POTENTIATION OF CYTOTOXIC CONJUGATES

Abstract: Cancer therapy and, more particularly, the potentiation or enhancement of the cytotoxic properties of immunotoxins by the co-administration of a second, unconjugated monoclonal antibody.

Description: POTENTIATION . QF ^ CYTOTOXIC CONJUGATE " BACKGROUND OF THE INVENTION Field of the Invention The present invention relates generally to cancer therapy and, more particularly, to the potentia- tion or enhancement of the cytotoxic properties of immuno- toxins by the co-administration of a second, unconjugated monoclonal antibody. Colorectal cancer is the second most- common cause of death from malignancy in the Western world. The American Cancer Society estimates that ther° were 138,000 new cases of colorectal cancer and 5^.900 pa¬ tients died from the disease in 1985. The oπtlook for patients with colorectal disease has remained essen¬ tially unaltered over the last 30 years, with the five- year survival being on the order of 30%. A major factor contributing to this is the lack of effective treatment for the disease once it has spread beyond the bnwel wall, since surgery during the early stages of the dis¬ ease offers the only prospect of cure. Unfortunately, the majority of patients have disseminated disease at the time of initial sur ery, primarily with overt or occult hepatic lymph node metastases. Despite numerous trials of chemotherapy, the only single agent shown to have any signif ant effect is 5-fluorouracil ; its response rate is inadequate and treatment with it rarely influences th<=> πH m p outcome. Combination chemotherapy and intrah^patir a er l infu¬ sion are being investigated but currently do not repre¬ sent effective treatments. Cancer of the ovary accounts foi rnurrbly 5% of all cancers in women and is the sixth leadinα cancer in women. Although surgery i curativ if t s lesion is detected early enough, the mortal ty associated with this disease has not improved, appreciably in the last 25 years. Ultrasound, laparoscopy or peri oneoscopy, and CAT scan are of limited value in the diagnosis of ovarian carcinoma. Serum markers such as carcinoembry- onic antigen and placental alkaline phosphatase as well as some newly defined antigens are found in the blood of some patients with adenocarcinoma, although there is no universal marker. Surgery with biopsy is the only definitive way of diagnosing ovarian carcinoma. Surgery is currently the only cure for ovarian carcinoma, and is only curative if the tumor has not spread. Radioisotope implants, x-ray irradiation, and chemotherapy are of limited use in the management of ovarian carcinoma. The most important prognostic indication is the extent of spread of the tumor at the time of diagno¬ sis and surgery. Stage I ovarian carcinoma (growth limited to the ovaries) has an overall 5-year survival rate of approximately 80%, Stage II (growth nvolving ovaries with pelvic extension) has a 5-year survival rate of 40%, Stage III (growth involv ng ovaries with extension to small bowel or omentLim) ' has a 5-year survi- val rate of 10%. and Stage IV (distant metastases) has a 5-year survival rate of less than 5%. The relative 5-year survival rate for ovarian carcinoma is 37% (1973-80), relatively the same as for 1960-63 (32%). Imaging studies of ovarian carcinoma with radiolabelled MoAbs have been performed to a limited degree in human and animal systems. Tumors as small as 1 mm in diameter in mice bearing xenoqraftp of human ovarian cancer have been imaged. The same ntudy also detected tumors in 8 of 10 patients w th ovarian cancer. Osteogenic sarcoma (OS) is the most common primary bone tumor. Although surgery can li curative if this lesion is detected early enough, f e usual course of this disease in 80%-85% of patients i .= ■ mul iple- pulmonary metastases and death within two years of diag¬ nosis. These metastases are often present but usually not large enough to be seen at diagnosis of the primary tumor. Amputation is the treatment of choice for OS. Limb salvage procedures have been performed, such as en-block resection and prosthetic replacement. Overall survival for limb salvage is either as poor or worse than with amputation. Radiation treatment has not been shown to prevent OS metastases. Most recent trials note survival rates of over 50% at 5 years for patients treated by widely di¬ verse adjunctive methods. The question is whether all of these widely diverse forms of treatment are effective or if there is a change in the natural history of the disease. Imaging studies of OS with radiolabelled MoAbs have been performed to a limited degree in human and animal systems. Human OS xenografts have been imaged in nude mice using an anti-OS MoAb labelled with 131 ' I . Because of the extent of these three forms of cancer, there is a need for new compositions and methods to treat primary, recurrent and metastatic disease. Description of the Relevant Literature Ramakrishnan and Houston, Science (1984) 223 : 58-61, describe the potentiation of immunotoxins directed against human acute lymphoblastic leukemia by chloroquine. Akiyama e_t a_l. , Cancer Res. (1985) 45:1005-07, report the potentiation of the cytotoxic activity against human tumor cells of toxic conjugates of Pseudσmonas exotoxin with anti-transferring receptor antibody or epidermal growth factor up to 10 to 20 fold by the cal¬ cium antagonists verapamil, D-600, and diltiazem and by the lysosomotropic agent β-glycylphenylnaphthy1 amide . Uckun et al. , Blut (1985) 50:19-23 describe the potentiation by mafosfamid (ASTA Z 7557) of the ex vivo efficacy of a T-cell directed immunotoxin contain¬ ing pokeweed antiviral protein (PAP). Erlich et al. , Mixing two monoclonal antibodies yields enhanced affinity for antigen, J. Immun. 