WO1999002660A1

WO1999002660A1 - Catalytic antibodies for the inactivation of herbicides

Info

Publication number: WO1999002660A1
Application number: PCT/IL1998/000305
Authority: WO
Inventors: Ehud Keinan; Avidor Shulman
Original assignee: Ehud Keinan; Avidor Shulman
Priority date: 1997-07-08
Filing date: 1998-06-30
Publication date: 1999-01-21
Also published as: AU7929498A; IL121257A0

Abstract

A catalytic antibody that is capable of catalyzing the inactivation of a herbicide, including an immunoglobulin that binds a stable transition state analog of a deactivation reaction of the herbicide.

Description

CATALYTIC ANTIBODIES FOR THE INACTIVATION OF HERBICIDES

FIELD OF THE INVENTION

The invention relates to the generation of catalytic antibodies capable of inactivating herbicides, in particular carbamate containing herbicides. It further relates to the isolation of a gene system for these antibodies, and to methods of expressing these antibodies, or active fragments thereof, in plant cells, thereby conferring herbicide resistance.

BACKGROUND OF THE INVENTION

There has been considerable interest of the chemical industry as well as the agricultural community in the development of crop cultivars with herbicide resistance. These efforts are motivated both by economic considerations as well as environmental aspects. The ability of a particular crop to tolerate a non-selective herbicide would eliminate the need for use of selective herbicides or mixtures of herbicides in order to combat weeds. Some nonselective herbicides may generally be more easily degraded, thus being less damaging to the environment. Herbicide resistance would also permit replanting in the presence of herbicides carried over from previous crops.

Herbicide resistant crops have been achieved in the past by conventional plant breeding, which involves the selection of individual specimens of the plant which show natural resistance to the herbicide.

Herbicide resistance has also been achieved by cell culture selection or protoplast fusion. Tissue culture techniques have been used to select for spontaneous mutations that occur, or to select somaclonal variations induced in the presence of a mutagen. It is possible to select cells that resist the selection pressure applied to the culture. Depending on the plant species it is sometimes possible to regenerate whole plants from the resistant cells International Patent Application WO 90/14000 discloses maize plants which are resistant to the effects of imidazoline and/or sulphonyl urea herbicides. These plants were produced by a pollen mutagenesis method, followed by pollination of recipient plants with the mutagenized pollen. Although mutagenesis may provide plants of a particular species which are resistant to a selected herbicide, the resistant trait cannot be easily transferred to other plant varieties.

Through modern techniques of molecular genetics it is now also possible to confer herbicide resistance on crops by the introduction of genes encoding resistance into plants. Genetic engineering methods may target the search for mutant plant genes which encode resistant plant proteins or enzymes, cloning these genes and introducing them into the genetic material of the crops.

Examples of this approach are found in EP 257,993 which discloses a general scheme of mutations to the gene encoding acetolactate synthase which can confer resistance to the sulphonylurea class of herbicides. International application WO 92/08794 discloses DNA coding for a mutant acetolactate synthase enzyme which confers on a plant into which it is introduced by transformation resistance to herbicides which inhibit wild-type acetolactate synthase, such as herbicides of the imidazoline and sulphonylurea families.

Another genetic engineering approach has centered on the detection of bacterial genes encoding enzymes capable of degrading certain herbicides which can confer resistance on crops. Genetically engineered crops such as tobacco with resistance to the herbicide phenmedipham have been generated through the cloning of such a bacterial gene, encoding a natural carbamate hydrolase enzyme, and its introduction into the crop's genome, as disclosed in US Patent 5,347,976.

The present invention discloses that it is possible to take advantage of an entirely different approach in order to achieve the goal of herbicide resistance, thus eliminating the previous necessity to rely on the haphazard occurrence of natural enzymes that may possess the catalytic activity capable of degrading the herbicide. This approach utilizes the ability of the immune system to produce antibodies capable of catalyzing the desired chemical reactions. This approach will enable the generation of tailor made molecules to catalyze the deactivation of the herbicide. This approach to herbicide resistance is neither taught nor suggested in the background art.

The first antibody catalyzed reactions were performed by Lerner and coworkers (Tramontano et al., Science 234, 1566-1570, 1986) and by Schultz and coworkers (Pollack et al., Science 234, 1570-1574, 1986), and since that time antibodies have been generated which catalyze a wide variety of chemical reactions (reviewed by Schultz and Lerner, Science 269, 1835-1842, 1995, Lerner et al., Science 252, 659-667, 1991;

Schultz, Science 240, 426-433, 1988) with specificity that rivals or even exceeds that of enzymes. In principle, catalytic antibodies are elicited against stable analogs of the transition state structures that are high energy intermediates of the reaction that is to be catalyzed. The transition state structure itself cannot be isolated since it is energetically unfavorable in comparison to the reactant or the product of the reaction.

