NZ225372A - Immunotoxin composition comprising purified ricin-a-chain species - Google Patents

Description

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Publication Date: .... 2J6l.A£R. .1991... P.O. Journal, No: ......1 fr*. £t[s3. t No.: Date: NEW ZEALAND PATENTS ACT, 1953 COMPLETE SPECIFICATION IMPROVED IMMUNOTOXIN THERAPIES UTILIZING PURIFIED RICIN A-CHAIN SPECIES i'/Wc, XOMA CORPORATION, incorporated in the State of Delaware, U.S.A., of 2910 Seventh Street, Berkeley, California 94710, U.S.A., hereby declare the invention for which—I-'/ we pray that a patent may be granted to mc/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - - 1 -(followed by page la) 22 5 372 ■let PATENT ArPLICjVEgQN OCOC-4» IMPROVED IMMUNOTOXIN THERAPIES UTILIZING PURIFIED RICIN A-CHAIN SPECIES Field of the Invention This invention relates generally to tjhe use of immunotoxins in chemotherapy and other human treatment applications and, more particularly, to improving the pharmacokinetics and toxicity characteristics of ricin-based immunotoxins.

BACKGROUND OF THE INVENTION The advent of monoclonal antibody technology in the mid-1970's was heralded as a major technical breakthrough for the fields of immunology and medicine. For the first time, researchers were able to transform B-cells to create hybrid cells, with immortal poten-20 tial, capable of secreting monoclonal antibodies, i.e., a collection of a single species of antibody reactive with a single epitope on a selected antigen. In one application, scientists contemplated that the remarkable specificity of these monoclonal antibodies could be 25 utilized to selectively deliver a toxic agent to a predetermined cell population, such as a tumor, in a patient. Thus, cancerous cells or other diseased eel-\ : lular material could be selectively killed without the nonspecific side-effects rampant with most common treatment regimes. This "magic bullet" combination of a monoclonal antibody conjugated to a toxin is known as an immunotoxin.

In spite of the enormous therapeutic potential apparent in the use of immunotoxins, very few med-35 ical successes have been reported, despite extensive research efforts. In practice, finding the appropriate combination of antibody and toxin that can actually 22 5 3 7 2 improve a chemotherapeutic or other therapy hak proven extremely difficult.

The most widely used toxin component of immu-notoxins is the ricin toxin A-chain. Ricin is a plant 5 lectin produced by castor beans (Ricinus communis) and consists of two polypeptides; chains A and B, linked by a single disulphide bond. Both chains are important in native ricin toxicity. The B-chain of ricin binds to glycoproteins and glycolipids on cell surfaces, and the 10 A-chain then penetrates the cell. Once incorporated into the cytosol, the A-chain can catalytically inactivate ribosomal protein synthesis, ultimately causing cell death. To improve specificity of ricin-based immunotoxins, researchers separate out the B-chain, and 15 conjugate just the A-chain to the antibody.

Cell culture experiments using an immunotoxin made with ricin toxin A-chain (RTA) have shown that RTA-based immunotoxins are highly specific cytotoxic agents, capable of removing more than 99% of the target 20 cells without damaging unrelated cells. Unfortunately, the in vivo utility of these RTA-based immunotoxins have generally been less than ideal, perhaps because of rapid clearance from the blood stream which would reduce the amount of immunotoxin available to interact 25 with the tumor.

Intravenous injections of ricin have been shown to accumulate into both the liver and spleen of test animals, causing severe damage to these two organs. Researchers have hypothesized that the rapid 30 clearance of RTA by cells of the reticuloendothelial system (RES), in general, is the major cause of rapid immunotoxin and RTA removal from the blood stream.

One proposed solution to overcome the recognition of RTA by the RES was to alter its glycosylation 35 pattern; typically by reacting RTA with chemicals, such as sodium metaperiodate and sodium cyanoborohydride, or through enzymatic deglycosylation treatment, such as 2 2 5 3 7 with alpha-mannosidase. These attempted modifications of the natural glycosylation of RTA have resulted in decreased in vivo blood clearance times of the modified !w*/" RTA. The treatments are generally undesirable, how ever, for a number of reasons. For example, any additional processing steps in the production of a pharmaceutical product, particularly those entailing removing certain moieties and thus altering naturally occurring proteins (such as ricin) require extensive monitoring of the reaction to ensure minimal heterogeneity in the final product. The added steps necessitated by the chemical reaction (particularly harsh oxidations), in conjunction with the extra purification steps, are burdensome and uneconomical, resulting in very low yields. 15 Moreover, the potential for quality control problems becomes greatly magnified.

