JPH06501705A - Synergistic treatment with a combination of anti-tumor antibodies and biologically active agents - 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] Field of Synergistic Therapeutic Inventions by Combinations of Anti-Tumor Antibodies and Biologically Active Agents The present invention is useful for the treatment of diseases, such as combinations of antibody therapy and biologically active agents into chemotherapy. Concerning the use of combinations. Tumor-bearing mammals treated with anti-tumor antibodies and chemotherapy Surprisingly achieving significantly higher remission when treated by a combined means consisting of It is based in part on recent discoveries. The method of the invention effectively eradicates tumor cells Unique for placing the immune system to act in contact with exogenous compounds provide a means to do so.

Background of the invention Layer weight 1171su C8 string 11 blood Tumor cells are absent or present in small amounts over their normal cellular counterparts Expresses a certain antigen. Most of them are shared by tumors and some embryonic cells. It is a differentiation antigen. ! ! A fleeting tumor that appears with sufficient selectivity in tumors. Antigens can serve as possible targets for therapeutic agents. This is this Report (Hellstromand [lellstrom, Accos 1is h　5cents　in　Cancer　Re5earch-1984Pr1z eYear, General Motors Cancer Re5earch Foundation + J, G, Fortner & J, E, Rhoads, eds, +J.B., Lippincott Company, Ph1ladel phfa 1985+ pages 216-240) Malignant melanoma, one of the tumors, and other tumors (Burchell and Ta ylor-Papadishtriou+RJ, Baldwin and V, S. Byers, eds, Monoclonal Antibodfes forTumor Detection and Dru Tar etin +Acadesic Press, 1985.1-15Bage i Kemshe ad, M, pp. 281-302).

Cells in which many antibodies are expressed in higher amounts by human tumors than by normal tissues Manufactured against surface antigens. Antibodies to cell surface antigens are produced in tumors in the presence of complement. May be cytotoxic to tumor cells (Hellstrom et al., 1962. Pr. og, Allergy 9:158-245), and some antibodies are antibody-dependent. can mediate persistent cell-mediated cytotoxicity (Perlmann et al.

1969, Adv, Immunol, Sat): 117-193; MacLen nan etc. +1969+Im+*unol.

17:897-910; 5 Kurzak et al., 1972. J, Exp, M ed, Minister: 997-1002; Po1lack et al., 1972. Int , J. Cancer, 9:316-323) is also well established. There is. In the first case, appropriate complement RC is commonly used in rabbits or guinea pigs. ), and in the latter case, the effector cell source (generally of murine origin). ) is required.

Antibodies against tumor-associated antigens kill human tumor cells in the presence of human effector cells. (Hellitro-et al., 1981. Int.

J, Cancer 27:2B1-285), and antibodies against such antigens. It was found that the body can kill tumor cells in the presence of human serum as a source of complement ( Hellstrom et al., 1985. Proc, Natl, Acad, Sci , 82: 1499-1502; Hellstrom et al., +1985+Mon oclonal Antibodies and Cancer Therap y+UCLA Symposia on Mo1ecular and Ce1 lular Bto1ology+No. 27tL, pages 149-164, Alan R0Liss+Inc, +NY).

An interesting approach to preparing anticancer drugs is the labeling of antibodies with radioactive isotopes. Ring (Larson et al., +1983.J, C11n, Invest, 72 :2101-2114;0rder, 1984. Compr, Tberapy 10:9-18; Carrasquillo et al.

1984, Cancer Treatment Reports 68:317 ~328; de Nardo et al., 1985゜Int, J, Radiat ion Oncology Biol, Phys, 11:335-34B), or toxins (Jangen etc., +1982.1g+nun+:+1.Rev , 62:185-216; Vitetta and Uhr.

1984, Tjansplant, 37:535-53B) or anticancer drugs (Gh ose et al., 1972. Br1t.

Mlld, J. i: 495-449; Hurwitz et al., 1975. Ca ncer Res, 35: 1175-1181; Rowland et al., +19 85+Cancer Immunol, Immunother, 19:1-7 ). The antibody confers specificity and the isotope or The drug provides the ability to destroy tumors. However, the drawbacks of this approach Both anticancer drugs and radioactive isotopes have high levels of toxicity to normal tissues. This is a fact. Therefore, non-specific symptoms in various organs such as kidney, liver or bone marrow. External ingestion would lead to substantial side effects.

