WO1983001785A1 - Monoclonal antibodies against leishmania - Google Patents

Abstract

Immortal, antibody-producing, hybridomally-produced clones which produce antibodies that react specifically with antigens produced by the protozoan parasite Leishmania without cross-reacting with other closely related protozoa. Also disclosed are methods of producing the clones and antibodies and methods of using the antibodies in diagnostic tests and pharmaceutical preparations.

Description

Description

Monoclonal Antibodies Against Leishmania

Technical Field

The present invention relates to hybridoma cell lines and to monoclonal antibodies produced thereby that are species and stage specific for members of the Human protozoan parasite Leishmania.

Background Art

Leishmaniasis is a complex of diseases caused by intracellular parasites of the genus Leishmania. The disease is transmitted by the sandfly (Phlebotomus) and has received various names according to the locality of its occurrence, such as Delhi sore, espundia, kala- azar, and tropical ulcer. The disease exists in several forms: visceral leishmaniasis, marked by fever, progressive anemia, and wasting, is caused L. donovani; mucocutaneous leishmaniasis, marked by ulceration of the mucous membranes of the nose and throat, is caused by L. braziliensis; and cutaneous leishmaniasis, marked by a papule which passes successively through the stage of tubercle, scab, and circumscribed ulcer, is caused by L. mexicana (western hemisphere) and L. tropica (eastern hemisphere) .

The initial symptoms of different types of leishmaniasis are easily confused with each other and with the initial sumptoms of other protozoan-caused disease, such as American trypanosomiasis caused by Trypanosoma cruzi (Chagas' disease). However, different protozoan diseases respond to different chemotherapeutic agents and cannot all be treated by the same drugs. Accordingly, rational treatment requires initial diagnosis of the genus and species of the infecting protozoan, and preferably even identification of the strain. Prior to the present invention leishmania braziliensis could be distinguished from L. mexicana by the bouyant density of nuclear and -kinetoplast DNA, by electrophoretic mobility of different isoenzymes, by differing characteristic growth patterns in both the sandfly and the hamster and in vitro, or by means of electron microscopy. However, all of these methods required extensive laboratory equipment or long periods of time, and a simple method for rapid and accurate diagnosis of fresh isolates of New World Leishmania species did not exist. Furthermore, immunologic cross reaction of the Leishmania species with T. cruzi, which is co-endemic with leishmania, has posed a serious problem in the use of serodiagnostic tests for leishmaniasis that are based on immunoassay, since immunological cross reactions often occur between closely related microbes (the Leishmania and Trypanosoma are both members of the family Trypanosomatidae) .

The advent of hybridomal techniques has brought about the possibility of producing homogeneous populations of highly specific antibodies against a variety of antigens. Koprowski et al, U.S. Patent 4,172,124, describes antibodies produced by somatic cell hybrids between myeloma cells and spleen or lymph cells that are specific for malignant tumors. Kuprowski et al, U.S. Patent 4,196,265, describes continuous cell lines of genetically stable fused cell hybrids capable of producing large amounts of IgG antibodies against specific viruses, such as influenza virus, rabies, mumps, SV40, and the like. Zurawski et al, Federation Proceedings 39:4922 (1980), discloses hybrodomas producing monoclonal IgG antibodies against tetanus toxin. Monoclonal antibodies have also been described against human tumor cells, Yeh et al, Proc. 5 Nat. Acad. Sci. 76:2927 (1979); human T lymphocyte subsets, Reinberz et al, Proc. Nat. Acad. Sci. 76:4062 (1979); and malaria parasites, Nussenzweig et al, Science 207:71 (1980). The parasite species most closely related to the Leishmania against which 0 monoclonal antibodies have been raised appears to be T. cruzi, Hudson, "Hybridoma Technology with Species Reference to Parasitic Disease", Chapter 22, World Health Organization (1979). No monoclonal antibodies against Leishmania appear to have been available prior -5 to the present invention.

The complex life cycle of Leishmania complicates the process of identifying these protozoa using highly specific monoclonal antibodies. Leishmania change in form when transmitted from insects to mammals. The amastigote stage (without flagellum) is found within white cells in the mammalian host, while the promastigote stage (with flagellum) is found in the insect host. When a sandfly bites a susceptible host, promastigotes are inoculated into the host where they convert to amastigotes and go on to produce disease.

When the sandfly bites a mammal with leishmaniasis, it acquires amastigotes which convert to promastigotes in its gut. In addition, amastigotes obtained from a mammalian host often convert to promastigotes when cultured. Antibodies prepared against the promastigote state may not be suitable in an assay for amastigotes normally present in infected mammals, and it was not known whether monoclonal antibodies produced in the laboratory would have suitable binding characteristics that would enable them to be used in imunological assays.

Disclosure of the Invention

Accordingly, it is an object of this invention to provide a rapid, immunological method of assaying for the presence of Leishmania in biological fluids.

It is a further object to provide a method of assaying for the presence of leishmania that does not cross-react to a significant extent with other genera of protozoa.

It is a still further object of this invention to provide a method of assaying for specific species of Leishmania.

It is a yet further object of this invention to provide a method of assaying for specific stages of the life cycle of leishmania.

These and other objects of the invention as will hereinafter become more readily apparent have been accomplished by providing an immortal antibody- producing, hybridomally-produced clone and an antibody produced thereby, wherein said antibody is an immunoglobulin specific for a species or strain of the genus Leishmania.

Brief Description of the Drawings A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same become better understood by reference to the following detailed description when considered in connection with the accompanying drawing, wherein: The FIGURE shows in graphical form the results of an indirect radioimmuno binding assay used to determine the reactivities of the culture supernatants from L. braziliensis-specific hybrids with L. braziliensis panamanensis and L. hertigi membrane antigens and the reactivities of culture supernatants from L. mexicana- specific hybrids with L. mexicana amazonensis and L. hertigi membrane antigens.