128:2709- 2713 (1982) describes an increase in sensitivity in antigen-binding assays using a mixture of monoclonal antibodies. Moyle et al. , Quantitative explanation for increased affinity shown by mixtures of monoclonal an¬ tibodies: importance of a circular complex, Molee. Immun. 20: 439-452 (1983) describes a mathematical model to predict the quantity of intermediates formed when pairs of IgGl mouse monoclonals are mixed with human chorionic gonadotropin. Wellerson R. and Kaplan, P., Enhanced binding activity observed between anti-carcinoembryonic mono¬ clonal antibodies, Hybridoma 5:199-213 (1986) describe the enhancement of binding when the antigen is exposed to a particular mixture of monoclonal antibodies. SUMMARY OF THE INVENTION The described invention relates to potentiat- ing the cytotoxicity of antibodies conjugated to a toxin, ie. , an imπvunotoxin. The enhancement of cytotoxicity is demonstrated by a cytotoxic cockta l containing an immunotoxin to which is added a second immunoglobulin that binds to a second epitope on the same antigen to which the immunotoxin binds. More specifically there is described herein a method for enhancing the cytotoxicity of an immunotoxin consisting of a first immunoglobulin conjugated to a toxin, said first immunoglobul n directed against a cell surface antigen, preferably a tumor associated antigen, wherein the method comprises exposing a target cell expressing the antigen with the immunotoxin and a second immunoglobulin in a dose stifficient to potentiate said immunotoxin, said second immunoglobulin able to bind to a different epitope on the antigen than that . bound by said first immunoglobulin. It is preferred that the first and second immunoglobulins are able to bind to an epitope on a carcinogenicembryonic antigen. Most preferably the epitope bound by the second immuno¬ globulin is within the normal cross-reacting antigen subsection of the carcinogenicembryonic antigen. I muno- globulins binding to T-cells ' both normal and leukemic are useful in the disclosed method. Particularly useful are immunoglobulins to the CD-5 antigen. Specific and preferred monoclonal antibodies for use in this inven¬ tion and their hybridomas are also described herein. In addition to the above methods, there is described herein a cytotoxic therapeutic cocktail com¬ prising: a conjugate including a toxin bound to a first immunoglobulin, said first immunoglobulin able to bind to an epitope on a cell surface antigen, preferably a tumor associated antigen; and a second immunoglobulin in a dose sufficient to potentiate said immunotoxin, said second immunoglobulin able to bind to a different epitope on the antigen than that bound by said first immunoglobulin. It is preferred that the cytotoxic therapeutic cocktail embrace first and second immuno¬ globulins that are able to bind to an epitope on the carcinogenicembryonic antigen. It is most preferred that the second immunoglobulin bind to an epitope that is located within the normal cross-reacting antigen subsection of the carcinogenicembryonic antigen. Cock¬ tails containing immunoglobulins binding to T-cells both normal and leukemic are also useful in this inven¬ tion. Particularly useful are immunoglobulins to the CD-5 antigen. It should be noted that it is not neces- sary to physically combine the immunotoxin and second immunoglobulin. Separate administration would also constitute a cocktail as defined herein. In addition to the above method and cocktails, there is described herein a method of ki lling cancer cells in humans by enhancing the cytotoxicity of an immunotoxin, which consists of a first immunoglobulin 5 conjugated to a toxin said immunoglobulin directed against a tumor associated antigen wherein the method comprises exposing cancer cells expressing the antigen to the immunotoxin and to a second immunoglobulin in a dose sufficient to potentiate said toxin, said second immuno- 0 globulin able to bind to a different epitope on the antigen than that bound by said first immunoglobulin. It is also preferred that the first and second immuno¬ globulins are able to bind to an epitope carcinogen¬ icembryonic antigen. It is most preferred that the 5 epitope bound by the second immunoglobulin is within the normal cross-reacting antigen subsection of the carcinocembryonic antigen. This method is useful against cancer cells selected from the group consisting of colo¬ rectal carcinoma cells, gastric cancer cells, pancreatic Q cancer cells, lung cancer cells, and breast cancer cells. Immunoglobulins binding to T-cells both normal and leu¬ kemic are useful in the disclosed method. Particularly useful are immunoglobulins to the CD-5 antigen. Prefer¬ red dosages are approximately 0.01 rag to 20.0 mg per kg 5 of host body weight per day and most preferably the doses are approximately 0.05 mg to 5.0 mg pei; kg of host body weight per day. The preferred ratio of said second immunoglobulin to said immunotoxin is 10,000 - 0.001 to 1 by weight and most preferably the ratio of 0 second immunoglobulin to said immunotoxin is 100 - 0.01 to 1 by weight. By a second epitope it is meant that the two antibodies bind to physically different locations on their target antigen. It is preferred that the binding- 5 not be competitive but a slight amount of competition due to close proximity is acceptable. Potentiation can be quantified and the effect of competi t.ion may be offset by appropriate changes in the ratios of immuno¬ toxin to immunoglobulin. Monoclonal antibodies are preferred for use in this invention. