Abstract 3324 This icon denotes a clinically relevant abstract

Replacement of the missing factor VIII (FVIII) is the current standard of care for patients with hemophilia A. However, the short half-life of FVIII makes frequent treatment necessary. Current efforts focus on the development of longer-acting FVIII concentrates by introducing chemical and genetic modifications to the protein. Any modification of the FVIII protein, however, risks increasing its immunogenic potential to induce neutralizing antibodies (FVIII inhibitors), and this is one of the major complications in current therapy. It would be highly desirable to identify candidates with a high risk for increased immunogenicity before entering clinical development to minimize the risk of exposing patients to such altered FVIII proteins. In the present study, we describe a transgenic mouse line that expresses a human F8 cDNA. This mouse is immunologically tolerant to therapeutic doses of native human FVIII but is able to mount an antibody response when challenged with a modified FVIII protein that possesses altered immunogenic properties. In this situation, immunologic tolerance breaks down and antibodies develop that recognize both the modified and the native human FVIII. The applicability of this new model for preclinical immunogenicity assessment of new FVIII molecules and its potential use for basic research are discussed.

Recombinant factor IX (rFIX) expressed in Chinese hamster ovary (CHO) cells has been shown to be safe and effective in clinical studies, but differs in pharmacokinetics from plasma-derived FIX (pdFIX). In clinical studies, CHO-derived rFIX had a 30–50 % lower in-vivo recovery when compared to pdFIX, whereas mean residence time and terminal half-life did not differ between preparations. Although rFIX shows high similarity to pdFIX in structure and function, differences in glycosylation and gamma-carboxylation degree can be detected. Moreover, although experimental proof has yet to be published, the lower degree of phosphorylation of amino acid serine 155, and the lower degree of sulfation of tyrosine 158 have been hypothesized to be causative for the lower in-vivo recovery of rFIX. These two modifications occur at less than 20 % for the tyrosine-sulfation and at less than 1 % for the serine phosphorylation in rFIX, whereas pdFIX has both modifications to more than 90 % completed. We identified human HEK293 cells to perform rFIX phosphorylation and sulfation to a higher extent than CHO cells. A rFIX-producing cell line derived from HEK293 cells was generated by stable transfection, and was adapted to suspension culture conditions to allow lab-scale fermentation. rFIX was produced and purified from a single fermentation run using two different down-stream process schemes: the first was able to enrich high-phosphorylated and -sulfated rFIX; the second to purify total rFIX from the supernatant at high yield. For pharmacokinetic comparison, these HEK293 materials, CHO-derived rFIX, and a pdFIX preparation were formulated in the same buffer. Determination of phosphorylation and sulfation by mass spectrometry showed a phosphorylation and sulfation degree of 50 % plus a 20 % single modification (phosphorylation or sulfation) for the HEK293-material purified by the modification enrichment method versus 15 % for both modifications plus a 15 % single modification for the material purified by the high-yield protocol. The values for CHO-derived rFIX and pdFIX were similar to those in the literature. Oligosaccharide mapping revealed glycosylation differences among CHO-, HEK293-, and pdFIX preparations, but high similarity between both HEK293-derived materials. We compared the pharmacokinetics of the various FIX preparations in FIX-knock-out mice. In-vivo recovery and area under the curve were statistically significantly higher for the high phosphorylated and sulfated HEK293-material than for total rFIX derived from HEK293 cells. However, these two parameters were lower for both HEK293-derived rFIX preparations than for CHO-derived rFIX, and lower for CHO-derived rFIX than for pdFIX. This may be due to glycosylation differences between these FIX preparations. Mean residence times and terminal half-lives were similar for all preparations. In summary, these findings emphasize that the degree of rFIX-sulfation and -phosphorylation influences the pharmacokinetic properties of rFIX.