Summary of the invention The present invention is useful for chemotherapy for inhibiting tumor cell proliferation, e.g. Administration of biologically active agents and anti-inflammatory agents to treat and inhibit diseases. Concerning the use of combinations of somatic therapy. Observations of the surprising effectiveness of treatment in combination Based in part on; several tumor-bearing mammals that received anti-tumor antibody 81196 The group achieved significantly higher inhibition of tumor growth in response to chemotherapy. II-like tumor Tumor-bearing mammals showed significantly lower levels of response to chemotherapy alone and There was no response at all to the body alone.

In particularly preferred embodiments of the invention, anti-tumor antibodies, such as preferably BR 96 monoclonal antibody is administered to a mammal, which is subsequently or simultaneously administered to a standard antibody. In a preferred embodiment of the invention, the chemotherapy is treated with chemotherapy of It is operated at the same time as body processing.

The effectiveness of the combination treatment makes malignant cells more susceptible to the toxic effects of chemotherapeutic agents. or chemotherapeutic drugs to elicit a synergistic immune response in the mammal. It is suggested that this may contribute to the development of antibodies at the tumor site.

Drawing description Figure 1 shows BR96 antibody or doxorubicin (Adr), and BR96 and Ad Tumors caused by treatment of nude mice (Ba1b/c nu/nu female) with the combination of r Human lung adenocarcinoma (H270?) tumor implants showing effects on tumor volume were into the right posterior flank of mice, and divide the mice into treatment groups. (8 mice/group), control mice (open circles) received PBS injection; The mice received only Adr (7tg/kg/injection, black square), and BR96 only (black square). BR96 and 5 wg/kg/injection Adr (white triangle) and BR96 and 7 wg/kg/injection of Adr (black triangles).

Figure 2 shows nudes treated with BR96 antibody and mitomycin C (MMC). Ba human lung adenocarcinoma H2707 tumor implants showing inhibition of tumor volume in mice. 1b/c into the right posterior flank of a nu/nu female mouse and the mouse was treated. divided into logical groups. Control mice (open circles) received PBS injections; remaining mice received M MC only (3■/kg/injection, black square), BR96 only (black circle) HBR96 and and MMC (white triangle) at 2 ■/kg/injection and BR96 and 3 ■/kg/injection. Received MMC (black triangle).

Detailed description of the invention The present invention relates to a therapeutic approach comprising treatment with anti-tumor antibodies and standard chemotherapy. In a preferred embodiment of the invention, the anti-tumor antibody binds to the antigen on the surface of tumor cells. reacts. In the most preferred embodiment, the anti-tumor antibody is a monoclonal antibody. Body BI? It is 96.

Although there is no obligation to explain the efficiency of antibody/chemotherapy tools in killing tumor cells, , antibodies that bind to the surface of tumor cells that are internalized, such as BR96, are likely to be used as drugs. It is suggested that increasing uptake makes cells more sensitive to chemotherapy killing. be measured. When given to immune-competing individuals other than nude mice, anti-tumor antibodies Treatments that make cells more sensitive to drugs or that make them more susceptible to the animal or patient's immune system Chemotherapy drugs such as doxol by making them more susceptible to response Bicine (adriamycin) and mitomycin C synergize to induce an immune response This can provide additional benefits.

For clarity of disclosure, the detailed description of the invention is divided into the following subsections: However, this does not limit the invention: (i) Characteristics of the antibody molecule of the present invention; (ii) preparation of monoclonal antibodies; and (iii) antitumor antibodies and biological activity. Tumor treatment with a combination of sex drugs.

of of of Antibodies of virtually any origin can be used in accordance with the invention, but are preferred. In some embodiments, the antibody defines a tumor-associated antigen. Monoclonal antibodies are , giving the advantage of continuous ampoule feeding. In fact, tumor-associated antigens can induce immunize and identify hybridomas that produce antibodies against such antigens. can react with and treat many types of tumors by can enable the rapid establishment of a panel of antibodies.

