AU8389698A - Antibodies and scfv immunotoxins specific to imported fire ants, and their application - Google Patents

Description

WO 99/02991 PCT/US98/14216 5 ANTIBODIES AND SCFV IMMUNOTOXINS SPECIFIC TO IMPORTED FIRE ANTS, AND THEIR APPLICATION 10 15 20 BACKGROUND OF THE INVENTION 25 Field of the Invention The present invention relates generally to immunology and genetic engineering technology. Specifically, the present invention relates to immunological engineering to produce novel 1 WO 99/02991 PCT/US98/14216 reagents that target poisons to cell surface molecules on the cells of microvilli in the midgut of imported fire ant queens. Description of the Related Art 5 Imported fire ants are an ecological and financial disaster in Texas as well as other states in the Southern United States. Imported fire ants were accidentally introduced into the U.S. in the 1930s. These pests completely upset and destroy natural ecosystems, and have detrimental economic effects in 10 agriculture (large mounds damage machinery), ranching (loss of newborn livestock), and recreation and tourism (loss of game birds and rendering park and resort areas uncomfortable at best). A specific problem of fire ant control is how one should control or eliminate imported insect species without 15 destroying native insect species. This problem pertains to numerous non-native animal species that have been introduced in all parts of the United States. Imported species often have a competitive advantage over native species since, in many cases, they have developed enhanced reproductive strategies and do not 20 have natural predators in their new environment (1). Thus, it is important to eliminate the foreign species. On the other hand, the native species should not be eliminated, as the proper balance of a particular ecosystem includes the presence of that native species. Presently, the art includes the various methods of fire 25 ant control. Chemical poisons, such as AMDRO are well known in the art and are used frequently. Such poisons, however, may pollute the environment, and indiscriminately eliminate native species as well as foreign species. 2 WO 99/02991 PCT/US98/14216 Thus, the prior art is deficient in an imported fire ant eradication product which is environmentally sound, specifically targeting and eliminating only imported fire ants. The present invention fulfills this long-standing need and desire in the art. 5 SUMMARY OF THE INVENTION The present invention is drawn to a safe, cost 10 effective, environmentally-friendly and ecologically-sound bioengineered product for managing imported fire ants, and a method of making this product. Immunological and genetic engineering techniques are used to generate monoclonal antibodies (mAbs) and resulting Fab fragments as well as viruses 15 (phage) that display antibody fragments, called single heavy or light chain V-gene fragments (scFv) or scFv heavy and light chain fragments (Fab), which exhibit high-avidity specific binding to cells of the microvilli of the midgut of imported fire ant queens. The specific monoclonal antibodies and phage displayed antibody 20 scFv and Fab fragments are conjugated to toxins (gelonin, bacterial endotoxins, or other toxins) or cDNA's coding for pro apoptotic inducers, cell cycle blockers, cell proliferation inhibitors, differentiation inducers are ligated to scFvFab fragments for targeted delivery and destruction of imported fire ant queens, but 25 not native species, thereby restoring the natural ecosystem. Furthermore, bispecific Fab's or scFv,with one arm of the Fab exhibiting specificity to the targeted cell membrane extracellular domain, and the other arm of the Fab exhibiting specificity to 3 WO 99/02991 PCT/US98/14216 gelonin, bacterial endotoxin or other toxins provides yet another novel method for specific targeted delivery of toxins. DNA sequence coding regions of the enzymatically active domain of gelonin, bacterial endotoxin or other toxins inserted into DNA 5 coding specific scFv heavy or light chain Ig fragments or Fab Ig fragments provides another method of targeted delivery of toxins. One object of the present invention is to provide a specific method for management and control of insect pests that is environmentally friendly and does not harm native animal 10 species. In an embodiment of the present invention, there is provided compositions which specifically deliver pro-apoptotic, cell cycle inhibitors to target cells. In yet another aspect of the present invention, there is 15 provided a composition to deliver toxins and cell growth inhibitors to target cells. In yet another aspect of the present invention, there is provided a method for producing reagents that direct poisons to target cells but not to non-target cells, comprising: immunizing an 20 animal with said target cells to produce monoclonal antibodies; harvesting, enriched spleen cells; hybridizing said spleen cells to a myeloma cell fusion partner using polyethylene glycol; selecting of hybridoma cells by growing on HAT media; screening hybridoma supernatants for production of murine antibodies using ELISA 25 technology and for antibody specificity to midgut microvilli antigens using immunohistological techniques; cloning by limiting dilution, screening hybridoma supernatants, expansion of clones and freezing of positive clones; and obtaining a stable, monoclonal 4 WO 99/02991 PCT/US98/14216 antibody producing hybridomas, produce supernatants or ascites fluid or prepare purified monoclonal antibody. In yet another aspect of the present invention, there is provided a method of killing a fire ant, comprising the step of 5 contacting said ant with the polypeptide produced by the method disclosed herein. Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments. These 10 embodiments are given for the purpose of disclosure. BRIEF DESCRIPTION OF THE DRAWINGS 15 So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof 20 which are illustrated in the appended drawings. these drawings form a part of the specification. It is to be noted however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope. 25 Figure 1 depicts schematically the methods of the present invention, including immune priming; cDNA preparation; creation of a phage library; phage selection; verification of specific scFvs and Fabs; and testing thereof. 5 WO 99/02991 PCT/US98/14216 DETAILED DESCRIPTION OF THE INVENTION 5 As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody comprised of heavy and light polypeptide chains with specificity to target cells and is generated and selected from a cloned antibody producing cell. As used herein, the term "antibody fragment" or "Fab" 10 refers to immunoglobulin based recognition units of minimum size. V-gene segments from immunoglobulin heavy and light chains that exhibit high affinity to target antigens. As used herein, the "scFv" fragment refers to immunoglobulin based recognition unit of minimum size, a single 15 heavy or light or combined heavy and light chain V-gene Ig fragment with high affinity to target cell. As used herein, the term "bispecific antibody" refers to either chemically derived or DNA technology derived Fab or scFv immunoglobulin fragments with specificity to two different 20 antigenic determinants, i.e., one arm of the Ig specificity unit reacting with targeted antigen and the other arm reacting specifically with toxins such as gelonin or bacterial endotoxins. As used herein the term "toxin" refers to any chemical that behaves in a toxic manner in that it kills cells when 25 incorporated into target cells, by being delivered by three distinct mechanisms: chemically linked to targeted Ig fragment, bispecific Fab technology, or by DNA technology providing scFv heavy chain toxin cytotoxic domain. A representative toxin is gelonin, a well 6 WO 99/02991 PCT/US98/14216 known ribosome inactivating protein or recombinant forms thereof. As used herein, the term "phage display library" refers to repertoire of up to 2x108 independent clones of 5 immunoglobulin Fab or scFv fragments. As used herein, the "pro-apoptotic", "cell cycle blockers", "cell proliferation inhibitors" and "cell proliferation agents" refer to cDNA from genes that control cell proliferation cell cycle, cell differentiation, and cell death that are ligated to 10 monoclonal Fab fragments or scFv heavy and/or light Ig fragments for specific delivery to target cell. As used herein, the term "phage displayed Fab" and "phage displayed scFv" refers to a repertoire of Fab or scFv heavy and/or light chain Ig fragments that are displayed on phage and 15 selected through antigen binding to target cells. In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, 20 Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual (1982); "DNA Cloning: A Practical Approach," Volumes I and II (D.N. Glover ed. 1985); "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid Hybridization" (B.D. Hames & S.J. Higgins eds. (1985)); "Transcription and Translation" (B.D. Hames & S.J. Higgins 25 eds. (1984)); "Animal Cell Culture" (R.I. Freshney, ed. (1986)); "Immobilized Cells And Enzymes" (IRL Press, (1986)); B. Perbal, "A Practical Guide To Molecular Cloning" (1984). 7 WO 99/02991 PCT/US98/14216 A "vector" is a replicon, such as a plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. A vector is said to be "pharmacologically acceptable" if its administration can 5 be tolerated by a recipient mammal. Such as agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a change in the physiology of a recipient mammal. For example, in the treatment 10 of retroviral infection, a compound which decreases the extent of infection or of physiologic damage due to infection, would be considered therapeutically effective. A cell has been "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the 15 cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells, for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in 20 which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the 25 transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations. 8 WO 99/02991 PCT/US98/14216 Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell. 5 A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. 