sirolimus and Hemophilia-A

Development of antibodies (inhibitors) against coagulation factor VIII (FVIII) is a major complication of intravenous replacement therapy in haemophilia A (HA). Current immune tolerance induction (ITI) regimens are not universally effective. Rituximab, a B cell-depleting antibody against CD20, has shown mixed results for inhibitor reversal in patients. This study aims to develop a combinatorial therapy for inhibitor reversal in HA, using anti-murine CD20 (anti-mCD20) antibody and rapamycin, which targets both B and T cell responses. Additionally, it extensively characterises the role of the IgG backbone in B cell depletion by anti-CD20 antibodies. For this, inhibitors were generated in BALB/c-HA mice by weekly IV injection of FVIII. Subsequently, anti-mCD20 (18B12) with IgG2a or IgG1 backbone was injected IV in two doses three weeks apart and B cell depletion and recovery was characterised. Rapamycin was administered orally 3x/week (for 1 month) while continuing FVIII injections. Altering the IgG backbone of anti-mCD20 from IgG2a to IgG1 reduced overall depletion of B cells (including memory B cells), and marginal zone, B-10, and B-1b cells were specifically unaffected. While neither antibody was effective alone, in combination with rapamycin, anti-mCD20 IgG2a but not IgG1 was able to reverse inhibitors in HA mice. This regimen was particularly effective for starting titres of ~10 BU. Although IgG1 anti-mCD20 spared potentially tolerogenic B cell subsets, IgG2a directed sustained hyporesponsiveness when administered in conjunction with rapamycin. This regimen represents a promising treatment for inhibitor reversal in HA, as both of these compounds have been extensively used in human patients.

Topics: Administration, Oral; Adoptive Transfer; Animals; Antibodies; Antibodies, Monoclonal; Antigens, CD20; B-Lymphocytes; Coagulants; Disease Models, Animal; Drug Administration Schedule; Drug Therapy, Combination; Factor VIII; Hemophilia A; Immune Tolerance; Immunoglobulin G; Immunosuppressive Agents; Injections, Intravenous; Male; Mice, Inbred BALB C; Mice, Knockout; Sirolimus; T-Lymphocytes, Regulatory; Time Factors

The immune response of hemophilia A patients to administered FVIII is a major complication that obviates this very therapy. We have recently described the use of synthetic, biodegradable nanoparticles carrying rapamycin and FVIII peptide antigens, to induce antigen-specific tolerance. Herein we test the tolerogenicity of nanoparticles that contains full length FVIII protein in hemophilia A mice, focusing on anti-FVIII humoral immune response. As expected, recipients of tolerogenic nanoparticles remained unresponsive to FVIII despite multiple challenges for up to 6 months. Furthermore, therapeutic treatments in FVIII-immunized mice with pre-existing anti-FVIII antibodies resulted in diminished antibody titers, albeit efficacy required longer therapy with the tolerogenic nanoparticles. Interestingly, durable FVIII-specific tolerance was also achieved in animals co-administered with FVIII admixed with nanoparticles encapsulating rapamycin alone. These results suggest that nanoparticles carrying rapamycin and FVIII can be employed to induce specific tolerance to prevent and even reverse inhibitor formation.

Topics: Animals; Antibodies, Neutralizing; Disease Models, Animal; Factor VIII; Hemophilia A; Immune Tolerance; Immunosuppressive Agents; Mice; Nanoparticles; Sirolimus; Vaccines, Synthetic

Current treatments to control pathological or unwanted immune responses often use broadly immunosuppressive drugs. New approaches to induce antigen-specific immunological tolerance that control both cellular and humoral immune responses are desirable. Here we describe the use of synthetic, biodegradable nanoparticles carrying either protein or peptide antigens and a tolerogenic immunomodulator, rapamycin, to induce durable and antigen-specific immune tolerance, even in the presence of potent Toll-like receptor agonists. Treatment with tolerogenic nanoparticles results in the inhibition of CD4+ and CD8+ T-cell activation, an increase in regulatory cells, durable B-cell tolerance resistant to multiple immunogenic challenges, and the inhibition of antigen-specific hypersensitivity reactions, relapsing experimental autoimmune encephalomyelitis, and antibody responses against coagulation factor VIII in hemophilia A mice, even in animals previously sensitized to antigen. Only encapsulated rapamycin, not the free form, could induce immunological tolerance. Tolerogenic nanoparticle therapy represents a potential novel approach for the treatment of allergies, autoimmune diseases, and prevention of antidrug antibodies against biologic therapies.

