David Lillicrap

Queen's University, Kingston, Ontario, Canada

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Publications (280)1424.53 Total impact

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    Full-text · Dataset · Jan 2016
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    Full-text · Dataset · Jan 2016
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    ABSTRACT: Anti-factor VIII (FVIII) antibodies is a major complication of FVIII replacement therapy for hemophilia A. We investigated the immune response to recombinant human factor VIII Fc (rFVIIIFc) in comparison to BDD-rFVIII and full-length rFVIII (FL-rFVIII) in hemophilia A mice. Repeated administration of therapeutically relevant doses of rFVIIIFc in these mice resulted in significantly lower antibody responses to rFVIII compared to BDD-rFVIII and FL-rFVIII and reduced antibody production upon subsequent challenge with high doses of rFVIIIFc. The induction of a tolerogenic response by rFVIIIFc was associated with higher percentage of regulatory T-cells, a lower percentage of pro-inflammatory splenic T-cells, and up-regulation of tolerogenic cytokines and markers. Disruption of Fc interactions with either FcRn or Fcγ receptors diminished tolerance induction, suggesting the involvement of these pathways. These results indicate that rFVIIIFc reduces immunogenicity and imparts tolerance to rFVIII demonstrating that recombinant therapeutic proteins may be modified to influence immunogenicity and facilitate tolerance.
    Preview · Article · Dec 2015 · Cellular Immunology
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    ABSTRACT: Recombinant FVIII manufacturing is characterized by poor product stability and low yields. Codon-optimization of transgenes accelerates translation by exploiting the synonymous codon usage bias of a species. However, this can alter the performance of the final product. Additionally, the effects of transgene design across diverse cell types are not well understood and are of interest for next-generation protein and gene therapies. To investigate the effects of transgene design across different host cells, B-domain-deleted (BDD) and modified codon-optimized (CO-N6) transgenes were inserted via lentiviral delivery into cBOECs, HEK293T, and MDCK cells. The CO-N6 cFVIII transgene produced threefold more protein per transgene in HEK293T cells, and sixfold more protein in the two canine cell lines. However, pharmacokinetic analysis in hemophilia A dogs demonstrated that cFVIII produced from cBOECs transduced with the CO-N6 transgene had significantly reduced in vivo recovery. Furthermore, this product showed reduced in vitro stability and activity on thrombin activation versus the BDD product. This trend was reversed in HEK293T lines. Overall, our results demonstrate the need for an integrated approach that not only assesses protein expression levels but also considers the influence that host-cells have on preserving the molecular and biochemical properties of the naturally occurring FVIII.
    Preview · Article · Nov 2015
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    ABSTRACT: The enigma that is factor VIII immunogenicity remains ever pertinent in the treatment of hemophilia A. Development of neutralizing antibodies against the therapeutic protein in 25-30% of patients likely depends on the appropriate activation of the innate immune response shortly following antigen encounter. Our understanding of this important immunological synapse remains ill-defined. In this review, we examine the three distinct factors contributing to the fate of factor VIII almost immediately after infusion: the characteristics of the protein, the cell, and the microenvironment. We propose a continuum between clearance and antigen presentation that facilitates removal of FVIII from circulation leading to either tolerance or immunity.
    No preview · Article · Nov 2015 · Cellular Immunology
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    ABSTRACT: The development of neutralizing anti-factor VIII (FVIII) antibodies (inhibitors) remains a major challenge for FVIII replacement therapy in hemophilia A patients. The adaptive immune response plays a crucial role in the development and maintenance of inhibitors. In this review, we focus on our current understanding of FVIII interactions with cells of the adaptive immune system and the phenotype of the resultant response. Additionally, we examine both current and novel FVIII tolerance induction methods that function at the level of the adaptive immune response.
    No preview · Article · Nov 2015 · Cellular Immunology
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    Full-text · Article · Nov 2015
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    David Lillicrap

