Subjects with Hereditary Angioedema Treated with Plasma-Derived C1-Esterase Inhibitor Concentrate (Berinert®)

245. Enhancing Factor VIII Expression in Platelets of Hemophilia A Mice Following Intraosseous Delivery of Lentiviral Vectors

In previous studies, we successfully demonstrated that following intraosseous (IO) infusion of lentiviral vectors (LVs) carrying a transgene encoding human factor VIII variant (hFVIII/N6) driven by a platelet-specific promoter (Gp1bα) into hemophilia A (HemA) mice without preconditioning, FVIII stored in platelet α-granules can partially correct phenotype over 5 months in hemophilia A mice with or without pre-existing inhibitors. In this study, we seek two strategies to further improve factor VIII expression levels in platelets. Firstly, a new human B domain-deleted FVIII variant (BDDFVIII-X10; a kind gift from Dr. Weidong Xiao) which contains a 10-amino acid change in the A1 domain of the FVIII heavy chain was generated. Following hydrodynamic injection of plasmids containing human FVIII variants (BDDFVIII-X10 or hFVIII/N6) driven by a human elongation factor-1 promoter (pEF1α-BDDFVIII-X10 or pEF1α-hFVIII/N6, 50 mg/mouse, n=6) into hemophilia A mice, BDDFVIII-X10 produced a 6-fold increase (532±18% vs 81±25%) in the clotting activity determined by an aPTT assay on day 1. Thus, four LVs containing BDDFVIII-X10 or hFVIII/N6 transgene driven by Gp1bα (G-F8X10-LV or G-F8-LV) or EF1α (E-F8X10-LV or E-F8-LV) promoter were constructed, respectively. Flow cytometry data showed that for human Dami megakaryocyte cells transduced with same dose of LVs, G-F8X10-LV produced a 2-fold increase of hFVIII+Dami cells (23.8% vs 13.4%) compared to G-F8-LV. In 293T cells, E-F8X10-LV produced a 3-fold increase of hFVIII+293T cells (42.6% vs 12.5%) compared to E-F8-LV. These results indicate that BDDFVIII-X10 may serve as a better variant candidate for gene therapy of hemophilia A. Secondly, we further explored the use of pharmacological agents to enhance lentiviral transduction efficiency by either suppressing the initial immune responses against LVs and LV-transduced cells or augmenting the frequency and number of viral entry and integration. The immune competent BL6 mice were pretreated with dexamethasone (Dex, IP injection, 5 mg/kg at -24h, -4h, 4h and 24h, n=9) or combination drugs (Dex, IP injection, 5 mg/kg at -24h, -4h, 4h and 24h; anti-CD8α antibody, IP injection, 4 mg/kg on day -1 and 2.5 mg/kg on day 4, 11, 16 and 21; rapamycin, IO infusion on day 1, 0.25 mg/kg), and IO infusion of GFP-LVs driven by an ubiquitous MND promoter (1.1×108i.f.u./mouse on day 0). On day 7, Dex only and combination drugs + LVs treated mice produced higher numbers of GFP+HSCs in the bone marrow (BM) compared with LV-only treated mice (62.4±5.0% and 50.6±5.7% vs 41.1±12.0%, P=0.003 and 0.06). Similarly, in the long term, higher numbers of GFP+HSCs were observed in BM of Dex only and combination drugs + LVs treated mice compared with LV-only treated mice (10.1±9.2% and 11.7±7.3% vs 6.0±1.6%, P=0.13 and 0.04). Taken together, these results indicate that IO infusion of LVs containing BDDFVIII-X10 into HemA mice pretreated with pharmacological drugs can be used to further enhance and prolong transgene expression levels in platelets for better correction of hemophilia A, in particular in patients with high risk of inhibitory antibody formation.