In US patent 4,888,281 it is disclosed that catalytic antibodies can bind a substrate, cause the conversion thereof to one or more products, and release the product. Catalytic antibodies may be prepared by immunological methods wherein they are elicited to antigens, as taught for example in US patent 4,888,281 or international patent application WO 89/10961. Smaller fragments of complete antibody molecules have also been shown to catalyze chemical reactions, as disclosed in international patent application WO 91/14769. The immune system produces a vast array of antibodies, in response to the exposure of a host animal to an appropriately designed synthetic antigen. After the generation period, during which the immune system improves the antibodies, these antibodies are screened, and antibodies which catalyze the reaction are isolated and generated in large amounts. The isolation, cloning and expression of selected antibodies are known methodologies.

The antibodies gain their catalytic activity through their ability to stabilize charged transition states, as well as by acting as entropic traps, and through catalytic groups and co-factors embedded in their binding sites. The relevant portion of the antibody molecule for the catalytic activity is the Fab fragment of the molecule. It may also be possible to obtain catalytic activity through the isolated Fv fragment of the antibody molecule, though generally the specificity of the smaller Fv fragment may be lower than that of the Fab fragment or the intact immunoglobulin.

The generation of catalytic antibodies capable of hydrolyzing carbamates has been accomplished previously, utilizing an antigen which incorporates a phosphonate or phosphonamidate group in place of the carbamate group (VanVranken et al., Tetrahedron Lett. 35, 3873-3876, 1994; Wentworth et al., Proc. Natl. Acad. Sci. USA 93, 799-803, 1996), however these reactions did not disclose the use of catalytic antibodies for inactivation of herbicides.

One of the advantages of the catalytic antibody approach is the ability to select the optimal activity for achieving the deactivation of herbicide. Furthermore, since the genetic material needed for the activity encodes two separate polypeptide chains there is diminished risk of inadvertently conferring the resistance trait on other plants in the environment.

Techniques for plant transformation are now well known and a number of foreign genes are capable of being introduced and expressed in transgenic plants. Transgenic plants that can be used to produce antibodies, particularly single chains of antibodies have been disclosed for example in EP657538. Transgenic plants that can be used to produce monoclonal antibodies such as catalytic antibodies have also been disclosed (reviewed by Hiatt and Ma, FEBS Letters, 307, 71-75, 1992; Ma et al., Eur. J. Immunol. 24, 131-138, 1994). These disclosures teach that plants are capable of synthesizing the two necessary gene products (i.e., the heavy and light chains of antibodies) and of assembling the two polypeptides correctly, in order to produce a functional antibody. However, they neither teach nor disclose the use of catalytic antibodies in order to impart herbicide resistance on a crop. SUMMARY OF THE INVENTION

It is an object of the present invention to provide antibodies that inactivate herbicides by utilizing the ability of the immune system to produce antibodies capable of catalyzing the desired chemical reaction. The antibodies of the invention are elicited against a stable transition state analog of the required chemical reaction.

It is another object of the present invention to provide cloned genetic material that encodes said antibodies. It is yet another object of the invention to provide transgenic plants that express said antibodies, thereby conferring herbicide resistance on said plants.

The present invention further provides methods for generating antibodies that catalyze chemical reactions resulting in the deactivation of known herbicides. Using the methods of the invention catalytic antibodies are generated against a synthetic antigen which is designed according to the specific herbicide to be deactivated.

The invention further provides methods for cloning of the genes that encode the catalytic antibodies into the genetic material of the selected crop plant, and to methods for expression of these genes, thus creating a crop which produces a protein whose function is to eliminate and deactivate a herbicide which is used to kill weeds surrounding this crop.

In a preferred embodiment, the invention is exemplified by antibodies that catalyze the hydrolysis reaction as a means for the deactivation of the herbicides which include a carbamate functional group.

One preferred embodiment provides antibodies that catalyze the hydrolysis of the carbamate herbicide chlorpropham, 1. Scheme 1 shows the chemical mechanism for this reaction: Chlorpropham

Scheme 1

According to a more preferred embodiment, a novel method is provided for the generation of plant species with resistance to the biscarbamate herbicide phenmedipham, 2..This biscarbamate herbicide, phenmedipham, 2, was selected as a more preferred embodiment because of its low toxicity and short life span after application. Utilization of this herbicide is therefore beneficial from the environmental standpoint.

Antibodies which catalyze the hydrolysis of carbamates are generated, utilizing an antigen which incorporates a phosphonate group in place of the carbamate group. Scheme 2 shows the chemical mechanism for this reaction incorporating a phosphora idate group:

slow fast spontaneous decomposition

Scheme 2

The synthetic antigen, 3, used to generate antibodies is analogous to the transition state which corresponds to the first step of the hydrolysis of phenmedipham, 2, and includes a phosphoramidate group in place of the carbamate group:

Transition State Analog 3

Further preferred embodiments provide monoclonal antibodies that impart resistance against important groups of herbicides including the urea herbicides, exemplified by diuron, 4, and the bipyridinium group of herbicides exemplified by paraquat, 5, and glyphosate, 6. These herbicides are deactivated through catalysis of different reactions and mechanisms.

