JPH01502195A - Method for enhancing cytotoxic conjugates - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Method for enhancing cytotoxic conjugates Background of the invention 1 skin 11 TECHNICAL FIELD This invention relates generally to the treatment of cancer, and more specifically to the treatment of cancer, and more particularly to the treatment of cancer. of potentiation or increase by co-administration of unconjugated monoclonal antibodies. .

Colorectal cancer is the second most common cause of death from malignant tumors in the Western world. Ru. The American Cancer Society estimates that in 1985 there were 138,000 new cases of colorectal cancer. It is estimated that 59,900 patients died from the disease.

For patients with colorectal cancer, the 5-year survival rate has been in the 30% range for the past 30 years. The fact remains that healing is the only way to heal. The hope is that surgery performed at an early stage of the disease, once the intestines have been Once it has spread beyond the wall, there is no effective treatment.

Unfortunately, most patients are diagnosed primarily with overt or occult symptoms at the time of their initial surgical procedure. The patient has widespread disease with extensive liver and lymph node metastases.

Despite numerous attempts at chemotherapy, it has been shown to have some significant effect. The only drug available is 5-fluorouracil, but its reaction rate is insufficient and its This treatment rarely affects the final outcome. II combination therapy and liver movement Intravenous injection has been investigated but currently does not represent an effective treatment.

Ovarian cancer accounts for approximately 5% of all cancers in women and is the sixth leading cancer in women.

If the disease is detected early enough, it can be treated with surgery, but the disease is fatal. Mortality rates have not improved significantly over the past 25 years.

Ultrasound/laparoscopy (laparoscopy or per toneos Copy) and CAT (computerized tomography) are used in the diagnosis of ovarian cancer. Carcinoembryonic antigen and placental alkaline phosphatase, as well as some Newly defined antigen-like serum markers found in the blood of certain adenocarcinoma patients However, there are no universal signs.

Surgery with biopsy is the only definitive way to diagnose ovarian cancer.

Surgery is currently the only treatment for ovarian cancer and is only treated if the tumor has not spread. It is possible. Radioisotope insertion, X-ray irradiation, and chemotherapy are used to treat ovarian cancer. It is of limited use.

The most important predictive sign is the extent of the tumor at the time of diagnosis and surgery. 1st Stage ovarian tumors (growth confined to the ovary) have an overall 5-year survival rate of almost 80%. Stage II (proliferation involving the ovaries with expansion to the pelvis) has a 5-year survival rate of 40%. In stage II (proliferation that includes the ovary with expansion to the ovary and retina), 10% survive for 5 days. ovarian tumors with a 5-year survival rate of less than 5% in stage II (distant metastasis). The relative 5-year survival rate was 37% in 1973-80 and 3% in 1960-63. Same as 2%.

Ovarian tumor detection using radioisotope-labeled monoclonal antibodies (MoAbs) Research to image ovarian tumors has been carried out to some extent in humans and animals. A small tumor 1-1 in diameter was imaged in a mouse with an explant. Also, according to the same study Tumors were detected in 8 out of 10 ovarian cancer patients.

Osteosalma II (OS) is the most common major bone tumor. This disease occurs early enough. If detected, it can be cured by surgery, but this disease occurs in 80-85% of patients. The disease course leads to multiple pulmonary metastases and death within 2 years of diagnosis. These metastases are Although often present at the time of diagnosis of the primary tumor, it is usually not large enough to be seen.

Amputation is an excellent treatment for osteosarcoma. Limb retrieval such as block resection and prosthetic replacement Law is done. The overall survival rate for limb retrieval is as poor as amputation or even worse.

Radiation therapy was not found to protect against osteosarcoma metastases.

More recent attempts have shown that patients treated with extensive cross-species therapy showed 50% success rate at 5 years. The survival rate was recorded. Are all of these different forms of broad-spectrum treatment effective? The question is whether there has been any change in the natural history of the disease.

Research to image osteosarcoma using monoclonal antibodies labeled with radioisotopes Studies have been conducted to some extent in humans and animals; 131 studies have been conducted on foreign osteosarcoma transplants in humans. Imaged in nude mice using labeled anti-osteosarcoma monoclonal antibodies .

New compositions and methods are needed to treat biometastatic disease.

Outstanding Nu II Ba 11 Ramakrishnan and Hauston 5science (1984); Volume 223.

On pages 58-61, chloroquine is used to treat acute limboblastic leukemia in humans. describes the enhancement of immunotoxins.

Akiyama et al. Cancer Re5 (1985) vol. 45, 1005- On page 1007, Pseudomonas exotoxin Anti-metastatic receptor antibodies or epidermal growth promoters and toxic conjugates against human tumor cells. The cytotoxic activity was similar to that of the calcium antagonist verapamil, D-600 and diltiazem. The socime agent β-glycylphenylnaphthylamide increases the It is reported that it will be strengthened.

In Blut (1985) volume 50, page 1-23, Uckun et al. Immunotoxin expression directed by Tffl cells containing pokeweed antiviral protein (PAP) Mahosfamide (ASTA Z 755) with ex vivo effect 7) describes the enhancement.

Ehrlich et al. bodyyields enhanced afTinity for a ntigen (two monoclonal antibodies with increased affinity for antibodies) Mixed)” (J, lm5un, (1982) vol. 128, p. 2709-2713 In a report titled 2), an antibody binding test using a mixture of monoclonal antibodies describes an increase in sensitivity in

Moyle et al. “Quantitative explanation for 　1ncreasedaffinity　showndy　m1xtures orson oclonal antib-odies:importa nee of a circular complex (monoclonal antibody Quantitative explanation of the increased affinity exhibited by mixtures: the importance of cyclic side coalescence)” (M olec, Immun, (1983) vol. 20.

