Production Of anti-FVIII Antibodies Research Articles

Marginal Zone B Cells Facilitate CD4 T Cell Proliferation in Response to Factor VIII in Mice with Hemophilia Α

BACKGROUND: The development of anti-factor VIII (FVIII) antibodies represents a significant barrier to FVIII replacement therapy in patients with hemophilia A. Despite this, the immune factors that regulate anti-FVIII antibody formation remain incompletely understood. We have previously shown that shortly after injection, FVIII co-localizes with marginal zone (MZ) B cells in the marginal sinus of the spleen in mice with hemophilia A. Moreover, mice depleted of MZ B cells did not produce detectable anti-FVIII IgG antibodies. Given the ability of MZ B cells to trap and rapidly respond to blood-borne antigens, produce both IgM and IgG antibodies and activate CD4 T cells, while also trafficking antigen to B cell follicles, these results suggest that MZ B cells likely play a critical role in the initiation and subsequent orchestration of an immune response to FVIII. In contrast to other model antigens, anti-FVIII antibody formation occurs only following multiple FVIII exposures in both human and mouse models. As CD4 T cells facilitate long-lasting antibody formation, and have previously been shown to play an important role in anti-FVIII antibody development (Bray GL, et al. Am J Hematol 1993; Reding MT, et al. Thromb Haemost 2000), it is possible that each FVIII exposure event eventually propagates a CD4 T cell response to a threshold necessary to generate an optimal antibody response. Consistent with this, we have previously shown that FVIII-specific CD4 T cell proliferation requires more than 1 prior exposure to FVIII. An improved understanding of the overall timing and factors influencing CD4 T cell activation in the immune response to FVIII is necessary for the development of strategies to prevent antibody formation. OBJECTIVE: Define the key factors that regulate CD4T cell activation following FVIII exposure and the number of exposure events required to observe FVIII-specific CD4 T cell proliferation. METHODS: Hemophilia A mice +/- CD4T cell depletion or MZ B cell depletion or S1PR1-TSS mice were challenged with 1-4 weekly exposures to FVIII. Anti-FVIII IgM and IgG was examined in plasma by enzyme-linked immunoassay (ELISA). To assess FVIII-specific CD4T cell proliferation, we engineered FVIII-OVA, which contains the ovalbumin peptide recognized by OTII T cell receptor transgenic T cells. CFSE-labeled OTII splenocytes were adoptively transferred into mice with hemophilia A, mice lacking B cells and MZ B cell-depleted mice, followed by administration of FVIII-OVA. RESULTS: Anti-FVIII IgG formation is CD4T cell-dependent, however IgM is readily produced in the absence of CD4T cells. In contrast, MZ B cell depletion prevented the formation of both IgM and IgG antibodies against FVIII. FVIII is unable to cause proliferation of CD4T cells or produce IgG antibodies following initial exposure, but instead significantly increases following the 3rd exposure to FVIII. Mapping the kinetics of antibody formation has uncovered that anti-FVIII IgM formation peaks and anti-FVIII IgG formation begins after the 3rd FVIII exposure, at the time of CD4 T cell proliferation. We next examined the influence of B cells on CD4 T cell proliferation, and observed that FVIII-specific CD4T cell proliferation is significantly impaired in B cell-deficient recipients. Moreover, in mice who have undergone MZ B cell depletion, there is likewise an absence of anti-FVIII IgM and IgG and a lack of measurable CD4 T cell proliferation. Finally, in S1PR1-TSS mice with MZ B cells that are unable to traffic antigen to the B cell follicle, anti-FVIII IgM and IgG formation occurs at the same rate as control mice. CONCLUSION: In summary, our findings suggest that MZ B cells are required for anti-FVIII IgM and IgG antibody formation as well as FVIII-specific CD4 T cell proliferation, which requires 3 antigen exposures. The finding that MZ B cells are not required to traffic antigen to the B cell follicle contributes to our hypothesis that MZ B cell production of IgM, with or without direct activation of CD4 T cells contributes to FVIII-specific CD4 T cell activation. These results suggest that targeted approaches designed to prevent anti-FVIII antibody formation may be most effectively achieved by defining key factors that regulate MZ B cell activation following initial FVIII exposure. In doing so, the present findings and future work will likely uncover important pathways to effectively target to reduce, or event prevent anti-FVIII antibody production.

IDO1 activity in selected immune cells controls antibody responses

Abstract Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme involved in the initial step of tryptophan degradation along the kynurenine pathway. It has immunosuppressive effects, linked to enzymic and non-enzymic regulatory functions. IDO1 is also crucial for sustaining the function of T regulatory cells (Treg), which are involved in the establishment of peripheral tolerance. We recently found that defective IDO1 induction is associated to anti-FVIII antibody production in patients with severe Hemophilia A1. In hemophilic mice, CpG- rich oligodeoxynucleotides (CpG-ODN) administration induced IDO1 expression in dendritic cells (DCs) that control FVIII antibody response. Recently we discovered that CpG-ODN selectively induced IDO1 in a subset of conventional DCs (cDCs). IDO1 was found to be highly expressed in gut CD11C+CD103+ DCs, which are required for establishment of oral tolerance2. Based on these data we analyzed the impact of IDO1 deficiency in controlling antibody responses, in two different experimental models (FVIII and ovalbumin, OVA, immunization). Interestingly, IDO1 deficiency resulted in a significant increase of FVIII-specific antibody production relative to wild type controls. Similarly, IDO KO mice showed a significant immunoglobulin production in a model of OVA-induced oral tolerance. These data suggest the regulatory role of IDO1 in controlling antibody responses to self and non-self antigens.

Circulating FVIII-specific IgG, IgA and IgM memory B cells from haemophilia A patients.