10 Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., 15 the strand having a sequence homologous to the mRNA). The present invention is directed to a fire ant eradication product which is environmentally sound, and specifically targets and eliminates only imported fire ants. It is contemplated additionally that the method of the present 20 invention can be used to specifically target any animal pest. Production and screening of monoclonal antibodies with high avidity to specific antigenic epitopes is a well established and standard laboratory procedure. DNA technology well known to those having ordinary skill in this art permits the 25 introduction of DNA coding for small immunoglobulin recognition units (called antibody fragments i.e., N-terminal variable domains of heavy and light immunoglobulin chains that exhibit the same antigenic specificity as the intact larger parent antibody) into 9 WO 99/02991 PCT/US98/14216 virus expression vectors (phage) that produce and display the scFv heavy and light chains and combinations of heavy and light chain Ig fragments on their surface (2-9). This technology has been used to specifically target tumor cells for selected 5 destruction; however, to date, this technology has not been applied to specifically target, for destruction, insect pests or other animal pests. The phage display method represents a major advance over traditional monoclonal antibodies in that large and diverse 10 repertories of scFv heavy or light and combinations of heavy and light chain Ig v-region genes can be generated and expressed on the surface of viruses; thereby permitting rapid screening and selection for high-avidity (tight binding) scFv Ig fragments with targeted specificity. Importantly, once specific phage-displayed 15 scFv Ig fragments have been selected for specificity to an antigenic epitope, the DNA that codes for the specific Fab fragment is available for genetically-engineered enzymatically active domain of gelonin, bacterial endotoxins, or other toxins, programmed cell death (apoptotic) genes as well as genes that 20 disrupt cell proliferation into the Fab DNA. Thus, a targeting system is produced which delivers specific toxins, apoptosis inducing agents or cell proliferation inhibitors, or cell differentiation inducing agents. Producing scFv or Fab Ig fragments with targeted specificity possessing enzymatically 25 active gelonin or bacterial endotoxins or other cell death inducing gene products provides a novel method for targeted delivery of cell death inducing products. 10 WO 99/02991 PCT/US98/14216 The present invention utilizes immunological and genetic engineering techniques to generate monoclonal antibodies and viruses (phage) that display antibody scFv and Fab Ig fragments selected to react specifically with the midgut microvilli 5 cells of imported fire ant queens, but not with microvilli cells of native fire ant queens. These Fab and scFv Ig fragments and specificity are not limited to the microvilli or imported fire ant queens, but encompass any cell, tissue or organ specifically targeted with Fab or scFv Ig fragments of any animal species in 10 which the destruction of such cells or tissues or organs will result in the containment or elimination of the animal species. These monoclonal antibodies and phage displayed scFv fragments are conjugated to a toxin, such as gelonin, a ribosome inactivating protein that has no mechanism for entering cells and is non-toxic, 15 unless specifically delivered inside cells, or other toxins, to deliver toxins to the digestive tract of imported fire ant queens. In yet another aspect of the present invention, there is provided a method of killing a fire ant, comprising the step of contacting said ant with the polypeptide produced by the method 20 disclosed herein. A person having ordinary skill in this art would readily be able to determine the optimum concentration of the novel polypeptides disclosed herein by routine experimentation according to the type and quantity of fire ant to be irradicated. The following examples are given for the purpose of 25 illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion: 11 WO 99/02991 PCT/US98/14216 EXAMPLE 1 Immunization of mice with midgut microvilli cells Balb/c mice are immunized with midgut preparations obtained from egg laying imported fire ant queens. Antigenically 5 complex minced midguts are used for immunizations, and then a dual selection procedure (see Example 4) and an immunohistochemical procedure (see Example 5) is performed for selecting either scFv heavy and/or light chain Ig fragments or monoclonal Fab fragments with specificity to cells of the microvilli 10 of the imported fire ant queens. Spleen cells from immunized mice are used to generate species-specific monoclonal antibodies, using standard cell culture procedures. The monoclonal antibodies have specificity to midgut microvilli antigens of imported fire ants. Resulting hybridoma 15 supernatants are selected for specificity using immunohistochemical procedures as described in Example 5. In addition, species-specific antibody phage displayed scFv heavy and/or light chain Ig fragments are generated which react with the extracellular domain of midgut microvilli cells. A 20 schematic of the step-by-step procedure in the development of phage displayed scFv heavy and/or light chain Ig fragments is presented in Figure 1. EXAMPLE 2 25 Preparation, Amplification and Purification of cDNA from total RNA obtained from immunized mice spleens Total RNA is extracted and purified from the spleens of three immune mice. The RNA is reverse transcribed (RT) into 12 WO 99/02991 PCT/US98/14216 cDNA employing a kit (Boehringer Mannheim) and using two sets of primers--one specific for IgG heavy chains and another