439-452), immunoglobulin Gl (IgG1) form when a pair of mouse monoclonal antibodies is mixed with human hair transplant gonadotropin. It describes a mathematical model that predicts the amount of intermediate material produced.

Wellerson, R., and Kaplan, P., “Enhanced bin Dingactivity observed between anti-c arcinoe + sbryonic monoclonal antibodi es (increased binding activity observed on the surface of multiple cancerous fetal monoclonal antibodies)” (H ybrido Soa (1986) 5 volumes.

(pages 199-213), the antigen was identified using a monoclonal antibody. describes an increase in binding upon exposure to mixtures of

Summary of the invention The described invention relates to enhancement of the cytotoxicity of antibodies conjugated to toxins, i.e. immunotoxins. . Increased cytotoxin binds to a second epitope of the same antigen that the immunotoxin binds to a second immunoglobulin that binds to a second epitope of the same antigen is added; evidenced by a cytotoxic cocktail containing immunotoxins.

More particularly, it includes cell surface antigens, preferably antigens with MgII. Immunotoxin cytotoxin consisting of a first immunoglobulin bound to the toxin 1. A method of increasing antigen-expressing antigen-expressing target cells with an immunotoxin. and a second immunoglobulin in an amount sufficient to potentiate the immunotoxin. The second immunoglobulin is bound to the first immunoglobulin on the antigen. A method is described that allows binding to epitope 1 different from that of the epitope. first and the second immunoglobulin are preferably capable of binding an epitope on the carcinoembryonic antigen. stomach.

The epitope bound by the second immunoglobulin is a normal cross-over of the carcinoembryonic antigen. Preferably, it is within a small portion of the reactive antigen.

Immunoglobulins that bind to both normal and leukemic T cells are useful in the disclosed methods. It is. Particularly useful are immunoglobulins directed against the CD-5 antigen. this invention Special and preferred monoclonal antibodies and their hybridomas for use in has been disclosed.

In addition to the above methods, the present invention also includes the use of epitopes on cell surface antigens, preferably tumor-associated antigens. The toxin is bound to the first immunoglobulin that can bind to the antigen. binds to a different epitope than said first immunoglobulin binds to. A cocktail for cytotoxic activity consisting of a conjugate containing a second immunoglobulin capable of is described. Cytotoxic therapeutic cocktails bind to epitopes on carcinoembryonic antigen. Preferably, the second immunoglobulin comprises a first and a second immunoglobulin that can be combined. Immunoglobulins are normally cross-reactive with a small fraction of the antigens in cancerous fetal immunoglobulins. Both normal and leukemic T, most preferably binding to an epitope. Cocktails containing immunoglobulins that bind to cells are useful in this invention. Special Useful are immunoglobulins directed against the CD-5 antigen. Immunotoxins and secondary immunity It should be noted that the epiglobulin must be physically bound. separately Also included in the cocktail defined herein is the administration of

Outside of the above methods and cocktails, here are some that are directed against tumor-associated antigens and toxins. Cytotoxicity and increased cytotoxicity of immunotoxins consisting of the first immunoglobulin associated with A method of killing human cancer cells by killing cancer cells expressing an antigen. The immunotoxin binds to a different epitope on the antigen than the first immunoglobulin. a second immunoglobulin in an amount sufficient to enhance said toxin's ability to bind to the toxin; A method is described consisting of exposure to phosphorus. First and second immunoglobulin is also preferably capable of binding to an epitope of a carcinoembryonic antigen. The epitope that this protein binds to is within a small fraction of the normal cross-reacting antigens of carcinoembryonic antigen. Most preferably, this method uses colorectal cancer cells, gastric cancer cells, pancreatic cancer cells, lung cancer cells. The present invention is useful for cancer cells selected from the group consisting of breast cancer cells, and breast cancer cells. normal and leukemia Immunoglobulins that bind to T cells of both sexes are useful in the disclosed methods. Especially C Immunoglobulins directed against the D-5 antigen are useful.

The preferred dosage is about 0.01 mg to 20 mg/kg host body weight per day, OB. Yes, most preferably 0.05B to 5.0mg per host body weight per day. It is. The ratio of the second immunoglobulin to the immunotoxin is 10 by weight; 000 to 0.001:1 and most preferably the second immune to the immunotoxin. The ratio of epidemic globulin is 100 to 0.01:1 by weight.

A second epitope is one that two antibodies bind to at physically different locations on their target antigen. It means that they match. Binding is preferably non-competitive but may occur through close proximity. A small amount of competition is accepted. Enhancement can be quantified, and the effects of competition are This can be offset by appropriately changing the ratio of toxin to immunoglobulin. I can do it. Monoclonal antibodies are preferably used in this invention.

Brief description of the drawing Figure 1 shows free RTA and X It is a graph section showing the influence of MMCO-228-RTA.

Detailed description of preferred embodiments This invention uses cytotoxic drugs to treat a variety of cancer types, including colorectal cancer, ovarian cancer, and osteosarcoma. Use elementary combinations. These novel conjugates are monoclonal molecules linked to cytotoxins. Antibodies or binding fragments thereof, collectively consisting of the named immunoglobulins. these The composition targets cancer cells to kill cancer cells while minimizing damage to normal tissue. Mainly administered.

