Antibody-assisted detoxication of reactive organophosphorus compounds

Abstract

Antibodies capable of accelerating the breakdown of reactive organophosphorus compounds, including organophosphorus pesticides and organophosphorus nerve agents, nucleotides encoding the heavy and light peptide sequences of the antibodies, vectors comprised of the nucleotide sequences, cells comprising the vector, haptens and constructs incorporating the haptens for inducing immunological responses that provide the antibodies and intermediates of the haptens and said construct are provided.

Description

ANTIBODY-ASSISTED DETOXICATION OF REACTIVE ORGANOPHOSPHORUS COMPOUNDSSTATEMENT OF GOVERNMENT RIGHTS

[0001] The invention was supported, at least in part, by a grant from the Government of the United States of America (National Institutes of Health (NIH) grant U01 NS112108). The US Government has certain rights to the invention.FIELD OF THE INVENTION

[0002] The present invention relates to antibodies and antigen binding fragments thereof that bind and / or convert to harmless products organophosphorus compounds as well as therapeutic and diagnostic methods of use and compositions comprising the described antibodies and antigen binding fragments.BACKGROUND OF THE INVENTION

[0003] Organophosphorus (OPs) compounds are a ubiquitous class of organic molecules that include various insecticides and highly toxic nerve agents. OP insecticides and OP nerve agents share a high degree of structure similarity in that both type of OP compound contain a central pentavalent phosphorous atom covalently attached to one double bonded group (W) and three single bonded groups X, Y, Z, one of which is an excellent leaving group which provides for a reactive molecule the general structure of which is as follows:

[0004] OP insecticides and OP nerve agents also share a common mechanism responsible for their toxicities in that both inactivate biologically-important enzymes thatare present in insects and mammals. Between 150,000 and 300,000 toxic incidences are attributed to exposures to OP insecticide annually just in the United States. As a result, there is great concern regarding exposures to such reactive OP compounds.

[0005] OP insecticides and OP nerve agents act by inhibiting the general cholinesterase (ChE) family of enzymes. Within that family of enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are the most well-known targets. In the case of AChE, the resulting OP adduct inactivates the enzyme, allows the neurotransmitter acetylcholine (ACh) to build up in the synapse, stimulatesautonomic receptors, and blocks neuromuscular junction receptors triggering neurotoxicity. The symptoms resulting from nerve agent exposure are primarily the consequence of accumulation of excess ACh at nerve junctions where ordinarily small amounts of ACh are needed for impulse transmission.

[0006] Exposure to a reactive OP compound results in the formation of OP- cholinesterase adducts that inhibit the cholinesterase function in the brain, central nervous system and blood. The OP-adducts are a covalent modification at an essential serine residue on the enzyme and the longer the OP remains attached as an adduct, the longer the enzyme is unable to function. If the OP-enzyme adduct is long lived; serious neurotoxicity problems including seizure / tremor can lead to death without medical intervention. The current standard of care (SOC) for OP exposures is administration of atropine, midazolam (or benzodiazepine) and an oxime in addition to ventilation and supportive action. Atropine is used to block muscarinic receptor overload resulting from surplus acetylcholine neurotransmitter. The midazolam or benzodiazepine is used to reduce or minimize seizure resulting from constant receptor innervation. The oxime is the only therapeutic of the three that is an actual antidote.

[0007] Oximes react directly with the phosphorus atom of OP groups that are covalently attached to the enzyme and displace it, and thereby ‘restore or reactivate’ the enzyme or protein activity. However, oximes are largely ineffective against most OP-enzyme adducts because they are too sterically congested to undergo reaction with the adduct, or the adduct has degraded to a different OP (i.e., ‘aging’). Another deficit to oxime antidotes is that they cannot access the essential enzyme pools in the central nervous system (CNS) due to poor biodistribution and an insufficient in vivo halflife. Most oxime antidotes are given multiple times in gram quantities in the first hours of intoxication to combat those deficiencies and unfortunately, high amounts of oxime is toxic. In the US, the oxime used therapeutically is 2-pyridine aldoxime methiodide (2-PAM), which remains as the only FDA-approvcd oxime.

[0008] The standard of care, which includes administering the aforementioned oxime, is sometimes life-saving, but often fails to eliminate circulating OP from the bloodstream. Simply stated, so long as OPs are present in the bloodstream, they will continue to inactivate target and non-target enzymes and p ro d uc e tox i ci ty . Once the SOC therapeutic dose limit is reached, supportive care is all that remains.

[0009] Therefore, there a need exists in the art for a therapeutic agent that safely and more efficiently clears or removes toxic OPs in subjects exposed to such compounds. A promising approach would be a therapeutic agent capable of scavenging or destroying OPs in the bloodstream as a cooperative SOC approach. Moreover, if the known reactivity of OPs toward oximes could be leveraged in the design of a novel therapeutic as a component of the SOC there would be more efficient destruction of the OP compound.

[0010] One therapeutic class of compounds that could be utilized as an antidote to OP poisoning isan antibody. However, standalone antibodies are subject to inactivation by an OP compound like any other susceptible protein. As a solution to that problem in the art antibodies are generated to accommodate both an oxime (e.g., 2-PAM) and an OP at a combined binding site. That would allow the oxime to more efficiently attack the OP and break it down before it is able to react with an antibody binding site residue.

[0011] Because SOC administration includes a large dose of an oxime antidote, a high circulating concentration of the oxime occurs. As a result, the oxime binding site of an antibody of the present invention is expected to be saturated upon co-administration of the antibody. In that context the oxime is reasonably considered to be a cofactor. Compared to a small molecule such as an oxime, antibodies are long-lived in the bloodstream, and since the co-administered oxime is protected from elimination by virtue of its selective binding to an antibody of the present invention, its half-life is also extended.

[0012] Thus, an antibody-based therapeutic to OP exposure as described herein would decrease OP toxicity without altering the current SOC treatment regime. This approach is even more important considering that many OPs will out-live the SOC oxime compound without the benefit of the antibodybased therapeutic approach described herein.

[0013] The antibodies described in this invention have in vitro and in vivo diagnostic, therapeutic and prophylactic utilities. For example, antibodies herein can be administered to cells or cell extracts in culture (in vitro or ex vivo) or in a subject (in vivo) to treat, prevent, and / or diagnose exposures to mono-, di- or triesters of organophosphorus (OP) compounds, including pesticides (e.g., insecticides, insecticide oxons, and herbicides), chemical warfare agents (e.g., ‘nerve agents’), flame retardants, lubricants, fuel additives, pharmaceuticals, plastics, solvents and OP-contaminated food products.

[0014] The antibodies or antigen binding fragments of the invention, or compositions comprising the antibodies or antigen binding fragments or mRNA or DNA that encodes the antibodies or fragments provided herein can be used to treat accidental or purposeful exposures, where an organophosphorus compound is assumed or identified as present. The provided antibody or antigen binding fragment(s) can bind, degrade or change in form OP compounds thereby reducing the exposure risk. Furthermore, the antibodies or antigen binding fragments provided and compositions comprising same can also be used to diagnose and determine exposure levels and subsequent treatments to be administered.

[0015] In one embodiment, the subject is a human or non-human subject and the antibody or an antigen binding fragment molecule of the invention can be administered for therapeutic purposes. The invention provides a method of sequestering, removing and / or degrading organophosphorus (OP) compounds in which the invention contacts and binds the OP compound.

[0016] In methods of sequestering an organophosphorus compound, the invention binds and removes or reduces levels of the organophosphorus compound from circulation but does not convert the organophosphorus compound. The invention may or may not cooperatively bind an oxime in this method.

[0017] In methods of degrading an organophosphorus compound, the invention acts alone or incombination with an oxime-reactivator (e.g., 2-pyridine aldoxime salt; previously or co-administered) to convert the organophosphorus compounds into less toxic components, e.g., hydrolyzed components.

[0018] The antibody invention described herein or antigen-binding fragments thereof or mRNA or DNA vaccine, may be used in combination with other therapies. For example, the combination therapy can include a composition of the present invention co-formulated with, and / or co-administered with, one or more additional therapeutic agents, an oxime cholinesterase reactivator (2-PAM, TMB-4, HI-6, obidoxime, pralidoxime) antidote, and / or atropine (post- synaptic muscarinic antagonist), and / or a muscle relaxant (midazolam, benzodiazepine) and / or other therapies. Methods of administering antibody therapeutics are well-known in the art. Suitable dosages of the antibody invention depend on the age and weight of the subject and the organophosphorus compound identified in the exposure.

[0019] In other embodiments, the antibodies are administered in combination with other therapeutic treatment modalities, including ventilation, supportive care (e.g., oxygen), anti-seizure therapeutics, and / or chemo therapeutics. Such combination therapies may advantageously lower acute and delayed toxicity, and reduce morbidity and mortality outcomes associated with exposure to organophosphorus compounds.

[0020] The antibodies described in this invention would be given: (a) in its own formulation separate from oxime or other therapeutics used to treat OP exposures; (b) formulated with 2-PAM or other oxime antidotes; (c) as a second formulation in combination with standard of care intervention, or oxime - antidote and / or midazolam.

[0021] In other embodiments, the haptens described in the invention may be used to elicit a defensive antibody response in the form of a vaccine that would reduce toxicity to unexpected or imminent OP exposures.SUMMARY OF THE INVENTION

[0022] Disclosed herein are compounds of Formula 1 and 2 ;

[0023] wherein R is hydrogen or alkyl and Ri and R2 independently are alkyl and X' is a suitable counterion;

[0024] In preferred embodiments the Formula 1 and Formula 2 compounds have the structures of Formula 3 and Formula 4, respectively:pyridinium hapten (4); X = counterion

[0025] In some embodiments compounds of Formula 1-4 are conjugated to a carrier protein through a linker, wherein the linker is attached to the Formula 1-4 compound’s aniline-NHz via an amide bond and to a carrier protein nucleophile through reaction of a reactive functional group of a linker precursor to provide an immunogenic construct.

[0026] Other haptens include those in which -the -OR1moiety of Formula 3 or Formula 4 hapten or the ethoxy group of the Formula 1 or Formula 2 hapten is replaced with a primary amine.

[0027] In principle embodiments the carrier protein is selected from the group consisting of serum albumin, KLH, ovalbumin (OVAL), human serum albumin (HSA), other antigenic peptides or other carrier proteins (CPs). The general structure of the immunogenic construct is therefore a carrier protein-linker-hapten.

[0028] The haptens describe herein resemble the transition state (i.e., are transition state analogs) in the mechanism for oxime detoxication resulting from addition of an oxyanion oxime nucleophile(e.g., 2-PAM) to an OP compound.

[0029] In some embodiments linkers include glutaric acid, succinic acid or polyethylene glycols- carboxylic acid (PEG-type) residues covalently attached to the OP-aniline of a Formula 1, Formula 2, Formula 3, or Formula 4 hapten to provide (a) a spacer between the OP and CP, and (b) a chemical moiety for attachment of the compound to the CP (e.g., a carboxylic acid).

[0030] In some embodiments CP is selected from the group of proteins typically used for coupling with compounds or other haptens and are not sufficiently large or complex on their own to induce an immune response that otherwise would produces ineffective antibodies. Representative examples are keyhole limpet hemocyanin (KLH), bovine or human serum albumin (BSA or HSA, respectively) and ovalbumin.

[0031] Also disclosed herein are methods of detoxication of OPs using antibodies elicited from immunization of a mammal using immunological constructs derived from hapten compounds of Formula 1, 2, 3 or 4.DESCRIPTION OF THE INVENTION

[0032] The present invention provides a chemical biology strategy for identification of a therapeutic antibody for use when humans or animals come into contact with OP reagents either deliberately or unintentionally. Described herein are immunogens that recognize OPs and accelerate the breakdown of OPs. The design of the immunogen is such that OP breakdown is accelerated by the elicited antibody and especially in the presence of 2-PAM (or other oximes) when they are in contact with an organophosphorus pesticide or nerve agent. While not limiting, the technology is also applicable to other OPs.

[0033] The antibodies of the present invention can be polyclonal antibodies, monoclonal antibodies, chimeric antibodies, human or humanized antibodies or antibody fragments, derivatives thereof including synthetic and genetically engineered variants of these are described by the present invention.

[0034] Monoclonal antibodies are produced by conventional murine monoclonal antibody methodology using somatic cell hybridization technique. For therapeutic applications, the antibodies of the present invention are based on mouse monoclonal antibodies (mAbs) but for use in humans mAbs would be humanized. These humanized mAbs can decrease or eliminate the presence of organophosphorus compounds following exposure, and decrease the toxic effects of such exposures.

