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 Barr 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 method for preparing a recombinant cell, the method comprising:
 (i) preparing an immortalized B cell clone, comprising transforming a human memory B lymphocyte using Epstein Barr virus (EBV) in the presence of a polyclonal B cell activator, wherein the polyclonal B cell activator is an agonist of a Pattern Recognition Receptor that is expressed on human memory B lymphocytes;   (ii) obtaining, from the immortalized B cell clone, a nucleic acid molecule that encodes an antibody of interest; and   (iii) inserting the nucleic acid molecule encoding the antibody of interest into an expression host cell,   thereby preparing a recombinant cell.   
     
     
         2 . The method of  claim 1 , wherein the expression host cell is a yeast cell, a plant cell, or an animal cell. 
     
     
         3 . The method of  claim 1 , wherein the nucleic acid molecule encoding the antibody of interest is modified to:
 (a) introduce one or more restriction site into the nucleic acid molecule;   (b) change usage of one or more codon in the nucleic acid molecule;   (c) introduce one or more transcription regulatory sequence into the nucleic acid molecule;   (d) introduce one or more translation regulatory sequence into the nucleic acid molecule; or   (e) any combination of (a)-(d).   
     
     
         4 . The method of  claim 1 , wherein the Pattern Recognition Receptor comprises a Toll Like Receptor (“TLR”) that is expressed on memory B cells. 
     
     
         5 . The method of  claim 4 , wherein the TLR is Toll Like Receptor 7 (TLR7), Toll Like Receptor 9 (TLR9) or Toll Like Receptor 10 (TLR10). 
     
     
         6 . The method of  claim 5 , wherein the agonist of the Pattern Recognition Receptor is CpG 2006 (SEQ ID NO:1). 
     
     
         7 . A method for preparing a nucleic acid molecule, the method comprising:
 (i) preparing an immortalized B cell clone, comprising transforming a human memory B lymphocyte using Epstein Barr virus (EBV) in the presence of a polyclonal B cell activator, wherein the polyclonal B cell activator is an agonist of a Pattern Recognition Receptor that is expressed on human memory B lymphocytes;   (ii) sequencing, from the immortalized B cell clone, a nucleic acid molecule that encodes an antibody of interest; and   (iii) using the sequence information from (ii) to prepare a nucleic acid molecule for insertion into an expression host cell in order to permit expression of the antibody of interest by the expression host cell,   thereby preparing the nucleic acid molecule.   
     
     
         8 . The method of  claim 7 , wherein the nucleic acid molecule encoding the antibody of interest is modified to:
 (a) introduce one or more restriction site into the nucleic acid molecule;   (b) change usage of one or more codon in the nucleic acid molecule;   (c) introduce one or more transcription regulatory sequence into the nucleic acid molecule;   (d) introduce one or more translation regulatory sequence into the nucleic acid molecule; or   (e) any combination of (a)-(d).   
     
     
         9 . A method for preparing a nucleic acid molecule that encodes an antibody of interest, the method comprising:
 (i) immortalizing human memory B lymphocytes, wherein the immortalizing comprises transforming human memory B lymphocytes using Epstein Barr virus (EBV) in the presence of a polyclonal B cell activator, wherein the polyclonal B cell activator is an agonist of a Pattern Recognition Receptor that is expressed on human memory B lymphocytes;   (ii) isolating an immortalized B cell clone capable of producing an antibody of interest; and   (iii) obtaining a nucleic acid molecule, from the immortalized B cell clone, that encodes the antibody of interest.   
     
     
         10 . The method of  claim 9 , further comprising:
 (iv) using the nucleic acid molecule to prepare an expression host cell capable of expressing the antibody of interest; and   (v) culturing or sub-culturing the expression host cell under conditions where the antibody of interest is expressed.   
     
     
         11 . The method of  claim 10 , further comprising:
 (vi) purifying the antibody of interest.   
     
     
         12 . The method of  claim 10 , wherein using the nucleic acid molecule to prepare the expression host cell in (iv) comprises inserting the nucleic acid molecule into the expression host cell. 
     
     
         13 . The method of  claim 10 , wherein using the nucleic acid molecule to prepare the expression host cell in (iv) comprises:
 (a) obtaining sequence information of the nucleic acid molecule encoding the antibody of interest; and   (b) using the sequence information to prepare a nucleic acid molecule encoding the antibody of interest for inserting into the expression host cell.   
     
     
         14 . The method of  claim 10 , wherein the nucleic acid molecule encoding the antibody of interest is modified to:
 (a) introduce one or more restriction site into the nucleic acid molecule;   (b) change usage of one or more codon in the nucleic acid molecule;   (c) introduce one or more transcription regulatory sequence into the nucleic acid molecule;   (d) introduce one or more translation regulatory sequence into the nucleic acid molecule; or   (e) any combination of (a)-(d).   
     
