WO2010123949A1 - Methods of detecting hpv strains using synergistic antibodies - Google Patents

Methods of detecting hpv strains using synergistic antibodies Download PDF

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Publication number
WO2010123949A1
WO2010123949A1 PCT/US2010/031821 US2010031821W WO2010123949A1 WO 2010123949 A1 WO2010123949 A1 WO 2010123949A1 US 2010031821 W US2010031821 W US 2010031821W WO 2010123949 A1 WO2010123949 A1 WO 2010123949A1
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protein
antibodies
hpv
synergistic
proteins
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PCT/US2010/031821
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French (fr)
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Kenneth Kopher
Adrien Malick
Julie Rosales
Erin Danaher
Peter Lu
Johannes Schweizer
Jon Silver
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Arbor Vita Corporation
Becton, Dickinson And Company
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Publication of WO2010123949A1 publication Critical patent/WO2010123949A1/en

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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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/084—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus

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 16 and 18 infection was examined by in situ hybridization and E6 protein expression by immunocytochemistry in 623 cervical tissue samples at various stages of tumor progression and a significant correlation was found between histological abnormality and HPV infection.
  • HPV infection is also associated with Netherton's syndrome (Weber et al. (2001) Br. J. Dermatol. 144:1044-1049) and epidermolysis verruciformis (Rubaie et al. (1998) Int. J. Dermatol. 37:766-771).
  • HPV can also be transmitted to a fetus by the mother (Smith et al. (2004) Sex. Transm. Dis. 31:57-62; Xu et al. (1998) Chin. Med.Sci. J. 13:29-31; Cason et al. (1998) Intervirology 41 :213-218).
  • the detection and diagnosis of disease is a prerequisite for the treatment of disease.
  • Literature of interest includes the following references: Zozulya et al., (Genome Biology 2:0018.1- 0018.12, 2001; Mombairts (Annu. Rev. Neurosci 22:487-509, 1999); Raming et al., (Nature 361 : 353-356, 1993); Belluscio et al., (Neuron 20: 69-81, 1988); Ronnet et al., (Annu. Rev. Physiol.
  • Lu et al. (Traffic 4: 416-533, 2003); Buck (Cell 100:611-618, 2000); Malnic et al., (Cell 96:713-723, 1999); Firestein (Nature 413:211-218, 2001); Zhao et al., (Science 279: 237-242, 1998); Touhara et al., (Proc. Natl. Acad. Sci. 96: 4040-4045, 1999); Sklar et al., (J. Biol. Chem 261:15538-15543, 1986); Dryer et al., (TiPS 20:413-417, 1999); Me et al., (J Neurobiol. 50:56-68, 2002); Munger (2002) Front. Biosci. 7:d641-9;
  • the present invention provides a method of detecting E6 protein of a HPV strain in a sample, the method comprising: (a) contacting an E6 protein binding partner with the sample; (b) contacting the E6 protein that is bound to the E6 protein binding partner with a plurality of synergistic antibodies that specifically bind to the E6 protein; and (c) detecting binding of the synergistic antibodies to the E6 protein, thereby detecting the E6 protein in the sample; wherein signal-to-noise ratio of detecting E6 protein using the plurality of synergistic antibodies is increased as compared to using each single antibody at a comparable amount to detect E6 protein.
  • the HPV strain is an oncogenic strain.
  • the synergistic antibodies specifically bind to E6 protein of one oncogenic HPV strain.
  • the synergistic antibodies may specifically bind to E6 proteins of more than one oncogenic HPV strain.
  • the synergistic antibodies specifically bind to E6 protein of HPV- 16, HPV- 18, or HPV-45.
  • the synergistic antibodies specifically bind to E6 proteins of HPV- 16 and HPV- 18.
  • the synergistic antibodies can also specifically bind to E6 proteins of HPV strains selected from the group consisting of HPV- 16, 18, 26, 30, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, and 82.
  • the E6 protein binding partner is immobilized.
  • the E6 protein binding partner is a PDZ domain polypeptide.
  • a PDZ domain polypeptide is MAGI- 1.
  • the PDZ domain polypeptide can be selected from the group consisting of CASK, MPPl, DLGl, DLG2, PSD95, NeDLG, TIP-33, SYNIa, TIP-43, LDP, LIM, LIMKl, LIMK2, MPP2, NOSl, AF6, PTN-4, prIL16, 41.8kD, KIAA0559, RGS12, KIAA0316, DVLl, TIP-40, TIAMl, MINTl, MAGI-I, MAGI-2, MAGI-3, KIAA0303, CBP, MINT3, TIP-2, KIAA0561, and TIP-I.
  • the sample is a cervical scrape, cervical biopsy, cervical lavage, blood or urine.
  • the sample can also be a histological sample.
  • the antibodies are monoclonal.
  • the antibodies are labeled.
  • the synergistic antibodies bind to different epitopes on E6 protein.
  • the sensitivity of detecting an E6 protein using the plurality of synergistic antibodies is increased as compared to using single antibody at a comparable amount to detect E6 protein.
  • the limit of detection (LOD) of an E6 protein using the plurality of synergistic antibodies is decreased as compared to using single antibody at a comparable amount to detect E6 protein.
  • the method is carried out via an enzyme immunoassay (EIA).
  • the assays used in the subject method is a sandwich enzyme-linked immunosorbent assay (ELISA).
  • the plurality of synergistic antibodies are used in immunoassays selected from the group consisting of lateral flow, flow-through, immunofiltration, chemiluminescent EIA, fluorescent EIA, and sandwich ELISA.
  • the subject method can be used as a part of a test for cervical cancer.
  • the present invention provides a method of screening for synergistic antibodies that bind to E6 protein of a HPV strain with enhanced sensitivity as compared to individual antibody specific against the E6 protein, the method comprising: (a) contacting an E6 binding partner with the E6 protein; (b) contacting the E6 protein that is bound to the E6 binding partner with at least two antibodies specific for the E6 protein; (c) detecting binding of the antibodies to the E6 protein that is bound to the E6 binding partner; and (d) selecting the combination of antibodies that has a higher signal-to-noise ratio as compared to the individual antibodies used alone at a comparable amount; wherein a higher signal-to-noise ratio indicates an enhanced sensitivity of E6 protein detection.
  • the method further comprises contacting the E6 protein with additional antibodies that are specific against the E6 protein.
  • the first antibody is immobilized to a substrate.
  • the HPV strain is an oncogenic strain.
  • the synergistic antibodies specifically bind to E6 protein of one oncogenic HPV strain.
  • the synergistic antibodies may specifically bind to E6 proteins of more than one oncogenic HPV strain.
  • the synergistic antibodies specifically bind to E6 protein of HPV- 16, HPV- 18, or HPV-45.
  • the synergistic antibodies specifically bind to E6 proteins of HPV- 16 and HPV- 18.
  • the synergistic antibodies can also specifically bind to E6 proteins of HPV strains selected from the group consisting of HPV- 16, 18, 26, 30, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, and 82.
  • the synergistic antibodies can be monoclonal or labeled.
  • the synergistic antibodies bind to different epitopes on E6 protein.
  • the sensitivity of detecting an E6 protein using the synergistic antibodies is increased as compared to using single antibody at a comparable amount to detect E6 protein.
  • the method is carried out via an enzyme-linked immunosorbent assay (ELISA).
  • the synergistic antibodies are used in immunoassays selected from the group consisting of lateral flow, flow-through, immunofiltration, chemiluminescent EIA, fluorescent EIA, and sandwich ELISA.
  • the invention comprises a method of detecting E6 protein of a HPV strain in a sample, the method comprising: (a) contacting the sample with a plurality of synergistic antibodies that specifically bind to the E6 protein; and (b) detecting binding of the synergistic antibodies to the E6 protein, thereby detecting the E6 protein in the sample; wherein signal-to-noise ratio of detecting E6 protein using the plurality of synergistic antibodies is increased as compared to using each single antibody at a comparable amount to detect E6 protein.
  • Figure 1 is a demonstration of synergistic effect of the paired antibodies in comparison to the antibodies tested alone. Bars represent signal to noise (S/N: SiHa to C33A- cells). Starred bars are those of the synergistic pairs of antibodies.
  • Figure 2 shows limit of detection (LOD) on SiHa cells using two different pairs of antibodies, one of which is 8Gl 1 monoclonal antibody (mAb), in comparison to an antibody tested alone in an enzyme immunoassay (EIA).
  • Figure 2a and 2b show a shift in LOD demonstrating increased sensitivity is seen with the 8Gl 1-6G6 and 4C6-6G6 paired monoclonal antibodies. No increase in sensitivity is noted with 8Gl 1- ICl and 4C6-1C1 paired monoclonal antibodies in this format.
  • FIG. 3 shows use of a HPV 16 N-terminal blocking peptide.
  • the results indicate that the 4C6 and 8Gl 1 Mabs recognize different E6 protein epitopes than that of the ICl and 6G6 Mabs.
  • the HPV- 16 E6 N- terminal peptide is designed to contain the HPV- 16 N-terminal 20 amino acid sequence. Both the 4C6 and 8G11 Mabs bind to an epitope located at the N-terminus of the HPV 16 E6 protein.
  • the present invention provides a method of detecting E6 protein of a HPV strain in a sample, the method comprising: (a) contacting an E6 protein binding partner with the sample; (b) contacting the E6 protein that is bound to the E6 protein binding partner with a plurality of synergistic antibodies that specifically bind to the E6 protein; and (c) detecting binding of the plurality of synergistic antibodies to the E6 protein, thereby detecting the E6 protein in the sample.
  • the present invention provides a method of detecting E6 protein of a HPV strain in a sample, the method comprising: (a) contacting the sample with a plurality of synergistic antibodies that specifically bind to the E6 protein; and (b) detecting binding of the synergistic antibodies to the E6 protein, thereby detecting the E6 protein in the sample; wherein signal-to-noise ratio of detecting E6 protein using the plurality of synergistic antibodies is increased as compared to using each single antibody at a comparable amount to detect E6 protein.
  • the HPV strain is an oncogenic strain.
  • the plurality of synergistic antibodies may bind to E6 protein of one oncogenic HPV strain or more than one oncogenic HPV strain.
  • the plurality of synergistic antibodies specifically binds to E6 protein of HPV- 16, HPV-18, HPV45, or a combination thereof.
  • the plurality of synergistic antibodies specifically binds to E6 protein of HPV- 16 and HPV- 18.
  • the plurality of synergistic antibodies specifically binds to E6 proteins of HPV strains selected from the group consisting of HPV- 16, 18, 26, 30, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, and 82.
  • the E6 protein binding partner of the method of the present invention can be a PDZ domain polypeptide.
  • the PDZ domain polypeptide is MAGI-I.
  • the PDZ domain polypeptide can be any of CASK, MPPl, DLGl, DLG2, PSD95, NeDLG, TIP-33, SYNIa, TIP- 43, LDP, LIM, LIMKl, LIMK2, MPP2, NOSl, AF6, PTN-4, prIL16, 41.8kD, KIAA0559, RGS12, KIAA0316, DVLl, TIP-40, TIAMl, MINTl, MAGI-I, MAGI-2, MAGI-3, KIAA0303, CBP, MINT3, TIP- 2, KIAA0561, or TIP-I.
  • specificity of detecting an E6 protein using the plurality of synergistic antibodies is increased as compared to using individual unpaired antibodies to detect E6 protein.
  • sensitivity of detecting an E6 protein using the plurality of synergistic antibodies is increased as compared to using individual unpaired antibodies to detect E6 protein.
  • Signal-to-noise ratio of detecting an E6 protein using the plurality of synergistic antibodies may also be increased as compared to using individual antibodies to detect E6 protein.
  • limit of detection (LOD) of an E6 protein using the plurality of synergistic antibodies is decreased as compared to using individual unpaired antibodies to detect E6 protein.
  • the present invention provides a method of screening antibodies for a plurality of synergistic antibodies that binds to E6 protein of a HPV strain with enhanced sensitivity as compared to a single individual antibody specific against the E6 protein, the method comprising: (a) contacting a first antibody of the antibody pair with the E6 protein; (b) contacting the E6 protein that is bound to the first antibody with a second antibody of the antibody pair; (c) detecting binding of the second antibody to the E6 protein that is bound to the first antibody of the antibody pair; and (d) selecting the antibody pair that has a higher signal-to-noise ratio as compared to the individual antibodies; wherein a higher signal-to-noise ratio indicates an enhanced sensitivity of E6 protein detection.
  • the first antibody is bound to another molecule immobilized to a substrate.
  • HPV Human Papilloma Virus
  • E6 oncoprotein Human papillomaviruses
  • HPVs are small double-stranded DNA viruses that induce hyperproliferative lesions in epithelial tissues. Genomic organization is a well conserved feature among papillomaviruses. There are three main regions in an HPV genome- early, late and the long control regions. In the early region (E) resides the transformation and immortalization potential of HPVs and consists of a number of regulatory genes for viral transcription and replication and cell cycle control.
  • the late region (L) codes for the two capsid genes and the long control region (LCR) contains all the cis-regulatory elements necessary for HPV transcription including the early promoter and the origin of replication (ori).
  • the HPV genome encodes 6 early (E) and two late (L) proteins.
  • El and E2 are the two viral proteins that are required for viral DNA replication, together with the host cell DNA replication machinery.
  • E4 and E5 are needed for amplification of the viral genome in the upper layers of the epithelium.
  • E6 and E7 proteins of high-risk HPV types are oncogenic. They cooperate to immortalize cells and also induce genomic instability.
  • E6 and E7 abrogate the activity of the cellular tumor suppressor proteins p53 and Rb, respectively.
  • E6 also increases telomerase activity.
  • Ll and L2 proteins form the viral capsid and are expressed late in infection in the upper layers of the epithelium.
  • the long- control-region (LCR) contains most of the regulatory DNA sequences needed for proper replication of the viral genome (origin of DNA replication) and for the expression of the viral genes (enhancer and promoter regions).
  • HPV high-risk HPV
  • HPV types i.e. strains have been separated into those that are more likely to develop into cancer and those that are less likely.
  • the so-called “high risk” HPV types are more likely to lead to the development of cancer, while “low-risk” viruses rarely develop into cancer.
  • Certain "high-risk" HPV strains infect epithelia in the anogenital region and are the etiological agents of cervical cancers.
  • the "low-risk" HPV strains include but are not limited to HPV 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81.
  • the sequence analysis of HPV E6 proteins from various HPV strains with regard to the oncogenic potential of the E6 proteins is shown in US Patent Nos. 7,312,041 and 7,399,467, both of which are herein incorporated by reference in their entirety.
  • an "oncogenic HPV strain” is an HPV strain that is known to cause cervical cancer as determined by the National Cancer Institute (NCI, 2001).
  • "Oncogenic E6 proteins” are E6 proteins encoded by the above oncogenic HPV strains. The sequences of exemplary oncogenic E6 proteins of interest are disclosed in US Patent Nos 7,312,041 and 7,399,467, which are herein incorporated by reference in their entirety.
  • HPV16-E6 GL9627100
  • HPV18-E6 GL9626069
  • HPV31-E6 GL9627109
  • HPV35-E6 GL9627127
  • HPV30-E6 GL9627320
  • HPV39-E6 GI:9627165
  • HPV45-E6 GL9627356
  • HPV51-E6 HPV16-E6: GL9627100
  • HPV18-E6 GL9626069
  • HPV31-E6 GL9627109
  • HPV35-E6 GL9627127
  • HPV30-E6 GL9627320
  • HPV39-E6 GI:9627165
  • HPV45-E6 GL9627356
  • HPV51-E6 HPV51-E6:
  • HPV65 GL9627155; HPV52-E6: GL9627370; HPV56-E6: GI:9627383; HPV59-E6: GL9627962; HPV58-E6: GL9626489; HPV33-E6: GI:9627118; HPV66-E6: GL9628582; HPV68b-E6: GL184383; HPV69-E6: GI:9634605; HPV26-E6: GL396956; HPV53-E6: GL9627377; HPV73: GL1491692; HPV82: GL9634614, HPV34 GL396989; HPV67 GL3228267; and HPV70 GI: 1173493.
  • E6 and E7 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 gene encodes for a small nuclear protein product of about 16-19 kD (Greenfield, L, et al. (1991) Proc.Natl.Acad.Sci.U.S.A. 88, 11217-11221).
  • the E6 protein contributes most significantly to the malignant conversion of the basal layer of the cervical epithelium.
  • 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).
  • Low-risk HPV types 6 and 11 E6 protein does not induce p53 degradation correlating with their weak transformation potential. Absence of functional p53 protein makes the cell highly susceptible to DNA damage and prevents the activation of p53 -mediated apoptosis. As a result of the activities of the E6 protein, keratinocytes reactivate DNA synthesis and this in turn alters the growth and differentiation of the basal epithelium anogenital mucosa, resulting in their immortalization.
  • a phenotypic characteristic of all high-risk oncogenic HPV E6 proteins is the presence of a conventional PDZ binding motif (X-S/T-X- ⁇ ) where X is any amino acid and ⁇ represents a hydrophobic amino acid.
  • PDZ domain refers to protein sequence of less than approximately 90 amino acids, (i.e., about 80-90, about 70-80, about 60-70 or about 50-60 amino acids), characterized by homology to the brain synaptic protein PSD-95, the Drosophila septate junction protein Discs-Large (DLG), and the epithelial tight junction protein ZOl (ZOl).
  • PDZ domains are also known as Discs-Large homology repeats ("DHRs") and GLGF repeats. PDZ domains generally appear to maintain a core consensus sequence (Doyle, D. A., 1996, Cell 85: 1067-76). PDZ domains are found in diverse membrane -associated proteins including members of the MAGUK family of guanylate kinase homologs, several protein phosphatases and kinases, neuronal nitric oxide synthase, tumor suppressor proteins, and several dystrophin-associated proteins, collectively known as syntrophins.
  • DHRs Discs-Large homology repeats
  • GLGF repeats generally appear to maintain a core consensus sequence (Doyle, D. A., 1996, Cell 85: 1067-76). PDZ domains are found in diverse membrane -associated proteins including members of the MAGUK family of guanylate kinase homologs, several protein phosphatases and kinases, neuronal
  • the present invention provides a method of detecting E6 protein of a HPV strain in a sample, the method comprising: (a) contacting an E6 protein binding partner with the sample; (b) contacting the E6 protein that is bound to the E6 protein binding partner with a plurality of synergistic antibodies that specifically bind to the E6 protein; and (c) detecting binding of the plurality of synergistic antibodies to the E6 protein, thereby detecting the E6 protein in the sample.
  • the E6 protein binding partner is a PDZ domain polypeptide.
  • An "E6 protein binding partner" can be any molecule that specifically binds to an oncogenic E6 protein.
  • Suitable oncogenic E6 protein binding partners include a PDZ domain (as described below), antibodies against oncogenic E6 proteins (such as those described below); other proteins that recognize oncogenic E6 protein (e.g., p53, E6-AP or E6-BP); DNA (i.e., cruciform DNA); and other partners such as aptamers.
  • an oncogenic E6 protein binding partner may be antibody that binds to these proteins, as described below, or a mixture of antibodies that each bind to a different proteins. As is known in the art, such binding partners may be labeled to facilitate their detection.
  • binding partners bind E6 with an binding affinity of less then 10 ⁇ 5 M, e.g., less than 10 ⁇ 6 , less than 10 ⁇ 7 , less than 10 ⁇ 8 M (e.g., less than 10 ⁇ 9 M, 10 ⁇ 10- 11 , 10 42 M, etc.).
  • PDZ domain refers to protein sequence of less than approximately 90 amino acids, (i.e., about 80-90, about 70-80, about 60-70 or about 50-60 amino acids), characterized by homology to the brain synaptic protein PSD-95, the Drosophila septate junction protein Discs-Large (DLG), and the epithelial tight junction protein ZOl (ZOl).
  • PDZ domains are also known as Discs-Large homology repeats ("DHRs") and GLGF repeats. PDZ domains generally appear to maintain a core consensus sequence (Doyle, D. A., 1996, Cell 85: 1067-76).
  • PDZ domains are found in diverse membrane -associated proteins including members of the MAGUK family of guanylate kinase homologs, several protein phosphatases and kinases, neuronal nitric oxide synthase, tumor suppressor proteins, and several dystrophin-associated proteins, collectively known as syntrophins.
  • PDZ domain-containing proteins and PDZ domain sequences may be found in U.S. Patent Nos. 7,312,041, and 7,399,467, which are herein incorporated by their entirety.
  • the term "PDZ domain” also encompasses variants (e.g., naturally occurring variants) of the sequences (e.g., polymorphic variants, variants with conservative substitutions, and the like) and domains from alternative species (e.g. mouse, rat).
  • PDZ domains are substantially identical to those shown in US Patent Applications 09/724553 and 10/938,249), e.g., at least about 70%, at least about 80%, or at least about 90% amino acid residue identity when compared and aligned for maximum correspondence.
  • PDZ domains can be mutated to give amino acid changes that can strengthen or weaken binding and to alter specificity, yet they remain PDZ domains (Schneider et al., 1998, Nat. Biotech. 17:170-5).
  • a reference to a particular PDZ domain e.g. a MAGI-I domain 2 is intended to encompass the particular PDZ domain and HPV E6-binding variants thereof.
  • a reference is also made to variants of that PDZ domain that bind oncogenic E6 protein of HPV, as described below.
  • the numbering of PDZ domains in a protein may change.
  • the MAGI-I domain 2 as referenced herein, may be referenced as MAGI- 1 domain 1 in other literature.
  • this reference should be understood in view of the sequence of that domain, as described herein, particularly in the sequence listing.
  • U.S. Patent Nos. 7,312,041, and 7,399,467 show the sequences, the names and Genbank accession numbers for various PDZ domains, where appropriate.
  • Further description of PDZ proteins, particularly a description of MAGI-I domain 2 protein is found in Serial No. 10/630,590, filed July 29, 2003 and published as US20040018487. This publication is incorporated by reference herein in its entirety for all purposes.
  • PDZ protein refers to a naturally occurring protein containing a PDZ domain.