The immunoglobulins of this invention target cytotoxic substances to specific cancer cells in a cellular host. used as a target substance for With this invention, 72 kilodaltons (KD) Immunoglobulins and carcinoembryonic antigen (CEA) that define epitopes on glycoprotein antigens ) can be used as a targeting agent. Wear. One or both of these antigens may be associated with cancer including colorectal cancer, ovarian cancer, and osteosarcoma. It is present in a variety of cancers, including but not limited to.

A variety of cytotoxic substances are suitable for use as immunotoxins. Possible with this invention Cytotoxic substances include iodine-131, yttrium-90, rhenium-188 and bis Radionuclides such as Mass-212; Vindesine, Methotrexate, Adriama Numerous chemotherapeutic agents such as cisin and cisplatinum; and pokeweed antiviral liposome inactivating proteins, including protein, abrin and ricin (or their AM) diphtheria toxin, Pseudomonas exotoxin A or recombinant derivatives, etc. can contain cytotoxic proteins. Generally rcbis+eric Toxi ns” (chimeric toxin), 01snee and Ph11. “Pharmi c, “Ther”, Volume 25.

pp. 355-381 (1982), and rMonoclona Antibo dies for Cancer Detection and Therapy y” (monoclonal antibodies for cancer detection and treatment), Baldwin and Bye rs, pages 159-179, 224-266, Academic P ress (published in 1985), and the descriptions of both documents are incorporated herein. shall be incorporated into the book.

Toxic lectins are of particular interest in this invention. Cellular effects of toxic lectins, especially rigid That of Ng and Ablin is well researched.

Toxic lectins are two molecules linked by disulfide bridge(s). It consists of the polypeptide chains A and B.

Cytotoxicity is associated with the A chain and its inhibition of protein synthesis in nucleated cells. Ru. The B chain recognizes polysaccharide units on the cell surface and associates such units with a high degree of affinity. Create harmonious interactions.

Once the B chain binds to the multi-W binding unit on the cell surface, the A chain is imported into the cell. This inhibits liposomal protein synthesis and ultimately leads to cell death. Ricin A Preferred for this invention is the use of U.S. Pat. No. 4,590,071. It is disclosed in the issue, and the literature is included in the reference example.

Norisin toxin for use in this invention, although not used in the examples shown later. The preferred form of the A chain is that in which substantially pure RTA-30 is used. . The term rRTA-30 is derived from Fulton et al. (J. Biol. Chew, (1999) (1985), Vol. 281, pp. 5314-5319) and Vidal et al. (InL, J.

C5neer (1985), Vol. 36, pp. 705-711). Refers to a type of ricin toxin A with a molecular weight of approximately 30 kD as described, For purposes of this invention, concentrations of about 75% or more of RTA-30 are included. The RTAi-prepared sample is considered to be substantially pure; The preparation of pure RTA-30 is described in U.S. Patent Application No. 074,824. , the description of which is incorporated herein by reference.

Immunoglobulins and toxins are generally linked covalently, more specifically by disulfide bonds. However, it is possible to bind the toxin by any chemical bond, thereby making it possible for the toxin to become an immune Together with Robulin, it allows it to be selected by target cells. to achieve such a union. Other methods are known in the art. The only criterion is that binding is immunoglobulin This must be achieved without significantly reducing the affinity for that epitope. That is to say, yes.

This cytotoxic conjugate may be a single or a composition of two or more conjugates, other chemical It can be administered to the cancer cell host as a cocktail containing therapeutic agents, compositions, etc.

Cocktails are used to treat heterogeneous tumor cells, where targeting multiple antigens is important. This is especially important when

The cytotoxic conjugates of this invention can be administered to a cancer cell host in any convenient manner. Pharmaceutical compositions using conjugates that can be administered parenterally, i.e., intravenously, intraperitoneally, It can be administered intracavitally. Accordingly, this invention provides an acceptable carrier, preferably or a pyrogen-free solution of the cytotoxic conjugate dissolved in an aqueous carrier; A composition for parenteral administration comprising a cocktail is provided.

Various aqueous carriers such as water, buffer, o, 4g saline, 0.3z glycine, etc. can be used. These solutions are sterile and generally free of particulate matter , these compositions can be sterilized by conventional, well-known hypersterilization techniques. child The composition contains the pH adjusting agents and buffering agents required for appropriate physiological conditions; Pharmaceutically acceptable auxiliary substances such as moderators, e.g. sodium acetate, sodium chloride, etc. May contain thorium, potassium chloride, calcium chloride, sodium lactate, etc. Wear.

The compositions of this invention may be lyophilized for reconstitution with a suitable carrier prior to storage and use. I can do it. This technique has been shown to be effective for common immunoglobulins. , known methods of lyophilization and reconstitution can be used. Lyophilization and reconstitution This results in varying degrees of loss of antibody activity, and the amount used must be adjusted to compensate. As will be understood by those skilled in the art.

Single or multiple administrations of the composition may be carried out with the dosage level and type selected by the physician. can. In either case, the pharmaceutical regimen is sufficient to effectively treat the cancer cell host. The amount of the inventive conjugate(s) should be given. conjugate or conjugate The dosage of Kuteru can vary widely and is generally about 0. OIB/kg/day～ 20.0 ug/ky/day, and more specifically 0.05 sog/kg/day to 5 ．． 0any/ky/day.

