Engineered Rabbit Antibody Variable Domains And Uses Thereof

Abstract

The present invention relates to methods for screening and producing polypeptides that immunospecifically bind to an antigen, which polypeptides comprise binding domains that are derived from rabbit immunoglobulin. Using rabbit antibody heavy-chain or light-chain scaffolds, the methods of the invention allow identification of novel CDR loops and framework regions that confer enhanced stability and/or affinity to isolated immunoglobulin variable domains, in particular, relative to those derived from rodent antibodies. The enhanced stability and/or affinity of the variable domains of the invention permit their use in the production research tools or therapeutic immunospecific polypeptides, including single domain immunospecific polypeptides, i.e., comprising one of a VH or VL domain.

What is claimed is: 
     
         1 . A method for producing a rabbit VL or VH domain that immunospecifically binds to an antigen, wherein said method is a first method comprising:
 a) selecting from a phage expression library a set of DNA sequences that encode VH or VL domains that immunospecifically bind to said antigen, said phage expression library prepared from DNA or cDNA sequences that encode VH or VL domains and that were obtained from a biological sample of a rabbit containing cells that express immunoglobulins; and   b) expressing said set of sequences, or a subset thereof, in bacteria as a fusion protein with CAT, selecting bacteria that have chloramphenicol resistance by virtue of CAT expression, and obtaining sequences encoding rabbit VH or VL domains from the selected bacteria, whereby said bacterial resistance to chloramphenicol conferred by said CAT-fusion correlates with solubility, stability, and/or lack of aggregation of said sequence fused to CAT;   
       or a second method comprising:
 a) expressing sequences of DNA or cDNA that encode VH or VL domains in bacteria as a fusion protein with CAT, wherein said DNA or cDNA sequences were obtained from a biological sample of a rabbit containing cells that express immunoglobulins; 
 b) selecting bacteria that have chloramphenicol resistance by virtue of CAT expression, and obtaining the set of sequences encoding rabbit VH or VL domains from the selected bacteria, whereby said bacterial resistance to chloramphenicol conferred by said CAT-fusion correlates with solubility, stability, and/or lack of aggregation of said sequence fused to CAT; and 
 c) selecting from a phage expression library DNA sequences that encode VH or VL domains that immunospecifically bind to said antigen, wherein said library is prepared from said set of sequences, or subset thereof. 
 
     
     
         2 . The method of  claim 1 , wherein said rabbit biological sample is from a rabbit that has been immunized with said antigen. 
     
     
         3 . The method of  claim 1 , wherein, in said first method, step a) is repeated one or more times or wherein, in said second method, step c) is repeated one or more times. 
     
     
         4 . The method of  claim 1 , wherein said biological sample comprises plasma cells, lymphoid, bone marrow, or appendix tissue. 
     
     
         5 . The method of  claim 4 , wherein said sample comprises lymphoid tissue and said lymphoid tissue is spleen tissue or lymph node tissue. 
     
     
         6 . An antibody comprising the VH or VL domain produced according to  claim 1  or antigen binding fragment thereof. 
     
     
         7 . The antibody or fragment of  claim 6 , wherein said antibody is a single domain antibody, a minibody, a diabody, a bispecific antibody, or a nanobody. 
     
     
         8 . A chimeric or humanized version of the antibody or fragment of  claim 6 . 
     
     
         9 . The antibody of  claim 6 , wherein said antibody or fragment is further conjugated to a therapeutic agent, an albumin-interacting peptide, a fibronectin interacting peptide, fibronectin, albumin, protein A, or protein G. 
     
     
         10 . The method of  claim 1 , wherein the selection is a selection at a chloramphenicol concentration of at least 1.8 nM; the recombinant Vh or VL domain as a Tm at least 54° C.; and/or the recombinant VH or VL domain exhibits a Kd for said antigen no greater than 20 nM. 
     
     
         11 . A polypeptide that immunospecifically binds an antigen, wherein said polypeptide comprises one or both of a rabbit VH or VL domain, and at least one of
 (a) a VH H FR1 domain having the amino acid sequence QEQLMETESGGGAEGGLVKPGASLTLTCTAS (SEQ ID NO: 57);   (b) a VH FR3 domain having the amino acid sequence YATWVNGRFTLSRDIDQSTGCLQLNSLTAADTATYYCAR (SEQ ID NO: 95);   (c) a VLFR1 domain having the amino acid sequence ELVLTQTPPSLSASVGETVRIRC (SEQ ID NO:150);   (d) a VL FR3 domain having the amino acid sequence GVSSRFKGSGSGTQFTLTISGVQCADAATYYC; (SEQ ID NO: 205); or   (e) a VL FR4 domain having the amino acid FAFGGGTELEIL (SEQ ID NO:210).   
     
     
         12 . The polypeptide of  claim 11  further comprising at least one of
 (f) a VH FR2 domain having the amino acid sequence WVRQAPGKGLEWIG (SEQ ID NO: 69); 
 (g) a VH FR4 domain having the amino acid sequence WGQGTLVTVSS (SEQ ID NO:139); 
 (h) a VL FR1 domain having the amino acid sequence ELVLTQTPSSVSAAVGGTVTINC (SEQ ID NO: 155); 
 (i) VL FR2 domain having the amino acid sequence WYQQKPEKPPTLLIS (SEQ ID NO: 174) or WYQQKPGQRPKLLIY (SEQ ID NO:181); 
 (j) a VL FR3 domain having the amino acid sequence GVPPRFSGSGSGTDYTLTIGGVQAEDVATYYC (SEQ ID NO: 183); or 
 (k) a VL FR4 domain having the amino acid sequence FGAGTNVEIK (SEQ ID NO:206). 
 
