GENERATION OF ANTIBODIES TO AN EPITOPE OF INTEREST THAT CONTAINS A PHOSPHOMIMETIC AMINO ACID
The invention provides methods of obtaining antibodies to an epitope of interest based on an anti-phosphoamino acid-focused library.
1 . A method of obtaining an antibody to an epitope of interest containing an aspartic acid or glutamic acid, the method comprising:
(a) obtaining an anti-phosphoamino acid-focused antibody library generated from a reference antibody, wherein the members of the library retain at least one minimal essential binding specificity determinant of a CDR from the reference antibody V H or V L ;
(b) screening the anti-phosphoamino acid-focused antibody library with a phosphoamino acid-containing epitope, wherein the epitope of interest has a phosphoamino acid substituted for the glutamic acid or for the aspartic acid;
(c) identifying members of the anti-phosphoamino acid-focused antibody library that exhibit better binding to the phosphoamino acid-containing epitope in comparison to the reference antibody, which defines a sublibrary of the anti-phosphoamino acid-focused library;
(d) screening the sublibrary of step (c) with the epitope of interest that has the glutamic acid or aspartic acid; and
(e) selecting an antibody from screening step (d) that binds to the epitope of interest.
2 . The method of claim 1 , wherein step (b) further comprises screening the anti-phosphoamino acid-focused antibody library with a phosphoamino acid comparator to identify members that exhibit better binding to the phosphamino acid-containing epitope than to the phosphamino acid comparator.
3 . The method of claim 1 , wherein the members of the anti-phosphoamino acid-focused library retain a minimal essential binding specificity determinant from the reference antibody CDR3 V H or V L region.
4 . The method of claim 1 , wherein the members of the anti-phosphoamino acid-focused library retain a minimal essential binding specificity determinant from the reference antibody heavy chain CDR3 and a minimal essential binding specificity determinant from the reference antibody light chain CDR3.
5 . The method of claim 4 , wherein the members of the anti-phosphoamino acid-focused library retain the reference antibody heavy chain CDR3 and the reference antibody light chain CDR3.
6 . The method of claim 1 , wherein the phosphoamino acid is phosphoserine or phosphothreonine.
7 . The method of claim 1 , wherein the aspartic acid or glutamic acid is a naturally occurring amino acid in the amino acid sequence of the epitope of interest.
8 . A method of obtaining an antibody to an epitope of interest containing an aspartic acid or glutamic acid, the method comprising:
(a) obtaining an anti-phosphoamino acid-focused antibody library generated from a reference antibody, wherein the members of the library retain at least one minimal essential binding specificity determinant of a CDR from the reference antibody V H or V L ;
(b) screening the anti-phosphoamino acid-focused antibody library with a phosphoamino acid-containing epitope, wherein the epitope of interest has a phosphoamino acid substituted for the glutamic acid or for the aspartic acid;
(c) identifying members of the anti-phosphoamino acid-focused antibody library that exhibit better binding to the phosphoamino acid-containing epitope in comparison to the reference antibody, which defines a sublibrary of the anti-phosphoamino acid-focused library;
(d) selecting one of the V regions of an antibody chain of an antibody identified in step (c) 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 identified in step (c);
(e) pairing the V region library of step (d) with a complementary V region, or a diverse library of complementary V regions, wherein the complementary V region or the diverse library of complementary V regions comprise an MEBSD from the reference antibody;
(f) screening the library of step (e) with the epitope of interest that has the glutamic acid or aspartic acid; and
(g) selecting an antibody that binds to the epitope wherein the antibody comprises an engineered V region.
9 . The method of claim 8 , wherein step (b) further comprises screening the anti-phosphoamino acid-focused antibody library with a phosphoamino acid comparator to identify members that exhibit better binding to the phosphamino acid-containing epitope than to the phosphamino acid comparator.
10 . The method of claim 8 , wherein the diverse library of complementary V regions in step (e) comprises members that have at least one exchange cassette exchanged with corresponding cassettes that have diverse sequences.
11 . The method of claim 8 , wherein the selected V region of step (d) is a heavy chain V region.
