Compositions and methods for cleaving IAP
OwnerChunying Du, Qiheng Yanginventors
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Abstract
The present invention relates to compositions and methods for making and using Omi-related and IAP-cleaving nucleotide sequences, mutant nucleotide sequences, and polypeptide sequences expressed therefrom, including both biologically active and inactive molecules. The present invention relates to cleaving IAP using an Omi polypeptide.
Claims86
A method for cleaving IAP, wherein the method comprises:
- contacting in vitro, isolated IAP with an amount of an isolated Omi family polypeptide, whereby upon contact, the Omi family polypeptide will cleave the IAP.
A method for cleaving IAP, wherein the method comprises:
- (a) isolating a population of cells, whereby the caspase found in the cells is bound by IAP;
- (b) contacting in vitro the isolated cell population with the Omi family polypeptide whereby upon contact, the Omi family polypeptide cleaves the IAP from the caspase.
A method for cleaving IAP comprising:
- (a) isolating and amplifying an Omi family member gene;
- (b) forming an Omi expression construct from the isolated and amplified Omi family member gene;
- (c) transfecting a cell population having caspase bound by IAP, with the Omi family member construct;
- and,
- (d) causing expression of the Omi vector, whereby the OMI family polypeptide cleaves IAP.
A method for cleaving LAP, wherein the method comprises:
- contacting isolated LAP with an amount of an isolated Omi family polypeptide, whereby upon contact, the Omi family polypeptide will cleave the IAP.
A polypeptide for cleaving IAP selected from the group consisting of Omi WT, OmiΔPDZ, OmiΔAVPS, Omi protease, Omi catalytic triad, and homologs thereof.
An isolated polypeptide for cleaving IAP selected from the group consisting of SEQ ID NOs. 44, 45, 48, 49, 54-57, 60-63, 66-75, and homologs thereof.
A polypeptide for cleaving IAP comprising a protease domain selected from the group consisting of SEQ ID NOs. 64-66 and 75-80, and homologs thereof.
A polypeptide having increased protease activity comprising a polypeptide selected from the group consisting of SEQ ID NOs. 48, 49, 55, 56, 57, 60-63, 66-75, and homologs thereof.
A polypeptide for binding to a BIR site on an IAP, comprising SEQ ID NO. 77 and homologs thereof.
A polypeptide for cleaving IAP comprising a polypeptide selected from the group consisting of SEQ ID NOs. 44 and 45, and homologs thereof.
A polypeptide which binds to IAP, but does not cleave IAP, selected from the group consisting of SEQ ID NOs. 46, 47, 50, 51, 58, 59, 64, 65, and homologs thereof.
A polypeptide which binds IAP but does not cleave LAP, comprising Omi SA.
An Omi serine protease for use in cleaving IAP.
An OmiΔPDZ for cleaving IAP.
A polypeptide molecule for cleaving an IAP comprising an amino acid sequence as set forth in C1n1-R1-C2n2-R2-C3n3-R3-C4n4, wherein:
- (a) R1 is a serine;
- (b) R2 is an amino acid residue selected from a group consisting of charged amino acid residues and aromatic amino acid residues;
- (c) R3 is an amino acid residue selected from a group consisting of charged amino acid residues and polar amino acid residues;
- and,
- (d) R1, R2 and R3 form a catalytic triad for cleavage of the IAP.
A polypeptide molecule comprising an amino acid sequence as set forth in SEQ ID NOs. 54 and 55.
A serine protease polypeptide molecule wherein the molecule is of the formula comprising C1n1-R1-C2n2-R2-C3n3-R3-C4n4, wherein:
- (a) R1 is a serine;
- (b) R2 is an amino acid residue selected from a group consisting of a charged amino acid residue and an aromatic amino acid residue;
- (c) R3 is an amino acid residue selected from a group consisting of a charged amino acid residue and a polar amino acid residue;
- (d) C1n1, C2n2, C3n3, and C4n4 are polypeptide chains;
- (e) n1 is an amino acid residue number ranging between 10 and 100;
- (f) n2 is an amino acid residue number ranging between 10 and 100;
- (g) n3 is an amino acid residue number ranging between 10 and 150;
- (h) n4 is an amino acid residue number ranging between 10 and 200;
- and,
- (i) R1, R2 and R3 form a catalytic triad for cleavage of a polypeptide substrate.
A polypeptide molecule comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 46, 47, and homologs thereof.
A polypeptide molecule comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 48, 49 and homologs thereof.
A polypeptide molecule comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 56, 57 and homologs thereof.
A polypeptide molecule comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 60 through 63 and homologs thereof.
A polypeptide molecule for inhibiting IAP cleavage comprising an amino acid sequence as set forth in C1n1-R1-C2n2-R2-C3n3-R3-C4n4, wherein:
- (a) R1 is an amino acid residue selected from a group consisting of an alanine, an arginine, an aspartic acid, an asparagine, a cysteine, a glutamic acid, a glutamine, a glycine, a histidine, an isoleucine, a leucine, a lysine, a methionine, a phenylalanine, a proline, a threonine, a tryptophan, a tyrosine, and a valine;
- (b) R2 is a histidine;
- (c) R3 is an aspartic acid;
- and,
- (d) an AVPS moiety binds to IP.