  • exemplary PDZ proteins include CASK, MPPl, DLGl, DLG2, PSD95, NeDLG, TIP-33, SYNIa, TIP-43, LDP, LIM, LIMKl, LIMK2, MPP2, NOSl, AF6, PTN-4, prIL16, 41.8kD, KIAA0559, RGS12, KIAA0316, DVLl, TIP-40, TIAMl, MINTl, MAGI-I, MAGI-2, MAGI-3, KIAA0303, CBP, MINT3, TIP- 2, KIAA0561, and TIP-I.
  • PL protein or "PDZ Ligand protein” refers to a protein that forms a molecular complex with a PDZ-domain, or to a protein whose carboxy -terminus, when expressed separately from the full length protein (e.g., as a peptide fragment of 4-25 residues, e.g., 8, 10, 12, 14 or 16 residues), forms such a molecular complex.
  • the molecular complex can be observed in vitro using a variety of assays described infra.
  • an oncogenic E6 protein from a high-risk HPV strain is a PL protein or PL ligand protein.
  • a "HPV E6-binding variant" of a particular PDZ domain is a PDZ domain variant that retains HPV E6 PDZ ligand binding activity.
  • Assays for determining whether a PDZ domain variant binds HPV E6 are described in great detail below, and guidance for identifying which amino acids to change in a specific PDZ domain to make it into a variant may be found in a variety of sources.
  • a PDZ domain may be compared to other PDZ domains described herein and amino acids at corresponding positions may be substituted, for example.
  • sequence a PDZ domain of a particular PDZ protein may be compared to the sequence of an equivalent PDZ domain in an equivalent PDZ protein from another species.
  • sequence of a PDZ domain from a human PDZ protein may be compared to the sequence of other known and equivalent PDZ domains from other species (e.g., mouse, rat, etc.) and any amino acids that are variant between the two sequences may be substituted into the human PDZ domain to make a variant of the PDZ domain.
  • the PDZ domain polypeptide used to capture E6 protein in a sample is MAGI-I.
  • the sequence of the human MAGI-I PDZ domain 2 may be compared to equivalent MAGI-I PDZ domains from other species (e.g. mouse Genbank GI numbers 7513782 and 28526157 or other homologous sequences) to identify amino acids that may be substituted into the human MAGI-I-PDZ domain to make a variant thereof.
  • Such method may be applied to any of the MAGI-I PDZ domains described herein.
  • Particular variants may have 1, up to 5, up to about 10, up to about 15, up to about 20 or up to about 30 or more, usually up to about 50 amino acid changes as compared to a sequence set forth in the sequence listing.
  • a GFG motif is present in a PDZ domain, in general, it should not be altered in sequence.
  • Exemplary PDZ peptide sequences are disclosed in US Patent Nos 7,312,041 and 7,399,467, which are herein incorporated by reference in their entirety.
  • variant PDZ domain polypeptides have a PDZ domain that has at least about 70 or 80%, usually at least about 90%, and more usually at least about 98% sequence identity with a variant PDZ domain polypeptide described herein, as measured by BLAST 2.0 using default parameters, over a region extending over the entire PDZ domain.
  • the E6 binding partner used in the method of the present invention may include, but are not be limited to, p53, E6-AP, E6-BP or engineered compounds that bind E6 oncoproteins.
  • the first E6 binding partner that captures E6 protein in a sample binds to the E6 protein at a location on the E6 protein that does not reduce the availability of the E6 protein for binding to the subject synergistic antibodies.
  • the E6 protein is a recombinant E6 fusion protein, for example, E6 fused to maltose binding protein (MBP), or glutathione-S transferase (GST).
  • MBP maltose binding protein
  • GST glutathione-S transferase
  • the E6 peptide or the relevant portion may 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. 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.).
  • MBP-E6 fusion proteins are prepared for use in the assays of the invention. PCR products containing E6 protein or a portion thereof are subcloned into an expression vector to permit expression of fusion proteins containing the E6 protein or a portion thereof and a heterologous domain (i.e., a MBP or GST).
  • a variety of host-expression vector systems may be utilized to express the recombinant E6 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.
  • antibody and “immunoglobulin” are used interchangeably herein to refer to a type capture agent that has at least an epitope binding domain of an antibody. These terms are well understood by those in the field, and refer to a protein containing one or more polypeptides that specifically binds an antigen.
  • One form of antibody constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of antibody chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions.
  • the recognized immunoglobulin polypeptides include the kappa and lambda light chains and the alpha, gamma (IgGi, IgG 2 , IgG 3 , IgG 4 ), delta, epsilon and mu heavy chains or equivalents in other species.
  • Full-length immunoglobulin "light chains" (of about 25 kDa or about 214 amino acids) comprise a variable region of about 110 amino acids at the NH 2 -terminus and a kappa or lambda constant region at the COOH- terminus.
  • Full-length immunoglobulin "heavy chains” (of about 50 kDa or about 446 amino acids), similarly comprise a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).
  • antibodies and immunoglobulin include antibodies or immunoglobulins of any isotype (IgM, IgG, IgD, IgE, or IgA), fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, peptides, peptidomimetics, peptoids, peptibodies, and fusion proteins comprising an antigen- binding portion of an antibody and a non-antibody protein.
  • the antibody may be in any suitable form e.g., monoclonal, polyclonal, or synthetic.
  • the antibodies may be detectably labeled, e.g. , with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like.
  • the antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like.
  • the antibodies may also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like. Also encompassed by the terms are Fab', Fv, F(ab') 2 , and or other antibody fragments that retain specific binding to antigen.
  • Antibodies may exist in a variety of other forms including, for example, Fv, Fab, and F(ab') 2 , as well as bi-functional (i.e. bi-specific) hybrid antibodies (e.g., Lanzavecchia et al.., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Huston et al.., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al.., Science, 242, 423-426 (1988), which are incorporated herein by reference).
  • bi-functional hybrid antibodies e.g., Lanzavecchia et al.., Eur. J. Immunol. 17, 105 (1987)
  • single chains e.g., Huston et al.., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird
  • the invention provides a combination of antibodies, particularly monoclonal antibodies, which bind to E6 of at least one strain of HPV, in some embodiments, at least one oncogenic HPV strain. In some embodiments, the antibodies bind to E6 of multiple oncogenic strains of HPV.
  • the invention provides antibodies that "recognize”, i.e., specifically bind to with K D of 10 ⁇ 6 M or less, multiple E6 proteins.
  • the subject antibodies each bind to (i.e., cross-react with) a plurality of different E6 proteins (i.e., at least 2, at least 3, at least 4, at least 5, at least 6 or at least 10, usually up to about 12, 15 or 20 or more different E6 proteins) from oncogenic, and, in certain embodiments, non-oncogenic strains of HPV.
  • the subject antibodies bind to amino acid motifs that are conserved between the E6 proteins of different HPV strains, and, accordingly, bind to E6 proteins that have this motif.
  • the antibodies bind at least the E6 proteins of HPV strains 16 and 18 (e.g. the E6 of HPV strains 16, 18, 31, 33 and 45; 16, 18 and 45; or, in other embodiments, the E6 proteins of all of the HPV strains mentioned herein). In other embodiments, the antibodies bind to at least the E6 proteins from HPV strains 16 and 45.
  • the subject antibodies may bind E6 from non-oncogenic strains of HPV (e.g., HPV strains 6 and/or 11) and, accordingly, the subject antibodies may bind to E6 proteins from oncogenic, as well as non-oncogenic, strains of HPV.
  • the plurality of antibodies directed against HPV E6 protein act synergistically in a detection assay for E6 protein, resulting in greater signals, and thus enhancing the sensitivity of the assay.
  • the term "plurality of antibodies” as used herein refers to two or more antibodies.
  • an antibody pair comprising two antibodies specific against an E6 protein is used in the subject method and the antibody pair has synergistic effects on increasing the signal-to-noise ratio and sensitivity of detecting E6 protein.
  • a plurality of antibodies of the present invention comprises more than two antibodies that have synergistic effects on binding and detecting E6 protein.
  • the term "synergistic” refers to a final signal enhancement output (measured by a spectrophotometer in the case of an EIA) or other instrumented or visual read (in the case of a lateral flow or flow through test) when two or more antibodies are used together.
  • the signal output is greater for the plurality of antibodies than for each of the individual antibodies alone at comparable doses, which leads to enhancement of the sensitivity of the assay.
  • the signal is greater for the plurality of antibodies used together than for each of the individual antibodies used alone at comparable doses.
  • the signal to noise (S/N) ratio is greater for the plurality of antibodies used together than for each of the individual antibodies used alone at comparable doses.
  • the present invention provides a method, wherein the sensitivity of detecting an E6 protein using the plurality of synergistic antibodies is increased as compared to using single antibodies alone to detect E6 protein. In some embodiments, the signal-to-noise ratio of detecting an E6 protein using the plurality of synergistic antibodies is increased as compared to using single antibodies to detect E6 protein.
  • the subject synergistic antibodies may bind to any portion or any length of the HPV E6 proteins.
  • the subject synergistic antibodies may bind to one of three sequence motifs found in HPV E6 proteins. These motifs generally correspond to regions of sequence similarity between E6 proteins from different strains of HPV. In general, therefore, a subject antibody binds to peptides having the following sequence: FQDPQERPRKLPQLCTELQTTIHDI and FEDPTRRPYKLPDLCTELNTSLQDI, corresponding to a first common sequence motif in the E6 proteins of HPV strains 16 and 18, respectively, LLIRCINCQKPLCPEEKQRHLDK and LLIRCLRCQKPLNPAEKLRHLNE, corresponding to a second common sequence motif in the E6 proteins of HPV strains 16 and 18, respectively, or RHLDKKQRFHNIRGRWTGRCMSCC and RHLNEKRRFHNIAGHYRGQCHSCC corresponding to a third common sequence motif in the E6 proteins of HPV strains 16 and 18, respectively.
  • a subject antibody binds to other E6 proteins, then it usually binds to the other E6 proteins at positions equivalent to those discussed above, where "positions equivalent to” generally means a stretch of contiguous amino acids that correspond to, i.e., are aligned with, the boxed amino acids when the sequence of the other E6 proteins are with those in Fig. 1 of U.S Patent No. 7,399,467, which is incorporated by reference herein. [0050] Accordingly, since antibodies generally recognize motifs smaller than those listed above, a subject antibody may recognize peptides that are smaller than and contained within the motifs described above.
  • a subject antibody may bind to a peptide having any 9 contiguous amino acids set forth in any one of SEQ NOS:l-6.
  • a subject antibody may recognize the sequences RPRKLPQLCTEL and RPYKLPDLCTEL, corresponding to sub-sequences of the first common sequences of E6 proteins of HPV strains 16 and 18, described above, LLIRCINCQKPL and LLIRCLRCQKPL corresponding to sub-sequences of the second common sequences of E6 proteins of HPV strains 16 and 18, as described above, or RHLDKKQRFHNI and RHLNEKRRFHNI corresponding to sub-sequences of the third common sequences of E6 proteins of HPV strains 16 and 18, as described above. Since these sub-sequences are generally conserved between different E6 proteins, as discussed above, antibodies that bind to the above-recited sequences generally bind to E6 proteins from other HPV strains.
  • the subject antibodies will bind to E6 proteins from HPV strains 16 and 45.
  • a subject antibody binds to peptides having the following sequence: FQDPQERPRKLPQLCTELQTTIHDI and FDDPKQRPYKLPDLCTELNTSLQDV, corresponding to a first common sequence motif in the E6 proteins of HPV strains 16 and 45, respectively,
  • LLIRCINCQKPLCPEEKQRHLDK and LLIRCLRCQKPLNPAEKRRHLKD corresponding to a second common sequence motif in the E6 proteins of HPV strains 16 and 45, respectively, or RHLDKKQRFHNIRGRWTGRCMSCC and RHLKDKRRFHSIAGQYRGQCNTCC corresponding to a third common sequence motif in the E6 proteins of HPV strains 16 and 45, respectively. If a subject antibody binds to other E6 proteins, then it usually binds to the other E6 proteins at positions equivalent to those discussed above, or boxed in Fig. 1 of US Patent No. 7,399,467, which is herein incorporated by reference.
  • a subject antibody may recognize peptides that are smaller than and contained within the motifs described above.
  • a subject antibody may bind to a peptide having any nine contiguous amino acids set forth in any one of SEQ NOS: 1, 3, 5, 57, 58 and 59.
  • a subject antibody may recognize the sequences RPRKLPQLCTEL and RPYKLPDLCTEL, corresponding to sub-sequences of the first common sequences of E6 proteins of HPV strains 16 and 45, described above, LLIRCINCQKPL and LLIRCLRCQKPL corresponding to sub-sequences of the second common sequences of E6 proteins of HPV strains 16 and 45, as described above, or RHLDKKQRFHNI and RHLKDKRRFHSI corresponding to sub-sequences of the third common sequences of E6 proteins of HPV strains 16 and 45, as described above.
  • antibodies that bind to the above -recited sequences generally bind to E6 proteins from other HPV strains.
  • cysteine residues can be replaced by serine residues to avoid disulfide bridge formation.
  • the subject antibodies may bind to the C-terminus of a HPV protein, for example, the C-terminus of E6 protein.
  • Antibodies specific for the HPV C-terminal PDZ ligand (PL) motif may be used for both capture and detection of E6 protein of at least one HPV strain, and for the treatment of HPV infection.
  • 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.
  • 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.
  • BCG Bacilli Calmette- Guerin
  • Corynebacterium parvum bacilli Calmette- Guerin
  • Monoclonal antibodies to a peptide may be prepared using any technique that 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 containing deletions of specific binding sites may be generated by known techniques.
  • 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.
  • 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., NY, Livingstone, 1974, Methods Enzymology: Imnmunoaffinity 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.
  • 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
  • any fragment of a longer full-length E6 protein that contains a subject common motif e.g., the full length protein
  • a full length E6 protein, or a fusion protein thereof may be used to make the subject antibodies.
  • a full length E6 protein, a peptide containing a recited sequence, or a chemically modified (e.g., conjugated) derivative or fusion thereof e.g., a MBP or GST fusion
  • a chemically modified (e.g., conjugated) derivative or fusion thereof e.g., a MBP or GST fusion
  • a nucleic acid encoding the polypeptide may be employed, or a mixture of different polypeptides (e.g., a mixture of E6 polypeptides, each polypeptide from a different HPV strain) may be used as an antigen (Michel (2002) Vaccine 20:A83- A88).
  • an antigen is mixed with an adjuvant, and a suitable non-human animal (e.g., a mouse, chicken, goat, rabbit, hamster, horse, rat or guinea pig, etc.) is immunized using standard immunization techniques (e.g., intramuscular injection) and once a specific immune response of the has been established, blood from the animal may be collected and polyclonal antisera that specifically binds to described peptides may be isolated.
  • a suitable non-human animal e.g., a mouse, chicken, goat, rabbit, hamster, horse, rat or guinea pig, etc.
  • standard immunization techniques e.g., intramuscular injection
  • cells from the spleen of the immunized animal are fused with a myeloma cell line, and, after fusion, the cells are grown in selective medium containing e.g., hypoxanthine, aminopterin, and thymidine (HAT), to select for hybridoma growth, and after 2-3 weeks, hybridoma colonies appear.
  • selective medium containing e.g., hypoxanthine, aminopterin, and thymidine (HAT)
  • HAT thymidine
  • Supernatants from these cultured hybridoma cells are screened for antibody secretion, usually by enzyme-linked immunosorbent assay (ELISA) or the like, and positive clones secreting monoclonal antibodies specific for the antigen can be selected and expanded according to standard procedures.
  • ELISA enzyme-linked immunosorbent assay
  • the peptides are shown as a "consensus" sequence (i.e. peptides in which one of several amino acids may exist at one or more positions) in order to indicate that any one or a mixture of different peptides that are described by the consensus could be used to make the subject antibodies. Accordingly, when a consensus sequence is described, every individual peptide that falls within the consensus should be considered explicitly described. In particular embodiments, exemplary species of peptide encompassed by the consensus sequences have a sequence found in a naturally-occurring HPV E6 protein.
  • Such exemplary sequences can be identified as sequences starting at the amino acid positions defined by the third column of Table 1, "Starting AA” of particular HPV types "HPV type”, and corresponding positions of other HPV E6 proteins (i.e., those positions that are aligned with the positions indicated in Table 1).
  • Antibodies that specifically bind to E7 protein can be generated via immunization method as described herein.
  • peptides having 9, 10, 11, 12, 13, 14, 15 or more, usually up to about 20 or more contiguous amino acids of any of the peptides described above may be used for immunizations.
  • a recited peptide sequence may be contained within a larger peptide that may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more, sometimes up to about 15 or 20 or more amino acids greater in size than a recited polypeptide.
  • a subject peptide may be from about 8 to about 30 amino acids in length.
  • a subject peptide is about 9-20 amino acids in length, and usually contains an amino acid sequence described above.
  • a suitable animal is immunized with a subject peptide or a mixture of subject peptides (e.g., a mixture of 2, 3, 4, 5 about 6 or more, about 10 or more or about 15 or more, usually up to about 20 or 30 or more peptides described above).
  • Antibodies are usually isolated from the animal and tested for binding to different HPV E6 proteins using standard methods (e.g., ELISA, western blot, etc.). In many embodiments, therefore, antibodies will be screened for binding to E6 and E7 proteins from HPV strains 16 and 18, HPV strains 16, 18, 31, 33 and 45, or, in certain embodiments, all of the HPV strains mentioned herein, and maybe others.
  • antibodies that bind to, i.e., cross- react with, E6 proteins from more than one strain of HPV may be identified, and permanent cell lines producing those antibodies may be established using known methods.
  • antibodies are usually tested for binding to more than one antigen, and those antigens are usually E6 proteins from various HPV strains, or fragments thereof.
  • the antibodies will be tested for binding to antigens in native and denatured states.
  • Antibodies that bind to a plurality of E6 proteins have desirable binding properties, and, accordingly, find use in the subject methods.
  • polynucleotides encoding E6 proteins of high-risk HPV types listed hereinabove may be chemically synthesized (DNA 2.0, Menlo Park, California) or cloned via RT-PCR from cervical cancer cell lines. Both maltose-binding-protein-E6 (MBP-E6) and glutathione-S-transferase-E6 (GST -E6) fusion protein types can be used. Production of GST-E6 and MBP-E6 proteins can be by standard protocols recommended by the suppliers (Amersham and New England Biolabs, respectively). Proteins are expressed in DH5 ⁇ E. coli using IPTG driven induction.
  • a 2-hour induction at 37° C yields GST-E6 or MBP-E6 recombinant proteins at about 1 mg/L, whereas induction overnight at 20° C and purification including rebinding of protein to the gel matrix may result in a final yield of 2-10mg/L.
  • Purity of MBP-E6 proteins is estimated to be > 90% based on PAGE analysis.
  • Recombinant E6 fusion proteins can be used as the immunogens.
  • mice can then be immunized with each of the HPV E6 proteins.
  • immunization protocols including varying antigen doses (100 ⁇ g-10 ⁇ g), adjuvants (CFA/IFA, poly(I)-poly(C), CpG+ Alum) and routes (subcutaneous, intraperitoneal) are tested. Immunization projects are set up with 5-15 mice each. Sera of immunized mice are tested in ELISA against the recombinant E6 protein. Mice showing sufficiently high titers (OD above 1 at 1 : 1000 dilution) against E6 in their sera are selected for fusions.
  • the recombinant E6 protein used in the final boost may contain a different tag from that used during the immunization, for example, GST-E6 is used in the boost when immunizations occurs with MBP-E6, and vice versa.
  • the subject antibodies may be conjugated to a detectable label, or may be part of a signal generating system, as described above.
  • an antibody composition for detecting a plurality of HPV E6 proteins is provided.
  • a mixture of different antibodies that recognize at least 5, 7, 9, 12, 15, 20 or 24 different strains of HPV may be employed.
  • the composition may contain a combination of antibodies that recognize at least 3 different oncogenic E6 proteins.
  • the composition may contain 1, 2, 3, 4, or 5 or more different antibodies, each antibody of the composition recognizing at least one (e.g., 2, 3, about 5, about 10, etc.) E6 proteins.
  • the antibodies bind to all or a portion of the E6 proteins from HPV strains mentioned herein, and, in certain embodiments, may also bind to non-oncogenic E6 proteins.
  • the antibodies may be mixed, or separate from each other, i.e., in different vessels.
  • hybridomas that produce the monoclonal antibodies described above and below may be deposited at the American Type Culture Collection (ATCC), for example, hybridoma cell lines 4E9.7 (PTA- 9679), 4E10.2 (PTA-9680, and 6H5.3 (PTA-9681). Any of the deposited hybridomas, the antibodies produced by those hybridomas, as well as other antibodies that bind the same epitopes as the antibodies produced by those hybridomas, are also embodiments of this invention and may be claimed herein. Such antibodies may be employed in any of the methods described herein. Detailed examples of generating anti-E6 monoclonal antibodies are disclosed in US Patent Nos 7,312,041 and 7,399,467, which are herein incorporated by reference in their entirety.
  • the present invention provides a method of detecting E6 protein of a HPV strain in a sample, the method comprising: (a) contacting an E6 protein binding partner with the sample; (b) contacting the E6 protein that is bound to the E6 protein binding partner with a plurality of synergistic antibodies that specifically bind to the E6 protein; and (c) detecting binding of the plurality of synergistic antibodies to the E6 protein, thereby detecting the E6 protein in the sample.
  • the method involves contacting a sample suspected of having HPV with an E6 protein binding partner that specifically binds to an E6 protein, contacting the E6 protein that is bound to the E6 binding partner in the first step with the plurality of synergistic antibodies of the present invention, and assessing any binding of the plurality of synergistic antibodies to the E6 protein.
  • binding of the plurality of synergistic antibodies to the E6 protein indicates the presence of HPV, for example, an oncogenic HPV strain, in the sample.
  • the plurality of synergistic antibodies specifically binds to E6 proteins of HPV16 or HPV18.
  • the plurality of synergistic antibodies specifically binds to E6 proteins of HPV strains selected from the group consisting of HPV- 16, 18, 26, 30, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, and 82.
  • the E6 protein binding partner is a PDZ domain containing polypeptide.