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a graphic illustration of the influence of free RTA and XMMCO-228-RTA on the growth of colorectal carcinoma LS174T cell xenografts . DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention employs cytotoxic conju¬ gates for the treatment of various forms of cancer, including colorectal carcinoma, ovarian carcinoma and osteogenic sarcoma. These novel conjugates are com- prised of monoclonal antibodies or binding fragments thereof, collectively termed immunoglobulins, bound to a cytotoxin. These compositions are administered to a cancer cell host in order to destroy cancer cells while doing minimal damage to normal tissue. The immunoglobulins of the present invention are employed as targeting agents for directing cytotoxic agents to specific cancer cells within a cancer cell host. According to the present invention, immunoglob¬ ulins which define an epitope on a 72 kilodalton (kD) glycoprotein antigen and immunoglobulins which define an epitope on carcinoembryonic antigen (CEA) are em¬ ployed as targeting agents. One or both of these anti¬ gens are present on a variety of cancers including, but not limited to, colorectal carcinoma, ovarian carcinoma and osteogenic sarcoma. A variety of cytotoxic agents are suitable for use in immunotoxins. The cytotox agents contem¬ plated by this invention can include radionuclides. such as Iodine-131, Yttrium-90, Rhenium-188, and Bismuth- 212; a number of chemotherapeutic drugs, such as vinde- sine, ethotrexate, adriamycin, and cisplatinum; and cytotoxic proteins such as ribσsomal inactivating proteins including, pokeweed antiviral protein, abrin and ricin (or their A-chains) , diphtheria toxin pseudo- monas exotoxin A or recombinant derivitives. etc. See generally, "Chimeric Toxins", Olsnee and Phil. Pharmac, Ther., 25:355-381 (1982), and "Monoclonal Antibodies for Cancer Detection and Therapy," eds. Baldwin and Byers, pp. 159-179, 224-266. Academic Press (1985), both of which are incorporated herein by reference. Toxic lectins are of particular interest in this invention. The cytotoxic action of toxic lectins, and especially that of ricin and abrin, has been well studied. It is known that toxic lectins consist of two polypeptide chains, A and B, linked by means of disul- fide bridge(s). Cytotoxicity is associated with the A chain and its inhibition of protein synthesis in nucle¬ ated cells. The B chain is essentially a delivery ve¬ hicle for the A chain. The B chain recognizes poly- saccharide units at the surface of cells and creates a high affinity interaction with such units. Once the B chain binds with polysaccharide units at the cell sur¬ face, the A chain is incorporated into the cell, block¬ ing ribosomal protein synthesis and ultimately leading to cell death. The use of ricin A chain is preferred in this invention and its use is as described in U.S. Patent No. 4,590,071, the disclosure of which are hereby incorporated by reference. Although not used in the examples provided, a preferred form of ricin toxin A chain for use in this invention is one wherein substantially pure RTA-30 is used. The term "RTA-30" refers 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. Cheπt. , 281:5314-5319 (1986) and Vidal et al. Int. J. Cancer, 36:705-711 (1985). For the purposes of this invention, RTA preparations containing concentrations of about 75% or more of RTA-30 are considered substan¬ tially pure. Preparation of substantially pure RTA-30 for use in conjunction with a MoAb is described in U.S. Patent Application Serial Number 074,824 which is incor¬ porated herein by reference. The immunoglobulin and toxin are generally bound by a covalent bond, more particularly a disulfide bond, but may be joined by any chemical bond which al¬ lows the toxin to travel to the target cell with the immunoglobulin. Other methods for achieving such a bond are well-known to those skilled in the art. The only criteria is that the bond must be achieved in a manner which does not significantly decrease the bind¬ ing affinity of the immunoglobulin for its epitope. The present cytotoxic conjugates may be admi¬ nistered to a cancer cell host either singly or in a cocktail containing two or more conjugate formulations, other chemotherapeutic agents, compositions, or the like. Cocktails are particularly important in the treat¬ ment * of heterogeneous tumor cell populations wherein targeting of multiple antigens is critical. The cytotoxic conjugates of the present inven¬ tion may be administered to a cancer cell host by any convenient method. Pharmaceutical compositions employ¬ ing the subject conjugates may be admini tered parenter- ally, i.e., intravenously, intraparitoneally, or the like. Thus, this invention provides compositions for parenteral administration which compri e a solution of pyrogen free, cytotoxic conjugates, or a cocktail there¬ of, dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine, or the like. These solutions are sterile and generally free of particulate matter. These composi¬ tions may be sterilized by conventional,, well-known filtration sterilization techniques. The compositions • may contain pharmaceutically acceptable auxiliary sub¬ stances as required to approximate physiological condi¬ tions such as pH adjusting and buffering agents, toxicity adjusting agents, or the like, for example, sodium acetate, sodium chloride, potassium chloride, potassium chloride, calcium chloride, sodium lactate,* etc. 5 The compositions of the present invention can be lyophilized for storage and reconstituted in a suit¬ able carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art-known lyophilization and reconstitution tech- 10 niques can be employed. It will be appreciated by those skilled in the art that lyophilization and reconstitu¬ tion can lead to varying degrees of antibody activity loss and that use levels may have to be adjusted to compensate. 