Collaborative Project #10: Toward Safer Gene Therapy for Hemophilia A

Collaborating Investigator: Roland Herzog, PhD 
Affiliation: Riley Children’s Foundation Professor of Immunology, Indiana University  

Funding Source: NIH
Funding: Number: P01HL160472 
Project Period: 02/05/22-01/31/27  

Significance

The X-linked bleeding disorder Hemophilia A is caused by a deficiency in the clotting protein factor VIII (FVIII), and FVIII gene therapy thus provides a promising strategy for disease treatment. Unfortunately, FVIII gene therapy frequently results in the emergence of anti-drug antibodies that lead to therapeutic resistance and result in severe autoimmune complications. Our collaborators in CP #10 have developed FVIII-targeted chimeric antigen receptor (CAR)-expressing regulatory T cells (Tregs) to counteract anti-drug antibody development. However, the persistence and activation of the engineered Treg cells requires interleukin-2 (IL-2) stimulation, which can also lead to pathogenic autoimmune responses through activation of effector T and NK cells. Thus, delivery of a biased IL-2 formulation that exclusively stimulates Tregs would be highly advantageous to prevent hemophilia A treatment complications. The overarching objective of this CP is to determine whether our lead immunosuppressive IL-2-based immunocytokine (denoted F5111 IC) will synergize with Treg therapy to reduce the burden of anti-drug antibodies, thereby mitigating anti-drug antibody formation. In vivo models of hemophilia will be used to advance translational development of this approach.

Approach

Aim 1: Produce engineered panel of F5111 IC variants and validate their binding and signaling properties. A previously designed panel of F5111 IC variants will be produced recombinantly by the NCBIB using a mammalian cell expression system and purified by protein G chromatography followed by size-exclusion chromatography using a fast liquid protein chromatography (FPLC) instrument. Each batch of protein will be functionally validated through biolayer interferometry-based binding studies using an Octet® machine and IL-2 signaling studies on commercial human peripheral blood mononuclear cells.

Aim 2: Test mitigation of immune-mediated FVIII gene therapy resistance by F5111 IC variants in a mouse model of hemophilia and identify the lead F5111 IC formulation. The purified and validated F5111 IC variants from Aim 1 will be provided to the Herzog Lab, which will administer these reagents in combination with engineered Treg cells to assess their therapeutic performance in mouse models of hemophilia A. This work will define the optimal cytokine/antibody affinity for preventing immune-mediated resistance to FVIII gene therapy, advancing the development of disease-specific ICs.

Push/Pull Interactions

Push: The NCBIB will design an IL-2 IC that specifically directs cytokine activity toward Treg cells and push this to our collaborators. Dr. Herzog will administer the IL-2 IC in tandem with CAR Treg therapy in mouse models of Hemophilia A and determine whether IL-2 IC enhances the performance of engineered cell therapy.

Pull: Based on the results of the Herzog Lab’s findings, the NCBIB will optimize formulation of the IL-2 IC by using a previously generated panel of mutants based on the Treg-potentiating F5111 IC with various intramolecular cytokine/antibody affinities. The Herzog Lab will be provided with these variants and will identify the variant that is most successful in promoting FVIII tolerance. The outcomes of this push-pull relationship will be valuable to the NCBIB in expanding the translational relevance of technologies developed in the Center and informing further applications of IC technologies to address a range of other medical challenges.