JP2001509397A - Antibodies and scFv immunotoxins specific for tofusia and their use - Google Patents

Abstract

  (57) [Summary] The present invention relates to a safe, cost-effective, environmentally harmless and genetically engineered product for controlling tofusia. Kind Code: A1 Monoclonal antibodies (mAbs) and scFv heavy and / or light chains exhibiting high affinity and specific binding to cells of the midgut microvilli of the queen Tofusiali using immunological and genetic processing techniques. Display Ig fragments and generate virus (phage). These specific monoclonal antibodies and phage display antibody Fab fragments are conjugated to toxins to reinstate Queen Tofusiali but not affect native species, restoring the natural ecosystem.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to immunology and genetic engineering. In particular, it relates to immunological engineering to create new drugs that target toxins to cell surface molecules on cells of the midgut microvilli of the queen Tofusiali.

[0002] Imported fire ant causes environmental and economic damage in Texas and other states in the southern United States. Tofsiali was brought to the United States in the 1930s by accident. These pests can upset and destroy natural ecosystems, adversely affecting agriculture (large ant mounds destroy machinery), adversely affecting ranch management (death of newborn livestock), and affecting recreation and tourism. Adverse effects (death of hunted birds and environmental degradation in parks and resort areas).

[0003] One particular problem with controlling fire ants is how to control or control introduced insect species without extinction of native insect species. This problem is related to a number of non-native species introduced into all parts of the United States. Introduced species often have enhanced competitive rehabilitation strategies and, because they do not have natural enemies in their new environment, have competitive advantages over native species (1). Therefore, it is important to control alien species. On the other hand, the proper balance of a particular ecosystem should include the presence of native species, which should not be controlled.

Currently, there are various methods for controlling ants. Chemical poisons such as AMDRO are well known in the prior art and are often used. However, these poisons pollute the environment and endanger native and non-native species as well.

[0005] Therefore, there is no Tofu sari removal product in the prior art that is environmentally safe and exerts an action only on Tofu sari to exterminate it. The present invention fulfills this long-awaited need.

The present invention relates to a safe, cost-effective, environmentally harmless and ecologically sound genetically engineered product for managing tofusia, and a method of making the product. Antibody fragments called single heavy or light chain V gene fragments (scFv) or scFv heavy and light chain fragments (Fab) exhibiting high activity that selectively bind to the cells of the midgut villi of the queen Tofsiali Immunological and genetic engineering techniques are used to generate monoclonal antibodies (mAbs) and consequently Fab fragments as well as viruses (phages). Specific monoclonal and phage display antibodies scFv and Fa
b fragment to a toxin (gelonin, bacterial endotoxin, or other toxin) or cDNA encoding a pro-apoptotic inducer, cell cycle blocker, cell growth inhibitor, differentiation inducer
Targeted transmission by ligating to the ab fragment will regenerate a natural ecosystem by controlling Queen Tofusiali but not native species. In addition, a bispecific Fab ′ in which one arm of the Fab exhibits specificity for the targeted extracellular region of the cell membrane and the other arm of the Fab exhibits specificity for gelonin, microbial endotoxin, or other toxins. The s or scFv also offers another new way to achieve specific targeted toxic transfer. DNA encoding scFv heavy or light chain Ig fragment or Fab Ig fragment specifically
Of the enzymatically active region of gelonin, bacterial endotoxin or other toxins inserted into
The A sequence coding region also provides another method for targeted transmission of the toxin.

One object of the present invention is to provide a specific method for the management and control of pests that are environmentally friendly and do not harm native animal species.

[0008] In one embodiment of the present invention, there is provided a composition that specifically delivers a pro-apoptotic cell cycle inhibitor to a target cell.

[0009] In another aspect of the present invention, there is provided a composition for providing a toxin and a cytostatic to target cells.

