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BACKGROUND: Hemophilia A arises from dysfunctional or deficient coagulation factor (F)VIII and leads to inefficient fibrin clot formation and uncontrolled bleeding events. The development of antibody inhibitors is a clinical complication in hemophilia A patients receiving FVIII replacement therapy. LE2E9 is an anti-C1 domain inhibitor previously isolated from a mild/moderate hemophilia A patient and disrupts FVIII interactions with von Willebrand factor and FIXa, though the intermolecular contacts that underpin LE2E9-mediated FVIII neutralization are undefined. OBJECTIVES: To determine the structure of the complex between FVIII and LE2E9 and characterize its mechanism of inhibition. METHODS: FVIII was bound to the antigen binding fragment (Fab) of NB2E9, a recombinant construct of LE2E9, and its structure was determined by cryogenic electron microscopy. RESULTS: This report communicates the 3.46 Å structure of FVIII bound to NB2E9, with its epitope comprising FVIII residues S2040 to Y2043, K2065 to W2070, and R2150 to H2155. Structural analysis reveals that the LE2E9 epitope overlaps with portions of the epitope for 2A9, a murine-derived inhibitor, suggesting that these residues represent a shared antigenic region on the C1 domain between FVIII-/- mice and hemophilia A patients. Furthermore, the FVIII:NB2E9 structure elucidates the orientation of the LE2E9 glycan, illustrating how the glycan sterically blocks interactions between the FVIII C1 domain and the von Willebrand factor D' domain. A putative model of the FVIIIa:FIXa complex suggests potential clashing between the NB2E9 glycan and FIXa light chain. CONCLUSION: These results describe an antigenic "hotspot" on the FVIII C1 domain and provide a structural basis for engineering FVIII replacement therapeutics with reduced antigenicity.
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Vγ9Vδ2 T cells represent a promising cancer therapy platform because the implementation of allogenic, off-the-shelf product candidates is possible. However, intravenous administration of human Vγ9Vδ2 T cells manufactured under good manufacturing practice (GMP)-compliant, serum-free conditions are not tested easily in most mouse models, mainly because they lack the ability to migrate from the blood to tissues or tumors. We demonstrate that these T cells do not migrate from the circulation to the mouse bone marrow (BM), the site of many malignancies. Thus, there is a need to better characterize human γδ T-cell migration in vivo and develop strategies to direct these cells to in vivo sites of therapeutic interest. To better understand the migration of these cells and possibly influence their migration, NSG mice were conditioned with agents to clear BM cellular compartments, i.e., busulfan or total body irradiation (TBI), or promote T-cell migration to inflamed BM, i.e., incomplete Freund's adjuvant (IFA), prior to administering γδ T cells. Conditioning with TBI, unlike busulfan or IFA, increases the percentage and number of γδ T cells accumulating in the mouse BM, and cells in the peripheral blood (PB) and BM display identical surface protein profiles. To better understand the mechanism by which cells migrate to the BM, mice were conditioned with TBI and administered γδ T cells or tracker-stained red blood cells. The mechanism by which γδ T cells enter the BM after radiation is passive migration from the circulation, not homing. We tested if these ex vivo-expanded cells can migrate based on chemokine expression patterns and showed that it is possible to initiate homing by utilizing highly expressed chemokine receptors on the expanded γδ T cells. γδ T cells highly express CCR2, which provides chemokine attraction to C-C motif chemokine ligand 2 (CCL2)-expressing cells. IFNγ-primed mesenchymal stromal cells (MSCs) (γMSCs) express CCL2, and we developed in vitro and in vivo models to test γδ T-cell homing to CCL2-expressing cells. Using an established neuroblastoma NSG mouse model, we show that intratumorally-injected γMSCs increase the homing of γδ T cells to this tumor. These studies provide insight into the migration of serum-free, ex vivo-expanded Vγ9Vδ2 T cells in NSG mice, which is critical to understanding the fundamental properties of these cells.
