WO2010127324A2 - Chimeric pdz domain-containing protein for viral detection - Google Patents

Chimeric pdz domain-containing protein for viral detection Download PDF

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WO2010127324A2
WO2010127324A2 PCT/US2010/033308 US2010033308W WO2010127324A2 WO 2010127324 A2 WO2010127324 A2 WO 2010127324A2 US 2010033308 W US2010033308 W US 2010033308W WO 2010127324 A2 WO2010127324 A2 WO 2010127324A2
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polypeptide
pdz
chimeric
pdz domain
domain
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PCT/US2010/033308
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French (fr)
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WO2010127324A3 (en
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Michael P. Belmares
Johannes Schweizer
Peter Lu
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Arbor Vita Corporation
Becton, Dickinson And Company
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • Cervical cancer is the second most common cancer diagnosis in women and is linked to high-risk human papillomavirus infection 99.7% of the time.
  • HPVs Human papillomaviruses
  • HPVs Human papillomaviruses
  • HPV 6 and 11 Specific human papillomavirus types, including HPV 6 and 11, frequently cause benign mucosal lesions, whereas other types such as HPV 16, 18, and a host of other strains, are predominantly found in high-grade lesions and cancer.
  • Individual types of human papillomaviruses (HPV) which infect mucosal surfaces have been implicated as the causative agents for carcinomas of the cervix, anus, penis, larynx and the buccal cavity, occasional periungal carcinomas, as well as benign anogenital warts.
  • HPV human papillomaviruses
  • the identification of particular HPV types is used for identifying patients with premalignant lesions who are at risk of progression to malignancy.
  • PCR polymerase chain reaction
  • the detection and diagnosis of disease is a prerequisite for the treatment of disease. Numerous markers and characteristics of diseases have been identified and many are used for the diagnosis of disease. Many diseases are preceded by, and are characterized by, changes in the state of the affected cells. Changes can include the expression of pathogen genes or proteins in infected cells, changes in the expression patterns of genes or proteins in affected cells, and changes in cell morphology. The detection, diagnosis, and monitoring of diseases can be aided by the accurate assessment of these changes. Inexpensive, rapid, early and accurate detection of pathogens can allow treatment and prevention of diseases that range in effect from discomfort to death.
  • the present invention provides a chimeric polypeptide comprising at least one PDZ domain or a PDZ ligand binding portion of a PDZ domain introduced from a first PDZ domain- containing polypeptide into a second PDZ domain- containing polypeptide, wherein the chimeric polypeptide has enhanced binding strength to a PDZ ligand as compared to the native second PDZ domain-containing polypeptide.
  • the first PDZ domain-containing polypeptide is MAGI-I polypeptide.
  • the introduced PDZ domain is PDZ domain 1 of MAGI-I polypeptide.
  • the introduced PDZ domain is selected from the group consisting of PDZ domains listed in Table 2. In some embodiments, the introduced PDZ domain has 70, 80, 90, 95, 96, 97, 98, or 99 % identity to a PDZ domain listed in Table 2. In some embodiments, at least one PDZ domain is introduced into the second PDZ domain- containing polypeptide. In some embodiments, the second PDZ domain-containing polypeptide is PSD95. In some embodiments, at least one PDZ domain of the second PDZ domain-containing polypeptide is substituted with the introduced PDZ domain from the first PDZ domain- containing polypeptide.
  • At least one PDZ domain of PSD95 is substituted with PDZ domain 1 of MAGI-I.
  • two PDZ domains of PSD95 protein are substituted with PDZ domain 1 of MAGI-I.
  • three PDZ domains of PSD95 protein are substituted with PDZ domain 1 of MAGI-I.
  • more than one PDZ domain e.g. 2, 3, 4, 5, 6, 7, or 8, PDZ domains are introduced into the second PDZ domain-containing polypeptide.
  • at least one PDZ domain from the first PDZ domain-containing polypeptide is recombined with non-PDZ portions of the second PDZ domain- containing polypeptide.
  • two PDZ domains from the first PDZ domain- containing polypeptide are recombined with the non-PDZ portions of PSD95.
  • three PDZ domains from the first PDZ domain-containing polypeptide are recombined with the non-PDZ portions of PSD95.
  • more than one PDZ domain e.g. 2, 3, 4, 5, 6, 7, or 8, PDZ domains are introduced into PSD95.
  • at least one PDZ domain 1 of MAGI-I is recombined with the non-PDZ portions of PSD95.
  • three PDZ domain 1 of MAGI-I are recombined with the non-PDZ portions of PSD95.
  • the chimeric polypeptide contains a PDZ ligand binding portion of a PDZ domain from the first PDZ domain-containing polypeptide. In some embodiments, the chimeric polypeptide contains a modified PDZ domain from the first PDZ domain-containing polypeptide. In some embodiments, the introduced PDZ domain is modified has 70, 80, 90, 95, 96, 97, 98, or 99 % identity to a PDZ domain from the first PDZ domain-containing polypeptide. In some embodiments, the PDZ ligand is an oncogenic protein from human papilloma virus (HPV).
  • HPV human papilloma virus
  • the oncogenic HPV strain is 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82.
  • the PDZ ligand is E6 protein from oncogenic HPV strain 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82.
  • the binding affinity of the chimeric polypeptide to a PDZ ligand is enhanced.
  • the binding avidity of the chimeric polypeptide to a PDZ ligand is enhanced.
  • the binding affinity and avidity of the chimeric polypeptide to a PDZ ligand are enhanced.
  • the binding strength of MAGI- 1 PDZ domain 1 to E6 protein is enhanced.
  • the binding strength of MAGI- 1 PDZ domain 1 to E6 protein is enhanced by 2 to 3 fold.
  • the present invention provides a chimeric polynucleotide construct comprising a polynucleotide sequence encoding a chimeric polypeptide which contains at least one PDZ domain or a portion of a PDZ domain introduced from a PDZ domain- containing polypeptide, wherein the chimeric polypeptide has enhanced binding to a PDZ ligand as compared to the native second PDZ domain- containing polypeptide.
  • the first PDZ domain-containing polypeptide is MAGI-I polypeptide.
  • the introduced PDZ domain is PDZ domain 1 of MAGI-I polypeptide.
  • the introduced PDZ domain is selected from the group consisting of PDZ domains listed in Table 2.
  • the modified PDZ domain has 70, 80, 90, 95, 96, 97, 98, or 99 % identity a PDZ domain listed in Table 2.
  • at least one PDZ domain is introduced into the second PDZ domain-containing polypeptide.
  • the second PDZ domain-containing polypeptide is PSD95.
  • at least one PDZ domain of the second PDZ domain-containing polypeptide is substituted with the introduced PDZ domain from the first PDZ domain-containing polypeptide.
  • At least one PDZ domain of PSD95 is substituted with PDZ domain 1 of MAGI-I. In some embodiments, three PDZ domains of PSD95 protein are substituted with PDZ domain 1 of MAGI-I. In some embodiments, more than one PDZ domain, e.g. 2, 3, 4, 5, 6, 7, or 8, are introduced into the second PDZ domain-containing polypeptide. In some embodiments, at least one PDZ domain from the first PDZ domain-containing polypeptide is recombined with non-PDZ portions of the second PDZ domain- containing polypeptide. In some embodiments, three PDZ domains from the first PDZ domain-containing polypeptide are recombined with the non-PDZ portions of PSD95.
  • the chimeric polypeptide contains a PDZ ligand binding portion of a PDZ domain from the first PDZ domain-containing polypeptide. In some embodiments, the chimeric polypeptide contains a modified PDZ domain from the first PDZ domain- containing polypeptide. In some embodiments, the PDZ ligand is an oncogenic protein from human papilloma virus (HPV).
  • HPV human papilloma virus
  • the oncogenic HPV strain is 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, and 82.
  • the PDZ ligand is E6 protein from oncogenic HPV strain 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82.
  • the binding affinity of the chimeric polypeptide to a PDZ ligand is enhanced.
  • the binding avidity of the chimeric polypeptide to a PDZ ligand is enhanced.
  • the binding affinity and avidity of the chimeric polypeptide to a PDZ ligand are enhanced.
  • the binding strength of MAGI- 1 PDZ domain 1 to E6 protein is enhanced.
  • the binding strength of MAGI- 1 PDZ domain 1 to E6 protein is enhanced by 2 to 3 fold.
  • the present invention provides a method for producing chimeric PDZ domain-containing polypeptide, the method comprising generating a chimeric polynucleotide construct encoding a chimeric polypeptide which contains at least one PDZ domain or a portion of a PDZ domain introduced from a PDZ domain- containing polypeptide, wherein the introduced PDZ domain in the chimeric polypeptide has enhanced binding to a PDZ ligand as compared to an isolated PDZ domain peptide.
  • the binding affinity of the chimeric polypeptide to a PDZ ligand is enhanced.
  • the binding avidity of the chimeric polypeptide to a PDZ ligand is enhanced. In some embodiments, the binding affinity and avidity of the chimeric polypeptide to a PDZ ligand are enhanced. In one example, the binding strength of MAGI-I PDZ domain 1 to E6 protein is enhanced by 2 to 3 fold. In some embodiements, the PDZ domain- containing polypeptide is MAGI-I polypeptide. In some embodiements, the PDZ domain- containing polypeptide is a modified MAGI-I polypeptide. In some embodiments, the PDZ domain- containing polypeptide is a PDZ domain listed in Table 2.
  • the PDZ domain- containing polypeptide is a modified PDZ domain listed in Table 2.
  • the present invention provides a method for detecting the presence of a PDZ ligand in a sample, the method comprising: (a) contacting a sample suspected of containing a PDZ ligand with a chimeric polypeptide containing at least one PDZ domain introduced from a PDZ domain- containing polypeptide, wherein the introduced PDZ domain in the chimeric polypeptide has enhanced binding to the PDZ ligand in the sample as compared to an isolated PDZ domain peptide; and (b) detecting binding of the PDZ ligand in the sample to the chimeric polypeptide, wherein binding of the PDZ ligand in the sample to the chimeric polypeptide indicates the presence of the PDZ ligand in the sample.
  • the present invention provides a method for determining if a subject is infected with an oncogenic strain of human papilloma virus (HPV), the method comprising detecting the presence of oncogenic HPV E6 protein in a sample from the subject using the subject oncogenic HPV E6 protein-binding chimeric polypeptide of the present invention, wherein the presence of oncogenic HPV E6 protein indicates that the subject is infected with an oncogenic strain of HPV.
  • HPV human papilloma virus
  • the present invention provides a method for purifying a PDZ ligand from a sample, the method comprising: (a) contacting a sample containing a PDZ ligand with the subject chimeric polypeptide of the present invention and (b) purifying the PDZ ligand from the sample, wherein the PDZ ligand is bound or not bound to the subject chimeric polypeptide.
  • kits for extracting HPV E6 protein from a sample comprising: (a) the subject chimeric polypeptide of the present invention, which binds E6 of an oncogenic HPV strain; (b) an extraction reagent that has a pH of at least about pH 10.0; (c) a neutralizing reagent and (d) instructions for using the kit.
  • the present invention also provides a kit for the detection and diagnosis of an E6 protein of an oncogenic HPV strain in a sample, comprising (a) the subject chimeric polypeptide of the present invention; (b) reagents for detection of the chimeric polypeptide bound to E6 protein; and (c) instructions for using the kit.
  • the present invention provides a method for producing a chimeric polypeptide having enhanced binding to a PDZ ligand, the method comprising: (a) generating the subject chimeric polynucleotide construct of the present invention, (b) expressing the chimeric polynucleotide construct in an expression vector, and (c) purifying the chimeric polypeptide encoded by the subject chimeric polynucleotide construct of the present invention.
  • Figure 1 is a schematic structure of an exemplary chimeric polypeptide PSD95-MAGI-1, which contains 3 PDZ domain 1 of MAGI-I and the non-PDZ domain portions of PSD95 as the backbone.
  • the 3 PDZ domains of PSD95 are all substituted with the PDZ domain 1 of MAGI-I.
  • the chimeric polypeptide is fused to GST at its C- terminus.
  • Figure 2 is an amino acid sequence of a fusion protein consisting of GST and PSD95-MAGI-1 chimeric PDZ domain- containing polypeptide. The first amino acid of the chimeric PDZ domain-containing polypeptide is shown in italics and bold.
  • Figure 3 shows a comparison between the PSD95-MAGI-1 chimeric PDZ domain-containing polypeptide and a single MAGI-I PDZ domain in terms of their binding strength to E6 protein from an oncogenic HPV strain, HPVl 6.
  • the comparison of E6 capture by a single MAGI-I PDZ domain versus the PSD95-MAGI-1 chimeric polypeptide is done by sandwich ELISA.
  • the binding signals at various concentrations of HPV16-E6 show enhanced binding of HPV16-E6 by the PSD-95-MAGI-1 chimeric PDZ domain-containing polypeptide over the single MAGI-I PDZ domain.
  • Figure 4 shows a comparison between the PSD95-MAGI-1 chimeric PDZ domain-containing polypeptide and a single MAGI-I PDZ domain in terms of their binding strength to E6 protein from an oncogenic HPV strain, HPVl 6.
  • the comparison of E6 capture by a single MAGI-I PDZ domain versus the PSD95-MAGI-1 chimeric polypeptide is done by a two-step lateral flow assay.
  • the binding signals at various concentrations of HPV16-E6 show enhanced binding of HPV16-E6 by the PSD-95-MAGI-1 chimeric PDZ domain- containing polypeptide over the single MAGI- 1 PDZ domain.
  • the present invention provides compositions and methods for detecting and diagnosing viral infection.
  • the compositions and methods for detection of viral infections are directed toward modulating the interactions between a PDZ ligand associated with viral infection and a chimeric protein containing at least one introduced PDZ domain that has higher binding strength to a PDZ ligand, more specifically, an E6 protein from an oncogenic strain of human papilloma virus.
  • HPV Human Papilloma Virus
  • HPV Oncogenic Proteins HPV
  • HPVs Human papillomaviruses
  • HPVs are small double-stranded DNA viruses that induce hyperproliferative lesions in epithelial tissues.
  • a subset of HPV strains infect epithelia in the anogenital region and are the etiological agents of cervical cancers.
  • These HPV strains are called "high-risk” and include but are not limited to HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82.
  • the oncogenic potential of these high-risk HPV strains is dependent on the cooperative action of the two early viral gene products, E6 and E7, which bind and alter the activity of cell cycle -regulatory proteins.
  • E6 forms a ternary complex composed of the tumor suppressor protein p53 and E6AP (E6-associated protein), a member of E3 ubiquitin ligase family of proteins, resulting in the ubiquitination and subsequent degradation of p53 (Huibregtse, J. M., et.al. 1991. EMBO J. 10:4129-4135).
  • E7 binds to and inactivates the retinoblastoma (pRb) family of proteins, thereby alleviating the pRb-mediated repression of E2F transcription factors that are responsible for transactivating many genes involved in progression into S phase (Cheng, S., et.al. 1995. Genes Dev. 9:2335-2349). Selective retention and expression of these two viral oncoproteins is essential for HPV-induced oncogenesis (Androphy, E. J., et.al. 1987. EMBO J. 6:989-992).
  • E6 The targeting of p53 for degradation by E6 is the most extensively studied function of E6. However, p53- independent functions of E6 are also necessary for transformation. For instance, several p53 degradation-defective E6 mutants are still able to immortalize mammary epithelial cells and transform 3Yl rat fibroblasts. In addition, activation of the human telomerase reverse transcriptase (hTERT) by E6 is more important for immortalization of epithelial cells than is inactivation of p53 (Kiyono, T., S. A. 1998. Nature 396:84-88). These observations indicate that E6 contributes to malignant conversion of HPV-infected cells through mechanisms in addition to inactivation of p53.
  • hTERT human telomerase reverse transcriptase
  • E6-interacting proteins contains multiple copies of a protein-protein interaction domain called PDZ (for "PSD-95/Discs Large/ZO-1"). All these PDZ domain- containing proteins are targeted for proteasome -mediated degradation by E6 in heterologous overexpression systems (Gardiol, D., et.al. 1999. Oncogene 18:5487-5496).
  • the high-risk HPV E6 proteins bind to these PDZ domain-containing proteins through a motif [X-(TVS)-X-V, where X is any amino acid] located at their extreme carboxy terminus, which is conserved among all high-risk HPV strains. In contrast, none of the low -risk HPV E6 proteins possesses such domains. Mutation of the second or fourth conserved amino acid residue [(T/S) or V] in this motif has been demonstrated to compromise the transforming activity of E6, suggesting that this PDZ domain-binding motif plays a critical role in E6-induced oncogenesis (Kiyono, T., A. 1997. Proc. Natl. Acad. Sci. USA 94:11612-11616). Additional studies of transgenic mice have demonstrated an important role for this motif in E6-mediated alterations in cellular proliferation in the eye lens.
  • Table IA E6 C-terminal sequences and oncogenicity. HPV variants are listed at the left. Sequences are identified from Genbank sequence records. PL Yes/No was defined by a match or non-match to the consenses determined at Arbor Vita and by Songyang et al. -X-(S/T)-X-(V/I/L). Oncogenicity data collected from National Cancer Institute. *Only found in oncogenic strains co-transfected with other oncogenic proteins.
  • Table IB E6 C-terminal sequences and oncogenicity. HPV variants are listed at the left. Sequences are identified from Genbank sequence records. PL Yes/No was defined by a match or non-match to the consenses determined at Arbor Vita and by Songyang et al. -X-(S/T)-X-(V/I/L). Oncogenicity data on new strains collected from N Engl J Med 2003; 348:518-527. II. PDZ Domain-Containing Proteins and PDZ Ligands
  • the present invention relates to the compositions and methods of using PDZ domain containing proteins for the detection and diagnosis of HPV.
  • the PDZ domain is a common structural domain of 80-90 amino-acids found in the signaling proteins of bacteria, yeast, plants, and animals.
  • PDZ domains are also referred to as DHR (Dig homologous region) or GLGF (glycine-leucine-glycine-phenylalanine) domains. These domains help anchor transmembrane proteins to the cytoskeleton and hold together signaling complexes (Ponting CP, et.al. Bioessays 1997; 19:469-479).
  • PDZ domain-containing proteins There are roughly 260 human PDZ domains, though since several PDZ domain containing proteins hold several domains, the actual number of PDZ proteins is closer to 180.
  • Table 2 lists non-limiting exemplary PDZ domain- containing proteins and PDZ domain sequences.
  • Some of the well-studied PDZ domain- containing proteins include include hDlg, hScrib, MAGI-I, MAGI-2, andMAGI-3 (for "Membrane-Associated Guanylate kinase homology proteins with an inverted domain structure"), PSD95, TIP-I and MUPPl.
  • MAGI-I Membrane associated guanylate kinase, WW and PDZ domain containing 1
  • MAGI-I membrane-associated guanylate kinase homologue
  • MAGUK membrane-associated guanylate kinase homologue
  • MAGI-I consists of six PSD95/T)iscLarge/ZO-l (PDZ) domains, a guanylate kinase domain and two WW domains flanked by the first and second PDZ domain (Dobrosotskaya, I. et.al 1997 J. Biol. Chem. 272, 31589-31597). Because PDZ domains are docking domains for PDZ-binding molecules, MAGI- 1 associates with a variety molecules such as NMDA (iV-methyl-D-aspartate) receptors, PTEN, BAI- 1 , ⁇ - catenin, mNETl, and ⁇ -catenin. These MAGI-I -associating molecules function at cell-cell contacts. MAGI-I, therefore, functions as a scaffold molecule by localizing to cell-cell contacts.
  • NMDA iV-methyl-D-aspartate
  • HPV E6 oncoproteins selectively complex with the widely-expressed cellular PDZ -protein MAGI-I, which is targeted for degradation in cells by high-risk HPV E6 proteins, suggesting that the transforming potentials of the viral oncoproteins depend partially on an ability to inactivate this cellular PDZ domain- containing protein.
  • HPV 16E6 and 18E6 mutant proteins having disrupted PDZ domain-binding motifs fail to bind MAGI- 1 and that the wild-type viral proteins do not complex with the related MAGUK proteins ZO-I and ZO-2.
  • PSD-95 is another member of the MAGUK- family of PDZ domain-containing proteins. Similar to all MAGUK- family proteins, the basic structure of PSD95 includes three PDZ domains, an IH3 domain, and a guanylate kinase-like domain (GK). It is almost exclusively located in the post synaptic density of neurons, and is involved in anchoring synaptic proteins. Its direct and indirect binding partners include neuroligin, NMDA receptor, AMPA receptor, and potassium channels (Sheng M, SaIa C (2001). Annu. Rev. Neurosci. 24: 1-29). PSD95 has also been identified as a target of high-risk HPV E6 proteins (Handa K, et.al., J.
  • TABLE 3 lists PDZ proteins and PDZ ligand (PL) proteins which the current inventors have identified as binding to one another.
  • Each page of TABLE 3 includes four columns. The columns in each section are number from left to right such that the left-most column in each section is column 1 and the right-most column in each section is column 4.
  • HPV Strain the various E6 proteins that contain the PDZ-Ligand sequences (PLs) that are examined (shown in parenthesis).
  • This column lists C- terminal four amino acids that correspond to the carboxyl-terminal end of a 20 amino acid peptide used in this binding study. All ligands are biotinylated at the amino-terminus and partial sequences are presented in TABLE 1.
  • the PDZ domain- containing protein (or proteins) that interact(s) with HPV E6— PL peptides are listed in the second column labeled "PDZ binding partner".
  • This column provides the gene name for the PDZ portion of the GST-PDZ fusion that interacts with the PDZ-ligand to the left.
  • the domain number is listed to the right of the PDZ (i.e., in column 4 labeled "PDZ Domain"), and indicates the PDZ domain number when numbered from the amino-terminus to the carboxy-terminus.
  • This table only lists interactions of a stronger nature, e.g., those that give a ⁇ 4 ⁇ or ⁇ 5 ⁇ classification in the G assay” .
  • “Classification” is a measure of the level of binding. In particular, it provides an absorbance value at 450 nm which indicates the amount of PL peptide bound to the PDZ protein.
  • the third and fourth columns of TABLE 3 are merely a repetition of the columns 1 and 2 with different E6 PLs tested and the PDZs bound by them at higher affinity.
  • TABLE 1 provides a listing of the partial amino acid sequences of peptides used in the assays.
  • HPV strain provides the HPV strain number used to refer to the E6 protein from that strain.
  • E6 C-terminal sequence provides the predicted sequence of the carboxy-terminal 10 amino acids of the E6 protein.
  • PL yes/no designates whether the E6-PL sequence contains sequence elements predicted to bind to PDZ domains.
  • oncogenic indicates that this HPV strain is known to cause cervical cancer as determined by the National Cancer Institute (NCI, 2001).
  • EXAMPLE 5 lists representative sequences of PDZ domains cloned into a vector (PGEX-3x vector) for production of GST-PDZ fusion proteins (Pharmacia).
  • An extended list of PDZ domains cloned into pGEX vectors for production of GST-PDZ fusion proteins is listed in U.S. Pat. No. 09/724553.
  • the PDZ proteins listed in TABLE 2 are naturally occurring proteins containing a PDZ domain. Only significant interactions are presented in this table. Thus, the present invention is particularly directed to the detection and modulation of interactions between a chimeric protein containing a PDZ domain and a PDZ ligand protein. In a similar manner, a chimeric protein containing PDZ domains that bind other pathogens can be used to diagnose infection. Additional examples of PDZ ligand proteins from pathogens suitable for diagnostic applications are included in TABLE 6, but are not intended to limit the scope of the invention.
  • a chimeric PDZ domain-containing protein is used to diagnose the presence of a PDZ ligand protein from a pathogenic organism.
  • pathogenic organisms with PL sequences include, but are not limited to, viruses such as Human Papillomaviruses, Hepatitus B virus, Adenovirus, Human T Cell Leukemia Virus, bacteria and fungi. TABLE 2
  • 26s subunit 9184389 1 RDMAEAHKEAMSRKLGQSESQGPPRAFAKVNSISPGSPSIAGL 1 p27 QVDDEIVEFGSVN TQNFQSLHNIGSWQHSEGALAPTILLSVSM
  • AIPC 12751451 1 LIRPSVISIIGLYKEKGKGLGFSIAGGRDCIRGQMGIFVKTIFPNG 3 SAAEDGRLKEGDEI LDVNGIPIKGLTFQEAIHTFKOIRSGLPVLTVRTKLVSPSLTNSS
  • AIPC 12751451 2 GISSLGRKTPGPKDRIVMEVTLNKEPRVGLGIGACCLALENSPP 4 GIYIHSLAPGSVAK
  • AIPC 12751451 QSENEEDVCFIVLNRKEGSGLGFSVAGGTDVEPKSITVHRVFS 5 QGAASQEGTMHRG
  • AIPC 12751451 4 LGRSVAVHDALCVEVLKTSAGLGLSLDGGKSSVTGDGPLVIK 6 RVYKGGAAEQAGIIE AGDEILAINGKPLVGLMHFDAWNIMKSVPEGPVQLLIRKHRNS
  • Atrophin- 1 2947231 3 HYKELDVHLRRMESGFGFRILGGDEPGQPILIGAVIAMGSADR 11
  • CARD 14 13129123 1 ILSQVTMLAFQGDALLEQISVIGGNLTGIFIHRVTPGSAADQMA 16 LRPGTQIVMVDYEA SEPLFKAVLEDTTLEEAVGLLRRVDGFCCLSVKVNTDGYKRL
  • CASK 3087815 1 TRVRLVQFQKNTDEPMGITLKMNELNHCIVARIMHGGMIHRQ 17 GTLHVGDEIREINGIS VANQTVEQLQKMLREMRGSITFKIVPSYRTQS
  • Connector 3930780 1 LEQKAVLEQVQLDSPLGLEIHTTSNCQHFVSQVDTQVPTDSRL 18 Enhancer QIQPGDEWQINEQ VWGWPRKNMVRELLREPAGLSLVLKKIPIP
  • DLG6 splice AB053303 1 PTSPEIQELRQMLQAPHFKGATIKRHEMTGDILVARIIHGGLAE 30 variant 2 RSGLLYAGDKLVEV NGVSVEGLDPEQVIHILAMSRGTIMFKWPVSDPPVNSS
  • GRIP l 4539083 1 WELMKKEGTTLGLTVSGGIDKDGKPRVSNLRQGGIAARSDQ 47 LDVGDYIKAVNGINLAKFRHDEIISLLKNVGERWLEVEYE
  • GRIP l 4539083 2 RSSVIFRTVEVTLHKEGNTFGFVIRGGAHDDRNKSRPWITCV 48 RPGGPADREGTIKPGDRLLSVDGIRLLGTRHAEAMSILKQCGQ EAALLIEYDVSVMDSVATASGNSS
  • GRIP l 4539083 3 HVATASGPLLVEVAKTPGASLGVALTTSMCCNKQVIVIDKIKS 49 ASIADRCGALHVGDHILSIDGTSMEYCTLAEATQFLANTTDQV KLEILPHHQTRLALKGPNS S
  • GRIP 1 4539083 4 TETTEWLTADPVTGFGIQLQGSVFATETLSSPPLISYIEADSPA 50 ERCGVLQIGDRVMAINGIPTEDSTFEEASQLLRDSSITSKVTLEI
  • GRIP 1 4539083 5 AESVIPSSGTFHVKLPKKHNVELGITISSPSSRKPGDPLVISDIKK 51 GSVAHRTGTLELGDKLLAIDNIRLDNCSMEDAVQILQQCEDLV KLKIRKDEDNSD
  • GRIP l 4539083 7 IMSPTPVELHKVTLYKDSDMEDFGFSVADGLLEKGVYVKNIRP 53 AGPGDLGGLKPYDRLLQVNHVRTRDFDCCLWPLIAESGNKL DLVISRNPLA
  • GTPase 2389008 1 SRGCETRELALPRDGQGRLGFEVDAEGFVTHVERFTFAETAGL 54
  • HTRA4 AL576444 1 HKKYLGLQMLSLTVPLSEELKMHYPDFPDVSSGVYVCKWEG 60 TAAQSSGLRDHDVIVNINGKPITTTTDWKALDSDSLSMAVLR GKDNLLLTVNSS
  • KIAA0380 2224700 1 QRCVIIQKDQHGFGFRVSGDRIVLVQSVRPGGAAMKAGVKEG 77 DRIIKVNGTMVTNSSHLEWKLIKSGAYVALTLLGSS
  • KIAA0545 14762850 1 LKVMTSGWETVDMTLRRNGLGQLGFHVKYDGTVAEVEDYG 80 FAWQAGLRQGSRLVEICKVAWTLTHDQMIDLLRTSVTVKW IIPPFEDGTPRRGW
  • KIAA0858 4240204 1 FSDMRISINQTPGKSLDFGFTIKWDIPGIFVASVEAGSPAEFSQL 86
  • KIAA1222 6330610 1 ILEKLELFPVELEKDEDGLGISIIGMGVGADAGLEKLGIFVKTV 93 TEGGAAQRDGRIQVNDQIVEVDGISLVGVTQNFAATVLRNTK GNVRFVIGREKPGQVS
  • IHTVANASGPLMVEIVKTPGSALGISLTTTSLRNKSVITIDRIKP 108 ASWDRSGALHPGDHILSIDGTSMEHCSLLEATKLLASISEKVR LEILPVPQSQRPL
  • LIMKl 4587498 1 TLVEHSKLYCGHCYYQTWTPVIEQILPDSPGSHLPHTVTLVSI 115 PASSHGKRGLSVSIDPPHGPPGCGTEHSHTVRVQGVDPGCMSP DVKNSIHVGDRILEINGTPIRNVPLDEIDLLIQETSRLLQLTLEH
  • LIM-RIL 1085021 1 IHSVTLRGPSPWGFRLVGRDFSAPLTISRVHAGSKASLAALCPG 117 DLIQAINGESTELMTHLEAQNRIKGCHDHLTLSVSRPE
  • MUPPl 2104784 1 QGRHVEVFELLKPPSGGLGFSWGLRSENRGELGIFVQEIQEGS 132 VAHRDGRLKETDQILAINGQALDQTITHQQAISILQKAXDTVQ LVIARGSLPQLV MUPPl 2104784 2 PVHWQHMETIELVNDGSGLGFGIIGGKATGVIVKTILPGGVA 133 DQHGRLCSGDHILKIGDTDLAGMSSEQVAQVLRQCGNRVKL MIARGAIEERTAPT MUPPl 2104784 3 QESETFDVELTKNVQGLGITIAGYIGDKKLEPSGIFVKSITKSSA 134 VEHDGRIQIGDQIIAVDGTNLQGFTNQQAVEVLRHTGQTVLLT LMRRGMKQEA MUPPl 2104784 4 LNYEIWAHVSKFSENSGLGISLEATVGHHFIRSVLPEGPVGHS 135 GKLFSGDELLEVNGITLLGENHQ
  • NeDLG 10863920 3 ILLHKGSTGLGFNIVGGEDGEGIFVSFILAGGPADLSGELRRGD 147 RILSVNGVNLRNATHEQAAAALKRAGQSVTIVAQYRPEEYSR FESKIHDLREQMMNSSMSSGSGSLRTSEKRSLE
  • NumbBindingPr AK056823 1 PDGEITSIKINRVDPSESLSIRLVGGSETPLVHIIIQHIYRDGVIAR 153 otein DGRLLPGDIILKVNGMDISNVPHNYAVRLLRQPCQVLWLTVM REQKFRSRNSS
  • Outer Membrane 7023825 1 LLTEEEINLTRGPSGLGFNIVGGTDQQYVSNDSGIYVSRIKENG 157 AAALDGRLQEGDKILSVNGQDLKNLLHQDAVDLFRNAGYAV SLRVQHRLQVQNGIHS p55T 12733367 1 PVDAIRILGIHKRAGEPLGVTFRVENNDLVIARILHGGMIDRQG 158 LLHVGDIIKEVNGHEVGNNPKELQELLKNISGSVTLKILPSYRD
  • PICKL 4678411 1 PTVPGKVTLQKDAQNLIGISIGGGAQYCPCLYIVQ VFDNTP AA 171
  • PTPLl 515030 1 PEREITLVNLKKDAKYGLGFQIIGGEKMGRLDLGIFISSVAPGG 180 PADFHGCLKPGDRLISVNSVSLEGVSHHAAIEILQNAPEDVTLV ISQPKEKISKVPSTPVHL
  • PTPLl 515030 4 ELEVELLITLIKSEKASLGFTVTKGNQRIGCYVHDVIQDPAKSD 183 GRLKPGDRLIKVNDTDVTNMTHTDAVNLLRAASKIVRLVIGR VLELPRIPMLPH
  • RGS 12 3290015 1 RPSPPRVRSVEVARGRAGYGFTLSGQAPCVLSCVMRGSPADF 185 VGLRAGDQILAVNEINVKKASHEDWKLIGKCSGVLHMVIAE GVGRFESCS
  • Shroom 18652858 1 IYLEAFLEGGAPWGFTLKGGLEHGEPLIISKVEEGGKADTLSSK 191 LQAGDEVVHINEVTLSSSRKEAVSLVKGSYKTLRLWRRDVC TDPGH
  • TIAM 1 4507500 HSIHIEKSDTAADTYGFSLSSVEEDGIRRLYVNSVKETGLASKK 204 GLKAGDEILEINNRAADALNSSMLKDPLSQPSLGLLVRTYPEL
  • TIP2 2613003 1 RKEVEVFKSEDALGLTITDNGAGYAFIKRIKEGSVIDHIHLISVG 207 DMIEAINGQSLLGCRHYEVARLLKELPRGRTFTLKLTEPRK TIP33 2613007 1 HSHPRWELPKTDEGLGFNVMGGKEQNSPIYISRIIPGGVAERH 208
  • HPV PDZ binding partner PDZ binding partner strain (signal 4 and 5 of 0-5)
  • HPV strain (signal 4 and 5 of 0-5)
  • HPV 35 Atrophin- 1 interact, prot. HPV 33 Magil (PDZ #2)
  • PTN-4 NeDLG (PDZ #1,2)
  • HPV 58 Atrophin- 1 interact, prot. (PDZ # HPV 66 DLGl (PDZ #1, 2)
  • TQV TQV 1
  • STV Session Detection Protocol 1
  • PDZ #2 HPV PDZ binding partner PDZ binding partner strain (signal 4 and 5 of 0-5)
  • HPV strain signal 4 and 5 of 0-5)
  • HPV strain denotes the strain from which the E6 C-terminal peptide sequence information was taken.
  • Peptides used in the assay varied from 18 to 20 amino acids in length, and the terminal four residues are listed in parenthesis.
  • the present invention provides a chimeric polypeptide containing at least one PDZ domain or a PDZ ligand binding portion of a PDZ domain introduced from a first PDZ domain containing polypeptide into a second PDZ domain containing polypeptide such that the chimeric polypeptide has enhanced binding strength than the native second PDZ domain containing polypeptide.
  • the binding strength of a chimeric PDZ domain to E6 protein is enhanced by greater than 1.2, 1.5, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, or 5 fold.
  • a "chimeric polypeptide” or a “chimeric polynucleotide” is an artificially constructed protein or polynucleotide comprising heterologous amino acid sequences or heterologous nucleic acid sequences, respectively.
  • Chimeric proteins are proteins created through the joining of two or more genes, or portions of two or more genes, which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with function properties derived from each of the original proteins.
  • Such chimeric proteins are created artificially by recombinant DNA technology well known in the art.
  • heterologous when used with reference to a protein or a nucleic acid indicates that the protein or the nucleic acid comprises two or more sequences or subsequences which are not found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid.
  • the nucleic acid has a promoter from one gene arranged to direct the expression of a coding sequence from a different gene.
  • the promoter is heterologous.
  • polypeptide peptide and protein are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • a native polypeptide is typically isolated from a naturally occurring source, in particular a mammalian or microbial source, such as a human source, or is produced recombinantly by use of a nucleotide sequence encoding the naturally occurring amino acid sequence.
  • native polypeptide refers to a naturally occurring polypeptide.
  • a “native" PDZ domain containing polypeptide refers to a naturally occurring polypeptide in which the PDZ domain is not modified in its amino acid residues.
  • a “variant” is a polypeptide, which has an amino acid sequence that differs from that of a native polypeptide in one or more amino acid residues. The variant is typically prepared by modification of a nucleotide sequence encoding the native polypeptide (e.g., to result in substitution, deletion or truncation of one or more amino acid residues of the polypeptide or by introduction (by addition or insertion) of one or more amino acid residues into the polypeptide) so as to modify the amino acid sequence constituting said native polypeptide.
  • the present invention provides a chimeric polypeptide in which at least one PDZ domain or a portion thereof is introduced from a first PDZ domain-containing polypeptide into a second PDZ domain- containing polypeptide, and that results in enhanced binding of such chimeric polypeptide to a PDZ ligand.
  • one PDZ domain is introduced from the first to the second PDZ domain- containing polypeptide.
  • two PDZ domains are introduced from the first to the second PDZ domain- containing polypeptide.
  • three PDZ domains are introduced from the first to the second PDZ domain-containing polypeptide.
  • more than three PDZ domains e.g.
  • PDZ domains are introduced from the first to the second PDZ domain-containing polypeptide.
  • an introduced PDZ domain from the first PDZ domain- containing polypeptide substitutes a PDZ domain naturally present in the second PDZ domain-containing polypeptide.
  • the present invention also includes a chimeric polypeptide in which a PDZ domain is introduced in addition without substituting a PDZ domain naturally present in the second PDZ domain-containing polypeptide.
  • the PDZ domain from the first PDZ domain- containing polypeptide can be introduced at any position in the second PDZ domain- containing polypeptide so long as the adjacent sequences of the second PDZ domain- containing polypeptide allows proper PDZ domain folding such that the chimeric polypeptide exhibits enhanced binding to a PDZ ligand than a native PDZ domain-containing polypeptide.
  • PDZ domains are introduced into more than one location in the second PDZ domain- containing polypeptide.
  • the chimeric polypeptide of the invention, the first PDZ domain-containing polypetide is a PDZ domain listed in Table 2 (SEQ ID No's: 1-220).
  • the first PDZ domain-containing polypeptide is MAGI-I and the second PDZ domain-containing polypeptide is PSD95.
  • Three PDZ domains of PSD95 are substituted with 3 PDZ domain 1 of MAGI-I, as shown in FIG 1.
  • the chimeric polypeptide contains 3 MAGI-I PDZ domain 1 with the non-PDZ portions of PSD95 as the backbone.
  • the non-PDZ domain portions of PSD95 when recombined with PDZ domain 1 of MAGI-I, allow correct PDZ folding, resulting in enhanced binding of the chimeric polypeptide to E6 protein from an oncogenic HPV strain.
  • the chimeric PSD95-MAGI-1 polypeptide is fused to GST at the C-terminus, and can be readily expressed in high quantities in an expression vector such as E. CoIi.
  • the amino acid sequence of the chimeric PSD95-MAGI-1 polypeptide fused to GST is shown in FIG 2.
  • the present invention also embodies a chimeric polypeptide in which the introduced PDZ domain may be modified, i.e. the introduced PDZ domain is a variant of the naturally occurring PDZ domain present in the native first PDZ domain-containing polypeptide.
  • the introduced PDZ domain is modified having at least 70, 80, 90, 95, 96, 97, 98, or 99 % identity to a PDZ domain.
  • Examples of PDZ domain variants include ones which have at least 80%, one which has at least 90%, or one which has at least 95% amino acid sequence identity to the native PDZ amino acid sequence, and which contain at least one functional or structural characteristic of a native PDZ domain, such as binding to the E6 oncogenic protein of HPV..
  • the invention also includes a chimeric polypeptide containing a PDZ ligand binding portion of a PDZ domain introduced from the first to the second PDZ domain- containing polypeptide.
  • a PDZ domain is generally composed of 80-90 amino-acids.
  • a PDZ ligand binding portion of a PDZ domain i.e. fragments of a PDZ domain
  • Such PDZ ligand binding portion of a PDZ domain may be generated entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding such PDZ domain or any fragment thereof.
  • the invention also encompasses chimeric polynucleotides which encode the chimeric polypeptides described herein.
  • the invention may also encompass a variant of a polynucleotide sequence encoding a chimeric polypeptide.
  • such a variant polynucleotide sequence will have at least 70, 80, 90, 95, 96, 97, 98, or 99 % identity to the polynucleotide sequence encoding a native PDZ domain from the first PDZ domain-containing polypeptide.
  • Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of a native PDZ domain- containing polypeptide.
  • the present invention also embodies polynucleotide sequences encoding a chimeric polypeptide containing at least one introduced PDZ domain or its variants possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the chimeric polypeptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • the chimeric polynucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter PDZ domain-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
  • oligonucleotide-mediated site -directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
  • one or more PDZ domains from the first PDZ domain containing polypeptiode are embedded in certain adjacent sequences of the second PDZ domain containing polypeptide such that the conformation of the chimeric polypeptide allows for higher binding of the chimeric polypeptide to a PDZ ligand as compared to the native second PDZ containing polypeptide.
  • a PDZ domain in its native polypeptide conformation has high binding affinity to a PDZ ligand but the same PDZ domain outside its native polypeptide conformation has been shown to have only marginal binding to a PDZ ligand.
  • an isolated PDZ domain or a PDZ domain outside its native polypeptide conformation would not be expected to have high binding ability to a PDZ ligand.
  • PDZ domain 2 of PSD95 captures high-risk HPV E6 to a marginal extent when represented as complete and correctly folded but isolated PDZ domain.
  • E6 capture via MAGI-I PDZ domain 1 is increased when MAGI-I-PDZ domain 1 is embedded in certain adjacent sequences of PSD95 polypeptide (sequences encompassing PDZ domains 1 through 3 of PSD95).
  • the sequences in the second PDZ domain polypeptide flanking the inserted PDZ domain confer enhanced PDZ ligand binding to the inserted PDZ domain.
  • the PSD95 non-PDZ protein portions confer enhanced E6 binding to the inserted PDZ domain 1 of MAGI-I.
  • sequences encoding a chimeric polypeptide containing at least one PDZ domain from a first PDZ containing polypeptide may be synthesized, in whole or in part, using chemical methods well known in the art.
  • chemical methods See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232.
  • such chimeric polypeptide may be synthesized using chemical methods.
  • peptide synthesis can be performed using various solid-phase techniques. (See, e.g., Roberge, J.
  • the peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z. Regnier (1990) Methods Enzymol. 182:392-421). The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York N.
  • the polynucleotide sequences encoding such chimeric polypeptide described herein may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
  • these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding the chimeric PDZ domain containing polypeptide.
  • Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding the chimeric PDZ domain containing polypeptide.
  • Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • sequences encoding a chimeric PDZ domain containing polypeptide and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
  • exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector.
  • Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic.
  • Enhancers appropriate for the particular host cell system used.
  • Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a chimeric PDZ domain containing polypeptide and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination as described in details under section XIX. (See, e.g., Sambrook, J. et al.
  • the present invention includes a chimeric PDZ domain containing polypeptide that has enhanced binding strength than a native PDZ domain containing polypeptide.
  • the binding strength of a PDZ domain containing polypeptide to a PDZ ligand includes binding affinity and binding avidity of such PDZ domain containing polypeptide to the PDZ ligand.
  • binding affinity refers to the equilibrium dissociation constant (expressed in units of concentration) associated with each PDZ domain-PDZ ligand binding interaction.
  • the binding affinity is directly related to the ratio of the kinetic off-rate (generally reported in units of inverse time, e.g., seconds-1) divided by the kinetic on-rate (generally reported in units of concentration per unit time, e.g., molar/second). In general it is not possible to unequivocally state whether changes in equilibrium dissociation constants are due to differences in on-rates, off-rates or both unless each of these parameters are experimentally determined (e.g., by BIACORE or SAPIDYNE measurements).
  • avidity is a term used to describe the combined strength of multiple bond interactions. Avidity is distinct from affinity, which is usually used to describe the binding strength of a single bond.
  • the chimeric polypeptide has synergistic binding to a PDZ ligand as compared to a native PDZ domain polypeptide.
  • "Enhanced” binding strength as used herein refers to increased, greater or higher binding affinity and/or avidity of a chimeric PDZ domain containing polypeptide to a PDZ ligand.
  • "diminished” binding strength refers to reduced, decreased or lesser binding affinity and/or avidity of a chimeric PDZ domain containing polypeptide to a PDZ ligand as compared to a native PDZ domain containing polypeptide or an isolated PDZ domain.
  • the chimeric polypeptide containing at least one PDZ domain or a PDZ ligand binding portion of a PDZ domain introduced from a first PDZ domain- containing polypeptide into a second PDZ domain- containing polypeptide has a higher binding affinity to a PDZ ligand, such as E6 protein, as compared to the native second PDZ domain-containing polypeptide.
  • a PDZ ligand such as E6 protein
  • the chimeric polypeptide containing at least one PDZ domain or a PDZ ligand binding portion of a PDZ domain introduced from a first PDZ domain-containing polypeptide into a second PDZ domain-containing polypeptide has a higher binding affinity to a PDZ ligand, such as E6 protein, as compared to the native first PDZ domain- containing polypeptide.
  • the native first PDZ domain-containing polypeptide does not have any modifications in its PDZ domains.
  • the chimeric polypeptide containing at least one PDZ domain or a PDZ ligand binding portion of a PDZ domain introduced from a first PDZ domain-containing polypeptide into a second PDZ domain-containing polypeptide has a higher binding affinity to a PDZ ligand, such as E6 protein, as compared to a single isolated PDZ domain.
  • a PDZ ligand such as E6 protein
  • a chimeric polypeptide in which three PDZ domains of the second PDZ domain-containing polypeptide PSD95 have been substituted with three PDZ domain 1 of MAGI shows enhanced binding to E6 protein from oncogenic HPV strain 16 over the single MAGI- 1 PDZ domain 1 as determined by sandwich ELISA and lateral flow assays (FIGS 3 and 4).
  • the chimeric polypeptide described herein may be particularly useful to increase the avidity of the chimeric PDZ domain containing polypeptide to a PDZ ligand, such as HPV E6 protein.
  • the present invention embodies a chimeric PDZ domain-containing polypeptide which has multiple introduced PDZ domains, thereby increasing the overall binding avidity and binding strength of such chimeric PDZ domain- containing polypeptide to a PDZ ligand.
  • the chimeric PDZ domain-containing polypeptide may have enhanced binding affinity and binding avidity to a PDZ ligand as compared to the native first PDZ domain-containing polypeptide.
  • the chimeric PDZ domain-containing polypeptide may have enhanced binding affinity and binding avidity to a PDZ ligand as compared to the native second PDZ domain-containing polypeptide.
  • the chimeric PDZ domain-containing polypeptide may have enhanced binding affinity and binding avidity to a PDZ ligand as compared to an isolated PDZ domain.
  • the binding properties of the molecules of the invention including binding affinity and binding avidity for a PDZ ligand protein may be initially determined using in vitro assays (biochemical or immunological based assays) known in the art for PDZ domain and PDZ ligand interactions, including but not limited to ELISA assay, surface plasmon resonance assay, immunoprecipitation assays, cytometric bead array and lateral flow assay.
  • in vitro assays biochemical or immunological based assays
  • the molecules of the invention have similar binding properties in in vivo models as those in in vitro based assays.
  • the present invention does not exclude molecules of the invention that do not exhibit the desired phenotype in in vitro based assays but do exhibit the desired phenotype in vivo.
  • the invention encompasses assays known in the art, and exemplified herein, to characterize the binding of a chimeric PDZ domain-containing polypeptide to a PDZ ligand.
  • a sandwich ELISA may be performed.
  • An exemplary assay may comprise the following: assay plates may be coated overnight at 4 0 C with a chimeric PDZ domain-containing polypeptide or a control polypeptide in coating buffer. The plates may then be washed and blocked. Following washing, an aliquot of human E6 protein may be added to each well and incubated for 2 hrs at room temperature.
  • an antibody specific for E6 and conjugated with peroxidase may be added to each well and incubated for 1 hour at room temperature.
  • the plate may again be washed with wash buffer and 100 ul of substrate buffer containing OPD (O-phenylenediamine dihydrochloride (Sigma)) may be added to each well.
  • OPD O-phenylenediamine dihydrochloride (Sigma)
  • the oxidation reaction observed by the appearance of a yellow color, may be allowed to proceed for 30 minutes and stopped by the addition of 100 ul of 4.5 NH2 SO4.
  • the absorbance may then read at (492-405) nm.
  • EC 50 half maximal effective concentration refers to the concentration of a molecule which induces a response halfway between the baseline and maximum.
  • EC 50 is a good measure of binding strength of a PDZ domain to a PL, and therefore useful in determining which PDZ domains from the first PDZ domain containing polypeptide to introduce to the second PDZ domain containing polypeptide to constitute the chimeric PDZ domain containing polypeptide that has enhanced binding strength to a PDZ ligand over a native PDZ containing polypeptide.
  • the molecules of the invention comprising a chimeric PDZ domain-containing polypeptide may be assayed using any surface plasmon resonance (SPR) based assays known in the art for characterizing the kinetic parameters of binding between the chimeric PDZ domain-containing polypeptide and a PDZ ligand.
  • SPR surface plasmon resonance
  • SPR instrument commercially available including, but not limited to, BIAcore Instruments, available from Biacore AB (Uppsala, Sweden); IAsys instruments available from Affinity Sensors (Franklin, Mass.); IBIS system available from Windsor Scientific Limited (Berks, UK), SPR-CELLIA systems available from Nippon Laser and Electronics Lab (Hokkaido, Japan), and SPR Detector Spreeta available from Texas Instruments (Dallas, Tex.) can be used in the instant invention.
  • BIAcore Instruments available from Biacore AB (Uppsala, Sweden
  • IAsys instruments available from Affinity Sensors (Franklin, Mass.)
  • IBIS system available from Windsor Scientific Limited (Berks, UK)
  • SPR-CELLIA systems available from Nippon Laser and Electronics Lab (Hokkaido, Japan)
  • SPR Detector Spreeta available from Texas Instruments (Dallas, Tex.
  • SPR based assays involve immobilizing a member of a binding pair on a surface, and monitoring its interaction with the other member of the binding pair in solution in real time.
  • SPR is based on measuring the change in refractive index of the solvent near the surface that occurs upon complex formation or dissociation.
  • the surface onto which the immobilization occur is the sensor chip, which is at the heart of the SPR technology; it consists of a glass surface coated with a thin layer of gold and forms the basis for a range of specialized surfaces designed to optimize the binding of a molecule to the surface.
  • a variety of sensor chips are commercially available especially from the companies listed supra, all of which may be used in the methods of the invention.
  • sensor chips examples include those available from BIAcore AB, Inc., e.g., Sensor Chip CM5, SA, NTA, and HPA.
  • a molecule of the invention may be immobilized onto the surface of a sensor chip using any of the immobilization methods and chemistries known in the art, including but not limited to, direct covalent coupling via amine groups, direct covalent coupling via sulfhydryl groups, biotin attachment to avidin coated surface, aldehyde coupling to carbohydrate groups, and attachment through the histidine tag with NTA chips.
  • the kinetic parameters of the binding of a chimeric PDZ domain-containing polypeptide to a PDZ ligand may be determined using a BIAcore instrument (e.g., BIAcore instrument 1000, BIAcore Inc., Piscataway, NJ.).
  • a BIAcore instrument e.g., BIAcore instrument 1000, BIAcore Inc., Piscataway, NJ.
  • the binding of a chimeric polypeptide containing 3 PDZ domain 1 of MAGI-I and the non-PDZ portions of PSD95 to E6 protein from an oncogenic HPV strain is determined using BIAcore.
  • the resulting binding curves are globally fitted using computer algorithms supplied by the SPR instrument manufacturer, e.g., BIAcore, Inc. (Piscataway, NJ.). These algorithms calculate both the K 0n and K off from which the apparent equilibrium binding constant, Ka is deduced as the ratio of the two rate constants (i.e., K o g-/K on ). More detailed treatments of how the individual rate constants are derived can be found in the BIAevaluaion Software Handbook (BIAcore, Inc., Piscataway, NJ.). The analysis of the generated data may be done using any method known in the art.
  • lateral flow assays are utilized to determine binding between a chimeric PDZ domain- containing polypeptide and a PDZ ligand.
  • Lateral flow devices work by applying fluid to a test strip that has been treated with specific biologicals. Carried by the liquid sample, phosphors labeled with corresponding biologicals flow through the strip and can be captured as they pass into specific zones. The amount of phosphor signal found on the strip is proportional to the amount of the target analyte.
  • a sample suspected of containing one of the viruses, bacteria or related microbes disclosed herein is added to a lateral flow device by some means, the sample is allowed to move by diffusion and a line or colored zone indicates the presence of the virus or bacteria.
  • the lateral flow typically contains a solid support (for example nitrocellulose membrane) that contains three specific areas: a sample addition area, a capture area containing one or more chimeric PDZ domain-containing polypeptides and/or antibodies immobilized, and a read-out area that contains one or more zones, each zone containing one or more labels.
  • a solid support for example nitrocellulose membrane
  • the lateral flow can also include positive and negative controls.
  • a lateral flow device in certain exemplary procedures would perform as follows: a viral or bacterial PDZ ligand protein is separated from other bacterial, viral and cellular proteins in a biological sample by bringing an aliquot of the biological sample into contact with one end of a test strip, and then allowing the proteins to migrate on the test strip, e.g., by capillary action such as lateral flow.
  • One or more PL binding agents such as chimeric PDZ domain- containing polypeptides, antibodies, and/or aptamers are included as capture and/or detect reagents.
  • a test strip comprises a proximal region for loading the sample (the sample- loading region) and a distal test region containing a chimeric PDZ domain-containing polypeptide as a capture agent and buffer reagents and additives suitable for establishing binding interactions between the chimeric PDZ domain- containing polypeptide and any viral or bacterial PL protein in the migrating biological sample.
  • the test strip comprises two test regions that contain different chimeric PDZ domain- containing polypeptides, i.e., each capable of specifically interacting with a different viral or bacterial PL protein analyte.
  • the lateral flow can include tests for a variety of different viruses and bacteria.
  • a and G Two complementary assays, termed "A" and "G", are developed to detect binding between a PDZ -domain polypeptide and candidate PDZ ligand.
  • binding is detected between a peptide having a sequence corresponding to the C-terminus of a protein anticipated to bind to one or more PDZ domains (i.e. a candidate PL peptide) and a PDZ-domain polypeptide (typically a fusion protein containing a PDZ domain).
  • a candidate PL peptide i.e. a PL peptide
  • a PDZ-domain polypeptide typically a fusion protein containing a PDZ domain.
  • the candidate PL peptide is immobilized and binding of a soluble chimeric PDZ-domain polypeptide to the immobilized peptide is detected (the "A"' assay is named for the fact that in one embodiment an avidin surface is used to immobilize the peptide).
  • the chimeric PDZ-domain polypeptide is immobilized and binding of a soluble PL peptide is detected (The "G” assay is named for the fact that in one embodiment a GST-binding surface is used to immobilize the PDZ-domain polypeptide).
  • GST-binding surface is used to immobilize the PDZ-domain polypeptide.
  • One embodiment of the present invention includes a chimeric PDZ domain- containing polypeptide fused to GST.
  • PCR products containing the chimeric PDZ domain polynucleotide are subcloned into an expression vector to permit expression of fusion proteins containing a chimeric polypeptide with at least one introduced PDZ domain and a heterologous domain (i.e., a glutathione-S transferase sequence, "GST").
  • PCR products i.e., DNA fragments
  • DNA fragments representing the chimeric polynucleotide with at least one introduced PDZ domain encoding DNA are extracted from agarose gels using the "Sephaglas" gel extraction system (Pharmacia) according to the manufacturer's re commendations .
  • PCR primers are designed to include endonuclease restriction sites to facilitate ligation of PCR fragments into a GST gene fusion vector (pGEX-3x; Pharmacia, GenBank accession no. XXUl 3852) in- frame with the glutathione-S transferase coding sequence.
  • This vector contains an IPTG inducible lacZ promoter.
  • the pGEX-3x vector was linearized using Bam HI and Eco RI or, in some cases, Eco RI or Sma I, and dephosphorylated. For most cloning approaches, double digestion with Bam HI and Eco RI was performed, so that the ends of the PCR fragments to clone are Bam HI and Eco RI.
  • restriction endonuclease combinations used are BgI II and Eco RI, Bam HI and Mfe I, or Eco RI only, Sma I only, or BamHI only.
  • the DNA portion cloned represents the PDZ domains and the cDNA portion located between individual domains.
  • DNA linker sequences between the GST portion and the chimeric PDZ containing polynucleotide portion vary slightly, dependent on which of the above described cloning sites and approaches are used.
  • the amino acid sequence of the GST-chimeric PDZ domain containing fusion protein varies in the linker region between GST and the chimeric PDZ domain-containing polypeptide.
  • Linker sequences corresponding to different cloning sites/approaches are shown below. Linker sequences (vector DNA encoded) are bold, sequences of the chimeric polynucleotides containing at least one introduced PDZ domain are in italics.
  • Ligation is performed for 4-10 hours at 7C using T 4 DNA ligase. It will be understood that some of the resulting constructs include very short linker sequences and that, when multiple PDZ domains are cloned, the constructs included some DNA located between individual PDZ domains.
  • the ligation products are transformed in DH5alpha or BL-21 E. coli bacteria strains. Colonies are screened for presence and identity of the cloned chimeric polynucleotide containing at least one introduced PDZ domain as well as for correct fusion with the glutathione S-transferase encoding DNA portion by PCR and by sequence analysis. Positive clones are tested in a small-scale assay for expression of the GST/chimeric PDZ domain fusion protein and, if expressing, these clones are subsequently grown up for large scale preparations of GST/chimeric PDZ domain fusion protein.