Approximately, 25% of haemophilia A (HA) patients treated by factor VIII (FVIII), develop antibodies, known as inhibitors, neutralizing the activity of infused FVIII. This immune response involves B cells (BC), including FVIII-specific memory B cells (MBC). Production of anti-FVIII antibodies after stimulation of FVIII-specific MBC suggests a role of these cells in the immune response to FVIII. Animal models allowed the study of circulating FVIII-specific cells, however few data are available on HA patients. In the present study, we simultaneously detected, via ELISpot assay, different isotypes of MBC in the blood of HA patients, after polyclonal activation. Patients included: three with active inhibitors; three with a history of inhibitors; six without any past or active inhibitor. FVIII-specific MBC were detected in peripheral blood of HA patients: (i) patients with active inhibitors (IgG: 4-5.2/10(6) BC; IgA: 2.9-4/10(6) BC) (ii) patients with a past of inhibitors (no IgG BC; IgA: 5-7.5/10(6) BC) (iii) patients without inhibitors (no IgG BC or IgA BC except one patient had two FVIII-specific IgA BC/10(6) BC). FVIII-specific IgA MBC were detected in HA patients with past and current immune responses against FVIII and FVIII-specific IgG MBC were found only in those with positive inhibitors. This study shows the possibility to detect and characterize easily and simultaneously the MBC from patient blood and that MBC seem different according to anti-FVIII immune history. It could be a useful tool to study anti-FVIII response and Immune Tolerance Induction cellular mechanisms.

434. Adoptive Treg Cell Therapy Using Factor VIII-Specific CAR Regulatory T Cells Regulates Anti-Factor VIII Immune Responses in Hemophilia A Mice

The immune response to factor VIII (FVIII; F8 in constructs) protein limits the effectiveness of treatments for hemophilia A (HemA) patients. Our previous studies demonstrated that regulatory T cells (Tregs) play a pivotal role in modulation of anti-FVIII immune responses. In particular, adoptive transfer of Tregs isolated from FVIII-primed HemA/Foxp3 mice attenuated anti-FVIII immune responses induced by gene transfer of FVIII plasmid in HemA mice. For developing adoptive Treg therapy, we successfully expanded FVIII-sensitized polyclonal Tregs using an FVIII-specific expansion protocol in vitro and showed that these cells had increased FVIII-specific suppressive activity compared with nonspecific Tregs. However, FVIII-specific Tregs in the polyclonal population are still in very small numbers. In this study, we explored the strategy to generate FVIII-specific Tregs using the chimeric antigen receptor (CAR) approach. Lentiviral vector (LV) incorporating a high-binding anti-FVIII antibody-derived variable region (scFv) linked to signaling and costimulatory moieties of immune receptors (third generation CAR) and fused with a murine Foxp3 cDNA (F8CAR-Foxp3-LV) was prepared and used to transduce murine CD4+T cells. Flow cytometry analysis confirmed extracellular scFv and intracellular Foxp3 expression in transduced cells (F8CAR-Tregs). In vitro suppressive assay showed that transduced CD4+T cells had significantly higher FVIII-specific suppressive activity than untransduced cells towards FVIII-specific CD4+ effector T cells (Teffs). In addition, 1×106 transduced cells and untransduced cells were adoptively transferred into HemA mice. One day after cell transfer, the treated mice were challenged with FVIII plasmid injected hydrodynamically. The anti-FVIII antibody titers are evaluated overtime. It is expected that F8CAR-Foxp3-LV transduced cells will prevent or decrease the production of anti-FVIII antibodies. We have also prepared a LV incorporating only the F8CAR region (F8CAR-LV). CD4+CD25− and CD4+CD25+ cells isolated from HemA mice were transduced with F8CAR-LV to generate FVIII-specific Teffs and Tregs, respectively. In vitro FVIII-specific suppressive assays using CD4+ Teffs from FVIII-primed HemA mice or F8CAR-LV transduced Teffs as responder cells are performed to compare the suppressive function of F8CAR-LV transduced CD4+CD25+ cells and F8CAR-Foxp3-LV transduced CD4+ cells. CD4+CD25+ cells isolated from FVIII-primed HemA mice and untransduced CD4+CD25+ and CD4+ cells from HemA mice are used as control cells. These experiments compare the extent of suppression towards specific F8CAR Teffs and polyclonal FVIII-specific Teffs as well as the potency of suppressive function between two differently engineered F8CAR Tregs. Finally, adoptive transfer experiments into HemA mice using the transduced and control cell populations are performed to evaluate their in vivo function to protect the HemA mice from anti-FVIII antibody production. We anticipate that compared with nonspecific and polyclonally expanded Tregs, FVIII-specific CAR Tregs will exert superior suppressive activity towards anti-FVIII immune responses without triggering systemic immune suppression.

Factor VIII-specific CAR regulatory T cells modulate murine anti-factor VIII immune responses

Abstract The immune response to factor VIII protein (FVIII; F8 in constructs) limits the effectiveness of treatments for hemophilia A (HemA) patients. Previously we demonstrated that regulatory T cells (Tregs) play a pivotal role in modulating anti-FVIII immune responses. For application of adoptive Treg therapy, we successfully expanded highly suppressive murine polyclonal Tregs antigen specifically in vitro. However, the FVIII-specific Tregs in the polyclonal population are still in very small numbers. Thus, we explored the strategy to generate FVIII-specific Tregs using the chimeric antigen receptor (CAR) approach. Lentiviral vector (LV) incorporating a high-binding anti-FVIII scFv linked to the CAR signaling domains and fused with a Foxp3 cDNA (F8CAR-Foxp3-LV) was prepared and used to transduce murine CD4+T cells. Flow cytometry analysis confirmed extracellular scFv and intracellular Foxp3 expression in transduced cells (F8CAR-Tregs). In vitro FVIII-specific suppressive assay showed that transduced cells had significantly higher suppressive activity than untransduced cells towards murine effector T cells. In addition, 1×106 transduced cells and untransduced cells were adoptively transferred into HemA mice and the treated mice were subsequently challenged with FVIII plasmid injected hydrodynamically. The anti-FVIII antibody titers are evaluated overtime. It is expected that F8CAR-Foxp3-LV transduced cells will prevent or decrease the production of anti-FVIII antibodies in HemA mice. We anticipate that compared with nonspecific or polyclonally expanded Tregs, FVIII-specific CAR Tregs will exert superior suppressive activity towards anti-FVIII immune responses without triggering systemic immune suppression.