Memory B cells specific for factor VIII (FVIII) are critical for maintaining FVIII inhibitors in patients with hemophilia A. They are precursors of anti-FVIII antibody-producing plasma cells and are highly efficient antigen-presenting cells for the activation of T cells. The eradication of FVIII-specific memory B cells will be a prerequisite for any successful new approach to induce immune tolerance in patients with FVIII inhibitors. Little is known about the regulation of these cells. Previously we showed that ligands for toll-like receptors (TLR) 7 and 9 are able to re-stimulate FVIII-specific memory B cells in the absence of T-cell help. However, alternative “helper cells” such as dendritic cells are essential for providing help to memory B cells under such conditions. Based on these findings, we asked which co-stimulatory interactions are required for the restimulation of memory B cells in the presence of dendritic cells and ligands for TLR and whether these co-stimulatory interactions are the same as those required for the restimulation of memory B cells in the presence of activated T cells. We used spleen cells from hemophilic mice treated with human FVIII to generate highly purified populations of memory B cells, CD4+ T cells and dendritic cells. The required purity was achieved by a combination of magnetic bead separation and fluorescence-activated cell sorting. The memory B cell compartment was specified by the expression of CD19 together with IgG and the absence of surface IgM and IgD. Memory B cells were cultured in the presence of FVIII to stimulate their differentiation into anti-FVIII antibody-producing plasma cells. Different combinations of CD4+ T cells, ligands for TLR 7 and 9 and dendritic cells were added to the memory-B-cell cultures. Blocking antibodies and competitor proteins were used to specify the co-stimulatory interactions required for the re-stimulation of memory B cells in the presence of either CD4+ T cells or dendritic cells and ligands for TLR 7 and 9. Our results demonstrate that the blockade of B7-1 and B7-2 as well as the blockade of CD40L inhibit the re-stimulation of FVIII-specific memory B cells and their differentiation into anti-FVIII antibody-producing plasma cells in the presence of T-cell help. Similar requirements apply for the re-stimulation of memory B cells in the presence of dendritic cells and ligands for TLR 7 or 9. Dendritic cells in the absence of ligands for TLR are not able to provide help for the re-stimulation of memory B cells, which indicates that dendritic cells need to be activated. Furthermore, ligands for TLR 7 or 9 were not able to re-stimulate memory B cells in the complete absence of dendritic cells. Based on these results we conclude that dendritic cells activated by ligands for TLR 7 or 9 can substitute for activated CD4+ T cells in providing co-stimulatory help for memory-B-cell re-stimulation. CD40-CD40L interactions seem to be the most important co-stimulatory interactions for the re-stimulation of memory B cells, not only in the presence of activated CD4+ T cells but also in the presence of ligands for TLR and dendritic cells.

Therapy of hemophilia A has greatly benefited from the development of safe recombinant and plasmatic factor VIII (FVIII) concentrates. Current efforts to improve products focus on the extension of half-life by chemical and/or molecular modifications of FVIII. However, any modification of the FVIII protein poses the risk of creating neo-antigens that might cause FVIII inhibitors to be induced in patients. Therefore it is important to monitor the potential creation of neo-antigens during preclinical and clinical phases of drug development. Currently available animal models for hemophilia A develop high titers of anti-FVIII antibodies when treated with human FVIII. Using these models, it is difficult to differentiate between immune responses against native human FVIII and immune responses against human FVIII that carries neo-antigens. Considering these limitations, our aim is to develop a new model for hemophilia A that does not respond with antibodies to native human FVIII but develops antibodies against human FVIII that carries neo-antigens. We created a series of hemophilic mouse lines that carry a transgene for human FVIII that was placed under the control of an albumin promoter to direct liver-specific expression. Transgenic founder mice were generated by direct microinjection of the vector into the male pronucleus of fertilized oocytes obtained from mated female C57BL/6J mice after superovulation. Transgenic mice were crossed with hemophilic mice and bred to homozygousity for the expression of the human FVIII transgene. We analyzed the expression of human FVIII by real time PCR in lung, kidney, liver, heart, muscle, spleen, lymph nodes and reproductive organs. Gene expression analysis of bone marrow and thymus are currently ongoing. We selected three sublines (E, G and I) that show different levels of liver-specific expression of human FVIII for further analysis. We did not detect any FVIII antigen in the circulation in any of these three sublines when we used two different ELISA systems with detection limits around 1 ng/ml. We treated mice of sublines E, G and I intravenously with eight weekly doses of 200 ng of human FVIII (Advate) and analyzed the potential development of antibodies against native human FVIII. Our results indicate that transgenic mice of sublines E and I are immunologically tolerant to native human FVIII. They do not develop anti-FVIII antibodies (about 90% of all mice tested) or develop low titers (below 1:80 in 10% of mice tested) only. In contrast, mice of subline G develop high titers of anti-FVIII antibodies indicating that they are not immunologically tolerant to human FVIII. Preliminary data suggest that the degree of immunological tolerance against human FVIII correlates to a certain extent with the expression levels of the human FVIII transgen in liver and/or thymus. We are in the process of verifying these preliminary data. Furthermore, we have started to analyze FVIII-specific T-cell responses to define potential differences in the repertoire of FVIIIspecific T cells between the three sublines. We conclude that transgenic expression of human FVIII under the control of an albumin promoter is able to induce immune tolerance to human native FVIII in hemophilic mice. However, a certain threshold level of gene expression might be required for the induction of immune tolerance.