[0035] Methods for making humanized and human mAbs are known in the art. One method for making human antibodies employs the use of transgenic animals, such as a transgenic mouse. Methods for making such transgenic animals are known. Also within the scope of the invention are humanized antibodies in which specific amino acids have been substituted, deleted or added to improve binding the antigen or processing the antigen. For example, binding framework residues of the invention can be replaced at or adjacent to the complementarity determining region (CDR) capable of interacting with a CDR.

[0036] Non-intact antibodies are also useful in this invention such as those derived from any of the antibody-producing methods described above. For example, antigen-binding fragments or full-length monomeric, dimeric or trimcric polypeptides derived from the above-described antibodies arc useful.

[0037] Examples of antibody homologs include: (a) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and Cnl domains; (b) a F(ab') 2 fragment or bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (c) a Fd fragment consisting of the VH and CHI domains; (d) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (e) a single domain functional heavy chain antibody, and (f) an isolated complementarity determining region containing CDRs together with sufficient framework to provide an antigen binding fragment.

[0038] I. Definitions

[0039] The term “OP” as used herein, refers to an organophosphorus compound capable of reacting with a protein nucleophile.

[0040] The term “OP-adduct”, as used herein, refers to the product of that is formed when an amino acid, peptide or protein is exposed to with an OP compound.

[0041] The term “conjugate”, as used herein, refers to the product from covalently attachment of a hapten to a carrier protein through an intervening linker.

[0042] “Substituted” indicates that one or more hydrogens on an atom of a chemical moiety indicated in the expression using “substituted” is replaced with a substituent selected from the indicated group(s), provided that the indicated atom’ s normal valency is not exceeded, and that the substitution results in a stable compound. Suitable substituents include, e.g., alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRyand / or C'OOR', wherein each Rxand Ryare independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy. When a substituent is oxo (i.e., =O) or thioxo (i.e., =S) group, then 2 hydrogen atoms on the atom that is substituted are replaced.

[0043] "Alkyl" refers to a Cl -Cl 8 hydrocarbon radical containing normal, secondary, tertiary or cyclic carbon atoms. Examples are methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1 -propyl (n-Pr, n- propyl,-CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1 -butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-l -propyl (i-Bu, i- butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2- methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1 -pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-.CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3- methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-l -butyl (-CH2CH2CH(CH3)2), 2-methyl-l -butyl (-CH2CH(CH3)CH2CH3), 1 -hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2 -methyl -2 -pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2- pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3-

[0044] In some aspects the alkyl group is unsubstituted. In other aspect the alkyl group is substituted with one or more of alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRyand / or COORX, wherein each Rxand Ryare independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.

[0045] In some aspects the alkyl group is substituted with one or more of alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle,cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRyand COORX, wherein each Rxand Ryare independently H, alkyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.

[0046] The term "alkoxy" refers to the group alkyl-O-, wherein alkyl is as defined herein. Preferred alkoxy groups include, e.g., methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, secbutoxy, n-pentoxy, n-hexoxy, and 1,2- dimethylbutoxy. In some aspects the alkoxy group is unsubstituted. In other aspects the alkoxy group is substituted with one or more alkyl, alkylidenyl, alkenylidenyl, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NR'R- and COORX, wherein each Rxand Ryare independently H, alkyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.

[0047] Preferred alkoxy groups include, e.g., methoxy, ethoxy, n-propoxy, iso- propoxy, n-butoxy, tert-butoxy, sec -butoxy, n-pentoxy, n-hexoxy, and 1,2- dimethylbutoxy. In some aspects the preferred alkoxy group is unsubstituted. In other aspects the alkoxy group is substituted with one or more of alkyl, alkylidenyl, alkenylidenyl, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRyand COORX, wherein each Rxand Ryare independently H, alkyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.

[0048] The term "aryl" refers to an unsaturated aromatic carbocyclic group from 6 to 20 carbon atoms having a single ring (e.g., phenyl) or multiple fused rings, wherein at least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Preferred aryls include phenyl and naphthyl.

[0049] In some aspect the aryl group is unsubstituted. In other aspects the aryl group is substituted with one or more of alkyl, alkenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRyand COORX, wherein each Rxand Ryare independently H, alkyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.

[0050] The term "cycloalkyl" refers to a non-aromatic cyclic alkyl group of from 3 to 20 carbon atoms having a single cyclic ring or multiple condensed rings. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl or cyclooctyl, , or multiple ring structures such as adamantanyl.

[0051] In some aspects the cycloalkyl group is unsubstituted. In other aspects the cycloalkyl groupis substituted with one or more alkyl, alkenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRyand COORX, wherein each Rxand Ryare independently H, alkyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.

[0052] The cycloalkyl group in some aspects is partially unsaturated, thereby providing a cycloalkenyl.

[0053] The term “heteroaryl” as defined herein, is as a monocyclic, bicyclic, or tricyclic ring system containing one, two, or three aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in the aromatic ring system, and which is some aspects is unsubstituted and in other aspects is substituted, for example, with one or more, and in particular one to three, substituents, including halo, alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, amino, alkylamino, acylamino, alkylthio, alkylsulfinyl, and alkylsulfonyl.

[0054] Examples of heteroaryl groups include, but are not limited to, 2H- pyrrolyl, 3 A / -i ndoly I , 4Hquinolizinyl, 4n77-carbazolyl, acridinyl, benzo [ / flthicnyl, benzothiazolyl, |3-carbolinyl, carbazolyl, chromenyl, cinnaolinyl, dibenzo [b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolisinyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, naptho[2,3-Z>], oxazolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, and xanthenyl.

[0055] In one aspect of the invention the term “heteroaryl” denotes a monocyclic aromatic ring containing five or six ring atoms containing carbon and 1, 2, 3, or 4 heteroatoms independently selected from the group non-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H, O, alkyl, phenyl or benzyl. In another aspect heteroaryl denotes an ortho-fused bicyclic heterocycle of eight to ten ring atoms, particularly a benz -derivative or one derived by fusing a propylene, or tetramethylene diradical thereto. In some aspects the hetero aryl is unsubstituted. In other aspects the heteroaryl is substituted with one or more of alkyl, alkenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRyand COORX, wherein each Rxand Ryare independently H, alkyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.

[0056] The term “acyloxy” refers to -O-C(=O)R, wherein R is an alkyl group as previously defined. Examples of acyloxy groups include, but are not limited to, acetoxy, propanoyloxy, butanoyloxy, and pentanoyloxy. Any alkyl group as defined above can be used to form an acyloxy group.

[0057] The term “amino” refers to -NH2, and the term “alkylamino” refers to NR2, wherein at leastone R is alkyl and the second R is alkyl or hydrogen.

[0058] The term “acylamino” refers to RC(=O)N, wherein R is alkyl or aryl.

[0059] The term “imino” refers to -C=NH.

[0060] The term “nitro” refers to -NO2.

[0061] The term “trifluoromethyl” refers to -CF3.

[0062] The term “trifluoromethoxy” refers to -OCF3.

[0063] The term “cyano” refers to -CN.

[0064] The term “hydroxy” or “hydroxyl” refers to -OH.

[0065] The term “oxy” refers to -O-.

[0066] The term “thio” refers to -S-.

[0067] The term “thioxo” refers to (=S).

[0068] The term “keto” or “oxo” refers to (=O).

[0069] The term “thiophosphoro” refers to (P=S).

[0070] As used herein, “amino acid” refers to a compound with an amine and a carboxylic acid separated by a CHR moiety. The R descriptor refers to the side chain in the amino acid. In some aspects the amino acid side chain R any one of the naturally occurring amino acids in nature or any of the unnatural amino acids such as ornithine. In some aspects the chiral atom of an amino acid is in the R stereochemical configuration. In other aspects the chiral atom of an amino acid is in the S stereochemical configuration.

[0071] As to any of the above groups, which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and / or synthetically non-feasible. In addition, the compounds of this invention include all stereochemical isomers arising from the substitution of these compounds unless indicated or implied by context.

[0072] Numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

[0073] II. Compounds of the Invention

[0074] In one embodiment of the invention, disclosed herein are compounds of Formula 1 and 2;

[0075] Preferred Formula 3 and 4 compounds have the structures of Formula 1 and Formula 2, respectively.

[0076] Formula 1-4 compounds are haptens for eliciting immunological responses to produce antibodies or fragments of antibodies that specifically bind these compounds. For that purpose, the carboxylic acid group of the Formula 1-4 compound-linker is conjugated to a carrier protein, wherein the carrier protein is selected from the group consisting of scrum albumin, KLH, ovalbumin, other antigenic peptides and other carrier proteins (CP). The general structure of the immunogens for eliciting the immunological response is represented by protein-linker-hapten.

[0077] Linker is glutaric acid, succinic acid or polyethylene glycols-carboxylic acid (PEG) that is attached to the OP-amine to provide (a) space between the OP and CP, and (b) a chemical moiety for attachment of the compound to CP (e.g., a carboxylic acid). Non-limiting representative examples of linkers include glutaric acid or succinic acid or polyethylene glycol-carboxylic acid (PEG), aminocaproic acid or cysteine.

[0078] CP is selected from a group of proteins used for coupling with compounds or other haptens that are not sufficiently large or complex on their own to induce an immune response, which produces ineffective antibodies. Representative examples are keyhole limpet hemocyanin (KLH), bovine and human serum albumin (BSA and HSA, respectively) or ovalbumin.

[0079] Also disclosed herein are methods to prepare haptens of the present inventions, methods of conjugation of the haptens to carrier protein and method of eliciting antibodies capable of detoxifying OP compounds in the presence of an oxime cofactor.

[0080] Also disclosed herein are methods of detoxication of OPs using the antibodies elicited from haptens of Formula 1, 2, 3 or 4.

[0081] Conjugates (i.e., hapten-linker-CP) containing compounds of Formula 1, 2, 3 and 4 are immunogens for the generation of antibodies. The conjugates are injected into mice but other animals (i.e., rabbits, or goats or other animals) are contemplated for producing antibodies of the present invention according to protocols adapted from those described herein.Antibodies were isolated that recognized the original hapten. Monoclonal antibodies (mAbs) were isolated using hybridoma techniques. Functional evaluation of hybridoma supernatant showed the supernatant accelerated the breakdown of OPs including paraoxon and nerve agent model compounds. Highly purified mAbs from hybridoma clones corresponding to these supernatants also accelerated the breakdown of OPs including paraoxon and nerve agent model compounds. In both cases, breakdown was accelerated by the presence of 2-PAM.

[0082] in. Methods of Use

[0083] The isolated antibodies are used as a therapeutic in detoxifying a human or animal exposed to toxic OP compounds. Co-administration of 2-PAM (or other oximes) accelerated the detoxication of OPs or nerve agents in vivo as shown by the in vitro results presented in the examples.

[0084] In summary, using a chemical biology approach, we have identified a method to efficiently detoxicate OP exposure in the presence of 2-PAM or other oximes. This technology provides a valuable tool to treat OP exposure especially long-lived OP exposure from pesticides and nerve agents with reduced toxicity from the treatment itself. This technology will help victims of OP exposure obtain more effective countermeasure treatment especially for OPs (e.g., nerve agents that do not currently have any medical countermeasures.

[0085] IV. Numbered embodiments

[0086] The following numbered embodiments are intended to further describe the invention and not to limit its scope in any manner.

[0087] Embodiment 1. An antibody or a fragment of an antibody comprising amino acid sequences for a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: a CDR-H1 amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 7; a CDR-H2 amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 8 and a CDR-H3 amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 9; and wherein the light chain variable region comprises: a CDR- LI amino acid sequence as shown in SEQ ID NO: 4; a CDR-L2 amino acid sequence as shown in SEQ ID NO:5; and a CDR-L3 amino acid sequence as shown in SEQ ID NO: 6,SEQ ID NO: 1 TYGVGVGSEQ ID NO:2 NIWWNDDKHYNSALKS SEQ ID NO:3 LGTPYSEQ ID NO:4 KASQDINKNIA SEQ ID NO:5 YTSTLQPSEQ ID NO:6 LQYDNLRT, SEQ ID NO:7 DSLIHSEQ ID NO:8 WMDSEDGESTYAPKFQD SEQ ID NO:9 GGTHYwherein the antibody or the fragment of an antibody is capable of specific binding to an organophosphorus compound and an oxime capable of breakdown of the organophosphorus compound, wherein the organophosphorus compound is an organophosphorus pesticide or nerve agent.