     
         15 . A method for obtaining a nucleic acid sequence that encodes an antibody of interest, the method comprising:
 (i) immortalizing human memory B lymphocytes, wherein the immortalizing comprises transforming human memory B lymphocytes using Epstein Barr virus (EBV) in the presence of a polyclonal B cell activator, wherein the polyclonal B cell activator is an agonist of a Pattern Recognition Receptor that is expressed on human memory B lymphocytes;   (ii) isolating an immortalized B cell clone capable of producing an antibody of interest; and   (ii) obtaining sequence information, of a nucleic acid molecule encoding the antibody of interest, from the immortalized B cell clone.   
     
     
         16 . The method of  claim 10 , wherein the antibody of interest is directed against respiratory syncytial virus (RSV), human immunodeficiency virus; hepatitis A virus; hepatitis B virus; hepatitis C virus; herpes simplex virus type I or type 2; SARS coronavirus; measles virus; mumps virus; rubella virus; rabies virus; ebola virus; influenza virus; papillomavirus; vaccinia virus; varicella-zoster virus; variola virus; polio virus; rhino virus; a human endogenous retrovirus;  P. falciparum; P. vivax; P. malariae; P. ovale; Corynebacterium diphtherias; Clostridium tetani; Clostridium botulinum; Bordetella pertussis; Haemophilus influenzae; Neisseria meningitidis;  serogroup A, B, C, W135 and/or Y;  Streptococcus pneumoniae; Streptococcus agalactiae; Streptococcus pyogenes; Staphylococcus aureus; Bacillus anthracis; Moraxella catarrhalis; Chlamydia trachomatis; Chlamydia pneumoniae; Yersinia pestis; Francisella tularensis; Salmonella  species;  Vibrio cholerae ; toxic  E. coli ; TNF-α, β-amyloid protein, SARS coronavirus spike protein, prion protein PrP, complement C5, CBL, CD147, IL-8, HIV glycoprotein gp120, VLA-4, CDI Ia, CD18, VEGF, CD40L, an ICAM, a VCAM, CD80, TPL2, Her2 or an integrin. 
     
     
         17 . A method of treating or preventing, in a subject, a condition characterized by expression of: serogroup A, B, C, W135 and/or Y; TNF-α; β-amyloid protein; SARS coronavirus spike protein; prion protein PrP; complement C5; CBL; CD147; IL-8; HIV glycoprotein gp120; VLA-4; CDI Ia; CD18; VEGF; CD40L; an ICAM; a VCAM; CD80; TPL2; Her2; or an integrin, the method comprising administering to the subject an antibody prepared according to the method of  claim 16 . 
     
     
         18 . A method of treating or preventing, in a subject, an infection by: respiratory syncytial virus (RSV); human immunodeficiency virus; hepatitis A virus; hepatitis B virus; hepatitis C virus; herpes simplex virus type I or type 2; SARS coronavirus; measles virus; mumps virus; rubella virus; rabies virus; ebola virus; influenza virus; papillomavirus; vaccinia virus; varicella-zoster virus; variola virus; polio virus; rhino virus; a human endogenous retrovirus;  P. falciparum; P. vivax; P. malariae; P. ovale; Corynebacterium  diphtherias;  Clostridium tetani; Clostridium botulinum; Bordetella pertussis; Haemophilus influenzae; Neisseria meningitidis; Streptococcus pneumoniae; Streptococcus agalactiae; Streptococcus pyogenes; Staphylococcus aureus; Bacillus anthracis; Moraxella catarrhalis; Chlamydia trachomatis; Chlamydia pneumoniae; Yersinia pestis; Francisella tularensis; Salmonella  species;  Vibrio cholerae ; or toxic  E. coli , the method comprising administering to the subject an antibody prepared according to the method of  claim 16 . 
     
     
         19 . A method of detecting RSV in a subject, the method comprising contacting a biological sample from the subject with an anti-RSV antibody prepared according to the method of  claim 16 , wherein the anti-RSV antibody is detectably labeled. 
     
     
         20 . A method of activating an isolated human B memory lymphocyte, the method comprising contacting an isolated human B memory lymphocyte with an agonist of a Pattern Recognition Receptor (PRR) expressed on memory B cells. 
     
     
         21 . The method of  claim 20 , wherein the PRR comprises a Toll-Like Receptor (TLR) which is expressed on memory B cells. 
     
     
         22 . The method of  claim 21 , wherein the agonist of the PRR comprises one or more of:
 (i) CpG (SEQ ID NO:1);   (ii) R-848   (iii) imidazoquinoline compounds that stimulate TLRs;   (iv) CD40L;   (v) BAFF;   (vi) a cell that expresses CD40L or BAFF;   (vi) a monoclonal antibody that mimics the effect of a polyclonal B cell activator; or   (vii) any combination of (i)-(vi).