  • the PDZ domain polypeptide may be MAGI-I or any of the PDZ domain polypeptide selected from the group consisting of CASK, MPPl, DLGl, DLG2, PSD95, NeDLG, TIP-33, SYNIa, TIP-43, LDP, LIM, LIMKl, LIMK2, MPP2, NOSl, AF6, PTN-4, prILl ⁇ , 41.8kD, KIAA0559, RGS12, KIAA0316, DVLl, TIP-40, TIAMl, MINTl, MAGI-I, MAGI- 2, MAGI-3, KIAA0303, CBP, MINT3, TIP-2, KIAA0561, and TIP-I, as described hereinabove.
  • the subject synergistic antibodies are monoclonal.
  • the plurality of synergistic antibodies can be unconjugated or directly or indirectly labeled.
  • the paired antibodies bind to different epitopes on an E6 protein.
  • E6 protein For example, a peptide corresponding to the N-terminal region of E6 has been used to demonstrate different binding specificities of the monoclonal antibodies of the present invention.
  • the monoclonal antibodies that act synergistically only one of the pair is inhibited by the N- terminal peptide, indicating the two antibody members of a synergistic antibody pair do not share the identical binding site on E6 protein.
  • the method of the present invention is carried out via an enzyme immunoassay (EIA).
  • the enzyme immunoassay is a sandwich enzyme-linked immunosorbent assay (ELISA).
  • ELISA sandwich enzyme-linked immunosorbent assay
  • the plurality of synergistic antibodies of the present invention can be used in many immunoassays and variations thereof including but not limited to lateral flow, flow-through, immunofiltration, chemiluminescent EIA, fluorescent EIA, and sandwich ELISA.
  • an enzyme based immunoassay test procedure HPV EIA is used as the detection assay to demonstrate the synergistic effect of the antibody pair of the present invention in binding and detecting E6 proteins in a sample.
  • the synergistic pair of antibodies can then be utilized in additional assay test formats such as lateral flow and "flow-through” (i.e. DirectigenTM RSV).
  • additional assay test formats such as lateral flow and "flow-through” (i.e. DirectigenTM RSV).
  • the immunoassays are well known techniques in the art and are described herein, infra.
  • the immunoassays which can be used for the subject methods include but are not limited to competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, immunohistochemistry, and cellular immuno staining (fixed or native) assays to name but a few.
  • competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assay
  • the method of the present invention utilizes an enzyme linked immunosorbent assay (ELISA), preferably a sandwich ELISA, to detect the presence of, for example, oncogenic E6 proteins bound to the plurality of synergistic antibodies of the present invention.
  • ELISA enzyme linked immunosorbent assay
  • sandwich ELISA an enzyme linked immunosorbent assay
  • the terms "sandwich”, “sandwich ELISA”, “sandwich diagnostic” and “capture ELISA” all refer to the concept of detecting a biological polypeptide with two different test agents.
  • ELISA is a well known technique in the art.
  • ELISA involves preparing antigen, coating the well of a 96 well multiwell plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
  • an antigen binding partner for example, a PDZ domain polypeptide
  • antibodies, for example, the subject synergistic antibodies, either unconjugated or conjugated to a detectable compound may be added following the capture of the antigen of interest by the first antigen binding partner.
  • the first antigen binding partner binds E6 protein at a location on the E6 protein that does not reduce the availability of the E6 protein for its binding to the plurality of synergistic antibodies of the present invention.
  • One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art.
  • the method of the present invention involves testing samples of human cervical tissue culture cell lines with plurality of synergistic antibodies of the present invention directed against HPV E6 protein. Results, for example, signal to noise ratios, are compared to results obtained with each of the individual antibodies.
  • limit of detection (LOD) testing of serially diluted extracted human cervical cell lines, for example, SiHa cells is performed with the identified plurality of synergistic antibodies and compared to results obtained from each single antibody to determine if endpoint sensitivity is improved.
  • LOD limit of detection
  • the EIA consists of a microwell plate coated with a PDZ domain-bearing protein to capture a recombinant MBP E6 fusion protein or extracted E6 derived from human cervical cell lines.
  • the assay works by coating the wells of the microwell plate overnight at 4°C with a PDZ domain -bearing protein. The wells are washed with an automatic microwell plate washer the next morning and blocked for two hours at 25°C. Wells are washed again and extracted human cervical cell line sample (E6 positive or negative) are added to the well and incubated for an hour at 25°C.
  • the antibodies are added to the plate as either pairs or by themselves and incubated for an hour at 25°C.
  • a suitable secondary antibody conjugated to an enzyme ie. Goat anti mouse horseradish peroxidase
  • Goat anti mouse horseradish peroxidase is added to the wells after another wash step and incubated again for an hour at 25°C.
  • One final wash step is performed and the corresponding soluble substrate (i.e. 3, 3', 5, 5'-Tetramethylbenzidine (TMB) is added to the wells and incubated for thirty minutes at 25°C.
  • TMB trimethylbenzidine
  • the corresponding stop solution diluted acid solution such as sulfuric acid
  • A450 absorbance of 450nm
  • signal to noise ratio for each of the plurality of synergistic antibodies can be determined from the A450 signals generated from extracted cervical cell lines that either contain (ie. SiHa) or do not contain (i.e. C33A-) the E6 protein.
  • the signal to noise ratio for the plurality of synergistic antibodies is compared to the signal to noise ratio determined for each individual antibody. An increase in the signal to noise for a given plurality of synergistic antibodies over that of the individual antibody demonstrates a synergistic effect of the plurality of synergistic antibodies.
  • Synergistic antibodies can then be run in the conventional HPV EIA.
  • serial dilutions of extracted HPV positive cells for example, SiHa cells are tested with the synergistic antibody pairs in comparison to the individual antibodies and the endpoint sensitivity of the assay is compared.
  • various substrates can be used for binding the antibodies; antibodies recognizing different E6 epitopes can be used; different labels for detecting antibody-E6 interactions can be employed; and different ways of detection can be used.
  • the method of the present invention can employ a variety of surfaces or substrates to bind the polypeptides, antigens, antibodies or antibody fragments.
  • solid phase support or “substrate” is intended any support capable of binding polypeptide, antigen or antibody.
  • supports or substrates include but are not limited to glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, nitrocellulose, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material can have virtually any possible structural configuration so long as the coupled molecule is capable of binding to a PDZ domain polypeptide or an E6 antibody.
  • the support configuration can be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface can be flat, such as a sheet, culture dish, test strip, etc.
  • a surface can be an "assay plate" which is formed from a material (e.g. polystyrene) which optimizes adherence of protein thereto.
  • a material e.g. polystyrene
  • 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.
  • the assay plate can be a "microliter" plate.
  • microliter 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 ⁇ l.
  • 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.
  • the plurality of synergistic antibodies, one or all members of the plurality of antibodies can be labeled.
  • a "detectable label” has the ordinary meaning in the art and refers to an atom (e.g., radionuclide), molecule (e.g., fluorescein), or complex, that is or can be used to detect (e.g., due to a physical or chemical property), indicate the presence of a molecule or to enable binding of another molecule to which it is covalently bound or otherwise associated.
  • label also refers to covalently bound or otherwise associated molecules (e.g., a biomolecule such as an enzyme) that act on a substrate to produce a detectable atom, molecule or complex.
  • Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Labels useful in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads.TM.), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, enhanced green fluorescent protein, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, or 32 P), enzymes (e.g., hydrolases, particularly phosphatases such as alkaline phosphatase, esterases and glycosidases, or oxidoreductases, particularly peroxidases such as horse radish peroxidase, and others commonly used in ELISAs), substrates, cofactors, inhibitors, chemiluminescent groups, chromogenic agents, and colorimetric labels such as colloidal
  • Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
  • Means of detecting such labels are well known to those of skill in the art.
  • radiolabels and chemiluminescent labels may be detected using photographic film or scintillation counters
  • fluorescent markers may be detected using a photodetector to detect emitted light (e.g., as in fluorescence-activated cell sorting).
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art.
  • Non-radioactive labels are often attached by indirect means.
  • a ligand molecule e.g., biotin
  • the ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal generating system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a signal generating system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • ligands and anti-ligands can be used.
  • a ligand has a natural anti-ligand, for example, biotin, thyroxine, and Cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands.
  • any haptenic or antigenic compound can be used in combination with an antibody.
  • the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
  • Means of detecting labels are well known to those of skill in the art.
  • means for detection include a scintillation counter, photographic film as in autoradiography, or storage phosphor imaging.
  • the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • CCDs charge coupled devices
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple colorimetric labels may be detected by observing the color associated with the label. It will be appreciated that when pairs of fluorophores are used in an assay, it is often preferred that they have distinct emission patterns (wavelengths) so that they can be easily distinguished.
  • Direct labels may include but are not limited to radioisotopes (e.g., 125 I; 35 S, and the like); enzymes whose products are detectable (e.g., luciferase, ⁇ -galactosidase, horseradish peroxidase, and the like); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, and the like); fluorescence emitting metals, e.g., 152 Eu, 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.
  • radioisotopes e.g., 125 I; 35 S, 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; a GFP from another species such as Renilla reniformis, Renilla mulleri, or Ptilosarcus guernyi, as described in, e.g.,
  • the label is 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, Ausubel, supra, for a review of techniques involving biotin-avidin conjugation and similar assays.
  • 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).
  • an anti-hapten antibody e.g. anti-digoxin antibody
  • 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
  • washing solution e.g., non-adhering cells, non-adhering capture agent, unbound ligand, receptor, receptor construct, cell lysate, or HRP antibody
  • a detergent e.g., Triton X
  • 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.
  • 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 an antigen, an antibody, or a PDZ-containing protein.
  • 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 the first antibody -E6-second antibody sandwich interactions.
  • Examples of enzyme-substrate combinations include but are not limited to, for example:
  • HRP Horseradish peroxidase
  • HRPO horseradish peroxidase
  • a dye precursor e.g. orthophenylene diamine [OPD] or 3,3',5,5'-tetramethyl benzidine hydrochloride [TMB]
  • OPD orthophenylene diamine
  • TMB 3,3',5,5'-tetramethyl benzidine hydrochloride
  • 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.
  • a chromogenic substrate e.g. p-nitrophenyl-Beta-D- galactosidase
  • fluorogenic substrate 4-methylumbelliferyl-Beta-D-galactosidase 4-methylumbelliferyl-Beta-D-galactosidase.
  • the E6 binding partner for example, the PDZ domain-containing polypeptides are immobilized on a solid surface.
  • the substrate to which the polypeptide is bound may be 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 semi-permeable membrane, and the like.
  • Suitable materials include glass, plastic (e.g., polyethylene, PVC, polypropylene, polystyrene, and the like), nitrocellulose, protein, paper, carbohydrate, lipid monolayer or supported lipid bilayer, films and other solid supports.
  • plastic e.g., polyethylene, PVC, polypropylene, polystyrene, and the like
  • nitrocellulose 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 PDZ domain containing polypeptides are organized as an array.
  • array refers to an ordered arrangement of immobilized proteins, in which particular different proteins (i.e., recognizing different E6 proteins of different HPV strains, or different epitopes of E6 protein) are located at different predetermined sites on the substrate. Because the location of particular antibodies on the array is known, binding at that location can be correlated with binding to the antigen situated at that location. Immobilization of antibodies on beads (individually or in groups) is another particularly useful approach. In one embodiment, individual antibodies 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 flow cytometry) 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 HPV protein may be determined.
  • 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).
  • 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.
  • 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.).
  • the immobilization domain is a GST moiety, as described herein.
  • the substrates may be printed with a hydrophobic (e.g., Teflon) mask to form wells.
  • a hydrophobic (e.g., Teflon) mask to form wells.
  • Preprinted glass slides with 3, 10 and 21 wells per 14.5 cm 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 ⁇ 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 ).
  • 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 10, at 4° C.) to 37° C. and from 1 hr to more than 24 hours. Typically mAbs are attached to plastic with carbonate buffers of pH 8.5 or 9.6.
  • buffer e.g., PBS, or 50 to 300 mM TRIS, MOPS, HEPES, PIPES, acetate buffers, pHs 6.5 to 10, at 4° C.
  • Proteins may be covalently bound or noncovalently attached through nonspecific bonding. If covalent bonding between the 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.
  • the PDZ domain containing polypeptide is immobilized and binding of the captured E6 protein to the plurality of synergistic antibodies of the present invention is detected.
  • the capture of the E6 protein by the PDZ domain containing polypeptide is carried out as follows: a PDZ-domain polypeptide is bound to a surface, e.g. a protein binding surface.
  • a PDZ-domain polypeptide is bound to a polystyrene 96-well plate.
  • the PDZ-domain polypeptide 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 PDZ-domain polypeptide to the plate does not alter the ligand-binding properties of the PDZ domain.
  • One example of an adequate binding to the plate can be achieved when: 50 ⁇ L per well of 5 ⁇ g/mL PDZ-domain polypeptide or, a non-PDZ containing polypeptide as a negative control, in PBS/BSA is added to a polystyrene 96-well plate for 2 hours at 4 0 C. The plate is again washed 3 times with PBS.
  • E6 peptides are introduced to the plate and allowed to react with the surface by addition of 50 uL per well of 20 uM solution of the E6 peptide in PBS/BSA for 10 minutes at 4 0 C followed by an additional 20 minute incubation at 25 0 C.
  • the plate is washed 3 times with ice cold PBS.
  • the binding of the E6 peptide to the PDZ-domain polypeptide can then be incubated with the plurality of synergistic antibodies of the present invention.
  • the binding and detection of E6 protein by the subject synergistic antibodies may be via any immunological based assay, such as immunoprecipitation, western blotting, enzyme immunoassays (EIA), RAMAN spectroscopy, lateral flow, flow-through, DirectigenTM, and cytometric bead array (CBA).
  • immunological based assay such as immunoprecipitation, western blotting, enzyme immunoassays (EIA), RAMAN spectroscopy, lateral flow, flow-through, DirectigenTM, and cytometric bead array (CBA).
  • Immunoprecipitation protocols generally involve lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-4O or Triton X-IOO, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/ or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4 0 C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer.
  • a lysis buffer such as RIPA buffer (1% NP-4O or Triton X-IOO,
  • the ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis.
  • One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads).
  • Western blot analysis generally involves preparation of protein samples followed by electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), and transfer of the separated protein samples from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon. Following transfer, the membrane is blocked in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washed in washing buffer (e.g., PBS-Tween 20), and incubated with primary antibody (the antibody of interest) diluted in blocking buffer.
  • blocking solution e.g., PBS with 3% BSA or non-fat milk
  • washing buffer e.g., PBS-Tween 20
  • the membrane is washed in washing buffer, incubated with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 1251), and after a further wash, the presence of the antigen may be detected.
  • a secondary antibody which recognizes the primary antibody, e.g., an anti-human antibody conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 1251), and after a further wash, the presence of the antigen may be detected.
  • an enzymatic substrate e.g., horseradish peroxidase or alkaline phosphatase
  • radioactive molecule e.g., 32P or 1251
  • Ramon spectroscopy nanotechnoiogy may be used to detect binding of the subject antibodies to oncogenic E6 proteins.
  • a cytometric bead array may be used to detect binding of the subject synergistic antibodies to oncogenic E6 proteins.
  • a Cytometric Bead Array (CBA), commonly referred to as a multiplexed bead assay, is a series of spectrally discrete particles that can be used to capture and quantitate soluble analytes, for example, an E6 protein. The analyte is then measured by detection of a fluorescence- based emission and flow cytometric analysis.
  • the basic "sandwich assay" schema and protocols for the CBA are available at BD Biosciences.
  • the CBA generates data that is comparable to ELISA based assays, but in a "multiplexed” or simultaneous fashion. Concentration of unknowns is calculated for the cytometric bead array as with any sandwich format assay, i.e. through the use of known standards and plotting unknowns against a standard curve.
  • an immuno-lateral flow assay may be used to detect binding of the subject synergistic antibodies to oncogenic E6 proteins in a sample.
  • oncogenic HPV E6 is separated on a test strip.
  • oncogenic HPV E6 may be detected using a detectably labeled antibody of the present invention that binds oncogenic HPV E6.
  • Oncogenic HPV E6 may be quantitated using a reflectance spectrophotometer, or by eye, for example.
  • E6 protein from one oncogenic HPV strain is present and detected on the test strip using the plurality of synergistic antibodies and the method of the present invention.
  • the antibody pair and the method of the present invention allows for detection of E6 proteins from at least two oncogenic HPV strains in a sample, for example, HPV16 and HPV18.
  • the plurality of synergistic antibodies specifically binds to E6 proteins of at least 3, 4, 5, 6, 7, 8, 9, 10 or more different oncogenic HPV strains including but not limited to HPV 16, 18, 26, 30, 31, 34, 39, 45, 51, 52, 53, 58, 59, 66, 68, 69, 70, 73, and 82.
  • HPV strain-specific E6 protein detection allows for a strip test for detecting the presence of E6 proteins, in which different HPV strains can be detected at distinct test lines on one test strip.
  • oncogenic HPV E6 is separated from other proteins in a 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, which are herein incorporated by reference in their entirety.
  • 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 a capture agent, which can be a PDZ domain containing polypeptide, for example.
  • a capture agent which can be a PDZ domain containing polypeptide, for example.
  • 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 PDZ domain polypeptide 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 anti-E6 plurality of synergistic antibodies of the present invention as detector.
  • the subject synergistic antibodies may be used as a detector antibody composition in a lateral flow assay.
  • one or both members of the detector anti-E6 antibody pair may be detectably labeled.
  • the detector antibody pair is specific for an epitope of E6 proteins that is common to all oncogenic E6 proteins, or a mixture of paired antibodies that can, together, bind to all oncogenic E6 proteins.
  • an immunohistochemical assay may be used to detect binding of the subject synergistic antibodies to oncogenic E6 proteins in a sample, for example, a histological sample.
  • the assay may be slide based detection of E6 proteins.
  • Immunohistochemistry or IHC refers to the process of locating proteins in cells of a tissue section exploiting the principle of antibodies binding specifically to antigens in biological tissues. Visualizing an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyze a color-producing reaction. Alternatively, the antibody can also be tagged to a fluorophore, such as FITC, rhodamine, Texas Red or Alexa Fluor. For IHC, the sample may be either thin (about 4-40 ⁇ m) slices taken of the tissue of interest, or the whole tissue if the tissue is not very thick and is penetrable. The sample used in IHC with the subject antibody for detection of oncogenic E6 proteins may be a cervical scrape or cervical biopsy.
  • a DirectigenTM assay format is used to detect HPV E6 protein.
  • DirectigenTM is a rapid chromatographic immunoassay for the direct and qualitative detection of viruses, such as influenza A and B viral antigens, from a biological sample such as nasopharyngeal washes/aspirates, nasopharyngeal swabs and throat swabs of symptomatic patients.
  • the DirectigenTM EZ Flu A+B test is a differentiated test, such that influenza A viral antigens can be distinguished from influenza B viral antigens from a single processed sample using a single device. The test is to be used as an aid in the diagnosis of influenza A and B viral infections.
  • the BD DirectigenTM EZ RSV (Respiratory syncytial virus) test utilizes a new, improved format that requires only one reagent and one device for rapid RSV detection.
  • the testing device employs innovative lateral flow technology, a method whereby dried reagents have already been applied to the testing strip. Workflow is reduced to a two-step process, with clear-cut results shown by the appearance of an easy- to-read double line, indicating a positive result for RSV, along with the Internal Control line.
  • These assay formats can all be used to carry out the method of the present invention for detection of E6 protein in a sample.
  • a sample may be contacted with a subject plurality of synergistic antibodies of the invention, and the presence of an oncogenic E6 protein may be detected using the PDZ domain containing polypeptide. Binding of an oncogenic HPV E6 protein to the PDZ domain polypeptide and a subject plurality of synergistic antibodies indicates the presence of an oncogenic HPV E6 protein in the sample, and thus the presence of an oncogenic HPV strain.
  • a "tagged" version of a PDZ domain containing polypeptide that specifically recognizes oncogenic E6 proteins can be used to detect the presence of oncogenic E6 protein in a sample. 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.
  • the present invention provides a method of screening antibodies for synergistic antibodies that bind to E6 protein of a HPV strain with enhanced sensitivity as compared to an individual antibody specific against the E6 protein, the method comprising: (a) contacting an E6 binding partner, for example, a PDZ domain containing polypeptide, with the E6 protein; (b) contacting the E6 protein that is bound to the E6 binding partner with at least two anti-E6 antibodies; (c) detecting binding of the anti-E6 antibodies to the E6 protein; and selecting the synergistic anti-E6 antibodies that have a higher signal-to- noise ratio as compared to the individual antibodies alone or a single anti-E6 antibody used at twice the amount; wherein a higher signal-to-noise ratio indicates an enhanced sensitivity of E6 protein detection.
  • an E6 binding partner for example, a PDZ domain containing polypeptide
  • the antibodies of the invention may be screened for immunospecific binding by any method known in the art.
  • the immunoassays which can be used in identifying synergistic antibodies include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and cellular immunostaining (fixed or native) assays to name but a few.
  • the binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3 H or 125 I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
  • the affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays.
  • the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3 H or 125 I) in the presence of increasing amounts of an unlabeled second antibody.
  • Antibodies of the invention may be screened using immunocytochemisty methods on cells (e.g., mammalian cells, such as CHO cells) transfected with a vector enabling the expression of an antigen or with vector alone using techniques commonly known in the art.
  • cells e.g., mammalian cells, such as CHO cells
  • Antibodies that bind antigen transfected cells, but not vector-only transfected cells, are antigen specific.
  • the assay is an antigen capture assay, and an array or microarray of antibodies may be employed for this purpose.
  • Methods for making and using microarrays of polypeptides are known in the art (see e.g. U.S. patents 6,372,483, 6,352,842, 6,346,416 and 6,242,266).
  • Various known methods can be used to purify the identified synergistic antibodies. These methods include but are not limited to immunofiltration or affinity purification of antibodies. Briefly, immunofiltration refers to purification of antibodies by mixing with specific antigen. The antigen is then removed from the antibody by treatment with soluble carriers.
  • hybridoma cells whose supernatants give the desired activity are selected for cloning.
  • cells are cloned by limiting dilution in a 96-well flat bottom plate.
  • Purification of antibodies from tissue culture supernatants can be performed by protein G and A affinity chromatography (Amersham). The isotype of the antibodies can be determined using Cytometric bead array.
  • Antibody purification methods and protocols are well established in the art and are within the knowledge of one skilled in the art.
  • Bio samples to be analyzed using the methods of the invention may be 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.
