UA97504C2 - Method of reducing the risk of occurrence or delaying the occurrence of cancer in a subject using an anti-vegf-specific antagonist - Google Patents

Abstract

Disclosed herein are methods of treating benign, pre-cancerous, or non- metastatic tumors using an anti-VEGF-specific antagonist. Also disclosed are methods of treating a subject at risk of developing benign, pre-cancerous, or non-metastatic tumors using an anti-VEGF-specific antagonist. Also disclosed are methods of treating or preventing recurrence of a tumor using an anti-VEGF-specific antagonist as well as use of VEGF-specific antagonists in neoadjuvant and adjuvant cancer therapy.

Description

reported the mitogenic effects of MESE on several non-endothelial cell types, such as retinal pigment epithelial cells, pancreatic ductal cells, and Schwann cells.

The recognition of MESE as the main regulator of angiogenesis in pathological conditions has led to numerous attempts to block the activity of MESE in those conditions that are accompanied by pathological angiogenesis.

MECE expression is upregulated in most malignancies, and overexpression of MECE correlates with more advanced stage or worse prognosis in many solid tumors. Accordingly, molecules that inhibit the MES signaling pathways have been used to treat relatively advanced solid tumors in which abnormal angiogenesis has been noted.

Despite evidence for a role for MESE in the development of conditions or diseases associated with abnormal angiogenesis, including late-stage and metastatic or invasive tumors, less is known about the role

MES at an early stage or with benign tumors, in the recurrence of tumors after a period of "hibernation", in the development of tumors in secondary places from dormant tumors, malignant tumors or micrometastases. The present invention addresses these and other needs, as will become apparent upon review of the following description.

The essence of the invention

The use of MECE-specific antagonists in combination with chemotherapy has been shown to be beneficial in patients with metastatic colorectal cancer and non-small cell lung carcinoma, among others, but little is known about the effect of MECE-specific antagonist therapy on benign, early-stage tumors; recurrence of tumors after a period of hibernation, surgical or other intervention; the development of tumors in secondary places from dormant tumors, malignant tumors or micrometastases; or in the adjuvant or neoadjuvant setting.

We report here results that demonstrate that MESE-specific antagonists can be used to treat tumors at an early stage of their development, including benign, precancerous, non-metastatic and operable tumors. These results also demonstrate that MECE-specific antagonists can be used for neoadjuvant cancer therapy (eg, benign or malignant cancers) or to prevent and/or reduce the likelihood of cancer recurrence (eg, benign or malignant cancers), including methods of adjuvant adjuvant therapy. This invention represents an important breakthrough in medicine, providing more effective, less toxic treatment for patients with cancer, including benign, early and operable types of cancer (both before and after surgery).

Accordingly, elements of the novelty of the present invention are methods of treating benign, precancerous or non-metastatic cancer in a subject, which include administering to the subject an effective amount of a MESIE-specific antagonist. In certain embodiments, the administration of a MESIE-specific antagonist prevents the transformation of a benign, precancerous or non-metastatic cancer into an invasive or metastatic cancer. For example, such a benign, precancerous or non-metastatic cancer can be stage 0 cancer, stage | or stage II, and administration of a MESiE-specific antagonist prevents the progression of a benign, precancerous, or non-metastatic cancer to the next stage(s), such as stage 0, stage I, stage II, stage II, or stage MI cancer.

In certain embodiments, the MECE-specific antagonist is administered for a time and in an amount sufficient to cure a benign, precancerous or non-metastatic tumor in a subject or to prevent the transformation of a benign, precancerous or non-metastatic tumor into an invasive or metastatic cancer . In certain embodiments, administration of a MECE-specific antagonist reduces tumor size, tumor mass, or the number of benign, precancerous, or non-metastatic tumors. A MESE-specific antagonist can also be administered for a time and in such an amount that is sufficient to reduce the density of vessels in a benign, precancerous or non-metastatic tumor.

As described herein, the methods of the present invention can be used to treat, for example, stage 0 cancer (eg, ip z carcinoma), stage | or in stage II. Methods of adjuvant and neoadjuvant therapy can be used to treat any type of cancer, for example, benign or malignant. In certain embodiments of the present invention, such cancer is an epithelial cell solid tumor, including, but not limited to, gastrointestinal cancer, colon cancer, breast cancer, prostate cancer, kidney cancer, lung cancer (eg, non-small cell lung carcinoma), melanoma, ovarian cancer, pancreatic cancer, head and neck cancer, liver cancer, and soft tissue cancer (eg, B-cell lymphomas such as non-Hodgkin's lymphoma, multiple myeloma, and leukemias such as chronic lymphocytic leukemia). In another embodiment, the benign, precancerous or non-metastatic tumor is a polyp, adenoma, fibroma, lipoma, gastrinoma, insulinoma, chondroma, osteoma, hemangioma, lymphangioma, meningioma, leiomyoma, rhabdomyoma, squamous cell papilloma, acoustic neuroma, neurofibroma, bile duct cystanoma, mesothelioma, teratoma, myxoma, trachoma, granuloma, hamartoma, transitional cell papilloma, pleomorphic salivary gland adenoma, desmoid tumor, dermoid cystpapilloma, cystadenoma, focal nodular hyperplasia or nodular regenerative hyperplasia. In another embodiment, the method of the present invention is preferably used for the treatment of adenoma. Non-limiting examples of adenomas include hepatocellular adenoma, renal adenoma, metanephric adenoma, bronchial adenoma, alveolar adenoma, adrenal adenoma, pituitary adenoma, parathyroid adenoma, pancreatic adenoma, salivary gland adenoma, gastrointestinal adenoma, tubular adenoma, and bile duct adenoma. .

Elements of novelty of the present invention are also methods that include the introduction of an effective amount to the subject

MES - a specific antagonist to prevent the development or recurrence of benign, precancerous or non-metastatic cancer in this subject. In certain embodiments of the present invention, the subject is at risk of developing cancer, polyps, or cancer syndrome. In one example, the subject has a family history of cancer, polyps, or a hereditary cancer syndrome (eg, multiple endocrine neoplasia type 1 (MENT)). In certain aspects of the present invention, the subject is at risk of developing a benign precancerous or non-metastatic gastrointestinal tumor, desmoid tumor, or adenoma (eg, gastrointestinal adenoma, pituitary adenoma, or pancreatic adenoma). In certain embodiments, a method of the present invention prevents the development or recurrence of a benign, precancerous, or non-metastatic cancer in a subject who has never had a tumor, in a subject who has never had clinically evident cancer, or in a subject who who had only a benign tumor.

In another aspect, this invention relates to a method of treating a gastrointestinal tumor at stage 0, stage | or stage II in the subject, which comprises administering to the subject an effective amount of the MECE-specific antagonist for a time and in such an amount that is sufficient to cure a stage 0, stage or stage II gastrointestinal tumor. Such a gastrointestinal tumor can be a cancer of the gastrointestinal system at stage 0, stage | or stage II, including anal cancer, colorectal cancer, rectal cancer, esophageal cancer, gallbladder cancer, stomach cancer, liver cancer, pancreatic cancer, and small bowel cancer. In one embodiment, such gastrointestinal tumor is a stage 0 (eg, high-grade adenoma) or stage I tumor. In one embodiment, the subject has not previously undergone resection for the treatment of the gastrointestinal tumor.

In another aspect, the present invention relates to a method of treating a subject at risk of developing a gastrointestinal tumor, which comprises administering to the subject a MECE-specific antagonist for a time and in an amount sufficient to prevent the development or recurrence of a gastrointestinal tumor. intestinal tumor in this subject.

Such a gastrointestinal tumor can be any gastrointestinal tumor, including, but not limited to, an adenoma, one or more polyps, or a cancer of the | or the 1st stage.

In certain embodiments of the methods of the present invention, the subject is over 50, has an inherited cancer syndrome, or has a family history of colon cancer or polyps. Non-limiting examples of inherited gastrointestinal cancer syndromes include familial adenomatous polyposis (FAP), familial adenomatous polyposis (FAP),

Gardner, pancreatic cancer and hereditary non-polyposis colorectal cancer (HNCCC). In certain embodiments, the subject has or has not previously undergone colonoscopy. In one embodiment, the MESE-specific antagonist is administered for a time and in an amount sufficient to reduce the number of adenomatous colorectal polyps in a subject having CAP.

In another aspect, the present invention relates to a method of preventing or reducing the likelihood of cancer recurrence in a subject, which comprises administering to the subject a MECE-specific antagonist for a time and in an amount sufficient to prevent or reduce the likelihood of cancer recurrence in the subject subject The present invention includes a method of preventing the recurrence of cancer in a subject with a tumor, which includes the steps of removing the tumor (for example, by using radical surgery) and the subsequent administration to the subject of a MESE-specific antagonist. The present invention includes methods of preventing regrowth of a tumor in a subject, which includes the steps of removing the tumor (eg, by using radical surgery) and then administering to the subject

MECE-specific antagonist In a related aspect, the present invention includes a method of preventing cancer recurrence in a subject or reducing the likelihood of cancer recurrence in a subject, which optionally comprises administering to the subject an effective amount of a MECE-specific antagonist prior to surgery, performing radical surgery, and administration of an effective amount of the MECE-specific antagonist after surgery, wherein the administration of the MECE-specific antagonist after surgery prevents recurrence of the cancer or reduces the likelihood of recurrence of the cancer. In another related aspect, the present invention relates to a method of preventing cancer recurrence in a subject or reducing the likelihood of cancer recurrence in a subject, which comprises administering to the subject an effective amount of an MECE-specific antagonist in the absence of any other anticancer therapeutic agent, in in which this administration prevents the recurrence of cancer or reduces the likelihood of recurrence of cancer in the subject.

For each of the above aspects, the tumor can be any type of tumor, including, but not limited to, solid tumors and, in particular, the tumors and adenomas described herein. A subject may have a dormant tumor or micrometastases that may or may not be clinically apparent. In one embodiment of this aspect, the MESE-specific antagonist is administered for a time and in an amount sufficient to reduce neovascularization of the dormant tumor or micrometastases. In another embodiment, the MESE-specific antagonist is administered for a time and in such an amount that is sufficient to prevent the development of a clinically manifested tumor or its metastases or to increase the duration of survival of the subject.

In one embodiment, the MESE-specific antagonist is used as monotherapy. In another embodiment, the subject has previously been treated for a tumor, for example by taking anti-cancer therapy. In one example, such anti-cancer therapy is surgery. In another embodiment, the subject may be additionally treated by receiving an anti-cancer therapy before, during (eg, concurrently) or after the subject is administered the MESE-specific antagonist. Examples of anticancer therapies include, but are not limited to, surgery, radiation therapy (radiotherapy), biotherapy, immunotherapy, chemotherapy, or a combination of these therapies.

In embodiments where the subject has previously undergone radical surgery, the MESE-specific antagonist is generally administered after a period of time necessary for post-operative recovery. This period of time may include the time required for wound healing or surgical dissection, the time required to reduce the risk of wound margin separation, or the time required for the subject to regain the level of health they had prior to surgery or better than before surgery. The period between the completion of radical surgery and the first administration of the MESE-specific antagonist may include a period required for drug rest when the subject needs a break or requests a break between treatment regimens.

In general, this period between the completion of radical surgery and the start of the administration of the MESE-specific antagonist can be less than a week, 1 week, 2 weeks, 3 weeks, 4 weeks (28 days), 5 weeks, 6 weeks, 7 weeks, 8 weeks , From months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, From years or more. In one embodiment, the period of time between the radical surgery and the administration of the MECE-specific antagonist is greater than 2 weeks and less than 1 year.

Each of the above aspects may further include monitoring the subject for cancer recurrence.

The present invention also relates to methods of neoadjuvant therapy prior to surgical removal of an operable cancer in a subject, such as a patient, which include administering to the patient an effective amount of an MESE-specific antagonist, such as bevacizumab, when the patient has already been diagnosed with a tumor or cancer. The MESE-specific antagonist can be administered alone or in combination with at least one chemotherapeutic drug.

The present invention also relates to a method of treating a subject with operable cancer, which includes administering to the subject an effective amount of a MESE-specific antagonist before surgery and after surgery to resect the cancer. In one embodiment, the method further includes the step of administering to the subject an effective amount of an MECE-specific antagonist after surgery to prevent cancer recurrence.

In another aspect, the present invention relates to a method of neoadjuvant therapy that includes administering to a subject with operable cancer an effective amount of a MESI-specific antagonist, such as bevacizumab, and at least one chemotherapy drug prior to radical surgery. This method can be used to extend the disease-free survival (DFS) or overall survival (38) of the subject. In one embodiment, VVH or ZV is assessed 2-5 years after the start of treatment.

In another aspect, the present invention relates to a method of reducing the size of a tumor in a subject having a tumor,

unresectable, which includes administering to the subject an effective amount of a MECE-specific antagonist, and this administration reduces the size of the tumor to such an extent that it is resectable. In one embodiment, the method further includes administering to the subject an effective amount of the MESE-specific antagonist after tumor resection.

In another aspect, the present invention relates to a method of treating cancer in a subject, which includes the following steps: a) a first step, which includes several cycles of treatment, each of which involves administering to the subject an effective amount of an MECE-specific antagonist, such as bevacizumab, and, at least one chemotherapeutic drug with a predetermined interval; b) radical surgical intervention to remove cancer; and c) the second stage, which involves several cycles of maintenance therapy, each of which includes administering to the subject an effective amount of an MECE-specific antagonist such as bevacizumab, without the administration of any chemotherapeutic drug at a predetermined interval. In one embodiment, the first stage includes a first block of treatment cycles with the introduction of a MESiE-specific antagonist, such as bevacizumab, and a first regimen of chemotherapy, followed by a second block of treatment cycles with the introduction of a MESE-specific antagonist, such as bevacizumab, and a second chemotherapy regimen. In one embodiment, when the cancer being treated is breast cancer, the first chemotherapy regimen includes doxorubicin and cyclophosphamide, and the second chemotherapy regimen includes paclitaxel.

The present invention relates to methods that involve administering to a subject with metastatic or non-metastatic cancer, following radical surgery, an effective amount of a MESE-specific antagonist, such as bevacizumab. In one embodiment, such a method additionally includes the use of at least one chemotherapeutic drug. This method can be used to extend the duration of VVH and ZV of the subject. In one embodiment, the subject remains free of the disease for at least 1 year and up to 5 years after treatment.

In one aspect, such a method includes the following steps: a) a first step, which includes several cycles of treatment, each of which involves administering to the subject an effective amount of a MESE-specific antagonist, such as bevacizumab, and at least one chemotherapy drug at a predetermined interval; and b) a second stage, which involves several cycles of maintenance therapy, each of which involves administering to the subject an effective amount of an MECE-specific antagonist such as bevacizumab, without the administration of any chemotherapeutic drug at a predetermined interval, with the combined first and second stages continuing for at least one year after the start of postoperative treatment. In one embodiment, the first stage includes a first block of treatment cycles with the introduction of a MESE-specific antagonist, such as bevacizumab, and a first regimen of chemotherapy, followed by a second block of treatment cycles with the introduction of a MESE-specific antagonist, such as bevacizumab, and a second chemotherapy regimen. When the cancer being treated is breast cancer, the first chemotherapy regimen includes doxorubicin and cyclophosphamide, and the second chemotherapy regimen includes paclitaxel.

In certain embodiments of each of the above aspects of the present invention, the MESE-specific antagonist is a compound that binds to MESE or reduces the expression of MESE or its biological activity. MESE-specific antagonist is one of the following indicative compounds: a polypeptide that specifically binds to

MESE, MESE-specific ribozyme, MESE-specific peptityl, oligomer with an antisense nucleic base, complementary to at least part of the nucleic acid molecule encoding the MEC polypeptide, small RNA molecule complementary to at least part of the nucleic acid molecule encoding the polypeptide MESE, or aptamer. A peptide that specifically binds to MESE can be a soluble MESE receptor protein or a fragment thereof that binds to MESE, or a chimeric MESE receptor protein, such as EI-1/Ps, KOV/Ps or RIIKOV/Es. A polypeptide that specifically binds to MESE can also be an anti-MESE antibody or its fragment that binds to an antigen. Such an anti-MESE antibody or antigen-binding fragment thereof may be a monoclonal antibody, a chimeric antibody, a fully human antibody, or a humanized antibody. Exemplary antibodies for use in the methods of the present invention include bevacizumab (AMABTIMF), (36-31, B20-4.1 and fragments thereof. The antibody or antibody-binding fragment thereof may also be an antibody lacking the Es portion, the structure of K(ab)", Bar or RU.

Depending on the type and severity of the disease, the dose of the MECE-specific antagonist, such as bevacizumab, can be from about 1 μg/kg to about 50 mg/kg, and preferably from about 5 mg/kg to about 15 mg/kg, including but not limited to limited to them, 7.5 mg/kg or 10 mg/kg. The frequency of administration depends on the type and severity of the disease. The introduction can be repeated and last several days or longer, depending on the condition; treatment is continued until the cancer is cured or until the desired therapeutic effect is achieved as evidenced by methods described herein or well known to those skilled in the art. In one example, the MESE-specific antagonist (e.g., antibody) of the present invention is administered once weekly, once every 2 weeks, or once every 3 weeks at a dose range of from about 5 mg/kg to about 15 mg/kg, inclusive. but not limited to 7.5 mg/kg or 10 mg/kg. However, other dosage regimens may also be used. The progress of the treatment of the present invention is easily monitored by conventional methods and assays.

In additional embodiments of each of the above aspects of the present invention, the MESE-specific antagonist is administered locally or systemically (eg, orally or intravenously). In one embodiment, treatment with the MECE-specific antagonist is continued until the patient is free of cancer, for a period of time selected from the group consisting of 1 year, 2 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years and 12 years.

Although the subject may be treated in a number of different ways before, during, or after administration of the MECE-specific antagonist, in one embodiment of each aspect of the present invention, the subject is treated without surgery or chemotherapy. In other embodiments, the MECE-specific antagonist is used as monotherapy or as monotherapy during treatment with the MECE-specific antagonist, as determined by the clinician or as described herein.

In other embodiments, treatment is with a MESE-specific antagonist in combination with additional anticancer therapy, including, but not limited to, surgery, radiation therapy, chemotherapy, differentiation therapy, biotherapy, immunotherapy, an angiogenesis inhibitor, and an antiproliferative compound. Treatment with a MESIE-specific antagonist may also include any combination of the types of therapeutic regimens listed above. In addition, cytotoxic drugs, antiangiogenic and antiproliferative drugs can be used in combination with a MESE-specific antagonist. In one embodiment, the anticancer therapy is chemotherapy. For example, the chemotherapeutic drug is selected from, for example, alkylating drugs, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, periwinkle alkaloids, epidophyllotoxins, antibiotics, I-asparaginase, topoisomerase inhibitor, interferons, platinum coordination complexes, anthracenedione substituted urea , derivatives of methylhydrazine, means for suppressing the cortical layer of the adrenal gland, adrenocorticosteroids, progestins, estrogens, antiestrogens, androgens, antiandrogens, analogues of gonadotropin-releasing hormone, etc. In some embodiments, such a chemotherapeutic drug and the MESE-specific antagonist are administered simultaneously.

In embodiments that provide additional anticancer therapy, the subject may receive this additional anticancer therapy before, during (eg, concurrently) or after administration of the MESI-specific antagonist. In one embodiment, the anticancer therapy is chemotherapy that includes administration of irinotecan, fluorouracil, leucovorin, gemcitabine, or combinations thereof. In one embodiment, the MESE-specific antagonist used as monotherapy or in combination with anticancer therapy may be administered as adjunctive therapy. In one embodiment, the anti-cancer therapy for prostate cancer, ovarian cancer, and breast cancer can be hormone therapy. In one exemplary embodiment, the MECE-specific antagonist is administered in combination with an anti-cancer therapy that does not include an anti-Neg2 antibody, fragments, or derivatives thereof (e.g., a Negg series of antibodies)).

The methods of this invention provide a particular advantage in the treatment and prevention of tumors at an early stage of development, preventing progression to more advanced stages and reducing morbidity and mortality rates associated with advanced cancer. The method of the present invention is also useful in preventing the recurrence or regrowth of a tumor, such as a dormant tumor that persists after removal of the primary tumor, as well as in preventing the development or proliferation of micrometastases.

In the methods of the present invention, the cancer may be a solid tumor, such as breast cancer, colorectal cancer, rectal cancer, lung cancer, renal cell carcinoma, glioma (eg, anaplastic astrocytoma, anaplastic oligoastrocytoma, anaplastic oligodendroglioma, glioblastoma multiforme), cancer kidney, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, carcinoid carcinoma, head and neck cancer, melanoma and ovarian cancer. In one embodiment, such cancer is gastrointestinal cancer.

In additional embodiments of each of the above aspects of the present invention, the MESE-specific antagonist is administered in such an amount and for such a time (e.g., within one particular therapeutic regimen) as to reduce (e.g., by 2095, 3095, 40905, 5095, 60905, 7095 , 8095 9095, 10095 or more) the number of cancer cells in a tumor, including but not limited to benign, precancerous, or non-metastatic cancers; to reduce the size of a tumor, polyp or adenoma; to reduce tumor mass; to inhibit (i.e. reduce to a certain extent and/or stop) the infiltration of cancer cells into peripheral organs; to reduce the secretion of hormones; to reduce the number of polyps; to reduce vascular density in a tumor, including, but not limited to, benign, precancerous, or non-metastatic cancerous tumors; to inhibit tumor metastasis; to slow or stop tumor growth or proliferation of tumor cells; to reduce or prevent the growth of a dormant tumor; to reduce or prevent the growth or proliferation of micrometastases; to reduce or prevent tumor regrowth after treatment or removal; to increase or prolong the VVH or ZV of a subject at risk of developing or diagnosed with a benign, precancerous or non-metastatic tumor; and/or to alleviate to some extent one or more symptoms associated with cancer. In one example, survival is defined as OS or OS and is estimated at 2-5 years after initiation of treatment. In some additional embodiments, the MECE-specific antagonist is used to prevent the development or recurrence of cancer in a subject. In one example, the prevention of cancer recurrence was evaluated in a population of subjects after a period of about 4 years to confirm the absence of disease recurrence in at least 8090 subjects. In another example, a MESE-specific antagonist was used to reduce the likelihood of recurrence of a tumor or cancer in a subject. In one example, cancer recurrence was assessed after approximately 3 years, and at least a 5095 reduction in the recurrence rate was demonstrated compared to patients who received chemotherapy alone.

The methods of the present invention may also include monitoring the subject for cancer or tumor recurrence.

Other features and advantages of this invention will become apparent from the following detailed description, drawings and claims.

A brief description of the drawings

Fig. 1A-1E are a series of photomicrographs showing the expression of MESE-A in adenomas of Ars"" and in a normal villous (protrusion on the surface of the mucosa). Hybridization of ip 5ish with a probe on an intestinal adenoma from the small (Fig. 1A, 10) and large intestine (Fig. 18, 1E), as well as on normal villi (Fig. 1C, 1E) of a 14-week-old Arstip/k mouse demonstrates the expression of MESEE -A in epithelial (arrows) and stromal cells (arrowhead). Field of vision of high illumination; Fig. TA-1C, low light field of view; Fig. 10-1E.

Fig. 2A-2E are a series of graphs showing that inhibition of MESE-A reduces tumor weight and prolongs survival.

Fig. 2A is a graph showing tumor mass in an individual mouse in a group. Tumor mass is shown as segments from the largest to the smallest tumor mass value. White crosses show group mean values. "P"0.008, thr"B5.Z x 1075, M means the number of animals.

Fig. 28 is a series of graphs showing the distribution of tumors by diameter and as a percentage of the total number of tumors. M stands for the number of tumors in the group.

Fig. 20 is a series of graphs showing an overlay of tumor size frequencies after 3 weeks of treatment (top) and after 6 weeks of treatment (inset). The graph below shows the overlap of the frequency of tumor sizes compared to day 0. The vertical bars illustrate the size less than or equal to which corresponds to a higher frequency of tumors in animals injected with tAbr (6-31; 1 mm for 3-week treatment and 1.2 mm in the b-week treatment group.

Fig. 20 is a graph showing the average diameter of a tumor depending on its location in the intestine.

M stands for the number of tumors per group in the first, second, third, and fourth quarters of the intestine, respectively. The Day 0 group consisted of 12 animals, the other groups contained 10 animals each. 5 - stomach; C - cecum; A - rectum. Bars represent the standard error of the mean (SEM). "Р"1.0 x 1079, "хр"б0.002 compared to tAb Сb-

З1 in 3 or so weeks.

Fig. 2E is a graph showing the mean tumor diameter in fourteen Arstith mice; MCSP-Ste (black bars) and Arstime; MESsEoch; MiCip-Ste (grey bars), presented in descending order. Intercepts represent standard deviation (50).

Fig. 2E is a graph showing Kaplan-Meier survival estimates for mice treated with tA a6-31 (gray line) or control Ida (black line). The empty arrow means the duration of processing (treatment).

Median survival is shown by gray arrows. "P" 2.4 x 103, M means the number of mice in the group.

Fig. ZA-3I show the effect of anti-MESE-A treatment on the change in tumor morphology, but not on the proliferative index.

Fig. ZA-Z3B are microphotographs of a segment of the jejunum stained with methylene blue of the small intestine.

Fi. 30-30 are photomicrographs showing a low-magnification section of the jejunum stained with hematoxylin and eosin.

Fi. ZE and ZE are photomicrographs in which a section of the tumor from the jejunum stained with hematoxylin and eosin is presented with high magnification.

Fig. 3 and 3 are photomicrographs showing tumor tissue and normal mucosa stained immunohistochemically with the Ki-67 antibody at low magnification. Contrast staining with hematoxylin and eosin.

Fig. ZK is a graph showing the proliferative index expressed as the percentage of Ki-67 positive nuclei relative to the total number of nuclei. Bars represent the standard error of the mean (5EM).

Figure 3I shows Western blotting of normal mucosal lysates from animals treated with control dae (M1-M4) or tAbB (26-31 (M5-M8) and tumor lysates from animals treated with control dae (T1-T4) or tAbB ( 46-31 (T5-T8).

Fig. 4A-4C show reduced tumor vessel density after treatment with tAb (36-31. Figs. 4A-4C are confocal images of 80-μm sections of tumors from the jejunum after immunohistochemical staining.

Green color - SOZ1, vascular endothelial cells; blue - E-cadherin, epithelial cells; red - smooth muscle actin.

Fig. 4C is a graph showing vessel density expressed as a percentage of the area positive for POP31 relative to the total area of the tumor being analyzed. Intercepts represent the standard error of the mean (SEM); n means the number of analyzed tumors.

Fig. BA-50 is a series of graphs showing how anti-MESIE-A treatment inhibits pituitary tumor growth.

Fig. BA is a graph showing the mean tumor volume in control da (black line) and tA (26-31 (gray line) treated groups after 9, 25, 39, 53 and 67 days of treatment. Bars represent standard error of the mean value (ZEM), M stands for the number of mice in the group.

Fig. 58 is a graph showing the volume of tumors in individual mice treated with control da (solid lines) and tA (46-31 (dashed lines). 7 mice were euthanized before the end of the study due to poor health (lines end to the mark of 67 days).

Fig. 5C is a graph showing the doubling of tumor-free survival in the control da (black line) and tAB (6-31 (gray line) groups, evaluated at 9, 25, 39, 53 and 67 days after the start of treatment.

Fig. 50 is a graph showing measurements of tumor volume in animals in which pituitary tumors were implanted under the skin, treated with control IDS (black line) and tAB (26-31 (gray line), after 1, 7, 14, 21, 28 and 35 days of treatment.

Intercepts represent the standard error of the mean (SEM); M stands for the number of mice in the group.

Fig. 6 is a graph showing that Mep177 pituitary and pituitary adenomas express MESE-A, MESEV-i, and MESEV-2. The relative expression of MESE-A, MESEV-1, and MESEV-2 is shown for wild-type pituitary (black bar), non-tumor pituitary tissue from Mep177 mice (grey), small untreated pituitary adenomas, and pituitary tumors treated with control Idsb; (red) and tAr (36-31 (blue). Bars represent the standard error of the mean value (SEM); M is unreliable.

Fig. 7 is a series of NMR images of representative pituitary tumors from Mep1"" mice. Coronal sections of pituitary adenoma in animals treated with control da and tAbB are shown (46-31 after 9, 39, and 67 days of treatment. For Day 9, the edges of pituitary adenoma were highlighted by yellow stars. Tumor volume after treatment with control Id was 23.2; 55.9 and 142.0 mm, and after treatment tA 26-31 - 18.9, 27.2 and 35.3 mm3 after 9, 39 and 67 days from the start of treatment, respectively.

Fig. 8A-8H are a series of images showing histological examination of pituitary and pancreatic tumors in Mep1-7 mice,

Fig. BA-88 shows pituitary tumors after staining with hematoxylin and eosin, and Fig. 8E-8E - pancreatic tumors after the same staining.

Fig. 80-80 show pituitary tumors after immunohistochemical staining with panendothelial marker MESA-32, and fig. 802-8H - tumors of the pancreas after immunohistochemical staining of ip with the panendothelial marker MESA-32.

Fig. 81 is a graph showing the results of vascular density assessment in pancreatic tumors in animals treated with control Idsi and anti-MESE. Intercepts represent the standard error of the mean (SEM); Me - unreliable.

Fig. 9A-90 is a series of images showing that pituitary tumors from Mep177 mice and pituitary tumor grafts stain positive for prolactin. Immunohistochemical staining with an anti-prolactin antibody is shown for hypophyseal tumors (Figs. 9A-9B) and subcutaneous pituitary tumor transplants (Figs. 90-90).

The image in Fig. 9E and 9E show that a pituitary tumor transplanted close to the mammary gland exhibits prolactin-induced secretory changes (left side of the image).