Paraquat 5

Glyphosate 6

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to antigens which are capable of eliciting through immunogenic methods antibodies which can catalyze the deactivation of a herbicide molecule. These antigens comprise a hapten, or a hapten linked to a suitable carrier molecule, wherein the hapten is chosen, inter alia, because it resembles a presumed transition state structure of the herbicide deactivation reaction, and is therefore defined herein and in the claims as a "transition state analog". A compound is defined as a "stable" analog if it is robust enough to withstand isolation in useful quantities. The antibodies elicited are capable of catalyzing a chemical reaction, and are therefore defined herein and in the claims as "catalytic antibodies". The catalytic antibodies according to the invention are designed to inactivate a herbicide by binding the presumed transition state structure in a rate limiting step of a deactivation reaction of the herbicide. The terms "inactivation" and "deactivation" are used herein interchangeably, and refer to any chemical reaction whereby the herbicide is rendered devoid of its herbicidal activity. Catalysts increase chemical reaction rates by lowering the activation energy of a reaction. Antibodies elicited to a hapten or immunogen according to the invention, should stabilize the energy of the transition state relative to the reactants and products. This approach has been successfully demonstrated in the generation of specific catalytic monoclonal antibodies. Catalytic antibodies elicited with haptens according to the invention are "specific" in that they are designed to catalyze only the cleavage or formation of certain chemical bonds having the selected structural elements or conformations.

Catalytic antibodies may be obtained by processes comprising generating a plurality of monoclonal antibodies to an antigen and screening the plurality of antibodies so generated to identify a monoclonal antibody that catalyzes the reaction of interest. Antibodies may be generated either in vitro or in vivo. In still a further and related process, an animal is immunized with an antigen thereby generating antibody producing lymphocytes in said animal, antibody producing lymphocytes are removed from the animal, said lymphocytes are fused with myeloma cells to produce a plurality of hybridoma cells each of which produces monoclonal antibodies, the plurality of monoclonal antibodies is screened to identify a monoclonal antibody that catalyzes the reaction, and the hybridoma producing the catalytic antibody is cloned.

As used herein the term "animal" refers to any organism with an immune system. Antibody and immunoglobulin refer to any of several classes of structurally related proteins that function as part of the immune response of an animal, which proteins include IgG, IgD, IgE, IgA, IgM and related proteins. Under normal physiological conditions antibodies are found in plasma and other body fluids and in the membrane of certain cells and are produced by lymphocytes of the type denoted B cells or their functional equivalent. Antibodies of the IgG class are made up of four polypeptide chains linked together by disulfide bonds. The four chains of intact IgG molecules are two identical heavy chains referred to as H-chains and two identical light chains referred to as L-chains. Known active fragments of catalytic immunoglobulin molecules will include the F(ab')₂ portion, Fab fragments and Fv fragments.

In still further related processes nucleic acids encoding the catalytic antibodies, or active fragments thereof, are isolated and cloned. These nucleic acids may be used to create cells which express in vivo catalytic antibodies. Cells may be taken from an organism and genetically engineered to express the catalytic antibodies or fragments thereof. The protein may be designed by known methods to remain within the cell, to remain on the cell surface or to be secreted from the cell.

In still further related processes the cloned nucleic acids are introduced into plant cells, via a suitable vector, enabling expression of the antibodies in plants, thereby conferring herbicide resistance on said plants expressing said antibodies.

I. Selection and synthesis of antigens

According to the present invention an antigen is selected from the group consisting of: a) a hapten comprising a stable transition state analog of the desired chemical reaction; or b) the hapten linked to a carrier molecule.

According to the present invention the hapten will be an analog of the presumed transition state structure in the deactivation pathway of a herbicide. These herbicide molecules can be subdivided according to the following families, and depicted by their appended generic formulae:

Herbicide Families

1. Salts of halogenated alkanoic acids

examples:

CICH₂CO₂Na CI₃CCO₂Na CBL_jCCL_jCO^

Sodium Chloroacetate TCA Dalapon-sodium

general formula I:

R X ,-, , CCO ^■_ n (3-n) 2

R = FL Alkyl, Aryl X = Halide M = Metal

2. (Arvioxyialkanoic acids and precursors

examples :

MCPA 2,4-D 2,4,5-T

Dichlorprop ecoprop

Triclopyr MCPB 2,4-DB general formula II:

R, R' = H, alkyl, aryl, heteroalkyl Ar = substituted aromatic or heteroaromatic groups, a chiral center may exist along the chain

3. Arylcarboxylic acids and their derivatives examples :

Chloramben Dicamba 2,3,6-TBA

Clopyralid Picloram Chlorthal-dimethyl

general formula HI:

Ar— COOR

Ar = substituted aromatic or heteroaromatic groups R = H, alkyl, aryl 4. Esters of aryloxyphenoxyalkanoic acids

examples:

Fenoxaprop-ethyl

Fluazifop-butyl

Quizalofop-ethyl

general formula IV:

Ar = substituted aromatic or heteroaromatic groups R, R', R" = H, alkyl, aryl, or various functional groups.

