Compositions and methods for detecting and regulating fibronectin-integrin interaction and signaling

Abstract

Provided are antibodies that include amino acid sequences of SEQ ID NOs: 2, 4, and 6-12, or amino acid sequences that are about 95% identical thereto, and fragments thereof Also provided are scFv peptides that include a VH segment having a first amino acid sequence of amino acids 4-113 of any one of SEQ ID NOs: 2 and 8-12, a VL segment having a second amino acid sequence having amino acids 113-237 of SEQ ID NOs. 2 and 8-12, or both; nucleic acids encoding the same; methods for using the same to detect and/or target conformational states of FN in samples; methods for treating diseases and/or disorders and/or for meliorating at least one symptom of consequence of a disease or disorder associated with abnormal expression of a force-induced conformational state of FN in subjects; and methods for screening for compounds having selective binding activities for conformational states of FN.

1 . An isolated and purified antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8-12, a fragment thereof, or an antibody having an amino acid sequence that is approximately 95% identical to the sequence of any one of SEQ ID NOs: 2, 4, 6, and 8-12, or a fragment thereof, wherein the isolated and purified antibody or the fragment thereof comprises a heavy chain CDR1 comprising SYAMS (SEQ ID NO: 24), a heavy chain CDR2 comprising DIYDGGGTNYADSVKG (SEQ ID NO: 25), a heavy chain CDR3 comprising TADNFY (SEQ ID NO: 26) or TADNFD (SEQ ID NO: 27), a light chain CDR1 comprising RASQSISSYLN (SEQ ID NO: 28), a light chain CDR2 comprising AASTLQS (SEQ ID NO: 29), and a light chain CDR3 comprising QQANSAPTT (SEQ ID NO: 30), and further wherein the isolated and purified antibody or the fragment thereof binds to a strained conformation of fibronectin (Fn). 
     
     
         2 . The isolated and purified antibody of  claim 1 , wherein the amino acid sequence comprises at least one modification selected from the group consisting of an amino acid deletion, an amino acid addition, an amino acid substitution, and combinations thereof. 
     
     
         3 . The isolated and purified antibody of  claim 1 , wherein the antibody or fragment thereof comprises an scFv fragment. 
     
     
         4 . The isolated and purified antibody of  claim 3 , wherein the scFv fragment is mammalian. 
     
     
         5 . The isolated and purified antibody of  claim 3 , wherein the scFv fragment is humanized. 
     
     
         6 . An isolated and purified nucleic acid sequence encoding the antibody of  claim 1 , or a fragment thereof. 
     
     
         7 . A single chain variable fragment (scFv) peptide, comprising a V H  segment comprising a first amino acid sequence selected from the group consisting of amino acids 4-113 of any one of SEQ ID NOs: 2 and 8-12, a V L  segment comprising a second amino acid sequence selected from the group consisting of amino acids 113-237 of SEQ ID NOs. 2 and 8-12, or a combination thereof. 
     
     
         8 . The scFv peptide of  claim 7 , wherein the V H  segment and V L  segment are coupled together with a linker peptide. 
     
     
         9 . The scFv peptide of  claim 8 , wherein the linker peptide is a glycine-rich peptide. 
     
     
         10 . The scFv peptide of  claim 9 , wherein the glycine-rich peptide comprises a concatemer of one, two, or three copies of SEQ ID NO: 17, a concatemer of one, two, or three copies of SEQ ID NO: 18, or a mixture of one, two, or three copies of SEQ ID NO: 17 and one, two, or three copies of SEQ ID NO: 18. 
     
     
         11 . The scFv peptide of  claim 7 , further comprising at least two pairs of the V H  segment and V L  segment, wherein the at least two pairs are linked to form a multivalent scFv. 
     
     
         12 . The scFv peptide of  claim 7 , wherein the scFv peptide is present in the pharmacologically acceptable carrier. 
     
     
         13 . The scFv peptide of  claim 7 , wherein the scFv peptide is grafted into a human or humanized antibody. 
     
     
         14 . A recombinant nucleic acid, comprising a first nucleic acid segment encoding a V H  segment having a first amino acid sequence selected from the group consisting of amino acids 4-113 of any one of SEQ ID NOs: 2 and 8-12, a second nucleic acid segment encoding a V L  segment having a second amino acid sequence selected from the group consisting of amino acids 113-227 of SEQ ID NOs. 2 and 8-12, or a combination thereof, wherein the first and second segments are optionally present in a same reading frame. 
     
     
         15 . The recombinant nucleic acid of  claim 14 , further comprising a third nucleic acid segment encoding a linker peptide coupling together the first and second segments in frame. 
     