    Preview · Article · Feb 2015 · Blood
  • Rydz N · Grabell J · Lillicrap D · James PD
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    ABSTRACT: In a normal population, VWF plasma levels (VWF:Ag) and VWF activity (VWF:RCo) increase by approximately 0.17 and 0.15 IU mL(-1) per decade, but the influence of age is unknown in patients with type 1 von Willebrand disease (VWD). In a retrospective cohort study, the medical records of 31 type 1 VWD patients over the age of 30, who had been followed for ≥5 years, were reviewed for baseline clinical data and previously performed VWF:Ag, VWF:RCo and factor VIII levels (FVIII:C). VWF multimer analysis was normal in 28/31 cases performed. Mean age at diagnosis was 33 (range 16-60 years), and duration of follow-up ranged from 5 to 26 years (mean 11 years). Patients had 2-10 time points of VWD testing (mean of 5.2). The mean VWF:Ag, VWF:RCo and FVIII:C at time of diagnosis were 0.44 IU mL(-1) 0.34 IU mL(-1) and 0.75 IU mL(-1) . At last follow-up, the mean VWF:Ag, VWF:RCo and FVIII:C were significantly increased to 0.71 IU L(-1) , 0.56 IU mL(-1) and 0.90 IU mL(-1) (P ≤ 0.001, <0.001, and 0.0081 respectively). Here 18/31 patients had VWF:Ag, VWF:RCo and FVIII: C levels that increased into the normal range. The rate of change in VWF:Ag, VWF:RCo and FVIII was 0.30 IU mL(-1) (0.21-0.39, CI 95%, P < 0.0001), 0.20 IU mL(-1) per decade (0.13-0.27, CI 95%, P = 0.0001) and 0.20 IU mL(-1) (0.11-0.29, CI 95%, P = 0.0011). Patients with type 1 VWD experience age-related increases to VWF:Ag and VWF:RCo which can result in normalization of VWF levels. Further studies are required to determine if the bleeding phenotype resolves with the increases in VWF:Ag and VWF:RCo levels. © 2015 John Wiley & Sons Ltd.
    No preview · Article · Feb 2015 · Haemophilia
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    ABSTRACT: The rising incidence of neutralizing antibodies (inhibitors) against therapeutic factor VIII prompted the conduct of studies to answer the question as to whether this rise is related to the introduction of recombinant factor VIII products. The present article summarizes current opinions and results of non-clinical and clinical studies on the immunogenic potential of recombinant compared to plasma-derived factor VIII concentrates. Numerous studies provided circumstantial evidence that von Willebrand factor, the natural chaperone protein present in plasma-derived factor VIII products, plays an important role in protecting exogenous factor VIII from uptake by antigen presenting cells and from recognition by immune effectors. However, the definite contribution of von Willebrand factor in reducing the inhibitor risk and in the achievement of immune tolerance is still under debate. Copyright© Ferrata Storti Foundation.
    Preview · Article · Feb 2015 · Haematologica
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    ABSTRACT: We report an 11-month-old boy with severe hemophilia A who had regular exposure to factor VIII (FVIII) intended to reduce the risk of developing an inhibitor. He developed a high-titer inhibitor (peak titer 19 BU) that disappeared within 6 weeks of starting immune tolerance induction (ITI). Anti-FVIII IgG4 peaked briefly compared with anti-FVIII IgG1 and the Bethesda titer. Neither rapid resolution of an inhibitor after prophylaxis nor this behavior of anti-FVIII IgG4 has been previously reported. Transient anti-FVIII IgG4 may be a marker of an attenuated anti-FVIII response induced by prophylactic FVIII therapy.
    No preview · Article · Nov 2014 · Journal of Pediatric Hematology/Oncology
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    David Lillicrap
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    ABSTRACT: Details of the pathophysiologic mechanisms that underlie complex disorders, such as the thrombo-occlusive events associated with myocardial infarction, stroke, and venous thromboembolism, are challenging to address. Recent advances have been made through the application of genome-wide association studies (GWAS) to identify genetic loci associated with plasma levels of procoagulant proteins and risk of thrombotic disease. GWAS have consistently identified the gene encoding syntaxin-binding protein 5 (STXBP5) in this context. STXBP5 is expressed in both endothelium and platelets, and SNPs within the STXBP5 locus have been associated with plasma levels of vWF and increased venous thrombosis risk. In this issue of the JCI, two complementary reports from the laboratories of Charles Lowenstein and Sidney Whiteheart describe studies that highlight the complexity of the function of STXBP5 in control of storage granule development and exocytosis in platelets and endothelium. Together, these studies demonstrate that STXBP5 differentially regulates exocytosis in these two cell types. While STXBP5 facilitates granule release from platelets, it inhibits secretion from the Weibel-Palade bodies (WPBs) of endothelial cells.
    Preview · Article · Sep 2014 · Journal of Clinical Investigation