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Neuroblastoma , Receptores de Antígenos de Linfócitos T gama-delta , Humanos , Camundongos , Animais , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Bussulfano , Quimiocinas , Receptores de QuimiocinasRESUMO
BACKGROUND: Laboratory resurrection of ancient coagulation factor (F) IX variants generated through ancestral sequence reconstruction led to the discovery of a FIX variant, designated An96, which possesses enhanced specific activity independent of and additive to that provided by human p.Arg384Lys, referred to as FIX-Padua. OBJECTIVES: The goal of the current study was to identify the amino acid substitution(s) responsible for the enhanced activity of An96 and create a humanized An96 FIX transgene for gene therapy application. METHODS: Reductionist screening approaches, including domain swapping and scanning residue substitution, were used and guided by one-stage FIX activity assays. In vitro characterization of top candidates included recombinant high-purity preparation, specific activity determination, and enzyme kinetic analysis. Final candidates were packaged into adeno-associated viral (AAV) vectors and delivered to hemophilia B mice. RESULTS: Five of 42 total amino acid substitutions in An96 appear sufficient to retain the enhanced activity of An96 in an otherwise human FIX variant. Additional substitution of the Padua variant further increased the specific activity 5-fold. This candidate, designated ET9, demonstrated 51-fold greater specific activity than hFIX. AAV2/8-ET9 treated hemophilia B mice produced plasma FIX activities equivalent to those observed previously for AAV2/8-An96-Padua, which were 10-fold higher than AAV2/8-hFIX-Padua. CONCLUSION: Starting from computationally inferred ancient FIX sequences, novel amino acid substitutions conferring activity enhancement were identified and translated into an AAV-FIX gene therapy cassette demonstrating high potency. This ancestral sequence reconstruction discovery and sequence mapping refinement approach represents a promising platform for broader protein drug and gene therapy candidate optimization.
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Fator IX , Hemofilia B , Humanos , Camundongos , Animais , Fator IX/metabolismo , Hemofilia B/terapia , Hemofilia B/tratamento farmacológico , Cinética , Terapia Genética , Substituição de Aminoácidos , Vetores Genéticos , Dependovirus/genética , Dependovirus/metabolismoRESUMO
The application of immunotherapies such as chimeric antigen receptor (CAR) T therapy or bi-specific T cell engager (BiTE) therapy to manage myeloid malignancies has proven more challenging than for B-cell malignancies. This is attributed to a shortage of leukemia-specific cell-surface antigens that distinguish healthy from malignant myeloid populations, and the inability to manage myeloid depletion unlike B-cell aplasia. Therefore, the development of targeted therapeutics for myeloid malignancies, such as acute myeloid leukemia (AML), requires new approaches. Herein, we developed a ligand-based CAR and secreted bi-specific T cell engager (sBite) to target c-kit using its cognate ligand, stem cell factor (SCF). c-kit is highly expressed on AML blasts and correlates with resistance to chemotherapy and poor prognosis, making it an ideal candidate for which to develop targeted therapeutics. We utilize γδ T cells as a cytotoxic alternative to αß T cells and a transient transfection system as both a safety precaution and switch to remove alloreactive modified cells that may hinder successful transplant. Additionally, the use of γδ T cells permits its use as an allogeneic, off-the-shelf therapeutic. To this end, we show mSCF CAR- and hSCF sBite-modified γδ T cells are proficient in killing c-kit+ AML cell lines and sca-1+ murine bone marrow cells in vitro. In vivo, hSCF sBite-modified γδ T cells moderately extend survival of NSG mice engrafted with disseminated AML, but therapeutic efficacy is limited by lack of γδ T-cell homing to murine bone marrow. Together, these data demonstrate preclinical efficacy and support further investigation of SCF-based γδ T-cell therapeutics for the treatment of myeloid malignancies.