Fig. TOA-100 show that serum levels of prolactin and growth hormone are elevated in mice with pituitary tumors and pituitary tumor transplants.

Fig. 10A is a graph showing the dependence of serum RAV level (ng/ml) on pituitary tumor volume (mm3) in 19 untreated tumor-bearing Mep!1-7 mice (positive correlation).

Fig. 108 is a graph that shows the dependence of the level of РЇ| (ng/ml) in serum from pituitary tumor volume (mm3) in mice treated with control dae (black triangles) and tAb (26-31 (gray circles), at the end of the study.

Fig. 100-100 are graphs showing serum levels of proactin (C) and growth hormone (0) in animals with pituitary adenoma transplants on Day 1 and Day 35 of treatment.

Fig. 11 is a graph showing the effects of anti-MESE treatment ("intervention") on early stage tumor progression in the Nir-TrAd mice model of islet tumorigenesis. This graph demonstrates the reduction in tumor angiogenesis, as judged by the mean number of angiogenic islets, after treatment with an anti-MESE antibody for 9-11 weeks, compared to treatment with an isotype-matched control monoclonal antibody.

Fig. 12A and 128 show the results of a regression analysis that revealed no significant difference in tumor weight or survival between treatment with an anti-MESE antibody and treatment with an isotype-matched control monoclonal antibody in a Vir-TrAd mouse model of islet of Langerhans tumor development.

Fig. 12A is a graph showing tumor weight in mice treated with an anti-MESE antibody compared to those treated with an isotype-matched control monoclonal antibody.

Fig. 128 is a graph showing the survival over time of mice injected with an anti-MESEE antibody compared to those treated with an isotype-matched control monoclonal antibody.

Fig. 13 is a graph showing the efficacy of prolonged anti-MESE therapy in inhibiting regrowth of tumors after cytoreduction with taxanes or gemcitabine.

Fig. 14A-140 is a series of images showing that an untreated primary pituitary adenoma (FIG. 14A, above the dotted line) is variably and weakly positive for growth hormone, compared to the adjacent normal anterior pituitary (below the dotted line). One of four transplanted pituitary tumors (treated with control Ids) was weakly positive for growth hormone (Fig. 148). Primary pituitary tumors from mice injected with tar (26-31 (Fig. 14C) or control dae (Fig. 140) were focally positive for growth hormone.

Detailed description of the invention

I. Definitions

The term "MESE" or "MESE-A" refers to human vascular endothelial cell growth factor of 165 amino acids, as well as related human vascular endothelial cell growth factors containing 121, 145, 189, and 206 amino acids, as described, for example, in EP oh bsiepse, 246:1306 (1989) and Noyshsk et al. My. Epdosiip., 5:1806 (1991), together with their natural allelic and processed forms. MESE-A is part of a gene family that includes MESE-B, MESB-C, MESB-O, MESB-E, MESB-E, and RIYAE. MESE-A binds mainly to two high-affinity receptor tyrosine kinases, MESEN-1 (Ei-1) and MESEV-2 (RIC-1/KOV), and the latter is the main transmitter of MESE-A mitogenic signals of vascular endothelial cells. In addition, neuropilin-1 has been identified as a receptor for heparin-binding isoforms of MECE-A, and it has been shown that it may play a role in vascular development. The term "MESE" or "MESE-A" also refers to vascular endothelial cell growth factors of other species, such as mice, rats, or primates. Sometimes the MES of a particular species is indicated by a corresponding designation, such as LMESE for human MESE or tuUESE for mouse MESE. The term "MESE" is also used to refer to truncated forms or fragments of this polypeptide containing from 8 to 109 or from 1 to 109 of the 165 amino acids of human vascular endothelial cell growth factor. References to such MES forms are easily recognizable in this application - for example, "MESE (8-109)", "MESE (1-109)" or "MESE:v5". The amino acid positions for the "truncated" native MESE are numbered as shown in the native MESE sequence. For example, amino acid position 17 (methionine) in truncated native MESEE is position 17 (methionine) also in native

MES. Truncated native MESE has an affinity for binding to KOV and EC-1 receptors, comparable to native

MES.

The term "MEC variant" as used herein refers to a MEC polypeptide that includes one or more amino acid mutations in the native MEC sequence. Optionally, such one or more amino acid mutations include an amino acid substitution. In order to conveniently designate the MECs of the variants described herein, the amino acid residue position numbers correspond to the amino acid sequence of the putative native MEC (see

Ye eshpad ei ai., vyrga and Noysk ei ai., zirga.).

A "native sequence" polypeptide is a polypeptide that has the same amino acid sequence as a naturally occurring polypeptide. Therefore, a polypeptide with a native sequence can have the amino acid sequence of a naturally occurring polypeptide from any mammal. Such a polypeptide with a native sequence can be isolated from nature or obtained by recombinant means or by synthesis.

The term "native sequence" polypeptide includes naturally occurring truncated or secreted forms of this polypeptide (eg, the extracellular domain sequence), naturally occurring variant forms (eg, alternatively spliced forms), and naturally occurring allelic variants of this polypeptide. "Variant" polypeptide means a biologically active polypeptide that has at least about 8095 amino acid sequence identity with the native sequence polypeptide. Such variants include, for example, polypeptides to which one or more amino acid residues have been added or removed from the M- and C-termini of the protein chain. Of course, the variant will have at least 8095 amino acid sequence matches, preferably 9095 amino acid sequence matches, and even more preferably approximately 9595 amino acid sequence matches to the native sequence polypeptide. "Biological activity of MESE" includes binding to any MESE receptor or any signaling activity of MESE, such as regulation of normal and abnormal angiogenesis and vasculogenesis (Eeitaga apa Oamiv-

Ztuy (1997) Epadosiipe VNem. 18:4-25; BRepaga (1999) 9). My. May 77:527-543); promotion of embryonic vasculogenesis and angiogenesis (Sapteii et al. (1996) Mashge 380:435-439; Reitaga et al. (1996) Mashge 380:439-442); and modulation of cyclic proliferation of blood vessels in the female reproductive tract, as well as for bone growth and cartilage formation (Ree!taga eyi ai!. (1998) Maishtge Mei. 4:336-340; Segreg ei ai. (1999) Maishge Med. 5 :623-628). In addition to being an angiogenic factor in angiogenesis and vasculogenesis, MESE as a pleiotropic growth factor demonstrates numerous biological effects in other physiological processes, such as endothelial cell survival, vascular permeability and vasodilatation, monocyte chemotaxis and calcium influx (Regtaga apa Oamiz-Ztuin (1997 ), Zirga apa Sere-Ztsage? Moreover, recent studies have reported the mitogenic effects of MESE on several types of non-endothelial cells, such as retinal pigment epithelial cells, pancreatic ductal cells, and Schwann cells (Spcehip et al.

(1995) In). Seii Rpuzioi. 164:385-394; Oregd-U/eivN ei ai. (1997) Mine. SeiI. Epdosiipoi. 126:125-132; bopai! her a. (1999)

U. Meshghozsi. 19:5731-5740). "MESE-specific antagonist" is described here in Section II. "Anti-MESE antibody" is an antibody that binds to MESE with sufficient affinity and specificity. Such a selected antibody will usually have a sufficiently strong affinity for binding to MESE; for example, such an antibody can bind to PMESE with a Ka of 100 nM to 1 pM. Antibody affinity can be assessed, for example, using a test based on surface plasmon resonance (such as the ViAsoge test, described in the publication of patent application PCT Me U/O02005/012359); immunoenzymatic solid phase analysis (EGI5A); and competitive analyzes (for example, VIA). In certain embodiments, an anti-MESE antibody of the present invention can be used as a therapeutic drug that targets and affects diseases or conditions associated with MESE activity. In addition, such an antibody can be subjected to other tests for biological activity, for example, in order to evaluate its effectiveness as a therapeutic agent. Such tests are known to those skilled in the art and depend on the target antigen and the purpose of the antibody. Examples include NOMES (human umbilical vein endothelial cells) inhibition assay, as will be described in the Examples below; tests for tumor cell growth inhibition (for example, as described in the publication U/UO 89/06692); antibody-mediated cellular cytotoxicity (AOSS) and complement-mediated cytotoxicity (COC) assays (US Pat.

Mo 5,500,362); as well as tests for agonistic activity or hematopoiesis (see UUHO publication 95/27062). Anti-

A MES antibody will not normally bind to other MES homologues, such as MES-B or MES-C, nor to other growth factors such as RICE, ROSE or VES. Additional information on anti-MESE antibodies can be found in Section II, A.

In this description and in the claims of the invention, the numbering of the residues in the immunoglobulin heavy chain is as in the catalog of the European Union, as described in Karbai sei ai!., Zediepsev oi Rgoivip5 oi Ittipoiodis! Ipiegevi, Biy Ey.

Ribiis Neay Zegmise, Maiopai! Ipviitsshev oi Neay, VeshShezaa, Ma. (1991), which are incorporated herein by reference. "Catalog of the European Union, as in Kabat" refers to the numbering of DS antibody residues! BO of a person.

The term "antibody" is used herein in its broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), as well as antibody fragments, as long as they exhibit the desired biological activity. The "Ka" or "Ka value", according to one embodiment of the present invention, is determined by a radiolabeled MES binding assay (RIA) performed on the Rab version of the antibody and the MES molecule, as described in the following affinity assay binding of the Gar5 solution with MESE by equilibration

Eab with a minimal concentration of (125I)-labeled MESE(109) in the presence of a series of titrations of unlabeled MESE followed by capture of bound MESE with a plate coated with an anti-Rab antibody (Spep, Yei Ai. (1999) U. Mo! Vio! 293: 865-881). To provide conditions for this assay, microtitrator plates (Oupech) are coated overnight with 5 μg/ml anti-Rar antibody (Sarre! Garz) in 50 mM sodium carbonate (pH 9.6) and then blocked with 295 (v/o) albumin of bovine serum in РВ5 (phosphate-saline buffer solution) for 2-5 hours at room temperature (approximately 23"С). On a non-adsorbing plate (Mips 269620) mix 100 pM or 26 pM

HCMESE(109) with serial dilutions of the corresponding Bar, for example Ear-12 (Rgevia ei ai., (1997) Sapseg Vez. 57:4593-4599). This Rar is then incubated overnight; however, incubation may be continued for 65 hours to ensure that equilibrium is reached. The mixtures are then transferred to a capture plate for incubation at room temperature for 1 hour. Then the solution is removed, and the plate is washed 8 times with 0.195 Tween in

RV5. After the plates have dried, 150 μl/well of the scintillator (Misgo5sipi-20; RaskKaga) is added and the Torsoshtspi gamma counter (RaskKaga) is turned on for 10 minutes. For use in competitive binding assays, select concentrations of each Rab that give 2095 or less of maximal binding. In accordance with another variant of implementation, KA or the value of Ka is determined using surface plasmon resonance using ViAsoge"m-2000 or ViAsoge"m-3000 (ViAsoge, Ips., Rivsagazhau, MO) at 25"C with CM5 chips on an immobilized NMESE (8-109) at "10 reaction units (RU). Briefly, biosensor chips made of carboxymethylated dextran are activated by M-ethyl-M'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EOS) and M-hydroxysuccinimide (MH) according to the manufacturers' instructions Human MSE is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (70.2 μM) before injection at a rate of 5 μl/min until approximately 10 reaction units are achieved (A) of attached protein. After injection of human MESE, 1M ethanol amine is administered, also by injection, to block unreacted groups. To measure kinetics, 2-fold serial dilutions of Rab (from 0.78 nM to 500 nM) in RV5 in injected with 0.0595 Tween 20 (RV5T) at 25"C at a rate of about 25 μl/minute. Coefficients of association (Kop) and coefficients of dissociation (Koi) are calculated with the help of a simple one-to-one binding model of Iapdtiig (ViIAsoge Emaitsayop Zoiymage megsiop 3.2) by simultaneous fitting of association and dissociation sensors. The equilibrium dissociation constant (Ka) was calculated as the Kok/Kop ratio (see, for example, Spep, U., et al., (1999).). My Vio! 293:865-881). When the rate of direct reaction exceeds 105

M" 87 in the above-described surface plasmon resonance analysis, it can be determined using the fluorescence quenching technique, which measures the increase or decrease in the intensity of the fluorescence emission (excitation - 295 nm; emission - 340 nm, 16-nm bandwidth) at 2570 20 nM anti- MESE antibodies (in the form

EPar) in РВ5, pH 7 2, in the presence of increasing concentrations of the short form (8-109) of human MESE or mouse MESE, for which a spectrophotometer with the possibility of stopping the flow (Amim Ipvigitepov) or a spectrophotometer is used

ZI M-Atipso series 8000 (TPeppob5resigopis) with a covette equipped with a stirrer. A "blocking" antibody or an "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. For example, a MESE-specific antagonist antibody binds MESE and inhibits the ability of MESE to induce the proliferation of vascular endothelial cells. The best blocking antibodies or antagonist antibodies completely suppress the biological activity of the antigen.

Unless otherwise indicated, the term "multivalent antibody" is used herein to refer to an antibody that contains three or more antigen-binding sites. For example, a multivalent antibody is designed to have three or more antigen binding sites and is generally not an antibody with a native IDM or IDA sequence. "Em" fragment is an antibody fragment that contains the complete antigen recognition and binding site. This region consists of a variable domain dimer of one heavy and one light chain in close association, which may be covalent in nature, for example in zsEU. It is in this configuration that the three OWLs (OWLs - complementarity determining region) of each variable domain interact, determining the antigen binding site on the surface of the Mn-Mi dimer. Taken together, the six OWLs or a subset of them determine the specificity of an antibody for antigen binding. However, even a single variable domain (or half of an Em containing only three antigen-specific OWLs) has the ability to recognize and bind antigen, although usually with lower affinity than the complete binding site.

The term "antibody variable domain" as used herein refers to the portions of the light and heavy chains of antibody molecules that include the amino acid sequences of the complementarity-determining regions (SORtI, SOR2, and SORZ) and the framework region (SR). Mn refers to the variable domain of the heavy chain. M refers to the variable domain of the light chain. In the methods used in the present invention, the amino acid positions attributed to SOVA and EB can be determined according to Cabrai

Ittipoiodisai! Ipiegevi (Mayopai! Ipziyshev oi Neatsy, Ve(Pezada, Ma., 1987 and 1991)). The numbering of amino acids of antibodies or their antigen-binding fragments also corresponds to Cabrai's numbering.

The term "areas that determine complementarity" (SOBI, SOB2 and SOVAZ) refers to the amino acid residues of the variable domain of the antibody, the presence of which is necessary for antigen binding. Each variable domain typically has three SOV regions identified as SOVI, SOV2, and SOVZ. Each complementarity-determining region may include amino acid residues from a "complementarity-determining region" as defined by Cabrai (ie, approximately residues 24-34 (11), 50-56 (12), and 89-97 (13) in the variable domain of chains and 31-35 (NO), 50-65 (H2) and 95-102 (NZ) in the variable domain of the heavy chain; Kabai ei ai., Zediepsev oi Rgoieip5 oi Ittipoiodis!

IpziShyshevz oi Neay, VeShevzaa, MO. (1991)); and/or such residues from the "hypervariable loop" (ie, approximately residues 26-32 (11), 50-52 (I 2) and 91-96 (I 3) in the variable domain of the light chain and 26-32 (NO) , 53-55 (H2) and 96-101 (NZ) in the variable domain of the heavy chain; Spoipia and Hev5k.). Moi. Viol. 196:901-917 (1987)). In some cases, the region that determines complementarity may include amino acids from the OWL region defined by

Kabai, and from a hypervariable loop. For example, antibody 405's heavy chain SOVNI includes amino acids 25 through 35. "Framework regions" (ER) are those residues of the variable domain that are not SOV residues. Each variable domain typically has four EVs, identified as EVT, EVN2, EVZ, and EV4. If the OWLs are determined according to Kabay, then the EB residues of the light chain are located approximately at residues 1-23 (I SEV1), 35-49 (I SEN), 57-88 (I SENVZ) and 98-107 (I SEN4), and EA residues of the heavy chain - approximately on residues 1-30 (NSENI1), 36-49 (NSENh), 66-94 (NSEAZ) and 103-113 (NSEN4) in the residues of the heavy chain. If SOVA contains amino acid residues from the hypervariable loop, the EB residues of the light chain are positioned approximately at residues 1-25 (I SEN), 33-49 (SENg), 53-90 (I SEVAZ) and 97-107 (I SEV4) in the light chain , and the BA residues of the heavy chain are approximately on residues 1-25 (NSENI1), 33-52 (NSENh), 56-95 (NSENZ) and 102-113 (NSEBA4) in the heavy chain residues. In certain cases where an OWL includes amino acids from both the KaBrae-defined OWL site and the hypervariable loop, the EB residues will be adjusted accordingly. For example, when SOVNI includes amino acids Н26-НЗ5, residues

EW! of the heavy chain are in positions 1-25, and EB2 residues are in positions 36-49.

The Rab fragment contains the variable and constant domains of the light chain, as well as the variable domain and the first constant domain (CDN) of the heavy chain. E(ar)2 antibody fragments include a pair of Rab fragments, which are usually covalently linked at their carboxy termini by intermediate hinges cysteines. Other chemical bonds between antibody fragments are also known to those skilled in the art. "Single-chain Em" or "5cEm" antibody fragments contain the Mn and Mi domains of the antibody, in which these domains are present in a single polypeptide chain. In general, the Em polypeptide additionally contains a polypeptide linker between the Mn and Mi domains, which enables the ssEm to form the desired structure for antigen binding. For review

HERE see Rishskiyip in the book Te Rinaptasiodu oi Moposiopa! Apiirodiech, Mo! 113, Noseprigd 5 Mooge ev.

Zrgipdeg-Mepad, Mem Khoik, pp. 269-315 (1994).

The term "diatyl" refers to small antibody fragments with two antigen-binding sites; these fragments contain the variable domain (Mn) of the heavy chain, connected to the variable domain (Mi) of the light chain in the same polypeptide chain (Mn and Mu). Using a linker that is too short to allow pairing between these two domains on the same chain forces the domains to pair with additional domains on the other chain and create antigen-binding sites. Such diatila are described in more detail, for example, in such sources as EP 404,097; MO 93/11161; and Noiiipdeg ei ai., Rgos. Mai). Asad 5bi.

O5A, 90:6444-6448 (1993).

The expression "linear antibodies" refers to the antibodies described in 7araiia ei ai., Rgoiviiep Epdo., 8(10):1057-1062 (1995). Briefly, these antibodies contain a pair of tandem Ba segments (Mn-Sni-Mn-Sni), which, together with additional light chain polypeptides, form a pair of antigen-binding sites. Linear antibodies can be bispecific or monospecific.

The term "monoclonal antibody" as used herein refers to an antibody derived from a population of substantially homogeneous antibodies, that is, the individual antibodies that make up that population are identical except for possible natural mutations that may be present in small amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Moreover, unlike conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on a given antigen. The modifier "monoclonal" indicates the nature of such an antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that such antibody be obtained by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be obtained by the hybridoma method first described by Coppieg et al., Maischge 256:495 (1975), or by a method that uses recombinant

DNA (see, for example, US Pat. No. 4,816,567). "Monoclonal antibodies" can also be selected from libraries of phage antibodies according to the method described, for example, in Siaskzop et al., Maishge 352:624-628 (1991) and Magk5 et al., 9. Moi. Vioi 222:581-597 (1991).

Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequences in the antibody,

derived from a particular species, or belonging to a particular class or subclass of antibodies, while the rest of the chain is identical or homologous to corresponding sequences in antibodies derived from another species, or belonging to another class or subclass of antibodies, and fragments such antibodies as long as they demonstrate the desired biological activity (US patent Mo 4,816,567; Motgpizop eyi ai., Rgos. Mai. Asad. 5si. OBA 81:6851-6855 (1984)). "Humanized" forms of non-human antibodies (eg, murine antibodies) are chimeric antibodies that contain minimal sequence derived from a non-human immunoglobulin.

Most often, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region of a non-human species (donor antibody), such as mouse, krill, or primate, have the desired specificity, affinity, and ability. In certain cases, the residues of the human immunoglobulin E framework region are replaced by corresponding residues that do not belong to humans. Moreover, humanized antibodies may include residues that are not found in either the recipient antibody or the donor antibody. Such modifications are intended to achieve even more improved characteristics of the antibody. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to such non-human immunoglobulin, and all or substantially all of the EV regions have a human immunoglobulin sequence. Such a humanized antibody can optionally also contain at least a part of the constant region of an immunoglobulin (EC), typically a human immunoglobulin. For more detailed information, see Uopez et al., Mashte 321:522-525 (1986); NViesptapp ei ai., Mashge 332:323-329 (1988); and Rhezia,

Sy. Or. Zygisi Cheers! 2:593-596 (1992). A "human antibody" is one that has an amino acid sequence corresponding to such an antibody produced by a human and/or obtained by any technique designed to generate human antibodies as described herein. This definition of a human antibody clearly excludes a humanized antibody that contains non-human antigen-binding residues. Human antibodies can be obtained using various techniques known to those skilled in the art. In one embodiment, the human antibody is selected from a phage library that expresses human antibodies (Masdnap ei ai. Mayishge Vioiespoiodu 14:309-314 (1996): ZNeviv ei aI. Rgosz. Mai). Asai. si. 95:6157-6162 (1998)); Noodeproot 8 M/ipieg, U. Moi. Biol., 227:381 (1991); Magkz ei ai|.,».

My. BioI., 222:581 (1991)). Human antibodies can be obtained by introducing human immunoglobulin loci into transgenic animals, such as mice, in which endogenous immunoglobulin genes are partially or completely inactivated. After that, the production of a human antibody is observed, which in all respects strongly resembles what is observed in humans, including the rearrangement of the gene, the assembly and the spectrum of the antibodies. This approach is described, for example, in US patents MoeMo 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, as well as in the following scientific publications: Magkz ei ai!., Vio/Thesppoiodu 10: 779-783 (1992); And oprego eyi a!., Maishge 368: 856-859 (1994); Moitisop, Maishge 368:812-13 (1994); Rizpma ei ai., Journal of Science and Technology 14: 845-51 (1996); Meiregodeg, Mashge

Physiology 14: 826 (1996); And opregd apa Nyvlag, Ipiet. German And so on. 13:65-93 (1995). Alternatively, a human antibody can be obtained by immortalizing human B lymphocytes, which gives an antibody directed against the target antigen (such B lymphocytes can be taken from the individual or have been immunized ip miigo). See, for example, Soye eyi aYi., Moposiopa! Apiirodiez apa Sapseg Tpegaru, Aiap NV. I iv5, p. 77 (1985); Woegpeg ei ai., ».

Ittypo)., 147 (1):86-95 (1991) and US Patent No. 5,750,373.

An "affinity mature" antibody is an antibody with one or more changes in one or more regions of its SOV that result in improved affinity of such antibody for an antigen, compared to a parent antibody that has not acquired the corresponding changes. The best mature affinity antibodies will have nanomolar or even picomolar affinity for the target antigen. Affinity-mature antibodies are prepared using methods known to those skilled in the art. MagKk5 ei ai. Vio/Tesppoiodu 10:779-783 (1992) describe affinity maturation by shuffling the MN and MI domains. Random mutagenesis of OWL and/or framework residues is described: Vagbaz Yei a. Rgos Mai. Asad 5si

O5BA 91:3809-3813 (1994); 5 spieg her am. Sbepe 169:147-155 (1995); Meiyup ey ay. U. Ittipoi. 155:1994-2004 (1995);

Uaskzop eyi ai., U. Ittipoi. 154(7):3310-9 (1995); NamkKipz ei ai., U. Moi. Axes 226:889-896 (1992). The "functional antigen binding site" of the antibody is able to bind the target antigen. The antigen-binding affinity of the antigen-binding site is not necessarily as strong as that of the parent antibody from which the antigen-binding site is derived, but the ability to bind the antigen should be amenable to determination by any of a variety of methods known to assess binding of antibody to antigen. Moreover, the antigen-binding affinity of each of the antigen-binding sites of the multivalent antibody does not necessarily have to be quantitatively the same. For the multivalent antibodies discussed herein, the number of functional antigen binding sites can be assessed using an ultracentrifugation method as described in Example 2 of US Patent Application Me 20050186208. According to this assay method, the target antigen and the multimeric antibody were combined in various ratios , and the average molecular weight of such complexes was calculated assuming a different number of functional binding sites. These theoretical numbers were compared with real experimental data obtained in order to estimate the number of functional binding sites.

An antibody having the "biological characteristic" of a defined antibody is one that possesses one or more biological characteristics that distinguish it from other antibodies that bind to the same antigen.

In order to screen for antibodies that bind to the epitope on the antigen bound by the antibody of interest, a routine epitope cross-competition assay can be performed, such as described in

Apiirodiev, A I aroghaiogu Mapiai, Soia 5riipd Nagrog I arogaiugu, Ea Napozh apa Vamid I ape (1988). A "species-specific antibody" is an antibody that has a stronger affinity for binding to an antigen from a first mammalian species than to a homologue of this antigen from a second mammalian species. Of course, such a species-specific antibody "specifically binds" to a human antigen (ie, has a binding affinity (Ka) of no more than about 1 x 107 M, preferably no more than about 1 x 103 M, and most preferably no more than about 1 x 109 M), but has an affinity for binding to a homologue of that antigen from a second non-human mammalian species that is at least 50-fold, or at least 500-fold, or at least 1000-fold weaker than the affinity to binding to a human antigen. Such a species-dependent antibody may belong to any of the various types of antibodies previously defined, but is typically a humanized or human antibody.

The term "antibody-mutant" or "antibody-variant", as used herein, refers to a variant of the amino acid sequence of a species-specific antibody in which one or more amino acid residues of that species-specific antibody have been modified. Such mutants necessarily have less than 10095 sequence identity or similarity with the species-dependent antibody. In one embodiment, the mutant antibody will have a sequence that is at least 7595 identical or similar to the amino acid sequence of the heavy or light chain variable domain of the species-specific antibody, preferably at least 8095, more preferably at least 8595, even better at least 9095, and most preferably at least 9595. Identity or similarity with respect to this sequence is defined here as the percentage of amino acid residues in the candidate sequence that are identical (i.e., the same) or similar (i.e., amino acid residues from the same group based on shared side chain properties - see below) with residues of a species-dependent antibody, after comparing these sequences and introducing intervals, if necessary, to achieve the maximum percentage of sequence identity. None of the M-termini, C-termini, or internal extensions, deletions, or insertions in the antibody sequence outside the variable domain will be considered to disrupt sequence identity or similarity.

To increase the half-life of antibodies or polypeptides containing the amino acid sequences of the present invention, a salvage receptor-binding antigenic determinant may be attached to this antibody (especially a fragment of the antibody) as described in US Pat. No. 5,739,277. For example, a nucleic acid molecule encoding such a rescue receptor-binding antigenic determinant can be linked by a framework to a nucleic acid encoding a polypeptide sequence of the present invention, so that a fusion protein expressed by such an engineered nucleic acid molecule will contain a rescue receptor-binding antigenic determinant and polypeptide sequence according to this invention. The term "rescue receptor-binding antigenic determinant" as used herein refers to the epitope of the Es region of the Ids molecule (eg, IdSy, Ids2, IdS3, or IdSya) that is responsible for increasing Ip beyond the molecule's serum half-life

Id. Antibodies with substitutions in their ES region and increased half-life in serum are also described in the following publications: UUO00/42072, UMO 02/060919;

ZpieiYidz ei ai., 9. Vioi. Spent 276:6591-6604 (2001); Nipyuop, u. Vioi Spent 279:6213-6216 (2004). In another embodiment, the half-life in serum can be increased, for example, by adding other polypeptide sequences. For example, antibodies or other polypeptides used in the methods of the present invention may be attached to serum albumin or to that portion of serum albumin that binds to the EsVP receptor, or to a serum albumin-binding peptide such that the albumin serum binds to such an antibody or polypeptide, as described in the publication UUO01/45746. In one embodiment, the serum albumin peptide that binds the antibody contains the amino acid sequence YUISI RAMUSSI MU. In another embodiment, these methods increase the half-life of Bar. For the amino acid sequences of the serum albumin peptide that is capable of binding the antibody, see also UOeppiv iai. 9U. Vioi Spent 277:35035-35043 (2002). A "chimeric MESE receptor protein" is a MESE receptor molecule that has amino acid sequences derived from at least two different proteins, one of which is the MESE receptor protein. In certain embodiments, such chimeric MESEE receptor protein is capable of binding to MESEE and inhibiting its biological activity. An "isolated" polypeptide or "isolated" antibody is one that has been identified and isolated and/or recovered from some component of its natural environment. Contaminant components of its natural environment are materials that could affect the diagnostic and therapeutic use of such a polypeptide or antibody and may include enzymes, hormones, and other solutes, similar or dissimilar to proteins. In certain embodiments, such a polypeptide or antibody will be purified (1) to greater than 9590 by weight of the polypeptide or antibody, as determined by Method I, and preferably greater than 9995 by weight; (2) to an extent sufficient to obtain at least 15 residues of the M-terminus or internal amino acid sequence using a spinning glass sequencer; (3) to homogeneity by 505-RACE under reducing or non-reducing conditions using Sootavie blue or silver staining.

The isolated polypeptide or antibody includes the polypeptide or antibody inside the recombinant cells, since at least one component of the natural environment of this polypeptide will not be present. However, usually the isolated polypeptide or antibody will be obtained using at least one purification step.

