AU626087B2 - Improved immunotoxin therapies utilizing purified ricin a-chain species - Google Patents

Description

W-AI -21227/88 WORLD INTELLECTUAi. PROPERTY ORGANIZATION internationl BU reu 0

PCT

INTERNATIONAL APPLICATION PUBLISHIED UN DER TH E PATENT COOPER 4'TION TREATY (PCT) International Patent Classification 4 (11) International Publication Number: WO 89/ 00583 C07K 15/00, 17/00, 3/12 A I C07 K 3/22, A61IK 39/395, 39/44 (43) International Publication Date: 26 January 1989 (26.01.89) (21) International Application Number: PCT/US8S/02343 (22) International Filing Date: (31) Priority Application Number: (32) Priority Date: (33) Priority Country: 12 July 1988 (12,07.88) (1,4,824 17 July 1987 (17.07.87) (81) Dcsignaied States: AT (European patent), AU, BE (Eu-, ropean patent), CH (European patent), DE (European patent), FR (European patent), GB (European patent), IT (European patent), JP, KR, LU (European pat NL (European patent), SE (European pa-: tent), Pu biisht~d Withi inflerno tio,;aI search report.

Before the expiration of/the time limitfir amnending Mhe claims and to be republished in tile event ol/the receipt of amnendmnens, (71) Applicant: XOMA CORPORATION (US/US]; 2910 Seventh Street, Berkeley, CA 947 10 (US), (72) Inventors: SCANNON, Patrick, 1. ;Routr. 1, Box 2141, Davis, CA 95616 KAWAHATA, 'Russell, T. 84 Berkeley Avenue, San Anselmo, CA 94960 (US), (74) Agent; SMIITH, William, NI.; Townsend and Townsend; 0-ne Niarket Plaza, 2000 Steuart Tower, San Francisco, CA 04105 (US).

A.D0. j, p, 6 APR VT9 PATE0,Y OFICEj 626087 (54)Title; INIPROVED ININUNOTOXIN THERAPIES UTILIZING PURIFIED RICIN A-CHAIN SPECIES PHA9A'REI 'NSV~ XNAXE- I!-RTA LZ (57) Abstract Improved immunotoxin th'irapies are obtained by ulilizing immunotoxins uo-imprising ricin enriched in purifled rictn A-chiin species in. conjunction with specific biintg components, particularly monoclonal antibodies speci~ically reactive with markers of predetermined cell popv'iations.

zo I- 200 400 800 00O 0,0 0CM '4X !6 RME AFTER :NJECTION NtROjTE$ FARAO0cET13 or 1111- XNXWE^,'-RTA 33 '.4 ME AVEIR RJEOT'OM 'WN'

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WO 89/00583 PCT/US88/02343 -1- IMPROVED IMMUNOTOXIN THERAPIES UTILIZING PURIFIED RICiN A-CHAIN SPECIES Field of the Invention This invention relates generally to the use of immunotoxins in chemotherapy and other human t-eatment applications and, more particularly, to improving the pharmacokinetics and toxicity characteristics of ricin-based inunmmunotoxins.

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 potential, 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 tiese monoclonal antibodies could be tilized to selectively deliver a toxic agent to a predetermined cell population, such as a tumor, in a Spatient. Thus, cancerous cells or other diseased cellular 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 medical successes have been reported, despite extensive research efforts. In practice, finding the appropriate combination of antibody and toxin that can actually I I I i i I 1L1 WO 89/00583 PCT/US88/02343 -2improve a chemotherapeutic or other therapy has proven extremely difficult.

The most widely used toxin component of immunotoxins is the ricin toxin A-chain. Ricin is a plant 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 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 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 tartet cells without damaging unrel'.d cells. Unfortunately, the in vivo utility of these RTA-based immunotoxins have generally been less thar ideal, perhaps because of rapid clearance from the blood stream which would reduce the amount of immunotoxin available to interact 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 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 pa'tern; typically by reacting RTA with chemicals, such as sodium metaperiodate and sodium cyanoborohydride, or through enzymatic deglycosylation treatment, such as T^-ii WO 89/00583 PCT/US88/02343 -3with alpha-mannosidase. The94 attempted modifications of the natural glycosylation pf RTA have resulted in decreased in vivo blood clearance times of the modified RTA. The treatments are genaeally undesirable, hewever, 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.

