# Factor VIII Immunogenicity-Biology and Structure: Project 4

> **NIH NIH U54** · CHILDREN'S HOSP OF PHILADELPHIA · 2020 · $305,571

## Abstract

Project 4-Abstract
We approach the investigation of the biochemical and structural basis for the inhibitory immune response to
FVIII, with a fresh perspective drawn from our extensive background in the biochemistry of the homologous
and related coagulation protein, FV. We will, for the first time, systematically address the implication from
human patient data that the antibody response to human FVIII differs fundamentally, in character and intensity,
to that of human FV. Using comparable B domain deleted variants of both proteins injected into wild type mice,
we propose to compare the total inhibitory antibody response. Further mapping of the differential response will
be performed using chimeric variants of FVIII or FV containing individual domains from the human protein on
the mouse background. Obvious differences will be further studied by examining the spectrum of the immune
response to FV by hybridoma technology in comparison to that previously established with FVIII. A second
hypothesis will investigate whether the differential response to the two proteins relates to mechanistic
differences employed to maintain functional quiescence in each procofactor. Studies under Aim 2 further
address whether the binding of FVIII to vWF plays a fundamental role in regulating the immune response to the
cofactor using immunization strategies with FVIII mutants with variously impaired vWF binding. A second
approach will test whether defined proteolytic derivatives of FVIII, normally produced during its life cycle, play a
previously unconsidered role in interrogating the immune system and determining the inhibitory response to
FVIII. Aim 3 draws on our structural advances with FV(a) derivatives to ask whether the structural framework
for considering the effects of inhibitory antibody binding to FVIII is compromised by geometric constraints
within the C domains imposed by crystal packing. We propose small angle scattering and high resolution Cryo-
EM of FVIII in solution and bound to membrane nanodiscs to address whether conformational flexibility within
the C domains underlies its solution conformation and when bound to membranes. We will use prototypic
antibody inhibitors to C1 and C2 as well as the recombinant D’D3 fragment of vWF to test for the commonality
of the idea that these ligands stabilize the C domains within FVIII in a sub-optimal for its high affinity interaction
with membranes. Finally drawing from structural advances with FV(a) and FXa, we will use x-ray
crystallography in combination with the other structural approaches to test the hypothesis that inhibitory
antibodies to the A2 domain segregate into those that impact the binding of the cofactor to FIXa, to FX or both
to some intermediate extent. Our proposed multi-pronged approach, within the larger context of this U54, will
shed new light on basic details of the biochemistry underlying the inhibitor response to FVIII infusions in
hemophilia A patients.

## Key facts

- **NIH application ID:** 9949786
- **Project number:** 5U54HL142012-03
- **Recipient organization:** CHILDREN'S HOSP OF PHILADELPHIA
- **Principal Investigator:** Rodney M Camire
- **Activity code:** U54 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $305,571
- **Award type:** 5
- **Project period:** — → —

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9949786

## Citation

> US National Institutes of Health, RePORTER application 9949786, Factor VIII Immunogenicity-Biology and Structure: Project 4 (5U54HL142012-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9949786. Licensed CC0.

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