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.

522. Targeting FVIII-Expression To Liver Sinusoidal Cells By Lentiviral Vectors Corrects the Bleeding Phenotype in Hemophilia A Overcoming Immunological Responses

Hemophilia A (HA) is an X-linked bleeding disorder due to mutations in clotting factor (F) VIII gene. To date the treatment for preventing major bleeding episodes is represented by replacement therapy with recombinant or plasma-derived FVIII. The two major concerns are high cost and development of FVIII neutralizing antibodies in 20-30% of patients.Several studies on gene transfer by direct injection of LV for HA have been recently published. Many efforts were focused on the improvement of LV, to obtain a selective targeting of transgene expression, or on the production of several bioengineered FVIII, in order to overcome some of the issues related to FVIII expression in HA animal models. However, in most cases, the immune responses associated with FVIII remain the major obstacle.We prepared LVs containing the B-domain deleted (BDD) hFVIII under the control of PGK, VEC or CD11b promoters with or without the addition of the miRTs used for initial GFP expression studies, and we then injected HA mice with 109 TU/mouse of these LVs (3 mice for LV PGK-hFVIII ±42; 4-9 mice for the other vectors) and assessed FVIII activity by aPTT assay.All mice injected with LV-VEC-hFVIII ± miRTs and LV-CD11b-hFVIII ± miRTs showed a FVIII activity between 3.5 and 5% one week after injection, while HA mice injected with LV-PGK-hFVIII± 42 showed a FVIII activity £1%. Moreover, starting from 2 weeks after LVs injection we evaluated the presence of anti-FVIII antibodies by a direct ELISA. We detected the presence of anti-FVIII antibodies in the plasma of mice injected with LV-PGK-hFVIII±miRT-142 1 month after LV injection. Interestingly, the antibody titer was significantly lower in mice injected with LV-PGK-hFVIII-miRT-142-3p. In all mice injected with LV-VEC-hFVIII±miRT-122-142-3pwe detected hFVIII activity by aPTT assay up to 52 weeks after injection without production of anti-FVIII antibodies. HA mice injected LV-CD11b-hFVIII±miRT-126 showed hFVIII activity up to 52 w as well; interestingly, 60% of mice injected with LV-CD11b-hFVIII produced anti-FVIII antibodies 10-16 weeks after LV injection, while no anti-FVIII antibodies were detected in plasma of injected mice with LV-CD11b-hFVIII-miRT-126.Genomic analysis on liver samples from mice 24 w after injection of LV-VEC-hFVIII±miRT-122-142-3p and LV-CD11b-hFVIII±miRT-126 demonstrated the presence of LV sequence integrated in the genome of injected mice. Immunofluorescence on liver sections showed that LSECs and KCs were positive for hFVIII. Next, to assess whether EC, in particular LSECs, are able to induce immunotolerance, we immunized mice with Refacto. Mice producing anti-FVIII Ab were then injected with 109 TU of LV-VEC-hFVIII-miRT-122-142-3p. We detected hFVIII activity in all injected mice and, noteworthy, antibody titer decreased over time in the plasma of these mice.In conclusion, LV expressing FVIII under the control of VEC or CD11b promoters combined with miRTs combinations were able to overcome FVIII off-target expression limiting immune responses and providing phenotypic correction in treated HA mice with FVIII expression by sinusoidal cells.

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.

A “Biomedical Analytics” Approach Suggests a Role For TNF Superfamily Signaling In Acquired Hemophilia Patients Who Do Not Respond To Rituximab Treatment