Best Mode for Carrying Out the Invention ^■ Cell lines prepared by the procedures described herein are exemplified by cultures now on deposit with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. These culturs were deposited on November 13, 1981, and are identified by ATCC # HB8104, ATCC # HB8103, and ATCC # HB8105, and in this application by B-13, M-2, and XXIV- 1A12-E5, respectively.

Although it was known prior to the present invention that monoclonal antibodies can be produced against various antigens, it was not known whether monoclonal antibodies produced against an antigen derived from a Leishmania protozoan would be specific since it was also known that different species of Leishmania share antigens with each other and with other closely related protozoa, such as T. cruzi. Surprisingly, applicants have discovered that monoclonal antibodies produced using antigens from the cell membrane of a species of Leishmania will react selectively with the same species of Leishmania but not with other protozoa, such as T. cruzi, and usually will not react with other species of Leishmania. Such antibodies will prove useful in the area of diagnostic medicine, as they will allow rapid immunological

- ^RE testing of biological fluids for the presence of Leishmania. Since specific species and stages can be identified without danger of misidentification, rational chemotherapy can be more rapidly and cheaply initiated than was previously possible using earlier methods of identification.

Monoclonal antibodies of the invention may be produced that are specific for any of the species of Leishmania. By specific is meant that antibodies bind to antigens derived from an organism to which the antibody is said to be specific so that at least three times as many antibodies bind to the specific organism as to any other protozoan for any given concentrations of antibody and protozoan antigen in which the concentration of antibody is the limiting factor.

Particularly preferred are antibodies specific for L. mexicana, L. braziliensis, L. tropica, or L. Donovania. Antibodies that are even more specific, for example, against specific subspecies or strains, are also within the scope of the invention. It is even possible to produce antibodies that are specific against a particular stage of the life cycle of leishmania. Antibodies induced initially by antigens derived from the amastigote stage of a particular species produce antibodies that react with the amastigote stage without reacting with the promastigote stage of the same species. Such antibodies are especially important, since it is the amastigote stage that exists in a mammalian host. Many different techniques of hybridoma formation and monoclonal antibody production are known and may be applied in carrying out the objects of the invention. In general, the process comprises sensitizing an animal with an antigen to induce an immune response, obtaining i mune cells from the animal, and fusing the immune cell with a malignant cell line using one of a variety of fusing agents. Resulting hybrids are then grown in a medium which precludes the expansion of the original malignant cell line. After an initial period during which non-hybrid cells die, growing hybrids may be observed microscopically. Each of these colonies is assayed for the immune function sought. Colonies which demonstrate antibody secretion against a species or stage of Leishmania are cloned. These clones are then grown in large quantity, for example by stepwise transfer to larger wells, flasks, and bottles. Each of these steps is discussed in somewhat greater detail in the following paragraphs. However, it will be recognized by a practitioner in the area of hybridoma technology that many modifications and additions may be made to the techniques included in this discussion, which is intended to be exemplary and not limiting, while remaining within the scope of the present invention.

The first step of raising monoclonal hybrids is generally immunization of an animal. Because fusion occurs preferentially with proliferating cells, it is preferred to schedule immunization to obtain as many immunoblasts as possible, with harvesting occurring about 3-4 days after the last inoculation being most preferred. In the present application suitable antigens for use in immunization include proteins, glycoproteins, lipoproteins,, and other macromolecules present on the surface of or excreted by any stage of the life cycle of a Leishmania species. Antigenic macromolecules may be used in purified form, but suitable results are also obtained using either whole protozoa or membrane preparations derived from whole protozoa. Whole protozoa are preferrably rendered non- viable by chemical treatment, e.g. formaldehyde fixing or irradiation, in order minimize biohazard problems. Membrane preparations can be obtained by any methods that disrupt the protozoa and allow purification of the membrane fraction. Suitable methods include sonication or nitrogen cavitation followed by differential centrification or density gradient purification. For particulate antigens, such as membrane preparations or whole protozoa, which are often strong immunogens, either intraperitoneal, intravenous, subcutaneous, or intra-foot pad injection can be used with success. The immunization schedule may entail two or more injections, at interavls of up to a few weeks, with the last injection being three to four days before the fusion. Adjuvants may be included if desired and are preferred when soluble antigens are -used. It is also possible to employ immune cells which have been sensitized naturally, to use cells stimulated by polyclonal activators such as lipopolysaccharides, or to carry out in vitro sensitization of either B or T lymphocytes.

The cell line chosen for hybridization should be capable of rapid growth, be deficient in its metabolism for a component of the growth medium, and have potential for good fusion frequency. The species from which the immortalizing line is derived should be closely related to the species from which the antibody- producing cell is obtained. Intraspecies fusions, particularly between like strains, work better than interspecies fusions. Especially preferred are plasmocytoma-derived cell lines obtained from the same species and strain as the immune cells.

Readily available cell lines, many of which are mutants selected for their inability to secrete immunoglobulin, include the following: 1. P3-NSl-l-Ag4-l, or NSl, a variant of the P₃ (M0PC2*L) mouse myeloma line. 2. MPC_ι;L-X45-6TG, or X45, a Balb/c mouse plasmacytoma. 3. P3-X63-Ag8, or X63, the mouse myeloma cell line originally used by Kohler and Milstein. Recent mutants have been developed (e.g. X63-Ag8.653) which no longer secrete immunoglobulins. 4. sp2/0-Agl4, another BALB/C mouse myeloma line.