  • Fusion protein of GST and chimeric PDZ-domain containing polypeptide is overexpressed following addition of IPTG to the culture medium and purified.
  • GST Gene Fusion System second edition, revision 2; published by Pharmacia.
  • a small culture (50 mis) containing a bacterial strain (DH5 ⁇ , BL21 or JM 109) with the fusion protein construct is grown overnight in 2xYT media at 37 0 C with the appropriate antibiotic selection (100 ug/ml ampicillin; a.k.a. 2x YT-amp).
  • the overnight culture is poured into a fresh preparation of 2xYT-amp (typically 1 liter) and grown until the optical density (OD) of the culture is between 0.5 and 0.9 (approximately 2.5 hours).
  • IPTG isopropyl ⁇ -D-thiogalactopyranoside
  • All following steps, including centrifugation, are performed on ice or at 4 0 C.
  • Bacteria are collected by centrifugation (450Ox g) and resuspended in Buffer A- (50 mM Tris, pH 8.0, 50 mM dextrose, 1 mM EDTA, 200 uM phenylmethylsulfonylfluoride).
  • Buffer A- 50 mM Tris, pH 8.0, 50 mM dextrose, 1 mM EDTA, 200 uM phenylmethylsulfonylfluoride.
  • Buffer A+ Buffer A-, 4 mg/ml lysozyme
  • Buffer B 10 mM Tris, pH 8.0, 50 mM KCl, 1 mM EDTA. 0.5% Tween- 20, 0.5% NP40 (a.k.a.
  • Fusion proteins are assayed for size and quality by SDS gel electrophoresis (PAGE) as described in "Sambrook.” Fusion protein aliquots are stored at minus 8O 0 C. and at minus 2O 0 C.
  • amino acid sequences provided in Table 2 may contain amino acids derived from a fusion protein, e.g., fusion protein of GST and chimeric PDZ-domain containing polypeptide of particular interest may be up to 20 amino acids shorter (e.g., 5, 8, 10, 12 or 15 amino acids shorter) than the sequence provided in Table 2.
  • a sequence may be shortened by up to 3, 6, 9, or 12 amino acids from the C-terminus, the N-terminus, or both termini.
  • Certain PDZ domains are bound by the C-terminal residues of PDZ-binding proteins.
  • the C-terminal residues of sequences are visually inspected for sequences that one might predict would bind to PDZ -domain containing proteins (see, e.g., Doyle et al., 1996, Cell 85, 1067; Songyang et al., 1997, Science 275, 73), including the additional consensus for PLs identified at Arbor Vita Corporation (U.S. patent application Ser. No. 60/360061). TABLE 1 lists some of these proteins, and provides corresponding C-terminal sequences.
  • Synthetic peptides of defined sequence can be synthesized by any standard resin-based method (see, e.g., U.S. Pat. No. 4,108,846; see also, Caruthers et al., 1980, Nucleic Acids Res. Symp. Ser., 215-223; Horn et al., 1980, Nucleic Acids Res. Symp. Ser., 225-232; Roberge, et al., 1995, Science 269:202).
  • the peptides used in the assays described herein are prepared by the FMOC (see, e.g., Guy and Fields, 1997, Meth. Enz.
  • peptides are labeled with biotin at the amino-terminus by reaction with a four-fold excess of biotin methyl ester in dimethylsulfoxide with a catalytic amount of base.
  • the peptides are cleaved from the resin using a halide containing acid (e.g. trifluoroacetic acid) in the presence of appropriate antioxidants (e.g. ethanedithiol) and excess solvent lyophilized.
  • peptides can be redissolved and purified by reverse phase high performance liquid chromatography (HPLC).
  • HPLC solvent system involves a Vydac C- 18 semi-preparative column running at 5 mL per minute with increasing quantities of acetonitrile plus 0.1% trifluoroacetic acid in a base solvent of water plus 0.1% trifluoroacetic acid.
  • the identities of the peptides are confirmed by MALDI cation-mode mass spectrometry.
  • the present invention also relates to the detection of the interaction between a chimeric PDZ domain- containing polypeptide and a PDZ ligand.
  • Various assay formats known in the art can be used to select ligands that are specifically reactive with a particular protein. For example, solid-phase ELISA immunoassays, immunoprecipitation, Biacore, and Western blot assays can be used to identify peptides that specifically bind PDZ- domain polypeptides.
  • two different, complementary assays are developed to detect PDZ-PL interactions.
  • one binding partner of a chimeric PDZ domain- containing polypeptide and PDZ ligand pair is immobilized, and the ability of the second binding partner to bind is determined.
  • These assays can be readily used to screen for hundreds to thousands of potential PDZ-ligand interactions in a few hours. Thus these assays can be used to identify yet more novel PDZ-PL interactions in cells. In addition, they can be used to identify antagonists of PDZ- PL interactions.
  • fusion proteins are used in the assays and devices of the invention. Methods for constructing and expressing fusion proteins are well known. Fusion proteins generally are described in Ausubel et al., Kroll et al., 1993, DNA Cell. Biol. 12:441, and Imai et al., 1997, Cell 91:521-30. Usually, the fusion protein includes a domain to facilitate immobilization of the protein to a solid substrate ("an immobilization domain"). Often, the immobilization domain includes an epitope tag (i.e., a sequence recognized by an antibody, typically a monoclonal antibody) such as polyhistidine (Bush et al, 1991, J.
  • an epitope tag i.e., a sequence recognized by an antibody, typically a monoclonal antibody
  • the immobilization domain is a GST coding region. It will be recognized that, in addition to the chimeric PDZ domain- containing polypeptide and the particular residues bound by an immobilized antibody, protein A, or otherwise contacted with the surface, the protein (e.g., fusion protein), will contain additional residues.
  • residues of synthetic e.g., poly(alanine)
  • heterologous origin e.g., spacers of, e.g., between 10 and 300 residues.
  • Chimeric PDZ domain-containing polypeptide used in the methods of the invention are typically made by (1) constructing a vector (e.g., plasmid, phage or phagemid) comprising a polynucleotide sequence encoding the desired polypeptide, (2) introducing the vector into an suitable expression system (e.g., a prokaryotic, insect, mammalian, or cell free expression system), (3) expressing the fusion protein and (4) optionally purifying the fusion protein.
  • a vector e.g., plasmid, phage or phagemid
  • an suitable expression system e.g., a prokaryotic, insect, mammalian, or cell free expression system
  • expression of the protein comprises inserting the coding sequence into an appropriate expression vector (i.e., a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence required for the expression system employed, e.g., control elements including enhancers, promoters, transcription terminators, origins of replication, a suitable initiation codon (e.g., methionine), open reading frame, and translational regulatory signals (e.g., a ribosome binding site, a termination codon and a polyadenylation sequence.
  • control elements including enhancers, promoters, transcription terminators, origins of replication, a suitable initiation codon (e.g., methionine), open reading frame, and translational regulatory signals (e.g., a ribosome binding site, a termination codon and a polyadenylation sequence.
  • a suitable initiation codon e.g., methionine
  • open reading frame e.g., open reading frame
  • translational regulatory signals e.g
  • the coding sequence of the fusion protein includes a chimeric PDZ domain-containing polynucleotide sequence and an immobilization domain as described herein.
  • Polynucleotides encoding the amino acid sequence for each domain can be obtained in a variety of ways known in the art; typically the polynucleotides are obtained by PCR amplification of cloned plasmids, cDNA libraries, and cDNA generated by reverse transcription of RNA, using primers designed based on sequences determined by the practitioner or, more often, publicly available (e.g., through GenBank).
  • the primers include linker regions (e.g., sequences including restriction sites) to facilitate cloning and manipulation in production of the fusion construct.
  • the polynucleotides corresponding to the PDZ and immobilization regions are joined in-frame to produce the fusion protein-encoding sequence.
  • the fusion proteins of the invention may be expressed as secreted proteins (e.g., by including the signal sequence encoding DNA in the fusion gene; see, e.g., Lui et al, 1993, PNAS USA, 90:8957-61) or as nonsecreted proteins.
  • the chimeric PDZ domain-containing polypeptides or PL polypeptides are immobilized on a solid surface.
  • the substrate to which the polypeptide is bound may in any of a variety of forms, e.g., a microtiter dish, a test tube, a dipstick, a microcentrifuge tube, a bead, a spinnable disk, a permeable or semipermeable membrane, and the like.
  • Suitable materials include glass, plastic (e.g., polyethylene, PVC, polypropylene, polystyrene, and the like), protein, paper, carbohydrate, lipid monolayer or supported lipid bilayer, films and other solid supports.
  • Other materials that may be employed include ceramics, metals, metalloids, semiconductive materials, cements and the like.
  • the chimeric PDZ domain-containing polypeptides and/or PL fusion proteins are organized as an array.
  • array refers to an ordered arrangement of immobilized fusion proteins, in which particular different fusion proteins (i.e., having different PDZ domains) are located at different predetermined sites on the substrate. Because the location of particular fusion proteins on the array is known, binding at that location can be correlated with binding to the chimeric PDZ domain-containing polypeptide situated at that location. Immobilization of fusion proteins on beads (individually or in groups) is another particularly useful approach. In one embodiment, individual fusion proteins are immobilized on beads. In one embodiment, mixtures of distinguishable beads are used.
  • Distinguishable beads are beads that can be separated from each other on the basis of a property such as size, magnetic property, color (e.g., using FACS) or affinity tag (e.g., a bead coated with protein A can be separated from a bead not coated with protein A by using IgG affinity methods). Binding to particular chimeric PDZ domain- containing polypeptide may be determined.
  • a property such as size, magnetic property, color (e.g., using FACS) or affinity tag (e.g., a bead coated with protein A can be separated from a bead not coated with protein A by using IgG affinity methods). Binding to particular chimeric PDZ domain- containing polypeptide may be determined.
  • Methods for immobilizing proteins are known, and include covalent and non-covalent methods.
  • One suitable immobilization method is antibody-mediated immobilization.
  • an antibody specific for the sequence of an "immobilization domain" of the PDZ-domain containing protein is itself immobilized on the substrate (e.g., by adsorption).
  • One advantage of this approach is that a single antibody may be adhered to the substrate and used for immobilization of a number of polypeptides (sharing the same immobilization domain). For example, an immobilization domain consisting of poly-histidine (Bush et al, 1991, J.
  • Biol Chem 266:13811-14 can be bound by an anti-histidine monoclonal antibody (R&D Systems, Minneapolis, Minn.); an immobilization domain consisting of secreted alkaline phosphatase (“SEAP") (Berger et al, 1988, Gene 66:1-10) can be bound by anti- SEAP (Sigma Chemical Company, St. Louis, Mo.); an immobilization domain consisting of a FLAG epitope can be bound by anti-FLAG.
  • SEAP secreted alkaline phosphatase
  • SEAP secreted alkaline phosphatase
  • SEAP secreted alkaline phosphatase
  • FLAG epitope can be bound by anti-FLAG.
  • Other ligand-antiligand immobilization methods are also suitable (e.g., an immobilization domain consisting of protein A sequences (Harlow and Lane, 1988, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory; Sigma Chemical Co., St.
  • the immobilization domain is a GST moiety, as described herein.
  • glass and plastic are especially useful substrates.
  • the substrates may be printed with a hydrophobic (e.g., Teflon) mask to form wells.
  • Preprinted glass slides with 3, 10 and 21 wells per 14.5 cm 2 slide "working area" are available from, e.g., SPI Supplies, West Chester, Pa.; also see U.S. Pat. No. 4,011,350).
  • a large format (12.4 cm x 8.3 cm) glass slide is printed in a 96 well format is used; this format facilitates the use of automated liquid handling equipment and utilization of 96 well format plate readers of various types (fluorescent, colorimetric, scintillation).
  • higher densities may be used (e.g., more than 10 or 100 polypeptides per cm 2 ). See, e.g., MacBeath et al, 2000, Science 289: 1760-63.
  • antibodies are bound to substrates (e.g., glass substrates) by adsorption.
  • Suitable adsorption conditions include incubation of 0.5-50 ug/ml (e.g., 10 ug/ml) mAb in buffer (e.g., PBS, or 50 to 300 mM Tris, MOPS, HEPES, PIPES, acetate buffers, pHs 6.5 to 8, at 4 0 C) to 37 0 C. and from lhr to more than 24 hours.
  • buffer e.g., PBS, or 50 to 300 mM Tris, MOPS, HEPES, PIPES, acetate buffers, pHs 6.5 to 8, at 4 0 C
  • Proteins may be covalently bound or noncovalently attached through nonspecific bonding. If covalent bonding between the fusion protein and the surface is desired, the surface will usually be polyfunctional or be capable of being polyfunctionalized. Functional groups which may be present on the surface and used for linking can include carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like. The manner of linking a wide variety of compounds to various surfaces is well known and is amply illustrated in the literature. Exemplary assays:
  • the invention provides an assay in which biotinylated candidate PL peptides are immobilized on an avidin-coated surface. The binding of PDZ-domain fusion protein to this surface is then measured.
  • the PDZ-domain fusion protein is a chimeric PDZ domain-containing protein fused to GST (GST-chimeric PDZ fusion protein) and the assay is carried out as follows:
  • Avidin is bound to a surface, e.g. a protein binding surface.
  • avidin is bound to a polystyrene 96 well plate (e.g., Nunc Polysorb (cat #475094) by addition of 100 uL per well of 20 ug/mL of avidin (Pierce) in phosphate buffered saline without calcium and magnesium, pH 7.4 ("PBS", GibcoBRL) at 4 0 C. for 12 hours.
  • PBS phosphate buffered saline without calcium and magnesium, pH 7.4
  • the plate is then treated to block nonspecific interactions by addition of 200 uL per well of PBS containing 2 g per 100 mL protease-free bovine serum albumin ("PBS/BSA”) for 2 hours at 4 0 C.
  • PBS/BSA protease-free bovine serum albumin
  • Biotinylated PL peptides are immobilized on the surface of wells of the plate by addition of 5O uL per well of 0.4 uM peptide in PBS/BSA for 30 minutes at 4 0 C.
  • each different peptide is added to at least eight different wells so that multiple measurements (e.g. duplicates and also measurements using different (GST/PDZ-domain fusion proteins and a GST alone negative control) can be made, and also additional negative control wells are prepared in which no peptide is immobilized.
  • the plate is washed 3 times with PBS.
  • GST-chimeric PDZ-domain fusion protein (prepared as described) is allowed to react with the surface by addition of 50 uL per well of a solution containing 5 ug/mL GST-chimeric PDZ-domain fusion protein in PBS/BSA for 2 hours at 4 0 C.
  • GST alone i.e. not a fusion protein
  • specified wells generally at least 2 wells (i.e. duplicate measurements) for each immobilized peptide.
  • the plate is washed 3 times with PBS to remove unbound fusion protein.
  • the binding of the GST-chimeric PDZ-domain fusion protein to the avidin-biotinylated peptide surface can be detected using a variety of methods, and detectors known in the art.
  • 50 uL per well of an anti-GST antibody in PBS/BSA e.g. 2.5 ug/mL of polyclonal goat-anti-GST antibody, Pierce
  • PBS/BSA polyclonal goat-anti-GST antibody, Pierce
  • HRP horseradish peroxidase
  • TMB horseradish peroxidase-conjugated polyclonal rabbit anti-goat immunoglobulin antibody
  • a specific or selective reaction will be at least twice background signal, more typically more than 5 times background, and most typically 10 or more times the background signal.
  • a statistically significant reaction will involve multiple measurements of the reaction with the signal and the background differing by at least two standard errors, more typically four standard errors, and most typically six or more standard errors.
  • a statistical test e.g. a T-test
  • comparing repeated measurements of the signal with repeated measurements of the background will result in a p-value ⁇ 0.05, more typically a p-value ⁇ 0.01, and most typically a p-value ⁇ 0.001 or less.
  • the signal from binding of a GST-chimeric PDZ-domain fusion protein to an avidin surface not exposed to (i.e. not covered with) the PL peptide is one suitable negative control (sometimes referred to as "B").
  • the signal from binding of GST polypeptide alone (i.e. not a fusion protein) to an avidin-coated surface that has been exposed to (i.e. covered with) the PL peptide is a second suitable negative control (sometimes referred to as "B2"). Because all measurements are done in multiples (i.e.
  • the arithmetic mean (or, equivalently, average) of several measurements is used in determining the binding, and the standard error of the mean is used in determining the probable error in the measurement of the binding.
  • the standard error of the mean of N measurements equals the square root of the following: the sum of the squares of the difference between each measurement and the mean, divided by the product of (N) and (N-I).
  • specific binding of the PDZ protein to the plate-bound PL peptide is determined by comparing the mean signal ("mean S") and standard error of the signal (“SE”) for a particular PL-PDZ combination with the mean Bl and/or mean B2.
  • G Assay Detection of PDZ-Ligand Binding Using Immobilized GST-chimeric PDZ-domain fusion protein
  • the invention provides an assay in which a GST-chimeric PDZ-domain fusion protein is immobilized on a surface ("G" assay). The binding of labeled PL peptide (e.g., as listed in TABLE 1) to this surface is then measured.
  • the assay is carried out as follows:
  • a GST-chimeric PDZ-domain fusion protein is bound to a surface, e.g. a protein binding surface.
  • a GST-chimeric PDZ-domain fusion protein containing one or more introduced PDZ domains is bound to a polystyrene 96-well plate.
  • the GST-chimeric PDZ-domain fusion protein can be bound to the plate by any of a variety of standard methods known to one of skill in the art, although some care must be taken that the process of binding the fusion protein to the plate does not alter the ligand-binding properties of the PDZ domain.
  • the GST-chimeric PDZ-domain fusion protein is bound via an anti-GST antibody that is coated onto the 96-well plate. Adequate binding to the plate can be achieved when:
  • Biotinylated PL peptides are allowed to react with the surface by addition of 50 uL per well of 20 uM solution of the biotinylated peptide in PBS/BSA for 10 minutes at 4 0 C, followed by an additional 20 minute incubation at 25 C. The plate is washed 3 times with ice cold PBS.
  • (4) Specific binding of a PL peptide and a PDZ domain polypeptide is determined by comparing the signal from the well(s) in which the PL peptide and PDZ domain polypeptide are combined, with the background signal(s).
  • the background signal is the signal found in the negative control(s).
  • a specific or selective reaction will be at least twice background signal, more typically more than 5 times background, and most typically 10 or more times the background signal.
  • a statistically significant reaction will involve multiple measurements of the reaction with the signal and the background differing by at least two standard errors, more typically four standard errors, and most typically six or more standard errors.
  • a statistical test e.g.
  • the signal from binding of a given PL peptide to immobilized (surface bound) GST polypeptide alone is one suitable negative control (sometimes referred to as "B 1"). Because all measurement are done in multiples (i.e. at least duplicate) the arithmetic mean (or, equivalently, average.) of several measurements is used in determining the binding, and the standard error of the mean is used in determining the probable error in the measurement of the binding.
  • the standard error of the mean of N measurements equals the square root of the following: the sum of the squares of the difference between each measurement and the mean, divided by the product of (N) and (N-I).
  • specific binding of the PDZ protein to the platebound peptide is determined by comparing the mean signal ("mean S") and standard error of the signal ("SE”) for a particular PL-PDZ combination with the mean Bl.
  • the "G” assay” is identical to the “G 0 assay” except that at step (2) the peptide concentration is 1 uM instead of 20 uM and the incubation is performed for 60 minutes at 25 0 C (rather than, e.g., 10 minutes at 4 0 C. followed by 20 minutes at 25 0 C). This results in lower sensitivity for interactions of low affinity, rapid dissociation rate, and/or affinity that is less at 25 0 C than at 4 0 C. Interactions will have lower affinity at 25 0 C than at 4 0 C. if (as we have found to be generally true for PDZ-ligand binding) the reaction entropy is negative (i.e.
  • the entropy of the products is less than the entropy of the reactants).
  • the PDZ-PL binding signal may be similar in the "G” assay” and the “G 0 assay” for interactions of slow association and dissociation rate, as the PDZ-PL complex will accumulate during the longer incubation of the "G” assay.”
  • comparison of results of the "G” assay” and the "G 0 assay” can be used to estimate the relative entropies, enthalpies, and kinetics of different PDZ-PL interactions.
  • thermodynamics and kinetics of PDZ-PL interactions can be used in the design of efficient inhibitors of the interactions.
  • PDZ domain (as evidenced by similar binding in the "G” assay” as in the “G 0 assay") may itself dissociate slowly and thus be of high affinity.
  • step (2) of the "G assay” variation of the temperature and duration of step (2) of the "G assay” can be used to provide insight into the kinetics and thermodynamics of the PDZ-ligand binding reaction and into design of inhibitors of the reaction.
  • the PDZ-PL detection assays can employ a variety of surfaces to bind the PL and/or PDZ-containing proteins.
  • a surface can be an "assay plate" which is formed from a material (e.g. polystyrene) which optimizes adherence of either the PL protein or PDZ-containing protein thereto.
  • the individual wells of the assay plate will have a high surface area to volume ratio and therefore a suitable shape is a flat bottom well
  • proteins of the assays are adherent.
  • Other surfaces include, but are not limited to, polystyrene or glass beads, polystyrene or glass slides, papers, dipsticks, plastics, films and the like.
  • the assay plate can be a "microtiter" plate.
  • microtiter plate when used herein refers to a multiwell assay plate, e.g., having between about 30 to 200 individual wells, usually 96 wells.
  • the assay plate is a 96 well polystyrene plate (such as that sold by
  • aqueous sample comprising buffers suspended therein will be added to each well of the assay plate.
  • the detectable labels of the invention can be any detectable compound or composition which is conjugated directly or indirectly with a molecule (such as described above).
  • the label can be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, can catalyze a chemical alteration of a substrate compound or composition which is detectable.
  • the preferred label is an enzymatic one which catalyzes a color change of a non-radioactive color reagent.
  • the label can be indirectly conjugated with the antibody.
  • the antibody can be conjugated with biotin and any of the categories of labels mentioned above can be conjugated with avidin, or vice versa (see also "A” and "G” assay above). Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner. See, Au subel, supra, for a review of techniques involving biotin-avidin conjugation and similar assays.
  • the antibody is conjugated with a small hapten (e.g.
  • Assay variations can include different washing steps.
  • washing is meant exposing the solid phase to an aqueous solution (usually a buffer or cell culture media) in such a way that unbound material (e.g., non-adhering cells, non-adhering capture agent, unbound ligand, receptor, receptor construct, cell lysate, or HRP antibody) is removed therefrom.
  • a detergent e.g., Triton X
  • the aqueous washing solution is decanted from the wells of the assay plate following washing.
  • washing can be achieved using an automated washing device.
  • several washing steps e.g., between about 1 to 10 washing steps.
  • blocking buffer refers to an aqueous, pH buffered solution containing at least one blocking compound which is able to bind to exposed surfaces of the substrate which are not coated with a PL or a chimeric PDZ domain containing polypeptide.
  • the blocking compound is normally a protein such as bovine serum albumin (BSA), gelatin, casein or milk powder and does not cross-react with any of the reagents in the assay.
  • BSA bovine serum albumin
  • the block buffer is generally provided at a pH between about 7 to 7.5 and suitable buffering agents include phosphate and TRIS.
  • enzyme-substrate combinations can also be utilized in detecting PDZ-PL interactions.
  • enzyme-substrate combinations include, for example: (i) Horseradish peroxidase (HRP or HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g.
  • orthophenylene diamine [OPD] or 3,3',5,5'-tetramethyl benzidine hydrochloride [TMB]) (as described above), (ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate, (iii) Beta-D-galactosidase (Beta D-GaI) with a chromogenic substrate (e.g. p-nitrophenyl-Beta-D-galactosidase) or fluorogenic substrate 4-methylumbelliferyl- Beta-D-galactosidase.
  • AP alkaline phosphatase
  • Beta-D-galactosidase Beta-D-galactosidase
  • a chromogenic substrate e.g. p-nitrophenyl-Beta-D-galactosidase
  • the present invention relates to the method of determining if a subject is infected with an oncogenic strain of HPV. Diagnosing the presence of pathogens requires collection of samples appropriate to the organism. For detection of oncogenic HPV E6 proteins, one could collect tissue for testing from the cervix, penis, anus, or throat using a scrape, swab or biopsy technique. For diagnosis of bloodborne pathogens such as HIV, collection of blood through standard means would be most appropriate. Diagnosis of fungal or viral infections that may have caused skin lesions would require the collection of a sample from the affected area.
  • This invention is not intended to cover sampling devices. However, it should be noted that since the invention is predicated on the detection of PDZ or PL proteins, appropriate care must be taken to collect a sufficient amount of sample to detect pathogen proteins and to maintain the integrity of proteins in the sample. The amount of sample to collect should be determined empirically for each diagnostic test. Factors in the decision may include, but not be limited to, the stage at which detection is desired, the amount of pathogen per unit sample, the amount of diagnostic protein per unit per unit sample, availability of diagnostic epitopes and the stability of diagnostic epitopes.
  • Exemplary collection devices for cervical tissue include, but are not limited to, those described in U.S. Pat. Nos. 6,241,687, 6,352,513, 6,336,905, 6,115,990 and 6,346,086. These collection devices would facilitate the collection of cervical tissue for the diagnosis of oncogenic human papillomavirus infection. These devices are predominantly collection of cervical cells or tissues through scraping; alternatively, one could use standard biopsy methods to collect samples from any tissues to be examined.
  • sample collection need not be limited to collection of proteins.
  • DNA could be collected from test samples, specific primers for oncogenic E6 proteins could be used to either amplify the DNA content (using a DNA polymerase) or transcribe and translate the sample into proteins that could be tested with methods disclosed herein. VII. Assays for Detecting Oncogenic E6 Proteins
  • One embodiment of the present invention includes the detection of oncogenic E6 proteins using a chimeric protein containing at least one introduced PDZ domain that has higher binding strength to E6 than an isolated PDZ domain.
  • Oncogenic E6 proteins can be detected by their ability to bind to chimeric PDZ domain containing polypeptides. This could be developed into a single detection stage approach or more favorably as a two-stage or "sandwich" approach for increased sensitivity and specificity.
  • a "tagged" version of a PDZ domain that specifically recognizes oncogenic E6 proteins can be used to directly probe for the presence of oncogenic E6 protein in a sample.
  • an example of this would be to attach the test sample to a solid support (for example, cervical cells or tissue could be coated on a slide and "fixed” to permeablize the cell membranes), incubate the sample with a tagged chimeric PDZ domain-containing polypeptide (PL detector, e.g. GST-chimeric PDZ fusion protein) under appropriate conditions, wash away unbound PL detector, and assay for the presence of the "tag" in the sample.
  • PL detector e.g. GST-chimeric PDZ fusion protein
  • chimeric PDZ domain-containing protein is coupled with a second method of either capturing or detecting captured proteins.
  • the second method could be using an antibody that binds to the E6 oncoprotein or a second compound or protein that can bind to E6 oncorproteins at a location on the E6 protein that does not reduce the availability of the E6 PDZ ligand.
  • Such proteins may include, but not be limited to, p53, E6-AP, E6-BP or engineered compounds that bind E6 oncoproteins.
  • the peptide may be attached to a suitable carrier, such as BSA or KLH, by means of a side chain functional group or linkers attached to a side chain functional group.
  • a suitable carrier such as BSA or KLH
  • Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.
  • Monoclonal antibodies to a peptide may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein, 1975, Nature 256:495-497, the human B-cell hybridoma technique, Kosbor et al., 1983, Immunology Today 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026- 2030 and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)).
  • Antibody fragments which contain deletions of specific binding sites may be generated by known techniques.
  • fragments include but are not limited to F(ab') 2 fragments, which can be produced by pepsin digestion of the antibody molecule and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed (Huse et al., 1989, Science 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for the peptide of interest.
  • the antibody or antibody fragment specific for the desired peptide can be attached, for example, to agarose, and the antibody-agarose complex is used in immunochromatography to purify peptides of the invention. See, Scopes, 1984, Protein Purification: Principles and Practice, Springer- Verlag New York, Inc., N. Y., Livingstone, 1974, Methods Enzymology: Immunoaffmity Chromatography of Proteins 34:723-731.
  • Antibodies can also be linked to other solid supports for diagnostic applications, or alternatively labeled with a means of detection such an enzyme that can cleave a colorimetric substrate, a fluorophore, a magnetic particle, or other measurable compositions of matter.
  • E6 proteins have historically been difficult to produce.
  • An example is to prepare the E6 antigen (to raise antibodies against) in the same manner that one would prepare tissue or cell samples for testing.
  • Another method that could be employed is to use peptides corresponding to antigenic regions of the E6 proteins as predicted by Major Histocompatibility Complex (MHC) and T Cell Receptor (TCR) consensus binding.
  • MHC Major Histocompatibility Complex
  • TCR T Cell Receptor
  • E6 proteins that have been captured by PDZ domains could be detected by several alternative methods. Several proteins are known to associate with E6 proteins. Any of them that had a reasonable affinity for E6 could be used to detect the presence of captured and concentrated E6 protein in a sample by one skilled in the art. In addition, new binding proteins or aptamers could be identified that bound to E6 proteins. Third, activity assays specific for E6 could be employed.
  • the detection assay itself could also be carried out using a variety of methods.
  • a standard ELISA using a PDZ to capture could be set up as a competition, where the PDZ domain is pre-loaded with a labeled PL that has lower affinity than the E6 proteins. Thus, in the presence of E6, the label is displaced and one sees a reduction of signal that corresponds to E6 presence.
  • Other variants that use aspects of competition and inhibition of binding are intended to be included as well.
  • One variant could even have the PL covalently attached to the PDZ domain through a linker such that the PL could bind its own PDZ domain.
  • the present invention provides a method for detecting PDZ-PL interaction with enhanced PDZ binding to PL using a chimeric protein containing at least one introduced PDZ domain from a PDZ domain-containing polypeptide.
  • the "A” and “G” assays of the invention can be used to determine the "apparent affinity" of binding of a PDZ ligand peptide to a chimeric PDZ domain-containing protein. Apparent affinity is determined based on the concentration of one molecule required to saturate the binding of a second molecule (e.g., the binding of a ligand to a receptor). Two particularly useful approaches for quantitation of apparent affinity of PDZ-ligand binding are provided infra.
  • the PL peptide is allowed to react with the bound GST/ chimeric PDZ fusion protein as well as the GST alone negative control) for 10 minutes at 4 0 C followed by 20 minutes at 25 0 C.
  • the plate is washed 3 times with ice cold PBS to remove unbound labeled peptide.
  • the net binding signal is determined by subtracting the binding of the peptide to GST alone from the binding of the peptide to the GST- chimeric PDZ fusion protein. The net binding signal is then plotted as a function of ligand concentration and the plot is fit (e.g.
  • a fixed concentration of a chimeric PDZ-domain containing polypeptide and increasing concentrations of a labeled PL peptide (labeled with, for example, biotin or fluorescein, see TABLE 1 for representative peptide amino acid sequences) are mixed together in solution and allowed to react.
  • preferred peptide concentrations are 0.1 uM, 1 uM, 10 uM, 100 uM, 1 mM.
  • appropriate reaction times can range from 10 minutes to 2 days at temperatures ranging from 4 0 C to 37 0 C.
  • the identical reaction can also be carried out using a non-PDZ domain-containing protein as a control (e.g., if the chimeric PDZ- domain containing polypeptide is a fusion protein, the fusion partner can be used).
  • (2) PDZ-ligand complexes can be separated from unbound labeled peptide using a variety of methods known in the art.
  • the complexes can be separated using high performance size-exclusion chromatography (HPSEC, gel filtration) (Rabinowitz et al., 1998, Immunity 9:699), affinity chromatography (e.g. using glutathione Sepharose beads), and affinity absorption (e.g., by binding to an anti-GST-coated plate as described supra).
  • HPSEC high performance size-exclusion chromatography
  • affinity chromatography e.g. using glutathione Sepharose beads
  • affinity absorption e.g., by binding to an anti-GST-coated plate as described supra.
  • the PDZ-ligand complex is detected based on presence of the label on the peptide ligand using a variety of methods and detectors known to one of skill in the art. For example, if the label is fluorescein and the separation is achieved using HPSEC, an in-line fluorescence detector can be used. The binding can also be detected as described supra for the G assay.
  • the invention provides a method of determining the apparent affinity of binding between a PDZ domain and a ligand by immobilizing a polypeptide comprising the PDZ domain and a non-PDZ domain on a surface, contacting the immobilized polypeptide with a plurality of different concentrations of the ligand, determining the amount of binding of the ligand to the immobilized polypeptide at each of the concentrations of ligand, and calculating the apparent affinity of the binding based on that data.
  • the chimeric polypeptide comprising at least one introduced PDZ domain is a fusion protein.
  • the e.g., fusion protein is GST-chimeric PDZ domain containing fusion protein, but other polypeptides can also be used (e.g., a fusion protein including a PDZ domain and any of a variety of epitope tags, biotinylation signals and the like) so long as the polypeptide can be immobilized.
  • a fusion protein including a PDZ domain and any of a variety of epitope tags, biotinylation signals and the like so long as the polypeptide can be immobilized.
  • a fusion protein including a PDZ domain and any of a variety of epitope tags, biotinylation signals and the like so long as the polypeptide can be immobilized.
  • a fusion protein including a PDZ domain and any of a variety of epitope tags, biotinylation signals and the like so long as the polypeptide can be immobilized.
  • the chimeric polypeptide is immobilized by binding the polypeptide to an immobilized immunoglobulin that binds the non-PDZ domain (e.g., an anti-GST antibody when a GST-chimeric PDZ fusion polypeptide is used).
  • an immobilized immunoglobulin that binds the non-PDZ domain e.g., an anti-GST antibody when a GST-chimeric PDZ fusion polypeptide is used.
  • the step of contacting the ligand and PDZ-domain polypeptide is carried out under the conditions provided supra in the description of the "G" assay. It will be appreciated that binding assays are conveniently carried out in multiwell plates (e.g., 24-well, 96-well plates, or 384 well plates).
  • the present method has considerable advantages over other methods for measuring binding affinities PDZ- PL affinities, which typically involve contacting varying concentrations of a GST-PDZ fusion protein to a ligand- coated surface.
  • some previously described methods for determining affinity e.g., using immobilized ligand and GST-PDZ protein in solution
  • the present invention provides methods for analysis of PDZ-ligand interactions using a chimeric protein containing at least one introduced PDZ domain that has enhanced binding strength to a PDZ ligand.
  • the chimeric polypeptide described herein can have multiple introduced PDZ domains.
  • the affinity is determined for a particular ligand and a plurality of PDZ domains.
  • the plurality of different PDZ proteins are from a particular tissue (e.g., central nervous system, spleen, cardiac muscle, kidney) or a particular class or type of cell, (e.g., a hematopoietic cell, a lymphocyte, a neuron) and the like.
  • the plurality of different PDZ proteins represents a substantial fraction (e.g., typically a majority, more often at least 80%) of all of the PDZ proteins known to be, or suspected of being, expressed in the tissue or cell(s), e.g., all of the PDZ proteins known to be present in lymphocytes.
  • the plurality is at least 50%, usually at least 80%, at least 90% or all of the PDZ proteins disclosed herein as being expressed in hematopoietic cells.
  • the binding of a ligand to the plurality of PDZ domains is determined. Using this method, it is possible to identify a particular PDZ domain bound with particular specificity by the ligand.
  • the binding may be designated as "specific” if the affinity of the ligand to the particular PDZ domain is at least 2- fold that of the binding to other PDZ domains in the plurality (e.g., present in that cell type).
  • the binding is deemed "very specific” if the affinity is at least 10-fold higher than to any other PDZ in the plurality or, alternatively, at least 10-fold higher than to at least 90%, more often 95% of the other PDZs in a defined plurality.
  • the binding is deemed “exceedingly specific” if it is at least 100-fold higher.
  • a ligand could bind to 2 different PDZs with an affinity of 1 uM and to no other PDZs out of a set 40 with an affinity of less than 100 uM. This would constitute specific binding to those 2 PDZs. Similar measures of specificity are used to describe binding of a PDZ domain to a plurality of PLs.
  • the introduced PDZ domain in the chimeric polypeptide has enhanced binding to a PDL ligand, more specifically to an E6 protein from an oncogenic HPV strain.
  • the binding of the chimeric polypeptide containing at least one introduced PDZ domain to E6 is enhanced by at least 2 folds as compared to an isolated PDZ domain.
  • the binding of the chimeric polypeptide containing at least one introduced PDZ domain to E6 is enhanced as compared to a PDZ domain present in a native polypeptide, in which the PDZ domain is not modified in any way. It should be recognized that high specificity PDZ-PL interactions represent potentially more valuable targets for achieving a desired biological effect.
  • an inhibitor or enhancer to act with high specificity is often desirable.
  • the most specific PDZ-ligand interactions are also the diagnostic targets, allowing specific detection of the interaction or disruption of an interaction. Enhancing the binding strength of a PDZ domain- containing protein to a PDZ ligand may improve the sensitivity of a diagnostic assay, such as cervical cancer diagnostic test.
  • the invention provides a method of identifying a high specificity interaction between a particular introduced PDZ domain in a chimeric polypeptide and a ligand known or suspected of binding at least one PDZ domain, by providing a plurality of different immobilized polypeptides, each of said polypeptides comprising a PDZ domain and a non-PDZ domain; determining the affinity of the ligand for each of said polypeptides, and comparing the affinity of binding of the ligand to each of said polypeptides, wherein an interaction between the ligand and a particular PDZ domain is deemed to have high specificity when the ligand binds an immobilized polypeptide comprising the particular PDZ domain with at least 2-fold higher affinity than to immobilized polypeptides not comprising the particular PDZ domain.
  • the affinity of binding of a specific PDZ domain to a plurality of ligands is determined.
  • the invention provides a method of identifying a high specificity interaction between a PDZ domain and a particular ligand known or suspected of binding at least one PDZ domain, by providing an immobilized chimeric polypeptide comprising the PDZ domain and a non-PDZ domain; determining the affinity of each of a plurality of ligands for the polypeptide, and comparing the affinity of binding of each of the ligands to the polypeptide, wherein an interaction between a particular ligand and the PDZ domain is deemed to have high specificity when the ligand binds an immobilized polypeptide comprising the PDZ domain with at least 2-fold higher affinity than other ligands tested.
  • the binding may be designated as "specific” if the affinity of the PDZ to the particular PL is at least 2-fold that of the binding to other PLs in the plurality (e.g., present in that cell type).
  • the binding is deemed “very specific” if the affinity is at least 10-fold higher than to any other PL in the plurality or, alternatively, at least 10-fold higher than to at least 90%, more often 95% of the other PLs in a defined plurality.
  • the binding is deemed “exceedingly specific” if it is at least 100-fold higher.
  • the plurality is at least 5 different ligands, more often at least 10.
  • interactions between PDZ proteins and PL proteins in cells may be disrupted or inhibited by the presence of pathogens.
  • Pathogens can be identified using screening assays described herein. Agonists and antagonists of PDZ-Pathogen PL interactions or PDZ-Cellular PL interactions can be useful in discerning or confirming specific interactions.
  • an agonist will increase the sensitivity of a PDZ-pathogen PL interaction.
  • an antagonist of a PDZ -pathogen PL interaction can be used to verify the specificity of an interaction.
  • the motifs disclosed herein are used to design inhibitors.
  • the antagonists of the invention have a structure (e.g., peptide sequence) based on the C- terminal residues of PL-domain proteins listed in TABLE 1. In some embodiments, the antagonists of the invention have a structure (e.g., peptide sequence) based on a PL motif disclosed herein or in U.S. patent application Ser. No. 09/724553.
  • the PDZ/PL antagonists and antagonists of the invention may be any of a large variety of compounds, both naturally occurring and synthetic, organic and inorganic, and including polymers (e.g., oligopeptides, polypeptides, oligonucleotides, and polynucleotides), small molecules, antibodies, sugars, fatty acids, nucleotides and nucleotide analogs, analogs of naturally occurring structures (e.g., peptide mimetics, nucleic acid analogs, and the like), and numerous other compounds.
  • polymers e.g., oligopeptides, polypeptides, oligonucleotides, and polynucleotides
  • small molecules antibodies, sugars, fatty acids, nucleotides and nucleotide analogs, analogs of naturally occurring structures (e.g., peptide mimetics, nucleic acid analogs, and the like), and numerous other compounds.
  • PDZ-PL interaction agonists can also be use
  • the assays described supra and other assays can also be used to identify the binding of other molecules (e.g., peptide mimetics, small molecules, and the like) to PDZ domain sequences.
  • other molecules e.g., peptide mimetics, small molecules, and the like
  • combinatorial and other libraries of compounds can be screened, e.g., for molecules that specifically bind to PDZ domains. Screening of libraries can be accomplished by any of a variety of commonly known methods.
  • screening can be carried out by contacting the library members with a chimeric PDZ-domain-containing polypeptide immobilized on a solid support (e.g. as described supra in the "G” assay) and harvesting those library members that bind to the protein.
  • a solid support e.g. as described supra in the "G” assay
  • panning techniques are described by way of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and in references cited hereinabove.
  • the two-hybrid system for selecting interacting proteins in yeast can be used to identify molecules that specifically bind to a chimeric PDZ domain- containing protein. Furthermore, the identified molecules are further tested for their ability to inhibit transmembrane receptor interactions with a PDZ domain.
  • antagonists of an interaction between a PDZ containing protein and a PL protein are identified. In one embodiment, a modification of the "A" assay described supra is used to identify antagonists.
  • a modification of the "G" assay described supra is used to identify antagonists.
  • screening assays are used to detect molecules that specifically bind to PDZ domains. Such molecules are useful as agonists or antagonists of PDZ -protein-mediated cell function (e.g., cell activation, e.g., T cell activation, vesicle transport, cytokine release, growth factors, transcriptional changes, cytoskeleton rearrangement, cell movement, chemotaxis, and the like).
  • cell activation e.g., T cell activation, vesicle transport, cytokine release, growth factors, transcriptional changes, cytoskeleton rearrangement, cell movement, chemotaxis, and the like.
  • such assays are performed to screen for leukocyte activation inhibitors for drug development. The invention thus provides sensitive assays to detect molecules that specifically bind to PDZ domain-containing proteins.
  • recombinant cells expressing PDZ domain-encoding nucleic acids can be used to produce PDZ domains in these assays and to screen for molecules that bind to the domains.
  • Molecules are contacted with the PDZ domain (or fragment thereof) under conditions conducive to binding, and then molecules that specifically bind to such domains are identified. Methods that can be used to carry out the foregoing are commonly known in the art.
  • antagonists are identified by conducting the A or G assays in the presence and absence of a known or candidate antagonist. When decreased binding is observed in the presence of a compound, that compound is identified as an antagonist. Increased binding in the presence of a compound signifies that the compound is an agonist.
  • a test compound in one assay, can be identified as an inhibitor (antagonist) of binding between a chimeric PDZ-domain-containing polypeptide and a PL protein by contacting a chimeric PDZ-domain- containing polypeptide and a PL peptide in the presence and absence of the test compound, under conditions in which they would (but for the presence of the test compound) form a complex, and detecting the formation of the complex in the presence and absence of the test compound. It will be appreciated that less complex formation in the presence of the test compound than in the absence of the compound indicates that the test compound is an inhibitor of a chimeric PDZ protein -PL protein binding.
  • the "G” assay is used in the presence or absence of a candidate inhibitor.
  • the "A” assay is used in the presence or absence of a candidate inhibitor.
  • one or more chimeric PDZ-domain-containing polypeptide- GST-fusion proteins are bound to the surface of wells of a 96-well plate as described supra (with appropriate controls including nonfusion GST protein). All fusion proteins are bound in multiple wells so that appropriate controls and statistical analysis can be done. A test compound in BSA/PBS (typically at multiple different concentrations) is added to wells.
  • a detectably labeled (e.g., biotinylated) peptide known to bind to the relevant PDZ domain is added in each of the wells at a final concentration of, e.g., between about 2 uM and about 40 uM, typically 5 uM, 15 uM, or 25 uM.
  • This mixture is then allowed to react with the PDZ fusion protein bound to the surface for 10 minutes at 4 0 C followed by 20 minutes at 25 0 C.
  • the surface is washed free of unbound peptide three times with ice cold PBS and the amount of binding of the peptide in the presence and absence of the test compound is determined.
  • the level of binding is measured for each set of replica wells (e.g. duplicates) by subtracting the mean GST alone background from the mean of the raw measurement of peptide binding in these wells.
  • the A assay is carried out in the presence or absence of a test candidate to identify inhibitors of PL-PDZ interactions.
  • a test compound is determined to be a specific inhibitor of the binding of an introduced PDZ domain (P) in a chimeric polypeptide and a PL (L) sequence when, at a test compound concentration of less than or equal to 1 mM (e.g., less than or equal to: 500 uM, 100 uM, 10 uM, 1 uM, 100 nM or 1 nM) the binding of P to L in the presence of the test compound less than about 50% of the binding in the absence of the test compound, (in various embodiments, less than about 25%, less than about 10%, or less than about 1%).
  • 1 mM e.g., less than or equal to: 500 uM, 100 uM, 10 uM, 1 uM, 100 nM or 1 nM
  • the net signal of binding of P to L in the presence of the test compound plus six (6) times the standard error of the signal in the presence of the test compound is less than the binding signal in the absence of the test compound.
  • assays for an inhibitor are carried out using a single chimeric PDZ-domain-containing polypeptide -PDZ ligand pair (e.g., a chimeric PDZ-GST fusion protein and a PL peptide).
  • the assays are carried out using a plurality of pairs, such as a plurality of different pairs listed in TABLE 3.
  • [00163] it is desirable to identify compounds that, at a given concentration, inhibit the binding of one PL-PDZ pair, but do not inhibit (or inhibit to a lesser degree) the binding of a specified second PL- PDZ pair.
  • These antagonists can be identified by carrying out a series of assays using a candidate inhibitor and different PL-PDZ pairs (e.g., as shown in the matrix of TABLE 3) and comparing the results of the assays. All such pairwise combinations are contemplated by the invention (e.g., test compound inhibits binding OfPL 1 to PDZ 1 to a greater degree than it inhibits binding of PL 1 to PDZ 2 or PL 2 to PDZ 2 ).
  • test compound inhibits binding OfPL 1 to PDZ 1 to a greater degree than it inhibits binding of PL 1 to PDZ 2 or PL 2 to PDZ 2 ).
  • the Ki (“potency") of an inhibitor of a PDZ-PL interaction can be determined.
  • Ki is a measure of the concentration of an inhibitor required to have a biological effect.
  • administration of an inhibitor of a PDZ-PL interaction in an amount sufficient to result in an intracellular inhibitor concentration of at least between about 1 and about 100 Ki is expected to inhibit the biological response mediated by the target PDZ-PL interaction.
  • the Kd measurement of PDZ-PL binding as determined using the methods supra is used in determining Ki.
  • the invention provides a method of determining the potency (Ki) of an inhibitor or suspected inhibitor of binding between a PDZ domain and a ligand by immobilizing a chimeric polypeptide comprising at least one introduced PDZ domain and a non-PDZ domain on a surface, contacting the immobilized polypeptide with a plurality of different mixtures of the ligand and inhibitor, wherein the different mixtures comprise a fixed amount of ligand and different concentrations of the inhibitor, determining the amount of ligand bound at the different concentrations of inhibitor, and calculating the Ki of the binding based on the amount of ligand bound in the presence of different concentrations of the inhibitor.
  • the polypeptide is immobilized by binding the polypeptide to an immobilized immunoglobulin that binds the non-PDZ domain.
  • This method which is based on the "G” assay described supra, is particularly suited for high-throughput analysis of the Ki for inhibitors of PDZ- ligand interactions. Further, using this method, the inhibition of the PDZ-ligand interaction itself is measured, without distortion of measurements by avidity effects. Typically, at least a portion of the ligand is detectably labeled to permit easy quantitation of ligand binding.
  • an enhancer (sometimes referred to as, augmentor or agonist) of binding between a PDZ domain and a ligand is identified by immobilizing a chimeric polypeptide comprising at least one introduced PDZ domain and a non-PDZ domain on a surface, contacting the immobilized polypeptide with the ligand in the presence of a test agent and determining the amount of ligand bound, and comparing the amount of ligand bound in the presence of the test agent with the amount of ligand bound by the polypeptide in the absence of the test agent.
  • At least two-fold (often at least 5-fold) greater binding in the presence of the test agent compared to the absence of the test agent indicates that the test agent is an agent that enhances the binding of the PDZ domain to the ligand.
  • agents that enhance PDZ-ligand interactions are useful for disruption (dysregulation) of biological events requiring normal PDZ-ligand function (e.g., cancer cell division and metastasis, and activation and migration of immune cells).
  • the present invention also embodies a chimeric polypeptide containing a modified PDZ domain introduced from another PDZ domain-containing polypeptide.
  • One embodiment of the invention includes increasing the specificity or sensitivity of a PDZ-PL interaction through mutagenesis and selection of high affinity or high specificity variants. Methods such as chemical (e.g., EMS) or biological mutagenesis (e.g. Molecular shuffling or DNA polymerase mutagenesis) can be applied to create mutations in DNA encoding PDZ domains or PL domains. Proteins can then be made from variants and tested using a number of methods described herein (e.g., ⁇ A ⁇ assay, ⁇ G ⁇ assay or yeast two hybrid).
  • PDZ domain- containing proteins are involved in a number of biological functions, including, but not limited to, vesicular trafficking, tumor suppression, protein sorting, establishment of membrane polarity, apoptosis, regulation of immune response and organization of synapse formation.
  • this family of proteins has a common function of facilitating the assembly of multi-protein complexes, often serving as a bridge between several proteins, or regulating the function of other proteins.
  • these proteins are found in essentially all cell types. Consequently, detection of inappropriate PDZ:PL interactions or abnormal interactions can be utilized to diagnose a wide variety of biological and physiological conditions.
  • detection of PL proteins from pathogenic organisms can be diagnosed using PDZ domains.
  • the peptides of the invention or analogues thereof may be prepared using virtually any art-known technique for the preparation of peptides and peptide analogues.
  • the peptides may be prepared in linear form using conventional solution or solid phase peptide syntheses and cleaved from the resin followed by purification procedures (Creighton, 1983, Protein Structures And Molecular Principles, W. H. Freeman and Co., N.Y.). Suitable procedures for synthesizing the peptides described herein are well known in the art.
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure and mass spectroscopy).
  • analogues and derivatives of the peptides can be chemically synthesized.
  • the linkage between each amino acid of the peptides of the invention may be an amide, a substituted amide or an isostere of amide.
  • Nonclassical amino acids or chemical amino acid analogues can be introduced as a substitution or addition into the sequence.
  • Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, ⁇ - amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, ⁇ -Abu, . ⁇ -Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl amino acids, and amino acid analogues in general.
  • the amino acid can be D (dextrorotary) or L (levo
  • the peptide or the relevant portion may also be synthesized using conventional recombinant genetic engineering techniques.
  • a polynucleotide sequence encoding a linear form of the peptide is inserted into an appropriate expression vehicle, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation.
  • the expression vehicle is then transfected into a suitable target cell which will express the peptide.
  • the expressed peptide is then isolated by procedures well- established in the art.
  • microorganisms such as bacteria transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungi expression vectors containing an appropriate coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an appropriate coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an appropriate coding sequence; or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungi expression vectors containing an appropriate coding sequence; insect cell systems infected with recomb
  • increasing the number of copies of a PL detector may be used to increase the specificity or sensitivity of detection.
  • An example of this is presented in EXAMPLE 4.
  • the TIP-TIP-IgG vector produces a fusion protein that has duplicated copies of the PDZ domain from TIP-I and the protein itself should dimerize on the basis of the IgG constant region backbone. Hence, a single protein contains 2-4 copies of the TIP-I PDZ domain.
  • addition tandem repeats of PL detectors could be fashioned.
  • different PDZ domains from different proteins could be engineered to express as a single protein (e.g., the PDZ domains of TIP-I and MAGI-I could be engineered to detect oncogenic HPV E6 proteins).
  • a different Ig backbone could be used to increase the avidity of a construct.
  • the IgG constant regions will dimerize with itself, but the IgM constant regions will form a complex often monomers.
  • the expression elements of the expression systems vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used in the expression vector.
  • inducible promoters such as pL of bacteriophage .lamda., plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used; when cloning in insect cell systems, promoters such as the baculovirus polyhedron promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the aden
  • sequences encoding the peptides of the invention may be driven by any of a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu et al., 1987, EMBO J. 3:17-311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J.
  • Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express the foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • a coding sequence may be cloned into non-essential regions (for example the polyhedron gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedron promoter).
  • Successful insertion of a coding sequence will result in inactivation of the polyhedron gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedron gene).
  • a number of viral based expression systems may be utilized.
  • a coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing peptide in infected hosts, (e.g., See Logan & Shenk, 1984, Proc. Natl.
  • the vaccinia 7.5 K promoter may be used, (see, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. USA 79:4927- 4931).
  • Tags and markers are frequently used to aid in purification of components or detection of biological molecules.
  • biological tags include, but are not limited to, glutathione-S-transferase, maltose binding protein, Immunoglobulin domains, Intein, Hemagglutinin epitopes, myc epitopes, etc.
  • chemical tags include, but are not limited to, biotin, gold, paramagnetic particles or fluorophores. These examples can be used to identify the presence of proteins or compounds they are attached to or can be used by those skilled in the art to purify proteins or compounds from complex mixtures.
  • the peptides and peptide analogues of the invention can be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography and the like.
  • the actual conditions used to purify a particular peptide or analogue will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those having skill in the art.
  • the purified peptides can be identified by assays based on their physical or functional properties, including radioactive labeling followed by gel electrophoresis, radioimmuno-assays, ELISA, bioassays, and the like. XII. Diagnostic Kit
  • the present invention also includes a kit for detection and diagnosis of HPV.
  • a subject kit usually contains a chimeric polypeptide containing at least one introduced PDZ domain that has high binding strength to a PDZ ligand.
  • the kit further includes a second PDZ ligand binding partner, which may be an antibody, for example, an antibody specific for E6 protein.
  • the second PDZ ligand binding partner is labeled with a detectable label.
  • a secondary labeling component such as a detectably labeled secondary antibody, is included.
  • a subject kit further comprises a means, such as a device or a system, for isolating oncogenic HPV E6 from the sample.
  • kits may optionally contain proteasome inhibitor.
  • a subject kit can further include, if desired, one or more of various conventional components, such as, for example, containers with one or more buffers, detection reagents or antibodies.
  • Printed instructions either as inserts or as labels, indicating quantities of the components to be used and guidelines for their use, can also be included in the kit.
  • the specified materials and conditions are important in practicing the invention but that unspecified materials and conditions are not excluded so long as they do not prevent the benefits of the invention from being realized. Exemplary embodiments of the diagnostic methods of the invention are described above in detail.
  • the oncogenic E6 detection reaction may be performed using an aqueous or solid substrate, where the kit may comprise reagents for use with several separation and detection platforms such as test strips, sandwich assays, etc.
  • the test strip kit has bound thereto a chimeric PDZ domain-containing polypeptide that specifically binds the PL domain of an oncogenic E6 protein and captures oncogenic E6 protein on the solid support.
  • the kit further comprises a detection antibody or antibodies, which is either directly or indirectly detectable, and which binds to the oncogenic E6 protein to allow its detection.
  • Kits may also include components for conducting western blots (e.g., pre-made gels, membranes, transfer systems, etc.); components for carrying out ELISAs (e.g., 96-well plates); components for carrying out immunoprecipitation (e.g. protein A); columns, especially spin columns, for affinity or size separation of oncogenic E6 protein from a sample (e.g. gel filtration columns, PDZ domain polypeptide columns, size exclusion columns, membrane cut-off spin columns etc.).
  • ELISAs e.g., 96-well plates
  • immunoprecipitation e.g. protein A
  • columns, especially spin columns, for affinity or size separation of oncogenic E6 protein from a sample e.g. gel filtration columns, PDZ domain polypeptide columns, size exclusion columns, membrane cut-off spin columns etc.
  • the subject kits may also contain control samples containing oncogenic or non-oncogenic E6, and/or a dilution series of oncogenic E6, where the dilution series represents a range of appropriate standards with which a user of the kit can compare their results and estimate the level of oncogenic E6 in their sample.
  • a dilution series may provide an estimation of the progression of any cancer in a patient. Fluorescence, color, or autoradiological film development results may also be compared to a standard curve of fluorescence, color or film density provided by the kit.
  • the subject kits typically further include instructions for using the components of the kit to practice the subject methods.
  • the instructions for practicing the subject methods are generally recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc.
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided.
  • An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
  • kits including at least a computer readable medium including programming as discussed above and instructions.
  • the instructions may include installation or setup directions.
  • the instructions may include directions for use of the invention with options or combinations of options as described above.
  • the instructions include both types of information.
  • kits may serve a number of purposes.
  • the combination may be packaged and purchased as a means for producing rabbit antibodies that are less immunogenic in a non-rabbit host than a parent antibody, or nucleotide sequences them.
  • the instructions are generally recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging), etc.
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, etc, including the same medium on which the program is presented.
  • a suitable computer readable storage medium e.g., CD-ROM, diskette, etc, including the same medium on which the program is presented.
  • kits for practicing the subject methods e.g., for extracting and purifying a protein from a protein extract, in certain embodiments, for testing the presence of a pathogen protein from a sample containing such pathogen.
