Emerging successful clinical data on gene therapy using adeno-associated viral (AAV) vector for hemophilia B (HB) showed that the risk of cellular immune response to vector capsid is clearly dose ...dependent. To decrease the vector dose, we explored AAV-8 (1-3 × 1012 vg/kg) encoding a hyperfunctional factor IX (FIX-Padua, arginine 338 to leucine) in FIX inhibitor-prone HB dogs. Two naïve HB dogs showed sustained expression of FIX-Padua with an 8- to 12-fold increased specific activity reaching 25% to 40% activity without antibody formation to FIX. A third dog with preexisting FIX inhibitors exhibited a transient anamnestic response (5 Bethesda units) at 2 weeks after vector delivery following by spontaneous eradication of the antibody to FIX by day 70. In this dog, sustained FIX expression reached ∼200% and 30% of activity and antigen levels, respectively. Immune tolerance was confirmed in all dogs after challenges with plasma-derived FIX concentrate. Shortening of the clotting times and lack of bleeding episodes support the phenotypic correction of the severe phenotype, with no clinical or laboratory evidence of risk of thrombosis. Provocative studies in mice showed that FIX-Padua exhibits similar immunogenicity and thrombogenicity compared with FIX wild type. Collectively, these data support the potential translation of gene-based strategies using FIX-Padua for HB.
•Liver-restricted expression of FIX-Padua induces immune tolerance to the transgene in hemophilia B inhibitor dog models.•Long-term toxicity studies show no increased risk of thrombogenicity of FIX-Padua in mice and dogs.
Recombinant canine B-domain deleted (BDD) factor VIII (FVIII) is predominantly expressed as a single-chain protein and exhibits greater stability after activation compared with human FVIII-BDD. We ...generated a novel BDD-FVIII variant (FVIII-RH) with an amino acid change at the furin cleavage site within the B domain (position R1645H) that mimics the canine sequence (HHQR vs human RHQR). Compared with human FVIII-BDD, expression of FVIII-RH protein revealed a 2.5-fold increase in the single-chain form. Notably, FVIII-RH exhibited a twofold increase in biological activity compared with FVIII-BDD, likely due to its slower dissociation of the A2-domain upon thrombin activation. Injection of FVIII-RH protein in hemophilia A (HA) mice resulted in more efficacious hemostasis following vascular injury in both the macro- and microcirculation. These findings were successfully translated to adeno-associated viral (AAV)-based liver gene transfer in HA mice. Expression of circulating FVIII-RH was approximately twofold higher compared with AAV-FVIII-BDD–injected mice. Moreover, FVIII-RH exhibits superior procoagulant effects compared with FVIII-BDD following a series of hemostatic challenges. Notably, the immunogenicity of FVIII-RH did not differ from FVIII-BDD. Thus, FVIII-RH is an attractive bioengineered molecule for improving efficacy without increased immunogenicity and may be suitable for both protein- and gene-based strategies for HA.
•The novel FVIII variant (FVIII-RH) has enhanced stability and procoagulant activity in both in vitro and in vivo models.•FVIII-RH is efficacious and safe; thus, it is an attractive molecule for protein replacement and as a transgene in gene-therapy strategies.
Intravascular delivery of adeno-associated virus (AAV) vector is commonly used for liver-directed gene therapy. In humans, the high prevalence of neutralizing antibodies to AAV-2 capsid and the wide ...cross-reactivity with other serotypes hamper vector transduction efficacy. Moreover, the safety of gene-based approaches depends on vector biodistribution, vector dose, and route of administration. Here we sought to characterize the safety of AAV-5 and AAV-6 for liver-mediated human factor IX (hFIX) expression in rabbits at doses of 1 × 10(12) or 1 × 10(13) viral genomes/kg. Circulating therapeutic levels of FIX were observed in both cohorts of AAV-6-hFIX, whereas for AAV-5-hFIX only the high dose was effective. Long-lasting inhibitory antibodies to hFIX were detected in three of the 10 AAV-6-injected animals but were absent in the AAV-5 group. Overall, vector shedding in the semen was transient and vector dose-dependent. However, the kinetics of clearance were remarkably faster for AAV-5 (3-5 weeks) compared with AAV-6 (10-13 weeks). AAV-6 vector sequences outside the liver were minimal at 20-30 weeks post-injection. In contrast, AAV-5 exhibited relatively high amounts of vector DNA in tissues other than the liver. Together these data are useful to further define the safety and potential for clinical translation of these AAV vectors.
