Methods and compositions involving fibrillizing polypeptides for nanofibers

Abstract

Embodiments of the invention are directed to fibrillar adjuvants. For example, epitopes assembled by a synthetic peptide domain into nanofibers comprising a β-fibrillization peptide may elicit high antibody titers in the absence of any adjuvant. In certain embodiments, multiple different antigens may be integrated into polypeptide nanofibers, providing biomaterials with modular and precise composition of bioactive proteins.

1 - 60 . (canceled) 
     
     
         61 . A method of inducing an immune response comprising administering an effective amount of a composition, wherein the composition comprises a β-sheet nanofiber complex composition comprising a β-sheet nanofiber structure comprising:
 a) a plurality of non-β-sheet peptide tags that undergo a transition from a non-β-sheet structure to a β-sheet structure in the presence of β-sheet peptides, wherein the non-β-sheet peptide tags comprise an amino acid sequence having at least 90% identity with the sequence of LVVLHSELHKLKSEL (SEQ ID NO:1), LVVLHSHLEKLKSEL (SEQ ID NO:2), LKVELEKLKSELVVLHSELHKLKSEL (SEQ ID NO:3), LKVELEKLKSELVVLHSHLEKLKSEL (SEQ ID NO:4), or LKVELKELKKELVVLKSELKELKKEL (SEQ ID NO:5), wherein at least one of the non-β-sheet peptide tags is attached to an antigen; and 
 b) a plurality of β-sheet peptides. 
 
     
     
         62 - 63 . (canceled) 
     
     
         64 . A method of culturing a cell, comprising incubating the cell in a cell culture medium comprising a β-sheet nanofiber complex composition comprising a β-sheet nanofiber structure comprising:
 a) a plurality of non-β-sheet peptide tags that undergo a transition from a non-β-sheet structure to a β-sheet structure in the presence of β-sheet peptides, wherein the non-β-sheet peptide tags comprise an amino acid sequence having at least 90% identity with the sequence of LVVLHSELHKLKSEL (SEQ ID NO:1), LVVLHSHLEKLKSEL (SEQ ID NO:2), LKVELEKLKSELVVLHSELHKLKSEL (SEQ ID NO:3), LKVELEKLKSELVVLHSHLEKLKSEL (SEQ ID NO:4), or LKVELKELKKELVVLKSELKELKKEL (SEQ ID NO:5), wherein at least one of the non-β-sheet peptide tags is attached to an antigen; and 
 b) a plurality of β-sheet peptides. 
 
     
     
         65 . The method of  claim 61 , wherein the non-β-sheet peptide tag comprises an amino acid sequence having at least 95% identity with the sequence of LVVLHSELHKLKSEL (SEQ ID NO:1), LVVLHSHLEKLKSEL (SEQ ID NO:2), LKVELEKLKSELVVLHSELHKLKSEL (SEQ ID NO:3), LKVELEKLKSELVVLHSHLEKLKSEL (SEQ ID NO:4), or LKVELKELKKELVVLKSELKELKKEL (SEQ ID NO:5). 
     
     
         66 . The method of  claim 61 , wherein the non-β-sheet peptide tag comprises the amino acid sequence of LVVLHSELHKLKSEL (SEQ ID NO:1), LVVLHSHLEKLKSEL (SEQ ID NO:2), LKVELEKLKSELVVLHSELHKLKSEL (SEQ ID NO:3), LKVELEKLKSELVVLHSHLEKLKSEL (SEQ ID NO:4), or LKVELKELKKELVVLKSELKELKKEL (SEQ ID NO:5). 
     
     
         67 . The method of  claim 61 , wherein the non-β-sheet peptide tag consists of the amino acid sequence of LVVLHSELHKLKSEL (SEQ ID NO:1), LVVLHSHLEKLKSEL (SEQ ID NO:2), LKVELEKLKSELVVLHSELHKLKSEL (SEQ ID NO:3), LKVELEKLKSELVVLHSHLEKLKSEL (SEQ ID NO:4), or LKVELKELKKELVVLKSELKELKKEL (SEQ ID NO:5). 
     
