ALBERT

Description: Platelet activation leads to the incorporation of 32[PO4(2-)] into bovine coagulation factor Va and recombinant human factor VIII. In the presence of the soluble fraction from thrombin-activated platelets and (gamma-32P) adenosine triphosphate, radioactivity is incorporated exclusively into the M(r) = 94,000 heavy chain (H94) of factor Va and into the M(r) = 210,000 to 90,000 heavy chains as well into the M(r) = 80,000 light chain of factor VIII. Proteolysis of the purified phosphorylated M(r) = 94,000 factor Va heavy chain by activated protein C (APC) gave products of M(r) = 70,000, 24,000, and 20,000. Only the intermediate M(r) = 24,000 fragment contained radioactivity. Because the difference between the M(r) = 24,000 and M(r) = 20,000 fragments is located on the COOH-terminal end of the bovine heavy chain, phosphorylation of H94 must occur within the M(r) = 4,000 peptide derived from the carboxyl-terminal end of H94 (residues 663 through 713). Exposure of the radioactive factor VIII molecule to thrombin ultimately resulted in a nonradioactive light chain and an M(r) = 24,000 radioactive fragment that corresponds to the carboxyl-terminal segment of the A1 domain of factor VIII. Based on the known sequence of human factor VIII, phosphorylation of factor VIII by the platelet kinase probably occurs within the acidic regions 337 through 372 and 1649 through 1689 of the procofactor. These acidic regions are highly homologous to sequences known to be phosphorylated by casein kinase II. Results obtained using purified casein kinase II gave a maximum observed stoichiometry of 0.6 mol of 32[PO4(2-)]/mol of factor Va heavy chain and 0.35 mol of 32[PO4(2-)]/mol of factor VIII. Phosphoamino acid analysis of phosphorylated factor Va by casein kinase II or by the platelet kinase showed only the presence of phosphoserine while phosphoamino acid analysis of phosphorylated factor VIII by casein kinase II showed the presence of phosphothreonine as well as small amounts of phosphoserine. The platelet kinase responsible for the phosphorylation of the two cofactors was found to be inhibited by several synthetic protein kinase inhibitors. Finally, partially phosphorylated factor Va was found to be more sensitive to APC inactivation than its native counterpart. Our findings suggest that phosphorylation of factors Va and VIIIa by a platelet casein kinase II- like kinase may downregulate the activity of the two cofactors.