5. GD-36-A.Agl lymphoblastoid cell line obtained by ' injection of SV40 virus into Syrian hamsters.

6. Subclone Y3 - Ag 1.2.3., or Y3, from the rat myeloma mutant 210.RCY3.Agl. With the growth of hybridoma technology, new immortal cell lines suitable for hybrid formation are continually being developed. Such cell lines are also suitable for carrying out the hybridization processes described in connection with the present invention. Once immune cells are obtained and a suitable immortal cell line chosen, the cells are fused to form the hybrid cell line. Various techniques are available for inducing fusion and include virus-induced fusion and polyethyleneglycol-induced fusion. Inactivated Sendai virus is preferred for virus-induced fusion.

Other viruses can induce fusion of somatic cells if the two parental cells are both susceptible to the virus . Among suitable viruses are HVJ and Epstein-Barr virus . Polyethyleneglycol (PEG) can also be used as a fusing agent. PEG itself is toxic for cells at high concentrations and various concentrations should be tested for effects on viability before attempting fusion. PEG having molecular weights varying from 1000 to 6000 may be used. PEG should be diluted with 30-50% saline or serum-free medium. Since PEG is toxic for the cells, the time exposure to PEG should be limited. Exposure to PEG for 1-10 minutes is best for many cell lines.

Other conditions that should be controlled for increased fusion "efficiency are temperature and cell ratios. Extremes of temperature should be avoided during fusion. Preincubation of each component of the fusion system at about 37° prior to fusion is preferred. The ratio between spleen cells and malignant cells should be optimized to avoid self- fusion among spleen cells. Myeloma/spleen-cell ratios ranging from 2:1 to 1:20 are suitable, with 1:8 to 1:10 being preferred.

The mixture of cells obtained after fusion contains hybrids, fused and unfused parental spleen cells, and malignant cells. Spleen cells cannot maintain growth in routine culture medium and will eventually die out. Malignant cells would keep on dividing and soon overgrow the hybrids unless a selective medium is used that will allow only the growth of hybrids. The malignant cell lines must therefore be selected so that they are unable to grow on the chosen culture medium. For example, several available cell lines are hypoxanthine guanine phosphoribosyl transferase (HGPRT) deficient and will not grow in aminopterine-containing medium because of their inability to synthesize purines from thymidine and hypoxanthine. Some HGPRT revertants may occur among the malignant cells and these should be periodically purged with 8-azaguanine. The selection medium used to allow only growth of hybrids is composed of hypoxanthine, 1 x 10 M; aminopterine, 4 x 10 M;

' and thymidine, 1.6 x 10^"5M, (HAT medium). Other culture media and deficient cell lines may also be used in the practice of the invention.

The fusion mixture can be grown in HAT medium immeditely after fusion or at a later time. The feeding schedules for the fused cells may vary, but obligatory feeding of HAT medium (or another deficient medium) at intervals, for example on days 1, 6, and 11, is required, followed by growth in either regular culture medium or a medium containing hypoxathine and thymidine.

Standard tissue culture medium may be used to support the growth of hybrids. Good results may be obtained with Iscove's medium, Dulbecco's modified Eagle's medium (DMEM), or HY medium: DMEM enriched with 4.5g glucose/liter, 10% NCTC 109, 20% serum, and 0.15% glutamine. HY medium is preferred.

Serum used in media should be tested for its ability to support the growth of the malignant cell line prior to use. Hybrids may grow in, for example, horse or calf serum, but fetal bovine serum has no im unoglobulin, an important consideration that makes screening for antibody-producing cells much easier, and is therefore preferred. Hybrids may also be grown in serum-free media supplemented with 10-20% of a serum albumin, e.g., bovine serum albumin, and trace elements.

Feeder cells may be used in the initial stages of cell growth to enhance the survivability of the isolated cells. Irradiated thymocytes, spleen cells, myeloma cell lines, and mouse peritoneal macrophages may be used for feeder layers. Preferred feeder cells for use with mouse hybrids are mouse pertinoneal macrophages from the same species as the spleen donor, obtained by washing the peritoneal cavity of a mouse with aqueous sucrose. Macrophages harvested and plated (about 5 x 10⁴/ml x 10^/ml) the day before fusion are preferred.

Rapid identification of antibody-producing hybrids is important in order to avoid expenditure of time and resources on cultivation of extraneous cells. Early detection of hybridoma antibodies may be performed using standard immunological assays, for example, where the antigen is bound to a solid support and allowed to react with hybridoma supernatants. The presence of antibodies may be detected by sandwich techniques using a variety of indicators, such as rabbit anti-mouse antibodies that are labelled with radioactive isotopes or enzymes. Most of the common methods for detecting immunological activity are sufficiently sensitive for use in detecting antibody-producing cells. Several assay systems are discussed in more detail in a later section.

If a solid phase assay system is used, several methods are available to bind the immunogen to a solid phase. For example, many soluble antigens bind by adsorbtion to plastic surfaces. Such plates or wells may be rigorusly washed without affecting antigen binding. When whole cells or particulate fractions comprise the antigen, glutaraldehyde or an underlayer of antibody can be used to fix cells or membranes to a plastic surface. Dessication of cells .under vacuum can also be used. This technique also perserves cells for prolonged periods of time if they are used as an antigen.

The reagents used to detect the presence of the antibody/immunogen complex are chosen according to the species involved in the fusion. For example, when mouse cells are used, anti-mouse immunoglobulins may be used. Protein A can also be used because of its ability to bind to the Fc portion of IgG. These reagents may be labeled with radioactive isotopes

(radio-immunoassay, RIA) or with enzymes (enzyme-linked immunoassay, ELISA or EIA) . Both RIA and ELISA can be used in automated screening procedures if desired.

