Methods and Compositions Related to Soluble Monoclonal Variable Lymphocyte Receptors of Defined Antigen Specificity

Abstract

Disclosed are compositions and methods related to variable lymphocyte receptors (VLRs). More particularly, disclosed are a variety of antigen specific polypeptides, including soluble, monoclonal, and multivalent forms, as well as methods of using the polypeptides, antibodies that bind the antigen specific polypeptides, and nucleic acids, vectors and expression systems that encode the polypeptides. Antigen specific polypeptides that selectively bind pathogens, like anthrax, and carbohydrates, like blood group determinants, are specifically disclosed.

1 . A method of making a soluble, monoclonal antigen specific polypeptide comprising
 a. isolating a cDNA clone encoding an antigen specific polypeptide, wherein the antigen specific polypeptide comprises an N-terminal leucine rich repeat (LRRNT), one or more leucine rich repeats (LRRs), a C-terminal leucine rich repeat (LRCCT), and a connecting peptide, wherein the connecting peptide comprises an alpha helix;   b. transfecting a cell in culture medium with the cDNA clone of step (a); and   C. isolating the antigen specific polypeptide from the culture medium.   
     
     
         2 . The method of  claim 1 , wherein the antigen specific polypeptide binds a target protein. 
     
     
         3 . The method of  claim 1 , wherein the antigen specific polypeptide binds a target carbohydrate. 
     
     
         4 . The method of  claim 1 , wherein the antigen specific polypeptide binds a target pathogen. 
     
     
         5 . The method of  claim 1 , further comprising the step of generating a stable cell line comprising the cDNA clone. 
     
     
         6 . A soluble, monoclonal antigen specific polypeptide made by the method of  claim 1 . 
     
     
         7 . The soluble, monoclonal antigen specific polypeptide of  claim 6 , wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:5, 47, 49, 51, 53, 55, 57, 59 and 61. 
     
     
         8 . The soluble, monoclonal antigen specific polypeptide of  claim 6 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:20. 
     
     
         9 . A multivalent protein comprising multiple antigen specific polypeptides, wherein each antigen specific polypeptide comprises
 a. a N-terminal leucine rich repeat (LRRNT),   b. one or more internal leucine rich repeats (LRRs),   c. a C-terminal leucine rich repeat (LRCCT), and   d. a connecting peptide, wherein the connecting peptide comprises an alpha helix.   
     
     
         10 . The multivalent protein of  claim 9 , comprising up to ten antigen specific polypeptides. 
     
     
         11 . The multivalent protein of  claim 9 , wherein the antigen specific polypeptides bind a target protein. 
     
     
         12 . The multivalent protein of  claim 9 , wherein the antigen specific polypeptides bind a target carbohydrate. 
     
     
         13 . The multivalent protein of  claim 9 , wherein the antigen specific polypeptides bind a target pathogen. 
     
     
         14 . The multivalent protein of  claim 9 , wherein the antigen specific polypeptides are soluble. 
     
     
         15 . An antigen specific polypeptide comprising
 a. a N-terminal leucine rich repeat (LRRNT),   b. one or more leucine rich repeats (LRRs),   c. a C-terminal leucine rich repeat (LRCCT), and   d. a connecting peptide, wherein the connecting peptide comprises an alpha helix and wherein the binding polypeptide specifically binds a target carbohydrate.   
     
     
         16 . The antigen specific polypeptide of  claim 15 , wherein the target carbohydrate is a blood group determinant. 
     
     
         17 . The antigen specific polypeptide of  claim 16 , wherein the blood group determinant is the H determinant. 
     
     
         18 . The antigen specific polypeptide of  claim 16 , wherein the antigen specific polypeptide has the amino acid sequence of SEQ ID NO:20. 
     
     
         19 . A method of typing blood comprising
 a. contacting a blood sample with the antigen specific polypeptide of  claim 16 , wherein the antigen specific polypeptide is detectably labeled; and   b. detecting labeled antigen specific polypeptide bound to one or more cells in the blood sample, the presence or absence of the label indicating the blood type.   
     