By "fragment" is meant a part of a polypeptide or a nucleic acid molecule that preferably contains at least 1095, 2095, 3095, 40905, 5095, 6090, 7095, 8095, 9095, 9595 or more of the full length of a reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100, 200, 300, 400, 500, 600 or more nucleotides or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 190, 200 amino acids or more. "Anti-angiogenesis drug" or "angiogenesis inhibitor" refers to a small molecular weight substance, polynucleotide, polypeptide, isolated protein, recombinant protein, antibody, or conjugates or fusion proteins thereof that inhibits, directly or indirectly, angiogenesis, vasculogenesis, or unwanted permeability vessels It should be understood that such an anti-angiogenic drug includes those drugs that bind and block the angiogenic activity of an angiogenic factor or its receptor. For example, an anti-angiogenic drug is an antibody or other antagonist to an angiogenic factor as previously defined, such as antibodies to MESE-A or to the MESE-A receptor (eg, KOV receptor or RI-1 receptor), anti-ROSEV inhibitors such as

Siemes"mMm (Imatinib mesylate). Anti-angiogenesis drugs also include native inhibitors of angiogenesis, for example, angiostatin, endostatin, etc. See, for example, Kiadegyp 5 B'Atoge, App. Veum. Rpuzioi!., 53:217-39 ( 1991); 5igei 5 Oyeitag, Opsodepe, 22:3172-3179 (2003) (for example, Table 3 lists the regimens of anti-angiogenic therapy in malignant melanoma); Reitaga 5 Aiiayu, Maishge Meaisipe 5:1359-1364 (1999); Topipi ei Ai., Opsodepe, 22:6549-6556 (2003) (eg, Table 2 lists known anti-angiogenic factors); and Ai. Ipi. 9. Siip. Opsoi., 8:200-206 (2003) e.g., in Table 1 listed anti-angiogenic drugs that have been used in clinical trials). "Treatment" refers to both therapeutic treatment and prophylactic or preventive measures. Those in need of treatment include those who already have a benign, precancerous, or non-metastatic tumor, and as well as those who need to prevent the development or recurrence of cancer.

The term "therapeutically effective amount" refers to an amount of an MECE-specific antagonist to treat or prevent a disease or disorder in a mammal. In the case of a precancerous, benign or early-stage tumor, a therapeutically effective amount of the MECE-specific antagonist can reduce the number of cancer cells; reduce the size of the primary tumor; suppress (that is, slow down to a certain extent, or rather stop) the infiltration of cancer cells into peripheral organs; inhibit (i.e. slow down to a certain extent, and better stop) tumor metastasis; suppress tumor growth to a certain extent; and/or alleviate to some extent one or more symptoms associated with the disorder. In the treatment of a dormant tumor or micrometastases, a therapeutically effective amount of a MECE-specific antagonist can reduce the number or proliferation of micrometastases; slow down or prevent the growth of a dormant tumor; or slow or prevent the recurrence of a tumor after treatment or removal (for example, after anti-cancer treatment such as surgery, radiation therapy or chemotherapy). Depending on the degree to which the drug can prevent the growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. The effectiveness of anticancer therapy can be determined, for example, by evaluating the duration of survival, the duration of the interval to disease progression (TPR), the frequency of response to treatment (AR), the duration of such response, the duration of remission and/or quality of life.

An effective amount can improve disease-free survival (OE5), improve overall survival (05), reduce the likelihood of recurrence, increase the interval to recurrence, increase the interval to distant recurrence (ie, recurrence outside the primary site), cure cancer, improve cancer symptoms ( for example, according to the results of cancer-specific examinations), reduce the probability of the occurrence of a second primary cancer, etc. "Operable" cancer is cancer that is limited to the primary organ and can be treated surgically. "Survival" refers to the length of time a patient remains alive and includes disease-free survival (OE5), progression-free survival (PE5), and overall survival (05). Survival can be estimated by the Kaplan-Meier method, and the difference in survival ratio is calculated using the stratified log-rank test. "Disease-free survival (DFS5)" refers to the length of time a patient remains alive without the cancer returning, of about 1 year, about 2 years, about C years, about 4 years, about 5 years, about 10 years, etc. d. from the onset of the disease or from the diagnosis. According to one aspect of the present invention, OB5 is analyzed based on the principle of taking at least one dose of the prescribed treatment. Events used in the JOB5 analysis may include local, regional, or distant cancer recurrence, occurrence of a second cancer, and death from any cause in patients without any previous event (eg, breast cancer recurrence or second primary cancer). "Overall survival" refers to the length of time a patient remains alive for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 10 years, etc. from the onset of the disease or from the diagnosis. In the studies underlying this invention, the phenomenon used in the survival analysis was death from any cause. "Prolonged survival" means an increase in OE5 and/or O5 in a treated patient relative to an untreated patient (ie, relative to a patient not treated with an MECE-specific antagonist, such as an MECE antibody), or relative to a control treatment protocol, such as treatment with only a chemotherapy drug such as paclitaxel. Survival is monitored for at least about 6 months, or at least about 12 months, or at least about 2 years, or at least about 3 years, or at least about 4 years, or at least about 5 years, or at least about 10 years, etc. after starting treatment or after diagnosis. The "hazard ratio" in survival analysis is the result of the difference between the two survival curves, which represents the reduction in the risk of death in the case of treatment, compared with control, over the period of follow-up. The risk ratio is a statistical characteristic for the intensity of the flow of phenomena. For the purposes of this invention, the risk ratio is determined by the probability of the phenomenon in the experimental group divided by the probability of the phenomenon in the control group, at some specific point in time.

The term "simultaneous" is used herein to describe the administration of two or more therapeutic agents where at least some of the administration overlaps in time. Accordingly, co-administration includes an administration regimen in which administration of one or more drugs is continued after the administration of one or more other drugs has been discontinued.

"Monotherapy" refers to a treatment regimen that involves the use of only one drug for the treatment of cancer or tumor during the course of therapy. Monotherapy with the use of a MESE-specific antagonist means that this MESE-specific antagonist is administered in the absence of additional anticancer therapy during the course of treatment.

"Maintenance therapy" means a treatment regimen designed to reduce the likelihood of relapse or progression of the disease. Maintenance therapy can be prescribed for any period of time, including lifelong. Maintenance therapy may be prescribed after the initial course of treatment or in combination with initial or additional therapy. Doses used for maintenance therapy may vary and may include reduced doses compared to doses used for other therapies.

As used herein, the terms "neoadjuvant therapy" or "preoperative therapy" refer to therapy administered before surgery. The goal of neoadjuvant therapy is to provide immediate systemic treatment, potentially capable of eradicating micrometastases, which otherwise, i.e. in the case of a standard sequence, when systemic therapy is prescribed after surgery, could reach the stage of proliferation. Neoadjuvant therapy can also help reduce the size of the tumor and, thereby, allow complete resection of previously inoperable tumors or save part of the organ and its function. Moreover, neoadjuvant therapy allows the assessment of IP beyond the effectiveness of the drug, which can help guide the choice of subsequent courses of treatment.

The term "adjuvant therapy," as used herein, refers to therapy given after surgery, when no evidence of residual disease is present, to reduce the risk of disease recurrence. Therefore, the goal of adjuvant therapy is to prevent cancer recurrence and, accordingly, reduce the likelihood of cancer-related death.

The term "standard chemotherapy" refers to chemotherapy drugs that are routinely used in the treatment of a given type of cancer.

The term "radical surgery" or "definitive operation" is used here as it is in the medical community. Radical surgery includes, for example, operations, surgical or otherwise, that result in the removal or resection of a tumor, including those that result in the removal or resection of the entire macroscopically visible tumor.

Radical surgery includes, for example, complete or clinical radical resection or complete macroscopic tumor resection. Radical surgery includes operations that are performed in one or more stages, and includes, for example, multistage surgical interventions, where one or more surgical or other procedures are performed before resection of the tumor. Radical surgery includes operations to remove or resect the tumor, including the affected organs, parts of organs and tissues, as well as surrounding organs such as lymph nodes, parts of organs or tissues.

The terms "cancer" and "cancerous" describe a physiological condition in mammals that is typically characterized by unregulated cell growth. This definition includes benign and malignant cancers, as well as dormant tumors or micrometastases. "Early-stage cancer" or "early-stage tumor" means cancer that is not invasive or metastatic or is classified as stage 0 cancer, | or II.

The term "precancerous" refers to a condition or growth that typically precedes or develops into cancer. A "precancerous" growth will have cells characterized by abnormal cell cycle regulation, proliferation, or differentiation. This is determined by appropriate markers of cell cycle regulation, cell proliferation or differentiation.

"Dysplasia" means abnormal growth or development of tissue, organ or cells. Dysplasia is often high-grade or precancerous.

"Metastasis" means the spread of cancer from its primary site to other places in the body. Cancer cells can break away from the primary tumor, enter the lymphatic and blood vessels, circulate with the bloodstream, and grow in a distant location (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant.

Metastasis is a process that is the result of tumor cells detaching from the primary tumor, moving with the bloodstream and stopping at a distant location. In a new place, such cells establish a blood supply and can grow, forming a life-threatening mass. Stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior; at the same time, the interaction between the tumor cell and the cells of the host in a remote location is also of significant importance.

"Micrometastasis" refers to a small number of cells that have spread from the primary tumor to other parts of the body. Micrometastases may (or may not) be detected during screening or diagnostic testing. "Non-metastatic" cancer is cancer that is benign, or that remains at the primary site and has not spread to the lymphatic and blood vessel systems or to tissues other than those of the primary site. In general, non-metastatic cancer is any cancer that can be classified as stage 0 cancer. or II, and sometimes cancer at the stage of IP.

Reference to a tumor or cancer as being "stage 0", "stage I", "stage II", "stage PI" or "stage

IM", corresponds to the classification used by specialists in this field. Although the actual stage of cancer depends on the type of cancer, in general, stage 0 cancer is lymph node involvement, stage I cancer is a small localized tumor, stage I and IS cancer is locally advanced tumor that shows local lymph node involvement, and stage IM cancer is - this is metastatic cancer. Specific stages for each type of tumor are known to an experienced clinician.

The term "tumor" as used herein refers to all types of neoplastic growth and proliferation of cells, both benign and malignant, as well as all precancerous and cancerous cells and tissues.

"Primary tumor" or "primary cancer" refers to the initial cancer and not to a metastatic lesion located in another tissue, organ, or site of the subject's body.

A "benign tumor" or "benign cancer" refers to a tumor that remains localized at the site of origin and does not have the ability to infiltrate, invade a distant site, or metastasize to a distant site. "Cancer recurrence" as used herein refers to the return of cancer after treatment and includes the return of cancer in the primary organ, as well as distal recurrence, where the cancer returns outside the primary organ. A "dormant tumor" refers to a prolonged quiescent state in which tumor cells are present, but tumor progression is not clinically evident. Dormant tumors may (or may not) be detected during screening or diagnostic testing. "Tumor mass" means the number of cancer cells, the size of the tumor, or the amount of cancer in the body. Tumor mass is also called tumor burden.

"Number of tumors" means the number of tumors.

"Subject" shall mean a mammal, including but not limited to humans and non-human mammals such as cattle, horses, dogs, sheep or cats. Mostly the subject is a person. "Subject population" means a group of subjects with cancer, such as in a clinical trial, or as seen by oncologists after FDA approval of a treatment for a specific indication, such as neoadjuvant therapy cancer In one embodiment, the population includes at least 3,000 subjects.

The term "anticancer therapy" refers to therapy used in the treatment of cancer. Examples of anticancer therapeutic agents include, but are not limited to, chemotherapeutic agents, growth inhibitors, cytotoxic agents, radiation therapy agents, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other cancer agents such as anti-

HEV-2 antibodies, anti-CO20 antibodies, epidermal growth factor receptor (EGF) antagonist (eg, tyrosine kinase inhibitor), NENIT/ESEA inhibitor (eg, erlotinib (Tagsema""M)), platelet-derived growth factor inhibitors (eg, Siemes" mM (Imatinib mesylate)), cyclooxygenase-2 (COX-2) inhibitor (eg, celecoxib), interferons, cytokines, antagonists (eg, neutralizing antibodies) that bind to one or more of the following targets EB2, EtB3, EpB4 .

The term "cytotoxic" drug, as used herein, refers to a substance that inhibits or prevents cell function and/or causes cell destruction. This term includes radioactive isotopes (eg, 1791, (125, UuzZ0 and Ne!86), chemotherapeutic drugs and toxins, such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or their fragments. A "chemotherapeutic drug" is a chemical a compound used in the treatment of cancer. Examples of chemotherapeutic drugs include alkylating agents such as thiotepa and SUTOHAMP, cyclophosphamide; alkyl sulfonals such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquine, methyredopa, and uredopa; ethyleneimines and methylmelamines; including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bulatacin and bulatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; SS-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duokarm icin (including synthetic analogues KM/U-2189 and SV1-TMI); eleuterobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquine, fenesterin, prednimustine, trophosphamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimpustine; antibiotics such as enediyne antibiotics (eg calicheamicin, especially calicheamicin gamma 11 and calicheamicin omega! 1 (see eg Adpem, Speth pi). Ed.

Epa!., 33: 183-186 (1994)); dinamycin, including dinamycin A; bisphosphonates such as clodronate, esperamycin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophyllin, chromomycins, dactinomycin, daunorubicin, detorubicin, b-diazo-5-oxo-III --orleucin , AOKIAMUSIMF, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, ezorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peptiromycin, potyromycin, purom , chelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, zinostatin, zuroubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-E)); folic acid and analogues such as denopterin, methotrexate, pteropterin, trimetrexate: purine analogues such as fludarabine, b6-mercaptopurine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, b-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostan, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; a folic acid supplement such as prolenic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisanthrene; edatrexate; Defofamine; demecolcin; diaziquin; elfornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; Lentinan; lonidine; maytansinoids such as maytansine and ansamitocins; mitoguasan; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2- ethyl hydrazide; procarbazine; ROKF polysaccharide complex (HO Maiishga! Rgodisi5, Edepe, OV); razoxane; risoxin; Sizophyran; spirogermanium; tenuazonic acid; triaziquin; 2,2'2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobromane; hacytosine; arabinoside ("Aga-C") ; cyclophosphamide; thiotepa; taxoids, for example TAHOII F, paclitaxel (VgisioI-Mueg5 5ditsiBio Opsoiodu, Riipseip, M.).), AVAACHAME "M without cremophor, paclitaxel preparation of nanotechnology (Ategpsap РNAPтасецииса! Rappeg5, Zspashtbrego, Shipoiz) and TACHOTEKEF doxetaxel ( KPppbpe-

Rocheps Rogeg, Apiopu, Egapse); Chloranbucil; SEM2ANF gemcitabine; b-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (MP-16); ifosfamide; mitoxantrone; vincristine; MAMEI VIMEF vinorelbin; novantrone; teniposide; edatrexan; daunomycin; aminopterin; xeloda; ibandronate; Irinotecan (Satriozag, CRT-11) (including a treatment regimen using irinotecan with 5-

ERC and leucovorin); VE5 2000 topoisomerase inhibitor; difluoromethylornithine (OMEO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (IM); oxaliplatin, including oxaliplatin regimen (ROI EOH); inhibitors of PKS-alpha, Vai, H-Vaz, ESEV (for example, erlotinib (Tagsema ""M)) and MECP-A, which reduce cell proliferation, as well as pharmaceutically acceptable salts, acids or derivatives of any of the above compounds.

Also included in this definition are antihormonal drugs that regulate or inhibit the action of hormones on the tumor, such as anti-estrogens and selective estrogen receptor modulators (SERM5), including, for example, tamoxifen (including MY MAOI tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen , trioxyphene; keoxifen, I 117018, onapristone and ЕАВЕ5ТОМ toremifene; aromatase inhibitors that inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MESABEF megestrol acetate, AVOMABIMF exemestane, formestanie, fadrozole, VIMIZONO vorozole, REEMANAF letrozole and AVIMIOEHF anastrozole; as well as anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (analogue of 1,3-dioxolane cytosine nucleoside); antisense oligonucleotides, in particular those that suppress the expression of genes in signaling pathways that are involved in impaired cell proliferation, such as, for example, PKS-alpha, Nai and H-Bav; ribozymes such as expression inhibitor

MES (for example, AMOIO2UMEF ribozyme) and an inhibitor of NEN2 expression; vaccines, such as gene therapy vaccines, such as ASGGOMESTIMF vaccine, GEOMESTIMF vaccine and MAHIOF vaccine; REOGISHKIMF pi--2; topoisomerase 1 inhibitor GOETOTESAMEIA; AVARKEGIHF Fri; vinorelbine and esperamycin (see US Pat. No. 4,675,187), as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above compounds. "Growth inhibitory agent" refers to a compound or composition that inhibits the growth of an ip myo and/or ip mimo cell. Therefore, a drug that suppresses growth can be such that it significantly reduces the percentage of cells in phase 5.

Examples of drugs that inhibit growth include drugs that block cell cycle progression (other than phase 5), such as drugs that cause C1 arrest and M-phase arrest. Classic M-phase blockers include periwinkle drugs (vincristine and vinblastine), TAHOI F, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those drugs that stop C11 are also involved in phase 5 arrest, for example, DNA-alkylating drugs such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. More detailed information can be found in the following sources:

Tne MoiIesshag Vaviv oi Sapseg, Mepavye!zoNnp 4. Ivgaei, edv., Chapter 1, entitled "Sei! susie gedshayop, opsodepev, apa apiipeoriaviis apidv" Bu Mygakati eyi a. (M/V Zaipage: RpyPaade!rnia, 1995), in particular p. 13.

The term "cytokine" is a generic name for proteins that are released by one population of cells and that act on other cells as intercellular mediators. Examples of such cytokines are lymphokines, monokines and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, human growth hormone M-methionyl, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle-stimulating hormone (E5H), thyroid-stimulating hormone (T5H) and luteinizing hormone (IN); epidermal growth factor; hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor alpha and beta; Mullerian inhibitory substance; murine gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TRO); nerve growth factors such as MOE-alpha; platelet growth factor; transforming growth factors (TGF), such as TAB-alpha and TavB-beta; insulin-like growth factor-i! and -III; erythropoietin (EPO); osteoinductive factors; interferons, such as interferon-alpha, -beta and -gamma; colony-stimulating factors (C5BE), such as macrophage C5E (M-C5E); granulocytomacrophage CE (2M-C5BE); and granulocyte C5E ((-C5BE); interleukins (I), such as 1-1, 1-1 alpha, 1-2, 1-3, 1-4, 1-5, 11 -6, 11 -7, 11 -8, P/ -9, I-10, 11-11, 11-12; tumor necrosis factor, such as TME-alpha or TME-beta; as well as other polypeptide factors, including HE and cat-ligand (KI) .The term "cytokine" as used herein includes proteins from natural sources or from recombinant cell cultures, as well as biologically active equivalents of cytokines with native sequences.

The term "prodrug", as used in this application, refers to a prodrug or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and is capable of being enzymatically activated or converted to a more active parent form (see for example, Umiitap, "Rgodgide ip Sapseg Spetoipegaru" Viospetis! Zosieiu Tgapsasiop5, 14, pp. 375-382; 6157

Meeeiiipd VeiGavi (1986), 5ieiYa eyii ai., "Rgodpide: A Spetisa! Arrgoasp yu Tagdeiiea Ota Oeiiimegu"; Oigesiy Od

Oeiimegu, Vogsepagai ei ai., (e9.), pp. 247-267, Nytapa Rge5z5 (1985). Prodrugs of the present invention include, but are not limited to, phosphate-containing prodrugs; prodrugs containing sulfate; prodrugs containing a peptide; prodrugs modified with SO-amino acid; glycosylated prodrugs; prodrugs containing p-lactam; prodrugs containing optionally substituted phenoxyacetamide or prodrugs containing optionally substituted phenylacetamide; 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted to a more active drug that does not have cytotoxicity. Examples of cytotoxic drugs that can be obtained in the form of prodrugs for use in accordance with the present invention include, but are not limited to, those chemotherapeutic drugs that have been described above.

"Radiation therapy" refers to the use of directed gamma rays or beta rays to cause sufficient damage to a cell to limit its ability to function normally or to destroy the cell completely. Specialists in this field know numerous methods of determining the dose and duration of such treatment.

Typically, it is carried out at a time in the range of doses from 10 to 200 units (Gray) per day. "Reduce or suppress" means the ability to cause a total reduction of 2,095 or more, preferably 5,095 or more, and most preferably 7,595, 85,905, 9,095, 9,595 or more. The reduction or suppression may relate to the symptoms of the disorder being treated, the presence or size of metastases or micrometastases, the size of the primary tumor, the presence or size of a dormant tumor, or the size or number of blood vessels in angiogenic disorders. "Antisense nucleic acid oligomer" means a nucleic acid oligomer, regardless of its length, that is complementary to at least a portion of the coding strand or mRNA of a gene.

"Nucleic base oligomer" means a compound that includes a chain of at least eight nucleic bases, preferably at least twelve, and most preferably at least sixteen bases joined together by linkage groups. This definition covers both naturally occurring and non-naturally occurring oligonucleotides, modified and unmodified, as well as oligonucleotide mimetics such as protein nucleic acids, closed nucleic acids and arabinonucleic acids. Numerous nucleic acid bases and linking groups can be used in the nucleic acid oligomers of the present invention, including those described in US Patent Publications No. 20030114412 (see, for example, paragraphs 27-45 of this publication) and Me 20030114407 (see, for example, paragraphs 35 -52 of this publication), which are considered included in this description by reference. A nucleic acid oligomer can also be targeted to translational start and end sites. In certain embodiments, the antisense nucleic acid oligomer contains from about 8 to about 30 nucleotides. An antisense oligomer with a nucleic acid can also contain at least 40, 60, 85, 120 or more consecutive nucleotides that are complementary to the mRNA or DNA of the MES and can be the length of the full mRNA or gene.

"Small RNA" means any RNA molecule, single- or double-stranded, that contains at least 15 nucleotides, and preferably 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34, or 35 nucleotides in length, or even 50 or 100 nucleotides in length (including all insertions in between). In certain embodiments, such a small RNA is capable of mediating RNA interference (RNAi). The term "mediates RNA interference" as used herein refers to the ability to distinguish which RNAs are to be degraded by the RNA interference machinery or process. The term small (or short) RNA includes "small interfering RNA" and "microRNA".

In general, miRNAs (tRNAs) are small (eg, 17-26 nucleotides), single-stranded non-coding RNAs that are derived from approximately 70-nucleotide hairpin RNA precursors by Dicer action. Small interfering

RNAs (siRNAs) are similar in size and are also non-coding; however, zivMAs are derived from long α5AMAs and are usually double-stranded. ziyMA can also include short hairpin RNAs in which both strands of the ziyMA duplex are inserted into a single RNA molecule. Small RNAs can be used to describe both types of RNA. These terms include double-stranded RNA, single-stranded RNA, isolated RNA (partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA), and modified RNA that differs from native RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such changes may include the addition of non-nucleotide material, such as at the end(s) of the small RNA or within (into one or more RNA nucleotides). Nucleotides in the RNA molecules of the present invention may also include non-standard nucleotides, including those that do not occur in nature, or deoxyribonucleotides. See “oligomers with a nucleic acid base” above for additional modifications of the nucleic acid molecule. In one embodiment, the RNA molecules contain a 3'-hydroxyl group.

The term "intravenous infusion" refers to the introduction of a drug into a vein of a patient, animal or person, over a period of time that exceeds approximately 5 minutes, and preferably is from 30 to 90 minutes, although, according to the present invention, the intravenous infusion can, alternatively, last 10 hours or less.

The term "intravenous bolus" or "intravenous push" refers to the administration of a drug into a vein of an animal or human in such a way that the body receives the drug in about 15 minutes or less, preferably 5 minutes or less.

The term "subcutaneous administration" refers to the introduction of a drug under the skin of a patient, animal, or human, preferably into a pocket between the skin and the underlying tissue, by relatively slow, sustained delivery from a container of the drug. Such a pocket can be formed by pinching or pulling the skin away from the fabric below.

The term "subcutaneous infusion" refers to the administration of a drug under the skin of a patient, animal, or human, preferably in a pocket between the skin and underlying tissue, by relatively slow, sustained delivery from a container of the drug over a period of time, including but not limited to 30 minutes or less, or 90 minutes or less. Optionally, such infusion may be accomplished by implanting under the skin of a patient, animal, or human, a drug delivery pump that delivers a set amount of drug at specified time intervals, such as 30 minutes, 90 minutes, or throughout the course of treatment.

The term "subcutaneous bolus" refers to the administration of a drug under the skin of a patient, animal, or human, when the drug is delivered in less than about 15 minutes, preferably less than 5 minutes, and more preferably less than 60 seconds. The drug is mainly injected into a pocket between the skin and the underlying tissue, which can be formed, for example, by pinching or pulling the skin away from the underlying tissue.

The word "label" as used herein refers to a detectable compound or composition that forms, directly or indirectly, a conjugate with a polypeptide. Such a label may be detectable by itself (eg, radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze a chemical change in a substrate, compound, or composition that is already detectable.

I. The use of MESIE-specific antagonists in the treatment of tumors at an early stage, as well as for neoadjuvant and adjuvant therapy

The present invention relates to the use of MESIE-specific antagonists in the treatment of a subject with a benign, precancerous or non-metastatic tumor; in the treatment of a subject with a dormant tumor or micrometastases; or in the treatment of a subject at risk of developing cancer. For example, by using two independent approaches to inhibit MES, namely monotherapy with a monoclonal antibody (tAbB) targeting

MESVB-A and genetic deletion of MESEB-A, we demonstrated in the model of early intestinal adenoma in mice

Arstip" that inhibition of the MESE signaling pathway is sufficient to halt tumor growth and provide a long-term survival benefit in this model. We also demonstrated using a monoclonal antibody (mAb) targeting MESE-A that inhibition of the MESE signaling pathway is sufficient to stop pituitary adenoma growth and reduce excess hormone secretion in a mouse model of multiple endocrine neoplasia type 1 (MEM). Moreover, we demonstrated in a murine islet of Langerhans tumor model (HIV-TrRAd) using a monoclonal antibody (tAbB) targeting MESE-A that inhibition

MES leads to a sharp decrease in tumor angiogenesis, and when used as therapy after cytoreduction by surgical intervention or the administration of chemotherapeutic drugs, suppresses the regrowth of tumors.

The present invention also relates to the use of MESEE antagonists in the neoadjuvant and adjuvant setting.

I. MESE-specific antagonists

A MEC-specific antagonist is a molecule (peptidyl or non-peptidyl) capable of binding to MEC, reducing the expression level of MEC, or neutralizing, blocking, inhibiting, abrogating, reducing, or impairing the biological activity of MEC, including the binding of MEC to one or more receptors. MESEE, MES-mediated angiogenesis, survival and proliferation of endothelial cells. Preferably, the MESE-specific antagonist reduces or suppresses, by at least 1095, 2095, 3095, 4090, 5090, 6090, 7095, 8090, 9090, or more, the level of expression or biological activity of MESE. Preferably, the MESE inhibited by the MESE-specific antagonist is any one isoform of USEE or several isoforms of MESE, such as MESE (8-109), MESE (1-109), MESE 65, MESE 21, MESE 145, MESE two, or MESEgov.

A MES-specific antagonist used in the methods of the present invention includes peptidyl or non-peptidyl compounds that specifically bind MES, such as anti-MESE antibodies and antigen-binding fragments thereof, variant antagonists of MESE polypeptides and fragments thereof , which specifically connect with

MES, as well as receptor molecules into derivatives that specifically bind to MES, thereby limiting its binding to one or more receptors (for example, soluble proteins of the MES receptor or fragments thereof that bind MESIE, or chimeric receptor proteins MESEE), fusion proteins (for example, MESEE-Tgar (Vedepegop)) and MES and21- gelonin (Regediipe). MESE-specific antagonists also include antisense oligomers with a nucleic base complementary to at least a fragment of a nucleic acid molecule encoding a polypeptide

MES; small RNAs complementary to at least a fragment of the nucleic acid molecule encoding the MESE polypeptide; ribozymes targeting MESE; peptyl to MES; and MES aptamers.

A. Anti-MESE antibodies

Anti-MESEE antibodies used in the methods of the present invention include any antibody or antigen-binding fragment thereof that binds to MESE with sufficient affinity and specificity and can reduce or inhibit the biological activity of MESE. An anti-MESE antibody will not normally bind to other MESE homologues, such as MESE-B or MESE-C, or to other growth factors such as RICE, ROS or BE.

In certain embodiments of the present invention, anti-MESE antibodies include, but are not limited to, a monoclonal antibody that binds to the same epitope as the monoclonal anti-MESE antibody A4.6.1 produced by hybridoma ATCC HB 10709; recombinant humanized anti-MESE monoclonal antibody obtained according to Rhezia et al. (1997) Sapseg Nev. 57:4593-4599, including, but not limited to, an antibody known as bevacizumab (BM; AmaziipF). Bevacizumab includes mutated framework regions (human DS and antigen-binding regions, which determine complementarity, from the murine anti-pnMESE monoclonal antibody A.4.6.1, which blocks the binding of human MESE to its receptors. Approximately 9395 amino acid sequence of bevacizumab, including most of the framework regions derived from human IDS! and about 7,905 sequences derived from murine antibody A4.6.1. Bevacizumab has a molecular weight of about 149,000 daltons and is glycosylated. Bevacizumab and other humanized anti-MESE antibodies are described in detail in US Pat. No. 6,884,879, issued Feb 26, 2005. Additional examples of antibodies include, but are not limited to,

antibodies of the b or 820 series (for example, (46-31, B20-4.1), as described in the PCT Mo patent application publication

MO2005/012359. For additional antibodies, see US Patent Nos. 7,060,269, 6,582,959, 6,703,020; 6, 342,219; 6,054,297; MO98/45332; UMO 96/30046; M/094/10202; ER 066686881; publication of US patent applications

MoMmo 2006009360, 20050186208, 20030206899, 20030190317, 20030203409 and 20050112126; and also Rorkom ei ai.,

Washpai oi Ittipoiodis! Meipoadv 288:149-164 (2004). Other examples of antibodies that may be used in accordance with the present invention include those that bind to a functional epitope on human MESA that contains residues E17,

M18, 019, 21, 25, 089, 191, K101, E103 and C104 or, alternatively, residues E17, 21, 022, x25, 063, I83 and 089.