[0088] Embodiment 2. The antibody or the fragment of the antibody of embodiment 1 further comprising an amino acid sequence for a heavy chain framework region, wherein the heavy chain framework region comprises a FR-H1 amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO: 14, a FR-H2 amino acid sequence as shown in SEQ ID NO: 11 or SEQ ID NO: 15, a FR-H3 amino acid sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 16 or a FR-H4 amino acid sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 17.SEQ ID NO: 10 QVTLKESGPGILQPSQTLSLTCSFSGFSLSSEQ ID NO: 11 WIRQPSGKGLEWLASEQ ID NO: 12 RLIISKDTSKKQVFLKISSVDTADTATYYCAQSEQ ID NO: 13 WGQGTLVTVSASEQ ID NO: 14 EVQLQQSGAELVRPGASVMLSCTVSGFNIKSEQ ID NO: 15 WVKQRPEQGLEWIGSEQ ID NO: 16 KATITADTSSNTAYLQLSRLTSEDTAIYYCTRSEQ ID NO: 17 WGQGTTLTVSS

[0089] Embodiment 3. The antibody or the fragment of the antibody of embodiment 1 or 2 further comprising an amino acid sequence for a light chain framework region, wherein the light chain framework region comprises a FR-L1 amino acid sequence as shown in SEQ ID NO: 18, a FR-L2 amino acid sequence as shown in SEQ ID NO: 19, a FR-L3 amino acid sequence as shown in SEQ ID NO:20 or a FR-L4 amino acid sequence as shown in SEQ ID NO:21.SEQ ID NO: 18 DIQMTQSPSSLSASLGDKVTITC SEQ ID NO:19 WYQHKPGKGPRLLIY SEQ ID NO:20 GIPSRFSGSGSGRDYSFSISNLEPEDIATYYC SEQ ID NO:21 FGGGTKLEIK

[0090] Embodiment 4. The antibody or the fragment of the antibody of embodiment 1, 2 or 3 further comprising an amino acid sequence for a heavy chain constant region wherein said amino acid sequence is shown in SEQ ID: 22SEQ ID NO:22 AKTTAPSVYPLAPVCGDTSGSSVTLGCLVKGYFPEPVTL TWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTK VDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVD VSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSG KEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCM VTDFMPEDIYVEWTNNGKTELNYKNTEPVLDFDGFYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.

[0091] Embodiment 5. The antibody or the fragment of the antibody of any one of embodiments 1- 4 further comprising an amino acid sequence for a light chain constant region wherein said amino acid sequence is shown in SEQ ID NO:23SEQ ID NO:23 RADAAPTVSTFPPSSEQLTSGGASVVCFLNNFYPKDTNVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEAT HKTSTSPIVKSFNRNEC.

[0092] Embodiment 6. The antibody or the fragment of the antibody of any one of embodiments 1-5, wherein the antibody is capable of hydrolyzing organophosphorus compounds including organophosphorus pesticides.

[0093] Embodiment 7. The antibody or the fragment of the antibody of any one of embodiments 1- 5, wherein the antibody is capable of hydrolyzing organophosphorus compounds including nerve agents.

[0094] Embodiment 8. The antibody or the fragment of the antibody of any one of embodiments 1- 9, wherein the oxime is 2-pyridine aldoxime methyl chloride (2-PAM)

[0095] Embodiment 9. The antibody or the fragment of the antibody of an antibody of embodiment 1, wherein the antibody or the fragment of the antibody is capable of selective binding to 4- { [ethoxy ([2-(pyridin-2-yl)ethyl]amino)phosphoryl]-methyl[ aniline.10.

[0096] Embodiment 10. The antibody or the fragment of the antibody of embodiment 1, wherein the antibody or the fragment of the antibody is capable of selective binding to N-mcthyl- 4-[(4- { [ethoxy ( [2- (pyridinium-2-y l)ethy 1] amino) -phosphoryl] methyl [aniline .

[0097] Embodiment 11. An isolated polynucleotide comprising a sequence encoding a heavy chain variable region and a light chain variable region of the antibody or the fragment of the antibody of embodiment 1.

[0098] Embodiment 12. The isolated polynucleotide of embodiment 11, wherein the isolated polynucleotide is comprised of the sequence encoding the heavy chain variable region as set forth by SEQ ID NO:24 or SEQ ID NO:25SEQ ID NO:24 CAGGTCACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCA GACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTATGGTGTGGGTGTAGGTTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTG GCCAACATTTGGTGGAATGATGATAAGCACTATAACTCAGCCCTGAAGAGC CGGCTCATTATCTCCAAGGATACCTCCAAGAAGCAGGTATTCCTCAAGATC TCCAGCGTGGACACTGCAGATACTGCCACATACTACTGTGCTCAACTGGGGACTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCASEQ ID NO: 25 GAAGTTCAACTGCAGCAGTCTGGGGCAGAACTTGTGAGGCCAGGGG CCTCAGTCATGTTGTCCTGCACAGTTTCTGGCTTCAACATTAAAGACAGCCT TATACACTGGGTGAAGCAGAGGCCTGAGCAGGGCCTGGAGTGGATTGGAT GGATGGATTCTGAGGATGGTGAAAGTACTTATGCCCCGAAGTTCCAGGACA AGGCCACTATTACTGCAGACACATCCTCCAATACAGCCTATCTGCAGCTCA GCAGGCTGACATCTGAGGACACTGCCATCTATTACTGTACTCGAGGTGGGA CACACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

[0099] Embodiment 13. The isolated polynucleotide of embodiment 11 or 12, wherein the isolated polynucleotide is comprised of the sequence encoding the light chain variable region as set forth by SEQ ID NO:26SEQ ID NO:26 ATGAGACCGTCTATTCAGTTCCTGGGGCTCTTGTTGTTCTGGCTTCAT GGTACTCAGTGTGACATCCAGATGACACAGTCTCCATCCTCACTGTCTGCATCTCTGGGAGGCAAAGTCACCATCACTTGCAAGGCAAGCCAAGACATTAATA AGTACATAGCTTGGTACCAACACAAGCCTGGAAAAAGTCCTAGGCTGCTCA TACATTACACATCTGCGTTAGAGCCAGGCATCCCATCAAGGTTCAGTGGAA GTGGGTCTGGGAGAGATTATTCCTTCAGCATCAGCAACCTGGAGCCTGAAG ATATTGCAACTTATTATTGTCTACAGTATGATAATCTTCGGACGTTCGGTGG AGGCACCAAACTGGAAATCAAA.

[0100] Embodiment 14. An isolated vector comprising the polynucleotide of embodiment 12 or 13.

[0101] Embodiment 15. An isolated host cell comprising the vector of embodiment 14.

[0102] Embodiment 16. A method of making the antibody or antigen binding fragment of the antibody of claim 1, wherein the method comprises: a) culturing the host cell of embodiment 15 under conditions suitable for expression of the polynucleotide encoding the antibody or the fragment of the antibody; and b) isolating the antibody or antigen binding fragment of the antibody.

[0103] Embodiment 17. A compound having the structure of:wherein R is hydrogen or alkyl, Ri and R: independently are alkyl and X is a suitable counterion.

[0104] Embodiment 18. The compound of embodiment 17, wherein R is hydrogen and Ri and R2 are each methyl.

[0105] Embodiment 19. A compound having the structure of:wherein R is hydrogen or alkyl, Ri and R2 independently are alkyl, X’ is a suitable counterion, L is a linker residue and RG is a reactive group capable of forming a covalent bond to a carrier protein.

[0106] Embodiment 20. The compound of embodiment 19, wherein -Linker-RG has the structure selected from the group consisting of -C(O)-R3-PEG-R4-CO2H, -C(O)-R3-CO2H, -C(O)-R3- NH2, - C(O)-R3-SH and salt and protected forms thereof, wherein R3 and R4 independently are alkyl and PEG is a polyethylene glycol residue having 2-12 repeating ethyleneoxy subunits.

[0107] Embodiment 21. The compound of embodiment 19, wherein -L-RG is selected from the group consisting of glutaric acid, succinic acid, aminocaproic acid and cysteine residues and salt and protected forms thereof.

[0108] Embodiment 22. The compound of embodiment 19, wherein the compound has the structure of:or wherein the compound structure has its carboxylic acid functional group in salt or protected form.

[0109] Embodiment 23. The compound of embodiment 19, wherein the compound has the structureof:or wherein the compound structure has its carboxylic acid functional group in salt or protected form.

[0110] Embodiment 24. A composition of matter represented by the formula:CP-Linker-hapten or salt thereof, wherein CP is a Carrier Protein, linker is a spacer molecule connecting hapten to carrier protein and hapten is an immunologically competent moiety having the structure ofOR-P-NR2OR wherein R directly attached to the phosphorous atom is a substituted or unsubstituted alkylarylamine residue in which a carbon atom of its alkyl moiety is covalently attached to the phosphorous atom and wherein the nitrogen atom of its amino group is covalently attached to the linker,-OR is an alkoxy group and-NR2 is a substituted or unsubstituted 2-(2-pyridyl)cthylamino moiety, wherein the nitrogen atom of the moiety’s amino group is covalently attached to the phosphorous atom.

[0111] Embodiment 25. The composition of embodiment 24, wherein the carrier protein is keyhole limpet hemocyanin (KLH), bovine or human serum albumin (BSA or HSA, respectively), or ovalbumin.

[0112] Embodiment 26. The composition of embodiment 24 or 25, wherein the hapten moiety corresponds in structure to a compound of embodiment 17 in which the aniline nitrogen atom of the compound is the site of covalent attachment to the linker.

[0113] Embodiment 27. The composition of any one of embodiments 24-26 wherein the Linker has the structure of -C(O)-R3-PEG-R4-C(O)- or -C(O)-R3-C(O)-,wherein R3 and R4 independently are alkyl and PEG is a polyethylene glycol residue having 2- 12 repeating ethyleneoxy subunits, wherein one carbonyl carbon atom of the structure is covalently attached to the nitrogen atom of the amino group of alkylarylamine residue and the other carbonyl carbon atom is covalently attached to a Lysine epsilon amino group of the carrier protein.

[0114] Embodiment 28. The composition of any one of embodiments 24-26 wherein the Linker has the structure -C(O)-Rs-NH-, -C(O)-R3-S- wherein one carbonyl carbon atom of the structure is covalently attached to the nitrogen atom of the amino group of alkylarylamine residue and the amino or thiol group is covalently attached to the carrier protein through and amide or disulfide bond, respectfully.

[0115] Embodiment 29. Use of an antibody of any one of embodiments 1-12 in preparation of a medicant for treating a subject exposed to an organophosphorus compound, wherein the organophosphorus compound is an organophosphorus pesticide or an organophosphorus nerve agent.

[0116] Embodiment 30. A vaccine comprising a hapten as defined in embodiment 24 and a pharmaceutically acceptable carrier and / or immuno-effective adjuvant.

[0117] Embodiment 31. A diagnostic method for determining exposure of a subject to a organophosphorus compound comprising the use of an antibody or fragment of an antibody defined by any one of embodiments 1-10.

[0118] The invention is further described by the following non-limiting examples.EXAMPLES

[0119] Design and Synthesis of Antigens.

[0120] Although there are numerous reports concerning the synthesis of organophosphorus (OP) compounds, prior to our work there were no reports in the literature concerning the synthesis of OP compounds attached to an oxime (e.g., 2-PAM) detoxication agent via a phosphonate P-C linkage). This new chemical structure represents the transition state for the detoxication of an OP by 2-PAM. Illustrative of this approach is the design and synthesis of transition state analogs for 2-PAM accelerating the breakdown of paraoxon. This unprecedented approach is also relevant to OP nerve agents and we provide data that shows the mAbs produced with this new approach is also applicable to OP nerve agent detoxication.

[0121] Preparation of Antigens and Conjugation of Antigens to Proteins. Pyridine hapten with PEG-t-butyl ester linker (tert-butyl-3-(2-{2-[(4-{ [ethoxy({ [2- (pyridin-2-yl)ethyl] amino}) phosphoryl]methyl} phenyl)carbamoyl]ethoxy} ethoxy )propanoate and pyridine hapten with glutaric acid linker were synthesized. Tn addition, the corresponding N-methyl pyridinyl haptens with PEG-t- butyl or glutaric acid or succinate acid linkers were synthesized.

[0122] Example 1. Mouse Immunizations.