The BR96 antibody is an antibody of the 1gG3 subclass. The antibodies are used in various organ types. type cancer cells, such as breast, lung, colon, and ovarian tumors; Furthermore, BR9 shows high specificity for cultured cell lines established from colon cancer. 6 Antibodies can be used to target other types of tumor cells, such as T-cell lymphoma cell lines, CEM and against MOLT-4, the B-cell lymphoma cell line P3HR-1 or the melanoma cell line. The B[196 antibody shows no binding, as shown by electron microscopy, for example. can be internalized into antigen-positive tumor cells; is toxic to cells, mediates ADCC and CDC activities, and surprisingly It is cytotoxic alone in its non-denatured form when applied at sufficiently high doses. be. The BR96 antibody is directed against fucosylated Le' antigens, or such entities. It appears to recognize closely related antigens.

As used herein, the term "BR96 antibody" refers to a complete intact protein. Reclonal and monoclonal antibody molecules, such as hybridoma ATCCN murine BR96 monoclonal antibody produced by ct HB10036, and and chimeric antibody molecules, such as hybridoma ATCCN111IB10460. This includes chimeric BR96 antibodies produced by. The above B[196 antibody is available in the art. Using well-established techniques in Any fragment, e.g. Fab, F(ab')z and Fv flag [For example, Rouseauz, +”Optimal Con dions For The Preparation of Prote alytic Fragments From 1lonoclonal IgG of Different Rat IgG 5ubclasses″B Methods　Enz　mol　、、匪：663～669(Academic Press 1986)) The BR96 antibody of the present invention can also be used as a fusion protein. Including quality.

In addition, the BR96 antibody can be used in major organs such as kidneys, lungs, liver, skin, lungs, and breasts. on normal human tissue from the chambers, colon, brain, thyroid, heart, lymph nodes or ovaries. The antibody exhibits no immunohistochemically detectable binding in the peripheral bloodstream. BR96 antibody, which does not react with lymphocytes, has some It exhibits limited binding to cells and has been shown to have limited binding to porcine cells in the pancreas, and to gastric and and binds to epithelial cells in the esophagus. Therefore, the BR96 antibody is Compared to most known antitumor drugs, it has a high degree of specificity for tumor cells. It is more effective than antibodies (for example, Heilstro-, etc.).

“Is-unological approaches To Tul+or Therapy: Monoclonal Antibodies, Tumor V accines, and Anti-1diotypes’+7 odifie d Anti ens and Antibodies In Dia nos is and TheraQuash/Rodwell (Publisher), 1-39 Page (Marcell Dekker, Inc., 1989) and Bagsh awe,”7w+1our Markers-Where Do He Go From Here', Br, J.

Cncer, 48:167-175 (1983)).

monoclonal ill According to the invention, the monoclonal antibody may be any of the monoclonal antibodies known in the art. method, such as Xohler and Milstein (1975, Nat. ure256:495-497) and the hybridoma technique originally developed by and trioma technique, human B-cell hybridoma technique (Kozborn et al. , 1983゜Immunology Today ↓ Near 2), EBV-High Bridema technique (Cole et al., +1985, Monoclonal Ant ibodies and Cancer Thera, Alan R, Li5 s+Inc, pages 77-96 and Husenato, 1989, 5science 246: 1275-1281).

as well as the chimeric antibody techniques discussed above; The law is not limited to that.

Although the present invention is demonstrated using mouse monoclonal antibodies, the present invention is limited to without limitation; in fact, human antibodies can be used and are found to be preferred. . Such antibodies were developed using human hybridomas (Cote, +1983 +Proc, Natl, Acad, Sci, +80: 2026-2030) or by in vitro transformation of human B cells with EBV virus. (Cole, etc., +1985+ Monoclonal Antibodies andCancer Yukoyu■, Alan R, Li5s+ pages 77-96 ),can get. Indeed, according to the present invention, techniques for the production of chimeric antibodies (M orrison, +1984+Proc, Natl, Acad, Sci, + 81: 6851-6855; Neuberger et al.

