WO2008140483A2 - Procédés et anticorps pour détecter un antigène protecteur - Google Patents

Procédés et anticorps pour détecter un antigène protecteur Download PDF

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WO2008140483A2
WO2008140483A2 PCT/US2007/023624 US2007023624W WO2008140483A2 WO 2008140483 A2 WO2008140483 A2 WO 2008140483A2 US 2007023624 W US2007023624 W US 2007023624W WO 2008140483 A2 WO2008140483 A2 WO 2008140483A2
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antibody
fragment
amino acid
antibodies
protective antigen
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PCT/US2007/023624
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WO2008140483A3 (fr
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Thi-Sau Migone
Christopher D. Ward
Theodore H. Hewit
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Human Genome Sciences, Inc.
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Publication of WO2008140483A2 publication Critical patent/WO2008140483A2/fr
Publication of WO2008140483A3 publication Critical patent/WO2008140483A3/fr

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    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1278—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Bacillus (G)
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • 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
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)

Definitions

  • the present invention relates to methods and compositions for detecting anthrax and/or anthrax related toxins using electrochemiluminescence (ECL).
  • ECL electrochemiluminescence
  • the present invention relates to an ECL detection method for detecting the protective antigen component of anthrax toxins.
  • the present invention further relates to antibodies or fragments, or variants thereof, or related molecules that specifically bind to the protective antigen (PA) of Bacillus anthracis (Anthrax).
  • PA protective antigen
  • Such antibodies have uses, for example, as capture and/or detection antibodies in the ECL method for the detection of anthrax and/or anthrax related toxins.
  • Bacillus anthracis is a Gram-positive, rod-shaped, aerobic and/or facultative anaerobic, spore forming bacterium that is responsible for the deadly disease anthrax.
  • There are three recognized routes of anthrax infection including cutaneous (through skin), gastrointestinal, and pulmonary (via inhalation) infection. Of the three ways to contract the disease, inhalation is the avenue that most frequently leads to the death of the patient.
  • the mortality caused by anthrax is predominantly due to three characterized virulence factors, encapsulation, and the production of two interlinked toxins.
  • the polyglutamate capsule presents phagocytosis of the bacterium.
  • the anthrax toxins are made from three proteins, lethal factor (LF), edema factor (EF), and protective antigen (PA).
  • the anthrax toxin is a bipartite toxin that contains A and B moieties, similar to that of diphtheria toxin and many clostridial toxins.
  • the LF and EF proteins function as enzymatic A moieties of the toxin, while the PA protein functions as the B, or binding, moiety.
  • PA binds to host cell surface receptors, (e.g., anthrax receptor (ATR) and/or capillary morphogenesis gene 2 (CMG2)) and is cleaved at the sequence RKKR by cell surface proteases such as furin.
  • ATR anthrax receptor
  • CMG2 capillary morphogenesis gene 2
  • PA63 cell surface proteases
  • Some cleavage to the PA63 form may be mediated by serum proteases and occur prior to PA, in this case PA63, binding to the cell surface.
  • PA63 fragment Release of the 20 kilodalton PA fragment enables the PA63 fragment to multimerize into a heptameric ring structure and exposes a site on PA63 to which LF and EF compete for and bind to with high affinity.
  • the complex is then internalized by receptor-mediated endocytosis. Acidification of the vesicle causes conformational changes in the pA63 heptamer that result in transportation of LF and EF toxins across the endosomal membrane, after which they are released into the cytosol where they exert their cytotoxic effects.
  • PA and EF combine to produce anthrax edema toxin (ET).
  • the edema factor (EF) component of edema toxin (EF+PA) is a calmodulin dependent adenylate cyclase whose action upsets cellular water homeostasis mechanisms, thereby resulting in swelling of infected tissues.
  • the lethal factor (LF) moiety of lethal toxin (LF+PA) is a zinc metalloproteinase that inactivates mitogen activated protein kinase kinase (MAPKKl and MAPKK2) in vitro resulting in inactivation of the MAPK signal transduction pathway. LF is the predominant cause of severe disease and death following inhalation spore exposure.
  • Lethal factor induces a hyperinflamrnatory condition in macrophages resulting in the production of proinflammatory cytokines including TNF-alpha and interleukin- lbeta, which are responsible for shock and death of anthrax patients.
  • the initial clinical signs and symptoms are nonspecific and may include malaise, headache, fever, nauseas, and vomiting.
  • signs and symptoms include, but are not limited to, sudden onset of respiratory distress with dyspnea, stridor, cyanosis and chest pain. The onset of respiratory distress is followed by shock and death, with close to 100% mortality.
  • Bacillus Anthracis infection and anthrax toxin please see, e.g., Critical Reviews in Microbiology (2001) 27:167-200, Medical Progress (1999) 341:815-826, and Microbes and Infection (1999) 2:131-139, each of which are hereby incorporated by reference in their entireties.
  • Several methods are currently available to detect and/or quantify the anthrax toxin, including, for example, the use of bacterial culture, enzyme-linked immunosorbent assays (ELISAs) and polymerase chain reaction (PCR). Bacterial culturing, although sensitive and capable of providing positive spore identification, does not allow for quantitative spore determination and may take upwards of 48 hours for results. [0011] PCR methods for the detection of anthrax spores have been described in scientific literature and/or are commercially available.
  • PCR methods while capable of detecting low levels of anthrax toxins, are unable to quantitate the level of anthrax toxin. Furthermore, due to assay protocols which require the need for incubation of samples to enable spore growth prior to testing using PCR, sample results may take upwards of 24 hours to obtain.
  • Sandwich ELISAs such as the PA sandwich ELISA described in Mabry et al, are prone to matrix/background interference due to the inclusion of a capture antibody, primary detection antibody and a secondary labeled antibody conjugate.
  • the primary detection antibody is a rabbit anti-PA polyclonal
  • the secondary labeled antibody conjugate is a goat anti-rabbit polyclonal HRP. Consequently, due to the presence of non-specific rabbit antibodies with the PA detection and capture antibodies, a rabbit serum sample tested in the PA sandwich ELISA would likely have significant effects.
  • Rabbit models are often used to test the efficacy of postexposure prophylaxis and/or postexposure small molecule- and protein based therapeutic approaches.
  • an ideal postexposure test protocol the animal would be exposed to the anthrax toxin and then sera samples would be taken from the animal to detect the toxin. Once the toxin is detected, the animal would be treated with the therapeutic.
  • One challenge in applying this protocol is that death in animal models generally occurs within a few hours after signs of anthrax exposure. Consequently, there is only a small window of opportunity between detecting the toxin, confirming anthrax exposure, and administering postexposure treatment. Thus, a sensitive, rapid detection method is needed for these type of protocols.
  • a less than ideal protocol is commonly used to overcome the lack of a sufficient method for detecting toxin in vivo.
  • animals are exposed to toxin, and, at a predetermined time following exposure, the animals are administered a test therapeutic.
  • This protocol has many disadvantages when compared to the previously described protocol.
  • the animals are often administered the test therapeutic prior to showing clinical signs of anthrax exposure.
  • Such early administration could generate therapeutic results that could not be duplicated and/or are not representative of a human exposure incident.
  • individuals would likely seek treatment only after onset of initial symptoms. In the 2001 anthrax incidents, the mean incubation period from the time of exposure to the initial onset of symptoms was five days.
  • the invention relates to an electrochemiluminescence (ECL) assay, which includes antibodies that specifically bind to protective antigen (PA), for detecting the protective antigen (PA) of anthrax toxin in a sample.
  • ECL electrochemiluminescence
  • the invention provides an ECL method for diagnosing PA in a sample including the following steps: immobilization of a PA capture antibody on a surface containing an electrode; capture of PA antigen present in a sample by the immobilized PA capture antibody; and detection of the PA antigen from a sample using an ECL labeled PA detection antibody that specifically binds to the protective antigen (PA).
  • the ECL protective antigen detection method of the present invention has several advantages over currently available detection methods.
  • the ECL method has rapid run times.
  • the method can be run as a quantitative assay, for example, in which results can be obtained after receiving a sample in less than or equal to about 3 hours, less than or equal to about 4 hours, less than or equal to about 5 hours, or less than or equal to about 10 hours.
  • the term "about” is used to refer to a range within the standard margin of error.
  • the method can be run as a screening assy in which results can be obtained after receiving a sample in less than or equal to about 1 hour, less than or equal to about 2 hours, less than or equal to about 3 hours, or less than or equal to about 5 hours. Consequently, during an exposure incident, confirmation of exposure and administration of a therapeutic can be made quickly and therefore significantly increase the likelihood of successful treatment.
  • the ECL method of the present invention is also very sensitive.
  • the ECL method has a lower limit of detection for PA from a sample of less than or equal to about 0.1 ng/mL, less than or equal to about 0.2 ng/mL, less than or equal to about 0.25 ng/mL, or less than or equal to about 0.4 ng/mL.
  • PA present in a sample can be detected at an early toxemia stage and thus a proper therapeutic can be quickly administered.
  • the ECL method of the present invention also has very little matrix effect. This is attributed to the specificity, purity and/or affinity of the PA detection antibody and the fact that very few biologies contribute to ECL signaling. Therefore, there is little interference from the other compounds contributing to the ECL signal and/or other compounds interfering with the binding of protective antigen.
  • the PA capture antibody is immobilized on an uncoated or coated surface containing an electrode.
  • an electrode Many suitable substance are known and can be used to coat the suface, for example, to help facilitate immobilization of the antibody.
  • the surface containing an electrode is coated with streptavidin.
  • the coated surface containing an electrode can be, for example, a streptavidin coated bead such as Dynabeads ® M-280 Streptavidin
  • the surface containing an electrode is precoated with streptavidin and the PA capture antibody is biotinylated.
  • biotinylation of antibodies as well as coupling methods of the biotinylated antibody to coated surfaces which can be used in the invention are known by one of skill in the art. See, e.g., Merrill et al., Anal. Biochem. 357 (2006) 181-187.
  • the immobilization of the capture PA antibody is therefore accomplished by the coupling of the biotinylated PA capture antibody to the avidin or streptavidin precoated surface containing an electrode.
  • Other mechanisms for coupling molecules to a surface are known and can be used in connection with the invention.
  • the immobilized PA capture antibody in the ECL assay includes an antibody that specifically binds to the protective antigen (PA).
  • PA protective antigen
  • the present invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that specifically bind to a PA polypeptide (SEQ ID NO:2) or polypeptide fragment or variant of PA.
  • the PA antibody includes a single chain Fv's (scFvs) that specifically bind PA polypeptide (SEQ ID NOS:48-65).
  • the PA capture antibody includes a polyclonal PA antibody. Methods for generating polyclonal antibodies are known.
  • Anti-PA polyclonal antibodies include, but are not limited to, human, rabbit, guinea pig, goat, and horse anti-PA polyclonal antibodies.
  • the polyclonal PA capture antibody includes a rabbit polyclonal.
  • the PA detection antibody used in the ECL method includes an antibody or fragment, or variant thereof, or related molecule that specifically binds to the protective antigen (PA) of Bacillus anthracis (Anthrax).
  • the PA detection antibody encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that specifically bind to a PA polypeptide (SEQ ID NO:2) or polypeptide fragment or variant of PA.
  • the PA detection antibody inlcudes a PA single chain Fv's (scFvs) identified in Table 1 (SEQ ID NOS:48-65).
  • the PA detection antibody of the present invention is coupled to a detectable label, such as an electrochemilurninescent label (ECL) or an electrochemiluminescent (ECL) TAG.
  • ECL label an electrochemilurninescent label
  • ECL TAG electrochemiluminescent TAG
  • the terms "ECL label” and “ECL TAG” are used interchangeably and refer generally to a label designed to emit light when electrochemically stimulated.
  • the coupling of the ECL label to the PA detection antibody can occur directly or indirectly. Methods for coupling labels, such as ECL labels, to a molecule, such as an antibody, are known.
  • Commonly used electrochemiluminescent labels which can be coupled to the PA detection antibodies include, but are not limited to, organometallic compounds.
  • ECL labels which can be used in ECL detection method of the present invention include, for example, ECL labels which are commercially available from Meso Scale Discovery (MSD ® ) (Gaithersburg, MD) and BioVeris TM Corporation (Gaithersburg, MD).
  • ECL labels from MSD ® include, but are not limited to, Ru(bpy) 3 2+ (MSD-Tag TM ).
  • ECL labels from BioVerisTM include, but are not limited to, Ru(bpy) 3 2+ (BV-TAGTM Label).
  • a BioVerisTM ECL label is coupled to the PA detection antibody and the PA ECL detection method uses the commercially avaialable BioVeris Technology TM .
  • the ECL labeled PA detection antibody specifically binds to the PA antigen captured by the capture antibody.
  • the detection of the PA antigen from a sample using an ECL labeled PA detection antibody can be performed using any commercially available ECL detection instrument.
  • ECL detection instruments are commercially available from, for example, Meso Scale Discovery (MSD ) (Gaithersburg, MD) and BioVerisTM Corporation (Gaithersburg, MD). Examples of commercial ECL detection instruments from MSD ® include, but are not limited to, the SECTORTM Imager 6000, SECTORTM Imager 2400, SECTOR PR TM 400 and the SECTOR PR TM 100. Examples of BioVerisTM Corp. detection instruments include, but are not limited to, the M-SERIES TM M8, M-SERIESTM 384, and M-SERIESTM MlM.
  • the antibodies which can be used in the ECL detection method of the present invention encompasses antibodies, or fragments or variants thereof, that bind to an epitope that comprises the RKKR sequence of amino acid residues 193 to 196 of SEQ ID NO:2.
