Animals, cells and methods for production of detectably-labeled antibodies

Abstract

Genetically-modified mammals and immune cells are provided which are capable of producing or secreting detectably-labeled immunoglobulin molecules as a result of genetic modifications of at least one immunoglobulin gene in the genome thereof, such that a fusion polynucleotide encoding a detectable protein or peptide and an immunoglobulin component molecule is present.

1 - 32 . (canceled)  
     
     
         33 . A hybridoma comprising a genetically-modified immune cell having a vector comprising a fusion polynucleotide, said fusion polynucleotide comprising a nucleic acid encoding an immunoglobulin molecule comprising two heavy chains and two light chains, a nucleic acid encoding one detectable protein, and a nucleic acid encoding a flexible linker peptide located between the nucleic acid encoding the immunoglobulin molecule and the nucleic acid encoding the one detectable protein, and wherein said immune cell is capable of expressing one detectable protein fused with said immunoglobulin molecule, with the flexible linker peptide between the immunoglobulin molecule and the one detectable protein, wherein antibodies secreted by said genetically-modified immune cell comprise said one detectable protein.  
     
     
         34 . A chimeric, detectably-labeled immunoglobulin molecule comprising at least one detectable protein or peptide fused with the kappa immunoglobulin light chain, the lambda immunoglobulin light chain, an immunoglobulin heavy chain, or any combination thereof.  
     
     
         35 . The chimeric, detectably-labeled immunoglobulin molecule of  claim 34  wherein said at least one detectable peptide or protein is present at the C-terminus of the gene product of said fusion polynucleotide.  
     
     
         36 . The chimeric, detectably-labeled immunoglobulin molecule of  claim 35  wherein a polynucleotide encoding said at least one detectable peptide or protein present at the C-terminus of the gene product of said fusion polynucleotide is located in exon G1.  
     
     
         37 . The chimeric, detectably-labeled immunoglobulin molecule of  claim 35  wherein said at least one detectable peptide or protein is present at the C-terminus of the gene product of said fusion polynucleotide with a flexible linker peptide therebetween.  
     
     
         38 . The chimeric, detectably-labeled immunoglobulin molecule of  claim 37  wherein a polynucleotide encoding said at least one detectable peptide or protein present at the C-terminus of the gene product of said fusion polynucleotide with a flexible linker therebetween is located in exon G1.  
     
     
         39 . The chimeric, detectably-labeled immunoglobulin molecule of  claim 34  wherein said immunoglobulin heavy chain gene is selected from the group consisting of IgG, IgM, IgD and IgA.  
     
     
         40 . The chimeric, detectably-labeled immunoglobulin molecule of  claim 34  comprising at least one detectable protein or peptide in the heavy chain of said immunoglobulin molecule.  
     
     
         41 . The chimeric, detectably-labeled immunoglobulin molecule of  claim 34  comprising at least one detectable protein or peptide in the light chain of said immunoglobulin molecule.  
     
     
         42 . The chimeric, detectably-labeled immunoglobulin molecule of  claim 34  comprising at least one detectable protein or peptide in the heavy chain and at least one detectable protein or peptide in the light chain of said immunoglobulin molecule.  
     
     
         43 . The chimeric, detectably-labeled immunoglobulin molecule of  claim 42  wherein at least one said detectable protein or polypeptide is capable of quenching fluorescence.  
     
     
         44 . The chimeric, detectably-labeled immunoglobulin molecule of  claim 34  wherein said at least one detectable protein or peptide is an autofluorescent protein or peptide, a visibly-detectable protein or peptide, an enzymatically active protein or peptide, a protein or peptide capable of interacting with another molecule to produce a detectable product, or any combination thereof.  
     
     
         45 . The chimeric, detectably-labeled immunoglobulin molecule of  claim 44  wherein said at least one detectable protein is an autofluorescent protein or peptide.  
     
     
         46 . (canceled)  
     
     
         47 . (canceled)  
     
     
         48 . The chimeric, detectably-labeled immunoglobulin molecule of  claim 44  wherein said at least one detectable protein is a combination of an autofluorescent protein or peptide and an enzymatically-active protein or peptide.  
     