  • the subject is one who is susceptible to HPV, (e.g., as determined by family history).
  • 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. In some embodiments a cervical scrape, biopsy, or lavage is obtained from a subject. In other embodiments, the sample is a blood or urine sample. In still other embodiments, the sample is a histological sample. [00117] 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 protease 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.
  • a suitable control may also include a sample taken from an individual with recurring HPV infections which clear spontaneously.
  • a sample is contacted to a solid support-bound PDZ domain polypeptide under conditions suitable for binding of the PDZ domain polypeptide to E6 proteins in the sample, and after separation of unbound sample proteins from the bound proteins, the bound proteins are detected using the subject synergistic antibodies using known methods.
  • Diagnosing the presence of pathogens requires collection of samples appropriate to the organism. For detection of HPV E6 proteins, one would collect tissue for testing from the cervix, penis, anus, head or neck region (e.g. in the nasal cavity, sinuses, lips, mouth, salivary glands, throat, or larynx) 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 E6 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. [00123] Although the diagnostic method disclosed in this application is directed at the detection of E6 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.
  • DNA could be collected from test samples, specific primers for oncogenic E6 and E7 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.
  • Subject refers to any animal, e.g., mammal, human or non-human.
  • the subject is a mammalian subject.
  • Exemplary subjects include, but are not necessarily limited to, humans, non-human primates, mice, rats, cattle, sheep, goats, pigs, dogs, cats, birds, deer, elk, rabbit, reindeer, deer, and horses, with humans being of particular interest.
  • the method of the present invention results in a higher signal and thus an increased signal to noise ratio of detecting an E6 protein of HPV as compared to using individual antibodies to detect the E6 protein.
  • the method of the present invention has higher sensitivity of detecting a HPV E6 protein as compared to using individual antibodies to detect the E6 protein.
  • the method of the present invention has higher specificity of detecting a HPV E6 protein as compared to using individual antibodies to detect the E6 protein.
  • the method of the present invention has lower false positive rate of detecting a HPV E6 protein as compared to using individual antibodies to detect the E6 protein.
  • the term “specific binding” or “specificity” refers to the ability of an antibody or a combination of antibodies to preferentially bind to a particular analyte or component that is present in a homogeneous mixture of different analytes or components in a biological sample.
  • the term “analyte” is used herein interchangeably and refers to a known or unknown component of a sample.
  • a specific binding interaction will discriminate between desirable and undesirable analytes in a sample, typically more than about 10 to 100-fold or more (e.g., more than about 1000- or 10,000-fold).
  • the affinity between a capture agent e.g.
  • an antibody or a polypeptide and an analyte in a sample when they are specifically bound in an antibody /antigen complex is at least 10 " , at least 10 " M, at least 10 " M, usually up to about 10 "10 M.
  • specificity refers to the proportion of people without HPV who have a negative test result.
  • the specificity of the HPV detection method of the present invention is increased as compared to using individual antibodies that bind to E6 protein to detect HPV. In some embodiments, the specificity of the HPV detection method of the present invention is increased to about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more than 99.9%.
  • the rate of erroneously detecting an HPV strain when such HPV strain is in fact absent in a sample is decreased as compared to using individual antibodies that bind to E6 protein to detect HPV.
  • the rate of erroneous detection of HPV e.g. the false positive rate
  • the rate of erroneous detection of HPV is decreased to about 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or less than 0.1%.
  • the signal to noise ratio of the HPV detection method using synergistic antibodies of the present invention is increased as compared to using individual antibodies that bind to E6 protein to detect HPV.
  • the signal to noise (S/N) ratio generally refers to a ratio of desired signal to undesired background signal.
  • the synergistic antibodies of the present invention result in a higher signal for detecting E6 protein without increasing the level of background noise.
  • the signal to noise ratio of detecting HPV E6 protein using the synergistic antibodies of the present invention is increased by about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.5, 5.0, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more than 1,000 folds, as compared to using each individual antibodies to detect E6 protein at comparable doses.
  • the signal to noise ratio of detecting HPV E6 protein using the synergistic antibodies of the present invention is increased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, as compared to using each individual antibodies to detect E6 protein at comparable doses.
  • antibodies which have S/N ratios that are at least 20% greater than the S/N ratios for the respective individual mAb are deemed to be synergistic.
  • the sensitivity of the HPV detection method using synergistic antibodies of the present invention is increased as compared to using individual antibodies that bind to E6 protein to detect
  • sensitivity refers to the proportion of people with HPV who have a positive test result. In other embodiments, sensitivity refers to the smallest amount of a substance, such as a protein in a sample, which a diagnostic test can detect.
  • a first E6 binding partner for example, a PDZ domain polypeptide such as MAGI-I or MUPPl
  • a PDZ domain polypeptide such as MAGI-I or MUPPl
  • a plurality of synergistic antibodies that specifically binds to the E6 protein bound to the first E6 binding partner for example a PDZ domain polypeptide such as MAGI-I or MUPPl
  • the sensitivity of correctly detecting an E6 protein or an oncogenic HPV strain using the method and the synergistic antibodies of the present invention is increased as compared to using individual antibodies or a PDZ domain containing polypeptide that binds to E6 protein for detection.
  • the sensitivity of correct HPV detection using the method of the present invention is increased to about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more than 99.9%.
  • Sensitivity can be measured by the number of E6 molecules that can be detected by the subject synergistic antibodies in a given volume of a sample. Sensitivity can also be measured by the number of HPV infected cells per given volume of a sample. Understanding the sensitivity of the synergistic antibodies of the present invention as compared to individual antibodies is essential because it helps to define the amount of tissue or cell sample that must be tested to obtain a definitive result.
  • Sensitivity of E6-antibody binding can be measured based on apparent affinity, which 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, in this case, binding of E6 to an anti-E6 antibody.
  • 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, in this case, binding of E6 to an anti-E6 antibody.
  • a fixed concentration of an anti-E6 antibody of the present invention and increasing concentrations of a labeled E6 peptide (labeled with, for example, biotin or fluorescein) are mixed together in solution and allowed to react.
  • Representative HPV E6 peptide amino acid sequences are disclosed herein and also in US Patent Nos.
  • preferred E6 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 nonspecific antibody as a control.
  • Antibody-E6 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 antibody-E6 complex is detected based on presence of the label on the E6 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 antibody-E6 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 [ i lgan d ] " 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:
  • binding affinity itself can be determined using any suitable equation (see Cantor and Schimmel (1980) BIOPHYSICAL CHEMISTRY W H Freeman & Co., San Francisco) or software. 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).
  • Signal-to-noise ratio typically compares the level of a desired signal, for example, specific binding to E6 protein, and the level of background noise, for example, any unspecific binding not to E6 protein. The higher the ratio, the less obtrusive the background noise is.
  • the method of the present invention enhances the signal-to-noise ratio of detecting an E6 protein by about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 500, 1,000 or 10,000 fold or more, as compared to using individual unpaired antibodies to detect the E6 protein.
  • the method of the present invention results in a lower false positive rate of erroneously detecting an HPV E6 protein as compared to using individual antibodies to detect the E6 protein.
  • the false positive rate for a test is the false-positive test results divided by all patients without the disease.
  • the false positive rate of detecting E6 protein of a HPV strain in a sample using the method of the present invention may be about 10%, 9%, 8%, 7, %, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less.
  • kits for carrying out the methods of the invention also includes kits for carrying out the methods of the invention.
  • a subject kit contains subject synergistic antibodies that specifically bind to E6 protein of an HPV strain.
  • the HPV strain can be oncogenic or non-oncogenic.
  • the kit further contains a PDZ domain polypeptide.
  • the kits contain a first and second binding partner, where the first binding partner is a PDZ domain polypeptide and the second binding partner is a subject plurality of synergistic antibodies that specifically binds to E6 protein.
  • the antibody pair 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 protein from the sample.
  • the kit may optionally contain protease 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.
  • kits may comprise reagents for use with several separation and detection platforms such as test strips, sandwich assays, etc.
  • 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.
  • Subject kits may also contain control samples containing oncogenic or non-oncogenic E6 proteins, and/or a dilution series of oncogenic E6 proteins, 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 proteins 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 standard curves 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.
  • the present invention discloses a method of detecting HPV E6 protein in a sample with high sensitivity.
  • the present invention discloses a method of screening antibodies for synergistic antibodies that bind to E6 protein of a HPV strain with enhanced sensitivity as compared to an individual antibody specific against the E6 protein.
  • the synergistic antibodies and methods of the instant invention are useful for a variety of diagnostic analyses.
  • the instant antibodies and methods are useful for diagnosing infection by an oncogenic strain of HPV in an individual; for determining the likelihood of having cancer; for determining a patient's response to treatment for HPV; for determining the severity of HPV infection in an individual; and for monitoring the progression of HPV in an individual.
  • the plurality of synergistic antibodies and the methods of the instant invention are useful in the diagnosis of infection with an oncogenic or a non-oncogenic strain of HPV associated with cancer, including cervical, ovarian, breast, anus, penis, prostate, larynx and the buccal cavity, tonsils, nasal passage, skin, bladder, head and neck squamous- cell, occasional periungal carcinomas, as well as benign anogenital warts.
  • the antibodies and the methods of the instant invention are useful in the diagnosis of infection with an oncogenic or a non-oncogenic strain of HPV associated with Netherton's syndrome, epidermolysis verruciformis, endometriosis, and other disorders.
  • the antibodies and the methods of the instant invention are useful in the diagnosis of infection with an oncogenic or a non-oncogenic strain of HPV in adult women, adult men, fetuses, infants, children, and immunocompromised individuals.
  • the subject methods may generally be performed on biological samples from living subjects.
  • a particularly advantageous feature of the invention is that the methods can simultaneously detect, in one reaction, several known oncogenic or non-oncogenic strains of HPV.
  • the synergistic antibodies of the invention may be employed in immunohistological examination of a sample.
  • This example describes an enzyme immunoassay (EIA) that is used to determine which pairs of anti- HPV-16 E6 monoclonal antibodies (mAbs) are synergistic.
  • EIA enzyme immunoassay
  • the assay was carried out as follows: the wells were coated with 50 ⁇ L of a 5 ⁇ g/mL Maji-1 PDZ (monomeric PDZ) solution (in PBS: 1OmM Na 2 HPO 4 , 1.8mM KH 2 PO 4 , 137mM NaCl, 3.9mM KCl, pH 7.4).
  • the plate was incubated overnight (ON) at 5 0 C and washed the next morning with a wash buffer containing 5OmM Tris-HCl, pH 8.0, 15OmM NaCl, 0.05% Tween-20). The wash was repeated 3 times using an automated washer (Biotek). The plate was blocked with 140 ⁇ L 2% BSA in PBS and incubated for 2 hours at 25 0 C. The wash step was repeated and either 2.5MM/mL (Millions per mL) extracted SiHa (HPV 16) or C33A- cells (diluted in AVC HPV lysis buffer) were added to the plate. The cells were incubated for 1 hour at 25 0 C.
  • 5 ⁇ g/mL mAb solutions were prepared in 2% BSA and 0.05% Tween in PBS.
  • the mAbs were incubated in the plate for 1 hour at 25 0 C.
  • the wash step was repeated and 50 ⁇ L of a 1:5000 dilution of Goat anti Mouse- HRP conjugate (diluted in 2% BSA and 0.05% Tween in PBS) was added to each well (GxM- HRP- Jackson Immunologicals 115-035-062) and incubated for 1 hour at 25 0 C.
  • the plate was washed for 5 times and 50 ⁇ L TMB (Neogen K-Blue 331177) was added to each well and incubated for 30 minutes at 25 0 C.
  • 50 ⁇ L EIA Stop Solution 1% Sulfuric Acid
  • the signal to noise ratio was calculated by dividing A450 of pertinent mAb (or mAb combination) in SiHa well by A450 of the same mAb (or mAb combination) in C33A- well.
  • the S/N of individual mAb results were compared to those of combination mAb results. mAbs which have S/N ratios that are at least 20% greater than the S/N ratios for the respective individual mAb were deemed to be synergistic.
  • Starred bars in FIG. 1 represent pairs of antibodies having synergistic effects. The results demonstrate the synergistic effect of the paired antibodies, which give higher signal to noise ratios as compared to using the individual antibodies alone for detection of E6 protein.
  • This example describes a titration enzyme immunoassay (EIA) that demonstrates that the titration curve of the synergistic pair, in comparison to the titration curve of the individual mAbs, is positively shifted, suggesting a greater signal with the combined antibody pair.
  • the assay was carried out as follows: the wells were coated with 50 ⁇ L of a 5 ⁇ g/mL Maji-1 PDZ (monomeric PDZ) solution (in PBS: 1OmM Na 2 HPO 4 , 1.8mM KH 2 PO 4 , 137mM NaCl, 3.9mM KCl, pH 7.4).
  • the plate was incubated overnight (ON) at 5 0 C and washed the next morning with a wash buffer containing 5OmM Tris-HCl, pH 8.0, 15OmM NaCl, 0.05% Tween-20). The wash was repeated 3 times using an automated washer (Biotek). The plate was blocked with 140 ⁇ L 2% BSA in PBS and incubated for 2 hours at 25 0 C. Serial dilutions of SiHa (10, 5, 2.5, 1.25, & 0.63MM/mL) were prepared in a "constant bed" of lMM/mL C33A- cell lysate (Lysate prepared using AVC lysis buffer).
  • mAb combinations or individual mAbs were added to wells of the serially diluted SiHa cell lysate and incubated for 1 hour at 25 0 C.
  • the wash step was repeated and 50 ⁇ L of a 1:5000 dilution of Goat anti Mouse- HRP conjugate (diluted in 2% BSA and 0.05% Tween in PBS) was added to each well (GxM- HRP- Jackson Immunologicals 115-035-062) and incubated for 1 hour at 25 0 C.
  • the plate was washed for 5 times and 50 ⁇ L TMB (Neogen K-Blue 331177) was added to each well and incubated for 30 minutes at 25 0 C.
  • This example describes an inhibition enzyme immunoassay (EIA) that demonstrates that the monoclonal antibody pairs have different binding specificities on E6 protein and the two members of a synergistic antibody pair do not share the same binding site on E6.
  • the assay was carried out as follows: the wells were coated with 50 ⁇ L of a 5 ⁇ g/mL Maji-1 PDZ (monomeric PDZ) solution (in PBS: 1OmM Na 2 HPO 4 , 1.8mM KH 2 PO 4 , 137mM NaCl, 3.9mM KCl, pH 7.4).
  • the plate was incubated overnight (ON) at 5 0 C and washed the next morning with a wash buffer containing 5OmM Tris-HCl, pH 8.0, 15OmM NaCl, 0.05% Tween-20). The wash was repeated 3 times using an automated washer (Biotek). The plate was blocked with 140 ⁇ L 2% BSA in PBS and incubated for 2 hours at 25 0 C. Serial dilutions of SiHa (10, 5, 2.5, 1.25, 0.63, 0.31, 0.16MM/mL) were prepared in a "constant bed" of lMM/mL C33A- cell lysate (Lysate prepared using AVC lysis buffer).
  • the individual mAbs were prepared with or without 10 ⁇ g/mL of an HPV 16 N-terminal peptide (NTP).
  • NTP HPV 16 N-terminal peptide
  • the mAb with the N-terminal peptide was pre-incubated for 20 minutes at 25 0 C and then added to the appropriate wells of the serially diluted SiHa cell lysate.
  • the wash step was repeated and 50 ⁇ L of a 1 :5000 dilution of Goat anti Mouse- HRP conjugate (diluted in 2% BSA and 0.05% Tween in PBS) was added to each well (GxM-HRP- Jackson Immunologicals 115-035-062) and incubated for 1 hour at 25 0 C.
  • the plate was washed for 5 times and 50 ⁇ L TMB (Neogen K-Blue 331177) was added to each well and incubated for 30 minutes at 25 0 C. 50 ⁇ L EIA Stop Solution (1% Sulfuric Acid) was then added to each well. The plate was read at OD450 in a microplate reader. A450 titration curves of the individual mAbs for both sets (with and without NTP) were plotted. If the titration curve of the mAb with NTP was suppressed, then that mAb was considered inhibited. The results show that mAbs 4C6 and 8Gl 1 were inhibited by the NTP whereas mAb 6G6 was not.
  • the 6G6 mAb is recognizing a different epitope for HPV 16 than either 8Gl 1 or 4C6 mAb.
  • the S/N of individual mAb results were compared to those of the combination mAb results. mAbs which have S/N ratios that are at least 20% greater than the S/N ratios for the respective individual mAb were deemed to be synergistic.
  • Fig. 3 shows use of a HPV 16 N-terminal blocking peptide.
  • the HPV E6 16 N-terminal peptide is designed to contain the HPV 16 N-terminal 20 amino acid sequence. The results indicate that the 8Gl 1 and
  • 4C6 mAbs are inhibited by an HPV 16 N-terminal peptide, whereas the 6G6 mAb is not inhibited.
  • Both the 4C6 and 8Gl 1 Mabs bind to an epitope located at the N-terminus of the HPV- 16 E6 protein. This indicates that the 6G6 mAb recognizes a different epitope on the E6 protein (i.e. not on the N-terminus of E6) than either the 8Gl 1 or 4C6 mAb.

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Abstract

In one aspect, the present invention provides a method of detecting E6 protein of a HPV strain in a sample with increased sensitivity, the method involving use of a plurality of synergistic antibodies that bind to E6 protein. In another aspect, the present invention discloses a method of screening for synergistic antibodies that bind to E6 protein of a HPV strain with enhanced sensitivity as compared to a single antibody specific against the E6 protein. The subject synergistic antibodies find use in a variety of diagnostic applications, including methods of diagnosing cancer. Kits for performing the subject methods and containing the subject synergistic antibodies are also provided.

Description

METHODS OF DETECTING HPV STRAINS USING SYNERGISTIC ANTIBODIES
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 61/171,017, filed April 20, 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. Further research demonstrated that the E6-E7 genes from HPV 16 are sufficient to immortalize human keratinocytes in culture. It was also 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. HPV 16 and 18 infection was examined by in situ hybridization and E6 protein expression by immunocytochemistry in 623 cervical tissue samples at various stages of tumor progression and a significant correlation was found between histological abnormality and HPV infection.
[0004] 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, breast (Yu et al. (1999) Anticancer Res. 19:55555057-5061; Liu et al. (2001) J. Hum. Virol. 44:329-334), anus, penis, prostate (De Villiers et al. (1989) Virology 171:248:253), larynx and the buccal cavity, tonsils (Snijders et al. (1994) J. Gen. Virol. 75(Pt 10):2769-2775), nasal passage (Trujillo et al. (1996) Virus Genes 12:165-178; Wu et al. (1993) Lancet 341:522-524), skin (Trenfield et al. (1993) Australas. J. Dermatol. 34:71-78), bladder (Baithun et al. (1998) Cancer Surv. 31 :17-27), head and neck squamous-cell carcinomas (Braakhuis et al. (2004) J. Natl. Cancer Inst. 96:978-980), 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] HPV infection is also associated with Netherton's syndrome (Weber et al. (2001) Br. J. Dermatol. 144:1044-1049) and epidermolysis verruciformis (Rubaie et al. (1998) Int. J. Dermatol. 37:766-771). HPV can also be transmitted to a fetus by the mother (Smith et al. (2004) Sex. Transm. Dis. 31:57-62; Xu et al. (1998) Chin. Med.Sci. J. 13:29-31; Cason et al. (1998) Intervirology 41 :213-218). [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 pathogens 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. Literature
[0007] Literature of interest includes the following references: Zozulya et al., (Genome Biology 2:0018.1- 0018.12, 2001; Mombairts (Annu. Rev. Neurosci 22:487-509, 1999); Raming et al., (Nature 361 : 353-356, 1993); Belluscio et al., (Neuron 20: 69-81, 1988); Ronnet et al., (Annu. Rev. Physiol. 64:189-222, 2002); Lu et al., (Traffic 4: 416-533, 2003); Buck (Cell 100:611-618, 2000); Malnic et al., (Cell 96:713-723, 1999); Firestein (Nature 413:211-218, 2001); Zhao et al., (Science 279: 237-242, 1998); Touhara et al., (Proc. Natl. Acad. Sci. 96: 4040-4045, 1999); Sklar et al., (J. Biol. Chem 261:15538-15543, 1986); Dryer et al., (TiPS 20:413-417, 1999); Me et al., (J Neurobiol. 50:56-68, 2002); Munger (2002) Front. Biosci. 7:d641-9;
Glaunsinger (2000) Oncogene 19:5270-80; Gardiol (1999) Oncogene 18:5487-96; Pim (1999) Oncogene 18:7403-8; Meschede (1998) J. Clin. Microbiol. 36:475-80; Kiyono (1997) Proc. Natl. Acad. Sci. 94:11612- 6; and Lee (1997) Proc. Natl. Acad. Sci. 94:6670-5; Banks (1987) J. Gen. Virol. 68:1351-1359; Fuchs et al., (Hum. Genet. 108:1-13, 2001); and Giovane et al. (1999) Journal of Molecular Recognition 12:141-152 and published US patent applications 20030143679 and 20030105285; and US patents 6,610,511, 6,492,143 6,410,249, 6,322,794, 6,344,314, 5,415,995, 5753233, 5,876,723, 5,648,459, 6,391,539, 5,665,535 and 4,777,239.
SUMMARY OF THE INVENTION
[0008] In one aspect, the present invention provides a method of detecting E6 protein of a HPV strain in a sample, the method comprising: (a) contacting an E6 protein binding partner with the sample; (b) contacting the E6 protein that is bound to the E6 protein binding partner with a plurality of synergistic antibodies that specifically bind to the E6 protein; and (c) detecting binding of the synergistic antibodies to the E6 protein, thereby detecting the E6 protein in the sample; wherein signal-to-noise ratio of detecting E6 protein using the plurality of synergistic antibodies is increased as compared to using each single antibody at a comparable amount to detect E6 protein.