  • the antibodies which can be used in the ECL detection method of the invention bind an epitope of PA and occlude access of proteases to the RKKR cleavage site of PA (amino acid residues 193 to 196 of SEQ ID NO:2).
  • PA antibodies which can be used in the ECL detection method of the invention are antibodies which are capable of neutralizing the ability of PA to bind to a cellular anthrax receptor, e.g., ATR (SEQ ID NO:3) or CMG2 (SEQ ID NO:42).
  • antibodies which can be used in the ECL detection method of the invention are capable of neutralizing the ability of the PA (particularly the PA63 form of PA) to form oligomers, and more specifically to form heptamers.
  • antibodies which can be used in the ECL detection method of the invention are capable of neutralizing the ability of PA (particularly the PA63 form of PA) to bind to either EF or LF (SEQ ID NOs:4 or 5, respectively).
  • antibodies which can be used in the ECL detection method of the invention bind specifically bind to PA but do not neutralize PA.
  • antibodies which can be used in the invention are capable of binding to either cellbound PA.
  • antibodies which can be used in the invention are capable of binding PA in the extracellular matrix.
  • antibodies which can be used in the invention are capable of binding spore-based PA.
  • the antibodies which can be used in the ECL detection method of the invention have a dissociation constant (K D ) of less than or equal to 10 "7 M, less than or equal to 10 "8 M, less than or equal to 10 ⁇ 9 M, less than or equal to 10 '10 M, less than or equal to 10 "11 M, or less than or equal to 10 ⁇ 12 M.
  • the antibodies used in the invention have a dissociation constant (K D ) of less than or equal to 10 "9 M.
  • antibodies which can be used in the ECL detection method of the invention have an off rate (k off ) of 10 '3 /sec or less. In one embodiment, antibodies which can be used in the ECL detection method of the invention have an off rate (k off ) of lO ⁇ sec or less. In other embodiments, antibodies which can be used in the ECL detection method of the invention have an off rate (k off ) of 10 '5 /sec or less.
  • the present invention also provides an ECL detection kit for screening and/or detecting protective antigen (PA).
  • the kit includes a capture antibody immobilized on a surface containing an electrode and an ECL labeled PA detection antibody that specifically binds to the protective antigen (PA).
  • the kit can be used in combination with a portable ECL analyzer.
  • ECL analyzers which can be used with the ECL detection kit for PA include, but are not limited to, the portable cartridge and reader system from MSD ® and BioVeris' M-SERIES ® MlM analyzer (BioVerisTM, Gaithersburg, MD).
  • the kit is particularly useful because it allows the detection of PA in a sample taken in the field or at any anthrax exposure site, in addition to the laboratory.
  • the ECL detection kit for PA is advantageous in that it provides an ECL labeled PA antibody that specifically binds to the PA antigen. Further, the ECL detection kit can be used as a screening method. Confirmatory results for the screeing assay can be obtained in, for example, less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, or less than about 5 hours.
  • Figure 1 depicts the 96-well plate set-up for performing the ECL detection method described in Example 3. Rows A and B, lanes 1-9 represent the standard curve. Rows C-H, lanes 1-12 represent various PA sample spikes in rabbit serum. Sample wells labeled as BLANK represent wells with 20% serum. Sample wells labeled zero (0) are 100% rabbit serum samples diluted 1:5 in sample diluent.
  • the invention provides an electrochemiluminescence (ECL) detection method for detecting the protective antigen (PA) of Bacillus anthracis (Anthrax) in a sample.
  • ECL electrochemiluminescence
  • the invention provides an ECL method for detecting PA in a sample which includes the following steps: immobilization of a PA capture antibody on an electrode surface; capture of PA antigen present in a sample by the immobilized PA capture antibody; and detection of the PA antigen from a sample using an ECL labeled PA detection antibody that specifically binds to the protective antigen (PA).
  • Detection methods have been developed which utilize electrochemiluminescence (ECL) technology.
  • Electrochemiluminescence (ECL) or electrogenerated chemiluminescence is a form of chemiluminescence (CL) in which the light emitting chemiluminescent reaction is preceded by an electrochemical reaction.
  • CL chemiluminescence
  • the electrochemical reaction allows the time and position of the light emitting reaction to be controlled. By controlling the time, light emission can be delayed until certain desired reaction occur, such as immune or enzyme catalyzed reactions.
  • similar control can be exercised over alternative detection methods, such as, for example, fluorescence detection, the equipment for these alternative detection methods is considerably more sophisticated and expensive. Control over position can be used to confine light emission to a region which is precisely located with respect to the detector, improving sensitivity by increasing the ratio of signal to noise.
  • ECL methods typically include a capture step, which includes the use of a capture agent bound to a surface having an incorporated electrode, and a detection step, which uses a detection agent coupled to an ECL label.
  • the ECL label is responsible for the light emission generated from the chemiluminscent reaction stimulated by the electrochemical reaction.
  • ECL labels are also commonly referred to as TAGs.
  • ECL labels include, but are not limited to, organometallic compounds where the metal is from, for example, the noble metals of group VIII, including Ru-containing and Os-containing organometallic compounds such as the Ru(2,2'-bipyridine) 3 2+ moiety (also referred to as "Rubpy” or "TAGl”, see e.g., U.S. Pat No. 5,238,808).
  • TAGl and Rubpy also refer to derivatives of Ru(2,2'-bipyridine) 3 2+ .
  • ECL-based detection systems Fundamental to ECL-based detection systems is the need for an electrical potential to excite the ECL label to emit light.
  • An electrical wavelength is applied across an electrode surface, typically a metal surface, and a counterelectrode (see e.g., U.S. Pat. Nos. 5,068,088, 5,093,268, 5,061,445, 5,238,808, 5,147,806, 5,247,243, 5,296,191, 5,310,687, 5,221,605 and 6,673,533, each of which is herein incorporated by reference).
  • the ECL label is promoted to an excited state as a result of a series of chemical reactions triggered by the electrical energy received from the working electrode.
  • a molecule such as, for example, oxalate or tripropylamine, is typically added during a detection method which promotes the chemical reaction and consequently the emission of measurable light from the ECL label.
  • ECL detection methods provide many advantages over other assay formats which use, for example, PCR and ELISA technology. ECL methods typically have greater sensitivity, lower background signal, greater signal to noise ratios, increased dynamic range, enable the use of complex matrices with minimal matrix effects, and provide shorter time to results when compared to other detection.
  • results from a recent publication by Guglielmo-Viret et ah which compared electrochemiluminescence (ECL) and ELISA immunoassay technologies, showed that the ECL assay was two to four times more sensitive than the ELISA (0.78-1.56 ng/ml versus 3.12 ng/ml) and was more rapid (2.5 hours versus 5 hours) in the detection of botulinum type B neurotoxin (BotNT B) (Guglielmo-Viret et al, Journal of Immunological Methods 301:164-172 (2005).
  • BotNT B botulinum type B neurotoxin
  • ECL electrochemiluminescence
  • the ECL protective antigen detection method of the present invention has several advantages over currently available detection methods.
  • the ECL method has rapid run times. The method can be run as a quantitative assay in which results can be obtained after receiving a sample in, for example, less than or equal to about 3 hours, less than or equal to about 4 hours, less than or equal to about 5 hours, or less than or equal to about 10 hours.
  • the method can be run as a screening assy in which results can be obtained after receiving a sample in, for example, less than or equal to about 1 hour, less than or equal to about 2 hours, less than or equal to about 3 hours, or less than or equal to about 5 hours. Consequently, during an exposure incident, confirmation of exposure and administration of a therapeutic can be made quickly and therefore significantly increase the likelihood of successful treatment.
  • the ECL method of the present invention is also sensitive.
  • the ECL method has a lower limit of detection of PA from a sample of, for example, less than or equal to about 0.1 ng/mL, less than or equal to about 0.2 ng/mL, less than or equal to about 0.25 ng/mL, or less than or equal to about 0.4 ng/mL.
  • PA present in a sample can be detected at an early toxemia stage and thus a proper therapeutic can be quickly administered.
  • the ECL method of the present invention also has very little matrix effect. This is attributed to the specificity, purity and/or affinity of the PA detection antibody and the fact that very few biologies contribute to ECL signaling. Therefore, there is little interference from the other compounds contributing to the ECL signal and/or other compounds interfering with the binding of protective antigen. Consequently, this allows for the use of a higher concentration of matrix in addition to better sensitivity.
  • the currenty detection method requires small sample volumes, for example, less than or equal to about 100 ⁇ l, less than or equal to about-50- ⁇ l,-less than or equal-to about 25 ⁇ l, or.less-than or : equal_to-about.l2.5_ ⁇ l
  • the ECL detection method of the present invention is advantageous because it provides an antibody that specifically binds to PA. Accordingly, the ECL detection method provides greater sensitivity, lower background signal, greater signal to noise ratios, increased dynamic range, enables the use of complex matrices with minimal matrix effects, and provides shorter time to results when compared to other detection methods for anthrax and/or anthrax toxins.
  • the term "detection method” refers in general to the ability to detect, diagnose, prognose, quantitate, or monitor the protective antigen (PA) of Bacillus anthracis (Anthrax).
  • the ECL detection method is useful for the detection and diagnosis of a disease or disorder associated with Bacillus anthracis or anthrax toxins in an animal, for example, a mammal, including a human.
  • the ECL method for detecting protective antigen is useful in an animal study protocol for determining the efficacy of postexposure prophylaxis and/or postexposure small molecule- and protein based therapeutics for treating anthrax exposure.
  • the increased sensitivity and rapid results of the ECL method enable the early detection of toxemia in a serum sample drawn from the animal.
  • the early detection of the toxemia can be important in animal studies because the progression from anthrax exposure to toxin secretion to late stage onset of symptoms and death occurs rapidly in animals. Consequently, there is only a small window in which to administer the test therapeutic.
  • the test therapeutic may not permit accurate evaluation.
  • the therapeutic is preferably administered to the study animals during a period which simulates the time in which administration to an exposed human would occur.
  • administration of the drug too early could provide data which overestimates the effectiveness of the drug.
  • administration of the drug too late could underestimate the effectiveness of the drug.
  • the early detection of the protective antigen by the ECL method permits more accurate evaluation of a test therapeutic by enabling administration of the therapeutic during the small window following onset of PA toxemia in animal models.
  • the ECL method for detecting protective antigen is also useful for the early diagnosis of anthrax exposure in humans.
  • the ECL method for detecting protective antigen would be useful for confirming anthrax exposure in the field.
  • the term "field" is used as ordinarily understood by one of skill in the art and includes, but is not limited to, a combat field as well other possible exposure sites such as, but not limited to, office buildings, schools, or post offices.
  • the ECL detection method could be used as a rapid screening method to provide positive or negative confirmation of exposure.
  • the steps of the ECL method could be performed simultaneously and the detection could be performed using- a portable detection instrument.
  • the term "surface” is used to refer to any surface to which an electrode can be attached. Examples of such surfaces, include, but are not limited to, multi-well plates, cassettes ⁇ see, e.g., U.S. Pat. No. 6,673,533, which is herein incorporated by reference), or individual test strips. Compositions of such surfaces include, but are not limited to, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene, hi general, any composition which do not interfere which the electrochemiluminescence of the detection method can be used. Typically, the surface will contain an electrode.
  • the term "surface” may also include a combination of surfaces.
  • the BioVeris TechnologyTM uses a primary surface on which the ECL labeled detection antibody is attached, such as a bead, and a secondary surface which magnetically captures the bead.
  • the electrode is typically present on the secondary surface.
  • the PA capture antibody is immobilized on an uncoated electrode surface.
  • Uncoated electrode surfaces which can be used in the present invention are known.
  • Example of electrode surfaces include, but are not limited to, MULTI- ARRAY ® 96- and 384-well plates from MSD ® (Gaithersburg, MD).
  • the electrode surface is coated with, for example, a compound that facilitates attachment of an antibody molecule to the surface, for example, streptavidin or avidin.
  • coated surfaces which can be used in the present invention are known and include, but are not limited to, streptavidin coated beads such as Dynabeads M-280 Streptavidin (BioVeris , Gaithersburg, MD) for use in the BioVeris (BV) Technology or a SECTORTM strepatavidin coated 96- or 384-well plate from Meso Scale Diagnostics, LLC. (Gaithersburg, MD).
  • the electrode surface is precoated with streptavidin and the PA capture antibody is biotinylated.
  • biotinylation of antibodies as well as coupling methods of the biotinylated antibody to coated surfaces which can be used in the invention are known by one of skill in the art. See, e.g., Merrill et al, Anal. Biochem. 357 (2006) 181-187.
  • the immobilization of the capture PA antibody occurs as a result of the coupling of the biotinylated PA capture antibody to the avidin or streptavidin precoated electrode surface.
  • the immobilization of the PA capture antibody is performed by adding a biotinylated PA antibody present in a buffer to a streptavidin coated plate.
  • the buffer serves as a blocking buffer which blocks any uncoated area of the plate and therefore reduces nonspecific background.
  • the blocking buffer includes bovine serum albumin.
  • the bovine serum albumin is present at a concentration of about 1% w/v to about 10% w/v, about 3% w/v to about 8% w/v, or about 4% w/v to about 6% w/v. In one embodiment, the bovine serum albumin is present at a concentration of 5% w/v.
  • the blocking buffer includes a surfactant.
  • Surfactants which can be used in the blocking buffer are known and include, but are not limited to ionic and non-ionic surfactants.