Processing by the proprotein convertase furin is believed to be critical for the biological activity of multiple proteins involved in hemostasis, including coagulation factor VIII (FVIII). This ...belief prompted the retention of the furin recognition motif (amino acids 1645-1648) in the design of B-domain-deleted FVIII (FVIII-BDD) products in current clinical use and in the drug development pipeline, as well as in experimental FVIII gene therapy strategies. Here, we report that processing by furin is in fact deleterious to FVIII-BDD secretion and procoagulant activity. Inhibition of furin increases the secretion and decreases the intracellular retention of FVIII-BDD protein in mammalian cells. Our new variant (FVIII-ΔF), in which this recognition motif is removed, efficiently circumvents furin. FVIII-ΔF demonstrates increased recombinant protein yields, enhanced clotting activity, and higher circulating FVIII levels after adeno-associated viral vector-based liver gene therapy in a murine model of severe hemophilia A (HA) compared with FVIII-BDD. Moreover, we observed an amelioration of the bleeding phenotype in severe HA dogs with sustained therapeutic FVIII levels after FVIII-ΔF gene therapy at a lower vector dose than previously employed in this model. The immunogenicity of FVIII-ΔF did not differ from that of FVIII-BDD as a protein or a gene therapeutic. Thus, contrary to previous suppositions, FVIII variants that can avoid furin processing are likely to have enhanced translational potential for HA therapy.
During intracellular processing, factor VIII (FVIII) undergoes proteolysis at multiple sites with the most predominant cleavage at a Paired basic Amino acid Cleaving Enzyme (PACE)-furin cleavage site ...at the carboxy-terminus of the B-domain at residue 1648 to give rise to two polypeptide chains, the heavy chain and the light chain. Through a metal ion dependent association, these chains form a heterodimer that is the secreted form of the protein. Previously, we observed that canine B-domain deleted FVIII (cFVIII-BDD) is secreted primarily as a single chain (SC) molecule (170kD) while human FVIII-BDD (hFVIII-BDD) is secreted predominantly as a heterodimer. In addition, cFVIII-BDD is more stable and has higher biological activity. Amino acid sequence analysis revealed a single amino acid difference between cFVIII (1645-HHQR-1648) and hFVIII (1645-RHQR-1648) at the PACE-furin cleavage recognition site. Characterization of hFVIII-R1645H demonstrated that this residue is responsible for the secretion of cFVIII predominantly as a single polypeptide chain and its inherent increased stability and specific activity. Furthermore, hFVIII-R1645H has enhanced hemostatic effects upon vascular injury and in the setting of AAV delivery was associated with increased circulating levels compared to wild type hFVIII-BDD. These data suggest that there is suboptimal cleavage of cFVIII and hFVIII-R1645H by PACE-furin. Here we tested whether deletion of part or all of the PACE-furin recognition sequence may further decrease the efficiency of cleavage at the site resulting in a larger portion of single SC molecules and thus may confer increased stability compared to hFVIII-R1645H. A series of hFVIII-BDD deletion variants (n=5) of the PACE-furin cleavage recognition site (1645-1648) were generated: del1645, del1645-46, del1645-47, del1645-48 and del1648. Stable BHK cell lines were established for each variant and protein was purified using ion exchange chromatography. On an SDS-PAGE gel under reducing conditions the deletion variants migrate in a similar manner, however, the proportion of the hFVIII in the single chain form compared to hFVIII-BDD (15% SC) was 3.5-fold higher for del1645-46 (57% SC), del1645-47 (53% SC) and del1645-48 (48% SC) which is similar to R1645H (52% SC). The single deletion variants on SDS-PAGE were 2-fold higher (del1645, 34% SC) or identical (del1648, 15% SC) to hFVIII-BDD. In a one-stage aPTT assay, all variants had activity similar to hFVIII-BDD. While in the two-stage aPTT assay, three variants (del1645-46, del1645-47, del1645-48) had 2-fold higher activity than hFVIII-BDD. Variants del1645 and