5. Nitriles (and precursors)

examples :

Dichlobenil

general formula V:

R

I Ar

R = Nitrile or a nitrile precursor

Ar = substituted aromatic or heteroaromatic groups

6. Amides examples:

Diphenamid Isoxaben

Tebutam general formula VI:

Ri, R₂, R₃ = H, alkyl, aryl, substituted aromatic or heteroaromatic groups.

7. Anilides (2-chloroacetanilides

examples :

Acetochlor Alachlor Butachlor

ffl^o

Metazacnlor Metolachlor Pretilachlor

O

^{^}CHCOΞL^

Propachlor

general formula VII:

Ri = alkyl, aryl, various functional groups... R₂ = alkyl, aryl, various functional groups... R₃ = substituted aromatic or heteroaromatic groups.

8. Anilides (esters of N-arylalanines

examples :

Beπzoylprop-ethyl Flamprop-methyl Flamprop-isopropyl general formula VΗI:

Ar_l5 Ar₂ = substituted aromatic or heteroaromatic groups. R = alkyls, esters, aryls

9. Anilides (miscellaneous)

examples :

Diflufenican Mefluidide Naptalam

Pentanochlor Perfluidone

Propanil general formula DC:

Ri = H, alkyl, substituted alkyl

R₂ = alkyl, substituted alkyl

Ar = substituted aromatic or heteroaromatic groups. 10. Phenols

examples:

Dinoseb DNOC Pentachlorophenol

general formula X:

Ar— OH

Ar = substituted aromatic or heteroaromatic groups.

11. Diphenyl ethers

examples :

Acifluorfen

Fluorodifen

Oxyfluorfen

general formula XT.

Ar O-Ar₂

Ari = substituted aromatic or heteroaromatic group Ar₂ = substituted aromatic or heteroaromatic group (usually a nitro group at the para position)

12. Nitroanilines examples:

Butralin Ethalfluralin

Nitralin Oryzalin

Pendimethalin Trifluralin

general formula XII:

R

Ar-N

Ri, R₂ = alkyls, aryls

Ar = substituted aromatic or heteroaromatic group (usually substituted with two nitro groups at the ortho positions) 13. Carbamates

examples :

>- HC0₂CH(CH₃)₂ <v Λ— NHC0₂CH(CH₃)₂

Cl Chlorpropham Propham

Asulam

general formula XTH:

R = H, alkyl, aryl Ri, R₂ = aliphatic, aromatic, substituted aromatic or heteroaromatic group

(may be substituted with carbamate group thus creating a biscarbamate)

14. Thiocarbarnates examples :

Thiobencarb Di-allate (E,Z)

[

(c^^CHi^cscx^c^cα_j

Tri-allate

general formula TV:

Ri, R₂, = H, substituted alkyls or aryls R₃ = substituted alkyls or aryls

15. Ureas

examples:

Chloroxuron

Cl — V-NHCNOCH₃

Isoproturon α Linuron

Methabenzthiazuron,

Monolinuron Monuron

Tebuthiuron

general formula XV:

Ar = substituted aromatic or heteroaromatic group R_1-3 = H, alkyls, substituted alkyls, aryls 16. Sulphonylureas examples:

Chlorsulfuron DPX L5300

Sulfometuron-methyl

Triasulfuron

general formula XVI:

Ri_-3 = H, alkyl, aryl, substituted aromatic and heteroaromatic group Ar = substituted aromatic and heteroaromatic group

17. Imidazolinones

examples :

Imazamethabenz-methyl Imazamethabenz-methyl (m-isomer) (p-isomer)

Imazapyr-isopropylammonium Imazaquin

Imazethapyr general formula XVLI:

Rι-3 = H, alkyl, aryl ...

Ar = substituted aromatic or heteroaromatic group

18. Pyrimidines examples

Bromacil Terbacil

general formula XVTLI:

Ri, R = H, halide, alkyl, aryl, substituted aromatic or heteroaromatic group R₃, R₄ = H, alkyl, aryl, substituted aromatic or heteroaromatic group

19. Pyridazines

examples :

Chloridazon

Norflurazon

Pyridate general formula XLX:

Rn = Aryls, alkyls, halides, various functional groups R' = H, alkyls, aryls

20. 1. 3. 5-triazines

examples:

Atrazine Cyanazine Simazine

Terbuthylazine Ametryn Aziprotryne

Desmetryn Prometryn Terbutryn

general formula XX:

Ri, R2, R₃ = aryls, alkyls, various functional groups

21. Triazinones

examples :

Hexazinone Metamitron Metribuzin

general formula XXI:

Rn = H, alkyl, aryls, various functional groups

22. Bipyridinium compounds

examples : ^lN* ⁺N' 2Br- CH. _ -NN++ vs — // +N- ^•CH₃ 2Cl^*

Diquat dibromide Paraquat dichloride

general formula XXH:

Rι,₂ = alkyl, aryl, substituted aromatic or heteroaromatic group X = halide ion or any other appropriate counter ions

23. Miscellaneous organic compounds

Amitrole Benazolin

Difenzoquat methyl Ethofumesate

Fluridone Methazole

Oxadiazon

24. O dmes

Alloxydim-sodium Sethoxydim-sodium

general formula XXTJT.

