Induction of tolerance to factor VIII inhibitors by gene therapy with immunodominant A2 and C2 domains presented by B cells as Ig fusion proteins

Abstract

Up to 30% of patients with hemophilia A given therapeutic factor VIII (fVIII) can make inhibitory antibodies, the majority of which are reactive with its C2 and A2 domains. We have previously demonstrated that antigen-specific tolerance to several antigens can be induced by lipopolysaccharide (LPS)-activated B-cell blasts transduced with immunoglobulin (IgG)-antigen fusion constructs. To apply this system to hemophilia A inhibitor formation, we created retroviral vectors expressing fVIII amino acids S2173-Y2332 (C2 domain) and S373-R740 (A2 domain) in frame with an IgG heavy chain backbone. These vectors were transduced into B-cell blasts to induce tolerance in both naive and fVIII-primed hemophilic (E16 fVIII(-/-)) mice. Thus, treatment of E16 fVIII(-/-) mice with B cells expressing fVIII C2 and A2 domains led to tolerance in terms of specific humoral response (including inhibitory antibody titers) and cellular responses to fVIII and its C2 or A2 domains. Moreover, a significant reduction in immune responses to fVIII could be achieved in immunized hemophilic mice with existing anti-fVIII titers. This hyporesponsive state persisted for at least 2 months and withstood additional challenge with fVIII. Further experiments, in which mice were treated with a depleting monoclonal anti-CD25, suggested that a regulatory T cell may be required for the tolerogenic effect of transduced B cells. These findings demonstrate that B-cell presentation of fVIII domains on an Ig backbone specifically prevents or decreases existing antibodies in hemophilia A mice.

Figure 1.

Linear map of C2-Ig/ or A2-IgG/MSCV retroviral vector.

Figure 2.

Recipients of B-cell blasts transduced with C2-IgG/MSCV, A2-IgG/MSCV, or both are hyporesponsive to fVIII and its C2 or A2 domains in naive E16 mice. (A-C) Naive hemophilia A mice were injected with B cells transduced with retroviral vectors expressing C2-IgG (□), A2-IgG (▵), both (▪), or control SAG-IgG (♦) and challenged repeatedly with human fVIII. Animals were euthanized at 5 days after final fVIII injection. T-cell proliferation of spleen cells from individual mice was measured in triplicate against C2 (A), A2 (B), and fVIII (C). Data represent the mean plus or minus SEM for individual mice of the cpm incorporated minus background. ^*P < .05 versus mock control (SAG-Ig B cells). ^**P < .01. Serum anti-C2 (D) and anti-fVIII (E) antibody (total IgG) titers were determined by ELISA. One set of 2 similar experiments is shown. Factor VIII inhibitor titers (F) were evaluated with a chromogenic assay as described in “Materials and methods.” Two independent experiments; ^*P < .05, ^**P < .01, ^***P < .001 versus mock control.

Figure 3.

Immunized E16 recipients of B-cell blasts transduced with C2-IgG/MSCV and A2-IgG/MSCV are hyporesponsive to fVIII and its C2 or A2 domain. Recipient E16 mice were primed by 4 weekly injections of human fVIII. LPS blasts were transduced with the indicated retroviral vectors, and then used as a source of B cells for tolerance induction on day 42. Mice were then boosted with 2 μg of human fVIII on day 49 and the animals euthanized 7 days later. T-cell proliferation was measured against C2 (A), A2 (B), and fVIII (C) as described in Figure 2. (A-C) ♦ indicates control SAG-Ig; □, C2-IgG plus A2-IgG. Total serum IgG anti-C2 (D) and anti-fVIII (E) antibody titers were determined by ELISA on days 28 and 56. One of 2 similar experiments is shown. Data represent the mean plus or minus SEM. ^*P < .05 or ^**P < .01 versus mock SAG-Ig control. Factor VIIII inhibitor titers are shown in panel F. ^*P < .05, ^**P < .01, or ^***P < .001 versus mock control or before gene therapy on day 28.

Figure 4.

Persistence of tolerance induced by B-cell blasts transduced with C2-IgG-MSCV and A2-IgG-MSCV in immunized E16 recipients. Protocol was as in Figure 3, except that mice received an additional booster with fVIII on day 102 and were euthanized on day 109. Recipient E16 mice were primed by 4 weekly injections of human fVIII. Total serum IgG anti-C2 (A) and anti-fVIII (B) antibody titers were determined by ELISA on days 28, 56, 102, and 109. Factor VIIII inhibitor titers are shown in panel C. The data represent the mean plus or minus SEM; ^*P < .05, ^**P < .01, P < .001 versus mock SAG-Ig control.

Figure 5.

Treatment with monoclonal anti-CD25 prevents the tolerogenic effects of B-cell gene therapy. E16 mice were treated with PC61 anti-CD25 antibody or normal rat IgG and injected with B cells transduced with retroviral vectors expressing C2-IgG plus A2-IgG or control SAG-IgG. The efficacy of PC61 depletion was validated by flow cytometric analysis of peripheral blood leukocytes obtained from treated mice on day 9. The percentage of CD25⁺ cells is indicated in the top right quadrant. (A). Mice were challenged repeatedly with human fVIII as in Figure 2. Animals were euthanized at 5 days after final fVIII injection and measures of cellular and humoral immunity were taken as described in “Materials and methods.” T-cell responses are depicted in panels B and C (B: ♦, C2-IgG + A2-IgG; ○, SAG-Ig; C: ▵, C2-IgG + A2-IgG; ▪, SAG), and ELISA titers are shown in panels D and E. Inhibitory antibody titers are shown in panel F. ^*P < .05 or ^**P < .01 versus mock control. Error bars indicate mean ± SEM for individual mice of the cpm incorporated minus background.