[0010] In yet another aspect of the invention, a method of producing a drug that directs a toxin to target cells but not non-target cells comprises immunizing an animal having the target cells to produce a monoclonal antibody. Removing the enriched spleen cells, crossing the spleen cells with a myeloma cell fusion partner using polyethylene glycol, growing on HAT medium to select hybridoma cells, and using ELISA technology. Screening the hybridoma supernatant for its ability to produce mouse antibodies, and for its specificity for midgut microvillous antigen using immunohistological techniques; and
Performing limited dilution, screening the hybridoma supernatant, expanding the clone, and cloning by freezing the positive clone;
A method for obtaining a stable monoclonal antibody for producing a hybridoma and producing a supernatant or ascites fluid or preparing a purified monoclonal antibody is provided.

[0011] In yet another aspect of the present invention, there is provided a method of killing termites comprising contacting the ant with a polypeptide produced by the methods disclosed herein.

[0012] From the following description of the preferred embodiments, further aspects, features,
The benefits will become apparent. These embodiments are provided for the purpose of disclosure.

As used herein, the term “monoclonal antibody” or “mAb” refers to an antibody composed of heavy and light polypeptide chains having specificity for a target cell, and is used for the production of cloned antibodies. Generated and selected from cells.

As used herein, “antibody fragment” or “Fab” refers to an immunoglobulin based on the smallest recognition unit, a V gene segment derived from an immunoglobulin heavy and light chain exhibiting affinity for a target cell. pointing.

As used herein, the term “scFv” fragment refers to an immunoglobulin based on the smallest recognition unit, a heavy or light chain having specificity for a target cell, or a combined heavy and light chain V Gene Ig fragment.

As used herein, the term “bispecific antibody” refers to two different antigenic determinants, one arm of the Ig specificity unit that reacts with the targeted antigen and gelonin or Chemically or Fab-derived Fab or sc with specificity for the other arm that specifically reacts with a toxin, such as a microbial endotoxin
Refers to Fv immunoglobulin fragments.

As used herein, the term “toxin” is used in three different mechanisms: chemically linked to the targeted Ig fragment, by bispecific Fab technology, or by scFv heavy chain. Refers to all chemicals that act in a toxic form in the sense that they kill cells when integrated into target cells by being transmitted by DNA technology that results in an area of toxin cytotoxicity. A representative toxin is gelonin, a known ribosome inactivating protein or a recombinant form thereof.

The “phage display library (phage display library)”
library)] is a maximum of 2 x 1 of immunoglobulin or scFv fragment
Refers to a repertoire of 08 independent clones.

As used herein, “pro-aporttic” and “cell cycle blocker”
The terms "cell growth inhibitor" and "cell growth agent" are used to control cell growth, cell cycle, cell differentiation, and cell death, and to transduce specifically to target cells. Refers to cDNA from the gene ligated to the chain and / or light chain Ig fragments.

As used herein, the terms “phage display Fab” and “phage display scFv” refer to Fab or scFv heavy and / or light chain Igs displayed on phage and selected through antigen binding to target cells. Points to the repertoire of the fragment.

According to the present invention, conventional molecular biology, microbiology, and genetic engineering techniques can be used within the prior art. Such techniques are explained fully in the literature. For example, Maniatis, Fritsch & Sambrook, "Molecular Cloni
ng: A Laboratory Manual (1982); “DNA Cloning: A Practical Approach
, "Volumes I and II (DN Glover eds., 1985);" Oligonucleotide Synthesi
s "(edited by MJ Gait, 1984);" Nucleic Acid Hybridization "(BD Hames &
S. J. Higgins, (1985)); "Transcription and Translation" (B.D.H.)
ames & S. J. Higgins, (1984)); "Animal Cell Culture" (RI Fres)
hney, (1986)); “Immobilized Cells and Enzymes” (IRL Press, (1986)).
B.); See Perbal, "A Practical Guide To Molecular Cloning" (1984).