Hybrids obtained by fusion are initially heterogenous colonies. In order to obtain a homogeneous cell line these colonies must be cloned. By this is meant the process of achieving growth of a cell line from a single parental cell. Cloning of hybrids is preferably performed after 5-16 days of cell growth in selective medium. Later cloning of hybrids usually results in colonies which are slow growers and low yielders of antibody.

Cloning is performed by the limiting dilution method in fluid phase or by directly selecting single cells growing in semi-solid agarose. For limiting dilutions, cell suspensions are diluted serially to yield samples which have a statistical probability of having only one cell per well. The agarose technique begins with seeding of cells in a semi-solid upper layer of agarose over a lower layer containing feeder cells. The colonies from the upper layer are picked up and transferred to individual wells. Feeder cells, such as peritoneal macrophages, can be used to improve the cloning efficiency. Antibody secreting hybrids grown in tissue culture flasks generally yield a supernatant with an antibody concentration in the range of 10-100 yg/ l. In order to obtain higher concentrations, hybrids may be transferred into animals with inflammatory ascites. Ascites may be induced, for example, by intraperitoneal injection of mineral oil or 2,6,10,14- tetamethylpentadecane (pristane) 5-30 days in advance of inoculation with hybridoma cells. Under these conditions, antibody-containing ascites can be harvested 7-30 days after intraperitoneal injection of f_ O about 10 to 10° cells. The ascites contains a higher concentration of antibodies (1-15 mg/ml) , but includes both monoclonal antibodies and immunoglobulins from the inflammtory ascites. Ascites are generally induced in an animal of the same species as the one from which the immune and immortal cells were derived since growth of hybrids in interspecies animals requires immunosuppression of the host, for example by total body irradiation and administration of antilymphocyte serum prior to hybrid injection, in addition to the procedure normally followed for intraspecies growth of hybrids. Athymic nude mice can be used as an immunodepressed host if desired. Antibodies may be purified by any of the standard techniques of protein separation such as differential precipitation using, for example, ammonium sulfate; electrophoresis; chromatographic separation based on molecular size, such as Sephadex chromatography; or various techniques based on binding properties of or to the antibodies, much as affinity chromatography. Complete purification is not required since only the desired immunoglobulin is present and other components do not generally interfere with its immunological action.

A particularly preferred sequence for the production of monoclonal antibodies is described in the following paragraphs. BALB/c mice are immunized with a homogenized membrane preparation from the amastigote stage of the Leishmania species or strain against which protection is desired. The mice are allowed to rest for at least three weeks and then immunized again. Their spleens are removed 3-4 days after the last innoculation in order to obtain dividing B cells for fusion.

A preferred myeloma cell line is P3-NSI/-AG 4-1 (NS1), which is an azaguanine resistant, non-secretor myeloma line previously described by Kohler and Milstein, Eur. J. Immunol. 6_:511 (1976). When fused it produces K chains. Sp210-Ag is also a preferred cell line. Thirty percent polyethylene glycol (PEG-1000, Gallard-Schlesinger) is preferred as a fusion promotor. -Spleen cells are fused with the NSI cell line at a ratio of 8-10 spleen cells to 1 NSI cell.

The NSl/spleen-cell mixture is washed in serum-free MEM medium and suspended in 30% PE.G in MEM buffered with 0.02M Hepes, pH 7.2, and centrifuged at 800 rpm for 6 in after a total of 8 min in the PEG-containing medium. The PEG medium is removed, and the cells are plated onto microtiter plates in Dulbecco's MEM with high glucose (4.5 g/l) supplemented with 20% fetal calf serum, 10% NCTC 109 medium, 0.150 mg/ l oxalacetate, 0.050 mg/ml pyruvate, 0.200 units/ml bovine insulin, and 20 mM glutamine and containing 1.6 x 10^" M thymidine and 10 M hypoxanthine. .An equal volume of the above medium containing 8 x 10 —7M am opterin is added 24-48 hr later to make hypoxanthine-aminopterin- thymidine (HAT) selective medium. Fourteen to twenty-one days later the supernatants are ready for an indirect radioimmunoassay. A - 1^*■*2^*■•5-'i- rabbit anti-mouse F(ab' )₂ with at least 2 x 10' cpm/ yg is used to detect the antibodies produced by the hybridoma that bind to the target parasite cell. Briefly, the assay begins with incubation of parasites or membrane antigens in ϋ-bottomed microliter wells, 2 to 5 X 10⁵/well with 20 l of hybridoma culture supernatant for 1 hour at 0°C. The wells are then washed twice in phosphate buffered saline solution containing 0.5% BSA and 20 mM Hepes and buffered at pH 7.2. ¹²⁵I-Rabbit anti-mouse F(ab' )₂, 10 cpm/well in 20yl assay buffer, is then added to each well, and the cells are further incubated for 1 hour at 0°C. The cells are then washed 3X and transferred to tubes for counting. As the clones grow, they are transferred to larger wells and finally to Falcon flasks (25 cm ) . The hybrids can be grown in tissue culture or as ascites cells in mice. For the latter, mice are prepared by injecting 0.5 cm Pristane (2,6,10,14- tetramethylpentadecane) intraperitoneally 1 to 4 weeks before injecting hybrid cells intraperitoneally. Karryotype analysis is carried out on the clones, and their products are subjected to isoelectric focussing to verify the presence of monoclonal antibodies.