[0009] In some embodiments, the HPV strain is an oncogenic strain. In some embodiments, the synergistic antibodies specifically bind to E6 protein of one oncogenic HPV strain. The synergistic antibodies may specifically bind to E6 proteins of more than one oncogenic HPV strain. In some embodiments, the synergistic antibodies specifically bind to E6 protein of HPV- 16, HPV- 18, or HPV-45. In some embodiments, the synergistic antibodies specifically bind to E6 proteins of HPV- 16 and HPV- 18. The synergistic antibodies can also specifically bind to E6 proteins of HPV strains selected from the group consisting of HPV- 16, 18, 26, 30, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, and 82. [0010] In some embodiments, the E6 protein binding partner is immobilized. In some embodiments, the E6 protein binding partner is a PDZ domain polypeptide. One example of a PDZ domain polypeptide is MAGI- 1. The PDZ domain polypeptide can be selected from the group consisting of CASK, MPPl, DLGl, DLG2, PSD95, NeDLG, TIP-33, SYNIa, TIP-43, LDP, LIM, LIMKl, LIMK2, MPP2, NOSl, AF6, PTN-4, prIL16, 41.8kD, KIAA0559, RGS12, KIAA0316, DVLl, TIP-40, TIAMl, MINTl, MAGI-I, MAGI-2, MAGI-3, KIAA0303, CBP, MINT3, TIP-2, KIAA0561, and TIP-I. In some embodiments, the sample is a cervical scrape, cervical biopsy, cervical lavage, blood or urine. The sample can also be a histological sample. In some embodiments, the antibodies are monoclonal. In some embodiments, the antibodies are labeled. In some embodiments, the synergistic antibodies bind to different epitopes on E6 protein. In some embodiments, the sensitivity of detecting an E6 protein using the plurality of synergistic antibodies is increased as compared to using single antibody at a comparable amount to detect E6 protein. In some embodiments, the limit of detection (LOD) of an E6 protein using the plurality of synergistic antibodies is decreased as compared to using single antibody at a comparable amount to detect E6 protein. In some embodiments, the method is carried out via an enzyme immunoassay (EIA). One example of the assays used in the subject method is a sandwich enzyme-linked immunosorbent assay (ELISA). In some embodiments, the plurality of synergistic antibodies are used in immunoassays selected from the group consisting of lateral flow, flow-through, immunofiltration, chemiluminescent EIA, fluorescent EIA, and sandwich ELISA. The subject method can be used as a part of a test for cervical cancer.
[0011] In one aspect, the present invention provides a method of screening for synergistic antibodies that bind to E6 protein of a HPV strain with enhanced sensitivity as compared to individual antibody specific against the E6 protein, the method comprising: (a) contacting an E6 binding partner with the E6 protein; (b) contacting the E6 protein that is bound to the E6 binding partner with at least two antibodies specific for the E6 protein; (c) detecting binding of the antibodies to the E6 protein that is bound to the E6 binding partner; and (d) selecting the combination of antibodies that has a higher signal-to-noise ratio as compared to the individual antibodies used alone at a comparable amount; wherein a higher signal-to-noise ratio indicates an enhanced sensitivity of E6 protein detection.
[0012] In some embodiments, the method further comprises contacting the E6 protein with additional antibodies that are specific against the E6 protein. In some embodiments, the first antibody is immobilized to a substrate. In some embodiments, the HPV strain is an oncogenic strain. In some embodiments, the synergistic antibodies specifically bind to E6 protein of one oncogenic HPV strain. The synergistic antibodies may specifically bind to E6 proteins of more than one oncogenic HPV strain. In some embodiments, the synergistic antibodies specifically bind to E6 protein of HPV- 16, HPV- 18, or HPV-45. In some embodiments, the synergistic antibodies specifically bind to E6 proteins of HPV- 16 and HPV- 18. The synergistic antibodies can also specifically bind to E6 proteins of HPV strains selected from the group consisting of HPV- 16, 18, 26, 30, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, and 82. The synergistic antibodies can be monoclonal or labeled. In some embodiments, the synergistic antibodies bind to different epitopes on E6 protein. In some embodiments, the sensitivity of detecting an E6 protein using the synergistic antibodies is increased as compared to using single antibody at a comparable amount to detect E6 protein. In some embodiments, the method is carried out via an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the synergistic antibodies are used in immunoassays selected from the group consisting of lateral flow, flow-through, immunofiltration, chemiluminescent EIA, fluorescent EIA, and sandwich ELISA.
[0013] In yet another embodiment, the invention comprises a method of detecting E6 protein of a HPV strain in a sample, the method comprising: (a) contacting the sample with a plurality of synergistic antibodies that specifically bind to the E6 protein; and (b) detecting binding of the synergistic antibodies to the E6 protein, thereby detecting the E6 protein in the sample; wherein signal-to-noise ratio of detecting E6 protein using the plurality of synergistic antibodies is increased as compared to using each single antibody at a comparable amount to detect E6 protein. 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 demonstration of synergistic effect of the paired antibodies in comparison to the antibodies tested alone. Bars represent signal to noise (S/N: SiHa to C33A- cells). Starred bars are those of the synergistic pairs of antibodies.
[0017] Figure 2 shows limit of detection (LOD) on SiHa cells using two different pairs of antibodies, one of which is 8Gl 1 monoclonal antibody (mAb), in comparison to an antibody tested alone in an enzyme immunoassay (EIA). Figure 2a and 2b show a shift in LOD demonstrating increased sensitivity is seen with the 8Gl 1-6G6 and 4C6-6G6 paired monoclonal antibodies. No increase in sensitivity is noted with 8Gl 1- ICl and 4C6-1C1 paired monoclonal antibodies in this format.
[0018] Figure 3 shows use of a HPV 16 N-terminal blocking peptide. The results indicate that the 4C6 and 8Gl 1 Mabs recognize different E6 protein epitopes than that of the ICl and 6G6 Mabs. The HPV- 16 E6 N- terminal peptide is designed to contain the HPV- 16 N-terminal 20 amino acid sequence. Both the 4C6 and 8G11 Mabs bind to an epitope located at the N-terminus of the HPV 16 E6 protein.
DETAILED DESCRIPTION OF THE INVENTION
[0019] Throughout this application, various publications, patents and published patent applications are cited. The disclosures of these publications, patents and published patent applications referenced in this application are hereby incorporated by reference in their entirety into the present disclosure. Citation herein by Applicant of a publication, patent, or published patent application is not an admission by Applicant of said publication, patent, or published patent application as prior art.
[0020] In one aspect, the present invention provides a method of detecting E6 protein of a HPV strain in a sample, the method comprising: (a) contacting an E6 protein binding partner with the sample; (b) contacting the E6 protein that is bound to the E6 protein binding partner with a plurality of synergistic antibodies that specifically bind to the E6 protein; and (c) detecting binding of the plurality of synergistic antibodies to the E6 protein, thereby detecting the E6 protein in the sample.
[0021] In one aspect, the present invention provides a method of detecting E6 protein of a HPV strain in a sample, the method comprising: (a) contacting the sample with a plurality of synergistic antibodies that specifically bind to the E6 protein; and (b) detecting binding of the synergistic antibodies to the E6 protein, thereby detecting the E6 protein in the sample; wherein signal-to-noise ratio of detecting E6 protein using the plurality of synergistic antibodies is increased as compared to using each single antibody at a comparable amount to detect E6 protein.
[0022] In some embodiments, the HPV strain is an oncogenic strain. The plurality of synergistic antibodies may bind to E6 protein of one oncogenic HPV strain or more than one oncogenic HPV strain. In some embodiments, the plurality of synergistic antibodies specifically binds to E6 protein of HPV- 16, HPV-18, HPV45, or a combination thereof. In one example, the plurality of synergistic antibodies specifically binds to E6 protein of HPV- 16 and HPV- 18. In some embodiments, the plurality of synergistic antibodies specifically binds to E6 proteins of HPV strains selected from the group consisting of HPV- 16, 18, 26, 30, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, and 82. The E6 protein binding partner of the method of the present invention can be a PDZ domain polypeptide. In one example, the PDZ domain polypeptide is MAGI-I. The PDZ domain polypeptide can be any of CASK, MPPl, DLGl, DLG2, PSD95, NeDLG, TIP-33, SYNIa, TIP- 43, LDP, LIM, LIMKl, LIMK2, MPP2, NOSl, AF6, PTN-4, prIL16, 41.8kD, KIAA0559, RGS12, KIAA0316, DVLl, TIP-40, TIAMl, MINTl, MAGI-I, MAGI-2, MAGI-3, KIAA0303, CBP, MINT3, TIP- 2, KIAA0561, or TIP-I. In some embodiments, specificity of detecting an E6 protein using the plurality of synergistic antibodies is increased as compared to using individual unpaired antibodies to detect E6 protein. In some embodiments, sensitivity of detecting an E6 protein using the plurality of synergistic antibodies is increased as compared to using individual unpaired antibodies to detect E6 protein. Signal-to-noise ratio of detecting an E6 protein using the plurality of synergistic antibodies may also be increased as compared to using individual antibodies to detect E6 protein. Additionally, in some embodiments, limit of detection (LOD) of an E6 protein using the plurality of synergistic antibodies is decreased as compared to using individual unpaired antibodies to detect E6 protein.
[0023] In another aspect, the present invention provides a method of screening antibodies for a plurality of synergistic antibodies that binds to E6 protein of a HPV strain with enhanced sensitivity as compared to a single individual antibody specific against the E6 protein, the method comprising: (a) contacting a first antibody of the antibody pair with the E6 protein; (b) contacting the E6 protein that is bound to the first antibody with a second antibody of the antibody pair; (c) detecting binding of the second antibody to the E6 protein that is bound to the first antibody of the antibody pair; and (d) selecting the antibody pair that has a higher signal-to-noise ratio as compared to the individual antibodies; wherein a higher signal-to-noise ratio indicates an enhanced sensitivity of E6 protein detection. In some embodiments, the first antibody is bound to another molecule immobilized to a substrate.
[0024] Accordingly, the methods of the present invention find use in a variety of diagnostic applications, including methods of diagnosing cancer, particularly cervical cancer. Human Papilloma Virus (HPV) and E6 oncoprotein [0025] Human papillomaviruses (HPVs) are small double-stranded DNA viruses that induce hyperproliferative lesions in epithelial tissues. Genomic organization is a well conserved feature among papillomaviruses. There are three main regions in an HPV genome- early, late and the long control regions. In the early region (E) resides the transformation and immortalization potential of HPVs and consists of a number of regulatory genes for viral transcription and replication and cell cycle control. The late region (L) codes for the two capsid genes and the long control region (LCR) contains all the cis-regulatory elements necessary for HPV transcription including the early promoter and the origin of replication (ori). The HPV genome encodes 6 early (E) and two late (L) proteins.
[0026] El and E2 are the two viral proteins that are required for viral DNA replication, together with the host cell DNA replication machinery. E4 and E5 are needed for amplification of the viral genome in the upper layers of the epithelium. E6 and E7 proteins of high-risk HPV types are oncogenic. They cooperate to immortalize cells and also induce genomic instability. E6 and E7 abrogate the activity of the cellular tumor suppressor proteins p53 and Rb, respectively. E6 also increases telomerase activity. Ll and L2 proteins form the viral capsid and are expressed late in infection in the upper layers of the epithelium. The long- control-region (LCR) contains most of the regulatory DNA sequences needed for proper replication of the viral genome (origin of DNA replication) and for the expression of the viral genes (enhancer and promoter regions).
[0027] There are over 100 different types of HPV, and these HPV types i.e. strains have been separated into those that are more likely to develop into cancer and those that are less likely. The so-called "high risk" HPV types are more likely to lead to the development of cancer, while "low-risk" viruses rarely develop into cancer. Certain "high-risk" HPV strains infect epithelia in the anogenital region and are the etiological agents of cervical cancers. These high-risk HPV strains include but are not limited to HPV- 16, HPV-18, HPV-26, HPV-30, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV53, HPV54, HPV- 56, HPV-58, HPV-59, HPV-66, HPV-68, HPV-69, HPV-73, and HPV-82. The "low-risk" HPV strains include but are not limited to HPV 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81. The sequence analysis of HPV E6 proteins from various HPV strains with regard to the oncogenic potential of the E6 proteins is shown in US Patent Nos. 7,312,041 and 7,399,467, both of which are herein incorporated by reference in their entirety.
[0028] An "oncogenic HPV strain" is an HPV strain that is known to cause cervical cancer as determined by the National Cancer Institute (NCI, 2001). "Oncogenic E6 proteins" are E6 proteins encoded by the above oncogenic HPV strains. The sequences of exemplary oncogenic E6 proteins of interest are disclosed in US Patent Nos 7,312,041 and 7,399,467, which are herein incorporated by reference in their entirety. The sequences of various HPV proteins are also found as database entries at NCBI's Genbank database, as follows: HPV16-E6: GL9627100; HPV18-E6: GL9626069; HPV31-E6: GL9627109; HPV35-E6: GL9627127; HPV30-E6: GL9627320; HPV39-E6: GI:9627165; HPV45-E6: GL9627356; HPV51-E6:
GL9627155; HPV52-E6: GL9627370; HPV56-E6: GI:9627383; HPV59-E6: GL9627962; HPV58-E6: GL9626489; HPV33-E6: GI:9627118; HPV66-E6: GL9628582; HPV68b-E6: GL184383; HPV69-E6: GI:9634605; HPV26-E6: GL396956; HPV53-E6: GL9627377; HPV73: GL1491692; HPV82: GL9634614, HPV34 GL396989; HPV67 GL3228267; and HPV70 GI: 1173493.
[0029] 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 gene encodes for a small nuclear protein product of about 16-19 kD (Greenfield, L, et al. (1991) Proc.Natl.Acad.Sci.U.S.A. 88, 11217-11221). The E6 protein contributes most significantly to the malignant conversion of the basal layer of the cervical epithelium. The E6 product from high-risk HPV- 16 and 18 interacts with the anti-oncogenic regulator p53 and the ubiquitin degradation pathway protein E6AP, leading to the degradation of the p53 protein (Werness, B.A., et al. (1990) Science 248, 76-79). Briefly, 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). Low-risk HPV types 6 and 11 E6 protein does not induce p53 degradation correlating with their weak transformation potential. Absence of functional p53 protein makes the cell highly susceptible to DNA damage and prevents the activation of p53 -mediated apoptosis. As a result of the activities of the E6 protein, keratinocytes reactivate DNA synthesis and this in turn alters the growth and differentiation of the basal epithelium anogenital mucosa, resulting in their immortalization.
[0030] A phenotypic characteristic of all high-risk oncogenic HPV E6 proteins is the presence of a conventional PDZ binding motif (X-S/T-X-φ) where X is any amino acid and φ represents a hydrophobic amino acid. As used herein, the term "PDZ domain" refers to protein sequence of less than approximately 90 amino acids, (i.e., about 80-90, about 70-80, about 60-70 or about 50-60 amino acids), characterized by homology to the brain synaptic protein PSD-95, the Drosophila septate junction protein Discs-Large (DLG), and the epithelial tight junction protein ZOl (ZOl). PDZ domains are also known as Discs-Large homology repeats ("DHRs") and GLGF repeats. PDZ domains generally appear to maintain a core consensus sequence (Doyle, D. A., 1996, Cell 85: 1067-76). PDZ domains are found in diverse membrane -associated proteins including members of the MAGUK family of guanylate kinase homologs, several protein phosphatases and kinases, neuronal nitric oxide synthase, tumor suppressor proteins, and several dystrophin-associated proteins, collectively known as syntrophins.
E6 Binding Partner for Capturing E6 Protein
[0031] In one aspect, the present invention provides a method of detecting E6 protein of a HPV strain in a sample, the method comprising: (a) contacting an E6 protein binding partner with the sample; (b) contacting the E6 protein that is bound to the E6 protein binding partner with a plurality of synergistic antibodies that specifically bind to the E6 protein; and (c) detecting binding of the plurality of synergistic antibodies to the E6 protein, thereby detecting the E6 protein in the sample. In some embodiments, the E6 protein binding partner is a PDZ domain polypeptide. [0032] An "E6 protein binding partner" can be any molecule that specifically binds to an oncogenic E6 protein. Suitable oncogenic E6 protein binding partners include a PDZ domain (as described below), antibodies against oncogenic E6 proteins (such as those described below); other proteins that recognize oncogenic E6 protein (e.g., p53, E6-AP or E6-BP); DNA (i.e., cruciform DNA); and other partners such as aptamers. In some embodiments, detection of more than 1 oncogenic E6 protein (e.g., all oncogenic E6 proteins, E6 proteins from HPV strains 16 and 18, or E6 proteins from HPV strains 16 and 45 etc.) is desirable, and, as such, an oncogenic E6 protein binding partner may be antibody that binds to these proteins, as described below, or a mixture of antibodies that each bind to a different proteins. As is known in the art, such binding partners may be labeled to facilitate their detection. In general, binding partners bind E6 with an binding affinity of less then 10~5 M, e.g., less than 10~6, less than 10~7, less than 10~8 M (e.g., less than 10~9 M, 10Λ 10-11, 1042 M, etc.).
[0033] As used herein, the term "PDZ domain" refers to protein sequence of less than approximately 90 amino acids, (i.e., about 80-90, about 70-80, about 60-70 or about 50-60 amino acids), characterized by homology to the brain synaptic protein PSD-95, the Drosophila septate junction protein Discs-Large (DLG), and the epithelial tight junction protein ZOl (ZOl). PDZ domains are also known as Discs-Large homology repeats ("DHRs") and GLGF repeats. PDZ domains generally appear to maintain a core consensus sequence (Doyle, D. A., 1996, Cell 85: 1067-76). PDZ domains are found in diverse membrane -associated proteins including members of the MAGUK family of guanylate kinase homologs, several protein phosphatases and kinases, neuronal nitric oxide synthase, tumor suppressor proteins, and several dystrophin-associated proteins, collectively known as syntrophins.
[0034] Exemplary PDZ domain-containing proteins and PDZ domain sequences may be found in U.S. Patent Nos. 7,312,041, and 7,399,467, which are herein incorporated by their entirety. The term "PDZ domain" also encompasses variants (e.g., naturally occurring variants) of the sequences (e.g., polymorphic variants, variants with conservative substitutions, and the like) and domains from alternative species (e.g. mouse, rat). Typically, PDZ domains are substantially identical to those shown in US Patent Applications 09/724553 and 10/938,249), e.g., at least about 70%, at least about 80%, or at least about 90% amino acid residue identity when compared and aligned for maximum correspondence. It is appreciated in the art that PDZ domains can be mutated to give amino acid changes that can strengthen or weaken binding and to alter specificity, yet they remain PDZ domains (Schneider et al., 1998, Nat. Biotech. 17:170-5). Unless otherwise indicated, a reference to a particular PDZ domain (e.g. a MAGI-I domain 2) is intended to encompass the particular PDZ domain and HPV E6-binding variants thereof. In other words, if a reference is made to a particular PDZ domain, a reference is also made to variants of that PDZ domain that bind oncogenic E6 protein of HPV, as described below. In this respect it is noted that the numbering of PDZ domains in a protein may change. For example, the MAGI-I domain 2, as referenced herein, may be referenced as MAGI- 1 domain 1 in other literature. As such, when a particular PDZ domain of a protein is referenced in this application, this reference should be understood in view of the sequence of that domain, as described herein, particularly in the sequence listing. U.S. Patent Nos. 7,312,041, and 7,399,467 show the sequences, the names and Genbank accession numbers for various PDZ domains, where appropriate. Further description of PDZ proteins, particularly a description of MAGI-I domain 2 protein, is found in Serial No. 10/630,590, filed July 29, 2003 and published as US20040018487. This publication is incorporated by reference herein in its entirety for all purposes.
[0035] As used herein, the term "PDZ protein" refers to a naturally occurring protein containing a PDZ domain. Exemplary PDZ proteins include CASK, MPPl, DLGl, DLG2, PSD95, NeDLG, TIP-33, SYNIa, TIP-43, LDP, LIM, LIMKl, LIMK2, MPP2, NOSl, AF6, PTN-4, prIL16, 41.8kD, KIAA0559, RGS12, KIAA0316, DVLl, TIP-40, TIAMl, MINTl, MAGI-I, MAGI-2, MAGI-3, KIAA0303, CBP, MINT3, TIP- 2, KIAA0561, and TIP-I.
[0036] As used herein, the term "PL protein" or "PDZ Ligand protein" refers to a protein that forms a molecular complex with a PDZ-domain, or to a protein whose carboxy -terminus, when expressed separately from the full length protein (e.g., as a peptide fragment of 4-25 residues, e.g., 8, 10, 12, 14 or 16 residues), forms such a molecular complex. The molecular complex can be observed in vitro using a variety of assays described infra. For example, an oncogenic E6 protein from a high-risk HPV strain is a PL protein or PL ligand protein.
[0037] In the case of the PDZ domains described herein, a "HPV E6-binding variant" of a particular PDZ domain is a PDZ domain variant that retains HPV E6 PDZ ligand binding activity. Assays for determining whether a PDZ domain variant binds HPV E6 are described in great detail below, and guidance for identifying which amino acids to change in a specific PDZ domain to make it into a variant may be found in a variety of sources. In one example, a PDZ domain may be compared to other PDZ domains described herein and amino acids at corresponding positions may be substituted, for example. In another example, the sequence a PDZ domain of a particular PDZ protein may be compared to the sequence of an equivalent PDZ domain in an equivalent PDZ protein from another species. For example, the sequence of a PDZ domain from a human PDZ protein may be compared to the sequence of other known and equivalent PDZ domains from other species (e.g., mouse, rat, etc.) and any amino acids that are variant between the two sequences may be substituted into the human PDZ domain to make a variant of the PDZ domain. In some embodiments, the PDZ domain polypeptide used to capture E6 protein in a sample is MAGI-I. For example, the sequence of the human MAGI-I PDZ domain 2 may be compared to equivalent MAGI-I PDZ domains from other species (e.g. mouse Genbank GI numbers 7513782 and 28526157 or other homologous sequences) to identify amino acids that may be substituted into the human MAGI-I-PDZ domain to make a variant thereof. Such method may be applied to any of the MAGI-I PDZ domains described herein. Particular variants may have 1, up to 5, up to about 10, up to about 15, up to about 20 or up to about 30 or more, usually up to about 50 amino acid changes as compared to a sequence set forth in the sequence listing. In making a variant, if a GFG motif is present in a PDZ domain, in general, it should not be altered in sequence. Exemplary PDZ peptide sequences are disclosed in US Patent Nos 7,312,041 and 7,399,467, which are herein incorporated by reference in their entirety.