A “vector” is a replicon, such as a plasmid, phage, or cosmid, to which other DNA segments can be attached, resulting in replication of the attached segment. A vector is said to be "pharmaceutically acceptable" if the recipient animal can tolerate its administration. If the dose is physiologically significant, the drug is said to be administered in a "pharmaceutically effective amount". An agent is physiologically significant if its presence alters the physiology of a recipient animal. For example, in the case of retroviral infection, compounds that reduce the extent of infection or the degree of physiological damage caused by the infection are considered to be pharmaceutically effective.

When such DNA is introduced inside a cell, the cell is said to have been "transformed" by exogenous or heterologous DNA. The transforming DNA may or may not be ligated (covalently linked) into the genome of the cell. For example,
In prokaryotes, yeast, and mammals, the transforming DNA may be retained on episomal elements, such as plasmids. In the case of a eukaryotic cell, stable transformation is one in which the transforming DNA is integrated with the chromosome and becomes inherited by daughter cells through chromosome replication. This stability is indicated by the ability of the eukaryotic cell to establish a cell line or a clone composed of a group of daughter cells containing the transforming DNA. "Clone" refers to a group of cells derived from one cell or a common ancestor by nuclear fission. `` Cell lineage ''
Is a clone of a primary cell that is capable of stable growth in vitro for many generations.

[0024] Transcription and translation control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, and terminators, that provide for the expression of a coding expression sequence in a host cell.

“DNA molecule” refers to a polymeric form of a deoxyribonucleotide (adenine, guanine, thymine, or cytosine) in either a single-stranded or double-stranded helix. The term refers only to the primary and secondary structure of the molecule, but does not limit the particular tertiary structure. Thus, the term specifically includes double-stranded DNA found in linear DNA molecules (eg, restriction fragments), viruses, plasmids, and chromosomes. In the following discussion, the term structure refers to the non-transcribed strand of DNA (
For example, a sequence in the 5'-3 'direction along a strand having a sequence homologous to mRNA) is to be used in accordance with ordinary terminology.

[0026] The present invention relates to a ant-control product that is environmentally safe and specifically targets only Tofu sari to combat it. It is also within the scope of the present invention to use it to specifically target any pest.

The preparation and screening of monoclonal antibodies with high affinity for a particular antigenic epitope is a well-established standard laboratory procedure. Using DNA techniques known to those skilled in the art, small immunoglobulin recognition units (referred to as antibody fragments, eg, the N-terminal regions of heavy and light immunoglobulin chains that exhibit the same antigenic properties as the larger parent antibody intact) ) Is expressed on the surface by scF
v create a combination of heavy and light chains and heavy and light chain Ig fragments;
It can be introduced into a viral expression vector (phage) to be displayed (2-9). Although this technique has been used to selectively destroy tumor cells, to date, this technique has been used to target specifically to destroy pests and other pests. Not.

The phage display method allows the generation and expression of large and diverse scFv heavy or light chains and combinations of heavy and light chain Ig v-region genes on the surface of the virus, thereby targeting High affinity (strong binding) scFv I with specificity
It is superior to conventional monoclonal antibodies in that it allows for quick selection and selection of g fragments. Importantly, once a particular phage-displayed scFv Ig fragment has been selected for its specificity for one antigenic epitope, DNA encoding that particular Fab fragment can be used to target gelonin, bacterial endotoxin, or other toxins. Enzymatically active regions, programmed cell death (suicide) genes, and genes that disrupt cell growth
A can be sent by genetic engineering. Therefore, it is possible to create a targeting system for transmitting a specific toxin, an agent for inducing cell death, or an agent for inhibiting cell proliferation, an agent for inducing cell differentiation, and the like. Creating scFv or Fab Ig fragments or other cell death-inducing gene products that have targeting specificity and process enzymatically active gelonin or bacterial endotoxin can be used for targeted delivery of cell death-inducing products. It offers a new way.