15 Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formulation should provide a quantity of conjugate(s) of this invention sufficient 2.Q. to effectively treat the cancer cell host. The dose of conjugate or conjugate cocktail will vary widely, gener¬ ally from about 0.01 g/kg/day to 20.0 mg/kg/day, usu¬ ally about 0.05 mg/kg/day to 10.0 mg/kg/day, and, more particularly, 0.05 mg/kg/day to 5.0 mg/kg/day. 25 The dose of unconjugated immunoglobulin will vary widely depending upon the immunotoxin employed, choice of unconjugated immunoglobulin, nature and extent of the tumor, and the like. The dose will generally be in the range of 0.001 mg/kg/day to 100.00 mg/kg/day, 3O usually about 0.01 mg/kg/day to 10.0 mg/kg/day and, more particularly, about 0.1 to 1 mg/kg/day. The ratio of unconjugated immunoglobulin to conjugate will also vary widely, generally from about 10,000-0.001 to 1, usually about: 1000-0.01 to 1 and, more particularly, 35 about 100-0.1 to 1. Kits may also be supplied for use with the subject conjugates or cocktails. Thus, the subject compositions may be provided, usually in lyophilized form, either alone or in conjunction with additional chemotherapeutic agents for cancer therapy. These kits may include buffers, such as phosphate buffered saline, other inert ingredients, or the like. The kits may also include specific instructions including suggested protocols for the administration of the subject compo¬ sitions to a cancer cell host. While the actual mechanism of endocytosis is poorly understood, it is known that most, if not all, immunotoxins must be endocytosed, following binding to target cell surface antigens, in order to be efficacious. It is believed that endocytosis is triggered by the binding of receptors on cell surface, antigens. According to the present invention, the effi¬ cacy of the subject immunotoxin compositions may be potentiated by the co-administration, via a similar route, of an unconjugated immunoglobulin in conjunction with the subject immunotoxins. Potentiation means that the efficacy of the immunotoxin, in terms of target cell kill, is increased for a given dose of immunotoxin. Although the following theory should not be deemed a limitation of this invention, it is believed that the enhanced cytotoxicity is due to an increased affinity for the antigen caused by the binding of the second immunoglobulin. This rationale will have wide ranging effects upon the use of immunoconjugates where the target cell antigen is defined and antibodies bind¬ ing to related epitopes are available. The following examples are offered by way of illustration and not limitation. EXPERIMENTAL EXAMPLE I - XMMCO-228-RTA CONJUGATES A. Production of Hybridomas Balb/c mice (Bantin & Kingman, U.K.) were immunized with carcinoembryonic antigen (CEA) derived from a perchlorate extract of a colon carcinoma liver metastasis. The immunization schedule consisted of 10 μg CEA in complete Freund' s adjuvant (CFA) given intra- peritoneally on Days 0 and 7, and 20 ug of CEA (Also in CFA) given intraperitoneally on Days 25, 56 and 63. Three days after the last antigen boost, spleen cells from an immunized mouse were aseptically removed. Following procedures as outlined elsewhere (Galfre et al (1977) Nature 266:550, which is incorporated by ref- erence), 10 spleen cells were fused with 10 cells of P3-NSI-Ag4-l (A.T.C.C. Accession No. TIB-18), a hypo- xanthine-methotrexate-thymidine (HMT) sensitive murine myeloma cell line. Using polyethylene glycol (PEG), hybrid cells were placed into 96-well culture plates, (Costar, Cambridge, MA #3596) on medium containing a 3 feeder layer of rat peritoneal exudate cells (2.5 x 10 cells/well). Cells were cultured in -Dulbecco ' s Modified Eagle's Medium (Flow Labs, Irvine, U.K.) containing 15% Fetal Calf Serum (Myoclone, Gibco, Paisley, U.K. ) and hypoxanthine (10 -4 M) , thymidine (1.6 x 10-5 M) (both from Sigma, Dorset, U.K.) and methotrexate (10 ~ M) (Lederle, Hampshire, U.K.). Within two weeks post fusion, cultures of hybridoma cells were tested for antibody binding to CEA by enzyme immunoassay (EIA) . Cultures that were posi¬ tive were cloned using limiting dilution, plating 1-3 cells/well into 96 well culture plates. Wells contain-- ing only one colony were identified by microscopic ex¬ amination, then tested for reactivity with CEΛ and nor¬ mal colon antigen (NCA) by EIA and radioimmunoassay (RIA), and for reactivity on extranuclear membranes from primary colorectal tumors and normal colon by EIA. The clone designated XMMCO-228 was found to stably se¬ crete immunoglobulin which was determined to be of the 5 IgG2a subclass by solid phase RIA using standard meth¬ ods. Hybridoma XMMCO-228 is presently on deposit with the American Type Culture Collection (A.T.C.C), 12301 Parklawn Dr., Rockville, MD 20852, USA. The deposit was made on August 14, 1986, and given A.T.C.C. Acces- 1.0 sion No. HB 9174. Balb/c mice (Bantin & Kingman, U.K. ), 6-10 weeks old, were used to culture the hybridoma perito- 7 neally. Approximately 10 hybridoma cells were injected into mice that had been pretreated 3 weeks earlier with 15 0.5 mis of pristane (Aldridge, Gillingha , Dorset. U.K.) injected intraperitoneally (i.p.). The resultant ascites fluid, collected 3 weeks after injection of the hybridomas, contained on average 5 mg/ml of the antibody as determined by measuring immunoglobulin level accord- 0 ing to the method of Price' and Baldwin I . CRS Med_.