Antibody responses against factor VIII (FVIII) are the major complication that arises when patients with hemophilia A are treated with factor VIII products. Therefore, understanding regulation of anti-FVIII immune responses is of outmost importance. Antibody responses are well established to result from differentiation of B cells into antibody-secreting plasma cells. B cells need help from activated CD4+ T cells to develop high-affinity antibody responses against protein antigens such as FVIII. Recently, naturally occurring CD4+CD25+ regulatory T cells have been shown to modulate antibody responses by either suppressing the function of CD4+ T helper cells or by directly acting on B cells. However, the potential importance of CD4+CD25+ T cells in regulating antibody responses to foreign protein antigens is controversial. Furthermore, the extent to which naturally occurring CD4+CD25+ T cells regulate antibody responses against exogenous proteins such as FVIII when these proteins are given to previously untreated patients is unclear. To obtain information on how important naturally occurring CD4+CD25+ T cells are under such conditions, we asked whether these cells regulate anti-FVIII antibody responses in murine hemophilia A. We studied E17 hemophilic mice with two different genetic backgrounds (C57BL/6J and Balb/c) and treated them with four intravenous doses of human FVIII given at weekly intervals. Before the first dose of FVIII, CD4+CD25+ T cells were depleted in vivo using an anti-CD25 antibody that has been shown to deplete naturally occurring CD4+CD25+ T cells in mice. In vivo depletion of regulatory T cells using the same antibody has been successfully applied in a variety of mouse studies to evaluate the significance of naturally occurring CD4+CD25+ T cells in different immunological systems. An isotype-matched control antibody was used as a negative control. A week after the second and the fourth dose of FVIII, plasma samples were taken and tested for anti-FVIII antibodies. We found differences in titers of anti-FVIII antibodies between mice treated with anti-CD25 antibodies and control mice in Balb/c mice but not in C57BL/6J mice. Hemophilic Balb/c mice that had been pre-treated with anti-CD25 antibodies developed higher titers of anti-FVIII antibodies than mice that had been pre-treated with an isotype-matched control antibody. Differences were seen as a statistical trend (p=0.091) after two doses of FVIII and reached statistical significance (p=0.024) after four doses of FVIII. No differences in antibody titers were observed in hemophilic C57BL/6J mice. Our results strongly indicate that the ability of naturally occurring regulatory T cells to modulate anti-FVIII antibody responses in hemophilic mice depends on the genetic background of these mice. Immunoregulatory factors such as cytokines or chemokines as well as differences in the number and functional activity of naturally occurring regulatory T cells that are found in secondary lymphoid organs are likely to determine the regulatory capacity of these cells. Based on our results we conclude that differences in number and functional activity of naturally occurring regulatory T cells should be considered in the search for risk factors associated with the development of FVIII inhibitors in patients.

The prevention of neutralizing anti-factor VIII (FVIII) antibodies remains the major challenge in the treatment of hemophilia A patients with FVIII products. Therefore, it is important to understand how B cells differentiate into antibody-producing plasma cells, how this process is regulated and how it can be inhibited. A number of inhibitory receptors have been identified that are expressed on B cells and might provide targets for the prevention of antibody development against FVIII. The inhibitory receptor FcγIIB has emerged as a key regulator of B cell responses in the later phase of antibody responses. It has been described as a potent inhibitor of B-cell-receptor (BCR) signaling when co-ligated to the BCR by engagement of antigen-containing immune complexes. Based on these findings we asked whether the inhibitory receptor FcγIIB is involved in the natural regulation of anti-FVIII antibody responses in murine models. If this receptor were an important negative regulator of anti-FVIII antibody responses, mice that do not express this receptor should develop higher titers of anti-FVIII antibodies than mice that express the receptor. We treated wildtype mice and FcγRIIB knockout mice, both on a C57BL/6J background, with four intravenous doses of 200ng of human FVIII (80U/kg) and analyzed titers of anti-FVIII antibodies after the second, the third and the fourth dose. Surprisingly, we did not see any significant difference in antibody titers between both strains of mice. We extended our study and asked under which conditions FcγRIIB knockout mice would develop higher titers of anti-FVIII antibodies than wildtype mice. We tested different application routes (i.v. and i.p.) of FVIII, compared low doses (200ng) and high doses (80μg) and combined the i.p. application with or without Freund’s adjuvant. Furthermore, we included KLH-TNP (i.p. application of 100μg with Freund’s adjuvant) as a positive control that was previously shown to induce different levels of antibodies in wildtype mice compared with FcγRIIB knockout mice. We found significant differences in the development of specific antibodies between both strains of mice when we used the positive control (i.p. KLH-TNP with adjuvants) and when we applied high dose FVIII (80μg) with Freund’s adjuvant i.p. We did not see any differences in antibody titers between both strains of mice with any of the other treatment schedules. Our results indicate that the inhibitory receptor FcγIIB is only active as a negative regulator for antibody responses against foreign proteins in vivo when a certain threshold level of antibodies is reached in the circulation. This critical threshold level is obviously not reached when mice are treated with clinically relevant doses of FVIII. Based on our results we conclude that the inhibitory receptor FcγIIB is not involved in the regulation of antibody responses against therapeutically relevant doses of human FVIII in murine models. Whether the same situation is true for patients with hemophilia A remains to be shown. Furthermore, the relevance of the inhibitory receptor FcγIIB might differ between patients who react to the FVIII product as a foreign protein and patients who react to FVIII as an altered self protein or self protein.