[0123] The procedures and care of animals used in this work conformed to the Guiding Principles in the Care and Use of Animals provided by the American Physiological Society, as well as all Federal and California regulations. Female Swiss Webster mice (7 weeks, 20 g) were used in immunization studies with the conjugated immunogens. After a 1-week stabilization period, 5 mice were immunized with each KLH-PEG conjugated hapten (i.e., 4- { [ethoxy ([2-(pyridin-2- yl)ethyl]amino)phosphoryl]- methyl} aniline) or KLH-PEG conjugated hapten (i.e., N-methyl 4- { [ethoxy ([2-(pyridin-2- yl)ethyl]amino)-phosphoryl]-methyl}aniline). The immunization protocol consisted of an initial i.p. injection of 100 pg of KLH- conjugated immunogen in 200 p l of an oil-in-water emulsion (PBS- containing Sigma Adjuvant System) followed by another boost 2 weeks later with OVAL-PEG conjugated immunogen. Based on ELISA results, after an additional 4 weeks, certain animals were injected with 100 pg KLH-conjugated immunogen in the tail vein and the spleens were harvested the next day. The remaining mice were treated with 100 pg OVAL-PEG conjugated immunogen as an i.p. injection (third boost). After 5 weeks these mice were injected with 100 pg KLH-conjugated immunogen in the tail vein and the spleens were harvested the next day.

[0124] During the immunization protocol, mouse blood (50-100 pl) was collected from each animal from the orbital sinus 7 days after each boost. Heparin (1- 3 IU), was added to each blood sample and mixed thoroughly, and plasma was separated from blood cells through centrifugation at 5,000 rpm for 10 minutes at 4°C. Each plasma sample was tested for the presence of anti-HSA-PEG conjugated immunogen antibodies by enzyme-linked immunosorbent assay (ELISA) (see below). Dilution studies were done. When the titer reached an EC50 or 1 :30,000-1:60,000 dilution, the mice were allowed to rest for at least 1 month. A final tail vein injection was made with KLH-conjugated immunogen (100 pg in PBS) 1-3 days before the mice were taken for splenectomy and cell fusion.

[0125] Example 2. Cell Culture and Cell Fusion.

[0126] Mouse myeloma cells NS-1 were seeded at 2 x 107 cells and 1 x 108 viable splenocytes and centrifuge. 1 mL of ClonaCell™-HY PEG was slowly added to the cell pellet dropwise using a 1 mL serological pipette. The mixture was grown in Medium A (Stemcell Technologies). After cell fusions, five 100-mm dishes were plated with 2-4 x 10^ cells / ml in methycellulose medium (Medium D; Stemcell Technologies) for 7-14 days. Single colonies of hybridoma cells were picked from methylcellulose medium and cultured in Medium E (Stemcell Technologies) in 96- well plates. Cell culture supernatants were tested for the presence of antibodies to HSA-conjugated immunogens using ELISA. Positive cultures were expanded and supernatants were tested against a panel of antigens to verify antibody selectivity before secondary cloning in methylcellulose Medium D or by limited dilution in Medium E.

[0127] Numerous (> 200) hybridoma clones were positively identified by ELISA. Hybridoma supernatant was used in competition (IC50) studies against haptens and controls. The results showed selectivity for the hapten. Four representative hybridoma cell lines showed EC50 range of 10'^ to 10'QM. The successful identification of positive hybridomas permitted selection of hybridoma clones for development of monoclonal antibodies (mAbs). Many of the other remaining colonies showing excellent response to the immunogens. Many of the colonies were co-screened with 2-PAM and those showing dual binding efficacy were developed further. From > 200 hybridoma clones, 9 clones (i.e., A-I) were used for examination of breakdown of paraoxon and nerve agent model compounds, in vitro.

[0128] After recloning of hybridoma cells for 2-3 rounds, the monoclonal cells afforded single heavy-chain and single light-chain clones and were deemed clones that generated mAbs and were isotyped with an isotyping kit. Most of the clones from the spleens that were cultured and fused isotyped as IgGl (kappa). For example, the “C” mAb eventually elected for extensive evaluation possessed this isotype. On the other hand, the “G” mAb eventually elected for extensive evaluation possessed the IgG2a (kappa) isotypc. These isotypcs arc characteristic of mAbs useful in therapeutic applications.

[0129] Example 3. Purification of Monoclonal Antibodies from Hybridoma Supernatant.

[0130] For hybridoma cells expressing the desired anti-hapten mAbs, cell cultures were expanded. Briefly, culture medium of the hybridoma cells was centrifuged at 1,000 rpm to separate cells from the supernatant, and the supernatant was filtered to remove residual debris. The culture medium supernatant was mixed thoroughly at room temperature prior to purification. For mAb purification, protein was dialyzed against PBS (pH 7.4) and mixed 1: 1 with binding buffer and then chromatographed on Protein G Sepharose 4B (Sigma) equilibrated with PBS (pH 7.4). Culture medium was loaded onto the column at 1 ml / min. The Protein G column was washed with PBS (pH 7.4). Bound antibodies were eluted with low pH elution buffer (pH 2.0) and immediately neutralized to pH ~7.5 with 1.0 M Tris. Protein-containing fractions were pooled based on optical density at 280 nm (OD280) absorbance and dialyzed against PBS (pH 7.4) for routine application. Purified antibodies were quantified by OD280 and were estimated to be >90% pure. These highly purified mAbs are characteristically useful to quantify functional activity.

[0131] Example 4. Ascites Fluid and Purification of mAbs Therefrom

[0132] Hybridoma cells were grown in 10% FBS IMDM media with 2% glutamine and 1% penicillin streptomycin at 37°C incubator with 5% CO2 supply. Cells were grown for 7-9 days from seeding to maturity and each mouse (Balb / c Retired breeder female) was injected with 0.5 ml of cells in DMEM (2.5 x 106 cell / mouse, i.p.). Immunoreactivity was monitored from ascites for several harvests. At maximum production a final harvest was done. For one clone, 11 mL of Ascites fluid (to be called mAb “C”) was obtained. From another clone, 10 ml of Ascites fluid (to be called mAb “G”) was obtained. In a typical preparation, 8-9 ml of Ascites fluid was placed on a 1.3 ml Protein A Sepharose 4B resin column previously equilibrated with 10-fold volumes of PBS, pH 7.4. Elution and analysis was done as described above (see Example 3). Monoclonal Abs were quantified by OD280. For mAb“C” highly purified IgG afforded 2.1 mg at a final concentration of 1.5 mg / ml. For mAh “G” highly purified IgG afforded 2.0 mg at a final concentration of 1.4 mg / mL. Mono clonal Abs were used directly without any preservatives. The mAbs were characterized by limiting ELISA and other functional assays. Those highly purified mAbs are useful to quantify functional activity.

[0133] Example 5. ELISA.

[0134] For quantification of immunoreactivity of serum from immunized mice or analysis of hapten binding of highly purified antibodies, ELISA was conducted.The wells of 96-well ELISA plates were coated with 0.01-2.0 pg of hapten. This was done a variety of ways: 1) hapten was coated onto polylysine plates or 2) immunogen-protein was coated onto other plates (Maxisorp or Micro fluor-2). Both plates afforded robust platforms for ELISA measurements.

[0135] A, Hapten-polylvsine ELISA plates. Haptens (activated pyridine carboxylate or N-methyl pyridinium carboxylates): (30.5 pmol) in DMSO was added 0.5M solution of sulfo-NHS followed by a 0.5M solution of EDCI. The mixture was stirred at RT for 30 min and the mixture was diluted with water to provide a 10 ii g / mL activated antigen solution that was used in the following step. To each well of poly-L-lysine coated plates, 60 pL of a 10 pg / mL solution of activated hapten was added. In some cases, the amount of antigen put on the plate was varied. The resulting plates were incubated for 1 hour and then stored at 4 °C overnight. On day 2, the excess solution was removed and each well was rinsed with PBST three times and then air dried.

[0136] B. Protein-immunogen ELISA plates. Immunogens with PEG- or glutaric acid or succinic acid linkers covalently linked between the pyridine carboxylate or N-methyl pyridinium carboxylates (30.5 pmol in water, shown as Hapten A or B, below) and HSA or OVAL or KLH was added directly to Maxisorp or Microfluor-2 plates. Below, the -NH- moiety is linked to the protein of interest. In some cases, the amount of protein-antigen put on the plate was varied. The resulting plates were incubated for 1 hour and then stored at 4 °C overnight. On day 2, the excess solution was removed and each well was rinsed with PBST three times and then air dried.

[0137] C. Procedure. Plates could be reliably stored at 40C for many months. 1 hour before use, PBS (without calcium or magnesium) at pH 7.4 was used to rinse the plates. After removing PBS, plates were rinsed with PBST (PBS with 0.1% Tween-20), and the 96-well plates blocked for 1 hour with blocking buffer [PBS containing 0.05% (v / v) Tween-20 (PBST) and 1% (w / v) bovine casein (PBSTC)].

[0138] After removing the blocking buffer, typically, 50-100 ml of hybridoma culture medium or purified antibody in PBSTC was applied to each well and allowed to incubate at 37°C for 1 hour. In some cases, dilutions were done to obtain an EC50 for dilution. After rinsing off excess primary antibody and washing, an aliquot of anti -mouse antibody with covalently attached HRP (1:4000 dilution in PBSTC) was added to each well and incubated al room temperature for 1 hour. After three rinses with PBST, bound antibody was detected by adding of chemiluminescent substrate (SuperSignal™ West Femto Maximum Sensitivity) to each well and reading the luminosity using a Synergy plate reader (BioTek). For evaluation of serum titers, plasma samples were diluted in PBST. Diluted plasma assayed ranged generally from 1: 10- to 1:300, 000-fold dilutions depending on the titer. Hybridoma supernatant or highly purified mAbs also worked well with these formats. Using either the hapten- based or protein-hapten-based ELISA procedure afforded highly reproducible and robust quantification of antibody.

[0139] Example 7. Pyridine aniline hapten (1). 4-{[ethoxy([2-(pyridin-2- yl)ethyl]amino) phosphoryl]-nitro precursor to hapten pyridine hapten (3)

[0140] P«ra-nilro hapten precursor (2.05 g) was placed in 50 mL of anhydrous methanol under nitrogen gas. 5-8 mg of Pd-C was added and the evac / nitrogen procedure re-conducted and the reaction vial fitted with a balloon filled with hydrogen, and opened to begin the reduction.

[0141] After 3-4 h, the reaction progress was 50-60% as assessed by TLC (sm Rf = 0.6; prod Rf = 0.45 MeOH / CHCh 1:4) and the hydrogen balloon was refilled, and stirred overnight at which point the starting material was gone and product. The reaction mixture was filtered through methanol-wetted Celite atop a fritted vacuum funnel, and evaporated under vacuum to afford 1 .78 g (5.51 mmol; 95%) of product as a single spot on TLC.

[0142] Flash chromatography using 5% MeOH / CHCh affords about 85% recovery from the crude (1.51 g; 4.67 mmol). *H NMR (499 MHz, CDCh): 5 8.47 (ddd, 7 = 4.9, 1.8, 0.9 Hz, 1H), 7.57 (td, J = 7.7, 1.9 Hz, 1H), 7.14 - 7.06 (m, 2H), 7.01 (dq, J = 9.1, 2.6 Hz, 2H), 6.61 - 6.53 (m, 3H), 3.99 (dp, J = 10.1, 7.1 Hz, 1H), 3.92 - 3.81 (m, 1H), 3.32 - 3.20 (m, 2H), 2.97 (d, J = 19.9 Hz, 3H), 2.85 (t, J = 6.4 Hz, 2H), 1.23 (t, 7= 7.1 Hz, 3H).13C NMR (126 MHz, CDCh): 3 159.37, 149.30, 145.05, 136.50, 130.59, 130.55, 123.63, 122.23 (dPCC, 7 = 8.8 Hz), 121.51, 115.34, 59.84 (d, 7 = 6.7 Hz), 40.39, 39.71 (d, 7poc = 5 Hz), 34.49 (d, 7PC = 126.4 Hz), 16.48, 16.43.31P NMR (202 MHz, CDCh) 8: 30.86.

[0143] Example 8. Pyridine hapten with glutaric acid linker.

[0144] 4-[(4-{[ethoxy([2-(pyridin-2-yl)ethyl]amino)phosphoryl]-methyl}phenyl)carbamoyl]-butanoic acid.

[0145] To methylene chloride (2.5 mL) was added the pyridine hapten aniline (90 mg), DMAP (4.0 mg), and stirred at room temperature for 1-15 min. To the solution was added glutaric anhydride (35 mg) in one portion as a solid. The reaction was stirred at rt for 15-20 min at which point TLC (20% MeOH / CHCh) showed disappearance of the starting material from Rf = 0.45 MeOH / CHCh (1:4) and formation of a new UV active streak at Rf= 0.15-0.35 that did not yellow on exposure to air. Addition of 1-5% (trace) HO AC to the TLC chamber resulted in a more defined spot at Rf = 0.36. The reaction mixture was evaporated to a white- yellowish semi-solid (133 mg). This crude material was taken up in warm chloroform containing a small amount of methanol and refrigerator resulting in a white solid ppt. This ppt showed a minor impurity spot on TLC, and the white solid was recrystallized from 100% hot chloroform to afford a uniform white semi- crystalline product (76 mg; 174 pmole; 64% ) of one spot on TLC.