     
         68 . The method of  claim 61 , wherein the non-β-sheet peptide tag is 14 to 56 amino acids in length. 
     
     
         69 . The method of  claim 61 , wherein the non-β-sheet peptide tags and β-sheet peptides have a molar ratio of about 1:10 to about 1:10,000. 
     
     
         70 . The method of  claim 61 , wherein one or more of the β-sheet peptides are 2 to 40 amino acids in length. 
     
     
         71 . The method of  claim 61 , wherein one or more of the β-sheet peptides comprise an amino acid sequence having at least 90% identity with the sequence of QQKFQFQFEQQ (SEQ ID NO. 6); QQKFQFQFHQQ (SEQ ID NO. 7); FKFEFKFE (SEQ ID NO. 8); KFQFQFE (SEQ ID NO. 9); QQRFQFQFEQQ (SEQ ID NO. 10); QQRFQWQFEQQ (SEQ ID NO. 11); FEFEFKFKFEFEFKFK (SEQ ID NO. 12); QQRFEWEFEQQ (SEQ ID NO. 13); QQXFXWXFQQQ (Where X denotes ornithine) (SEQ ID NO. 14); FKFEFKFEFKFE (SEQ ID NO. 15); FKFQFKFQFKFQ (SEQ ID NO. 16); AEAKAEAKAEAKAEAK (SEQ ID NO. 17); AEAEAKAKAEAEAKAK (SEQ ID NO. 18); AEAEAEAEAKAKAKAK (SEQ ID NO. 19); RADARADARADARADA (SEQ ID NO. 20); RARADADARARADADA (SEQ ID NO. 21); SGRGYBLGGQGAGAAAAAGGAGQGGYGGLGSQG (SEQ ID NO. 22); EWEXEXEXEX (Where X=V, A, S, or P) (SEQ ID NO. 23); WKXKXKXKXK (Where X=V, A, S, or P) (SEQ ID NO. 24); KWKVKVKVKVKVKVK (Where X=V, A, S, or P) (SEQ ID NO. 25); LLLLKKKKKKKKLLLL (SEQ ID NO. 26); VKVKVKVKVDPPTKVKVKVKV (SEQ ID NO. 27); VKVKVKVKVDPPTKVKTKVKV (SEQ ID NO. 28); KVKVKVKVKDPPSVKVKVKVK (SEQ ID NO. 29); VKVKVKVKVDPPSKVKVKVKV (SEQ ID NO. 30); VKVKVKTKVDPPTKVKTKVKV (SEQ ID NO. 31); Fmoc-FF; Fmoc-GG; Fmoc-FG; KKSLSLSLSLSLSLKK (SEQ ID NO. 32); or YTIAALLSPY (SEQ ID NO. 33). 
     