  • the surfactant is polysorbate, for example, Tween-20TM.
  • the blocking buffer includes an acid or base salt solution.
  • Acid and base salt solutions which can be used in the blocking buffer are known and include, but are not limited to, succinate, histidine, citrate -and/or phosphate— M-one-embodimenty-the-blocking-buf-fer-contains-a-phosphate solution.
  • the immobilized PA capture antibody in the ECL assay includes an antibody that specifically binds to the protective antigen (PA).
  • the present invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that specifically bind to a PA polypeptide (SEQ ID NO:2) or polypeptide fragment or variant of PA.
  • the PA antibody is a single chain Fv's (scFvs) that specifically bind PA polypeptide (SEQ ID NOS:48-65).
  • the PA capture antibody is a polyclonal PA antibody.
  • Anti-PA polyclonal antibodies include, but are not limited to, rabbits, guinea pig, goat, and horse anti-PA polyclonal antibodies.
  • the polyclonal PA capture antibody is a rabbit polyclonal.
  • ECL detection method includes the capture of PA antigen present in a sample.
  • sample includes both biological and non-biological samples.
  • a "biological sample” can include, but is not limited to, any fluid(s) and/or cells obtained from an individual, body fluid, body tissue, body cell, cell line, tissue culture, bacterial culture, or other source which may contain a PA polypeptide protein or mRNA.
  • Biological fluids include, but are not limited to, fluids from fermentation broth, cell cultures supernatants, conditioned cell culture medium, cell lysates, cleared cell lysates, cell extracts, tissue extracts, blood, plasma, serum, sputum, urine, semen, mucus, milk, synovial fluid, pleural fluid, edema fluid, spinal fluid, saliva and fractions thereof that contain protein.
  • the protective antigen (PA) present in a sample is added to the plate with the capture antibody immobilized on the electrode surface.
  • the protective antigen present in the sample binds to and is "captured" by the capture antibody, hi one aspect of this embodiment, the sample is diluted with a sample diluent.
  • a sample diluent may be desirable when the PA concentration in the sample is outside the dynamic range of the ECL detection method.
  • the sample diluent can include a blocking agent such as, but not limited to, bovine serum albumin.
  • the blocking agent is present in the sample diluent at a concentration of about 1% w/v to about 10% w/v, about 3% w/v to about 8% w/v, or about 4% w/v to about 6% w/v.
  • the sample diluent includes a surfactant.
  • Surfactants which can be used in the sample diluent are known and include, but are not limited to ionic and non-ionic surfactants.
  • the surfactant is polysorbate, for example,- Tween-20TM.
  • the surfactant is present in the sample diluent at a concentration of, for example, between about 0.01% v/v to about 1% v/v or, for example, between about 0.05% v/v to about 0.5% v/v.
  • the sample diluent includes an acid or base salt solution. Acid and base salt solutions which can be used in the sample diluent are known and include, but are not limited to, succinate, histidine, citrate and/or phosphate.
  • the sample diluent contains a phosphate solution.
  • the sample is diluted with the sample diluent at a sample: diluent ratio of, for example, less than or equal to about 1 :2, less than or equal to about 1:3, less than or equal to about 1:4, less than or equal to about 1 :5, less than or equal to about 1 :10, or less than or equal to about 1 :20.
  • the sample is further diluted with a second sample diluent.
  • the second sample diluent includes pooled rabbit serum.
  • the pooled rabbit serum can be obtained from Bioreclaimation (Hicksville, NY, USA).
  • the second sample diluent includes pooled serum from a source including, but are not limited to, human, guinea pig, goat, or horse serum.
  • the second sample diluent including the serum is added so that the amount of serum in the sample matches the amount of serum in the standard curve.
  • the pooled serum is present in the second sample diluent at a concentration of less than or equal to about 5% v/v, less than or equal to about 10% v/v, less than or equal to about 15% v/v, less than or equal to about 20% v/v, or less than or equal to about 25% v/v.
  • the sample is diluted with the second sample diluent at a sample:diluent ratio of, for example, less than or equal to about 1:2, less than or equal to about 1:3, less than or equal to about 1 :4, less than or equal to about 1 :5, or less than or equal to about 1 :10.
  • the ECL detection method also includes a detection step.
  • the detection step includes: binding of an ECL labeled PA detection antibody to the captured protective antigen; electrochemically inducing the ECL to emit light; and detecting and measuring the emitted light.
  • the PA detection antibody is coupled to a detectable label, such as an electrochemiluminescent label (ECL) or an electrochemiluminescent (ECL) TAG.
  • ECL label and “ECL TAG” are used interchangeably and refer generally to a label designed to emit light when electrochemically stimulated.
  • the coupling of the ECL label to the PA detection antibody can be direct or indirect.
  • ECL labels confer many advantages over labels used in traditional detection methods. ECL labels are stable for long periods of time, can be attached efficiently to a wide variety of biological materials, are safe, inexpensive, and give a highly characteristic signal that does not occur in nature. Furthermore, detection based on ECL is sensitive, fast, reproducible, utilizes simple instrumentation, and results in littler interference by components such as phosphate buffered saline (PBS), surfactants, or serum. Commonly used electrochemiluminescent (ECL) labels which can be coupled to the PA detection antibodies include, but are not limited to, organometallic compounds.
  • the ECL label is, for example, a ruthenium-containing or osmium-containing luminescent organometallic compound (See, e.g., U.S. Pat. No. 5,310,687, which is herein incorporated by reference).
  • ECL labels which can be used in the present invention include, for example, ECL labels which are commercially available from Meso Scale Discovery (MSD ® ) (Gaithersburg, MD) and BioVerisTM Corporation (Gaithersburg, MD). Examples of ECL labels available from MSD ® include, but are not limited to, Ru(bpy) 3 2+ (MSD- TagTM).
  • Example of ECL labels from BioVeris TM include, but are not limited to, Ru(bpy) 3 2+ (BV-TAGTM Label). According to one embodiment, when a BioVeris TM ECL label is coupled to the PA detection antibody, the PA ECL detection method of the present
  • TM invention uses the BioVeris Technology .
  • the ECL labeled PA detection antibody is added to the plate, which includes the PA antigen captured by the immobilized capture antibody.
  • the ECL labeled PA detection antibody then specifically binds to the PA antigen.
  • a read buffer is added to the plate and electrical energy is generated by the working electrode of the plate which results in the electrochemical stimulation of the ECL label.
  • the electrochemical stimulation of the ECL label by electrochemical energy causes an excited state of ECL label which results in the emission of electromagnetic radiation.
  • the read buffer used in the invention may include, for example, tripropylamine, oxalate, pyruvate, lactate, manlonate, citrate, tartrate or peroxydisulfate.
  • the read buffer may be a phosphate based buffer, tris-based buffer, or a glycyl-glycine based buffer.
  • the read buffer may also include a surfactant, for example, Triton X-100.
  • Examples of read buffers which can be used in the invention are commercially available from Meso Scale Discvoery (MSD ® ) (Gaithersburg, MD) and include Read Buffer T ® , Read Buffer S ® , Read Buffer P ® , and Read Buffer G ® .
  • Examples of read buffers which can be used in the invention are commercially available from BioVerisTM Corporation (Gaithersburg, MD) and include BV-GLO ® read buffers.
  • the light emitted upon electrochemical stimulation is detected and/or measured.
  • the detection can be performed using any commercially available ECL detection instrument.
  • ECL detection instruments which can be used in the invention are available from, for example, Meso Scale Discovery (MSD ® ) (Gaithersburg, MD) and BioVerisTM Corporation (Gaithersburg, MD). Examples of commercial ECL detection instruments from MSD ® include, but are not limited to, the SECTOR TM Imager 6000, SECTOR TM Imager 2400, SECTOR PRTM 400 and the SECTOR PRTM 100. Examples of BioVeris TM Corp.
  • the invention may include an incubation period after the addition of the capture antibody, after the addition of the sample, or after the addition of the ECL labeled detection antibody.
  • the incubation period can be from about 15 minutes to about 150 minutes, from about 30 minutes to about 120 minutes, or from about 45 minutes to about 90 minutes.
  • the incubation period after the addition of the capture antibody is from about 45 minutes to about 90 minutes.
  • the incubation period after the addition of the sample is from about 90 minutes to about 150 minutes. In one aspect of this embodiment, the incubation period after the addition of the ECL labeled detection antibody is about 45 minutes to about 90 minutes. According to another aspect of this embodiment, the temperature during the incubation period may be about 18 0 C to about 39°C. In one embodiment, the incubation temperature after the addition of the capture antibody is about 2O 0 C to about 25 0 C. In one embodiment, the incubation temperature after the addition of the sample is about 35 0 C to about 39 0 C. In one embodiment, the incubation temperature after the addition of the ECL labeled detection antibody is about 2O 0 C to about 25°C.
  • the plate may be agitated or shaken during the incubation period.
  • the plate is agitated.
  • Methods for agitating the plate are known by one of skill in the art and include, but are not limited to, the use of a shaker or a rocker.
  • the shaker is set at a rate of, for example, less than or equal to about 100 rpm, less than or equal to about 200 rpm, less than or equal to about 300 rpm, less than or equal to about 400 rpm, or less than or equal to about 500 rpm.
  • the shaker is set at a rate of less than or equal to about 200 rpm.
  • the ECL detection method may include a wash step after the addition of the capture antibody, after the addition of the sample, or after the addition of the ECL labeled detection antibody.
  • a wash step is performed after each step of the ECL detection method.
  • the purpose of the wash step is to remove, or wash away, any unbound capture antibody, sample, or ECL labeled detection antibody added to the plate.
  • the wash step is performed using a wash buffer.
  • the wash buffer includes a surfactant and an acid and/or base salt solution.
  • Surfactants which can be used in the wash buffer are known and include, but are not limited to ionic and non-ionic surfactants.
  • the surfactant is polysorbate, for example, Tween-20TM.
  • the surfactant is present in the washing buffer at a concentration of between about 0.01% v/v to about 1% v/v or between about 0.05% v/v to about 0.5% v/v.
  • the blocking buffer includes an acid and/or base salt solution. Acid and/or base salt solutions which can be used in the washing buffer are known and include, but are not limited to, succinate, histidine, citrate and/or phosphate. In one embodiment, the blocking buffer contains a phosphate solution. Antibodies
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen.
  • antibody encompasses not only whole antibody molecules, but also antibody multimers and antibody fragments, as well as variants (including derivatives) of antibodies, antibody multimers and antibody fragments.
  • Molecules which are described by the term “antibody” herein include, for example, but are not limited to: single chain Fvs (scFvs), Fab fragments, Fab' fragments, F(ab') 2 , disulfide linked Fvs (sdFvs), Fvs, and fragments comprising or alternatively consisting of, either a VL or a VH domain.
  • scFvs single chain Fvs
  • Fab fragments fragments
  • Fab' fragments fragments
  • F(ab') 2 disulfide linked Fvs
  • Fvs fragments comprising or alternatively consisting of, either a VL or a VH domain.
  • sdFvs disulfide linked Fvs
  • Antibodies which are included as capture and/or detection antibodies include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intracellularly-made antibodies (i.e., intrabodies), and epitope-binding fragments of any of the above.
  • the immunoglobulin molecules which are included can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , IgA 1 and IgA 2 ) or subclass of immunoglobulin molecule.
  • an antibody included comprises, or alternatively consists of, a VH domain, VH CDR, VL domain, or VL CDR having an amino acid sequence of any one of the cell lines in the ATCC Deposits referred to referred to in Table 1, or a fragment or variant thereof.
  • the immunoglobulin is an IgGl isotype.
  • the immunoglobulin is an IgG4 isotype.
  • Immunoglobulins may have both a heavy and light chain.
  • An array of IgG, IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with a light chain of the kappa or lambda forms.
  • isolated antibody an antibody removed from its native environment.
  • an antibody produced by, purified from and/or contained within a hybridoma and/or a recombinant host cell is considered isolated.
  • specific binding by an antibody to PA means that an antibody binds PA but does not significantly bind to (i.e., cross react with) proteins other than PA, such as other proteins in the same family of proteins.
  • An antibody that binds PA protein and does not cross-react with other proteins is not necessarily an antibody that does not bind said other proteins in all conditions; rather, the PA-specific antibody included in the invention preferentially binds PA compared to its ability to bind said other proteins such that it will be suitable for use in at least one type of assay or treatment, i.e., give low background levels or result in no unreasonable adverse effects in treatment.
  • the portion of a protein bound by an antibody is known as the epitope.
  • An epitope may either be linear (i.e., comprised of sequential amino acids residues in a protein sequences) or conformational (i.e., comprised of one or more amino acid residues that are not contiguous in the primary structure of the protein but that are brought together by the secondary, tertiary or quaternary structure of a protein).
  • an antibody that specifically binds PA may or may not bind fragments of PA and/or variants of PA (e.g., proteins that are at least 90% identical to PA) depending on the presence or absence of the epitope bound by a given PA-specific antibody in the PA fragment or variant.
  • PA-specific antibodies may bind species orthologues of PA (including fragments thereof) depending on the presence or absence of the epitope recognized by the antibody in the orthologue.
  • PA-specific antibodies may bind modified forms of PA, for example, PA fusion proteins. In such a case when antibodies bind PA fusion proteins, the antibody must make binding contact with the PA moiety of the fusion protein in order for the binding to be specific.
  • Antibodies that specifically bind to PA can be identified, for example, by immunoassays or other techniques known to those of skill in the art, e.g., the immunoassays described in the Examples below.