Engineered antigen-specific human regulatory T cells: immunosuppression of FVIII-specific T- and B-cell responses

AbstractExpansion of human regulatory T cells (Tregs) for clinical applications offers great promise for the treatment of undesirable immune responses in autoimmunity, transplantation, allergy, and antidrug antibody responses, including inhibitor responses in hemophilia A patients. However, polyclonal Tregs are nonspecific and therefore could potentially cause global immunosuppression. To avoid this undesirable outcome, the generation of antigen-specific Tregs would be advantageous. Herein, we report the production and properties of engineered antigen-specific Tregs, created by transduction of a recombinant T-cell receptor obtained from a hemophilia A subject's T-cell clone, into expanded human FoxP3+ Tregs. Such engineered factor VIII (FVIII)-specific Tregs efficiently suppressed the proliferation and cytokine production of FVIII-specific T-effector cells. Moreover, studies with an HLA-transgenic, FVIII-deficient mouse model demonstrated that antibody production from FVIII-primed spleen cells in vitro were profoundly inhibited in the presence of these FVIII-specific Tregs, suggesting potential utility to treat anti-FVIII inhibitory antibody formation in hemophilia A patients.

Strategies for inhibitor treatment and costs in the short and long term: a critical evaluation of recent clinical studies.

One important complication of patients with severe haemophilia A is the formation of inhibitory antibodies to factor VIII (FVIII). Immune tolerance induction (ITI) is the treatment of choice for patients with inhibitors, but this approach is successful in about 60% of patients. Treatment of acute bleeding in patients with inhibitors is one of the greatest challenges in haemophilia management and is costly. Bypassing agents are the mainstay of treatment in these patients. The aims of this study were to review the most recent publications concerning the costs of inhibitor treatment. We conducted a literature review using PubMed which yielded 63 papers analysing the costs of inhibitor management of which 12 were suitable for our study. Four of eight studies supported the use of activated prothrombin complex concentrate (aPCC) with lower costs, but the remaining four studies showed that recombinant factor VIIa (rFVIIa) had a lower average treatment cost. Of four ITI studies, two supported lifelong cost-effectiveness of ITI vs. bypassing agents and the remaining two papers showed a high cost of inhibitor treatment. Dosages, time between onset of bleeding and treatment, patient characteristics and the price of drugs are some of the important issues that should be considered for further studies.

Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells

AbstractHemophilia A is the X-linked bleeding disorder caused by deficiency of coagulation factor VIII (FVIII). To address serious complications of inhibitory antibody formation in current replacement therapy, we created tobacco transplastomic lines expressing FVIII antigens, heavy chain (HC) and C2, fused with the transmucosal carrier, cholera toxin B subunit. Cholera toxin B-HC and cholera toxin B-C2 fusion proteins expressed up to 80 or 370 µg/g in fresh leaves, assembled into pentameric forms, and bound to GM1 receptors. Protection of FVIII antigen through bioencapsulation in plant cells and oral delivery to the gut immune system was confirmed by immunostaining. Feeding of HC/C2 mixture substantially suppressed T helper cell responses and inhibitor formation against FVIII in mice of 2 different strain backgrounds with hemophilia A. Prolonged oral delivery was required to control inhibitor formation long-term. Substantial reduction of inhibitor titers in preimmune mice demonstrated that the protocol could also reverse inhibitor formation. Gene expression and flow cytometry analyses showed upregulation of immune suppressive cytokines (transforming growth factor β and interleukin 10). Adoptive transfer experiments confirmed an active suppression mechanism and revealed induction of CD4+CD25+ and CD4+CD25− T cells that potently suppressed anti-FVIII formation. In sum, these data support plant cell-based oral tolerance for suppression of inhibitor formation against FVIII.

