Monoclonal antibody production by ebv transformation of b cells

Abstract

A method for producing a clone of an immortalised human B memory lymphocyte, comprising the step of transforming human B memory lymphocytes using Epstein Barr virus (EBV) in the presence of a polyclonal B cell activator. The method is particularly useful in a method for producing a clone of an immortalised human B memory lymphocyte capable of producing a human monoclonal antibody with a desired antigen specificity, comprising the steps of: (i) selecting and isolating a human memory B lymphocyte subpopulation; (ii) transforming the subpopulation with Epstein Ban virus (EBV) in the presence of a polyclonal B cell activator; (iii) screening the culture supernatant for antigen specificity; and (iv) isolating an immortalised human B memory lymphocyte clone capable of producing a human monoclonal antibody having the desired antigen specificity.

1 . A monoclonal antibody, or an antigen binding fragment thereof, produced by a method comprising:
 (i) contacting a memory B lymphocyte with EBV and an agonist of a Pattern Recognition Receptor that is expressed on memory B cells;   (ii) culturing said memory B lymphocyte; and   (iii) obtaining, and optionally, isolating said monoclonal antibody or fragment thereof,   wherein said monoclonal antibody or fragment thereof is directed against respiratory syncytial virus (RSV).   
     
     
         2 . A monoclonal antibody, or an antigen binding fragment thereof, produced by a method comprising:
 (i) contacting memory B lymphocytes with EBV and an agonist of a Pattern Recognition Receptor that is expressed on memory B cells;   (ii) culturing said memory B lymphocytes;   (iii) screening said memory B lymphocytes for specificity against RSV;   (iv) isolating an immortalized memory B lymphocyte clone capable of producing a monoclonal antibody having said specificity;   (v) (a) (1) culturing said immortalized memory B lymphocyte clone; and
 (2) obtaining, and optionally, isolating said monoclonal antibody or fragment thereof, or 
   (b) (1) obtaining and/or sequencing nucleic acid that encodes an antibody, or fragment thereof, from said immortalized memory B lymphocyte clone;
 (2) inserting said nucleic acid into an expression host, or using said nucleic acid sequence to prepare an expression host, in order to permit expression of said antibody, or fragment thereof, in said host; 
 (3) expressing said antibody, or fragment thereof, in said host; and 
 (4) obtaining, and optionally, isolating said antibody or fragment thereof, 
   wherein said monoclonal antibody or fragment thereof is directed against RSV.   
     
     
         3 . A monoclonal antibody, or an antigen binding fragment thereof, produced by a method comprising:
 (i) producing an immortalized memory B lymphocyte comprising the steps of:
 (a) incubating or pulsing a population of cells comprising a memory B lymphocyte with EBV; 
 (b) diluting said population of cells with culture medium; and 
 (c) contacting said diluted population of cells with a polyclonal B cell activator, wherein said polyclonal B cell activator is an agonist of a Pattern Recognition Receptor that is expressed on memory B cells; 
   (ii) (a) (1) culturing said immortalized memory B lymphocyte; and
 (2) obtaining, and optionally, isolating said monoclonal antibody or fragment thereof from said immortalized memory B lymphocyte, or 
   (b) (1) obtaining and/or sequencing nucleic acid that encodes an antibody, or fragment thereof, from said immortalized memory B lymphocyte;
 (2) inserting said nucleic acid into an expression host, or using said nucleic acid sequence to prepare an expression host, in order to permit expression of said antibody, or fragment thereof, in said host; 
 (3) expressing said antibody, or fragment thereof, in said host; and 
 (4) obtaining, and optionally, isolating said antibody or fragment thereof, 
   wherein said monoclonal antibody or fragment thereof is directed against RSV.   
     
     
         4 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 3 , wherein the method comprises the step of: increasing the concentration of memory B lymphocytes in said population of cells comprising memory B lymphocytes, or depleting undesired cells from said population of cells comprising memory B lymphocytes prior to the step of incubating or pulsing said population of cells with EBV. 
     
     
         5 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 2 , wherein said nucleic acid or nucleic acid sequence is manipulated before said inserting step to introduce restriction sites, to change codon usage, or optimize transcription or translation regulatory sequences. 
     
     
         6 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 3 , wherein said nucleic acid or nucleic acid sequence is manipulated before said inserting step to introduce restriction sites, to change codon usage, or optimize transcription or translation regulatory sequences. 
     
     
         7 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 1 , wherein said Pattern Recognition Receptor is a Toll Like Receptor (“TLR”) that is expressed on memory B cells. 
     