Acquired hemophilia (AH) is a rare, life-threatening, bleeding disorder caused by autoantibodies that inhibit the clotting function of FVIII (Inhibitors). A major challenge in AH management is that patients vary in their responses to treatments (immunosuppressive agents such as cyclophosphamide, corticosteriods, azothioprine, and rituximab; alone or in combination). In-depth research of improved methods of treatment has been hampered by the very low incidence of this disease (1:1x106), as well as the presence of co-morbidities including autoimmune disorders and malignancies. To overcome these limitations, we employed a novel analytic approach that takes into account individual patient characteristics and response to treatment over time to predict biologic mechanisms that may contribute to outcome.As part of our IRB approved study to determine immune factors associated with outcome of anti FVIII inhibitor antibody treatment in AH patients, we measured cytokine secretion in plasma collected at the start of therapy and at various time points during treatment and follow-up. Of 12 patients, 11 were treated with rituximab (anti-CD20), either alone or in combination with cyclophosphamide and/or corticosteroids. One patient achieved spontaneous remission. Data were stratified according to time post-treatment and plasma measurements of cytokines were compared between non-responders (NR; n=6) and complete responders (CR; n=6) using logistic regression analysis. Statistical significance was confirmed via two-sample Mann-Whitney test (Stata/IC). A higher baseline (time 0) secretion of plasma GCSF, an increase in CCL2 within the first 30 days of treatment (time 1), and increased BAFF > 181 days post treatment (time 5) were associated with NR. We next determined the pmol/L concentrations of these cytokines (termed our “significance set”) measured in individual patients (n=10) at times 0, 1, and 5. Each patient's significance set was used as input in Ingenuity Pathway Analysis, and patient-specific networks of predicted molecular interactions (set to the 35 closest neighbors) were generated at each time. We then compared the generated networks across time and between patients to determine similarities and differences in predicted molecules.The majority of the predicted outcomes were shared among all patients, and included members of the TNF superfamily, immunoglobulins, co-stimulatory molecules, and chemokines. As members of the TNF superfamily are crucial for B cell survival and signaling, we focused on the predicted appearance and change over time of these molecules. BCMA (TNFRSF 17), BAFF-R (TNFRSF 13C), and APRIL (TNFSF 13) were predicted to be expressed by all patients at time 1, corresponding to the period of time where B cells are depleted by rituximab and survival signals are required for repopulation. This expression was predicted to continue through time 5. At the latter time, another TNF superfamily ligand, LTα1β2, was predicted to occur in 3/5 NR, but only 1/5 CR. The LTα1β2 receptor, LTβR, was also predicted to be expressed at this time. Intriguingly, LIGHT (TNFSF 14), which also binds LTβR, was predicted to be expressed in 6/10 patients at baseline. As signaling through LTβR can activate both the classical and noncanonical NFκb pathways and exacerbate inflammation, it is intriguing to speculate that LIGHT/LTα1β2/LTβR may be associated with development of AH. Moreover, stimulation of B cells via this pathway, in addition to signaling through the BAFF-R may render B cells less responsive to rituximab treatment. Therefore, drugs that target TNF superfamily members might represent a new treatment option for AH patients who do not respond to conventional therapies. Disclosures:Off Label Use: Patients with acquired hemophilia were treated with rituximab cyclophosphamide and/or corticosteroids to decrease anti-FVIII antibody production.

Development of Direct Intra-Bone Marrow Gene Transfer of Lentiviral Vectors Containing a B-Domain Variant of Human Factor VIII for Hemophilia A Treatment

Abstract 4710Lentiviral vectors (LVs) directly delivered to the bone marrow by intra-bone marrow (iBM) injection can efficiently transduce hematopoietic stem cells (HSCs) for treating genetic blood disorders. Our previous study showed that FVIII activity in the plasma of hemophilia A (HemA) mice achieved nearly normal levels one week after being treated with iBM injection of LVs containing a B-domain variant human factor VIII (hFVIII/N6) gene driven by a human elongation factor-1 promoter (E-LV). However, plasma FVIII activity fell very quickly with concomitant generation of anti-FVIII antibodies. In this study, to prevent anti-FVIII immune responses in the treated HemA mice, we treated three groups of mice with intraperitoneal injection of 200 μL of immunomodulatory protocols including IL-2/IL-2 mAb complexes (IL-2 Complex, 1 μg IL-2 mixed with 5 μg anti-IL-2 mAb per mouse on day −5, −4, and −3), anti-mouse CD3ε mAb (20 μg per mouse on day −2, −1, 0, 1 and 2), and rabbit anti-mouse thymocyte IgG (ATG, 0.6 mg per mouse on day −1 and 0), respectively. The mice were treated with 5 μL E-LV (7 × 107 TU/mL) on day 0. The peak plasma FVIII levels in 0/4 E-LV only treated mice, 3/5 IL-2 Complex pretreated mice, 2/3 anti-CD3ε pretreated mice and 4/5 ATS pretreated mice showed up one month after treatment. 2/4 E-LV only treated mice, 2/5 IL-2 Complex pretreated mice, 1/3 anti-CD3ε pretreated mice and 3/5 ATS pretreated mice generated anti-FVIII antibodies two months after treatment. These data indicated that all three immunomodulatory protocols protected E-LV transduced cells from eradication mediated by cytotoxic T cells in the early stage after treatment; however these protocols did not prevent the production of anti-FVIII antibodies at later time points. Next, we employed 10 HemA/Foxp3-Tg mice in which all T cells over-expressed Foxp3 as an immunosuppressive mouse model and treated them with low (5 μL) or high (25 μL) dosage of E-LVs, respectively. None of the 10 treated HemA/Foxp3-Tg produced anti-FVIII antibodies however plasma FVIII levels in all 10 mice fell to low levels one month after treatment. Thus, increase of regulatory T cells could not totally inhibit cytotoxic T cells to lyse transduced cells, whereas they prevented anti-FVIII antibody production. In order to examine whether LV-transduced cells were destroyed in the early stage post-injection, we treated HemA mice with 3 μL of LVs (4.6 × 1010 TU/mL) containing GFP gene driven by a modified myeloid proliferative sarcoma virus (MND) promoter and tracked GFP expression in bone marrow cells overtime. The percentage of GFP positive cells reached highest on day 15 post-injection, and then fell quickly afterwards, while GFP positive cells (0.04% of total bone marrow cells) were still detectable on day 94. Therefore, in order to achieve persistent transgene expression of hFVIII in the HemA mice treated with intra-bone marrow injection of E-LVs, cytotoxic-T-cell-mediated elimination of the transduced cells needs to be inhibited in the early stage and formation of anti-FVIII antibody can be eliminated by increasing FVIII-specific Treg cell populations. Disclosures:No relevant conflicts of interest to declare.