Topics: Animals; Antigens; CD4-Positive T-Lymphocytes; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Factor VIII; Female; Hemocyanins; Hemophilia A; Humans; Hypersensitivity, Delayed; Immune Tolerance; Immunity, Humoral; Immunosuppression Therapy; Immunosuppressive Agents; Lactic Acid; Mice; Mice, Inbred BALB C; Nanocapsules; Nanoparticles; Oligodeoxyribonucleotides; Ovalbumin; Peptide Fragments; Peptides; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Proteins; Recombinant Proteins; Sirolimus

Formation of inhibitory antibodies is a frequent and serious complication of factor (F) VIII replacement therapy for the X-linked bleeding disorder hemophilia A. Similarly, hemophilia A mice develop high-titer inhibitors to recombinant human FVIII after a few intravenous injections.. Using the murine model, the study sought to develop a short regimen capable of inducing tolerance to FVIII.. A 1-month immunomodulatory protocol, consisting of FVIII administration combined with oral delivery of rapamycin, was developed.. The protocol effectively prevented formation of inhibitors to FVIII upon subsequent intravenous treatment (weekly for 3.5 months). Control mice formed high-titer inhibitors and had CD4(+) T effector cell responses characterized by expression of IL-2, IL-4 and IL-6. Tolerized mice instead had a CD4(+)CD25(+)FoxP3(+) T cell response to FVIII that suppressed antibody formation upon adoptive transfer, indicating a shift from Th2 to Treg if FVIII antigen was introduced to T cells during inhibition with rapamycin. CD4(+) T cells from tolerized mice also expressed TGF-β1 and CTLA4, but not IL-10. The presence of FVIII antigen during the time of rapamycin administration was required for specific tolerance induction.. The study shows that a prophylactic immune tolerance protocol for FVIII can be developed using rapamycin, a drug that is already widely in clinical application. Immune suppression with rapamycin was mild and highly transient, as the mice regained immune competence within a few weeks.

Topics: Adoptive Transfer; Animals; Antibodies; CD4-Positive T-Lymphocytes; Cells, Cultured; Coagulants; CTLA-4 Antigen; Disease Models, Animal; Drug Administration Schedule; Factor VIII; Forkhead Transcription Factors; Hemophilia A; Humans; Immune Tolerance; Immunosuppressive Agents; Interleukin-2; Interleukin-2 Receptor alpha Subunit; Interleukin-4; Interleukin-6; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Recombinant Proteins; Sirolimus; Time Factors; Transforming Growth Factor beta1

Topics: Animals; Antibodies; CD4-Positive T-Lymphocytes; Coagulants; Factor VIII; Hemophilia A; Humans; Immune Tolerance; Immunosuppressive Agents; Male; Sirolimus

Gene and protein replacement therapies for inherited protein deficiencies such as hemophilia or lysosomal storage disorders are limited by deleterious immune responses directed against their respective therapeutic proteins. Therefore, the development of protocols preventing such responses is key to providing successful long-term therapy.. We sought to develop a protocol, utilizing a drug/peptide cocktail, that would effectively shift the antigen-specific CD4+ T-cell population, tipping the balance from effector T cells (Teffs) towards regulatory T cells (Tregs).. Treg-deficient (DO11.10-tg Rag2(-/-)) BALB/c mice were used to screen for an optimal protocol addressing the aforementioned goal and to study the mechanisms underlying in vivo changes in T-cell populations. Muscle-directed gene transfer to hemophilia B mice was also performed in order to test the optimal protocol in a therapeutically relevant setting.. Specific antigen administration (4-week repeated dosing) combined with rapamycin and interleukin-10 led to substantial reductions in Teffs, via activation-induced cell death, and induced CD4+CD25+FoxP3+ Tregs to a large extent in multiple organs. The proportion of apoptotic T cells also increased over time, whereas Teffs and Tregs were differentially affected. When applied to a model of protein deficiency (gene therapy for hemophilia B), the protocol successfully prevented inhibitor formation, whereas non-specific immunosuppression was only marginally effective.. It is feasible to provide a short-term, prophylactic protocol allowing for the induction of immune tolerance. This protocol may provide a marked advance in efforts seeking to improve clinical outcomes in disorders involving therapeutic protein replacement.

Topics: Animals; Antigens; CD4-Positive T-Lymphocytes; Factor IX; Forkhead Transcription Factors; Genetic Therapy; Hemophilia A; Humans; Immune Tolerance; Interleukin-10; Interleukin-2 Receptor alpha Subunit; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Sirolimus; T-Lymphocytes, Regulatory