     
         8 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 2 , wherein said Pattern Recognition Receptor is a Toll Like Receptor (“TLR”) that is expressed on memory B cells. 
     
     
         9 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 3 , wherein said Pattern Recognition Receptor is a Toll Like Receptor (“TLR”) that is expressed on memory B cells. 
     
     
         10 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 4 , wherein said Pattern Recognition Receptor is a Toll Like Receptor (“TLR”) that is expressed on memory B cells. 
     
     
         11 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 1 , wherein said Pattern Recognition Receptor is Toll Like Receptor 7 (TLR7), Toll Like Receptor 9 (TLR9) and/or Toll Like Receptor 10 (TLR10). 
     
     
         12 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 2 , wherein said Pattern Recognition Receptor is Toll Like Receptor 7 (TLR7), Toll Like Receptor 9 (TLR9) and/or Toll Like Receptor 10 (TLR10). 
     
     
         13 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 3 , wherein said Pattern Recognition Receptor is Toll Like Receptor 7 (TLR7), Toll Like Receptor 9 (TLR9) and/or Toll Like Receptor 10 (TLR10). 
     
     
         14 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 4 , wherein said Pattern Recognition Receptor is Toll Like Receptor 7 (TLR7), Toll Like Receptor 9 (TLR9) and/or Toll Like Receptor 10 (TLR10). 
     
     
         15 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 1 , wherein said agonist of said Pattern Recognition Receptor is selected from the group consisting of: CpG oligodeoxynucleotides that stimulate TLR9; imiquimod, R-848, and other imidazoquinoline compounds that stimulate TLR7 and TLR8; loxoribine, 7-thia-8-oxoguanosine, 7-deazaguanosine that stimulate TLR7 and TLR8; monoclonal antibodies that mimic the effects of these activators and other synthetic compounds that trigger TLR7, TLR8 or TLR9. 
     
     
         16 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 2 , wherein said agonist of said Pattern Recognition Receptor is selected from the group consisting of: CpG oligodeoxynucleotides that stimulate TLR9; imiquimod, R-848, and other imidazoquinoline compounds that stimulate TLR7 and TLR8; loxoribine, 7-thia-8-oxoguanosine, 7-deazaguanosine that stimulate TLR7 and TLR8; monoclonal antibodies that mimic the effects of these activators and other synthetic compounds that trigger TLR7, TLR8 or TLR9. 
     
     
         17 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 3 , wherein said agonist of said Pattern Recognition Receptor is selected from the group consisting of: CpG oligodeoxynucleotides that stimulate TLR9; imiquimod, R-848, and other imidazoquinoline compounds that stimulate TLR7 and TLR8; loxoribine, 7-thia-8-oxoguanosine, 7-deazaguanosine that stimulate TLR7 and TLR8; monoclonal antibodies that mimic the effects of these activators and other synthetic compounds that trigger TLR7, TLR8 or TLR9. 
     
     
         18 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 4 , wherein said agonist of said Pattern Recognition Receptor is selected from the group consisting of: CpG oligodeoxynucleotides that stimulate TLR9; imiquimod, R-848, and other imidazoquinoline compounds that stimulate TLR7 and TLR8; loxoribine, 7-thia-8-oxoguanosine, 7-deazaguanosine that stimulate TLR7 and TLR8; monoclonal antibodies that mimic the effects of these activators and other synthetic compounds that trigger TLR7, TLR8 or TLR9. 
     
     
         19 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 1 , wherein the agonist of said Pattern Recognition Receptor is CpG 2006. 
     
     
         20 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 2 , wherein the agonist of said Pattern Recognition Receptor is CpG 2006. 
     
     
         21 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 3 , wherein the agonist of said Pattern Recognition Receptor is CpG 2006. 
     
     
         22 . The monoclonal antibody, or an antigen binding fragment thereof, of  claim 4 , wherein the agonist of said Pattern Recognition Receptor is CpG 2006. 
     
     
         23 . A pharmaceutical composition comprising the monoclonal antibody, or an antigen binding fragment thereof, of  claim 1 , and a pharmaceutically acceptable excipient. 
     
     
         24 . A pharmaceutical composition comprising the monoclonal antibody, or an antigen binding fragment thereof, of  claim 2 , and a pharmaceutically acceptable excipient. 
     
     
         25 . A pharmaceutical composition comprising the monoclonal antibody, or an antigen binding fragment thereof, of  claim 3 , and a pharmaceutically acceptable excipient. 
     
     
         26 . A pharmaceutical composition comprising the monoclonal antibody, or an antigen binding fragment thereof, of  claim 4 , and a pharmaceutically acceptable excipient.