del1648 had similar activity to hFVIII-BDD. Using an A2 domain dissociation clotting-based assay, we found that del1645-46, del1645-47, del1645-48 and del1648 were more stable following thrombin (IIa) activation compared to hFVIII-BDD suggesting that the A2 domain of these variants may dissociate more slowly than the wild type human A2 domain following IIa cleavage. These findings explain, at least in part, the superior clotting activity determined in multiple assays. The hFVIII PACE-furin variants were also introduced into an adeno-associated viral vector serotype 8 (AAV8-hAAT-hFVIII-BDD) for liver targeted delivery. Each AAV8-hAAT-hFVIII-P/F variant was delivered to hemophilia A mice (5x1011vector genomes/mouse)(n=6/group). At 4 weeks post vector administration, the hFVIII expression of del1645-47 was 3-fold higher than hFVIII-BDD followed by del1645, del1645-48, del1645-46 while del1648 was similar to hFVIII-BDD. In summary, these in vitro data demonstrate that deletion of residues 1645-46, 1645-47 or 1645-48 of the PACE-furin cleavage recognition site confers 2-3-fold higher biological activity than wild type hFVIII-BDD. In vivo these variants result in 2-3 fold higher levels of hFVIII expression after AAV delivery compared to wild type hFVIII-BDD. Together these data suggest that the hFVIII-P/F deletion variants have enhanced hemostatic function and are superior to wild type hFVIII. In the setting of gene-based therapeutics for hemophilia A, these novel variants provide a unique strategy for increasing factor VIII expression which will allow the use of a lower vector dose which is a critical improvement for translation of this approach.
Nguyen:Pfizer, Inc.: Research Funding. Camire:Pfizer: Patents & Royalties, Research Funding. Sabatino:Spark Therapeutics: Research Funding; Pfizer, Inc.: Research Funding.
The paired basic amino acid cleaving enzyme (PACE)/Furin is a protein convertase system that plays a vital role in several biological processes, including coagulation. The propeptide processing of ...human FIX by PACE/Furin is a critical posttranslational modification, so cells co-expressing PACE/Furin and FIX are used for production of clinical recombinant protein. In the development of recombinant B-domain deleted (BDD) FVIII for hemophilia A (HA), a 14 amino acid B-domain sequence containing a putative cleavage site for PACE/Furin was retained because it was believed to be critical for intracellular processing and secretion. In contrast to FIX, we report here a surprising detrimental effect of PACE/Furin in FVIII activity and intracellular processing and secretion.
We engineered a human FVIII variant where the PACE/Furin site at residues 1645-1648 was deleted from FVIII-BDD (FVIII-ΔP/F). Notably, FVIII-ΔP/F exhibits a 3-fold increased activity over FVIII-BDD (p=0.0004) in a 2-stage APTT assay. Moreover, the A2-domain dissociation of activated FVIII-ΔP/F was also 3-fold longer compared to FVIII-BDD, suggesting a more stable activated FVIII molecule. The amount of FVIII secreted from stably transduced BHK cells was about 3-fold higher for FVIII-ΔP/F than for FVIII-BDD. Conversely, the amount of intracellular FVIII antigen was lower for FVIII-ΔP/F than for FVIII-BDD.
To confirm that PACE/Furin was implicated in the underlying mechanisms for the observed differences in FVIII secretion, we inhibited PACE/Furin in FVIII-BDD producing BHKs by transducing them with vectors expressing an engineered α1-antitrypsin variant (haat-PDX) that specifically inhibits PACE/Furin. This resulted in a 40% increase in FVIII secretion (p=0.017) and a decrease in intracellular FVIII-BDD, whereas transduction with the haat-wild type control, which does not inhibit PACE/Furin, did not significantly change the amount of FVIII secreted (p=0.32). Importantly, the secretion and intracellular levels of FVIII-ΔP/F were not affected by the inhibition of PACE/Furin by haat-PDX, indicating that the secretion of this FVIII variant does not benefit from further inhibition of PACE/Furin cleavage. Together these data suggest that the increased secretion of FVIII-ΔP/F compared to FVIII-BDD is due to the former circumventing PACE/Furin.