Moreover, the potential for quality control problems becomes greatly magnified.

Thus, there is a significant need for improved RTA-basc.d immunotoxins exhibiting superior in vivo properties. The immunotoxin should retain high specific 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 preferred to minimize nonspecific toxicity in the host. The present invention fulfills these needs.

I SUMMARY OF THE INVENTION I ~The present invention provides novel methods for the in vivo treatment of a patient utilizing immunotoxins comprising a specific bindinq component complexed 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 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

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WO 89/00583 PCT/US88/02343 -4techniques 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 harmfr' cell populations from a patient, with minimal nonspecific toxicity. Pharmaceutical compositions are also provided for use in the treatments.

BRIE' DESCRIPTION OF THE FIGURES 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 studies ltilizing immunotoxins having different RTA species.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Novel methods are provided for improving 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 tound in naturally-occur ting ricin, increased blood residence time of the immunotoxin is achieved, without significantly increasing nonspecific toxicity. In general, the immunotoxins of the pnesent invention have less non-specific toxicity.

Thus, the methods and compositions of the present invention provide means for substantially improved treatments for, 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" rmfrs to a 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 -a I WO 89/00583 PCT/US88/02343 (1986) and Vidal et al. Int. J. Cancer, 36:705L711 (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 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 between the two species is that RTA-30 experimentally exhibits lower glycosylation than RTA-33, with the RTAspecies having a single complex oligosaccharide, and the RTA-33 having a high mannose type oligosaccharide in addition to the complex unit found on the (see, Foxwell et al., Biochem. Biophvs. 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, 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 Foxwell et al., suora). These can include gel filtration, anAon or cation exchange chromatography, electrophoresis, hydrophobic chromatography, affinity chromatography, and the like. A p-eferred means of separation is based on the different glycosylation patterns between RTA-30 and RTA-33. Carboxymethylcellulose 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 different affinities for RTA-33 and RTA-30. It will be readily apparent to those skilled in the art that these se~ration procedures are reproducible and economical, :I ii WO 89/00583 PCT/US88/02343 -6and do not produce contaminating by-products, unlike many chemical modification processes.

By following the above procedures, concentrations in the immunotoxin preparations may be increased well above the level in naturally occurring ricin about 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 in 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 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 eisure that the toxic agent is not separated from the' binding agent until attachment of t:he entire immunotoxin to a cell within a preselected cell population. The two components are usually chemii 25 cally bonded together by any of a variety of well-known chemical procedures.

For example, when the cytotoxic agent is RTAand the second component is an intact immunoglobu- A lin, such as a monoclonal antibody, the linkage may be by way of heterobifunctional linkers, such as, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) carbodiimide, gluteraldehyde, 2-iminothinlane or the like, to form peptide, amide, ester, thioester, disulfide bridees 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

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WO s39100583 PCT/US88/02343 -7about 1-2 RTA-30 moieties, preferably 2-3 or more, 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 Conjugates; 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 delivery vehicle for the cytotoxic agent in the immunotoxin, can be obtained from a number of sources. Pre- I ferably, intact immunoglobulins or their fragments, such as Fv, Fab, F(ab 2 half antibody molecules, a single heavy/light c.ain 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 capa- Sble of binding to markers, including growth factor or 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 techniques. Recent technical advances have provided additional forms of immunoglobulins and methods of making them. For example, the utilization of recombinant DNA technology; has produced functional, assembled immunoglobulins or hybrid immunoglobulins the constant region from human monoclonal antibodies combine with mouse variable regions) suitable for use in immutioxins (see, EPA 84302368.0, which is incorporated herein by reference).

Typically, the antibodies are capable of binding to epitopes of markers on selected cell populations, such as neoplastic calls or T-cells. The marker is generally a unique surfaci protein, but a large WO 89/00583 PCT/US88/02343 -8variety of markers, such as other proteins, glycoproteins, lipnproteins, polysaccharides and the like, which are produced by or displayed by the cells to be recognized by the immunotoxin, can be utilized in accordance 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 and do not form part of the present invention.

The immunotoxin may be utilized in prophylactic 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 specific binding protein of a immunotoxin may recogiize markers on tumor cells, immune cells T-cells or B-cells), hormone responsive celln to insulin) and growth factor responsive cells to interleukins), fungi, bacteria, parasites, or virus infected cells. Blood from the mammal may be combined extracorporeally 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 dosages for the treatment of human melanoma as described in U.S. Patent No. 4,590,071. Hybridoma cell line XMIME-001 was deposited with the American Type Culture Collection (ATCC) and given ATCC Accession No. HB 8759.