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    ABSTRACT: Background Routine prophylaxis with replacement factor VIII (FVIII)—the standard of care for severe hemophilia A—often requires frequent intravenous infusions (3–4 times per week). A FVIII molecule with an extended half-life could reduce infusion frequency. The A-LONG study established the safety, efficacy and prolonged pharmacokinetics of recombinant FVIII Fc fusion protein (rFVIIIFc) in previously treated adolescents and adults with severe hemophilia A.Objective In this post-hoc analysis, we investigated the relationship between subjects’ prestudy (FVIII) and on-study (rFVIIIFc) regimens.Methods We analyzed two subgroups of subjects: prior prophylaxis and on-study individualized prophylaxis (n=80), and prior episodic treatment and on-study weekly prophylaxis (n=16). Subjects’ prestudy dosing regimens and bleeding rates were compared with their final rFVIIIFc regimen and annualized bleeding rate (ABR) in the last 3 months on study. Dosing regimen simulations based on population pharmacokinetics models for rFVIII and rFVIIIFc were performed.ResultsCompared with their prestudy regimen, 79/80 (98.8%) subjects on individualized rFVIIIFc prophylaxis decreased their infusion frequency. Overall ABRs were low, with comparable factor consumption. Longer dosing intervals, including 5-day dosing, were associated with higher baseline von Willebrand factor antigen levels. Simulated dosing regimens predicted a greater proportion of subjects with steady-state FVIII activity trough levels ≥1 IU/dL (1%) with rFVIIIFc versus equivalent rFVIII regimens.Conclusion These results suggest that patients on rFVIIIFc prophylaxis can reduce their infusion frequency compared with their prior FVIII regimen while maintaining low bleeding rates, affording more patients trough levels ≥1 IU/dL compared with rFVIII products utilizing more frequent dosing regimens.This article is protected by copyright. All rights reserved.
    No preview · Article · Sep 2014 · Journal of Thrombosis and Haemostasis
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    ABSTRACT: Rare missense mutations in the von Willebrand factor (VWF) A3 domain that disrupt collagen binding, have been found in patients with a mild bleeding phenotype. However, the analysis of these aberrant VWF-collagen interactions has been limited. Here, we have developed mouse models of collagen binding mutants and analyzed the function of the A3 domain using comprehensive in vitro and in vivo approaches. 5 loss-of-function (p.S1731T, p.W1745C, p.S1783A, p.H1786D, A3 deletion) and 1 gain-of-function (p.L1757A) variant were generated in the mouse VWF cDNA. The results of these various assays were consistent, although the magnitude of the effects were different: the gain-of-function (p.L1757A) variant showed consistent enhanced collagen binding while the loss-of-function mutants showed variable degrees of functional deficit. We further analyzed the impact of direct platelet-collagen binding by blocking glycoprotein VI (GPVI) and integrin α2β1 in our ferric chloride murine thrombosis model. The inhibition of GPVI demonstrated a comparable functional defect in thrombosis formation to the VWF(-/-) mice whereas α2β1 inhibition demonstrated a milder bleeding phenotype. Furthermore, a delayed and markedly reduced thrombogenic response was still evident in VWF(-/-), GPVI and α2β1 blocked animals, suggesting that alternative primary hemostatic mechanisms can partially rescue the bleeding phenotype associated with these defects.
    Preview · Article · Jul 2014 · Blood
  • Laura L Swystun · David Lillicrap
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    ABSTRACT: In this issue of Blood, Yee et al1 have demonstrated that expression or infusion of a truncated von Willebrand factor (VWF) fragment containing the factor VIII (FVIII)-binding D′D3 region of VWF is sufficient to stabilize endogenous FVIII levels in VWF-deficient mice. In the absence of the carrier function of VWF, FVIII is susceptible to rapid proteolysis and clearance resulting in markedly reduced plasma levels of FVIII that contribute to a bleeding diathesis.
    No preview · Article · Jul 2014 · Blood
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    ABSTRACT: The Canadian Hemophilia Assessment and Resource Management System (CHARMS) tracks factor concentrates (FC) from the sole suppliers, Canadian Blood Services (CBS) and Hema-Quebec (HQ), to hospitals and to patients' homes. Patients FC infusion data are entered into CHARMS at Canadian Hemophilia Treatment Centres (HTCs) then exported to the national database (CentrePoint). From 2000 to 2009, 2260 registered haemophilia A or B patients received FVIII (1 009 097 765 IU) and FIX (272 406 859 IU). Over 91% of FVIII and over 84% of FIX was infused at home. Utilization of FVIII progressively increased; this was accounted for by an increase in the number of patients treated (r = 0.97; P < 0.001), there being a linear relationship between the increase in utilization and the increase in number of patients treated (P < 0.001). There was also a correlation with the annual amount used per patient (r = 0.95; P < 0.001). Utilization of FIX did not increase over time. The highest proportional utilization of both FVIII and FIX was for prophylaxis, and this proportion progressively increased being, in year 10 (2009), 77% and 66% for FVIII and FIX respectively. The proportion used for bleeding remained steady; in year 10 that proportion was 14% for FVIII and 26% for FIX, the use per patient for bleeding decreasing. The HTC-based CHARMS tracking system is essential, in Canada, for analysing indications for infusion, for predicting utilization and planning for future needs.