Description: Background: T effector cells (Teff) within the stem cell graft in allogeneic hematopoietic stem cell transplantation (HSCT) can elicit disabling acute graft-versus-host disease (aGvHD) contributing to transplant-related mortality. Teff as donor lymphocyte infusion (DLI) are a therapeutic option to re-induce complete remission (CR) after leukemia relapse. Usually DLIs are given in dose escalating regimens until CR is achieved or first signs of aGvHD develop. To monitor the DLI induced allo-immune response and the efficacy of aGvHD treatment is a clinical challenge, since no established biomarkers are available. T regulatory cells (Tregs) are thought to play an important role in balancing immune responses and studies have shown effects of adoptive Treg transfer as a therapeutic option in GvHD. T cell receptor (TCR) sequencing of T cell subsets, such as Tregs, after DLI might allow the identification of clones inducing control of GvHD. Here we report data of 29 DLI patients with a median follow-up of 〉1 year allowing us to thoroughly analyze differences of the TCR repertoire in patients with or without aGvHD. Aims: We aimed to analyze Treg TCR diversity and clonality changes over time as a potential biomarker for development and treatment response of aGvHD following DLI. Patients and Methods: The study cohort consisted of 29 leukemia patients after HSCT who received DLI treatment for recurrence of disease, molecular relapse or high risk phenotype. Blood samples were taken before and after DLI. A median of 4 (range 2-6) blood samples per patient were available for TCR-sequencing. The last sample was taken at a median of 123 (27-530) days post DLI. After generation of PBMCs CD4+CD127-CD25+ Tregs were FACS-sorted for cDNA-based CDR3-region amplification of the TCR-β chain. CDR3 amplicons were sequenced on the Illumina MiSeq platform and annotated using the IMGT.org database. Further bioinformatic analyses were based on VDJtools and the tcR R-package. Results: In 18/29 patients we observed clinical symptoms of aGvHD, with blood samples available of the acute onset in 12/18 cases. Treg frequencies and absolute numbers did not differ between aGvHD and noGvHD samples. Treg TCR diversity, assessed via inverse Simpson's diversity index (1/D) increased on average by 322% at the first occurrence of aGvHD compared to the previous sample (Figure 1A, B). Stratifying for aGvHD severity (total grade 1-2 vs. 3-4) did not reveal any group differences. However, stratifying by organ involvement (skin vs. GI/liver) showed a more pronounced increase of 1/D in patients with aGvHD of the GI tract and/or the liver. Moreover, in 11 subjects blood samples were available a median of 7 (3-14) days prior to aGvHD diagnosis. Already at this preclinical time point we detected an increased 1/D of +361% (Figure 1A, B). Again, aGvHD organ involvement significantly affected the result, with GI/liver involvement mainly driving this effect (+567% vs. +1% 1/D). In contrast, patients who did not develop aGvHD at any time after DLI showed on average a slight decrease of -8% 1/D compared to the previous time point. Next, we analyzed all aGvHD patients with at least one sample available after initiation of treatment (local and/or systemic steroids). Control or remission of aGvHD symptoms was accompanied by decreased 1/D of on average -58% compared to the previous samples (Figure 2). In 9/11 patients we saw a focusing of the Treg TCR repertoire; in the other two (Figure 2, red lines) no or only partial clinical response to systemic steroids was reported. Conclusion: Our data describe detailed changes in the Treg compartment on the TCR level following development and treatment of aGvHD. Currently, aGvHD diagnosis following DLI treatment relies solely on clinical symptoms, deciding whether further dose increments of DLI can be administered. As our data suggest, Treg TCR sequencing may support transplant specialists with (1) detection of patients at risk for aGvHD, even prior to clinical symptoms, (2) identification of patients eligible for further dose increments (compare Figure 1B), and (3) assessment of aGvHD treatment with guidance whether higher dosage of steroids and/or alternative immunosuppressive treatment might be required (compare Figure 2, red lines). Future prospective studies are needed to replicate these findings in a large cohort, potentially enabling identification of specific Treg TCR clones controlling the allo-immune response in aGvHD. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees. Koenecke:BMS: Consultancy; abbvie: Consultancy; Amgen: Consultancy; Roche: Consultancy.

Description: Memory B cells are essential for maintaining long-term antibody responses. They can persist for years even in the absence of antigen and are rapidly re-stimulated to differentiate into antibody-producing plasma cells when they encounter their specific antigen. Previously we demonstrated that ligands for TLR 7 and 9 amplify the differentiation of FVIII-specific memory B cells into anti-FVIII antibody-producing plasma cells at low concentrations of FVIII and prevent the inhibition of memory-B-cell differentiation at high concentrations of FVIII. The modulation of FVIII-specific memory-B-cell responses by agonists for TLR is highly relevant for the design of new immunotherapeutic approaches in patients with FVIII inhibitors because TLR are activated by a range of different viral and bacterial components. Specifically, TLR 7 is triggered by single-stranded RNA derived from viruses and TLR 9 is triggered by bacterial DNA containing unmethylated CpG motifs. We further explored the modulation of FVIII-specific memory-B-cell responses by agonists for TLRs by studying a broad range of concentrations of CpG DNA, a ligand for TLR 9, both in vitro and in vivo using the murine E17 model of hemophilia A. We used CpG-DNA in concentrations ranging from 0.1 to 10,000 ng/ml to study the modulation of FVIII-specific memory-B-cell responses in vitro and verified the specificity of the effects observed by including a blocking agent for TLR 9 and GpC-DNA, a non-stimulating negative control for CpG DNA. Furthermore, we used doses of CpG DNA ranging from 10 to 50,000 ng per dose to study the modulation of FVIII-specific memory-B-cell responses in vivo. E17 hemophilic mice were treated with a single intravenous dose of 200 ng FVIII to stimulate the generation of FVIII-specific memory B cells and were subsequently treated with another dose of FVIII that was given together with CpG DNA. We analyzed titers of anti-FVIII antibodies in the circulation of these mice one week after the second dose of FVIII. Previously we had shown that a single dose of 200 ng FVIII, given intravenously to E17 hemophilic mice, stimulates the formation of FVIII-specific memory B cells but is not sufficient to induce anti-FVIII antibodies that would be detectable in the circulation. Our results demonstrate a biphasic effect of CpG DNA on the re-stimulation of FVIII-specific memory B cells and their differentiation into antibody-producing plasma cells. Both in vitro and in vivo studies show that CpG DNA at high doses inhibits the re-stimulation and differentiation of FVIII-specific memory B cells. However, CpG DNA at low doses amplifies these processes. Amplification and inhibition of memory-B-cell responses are due to specific interactions of CpG DNA with TLR 9. Both effects are blocked by addition of a blocking agent for TLR 9 in vitro. We conclude that triggering of TLR 9 by bacterial DNA has a substantial influence on FVIII-specific memory-B-cell responses. The consequence of TLR 9 triggering can be inhibitory or stimulatory, depending on the actual concentration of the bacterial DNA. Our findings demonstrate the potential modulatory effects of bacterial infections on the regulation of FVIII inhibitor development.