The dose of unconjugated immunoglobulin depends on the immunotoxin used, the unconjugated immunoglobulin It varies greatly depending on the choice of epiglobulin, the nature and spread of the tumor, etc. The dosage is Generally O/OO1why/kg/day to 100.00H/kf/day, Usually about 0.01my/ky/day to 10.0m1F/97 days, and more special It is about 0.1 to 1 ady/kg/day. Clusters of unbound immunoglobulin The ratio in the compound also varies widely and is generally about 10,000 to 0.001:1. and usually about 1000 to 0.01:1, and more particularly about 100 to 0.1:1. It is 1.

Kits can also be provided for using the subject conjugates or cocktails. write As such, the compositions in question are usually in lyophilized form, either alone or in addition to chemotherapy for the treatment of cancer. Can be given in combination with legal agents. These kits contain phosphorus-a buffered food. Buffers such as saline, other inert ingredients, etc. may be included. Also, this kit Special instructions containing suggested protocols for adding the composition of interest to the cancer cell host It may also include an indication.

The actual mechanism of endocytosis is poorly understood. However, most, if not all, immunotoxins must be effective against their target cells. After binding to the surface antigen, endocytosis must be taken up by the receptor is believed to be triggered by binding to surface antigens.

With this invention, the effects of a subject immunotoxin composition are combined with a subject immunotoxin. It can be enhanced by co-administering immune globulins that are not present by a similar route. Wear. Potentiation is the administration of an immunotoxin that has been given the immunotoxin's effect of killing target cells. It means increasing with a given amount.

Although the following theory should not be considered a limitation of this invention, increased cytotoxins are believed to be due to increased affinity for the antigen resulting from immunoglobulin binding . The rationale for this is that the target cell antigen is limited and relies on antibody binding to related epitopes. If possible, the use of immunoconjugates would have wide-ranging effects.

The following examples are presented for illustrative purposes and for limitation purposes only in Ba1b/c mice. (Bantin & King+man, U, K,) for the treatment of cancer liver metastasis. Immunization was carried out with carcinoembryonic anti-1ii (CEA) derived from chloric acid extract. Immunization schedule were given 10 μs CEA in complete Freund's adjuvant (CFA) on days 0 and 7. intraluminally and 20 μg of CEA (same (in CFA) on days 25, 56, and 63). It consists of intraperitoneal administration.

Three days after the final antigen administration, spleen cells were aseptically removed from the immunized mice. alfre et al., Nature (I 977), vol. 266.

It is described on page 550, and the description of said document is incorporated into this document. ) to Following the method outlined, 10' tile m cells were treated with 106 voxanthine supplements. Xetothymidine (HMT) sensitive murine myeloma cell line, P3-NS I -Cell fusion with Ag4-1 (A, T, C, C, usage number TIB-18).

Using polyethylene glycol (PEG), hybrid cells were cultured in a 96-chamber culture plate (Cast Rat peritoneal exudate cells in ER, Gambridge, MA #3596) The cells were placed on a medium containing one layer of feeders (7 chambers of 2.5×10 3 N cells).

The cells were prepared using 15% Fetal Calf Serum (Myoelo). ne.

G 1bco, P aisley, U, K, ) and hyboxanthin ( 10-'M), thymidine (1,6 x 10-5M) (both Sigma, Dorse (Purchased from T, U, K,) and methotrexate (10-5M) (Lederle , Hawpshire.

Dulbecco's modified Eagle's medium (Mo defined Eagle’s Medium) (F low Labs, I rvine.

It was cultured in U, Ni, ).

Within 2 weeks of fusion, cultures of hybrid cells were subjected to enzyme immunoassay (EIA). Antibody binding was tested by. Positive cultures were plated at 1-3 cells/chamber in a 96-chamber culture plate. Cloning was performed using limited dilution plate culture. A room containing only one colony This is confirmed by microscopic examination, and then the reaction between CEA and normal colon antigen (NCA') is confirmed. Responsiveness was determined by ETA and radioimmunoassay (RIA), and next colorectal tumor The reactivity between the nuclear membrane and the outer nuclear membrane from normal colon was tested by EIA. XMMCO-22 The clone labeled 8 was found to stably secrete immunoglobulin; Immunoglobulins were determined in subclass IH02g by solid-phase RIA using standard methods. It was decided that there was. Hybridoma XMMCO-228 is at 12301 Park lown Dr., Rockville, MD 20852゜tJsA American Type Cu1ture Co11ection (A , T, C, C,). The deposit was made on August 14, 1986, and A. T, C, C, deposit number HB 9174 was given.

6-10 week old Ba1b/c mice (Banten & K ing+man.

Hybridomas were cultured intraperitoneally using U. 3 weeks early (0,5 mN pristane (A ldidge, G 1llir+gha) −.

Dorset, U, K, ) was injected intraperitoneally (i, p, ) for pretreatment. Mice were injected with approximately 107 hybridoma cells. Hybridization of ascites that occurs 3 weeks after injection, and collected using Pr1ce and Baldwin's method (ICR3 Med, Sei, (1984) 12 volumes.

It is described on pages 1000 to 01, and the description of the said document is incorporated into this document. Ru. ), immunoglobulin measurements revealed that it contained 5my7'al antibodies.

Antibodies in ascites can be isolated using S-epharose-Pro using methods known to those skilled in the art. It was purified by affinity chromatography using teinA.