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Leucemia Mieloide Aguda , Camundongos , Animais , Ligantes , Receptores Proteína Tirosina Quinases , Proteínas Proto-Oncogênicas c-kit/genética , Imunoterapia Adotiva , Fator de Células-TroncoRESUMO
Hemophilia A is the most common X-linked bleeding disorder affecting more than half-a-million individuals worldwide. Persons with severe hemophilia A have coagulation FVIII levels <1% and experience spontaneous debilitating and life-threatening bleeds. Advances in hemophilia A therapeutics have significantly improved health outcomes, but development of FVIII inhibitory antibodies and breakthrough bleeds during therapy significantly increase patient morbidity and mortality. Here we use sheep fetuses at the human equivalent of 16-18 gestational weeks, and we show that prenatal transplantation of human placental cells (107-108/kg) bioengineered to produce an optimized FVIII protein, results in considerable elevation in plasma FVIII levels that persists for >3 years post-treatment. Cells engraft in major organs, and none of the recipients mount immune responses to either the cells or the FVIII they produce. Thus, these studies attest to the feasibility, immunologic advantage, and safety of treating hemophilia A prior to birth.
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Hemofilia A , Humanos , Animais , Feminino , Gravidez , Ovinos , Hemofilia A/genética , Fator VIII/genética , Fator VIII/metabolismo , Placenta/metabolismo , Coagulação Sanguínea , Feto/metabolismoRESUMO
Adoptive cell therapy (ACT) utilizing γδ T cells is becoming a promising option for the treatment of cancer, because it offers an off-the-shelf allogeneic product that is safe, potent, and clinically effective. Approaches to engineer or enhance immune-competent cells for ACT, like expression of chimeric antigen receptors (CARs) or combination treatments with bispecific T cell engagers, have improved the specificity and cytotoxic potential of ACTs and have shown great promise in preclinical and clinical settings. Here, we test whether electroporation of γδ T cells with CAR or secreted bispecific T cell engager (sBite) mRNA is an effective approach to improve the cytotoxicity of γδ T cells. Using a CD19-specific CAR, approximately 60% of γδ T cells are modified after mRNA electroporation and these cells show potent anticancer activity in vitro and in vivo against two CD19-positive cancer cell lines. In addition, expression and secretion of a CD19 sBite enhances γδ T cell cytotoxicity, both in vitro and in vivo, and promotes killing of target cells by modified and unmodified γδ T cells. Taken together, we show that transient transfection of γδ T cells with CAR or sBite mRNA by electroporation can be an effective treatment platform as a cancer therapeutic.
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GD2-targeting immunotherapies have improved survival in children with neuroblastoma, yet on-target, off-tumor toxicities can occur and a subset of patients cease to respond. The majority of neuroblastoma patients who receive immunotherapy have been previously treated with cytotoxic chemotherapy, making it paramount to identify neuroblastoma-specific antigens that remain stable throughout standard treatment. Cell surface glycoproteomics performed on human-derived neuroblastoma tumors in mice following chemotherapy treatment identified protein tyrosine kinase 7 (PTK7) to be abundantly expressed. Furthermore, PTK7 shows minimal expression on pediatric-specific normal tissues. We developed an anti-PTK7 chimeric antigen receptor (CAR) and find PTK7 CAR T cells specifically target and kill PTK7-expressing neuroblastoma in vitro. In vivo, human/murine binding PTK7 CAR T cells regress aggressive neuroblastoma metastatic mouse models and prolong survival with no toxicity. Together, these data demonstrate preclinical efficacy and tolerability for targeting PTK7 and support ongoing investigations to optimize PTK7-targeting CAR T cells for neuroblastoma.