  • variant refers to a polypeptide that possesses a similar or identical amino acid sequence as a PA polypeptide, a fragment of a PA polypeptide, an anti-PA antibody or antibody fragment thereof.
  • a variant having a similar amino acid sequence refers to a polypeptide that satisfies at least one of the following: (a) a polypeptide comprising, or alternatively consisting of, an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of PA polypeptide (SEQ E) NO:2), a fragment of a PA polypeptide, an anti-PA antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one or more
  • a polypeptide with similar structure to a PA polypeptide, a fragment of a PA polypeptide, an anti-PA antibody or antibody fragment thereof, described herein refers to a polypeptide that has a similar secondary, tertiary or quaternary structure of a PA polypeptide, a fragment of a PA polypeptide, an anti-PA antibody, or antibody fragment thereof, described herein.
  • the structure of a polypeptide can determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy.
  • a variant PA polypeptide, a variant fragment of a PA polypeptide, or a variant anti-PA antibody and/or antibody fragment possesses similar or identical function and/or structure as the reference PA polypeptide, the reference fragment of a PA polypeptide, or the reference anti-PA antibody and/or antibody fragment, respectively.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art.
  • An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-2268(1990), modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-5877(1993).
  • the BLASTn and BLASTx programs of Altschul, et al. J. MoI. Biol. 215:403-410(1990) have incorporated such an algorithm.
  • Gapped BLAST can be utilized as described in Altschul et al. Nucleic Acids Res. 25:3589-3402(1997).
  • PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • derivative refers to a variant polypeptide of the invention that comprises, or alternatively consists of, an amino acid sequence of a PA polypeptide, a fragment of a PA polypeptide, or an antibody of the invention that specifically binds to a PA polypeptide, which has been altered by the introduction of amino acid residue substitutions, deletions or additions.
  • derivative also refers to a PA polypeptide, a fragment of a PA polypeptide, an antibody that specifically binds to a PA polypeptide which has been modified, e.g., by the covalent attachment of any type of molecule to the polypeptide.
  • a PA polypeptide, a fragment of a PA polypeptide, or an anti-PA antibody may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.
  • a derivative of a PA polypeptide, a fragment of a PA polypeptide, or an anti-PA antibody may be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.
  • a derivative of a PA polypeptide, a fragment of a PA polypeptide, or an anti-PA antibody may contain one or more non-classical amino acids.
  • a polypeptide derivative possesses a similar or identical function as a PA polypeptide, a fragment of a PA polypeptide, or an anti-PA antibody, described herein.
  • fragment refers to a polypeptide comprising an amino acid sequence of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 35 amino acid residues, at least 40 amino acid residues, at least 45 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues, of the amino acid sequence of PA, or an anti- PA antibody (including molecules such as scFv's, that comprise, or alternatively consist of, antibody fragments or variants thereof) that specifically binds to PA.
  • an anti- PA antibody including molecules such as scFv's, that comprise, or alternative
  • the term "host cell” as used herein refers to the particular subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.
  • Antibodies are preferably provided in an isolated form, and preferably are substantially purified. By “isolated” is intended an antibody removed from its native environment. Thus, for example, an antibody produced and/or contained within a recombinant host cell is considered isolated.
  • the basic antibody structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kilodalton) and one "heavy” chain (about 50-70 kilodalton).
  • the amino- terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms "heavy chain” and “light chain” refer to the heavy and light chains of an antibody unless otherwise specified.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains.
  • Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes).
  • the variable regions of each light/heavy chain pair form the antibody binding site.
  • an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same.
  • the chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs.
  • the CDRs from the heavy and the light chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FRl, CDRl, FR2, CDR2, FR3, CDR3 and FR4.
  • a bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann CHn. Exp. Immunol. 79: 315-321 (1990), Kostelny et al. J Immunol. 148:1547 1553 (1992).
  • bispecific antibodies may be formed as "diabodies" (Holliger et al.
  • the present invention includes the use of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that specifically bind to a polypeptide or a polypeptide fragment of PA.
  • antibodies that specifically bind to a polypeptide or a polypeptide fragment of PA include antibodies corresponding to the scFvs referred to in Table 1.
  • Such scFvs may routinely be "converted" to immunoglobulin molecules by inserting, for example, the nucleotide sequences encoding the VH and/or VL domains of the scFv into an expression vector containing the constant domain sequences and engineered to direct the expression of the immunoglobulin molecule.
  • antibodies that are known in the art to specifically bind to the PA antibodies for example, but not limited to, Valtorim TM (MDX-1303) a fully human antibody, AnthimTM, a monoclonal antibody, a rabbit polyclonal antibody (Imgenex ® Corp., Cat. Nos. IMG-660 and IMG-659), mouse monoclonal (BAP0105) to PA (Cat No. GTX21992, Genetex ® , Inc., San Antonio, TX), mouse monoclonal (C3) to PA (Cat. No. GTX28240, Genetex ® , Inc., San Antonio, TX), and mouse monoclonal (BAPOlOl) to PA (Cat. No. GTX21988, Genetex ® , Inc., San Antonio, TX) can be used as capture and/or detection antibodies in the ECL detection method.
  • Valtorim TM MDX-1303
  • AnthimTM a monoclonal antibody
  • a rabbit polyclonal antibody Imgenex ®
  • scFvs single chain antibody molecules
  • scFvs single chain antibody molecules
  • Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs (e.g., VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of the corresponding region of the antibody expressed by a cell line contained in an ATCC Deposit referred to in Table 1), that specifically bind to PA (or fragments or variants thereof) can be used with the invention, as are nucleic acid molecules that encode these scFvs, and/or molecules.
  • the invention includes the use of scFvs as detection and/or capture antibodies in the ECL method, wherein the scFvs include an amino acid sequence selected from the group consisting of SEQ ID NOs: 48-56, preferably SEQ ID NOs:50 and 53 as referred to in Table 1 below.
  • Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs (e.g., VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those referred to in Table 1), that specifically bind to PA are also included in the invention, as are nucleic acid molecules that encode these scFvs, and/or molecules (e.g., SEQ ID NOs:57-65).
  • NSO cell lines that express IgGl antibodies that comprise the VH and VL domains of scFvs which can be used in the invention have been deposited with the American Type Culture Collection ("ATCC") on the dates listed in Table 1 and given the ATCC Deposit Numbers identified in Table 1.
  • ATCC American Type Culture Collection
  • the ATCC is located at 10801 University Boulevard, Manassas, VA 20110-2209, USA.
  • the ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.
  • the invention includes the use of antibodies that comprise the VH and VL domains of scFvs.
  • an antibody included in the invention is an antibody expressed by cell line NSO PA 2973 (PWD0587) #240-22 (See Table 1).
  • PA protein is a 764 amino acid protein (SEQ ID NO:2) comprising a signal sequence from amino acid residues 1-29, and a 735 amino acid secreted protein which undergoes further process upon binding to an anthrax receptor, (e.g., ATR or CMG2) on the cell surface.
  • anthrax receptor e.g., ATR or CMG2
  • the 735 amino acid secreted protein also known as PA83 because it has a molecular weight of approximately 83 kilodaltons, has a structure that is largely made up of antiparallel beta pleated sheets with only a few short alpha-helices.
  • the protein can be divided into four domains: Domain I (amino acid residues 30-287 of SEQ BD NO:2), Domain ⁇ (amino acid residues 288-516 of SEQ ID NO:2), Domain III (amino acid residues 517-624 of SEQ ID NO:2), and Domain IV (amino acid residues 625-764) of SEQ ED NO:2).
  • Domain I contains two calcium ions and the protease cleavage site RKKR at amino acid residues 193-196 of SEQ ID NO:2.
  • Domain I contains the entire 20 kilodalton fragment (PA20, amino acid residues 30-196 of SEQ ID NO:2) that is cleaved off of PA upon binding to an anthrax receptor (e.g., ATR or CMG2) at the cell surface. That portion of Domain I that remains after cleavage of PA20 forms the N terminus of active PA63 and may be involved in binding LF and EF.
  • Domain II is the heptamerization domain and also contains a large flexible loop that is implicated in membrane insertion. Domain III, is small and its function is not clearly understood.
  • Domain IV is the receptor binding domain.
  • antibodies which can be used may bind the intact 735 amino acid secreted form of PA (PA83), polypeptides that comprise or alternatively consist of the PA63 protein, the PA20 fragment, and/or any one or more of domains I, II, III, or FV.
  • PA83 the intact 735 amino acid secreted form of PA
  • antibodies which can be used bind PA83 and prevent its cleavage of the PA20 fragment from the PA63 fragment by proteases.
  • antibodies may bind the PA63 form of PA and prevent oligomerization, and in particular heptamerization of PA63.
  • the antibodies may specifically bind PA polypeptide.
  • An antibody that specifically binds PA may, in some embodiments, bind fragments, variants (including species orthologs of PA), multimers or modified forms of PA.
  • an antibody specific for PA may bind the PA moiety of a fusion protein comprising all or a portion of PA.
  • PA proteins may be found as monomers or multimers (i.e., dimers, trimers, tetramers, and higher multimers). Accordingly, the present invention includes the use of antibodies that bind PA proteins found as monomers or as part of multimers. In one embodiment, antibodies may bind PA monomers, dimers, trimers or heptamers. In additional embodiments, antibodies may bind at least dimers, at least trimers, or at least tetramers containing one or more PA polypeptides.
  • Antibodies used in the invention may bind PA homomers or heteromers.
  • the term homomer refers to a multimer containing only PA proteins (including PA fragments such as PA63, variants, and fusion proteins, as described herein). These homomers may contain PA proteins having identical or different polypeptide sequences.
  • a homomer is a multimer containing only PA proteins having an identical polypeptide sequence.
  • antibodies may bind PA homomers containing PA proteins having different polypeptide sequences.
  • antibodies may bind a PA homodimer (e.g., containing PA proteins having identical or different polypeptide sequences).
  • antibodies may bind at least a homodimer, at least a homotrimer, or at least a homotetramer of PA. [0092] In specific embodiments antibodies used in the present invention bind PA homohep tamers.
  • heteromer refers to a multimer containing heterologous proteins (i.e., proteins containing polypeptide sequences that do not correspond to a polypeptide sequences encoded by the PA gene) in addition to the PA proteins.
  • antibodies used in the invention bind a heterodimer, a heterotrimer, or a heterotetramer.
  • the antibodies may bind at least a heterodimer, at least a heterotrimer, or at least a -heterotetramer containing one or more PA polypeptides.
  • antibodies may bind a PA heteroheptamer.
  • Antibodies included in the invention may bind PA multimers that are the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
  • PA multimers such as, for example, homoheptamers, that are formed when PA proteins (such as PA63 polypeptide monomers) contact one another in solution.
  • PA proteins such as PA63 polypeptide monomers
  • heteromultimers such as, for example, heteroheptamers, that are formed when proteins of the invention contact antibodies to the PA polypeptides (including antibodies to the heterologous polypeptide sequence in a fusion protein) in solution. Multimers are formed by covalent associations with and/or between PA proteins.
  • covalent associations may involve one or more amino acid residues contained in the polypeptide sequence of the protein (e.g., the polypeptide sequence recited in SEQ ID NO:2).
  • the covalent associations are cross- linking between cysteine residues located within the polypeptide sequences of the proteins which interact in the native (i.e., naturally occurring) polypeptide.
  • the covalent associations are the consequence of chemical or recombinant manipulation.
  • such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a PA fusion protein.
  • covalent associations are between the heterologous sequence contained in a fusion protein (see, e.g., US Patent Number 5,478,925).
  • the covalent associations are between the heterologous sequence contained in a PA-Fc or PA-human serum albumin (PA-HSA) fusion protein (as described herein).
  • Antibodies included in the present invention may bind PA polypeptide fragments comprising or alternatively, consisting of, an amino acid sequence contained in SEQ ID NO:2. Protein fragments may be "free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
  • Antibodies may bind polypeptide fragments, including, for example, fragments that comprise or alternatively, consist of from about amino acid residues: 1 to 29, 30 to 59, 60 to 89, 90 to 119, 120 tol49, 150 to 175, 176 to 196, 197 to 226, 227 to 256, 257 to 287, 288 to 312, 313 to 337, 338 to 362, 363 to 387, 388 to 412, 413 to 437, 438 to 462, 463 to 487, 488 to 516, 517 to 542, 543 to 569, 570 to 569, 570 to 596, 597 to 624, 625 to 652, 653 to 680, 681 to 708, 709 to 736, and/or 737 to 764 of SEQ ID NO:2.
  • polypeptide fragments that antibodies of the invention may bind can be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acids in length.
  • about includes the particularly recited value, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
  • Antibodies may bind polypeptide fragments selected from the group: a polypeptide comprising or alternatively, consisting of, the full length PA polypeptide (amino acid residues 1 to 764 in SEQ ID NO:2); a polypeptide comprising or alternatively, consisting of, the secreted form of PA (amino acid residues 30 to 764 in SEQ DD NO:2); a polypeptide comprising or alternatively, consisting of, the PA20 fragment (amino acid residues from about 30 to about 196 in SEQ ID NO:2); a polypeptide comprising or alternatively, consisting of, the PA63 fragment (amino acid residues from about 197 to about 764 in SEQ ID NO:2); a polypeptide comprising or alternatively, consisting of, PA domain I (amino acid residues 30 to 287 of SEQ ID NO:2); a polypeptide comprising or alternatively, consisting of, PA domain II (amino acid residues 288 to 516 of S
  • Domain I contains the proteolytic cleavage site. When the secreted form of PA is cleaved at this site, a 20 kilodalton fragment (PA20) is released from PA, generating the biologically active 63 kilodalton PA63 fragment.