__S_ci. (1984) 12:1000-01, which is incorporated by reference. The antibody in ascites fluid was purified by affinity chromatography using a Sepharose - Protein A column using methods well known by those skilled in the 5 art. The hybridoma was grown in vitro in Dulbecco ' s Minimal Essential Medium (Flow Labs, Irvine, U.K. ) with 10% Fetal Calf Serum (Myoclone, Gibco, Paisley. U.K.) and hypoxanthine (10 -4 M) , thymidine (1.6 x 10-"5 M) 0 (both from Sigma, Dorset, U.K. ) and methotrexate ( 10 ~ M) (Lederle, Hampshire, U.?.) in plastic 300 ml bottles. Cell concentration was 10 cells/ml medium over a cultur- ing period of 4-5 days, with a MoAb concentration of 4 μg/ml medium, and a doubling time of 12 hours. 5 B. In Vitro Antibody Binding to Cell Lines Measured by Flow Cytometry The binding of XMMCO-228 to MKN45 cells was determined by flow cytometry employing methods well known to those skilled in the art and described above. The gastric carcinoma cell line MKN45 expresses both the 72kD antigen with which the XMMCO-791 MoAb reacts and the CEA antigen with which XMMCO-228 reacts. B14 is a MoAb directed against breast cancer, which reacts with the normal cross-reacting antigen (NCA) subsection of the CEA antigen. It is described in Example II. Normal mouse serum was used as a control. The results are summarized in Table I. TABLE I Binding to MKN45 Cells by Flow Cytometry Mean Fluorescence MoAb Intensity XMMCO-228 987 B14 790 Control 15 C. In Vivo Antibody Binding to Normal Tissues Measuxed by Immunoperoxidase XMMCO-228 was evaluated for crossreactivity with normal tissues using the indirect immunoperoxidase conjugate technique. As a control, tissues were simul¬ taneously tested with a purified murine MoAb in the same concentration. The control immunoglobulins used for these purposes were an IgG2 mouse myeloma protein (UPC 10, Litton Bionetics, Kensington, MD) or a MoAb with reactivity to sheep red blood cells (SRBC). Fresh tissue slices (2-3mm thickness and up to 2.0 cm in width) was generously coated with Optimal Cutting Temperature Compound (OCT) , (Ames Co. , Elkhar, Indiana) and wrapped in aluminum foil. The specimen was "snap" frozen in liquid nitrogen-cooled isopentarre (2-methylbutane, VWR, Norwald, CA) at controlled temper- ature (-120 to -150°C) for 10-15 seconds. The tissue was kept frozen for long-term storage at -70°C, or for short-term storage at -20°C, in a tightly sealed con¬ tainer to prevent evaporation and drying of tissues. Frozen tissues were allowed to equilibrate slowly overnight by placing in a -35°C cryostat prior to cutting the frozen sections. Cut sections were dried overnight at room temperature prior to staining. A circle was etched on each slide around the tissue using a diamond pencil. Slides were then fixed in acetone for 1 minute, followed by a 10 minute PBS wash. Excess fluid was removed and the primary anti¬ body applied for one hour while making sure that the tissue was covered and not allowed to dry out, followed by a 10 minute PBS wash. Excess fluid was removed and peroxidase-con- jugated goat anti-mouse IgG (Tago/product code 6450) (diluted 1:10 in PBS) applied for 30 minutes, followed by a 10 minute PBS wash. The slides were developed in aminoethylcar- bazole (AEC), (0.5 ml AEC stock 120 mg AEC in 15 ml dimethylformamide) , 9.5 ml acetate buffer (79 ml of 0.1 M sodium acetate, 21 ml of 0.1 N acetic acid), and 0.05 ml 3% H_0_) for 5 minutes, followed by a 5 minute H_0 wash. The slides were then counterstained with fresh Mayer ' s Hemtoxylin for 5 minutes, followed by a 5 minute wash in running tap water and coverslipped with glycerine mounting media. Table II is a summary of the results: TABLE II Antibody Binding to Normal Tissues Measured by Immunoperoxidase COLORECTAL CANCER: 5/5 intense staining of Ca 9O° + of population staining +) NORMAL COLON: 5/5 intense staining of glandular epith., especially intense apical staining AUTOPSY TISSUE: Negative tissue- Kidney Pancreas Muscle Liver Skin Heart Testis Spleen Spinal Cord Stomach Aorta Cerebellum Lymph Node Questionable (+/-) Lung Positive- Esophagus(+ squamσπs ppitli) NOTE: glucose oxidase method showed that Λb. 22R does not stain granulocytes. Almost every other antibody that we have studied that is similar to CF.