Allergen-binding IgE monoclonal antibodies and method for preparing hypoallergens
The present invention relates to human IgE antibodies and derivatives thereof, which bind non-continuous planar allergenic epitope, such as in β-lactoglobulin, with high affinity and specificity. The present invention also relates to processes for making and engineering such allergen binding monoclonal antibodies and to methods for using these antibodies and derivatives thereof in the field of immunodiagnostics and immunotherapy.
1. A method for selecting a modified β-lactoglobulin as a hypoallergen candidate, the method comprising the steps of:
(a) modifying an unmodified nucleic acid sequence encoding unmodified β-lactoglobulin represented by SEQ ID NO: 8 to produce a modified nucleic acid sequence encoding a modified β-lactoglobulin, wherein the modified β-lactoglobulin comprises an amino acid mutation in 1-5 amino acids in one or more of the following amino acid regions of SEQ ID NO:8: 18-20, 42-47, 55-59, 65-70, 125-127, and 154-161;
(b) expressing or producing the modified β-lactoglobulin from the modified nucleic acid sequence;
(c) contacting the modified β-lactoglobulin with an IgE antibody specific for unmodified β-lactoglobulin;
(d) comparing binding affinity of the modified β-lactoglobulin to the IgE antibody to binding affinity of unmodified β-lactoglobulin to the IgE antibody; and
(e) selecting the modified β-lactoglobulin as a hypoallergen candidate if the binding affinity of the modified β-lactoglobulin to the IgE antibody is decreased by at least tenfold compared to the binding affinity of the IgE antibody to the unmodified β-lactoglobulin.
2. A method for producing a modified allergenic polypeptide, the method comprising the steps of:
(a) modifying an unmodified nucleic acid sequence encoding an unmodified β-lactoglobulin polypeptide (SEQ ID NO:8) to produce a modified nucleic acid sequence encoding a modified β-lactoglobulin polypeptide wherein the modified β-lactoglobulin polypeptide comprises a T18Y mutation or a T18Y/E45Y/L57Y mutation; and
(b) expressing or producing the modified β-lactoglobulin polypeptide from the modified nucleic acid.
3. A method according to claim 1 , wherein the modified β-lactoglobulin comprises an amino acid mutation in at least amino acid region 18-20.
4. A method according to claim 1 , wherein the modified β-lactoglobulin comprises an amino acid mutation in at least amino acid region 42-47.
5. A method according to claim 1 , wherein the modified β-lactoglobulin comprises an amino acid mutation in at least amino acid region 55-59.
6. A method according to claim 1 , wherein the modified β-lactoglobulin comprises an amino acid mutation in at least amino acid region 65-70.
7. A method according to claim 1 , wherein the modified β-lactoglobulin comprises an amino acid mutation in at least amino acid region 154-161.