[0146] *H NMR (500 MHz, CDCh) 5 9.27 (s, 1H), 8.53 (d, J = 4.6 Hz, 1H), 7.71 (td, J = 7.7, 1.8 Hz, 1H), 7.37 (d, J = 8.2 Hz, 2H), 7.25 (m, 2H), 7.03 (m, 2H), 3.88 (dp, J = 10.2, 7.1 Hz, 1H), 3.75 - 3.66 (m, 1H), 3.04 - 2.96 (m, 2H), 2.92 (dd, J = 20.1, 5.0 Hz, 1H), 2.84 (d, J = 14.9 Hz, 1H), 2.46 (t, J = 7.5 Hz, 2H), 2.42 - 2.36 (m, 2H), 2.06 - 2.00 (m, 2H), 1.15 (t, J = 7.0 Hz, 3H).13C NMR (126 MHz, CDCh) 5 176.35 (C=O), 171.74 (C=O), 158.95, 148.33, 137.65, 137.50, 130.01 (d, J = 6.1 Hz), 126.87, 124.07, 122.12, 120.25, 60.10 (d, J = 6.9 Hz), 41.15, 38.92 (d, JPOc = 5 Hz), 36.16, 34.16 (d, JPC = 126.5 Hz), 33.70, 29.73, 21.15, 16.45 (d, JPOcc = 5.4 Hz).31P NMR (202 MHz, CDCh) 5 30.68. LRMS: m / z 434 (M + H), 456 (m + Na).

[0147] Example 9. N-Methyl pyridinium hapten with glutaric acid linker.

[0148] N-methyl-4-[(4-{[ethoxy([2-(pyridinium-2-yl)ethyl]amino)-phosphoryl]methyl}phenyl) carbamoyl] butanoic acid was synthesized by treating the pyridine hapten with glutaric acid linker (i.e., 4-[(4- { [ethoxy([2-(pyridin-2-yl)ethylJamino)-phosphoryl]methyl }phenyl)carbamoyl]butanoic acid, shown above) with methyl iodide. After purification, the analytical results confirmed thepresence of the N-methyl pyridinium hapten with glutaric acid linker.

[0149] ' H NMR (500 MHz, DMSO) 8 12.05 (bs, 1H), 9.83 (s, 1H), 8.91 (d, 7 = 5.6 Hz, 1H), 8.44 (t, J = 7.3 Hz, 1H), 7.96 - 7.90 (m, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.13 (dd, J = 8.6, 2.2 Hz, 2H), 4.85 - 4.76 (m, 1H), 4.21 (s, 3H), 3.84 - 3.71 (m, 3H), 3.24 - 3.14 (tn, 4H), 3.10 (t, J = 6.3 Hz, 2H), 3.06 - 2.91 (m, 2H), 2.34 (t, J = 1A Hz, 2H), 2.30 - 2.21 (m, 2H), 1.80 (p, J = 7.4 Hz, 2H), 1.14 (t, J = 7.0 Hz, 3H).31P NMR (202 MHz, DMSO) 8 30.77.

[0150] Example 10. Pyridine hapten (1) with PEG linker.pyridine hapten with PEG-linker

[0151] Pyridine hapten (1 ), below was stored as a PEG-linked t-butyl ester and treated with 50% TFA to generate the free carboxylic acid. After evaporation of TFA, the pyridine hapten conjugated with PEG was used to conjugate proteins. The analytical results confirmed the presence of the pyridine hapten with PEG linker as the t-butyl ester.

[0152] *H NMR (499 MHz, CDC13) 8 8.55 - 8.63 (m, 1H), 8.46 - 8.55 (m, 1H), 7.60 - 7.73 (m, 1H), 7.41 - 7.51 (m, 2H), 7.10 - 7.24 (m, 4H), 3.95 - 4.07 (m, 1H), 3.84 - 3.95 (m, 1H), 3.82 (s, 2H), 3.72 - 3.77 (m, 2H), 3.68 (d, J = 5.48 Hz, 4H), 3.26 - 3.36 (m, 2H), 3.12 - 3.20 (m, 1H), 3.06 (d, J = 20.16 Hz, 2H), 2.87 - 2.96 (m, 2H), 2.60 - 2.69 (m, 2H), 2.44 - 2.52 (m, 2H), 1.44 (s, 9H), 1.25 (t, 3H).

[0153] Mass spectrum shows signals for C28H42N3O7P + H: 564.1 (MH+) and C28H42N3O7P + Na: 586.2 (MNa+) and a signal for C24H34N3O7P+H: 508.1 (MH-C4H8)+

[0154] Example 11. N-Methyl pyridinium hapten with PEG linker.pyridinium hapten with PEG-linker

[0155] N-methyl-4-[(4-{ [ethoxy([2-(pyridinium-2-yl)ethyl]amino)-phosphoryl]methyl}phenyl) carbamoyl] PEG carboxylic acid was obtained in an analogous fashion to that described above for the glutarate compound. The pyridine hapten (1) as the t-butyl ester was treated with 50% TFA and, after evaporation, the carboxylic acid (pyridinium hapten (2) PEG-linked) was treated with proteins as above to form the immunogen. The analytical results confirmed the presence of the N-methyl pyridinium hapten with PEG linker as the t-butyl ester.

[0156] *H NMR (499 MHz, CDCh) 8 8.91 (broad s, 1H), 8.69 - 8.75 (m, 1H), 8.17 - 8.26 (m, 1H),7.91 - 7.99 (m, 1H), 7.73 - 7.80 (m, 1H), 7.38 (d, J = 8 Hz, 2H), 7.27 (d, J = 8 Hz, 2H), 4.49 - 4.64 (m,1H), 4.16 (s, 3H), 4.02 - 4.12 (m, 2H), 3.81 - 3.95 (m, 3H), 3.69 (s, 7H), 3.43 - 3.65 (m, 4H), 3.12 - 3.33 (m, 3H), 2.99 - 3.12 (m, 3H), 2.64 - 2.84 (m, 3H), 2.39 (s, 2H), 1.41 (s, 10H), 1.33 (t, 4H). MS: 578.1 (Calcd. for C29H45N3O?P)+: 578.3

[0157] As described above, purity of each hapten was >95% as judged by NMR, HPLC, and the structure was confirmed by MS. Each hapten was conjugated to keyhole limpet hemocyanin (KLH) or chicken egg white ovalbumin (OVAL) or human serum albumin (HSA) using ethyldiisopropylcarbodiimide and sulfo-N-hydroxysuccinimide (1 :1). Highly purified conjugated haptencarrier protein immunogen was obtained after removing excess hapten and reagents by filtration through a spin column and dialysis against phosphate-buffered saline (PBS) buffer (pH 7.4) through repeated filtration with Amicon Ultra 4 centrifugation tubes. Protein concentration was determined by a BCA assay. The structure of the conjugated hapten-carrier protein immunogen (i.e., OVAL and HSA) was confirmed by MALDf TOF mass spectrometry (MS). MS data showed that 7-9 haptens / HSA protein were present. The structure of conjugated hapten-carrier protein immunogen of KLH was confirmed by UV-VIS studies. Chemical stability of pyridine- and N-methyl-pyridinyL PEG antigen was tested by incubating the antigen at pH 6.5 and 8.0, room temperature, and analyzing aliquots of the incubation taken over time by31P NMR. The antigens examined possessed half-life values with estimated pseudo 1storder half-lives > 220 days. In the31P experiments, we did not observe any new signals that may have indicated the loss of phosphonate or related groups from the antigens over the course of the stability tests.

[0158] Example 12. Synthesis of Organophosphorus Nerve Agent Model Compounds.

[0159] Briefly, para nitrophenol was combined with monochloridates of nerve agent model compounds to afford para nitrophenol nerve agent model compounds. The monochloridates were prepared as described before. The structures are shown below alongside to 2-PAM. Synthesis of p- nitrophenyl esters of nerve agent model compounds was accomplished by combining the corresponding nerve agent monochloridates with triethylamine and dimethylamino pyridine at 4°C in CH2CI2. After addition of p-nitrophenol and stirring at room temperature for 5 hours, the mixture was cooled to 4°C and made basic, and the separated organic fraction was evaporated to dryness and chromatographed to afford the desired products in 78%- 91% yield. Each product was fully characterized by NMR.

[0160] Example 13. Conversion of paraoxon to p-nitrophenol by hybridoma supernatants A-I ± 2-PAM as judged by HPLC.N-methvI 2-pyridinium p-mtrophenoxy sarin p-mtrophenoxy soman aldoxime (2-PAM)

[0161] Paraoxon (12.5 nmol / incubation) was combined with 225 u L supernatants A-I and incubated at 37 degrees for 1 hour. Thereafter, incubation was stopped by addition of ethyl acetate containing 0.5% TFA, (0.5 mL) and 20 mg NaCl with mixing, centrifugation, decanting and evaporation. The samples were taken up in 200 pL MeOH for HPLC. As controls, paraoxon alone or paraoxon + PBS (pH 7.5) was run in parallel.

[0162] In another experiment, the effect of 2-PAM on the conversion of paraoxon to p-nitrophenol by hybridoma supernatants A-l was examined (Table 1). Paraoxon (12.5 nmol / incubation) was combined with 225 pL hybridoma supernatants A-I + 2- PAM (0.5 mM final cone.) and incubated at 37 degrees for 1 hour. Thereafter, incubations were stopped by addition of ethyl acetate containing 0.5% TFA (0.5 mL) and 20 mg NaCl was added with mixing, centrifugation, decanting and evaporation. The evaporated samples were taken up in 200 pL MeOH for HPLC. As controls, paraoxon alone or paraoxon + PBS (pH 7.5) was run in parallel.

[0163] Conclusions: Very little p-nitrophenol was observed in the 100% extraction (Entry 1) and very low amounts of p-nitrophenol was observed in the pH 7.5 control group (Entry 2). 2-PAM had a slight effect on background breakdown at pH 7.5 (compare entry 2 columns 5 and 6). A comparison of column 5 (pg p-nitrophenol formed / incubation for 1 hour) with columns 6 (pg p-nitrophenol formed / incubation for 1 hour in the presence of 2-PAM) showed that in most hybridoma supernatant cases (Entries 3-11), the amount of p-nitrophenol formed in the presence of 2-PAM was significantly greater.

[0164] Select incubation extracts were examined by LCMS. LCMS confirmed that the -nitrophenol quantified in HPLC runs was in fact, p-nitrophenol. Monoclonal hybridoma supernatants obtained from hybridomas elicited from animals inoculated with immunogens described above were examined:

[0165] For N-Methyl pyridinium hapten with PEG linker attached to KLH (i.e., N- methyl-4-[(4- { [ethoxy([2-(pyridinium-2-yl)ethyl]amino)- phosphoryl]methyl}phenyl)carbamoyl]) or pyridine hapten with PEG linker attached to KLH (i.e., 4-[(4-{ [ethoxy([2-(pyridinium-2- yl)ethyl]amino)phosphoryl]- methyl }phenyl)carbamoyl] and immunogens boosted with OVAL- immunogens (described in EXAMPLES 1-3), significant acceleration of paraoxon breakdown was observed in the presence of hybridoma supernatants. In most cases, even greater acceleration of OP breakdown was observed in the presence of hybridoma supernatants in the presence of 2-PAM. The data shows the hybridoma supernatants induced breakdown of paraoxon in the presence of 2-PAM.

[0166] Example 14. Binding Selectivity for mAb “C” and “G”. Protein (150 pg of protein in 100 pL of water) was placed into each well of a Maxisorp plate for 1 hour at RT. The plate was emptied and washed 3 times (PBST, 10 mins each, RT). As custom, each well was then treated with 100 uL of 1% Casein protein as a general blocker. The plates were emptied and washed as above 3 times (PBST, 10 mins, RT). After the plates were air dried for 10 mins, mAh C or G was added to each well (1.5 ug in 100 uL and dilutions 1 :10, 1: 100, 1: 1000, 1:10,000 and 1: 100,000) for 1 hour at RT. As standard, after 1 hour, the plate was emptied washed as above 3 times (PBST, 10 mins, RT). Dried plates (10 mins) were treated with secondary antibody and washed as above 3 times (PBST, 10 mins, RT). Thereafter, substrate was added and the plates were read.

[0167] For comparison, a standard ELISA plate was run under standard conditions for highly purified C and G antibodies. Signal versus dilution curves were plotted and showed normal sigmoidal plots. Accordingly, the signal at a 1 : 10 dilution of mAb (0.15 pg mAb) C or G was compared to the signal observed for incubation of 150 pg of protein (HSA, KLH or Ovalbumin). Generally, the plots of signal versus mAb dilution were flat. Under these conditions, very little amount of binding to protein by the highly purified mAb was observed.

