Immunoglobulin DNA cassette molecules, monobody constructs, methods of production, and methods of use therefor

Abstract

Featured are DNA molecules, expression vectors, and host cells useful for creation of immunoglobulins, as well as novel immunoglobulin molecules termed monobodies. Additionally provided are methods of production of immunoglobulins, including monobodies, as well as methods of using the disclosed immunoglobulin and monobody constructs, expression vectors and host cells containing DNA encoding molecules for production of immunoglobulin and monobody proteins.

1 . An isolated nucleic acid molecule comprising an immunoglobulin cassette construct, wherein the immunoglobulin cassette comprises an immunoglobulin leader molecule operably linked to a stable immunoglobulin domain region.  
     
     
         2 - 9 . (canceled)  
     
     
         10 . An isolated nucleic acid molecule of  claim 1  comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, or a sequence at least 85%, identical thereto.  
     
     
         11 . An isolated nucleic acid molecule comprising immunoglobulin cassette insert sequences selected from the group consisting of SEQ ID NO:83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO 87, SEQ ID NO:88, or a sequence at least 85% identical thereto.  
     
     
         12 . An isolated DNA cassette expression vector comprising one or more DNA immunoglobulin cassette molecules of  claim 1 .  
     
     
         13 - 14 . (canceled)  
     
     
         15 . A recombinant host cell comprising the expression vector of  claim 12 .  
     
     
         16 - 17 . (canceled)  
     
     
         18 . A method of synthesizing a DNA vector capable of encoding an immunoglobulin comprising: 
 synthesizing one or more immunoglobulin DNA cassettes wherein an immunoglobulin leader sequence is operably linked to a stable immunoglobulin domain, wherein the operable linkage includes a cloning site;    introducing one or more immunoglobulin DNA cassettes into an expression vector; and    incorporating insert sequences into the cloning site of one or more immunoglobulin DNA cassettes;    wherein incorporation of insert sequences results in formation of DNA sequences encoding a desired immunoglobulin molecule.    
     
     
         19 . The method of  claim 18 , wherein two immunoglobulin DNA cassettes are incorporated into the expression vector, wherein a first immunoglobulin DNA cassette comprises a heavy chain immunoglobulin and a second immunoglobulin DNA cassette comprises a light chain immunoglobulin.  
     
     
         20 . A DNA expression vector comprising nucleic acid sequences encoding one or more immunoglobulin molecules wherein the DNA vector is synthesized by the method of  claim 18 .  
     
     
         21 . A transfected cell line capable of producing an antibody comprising a DNA expression vector of claim  8 .  
     
     
         22 . An improved method of antibody production, comprising: 
 providing a first nucleic acid encoding a light chain immunoglobulin comprising a light chain leader sequence operably linked to a light chain variable region operably linked to a light chain constant region; and    providing a second nucleic acid encoding a heavy chain immunoglobulin comprising a heavy chain leader sequence operably linked to a heavy chain variable region operably linked to a heavy chain constant region immunoglobulin; and    introducing the nucleic acids into a host cell under conditions that allow expression and assembly of said light and heavy chain immunoglobulins to produce a functional antibody preparation.    
     
     
         23 - 26 . (canceled)  
     
     
         27 . A method of production of a battery of antibodies having identical antigen specificity and variable effector functions comprising: 
 isolating immunoglobulin light and heavy chain DNA variable domain insert sequences having the desired antigen specificity;    providing a selection of expression vectors comprising immunoglobulin DNA cassette vectors, wherein the selection includes cassette vectors having different stable immunoglobulin light and heavy chain constant domains having variable effector functions;    incorporating the immunoglobulin DNA variable domain insert sequences into the cloning site of the DNA cassettes; and    introducing the resulting DNA expression vectors into a suitable host cell under conditions that allow expression and assembly of resulting light and heavy chain immunoglobulins to produce functional antibody preparation;    wherein resultant expressed antibodies comprises a battery of antibodies having identical antigen specificity and variable effector function.    
     
     
         28 - 31 . (canceled)  
     
     
         32 . A method of production of a battery of antibodies having identical effector function and variable antigen specificity comprising: 
 isolating immunoglobulin light and heavy chain DNA variable domain insert sequences having variable antigen specificity;    providing expression vector comprising immunoglobulin DNA cassette vectors, wherein the vector comprises stable immunoglobulin light and heavy chain constant domains having a desired effector function;    incorporating the immunoglobulin DNA variable domain insert sequences into the cloning site of the DNA cassettes; and    introduction of resulting DNA expression vectors into a suitable host cell under conditions that allow expression and assembly of resulting light and heavy chain immunoglobulins to produce functional antibody preparation;    wherein resultant expressed antibodies comprises a battery of antibodies having variable antigen specificity and identical effector function.    
     