  • the subject kits at least include a chimeric polypeptide containing at least one introduced PDZ domain having high binding strength to a PDZ ligand protein for the capture of such PDZ ligand in a sample.
  • the kits may include an extraction reagent that has a pH of at least about pH 10.0, and a neutralizing reagent.
  • the extraction reagent and/or the neutralizing reagent contain a non-ionic detergent.
  • kits may also include buffers and detection reagents for detecting that protein using the capture chimeric PDZ domain- containing polypeptide.
  • the above components may be present in separate containers or one or more components may be combined into a single container, e.g., a glass or plastic vial.
  • the subject kits may further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc.
  • Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded.
  • Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the
  • the present invention provides methods of detecting oncogenic HPV E6 protein in a sample and finds utility in diagnosing HPV infection in a subject.
  • a biological sample is obtained from a subject, and, the presence of oncogenic HPV E6 protein in the sample is determined.
  • the presence of a detectable amount of oncogenic HPV E6 protein in a sample indicates that the individual is infected with a oncogenic strain of HPV.
  • the level of oncogenic HPV E6 protein in a biological sample is determined, and compared to the amount of a control in the sample.
  • the relative amount of oncogenic HPV E6 protein in a sample indicates the severity of the infection by HPV.
  • the methods generally involve two binding partners of oncogenic HPV E6 protein, one of which is a chimeric polypeptide containing at least one introduced PDZ domain, as described above.
  • the methods involve a) isolating the oncogenic HPV E6 protein from a sample using one of the binding partners, and b) detecting the oncogenic HPV E6 protein with the other binding partner.
  • only the chimeric PDZ domain-containing polypeptide or a variant of the chimeric PDZ domain- containing polypeptide is used for determining if a subject is injected with an oncogenic strain of HPV.
  • methods of the invention involve at least partially separating (i.e., isolating) native oncogenic HPV E6 protein from other proteins in a sample. This separation is usually achieved using a first binding partner for the oncogenic HPV E6.
  • the first binding partner is a chimeric polypeptide containing at least one introduced PDZ domain, or, in other embodiments an anti-HPV E6 antibody or mixture of antibodies.
  • only the chimeric PDZ domain-containing polypeptide or a variant of the chimeric PDZ domain-containing polypeptide e.g.
  • labeled chimeric PDZ domain-containing polypeptide is used for isolating the E6 protein from an oncogenic strain of HPV.
  • one of the oncogenic HPV E6 binding partners is bound, directly or via a linker, to an insoluble support.
  • Insoluble supports are known in the art and include, but are not limited to, a bead (e.g, magnetic beads, polystyrene beads, and the like); a membrane; and the like.
  • a chimeric polypeptide containing at least one introduced PDZ domain is bound to a magnetic bead.
  • the chimeric PDZ domain-containing polypeptide bound to the magnetic bead is contacted with the sample, and, after a complex is formed between the antibody and any E6 protein in the sample, a magnetic field is applied, such that the complex is removed from the sample.
  • a magnetic field is applied, such that the complex is removed from the sample.
  • E6 protein bound to the chimeric PDZ domain-containing polypeptide is removed from the sample by removing the membrane, or by transferring the sample to a separate container.
  • the E6 protein bound to the bead is removed from the sample by centrifugation or filtration.
  • a different E6 binding partner e.g., an anti-E6 antibody.
  • a suitable separation means is used with a suitable platform for performing the separation.
  • a suitable platform for performing the separation.
  • the separation is performed using any of a variety of platforms, including, but not limited to, affinity column chromatography, capillary action or lateral flow test strips, immunoprecipitation, etc.
  • oncogenic HPV E6 is separated from other proteins in the sample by applying the sample to one end of a test strip, and allowing the proteins to migrate by capillary action or lateral flow.
  • Methods and devices for lateral flow separation, detection, and quantitation are known in the art. See, e.g., U.S. Pat. Nos. 5,569,608; 6,297,020; and 6,403,383.
  • a test strip comprises, in order from proximal end to distal end, a region for loading the sample (the sample-loading region) and a test region containing an oncogenic E6 protein binding partner, e.g., a region containing a chimeric PDZ domain-containing polypeptide or, in other embodiments, a region containing an anti-E6 antibody.
  • an oncogenic E6 protein binding partner e.g., a region containing a chimeric PDZ domain-containing polypeptide or, in other embodiments, a region containing an anti-E6 antibody.
  • detection of captured E6 proteins is carried out using detectably labeled antibody specific for an epitope of E6 proteins that is common to all oncogenic E6 proteins, or a mixture of antibodies that can, together, bind to all oncogenic E6 proteins.
  • an E6 antibody may be present in the test region and detection of oncogenic E6 bound to the E6 antibody uses a labeled chimeric PDZ domain- containing polypeptide.
  • oncogenic E6 protein is separated from other proteins in the sample, oncogenic E6 protein is detected and/or the level or amount of oncogenic E6 protein is determined (e.g., measured).
  • oncogenic E6 protein is generally detected using a binding partner, e.g. an antibody or antibodies specific to E6, or a chimeric polypeptide containing at least one introduced PDZ domain as described herein.
  • Detection with a specific antibody is carried out using well-known methods.
  • the binding partner is detectably labeled, either directly or indirectly.
  • Direct labels include radioisotopes (e.g., .sup.1251; .sup.35S, and the like); enzymes whose products are detectable (e.g., luciferase, .beta.-galactosidase, horse radish peroxidase, and the like); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, and the like); fluorescence emitting metals, e.g., .sup.152Eu, or others of the lanthanide series, attached to the antibody through metal chelating groups such as EDTA; chemiluminescent compounds, e.g., luminol, isoluminol, acridinium salts, and the like; bioluminescent compounds,
  • Fluorescent proteins include, but are not limited to, a green fluorescent protein (GFP), including, but not limited to, a "humanized” version of a GFP, e.g., wherein codons of the naturally-occurring nucleotide sequence are changed to more closely match human codon bias; a GFP derived from Aequoria victoria or a derivative thereof, e.g., a "humanized” derivative such as Enhanced
  • GFP which are available commercially, e.g., from Clontech, Inc.; a GFP from another species such as Renilla reniformis, Renilla mulleri, or Ptilosarcus guernyi, as described in, e.g., WO 99/49019 and Peelle et al. (2001) J.
  • Indirect labels include second antibodies specific for E6-specific antibodies, wherein the second antibody is labeled as described above; and members of specific binding pairs, e.g., biotin-avidin, and the like.
  • a level of oncogenic E6 is quantitated. Quantitation can be carried out using any known method, including, but not limited to, enzyme-linked immunosorbent assay (ELISA); radioimmunoassay
  • RIA RIA
  • quantitation is accomplished by comparing the level of expression product detected in the sample with a standard curve.
  • oncogenic HPV E6 is separated on a test strip, as described above.
  • oncogenic HPV E6 is detected using a detectably labeled binding partner (e.g. a labeled chimeric polypeptide containing at least one introduced PDZ domain) that binds oncogenic HPV E6.
  • a detectably labeled binding partner e.g. a labeled chimeric polypeptide containing at least one introduced PDZ domain
  • Oncogenic HPV E6 may be quantitated using a reflectance spectrophotometer, or by eye, for example.
  • Bio samples to be analyzed using the methods of the invention are obtained from any mammal, e.g., a human or a non-human animal model of HPV.
  • the biological sample is obtained from a living subject.
  • the subject from whom the sample is obtained is apparently healthy, where the analysis is performed as a part of routine screening. In other embodiments, the subject is one who is susceptible to
  • HPV HPV
  • the subject has symptoms of HPV (e.g., cervical warts, or the like).
  • the subject has been provisionally diagnosed as having HPV (e.g. as determined by other tests based on e.g., PCR).
  • the biological sample may be derived from any tissue, organ or group of cells of the subject.
  • a cervical scrape, biopsy, or lavage is obtained from a subject.
  • the biological sample is processed, e.g., to remove certain components that may interfere with an assay method of the invention, using methods that are standard in the art.
  • the biological sample is processed to enrich for proteins, e.g., by salt precipitation, and the like.
  • the sample is processed in the presence proteasome inhibitor to inhibit degradation of the E6 protein.
  • the level of E6 protein in a sample may be quantified and/or compared to controls.
  • Suitable control samples are from individuals known to be healthy, e.g., individuals known not to have HPV. Control samples can be from individuals genetically related to the subject being tested, but can also be from genetically unrelated individuals.
  • a suitable control sample also includes a sample from an individual taken at a time point earlier than the time point at which the test sample is taken, e.g., a biological sample taken from the individual prior to exhibiting possible symptoms of HPV.
  • XVIII Purification of PDZ Ligand Proteins
  • the present invention provides a method for purifying a PDZ ligand protein from a protein extract.
  • the methods involve two steps: a) contacting a sample containing a PDZ ligand protein with a chimeric polypeptide containing at least one introduced PDZ domain which has higher binding strength to a PDZ ligand as described above; and purifying the PDZ ligand protein from the sample.
  • the method may optionally include contacting the subject sample with an extraction reagent having a pH that is greater than about pH. 10.0 followed by contacting the subject sample with a neutralizing reagent to produce a protein extract.
  • the extraction reagent and/or the neutralizing reagent contains a non-ionic detergent.
  • the resultant protein extract contains a non-ionic detergent and has a pH that is neutral (i.e., between about pH 7.0 and about pH 8.0).
  • the methods generally produce a protein extract containing proteins that are readily detectable using capture agents for those proteins, for example, using a chimeric PDZ domain- containing protein for capturing a PDZ ligand.
  • a protein extract produced by the instant methods are generally suitable for use in binding assays, e.g., immunological assays, for detection of those proteins.
  • the methods may include: increasing the pH of the fixed cells to a pH of at least about pH 10.0 to produce an intermediate composition, and then, in the presence of a non-ionic detergent, neutralizing the pH of the intermediate composition to produce the protein extract.
  • certain embodiments of the instant methods include: a) contacting a sample with an extraction reagent to produce an intermediate composition having a pH of at least about pH 10.0; and b) contacting the intermediate composition with a neutralizing reagent comprising a non-ionic detergent; to neutralize said pH of the intermediate composition and produce the protein extract.
  • the method may include: a) contacting the sample with an extraction reagent comprising a non-ionic detergent to produce an intermediate composition having a pH of at least about pH 10.0; and, b) contacting the intermediate composition with a neutralizing reagent; to neutralize the pH of the intermediate composition and produce the protein extract.
  • the protein extract produced by the instant methods may contain more protein that is accessible to capture agents (e.g. a chimeric PDZ domain-containing polypeptide) than a protein extract made using other methods, e.g., methods that do not employ: a high pH extraction step (i.e., a step that increases pH to greater than about pH 10.0 or pH 11.0), a neutralizing step (i.e., a step that increases pH to about pH 7.0 to about pH 8.0) and a non-ionic detergent. Neither high pH alone nor non-ionic detergent alone produces such a protein extract.
  • a high pH extraction step i.e., a step that increases pH to greater than about pH 10.0 or pH 11.0
  • a neutralizing step i.e., a step that increases pH to about pH 7.0 to about pH 8.0
  • a non-ionic detergent Neither high pH alone nor non-ionic detergent alone produces such a protein extract.
  • the high pH extraction reagent solubilizes proteins in the fixed cells, whereas the non- ionic detergent prevents the solubilized proteins in the intermediate composition from re-aggregating or precipitating as the pH of the intermediate composition is neutralized.
  • the non- ionic detergent prevents the solubilized proteins in the intermediate composition from re-aggregating or precipitating as the pH of the intermediate composition is neutralized.
  • the subject protein extract contain solubilized HPV E6 protein (particularly E6 protein from oncogenic strains of HPV) that is accessible to and readily detectable by a binding agent, e.g. a chimeric PDZ domain- containing polypeptide without further treatment of the protein extract (e.g., without further addition of denaturant, pH changes or heating).
  • the protein extract may also contain solubilized or insoluble membranes, proteins other than HPV E6 protein, and other cellular contents such as DNA, RNA, carbohydrates, etc. Other contaminants such as those derived from mucal contamination of the original cellular sample may also be present.
  • the components of the protein extract generally do not contain whole (i.e., cytologically intact) cells.
  • the protein extract may be used immediately, or stored, e.g., in frozen form, before use.
  • E6 protein may be concentrated from the cell extract by incubating the extract with particles containing binder for the E6.
  • the binder may comprise a chimeric PDZ domain containing polypeptide, E6 associated protein (E6AP) or fragments thereof, or E6 binding protein (E6BP) or fragments thereof.
  • E6AP E6 associated protein
  • E6BP E6 binding protein
  • chimeric polynucleotide construct Once the chimeric polynucleotide construct has been created, it is then introduced into a suitable host cell by techniques known in the art. A variety of expression vector/host systems may be utilized to contain and express sequences encoding the chimeric polypeptide described herein.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transfonmed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transfonmed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmi
  • cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding the chimeric PDZ domain-containing polypeptide.
  • routine cloning, subcloning, and propagation of polynucleotide sequences encoding CJPDZ can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla Calif.) or pSPORTl plasmid (Life Technologies). Ligation of sequences encoding CJPDZ into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
  • these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
  • vectors which direct high level expression of chimeric PDZ domain-containing polypeptide may be used.
  • vectors containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of the chimeric PDZ domain-containing polypeptide.
  • a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
  • Plant systems may also be used for expression of the chimeric PDZ domain- containing polypeptide. Transcription of sequences encoding a chimeric PDZ domain- containing polypeptide may be driven by viral promoters, e.g., the 35S and 19S promoters of CAMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al.
  • a number of viral-based expression systems may be utilized.
  • sequences encoding a chimeric PDZ domain-containing polypeptide may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses the chimeric PDZ domain-containing polypeptide in host cells.
  • sequences encoding a chimeric PDZ domain-containing polypeptide may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses the chimeric PDZ domain-containing polypeptide in host cells.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • SV40 or EBV -based vectors may also be used for high-level protein expression.
  • HACs Human artificial chromosomes
  • HACs may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid.
  • HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J. J. et al. (1997) Nat Genet. 15:345-355.)
  • sequences encoding a chimeric PDZ domain-containing polypeptide can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
  • Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk.sup.- or apr.sup.- cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides, neomycin and G- 418
  • als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites.
  • Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), ⁇ -glucuronidase and its substrate .beta.- glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, C. A. (1995) Methods MoI. Biol.
  • marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
  • sequence encoding a chimeric PDZ domain- containing polypeptide is inserted within a marker gene sequence, transformed cells containing sequences encoding the chimeric PDZ domain-containing polypeptide can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding a chimeric PDZ domain- containing polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain the nucleic acid sequence encoding a chimeric PDZ domain-containing polypeptide and that express the chimeric PDZ domain- containing polypeptide may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA- RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. [00223] Immunological methods for detecting and measuring the expression of the chimeric PDZ domain- containing polypeptide using either specific polyclonal or monoclonal antibodies are known in the art.
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated cell sorting
  • a wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding a chimeric PDZ domain-containing polypeptide include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • the sequences encoding CJPDZ, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • T7, T3, or SP6 RNA polymerase
  • reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates., cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding a chimeric PDZ domain-containing polypeptide may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode a chimeric PDZ domain-containing polypeptide may be designed to contain signal sequences which direct secretion of the chimeric PDZ domain-containing polypeptide through a prokaryotic or eukaryotic cell membrane.
  • a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post- translational activities (e.g., CHO, HeLa, MDCK, HEK293, and W138), are available from the American Type Culture Collection (ATCC, Bethesda Md.) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • nucleic acid sequences encoding a chimeric PDZ domain-containing polypeptide may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a fusion protein comprising a chimeric PDZ domain- containing polypeptide and a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of the chimeric PDZ domain-containing polypeptide activity.
  • Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices.
  • Such moieties include, but are not limited to, glutathione S- transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the CJPDZ encoding sequence and the heterologous protein sequence, so that CJPDZ may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
  • synthesis of radiolabeled chimeric PDZ domain- containing polypeptide may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract systems (Promega). These systems couple transcription and translation of protein- coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, preferably 35 S -methionine.
  • Fragments of a chimeric PDZ domain-containing polypeptide may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin-Elmer). Various fragments of a chimeric PDZ domain- containing polypeptide may be synthesized separately and then combined to produce the full length molecule.
  • the present invention provides a method of treating oncogenic HPV in a subject.
  • the oncogenic HPV strain of the method is strain 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82.
  • the present invention provides a method of treating HPV in a subject with a polypeptide containing a PDZ domain.
  • the present invention provides a method of treating HPV in a subject with a chimeric polypeptide containing a PDZ domain.
  • the polypeptide of the invention binds to E6 protein from oncogenic HPV strain 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82. In one embodiment, the polypeptide of the invention binds to E6 protein from multiple oncogenic HPV strains. In one embodiment, the present invention provides a method of treating HPV in a subject with a chimeric polypeptide containing a PDZ domain that binds the oncogenic E6 protein of HPV.
  • the method comprises administering to the subject in need thereof an effective amount of a PDZ domain- or PDZ domain fragment-containing chimeric polypeptide of the present invention, thereby interfering with the effects of oncogenic E6 protein on cellular function.
  • the method of the present invention prevents growth of cancer caused by HPV in a patient. In one embodiment, the method of the present invention prevents growth of cancer in a patient for which oncogenic E6 protein is detected.
  • the methods of the present invention slow or stop growth of cancer, such as cerical, in others, either in a patient or subject through administration of a PDZ domain- containing polypetide.
  • the PDZ domain- containing polypeptide is a chimeric polypeptide.
  • a reversal of cervical cancer growth results in the reduction in size of the tumor. Size reduction can be more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, compared to pretreatment levels.
  • administration of the PDZ domain-containing chimeric polypeptide continues after the tumor is reduced in size by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. Administrations of the PDZ domain-containing chimeric polypeptide can continue until or beyond the point where a therapeutic endpoint is reached, as determined by a physician.
  • Non-limiting examples of therapeutic endpoints include partial remission, complete remission, a reduction in tumor size, stable tumor size, slowing of tumor growth, reducing the frequency of metastasis, prevention of metastasis for a period exceeding at least 3 months, at least 6 months, at least 9 months or at least 12 months, extension of expected life expectancy, prevention of recurrence of a cancer, extension of the expected time necessary for recurrence of cancer, and reducing the frequency or severity of one or more sequelae of cancer, such as pain, edema, etc.
  • Continued administration beyond a given therapeutic endpoint can be utilized as a maintenance therapy to prevent cancer relapse.
  • treatment can continue beyond a selected therapeutic endpoint for more than 1 month, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years, or longer, or for the life of the subject.
  • administration of the PDZ domain- containing chimeric polypeptide is combined with the administration of an additional therapeutic agent as part of a therapeutic regimen.
  • the additional therapeutic agent can be administered before, during, or after the administration of the PDZ domain-containing chimeric polypeptide.
  • Agents administered during the administration of PDZ domain- containing chimeric polypeptide can be co-administered as a single composition and delivery or delivered as part of the same procedure, administered at about the same time in separate administration events.
  • Agents administered before or after administration of the PDZ domain-containing chimeric polypeptide can be administered in time frames preceding or following PDZ domain- containing chimeric polypeptide administration that include the following, without limitation: less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or 22 hours; less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days; less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 weeks; less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 months; and less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 years.
  • the additional therapeutic agent is conjugated to the PDZ domain- containing chimeric polypeptide.
  • agents of the present invention can be combined with anti-tumor or anti-cancer therapeutics capable of decreasing or preventing a further increase in tumor growth.
  • Non-limiting examples are chemotherapeutic agents, cytotoxic agents, and non-peptide small molecules such as Gleevec® (Imatinib Mesylate), Velcade® (bortezomib), Casodex (bicalutamide), Iressa® (gefitinib), and Adriamycin; alkylating agents such as thiotepa and cyclosphosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophos
  • paclitaxel TAXOLTM, Bristol-Myers Squibb Oncology, Princeton, NJ.
  • docetaxel TAXOTERETM, Rhone-Poulenc Rorer, Antony, France
  • retinoic acid esperamicins
  • capecitabine ecitabine
  • pharmaceutically acceptable salts, acids or derivatives of any of the above TAXOLTM, Bristol-Myers Squibb Oncology, Princeton, NJ.
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens including for example tamoxifen, (NolvadexTM), raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti- androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; chlorambucil; gemcitabine; 6 -thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine;
  • anti-estrogens including for example t
  • the compounds or pharmaceutical composition of the present invention can be used in combination with commonly prescribed anti-cancer drugs such as Herceptin®, Avastin®, Erbitux®, Rituxan®, Taxol®, Arimidex®, Taxotere®, ABVD, AVICINE, Abagovomab, Acridine carboxamide, Adecatumumab, 17-N- Allylamino-17-demethoxygeldanamycin, Alpharadin, Alvocidib, 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone, Amonaf ⁇ de, Anthracenedione, Anti-CD22 immunotoxins, Antineoplastic, Antitumorigenic herbs, Apaziquone, Atiprimod, Azathioprine, Belotecan, Bendamustine, BIBW 2992, Biricodar, Brostallicin, Bryostatin, Buthionine sulfoximine, CBV (chemotherapy), Calycul
  • Combinations can be made with one or more than one of the above.
  • Various delivery systems are known and can be used to administer a biologically active agent of the invention, non-limiting examples of which include liposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated endocytosis (see, e.g., Wu and Wu, (1987), J. Biol. Chem. 262:4429-4432), construction of a therapeutic nucleic acid as part of a retroviral or other vector, liquid suspension, solid or semi-solid compositions, and the like.
  • Methods of delivery include but are not limited to intra-arterial, intra-muscular, intravenous, intranasal, subcutaneous, intraperitoneal, intracerobrospinal, intra- articular, intrasynovial, intrathecal, intratumoral, peritumoral, and oral routes.
  • the agents are delivered to a tissue comprising cancerous tissue in the subject. [00235] Administration of the selected agent can be effected in one dose, continuously or intermittently throughout the course of treatment.
  • a fixed dose of the PDZ domain-containing chimeric polypeptide can be in the range from about 1 mg to about 2000 mg.
  • the fixed dose may be approximately 420 mg, approximately 525 mg, approximately 840 mg, or approximately 1050 mg of the chimeric polypeptide.
  • an initial candidate dose of PDZ domain-containing chimeric polypeptide can include about 1 ⁇ g/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) for administration to the subject, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily dosage can range, for example, from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment is sustained until a desired amelioration of disease occurs. The progress of this therapy is easily monitored by conventional techniques and assays.
  • Dosage regimens may include an initial dose of PDZ domain- containing chimeric polypeptide of 6 mg/kg, 8 mg/kg, or 12 mg/kg delivered by intravenous or subcutaneous infusion, followed by subsequent weekly maintenance doses of 2 mg/kg by intravenous infusion, intravenous bolus injection, subcutaneous infusion, or subcutaneous bolus injection. Where the antibody is well-tolerated by the patient, the time of infusion may be reduced.
  • the invention includes an initial dose of 12 mg/kg PDZ domain- containing chimeric polypeptide, followed by subsequent maintenance doses of 6 mg/kg once per 3 weeks.
  • Another dosage regimen involves an initial dose of 8 mg/kg PDZ domain-containing chimeric polypeptide, followed by 6 mg/kg once per 3 weeks. Still another dosage regimen involves an initial dose of 8 mg/kg PDZ domain- containing chimeric polypeptide, followed by subsequent maintenance doses of 8 mg/kg once per week or 8 mg/kg once every 2 to 3 weeks. As an alternative regimen, initial doses of 4 mg/kg PDZ domain-containing chimeric polypeptide may be administered on each of days 1 , 2 and 3 , followed by subsequent maintenance doses of 6 mg/kg once per 3 weeks.
  • An additional regimen involves an initial dose of 4 mg/kg PDZ domain- containing chimeric polypeptide, followed by subsequent maintenance doses of 2 mg/kg twice per week, wherein the maintenance doses are separated by 3 days.
  • the invention may include a cycle of dosing in which delivery of PDZ domain- containing chimeric polypeptide is 2-3 times per week for 3 weeks. The 3 week cycle is preferably repeated as necessary to achieve suppression of disease symptoms.
  • the invention further includes a cyclic dosage regimen in which delivery of PDZ domain- containing chimeric polypeptide is daily for 5 days. According to the invention, the cycle can be repeated as necessary to achieve amelioration of disease.
  • the PDZ domain-containing chimeric polypeptide of the invention can also be administered at time intervals indicated based on the non-limiting factors listed above, as can be determined by one skilled in the art.
  • the PDZ domain-containing chimeric polypeptide can be administered every day, every week, every two weeks, every three weeks, every four weeks, or every five weeks.
  • the fixed doses may, for example, continue to be administered until disease progression, adverse event, or other time as determined by a physician is reached. For example, from two, three, or four, up to 20 or more fixed doses may be administered.
  • one or more loading dose(s) of the binding agent are administered, followed by one or more maintenance dose(s) of the binding agent.
  • a plurality of the same fixed dose are administered to the subject.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the PDZ domain-containing chimeric polypeptide, an anti-cancer therapeutic agent, and a pharmaceutically acceptable carrier.
  • binding agents and anti-cancer therapeutic agents are described above.
  • the preparation of pharmaceutical compositions of this invention is conducted in accordance with generally accepted procedures for the preparation of pharmaceutical preparations. See, for example, Remington's Pharmaceutical Sciences 18th Edition (1990), E.W. Martin ed., Mack Publishing Co., PA.
  • Appropriate processing may include mixing with appropriate non-toxic and non-interfering components, sterilizing, dividing into dose units, and enclosing in a delivery device.
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • compositions for oral, intranasal, or topical administration can be supplied in solid, semisolid or liquid forms, including tablets, capsules, powders, liquids, and suspensions.
  • Compositions for injection can be supplied as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolution or suspension in liquid prior to injection.
  • a preferred composition is one that provides a solid, powder, or aerosol when used with an appropriate aerosolizer device.
  • Liquid pharmaceutically acceptable compositions can, for example, be prepared by dissolving or dispersing a polypeptide embodied herein in a liquid excipient, such as water, saline, aqueous dextrose, glycerol, or ethanol.
  • a liquid excipient such as water, saline, aqueous dextrose, glycerol, or ethanol.
  • the composition can also contain other medicinal agents, pharmaceutical agents, adjuvants, carriers, and auxiliary substances such as wetting or emulsifying agents, and pH buffering agents.
  • the PDZ domain- containing chimeric polypeptide can be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle Such vehicles are inherently nontoxic, and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate can also be used. Liposomes may be used as carriers.
  • the vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.
  • the pharmaceutical compositions can be formulated in slow release or sustained release forms, whereby a relatively consistent level of the active compound are provided over an extended period.
  • sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
  • LUPRON DEPOT® injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate
  • poly-D-(-)-3-hydroxybutyric acid poly-D-(-)-3-hydroxybutyric acid.
  • Therapeutic agents can be delivered as a therapeutic or as a prophylactic (e.g., inhibiting or preventing onset of neurodegenerative diseases).
  • therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated.
  • the agents may be administered to a patient at risk of developing a disease or to a patient reporting one or more of the physiological symptoms of such a disease, even though a diagnosis may not have yet been made.
  • prophylactic administration may be applied to avoid the onset of the physiological symptoms of the underlying disorder, particularly if the symptom manifests cyclically.
  • the therapy is prophylactic with respect to the associated physiological symptoms instead of the underlying indication.
  • the actual amount effective for a particular application will depend, inter alia, on the condition being treated and the route of administration.
  • the treatment duration and regimen can vary depending on the particular condition and subject that is to be treated.
  • a therapeutic agent can be administered by the subject method over at least 1, 7, 14, 30, 60, 90 days, or a period of months, years, or even throughout the lifetime of a subject.
  • Treatment can also be designed to reach a suitable positive outcome, non-limiting examples of which include partial remission, complete remission, a reduction in tumor size, stable tumor size, slowing of tumor growth, reducing the frequency of metastasis, prevention of metastasis for a period exceeding at least 3 months, at least 6 months, at least 9 months or at least 12 months, extension of expected life expectancy, prevention of recurrence of a cancer, extension of the expected time necessary for recurrence of cancer, and reducing the frequency or severity of one or more sequelae of cancer, such as pain, edema, etc.
  • a suitable positive outcome non-limiting examples of which include partial remission, complete remission, a reduction in tumor size, stable tumor size, slowing of tumor growth, reducing the frequency of metastasis, prevention of metastasis for a period exceeding at least 3 months, at least 6 months, at least 9 months or at least 12 months, extension of expected life expectancy, prevention of recurrence of a cancer, extension of the
  • administration of the PDZ domain- containing chimeric polypeptide is combined with the administration of an adjuvant.
  • adjuvant is used to refer to any agent that enhances the response of the immune system.
  • the adjuvant can be administered before, during, or after the administration of the PDZ domain- containing chimeric polypeptide.
  • Adjuvants administered during the administration of the PDZ domain-containing chimeric polypeptide can be co-administered as a single composition and delivery or delivered as part of the same procedure, administered at about the same time in separate administration events.
  • Adjuvants administered before or after administration of the PDZ domain- containing chimeric polypeptide can be administered in time frames preceding or following PDZ domain- containing chimeric polypeptide administration that include the following, without limitation: less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or 22 hours; less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days; less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 weeks; and less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 months.
  • adjuvants examples include but are not limited to aluminum compounds; Bacillus Calmette- Guerin (BCG); various toll-like receptor (TLR) stimulants such as CpG, Lipopolysaccharide (LPS), poly-IC; and cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF).
  • BCG Bacillus Calmette- Guerin
  • TLR toll-like receptor
  • CpG CpG
  • LPS Lipopolysaccharide
  • poly-IC poly-IC
  • cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF).
  • kits that can be used in the above-described methods.
  • a kit comprises a composition of the invention, in one or more containers.
  • the invention provides kits comprising a PDZ domain-containing chimeric polypeptide.
  • the binding agents can be, without limitation, any of those described above.
  • the binding agents can further be provided, without limitation, in any of the formulations and/or doses described above.
  • the kits may further comprise additional agents, such as those described above, for use according to the methods of the invention.
  • the agents can be provided in any suitable container, including but not limited to test tubes, vials, flasks, bottles, ampules, syringes, or the like.
  • the agents can be provided in a form that may be directly administered to a subject, or in a form that requires preparation prior to administration, such as in the reconstitution of lyophilized agents. Agents may be provided in aliquots of single-doses or as stocks from which multiple doses may be obtained.
  • the diagnostic test may evaluate expression of the lab gene (including overexpression) or the abundance of Lab protein.
  • a sample may be obtained from a patient in need of therapy. Where the subject has cancer, the sample is generally a tumor sample.
  • the biological sample herein may be a fixed sample, e.g., a formalin fixed, paraffin-embedded (FFPE) sample, or a frozen sample.
  • FFPE formalin fixed, paraffin-embedded
  • mRNA analysis methods include but are not limited to hydridization methods, such as Northern blot analysis, and amplification methods, such as real-time PCR.
  • Determining the protein level typically involves a) contacting the protein contained in a biological sample comprising cancer cells with an agent that specifically binds to Lab protein; and (b) identifying any agentprotein complex so formed.
  • the agent that specifically binds a cancer related protein is an antibody, preferably a monoclonal antibody. Contacting the sample can be performed on cells removed from a subject, as in a biopsy or cell culture, or on cells within the body of a subject.
  • the effect of PDZ domain-containing chimeric polypeptide on HPV infection and cervical cancer in a subject can be assessed in a number of ways, taking advantage of means for identifying cervical cancer in a sample.
  • the effect of an agent on a cancer cell or population of cells comprising cancer cells can be determined by assaying for a difference in the mRNA levels of a target gene between the test cancer cell and a control cell, when they are contacted with a candidate agent.
  • the differential expression of the cancer related gene is determined by detecting a difference in the level of the encoded polypeptide or gene product. Effect over time can also be evaluated by comparing the results of cancer gene expression levels from samples isolated before treatment to samples isolated subsequent to the start of treatment.
  • Genes of interest may be those associated with cancer cells in general, or specific cervical cancers. Reduction of gene expression, either at the nucleotide or protein level, in genes associated with cancer cells can be used as an indication of a corresponding reduction in the presence of the associated cell type within the sample, and a favorable response to treatment.
  • nucleic acid contained in a biological sample comprising cancer cells is first extracted according to standard methods in the art. For instance, mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al.
  • amplification means any method employing a primer and a polymerase capable of replicating a target sequence with reasonable fidelity.
  • Amplification may be carried out by natural or recombinant DNA polymerases such as TaqGoldTM, T7 DNA polymerase, Klenow fragment of E.coli DNA polymerase, and reverse transcriptase.
  • a preferred amplification method is PCR.
  • the isolated RNA can be subjected to a reverse transcription assay that is coupled with a quantitative polymerase chain reaction (RT- PCR) in order to quantify the expression level of a cancer related gene.
  • RT- PCR quantitative polymerase chain reaction
  • Detection of the gene expression level can be conducted in real time in an amplification assay.
  • the amplified products can be directly visualized with fluorescent DNA-binding agents including but not limited to DNA intercalators and DNA groove binders. Because the amount of the intercalators incorporated into the double-stranded DNA molecules is typically proportional to the amount of the amplified DNA products, one can conveniently determine the amount of the amplified products by quantifying the fluorescence of the intercalated dye using conventional optical systems in the art.
  • DNA-binding dye suitable for this application include SYBR green, SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidium bromide, acridines, proflavine, acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyls, anthramycin, and the like.
  • probe-based quantitative amplification relies on the sequence-specific detection of a desired amplified product. It utilizes fluorescent, target-specific probes (e.g., TaqMan® probes) resulting in increased specificity and sensitivity. Methods for performing probe-based quantitative amplification are well established in the art and are taught in U.S. Patent No. 5,210,015.
  • probes are allowed to form stable complexes with the target polynucleotides (e.g., cancer related genes) contained within the biological sample derived from the test subject in a hybridization reaction.
  • target polynucleotides e.g., cancer related genes
  • the target polynucleotides provided in the sample are chosen to be complementary to sequences of the antisense nucleic acids.
  • the nucleotide probe is a sense nucleic acid
  • the target polynucleotide is selected to be complementary to sequences of the sense nucleic acid.
  • hybridization can be performed under conditions of various stringencies. Suitable hybridization conditions for the practice of the present invention are such that the recognition interaction between the probe and target cancer related gene is both sufficiently specific and sufficiently stable. Conditions that increase the stringency of a hybridization reaction are widely known and published in the art. See, for example, (Sambrook, et al., (1989), supra; Nonradioactive In Situ Hybridization Application Manual, Boehringer Mannheim, second edition).
  • the hybridization assay can be formed using probes immobilized on any solid support, including but are not limited to nitrocellulose, glass, silicon, and a variety of gene arrays. A preferred hybridization assay is conducted on high-density gene chips as described in U.S. Patent No.
  • the nucleotide probes are conjugated to a detectable label.
  • Detectable labels suitable for use in the present invention include any composition detectable by photochemical, biochemical, spectroscopic, immunochemical, electrical, optical, or chemical means.
  • a wide variety of appropriate detectable labels are known in the art, which include fluorescent or chemiluminescent labels, radioactive isotope labels, enzymatic or other ligands.
  • a fluorescent label or an enzyme tag such as digoxigenin, ⁇ - galactosidase, urease, alkaline phosphatase or peroxidase, avidin/biotin complex.
  • the detection methods used to detect or quantify the hybridization intensity will typically depend upon the label selected above.
  • radiolabels may be detected using photographic film or a phosphoimager.
  • Fluorescent markers may be detected and quantified using a photodetector to detect emitted light.
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and measuring the reaction product produced by the action of the enzyme on the substrate; and finally colorimetric labels are detected by simply visualizing the colored label.
  • An agent- induced change in expression of cancer related genes in a cell or cell population can also be determined by examining the corresponding gene products. Determining the protein level typically involves a) contacting the protein contained in a biological sample comprising cancer cells with an agent that specifically bind to the cancer related protein; and (b) identifying any agent:protein complex so formed.
  • the agent that specifically binds a cancer related protein is an antibody, as in a biopsy or cell culture, or on cells within the body of a subject.
  • the reaction is performed by contacting the agent with a sample of the cancer related proteins derived from the test samples under conditions that will allow a complex to form between the agent and the cancer related proteins.
  • the formation of the complex can be detected directly or indirectly according to standard procedures in the art.
  • the agents are supplied with a detectable label and unreacted agents may be removed from the complex; the amount of remaining label thereby indicating the amount of complex formed.
  • an indirect detection procedure requires the agent to contain a label introduced either chemically or enzymatic ally.
  • a desirable label generally does not interfere with binding or the stability of the resulting agent:polypeptide complex.
  • the label is typically designed to be accessible to an antibody for an effective binding and hence generating a detectable signal.
  • a wide variety of labels suitable for detecting protein levels are known in the art. Non-limiting examples include radioisotopes, enzymes, colloidal metals, fluorescent compounds, bioluminescent compounds, and chemiluminescent compounds.
  • agent:polypeptide complexes formed during the binding reaction can be quantified by standard quantitative assays. As illustrated above, the formation of agent:polypeptide complex can be measured directly by the amount of label remained at the site of binding. In an alternative, the cancer related protein is tested for its ability to compete with a labeled analog for binding sites on the specific agent. In this competitive assay, the amount of label captured is inversely proportional to the amount of cancer related protein present in a test sample. [00262] A number of techniques for protein analysis based on the general principles outlined above are available in the art.
  • Radioimmunoassays include but are not limited to radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunofluorescent assays, and SDS-PAGE.
  • ELISA enzyme linked immunosorbent assay
  • immunoradiometric assays immunoradiometric assays
  • in situ immunoassays using e.g., colloidal gold, enzyme or radioisotope labels
  • western blot analysis immunoprecipitation assays
  • immunofluorescent assays include SDS-PAGE.
  • Antibodies that specifically recognize or bind to cancer related proteins are preferable for conducting the aforementioned protein analyses. Where desired, antibodies that recognize a specific type of post-translational modifications can be used.
  • Post-translational modifications include but are not limited to glycosylation, lipidation, acetylation, and phosphorylation. These antibodies may be purchased from commercial vendors. Alternatively, these antibodies can be generated using conventional polyclonal or monoclonal antibody technologies by immunizing a host animal or an antibody-producing cell with a target protein that exhibits the desired post- translational modification.
  • compositions and methods described hereinabove are readily employed in a variety of research and diagnostic methods, including methods of diagnosing a particular disease or condition, or infection by an infections agent, such as a virus or bacteria.
  • the method is employed as part of a diagnostic for detecting HPV infected cells. Since the presence of oncogenic strains of HPV is associated with cancerous and pre-cancerous cells, the instant methods may be employed to detect cancerous or pre-cancerous cervical cells.
  • HPV is known to be a causative agent in the following diseases: epidermodysplasia verruciformis (EV), a lifelong skin disorder that results in high risk for skin (e.g., squamocellar) cancer; cervical neoplasias such as cervical intraepithelial neoplasia (CIN) and invasive cervical carcinoma (ICC); viginal neoplasias such as vaginal intraepithelial neoplasia (VAIN) and vaginal carcinoma (VC); vulval neoplasias such as vulvar intraepithelial neoplasia (VIN) and vulvar carcinoma; penile carcinoma (including Bowenoid papulosis); anal (AC) and perianal carcinomas (PC); oropharyngeal carcinomas (OS); esophageal carcinomas (EC); non-melanoma skin cancers (e.g., basal cell carcinoma-BCC and
  • the instant methods may be employed as a diagnostic for any of these diseases.
  • cells are obtained (e.g., exfoliated or dissected) from a subject and deposited into a liquid medium containing a fixative that, in certain embodiments, may be a transport medium for cytological test.
  • the cells are usually obtained in doctor's office or clinic, the cellular sample is forwarded to and received by a testing facility in which the above-recited protein detection methods and, optionally, cytology assays are performed. Results from the testing are communicated to the subject, in some embodiments via the doctor and an associate thereof.
  • the subject from which cells are employed may be a mammal, e.g., a dog or cat, a rodent (e.g., mouse, guinea pig, or rat), or primate (e.g., a human, chimpanzee, or monkey).
  • the subject will be a human, particularly a male or female.
  • the subject may show symptoms of HPV infection (e.g., may have warts on one or more parts of the body), may be suspected of being infected by HPV (e.g., may contain cells that are cytologically consistent with such an infection) or may have already tested positive for HPV.
  • the subject may have no indication of HPV infection, and the above methods may be employed as part of a routine screen.
  • the instant methods may be employed to detect any strain of oncogenic HPV, e.g., HPV 26, HPV 53, HPV 66, HPV 73, HPV 82, HPV 16, HPV 18, HPV 31, HPV 35, HPV 30, HPV 39, HPV 45, HPV 51, HPV 52, HPV 56, HPV 59, HPV 58, HPV 33, HPV 66, HPV 68 or HPV 69, (particularly any of the most prevalent HPV strains, e.g., HPV 16, HPV 18, HPV 31, HPV 33 and HPV 45) by detecting the E6 protein from that strain.
  • HPV 26 HPV 53, HPV 66, HPV 73, HPV 82
  • HPV 16 HPV 18, HPV 31, HPV 35, HPV 30, HPV 39
  • HPV 45 HPV 51, HPV 52, HPV 56, HPV 59, HPV 58, HPV 33, HPV 66, HPV 68 or HPV 69
  • the fixed cells contain oncogenic E6 protein or which strain an oncogenic E6 protein is from. If a detection assay indicates the presence of an oncogenic E6 protein in fixed cells, then the identity of the strain of HPV that infected those cells can be determined by other molecular assays, e.g., those that employ antibodies specific to a particular E6 protein or other protein encoded by the virus, or by sequencing viral DNA. [00270] The following examples are offered by way of illustration and not by way of limitation.
  • a chimeric PSD95-MAGI-1 chimeric PDZ domain- containing polypeptide was generated, in which all three PDZ domains of PSD95 are substituted with the PDZ domain 1 of MAGI-I.
  • the methods of generating chimeric polynucleotide and chimeric polypeptide are well known in the art (see, e.g., Maniatis et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N. Y.).
  • the amino acid sequence of this chimeric polypeptide is shown below.
  • the chimeric PDZ domain-containing polypeptide may contain only one introduced PDZ domain from the first PDZ domain-containing polypeptide.
  • a single MAGI-I PDZ domain 1 is introduced into PSD95 polypeptide and the MAGI-I PDZ domain insert is embedded in certain adjacent sequences of PSD95 such that the conformation of the chimeric polypeptide allows for higher binding of the chimeric polypeptide to a PDZ domain as compared to the native PSD95 polypeptide.
  • the methods of generating the chimeric polypeptide are well known in the art.
  • This example provides a chimeric polypeptide in which two of the three PDZ domains in PSD95 are substituted with a PDZ domain from the first PDZ domain containing polypeptide TIP- 1.
  • Two TIP- 1 PDZ domains are introduced into PSD95 polypeptide and each of the TIP-I PDZ domain inserts is embedded in certain adjacent sequences of PSD95 such that the conformation of the chimeric polypeptide allows for higher binding of the chimeric polypeptide to a PDZ domain as compared to the native PSD95 polypeptide.
  • the chimeric PSD95-TIP-1 polypeptide exhibits enhanced binding strength to HPVl 6-E6 than a native PSD95 polypeptide in a capture ELISA.
  • This example describes the cloning of chimeric PDZ domain containing genes or portions of chimeric PDZ domain containing genes are into eukaryotic expression vectors in fusion with a number of protein tags, including but not limited to Glutathione S-Transferase (GST), Enhanced Green Fluorescent Protein (EGFP), or Hemagglutinin (HA).
  • GST Glutathione S-Transferase
  • EGFP Enhanced Green Fluorescent Protein
  • HA Hemagglutinin
  • DNA fragments corresponding to chimeric PDZ domain containing genes are generated by RT-PCR from RNA from a library of individual cell lines (CLONTECH Cat#K4000-l) derived RNA, using random (oligonucleotide) primers (Invitrogen Cat.#48190011).
  • DNA fragments corresponding to chimeric PDZ domain containing genes or portions of chimeric PDZ domain containing genes are generated by standard PCR, using above purified cDNA fragments and specific primers (see Table 5). Primers used are designed to create restriction nuclease recognition sites at the PCR fragment's ends, to allow cloning of those fragments into appropriate expression vectors.
  • DNA samples are submitted to agarose gel electrophoresis. Bands corresponding to the expected size are excised. DNA was extracted by Sephaglas Band Prep Kit (Amersham Pharmacia Cat#27-9285-01) and digested with appropriate restriction endonuclease. Digested DNA samples are purified once more by gel electrophoresis, according to the same protocol used above. Purified DNA fragments are coprecipitated and ligated with the appropriate linearized vector. After transformation into E. coli, bacterial colonies are screened by colony PCR and restriction digest for the presence and correct orientation of insert. Positive clones are innoculated in liquid culture for large scale DNA purification. The insert and flanking vector sites from the purified plasmid DNA are sequenced to ensure correct sequence of fragments and junctions between the vectors and fusion proteins.
  • All PDZ domain- containing genes are cloned into the vector pGEX-3X (Amersham Pharmacia #27-4803- 01, Genemed Acc#U13852, GI#595717), containing a tac promoter, GST, Factor Xa, ⁇ -lactamase, and lac repressor.
  • the amino acid sequence of the pGEX-3X coding region including GST, Factor Xa, and the multiple cloning site is listed below. Note that linker sequences between the cloned inserts and GST-Factor Xa vary depending on the restriction endonuclease used for cloning. Amino acids in the translated region below that may change depending on the insertion used are indicated in small caps, and are included as changed in the construct sequence listed in (C).
  • TIPl TAX Interacting Protein 1
  • CD5.gamma both provided by the laboratory of Dr. Brian Seed at Harvard University and generated by recombinant DNA technology, containing an IgG region
  • MIN a derivative of MSCV, containing IRES and NGFR, generated by recombinant DNA technology
  • 280 TIPG3-1
  • GCTGCACGG Generates an EcoRl site downstsream (3 T ) of the stop codon. Used for cloning into pGEX-3X.
  • CTAGGATCCGGGCC Forward (5' to 3') primer corresponding to TIPl.
  • AGCCCGGTCACC Generates a Bam Hl site upstream (5 T ) of the PDZ boundary. Used for cloning into PEAKlO or CD5y.
  • TIPl TAX Interacting Protein 1
  • GI# 11908159
  • Construct TIP 1, PDZ domain 1 of l-pGEX-3X, Primers: 399& 400, Vector Cloning Sites(573 T ): Bam Hl/EcoRl, Insert Cloning Sites(573 T ):
  • constructs using pGEX-3X expression vector are used to make fusion proteins according to the protocol outlined in the GST Fusion System, Second Edition, Revision 2, Pharmacia Biotech. Method II and was optimized for a IL LgPP.
  • the constructs using the CD5 gamma or PeaklOIgG expression vectors are used to make fusion protein.
  • Purified DNA vectors are transfected into 293 EBNA T cells under standard growth conditions (DMEM +10% FCS) using standard calcium phosphate precipitation methods (Sambrook, Fritsch and Maniatis, Cold Spring Harbor Press) at a ratio of about 1 ⁇ g vector DNA for 1 million cells. This vector results in a fusion protein that is secreted into the growth medium. Transiently transfected cells are tested for peak expression, and growth media containing fusion protein is collected at that maximum (usually 1-2 days). Fusion proteins are either purified using Protein A chromatography or frozen directly in the growth media without addition.
  • the PSD95-MAGI-1 chimeric PDZ domain-containing polypeptide and a single MAGI-I PDZ domain are tested to determine their relative binding strength to the PL of the HPV16 E6 protein.
  • Peptide corresponding to the PDZ ligand (PL) of HPV16 E6 was titrated against a constant amount of the PSD95-MAGI-1 chimeric PDZ domain- containing polypeptide or the single MAGI-I PDZ domain, and the results are shown in FIGS 3 and 4.
  • HPV16-infected cervical cancer cell lines SiHa and CasKi are washed with cold PBS and resuspended in HEPES lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, 0.5% triton X-100, 1 mg/ml BSA, one pellet protease inhibitor cocktail (Roche), and 1 mM PMSF) at 2 xlO 7 cells/ml. Lysis proceeds on ice for 30 min. and lysates are cleared by centrifugation at 14,00Ox g for 5 minutes at 4 0 C.
  • HEPES lysis buffer 50 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, 0.5% triton X-100, 1 mg/ml BSA, one pellet protease inhibitor cocktail (Roche), and 1 mM PMSF
  • E6 proteins are immunoprecipitated with a mouse anti-E6 antibody (clone 6F4) and protein G beads (Pharmacia, Piscataway, N.J.). After 2 hours incubation at 4 0 C. with rotation, beads are washed 3 times with washing Buffer [50 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, protease inhibitor cocktail (CALBIOCHEM), 1 mM PMSF]. Pellets are resuspended in SDS-PAGE sample buffer and analyzed by immuno blotting using 6F4 anti-E6 antibody and anti-mouse-IgG- HRP conjugated (Jackson Immuno Research).
  • SiHa and CasKi cervical cancer cell lines are lysed at 2xlO 7 cells/ml in lysis buffer 30 min. on ice. Lysates corresponding to approx. 10 6 cells are immediately resolved on a 12% SDS-PAGE gel followed by transfer to a PVDF membrane. E6 proteins are detected with 6F4 anti-E6 HPVl 6 antibody and anti-mouse-IgG-HRP conjugated (Jackson Immuno Research). C) Results:
  • E6 protein As present in cervical cancer cells upon infection with HPVl 6 and to ensure that an anti-E6 monoclonal antibody-specific band seen in PAGE represents viral E6 protein, 293 EBNA-T cells are transfected with a construct expressing untagged E6 protein of HPV type 16. Cell lysates are prepared of those cells, and HPV infected SiHa cervical cancer cells. E6 protein from both lysates (transfected and HPV infected) was immunoprecipitated by use of an anti E6-specific monoclonal antibody. Both lysates are analyzed side by side using PAGE technology.
  • the E6-specific band obtained for transfected E6 migrates in PAGE at the same level as the anti E6 antibody specific band from SiHa cervical cancer cell lines, thus most strongly suggesting that the product immunoprecipitated with anti E6-specific monoclonal antibody represent viral E6 protein.
  • Using the specific E6 monoclonal antibody a band of the same size was detected in HPVl 6 infected cervical cancer cell type CasKi.
  • E6 protein in lysates are measured by comparing E6-Specific signal in PAGE with signals obtained by MBP-E6 (HPVl 6) fusion protein loaded onto the same gel.
  • MBP-E6 fusion protein was digested with factor X to release the E6 portion only.
  • Signal intensity comparison studies demonstrated, that cervical cancer derived cell lines injected with HPV16 (SiHa, CasKi) contain E6 at a concentration of 0.3 to 3 ng per IxIO 6 cells. It is concluded, that quantities and stability of E6 are such that detection by an E6-specific (ELISA-) assay will be feasible.
  • Fusion protein of GST and the chimeric PSD95-MAGI-1 PDZ domain containing polypeptide is tested in pull down experiments. Briefly, 10 ⁇ g recombinant GST-chimeric PDZ protein is incubated with 30 ⁇ l of glutathione-sepharose beads in 1 ml of buffer [50 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, 0.1% Triton X- 100, protease inhibitor cocktail, 1 mM PMSF] for 1 h at 4 0 C with rotation.
  • buffer 50 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, 0.1% Triton X- 100, protease inhibitor cocktail, 1 mM PMSF
  • cell lysates of 10 7 293 cells transiently transfected with either pMKit-HA-HPV16-E6 or pMKit-HA vector alone are incubated with the beads bound to the fusion PDZ proteins for 3 h at 4 0 C with rotation. Beads are washed and analyzed in 12% SDS- PAGE gel electrophoresis followed by Western blotting. Membranes are probed with biotin conjugated anti-HA antibodies (clones 3F10, or 12CA5, Boebringer Mannheim) and HRP-Streptavidin (Zymed).
  • cell lysates from 293 cells transiently transfected with pmKit-HA, pmkit-HPV16-HA-E6 or pmKit-HA-HPV16 E6-delta-PL are incubated with recombinant GST-chimeric PDZ protein and immobilized on glutathione-sepharose beads and bound fractions are immunoblotted with anti HA antibodies.
  • lysates are immunoprecipitated and detected with anti-HA antibodies.
  • PDZ domains or PDZ domain containing proteins for example, a native PSD95, a native MAGI-I, a single MAGI-I PDZ domain 1, and a single PSD95 PDZ domain 2, are tested for pull down of endogenous over expressed E6 from cell lysate.
  • Lysates of cells transfected with HA-tagged E6 HPV- 16 are incubated with GST- chimeric PSD95-MAGI-1 protein and the other GST fusion proteins representing the above PDZ domains bound to Sepharose beads.
  • Control cell samples are transfected with HA expressing constructs.
  • Detection with anti HA monoclonal antibody demonstrates that E6 is selectively pulled out of cell lysates via the PDZ domain represented by the oncogenic E6-PL-detector of all GST-PDZ proteins tested (native PSD95, native MAGI-I, single MAGI-I PDZ domain 1, single PSD95 PDZ domain 2).
  • Results demonstrate that the GST fusion protein containing the chimeric PSD95-MAGI-1 polypeptide associates with HA-E6 but not with HA- E6.DELTA.PL (lacking the 3 C-terminal PDZ domain binding amino acids).
  • the GST fusion protein containing the chimeric PSD95-MAGI-1 polypeptide binds E6 PL with the highest binding strength than the other PDZ domains tested, i.e. native PSD95, native MAGI-I, single MAGI-I PDZ domain 1, and single PSD95 PDZ domain 2.
  • This method can be used to determine whether a chimeric PDZ domain containing polypeptide has the capacity of specific E6 binding and the relative binding strength to E6 protein.
  • Endogenous E6 Protein of HPV Infected Cervical Cancer Cell Lines can be Detected in a Sandwich ELISA Via a Chimeric PDZ Domain Containing Polypeptide
  • a chimeric PDZ domain containing polypeptide is used to selectively detect presence of E6 protein in HPV infected cells via a sandwich ELISA.
  • the specific capturing of oncogenic E6 but not non-oncogenic E6 demonstrates that the chimeric PDZ domain containing polypeptide can be applied for a E6 detection based diagnostic test for HPV infection and/or cervical cancer test.
  • Sandwich type 1 ELISA Anti-E6 antibody is coated onto a 96-well Polysorp or Maxysorp ELISA plate at 5 ⁇ g/ml in PBS (100 ⁇ l/well) overnight at 4 0 C. Plates are washed with PBS and blocked with 200 ⁇ l PBS/2% BSA for 2 hours at 4 C. Cell lysates diluted in PBS/2% BSA are added and incubated at room temperature for 1 hour.
  • a chimeric PDZ domain containing polypeptide for example a chimeric PSD95 substituted with MAGI- 1 PDZ domain 1 or the same chimeric polypeptide fused to GST
  • a chimeric PDZ domain containing polypeptide for example a chimeric PSD95 substituted with MAGI- 1 PDZ domain 1 or the same chimeric polypeptide fused to GST
  • PBS/2% BSA 100 ⁇ l of a chimeric PDZ domain containing polypeptide (for example a chimeric PSD95 substituted with MAGI- 1 PDZ domain 1 or the same chimeric polypeptide fused to GST) at 5 ⁇ g/ml is added in PBS/2% BSA, and plates are incubated at room temperature for 45 min. Plates are then washed 3 times with PBS and incubated with anti-hlgG-HRP (Jackson Immuno Research) or anti-GST-HRP (Pharmacia) at the appropriate concentration in PBS/2% BSA at room temperature
  • cell lysates are preincubated with a chimeric PDZ domain containing polypeptide at 2.5-5 ug/ml final concentration, for 1-2 hours at 4 0 C, prior to adding to the anti-E6 antibody coated plate.
  • Sandwich type 2 ELISA In sandwich 2, reagents and procedures mostly correspond to those used in sandwich 1. In contrast to sandwich 1, 100 ⁇ l of a chimeric PDZ domain containing polypeptide is coated onto the ELISA plate and the anti-E6 antibody is used for detection of the chimeric PDZ domain containing polypeptide - bound E6, followed by anti-mouse IgG-HRP (Jackson Immuno Research). In a modified version of sandwich 2, biotinylated reagents (anti-E6 antibody or the chimeric PDZ domain containing polypeptide) will be used followed by streptavidin-HRP to further diminish background and to increase sensitivity.
  • a sandwich ELISA is conceived in two different variations.
  • oncogenic E6 protein is captured via the chimeric PDZ domain containing polypeptide to the solid phase and E6 detection occurs via a specific E6 antibody or another E6 binding specific agent like nucleic acid based binding compounds, chemicals binding E6, E6 binding proteins or a combination of those compounds.
  • Cells are lysed directly on a tissue culture plate and lysates are precleared by centrifugation from insoluble components.
  • Lysates are preincubated at 4 0 C. with a chimeric PDZ domain containing polypeptide, a fusion protein of GST and the chimeric PSD95 substituted with MAGI-I PDZ domain 1. Subsequently, lysates are loaded onto E6-specific antibody coated ELISA plates. Detection occurred via addition of HRP conjugated GST-specific antibody and addition of the HRP substrate TMB after appropriate washes between different incubation steps. Detection signal is constituted by a colorimetric change that is quantified using absorbance measurements at 450 nm.
  • HPV 16-E6 of over expressing E6 transfected 293 EBNA-T cells and of HPVl 6 infected cervical cancer derived cell lines is detected.
