Method of isolating human antibodies
Provided is a novel method of isolating and producing human antibodies with desired specificity from human B cells. In particular, a method of isolating human antibodies from memory B cells obtained from patients which suffer from a disease which is caused by or involves activation of the immune system, for example autoimmune and inflammatory disorders is described.
1. A method of producing a human antibody or binding fragment thereof with desired specificity characterized by isolating B cells from short term oligoclonal cultures of activated B cells that secrete antibodies of IgG isotype comprising:
(a) selecting memory B cells that express antibodies against a protein of interest from one or more biological samples derived from a subject suffering from an autoimmune disease, whose sera is screened for the presence of auto-antibodies against a protein of interest, on the basis of the expression of at least one of CD22 cell-surface membrane marker and antigen binding;
(b) stimulating the selected cells for about three to five hours with a first polyclonal B cell activator which is Epstein-Barr virus (EBV) under cell culture conditions and in the absence of a second polyclonal B cell activator;
(c) separating the cells from said activator;
(d) activating the stimulated cells with a second polyclonal B cell activator which is a CpG-based oligonucleotide under cell culture conditions; and,
(e) screening the activated cells to identify cells that express IgG isotype antibodies of interest, wherein the culture conditions of step (d) do not comprise a cytokine.
2. The method of claim 1 , wherein the culture conditions in step (b) do not comprise a cytokine.
3. The method of claim 1 , wherein in step (c) the first polyclonal activator is removed by diluting or washing out.
4. The method of claim 1 , wherein in step (d) the transferred new selected cells are exposed to the second polyclonal activator for about eight to fourteen days.
5. The method of claim 1 , wherein in at least one of step (d) and (e) the cells are cultured under oligoclonal conditions with about ten cells per well in eight to fourteen day cultures.
6. The method of claim 1 , wherein the method comprises
(i) obtaining mRNA from bulk or single B cells sorted from short term oligoclonal cultures of activated B cells secreting the antibody of the invention;
(ii) obtaining cDNA from the mRNA of step (i);
(iii) using a primer extension reaction to amplify from said cDNA the gene repertoire corresponding to the heavy chains (HC) and the light chains (LC) and optionally constant domain of said antibodies;
(iv) using said repertoire to express said antibody or an antigen-binding fragment thereof in a host cell;
(v) identifying the antibody clone presumably responsible for the reactivity of the parental B cell culture; and
(vi) isolating the monoclonal antibody or an antigen-binding fragment thereof.
7. The method of claim 6 , wherein the host cell is an animal cell.
8. The method of claim 6 , wherein the DNA is manipulated between steps (iii) and (iv) in a manner selected from the group consisting of introducing at least one restriction site, changing codon usage, introducing coding sequences for functional domains or peptide linkers; adding or optimizing transcription or translation regulatory sequences, and combinations thereof.
9. The method of claim 1 , wherein the protein of interest is selected from the group consisting of extracellular proteins, polysaccharides, lipopolyproteins and lipopolysaccharides, which are secreted, associated or attached to a membrane or transmembranous.
10. The method of claim 9 , wherein the antigen is selected from the group consisting of leukotrienes, lymphokines, cytokines, interleukins, interferons and chemokines.
11. The method of claim 1 further comprising the step of admixing the isolated monoclonal antibody or antigen binding fragment thereof with a pharmaceutically acceptable carrier.
12. The method of claim 1 , wherein the antibody or binding fragment thereof is attached to a functional domain or drug, or a detectable label selected from the group consisting of an enzyme, radioisotope, a fluorophore and a heavy metal.
13. The method of claim 1 , wherein the B cells are depleted in IgM and IgD isotypes.
14. The method of claim 1 , further comprising:
single-cell harvesting of oligoclonal cultures producing the antibody of interest; and,
sequencing or cloning the cDNA of at least one of the variable light, the heavy chain regions and the constant region of the antibody of interest.