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Neuroblastoma , Receptores de Antígenos Quiméricos , Humanos , Criança , Animais , Camundongos , Neuroblastoma/terapia , Neuroblastoma/patologia , Imunoterapia , Receptores de Antígenos Quiméricos/genética , Proteínas Tirosina QuinasesRESUMO
The development of pathogenic antibody inhibitors against coagulation factor VIII (FVIII) occurs in â¼30% of patients with congenital hemophilia A receiving FVIII replacement therapy, as well as in all cases of acquired hemophilia A. KM33 is an anti-C1 domain antibody inhibitor previously isolated from a patient with severe hemophilia A. In addition to potently blocking FVIII binding to von Willebrand factor and phospholipid surfaces, KM33 disrupts FVIII binding to lipoprotein receptor-related protein 1 (LRP1), which drives FVIII hepatic clearance and antigen presentation in dendritic cells. Here, we report on the structure of FVIII bound to NB33, a recombinant derivative of KM33, via single-particle cryo-electron microscopy. Structural analysis revealed that the NB33 epitope localizes to the FVIII residues R2090-S2094 and I2158-R2159, which constitute membrane-binding loops in the C1 domain. Further analysis revealed that multiple FVIII lysine and arginine residues, previously shown to mediate binding to LRP1, dock onto an acidic cleft at the NB33 variable domain interface, thus blocking a putative LRP1 binding site. Together, these results demonstrate a novel mechanism of FVIII inhibition by a patient-derived antibody inhibitor and provide structural evidence for engineering FVIII with reduced LRP1-mediated clearance.
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Hemofilia A , Hemostáticos , Humanos , Fator VIII/metabolismo , Microscopia Crioeletrônica , Domínios Proteicos , Fator de von Willebrand/metabolismoRESUMO
Humoral immunity to factor VIII (FVIII) represents a significant challenge for the treatment of patients with hemophilia A. Current paradigms indicate that neutralizing antibodies against FVIII (inhibitors) occur through a classical CD4 T cell, germinal center (GC) dependent process. However, clinical observations suggest that the nature of the immune response to FVIII may differ between patients. While some patients produce persistent low or high inhibitor titers, others generate a transient response. Moreover, FVIII reactive memory B cells are only detectable in some patients with sustained inhibitor titers. The determinants regulating the type of immune response a patient develops, let alone how the immune response differs in these patients remains incompletely understood. One hypothesis is that polymorphisms within immunoregulatory genes alter the underlying immune response to FVIII, and thereby the inhibitor response. Consistent with this, studies report that inhibitor titers to FVIII differ in animals with the same F8 pathogenic variant but completely distinct backgrounds; though, how these genetic disparities affect the immune response to FVIII remains to be investigated. Given this, we sought to mechanistically dissect how genetics impact the underlying immune response to FVIII. In particular, as the risk of producing inhibitors is weakly associated with differences in HLA, we hypothesized that genetic factors other than HLA influence the immune response to FVIII and downstream inhibitor formation. Our data demonstrate that FVIII deficient mice encoding the same MHC and F8 variant produce disparate inhibitor titers, and that the type of inhibitor response formed associates with the ability to generate GCs. Interestingly, the formation of antibodies through a GC or non-GC pathway does not appear to be due to differences in CD4 T cell immunity, as the CD4 T cell response to an immunodominant epitope in FVIII was similar in these mice. These results indicate that genetics can impact the process by which inhibitors develop and may in part explain the apparent propensity of patients to form distinct inhibitor responses. Moreover, these data highlight an underappreciated immunological pathway of humoral immunity to FVIII and lay the groundwork for identification of biomarkers for the development of approaches to tolerize against FVIII.
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Hemofilia A , Hemostáticos , Animais , Anticorpos Neutralizantes , Fator VIII , Centro Germinativo/metabolismo , Humanos , CamundongosRESUMO
γδ T lymphocytes represent an emerging class of cellular immunotherapy with preclinical promise to treat cancer, notably neuroblastoma. The innate-like immune cell subset demonstrates inherent cytoxicity toward tumor cells independent of MHC recognition, enabling allogeneic administration of healthy donor-derived γδ T cell therapies. A current limitation is the substantial interindividual γδ T cell expansion variation among leukocyte collections. Overcoming this limitation will enable realization of the full potential of allogeneic γδ T-based cellular therapy. Here, we characterize γδ T cell expansions from healthy adult donors and observe that highly potent natural killer (NK) lymphocytes expand with γδ T cells under zoledronate and IL-2 stimulation. The presence of NK cells correlates with both the expansion potential of γδ T cells and the overall potency of the γδ T cell therapy. However, the potency of the cell therapy in combination with an antibody-based immunotherapeutic, dinutuximab, appears to be independent of γδ T/NK cell content both in vitro and in vivo, which minimizes the implication of interindividual expansion differences toward efficacy. Collectively, these studies highlight the utility of maintaining the NK cell population within expanded γδ T cell therapies and suggest a synergistic action of combined innate cell immunotherapy toward neuroblastoma.