The present invention provides antibodies scFv and Fab Ig fragments selected to specifically react with the midgut microvillous cells of the queen Tofusiali and not with those of the native fire ant queen Immunological and genetic engineering techniques are used to generate monoclonal antibodies and viruses (phages) that display
These Fab and scFv Ig fragments and specificities are not limited to the imported flame queen villi, but all such that destruction of the cells, tissues, or organs would lead to containment or extinction of the animal species. All cells, tissues or organs specifically targeted by the Fab or scFv Ig fragment of the animal species of the above are also included in the scope. These monoclonal antibodies and phage-expressed scFv fragments contain toxins such as gelonin, ribosome-inactivating proteins that do not have a mechanism to enter cells, and are non-toxic unless specifically transmitted into cells, or other toxins. Joined to
Transmits toxins to the digestive glands of Queen Tofsiali ants.

In another aspect of the present invention, there is provided a method for killing ants, comprising the step of contacting the ant with a polypeptide produced by a method disclosed herein. One skilled in the art will readily be able to determine, with routine experimentation, the optimal concentration of the novel polypeptides disclosed herein based on the type and amount of termite to be controlled.

The following examples are given for the purpose of illustrating various embodiments of the present invention and are not intended to limit the invention in any way.

Example 1 Immunization of Mice with Midgut Microvillous Cells Balb / c mice are immunized with a midgut preparation obtained from incubating Queen Tofusiali ants. For the immunization, the antigenically conjugated cut intestine was used, followed by a double selection procedure (see Example 4) and an immunohistological procedure (see Example 5), A scFv heavy chain and / or light chain Ig fragment or a monoclonal antibody Fab fragment having specificity for the cells of the microvilli of the queen ant is selected.

The spleen cells of the immunized mice are used to generate species-specific monoclonal antibodies using standard cell culture techniques. These monoclonal antibodies have specificity for the midgut microvillus antigen of the queen Tofusiali. The resulting hybridoma supernatant is selected for specificity using an immunohistological procedure as described in Example 5.

In addition, species-specific antibody phage display scFv heavy and / or light chain Ig fragments are generated that react with the extracellular region of midgut microvillous cells. A schematic of the step-by-step procedure in the development of phage display scFv heavy and / or light chain Ig fragments is shown in FIG.

[0035] Preparation of cDNA from total RNA obtained from Example 2 spleens of immunized mice, amplified, extracted 及 beauty purified total RNA from three mice spleens were purified. RNA kit (Boehri
nger Mannheim) and reverse transcribed (RT) using two additional primers; one specific for the IgG heavy chain and the other specific for the kappa light chain ( 4). Each cDNA has been published so far (4,
Amplify by polymerase chain reaction (PCR) procedure using IgG heavy chain and kappa light chain specific primers and thermocycling conditions. The PCR amplification products are separated by size by gel electrophoresis.

[0036] scFv heavy chain was displayed on the surface of Example 3 linear phage and / or light chain Ig fragment combinatorial library of building already stated two of stages sequences forming and Step (4) and Strategene Inc. Made by ImmunoZ
The combined scFv heavy and light chain Ig fragment libraries are constructed in a linear phage expression vector using the AP kit. In summary, gel purified PC
The R product is enzymatically cleaved and the PCR product ligated into heavy chain vector-1Hc2 and light chain vector-ILc. The constructed heavy and light chain libraries are scFv
Consists of fragments. Next, the heavy and light chain components are combined to produce Fa
b. Create a combination composition. The E. coli cells are then transformed with a phage library and infected with helper phage (VCSN13, Strategene) to produce sufficient quantities of phage display antibody fragments for selection and selection purposes (4). The scFv heavy and light chains from the immunoglobulin heavy and light chains, as well as the heavy and light chain Ig V region-specific fragments, were converted to single-chain V fragments (scFv) with high binding affinity for Used as either a binding heavy / light chain V fragment (Fab) with binding affinity.