  • PA20 20 kilodalton fragment
  • antibodies may bind an epitope at or near this cleavage site and prevent the cleavage of the secreted form of PA that results in the generation of PA20 and PA63.
  • antibodies may prevent cleavage of PA into PA20 and PA63 may bind one or more PA peptides (as well as the native amino acid secreted form of the protein, PA83, see, e.g., Example 2) selected from the group consisting of: (a) amino acid residues 190 to 209 of SEQ DD NO:2; (b) amino acid residues 181 to 201 of SEQ DD NO:2; (c) amino acid residues 198 to 212 of SEQ DD NO:2; (d) amino acid residues 196 to 212 of SEQ DD NO:2; (e) amino acid residues 194 to 212 of SEQ DD NO:2; (f) amino acid residues 192 to 212 of SEQ DD NO:2; (g) amino acid residues 190 to 212 of SEQ DD NO:2; (h) amino acid residues 188 to 212 of SEQ DD NO:2; (i) amino acid residues 186 to 212 of SEQ DD NO
  • Domain IV of PA is important for interactions between PA and its receptor (e.g., ATR (SEQ DD NO:3) or CMG2 (SEQ DD NO:42)).
  • PA e.g., ATR (SEQ DD NO:3) or CMG2 (SEQ DD NO:42)
  • antibodies included in the present invention bind PA polypeptide fragments comprising, or alternatively consisting of amino acid residues 625 to 764 of SEQ ID NO:2.
  • the antibodies may bind all or a portion of domain IV of PA prevent PA from binding to ATR and/or CMG2.
  • the antibodies may bind all or a portion of domain IV of PA protect cells from death induced by anthrax toxins.
  • Antibodies may also bind fragments comprising, or alternatively, consisting of structural or functional attributes of PA.
  • Such fragments include amino acid residues that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet-forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, surface forming regions, and high antigenic index regions (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson- Wolf program) of complete (i.e., full-length) PA.
  • Antibodies included in the invention may also bind regions of PA that are essential for PA function. Antibodies included in the present invention may also bind regions of PA that are essential for PA function and inhibit or abolish PA function.
  • protein engineering may be employed to improve or alter the characteristics of PA polypeptides. Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides can show, e.g., enhanced activity or increased stability. In addition, they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions. Antibodies of the present invention may bind such modified PA polypeptides.
  • Non-naturally occurring variants of PA may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter et al, Nucl. Acids Res. 73:4331 (1986); and Zoller et al, Nucl. Acids Res. 70:6487 (1982)), cassette mutagenesis (see e.g., Wells et al, Gene 34:315 (1985)), restriction selection mutagenesis (see e.g., Wells et al, Philos. Trans. R. Soc.
  • the invention also encompasses antibodies that bind PA derivatives and analogs that have one or more amino acid residues deleted, added, and/or substituted.
  • cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges; N-linked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N- linked sites.
  • PA polypeptides e.g., arginine and lysine residues
  • amino acid residues of PA polypeptides may be deleted or substituted with another residue to eliminate undesired processing by proteases such as, for example, furins or kexins.
  • the antibodies used in the invention may also include antibodies that bind a polypeptide comprising, or alternatively, consisting of a polypeptide comprising, or alternatively, consisting of the polypeptide of SEQ ID NO:2 including the leader; a polypeptide comprising, or alternatively, consisting of the polypeptide of SEQ ID NO:2 minus the amino terminal methionine; a polypeptide comprising, or alternatively, consisting of the polypeptide of SEQ ID NO:2 minus the leader; a polypeptide comprising, or alternatively, consisting of the PA domain I; a polypeptide comprising, or alternatively, consisting of the PA domain II; a polypeptide comprising, or alternatively, consisting of the PA domain III; a polypeptide comprising, or alternatively, consisting of the PA domain IV; a polypeptide comprising, or alternatively, consisting of the PA20 fragment; a polypeptide comprising, or alternatively, consisting of the PA63 fragment; as well as polypeptide
  • a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a PA polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the PA polypeptide.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • whether any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in SEQ ED NO:2 can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711.
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
  • the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.
  • a determination of whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of this embodiment.
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%.
  • a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of this embodiment.
  • Antibodies of the invention may bind modified PA Polypeptides
  • Antibodies included in the present invention may bind modified forms of PA proteins SEQ ID NO:2).
  • antibodies included in the present invention bind PA polypeptides (such as those described above) including, but not limited to naturally purified PA polypeptides, PA polypeptides produced by chemical synthetic procedures, and PA polypeptides produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells using, for example, the recombinant compositions and methods described above. Depending upon the host employed in a recombinant production procedure, the polypeptides may be glycosylated or non-glycosylated.
  • PA polypeptides may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • antibodies included in the present invention may bind PA proteins that were chemically synthesized using techniques known in the art (e.g., see Creighton, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y. (1983), and Hunkapiller, et ah, Nature 370:105-111 (1984)).
  • a peptide corresponding to a fragment of a PA polypeptide can be synthesized by use of a peptide synthesizer.
  • nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the PA polypeptide sequence.
  • Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.
  • the amino acid can be D (dextrorotary) or L (levorotary).
  • the antibodies included in the invention additionally encompasses antibodies that may bind PA polypeptides that are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derealization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
  • PA polypeptides for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression.
  • the polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
  • antibodies that may bind chemically modified derivatives of PA polypeptides which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U. S. Patent No. 4,179,337).
  • the chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
  • PA polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given PA polypeptide.
  • PA polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic PA polypeptides may result from posttranslational natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • the invention includes antibodies (e.g., antibodies comprising two heavy chains and two light chains linked together by disulfide bridges) that specifically bind PA (SEQ ID NO:2) or fragments or variants thereof, wherein the amino acid sequence of the heavy chain and the amino acid sequence of the light chain are the same as the amino acid sequence of a heavy chain and a light chain of one or more scFvs or cell lines referred to in Table 1.
  • antibodies e.g., antibodies comprising two heavy chains and two light chains linked together by disulfide bridges
  • PA SEQ ID NO:2
  • fragments or variants thereof wherein the amino acid sequence of the heavy chain and the amino acid sequence of the light chain are the same as the amino acid sequence of a heavy chain and a light chain of one or more scFvs or cell lines referred to in Table 1.
  • the invention includes antibodies (each consisting of two heavy chains and two light chains linked together by disulfide bridges to form an antibody) that specifically bind PA or fragments or variants thereof, wherein the amino acid sequence of the heavy chain or the amino acid sequence of the light chain are the same as the amino acid sequence of a heavy chain or a light chain of one or more scFvs or cell lines referred to in Table 1.
  • Immunospecific binding to PA polypeptides may be determined by immunoassays known in the art or described herein for assaying specific antibody-antigen binding.
  • Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies that specifically bind to PA are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies molecules, fragments and/or variants (SEQ ID NOS:57-65).
  • the invention includes antibodies that specifically bind to a PA or a fragment or variant thereof, comprise a polypeptide having the amino acid sequence of a heavy chain of at least one of the scFvs referred to in Table 1 or cell lines contained in the ATCC Deposits referred to in Table 1 and/or a light chain of at least one of the scFvs referred to in Table 1 or cell lines contained in the ATCC Deposits referred to in Table 1.
  • antibodies that specifically bind to PA or a fragment or variant thereof comprise a polypeptide having the amino acid sequence of any one of the VH domains of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1 and/or any one of the VL domains of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1.
  • antibodies comprise the amino acid sequence of a VH domain and VL domain from a single scFv referred to in Table 1 or single recombinant antibody expressed by a cell line contained in an ATCC Deposit referred to in Table 1.
  • antibodies comprise the amino acid sequence of a VH domain and a VL domain from different scFvs referred to in Table 1 or different recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1.
  • Molecules comprising, or alternatively consisting of, antibody fragments or variants of the VH and/or VL domains of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1 that specifically bind to PA are also inlcuded in the invention, as are nucleic acid molecules encoding these VH and VL domains, molecules, fragments and/or variants (SEQ ID NOS:57-65).
  • the present invention also includes the use antibodies that specifically bind to a polypeptide, or polypeptide fragment or variant of PA, wherein said antibodies comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one, two, three, or more of the VH CDRs contained in a VH domain of one or more scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1.
  • the invention includes antibodies that specifically bind PA or fragments or variants thereof, comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of a VH CDRl contained in a VH domain of one or more scFvs or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1.
  • antibodies that specifically bind PA comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VH CDR2 contained in a VH domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1.
  • antibodies that specifically bind PA or fragments or variants thereof comprise, or alternatively consist of a polypeptide having the amino acid sequence of a VH CDR3 contained in a VH domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1.
  • Molecules comprising, or alternatively consisting of, these antibodies, or antibody fragments or variants thereof, that specifically bind to PA or a PA fragment or variant thereof are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments and/or variants (SEQ ID NOS:57-65).
  • the present invention also includes the use of antibodies that specifically bind to a PA polypeptide or a polypeptide fragment or variant of PA, wherein said antibodies comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one, two, three, or more of the VL CDRs contained in a VL domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1.
  • the invention includes antibodies that specifically bind PA or a fragment or variant thereof, comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of a VL CDRl contained in a VL domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1.
  • antibodies that specifically bind PA or a fragment or variant thereof comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VL CDR2 contained in a VL domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1.
  • antibodies that specifically bind PA or a fragment or variant thereof comprise, or alternatively consist of a polypeptide having the amino acid sequence of a VL CDR3 contained in a VL domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1.
  • Molecules comprising, or alternatively consisting of, these antibodies, or antibody fragments or variants thereof, that specifically bind to PA or a PA fragment or variant thereof are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments and/or variants (SEQ ID NOS:57-65).
  • the present invention also includes the use antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants) that specifically bind to PA polypeptide or a fragment or variant of a PA, wherein said antibodies comprise, or alternatively consist of, one, two, three, or more VH CDRs and one, two, three or more VL CDRs, as contained in a VH domain or VL domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1.
  • the invention includes the use of antibodies that specifically bind to a PA polypeptide or polypeptide fragment or variant of PA, wherein said antibodies comprise, or alternatively consist of, a VH CDRl and a VL CDRl, a VH CDRl and a VL CDR2, a VH CDRl and a VL CDR3, a VH CDR2 and a VL CDRl, VH CDR2 and VL CDR2, a VH CDR2 and a VL CDR3, a VH CDR3 and a VH CDRl, a VH CDR3 and a VL CDR2, a VH CDR3 and a VL CDR3, or any combination thereof, of the VH CDRs and VL CDRs contained in a VH domain or VL domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1.
  • one or more of these combinations are from the same scFv or the same recombinant antibody expressed by cell line contained in an ATCC deposit as disclosed in Table 1.
  • Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies, that specifically bind to PA or a fragment or variant thereof are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants (SEQ ID NOS:57-65).
  • Antibodies shown in Table 1, which can be used in the invention, were prepared via the utilization of a phage scFv display library. Technologies utilized for achieving the same are disclosed in the patents, applications, and references disclosed herein.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues) or synthetic cDNA libraries.
  • the DNA encoding the VH and VL domains are joined together by an scFv linker by PCR and cloned into a phagemid vector (e.g., pCANTAB 6 or pComb 3 HSS).
  • the vector is electroporated in E. coli and the E. coli is infected with helper phage.
  • Phage used in these methods are typically filamentous phage including fd and M 13 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII.
  • Phage expressing an antigen binding domain that binds to an antigen of interest i.e., a PA polypeptide or a fragment thereof
  • an antigen of interest i.e., a PA polypeptide or a fragment thereof
  • Examples of phage display methods that can be used to make the antibodies of the present invention include, but are not limited to, those disclosed in Brinkman et al, J. Immunol. Methods 182:41-50 (1995); Ames et al, J. Immunol.
  • the cDNAs encoding the VH and VL domains of the scFvs referred to in Table 1 or recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1 may be expressed in all possible combinations using a phage display library, allowing for the selection of VH/VL combinations that bind PA polypeptides with preferred binding characteristics such as improved affinity or improved off rates.
  • VH and VL segments — and in particular, the CDR regions of the VH and VL domains of the scFvs referred to in Table 1 or recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1, in particular, may be mutated in vitro. Expression of VH and VL domains with "mutant" CDRs in a phage display library allows for the selection of VH/VL combinations that bind PA polypeptides with preferred binding characteristics such as improved affinity or improved off rates.
  • antibodies used in the invention comprise the VH and VL domains of the PWD0587 scFv wherein the VH domain contains one or more of the following mutations (using amino acid numbering according to that of SEQ ID NO: 53): Q13R, S31W, HOOV, and/or E105D.
  • An antibody comprising the PWD0587 VH domain with the Q13R, S31W and HOOV mutations and the PWD0587 VL domain had an approximately 11 fold increase in affinity for the PA antigen compared to an antibody comprising the PWD0587 heavy and light chains.
  • an antibody which is included in the invention comprises the PWD0587 VH domain with the Q13R, S3 IW and Il 00V mutations and the PWD0587 VL domain.
  • an antibody comprising the PWD0587 VH domain with the Ql 3R and S31W mutations and the PWD0587 VL domain had an approximately 68 fold increase in affinity for the PA antigen compared to an antibody comprising the PWD0587 heavy and light chains.
  • an antibody which can be used in the invention comprises the PWD0587 VH domain with the Q13R and S31W mutations and the PWD0587 VL domain.