A series 6 antibody according to the present invention is an anti-MESE antibody derived from the sequence (6 antibody or an antibody derived from (56) according to any of Figures 7, 24-26 and 34-35 of the PCT application publication

Me MUO2005/012359. In one embodiment, an antibody of the (46) series binds to a functional epitope on

Human MES, which contains residues E17, 21, 022, 25, 063, I83 and 089.

The B20 antibody of the present invention is an anti-MESE antibody derived from the antibody sequence 820 or an antibody derived from B20 according to any of figures 27-29 of PCT application publication Mo

MO2005/012359. In one embodiment, the B20-series antibody binds to a functional epitope on human MESE that contains residues E17, M18, 019, 21, 25, 089, 191, K1T01, ET1OZ3 and C104., "Functional epitope" according to the present invention refers to the amino acid residues of the antigen, which energetically contribute to the binding of the antibody. Mutation of any of the energetically important residues of the antigen (for example, mutation of wild-type MESE to alanine or a homologous mutation) will disrupt the binding of the antibody, so that the ratio of the relative affinity (ISvo of the mutant MESE/Svo of the wild-type MESE) of the antibody will exceed 5 (see

Example 2 in the publication UUO2005/012359). In one embodiment, the relative affinity ratio is determined using a solid-phase enzyme-linked immunosorbent assay (ELISA), which shows phage binding in solution. Briefly, Mahizogr 96-well immunoplates (MOMS) are coated overnight at 4C with the Rab form of the antibody under test at a concentration of 2 μg/ml in RV5 and block RV5, 0.595 VBA, and 0.0595 Tween 20 (RVT) for 2 hours at room temperature Serial dilutions of phage with alanine point mutations of NMESE (residues 8-109) or wild-type NMESE (8-109) in RVT are first incubated on Bar-coated plates for 15 minutes at room temperature and then washed with PB5 , 0.0595 Tween 20 (РВ5Т). The bound phage is detected by the conjugate of the anti-M13 monoclonal antibody with horseradish peroxidase (AtezhNpat RIaptasia), diluted 1:5000 in RVT, developed by 3,3", 5,5'-tetramethylbenzidine (TMV, Kiikedaaga 5 Reigtu Harz, Saipegsrigo, MO) as a substrate for about 5 minutes, quenching with 1.0M NzRO»- and reading on a spectrophotometer at 450 nm. The ratio of ISvo values (ISvo, alanine/ISvzo, wild type) represents the fold reduction in binding affinity (relative binding affinity).

B. MESE receptor molecule

The two best-characterized MESE receptors are MESENI (also known as EP-1), Her MESEN2 (also known as COMA, and RI/K-1 for the murine homologue). The specificity of each receptor for each member of the MESE family is different, but MESE-A binds to both RI-1 and KOA. The full-length PI-1 receptor includes an extracellular domain that has seven Id domains, a transmembrane domain, and an intracellular domain with tyrosine kinase activity. The extracellular domain is involved in the binding of MESE, and the intracellular domain is involved in signal transduction.

MESE receptor molecules or fragments thereof that specifically bind to MESE can be used in the methods of the present invention to bind and exclude the MESE protein, thereby preventing its signaling function. In certain embodiments, the MESE receptor molecule or its MESE-binding fragment has a soluble form, such as 5E-1. The soluble form of the receptor exerts an inhibitory effect on the biological activity of the MESE protein, thereby preventing its binding to natural receptors present on the surface of target cells. Also included in this definition are MESE receptor fusion proteins, examples of which will be given below.

A chimeric MESEE receptor protein is a receptor molecule having amino acid sequences derived from at least two different proteins, at least one of which is a MESEE receptor protein (eg, I-1 or

KOA receptor), capable of binding to MES and suppressing its biological activity. In certain embodiments, chimeric MESEE receptor proteins of the present invention consist of amino acid sequences derived only from two different MESEE receptor molecules; however, amino acid sequences containing one, two, three, four, five, six, or all seven Id-like domains from the extracellular region of the MP-1 receptor and/or the ligand-binding KOH can be linked to amino acid sequences from other unrelated proteins, for example with immunoglobulin sequences. Other amino acid sequences with which the Id-like domains are combined will be readily apparent to those skilled in the art. Examples of chimeric receptor proteins

MEs include, for example, soluble EI-1/Ps, KOV/Ps or EK-1/KOV/Es (also known as MESE Tgar). (See, for example, the publication of the patent application PCT Me MUO97/44453).

Soluble MEC receptor protein or chimeric receptor proteins of the present invention include MEC receptor proteins that are not anchored to the cell surface via a transmembrane domain. As such, soluble forms of the MES receptor, including chimeric receptor proteins, although able to bind to and inactivate MESIE, do not contain a transmembrane domain, and therefore do not become associated with the membrane of cells that express this molecule.

S. Ribozymes

Ribozymes are enzymatic RNA molecules capable of catalyzing specific RNA cleavage. Ribozymes act by sequence-specific hybridization to a complementary RNA target followed by endonucleotic cleavage. Specific ribozyme cleavage sites in a potential RNA target can be identified by known methods. For more detailed information see, for example, Nozvi, Sigtepi

Vioiyudu, 4:469-471 (1994) and publication of patent application PCT Mo UMO 97/33551. One exemplary ribozyme that targets MESE expression is Apdiolgute. (See, e.g., US Patent Application Publication No. 20060035278.)

Or. Aptamers

Aptamers are nucleic acid molecules that form tertiary structures that specifically bind to a target molecule, such as the MESE polypeptide. The production and therapeutic use of aptamers is well established in the art. See, for example, US Patent No. 5,475,096. The MESE aptamer is a pegylated modified oligonucleotide that adopts a three-dimensional conformation that enables it to bind to extracellular MESE. One example of a therapeutically effective aptamer targeting MESE that has found use in the treatment of age-related macular degeneration is pegaptanib (Masidep"Mm, Eueiesp, Mem/Hogk). Additional information on aptamers can be found in US Patent Application Publication No. 20060148748.

E. Peptitila

Peptide is a peptide sequence linked to an amino acid sequence encoding a fragment or part of an immunoglobulin molecule. Polypeptides can be derived from randomized sequences selected by any method for specific binding, including, but not limited to, phage display technology. In one embodiment, the selected polypeptide can be linked to the amino acid sequence encoding the EC part of the immunoglobulin. Peptides that specifically bind to and antagonize MES may also find use in the methods of this invention.

ER. MEVE-specific antagonistic nucleic acid molecules

The methods of this invention also relate to the use of MESiE-specific antagonistic nucleic acid molecules, including antisense oligomers with a nucleic base and small RNAs.

In one embodiment, the present invention relates to the use of nucleic acid-based antisense oligomers targeting MES RNA. By binding to a complementary nucleic acid sequence (sense or coding strand), antisense oligomers with a nucleic base are able to inhibit protein expression, possibly through enzymatic cleavage of this RNA strand by RNase H.

One example of an antisense nucleic acid oligomer particularly useful in the methods and compositions of the present invention is a morpholino oligomer. Morpholinos are used to block the access of other molecules to specific sequences within nucleic acid molecules. They can block the access of other molecules to small (725 bases) areas of ribonucleic acid (RNA). Morpholino is sometimes called PMO, which is an abbreviation of phosphorodiamidate morpholino oligo.

Morpholines are used to disrupt gene function by preventing cells from producing a target protein or modifying pre-mRNA splicing. Morpholins are synthetic molecules that bind to complementary RNA sequences through standard nucleic acid base pairing (according to the principle of complementarity). Although morpholines have standard nucleic acid bases, these bases are linked to morpholine rings instead of deoxyribose rings, and not through phosphates, but through phosphorodiamidate groups. Substitution of anionic phosphates by uncharged phosphorodiamidate groups prevents ionization in the normal physiological pH range, so that morpholines in organisms and cells are uncharged molecules.

Morpholines work by "space-locking" or binding to a target sequence in RNA and blocking molecules that could interact with the RNA. Due to their completely unnatural structure, morpholines are not recognized by cellular proteins. Nucleases do not destroy morpholines, and morpholines do not activate IyI-like receptors (receptors of the immune system, similar to the receptor protein of the fruit fly

Ogozorpya - approx. trans.), so that they do not activate genetically determined immune responses, such as the interferon system or ME-kV-mediated inflammatory response. It is also not known that morpholines modify DNA methylation. Therefore, morpholines that target any part of the MESE that can reduce or inhibit the expression levels or biological activity of the MESE are particularly useful in the methods and compositions of the present invention.

The present invention also relates to the use of RNA interference (RNAI) to inhibit the expression of MES.

AMAi is a form of post-transcriptional gene silencing initiated by the introduction of double-stranded RNA (y58MA).

Short (from 15 to 32 nucleotides) double-stranded RNAs, commonly known as "5ikMA", "small RNAs" or "microRNAs", are effective in down-regulating gene expression in nematodes (7atoge ei ai., Seii! 101: 25-33) and in mammalian tissue culture cell lines. Further therapeutic efficacy of this approach in mammals will be demonstrated by Mesaigue et al. (Mayige 418:38-39. 2002). Small RNAs have at least 15 nucleotides, preferably 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, in length and even up to 50 or 100 nucleotides in length (including all insertions in between). Such small RNAs that are substantially identical or complementary to any region of MESE are included in the list of MESE-specific antagonists of the present invention.

The specific requirements and modifications of small RNA are known to specialists in this field and are described, for example, in PCT patent application publication MoU UUO01/75164 and in US patent application publications MoMo 20060134787, 20050153918, 20050058982, 20050037988 and 20040203145, the relevant parts of which are considered to be included in this description. by link In specific embodiments, azAMA can be synthesized or generated by processing longer double-stranded RNAs, for example in the presence of a Dicer enzyme under conditions in which azAMA is converted into RNA molecules of about 17 to about 26 nucleotides in length. ziiMAs can also be obtained by expression of a suitable DNA fragment (for example, a hairpin DNA construct). In general, ZyMA has 2- or 3-nucleotide 3' cantilever ends, which are preferably (2'i-deoxy)thymidine or uracil. viviMAs typically contain a 33' hydroxyl group. In certain embodiments, single-stranded zIMA or a5AMA with blunt ends are used. In order to further enhance the stability of the RNA, the 3" cantilevers are stabilized against degradation. In one embodiment, the RNA is stabilized by the inclusion of purine nucleotides such as adenosine or guanosine. Alternatively, substitution of pyrimidine nucleotides with modified analogues is allowed, for example, substitution of uridine 2-nucleotide cantilevers by (2 '-deoxy) thymine, which does not affect the effectiveness of AMAI. The absence of the 2" hydroxyl group significantly increases the resistance of the console to the action of nuclease in the tissue culture medium.

zivMA molecules can be obtained by various protocols, including chemical synthesis or recombinant production using the Ogozorpia ip migo system. They can be purchased from companies such as Onagtasop

Vezeagsi Ips. or Hegadop Ips., or synthesize using industrial production kits such as

Ziiepseg"M vivMA Sopvigisiyop Kii from Atriop (catalogue number 1620) or Nibspre"m AMAI Tgapzsepriip Kii from

Meme Epdiapa Vio! arz (catalogue number E2O00O5).

Alternatively, vIMA can be prepared using standard RNA transcription and O5AMA renaturation techniques, such as those described in EIBazpig et al. (bdepe5 5 YuOeem. 15:188-200, 2001), Sigaga eyi a. (Mashge 442:199-202 (2006)), Agamip Yei a. (Mashgte 442:203-207 (2006)), Ssimpa ei a. (sepe5 YuOeum. 20:1709-1714 (2006)) and I ay eyi a!. (Zsiepse 313:363-367 (2006)).

Short hairpin RNAs (spAMAZv) as described in Mi ei ai. (Rhos. Maiy!. Asad. 5si OBA, 99:6047-6052, 2002) or

Radaizop eyi a. (Sepev 4 JuOem, 16:948-958, 2002), may also find use in the methods of this invention.

ZPAMA are designed in such a way that both sense and antisense strands are included in one RNA molecule and connected by a loop of nucleotides (3 or more). 56pIAMA can be synthesized and purified using standard IP migo T7 transcriptional synthesis as described above, as well as in Mi ei ai., zirga. ZPAMA can also be subcloned into an expression vector having murine Ob promoter sequences, which can then be introduced into cells and used to overexpress ZPAMA.

There are many methods for transfecting or introducing a5STRAIN into mammalian cells. For example, the industry produces several transfection reagents that are suitable for transfection of lipid-based mAbs, including, but not limited to, Tgap5iIt-TKO"m (Migy5, MIV catalog number 2150), Ttsapetezzepdeg"m (diadep, catalog number 301525 ), OiIidogGesiatipe "M apa Giroiesiatipe "m (Ipiygodep, catalog number MIV 12252-011 and catalog number 13778-075), viROKT "m (Atrbriop, catalog number 1631), OpappagESTt m (Rizpeg Zsiepiys, catalog number T -2001-01).The industry also produces preparations for electroporation-based (electropulse opening of cell pores) zivMA transfection methods, such as in ROKTeg"m (Atrbriop Ips. catalog number 1629). Microinjection methods can also be used. A small RNA can also be transcribed from an expression element introduced into cells, when this expression element includes a coding sequence for transcription of a small RNA operably linked to one or more transcriptional regulatory sequences. When desired, plasmids, vectors, or viral vectors may also be used to deliver a5zAMA or zyMA, and such vectors are known to those skilled in the art. Protocols for each transfection reagent are available from the manufacturer.

IM. Therapeutic applications

Despite extensive information regarding the role of MESEE in angiogenesis, relatively little is known about the role of MESEE in benign, precancerous, or non-metastatic cancers or in the adjuvant or neoadjuvant setting. We found that MESE-specific antagonists can be used in the treatment of benign, precancerous or non-metastatic types of cancer; in the treatment of dormant tumors or micrometastases; to prevent recurrence or regrowth of tumors; or to treat or prevent cancer in a subject at risk of developing it. MESE-specific antagonists can also be used for adjuvant therapy for the treatment of a subject with non-metastatic cancer, following radical surgery, or for neoadjuvant therapy for the treatment of a subject with operable cancer, when such therapy is contemplated before surgical removal of operable cancer. cancer in the subject. Although therapeutic applications are further divided into therapy, prophylaxis, adjuvant therapy, and neoadjuvant therapy, it should be understood by those skilled in the art that these categories are not necessarily mutually exclusive.

A. Classification of tumors

The term cancer encompasses a group of proliferative disorders that include, but are not limited to, precancerous growths, benign tumors, malignant tumors, and dormant tumors. Benign tumors remain localized to their site of origin and do not have the ability to infiltrate, invade distant sites, or metastasize to distant sites. Malignant tumors will invade and damage other tissues around them. They can also acquire the ability to break away from their original site and spread to other parts of the body (metastasize), usually with the bloodstream or through the lymphatic system to where the lymph nodes are located.

Dormant tumors are quiescent tumors in which tumor cells are present, but tumor progression is not clinically evident.

Primary tumors are classified by the type of tissue from which they originate; metastatic tumors are classified by the type of tissue from which the cancer cells originate. Over time, the cells of a malignant tumor become more and more abnormal and look less like normal cells. This change in the appearance of cancer cells is called the grade of the cancer, and cancer cells are described as well differentiated (low grade), moderately differentiated, poorly differentiated, or undifferentiated (high grade). Well-differentiated cells have a completely normal appearance and are similar to the normal cells from which they originate. Undifferentiated cells are cells that have become so abnormal that the ability to determine the origin of these cells is lost.

Cancer staging systems describe how far the cancer has spread anatomically and attempt to group patients with a similar prognosis and treatment into the same stage. Several tests may be done to help determine the stage of the cancer, including a biopsy and certain other imaging studies, such as a chest X-ray, mammogram, bone scan, CT scan, and MRI scan. Blood tests and clinical examination are also used to assess the general health of the patient and determine whether the cancer has spread to certain organs.

To stage a cancer, the American Joint Committee on Cancer first assigns cancer, particularly solid tumors, a letter category according to the TMM classification system. Cancer types are indicated by the letter T (tumor size), M (palpable nodes), and/or M (metastases). T1, T2, TZ and T4 describe the increasing size of the primary lesion; MO, M1, M2, MZ indicate a gradual increase in node damage; MO and M! reflect the absence or presence of distant metastases.

According to the second method of cancer staging, also known as General Staging or Roman Numeral Staging, cancer is divided into stages from 0 to IM, taking into account the size of the primary lesion, as well as the presence of spread to nodes and distant metastases. According to this system, all cases are grouped into four stages, which are indicated by Roman numerals from | to MI, or are classified as "recurrent". For certain types of cancer, stage 0 means "low" or "Ti," such as low-grade ductal carcinoma or high-grade lobular carcinoma for breast cancer. High-grade adenomas can also be classified as stage 0. In general, stage | cancer are small, localized tumors that are usually curable, while stage MI usually represents unresectable or metastatic cancer. Stage I cancer! and AND are usually locally advanced and/or demonstrate spread to local lymph nodes. In general, the higher the stage number, the more extensive the disease, including the larger size of the tumor and/or the spread of the cancer to nearby lymph nodes and/or organs adjacent to the primary tumor. These stages are precisely defined, but the definitions are different for each species cancer and are known to those skilled in the art.

Many cancer registries, such as the Surveillance, Epidemiology and Outcomes Program (5EEV)

of the National Cancer Institute of the United States, use a cumulative division in stage. This system is used for all types of cancer. She divides all cases of cancer into five main categories:

Ip vish is an early stage of cancer that is present only in the layer of cells in which it started.

Localized is a cancer that is limited to the organ in which it started, with no signs of spread.

Regional is a cancer that has spread beyond the initial (primary) site to nearby lymph nodes or organs and tissues.

Distal is cancer that has spread from the primary site to distant organs or distant lymph nodes.

Unknown - this term is used when there is not enough information to determine the stage.

In addition, it is common for cancer to return months or years after the original tumor is removed. Cancer that returns after eradication of the entire visible tumor is called recurrent disease. Disease that returns at the site of the primary tumor is locally recurrent, and disease that returns as metastases is called distal recurrence. A dormant tumor is a tumor in a quiescent state in which tumor cells are present, but tumor progression is not clinically evident. Micrometastases are small metastases or some number of cells that have spread from the primary tumor to other parts of the body.

Micrometastases may or may not be detected by screening or diagnostic testing.

The methods of the present invention are useful for preventing the occurrence of dormant tumors, or micrometastases, or tumor recurrence, for example, in a setting where a dormant tumor or micrometastasis is present but may not always be clinically detectable.

The methods of the present invention are also useful for the treatment of early-stage cancers, including, but not limited to, benign, precancerous or non-metastatic tumors. This includes any tumor stage

O, I or II, any non-metastatic stage II tumor, any condition that typically precedes cancer or develops into cancer, including but not limited to dysplasia, and any tumor that remains localized at the site of origin without infiltration, invasion of distant sites, or metastasis to distant sites. Examples of benign, precancerous or non-metastatic tumors include polyp, adenoma, fibroma, lipoma, gastrinoma, insulinoma, chondroma, osteoma, hemangioma, lymphangioma, meningioma, leiomyoma, rhabdomyoma, squamous cell papilloma, acoustic neuroma, neurofibroma, bile duct cystoma, mesothelioma, teratoma , myxomas, trachomas, granulomas, hamartoma, transitional cell papilloma, pleomorphic salivary gland adenoma, desmoid tumor, dermoid cystpapilloma, cystadenoma, focal nodular hyperplasia and nodular regenerative hyperplasia.

Since angiogenesis is involved in both primary tumor growth and metastasis, the anti-angiogenic treatment provided by the present invention is capable of suppressing neoplastic growth of the tumor at the primary site, as well as preventing tumor metastasis at secondary sites, thereby opening the front to attack the tumor by other therapeutic means. Examples of cancers that are treated with the methods and compositions of this invention include both solid and non-solid or soft tissue tumors. Examples include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, women's cancer external genitalia, thyroid cancer, liver carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low-grade/follicular non-Hodgkin lymphoma (NHL), intermediate/follicular MNL, intermediate-grade diffuse MNL, high-grade immunoblastic MNI, high-grade lymphoblastic MNI, high-grade small-cell MNI with unsplit nuclei, massive ur agitating

MNI., lymphoma from the cells of the mantle zone, lymphoma associated with AIDS, Waldenström's macroglobulinemia, chronic lymphocytic leukemia (CL/), acute lymphoblastic leukemia (AL/), hairy cell leukemia, chronic myeloblastic leukemia, post-transplantation lymphoproliferative disorder ( RTIO), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as edema associated with brain tumors), and Meigs syndrome. More specifically, cancers amenable to treatment with the MECE-specific antagonists of the present invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), kidney cancer, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, brain tumors, gliomas (eg, anaplastic astrocytoma, anaplastic oligoastrocytoma, anaplastic oligodendroglioma, glioblastoma multiforme), melanoma, ovarian cancer, mesothelioma, and multiple myeloma

Preferably, the methods of the present invention are used to treat colorectal cancer in humans.

B. Adenomas

In one embodiment, the methods of the present invention are used to treat benign epithelial cells known as adenomas. Adenoma is a benign tumor of glandular origin. Adenomas typically originate from epithelial cells used for secretion. Epithelial cells are distributed throughout the body, but only a subset of such cells are used for secretion.

Epithelial cells used for secretion make up specific parts of the body called glands. The function of the glands in the body is to produce a number of substances, including but not limited to sweat, saliva, breast milk, mucus, and hormones. Adenoma can form from most glandular cells in the body.

Adenoma can form like a malignant or cancerous tumor. Adenomas are benign, and therefore do not metastasize or spread to other organs or tissues. Although most adenomas remain benign, some adenomas develop into malignancy, and when this happens, the malignant adenoma is called an adenocarcinoma. For example, colon and rectal cancer may begin as an adenoma or polyp, and later develop into adenocarcinoma; bronchial adenomas can develop into lung cancer.

Often, adenomas have a noticeable effect on the organs or tissue of the gland in which they develop. Adenomas often secrete excessive levels of hormones. When this happens, the consequences can be quite desperate for the affected individual. In certain situations, these consequences can be life-threatening. However, some adenomas develop without any noticeable effects.

There are certain types of adenomas that are more common in women, such as liver adenomas. Others, such as colon adenomas, are most common in older adults, such as those in their fifties. In addition, there are factors that make the patient prone to the development of adenoma. For example, women who use oral contraceptives may have an increased risk of liver adenoma. Certain types of adenoma,

for example colon adenoma, can be inherited.

Symptoms associated with adenomas vary widely. For example, a breast adenoma, called a fibroadenoma, typically does not cause symptoms and may be so small that the affected individual is unable to detect it. However, other breast adenomas can be quite large and palpable. On the other hand, pulmonary adenoma can cause fever, chills, shortness of breath and coughing up blood.

Adenomas are diagnosed by various methods, including taking blood and urine samples, ultrasound, computed tomography (CT), and nuclear magnetic resonance (NMR). A biopsy is typically used to determine whether a tumor is benign or malignant. The methods of the present invention are particularly useful for the treatment of adenoma, the treatment and prevention of adenoma recurrence, for example in a subject having a dormant tumor or micrometastases, or for preventing the development of adenoma in a subject having any of the following risk factors: associated with adenoma.

S. Gastrointestinal tumors

In another embodiment, the methods of the present invention are used to treat benign, precancerous, non-metastatic or dormant gastrointestinal tumors or micrometastases from a gastrointestinal tumor. This includes any stage 0, I or II tumor; any non-metastatic stage I tumor!; any condition that typically precedes a gastrointestinal tumor or develops into a gastrointestinal tumor that remains localized at the site of origin without infiltration, invasion of distant sites, or metastasis to distant sites. Gastrointestinal tumors include any polyp, adenoma, tumor, or cancer of the digestive system, including the esophagus, stomach, liver, biliary tract (gall bladder, bile ducts, ampulla of pharynx), intestine, pancreas, colon, rectum, and anus. . They will be described in more detail below. (Y) Cancer of the anal canal

Anal cancer is a malignant tumor of the anal canal or the edge of the anus. Anal cancers often begin as anal dysplasia. Anal dysplasia consists of cells in the anus that have undergone abnormal changes but show no signs of invasion into surrounding tissue. The most severe form of anal dysplasia is carcinoma of the viscera, when the cells look like cancer cells, but do not go beyond the place where normal cells lie. Over time, anal dysplasia changes to the point where the cells become invasive and gain the ability to metastasize or travel to other parts of the body. Anal dysplasia is sometimes called anal intraepithelial neoplasia (AIM). When anal cancer does spread, it is usually through direct invasion into surrounding tissues or through the lymphatic system. The spread of anal cancer through blood is less common, although it can occasionally occur.

Several factors have been linked to the development of anal cancer. Most importantly, an association between human papillomavirus (HPV) and anal cancer and some other cancers, including cervical cancer and head and neck cancer, has been shown. Another sexually transmitted virus, the human immunodeficiency virus (HIV), has been associated with anal cancer, and HIV-infected individuals are at increased risk for human papillomavirus infection. Because anal cancer begins as anal dysplasia before progressing to anal cancer, patients with AIM appear to be at increased risk of developing anal cancer. In addition, anal cancer occurs with increased frequency in patients who have such benign anal conditions as anal fistulas, anal fissures, perianal abscesses, and hemorrhoids.

The methods of the present invention are particularly useful for treating early-stage anal cancer, treating or preventing recurrence of anal cancer, for example, in a subject having a dormant tumor or micrometastases, or for preventing the development of anal cancer in a subject having any which of the risk factors associated with anal cancer. (u) Colorectal cancer and rectal cancer

The colon is the longest part of the large intestine, also known as the colon. Colon cancer is the third most common cancer in both men and women in America. Colon cancer rates are highest among African Americans, who are also more likely to die from the disease.

The risk of developing colon cancer increases significantly after the age of 50, but many cases are reported among younger people each year. In general, colon cancer and rectal cancer form the same group and share the same risk factors. Individuals with a personal or family history of colon cancer, polyps, or hereditary colon cancer syndromes (eg, EAR and NMRCC), as well as patients with ulcerative colitis or Crohn's disease, are all at increased risk and may require earlier screening aged than the general population. A person with one first-degree relative (a parent, sibling, or child) with colon cancer is 2 to 3 times more likely to develop the cancer than a person without such an affected relative.

Colon cancer can be diagnosed by various methods well known to clinicians. Screening tests are the most effective way to diagnose colon cancer in its early stages (such as polyps or adenomas) because these stages are often not associated with any symptoms. Over time, a polyp can grow into a tumor that can bleed or block the colon, causing symptoms. These symptoms include bleeding from the rectum, blood in the stool or in the toilet after a bowel movement, a change in the shape of the stool (ie, thinning), sharp abdominal pain, and feeling the need to have a bowel movement when there is no need. In the case of tumors and polyps that bleed occasionally, blood can be detected in stool samples using an occult blood test (OCT). By itself, EOVT detects about 2,495 cases of cancer.

In general, colorectal cancer is divided into stages as follows:

Stage 0 (also called colon carcinoma) - the cancer is limited to the outermost part of the colon wall

Stage | - cancer that has spread to the second and third layers of the colon wall, but not to the outer wall of the colon or beyond it. This stage is also called Dukes A colon cancer.

Stage II is cancer that has spread through the wall of the colon, but has not yet invaded any of the lymph nodes (small structures that help fight infection or disease). This stage is also called Dukes B colon cancer.

Stage I is cancer that has spread through the wall of the colon, reached the lymph nodes, but has not yet spread to other parts of the body. This stage is also called Dukes C colon cancer.

Stage MI is cancer that has spread to other parts of the body (for example, the liver and lungs). This stage is also called Dukes Yu's colon cancer.

The methods of the present invention are particularly useful in the treatment of early stages of colorectal cancer, in the treatment or prevention of recurrence of colorectal cancer, such as in a subject having a dormant tumor or micrometastases, or in the prevention of the development of colorectal cancer in a subject having any of the risk factors for the development of colorectal cancer, as they were previously described. (iii) Esophageal cancer

The esophagus is a muscular tube that connects the throat to the stomach. In the vast majority of cases, esophageal cancer develops from the inner lining (mucosal) of the esophagus, and not from the muscle or cartilage cells that make up the rest of the esophagus. The lining of the esophagus is somewhat unique in that it changes direction from the throat to the stomach. In the upper (proximal) esophagus, the lining resembles the lining of the throat and consists of squamous cells. Accordingly, when cancer develops in this area, it is usually squamous cell carcinoma. In the lower (distal) esophagus, the more common type of cancer is adenocarcinoma.

In addition to invasive types of cancer, patients are sometimes diagnosed with precancerous lesions, which are called IP zysh carcinoma. Such precancerous lesions can be seen before the development of squamous cell carcinoma or adenocarcinoma. Esophageal carcinoma occurs when the lining of the esophagus undergoes cancerous changes without any invasion into deeper tissues. Accordingly, although the cells themselves have cancer-like qualities, there is no risk of spread because no invasion occurs. Another type of lesion that is considered a precursor to cancer itself is Barrett's esophagus, which will be discussed in more detail below.

Esophageal cancer affects approximately 13,500 Americans each year, causing 12,500 deaths. Most patients are diagnosed after 50 or 60 years of age, and men are about 4 times more likely. In the past, the vast majority (-8595) of diagnosed esophageal cancer was squamous cell carcinoma, which occurs in the upper esophagus. Risk factors for this type of cancer include smoking and alcohol consumption. Although these factors are thought to be independent (with smoking being more important), there appears to be a synergistic effect between the two factors in the development of esophageal cancer. Other potential carcinogens for the development of esophageal squamous cell carcinoma are nitrosamines, asbestos fibers, and petroleum products.