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Neuroblastoma , Receptores de Antígenos de Linfócitos T gama-delta , Proliferação de Células , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Imunoterapia , Neuroblastoma/terapiaRESUMO
The intrinsic tenase (Xase) complex, formed by factors (f) VIIIa and fIXa, forms on activated platelet surfaces and catalyzes the activation of factor X to Xa, stimulating thrombin production in the blood coagulation cascade. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering our understanding of the structural underpinnings that guide Xase complex assembly. Here, we aimed to characterize the Xase complex bound to a lipid nanodisc with biolayer interferometry (BLI), Michaelis-Menten kinetics, and small-angle X-ray scattering (SAXS). Using immobilized lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. Enzyme kinetic measurements demonstrated the assembly of an active enzyme complex in the presence of lipid nanodiscs. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein-protein interactions. Our results highlight multiple points of contact between fVIIIa and fIXa, including a novel interaction with fIXa at the fVIIIa A1-A3 domain interface. Lastly, we identified hemophilia A/B-related mutations with varying severities at the fVIIIa/fIXa interface that may regulate Xase complex assembly. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex.
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Fator IXa , Fator VIIIa/metabolismo , Lipídeos , Cisteína Endopeptidases , Fator IXa/química , Fator IXa/genética , Fator IXa/metabolismo , Proteínas de Neoplasias , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Advances in the development of novel treatment options for hemophilia A are prevalent. However, the anti-factor VIII (FVIII) neutralizing antibody (inhibitor) response to existing FVIII products remains a major treatment challenge. Although some novel products are designed to function in the presence of inhibitors, they do not specific address the immunogenicity risk or mechanistic causes of inhibitor development, which remain unclear. Furthermore, most preclinical studies supporting clinical gene therapy programs have reported immunogenicity signals in animal models, especially at higher vector doses and sometimes using multiple vector designs. In these settings, immunogenicity risk factor determination, comparative immunogenicity of competing vector designs, and the potential for obtaining meaningful prognostic data remain relatively unexplored. Additionally, there remains the opportunity to investigate clinical gene therapy as an alternative to standard immune tolerance induction therapy. The current study was designed to address these issues through longitudinal dose-response evaluation of 4 adeno-associated viral (AAV) vector candidates encoding 2 different FVIII transgenes in a murine model of hemophilia A. Plasma FVIII activity and anti-FVIII antibody data were used to generate a pharmacokinetic model that (1) identifies initial AAV-FVIII product expression kinetics as the dominant risk factor for inhibitor development, (2) predicts a therapeutic window where immune tolerance is achieved, and (3) demonstrates evidence of gene therapy-based immune tolerance induction. Although there are known limitations to the predictive value of preclinical immunogenicity testing, these studies can uncover or support the development of design principles that can guide the development of safe and effective genetic medicines.