[0038] In general, variant PDZ domain polypeptides have a PDZ domain that has at least about 70 or 80%, usually at least about 90%, and more usually at least about 98% sequence identity with a variant PDZ domain polypeptide described herein, as measured by BLAST 2.0 using default parameters, over a region extending over the entire PDZ domain.
[0039] In some embodiments, the E6 binding partner used in the method of the present invention may include, but are 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. In most embodiments, the first E6 binding partner that captures E6 protein in a sample binds to the E6 protein at a location on the E6 protein that does not reduce the availability of the E6 protein for binding to the subject synergistic antibodies.
[0040] In some embodiments, the E6 protein is a recombinant E6 fusion protein, for example, E6 fused to maltose binding protein (MBP), or glutathione-S transferase (GST). The E6 peptide or the relevant portion may 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.). In one example, MBP-E6 fusion proteins are prepared for use in the assays of the invention. PCR products containing E6 protein or a portion thereof are subcloned into an expression vector to permit expression of fusion proteins containing the E6 protein or a portion thereof and a heterologous domain (i.e., a MBP or GST).
[0041] A variety of host-expression vector systems may be utilized to express the recombinant E6 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.
Antibody Compositions
[0042] The terms "antibody" and "immunoglobulin" are used interchangeably herein to refer to a type capture agent that has at least an epitope binding domain of an antibody. These terms are well understood by those in the field, and refer to a protein containing one or more polypeptides that specifically binds an antigen. One form of antibody constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of antibody chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions.
[0043] The recognized immunoglobulin polypeptides include the kappa and lambda light chains and the alpha, gamma (IgGi, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains or equivalents in other species. Full-length immunoglobulin "light chains" (of about 25 kDa or about 214 amino acids) comprise a variable region of about 110 amino acids at the NH2-terminus and a kappa or lambda constant region at the COOH- terminus. Full-length immunoglobulin "heavy chains" (of about 50 kDa or about 446 amino acids), similarly comprise a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).
[0044] The terms "antibodies" and "immunoglobulin" include antibodies or immunoglobulins of any isotype (IgM, IgG, IgD, IgE, or IgA), fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, peptides, peptidomimetics, peptoids, peptibodies, and fusion proteins comprising an antigen- binding portion of an antibody and a non-antibody protein. The antibody may be in any suitable form e.g., monoclonal, polyclonal, or synthetic. The antibodies may be detectably labeled, e.g. , with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like. The antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like. The antibodies may also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like. Also encompassed by the terms are Fab', Fv, F(ab')2, and or other antibody fragments that retain specific binding to antigen. [0045] Antibodies may exist in a variety of other forms including, for example, Fv, Fab, and F(ab')2, as well as bi-functional (i.e. bi-specific) hybrid antibodies (e.g., Lanzavecchia et al.., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Huston et al.., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al.., Science, 242, 423-426 (1988), which are incorporated herein by reference). (See, generally, Hood et al, Immunology, Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986). Monoclonal antibodies, polyclonal antibodies, and "phage display" antibodies are well known in the art and encompassed by the term "antibodies". [0046] The invention provides a combination of antibodies, particularly monoclonal antibodies, which bind to E6 of at least one strain of HPV, in some embodiments, at least one oncogenic HPV strain. In some embodiments, the antibodies bind to E6 of multiple oncogenic strains of HPV. In other words, the invention provides antibodies that "recognize", i.e., specifically bind to with KD of 10~6 M or less, multiple E6 proteins. In other words, the subject antibodies each bind to (i.e., cross-react with) a plurality of different E6 proteins (i.e., at least 2, at least 3, at least 4, at least 5, at least 6 or at least 10, usually up to about 12, 15 or 20 or more different E6 proteins) from oncogenic, and, in certain embodiments, non-oncogenic strains of HPV. In general, the subject antibodies bind to amino acid motifs that are conserved between the E6 proteins of different HPV strains, and, accordingly, bind to E6 proteins that have this motif. In certain embodiments, the antibodies bind at least the E6 proteins of HPV strains 16 and 18 (e.g. the E6 of HPV strains 16, 18, 31, 33 and 45; 16, 18 and 45; or, in other embodiments, the E6 proteins of all of the HPV strains mentioned herein). In other embodiments, the antibodies bind to at least the E6 proteins from HPV strains 16 and 45. The subject antibodies may bind E6 from non-oncogenic strains of HPV (e.g., HPV strains 6 and/or 11) and, accordingly, the subject antibodies may bind to E6 proteins from oncogenic, as well as non-oncogenic, strains of HPV.
[0047] In some embodiments, the plurality of antibodies directed against HPV E6 protein act synergistically in a detection assay for E6 protein, resulting in greater signals, and thus enhancing the sensitivity of the assay. The term "plurality of antibodies" as used herein refers to two or more antibodies. In some embodiments, an antibody pair comprising two antibodies specific against an E6 protein is used in the subject method and the antibody pair has synergistic effects on increasing the signal-to-noise ratio and sensitivity of detecting E6 protein. In other embodiments, a plurality of antibodies of the present invention comprises more than two antibodies that have synergistic effects on binding and detecting E6 protein. [0048] The term "synergistic" refers to a final signal enhancement output (measured by a spectrophotometer in the case of an EIA) or other instrumented or visual read (in the case of a lateral flow or flow through test) when two or more antibodies are used together. The signal output is greater for the plurality of antibodies than for each of the individual antibodies alone at comparable doses, which leads to enhancement of the sensitivity of the assay. In some embodiments, the signal is greater for the plurality of antibodies used together than for each of the individual antibodies used alone at comparable doses. In other embodiments, the signal to noise (S/N) ratio is greater for the plurality of antibodies used together than for each of the individual antibodies used alone at comparable doses. For example, if a pair of antibodies is used to detect E6, the signal-to-noise ratio of E6 detection using the antibody pair is greater than that of each single antibody used alone at twice the concentration. In some embodiments, the present invention provides a method, wherein the sensitivity of detecting an E6 protein using the plurality of synergistic antibodies is increased as compared to using single antibodies alone to detect E6 protein. In some embodiments, the signal-to-noise ratio of detecting an E6 protein using the plurality of synergistic antibodies is increased as compared to using single antibodies to detect E6 protein. [0049] The subject synergistic antibodies may bind to any portion or any length of the HPV E6 proteins. In some embodiments, the subject synergistic antibodies may bind to one of three sequence motifs found in HPV E6 proteins. These motifs generally correspond to regions of sequence similarity between E6 proteins from different strains of HPV. In general, therefore, a subject antibody binds to peptides having the following sequence: FQDPQERPRKLPQLCTELQTTIHDI and FEDPTRRPYKLPDLCTELNTSLQDI, corresponding to a first common sequence motif in the E6 proteins of HPV strains 16 and 18, respectively, LLIRCINCQKPLCPEEKQRHLDK and LLIRCLRCQKPLNPAEKLRHLNE, corresponding to a second common sequence motif in the E6 proteins of HPV strains 16 and 18, respectively, or RHLDKKQRFHNIRGRWTGRCMSCC and RHLNEKRRFHNIAGHYRGQCHSCC corresponding to a third common sequence motif in the E6 proteins of HPV strains 16 and 18, respectively. If a subject antibody binds to other E6 proteins, then it usually binds to the other E6 proteins at positions equivalent to those discussed above, where "positions equivalent to" generally means a stretch of contiguous amino acids that correspond to, i.e., are aligned with, the boxed amino acids when the sequence of the other E6 proteins are with those in Fig. 1 of U.S Patent No. 7,399,467, which is incorporated by reference herein. [0050] Accordingly, since antibodies generally recognize motifs smaller than those listed above, a subject antibody may recognize peptides that are smaller than and contained within the motifs described above. For example, a subject antibody may bind to a peptide having any 9 contiguous amino acids set forth in any one of SEQ NOS:l-6. In particular, a subject antibody may recognize the sequences RPRKLPQLCTEL and RPYKLPDLCTEL, corresponding to sub-sequences of the first common sequences of E6 proteins of HPV strains 16 and 18, described above, LLIRCINCQKPL and LLIRCLRCQKPL corresponding to sub-sequences of the second common sequences of E6 proteins of HPV strains 16 and 18, as described above, or RHLDKKQRFHNI and RHLNEKRRFHNI corresponding to sub-sequences of the third common sequences of E6 proteins of HPV strains 16 and 18, as described above. Since these sub-sequences are generally conserved between different E6 proteins, as discussed above, antibodies that bind to the above-recited sequences generally bind to E6 proteins from other HPV strains.
[0051] In certain alternative embodiments, the subject antibodies will bind to E6 proteins from HPV strains 16 and 45. In general, therefore, a subject antibody binds to peptides having the following sequence: FQDPQERPRKLPQLCTELQTTIHDI and FDDPKQRPYKLPDLCTELNTSLQDV, corresponding to a first common sequence motif in the E6 proteins of HPV strains 16 and 45, respectively,
LLIRCINCQKPLCPEEKQRHLDK and LLIRCLRCQKPLNPAEKRRHLKD, corresponding to a second common sequence motif in the E6 proteins of HPV strains 16 and 45, respectively, or RHLDKKQRFHNIRGRWTGRCMSCC and RHLKDKRRFHSIAGQYRGQCNTCC corresponding to a third common sequence motif in the E6 proteins of HPV strains 16 and 45, respectively. If a subject antibody binds to other E6 proteins, then it usually binds to the other E6 proteins at positions equivalent to those discussed above, or boxed in Fig. 1 of US Patent No. 7,399,467, which is herein incorporated by reference. [0052] Accordingly, since antibodies generally recognize motifs smaller than those listed above, a subject antibody may recognize peptides that are smaller than and contained within the motifs described above. For example, a subject antibody may bind to a peptide having any nine contiguous amino acids set forth in any one of SEQ NOS: 1, 3, 5, 57, 58 and 59. In particular, a subject antibody may recognize the sequences RPRKLPQLCTEL and RPYKLPDLCTEL, corresponding to sub-sequences of the first common sequences of E6 proteins of HPV strains 16 and 45, described above, LLIRCINCQKPL and LLIRCLRCQKPL corresponding to sub-sequences of the second common sequences of E6 proteins of HPV strains 16 and 45, as described above, or RHLDKKQRFHNI and RHLKDKRRFHSI corresponding to sub-sequences of the third common sequences of E6 proteins of HPV strains 16 and 45, as described above. Since these subsequences are generally conserved between different E6 proteins, as discussed above, antibodies that bind to the above -recited sequences generally bind to E6 proteins from other HPV strains. In certain embodiments, cysteine residues can be replaced by serine residues to avoid disulfide bridge formation. [0053] In some embodiments, the subject antibodies may bind to the C-terminus of a HPV protein, for example, the C-terminus of E6 protein. Antibodies specific for the HPV C-terminal PDZ ligand (PL) motif may be used for both capture and detection of E6 protein of at least one HPV strain, and for the treatment of HPV infection.
Methods of Generating Antibodies
[0054] 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.
[0055] Monoclonal antibodies to a peptide may be prepared using any technique that 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.
[0056] Antibody fragments containing 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.
[0057] 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., NY, Livingstone, 1974, Methods Enzymology: Imnmunoaffinity 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.
[0058] 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.
[0059] Methods for making antibodies, particular monoclonal antibodies, are well known in the art and described in various well known laboratory manuals (e.g., Harlow et al., Antibodies: A Laboratory Manual, First Edition (1988) Cold spring Harbor, N.Y.; Harlow and Lane, Using Antibodies : A Laboratory Manual, CSHL Press (1999) and Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, (1995)). Accordingly, given the peptide sequences, methods for making the subject antibodies do not need to be described herein in any great detail. Any fragment of a longer full-length E6 protein that contains a subject common motif (e.g., the full length protein), a full length E6 protein, or a fusion protein thereof may be used to make the subject antibodies. In certain embodiments, a full length E6 protein, a peptide containing a recited sequence, or a chemically modified (e.g., conjugated) derivative or fusion thereof (e.g., a MBP or GST fusion), may be used as an antigen. In certain embodiments, a nucleic acid encoding the polypeptide may be employed, or a mixture of different polypeptides (e.g., a mixture of E6 polypeptides, each polypeptide from a different HPV strain) may be used as an antigen (Michel (2002) Vaccine 20:A83- A88). Accordingly an antigen is mixed with an adjuvant, and a suitable non-human animal (e.g., a mouse, chicken, goat, rabbit, hamster, horse, rat or guinea pig, etc.) is immunized using standard immunization techniques (e.g., intramuscular injection) and once a specific immune response of the has been established, blood from the animal may be collected and polyclonal antisera that specifically binds to described peptides may be isolated. In many cases, cells from the spleen of the immunized animal are fused with a myeloma cell line, and, after fusion, the cells are grown in selective medium containing e.g., hypoxanthine, aminopterin, and thymidine (HAT), to select for hybridoma growth, and after 2-3 weeks, hybridoma colonies appear. Supernatants from these cultured hybridoma cells are screened for antibody secretion, usually by enzyme-linked immunosorbent assay (ELISA) or the like, and positive clones secreting monoclonal antibodies specific for the antigen can be selected and expanded according to standard procedures. [0060] Exemplary peptides of E6 suitable for immunizations are described in Table 1. The peptides are shown as a "consensus" sequence (i.e. peptides in which one of several amino acids may exist at one or more positions) in order to indicate that any one or a mixture of different peptides that are described by the consensus could be used to make the subject antibodies. Accordingly, when a consensus sequence is described, every individual peptide that falls within the consensus should be considered explicitly described. In particular embodiments, exemplary species of peptide encompassed by the consensus sequences have a sequence found in a naturally-occurring HPV E6 protein. Such exemplary sequences can be identified as sequences starting at the amino acid positions defined by the third column of Table 1, "Starting AA" of particular HPV types "HPV type", and corresponding positions of other HPV E6 proteins (i.e., those positions that are aligned with the positions indicated in Table 1). Antibodies that specifically bind to E7 protein can be generated via immunization method as described herein.
[0061] Accordingly, peptides having 9, 10, 11, 12, 13, 14, 15 or more, usually up to about 20 or more contiguous amino acids of any of the peptides described above may be used for immunizations. In some embodiments, a recited peptide sequence may be contained within a larger peptide that may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more, sometimes up to about 15 or 20 or more amino acids greater in size than a recited polypeptide. Accordingly, a subject peptide may be from about 8 to about 30 amino acids in length. In certain embodiments, a subject peptide is about 9-20 amino acids in length, and usually contains an amino acid sequence described above.
[0062] Accordingly, depending on the antibodies desired, a suitable animal is immunized with a subject peptide or a mixture of subject peptides (e.g., a mixture of 2, 3, 4, 5 about 6 or more, about 10 or more or about 15 or more, usually up to about 20 or 30 or more peptides described above). Antibodies are usually isolated from the animal and tested for binding to different HPV E6 proteins using standard methods (e.g., ELISA, western blot, etc.). In many embodiments, therefore, antibodies will be screened for binding to E6 and E7 proteins from HPV strains 16 and 18, HPV strains 16, 18, 31, 33 and 45, or, in certain embodiments, all of the HPV strains mentioned herein, and maybe others. Accordingly, antibodies that bind to, i.e., cross- react with, E6 proteins from more than one strain of HPV may be identified, and permanent cell lines producing those antibodies may be established using known methods. In other words, antibodies are usually tested for binding to more than one antigen, and those antigens are usually E6 proteins from various HPV strains, or fragments thereof. In most embodiments, the antibodies will be tested for binding to antigens in native and denatured states. Antibodies that bind to a plurality of E6 proteins have desirable binding properties, and, accordingly, find use in the subject methods.
[0063] In one example, polynucleotides encoding E6 proteins of high-risk HPV types listed hereinabove may be chemically synthesized (DNA 2.0, Menlo Park, California) or cloned via RT-PCR from cervical cancer cell lines. Both maltose-binding-protein-E6 (MBP-E6) and glutathione-S-transferase-E6 (GST -E6) fusion protein types can be used. Production of GST-E6 and MBP-E6 proteins can be by standard protocols recommended by the suppliers (Amersham and New England Biolabs, respectively). Proteins are expressed in DH5α E. coli using IPTG driven induction. A 2-hour induction at 37° C yields GST-E6 or MBP-E6 recombinant proteins at about 1 mg/L, whereas induction overnight at 20° C and purification including rebinding of protein to the gel matrix may result in a final yield of 2-10mg/L. Purity of MBP-E6 proteins is estimated to be > 90% based on PAGE analysis. Recombinant E6 fusion proteins can be used as the immunogens.
[0064] Mice can then be immunized with each of the HPV E6 proteins. A variety of immunization protocols including varying antigen doses (100 μg-10 μg), adjuvants (CFA/IFA, poly(I)-poly(C), CpG+ Alum) and routes (subcutaneous, intraperitoneal) are tested. Immunization projects are set up with 5-15 mice each. Sera of immunized mice are tested in ELISA against the recombinant E6 protein. Mice showing sufficiently high titers (OD above 1 at 1 : 1000 dilution) against E6 in their sera are selected for fusions. To increase the frequency of hybridomas secreting of anti-E6 antibodies, the recombinant E6 protein used in the final boost may contain a different tag from that used during the immunization, for example, GST-E6 is used in the boost when immunizations occurs with MBP-E6, and vice versa.
[0065] As is well known in the art, the subject antibodies may be conjugated to a detectable label, or may be part of a signal generating system, as described above.
[0066] Accordingly, using the methods set forth above, an antibody composition for detecting a plurality of HPV E6 proteins is provided. In certain embodiments, a mixture of different antibodies that recognize at least 5, 7, 9, 12, 15, 20 or 24 different strains of HPV may be employed. The composition may contain a combination of antibodies that recognize at least 3 different oncogenic E6 proteins. The composition may contain 1, 2, 3, 4, or 5 or more different antibodies, each antibody of the composition recognizing at least one (e.g., 2, 3, about 5, about 10, etc.) E6 proteins. Collectively, the antibodies bind to all or a portion of the E6 proteins from HPV strains mentioned herein, and, in certain embodiments, may also bind to non-oncogenic E6 proteins. The antibodies may be mixed, or separate from each other, i.e., in different vessels.
[0067] Any of the above-described antibodies may bind to an epitope set forth in Table 1. Table 1: Epitopes
Figure imgf000020_0001
[0068] Certain hybridomas that produce the monoclonal antibodies described above and below may be deposited at the American Type Culture Collection (ATCC), for example, hybridoma cell lines 4E9.7 (PTA- 9679), 4E10.2 (PTA-9680, and 6H5.3 (PTA-9681). Any of the deposited hybridomas, the antibodies produced by those hybridomas, as well as other antibodies that bind the same epitopes as the antibodies produced by those hybridomas, are also embodiments of this invention and may be claimed herein. Such antibodies may be employed in any of the methods described herein. Detailed examples of generating anti-E6 monoclonal antibodies are disclosed in US Patent Nos 7,312,041 and 7,399,467, which are herein incorporated by reference in their entirety.
Synergistic Antibodies for Detecting HPV E6 Proteins in a Sample
[0069] In one aspect, the present invention provides a method of detecting E6 protein of a HPV strain in a sample, the method comprising: (a) contacting an E6 protein binding partner with the sample; (b) contacting the E6 protein that is bound to the E6 protein binding partner with a plurality of synergistic antibodies that specifically bind to the E6 protein; and (c) detecting binding of the plurality of synergistic antibodies to the E6 protein, thereby detecting the E6 protein in the sample.
[0070] In general, the method involves contacting a sample suspected of having HPV with an E6 protein binding partner that specifically binds to an E6 protein, contacting the E6 protein that is bound to the E6 binding partner in the first step with the plurality of synergistic antibodies of the present invention, and assessing any binding of the plurality of synergistic antibodies to the E6 protein. In most embodiments, binding of the plurality of synergistic antibodies to the E6 protein indicates the presence of HPV, for example, an oncogenic HPV strain, in the sample. In some embodiments, the plurality of synergistic antibodies specifically binds to E6 proteins of HPV16 or HPV18. In some embodiments, the plurality of synergistic antibodies specifically binds to E6 proteins of HPV strains selected from the group consisting of HPV- 16, 18, 26, 30, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, and 82. In some embodiments, the E6 protein binding partner is a PDZ domain containing polypeptide. The PDZ domain polypeptide may be MAGI-I or any of the PDZ domain polypeptide selected from the group consisting of CASK, MPPl, DLGl, DLG2, PSD95, NeDLG, TIP-33, SYNIa, TIP-43, LDP, LIM, LIMKl, LIMK2, MPP2, NOSl, AF6, PTN-4, prILlό, 41.8kD, KIAA0559, RGS12, KIAA0316, DVLl, TIP-40, TIAMl, MINTl, MAGI-I, MAGI- 2, MAGI-3, KIAA0303, CBP, MINT3, TIP-2, KIAA0561, and TIP-I, as described hereinabove. [0071] In some embodiments, the subject synergistic antibodies are monoclonal. The plurality of synergistic antibodies can be unconjugated or directly or indirectly labeled. In some embodiments, the paired antibodies bind to different epitopes on an E6 protein. For example, a peptide corresponding to the N-terminal region of E6 has been used to demonstrate different binding specificities of the monoclonal antibodies of the present invention. Of the monoclonal antibodies that act synergistically, only one of the pair is inhibited by the N- terminal peptide, indicating the two antibody members of a synergistic antibody pair do not share the identical binding site on E6 protein.