Λ has stained granulocytes. It is significant that XMMCO-228 MoAbs do not bind to granulocytes. Normal colon antigen (NCA) ap¬ pears on such diverse tissues as normal colon, granulo¬ cytes, and progenitor bone marrow cells. Because the CEA antigen has a NCA subsection, many anti-CEA MoAbs also bind the NCA on various normal tissues, which XMMCO- 228 MoAbs do not. D. . XMMCO-228 Ricin Toxin A Chain (RTA)__Con ugates The conjugation technique, including purifi¬ cation of the A chain of ricin, is disclosed in U.S. Patent No. 4,590,071, the disclosures of which are hereby incorporated by reference. E. XMMCO-228-RTA In Vitro Cytotoxicity In vitro cytotoxicity of XMMCO-228-RTA conju- gates was determined by the 75Se-Selenomethionine As¬ say. Single cell suspensions of gastric carcinoma de¬ rived MKN45 cells were inoculated with immunotoxin or RTA for 15 minutes, washed and added to flat bottomed microtiter plates at a concentration of 10 cells/0.2 ml of immunotoxin or RTA in RPMI medium containing 10% fetal calf serum. Various amounts of immunotoxin were added to each well. Twenty-four hours later, 75Se-Sele- nomethionine was added to each well. Cultures were further incubated for 16 hours, washed, dried on the bottom of the wells, and the wells were counted in a gamma counter. The results are summarized in Table III, expressed in terms of 50% inhibition concentra- tion. TABLE III In Vitro Cytotoxicity Against MKN45 Cells Agent IC 50 (ng/ml) RTA 2238 XMMCO-228-RTA 600 Agent IC 5 (molarity)* RTA 1 x 10 ~8 M XMMCO-228-RTA 1 x 10 "10 M * djusted for equivalent RTA molarity F. XMMCO-228-RTA Therapy of Human Tumor Xenografts For each data point, 7-10 nude mice were im¬ planted with LS174T cells, a human colon adenocarcinoma derived cell line (A.T.C.C. Accession No. CL 188). Three days post-implant, the first dose of the treat- ent compound was administered, followed by 10-20 doses until the schedule dose was completed. The results are summarized in Table IV, expressed as a ration of the tumor weight in treated mice divided by the tumor weight in control (untreated) mice. The results, in terms of mean tumor diameter, are summarized graphically in Figure I. TABLE IV Therapy of Human Tumor Xenogrπfts With XMMCO-228-RTA 5 Expt. Xenograft Treatment Schedule Response: Tumor Compound Dose Tumor Weight Treated mg/kg Tumor Weight Control LS 17 T RTA 20 0.70 LS 174T XMMCO-228 80 1.18 1.0 LS 17 T XMMCO-228-RTA 56 0.36 These results demonstrate the. efficacy of XMMCO-228- RTA in the therapy of human tumor xenografts. 5 EXAMPLE II XMMC0-791-RTA AND XMM0C-228-RTA COCKTAILS A. XMMCO-791-RTA + XMMCO-228-RTA Conjugate Cockt_ail_s A cocktail of XMMCO-791-RTA (Scand. J. Immuno. 18:411-420 (1983) and XMMCO-228-RTA consists of a mix¬ 0 ture of XMMCO-791-RTA and XMMC0-228-RTA. in any ratio, and administered to a cancer cell host in a dose range of about 0.01 mg/kg/day to 20.0 mg/kg/day. The cocktail is administered parenterally, generally by intravenous infusion or intrapercitoneal injection in a suitable 5 vehicle, such as phosphate buffered saline or the like. B. In Vitro Cytotoxicity of Conjugate Cocktails In vitro cytotoxicity of XMMC0-228-RTA conju- gates was determined by the 75Se-Selenomethionine as¬ 0 say. Single cell suspensions of gastric carcinoma de¬ rived MKN45 cells were inoculated with immunotoxin or RTA for 15 minutes, washed and added to flat bottomed microtiter plates at a concentration of 10 " cells/0.2 ml of immunotoxin or RTA in RPMI medium containing 10% 5 fetal calf serum. Various amounts of immunotoxin were added to each well. Twenty-four hours later. 75Se-Selenomethionine was added to each well. Cultures were further incubated for 16 hours, washed, dried on the bottom of the wells, and the wells were counted in a gamma counter. The results are summarized in Table IV above, expressed in terms of 50% inhibition concentra¬ tion. These data indicate that when XMMCO-791-RTA and XMMCO-228-RTA are administered in a "cocktail" form, the cytotoxicity is at least additive, and possibly synergistic. They also show the efficacy of immunotoxin (or antibody) directed RTA compared to RTA alone. C. XMMC0-791-RTA + XMMCO-228-RTA Conjugate Cocktail Therapy of Human Tumor Xenografts For each data point, 7-10 nude mice were im¬ planted with MKN 45 cells, derived cell line. Three days post-implant, the first dose of the treatment com¬ pound was administered, followed by 10-20 doses until the scheduled dose was completed. The results are sum- marized in Table V, expressed as a ratio of the tumor weight in treated mice divided by the tumor weight in control (untreated) mice. TABLE V Therapy of Human Tumor Xenografts With XMMC0-228-RTA 5 Expt. Xenograft Treatment Schedule Response: Tumor Compound Dose Xy or Weight Treated mg/kg Tπmor Weight Control 1 MKN 45 XMMCO-228-RTA 25 0.74 XMMCO-228 25 1.1 1.0 2 MKN 45 XMMCO-228-RTA 0.56 + XMMCO-791-RTA *25 mg/kg each of XMMC0-228-RTA and XMMCO-791 -RTA admin i stered as a mixture . 5 These data indicate that when XMMCO-791-RTA and XMMCO-228-RTA are administered in a "cocktai l " form , the cytotoxicity i s at least additive , and possibly synergistic . They also show the efficacy of a cocktai l consi sting of XMMCO-791-RTA and XMMCO- 228-RTA in treat¬ 0 ing cancer cells in a host . EXAMPLE III POTENTIATION OF XMMCO-228-RTA BY XMMBR-B14 MOABS 5 A. Production of XMMBR-B14 Hybridomas Balb/c mice were immunized with 521AM whole cells derived from ascites breast metastasis. The immu¬ nization schedule consisted of 6 intraperitoneal injec¬ tions of 10 cells at weekly intervals. A final intra¬ 0 venous boost of 3x10 cells was given 3 days prior to fusion. Three days after the last antigen boost, spleen cells from an immunized mouse were aseptically removed. A single cell suspension was obtained using an 80 mesh 5 wire screen grid #1985-00080 (Bellco). Cells were washed with Iscoves Complete Medium (Grand Island. M.Y. , N.Y. ) and counted. Sp2/0-Agl4 cells (A.T.C.C. Accession No. CRL 1581), a hypoxanthine-aminopterin-thymidine (HAT) sensitive murine myeloma cell line, were washed 3 times and counted. Using polyethylene glycol (PEG 4000 