Thus, there is a significant need for improved RTA-based immunotoxins exhibiting superior in vivo properties. The immunotoxin should retain high speci-20 fic cytotoxicity, yet minimize the host's nonspecific toxicity. It should also be relatively simple and inexpensive to manufacture reproducibly. Ideally, the immunotoxin will still retain certain natural clearance properties, however, because some clearance is prefer-25 red to minimize nonspecific toxicity in the host. The present invention fulfills these needs.

» SUMMARY OF THE INVENTION The present invention provides novel methods 30 for the in vivo treatment of a patient utilizing immunotoxins comprising a specific bind.inq component com-plexed with a ricin toxin A-chain (RTA) component, wherein the relative amount of RTA-30 species within the RTA of the immunotoxins is increased over the 35 amount of RTA-30 species found in naturally-occurring ricin. The RTA-30 species may be separated from other RTA species found in ricin by standard chromatographic 2 2 5 3 7 & tM. — 4 — techniques to achieve RTA-30 concentrations up^to substantial homogeneity, about 95% or more. In particular, the novel RTA-30-based immunotoxins can be utilized to selectively remove harmful cell populations from 5 a patient, with minimal nonspecific toxicity. Pharmaceutical compositions are also provided for use in the treatments.

BRIEF DESCRIPTION OF THE FIGURES 10 Figure 1 shows the pharmacokinetics of immunotoxins having different RTA species.

Figures 2-4 show the biodistribution of immunotoxins having different RTA species.

Figures 5 and 6 show the results of perfusion 15 studies utilizing immunotoxins having different RTA species.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Novel methods are provided for improving 20 ricin toxin A-chain (RTA)-based immunotoxin therapy in human patients by utilizing an enriched concentration of the RTA-30 species of RTA as a toxic component of immunotoxins. By utilizing RTA-30 species in concentrations higher than touncl in naturally-occurring 25 ricin, increased blood residence time of the immunotoxin is achieved, without significantly increasing nonspecific toxicity. In general, the immunotoxins of the puesent invention have less non-specific toxicity. Thus, the methods and compositions of the present in-30 vention provide means for substantially improved treatments for, e.g., the removal of undesired cell populations from a patient, such as cancerous cells in tumors or cells responsible for graft versus host disease.

As used herein, the term "RTA-30" refers to a 35 species of ricin toxin A-chain having a molecular weight of approximately 30 kD, such as described in detail by Fulton et al. J. Biol. Chem., 281:5314-5319 22 5 3 72 (1986) and Vidal et: a^. Int. J. Cancer, 36:705^-711 (1985) , both of which are incorporated herein by reference. Depending on the source, RTA-30 typically comprises about 65% of the protein obtained from naturally 5 occurring ricin, with RTA-33 (about 33 kD) comprising most of the remaining protein. The two species have the same isoelectric point (about 7.6) and exhibit similar in vitro activities, such as protein synthesis inhibition and cell toxicity. A substantial difference 10 between the two species is that RTA-30 experimentally exhibits lower glycosylation than RTA-33, with the RTA-30 species having a single complex oligosaccharide, and the RTA-33 having a high mannose type oligosaccharide in addition to the complex unit found on the RTA-30 15 (see, Foxwell et aJL. , Biochem. Biophys. Acta., 840:193-203 (1985), which is incorporated herein by reference). The lower carbohydrate content of RTA-30 provides longer blood clearance for the immunotoxin. The presence of some sugars can provide a reasonable clearance rate, 20 however, minimizing kidney damage and other nonspecific toxicity.

Separation of RTA-30 from other RTA's may be accomplished by a variety of well known separation procedures (see, e.g., Fulton et al., Vidal et al., and 25 Foxwell et. al. , supra) . These can include gel filtration, anion or cation exchange chromatography, electrophoresis, hydrophobic chromatography, affinity chroma-V tography, and the like. A preferred means of RTA-30 separation is based on the different glycosylation pat-30 terns between RTA-30 and RTA-33. Carboxymethylcellu- lose columns run with a sodium chloride gradient readily separate the two predominant species. Alternatively, Concanavalin A may allow for separation when used in an affinity chromatography procedure, because of 35 different affinities for RTA-33 and RTA-30. It will be readily apparent to those skilled in the art that these separation procedures are reproducible and economical, /""A 22 5 3 72 and do not produce contaminating by-products, linlike many chemical modification processes.