     
         72 . The method of  claim 61 , wherein one or more of the β-sheet peptides comprise an amino acid sequence having at least 95% identity with the sequence of QQKFQFQFEQQ (SEQ ID NO. 6); QQKFQFQFHQQ (SEQ ID NO. 7); FKFEFKFE (SEQ ID NO. 8); KFQFQFE (SEQ ID NO. 9); QQRFQFQFEQQ (SEQ ID NO. 10); QQRFQWQFEQQ (SEQ ID NO. 11); FEFEFKFKFEFEFKFK (SEQ ID NO. 12); QQRFEWEFEQQ (SEQ ID NO. 13); QQXFXWXFQQQ (Where X denotes ornithine) (SEQ ID NO. 14); FKFEFKFEFKFE (SEQ ID NO. 15); FKFQFKFQFKFQ (SEQ ID NO. 16); AEAKAEAKAEAKAEAK (SEQ ID NO. 17); AEAEAKAKAEAEAKAK (SEQ ID NO. 18); AEAEAEAEAKAKAKAK (SEQ ID NO. 19); RADARADARADARADA (SEQ ID NO. 20); RARADADARARADADA (SEQ ID NO. 21); SGRGYBLGGQGAGAAAAAGGAGQGGYGGLGSQG (SEQ ID NO. 22); EWEXEXEXEX (Where X=V, A, S, or P) (SEQ ID NO. 23); WKXKXKXKXK (Where X=V, A, S, or P) (SEQ ID NO. 24); KWKVKVKVKVKVKVK (Where X=V, A, S, or P) (SEQ ID NO. 25); LLLLKKKKKKKKLLLL (SEQ ID NO. 26); VKVKVKVKVDPPTKVKVKVKV (SEQ ID NO. 27); VKVKVKVKVDPPTKVKTKVKV (SEQ ID NO. 28); KVKVKVKVKDPPSVKVKVKVK (SEQ ID NO. 29); VKVKVKVKVDPPSKVKVKVKV (SEQ ID NO. 30); VKVKVKTKVDPPTKVKTKVKV (SEQ ID NO. 31); Fmoc-FF; Fmoc-GG; Fmoc-FG; KKSLSLSLSLSLSLKK (SEQ ID NO. 32); or YTIAALLSPY (SEQ ID NO. 33). 
     
     
         73 . The method of  claim 61 , wherein one or more of the β-sheet peptides comprise the amino acid sequence of QQKFQFQFEQQ (SEQ ID NO. 6); QQKFQFQFHQQ (SEQ ID NO. 7); FKFEFKFE (SEQ ID NO. 8); KFQFQFE (SEQ ID NO. 9); QQRFQFQFEQQ (SEQ ID NO. 10); QQRFQWQFEQQ (SEQ ID NO. 11); FEFEFKFKFEFEFKFK (SEQ ID NO. 12); QQRFEWEFEQQ (SEQ ID NO. 13); QQXFXWXFQQQ (Where X denotes ornithine) (SEQ ID NO. 14); FKFEFKFEFKFE (SEQ ID NO. 15); FKFQFKFQFKFQ (SEQ ID NO. 16); AEAKAEAKAEAKAEAK (SEQ ID NO. 17); AEAEAKAKAEAEAKAK (SEQ ID NO. 18); AEAEAEAEAKAKAKAK (SEQ ID NO. 19); RADARADARADARADA (SEQ ID NO. 20); RARADADARARADADA (SEQ ID NO. 21); SGRGYBLGGQGAGAAAAAGGAGQGGYGGLGSQG (SEQ ID NO. 22); EWEXEXEXEX (Where X=V, A, S, or P) (SEQ ID NO. 23); WKXKXKXKXK (Where X=V, A, S, or P) (SEQ ID NO. 24); KWKVKVKVKVKVKVK (Where X=V, A, S, or P) (SEQ ID NO. 25); LLLLKKKKKKKKLLLL (SEQ ID NO. 26); VKVKVKVKVDPPTKVKVKVKV (SEQ ID NO. 27); VKVKVKVKVDPPTKVKTKVKV (SEQ ID NO. 28); KVKVKVKVKDPPSVKVKVKVK (SEQ ID NO. 29); VKVKVKVKVDPPSKVKVKVKV (SEQ ID NO. 30); VKVKVKTKVDPPTKVKTKVKV (SEQ ID NO. 31); Fmoc-FF; Fmoc-GG; Fmoc-FG; KKSLSLSLSLSLSLKK (SEQ ID NO. 32); or YTIAALLSPY (SEQ ID NO. 33). 
     