The hybridomally-produced anti-Leishmania antibodies of the present invention can be used in any of the array of available immunoassay techniques which utilize the binding interaction between the antibody and an antigen. The present invention is not limited to any of these techniques in particular. The most common of these is radioimmunoassay (RIA). RIA is a well- known technique and will not be described in detail here. For particulars, reference is made to Chard, "An Introduction to Radioimmunoassy and Related

Techniques", North-Holland Publishing Company, 1978, which is herein incorporated by reference. Any of the many variations of RIA can be used, such as homogenous phase RIA, heterogeneous or solid phase RIA, single antibody methods or double antibody methods, and direct (forward) or reverse sandwich assays. Particularly preferred are solid phase systems wherein the antibody (IgG or IgM) is covalently coupled to an insoluble support so that both the antibody and the bound complex after incubation can be readily separated from the soluble free fraction. A wide variety of solid phase supports have been described, which include particles of dextran, cellulose, continuous surfaces such as polystyrene or polypropylene discs, walls of plastic tubes, glass discs, glass particles, and the like. Particulate solid phases are widely used for a variety of different assays and are included in the present invention. Antibodies are attached to the particles by any of a number of techniques designed to yield a non- reversible covalent or non-covalent link between protein and particle, for example, directly or by cyanogen bromide activation. Other alternatives are the use of antibodies entrapped in the interstices of a polyacrylamide gel or bound to magnetic particles. An assay tube is set up containing either sample or standard, along with the tracer and an appropriate amount of solid phase bound antibody, plus a detergent to prevent aggreg ion of the particles and non-specific absorption of the tracer. After an incubation period during which the tubes are continuously mixed, the solid phase is sedimented by centrifugation; the supernatant is removed and the solid phase subject to two or more washes with buffer in order to remove free tracer trapped within and between the particles. The counts on the solid phase (bound fraction) are then measured. Immunoradiometric assays, as described in Chards at page 423, can also be used. When a second antibody radioimmunoassay system is used, the second antibody may be IgM or may be IgG. Another immunoassay technique useful with the antibodies of the present invention is enzyme immunoassay. This technique is also well known to the art and reference is made to Schuurs and VariWeemen, Clinica Chimica Acta, 81:1-40 (1977), which is herein incoporated by reference. In this technique, enzymes are applied as labels on antigen or antibodies for identification -and localization of the immunoreactants. Any method in which the extent of binding of enzyme-labeled antigen or enzyme-labeled antibody to its i munoreactant is measured is included in this invention. Enzyme immunoassays can be classified as homogenous or heterogeneous, depending on whether the labeled reagent behaves differently or identically whether or not it is bound to specific counterparts in the immunoreaction, and which therefore may or may not require physical separation of the reactants into two fractions. The variety of enzymes used, methods of linking enzymes to the immunological components, purification of the conjugates, as well as various assay principles and methods are well described in the aforementioned Schuurs and VanWeemen article. Needless to say, any enzyme immunoassay which has used antibodies in the past can be used with the specific, high-affinity, monoclonal antibodies of the present invention.

Another immunoassay method useful in the present invention is the latex agglutination method. In this method, latex particles are coated with antigen derived from leishmania and incubated with hybridomally produced IgM antibodies. Inhibition of agglutination will occur when a sample of physiological fluid containing the same Leishmania antigen is incubated with this mixture. The inhibition of agglutination can

O PI either be followed with a counter or by recently developed infrared absorption techniques. An alternative is to coat the latex particles with the hybridomally-produced anti-Leishmania antibodies. Incubation of these coated particles with physiological fluid comprising leishmania antigen will cause aggutination. Instead of latex particles, animal cells such as red blood cells can of course be used. In this case, the technique becomes a variation of the well known hemagglutination technique used with IgG antibodies and red blood cells.

Other useful immunoassay techniques are those using other labeling techniques that result in other types of detectably-tagged antigens such as: fluorescent dyes, Aalbeses, R.C., Clin. Chim. Acta _48:109-111 (1973); electron-dense compounds (such as ferritin) , Singer, S.J. et al, J. Biophys. Biochem. Cyto. _9_: 519- 537 (1961); protein - bacteriophage conjugates, Haimovich, J. et al, Biochim. Biophys. Acta 207: 115-124 (1970); or stable free radicals, Bastiani, R.J. , et al, Am. J. Med. Technol. 3_9: 211-216 (1973).

The previously described immunoassay systems are used to assay for the presence of antigens from

Leishmania in biological fluids, such as serum, or fluid obtained directly from the site of an apparent parasitic infection. It is also possible to use the detectably-tagged monoclonal antibodies in epidemiology studies of the sandfly vector or other aspects of the life cycle of Laishmania. The detectably-tagged antibodies of the invention can be prepared in kit form ready for use in an assay procedure. The kit will

/O EΛ contain the antibody in any stable form, for example, lypholized, frozen, or in solution, along with any other necessary reagents and accessories, such as Leishmania antigen standards, reaction vessels, such as plastic tubes with or without antigen or antibody coatings, and sample transfering devices, such as pipets. Many s-uch kits are now available for other immunoassays, and the manufacturing of such kits is now standard practice. The monoclonal antibodies of the invention may also be used in the chemotherapy of leishmaniasis as carriers of drugs having antileishmania activity. The antibody is attached chemically, usually by a covalent bond, to a drug which is administered at the normal rate for that drug, usually intravenously. The antibody acts to concentrate the drug at the location of the protozoan infection and thereby tq increase its effectiveness. Accordingly, in many cases it is possible to reduce the amount of drug administered in this manner.