The hybridoma is a 10% tallow baby serum in a plastic 8300ml bottle. (Myoclone, Gibco, Pa1sley, U, K,) Toshihokisa Thymidine (1,6 x 1O-58) (both from Sigma).

Dorset, U.K.) and methotrexate (10-'M) (purchased from Leder! Dulbecco's minimum requirement including e, Hampsbire, U, K, Fiow Labs, Irvine.

The concentration of 0m cells grown in vitro in U, Ni, ) over a 4-5 day culture period is 105 cells 7'al culture medium, and the concentration of monoclonal antibody is 4 μg/s. rl medium, doubling time was 12 hours.

The binding of XMMCO-228 to MKN45al cells is known to those skilled in the art and described above. It was measured by flow cytometry (flowcyromeLry).

Gastric cancer cell line MKN45 reacts with XMMCO-791 monoclonal antibody. Expresses the CEA antigen that reacts with the 72 kD antigen and XMMCO-228.

B14 is a monoclonal antibody directed against breast cancer, and is a monoclonal antibody directed against breast cancer. Reacts with a small portion of cross-reacting antigen (NCA).

It is described in Example ■. Normal mouse serum was used as a control. Conclusion The results are summarized in Table 1.

caged people MKN45's Kano Mumono Ronal Royal 1st Light Branch by Flow Itometo 1- X M M CO-228987 1 to 4':A XMMCO-228 pecked normal cells using indirect immunoperoxidase conjugate method. Cross-reactivity with cells was evaluated. As a control, murine monochrome tissue purified simultaneously tested at the same concentration with null antibodies. Control immunoglobulins used for this purpose The protein is IgG2 mouse myeloma protein (UPC10, LittonBionet ics, Kensington, MD) or sheep red blood cell cells (SRBC). It was a monoclonal antibody that was reactive.

Cut fresh tissue sections (up to 2-3 mm thick and 2c+s wide) into obtimal cutters. Optimal Cutting Tem perature Compound) (OCT) (A'mes Co,.

Loosely cover with E 1 khar, I ndiana) and wrap with aluminum foil. is.

Specimens were prepared using liquid nitrogen cooled isopentane (2-methylbutane, VWR).

Norwald, CA) at controlled temperatures (-120"C to -150"C). The 0 tissue that was flash frozen for 10-15 seconds was hermetically sealed to prevent tissue evaporation and drying. It was stored frozen in a container at -70°C for a long period of time and -20''C for a short period of time.

The frozen composite fibers were left in a -35°C freezer overnight before cutting out the frozen pieces. Balance properly. Sections were dried overnight at room temperature prior to staining.

Use a diamond pencil to etch a circle around the section of each glass slide. ing. Slides were then fixed in acetone for 1 minute, followed by addition of phosphate buffer. Washed with saline (PBS) for 10 minutes. Remove excess fluid and cover the tissue to keep it dry. Apply the next antibody for 1 hour while ensuring that Washed for minutes.

Excess fluid was removed and peroxidase-conjugated sheep anti-mouse immunoglobulin G (T ago/product code 6450) (diluted 1=10 in PBS) applied for 330 minutes. , and then washed with PBS for 10 minutes.

Aminoethyl carbazole slides 0.5 mj of stock solution of 1201 AEC in methylformamide! ,9.5-l acetate buffer (79 ml of 0.1M sodium acetate, 0.IN acetic acid from 21 companies), and 0,05 mf of 3% H20□) for 5 minutes, then washed with water for 5 minutes. did. The slides were then coated with fresh Mayer's hematoxylin (Mayer's H aematoxylin) for 5 minutes, then washed in running water for 5 minutes, and Gently cover with lyserine mounting medium. Table ■ summarizes the results.

Antibody penetration into normal tissues measured by immunoperoxidase Approximately 9 oz of the colorectal cancer 515 staining positive group was strongly stained 55 and 7 Autopsy tissue negative tissue kidney pancreas muscle liver Skin heart Midorimaru pancreas The marrow kidney aorta cerebellum lymph nodes Doubt (+/-) lungs Positive- 1+ Note: In the glucose oxidase method, antibody 228 may not stain granulocytes. It was shown. All of the other antibodies similar to CEA that we studied target granulocytes. Stained.

Importantly, XMMCO-228 does not bind to granulocytes. normal colon anti [(NCA) is present in diverse tissues such as normal colon, granulocytes, and ancestral bone marrow cells. Since the CEA antigen has a small NCA fragment, anti-CEA monoclonal antibodies are available in a variety of ways. XMMCO-228 monoclonal antibody binds NCA on normal tissues. you do not.

D. XMMCO-228 Ricin Toxin-A RTA+Muridine AM Purification Included The legal one is US Patent No. 4.590.

No. 071, the disclosure of which is incorporated herein by reference. The in vitro cytotoxicity of 8-RTA conjugates was determined by 75Se-selenomethionine. It was measured using the analytical method. Immunotoxicity in single cell suspension of MKN45 cells derived from gastric cancer Inoculate with RTA for 15 minutes, wash, and add 10% tallow serum to a flat-bottomed micrometer plate. A concentration of 10 cells for 0.2 d of immunotoxin or RTA in RPMI medium containing It was added with Various amounts of immunotoxin were added to each chamber. 24 hours later? SSease Renomethionine was added to each chamber. Cultures were kept at room temperature for an additional 16 hours, washed, and The sample was dried on the bottom of the cell, and the chamber was calculated using a gamma counter. Results are expressed as 50% inhibitory concentration. They are summarized in Table ■.

nth In vitro 6 on MKN451 1-1 1st work discharge/processing■ RT A 2238 XMMCO-228-RTA 600 1-I IC0 Mol Agency 1 RTA IX 10-”M XMMCO-228-RTA lXl0-”Adjust to an RTA molar concentration equivalent to M1. Ta.