[0072] In some embodiments, the method of the present invention is carried out via an enzyme immunoassay (EIA). In one example, the enzyme immunoassay is a sandwich enzyme-linked immunosorbent assay (ELISA). The plurality of synergistic antibodies of the present invention can be used in many immunoassays and variations thereof including but not limited to lateral flow, flow-through, immunofiltration, chemiluminescent EIA, fluorescent EIA, and sandwich ELISA. In one example, an enzyme based immunoassay test procedure (HPV EIA) is used as the detection assay to demonstrate the synergistic effect of the antibody pair of the present invention in binding and detecting E6 proteins in a sample. The synergistic pair of antibodies can then be utilized in additional assay test formats such as lateral flow and "flow-through" (i.e. Directigen™ RSV). The immunoassays are well known techniques in the art and are described herein, infra. [0073] The immunoassays which can be used for the subject methods include but are not limited to competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, immunohistochemistry, and cellular immuno staining (fixed or native) assays to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
[0074] In some embodiments, the method of the present invention utilizes an enzyme linked immunosorbent assay (ELISA), preferably a sandwich ELISA, to detect the presence of, for example, oncogenic E6 proteins bound to the plurality of synergistic antibodies of the present invention. As used herein, the terms "sandwich", "sandwich ELISA", "sandwich diagnostic" and "capture ELISA" all refer to the concept of detecting a biological polypeptide with two different test agents. ELISA is a well known technique in the art. Briefly, ELISA involves preparing antigen, coating the well of a 96 well multiwell plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISA, the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. In another variation, an antigen binding partner, for example, a PDZ domain polypeptide, may be coated to the well. In this case, antibodies, for example, the subject synergistic antibodies, either unconjugated or conjugated to a detectable compound may be added following the capture of the antigen of interest by the first antigen binding partner. The first antigen binding partner binds E6 protein at a location on the E6 protein that does not reduce the availability of the E6 protein for its binding to the plurality of synergistic antibodies of the present invention. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art.
[0075] In some embodiments, the method of the present invention involves testing samples of human cervical tissue culture cell lines with plurality of synergistic antibodies of the present invention directed against HPV E6 protein. Results, for example, signal to noise ratios, are compared to results obtained with each of the individual antibodies. In some embodiments, limit of detection (LOD) testing of serially diluted extracted human cervical cell lines, for example, SiHa cells, is performed with the identified plurality of synergistic antibodies and compared to results obtained from each single antibody to determine if endpoint sensitivity is improved. [0076] In one example, for illustrative purposes without any limitation to a particular embodiment, the EIA consists of a microwell plate coated with a PDZ domain-bearing protein to capture a recombinant MBP E6 fusion protein or extracted E6 derived from human cervical cell lines. The assay works by coating the wells of the microwell plate overnight at 4°C with a PDZ domain -bearing protein. The wells are washed with an automatic microwell plate washer the next morning and blocked for two hours at 25°C. Wells are washed again and extracted human cervical cell line sample (E6 positive or negative) are added to the well and incubated for an hour at 25°C. Following another wash step, the antibodies are added to the plate as either pairs or by themselves and incubated for an hour at 25°C. A suitable secondary antibody conjugated to an enzyme (ie. Goat anti mouse horseradish peroxidase) is added to the wells after another wash step and incubated again for an hour at 25°C. One final wash step is performed and the corresponding soluble substrate (i.e. 3, 3', 5, 5'-Tetramethylbenzidine (TMB) is added to the wells and incubated for thirty minutes at 25°C. The corresponding stop solution (diluted acid solution such as sulfuric acid) is added to the wells and the microplate is then read at an absorbance of 450nm (A450) in a standard microplate UV/Vis plate reader.
[0077] For a sandwich EIA, signal to noise ratio for each of the plurality of synergistic antibodies can be determined from the A450 signals generated from extracted cervical cell lines that either contain (ie. SiHa) or do not contain (i.e. C33A-) the E6 protein. The signal to noise ratio for the plurality of synergistic antibodies is compared to the signal to noise ratio determined for each individual antibody. An increase in the signal to noise for a given plurality of synergistic antibodies over that of the individual antibody demonstrates a synergistic effect of the plurality of synergistic antibodies.
[0078] Synergistic antibodies can then be run in the conventional HPV EIA. In some embodiments, serial dilutions of extracted HPV positive cells, for example, SiHa cells are tested with the synergistic antibody pairs in comparison to the individual antibodies and the endpoint sensitivity of the assay is compared. [0079] 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 antibodies; antibodies recognizing different E6 epitopes can be used; different labels for detecting antibody-E6 interactions can be employed; and different ways of detection can be used.
[0080] The method of the present invention can employ a variety of surfaces or substrates to bind the polypeptides, antigens, antibodies or antibody fragments. By "solid phase support" or "substrate" is intended any support capable of binding polypeptide, antigen or antibody. Well-known supports or substrates, include but are not limited to glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, nitrocellulose, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material can have virtually any possible structural configuration so long as the coupled molecule is capable of binding to a PDZ domain polypeptide or an E6 antibody. Thus, the support configuration can be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface can be flat, such as a sheet, culture dish, test strip, etc. Those skilled in the art will know many other suitable carriers for binding antibody, peptide or antigen, or can ascertain the same by routine experimentation.
[0081] In another example, a surface can be an "assay plate" which is formed from a material (e.g. polystyrene) which optimizes adherence of 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.
[0082] For example, the assay plate can be a "microliter" plate. The term "microliter" 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 μl. 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 μl to 300 μl, more preferably 100 μl to 200 μl, of an aqueous sample comprising buffers suspended therein will be added to each well of the assay plate. [0083] In some embodiments, the plurality of synergistic antibodies, one or all members of the plurality of antibodies can be labeled. As used herein, a "detectable label" has the ordinary meaning in the art and refers to an atom (e.g., radionuclide), molecule (e.g., fluorescein), or complex, that is or can be used to detect (e.g., due to a physical or chemical property), indicate the presence of a molecule or to enable binding of another molecule to which it is covalently bound or otherwise associated. The term "label" also refers to covalently bound or otherwise associated molecules (e.g., a biomolecule such as an enzyme) that act on a substrate to produce a detectable atom, molecule or complex. Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads.TM.), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, enhanced green fluorescent protein, and the like), radiolabels (e.g., 3H, 1251, 35S, 14C, or 32P), enzymes (e.g., hydrolases, particularly phosphatases such as alkaline phosphatase, esterases and glycosidases, or oxidoreductases, particularly peroxidases such as horse radish peroxidase, and others commonly used in ELISAs), substrates, cofactors, inhibitors, chemiluminescent groups, chromogenic agents, and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels and chemiluminescent labels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted light (e.g., as in fluorescence-activated cell sorting). Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal generating system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, for example, biotin, thyroxine, and Cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody. The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter, photographic film as in autoradiography, or storage phosphor imaging. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Also, simple colorimetric labels may be detected by observing the color associated with the label. It will be appreciated that when pairs of fluorophores are used in an assay, it is often preferred that they have distinct emission patterns (wavelengths) so that they can be easily distinguished.
[0084] Direct labels may include but are not limited to radioisotopes (e.g., 125I; 35S, and the like); enzymes whose products are detectable (e.g., luciferase, β-galactosidase, horseradish peroxidase, and the like); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, and the like); fluorescence emitting metals, e.g., 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; 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.
[0085] Sometimes, the label is 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, Ausubel, 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.
[0086] 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.
[0087] Various buffers can also be used in the sandwich detection assays of the present invention. 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 an antigen, an antibody, or a PDZ-containing protein. 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.
[0088] Various enzyme-substrate combinations can also be utilized in detecting the first antibody -E6-second antibody sandwich interactions. Examples of enzyme-substrate combinations include but are not limited to, for example:
[0089] (i) Horseradish peroxidase (HRP or HRPO) with hydrogen peroxide as a substrate, wherein the hydrogen peroxide oxidizes a dye precursor (e.g. orthophenylene diamine [OPD] or 3,3',5,5'-tetramethyl benzidine hydrochloride [TMB]) (as described above).
[0090] (ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate.
[0091] (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. [0092] 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 in their entirety.
[0093] Preferred embodiments of the antibody ELISA are described in detail herein. 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. In some embodiments, the E6 binding partner, for example, the PDZ domain-containing polypeptides are immobilized on a solid surface. The substrate to which the polypeptide is bound may be 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 semi-permeable membrane, and the like. Suitable materials include glass, plastic (e.g., polyethylene, PVC, polypropylene, polystyrene, and the like), nitrocellulose, 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.
[0094] In some embodiments, the PDZ domain containing polypeptides are organized as an array. The term "array," as used herein, refers to an ordered arrangement of immobilized proteins, in which particular different proteins (i.e., recognizing different E6 proteins of different HPV strains, or different epitopes of E6 protein) are located at different predetermined sites on the substrate. Because the location of particular antibodies on the array is known, binding at that location can be correlated with binding to the antigen situated at that location. Immobilization of antibodies on beads (individually or in groups) is another particularly useful approach. In one embodiment, individual antibodies 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 flow cytometry) 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 HPV protein may be determined. [0095] 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. [0096] 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 cm 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 cmχ8.3 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 10, at 4° C.) to 37° C. and from 1 hr to more than 24 hours. Typically mAbs are attached to plastic with carbonate buffers of pH 8.5 or 9.6. Proteins may be covalently bound or noncovalently attached through nonspecific bonding. If covalent bonding between the 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.
[0097] In some embodiments, the PDZ domain containing polypeptide is immobilized and binding of the captured E6 protein to the plurality of synergistic antibodies of the present invention is detected. In one example, the capture of the E6 protein by the PDZ domain containing polypeptide is carried out as follows: a PDZ-domain polypeptide is bound to a surface, e.g. a protein binding surface. In a preferred embodiment, a PDZ-domain polypeptide is bound to a polystyrene 96-well plate. The PDZ-domain polypeptide 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 PDZ-domain polypeptide to the plate does not alter the ligand-binding properties of the PDZ domain. One example of an adequate binding to the plate can be achieved when: 50 μL per well of 5 μg/mL PDZ-domain polypeptide or, a non-PDZ containing polypeptide as a negative control, in PBS/BSA is added to a polystyrene 96-well plate for 2 hours at 40C. The plate is again washed 3 times with PBS. E6 peptides are introduced to the plate and allowed to react with the surface by addition of 50 uL per well of 20 uM solution of the E6 peptide in PBS/BSA for 10 minutes at 40C followed by an additional 20 minute incubation at 250C. The plate is washed 3 times with ice cold PBS. The binding of the E6 peptide to the PDZ-domain polypeptide can then be incubated with the plurality of synergistic antibodies of the present invention. The binding and detection of E6 protein by the subject synergistic antibodies may be via any immunological based assay, such as immunoprecipitation, western blotting, enzyme immunoassays (EIA), RAMAN spectroscopy, lateral flow, flow-through, Directigen™, and cytometric bead array (CBA). These assays are well known in the art and are briefly described herein, infra. [0098] Immunoprecipitation protocols generally involve lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-4O or Triton X-IOO, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/ or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 40C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads).
[0099] Western blot analysis generally involves preparation of protein samples followed by electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), and transfer of the separated protein samples from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon. Following transfer, the membrane is blocked in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washed in washing buffer (e.g., PBS-Tween 20), and incubated with primary antibody (the antibody of interest) diluted in blocking buffer. After this incubation, the membrane is washed in washing buffer, incubated with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 1251), and after a further wash, the presence of the antigen may be detected. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise.
[00100] Surface-enhanced Raman scattering nanotechnoiogy has been a valuable tool for surface and interfacial research. The enhancement associated with this process overcomes the low traditional low sensitivity problem in the normal RAMAN scattering. One of the specific applications is that it makes detection of minute quantities of biochemical chemicals, e.g., DNA, RNA, proteins, on metal surfaces feasible for bio-detection and bio-fingerprinting. In some embodiments, Ramon spectroscopy nanotechnoiogy may be used to detect binding of the subject antibodies to oncogenic E6 proteins. [00101] In some embodiments, a cytometric bead array (CBA) may be used to detect binding of the subject synergistic antibodies to oncogenic E6 proteins. A Cytometric Bead Array (CBA), commonly referred to as a multiplexed bead assay, is a series of spectrally discrete particles that can be used to capture and quantitate soluble analytes, for example, an E6 protein. The analyte is then measured by detection of a fluorescence- based emission and flow cytometric analysis. The basic "sandwich assay" schema and protocols for the CBA are available at BD Biosciences. The CBA generates data that is comparable to ELISA based assays, but in a "multiplexed" or simultaneous fashion. Concentration of unknowns is calculated for the cytometric bead array as with any sandwich format assay, i.e. through the use of known standards and plotting unknowns against a standard curve.
[00102] In some embodiments, an immuno-lateral flow assay may be used to detect binding of the subject synergistic antibodies to oncogenic E6 proteins in a sample. In some embodiments, oncogenic HPV E6 is separated on a test strip. For example, oncogenic HPV E6 may be detected using a detectably labeled antibody of the present invention that binds oncogenic HPV E6. Oncogenic HPV E6 may be quantitated using a reflectance spectrophotometer, or by eye, for example. Methods and compositions for analyte detection are disclosed in US Patent Application Publication No. US20080199851, which is herein incorporated by reference in its entirety.
[00103] In some embodiment, E6 protein from one oncogenic HPV strain is present and detected on the test strip using the plurality of synergistic antibodies and the method of the present invention. In other embodiments, the antibody pair and the method of the present invention allows for detection of E6 proteins from at least two oncogenic HPV strains in a sample, for example, HPV16 and HPV18. In some embodiments, the plurality of synergistic antibodies specifically binds to E6 proteins of at least 3, 4, 5, 6, 7, 8, 9, 10 or more different oncogenic HPV strains including but not limited to HPV 16, 18, 26, 30, 31, 34, 39, 45, 51, 52, 53, 58, 59, 66, 68, 69, 70, 73, and 82. HPV strain-specific E6 protein detection allows for a strip test for detecting the presence of E6 proteins, in which different HPV strains can be detected at distinct test lines on one test strip.
[00104] In many embodiments, oncogenic HPV E6 is separated from other proteins in a 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, which are herein incorporated by reference in their entirety. 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 a capture agent, which can be a PDZ domain containing polypeptide, for example. 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 PDZ domain polypeptide 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 anti-E6 plurality of synergistic antibodies of the present invention as detector. The subject synergistic antibodies may be used as a detector antibody composition in a lateral flow assay. For example, one or both members of the detector anti-E6 antibody pair may be detectably labeled. In some embodiments, the detector antibody pair is specific for an epitope of E6 proteins that is common to all oncogenic E6 proteins, or a mixture of paired antibodies that can, together, bind to all oncogenic E6 proteins. [00105] In some embodiments, an immunohistochemical assay may be used to detect binding of the subject synergistic antibodies to oncogenic E6 proteins in a sample, for example, a histological sample. In some embodiments, the assay may be slide based detection of E6 proteins. Immunohistochemistry or IHC refers to the process of locating proteins in cells of a tissue section exploiting the principle of antibodies binding specifically to antigens in biological tissues. Visualizing an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyze a color-producing reaction. Alternatively, the antibody can also be tagged to a fluorophore, such as FITC, rhodamine, Texas Red or Alexa Fluor. For IHC, the sample may be either thin (about 4-40 μm) slices taken of the tissue of interest, or the whole tissue if the tissue is not very thick and is penetrable. The sample used in IHC with the subject antibody for detection of oncogenic E6 proteins may be a cervical scrape or cervical biopsy.
[00106] In some embodiment, a Directigen™ assay format is used to detect HPV E6 protein. Directigen™ is a rapid chromatographic immunoassay for the direct and qualitative detection of viruses, such as influenza A and B viral antigens, from a biological sample such as nasopharyngeal washes/aspirates, nasopharyngeal swabs and throat swabs of symptomatic patients. The Directigen™ EZ Flu A+B test is a differentiated test, such that influenza A viral antigens can be distinguished from influenza B viral antigens from a single processed sample using a single device. The test is to be used as an aid in the diagnosis of influenza A and B viral infections. The BD Directigen™ EZ RSV (Respiratory syncytial virus) test utilizes a new, improved format that requires only one reagent and one device for rapid RSV detection. The testing device employs innovative lateral flow technology, a method whereby dried reagents have already been applied to the testing strip. Workflow is reduced to a two-step process, with clear-cut results shown by the appearance of an easy- to-read double line, indicating a positive result for RSV, along with the Internal Control line. These assay formats can all be used to carry out the method of the present invention for detection of E6 protein in a sample.
[00107] In alternative embodiments, a sample may be contacted with a subject plurality of synergistic antibodies of the invention, and the presence of an oncogenic E6 protein may be detected using the PDZ domain containing polypeptide. Binding of an oncogenic HPV E6 protein to the PDZ domain polypeptide and a subject plurality of synergistic antibodies indicates the presence of an oncogenic HPV E6 protein in the sample, and thus the presence of an oncogenic HPV strain. In this case, a "tagged" version of a PDZ domain containing polypeptide that specifically recognizes oncogenic E6 proteins can be used to detect the presence of oncogenic E6 protein in a sample. 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.
Identification of Synergistic Antibodies
[00108] In one aspect, the present invention provides a method of screening antibodies for synergistic antibodies that bind to E6 protein of a HPV strain with enhanced sensitivity as compared to an individual antibody specific against the E6 protein, the method comprising: (a) contacting an E6 binding partner, for example, a PDZ domain containing polypeptide, with the E6 protein; (b) contacting the E6 protein that is bound to the E6 binding partner with at least two anti-E6 antibodies; (c) detecting binding of the anti-E6 antibodies to the E6 protein; and selecting the synergistic anti-E6 antibodies that have a higher signal-to- noise ratio as compared to the individual antibodies alone or a single anti-E6 antibody used at twice the amount; wherein a higher signal-to-noise ratio indicates an enhanced sensitivity of E6 protein detection. [00109] The antibodies of the invention may be screened for immunospecific binding by any method known in the art. The immunoassays which can be used in identifying synergistic antibodies include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and cellular immunostaining (fixed or native) assays to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
[00110] The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.
[00111] Antibodies of the invention may be screened using immunocytochemisty methods on cells (e.g., mammalian cells, such as CHO cells) transfected with a vector enabling the expression of an antigen or with vector alone using techniques commonly known in the art. Antibodies that bind antigen transfected cells, but not vector-only transfected cells, are antigen specific.
[00112] In certain embodiments, however, the assay is an antigen capture assay, and an array or microarray of antibodies may be employed for this purpose. Methods for making and using microarrays of polypeptides are known in the art (see e.g. U.S. patents 6,372,483, 6,352,842, 6,346,416 and 6,242,266). [00113] Various known methods can be used to purify the identified synergistic antibodies. These methods include but are not limited to immunofiltration or affinity purification of antibodies. Briefly, immunofiltration refers to purification of antibodies by mixing with specific antigen. The antigen is then removed from the antibody by treatment with soluble carriers. Alternatively, hybridoma cells whose supernatants give the desired activity are selected for cloning. In one example, cells are cloned by limiting dilution in a 96-well flat bottom plate. Purification of antibodies from tissue culture supernatants can be performed by protein G and A affinity chromatography (Amersham). The isotype of the antibodies can be determined using Cytometric bead array. Antibody purification methods and protocols are well established in the art and are within the knowledge of one skilled in the art.
Sample Collection
[00114] Biological samples to be analyzed using the methods of the invention may be 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.
[00115] 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). 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).
[00116] 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. In other embodiments, the sample is a blood or urine sample. In still other embodiments, the sample is a histological sample. [00117] 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 protease inhibitor to inhibit degradation of the E6 protein.
[00118] 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. A suitable control may also include a sample taken from an individual with recurring HPV infections which clear spontaneously.
[00119] In certain embodiments, a sample is contacted to a solid support-bound PDZ domain polypeptide under conditions suitable for binding of the PDZ domain polypeptide to E6 proteins in the sample, and after separation of unbound sample proteins from the bound proteins, the bound proteins are detected using the subject synergistic antibodies using known methods.
[00120] Diagnosing the presence of pathogens requires collection of samples appropriate to the organism. For detection of HPV E6 proteins, one would collect tissue for testing from the cervix, penis, anus, head or neck region (e.g. in the nasal cavity, sinuses, lips, mouth, salivary glands, throat, or larynx) 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.
[00121] This invention is not intended to cover sampling devices. However, it should be noted that since the invention is predicated on the detection of E6 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.
[00122] 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. [00123] Although the diagnostic method disclosed in this application is directed at the detection of E6 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 and E7 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.
[00124] "Subject", "individual," "host" and "patient" are used interchangeably herein, to refer to any animal, e.g., mammal, human or non-human. Generally, the subject is a mammalian subject. Exemplary subjects include, but are not necessarily limited to, humans, non-human primates, mice, rats, cattle, sheep, goats, pigs, dogs, cats, birds, deer, elk, rabbit, reindeer, deer, and horses, with humans being of particular interest.
Specificity and Sensitivity
[00125] In some embodiments, the method of the present invention results in a higher signal and thus an increased signal to noise ratio of detecting an E6 protein of HPV as compared to using individual antibodies to detect the E6 protein. In some embodiments, the method of the present invention has higher sensitivity of detecting a HPV E6 protein as compared to using individual antibodies to detect the E6 protein. In some embodiments, the method of the present invention has higher specificity of detecting a HPV E6 protein as compared to using individual antibodies to detect the E6 protein. In other embodiments, the method of the present invention has lower false positive rate of detecting a HPV E6 protein as compared to using individual antibodies to detect the E6 protein.
[00126] In some embodiments, the term "specific binding" or "specificity" refers to the ability of an antibody or a combination of antibodies to preferentially bind to a particular analyte or component that is present in a homogeneous mixture of different analytes or components in a biological sample. The term "analyte" is used herein interchangeably and refers to a known or unknown component of a sample. Typically, a specific binding interaction will discriminate between desirable and undesirable analytes in a sample, typically more than about 10 to 100-fold or more (e.g., more than about 1000- or 10,000-fold). Typically, the affinity between a capture agent e.g. an antibody or a polypeptide and an analyte in a sample when they are specifically bound in an antibody /antigen complex is at least 10" , at least 10" M, at least 10" M, usually up to about 10"10 M. In some embodiments, specificity refers to the proportion of people without HPV who have a negative test result.
[00127] In some embodiments, the specificity of the HPV detection method of the present invention is increased as compared to using individual antibodies that bind to E6 protein to detect HPV. In some embodiments, the specificity of the HPV detection method of the present invention is increased to about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more than 99.9%. In some embodiments, the rate of erroneously detecting an HPV strain when such HPV strain is in fact absent in a sample is decreased as compared to using individual antibodies that bind to E6 protein to detect HPV. In some embodiments, the rate of erroneous detection of HPV, e.g. the false positive rate, is decreased to about 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or less than 0.1%.