specific for kappa light chains (4). cDNAs are amplified by the polymerase chain reaction (PCR) procedure using IgG heavy and kappa light 5 chain specific primers, respectively, and thermal cycling conditions published previously (see 4,5,6). The PCR-amplified products are separated according to size by gel electrophoresis. EXAMPLE 3 10 Construction of a scFv heavy and/or light chain Ig fragment combinatorial library displayed on the surface of filamentous phage Combinatorial scFv heavy and light chain Ig fragment 15 libraries are constructed in a filamentous phage expression vector using a two-step sequential ligation procedure as described (4) and an ImmunoZAP kit from Stratagene. Briefly, the gel-purified PCRproducts are enzymatically cleaved and the PCRproducts are ligated into the heavy chain vector - lHc2, and the light chain 20 vector-lLc. Constructed heavy and light chain libraries consist of scFv fragments. Next, heavy and light chain constructs are combined to yield Fab combinatorial constructs. E. coli cells are then transformed with the phage library and infected with helper phage (VCSM13, Stratagene) to produce sufficient amounts of 25 phage displaying antibody fragments for screening and selection purposes (4). scFv heavy and light as well as heavy and light chain Ig V-region specificity fragments from immunoglobulin heavy and light chain are utilized as either single chain V 13 WO 99/02991 PCT/US98/14216 fragments (scFv) of high binding affinity to target cells or as a combined heavy/light chain V-fragments (Fab) with high binding affinity to target cells. 5 EXAMPLE 4 Two-step selection for identifying antibody scFv or Fab fragments with specificity to midgut membrane antigens from imported fire ant queens 10 The first selection step involves reacting the entire antibody scFv or Fab fragment-expressing phage library with midguts opened to expose microvilli cells or cells dissociated, obtained from imported fire ant queens. Ig displayed phage failing to bind are washed away while phage displaying scFv or 15 Fab Ig fragments that bind to imported fire ant midgut antigens with high affinity are eluted and saved. The second selection is conducted using midgut preparations from native fire ant queens. Only phage-displayed scFv or Fab fragments that fail to react with midgut membrane antigens from native fire ant queens are 20 collected. This selection procedure is repeated three times to provide specific Ig fragments with high avidity and affinity for cloning. The selected phage (those that react with midgut membrane antigens of imported fire ant queens but not native fire ant queens) are genetically engineered so that they no longer 25 are displayed, but are secreted when produced in E. coli. The selection procedure is not limited to the above sequence, in that, Ig displayed phage can be first reacted with microvilli antigens of native fire ant queens, and Ig displayed phage not reacting with 14 WO 99/02991 PCT/US98/14216 the native fire ant queen microvilli antigens can be selected for reaction with microvilli antigens of imported fire ant queens. EXAMPLE 5 5 Verification that selected phage displayed antibody scFv or Fab fragments and monoclonal antibodies react with imported fire ant midgut microvilli cells The selected phage-displayed scFv or Fab fragments 10 (from Example 4, above) react with a variety of midgut cellular membrane antigens, including antigens of the microvilli cells. Immunohistochemical staining of sectioned midgut tissue is used to identify cloned phage-displayed scFv heavy and light chains or combinations of heavy and light chain Ig framments that react 15 with midgut microvilli cells of imported fire ant queens exclusively. Midgut sectioned tissue is reacted with cloned scFv or Fab fractions, and assayed for the presence of the phage using the sensitive immunoperoxidase VECTASTAIN Elite ABC system (Vector Laboratories). Clones that give a positive reaction with 20 microvilli cells of imported fire ant queens and fail to react with native fire ant microvilli cells are amplified in E. coli and used as described in Example 6 and 7. For selection of monoclonal antibodies, midgut tissue sections are reacted with hybridoma supernatants, washed, and reacted with alkaline phosphatase 25 conjugated rabbit secondary antibody to mouse Ig. Substrate is then added and slides are examined for positive reactions to the midgut microvilli antigens of imported fire ant queens but not the midgut microvilli antigens of native fire ant queens. 15 WO 99/02991 PCT/US98/14216 EXAMPLE 6 Verification that the selected phage-displayed antibody fragments, as well as monoclonal antibodies, are effective in vivo 5 Monoclonal antibodies and scFv heavy and/or light chain Ig fragments amplified in E. coli that are selected for specificity and high affinity binding to the microvilli of imported fire ants are tested for ability to be internalized by microvilli cells when administered orally. Ant colonies (imported and native) are 10 established in a controlled envronment with each colony consisting of a single egg laying imported fire ant queen, 50 virgin winged fire ant Queens and approximately 5,000 worker ants. The colonies are fed either monoclonal antibodies or scFv or Fab fragments mixed in soybean meal. The egg laying queen and 15 virgin