Since the antibody is a protein, any method of attachment to the drug that does not destroy the medicinal properties of the drug or the antigen- antibody binding properties of the antibody is suitable. Suitable methods depend on the structure of the drug and can normally be accomplished by a reaction joining a non-essential functional group of the drug with a non-essential amino acid of the antibody. Several examples of attachment for specific drugs are given later. Suitable drugs include the antimonials, diamidines, and polyene antibiotics. Specific examples, not intended to be limiting, are sodium antimony tartrate, lithium antimony thiomalate, antimony bis(pyrocatechol-3,5-disulfonate), sodium stibogluconate, meglumine, stilbamidine, hydroxystilbamidine, cycloguanil pamoate, amphotericin B, mystatin, 2-dehydroemetine, and berberine chloride. Sodium stibogluconate and other drugs containing a carboxylate group can be attached to antibodies by the formation of a peptide bond with an amino group of the antibody. Since only a fraction of the surface of the antibody is involved in binding with the Lsishmania antigen, random attachment will generally preserve binding affinity. Amino-containing drugs, such as cycloguanil pamoate and others, can likewise be attached by peptide bonds to carboxyl groups in the antibody.

The antibody with the drug- attached may be made up into a pharmaceutical composition ready for use, comprising the drug-antibody combination either alone or in the presence of a pharmaceuticaly acceptble carrier. The pharmaceutical carrier in which the composition is suspended or dissolved may be any solvent or solid that is non-toxic to the inoculated animal and chemically compatable with the drug-antibody complex. Suitable pharmaceutical carriers include liquid carriers, such as normal saline and other non- toxic salts at or near physiological concentrations, and solid carriers, such as talc or sucrose. It is also possible to deliver the drugs using liposomes to which the antibody has been attached.

Having now generally described this invention, the same will be better understood by reference to certain specific examples which are included herein for purposes of illustration only and are not intended to be limiting of the invention or any embodiment thereof, unless specified. Example 1.

Female BALB/c mice were injected with membrane rich preparations isolated from either L. braziliensis panamanensi or L. mexicana amazonensis promastigotes. The membrane preparations were obtained by disrupting the promastigotes by nitrogen cavitation, followed by subfractionation by differential centrifugation and purification on sucrose density gradients. The method of fusion was that of Kennett et al, in Current Topics in Microbiology and Immunology, Vol. 81, 77, Melchers et al, eds., Springer-Verlag, New York (1978), which is herein incorporated by reference. Antibody production was evaluated between days 14 to 17 after fusion by an indirect radioactive binding assay utilizing leishmanial membranes hydrophobically fixed to polyvinylchloride microtiter plates. Sonicated leishmanial membranes diluted in phosphate-buffered saline (PBS) containing 0.02% Na g were plated in Cooke ^■TJ*-bottom 96-well PVC plates overnight at 5^βC. The plates were washed six times in PBS containing 5% horse serum at 0.02% Na g. Culture medium supernatants containing secreted antibodies were incubated with the antigen plates for a minimum of 2h at 5^βC. Immune mouse serum (diluted 1:20) and cell culture medium were used as positive and negative controls, respectively. The plates were again washed six times with PBS containing 5% horse serum and 0.02% NaNg. Affinity- purified ¹²⁵I-labelled rabbit F(ab^* )₂ anti-mouse immunoglobulin (5-10 ng per well; 10 c.p.m.) was added to the wells and incubated for lh at 0^βC. Excess antibody was again removed by washing five times. The plates were then air dried and the radioactivity bound to each well measured using a Packard Auto-Gamma counter. Antibody secretion was indicated by an elevation of bound radioactivity over background radioactivity measured by the negative control. Cultures secreting antibody were then evaluated for crossreactivity with L. hertigi or L. enrietti antigens. Selected wells were cloned by limiting diluation.

The average values for 6 fusions were: 73% of the total wells plated showed hybrid growth, 18% of these hybrid-containing wells produced antibodies to leishmania antigens, and 0.8% of the hybrid-containing wells were producing species-specific antibody. The specificity of the monoclonal Leishmania antibodies produced is seen in the FIGURE. Monoclonal antibodies V-3B9, V-1A12 and 1X-1G3 are crossreactive positive controls. The amount of radioiodinated rabbit anti- mouse immunoglobulin that bound to plates containing the L. braziliensis panamanensis membrane antigens, previously incubated with the L. braziliensis species- specific hybrid supernatants, was 17 to 21 times greater than that which bound after preincubation with control medium. After incubation with L. mexicana species-specific hybrid supernatants, the amount of radioiodinated rabbit anti-mouse immunoglobulin that bound to plates containing L. mexicana amazonensis membrane antigens was 6.1 to 38 times greater than that which bound to the medium controls. When L. hertigi membrane antigens were used, the amount of radioiodinated rabbit anti-mouse immunoglobulin bound in the case of either L. braziliensis or L. mexicana species-specific supernatants was onl 0.7 to 3.0 (average value 1.3) times that bound in the medium controls. Similar specificity was seen if the L. braziliensis hybrids were assayed for reactivity with L. enrietti, L. mexicana amazonensis, or L. hertigi, or if the L. mexicana hybrids were assayed for reactivity with L. braziniensis braziliensis or L. hertigi (data not shown) .

The reactivities of the culture supernatants of cloned hybrids with external membrane components of Leishmania were determined in an indirect binding assay using glutaraldehyde fixed Leishmania (Table 1). The assay procedure was essentially as described above for evaluation of antibody production by hybrids except that 'V^*-bottom microtitre plates (Cooke) were used and the leishmania (3-5x10 per well) were washed only three times after being pelleted at 400g for 10 min. after each incubation with antibody. On completion of the assay, the Leishmania were resuspended after the final wash i lOOyl of 0.5M NaOH and 60yl was taken for radioactivity measurement.