A different situation is observed in a group of patients with a risk of developing adenocarcinoma, usually of the lower part of the esophagus. Adenocarcinoma used to be a less common disease than squamous cell carcinoma.

However, it has now become even more common than squamous cell carcinoma. In general, adenocarcinoma is thought to arise in the setting of Barrett's esophagus, which is a condition where the normal lining of the esophagus is replaced by a lining that resembles the stomach. Barrett's esophagus is diagnosed with an endoscopy, where a fiberoptic camera is used to look into the esophagus and take a sample for biopsy. Barrett's esophagus is thought to develop as a result of the chronic action of stomach acid on the lower esophagus. This occurs in patients with gastroesophageal reflux disease (GERD), which causes symptoms such as heartburn, bloating, loss of appetite, or stomach pain when eating or at night when sleeping.

Patients with SENO have an increased risk of developing Barrett's esophagus and, accordingly, an increased risk of developing esophageal adenocarcinoma.

Although Barrett's esophagus, by definition, occurs when the lining of the esophagus becomes abnormal, varying degrees of these abnormalities can occur. They are scored for dysplasia, which is used to determine the likelihood of Barrett's esophagus progressing to cancer. Patients with Barrett's esophagus and high-grade dysplasia should be examined endoscopically every 3 months or treated, as these changes are considered precancerous and have a high probability of progression to cancer. The most sensitive test for documenting localized esophageal cancer or dysplasia is endoscopy. During endoscopy, the suspicious area of the esophagus can be seen directly with the help of a fiber-optic camera, as well as the location of abnormal changes, the presence or absence of bleeding, and the degree of obstruction.

Laryngoscopy or bronchoscopy may also be necessary depending on the location and degree of esophageal cancer. Today's standard of medical care also includes performing an ultrasound during endoscopy, i.e. endoscopic ultrasound (OB). CT scans, barium swallow tests, x-rays, and other, more routine tests, including blood screening tests, are typically done to make a proper diagnosis and stage the cancer.

The methods of the present invention are particularly useful for treating early-stage esophageal cancer, for treating or preventing recurrence of esophageal cancer, for example, in a subject who has a dormant tumor or micrometastases, or for preventing esophageal cancer in a subject who has any from the risk factors associated with the development of esophageal cancer, such as those described earlier. (m) Cancer of the gallbladder

The gallbladder is a small pear-shaped organ that stores and concentrates bile. The gallbladder and liver are connected by the bile duct. Primary gallbladder cancer affects about 6,000 adults in the United States each year.

In most cases, these are adenocarcinomas of such subtypes as papillary, nodular and tubular (tubular), depending on the appearance of tumor cells under a microscope. Less common subtypes include squamous cell, signet ring cell, and adenosquamous (adenoacanthoma) adenocarcinoma.

Gallbladder cancer is most common in elderly patients with an average age at diagnosis of 62-66 years. It affects women more often - the ratio of women to men is about 3:1.

The cause of gallbladder cancer is unknown, although it has been associated with gallstones, high estrogen levels, cigarette smoking, alcohol, obesity, and female sex. In addition, patients with inflammatory bowel disease (ulcerative colitis and Crohn's disease) are 10 times more likely to develop extrahepatic biliary tract cancer.

In general, gallbladder cancer is diagnosed through history, physical examination, and laboratory tests that include metabolic chemistry and liver function tests to detect abnormal levels of various substances in the blood that may indicate underlying hepatobiliary disease. A urinalysis to assess urinary levels of some of these substances is also usually provided. Additional methods can be used, such as ultrasound,

MRI, cholangiography and CT scan.

The methods of the present invention are particularly useful for the treatment of early-stage gallbladder cancer, for the treatment or prevention of recurrence of gallbladder cancer, such as in a subject who has a dormant tumor or micrometastases, or for the prevention of gallbladder cancer in a subject who has any of the risk factors associated with the development of gallbladder cancer, such as those described earlier. (y) Stomach cancer

In the United States, stomach cancer is now the 14th most common type of cancer. Stomach cancer can rarely be found before the age of 40, and after this age its frequency increases.

Over 9,095 cases of stomach cancer arise from the lining of the stomach. Because this lining has glands, cancer originating from it is called an adenoma, and more advanced forms are called adenocarcinoma. Although there are other types of cancer that can arise in the stomach (lymphoma - from lymphatic tissue, leiomyosarcoma - from muscle tissue, squamous cell carcinoma - from lining without glands), the vast majority are adenocarcinomas.

Studies have linked Neiijsobasieg rugi infection with stomach cancer. N. ruyugi is associated with gastric ulcers and chronic atrophic gastritis, which may explain the high incidence of gastric cancer in patients infected

N. ruogii. However, the exact role of N. ruyogi in the development of gastric cancer remains unclear.

A variety of tests are used to accurately identify the type of stomach cancer, including double-contrast X-rays and upper gastric endoscopy. Other methods are used to diagnose stomach cancer, such as CT scan, PET scan and laparoscopy.

The methods of the present invention are particularly useful for the treatment of early-stage gastric cancer, for the treatment or prevention of recurrence of gastric cancer, for example, in a subject who has a dormant tumor or micrometastases, or for the prevention of gastric cancer in a subject who has any which of the risk factors associated with the development of stomach cancer, such as those described earlier. (mi) Liver cancer

There are a number of benign liver tumors. Hemangiomas are the most common benign liver tumors, when a blood-filled tumor forms inside the liver. Other benign tumors include adenomas and focal nodular hyperplasia. Although these tumors do not affect the surrounding tissues and do not metastasize, it is often difficult to tell the difference between benign and malignant tumors from radiographs.

Hepatocellular carcinoma (HCC), a cancer arising from hepatocytes, is the most common type of primary liver cancer, accounting for about 7,095 of all liver cancers. Cancer that arises from the bile ducts in the liver is known as cholangiocarcinoma; its fate is 10-2095 of all types of liver cancer. Such cancer can arise from the bile ducts inside the liver (then this is intrahepatic cholangiocarcinoma) or from the bile ducts that leave the liver (then this is extrahepatic cholangiocarcinoma). Other rare types of cancer can also develop in the liver. These include hemangiosarcomas (malignant, blood-filled tumors) and hepatoblastomas (a rare cancer that develops in very young children).

There are a number of risk factors associated with liver cancer. In the United States, the most common risk factor for liver cancer is cirrhosis. Chronic hepatitis C virus (HCV) infection is also a common cause of liver cancer in the United States. In the rest of the world, other risk factors are more common, such as chronic hepatitis B virus (HBV) infection and food contamination with aflatoxin B1.

Several screening tests are used to detect liver cancer. One potential screening test involves detecting blood levels of alpha-fetoprotein (AFP). AFP is a protein that is found in high concentrations in fetal blood, but usually disappears after birth. The level of AFP increases in the presence of hepatocellular carcinoma and can serve as a marker for the development of liver cancer. Although some patients at high risk of developing liver cancer are routinely tested for AFP levels, not all types of liver cancer produce high levels of AFP in the blood, and by the time most patients have high levels of AFP, their tumor is already at an advanced stage. Other blood proteins can also be used as a screening tool for liver cancer.

Several studies have shown that the proteins des-gamma-carboxy prothrombin (OSP) and agglutinin-reactive fraction of GI epv cytophages (AER-I 3) can also be used as markers of liver cancer formation; however, they are rarely used in practice.

In addition, when liver cancer is suspected, ultrasound examination, CT scan, NMR scan, angiography, fluorodeoxyglucose-positron emission tomography (EOS-PET), biopsy and exploratory laparotomy are performed to clarify the diagnosis and establish the stage of liver cancer.

The methods of the present invention are particularly useful for the treatment of early-stage liver cancer, for the treatment or prevention of recurrence of liver cancer, for example, in a subject who has a dormant tumor or micrometastases, or for the prevention of liver cancer in a subject who has any which of the risk factors associated with the development of liver cancer, such as those described earlier. (my) Pancreatic cancer

The pancreas is a pear-shaped gland about six inches long, located deep in the abdomen, between the stomach and the spine. It is divided into three parts: the widest part is called the head, the middle part is the body, and the thin end is the tail. The pancreas is responsible for producing hormones, including insulin, which helps regulate blood sugar, and enzymes used by the intestines to digest food. These enzymes are transported through ducts within the pancreas and enter the common bile duct, which carries them to the intestines. The incidence of pancreatic cancer is highest between the ages of 60 and 80; it is rarely observed in people under 40 years of age. Pancreatic cancer occurs at about the same rate in men and women, although the incidence in women has increased in recent years. Smoking cigarettes increases the probability of pancreatic cancer by 2-3 times. The risk triples if a mother, father, or sibling has the disease. Having a family history of breast or colon cancer also increases the risk. This is due to hereditary mutations in the genes that cause cancer. The exact cause of this disease is unknown, but it is believed to be the result of a combination of inherited genetic and environmental changes.

When a doctor suspects that a patient has pancreatic cancer, ultrasound, CT scan, and endoscopy are used to clarify the diagnosis and determine the stage of the cancer. Some patients with pancreatic cancer may have elevated levels of carbohydrate antigen 19-9 (CA 19-9). In patients who have elevated levels, it is useful to confirm the diagnosis, in combination with other tests, and to monitor the disease during treatment. This level can be checked periodically during treatment to see if the cancer is stable or getting worse.

The methods of the present invention are particularly useful for the treatment of early-stage pancreatic cancer, for the treatment or prevention of recurrence of pancreatic cancer, such as in a subject who has a dormant tumor or micrometastases, or for the prevention of pancreatic cancer in a subject who has any of the risk factors associated with the development of pancreatic cancer, such as those described previously. (mii) Cancer of the small intestine

The small intestine, also known as the small intestine, is the part of the digestive tract that connects the stomach to the large intestine. It has three distinct parts: 1) duodenum, 2) jejunum and 3) ileum. Surprisingly, despite the impressively large length of the small intestine compared to the rest of the digestive tract, small bowel cancer is very rare. It includes cases that start in that gut, or cases where it has spread here from somewhere else in the body. Small bowel cancer accounts for less than 5,905 of all bowel cancers and 0.595 of all cancers diagnosed in the United States.

The cause of most cases of small bowel cancer is unknown. However, there are certain possible risk factors that can increase the likelihood of developing small bowel cancer. Some examples include Crohn's disease, celiac sprue, Peitz-Egers syndrome, and intestinal polyposis.

There are four main types of small bowel cancer, depending on how it looks under the microscope and the cells from which it originates. The most common type is adenocarcinoma. It typically begins in the lining or inner layer of the intestine and usually occurs in the duodenum. Another type is sarcoma and its typical subtype is leiomyosarcoma, which begins in the muscular wall of the small intestine and usually occurs in the ileum.

The third type is carcinoid, which starts in special hormone-producing cells in the small intestine and usually occurs in the small intestine, sometimes in the appendix (which is the first part of the large intestine).

The fourth type is lymphoma, which begins in the lymphatic tissue of the small intestine and usually occurs in the ileum. The most typical subtype of lymphoma is non-Hodgkin's lymphoma. A less common subtype of small bowel cancer is gastrointestinal stromal tumor, which can occur in any of the three parts of the small intestine.

Small bowel cancer is usually diagnosed with a complete medical history, physical examination, stool sample examination, endoscopy or colonoscopy, CT scan, ultrasound, and X-ray examination.

The methods of the present invention are particularly useful for the treatment of early-stage small bowel cancer, for the treatment or prevention of recurrence of small bowel cancer, such as in a subject who has a dormant tumor or micrometastases, or for the prevention of small bowel cancer in a subject who has any of the risk factors associated with the development of small bowel cancer, such as those described previously.

M. Prevention

As a further aspect of the present invention, we have found that MESI-specific antagonists can be used to treat benign, precancerous or early stage cancer or to treat or prevent cancer recurrence. The methods of the present invention can be used to treat cancer itself or to prevent the progression of cancer to a metastatic or invasive stage or to a higher grade or stage. For example, they can be used to treat a subject with stage 0 cancer or polyps in order to prevent progression to a stage I or higher tumor stage. Similarly, in a patient with stage II cancer, these methods can be used to prevent progression to stage III or stage II cancer.

MES-specific antagonists can be used to prevent tumor recurrence. For example, if a tumor has been identified and treated (eg with chemotherapy or surgical removal), MESiE-specific antagonists can be used to prevent colorectal tumor recurrence either locally or colorectal tumor metastasis. To prevent tumor recurrence, MECE-specific antagonists can be used, for example, to treat a dormant tumor or micrometastases or to prevent the growth or regrowth of a dormant tumor or micrometastases that may not be clinically apparent.

We have also found that MECE-specific antagonists can be used to prevent the development of cancer in a subject who has never had cancer or is at risk of developing it. There is a wide range of risk factors associated with cancer, many of which have been described previously. Indicative risk factors include advanced age (ie, over 50 years), family history of cancer, viral infection

NRM, NIM (HIV), NVM and NOM, use of oral contraceptives, cirrhosis, ulcerative colitis, esophagus

Barrett's infection with N. ruiogi and the presence of polyps or dysplasia. In addition, when the subject has hereditary cancer syndrome, the risk of developing cancer increases. Non-limiting examples of such syndromes include ARS, NMRCOS, Gardner syndrome, and MEM1. Additional risk factors for developing cancer may be identified after clinical examination and include elevated levels of hormones or blood proteins such as RB5A (prostate cancer), CA-125 (ovarian cancer), AER (liver cancer), OSR (liver cancer) and CA 19-9 (pancreatic cancer).

E. Neoadjuvant therapy

The present invention relates to a method of neoadjuvant therapy that precedes the surgical removal of an operable cancer in a subject, such as a patient, and comprises administering to the patient (e.g., when the patient has been diagnosed with a tumor and/or cancer) an effective amount of an MESE-specific antagonist, such as an MESE antibody . Optionally, the MESIE-specific antagonist is administered in combination with at least one chemotherapeutic drug. The additional step of administering to the subject an effective amount of an MACE-specific antagonist after surgery may also be used with the neoadjuvant therapy described herein. In methods that include administering to the subject an effective amount of an MACE-specific antagonist after surgery. Any of the methods of adjuvant therapy described here can be used.

For example, one method includes treating cancer in a subject, which comprises the following steps a) a first step consisting of multiple treatment cycles, each of which involves administering to the subject an effective amount of an MECE-specific antagonist, such as bevacizumab, and optionally at least one chemotherapy drug at a defined interval, b) radical surgery to remove the cancer, and optionally c) a second stage consisting of several cycles of maintenance treatment, each of which involves administering to the subject an effective amount of an MECE-specific antagonist, such as bevacizumab, with a chemotherapeutic the drug after a certain interval or without it.

For neoadjuvant therapy, the MECE-specific antagonist can be administered in an amount and for such a time (eg, as part of a specific regimen) to reduce (eg, by 2095, 30905, 4090, 50905, 6090, 7095, 8095, 90905, 100905 or more) the number of cancer cells in the tumor, reduce the size of the tumor (for example, to enable resection), reduce the mass of the tumor, suppress (ie, reduce to some extent and/or stop) the infiltration of cancer cells into peripheral organs, reduce the density of vessels in the tumor, suppress tumor metastasis, slow down or suppress the growth of a tumor or the proliferation of its cells, reduce or prevent the growth of a dormant tumor, reduce or prevent the growth or proliferation of micrometastases, increase or prolong the VVX or ZV of a subject susceptible to the development of a tumor or a subject diagnosed with benign , precancerous or non-metastatic tumor; and/or alleviate to some extent one or more symptoms associated with cancer.

In one example, neoadjuvant therapy is administered with the goal of prolonging VVX or ZV, where VVX and VV are assessed approximately 2-5 years after the first administration of the antibody. In certain embodiments, VVH and ZV are assessed at about 3-5 years, at about 4-5 years, at least 4 years, or at least 5 years from the start of treatment or from diagnosis. Typically, the MEC-specific antagonist is a MEC antibody such as bevacizumab.

In another example, administration of the antibody and/or chemotherapy can reduce the rate of disease recurrence (cancer recurrence in the primary organ and/or distal recurrence) in a subject population by about 5095 over a 3-year period (where "about 5095" includes a range of about 4595 to about 7095), for example, reduces recurrence in the primary organ by about 5295 over a 3-year period and/or reduces distal recurrence by about 5395, compared to subjects treated with chemotherapy alone (eg, a taxoid such as paclitaxel.

A MESE-specific antagonist, e.g., an MESE antibody, is administered to a subject, e.g., a patient, according to known methods, such as intravenous administration, e.g., as a bolus or continuous infusion over a period of time, intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, by intra-articular, intrasynovial, intrathecal, oral, local or inhalation routes. Intravenous administration of the antibody is preferred.

Although a MESE-specific antagonist, such as an MESE antibody, can be administered as a single agent, a patient can optionally be treated with a combination of an MESE antibody and one or more chemotherapy drugs. In one embodiment, at least one of the chemotherapeutic agents is a taxoid. Such combined administration includes the simultaneous administration of individual drugs or a single pharmaceutical form of these drugs, as well as their sequential administration in any order, but it is better that both (or all) active compounds simultaneously realize their biological activity over a period of time. Therefore, the chemotherapeutic drug can be administered before or after the administration of an MESE-specific antagonist, such as an MESE antibody. In this embodiment, the period of time between at least one administration of a chemotherapeutic drug and at least one administration of an MESE-specific antagonist, such as an MESE antibody, is preferably about 1 month or less, and most preferably about 2 weeks or less. Alternatively, a chemotherapeutic drug and an MES-specific antagonist, such as a MES antibody, are administered to the patient simultaneously in the same dosage form or in different dosage forms. Treatment with a combination of a chemotherapy drug (eg, Taxoid) and a MESE antibody (eg, bevacizumab) can provide a synergistic, i.e. greater than the sum, therapeutic effect for the patient.

The chemotherapeutic drug, if administered, is usually administered in doses known for this purpose or optionally reduced to take into account the combined effects of the drugs or adverse side effects attributed to the administration of the antimetabolite chemotherapy drug. Preparation and dosage regimens for such chemotherapy drugs should follow the manufacturer's instructions or be determined empirically by an experienced practitioner. When the chemotherapy drug is paclitaxel, it is best given weekly (eg, at a dose of 80 mg/m) or every 3 weeks (eg, at a dose of 175 mg/m? or 135 mg/mU). Acceptable doses of docetaxel include 60 mg/m , 70 mg/m, 75 mg/m, 100 mg/m? (every 3 weeks); or 35 mg/m? or 40 mg/m? (weekly).

Various chemotherapy drugs that can be combined have been described previously. In certain embodiments of the present invention, chemotherapeutic agents for combination with an MEC-specific antagonist, such as an MEC antibody, include, but are not limited to, for example: a taxoid (including docetaxel and paclitaxel), periwinkle drugs (such as vinorelbine or vinblastine), platinum compounds (such such as carboplatin or cisplatin), an aromatase inhibitor (such as letrozole, anastrazole, or exemestane), an anti-estrogen (such as fulvestrant or tamoxifen), etoposide, thiotepa, cyclophosphamide, methotrexate, liposomal doxorubicin, pegylated liposomal doxorubicin, capecitabine, gemcitabine, a cyclooxygenase inhibitor -2 (COX-2) (eg, celecoxib) or a proteosome inhibitor (eg, ROZ42).

When an anthracycline (eg, doxorubicin or epirubicin) is administered to a subject, it is best to do so before or after the administration of an MECE-specific antagonist, such as bevacizumab. However, a modified anthracycline, such as liposomal doxorubicin (TC 0-99 (MUOSETF)), pegylated liposomal doxorubicin (SAEI UHF), or epirubicin with reduced cardiac toxicity, can be combined with a MESE-specific antagonist, such as bevacizumab.

In one embodiment of the administration regimen, the neoadjuvant therapy of the present invention comprises a first step in which a MESE-specific antagonist, such as bevacizumab, and one or more chemotherapy agents are administered to the patient in multiple neoadjuvant cycles, followed by surgery for radical tumor removal. The duration of each neoadjuvant cycle is from one to three weeks, depending on the specific treatment plan. For example, a treatment cycle can be 3 weeks, which means that patients receive one dose of chemotherapy and one dose of bevacizumab every two weeks. The entire first stage of neoadjuvant treatment may include 4-8 cycles. In certain embodiments of the present invention, neoadjuvant therapy lasts less than one year; in one version - less than 6 months preceding the operation. Depending on the type and severity of the disease, doses of the MESI-specific antagonist, such as bevacizumab, are preferably selected in the range of from 1 μg/kg to about 50 mg/kg, and preferably from about 5 mg/kg to about mg/kg, including 7, 5 mg/kg or 10 mg/kg. In certain aspects, chemotherapy regimens traditionally involve intermittent administration of high doses. In certain other aspects, chemotherapy drugs are administered in smaller doses, but more frequently, without scheduled intervals ("metronomic chemotherapy"). The progress of the treatment according to this invention is easily monitored by conventional methods and tests.

In addition to MES antibodies and a chemotherapeutic drug, the combination may also include other treatment regimens. For example, the second (third, fourth, etc.) chemotherapy drug to be administered is either a different taxoid chemotherapy drug or a non-taxoid chemotherapy drug.

For example, the second chemotherapy drug can be a taxoid (such as docetaxel or paclitaxel), a periwinkle drug (such as vinorelbine), a platinum compound (such as carboplatin or cisplatin), an antihormonal drug (such as an aromatase inhibitor or an antiestrogen), gemcitabine, capecitabine, etc. .p.

Exemplary combinations include taxoid/platinum compound, gemcitabine/taxoid, gemcitabine/vinorelbine, vinorelbine/taxoid, capecitabine/taxoid, and the like. "Cocktails" of various chemotherapeutic drugs can be administered.

Other therapeutic agents that can be combined with a MESE antibody include any or more of: another MESE antagonist or MESE receptor antagonist, such as a second anti-MESE antibody, MESE variants, soluble fragments of the MESE receptor, aptamers capable of blocking MESE or MESEV , neutralizing anti-MESE antibodies, MESEA tyrosine kinase inhibitors and any combination thereof. Other therapeutic agents suitable for combination antitumor therapy using the antibody of the present invention include antagonists of other factors involved in tumor growth, such as ESEN, Eva (also known as Neg2), EtOB3, EgOBBA, or TME. In one exemplary embodiment, the composition for the MESE-specific antagonist does not include an anti-EgpVa antibody, fragment, or derivative thereof (eg, a Negseriipb antibody). In certain embodiments of the present invention, an anti-MESE antibody can be used in combination with small molecule receptor tyrosine kinase inhibitors (SMRs) that target one or more tyrosine kinase receptors, such as MESE receptors, ECE receptors, ECE receptors, and ROSE receptors. Many therapeutic small molecule AKIs are known to those skilled in the art, including, but not limited to, vatalanib (RTK787), erlotinib (TAVSEMAF), O51I-7904, 206474 (2ASTIMAF), 206126 (АМС453), 201839, sunitinib (ZOTEMTF)), semaxanib (505416), АМО706, АСО13736, Imatinib (SIEEMESF), MI M-518, SER-701, RKOS-412, Lapatinib (25K572016), ME! SAOEF, A2O2171, sorafenib (Mekhamanf)), ХИ 880 and SNIV-265.

Suitable doses of any of the above drugs for simultaneous administration are those currently used, or may be reduced, taking into account the combined effect (synergism) of these drugs and the MESE antibody.

In addition to the above treatment regimens, the patient may be prescribed radiation therapy.

In certain embodiments of the present invention, the injected MES antibody is an intact, naked antibody. However

MES antibody can be conjugated with a cytotoxic drug. In certain embodiments, such conjugated antibody or antigen to which it binds is internalized by the cell, which provides increased therapeutic efficacy of such conjugate in killing the cancer cell to which it binds. In one embodiment, the cytotoxic drug targets or damages nucleic acid in the cancer cell. Examples of such cytotoxic drugs include maytansinoids, calicheamicins, ribonucleases, and DNA endonucleases.

MI. Adjuvant therapy

The present invention relates to a method of adjuvant therapy, which includes the administration of a subject with non-metastatic cancer, after a radical surgical intervention, MESE-specific antagonist, for example MESE antibodies.

For example, the method may include the following stages: a) a first stage consisting of several cycles of treatment, each of which involves administering to the subject an effective amount of an MECE-specific antagonist, such as bevacizumab, and optionally at least one chemotherapy drug at a specified interval; b) the second stage, consisting of several cycles of maintenance treatment, each of which involves administering to the subject an effective amount of an MECE-specific antagonist, such as bevacizumab, without a chemotherapy drug at a specified interval. At the same time, the combined first and second stages last at least 1 year after the initial postoperative treatment. In one embodiment, the first stage includes a first block of treatment cycles in which a MESE-specific antagonist, such as bevacizumab, and a first chemotherapy regimen are administered, and the first block is followed by a second block of treatment cycles in which a MESE-specific antagonist, such as bevacizumab, is administered and a second regimen chemotherapy.

For adjuvant therapy, the MECE-specific antagonist can be administered in an amount and for such a time (eg, as part of a specific treatment regimen) to reduce (eg, by 20905, 3095, 4095, 5095, 6095, 7090, 8095, 9095, 10095 or more) the number of cancer cells in the tumor; reduce the size of the tumor (for example, to enable resection); reduce tumor mass; suppress (that is, reduce to a certain extent and/or stop) the infiltration of cancer cells into peripheral organs; reduce the density of blood vessels in the tumor; suppress tumor metastasis; to slow down or suppress the growth of a tumor or the proliferation of its cells; reduce or prevent the growth of a dormant tumor; reduce or prevent the growth or proliferation of micrometastases; and/or alleviate to some extent one or more symptoms associated with cancer. In some additional embodiments, the MESE-specific antagonist can be used to prevent the occurrence or recurrence of cancer in a subject. In one example, prevention of cancer recurrence was evaluated in a population of subjects after approximately 4 years. The absence of disease recurrence was confirmed in about 8095 subjects of this population. In another example, the prevention of disease recurrence was evaluated after about 3 years. The recurrence rate was reduced by at least 5,095, compared with subjects who received chemotherapy alone.

In general, the MESI-specific antagonist is administered after a period of time sufficient for the subject to recover from surgery. This period may include the time required for the wound or surgical incision to heal, the time required to reduce the risk of dehiscence, or the time required for the subject to return to a level of health substantially similar to or better than before surgery . The period of time between the completion of radical surgery and the first administration of the MESE-specific antagonist may also include the time required to rest from the medication when the subject needs or requests an interval between treatment regimens.

In general, the period of time between the completion of radical surgery and the initiation of therapy with an MACE-specific antagonist can be less than 1 week, 1 week, 2 weeks, C weeks, 4 weeks (28 days), 5 weeks, b weeks, 7 weeks, 8 weeks, From months, 4 months, 5 months, 6 months, 7 months, From months, 9 months, 10 months, 11 months, 1 year, 2 years, From years or more. In one embodiment, the period of time between the radical surgery and the administration of the MESE-specific antagonist is greater than 2 weeks but less than 1 year.

In one example, a MESE-specific antagonist, such as an MESE antibody, is administered in an amount effective to prolong disease-free survival (DFS) or overall survival (OS), wherein DFS and OS are assessed approximately 2-5 years after the first administration of the antibody . In certain embodiments, the VVH and ZV of the subject are evaluated at about 3-5 years, at about 4-5 years, or at least 4 years, or at least 5 years after initiation of treatment or after diagnosis.

A MESE-specific antagonist, e.g., an MESE antibody, is administered to a subject, e.g., a patient, according to known methods, such as intravenous administration, e.g., as a bolus or continuous infusion over a period of time, intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, by intra-articular, intrasynovial, intrathecal, oral, local or inhalation routes. Intravenous administration of the antibody is preferable.

A MESE-specific antagonist can be administered as a single therapeutic agent. In other embodiments, the patient is treated with a combination of a MESE-specific antagonist and one or more chemotherapy drugs. In some embodiments, at least one of the chemotherapy drugs is a taxoid. Combined administration includes the simultaneous administration of individual drugs or a single pharmaceutical form of these drugs, as well as their sequential administration in any order, but it is better that both (or all) active compounds simultaneously realize their biological activity over a period of time. Therefore, the chemotherapeutic drug can be administered before or after the administration of an MESE-specific antagonist, such as an MESE antibody. In this embodiment, the time period between at least one administration of a chemotherapeutic drug and at least one administration of an MESE-specific antagonist, such as an MESE antibody, is preferably about 1 month or less, and preferably about 2 weeks or less. As an option, the chemotherapeutic drug and the MESE antibody are administered to the patient simultaneously in the same dosage form or in different dosage forms. Treatment with a combination of a chemotherapy drug (eg, Taxoid) and a MESE antibody (eg, bevacizumab) can provide a synergistic, ie greater than the sum, therapeutic effect for the patient. The chemotherapeutic drug, if administered, is usually administered in doses known for this purpose or optionally reduced to take into account the combined effects of the drugs or adverse side effects attributed to the administration of the antimetabolite chemotherapy drug. Preparation and dosage regimens for such chemotherapy drugs should follow the manufacturer's instructions or be determined empirically by an experienced practitioner. When the chemotherapy drug is paclitaxel, it is preferred that it be administered weekly (eg at a dose of 80 mg/m2) or every 3 weeks (eg at a dose of 175 mg/m2 or 135 mg/m2). Acceptable doses of docetaxel include 60 mg/m, 70 mg/m, 75 mg/m2, 100 mg/m? (every 3 weeks); or 35 mg/m? or 40 mg/m (weekly).