Furin is ubiquitously expressed in mammal tissues. In a stringent cellular model, we used LoVo, a unique human cell line that lacks functional Furin to determine whether expression of FVIII-ΔP/F and FVIII-BDD would differ, as we have observed in cells expressing Furin. Interestingly, the secretion of FVIII-ΔP/F and FVIII-BDD were comparable. This result confirms that FVIII-BDD is secreted better in the absence of Furin. In summary, our novel variant FVIII-ΔP/F exhibits enhanced secretion primarily by bypassing PACE/Furin cleavage; inhibiting this cellular process also enhances the secretion of FVIII. Futhermore in vivo experiments also demonstrated a beneficial effect of FVIII-ΔP/F: HA mice (n=4-7/dose) given adeno-associated viral 8 (AAV8) vectors for liver gene expression of FVIII-ΔP/F resulted in a 3-fold higher circulating FVIII levels than FVIII-BDD-expressing mice (p=0.025).
These exciting results from human FVIII-ΔP/F prompt us to test this variant HA canine model. First we found that recombinant canine FVIII with the entire PACE/Furin site deleted (cFVIII-ΔP/F) had increased activity in a 2-stage aPTT assay compared to wild-type cFVIII-BDD. Injection of cFVIII-ΔP/F effectively corrects the hemophilia coagulopathy in two HA dogs. Further, AAV8 liver gene therapy with cFVIII-ΔP/F in additional two HA dogs at doses of ~6 x 1012 vg/kg, a log lower than previously used for canine FVIII-BDD AAV8 gene therapy, resulted in therapeutic levels of cFVIII and shortening of clotting times. Preliminary data on injection of cFVIII-BDD protein was well tolerated in cFVIII-ΔP/F-expressing dogs.
In conclusion, these data suggest that PACE/Furin cleavage of FVIII hampers protein biological activity. FVIII variants lacking PACE/Furin recognition sequences are secreted more efficiently and exhibit improved hemostatic effects in both protein- and gene-based strategies. Inhibition of PACE/Furin in manufacturing systems for recombinant human FVIII may increase the yields of protein production. Thus these strategies have a strong rationale for translation to HA therapy.
No relevant conflicts of interest to declare.
Abstract 2208
We previously reported that recombinant canine B-domain deleted factor VIII (FVIII) was expressed predominantly (>75%) as a single-chain protein and, upon activation with thrombin, ...yielded an active cofactor species with increased stability due to delayed A2 domain dissociation. We hypothesized that this could be in part due to a unique recognition sequence (HHQR) for intracellular PACE/Furin protease present in the canine FVIII, but absent in other mammals including human, porcine and murine FVIII where the sequence is RHQR. Here, we changed position 1645 from R to H in the B-domain deleted human FVIII (hFVIII-RH) and found that much more of the protein was expressed in a single chain form (3-fold) compared to hFVIII-wild type (hFVIII-WT). Importantly, the variant hFVIII-RH was biologically active exhibiting a 2-fold higher activity measured in a 2-stage activated partial thromboplastin time (p<0.05) likely due to slower dissociation of the A2-domain upon thrombin activation compared to hFVIII-WT (t½ 3.48 vs. 1.48 minutes, respectively).