Table 1 Relative reactivities of leishmania monoclonal antibodies with epi astigotes of T. cruzi

Tfetio: CPMbound-βntibody/CPMbound-control

obde done L. mexicana L. B_raziliensi T. cruzi amazonensis pana ensi

M-l IX-2B8-D2 21.0 1.1

M-2 IX-2H7-E10 23.0 0.7

M-3 IX-5H9-C1 17.9 0.9

M-4 ---X-1F9-A9 10.6 0.9

M-5 -EX-1E2-D12 8.7 1.3

M-6 IX-2E3-C10 2.4 1.0

M-7 IX-5D1-C10 1.4 1.1

B-l VI-4M-A8 1.0 40.0 1.4 B-2 VI-4Θ-D10 1.2 59.7 1.0

B-3 VI-4D10-D12 1. 5 39.9 1.3

B-4 VI-2A5-A4 1.4 42.0 1.2

B-5 VII-2C5-C5 1.2 49.0 0.8

B-6 VII-1C4-H6 1.4 45.0 0.9

B-7 VI-2A4-E3 1.2 40.7 1.7

B-8 VII-3E12-D3 1.2 43.0 0.9

B-9 VII-4F11-A6 1.6 38.8 1.0

B-10 VII-4D6-E8 1.2 62.9 0.8

B-ll VI-5G3-F3 1.1 50.0 0.9

V-1A12-G2 21.5 14.8 14.2

V-3G5-06 21.5 8.2 12.4

All the specific hybridoma antibodies tested, except M-7, bound to the appropriate glutaraldehyde- fixed leishmania species. Since nearly all the antibodies of interest appeared to react with external surface components, glutaraldehyde-fixed organisms were used in the indirect binding assay. In addition, this assay was chosen because it avoids proteolysis, which can occur during membrane isolation. In the case of clone M-7, the antigen recognized is either destroyed by glutaraldehyde or the antigen is an internal membrane associated component.

No significant reactivity was found for any of the species-specific monoclonal antibodies with Trypanosoma cruzi (strain Y) epimastigotes (Table 1). Clones V- 1A12-G2 and V-3G5-C6 produce crossreactive Leishmania monoclonal antibodies that are clearly crossreactive with the T. cruzi epimastigotes. The levels of reactivity of these clones with the T. cruzi epimastigotes are comparable to those with L. mexicana amazonensis and L. braziliensis panamanensi. The species-specific monoclonal antibodies were evaluated for their reactivity against a panel of L. mexicana and L. braziliensis strains (Table 2), using the same procedure used for assaying hybrids for antibody production described above. Each strain designation had been recently determined by isoenzyme analysis. Isolates designated as WR303, Tres Bracoc 04 and Tres Brocos 11 are L. mexicana; M2903 and Tres Bracos 05, 08 and 13 are L. braziliensis.

- //d//

Table 2

Comparative reactivities of Monoclonal

Antibodies to Strains of L. mexicana and L. braziliensis Ratio: CPM Bound-Antibody/CPM Bound-Control mexicana L. braziliensi

Tres Tres Tres Tres Tres

Bracos Bracos Lbb Bracos Bracos Bracos

Clone WR303 04 11 M2903 05 13 08

IX-2B8-D2 19.6 14.7 21.1 0.8 1.0 1.1 0.9

IX-2H7-E10 21.4 14.8 17.9 0.6 0.9 0.7 0.7

IX-5H9-C1 16.0 7.2 10.3 0.6 0.9 0.8 0.7 I

IX-1F9-A9 9.3 5.5 7.7 0.6 0.9 0.9 0.8 to

I

IX-IE2-D12 8.1 5.1 7.3 0.8 1.2 1.2 1.0

VI-4A4-A8 1.0 2.8 ND 15.5 ND ND ND

VI-4B9-D10 1.2 0.9 1.2 7.3 29.5 14.0 13.1

VI-4D10-D12 1.5 1.9 1.5 60.4 30.9 13.1 12.8

VI-2A5-A4 1.4 1.2 2.6 7.5 20.4 10.7 6.6

VII-2C5-C5 1.1 0.7 1.3 78.6 55.2 7.4 14.5

VII-1C4-H6 1.4 1.1 2.1 5.9 30.3 7.6 10.9

VI-2A4-E3 1.2 1.6 12.8 10.8 30.6 16.4 14.4

VII-3E12-D3 1.1 0.9 9.6 63.8 27.5 9.7 11.8

VII-4F11-A6 1.6 1.2 13.7 4.3 24.5 10.0 93

VII-4D6-E8 1.2 0.9 5.2 80.2 29.6 14.4 15.7

VI-5G3-F3 1.1 0.7 1.7 0.9 1.4 5.4 0.4

Clones M-l, M-2, M-3^', M-4, and M-5 are reactie with the leishmania mexicana strains but are unreactive with the leishmania braziliensis strains. In contrast, clones B-l, B-2, B-3, B-4, B-5, B-6 and B-ll are reactive with the leishmania braziliensis strains, but not Laishmania mexicana strains. Clones B-7, B-8, B-9 and B-10 are reactive with all the leishmania braziliensis strains and not the Leishmania mexicana strains L. mexicana amazonensis WR303 and Tres Bracos 04; these, however, are reactive with the leishmania mexicana strain Tres bracos 11. Clone B-ll appears to be specific for a subgroup of Leishmania braziliensis, reacting only with the leishmania strains L. braziliensis (Tres Brcos 13) and L. braziliensis panamanensis.

Example 2

Clones producing antibodies against L. braziliensis were produced as described in Example 1 and tested for cross-reactivity against subspecies. The original immune response was elicited against L. braziliensis braziliensis promastigates for Clones B-12 through B-l6. Comparative reactivities are shown in Table 3.

Table 3

Gbπparative reactivities of itionoclonal antibodies to subspecies of L. braziliensis promastigates.

