GENERATION OF ANTIBODIES TO AN EPITOPE OF INTEREST
The invention provides methods of obtaining antibodies to an epitope of interest based on an anti-hapten focused library.
1 . A method of obtaining an antibody to an epitope of interest, the method comprising:
(a) screening an anti-hapten focused library with a hapten-labeled epitope comprising the epitope of interest joined to a hapten;
(b) identifying members of the anti-hapten focused library that bind to the hapten-labeled epitope to generate a sublibrary of the anti-hapten focused library;
(c) screening the sublibrary of step (b) with the epitope of interest that is not attached to the hapten; and
(d) selecting an antibody that binds to the epitope, thereby obtaining an antibody to the epitope of interest.
2 . The method of claim 1 , wherein the members of the anti-hapten focused library retain a minimal essential binding specificity determinant from the reference antibody CDR1 or CDR2V H or V L region.
3 . The method of claim 1 , wherein the members of the anti-hapten focused library retain a minimal essential binding specificity determinant from the reference antibody CDR3V H or V L region.
4 . The method of claim 1 , wherein the members of the anti-hapten focused library retain a minimal essential binding specificity determinant from the reference antibody heavy chain CDR2 and a minimal essential binding specificity determinant from the reference antibody light chain CDR3.
5 . The method of claim 1 , wherein the step of screening the anti-hapten antibody library with the hapten-labeled epitope further comprises screening the anti-hapten antibody library with a hapten comparator molecule; and selecting an antibody that exhibits increased binding to the hapten-labeled epitope relative to the binding to the hapten comparator molecule.
6 . The method of claim 1 , wherein the hapten is a modified tyrosine residue.
7 . The method of claim 1 , wherein the hapten is a naturally occurring modified amino acid.
8 . The method of claim 1 , wherein the hapten is a phosphorylated amino acid.
9 . The method of claim 8 , wherein the phosphorylated amino acid is phosphotyrosine, phosphoserine, or phosphothreonine.
10 . The method of claim 9 , wherein the phosphorylated amino acid is phosphotyrosine.
11 . The method of claim 1 , wherein the hapten-labeled epitope is a peptide of from 15 to 50 amino acids in length.
12 . The method of claim 1 , wherein the hapten-labeled epitope is a protein antigen comprising the epitope of interest.
13 . The method of claim 1 , wherein about 10 5 or fewer colonies of the anti-hapten focused library are screened.
14 . The method of claim 1 , further comprising:
(e) selecting one of the V regions of the antibody selected in (d) and exchanging a cassette of the selected V region with a library of corresponding cassettes to provide a library of engineered V regions, wherein the selected V region retains at least one minimal essential binding specificity determinant of a CDR from the antibody selected in (d);
(f) pairing the V region library of step (e) with the complementary V region from the antibody selected in step (d) to form a library of antibodies;
(g) screening the library of step (f) with the epitope of interest that is not attached to the hapten; and
(h) selecting an antibody that binds to the epitope wherein the antibody comprises an engineered V region.
15 . The method of claim 14 , wherein the antibody selected in step (h) no longer binds to the hapten.
16 . The method of claim 14 , wherein the selected V region is a heavy chain V region.
17 . The method of claim 14 , wherein the at least one minimal binding specificity determinant retained is from a CDR3.
18 . The method of claim 14 , wherein the cassette that is exchanged in step (e) is a CDR3-FR4 cassette.
19 . The method of claim 14 , further comprising:
(i) selecting the engineered V region from the antibody selected in step (h) and exchanging another cassette of the engineered V region with a library of corresponding cassettes, wherein the selected V region retains at least one minimal essential binding specificity determinant from a CDR from the antibody selected in (h);
(j) pairing the V region library of step (i) with the complementary V region of the antibody selected in (h) to form an antibody library;
(k) screening the antibody library of step (j) with an epitope of interest that is not attached to the hapten; and
(l) selecting an antibody that binds to the epitope, thereby obtaining an antibody to an epitope of interest.
20 . The method of claim 1 , wherein the antibody is a Fab.
21 . The method of claim 20 , wherein the antibody is secreted.
22 . The method of claim 1 , wherein the anti-hapten focused library is a display library.
23 . The method of claim 22 , wherein the display library is a phage display library.
24 . The method of claim 1 , wherein the anti-hapten focused library that comprises binding members that:
retain the binding specificity of a reference anti-hapten antibody and comprise at least one heavy chain CDR minimal essential binding specificity determinant from the reference anti-hapten antibody and at least one light chain CDR minimal essential binding specificity determinant from the reference anti-hapten antibody; and have at least one diverse exchange cassette.