Allnu Lu -i-nIcr~2rpr -'he p ecifc bt ng Scomponent and RTA as the toxin _t een sed in FDA-approved Phase X an- I Clinical Trials, the proto- -Md r suLts of which are descri bed in detail in WO 89/00583 PCT/US88/02343 -9- SBriefly, the immunotoxin was administered to patients in the form of intravenous injections of 0.4 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 oncoloa4 dose of an immunosuppressive agent, such as methotrexate, cyclophosphamide, prednisone, or cyclosporine, in order to Dlunt the immune system to prevent 'immune response against the immunotoxin. Depending on the immune response mounted against the imrnunotoxin, the treatment may be repeated, up to three times. Immunotoxins incorporating enhanced levels of RTA-30 will require substantially smaller dosages to be effective, typically at least about 10-25% less, but in some therapies abotit to less.

In accordance with another embodiment of the present invention, iminunotoxins are utilized prophy2,ac- 2 tically in improving bone marrow transplantation, by reducing the likelihood of egraft versus host disease (GVHD) ,as follows: Patients can receive bone marrvw transplantations (BMT) in order to treat a variety of diseases, such as hemnatological malignancies, aplastic anemia, Severe combined immunodeficiency (SCID) or variants, certain inborn errors of metabol~sm, or certain solid tumors. In some situations, bcne marrow donors fall into categories of genotypically hapiotype matched or unrelated partially-phenotypic lILA mat-hed. These categories of donors result in a 100% incid3encpa of GVHiD in the recipient. BMT treatment with allooeneically matched sibling donors results in an incidence Of GV110 Of about 30 or more.

3$ After MIT, at a time following Qevidencoe of hematopoietic recovery and pri~r t* rapid, proliferation~ J~l~ of Gvnl-produtinra cells, ar, iunoctodn Iveactive with WO 89/00583 PCT/US88/02343 GVHD-producing mature T cells is infused. A preferred immunotoxin, Xr4MLY-H65-RTA-30, consists of an (pan T lymphocyte) specific monoclonal antibody conjugated to RTA-30. The iicmunoto.Nin can be infused starting 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 immrunotoxin is described in U.S. Serial No. 938,855, which is incorporated by refcrence herein. This application also describes typical protocols for the treatment of GVHD in BMT recipients, as well as characterization of the monoclonal antibody. The hybridoma produicing was deposited with the ATCC and given ATCC Accession No. 11B 9286.

In vitro studies have demonstrated that immunotoxin will kill T-cells when incubated with human marrow without causing toxicity to hematopoietic pxr-genitor cells, The biolog'ic activity of this pan-T-cell iminunotoxin indicates thatc it can be a potent anti-T-cell cytotoxinp able to abrogate T-ce'l reactions contributing to the pathogenesis of Gym.I), particularly when the ElTA component is enriched with Pre-clinical in vitro, studies in animal models has indicated that the toxicity of PTA immunotoxins was low. Rats were given 14 consecutive doses of 2.4 RTA.-imrnunotoxin and the principal toxicity was transient hypoalbuminemia and occasional Mild elevati-on in liver enzymos, Monkeys treated with the immunotoxin 3 0 experienced various side-effects that were reversible.

Since XMLY-1165 is not known to bind to any non-human tissues (except weakly to monkey 5ranulocytes) this, toxicity, at high dogses, is considered non-directed and non-specii,: possibly U to RTA The use 0O RTA~ en'riched with RTA-30 wilL robstanti~ally lessen this nonspecific tox~icity.

WO 89/00583 PCT/US88/02343 -11- Depending upon the particular therapy, the immunotoxins of the present invention will be commonly incorporated as components of pharmaceutical compositions. Such compo ns will contain a therapeutic amount of the immunotuxins 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 dater (with or without 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 be used as separately administered compositions or in conjunction with other cytotoxic agents. These can include various immunotoxins and chemotherapeutic drugs, such as vindesine, methotre.ate, adriamycin, and cisplatinum, various radionuclides, and the like. Pharmaceutical 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 normal saline and about 0.1 mg of immunotoxin.