Blood I by XMMCO-228-RTA of F. Cellular cells derived from human colon adenocarcinoma were administered to 7 to 10 nude mice at each data point. In (A, T, C, C, accession number CL188) LS174T cells were transplanted. Ta. First dose of therapeutic compound on day 3 post-transfer followed by completion of scheduled dosing. The administration was repeated 10 to 20 times. The results are summarized in Table ■, and the control ( Expressed as the ratio of tumor weight in treated mice divided by tumor weight in untreated mice.

The results, expressed as mean tumor diameter, are summarized graphically in Table 1.

shaver ゛　2　Sunday　Evening゛　Response to Outpatient transplantation Scheduled administration Tumor weight in treatment area 2 LS 174T XMHCO-22 8801, 183LS 174T XMMCO-228-RTA 56 0, 3. These results demonstrate the effectiveness of XMMCO-228-RT in the treatment of human tumor outpatient transplants. This proves the result of A.

II: XMMCO-791-RTA and XMMCO-228-RTA - le A, XMMCO-791-RTA + XMMCO-228-RTA officer XMMCO-228RTA (Scand, J. Immuno.

(1983) Vol. 18, pp. 411-420) and X M M C○-228- RTA's cocktail is XMHCO-791-R, TA and X　M　C○-22 8-RTA at a ratio of approximately 0.01 ml to the cancer cell host. 1 on 9/? / day ~ 20. A dosage range of OB/kg/day can be administered. mosquito Qutel can be administered parenterally or commonly in a suitable vehicle such as phosphate buffered saline. It is typically administered by intravenous infusion or intraperitoneal injection.

B, JfflX M M C○-228-R in vitro In vitro cytotoxicity of TA conjugates was determined by 75Se-selenomethionine analysis. It was measured. Single cell suspension of MKN45 cells derived from gastric cancer was treated with immunotoxin or RT. Leave A for 15 minutes, wash, and add RPM containing 10% tallow serum to a flat-bottomed micrometer plate. Added at a concentration of 105:f3 cells per 0.2 ml of immunotoxin or RTA in I medium. added. Various amounts of immunotoxin were added to each chamber. 24 hours later, 1 S S e- Selenomethionine was added to each chamber. Cultures were kept at room temperature for an additional 16 hours, washed, and Dry on the bottom of the chamber and count on a chamber 3 gamma counter. Results are expressed as 50% inhibitory concentration. These are summarized in Table ■.

These data are XMMCO-791-RTA and XMMCO-228-R When TA is administered in “cocktail” form, cytotoxicity is at least additive, and This indicates that it is probably synergistic. They also offer greater immunity compared to RTA alone. The effect of RTA directed by epizootic toxin (or antibody) is also shown.

C-8's XMMCO-791-RTA+XMMCO-228-RTA'' 5. At each data point, cell lines were induced in 7 to 10 nude mice. MKN45 cells were transplanted. The first dose of therapeutic compound was given at 38 days post-transplant. followed by 10-20 doses until the scheduled dose was completed. Result is The weight of treated mice divided by the tumor weight of control (untreated) mice is summarized in Table 7. It is expressed as a ratio of the tumor weight of each tumor.

LL friend XMMCO-228-RTA human tumor outpatient transplant response Outpatient transplant treatment, scheduled administration, tumor weight in treatment area! ! - Senkura 1 - Otsu - - Fu Ran Punch! I MKN 45 XMMCO-228-RTA25 0.74χMMCO-2 28-25 1.1 2 HKN 45 XMMCO-228-RTA Army 0.56% MMCO-79 1-RTA 1 XMMCO-228-RTA and XMMCO-7 at 25 mL/kIF each 91-RTA was administered as a mixture.

These data are X! LMCO-791-RTA and XMMCo-228-RTA When administered in “cocktail” form, cytotoxicity is at least additive, and possibly It has been shown that they are highly synergistic. They are XMMCO-791-RTA and Effect of a cocktail consisting of MM CO-228-4TA on tumor cells in the host is also shown.

: XMMCO-228-RTA's X M M B R-B14 monoclonal Miyako ζL beg 11 A. Immunizations were made with 521AM whole cells derived from hydrophilic thoracic metastases. Immunization schedule is for 1 week It consisted of six intraperitoneal injections of 105 cells at intervals. ! & final 3x10 Intravenous administration of cells was performed 3 days before fusion. Three days after the final antigen administration, immunization The pancreatic cells of the mouse were aseptically removed. Sift the single cell suspension through a 80-mesh W4 mesh sieve. #1985-00080 (Belleo). iscove cells Iscoves Complete Medium (Grand 15land, N, Y,, N, Y,) and counted. Hyboxanthin S, an aminopriten-thymidine (HAT) sensitive murine myeloma cell line p2/A g14RB cells (A, T, C, C, usage number CRL1581) Washed three times and counted. 30% (V/V) polyethylene glycol (PEG40 00 Merck), 10% dimethyl sulfoxide and 60% Iscobus Using a medium, cells were fused at a ratio of 1 myeloma cell to 2 pancreatic cells. fusion The products were added to 96 chamber culture plates at a concentration of 105 myeloma cells/chamber. 20 cells % fetal bovine serum and HAT medium (hypoxanthine 136 my/100 ral.