  • the detection limit using MAGI-I PDZ domain 2 is at approximately 250,000 cells. It is predicted that binding or detection via a chimeric PDZ domain containing polypeptide, such as PSD95-MAGI-1 PDZl as described herein, will increase sensitivity to 25,000 cells or less. Background reduction can be achieved by optimizing choice and concentrations of all components in the system, as well as by additional component purification or addition of size exclusion or filtering procedures.
  • Detection signal can be enhanced by use of more sensitive detection systems, for example luminescence based technologies.
  • E6:PDZ binding can be enhanced by choosing a PDZ domain with higher E6-binding affinity for the chimeric PDZ domain containing polypeptide, and by treating the E6 containing lysates with phosphatases, thus freeing all E6-PL sites from any phosphate that might interfere with, diminish or abrogate E6-PI-specific binding to the chimeric PDZ domain containing polypeptide.
  • Endogenous E6 Protein of HPV Infected Cervical Cancer Cell Lines can be Detected via a Membrane Bound Chimeric PDZ Domain Containing Polypeptide.
  • Membrane Based Detection can be used to Enhance Sensitivity of Chimeric PDZ domain Containing Polypeptide Based Assay.
  • wash 3x with PBS about 5-10 minutes/wash, 1 ml/wash, aspirate directly first wash. It is proper to wash at room temperature. Incubate membrane with cell lysate, about 300 ⁇ l, 3 million cells total, for 30 minutes at room temperature (rock solutions). Also perform 1:10 dilutions (3 million, 300,000, 30,000, 3,000) in PBS/2% BSA (33.33 ⁇ l sample, 300 ⁇ l PBS/2% BSA). Wash 3x with PBS, 3- 57wash, all at 4 0 C, 1 ml/wash. Then incubate membrane with anti-E6 (6F4) for 30 minutes at 4 0 C.
  • anti-E6 6F4

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Abstract

This invention provides compositions and methods for detecting pathogen infections in human samples and purifying pathogen proteins from human samples. The detection and purification utilize specific chimeric PDZ domain-containing proteins to detect the presence of pathogen proteins or abnormal expression of human proteins resulting from pathogen infections, and purify such pathogen proteins. Specific methods, compositions, and kits are disclosed herein for the detection and purification of oncogenic human papilloma virus E6 proteins in clinical samples.

Description

CHIMERIC PDZ DOMAIN-CONTAINING PROTEIN FOR VIRAL DETECTION
CROSS REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application No. 61/174,416, filed April 30, 2009, which is incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
[0002] Cervical cancer is the second most common cancer diagnosis in women and is linked to high-risk human papillomavirus infection 99.7% of the time. Currently, 12,000 new cases of invasive cervical cancer are diagnosed in US women annually, resulting in 5,000 deaths each year. Furthermore, there are approximately 400,000 cases of cervical cancer and close to 200,000 deaths annually worldwide. Human papillomaviruses (HPVs) are one of the most common causes of sexually transmitted disease in the world. Overall, 50-75% of sexually active men and women acquire genital HPV infections at some point in their lives. An estimated 5.5 million people become infected with HPV each year in the US alone, and at least 20 million are currently infected. The more than 100 different isolates of HPV have been broadly subdivided into high-risk and low-risk subtypes based on their association with cervical carcinomas or with benign cervical lesions or dysplasias.
[0003] A number of lines of evidence point to HPV infections as the etiological agents of cervical cancers. Multiple studies in the 1980's reported the presence of HPV variants in cervical dysplasias, cancer, and in cell lines derived from cervical cancer. Further research demonstrated that the E6-E7 region of the genome from oncogenic HPV 18 is selectively retained in cervical cancer cells, suggesting that HPV infection could be causative and that continued expression of the E6-E7 region is required for maintenance of the immortalized or cancerous state. The following year, Sedman et al demonstrated that the E6-E7 genes from HPV 16 are sufficient to immortalize human keratinocytes in culture. Barbosa et al demonstrated that although E6-E7 genes from high risk HPVs could transform cell lines, the E6-E7 regions from low risk, or non-oncogenic variants such as HPV 6 and HPV 11 are unable to transform human keratinocytes. More recently, Pillai et al examined HPV 16 and 18 infection by in situ hybridization and E6 protein expression by immunocytochemistry in 623 cervical tissue samples at various stages of tumor progression and found a significant correlation between histological abnormality and HPV infection. [0004] Current treatment paradigms are focused on the actual cervical dysplasia rather than the underlying infection with HPV. Women are screened by physicians annually for cervical dysplasia and are treated with superficial ablative techniques, including cryosurgery, laser ablation and excision. As the disease progresses, treatment options become more aggressive, including partial or radical hysterectomy, radiation or chemotherapy. A significant unmet need exists for early and accurate diagnosis of oncogenic HPV infection as well as for treatments directed at the causative HPV infection, preventing the development of cervical cancer by intervening earlier in disease progression. Human papillomaviruses characterized to date are associated with lesions confined to the epithelial layers of skin, or oral, pharyngeal, respiratory, and, most importantly, anogenital mucosae. Specific human papillomavirus types, including HPV 6 and 11, frequently cause benign mucosal lesions, whereas other types such as HPV 16, 18, and a host of other strains, are predominantly found in high-grade lesions and cancer. Individual types of human papillomaviruses (HPV) which infect mucosal surfaces have been implicated as the causative agents for carcinomas of the cervix, anus, penis, larynx and the buccal cavity, occasional periungal carcinomas, as well as benign anogenital warts. The identification of particular HPV types is used for identifying patients with premalignant lesions who are at risk of progression to malignancy. Although visible anogenital lesions are present in some persons infected with human papillomavirus, the majority of individuals with HPV genital tract infection do not have clinically apparent disease, but analysis of cytomorphological traits present in cervical smears can be used to detect HPV infection. Papanicolaou tests are a valuable screening tool, but they miss a large proportion of HPV- infected persons due to the unfortunate false positive and false negative test results. In addition, they are not amenable to worldwide testing because interpretation of results requires trained pathologists. [0005] Conventional viral detection assays, including serologic assays, sandwich ELISA assays and growth in cell culture, are not commercially available and/or are not suitable for the diagnosis and tracking of HPV infection. Recently, several PCR (polymerase chain reaction)-based tests for HPV infections have become available. Though the tests provide the benefit of differentiating oncogenic from non-oncogenic infections, they are fairly expensive to administer and require highly trained technicians to perform PCR and/or luminometer assays. In addition, PCR has a natural false positive rate that may invoke further testing or procedures that are not required. Since the oncogenicity of HPV has been shown to be protein based, early detection of HPV DNA or RNA may lead to unnecessary medical procedures that the body's immune system may solve naturally.
[0006] The detection and diagnosis of disease is a prerequisite for the treatment of disease. Numerous markers and characteristics of diseases have been identified and many are used for the diagnosis of disease. Many diseases are preceded by, and are characterized by, changes in the state of the affected cells. Changes can include the expression of pathogen genes or proteins in infected cells, changes in the expression patterns of genes or proteins in affected cells, and changes in cell morphology. The detection, diagnosis, and monitoring of diseases can be aided by the accurate assessment of these changes. Inexpensive, rapid, early and accurate detection of pathogens can allow treatment and prevention of diseases that range in effect from discomfort to death.
SUMMARY OF THE INVENTION
[0007] In one aspect, the present invention provides a chimeric polypeptide comprising at least one PDZ domain or a PDZ ligand binding portion of a PDZ domain introduced from a first PDZ domain- containing polypeptide into a second PDZ domain- containing polypeptide, wherein the chimeric polypeptide has enhanced binding strength to a PDZ ligand as compared to the native second PDZ domain-containing polypeptide. In some embodiments, the first PDZ domain-containing polypeptide is MAGI-I polypeptide. In some embodiments, the introduced PDZ domain is PDZ domain 1 of MAGI-I polypeptide. In some embodiments, the introduced PDZ domain is selected from the group consisting of PDZ domains listed in Table 2. In some embodiments, the introduced PDZ domain has 70, 80, 90, 95, 96, 97, 98, or 99 % identity to a PDZ domain listed in Table 2. In some embodiments, at least one PDZ domain is introduced into the second PDZ domain- containing polypeptide. In some embodiments, the second PDZ domain-containing polypeptide is PSD95. In some embodiments, at least one PDZ domain of the second PDZ domain-containing polypeptide is substituted with the introduced PDZ domain from the first PDZ domain- containing polypeptide. In some embodiments, at least one PDZ domain of PSD95 is substituted with PDZ domain 1 of MAGI-I. In some embodiments, two PDZ domains of PSD95 protein are substituted with PDZ domain 1 of MAGI-I. In some embodiments, three PDZ domains of PSD95 protein are substituted with PDZ domain 1 of MAGI-I. In some embodiments, more than one PDZ domain, e.g. 2, 3, 4, 5, 6, 7, or 8, PDZ domains are introduced into the second PDZ domain-containing polypeptide. In some embodiments, at least one PDZ domain from the first PDZ domain-containing polypeptide is recombined with non-PDZ portions of the second PDZ domain- containing polypeptide. In some embodiments, two PDZ domains from the first PDZ domain- containing polypeptide are recombined with the non-PDZ portions of PSD95. In some embodiments, three PDZ domains from the first PDZ domain-containing polypeptide are recombined with the non-PDZ portions of PSD95. In some embodiments, more than one PDZ domain, e.g. 2, 3, 4, 5, 6, 7, or 8, PDZ domains are introduced into PSD95. In some embodiments, at least one PDZ domain 1 of MAGI-I is recombined with the non-PDZ portions of PSD95. In some embodiments, three PDZ domain 1 of MAGI-I are recombined with the non-PDZ portions of PSD95. In some embodiments, the chimeric polypeptide contains a PDZ ligand binding portion of a PDZ domain from the first PDZ domain-containing polypeptide. In some embodiments, the chimeric polypeptide contains a modified PDZ domain from the first PDZ domain-containing polypeptide. In some embodiments, the introduced PDZ domain is modified has 70, 80, 90, 95, 96, 97, 98, or 99 % identity to a PDZ domain from the first PDZ domain-containing polypeptide. In some embodiments, the PDZ ligand is an oncogenic protein from human papilloma virus (HPV). In some embodiments, the oncogenic HPV strain is 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82. In some embodiments, the PDZ ligand is E6 protein from oncogenic HPV strain 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82. In some embodiments, the binding affinity of the chimeric polypeptide to a PDZ ligand is enhanced. In some embodiments, the binding avidity of the chimeric polypeptide to a PDZ ligand is enhanced. In some embodiments, the binding affinity and avidity of the chimeric polypeptide to a PDZ ligand are enhanced. In some embodiments, the binding strength of MAGI- 1 PDZ domain 1 to E6 protein is enhanced. For example, the binding strength of MAGI- 1 PDZ domain 1 to E6 protein is enhanced by 2 to 3 fold. [0008] In another aspect, the present invention provides a chimeric polynucleotide construct comprising a polynucleotide sequence encoding a chimeric polypeptide which contains at least one PDZ domain or a portion of a PDZ domain introduced from a PDZ domain- containing polypeptide, wherein the chimeric polypeptide has enhanced binding to a PDZ ligand as compared to the native second PDZ domain- containing polypeptide. In some embodiments, the first PDZ domain-containing polypeptide is MAGI-I polypeptide. In some embodiments, the introduced PDZ domain is PDZ domain 1 of MAGI-I polypeptide. In some embodiments, the introduced PDZ domain is selected from the group consisting of PDZ domains listed in Table 2. In some embodiments, the modified PDZ domain has 70, 80, 90, 95, 96, 97, 98, or 99 % identity a PDZ domain listed in Table 2. In some embodiments, at least one PDZ domain is introduced into the second PDZ domain-containing polypeptide. In some embodiments, the second PDZ domain-containing polypeptide is PSD95. In some embodiments, at least one PDZ domain of the second PDZ domain-containing polypeptide is substituted with the introduced PDZ domain from the first PDZ domain-containing polypeptide. In some embodiments, at least one PDZ domain of PSD95 is substituted with PDZ domain 1 of MAGI-I. In some embodiments, three PDZ domains of PSD95 protein are substituted with PDZ domain 1 of MAGI-I. In some embodiments, more than one PDZ domain, e.g. 2, 3, 4, 5, 6, 7, or 8, are introduced into the second PDZ domain-containing polypeptide. In some embodiments, at least one PDZ domain from the first PDZ domain-containing polypeptide is recombined with non-PDZ portions of the second PDZ domain- containing polypeptide. In some embodiments, three PDZ domains from the first PDZ domain-containing polypeptide are recombined with the non-PDZ portions of PSD95. In some embodiments, at least one PDZ domain 1 of MAGI-I is recombined with the non-PDZ portions of PSD95. In some embodiments, three PDZ domain 1 of MAGI-I are recombined with the non-PDZ portions of PSD95. In some embodiments, the chimeric polypeptide contains a PDZ ligand binding portion of a PDZ domain from the first PDZ domain-containing polypeptide. In some embodiments, the chimeric polypeptide contains a modified PDZ domain from the first PDZ domain- containing polypeptide. In some embodiments, the PDZ ligand is an oncogenic protein from human papilloma virus (HPV). In some embodiments, the oncogenic HPV strain is 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, and 82. In some embodiments, the PDZ ligand is E6 protein from oncogenic HPV strain 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82. In some embodiments, the binding affinity of the chimeric polypeptide to a PDZ ligand is enhanced. In some embodiments, the binding avidity of the chimeric polypeptide to a PDZ ligand is enhanced. In some embodiments, the binding affinity and avidity of the chimeric polypeptide to a PDZ ligand are enhanced. In some embodiments, the binding strength of MAGI- 1 PDZ domain 1 to E6 protein is enhanced. In one example, the binding strength of MAGI- 1 PDZ domain 1 to E6 protein is enhanced by 2 to 3 fold.
[0009] In another aspect, the present invention provides a method for producing chimeric PDZ domain-containing polypeptide, the method comprising generating a chimeric polynucleotide construct encoding a chimeric polypeptide which contains at least one PDZ domain or a portion of a PDZ domain introduced from a PDZ domain- containing polypeptide, wherein the introduced PDZ domain in the chimeric polypeptide has enhanced binding to a PDZ ligand as compared to an isolated PDZ domain peptide. In some embodiments, the binding affinity of the chimeric polypeptide to a PDZ ligand is enhanced. In some embodiments, the binding avidity of the chimeric polypeptide to a PDZ ligand is enhanced. In some embodiments, the binding affinity and avidity of the chimeric polypeptide to a PDZ ligand are enhanced. In one example, the binding strength of MAGI-I PDZ domain 1 to E6 protein is enhanced by 2 to 3 fold. In some embodiements, the PDZ domain- containing polypeptide is MAGI-I polypeptide. In some embodiements, the PDZ domain- containing polypeptide is a modified MAGI-I polypeptide. In some embodiments, the PDZ domain- containing polypeptide is a PDZ domain listed in Table 2. In some embodiments, the PDZ domain- containing polypeptide is a modified PDZ domain listed in Table 2. In yet another aspect, the present invention provides a method for detecting the presence of a PDZ ligand in a sample, the method comprising: (a) contacting a sample suspected of containing a PDZ ligand with a chimeric polypeptide containing at least one PDZ domain introduced from a PDZ domain- containing polypeptide, wherein the introduced PDZ domain in the chimeric polypeptide has enhanced binding to the PDZ ligand in the sample as compared to an isolated PDZ domain peptide; and (b) detecting binding of the PDZ ligand in the sample to the chimeric polypeptide, wherein binding of the PDZ ligand in the sample to the chimeric polypeptide indicates the presence of the PDZ ligand in the sample.
[0010] In yet another aspect, the present invention provides a method for determining if a subject is infected with an oncogenic strain of human papilloma virus (HPV), the method comprising detecting the presence of oncogenic HPV E6 protein in a sample from the subject using the subject oncogenic HPV E6 protein-binding chimeric polypeptide of the present invention, wherein the presence of oncogenic HPV E6 protein indicates that the subject is infected with an oncogenic strain of HPV.
[0011] In still another aspect, the present invention provides a method for purifying a PDZ ligand from a sample, the method comprising: (a) contacting a sample containing a PDZ ligand with the subject chimeric polypeptide of the present invention and (b) purifying the PDZ ligand from the sample, wherein the PDZ ligand is bound or not bound to the subject chimeric polypeptide.
[0012] Also provided by the present invention includes a kit for extracting HPV E6 protein from a sample, the kit comprising: (a) the subject chimeric polypeptide of the present invention, which binds E6 of an oncogenic HPV strain; (b) an extraction reagent that has a pH of at least about pH 10.0; (c) a neutralizing reagent and (d) instructions for using the kit. The present invention also provides a kit for the detection and diagnosis of an E6 protein of an oncogenic HPV strain in a sample, comprising (a) the subject chimeric polypeptide of the present invention; (b) reagents for detection of the chimeric polypeptide bound to E6 protein; and (c) instructions for using the kit. [0013] In another aspect, the present invention provides a method for producing a chimeric polypeptide having enhanced binding to a PDZ ligand, the method comprising: (a) generating the subject chimeric polynucleotide construct of the present invention, (b) expressing the chimeric polynucleotide construct in an expression vector, and (c) purifying the chimeric polypeptide encoded by the subject chimeric polynucleotide construct of the present invention.
INCORPORATION BY REFERENCE
[0014] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0016] Figure 1 is a schematic structure of an exemplary chimeric polypeptide PSD95-MAGI-1, which contains 3 PDZ domain 1 of MAGI-I and the non-PDZ domain portions of PSD95 as the backbone. The 3 PDZ domains of PSD95 are all substituted with the PDZ domain 1 of MAGI-I. The chimeric polypeptide is fused to GST at its C- terminus.
[0017] Figure 2 is an amino acid sequence of a fusion protein consisting of GST and PSD95-MAGI-1 chimeric PDZ domain- containing polypeptide. The first amino acid of the chimeric PDZ domain-containing polypeptide is shown in italics and bold.
[0018] Figure 3 shows a comparison between the PSD95-MAGI-1 chimeric PDZ domain-containing polypeptide and a single MAGI-I PDZ domain in terms of their binding strength to E6 protein from an oncogenic HPV strain, HPVl 6. The comparison of E6 capture by a single MAGI-I PDZ domain versus the PSD95-MAGI-1 chimeric polypeptide is done by sandwich ELISA. The binding signals at various concentrations of HPV16-E6 show enhanced binding of HPV16-E6 by the PSD-95-MAGI-1 chimeric PDZ domain-containing polypeptide over the single MAGI-I PDZ domain.
[0019] Figure 4 shows a comparison between the PSD95-MAGI-1 chimeric PDZ domain-containing polypeptide and a single MAGI-I PDZ domain in terms of their binding strength to E6 protein from an oncogenic HPV strain, HPVl 6. The comparison of E6 capture by a single MAGI-I PDZ domain versus the PSD95-MAGI-1 chimeric polypeptide is done by a two-step lateral flow assay. The binding signals at various concentrations of HPV16-E6 show enhanced binding of HPV16-E6 by the PSD-95-MAGI-1 chimeric PDZ domain- containing polypeptide over the single MAGI- 1 PDZ domain.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention provides compositions and methods for detecting and diagnosing viral infection. In general, the compositions and methods for detection of viral infections are directed toward modulating the interactions between a PDZ ligand associated with viral infection and a chimeric protein containing at least one introduced PDZ domain that has higher binding strength to a PDZ ligand, more specifically, an E6 protein from an oncogenic strain of human papilloma virus. I. Human Papilloma Virus (HPV) and HPV Oncogenic Proteins
[0021] Human papillomaviruses (HPVs) are small double-stranded DNA viruses that induce hyperproliferative lesions in epithelial tissues. A subset of HPV strains infect epithelia in the anogenital region and are the etiological agents of cervical cancers. These HPV strains are called "high-risk" and include but are not limited to HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82. The oncogenic potential of these high-risk HPV strains is dependent on the cooperative action of the two early viral gene products, E6 and E7, which bind and alter the activity of cell cycle -regulatory proteins. E6 forms a ternary complex composed of the tumor suppressor protein p53 and E6AP (E6-associated protein), a member of E3 ubiquitin ligase family of proteins, resulting in the ubiquitination and subsequent degradation of p53 (Huibregtse, J. M., et.al. 1991. EMBO J. 10:4129-4135). E7 binds to and inactivates the retinoblastoma (pRb) family of proteins, thereby alleviating the pRb-mediated repression of E2F transcription factors that are responsible for transactivating many genes involved in progression into S phase (Cheng, S., et.al. 1995. Genes Dev. 9:2335-2349). Selective retention and expression of these two viral oncoproteins is essential for HPV-induced oncogenesis (Androphy, E. J., et.al. 1987. EMBO J. 6:989-992).
[0022] The targeting of p53 for degradation by E6 is the most extensively studied function of E6. However, p53- independent functions of E6 are also necessary for transformation. For instance, several p53 degradation-defective E6 mutants are still able to immortalize mammary epithelial cells and transform 3Yl rat fibroblasts. In addition, activation of the human telomerase reverse transcriptase (hTERT) by E6 is more important for immortalization of epithelial cells than is inactivation of p53 (Kiyono, T., S. A. 1998. Nature 396:84-88). These observations indicate that E6 contributes to malignant conversion of HPV-infected cells through mechanisms in addition to inactivation of p53.
[0023] Numerous cellular factors have been identified as binding partners for E6. These factors are involved in a variety of cellular processes such as calcium signaling, cell adhesion, transcriptional control, DNA synthesis, apoptosis, cell cycle control, DNA repair, and small-G-protein signaling. In particular, one group of the E6- interacting proteins contains multiple copies of a protein-protein interaction domain called PDZ (for "PSD-95/Discs Large/ZO-1"). All these PDZ domain- containing proteins are targeted for proteasome -mediated degradation by E6 in heterologous overexpression systems (Gardiol, D., et.al. 1999. Oncogene 18:5487-5496). The high-risk HPV E6 proteins bind to these PDZ domain-containing proteins through a motif [X-(TVS)-X-V, where X is any amino acid] located at their extreme carboxy terminus, which is conserved among all high-risk HPV strains. In contrast, none of the low -risk HPV E6 proteins possesses such domains. Mutation of the second or fourth conserved amino acid residue [(T/S) or V] in this motif has been demonstrated to compromise the transforming activity of E6, suggesting that this PDZ domain-binding motif plays a critical role in E6-induced oncogenesis (Kiyono, T., A. 1997. Proc. Natl. Acad. Sci. USA 94:11612-11616). Additional studies of transgenic mice have demonstrated an important role for this motif in E6-mediated alterations in cellular proliferation in the eye lens.
TABLE IA Correlation of E6 PDZ-ligands and oncogenicity
E6 C-terminal HPV strain sequence PL yes/no oncogenic Seq ID No
HPV 4 GYCRNCIRKQ NO NO 221
HPV I l WTTCMEDLLP NO NO 222
HPV 20 GICRLCKHFQ NO NO 223 E6 C-terminal
HPV strain sequence PL yes/no oncogenic Seq ID No
HPV 24 KGLCRQCKQI No No 224
HPV 28 WLRCTVRIPQ No No 225
HPV 36 RQCKHFYNDW No No 226
HPV 48 CRNCISHEGR No No 227
HPV 50 CCRNCYEHEG No No 228
HPV 16 SSRTRRETQL Yes Yes 229
HPV 18 RLQRRRETQV Yes Yes 230
HPV 31 WRRPRTETQV Yes Yes 231
HPV 35 WKPTRRETEV Yes Yes 232
HPV 30 RRTLRRETQV Yes Yes 233
HPV 39 RRLTRRETQV Yes Yes 234
HPV 45 RLRRRRETQV Yes Yes 235
HPV 51 RLQRRNETQV Yes Yes 236
HPV 52 RLQRRRVTQV Yes Yes 237
HPV 56 TSREPRESTV Yes Yes 238
HPV 59 QRQARSETLV yes Yes 239
HPV 58 RLQRRRQTQV Yes Yes 240
HPV 33 RLQRRRETAL Yes Yes 241
HPV 66 TSRQATESTV Yes Yes* 242
HPV 68 RRRTRQETQV Yes Yes 243
HPV 69 RRREATETQV Yes Yes 244
Table IA: E6 C-terminal sequences and oncogenicity. HPV variants are listed at the left. Sequences are identified from Genbank sequence records. PL Yes/No was defined by a match or non-match to the consenses determined at Arbor Vita and by Songyang et al. -X-(S/T)-X-(V/I/L). Oncogenicity data collected from National Cancer Institute. *Only found in oncogenic strains co-transfected with other oncogenic proteins.
TABLE IB
Correlation of recently identified oncogenic E6 proteins
E6 C-termmal
HPV strain sequence PL yes/no oncogenic Seq ID No
HPV 26 RPRRQTETQV Yes Yes 245
HPV 53 RHTTATESAV Yes Yes 246
HPV 66 TSRQATESTV Yes Yes 247
HPV 73 RCWRPSATW Yes Yes 248
HPV 82 PPRQRSETQV Yes Yes 249
Table IB: E6 C-terminal sequences and oncogenicity. HPV variants are listed at the left. Sequences are identified from Genbank sequence records. PL Yes/No was defined by a match or non-match to the consenses determined at Arbor Vita and by Songyang et al. -X-(S/T)-X-(V/I/L). Oncogenicity data on new strains collected from N Engl J Med 2003; 348:518-527. II. PDZ Domain-Containing Proteins and PDZ Ligands
[0024] The present invention relates to the compositions and methods of using PDZ domain containing proteins for the detection and diagnosis of HPV. The PDZ domain is a common structural domain of 80-90 amino-acids found in the signaling proteins of bacteria, yeast, plants, and animals. PDZ domains are also referred to as DHR (Dig homologous region) or GLGF (glycine-leucine-glycine-phenylalanine) domains. These domains help anchor transmembrane proteins to the cytoskeleton and hold together signaling complexes (Ponting CP, et.al. Bioessays 1997; 19:469-479). There are roughly 260 human PDZ domains, though since several PDZ domain containing proteins hold several domains, the actual number of PDZ proteins is closer to 180. Table 2 lists non-limiting exemplary PDZ domain- containing proteins and PDZ domain sequences. Some of the well-studied PDZ domain- containing proteins include include hDlg, hScrib, MAGI-I, MAGI-2, andMAGI-3 (for "Membrane-Associated Guanylate kinase homology proteins with an inverted domain structure"), PSD95, TIP-I and MUPPl. [0025] MAGI-I (Membrane associated guanylate kinase, WW and PDZ domain containing 1), is a member of the membrane-associated guanylate kinase homologue (MAGUK) family. MAGUK proteins participate in the assembly of multiprotein complexes on the inner surface of the plasma membrane at regions of cell-cell contact. MAGI-I protein may play a role as scaffolding protein at cell-cell junctions. There are alternatively spliced transcript variants encoding different isoforms of MAGI-I protein. MAGI-I consists of six PSD95/T)iscLarge/ZO-l (PDZ) domains, a guanylate kinase domain and two WW domains flanked by the first and second PDZ domain (Dobrosotskaya, I. et.al 1997 J. Biol. Chem. 272, 31589-31597). Because PDZ domains are docking domains for PDZ-binding molecules, MAGI- 1 associates with a variety molecules such as NMDA (iV-methyl-D-aspartate) receptors, PTEN, BAI- 1 , δ- catenin, mNETl, and β-catenin. These MAGI-I -associating molecules function at cell-cell contacts. MAGI-I, therefore, functions as a scaffold molecule by localizing to cell-cell contacts.
[0026] All high-risk HPV E6 oncoproteins selectively complex with the widely-expressed cellular PDZ -protein MAGI-I, which is targeted for degradation in cells by high-risk HPV E6 proteins, suggesting that the transforming potentials of the viral oncoproteins depend partially on an ability to inactivate this cellular PDZ domain- containing protein. HPV 16E6 and 18E6 mutant proteins having disrupted PDZ domain-binding motifs fail to bind MAGI- 1 and that the wild-type viral proteins do not complex with the related MAGUK proteins ZO-I and ZO-2. When E6 complexes with the PDZ domains on the MAGI proteins including MAGI-I, MAGI-2, and MAGI-3, it distorts their shape and thereby impedes their function. Such specific and selective interactions between HPV E6 proteins and MAGI proteins suggest that the ability of high-risk HPV E6 oncoproteins to associate with MAGI PDZ domains in cells contribute to the development of HPV-associated cancers in people. More specifically, a dimer has been reported between MAGI- 1 PDZ domain 1 and E6 peptide from an oncogenic HPV strain in the cocrystal structure (Zhang Y, et.al,. J. Virology, vol 81, 2007).
[0027] PSD-95 is another member of the MAGUK- family of PDZ domain-containing proteins. Similar to all MAGUK- family proteins, the basic structure of PSD95 includes three PDZ domains, an IH3 domain, and a guanylate kinase-like domain (GK). It is almost exclusively located in the post synaptic density of neurons, and is involved in anchoring synaptic proteins. Its direct and indirect binding partners include neuroligin, NMDA receptor, AMPA receptor, and potassium channels (Sheng M, SaIa C (2001). Annu. Rev. Neurosci. 24: 1-29). PSD95 has also been identified as a target of high-risk HPV E6 proteins (Handa K, et.al., J. Virology, 2007 Feb;81(3):1379-89). Furthermore, E6 protein capture via PSD95 domain 2 is increased when PSD95 PDZ domain 2 is embedded in certain adjacent sequences of PSD95 encompassing PDZ domains 1 through 3 of PSD95. [0028] TABLE 3 lists PDZ proteins and PDZ ligand (PL) proteins which the current inventors have identified as binding to one another. Each page of TABLE 3 includes four columns. The columns in each section are number from left to right such that the left-most column in each section is column 1 and the right-most column in each section is column 4. Thus, the first column in each section is labeled "HPV Strain" and lists the various E6 proteins that contain the PDZ-Ligand sequences (PLs) that are examined (shown in parenthesis). This column lists C- terminal four amino acids that correspond to the carboxyl-terminal end of a 20 amino acid peptide used in this binding study. All ligands are biotinylated at the amino-terminus and partial sequences are presented in TABLE 1. [0029] The PDZ domain- containing protein (or proteins) that interact(s) with HPV E6— PL peptides are listed in the second column labeled "PDZ binding partner". This column provides the gene name for the PDZ portion of the GST-PDZ fusion that interacts with the PDZ-ligand to the left. For PDZ domain-containing proteins with multiple domains the domain number is listed to the right of the PDZ (i.e., in column 4 labeled "PDZ Domain"), and indicates the PDZ domain number when numbered from the amino-terminus to the carboxy-terminus. This table only lists interactions of a stronger nature, e.g., those that give a Λ4Λ or Λ5Λ classification in the G assay" . "Classification" is a measure of the level of binding. In particular, it provides an absorbance value at 450 nm which indicates the amount of PL peptide bound to the PDZ protein. The following numerical values have the following meanings: T— A450 Hm 0-1; Λ2Λ— A450nm 1-2; Λ3Λ— A450nm 2-3; Λ4Λ— A450nm 3-4; Λ5Λ— A450nm of 4 more than 2x repeated; Λ0Λ— A450 nm 0, i.e., not found to interact. The third and fourth columns of TABLE 3 are merely a repetition of the columns 1 and 2 with different E6 PLs tested and the PDZs bound by them at higher affinity. [0030] Further information regarding these PDZ ligand proteins and PDZ domain- containing proteins is provided in TABLES 1 and 2 and EXAMPLE 5. In particular, TABLE 1 provides a listing of the partial amino acid sequences of peptides used in the assays. When numbered from left to right, the first column labeled "HPV strain" provides the HPV strain number used to refer to the E6 protein from that strain. The column labeled "E6 C-terminal sequence" provides the predicted sequence of the carboxy-terminal 10 amino acids of the E6 protein. The third column labeled "PL yes/no" designates whether the E6-PL sequence contains sequence elements predicted to bind to PDZ domains. The final column labeled "oncogenic" indicates that this HPV strain is known to cause cervical cancer as determined by the National Cancer Institute (NCI, 2001).
[0031] EXAMPLE 5 lists representative sequences of PDZ domains cloned into a vector (PGEX-3x vector) for production of GST-PDZ fusion proteins (Pharmacia). An extended list of PDZ domains cloned into pGEX vectors for production of GST-PDZ fusion proteins is listed in U.S. Pat. No. 09/724553.
[0032] As discussed in detail herein, the PDZ proteins listed in TABLE 2 are naturally occurring proteins containing a PDZ domain. Only significant interactions are presented in this table. Thus, the present invention is particularly directed to the detection and modulation of interactions between a chimeric protein containing a PDZ domain and a PDZ ligand protein. In a similar manner, a chimeric protein containing PDZ domains that bind other pathogens can be used to diagnose infection. Additional examples of PDZ ligand proteins from pathogens suitable for diagnostic applications are included in TABLE 6, but are not intended to limit the scope of the invention. [0033] In certain embodiments of the invention, a chimeric PDZ domain-containing protein is used to diagnose the presence of a PDZ ligand protein from a pathogenic organism. Examples of pathogenic organisms with PL sequences include, but are not limited to, viruses such as Human Papillomaviruses, Hepatitus B virus, Adenovirus, Human T Cell Leukemia Virus, bacteria and fungi. TABLE 2
PDZ Domains Used in Assays of the Invention
Gene Name Gl or PDZ Sequence fused to GST Construct Seq
Acc# # ID
26s subunit 9184389 1 RDMAEAHKEAMSRKLGQSESQGPPRAFAKVNSISPGSPSIAGL 1 p27 QVDDEIVEFGSVN TQNFQSLHNIGSWQHSEGALAPTILLSVSM
AF6 430993 1 LRKEPEIITVTLKKQNGMGLSIVAAKGAGQDKLGIYVKSWKG 2 GAADVDGRLAAGDQ LLSVDGRSLVGLSQERAAELMTRTSSWTLEVAKOG
AIPC 12751451 1 LIRPSVISIIGLYKEKGKGLGFSIAGGRDCIRGQMGIFVKTIFPNG 3 SAAEDGRLKEGDEI LDVNGIPIKGLTFQEAIHTFKOIRSGLPVLTVRTKLVSPSLTNSS
AIPC 12751451 2 GISSLGRKTPGPKDRIVMEVTLNKEPRVGLGIGACCLALENSPP 4 GIYIHSLAPGSVAK
MESNLSRGDQILEVNSVNVRHAALSKVHAILSKCPPGPVRLVI GRHPNPKVSEQEMD EVIARSTYQESKEANSS
AIPC 12751451 3 QSENEEDVCFIVLNRKEGSGLGFSVAGGTDVEPKSITVHRVFS 5 QGAASQEGTMHRG
DFLLSVNGASLAGLAHGNVLKVLHQAQLHKDALWIKKGMD QPRPSNSS
AIPC 12751451 4 LGRSVAVHDALCVEVLKTSAGLGLSLDGGKSSVTGDGPLVIK 6 RVYKGGAAEQAGIIE AGDEILAINGKPLVGLMHFDAWNIMKSVPEGPVQLLIRKHRNS
S alpha actinin-2 2773059 1 QTVILPGPAAWGFRLSGGIDFNQPLVITRITPGSKAAAANLCPG 7 associated DviLAiDGFGTESMT HADGQDRIKAAEFΓV
LIM protein
APXL-I 13651263 1 ILVEVQLSGGAPWGFTLKGGREHGEPLVITKIEEGSKAAAVDK 8 LLAGDEIVGINDIGLS GFRQEAICLVKGSHKTLKLWKENSS
Atrophin- 1 2947231 1 REKPLFTRDASQLKGTFLSTTLKKSNMGFGFTIIGGDEPDEFLQ 9
Interacting VKSVIPDGPAAQD
Protein GKMETGDVIVYINEVCVLGHTHADWKLFQSVPIGQSVNLVL CRGYP
Atrophin- 1 2947231 2 LSGATQAELMTLTIVKGAQGFGFTIADSPTGQRVKQILDIQGCP 10
Interacting GLCEGDLIVEINQQ
Protein NVQNLSHTEWDILKDCPIGSETSLIIHRGGFF
Atrophin- 1 2947231 3 HYKELDVHLRRMESGFGFRILGGDEPGQPILIGAVIAMGSADR 11
Interacting DGRLHPGDELVYVD
Protein GIPVAGKTHRYVIDLMHHAARNGQVNLTVRRKVLCG
Atrophin- 1 2947231 4 EGRGISSHSLQTSDAVIHRKENEGFGFVIISSLNRPESGSTITVPH 12
Interacting KIGRIIDGSPADR
Protein CAKLKVGDRILAVNGQSIINMPHADIVKLIKDAGLSVTLRIIPQ
EEL
Atrophin- 1 2947231 5 LSDYRQPQDFDYFTVDMEKGAKGFGFSIRGGREYKMDLYVLR 13
Interacting LAEDGPAIRNGRM
Protein RVGDQIIEINGESTRDMTHARAIELIKSGGRRVRLLLKRGTGQ
Atrophin- 1 2947231 6 HESVIGRNPEGQLGFELKGGAENGQFPYLGEVKPGKVAYESG 14
Interacting SKLVSEELLLEVNE
Protein TPVAGLTIRDVLAVIKHCKDPLRLKCVKQGGIHR
CARDI l 12382772 r NLMFRKFSLERPFRPSVTSVGHVRGPGPSVQHTTLNGDSLTSQ 15
LTLLGGNARGSFV
HSVKPGSLAEKAGLREGHQLLLLEGCIRGERQSVPLDTCTKEE AHWTIQRCSGPVTL HYKVNHEGYRKLV Gene Name Gl or PDZ Sequence fused to GST Construct Seq
Acc# # ID
CARD 14 13129123 1 ILSQVTMLAFQGDALLEQISVIGGNLTGIFIHRVTPGSAADQMA 16 LRPGTQIVMVDYEA SEPLFKAVLEDTTLEEAVGLLRRVDGFCCLSVKVNTDGYKRL
CASK 3087815 1 TRVRLVQFQKNTDEPMGITLKMNELNHCIVARIMHGGMIHRQ 17 GTLHVGDEIREINGIS VANQTVEQLQKMLREMRGSITFKIVPSYRTQS
Connector 3930780 1 LEQKAVLEQVQLDSPLGLEIHTTSNCQHFVSQVDTQVPTDSRL 18 Enhancer QIQPGDEWQINEQ VWGWPRKNMVRELLREPAGLSLVLKKIPIP
Cytohesin 3192908 1 QRKLVTVEKQDNETFGFEIQSYRPQNQNACSSEMFTLICKIQE 19 Binding Protein DSPAHCAGLQAGD
VLAMNGVSTEGFTYKQWDLIRSSGNLLTIETLNG
Densin lδO 16755892 1 RCLIQTKGQRSMDGYPEQFCVRIEKNPGLGFSISGGISGQGNPF 20
KPSDKGIFVTRVQ
PDGPASNLLQPGDKILQANGHSFVHKEHEKAVLLLKSFQNTV
DLVIQRELTV
DLGl 475816 1 IQVNGTDADYEYEEITLERGNSGLGFSIAGGTDHPHIGDDSSIFI 21 TKIITGGAAAQDGR LRVNDCILQVNEVDVRDVTHSKAVEALKEAGSIVRLYVKRRN
DLGl 475816 2 IQLIKGPKGLGFSIAGGVGNQHIPGDNSIYVTKIIEGGAAHKDG 22 KLQIGDKLLAVMNVC LEEVTHEEAVTALKNTSDFVYLKVAKPTSMYMNDGN
DLGl 475816 3 ILHRGSTGLGFNIVGGEDGEGIFISFILAGGPADLSGELRKGDRII 23 SVHSVDLRAASHE QAAAALKNAGQAVTIVAQYRPEEYSR
DLG2 475816 1 ISYVNGTEIEYEFEEITLERGNSGLGFSIAGGTDNPHIGDDPGIFI 24 TKIIPGGAAAEDGR LRVHDCILRVNEVDVSEVSHSKAVEALKEAGSIVRLYVRRR
DLG2 12736552 ISWEIKLFKGPKGLGFSIAGGVGNQHIPGDNSIYVTKIIDGGAA 25
QKDGRLQVGDRLL
MVNNYSLEEVTHEEAVAILKNTSEWYLKVGNPTTI
DLG2 12736552 IWAVSLEGEPRKWLHKGSTGLGFNIVGGEDGEGIFVSFILAGG 26
PADLSGELQRGDQ
ILSVNGIDLRGASHEQAAAALKGAGQTVTIIAQYQPED
DLG5 3650451 1 GIPYVEEPRHVKVQKGSEPLGISIVSGEKGGIVSKVTVGSIAHQ 27 AGLEYGDQLLEFN GINLRSATEQQARLIIGQQCDTITILAQYNPHVHQLRNSSZLTD
DLG5 3650451 2 GILAGDANKKTLEPRWFIKKSQLELGVHLCGGNLHGVFVAE 28 VEDDSPAKGPDGLVP GDLILEYGSLDVRNKTVEEVYVEMLKPRDGVRLKVQYRPEEFI
VTD
DLG6, splice 14647140 1 PTSPEIQELRQMLQAPHFKALLSAHDTIAQKDFEPLLPPLPDNIP 29 variant 1 ESEEAMRIVCLVKN
QQPLGATIKRHEMTGDILVARIIHGGLAERSGLLYAGDKLVEV NGVSVEGLDPEQVIH ILAMSRGTIMFKWPVSDPPVNSS
DLG6, splice AB053303 1 PTSPEIQELRQMLQAPHFKGATIKRHEMTGDILVARIIHGGLAE 30 variant 2 RSGLLYAGDKLVEV NGVSVEGLDPEQVIHILAMSRGTIMFKWPVSDPPVNSS
DVLl 2291005 1 LNIVTVTLNMERHHFLGISIVGQSNDRGDGGIYIGSIMKGGAV 31 AADGRIEPGDMLLQV NDVHFENMSNDDAVRVLREIVSQTGPISLTVAKCW
DVL2 2291007 1 LNIITVTLNMEKYNFLGISIVGQSNERGDGGIYIGSIMKGGAVA 32 ADGRIEPGDMLLQVN DMNFENMSNDDAVRVLRDIVHKPGPIVLTVAKCWDPSPQNS Gene Name Gl or PDZ Sequence fused to GST Construct Seq
Acc# # ID
DVL3 6806886 1 IITVTLNMEKYNFLGISIVGQSNERGDGGIYIGSIMKGGAVAAD 33
GRIEPGDMLLQVNEI
NFENMSNDDAVRVLREIVHKPGPITLTVAKCWDPSP
ELFIN l 2957144 1 TTQQIDLQGPGPWGFRLVGRKDFEQPLAISRVTPGSKAALANL 34
CIGDVITAIDGENTS
NMTHLEAQNRIKGCTDNLTLTVARSEHKVWSPLV
ENIGMA 561636 1 IFMDSFKWLEGPAPWGFRLQGGKDFNVPLSISRLTPGGKAAQ 35 AGVAVGDWVLSID GENAGSLTHIEAQNKIRACGERLSLGLSRAQPV
ERBIN 8923908 1 QGHELAKQEIRVRVEKDPELGFSISGGVGGRGNPFRPDDDGIF 36 VTRVQPEGPASKLLQPGDKIIQANGYSFINIEHGQAVSLLKTFQ NTVELIIVREVSS
EZRIN Binding 3220018 1 ILCCLEKGPNGYGFHLHGEKGKLGQYIRLVEPGSPAEKAGLLA 37 Protein 50 GDRLVEVNGENVEEGKETHQQWSRIRAALNAVRLLWDPEF IVTD
EZRIN Binding 3220018 2 IRLCTMKKGPSGYGFNLHSDKSKPGQFIRSVDPDSPAEASGLR 38 Protein 50 AQDRIVEVNGVCMEGKQHGDWSAIRAGGDETKLLWDRET DEFFMNSS
FLJOOOI l 10440352 1 KNPSGELKTVTLSKMKQSLGISISGGIESKVQPMVKIEKIFPGG 39 AAFLSGALQAGFELVAVDGENLEQVTHQRAVDTIRRAYRNKA REPMELWRVPGPSPRPSPSD
FLJl 1215 11436365 1 EGHSHPRWELPKTEEGLGFNIMGGKEQNSPIYISRIIPGGIADR 40 HGGLKRGDQLLSVNGVSVEGEHHEKAVELLKAAQGKVKLW RYTPKVLEEME
FLJ12428 BC012040 1 PGAPYARKTFTIVGDAVGWGFWRGSKPCHIQAVDPSGPAAA 41 AGMKVCQFWSVNGLNVLHVDYRTVSNLILTGPRTIVMEVME ELEC
FLJ12615 10434209 i GQYGGETVKΓVRIEKARDIPLGATVRNEMDSVΠSRIVKGGAAE 42 KSGLLHEGDEVLEINGIEIRGKDVNEVFDLLSDMHGTLTFVLIP SQQIKPPPA
FLJ20075 7019938 1 ILAHVKGIEKEVNVYKSEDSLGLTITDNGVGYAFIKRIKDGGVI 43 DSVKTICVGDHIESINGENIVGWRHYDVAKKLKELKKEELFTM KLIEPKKAFEI
FLJ21687 10437836 1 KPSQASGHFSVELVRGYAGFGLTLGGGRDVAGDTPLAVRGLL 44 KDGPAQRCGRLEVGDLVLHINGESTQGLTHAQAVERIRAGGP QLHLVIRRPLETHPGKPRGV
FLJ31349 AK055911 1 PVMSQCACLEEVHLPNIKPGEGLGMYIKSTYDGLHVITGTTEN 45 SPADRSQKIHAGDEVIQVNQQTWGWQLKNLVKKLRENPTG WLLLKKRPTGSFNFTPEFIVTD
FLJ32798 AK057360 1 LDDEEDSVKIIRLVKNREPLGATIKKDEQTGAIIVARIMRGGAA 46 DRSGLIHVGDELREVNGIPVEDKRPEEIIQILAQSQGAITFKIIPG SKEETPSNSS
GRIP l 4539083 1 WELMKKEGTTLGLTVSGGIDKDGKPRVSNLRQGGIAARSDQ 47 LDVGDYIKAVNGINLAKFRHDEIISLLKNVGERWLEVEYE
GRIP l 4539083 2 RSSVIFRTVEVTLHKEGNTFGFVIRGGAHDDRNKSRPWITCV 48 RPGGPADREGTIKPGDRLLSVDGIRLLGTRHAEAMSILKQCGQ EAALLIEYDVSVMDSVATASGNSS
GRIP l 4539083 3 HVATASGPLLVEVAKTPGASLGVALTTSMCCNKQVIVIDKIKS 49 ASIADRCGALHVGDHILSIDGTSMEYCTLAEATQFLANTTDQV KLEILPHHQTRLALKGPNS S
GRIP 1 4539083 4 TETTEWLTADPVTGFGIQLQGSVFATETLSSPPLISYIEADSPA 50 ERCGVLQIGDRVMAINGIPTEDSTFEEASQLLRDSSITSKVTLEI
EFDVAES Gene Name Gl or PDZ Sequence fused to GST Construct Seq
Acc# # ID
GRIP 1 4539083 5 AESVIPSSGTFHVKLPKKHNVELGITISSPSSRKPGDPLVISDIKK 51 GSVAHRTGTLELGDKLLAIDNIRLDNCSMEDAVQILQQCEDLV KLKIRKDEDNSD
GRIP 1 4539083 6 IYTVELKRYGGPLGITISGTEEPFDPIIISSLTKGGLAERTGAIHIG 52 DRILAINSSSLKGKPLSEAIHLLQMAGETVTLKIKKQTDAQSA
GRIP l 4539083 7 IMSPTPVELHKVTLYKDSDMEDFGFSVADGLLEKGVYVKNIRP 53 AGPGDLGGLKPYDRLLQVNHVRTRDFDCCLWPLIAESGNKL DLVISRNPLA
GTPase 2389008 1 SRGCETRELALPRDGQGRLGFEVDAEGFVTHVERFTFAETAGL 54
Activating RPGARLLRVCGQTLPSLRPEAAAQLLRSAPKVCVTVLPPDESG Enzyme RP
Guanine 6650765 1 AKAKWRQWLQKASRESPLQFSLNGGSEKGFGIFVEGVEPGS 55 Exchange Factor KAADSGLKRGDQIMEVNGQNFENITFMKAVEILRNNTHLALT VKTNIFVFKEL
HEMBA 10436367 1 LENVIAKSLLIKSNEGSYGFGLEDKNKVPIIKLVEKGSNAEMA 56 1000505 GMEVGKKIFAINGDLVFMRPFNEVDCFLKSCLNSRKPLRVLVS TKP
HEMBA 10436367 2 PRETVKIPDSADGLGFQIRGFGPSWHAVGRGTVAAAAGLHPG 57 1000505 QCIIKVNGINVSKETHASVIAHVTACRKYRRPTKQDSIQ
HEMBA 7022001 1 EDFCYVFTVELERGPSGLGMGLIDGMHTHLGAPGLYIQTLLPG 58
1003117 SPAAADGRLSLGDRILEVNGSSLLGLGYLRAVDLIRHGGKKM RFLVAKSDVETAKKI
HTRA3 AY040094 1 LTEFQDKQIKDWKKRFIGRMRTITPSLVDELKASNPDFPEVSS 59 GIYVQEVAPNSPSQRGGIQDGDIIVKVNGRPLVDSSELQEAVLT ESPLLLEVRRGNDDLLFSNSS
HTRA4 AL576444 1 HKKYLGLQMLSLTVPLSEELKMHYPDFPDVSSGVYVCKWEG 60 TAAQSSGLRDHDVIVNINGKPITTTTDWKALDSDSLSMAVLR GKDNLLLTVNSS
INADL 2370148 1 IWQIEYIDIERPSTGGLGFSWALRSQNLGKVDIFVKDVQPGSV 61 ADRDQRLKENDQILAINHTPLDQNISHQQAIALLQQTTGSLRLI VAREPVHTKSSTSSSE
INADL 2370148 2 PGHVEEVELINDGSGLGFGIVGGKTSGVWRTIVPGGLADRDG 62 RLQTGDHILKIGGTNVQGMTSEQVAQVLRNCGNSS INADL 2370148 3 PGSDSSLFETYNVELVRKDGQSLGIRIVGYVGTSHTGEASGIYV 63 KSIIPGSAAYHNGHIQVNDKIVAVDGVNIQGF ANHD WEVLRN AGQVVHLTLVRRKTSSSTSRIHRD
INADL 2370148 4 NSDDAELQKYSKLLPIHTLRLGVEVDSFDGHHYISSIVSGGPVD 64 TLGLLQPEDELLEVNGMQLYGKSRREAVSFLKEVPPPFTLVCC RRLFDDEAS INADL 2370148 LSSPEVKIVELVKDCKGLGFSILDYQDPLDPTRSVIVIRSLVAD 65
GVAERSGGLLPGDRLVSVNEYCLDNTSLAEAVEILKAVPPGLV