1984, Nature 312:604-608; Takeda et al., 1 985. Nature 314: 452-454) has the appropriate biological activity. Genes from mouse antibody molecules of appropriate antigen specificity along with genes from human antibody molecules Developed by splicing the child.

The subsections below describe how the antibodies used in the following examples are prepared. I will post it.

The monoclonal antibody of the present invention, designated as BR96, can be used as an immunogen in breast cancer cells. It was produced by the hybridoma technique described below using cell line H3396. listed below BR96 hybrid prepared as described above and producing a BR96 antibody was deposited with the ATCC on February 22, 1989, and identified as: Received: B1166 ATCC Accession Number I HB 10036゜Another aspect According to BR96 was purified as described (Nisonoff et al.).

“The Antibody Mo1ecule”, Academic Pre ss, New York (1975)) by pepsin digestion. Tumor F(ab')z fragment to tumor (13396) and l'1cF7 cells. The binding was shown to be comparable to that of the complete R96 monoclonal antibody. .

Furthermore, chimeric (murine/human) antibodies such as Fe1l-r Proc, Na kl.

Acad, Sci, USA 86:8507-8511 (1989), and 19 U, S co-pending application No. 243,873 filed on September 14, 1988; U, S Application No. 468, No. 035 filed on June 22, 1990 (this publication) The two-step homologous recombination method as described in the application (assigned to the same assignee) the disclosures of all such documents are incorporated herein by reference. be caught. This two-step method uses BR96 containing human Ig-Ggammal f+ chain. To generate hybridomas expressing chimeric antibodies, murine BR96 monoclonal Mouse hybridoma cell lines expressing local antibodies (hybridoma ATCC human I to transfect Nct) 1810036).

Next, this technology involves the use of a targeting vector encoding the Ggammal H chain. Hybridomas are murine hives expressing BR96 chimeric antibodies containing human KL chains. To generate ridomas, a standard containing DNA encoding human kappa (K) light chain is used. transfected with a specific vector. Transfect the hybridoma The targeting vector used to mmal HC-DDa vector (Oncogen.

5eattle, -^) and HIndI [[digested psVzgpt/Ck vector (Oncogen+5eattle.

WA).

Prepared as follows and generates chimeric human/murine BR96 antibody , a chimeric BR96 hybrid identified herein as ChiBR96 was deposited with the ATCC on May 23, 1990, and identified as: Chimeric antibody: ChiBR96ATCC accession number: HB 10460° Immediately after an expressing hybridoma is identified, the hybridoma is cultured. , and the desired chimeric molecule is prepared using techniques in the art to isolate monoclonal antibodies. It is isolated from cell culture supernatant using techniques well known in the art.

Furthermore, the present invention can bind the same antigenic determinants as the BR96 antibody, and encapsulates an antibody that can compete with the antibody for binding. They are the same as the BR96 antibody. have the same antigenic specificity, but different species origin, isotope, binding affinity or biological function. (e.g., cytotoxicity). For example, B1196 anti Classes, isotopes and other variants of the antibodies of the invention having different antigen-binding regions are known in the art. can be constructed using class switch recombination and fusion techniques known in the art. (For example, Thai + 5ana, etc., "Immunoglobalin Heavy Chain ClassSwitch From IgM to IgG In A [1ybridosa', Eur, J, Ig+muno1. +13:614 (1983); 5pira et al.,’The Identif cation of Monoclonal ClassSwitch Va riants By 5ubsselection And ELISA As5 ay”, J, 1m+munol.

Other chimeric antibodies or other recombinant antibodies with the same binding specificity (e.g., if the antibody is fusion in combination with two proteins, such as lymphokines or tumor inhibitory growth factors. synthetic proteins) are within the scope of the invention.

By the name of the name 5 The present invention provides treatment with anti-tumor antibodies and bioactive agents in standard chemotherapy. Provides a combination treatment comprising treatments that include: In a preferred embodiment of the invention chemotherapy is given at the same time as antibody treatment.