Rl-3 = ^: alkyl, aryl, substituted aromatic and heteroaromatic group

25. Organophosphi arus compounds

Bensulide Butamifos

NH l.₂CO

Fosamine (ammonium) Glufosinate (ammonium) Glyphosate

general formula XXTV:

= H, alkyl, substituted aromatic or heteroaromatic group

26. Organoarsenic compounds

examples :

Dimethylarsinic acid Methylarsonic acid

Sodium hydrogen methylarsonate Disodium methyl arsonat

general formula XXV:

Ri = alkyls, aryls, various functional groups. R₂, R₃ = H, metals, alkyls

27. Fumigants examples :

Chloropicrin Dazomet Methyl bromide

CBL_jNCS CB^NHCSNa

Methyl isothiocyanate Metham-sodium

general description : volatile, toxic chemicals

Transition state analogs are designed to mimic the presumed transition structures of the reaction to be catalyzed. Examples of such transition state analogs are well known in the art, and have been reviewed extensively (see for example Lerner et al. Science 252, 659- 667, 1191).

By way of example, according to a specific embodiment of the present invention, a stable transition state analog, II, of a herbicide from the family of carbamate herbicides (family 13 in the above list) is synthesized according to scheme 3:

Scheme 3

wherein:

Ri : halide, alkyl, aryl, heteroatom functional groups

R₂ : Alkyl, aryl, easily removed protecting groups

R₃ : H, alkyl, aryl, various functional groups

R__t : Alkyl, aryl, easily removed protecting groups

R₅ : Alkyl, aryl

R_<5 : alkyl, aryl, substituted aromatic or heteroaromatic groups - carrying at least two hydroxy/amino groups. a) Esterification of the acid with a good leaving group such as a benzylic group.

For example using aryl benzoic acid, K₂C0₃, PolyEthylene Glycol 1000, acetonitrile, reflux. According to procedures in literature (Synth. Comm. 18, 1167, 1988). b) Attachment of an alkyl, aryl or various functional groups to the amine moiety, for example using glacial acetic acid, NaBFL according to procedures in literature (JACS 96, 7812, 1974).

c-g) THF, pyridine, O°C.

π. Generation of monoclonal catalytic antibodies

Methods for the generation and selection of monoclonal antibodies are well known in the art, as summarized for example in reviews such as Tramontano and Schloeder, Methods in

Enzymology 178, 551-568, 1989. The haptens of the present invention may be used to generate antibodies in vitro. More preferably, the haptens will be used to elicit antibodies in vivo, by coupling the hapten to a suitable carrier to produce an immunogen.

In general, a suitable host animal is immunized with the immunogen of choice.

Advantageously, the animal host used will be a mouse of an inbred strain. According to a preferred embodiment of the present invention the animal(s) will be immunized with an immunogen which comprises one or more molecules of the hapten of interest covalently linked to a suitable carrier. The carrier of choice may be selected from the group of proteins, natural or synthetic peptides or polypeptides, or any other suitable carrier moiety.

Well known examples of such carriers include but are not limited to proteins such as bovine serum albumin, human serum albumin, keyhole limpet hemocyanin, linear or branched copolymers of amino acids, and the like.

Animals are typically immunized with a mixture comprising a solution of the immunogen in a physiologically acceptable vehicle, and any suitable adjuvant, which achieves an enhanced immune response to the immunogen. By way of example, the primary immunization conveniently may be accomplished with a mixture of a solution of the immunogen and Freund's complete adjuvant, said mixture being prepared in the form of a water in oil emulsion. Typically the immunization may be administered to the animals intramuscularly, intradermally, subcutaneously, intraperitoneally , into the footpads, or by any appropriate route of administration.

The immunization schedule of the immunogen may be adapted as required, but customarily involves several subsequent or secondary immunizations using a milder adjuvant such as Freund's incomplete adjuvant.

Antibody titers and specificity of binding to the hapten can be determined during the immunization schedule by any convenient method including by way of example radioimmunoassay, or enzyme linked immunoassay. Antibody activity assays can be based on detection of the reaction product of the catalytic inactivation of the herbicide, assays based on the disappearance of the intact herbicide, and in terms of catalytic efficiency in the desired deactivation reaction such as hydrolysis of the herbicide.

Antibody activity assays based on the detection of reaction products or on the disappearance of substrate may conveniently be followed by spectrophotometric.means , by high pressure liquid chromatography (HPLC) or by means of any other suitable detection system.

When suitable antibody titers are achieved, antibody producing lymphocytes from the immunized animals are obtained, and these are cultured, selected and cloned, as is known in the art. Typically, lymphocytes may be obtained in large numbers from the spleens of immunized animals, but they may also be retrieved from the circulation, the lymph nodes or other lymphoid organs. Lymphocytes are then fused with any suitable myeloma cell line, to yield hybridomas, as is well known in the art.

Alternatively, lymphocytes may also be stimulated to grow in culture, and may be immortalized by methods known in the art including the exposure of these lymphocytes to a virus, a chemical or a nucleic acid such as an oncogene, according to established protocols.