     
         33 - 36 . (canceled)  
     
     
         37 . An antibody or antigen binding fragment thereof made by introducing a DNA expression vector comprising a nucleic acid sequence encoding a human IgG1 heavy chain constant region sequence of SEQ ID NO:10, and a nucleic acid sequence encoding a humanized 1D9 heavy chain variable region into a host cell under conditions that allow expression and assembly of said immunoglobulins.  
     
     
         38 . The antibody or antigen binding fragment thereof of  claim 37 , wherein the expression vector further comprises a nucleic acid sequence encoding a human light chain constant region.  
     
     
         39 . The antibody or antigen binding fragment thereof of  claim 38 , wherein the human light chain constant region is a kappa light chain constant region.  
     
     
         40 . The antibody or antigen binding fragment thereof of  claim 37 , wherein the expression vector further comprises a nucleic acid sequence encoding a humanized 1D9 light chain variable region.  
     
     
         41 . The antibody or antigen binding fragment thereof of  claim 40 , wherein the expression vector further comprises a nucleic acid sequence encoding a human light chain constant region.  
     
     
         42 . The antibody or antigen binding fragment thereof of  claim 41 , wherein the human light chain constant region is a kappa light chain constant region.  
     
     
         43 . The antibody or antigen binding fragment thereof of  claim 37 , wherein the IgG1 heavy chain constant region nucleic acid sequence comprises the heavy chain constant region encoding nucleotide sequence of SEQ ID NO:9.  
     
     
         44 . The antibody or antigen binding fragment thereof of  claim 38  or  42 , wherein the kappa light chain constant region nucleic acid sequence encodes the light chain constant region sequence of SEQ ID NO:12.  
     
     
         45 . The antibody or antigen binding fragment thereof of  claim 44 , wherein the kappa light chain constant region nucleic acid sequence comprises the light chain constant region encoding nucleotide sequence of SEQ ID NO:11.  
     
     
         46 . The antibody or antigen binding fragment thereof of  claim 37 , wherein the nucleic acid sequence encoding the heavy chain constant region and the nucleic acid encoding the humanized heavy chain variable region are operably linked to a promoter selected from the group consisting of a EF-1a promoter, a CMV promoter, an SV40 promoter, and an adenovirus major late promoter.  
     
     
         47 . The antibody or antigen binding fragment thereof of  claim 46 , wherein the promoter is a EF-1a promoter.  
     
     
         48 . The antibody or antigen binding fragment thereof of  claim 47 , wherein the nucleic acid sequence encoding the heavy chain constant region and the nucleic acid sequence encoding the humanized heavy chain variable region are under control of the same promoter.  
     
     
         49 . The antibody or antigen binding fragment thereof of  claim 47  or  48 , wherein the vector further comprises a BGH poly A sequence.  
     
     
         50 . The antibody or antigen binding fragment thereof of  claim 49 , wherein the BGH polyA sequence is downstream from the sequence encoding the heavy chain constant region.  
     
     
         51 . The antibody or antigen binding fragment thereof of  claim 37 , wherein the nucleic acid sequence encoding the heavy chain constant region further comprises an immunoglobulin leader sequence.  
     
     
         52 . The antibody or antigen binding fragment thereof of  claim 39  or  42 , wherein the nucleic acid sequence encoding the light chain constant region further comprises an immunoglobulin leader sequence.  
     
     
         53 . The antibody or antigen binding fragment thereof of  claim 37 , wherein the nucleic acid sequence encoding the humanized heavy chain variable region further comprises an immunoglobulin leader sequence.  
     
     
         54 . The antibody or antigen binding fragment thereof of  claim 40  or  42 , wherein the nucleic acid sequence encoding the humanized light chain variable region further comprises an immunoglobulin leader sequence.  
     
     
         55 . An antibody or antigen binding fragment thereof made by introducing a DNA expression vector comprising a nucleic acid sequence encoding the heavy chain constant region sequence of SEQ ID NO:10, a nucleic acid sequence encoding a humanized 1D9 heavy chain variable region, a nucleic acid encoding the light chain constant region sequence of SEQ ID NO:12, and a nucleic acid sequence encoding a humanized 1D9 light chain variable region into a host cell under conditions that allow expression and assembly of said immunoglobulins.  
     
     
         56 . The antibody or antigen binding fragment thereof of  claim 55 , wherein the nucleic acid sequence encoding the heavy chain constant region and the nucleic acid sequence encoding the humanized heavy chain variable region are operably linked to an EF-1a promoter.  
     