In Vivo Expansion of Treg Cells Prevents Anti-FVIII Antibody Production Following FVIII Protein Replacement Therapy in Hemophilia A Mice

Abstract 23▪▪This icon denotes a clinically relevant abstractInhibitory antibodies formation is a major complication following protein replacement therapy for hemophilia A. Interleukin (IL)-2 mixed with a particular IL-2 monoclonal antibody (mAb, JES6-1) can induce selective expansion of regulatory T (Treg) cells. In order to address the question whether in vivo expansion of Treg cells can modulate FVIII-specific immune responses following FVIII protein replacement therapy, we treated hemophilia A mice with IL-2/IL-2 mAb complexes (6 μg/mouse) three times per week for 4 weeks, and concurrently with BDD-FVIII protein (0.3U/mouse) three times per week for 4 weeks, followed by BDD-FVIII (1U/mouse) once per week consecutively for another 14 weeks. Compared to control mice (n=3) which produced high-titer anti-FVIII antibodies after treatment with BDD-FVIII protein only for 4 weeks, mice treated with IL-2/IL-2 mAb complexes + BDD-FVIII protein produced no inhibitory antibody titers against FVIII over time. PC61 is an anti-murine CD25 antibody which depletes CD4+ CD25+ Treg cells. Mice (n=4) treated with IL-2 complexes + BDD-FVIII protein + PC61 for 4 weeks produced high-titer inhibitory antibodies. These data indicate that immunomodulation by 4-week treatment of IL-2/IL-2 mAb complexes successfully suppressed anti-FVIII immune responses by in vivo expanded Treg cells, and induced long-term tolerance to FVIII. A marked 5–7-fold increase in percentages and total numbers of CD4+ Foxp3+ Treg cells and Foxp3+ Helios+ T cells were observed in the peripheral blood, spleen and lymph nodes on the peak days during IL-2/IL-2 mAb complexes treatment indicating that most of the expanded Treg cells are thymically derived natural Treg (nTreg) cells instead of peripherally induced adaptive Treg (aTreg) cells. Treg cells maintained at high levels for 4 weeks, and these levels gradually returned to normal within the next 7–14 days. Also, the expanded Treg cells showed a considerable increase in the expression of molecules crucial to their suppressive function, including CD25, glucocorticoid induced TNFR (GITR) and cytotoxic T lymphocyte antigen 4 (CTLA-4) relative to Treg cells from the control mice. The expansion of CD4+ Foxp3+ populations and their suppressive functions were examined and supported by suppressive, proliferative and cytokines assays using splenic cells isolated from IL-2/IL-2 mAb + FVIII treated mice. In a separate experiment, mice treated with IL-2/IL-2 mAb complexes + full-length FVIII protein for consecutive 4 weeks generated neither inhibitory nor non-inhibitory antibodies against FVIII up to day 60 post treatment. However, these mice produced anti-FVIII antibodies following a second challenge of 4 week infusions of full-length FVIII protein at a lower titer than mouse groups treated with IgG2a isotype control antibody + FVIII and FVIII only. Our results demonstrate the important role of Treg cells in suppressing anti-FVIII immune responses. Treatment of IL-2/IL-2 mAb complexes represents a highly promising strategy for prevention of anti-FVIII antibody formation following either the BDD-FVIII or full-length FVIII protein replacement therapy in hemophilia A mice. Disclosures:No relevant conflicts of interest to declare.

Potential Immunogenicity of Amino Acid Sequences Encoded by Ns-SNPs in Factor VIII,

Abstract 3329Development of inhibitory antibodies that block factor VIII (FVIII) pro-coagulant function requires T-cell help. In order for T-cell stimulation to occur, one or more FVIII-derived peptides must first bind effectively to MHC Class II (e.g. HLA-DRB1) receptors on the surface of antigen presenting cells. If this Class II-peptide complex is recognized by a receptor on a helper T cell, then high-avidity Class II-peptide-T-cell receptor interactions can (but may not) cause proliferation and cytokine secretion leading to anti-FVIII antibody production. Thus the initial binding of a FVIII peptide to a Class II receptor can initiate an HLA-restricted cascade of events that leads to inhibitor formation in hemophilia A (HA) patients. Non-hemophilic, non-synonymous single nucleotide polymorphisms (ns-SNPs) in the human F8 gene were identified previously (Viel et al., Blood 109:3713–24, 2007) that result in the FVIII sequence variations R484H, R776G, D1241E and M2238V. Recombinant FVIII proteins with R484, R776, either D1241 or E1241, and M2238 are currently used therapeutically. The potential immunogenicity of FVIII peptides corresponding to these sequence variations is now being analyzed directly using in vitro assays employing cellular fractions of HA blood, recombinant proteins and synthetic peptides. These studies are motivated by a concern that some HA patients who express even very low levels of their endogenous, hemophilic FVIII, which should tend to promote immune tolerance to exogenous FVIII, could in principle develop inhibitors provoked solely by amino acid sequence mismatch(es) at sites in FVIII encoded by ns-SNPs. This could happen in patients with specific HLA types if their corresponding Class II receptors presented a FVIII peptide that then stimulated a helper T-cell response.Synthetic FVIII peptides containing both alternate sequences encoded by these 4 ns-SNPs were tested for binding to the monomeric extracellular domains of 10 common DRB1 alleles using competitive binding assays. The results are as follows: (1) 484: R484 peptides had strong affinity (IC50 < 1 mM) for DRB1*15:01 and DRB1*03:01; H484 peptides had moderate affinity (IC50 = 1–10 mM) for DRB1*03:01 and DRB1*15:01; both R484 and H484 peptides bound with moderate affinity to DRB1*11:04 and DRB1*04:04; (2) 776: R776 and G776 peptides had moderate affinity for DRB1*09:01; (3) 1241: E1241 peptides had strong affinity for DRB1*09:01 and moderate affinity for DRB1*01:01, D1241 peptides bound weakly (IC50 = 30–50 mM) to DRB1*09:01 and DRB1*01:01; (4) 2238: Both M2238 and V2238 peptides had strong affinity for DRB1*03:01 and moderate affinity binding to DRB1*01:01, DRB1*11:04, DRB1*11:01 and DRB1*04:04. Results of the binding assays compared reasonably well with computer-predicted affinities of the peptides for some of the better-characterized HLA-DRB1 alleles.T-cell stimulation and MHC Class II tetramer staining experiments (which can unambiguously identify antigen-specific T cells) were next carried out using CD4 T cells from (1) 4 HA subjects (1 with a current inhibitor, 1 with a previous inhibitor and 2 without inhibitors) whose F8 gene encoded the V2238 variant and therefore had been treated with a therapeutic FVIII produce that was mismatched at this site with respect to the endogenous sequence encoded by their hemophilic F8 gene; and (2) from 4 HA subjects with the M2238 variant. All 8 patients expressed either the DRB1*03:01 or DRB1*11:01 alleles (one expressed both). No peptides corresponding to regions encoded by either version of this ns-SNP were recognized as T-cell epitopes by CD4 cells from these subjects, despite moderate to strong binding affinity of the peptides for DR0301 and DR1101 proteins. This suggests that the immunogenicity of these FVIII regions in HA patients who express these F8 and HLA genotype combinations may be less than might have been anticipated based on the binding data.HA patients with HLA-DRB1 receptors that do not effectively bind peptides encoded by ns-SNPs in the F8 gene would be expected to have a low risk of developing helper T-cell responses to FVIII sequences encoded by these ns-SNPs. Thus, the negative as well as positive peptide-binding assay results have significant clinical and pharmaceutical relevance. Results of assays such as those described above could be useful in developing improved methods to assess the relative risk of individual HA patients developing an inhibitor. Disclosures:Pratt:Bayer, CSL Behring, Pfizer: Research Funding; Puget Sound Blood Center: Patents & Royalties.