Integration-deficient Lentiviral Vectors Expressing Codon-optimized R338L Human FIX Restore Normal Hemostasis in Hemophilia B Mice

Integration-deficient lentiviral vectors (IDLVs) have been shown to transduce a wide spectrum of target cells and organs in vitro and in vivo and to maintain long-term transgene expression in nondividing cells. However, epigenetic silencing of episomal vector genomes reduces IDLV transgene expression levels and renders these safe vectors less efficient. In this article, we describe for the first time a complete correction of factor IX (FIX) deficiency in hemophilia B mice by IDLVs carrying a novel, highly potent human FIX cDNA. A 50-fold increase in human FIX cDNA potency was achieved by combining two mechanistically independent yet synergistic strategies: (i) optimization of the human FIX cDNA codon usage to increase human FIX protein production per vector genome and (ii) generation of a highly catalytic mutant human FIX protein in which the arginine residue at position 338 was substituted with leucine. The enhanced human FIX activity was not associated with liver damage or with the formation of human FIX-directed inhibitory antibodies and rendered IDLV-treated FIX-knockout mice resistant to a challenging tail-clipping assay. A novel S1 nuclease-based B1-quantitative polymerase chain reaction assay showed low levels of IDLV integration in mouse liver. Overall, this study demonstrates that IDLVs carrying an improved human FIX cDNA safely and efficiently cure hemophilia B in a mouse model.

Ex vivo expanded autologous polyclonal regulatory T cells suppress inhibitor formation in hemophilia

Adoptive cell therapy utilizing ex vivo expanded polyclonal CD4+CD25+FOXP3+ regulatory T cells (Treg) is in use in clinical trials for the treatment of type 1 diabetes and prevention of graft versus host disease in bone marrow transplantation. Here, we seek to evaluate this approach in the treatment of inherited protein deficiencies, i.e., hemophilia, which is often complicated by antibody formation against the therapeutic protein. Treg from mice that express green fluorescent protein–marked FoxP3 were highly purified by two-step magnetic/flow sorting and ex vivo expanded 50- to 100-fold over a 2-week culture period upon stimulation with antibody-coated microbeads. FoxP3 expression was maintained in >80% of expanded Treg, which also expressed high levels of CD62L and CTLA-4. Transplanted Treg suppressed inhibitory antibody formation against coagulation factors VIII and IX in protein and gene therapies in strain-matched hemophilia A and B mice, including in mice with pre-existing antibodies. Although transplanted Treg became undetectable within 2 weeks, suppression persisted for >2 months. Additional studies suggested that antigen-specific suppression emerged due to induction of endogenous Treg. The outcomes of these studies support the concept that cell therapy with ex vivo expanded autologous Treg can be used successfully to minimize immune responses in gene and protein replacement therapies.

Targeting Long-Lived Plasma Cells Is Essential For Treating Hemophilia A Inhibitors

The most critical and challenging problem for therapeutic treatment in hemophilia A patients is due to the formation of Inhibitory antibodies. It is hypothesized that long-lived plasma cells (LLPCs) play an important role in the persistent production of anti-FVIII antibodies in hemophilia A (HemA) inhibitor patients. The migration of plasma cells to the BM, where they become the LLPCs, is largely controlled by an interaction between the C-X-C type chemokine ligand 12 (CXCL12) produced by bone marrow (BM) stromal cells and its receptor CXC receptor4 (CXCR4; CD184) on plasma cells surface. Our previous data showed that administration of anti-murine CD20 (IgG2a) alone can deplete B cells significantly and reduce anti-FVIII inhibitor titers transiently in FVIII plasmid-treated HemA mice with pre-existing inhibitors, however, complete tolerance to FVIII was not achieved probably due to the persistence of LLPCs. We sought novel therapeutic strategies that target CXCL12/CXCR4 pathway to reduce/eliminate LLPCs and achieve the goal for long-term tolerance to FVIII in the HemA inhibitor mice. AMD3100, the CXCR4 antagonist, plus G-CSF inhibit the interaction of CXCL12 and CXCR4, thus facilitating the mobilization of stem cells and blocking the homing and retention of LLPCs. The combined treatment strategy used anti-CD20, G-CSF and AMD3100 to target B cells plus with the specific IL-2/IL-2mAb (JES6-1) complexes to target T cells for preventing both T and B cell-dependent anti-FVIII immune responses. Two groups of FVIII-primed inhibitor mice were treated with different combined immunomodulation regimens: (1) IL-2 complexes+AMD3100+G-CSF+anti-CD20, (2) AMD3100+G-CSF+anti-CD20. Control mouse groups were treated with each of the single regimens and FVIII only, or untreated as the naive control. All the treatments were administered one cycle per two weeks for 6 weeks and the therapeutic effects (FVIII activities) as well as immune responses (anti-FVIII inhibitors) were evaluated at different time points after treatment. Significant expansion of Treg cells reaching a 5∼7-fold increase on the peak days (day 3-7 after treatment) was observed in the IL-2/IL-2mAb complexes treated groups, whereas ∼95% of B cell populations were depleted in the anti-CD20 treated groups. In addition, administration of AMD3100 plus G-CSF significantly reduced circulating and bone marrow CXCR4+ plasma cells by flow cytometry analysis as well as decreased FVIII-specific plasma cells in bone marrow by ELISPOT assay. Except for the control groups, the two mouse groups treated with combined immunosuppressive regimens showed a significant reduction of inhibitory titers following the treatment. Long-term responses are being followed and second challenge with FVIII plasmid will be used to evaluate the induction of long term tolerance to FVIII. These combination regimens are highly promising in modulating/eliminating pre-existing anti-FVIII antibodies and inducing long-term tolerance in FVIII primed subjects. Disclosures:No relevant conflicts of interest to declare.