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Hemofilia A , Hemostáticos , Animais , Fator VIII/genética , Fator VIII/uso terapêutico , Terapia Genética , Hemofilia A/genética , Hemofilia A/terapia , Camundongos , TransgenesRESUMO
Introduction: Placenta-derived mesenchymal cells (PLCs) endogenously produce FVIII, which makes them ideally suited for cell-based fVIII gene delivery. We have previously reported that human PLCs can be efficiently modified with a lentiviral vector encoding a bioengineered, expression/secretion-optimized fVIII transgene (ET3) and durably produce clinically relevant levels of functionally active FVIII. The objective of the present study was to investigate whether CRISPR/Cas9 can be used to achieve location-specific insertion of a fVIII transgene into a genomic safe harbor, thereby eliminating the potential risks arising from the semi-random genomic integration inherent to lentiviral vectors. We hypothesized this approach would improve the safety of the PLC-based gene delivery platform and might also enhance the therapeutic effect by eliminating chromatin-related transgene silencing. Methods: We used CRISPR/Cas9 to attempt to insert the bioengineered fVIII transgene "lcoET3" into the AAVS1 site of PLCs (CRISPR-lcoET3) and determined their subsequent levels of FVIII production, comparing results with this approach to those achieved using lentivector transduction (LV-lcoET3) and plasmid transfection (Plasmid-lcoET3). In addition, since liver-derived sinusoidal endothelial cells (LSECs) are the native site of FVIII production in the body, we also performed parallel studies in human (h)LSECs). Results: PLCs and hLSECs can both be transduced (LV-lcoET3) with very high efficiency and produce high levels of biologically active FVIII. Surprisingly, both cell types were largely refractory to CRISPR/Cas9-mediated knockin of the lcoET3 fVIII transgene in the AAVS1 genome locus. However, successful insertion of an RFP reporter into this locus using an identical procedure suggests the failure to achieve knockin of the lcoET3 expression cassette at this site is likely a function of its large size. Importantly, using plasmids, alone or to introduce the CRISPR/Cas9 "machinery", resulted in dramatic upregulation of TLR 3, TLR 7, and BiP in PLCs, compromising their unique immune-inertness. Discussion: Although we did not achieve our primary objective, our results validate the utility of both PLCs and hLSECs as cell-based delivery vehicles for a fVIII transgene, and they highlight the hurdles that remain to be overcome before primary human cells can be gene-edited with sufficient efficiency for use in cell-based gene therapy to treat HA.
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Hemofilia A , Células-Tronco Mesenquimais , Feminino , Humanos , Gravidez , Hemofilia A/terapia , Fator VIII , Células Endoteliais/metabolismo , Placenta/metabolismo , Células-Tronco Mesenquimais/metabolismoRESUMO
While targeting CD19+ hematologic malignancies with CAR T cell therapy using single chain variable fragments (scFv) has been highly successful, novel strategies for applying CAR T cell therapy with other tumor types are necessary. In the current study, CAR T cells were designed using a ligand binding domain instead of an scFv to target stem-like leukemia cells. Thrombopoietin (TPO), the natural ligand to the myeloproliferative leukemia protein (MPL) receptor, was used as the antigen binding domain to engage MPL expressed on hematopoietic stem cells (HSC) and erythropoietic and megakaryocytic acute myeloid leukemias (AML). TPO-CAR T cells were tested in vitro against AML cell lines with varied MPL expression to test specificity. TPO-CAR T cells were specifically activating and cytotoxic against MPL+ leukemia cell lines. Though the TPO-CAR T cells did not extend survival in vivo, it successfully cleared the MPL+ fraction of leukemia cells. As expected, we also show the TPO-CAR is cytotoxic against MPL expressing bone marrow compartment in AML xenograft models. The data collected demonstrate preclinical potential of TPO-CAR T cells for stem-like leukemia through assessment of targeted killing of MPL+ cells and may facilitate subsequent HSC transplant under reduced intensity conditioning regimens.