The antibodies used in the present invention are anti-tumor antibodies, preferably monoclonal antibodies. In certain embodiments of the invention, the complete antibody molecule may be used. is desired, but in other embodiments Fv, F(ab) and F( It is possible to use fragments of antibody molecules, including ab')z fragments. Any such fragments as desired (but not limited to said fragments) The drug binds to tumor cells and makes the cells more sensitive to chemotherapeutic agents. and minimize the immune functions associated with the Fc region of the antibody molecule, and Minimizes the generation of immune responses directed at the same Fc5I region. On the other hand, regarding the Fc@l region, Monoclonal to include the human PCEI range to maximize associated immune function. It is desirable to construct antibodies. Therefore, the present invention provides antibody therapy for specific situations. Better adapt the means.

The chemotherapeutic means used in the present invention are suitable for the treatment of tumors or malignancies. Includes any possible means. Different malignancies are associated with specific anti-tumor antibodies and requires the use of certain chemotherapeutic means, and said use will be determined on a case-by-case basis. The present invention is directed to the treatment of any malignant condition, such as adenocarcinoma, such as breast cancer and colon cancer, Non-small cell lung cancer, leukemia, lymphoma and neuroectodermal tumors such as melanoma, including, but not limited to, dyscytoma and glioblastoma multiforme.

The use of a combination treatment of anti-tumor antibody treatment and chemotherapy is shown in the following example. According to the authors, it is desirable to ensure that anti-tumor antibodies can contact their tumor cell targets. be done. Therefore, tumors that are relatively inaccessible to exogenously administered antibodies, even For example, in mammals bearing brain tumors, antibodies may be administered locally into the tumor or In the case of brain tumors, for example, permeability modifiers can make the blood-brain barrier more permeable. or administering the antibody or antibody fragment into the cerebrospinal fluid or through the carotid artery. is desired.

Having described the invention generally, the following illustrative examples will provide a further understanding of the invention. This is for the purpose of limiting, but not limiting.

Example 1 Sign on the nude mouse Nude mice (Balb/c nu/nu female) were divided into eight specific treatment groups (8 0 individual groups were divided into 82707 tumor (Onc mice/group). (established from human lung knee cancer metastases established in culture with Received a graft of ×3IIll fragment; the tumor fragment was inserted into the right posterior flank. Ta. Tumors grew in all mice.

13.17 and 21 days after tumor implantation, mice were identified based on their group. received treatment means.

A control group of mice received an injection of phosphate buffered saline (PBS, 0.2 m . The remaining group of mice received BR96 (0.5 μ/injection in PBS O, 2 d) or received injections of chemotherapy drugs or a combination of both. Chemotherapy drugs are approximately 0.2 Doxorubicin (Adriamycin) was administered at a volume of 5 μg/kg/injection. or 7 kg/injection, and mitomycin C is It was administered at 2 g/kg/injection and 3 g/kg/injection. All treatments The experiments were carried out on 13.17 and 21 days after transplantation. The tumor volume was 13°20. after transplantation. Determined on 26.36.43 and 50th day. The results of this study are shown in Figures 1 and 2. be done.

At a dose of 7 kg/injection, adriamycin is toxic to mice; RW all 8 mice in the group by day 50. BR96 is 7■ of that group when administered with adriamycin at a dose of /kg/injection. All mice died by day 42.

5■ / → / Injection of adriamycin dose reduction is BR96 continued to produce some toxicity and by day 42, 2 out of 8 animals in the group This resulted in the death of one mouse.

, administration of mitomycin C (3 kg/injection) was given along with administration of 81196. When treated, this resulted in a significant reduction in tumor volume. Mitomycin C (3g/k Administration of B[19 g/injection) alone inhibits tumor growth, but in combination B[19 6/Mytocin C treatment but not to the same extent as found for treatment.

The dose of mitomycin C was reduced to 2 kg/injection and together with BR96 When administered, a small increase in antitumor activity was found for administration of BR96 alone. was found to have more activity than The toxic effects of mitomycin C are No amount was found in any animal.

BR96 itself has antitumor activity, but as shown above, monochrome The anti-tumor activity of the null antibody was demonstrated in mice at the doses and time points used in the present study. If given, not detected.