     
         49 . The chimeric, detectably-labeled immunoglobulin molecule of  claim 48  wherein said at least one detectable protein is a combination of green fluorescent protein and alkaline phosphatase.  
     
     
         50 . A method for producing a quantity of detectably-labelled polyclonal antibodies comprising the steps of 
 a) providing a genetically-modified mammal in accordance with claim  1 ;    b) immunizing said genetically-modified mammal with a preselected immunogen, wherein said genetically-modified mammal generates antibodies to said immunogen, wherein antibodies secreted by immune cells of said genetically-modified mammal comprise said at least one detectable protein or peptide; and    c) isolating said detectably-labelled antibodies from said genetically-modified mammal.    
     
     
         51 . A method for producing a quantity of detectably-labelled monoclonal antibodies comprising the steps of 
 a) preparing a genetically-modified mammal in accordance with claim  1 ;    b) immunizing said genetically-modified mammal with a preselected immunogen, wherein immune cells of said genetically-modified mammal generate antibodies to said immunogen, wherein antibodies secreted by said immune cells comprise said at least one detectable protein or peptide; and    c) immortalizing antibody-producing immune cells isolated from said genetically-modified mammal;    d) selecting immortalized immune cells isolated from said genetically-modified mammal that secrete antibodies specific to said immunogen; and    e) preparing a quantity of detectably-labeled monoclonal antibodies from said selected immune cells.    
     
     
         52 . A genetically-modified non-human mammal capable of producing a detectably-labeled antibody in response to immunization by an antigen, the genome of said non-human mammal comprising at least one fusion polynucleotide comprising a nucleic acid encoding at least one detectable protein or peptide and a nucleic acid encoding an immunoglobulin molecule comprising two heavy chains and two light chains, wherein an antibody secreted by an immune cell of said genetically-modified non-human mammal comprises said at least one detectable protein or peptide.  
     
     
         53 . A chimeric, detectably-labeled immunoglobulin molecule comprising at least one fluorescent protein or peptide and at least one fluorescence-quenching protein or peptide fused with a component of said immunoglobulin molecule independently selected from the group consisting of the kappa immunoglobulin light chain, the lambda immunoglobulin light chain, an immunoglobulin heavy chain, and any combination thereof.  
     
     
         54 . A genetically-modified non-human mammal whose genome comprises a vector comprising a fusion polynucleotide, said fusion polynucleotide comprising a nucleic acid encoding an immunoglobulin molecule comprising two heavy chains and two light chains, and a nucleic acid encoding at least one detectable protein, wherein said non-human mammal is capable of expressing said at least one detectable protein fused with said immunoglobulin molecule wherein an antibody secreted by an immune cell of said genetically-modified non-human mammal comprises said at least one detectable protein.  
     
     
         55 . The genetically-modified non-human mammal of  claim 54  wherein said at least one detectable protein encoded by the fusion polynucleotide is present at the C-terminus of the gene product of said fusion polynucleotide.  
     
     
         56 . The genetically-modified non-human mammal of  claim 55  wherein said nucleic acid encoding said at least one detectable protein present at the C-terminus of the gene product of said fusion polynucleotide is located in exon G1.  
     
     
         57 . The genetically-modified non-human mammal of  claim 54  wherein said at least one detectable protein is present at the C-terminus of the gene product of said fusion polynucleotide with a flexible linker peptide located between the one detectable protein and the immunoglobulin molecule.  
     
     
         58 . The genetically-modified non-human mammal of  claim 54  wherein an antibody secreted by an immune cell of said genetically-modified non-human mammal comprises at least one detectable protein in the heavy chain of said antibody.  
     
     
         59 . The genetically-modified non-human mammal of  claim 54  wherein an antibody secreted by an immune cell of said genetically-modified non-human mammal comprises at least one detectable protein in the light chain of said antibody.  
     
