BPAC 2010 Laboratory of Hemostasis. Basil Golding M.D. Director, Division of Hematology OBRR/CBER/FDA. Laboratory of Hemostasis. Research/Regulator Chava Kimchi-Sarfaty, Ph.D., Senior Staff Fellow Nobuko Katagiri, Ph.D., Staff Fellow Mikhail Ovanesov, Ph.D., Visiting Scientist
Basil Golding M.D.
Director, Division of Hematology
Full-time Regulatory Reviewers
( Retired in June 2010)
Chava Kimchi-Sarfaty, Ph.D.
Laboratory of Hemostasis
DH / CBER / FDA
New concept paper/guidance on clinical pharmacogenomics discusses the following issues:
However, currently there are no regulations or guidelines vis à vis the choice of sequence in the development of a recombinant therapeutic protein
SNPs in the ADAMTS13 protein that were investigated in our laboratory
CUB1 and 2
SNP studied by our lab
I. ADAMTS13, the von Willebrand Factor (VWF) cleaving protease:
We have completed the in silico studies of twelve Single Nucleotide Polymorphisms (SNPs) in ADAMTS13, located in various domains of the protein:
The results of these in silico studies were compared to in vitro studies
Intracellular protein levels & conformation changes
Protein conformational changes
Intracellular and extracellular protein levels
In silico and in vitro studies of synonymous mutation Val107Val in F9 gene, found in five unrelated Swedish Hemophilia B patients:
In silico and in vitro assays performed as described previously for ADAMTS13
Zuben E. Sauna Ph.D.
Evaluate genetic variability in the endogenous F8 gene vis- à-vis polymorphisms as a risk-factor for immunogenicity
Evaluate HLA restriction of patients (in the context of sequence mismatch) as a risk-factor for immunogenicity
A pharmacogenetic approach, based on individual patients, is necessary for the accurate prediction of immunogenicity
The extent and position of sequence mismatch between the endogenous and infused FVIII (due to disease causing mutations and SNPs) and the HLA restriction of the patient are important risk-factors for immunogenicity
Study of the regulation of blood coagulation kinetics
2009 2010 2011 2012 2013 2014 years
Thrombin generation test
Mechanism of Action,
Clot growth assay
Coagulation in mouse
(deep tissue imaging)
Control non-implicated lots
Negative control (buffer)
Identification of Binding Sites on Coagulation Factor VIII (FVIII) for its Catabolic Receptors:
Low-Density Lipoprotein Receptor (LDLR)
Low-Density Lipoprotein Receptor-Related Protein (LRP)
LRP and LDLR: Expressed in the liver, and catabolize various ligands (plasma lipoproteins etc). Cooperate in clearance of FVIII: deficiency in both receptors results in prolongation of the half-life of FVIII by 5 times in mice
The ongoing project involves: establishing expression of FVIII, LRP and LDL fragments, performing scanning mutagenesis of FVIII, testing interaction of the FVIII mutants with LRP and LDL in various in vitro and in vivo assays to determine the binding epitope(s) on FVIII.
Future Plans: Testing possibility of generation of "improved" (long-lasting) FVIII for better treatment of Hemophilia A. The “improved” FVIII would have a decrease in the affinities for LDLR and LRP, while retaining activity, but not more immunogenic
Selection of FVIII amino acid residues for scanning mutagenesis
Expression of fragments of FVIII, LDLR, LRP and FVIII mutants
Performing binding assays for FVIII variants and receptors’ fragments
Testing receptor-mediated internalization of FVIII variants in cell culture
Testing clearance of FVIII variants
in mice (normal, FVIII- and vWF-deficient)
Determination of critical residues of FVIII for binding to LRP
I. Ongoing Experiments: Expression of FVIII and its variants
LRP (LDLR) -binding region
Constructs to be Generated
Introduction of point mutations
Light Chain variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
O-linked sugar domain
II. Ongoing Experiments: Expression of LDLR Fragments
(a minimal ligand-binding motif is composed by two adjacent CRs)
1) CR 1-7 . . . .
2) CR 1-2 . . . .
3) CR 2-3. . . . . . .
4) CR 3-4 . . . . . . . . .
5) CR 4-5 . . . . . . . . . . .
6) CR 5-6 . . . . . . . . . . . . . .
7) CR 6-7 . . . . . . . . . . . . . . . .
Generation of FVIII having a decrease in affinities for LDLR and LRP yet retaining the functional activity, and not increased immunogenicity.
The “improved” FVIII would have a longer half-life in the circulation, thus requiring less frequent infusions into Hemophilia A patients, and a reduced cost.