We next sought to determine the potential in vivo efficacy and safety of circulating hFVIII-RH in murine models of hemophilia A (HA) by hepatocyte-restricted transgene expression using adeno-associated viral (AAV serotype 8) vector and by exogenous administration of the recombinant protein. HA mice received varying doses (8×1012-4×1013 vg/kg) of AAV-hFVIII-WT or AAV-hFVIII-RH and exhibited a corresponding dose-dependent response of FVIII with plateau antigen levels 2–3 fold higher in hFVIII-RH than hFVIII-WT expressing mice (n = 5/group, p < 0.05 at all doses). At the low dose cohort, circulating FVIII antigen levels were 79±6.6 and 50±9 ng/ml for the FVIII-RH and FVIII-WT, respectively. In the mid dose cohort, FVIII levels were 165±54 and 84±9 ng/ml and in the high dose 274±39 and 120±35 ng/ml. We monitored the blood loss following tail-clip assay in groups of mice stratified into groups determined by expression to be low (28–60 ng/mL), mid (60–100 ng/mL), or high (above 100 ng/mL) for analysis (n= 3–9/group). Expression of either hFVIII variant was capable of decreasing the blood loss of mice in the low group compared to untreated HA mice, but did not reach that of hemostatically normal mice. Notably, at the mid expression group only hFVIII-RH expressing mice had corrected blood loss to normal hemostasis (hFVIII-RH vs hFVIII-WT p < 0.02). In the high dose all mice (FVIII-RH and FVIII-WT) exhibited blood loss similar to that of hemostatically normal mice. The increased effect of hFVIII-RH is a result of more stable clot as seen in the FeCl3- carotid artery injury model. At comparable FVIII levels, 15/16 mice expressing hFVIII-RH form stable occlusive thrombi whereas only 14/21 in hFVIII-WT group.
Laser-induced injury at microcirculation resulted in increased fibrin deposition by 2–3 fold in HA mice expressing FVIII-RH (n=4 mice, 20 injuries) compared to those expressing FVIII-WT (n=3, 10 injuries). Similar findings were obtained upon injection of recombinant protein to achieve ∼40% of normal for both FVIII-RH (n=3, 13 injuries) and FVIII-WT (n=3, 18 injuries). Moreover, we assessed the immunogenicity of hFVIII-RH by administering AAV vectors to transgenic HA mice tolerant to B-domain deleted hFVIII-WT. At 4 weeks post AAV administration all mice exhibited stable hFVIII-WT/hFVIIl-RH levels (n=5/group) with no evidence of inhibitor by Bethesda assay or by mouse specific-hFVIII-IgG titers. In summary, FVIII-RH is superior to FVIII-WT in terms of expression levels and total hemostastic function without evidence of increased immunogenicity. Therefore FVIII-RH is an attractive molecule for protein replacement and gene/cell-based strategies for HA.
No relevant conflicts of interest to declare.
Emerging data from early phase clinical studies of AAV gene therapy for hemophilia B (HB) (factor IX FIX deficiency) show sustained expression of therapeutic levels of FIX and phenotypic improvement. ...However, the safety and efficacy of in vivo gene therapy is limited by the vector dose. Recently, we reported a naturally occurring, hyperfunctional FIX (FIX Padua) caused by a single amino acid change of arginine 338 to leucine that exhibits an 8-fold increase in specific activity in humans (N Engl J Med 2009), making it a potential candidate for HB gene therapy with reduced vector doses. However, to take advantage of FIX Padua for HB gene therapy, it is critical to first define the risk of immunogenicity of this variant in preclinical models of severe HB.
We have previously shown that delivery of AAV-cFIX-Padua to skeletal muscle in HB dogs with a missense mutation in the canine (c) F9 gene resulted in no anti-FIX neutralizing antibodies (inhibitors), non-neutralizing antibodies (IgG) or FIX-specific T-cell response (Blood 2012). While promising, these dogs express FIX RNA and have a pre-existing tolerance to cFIX due to the nature of their mutation, and so do not represent the most rigorous model for immunogenicity studies.
Here, we tested the efficacy and immunogenicity of cFIX Padua in a severe HB dog colony with an early stop codon mutation. This mutation results in no FIX RNA transcript, and the dogs are prone to develop cFIX inhibitors upon exposure to protein concentrates. Three dogs were infused peripherally with a liver-specific AAV8-cFIX-Padua at two different doses, and monitored for cFIX antigen and activity levels and inhibitors. The first dog, which received 3 x 1012 vg/kg, showed average plateaued expression levels of 3.98 ± 1.44% antigen and 24.5 ± 4.1% activity, with no development of anti-cFIX inhibitors or IgG antibodies. Whole blood clotting time (WBCT) and aPTTs returned to normal by day 3 post-vector administration and have remained stable for >20 months (ongoing observations). A second dog was treated with a lower dose of 1 x 1012 vg/kg and showed average plateaued expression levels of 2.41 ± 0.05% antigen and 22.0 ± 0.4% activity, with no development of anti-cFIX inhibitors or IgG antibodies. WBCT and aPTTs returned to normal by day 3 post-vector administration and have remained stable for >3 months.