550. Depletion of B Cells by Anti-CD20 Partially Regulates Anti-FVIII Antibody Production in the Nonviral Gene Therapy Model

550. Depletion of B Cells by Anti-CD20 Partially Regulates Anti-FVIII Antibody Production in the Nonviral Gene Therapy Model

Reduced Immunogenicity of FVIII Peptides by Rational Modifications of An Immunodominant T-Cell Epitope.

Abstract 220 Introduction.The development of antibodies that interfere with factor VIII (FVIII) pro-coagulant activity, often referred to as “inhibitors”, can complicate the treatment of hemophilia A. These alloimmune responses, as well as the rare development of autoimmune FVIII inhibitors, are associated with significant morbidity and mortality. The production of anti-FVIII antibodies follows stimulation of helper T cells by epitopes in FVIII. An immunodominant HLA-DRB1*0101-restricted T-cell epitope was recognized by CD4+ T cells from a mild hemophilia A inhibitor subject and from his brother, who had a sub-clinical inhibitor (James et al., J Thromb Haemost 5: 2399-2407, 2007). Their CD4+ T cells recognized overlapping synthetic peptides with sequences corresponding to FVIII residues 2186-2205, 2187-2205 and 2194-2213. Nineteen T-cell clones recognizing this epitope were isolated, with phenotypes representing four distinct T-cell lineages. Aims:(1) to evaluate the promiscuity/immunodominance of an HLA-DRB1*0101-restricted T-cell epitope in FVIII; (2) to introduce amino acid substitutions that will prevent presentation of this epitope to the immune system by DR0101 and by other DR alleles. Methods.The minimal epitope and MHC Class II (DR0101) “anchor” residues were determined using a competition assay measuring displacement of a labeled peptide having high affinity for recombinant DR0101 by a series of FVIII peptides. Peptide concentrations at which 50% inhibition of the labeled peptide binding occurred (IC50) were obtained by regression analysis. Binding of the peptides to five additional DR alleles was evaluated directly using recombinant proteins; predicted binding of peptides to additional DR alleles was evaluated using the program ProPred. Proliferation and cytokine production by the clones in response to wild-type and modified peptides were measured, and the concentrations at which half-maximal T-cell responses (EC50) to the FVIII peptides occurred were determined. Results.Binding of truncated peptides to DR0101 identified FVIII2194-2205 as the minimal epitope. Binding of FVIII2194-2205 peptides with single Arg substitutions identified F2196, M2199, A2201 and S2204 as anchor residues at positions 1, 4, 6 and 9, respectively, corresponding to peptide-binding pockets seen in the crystal structure of a DR0101-peptide complex. The relative binding of Ala-substituted peptides confirmed that F2196 and M2199 are anchor residues. T-cell stimulation requires recognition of peptides by both the Class II receptor and the T-cell receptor (TCR). Sequences of TCR variable regions (TCRBVs) expressed by the clones were identified as TCRBV20-1*01 (3 VDJ combinations), TCRBV6-6*01, TCRBV5-1*01, and TCRBV6-1*01, indicating at least six different T-cell progenitors recognized this epitope. The clones were next stimulated with peptides having modified epitopes. Strikingly, none proliferated or secreted cytokines when stimulated by FVIII2194-2205, F2196A, which also showed an IC50 > 10 μM when tested for binding to DR0101, DR0301, DR0401, DR1101, DR1104, and DR1501. Substitutions at other anchor positions affected binding to some but not all of the DR proteins. Predicted binding of the F2196A variant to 51 DR alleles was analyzed using ProPred; none bound at a threshold stringency of 10% (low stringency, thus the predicted epitopes included those with lower calculated affinities). In preparation for directly testing the immunogenicity of additional substitutions, all possible amino acid substitutions at position 2196 were evaluated using ProPred. 13 of 19 possible substitutions were predicted to prevent FVIII2194-2205 binding to all 51 DR alleles included in the algorithm (with a 3% threshold = intermediate stringency). Conclusions.MHC class II anchor residues and TCR contact sites for an immunodominant HLA-DRB1*0101-restricted T-cell epitope have been mapped precisely. Both measured and predicted effects of amino acid substitutions indicated that this F2196 is essential for effective presentation of this epitope by multiple DR alleles. Effects of various sequence modifications on FVIII function, conformation and immunogenicity are currently being evaluated using recombinant FVIII and FVIII C2 domain proteins to indicate their possible therapeutic potential. Disclosures:Pratt:CSL Behring: Research Funding; Bayer Healthcare: Research Funding; Baxter: Honoraria; Grifols: Honoraria.