queens from each colony are sacrificed and the midguts are removed. Frozen tissue sections are made from the midgut, and these sections are analyzed for the presence and internalization of scFv heavy and/or light chain Ig fragments by immunohistochemical staining as described above. Analyses of 20 monoclonal Fab fragments are conducted using this methodology. EXAMPLE 7 Verification that phage-displayed generated scFv Ig fragments 25 and monoclonal Fab fragments are an effective delivery system for targeted destruction of imported fire ants: The ribosome inactivating protein, gelonin, bacterial endotoxins, or other toxins are attached to monoclonal Fab 16 WO 99/02991 PCT/US98/14216 fragments and scFv heavy and/or light chain Ig fragments by the following technologies: by well established chemical conjugation or attachment procedures that have been successful in conjugation of toxins to monoclonal antibody Fab fragments or to scFv heavy 5 chain Ig fragments; by bispecific (Fab)2 or scFv heavy chain heterodimers with specificity to targeted antigens and specificity to toxin generated in vitro by establishing a stable thioether linkage using established procedures (10-12); in vivo by using DNA technology and genetic engineering technologies to generate 10 dimerization peptides to produce bivalent dimers (with specificities to targeted antigen and toxin) in E coli or mammalian cells, using described techniques (13), and by genetically engineering scFv heavy chain-enzymatically active domain of toxin, expressed and secrteted by bacteria using described 15 technology (14). The testing "feeding" of scFv heavy and/or light chain or monoclonal Fab fragments for ability to bind to midgut microvilli antigens of imported fire ant queens but not midgut microvilli antigens of native fire ant queens in controlled 20 established laboratory colonies of imported and native fire ants, utilizing immunohistological techniques to establish attachment and internalization of Ig fragments to midgut microvillli antigens is performed. Testing of targeted scFv heavy and/or light chains and monoclonal Fab fragments + toxin in laboratory established 25 ant colonies as well as in field conditions for ability to selectively kill imported fire ants is performed. The following references were cited herein: 17 WO 99/02991 PCT/US98/14216 1. Holldobler, B. and E. O. Wilson 1990. The Ants. The Belknap Press, Cambridge, Mass. 2. Barbas, C. and R. Lerner. 1991. Combinatorial immunoglobulin libraries on the surface of phage (phabs): rapid selection of 5 antigen-specific. Fab. Methods: Comp. Met. Enzym.2:119. 3. Ames, R., M. Tornetta, C. Jones and P. Tsui. 1994. Isolation of neutralizing anti-C5a antibodies from a filamentous phage monovalent Fab display library. J. Immun. 152:4572. 4. Ames, et al (15 co-authors). 1995. Neutralizing murine 10 monoclonal antibodies to human IL-5 isolated from hybridomas and a filamentous phage Fab display library. J. Immunol. 154:6355. 5. Winter, G., A. D. Griffiths, R. E. Hawkins and H. R. Hoogenboom. 1994. Making antibodies by phage display technology. Ann. Rev. 15 Immunol. 12:433. 6. Vaughan, T. J., et al., 1996. Human antibodies with sub nanomolar affinities isolated from a large non-immunized phage display library. Nature Biotech. 14: 309. 7. Kruif, J. de, et al., 1996. New perspectives on recombinant 20 human antibodies. Immunology Today 17:453. 8. Kruif, J. de, and T. Logtenberg. 1996. Leucine zipper dimerized bivalent and bispecific scFv antibodies from a semi-synthetic antibody phage display library. J. Bio. Chem. 271:7630. 9. Davies, J. and L. Riechmann. 1995. Antibody VH domains as 25 small recognition units. Biotechnology 13:475. 10. French, R, C. Penney, A. Browning, F. Stirpe, A. George and M. Glennie. 1995. Delivery of the ribosome-inactivating protein, 18 WO 99/02991 PCT/US98/14216 gelonin, to lymphoma cells via CD22 and CD38 using bispecific antibodies. Brit. J. Cancer 71:986. 11. Better, M., S. Bernhard, D. Fishwild, P. Nolan, R. Bauer, A. Kung, and S. Carroll. 1994. Gelonin analogs with engineered cysteine 5 residues form antibody immunoconjugates with unique properties. J. Biol. Chem. 269:9644. 12. Glennie, M.J., H. M. McBride, A. T. Worth, and G. T. Stevenson. Preparation and performance of bispecific F(ab') 2 antibody containing thioether-linked Fab'_ fragments. J. Immunol. 10 139:2367-2375, 1987. 13. Holliger, P., and G. Winter. Engineering bispecific antibodies. Current Opinion in Biotechnology 4: 446-449, 1993. 14. Maurer-Gebhard, M., M. Schmidt, M. Azemar, U. Altenschmidt, E. Stocklin, W. Wels, and B. Groner. Systemic treatment with a 15 recombinant erbB-2 receptor-specific tumor toxin efficiently reduces pulmonary metastases in mice injected with genetically modified carcinoma cells. Cancer Res. 58:2661-2666, 1998. Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art 20 to which the invention pertains. Further, these patents and publications are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. One skilled in the art will appreciate readily that the 25 present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods, procedures, treatments, 19 WO 99/02991 PCT/US98/14216 molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which 5 are encompassed within the spirit of the invention as defined by the scope of the claims. 20