Description: 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.

Description: Abstract 4356 Baxter and Nektar have developed a longer acting recombinant FVIII (BAX 855), which is manufactured by coupling stable PEG using Nektar technology to Baxter’s full-length rFVIII bulk drug substance from the protein-free ADVATE™ manufacturing process. BAX 855 was characterized by a number of analytical methods, focusing on the elucidation of the primary structure, posttranslational modifications, PEGylation site distribution and three-dimensional structure. The primary structure of BAX 855 was investigated using a peptide mapping approach. Samples were digested with trypsin and the resulting peptides were separated by reversed phase chromatography and detected with on-line electrospray ionization mass spectrometry. This approach resulted in a sequence coverage of 94% with good consistency demonstrated between different BAX 855 batches. Protein-bound oligosaccharides were determined by normal phase HPLC of fluorescence labeled N-glycans released by PNGase F treatment. The composition of the N-linked oligosaccharides showed a similar pattern between BAX 855 and unmodified rFVIII, confirming that the N-glycosylation pattern remained intact during the PEGylation process. PEGylation site distribution and detailed analysis of the consistency of PEGylation was investigated by activating BAX 855 with thrombin. The resulting PEGylated and non-PEGylated fragments were separated using a RP-HPLC approach and the bound PEG was measured for each thrombin fragment. The distribution of PEG among the different thrombin fragments of rFVIII was shown to be consistent between several BAX 855 batches. Moreover, RP-HPLC of native BAX 855 showed reproducible subunit composition and PEGylation. SDS-PAGE and Western blot analysis of BAX 855 revealed changes in the electrophoretic pattern due to PEGylation without the appearance of any relevant degradation products. The random PEGylation of rFVIII was shown using antibodies with different epitope specificities. Dynamic light scattering and Fourier-transformed infrared spectroscopy (FTIR) were used to monitor the consistency of three dimensional structures. The mean hydrodynamic diameter of BAX 855 was between 30 and 40 nm, which is a characteristic size for a ∼300-kDa protein. Several BAX 855 batches showed almost overlapping FTIR absorbance spectra, especially in the conformationally susceptible amide I and amide II peak at 1650 and 1550 cm−1, respectively, indicative for good consistency of the manufacturing process. In summary, BAX 855, a PEGylated rFVIII derivative, can be manufactured reproducibly without changes to the protein structure characteristic for a fully functional FVIII molecule. Disclosures: Kaliwoda: Baxter Innovations GmbH: Employment. Graninger:Baxter Innovations GmbH: Employment. Matthiessen:Baxter Innovations GmbH: Employment. Schrenk:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Mitterer:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

