Synthetic antibodies to BAX and uses thereof
Synthetic fragment antigen-binding (Fab) antibodies are disclosed that bind to an N-terminal activation site of BCL-2-associated X-protein (BAX) and inhibit BAX activation. Also disclosed are methods of using the Fabs for measuring inactive monomeric BAX levels, screening for small molecules that bind to an N-terminal activation site of BAX, inhibiting apoptotic cell death, and predicting the ability of a cancer therapy to promote apoptotic cell death.
1. A synthetic fragment antigen-binding (Fab) antibody that binds to an N-terminal activation site of BCL-2-associated X-protein (BAX) and inhibits BAX activation, wherein the synthetic fragment antigen-binding (Fab) antibody is selected from the group consisting of
3E8 Fab, having a light chain comprising a CDR L1, a CDRL2, and a CDRL3 region comprising the amino acid sequence QSSYSLI (SEQ ID NO: 5), a CDRH1 region comprising the amino sequence LSYYSM (SEQ ID NO: 6), a CDRH2 region comprising the amino acid sequence SISPYYGYTY (SEQ ID NO: 7), and a CDRH3 region comprising the amino acid sequence RGGAYYFGYYGSGSYAMD (SEQ ID NO: 8);
3G9 Fab, having a light chain comprising a CDR L1, a CDRL2, and a CDRL3 region comprising the amino acid sequence QHYYYSPWPI (SEQ ID NO: 17), a CDRH1 region comprising the amino sequence LYSYYI (SEQ ID NO: 18), a CDRH2 region comprising the amino acid sequence SISPYYSSTY (SEQ ID NO: 19), and a CDRH3 region comprising the amino acid sequence RSSYSYAGMD (SEQ ID NO: 20); and
2C11 Fab, having a light chain comprising a CDR L1, a CDRL2, and a CDRL3 region comprising the amino acid sequence QSYVSPI (SEQ ID NO: 21), a CDRH1 region comprising the amino sequence ISSYYI (SEQ ID NO: 22), a CDRH2 region comprising the amino acid sequence SISSYYSSTY (SEQ ID NO: 23), and a CDRH 3 region comprising the amino acid sequence RVSYGHAYVGYSSGMD (SEQ ID NO: 24).
2. The synthetic Fab antibody of claim 1 , wherein the antibody maintains BAX in its inactive, monomeric form.
3. The synthetic Fab antibody of claim 1 , wherein the antibody binds to residues of helices α1 and α6, and to residues of α1-α2 loop of BAX.
4. The synthetic Fab antibody of claim 1 , wherein the antibody binds to BAX with a half-maximal binding (EC 50 ) affinity of 2nM-70nM.
5. The synthetic Fab antibody of claim 1 , wherein the antibody blocks the interaction of BAX with a BAX activating partner.
6. The synthetic Fab antibody of claim 5 , wherein the BAX activating partner is selected from the group consisting of tBID, PUMA, BIM and NOXA.
7. The synthetic Fab antibody of claim 1 , wherein the synthetic Fab antibody is conjugated to a therapeutic agent.
8. The synthetic Fab antibody of claim 1 , wherein the synthetic Fab antibody is conjugated to an agent that facilitates transport across a cell membrane.
9. The synthetic Fab antibody of claim 1 , wherein the synthetic Fab antibody is labeled with a fluorescent label or a radioactive label.