    Full-text · Article · Jul 2014 · Haemophilia
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    ABSTRACT: The objective of this study was to evaluate the inhibitor development (ID) in previously untreated patients (PUPs) with severe haemophilia A (FVIII ≤ 0.01 IU mL−1). All Canadian Haemophilia Treatment Centres completed a questionnaire on patients born between September 2005 and August 2010 and followed for up to 7 years. Eligible patients had at least 20 exposure days (ED) or had developed an inhibitor. The odds ratio (OR) and 95% confidence intervals (95% CI) for risk factors to develop an inhibitor were estimated using unconditional logistic regression. A total of 99 haemophilia A PUPs were studied. Thirty-four (34%) developed an inhibitor (24/34 of high titre). Inhibitors developed in 25/63 (40%) patients with a high-risk mutation. ID was most frequent in Aboriginals (86%). Dose intensity (IU kg−1 day−1 X number of ED) at first exposure to factor VIII (FVIII) was associated with a crude OR increase of 1.10 (95% CI: 0.99–1.23) with each increase of 100 dose-intensity units. Haemarthrosis and intracranial bleeding as the indication for first exposure to FVIII concentrate were associated with a crude OR for ID of 7.63 (95% CI: 2.14–27.17) and 5.08 (95% CI: 1.11–23.31) respectively. ID according to FVIII concentrate used was: Advate ® 18/50 (36%), Kogenate FS® or Helixate FS® 15/36 (42%), Wilate® 0/11 and Xyntha® 1/2. In multivariate analysis, Aboriginal ethnicity (OR = 11.69; 95% CI: 1.11–122.86) and haemarthrosis (OR = 4.49; 95% CI: 1.08–18.61) were statistically significant. The cumulative incidence of ID in severe haemophilia A PUPs was 34% and varied according to ethnicity, type of bleeding at first ED, type of FVIII product and dose intensity at first exposure.
    No preview · Article · Jun 2014 · Haemophilia
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    ABSTRACT: Ex vivo gene therapy strategies avoid systemic delivery of viruses thereby mitigating the risk of vector associated immunogenicity. Previously, we delivered autologous FVIII-expressing blood outgrowth endothelial cells (BOECs) to hemophilia A mice and showed that these cells remain sequestered within the implanted matrix and provide therapeutic levels of FVIII. Prior to translating this strategy into the canine model of hemophilia A, we increased cFVIII transgene expression by at least 100-fold with the use of the EF1α promoter and a strong endothelial enhancer element. BOECs isolated from hemophilia A dogs transduced with this lentiviral vector express levels of cFVIII ranging between 1.0 to 1.5 U/mL/10(6) cells/24 hours. Autologous BOECs have been implanted into the omentum of 2 normal and 3 hemophilia A dogs. These implanted cells formed new vessels in the omentum. All three hemophilia A dogs treated with FVIII-expressing autologous BOECs developed anti-FVIII IgG2 antibodies, but in only two of the dogs were these antibodies inhibitory (≤5 BU). Factor VIII antigen levels >40%, in the absence of FVIII coagulant function, were detected in the circulation for up to a year after a single gene therapy treatment, indicating prolonged cellular viability and synthesis of FVIII.
    Full-text · Article · May 2014 · Blood
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    K. H. High · A. Nathwani · T. Spencer · D. Lillicrap
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    ABSTRACT: After many reports of successful gene therapy studies in small and large animal models of haemophilia, we have, at last, seen the first signs of success in human patients. These very encouraging results have been achieved with the use of adeno-associated viral (AAV) vectors in patients with severe haemophilia B. Following on from these initial promising studies, there are now three ongoing trials of AAV-mediated gene transfer in haemophilia B all aiming to express the factor IX gene from the liver. Nevertheless, as discussed in the first section of this article, there are still a number of significant hurdles to overcome if haemophilia B gene therapy is to become more widely available. The second section of this article deals with the challenges relating to factor VIII gene transfer. While the recent results in haemophilia B are extremely encouraging, there is, as yet, no similar data for factor VIII gene therapy. It is widely accepted that this therapeutic target will be significantly more problematic for a variety of reasons including accommodating the larger factor VIII cDNA, achieving adequate levels of transgene expression and preventing the far more frequent complication of antifactor VIII immunity. In the final section of the article, the alternative approach of lentiviral vector-mediated gene transfer is discussed. While AAV-mediated approaches to transgene delivery have led the way in clinical haemophilia gene therapy, there are still a number of potential advantages of using an alternative delivery vehicle including the fact that ex vivo host cell transduction will avoid the likelihood of immune responses to the vector. Overall, these are exciting times for haemophilia gene therapy with the likelihood of further clinical successes in the near future.
    Preview · Article · May 2014 · Haemophilia
  • S. Albánez · K. Ogiwara · J. Grabell · P. James · D. Lillicrap