By following the above procedures, RTA-30 concentrations in the immunotoxin preparations may be increased well above the level in naturally occurring ricin (i.e., about 65%). Purified RTA-30 of concentrations of about 75% or greater are preferred, with concentrations of 85 to 95%, or more, most preferred. As desired, other species of RTA may be added to purified RTA-30 to control the relative species concentrations. Preferably, the RTA species will be utilized at concentrations that maximize iri vivo localization, yet minimize nonspecific toxicity.

As used herein, the terms "immunotoxin" refers to the combination of a specific binding component complexed with a cytotoxic agent (e.g., RTA-30). The specific binding component provides the means for delivering the toxic agent to a particular cell type, typically preselected, such as cells forming a carcinoma. The two components are complexed in a manner that is likely to ensure that the toxic agent is not separated from the binding agent until attachment of the entire immunotoxin to a cell within a preselected cell population. The two components are usually chemically bonded together by any of a variety of well-known chemical procedures.

For example, when the cytotoxic agent is RTA-30 and the second component is an intact immunoglobulin, such as a monoclonal antibody, the linkage may be by way of heterobifunctional linkers, such as, N-succi-nimidyl 3-(2-pyridyldithio)propionate (SPDP), carbodi-imide, gluteraldehyde, 2-iminothiolane or the like, to form peptide, amide, ester, thioester, disulfide bridges or other bonds. The linkage may also be between amino acid and sugar moieties of the two components, depending upon the particular application. On the average, each immunoglobulin will contain at least o w about 1-2 RTA-30 moieties, preferably 2-3 or mbre, and, most preferably, about 2.6. Production of various immunotoxins is well-known within the art and can be found, for example, in "Monoclonal Antibody-Toxin Con-5 jugates; Aiming the Magic Bullet," Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190 (1982), and U.S. Patents 4,671,958, and 4,590,071, all of which are incorporated herein by reference .

The specific binding agent, acting as the de livery vehicle for the cytotoxic agent in the immunotoxin, can be obtained from a number of sources. Preferably, intact immunoglobulins or their fragments, such as Fv, Fab, F(ab2), half antibody molecules, 15 (i.e., a single heavy/light chain pair), will be used. Most preferably, immunoglobulins are monoclonal antibodies of the IgM or IgG isotype, of mouse, human or other mammalian origin. Other proteins or agents capable of binding to markers, including growth factor or 20 hormone receptors, on selected cell populations may be utilized.

Common sources of monoclonal antibodies are immortalized murine or human cell lines that may be cloned and screened in accordance with conventional 25 techniques. Recent technical advances have provided additional forms of immunoglobulins and methods of making them. For example, the utilization of recombinant BNA technology has produced functional, assembled immunoglobulins or hybrid immunoglobulins (e.g., the 30 constant region from human monoclonal antibodies combined with mouse variable regions), suitable for use in immunotoxins (see, e.g., EPA 84302368.0, which is incorporated herein by reference).

Typically, the antibodies are capable of 35 binding to epitopes of markers on selected cell populations, such as neoplastic cells or T-cells. The marker is generally a unique surface protein, but a large 0 9 R X 7 9 VaJ" g WW v&V3 variety of markers, such as other proteins, glycoproteins, lipoproteins, polysaccharides and the like, which are produced by or displayed by the cells to be recognized by the immunotoxin, can be utilized in ac-5 cordance with the present invention. The general immunization, fusion, screening and expansion methods of monoclonal antibody technology, as well as the choice of markers, are well known to those skilled in the art W* and do not form part of the present invention.

The immunotoxin may be utilized in prophylac tic and therapeutic settings to aid in the killing or removal of a wide variety of predetermined cell populations in a mammal, including infectious organisms, depending upon the disease state. By way of example, the 15 specific binding protein of a immunotoxin may recognize markers on tumor cells, immune cells (e.g., T-cells or B-cells), hormone responsive cells (e.g., to insulin) and growth factor responsive cells (e.g., to interleu-kins), fungi, bacteria, parasites, or virus infected 20 cells. Blood from the mammal may be combined extracor-poreally with the RTA-30 enriched ricin-based immunotoxins, whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal.