[0168] Even under conditions of 1,000-fold excess protein (HSA, KLH or Ovalbumin) over mAb,mAb recognized only a small percent of binding (Table 2). The conclusion is that both mAb C and G did not recognize or bind to standard proteins. Accordingly, the data suggests the immunization and monoclonal hybridoma workup did not afford non-specific protein binders. Rather, the mAbs C and G were highly selective for the hapten and not the carrier protein.

[0169] Example 15. Conversion of Nerve Agent Model Compounds to p- nitro phenol by hybridoma supernatants A-I ± 2-PAM as judged by HPLC.

[0170] Previously, we have shown that nerve agent model compounds recapitulate the molecular events and toxicity of actual nerve agents. The difference is that nerve agent model compounds generally are less toxic than actual nerve agents. United States treaties prohibit the use of actual nerve agents in non-government facilities. Nevertheless, nerve agent model compounds provide an excellent means to assess countermeasures. One such nerve agent model compound comprises replacement of leaving groups like fluorine with p-nitrophenol. Accordingly, / j-nitrophenol nerve agent model compounds (see Example 12) were synthesized and characterized.

[0171] Conversion of p-nitrophenol nerve agent model compounds of sarin and of soman by hybridoma supernatant G was done judged by HPLC. Para -nitrophenol nerve agent model compound (12.5 nmol / incubation) was combined with 225 LI L hybridoma supernatant G and incubated at 37 degrees for 1 hour. Thereafter, incubation was stopped by addition of ethyl acetate / (0.5% TFA, 0.5 mL) and 20 mg NaCl with mixing, centrifugation, decanted and evaporated. The samples were taken up in 200 LI L MeOH for HPLC. Controls (p -nitrophenol nerve agent model compounds alone + PBS (pH 7.5), p-nitrophenol nerve agent model compound + 2- PAM (0.5 mM) were run and compared to p-nitrophcnol nerve agent model compound + mAb G and p-nitrophenol nerve agent model compound + mAb G + 2- PAM (0.5 mM).

[0172] The results showed (Table 3) hybridoma supernatant G accelerated the breakdown of the sarin model compound 5-fold (compared to pH 7.5 alone) in the presence of 2-PAM. In contrast, breakdown of the soman model compound (compared to pH 7.5 alone) accelerated breakdown 1.1- fold in the presence of 2-PAM. Detectable selectivity was observed likely due to the steric bulk of the soman compound vs. the sarin compound.

[0173] Breakdown of Sarin model compound by hybridoma supernatant “G”

[0174] Breakdown of Soman model compound by hybridoma supernatant “G”

[0175] Example 16. Highly Purified mAbs “C” and “G” accelerate breakdown of paraoxon especially in the presence of 2-PAM on the basis of analysis by HPLC.

[0176] Incubations. A typical incubation was conducted in a total volume of 0.5 mL. Incubation mixtures contained 0.05 M potassium phosphate buffer (pH 7.5) or potassium phosphate buffer plus 2-PAM (500 p M final concentration) or highly purified 0.6 mg mAb “C” or “G” (see Example 4) alone in phosphate buffer or 0.6 mg mAb “C” or “G” in phosphate buffer plus 2-PAM (500 pM final concentration). Incubations were prepared at room temperature. Incubations were initiated by the addition of paraoxon (50 pM final concentration) to the incubation and placed at 37 °C with constant shaking. After 1 hour, incubations were stopped by the addition of mL cold acetonitrile containing 0.5% trifluoro acetic acid (TFA). Before LC analysis, an acetonitrile solution containing an internal standard (C-POX, 2.8 pM final concentration) was added to the sample as a calibration standard. One mL of the cold mixture was treated with 20-30 mg of NaCl and centrifuged at 3,500 rpm to separate the organic and aqueous fractions. The organic fraction was decanted and dried under a stream of nitrogen. Before LC analysis, the residue was taken up in 100 p L acetonitrile, centrifuged at 12,000 rpm and decanted into LC vials for analysis.

[0177] Analytical. Prior to analysis, samples were stored at -20 ° C. Analysis of samples was conducted using a LC analysis method. The organic material (80 pL) was used for LC analysis. The LC was an Agilent LC. The LC was run on a RP column (i.e., Luna C-18, 5 pm, 150 mm x 4.6 mm i.d.) equilibrated with isocratic 30% acetonitrile 70% water containing 0.05% TFA in an isocratic elution. Typical LC retention times for this system were: p-nitrophenol (10.2 mins), C-POX internal standard (15.5 mins) and paraoxon (20.6 mins) and were all simultaneously analyzed by HPLC via multiple wavelength monitoring.

[0178] Example 17. Breakdown of Paraoxon by Highly Purified Antibodies “C” and “G” as determined by HPLC product analysis

[0179] The results showed that highly purified mAbs “C” and “G” accelerated the breakdown of paraoxon. Compared to paraoxon at pH 7.5 and 2-PAM (0.5 mM), in the presence of highly purified mAb “C” or “G”, about a 5-fold acceleration of the breakdown of paraoxon was observed (Entry 4 or 6 versus Entry 2). mAbs “C” and “G” alone showed a modest acceleration of breakdown in the absence of 2-PAM (Entries 3 and 5 versus Entry 1). The conclusion is that 2-PAM and paraoxon is recognized by the mAb and combines with the mAb to afford a powerful accelerant for efficient breakdown of paraoxon.

[0180] Example 18. Sequence Examples

[0181] Antibody protein and nucleotide sequences for 1A2E7H12 and 5D8A6A4 are provided below with CDR domains indicated by bold, Frame Regions indicated by an underline and Constant Regions indicated by plain text. For the nucleotide sequences the signal sequences and the stop codons are indicated by italics as are the signal sequences for the peptide sequences. IgBLAST analyses of V(D)J junctions are provided in tables following the sequence data.

[0182] A. Antibody sequences of 1A2E7H12

[0183] Heavy chain DNA sequence (1395 bp): Signal sequence- FR1-CDR1- FR2-CDR2-FR3-CDR3-FR4-Constant Region-Stop codon[001S4] ATGGGCAGGCTTACTTCTTCATTCCTGCTACTGATTGTCCCTGCATAT G7CC7G7CCCAGGTCACTCTGAAAGAGTCTGGCCCTGGGATATTGCAG CCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGC ACTTATGGTGTGGGTGTAGGTTGGATTCGTCAGCCTTCAGGGAAGGGTCT GGAGTGGCTGGCCAACATTTGGTGGAATGATGATAAGCACTATAACTC AGCCCTGAAGAGCCGGCTCATTATCTCCAAGGATACCTCCAAGAAGCAGGTATTCCTCAAGATCTCCAGCGTGGACACTGCAGATACTGCCACATACTACTGTGCTCAACTGGGGACTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAAGTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTTTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTTTGATGGTTTTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTTTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGG GTAAA7G / V

[0185] Heavy chain: Amino acid sequence (464 aa). Signal pepritfe-FRl-CDRl- FR2-CDR2-FR3-CDR3-FR4-Constant Region. r001861MGRLr5SFLLLZVPAymOVTLKESGPGILOPSQTLSLTCSFSGFSLSTYGVGVGWIRQPSGKGLEWLANIWWNDDKHYNSALKSRLnSKDTSKKOVFLKISSVDTADTATYYCAQLGTPYWGOGTLVTVSAAKTTAPSVYPLAPVCGDTSGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGP SVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTH REDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTE PVLDFDGFYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG KTGA

[0187] Light chain: DNA sequence (702 bp). Signal ^e^; / ence-FRl-CDRl-FR2-CDR2 FR3-CDR3-FR4-Constant Region-Stop codon\G01&S\ATGAGACCGTCTATTCAGTTCCTGGGGCTCTTGTTGTTCTGGCTTCATOGrACrCAGTOrGACATCCAGATGACACAGTCTCCATCCTCACTGTCTGCATCTCTGGGAGGCAAAGTCACCATCACTTGCAAGGCAAGCCAAGACATTAATAAGTACATAGCTTGGTACCAACACAAGCCTGGAAAAAGTCCTAGGCTGCTCATACATTACACATCTGCGTTAGAGCCAGGCATCCCATCAAGGTTCAGTGGAAGTGGGTCTGGGAGAGATTATTCCTTCAGCATCAGCAACCTGGAGCCTGAAGATATTGCAACTTATTATTGTCTACAGTATGATAATCTTCGGACGTTCGGTGGAGGCACCAAACTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT7AG.

[0189] Light chain: Amino acid sequence (234 aa). Signal peptide-FRl-CDRl-FR2-CDR2-FR3- CDR3-FR4-Constant region r001901M7?PS / GFLGLLLFWLHGGGCDIOMTOSPSSLSASLGDKVTITCKASODINKNIAWYQHKPGKGPRLLIYYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLRTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0191] B. Antibody sequences of 5D8A6A4:

[0192] Heavy chain: DNA sequence (1374 bp). Signal sequence-FR 1 -CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant Region-Stop codon

[0193] ATGAAATGCAGCTGGGTCATCTTCTTCCTGATGGCAGTGGTTATAGGGG7CAA7TCAGAAGTTCAACTGCAGCAGTCTGGGGCAGAACTTGTGAGGCCAGGGGCCTCAGTCATGTTGTCCTGCACAGTTTCTGGCTTCAACATTAAAGACAGCCTTATACACTGGGTGAAGCAGAGGCCTGAGCAGGGCCTGGAGTGGATTGGATGGATGGATTCTGAGGATGGTGAAAGTACTTATGCCCCGAAGTTCCAGGACAAGGCCACTATTACTGCAGACACATCCTCCAATACAGCCTATCTGCAGCTCAGCAGGCTGACATCTGAGGACACTGCCATCTATTACTGTACTCGAGGTGGGACACACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCATCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAArGA

[0194] Heavy chain: Amino acid sequence (457 aa). Signal peptide-FRl-CDR1 -FR2- CDR2-FR3 - CDR3-FR4-Constant region-Srop codon r001951MACSWWFFLAMW7GVASEVOLQOSGAELVRPGASVMLSCTVSGFNIKDSLIHWVKORPEOGLEWIGWMDSEDGESTYAPKFQDKATITADTSSNTAYLQLSRLTSEDTAIYYCTRGGTHYWGOGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFIYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0196] Light chain: DNA sequence (702 bp): Signal vegMerace-FRl-CDRl-FR2-CDR2-FR3-CDR3-FR4-Constant Region-Stop codon

[0197] ATGAGACCGTCTATTCAGTTCCTGGGGCTCTTGTTGTTCTGGCTTCATGGTGGTCAGTGTGACATCCAGATGACACAGTCTCCATCCTCACTGTCTGCATCTCTGGGAGACAAAGTCACCATCACTTGCAAGGCAAGTCAAGACATTAATAAGAATATAGCATGGTACCAACACAAGCCTGGAAAAGGTCCTAGGCTGCTCATATATTACACGTCTACATTACAACCAGGCATCCCATCAAGGTTCAGTGGAAGTGGATCTGGGAGAGATTATTCCTTCAGCATCAGCAACCTGGAGCCTGAAGATATTGCAACTTATTATTGTCTCCAGTATGATAATCTTCGGACGTTCGGTGGAGGCACCAAACTGGAGATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAAT GAGTGT7AG

[0198] Light chain: Amino acid sequence (233 aa). Signal peptide-FRl-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region.1001991, t7A7JS7077.G / 77.7 VV77 / GGGCDIOMTOSPSSI.SASI.GF>KVT[TCKASODINKNIAWYQHKPGKGPRLLIYYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLOYDNLRTFGGGTKLEIKRADAAPTVSIFPPSSEOLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0200] Example 19. Positron emission tomography (PET) showed that anti-OP antibodies reduceparaoxon POX tissue radioactivity levels in vivo.

[0201] Carbon- 11 paraoxon ([' 'C ]-POX) was synthesized by reaction of CI L[ C|CI LI with a mixture of CS2CO3 and O-ethyl, O-p-nitrophenyl phosphate in DMF. The product [nC]-POX was purified from the reaction mixture using a semi-preparative HPLC column (IR = 5.7 min, Luna C18(2) 250 x 10 mm, 50% PB pH 6.8, 50% MeCN eluent at 8 mL / min). [nC]POX was prepared in 10.3 ± 2.0% decay corrected yield with a molar activity of 4.96 ± 0.74 GBq / u mol (134 + 20 Ci / mmol).