    No preview · Article · May 2014 · Thrombosis Research

Publication Stats

7k Citations
1,424.53 Total Impact Points

Institutions

  • 1987-2015
    • Queen's University
      • • Department of Pathology and Molecular Medicine
      • • Department of Medicine
      • • Department of Biology
      Kingston, Ontario, Canada
  • 2006-2014
    • Queen's University Belfast
      Béal Feirste, N Ireland, United Kingdom
  • 1999-2014
    • Queens University of Charlotte
      Charlotte, North Carolina, United States
  • 2013
    • Kingston University
      England, United Kingdom
  • 2010
    • The Children's Hospital of Philadelphia
      • Department of Pediatrics
      Philadelphia, Pennsylvania, United States
  • 2005
    • Dalhousie University
      • Department of Chemistry
      Halifax, Nova Scotia, Canada
  • 2002
    • The University of Calgary
      • Department of Paediatrics
      Calgary, Alberta, Canada
    • Radboud University Medical Centre (Radboudumc)
      • Department of Human Genetics
      Nymegen, Gelderland, Netherlands
  • 1993
    • Royal Berkshire NHS Foundation Trust
      Reading, England, United Kingdom
    • Howard Hughes Medical Institute
      Ашбърн, Virginia, United States
  • 1986-1993
    • Kingston General Hospital
      Kingston, Ontario, Canada
  • 1989
    • University of Ottawa
      • Department of Medicine
      Ottawa, Ontario, Canada
  • 1985-1989
    • University of Wales
      • College of Medicine
      Cardiff, Wales, United Kingdom