[00128] In some embodiments, the signal to noise ratio of the HPV detection method using synergistic antibodies of the present invention is increased as compared to using individual antibodies that bind to E6 protein to detect HPV. The signal to noise (S/N) ratio generally refers to a ratio of desired signal to undesired background signal. In some embodiments, the synergistic antibodies of the present invention result in a higher signal for detecting E6 protein without increasing the level of background noise. In some embodiments, the signal to noise ratio of detecting HPV E6 protein using the synergistic antibodies of the present invention is increased by about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.5, 5.0, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more than 1,000 folds, as compared to using each individual antibodies to detect E6 protein at comparable doses. In some embodiments, the signal to noise ratio of detecting HPV E6 protein using the synergistic antibodies of the present invention is increased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, as compared to using each individual antibodies to detect E6 protein at comparable doses. In one embodiment, antibodies which have S/N ratios that are at least 20% greater than the S/N ratios for the respective individual mAb are deemed to be synergistic. [00129] In some embodiments, the sensitivity of the HPV detection method using synergistic antibodies of the present invention is increased as compared to using individual antibodies that bind to E6 protein to detect
HPV. In some embodiments, sensitivity refers to the proportion of people with HPV who have a positive test result. In other embodiments, sensitivity refers to the smallest amount of a substance, such as a protein in a sample, which a diagnostic test can detect. Having a first E6 binding partner, for example, a PDZ domain polypeptide such as MAGI-I or MUPPl, to capture E6 protein and a plurality of synergistic antibodies that specifically binds to the E6 protein bound to the first E6 binding partner, for example a PDZ domain polypeptide such as MAGI-I or MUPPl, may increase the sensitivity of detecting E6 protein including oncogenic E6 protein, thus oncogenic HPV in a sample, especially with the amount of sample or viral titer is low. In some embodiments, the sensitivity of correctly detecting an E6 protein or an oncogenic HPV strain using the method and the synergistic antibodies of the present invention is increased as compared to using individual antibodies or a PDZ domain containing polypeptide that binds to E6 protein for detection. In some embodiments, the sensitivity of correct HPV detection using the method of the present invention is increased to about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more than 99.9%. [00130] Sensitivity can be measured by the number of E6 molecules that can be detected by the subject synergistic antibodies in a given volume of a sample. Sensitivity can also be measured by the number of HPV infected cells per given volume of a sample. Understanding the sensitivity of the synergistic antibodies of the present invention as compared to individual antibodies is essential because it helps to define the amount of tissue or cell sample that must be tested to obtain a definitive result.
[00131] Sensitivity of E6-antibody binding can be measured based on apparent affinity, which 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, in this case, binding of E6 to an anti-E6 antibody. In one example, a fixed concentration of an anti-E6 antibody of the present invention and increasing concentrations of a labeled E6 peptide (labeled with, for example, biotin or fluorescein) are mixed together in solution and allowed to react. Representative HPV E6 peptide amino acid sequences are disclosed herein and also in US Patent Nos. 7,312,041 and 7,399,467, which are both incorporated by reference in their entirety. In one embodiment, preferred E6 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 nonspecific antibody as a control. Antibody-E6 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). The antibody-E6 complex is detected based on presence of the label on the E6 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 antibody-E6 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[ilgand]=Saturation Binding x ([ligand]/([ligand+Kd])
[00132] The calculation of binding affinity itself can be determined using any suitable equation (see Cantor and Schimmel (1980) BIOPHYSICAL CHEMISTRY W H Freeman & Co., San Francisco) or software. 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).
[00133] It will be recognized that high specificity and high sensitivity interactions between an E6-binding partner and E6 represent potentially more valuable system for detecting oncogenic HPV strains in a sample. Signal-to-noise ratio typically compares the level of a desired signal, for example, specific binding to E6 protein, and the level of background noise, for example, any unspecific binding not to E6 protein. The higher the ratio, the less obtrusive the background noise is. In some embodiments, the method of the present invention enhances the signal-to-noise ratio of detecting an E6 protein by about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 500, 1,000 or 10,000 fold or more, as compared to using individual unpaired antibodies to detect the E6 protein. [00134] In some embodiments, the method of the present invention results in a lower false positive rate of erroneously detecting an HPV E6 protein as compared to using individual antibodies to detect the E6 protein. The false positive rate for a test is the false-positive test results divided by all patients without the disease. The false positive rate of detecting E6 protein of a HPV strain in a sample using the method of the present invention may be about 10%, 9%, 8%, 7, %, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less.
Kits
[00135] The present invention also includes kits for carrying out the methods of the invention. In some embodiments, a subject kit contains subject synergistic antibodies that specifically bind to E6 protein of an HPV strain. The HPV strain can be oncogenic or non-oncogenic. In some embodiments, the kit further contains a PDZ domain polypeptide. In many embodiments, the kits contain a first and second binding partner, where the first binding partner is a PDZ domain polypeptide and the second binding partner is a subject plurality of synergistic antibodies that specifically binds to E6 protein. In some embodiments, the antibody pair 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 protein from the sample. The kit may optionally contain protease inhibitor. [00136] 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. [00137] In a subject kit, the oncogenic E6 protein 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. 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.). [00138] Subject kits may also contain control samples containing oncogenic or non-oncogenic E6 proteins, and/or a dilution series of oncogenic E6 proteins, 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 proteins 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 standard curves of fluorescence, color or film density provided by the kit.
[00139] 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.
Utility
[00140] In one aspect, the present invention discloses a method of detecting HPV E6 protein in a sample with high sensitivity. In another aspect, the present invention discloses a method of screening antibodies for synergistic antibodies that bind to E6 protein of a HPV strain with enhanced sensitivity as compared to an individual antibody specific against the E6 protein. The synergistic antibodies and methods of the instant invention are useful for a variety of diagnostic analyses. The instant antibodies and methods are useful for diagnosing infection by an oncogenic strain of HPV in an individual; for determining the likelihood of having cancer; for determining a patient's response to treatment for HPV; for determining the severity of HPV infection in an individual; and for monitoring the progression of HPV in an individual. The plurality of synergistic antibodies and the methods of the instant invention are useful in the diagnosis of infection with an oncogenic or a non-oncogenic strain of HPV associated with cancer, including cervical, ovarian, breast, anus, penis, prostate, larynx and the buccal cavity, tonsils, nasal passage, skin, bladder, head and neck squamous- cell, occasional periungal carcinomas, as well as benign anogenital warts. The antibodies and the methods of the instant invention are useful in the diagnosis of infection with an oncogenic or a non-oncogenic strain of HPV associated with Netherton's syndrome, epidermolysis verruciformis, endometriosis, and other disorders. The antibodies and the methods of the instant invention are useful in the diagnosis of infection with an oncogenic or a non-oncogenic strain of HPV in adult women, adult men, fetuses, infants, children, and immunocompromised individuals.
[00141] The subject methods may generally be performed on biological samples from living subjects. A particularly advantageous feature of the invention is that the methods can simultaneously detect, in one reaction, several known oncogenic or non-oncogenic strains of HPV. In particular embodiments, the synergistic antibodies of the invention may be employed in immunohistological examination of a sample.
EXAMPLES
[00142] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Example 1
Detection of E6 using paired antibody results in higher signal to noise ratio
[00143] This example describes an enzyme immunoassay (EIA) that is used to determine which pairs of anti- HPV-16 E6 monoclonal antibodies (mAbs) are synergistic. The assay was carried out as follows: the wells were coated with 50 μL of a 5 μg/mL Maji-1 PDZ (monomeric PDZ) solution (in PBS: 1OmM Na2HPO4, 1.8mM KH2PO4 , 137mM NaCl, 3.9mM KCl, pH 7.4). The plate was incubated overnight (ON) at 50C and washed the next morning with a wash buffer containing 5OmM Tris-HCl, pH 8.0, 15OmM NaCl, 0.05% Tween-20). The wash was repeated 3 times using an automated washer (Biotek). The plate was blocked with 140 μL 2% BSA in PBS and incubated for 2 hours at 250C. The wash step was repeated and either 2.5MM/mL (Millions per mL) extracted SiHa (HPV 16) or C33A- cells (diluted in AVC HPV lysis buffer) were added to the plate. The cells were incubated for 1 hour at 250C. The wash step was repeated and either mixture of mAb (25 μL of 5 μg/mL per mAb per well- 2 mAb per well = 50 μL total volume) or the individual control mAb (25 μL of 5 μg/mL mAb + 25 μL 2% BSA in PBS) was added to the appropriate wells. 5 μg/mL mAb solutions were prepared in 2% BSA and 0.05% Tween in PBS. The mAbs were incubated in the plate for 1 hour at 250C. The wash step was repeated and 50 μL of a 1:5000 dilution of Goat anti Mouse- HRP conjugate (diluted in 2% BSA and 0.05% Tween in PBS) was added to each well (GxM- HRP- Jackson Immunologicals 115-035-062) and incubated for 1 hour at 250C. The plate was washed for 5 times and 50 μL TMB (Neogen K-Blue 331177) was added to each well and incubated for 30 minutes at 250C. 50 μL EIA Stop Solution (1% Sulfuric Acid) was then added to each well. The plate was read at OD450 in a microplate reader. The signal to noise ratio (S/N) was calculated by dividing A450 of pertinent mAb (or mAb combination) in SiHa well by A450 of the same mAb (or mAb combination) in C33A- well. The S/N of individual mAb results were compared to those of combination mAb results. mAbs which have S/N ratios that are at least 20% greater than the S/N ratios for the respective individual mAb were deemed to be synergistic.
[00144] Starred bars in FIG. 1 represent pairs of antibodies having synergistic effects. The results demonstrate the synergistic effect of the paired antibodies, which give higher signal to noise ratios as compared to using the individual antibodies alone for detection of E6 protein.
Example 2
Detection of E6 using paired antibody results in lower limit of detection (LOD)
[00145] This example describes a titration enzyme immunoassay (EIA) that demonstrates that the titration curve of the synergistic pair, in comparison to the titration curve of the individual mAbs, is positively shifted, suggesting a greater signal with the combined antibody pair. The assay was carried out as follows: the wells were coated with 50 μL of a 5 μg/mL Maji-1 PDZ (monomeric PDZ) solution (in PBS: 1OmM Na2HPO4, 1.8mM KH2PO4 , 137mM NaCl, 3.9mM KCl, pH 7.4). The plate was incubated overnight (ON) at 50C and washed the next morning with a wash buffer containing 5OmM Tris-HCl, pH 8.0, 15OmM NaCl, 0.05% Tween-20). The wash was repeated 3 times using an automated washer (Biotek). The plate was blocked with 140 μL 2% BSA in PBS and incubated for 2 hours at 250C. Serial dilutions of SiHa (10, 5, 2.5, 1.25, & 0.63MM/mL) were prepared in a "constant bed" of lMM/mL C33A- cell lysate (Lysate prepared using AVC lysis buffer). mAb combinations or individual mAbs were added to wells of the serially diluted SiHa cell lysate and incubated for 1 hour at 250C. The wash step was repeated and 50 μL of a 1:5000 dilution of Goat anti Mouse- HRP conjugate (diluted in 2% BSA and 0.05% Tween in PBS) was added to each well (GxM- HRP- Jackson Immunologicals 115-035-062) and incubated for 1 hour at 250C. The plate was washed for 5 times and 50 μL TMB (Neogen K-Blue 331177) was added to each well and incubated for 30 minutes at 250C. 50 μL EIA Stop Solution (1% Sulfuric Acid) was then added to each well. The plate was read at OD450 in a microplate reader. A450 titration curve of the combination mAb pair was plotted and compared to the titration curve of the individual mAbs. The signal to noise ratio (S/N) was calculated by dividing A450 of pertinent mAb (or mAb combination) in SiHa well by A450 of the same mAb (or mAb combination) in
C33A- well. The S/N of individual mAb results were compared to those of the combination mAb results. mAbs which have S/N ratios that are at least 20% greater than the S/N ratios for the respective individual mAb were deemed to be synergistic.
[00146] As shown in FIG. 2a and 2b, a shift in limit of detection (LOD) demonstrating increased sensitivity is seen with the 8Gl 1-6G6 and 4C6-6G6 paired monoclonal antibodies as compared to using individual monoclonal antibodies 8Gl 1 alone or 4C6 alone. No significant increase in sensitivity is noted with 8Gl 1- ICl and 4C6-1C1 paired monoclonal antibodies in this assay. The results suggest that mAb pair 8Gl 1-6G6 and mAb pair 4C6-6G6 have synergistic effects in detecting E6 protein, lowering the LOD and enhancing the sensitivity of E6 detection.
Example 3
Paired antibodies recognize different epitopes on E6 protein
[00147] This example describes an inhibition enzyme immunoassay (EIA) that demonstrates that the monoclonal antibody pairs have different binding specificities on E6 protein and the two members of a synergistic antibody pair do not share the same binding site on E6. The assay was carried out as follows: the wells were coated with 50 μL of a 5 μg/mL Maji-1 PDZ (monomeric PDZ) solution (in PBS: 1OmM Na2HPO4, 1.8mM KH2PO4 , 137mM NaCl, 3.9mM KCl, pH 7.4). The plate was incubated overnight (ON) at 50C and washed the next morning with a wash buffer containing 5OmM Tris-HCl, pH 8.0, 15OmM NaCl, 0.05% Tween-20). The wash was repeated 3 times using an automated washer (Biotek). The plate was blocked with 140 μL 2% BSA in PBS and incubated for 2 hours at 250C. Serial dilutions of SiHa (10, 5, 2.5, 1.25, 0.63, 0.31, 0.16MM/mL) were prepared in a "constant bed" of lMM/mL C33A- cell lysate (Lysate prepared using AVC lysis buffer). The individual mAbs were prepared with or without 10 μg/mL of an HPV 16 N-terminal peptide (NTP). The mAb with the N-terminal peptide was pre-incubated for 20 minutes at 250C and then added to the appropriate wells of the serially diluted SiHa cell lysate. The wash step was repeated and 50 μL of a 1 :5000 dilution of Goat anti Mouse- HRP conjugate (diluted in 2% BSA and 0.05% Tween in PBS) was added to each well (GxM-HRP- Jackson Immunologicals 115-035-062) and incubated for 1 hour at 250C. The plate was washed for 5 times and 50 μL TMB (Neogen K-Blue 331177) was added to each well and incubated for 30 minutes at 250C. 50 μL EIA Stop Solution (1% Sulfuric Acid) was then added to each well. The plate was read at OD450 in a microplate reader. A450 titration curves of the individual mAbs for both sets (with and without NTP) were plotted. If the titration curve of the mAb with NTP was suppressed, then that mAb was considered inhibited. The results show that mAbs 4C6 and 8Gl 1 were inhibited by the NTP whereas mAb 6G6 was not. Therefore the 6G6 mAb is recognizing a different epitope for HPV 16 than either 8Gl 1 or 4C6 mAb. The S/N of individual mAb results were compared to those of the combination mAb results. mAbs which have S/N ratios that are at least 20% greater than the S/N ratios for the respective individual mAb were deemed to be synergistic.
[00148] Fig. 3 shows use of a HPV 16 N-terminal blocking peptide. The HPV E6 16 N-terminal peptide is designed to contain the HPV 16 N-terminal 20 amino acid sequence. The results indicate that the 8Gl 1 and
4C6 mAbs are inhibited by an HPV 16 N-terminal peptide, whereas the 6G6 mAb is not inhibited. Both the 4C6 and 8Gl 1 Mabs bind to an epitope located at the N-terminus of the HPV- 16 E6 protein. This indicates that the 6G6 mAb recognizes a different epitope on the E6 protein (i.e. not on the N-terminus of E6) than either the 8Gl 1 or 4C6 mAb.
[00149] 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 method of detecting E6 protein of a HPV strain in a sample, the method comprising:
(a) contacting an E6 protein binding partner with the sample;
(b) contacting the E6 protein that is bound to the E6 protein binding partner with a plurality of synergistic antibodies that specifically bind to the E6 protein; and
(c) detecting binding of the synergistic antibodies to the E6 protein, thereby detecting the E6 protein in the sample; wherein signal-to-noise ratio of detecting E6 protein using the plurality of synergistic antibodies is increased as compared to using each single antibody at a comparable amount to detect E6 protein.
2. The method of claim 1, wherein the HPV strain is an oncogenic strain.
3. The method of claim 1, wherein the synergistic antibodies specifically bind to E6 protein of one oncogenic HPV strain.
4. The method of claim 1, wherein the synergistic antibodies specifically bind to E6 proteins of more than one oncogenic HPV strain.
5. The method of claim 1, wherein the synergistic antibodies specifically bind to E6 protein of HPV- 16.
6. The method of claim 1, wherein the synergistic antibodies specifically bind to E6 protein of HPV- 18.
7. The method of claim 1, wherein the synergistic antibodies specifically bind to E6 protein of HPV-45.
8. The method of claim 1, wherein the synergistic antibodies specifically bind to E6 proteins of HPV-16 and HPV-18.
9. The method of claim 1, wherein the synergistic antibodies specifically bind to E6 proteins of HPV strains selected from the group consisting of HPV- 16, 18, 26, 30, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, and 82.
10. The method of claim 1, wherein the E6 protein binding partner is immobilized.
11. The method of claim 1, wherein the E6 protein binding partner is a PDZ domain polypeptide.
12. The method of claim 11, wherein the PDZ domain polypeptide is MAGI-I.
13. The method of claim 11, wherein the PDZ domain polypeptide is selected from the group consisting of CASK, MPPl, DLGl, DLG2, PSD95, NeDLG, TIP-33, SYNIa, TIP-43, LDP, LIM, LIMKl, LIMK2, MPP2, NOSl, AF6, PTN-4, prIL16, 41.8kD, KIAA0559, RGS12, KIAA0316, DVLl, TIP-40, TIAMl, MINTl, MAGI-I, MAGI-2, MAGI-3, KIAA0303, CBP, MINT3, TIP-2, KIAA0561, and TIP-I.
14. The method of claim 1, wherein the sample is a cervical scrape, cervical biopsy, cervical lavage, blood or urine.
15. The method of claim 1, wherein the sample is a histological sample.
16. The method of claim 1, wherein the antibodies are monoclonal.
17. The method of claim 1, wherein the antibodies are labeled.
18. The method of claim 1, wherein the synergistic antibodies bind to different epitopes on E6 protein.
19. The method of claim 1, wherein sensitivity of detecting an E6 protein using the plurality of synergistic antibodies is increased as compared to using single antibody at a comparable amount to detect E6 protein.
20. The method of claim 1, wherein limit of detection (LOD) of an E6 protein using the plurality of synergistic antibodies is decreased as compared to using single antibody at a comparable amount to detect E6 protein.
21. The method of claim 1, wherein the method is carried out via an enzyme immunoassay (EIA).
22. The method of claim 1, wherein the method is carried out via a sandwich enzyme-linked immunosorbent assay (ELISA).
23. The method of claim 1, wherein the plurality of synergistic antibodies are used in immunoassays selected from the group consisting of lateral flow, flow-through, immunofiltration, chemiluminescent EIA, fluorescent EIA, and sandwich ELISA.
24. The method of claim 1, wherein the method can be used as a part of a test for cervical cancer.
25. A method of screening for synergistic antibodies that bind to E6 protein of a HPV strain with enhanced sensitivity as compared to individual antibody specific against the E6 protein, the method comprising:
(a) contacting an E6 binding partner with the E6 protein;
(b) contacting the E6 protein that is bound to the E6 binding partner with at least two antibodies specific for the E6 protein;
(c) detecting binding of the antibodies to the E6 protein that is bound to the E6 binding partner; and
(d) selecting the combination of antibodies that has a higher signal-to-noise ratio as compared to the individual antibodies used alone at a comparable amount; wherein a higher signal-to-noise ratio indicates an enhanced sensitivity of E6 protein detection.
26. The method of claim 25 further comprising contacting the E6 protein with additional antibodies that are specific against the E6 protein.
27. The method of claim 25, wherein the first antibody is immobilized to a substrate.
28. The method of claim 25, wherein the HPV strain is an oncogenic strain.
29. The method of claim 25, wherein the synergistic antibodies specifically bind to E6 protein of one oncogenic HPV strain.
30. The method of claim 25, wherein the synergistic antibodies specifically bind to E6 proteins of more than one oncogenic HPV strain.
31. The method of claim 25, wherein the synergistic antibodies specifically bind to E6 protein of HPV- 16.
32. The method of claim 25, wherein the synergistic antibodies specifically bind to E6 protein of
HPV- 18.
33. The method of claim 25, wherein the synergistic antibodies specifically bind to E6 protein of
HPV-45.
34. The method of claim 25, wherein the synergistic antibodies specifically bind to E6 proteins of HPV-16 and HPV-18.
35. The method of claim 25, wherein the synergistic antibodies specifically bind to E6 proteins of HPV strains selected from the group consisting of HPV- 16, 18, 26, 30, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, and 82.
36. The method of claim 25, wherein the synergistic antibodies are monoclonal.
37. The method of claim 25, wherein the synergistic antibodies are labeled.
38. The method of claim 25, wherein the synergistic antibodies bind to different epitopes on E6 protein.
39. The method of claim 25, wherein sensitivity of detecting an E6 protein using the synergistic antibodies is increased as compared to using single antibody at a comparable amount to detect E6 protein.
40. The method of claim 25, wherein the method is carried out via an enzyme -linked immunosorbent assay (ELISA).
41. The method of claim 25, wherein the synergistic antibodies are used in immunoassays selected from the group consisting of lateral flow, flow-through, immunofiltration, chemiluminescent EIA, fluorescent EIA, a inndd ssaannddwwiicchh EELLIISSAA..
42. A L method of detecting E6 protein of a HPV strain in a sample, the method comprising:
(a ι)) ccoonnttaaccttiinngg tthhee Ss£ample with a plurality of synergistic antibodies that specifically bind to the E6 protein; and
(b) detecting binding of the synergistic antibodies to the E6 protein, thereby detecting the E6 protein in the sample; wherein signal-to-noise ratio of detecting E6 protein using the plurality of synergistic antibodies is incrreeaasseedd aass compared to using each single antibody at a comparable amount to detect E6 protein.
PCT/US2010/031821 2009-04-20 2010-04-20 Methods of detecting hpv strains using synergistic antibodies WO2010123949A1 (en)

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CN102495207A (en) * 2011-12-26 2012-06-13 华东交通大学 High-sensitivity enzyme-linked immunoassay method
CN103116021A (en) * 2013-02-06 2013-05-22 北京工业大学 ELISA (enzyme-linked immunosorbent assay) kit for detecting cochinchinenin A
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