An additional dog, upon previous exposure to recombinant human (h) FIX protein, had developed inhibitors that cross-reacted with cFIX. This immune response was ongoing at the time of vector administration (3 x 1012 vg/kg). Anti-cFIX antibodies peaked at day 14 post-AAV, with 4.7 BUs and 3643 ng/mL IgG2, but dropped to undetectable levels by day 70. There was a concurrent rise in cFIX Padua expression levels, suggesting successful tolerization to the cFIX Padua. Antigen levels plateaued at 14.6 ± 4.3% and activity at 51.7 ± 23.5%, with ongoing normalization of WBCT and aPTTs for >18 months. In all three dogs, cholesterol, albumin and total protein were within normal limits with no clinical or laboratory evidence of nephrotic syndrome (a potential complication in FIX inhibitor patients that have undergone immune tolerance induction with frequent FIX protein injections).
The safety of FIX Padua was further confirmed using a mouse model of HB. Mice (n=8-12/group) were treated with 5 x 1010 vg/kg liver-directed AAV8-hFIX-WT or AAV8-hFIX-Padua, resulting in expression levels of 1076 ± 343 ng/mL (21.5 ± 6.9% antigen, 67.5 ± 10.1% activity) and 797 ± 255 ng/mL (15.9 ± 5.1% antigen, 274.8 ± 73.8% activity), respectively. In cross-over experiments, the mice were then were immunologically challenged 10-15 weeks after gene delivery with 100 ug/kg of the reciprocal recombinant protein (ie mice expressing hFIX Padua were challenged with hFIX WT, and visa versa). Challenges were administered subcutaneously alone or with adjuvant (CFA) weekly for 4 weeks. In no instance did mice develop antibodies to either FIX, suggesting that tolerance was successfully induced in all cases.
Together, these date show that FIX Padua shows no increase in immunogenicity compared to FIX WT and is capable not only of preventing inhibitor formation, but also of eradicating pre-existing inhibitory antibodies to FIX in an inhibitor-prone HB dog model. Thus, FIX Padua is an attractive transgene that will allow for decreased vector doses in human HB gene therapy, improving the safety profile of AAV liver gene therapy without increased immunogenicity.
High:Alnylam Pharmaceuticals: Consultancy; BioMarin: Consultancy; bluebirdbio, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; BristolMyersSquibb: Consultancy, membership on a Data Safety and Monitoring Board, membership on a Data Safety and Monitoring Board Other; Elsevier, Inc.: royalties from textbook, royalties from textbook Patents & Royalties; Genzyme, Inc.: Membership on an entity's Board of Directors or advisory committees; Intrexon: Consultancy; Novo Nordisk: Consultancy, Member of a grant review committee, Member of a grant review committee Other; Shire : Consultancy; Benitec: Consultancy.
Abstract 3350
Cancer is frequently associated with activation of coagulation, and a procoagulant state facilitates tumor metastasis. Recent studies have suggested that the activated protein C (aPC) ...pathway plays a role in modulating tumor metastasis, and this protection likely requires both the anticoagulant and cytoprotective effects of aPC. Notably, our early work revealed that the inactive precursor, zymogen PC (zyPC), can even more effectively protect against metastasis. The aim of this study was therefore to explore mechanisms through which zyPC could prevent metastatic cancer progression in a murine cancer model.
A liver gene transfer model using viral vectors was utilized to achieve a wide range of sustained expression of wildtype (WT) or mutant murine zyPCs. C57BL/6 experimental mice expressing stable levels of zyPCs and age and gender matched control mice receiving PBS were injected intravenously with 2.5×105 murine melanoma B16F10 cells, which metastasize to the lungs. After 3 weeks the number of pulmonary tumors was determined.