     
         57 . The antibody or antigen binding fragment thereof of  claim 55 , wherein the nucleic acid sequence encoding the light chain constant region and the nucleic acid sequence encoding the humanized light chain variable region are operably linked to an EF-1a promoter.  
     
     
         58 . The antibody or antigen binding fragment thereof of  claim 56 , wherein the nucleic acid sequence encoding the light chain constant region and the nucleic acid sequence encoding the humanized light chain variable region are operably linked to an EF-1a promoter.  
     
     
         59 . The antibody or antigen binding fragment thereof of  claim 55 ,  56 ,  57  or  58 , wherein the vector further comprises a BGH poly A sequence.  
     
     
         60 . The antibody or antigen binding fragment thereof of  claim 59 , wherein the BGH polyA sequence is downstream from the sequence encoding the heavy chain constant region.  
     
     
         61 . The antibody or antigen binding fragment thereof of  claim 55 , wherein the nucleic acid encoding the humanized heavy chain variable region further comprises an immunoglobulin leader sequence.  
     
     
         62 . The antibody or antigen binding fragment thereof of  claim 55 , wherein the nucleic acid sequence encoding the humanized light chain variable region further comprises an immunoglobulin leader sequence.  
     
     
         63 . An antibody or antigen binding fragment thereof made by: 
 i) providing a first nucleic acid encoding a light chain immunoglobulin comprising a light chain leader sequence operably linked to a humanized 1D9 light chain variable region operably linked to the light chain constant region encoding nucleotide sequence of SEQ ID NO:11;    ii) providing a second nucleic acid encoding a heavy chain immunoglobulin comprising a heavy chain leader sequence operably linked to a humanized 1D9 heavy chain variable region operably linked to the human IgG1 heavy chain constant region encoding nucleotide sequence of SEQ ID NO:9; and    iii) introducing the nucleic acids into a host cell under conditions that allow expression and assembly of said light and heavy chain immunoglobulins.    
     
     
         64 . The antibody or antigen binding fragment thereof of  claim 37 ,  55  or  63 , wherein the host cell is selected from the group consisting of a bacterial, a yeast and a mammalian cell.  
     
     
         65 . The antibody or antigen binding fragment thereof of  claim 64 , wherein the cell is a mammalian cell.  
     
     
         66 . The antibody or antigen binding fragment thereof of  claim 65 , wherein the mammalian cell is selected from the group consisting of a lymphocytic cell line, CHO, and COS cells.  
     
     
         67 . The antibody or antigen binding fragment thereof of  claim 65 , wherein the mammalian cell is a CHO cell.  
     
     
         68 . The antibody or antigen binding fragment thereof of  claim 67 , wherein the CHO cell is a DHFR deficient CHO cell.  
     
     
         69 . An antibody or antigen binding fragment thereof comprising a human IgG1 heavy chain constant region of SEQ ID NO:10, or a portion thereof, and a humanized 1D9 heavy chain variable region.  
     
     
         70 . The antibody or antigen binding fragment thereof of  claim 69 , further comprising a human light chain constant region.  
     
     
         71 . The antibody or antigen binding fragment thereof of  claim 70 , wherein the human light chain constant region is a kappa light chain constant region.  
     
     
         72 . The antibody or antigen binding fragment thereof of  claim 69 , further comprising a humanized 1D9 light chain variable region.  
     
     
         73 . The antibody or antigen binding fragment thereof of  claim 72 , further comprising a human light chain constant region.  
     
     
         74 . The antibody or antigen binding fragment thereof of  claim 73 , wherein the human light chain constant region is a kappa light chain constant region.  
     
     
         75 . The antibody or antigen binding fragment thereof of  claim 69 , wherein the heavy chain constant region comprises the heavy chain constant region encoded by a nucleotide sequence of SEQ ID NO:9.  
     
     
         76 . The antibody or antigen binding fragment thereof of  claim 74 , wherein the light chain constant region is the light chain constant region of SEQ ID NO:12, or a portion thereof.  
     
     
         77 . The antibody or antigen binding fragment thereof of  claim 76 , wherein the light chain constant region comprises the light chain constant region encoded by a nucleotide sequence of SEQ ID NO:11.  
     
     
         78 . An antibody or antigen binding fragment thereof, wherein the constant region is: 
 i) a heavy chain constant region sequence of SEQ ID NO:10, or portion thereof; and    ii) a light chain constant region sequence of SEQ ID NO:12, or a portion thereof;    and wherein the variable region is:    a) a humanized 1D9 heavy chain variable region; and    b) a humanized 1D9 light chain variable region.