Description: Abstract 4345 Baxter and Nektar have developed BAX 855, a PEGylated form of Baxter’s recombinant FVIII (rFVIII) product based on the ADVATE™ manufacturing process. The objective of this preclinical study program was to evaluate the safety of BAX 855 in different species. The systemic toxic potential of BAX 855 was tested in rats and cynomolgus monkeys. The product was given intravenously at 350 or 700 U/kg to rats every other day for 28 days. Monkeys were treated at doses of 150, 350 or 700 U/kg every five days for one month. To demonstrate consistent safety between different batches, two lots of BAX 855 were used in each study. Assessment of toxicity was based on mortality, clinical observations, ophthalmic examination, clinical pathology, assessment on male fertility in rats, organ weights and pathology evaluations. In addition to safety endpoints, toxicokinetics and the formation of anti-product antibodies were assessed. In rats, no drug-related changes with respect to body weight, food consumption, ophthalmoscopy, hematology cellular variables, clinical chemistry, urinalysis, seminology or organ weights were noted. In addition, there were no treatment-related adverse effects on the coagulation markers. Macroscopic as well as microscopic examination and organ weights revealed no test-item related adverse effects. In monkeys, there were no signs of toxicity or mortalities throughout the experimental period at any dose level. Minor findings in red blood cell variables as well as a corresponding increase in reticulocyte counts together with bloody emesis or hematoma in single animals and a prolonged APTT in all animals treated with BAX 855 were noted during the last week of dosing. These observations were likely caused by the development of cross-reactive neutralizing anti-FVIII antibodies against endogenous FVIII which probably caused a partial reduction in cynomolgus monkey FVIII activity. A partial recovery for APTT was noted until day 12 of the recovery phase. The formation of binding and neutralizing antibodies was also reflected in a statistically significant decrease in exposure observed during toxicokinetic analysis at the study end. The formation of antibodies against BAX 855 is an expected immune reaction after repeated application of heterologous human proteins to animals, which is also well known for non-PEGylated FVIII products. In conclusion, intravenous administration of BAX 855 for 28 days did not result in any evidence of systemic effect. Therefore, 700 U/kg was considered the No Observed Adverse Effect level (NOAEL) in these studies. The good safety profile of BAX 855 during the preclinical program provides the basis for proceeding with human trials. Disclosures: Dietrich: Baxter Innovations GmbH: Employment. Spatzenegger:Baxter Innovations GmbH: Employment. Stidl:Baxter Innovations GmbH: Employment. Wolfsegger:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment.

Description: Abstract 4357 Baxter has developed a PEGylated recombinant human factor VIII conjugate by modifying the FVIII molecule with polyethylene glycol (PEGylation). The product is derived from a CHO cell line using a plasma-protein-free method and a virus inactivation step. The objective of this preclinical study-program was to evaluate the safety of Baxter’s longer acting rFVIII in different species. The thrombogenic potential of Baxter’s longer acting rFVIII was evaluated in the rabbit stasis model developed by Wessler. Two lots of Baxter’s longer acting rFVIII were used to demonstrate consistent safety between different batches. Advate served as the control item. Baxter’s longer acting rFVIII was not thrombogenic in this preclinical model at a dose of 900 U/kg. There was no evidence of thrombogenic potential after intravenous treatment with either the test item or Advate, the active reference item. The effects of Baxter’s longer acting rFVIII on body temperature, heart rate, blood pressure, respiratory variables or QT, QTcf, PR and/or QRS intervals and respiratory system (intra-thoracic pressure) was evaluated in eight male, conscious telemetered cynomolgus monkeys. Baxter’s longer acting rFVIII was given at 150 and 600 U/kg/day to male monkeys by intravenous administration. Baxter’s longer acting rFVIII did not cause any adverse clinical, respiratory or cardiovascular effects and was very well tolerated at all dose levels tested. In conclusion, Baxter’s longer acting rFVIII did not cause any thrombocenic events or adverse clinical, respiratory or cardiovascular effects and was very well tolerated at all dose levels. Disclosures: Dietrich: Baxter Innovations GmbH: Employment. Kubik:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment.

Description: We studied a patient with large granular lymphocyte (LGL) leukemia for evidence of human T-cell leukemia/lymphoma virus (HTLV) infection. Serum from this patient was positive for HTLV-I/II antibodies by enzyme- linked immunosorbent assay (ELISA) and was confirmed positive in Western blot and radioimmunoprecipitation assays. Results of a synthetic peptide-based ELISA showed that the seropositivity was caused by HTLV-II and not HTLV-I infection. Analyses of enzymatic amplification of DNA from bone marrow sections using the polymerase chain reaction (PCR) were positive for HTLV-II specific gag, pol, env, and pX gene sequences. Cloning and sequencing of amplified products showed that the HTLV-II pol and pX sequences in patient DNA differed from the sequences of 17 other HTLV-II isolates examined in our laboratory. HTLV infection may have a role in some patients in the pathogenesis of LGL leukemia.