Expression of WT zyPC in C57BL/6s decreased the rates of metastasis in a dose-dependent manner compared to PBS controls (p<0.01; n=8–18/group). These effects were noted even in mice injected with low vector dose (200% zyPC expression). Conversely, when PC-deficient mice (3% of normal, n=7) were administered B16F10s without zyPC-expression, they did not survive the full 3 weeks, while their littermate controls (PC > 50% of normal, n=6) did despite high rates of metastasis. These data clearly demonstrate the protective role of zyPC in tumor progression.
We then tested modified zyPCs to identify the critical functions responsible for our observations in this tumor model. Two mutants with minimal anticoagulant function, R15Q and S195A, were generated. zyPC-R15Q is unable to dock to the thrombin-thrombomodulin complex active site and so cannot be converted to aPC. Compared to PBS controls (n=7), mice expressing zyPC-R15Q still showed a significant decrease in the number of tumor foci (p<0.001; 75–99% reduction; n=13) similar to the WT zyPC (p=0.28; n=8). Mice expressing zyPC-S195A (n=12), which has a mutation in the serine protease active site, also showed a significant decrease in the number of tumor foci compared to PBS controls (n=8; p<0.05; 90–99% reduction). As with the R15Q, mutating the S195 did not affect the ability of zyPC to protect against metastasis (p=0.22).
Next, we tested the importance of the main PC/aPC cellular receptors in our model. Binding to endothelial protein C receptor (EPCR) enhances activation of PC. We inhibited this binding by injecting anti-EPCR blocking antibody 1560 (J Thromb Haemost. 2005 3:1351) intraperitoneally one hour prior to the B16F10 cells. zyPC-expressing mice that received anti-EPCR antibody (n=22) still had a significant reduction in tumor rates compared to PBS controls (n=10; p<0.01; 45–75% reduction). Moreover, mice expressing zyPC had similar levels of protection whether they received the anti-EPCR antibody or an isotype control (n=22–24; p=0.31). EPCR binding not only increases activation of PC, it also mediates the cytoprotective effect by clustering with and facilitating the activation of the signaling protease-activated receptor 1 (PAR1). PAR1 −/− mice expressing zyPC (n=21) challenged with B16F10 cells still had reduced rates of metastasis compared to PAR1 −/− PBS controls (n=15; p<0.01; 67% reduction). The zyPC protection in PAR1 null mice was comparable to that in PAR1 +/− littermate controls (n=10; p=0.619). Collectively, these findings suggest a distinct mechanism by which zyPC modulates tumor progression independent of EPCR and PAR1, both of which are required for aPC-mediated protection.
Despite elevated circulating levels of PC, zyPC-expressing mice did not suffer from increased blood loss following tail clipping or show prolonged activated partial thromboplastin times (aPTTs) compared to hemostatically normal mice.
In conclusion, zyPC protects against metastatic cancer progression in a dose-dependent manner. Our data show for the first time that this zyPC effect is independent of its anticoagulant function. Furthermore, protection is not mediated through EPCR or PAR1, suggesting an alternative pathway by which zyPC limits tumor progression. These findings suggest that WT zyPC and variants with little to no anticoagulant function are safe and efficacious in preventing metastatic cancer progression.
Van Sluis:PCT patent pending: Protein C: A Zymogen for Anti-Cancer Treatment Patents & Royalties. High:PCT patent pending: Protein C: A Zymogen for Anti-Cancer Treatment Patents & Royalties. Spek:PCT patent pending: Protein C: A Zymogen for Anti-Cancer Treatment Patents & Royalties. Arruda:PCT patent pending: Protein C: A Zymogen for Anti-Cancer Treatment Patents & Royalties.
DANGEROUS DROPS: A CASE OF HOMEOPATHIC DIGITALIS Mock, Gabrielle; Frenchu, Kiersten; Siner, Joshua
Journal of the American College of Cardiology,
03/2019, Letnik:
73, Številka:
9
Journal Article
PDF