     
         60 . The genetically-modified non-human mammal of  claim 54  wherein an antibody secreted by an immune cell of said genetically-modified non-human mammal comprises at least one detectable protein in the heavy chain and at least one detectable protein in the light chain of said antibody.  
     
     
         61 . The genetically-modified non-human mammal of  claim 54  wherein the one detectable protein is capable of quenching fluorescence.  
     
     
         62 . The genetically-modified non-human mammal of  claim 54  wherein the one detectable protein is an autofluorescent protein, a visibly-detectable protein, an enzymatically active protein, or a protein capable of interacting with another molecule to produce a detectable product.  
     
     
         63 . The genetically-modified non-human mammal of  claim 62  wherein said one detectable protein is an autofluorescent protein.  
     
     
         64 . The genetically-modified non-human mammal of  claim 60  wherein said one detectable protein is a combination of an autofluorescent protein and an enzymatically-active protein.  
     
     
         65 . The genetically-modified non-human mammal of  claim 64  wherein said one detectable protein is a combination of green fluorescent protein and alkaline phosphatase.  
     
     
         66 . A genetically-modified immune cell having a vector comprising a fusion polynucleotide, said fusion polynucleotide comprising a nucleic acid encoding an immunoglobulin molecule comprising two heavy chains and two light chains, a nucleic acid encoding at least one detectable protein, and a nucleic acid encoding a flexible linker peptide located between the nucleic acid encoding the immunoglobulin molecule and the nucleic acid encoding the at least one detectable protein, and wherein said immune cell is capable of expressing at least one detectable protein fused with said immunoglobulin molecule, with the flexible linker peptide between the immunoglobulin molecule and the one detectable protein, wherein antibodies secreted by said genetically-modified immune cell comprise said one detectable protein.  
     
     
         67 . The genetically-modified immune cell of  claim 66  wherein said one detectable protein is present at the C-terminus of the gene product of said fusion polynucleotide.  
     
     
         68 . The genetically-modified immune cell of  claim 66 , wherein said nucleic acid encoding said one detectable protein present at the C-terminus of the gene product of said fusion polynucleotide is located in exon G1.  
     
     
         69 . The genetically-modified immune cell of  claim 66  wherein an antibody secreted by said immune cell comprises at least one detectable protein in the heavy chain of said antibody.  
     
     
         70 . The genetically-modified immune cell of  claim 66  wherein an antibody secreted by said genetically-modified immune cells comprises at least one detectable protein in the light chain of said antibody.  
     
     
         71 . The genetically-modified immune cell of  claim 66  wherein an antibody secreted by said genetically-modified immune cells comprises at least one detectable protein in the heavy chain and at least one detectable protein in the light chain of said antibody.  
     
     
         72 . The genetically-modified immune cell of  claim 66  wherein the one detectable protein is capable of quenching fluorescence.  
     
     
         73 . The genetically-modified immune cell of  claim 66  wherein the one detectable protein is an autofluorescent protein or peptide, a visibly-detectable protein or peptide, an enzymatically active protein or peptide, or a protein or peptide capable of interacting with another molecule to produce a detectable product.  
     
     
         74 . The genetically-modified immune cell of  claim 73  wherein the one detectable protein is an autofluorescent protein.  
     
     
         75 . The genetically-modified immune cell of  claim 73  wherein said one detectable protein is a combination of an autofluorescent protein and an enzymatically-active protein.  
     
     
         76 . The genetically-modified immune cell of  claim 75  wherein said one detectable protein is a combination of green fluorescent protein and alkaline phosphatase.  
     
     
         77 . The genetically modified non-human mammal of  claim 62  or the genetically modified immune cell of  claim 73 , or the chimeric detectably labeled immunoglobulin molecule of  claim 44 , wherein said protein capable of interacting with another molecule to produce a detectable product is selected from the group consisting of an intein, a biotin-binding subunit of streptavidin or avidin, a His tag, or a chitin-binding domain, or any combination thereof, and wherein said protein capable of interacting with another molecule to produce a detectable product may also be used to facilitate purification of said detectable product.