Limited Promiscuity of HLA-DRB1 Presented Peptides Derived of Blood Coagulation Factor VIII

The formation of inhibitory antibodies directed against coagulation factor VIII (FVIII) is a severe complication in the treatment of hemophilia A patients. The induction of anti-FVIII antibodies is a CD4+ T cell-dependent process. Activation of FVIII-specific CD4+ T cells is dependent on the presentation of FVIII-derived peptides on MHC class II by antigen-presenting cells. Previously, we have shown that FVIII-pulsed human monocyte-derived dendritic cells can present peptides from several FVIII domains. In this study we show that FVIII peptides are presented on immature as well as mature dendritic cells. In immature dendritic cells half of the FVIII-loaded MHC class II molecules are retained within the cell, whereas in LPS-matured dendritic cells the majority of MHC class II/peptide complexes is present on the plasma membrane. Time-course studies revealed that presentation of FVIII-derived peptides was optimal between 12 and 24 hours after maturation but persisted for at least 96 hours. We also show that macrophages are able to internalize FVIII as efficiently as dendritic cells, however FVIII was presented on MHC class II with a lower efficiency and with different epitopes compared to dendritic cells. In total, 48 FVIII core-peptides were identified using a DCs derived of 8 different donors. Five HLA-promiscuous FVIII peptide regions were found – these were presented by at least 4 out of 8 donors. The remaining 42 peptide core regions in FVIII were presented by DCs derived from a single (30 peptides) or two to three donors (12 peptides). Overall, our findings show that a broad repertoire of FVIII peptides can be presented on HLA-DR.

Antigenic liposomes displaying CD22 ligands induce antigen-specific B cell apoptosis

Antibodies confer humoral immunity but can also be harmful when they target an autoantigen, alloantigen, allergen, or biotherapeutic. New strategies are needed for antigen-specific suppression of undesired antibody responses, particularly to T cell-dependent protein antigens, because they elicit T cell help. Here we show that liposomal nanoparticles, displaying both antigen and glycan ligands of the inhibitory coreceptor CD22, induce a tolerogenic program that selectively causes apoptosis in mouse and human B cells. These SIGLEC-engaging tolerance-inducing antigenic liposomes (STALs, where SIGLEC is defined as sialic acid-binding Ig-like lectin) induced robust antigen-specific tolerance to protein antigens in mice, preventing subsequent immune response to challenge with the same antigen. Since development of inhibitory antibodies to FVIII is a serious problem in treatment of hemophilia A patients, we investigated the potential of this approach for inducing tolerance to FVIII in a hemophilia mouse model. STALs prevented formation of inhibitory FVIII antibodies, allowing for effective administration of FVIII to hemophilia mice to prevent bleeding. These findings suggest that STALs could be used to eliminate or prevent harmful B cell-mediated immune responses.