In an embodiment of the present invention, immunotoxins are utilized in cancer therapy as follows: Antimelanoma immunotoxin XMMME-001-RTA-30 can be prepared and then tested extensively in vitro, on human tissues, and in animals to establish precise 30 dosages for the treatment of human melanoma as described in U.S. Patent No. 4,590,071. Hybridoma cell line XMMME-001 was deposited with the American Type Culture Collection (ATCC) and given ATCC Accession No. HB 8759. An immunotoxin incorporating it as the specific binding 35 component and RTA as the toxin has been used in FDA-approved Phase I and Phase II Clinical Trials, the protocols and results of which are described in detail in SSI*-, 22 5372 'f commonly-owned U.S. Application No. 053,189, filed May 20, 1987, which is incorporated by reference herein. Briefly, the immunotoxin was administered to patients in the form of intravenous injections of 0.4 5 mg/kg/day for 5 days. In a Phase I/II Trial, patients were given a single 0.4 mg/kg does of XMMME-001-RTA in conjunction with a standard oncologic dose of an immu-nosuppressive agent, such as methotrexate, cyclophos-phamide, prednisone, or cyclosporine, in order to blunt 10 the immune system to prevent immune response against the immunotoxin. Depending on the immune response mounted against the immunotoxin, the treatment may be repeated, up to three times. Immunotoxins incorporating enhanced levels of RTA-30 will require substantial-15 ly smaller dosages to be effective, typically at least about 10-25% less, but in some therapies about 30 to 50% less.

In accordance with another embodiment of the present invention, immunotoxins are utilized prophylac-20 tically in improving bone marrow transplantation, by reducing the likelihood of graft versus host disease (GVHD), as follows: Patients can receive bone marrow transplantations (BMT) in order to treat a variety of diseases, 25 such as hematological malignancies, aplastic anemia, Severe Combined Immunodeficiency (SCID) or variants, certain inborn errors of metabolism, or certain solid tumors. In some situations, bone marrow donors fall into categories of genotypically haplotype matched or 30 unrelated partially-phenotypic HLA matched. These categories of donors result in a 100% incidence of GVHD in the recipient. BMT treatment with allogeneically matched sibling donors results in an incidence of GVHD of about 30% or more.

After BMT, at a time following evidence of hematopoietic recovery and prior to rapid proliferation of GVHD-producing cells, an immunotoxin reactive with 22 5 3 7 2 GVHD-producing mature T cells is infused. A preferred immunotoxin, XMMLY-H65-RTA-30, consists of an anti-CD5 (pan T lymphocyte) specific monoclonal antibody conjugated to RTA-30. The immunotoxin can be infused start-5 ing on day 10 post-transplant for 7 consecutive days (days 10-17) at a dose of about 0.05 to 0.1 mg/kg/day.

Preparation of the immunotoxin XMMLY-H65-RTA is described in U.S. Serial No. 938,855, which is in-corporated by reference herein. This application also 10 describes typical protocols for the treatment of GVHD in BMT recipients, as well as characterization of the H65 monoclonal antibody. The hybridoma producing XMMLY-H65 was deposited with the ATCC and given ATCC Accession No. HB 9286.

In vitro studies have demonstrated that XMMLY-H65-RTA immunotoxin will kill T-cells when incubated with human marrow without causing toxicity to hematopoietic progenitor cells. The biologic activity of this pan-T-cell immunotoxin indicates that it can be 20 a potent anti-T-cell cytotoxin, able to abrogate T-cell reactions contributing to the pathogenesis of GVHD, particularly when the RTA component is enriched with RTA-30.

Pre-clinical in vitro studies in animal mod- O els has indicated that the toxicity of RTA immunotoxins was low. Rats were given 14 consecutive doses of 2.4 mg/kg RTA-immunotoxin and the principal toxicity was transient hypoalbuminemia and occasional mild elevation in liver enzymes. Monkeys treated with the immunotoxin 30 experienced various side-effects that were reversible. Since XMMLY-H65 is not known to bind to any non-human tissues (except weakly to monkey granulocytes) this toxicity, at high doses, is considered non-directed and non-specific, possibly due to RTA. The use of RTA en-35 riched with RTA-30 will substantially lessen this nonspecific toxicity. m 22 5 3 7 2 Depending upon the particular therap^, the immunotoxins of the present invention will be commonly incorporated as components of pharmaceutical compositions. Such compositions will contain a therapeutic 5 amount of the immunotoxins of the present invention with a pharmaceutically effective carrier.

A pharmaceutical carrier can be any compatible nontoxic substance suitable to deliver the immunotoxins to the patients. Sterile water (with or without 10 excipients), alcohol, fats, waxes and inert cells may be used as the carrier, often in conjunction with acceptable adjuvants, such as buffering agents, dispersing agents, and the like.

The immunotoxins of the present invention may 15 be used as separately administered compositions or in conjunction with other cytotoxic agents. These can include various immunotoxins and chemotherapeutic drugs, such as vindesine, methotrexate, adriamycin, and cis-platinum, various radionuclides, and the like. Pharma-20 ceutical compositions can include "cocktails" of various immunotoxins with cytotoxic agents in conjunction with the immunotoxins of the present invention. Thus, a typical pharmaceutical composition for intravenous infusion could be made up to contain about 150 ml of 25 normal saline and about 0.1 mg of immunotoxin.