HLGICKPLVEFΓVTD INADL 2370148 6 PNFSHWGPPRIVEIFREPNVSLGISIWGQTVIKRLKNGEELKG 66 IFIKQVLEDSPAGKTNALKTGDKILEVSGVDLQNASHSEAVEAI KNAGNPWFIVQSLSSTPRVIPNVHNKANSS INADL 2370148 7 PGELHIIELEKDKNGLGLSLAGNKDRSRMSIFWGINPEGPAAA 67 DGRMRIGDELLEINNQILYGRSHQNASAIIKTAPSKVKLVFIRN EDAVNQMANSS INADL 2370148 8 PATCPIVPGQEMIIEISKGRSGLGLSIVGGKDTPLNAIVIHEVYE 68 EGAAARDGRLWAGDQILEVNGVDLRNSSHEEAITALRQTPQ
KVRLWY KIAAO 147 1469875 1 ILTLTILRQTGGLGISIAGGKGSTPYKGDDEGIFISRVSEEGPAA 69 RAGVRVGDKLLEVNGVALQGAEHHEAVEALRGAGTAVQMR Gene Name Gl or PDZ Sequence fused to GST Construct Seq
Acc# # ID
VWRERMVEPENAEFIVTD
KIAAO 147 1469875 PLRQRHVACLARSERGLGFSIAGGKGSTPYRAGDAGIFVSRIAE 70
GGAAHRAGTLQVGDRVLSINGVDVTEARHDHAVSLLTAASPT
IALLLEREAGG
KIAAO 147 1469875 ILEGPYPVEEIRLPRAGGPLGLSIVGGSDHSSHPFGVQEPGVFIS 71
KVLPRGLAARSGLRVGDRILAVNGQDVRDATHQEAVSALLRP
CLELSLLVRRDPAEFIVTD
KIAAO 147 1469875 4 RELCIQKAPGERLGISIRGGARGHAGNPRDPTDEGIFISKVSPTG 72 AAGRDGRLRVGLRLLEVNQQSLLGLTHGEAVQLLRSVGDTLT VLVCDGFEASTDAALEVS
KIAA0303 2224546 1 PHQPIVIHS SGKNYGFTIRAIRVYVGDSDIYTVHHIVWNNVEEG 73 SPACQAGLKAGDLITHINGEPVHGLVHTEVIELLLKSGNKVSIT TTPF
KIAA0313 7657260 1 ILACAAKAKRRLMTLTKPSREAPLPFILLGGSEKGFGIFVDSVD 74 SGSKATEAGLKRGDQILEVNGQNFENIQLSKAMEILRNNTHLSI TVKTNLFVFKELLTNSS
KIAA0316 6683123 1 IPPAPRKVEMRRDPVLGFGFVAGSEKPVWRSVTPGGPSEGKL 75 IPGDQIVMINDEPVSAAPRERVIDLVRSCKESILLTVIQPYPSPK
KIAA0340 2224620 1 LNKRTTMPKDSGALLGLKWGGKMTDLGRLGAFITKVKKGS 76
LADWGHLRAGDEVLEWNGKPLPGATNEEVYNIILESKSEPQ VEIIVSRPIGDIPRIHRD
KIAA0380 2224700 1 QRCVIIQKDQHGFGFRVSGDRIVLVQSVRPGGAAMKAGVKEG 77 DRIIKVNGTMVTNSSHLEWKLIKSGAYVALTLLGSS
K1AA0382 7662087 1 ILVQRCVIIQKDDNGFGLTVSGDNPVFVQSVKEDGAAMRAGV 78 QTGDRIIKVNGTLVTHSNHLEVVKLIKSGSYVALTVQGRPPGN
SS
KIAA0440 2662160 1 SVEMTLRRNGLGQLGFHVNYEGIVADVEPYGYAVVQAGLRQ 79 GSRLVEICKVAVATLSHEQMIDLLRTSVTVKWIIPPHD
KIAA0545 14762850 1 LKVMTSGWETVDMTLRRNGLGQLGFHVKYDGTVAEVEDYG 80 FAWQAGLRQGSRLVEICKVAWTLTHDQMIDLLRTSVTVKW IIPPFEDGTPRRGW
KIAA0559 3043641 1 HYIFPHARIKITRDSKDHTVSGNGLGIRIVGGKEIPGHSGEIGAY 81 IAKILPGGSAEQTGKLMEGMQVLEWNGIPLTSKTYEEVQSIISQ QSGEAEICVRLDLNML
KIAA0561 3043645 1 LCGSLRPPIVIHSSGKKYGFSLRAIRVYMGDSDVYTVHHWWS 82 VEDGSPAQEAGLRAGDLITHINGESVLGLVHMDWELLLKSG NKISLRTTALENTSIKVG
KIAA0613 3327039 1 SYSVTLTGPGPWGFRLQGGKDFNMPLTISRITPGSKAAQSQLS 83 QGDLWAIDGVNTDTMTHLEAQNKIKSASYNLSLTLQKSKNS
S
KIAA0751 12734165 1 ISRDSGAMLGLKVVGGKMTESGRLCAFITKVKKGSLADTVGH 84
LRPGDEVLEWNGRLLQGATFEEVYNIILESKPEPQVELWSRP IAIHRD
KIAA0807 3882334 1 ISALGSMRPPIIIHRAGKKYGFTLRAIRVYMGDSDVYTVHHMV 85
WHVEDGGPASEAGLRQGDLITHVNGEPVHGLVHTEVVELILK
SGNKVAISTTPLENSS
KIAA0858 4240204 1 FSDMRISINQTPGKSLDFGFTIKWDIPGIFVASVEAGSPAEFSQL 86
QVDDEIIAINNTKFSYNDSKEWEEAMAKAQETGHLVMDVRR
YGKAGSPE
KIAA0902 4240292 1 QSAHLEVIQLANIKPSEGLGMYIKSTYDGLHVITGTTENSPADR 87 CKKIHAGDEVIQVNHQTWGWQLKNLVNALREDPSGVILTLK KRPQSMLTSAPA
KIAA0967 4589577 1 ILTQTLIPVRHTVKIDKDTLLQDYGFHISESLPLTWAVTAGGS 88 Gene Name Gl or PDZ Sequence fused to GST Construct Seq
Acc# # ID
AHGKLFPGDQILQMNNEPAEDLSWERAVDILREAEDSLSITV VRCTSGVPKSSNSS
KIAA0973 4589589 1 GLRSPITIQRSGKKYGFTLRAIRVYMGDTDVYSVHHIVWHVEE 89 GGPAQEAGLCAGDLITHVNGEPVHGMVHPEWELILKSGNKV AVTTTPFE
KIAA1095 5889526 1 QGEETKSLTLVLHRDSGSLGFNIIGGRPSVDNHDGSSSEGIFVS 90 KIVDSGPAAKEGGLQIHDRIIEVNGRDLSRATHDQAVEAFKTA KEPIWQVLRRTPRTKMFTP
KIAAl 09S 5889526 2 QEMDREELELEEVDLYRMNSQDKLGLTVCYRTDDEDDIGIYIS 91 EIDPNSIAAKDGRIREGDRIIQINGIEVQNREEAVALLTSEENKN FSLLIARPELQLD
KIAA1202 6330421 1 RSFQYVPVQLQGGAPWGFTLKGGLEHCEPLTVSKIEDGGKAA 92 LSQKMRTGDELVNINGTPLYGSRQEALILIKGSFRILKLIVRRR
NAPVS
KIAA1222 6330610 1 ILEKLELFPVELEKDEDGLGISIIGMGVGADAGLEKLGIFVKTV 93 TEGGAAQRDGRIQVNDQIVEVDGISLVGVTQNFAATVLRNTK GNVRFVIGREKPGQVS
KIAA1284 6331369 1 KDVNVYVNPKKLTVIKAKEQLKLLEVLVGIIHQTKWSWRRTG 94 KQGDGERLVVHGLLPGGSAMKSGQVLIGDVLVAVNDVDVTT ENIERVLSCIPGPMQVKLTFENAYDVKRET
KIAA1389 7243158 1 TRGCETVEMTLRRNGLGQLGFHVNFEGIVADVEPFGFAWKAG 95 LRQGSRLVEICKVAVATLTHEQMIDLLRTSVTVKWIIQPHDD GSPRR
KIAA1415 7243210 1 VENILAKRLLILPQEEDYGFDIEEKNKAVWKSVQRGSLAEVA 96 GLQVGRKIYSINEDLVFLRPFSEVESILNQSFCSRRPLRLLVATK AKEIIKIP
KIAA1526 5817166 1 PDSAGPGEVRLVSLRRAKAHEGLGFSIRGGSEHGVGIYVSLVE 97 PGSLAEKEGLRVGDQILRVNDKSLARVTHAEAVKALKGSKKL VLSVYSAGRIPGGYVTNH
KIAA1526 5817166 2 LQGGDEKKVNLVLGDGRSLGLTIRGGAEYGLGIYITGVDPGSE 98 AEGSGLKVGDQILEVNWRSFLNILHDEAVRLLKSSRHLILTVK DVGRLPHARTTVDE
KIAA1526 5817166 3 WTSGAHVHSGPCEEKCGHPGHRQPLPRIVTIQRGGSAHNCGQ 99 LKVGHVILEVNGLTLRGKEHREAARIIAEAFKTKDRDYIDFLD
SL
KIAA1620 10047316 1 ELRRAELVEIIVETEAQTGVSGINVAGGGKEGIFVRELREDSPA 100 ARSLSLQEGDQLLSARVFFENFKYEDALRLLQCAEPYKVSFCL KRTVPTGDLALRP
KIAA1634 10047344 1 PSQLKGVLVRASLKKSTMGFGFTIIGGDRPDEFLQVKNVLKQG 101 PAAQDGKIAPGDVIVDINGNCVLGHTHADVVQMFQLVPVNQ YVNLTLCRGYPLPDDSED
KIAA1634 10047344 2 ASSGSSQPELVTIPLIKGPKGFGFAIADSPTGQKVKMILDSQVV 102 CQGLQKGDIIKEIYHQNVQNLTHLQWEVLKQFPVGADVPLLI LRGGPPSPTKTAKM
KIAA1634 10047344 3 LYEDKPPLTNTFLISNPRTTADPRILYEDKPPNTKDLDVFLRKQ 103 ESGFGFRVLGGDGPDQSIYIGAIIPLGAAEKDGRLRAADELMCI DGIPVKGKSHKQVLDLMTRAARNGHVLLTVRRKIFYGEKQPE DDSGSPGIHRELT
KIAA1634 10047344 4 PAPQEPYDWLQRKENEGFGFVILTSKNKPPPGVIPHKIGRVIE 104 GSPADRCGKLKVGDHISAVNGQSΓVELSHDNIVQLIKDAGVTV TLTVIAEEEHHGPPS
KIAA1634 10047344 5 QNLGCYPVELERGPRGFGFSLRGGKEYNMGLFILRLAEDGPAI 105 KDGRIHVGDQIVEINGEPTQGITHTRAIELIQAGGNKVLLLLRP Gene Name Gl or PDZ Sequence fused to GST Construct Seq
Acc# # ID
GTGLIPDHGLA
KIAA1719 1267982 0 ITWELIKKEGSTLGLTISGGTDKDGKPRVSNLRPGGLAARSDL 106 LNIGDYIRSVNGIHLTRLRHDEIITLLKNVGERWLEVEY
KIAA1719 1267982 1 ILDVSLYKEGNSFGFVLRGGAHEDGHKSRPLVLTYVRPGGPA 107 DREGSLKVGDRLLSVDGIPLHGASHATALATLRQCSHEALFQV EYDVATP
KIAA1719 1267982 2 IHTVANASGPLMVEIVKTPGSALGISLTTTSLRNKSVITIDRIKP 108 ASWDRSGALHPGDHILSIDGTSMEHCSLLEATKLLASISEKVR LEILPVPQSQRPL
KIAAl 719 1267982 3 IQIVHTETTEWLCGDPLSGFGLQLQGGIFATETLSSPPLVCFIEP 109 DSPAERCGLLQVGDRVLSINGIATEDGTMEEANQLLRDAALA HKWLEVEFDVAESV
KIAA1719 1267982 4 IQFDVAESVIPSSGTFHVKLPKKRSVELGITISSASRKRGEPLIIS 110 DIKKGSVAHRTGTLEPGDKLLAIDNIRLDNCPMEDAVQILRQC EDLVKLKIRKDEDN
KIAAl 719 1267982 5 IQTTGAVSYTVELKRYGGPLGITISGTEEPFDPIVISGLTKRGLA 111 ERTGAIHVGDRILAINNVSLKGRPLSEAIHLLQVAGETVTLKIK KQLDR
KIAAl 719 1267982 6 ILEMEELLLPTPLEMHKVTLHKDPMRHDFGFSVSDGLLEKGV 112
YVHTVRPDGPAHRGGLQPFDRVLQVNHVRTRDFDCCLAVPLL AEAGDVLELIISRKPHTAHSS
LIM Mystique 12734250 1 MALTVDVAGPAPWGFRITGGRDFHTPIMVTKVAERGKAKDA 113 DLRPGDIΓVAINGESAEGMLHAEAQSKIRQSPSPLRLQLDRSQA
TSPGQT
LIM Protein 3108092 1 SNYSVSLVGPAPWGFRLQGGKDFNMPLTISSLKDGGKAAQAN 114 VRIGDWLSIDGINAQGMTHLEAQNKIKGCTGSLNMTLQRAS
LIMKl 4587498 1 TLVEHSKLYCGHCYYQTWTPVIEQILPDSPGSHLPHTVTLVSI 115 PASSHGKRGLSVSIDPPHGPPGCGTEHSHTVRVQGVDPGCMSP DVKNSIHVGDRILEINGTPIRNVPLDEIDLLIQETSRLLQLTLEH
D
LIMK2 1805593 1 PYSVTLISMPATTEGRRGFSVSVESACSNYATTVQVKEVNRM 116 HISPNNRNAIHPGDRILEINGTPVRTLRVEEVEDAISQTSQTLQL LIEHD
LIM-RIL 1085021 1 IHSVTLRGPSPWGFRLVGRDFSAPLTISRVHAGSKASLAALCPG 117 DLIQAINGESTELMTHLEAQNRIKGCHDHLTLSVSRPE
LU-I U52111 1 VCYRTDDEEDLGIYVGEVNPNSIAAKDGRIREGDRIIQINGVDV 118 QNREEAVAILSQEENTNISLLVARPESQLA
MAGIl 3370997 1 IQKKNHWTSRVHECTVKRGPQGELGVTVLGGAEHGEFPYVG 119 AVAAVEAAGLPGGGEGPRLGEGELLLEVQGVRVSGLPRYDVL GVIDSCKEAVTFKAVRQGGR
MAGIl 3370997 2 PSELKGKFIHTKLRKSSRGFGFTWGGDEPDEFLQIKSLVLDGP 120 AALDGKMETGDVIVSVNDTCVLGHTHAQWKIFQSIPIGASVD LELCRGYPLPFDPDDPN
MAGIl 3370997 3 PATQPELITVHIVKGPMGFGFTIADSPGGGGQRVKQIVDSPRCR 121 GLKEGDLIVEVNKKNVQALTHNQWDMLVECPKGSEVTLLV QRGGNLS
MAGIl 3370997 4 PDYQEQDIFLWRKETGFGFRILGGNEPGEPIYIGHIVPLGAADT 122 DGRLRSGDELICVDGTPVIGKSHQLWQLMQQAAKQGHVNLT VRRKWFAVPKTENSS
MAGIl 3370997 GWSTWQPYDVEIRRGENEGFGFVIVSSVSRPEAGTTFAGNA 123
CVAMPHKIGRIIEGSPADRCGKLKVGDRILAVNGCSITNKSHSD
IVNLIKEAGNTVRLRIIPGDESSNA
MAGIl 3370997 6 QATQEQDFYTVELERGAXGFGFSLRGGREYNMDLYVLRLAE 124 Gene Name Gl or PDZ Sequence fused to GST Construct Seq
Acc# # ID
QGPAERCGKMRIGDEILEINGETTKNMKHSRAIELIKNGGRRV RLFLKRG
MGC5395 BC012477 1 PAXMEKEETTRELLLPNWQGSGSHGLTIAQRDDGVFVQEVTQ 125 NSPAARTGWKEGDQIVGATIYFDNLQSGEVTQLLNTMGHHT VGLKLHRKGDRSPNSS
MINTl 2625024 1 SENCKdVFIEKQKGEILGWIVESGWGSILPTVIIANMMHGGPA 126 EKSGKLNIGDQIMSINGTSLVGLPLSTCQSIIKGLKNQSRVKLNI VRCPPVNSS
MINTl 2625024 2 LRCPPVITVLIRRPDLRYQLGFSVQNGIICSLMRGGIAERGGVR 127 VGHRIIEINGQSWATPHEKIVHILSNAVGEIHMKTMPAAMYR LLNSS
MINT3 3169808 1 LSNSDNCREVHLEKRRGEGLGVALVESGWGSLLPTAVIANLL 128 HGGPAERSGALSIGDRLTAINGTSLVGLPLAACQAAVRETKSQ TSVTLSIVHCPPVTTAIM MINT3 3169808 2 LVHCPPVTTAIIHRPHAREQLGFCVEDGIICSLLRGGIAERGGIR 129 VGHRIIEINGQSWATPHARIIELLTEAYGEVHIKTMPAATYRL
LTG
MPPl 189785 1 RKVRLIQFEKVTEEPMGITLKLNEKQSCTVARILHGGMIHRQG 130 SLHVGDEILEINGTNVTNHSVDQLQKAMKETKGMISLKVIPNQ MPP2 939884 1 PVPPDAVRMVGIRKTAGEHLGVTFRVEGGELVIARILHGGMV 131 AQQGLLHVGDIIKEVNGQPVGSDPRALQELLRNASGSVILKILP NYQ
MUPPl 2104784 1 QGRHVEVFELLKPPSGGLGFSWGLRSENRGELGIFVQEIQEGS 132 VAHRDGRLKETDQILAINGQALDQTITHQQAISILQKAXDTVQ LVIARGSLPQLV MUPPl 2104784 2 PVHWQHMETIELVNDGSGLGFGIIGGKATGVIVKTILPGGVA 133 DQHGRLCSGDHILKIGDTDLAGMSSEQVAQVLRQCGNRVKL MIARGAIEERTAPT MUPPl 2104784 3 QESETFDVELTKNVQGLGITIAGYIGDKKLEPSGIFVKSITKSSA 134 VEHDGRIQIGDQIIAVDGTNLQGFTNQQAVEVLRHTGQTVLLT LMRRGMKQEA MUPPl 2104784 4 LNYEIWAHVSKFSENSGLGISLEATVGHHFIRSVLPEGPVGHS 135 GKLFSGDELLEVNGITLLGENHQDWNILKELPIEVTMVCCRR
TVPPT MUPPl 2104784 5 WEAGIQHIELEKGSKGLGFSILDYQDPIDPASTVIIRSLVPGGIA 136 EKDGRLLPGDRLMFVNDVNLENSSLEEAVEALKGAPSGTVRI GVAKPLPLSPEE MUPPl 2104784 6 RNVSKESFERTINIAKGNSSLGMTVSANKDGLGMIVRSIIHGGA 137 ISRDGRIAIGDCILSINEESTISVTNAQARAMLRRHSLIGPDIKIT YVPAEHLEE MUPPl 2104784 7 LNWNQPRRVELWREPSKSLGISRVGGRGMGSRLSNGEVMRGIF 138 IKHVLEDSPAGKN
GTLKPGDRIVEVDGMDLRDASHEQAVEAIRKAGNPWFMVQS IINRPRKSPLPSLL
MUPPl 2104784 8 LTGELHMIELEKGHSGLGLSLAGNKDRSRMSVFIVGIDPNGAA 139 GKDGRLQIADELLEI NGQILYGRSHQNASSIIKCAPSKVKIIFIRNKDAVNQ MUPPl 2104784 9 LSSFKNVQHLELPKDQGGLGIAISEEDTLSGVIIKSLTEHGVAA 140 TDGRLKVGDQILAVD DEIWGYPIEKFISLLKTAXMTVKLTIHAENPDSQ MUPPl 2104784 10 LPGCETTIEISKGRTGLGLSIVGGSDTLLGAIIIHEVYEEGAACK 141 DGRLWAGDQILEVN GIDLRKATHDEAINVLRQTPQRVRLTLYRDEAPYKE Gene Name Gl or PDZ Sequence fused to GST Construct Seq
Acc# # ID
MUPPl 2104784 11 KEEEVCDTLTIELQKKPGKGLGLSIVGKRNDTGVFVSDIVKGGI 142 ADADGRLMQGDQIL MVNGEDVRNATQEAVAALLKCSLGTVTLEVGRIKAGPFHS
MUPPl 2104784 12 LQGLRTVEMKKGPTDSLGISIAGGVGSPLGDVPIFIAMMHPTG 143 VAAQTQKLRVGDRI VTICGTSTEGMTHTQAVNLLKNASGSIEMQWAGGDVSV
MUPPl 2104784 13 LGPPQCKSITLERGPDGLGFSIVGGYGSPHGDLPIYVKTVFAKG 144 MSEDGRLKRGDQ IIAVNGQSLEGVTHEEAVAILKRTKGTVTLMVLS
NeDLG 10863920 1 IQYEEIVLERGNSGLGFSIAGGIDNPHVPDDPGIFITKIIPGGAAA 145 MDGRLGVNDCVLR VNEVEVSEWHSRAVEALKEAGPWRLWRRRQN
NeDLG 10863920 2 ITLLKGPKGLGFSIAGGIGNQHIPGDNSIYITKIIEGGAAQKDGR 146 LQIGDRLLAVNNTNLQDVRHEEAVASLKNTSDMVYLKVAKP
GSLE
NeDLG 10863920 3 ILLHKGSTGLGFNIVGGEDGEGIFVSFILAGGPADLSGELRRGD 147 RILSVNGVNLRNATHEQAAAALKRAGQSVTIVAQYRPEEYSR FESKIHDLREQMMNSSMSSGSGSLRTSEKRSLE
Neurabin II AJ401189 1 CVERLELFPVELEKDSEGLGISIIGMGAGADMGLEKLGIFVKTV 148 TEGGAAHRDGRIQVNDLLVEVDGTSLVGVTQSFAASVLRNTK GRVRFMIGRERPGEQSEVAQRIHRD
NOSl 642525 1 IQPNVISVRLFKRKVGGLGFLVKERVSKPPVIISDLIRGGAAEQS 149 GLIQAGDIILAVNGRPLVDLSYDSALEVLRGIASETHWLILRG P novel PDZ gene 7228177 1 QANSDESDIIHSVRVEKSPAGRLGFSVRGGSEHGLGIFVSKVEE 150 GSSAERAGLCVGDKITEVNGLSLESTTMGSAVKVLTSSSRLHM MVRRMGRVPGIKFSKEKNSS novel PDZ gene 7228177 2 PSDTSSEDGVRRIVHLYITSDDFCLGFNIRGGKEFGLGIYVSKV 151 DHGGLAEENGIKVGDQVLAAIGVRFDDISHSQAVEVLKGQTHI MLTIKETGRYPAYKEMNSS
Novel Serine 1621243 1 KIKKFLTESHDRQAKGKAITKKKYIGIRMMSLTSSKAXELKDR 152 Protease HRDFPDVISGAYIIEVIPDTPAEAGGLKENDVIISINGQSWSAN DVSDVIKRESTLNMWRRGNEDIMITV
NumbBindingPr AK056823 1 PDGEITSIKINRVDPSESLSIRLVGGSETPLVHIIIQHIYRDGVIAR 153 otein DGRLLPGDIILKVNGMDISNVPHNYAVRLLRQPCQVLWLTVM REQKFRSRNSS
Numb Binding AK056823 2 HRPRDDSFHVILNKSSPEEQLGIKLVRKVDEPGVFIFNVLDGGV 154 Protein AYRHGQLEENDRVLAINGHDLRYGSPESAAHLIQASERRVHL WSRQVRQRSPENSS
Numb Binding AK056823 3 PTITCHEKWNIQKDPGESLGMTVAGGASHREWDLPIYVISVE 155 Protein PGGVISRDGRIKTGDILLNVDGVELTEVSRSEAVALLKRTSSSI VLKALEVKEYEPQEFIV
Numb Binding AK056823 4 PRCLYNCKDIVLRRNTAGSLGFCIVGGYEEYNGNKPFFIKSIVE 156 Protein GTPAYNDGRIRCGDILLAVNGRSTSGMIHACLARLLKELKGRI TLTΓVSWPGTFL
Outer Membrane 7023825 1 LLTEEEINLTRGPSGLGFNIVGGTDQQYVSNDSGIYVSRIKENG 157 AAALDGRLQEGDKILSVNGQDLKNLLHQDAVDLFRNAGYAV SLRVQHRLQVQNGIHS p55T 12733367 1 PVDAIRILGIHKRAGEPLGVTFRVENNDLVIARILHGGMIDRQG 158 LLHVGDIIKEVNGHEVGNNPKELQELLKNISGSVTLKILPSYRD
TITPQQ
PAR3 8037914 1 DDMVKLVEVPNDGGPLGIHVVPFSARGGRTLGLLVKRLEKGG 159 KAEHENLFRENDCIVRINDGDLRNRRFEQAQHMFRQAMRTPII Gene Name Gl or PDZ Sequence fused to GST Construct Seq Acc# # ID
WFHWPAA
PAR3 8037914 2 GKRLNIQLKKGTEGLGFSITSRDVTIGGSAPIYVKNILPRGAAIQ 160
DGRLKAGDRLIEVNGVDLVGKSQEEWSLLRSTKMEGTVSLL VFRQEDA
PAR3 8037914 3 TPDGTREFLTFEVPLNDSGSAGLGVSVKGNRSKENHADLGIFV 161
KSIINGGAASKDGRLRVNDQLIAVNGESLLGKTNQDAMETLR RSMSTEGNKRGMIQLΓVA
PAR6 2613011 1 LPETHRRVRLHKHGSDRPLGFYIRDGMSVRVAPQGLERVPGIF 162
ISRLVRGGLAESTGLLAVSDEILEVNGIEVAGKTLDQVTDMMV AISHNLIVTVKPANQR
PAR6 GAMMA 13537118 1 IDVDLVPETHRRVRLHRHGCEKPLGFYIRDGASVRVTPHGLEK 163
VPGIFISRMVPGGLAESTGLLAVNDEVLEVNGIEVAGKTLDQV TDMMIAISHNLΓVTVKPANQRNNW
PDZ-73 5031978 1 RSRKLKEVRLDRLHPEGLGLSVRGGLEFGCGLFISHLIKGGQA 164
DSVGLQVGDEIVRINGYSISSCTHEEVINLIRTKKTVSIKVRHIG LIPVKSSPDEFH
PDZ-73 5031978 2 IPGNRENKEKKVFISLVGSRGLGCSISSGPIQKPGIFISHVKPGSL 165
SAEVGLEIGDQIVEVNGVDFSNLDHKEAVNVLKSSRSLTISIVA AAGRELFMTDEF
PDZ-73 5031978 3 PEQIMGKDVRLLRIKKEGSLDLALEGGVDSPIGKVWSAVYER 166
GAAERHGGIVKGDEIMAINGKIVTDYTLAEADAALQKAWNQ GGDWIDLWAVCPPKEYDD
PDZKL 2944188 1 LTSTFNPRECKLSKQEGQNYGFFLRIEKDTEGHLVRWEKCSP 167
AEKAGLQDGDRVLRINGVFVDKEEHMQWDLVRKSGNSVTL LVLDGDSYEKAGSPGIHRD
PDZKL 2944188 2 RLCYLVKEGGSYGFSLKTVQGKKGVYMTDITPQGVAMRAGV 168
LADDHLIEVNGENVEDASHEEVVEKVKKSGSRVMFLLVDKET DKREFΓVTD
PDZKL 2944188 3 QFKRETASLKLLPHQPRIVEMKKGSNGYGFYLRAGSEQKGQII 169
KDIDSGSPAEEAGLKNNDLWAVNGESVETLDHDSWEMIRK GGDQTSLLWDKETDNMYRLAEFIVTD
PDZKL 2944188 4 PDTTEEVDHKPKLCRLAXGENGYGFHLNAIRGLPGSFIKEVQK 170
GGPADLAGLEDEDVIIEVNGVNVLDEPYEKWDRIQSSGKNVT LLVZGKNSS
PICKL 4678411 1 PTVPGKVTLQKDAQNLIGISIGGGAQYCPCLYIVQ VFDNTP AA 171
LDGTVAAGDEITGVNGRSIKGKTKVEVAKMIQEVKGEVTIHY
NKLQ
PIST 98374330 1 SQGVGPIRKVLLLKEDHEGLGISITGGKEHGVPILISEIHPGQPA 172
DRCGGLHVGDAILAVNGVNLRDTKHKEAVTILSQQRGEIEFEV VYVAPEVDSD prIL16 1478492 1 IHVTILHKEEGAGLGFSLAGGADLENKVIWHRVFPNGLASQE 173
GTIQKGNEVLSINGKSLKGTTHHDALAILRQAREPRQAVIVTR KLTPEEFIVTD prIL16 1478492 2 TAEATVCTVTLEKMSAGLGFSLEGGKGSLHGDKPLTINRIFKG 174
AASEQSEEVQPGDEILQLGGTAMQGLTRFEAWNIIKALPDGPV TIVIRRKSLQSK
PSD95S 3318652 1 LEYEeITLERGNSGLGFSIAGGTDNPHIGDDPSIITKIIPGGAAAQ 175
DGRLRVNDSILFVNEVDVREVTHSAAVEALKEAGSIVRLYVM RRKPPAENSS
P5D95 3318652 2 HVMRRKPPAEKVMEIKLIKGPKGLGFSIAGGVGNQHIPGDNSI 176
YVTKIIEGGAAHKDGRLQIGDKILAVNSVGLEDVMHEDAVAA LKNTYDWYLKVAKPSNAYL
P5D95 3318652 3 REDIPREPRRIVIHRGSTGLGFNIVGGEDGEGIFISFILAGGPADL 177 Gene Name Gl or PDZ Sequence fused to GST Construct Seq
Acc# # ID
SGELRKGDQILSVnGVDLRNASHEQAAIALKNAGQTVTIIAQY KPEFIVTD
PTN-3 179912 1 LIRITPDEDGKFGFNLKGGVDQKMPLWSRINPESPADTCIPKL 178 NEGDQIVLINGRDISEHTHDQWMFIKASRESHSRELALVIRRR PTN-4 190747 1 IRMKPDENGRFGFNVKGGYDQKMPVIVSRVAPGTPADLCVPR 179 LNEGDQWLINGRDIAEHTHDQWLFIKASCERHSGELMLLVR PNA
PTPLl 515030 1 PEREITLVNLKKDAKYGLGFQIIGGEKMGRLDLGIFISSVAPGG 180 PADFHGCLKPGDRLISVNSVSLEGVSHHAAIEILQNAPEDVTLV ISQPKEKISKVPSTPVHL
PTPLl 515030 2 GDIFEVELAKNDNSLGISVTGGVNTSVRHGGIYVKAVIPQGAA 181 ESDGRIHKGDRVLAVNGVSLEGATHKQAVETLRNTGQWHLL LEKGQSPTSK
PTPLl 515030 3 TEENTFEVKLFKNSSGLGFSFSREDNLIPEQINASIVRVKKLFAG 182 QPAAESGKIDVGDVILKVNGASLKGLSQQEVISALRGTAPEVF LLLCRPPPGVLPEIDT
PTPLl 515030 4 ELEVELLITLIKSEKASLGFTVTKGNQRIGCYVHDVIQDPAKSD 183 GRLKPGDRLIKVNDTDVTNMTHTDAVNLLRAASKIVRLVIGR VLELPRIPMLPH
PTPLl 515030 5 MLPHLLPDITLTCNKEELGFSLCGGHDSLYQWYISDINPRSVA 184 AIEGNLQLLDVIHYVNGVSTQGMTLEEVNRALDMSLPSLVLK ATRNDLPV
RGS 12 3290015 1 RPSPPRVRSVEVARGRAGYGFTLSGQAPCVLSCVMRGSPADF 185 VGLRAGDQILAVNEINVKKASHEDWKLIGKCSGVLHMVIAE GVGRFESCS
RGS3 18644735 1 LCSERRYRQITIPRGKDGFGFTICCDSPVRVQAVDSGGPAERAG 186 LQQLDTVLQLNERPVEHWKCVELAHEIRSCPSEIILLVWRMV PQVKPGIHRD
Rhohilin-like 14279408 1 ISFSANKRWTPPRSIRFTAEEGDLGFTLRGNAPVQVHFLDPYCS 187 ASVAGAREGDYIVS IQLVDCKWLTLSEVMKLLKSFGEDEIEMKWSLLDSTSSMHN
KSAT
Serine Protease 2738914 1 RGEKKNSSSGISGSQRRYIGVMMLTLSPSILAELQLREPSFPDV 188 QHGVLIHKVILGSPAHRAGLRPGDVILAIGEQMVQNAEDVYE AVRTQSQLAVQIRRGRETLTLYV Shank 1 6049185 1 EEKTWLQKKDNEGFGFVLRGAXADTPIEEFTPTPAFPALQYL 189 ESVDEGGVAWQAGLRTGDFLIEVNNENWKVGHRQWNMIR QGGNHLVLKWIVTRNLDPDDTARKKA Shank 3 1 SDYVIDDKVAVLQKRDHEGFGFVLRGAKAETPIEEFTPTPAFP 190 ALQYLE
SVDVEGVAWRAGLRTGDFLIEVNGVNWKVGHKQWALIRQ GGNRLVMKWSVTRKPEEDG
Shroom 18652858 1 IYLEAFLEGGAPWGFTLKGGLEHGEPLIISKVEEGGKADTLSSK 191 LQAGDEVVHINEVTLSSSRKEAVSLVKGSYKTLRLWRRDVC TDPGH
SIPl 2047327 1 IRLCRLVRGEQGYGFHLHGEKGRRGQFIRRVEPGSPAEAAALR 192 AGDRLVEVNGVNVEGETHHQVVQRIKAVEGQTRLLVVDQN
SIPl 2047327 2 IRHLRKGPQGYGFNLHSDKSRPGQYIRSVDPGSPAARSGLRAQ 193 DRLIEVNGQNVEGLRHAEWASIKAREDEARLLWDPETDE
SITAC-18 8886071 1 PGVREIHLCKDERGKTGLRLRKVDQGLFVQLVQANTPASLVG 194 LRFGDQLLQIDGRDCAGWSSHKAHQWKKASGDKIVVWRD RPFQRTVTM
SITAG-18 8886071 2 PFQRTVTMHKDSMGHVGFVIKKGKIVSLVKGSSAARNGLLTN 195 Gene Name Gl or PDZ Sequence fused to GST Construct Seq
Acc# # ID
HYVCEVDGQNVIGL KDKKIMEILATAGNWTLTΠPSVIYEHIVEFΓV
SSTRIP 7025450 LKEKWLLQKKDSEGFGFVLRGAKAQTPIEEFTPTPAFPALQY 196
LESVDEGGVAWRAGLRMGDFLIEVNGQNWKVGHRQWNMI
RQGGNTLMVKWMVTRHPDMDEAVQ
SYNTENIN 2795862 LEIKQGIREVILCKDQDGKIGLRLKSIDNGIFVQLVQANSPASL 197 VGLRFGDQVLQINGENCAGWSSDKAHKVLKQAFGEKITMRIH
RD
SYNTENIN 2795862 RDRPFERTITMHKDSTGHVGFIFKNGKITSIVKDSSAARNGLLT 198 EHNICEINGQNVIGLKDSQIADILSTSGNSS
Syntrophin 1 1145727 QRRRVTVRKADAGGLGISIKGGRENKMPILISKIFKGLAADQT 199 alpha EALFVGDAILSVNGEDLSSATHDEAVQVLKKTGKEWLEVKY
MKDVSPYFK
Syntrophin beta i 476700 IRWKQEAGGLGISIKGGRENRMPILISKIFPGLAADQSRALRL 200
2 GDAILSVNGTDLRQA
THDQAVQALKRAGKEVLLEVKFIREFIVTD
Syntrophin 9507164 EPFYSGERTVTIRRQTVGGFGLSIKGGAEHNIPVWSKISKEQR 201 gamma 1 AELSGLLFIGDAILQINGINVRKCRHEEWQVLRNAGEEVTLTV
SFLKRAPAFLKLP
Syntrophin 9507164 1 SHQGRNRRTVTLRRQPVGGLGLSIKGGSEHNVPWISKIFEDQ 202 gamma 2 AADQTGMLFVGDAVLQVNGIHVENATHEEWHLLRNAGDEV T ITVEYLREAPAFLK
TAX2-like 3253116 RGETKEVEVTKTEDALGLTITDNGAGYAFIKRIKEGSIINRIEAV 203 protein CVGDSIEAINDHSIVGCRHYEVAKMLRELPKSQPFTLRLVQPK
RAF
TIAM 1 4507500 HSIHIEKSDTAADTYGFSLSSVEEDGIRRLYVNSVKETGLASKK 204 GLKAGDEILEINNRAADALNSSMLKDPLSQPSLGLLVRTYPEL
E
TIAM 2 6912703 1 PLNVYDVQLTKTGSVCDFGFAVTAQVDERQHLSRIFISDVLPD 205 GLAYGEGLRKGNEIMTLNGEAVSDLDLKQMEALFSEKSVGLT L IARPPDTKATL TIPl 2613001 1 QRVEIHKLRQGENLILGFSIGGGIDQDPSQNPFSEDKTDKGIYV 206
TRVSEGGPAEIAGLQ
IGDKIMQVNGWDMTMVTHDQARKRLTKRSEEWRLLVTRQS
LQK
TIP2 2613003 1 RKEVEVFKSEDALGLTITDNGAGYAFIKRIKEGSVIDHIHLISVG 207 DMIEAINGQSLLGCRHYEVARLLKELPRGRTFTLKLTEPRK TIP33 2613007 1 HSHPRWELPKTDEGLGFNVMGGKEQNSPIYISRIIPGGVAERH 208
GGLKRGDQLLSVNGVSVEGEHHEKAVELLKAAKDSVKLWR
YTPKVL
TIP43 2613011 1 ISNQKRGVKVLKQELGGLGISIKGGKENKMPILISKIFKGLAAD 209
QTQALYVGDAILSVNGADLRDATHDEAVQALKRAGKEVLLE
VKYMREATPYV
X-Il beta 3005559 1 IHFSNSENCKELQLEKHKGEILGVVWESGWGSILPTVILANM 210
MNGGPAARSGKLSIG
DQIMSINGTSLVGLPLATCQGIIKGLKNQTQVKLNIVSCPPVTT
VLIKRNSS
X-Il beta 3005559 2 IPPVTTVLIKRPDLKYQLGFSVQNGIICSLMRGGIAERGGVRVG 211
HRIIEINGQSWATA
HEKIVQALSNSVGEIHMKTMPAAMFRLLTGQENSS ZO-I 292937 1 IWEQHTVTLHRAPGFGFGIAISGGRDNPHFQSGETSIVISDVLK 212
GGPAEGQLQENDR
VAMVNGVSMDNVEHAFAVQQLRKSGKNAKITIRRKKKVQIP Gene Name Gl or PDZ Sequence fused to GST Construct Seq
Acc# # ID
NSS
ZO-I 292937 ISSQPAKPTKVTLVKSRKNEEYGLRLASHIFVKEISQDSLAARD 213
GNIQEGDWLKINGT
VTENMSLTDAKTLIERSKGKLKMWQRDRATLLNSS
ZO-I 292937 IRMKLVKFRKGDSVGLRLAGGNDVGIFVAGVLEDSPAAKEGL 214
EEGDOILRVNNVDFTNI
IREEAVLFLLDLPKGEEVTILAQKKKDVFSN
ZO-2 12734763 LIWEQYTVTLQKDSKRGFGIAVSGGRDNPHFENGETSIVISDVL 215
PGGPADGLLQEND
RWMVNGTPMEDVLHSFAVQQLRKSGKVAAIWKRPRKV
ZO-2 12734763 RVLLMKSRANEEYGLRLGSQIFVKEMTRTGLATKDGNLHEGD 216 IILKINGTVTENMSLT DARKLIEKSRGKLQLWLRDS
ZO-2 12734763 HAPNTKMVRFKKGDSVGLRLAGGNDVGIFVAGIQEGTSAEQE 217
GLQEGDQILKVNTQ
DFRGLVREDAVLYLLEIPKGEMVTILAQSRADVY
ZO-3 10092690 IPGNSTIWEQHTATLSKDPRRGFGIAISGGRDRPGGSMWSDV 218
VPGGPAEGRLQTG
DHIVMVNGVSMENATSAFAIQILKTCTKMAINTVKRPRRIHLP
AEFIVTD
ZO-3 10092690 2 QDVQMKPVKSVLVKRRDSEEFGVKLGSQIFIKHITDSGLAARH 219 RGLQEGDLILQINGV SSQNLSLNDTRRLIEKSEGKLSLLVLRDRGQFLVNIPNSS
ZO-3 10092690 3 RGYSPDTRWRFLKGKSIGLRLAGGNDVGIFVSGVQAGSPAD 220 GQGIQEGDQILQVN
DVPFQNLTREEAVQFLLGLPPGEEMELVTQRKQDIFWKMVQ SEFIVTD
TABLE 3
Higher affinity interactions between HPV E6 PLs and PDZ domains
HPV PDZ binding partner PDZ binding partner strain (signal 4 and 5 of 0-5) HPV strain (signal 4 and 5 of 0-5)
HPV 35 Atrophin- 1 interact, prot. HPV 33 Magil (PDZ #2)
(TEV) (PDZ # 1, 3, 5) (TAL) TIPl
Magil (PDZ # 2, 3, 4, 5) DLGl
Lim-Ril Vartul (PDZ #1)
FLJ 11215 KIAA 0807
MUPP-I (PDZ #10) KIAA 1095 (Semcap3) (PDZ #1)
KIAA1095 (PDZ #1) KIAA 1934 (PDZ #1)
PTN-4 NeDLG (PDZ #1,2)
INADL (PDZ #8) Rat outer membrane (PDZ #1)
Vartul (PDZ # 1, 2,3) PSD 95 (PDZ #3 and 1-3)
Syntrophin-1 alpha
Syntrophin gamma- 1
TAX IP2
KIAA 0807
KIAA 1634 (PDZ #1)
DLGl (PDZl, 2)
NeDLG (1, 2, 3,)
Sim. Rat outer membrane (PDZ
#1)
MUPP-I (PDZ #13)
PSD 95 (1,2,3)
HPV 58 Atrophin- 1 interact, prot. (PDZ # HPV 66 DLGl (PDZ #1, 2)
(TQV) 1) (STV) NeDLG (PDZ #2) HPV PDZ binding partner PDZ binding partner strain (signal 4 and 5 of 0-5) HPV strain (signal 4 and 5 of 0-5)
Magil (PDZ #2) PSD 95 (PDZ #1, 2, 3)
DLGl (PDZl, 2) Magil (PDZ #2)
DLG2 (PDZ #2) KIAA 0807
KIAA 0807 KIAA 1634 (PDZ #1)
KIAA 1634 (PDZ #1) DLG2 (PDZ #2)
NeDLG (1, 2) Rat outer membrane (PDZ #1)
Sim. Rat outer membrane (PDZ NeDLG (1, 2)
#1) TIP-I
PSD 95 (1,2,3)
INADL (PDZ #8)
TIP-I
HPV 16* TIP-I HPV 52 Magil (PDZ #2)
(TQL) Magil (PDZ #2) (TQV)
HPV 18* TIPl
(TQV) Magi 1 (PDZ #2)
Table 3: Interactions between the E6 C-termini of several HPV variants and human PDZ domains. HPV strain denotes the strain from which the E6 C-terminal peptide sequence information was taken. Peptides used in the assay varied from 18 to 20 amino acids in length, and the terminal four residues are listed in parenthesis. Names to the right of each HPV E6 variant denote the human PDZ domain(s) (with domain number in parenthesis for proteins with multiple PDZ domains) that saturated binding with the E6 peptide in the G assay (See Description of the Invention), denotes that the PDZ domains of hDlgl are not tested against these proteins yet due to limited material, although both have been shown to bind hDlgl in the literature.
III. Chimeric PDZ Domain-Containing Polypeptide
[0034] The present invention provides a chimeric polypeptide containing at least one PDZ domain or a PDZ ligand binding portion of a PDZ domain introduced from a first PDZ domain containing polypeptide into a second PDZ domain containing polypeptide such that the chimeric polypeptide has enhanced binding strength than the native second PDZ domain containing polypeptide. In some embodiments, the binding strength of a chimeric PDZ domain to E6 protein is enhanced by greater than 1.2, 1.5, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, or 5 fold. [0035] A "chimeric polypeptide" or a "chimeric polynucleotide" is an artificially constructed protein or polynucleotide comprising heterologous amino acid sequences or heterologous nucleic acid sequences, respectively. Chimeric proteins are proteins created through the joining of two or more genes, or portions of two or more genes, which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with function properties derived from each of the original proteins. Such chimeric proteins are created artificially by recombinant DNA technology well known in the art. The term "heterologous" when used with reference to a protein or a nucleic acid indicates that the protein or the nucleic acid comprises two or more sequences or subsequences which are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid. For example, in one embodiment, the nucleic acid has a promoter from one gene arranged to direct the expression of a coding sequence from a different gene. Thus, with reference to the coding sequence, the promoter is heterologous.
[0036] The terms "polypeptide" "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. A native polypeptide is typically isolated from a naturally occurring source, in particular a mammalian or microbial source, such as a human source, or is produced recombinantly by use of a nucleotide sequence encoding the naturally occurring amino acid sequence. The term "native polypeptide" as used herein refers to a naturally occurring polypeptide. A "native" PDZ domain containing polypeptide refers to a naturally occurring polypeptide in which the PDZ domain is not modified in its amino acid residues. A "variant" is a polypeptide, which has an amino acid sequence that differs from that of a native polypeptide in one or more amino acid residues. The variant is typically prepared by modification of a nucleotide sequence encoding the native polypeptide (e.g., to result in substitution, deletion or truncation of one or more amino acid residues of the polypeptide or by introduction (by addition or insertion) of one or more amino acid residues into the polypeptide) so as to modify the amino acid sequence constituting said native polypeptide. [0037] The present invention provides a chimeric polypeptide in which at least one PDZ domain or a portion thereof is introduced from a first PDZ domain-containing polypeptide into a second PDZ domain- containing polypeptide, and that results in enhanced binding of such chimeric polypeptide to a PDZ ligand. In one embodiment, one PDZ domain is introduced from the first to the second PDZ domain- containing polypeptide. In another embodiment, two PDZ domains are introduced from the first to the second PDZ domain- containing polypeptide. In still another embodiment, three PDZ domains are introduced from the first to the second PDZ domain-containing polypeptide. In yet another embodiment, more than three PDZ domains, e.g. 4, 5, 6, 7, or 8 PDZ domains are introduced from the first to the second PDZ domain-containing polypeptide. In some embodiments, an introduced PDZ domain from the first PDZ domain- containing polypeptide substitutes a PDZ domain naturally present in the second PDZ domain-containing polypeptide. However, the present invention also includes a chimeric polypeptide in which a PDZ domain is introduced in addition without substituting a PDZ domain naturally present in the second PDZ domain-containing polypeptide. The PDZ domain from the first PDZ domain- containing polypeptide can be introduced at any position in the second PDZ domain- containing polypeptide so long as the adjacent sequences of the second PDZ domain- containing polypeptide allows proper PDZ domain folding such that the chimeric polypeptide exhibits enhanced binding to a PDZ ligand than a native PDZ domain-containing polypeptide. In some embodiments, PDZ domains are introduced into more than one location in the second PDZ domain- containing polypeptide. In some embodiments, the chimeric polypeptide of the invention, the first PDZ domain-containing polypetide is a PDZ domain listed in Table 2 (SEQ ID No's: 1-220).
[0038] In a specific embodiment, the first PDZ domain-containing polypeptide is MAGI-I and the second PDZ domain-containing polypeptide is PSD95. Three PDZ domains of PSD95 are substituted with 3 PDZ domain 1 of MAGI-I, as shown in FIG 1. The chimeric polypeptide contains 3 MAGI-I PDZ domain 1 with the non-PDZ portions of PSD95 as the backbone. The non-PDZ domain portions of PSD95, when recombined with PDZ domain 1 of MAGI-I, allow correct PDZ folding, resulting in enhanced binding of the chimeric polypeptide to E6 protein from an oncogenic HPV strain. The chimeric PSD95-MAGI-1 polypeptide is fused to GST at the C-terminus, and can be readily expressed in high quantities in an expression vector such as E. CoIi. The amino acid sequence of the chimeric PSD95-MAGI-1 polypeptide fused to GST is shown in FIG 2.
[0039] The present invention also embodies a chimeric polypeptide in which the introduced PDZ domain may be modified, i.e. the introduced PDZ domain is a variant of the naturally occurring PDZ domain present in the native first PDZ domain-containing polypeptide. In some embodiments, the introduced PDZ domain is modified having at least 70, 80, 90, 95, 96, 97, 98, or 99 % identity to a PDZ domain. Examples of PDZ domain variants include ones which have at least 80%, one which has at least 90%, or one which has at least 95% amino acid sequence identity to the native PDZ amino acid sequence, and which contain at least one functional or structural characteristic of a native PDZ domain, such as binding to the E6 oncogenic protein of HPV..
[0040] The invention also includes a chimeric polypeptide containing a PDZ ligand binding portion of a PDZ domain introduced from the first to the second PDZ domain- containing polypeptide. As described hereinabove, a PDZ domain is generally composed of 80-90 amino-acids. A PDZ ligand binding portion of a PDZ domain (i.e. fragments of a PDZ domain) may consist of a minimum number of amino acid residues required for PDZ ligand binding. Such PDZ ligand binding portion of a PDZ domain may be generated entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding such PDZ domain or any fragment thereof.
[0041] The invention also encompasses chimeric polynucleotides which encode the chimeric polypeptides described herein. The invention may also encompass a variant of a polynucleotide sequence encoding a chimeric polypeptide. In particular, such a variant polynucleotide sequence will have at least 70, 80, 90, 95, 96, 97, 98, or 99 % identity to the polynucleotide sequence encoding a native PDZ domain from the first PDZ domain-containing polypeptide. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of a native PDZ domain- containing polypeptide. [0042] It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding such chimeric polypeptide as described herein, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring PDZ domain, and all such variations are to be considered as being specifically disclosed.
[0043] The present invention also embodies polynucleotide sequences encoding a chimeric polypeptide containing at least one introduced PDZ domain or its variants possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the chimeric polypeptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding chimeric polypeptide containing at least one introduced PDZ domain or its variants without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence.
[0044] The chimeric polynucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter PDZ domain-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site -directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
[0045] In some embodiments, one or more PDZ domains from the first PDZ domain containing polypeptiode are embedded in certain adjacent sequences of the second PDZ domain containing polypeptide such that the conformation of the chimeric polypeptide allows for higher binding of the chimeric polypeptide to a PDZ ligand as compared to the native second PDZ containing polypeptide. A PDZ domain in its native polypeptide conformation has high binding affinity to a PDZ ligand but the same PDZ domain outside its native polypeptide conformation has been shown to have only marginal binding to a PDZ ligand. Thus, an isolated PDZ domain or a PDZ domain outside its native polypeptide conformation would not be expected to have high binding ability to a PDZ ligand. For example, PDZ domain 2 of PSD95 captures high-risk HPV E6 to a marginal extent when represented as complete and correctly folded but isolated PDZ domain. In one example of the present invention, E6 capture via MAGI-I PDZ domain 1 is increased when MAGI-I-PDZ domain 1 is embedded in certain adjacent sequences of PSD95 polypeptide (sequences encompassing PDZ domains 1 through 3 of PSD95). The sequences in the second PDZ domain polypeptide flanking the inserted PDZ domain confer enhanced PDZ ligand binding to the inserted PDZ domain. In one example, the PSD95 non-PDZ protein portions confer enhanced E6 binding to the inserted PDZ domain 1 of MAGI-I.
[0046] In another embodiment, sequences encoding a chimeric polypeptide containing at least one PDZ domain from a first PDZ containing polypeptide may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232.) Alternatively, such chimeric polypeptide may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solid-phase techniques. (See, e.g., Roberge, J. Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431 A Peptide Synthesizer (Perkin-Elmer). Additionally, the amino acid sequence of a chimeric polypeptide, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
[0047] The peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z. Regnier (1990) Methods Enzymol. 182:392-421). The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York N. Y.) In order to express a biologically active chimeric polypeptide, the polynucleotide sequences encoding such chimeric polypeptide described herein may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding the chimeric PDZ domain containing polypeptide. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding the chimeric PDZ domain containing polypeptide. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding a chimeric PDZ domain containing polypeptide and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162.) [0048] Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a chimeric PDZ domain containing polypeptide and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination as described in details under section XIX. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., ch. 9, 13, and 16.) IV. Binding of Chimeric PDZ Domain-Containing Protein to PDZ Ligand
[0049] The present invention includes a chimeric PDZ domain containing polypeptide that has enhanced binding strength than a native PDZ domain containing polypeptide. The binding strength of a PDZ domain containing polypeptide to a PDZ ligand includes binding affinity and binding avidity of such PDZ domain containing polypeptide to the PDZ ligand. The term "binding affinity" refers to the equilibrium dissociation constant (expressed in units of concentration) associated with each PDZ domain-PDZ ligand binding interaction. The binding affinity is directly related to the ratio of the kinetic off-rate (generally reported in units of inverse time, e.g., seconds-1) divided by the kinetic on-rate (generally reported in units of concentration per unit time, e.g., molar/second). In general it is not possible to unequivocally state whether changes in equilibrium dissociation constants are due to differences in on-rates, off-rates or both unless each of these parameters are experimentally determined (e.g., by BIACORE or SAPIDYNE measurements). In proteins, avidity is a term used to describe the combined strength of multiple bond interactions. Avidity is distinct from affinity, which is usually used to describe the binding strength of a single bond. As such, avidity is the combined synergistic binding strength of bond affinities rather than the sum of bonds. In some embodiments, the chimeric polypeptide has synergistic binding to a PDZ ligand as compared to a native PDZ domain polypeptide. "Enhanced" binding strength as used herein refers to increased, greater or higher binding affinity and/or avidity of a chimeric PDZ domain containing polypeptide to a PDZ ligand. Similarly, "diminished" binding strength refers to reduced, decreased or lesser binding affinity and/or avidity of a chimeric PDZ domain containing polypeptide to a PDZ ligand as compared to a native PDZ domain containing polypeptide or an isolated PDZ domain.
[0050] In one embodiment of the present invention, the chimeric polypeptide containing at least one PDZ domain or a PDZ ligand binding portion of a PDZ domain introduced from a first PDZ domain- containing polypeptide into a second PDZ domain- containing polypeptide has a higher binding affinity to a PDZ ligand, such as E6 protein, as compared to the native second PDZ domain-containing polypeptide. The native second PDZ domain-containing polypeptide does not have any modifications in its PDZ domains.
[0051] In another embodiment, the chimeric polypeptide containing at least one PDZ domain or a PDZ ligand binding portion of a PDZ domain introduced from a first PDZ domain-containing polypeptide into a second PDZ domain-containing polypeptide has a higher binding affinity to a PDZ ligand, such as E6 protein, as compared to the native first PDZ domain- containing polypeptide. The native first PDZ domain-containing polypeptide does not have any modifications in its PDZ domains.
[0052] In yet another embodiment, the chimeric polypeptide containing at least one PDZ domain or a PDZ ligand binding portion of a PDZ domain introduced from a first PDZ domain-containing polypeptide into a second PDZ domain-containing polypeptide has a higher binding affinity to a PDZ ligand, such as E6 protein, as compared to a single isolated PDZ domain. For instance, a chimeric polypeptide in which three PDZ domains of the second PDZ domain-containing polypeptide PSD95 have been substituted with three PDZ domain 1 of MAGI shows enhanced binding to E6 protein from oncogenic HPV strain 16 over the single MAGI- 1 PDZ domain 1 as determined by sandwich ELISA and lateral flow assays (FIGS 3 and 4).
[0053] The chimeric polypeptide described herein may be particularly useful to increase the avidity of the chimeric PDZ domain containing polypeptide to a PDZ ligand, such as HPV E6 protein. The present invention embodies a chimeric PDZ domain-containing polypeptide which has multiple introduced PDZ domains, thereby increasing the overall binding avidity and binding strength of such chimeric PDZ domain- containing polypeptide to a PDZ ligand.
[0054] In another embodiment, the chimeric PDZ domain- containing polypeptide may have enhanced binding affinity and binding avidity to a PDZ ligand as compared to the native first PDZ domain-containing polypeptide. In yet another embodiment, the chimeric PDZ domain-containing polypeptide may have enhanced binding affinity and binding avidity to a PDZ ligand as compared to the native second PDZ domain-containing polypeptide. In still another embodiment, the chimeric PDZ domain-containing polypeptide may have enhanced binding affinity and binding avidity to a PDZ ligand as compared to an isolated PDZ domain.
[0055] The binding properties of the molecules of the invention including binding affinity and binding avidity for a PDZ ligand protein may be initially determined using in vitro assays (biochemical or immunological based assays) known in the art for PDZ domain and PDZ ligand interactions, including but not limited to ELISA assay, surface plasmon resonance assay, immunoprecipitation assays, cytometric bead array and lateral flow assay. In other embodiments, the molecules of the invention have similar binding properties in in vivo models as those in in vitro based assays. However, the present invention does not exclude molecules of the invention that do not exhibit the desired phenotype in in vitro based assays but do exhibit the desired phenotype in vivo.
[0056] The invention encompasses assays known in the art, and exemplified herein, to characterize the binding of a chimeric PDZ domain-containing polypeptide to a PDZ ligand. To determine such binding, a sandwich ELISA may be performed. An exemplary assay may comprise the following: assay plates may be coated overnight at 40C with a chimeric PDZ domain-containing polypeptide or a control polypeptide in coating buffer. The plates may then be washed and blocked. Following washing, an aliquot of human E6 protein may be added to each well and incubated for 2 hrs at room temperature. Following a further wash, an antibody specific for E6 and conjugated with peroxidase may be added to each well and incubated for 1 hour at room temperature. The plate may again be washed with wash buffer and 100 ul of substrate buffer containing OPD (O-phenylenediamine dihydrochloride (Sigma)) may be added to each well. The oxidation reaction, observed by the appearance of a yellow color, may be allowed to proceed for 30 minutes and stopped by the addition of 100 ul of 4.5 NH2 SO4. The absorbance may then read at (492-405) nm.
[0057] Using the G-assay described herein, several GST-PDZ domain fusion proteins have been tested to determine their relative binding strength to the PL of the HPVl 6 E6 protein (TABLE 4). The term half maximal effective concentration (EC50) refers to the concentration of a molecule which induces a response halfway between the baseline and maximum. EC50 is a good measure of binding strength of a PDZ domain to a PL, and therefore useful in determining which PDZ domains from the first PDZ domain containing polypeptide to introduce to the second PDZ domain containing polypeptide to constitute the chimeric PDZ domain containing polypeptide that has enhanced binding strength to a PDZ ligand over a native PDZ containing polypeptide. TABLE 4
EC50 values for HPVl 6 E6 protein with various PDZ domains
RNA expression(Cervi- PDZ gene EC50a [uM] cal cell lines)
MagilC (PDZ2) 0.056 ++
Magi3 (PDZl) 0.31 neg.
SASTl KIAA 0.58 neg.
TIPl 0.75 +++
VARTUL 0.94 +
DLGl (PDZ2) ND ++++
PSD95 (PDZ 1-3) 1.0 ND
SAST2 1.2 ND
DLG2 (PDZ3) 1.6 ND
DLG3 (PDZ1-2) 3.8 ND
PSD95 (PDZ2) 6.8 ND
SIPl (PDZl) 7.5 ND
Table 4 legend: ND = not done.
[0058] The molecules of the invention comprising a chimeric PDZ domain-containing polypeptide may be assayed using any surface plasmon resonance (SPR) based assays known in the art for characterizing the kinetic parameters of binding between the chimeric PDZ domain-containing polypeptide and a PDZ ligand. Any SPR instrument commercially available including, but not limited to, BIAcore Instruments, available from Biacore AB (Uppsala, Sweden); IAsys instruments available from Affinity Sensors (Franklin, Mass.); IBIS system available from Windsor Scientific Limited (Berks, UK), SPR-CELLIA systems available from Nippon Laser and Electronics Lab (Hokkaido, Japan), and SPR Detector Spreeta available from Texas Instruments (Dallas, Tex.) can be used in the instant invention. For a review of SPR-based technology see Mullet et al., 2000, Methods 22: 77-91 ; Dong et al., 2002, Review in MoI. Biotech., 82: 303-23; Fivash et al., 1998, Current Opinion in Biotechnology 9: 97-101; Rich et al., 2000, Current Opinion in Biotechnology 11: 54-61; all of which are incorporated herein by reference in their entirety. Additionally, any of the SPR instruments and SPR based methods for measuring protein-protein interactions described in U.S. Pat. Nos. 6,373,577; 6,289,286; 5,322,798; 5,341,215; 6,268,125 are contemplated in the methods of the invention, all of which are incorporated herein by reference in their entirety. [0059] Briefly, SPR based assays involve immobilizing a member of a binding pair on a surface, and monitoring its interaction with the other member of the binding pair in solution in real time. SPR is based on measuring the change in refractive index of the solvent near the surface that occurs upon complex formation or dissociation. The surface onto which the immobilization occur is the sensor chip, which is at the heart of the SPR technology; it consists of a glass surface coated with a thin layer of gold and forms the basis for a range of specialized surfaces designed to optimize the binding of a molecule to the surface. A variety of sensor chips are commercially available especially from the companies listed supra, all of which may be used in the methods of the invention. Examples of sensor chips include those available from BIAcore AB, Inc., e.g., Sensor Chip CM5, SA, NTA, and HPA. A molecule of the invention may be immobilized onto the surface of a sensor chip using any of the immobilization methods and chemistries known in the art, including but not limited to, direct covalent coupling via amine groups, direct covalent coupling via sulfhydryl groups, biotin attachment to avidin coated surface, aldehyde coupling to carbohydrate groups, and attachment through the histidine tag with NTA chips.
[0060] In some embodiments, the kinetic parameters of the binding of a chimeric PDZ domain-containing polypeptide to a PDZ ligand may be determined using a BIAcore instrument (e.g., BIAcore instrument 1000, BIAcore Inc., Piscataway, NJ.). In a specific embodiment, the binding of a chimeric polypeptide containing 3 PDZ domain 1 of MAGI-I and the non-PDZ portions of PSD95 to E6 protein from an oncogenic HPV strain is determined using BIAcore.
[0061] Once an entire data set is collected, the resulting binding curves are globally fitted using computer algorithms supplied by the SPR instrument manufacturer, e.g., BIAcore, Inc. (Piscataway, NJ.). These algorithms calculate both the K0n and Koff from which the apparent equilibrium binding constant, Ka is deduced as the ratio of the two rate constants (i.e., Kog-/Kon). More detailed treatments of how the individual rate constants are derived can be found in the BIAevaluaion Software Handbook (BIAcore, Inc., Piscataway, NJ.). The analysis of the generated data may be done using any method known in the art.