     
         16 . The recombinant nucleic acid of  claim 14 , further comprising one or more additional nucleic acid segments that encode one or more subsequences of an intact antibody, such that the recombinant nucleic acid encodes a recombinant intact antibody. 
     
     
         17 . A method for targeting a conformational state of fibronectin (FN) in a sample, optionally a biological sample isolated from or present within a subject, the method comprising contacting the sample with a composition having a selective binding activity for a conformational state of FN comprising FnIII9-4G-10 (4G), whereby the conformational state is targeted. 
     
     
         18 . The method of  claim 17 , wherein the sample comprises or is suspected to comprise a tissue undergoing tissue repair, a tissue that is diseased, a tissue that suffers from a disorder, or any combination thereof. 
     
     
         19 . A method for detecting a conformational state of fibronectin (FN) in a sample, the method comprising contacting the sample with a composition having a selective binding activity for a conformational state of FN comprising FnIII9-4G-10 (4G); and detecting the binding of the composition, whereby the conformational state of FN is detected. 
     
     
         20 . The method of  claim 19 , wherein the sample comprises or is suspected to comprise a tissue undergoing tissue repair, a tissue that is diseased, a tissue that suffers from a disorder, or a combination thereof. 
     
     
         21 . The method of  claim 20 , wherein the sample comprises or is suspected to comprise a pathologic extracellular matrix (ECM). 
     
     
         22 . The method of  claim 20 , wherein the sample comprises or is suspected to comprise tumor stroma, a fibrotic ECM, or a combination thereof. 
     
     
         23 . The method of  claim 19 , wherein detecting the binding of the composition comprises detecting a binding ratio of composition to FN. 
     
     
         24 . The method of  claim 19 , wherein detecting the binding of the composition comprises distinguishing normal from diseased tissue. 
     
     
         25 . The method of  claim 19 , wherein detecting the binding of the composition comprises determining severity of fibrosis in the sample. 
     
     
         26 . The method of  claim 19 , wherein detecting the binding of the composition comprises detecting a transient, force-induced conformational change in FN. 
     
     
         27 . The method of  claim 19 , wherein detecting the binding of the composition comprises extracting structural information for an ECM in the sample. 
     
     
         28 . The method of  claim 27 , wherein extracting structural information for an ECM in the sample comprises delineating regions of high ECM strain. 
     
     
         29 . The method of  claim 28 , wherein the high ECM strain is associated with enhanced av integrin binding character. 
     
     
         30 . The method of  claim 19 , further comprising determining a type of treatment to be administered to the subject based on the detecting of the binding of the composition. 
     
     
         31 . A method for treating a disease or disorder in a subject, the method comprising administering to a subject in need there of a therapeutically effective amount of a composition having a selective binding activity for a conformational state of FN comprising FnIII9-4G-10 (4G), whereby treatment is accomplished. 
     
     
         32 . The method of  claim 31 , wherein the disease or disorder has a characteristic selected from the group consisting of a tissue undergoing tissue repair, a tissue that is diseased, a tissue that suffers from a disorder, and any combination thereof. 
     
     
         33 . The method of  claim 32 , wherein the characteristic is a pathologic extracellular matrix (ECM). 
     
     
         34 . The method of  claim 32 , wherein the characteristic is tumor stroma, a fibrotic ECM, or a combination thereof. 
     
     
         35 . The method of  claim 17 , wherein the composition having a selective binding activity for a conformational state of FN comprising FnIII9-4G-10 (4G) is an isolated and purified antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6-12, a fragment thereof, or an antibody have a sequence approximately 95% identical to a sequence of SEQ ID NOs: 2, 4, and 6-12, or a fragment thereof. 
     
     
         36 . The method of  claim 35 , wherein the amino acid sequence comprises at least one modification selected from the group consisting of an amino acid deletion, an amino acid addition, an amino acid substitution, and combinations thereof. 
     
     
         37 . The method of  claim 35 , wherein the antibody or fragment thereof comprises a scFv fragment. 
     
     
         38 . The method of  claim 37 , wherein the scFv fragment is mammalian. 
     
     
         39 . The method of  claim 37 , wherein the scFv fragment is humanized. 
     
     
         40 . A method for screening for a compound having a selective binding activity for a conformational state of FN comprising FnIII9-4G-10 (4G), the method comprising:
 (a) providing a sample comprising a conformational state of FN comprising FnIII9-4G-10 (4G);   (b) contacting the sample with a candidate compound; and   (c) detecting binding of the candidate compound to the sample.   
     