After fusion, the hybridomas are cultured under suitable culture conditions, for example in multiwell plates, and the culture supernatants are screened to identify cultures containing antibodies that recognize the hapten of choice. Hybridomas that secrete antibodies that recognize the hapten of choice are cloned by limiting dilution and expanded, under appropriate culture conditions. Monoclonal antibodies are purified and characterized in terms of immunoglobulin type, binding affinity and in terms of efficiency of catalysis of the hydrolysis reaction of the herbicide.

HI. Cloning of nucleic acids for catalytic antibodies

Cloning of cDNA encoding catalytic antibodies or fragments thereof may be accomplished by several approaches known in the art. In the preferred approach, mRNA from clonal hybridoma cell lines which produce catalytic antibodies is employed as starting material. - The cells are harvested and mRNA is extracted by standard methods known in the art. The cDNA is prepared by reverse transcription of the mRNA by standard methods known in the art. The cDNA for each chain of the immunoglobulin is cloned separately, and may be amplified by polymerase chain reaction using appropriate primers. The cDNA is then ligated into appropriate vectors by standard methods. The cDNA may be cloned into expression vectors and expressed separately in any convenient expression system, so that the properties of the expressed single chains of the antibodies may be determined. Alternatively, the individual chains may be expressed in the same cells which are then screened for the production of recombinant active antibodies. The method of using the invention will be modified in accordance with the system that is selected according to the current principles that are known in the art of recombinant protein production.

According to the present invention the genetic information for the production of the catalytic antibody of interest is introduced into the host plant cells by an appropriate plant cell vector as is known in the art. The present invention provides information that will enable the skilled artisan to prepare constructs of genetic material comprising an open reading frame that encodes at least one chain of a novel catalytic antibody capable of inactivating the herbicide of choice.

The most commonly used method for DNA mediated transformation of plants is to employ Agrobacterium tumefaciens as the delivery vehicle for introduction of recombinant vectors to the plant cell nucleus(Bevan and Chilton, Ann. Rev. Genetics 16, 357-384, 1982). Plant expression vectors are generally large and contain only one promoter and one polylinker region, and therefore it is useful to express only one immunoglobulin in each vector and to transform plants separately with individual heavy and light chain expressing vectors. Plants producing functional antibodies are produced in the progeny of a cross pollination between the individual heavy of light chain containing plants. In certain embodiments it will suffice to produce an active fragment of the catalytic activity, for instance a Fab fragment of the intact antibody or even an Fv fragment thereof. In addition to the nucleic acid encoding the protein or polypeptide of choice, the constructs of the invention may comprise the following elements: a selectable marker, an origin of replication, a transcriptional promoter, a translation start site, a signal sequence for secretion of the product. The novelty of the invention resides in the DNA which encodes at least part of one chain of the catalytic antibody that inactivates a herbicide, optionally in any combination with the elements recited above. The present disclosure also teaches various means to exploit the commercial potential of these catalytic antibodies by conferring herbicide resistance on the host plant. Vector of choice:

In a preferred embodiment according to the present invention, the vector that is used to introduce the encoded protein into the host cells of the plant will comprise an appropriate selectable marker. In a more preferred embodiment according to the present invention the vector is a plant expression vector comprising both a selectable marker and an origin of replication. In another most preferred embodiment according to the present invention the vector will be a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for propagation or integration in the genome of the organism of choice. In yet another embodiment, the construct comprising the promoter of choice, and the gene of interest is placed in a viral vector which is used to infect the cells. This virus may be integrated in the genome of the organism of choice or may remain non-integrated.

Transcriptional promoter of choice:

While the DNA encoding at least part of one chain of the catalytic antibody is an essential element of the invention, it is modular and can be used in different contexts. The promoter of choice that is used in conjunction with this invention is of secondary importance, and will comprise any suitable promoter. It will be appreciated by one skilled in the art, however, that it is necessary to make sure that the transcription start site(s) will be located upstream of open reading frame. In a preferred embodiment of the present invention, the promoter that is selected will comprise an element that is active in the particular host plant cells of interest.

These elements may be selected from transcriptional regulators that activate the transcription of genes essential for the survival of these cells in conditions of stress or starvation, including the heat shock proteins. Promoters containing this type of sequence may advantageously be used according to the present invention. Most preferably, the transcriptional promoter will be selected from those that are active in growing plant cells. Translational start site:

DNA sequences encoding the translational start site (ATG) of the gene to be expressed, will be placed downstream of the transcription start site(s). Any equivalent functional element selected from similar elements in this or other organisms may be used as appropriate in the organism of choice. Equivalent functional elements will include elements with synthetic bases, or elements found in other genes of plants as well as elements found in genes of other unicellular or multicellular organisms. Signal sequence: According to one embodiment of the present invention secretion of the protein out of the cell is preferred. In this embodiment the construct will comprise a signal sequence to effect secretion as is known in the art. A signal sequence that is recognized in the active growth phase will be most preferred. As will be recognized by the skilled artisan, the appropriate signal sequence should be placed immediately downstream of the translational start site (ATG), and in frame with the coding sequence of the gene to be expressed. Introduction of the construct into the microorganism or cell line of choice: Introduction of the construct into the cells is accomplished by any conventional method for transfection, infection or the like as is known in the art. In constructs comprising a selectable marker the cells may be selected for those bearing functional copies of the construct. If the plasmid comprising the gene of interest is episomal the appropriate selective conditions will be used during growth. Stable transfectants and stable cell lines may be derived from the transfected cells in appropriate cases, in order to conveniently maintain the genotype of interest. Cell growth is accomplished in accordance with the cell type, using any standard growth conditions as may be suitable to support the growth of the specific cell line.