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Leucemia Mieloide Aguda , Trombopoetina , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Ligantes , Proteínas de Neoplasias , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Citocinas , Receptores de Trombopoetina/genética , Linfócitos T/metabolismo , Trombopoetina/metabolismoRESUMO
Recombinant factor VIII (FVIII) products represent a life-saving intervention for patients with hemophilia A. However, patients can develop antibodies against FVIII that prevent its function and directly increase morbidity and mortality. The development of anti-FVIII antibodies varies depending on the type of recombinant product used, with previous studies suggesting that second-generation baby hamster kidney (BHK)-derived FVIII products display greater immunogenicity than do third-generation Chinese hamster ovary (CHO)-derived FVIII products. However, the underlying mechanisms responsible for these differences remain incompletely understood. Our results demonstrate that BHK cells express higher levels of the nonhuman carbohydrate α1-3 galactose (αGal) than do CHO cells, suggesting that αGal incorporation onto FVIII may result in anti-αGal antibody recognition that could positively influence the development of anti-FVIII antibodies. Consistent with this, BHK-derived FVIII exhibits increased levels of αGal, which corresponds to increased reactivity with anti-αGal antibodies. Infusion of BHK-derived, but not CHO-derived, FVIII into αGal-knockout mice, which spontaneously generate anti-αGal antibodies, results in significantly higher anti-FVIII antibody formation, suggesting that the increased levels of αGal on BHK-derived FVIII can influence immunogenicity. These results suggest that posttranslational modifications of recombinant FVIII products with nonhuman carbohydrates may influence the development of anti-FVIII antibodies.
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Anticorpos , Formação de Anticorpos , Inibidores dos Fatores de Coagulação Sanguínea , Fator VIII , Polissacarídeos , Processamento de Proteína Pós-Traducional/imunologia , Animais , Anticorpos/genética , Anticorpos/imunologia , Inibidores dos Fatores de Coagulação Sanguínea/genética , Inibidores dos Fatores de Coagulação Sanguínea/imunologia , Células CHO , Cricetinae , Cricetulus , Fator VIII/imunologia , Fator VIII/farmacologia , Hemofilia A/genética , Hemofilia A/imunologia , Camundongos , Camundongos Knockout , Polissacarídeos/genética , Polissacarídeos/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologiaRESUMO
Hepatic gene transfer with adeno-associated viral (AAV) vectors shows much promise for the treatment of the X-linked bleeding disorder hemophilia B in multiple clinical trials. In an effort to further innovate this approach and to introduce alternative vector designs with potentially superior features into clinical development, we recently built a vector platform based on AAV serotype 3 because of its superior tropism for human hepatocytes. A vector genome with serotype-matched inverted terminal repeats expressing hyperactive human coagulation factor IX (FIX)-Padua was designed for clinical use that is optimized for translation using hepatocyte-specific codon-usage bias and is depleted of immune stimulatory CpG motifs. Here, this vector genome was packaged into AAV3 (T492V + S663V) capsid for hepatic gene transfer in non-human primates. FIX activity within or near the normal range was obtained at a low vector dose of 5 × 1011 vector genomes/kg. Pre-existing neutralizing antibodies, however, completely or partially blocked hepatic gene transfer at that dose. No CD8+ T cell response against capsid was observed. Antibodies against the human FIX transgene product formed at a 10-fold higher vector dose, albeit hepatic gene transfer was remarkably consistent, and sustained FIX activity in the normal range was nonetheless achieved in two of three animals for the 3-month duration of the study. These results support the use of this vector at low vector doses for gene therapy of hemophilia B in humans.
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Orthologous proteins contain sequence disparity guided by natural selection. In certain cases, species-specific protein functionality predicts pharmacological enhancement, such as greater specific activity or stability. However, immunological barriers generally preclude use of nonhuman proteins as therapeutics, and difficulty exists in the identification of individual sequence determinants among the overall sequence disparity. Ancestral sequence reconstruction (ASR) represents a platform for the prediction and resurrection of ancient gene and protein sequences. Recently, we demonstrated that ASR can be used as a platform to facilitate the identification of therapeutic protein variants with enhanced properties. Specifically, we identified coagulation factor VIII (FVIII) variants with improved specific activity, biosynthesis, stability, and resistance to anti-human FVIII antibody-based inhibition. In the current study, we resurrected a panel of ancient mammalian coagulation factor IX (FIX) variants with the goal of identifying improved pharmaceutical candidates. One variant (An96) demonstrated 12-fold greater FIX activity production than human FIX. Addition of the R338L Padua substitution further increased An96 activity, suggesting independent but additive mechanisms. after adeno-associated virus 2 (AAV2)/8-FIX gene therapy, 10-fold greater plasma FIX activity was observed in hemophilia B mice administered AAV2/8-An96-Padua as compared with AAV2/8-human FIX-Padua. Furthermore, phenotypic correction conferred by the ancestral variant was confirmed using a saphenous vein bleeding challenge and thromboelastography. Collectively, these findings validate the ASR drug discovery platform as well as identify an ancient FIX candidate for pharmaceutical development.
