Prophylactic and therapeutic induction of antigen-specific immune tolerance has potential value in protein replacement therapies, cell transplant therapies, and prevention and treatment of autoimmunity. For example, in protein replacement therapy, a patient usually expresses a mutant form of the protein of interest (for example, factor VIII in hemophilia A), or expresses no protein at all, and as such the patient has not developed immunological tolerance to the replacement form and frequently develops immunity versus the replaced protein (approx. 30% incidence in factor VIII). As another example, in type 1 diabetes, the patient developed humoral and more importantly cellular autoimmunity versus pancreatic islet beta cell antigens. Thus, there exists a pressing need for antigen-specific tolerogenic vaccine technology to deliver such antigens (e.g., factor VIII prophylactically to induce pre-treatment tolerance; islet antigens therapeutically to re-introduce tolerance after early presentation of the disease).

Antigens derived from apoptotic cells are known to drive tolerance via deletion or anergy of reactive T cells. Reasoning that a large number of erythrocytes become apoptotic (eryptotic) and are cleared each day, we engineered a strategy to bind antigen to erythrocytes in situ with high affinity and specificity after intravenous injection, conjugating a glycophorin-A-binding peptide that we discovered by phage display to the antigen. Using a transgenic T cell model in the mouse sensitive to the model antigen ovalbumin, we demonstrate that erythrocyte-binding antigen bioconjugate (ERY1-OVA) induces extensive CD8⁺ T cell cross-priming, leading to an apoptotic and exhausted fate.  Furthermore, mice tolerized with ERY1-OVA induced far fewer OVA-specific cytotoxic IFN-γ⁺ CD8⁺ T cell responses compared with OVA-tolerized mice following antigen challenge with a potent adjuvant. Tolerization with ERY1-OVA also promoted growth of OVA-expressing transplanted cells in mice immunized against OVA, indicating functional inhibition of CTL generation. 

Erythrocyte binding also attenuated humoral immune reactions. The E. coli protein asparaginase (ASNase) is used to treat acute lymphoblastic leukemia, yet immune recognition of the microbial protein limits its efficacy and safety. ERY1-ASNase induced at least 4 orders of magnitude lower humoral immune response after multiple dosing in mice than the wild-type ASNase. Moreover, pre-tolerization with ERY1-ASNase ameliorated immunity to subsequent treatment, resulting in 6000-fold lower antibody titers than in the absence of pre-tolerization.

Thus, we report a novel biomolecular approach to hijack the body’s mechanisms in maintenance of tolerance to apoptotic erythrocytes to induce deletional tolerance to an exogenous erythrocyte-binding antigen to create a technology for antigen-specific tolerogenic vaccination. Prophylactic and therapeutic induction of antigen-specific immune tolerance has potential value in protein replacement therapies, cell transplant therapies, and prevention and treatment of autoimmunity. For example, in protein replacement therapy, a patient usually expresses a mutant form of the protein of interest (for example, factor VIII in hemophilia A), or expresses no protein at all, and as such the patient has not developed immunological tolerance to the replacement form and frequently develops immunity versus the replaced protein (approx. 30% incidence in factor VIII). As another example, in type 1 diabetes, the patient developed humoral and more importantly cellular autoimmunity versus pancreatic islet beta cell antigens. Thus, there exists a pressing need for antigen-specific tolerogenic vaccine technology to deliver such antigens (e.g., factor VIII prophylactically to induce pre-treatment tolerance; islet antigens therapeutically to re-introduce tolerance after early presentation of the disease).

Antigens derived from apoptotic cells are known to drive tolerance via deletion or anergy of reactive T cells. Reasoning that a large number of erythrocytes become apoptotic (eryptotic) and are cleared each day, we engineered a strategy to bind antigen to erythrocytes in situ with high affinity and specificity after intravenous injection, conjugating a glycophorin-A-binding peptide that we discovered by phage display to the antigen. Using a transgenic T cell model in the mouse sensitive to the model antigen ovalbumin, we demonstrate that erythrocyte-binding antigen bioconjugate (ERY1-OVA) induces extensive CD8⁺ T cell cross-priming, leading to an apoptotic and exhausted fate.  Furthermore, mice tolerized with ERY1-OVA induced far fewer OVA-specific cytotoxic IFN-γ⁺ CD8⁺ T cell responses compared with OVA-tolerized mice following antigen challenge with a potent adjuvant. Tolerization with ERY1-OVA also promoted growth of OVA-expressing transplanted cells in mice immunized against OVA, indicating functional inhibition of CTL generation. 

Erythrocyte binding also attenuated humoral immune reactions. The E. coli protein asparaginase (ASNase) is used to treat acute lymphoblastic leukemia, yet immune recognition of the microbial protein limits its efficacy and safety. ERY1-ASNase induced at least 4 orders of magnitude lower humoral immune response after multiple dosing in mice than the wild-type ASNase. Moreover, pre-tolerization with ERY1-ASNase ameliorated immunity to subsequent treatment, resulting in 6000-fold lower antibody titers than in the absence of pre-tolerization.

Thus, we report a novel biomolecular approach to hijack the body’s mechanisms in maintenance of tolerance to apoptotic erythrocytes to induce deletional tolerance to an exogenous erythrocyte-binding antigen to create a technology for antigen-specific tolerogenic vaccination.