Anti-cleaved icaspase substrate antibodies and methods of use

An antibody that binds to a cleaved inflammatory caspase (iCaspase) substrate, wherein the antibody specifically binds to a peptide comprising the amino acid sequence P4-P3-P2-D at the C-terminus of the peptide, wherein the antibody does not bind to peptides comprising the amino acid sequence D-X-X-D or E-X-X-D at the C-terminus, wherein X is any amino acid,

• wherein the antibody comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the antibody comprises:
• a) a CDRH1, a CDRH2, and a CDRH3 of a VH chain comprising the amino acid sequence set forth in SEQ ID NO:
• 1 and a CDRL1, CDRL2, and CDRL3 of a VL chain comprising the amino acid sequence set forth in SEQ ID NO:
• 2, or
• b) a CDRH1, a CDRH2, and a CDRH3 of a VH chain comprising the amino acid sequence set forth in SEQ ID NO:
• 9 and a CDRL1, CDRL2, and CDRL3 of a VL chain comprising the amino acid sequences set forth in SEQ ID NO:
• 10.

A method of screening for an antibody that binds to a cleaved iCaspase substrate, wherein the antibody specifically binds to a peptide comprising the amino acid sequence P4-P3-P2-D at the C-terminus of the peptide, wherein the antibody does not bind to peptides comprising the amino acid sequence D-X-X-D at the C-terminus, wherein X is any amino acid, comprising

• i) providing an antibody library;
• ii) positively selecting antibodies that bind to a peptide comprising the amino acid sequence P4-P3-P2-D motif at the C-terminus;
• and
• iii) negatively selecting antibodies that bind to a peptide comprising the amino acid sequence D-X-X-D at the C-terminus,
• thereby producing an antibody that specifically binds to a peptide comprising the amino acid P4-P3-P2-D at the C-terminus, and does not bind to peptides comprising the amino acid sequence D-X-X-D at the C-terminus.

A method of enriching cleaved iCaspase substrates in a sample comprising a mixture of polypeptides

• i) contacting the sample with an anti-cleaved iCaspase substrate antibody;
• and
• ii) selecting antibody-bound polypeptides from the sample,
• wherein the anti-cleaved iCaspase substrate antibody specifically binds to a peptide comprising the amino acid sequence P4-P3-P2-D at the C-terminus of the peptide, wherein the antibody does not bind to peptides comprising the amino acid sequence D-X-X-D or E-X-X-D at the C-terminus, wherein X is any amino acid.