     
         74 . The method of  claim 61 , wherein one or more of the β-sheet peptides consist of the amino acid sequence of QQKFQFQFEQQ (SEQ ID NO. 6); QQKFQFQFHQQ (SEQ ID NO. 7); FKFEFKFE (SEQ ID NO. 8); KFQFQFE (SEQ ID NO. 9); QQRFQFQFEQQ (SEQ ID NO. 10); QQRFQWQFEQQ (SEQ ID NO. 11); FEFEFKFKFEFEFKFK (SEQ ID NO. 12); QQRFEWEFEQQ (SEQ ID NO. 13); QQXFXWXFQQQ (Where X denotes ornithine) (SEQ ID NO. 14); FKFEFKFEFKFE (SEQ ID NO. 15); FKFQFKFQFKFQ (SEQ ID NO. 16); AEAKAEAKAEAKAEAK (SEQ ID NO. 17); AEAEAKAKAEAEAKAK (SEQ ID NO. 18); AEAEAEAEAKAKAKAK (SEQ ID NO. 19); RADARADARADARADA (SEQ ID NO. 20); RARADADARARADADA (SEQ ID NO. 21); SGRGYBLGGQGAGAAAAAGGAGQGGYGGLGSQG (SEQ ID NO. 22); EWEXEXEXEX (Where X=V, A, S, or P) (SEQ ID NO. 23); WKXKXKXKXK (Where X=V, A, S, or P) (SEQ ID NO. 24); KWKVKVKVKVKVKVK (Where X=V, A, S, or P) (SEQ ID NO. 25); LLLLKKKKKKKKLLLL (SEQ ID NO. 26); VKVKVKVKVDPPTKVKVKVKV (SEQ ID NO. 27); VKVKVKVKVDPPTKVKTKVKV (SEQ ID NO. 28); KVKVKVKVKDPPSVKVKVKVK (SEQ ID NO. 29); VKVKVKVKVDPPSKVKVKVKV (SEQ ID NO. 30); VKVKVKTKVDPPTKVKTKVKV (SEQ ID NO. 31); Fmoc-FF; Fmoc-GG; Fmoc-FG; KKSLSLSLSLSLSLKK (SEQ ID NO. 32); or YTIAALLSPY (SEQ ID NO. 33). 
     
     
         75 . The method of  claim 61 , wherein one or more of the β-sheet peptides comprise a plurality of self-assembling peptides. 
     
     
         76 . The method of  claim 61 , wherein the antigen is an enzyme, fluorescent protein, cell binding domain, cell adhesion domain, extracellular matrix domain, reporter protein, cytokine, antigen, signaling domain, immunomodulating protein, cross-linking protein, hormone, hapten, or a combination thereof. 
     
     
         77 . The method of  claim 61 , wherein at least one of the non-β-sheet peptide tags attached to an antigen is further defined as a fusion protein. 
     
     
         78 . The method of  claim 61 , wherein one or more of the non-β-sheet peptide tag are attached to the amino-terminus of an antigen peptide. 
     
     
         79 . The method of  claim 61 , wherein the β-sheet nanofiber complex composition has a length of at least about 0.2 to about 100 μm. 
     
     
         80 . The method of  claim 61 , wherein the β-sheet nanofiber complex composition has a molecular weight of at least about 10,000 da to about 7×10 8  da. 
     
     
         81 . A method of preparing a nanofiber complex composition, comprising mixing the following:
 a) a plurality of non-β-sheet peptide tags, wherein the non-β-sheet peptide tags comprise an amino acid sequence having at least 90% identity with the sequence of LVVLHSELHKLKSEL (SEQ ID NO:1), LVVLHSHLEKLKSEL (SEQ ID NO:2), LKVELEKLKSELVVLHSELHKLKSEL (SEQ ID NO:3), LKVELEKLKSELVVLHSHLEKLKSEL (SEQ ID NO:4), or LKVELKELKKELVVLKSELKELKKEL (SEQ ID NO:5), wherein a non-β-sheet peptide tag is attached to an antigen, and   b) a plurality of β-sheet peptides, under conditions that allow the non-β-sheet peptide tags to transition from a non-β-sheet structure to a β-sheet structure,   
       thereby preparing a nanofiber complex composition that forms a β-sheet structure comprising the non-β-sheet peptide tags and β-sheet peptides. 
     
     
         82 . The method of  claim 81 , further comprising shaking the mixture, thereby forming a microgel.