Various chemotherapy drugs that can be combined have been described previously. In certain embodiments of the present invention, chemotherapeutic drugs for combination with an MESE antibody include, but are not limited to, for example: a taxoid (including docetaxel and paclitaxel), periwinkle drugs (such as vinorelbine or vinblastine), platinum compounds (such as carboplatin or cisplatin), an inhibitor aromatases (such as letrozole, anastrazole, or exemestane), anti-estrogens (such as fulvestrant or tamoxifen), etoposide, thiotepa, cyclophosphamide, methotrexate, liposomal doxorubicin, pegylated liposomal doxorubicin, capecitabine, gemcitabine, cyclooxygenase-2 (COX-2) inhibitor (eg, celecoxib) or a proteosome inhibitor (eg, P5ZZ42).

When an anthracycline (eg, doxorubicin or epirubicin) is administered to a subject, it is best to do so before and/or after the administration of the MESE antibody, as provided by the protocols described in the Example below, where the anthracycline/cyclophosphamide combination was administered to the subject after surgery but before administration to him MES antibodies and taxoid. However, a modified anthracycline such as liposomal doxorubicin (TS 0-99 (MHOSETP), pegylated liposomal doxorubicin (SAEI UHF) or epirubicin with reduced cardiac toxicity can be combined with the MESE antibody.

In one administration scheme, the adjuvant therapy of the present invention comprises a first step in which an MEC-specific antagonist, e.g., an MEC antibody, and one or more chemotherapeutic agents are administered to patients as multiple treatment cycles, and a second step in which a MEC-specific antagonist, e.g. MESE antibody, used as the only drug in several cycles of maintenance therapy Each treatment cycle lasts from one to three weeks, depending on the specific treatment plan For example, a treatment cycle may include bevacizumab as a MESE-specific antagonist and last three weeks, which means that patients receive one dose of chemotherapy and one dose of bevacizumab every three weeks A treatment cycle can last two weeks, meaning patients receive one dose of chemotherapy and one dose of bevacizumab every two weeks The entire first stage of treatment can consist of 4-8 cycles During the second , the maintenance stage of bevacizumab is given once every two weeks or once every three weeks in the hall depends on the length of a particular cycle, and the total number of cycles is 30-50. In certain embodiments, adjuvant therapy lasts at least one year from the start of treatment, and the progress of the subject's condition will be observed after that time. Depending on the type and severity of the disease, the dose MESIE antibodies are preferably selected in the range of from 1 μg/kg to about 50 mg/kg, and more preferably from about mg/kg to about 15 mg/kg, including 7.5 mg/kg or 10 mg/kg In certain aspects, the regimen chemotherapy traditionally involves the intermittent administration of high doses In certain other aspects, chemotherapy drugs are administered in smaller doses, but more frequently, without scheduled intervals ("metronomic chemotherapy") The progress of treatment according to the present invention is easily monitored by conventional methods and assays

Administration of the antibody and chemotherapy can reduce the probability of disease recurrence (cancer recurrence in the primary organ and/or distant recurrence) in a subject population by about 5095 over 3 years (where "about 5095" includes a range of about 4595 to about 7095) This can be, for example, a reduction in the primary organ recurrence rate of approximately 5295 over three years and/or a reduction in the distal recurrence rate of approximately 5395 over three years, compared to subjects who received chemotherapy alone (eg, a taxoid such as paclitaxel)

This invention relates to a method of treating non-metastatic cancer in a population of non-metastatic cancer patients, which comprises administering to the patients an effective amount of an MECE-specific antagonist, such as an MECE antibody, and at least one chemotherapeutic drug after radical surgery, and evaluating the patients 4 or more years later , to confirm the absence of disease recurrence after approximately 4 years in at least about 8095 (preferably at least about 8595) of these patients. Such a population may include 3,000 or more patients.

This invention also relates to a method of reducing the probability of disease recurrence in a population of patients with non-metastatic cancer, which comprises administering to the patients an effective amount of bevacizumab and at least one chemotherapy drug after radical surgery, and according to which the probability of disease recurrence is reduced by at least 5095 over 3 years, compared to patients treated only with taxoid.

In addition to MES antibodies and a chemotherapeutic drug, the combination may also include other treatment regimens. For example, the second (third, fourth, etc.) chemotherapy drug to be administered is either a different taxoid chemotherapy drug or a non-taxoid chemotherapy drug.

For example, the second chemotherapy drug can be a taxoid (such as docetaxel or paclitaxel), a periwinkle drug (such as vinorelbine), a platinum compound (such as carboplatin or cisplatin), an antihormonal drug (such as an aromatase inhibitor or an antiestrogen), gemcitabine, capecitabine, etc. .p.

Exemplary combinations include taxoid/platinum compound, gemcitabine/taxoid, gemcitabine/vinorelbine, vinorelbine/taxoid, capecitabine/taxoid, and the like. "Cocktails" of various chemotherapeutic drugs can be administered. other therapeutics that can be combined with a MESE antibody include any or more of: another MESE antagonist or MESE receptor antagonist, such as a second anti-MESE antibody, MESE variants, soluble fragments of the MESE receptor, aptamers capable of blocking MESE or MESEV , neutralizing anti-MESE antibodies, MESEA tyrosine kinase inhibitors and any combination thereof. Other therapeutic agents suitable for combination antitumor therapy using the antibody of the present invention include antagonists of other factors involved in tumor growth, such as ESEN, Eva (also known as Neg2), EGOV3, EGBA, or TME. In one exemplary embodiment, the composition for the MESE-specific antagonist does not include an anti-EgpVa antibody, fragment, or derivative thereof (eg, a Negseriipb antibody). In certain embodiments of the present invention, an anti-MESE antibody can be used in combination with small molecule receptor tyrosine kinase inhibitors (SMRs) that target one or more tyrosine kinase receptors, such as MESE receptors, ECE receptors, ECE receptors, and ROSE receptors.

Many therapeutic small molecule AKIs are known to those skilled in the art including, but not limited to, vatalanib (RTK787), erlotinib (TAASEMAF), O51I-7904, 206474 (2ASTIMAF), 206126 (AMO453), 201839, sunitinib (FZOTEMTF), semaxanib ( 505416), AMO706, ASO13736, Imatinib (ZEEEEMESF), MISM-518, SER-701, RKS-412, Lapatinib (55K572016), ME SAOEF, A2O2171, sorafenib (MEHAMAKF)), ХИ 880 and SNIV-265.

Suitable doses of any of the above drugs for simultaneous administration are those currently used, or may be reduced, taking into account the combined effect (synergism) of these drugs and the MESE antibody.

In addition to the above treatment regimens, the patient may be prescribed radiation therapy.

In certain embodiments of the present invention, the injected MES antibody is an intact, naked antibody. However

MES antibody can be conjugated with a cytotoxic drug. In certain embodiments, such conjugated antibody or antigen to which it binds is internalized by the cell, which provides increased therapeutic efficacy of such conjugate in killing the cancer cell to which it binds. In one embodiment, the cytotoxic drug targets or damages nucleic acid in the cancer cell. Examples of such cytotoxic drugs include maytansinoids, calicheamicins, ribonucleases, and DNA endonucleases.

E. Doses, drugs and duration of treatment

Compositions based on the MESE-specific antagonist will be prepared, dosed and administered in accordance with the requirements of good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular subject being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the drug, the method of administration, the schedule of administration, and others known to the medical practitioner. The "therapeutically effective amount" of the MECE-specific antagonist to be administered is determined by the following considerations and is the minimum amount necessary to prevent, ameliorate, treat, or stabilize a benign, precancerous, or early-stage cancer; or to treat or prevent the occurrence or recurrence of a tumor, dormant tumor, or micrometastases, such as in the neoadjuvant or adjuvant setting. The MESE-specific antagonist is not necessarily, but optionally, combined with one or more drugs that are currently used to prevent or treat cancer or reduce the risk of developing cancer. The effective amount of such other drugs depends on the amount of MECE-specific antagonist present in the combination, the type of disorder or treatment, and other factors mentioned above. In general, they are used in the same doses and by the same routes of administration as before, or between 1 and 9995 of the doses used before.

Depending on the type and severity of the disease, about 1 mcg/kg to 100 mg/kg (e.g., 0.1-20 mg/kg) of the MESE-specific antagonist constitutes an approximate dose for administration to a patient, for example in one or more separate administrations or by continuous infusion. A typical daily dose would range from about 1 mcg/kg to about 100 mg/kg or more, depending on the factors listed above. Particularly preferred doses are 7.5 mg/kg, 10 mg/kg, and 15 mg/kg. In the case of repeated administration over several days or longer, depending on the condition, therapy is continued until the cancer is cured, as determined by the methods described above and known to those skilled in the art. However, other drug administration schedules may be used. In one examples when

A MES-specific antagonist is an antibody, the antibody of the present invention is administered once weekly, once every two weeks, or once every three weeks at a dose range of from about 5 mg/kg to about 15 mg/kg, including but not limited to 7.5 mg/kg or 10 mg/kg. The progress of the therapy according to this invention is easily monitored by conventional methods and analyses.

In one example, the MESE-specific antagonist is bevacizumab. For therapeutic use, bevacizumab is available in 100-mg and 400-mg single-use vials, without preservatives, yielding 4 mL and 16 mL of bevacizumab (25 mg/mL). To prepare 100 mg of the product, the recipe is as follows: 240 mg of α,α-trehalose dehydrate, 23.2 mg of sodium phosphate (monobasic, monohydrate), 4.8 mg of sodium phosphate (dibasic, anhydrous), 1.6 mg of polysorbate 20 and water for FOSHA injections. To prepare 400 mg of the product, the recipe is as follows: 960 mg of c,a-trehalose dehydrate, 92.8 mg of sodium phosphate (monobasic, monohydrate), 19.2 mg of sodium phosphate (dibasic, anhydrous), 6.4 mg of polysorbate 20 and water for FOSHA injections.

Duration of therapy is as medically indicated or until the desired therapeutic effect (eg, as described herein) is achieved. In certain embodiments, the therapy with the MESE-specific antagonist continues for 2 months, 4 months, 6 months, 8 months, 10 months, 1 year, 2 years, 3 years, 4 years, 5 years, or throughout the life of the subject.

In general, the amelioration or treatment of benign, precancerous or early stage cancer or adjuvant or neoadjuvant therapy for cancer (benign or malignant) involves alleviating one or more symptoms or one or more medical problems associated with the cancer. A therapeutically effective amount of the drug may provide one or a combination of the following: reduce (eg, by 2095, 3095, 40905, 5090, 7095, 8090, 9095, 10095, or more) the number of cancer cells in the tumor; reduce the size of the tumor; reduce tumor mass; suppress (that is, reduce to a certain extent and/or stop) the infiltration of cancer cells into peripheral organs; reduce the density of blood vessels in the tumor; suppress tumor metastasis; reduce or suppress tumor growth or tumor cell proliferation; reduce or prevent the growth of a dormant tumor; reduce or prevent the growth or proliferation of micrometastases; reduce or prevent regrowth of the tumor after treatment or removal (for example, due to adjuvant therapy); to increase or prolong the VVH or ZV of a subject prone to the development of a tumor or one who has been diagnosed with a benign, precancerous or non-metastatic or malignant tumor; reduce the size of the tumor to enable its surgical treatment (for example, due to neoadjuvant therapy); to alleviate to some extent one or more symptoms associated with cancer. In some additional embodiments, the MECE-specific antagonist can be used to prevent the occurrence or recurrence of cancer in a subject. In one example, prevention of cancer recurrence was evaluated in a population of subjects after approximately 4 years. The absence of disease recurrence was confirmed in about 8095 subjects of this population. In another example, the prevention of disease recurrence was evaluated after approximately 3 years. The recurrence rate was reduced by at least 5095, compared with subjects who received chemotherapy alone.

In one example, a MESE-specific antagonist, such as an MESE antibody, is administered in an effective amount to prolong VVX or ZV, with VVX or VV being assessed at 2-5 years after the first administration of the antibody. In certain embodiments, the subject's VVH or VV is estimated at about 3-5 years, about 4-5 years, or at least about 4 years or at least about 5 years after initiation of treatment or after diagnosis.

In one embodiment, the present invention can be used to prolong the survival of a subject prone to tumor development or diagnosed with a benign, precancerous, or non-metastatic tumor. Duration of survival is defined as the time from the first administration of the drug to death.

Duration of survival can also be estimated using the stratified hazard ratio (HR) for the treatment group versus the control group, which represents the patient's risk of death during treatment.

In yet another embodiment, the treatment of the present invention significantly increases the response rate to treatment in a group of subjects, such as cancer-prone or cancer-diagnosed patients receiving various anti-cancer therapy regimens. The response rate is defined as the percentage of patients who responded to treatment. In one aspect, the combination treatment of the present invention with an MACE-specific antagonist and surgery, radiation therapy, or one or more chemotherapy agents significantly increases the response rate compared to a group of patients treated with surgery, radiation therapy, and chemotherapy alone, and this increase has p value according to the chi-square test, less than 0.005.

Treating or preventing the occurrence or recurrence of a tumor, dormant tumor, or micrometastases involves preventing the formation of a tumor or metastases, generally after the initial treatment or removal of the tumor (eg, such anticancer therapy as surgery, chemotherapy, or radiation therapy). Surgery may leave behind residual tumor cells or dormant micrometastatic nodules that have the potential to reactivate the “angiogenic program” and enable more exponential tumor growth. Although the presence of a dormant tumor or micrometastases is not necessarily detectable during clinical studies and screening tests, a therapeutically effective amount is one that is sufficient to prevent or reduce the detection of a dormant tumor, micrometastases, metastases, or tumor recurrence by methods known to the clinician. In one example, a subject who is being treated for a tumor by surgical removal is then given a MESE-specific antagonist and is further monitored for dormant tumor, micrometastases, or tumor recurrence. The MECE-specific antagonist may be administered in combination with other anticancer therapy (eg, before or after the MECE-specific antagonist) and one or both therapies may be continued as maintenance therapy.

Additional methods for evaluating therapeutic efficacy in the treatment of cancer are described in the patent application publication

USA Me 20050186208.

Therapeutic preparations are prepared by standard methods known to specialists in this field - by mixing the active ingredient, which has the desired degree of purity, with optional, physiologically acceptable carriers, fillers or stabilizers (VKetipdiopye Rnpaptaseshiisa! Zsivepsev5 (207 eyyiop), ea. A. Seppago, 2000,

Prripsoy, UMiShatve 8 UMiKipv, RNPaade!rnia, RA). Acceptable carriers include saline or buffers such as phosphate, citrate, or other organic acid-based; antioxidants, including ascorbic acid; low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagines, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating drugs such as EOTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants, such as TM/EEM M, P HOBOMISZ5 M or REYA.

Optionally, but it is preferred, the preparation contains a pharmaceutically acceptable salt, typically, for example, sodium chloride, and preferably in an approximately physiological concentration. Optionally, the preparations of the present invention may contain a pharmaceutically acceptable preservative. In certain embodiments, the preservative concentration is from 0.1 to 2.095, typically 0/0. Acceptable preservatives include those known in the pharmaceutical industry.

Examples of preservatives are benzyl alcohol, phenol, t-cresol, methylparaben and propylparaben.

Optionally, the preparations of this invention may include a pharmaceutically acceptable surfactant in a concentration of 0.005 to 0.02905.

The preparations of this invention may contain more than one active compound as dictated by the indication being treated, preferably one that has complementary activity and does not adversely affect the other compounds. Such molecules are usually present in combination in amounts effective for the purposes for which they are intended.

The active ingredients can be placed in a microcapsule obtained, for example, by methods of coacervation or polymerization at the interface of phase separation, for example, hydroxymethylcellulose, or in a microcapsule made of gelatin or polymethyl methacrylate, in a colloidal drug delivery system (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules ) or in a macroemulsion. Such methods are described in Ketipdiopye

RPpagtaseshisai! bsiepsevz, zirga.

You can prepare preparations with a delayed release of the active compound. For this purpose, semipermeable matrices of solid hydrophobic polymers containing the antibody can be used in the form of formed products, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly(2-hydroxyethyl methacrylate) or polyvinyl alcohol), polyactides (US Pat.

Mo 3,773,919), copolymers of I-glutamic acid and y-ethyl-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as GYURKOM OEROT "M (microspheres for injection from copolymer lactic acid - glycolic acid and leuprolide acetate), and poly-0-(-)-3-hydroxybutyric acid. While polymers such as ethylene vinyl acetate and lactic acid - glycolic acid provide release of molecules for more than 100 days, certain hydrogels release proteins are not such significant periods of time. When encapsulated antibodies remain in the body for a long time, they can change their natural properties or aggregate under the influence of moisture and temperature of 37"C, losing biological activity and possibly changing immunogenicity. Rational stabilization techniques must take into account the mechanism involved. For example, if it is established that the mechanism of aggregation is the formation of an intermolecular 5-5 bond through thio-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilization from acid solutions, controlling moisture content, using appropriate additives, and developing a special composition of the polymer matrix .

MES-specific antagonists of the present invention are administered to a subject, e.g., a patient, according to known methods, such as intravenous administration, e.g., as a bolus or continuous infusion over a period of time, intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, intra- articular, intrasynovial, intrathecal, oral, local or inhalation routes. Topical administration is particularly desirable when toxicity or extensive side effects are associated with MESE antagonism. For therapeutic applications, an ex mimo strategy may be used. The ex mimo strategy involves the transfection or transduction of cells obtained from the subject with a polypeptide that encodes the MESE of the antagonist. After such transfection or transduction, the cells are returned to the subject.

THESE cells can be any of a wide variety of types, including, without limitation, hematopoietic cells (eg, bone marrow cells, macrophages, monocytes, dendritic cells, T cells, B cells), fibroblasts, epithelial cells, endothelial cells, keratinocytes, or muscle cells.

For example, when the MESE-specific antagonist is an antibody, the antibody is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal. If desired for local immunosuppressive treatment, the antibody can be administered directly into the lesion.

Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal and subcutaneous administration. In addition, the antibody is occasionally administered as a pulse infusion, especially when antibody doses are reduced.

It is preferable that the drug be administered by injection, preferably intravenously or subcutaneously, depending in part on whether the administration is short-term or chronic.

In another example, the MESE-specific antagonist is administered locally, for example by direct injections, when the disorder being treated or the location of the tumor allows it, and such injections can be periodically repeated. The MESE-specific antagonist can also be delivered to a subject systemically or directly to tumor cells, such as a tumor or tumor bed after surgical resection of a tumor, in order to prevent or reduce the likelihood of local recurrence or metastasis, such as a dormant tumor or micrometastases.

Alternatively, inhibitory nucleic acid molecules or a polynucleotide containing a nucleic acid sequence encoding a MESE-specific antagonist may be delivered to the appropriate cells in the subject. In certain embodiments, the nucleic acid may be targeted to the tumor itself. The nucleic acid can be introduced into the cells by any means suitable for the vector used. Many such methods are well known to specialists in this field (Zatogook ei ai., zirga; Maizop ei ai!., Vesotbipapi OMA, Sparieg 12, 2a eaiiop, Zsiepiys Ategisap VookK5, 1992). Examples of gene delivery methods include liposome-mediated transfection, electroporation, methods based on the use of calcium phosphate/OEAE dextran, gene gun, and microinjection.

THEIR. Combination therapy

The present invention also relates to the use of a combination of two or more MESE-specific antagonists of the present invention or a combination of at least one MESiE-specific antagonist with one or more additional anticancer therapies. Examples of anticancer therapy include, without limitation, surgery, radiation therapy (radiotherapy), biotherapy, immunotherapy, chemotherapy, or any combination thereof. In addition, cytotoxic, anti-angiogenic and anti-proliferative drugs can be used in combination with a MESE-specific antagonist.

In one example, the MESE-specific antagonist is used as a neoadjuvant therapy for the treatment of non-metastatic cancer after radical surgery. In this example, the MESE-specific antagonist can be administered with or without at least one additional chemotherapeutic drug.

In another example, the MESE-specific antagonist is used as a neoadjuvant therapy for the treatment of operable cancer before surgery. In this example, the MESE-specific antagonist can be administered preoperatively with or without at least one additional chemotherapy drug. In one example, the present invention relates to the use of a MESE-specific antagonist with one or more chemotherapeutic drugs (eg, a cocktail). Non-limiting examples of such chemotherapeutic agents include irinotecan, fluorouracil, leucovorin, or any combination thereof.

Combined administration includes the simultaneous administration of individual drugs or a single pharmaceutical form of these drugs, as well as their sequential administration in any order, but it is better that both (or all) active compounds simultaneously realize their biological activity over a period of time. Preparation and administration schemes of such chemotherapeutic drugs should be carried out in accordance with the manufacturer's instructions or determined empirically by an experienced practitioner. Preparation and administration schemes for chemotherapy are also described in the book: Spetoipegaru Begiise E., M.S. Reitu, MMiShate and UMiKip5, Vaitogye, Ma. (1992). The chemotherapeutic drug can be administered before, after, or simultaneously with the administration of the MESE-specific antagonist.

The drug according to the present invention may also contain more than one active compound, if it is necessary for the indication being treated, preferably with a complementary property and without a negative effect on each other.

For example, it may be desirable to additionally introduce antibodies that bind to ESEN, MESEE (for example, an antibody that binds different epitopes on MESEE), MESEA or EbVa2 (eg, NegseriipF), in the same preparation. In one exemplary embodiment, a composition for an MECE-specific antagonist does not include an anti-EgpVa antibody, fragment, or derivative thereof (eg, a NegserIPF antibody. Alternatively or additionally, such a composition may contain a cytotoxic drug, a cytokine, a growth inhibitor, and/or an antagonist of small of MESEV molecules Such molecules are randomly present in combination in amounts effective for a given purpose.

For the prevention or treatment of the disease, the appropriate dose of the MECE-specific antagonist will depend on the type of disease being treated, as previously determined, the severity and course of the disease, whether the MECE-specific antagonist is administered prophylactically or therapeutically, on previous therapy, from the patient's clinical history and reaction to the MESE-specific antagonist, as well as from the physician's discretion. The MESE-specific antagonist is randomly administered to the patient once or several times. In a combination therapy regimen, an MES-specific antagonist and one or more anticancer therapeutics are administered in a therapeutically effective or synergistic amount. A therapeutically effective amount, as used herein, is such an amount that the simultaneous administration of the MECE-specific antagonist and one or more other therapeutic agents, or the administration of a composition of the present invention, results in the reduction or inhibition of cancer as previously described.

A therapeutically synergistic amount is that amount of the MESE-specific antagonist and one or more other therapeutic drugs that is necessary to synergistically or substantially reduce or eliminate conditions or symptoms associated with a given disease.

The MESVE-specific antagonist and one or more other therapeutic agents can be administered simultaneously or sequentially in an amount and for a time sufficient to reduce or eliminate the occurrence or recurrence of a tumor, dormant tumor, or micrometastases. The MESE-specific antagonist and one or more other therapeutic agents may be administered as maintenance therapy to prevent or reduce the likelihood of tumor recurrence.

The MESE-specific antagonist may be packaged alone or in combination with other anticancer therapeutic compounds in a kit form. Such a kit may include optional components that help administer a single dose to the patient, such as vials for resuspending powder forms, syringes for injection, systems for intravenous administration, inhalers, etc. Additionally, a unit dose kit may contain instructions for the preparation and administration of such compositions. The kit can be made for one-time use by one patient, for repeated use by a specific patient (with a constant dose or a dose of individual compounds that changes during the course of treatment); or the kit may contain several different doses suitable for administration to several patients. Kit components can be contained in cardboard boxes, blister packs, bottles, tubes, etc.

H. Ready preparations

In another embodiment, the present invention provides a ready-made preparation containing materials for the treatment of the disorders described above. Such a ready-made drug contains a container, a label and an insert with information for the patient. Suitable containers include, for example, bottles, vials, syringes, etc. Containers can be made of different materials, such as glass and plastic. The container contains a composition effective in treating the condition and may have a sterile access port (for example, the container may be a bag containing an intravenous solution or a vial, the stopper of which is pierced by a hypodermic needle).

At least one active compound in the composition is an anti-MESE antibody. A label on or in association with the container indicates that the composition is being used to treat the condition of choice. The finished preparation may also contain a second container with a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It may additionally include other materials desired from both a commercial and consumer perspective, including other buffers, diluents, filters, needles and syringes. In addition, the finished preparation contains an insert with instructions for use, which contain, for example, a warning that this composition should not be used in combination with another composition, or a recommendation that the composition with the MESEE antibody should be administered alone or in combination with an anti-cancer composition The term "instructions for use" means instructions for applied therapy, medicinal product, treatment, treatment regimens, etc. by any means, such as in writing, such as a package insert and other written advertising material.

H. Deposit of materials

The following line of hybridoma cells was deposited in accordance with the provisions of the Budapest Treaty in

American Type Culture Collection (ATSS), Mapawzaz, USA:

EXAMPLES

Example 1. Inhibition of MESINE-A leads to inhibition of intestinal adenoma growth and long-term survival in Arstth mice

Familial adenomatous polyposis syndrome (FAP) and most cases of sporadic colorectal cancer are caused by mutations in the ARS gene. Patients with EAR develop hundreds and thousands of adenomatous polyps in the lower gastrointestinal tract, in addition to tumors outside the colon, which include dermoids and tumors of the upper gastrointestinal tract. Ars"p" mice with a heterozygous truncated allele in codon 850 imitate certain characteristic features of patients with EAR with a germline mutation of ARS (Mozeg vy ai., Zsiepse 247 322-324 (1990), bi ei aiY., Zsiepse 256 668-670 ( 1992)). The onset of tumor formation in Arsi mice occurs at a young age, and animals typically develop 60-150 intestinal polyps on a genetic background

C57VI/6. Tumor development results in greatly reduced lifespan in mice, with death usually occurring from anemia and/or hypoproteinemia (Mozeg et al., Zsiepse 247 322-324 (1990)) at approximately 5 months of age.

While humans with BAR typically develop colonic adenomas, in mice, for reasons that are not fully understood, the vast majority of polyps develop in the small intestine. These polyps reach 1-2 mm in diameter, while larger polyps (up to 4 mm in diameter) occur less often. Only isolated adenomas of the colon are observed, usually 0-3 per animal.

Ars has been reported to be involved in cellular processes, including proliferation, apoptosis, cell migration, cell adhesion, microtubule assembly, signal transduction, and chromosome segregation (for review, see Mayipke,

Apps. Neu. SeiI. Omg Vio!. 20 337-366 (2004)).

The best-studied function of Ars is its role as a regulator of beta-catenin in the M/pi signaling pathway (for review, see Maike, Moi. Raipoi. 52:169-173 (1999)). Briefly, in the absence of M/pi signals, Ars binds to axin and (y55K-Zbeta kinase) with the formation of a destruction complex for cytoplasmic beta-catenin, thereby preventing its nuclear translocation and subsequent activation of the T-cell factor/lymphoid enhancer family. factor (TCE/L ER) Transcriptional targets of TCE/EE include molecules involved in the cellular pathways previously mentioned.

The study of the mechanisms of tumor growth on xenografts has certain limitations, since these models do not repeat tumor development in a natural setting. To study the effects of anti-angiogenic therapy in a natural, genetically predisposed model of a non-malignant tumor, we investigated the Ars""" model of intestinal adenomatosis.

In the following example, the tumor phenotype of Ars mice was analyzed after short-term and long-term treatment with an indicative MESE-specific antagonist, anti-MESE-A tAB, as well as after genetic deletion of MESE-

And with the help of Ste-I ochR technology in intestinal epithelial cells.

For the experiments described below, mice Ars" (registration number 002020, 5) and 12.4KoMiISTe mice (registration number 004586, hereinafter MiPpStge, Maaizop ei ai., 9. Vioi. Speth. 277:33275-33283 (2002)) were obtained from Te daskvop I arogayugu (Vag Nagroi, ME). The results for MESEomoh mice (hereafter MES OH) were previously published (Segbeg ei aI., Oemeyuortepi 126:1149-1159 (1999)). Mice were kept in isolated microcages in a room with animal housing and experimental protocols were in compliance with federal regulations and approved by the Institutional Animal Care and Use Committee.

Expression of MESE-A in intestinal adenomas of Ars'it mice

In order to investigate the pattern of MESE-A expression in intestinal tumors of Ars mice, we performed IP hybridization on adenomas from 14-week-old mice. Hybridization of ip 5yi for these experiments was performed as previously described (Reitaga ei ai., At. 9). Heaven 162:1881-1893 (2003)). Briefly, neutral buffered formalin-fixed, dehydrated, and paraffin-embedded sections of intestinal tissue were deparaffinized and hydrated prior to deproteinization in 20 μg/ml proteinase K for 15 min at 3770.

IZR|OTR-labeled sense and antisense riboprobes were hybridized at 55°C overnight, then washed very thoroughly at 55°C in 0.1X standard saline citrate for 2 hours. Dry glass slides were exposed for 3 days at room temperature on Kodak VioMmax MA autoradiographic film (Eazitap Kodak Co., NKospevieg, MU), after which they were immersed in MTV2 emulsion for tracking nuclear particle tracks (Eazitap Kodak Co.), kept in airtight plastic boxes for slides, which contained a moisture absorber, for 28 days at 4"С, developed and contrast stained with hematoxylin and eosin (NUE).

The MESE-A probe was prepared as previously described (Ree!taga ei ai., At. U. Raipoi. 162:1881-1893 (2003)).

The length of the MESE-A probe was 349 nucleotides, which corresponded to nucleotides 297-645 of MM 031836. The upper primer had the sequence 5'- САА СОТ САС TАТ СА САТ САТ ССО (5EO IЮО MO: 1); lower primer - 5- OT

STA TT SSS PAA ASS Sta da (5EO IU MO: 2).

In these iPSC hybridization experiments, MESE-A expression was observed in epithelial cells with variable intensity compared to normal intestinal villous epithelium, although focally expressed signal was noted in adenoma stromal cells as well as in normal villous stroma (Fig. 1A-E).