     
         20 . A method of typing blood comprising
 a. contacting a blood sample with a first antigen specific polypeptide of  claim 16 , wherein the first antigen specific polypeptide is detectably labeled with a first label and wherein the first antigen specific polypeptide is specific for a first blood determinant;   b. contacting the blood sample with a second antigen specific polypeptide, wherein the second antigen specific polypeptide is detectably labeled with a second label and wherein the second antigen specific polypeptide is specific for a second blood determinant; and   c. detecting labeled first and second antigen specific polypeptides bound to one or more cell(s) in the blood sample, the presence or absence of the first and second labels indicating the blood type.   
     
     
         21 . A method of making the antigen specific polypeptide of  claim 15  comprising
 a. administering to a lamprey or hagfish the target carbohydrate; 
 b. isolating an antigen specific protein-encoding RNA from lymphocytes of the lamprey or hagfish; 
 c. amplifying antigen specific protein encoding cDNA from the isolated RNA of step (b); 
 d. cloning the cDNA of step (c) into an expression vector; 
 e. expressing the cDNA of step (d) in a host cell transformed with the expression vector; 
 f. isolating a cDNA clone of step (e); 
 g. transfecting a cultured cell with the cDNA clone of step (f); 
 h. screening the culture supernatant for an ability to bind the target carbohydrate, and 
 i. isolating the antigen specific protein from the supernatant that binds the target carbohydrate. 
 
     
     
         22 . The antigen specific protein made by the method of  claim 21 . 
     
     
         23 . A nucleic acid that encodes the antigen specific protein of  claim 22 . 
     
     
         24 . An expression vector comprising the nucleic acids of  claim 23 . 
     
     
         25 . A cultured cell comprising the expression vector of  claim 24 . 
     
     
         26 . An antigen specific polypeptide comprising
 a. a N-terminal leucine rich repeat (LRRNT),   b. one or more leucine rich repeats (LRRs),   c. a C-terminal leucine rich repeat (LRCCT), and   d. a connecting peptide, wherein the connecting peptide comprises an alpha helix and wherein the binding polypeptide specifically binds a  Bacillus anthracis  cell surface polypeptide.   
     
     
         27 . The antigen specific polypeptide of  claim 26 , wherein the  Bacillus anthracis  cell surface polypeptide is BclA. 
     
     
         28 . The antigen specific polypeptide of  claim 26 , wherein the binding polypeptide has the amino acid sequence selected from the group consisting of SEQ ID NOs:5, 22, 47, 49, 51, 53, 55, 57, 59 and 61. 
     
     
         29 . A method of detecting the presence of  Bacillus anthracis  in a sample, comprising
 a. contacting the sample with the antigen specific polypeptide of  claim 26 , wherein the antigen specific polypeptide is detectably labeled; and   b. detecting labeled antigen specific polypeptide bound to the sample, the presence of the label indicating the presence of  Bacillus anthracis  in the sample.   
     
     
         30 . A method of reducing pathogenicity of  Bacillus anthracis  in a subject comprising administering to the subject the antigen specific polypeptide of  claim 26 . 
     
     
         31 . A method of making the antigen specific polypeptide of  claim 26  comprising
 a. administering to a lamprey or hagfish the cell surface  Bacillus anthracis  polypeptide 
 b. isolating an antigen specific protein-encoding RNA from lymphocytes of the lamprey or hagfish; 
 c. amplifying antigen specific protein encoding cDNA from the isolated RNA of step (b); 
 d. cloning the cDNA of step (c) into an expression vector; 
 e. expressing the cDNA of step (d) in a host cell transformed with the expression vector; 
 f. isolating a cDNA clone of step (e); 
 g. transfecting a cultured cell with the cDNA clone of step (f); 
 h. screening the culture supernatant for an ability to bind the cell surface  Bacillus anthracis  polypeptide, and 
 i. isolating the antigen specific protein from the supernatant that binds the cell surface  Bacillus anthracis  polypeptide. 
 