Aminopterin 0.018B/100ml, Thymidine 136B/100 theory 1 ) was cultured in Iscove's medium supplemented with β-mercaptoethanol.

Within two weeks after fusion, cultures of hybridoma cells were incubated with Vec to test for reactivity. 200μg of nitrocellulose using tor L ads A B C kit Antibody binding to 521AM tumor cells was tested by spotting onto membranes. . Next, go to the chamber with the blue dot and select it for the fibroblast membrane extract from the same patient. A separate test was conducted. Plate positive cultures at 1-3 cells/chamber in 96-chamber culture plates. Cloning was performed using limited dilution. Microscopic examination of a chamber containing only one colony was confirmed, and then the reactivity was examined. Clone labeled XMMBR-B14 is recognized to stably secrete immunoglobulin, and the immunoglobulin is Ig It was determined to be a subclass of G1. Hybridoma XMMBR-B14 is currently Currently located at 12301 Parklawn Dr.

All1eri located in Rockville, MD 20852, USA canType　Culture　Co11ection(A,　T,　C,C ,). The deposit was made on January 14, 1987, A, T, C, C.

Deposit number HB9308 was given.

6-10 week old B alb/mice (Bantin & Kingman, U/ The hybridomas were cultured intraperitoneally using ni/). 3 weeks early (0.5ml bottle) pristane (A Idridge, G if l in Gham.

Dorset, U, K, ) was injected intraperitoneally (i, p, ) for pretreatment. Mice were injected with approximately 107 hybridoma cells. Hybridization of ascites that occurs 3 weeks after injection, and were collected using the method of Pr1ce and Baldwin (ICR3). Med, Sei, (1984) 12 volumes.

1000-01, which document is incorporated by reference. )yo Measurement of immunoglobulin revealed that the sample contained 5 tsg/va1 antibodies.

Antibodies in ascites can be isolated using S-epharose-Pro using methods known to those skilled in the art. Purified by affinity chromatography using teinA.

Hybridomas are made with 20% tallow serum in a plastic bottle made by 300 companies. Grown in vitro in Iscove's medium supplemented with β-mercaptoethanol. I cloned it. Cell concentration was 105 cells/company over a 4-5 day culture period. ・Medium, concentration of monoclonal antibody is 4 μg 7'nl ・Medium, doubling The time was 12 hours.

B. In vitro analysis of XMMBR-B14 by flow cytometry work for The binding of XMMBR-814 to cell lines and primary cancer-inducing cells is known to those skilled in the art. were measured by flow cytometry as described above. XMMBR-B14 is Tests to detect antigens expressed on breast cancer and colon cancer cell lines (Table ■) It was shown more. Another study using purified protein preparations showed that XMMBR-81 The epitope defined in 4 was CEA/NCA (carcinoembryonic antigen/normal cross-reactive anti-y It was demonstrated that this protein is recognized on a class of antigens named K). This is exposed normality It is the part of the CEA molecule that also cross-reacts with cross-reacting antigens. It has two primary colons It also reacts with cells derived from cancer. Normal mouse immunoglobulin (NMl) or used normal mouse serum (NMS) as a control.

schemer Flow Itometo 1-to 4'4:A Fluorescence Sarazen JLIII m 5"μ"i [directly above mu Colon cancer C170NMIg 19.3 - (Low CEA) B14B8 60 ．． 4±Colon cancer LS174T NM IE 22.2 - (Line 1) 81 4B8 2641.74 + Keidamagan LS174T NM rg 18.6 - (L ine 2) 814BS 1665.63 ÷ Breast cancer BT474-3 NM Ig 17.3 -814B8 316.41-2+ 2. -゛''rash- C212NMS 31.4- 814B8 607.53+ C213NMS 33.8- 814B! 3 836.84+ C, XMMBR-B 4-44 according to EIA: , standard EIA method known in the art for XMMBR-B14 of F4+ was used to test the reactivity between primary colon cancer membrane and normal colon mucosa.

The studies summarized in Table 1 show that XMMBR-B14 reacts with colon cancer membranes. There is.

1st JL EIA’s contribution to 4+“4 Skin 1l--ELIS^uni...0DCEA preparation sample Normal conjunctival membrane Colon cancer membrane (NP,) (TP,) (T186) (B2O33)XMMBR-B14 0.130 0.581 0.758 0.665 TP, - Pooled primary colon Membrane preparation sample from cancer NP, - Membrane preparation sample from pooled normal colon mucosa (colon cancer patient) Membrane preparation sample from T186 primary carcinoma T186 CEAB4058-type, P. System CE＾・;D, XMMB to CEA NCA of XMMBR-B14 Using solid-phase radioimmunoassay to measure the reactivity of R-B14 with CEA and NCA prepared samples. It was measured from Briefly, the antigen preparation sample is coated inside the chamber of a micrometer plate. Add the monoclonal antibody at 100 ml, keep at room temperature for 1 to 2 hours, and wash well. was labeled with 125 to detect murine monoclonal antibodies bound to Rabbit anti-mouse IgG F(ab)2I! 7r pieces were added. Table 1.

In the first test, semi-purified CE A (Ro(ers) and NCA (B399 The binding of monoclonal antibodies to 1) was compared. XMMBR-B14 is NCA and It bound to both CEA (NCA:CEA ratio is 1.2 near).