Merck) 30% v/v, 10% DMSO and 60% Iscoves, the two cell types were fused at a ratio of 2 spleen cells per myeloma cell. The fusion products were plated into 96-well culture plates at a concentration of 10 myeloma cells per well. Cells were cultured in Iscoves with 20% fetal bovine serum and β mercaptoethanol with HAT medium (hypo- xanthine 136 mg/100 ml, aminopterin .018 mg/100 ml, thymidine 136 mg/100 ml) . Within two weeks post fusion, cultures of hybridoma cells were tested for antibody binding to 521 AM tumor cell membranes by dotting onto nitrocellulosee membranes at 200 ng using a Vector Labs ABC kit to test for reactivity. Wells that gave a blue dot were then screened against fibroblast membrane extracts from the same patient. Cultures that were positive were cloned using limiting dilution, plating 1-3 cells/well into 96 well culture plates. Wells containing only one colony were identified by microscopic examination, then tested for reactivity. The clone designated XMMBR-B14 was found to stably secrete immunoglobulin which was deter- mined to be of the IgGl subclass. Hybridoma XMMBR-B14 is presently on deposit with the American Type Culture Collection (A.T.C.C), 12301 Parklawn Dr., Rockville, MD 20852, USA. The deposit was made on January 14, 1987, and given A.T.C.C. Accession No. HB 9308. Balb/c mice (Bantin & Kingman, U.K. ), 6-10 weeks old, were used to culture the hybridoma peritone- 7 ally. Approximately 10 hybridoma cells were injected into mice that had been pretreated 3 weeks earlier with 0.5 mis of pristane (Aldridge, Gillingham, Dorset,. U.K.) injected intraperitoneally (i.p.). The resultant ascites fluid, collected 3 weeks after injection of the hybridomas, contained on average 5 mg/ml of the antibody as determined by measuring immunoglobulin level accord¬ ing to the method of Price and Baldwin I_CRS Med. Sci . (1984) 12:1000-01, which is incorporated by reference-. The antibody in ascites fluid was purified by affinity chromatography using a Sepharose - Protein A column using methods well known by those skilled in the art. The hybridoma was grown and cloned in vitro in Iscoves Medium with 20% fetal bovine serum and 6 mercaptoethanol in plastic 300 ml bottles. Cell concen- 5 tration was 10 cells/ml medium over a culturing period of 4-5 days, with a MoAb concentration of 4 μg/ml medium, and a doubling time of 12 hours. B. In Vitro XMMBR-B14 Binding to Cells Measured by Flow Cytometry The binding of XMMBR-B14 to cell lines and primary carcinoma-derived cells was determined by flow cytometry employing methods well known by those skilled in the art and described above. The tests (Table VI) showed that XMMBR-B14 detects an antigen expressed on breast and colon carcinoma cell lines. Further studies using purified protein preparations have established that the epitope defined by XMMBR-B14 is found on an antigen of the classification termed CEA/NCA (carcino- embryonic antigen/normal cross-reacting antigen) . This is the portion of the CEA molecule that also cross-reacts with exposed normal cross-reacting antigen. It also reacts with cells derived from two primary colon carci- nomas. Normal mouse immunoglobulin (NMIg) or normal mouse serum (NMS) were used as controls. TABLE VI Binding to Various Cells by Flow Cytometry Target Cell. Reagent Fluorescence Reaction Units/cell 1. Cell Lines Colon Carcinoma C170 NM Ig 19.3 (Low CEA) B14B8 60.4 ± Colon Carcinoma LS174T NM Ig 22.2 _ (Line 1) B14B8 2641.7 4+ Colon Carcinoma LR174T NM Ig 18.6 _ (Line 2) B14B8 1665.6 3+ Breast Carcinoma BT474-3 NM Ig 17.3 _ B14B8 316.4 1-2+ 2. Primary Colon Carcinoma - Derived Cells C212 NMS 31 .4 B14B8 607. 5 3+ C213 NMS 33. 8 B14B8 836. 8 4+ C . XMMBR-B14 Reactivity With Colon Carcinoma and Normal Colonic Mucosa Measured by Enzyme Immunoassay ( E IA ) XMMBR-B14 wa s tested for reactivity with pri¬ mary colon carcinoma membrane and normal colonic mucosa using standard EIA methods well known by those skilled in the art . The test , summarized in Table VI I , shows that XMMBR-B14 reacts with colon carci noma membrane . TABLE VII Binding to Colon Membrane by F.IΛ Reagent ELISA Units (OP) Normal Colon Colon Carcinoma Membrane CEA membrane Prep. (NP ) (TP ) (T186) (B4058) XMMBR-B14 0.130 0.581 0.758 0.665 TP. - membrane preparation from pooled primary colon carcinoma. 1.0 NP. - membrane preparation from pooled normal colonic mucosa (from colon cancer patient). T186 - membrane preparation - primary colon carcinoma T186. CEA(B4058) - CEA preparation - semi-purified from liver metastasis of colon carcinoma 5 D. XMMBR-B14 Binding to CEA and NCA The Reactivity of XMMBR-B14 with CEA and NCA preparations was determined by a solid phase radioimmuno- assay. Briefly, antigen preparations were coated into 0 wells of microtest plates. Monoclonal antibody was then added, incubated 1 to 2 hours and wells washed. 125 I-labelled (F(ab)'2 fragments of rabbit anti-mouse IgG were then added to detect bound murine MoAb. Table VIII. 5 In the first test MoAb binding to semi-puri¬ fied CEA (Rogers) and NCA (B3991) was compared. XMMBR- B14 bound to both NCA and CEA (NCA:CEA ratio 1.2:7). 0 5 TABLE VIII Binding of XMMBR-B14 to CEA and NCA Antibody Mean CPM ± SD (- Background) Bound to: CEA (Rogers ) NCA (B3991 ) Ant i -CEA 1427 ± 222 4 ± 48 XMMBR-B14 2558 ± 114 955 ± 140 E. XMMBR-B14 Dose Response on XMMCO-228-RTA Cytotoxicity Against MNK45 Cells Table IX shows the increase in cytotoxicity of XMMCO-228-RTA immunotoxin against MNK45 cells when increasing amounts of XMMBR-B14 are added. Six different curves were generated using concentrations of XMMCO-228-RTA ranging from 1 ng/ml to 100 ng/ml. To these concentrations were added varying amounts of XMMBR-B14 ranging from 0 to 10 μg/ml. The calculated IC-.