Recombinant and plasma-derived factor VIII products induce distinct splenic cytokine microenvironments in hemophilia A mice

AbstractThe use of plasma-derived factor VIII (pdFVIII) concentrates in hemophilia A has been reported to result in reduced anti-FVIII antibody formation. In this study, we have investigated whether the cytokine microenvironment induced by pdFVIII has an influence on reducing anti-FVIII antibody titers in hemophilic mice. Microarray and confirmatory quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments show that pdFVIII infusion causes a different transcriptional profile in dendritic cells than recombinant FVIII (rFVIII). Both treatments caused up-regulation of proinflammatory gene expression, but rFVIII and pdFVIII treatments promote expression of genes that induce Th1 and Th2 responses, respectively. Moreover, administration of rFVIII or pdFVIII concentrates resulted in distinct T-cell splenic cytokine microenvironments. rFVIII induced the release of Th1 cytokines and IL-10, whereas pdFVIII induced the release of Th2 cytokines and transforming growth factor-β. We have also observed high titers of anti–human von Willebrand factor (VWF) antibodies in the pdFVIII-treated mice and propose that this results from antigenic competition. We further investigated the role of this phenomenon using infusions of FVIII and increasing concentrations of recombinant human factor IX (FIX). These studies show an inverse relationship between increasing concentrations of FIX and the production of anti-FVIII antibodies. In summary, these studies report new mechanisms that contribute to reduced anti-FVIII antibody development in hemophilia A after pdFVIII infusions.

Investigating the Mechanisms Underlying FVIII Antibody Production in Hemophilic Mice Following Recombinant and Plasma-Derived FVIII Infusion

The formation of Factor VIII (FVIII) antibodies after FVIII treatment in hemophilic patients is currently the major treatment-related complication faced by hemophilic patients. It has been reported that the use of plasma-derived FVIII (pdFVIII) in patients and animal models of hemophilia A will result in reduced anti-FVIII antibody formation compared to recombinant FVIII (rFVIII). In an attempt to explain this phenomenon, an initial hypothesis relating to the uptake of FVIII by antigen presenting cells has been formulated. In contrast, here we examine the hypothesis that the cytokine microenvironment induced by pdFVIII has a critical influence on the reduction of anti-FVIII antibody titers in hemophilia A mice. Micro-array and confirmatory quantitative RT-PCR experiments performed on CD11c+ dendritic cells, isolated 24 hours after rFVIII or pdFVIII infusion, show that pdFVIII causes a different immune gene expression profile than rFVIII administration. Both treatments caused the up regulation of pro-inflammatory gene expression. However, the rFVIII and pdFVIII treatments caused the up regulation of genes that induce Th1 and Th2 responses, respectively. Moreover, after administering rFVIII or pdFVIII concentrates to mice, we observed that each treatment induced a distinct T cell splenic cytokine micro-environment. Recombinant FVIII induced the release of Th1 cytokines and IL10, while pdFVIII induced the release of Th2 cytokines and TGF-α. In this mouse model, we observed the formation of high titers of anti-human VWF antibodies in the pdFVIII-treated mice and we propose that in these animals, VWF competes with FVIII for antigenic presentation. We further investigated the potential of antigenic competition by treating mice with FVIII and increasing concentrations of another unrelated protein, recombinant human factor IX (FIX). Our studies have shown an inverse relationship between increasing concentrations of FIX and the production of anti-FVIII antibodies. In summary, these studies allude to new and additional mechanisms that may contribute to the reduction of anti-FVIII antibody development in hemophilia A mice treated with pdFVIII.

HLA-DRB1*1101-Restricted T Cells from Two Unrelated Mild Hemophilia a Inhibitor Subjects with Missense Genotype R593C Respond to the Same Minimal Epitope.