1st = XMMBR-B14's CEA to NCA Average binding CPM standard deviation difference -Batland CEA Roers NCA (B3991 anti-CE^ 1427±222 44 3±48XMMBR-8142558±114 955±140E0MNK45 4. X M M CO-228-RT A thin XMMBR-B14 “Response” Table 1 shows the amount of XMMBR-B14 added and increased stimulation of MNK45 cells. XMMCO228RTA shows increased cytotoxicity of the immunotoxin.

XMMCO-228-RT ranging from 1 ny/va1 to 100 ng/ml Six different curves were generated using the concentration of A. 0-10 for these concentrations Various amounts of XMMBR-814 over μ3/ml were added. For each curve Table 1 shows the calculated IC5o values.

No. JAIL XMMCO-228-RT, a monoclonal antibody produced by XMMBR-814 Dose response to AMI cell toxicity MNK45 Cells XMMBR-814t)') U-fk antibody against MKN45 cells XMMC Cytotoxicity of O-228-RTA (μy/mi’) (ICsony/m/) 0.001 113 0.00 155 The present invention provides novel formulations and methods that are effective in treating various cancers. Cytotoxicity The compound is a cocktail form or the presence of certain unconjugated monoclonal antibodies. It is particularly effective under

The present invention has been described in some detail by way of illustration and examples for purposes of clarity of understanding. It is evident that certain changes and modifications may be practiced within the scope of the appended claims. Ru.

Qianjiao Kishiki (Sunday) international search report

Claims (24)

[Claims] 1. an immunotoxin and an amount sufficient to potentiate the immunotoxin to target cells expressing the antigen; , to a different epitope on said antigen than the first immunoglobulin binds to. said target cell comprising exposing said target cell to a second immunoglobulin which can be combined with said target cell. said first immunoglobulin directed against a cell surface antigen on the cell and bound to a toxin; A method of enhancing cytotoxicity of said immunotoxin comprising Robulin. 2. 2. The method according to claim 1, wherein the antigen is an antigen associated with a tumor. 3. The first and second immunoglobulins target epitopes on tumor cells expressing carcinoembryonic antigens. 3. The method of claim 2, wherein the method can be combined into a group. 4. The epitope to which the second immunoglobulin binds is a normal cross-reactivity of the carcinoembryonic antigen. 4. The method of claim 3 in which the antigen is present. 5. The first immunoglobulin is XXMMCO-228 conjugated to RTA; 5. The method according to claim 4, wherein the second immunoglobulin is XXMBR-B14. 6. Claim that the first and second immunoglobulins are capable of binding to an antigen on a T cell The method described in item 1. 7. 2. The method according to claim 1, wherein the antigen is CD-5. 8. 2. The method of claim 1, wherein the antigen is on leukemic T cells. 9. the first immunoglobulin capable of binding epitopes on cell surface antigens The toxin bound to the antigen is different from the first immunoglobulin bound to the antigen. and a second immunoglobulin capable of binding to an epitope. A cytotoxic therapeutic cocktail consisting of: 10. 10. The cocktail according to claim 9, wherein the antigen is an antigen associated with a tumor. 11. The first and second immunoglobulins are antigens on tumor cells that express cancerous fetal antigens. 10. A cocktail according to claim 9, which can be combined with a tobe. 12. The epitope to which the second immunoglobulin binds is a normal cross-reactor of the carcinoembryonic antigen. 12. The cocktail according to claim 11, wherein the cocktail is in a reactive antigen. 13. The first immunoglobulin is XXMMCO-228 conjugated to RTA; The method according to claim 12, wherein the second immunoglobulin is XXMBR-B14. Kutel. 14. The first and second immunoglobulins are capable of binding antigens on T cells. The cocktail according to item 9 of the desired scope. 15. 15. The cocktail according to claim 14, wherein the antigen is CD-5. 16. The use of immunotoxins to enhance cancer cells expressing tumor-associated antigens with immunotoxins a sufficient amount of the first immunoglobulin on the tumor-associated antigen to bind to the antigen. exposure to a second immunoglobulin capable of binding to the epitope said first molecule directed against said tumor-associated antigen and conjugated to a toxin. in humans by enhancing the cytotoxicity of the immunotoxin, which consists of immunoglobulins. How to kill cancer. 17. The first and second immunoglobulins are epitopes on tumor cells that express oncofetal antigens. 17. The method of claim 16, which can be coupled to a stove. 18. The epitope to which the second immunoglobulin binds is a normal cross-reactor of the carcinoembryonic antigen. 18. The method of claim 17, wherein the antigen is in a small portion. 19. The first immunoglobulin is XXMMCO-228 conjugated to RTA; The method according to claim 18, wherein the second immunoglobulin is XXMBR-B14. Law. 20. Cancer cells include colorectal cancer cells, gastric cancer cells, pancreatic cancer cells, lung cancer cells, and breast cancer cells. 17. The method of claim 16, wherein the method is selected from the group consisting of: 21. The first and second immunoglobulins are capable of binding antigens on T cells. Scope of Claim 16. The method according to item 16. 22. 22. The method of claim 21, wherein the antigen is CD-5. 23. 22. The method of claim 21, wherein the antigen is on leukemic T cells. 24. The ratio of the second immunoglobulin to the first immunoglobulin is 10 by weight. 17. The method of claim 16, wherein the ratio is 0 to 0.01:1.