An amount adequate to accomplish at least partial killing of a cell population is defined as a "therapeutically effective dose." Amounts needed to achieve this dosage will depend upon the severity of 30 the disease and the general state of the patient's own immune system, but generally range from about 0.005 to about 5.0 mg of immunotoxin per kilogram of body weight, with doses of about 0.05 to 2.0 mg/kg/dose being more commonly used. For prophylactic applica-35 tions, compositions containing the present immunotoxins or cocktails, may also be administered in similar dosages. 22 5 3 For treatment of melanomas, preferred dose regimens are about 0.4 mg/kg, administered daily for five days, or about 0.1 mg/kg to 2.0 mg/kg in a single dose. In general, systemic toxicity and the immune re-5 sponse are limiting factors to the size of the dose, and the highest dose and total cumulative dose must be considered. In graft versus host disease, immunotoxin dosages are preferably 0.05-0.3 mg/kg/day for up to about 14 days. Doses may be repeated as often as tol-10 erated. Actual methods for preparing and administering pharmaceutical compositions, including preferred dilution techniques for injections of the present compositions, are well known or will be apparent to those skilled in the art and are described in more detail in, 15 for example, Remington's Pharmaceutical Science, 16th Ed.f Mack Publishing Co., Pennsylvania (1982), which is incorporated herein by reference.

Kits can also be supplied utilizing the subject immunotoxins in the treatment of various disease 20 states. Thus, the subject immunotoxins of the present invention may be provided in containers, usually in a lyophilized form, either alone or in conjunction with O additional immunotoxins or non-complexed antibodies specific for desired epitopes. The immunotoxins and 25 antibodies, which may be conjugated to a label or unconjugated, are included in the kits with physiologically acceptable buffers, in accordance with the teach-ing of the art. Generally, these materials will be present in less than about 5% wt. based on the amount 30 of active ingredient, and usually present in total amount of at least about 0.001% wt. based again on the active ingredient concentration. Frequently, it will be desirable to include an inert extender or excipient to dilute the active ingredients, where the excipient 35 may be present in from about 1% to 99% wt. of the total composition. 22 5 3 e> The following examples are offered way of illustration and not limitation.

EXPERIMENTAL 5 A. Preparation of RTA Immunotoxins A preferred production process for RTA-based immunotoxins is described in U.S. Patent No. 4,590,071, which is incorporated herein by reference. The follow-'****' ing experimental process is based on that patent, and includes processing steps that separate RTA-30 from RTA-33. 1. Ricin Extraction from Whole Castor Beans.

Whole Castor beans are mechanically ground, and ricin extracted from the meal with a solution of 15 0.9% saline. This solution was filtered from the bean pellet and lipid layer using a Celite Filter Aid and Aerosil Adsorbent (Manville, Denver, CO; Degussa, Frankfurt). The filtrate was concentrated and then diafiltered against Tris Lactose, pH 7.8 (50mM lactose, 20 lOmM Tris pH 7.8, 50mM NaCl), and passed through a QAE ZETA prep cartridge (AMF-Cuno, LKB Instruments, Pleasant Hill, CA). The resultant material was diafil-tered against a Tris saline solution (lOmM Tris, 0.9% NaCl, pH 7.8) . 2. Ricin Toxin A-Chain Separation.

The diafiltrate is applied to a Sepharose 4B column (Pharmacia Fine Chemicals, Piscataway, N.J.) and the nonbinding flow-through containing ricin was loaded onto an acid-treated Sepharose column in order to sepa-30 rate the ricin toxin A-chain from the whole ricin (as described in U.S. 4,590,071, column 3, lines 26-52). The eluant thus obtained was diafiltered against Tris buffer (lOmM Tris, lOmM NaCl), and the resulting filtrate was passed through a QAE Sepharose Fast Flow col-35 umn (Pharmacia Fine Chemicals) equilibrated to the same buffer. The RTA obtained above was adjusted in NaCl concentration to 0.9 wt.%, and purified to remove toxin 22 5 3 72 B-chain impurities by applying to a Sepharose Column previously coupled to goat anti-RTB antibodies. 3. RTA Species Separation.