Inhibition of MESE-A reduces tumor weight in Arstip mice

To determine whether anti-MESE-A therapy could be effective in reducing the mass of benign intestinal tumors in mice, we treated Arsip mice with anti-MESE-A tAB (6-31 in a mouse-human chimeric format, which helped reduce the possibility of inducing an immune response. Anti-MESE-A tAr (36-31 was obtained from the human Rar phage library as described (Iapd eii ai., U. Vioi. Speth. 281:951-961 (2006)). For to create an antibody suitable for long-term administration to mice, the variable domains were transplanted into the murine constant domain of IdSeg.tAB (6-31 (Papod ei ai., 9. Vioi. Snet. 281:951-961 (2006)) or a closely related murine Idsga (anti-CP120), both at a dose of 5 mg/kg, were administered intraperitoneally once a week in a volume of 90-140 μl in RV 5. Treatment was continued for 3 weeks, 2 weeks, up to one year or until the mice Treatment of 5-14 mice per group began at the age of 9153 days.

We chose TAB 26-31 because of its ability to potently block both mouse and human MESE-A (I iapod eii ai., 9.

Axes Spent 281:951-961 (2006)). This distinguishes it from the well-characterized anti-MESE tAbB A.4.6.1, which inhibits human but not mouse MESE-A (Seppbeg et al., Sapseg Vez. 60:6253-6258 (2000), Papo et al., 9 . Vioi. Speth. 281:951-961 (2006)). To assess the short-term effect of tAr(6-31) on tumor weight, treatment of 10 mice per cohort was initiated at 13 weeks of age and continued for 3 or 6 weeks. To determine the tumor phenotype at the age of initiation of treatment, a control group of 12 untreated mice was examined in a 13-week age (Day 0).

Treatment with anti-MESE-A tAbB for 3 or 6 weeks significantly reduced the total tumor burden.

On Day 0, the average tumor volume in mice A Ars"'":" was 39.3 mm? (in the range from 12.3 mm? to 97.0 mm3) (Fig. 2A). The mean tumor volume in mice treated with control dO for 3 weeks was 96.8 mm (47 1 to 299.9 mm), while the mean tumor volume in mice treated with tA for 3 weeks C6-31, was 23.5 mm (4.5-58.2 mm). This was a statistically significant 7695 or 4-fold decrease in the average tumor burden after treatment with the drug tAr (6-31 (p«e0.008). After b-week administration of control Ids, the average tumor volume reached 198.6 mm (40.5-315.7 mm), while in mice injected with tAr (6-31, it remained low, at the level of 28.4 mm (3.2-75.9 mm3), demonstrating a statistically significant 8695 or 7-fold decrease in mean tumor volume (re5.3 x 1075) (Fig. 2A).

Such a noticeable decrease in the volume of the tumor after 3 and 6 weeks of treatment with the drug tAB 26-31 was due to the reduced size of the adenoma. And not with a reduced number of adenomas. After 3 weeks of treatment with control Id, the average number of tumors was 11639 (SP), while after the introduction of tAbB (46-31) it was 107511 (re0.28).

After 6 weeks of treatment with control Ids, the average number of tumors was 12011, while after administration of tA (26-31) it was 1003210 (re0.09). On Day 0, mice had an average of 10029 tumors.

To analyze the size and number of tumors, the intestinal tract was opened longitudinally from the glandular stomach to the rectum, washed and spread on filter paper. After overnight fixation with Moich Niviu Rihaime fixative (Zsiepiyis YuOevidp I arogaiogu Ips., Oev5 Riaipez, I) and staining with methylene blue (0.190 aqueous solution), the number, location and diameter of each intestinal adenoma of the small and large intestine were recorded by one observer who was unaware of method of treatment used, using an eyepiece with a 20-fold magnification on an Ieis dissecting microscope. This method made it possible to reliably fix polyps with a diameter of 0.3 mm or more. Tumor volumes were calculated as hemispheres. The tumor burden of each mouse was calculated as the sum of the volumes of its tumors. P-values were calculated using the Student's two-sided test. Mice in the untreated group (Day 0) were analyzed at the age of initiation of treatment (13 weeks) and served as controls for the antibody-treated mice.

After 3 or 6 weeks of treatment, there was no evidence that adenoma growth evaded anti-MESE-A treatment: tumors in mice injected with tABb C6-31 had a more compact size distribution (Figure 28, middle and bottom graphs), compared with a wide distribution of tumor sizes in mice injected with control IDS (Fig. 28, second and fourth graphs from the top). The average diameter of polyps in mice that were injected with the control Ida for 3 weeks was 1.28 mm, and in the tABr group (36-31 - 0.85 mm (re«9.2 x 10717). The corresponding figures after b-week treatment were 1.64 mm and 0.86 mm (re«e2.7 x 102153, The average diameter of the tumor on Day 0 was 0.97 mm.

Interestingly, anti-MESE-A treatment apparently inhibited the growth of tumors of all sizes. After 3-week treatment with the drug tAr (46-31 the frequency of small tumors with a diameter of 0.3-1.0 mm (for b-week treatment 0.3-1.2 mm) was higher in the control group, while the frequency of tumors larger than 1.0 mm in diameter (21.2 mm for b-week treatment) decreased (Fig. 2C, top and middle graphs).

Day 0 (Fig. 2C, bottom graph) shows that adenoma growth essentially stopped after the initiation of tAB administration (36-31.

Moreover, anti-MESE-A treatment with tAB (36-31) was effective in suppressing the growth of adenomas in all areas of the small intestine. Significantly smaller mean diameter of tumors was observed after treatment with tAb Cc6-31, compared to treatment with control Idas, and after three, and after six weeks of therapy (Fig. 203. In addition, the average diameter of the adenoma in the first quarter of the intestine of mice injected with tAb (46-31) was significantly reduced compared to that observed on Day 0 (double asterisk in Fig. 20). The decrease in the mean diameter of colon adenomas did not reach the level of statistical significance (Fig. 20). The mean diameter of colon polyps in mice treated with tAbr (36-31) for three weeks was 1,320.3 mm, while in control mice group it was 2,540.4 mm (p«e0,064). The average diameter of colon polyps in mice that were injected with tAB (6-31) for 6 weeks was 2,220.3 mm against 2,620.3 mm after the introduction of the control da (p "e0.37).

Deletion of MESER-A in intestinal epithelial cells reduces the mean diameter of tumors

As a next step, we wanted to dissect the contribution of MESE-A from intestinal epithelial sources to adenoma development in the Arstipia model. To this end, tumor diameter and number were assessed as described above in 13-week-old Arstipia mice and compared with mice in in which MESE-A was specifically extracted from intestinal epithelial cells using the SteLoch?R technology (MESEH mice; MiMp-Ste). Misha Arstitje; MiTsip-Ste and Arstit; HONEY

MiCShp-Ste were analyzed at 13 weeks of age.

The expression of Minp, an actin-binding protein and the main structural component of the brush border of specialized absorptive cells, begins during embryogenesis in the intestinal endoderm of the hindgut, and later spreads to the endoderm of the small intestine and colon (Vgashypvieip ei ai!., Oem. Yuup. 224 :90-102 (2002), E27ei!

Oemeiyurtepi 106:407-419 (1989), Mashyposhgu ei aI., EMVO 9. 7:3321-3329 (1988); Maiposhgu ei ai., Oeemeyuortepi 115:717-728 (1992)). In adults, the distribution of Miyip becomes diffuse with moderate apical polarization in the brush borders of fully differentiated cells that line the small intestine (Nobipe ei ai., Rgos. Mai).

Asad 5 82:8488-8492 (1985)). The expression of the recombinase Ste, driven by the MiPp promoter (MiPp-Ste), was previously characterized as reproducing the expression pattern of the MiPp gene in every cell of the intestinal epithelium from the crypt to the tip of the villi and the duodenum throughout the colon (Maadighop et al., 9 . Axes. Speth 277:33275-33283 (2002)).

Phenotype analysis revealed that the mean tumor diameter in control Arstipie mice; MiCp-Ste was 1.0250.3 mm, while in Arspip mice; UBB MiTsip-Ste it was 0.82250.3 mm (Fig. 2E), showing a decrease of 19.895 (r«e7.2 x 107). The groups did not differ significantly in the number of tumors. If Ars"ipk; MiCyp-Ste mice had 137411 intestinal adenomas, then Ars"tip mice; MESEoh; In Miyip-Ste, their number was 150,217 (r«0.27).

These data demonstrate that deletion of MESE-A from all intestinal epithelial cells from the duodenum to the colon leads to a significant inhibition of tumor growth, although not to the extent achieved by systemic administration of an anti-MESE-A antibody. Therefore, the obtained data allow us to state that there are epithelial sources

MESB-A contribute to the growth of intestinal adenomas in Ars""" mice.

Suppression of MESE-A prolongs the average survival time of Ars'ip mice

Given the efficacy of anti-MESE-A treatment in inhibiting tumor growth, we wanted to investigate whether treatment with tAbB (26-31) could provide long-term benefit to Ars mice. To this end, administration of tA (6-31 and control Id was continued up to 52 weeks or until the mice were seen moribund.

Interestingly, treatment with tAb (26-31) increased median survival from 24.0 weeks in controls to 33.6 weeks in mice treated with tAb (36-31) with a logarithm of 2.4 x 103 (Fig. 2E).

The tumor phenotype of four mice injected with tAB (6-31) was analyzed after euthanasia at 32, 51, 64, and 66 weeks of age (after 19, 38, 51, and 53 weeks of treatment with tAB (36-31, respectively). How shown in Table 1, the mean tumor diameter remained close to that of 19-week-old mice (1.64 mm in control Ids-treated mice: at 6 weeks). Similarly, the number of tumors remained comparable to that of 13 -week-old mice (on Day 0, mice had 59 to 161 adenomas; average 100). Three (one from each mouse 2, C, and 4 from Table 1) with 15 colonic adenomas (the total number of such tumors found in mice 1-4), which fell into the cut plane, did not show malignant transformation during histological analysis.

Table 1

Characteristics of tumors in mice after long-term treatment with drug 6-31

Misha Me| Age (week) | Week 46-31 | Number of tumors | Average diameter (mm) (Volume of tumors (mm?) 4 66 | шщущК5з 17150 | -ррсл63 | 2822 x Healthy animal subjected to euthanasia at the end of the study.

In summary, long-term anti-MESE-A treatment was generally well tolerated and provided increased survival of Ars mice. Moreover, tumor numbers and mean tumor diameters in mice treated long-term with tA (56-31) remained strikingly low, consistent with inhibition of new adenoma formation and adenoma growth.

Normal levels of total protein, albumin, and triglycerides in serum and reduced extramedullary splenic hematopoiesis in Ars"p/" mice on anti-MESE-A therapy

A general observation was that Arsp mice injected with tAr (36-31) appeared significantly more active and reactive than mice injected with control Id. Moreover, pale paws, which indicated progressive anemia, as reported by Mozeg ei ai., (Mozeg ei ai., Zsiepse 247:322-324 (1990)), were regularly observed in animals injected with control IDS, and were not observed in mice on tABr (6-31. This observation was confirmed by the fact that the average serum total protein and serum albumin in control da animals were reduced, while in tAbr (6-31) mice these values were within the normal range (Table 2).

Table 2

Chemical analysis of serum "control numbers 3.9-5.5 g/dp..////Ї77777777111111111111111Ї11111111111111Ї111111111111111 se control numbers 23-32 hydlo/////////17711111111111Ї111111111111111Ї11111111111111111111111111111111111111111111111111111111111111111111111111111112 /Ї777777777111111111111Ї1111111111111111С1111111111111 iii PNYA NNYA PNYA (SPO).

As reported for Ars mice (Moveg et al. Zsiepse 247:322-324 (1990)) and consistent with hypoproteinemia, mean triglyceride levels were elevated in control Ids-treated animals, although they were reduced to levels comparable to controls. number after treatment with the drug tAr C6-31 (Table 2).

Although no treatment-related difference in body weight was observed after 3 or 6 weeks of treatment, mean spleen weight was significantly increased (p<2.3 x 103) in control dO-treated mice. After 3 weeks of administration of control IDS, mice had an average spleen weight of 0.26 g or 1.1795 of body weight, while after treatment with tAr (56-31 for 3 weeks this figure was 0.11 g (0.4995 of body weight ).This increase in mean spleen weight in control Ida-injected mice is consistent with extramedullary hematopoiesis (EMG, compensatory erythropoiesis, in this case secondary to intestinal bleeding), as confirmed by histological examination of the spleens. Ten of 10 mice, which within weeks administered the control IDS, demonstrated pronounced EMG, while in the tAB group (46-31 2 mice had moderate EMG, five - weak EMG, and three had no diagnostic changes in the spleen.

The reduced degree of EMG in the spleen of mice that were short-term injected with tABb (6-31) suggests that anti-MESE-A therapy has a beneficial effect on reducing intestinal bleeding.

Changes in the kidneys after long-term treatment with the drug tAbB (46-31

To investigate the potential toxicity associated with administration of anti-MESE-A high affinity tAb (46-31), pancreas, liver and kidney were analyzed histologically after short-term (3-6 weeks) and long-term (18-53 weeks) treatment. For histological analysis, intestinal tissue fixed in Notox was dehydrated and embedded in paraffin, sections were made and stained with hematoxylin and eosin according to standard protocols.

No significant toxicity was observed in animals treated for 3-6 weeks. After long-term treatment with tAB (26-31), five of five mice exhibited varying degrees (mild to severe) of diffuse global glomerulosclerosis and moderate pancreatic stromal edema (reflecting hypoproteinemia). These observations were consistent with toxicity that was observed earlier, as a result of long-term administration of tA (6-31. Importantly, these adverse effects were outweighed by the general improvement in health, which was evidenced by the increased average survival.

The changed tumor morphology after tAr treatment (46-31 was not accompanied by a change in the proliferative index

For further characterization of intestinal polyps in Ars mice treated with the anti-MESE-A drug tABr C6-31, macroscopic and histological analyzes were performed as described above. The macroscopic morphology of polyps after treatment with the drug tAr 26-31 was markedly different from the morphology of polyps after treatment control yes (Fig. ZA-B). If tumors from mice injected with control Ids typically had a relatively intact, smooth surface, then tumors from animals treated with the drug tAr (6-31) had deep intussusceptions on their surface. Histological analysis showed that the tumors from animals treated with the control da and the tAbB drug (6-31) are tubular adenomas (Fig. 3C-E). Adenomas from mice treated with the control

Ida, had marked intravillous epithelial proliferation with vertical and lateral extension and were typically extended more than twice from the base to the luminal surface. The stroma was minimally fibrous.

Adenomas from mice treated with the drug tAB (6-31) had characteristically fewer intravillous epithelial cells, were less wide at the exit into the lumen, more benign and captured fewer adjacent villi.

Histological analysis of colon polyps in both groups revealed tubular adenomas on legs with abundant fibrovascular stroma and a variable amount (up to 10,095) of dysplastic epithelium.

In order to evaluate the degree of proliferation in the tumor tissue and in the normal mucous membrane, indirect immunohistochemical staining with Ki-67 antibody was performed (Fig. 305-U). For these experiments, notox-fixed, dehydrated, and paraffin-embedded sections of intestinal tissue were deparaffinized and hydrated before incubation in Tagdei Neiiiieema! (AKO, Spozipir, Denmark) at 9970 with subsequent quenching of endogenous peroxidase activity and blocking of avidin and biotin (Mesiog, Vypipdate, USA). Sections were further blocked for 30 minutes with 1095 blocking serum in PB5 with 395 bovine serum albumin. Tissue sections were incubated for 60 minutes with primary antibodies diluted in blocking serum, washed with TV5T Wieg (RAKO) and incubated with secondary antibodies for 30 minutes, washed with TV5T and incubated in ABC Eiye Neadepi (Mesiog) for 30 minutes, then incubated in Meia! Eppapsey BAV (Riegse, Noskooga, II) and counterstained with hematoxylin

Meyer. A rabbit polyclonal antibody against Ki-67 (5Rb, 1:200, ar Misiop, Egetopi, USA) was used as the primary antibody. The secondary antibody was a biotinylated goat anti-rabbit antibody (7.5 μg/ml,

Mesiog). All steps were performed at room temperature.

Quantitative analysis revealed similar numbers of Ki-67-positive cells in tumors from mice treated with control IdS or tAB (6-31. Similarly, the proliferative index of normal adjacent mucosa was comparable in both groups (Fig. 3K). Proliferative index was calculated from images of 5-μm paraffin sections of tumor tissue and normal mucosa obtained with an Agpio!5150 scanning microscope slide (Arriei Itadipo, Ips., Zaporozhye, USA) using Kizidny analysis (Apo!Vemiem/ (m2.6 )).

Areas of tumor tissue and normal mucosa were identified and outlined manually by a researcher who did not know which drug was administered to the animals. The proliferative index, which was defined as the percentage of positive relative

The Ki-67 of nuclei relative to the total number of nuclei was calculated in a semi-automated manner based on the color of the nucleus defined as a positive threshold. Determination of the proliferative index included the analysis of 29 tumors in the tAb group (26-31 (n-3) and 44 tumors in the control da group (n-3); all animals were treated for 6 weeks.

To check whether growth inhibition by the drug tAB (6-31) is accompanied by changes in the expression level of molecules involved in the main signal transduction pathways, Western blotting was performed. 31 and the control antibody were removed with a scalpel and mechanically homogenized in an OMMI TNI15 homogenizer in a buffer for HIV lysis.

Rabbit polyclonal antibodies against rZ8 MARK, phosphorus-rzv were used as primary antibodies

MARK, p42/p44 MARK, phosphorus-p42/p44 MARK, RTEM, AKI, phosphorus-AKI and phosphorus-a5KZalpha/beta (all 1:1000, Seii!

Zidpaiipo, OSapmegye, USA). Horseradish peroxidase-conjugated anti-rabbit antibody (1:5000, Spetisop) was used as a secondary antibody.

Although the expression level of many of the tested molecules after treatment with tAB (6-31) remained unchanged, in three of four tumor samples there was a moderate restoration of the level of phosphor-p38 MAPK to the level established in normal mucosa (Fig. 3I; p-p38, compare T5-T8 with M5-M8).

Decreased density of blood vessels in tumors under the influence of the drug tAb (26-31

Given that MESE-A is a known mitogen for vascular endothelial cells through a signaling pathway

MESEV-2, we studied the vascular system of tumors in mice injected with tAr (46-31 and control IDS, using immunohistochemical staining of thick tissue sections with an anti-CO31 antibody (Fig. 4A-B). In order to obtain confocal images on a microscope, we carried out perfusion of mice with 195 paraformaldehyde (PEA) in PBO5 under isofluorane anesthesia, the intestinal tract was flattened on filter paper, postfixed with 495 PEA and immersed in 3095 sucrose in PBO5 overnight at 4"C before pouring into OS... and freezing on dry ice . Cryosections were made 80 μm thick, fixed and 495 REA for 10 minutes, treated for greater permeability with 0.295 Tpop-X-100 in РВ5 and blocked for 30 minutes with 595 normal goat serum in РВ5 with 0.295 Tpop-X-100. Primary antibodies in blocking buffers were incubated overnight, secondary antibodies -5-6 hours, washed with PB5 and counterstained with Noespvi 33342 (0.5 mg/ml; Zidta, 51. Iocie, USA).As primary antibodies il used hamster monoclonal antibodies against SOC1 (1:500, Spetisop, Tetesia, USA), rat monoclonal antibodies, monoclonal antibodies against E-cadherin (1:2500, 7utea, bBoshii Zap Egapsizo, USA) and SuZ conjugated mouse monoclonal antibodies against smooth muscle actin (1:1000, bidta). The secondary antibodies used were Su5-conjugated anti-American hamster antibodies (1:500, in Chaskwop Ittipogezeagsi, SatrgiddevNigye, Velika

Britain) and AG EXA 488 conjugated anti-rat antibodies (1:500, MoIesshag Rgobez, Eideppe, USA).

The density of blood vessels in tumors from Ars mice was quantified from digital images taken with a CSO camera on a 76iz5 Achioriap2 fluorescent microscope (Tropical, USA). Each of the four groups (mice that were injected with control IdsCi or tAbB for 3 or 3 weeks ( 26-31) was represented by two animals, 11-22 tumors from each group were analyzed. The area of vessels on 80-μm tumor sections was calculated by marginal segmentation of positive fluorescence of SOZ1 using Itadeu m. 1.36 (pEr://t5B.ipyu.pip. house//).

Vascular density was then calculated as the ratio of POP1 positive pixels (image elements) to the total tumor area. The data obtained for all tumors from a given group of animals were averaged to have a mean value for each group.

Quantification of vascular density showed that the vascular component of tumors from mice injected with tAb (16-31) was reduced compared to that from mice injected with control da (Fig. 4C). After 3-week administration of control Ids, the average vascular area tumors was 23.295, while after the 3-week administration of tAb (46-31) it decreased to 18.695. The average area of blood vessels in the tumors of mice after the b-week administration of the control da was 25.595, and after the same duration of administration of tAbB (46-31 -19.795 .

Discussion

We used Ars"" mice to investigate the role of MESB-A in benign intestinal tumorigenesis. In the first part of the experiments, the aim was to evaluate the effects of short-term and long-term anti-MESE-A treatment in relation to established intestinal adenomas during their active growth. We have shown that treatment with the anti-MESE-A drug tA (6-31) significantly reduces the tumor burden and prolongs the survival of Areya mice,

Several studies have addressed the effects of food and chemopreventive drugs, including non-steroidal anti-inflammatory drugs (NSAIDs), on tumor burden in Ars mice (for a review, see

Warm up! yei ai!., Sapseg Eridetio!. Viotaikegz Rgeu. 12:391-400 (2003), the updated version of the list can be viewed at pyr//sogrei.pe/tip). Some of these studies report tumor reduction. NSAIDs such as piroxicam and sulindac, which target both COX-1 and COX-2, were among the most potent drugs in inhibiting tumorigenesis in Ars mice (Wooibroi! !., Sapseg Vevz. 56:2556-2560 (1996), Spii eyi a!., Sapseg

Transportation 57:4267-4273 (1997), Napsep-ReikK eyi a!., Sapseg I ey. 175:157-163 (2002), Ayshapa et al., Sagsipodepeviv 20:51-58 (1999)), in addition to selective COX-2 inhibitors, such as celecoxib (dasobu et al., Sapseg Nev. 60:5040 - 5044 (2000)) and A-285969 (M/adepaag-MiPeg eyi a/!., Vg. U. Sapseg 88 1445-1452 (2003)).

Combination treatment regimens have also been used successfully to reduce the number of tumors. Toiitapse eyi ai. used a specific inhibitor of the epidermal growth factor receptor, EKI-785 in combination with sulindac and showed almost complete elimination of tumors (Toitapse ei ai!., Mai. Med. 6 1024-1028 (2000)). Similarly, the combined effect of chemotherapy drugs raltitrexed (ATX) and 5-fluorouracil (B) provided a significant (3795) reduction in the number of tumors in mice Ars"": (Migrnnu ei ai!., Sapseg Vioi. Teg 3:1169-1176 ( 2004)).

Not so long ago, short-term administration of the receptor tyrosine kinase (ATK) inhibitor A2O2171 demonstrated a reduction in tumor burden in Ars mice ((Zoodiai ei a!., Sagsipodepeviv 27:2133-2139 (2006)). They noted that an earlier start of treatment (due to b weeks) with the inhibitor A2O2171 was able to reduce the number of tumors, while a later onset (after 10 weeks) reduced only the size of tumors (Xuodiai ei ai., zirga). However, this treatment had no effect on vessel density (Xuoodiai ei ai!. , virga).A2O2171 inhibits several receptor tyrosine kinases, including, but not limited to, MESEV-1, -2, and -3Z (M/yedde sei ai., Sapseg

Nez. 65:4389-4400(2005)).

We observed that the anti-MESE-A drug tAB (6-31) administered after 13 weeks did not reduce the number of existing tumors, although it did reduce the size of tumors and apparently inhibited the formation of new adenomas. These results were correlated with a marked increase in survival .However, we believe that it is possible that an anti-MESE-specific tumor prevention approach (with an earlier onset of treatment) could potentially be more effective in reducing the number of tumors than an interventional approach to pre-existing tumors (with a later onset of treatment ), which was used in our study. Nevertheless, anti-

MES-specific inhibition was effective at all stages of tumor growth.

Our study convincingly shows that targeting MESE-A is sufficient to achieve pronounced therapeutic effects in the Ars model: Comparison of systemic inhibition of MESE-A by the drug tA C6-31 with genetic deletion of MESE-A only in the intestinal epithelial compartment in mice Ars""x, MESEEeh, miTsip-

Ste shows that, in addition to epithelial cells, other cellular sources of MESE-A play an important role in adenoma growth

Arstipia, These additional sources of MESE-A potentially include mononuclear cells (bZipauata eyi ai.,

Sagsipodepevziv 23:1351-1359 (2002)) and stromal fibroblasts (Zepo eyi ai., Sapseg Nev. 5. 62:506-511 (2002), (UMiyatve eyi ai., 9. Siip. Ipuevi. 105:1589-1594 (2000) )).Our analysis of Ip ZISH evidences extra-epithelial expression

MESB-A in adenomas and normal villi, supporting this observation.

Based on the extensive amount of data, it can be considered proven that many of the observed antitumor effects of tA (46-31) are mediated by the inhibition of angiogenesis (M/ize ei a!., Rgos. Maii). Asad. si OBA 96: 3071-3076 (1999), 7asnagu eyi a)yi., Sagaiiomass Nez. 49:568-581 (2001)). Indeed, reduced vascular supply in response to anti-MESE-A monoclonal antibody was observed in tumor xenotransplantation studies (Vogodvigot et al., Rgoviage 35:1-10 (1998)). In accordance with this, a decrease in the area of vessels in intestinal adenomas of Arsip was observed after three and six weeks of administration of the drug tAr (6-31), compared to tumors from mice that were injected with the control Ida.

The significant accumulation of adenomas smaller than 1 mm, which was observed after inhibition of MESE-A, suggests that in the intestinal adenomas of Ars""" mice, the angiogenic switch may occur earlier than previously thought for tumor development and as shown in the Arseia716 model (Zepo et al. AI., Sapseg Rev. 62:506-511 2002)).

An important and unexpected finding of our study is that anti-angiogenic therapy targeting a single angiogenic factor can be highly effective in inhibiting tumor growth and may confer a survival benefit. This is apparently contrary to the view, which has developed mainly in studies of malignant tumors, that the main benefit of such therapy is the "normalization" of the blood vessels of the tumor to enable the delivery of chemotherapy (Chaip et al., May. May. 7:987-989 (2001 )). It can be hypothesized that a reduced propensity of benign tumors to acquire mutations, potentially leading to treatment resistance, may be responsible, at least in part, for this difference. Therefore, our data indicate the possibility of non-surgical treatment of benign tumors without the need to use chemotherapeutic drugs.

Example 2. Anti-MESE-A monoclonal antibody inhibits pituitary adenoma growth and reduces serum levels of prolactin and growth hormone in a model of multiple endocrine neoplasia in mice

Multiple endocrine neoplasia (MEM) is a disorder characterized by the occurrence of tumors affecting two or more endocrine glands. A patient is classified as having MEM type 1 (MEM'I) when the combined occurrence of tumors in the parathyroid glands, pancreatic islet cells, and anterior pituitary is identified. Mutations in the MEMT gene, which lead to truncation or absence of the menin protein, have been found to underlie this disorder (for review, see Rappei et al., Epadosg. Neiai. Sapseg 6:449-473 (1999)). When data on the frequent loss of the residual allele in tumors were added to this (Vuzygot ei ai., Rgos.

May). Asad 5si OBA 87:1968-1972 (1990), Oereiepko ei a!., Sapseg Nev. 57:2238-2243 (1997), I atesop ei ai., Mashtge 332:85-87 (1988)), MEMI was classified as a tumor suppressor gene. Although MEMI1 is mainly inherited as an autosomal dominant disorder, de poho mutations in the MEMI gene have been identified as the cause of sporadic cases of MEM.

The function of menin remains largely unexplained. Being ubiquitously expressed, this predominantly nuclear 610-amino acid protein is hypothesized to be involved in transcriptional regulation, DNA processing and repair, and cytoskeletal organization through its ip and mio interactions with the protein portion of the aforementioned pathways (for review, see Adagma! ei aY. , Nopt. Meiar. Nez. 37:369-374 (2005)). However, none of the interactions of this protein identified to date provide an explanation for tumorigenicity in MEM.

The current standard of care for pancreatic tumors, of which more than 5095 are gastrinomas and 10-3095 are insulinomas, is to reduce basal acid production in gastrinomas, whereas surgery is the optimal treatment for insulinomas. Treatment for pituitary tumors is elective surgery with various drug therapies, depending on the hormonal profile, while the treatment of choice for parathyroid tumors is surgical removal of the overactive gland. However, there is variability in the extent and exact timing of parathyroidectomy (for a review, see Vgapai et al., U. Siip. Epdosgipo!. Meiab. 86:5658-5671 (2001)). Recent advances in the development of new approaches to the diagnosis and treatment of MEMI have recently been reviewed (Mioia et al., Sy. Orip. Opsoi. 17:24-27 (2005)).

By means of homologous recombination, exons 3-8 of the mouse Mep1 gene were targeted for deletion (Stabigeye ei ai!., Rgos.

Mai! Asad 5 BOTH 98:1118-1123 (2001)). It was reported that by 9 months of age in heterozygous mice Mep! pancreatic islet lesions develop with additional frequent observation of parathyroid adenomas.

Larger and more numerous tumors in the pancreatic islets, parathyroid glands, thyroid gland, adrenal gland cortex, and pituitary gland appear before the age of 16 months (Stabigeye ei ai., Rgos. Mai). Asad si OBA 98:1118-1123 (2001)), having characteristics very similar to such a disorder in humans.