An OmiΔPDZ expressed intra-cellularly by an OmiΔPDZ vector in a cell.
A nucleic acid sequence, which expresses a polypeptide which cleaves IAP, wherein the nucleic acid sequence is selected from the group consisting of Omi family member nucleic acid sequences, degenerate variants of the Omi family member, and homologous sequences to the Omi family member.
A nucleic acid sequence which expresses isolated polypeptide for cleaving IAP, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NOs. 1-43, and homologos sequences thereof.
A nucleic acid sequence which expresses a polypeptide for cleaving IAP, wherein the nucleic acid sequence expresses a protease domain, selected from the group consisting of SEQ ID NOs. 44, 45, 48 and 49, and homologos sequences thereof.
A nucleic acid sequence which expresses a polypeptide having increased protease activity comprising a polypeptide selected from the group consisting of SEQ ID NOs. 48, 49, 50, 56, 57, 58, 59, 60, 61, and homologos sequences thereof.
A nucleic acid sequence which expresses a polypeptide for binding to a BIR site on an IAP, comprising SEQ ID NO. 82 and homologos sequences thereof.
A nucleic acid sequence which expresses a polypeptide for cleaving IAP comprising a polypeptide selected from the group consisting of SEQ ID NOs. 44, 45, and homologous sequences thereof.
A nucleic acid sequence, which expresses a polypeptide, which binds to LAP, but does not cleave LAP, selected from the group consisting of SEQ ID NOs. 4, 5, 9, 10, 20, 21, 28, and 29, and homologs thereof.
A nucleic acid sequence which expresses a polypeptide which binds IAP but does not cleave IAP, comprising Omi SA.
An expression vector comprising a nucleic acid that expresses a molecule for cleaving IAP selected from a group consisting of SEQ ID NOs. 1-43, and homologous sequences thereof.
An Omi expression vector, comprising an Omi family nucleic acid sequence and a vector selected from the group consisting of eukaryotic vectors MSCV, Harvey murine sarcoma virus, pFastBac, pFastBac HT, pFastBac DUAL, pSFV, pTet-Splice, pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, YACneo, pSVK3, pSVL, pMSG, pCH110, pKK232-8, p3'SS, pBlueBacIII, pCDM8, pcDNA1, pZeoSV, pcDNA3, pREP4, pET21b, pCEP4, and pEBVHis vectors.
A transfected cell comprising:
- (a) an expression vector that expresses an Omi family member polypeptide;
- and,
- (b) a promoter.
A pharmaceutical composition for treatment of a hyperproliferative disorder in an animal which comprises a pharmacologically acceptable carrier and a therapeutically effective amount of the liposomes containing Omi family member polypeptides of SEQ ID NOs. 44, 45, 48, 49, 52-57, 60-63, and 66-75.
A hybridization kit for detecting an Omi wild-type gene, wherein the kit comprises:
- (a) a container;
- and,
- (b) a nucleic acid molecule comprising a nucleotide molecule selected from a group consisting of Omi family nucleic acid sequences.
A hybridization kit for detecting an Omi mutant gene, wherein the kit comprises:
- (a) a container;
- and,
- (b) a nucleic acid molecule comprising a molecule selected from a group consisting of SEQ ID NOs. 1-41, and homologous sequences thereof.
A kit for detecting an Omi gene comprising:
- (a) PCR primers spanning an Omi family gene, a positive control;
- and,
- (b) sequencing products.
A kit for detecting an Omi polypeptide, wherein the kit comprises:
- (a) a container;
- and,
- (b) an antibody derived from polypeptide selected from a group consisting of SEQ ID NOs. 44-77, and homologs thereof.
An antibody which binds to the Omi serine.
An antibody which binds to a protease.
A mammalian cell consisting essentially of:
- (a) a cell transfected by an Omi expression vector;
- (b) the transfected cell producing an IAP-cleaving molecule selected from the group consisting of said IAP-cleaving molecules;
- and,
- (c) a promoter controlling transcription and the quantity of production of said IAP-cleaving molecule.
A method of detecting and identifying an IAP-cleaving molecule comprising:
- (a) binding an antibody directed against an antigen associated with the IAP-cleavage site on said IAP-cleaving molecule to a solid phase adsorbent surface;
- (b) adding a specimen containing a plurality of unlabelled IAP-cleaving molecules;
- (c) adding a plurality of labeled IAP-cleaving molecules;
- (d) detecting the bound labeled IAP-cleaving molecules;
- and,
- (e) calculating the amount of bound unlabeled IAP-cleaving molecules.
An Omi expression vector, comprising an Omi family nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-41, and homologs thereof, and a vector selected from the group consisting of eukaryotic vectors MSCV, Harvey murine sarcoma virus, pFastBac, pFastBac HT, pFastBac DUAL, pSFV, pTet-Splice, pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, YACneo, pSVK3, pSVL, pMSG, pCH110, pKK232-8, p3'SS, pBlueBacIII, pCDM8, pcDNA1, pZeoSV, pcDNA3, pREP4, pET21b, pCEP4, and pEBVHis vectors.
Dependencies are inferred from the claim text.