[0062] In another embodiment of the present invention, lateral flow assays are utilized to determine binding between a chimeric PDZ domain- containing polypeptide and a PDZ ligand. Lateral flow devices work by applying fluid to a test strip that has been treated with specific biologicals. Carried by the liquid sample, phosphors labeled with corresponding biologicals flow through the strip and can be captured as they pass into specific zones. The amount of phosphor signal found on the strip is proportional to the amount of the target analyte. [0063] A sample suspected of containing one of the viruses, bacteria or related microbes disclosed herein is added to a lateral flow device by some means, the sample is allowed to move by diffusion and a line or colored zone indicates the presence of the virus or bacteria. The lateral flow typically contains a solid support (for example nitrocellulose membrane) that contains three specific areas: a sample addition area, a capture area containing one or more chimeric PDZ domain-containing polypeptides and/or antibodies immobilized, and a read-out area that contains one or more zones, each zone containing one or more labels. The lateral flow can also include positive and negative controls. Thus, for example a lateral flow device in certain exemplary procedures would perform as follows: a viral or bacterial PDZ ligand protein is separated from other bacterial, viral and cellular proteins in a biological sample by bringing an aliquot of the biological sample into contact with one end of a test strip, and then allowing the proteins to migrate on the test strip, e.g., by capillary action such as lateral flow. One or more PL binding agents such as chimeric PDZ domain- containing polypeptides, antibodies, and/or aptamers are included as capture and/or detect reagents. Methods and devices for lateral flow separation, detection, and quantification are known in the art, e.g., U.S. Pat. Nos. 5,569,608, 6,297,020; and 6,403,383 incorporated herein by reference in their entirety. In one non-limiting example, a test strip comprises a proximal region for loading the sample (the sample- loading region) and a distal test region containing a chimeric PDZ domain-containing polypeptide as a capture agent and buffer reagents and additives suitable for establishing binding interactions between the chimeric PDZ domain- containing polypeptide and any viral or bacterial PL protein in the migrating biological sample. In alternative exemplary procedures, the test strip comprises two test regions that contain different chimeric PDZ domain- containing polypeptides, i.e., each capable of specifically interacting with a different viral or bacterial PL protein analyte. Optionally, the lateral flow can include tests for a variety of different viruses and bacteria. V. Detection of the Chimeric PDZ Domain-Containing Polypeptide or PDZ Ligand (PL) [0064] Two complementary assays, termed "A" and "G", are developed to detect binding between a PDZ -domain polypeptide and candidate PDZ ligand. In each of the two different assays, binding is detected between a peptide having a sequence corresponding to the C-terminus of a protein anticipated to bind to one or more PDZ domains (i.e. a candidate PL peptide) and a PDZ-domain polypeptide (typically a fusion protein containing a PDZ domain). In the "A" assay, the candidate PL peptide is immobilized and binding of a soluble chimeric PDZ-domain polypeptide to the immobilized peptide is detected (the "A"' assay is named for the fact that in one embodiment an avidin surface is used to immobilize the peptide). In the "G" assay, the chimeric PDZ-domain polypeptide is immobilized and binding of a soluble PL peptide is detected (The "G" assay is named for the fact that in one embodiment a GST-binding surface is used to immobilize the PDZ-domain polypeptide). Preferred embodiments of these assays are described in detail infra. However, it will be appreciated by ordinarily skilled practitioners that these assays can be modified in numerous ways while remaining useful for the purposes of the present invention.
A. Production of Fusion Proteins Containing PDZ-Domains
[0065] One embodiment of the present invention includes a chimeric PDZ domain- containing polypeptide fused to GST. PCR products containing the chimeric PDZ domain polynucleotide are subcloned into an expression vector to permit expression of fusion proteins containing a chimeric polypeptide with at least one introduced PDZ domain and a heterologous domain (i.e., a glutathione-S transferase sequence, "GST"). PCR products (i.e., DNA fragments) representing the chimeric polynucleotide with at least one introduced PDZ domain encoding DNA are extracted from agarose gels using the "Sephaglas" gel extraction system (Pharmacia) according to the manufacturer's re commendations .
[0066] PCR primers are designed to include endonuclease restriction sites to facilitate ligation of PCR fragments into a GST gene fusion vector (pGEX-3x; Pharmacia, GenBank accession no. XXUl 3852) in- frame with the glutathione-S transferase coding sequence. This vector contains an IPTG inducible lacZ promoter. The pGEX-3x vector was linearized using Bam HI and Eco RI or, in some cases, Eco RI or Sma I, and dephosphorylated. For most cloning approaches, double digestion with Bam HI and Eco RI was performed, so that the ends of the PCR fragments to clone are Bam HI and Eco RI. In some cases, restriction endonuclease combinations used are BgI II and Eco RI, Bam HI and Mfe I, or Eco RI only, Sma I only, or BamHI only. When more than one PDZ domain was cloned, the DNA portion cloned represents the PDZ domains and the cDNA portion located between individual domains. DNA linker sequences between the GST portion and the chimeric PDZ containing polynucleotide portion vary slightly, dependent on which of the above described cloning sites and approaches are used. As a consequence, the amino acid sequence of the GST-chimeric PDZ domain containing fusion protein varies in the linker region between GST and the chimeric PDZ domain-containing polypeptide. Protein linker sequences corresponding to different cloning sites/approaches are shown below. Linker sequences (vector DNA encoded) are bold, sequences of the chimeric polynucleotides containing at least one introduced PDZ domain are in italics. [0067] 1) GST— BamHI/BamHI — chimeric polynucleotide insert; Gly— He— chimeric polynucleotide insert [0068] 2) GST— BamHI/Bglll— chimeric polynucleotide insert; Gly— He— chimeric polynucleotide insert [0069] 3) GST— EcoRFEcoRI— chimeric polynucleotide insert; Gly— He— Pro— Gly— Asn— chimeric polynucleotide insert [0070] 4) GST— Smal/Smal— chimeric polynucleotide insert; Gly— He— Pro— chimeric polynucleotide insert [0071] The chimeric polynucleotide PCR fragment and linearized pGEX-3x vector are ethanol precipitated and resuspended in 10 ul standard ligation buffer. Ligation is performed for 4-10 hours at 7C using T 4 DNA ligase. It will be understood that some of the resulting constructs include very short linker sequences and that, when multiple PDZ domains are cloned, the constructs included some DNA located between individual PDZ domains. [0072] The ligation products are transformed in DH5alpha or BL-21 E. coli bacteria strains. Colonies are screened for presence and identity of the cloned chimeric polynucleotide containing at least one introduced PDZ domain as well as for correct fusion with the glutathione S-transferase encoding DNA portion by PCR and by sequence analysis. Positive clones are tested in a small-scale assay for expression of the GST/chimeric PDZ domain fusion protein and, if expressing, these clones are subsequently grown up for large scale preparations of GST/chimeric PDZ domain fusion protein.
[0073] Fusion protein of GST and chimeric PDZ-domain containing polypeptide is overexpressed following addition of IPTG to the culture medium and purified. Detailed procedure of small scale and large-scale fusion protein expression and purification are described in "GST Gene Fusion System" (second edition, revision 2; published by Pharmacia). In brief, a small culture (50 mis) containing a bacterial strain (DH5α, BL21 or JM 109) with the fusion protein construct is grown overnight in 2xYT media at 370C with the appropriate antibiotic selection (100 ug/ml ampicillin; a.k.a. 2x YT-amp). The overnight culture is poured into a fresh preparation of 2xYT-amp (typically 1 liter) and grown until the optical density (OD) of the culture is between 0.5 and 0.9 (approximately 2.5 hours). IPTG (isopropyl β-D-thiogalactopyranoside) is added to a final concentration of 1.0 mM to induce production of GST fusion protein, and culture is grown an additional 1 hour. All following steps, including centrifugation, are performed on ice or at 40C. Bacteria are collected by centrifugation (450Ox g) and resuspended in Buffer A- (50 mM Tris, pH 8.0, 50 mM dextrose, 1 mM EDTA, 200 uM phenylmethylsulfonylfluoride). An equal volume of Buffer A+ (Buffer A-, 4 mg/ml lysozyme) is added and incubated on ice for 3 min to lyse bacteria, or until lysis had begun. An equal volume of Buffer B (10 mM Tris, pH 8.0, 50 mM KCl, 1 mM EDTA. 0.5% Tween- 20, 0.5% NP40 (a.k.a. IGEPAL CA-630), 200 uM phenylmethylsulfonylfluoride) is added and incubated for an additional 20 min on ice. The bacterial cell lysate is centrifuged (x20,000 g), and supernatant is run over a column containing 20 ml Sepharose CL-4B (Pharmacia) "precolumn beads," i.e., sepharose beads without conjugated glutathione that had been previously washed with 3 bed volumes PBS. The flow-through is added to glutathione Sepharose 4B (Pharmacia, cat no. 17-0765-01) previously swelled (rehydrated) in Ix phosphate-buffered saline (PBS) and incubated while rotating for 30 min-1 hr. The supernatant- Sepharose slurry is poured into a column and washed with at least 20 bed volumes of Ix PBS. GST fusion protein is eluted off the glutathione sepharose by applying 0.5-1.0 ml aliquots of 5 mM glutathione and collected as separate fractions. Concentrations of fractions are determined by reading absorbance at 280 nm and calculating concentration using the absorbance and extinction coefficient. Those fractions containing the highest concentration of fusion protein are pooled and an equal volume of 70% glycerol is added to a final concentration of 35% glycerol. Fusion proteins are assayed for size and quality by SDS gel electrophoresis (PAGE) as described in "Sambrook." Fusion protein aliquots are stored at minus 8O0C. and at minus 2O0C.
[0074] The amino acid sequences provided in Table 2 may contain amino acids derived from a fusion protein, e.g., fusion protein of GST and chimeric PDZ-domain containing polypeptide of particular interest may be up to 20 amino acids shorter (e.g., 5, 8, 10, 12 or 15 amino acids shorter) than the sequence provided in Table 2. For example, a sequence may be shortened by up to 3, 6, 9, or 12 amino acids from the C-terminus, the N-terminus, or both termini.
B. Identification of Candidate PL Proteins and Synthesis of Peptides
[0075] Certain PDZ domains are bound by the C-terminal residues of PDZ-binding proteins. To identify PL proteins, the C-terminal residues of sequences are visually inspected for sequences that one might predict would bind to PDZ -domain containing proteins (see, e.g., Doyle et al., 1996, Cell 85, 1067; Songyang et al., 1997, Science 275, 73), including the additional consensus for PLs identified at Arbor Vita Corporation (U.S. patent application Ser. No. 60/360061). TABLE 1 lists some of these proteins, and provides corresponding C-terminal sequences. [0076] Synthetic peptides of defined sequence (e.g., corresponding to the carboxyl-termini of the indicated proteins) can be synthesized by any standard resin-based method (see, e.g., U.S. Pat. No. 4,108,846; see also, Caruthers et al., 1980, Nucleic Acids Res. Symp. Ser., 215-223; Horn et al., 1980, Nucleic Acids Res. Symp. Ser., 225-232; Roberge, et al., 1995, Science 269:202). The peptides used in the assays described herein are prepared by the FMOC (see, e.g., Guy and Fields, 1997, Meth. Enz. 289:67-83; Wellings and Atherton, 1997, Meth. Enz. 289:44-67). In some cases (e.g., for use in the A and G assays of the invention), peptides are labeled with biotin at the amino-terminus by reaction with a four-fold excess of biotin methyl ester in dimethylsulfoxide with a catalytic amount of base. The peptides are cleaved from the resin using a halide containing acid (e.g. trifluoroacetic acid) in the presence of appropriate antioxidants (e.g. ethanedithiol) and excess solvent lyophilized. [0077] Following lyophilization, peptides can be redissolved and purified by reverse phase high performance liquid chromatography (HPLC). One appropriate HPLC solvent system involves a Vydac C- 18 semi-preparative column running at 5 mL per minute with increasing quantities of acetonitrile plus 0.1% trifluoroacetic acid in a base solvent of water plus 0.1% trifluoroacetic acid. After HPLC purification, the identities of the peptides are confirmed by MALDI cation-mode mass spectrometry.
C. Detecting Interaction between a Chimeric PDZ Domain-Containing Polypeptide and a PDZ Ligand
[0078] The present invention also relates to the detection of the interaction between a chimeric PDZ domain- containing polypeptide and a PDZ ligand. Various assay formats known in the art can be used to select ligands that are specifically reactive with a particular protein. For example, solid-phase ELISA immunoassays, immunoprecipitation, Biacore, and Western blot assays can be used to identify peptides that specifically bind PDZ- domain polypeptides. As discussed herein, two different, complementary assays are developed to detect PDZ-PL interactions. In each, one binding partner of a chimeric PDZ domain- containing polypeptide and PDZ ligand pair is immobilized, and the ability of the second binding partner to bind is determined. These assays can be readily used to screen for hundreds to thousands of potential PDZ-ligand interactions in a few hours. Thus these assays can be used to identify yet more novel PDZ-PL interactions in cells. In addition, they can be used to identify antagonists of PDZ- PL interactions.
[0079] In various embodiments, fusion proteins are used in the assays and devices of the invention. Methods for constructing and expressing fusion proteins are well known. Fusion proteins generally are described in Ausubel et al., Kroll et al., 1993, DNA Cell. Biol. 12:441, and Imai et al., 1997, Cell 91:521-30. Usually, the fusion protein includes a domain to facilitate immobilization of the protein to a solid substrate ("an immobilization domain"). Often, the immobilization domain includes an epitope tag (i.e., a sequence recognized by an antibody, typically a monoclonal antibody) such as polyhistidine (Bush et al, 1991, J. Biol Chem 266:13811-14), SEAP (Berger et al, 1988, Gene 66: 1-10), or Ml and M2 flag (see, e.g, U.S. Pat. Nos. 5,011,912; 4,851,341; 4,703,004; 4,782,137). In an embodiment, the immobilization domain is a GST coding region. It will be recognized that, in addition to the chimeric PDZ domain- containing polypeptide and the particular residues bound by an immobilized antibody, protein A, or otherwise contacted with the surface, the protein (e.g., fusion protein), will contain additional residues. In some embodiments these are residues naturally associated with the PDZ-domain (i.e., in a particular PDZ-protein) but they may include residues of synthetic (e.g., poly(alanine)) or heterologous origin (e.g., spacers of, e.g., between 10 and 300 residues).
[0080] Chimeric PDZ domain-containing polypeptide used in the methods of the invention (e.g., PDZ fusion proteins) of the invention are typically made by (1) constructing a vector (e.g., plasmid, phage or phagemid) comprising a polynucleotide sequence encoding the desired polypeptide, (2) introducing the vector into an suitable expression system (e.g., a prokaryotic, insect, mammalian, or cell free expression system), (3) expressing the fusion protein and (4) optionally purifying the fusion protein.
[0081] In one embodiment, expression of the protein comprises inserting the coding sequence into an appropriate expression vector (i.e., a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence required for the expression system employed, e.g., control elements including enhancers, promoters, transcription terminators, origins of replication, a suitable initiation codon (e.g., methionine), open reading frame, and translational regulatory signals (e.g., a ribosome binding site, a termination codon and a polyadenylation sequence. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used.
[0082] The coding sequence of the fusion protein includes a chimeric PDZ domain-containing polynucleotide sequence and an immobilization domain as described herein. Polynucleotides encoding the amino acid sequence for each domain can be obtained in a variety of ways known in the art; typically the polynucleotides are obtained by PCR amplification of cloned plasmids, cDNA libraries, and cDNA generated by reverse transcription of RNA, using primers designed based on sequences determined by the practitioner or, more often, publicly available (e.g., through GenBank). The primers include linker regions (e.g., sequences including restriction sites) to facilitate cloning and manipulation in production of the fusion construct. The polynucleotides corresponding to the PDZ and immobilization regions are joined in-frame to produce the fusion protein-encoding sequence. The fusion proteins of the invention may be expressed as secreted proteins (e.g., by including the signal sequence encoding DNA in the fusion gene; see, e.g., Lui et al, 1993, PNAS USA, 90:8957-61) or as nonsecreted proteins. [0083] In some embodiments, the chimeric PDZ domain-containing polypeptides or PL polypeptides are immobilized on a solid surface. The substrate to which the polypeptide is bound may in any of a variety of forms, e.g., a microtiter dish, a test tube, a dipstick, a microcentrifuge tube, a bead, a spinnable disk, a permeable or semipermeable membrane, and the like. Suitable materials include glass, plastic (e.g., polyethylene, PVC, polypropylene, polystyrene, and the like), protein, paper, carbohydrate, lipid monolayer or supported lipid bilayer, films and other solid supports. Other materials that may be employed include ceramics, metals, metalloids, semiconductive materials, cements and the like.
[0084] In other embodiments, the chimeric PDZ domain-containing polypeptides and/or PL fusion proteins are organized as an array. The term "array," as used herein, refers to an ordered arrangement of immobilized fusion proteins, in which particular different fusion proteins (i.e., having different PDZ domains) are located at different predetermined sites on the substrate. Because the location of particular fusion proteins on the array is known, binding at that location can be correlated with binding to the chimeric PDZ domain-containing polypeptide situated at that location. Immobilization of fusion proteins on beads (individually or in groups) is another particularly useful approach. In one embodiment, individual fusion proteins are immobilized on beads. In one embodiment, mixtures of distinguishable beads are used. Distinguishable beads are beads that can be separated from each other on the basis of a property such as size, magnetic property, color (e.g., using FACS) or affinity tag (e.g., a bead coated with protein A can be separated from a bead not coated with protein A by using IgG affinity methods). Binding to particular chimeric PDZ domain- containing polypeptide may be determined.
[0085] Methods for immobilizing proteins are known, and include covalent and non-covalent methods. One suitable immobilization method is antibody-mediated immobilization. According to this method, an antibody specific for the sequence of an "immobilization domain" of the PDZ-domain containing protein is itself immobilized on the substrate (e.g., by adsorption). One advantage of this approach is that a single antibody may be adhered to the substrate and used for immobilization of a number of polypeptides (sharing the same immobilization domain). For example, an immobilization domain consisting of poly-histidine (Bush et al, 1991, J. Biol Chem 266:13811-14) can be bound by an anti-histidine monoclonal antibody (R&D Systems, Minneapolis, Minn.); an immobilization domain consisting of secreted alkaline phosphatase ("SEAP") (Berger et al, 1988, Gene 66:1-10) can be bound by anti- SEAP (Sigma Chemical Company, St. Louis, Mo.); an immobilization domain consisting of a FLAG epitope can be bound by anti-FLAG. Other ligand-antiligand immobilization methods are also suitable (e.g., an immobilization domain consisting of protein A sequences (Harlow and Lane, 1988, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory; Sigma Chemical Co., St. Louis, Mo.) can be bound by IgG; and an immobilization domain consisting of streptavidin can be bound by biotin (Harlow & Lane, supra; Sigma Chemical Co., St. Louis, Mo.). In a preferred embodiment, the immobilization domain is a GST moiety, as described herein. [0086] When antibody-mediated immobilization methods are used, glass and plastic are especially useful substrates. The substrates may be printed with a hydrophobic (e.g., Teflon) mask to form wells. Preprinted glass slides with 3, 10 and 21 wells per 14.5 cm2 slide "working area" are available from, e.g., SPI Supplies, West Chester, Pa.; also see U.S. Pat. No. 4,011,350). In certain applications, a large format (12.4 cm x 8.3 cm) glass slide is printed in a 96 well format is used; this format facilitates the use of automated liquid handling equipment and utilization of 96 well format plate readers of various types (fluorescent, colorimetric, scintillation). However, higher densities may be used (e.g., more than 10 or 100 polypeptides per cm2). See, e.g., MacBeath et al, 2000, Science 289: 1760-63. Typically, antibodies are bound to substrates (e.g., glass substrates) by adsorption. Suitable adsorption conditions are well known in the art and include incubation of 0.5-50 ug/ml (e.g., 10 ug/ml) mAb in buffer (e.g., PBS, or 50 to 300 mM Tris, MOPS, HEPES, PIPES, acetate buffers, pHs 6.5 to 8, at 40C) to 370C. and from lhr to more than 24 hours.
[0087] Proteins may be covalently bound or noncovalently attached through nonspecific bonding. If covalent bonding between the fusion protein and the surface is desired, the surface will usually be polyfunctional or be capable of being polyfunctionalized. Functional groups which may be present on the surface and used for linking can include carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like. The manner of linking a wide variety of compounds to various surfaces is well known and is amply illustrated in the literature. Exemplary assays:
"A Assay" Detection of PDZ-Ligand Binding Using Immobilized PL Peptide.
[0088] In one aspect, the invention provides an assay in which biotinylated candidate PL peptides are immobilized on an avidin-coated surface. The binding of PDZ-domain fusion protein to this surface is then measured. In a preferred embodiment, the PDZ-domain fusion protein is a chimeric PDZ domain-containing protein fused to GST (GST-chimeric PDZ fusion protein) and the assay is carried out as follows:
[0089] (1) Avidin is bound to a surface, e.g. a protein binding surface. In one embodiment, avidin is bound to a polystyrene 96 well plate (e.g., Nunc Polysorb (cat #475094) by addition of 100 uL per well of 20 ug/mL of avidin (Pierce) in phosphate buffered saline without calcium and magnesium, pH 7.4 ("PBS", GibcoBRL) at 40C. for 12 hours. The plate is then treated to block nonspecific interactions by addition of 200 uL per well of PBS containing 2 g per 100 mL protease-free bovine serum albumin ("PBS/BSA") for 2 hours at 40C. The plate is then washed 3 times with PBS by repeatedly adding 200 uL per well of PBS to each well of the, plate and then dumping the contents of the plate into a waste container and tapping the plate gently on a dry surface.
[0090] (2) Biotinylated PL peptides (or candidate PL peptides listed in TABLE 1) are immobilized on the surface of wells of the plate by addition of 5O uL per well of 0.4 uM peptide in PBS/BSA for 30 minutes at 40C. Usually, each different peptide is added to at least eight different wells so that multiple measurements (e.g. duplicates and also measurements using different (GST/PDZ-domain fusion proteins and a GST alone negative control) can be made, and also additional negative control wells are prepared in which no peptide is immobilized. Following immobilization of the PL peptide on the surface, the plate is washed 3 times with PBS.
[0091] (3) GST-chimeric PDZ-domain fusion protein (prepared as described) is allowed to react with the surface by addition of 50 uL per well of a solution containing 5 ug/mL GST-chimeric PDZ-domain fusion protein in PBS/BSA for 2 hours at 40C. As a negative control, GST alone (i.e. not a fusion protein) is added to specified wells, generally at least 2 wells (i.e. duplicate measurements) for each immobilized peptide. After the 2 hour reaction, the plate is washed 3 times with PBS to remove unbound fusion protein.
[0092] (4) The binding of the GST-chimeric PDZ-domain fusion protein to the avidin-biotinylated peptide surface can be detected using a variety of methods, and detectors known in the art. In one embodiment, 50 uL per well of an anti-GST antibody in PBS/BSA (e.g. 2.5 ug/mL of polyclonal goat-anti-GST antibody, Pierce) is added to the plate and allowed to react for 20 minutes at 40C. The plate is washed 3 times with PBS and a second, detectably labeled antibody is added. In one embodiment, 50 uL per well of 2.5 ug/mL of horseradish peroxidase (HRP)-conjugated polyclonal rabbit anti-goat immunoglobulin antibody is added to the plate and allowed to react for 20 minutes at 40C. The plate is washed 5 times with 50 mM Tris pH 8.0 containing 0.2% Tween 20, and developed by addition of 100 uL per well of HRP-substrate solution (TMB, Dako) for 20 minutes at room temperature (RT). The reaction of the HRP and its substrate is terminated by the addition of 100 uL per well of IM sulfuric acid and the absorbance (A) of each well of the plate is read at 450 nm.
[0093] (5) Specific binding of a PL peptide and a chimeric PDZ-domain containing polypeptide is detected by comparing the signal from the well(s) in which the PL peptide and the chimeric PDZ domain polypeptide are combined with the background signal(s). The background signal is the signal found in the negative controls. Typically a specific or selective reaction will be at least twice background signal, more typically more than 5 times background, and most typically 10 or more times the background signal. In addition, a statistically significant reaction will involve multiple measurements of the reaction with the signal and the background differing by at least two standard errors, more typically four standard errors, and most typically six or more standard errors. Correspondingly, a statistical test (e.g. a T-test) comparing repeated measurements of the signal with repeated measurements of the background will result in a p-value <0.05, more typically a p-value <0.01, and most typically a p-value <0.001 or less.
[0094] As noted, in an embodiment of the "A" assay, the signal from binding of a GST-chimeric PDZ-domain fusion protein to an avidin surface not exposed to (i.e. not covered with) the PL peptide is one suitable negative control (sometimes referred to as "B"). The signal from binding of GST polypeptide alone (i.e. not a fusion protein) to an avidin-coated surface that has been exposed to (i.e. covered with) the PL peptide is a second suitable negative control (sometimes referred to as "B2"). Because all measurements are done in multiples (i.e. at least duplicate) the arithmetic mean (or, equivalently, average) of several measurements is used in determining the binding, and the standard error of the mean is used in determining the probable error in the measurement of the binding. The standard error of the mean of N measurements equals the square root of the following: the sum of the squares of the difference between each measurement and the mean, divided by the product of (N) and (N-I). Thus, in one embodiment, specific binding of the PDZ protein to the plate-bound PL peptide is determined by comparing the mean signal ("mean S") and standard error of the signal ("SE") for a particular PL-PDZ combination with the mean Bl and/or mean B2.
"G Assay"— Detection of PDZ-Ligand Binding Using Immobilized GST-chimeric PDZ-domain fusion protein [0095] In one aspect, the invention provides an assay in which a GST-chimeric PDZ-domain fusion protein is immobilized on a surface ("G" assay). The binding of labeled PL peptide (e.g., as listed in TABLE 1) to this surface is then measured. In a preferred embodiment, the assay is carried out as follows:
[0096] (I) A GST-chimeric PDZ-domain fusion protein is bound to a surface, e.g. a protein binding surface. In a preferred embodiment, a GST-chimeric PDZ-domain fusion protein containing one or more introduced PDZ domains is bound to a polystyrene 96-well plate. The GST-chimeric PDZ-domain fusion protein can be bound to the plate by any of a variety of standard methods known to one of skill in the art, although some care must be taken that the process of binding the fusion protein to the plate does not alter the ligand-binding properties of the PDZ domain. In one embodiment, the GST-chimeric PDZ-domain fusion protein is bound via an anti-GST antibody that is coated onto the 96-well plate. Adequate binding to the plate can be achieved when:
[0097] a. 100 uL per well of 5 ug/mL goat anti-GST polyclonal antibody (Pierce) in PBS is added to a polystyrene 96-well plate (e.g., Nunc Polysorb) at 40C for 12 hours, b. The plate is blocked by addition of 200 uL per well of PBS/BSA for 2 hours at 40C. c. The plate is washed 3 times with PBS. d. 50 uL per well of 5 ug/mL GST-chimeric PDZ-domain fusion protein) or, as a negative control, GST polypeptide alone (i.e. not a fusion protein) in PBS/BSA is added to the plate for 2 hours at 40C. e. The plate is again washed 3 times with PBS.
[0098] (2) Biotinylated PL peptides are allowed to react with the surface by addition of 50 uL per well of 20 uM solution of the biotinylated peptide in PBS/BSA for 10 minutes at 40C, followed by an additional 20 minute incubation at 25 C. The plate is washed 3 times with ice cold PBS.
[0099] (3) The binding of the biotinylated peptide to the GST-chimeric PDZ-domain fusion protein surface can be detected using a variety of methods and detectors known to one of skill in the art. In one embodiment, 100 uL per well of 0.5 ug/mL streptavidin-horse radish peroxidase (HRP) conjugate dissolved in BSA/PBS is added and allowed to react for 20 minutes at 40C. The plate is then washed 5 times with 50 mM Tris pH 8.0 containing 0.2% Tween 20, and developed by addition of 100 uL per well of HRP-substrate solution (TMB, Dako) for 20 minutes at room temperature (RT). The reaction of the HRP and its substrate is terminated by addition of 100 uL per well of IM sulfuric acid, and the absorbance of each well of the plate is read at 450 nm.
[00100] (4) Specific binding of a PL peptide and a PDZ domain polypeptide is determined by comparing the signal from the well(s) in which the PL peptide and PDZ domain polypeptide are combined, with the background signal(s). The background signal is the signal found in the negative control(s). Typically a specific or selective reaction will be at least twice background signal, more typically more than 5 times background, and most typically 10 or more times the background signal. In addition, a statistically significant reaction will involve multiple measurements of the reaction with the signal and the background differing by at least two standard errors, more typically four standard errors, and most typically six or more standard errors. Correspondingly, a statistical test (e.g. a T-test) comparing repeated measurements of the signal with —repeated measurements of the background will result in a p-value <0.05, more typically a p-value <0.01, and most typically a p-value <0.001 or less. As noted, in an embodiment of the "G" assay, the signal from binding of a given PL peptide to immobilized (surface bound) GST polypeptide alone is one suitable negative control (sometimes referred to as "B 1"). Because all measurement are done in multiples (i.e. at least duplicate) the arithmetic mean (or, equivalently, average.) of several measurements is used in determining the binding, and the standard error of the mean is used in determining the probable error in the measurement of the binding. The standard error of the mean of N measurements equals the square root of the following: the sum of the squares of the difference between each measurement and the mean, divided by the product of (N) and (N-I). Thus, in one embodiment, specific binding of the PDZ protein to the platebound peptide is determined by comparing the mean signal ("mean S") and standard error of the signal ("SE") for a particular PL-PDZ combination with the mean Bl.
"G' assay" and "G" assay"
[00101] Two specific modifications of the specific conditions described supra for the "G assay" are particularly useful. The modified assays use lesser quantities of labeled PL peptide and have slightly different biochemical requirements for detection of PDZ-ligand binding compared to the specific assay conditions described supra. [00102] For convenience, the assay conditions described in this section are referred to as the "G' assay" and the "G" assay," with the specific conditions described in the preceding section on G assays being referred to as the "G0 assay." The "G' assay" is identical to the "G0 assay" except at step (2) the peptide concentration is 10 uM instead of 20 uM. This results in slightly lower sensitivity for detection of interactions with low affinity and/or rapid dissociation rate. Correspondingly, it slightly increases the certainty that detected interactions are of sufficient affinity and half-life to be of biological importance and useful therapeutic targets.
[00103] The "G" assay" is identical to the "G0 assay" except that at step (2) the peptide concentration is 1 uM instead of 20 uM and the incubation is performed for 60 minutes at 250C (rather than, e.g., 10 minutes at 40C. followed by 20 minutes at 250C). This results in lower sensitivity for interactions of low affinity, rapid dissociation rate, and/or affinity that is less at 250C than at 40C. Interactions will have lower affinity at 250C than at 40C. if (as we have found to be generally true for PDZ-ligand binding) the reaction entropy is negative (i.e. the entropy of the products is less than the entropy of the reactants). In contrast, the PDZ-PL binding signal may be similar in the "G" assay" and the "G0 assay" for interactions of slow association and dissociation rate, as the PDZ-PL complex will accumulate during the longer incubation of the "G" assay." Thus comparison of results of the "G" assay" and the "G0 assay" can be used to estimate the relative entropies, enthalpies, and kinetics of different PDZ-PL interactions. (Entropies and enthalpies are related to binding affinity by the equations delta G=RT 1 n (Kd)=delta H-T delta S where delta G, H, and S are the reaction free energy, enthalpy, and entropy respectively, T is the temperature in degrees Kelvin, R is the gas constant, and Kd is the equilibrium dissociation constant). In particular, interactions that are detected only or much more strongly in the "G.sup.O assay" generally have a rapid dissociation rate at 250C
(tl/2<10 minutes) and a negative reaction entropy, while interactions that are detected similarly strongly in the "G" assay" generally have a slower dissociation rate at 250C (tl/2>10 minutes). Rough estimation of the thermodynamics and kinetics of PDZ-PL interactions (as can be achieved via comparison of results of the "G0 assay" versus the "G" assay" as outlined supra) can be used in the design of efficient inhibitors of the interactions.
For example, a small molecule inhibitor based on the chemical structure of a PL that dissociates slowly from a given
PDZ domain (as evidenced by similar binding in the "G" assay" as in the "G0 assay") may itself dissociate slowly and thus be of high affinity.
[00104] In this manner, variation of the temperature and duration of step (2) of the "G assay" can be used to provide insight into the kinetics and thermodynamics of the PDZ-ligand binding reaction and into design of inhibitors of the reaction.
Assay Variations
[00105] As discussed supra, it will be appreciated that many of the steps in the above -described assays can be varied, for example, various substrates can be used for binding the PL and PDZ-containing proteins; different types of PDZ containing fusion proteins can be used; different labels for detecting PDZ/PL interactions can be employed; and different ways of detection can be used.
[00106] The PDZ-PL detection assays can employ a variety of surfaces to bind the PL and/or PDZ-containing proteins. For example, a surface can be an "assay plate" which is formed from a material (e.g. polystyrene) which optimizes adherence of either the PL protein or PDZ-containing protein thereto. Generally, the individual wells of the assay plate will have a high surface area to volume ratio and therefore a suitable shape is a flat bottom well
(where the proteins of the assays are adherent). Other surfaces include, but are not limited to, polystyrene or glass beads, polystyrene or glass slides, papers, dipsticks, plastics, films and the like.
[00107] For example, the assay plate can be a "microtiter" plate. The term "microtiter" plate when used herein refers to a multiwell assay plate, e.g., having between about 30 to 200 individual wells, usually 96 wells.
Alternatively, high-density arrays can be used. Often, the individual wells of the microtiter plate will hold a maximum volume of about 250 ul. Conveniently, the assay plate is a 96 well polystyrene plate (such as that sold by
Becton Dickinson Labware, Lincoln Park, NJ.), which allows for automation and high throughput screening. Other surfaces include polystyrene microtiter ELISA plates such as that sold by Nunc Maxisorp, Inter Med, Denmark.
Often, about 50 ul to 300 ul, more preferably 100 ul to 200 ul, of an aqueous sample comprising buffers suspended therein will be added to each well of the assay plate.
[00108] The detectable labels of the invention can be any detectable compound or composition which is conjugated directly or indirectly with a molecule (such as described above). The label can be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, can catalyze a chemical alteration of a substrate compound or composition which is detectable. The preferred label is an enzymatic one which catalyzes a color change of a non-radioactive color reagent.
[00109] The label can be indirectly conjugated with the antibody. One of skill is aware of various techniques for direct and indirect conjugation. For example, the antibody can be conjugated with biotin and any of the categories of labels mentioned above can be conjugated with avidin, or vice versa (see also "A" and "G" assay above). Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner. See, Au subel, supra, for a review of techniques involving biotin-avidin conjugation and similar assays. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody is conjugated with a small hapten (e.g. digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g. anti-digoxin antibody). Thus, indirect conjugation of the label with the antibody can be achieved. [00110] Assay variations can include different washing steps. By "washing" is meant exposing the solid phase to an aqueous solution (usually a buffer or cell culture media) in such a way that unbound material (e.g., non-adhering cells, non-adhering capture agent, unbound ligand, receptor, receptor construct, cell lysate, or HRP antibody) is removed therefrom. To reduce background noise, it is convenient to include a detergent (e.g., Triton X) in the washing solution. Usually, the aqueous washing solution is decanted from the wells of the assay plate following washing. Conveniently, washing can be achieved using an automated washing device. Sometimes, several washing steps (e.g., between about 1 to 10 washing steps) can be required.
[00111] Various buffers can also be used in PDZ-PL detection assays. For example, various blocking buffers can be used to reduce assay background. The term "blocking buffer" refers to an aqueous, pH buffered solution containing at least one blocking compound which is able to bind to exposed surfaces of the substrate which are not coated with a PL or a chimeric PDZ domain containing polypeptide. The blocking compound is normally a protein such as bovine serum albumin (BSA), gelatin, casein or milk powder and does not cross-react with any of the reagents in the assay. The block buffer is generally provided at a pH between about 7 to 7.5 and suitable buffering agents include phosphate and TRIS.
[00112] Various enzyme-substrate combinations can also be utilized in detecting PDZ-PL interactions. Examples of enzyme-substrate combinations include, for example: (i) Horseradish peroxidase (HRP or HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g. orthophenylene diamine [OPD] or 3,3',5,5'-tetramethyl benzidine hydrochloride [TMB]) (as described above), (ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate, (iii) Beta-D-galactosidase (Beta D-GaI) with a chromogenic substrate (e.g. p-nitrophenyl-Beta-D-galactosidase) or fluorogenic substrate 4-methylumbelliferyl- Beta-D-galactosidase. Numerous other enzyme-substrate combinations are available to those skilled in the art. For a general review of these, see U.S. Pat. Nos. 4,275,149 and 4,318,980, both of which are herein incorporated by reference.
[00113] Further, it will be appreciated that, although, for convenience, the present discussion primarily refers to detection of interaction between a PDZ ligand and a chimeric PDZ domain containing polypeptide, agonists or antagonists of PDZ-PL interactions can be used to diagnose cellular abnormalities. VI. Clinical Sample Collection
[00114] The present invention relates to the method of determining if a subject is infected with an oncogenic strain of HPV. Diagnosing the presence of pathogens requires collection of samples appropriate to the organism. For detection of oncogenic HPV E6 proteins, one could collect tissue for testing from the cervix, penis, anus, or throat using a scrape, swab or biopsy technique. For diagnosis of bloodborne pathogens such as HIV, collection of blood through standard means would be most appropriate. Diagnosis of fungal or viral infections that may have caused skin lesions would require the collection of a sample from the affected area.
[00115] This invention is not intended to cover sampling devices. However, it should be noted that since the invention is predicated on the detection of PDZ or PL proteins, appropriate care must be taken to collect a sufficient amount of sample to detect pathogen proteins and to maintain the integrity of proteins in the sample. The amount of sample to collect should be determined empirically for each diagnostic test. Factors in the decision may include, but not be limited to, the stage at which detection is desired, the amount of pathogen per unit sample, the amount of diagnostic protein per unit per unit sample, availability of diagnostic epitopes and the stability of diagnostic epitopes.
[00116] Exemplary collection devices for cervical tissue include, but are not limited to, those described in U.S. Pat. Nos. 6,241,687, 6,352,513, 6,336,905, 6,115,990 and 6,346,086. These collection devices would facilitate the collection of cervical tissue for the diagnosis of oncogenic human papillomavirus infection. These devices are predominantly collection of cervical cells or tissues through scraping; alternatively, one could use standard biopsy methods to collect samples from any tissues to be examined.
[00117] Although the diagnostic method disclosed in this application is directed at the detection of PL proteins, sample collection need not be limited to collection of proteins. One could alternatively collect RNA from tissue samples, use an in vitro translation kit to produce protein from collected templates, and then assay using methods disclosed herein. In a similar manner, DNA could be collected from test samples, specific primers for oncogenic E6 proteins could be used to either amplify the DNA content (using a DNA polymerase) or transcribe and translate the sample into proteins that could be tested with methods disclosed herein. VII. Assays for Detecting Oncogenic E6 Proteins
[00118] One embodiment of the present invention includes the detection of oncogenic E6 proteins using a chimeric protein containing at least one introduced PDZ domain that has higher binding strength to E6 than an isolated PDZ domain. Oncogenic E6 proteins can be detected by their ability to bind to chimeric PDZ domain containing polypeptides. This could be developed into a single detection stage approach or more favorably as a two-stage or "sandwich" approach for increased sensitivity and specificity.
[00119] For single stage approaches, a "tagged" version of a PDZ domain that specifically recognizes oncogenic E6 proteins, such as those disclosed in TABLES 1 and 3, can be used to directly probe for the presence of oncogenic E6 protein in a sample. As noted supra, an example of this would be to attach the test sample to a solid support (for example, cervical cells or tissue could be coated on a slide and "fixed" to permeablize the cell membranes), incubate the sample with a tagged chimeric PDZ domain-containing polypeptide (PL detector, e.g. GST-chimeric PDZ fusion protein) under appropriate conditions, wash away unbound PL detector, and assay for the presence of the "tag" in the sample. In addition, even without a tag, one could measure the physical properties of the chimeric PDZ domain- containing protein and the chimeric PDZ domain-containing protein bound to an E6 protein. Techniques such as surface plasmon resonance, circular dichoism, and other techniques that directly assess binding could be used to detect the presence of oncogenic E6 proteins. One should note, however, that PDZ domains may also bind endogenous cellular proteins. Thus, frequency of binding must be compared to control cells that do not contain E6 oncoproteins or the "PL detector" should be modified such that it is significantly more specific for the oncogenic E6 proteins (see section on "binding sensitivity").
[00120] For two-stage or sandwich approaches, use of the chimeric PDZ domain-containing protein is coupled with a second method of either capturing or detecting captured proteins. The second method could be using an antibody that binds to the E6 oncoprotein or a second compound or protein that can bind to E6 oncorproteins at a location on the E6 protein that does not reduce the availability of the E6 PDZ ligand. Such proteins may include, but not be limited to, p53, E6-AP, E6-BP or engineered compounds that bind E6 oncoproteins. Alternatively, one could also use DNA binding or Zn2+ binding to assay for the presence of captured E6 protein, since oncogenic E6 proteins are known to bind certain DNA structures through the use of divalent cations. Additionally, one could use the PDZ- captured E6 protein in an activity assay, since E6 is known to degrade DNA and certain proteins including p53 in the presence of a reticulocyte lysate.
Antibodies
[00121] Many biological assays are designed as a "sandwich", where an antibody constitutes one side of the sandwich. This method can improve the signal to noise ratio for a diagnostic by reducing background signal and amplifying appropriate signals. Antibodies can be generated that specifically recognize the diagnostic protein. Since this invention discloses the method of using a chimeric PDZ domain-containing protein or a PL to diagnose pathogen infections, antibodies should be generated that do not conflict with the PDZ:PL interaction. [00122] For the production of antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., may be immunized by injection with a peptide. The peptide may be attached to a suitable carrier, such as BSA or KLH, by means of a side chain functional group or linkers attached to a side chain functional group. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.
[00123] Monoclonal antibodies to a peptide may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein, 1975, Nature 256:495-497, the human B-cell hybridoma technique, Kosbor et al., 1983, Immunology Today 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026- 2030 and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)). In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce peptide-specific single chain antibodies.
[00124] Antibody fragments which contain deletions of specific binding sites may be generated by known techniques. For example, such fragments include but are not limited to F(ab')2 fragments, which can be produced by pepsin digestion of the antibody molecule and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., 1989, Science 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for the peptide of interest.
[00125] The antibody or antibody fragment specific for the desired peptide can be attached, for example, to agarose, and the antibody-agarose complex is used in immunochromatography to purify peptides of the invention. See, Scopes, 1984, Protein Purification: Principles and Practice, Springer- Verlag New York, Inc., N. Y., Livingstone, 1974, Methods Enzymology: Immunoaffmity Chromatography of Proteins 34:723-731. Antibodies can also be linked to other solid supports for diagnostic applications, or alternatively labeled with a means of detection such an enzyme that can cleave a colorimetric substrate, a fluorophore, a magnetic particle, or other measurable compositions of matter.
[00126] Specific antibodies against E6 proteins have historically been difficult to produce. In conjunction with the methods describe supra, one could employ a number of techniques to increase the likelihood of producing or selecting high affinity antibodies. An example is to prepare the E6 antigen (to raise antibodies against) in the same manner that one would prepare tissue or cell samples for testing. Alternatively, one could immunize with E6 fusion protein prepared in one manner, and screen for specific E6 antibodies using a second E6 protein prepared in a different manner. This should select for antibodies that recognize E6 epitopes that are conserved under different sample collection and preparation procedures. In another example, one could immunize animals with E6 antigen that has been rapidly denatured and renatured, such that epitopes that are insensitive to preparation conditions are selected for. Another method that could be employed is to use peptides corresponding to antigenic regions of the E6 proteins as predicted by Major Histocompatibility Complex (MHC) and T Cell Receptor (TCR) consensus binding.
Alternative Detection Methods for Captured E6 Protein
[00127] E6 proteins that have been captured by PDZ domains could be detected by several alternative methods. Several proteins are known to associate with E6 proteins. Any of them that had a reasonable affinity for E6 could be used to detect the presence of captured and concentrated E6 protein in a sample by one skilled in the art. In addition, new binding proteins or aptamers could be identified that bound to E6 proteins. Third, activity assays specific for E6 could be employed.
[00128] The detection assay itself could also be carried out using a variety of methods. A standard ELISA using a PDZ to capture could be set up as a competition, where the PDZ domain is pre-loaded with a labeled PL that has lower affinity than the E6 proteins. Thus, in the presence of E6, the label is displaced and one sees a reduction of signal that corresponds to E6 presence. Other variants that use aspects of competition and inhibition of binding are intended to be included as well. One variant could even have the PL covalently attached to the PDZ domain through a linker such that the PL could bind its own PDZ domain. Using donor quenching dyes, one would only see an increase in signal when the PL of an oncogenic E6 protein was able to displace the labeled PL. All such competition methods must be measured against controls that assess the amount of endogenous PL proteins that can bind the PDZ domain used to assess the presence of oncogenic E6 proteins. VIII. Measurements of Assay Sensitivity
[00129] The present invention provides a method for detecting PDZ-PL interaction with enhanced PDZ binding to PL using a chimeric protein containing at least one introduced PDZ domain from a PDZ domain-containing polypeptide. The "A" and "G" assays of the invention can be used to determine the "apparent affinity" of binding of a PDZ ligand peptide to a chimeric PDZ domain-containing protein. Apparent affinity is determined based on the concentration of one molecule required to saturate the binding of a second molecule (e.g., the binding of a ligand to a receptor). Two particularly useful approaches for quantitation of apparent affinity of PDZ-ligand binding are provided infra. These methods can be used to compare the sensitivity and affinity of different chimeric PDZ domain- containing protein constructs. Understanding the sensitivity of the PDZ for pathogen PLs is essential because it helps to define the amount of tissue or cell sample that must be tested to obtain a definitive result. [00130] (1) A GST/ chimeric PDZ fusion protein, as well as GST alone as a negative control, are bound to a surface (e.g., a 96-well plate) and the surface blocked and washed as described supra for the "G" assay. [00131] (2) 50 μL per well of a solution of biotinylated PL peptide (e.g. as shown in TABLE 1) is added to the surface in increasing concentrations in PBS/BSA (e.g. at 0.1 uM, 0.33 uM, 1 uM, 3.3 uM, 10 uM, 33 uM, and 100 uM). In one embodiment, the PL peptide is allowed to react with the bound GST/ chimeric PDZ fusion protein as well as the GST alone negative control) for 10 minutes at 40C followed by 20 minutes at 250C. The plate is washed 3 times with ice cold PBS to remove unbound labeled peptide.
[00132] (3) The binding of the PL peptide to the immobilized chimeric PDZ domain-containing polypeptide is detected as described supra for the "G" assay.
[00133] (4) For each concentration of peptide, the net binding signal is determined by subtracting the binding of the peptide to GST alone from the binding of the peptide to the GST- chimeric PDZ fusion protein. The net binding signal is then plotted as a function of ligand concentration and the plot is fit (e.g. by using the Kaleidagraph software package curve fitting algorithm; Synergy Software) to the following equation, where "Signal[ilgand]" is the net binding signal at PL peptide concentration "[ligand]," "Kd" is the apparent affinity of the binding event, and "Saturation Binding" is a constant determined by the curve fitting algorithm to optimize the fit to the experimental data: Signal[ilgand]=Saturation Binding x ([ligand]/([ligand]+Kd))
[00134] For reliable application of the above equation it is necessary that the highest peptide ligand concentration successfully tested experimentally be greater than, or at least similar to, the calculated Kd (equivalently, the maximum observed binding should be similar to the calculated saturation binding). In cases where satisfying the above criteria proves difficult, an alternative approach (infra) can be used.
Approach 2:
[00135] (1) A fixed concentration of a chimeric PDZ-domain containing polypeptide and increasing concentrations of a labeled PL peptide (labeled with, for example, biotin or fluorescein, see TABLE 1 for representative peptide amino acid sequences) are mixed together in solution and allowed to react. In one embodiment, preferred peptide concentrations are 0.1 uM, 1 uM, 10 uM, 100 uM, 1 mM. In various embodiments, appropriate reaction times can range from 10 minutes to 2 days at temperatures ranging from 40C to 370C. In some embodiments, the identical reaction can also be carried out using a non-PDZ domain-containing protein as a control (e.g., if the chimeric PDZ- domain containing polypeptide is a fusion protein, the fusion partner can be used).
[00136] (2) PDZ-ligand complexes can be separated from unbound labeled peptide using a variety of methods known in the art. For example, the complexes can be separated using high performance size-exclusion chromatography (HPSEC, gel filtration) (Rabinowitz et al., 1998, Immunity 9:699), affinity chromatography (e.g. using glutathione Sepharose beads), and affinity absorption (e.g., by binding to an anti-GST-coated plate as described supra).
[00137] (3) The PDZ-ligand complex is detected based on presence of the label on the peptide ligand using a variety of methods and detectors known to one of skill in the art. For example, if the label is fluorescein and the separation is achieved using HPSEC, an in-line fluorescence detector can be used. The binding can also be detected as described supra for the G assay.
[00138] (4) The PDZ-ligand binding signal is plotted as a function of ligand concentration and the plot is fit. (e.g., by using the Kaleidagraph software package curve fitting algorithm) to the following equation, where "Signal[ilgand]" is the binding signal at PL peptide concentration "[ligand]," "Kd" is the apparent affinity of the binding event, and "Saturation Binding" is a constant determined by the curve fitting algorithm to optimize the fit to the experimental data: Signal[Llgand]=Saturation Binding x ([ligand]/([ligand+Kd]) [00139] Measurement of the affinity of a labeled peptide ligand binding to a chimeric PDZ-domain containing polypeptide is useful because knowledge of the affinity (or apparent affinity) of this interaction allows rational design of inhibitors of the interaction with known potency. The potency of inhibitors in inhibition would be similar to (i.e. within one-order of magnitude of) the apparent affinity of the labeled peptide ligand binding to the introduced PDZ domain.
[00140] Thus, in one aspect, the invention provides a method of determining the apparent affinity of binding between a PDZ domain and a ligand by immobilizing a polypeptide comprising the PDZ domain and a non-PDZ domain on a surface, contacting the immobilized polypeptide with a plurality of different concentrations of the ligand, determining the amount of binding of the ligand to the immobilized polypeptide at each of the concentrations of ligand, and calculating the apparent affinity of the binding based on that data. Typically, the chimeric polypeptide comprising at least one introduced PDZ domain is a fusion protein. In one embodiment, the e.g., fusion protein is GST-chimeric PDZ domain containing fusion protein, but other polypeptides can also be used (e.g., a fusion protein including a PDZ domain and any of a variety of epitope tags, biotinylation signals and the like) so long as the polypeptide can be immobilized. In an orientation that does not abolish the ligand binding properties of the PDZ domain, e.g, by tethering the polypeptide to the surface via the non-PDZ domain via an anti-domain antibody and leaving the PDZ domain as the free end. It was discovered, for example, reacting a PDZ-GST fusion polypeptide directly to a plastic plate provided suboptimal results. The calculation of binding affinity itself can be determined using any suitable equation (e.g., as shown supra; also see Cantor and Schimmel (1980) BIOPHYSICAL CHEMISTRY W H Freeman & Co., San Francisco) or software.
[00141] Thus, in a preferred embodiment, the chimeric polypeptide is immobilized by binding the polypeptide to an immobilized immunoglobulin that binds the non-PDZ domain (e.g., an anti-GST antibody when a GST-chimeric PDZ fusion polypeptide is used). In a preferred embodiment, the step of contacting the ligand and PDZ-domain polypeptide is carried out under the conditions provided supra in the description of the "G" assay. It will be appreciated that binding assays are conveniently carried out in multiwell plates (e.g., 24-well, 96-well plates, or 384 well plates).
[00142] The present method has considerable advantages over other methods for measuring binding affinities PDZ- PL affinities, which typically involve contacting varying concentrations of a GST-PDZ fusion protein to a ligand- coated surface. For example, some previously described methods for determining affinity (e.g., using immobilized ligand and GST-PDZ protein in solution) did not account for oligomerization state of the fusion proteins used, resulting in potential errors of more than an order of magnitude.
[00143] Although not sufficient for quantitative measurement of PDZ-PL binding affinity, an estimate of the relative strength of binding of different chimeric PDZ domain containing polypeptide -PL pairs can be made based on the absolute magnitude of the signals observed in the "G assay." This estimate will reflect several factors, including biologically relevant aspects of the interaction, including the affinity and the dissociation rate. For comparisons of different ligands binding to a given PDZ domain- containing protein, differences in absolute binding signal likely relate primarily to the affinity and/or dissociation rate of the interactions of interest. IX. Measurements of Assay Specificity
[00144] The present invention provides methods for analysis of PDZ-ligand interactions using a chimeric protein containing at least one introduced PDZ domain that has enhanced binding strength to a PDZ ligand. The chimeric polypeptide described herein can have multiple introduced PDZ domains. In one embodiment of the invention, the affinity is determined for a particular ligand and a plurality of PDZ domains. In a preferred embodiment, the plurality of different PDZ proteins are from a particular tissue (e.g., central nervous system, spleen, cardiac muscle, kidney) or a particular class or type of cell, (e.g., a hematopoietic cell, a lymphocyte, a neuron) and the like. In a most preferred embodiment, the plurality of different PDZ proteins represents a substantial fraction (e.g., typically a majority, more often at least 80%) of all of the PDZ proteins known to be, or suspected of being, expressed in the tissue or cell(s), e.g., all of the PDZ proteins known to be present in lymphocytes. In an embodiment, the plurality is at least 50%, usually at least 80%, at least 90% or all of the PDZ proteins disclosed herein as being expressed in hematopoietic cells.
[00145] In one embodiment of the invention, the binding of a ligand to the plurality of PDZ domains is determined. Using this method, it is possible to identify a particular PDZ domain bound with particular specificity by the ligand. The binding may be designated as "specific" if the affinity of the ligand to the particular PDZ domain is at least 2- fold that of the binding to other PDZ domains in the plurality (e.g., present in that cell type). The binding is deemed "very specific" if the affinity is at least 10-fold higher than to any other PDZ in the plurality or, alternatively, at least 10-fold higher than to at least 90%, more often 95% of the other PDZs in a defined plurality. Similarly, the binding is deemed "exceedingly specific" if it is at least 100-fold higher. For example, a ligand could bind to 2 different PDZs with an affinity of 1 uM and to no other PDZs out of a set 40 with an affinity of less than 100 uM. This would constitute specific binding to those 2 PDZs. Similar measures of specificity are used to describe binding of a PDZ domain to a plurality of PLs.
[00146] In the present invention, the introduced PDZ domain in the chimeric polypeptide has enhanced binding to a PDL ligand, more specifically to an E6 protein from an oncogenic HPV strain. In one embodiment, the binding of the chimeric polypeptide containing at least one introduced PDZ domain to E6 is enhanced by at least 2 folds as compared to an isolated PDZ domain. In another embodiment, the binding of the chimeric polypeptide containing at least one introduced PDZ domain to E6 is enhanced as compared to a PDZ domain present in a native polypeptide, in which the PDZ domain is not modified in any way. It should be recognized that high specificity PDZ-PL interactions represent potentially more valuable targets for achieving a desired biological effect. The ability of an inhibitor or enhancer to act with high specificity is often desirable. In particular, the most specific PDZ-ligand interactions are also the diagnostic targets, allowing specific detection of the interaction or disruption of an interaction. Enhancing the binding strength of a PDZ domain- containing protein to a PDZ ligand may improve the sensitivity of a diagnostic assay, such as cervical cancer diagnostic test.
[00147] In one embodiment, the invention provides a method of identifying a high specificity interaction between a particular introduced PDZ domain in a chimeric polypeptide and a ligand known or suspected of binding at least one PDZ domain, by providing a plurality of different immobilized polypeptides, each of said polypeptides comprising a PDZ domain and a non-PDZ domain; determining the affinity of the ligand for each of said polypeptides, and comparing the affinity of binding of the ligand to each of said polypeptides, wherein an interaction between the ligand and a particular PDZ domain is deemed to have high specificity when the ligand binds an immobilized polypeptide comprising the particular PDZ domain with at least 2-fold higher affinity than to immobilized polypeptides not comprising the particular PDZ domain.
[00148] In a related aspect, the affinity of binding of a specific PDZ domain to a plurality of ligands (or suspected ligands) is determined. For example, in one embodiment, the invention provides a method of identifying a high specificity interaction between a PDZ domain and a particular ligand known or suspected of binding at least one PDZ domain, by providing an immobilized chimeric polypeptide comprising the PDZ domain and a non-PDZ domain; determining the affinity of each of a plurality of ligands for the polypeptide, and comparing the affinity of binding of each of the ligands to the polypeptide, wherein an interaction between a particular ligand and the PDZ domain is deemed to have high specificity when the ligand binds an immobilized polypeptide comprising the PDZ domain with at least 2-fold higher affinity than other ligands tested. Thus, the binding may be designated as "specific" if the affinity of the PDZ to the particular PL is at least 2-fold that of the binding to other PLs in the plurality (e.g., present in that cell type). The binding is deemed "very specific" if the affinity is at least 10-fold higher than to any other PL in the plurality or, alternatively, at least 10-fold higher than to at least 90%, more often 95% of the other PLs in a defined plurality. Similarly, the binding is deemed "exceedingly specific" if it is at least 100-fold higher. Typically the plurality is at least 5 different ligands, more often at least 10.
Agonists and Antagonists of PDZ-PL Interactions
[00149] As described herein, interactions between PDZ proteins and PL proteins in cells (e.g., cervical cells) may be disrupted or inhibited by the presence of pathogens. Pathogens can be identified using screening assays described herein. Agonists and antagonists of PDZ-Pathogen PL interactions or PDZ-Cellular PL interactions can be useful in discerning or confirming specific interactions. In some embodiments, an agonist will increase the sensitivity of a PDZ-pathogen PL interaction. In other embodiments, an antagonist of a PDZ -pathogen PL interaction can be used to verify the specificity of an interaction. In one embodiment, the motifs disclosed herein are used to design inhibitors. In some embodiments, the antagonists of the invention have a structure (e.g., peptide sequence) based on the C- terminal residues of PL-domain proteins listed in TABLE 1. In some embodiments, the antagonists of the invention have a structure (e.g., peptide sequence) based on a PL motif disclosed herein or in U.S. patent application Ser. No. 09/724553.