An amount adequate to accomplish at least partial killing of a cell popul.at.n 6s defined as a "therapeutically effective dose." amounts needed to achieve this dosage will depend upon the severity of 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 applications, compositions containing the present immunotoxins or Cocktails, may also be administered in similar dosaaes.

II WO 89/00583 PCT/US88/02343 -12- 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 response 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 tolerated. 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, for example, Remington's Pharmaceutical Science, 16th Ed., 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 states. Thus, the subject immunotoxins of the present Sinvention may be provided in containers, usually in a lyophilized form, either alone or in conjunction with additional immunotoxins or non-complexed antibodies specific for desired epitopes. The immunotoxins and antibodies, which may be conjugated to a label or unconjugated, are included in the ],its with physiologicaily acceptable buffers, in accordance with the teaching of the art. Generally, these materials will be h present in less than about 5% wt. based on the amount 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 may be present ih from about 1% to 99% wt. of the total composition.

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WO 89/00583 PCT/US88/02343 -13- The following examples are offered by way of illustration and not limitation.

EXPERIMENTAL

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 following 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 0.9% saline. This solution was filtered from the bean p..3let and lipid layer using a Celite Filter Aid and Aerosil Adsorbent (Manville; Denver, CO; Degussa, Frankfurt). The filtrate was concentrated and the;n diafiltered against Tris Lactose, pH 7.8 (50mM lactose, 10mM Tris pH 7.8, 50mM NaC1) and passed through a QAE ZETA prep cartridge (AMF-Cuno, LKB Instruments, Pleasant Hill, CA), The resultant material was diafiltered against a Tris saline solution (10mM 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, and the nonbinding flow-through containing ricin was loaded h onto an acid-treated Sepharose column in order to sepirate 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 dianiltered against Tris buffer (10mM Tris, 10mM NaCl) and the resulting filtrate was passed through a QAE Sepharose Fast Flow column (Pharmacia Fine Chemicals) equilibrated to the same buffer. The RTA obtained above w'as adjusted in NaCI concentration to 0.9 and ourified to remove toxin II ~I WO 89/00583 PCT/US88/02343 -14- 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 acetate, pH 5.5, the r(TA solution was bound to an S-Sepharose column, and eluted using a dual pH/salt grad.ent (pH 5.5-7.5, 0-0.09 M NaC1). The resulting two peaks represent substantially pure RTA-33 and R'A-30, consisting of molecular weight species of 33 and 30 kD, respectively. The solution was concentrated and glycerol add'ed to 10% for storage. This RTA solution was reducew with dithiothrei.tol (DTT) (as described in U.S.

4,590,071, column 4, lines 33-48, except that the buffer contained 5% dextrose instead of azide).

4. Immunotoxin Preparations The cell line XMMME-001, which secretes a numan melanoma specific monoclonal antibody, was depos-.

ited with the A.T.C.C. and designated Accession No.

HB8759. Immunotoxins utilizing that monoclonal antibody were prepared as detailed in U.S. Patent No.

4,590,071, except that RTA-30 or RTA-33 was substituted for RTA.

Another immunotoxin utilizing the H-65 antibody No. HB9286) was prepared as follows: An H-65 tissue culture harvest was concentrated 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 10mM Hepes Buffer, 0.25 M NaCl, pH 7.3, and then applied to a QAE Sepharoso Fast Flow column. The antibody passed through the column, and was diafiltered against PRS, 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 55, column 5, line 5, except that the buffer contained d<."trose instead of azide).

WO 89/00583 PCT/US88/02343 A concentrated RTA-30 solution and the H-6, solution were placed together in a formulation buffer consisting of 10mM P04, pH 7.0, 0.15 M NaC1, arid dextrose. This solution was applied to a Sephacryl S-200 HR column (Pharmacia Fine Chemicals), which had been pre-equilibrated with PBS containing 5% dextrose, and the immunotoxin eluted as fractions (as described in U.S. 4,590,071, column 5, lines 15-24). TWEEN was added up to 0.1% in the final solution.