IV. Catalytic antibody expression in transgenic plant cells

The production of monoclonal antibodies in plants may be accomplished by plant transformation and regeneration, as is well known in the art.

Alternatively, vectors encoding immunoglobulin heavy and light chains can be introduced into plant cell protoplasts by methods known in the art such as by means of eletroporation, or by using polyethylene glycol as a facilitator. Transient expression will result in the synthesis, assembly and secretion of functional antibodies, This system is more rapid than plant transformation and regeneration and may advantageously be used to optimize the parameters of the vector constructs. Thus, this system will be useful to optimize the type and arrangement of promoters

Analysis of regenerated plants expressing heavy and light chain constructs have shown that the native signal sequence on the transcript contributes significantly to the accumulation of the heavy or light chains while expression of individual chains was barely detectable in constructs without signal sequences (Hiatt et al., Nature 342, 76-78, 1989). The characterization of antibodies from plants has previously described antibodies which have been targeted for secretion through the plasma membrane. Alternative techniques for the efficient production of intracellular antibodies might include the single chain antibody construct (Chaudhary et al., Proc. Natl. Acad. Sci. USA 87, 1066-1069, 1990) in which endomembrane associated assembly of heavy and light chains is replaced by refolding of variable regions joined by a peptide linker.

EXAMPLES

In one specific embodiment of the present invention a stable transition state analog of the herbicide phenmedipham, 2, is synthesized according to the following scheme:

Scheme 4 a) Benzyl bromide, K₂CO₃, PolyEthylene Glycol 1000, acetonitrile, reflux , 24 hr. According to procedures in literature (Synth. Comm. 18, 1167, 1988). b) Glacial acetic acid, NaBE , 14 hr.

According to procedures in literature (JACS 96, 7812, 1974).

c-g) THF, pyridine, O°C, 24 hr. These reactions are known in the literature.

The skilled artisan will appreciate that many modifications and variations are possible within the scope of the dislosed invention, which is not intended to be defined by the limited specific embodiments and examples disclosed herein but rather by the scope of the claims that follow.

Claims

1. A catalytic antibody that is capable of catalyzing the inactivation of a herbicide, comprising an immunoglobulin that binds a stable transition state analog of a deactivation reaction of said herbicide.

2. The catalytic antibody of claim 1 wherein the herbicide is selected from the group of salts of halogenated alkanoic acids, aryloxy alkanoic acids, precursors of aryloxy alkanoic acids, arylcarboxylic acids, substituted arylcarboxylic acids, esters of aryloxyphenoxyalkanoic acids, substituted aromatic nitriles, substituted aromatic nitrile precursors, substituted heteroaromatic nitriles, substituted heteroaromatic nitrile precursors; alkyl derivatives of amides, aryl derivatives of amides, aromatic derivatives of amides, heteroaromatic derivatives of amides, 2-chloroacetanilides, esters of N- arylalanines, anilides, substituted phenols, diphenyl ethers, nitroanilines, carbamates, thiocarbamates, substituted aromatic ureas, substituted heteroaromatic ureas, sulphonylureas, imidazolinones, pyrimidines, pyridazines, 1,3,5-triazines; triazinones, bipyridinium compounds, oximes, organophosphorous compounds, organoarsenic compounds, and organic fumigants.

3. The catalytic antibody of claim 2 wherein the herbicide comprises at least one carbamate functional group.

4. The catalytic antibody of claim 3 wherein the herbicide comprises a compound of the general formula XTJI:

Wherein R is selected from the group of H, alkyl, or aryl ; and a) Ri, R₂ are independently selected from the group of aliphatic, aromatic, substituted aromatic or heteroaromatic groups; or b) Ri, R₂ are independently selected from the group of aliphatic, aromatic, substituted aromatic or heteroaromatic groups, wherein one of the substituents is a second carbamate group.

5. The catalytic antibody of claim 3 wherein the herbicide is selected from the group consisting of chlorbufam, chlorpropham, propham, carbetamide, desmedipham, asulam, phenisopham and phenmedipham.

6. The catalytic antibody of claim 1 wherein the stable transition state analog is a phosphora idate derivative of an intermediate in the deactivation of the herbicide.

7. The catalytic antibody of claim 3 wherein the stable transition state analog comprises a phosphoramidate group in place of the carbamate functional group.