  • an antibody comprising the PWD0587 VH domain with the Q13R, S3 IW, I100V and E105D mutations and the PWD0587 VL domain had an approximately 121 fold increase in affinity for the PA antigen compared to an antibody comprising the PWD0587 heavy and light chains.
  • an antibody which can be used in the invention comprises the PWD0587 VH domain with the Ql 3R, S3 IW, Il 00V and E105D mutations and the PWD0587 VL domain.
  • an antibody comprising the PWD0587 VH domain with the Q13R, S31W and E105D mutations and the PWD0587 VL domain had an approximately 665 fold increase in affinity for the PA antigen compared to an antibody comprising the PWD0587 heavy and light chains.
  • an antibody which can be used in the invention comprises the PWD0587 VH domain with the Q13R, S31W and E105D mutations and the PWD0587 VL domain.
  • Antibodies which can be used in the invention can be produced by any method known in the art.
  • antibodies in accordance with the present invention can be expressed in cell lines including, but not limited to, myeloma cell lines and hybridoma cell lines.
  • Sequences encoding the cDNAs or genomic clones for the particular antibodies can be used for transformation of a suitable mammalian or nonmamrnalian host cells or to generate phage display libraries, for example.
  • polypeptide antibodies of the invention may be chemically synthesized or produced through the use of recombinant expression systems.
  • One way to produce the antibodies used in the invention would be to clone the VH and/or VL domains of an scFv referred to in Table 1 or recombinant antibody expressed by the cell lines contained in the ATCC Deposits referred to in Table 1.
  • PCR primers complementary to VH or VL nucleotide sequences See Example 6 may be used to amplify the VH and VL sequences.
  • the PCR products may then be cloned using vectors, for example, which have a PCR product cloning site consisting of a 5' and 3' single T nucleotide overhang, that is complementary to the overhanging single adenine nucleotide added onto the 5' and 3' end of PCR products by many DNA polymerases used for PCR reactions.
  • the VH and VL domains can then be sequenced using conventional methods known in the art.
  • the VH and VL domains may be amplified using vector specific primers designed to amplify the entire scFv, (i.e. the VH domain, linker and VL domain.)
  • the cloned VH and VL genes may be placed into one or more suitable expression vectors.
  • PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site may be used to amplify the VH or VL sequences.
  • the PCR amplified VH domains may be cloned into vectors expressing the appropriate immunoglobulin constant region, e.g., the human IgGl or IgG4 constant region for VH domains, and the human kappa or lambda constant regions for kappa and lambda VL domains, respectively.
  • the vectors for expressing the VH or VL domains comprise a promoter suitable to direct expression of the heavy and light chains in the chosen expression system, a secretion signal, a cloning site for the immunoglobulin variable domain, immunoglobulin constant domains, and a selection marker such as neomycin.
  • the VH and VL domains may also be cloned into a single vector expressing the necessary constant regions.
  • the heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art (See, for example, Guo et al., J. Clin.
  • the invention provides polynucleotides comprising, or alternatively consisting of, a nucleotide sequence encoding an antibody included in the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof).
  • the invention also provides polynucleotides that hybridize under high stringency, or alternatively, under intermediate or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides complementary to nucleic acids having a polynucleotide sequence that encodes an antibody of the invention or a fragment or variant thereof.
  • the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
  • nucleotide sequences encoding these antibodies can be determined using methods well known in the art, i.e., the nucleotide codons known to encode the particular amino acids are assembled in such a way to generate a nucleic acid that encodes the antibody, of the invention.
  • Such a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides ⁇ e.g., as described in Kutmeier et al, BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source ⁇ e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells or Epstein Barr virus transformed B cell lines that express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a
  • nucleotide sequence of the antibody including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof
  • the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.
  • VH and VL domains of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1, or fragments or variants thereof, are inserted within framework regions using recombinant DNA techniques known in the art.
  • one, two, three, four, five, six, or more of the CDRs of a VH and/or a VL domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1, or fragments or variants thereof, are inserted within framework regions using recombinant DNA techniques known in the art.
  • the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. MoI. Biol. 278: 457-479 (1998) for a listing of human framework regions, the contents of which are hereby incorporated by reference in its entirety).
  • the polynucleotides generated by the combination of the framework regions and CDRs encode an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that specifically binds to a PA polypeptide.
  • an antibody including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof
  • polynucleotides encoding variants of antibodies or antibody fragments having one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions do not significantly alter binding of the antibody to its antigen.
  • such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules, or antibody fragments or variants, lacking one or more intrachain disulfide bonds.
  • Other alterations to the polynucleotide are encompassed by the present invention and fall within the ordinary skill of the art.
  • Monoclonal antibodies specific for PA polypeptides may be prepared using hybridoma technology.
  • Kohler et al. Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-CeIl Hybridomas, Elsevier, N. Y., pp. 571-681 (1981)).
  • XenoMouseTM mice may be immunized with PA polypeptides.
  • the splenocytes of such mice are extracted and fused with a suitable myeloma cell line.
  • a suitable myeloma cell line such as the myeloma cell line (SP20), available from the ATCC, may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP20), available from the ATCC.
  • the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the PA polypeptides.
  • an antibody including antibody fragments or variants thereof (e.g., a heavy or light chain of an antibody included in the invention)
  • an expression vector(s) containing a polynucleotide that encodes the antibody Once a polynucleotide encoding an antibody molecule (e.g., a whole antibody, a heavy or light chain of an antibody, or portion thereof (preferably, but not necessarily, containing the heavy or light chain variable domain)), has been obtained, the vector(s) for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
  • the invention thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule included in the invention (e.g., a whole antibody, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody, or a portion thereof, or a heavy or light chain CDR, a single chain Fv, or fragments or variants thereof), operably linked to a promoter.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464, the contents of each of which are hereby incorporated by reference in its entirety) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.
  • the expression vector(s) is(are) transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.
  • the invention provides host cells containing polynucleotide(s) encoding an antibody used in the invention (e.g., whole antibody, a heavy or light chain thereof, or portion thereof, or a single chain antibody, or a fragment or variant thereof), operably linked to a heterologous promoter.
  • the expression of entire antibody molecules, vectors encoding both the heavy and light chains may be co- expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • host-expression vector systems may be utilized to express antibody molecules.
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule.
  • These include, but are not limited to, bacteriophage particles engineered to express antibody fragments or variants thereof (single chain antibodies), microorganisms such as bacteria (e.g., E. coli, B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3, NSO cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from
  • bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO)
  • CHO Chinese hamster ovary cells
  • a vector such as the major intermediate early gene promoter element from human cytomegalovirus
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO 1. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pFN vectors (Inouye & Inouye, Nucleic Acids Res.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5- transferase (GST).
  • GST glutathione 5- transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • Autographa californica nuclear polyhedrosis virus may be used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • Antibody coding sequences may be cloned individually into non-essential regions (for example, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedrin promoter).
  • an AcNPV promoter for example, the polyhedrin promoter
  • the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome ⁇ e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 8 1:355-359 (1984)).
  • Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al, Methods in Enzymol. 153:51-544 (1987)).
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, HeLa, COS, NSO, MDCK, 293, 3T3, Wl 38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT2O and T47D, and normal mammary gland cell line such as, for example, CRL7O3O and HsS78Bst.
  • breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT2O and T47D
  • normal mammary gland cell line such as, for example, CRL7O3O and HsS78Bst.
  • stable expression is preferred.
  • cell lines which stably express the antibody may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the antibody molecule.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.
  • a number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al, Cell 11:223 (1977)), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al, Cell 22:8 17 (1980)) genes can be employed in tk-, hgprt- or aprt- cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al, Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci.
  • the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, "The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells” in DNA Cloning, Vol.3. (Academic Press, New York, 1987)).
  • vector amplification for a review, see Bebbington and Hentschel, "The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells" in DNA Cloning, Vol.3. (Academic Press, New York, 1987)).
  • a marker in the vector system expressing antibody is amplifiable
  • increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the coding sequence of the antibody, production of the antibody will also increase (Crouse et al, MoI. Cell. Biol. 3:257 (1983)).
  • Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively.
  • An advantage of glutamine synthase based vectors are the availability of cell lines (e.g., the murine myeloma cell line, NSO) which are glutamine synthase negative.
  • Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene.
  • a glutamine synthase expression system and components thereof are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657 which are incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors that may be used are commercially available from suppliers, including, for example Lonza Biologies, Inc. (Portsmouth, NH). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al, Bio/technology 10:169(1992) and in Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are incorporated in their entireties by reference herein.
  • the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides.
  • the light chain is preferably placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2 197 (1980)).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • an antibody molecule including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof
  • it may be purified by any method known in the art for purification of an immunoglobulin molecule, or more generally, a protein molecule, such as, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • Antibodies may be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
  • Antibodies may include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the antibodies may be glycosylated or may be non-glycosylated. In addition, antibodies may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • Antibodies may be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller, M., et al., 1984, Nature 310:105-111).
  • a peptide corresponding to a fragment of an antibody included in the invention can be synthesized by use of a peptide synthesizer.
  • nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the antibody polypeptide sequence.
  • Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t- butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na- methyl amino acids, and amino acid analogs in general. Furthermore, the amino
  • the invention encompasses the use of antibodies which may be differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.
  • Additional antibodies may have post-translational modifications, for example, e.g., N-linked or 0-lmked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or 0-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.
  • post-translational modifications for example, e.g., N-linked or 0-lmked carbohydrate chains, processing of N-terminal or C-terminal ends
  • attachment of chemical moieties to the amino acid backbone e.g., chemical modifications of N-linked or 0-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.
  • Antibodies included in the present invention may also be described or specified in terms of their binding to PA polypeptides or fragments or variants of PA polypeptides.
  • antibodies used in the invention may bind PA polypeptides, or fragments or variants thereof, with a dissociation constant or KQ of less than or equal to 5 X 10 "2 M, 10 "2 M, 5 X 10 '3 M, 10 "3 M, 5 X 10 "4 M, 10 "4 M, 5 X 10 " 5 M, or 10 "5 M.
  • antibodies used in the invention may bind PA polypeptides or fragments or variants thereof with a dissociation constant or K D less than or equal to 5 X 10 "6 M, 10 '6 M, 5 X 10 "7 M, 10 '7 M, 5 X 10 "8 M, or 10 "8 M.
  • antibodies used in the invention may bind PA polypeptides or fragments or variants thereof with a dissociation constant or K D less than or equal to 5 X 10 "9 M, 10 "9 M, 5 X 10 "10 M, 10 “10 M, 5 X 10 "11 M, 10 "11 M, 5 X 10 "12 M, 10 “12 M, 5 X “13 M, W 13 M, 5 X 10 "14 M, 10 “14 M, 5 X 10 "15 M, or 10 "15 M.
  • the invention encompasses the use of antibodies that may bind PA polypeptides with a dissociation constant or K D that is within any one of the ranges that are between each of the individual recited values.
  • antibodies used in the invention may bind PA polypeptides or fragments or variants thereof with an off rate (k o ff) of less than or equal to 5 X 10 "2 sec “ ⁇ 10 "2 sec “1 , 5 X 10 "3 sec “1 or 10 "3 sec “1 .
  • antibodies used in the invention may bind PA polypeptides or fragments or variants thereof with an off rate (k off ) less than or equal to 5 X 10 "4 sec “1 , 10 "4 sec “1 , 5 X 10 "5 sec “1 , or 10 "5 sec “1 5 X 10 "6 sec “1 , 10 “6 sec “1 , 5 X 10 "7 sec “1 or 10 "7 sec “1 .
  • the invention encompasses the use of antibodies that may bind PA polypeptides with an off rate (koff) that is within any one of the ranges that are between each of the individual recited values.
  • antibodies used in the invention may bind PA polypeptides or fragments or variants thereof with an on rate (k on ) of greater than or equal to 10 3 M '1 sec “1 , 5 X 10 3 M “1 sec “1 , 10 4 M '1 sec “1 or 5 X 10 4 M “1 sec “1 .
  • antibodies used in the invention may bind PA polypeptides or fragments or variants thereof with an on rate (Ic 0n ) greater than or equal to 10 5 M "1 sec “1 , 5 X 10 5 M “1 sec “1 , 10 6 M “1 sec “ l, or 5 X 10 6 M “1 sec “1 or 10 7 M “1 sec “1 .
  • the invention encompasses the use of antibodies that may bind PA polypeptides with on rate (Ic 0n ) that is within any one of the ranges that are between each of the individual recited values.
  • the antibodies used in the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), specifically bind to PA polypeptides and do not cross-react with any other antigens.
  • the antibodies used in the invention specifically bind to PA polypeptides (e.g., SEQ ID NO:2 or fragments or variants thereof) and do not cross-react with other bacterial binary toxins (A-B toxins) such as those from Clostridum difficile, Clostridium perfringens, Clostridium spiroforme, Clostridium botulinum, Bacillus cereus and/or Bacillus thuringiensis.
  • PA polypeptides e.g., SEQ ID NO:2 or fragments or variants thereof
  • A-B toxins bacterial binary toxins
  • the antibodies specifically bind to PA polypeptides and cross-react with other antigens.
  • the antibodies specifically bind to PA polypeptides (e.g., SEQ ID NO:2 or fragments or variants thereof) and cross-react with other bacterial binary toxins (A-B toxins) such as those from Clostridum difficile, Clostridium perfringens, Clostridium spiroforme, Clostridium botulinum, Bacillus cereus and/or Bacillus thuringiensis.