Example 4 Two-Stage Screening to Identify Antibody scFv or Fab Fragments with Specificity for Midgut Membrane Antigen from Queen Tofusiali Ant The first screening step is a whole antibody scFv or Fab fragment display phage library And reacting with the dissected cells of the midgut or the dissected cells to expose the villus cells of the queen Tofsiali. Unbound Ig display phage is washed away, and phage display scFv or Fab Ig fragments that bind with high affinity to the tofusia midgut antigen are eluted and stored. The second selection step is performed using a midgut preparation from a native Queen ants. Only phage-displayed scFv or Fab fragments that do not react with the midgut membrane antigen from native species of the queen ants are recovered. By repeating this selection procedure three times, specific Ig having high affinity or affinity for cloning can be obtained.
A fragment is obtained. The selected phage (which reacts with the mesenteric antigen of the queen Tofusiali but does not react with that of the native Queen ants) is no longer displayed, but is secreted when produced in E. coli. It is processed by genetic engineering. In this selection step, the Ig display phage can first react with the microvilli antigens of the native queen ants, and the Ig display phage that does not react with the native ants microvilli antigens are those of the queen Tofsiali. It is not limited to the above sequences in that it can be selected for reaction with the villous antigen.

[0038] Example 5 selected phage display antibody scFv or Fab fragments and verification selected phage display scFv or Fab fragments of the monoclonal antibody reacts with Tofushiari midgut microvilli cells (obtained in Example 4 Reacts with various midgut cell membrane antigens, including villous cell antigens. Shredded midgut tissue is used to identify cloned phage display scFv heavy and light chains or a combination of heavy and light chain Ig fragments, each of which reacts with the midgut microvillous cells of the queen Tofusiali. Midgut minced tissue was reacted with the cloned scFv or Fab fractions and the sensitive imunoperoxidase VECTASTAIN Elite ABC system (Vector Laboratori)
es) is used to check for the presence of phage. Clones that show a positive reaction with Tofushi ant villus cells but do not react with native species microglial cells are amplified inside E. coli and used as described in Examples 6 and 7. To screen for monoclonal antibodies, midgut shreds are reacted with the hybridoma supernatant, washed, and then reacted with alkaline phosphatase-conjugated rabbit secondary antibody to mouse Ig. Substrates are then added and the slides are examined for a positive response to the midgut microvillus antigen of Queen Tofushiari ants but not to the midgut microvillus antigen of the native Queen ants.

[0039] Phage display antibody fragments and monoclonal antibodies Example 6 selected is amplified in the effective is sorted by specificity and high affinity binding to the microvilli of the verification Tofushiari things in E. coli Lee down vivo monoclonal Antibodies and scFv and Fab heavy and / or light chain I
The g-fragment is tested for its ability to be internalized by villus cells when administered orally. Ant colonies (Tofushiari and native species) are created in a controlled environment, each colony consisting of one incubated Queen Tofsiali ant, fifty uninfested queen ants, and about 5,000 worker ants. To be configured. These colonies are fed with monoclonal antibodies or scFv or Fab fragments mixed into soybean meal. Incubated and unincubated queens from each colony are killed and the midgut is removed. Frozen tissue sections are made from the removed midgut and these strips are examined by immunohistological staining as described above to determine if the scFv heavy and / or light chain Ig fragments are present and internalized. Find out. Monoclonal Fab fragments are analyzed in this manner.

Example 7 Validation of Phage Display Generated scFv Ig Fragments and Monoclonal Fab Fragments as Effective Transmission Systems for the Control of Tofusia Ribosome Inactivating Proteins, Gelonin, Bacterial Endotoxins, or Other Toxins The monoclonal Fab fragment and scFv heavy chain and / or
Or attach to the light chain Ig fragment. By a well-established chemo-inoculation or attachment technique that has been successful in inoculating toxins into monoclonal antibody Fab fragments or scFv heavy chain Ig fragments; ) By using a bispecific (Fab) 2 or scFv heavy chain heterodimer with specificity for in vitro generated toxin by establishing a stable thioether bond; DNA technology in vivo and Genetic dimerization peptides are generated using genetic engineering techniques, and divalent dimers (having specificity for targeted antigens and toxins) in Escherichia coli or mammalian cells are generated using previously published techniques (13). By creating; and using all published techniques (14) Expressed by A, the scFv heavy chain enzymatically active region of the secreted toxin is by treating genetic engineering.