Example 2 A combination of doxorubicin and BR96 with Muse no Roku Or + cogen Human breast cancer cells (83396 and H3630) established in tissue culture were Density of 104 cells/well in 100μi of IMDl with % fetal bovine serum into the wells of a 96-well flat bottom plate. plate, Maintained at 37°C for 12-16 hours to allow attachment of cells to wells. Next, remove the medium from individual wells and add 100uf of fresh medium or BR9. 6 and/or doxorubicin (Adriamycin) with 100 μl of medium containing Exchanged. Next, various concentrations of BR96 and/or doxorubicin were studied. Cells were maintained at 37°C for 18 hours, at which point 1 μCi of [3H]-thymidine (“H-TdR)” was added to individual wells and the cells were incubated at 37°C for an additional 6 time, maintained. The plates were then frozen at -20°C for 6 hours, thawed, Individual wells were then harvested onto glass fiber filters, washed, and the DNA The radioactivity corresponding to the synthesis was determined using a scintillation counter and is shown in Table 1. The results presented demonstrate that BR96 and dosing at low concentrations for the individual breast cancer cell lines tested Higher ( (more toxic) concentrations of Adr are required to inhibit cell proliferation. Adr's In its absence, high concentrations of BR96 are required for significant inhibition of cell proliferation. .

Table 1 Concentration Oug/1ug/lOug/1100u/1000μg/-(, l-i la one ml g+1 ■1 ml cabinet lo, oooo o% ~33% 0% 24% 48% 0.0025-17% -14% 21% 39% 56 %0.0100 -3% -13% 23% 38% 58% 0.0400 1 5% -8% 29% 40% 54% Concentration Ottg/Iμg/LOug /　1oot! g/100011g/4-1 ta l ml m 1 mlo, oooo o% -27% -7% 15% 44% 0.0025 -8% -1% 15% 24% 49% 0.0100 3% 0% 14% 26% 52% 0.0400 15° 7° 7° 29’ 58’ BR96 and The combination of BR96 and Adr produced significant cell growth inhibition at low concentrations of BR96 and Adr. ; the anti-tumor effect for the combined treatment is due to the BR96 and significantly higher than the additive effect of Adr.

The foregoing description and examples are illustrative of the invention and are not intended to limit it. There isn't. Various changes and modifications may be made within the scope of the invention.

Mouth gura 1 Figure 2 international search report Continuation of front page (51) Int, C1,5 identification symbol Internal office reference number A61K 39/395 T9284-4C (72) Inventor Schreiber, George United States , Washington 98052. Redmond, One Hundred Seventeen Second Avenue Northeast I

Claims (12)

[Claims] 1. A method for inhibiting the growth of tumor cells, the method comprising: an antibody that binds to the cells; and contacting said cells with a biologically active agent capable of modifying the growth of said cells. A method comprising: 2. 2. The method of claim 1, wherein said biologically active agent is a chemotherapeutic drug. 3. said chemotherapeutic drug selected from the group consisting of doxorubicin and mitomycin C; The method according to claim 2, wherein the method is selected. 4. The antibody has been deposited with the ATCC under accession number HB10036. BR96 is a monoclonal antibody with binding specificity substantially similar to that of BR96. The method described in item 1 of the scope of the request. 5. 2. The method of claim 1, wherein said tumor cells are human tumor cells. 6. The tumor cells are treated with the antibody and the biologically active agent in vivo in a mammal. A method according to claim 1, which is contacted with. 7. A method for treating tumor growth in a mammal, the method comprising: an antibody that binds and a biologically active agent capable of modifying the growth of said tumor cells. The method comprises administering to said mammal. 8. 8. The method of claim 7, wherein said biologically active agent is a chemotherapeutic drug. 9. said chemotherapeutic drug selected from the group consisting of doxorubicin and mitomycin C; 9. The method according to claim 8, wherein the method is selected. 10. The antibody has been deposited with the ATCC under accession number HB10036. BR96 is a monoclonal antibody with binding specificity substantially similar to that of BR96. The method described in item 7 of the scope of the request. 11. 8. The method of claim 7, wherein the tumor cells are lung adenocarcinoma cells. 12. 8. The method of claim 7, wherein the tumor cells are breast cancer cells.