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Fator IX , Hemofilia B , Animais , Testes de Coagulação Sanguínea , Fator IX/genética , Terapia Genética , Hemofilia B/genética , Hemofilia B/terapia , Hemorragia , CamundongosRESUMO
Patients with the severe form of hemophilia A (HA) present with a severe phenotype, and can suffer from life-threatening, spontaneous hemorrhaging. While prophylactic FVIII infusions have revolutionized the clinical management of HA, this treatment is short-lived, expensive, and it is not available to many A patients worldwide. In the present study, we evaluated a panel of readily available cell types for their suitability as cellular vehicles to deliver long-lasting FVIII replacement following transduction with a retroviral vector encoding a B domain-deleted human F8 transgene. Given the immune hurdles that currently plague factor replacement therapy, we focused our investigation on cell types that we deemed to be most relevant to either prenatal or very early postnatal treatment and that could, ideally, be autologously derived. Our findings identify several promising candidates for use as cell-based FVIII delivery vehicles and lay the groundwork for future mechanistic studies to delineate bottlenecks to efficient production and secretion of FVIII following genetic-modification.
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Hematopoietic stem and progenitor cell (HSPC) lentiviral gene therapy is a promising strategy toward a lifelong cure for hemophilia A (HA). The primary risks associated with this approach center on the requirement for pre-transplantation conditioning necessary to make space for, and provide immune suppression against, stem cells and blood coagulation factor VIII, respectively. Traditional conditioning agents utilize genotoxic mechanisms of action, such as DNA alkylation, that increase risk of sterility, infection, and developing secondary malignancies. In the current study, we describe a non-genotoxic conditioning protocol using an immunotoxin targeting CD117 (c-kit) to achieve endogenous hematopoietic stem cell depletion and a cocktail of monoclonal antibodies to provide transient immune suppression against the transgene product in a murine HA gene therapy model. This strategy provides high-level engraftment of hematopoietic stem cells genetically modified ex vivo using recombinant lentiviral vector (LV) encoding a bioengineered high-expression factor VIII variant, termed ET3. Factor VIII procoagulant activity levels were durably elevated into the normal range and phenotypic correction achieved. Furthermore, no immunological rejection or development of anti-ET3 immunity was observed. These preclinical data support clinical translation of non-genotoxic antibody-based conditioning in HSPC LV gene therapy for HA.
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Factor VIII (fVIII) is a procoagulant protein that binds to activated factor IX (fIXa) on platelet surfaces to form the intrinsic tenase complex. Due to the high immunogenicity of fVIII, generation of antibody inhibitors is a common occurrence in patients during hemophilia A treatment and spontaneously occurs in acquired hemophilia A patients. Non-classical antibody inhibitors, which block fVIII activation by thrombin and formation of the tenase complex, are the most common anti-C2 domain pathogenic inhibitors in hemophilia A murine models and have been identified in patient plasmas. In this study, we report on the X-ray crystal structure of a B domain-deleted bioengineered fVIII bound to the non-classical antibody inhibitor, G99. While binding to G99 does not disrupt the overall domain architecture of fVIII, the C2 domain undergoes an ~8 Å translocation that is concomitant with breaking multiple domain-domain interactions. Analysis of normalized B-factor values revealed several solvent-exposed loops in the C1 and C2 domains which experience a decrease in thermal motion in the presence of inhibitory antibodies. These results enhance our understanding on the structural nature of binding non-classical inhibitors and provide a structural dynamics-based rationale for cooperativity between anti-C1 and anti-C2 domain inhibitors.