[0202] Carbon- 11 paraoxon positron emission tomography (PET) in vivo evaluations were conducted with highly purified anti-OP antibodies obtained from ascites with defined selectivity > 99% (as determined by ELISA) for hapten-protein conjugates as compared to controls including: carrier protein (KLH), ovalbumin, structurally-similar haptens and unmodified plates.

[0203] The efficacy of the anti-OP antibody to breakdown paraoxon (POX) in vivo, was evaluated in three rat tissues using the positron emission tomography tracer, [nC]-POX. [nC]-POX directly informs on the biodistribution (bioD) of POX. Antibodies that bind POX or promote the destruction of POX decreased the amount of ([nC]-POX distributed into tissues. Naive (control) rats were given [nC]-POX tracer alone and the biodistribution determined.

[0204] A. Tissue radioactivity levels were decreased in vivo by anti-OP antibodies at 1.0 h. The mAb dose was given at 6 mg / kg (e.g., a 250 g male rat = 620 11L of a 2.42 g / ml) in phosphate buffer 7.4 solution at t = 0 min. At 1.0 h and 24.0 h, the [nC]-POX tracer was given to rats to evaluate changes in the [nC]-POX levels due to the presence of the antibody.

[0205] Results showed naive rats given only [UC]-POX tracer afforded baseline radioactivity values defined in PET as maxima standard uptake values (SUVmax). The tissue values were determined following co-registration using computer tomography (CT). The various SUVm;istissues levels in naive rats exposed to [HC]-POX were brain = 2.8 ± 0.69, heart = 7.2 + / - 0.93 and lung = 9.2 + / - 2.2.

[0206] Rats given the antibody defined in this invention 1.0 h prior to the tracer showed SUVmaxbrain = 2.1 + / - 0.23, heart = 6.5 + / - 1.1 and lung = 7.6 + / - 2.2. All three tissues showed a decrease in radioactivity as the result of the antibody being present. Compared to naive rat levels, brain tissue levels dropped ~ 25%, heart levels dropped ~ 10 %, and lung levels dropped ~ 18% in antibody-treated rats.

[0207] In conclusion, one hour after the anti-OP antibody was given, radioactivity levels were lower in brain, heart and lung compared to naive subject levels. Because [UC]-POX covalently modifies its targets with retention of the radiolabel, the majority of radioactivity at one hour is associated with proteins, particularly enzymes. The greatest reduction in tissue radioactivity was observed in brain, where lower radioactivity tissues levels likely occurred as a result of less radioactive POX available to modify brain acetylcholinesterase (AChE) because of antibody binding and / or Ab-promoted breakdown of [nC]-POX. A large reduction in lung radioactivity levels was also observed relative to naive levels, due to lower amounts of available [nC]-POX. The presence of anti-OP antibodies led to lower radioactivity levels in heart as compared to naive levels but was the lowest reduction of the three tissues.

[0208] B. Tissue radioactivity levels were decreased in vivo by anti-OP antibodies at 24.0 h.

[0209] The anti-OP antibodies described in the invention were given 24.0 h prior to the introduction of [nC]-POX tracer to establish that the mAb function was sustained beyond 1.0 h.

[0210] Naive rats given only [nC]-POX tracer afforded standard uptake values (SUVmax) as indicated in Example 18A: brain = 2.8 + / - 0.69, heart = 7.2 + / - 0.93 and lung = 9.2 + / - 2.2. Rats given the antibody defined in this invention 24.0 h prior to the tracer showed SUVmax tissue levels: brain = 1.9 ± 0.055, heart = 5.4 + / - 0.99 and lung = 6.2 + / - 1.4. Each of the three tissues had lower radioactivity levels after 24.0 h antibody dosing, and were lower than levels determined at 1 .0 h. Compared to naive rats, brain tissue levels decreased - 31%, heart levels dropped - 26 %, and lung levels dropped - 33% in antibody- treated rats.

[0211] Compared to 1 h data, at 24.0 h after anti-OP mAb was given, tissue radioactivity levels were - 25-35% lower than naive subject in brain, heart and lung as compared to the 1.0 h rat tissue levels. Compared to biodistribution for animals administered mAb 1.0 h before, for studies done with administration 24.0 h before, antibody levels achieved in vivo equilibrium that differed. When [nC]- POX was given 24.0 h after the mAb dose, lower tissue radioactivity levels were found than at 1.0 h because less [nC]-POX was available to covalently modify targets presumably due to mAb binding and / or breakdown of the [nC]-POX in the bloodstream. But at 24 h the mAb entered and was present in tissues and other biological pools extending protection against [UC]-POX. Heart and lung dropped an additional 15-16% in tissue radioactivity at 24.0 h versus 1.0 h levels, and brain radioactivity levels were also decreased further (6%) indicating the lasting efficacy and stability of the mAb (agreement with Example 18A). The anti-OP mAb described in this invention not only confers protection from paraoxon at 24.0 h, but also indicates an improvement versus Ab given 1.0 h prior to exposure.

[0212] C. Tissue radioactivity levels were reduced in vivo by anti-OP antibodies when coadministered with 2-pyridine aldoxime methiodide (2-PAM) at 1.0 h.

[0213] As described by this invention, the anti-OP antibodies bound a hybrid structure of an organophosphorus compound and 2-PAM. The design produced antibodies that were able to bind both an organophosphorus compound such as POX and also 2-PAM simultaneously.

[0214] In this example, changes in tissue radioactivity levels due to [nC]-POX exposure were examined in the presence of 2-PAM (control) and both antibody and 2-PAM. In this example, rats were given [nC]-POX 1.0 h after 2-PAM alone (control) or given [nC]-POX 1.0 h after 2-PAM and mAb and the tissue radioactivity levels assessed. Because the half-life of 2-PAM is less than two hours, the 24.0 h timepoint was not evaluated. Tissue radioactivity levels were also compared to naive animals given the [nC]-POX tracer only and to rats given the anti-OP antibodies only.

[0215] Naive rats given only [nC]-POX tracer afforded standard uptake values (SUVmax) as indicated in Example 18A: brain = 2.8 + / - 0.69, heart = 7.2 + / - 0.93 and lung = 9.2 + / - 2.2. Control rats were given [nC]-POX tracer 1.0 h after 2-PAM (control) and afforded standard uptake values (SUVmax):brain = 2.4 (-12%), heart = 7.3 (-1.1 %), and lung = 7.0 (-24%). Lung tissue radioactivity levels were most affected by the presence of 2-PAM alone. The combination of 2-PAM and anti-OP mAb both administered 1.0 h prior to [' 'C]-POX tracer dose afforded SUVmaxvalues: brain = 2.2 + / - 0.12 (-20%), heart = 6.1 + / - 0.53 (-15%) and lung = 6.3 + / - 1.2 (-32%). The tissue radioactivity levels were lower as compared to 2-PAM only (control) and naive (tracer only) subjects.

[0216] Compared to naive (tracer only) rats, tissue radioactivity levels at 1.0 h were lower in rat brain and lung but not heart when given 2-PAM alone. When 2-PAM and anti-OP antibodies were coadministered 1. Oh prior to [nCJ -POX tracer, the tissue radioactivity levels were all lower than the tissue levels for the 2-PAM only dose suggesting a cooperative effect. This is supported, in part, by the tissue radioactivity levels when rats were given only anti-OP antibodies 1.0 h prior to tracer (SUVmaxbrain = 2.1 + / - 0.23, heart = 6.5 + / - 1.1 and lung = 7.6 + / - 2.2). In comparison, brain radioactivity levels were similar for both mAb dosing and mAb / 2-PAM co-administration at 1.0 h. However, heart and lung tissue radioactivity levels dropped an additional 5% and 14%, respectively, when 2-PAM was given with the mAb. The results confirmed that the combination of mAb and 2-PAM decreased tissue radioactivity levels in heart and lung but did not change brain levels as compared to mAh alone dosing.

[0217] Example 20. Pharmacokinetic Evaluation of Monoclonal Antibody “C”.Jugular vein catheterized adult male Sprague Dawley rats weighing 186-212 grams were housed individually and maintained in a temperature-controlled environment on a 12 hour: 12 hour light cycle (0600 hour on- 1800 hour off) upon arrival to the laboratory. Animals were given free access to food and water during a one-week habituation period to the laboratory. For PK studies, highly purified mAb “C” (Tow dose’ at 0.72 mg / animal in 0.15 mol / kg NaCl to mimic osmolality of human plasma, 300 mOsm / kg) was administered via the tail vein to three individually housed, food deprived, jugular-cannulated Sprague Dawley rats. A second group of 3 animals was treated at a ‘high dose’ of mAb “C” (1.44 mg / animal) via the tail vein. At time points over 26 days, blood was withdrawn through the jugular vein catheter, cooled and centrifuged to obtain serum.

[0218] Sera were stored at 4 °C until diluted with water for ELISA assays that were run along with standard curves of highly purified antibody “C” to quantify the pg antibody / ml of serum. A plot of pg antibody / ml of serum vs. time afforded PK profiles (Table 8). The data obtained yielded a “high dose” half-life of 1.3 days of mAb “C”, however, significant amounts of mAb “C” remained in rat serum even 25 days after initial administration (Table 8). The conclusion is that monoclonal antibody “C” is quite stable and long-lived.

[0219] Example 21 In Vitro Stability of Pyridine and Pyridinium Haptens.,

[0220] The aqueous hydrolytic stability of pyridine and pyridinium haptens was directly assessed by P-31 NMR. Hapten stability in aqueous media is required to elicit a robust immune response and the resultant antibodies produced recognize the desired hapten. The stability of pyridine and pyridinium haptens (35 mM) was assessed at pH 6.5 and 8.0 buffer to simulate in vivo pH extremes and validate the stability in aqueous media as a predictor of survival in vivo. P-31 was deemed the easiest and most sensitive analytical measure in two non-phosphate buffer systems at pH 6.5 and 8.0. Each hapten showed a distinctive chemical shift: pyridine (34.9 ppm) and pyridinium (35.0 ppm) with the / -butyl PEG monoacid linker attached. This assessment also tested the stability of the PEG linker. P-31 NMR scans were taken at various time points (i.e., 0, 60, 420 and 10,080 minutes) to determine amounts of hapten hydrolyzed.

[0221] At both pH values, P-31 NMR scans showed very little of either hapten hydrolyzed over time. For example, at 420 min ~ 0.06% of the pyridine hapten was hydrolyzed. By seven days (10,080 min) no additional pyridine hapten hydrolysis products were detectable. At seven days the pyridine hapten afforded minor peaks at 1.5 (0.58%) ppm, 12.6 (0.55 %) and -24.2 ppm (0.64 %). Under the conditions examined, the pyridinium hapten showed no evidence of any hydrolysis.

[0222] For the pyridine hapten, when all byproducts were considered in the P-31 analysis, the parent was present at 98.3% and byproducts summed to approximately 1.7% of the total P-31 signal at seven days. A plot of the P-31 peak height of the pyridine hapten versus time provideda linear plot. From the slope of the plot a pseudo first order rate constant was obtained and a half-life of 229 days was calculated. The pyridinium hapten showed no measurable evidence of hydrolysis. For the pyridinium hapten, it was concluded that the pseudo first order rate constant was very low and hydrolytic half-life was longer than 229 days at the pH values measured.

[0223] B. Stability of Highly Purified Monoclonal Antibody “C” in Rat Blood.The stability of highly purified monoclonal antibody “C” was examined in the presence of whole blood from Sprague Dawley rats. As a control, the stability of highly purified monoclonal antibody “C” was studied in pH 7.4 buffer. A time course study of highly purified mAb in blood and at pH 7.4 buffer over 3 hours at 37° C with serial sampling at 1, 2 and 3 hours was conducted. At 1, 2 and 3 hours, aliquots were taken from incubations and after dilutions, samples were directly placed on ELISA plates designed to recognize highly purified monoclonal antibody “C”. Triplicate samples were run. Blood samples were centrifuged at 12,800 x g for 3 minutes to separate serum from blood cells and taken up in water. A standard curve of highly purified mAb “C” in various dilutions, in triplicate was also run. The time course showed that over the course of 3 hours, no significant change in values of pg / well of mAb was observed for either incubations done in pH 7.4 buffer (1-1.26 pg “C”) or blood (10- 15.2 pg “C”). The study also determined that there were no interferences or confounders that would alter ELISA analyses.

[0224] On the basis of ELISA studied, the conclusion is that highly purified monoclonal antibody “C” is stable in rat blood and pH 7.4 buffer for at least 3 hours. Highly purified monoclonal antibody “C” did not recognize carrier proteins (i.e., KLH or Ovalbumin) or another protein (i.e., BSA) and is thus highly selective for the desired hapten. In addition, on the basis of P-31 NMR studies, pyridine and pyridinium haptens were stable for many weeks in aqueous solution. As such, a vaccine strategy using haptens would be successful in stimulating the immune system of animals to support protection from OP exposure. The data also suggested highly purified monoclonal antibody “C” was highly stable and would be anticipated to be long-lived for extended protection from OPs. Such a conclusion was validated by pharmacokinetic studies in small animals.

