Hemophilia A (HA) is a X-linked bleeding disorder caused by deficiency of coagulation factor VIII (FVIII). Optimal clinical management centers on FVIII protein concentrate replacement. However, up to 30% of patients with severe HA develop neutralizing antibodies to FVIII (inhibitors) upon exposure to therapeutic FVIII. Inhibitors neutralize the infused FVIII and, thus, pose a significant challenge in the management of these patients. Immune tolerance induction (ITI) using high-dose FVIII infusions can eliminate inhibitors but is not effective at generating long-term eradication in all patients. Thus, we developed an immunotherapy to more effectively eliminate FVIII-specific B cells to induce lasting eradication of inhibitors.

Adoptive T cell immunotherapies using chimeric antigen receptor (CAR)-modified T cells (CARTs) are showing encouraging results in the treatment of cancer. We previously reported a proof-of-concept CART approach to eliminate autoantibodies to desmoglein-3, implicated in pemphigus vulgaris, by targeting non-malignant B cells. Here, using a similar strategy, we constructed two FVIII-based CARs using isolated FVIII A2 and C2 domains, which are commonly targeted by inhibitors

Both FVIII fragments were used as the CAR extracellular domain followed by CD8a-derived hinge and transmembrane domains and intracellular signaling domains derived from 4-1BB and CD3z (BBz), as has been used in a recently FDA-approved CD19-targeted CAR (CART19) that have shown efficacy in patients. A2- and C2-CARs were introduced into primary human T cells using lentivirus vectors and were found to express on the cell surface. Primary expansion following anti-CD3/CD28 activation and lentivirus transduction yielded comparable population doublings of CART19 and A2-CARTs over 9-11 days (mean, 4.3 vs. 3.7; p=0.16). However, C2-CARTs consistently achieved fewer doublings compared to CART19 (mean 4.3 vs. 2.6; p=0.01). Additionally, C2-CARTs maintained larger cell volumes following initial activation compared to A2 and CART19, reminiscent of other CARs that demonstrate a high-level of ligand-independent basal activation. Consistent with hyper-activation and activation-induced cell death, flow cytometric analysis of C2-CART cultures showed prolonged expression of CD69, increased levels of cell death, and gradual loss of CAR+ cells compared to A2 and CART19 cultures. The FVIII C2 domain contains hydrophobic surfaces involved in binding to phospholipid membranes and von Willebrand Factor. We hypothesized that these hydrophobic regions may cause unfavorable interactions that result in CAR clustering and, in-turn, ligand-independent signaling. However, since these regions are also targeted by FVIII inhibitors, mutation of hydrophobic residues to improve CAR function would likely result in loss of binding to intended targets. Thus, we tested two independent strategies to improve function of C2-CARTs, both of which maintain native FVIII domain sequences. First, to block unfavorable interactions with C2 hydrophobic surfaces, we tested whether addition of C2 domain specific antibodies to the CART culture would improve expansion. Indeed, addition of anti-C2 antibodies improved C2-CART expansion by approximately one population doubling and reduced both cell volume and CD69 expression in C2-CARTs. As an alternate and perhaps more easily translatable approach, we replaced the BBz CAR signaling domain with a killer immunoglobulin receptor (KIR)-based multi-chain signaling system, which we previously utilized to improve function of a mesothelin-targeted CAR. KIR-based signaling effectively "rescued" the C2 CAR, and showed an expansion and activation profile comparable to CART19. Lastly, KIR-based FVIII CARs, demonstrated specific lysis of and cytokine response to target cells engineered to express surface anti-FVIII, even in the presence of soluble inhibitors.

These data indicate that unfavorable interactions may be ameliorated directly by blocking agents or, indirectly, by altering intracellular signal transduction domains. These strategies have allowed us to construct optimized FVIII-based CARTs to target FVIII-specific B cells that are responsible for producing FVIII inhibitors in patients with HA.


Doshi:Bayer Hemophilia Awards Program: Research Funding. Milone:Novartis: Patents & Royalties.

Author notes


Asterisk with author names denotes non-ASH members.