Using immunohistological techniques to tightly attach and internalize the Ig fragment to the midgut microvillous antigen, tofushiarii and a native species of ants are controlled in a well-established laboratory colony of tofushiarii. To provide the ability to bind to the midgut microvillus antigen of the queen ant but not to the midgut villus antigen of the native queen ant, the scFv heavy and / or light chains or the monoclonal Fab fragment Test "donation" was conducted. Tests are performed to determine whether the ability to selectively kill Tofusia only using a target scFv heavy and / or light chain and a monoclonal Fab fragment + toxin under laboratory established ant colonies and wild conditions.

The references cited herein are as follows: 1． Holldobler, B .; and E. O. Wilson 1990. The Ants. The Belknap Press, C
ambridge, Mass. 2． Barbas. C. and R. Lerner. 1991. Combinatorial immunoglobulin librari
es on the surface of phage (phabs): rapid selection of antigen-specific
. Fab. Methods: Comp. Met. Enzym. 2: 119. 3． Ames, R., M. Tornetta, C .; Jones and P.S. Tsui. 1994. Isolation of neut
ralizing 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 monoclonal
antibodies to human IL-5 isolated from hybridomas and a filamentous p
hage Fab display library. J. Immunol 154: 6355. Five. Winter, G., A. D. Griffiths, R.S. E. Hawkins and H. R. Hoogenboom. 199
Four. Making antibodies by phage display technology. Ann. Rev. Immunol. 12: 433. 6． Vaughan, T .; J., et al., 1996. Human antibodies with subnanomolar aff
inities isolated from a large non-immunized phage display library. Natur
e Biotech. 14: 309 7. Kruif, J .; de, et al., 1996. New perspectives on recombinant human an
tibodies. Immunolog Today 17: 453. 8． Kruif, J .; de, and T. Logtengerg. 1996. Leucine zipper dimerized biva
lent 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 small reco
gnition units. Biotechnology 13: 475. Ten. French, R, C. Penney, A. Browning, F. Stirpe, A .; George and M. Glenn
ie. 1995. Delivery of the ribosome-inactivating protein, gelonin, to lym
phoma cells via CD22 and CD38 using bispecific antibodies. Brit. J.
Cancer 71: 986. 11． Better, M., S. Bernhard, D.S. Fishwild, P.S. Nolan, R .; Bauer, A .; Kung, a
nd S. Caroll. 1994. Gelonon analogs with engineered cysteine residues fo
rm antibody immunoconjugates with unique properties. J. Biol. Chem. 269; 9644 12. Glennie, M. J., H. M. McBride, A. T. Worth, and G. T. Stevenson. Pre
paration and performance of bispecific F (ab ^, _- ) ₂ antibody contataining th
ioether-linked ^Fab, _- frabments. J. Immunol. 139: 2367-2375, 1987. 13. Holliger, P., and G. Winter. Engineering bispecific antibodies. Curr
ent Opinion in Biotechnology 4: 446-449, 1993. 14. Maurer-Gebhard, M., M. Schmidt, M .; Azemar, U.S. Altenschmidt, E .; Stock
lin, W .; Wels, and B. Groner. Systemic treatment with a recombinant erbB
-2 receptor-specific tumor toxin efficiently reduces pulmonary metast
ases in mice injected with genetically modified carcinoma cells. Cancer
Res. 58: 2661-2666, 1998.

All patents and publications mentioned herein are indicative of the level of those skilled in the art to which the invention pertains. Further, these patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually incorporated by reference.