After diafiltration against 20mM sodium ace-5 tate , pH 5.5, the RTA solution was bound to an S-Sepha-rose column, and eluted using a dual pH/salt gradient (pH 5.5-7.5, 0-0.09 M NaCl). The resulting two peaks represent substantially pure RTA-33 and RTA-30, con-'w' sisting of molecular weight species of 33 and 30 kD, respectively. The solution was concentrated and glycerol added to 10% for storage. This RTA solution was reduced with dithiothreitol (DTT) (as described in U.S. 4,590,071, column 4, lines 33-48, except that the buffer contained 5% dextrose instead of azide). 15 4. Immunotoxin Preparations The cell line XMMME-001, which secretes a human melanoma specific monoclonal antibody, was deposited with the A.T.C.C. and designated Accession No. HB8759. Immunotoxins utilizing that monoclonal anti-20 body were prepared as detailed in U.S. Patent No. 4,590,071, except that RTA-30 or RTA-33 was substituted for RTA.

O Another immunotoxin utilizing the H-65 anti body (A.T.C.C. No. HB9286) was prepared as follows: 25 An H-65 tissue culture harvest was concen trated and the pH adjusted to 8.5. The solution was applied to an immobilized Staph. Protein A Column and eluted with 0.1 M Citrate, pH 4.5. The eluate was diafiltered against lOmM Hepes Buffer, 0.25 M NaCl, 30 pH 7.3, and then applied to a QAE Sepharose Fast Flow column. The antibody passed through the column, and was diafiltered against PBS, pH 7.0, 5% dextrose. The antibody was activated for coupling to the RTA with SPDP (as described in U.S. 4,590,071, column 4, line 35 55, column 5, line 5, except that the buffer contained 5% dextrose instead of azide). 22 5 3 o A concentrated RTA-30 solution and the H-65 solution were placed together in a formulation buffer consisting of lOmM P04, pH 7.0, 0.15 M NaCl, and 5% dextrose. This solution was applied to a Sephacryl S-200 HR column (Pharmacia Fine Chemicals), which had been pre-equilibrated with PES containing 5% dextrose, and the immunotoxin eluted as fractions (as described in U.S. 4,590,071, column 5, lines 15-24). TWEEN 80 v*/ was added up to 0.1% in the final solution.

B. Pharmacokinetics and Tissue Distribution of RTA-30 and RTA-30 Immunotoxins. 1. Immunotoxin Radioiodination.

XMMME-0 01-RTA-30 and XMMME-0 01-RTA-33 , puri-TM fied on Cibacron blue (Ciba-Geigy, Los Angeles, CA) 125 coupled to Sepharose, were radiolabeled with I and 131 I, respectively, using 1,3,4,6-tetrachloro-3a,6a-di- phenylglycouril (Sigma Chemical Co.; Iodo-Gen, Pierce Chemical Co.) in an adaptation of the method of Mark- well, Pierce Bio-Research Products Technical Bulletin (1983). Iodo-Gen was dissolved in dichloromethane to a concentration of 1.0 mg/ml, and 50ml (i.e., lOmg/lOOmg protein) was dried onto the bottom of each reaction (^} vial under a stream of N2- The vials were rinsed once with lOmM phosphate-buffered saline (PBS), pH 7.0.

XMMME-001-RTA-30, 0.50mg in 0.40ml, was then added to 125 one of the vials, followed by 0.50mCi of I. XMMME- 001-RTA-33, 0.50mg in 0.33ml, was added to the other ^ 131 vial, ifollowed by 0.50mCi of I. Both reactions were carried out at room temperature for 30-45 minutes with occasional agitation. The radiolabeled immunotoxins were then separated from free radiolabel on 2ml columns of Sephadex G-25 in PBS, pH 7.0, by brief centrifuga- tion (Tuszyaski e_t al_. , Anal. Biochem. 106:118-122 (1980)) . Specific activity of XMMME-0 01-RTA-30 6 131 was determined to be 1.85x10 cpm/mg and that of I- XMMME-001-RTA-33 was determined to be 1.65x10^ cpm/mg.

In both samples, greater than 99% of radioactivity was 2 5 3 7 2 protein bound (precipitable by trichloroacetic'^ acid) . The labelled immunotoxins were diluted in PBS, pH 7.4 with human serum albumin (lmg/ml) to contain approximately 0.07mCi/ml. 2. Animal Preparation.