  • A-B toxins bacterial binary toxins
  • antibodies may preferentially bind PA (SEQ ID NO:2), or fragments and variants thereof relative to their ability to bind other antigens (e.g., other bacterial binary toxins (A-B toxins) such as those from Clostridum difficile, Clostridium perfringens, Clostridium spiroforme, Clostridium botulinum, Bacillus cereus and/or Bacillus thuringiensis).
  • A-B toxins other bacterial binary toxins
  • An antibody's ability to preferentially bind one antigen compared to another antigen may be determined using any method known in the art.
  • an antibody may be considered to bind a first antigen preferentially if it binds said first antigen with a dissociation constant (K D ) that is less than the antibody's K D for the second antigen.
  • an antibody may be considered to bind a first antigen preferentially if it binds said first antigen with an affinity (i.e., K D ) that is at least one order of magnitude less than the antibody's K D for the second antigen.
  • an antibody may be considered to bind a first antigen preferentially if it binds said first antigen with an affinity (i.e., KD) that is at least two orders of magnitude less than the antibody's K D for the second antigen.
  • KD an affinity
  • an antibody may be considered to bind a first antigen preferentially if it binds said first antigen with an off rate (k off ) that is less than the antibody's k off for the second antigen.
  • an antibody may be considered to bind a first antigen preferentially if it binds said first antigen with a k off that is at least one order of magnitude less than the antibody's koff for the second antigen.
  • an antibody may be considered to bind a first antigen preferentially if it binds said first antigen with a k off that is at least two orders of magnitude less than the antibody's k Off for the second antigen.
  • the invention also includes the use of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that have one or more of the same biological characteristics as one or more of the antibodies described herein.
  • biological characteristics is meant, the in vitro or in vivo activities or properties of the antibodies, such as, for example, the ability to bind to PA polypeptides (e.g., either the PA83 or PA63 form of PA); or the ability to inhibit the cleavage of the PA83 into PA20 and PA63 by proteases such as trypsin or furin.
  • antibodies may: prevent oligomerization of PA63, especially heptamerization of PA63; inhibit or abolish the ability of PA63 to bind to an anthrax receptor, e.g., ATR and/or CMG2; inhibit or abolish the ability of PA63 to bind LF or EF; inhibit or abolish the ability of PA63 to form pores in membranes; inhibit or abolish the ability of lethal toxin (LT) to kill cells, such as macrophages, or animals; or inhibit or abolish the ability of PA heptamers to translocate LF or EF across a membrane.
  • the antibodies will bind to the same epitope as at least one of the antibodies specifically referred to herein.
  • the present invention also includes the use of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that inhibit or abolish biological activities of PA.
  • biological activities of PA is meant, for example, the ability of PA83 to be cleaved by proteases into PA20 and PA63 fragments; the ability of PA to bind to ATR and/or CMG2; the ability of PA or PA63 to oligomerize, especially to heptamerize; the ability of PA63 to bind LF or EF; the ability of PA63 heptamers to form pores in a membrane; and/or the ability of PA heptamers to translocate EF or LF across a membrane.
  • an antibody that inhibits or abolishes biological activities of PA comprises, or alternatively consists of a VH and/or a VL domain of at least one of the scFvs referred to in Table 1 or recombinant antibodies expressed by the cell lines referred to in Table 1 , or a fragment or variant thereof.
  • an antibody that inhibits or abolishes biological activities of PA comprises, or alternatively consists of a VH and a VL domain of any one of the scFvs referred to in Table 1 or recombinant antibodies expressed by the cell lines referred to in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
  • the present invention also includes the use of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that inhibit the cleavage of the PA83 into PA20 and PA63 by proteases such as trypsin or furin.
  • An antibody that binds peptides that span the RKKR cleavage site of PA may be predictive of an antibody's ability to inhibit the cleavage of PA by proteases.
  • a PA cleavage assay is described in J. Biol. Chem.
  • an antibody that inhibits the cleavage of the PA83 into PA20 and PA63 comprises, or alternatively consists of a VH and/or a VL domain of at least one of the scFvs referred to in Table 1 or recombinant antibodies expressed by the cell lines referred to in Table 1, or a fragment or variant thereof, hi one embodiment, an antibody that inhibits the cleavage of the PA83 into PA20 and PA63 comprises, or alternatively consists of a VH and a VL domain of any one of the scFvs referred to in Table 1 or recombinant antibodies expressed by the cell lines referred to in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
  • the present invention also includes the use of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that block or inhibit the binding of PA to ATR and/or CMG2 (e.g., see Example 3).
  • an antibody that blocks or inhibits the binding of PA to ATR and/or CMG2 comprises, or alternatively consists of a VH and/or a VL domain of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1, or a fragment or variant thereof.
  • an antibody that blocks or inhibits the binding of PA to ATR and/or CMG2 comprises, or alternatively consists of a VH and a VL domain of any one of the scFvs referred to in Table 1 or any one of the recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also provided in the invention.
  • the present invention also includes the use of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that block or inhibit the ability of PA or PA63 to heptamerize.
  • an antibody that blocks or inhibits the ability of PA or PA63 to heptamerize comprises, or alternatively consists of a VH and/or a VL domain of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1, or a fragment or variant thereof.
  • an antibody that blocks or inhibits the ability of PA63 to heptamerize comprises, or alternatively consists of a VH and a VL domain of any one of the scFvs referred to in Table 1 or any one of the recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also provided in the invention. [0166] The present invention also includes the use of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that block or inhibit the ability of PA63 to bind EF or LF.
  • an antibody that blocks or inhibits the ability of PA63 to bind EF or LF comprises, or alternatively consists of a VH and/or a VL domain of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1, or a fragment or variant thereof.
  • an antibody that blocks or inhibits the ability of PA63 to bind EF or LF comprises, or alternatively consists of a VH and a VL domain of any one of the scFvs referred to in Table 1 or any one of the recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also provided in the invention.
  • the present invention also includes the use of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that block or inhibit the ability of PA63 heptamers to form pores in membranes.
  • an antibody that blocks or inhibits the ability of PA63 heptamers to form pores in membranes comprises, or alternatively consists of a VH and/or a VL domain of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1, or a fragment or variant thereof,
  • an antibody that blocks or inhibits the ability of PA63 heptamers to form pores in membranes comprises, or alternatively consists of a VH and a VL domain of any one of the scFvs referred to in Table 1 or any one of the recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1, or a fragment or variant thereof.
  • Nucleic acid molecules encoding these antibodies are also provided in the invention.
  • an antibody that blocks or inhibits the ability of PA63 heptamers to translocate EF or LF across membranes comprises, or alternatively consists of a VH and/or a VL domain of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1, or a fragment or variant thereof, hi one embodiment, an antibody that blocks or inhibits the ability of PA63 heptamers to translocate EF or LF across membranes comprises, or alternatively consists of a VH and a VL domain of any one of the scFvs referred to in Table 1 or any one of the recombinant antibodies expressed by the cell lines contained
  • the present invention also includes the use of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that block or inhibit the ability of anthrax lethal toxin to kill cells or animals, hi one embodiment, an antibody that blocks or inhibits the ability of anthrax lethal toxin to kill cells or animals comprises, or alternatively consists of a VH and/or a VL domain of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1, or a fragment or variant thereof.
  • an antibody that blocks or inhibits the ability of anthrax lethal toxin to kill cells or animals comprises, or alternatively consists of a VH and a VL domain of any one of the scFvs referred to in Table 1 or any one of the recombinant antibodies expressed by the cell lines contained in the ATCC Deposits referred to in Table 1 , or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also provided in the invention.
  • Antibodies used in the present invention may be characterized in a variety of ways.
  • antibodies and related molecules may be assayed for the ability to specifically bind to PA or a fragment or variant of PA, using techniques described herein or routinely modifying techniques known in the art.
  • Assays for the ability of the antibodies of the invention to specifically bind PA or a fragment or variant of PA may be performed in solution (e.g., Houghten, Bio/Techniques 13:412-421(1992)), on beads (e.g., Lam, Nature 354:82-84 (1991)), on chips (e.g., Fodcr, Nature 364:555-556 (1993)), on bacteria (e.g., U.S.
  • Patent No. 5,223,409 on spores (e.g., Patent Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (e.g., Cull et al., Proc. Natl. Acad. Sci. USA 89:1865-1869 (1992)) or on phage (e.g., Scott and Smith, Science 249:386-390 (1990); Devlin, Science 249:404-406 (1990); Cwirla et al., Proc. Natl. Acad. Sci. USA 87:7178-7182 (1990); and Felici, J. MoI. Biol.
  • Antibodies that have been identified to specifically bind to PA or a fragment or variant of PA can then be assayed for their specificity and affinity for PA using or routinely modifying techniques described herein or otherwise known in the art (see, e.g., Examples 1 and 2).
  • the antibodies may be assayed for specific binding to PA polypeptides and cross-reactivity with other antigens by any method known in the art.
  • Immunoassays which can be used to analyze specific binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as BIAcore analysis, FACS (fluorescence activated cell sorter) analysis, immunofluorescence, immunocytochemistry, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, western blots, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays, to name but a few.
  • ELISAs comprise preparing antigen, coating the well of a 96-well microti ter plate with the antigen, washing away antigen that did not bind the wells, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the wells and incubating for a period of time, washing away unbound antibodies or non-specifically bound antibodies, and detecting the presence of the antibodies specifically bound to the antigen coating the well.
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • the antibody 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.
  • the antigen need not be directly coated to the well; instead the ELISA plates may be coated with an anti-Ig Fc antibody, and the antigen in the form or a PA-Fc fusion protein, may be bound to the anti- Ig Fc coated to the plate. This may be desirable so as to maintain the antigen protein (e.g., the PA polypeptides) in a more native conformation than it may have when it is directly coated to a plate.
  • the antibody instead of coating the well with the antigen, the antibody may be coated to the well.
  • the detectable molecule could be the antigen conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase).
  • the binding affinity of an antibody (including an scFv or other molecule comprising, or alternatively consisting of, antibody fragments or variants thereof) 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., antigen labeled with 3 H or 125 I), or fragment or variant thereof 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.
  • labeled antigen e.g., antigen labeled with 3 H or 125 I
  • the affinity of the antibody of the present invention for PA 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.
  • a PA polypeptide is incubated with an antibody of the present invention conjugated to a labeled compound (e.g., compound labeled with 3 H or 125 I) in the presence of increasing amounts of an unlabeled second anti-PA antibody.
  • a labeled compound e.g., compound labeled with 3 H or 125 I
  • Assays for determining the ability of one antibody to competitively inhibit the binding of another antibody are known in the art (See, for example, Harlow, Ed & David Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory, 1988. pp. 567-569.)
  • This kind of competitive assay between two antibodies may also be used to determine if two antibodies bind the same, closely associated (e.g., overlapping) or different epitopes.
  • BIAcore kinetic analysis is used to determine the binding on and off rates of antibodies (including antibody fragments or variants thereof) to PA, or fragments of PA.
  • Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 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 to 4 hours) at 40 degrees C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 40 degrees C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer.
  • a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium de
  • 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).
  • immunoprecipitation protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
  • Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane 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., 32 P or 125 I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the anti
  • the present invention is an ECL detection kit for screening and/or detecting protective antigen (PA).
  • the kit includes a capture antibody immobilized on an surface containing an electrode and an ECL labeled PA detection antibody that specifically binds to the protective antigen (PA).
  • the kit can be used in combination with a portable ECL analyzer.
  • ECL analyzers which can be used with the ECL detection kit for PA include, but are not limited to, BioVeris' M-SERIES ® MlM analyzer. (BioVerisTM, Gaithersburg, MD)
  • the kit is useful in that it provides for the detection of PA in a sample taken in the field or at any possible anthrax exposure site in addition to the laboratory.
  • the ECL detection kit for PA is advantageous in that it provides an ECL labeled PA antibody that specifically binds to the PA antigen.
  • Example 1 Isolation and characterization of scFvs referred to in Table 1 [0182] Maxisorp tubes (Nunc) were coated overnight with 10 micrograms/ml of PA83 protein in PBS at 4°C. Unbound PA was removed by washing the tubes with IX PBST and IX PBS followed by filling the tubes with a 3% milk solution in IX PBS for one hour to block any exposed tube surface. Approximately 10 13 TU of phage from phage display libraries available from Cambridge Antibody Technology (Cambridgshire, United Kingdom) diluted in 3%milk/lXPBS was applied to the tube and incubated for at least 60 minutes at room temperature.
  • Phage are eluted by adding 1 ml of 100 mM triethylamine with gentle shaking after which the solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage were then used to infect 10 ml of mid-log E. coli TGl by incubating eluted phage with bacteria for 30 minutes at 37 0 C. The E. coli were then plated on 2XYT plates containing 2% glucose and 100 micrograms/ml ampicillin. The resulting bacterial library was then rescued with delta gene 3 helper phage to prepare phage for a subsequent round of selection.
  • This process is usually repeated for a total of 2-4 rounds of affinity purification. Specific enrichment of PA binding phage can be monitored during the selection process. Individual clones from both the second and the third rounds of selections were screened for the ability to bind to PA protein using the assay protocol described below.
  • PA83 Purified full-length PA protein (PA83) was labeled with Biotin-LC-Sulfo -NHS (Pierce) at a molar challenge ratio of 8:1 in PBS, 8.0 for 60 minutes at 23°C. Protein was separated from free label using a NAP 5 gel filtration column (Amersahm-Pharmacia Biotech) following manufacturer's protocol.