12 . The method of claim 8 , wherein the cassette that is exchanged in step (d) is a CDR3-FR4 cassette.
13 . The method of claim 8 , wherein the members of the anti-phosphoamino acid-focused library retain a minimal essential binding specificity determinant from the reference antibody CDR3 V H or V L region.
14 . The method of claim 8 , wherein the members of the anti-phosphoamino acid-focused library retain a minimal essential binding specificity determinant from the reference antibody heavy chain CDR3 and a minimal essential binding specificity determinant from the reference antibody light chain CDR3.
15 . The method of claim 14 , wherein the members of the anti-phosphoamino acid-focused library retain the reference antibody heavy chain CDR3 and the reference antibody light chain CDR3.
16 . The method of claim 8 , wherein the phosphoamino acid is phosphoserine or phosphothreonine.
17 . The method of claim 8 , wherein the aspartic acid or glutamic acid is a naturally occurring amino acid in the amino acid sequence of the epitope of interest.
18 . A method of obtaining an antibody to an epitope of interest containing an aspartic acid or glutamic acid, the method comprising:
(a) obtaining an anti-phosphoamino acid-focused antibody library generated from a reference antibody, wherein the members of the library retain at least one minimal essential binding specificity determinant of a CDR from the reference antibody V H or V L ;
(b) screening the anti-phosphoamino acid-focused antibody library with a phosphoamino acid-containing epitope, wherein the epitope of interest has a phosphoamino acid substituted for the glutamic acid or for the aspartic acid;
(c) identifying members of the anti-phosphoamino acid-focused antibody library that exhibit better binding to the phosphoamino acid-containing epitope in comparison to the reference antibody, which defines a sublibrary of the anti-phosphoamino acid-focused library;
(d) selecting one of the V regions of an antibody chain of an antibody identified in step (c) and pairing the V region with a diverse library of complementary V regions to form a library of antibodies;
(e) screening the library of step (d) with the epitope of interest that has the glutamic acid or aspartic acid; and
(f) selecting an antibody that binds to the epitope of interest.
19 . The method of claim 18 , wherein step (b) further comprises screening the anti-phosphoamino acid-focused antibody library with a phosphoamino acid comparator to identify members that exhibit better binding to the phosphamino acid-containing epitope than to the phosphamino acid comparator.
20 . The method of claim 18 , wherein the V region selected in step (d) is a V H region.
21 . The method of claim 18 , wherein the members of the anti-phosphoamino acid-focused library retain a minimal essential binding specificity determinant from the reference antibody CDR3 V H or V L region.
22 . The method of claim 18 , wherein the members of the anti-phosphoamino acid-focused library retain a minimal essential binding specificity determinant from the reference antibody heavy chain CDR3 and a minimal essential binding specificity determinant from the reference antibody light chain CDR3.
23 . The method of claim 22 , wherein the members of the anti-phosphoamino acid-focused library retain the reference antibody heavy chain CDR3 and the reference antibody light chain CDR3.
24 . The method of claim 18 , wherein the phosphoamino acid is phosphoserine or phosphothreonine.
25 . The method of claim 18 , wherein the aspartic acid or glutamic acid is a naturally occurring amino acid in the amino acid sequence of the epitope of interest.
26 . The method of claim 1 , wherein the anti-phosphoamino acid focused library comprises binding members that:
bind to the phosphoamino acid to which the reference antibody binds and comprise at least one heavy chain CDR minimal essential binding specificity determinant from the reference anti-phosphoamino acid antibody and at least one light chain CDR minimal essential binding specificity determinant from the reference anti-phosphoamino acid antibody; and have at least one exchange cassette for which members of the library comprise corresponding cassettes that have different sequences.
27 . A method of obtaining an antibody to an epitope of interest, the method comprising:
(a) immunizing an animal with a peptide antigen epitope of interest in which a phosphoamino acid has been substituted for a naturally occurring glutamic acid or aspartic acid;
(b) isolating a monoclonal antibody that (i) binds the phosphoamino acid-containing peptide antigen and (ii) binds the epitope of interest that comprises the aspartic acid or glutamic acid.