Male, Balb/C mice, weighing 20-25 grams, were divided into 7 groups of three animals each. At T=0, each animal received a single intravenous dose (150 ml, 2.5 mCi/isotope, tail vein) containing both the labeled 10 samples. Three animals from each group were necropsied at T=3, 30, 90, 180, 360, 1080, and 1440 minutes. A blood sample was taken by cardiac puncture prior to necropsy. The following organs were weighted and 125 131 counted for Iodine and Iodine: liver, spleen, kidneys, serum (100 ml), and a portion of the carcass (hindquarter). The isotopes were counted using an LKB Autogamma counter, set for dual isotope counting and automatic decay and spillover correction. These data were used to calculate the percent of the injected dose 20 and the percent of dose/gm in the serum and organs.

Other animals were perfused with heparinized PBS in order to remove the majority of the blood from the tissues. In this procedure, the animals were anesthetized, and a blood sample taken by cardiac puncture. 25 The chest cavity was opened, and a 27 gauge butterfly was inserted into the left ventricle. After opening the right atrium with iris scissors, the animal was perfuied through the left ventricle with 20 mis of cold PBS containing heparin (1 U/ml). The following organs 125 131 were .removed, weighed and counted for I and I: spleen, kidneys, liver, and a portion of the carcass. 3. Results.

The labelled immunotoxins were run on a 3-12% gradient SDS-PAGE gel with molecular weight markers. 35 The gels were stained with Coomassie blue and auto-radiographed, then cut into sections for counting.

/"-N O 3 n There were four bands visible on the^ stained gradient gels. These bands corresponded to an albumin band and three immunotoxin bands representing antibody conjugated to 1, 2, or 3 RTA chains. Autoradiography 5 of these gels indicated that a majority of the radioactivity was associated with the immunotoxin bands.

When the gels were cut and counted, 88% of the activity ^ was recovered in the immunotoxin bands (approximately 27% in each band) . The remaining activity was associ-10 ated with an area of the gel corresponding to free antibody (6%) or the area between free antibody and the dye front (6%).

The pharmacokinetics of the immunotoxins are shown in Figure 1. The plasma clearance curve of each 15 isotope was biphasic, showing an initial rapid decrease followed by a slower phase. The biodistribution data are shown in Figures 2-4. During the initial phase, the XMMME-OOl-RTA-33 was removed from the plasma compartment more rapidly than the XMMME-001-RTA-30 (47.5 20 vs 81.9% of the injected dose at T=3 minutes). The percentage of the injected dose in the plasma compartment was two-fold higher than that of the XMMME-001-RTA-33 during the study period. These differences could be attributed to a higher localization of the 25 XMMME-OOl-RTA-33 in the liver as compared to the XMMME-001-RTA-30. At T=3 through 360 minutes, the liver ^ localization of the XMMME-OOl-RTA-33 was up to two-fold higher than that of the XMMME-001-RTA-30. The distribution of the conjugates was not significantly dif-30 ferent in any of the other tissues examined.

As compared to the unperfused animals, the PBS perfusion of the animal prior to necropsy reduced the activity in the liver, kidneys, and carcass. These results are shown in Figure 5-6. The perfusion of the 35 animals reduced the activity (expressed as percent of dose) in the carcass by approximately 50%. The activity in the kidneys was reduced to nearly undetectable 22 5 3 7 2 r5 G levels and liver activity (Figure 6) was reduced by about 30%.

The data indicated that there were significant differences in the tissue distribution and the pharmacokinetics of the immunotoxins made with RTA-3 0 or RTA-33. The plasma residence times were significantly increased and the localization in the liver was significantly decreased with the XMMME-001-RTA-30 as compared to the XMMME-OOl-RTA-33. These results are consistent with the hypothesis that the in vivo clearance of these samples is mediated by carbohydrate residues recognized by receptors in the reticuloendothelial system. The carbohydrate residues remaining on RTA-30 ensure that the blood half-life will not be so large as 15 to unduly increase nonspecific toxicity.

An increased plasma residence time of the immunotoxin results in increased tumor localization. This, coupled with the similarity in specific toxicities between the RTA-30 and RTA-33 immunotoxins, pro-20 vides substantially improved immunotoxin-based therapies.

From the foregoing, it will be appreciated that the use of immunotoxins enriched in RTA-30 for in vivo therapy substantially reduces the blood clear-25 ance time of the immunotoxin, without interfering with the immunotoxin's specific toxicity. Thus, smaller doses of immunotoxin treatments are feasible, which reduce the side effects of immunotoxin therapy and improve the patient's prognosis for the entire treat-30 ment". Moreover, allergic reactions and other harmful aspects of an immune response generated against the immunotoxin are diminished. The production of RTA-30 enriched immunotoxins remains substantially the same as for prior RTA-based immunotoxins, minimizing additional 35 quality control and economic considerations.

