Engineered antibodies for the detection of phosphorylated tyrosine
View Patent ↗Presented herein are novel phosphotyrosine binding compositions, including antibodies and antibody fragments. The inventors of the present disclosure have resolved the crystal structures of two widely utilized pan-specific pY antibodies, PY20 and 4G10. These two known antibodies, although developed independently from animal immunizations, have surprisingly similar modes of recognition of the phosphate group, and revealed a generic binding structure among pan-specific pY antibodies. Based on this newly discovered convergent structure, engineered CDR-L3 loops were developed that impart greatly improved affinity to pY binding antibodies and other pY binding compositions. The inventions disclosed herein include antibodies and antibody fragments bearing these novel CDR-L3 sequences and methods of using such pY binding compositions for the detection of phosphotyrosine-bearing proteins.
1. A phosphotyrosine-binding composition, comprising
an antibody, or phosphotyrosine-binding fragment thereof, wherein the antibody or antibody fragments comprises:
a body scaffold comprising heavy and light chain sequences;
a CDR L1 sequence comprising SEQ ID NO: 24;
a CDR L2 sequence comprising SEQ ID NO: 25;
a CDR H1 sequence comprising SEQ ID NO: 26;
a CDR H2 sequence comprising SEQ ID NO: 27;
a CDR H3 sequence comprising SEQ ID NO: 28; and
a CDR L3 sequence comprising
a sequence selected from the group consisting of: SEQ ID NO: 6-SEQ ID NO: 23.
2. The phosphotyrosine binding composition of claim 1 , wherein
the antibody or fragment thereof comprises a whole antibody, a Fab, a Fab′, a F(ab) 2 , or a scFv.
3. The phosphotyrosine binding composition of claim 1 , wherein
the antibody or fragment thereof comprises one or more functional moieties.
4. The phosphotyrosine binding composition of claim 3 , wherein
the one or more functional moieties comprises an affinity tag, a fluorescent label, or an epitope for binding by secondary antibodies.
5. The phosphotyrosine binding composition of claim 1 , wherein
the CDR L3 sequence comprises SEQ ID NO: 15.
6. The phosphotyrosine binding composition of claim 1 , wherein
the CDR L3 sequence comprises SEQ ID NO: 22.
7. The phosphotyrosine binding composition of claim 1 , wherein
the CDR L3 sequence comprises SEQ ID NO: 10.
8. The phosphotyrosine binding composition of claim 1 , wherein
the CDR L3 sequence comprises SEQ ID NO:12.
9. The phosphotyrosine binding composition of claim 1 , wherein
the CDR L3 sequence comprises SEQ ID NO: 14.
10. The phosphotyrosine binding composition of claim 1 , wherein
the antibody, or phosphotyrosine binding fragment thereof, comprises:
a light chain comprising a sequence of at least 95% sequence identity to SEQ ID NO: 39, wherein the CDR L3 sequence thereof comprises a sequence selected from the group consisting of SEQ ID NO: 6-SEQ ID NO: 23; and
a heavy chain comprising a sequence of at least 95% sequence identity to SEQ ID NO: 43.
11. The phosphotyrosine binding composition of claim 10 , comprising
a light chain comprising a sequence of at least 95% sequence identity to SEQ ID NO: 40; and
a heavy chain comprising a sequence of at least 95% sequence identity to SEQ ID NO: 43.
12. The phosphotyrosine binding composition of claim 10 , comprising
a light chain comprising a sequence of at least 95% sequence identity to SEQ ID NO: 41; and
a heavy chain comprising a sequence of at least 95% sequence identity to SEQ ID NO: 43.