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:
3H4 Fab, having a light chain comprising a CDR L1, a CDRL2, and a CDRL3 region comprising the amino acid sequence of SEQ.ID.No:69, wherein the CDRL3 region comprises QSWYYSYPI (SEQ ID NO: 25), and a heavy chain CDRH1 region comprising the amino sequence LSYSSM (SEQ ID NO: 26), a CDRH2 region comprising the amino acid sequence SISSYYSYTS (SEQ ID NO: 27), and a CDRH3 region comprising the amino acid sequence RYYGYGGGID (SEQ ID NO: 28);
2A6 Fab, having a light chain comprising a CDR L1, a CDRL2, and a CDRL3 region comprising the amino acid sequence of SEQ.ID.NO:75, wherein the CDRL3 region comprises QSAGGYPLI (SEQ ID NO: 29), and a heavy chain CDRH1 region comprising the amino sequence IYYSSM (SEQ ID NO: 30), a CDRH2 region comprising the amino acid sequence SISPYSSYTS (SEQ ID NO: 31), and a CDRH3 region comprising the amino acid sequence RSFGYGWAFD (SEQ ID NO: 32);
3H1 Fab, having a light chain comprising a CDR L1, a CDRL2, and a CDRL3 region comprising the amino acid sequence of SEQ.ID.NO:67, wherein the CDRL3 region comprises QHSYPI (SEQ ID NO: 33), and a heavy chain CDRHI region comprising the amino sequence ISYSSI (SEQ ID NO: 34), a CDRH2 region comprising the amino acid sequence SIYSYSGSTY (SEQ ID NO: 35), and a CDRH3 region comprising the amino acid sequence RYGAMD (SEQ ID NO: 36).
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 2 nM-70 nM.
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.