B. Pharmacokinetics and Tissue Distribution of and RTA-30 Immunotoxins.

1. Immunotoxin Radioiodination.

XMMME-001-RTA-30 and XMMME-001-RTA-33, purified on CibacranTM blue (Ciba-Geigy, Los Angeles, CA) 125 coupled to Sepharose, were radiolabeled with 125I and 131 I, respectively, using 1,3,4,6-tetrachloro-3a,6a-diphenylglycouril (Sigma Chemical Co.; lodo-Gen, Pierce Chemical Co.) in an adaptation of the method of Markwell, Pierce Bio-Research Products Technical Bulletin (1983). Iodo-Gen was dissolved in dichloromethane to a concentra. ion of 1.0 mg/ml, and 50ml 10mg/100mg protein) was dried onto the bottom of each reaction vial under a stream of N 2 The vials were rinsed once with 10mM phosphate-buffered saline (PBS), pH XMMME-001-RTA-30, 0.50mg in 0.40ml, was then added to one of the vials, followed by 0.50mCi of 125I. XMME- 001-RTA-33, 0.50mng in 0.33ml, was added to the other vial, followed by 0.5OmCi of 131I. 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 centrifugation (Tuszyaski et al., Anal. Biochem. 106:118-122 (1980)). Specil'ic activity of 15I XMMME-001-RTA-30 was determined to be 1.85x10 cpm/mg and that of 13 XMME-001-RTA-33 was determined to be 1.65x10 6 cpm/mc.

In both samples, greater than 993 oi radioactivit was WO 89/00583 PCT/US8S/02343 -16protein bound (precipitable by trichloroacetic acid) The labelled immunotoxins were diluted in PBS, pH 7.4 with human serum albumin (Img/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 rl, mCi/isotope, tail vein) containing both the labeled 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 131 Iodine: liver, spleen, kidneys, serum (100 ml), and a portion of tbo 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 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, thet animals were anesthetized, and a blood sample taken by cardiac puncture.

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 were removed, weighed and counted for 125I and 131: 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.

The gels were stained with Coomassie blue and autoradiographed, then cut into sections for counting.

.4 WO 89/00583 PrCT/US88/02343 -17- 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 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 associated with an area of the gel corresponding to free antibody or the area between free antibody and the dye front The pharmacokinetics of the immunotoxins are shown in Figure 1. The plasma clearance curve of each 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-001-RTA-33 was removed from the plasma compartment more rapidly than the XMMME-001-RTA-30 (47.5 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 XMMME-001-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-001-RTA-33 was up to two-fold higher than chat of the XMMME-001-RTA-30. The distribution of the conjugates was not significantly different 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 animals reduced the activity (expressed as percent of dose) in the carcass by approximately 50%. The activit, in the kidneys was reduced to nearly undetectable WO 89/00583 PCT/US88/02343 -18levels and liver activity (Figure 6) was reduced by about The data indicated that there were significant differences in the tissue distribution and the pharmacokinetics of the immunotoxins made with 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-001-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 ensure that the blood half-life will not be so large as 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, provides substantially improved immunoti.in-based therapies.

From the foregoing, it will be appreciated that the use of immunotoxins enriched in RTA-30 for in vi-'o therapy substantially reduces the blood clearance 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 treatment. Moreover, allergic reactions and other harmful aspects of an immune response generated against the immunotoxin are diminished. The production of enriched immunotoxins remains substantially the same as for prior RTA-based immunotoxins, minimizina additional quality control and economic considerations.

Although the present invention has been described in some detail by way of example for purposes WO 89/00583 WO 89/u)583PCT/US88/02343 -'19of clarity aned understanding, it will be apparent that certain chaeiges and modifications may be practiced within the scope of the appended claims.

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Claims (22)

1. A method for inhibiting the expansion or activity of a predetermined cell population in a pa- tient, said method comprising administering to said pa- tient, or fluids from said patient, an effective dose of an immnunotoxin preparation comprising a bindLng com- ponent capable of attaching to said cells and a ricin A-chain (RTA),complexed with the binding component, wherein the concentration of RTA utilized in the immu- notoxin preparation is substantially enriched in over naturally-occurring ricin concentrations.

2. A method according to Claim 1, wherein the RTA-30 concentration in the immunotoxin preparation is at least about

3. A method according to Claim 1, wherein the RTA-30 concentration in the immunotoxin preparation is at least about 85% to

4. A method according to Claim 1, wherein the binding component is an immunoglobulin or binding fragment thereof. A method according to Claim 4, wherein the immunoglobulin is a monoclonal antibody.