8. A synthetic hapten used to generate antibodies ^"capable of catalyzing the deactivation of a herbicide comprising a stable transition state analog of a deactivation reaction of said herbicide.

9. A synthetic antigen used to generate antibodies capable of catalyzing the hydrolysis of a herbicide, comprising a synthetic hapten comprising a stable transition state analog of the deactivation of said herbicide.

10. The synthetic antigen of claim 9 wherein the synthetic hapten comprising a stable transition state analog is covalently bound to a carrier molecule.

11. The synthetic antigen of claim 9 wherein the hapten comprises a phosphoramidate derivative as a stable analog of a transition state of the deactivation of a herbicide with at least one carbamate functional group.

12. The synthetic antigen of claim 11 wherein the hapten analog comprises at least one phosphoramidate derivative as a stable transition state analog of a deactivation reaction of a herbicide selected from the group consisting of chlorbufam, chlorpropham, propham, carbetamide, desmedipham, asulam, phenisopham and phenmedipham.

13. The synthetic antigen of claim 10 wherein the hapten analog comprises a phosphoramidate derivative as a stable analog of a transition state of a deactivation reaction of a herbicide with at least one carbamate functional group.

14. The synthetic antigen of claim 13 wherein the hapten analog comprises a phosphoramidate derivative having the general formula:

wherein

Ri is selected from a halide, heteroatom functional group, alkyl, aryl R₃ is selected from H, alkyl, aryl, various functional groups R5 is selected from alkyl, aryl

R$ is selected from alkyl, aryl, substituted aromatic or heteroaromatic groups substituted with at least two hydroxy or amino groups.

15. A component part of an antibody which is capable of catalyzing the deactivation of a herbicide, said component part selected from the group consisting of an F(ab')₂ fragment, an Fab fragment, an Fv fragment, a heavy chain, a light chain, an unassociated mixture of a heavy chain and a light chain, a heterodimer consisting of a heavy chain and a light chain, a catalytic domain of a heavy chain, a catalytic domain of a light chain, a variable fragment of a light chain, a variable fragment of a heavy chain, and a singlechain variant of said antibody.

16. A recombinant nucleic acid molecule comprising a nucleic acid sequence coding for an antibody according to claim 1.

17. A recombinant nucleic acid molecule comprising a nucleic acid sequence coding for a component part of an antibody according to claim 15.

18. The recombinant nucleic acid molecule of claim 16 or claim 17 wherein the transcription of the antibody coding sequence is under the control of a transcriptional promoter effective in a suitable host cell.

19. The recombinant nucleic acid molecule of claim 18 that further comprises a nucleic acid sequence encoding a signal peptide suitable to promote secretion of the translated antibody.

20. The recombinant nucleic acid molecule of claim 18 that further comprises a selectable marker suitable for selecting host cells expressing the marker.

21. The recombinant nucleic acid molecule of claim 18 that is selected from the group consisting of a deoxyribonucleic acid molecule and a ribonucleic acid molecule.

22. The recombinant nucleic acid molecule of claim 18 that is selected from the group consisting of a naked nucleic acid molecule, a phage vector, a plasmid vector, a cosmid vector, a shuttle vector, a viral vector and a yeast artificial chromosome.

23. The recombinant nucleic acid molecule of claim 18 wherein the antibody is expressed in a host cell selected from the group consisting of a eukaryotic host cell and a prokaryotic host cell.

24. The recombinant nucleic acid molecule of claim 23 wherein the eukaryotic host cell is selected from the group consisting of an animal cell, a plant cell, a fungal cell and a yeast cell.

25. The recombinant nucleic acid molecule of claim 24 wherein the eukaryotic host cell is a plant cell.

26. Recombinant cells transfected with the recombinant nucleic acid of claim 18.

27. Recombinant cells expressing the recombinant nucleic acid of claim 18

28. Recombinant plants expressing the recombinant nucleic acid of claim 18.

29. Recombinant cells producing a catalytic antibody according to claim 1.

30. Recombinant cells producing a component part of a catalytic antibody according to claim 15.

31. Recombinant plants expressing a catalytic antibody according to claiml.

32. Recombinant plants expressing a catalytic antibody according to claiml 5.

33. A method of generating a catalytic antibody capable of catalyzing the deactivation of a herbicide comprising: synthesizing a hapten which is a stable transition state analog of a deactivation reaction of said herbicide; and eliciting antibodies to an antigen comprising said hapten.

34. A method of generating a monoclonal antibody capable of catalyzing the deactivation of a herbicide comprising: synthesizing a hapten which is a stable transition state analog of a deactivation reaction of said herbicide; immunizing an animal with an antigen comprising said hapten; isolating immunocytes from said immunized animal; immortalizing said immunocytes by means of a virus, a mutagen or fusion with a myeloma cell line; selecting cells secreting the catalytic antibody; and cloning and propagating a line of cells secreting said catalytic antibody.

35. A method of conferring herbicide resistance on a cell, comprising expressing in said cell a recombinant nucleic acid according to claim 18.

36. A method of conferring herbicide resitance on a plant, comprising expressing in the plant a recombinant nucleic acid according to claim 18.