Description: Von Willebrand factor (VWF) is composed of a series of multimers, the sizes of which are regulated by the plasma metalloprotease ADAMTS13. Reports suggest that a transient increase in VWF levels, triggered for instance by DDAVP treatment, will result in a decrease in ADAMTS13 activity. This phenomenon is widely believed to be due to ADAMTS13 being exhausted when confronted with an excess of substrate. Treating patients who have type 3 von Willebrand disease with a recombinant human von Willebrand factor (rVWF) that has not yet been exposed to ADAMTS13 might thus cause a drop in ADAMTS13 levels. We therefore tested whether a rise in VWF concentrations to supraphysiological levels caused by administration of rVWF could influence endogenous ADAMTS13 levels in animal models. Various doses of human rVWF (300, 600, and 1200 RCo IU/kg BW) were injected into rabbits and cynomolgus monkeys and plasma samples were collected at a range of time points. As expected, VWF antigen rose sharply in a dose-dependent manner (~25 IU/ml VWF:Ag for the highest dose, 15 min after injection) and then declined gradually (~7 IU/ml VWF:Ag for the highest dose, 18 hours after injection). When these samples were tested for ADAMTS13 activity using the FRETS assay, no relevant changes were observed throughout the entire test period in the rabbit or in the monkey samples, indicating that rVWF, even at high doses, did not compromise ADAMTS13 activity. Both rabbit and cynomolgus ADAMTS13 recognized human rVWF as the specific cleavage products were detectable by electrophoresis at all doses administered. These animal studies thus indicate that an excess of intravenously administered rVWF leading to supraphysiological levels does not exhaust ADAMTS13.

Description: rVWF expressed in Chinese hamster ovary (CHO) cells can be hardly distinguished from pdVWF. This holds true for the primary amino acid sequence and for posttranslational modifications including N- and O-linked glycans. However, because CHO cells lack a a2,6-sialyltransferase, the enzyme attaching N-acetylneuraminic acid (Neu5Ac) in a a2,6- linkage to the galactose residues of complex N-glycans, Neu5Ac will be found in CHO-cell derived glycoproteins only in a2,3-linkage. We used this minute difference in glycosylation to develop a lectin-based immunoassay that allows the selective measurement of human rVWF in human plasma based on the lack of a2,6-bound Neu5Ac on rVWF. At equal VWF:Ag concentrations, mixtures of rVWF and pdVWF will show lower ratios of a2,6-bound Neu5Ac to VWF:Ag than those in pdVWF alone. The difference in ratio is directly related to the concentration of rVWF in the mixture. We used the Sambucs nigra agglutinin (SNA) to measure VWF-bound a2,6-linked Neu5Ac. This lectin binds to Neu5Ac on N-glycans only when Neu5Ac is a2,6-linked to galactose. rVWF/pdVWF was captured from the sample by a plate-immobilized polyclonal rabbit anti-VWF antibody. a2,6-bound Neu5Ac was then specifically detected using biotinylated SNA and streptavidin peroxidase. The assay setup resulted in a linear dose-response curve ranging from 0.2 to 2.9 mU pdVWF:Ag/mL in human plasma. In contrast, rVWF showed no binding, even when tested at concentrations about 100-times higher. Subjecting plate-adsorbed pdVWF to enzymatic desialylation with neuraminidase completely abrogated the binding of SNA. The linkage specificity of the SNA binding was shown in an inhibition study using the trisaccharides 3′-sialyllactose and 6′-sialyllactose as hapten sugars. As expected only 6′-sialyllactose proved able to inhibit the binding of SNA to plate-immobilized pdVWF. This result confirmed the linkage specificity of the SNA binding essential for the discrimination of plasma-derived and rVWF. To demonstrate the feasibility of this approach we spiked a sample of a normal human plasma pool with rVWF in several concentrations ranging from 0 to 1.5 IU/ml. In these samples the VWF:Ag concentrations and the levels of VWF-bound a2,6-Neu5Ac with the SNA binding assay were measured. Then, we calculated the ratio of SNA binding to VWF:Ag on the assumption that all VWF was pdVWF, i.e. contained only a2,6-linked Neu5Ac. The difference between the expected and the actually found ratios of SNA binding to VWF:Ag correlated (R2=0.99) with the amount of rVWF present in the mixtures. Thus, we obtained a calibration curve that allowed the specific measurement of rVWF in a range of 0.2 to 1.5 IU/ml in the presence of 1 IU/ml pdVWF. Both assays, the VWF:Ag ELISA and the SNA-based immunoassay for the measurement of VWF-bound a2,6-bound Neu5Ac, showed acceptable precision. The data show that rVWF in a human plasma sample can readily be differentiated from endogenous pdVWF based on minute, linkage-specific differences of the N-glycan structures. This type of lectin-based immunoassay could be useful in the clinical setting to determine the circulating concentration of therapeutic CHO-cell-derived rVWF and to specifically measure its concentration in the presence of the endogenous glycoprotein.