[00150] The PDZ/PL antagonists and antagonists of the invention may be any of a large variety of compounds, both naturally occurring and synthetic, organic and inorganic, and including polymers (e.g., oligopeptides, polypeptides, oligonucleotides, and polynucleotides), small molecules, antibodies, sugars, fatty acids, nucleotides and nucleotide analogs, analogs of naturally occurring structures (e.g., peptide mimetics, nucleic acid analogs, and the like), and numerous other compounds. Although, for convenience, the present discussion primarily refers antagonists of PDZ- PL interactions, it will be recognized that PDZ-PL interaction agonists can also be use in the methods disclosed herein.
X. Methods of Optimizing a PL Detector
[00151] Although described supra primarily in terms of identifying interactions between PDZ -domain polypeptides and PL proteins, the assays described supra and other assays can also be used to identify the binding of other molecules (e.g., peptide mimetics, small molecules, and the like) to PDZ domain sequences. For example, using the chimeric PDZ domain- containing polypeptide with high binding strength to a PDZ ligand as disclosed herein, combinatorial and other libraries of compounds can be screened, e.g., for molecules that specifically bind to PDZ domains. Screening of libraries can be accomplished by any of a variety of commonly known methods. See, e.g., the following references, which disclose screening of peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390; Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell 76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner et al.; Rebar and Pabo, 1993, Science 263:671-673; and PCT Publication No. WO 94/18318.
[00152] In a specific embodiment, screening can be carried out by contacting the library members with a chimeric PDZ-domain-containing polypeptide immobilized on a solid support (e.g. as described supra in the "G" assay) and harvesting those library members that bind to the protein. Examples of such screening methods, termed "panning" techniques are described by way of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and in references cited hereinabove. [00153] In another embodiment, the two-hybrid system for selecting interacting proteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien et al., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used to identify molecules that specifically bind to a chimeric PDZ domain- containing protein. Furthermore, the identified molecules are further tested for their ability to inhibit transmembrane receptor interactions with a PDZ domain. [00154] In one aspect of the invention, antagonists of an interaction between a PDZ containing protein and a PL protein are identified. In one embodiment, a modification of the "A" assay described supra is used to identify antagonists. In one embodiment, a modification of the "G" assay described supra is used to identify antagonists. [00155] In one embodiment, screening assays are used to detect molecules that specifically bind to PDZ domains. Such molecules are useful as agonists or antagonists of PDZ -protein-mediated cell function (e.g., cell activation, e.g., T cell activation, vesicle transport, cytokine release, growth factors, transcriptional changes, cytoskeleton rearrangement, cell movement, chemotaxis, and the like). In one embodiment, such assays are performed to screen for leukocyte activation inhibitors for drug development. The invention thus provides sensitive assays to detect molecules that specifically bind to PDZ domain-containing proteins. For example, recombinant cells expressing PDZ domain-encoding nucleic acids can be used to produce PDZ domains in these assays and to screen for molecules that bind to the domains. Molecules are contacted with the PDZ domain (or fragment thereof) under conditions conducive to binding, and then molecules that specifically bind to such domains are identified. Methods that can be used to carry out the foregoing are commonly known in the art.
[00156] It will be appreciated by the ordinarily skilled practitioner that, in one embodiment, antagonists are identified by conducting the A or G assays in the presence and absence of a known or candidate antagonist. When decreased binding is observed in the presence of a compound, that compound is identified as an antagonist. Increased binding in the presence of a compound signifies that the compound is an agonist. [00157] For example, in one assay, a test compound can be identified as an inhibitor (antagonist) of binding between a chimeric PDZ-domain-containing polypeptide and a PL protein by contacting a chimeric PDZ-domain- containing polypeptide and a PL peptide in the presence and absence of the test compound, under conditions in which they would (but for the presence of the test compound) form a complex, and detecting the formation of the complex in the presence and absence of the test compound. It will be appreciated that less complex formation in the presence of the test compound than in the absence of the compound indicates that the test compound is an inhibitor of a chimeric PDZ protein -PL protein binding.
[00158] In one embodiment, the "G" assay is used in the presence or absence of a candidate inhibitor. In one embodiment, the "A" assay is used in the presence or absence of a candidate inhibitor. [00159] In one embodiment (in which a G assay is used), one or more chimeric PDZ-domain-containing polypeptide- GST-fusion proteins are bound to the surface of wells of a 96-well plate as described supra (with appropriate controls including nonfusion GST protein). All fusion proteins are bound in multiple wells so that appropriate controls and statistical analysis can be done. A test compound in BSA/PBS (typically at multiple different concentrations) is added to wells. Immediately thereafter, 30 uL of a detectably labeled (e.g., biotinylated) peptide known to bind to the relevant PDZ domain (see, e.g., TABLE 3) is added in each of the wells at a final concentration of, e.g., between about 2 uM and about 40 uM, typically 5 uM, 15 uM, or 25 uM. This mixture is then allowed to react with the PDZ fusion protein bound to the surface for 10 minutes at 40C followed by 20 minutes at 250C. The surface is washed free of unbound peptide three times with ice cold PBS and the amount of binding of the peptide in the presence and absence of the test compound is determined. Usually, the level of binding is measured for each set of replica wells (e.g. duplicates) by subtracting the mean GST alone background from the mean of the raw measurement of peptide binding in these wells.
[00160] In an alternative embodiment, the A assay is carried out in the presence or absence of a test candidate to identify inhibitors of PL-PDZ interactions.
[00161] In one embodiment, a test compound is determined to be a specific inhibitor of the binding of an introduced PDZ domain (P) in a chimeric polypeptide and a PL (L) sequence when, at a test compound concentration of less than or equal to 1 mM (e.g., less than or equal to: 500 uM, 100 uM, 10 uM, 1 uM, 100 nM or 1 nM) the binding of P to L in the presence of the test compound less than about 50% of the binding in the absence of the test compound, (in various embodiments, less than about 25%, less than about 10%, or less than about 1%). Preferably, the net signal of binding of P to L in the presence of the test compound plus six (6) times the standard error of the signal in the presence of the test compound is less than the binding signal in the absence of the test compound. [00162] In one embodiment, assays for an inhibitor are carried out using a single chimeric PDZ-domain-containing polypeptide -PDZ ligand pair (e.g., a chimeric PDZ-GST fusion protein and a PL peptide). In a related embodiment, the assays are carried out using a plurality of pairs, such as a plurality of different pairs listed in TABLE 3. [00163] In some embodiments, it is desirable to identify compounds that, at a given concentration, inhibit the binding of one PL-PDZ pair, but do not inhibit (or inhibit to a lesser degree) the binding of a specified second PL- PDZ pair. These antagonists can be identified by carrying out a series of assays using a candidate inhibitor and different PL-PDZ pairs (e.g., as shown in the matrix of TABLE 3) and comparing the results of the assays. All such pairwise combinations are contemplated by the invention (e.g., test compound inhibits binding OfPL1 to PDZ1 to a greater degree than it inhibits binding of PL1 to PDZ2 or PL2 to PDZ2). Importantly, it will be appreciated that, based on the data provided in TABLE 3 and disclosed herein (and additional data that can be generated using the methods described herein) inhibitors with different specificities can readily be designed.
[00164] For example, according to the invention, the Ki ("potency") of an inhibitor of a PDZ-PL interaction can be determined. Ki is a measure of the concentration of an inhibitor required to have a biological effect. For example, administration of an inhibitor of a PDZ-PL interaction in an amount sufficient to result in an intracellular inhibitor concentration of at least between about 1 and about 100 Ki is expected to inhibit the biological response mediated by the target PDZ-PL interaction. In one aspect of the invention, the Kd measurement of PDZ-PL binding as determined using the methods supra is used in determining Ki.
[00165] In one aspect, the invention provides a method of determining the potency (Ki) of an inhibitor or suspected inhibitor of binding between a PDZ domain and a ligand by immobilizing a chimeric polypeptide comprising at least one introduced PDZ domain and a non-PDZ domain on a surface, contacting the immobilized polypeptide with a plurality of different mixtures of the ligand and inhibitor, wherein the different mixtures comprise a fixed amount of ligand and different concentrations of the inhibitor, determining the amount of ligand bound at the different concentrations of inhibitor, and calculating the Ki of the binding based on the amount of ligand bound in the presence of different concentrations of the inhibitor. In an embodiment, the polypeptide is immobilized by binding the polypeptide to an immobilized immunoglobulin that binds the non-PDZ domain. This method, which is based on the "G" assay described supra, is particularly suited for high-throughput analysis of the Ki for inhibitors of PDZ- ligand interactions. Further, using this method, the inhibition of the PDZ-ligand interaction itself is measured, without distortion of measurements by avidity effects. Typically, at least a portion of the ligand is detectably labeled to permit easy quantitation of ligand binding.
[00166] In another aspect of the invention, an enhancer (sometimes referred to as, augmentor or agonist) of binding between a PDZ domain and a ligand is identified by immobilizing a chimeric polypeptide comprising at least one introduced PDZ domain and a non-PDZ domain on a surface, contacting the immobilized polypeptide with the ligand in the presence of a test agent and determining the amount of ligand bound, and comparing the amount of ligand bound in the presence of the test agent with the amount of ligand bound by the polypeptide in the absence of the test agent. At least two-fold (often at least 5-fold) greater binding in the presence of the test agent compared to the absence of the test agent indicates that the test agent is an agent that enhances the binding of the PDZ domain to the ligand. As noted supra, agents that enhance PDZ-ligand interactions are useful for disruption (dysregulation) of biological events requiring normal PDZ-ligand function (e.g., cancer cell division and metastasis, and activation and migration of immune cells).
[00167] The present invention also embodies a chimeric polypeptide containing a modified PDZ domain introduced from another PDZ domain-containing polypeptide. One embodiment of the invention includes increasing the specificity or sensitivity of a PDZ-PL interaction through mutagenesis and selection of high affinity or high specificity variants. Methods such as chemical (e.g., EMS) or biological mutagenesis (e.g. Molecular shuffling or DNA polymerase mutagenesis) can be applied to create mutations in DNA encoding PDZ domains or PL domains. Proteins can then be made from variants and tested using a number of methods described herein (e.g., ΛAΛ assay, ΛGΛ assay or yeast two hybrid). In general, one would assay mutants for high affinity binding between the mutated PDZ domain and a test sample (such as an oncogenic E6 protein) that have reduced affinity for other cellular PLs (as described in section IX). These methods are known to those skilled in the art and examples herein are not intended to be limiting. [00168]
XI. Detection of PDZ Ligand Protein
[00169] As indicated in the Background section, PDZ domain- containing proteins are involved in a number of biological functions, including, but not limited to, vesicular trafficking, tumor suppression, protein sorting, establishment of membrane polarity, apoptosis, regulation of immune response and organization of synapse formation. In general, this family of proteins has a common function of facilitating the assembly of multi-protein complexes, often serving as a bridge between several proteins, or regulating the function of other proteins. Additionally, as also noted supra, these proteins are found in essentially all cell types. Consequently, detection of inappropriate PDZ:PL interactions or abnormal interactions can be utilized to diagnose a wide variety of biological and physiological conditions. In particular, detection of PL proteins from pathogenic organisms can be diagnosed using PDZ domains. Most, but not all, embodiments of this invention, require the addition of a detectable marker to the PDZ or PL protein used for detection. Examples are given below. A. Chemical Synthesis
[00170] The peptides of the invention or analogues thereof, may be prepared using virtually any art-known technique for the preparation of peptides and peptide analogues. For example, the peptides may be prepared in linear form using conventional solution or solid phase peptide syntheses and cleaved from the resin followed by purification procedures (Creighton, 1983, Protein Structures And Molecular Principles, W. H. Freeman and Co., N.Y.). Suitable procedures for synthesizing the peptides described herein are well known in the art. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure and mass spectroscopy).
[00171] In addition, analogues and derivatives of the peptides can be chemically synthesized. The linkage between each amino acid of the peptides of the invention may be an amide, a substituted amide or an isostere of amide. Nonclassical amino acids or chemical amino acid analogues can be introduced as a substitution or addition into the sequence. Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, α- amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, .ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogues in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).
B. Recombinant Synthesis
[00172] If the peptide is composed entirely of gene-encoded amino acids, or a portion of it is so composed, the peptide or the relevant portion may also be synthesized using conventional recombinant genetic engineering techniques. For recombinant production, a polynucleotide sequence encoding a linear form of the peptide is inserted into an appropriate expression vehicle, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation. The expression vehicle is then transfected into a suitable target cell which will express the peptide. Depending on the expression system used, the expressed peptide is then isolated by procedures well- established in the art. Methods for recombinant protein and peptide production are well known in the art (see, e.g., Maniatis et al., 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N. Y.). [00173] A variety of host-expression vector systems may be utilized to express the peptides described herein. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungi expression vectors containing an appropriate coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an appropriate coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an appropriate coding sequence; or animal cell systems.
[00174] In some embodiments, increasing the number of copies of a PL detector may be used to increase the specificity or sensitivity of detection. An example of this is presented in EXAMPLE 4. The TIP-TIP-IgG vector produces a fusion protein that has duplicated copies of the PDZ domain from TIP-I and the protein itself should dimerize on the basis of the IgG constant region backbone. Hence, a single protein contains 2-4 copies of the TIP-I PDZ domain. In a similar manner, addition tandem repeats of PL detectors could be fashioned. In some embodiments, different PDZ domains from different proteins could be engineered to express as a single protein (e.g., the PDZ domains of TIP-I and MAGI-I could be engineered to detect oncogenic HPV E6 proteins). In a similar manner, a different Ig backbone could be used to increase the avidity of a construct. For example, the IgG constant regions will dimerize with itself, but the IgM constant regions will form a complex often monomers. [00175] The expression elements of the expression systems vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used in the expression vector. For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage .lamda., plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used; when cloning in insect cell systems, promoters such as the baculovirus polyhedron promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5 K promoter) may be used; when generating cell lines that contain multiple copies of expression product, SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker.
[00176] In cases where plant expression vectors are used, the expression of sequences encoding the peptides of the invention may be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu et al., 1987, EMBO J. 6:307-311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglie et al., 1984, Science 224:838-843) or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B (Gurley et al., 1986, MoI. Cell. Biol. 6:559-565) may be used. These constructs can be introduced into planleukocytes using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. For reviews of such techniques see, e.g., Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.
[00177] In one insect expression system that may be used to produce the peptides of the invention, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express the foreign genes. The virus grows in Spodoptera frugiperda cells. A coding sequence may be cloned into non-essential regions (for example the polyhedron gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedron promoter). Successful insertion of a coding sequence will result in inactivation of the polyhedron gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedron gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed, (e.g., see Smith et al., 1983, J. Virol. 46:584; Smith, U.S. Pat. No. 4,215,051). Further examples of this expression system may be found in Current Protocols in Molecular Biology, Vol. 2, Ausubel et al., eds., Greene Publish. Assoc. & Wiley Interscience.
[00178] In mammalian host cells, a number of viral based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, a coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing peptide in infected hosts, (e.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Alternatively, the vaccinia 7.5 K promoter may be used, (see, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. USA 79:4927- 4931).
[00179] Other expression systems for producing linear peptides of the invention will be apparent to those having skill in the art.
C. Tags or Markers
[00180] Tags and markers are frequently used to aid in purification of components or detection of biological molecules. Examples of biological tags include, but are not limited to, glutathione-S-transferase, maltose binding protein, Immunoglobulin domains, Intein, Hemagglutinin epitopes, myc epitopes, etc. Examples of chemical tags include, but are not limited to, biotin, gold, paramagnetic particles or fluorophores. These examples can be used to identify the presence of proteins or compounds they are attached to or can be used by those skilled in the art to purify proteins or compounds from complex mixtures.
D. Purification of the Peptides and Peptide Analogues
[00181] The peptides and peptide analogues of the invention can be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography and the like. The actual conditions used to purify a particular peptide or analogue will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those having skill in the art. The purified peptides can be identified by assays based on their physical or functional properties, including radioactive labeling followed by gel electrophoresis, radioimmuno-assays, ELISA, bioassays, and the like. XII. Diagnostic Kit
[00182] The present invention also includes a kit for detection and diagnosis of HPV. A subject kit usually contains a chimeric polypeptide containing at least one introduced PDZ domain that has high binding strength to a PDZ ligand. In one embodiment, the kit further includes a second PDZ ligand binding partner, which may be an antibody, for example, an antibody specific for E6 protein. In some embodiments, the second PDZ ligand binding partner is labeled with a detectable label. In other embodiments, a secondary labeling component, such as a detectably labeled secondary antibody, is included. In some embodiments, a subject kit further comprises a means, such as a device or a system, for isolating oncogenic HPV E6 from the sample. The kit may optionally contain proteasome inhibitor. [00183] A subject kit can further include, if desired, one or more of various conventional components, such as, for example, containers with one or more buffers, detection reagents or antibodies. Printed instructions, either as inserts or as labels, indicating quantities of the components to be used and guidelines for their use, can also be included in the kit. In the present disclosure it should be understood that the specified materials and conditions are important in practicing the invention but that unspecified materials and conditions are not excluded so long as they do not prevent the benefits of the invention from being realized. Exemplary embodiments of the diagnostic methods of the invention are described above in detail.
[00184] In a subject kit, the oncogenic E6 detection reaction may be performed using an aqueous or solid substrate, where the kit may comprise reagents for use with several separation and detection platforms such as test strips, sandwich assays, etc. In many embodiments of the test strip kit, the test strip has bound thereto a chimeric PDZ domain-containing polypeptide that specifically binds the PL domain of an oncogenic E6 protein and captures oncogenic E6 protein on the solid support. In some embodiments, the kit further comprises a detection antibody or antibodies, which is either directly or indirectly detectable, and which binds to the oncogenic E6 protein to allow its detection. Kits may also include components for conducting western blots (e.g., pre-made gels, membranes, transfer systems, etc.); components for carrying out ELISAs (e.g., 96-well plates); components for carrying out immunoprecipitation (e.g. protein A); columns, especially spin columns, for affinity or size separation of oncogenic E6 protein from a sample (e.g. gel filtration columns, PDZ domain polypeptide columns, size exclusion columns, membrane cut-off spin columns etc.).
[00185] The subject kits may also contain control samples containing oncogenic or non-oncogenic E6, and/or a dilution series of oncogenic E6, where the dilution series represents a range of appropriate standards with which a user of the kit can compare their results and estimate the level of oncogenic E6 in their sample. Such a dilution series may provide an estimation of the progression of any cancer in a patient. Fluorescence, color, or autoradiological film development results may also be compared to a standard curve of fluorescence, color or film density provided by the kit.
[00186] In addition to above-mentioned components, the subject kits typically further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
[00187] Also provided by the subject invention are kits including at least a computer readable medium including programming as discussed above and instructions. The instructions may include installation or setup directions. The instructions may include directions for use of the invention with options or combinations of options as described above. In certain embodiments, the instructions include both types of information.
[00188] Providing the software and instructions as a kit may serve a number of purposes. The combination may be packaged and purchased as a means for producing rabbit antibodies that are less immunogenic in a non-rabbit host than a parent antibody, or nucleotide sequences them.
[00189] The instructions are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging), etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, etc, including the same medium on which the program is presented. XIII. Pathogen Protein Extraction Kit
[00190] In yet another aspect, the present invention provides kits for practicing the subject methods, e.g., for extracting and purifying a protein from a protein extract, in certain embodiments, for testing the presence of a pathogen protein from a sample containing such pathogen. The subject kits at least include a chimeric polypeptide containing at least one introduced PDZ domain having high binding strength to a PDZ ligand protein for the capture of such PDZ ligand in a sample. The kits may include an extraction reagent that has a pH of at least about pH 10.0, and a neutralizing reagent. The extraction reagent and/or the neutralizing reagent contain a non-ionic detergent. In addition, the kits may also include buffers and detection reagents for detecting that protein using the capture chimeric PDZ domain- containing polypeptide. The above components may be present in separate containers or one or more components may be combined into a single container, e.g., a glass or plastic vial. [00191] In addition to the above components, the subject kits may further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the
XIV. Methods for Determining if a Subject is Infected with an Oncogenic Strain of HPV
[00192] The present invention provides methods of detecting oncogenic HPV E6 protein in a sample and finds utility in diagnosing HPV infection in a subject. In many embodiment, a biological sample is obtained from a subject, and, the presence of oncogenic HPV E6 protein in the sample is determined. The presence of a detectable amount of oncogenic HPV E6 protein in a sample indicates indicates that the individual is infected with a oncogenic strain of HPV. In other embodiments, the level of oncogenic HPV E6 protein in a biological sample is determined, and compared to the amount of a control in the sample. The relative amount of oncogenic HPV E6 protein in a sample indicates the severity of the infection by HPV.
[00193] The methods generally involve two binding partners of oncogenic HPV E6 protein, one of which is a chimeric polypeptide containing at least one introduced PDZ domain, as described above. In general, the methods involve a) isolating the oncogenic HPV E6 protein from a sample using one of the binding partners, and b) detecting the oncogenic HPV E6 protein with the other binding partner. However, in one embodiment of the present invention, only the chimeric PDZ domain-containing polypeptide or a variant of the chimeric PDZ domain- containing polypeptide is used for determining if a subject is injected with an oncogenic strain of HPV.
XV. Isolating Oncogenic HPV E6 Protein
[00194] In general, methods of the invention involve at least partially separating (i.e., isolating) native oncogenic HPV E6 protein from other proteins in a sample. This separation is usually achieved using a first binding partner for the oncogenic HPV E6. In many embodiments, the first binding partner is a chimeric polypeptide containing at least one introduced PDZ domain, or, in other embodiments an anti-HPV E6 antibody or mixture of antibodies. In yet another embodiment, only the chimeric PDZ domain-containing polypeptide or a variant of the chimeric PDZ domain-containing polypeptide (e.g. labeled chimeric PDZ domain-containing polypeptide) is used for isolating the E6 protein from an oncogenic strain of HPV. [00195] In certain embodiments, one of the oncogenic HPV E6 binding partners is bound, directly or via a linker, to an insoluble support. Insoluble supports are known in the art and include, but are not limited to, a bead (e.g, magnetic beads, polystyrene beads, and the like); a membrane; and the like. In one non-limiting example, a chimeric polypeptide containing at least one introduced PDZ domain is bound to a magnetic bead. The chimeric PDZ domain-containing polypeptide bound to the magnetic bead is contacted with the sample, and, after a complex is formed between the antibody and any E6 protein in the sample, a magnetic field is applied, such that the complex is removed from the sample. Where the PDZ domain polypeptide is bound to an insoluble support, such as a membrane, E6 protein bound to the chimeric PDZ domain-containing polypeptide is removed from the sample by removing the membrane, or by transferring the sample to a separate container. Where the chimeric PDZ domain- containing polypeptide is bound to a bead, the E6 protein bound to the bead is removed from the sample by centrifugation or filtration. Such embodiments are envisioned using a different E6 binding partner, e.g., an anti-E6 antibody.
[00196] In general, a suitable separation means is used with a suitable platform for performing the separation. For example, where oncogenic HPV E6 is separated by binding to chimeric PDZ domain-containing polypeptides, the separation is performed using any of a variety of platforms, including, but not limited to, affinity column chromatography, capillary action or lateral flow test strips, immunoprecipitation, etc.
[00197] In many embodiments, oncogenic HPV E6 is separated from other proteins in the sample by applying the sample to one end of a test strip, and allowing the proteins to migrate by capillary action or lateral flow. Methods and devices for lateral flow separation, detection, and quantitation are known in the art. See, e.g., U.S. Pat. Nos. 5,569,608; 6,297,020; and 6,403,383. In these embodiments, a test strip comprises, in order from proximal end to distal end, a region for loading the sample (the sample-loading region) and a test region containing an oncogenic E6 protein binding partner, e.g., a region containing a chimeric PDZ domain-containing polypeptide or, in other embodiments, a region containing an anti-E6 antibody. The sample is loaded on to the sample-loading region, and the proximal end of the test strip is placed in a buffer. Oncogenic E6 protein is captured by the bound antibody in the first test region. Detection of the captured oncogenic E6 protein is carried out as described below. For example, detection of captured E6 proteins is carried out using detectably labeled antibody specific for an epitope of E6 proteins that is common to all oncogenic E6 proteins, or a mixture of antibodies that can, together, bind to all oncogenic E6 proteins. In alternative embodiments, an E6 antibody may be present in the test region and detection of oncogenic E6 bound to the E6 antibody uses a labeled chimeric PDZ domain- containing polypeptide. XVI. Detecting and Quantitating Oncogenic E6 Protein
[00198] Once oncogenic E6 protein is separated from other proteins in the sample, oncogenic E6 protein is detected and/or the level or amount of oncogenic E6 protein is determined (e.g., measured). As discussed above, oncogenic E6 protein is generally detected using a binding partner, e.g. an antibody or antibodies specific to E6, or a chimeric polypeptide containing at least one introduced PDZ domain as described herein.
[00199] Detection with a specific antibody is carried out using well-known methods. In general, the binding partner is detectably labeled, either directly or indirectly. Direct labels include radioisotopes (e.g., .sup.1251; .sup.35S, and the like); enzymes whose products are detectable (e.g., luciferase, .beta.-galactosidase, horse radish peroxidase, and the like); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, and the like); fluorescence emitting metals, e.g., .sup.152Eu, or others of the lanthanide series, attached to the antibody through metal chelating groups such as EDTA; chemiluminescent compounds, e.g., luminol, isoluminol, acridinium salts, and the like; bioluminescent compounds, e.g., luciferin; fluorescent proteins; and the like. Fluorescent proteins include, but are not limited to, a green fluorescent protein (GFP), including, but not limited to, a "humanized" version of a GFP, e.g., wherein codons of the naturally-occurring nucleotide sequence are changed to more closely match human codon bias; a GFP derived from Aequoria victoria or a derivative thereof, e.g., a "humanized" derivative such as Enhanced
GFP, which are available commercially, e.g., from Clontech, Inc.; a GFP from another species such as Renilla reniformis, Renilla mulleri, or Ptilosarcus guernyi, as described in, e.g., WO 99/49019 and Peelle et al. (2001) J.
Protein Chem. 20:507-519; "humanized" recombinant GFP (hrGFP) (Stratagene); any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973; and the like.
[00200] Indirect labels include second antibodies specific for E6-specific antibodies, wherein the second antibody is labeled as described above; and members of specific binding pairs, e.g., biotin-avidin, and the like.
[00201] In some embodiments, a level of oncogenic E6 is quantitated. Quantitation can be carried out using any known method, including, but not limited to, enzyme-linked immunosorbent assay (ELISA); radioimmunoassay
(RIA); and the like. In general, quantitation is accomplished by comparing the level of expression product detected in the sample with a standard curve.
[00202] In some embodiments, oncogenic HPV E6 is separated on a test strip, as described above. In these embodiments, oncogenic HPV E6 is detected using a detectably labeled binding partner (e.g. a labeled chimeric polypeptide containing at least one introduced PDZ domain) that binds oncogenic HPV E6. Oncogenic HPV E6 may be quantitated using a reflectance spectrophotometer, or by eye, for example.
XVII. Biological Samples
[00203] Biological samples to be analyzed using the methods of the invention are obtained from any mammal, e.g., a human or a non-human animal model of HPV. In many embodiments, the biological sample is obtained from a living subject.
[00204] In some embodiments, the subject from whom the sample is obtained is apparently healthy, where the analysis is performed as a part of routine screening. In other embodiments, the subject is one who is susceptible to
HPV, (e.g., as determined by family history; exposure to certain environmental factors; etc.). In other embodiments, the subject has symptoms of HPV (e.g., cervical warts, or the like). In other embodiments, the subject has been provisionally diagnosed as having HPV (e.g. as determined by other tests based on e.g., PCR).
[00205] The biological sample may be derived from any tissue, organ or group of cells of the subject. In some embodiments a cervical scrape, biopsy, or lavage is obtained from a subject.
[00206] In some embodiments, the biological sample is processed, e.g., to remove certain components that may interfere with an assay method of the invention, using methods that are standard in the art. In some embodiments, the biological sample is processed to enrich for proteins, e.g., by salt precipitation, and the like. In certain embodiments, the sample is processed in the presence proteasome inhibitor to inhibit degradation of the E6 protein.
[00207] In the assay methods of the invention, in some embodiments, the level of E6 protein in a sample may be quantified and/or compared to controls. Suitable control samples are from individuals known to be healthy, e.g., individuals known not to have HPV. Control samples can be from individuals genetically related to the subject being tested, but can also be from genetically unrelated individuals. A suitable control sample also includes a sample from an individual taken at a time point earlier than the time point at which the test sample is taken, e.g., a biological sample taken from the individual prior to exhibiting possible symptoms of HPV. XVIII. Purification of PDZ Ligand Proteins
[00208] The present invention provides a method for purifying a PDZ ligand protein from a protein extract. In general, the methods involve two steps: a) contacting a sample containing a PDZ ligand protein with a chimeric polypeptide containing at least one introduced PDZ domain which has higher binding strength to a PDZ ligand as described above; and purifying the PDZ ligand protein from the sample. The method may optionally include contacting the subject sample with an extraction reagent having a pH that is greater than about pH. 10.0 followed by contacting the subject sample with a neutralizing reagent to produce a protein extract. The extraction reagent and/or the neutralizing reagent contains a non-ionic detergent. The resultant protein extract contains a non-ionic detergent and has a pH that is neutral (i.e., between about pH 7.0 and about pH 8.0). The methods generally produce a protein extract containing proteins that are readily detectable using capture agents for those proteins, for example, using a chimeric PDZ domain- containing protein for capturing a PDZ ligand. As such, a protein extract produced by the instant methods are generally suitable for use in binding assays, e.g., immunological assays, for detection of those proteins.
[00209] In certain embodiments the methods may include: increasing the pH of the fixed cells to a pH of at least about pH 10.0 to produce an intermediate composition, and then, in the presence of a non-ionic detergent, neutralizing the pH of the intermediate composition to produce the protein extract. Since, as mentioned above, the non-ionic detergent may be present in either the extraction reagent or the neutralizing reagent (or in both the extraction reagent and the neutralizing reagent), certain embodiments of the instant methods include: a) contacting a sample with an extraction reagent to produce an intermediate composition having a pH of at least about pH 10.0; and b) contacting the intermediate composition with a neutralizing reagent comprising a non-ionic detergent; to neutralize said pH of the intermediate composition and produce the protein extract. In other embodiments, the method may include: a) contacting the sample with an extraction reagent comprising a non-ionic detergent to produce an intermediate composition having a pH of at least about pH 10.0; and, b) contacting the intermediate composition with a neutralizing reagent; to neutralize the pH of the intermediate composition and produce the protein extract.
[00210] In certain embodiments, the protein extract produced by the instant methods may contain more protein that is accessible to capture agents (e.g. a chimeric PDZ domain-containing polypeptide) than a protein extract made using other methods, e.g., methods that do not employ: a high pH extraction step (i.e., a step that increases pH to greater than about pH 10.0 or pH 11.0), a neutralizing step (i.e., a step that increases pH to about pH 7.0 to about pH 8.0) and a non-ionic detergent. Neither high pH alone nor non-ionic detergent alone produces such a protein extract. In particular embodiments, the high pH extraction reagent solubilizes proteins in the fixed cells, whereas the non- ionic detergent prevents the solubilized proteins in the intermediate composition from re-aggregating or precipitating as the pH of the intermediate composition is neutralized. As would be apparent from the above, a variety of different denaturants, detergents, buffers, pHs and component concentrations may be employed in the reagents described above. The optimal denaturant, detergent, buffer or pH, or component concentration in any reagent is readily determined using routine methods.
[00211] In certain embodiments, the subject protein extract contain solubilized HPV E6 protein (particularly E6 protein from oncogenic strains of HPV) that is accessible to and readily detectable by a binding agent, e.g. a chimeric PDZ domain- containing polypeptide without further treatment of the protein extract (e.g., without further addition of denaturant, pH changes or heating). The protein extract may also contain solubilized or insoluble membranes, proteins other than HPV E6 protein, and other cellular contents such as DNA, RNA, carbohydrates, etc. Other contaminants such as those derived from mucal contamination of the original cellular sample may also be present. The components of the protein extract generally do not contain whole (i.e., cytologically intact) cells. The protein extract may be used immediately, or stored, e.g., in frozen form, before use.
[00212] After neutralization of the cell extract, E6 protein may be concentrated from the cell extract by incubating the extract with particles containing binder for the E6. The binder may comprise a chimeric PDZ domain containing polypeptide, E6 associated protein (E6AP) or fragments thereof, or E6 binding protein (E6BP) or fragments thereof. After E6 is captured by the particles, the particles are washed and E6 is then released from the particles by incubation with buffer at pH greater than 10. The particles are separated from the eluting solution and the remaining solution is then neutralized by the procedures described previously. Alternatively, E6 protein may be detected without release form the capture particles.
XIX. Production of Chimeric PDZ Domain-Containing Polypeptide
[00213] Once the chimeric polynucleotide construct has been created, it is then introduced into a suitable host cell by techniques known in the art. A variety of expression vector/host systems may be utilized to contain and express sequences encoding the chimeric polypeptide described herein. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transfonmed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. The invention is not limited by the host cell employed.
[00214] In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding the chimeric PDZ domain-containing polypeptide. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding CJPDZ can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla Calif.) or pSPORTl plasmid (Life Technologies). Ligation of sequences encoding CJPDZ into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When large quantities of a chimeric PDZ domain-containing polypeptide are needed, vectors which direct high level expression of chimeric PDZ domain-containing polypeptide may be used. For example, vectors containing the strong, inducible T5 or T7 bacteriophage promoter may be used. [00215] Yeast expression systems may be used for production of the chimeric PDZ domain-containing polypeptide. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation. (See, e.g., Ausubel, 1995, supra; Grant et al. (1987) Methods Enzymol. 153:516-54; and Scorer, C. A. et al. (1994) Bio/Technology 12:181-184.)
[00216] Plant systems may also be used for expression of the chimeric PDZ domain- containing polypeptide. Transcription of sequences encoding a chimeric PDZ domain- containing polypeptide may be driven by viral promoters, e.g., the 35S and 19S promoters of CAMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York N.Y., pp. 191- 196.)
[00217] In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding a chimeric PDZ domain-containing polypeptide may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses the chimeric PDZ domain-containing polypeptide in host cells. (See, e.g., Logan, J. and T. Shenk (19:34) Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV -based vectors may also be used for high-level protein expression.
[00218] Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J. J. et al. (1997) Nat Genet. 15:345-355.)
[00219] For long term production of recombinant proteins in mammalian systems, stable expression of a chimeric PDZ domain-containing polypeptide in cell lines is preferred. For example, sequences encoding a chimeric PDZ domain-containing polypeptide can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
[00220] Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk.sup.- or apr.sup.- cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides, neomycin and G- 418; and als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. MoI. Biol. 150: 1- 14.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), β-glucuronidase and its substrate .beta.- glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, C. A. (1995) Methods MoI. Biol. 55: 121-131.) [00221] Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding a chimeric PDZ domain- containing polypeptide is inserted within a marker gene sequence, transformed cells containing sequences encoding the chimeric PDZ domain-containing polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a chimeric PDZ domain- containing polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
[00222] In general, host cells that contain the nucleic acid sequence encoding a chimeric PDZ domain-containing polypeptide and that express the chimeric PDZ domain- containing polypeptide may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA- RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. [00223] Immunological methods for detecting and measuring the expression of the chimeric PDZ domain- containing polypeptide using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the chimeric PDZ domain-containing polypeptide is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul Minn., Sect. IV; Coligan, J. E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York N.Y.; and Pound, J. D. (1998) Immunochemical Protocols, Humana Press, TotowaNJ.).
[00224] A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding a chimeric PDZ domain-containing polypeptide include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding CJPDZ, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison Wis.), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates., cofactors, inhibitors, magnetic particles, and the like.
[00225] Host cells transformed with nucleotide sequences encoding a chimeric PDZ domain-containing polypeptide may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode a chimeric PDZ domain-containing polypeptide may be designed to contain signal sequences which direct secretion of the chimeric PDZ domain-containing polypeptide through a prokaryotic or eukaryotic cell membrane. [00226] In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the protein may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post- translational activities (e.g., CHO, HeLa, MDCK, HEK293, and W138), are available from the American Type Culture Collection (ATCC, Bethesda Md.) and may be chosen to ensure the correct modification and processing of the foreign protein.
[00227] In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding a chimeric PDZ domain-containing polypeptide may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a fusion protein comprising a chimeric PDZ domain- containing polypeptide and a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of the chimeric PDZ domain-containing polypeptide activity. Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S- transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the CJPDZ encoding sequence and the heterologous protein sequence, so that CJPDZ may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
[00228] In a further embodiment of the invention, synthesis of radiolabeled chimeric PDZ domain- containing polypeptide may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract systems (Promega). These systems couple transcription and translation of protein- coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, preferably 35S -methionine.
[00229] Fragments of a chimeric PDZ domain-containing polypeptide may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin-Elmer). Various fragments of a chimeric PDZ domain- containing polypeptide may be synthesized separately and then combined to produce the full length molecule. XX. Treatment with PDZ Domain-Containing Polypeptide
[00230] In one aspect, the present invention provides a method of treating oncogenic HPV in a subject. In some embodiments, the oncogenic HPV strain of the method is strain 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82. In one embodiment, the present invention provides a method of treating HPV in a subject with a polypeptide containing a PDZ domain. In one embodiment, the present invention provides a method of treating HPV in a subject with a chimeric polypeptide containing a PDZ domain. In some embodiments, the polypeptide of the invention binds to E6 protein from oncogenic HPV strain 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82. In one embodiment, the polypeptide of the invention binds to E6 protein from multiple oncogenic HPV strains. In one embodiment, the present invention provides a method of treating HPV in a subject with a chimeric polypeptide containing a PDZ domain that binds the oncogenic E6 protein of HPV. In one embodiment, the method comprises administering to the subject in need thereof an effective amount of a PDZ domain- or PDZ domain fragment-containing chimeric polypeptide of the present invention, thereby interfering with the effects of oncogenic E6 protein on cellular function. In one embodiment, the method of the present invention prevents growth of cancer caused by HPV in a patient. In one embodiment, the method of the present invention prevents growth of cancer in a patient for which oncogenic E6 protein is detected.
[00231] In one embodiment, the methods of the present invention slow or stop growth of cancer, such as cerical, in others, either in a patient or subject through administration of a PDZ domain- containing polypetide. In further embodiments, the PDZ domain- containing polypeptide is a chimeric polypeptide. In further embodiments, a reversal of cervical cancer growth results in the reduction in size of the tumor. Size reduction can be more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, compared to pretreatment levels. In some embodiments, administration of the PDZ domain-containing chimeric polypeptide continues after the tumor is reduced in size by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. Administrations of the PDZ domain-containing chimeric polypeptide can continue until or beyond the point where a therapeutic endpoint is reached, as determined by a physician. Non-limiting examples of therapeutic endpoints include partial remission, complete remission, a reduction in tumor size, stable tumor size, slowing of tumor growth, reducing the frequency of metastasis, prevention of metastasis for a period exceeding at least 3 months, at least 6 months, at least 9 months or at least 12 months, extension of expected life expectancy, prevention of recurrence of a cancer, extension of the expected time necessary for recurrence of cancer, and reducing the frequency or severity of one or more sequelae of cancer, such as pain, edema, etc. Continued administration beyond a given therapeutic endpoint can be utilized as a maintenance therapy to prevent cancer relapse. For example, treatment can continue beyond a selected therapeutic endpoint for more than 1 month, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years, or longer, or for the life of the subject. [00232] In some embodiments, administration of the PDZ domain- containing chimeric polypeptide is combined with the administration of an additional therapeutic agent as part of a therapeutic regimen. The additional therapeutic agent can be administered before, during, or after the administration of the PDZ domain-containing chimeric polypeptide. Agents administered during the administration of PDZ domain- containing chimeric polypeptide can be co-administered as a single composition and delivery or delivered as part of the same procedure, administered at about the same time in separate administration events. Agents administered before or after administration of the PDZ domain-containing chimeric polypeptide can be administered in time frames preceding or following PDZ domain- containing chimeric polypeptide administration that include the following, without limitation: less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or 22 hours; less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days; less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 weeks; less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 months; and less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 years. In some embodiments, the additional therapeutic agent is conjugated to the PDZ domain- containing chimeric polypeptide. [00233] In some embodiments, agents of the present invention can be combined with anti-tumor or anti-cancer therapeutics capable of decreasing or preventing a further increase in tumor growth. Non-limiting examples are chemotherapeutic agents, cytotoxic agents, and non-peptide small molecules such as Gleevec® (Imatinib Mesylate), Velcade® (bortezomib), Casodex (bicalutamide), Iressa® (gefitinib), and Adriamycin; alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, Casodex™, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK.R™ ; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, NJ.) and docetaxel (TAXOTERE™, Rhone-Poulenc Rorer, Antony, France); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included as suitable chemotherapeutic cell conditioners are anti -hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, (NolvadexTM), raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti- androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; chlorambucil; gemcitabine; 6 -thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; camptothecin-11 (CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO). Where desired, the compounds or pharmaceutical composition of the present invention can be used in combination with commonly prescribed anti-cancer drugs such as Herceptin®, Avastin®, Erbitux®, Rituxan®, Taxol®, Arimidex®, Taxotere®, ABVD, AVICINE, Abagovomab, Acridine carboxamide, Adecatumumab, 17-N- Allylamino-17-demethoxygeldanamycin, Alpharadin, Alvocidib, 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone, Amonafϊde, Anthracenedione, Anti-CD22 immunotoxins, Antineoplastic, Antitumorigenic herbs, Apaziquone, Atiprimod, Azathioprine, Belotecan, Bendamustine, BIBW 2992, Biricodar, Brostallicin, Bryostatin, Buthionine sulfoximine, CBV (chemotherapy), Calyculin, cell-cycle nonspecific antineoplastic agents, Dichloroacetic acid, Discodermolide, Elsamitrucin, Enocitabine, Epothilone, Eribulin, Everolimus, Exatecan, Exisulind, Ferruginol, Forodesine, Fosfestrol, ICE chemotherapy regimen, IT-IOl, Imexon, Imiquimod, Indolocarbazole, Irofulven, Laniquidar, Larotaxel, Lenalidomide, Lucanthone, Lurtotecan, Mafosfamide, Mitozolomide, Nafoxidine, Nedaplatin, Olaparib, Ortataxel, PAC-I, Pawpaw, Pixantrone, Proteasome inhibitor, Rebeccamycin, Resiquimod, Rubitecan, SN-38, Salinosporamide A, Sapacitabine, Stanford V, Swainsonine, Talaporfin, Tariquidar, Tegafur-uracil, Temodar, Tesetaxel, Triplatin tetranitrate, Tris(2-chloroethyl)amine, Troxacitabine, Uramustine, Vadimezan, Vinflunine, ZD6126, and Zosuquidar; Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl; Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-Ib; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfm; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride; Taxol; thiosemicarbazone derivatives; telomerase inhibitors; arsenic trioxide; planomycin; sulindac sulfide; cyclopamine; purmorphamine; gamma-secretase inhibitors; CXCR4 inhibitors; HH signaling inhibitors; Bmi-1 inhibitors; Bcl-2 inhibitors; Notch- 1 inhibitors; DNA checkpoint protein inhibitors; ABC transporter inhibitors; mitotic inhibitors; intercalating antibiotics; growth factor inhibitors; cell cycle modulators; enzymes; topoisomerase inhibitors; biological response modifiers; angiogenesis inhibitors; DNA repair inhibitors; and small G-protein inhibitors. Combinations can be made with one or more than one of the above. [00234] Various delivery systems are known and can be used to administer a biologically active agent of the invention, non-limiting examples of which include liposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated endocytosis (see, e.g., Wu and Wu, (1987), J. Biol. Chem. 262:4429-4432), construction of a therapeutic nucleic acid as part of a retroviral or other vector, liquid suspension, solid or semi-solid compositions, and the like. Methods of delivery include but are not limited to intra-arterial, intra-muscular, intravenous, intranasal, subcutaneous, intraperitoneal, intracerobrospinal, intra- articular, intrasynovial, intrathecal, intratumoral, peritumoral, and oral routes. In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, by injection, or by means of a catheter. In certain embodiment, the agents are delivered to a tissue comprising cancerous tissue in the subject. [00235] Administration of the selected agent can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Administration may be tailored to effect one or more of treatment, diagnosis, prognosis, or theranosis of a condition. A fixed dose of the PDZ domain-containing chimeric polypeptide can be in the range from about 1 mg to about 2000 mg. For example, the fixed dose may be approximately 420 mg, approximately 525 mg, approximately 840 mg, or approximately 1050 mg of the chimeric polypeptide. Depending on the type and severity of the disease, an initial candidate dose of PDZ domain-containing chimeric polypeptide can include about 1 μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) for administration to the subject, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage can range, for example, from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired amelioration of disease occurs. The progress of this therapy is easily monitored by conventional techniques and assays.
[00236] The following are non-limiting examples of dosing regimens preferably for use in emodiments of the invention. Dosage regimens may include an initial dose of PDZ domain- containing chimeric polypeptide of 6 mg/kg, 8 mg/kg, or 12 mg/kg delivered by intravenous or subcutaneous infusion, followed by subsequent weekly maintenance doses of 2 mg/kg by intravenous infusion, intravenous bolus injection, subcutaneous infusion, or subcutaneous bolus injection. Where the antibody is well-tolerated by the patient, the time of infusion may be reduced. Alternatively, the invention includes an initial dose of 12 mg/kg PDZ domain- containing chimeric polypeptide, followed by subsequent maintenance doses of 6 mg/kg once per 3 weeks. Another dosage regimen involves an initial dose of 8 mg/kg PDZ domain-containing chimeric polypeptide, followed by 6 mg/kg once per 3 weeks. Still another dosage regimen involves an initial dose of 8 mg/kg PDZ domain- containing chimeric polypeptide, followed by subsequent maintenance doses of 8 mg/kg once per week or 8 mg/kg once every 2 to 3 weeks. As an alternative regimen, initial doses of 4 mg/kg PDZ domain-containing chimeric polypeptide may be administered on each of days 1 , 2 and 3 , followed by subsequent maintenance doses of 6 mg/kg once per 3 weeks. An additional regimen involves an initial dose of 4 mg/kg PDZ domain- containing chimeric polypeptide, followed by subsequent maintenance doses of 2 mg/kg twice per week, wherein the maintenance doses are separated by 3 days. Alternatively, the invention may include a cycle of dosing in which delivery of PDZ domain- containing chimeric polypeptide is 2-3 times per week for 3 weeks. The 3 week cycle is preferably repeated as necessary to achieve suppression of disease symptoms. The invention further includes a cyclic dosage regimen in which delivery of PDZ domain- containing chimeric polypeptide is daily for 5 days. According to the invention, the cycle can be repeated as necessary to achieve amelioration of disease.
[00237] Repeated administrations of the PDZ domain- containing chimeric polypeptide of the invention can also be administered at time intervals indicated based on the non-limiting factors listed above, as can be determined by one skilled in the art. For example, the PDZ domain-containing chimeric polypeptide can be administered every day, every week, every two weeks, every three weeks, every four weeks, or every five weeks. The fixed doses may, for example, continue to be administered until disease progression, adverse event, or other time as determined by a physician is reached. For example, from two, three, or four, up to 20 or more fixed doses may be administered. In one embodiment, one or more loading dose(s) of the binding agent are administered, followed by one or more maintenance dose(s) of the binding agent. In another embodiment, a plurality of the same fixed dose are administered to the subject.
[00238] In another aspect, the present invention provides a pharmaceutical composition comprising the PDZ domain-containing chimeric polypeptide, an anti-cancer therapeutic agent, and a pharmaceutically acceptable carrier. Non-limiting examples of binding agents and anti-cancer therapeutic agents are described above. The preparation of pharmaceutical compositions of this invention is conducted in accordance with generally accepted procedures for the preparation of pharmaceutical preparations. See, for example, Remington's Pharmaceutical Sciences 18th Edition (1990), E.W. Martin ed., Mack Publishing Co., PA. Depending on the intended use and mode of administration, it may be desirable to process the active ingredient further in the preparation of pharmaceutical compositions. Appropriate processing may include mixing with appropriate non-toxic and non-interfering components, sterilizing, dividing into dose units, and enclosing in a delivery device.
[00239] The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
[00240] Pharmaceutical compositions for oral, intranasal, or topical administration can be supplied in solid, semisolid or liquid forms, including tablets, capsules, powders, liquids, and suspensions. Compositions for injection can be supplied as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolution or suspension in liquid prior to injection. For administration via the respiratory tract, a preferred composition is one that provides a solid, powder, or aerosol when used with an appropriate aerosolizer device.
[00241] Liquid pharmaceutically acceptable compositions can, for example, be prepared by dissolving or dispersing a polypeptide embodied herein in a liquid excipient, such as water, saline, aqueous dextrose, glycerol, or ethanol. The composition can also contain other medicinal agents, pharmaceutical agents, adjuvants, carriers, and auxiliary substances such as wetting or emulsifying agents, and pH buffering agents.
[00242] For parenteral administration, the PDZ domain- containing chimeric polypeptide can be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently nontoxic, and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate can also be used. Liposomes may be used as carriers. The vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The antibodies will typically be formulated in such vehicles at concentrations of about 1 mg/ml to 10 mg/ml. [00243] Where desired, the pharmaceutical compositions can be formulated in slow release or sustained release forms, whereby a relatively consistent level of the active compound are provided over an extended period. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
[00244] Therapeutic agents can be delivered as a therapeutic or as a prophylactic (e.g., inhibiting or preventing onset of neurodegenerative diseases). By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. For prophylactic benefit, the agents may be administered to a patient at risk of developing a disease or to a patient reporting one or more of the physiological symptoms of such a disease, even though a diagnosis may not have yet been made. Alternatively, prophylactic administration may be applied to avoid the onset of the physiological symptoms of the underlying disorder, particularly if the symptom manifests cyclically. In this latter embodiment, the therapy is prophylactic with respect to the associated physiological symptoms instead of the underlying indication. The actual amount effective for a particular application will depend, inter alia, on the condition being treated and the route of administration.
[00245] The treatment duration and regimen can vary depending on the particular condition and subject that is to be treated. For instance, a therapeutic agent can be administered by the subject method over at least 1, 7, 14, 30, 60, 90 days, or a period of months, years, or even throughout the lifetime of a subject. Treatment can also be designed to reach a suitable positive outcome, non-limiting examples of which include partial remission, complete remission, a reduction in tumor size, stable tumor size, slowing of tumor growth, reducing the frequency of metastasis, prevention of metastasis for a period exceeding at least 3 months, at least 6 months, at least 9 months or at least 12 months, extension of expected life expectancy, prevention of recurrence of a cancer, extension of the expected time necessary for recurrence of cancer, and reducing the frequency or severity of one or more sequelae of cancer, such as pain, edema, etc.
[00246] In one embodiment, administration of the PDZ domain- containing chimeric polypeptide is combined with the administration of an adjuvant. As used herein, the term "adjuvant" is used to refer to any agent that enhances the response of the immune system. The adjuvant can be administered before, during, or after the administration of the PDZ domain- containing chimeric polypeptide. Adjuvants administered during the administration of the PDZ domain-containing chimeric polypeptide can be co-administered as a single composition and delivery or delivered as part of the same procedure, administered at about the same time in separate administration events. Adjuvants administered before or after administration of the PDZ domain- containing chimeric polypeptide can be administered in time frames preceding or following PDZ domain- containing chimeric polypeptide administration that include the following, without limitation: less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or 22 hours; less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days; less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 weeks; and less than or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 months. Examples of adjuvants that can be used in the methods of the present invention include but are not limited to aluminum compounds; Bacillus Calmette- Guerin (BCG); various toll-like receptor (TLR) stimulants such as CpG, Lipopolysaccharide (LPS), poly-IC; and cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF).
[00247] In one aspect, the present invention provides kits that can be used in the above-described methods. In one embodiment, a kit comprises a composition of the invention, in one or more containers. In yet another embodiment, the invention provides kits comprising a PDZ domain-containing chimeric polypeptide. The binding agents can be, without limitation, any of those described above. The binding agents can further be provided, without limitation, in any of the formulations and/or doses described above. The kits may further comprise additional agents, such as those described above, for use according to the methods of the invention. The agents can be provided in any suitable container, including but not limited to test tubes, vials, flasks, bottles, ampules, syringes, or the like. The agents can be provided in a form that may be directly administered to a subject, or in a form that requires preparation prior to administration, such as in the reconstitution of lyophilized agents. Agents may be provided in aliquots of single-doses or as stocks from which multiple doses may be obtained.
[00248] Prospective patients can be subjected to a diagnostic test prior to receiving therapy so as to identify Lab- positive cancerous tissue. For example, the diagnostic test may evaluate expression of the lab gene (including overexpression) or the abundance of Lab protein. Generally, if a diagnostic test is performed, a sample may be obtained from a patient in need of therapy. Where the subject has cancer, the sample is generally a tumor sample. The biological sample herein may be a fixed sample, e.g., a formalin fixed, paraffin-embedded (FFPE) sample, or a frozen sample. Gene expression can be evaluated using mRNA extracted from a tissue sample using standard methods in the art. mRNA analysis methods include but are not limited to hydridization methods, such as Northern blot analysis, and amplification methods, such as real-time PCR. Determining the protein level typically involves a) contacting the protein contained in a biological sample comprising cancer cells with an agent that specifically binds to Lab protein; and (b) identifying any agentprotein complex so formed. In one aspect of this embodiment, the agent that specifically binds a cancer related protein is an antibody, preferably a monoclonal antibody. Contacting the sample can be performed on cells removed from a subject, as in a biopsy or cell culture, or on cells within the body of a subject.
[00249] The effect of PDZ domain-containing chimeric polypeptide on HPV infection and cervical cancer in a subject can be assessed in a number of ways, taking advantage of means for identifying cervical cancer in a sample. In one embodiment, the effect of an agent on a cancer cell or population of cells comprising cancer cells can be determined by assaying for a difference in the mRNA levels of a target gene between the test cancer cell and a control cell, when they are contacted with a candidate agent. Alternatively, the differential expression of the cancer related gene is determined by detecting a difference in the level of the encoded polypeptide or gene product. Effect over time can also be evaluated by comparing the results of cancer gene expression levels from samples isolated before treatment to samples isolated subsequent to the start of treatment. Genes of interest may be those associated with cancer cells in general, or specific cervical cancers. Reduction of gene expression, either at the nucleotide or protein level, in genes associated with cancer cells can be used as an indication of a corresponding reduction in the presence of the associated cell type within the sample, and a favorable response to treatment. [00250] To assay for an agent- induced alteration in the level of mRNA transcripts or corresponding polynucleotides, nucleic acid contained in a biological sample comprising cancer cells is first extracted according to standard methods in the art. For instance, mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. (1989), supra, or extracted by nucleic-acid-binding resins following the accompanying instructions provided by the manufacturers. The mRNA contained in the extracted nucleic acid sample is then detected by amplification procedures or conventional hybridization assays (e.g. Northern blot analysis) according to methods widely known in the art or based on the methods exemplified herein. [00251] For the purpose of this invention, amplification means any method employing a primer and a polymerase capable of replicating a target sequence with reasonable fidelity. Amplification may be carried out by natural or recombinant DNA polymerases such as TaqGold™, T7 DNA polymerase, Klenow fragment of E.coli DNA polymerase, and reverse transcriptase. A preferred amplification method is PCR. In particular, the isolated RNA can be subjected to a reverse transcription assay that is coupled with a quantitative polymerase chain reaction (RT- PCR) in order to quantify the expression level of a cancer related gene.
[00252] Detection of the gene expression level can be conducted in real time in an amplification assay. In one aspect, the amplified products can be directly visualized with fluorescent DNA-binding agents including but not limited to DNA intercalators and DNA groove binders. Because the amount of the intercalators incorporated into the double-stranded DNA molecules is typically proportional to the amount of the amplified DNA products, one can conveniently determine the amount of the amplified products by quantifying the fluorescence of the intercalated dye using conventional optical systems in the art. DNA-binding dye suitable for this application include SYBR green, SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidium bromide, acridines, proflavine, acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyls, anthramycin, and the like.
[00253] In another aspect, other fluorescent labels such as sequence specific probes can be employed in the amplification reaction to facilitate the detection and quantification of the amplified products. Probe-based quantitative amplification relies on the sequence-specific detection of a desired amplified product. It utilizes fluorescent, target-specific probes (e.g., TaqMan® probes) resulting in increased specificity and sensitivity. Methods for performing probe-based quantitative amplification are well established in the art and are taught in U.S. Patent No. 5,210,015.