     
     
         32 . A nucleic acid that encodes the antigen specific polypeptide of  claim 26 . 
     
     
         33 . An expression vector comprising the nucleic acids of  claim 32 . 
     
     
         34 . A cultured cell comprising the expression vector of  claim 33 . 
     
     
         35 . An antigen specific polypeptide comprising
 a. a N-terminal leucine rich repeat (LRRNT),   b. one or more leucine rich repeats (LRRs),   c. a C-terminal leucine rich repeat (LRCCT), and   d. a connecting peptide, wherein the connecting peptide comprises an alpha helix and wherein the binding polypeptide specifically binds a viral antigen.   
     
     
         36 . The antigen specific polypeptide of  claim 35 , wherein the viral antigen is human immunodeficiency virus (HIV). 
     
     
         37 . The antigen specific polypeptide of  claim 35 , wherein the viral antigen is HIV envelope protein gp120. 
     
     
         38 . The antigen specific polypeptide of  claim 35 , wherein the viral antigen is influenza. 
     
     
         39 . A method of detecting the presence of a virus in a sample, comprising
 a. contacting the sample with the antigen specific polypeptide of  claim 35 , wherein the antigen specific polypeptide is detectably labeled; and   b. detecting labeled antigen specific polypeptide bound to the sample, the presence of the label indicating the presence of the virus in the sample.   
     
     
         40 . A method of reducing pathogenicity of a virus in a subject comprising administering to the subject the antigen specific polypeptide of  claim 35 . 
     
     
         41 . A method of making the antigen specific polypeptide of  claim 35  comprising
 a. administering to a lamprey or hagfish the viral antigen 
 b. isolating an antigen specific protein-encoding RNA from lymphocytes of the lamprey or hagfish; 
 c. amplifying antigen specific protein encoding cDNA from the isolated RNA of step (b); 
 d. cloning the cDNA of step (c) into an expression vector; 
 e. expressing the cDNA of step (d) in a host cell transformed with the expression vector; 
 f. isolating a cDNA clone of step (e); 
 g. transfecting a cultured cell with the cDNA clone of step (f); 
 h. screening the culture supernatant for an ability to bind the viral antigen, and 
 i. isolating the antigen specific protein from the supernatant that binds the viral antigen. 
 
     
     
         42 . A nucleic acid that encodes the antigen specific protein of  claim 35 . 
     
     
         43 . An expression vector comprising the nucleic acids of  claim 42 . 
     
     
         44 . A cultured cell comprising the expression vector of  claim 43 . 
     
     
         45 . An antibody that selectively binds an antigen specific polypeptide comprising
 a. a N-terminal leucine rich repeat (LRRNT),   b. one or more leucine rich repeats (LRRs),   C. a C-terminal leucine rich repeat (LRCCT), and   d. a connecting peptide, wherein the connecting peptide comprises an alpha helix.   
     
     
         46 . The antibody of  claim 45 , wherein the antibody is labeled with a detectable moiety. 
     
     
         47 . The antibody of  claim 45 , wherein the antibody is 4C4 or 6C3. 
     
     
         48 . A method of making an antigen specific polypeptide comprising
 a. administering to a lamprey or hagfish a target antigen   b. isolating an antigen specific protein-encoding RNA from lymphocytes of the lamprey or hagfish;   c. amplifying antigen specific protein encoding cDNA from the isolated RNA of step (b);   d. cloning the cDNA of step (c) into an expression vector;   e. expressing the cDNA of step (d) in a host cell transformed with the expression vector;   f. isolating a cDNA clone of step (e);   g. transfecting a cultured cell with the cDNA clone of step (f);   h. screening the culture supernatant for an ability to bind the antigen, and   i. isolating the antigen specific protein from the supernatant that binds the antigen.   
     
     
         49 . The method of  claim 48 , wherein the antigen is selected from the group consisting of a protein, a pathogen, a carbohydrate, a lipid, a glycolipid and a glycoprotein.