The development of inhibitory anti-FVIII antibodies in hemophilia A patients, referred to clinically as “inhibitors”, results from stimulation of T cells when the infused FVIII is recognized as foreign. T-cell stimulation follows the binding of specific epitopes within FVIII-derived peptides to HLA-DR molecules on the surface of antigen-presenting cells. We are investigating the HLA dependence of immune responses to FVIII and characterizing T-cell epitopes to better understand the process of T-cell stimulation leading to anti-FVIII antibody production. Inhibitors are less common in mild/moderately severe patients than in severe hemophilia A, but an elevated risk of inhibitor formation has been noted for patients with missense mutation R593C in the FVIII A2 domain. CD4+ T cells were isolated from two unrelated hemophilic subjects with this missense genotype, one Dutch and one American. Both had a DRB1*1101 haplotype and longstanding low- to moderate-titer inhibitors. Antigen-specific T cells from both subjects were identified by staining with fluorescent, peptide-loaded MHC Class II tetramers. The tetramer staining was carried out after stimulating the cells with synthetic peptides corresponding to the FVIII A2 domain sequence. Both hemophilic subjects, but not an HLA-matched healthy control, had a DRB1*1101-restricted response to peptide FVIII589-608, which contained the wild-type R593 sequence. Single-cell sorting and expansion with PHA facilitated the isolation of A2-specific T-cell clones from both subjects. A polyclonal population of A2-specific T cells was also isolated from the American subject's CD4+ T cells. All T-cell clones and the polyclonal T-cell line proliferated in response to the FVIII589-608 peptide and to FVIII. However, the cytokine profiles were somewhat different for peptide-stimulated cells from the two subjects. The single clone obtained from the Dutch subject produced modest levels (<750 pg/mL) of IFN-g, TNF-a, IL-4 and IL-10, while the cloned and polyclonal T cells from the American subject secreted similar levels of the latter three cytokines but had a much more pronounced Th1 polarization, secreting >10,000 pg/mL of IFN-g. Somewhat surprisingly, all of the cloned and polyclonal A2-specific cultured cells proliferated in response to the hemophilic peptide FVIII589-608,593C, despite the fact that this peptide elicited no response from primary T-cell cultures isolated from the American subject. FVIII589-608 bound to recombinant, monomeric DRB1*1101 protein with high affinity, while the affinity of the hemophilic peptide FVIII589-608,593C was threefold lower. FVIII residues 594-602 (FLPNPAGVQ) comprise a predicted high-affinity DRB1*1101-binding motif. Proliferation assays carried out with truncated peptides (FVIII592-608, FVIII593-608, and FVIII594-608) confirmed the responsiveness of the clones and polyclonal T-cell line to peptides containing this predicted motif, but proliferation decreased markedly when residue R593 was removed. The immunogenicity of this FVIII region with respect to other HLA types was evaluated by binding assays utilizing several recombinant DR molecules, in order to determine the relative promiscuity of this T-cell epitope. The effects of modifying amino acid residues within this epitope are also under investigation, as substitutions at “anchor” positions should decrease the immunogenicity of the corresponding peptides. Similar substitutions in recombinant FVIII may allow development of less immunogenic variants of human FVIII proteins.

Preventing restimulation of memory B cells in hemophilia A: a potential new strategy for the treatment of antibody-dependent immune disorders

AbstractMemory B cells are responsible for the rapidly emerging antibody response after antigen reexposure. The signals required for the restimulation of memory B cells have not been fully explained. We used a murine model of anti–factor VIII (FVIII) antibody responses in hemophilia A to study the requirements for the restimulation of FVIII-specific memory B cells and their differentiation into anti-FVIII antibody-producing cells. We were particularly interested in the significance of activated T cells and costimulatory interactions. Our results indicate that the restimulation of FVIII-specific memory B cells is strictly dependent on interactions with activated T cells. These activated T cells can be specific for either FVIII or third-party antigens. Restimulation by T cells specific for third-party antigens requires the presence of FVIII, indicating that signals induced by B-cell receptor (BCR) triggering and by interactions with activated T cells are important. The blockade of B7-1 or B7-2 as well as the blockade of CD40L inhibits the restimulation and differentiation of FVIII-specific memory B cells in vitro and in vivo. The interference with inducible costimulator–inducible costimulator ligand (ICOS-ICOSL) interactions, however, does not cause any modulation. As expected, the production of anti-FVIII antibodies by plasma cells is not dependent on any of the costimulatory interactions tested.

In Hemophilia A and Autoantibody Inhibitor Patients: The Factor VIII A2 Domain and Light Chain Are Most Immunogenic

Factor VIII (fVIII) is a protein cofactor essential for blood coagulation, and it binds in the factor Xase complex to factors IXa, X, and phospholipid. In about 30% of severe hemophilia A patients, treatment with fVIII leads to production of anti-fVIII antibodies. Anti-fVIII autoantibodies also rarely appear in normal individuals. Those antibodies that inactivate fVIII (inhibitors) prevent optimal fVIII therapy. Inhibitor epitopes were previously localized to the fVIII A2, A3, and C2 domains and to an acidic amino acid region between A1 and A2. Such anti-fVIII antibodies interfere with fVIII binding to components of the factor Xase complex and prevent blood coagulation. When total anti-fVIII titers were determined for each fVIII domain in 43 inhibitor plasmas by immunoprecipitation (IP) and inhibitor neutralization assays, the anti-light chain (LCh) antibody titer was highest, anti-A2 was intermediate, and anti-A1 and anti-B were low. The relative immunogenicity of the fVIII domains in hemophilic and autoantibody inhibitor patients was similar.

Humanized Severe Combined Immunodeficient Mice as a Potential Model for the Study of Tolerance to Factor VIII

A Severe Combined Immunodeficient (SCID) mouse model has been established to evaluate experimental conditions leading to the production of factor VIII (FVIII) autoantibodies. To this end, we humanized 10 groups of 7 mice with peripheral blood mononuclear cells of 10 unrelated healthy blood donors (15 x 10(6) cells/mouse). Mice were injected with saline or immunized i.p. with 50 IU of a plasma derived human FVIII 24 h after reconstitution. Further immunization was made with 25 IU of FVIII every fortnight during 6 weeks and animals were sacrificed after 8 weeks. All reconstituted mice showed a spontaneous production of anti-FVIII antibodies in the absence of immunization with the corresponding antigen. However, no differences were observed regarding the quantity or the quality of these antibodies produced in the immunized or the saline group, indicating that tolerance to FVIII had been transferred with cell reconstitution. Affinity purified FVIII specific antibodies were capable of inhibiting FVIII activity and preventing the binding of FVIII to phospholipids in a dose-dependent manner. Immunoprecipitation experiments showed that the antibodies recognized only the C1 and C2 light chain domains. Since antibodies of interest can be found in the SCID mouse model and, moreover, since they are qualitatively comparable with the source donor's antibodies, this model provides a tool to study the regulation of tolerance against self antigens in normal subjects and in acquired haemophilia patients.