In the last years evidence has been provided for the importance of B cells in the pathogenesis of rheumatoid arthritis (RA). Several studies have supported the concept that humoral immunity, manifested by the production of autoantibodies, such as rheumatoid factors (RFs), plays a significant role in the course of the disease. Specific targeting of autoantibody-producing B cells, such as RF-producing B cells, should therefore be a promising new approach in the treatment of RA. We used a mouse model to induce human RF responses and asked the question whether oral treatment with the antigen (human IgG) recognized by RFs could induce immune tolerance to RF responses. Balb/c mice were orally treated with polyvalent human IgG before and after immunization with insoluble immune complexes (ICs) that triggered the induction of RFs. Serum titers of RFs were significantly reduced after both primary and booster immunization when human IgG was given as a single oral dose or continuously in drinking water. Continuous treatment with human IgG even prevented booster effects on RFs when treatment started after primary immunization. Treatment with IgG fragments provided evidence that the observed effect of human IgG was mediated by the Fc part and not the Fab part of IgG. Furthermore, transfer of spleen cells obtained from mice after oral treatment with human IgG suppressed RF responses in recipient mice. These data give promising indications that oral human IgG might represent an alternative approach for immunosuppressive B-cell targeted therapies in RA.

PURPOSE: Recently the characteristic pattern of A1PI seen on high resolution isoelectric focusing (IEF) gels was elucidated by the description of a new A1PI isoform missing the C-terminal amino acid, lysine. As this modification of A1PI could be induced in vitro by the action of basic carboxypeptidases we were interested in whether it can also be found in vivo. After intravenous application of intact human A1PI in rats, we determined the IEF pattern of A1PI in broncheoalveolar lavage (BAL) and checked for the presence of the C-terminal truncated peptide using mass spectrometry (MS).

PURPOSE: Eight in vivo preclinical protocols were conducted to assess the profile of A1PI Fraction (Fr.) IV-1 paste in comparison to A1PI using Fr. IV-1+4 paste regarding biocomparability.

Clinical manifestations of sickle cell disease are related to vaso-occlusive (VOC) events, which are responsible for acute and chronic organ damages. We have recently shown the beneficial effects of inhaled nitric oxide (NO) in both transgenic sickle cell (SAD) mice exposed to hypoxia/reoxygenation (H/R) and in sickle cell children with acute painful crisis (Blood 102: 1097, 2003; JAMA 289: 1136, 2003). Polynitroxyl-albumine (PNA), nitrosylated at position 34, may represent a new therapeutic tool in acute sickle cell VOCs. In SAD mice, PNA was intraperitoneally (ip) administrated and a dose-response curve was determined. Methemoglobin (MetHb) levels and blood pressure were measured under normoxia. PNA, 300 mg/Kg twice a day (equal to 4.5 mM/Kg twice a d.), induced a significant but transitory decrease in blood pressure 60 min after ip injection, while MetHb levels did not change. The effects of PNA and human serum albumine (hsAlb) on ischemia/reperfusion (H/R) lung injury related to sickle cell disease were then studied. SAD mice were divided into 4 groups of 6 animals each: one served as normoxia untreated control. The other groups were exposed to 46 hrs hypoxia (8% O2) followed by 2 hrs normoxia: one served as untreated hypoxic group, one was treated with hsAlb (300 mg/Kg twice a day) and one with PNA (300 mg/Kg twice a day). We evaluated lung histopathology, complete blood counts, bronchoalveolar (BAL) fluid neutrophil counts, cytokine levels, which are activated by H/R stimulus and are modulated by inhaled NO (Blood 102:1087, 2003).