Oral Anti-CD3 and Bioencapsulated FVIII Therapy Exhibit Distinct Mechanisms That Suppress Inhibitor Formation in Hemophilia a

Background Inhibitory antibodies against coagulation factor VIII (FVIII) present a serious complication to treatment of the X-linked coagulation disorder hemophilia A (HA). Oral tolerance represents unresponsiveness to subsequent parenteral antigen injections, which can be explored as a standalone or adjunctive prophylactic tolerance approach to prevent inhibitor formation. Although several mechanisms have been proposed, from anergy and/or deletion of antigen-specific T cells, to active suppression by regulatory T cells (Tregs), substantial knowledge gaps on the phenotype and function of such cells remain.

Hypothesis

We compared 2 methods to induce oral tolerance to FVIII: delivery of anti-CD3 treatment or a bioencapsulated FVIII cholera toxin B (CTB) subunit fusion product expressed in chloroplasts of lettuce cells (FVIII lettuce). Bioencapsulation prevents premature digestion of FVIII prior to uptake by the gut immune system, whereas CTB fusion ensures translocation across the intestinal epithelium. We previously established that oral uptake of FVIII lettuce suppresses inhibitor formation to subsequent intravenous (IV) FVIII injections [Blood 124:1659].

Methods

HA mice on a BALB/c background (n=≥8/group) received anti-mouse CD3 (full length or F(ab')₂, 0.5mg or 5mg) by oral gavage, 1X/day X 5 consecutive days. Concurrently, mice received 5 weekly IV injections of 1.5 international units (IU) of human FVIII (Xyntha). Control mice received FVIII injections without oral gavage. Alternatively, mice received FVIII lettuce (CTB-FVIII-HC/C2 domains, 1.5 µg/mouse) 2x/week for 9 weeks. 4 weeks into the experiment, 1.5 IU of human FVIII was IV administered 1X/week X 5 weeks. In a third experiment, mice received both oral anti-CD3 and FVIII lettuce.

Spleens from the FVIII lettuce group were isolated, magnetically enriched for CD4+ T cells and sorted into 3 populations: CD25+FoxP3+LAP- (nTreg), CD25+FoxP3+LAP+ (activated Treg), and CD25-FoxP3-LAP+ (LAP Treg). Single cell 3' RNA-seq was performed using the Chromium single cell system and Illumina sequencing. Differential gene expression analysis was performed using LTMG method to handle batch effects; unsupervised clustering was done using the Seurat pipeline and visualized using UMAP.

Results

Oral anti-CD3 F(ab')₂ (0.5 µg) administered concurrently with IV FVIII injections or oral FVIII lettuce each significantly reduced inhibitor formation in subsequently IV delivery of FVIII by 2-3-fold (p=0.01, 1-way ANOVA). Combining these oral therapy treatments, however, did not represent an improvement over monotherapy with either anti-CD3 or FVIII lettuce, indicating non-synergistic mechanisms of suppression. Oral anti-CD3 therapy caused splenic CD4+CD25+FoxP3+LAP+ activated Tregs to markedly upregulate immune checkpoint receptors PD1 and TIGIT independent of IL-10 production. Both PD1 and TIGIT expression represent a highly activated Treg profile with superior suppressive capacity. Phenotype and cell frequencies of nTregs and LAP Tregs remained unchanged in the anti-CD3 treatment group.

In contrast, FVIII lettuce treatment resulted in a significant increase in LAP Tregs (1.73% of CD4+) over control (0.55% of CD4+, p=0.0011, 1-way ANOVA), with a concurrent increase in IL-10 production (0.34% of CD4+) over control (0.018% of CD4+, p<0.0001, 2-way ANOVA). LAP Tregs from lettuce-fed mice showed high expression of ICOS, CD69 and Ki-67, but not CTLA-4.

Clustering analysis on single cell RNA sequencing data of nTreg, activated Treg, and LAP Tregs from FVIII lettuce-fed mice confirmed distinct clusters between the nTreg and LAP Treg subsets, with activated Tregs sharing properties of both. All 3 populations were highly heterogenous. We observed 1320 differentially expressed genes (fold change >2) between LAP Treg and nTreg (916 upregulated and 404 downregulated). Canonical nTreg markers such as FoxP3, CD25, and GITR were significantly downregulated in LAP Tregs. LAP expression highly correlated with expression of TGFb1 and IL-10 cytokines, increased expression of chemokine receptors and their ligands and of integrin family members, and showed an inflammation-induced profile characterized by upregulation of IL1B, granzymes, Jun Fos pathway, among others.

We conclude that LAP Tregs are a phenotypically distinct cell subset with a unique gene signature, allowing us to identify correlates of suppression in this population.

Biswas:Pfizer: Consultancy. Rana:Bayer Pharmaceutic company: Research Funding. Herzog:Spark Therapeutics: Research Funding; Regeneron Pharma/Intellia Therapeutics collaboration: Consultancy; Pfizer: Consultancy; BioMarin: Consultancy.