-. values for each curve are shown in Table IX. TABLE IX XMMBR-B14 Produced Monoclonal Antibody Dose Response on XMMC0-228-RTA Cytotoxicity (MKN45 Cells) XMMBR-B14 MoA Cytotoxicity of XMMCO-228-RTA (μg/ml) for MKN45 Cells ( IC-.- ng/ml) 10 29 1 47 0 1 60.8 0 01 87 0.001 113 0.00 155 The present invention provides efficacious, novel compounds and methods for the therapy of various cancer cells. The cytotoxic conjugates are particular¬ ly efficacious when administered in cocktail form or in the presence of certain unconjugated monoclonal anti¬ bodies. Although the present invention has been de¬ scribed in some detail by way of illustration and exam¬ ple for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims: WHAT IS CLAIMED IS: 1. A method for enhancing the cytotoxicity of an immunotoxin consisting of a first immunoglobulin conjugated to a toxin said first immunoglobulin directed against a cell surface antigen on a target cell - wherein the method comprises exposing a target cell expressing the antigen to the immunotoxin and a second immunoglob¬ ulin in a dose sufficient to potentiate said immunotoxin, said second immunoglobulin able to bind to a different epitope on the antigen than that bound by said first immunoglobulin. 2. A method according to claim 1, wherein the antigen is a tumor associated antigen. 3. A method according to claim 2, wherein the first and second immunoglobulins are able to bind to an epitope on a tumor cell expressing carcinoembry- onic antigen. 4. A method according to claim 3. wherein the epitope bound by the second immunoglobulin is within the normal cross-reacting antigen sub ' section of the carcinoembryonic antigen. 5. A method according to claim 4, wherein the first immunoglobulin is XXMMCO-228 bound to RTA and the second immunoglobulin is XXMBR-B14. 6. The method of claim 1, wherein the first and second immunoglobulins are able to bind to an anti¬ gen on a T cell. 7. The immunoglobulins of claim 1. wherein the antigen is CD-5. 8. The immunoglobulins of claim 1, wherein the antigen is on a leukemic T cell. 9. A cytotoxic therapeutic cocktail com- prising: a conjugate comprising a toxin linked to a first immunoglobulin, said first immunoglobulin able to bind to an epitope on a cell surface antigen; and a second immuloglobulin in a dose sufficient to potentiate said immunotoxin, said second immunoglob¬ ulin able to bind to a different epitope on the antigen than that bound by said first immunoglobulin. 10. A cocktail according to claim 9, wherein the antigen is a tumor associated antigen. 11. A cytotoxic therapeutic cocktail accord¬ ing to claim 9, wherein the first and second immuno¬ globulins are able to bind to an epitope on a tumor cell expressing carcinoembryonic antigen. 12. A cytotoxic therapeutic cocktail accord¬ ing to claim 11, wherein the epitope bound by the second immunoglobulin is within the normal cross-reacting an- tigen subsection of the carcinoembryonic antigen. 13. A cytotoxic therapeutic cocktail according to claim 12, wherein the first immunoglobulin is XXMMCO- 228 bound to RTA and the second immunoglobulin is XXMBR- B14. 14. A cytotoxic therapeutic cocktail accord¬ ing to claim 9, wherein the first and second immuno¬ globulins are able to bind to an antigen on a T cell.. 15. The immunoglobulins of claim 14, wherein the antigen is CD-5. 16. A method of killing cancer cells in humans by enhancing the cytotoxicity of an immunotoxin which consists of a first immunoglobulin conjugated to a toxin said first immunoglobulin directed against a tumor as- sociated antigen - wherein the method comprises exposing cancer cells expressing the tumor associated antigen to the immunotoxin and to a second immunoglobulin. in a dose sufficient to potentiate said toxin, said second immunoglobulin able to bind to a different epitope on the tumor associated antigen than that bound by said first immunoglobulin. 17. A method according to claim 16, wherein the first and second immunoglobulins are able to bind to an epitope on a tumor cell expressing carcinoembry¬ onic antigen. 18. A method according to claim 17, wherein the epitope bound by the second immunoglobulin is within the normal cross-reacting antigen stibsectiσn of the carcinoembryonic antigen. 19. A method according to claim 18, wherein the first immunoglobulin is XXMMCO-228 bound to RTA and the second immunoglobulin is XXMBR-B14. 20. A method according to claim 16, wherein the cancer cells are selected from the group consisting of colorectal carcinoma cells, gastric cancer cells, pancreatic cancer cells, lung cancer cells, and breast cancer cells. 21. The method of claim 16. wherein the first and second immunoglobulins are able to bind to an anti-- gen on a T cell. 22. The immunoglobulins of claim 21, wherein the antigen is CD-5. 23. The immunoglobulins of claim 21, wherein the antigen is on a leukemic T cell. 24. A method according to claim 16 wherein the ratio of said second immunoglobulin to said immuno¬ toxin is 100 - 0.01 to 1 by weight.

Inventors: BALDWIN ROBERT W, BYERS VERA S, SCANNON PATRICK J, BALDWIN, ROBERT, W., BYERS, VERA, S., SCANNON, PATRICK, J.
Applicants: XOMA CORP, XOMA CORPORATION
Application year: 1988
Main classification: A61K39
IPC codes: A61K38, A61K39, A61K47, A61P35, C07K16, C12P21, G01N33