Those skilled in the art will readily appreciate that the present invention is well adapted to achieve the objects and advantages set forth herein and the objects and advantages inherent therein. . The examples presented here demonstrate the methods, procedures, molecules,
And at the present time preferred examples, together with certain compounds, for illustrative purposes and are not intended to limit the scope of the invention. Modifications and other uses which come within the spirit of the invention, as set forth in the following claims, will be readily apparent to those skilled in the art.

In order that the above, and other features, advantages, and objects of the invention may be better understood, some embodiments are briefly illustrated in the following drawings. These drawings constitute a part of the specification. The attached drawings show preferred embodiments of the present invention, and do not limit the scope of the present invention.

[Brief description of the drawings]

FIG. 1 shows immunization, cDNA preparation, phage library construction, phage selection,
1 schematically illustrates the method according to the invention, including the verification of specific scFvs and Fabs, and their tests.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 15/02 G01N 33/53 Z C12P 21/08 G G01N 33/53 C12N 15/00 AC (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM) , AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, C U, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR , LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Clean, Kimberly United States 78703 Texas, Austin, McCallum 2505

Claims (12)

[Claims] 1. A method for preparing a drug that is specific for one target antigen and an active region of a toxin, comprising: creating a phage display library expressing scFv heavy and / or light chain Ig fragments; Reacting the heavy and / or light chain Ig fragment expressing phage display library with target cells; washing the reacted target cells to remove unbound scFv Ig fragment expressing phage; Eluting the scFv Ig fragment-expressing phage bound to the non-target cells; and reacting the eluted scFv Ig fragment-expressing phage with the non-target cells; and removing the unbound, eluted scFv Ig fragment-expressing phage from the non-target cells. Washing the cells; Engineering the scFv for image display, amplifying the scFv heavy and / or light chain Ig fragment expression phage display library inside E. coli, and recovering the secreted scFv Ig fragment. And creating the scFv heavy chain enzyme active region of the toxin and attaching the scFv Ig fragment to the toxin to create a single polypeptide having specificity for the target antigen and the active region of the toxin. 2. The scF, wherein the attachment is specific for a target antigen and a toxin.
2. The method of claim 1, wherein the method is performed by v-chain heavy chain and light chain bispecific chemical cross-linking or genetic engineering. 3. The method of claim 1, wherein the animal is a mouse. 4. The method of claim 1, wherein the target is a cell surface molecule of a target cell. 5. The method according to claim 1, wherein the target cells are cells of the microvilli of the midgut region of imported fire ant. 6. The method of claim 1, wherein the step of creating a library is performed by ligating the cDNA to a heavy chain vector and a light chain vector. 7. A polypeptide produced by the method of claim 1. 8. A method of producing a monoclonal antibody and a Fab fragment specific for the midgut microvillous antigen of imported fire ant, comprising immunizing an animal with target cells to produce the monoclonal antibody. When,
Harvesting the enriched spleen cells, crossing the spleen cells with a myeloma cell fusion partner using polyethylene glycol, growing on HAT medium and selecting the crossed cells, using an ELISA technique. Have the ability to produce mouse antibodies,
Using immunohistological techniques, the antibody is specific for the midgut microvillus antigen of the queen ant (imported fire ant), but the native fireflies (native fire ant)
ant) screening hybridoma supernatants for antibody specificity to queen midgut antigen; limiting dilution, screening hybridoma supernatants, expanding clones, and freezing positive clones And purifying monoclonal antibodies and Fab fragments specific for the midgut microvillus antigen of the imported fire ant queen. 9. The method of claim 8, wherein the animal is a mouse. 10. The method of claim 8, wherein the target cells are cells of the microvilli in the midgut region of the queen ant of imported fire ant. 11. A monoclonal antibody or Fa produced by the method of claim 8.
b fragment. 12. A method for killing ants, comprising the step of contacting an ant with the polypeptide produced by the method of claim 1.