Factor IX Delivery to the Skin Primes Inhibitor Formation and Sensitizes Hemophilia B Mice to Systemic Factor IX Administration

Inhibitor formation is the most serious complication of factor (F)IX replacement therapy for hemophilia B, exacerbated by anaphylactoid reactions occurring in up to 50% of inhibitor patients. Low success rates and a high burden of immune tolerance induction (ITI) therapy necessitate the search for novel immune tolerance therapies. Skin-associated lymphoid tissues (SALT) have been successfully targeted in allergen-specific immunotherapies. In this study, we aimed to develop a prophylactic immune tolerance protocol based on intradermal (ID) administration of rFIX.

In a dose-finding experiment, hemophilia B mice on C3H/HeJ genetic background (C3H/HeJ-F9^tm1Dws) received twice weekly ID injections of 0.01, 0.1 or 1 IU rFIX only for four weeks, followed by one intraperitoneal (IP) and four weekly intravenous (IV) injections of 1 IU FIX co-injected with anti-allergic agents (triprolidine and ABT-491). Control animals received the IP/IV doses only. One week after the last injection, plasma and/or lymph nodes (LNs) were collected for Bethesda assay, ELISA and flow cytometry analyses.

Unexpectedly, all animals developed significantly (8.9-17.6-fold, p<0.05) higher FIX inhibitor titers than the control group (mean 4.4, 3.9, 8.5 and 0.6 BU/ml in the 0.01, 0.1, 1 IU and control group, respectively). Anti-FIX IgG1 antibody levels were also significantly higher (2.7-3.4-fold, p<0.001) in all ID treated groups (mean 54.8, 68.8, 69.3 and 20.1 µg/ml in the 0.01, 0.1, 1 IU and control group, respectively). This apparent enhancement of inhibitor formation prompted analyses of plasma samples collected after four weeks of ID FIX injections only to find out whether ID treatment alone could elicit FIX inhibitor formation. Two (0.1 and 1 IU) of the three evaluated ID FIX doses led to inhibitor formation of a similar magnitude to the full-length ID/IP/IV regimen. Although most (seven out of nine) mice treated with the lowest ID dose of FIX (0.01 IU) had no detectable inhibitors, the same dose, when followed by the IP/IV treatment, resulted in a similar inhibitor response to the two higher doses (0.1 and 1 IU), suggesting that the lowest dose primed the immune responses, which were further amplified by the IP/IV regimen. Also, all ID treated animals had readily detectable anti-FIX IgG1, but the levels were smaller than after the full-length treatment (mean 17.8, 35.6 and 33 µg/ml in the 0.01, 0.1 and 1 IU groups, respectively). This suggests that the IP/IV regimen following the ID treatment enhanced mainly the total anti-FIX IgG1 response, with a lesser impact on the neutralizing antibody development.

Inhibitor formation following ID treatment seemed to be driven by T follicular helper (PD1 ⁺CXCR5 ⁺CD4 ⁺) and Germinal Center B cell (GL7 ⁺CD95 ⁺CD19 ⁺) responses in inguinal LNs, the frequencies of which were 1.75-fold and 4-fold (p<0.05) elevated in animals treated with 1 IU FIX ID compared to control mice that received PBS only.

Notably, none of the ID treated mice died of anaphylaxis despite receiving eight ID injections of FIX without anti-allergic agents. IV administration of FIX alone in C3H/HeJ-F9^tm1Dws hemophilia B mice results in fatal IgE-dependent anaphylaxis beginning after fourth injection with ~20% mortality and rising with subsequent injections in the surviving mice, which necessitates the use of anti-allergic agents. Interestingly, the ID treated mice had elevated levels of anti-FIX IgE antibodies. Animals that received ID injections only or followed by IP/IV treatment had 1.5- and 3.3-fold higher (p<0.01) anti-FIX IgE levels than mice that received the IP/IV treatment only. The absence of mortality in the ID treated animals suggested that ID FIX delivery might have a neutral or protective effect against anaphylaxis despite anti-FIX IgE development. To test this hypothesis, hemophilia B mice received eight twice weekly ID doses of 1 IU FIX followed by IV or IV only, with neither group receiving anti-allergic agents at any time (n=15/group). After the first IV injection, 33.3% and 0% animals died in the ID treated and the IV only group, respectively, prompting early termination of the experiment for humane reasons.

In conclusion, ID administration of FIX primes or produces strong inhibitor responses in a wide range of doses and sensitizes hemophilia B mice to systemic delivery of FIX, suggesting that the skin-associated lymphoid tissue may not be amenable to FIX tolerance induction.