I tio: CPM Bound-Antibody/< C-PM Bound-€ontrol

Ibb Ibb Ibp Ibq Ima done ϊ£903 LTBD042 WR120 ML176 LTED016

B-12 XIII-1H4-B3 14.0 38.1 0.9 0.9 1.1 B-13 XIV-2A5-A10 11. 9 34. 5 0.9 0.7 1.0 B-14 XHI-3F4-F6 14.6 48.0 0.9 1.2 1.1

B-15 XIII-3H6→ 2 24.8 40.1 25.3 9.4 2.2

B-16 XIII-4B5-F1 22.6 51.9 69.0 — 2.4

B-2 VI-4B9-D10 7.3 32.4 56.0 55.9 1.2

B-5 VII-2C5~C5 5.9 30.3 54.7 1.2 0.7

B-8 VII-3E12-D3 63.8 27.5 48.2 1.7 0.9

B-ll VII-5G3-F3 0.9 1.4 55.6 1.0 0.7

The species designation by isoenzyme analysis of the

Leishmania isolates are as follows: 1) M2903 and LTB0042 are L. braziliensis braziliensis

2) WR120 is L. braziliensis panamanensis

3) M1176 is L. braziliensis guanyensis

4) LBT0016 is L. mexicana amazonensis Several clones, i.e., B-12, B-13 and B-14, showed subspecies specificity for L. braziliensis braziliensis, indicating that a high degree of accuracy can be attained in identifying even subspecies using selected monoclonal antibodies. Likewise, clone B-ll, which was described in Example 1, was specific for L. braziliensis panamanensis.

Example 3

Monoclonal antibodies against the amastigote stage of leishmania have also been produced. Female Balb/c mice were immunized with either whole, irradiated

(15,000 rad) L. mexicana (Tres Bracos 16) amastigotes or with membrane-rich preparations of amastigotes . Amastogotes were purified from either, .hamster lesions or from an infected mouse macrophage cell line (J 774) . Standard methods of fusion were employed and antibody production was detected using an indirect radioactive binding assay with amastigotes covalently bound (using glutaraldehyde) to poly-L-lysine-coated polyvinylchloride (PVC) microtiter plates as previously described. Supernatants that were secreting antibody to L. mexicana amastigotes were then tested for crossreactivity with L. mexicana promastigotes and L. enrietti amastigotes.

Five fusions with the following average values were completed: 70% of the total wells showed hybrid growth, 10% of these wells produced antibodies to leishmania antigens, 0.5% of the hybrid-containing wells produced species—specific antibody, and 0.1% of the wells produced antibody that was both stage and species specific. Three hybrids that secrete stage- and species- specific antibody were successfully grown and placed into mice for ascites production. The specificity of these monoclonal antibodies was determined as before. The amount of ¹²⁵I-rabbit anti-mouse immunoglobuilin (RαMIg) that bound to plates coated with L. mexicana amastigotes previously incubated with these hybrid supernatants was 3.6 to 17 times greater than that which bound after preincubation with control media. When L. mexicana promatigotes or L. enrietti amastigotes were bound to plates, the amount of C 12513-RαMIg bound was 1.1 to 1.8 and 1.1 to 1.2, respectively, times that bound in the media controls.

The results are shown in Table 4. Table 4

Msnoclonal antibodies specific for L. mexicana amastigotes

Amastigote Prcmastigote

L. L. L. L. L.

Clone mexicana enrietti braziliensis mexicana braziliensi

XXI-C10-C11 3.6 - - 1.1 0.7

XIV-1AL2-E5 17.0 0.8 1.2 1.2 0.8

XIV-2D5-B10Ξ4 6.7 1.2 1.5 1.8 1.0

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein.

^■ ^ J

Claims

Claims

1. An immortal, antibody producing, hybridomally- produced clone, wherein said antibody is specific for the protozoan genus Lsishmania.

2. The clone of Claim 1, wherein said antibody is specific for one species of the genus Leishmania.

3. The clone of Claim 2, wherein said species is L. braziliensis, L. mexicana, L. tropica, or L. donovani.

4. The clone of Claim 1 or 3, wherein said antibody reacts with the amastigote stage of said protozoan.

5. The clone of Claim 1, wherein said clone is ATCC # HB8103, ATCC #HB8104, or ATCC #HB8105.

6. A monoclonal antibody against a Leishmania protozoan, wherein said antibody is a hybridoma- produced monoclonal immunoglobulin specific for the protozoan genus leishmania.

7. The antibody of Claim 6, wherein said antibody is specific for one species of the genus leishmania.

8. The antibody of Claim 7, wherein said species is L. braziliensis, L. mexicana, L. tropica, or L. donovani.

9. The antibody of Claim 6 or 8, wherein said antibody reacts with the amastagote stage of said protozoan.

10. The antibody of Claim 9, wherein said antibody is detectably-tagged.

11. The antibody of Claim 9, wherein said antibody is attached to a drug having antileismania activity .

12. A pharmaceutical composition comprising the antibody of Claim 11.

13. The pharmaceutical composition of Claim 12, wherein said composition further comprises a pharmaceutically acceptable carrier.

14. The antibody of Claim 6, wherein said antibody is produced by a cell line selected from ATCC SHB8103, ATCC #HB8104 or ATCC #HB8105.

15. An immunoassay system utilizing an antibody to assay for a Leishmania protozoan, wherein said antibody is a hybridoma-produced monoclonal immunoglobulin specific for the genus Leishmania.

16. The immunoassay^'system of Claim 15, wherein said antibody is specific for one species of the genus

Leishmania.

17. The immunoassay system of Claim 16, wherein said species is L. braziliensis, L. mexicana, L. tropica, or L. denovani♦

18. The immunoassay system of Claim 15 or 17, wherein said antibody reacts with the amastigote stage of said protozoan.

19. The immunoassay system of Claim 15, wherein said antibody is produced by a cell line selected from ATCC #HB8103, ATCC #8104, or ATCC #HB8105.

20. An immunoassay kit comprising the monoclonal antibody of Claim 6.