  • a polyclonal antibody specific for the Ml 3 phage coat (Amersham-Pharmacia Biotech) was labeled with the electrochemi- luminescent reporter Origen-T AG-NHS ((Ori-TAG), IGEN International, Inc.) at a molar challenge ratio of 5:1 in PBS, 8.0 buffer for 60 minutes at 23°C. Protein was separated from free label using a NAP 5 gel filtration column (Amersham-Pharmacia Biotech) following manufacturer's protocol. The amount of incorporated Origen -TAG label was determined by measuring the absorbance of the undiluted labeling reaction at 455 nm in a
  • the supernatants containing scFv-phage were screened for binding to PA83 using the following protocol: In a 96 well plate, 5 microliters of scFv-phage were combined with 150 microliters of 0.5 micrograms/ml Biotin-PA83 and 0.5 micrograms/ml Origen-Tag labeled anti-M13 polyclonal antibody and 20 micrograms of Streptavidin coated magnetic beads (Dynal M280 beads). The plate was sealed and mixed vigorously for 60 minutes at room temperature. The electrochemiluminescent (ECL) signal was measured in each well of the plate using an Origen M8 series ECL analyzer (IGEN International, Inc).
  • ECL electrochemiluminescent
  • antibody concentration at half-maximal antigen binding is a measure of affinity. In practical terms it can be used to rank the affinities of antibodies to quickly identify best binders. The lower the antibody concentration required for 50% of plateau binding, the higher is the affinity of the antibody for antigen.
  • a conventional ELISA is used to generate binding isotherms for PA antibodies in order to derive their EC- 50 values. Additionally, antibodies may be tested for their ability to bind peptides that span the RKKR cleavage site in PA.
  • substrate solution is prepared by dissolving 1 tablet of TMB (Sigma cat# T3405) in 5 ml of water. After the tablet is dissolved, 5 ml of the substrate buffer (0.1 M Na 2 PO 4 , 0.05 M Citric acid) and 2 microliters of 30% H 2 O 2 is added.
  • Plates are washed 4 times with PBST and 100 microliters of substrate is added to each well. Plates are incubated for 10 min at room temperature and the Absorption at 450 nm is measured on SpectraMax 3000 (Molecular Devices).
  • biotinylated peptides are: sp-186: biotin-SNSRKKRSTSAGPTVPDRDN (amino acids 190-206 of SEQ ID NO:2); sp-187: biotin-QLPELKQKSSNSRKKRSTSAG (amino acids 181-201 of SEQ ID NO:2); and s ⁇ -189: biotin-QLPELKQKS SNSRKK (amino acids 181- 195 of SEQ ID NO:2).
  • the plates are then washed 4 times with PBST.
  • substrate solution is prepared by dissolving 1 tablet of TMB (Sigma, Cat# T3405) in 5 ml of water. After the tablet is dissolved, 5 ml of the substrate buffer (0.1 M Na 2 PO 4 , 0.05 M Citric acid) and 2 ml of 30% H 2 O 2 are added. Plates are washed 4 times with PBST and 100 microliters of substrate is added to each well. Plates are incubated for 15 minutes at room temperature and the absorption (at 450 nm) is measured on SpectraMaxPlus (Molecular Devices).
  • Example 3 Method of Detecting PA in an ECL Detection Assay
  • a 0.1% PBS-Tween (PBS-T) wash buffer solution was prepared by mixing together 2 L of 1OX PBS (Quality Biological, Inc., Gaithersburg, MD, Cat. No. 130-069-161, Lot No. 714897), 100 mL 20% Tween-20 (Sigma-Aldrich, St. Louis, MO, Cat. No. P1379), and 17.9 L distilled water.
  • a 5% bovine serum albumin (BSA), 0.1% PBS-T blocking buffer was prepared by adding 5 g of BSA (Sigma, St. Louis, A3059-5009) to 100 L of wash buffer.
  • a 1% BSA, 0.1% PBST sample diluent solution was prepared by mixing 40 mL of blocking buffer with 160 mL of wash buffer solution.
  • a 20% serum solution was prepared by adding rabbit serum (New Zealand White, Cat. No. RABSRM, Lot No. RABBREC.8636) to sample diluent.
  • the read buffer used in the experiment was MSD ® Read Buffer T, Cat. No. R92TC-1 purchased from Meso Scale Discovery, Gaithersburg, MD.
  • the read buffer is prepared by adding 50 mL of 4X Read Buffer T to 150 mL of dH2O.
  • PA Sample Spikes Protective antigen sample spikes ranging from 100 ng/mL to 0.1 ng/mL were prepared in 20% rabbit serum sample spikes and neat rabbit serum sample spikes by diluting 0.45 mg/mL stock solution of protective antigen (Human Genome Sciences, Inc., Lot#H6504801-El) into a sample diluent.
  • protective antigen Human Genome Sciences, Inc., Lot#H6504801-El
  • Preparation of 20% rabbit serum sample spikes The 0.45 mg/mL PA stock solution was initially diluted 1 :90 to a concentration of 5000 ng/mL by adding 4.44 ⁇ L of PA stock solution to 395.56 ⁇ L of 20% serum solution.
  • the 5000 ng/mL solution was further diluted 1:100 to a concentration of 50 ng/mL by adding 4.0 ⁇ L of the 5000 ng/mL solution to 396 ⁇ L of 20% serum solution.
  • the 20% rabbit serum PA sample spikes were then prepared as shown in Table 3. Table 3 Preparation of 20% Rabbit Serum PA Sample Spikes
  • a 200 ng/mL PA solution is initially prepared by diluting 4.4 ⁇ L of 450000 ng/ML PA stock solution into 9895.6 ⁇ L of sample diluent.
  • the 200 ng/mL PA solution is then diluted 1 :2 in 40% rabbit serum for a final concentration of 100 ng/mL in 20% rabbit serum.
  • a 1:3 serial dilution is performed in 20% rabbit serum for a final standard curve ranging from 100 ng/mL to 0.0457 ng/mL.
  • Duplicate 25 ⁇ L aliquots were then added to the 96-well plate. As shown in Figure 1, the standard curve was added to rows A and B, lanes 1-8.
  • PA and PA heptamer were immobilized on individual flow cells of a BIAcore CM5 sensor chip.
  • the PA monoclonal antibodies, PWD0283 and PWD0587 (IgGl format), were diluted from 50 ⁇ g/mL (333 nM) to 0.625 ⁇ g/mL (4.1 nM). Each concentration was in contact with the PA proteins during a 4-minute association phase.
  • the off-rate of the anti-PA monoclonal antibodies was determined by washing the complex in the presence of buffer for 5 minutes. The binding data were analyzed using the BIAevaluation software, Version 3.1.
  • RNA is isolated from the cell lines and used as a template for RT-PCR designed to amplify the VH and VL domains of the antibodies expressed by the EBV cell lines.
  • Cells may lysed in the TRIzol® reagent (Life Technologies, Rockville. MD) and extracted with one fifth volume of chloroform. After addition of chloroform, the solution is allowed to incubate at room temperature for 10 minutes, and the centrifuged at 14,000 rpm for 15 minutes at 4 0 C in a tabletop centrifuge.
  • RNA is precipitated using an equal volume of isopropanol.
  • Precipitated RNA is pelleted by centrifuging at 14,000 rpm for 15 minutes at 4°C in a tabletop centrifuge. Following centrifugation, the supernatant is discarded and washed with 75% ethanol. Following washing, the RNA is centrifuged again at 800 rpm for 5 minutes at 4°C. The supernatant is discarded and the pellet allowed to air dry.
  • RNA is the dissolved in DEPC water and heated to 60 0 C for 10 minutes. Quantities of RNA can determined using optical density measurements.
  • cDNA may be synthesized, according to methods well-known in the art, from 1.5-2.5 micrograms of RNA using reverse transcriptase and random hexamer primers. cDNA is then used as a template for PCR amplification of VH and VL domains. Primers used to amplify VH and VL genes are shown in Table 9. Typically a PCR reaction makes use of a single 5' primer and a single 3' primer. Sometimes, when the amount of available RNA template is limiting, or for greater efficiency, groups of 5' and/or 3' primers may be used. For example, sometimes all five VH-5' primers and all JH3' primers are used in a single PCR reaction.
  • the PCR reaction is carried out in a 50 microliter volume containing IX PCR buffer, 2mM of each dNTP, 0.7 units of High Fidelity Taq polymerase, 5' primer mix, 3' primer mix and 7.5 microliters of cDNA.
  • the 5' and 3' primer mix of both VH and VL can be made by pooling together 22 pmole and 28 pmole, respectively, of each of the individual primers.
  • PCR conditions are: 96 0 C for 5 minutes; followed by 25 cycles of 94 0 C for 1 minute, 5O 0 C for 1 minute, and 72 0 C for 1 minute; followed by an extension cycle of 72 0 C for 10 minutes. After the reaction is completed, sample tubes were stored 4 0 C.
  • PCR samples are then electrophoresed on a 1.3% agarose gel. DNA bands of the expected sizes (-506 base pairs for VH domains, and 344 base pairs for VL domains) can be cut out of the gel and purified using methods well known in the art. Purified PCR products can be ligated into a PCR cloning vector (TA vector from Invitrogen Inc., Carlsbad, CA). Individual cloned PCR products can be isolated after transfection of E. coli and blue/white color selection. Cloned PCR products may then be sequenced using methods commonly known in the art.
  • TA vector from Invitrogen Inc., Carlsbad, CA
  • the applicant hereby requests that the application has been laid open to public inspection (by the Norwegian Patent Office), or has been finally decided upon by the Norwegian Patent Office without having been laid open inspection, the furnishing of a sample shall only be effected to an expert in the art.
  • the request to this effect shall be filed by the applicant with the Norwegian Patent Office not later than at the time when the application is made available to the public under Sections 22 and 33(3) of the Norwegian Patents Act. If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on the list of recognized experts drawn up by the Norwegian Patent Office or any person approved by the applicant in the individual case.
  • the applicant hereby requests that, until the application has been laid open to public inspection (by the Danish Patent Office), or has been finally decided upon by the Danish Patent office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art.
  • the request to this effect shall be filed by the applicant with the Danish Patent Office not later that at the time when the application is made available to the public under Sections 22 and 33(3) of the Danish Patents Act. If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Danish Patent Office or any person by the applicant in the individual case.
  • the applicant hereby requests that, until the application has been laid open to public inspection (by the Swedish Patent Office), or has been finally decided upon by the Swedish Patent Office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art.
  • the request to this effect shall be filed by the applicant with the International Bureau before the expiration of 16 months from the priority date (preferably on the Form PCT/RO/134 reproduced in annex Z of Volume I of the PCT Applicant's Guide). If such a request has been filed by the applicant any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Swedish Patent Office or any person approved by a applicant in the individual case.
  • the applicant hereby requests that until the date of a grant of a Netherlands patent or until the date on which the application is refused or withdrawn or lapsed, the microorganism shall be made available as provided in the 31F(I) of the Patent Rules only by the issue of a sample to an expert.
  • the request to this effect must be furnished by the applicant with the Netherlands Industrial Property Office before the date on which the application is made available to the public under Section 22C or Section 25 of the Patents Act of the Kingdom of the Netherlands, whichever of the two dates occurs earlier.

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Abstract

La présente invention porte sur un procédé par électrochimioluminescence (ECL) pour détecter l'antigène protecteur des toxines de l'anthrax à l'aide d'anticorps et de fragments, ou de variantes de ceux-ci, ou de molécules apparentées qui se lient spécifiquement à l'antigène protecteur (PA) de Bacillus anthracis (Anthrax). De tels procédés de détection par ECL ont des utilisations, par exemple, dans la détection d'un antigène protecteur dans un échantillon provenant d'un animal, de préférence un mammifère et, de la façon que l'on préfère le plus, un être humain, permettant ainsi l'administration d'un traitement préventif après exposition et/ou d'une thérapeutique à base de petites molécules et de protéines après exposition, pour traiter une exposition à l'anthrax.
PCT/US2007/023624 2006-11-09 2007-11-09 Procédés et anticorps pour détecter un antigène protecteur WO2008140483A2 (fr)

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CN106554418A (zh) * 2016-10-21 2017-04-05 中国人民解放军第三军医大学第附属医院 炭疽毒素受体2单克隆抗体及制备方法和应用
WO2018075758A1 (fr) * 2016-10-19 2018-04-26 Alexion Pharmaceuticals, Inc. Procédé de quantification de c5 non liée dans un échantillon
US11828683B2 (en) 2016-10-19 2023-11-28 Alexion Pharmaceuticals, Inc. Method of quantitating unbound C5a in a sample

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US20050267294A1 (en) * 2002-07-15 2005-12-01 Harvey Barrett R Antibodies with increased affinities for anthrax antigens
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018075758A1 (fr) * 2016-10-19 2018-04-26 Alexion Pharmaceuticals, Inc. Procédé de quantification de c5 non liée dans un échantillon
JP2019537710A (ja) * 2016-10-19 2019-12-26 アレクシオン ファーマシューティカルズ， インコーポレイテッド 試料中の非結合ｃ５の定量化方法
JP7096240B2 (ja) 2016-10-19 2022-07-05 アレクシオン ファーマシューティカルズ， インコーポレイテッド 試料中の非結合ｃ５の定量化方法
US11828683B2 (en) 2016-10-19 2023-11-28 Alexion Pharmaceuticals, Inc. Method of quantitating unbound C5a in a sample
US11965884B2 (en) 2016-10-19 2024-04-23 Alexion Pharmaceuticals, Inc. Method of quantitating unbound C5 in a sample
CN106554418A (zh) * 2016-10-21 2017-04-05 中国人民解放军第三军医大学第附属医院 炭疽毒素受体2单克隆抗体及制备方法和应用

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