6. A method according to Claim 1 wherein said cells are immune cells.

7. A method according to Claim 6, wherein the immune cells are from a bone marrow transplant donor.

8. A method accovding to Claim 1, wherein said cells are tumor cells. WO 89/00583 PCT/US88/02343 -21-

9. A pharmaceutical composition for use in the in vivo treatment of a patient comprising, immuno- toxins admixed in a pharmaceutically acceptable car- rier, a plurality of said immunotoxins comprising a specific binding component complexed with a ricin toxin A-chain (RTA) component, wherein the relative amount of species in the RTA of said immunotoxins is in- creased over the amount of the RTA-30 species in natu- rally-occurring ricin. A pharmaceutical composition according to Claim 9, wherein the RTA-30 species comprises at least about 7!5% of the RTA in said immunotoxin. A pharmaceutical composition according to Claim 9, wherein the RTA-30 species comprises at least about 85% to 95% of the RTA in said immunotoxin.

12. A composition according to Claim 9, wherein the RTA component in the immunotoxins is puri- fied from ricin with an immunoaffinity column.

13. A composition according to Claim 9, wherein the immunoaffinity column comprises antibodies specifically reactive with ricin B-chains.

14. A composition according to Claim 9, whern the specific binding component is reactive With a cellular marker. A composition according to Claim 14, wherein the marker is a cell surface antigen.

16. A composition according to Claim 9, wherein the specific binding component is an immuno- globulin or a binding fragment the!.eof. WO 89/00583 PCT/US88/02343 -22-

17. A composition according to Claim 16, wherein the immunoglobulin or binding fragment thereof is covalently bound to said immunotoxin.

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

19. A composition according to Claim 16, wherein the immunoglobulin is a monoclonal antibody. A composition according to Claim 9, wherein the specific binding component is complexed to the RTA component through a carbohydrate moiety of one of the components.

21. 'A method for treating a patient with a disease state susceptible to immunotoxin therapy, said method comprising administering to said patient an ef- fective amount of a composition according to Claim 9.

22. A method of treating blood of a mammal extracorporeally tc remove a predetermined cell popula- tion comprising the steps of: removing the blood from the mammal under conditions which prevent clotting; contacting the blood with an immunotoxin comptising a monoclonal antibody which reacts with markers specific for the cell population and is com- plexed with a ricin toxin A-chain preparation enriched with RTA-30) whereby the immunotoxin binds to cells of the cell population; separating the bound cells from the blood, and returning the blood to the mammal. I I i I 23

23. A method of increasing the efficacy of ricin-based immunotoxin therapy, said method comprising removing, from ricin, species of ricin toxin A-chain (RTA) other than RTA-30 and conjugating the RTA to a specific binding component.

24. A method according to Claim 23, wherein the removal is performed with ion exchange chromatography. A method for enhancing the effectiveness of ricin toxin A-chain (RTA)-based immunotoxin therapy to improve a disease condition in a patient, said method comprising administering to the patient an immunotoxin preparation comprising substantially pure RTA-30 complexed with an immunoglobulin reactive with cells in said patient at least partially responsible for said disease condition. 26, A kit for use in the diagnosis or treatment of a disease state, said Kit comprising a ontalner of lyophilized immunotoxin having 15 a ricin-toxin A-chain (RTA) component conjugated to a specific binding component, wherein the ricln A-chain component comprises substantially pure 27, A method for inhibiting the expansion or activity of a predetermined cell population in a patient said method as set out in claim 1 and substantially as hereinbefore described, 28, A pharmaceutical composition for use in the ln vivo treatment of a patient said composition has set out in claim 9 and substantially as hereinbefore described,

29. A method of treating blood of a mammal extracorporeally to remove a predetermined cell population said method as set out In claim 22 and substantially as hereinbefore dscribed, 30, A method of increasing the efficacy of a ricin-based mniunotoxln therapy said method as set out in claim 23 and substantially as herelnbefore described.

31. A method for enhancing the effectiveness of ricin toxin A-chain (RTA)-based immunotoxin therapy said method as set out Ir claim and 'ubstantially as hereinbefore described. CI 24

32. A kit for use in the diagnosis or treatment of a disease state said kit as set out in claim 26 and substantially as hereinbefore described. DATED this THENTY-EIGHTH day of APRIL 1992 Xoma Corporation Patent Attorneys for the Applicant SPRUSON FERGUSON