     
     
         13 . The polypeptide of  claim 11  or  12 , wherein said polypeptide further comprises at least one of
 (a) in the VH FR2 domain, a phenylalanine at position 46; 
 (b) in the VH FR2 domain, a glutamic acid at position 53; 
 (c) in the VH FR2 domain, an arginine at position 54; 
 (d) in the VH FR2 domain, a glycine at position 56; 
 (e) in the VH FR2 domain, an alanine at position 58; 
 (f) in the VH CDR1 domain, a cysteine at position 44; 
 (g) in the VH CDR2 domain, a cysteine at position 59; 
 (h) in the VH FR4 domain, an arginine at position 126; 
 (i) in the VL FR2 domain, a phenylalanine or a tyrosine at position. 39; 
 (j) in the VL FR2 domain, a lysine at position 45; or 
 (k) in the VL FR3 domain; a cysteine at position 91, 
 
       wherein said positions are according the positions of the VH domain or VL domain sequence alignments in  FIGS. 3 and 4 , respectively. 
     
     
         14 . A polypeptide that immunospecifically binds an antigen, wherein said polypeptide comprises one or both of a rabbit VH or VL domain, and at least one of
 (f) a VH FR2 domain having the amino acid sequence WVRQAPGKGLEWIG (SEQ ID NO: 69);   (g) a VH FR4 domain having the amino acid sequence WGQGTLVTVSS (SEQ ID NO:139);   (h) a VL FR1 domain having the amino acid sequence ELVLTQTPSSVSAAVGGTVTINC (SEQ ID NO: 155);   (i) VL FR2 domain having the amino acid sequence WYQQKPEKPPTLLIS (SEQ ID NO: 174) or WYQQKPGQRPKLLIY (SEQ ID NO:181);   (j) a VL FR3 domain having the amino acid sequence GVPPRFSGSGSGTDYTLTIGGVQAEDVATYYC (SEQ ID NO: 183); or   (k) a VL FR4 domain having the amino acid sequence FGAGTNVEIK (SEQ ID NO:206); and   wherein said polypeptide further comprises at least one of   (a) in the VH FR2 domain, a phenylalanine at position 46;   (b) in the VH FR2 domain, a glutamic acid at position 53;   (c) in the VH FR2 domain, an arginine at position 54;   (d) in the VH FR2 domain, a glycine at position 56;   (e) in the VH FR2 domain, an alanine at position 58;   (f) in the VH CDR1 domain, a cysteine at position 44;   (g) in the VH CDR2 domain, a cysteine at position 59;   (h) in the VH FR4 domain, an arginine at position 126;   (i) in the VL FR2 domain, a phenylalanine or a tyrosine at position. 39;   (j) in the VL FR2 domain, a lysine at position 45; or   (k) in the VL FR3 domain; a cysteine at position 91   
       wherein said positions are according the positions of the VH domain or VL domain sequence alignments in  FIGS. 3 and 4 , respectively. 
     
     
         15 . An antibody comprising the polypeptide according to  claim 11  or  14  or an antigen binding fragment thereof; wherein a VH or VL domain of said polypeptide folds to form a soluble, non-aggregative polypeptide that allows selection at a chloramphenicol concentration of at least 1.8 mM when fused to CAT; and wherein the VH or VL domain has a Tm of at least 54° C. 
     
     
         16 . The antibody or fragment of  claim 6 , wherein said antibody is a single chain antibody, a single domain antibody, a minibody, a diabody, a bispecific antibody, or a nanobody; or wherein said antibody is an antibody fragment that is a Fab, Fab′, F(ab′)2 or Fv fragment. 
     
     
         17 . The polypeptide according to  claim 11  or  14 , wherein said polypeptide does not have one or more of a CH1 domain, CH2 domain, CH3 domain or CL domain; or does not have one of a VH or VL domain; and wherein a VH or VL domain of said polypeptide folds to form a soluble, non-aggregative polypeptide that allows selection at a chloramphenicol concentration of at least 1.8 mM when fused to CAT; and wherein the VH or VL domain has a Tm of at least 54° C. 
     
     
         18 . The polypeptide of  claim 17  which does not comprise a rabbit VH domain. 
     
     
         19 . The polypeptide of  claim 17 , which does not comprise a VH domain of a species other than rabbit. 
     
     
         20 . The polypeptide of  claim 17 , which does not comprise a rabbit VL domain. 
     
     
         21 . The polypeptide of  claim 17 , which does not comprise a VL domain of a species other than rabbit. 
     
     
         22 . The polypeptide of any one of  claims 17 - 21 , which does not comprise a CH2 domain. 
     
     
         23 . The polypeptide of any one of  claims 17 - 21 , which does not comprise a CH3 domain. 
     
     
         24 . The polypeptide of any one of  claims 17 - 21 , which does not comprise a CH1 domain. 
     
     
         25 . The polypeptide of  claim 17 , wherein said polypeptide is further conjugated to a therapeutic agent, an albumin interacting peptide, a fibronectin interacting peptide, fibronectin, albumin, protein A, or protein G.