[0225] Example 22. Serum clinic analysis of low and high dose-treated rats.

[0226] We examined serum clinic parameters to investigate the safety of anti-paraoxon mAb “C” in rats. We compared low dose (0.72 mg / rat) and high dose (1.44 mg / rat) values. Male rats were treated with mAb via tail vein injection (see Table 9) and blood from each animal was collected via jugular vein catheter. Serum was separated from blood cells via centrifugation(12,000 x rpm, 2 minutes). Serum from multiple animals at each time point was pooled (n = 3) and pooled samples were sent for serum clinic analysis done by a commercial lab. The serum clinical data for the effect of the mAb treatment was compared to the mean and standard deviation values for literature controls, see Table 10.

[0227] In conclusion, no significant differences in serum clinical values were observed for rats administered mAb “C” (at either low or high doses) at 270 minutes or 31.5 hours compared to the reference values from the literature. For the two time points examined, no detectable hepatic toxicity was observed on the basis of serum clinical values (ALT, AST, bilirubin). Based on serum clinical values (BUN, creatinine, salts), no detectable kidney toxicity was observed. We conclude mAb “C” is safe and mAb “C” mAb fragments would also be safe to administer to animals.

[0228] Table 10. Serum clinic values for mAba-treated rats

[0229] All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

Claims

WHAT IS CLAIMED IS:

1. An antibody or a fragment of an antibody comprising amino acid sequences for a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: a CDR-H1 amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 7; a CDR-H2 amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 8 and a CDR-H3 amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 9; and wherein the light chain variable region comprises: a CDR- LI amino acid sequence as shown in SEQ ID NO: 4; a CDR-L2 amino acid sequence as shown in SEQ ID NO:5; and a CDR-L3 amino acid sequence as shown in SEQ ID NO: 6,SEQ ID NO:1 TYGVGVGSEQ ID NO:2 NIWWNDDKHYNSALKS SEQ ID NO:3 LGTPYSEQ ID NO: 4 KASQDINKNIA SEQ ID NO: 5 YTSTLQPSEQ ID NO:6 LQYDNLRT, SEQ ID NO:7 DSLIHSEQ ID NO:8 WMDSEDGESTYAPKFQD SEQ ID NO:9 GGTHY wherein the antibody or the fragment of an antibody is capable of specific binding to an organophosphorus compound and an oxime capable of breakdown of the organophosphorus compound, wherein the organophosphorus compound is an organophosphorus pesticide or nerve agent.

2. The antibody or the fragment of the antibody of claim 1 further comprising an amino acid sequence for a heavy chain framework region, wherein the heavy chain framework region comprises a FR-H1 amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO: 14, a FR-H2 amino acid sequence as shown in SEQ ID NO: 11 or SEQ ID NO: 15, a FR-H3 amino acid sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 16 or a FR-H4 amino acid sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 17.SEQ ID NO: 10 QVTLKESGPGILQPSQTLSLTCSFSGFSLSSEQ ID NO: 11 WIRQPSGKGLEWLASEQ ID NO: 12 RLIISKDTSKKQVFLKISSVDTADTATYYCAQSEQ ID NO: 13 WGQGTLVTVSASEQ ID NO: 14 EVQLQQSGAELVRPGASVMLSCTVSGFNIKSEQ ID NO: 15 WVKQRPEQGLEWIGSEQ ID NO: 16 KATITADTSSNTAYLQLSRLTSEDTAIYYCTRSEQ ID NO: 17 WGQGTTLTVSS3. The antibody or the fragment of the antibody of claim 1 further comprising an amino acid sequence for a light chain framework region, wherein the light chain framework regioncomprises a FR-L1 amino acid sequence as shown in SEQ ID NO: 18, a FR-L2 amino acid sequence as shown in SEQ ID NO: 19, a FR-L3 amino acid sequence as shown in SEQ ID NO: 20 or a FR-L4 amino acid sequence as shown in SEQ ID NO:21.SEQ ID NO: 18 DIQMTQSPSSLSASLGDKVTITC SEQ ID NO: 19 WYQHKPGKGPRLLIY SEQ ID NO:20 GIPSRFSGSGSGRDYSFSISNLEPEDIATYYCSEQ ID NO:21 FGGGTKLEIK4. The antibody or the fragment of the antibody of claim 1 further comprising an amino acid sequence for a heavy chain constant region wherein said amino acid sequence is shown in SEQ ID:22SEQ ID NO:22 AKTTAPSVYPLAPVCGDTSGSSVTLGCLVKGYFPEPVTL TWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTK VDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVD VSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSG KEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCM VTDFMPEDIYVEWTNNGKTELNYKNTEPVLDFDGFYFMYSKLRVEKKNWVE RNSYSCSVVHEGLHNHHTTKSFSRTPGK.

5. The antibody or the fragment of the antibody of claim 1 further comprising an amino acid sequence for a light chain constant region wherein said amino acid sequence is shown in SEQ ID NO: 23SEQ ID NO:23 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINV KWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEAT HKTSTSPIVKSFNRNEC.

6. The antibody or the fragment of the antibody of claim 1 wherein the antibody or the fragment of the antibody is capable of hydrolyzing a organophosphorus compound wherein the organophosphorus compound is an organophosphorus pesticide.

7. The antibody or the fragment of the antibody of claim 1, wherein the antibody or the fragment of the antibody is capable of hydrolyzing organophosphorus compounds wherein the organophosphorus compound is a nerve agent.

8. The antibody or the fragment of the antibody of claim 1, wherein the oxime is 2-pyridine aldoxime methyl chloride (2-PAM)9. The antibody or the antibody fragment of the antibody of claim 1, wherein the antibody or the fragment of the antibody is capable of selective binding to 4-{[ethoxy([2-(pyridin-2- yl)ethyl]amino)phosphoryl]-methyl (aniline.10.

10. The antibody or the antibody fragment of the antibody of claim 1, wherein the antibody or the fragment of the antibody is capable of selective binding to N-methyl- 4-[(4-{[ethoxy([2- (py ridinium-2-y l)ethy 1] amino) -pho sphory 1] methyl } aniline .

11. An isolated polynucleotide comprising a sequence encoding a heavy chain variable region and a light chain variable region of the antibody or the fragment of the antibody of claim 1.

12. The isolated polynucleotide of claim 13, wherein the isolated polynucleotide is comprised of the sequence encoding the heavy chain variable region as set forth by SEQ ID NO:24 or SEQ ID NO:25SEQ ID NO:24CAGGTCACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTATGGT GTGGGTGTAGGTTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTG GCCAACATTTGGTGGAATGATGATAAGCACTATAACTCAGCCCTGAAGAGC CGGCTCATTATCTCCAAGGATACCTCCAAGAAGCAGGTATTCCTCAAGATC TCCAGCGTGGACACTGCAGATACTGCCACATACTACTGTGCTCAACTGGGG ACTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCASEQ ID NO: 25GAAGTTCAACTGCAGCAGTCTGGGGCAGAACTTGTGAGGCCAGGGGCCTCAGTCATGTTGTCCTGCACAGTTTCTGGCTTCAACATTAAAGACAGCCT TATACACTGGGTGAAGCAGAGGCCTGAGCAGGGCCTGGAGTGGATTGGAT GGATGGATTCTGAGGATGGTGAAAGTACTTATGCCCCGAAGTTCCAGGACA AGGCCACTATTACTGCAGACACATCCTCCAATACAGCCTATCTGCAGCTCA GCAGGCTGACATCTGAGGACACTGCCATCTATTACTGTACTCGAGGTGGGA CACACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

13. The isolated polynucleotide of claim 11 wherein the isolated polynucleotide is comprised of the sequence encoding the light chain variable region as set forth by SEQ ID NO:26SEQ ID NO:26ATGAGACCGTCTATTCAGTTCCTGGGGCTCTTGTTGTTCTGGCTTCATGGTACTCAGTGTGACATCCAGATGACACAGTCTCCATCCTCACTGTCTGCAT CTCTGGGAGGCAAAGTCACCATCACTTGCAAGGCAAGCCAAGACATTAATA AGTACATAGCTTGGTACCAACACAAGCCTGGAAAAAGTCCTAGGCTGCTCA TACATTACACATCTGCGTTAGAGCCAGGCATCCCATCAAGGTTCAGTGGAA GTGGGTCTGGGAGAGATTATTCCTTCAGCATCAGCAACCTGGAGCCTGAAG ATATTGCAACTTATTATTGTCTACAGTATGATAATCTTCGGACGTTCGGTGG AGGCACCAAACTGGAAATCAAA14. An isolated vector comprising the polynucleotide of claim 12 or claim 13.

15. An isolated host cell comprising the vector of claim 14.

16. A method of making the antibody or the fragment of the antibody of claim 1, wherein the method comprises: a) culturing the host cell of claim 15 under conditions suitable for expression of the polynucleotide encoding the antibody or the fragment of the antibody; and b) isolating the antibody or antigen binding fragment of the antibody.

17. A compound having the structure of:wherein R is hydrogen or alkyl, Ri and R2independently are alkyl and X’ is a suitable counterion.

18. The compound of claim 17, wherein R is hydrogen and Ri and R2are each methyl.

19. A compound having the structure of:wherein R is hydrogen or alkyl, Ri and R2 independently are alkyl, X" is a suitable counterion, L is a linker residue and RG is a reactive group capable of forming a covalent bond to a carrier protein.

20. The compound of claim 19, wherein -Linker-RG has the structure selected from the group consisting of -C(O)-R3-PEG-R4-CO2H, -C(O)-R3-CO2H, -C(O)-R3- NH2, -C(O)-R3-SH and salt and protected forms thereof, wherein R3and R4 independently are alkyl and PEG is a polyethylene glycol residue having 2-12 repeating ethyleneoxy subunits.

21. The compound of claim 19, wherein -L-RG is selected from the group consisting ofglutaric acid, succinic acid, aminocaproic acid and cysteine residues and salt and protected forms thereof.

22. The compound of claim 20, wherein the compound has the structure of:or wherein the compound structure has its carboxylic acid functional group in salt or protected form.

23. The compound of claim 21, wherein the compound has the structure of:or wherein the compound structure has its carboxylic acid functional group in salt or protected form.

24. A composition of matter represented by the formula:CP-Linker-hapten or salt thereof, wherein CP is a Carrier Protein, linker is a spacer molecule connecting hapten to carrier protein and hapten is an immunologically competent moiety having the structure ofwherein R directly attached to the phosphorous atom is a substituted or unsubstituted alkylarylamine residue in which a carbon atom of its alkyl moiety is covalently attached tothe phosphorous atom and wherein the nitrogen atom of its amino group is covalently attached to the linker,-OR is an alkoxy group and-NR2 is a substituted or unsubstituted 2-(2-pyridyl)ethylamino moiety, wherein the nitrogen atom of its amino group is covalently attached to the phosphorous atom.

25. The composition of claim 24, wherein the carrier protein is keyhole limpet hemocyanin (KLH), bovine or human serum albumin (BSA or HSA, respectively), or ovalbumin.

26. The composition of claim 24, wherein the hapten moiety corresponds in structure to a compound of claim 17 in which the aniline nitrogen atom of the compound is the site of covalent attachment to the linker.

27. The composition of claim 24 wherein the Linker has the structure of -C(O)-Rs-PEG-R4- C(O)- or -C(O)-R3-C(O)-, wherein R3 and R4 independently are alkyl and PEG is a polyethylene glycol residue having 2-12 repeating ethyleneoxy subunits, wherein one carbonyl carbon atom of the structure is covalently attached to the nitrogen atom of the amino group of alkylarylamine residue and the other carbonyl carbon atom is covalently attached to a Lysine epsilon amino group of the carrier protein.

28. The composition of claim 26 wherein the Linker has the structure -C(O)-R3-NH-, -C(O)- R3-S- wherein one carbonyl carbon atom of the structure is covalently attached to the nitrogen atom of the amino group of alkylarylamine residue and the amino or thiol group is covalently attached to the carrier protein through and amide or disulfide bond, respectfully.

29. Use of an antibody or a fragment of an antibody of claim 1 in preparation of a medicant for treating a subject exposed to an organophosphorus compound, wherein the organophosphorus compound is an organophosphorus pesticide or an organophosphorus nerve agent.

30. A vaccine comprising the hapten as defined in claim 24 and a pharmaceutically acceptable carrier and / or immuno-effective adjuvant.

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