Claims (12)

1. A method of producing a reagent with specificity 5 to a target antigen and an active domain of a toxin, comprising the steps of: creating an scFv heavy and/or light chain Ig fragment expressing phage display library; reacting said scFv heavy and/or light chain Ig fragment 10 expressing phage display library with said target cells; washing said reacted target cells to remove unbound scFv Ig fragment-expressing phage; eluting bound scFv Ig fragment-expressing phage from said target cells; 15 reacting said eluted scFv Ig fragment-expressing phage with said non-target cells; washing said reacted non-target cells to remove unbound, eluted scFv Ig fragment-expressing phage; engineering scFv for non-phage dislay; 20 amplifying scFv heavy and/or light chain Ig fragments in E. coli; collecting secreted scFv Ig fragments; attaching said scFv Ig fragments to a toxin to produce scFv heavy chain-enzymatically active domain of toxin thus producing 25 a single polypeptide with specificity to targeted antigen and active domain of toxin. 21 WO 99/02991 PCT/US98/14216

2. The method of claim 1, wherein said attaching is by chemical cross-linking, bispecific scFv heavy and light chain, with specificity to targeted antigen and toxin, or genetic engineering. 5

3. The method of claim 1, wherein said animal is a mouse.

4. The method of claim 1, wherein said target is a 10 cell surface molecule of a target cell.

5. The method of claim 1, wherein said target cell is a cell in the microvilli of the midgut region of an imported fire ant. 15

6. The method of claim 1, wherein said creating step is carried out by ligating cDNA into heavy chain vectors and light chain vectors.

7. A polypeptide produced by the method of claim 20 1.

8. A method for producing monoclonal antibody and Fab fragments with specificity to midgut microvilli antigens of imported fire ant queens, comprising: 25 immunizing an animal with said target cells to produce monoclonal antibodies; harvesting enriched spleen cells; 22 WO 99/02991 PCT/US98/14216 hybridizing said spleen cells to a myeloma cell fusion partner using polyethylene glycol; selecting of hybridoma cells by growing on HAT media; screening hybridoma supernatants for production of 5 murine antibodies using ELISA technology and for antibody specificity to midgut microvilli antigens of imported fire ant queens but not midgut antigens of native fire ant queens using immunohistological techniques; cloning by limiting dilution, screening hybridoma 10 supernatants, expansion of clones and freezing of positive clones; and purifying monoclonal antibody and Fab fragments with specificity to midgut microvilli antigens of imported fire ant queens. 15

9. The method of claim 8, wherein said animal is a mouse.

10. The method of claim 8, wherein said target cell is 20 a cell in the microvilli of the midgut region of an imported fire ant.

11. A monoclonal antibody or Fab fragment produced by the method of claim 8. 25

12. A method of killing a fire ant, comprising the step of contacting said ant with the polypeptide produced by the method of claim 1. 23