Compositions and methods for suppression of inhibitor formation against coagulation factors in hemophilia patients

Abstract

Protein replacement therapy for patients with haemophilia or other inherited protein deficiencies is often complicated by pathogenic antibody responses, including antibodies that neutralize the therapeutic protein or that predispose to potentially life-threatening anaphylactic reactions by formation of IgE. Using murine and canine haemophilia as a model, we have developed a prophylactic protocol. Against such responses that is non-invasive and does not include immune suppression mor genetic manipulation of the patient's cells. Oral delivery of a coagulation factor expressed in chloroplasts, bioencapsulated in plant cells, effectively blocked formation of inhibitory antibodies in protein replacement therapy. Inhibitor titers were mostly undetectable and up to 100-fold lower in treated subjects when compared to controls. Moreover, this treatment eliminated fatal anaphylactic reactions that occurred after four to six exposures to intravenous coagulation factor protein. Finally, the method can effectively be used to reverse or reduce undesirable pre-existing inhibitor titers.

1 - 19 . (canceled) 
     
     
         20 . A composition comprising lyophilized plant material, said material comprising a coagulation protein, or immunogenic fragments thereof, produced in chloroplasts within said plant, said coagulation protein or immunogenic fragment retaining immunogenicity in lyophilized form, which upon oral administration to a mammal in need thereof is effective to produce oral tolerance to said protein. 
     
     
         21 . The composition of  claim 20  wherein said coagulation factor is F.II, F.III, F.IV, F.V, F.VI, F.VII, F.VIII, FIX, F.X, F.XI, F.XII, or F XIII, or a polypeptides having at least 90 percent identity therewith. 
     
     
         22 . The composition of  claim 21  comprising a therapeutic fusion protein comprising an immunogenic fragment of a coagulation factor operably linked to non toxic cholera toxin B subunit (CTB), said fusion protein inducing tolerance to said fragment in said mammal upon oral administration. 
     
     
         23 . The composition of  claim 21 , wherein said coagulation factor fragment is obtained from FIX. 
     
     
         24 . The composition of  claim 21 , wherein said coagulation factor is FVIII and at least one immunogenic fragment is a domain of said FVIII selected from the group consisting of A1, A2, A3, B, Cl, C2 or heavy chain (HC) fragments. 
     
     
         25 . The composition of  claim 21 , wherein said therapeutic protein is at least one immunological fragment of FVIII consisting of a C2 domain and/or a HC domain, each fused to cholera non toxic B subunit (CTB); and said orally administered fragments inducing tolerance to said coagulation protein by suppressing inhibitory antibody formation. 
     
     
         26 . The composition of  claim 25 , wherein said compositions is effective to induce expression of TGF-β producing CD4 + CD25 − LAP +  regulatory T cells in spleen, MLN, and Peyer's patches. 
     
     
         27 . The composition of  claim 20 , wherein said plant is selected from the group consisting of lettuce, carrots, cauliflower, cabbage, low-nicotine tobacco, spinach, kale, and cilantro. 
     
     
         28 . The composition of  claim 22 , wherein said fusion protein contains a hinge peptide and furin cleavage site between said CTB and said FVIII or at least one immunological fragment thereof. 
     
     
         29 . The composition of  claim 25 , wherein said a C2-CTB and an HC-CTB fusion protein are administered together. 
     
     
         30 . The composition of  claim 21 , wherein said coagulation factor is FVIII which is effective to reduce inhibitor formation against FVIII in hemophilia A subjects. 
     
     
         31 . A method for the treatment of Hemophilia A in a subject in need thereof comprising administration of an effective amount of the composition of  claim 22  to a subject in need thereof, said composition being effective to suppress formation of inhibitors of FVIII in said subject and induce expression of TGF-β producing CD4 + CD25 − LAP +  regulatory T cells in spleen, MLN, and Peyer's patches. 
     
     
         32 . A method for the treatment of Hemophilia A in a subject in need thereof comprising administration of an effective amount of the composition of  claim 25  to a subject in need thereof, said composition being effective to suppress formation of inhibitors of FVIII in said subject and induce expression of TGF-r3 producing CD4 + CD25 − LAP +  regulatory T cells in spleen, MLN, and Peyer's patches. 
     
     
         33 . A method for the treatment of Hemophilia B in a subject in need thereof comprising administration of an effective amount of the composition of  claim 23  to a subject in need thereof, said composition being effective to suppress formation of inhibitors of FIX in said subject and induce expression of TGF-r3 producing CD4 + CD25 − LAP +  regulatory T cells in spleen, MLN, and Peyer's patches. 
     
     
         34 . The method of  claim 31 , wherein said subject has pre-existing antibody inhibitors to said F VIII. 
     
     
         35 . The method of  claim 33 , wherein said subject has pre-existing antibody inhibitors to said FIX.