[00254] In yet another aspect, conventional hybridization assays using hybridization probes that share sequence homology with cancer related genes can be performed. Typically, probes are allowed to form stable complexes with the target polynucleotides (e.g., cancer related genes) contained within the biological sample derived from the test subject in a hybridization reaction. It will be appreciated by one of skill in the art that where antisense is used as the probe nucleic acid, the target polynucleotides provided in the sample are chosen to be complementary to sequences of the antisense nucleic acids. Conversely, where the nucleotide probe is a sense nucleic acid, the target polynucleotide is selected to be complementary to sequences of the sense nucleic acid. [00255] As is known to one skilled in the art, hybridization can be performed under conditions of various stringencies. Suitable hybridization conditions for the practice of the present invention are such that the recognition interaction between the probe and target cancer related gene is both sufficiently specific and sufficiently stable. Conditions that increase the stringency of a hybridization reaction are widely known and published in the art. See, for example, (Sambrook, et al., (1989), supra; Nonradioactive In Situ Hybridization Application Manual, Boehringer Mannheim, second edition). The hybridization assay can be formed using probes immobilized on any solid support, including but are not limited to nitrocellulose, glass, silicon, and a variety of gene arrays. A preferred hybridization assay is conducted on high-density gene chips as described in U.S. Patent No. 5,445,934. [00256] For a convenient detection of the probe-target complexes formed during the hybridization assay, the nucleotide probes are conjugated to a detectable label. Detectable labels suitable for use in the present invention include any composition detectable by photochemical, biochemical, spectroscopic, immunochemical, electrical, optical, or chemical means. A wide variety of appropriate detectable labels are known in the art, which include fluorescent or chemiluminescent labels, radioactive isotope labels, enzymatic or other ligands. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as digoxigenin, β- galactosidase, urease, alkaline phosphatase or peroxidase, avidin/biotin complex.
[00257] The detection methods used to detect or quantify the hybridization intensity will typically depend upon the label selected above. For example, radiolabels may be detected using photographic film or a phosphoimager. Fluorescent markers may be detected and quantified using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and measuring the reaction product produced by the action of the enzyme on the substrate; and finally colorimetric labels are detected by simply visualizing the colored label.
[00258] An agent- induced change in expression of cancer related genes in a cell or cell population can also be determined by examining the corresponding gene products. Determining the protein level typically involves a) contacting the protein contained in a biological sample comprising cancer cells with an agent that specifically bind to the cancer related protein; and (b) identifying any agent:protein complex so formed. In one aspect of this embodiment, the agent that specifically binds a cancer related protein is an antibody, as in a biopsy or cell culture, or on cells within the body of a subject.
[00259] The reaction is performed by contacting the agent with a sample of the cancer related proteins derived from the test samples under conditions that will allow a complex to form between the agent and the cancer related proteins. The formation of the complex can be detected directly or indirectly according to standard procedures in the art. In the direct detection method, the agents are supplied with a detectable label and unreacted agents may be removed from the complex; the amount of remaining label thereby indicating the amount of complex formed. For such method, it is preferable to select labels that remain attached to the agents even during stringent washing conditions. It is preferable that the label does not interfere with the binding reaction. In the alternative, an indirect detection procedure requires the agent to contain a label introduced either chemically or enzymatic ally. A desirable label generally does not interfere with binding or the stability of the resulting agent:polypeptide complex. However, the label is typically designed to be accessible to an antibody for an effective binding and hence generating a detectable signal. [00260] A wide variety of labels suitable for detecting protein levels are known in the art. Non-limiting examples include radioisotopes, enzymes, colloidal metals, fluorescent compounds, bioluminescent compounds, and chemiluminescent compounds.
[00261] The amount of agent:polypeptide complexes formed during the binding reaction can be quantified by standard quantitative assays. As illustrated above, the formation of agent:polypeptide complex can be measured directly by the amount of label remained at the site of binding. In an alternative, the cancer related protein is tested for its ability to compete with a labeled analog for binding sites on the specific agent. In this competitive assay, the amount of label captured is inversely proportional to the amount of cancer related protein present in a test sample. [00262] A number of techniques for protein analysis based on the general principles outlined above are available in the art. They include but are not limited to radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunofluorescent assays, and SDS-PAGE. [00263] Antibodies that specifically recognize or bind to cancer related proteins are preferable for conducting the aforementioned protein analyses. Where desired, antibodies that recognize a specific type of post-translational modifications can be used. Post-translational modifications include but are not limited to glycosylation, lipidation, acetylation, and phosphorylation. These antibodies may be purchased from commercial vendors. Alternatively, these antibodies can be generated using conventional polyclonal or monoclonal antibody technologies by immunizing a host animal or an antibody-producing cell with a target protein that exhibits the desired post- translational modification.
[00264] In practicing the subject method, it may be desirable to discern the expression pattern of cancer related protein in different bodily tissue, in different cell types, and/or in different subcellular structures. These studies can be performed with the use of tissue-specific, cell-specific or subcellular structure specific antibodies capable of binding to protein markers that are preferentially expressed in certain tissues, cell types, or subcellular structures. To detect and quantify the immunospecific binding, digital image systems including but not limited to confocal microscope can be employed. XXl. Utility
[00265] The compositions and methods described hereinabove are readily employed in a variety of research and diagnostic methods, including methods of diagnosing a particular disease or condition, or infection by an infections agent, such as a virus or bacteria. In one embodiment, the method is employed as part of a diagnostic for detecting HPV infected cells. Since the presence of oncogenic strains of HPV is associated with cancerous and pre-cancerous cells, the instant methods may be employed to detect cancerous or pre-cancerous cervical cells. [00266] HPV is known to be a causative agent in the following diseases: epidermodysplasia verruciformis (EV), a lifelong skin disorder that results in high risk for skin (e.g., squamocellar) cancer; cervical neoplasias such as cervical intraepithelial neoplasia (CIN) and invasive cervical carcinoma (ICC); viginal neoplasias such as vaginal intraepithelial neoplasia (VAIN) and vaginal carcinoma (VC); vulval neoplasias such as vulvar intraepithelial neoplasia (VIN) and vulvar carcinoma; penile carcinoma (including Bowenoid papulosis); anal (AC) and perianal carcinomas (PC); oropharyngeal carcinomas (OS); esophageal carcinomas (EC); non-melanoma skin cancers (e.g., basal cell carcinoma-BCC and squamous cell carcinoma-SCC); and melanoma. As such, in one embodiment, the instant methods may be employed as a diagnostic for any of these diseases. [00267] In one embodiment, cells are obtained (e.g., exfoliated or dissected) from a subject and deposited into a liquid medium containing a fixative that, in certain embodiments, may be a transport medium for cytological test. The cells are usually obtained in doctor's office or clinic, the cellular sample is forwarded to and received by a testing facility in which the above-recited protein detection methods and, optionally, cytology assays are performed. Results from the testing are communicated to the subject, in some embodiments via the doctor and an associate thereof.
[00268] The subject from which cells are employed may be a mammal, e.g., a dog or cat, a rodent (e.g., mouse, guinea pig, or rat), or primate (e.g., a human, chimpanzee, or monkey). In many embodiments, the subject will be a human, particularly a male or female. In certain embodiments, the subject may show symptoms of HPV infection (e.g., may have warts on one or more parts of the body), may be suspected of being infected by HPV (e.g., may contain cells that are cytologically consistent with such an infection) or may have already tested positive for HPV. In certain embodiments, the subject may have no indication of HPV infection, and the above methods may be employed as part of a routine screen.
[00269] In one embodiment, the instant methods may be employed to detect any strain of oncogenic HPV, e.g., HPV 26, HPV 53, HPV 66, HPV 73, HPV 82, HPV 16, HPV 18, HPV 31, HPV 35, HPV 30, HPV 39, HPV 45, HPV 51, HPV 52, HPV 56, HPV 59, HPV 58, HPV 33, HPV 66, HPV 68 or HPV 69, (particularly any of the most prevalent HPV strains, e.g., HPV 16, HPV 18, HPV 31, HPV 33 and HPV 45) by detecting the E6 protein from that strain. In one embodiment, at the point of initiating the instant methods, it is not known if the fixed cells contain oncogenic E6 protein or which strain an oncogenic E6 protein is from. If a detection assay indicates the presence of an oncogenic E6 protein in fixed cells, then the identity of the strain of HPV that infected those cells can be determined by other molecular assays, e.g., those that employ antibodies specific to a particular E6 protein or other protein encoded by the virus, or by sequencing viral DNA. [00270] The following examples are offered by way of illustration and not by way of limitation.
EXAMPLES Example 1
Generation of PSD95-MAGI-1 chimeric PDZ domain-containing polypeptide
[00271] As described herein, a chimeric PSD95-MAGI-1 chimeric PDZ domain- containing polypeptide was generated, in which all three PDZ domains of PSD95 are substituted with the PDZ domain 1 of MAGI-I. The methods of generating chimeric polynucleotide and chimeric polypeptide are well known in the art (see, e.g., Maniatis et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N. Y.). The amino acid sequence of this chimeric polypeptide is shown below.
[00272] PRGSPSELKGKFIHTKLRKSSRGFGFTVVGGDEPDEFLQIKSLVLDGPAALDGKMETGDVIVSVND TCVLGHTHAQWKIFQSIPIGASVDLELCRGYPPAEKFIHTKLRKSSRGFGFTWGGDEPDEFLQIKSLVLDG PAALDGKMETGDVIVSVNDTCVLGHTHAQVVKIFQSIPIGASVDLELCRGYPSNAYLSDSYAPPDITTSYSQ HLDNEISHSSYLGTDYPTAMTPTSPRRYSPVAKDLLGEEDIPKGKFIHTKLRKSSRGFGFTWGGDEPDEFL QIKSLVLDGPAALDGKMETGDVIVSVNDTCVLGHTHAQVVKIFQSIPIGASVDLELCRGYPLPFDPD Example 2
Generation of a chimeric PDZ domain-containing polypeptide with one PDZ domain substitution [00273] The chimeric PDZ domain- containing polypeptide may contain only one introduced PDZ domain from the first PDZ domain-containing polypeptide. A single MAGI-I PDZ domain 1 is introduced into PSD95 polypeptide and the MAGI-I PDZ domain insert is embedded in certain adjacent sequences of PSD95 such that the conformation of the chimeric polypeptide allows for higher binding of the chimeric polypeptide to a PDZ domain as compared to the native PSD95 polypeptide. The methods of generating the chimeric polypeptide are well known in the art. Example 3
Generation of PSD95-TIP-1 chimeric PDZ domain-containing polypeptide
[00274] This example provides a chimeric polypeptide in which two of the three PDZ domains in PSD95 are substituted with a PDZ domain from the first PDZ domain containing polypeptide TIP- 1. Two TIP- 1 PDZ domains are introduced into PSD95 polypeptide and each of the TIP-I PDZ domain inserts is embedded in certain adjacent sequences of PSD95 such that the conformation of the chimeric polypeptide allows for higher binding of the chimeric polypeptide to a PDZ domain as compared to the native PSD95 polypeptide. The chimeric PSD95-TIP-1 polypeptide exhibits enhanced binding strength to HPVl 6-E6 than a native PSD95 polypeptide in a capture ELISA. Example 4
Generation of Eukaryotic Expression Constructs Bearing DNA Fragments that Encode a Chimeric PDZ
Domain Containing Polypeptide or Portions of a Chimeric PDZ Domain Containing Polypeptide [00275] This example describes the cloning of chimeric PDZ domain containing genes or portions of chimeric PDZ domain containing genes are into eukaryotic expression vectors in fusion with a number of protein tags, including but not limited to Glutathione S-Transferase (GST), Enhanced Green Fluorescent Protein (EGFP), or Hemagglutinin (HA). A. Strategy
[00276] DNA fragments corresponding to chimeric PDZ domain containing genes are generated by RT-PCR from RNA from a library of individual cell lines (CLONTECH Cat#K4000-l) derived RNA, using random (oligonucleotide) primers (Invitrogen Cat.#48190011). DNA fragments corresponding to chimeric PDZ domain containing genes or portions of chimeric PDZ domain containing genes are generated by standard PCR, using above purified cDNA fragments and specific primers (see Table 5). Primers used are designed to create restriction nuclease recognition sites at the PCR fragment's ends, to allow cloning of those fragments into appropriate expression vectors. Subsequent to PCR, DNA samples are submitted to agarose gel electrophoresis. Bands corresponding to the expected size are excised. DNA was extracted by Sephaglas Band Prep Kit (Amersham Pharmacia Cat#27-9285-01) and digested with appropriate restriction endonuclease. Digested DNA samples are purified once more by gel electrophoresis, according to the same protocol used above. Purified DNA fragments are coprecipitated and ligated with the appropriate linearized vector. After transformation into E. coli, bacterial colonies are screened by colony PCR and restriction digest for the presence and correct orientation of insert. Positive clones are innoculated in liquid culture for large scale DNA purification. The insert and flanking vector sites from the purified plasmid DNA are sequenced to ensure correct sequence of fragments and junctions between the vectors and fusion proteins.
B. Vectors:
[00277] All PDZ domain- containing genes are cloned into the vector pGEX-3X (Amersham Pharmacia #27-4803- 01, Genemed Acc#U13852, GI#595717), containing a tac promoter, GST, Factor Xa, β-lactamase, and lac repressor. [00278] The amino acid sequence of the pGEX-3X coding region including GST, Factor Xa, and the multiple cloning site is listed below. Note that linker sequences between the cloned inserts and GST-Factor Xa vary depending on the restriction endonuclease used for cloning. Amino acids in the translated region below that may change depending on the insertion used are indicated in small caps, and are included as changed in the construct sequence listed in (C).
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGL EFPNLPYYIDGDVKLTQSMAIIRYIADKHWLGGCPKERAEISMLEGAVLD IRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHP DFMLYDALDWLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAW PLQGWQATFGGGDHPPKSDLIEGRgipgnss
[00279] In addition, TAX Interacting Protein 1 (TIPl), in whole or part, was cloned into many other expression vectors, including but not limited to CD5.gamma., PEAKlO (both provided by the laboratory of Dr. Brian Seed at Harvard University and generated by recombinant DNA technology, containing an IgG region), and MIN (a derivative of MSCV, containing IRES and NGFR, generated by recombinant DNA technology).
C. Constructs:
[00280] Primers used to generate DNA fragments by PCR are listed in Table 5. PCR primer combinations and restriction sites for insert and vector are listed below, along with amino acid translation for insert and restriction sites. Non-native amino acid sequences are shown in lower case. TABLE 5
Primers used in cloning of DLG 1 (domain 2 of 3), MAGI 1 (domain 2 of 6), and TIP 1 into representative expression vectors.
ID# (Primer Seq Name) Primer Sequence Description ID
1928 AATGGGGATCCAGC Forward (5' to 3') primer corresponding to DLG 1, 273 (654DL12F) TCATTAAGG domain 2 of 3. Generates a Bam Hl site upstream (5T) of the PDZ boundary. Used for cloning into pGEX-3X.
1453 CACGGATCCCTTTCTG Forward (5' to 3') primer corresponding to MAGI 1, 275 (43SBAF) AGTTGAAGGC domain 2 of 6. Generates a BamHl site upstream (5T) of the PDZ boundary. Used for cloning into pGEX-3X.
1454 TATGAATTCCATCTG Reverse (3' to 5') primer corresponding to MAGI 1, 276 (436BAR) GATCAAAAGGCAAT domain 2 of 6. Generates an EcoRl site G downstream (3T) of the PDZ boundary. Used for cloning into pGEX-3X.
399 CAGGGATCCAAAGA Forward (5' to 3') primer corresponding to TIPl. 277 (86TAF) GTTGAAATTCACAA Generates a Bam Hl site upstream (5T) of the PDZ GC boundary. Used for cloning into pGEX-3X.
400 ACGGAATTCTGCAG Reverse (3' to 5') primer corresponding to TIPl. 278 (87TAR) CGACTGCCGCGTC Generates an EcoRl site downstream (3T) of the PDZ boundary. Used for cloning into pGEX-3X.
1319 AGGATCCAGATGTC Forward (5' to 3') primer corresponding to TIPl. 279 (TIPG5-1) CTACATCCC Generates a Bam Hl site upstream (5T) of the start codon. Used for cloning into pGEX-3X.
1320 GGAATTCATGGACT Reverse (3' to 5') primer corresponding to TIPl. 280 (TIPG3-1) GCTGCACGG Generates an EcoRl site downstsream (3T) of the stop codon. Used for cloning into pGEX-3X.
2753 AGAGAATTCTCGAG Forward (5' to 3') primer corresponding to TIPl. 281 (1109TIF) ATGTCCTACATCCC Generates an EcoRl site upstream (5T) of the start codon. Used for cloning into MN.
2762 TGGGAATTCCTAGG Reverse (3' to 5') primer corresponding to TIPl. 282 (1117TIR) ACAGCATGGACTG Generates an EcoRl site downstream (3T) of the stop codon. Used for cloning into MN.
2584 CTAGGATCCGGGCC Forward (5' to 3') primer corresponding to TIPl. 283 (1080TIF) AGCCCGGTCACC Generates a Bam Hl site upstream (5T) of the PDZ boundary. Used for cloning into PEAKlO or CD5y.
2585 GACGGATCCCCCTG Reverse (3' to 5') primer corresponding to TIPl. 284 (1081TIR) CTGCACGGCCTTCTG Generates a Bam Hl site downstream (3T) of the PDZ boundary. Used for cloning into PEAKlO or CD5y.
2586 GACGAATTCCCCTG Reverse (3' to 5') primer corresponding to TIPl. 285 (1082TIR) CTGCACGGCCTTCTG Generates an EcoRl site downstream (3T) of the PDZ boundary. Used for cloning into PEAKlO or CD5y.
2587 CTAGAATTCGGGCC Forward (5' to 3') primer corresponding to TIPl. 286 (1083TIF) AGCCGGTCACC Generates an Eco Rl site upstream (5T) of the PDZ boundary. Used for cloning into PEAKlO or CD5y.
[00281] 1. DLG 1, PDZ domain 2 of 3: Acc#:U13897 GI#:558437 Construct: DLG 1, PDZ domain 2 of 3-pGEX- 3X, Primers: 1928 & 1929, Vector Cloning Sites(573T): Bam Hl/EcoRl, Insert Cloning Sites(573T): BamHl/EcoRl aa 1-aa 88 (SEQ ID NO: 287) giqLIKGPKGLGFSIAGGVGNQHIPGDNSIYVTKIIEGGAAHKDGKLQIG DKLLAVNNVCLEEVTHEEAVTALKNTSDFVYLKVAnss [00282] 2. MAGI 1. Acc#:AB010894 GI#:3370997 Construct: MAGI 1, PDZ domain 2 of 6-pGEX-3X Primers:
1453 & 1454, Vector Cloning Sites(573T): Bam Hl/EcoRl, Insert Cloning Sites(5'/3T): BamHl/EcoRl aa 1-aa 108 (SEQ ID NO: 288) giPSELKGKFIHTKLRKSSRGFGFTWGGDEPDEFLQIKSLVLDGPAALD
GKMETGDVIVSVNDTCVLGHTHAQWKIFQSIPIGASVDLELCRGYPLPF DPDgihrd
[00283] 3. TAX Interacting Protein 1 (TIPl): Acc#:AF028823.2 GI#: 11908159 Construct: TIP 1, PDZ domain 1 of l-pGEX-3X, Primers: 399& 400, Vector Cloning Sites(573T): Bam Hl/EcoRl, Insert Cloning Sites(573T):
BamHl/EcoRl aa 1-aa 107 (SEQ ID NO: 289) giQRVEIHKLRQGENLILGFSIGGGIDQDP SQNPF SEDKTDKGIYVTRVS
EGGPAEIAGLQIGDKIMQVNGWDMTMVTHDQARKRLTKRSEEWRLLVT RQSLQnss
[00284] Construct: TIP l-pGEX-3X, Primers: 1319& 1320,Vector Cloning Sites(5'/3T): Bam Hl/EcoRl, Insert
Cloning Sites(573T): BamHl/EcoRl aa 1-aa 128 (SEQ ID NO: 290) giqMSYIPGQPVTAWQRVEIHKLRQGENLILGFSIGGGIDQDPSQNPFS
EDKTDKGIYVTRVSEGGPAEIAGLQIGDKIMQVNGWDMTMVTHDQARKRL
TKRSEEWRLLVTRQSLQKAVQQSMnss
[00285] Construct: TIPl-MIN, Primers: 2753& 2762, Vector Cloning Sites(5'/3T): EcoRl/EcoRl, Insert Cloning
Sites(5'/3T): EcoRl/EcoRl aa 1-aa 129 (SEQ ID NO: 291) agilEMSYIPGQPVTAWQRVEIHKLRQGENLILGFSIGGGIDQDPSQNP
FSEDKTDKGIYVTRVSEGGPAEIAGLQIGDKIMQVNGWDMTMVTHDQARK IRLTKRSEEWRLLVTRQSLQKAVQQSMLS
[00286] Construct: TIP 1-CD5.gamma. Primers: 2584& 2585, Vector Cloning Sites(5'/3T): Bam Hl/Bam Hl, Insert
Cloning Sites(5'/3T): BamHl/Bam Hl aa 1-aa 122 (SEQ ID NO: 292) adPGQPVTAWQRVEIHKLRQGENLILGFSIGGGIDQDPSQNPFSEDKTD
KGIYVTRVSEGGPAEIAGLQIGDKIMQVNGWDMTMVTHDQARKRLTKRSE
EWRLLVTRQSLQKAVQQSdpe
D. GST Fusion Protein Production and Purification
[00287] The constructs using pGEX-3X expression vector are used to make fusion proteins according to the protocol outlined in the GST Fusion System, Second Edition, Revision 2, Pharmacia Biotech. Method II and was optimized for a IL LgPP.
[00288] Purified DNA was transformed into E. coli and allowed to grow to an OD600 of 0.4-0.8 (600λ). Protein expression was induced for 1-2 hours by addition of IPTG to cell culture. Cells are harvested and lysed. Lysate was collected and GS4B beads (Pharmacia Cat#17-0756-01) are added to bind GST fusion proteins. Beads are isolated and GST fusion proteins are eluted with GEB II. Purified proteins are stored in GEB II at -80C. Purified proteins are used for ELISA-based assays and antibody production.
E. IgG Fusion Protein Production and Purification
[00289] The constructs using the CD5 gamma or PeaklOIgG expression vectors are used to make fusion protein. Purified DNA vectors are transfected into 293 EBNA T cells under standard growth conditions (DMEM +10% FCS) using standard calcium phosphate precipitation methods (Sambrook, Fritsch and Maniatis, Cold Spring Harbor Press) at a ratio of about 1 μg vector DNA for 1 million cells. This vector results in a fusion protein that is secreted into the growth medium. Transiently transfected cells are tested for peak expression, and growth media containing fusion protein is collected at that maximum (usually 1-2 days). Fusion proteins are either purified using Protein A chromatography or frozen directly in the growth media without addition. EXAMPLE 5
DETERMINATIONS OF PDZ DOMAIN BINDING STRENGTH TO HPVl 6 E6
[00290] Using the sandwich ELISA and lateral flow assay described above, the PSD95-MAGI-1 chimeric PDZ domain-containing polypeptide and a single MAGI-I PDZ domain are tested to determine their relative binding strength to the PL of the HPV16 E6 protein. Peptide corresponding to the PDZ ligand (PL) of HPV16 E6 was titrated against a constant amount of the PSD95-MAGI-1 chimeric PDZ domain- containing polypeptide or the single MAGI-I PDZ domain, and the results are shown in FIGS 3 and 4. These results demonstrate that the PSD95-MAGI- 1 chimeric PDZ domain- containing polypeptide has enhanced binding of HPV16-E6 protein over the single MAGI- 1 PDZ domain, suggesting that the PSD95-MAGI-1 chimeric PDZ domain- containing polypeptide is a more sensitive capture/detection agent for oncogenic HPV infections. EXAMPLE 6
Pathogen Proteins
[00291] Many other proteins from pathogens can be detected using proteins or compounds directed at detection of a PDZ:PL interaction. Table 6 contains some exemplary proteins that could be detected using technology disclosed herein, but is not meant to be limiting in any manner.
TABLE 6 Example Pathogens amenable to PDZ: PL diagnostics
Gi or ACC PL/ Pathogen Protein number PDZ
Adenovirus E4 19263371 PL
Hepatitis B Protein X 1175046 PL virus
Human T TAX 6983836 PL
Cell Leukemia
Virus
Herpesvirus DNA polymerase 18307584 PL
Herpesvirus US2 9629443 PL
EXAMPLE 7
Quantification of Endogenous E6 Protein in Cells Infected with HPVl 6
A) Abstract:
[00292] Experiments are designed and performed to determine quantities of endogenous E6 protein in HPV 16 infected cervical cancer cell lines. Results demonstrate that HPVl 6 infected cervical cancer cell lines contain in the order of 10,000 to 100,000 molecules E6. From this finding is concluded, that E6 protein can be used as a diagnostic or prognostic marker for cellular HPV infection. Use of protein degradation pathway inhibitors may facilitate such an assay.
B) Methods:
Immunoprecipitation of E6 protein:
[00293] HPV16-infected cervical cancer cell lines SiHa and CasKi are washed with cold PBS and resuspended in HEPES lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, 0.5% triton X-100, 1 mg/ml BSA, one pellet protease inhibitor cocktail (Roche), and 1 mM PMSF) at 2 xlO7 cells/ml. Lysis proceeds on ice for 30 min. and lysates are cleared by centrifugation at 14,00Ox g for 5 minutes at 40C. E6 proteins are immunoprecipitated with a mouse anti-E6 antibody (clone 6F4) and protein G beads (Pharmacia, Piscataway, N.J.). After 2 hours incubation at 40C. with rotation, beads are washed 3 times with washing Buffer [50 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, protease inhibitor cocktail (CALBIOCHEM), 1 mM PMSF]. Pellets are resuspended in SDS-PAGE sample buffer and analyzed by immuno blotting using 6F4 anti-E6 antibody and anti-mouse-IgG- HRP conjugated (Jackson Immuno Research).
Detection of E6 Protein from Cervical Cancer Cell Lysates by Western Technology:
[00294] SiHa and CasKi cervical cancer cell lines are lysed at 2xlO7 cells/ml in lysis buffer 30 min. on ice. Lysates corresponding to approx. 106 cells are immediately resolved on a 12% SDS-PAGE gel followed by transfer to a PVDF membrane. E6 proteins are detected with 6F4 anti-E6 HPVl 6 antibody and anti-mouse-IgG-HRP conjugated (Jackson Immuno Research). C) Results:
[00295] To determine the apparent molecular weight of endogenous E6 protein as present in cervical cancer cells upon infection with HPVl 6 and to ensure that an anti-E6 monoclonal antibody-specific band seen in PAGE represents viral E6 protein, 293 EBNA-T cells are transfected with a construct expressing untagged E6 protein of HPV type 16. Cell lysates are prepared of those cells, and HPV infected SiHa cervical cancer cells. E6 protein from both lysates (transfected and HPV infected) was immunoprecipitated by use of an anti E6-specific monoclonal antibody. Both lysates are analyzed side by side using PAGE technology. The E6-specific band obtained for transfected E6 migrates in PAGE at the same level as the anti E6 antibody specific band from SiHa cervical cancer cell lines, thus most strongly suggesting that the product immunoprecipitated with anti E6-specific monoclonal antibody represent viral E6 protein. Using the specific E6 monoclonal antibody, a band of the same size was detected in HPVl 6 infected cervical cancer cell type CasKi.
[00296] In a different experimental procedure, endogenous viral E6 protein of HPV 16 infected cervical cancer cell line SiHa and CasKi was directly detected from their cell lysates. Bands that are dependent on E6-specific monoclonal antibody ran in the same way as the band for cells transfected with E6 encoding vector. [00297] To test whether E6 in vivo stability can be enhanced by selectively blocking proteasome involved in protein degradation, cell lysates of some samples are treated with proteasome inhibitor MGl 32. In those samples, the E6 specific band is about 2-3 times more intense. This demonstrates, that addition of an appropriate mixture of protein degradation pathway inhibiting agents can be used to increase the signal specific to E6 protein by augmenting its accumulation temporarily in cells.
[00298] Quantities of E6 protein in lysates are measured by comparing E6-Specific signal in PAGE with signals obtained by MBP-E6 (HPVl 6) fusion protein loaded onto the same gel. In some cases, MBP-E6 fusion protein was digested with factor X to release the E6 portion only. Signal intensity comparison studies demonstrated, that cervical cancer derived cell lines injected with HPV16 (SiHa, CasKi) contain E6 at a concentration of 0.3 to 3 ng per IxIO6 cells. It is concluded, that quantities and stability of E6 are such that detection by an E6-specific (ELISA-) assay will be feasible. EXAMPLE 8
Chimeric PDZ Domain Containing Polypeptides Bind Selectively Endogenous HPV6E6 Proteins Present in
Cell Lysates and can be used to Separate Endogenous E6 Protein from Other Components Present in a Cell
Lysate A) Abstract
[00299] Experiments are undertaken to test, whether chimeric PDZ domain containing polypeptides will selectively bind endogenous E6 of cells transfected with E6 encoding vector. Moreover, it is tested whether the chimeric PDZ domain containing polypeptides can be used to separate E6 from other molecules in the cell lysate subsequent to binding. Findings demonstrate that chimeric PDZ domain containing polypeptide is selective and can be applied to separate E6 protein from the complex mixture of cell lysate molecules.
B) Methods
Pull Down of E6 Protein with GST-chimeric PDZ Proteins:
[00300] Fusion protein of GST and the chimeric PSD95-MAGI-1 PDZ domain containing polypeptide is tested in pull down experiments. Briefly, 10 μg recombinant GST-chimeric PDZ protein is incubated with 30 μl of glutathione-sepharose beads in 1 ml of buffer [50 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, 0.1% Triton X- 100, protease inhibitor cocktail, 1 mM PMSF] for 1 h at 40C with rotation. Subsequently, cell lysates of 107 293 cells transiently transfected with either pMKit-HA-HPV16-E6 or pMKit-HA vector alone are incubated with the beads bound to the fusion PDZ proteins for 3 h at 40C with rotation. Beads are washed and analyzed in 12% SDS- PAGE gel electrophoresis followed by Western blotting. Membranes are probed with biotin conjugated anti-HA antibodies (clones 3F10, or 12CA5, Boebringer Mannheim) and HRP-Streptavidin (Zymed). [00301] Alternatively, cell lysates from 293 cells transiently transfected with pmKit-HA, pmkit-HPV16-HA-E6 or pmKit-HA-HPV16 E6-delta-PL, are incubated with recombinant GST-chimeric PDZ protein and immobilized on glutathione-sepharose beads and bound fractions are immunoblotted with anti HA antibodies. In parallel, lysates are immunoprecipitated and detected with anti-HA antibodies.
C) Results
[00302] In a "pull down" experiment, different PDZ domains or PDZ domain containing proteins, for example, a native PSD95, a native MAGI-I, a single MAGI-I PDZ domain 1, and a single PSD95 PDZ domain 2, are tested for pull down of endogenous over expressed E6 from cell lysate. Lysates of cells transfected with HA-tagged E6 HPV- 16 are incubated with GST- chimeric PSD95-MAGI-1 protein and the other GST fusion proteins representing the above PDZ domains bound to Sepharose beads. Control cell samples are transfected with HA expressing constructs. Detection with anti HA monoclonal antibody demonstrates that E6 is selectively pulled out of cell lysates via the PDZ domain represented by the oncogenic E6-PL-detector of all GST-PDZ proteins tested (native PSD95, native MAGI-I, single MAGI-I PDZ domain 1, single PSD95 PDZ domain 2). Results demonstrate that the GST fusion protein containing the chimeric PSD95-MAGI-1 polypeptide associates with HA-E6 but not with HA- E6.DELTA.PL (lacking the 3 C-terminal PDZ domain binding amino acids). In addition, the GST fusion protein containing the chimeric PSD95-MAGI-1 polypeptide binds E6 PL with the highest binding strength than the other PDZ domains tested, i.e. native PSD95, native MAGI-I, single MAGI-I PDZ domain 1, and single PSD95 PDZ domain 2. This method can be used to determine whether a chimeric PDZ domain containing polypeptide has the capacity of specific E6 binding and the relative binding strength to E6 protein. The conclusion is made, that competition by potentially PDZ binding proteins represented by the complex mixture of cell lysates and E6 for binding to PDZs can be shifted towards selective binding of E6 by appropriate choice of the specific PDZ domain that constitutes the chimeric PDZ domain containing polypeptide described herein. EXAMPLE 9
Endogenous E6 Protein of HPV Infected Cervical Cancer Cell Lines can be Detected in a Sandwich ELISA Via a Chimeric PDZ Domain Containing Polypeptide
A) Abstract:
[00303] Experiments are described, in which a chimeric PDZ domain containing polypeptide is used to selectively detect presence of E6 protein in HPV infected cells via a sandwich ELISA. The specific capturing of oncogenic E6 but not non-oncogenic E6 demonstrates that the chimeric PDZ domain containing polypeptide can be applied for a E6 detection based diagnostic test for HPV infection and/or cervical cancer test.
B) Methods:
[00304] Sandwich type 1 ELISA: Anti-E6 antibody is coated onto a 96-well Polysorp or Maxysorp ELISA plate at 5 μg/ml in PBS (100 μl/well) overnight at 40C. Plates are washed with PBS and blocked with 200 μl PBS/2% BSA for 2 hours at 4 C. Cell lysates diluted in PBS/2% BSA are added and incubated at room temperature for 1 hour. After 3 washes with PBS, 100 μl of a chimeric PDZ domain containing polypeptide (for example a chimeric PSD95 substituted with MAGI- 1 PDZ domain 1 or the same chimeric polypeptide fused to GST) at 5 μg/ml is added in PBS/2% BSA, and plates are incubated at room temperature for 45 min. Plates are then washed 3 times with PBS and incubated with anti-hlgG-HRP (Jackson Immuno Research) or anti-GST-HRP (Pharmacia) at the appropriate concentration in PBS/2% BSA at room temperature for 45 minutes. After 5 washes with 50 mM Tris/0.2% Tween- 20, plates are incubated with 100 μl /well TMB substrate (Dako Industries). The colorimetric reaction is stopped at appropriate times (usually after 20 minutes) by addition of 100 μl of 0.1 M H2SO4 and plates read at A450 nm in an ELISA plate reader.
[00305] In a variant of sandwich 1 ELISA, cell lysates are preincubated with a chimeric PDZ domain containing polypeptide at 2.5-5 ug/ml final concentration, for 1-2 hours at 40C, prior to adding to the anti-E6 antibody coated plate.
[00306] Sandwich type 2 ELISA: In sandwich 2, reagents and procedures mostly correspond to those used in sandwich 1. In contrast to sandwich 1, 100 μl of a chimeric PDZ domain containing polypeptide is coated onto the ELISA plate and the anti-E6 antibody is used for detection of the chimeric PDZ domain containing polypeptide - bound E6, followed by anti-mouse IgG-HRP (Jackson Immuno Research). In a modified version of sandwich 2, biotinylated reagents (anti-E6 antibody or the chimeric PDZ domain containing polypeptide) will be used followed by streptavidin-HRP to further diminish background and to increase sensitivity.
C) Results:
[00307] A sandwich ELISA is conceived in two different variations. In Type 1 sandwich ELISA, E6 protein present in cell lysates in captured by E6-specific monoclonal antibody, and detection of specifically oncogenic variants occurs via the chimeric PDZ domain containing polypeptide. In the type 2 ELISA set up, oncogenic E6 protein is captured via the chimeric PDZ domain containing polypeptide to the solid phase and E6 detection occurs via a specific E6 antibody or another E6 binding specific agent like nucleic acid based binding compounds, chemicals binding E6, E6 binding proteins or a combination of those compounds. Cells are lysed directly on a tissue culture plate and lysates are precleared by centrifugation from insoluble components. Lysates are preincubated at 40C. with a chimeric PDZ domain containing polypeptide, a fusion protein of GST and the chimeric PSD95 substituted with MAGI-I PDZ domain 1. Subsequently, lysates are loaded onto E6-specific antibody coated ELISA plates. Detection occurred via addition of HRP conjugated GST-specific antibody and addition of the HRP substrate TMB after appropriate washes between different incubation steps. Detection signal is constituted by a colorimetric change that is quantified using absorbance measurements at 450 nm.
[00308] HPV 16-E6 of over expressing E6 transfected 293 EBNA-T cells and of HPVl 6 infected cervical cancer derived cell lines is detected. For HPV infected cells, the detection limit using MAGI-I PDZ domain 2 is at approximately 250,000 cells. It is predicted that binding or detection via a chimeric PDZ domain containing polypeptide, such as PSD95-MAGI-1 PDZl as described herein, will increase sensitivity to 25,000 cells or less. Background reduction can be achieved by optimizing choice and concentrations of all components in the system, as well as by additional component purification or addition of size exclusion or filtering procedures. Detection signal can be enhanced by use of more sensitive detection systems, for example luminescence based technologies. E6:PDZ binding can be enhanced by choosing a PDZ domain with higher E6-binding affinity for the chimeric PDZ domain containing polypeptide, and by treating the E6 containing lysates with phosphatases, thus freeing all E6-PL sites from any phosphate that might interfere with, diminish or abrogate E6-PI-specific binding to the chimeric PDZ domain containing polypeptide. EXAMPLE 10
Endogenous E6 Protein of HPV Infected Cervical Cancer Cell Lines can be Detected via a Membrane Bound Chimeric PDZ Domain Containing Polypeptide. Membrane Based Detection can be used to Enhance Sensitivity of Chimeric PDZ domain Containing Polypeptide Based Assay.
A) Abstract:
[00309] Experiments are conducted to demonstrate that the cervical cancer ELISA test types 1 and 2 can be performed using a membrane based format. In the membrane -based form of the cervical cancer diagnostic kit, the principles of the traditional ELISA based sandwich 1 and 2 are maintained, especially with regard to the capturing or detection of exclusively the oncogenic forms of E6. Sensitivity is found to be largely increased in the membrane based assay versus the traditional ELISA.
B) Methods:
[00310] Preblock 12 well coming plates (tissue culture treated with lid, polystyrene, 22 mm well diameter) with 2 ml PBS/2% BSA and then rinse 3x with 2 ml PBS Spot nitrocellulose membrane with 2 μl GST-Magil domain 2 solution (88.6, 0.17 mg/ml) using 2 μl pipetman (duplicate spots in 1x1.5 cm membrane, transblot, transfer medium, supported nitrocellulose membrane, catalog no. 162-0097 (0.2 μM), Lot No. 8934). Allow to air dry for about 5-10 minutes. Hydrate membrane with 1 ml PBS for a couple of minutes in plate. Block membrane in each well with 1 ml PBS/2% BSA for 30 minutes at room temperature while rocking. Wash 3x with PBS about 5-10 minutes/wash, 1 ml/wash, aspirate directly first wash. It is proper to wash at room temperature. Incubate membrane with cell lysate, about 300 μl, 3 million cells total, for 30 minutes at room temperature (rock solutions). Also perform 1:10 dilutions (3 million, 300,000, 30,000, 3,000) in PBS/2% BSA (33.33 μl sample, 300 μl PBS/2% BSA). Wash 3x with PBS, 3- 57wash, all at 40C, 1 ml/wash. Then incubate membrane with anti-E6 (6F4) for 30 minutes at 40C. (1:5000 dilution, or 1:50 of 1: 100 6F4 in PBS/2% BSA). (Need 0.4 ml/well, and for 36 wells need 16 ml a) 1) 320 ul of 1:100 6F4, 15.68 ml PBS/2% BSA. Wash 3x with PBS, at 40C, .about 5-10 minutes/wash. Then incubate with HRP-anti-mouse (1: 1000) for 30 minutes at 40C while rocking (HRP-anti-Mouse Ig Horseradish peroxidase linked whole antibody from Sheep, Amersham, NA93 IV, lot 213295. Use 400 μl per well. For 36 wells would need a) 16 μl HRP-anti- mouse, 16 ml PBS/2% BSA. Wash 5x with PBS at 40C, about 5-10 minutes rocking/wash, last wash 10 minutes. Then aspirate last wash, and add 1 ml fresh PBS to each well. Finally develop with ECL+ system in Petri dish and expose in Kodak film. C) Results:
[00311] In a sandwich type 2 setup, GST- chimeric PDZ domain containing polypeptide is spotted on a membrane and decreasing quantities of HPVl 1 and HPVl 6 MBP -E6 fusion proteins are added for binding. Detection with E6 specific antibodies demonstrates specificity of signal for oncogenic (HPVl 6), but not non-oncogenic E6 (HPVl 1). Upon longer exposure (5 minutes), HPV16 MBP-E6 quantities of 0.1 nanogram total are readily detectable. [00312] In the same experiment, lysates of HPV16-E6 transfected cells and mock transfected cells are applied to a membrane based S2 test. E6-specific signal is obtained only for the E6 expressing cells, not for mock transfected cells. These results demonstrate that the membrane based cervical cancer test can be executed in a membrane -based format.
[00313] In a subsequent experiment, lysates of HPV infected cells are tested. Only the HPVl 6-E6 expressing cells are yielding signal (SiHa and CasKi), but not the HPV negative but cervical cancer positive cell line C33. Previously it has been determined that E6-specific signal detected via GST-MAGIl domain 2 is obtained at 300,000 cells, suggesting that an optimized form using the chimeric PDZ domain containing polypeptide, such as the chimeric PSD95-MAGI1 PDZ domain 1 polypeptide, may detect HPV-E6 proteins of substantially lower cell numbers. [00314] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A chimeric polypeptide comprising at least one PDZ domain or a PDZ ligand binding portion of a PDZ domain from a first PDZ domain- containing polypeptide and a second PDZ domain-containing polypeptide.
2. The chimeric polypeptide of claim 1, wherein said first PDZ domain-containing polypeptide is MAGI-I polypeptide.
3. The chimeric polypeptide of claim 1 , wherein said first PDZ domain-containing polypeptide is PDZ domain 1 of MAGI-I polypeptide.
4. The chimeric polypeptide of claim 1, wherein said first PDZ domain-containing polypetide is a PDZ domain listed in Table 2 (SEQ ID No's: 1-220).
5. The chimeric polypeptide of claim 1, wherein said second PDZ domain-containing polypeptide is PSD95.
6. The chimeric polypeptide of claim 1, wherein said first PDZ domain-containing polypeptide is MAGI-I polypeptide and said second PDZ domain- containing polypeptide is PSD95.
7. The chimeric polypeptide of claim 1, further comprising at least one PDZ domain from said first PDZ domain-containing polypeptide located in a non-PDZ portion of said second PDZ domain-containing polypeptide.
8. The chimeric polypeptide of claim 1, wherein said first PDZ domain-containing polypeptide is MAGI-I polypeptide located in a non-PDZ portion of said second PDZ domain-containing polypeptide.
9. The chimeric polypeptide of claim 1, whereing said first PDZ domain-containing polypeptide is MAGI-I polypeptide located in a non-PDZ portion of said second PDZ domain-containing polypeptide which is PSD95.
10. The chimeric polypeptide of claim 1, further comprising three PDZ domains from said first PDZ domain-containing polypeptide located in non-PDZ portions of PSD95.
11. The chimeric polypeptide of claim 1 , wherein said PDZ domain of said first PDZ domain- containing polypeptide contains a PDZ ligand binding portion.
12. The chimeric polypeptide of claim 1, wherein said PDZ domain of said first PDZ domain- containing polypeptide contains a modified PDZ domain.
13. The chimeric polypeptide of claim 1 , wherein a ligand of said PDZ domain of said first PDZ domain-domain containing polypeptide is an oncogenic protein.
14. The chimeric polypeptide of claim 1, wherein a ligand of said first PDZ domain-domain containing polypeptide is an oncogenic protein from human papilloma virus (HPV).
15. The chimeric polypeptide of claim 1 , wherein a ligand of said first PDZ domain-domain containing polypeptide is an oncogenic protein from human papilloma virus (HPV) strain 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82.
16. The chimeric polypeptide of claim 1 , wherein a ligand of said first PDZ domain-domain containing polypeptide is E6 protein from oncogenic human papilloma virus (HPV) strain 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82.
17. The chimeric polypeptide of claim 1 , wherein the binding affinity of said chimeric polypetide to a PDZ ligand is enhanced by greater than 1.2 to 5 fold.
18. The chimeric polypeptide of claim 1 , wherein the binding avidity of said chimeric polypetide to a PDZ ligand is enhanced by greater than 1.2 to 5 fold.
19. The chimeric polypeptide of claim 1 , wherein the binding avidity and binding affinity of said chimeric polypetide to a PDZ ligand is enhanced by greater than 1.2 to 5 fold.
20. The chimeric polypeptide of claim 1, wherein said chimeric polypeptide has enhanced binding affinity and/or binding avidity to a PDZ ligand as compared to the native said second PDZ domain- containing polypeptide.
21. The chimeric polypeptide of claim 1, wherein the binding avidity and/or binding affinity to E6 protein is enhanced by greater than 1.2 to 5 fold.
22. The chimeric polypeptide of claim 1, wherein said first PDZ domain-containing polypeptide is MAGI-I polypeptide and the binding strength of MAGI-I PDZ domain 1 to E6 protein is enhanced by greater than 1.2 to 5 fold.
23. A chimeric polypeptide comprising at least one PDZ domain- containing polypeptide or PDZ ligand binding portion of a PDZ domain from a MAGI-I polypeptide and a PSD95 polypeptide.
24. The chimeric polypeptide of claim 23, further comprising a PDZ domain-containing polypeptide from said MAGI-I polypeptide.
25. The chimeric polypeptide of claim 23, further comprising a PDZ ligand binding portion of a PDZ domain from said MAGI-I polypeptide.
26. The chimeric polypeptide of claim 23, further comprising at least one PDZ domain-containing polypeptide from said MAGI-I polypeptide located in a non-PDZ domain-containing portion of said PSD95 polypeptide.
27. The chimeric polypeptide of claim 23, wherein three PDZ domains of said PSD95 polypeptide contain PDZ domain- containing polypeptides from said MAGI-I polypeptide.
28. The chimeric polypeptide of claim 23, wherein the PDZ domain-containing polypeptide from said MAGI-I polypeptide contains a modified PDZ domain.
29. The chimeric polypeptide of claim 23, wherein a ligand of the PDZ domain- containing polypeptide from said MAGI-I polypeptide is an oncogenic protein.
30. The chimeric polypeptide of claim 23, wherein a ligand of the PDZ domain- containing polypeptide from said MAGI-I polypeptide is an oncogenic protein from human papilloma virus (HPV).
31. The chimeric polypeptide of claim 23 , wherein a ligand of the PDZ domain- containing polypeptide from said MAGI-I polypeptide is an oncogenic protein from human papilloma virus (HPV) strain 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82.
32. The chimeric polypeptide of claim 23, wherein a ligand of the PDZ domain- containing polypeptide from said MAGI-I polypeptide is E6 protein from an oncogenic human papilloma virus (HPV) strain 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82.
33. The chimeric polypeptide of claim 23, wherein the binding avidity of said chimeric polypetide to a PDZ ligand is enhanced by greater than 1.2 to 5 fold.
34. The chimeric polypeptide of claim 23, wherein the binding affinity of said chimeric polypetide to a PDZ ligand is enhanced by greater than 1.2 to 5 fold.
35. The chimeric polypeptide of claim 23, wherein the binding avidity and binding affinity of said chimeric polypetide to a PDZ ligand is enhanced by greater than 1.2 to 5 fold.
36. The chimeric polypeptide of claim 23, wherein the binding strength of MAGI-I PDZ domain 1 to E6 protein is enhanced by greater than 1.2 to 5 fold.
37. The chimeric polypeptide of claim 23, wherein said chimeric polypeptide has enhanced binding affinity and/or binding avidity to a PDZ ligand as compared to native said PSD95 polypeptide.
38. A chimeric polynucleotide construct encoding for a chimeric polypeptide comprising at least one PDZ domain or a PDZ ligand binding portion of a PDZ domain from a first PDZ domain- containing polypeptide and a second PDZ domain- containing polypeptide.
39. The chimeric polynucleotide of claim 38, wherein said first PDZ domain-containing polypeptide is MAGI-I polypeptide.
40. The chimeric polynucleotide of claim 38, wherein said first PDZ domain-containing polypeptide is PDZ domain 1 of MAGI-I polypeptide.
41. The chimeric polynucleotide of claim 38, wherein said first PDZ domain-containing polypetide is a PDZ domain listed in Table 2 (SEQ ID No's: 1-220).
42. The chimeric polynucleotide of claim 38, wherein said second PDZ domain-containing polypeptide is PSD95.
43. The chimeric polynucleotide of claim 38, wherein said first PDZ domain-containing polypeptide is MAGI-I polypeptide and said second PDZ domain- containing polypeptide is PSD95.
44. The chimeric polynucleotide of claim 38, further comprising at least one PDZ domain from said first PDZ domain-containing polypeptide located in a non-PDZ portion of said second PDZ domain-containing polypeptide.
45. The chimeric polynucleotide of claim 38, wherein said first PDZ domain-containing polypeptide is MAGI-I polypeptide located in a non-PDZ portion of said second PDZ domain-containing polypeptide.
46. The chimeric polynucleotide of claim 38, whereing said first PDZ domain- containing polypeptide is MAGI-I polypeptide located in a non-PDZ portion of said second PDZ domain- containing polypeptide which is PSD95.
47. The chimeric polynucleotide of claim 38, further comprising three PDZ domains from said first PDZ domain- containing polypeptide located in non-PDZ portions of PSD95.
48. The chimeric polynucleotide of claim 38, wherein said PDZ domain of said first PDZ domain- containing polypeptide contains a PDZ ligand binding portion.
49. The chimeric polynucleotide of claim 38, wherein said PDZ domain of said first PDZ domain- containing polypeptide contains a modified PDZ domain.
50. The chimeric polynucleotide of claim 38, wherein a ligand of said PDZ domain of said first PDZ domain-domain containing polypeptide is an oncogenic protein.
51. The chimeric polynucleotide of claim 38, wherein a ligand of said first PDZ domain-domain containing polypeptide is an oncogenic protein from human papilloma virus (HPV).
52. The chimeric polynucleotide of claim 38, wherein a ligand of said first PDZ domain-domain containing polypeptide is an oncogenic protein from human papilloma virus (HPV) strain 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82.
53. The chimeric polynucleotide of claim 38, wherein a ligand of said first PDZ domain-domain containing polypeptide is E6 protein from oncogenic human papilloma virus (HPV) strain 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82.
54. The chimeric polynucleotide of claim 38, wherein the binding affinity of said chimeric polypetide to a PDZ ligand is enhanced by greater than 1.2 to 5 fold.
55. The chimeric polynucleotide of claim 38, wherein the binding avidity of said chimeric polypetide to a PDZ ligand is enhanced by greater than 1.2 to 5 fold.
56. The chimeric polynucleotide of claim 38, wherein the binding avidity and binding affinity of said chimeric polypetide to a PDZ ligand is enhanced by greater than 1.2 to 5 fold.
57. The chimeric polynucleotide of claim 38, wherein said chimeric polypeptide has enhanced binding affinity and/or binding avidity to a PDZ ligand as compared to the native said second PDZ domain- containing polypeptide.
58. The chimeric polynucleotide of claim 38, wherein the binding avidity and/or binding affinity to E6 protein is enhanced by greater than 1.2 to 5 fold.
59. The chimeric polynucleotide of claim 38, wherein said first PDZ domain-containing polypeptide is MAGI-I polypeptide and the binding strength of MAGI-I PDZ domain 1 to E6 protein is enhanced enhanced by greater than 1.2 to 5 fold.
60. A chimeric polynucleotide construct encoding for a chimeric polypeptide comprising at least one PDZ domain-containing polypeptide or PDZ ligand binding portion of a PDZ domain from a MAGI-I polypeptide and a PSD95 polypeptide.
61. The chimeric polynucleotide of claim 60, further comprising a PDZ domain- containing polypeptide from said MAGI-I polypeptide.
62. The chimeric polynucleotide of claim 60, further comprising a PDZ ligand binding portion of a PDZ domain from said MAGI-I polypeptide.
63. The chimeric polynucleotide of claim 60, further comprising at least one PDZ domain-containing polypeptide from said MAGI-I polypeptide located in a non-PDZ domain-containing portion of said PSD95 polypeptide.
64. The chimeric polynucleotide of claim 60, wherein three PDZ domains of said PSD95 polypeptide contain PDZ domain- containing polypeptides from said MAGI-I polypeptide.
65. The chimeric polynucleotide of claim 60, wherein the PDZ domain- containing polypeptide from said MAGI- 1 polypeptide contains a modified PDZ domain.
66. The chimeric polynucleotide of claim 60, wherein a ligand of the PDZ domain-containing polypeptide from said MAGI-I polypeptide is an oncogenic protein.
67. The chimeric polynucleotide of claim 60, wherein a ligand of the PDZ domain-containing polypeptide from said MAGI-I polypeptide is an oncogenic protein from human papilloma virus (HPV).
68. The chimeric polynucleotide of claim 60, wherein a ligand of the PDZ domain-containing polypeptide from said MAGI-I polypeptide is an oncogenic protein from human papilloma virus (HPV) strain 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82.
69. The chimeric polynucleotide of claim 60, wherein a ligand of the PDZ domain-containing polypeptide from said MAGI-I polypeptide is E6 protein from an oncogenic human papilloma virus (HPV) strain 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82.
70. The chimeric polynucleotide of claim 60, wherein the binding avidity of said chimeric polypetide to a PDZ ligand is enhanced by greater than 1.2 to 5 fold.
71. The chimeric polynucleotide of claim 60, wherein the binding affinity of said chimeric polypetide to a PDZ ligand is enhanced by greater than 1.2 to 5 fold.
72. The chimeric polynucleotide of claim 60, wherein the binding avidity and binding affinity of said chimeric polypetide to a PDZ ligand is enhanced by greater than 1.2 to 5 fold.
73. The chimeric polynucleotide of claim 60, wherein the binding strength of MAGI-I PDZ domain 1 to E6 protein is enhanced by greater than 1.2 to 5 fold.
74. The chimeric polynucleotide of claim 60, wherein said chimeric polypeptide has enhanced binding strength to a PDZ ligand as compared to native said PSD95 polypeptide.
75. A method for producing chimeric PDZ domain- containing polypeptides, the method comprising generating a chimeric polynucleotide construct encoding a chimeric polypeptide which contains at least one PDZ domain or a portion of a PDZ domain from a PDZ domain- containing polypeptide, wherein the PDZ domain added to the chimeric polypeptide has enhanced binding to a PDZ ligand as compared to an isolated PDZ domain peptide.
76. The method of claim 75, wherein said PDZ domain-containing polypeptide is MAGI-I polypeptide.
77. The method of claim 75, wherein said PDZ domain- containing polypeptide is a modified MAGI-I polypeptide.
78. The method of claim 75, wherein said PDZ domain- containing polypeptide is a PDZ domain listed in Table 2 (SEQ ID No's: 1-220).
79. The method of claim 75, wherein said PDZ domain- containing polypeptide is a modified polypeptide of a PDZ domain listed in Table 2 (SEQ ID No's: 1-220).
80. A method for detecting the presence of a PDZ ligand in a sample, the method comprising:
(a) contacting a sample suspected of containing a PDZ ligand with the chimeric polypeptide from any of claims 1-37; and
(b) detecting binding of the PDZ ligand in the sample to said chimeric polypeptide, wherein binding of the PDZ ligand in the sample to the chimeric polypeptide indicates the presence of the PDZ ligand in the sample.
81. The method of claim 80, wherein said chimeric polypeptide has enhanced binding to the PDZ ligand in the sample as compared to an isolated PDZ domain peptide
82. A method for determining if a subject is infected with an oncogenic strain of human papilloma virus (HPV), the method comprising detecting the presence of oncogenic HPV E6 protein in a sample from the subject using an oncogenic HPV E6 protein-binding chimeric polypeptide from any of claims 1-37, wherein the presence of oncogenic HPV E6 protein indicates that the subject is infected with an oncogenic strain of HPV.
83. The method of claim 82, wherein said detecting step further comprisies detecting the presence of said oncogenic HPV E6 protein using an antibody that specifically binds to said oncogenic HPV E6 protein.
84. The method of claim 82, wherein said sample is a cervical scrape, biopsy, or lavage.
85. The method of claim 84, wherein said method is an ELISA or a sandwich assay.
86. The method of claim 82, wherein said sample is prepared in the presence of a proteasome inhibitor.
87. The method of claim 82, wherein said method is performed as a part of a test for cervical cancer.
88. The method of claim 82, wherein the oncogenic HPV is HPV strain 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 66, 73, or 82.
89. A method for purifying a PDZ ligand from a sample, the method comprising:
(a) contacting a sample containing a PDZ ligand with the subject chimeric polypeptide from any of claims 1-37; and (b) purifying the PDZ ligand from the sample, wherein the PDZ ligand is bound or not bound to the subject chimeric polypeptide.
90. A kit for extracting HPV E6 protein from a sample, the kit comprising:
(a) the subject chimeric polypeptide from any of claims 1-37, which binds E6 of an oncogenic HPV strain;
(b) an extraction reagent that has a pH of at least about pH 10.0;
(c) a neutralizing reagent; and
(d) instructions for using the kit.
91. A kit for the detection and diagnosis of an E6 protein of an oncogenic HPV strain in a sample, comprising:
(a) the subject chimeric polypeptide from any of claims 1-37;
(b) reagents for detection of the chimeric polypeptide bound to E6 protein; and
(c) instructions for using the kit.
92. A method for producing a chimeric polypeptide having enhanced binding to a PDZ ligand, the method comprising:
(a) generating the subject chimeric polynucleotide from any of claims 38-74,
(b) expressing the chimeric polynucleotide construct in an expression vector, and
(c) purifying the chimeric polypeptide encoded by the subject chimeric polynucleotide construct of the present invention.
93. A method for treating HPV in a subject with a chimeric polypeptide containing a PDZ domain.
PCT/US2010/033308 2009-04-30 2010-04-30 Chimeric pdz domain-containing protein for viral detection WO2010127324A2 (en)

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