     
         41 . The method of  claim 40 , wherein the candidate compound is a member of a library of compounds. 
     
     
         42 . The method of  claim 40 , wherein the candidate compound is a small molecule or an antibody. 
     
     
         43 . The method of  claim 40 , wherein the conformational state of FN is a force-induced conformational change in Fn. 
     
     
         44 . A compound identified by the method of  claim 40 . 
     
     
         45 . A method for treating a disease or disorder in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising the scFv peptide of  claim 7 , whereby treatment is accomplished. 
     
     
         46 . A method for ameliorating at least one symptom of consequence of a disease or disorder associated with abnormal expression of a force-induced conformational state of FN comprising FnIII9-4G-10 (4G) in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising the scFv peptide of  claim 7 , wherein at least one symptom of consequence of a disease or disorder associated with abnormal expression of a force-induced conformational state of FN comprising FnIII9-4G-10 (4G) is ameliorated. 
     
     
         47 . The method of  claim 45 , wherein the disease or disorder is associated with a tissue undergoing tissue repair, a tissue that is diseased, a tissue that suffers from a disorder, or any combination thereof. 
     
     
         48 . The method of  claim 45 , wherein the disease or disorder is associated with a pathologic extracellular matrix (ECM). 
     
     
         49 . The method of  claim 45 , wherein the disease or disorder is associated with tumor stroma, a fibrotic ECM, or a combination thereof. 
     
     
         50 . A single chain variable fragment (scFv) peptide comprising a heavy chain CDR1 of sequence SYAMS (SEQ ID NO: 24), a heavy chain CDR2 of sequence DIYDGGGTNYADSVKG (SEQ ID NO: 25), a heavy chain CDR3 of sequence TADNFY (SEQ ID NO: 26) or TADNFD (SEQ ID NO: 27), a light chain CDR1 of sequence RASQSISSYLN (SEQ ID NO: 28), a light chain CDR2 of sequence AASTLQS (SEQ ID NO: 29), and a light chain CDR3 of sequence QQANSAPTT (SEQ ID NO: 30). 
     
     
         51 . The scFv peptide of  claim 50 , wherein the scFv compriuses a V H  segment and a V L  segment coupled together with a linker peptide. 
     
     
         52 . The scFv peptide of  claim 51 , wherein the linker peptide is a glycine-rich peptide. 
     
     
         53 . The scFv peptide of  claim 52 , wherein the glycine-rich peptide comprises a concatemer of one, two, or three copies of SEQ ID NO: 17, a concatemer of one, two, or three copies of SEQ ID NO: 18, or a mixture of one, two, or three copies of SEQ ID NO: 17 and one, two, or three copies of SEQ ID NO: 18. 
     
     
         54 . The scFv peptide of  claim 50 , further comprising at least two pairs of the V H  segment and V L  segment, wherein the at least two pairs are linked to form a multivalent scFv. 
     
     
         55 . The scFv peptide of  claim 50 , wherein the scFv peptide is present in the pharmacologically acceptable carrier. 
     
     
         56 . The scFv peptide of  claim 50 , wherein the scFv peptide is grafted into a human or humanized antibody. 
     
     
         57 . The scFv peptide of  claim 50 , wherein the scFv peptide further comprises a modification at its N-terminus, its C-terminus, or both. 
     
     
         58 . The scFv peptide of  claim 57 , wherein the modification comprises addition of a peptide tag, a SARAH domain, or a combination thereof. 
     
     
         59 . The scFv peptide of  claim 58 , wherein the tag comprises a his tag, a myc tag, a VSV tag, an HA tag, a SortaseA tag, a PelB sequence, or any combination of one or more thereof, and further wherein the tag comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-36. 
     
     
         60 . The scFv peptide of  claim 58 , wherein the SARAH domain comprises a sequence selected from the group consisting of SEQ ID NOs: 19-23. 
     
     
         61 . (canceled) 
     
     
         62 . The isolated and purified antibody of  claim 1 , wherein the antibody further comprises a modification at its N-terminus, its C-terminus, or both. 
     
     
         63 . The isolated and purified antibody of  claim 62 , wherein the modification comprises addition of a peptide tag, a SARAH domain, or a combination thereof. 
     
     
         64 . The isolated and purified antibody of  claim 63 , wherein the tag comprises a his tag, a myc tag, a VSV tag, an HA tag, a SortaseA tag, a PelB sequence, or any combination of one or more thereof. 
     
     
         65 . The isolated and purified antibody of  claim 63 , wherein the SARAH domain comprises a sequence selected from the group consisting of SEQ ID NOs: 19-23.