Although the present invention has been described in some detail by way of example for purposes 2 5372 of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (25)

22537 -20- WHA7/vVE

1. A method for inhibiting the expansion or activity of a predetermined cell population in a non-human patient, said method comprising administering to said patient, or fluids from said patient, an effective dose of an immunotoxin preparation comprising a binding component capable of attaching to said cells and a ricin A-chain (RTA) complexed with the binding component, 10 wherein the concentration of RTA utilized in the.immunotoxin preparation comprises at least 75% RTA-30. 15

2. A method according to Claim 1, wherein the RTA-30 concentration in the immunotoxin preparation •' 20 is at least 85% to 95% of the KEA in said iirmunotoxin.

3. A method according to Claim 1, wherein the binding component is an immunoglobulin or binding fragment thereof- 25

4. A method according, to Claim 3, wherein the immunoglobulin is a monoclonal antibody.

5. A method according to Claim 1 wherein 30 said-cells are immune cells. i

6. A method according to Claim 5,^whereiri^^. the immune cells are from a bone marrow transplant donor. 35

7# A method according to Claim 1, wherein said cells are tumor cells. 2^5372 -21-

A pharmaceutical composition ~~~~ i comprising immunotoxins admixed in a pharmaceutically acceptable carrier, --wherein a plurality of said inmunotoxins comprise a 5 specific binding component: complexed with a ricin toxin A-chain (RTA) component, wherein the relative amount of RTA-30 species in the RTA of said immunotoxins comprises at least 75% of the RTA in said immunotoxin. 10 15

9. A pharmaceutical composition according to Claim 8, wherein the RTA-3 0 species comprises at least 85% to 95% of the RTA in said immunotoxin.

10. A composition according to Claim 8S 20 wherein the RTA component in the immunotoxins is purified from ricin with an immunoaffinity column.

11. A composition according to Claim 8 , wherein the immunoaffinity column comprises antibodies 25 specifically reactive with ricin B-chains.

12. A composition according to Claim 8, wherein 'the specific binding component is reactive »«*th a cellular marker. 30

13. A composition according to Claim 12, wherein the marker is a cell surface antigen.

14. A composition according to Claim 8» 35 wherein the specific binding component is anyimmuno-globulin or a binding fragment thereof. ' vVJ , m 3~ i\ Y+-' O (- U r—' -22-

15 . A composition according to Clai^i 14 , wherein the immunoglobulin or binding fragment thereof is covalently bound to said immunotoxin. 5

16 . A composition according to Claim 14a wherein the covalent bond is a disulfide bridge or a peptide bond.

17. A composition according to Claim"_14, 10 wherein the immunoglobulin is a monoclonal antibody.

28. A composition according to Claim 8, wherein the specific binding component is complexed to the RTA component through a carbohydrate moiety of one 15 of the components.

19. A method for treating a non-human patient with a disease state susceptible to immunotoxin therapy, said method comprising administering to said patient an ef- 20 fective amount of a composition according to Claim 8.

20. A method of treating blood of a non-human mairmal extracorporeally to remove a predetermined cell population comprising the steps of: 25 (a) removing the blood from the mammal under conditions which prevent clotting; (b) contacting the blood with an immunotoxin comprising a monoclonal antibody which reacts with markers specific for the cell population and is com- 30 plexed with a ricin toxin A-chain preparation enriched with RTA-30 whereby the immunotoxin binds to cells of the.cell population; (c) separating the bound cells from the blood; and 35 (d) returning the blood ta^-sfa&e mammal. 225872 10 -23-

21. A method of increasing the effitacy of ricin-based immunotoxin therapy, said method comprising removing, from ricin, species of ricin toxin A-chain (RTA) other than RTA-30 prior to conjugation of the RTA to a specific binding component.

22. A method according to Claim 21 > wherein the removal is performed with ion exchange chromatography.

23. A method for enhancing the effectiveness of ricin toxin A-chain (RTA)-based immunotoxin therapy to improve a disease condition in a non-human patientj said method comprising administering to the patient an immuno-15 toxin preparation comprising substantially pure RTA-30 complexed with an immunoglobulin reactive with cells in said patient at least partially responsible for said disease condition. 20

24. A kit for use in the diagnosis or treat ment of a disease state, said kit comprising a container of lyophilized immunotoxin having a ricin-toxin A-chain (RTA) component, wherein the ricin A-chain component comprises substantially pure RTA-30. 25

25. A method for inhibiting the expansion of activity of a predetermined cell population in a non-human patient substantially as herein described with reference to the examples. i By I ii!/W»i/Their Authorised Aaerrts 30 " A. J. PARK & SON Per