There is enough evidence that blocking MESE-A-mediated angiogenesis leads to tumor suppression (Sepeg et al., Sapseg VNev5. 60:6253-6258 (2000), Noiazpi et al., Rgos. May). Asad 5 OBA 99:11393-11398 (2002), MiPatsyeg ei a!., Mayte 367:576-579 (1994), Rgezhei ei aI., Sapseg Nev. 59:5209-5218 (1999),

Mooa ei aiI., Sapseg No. 60:2178-2189 (2000)), as anti-MESE-A approaches have been used in various preclinical models derived from human malignant cancer cell lines (for a review, see Seregi et al., Sapseg

Vev5. 60:2178-2189 (2000). However, xenotransplanted tumors poorly replicate tumor development in a natural setting. Moreover, to date there have been no attempts to use anti-MESE-A antibody therapy to suppress the growth of benign tumors or tumors of endocrine origin. To investigate the role of MESE-A in the development of adenomas specific for endocrine tissue, we studied the effects of anti-angiogenic therapy in a natural, non-malignant tumor model - the MEM model! in the Mep177 mouse. The volume of tumors (adenomas) in Mep1-7 mice, as well as subcutaneous transplants of pituitary tumors in nude Vair/c mice were analyzed after short-term treatment with an indicative MESE-specific antagonist - anti-MESE-A monoclonal antibody (tABb).

In addition, the possibility of reducing the elevated levels of hormones associated with MEMI by introducing anti-MESE-A TAB was investigated.

For the experiments described below, Mep17" mice (registration number 004066) were obtained from TNne daskwap

Y arogayugu (Wag Nagbrog, USA), and naked mice Vair/s - from Spazez Wimeg I arogayugies Ips. (ULiItipoyup, USA).

Experimental female Mep177 mice of mixed 129-EMV origin were obtained by crossing Mep177 males and females. Mice were kept in isolated microcages in a room with limited access and received food ad libitum. Animal housing and experimental protocols were in compliance with federal law and approved by the Institutional Animal Care and Use Committee.

Treatment with antibody tABr (36-31 inhibits the growth of pituitary adenoma Mep177

To investigate whether anti-MESE-A treatment is effective in inhibiting pituitary adenoma growth, 125 female Mep!1177 mice aged 11 to 13 months were subjected to MRI to identify mice with pituitary tumors. Tumor-bearing mice were again subjected to a diagnostic imaging study after 14 and 28 days to determine the rate of adenoma growth. A group of 9 mice with an average tumor growth rate of 12.495 per day and an average tumor volume of 15.58244.0 mm3 at baseline received control IDS, and a group of 8 mice with an average tumor growth rate of 10.295 per day and an average tumor volume of 16.7055.7 mm3 at the beginning of the study received anti-MESE-A TAB 26-31 for 67 days or until they were found dying.

For treatment with TAB antibodies (6-31 and control and intraperitoneal injection of 5 mg/kg of anti-MESE tA (6-31 (Papo ei ai!., 9. Vioi. Spet. 281:951-961 (2005)) or close by isotype to the control Idse (anti-CP120) was given once a week in a volume of 100-200 μl in PB5. Administration of eight (tAbB (6-31)) or nine (control

Id) to Mep!1"7 mice with pituitary adenoma ip 5ish began at the age of 13.5-14.5 months and continued for 67 days or until the mice were found dying. Treatment 23 (control Ids) and 35 (tA (6-31 of nude Wair/s mice with subcutaneous grafts of pituitary adenomas were initiated 4 months after transplantation and continued for 35 days or until the mice were found dying or until the tumor volume reached 3000 mm3.

The animals were examined using MRI every two weeks to monitor the growth of the pituitary adenoma ip mimo. NMR images were obtained on a magnet with a horizontal channel 9.47 (Ohoga Ipvipitepiv Tsa., Ohio,

Great Britain) and a console control panel (Magiap, Ips., Rayu Ayo, USA) using a 3 cm bulk winding for transmission and reception (Maiap, Ips.). A fast spin echo imaging sequence was used with a repetition time of 4 seconds, an echo echelon length of 8, an interval of 12 ms, an effective time of 48 ms, and an average of six. The image matrix was 1282, with a field of view (20 mm): and a slice thickness of 0.5 mm.

Mice were fixed in the supine position with 295 isoflurane in medical air; body temperature was monitored with a rectal probe and maintained at 37"C with warm air during the 15-minute image acquisition time. After image acquisition, the animals were allowed to oscillate on a warm surface, after which they were returned to the place of confinement. The volume of the primary pituitary tumor was calculated from NMR data with using three-dimensional zones of interest introduced in the Apaiuge software (Apaiugebigesi, Ips.,

This era, KB).

After 39 days of treatment, a statistically significant decrease in the average volume of the pituitary tumor was observed in the group treated with the antibody tAbr (6-31, compared to the group treated with the control da (Fig.

BA). At the end of the study (after 67 days), a statistically significant 7295 or 3.7-fold decrease in the average volume of the pituitary tumor was noted after treatment with the tAbB antibody (36-31 (p«e0.016). If the majority (6 out of 9) of the tumors

Mep!-7, which were treated with control IDS, continued to grow actively throughout the treatment period, then growth

7 of 8 pituitary adenomas treated with tAr antibody (46-31) were significantly slowed (Fig. 58). Four mice on control IdSi and three on tABr (26-31) were euthanized before the end of the study due to poor health, including one mice on control IDS before imaging on treatment Day 25. Tumor-doubling-free survival was significantly increased in the tAbB group (6-31 with a log-rank re of 0.019 (Fig. 5C), indicating improved health in these mice due to inhibition of tumor growth In two mice in the ip tABr group (6-31), the tumor volume did not double even on Day 67 of treatment.

Anti-MESVB-A antibody inhibits the growth of subcutaneous pituitary adenoma transplants

To test the effectiveness of anti-MESE-A antibody treatment on the Mep11-7 model of transplanted pituitary adenoma, subcutaneous tumors were induced in 6-8-week-old female Vair/s nude mice using the following method. For these experiments, one ip 5ish pituitary adenoma from a Mep17" mouse was removed and minced into pieces of approximately 1 mm3, mixed with the basal membrane of the VO Mayde! Mayih Vasetepi Metbgape matrix (VO, Vega, USA) and planted in a volume of 200 μl under skin on the dorsal side of nude VaiB/s mice.Four months later, one subcutaneous tumor (approximately 900 mm3 volume) was removed, minced, mixed with Mayde!, and grafted as described above to establish a cohort of mice with pituitary adenoma transplants.

The size of subcutaneous grafts of pituitary adenoma was measured with a caliper (Egei M. Romeg So. Ips.,

Meulop, USA) as the largest diameter of the tumor and the diameter in the direction perpendicular to it. The volume of the tumor was calculated according to the following formula: M-parg/6 (a - the largest diameter of the tumor, B - the perpendicular diameter).

A group of 35 mice with an average tumor volume of 515242 mm? at the beginning of treatment received tA C6-31, and a group of 23 mice with an average tumor volume of 5272464 mm at the beginning of treatment received control ds for 35 days using the methods described above. At the end of the study, tumors treated with control IDS increased their volume almost fourfold, to an average of 2071 2152 mm", while in mice treated with the antibody tAB (46-31, tumor growth essentially stopped; after 35 days, the average size their tumor volume was 556289 mm (Fig. 50). A statistically significant 7395 or 3.7-fold decrease in the average tumor volume was noted after treatment with the antibody tAB 26-31 (re«1.9 x 1072),

These data prove that anti-MESE-A tAB (46-31) is effective in inhibiting the growth of pituitary adenoma, as well as subcutaneous transplants of pituitary adenoma, the susceptibility to which was determined by Mep heterozygosity.

Expression of MESE-A, MESEV-1 and MESEV-2 in normal pituitary gland and pituitary tumor tissue

To investigate the level of expression of MESE-A, MESEV-1 and MESEV-2 in the pituitary tissue and to check how this level is affected by treatment with the TAr antibody (26-31, we compared the relative expression of MESE-A, MESEV-1 and MESEV-2 in p in five adenomas of the pituitary gland of the lower lobe treated with the control Ids (average volume 96.228.7 mm), in five adenomas of the pituitary gland with the lower lobe treated with the antibody tA C6-31 (average volume 35.2344.0 mm) , as well as in five untreated, near-aged, small pituitary adenomas (9,732.9 mm3), four near-aged normal pituitaries from Mep1:7 mice, and eight near-aged wild-type pituitary samples. 1 and MESEV-2 in five pituitary tumor transplants treated with control IDS (average volume 20632205 mm3) and in five transplants treated with tABr antibody (6-31 (average volume 577245 mm3).

For these experiments, DNA-free total RNA was prepared from snap-frozen pituitary adenomas or normal pituitary glands using the VMeavu Kii kit (Oiadep, Niidep, Germany) according to the manufacturer's protocol. One-step quantitative RT-PCR was carried out in a total volume of 50 μl using the Zyregospri PI Riaypit Ope-5ier ZAT-PCR kit of Kyiv (Ipmygodep, Sapzrad, USA), with 100 ng of total RNA, 45 nM of each ROSA primer and 12.5 nM probe Tadtap. To detect the expression of genes of interest, the following primers Tadtap Sepe Ehrghezviop Avgzau and a mixture of probes (Arriei

Viozuvzietve, Roviei Spu, USA): MESE-A (Azzau IU: Mt00437304 t!), MESEV-1 (Avzau IU: Mto00438980 ti) and

MESEV-2 (Avzau IU: Mt00440099 ti!i). SARON expression was detected using Tadtap primers and probes synthesized in our own laboratory (direct primer sequence: АТатТТОСАСТ АТОАСТОСАС TСАСаЙ (5EO

IO MO: 3); reverse primer sequence: ZAASHASASSA STASASTOSA SOASA (5EO IU MO: 4); the sequence of the Tadtap probe: AABSSSATSA SSATSTTOSA SSIASSSOAIA (5EO IYU MO: 5)).

The reactions were carried out in a real-time system of Arriea Viozuzietv 7500 NeaI-Tite RSA was run under the following conditions: the reverse transcription stage (15 minutes at 48"C), the next denaturation stage (2 minutes at 957) and 40 cycles of 15 seconds at 95"7C and 1 minute at 60°C. The level of gene expression in each sample was determined by the method of relative quantitative analysis (YAYSI method) using the SARON gene as an endogenous control and total RNA of the mouse placenta (Siopiesy, Moscow, USA) as a standard.

Noteworthy is the fact that the mean relative expression of MESE-A was significantly increased in adenomas of the ip v5ish treated with tAB (46-31, compared with pituitary tumors treated with control Ida, small tumors that were not treated, and normal pituitary from wild-type or Mep1"- mice (Fig. 6).

The expression of MESE-A was relatively high in both subcutaneous tumor graft samples. High level

The MESE-A transcript, which was observed in tumors treated with the antibody tAr (46-31), is potentially the result of a compensatory response to the systemic sequestration of MESE-A by the antibody tAB (36-31.

However, it is clear that increased MECE-A was not sufficient to trigger tumor growth.

While the average relative expression of MESEV-1 appeared to be quite unchanged in samples of different tissues, the average relative expression of MESEV-2 was obviously lower in tumor samples of i.v. treated with control Idsi or tA (26-31, compared to the expression in tumor grafts, small untreated tumors and in normal pituitary from wild-type or Mep1- mice (Fig. 6).

NMR allows you to monitor the growth of the tumor

MRI was used, as described above, to monitor the growth of the pituitary adenoma of the pterygium during the entire treatment period at a frequency of 1 time per two weeks. Graphs of a coronal section of the brain with a demonstrable adenoma from two Mep1"" mice, one of which was treated with the control IdS, and the other with the tAB antibody (6-31, shown in Fig. 7 for 9, 39 and 67 days after the start of treatment.

Histology of pituitary and pancreatic tumors

Pituitary adenomas of Mep17" were histologically similar in mice treated with tAb (36-31) and control dae (Fig. 8A-B). For these experiments, formalin-fixed tissue was dehydrated and embedded in paraffin, sections were made and stained with hematoxylin and eosin (NeE ) for histological analysis according to standard protocols.Typically, tumor cells were small (10 μm in diameter), with a high nuclear/cytoplasmic ratio, often mitotically active, with up to 40 mitotic figures per ten 625-μm diameter fields.

The tumors were variably solid or cystic, with multiple endothelial cell-lined vessels, acutely hemorrhagic areas (intact RBCs in areas without endothelial lining, no fibrin or cellular organization), and scattered hemosiderin-laden macrophages, suggestive of previous hemorrhage. Variable necrosis of individual cells and minimal fibrosis were noted.

Tumor vessels were irregularly arranged, typically 5-10 μm in diameter, although rarely up to 50 μm, with a small number of non-tumor perivascular stromal cells.

The vascular pattern in tumors treated with control da or tAbr (46-31) was observed using indirect immunohistochemical staining with the panendothelial cell marker antibody MESA-32 (Fig. 80-

AT). For these experiments, formalin-fixed, paraffin-embedded tissue sections were deparaffinized before quenching endogenous peroxidase activity and blocking with avidin and biotin (Mesiog, Vigpipedate, USA). Sections were blocked for 30 minutes with 1095 normal rabbit serum in RV5 with 395 B5A. The tissue sections were then incubated with primary antibodies for 60 minutes, biotinylated secondary antibodies for 30 minutes, incubated in ABC reagent (Mesiog, Vigipdate, USA) for 30 minutes, followed by a 5-minute incubation in metal-enriched Meia! Eppapsey SAV (Riegse, Nosktoga, USA). After that, the sections were counterstained with Meyer's hematoxylin. The primary antibody used was goat anti-mouse prolactin, clone MESAZ2, at 2 µg/ml (VO Viozsiepsez, Zap Doze, USA). The secondary antibodies used were biotinylated rabbit anti-goat antibody at 7.5 μg/ml (Mesiog, Vipipdate, USA) and biotinylated rabbit anti-rat antibody at 2.5 μg/ml (Mesiog, Vipipdate, USA). Staining of panendothelial cell antigen required pretreatment with Tagodei Neihem's reagent! (Bako, Sagrepiepa, USA) at 997C for 20 minutes.

All other steps were performed at room temperature.

The density of blood vessels significantly decreased under the influence of tar c6-31 treatment - to 4695 of the density of blood vessels in tumors treated with the control Ids (p - 0.009; Fig. 81). In both groups, the vascularity was less than in the normal adjacent anterior pituitary gland. To quantify vascular density, sections stained with MESA-32 were analyzed using the Agpo slide scanning platform! 51-50 (Arriey Itadipo; Zap dose, USA) and a 10x objective. Pituitary tumor sites were identified and delineated manually. Pixel colors corresponding to MESA-32 staining were determined and vessel area was measured accordingly. Tumor cell nuclei were identified by pixel color and object shape. The vessel area was then normalized to the number of pituitary tumor cells. Pancreatic islets were analyzed similarly, except that the area of MESA-32 staining was normalized to the area of the islet tumor.

In addition to pituitary tumors, pancreatic islet tumors were frequently identified in Mep!i"" mice and examined histologically at the end of treatment.

Islet tumors (defined as those greater than 105 μm? in the plane of section) were typically solid, without significant hemorrhage or necrosis. The mean area of tumors from six animals treated with anti-

MESE-A Mar (46-31 (n-32 tumors) was only 3995 of the average area of tumors from five animals treated with control IDS; (n-45 tumors; p - 0.026). Pancreatic adenomas (Fig. 8E -E), which were treated with control Ids, appeared to be generally larger (up to 7.0 mm in the plane of section) and more vascular than adenomas treated with tAB (46-31 (tumor diameter up to 5.0 mm in the plane of section). In in four of seven mice treated with control IdSy, pancreatic tumors contained dilated, blood-filled, thin-walled, endothelial cell-lined cavities up to 300 μm in diameter, whereas mice treated with tAB 26-31 often had no apparent vascularity (Fig. 82-H). Pancreatic tumors from one of eight mice treated with tAr (6-31) showed dilated vessels. Hemosiderin-laden macrophages were occasionally detected in pancreatic tumors in both groups of animals, indicating islet hemorrhage .

The density of vessels in islet tumors significantly decreased under the influence of treatment (46-31 - to 5695 density in islet tumors treated with the control Ids (p - 271077). In addition, "normal" islets (less than 109 μm? in the plane of section) from mice treated with tA C6-31 had reduced vascular density, but to a lesser extent to 7695 of the density in animals treated with control IdS (p - 47107) (see Fig. 8). The only male treated with control Ids had a single a tumor of the adrenal cortex with a diameter of 12 mm, which is a known type of tumor in the glands in MEM syndrome.

The histology of subcutaneous transplants of pituitary adenoma was comparable to the histology of hypophysis tumors, which indicates successful reproduction of endocrine tumor growth in distal locations.

Mep1 pituitary adenomas are prolactinomas

Approximately 6095 pituitary adenomas in MEM! patients secrete prolactin (RA), less than 2595 - growth hormone (GH) and 595 - adenocorticotropic hormone (ASTN, Tipitr eii ai., OOOUM 89:653-669 (1996)). To investigate whether pituitary adenomas of Mepi!77 mice secrete RAV, immunohistochemical anti-RVI staining was performed. antibodies on pituitary adenomas of ip zyts treated with control Ids and tAB (26-31, as well as on pituitary tumor transplants. Six of six pituitary adenomas treated with control da and five of five pituitary adenomas treated with tA (46-31, demonstrated specific positive staining for prolactin in approximately 50-9590 cells, which defines them as prolactinomas (Fig. 9A-98). Corresponding with this result is the report

Stgarigee her a. about the fact that Mepi"5M" pituitary tumors are positive for RVI. in immunohistochemical staining (Stabigye ei ai., Rhos. Mai. si OBA 98:1118-1123 (2001)). Prolactin staining was also positive in adenoma transplants of the pituitary gland in both groups (Fig. 90-90). Consistent with the functional secretion of prolactin from these tumors, the mammary gland tissue of female mice carrying pituitary adenomas transplanted consistently showed moderate to marked lactational changes in both the control and control groups on anti-MESE-A treatment (Fig. 9E, E).

Based on immunohistochemical staining, 6 of 11 untreated primary pituitary tumors were focally and weakly positive for growth hormone (Figure 14A), whereas only one of four transplanted pituitary tumors showed focal weak staining (Figure 148). The expression of growth hormone was present in the pituitary tumors of the ip 5th, which were treated with both the control IDS and the antibody (46-31 (fig. 6C, 0).

Normal tissue of the anterior pituitary showed strong reactivity in -20-3090o cells (Fig 14A).

Serum prolactin levels correlate with pituitary tumor volume in untreated or control-treated mice and are reduced by tAb treatment (26-31

Given that all examined pituitary adenomas from Mep!1-7 mice treated with both control Ide and tA (6-31) were positive for prolactin by immunohistochemical analysis, we investigated whether increased levels

RVI in the serum of Mep1:7 mice, which are carriers of pituitary adenomas. To this end, we first analyzed 46 untreated female Mepi mice for pituitary tumor status and serum RVI levels and five wild-type controls from the same littermate for serum RVI levels. The level of prolactin in the serum was analyzed using the Maiopa! Noppope 4 Reriide Rgodgat in Nagbog OSI A (Toitapse, USA).

Mice ranged in age from 10.5 to 15.6 months (mean age 13.3 months). The mean serum prolactin level in wild-type mice was 43.8x425.3 ng/ml. 27 out of 46 Mepi mice did not have a pituitary tumor on MRI. The mean serum prolactin level in these mice was 69.02424.6 ng/ml. . A group of 10 mice had small pituitary tumors with an average volume of 1,720.6 mm3. The serum prolactin level in these mice was elevated and averaged 188.7361.9 ng/ml. In 9 mice with large pituitary tumors (average volume 83.15423.8 mmu) mean serum prolactin level was 13239.823466.5 ng/ml. These data show that in Mep177 mice there is a positive correlation between serum prolactin level and tumor volume (Fig. 10A ) with the Pearson correlation coefficient A: -0.94, which indicates that the level of RAI in the serum can be used as a diagnostic tool in determining the status of a pituitary tumor.

To test whether anti-MESE-A treatment has an effect on serum prolactin levels, we analyzed the serum of seven Mep1:" mice with pituitary adenomas and ip mice treated with control IdS and seven of the same mice treated with tAb antibody (36- 31, at the end of the study (Day 67).In control Id mice, mean serum prolactin levels increased to 12566.723047.4 ng/ml and were generally higher as tumor volume increased (mean volume 116 2218 .5 mmu) with A-0.80 (p«0.03). In mice on tAr (26-31 the level of prolactin in the serum remained low, at an average level of 5163.7-41608.9 ng/ml, however, statistical correlation with tumor volume was not (mean volume 35.3246.5 mm3y), B-0.12 (p«0.80) (Fig. 108). Nevertheless, these data prove that tAr (46- 31 not only suppresses the growth of pituitary adenoma, but also leads to a decrease in the average level of prolactin in the serum, compared to the control Ida (p«e0.053).

Anti-MESE-A treatment reduces serum prolactin levels in mice with subcutaneous pituitary tumor transplants

Although the above data show that anti-MESE-A antibody treatment reduces serum prolactin levels in Mep177 tumor-bearing mice, we investigated this aspect further in the context of pituitary adenoma subcutaneous grafts in VaiYr/s nude mice. Prolactin in serum was determined in samples from 23 mice on control and 35 mice on tABr 26-31, taken at the beginning of treatment (Day 1) and at the end of the study (Day 35).

While on Day 1 the mean serum prolactin level was comparable between the two groups, on Day 35 treatment with tAbB (6-31) significantly reduced the prolactin level (Fig. 10C and 100, respectively).

Since the current treatment of MEM1 with prolactin includes drug therapy or planned hypophysectomy followed by radiation therapy, our data show that treatment with the antibody tAB (6-31) provides a decrease in serum prolactin levels with a marked inhibition of tumor growth, which is a possible new approach to the treatment of MEM patients! .

Serum insulin levels are elevated in Mep127 mice

To test whether serum insulin levels were elevated in Mep177 mice, serum samples from 6 non-starved mice treated with control da and b non-starved mice treated with tAr (36-31; all animals had histologically confirmed pancreatic lesions. Level insulin was also analyzed in 5 non-starved wild-type and young mice using the Shigazepime kit

Moivze Ipzcip EGIZA Kiy according to the manufacturer's instructions (Megsodia, Orrzaia, Sweden). No correlation with the type of treatment was observed, but the mean insulin level was markedly elevated in Mep1-" mice (control Id, 3.8:22.6 ng/ml; tAB (6-31, 3,722.4 ng/ml), compared with wild-type mice (1,420.7 ng/ml).

Discussion

Our data suggest that MESE-A is necessary for the growth of benign pituitary adenomas in the MEMI mouse model, as anti-MESE-A monoclonal antibody therapy was shown to be sufficient to inhibit tumor growth.

Based on the available literature, it can be considered possible that many of the antitumor effects of tAb (46-31) are mediated by inhibition of MESEV-2-dependent angiogenesis (M/ize ei ai!., Rgos. Mai). Asai. 5si. 96:3071- 3076 (1999) and 7aspagu eyi ai., Sagaiomazs. Vez. 49:568-581 (2001)). Although anti-MESEN-A treatment did not significantly affect the relative mean expression of MESEN-2 mRNA in large pituitary adenomas (Fig. 6), immunohistochemical staining for MESEN-32 showed a highly significant decrease in vascularity (by approximately 5095) in pituitary and pancreatic tumors under the effect of tAbB antibody treatment (6-31. A corresponding decrease in tumor growth under the effect of anti-MESE-A treatment was observed both in the case of pituitary adenoma and in the case of pancreatic adenoma. The density of vessels in normal pancreatic islets was also significantly reduced by anti-MESE- And the treatment, although the magnitude of such changes (2595 reduction compared to control IDS treatment) was smaller than that observed in pancreatic adenomas.

The high level of MESE-A transcript observed in tumors treated with the antibody tAB (26-31) may be the result of a compensatory response to the sequestration of MESE-A by the antibody tAB C6-31. However, it is clear that such increased MESE-A is not sufficient to trigger tumor growth.

In addition to demonstrating that treatment with anti-MESE-A tAbBr (26-31) as monotherapy significantly reduces the tumor burden in Mep17 mice due to effective inhibition of adenoma growth, we showed that serum prolactin levels are also reduced. Thus, our data suggest the possibility of non-surgical treatment of benign tumors of the endocrine system with cessation of disease progression without the use of chemotherapeutic drugs. Alternatively, such blockade of MESE-A can be combined with an appropriate pharmacological drug. For example, in the case of prolactin-secreting adenomas, an antagonist of MESE-A can be combined with dopamine agonist.

Example 3. Effectiveness of anti-MESE intervention and regression/survival efficacy in the HIV-TVAD model of multistage carcinogenesis

In order to better understand the role of anti-MESEE therapy at different stages of tumor growth, we turned to a number of preclinical tumor models, including HIV-TrAd. AIR-TrAd (Ekheichyz, Ips.) is a conditional version of the pancreatic islet tumor model in mice, which is triggered by transgenic expression of the antigen 5M40 I agde T (TAd)

(targeting the p-cell of the pancreas, when Tad functions as a strong oncogene by binding p52 and AB). HIV-

TrVAd is phenotypically similar to the HIV-Tad model previously described (Napanap, Maite 315:115-122 (1985);

Vegdegs ei ai., 5siepse 284 (808-811), 1999). We found that this model is capable of representing progression through a series of increasingly aggressive stages, including activation of the MESE signaling system and the "angiogenic switch" (ie, initiation of the process of new blood vessel formation) in approximately 5 weeks. Small tumors are formed in 10 weeks, which coincides with the beginning of their malignant transformation. Large invasive carcinomas are formed in 12 weeks. Thus, until 10 weeks of age, the growth of cells in mice cannot be malignant or metastatic. The age period between 10 and 12 weeks generally includes the formation of invasive cancer.

For these experiments, HIV-TrAd mice were maintained in accordance with standard IASIS recommendations; they received a high-sugar diet and 59o sucrose water to alleviate symptoms of hyperinsulinemia caused by an increase in insulin-secreting beta cells in the pancreas. At the age of 9-9.5 or 11-12 weeks, mice were injected intraperitoneally twice a week with 5 mg/kg anti-MESE antibody or an isotype-related anti-pollen control monoclonal antibody in sterile, phosphate-buffered saline. In the "intervention" experiment, mice aged 9-9.5 weeks were treated for 14 days, after which they were examined. In the "regression" experiment, 11-12-week-old mice were studied after 7, 14 and 21 days of treatment.

To assess survival, another group of mice was treated until the mice became ill or died. After a specified interval in the "intervention" and "regression" experiments, the pancreas and spleen of each mouse were removed and photographed. The number of tumors in the pancreas was determined by excision of each spherical tumor and counting. The volume of tumors was determined by measuring the two largest diameters of each tumor, after which the formula 0.52 (xu) was used. To determine the total tumor burden, the volumes of all detected tumors were calculated. Means and standard deviations were calculated and data were presented graphically using Misgozoy Ehse! m11.3.3 (Mysgozoi, Ips.). Statistical comparisons between the number and volume of tumors in different groups were performed using Her Student's test. Kaplan-Meier curves for survival analysis were constructed using UMR 6.0 (5A5 Ipeyshie, Ips.), statistical comparisons were performed using log-rank analysis.

Treatment of 9-week-old animals ("intervention" experiment) with anti-MESE antibodies resulted in a sharp decrease in tumor angiogenesis (Fig. 11). Treatment of 11-week-old animals ("regression" experiment) led to a decrease in the vascularity and proliferation of tumors (Fig. 12A). However, unlike the "intervention" experiment, only a temporary reduction in tumor growth was detected without any effect on survival (Fig. 128). These studies have demonstrated that early tumors are more sensitive to anti-MESE targeted therapy compared to advanced tumors.

Example 4. Anti-MESEE and chemotherapy are effective in preclinical models

Surgery may leave behind residual tumor cells or dormant micrometastatic nodules that have the potential to reactivate the “angiogenic program” and promote more exponential tumor growth.

We used genetic mouse tumors and human xenografts to model tumor dormancy, followed by heavy chemotherapy regimens to “cytoreduce” the tumor concurrently or sequentially with anti-MESE therapy and subsequent maintenance therapy with anti-MESE monoclonal antibodies.

Therefore, we are more likely to treat an animal to prevent tumor recurrence than to pursue a tumor after it has been removed or reformed, including, in certain cases, a tumor that is refractory, recurrent, or resistant to other therapy regimens, including targeted anti-MESE therapy regimens. Fig. 13 demonstrates the observation that docetaxel is effective in reducing tumor burden, but dormant cells begin to regrow after approximately 2 months. At the same time, simultaneous treatment with anti-MESE suppresses tumor regrowth, despite the fact that it alone has little effect on the growth of larger, more established tumors. Additional data demonstrate that prolonged anti-MESE therapy inhibits tumor regrowth even after cytoreduction with taxanes or gemcitabine. These results demonstrate that anti-MESE can be used to effectively block the growth or regrowth of dormant tumors or micrometastases. These data are consistent with the fact that neovascularization is a necessary prerequisite for the rapid clonal expansion associated with macroscopic tumorigenesis and support the use of MESE-specific antagonists (eg, anti-MESE antibodies, including, but not limited to, b-series antibodies or B20) in maintaining the dormant state of the tumor and suppressing the regrowth of the tumor after the initial treatment of the tumor and before the formation of new tumors, reactivation of dormant tumors, malignant tumors or micrometastases.

Example 5. Neoadjuvant therapy using bevacizumab

This example illustrates the use of bevacizumab in the neoadjuvant therapy of patients with operable, metastatic breast cancer.

Neoadjuvant therapy has been widely used in the treatment of locally advanced or potentially operable large breast tumors. Randomized trials have shown that neoadjuvant therapy reduces the need for mastectomy (thus conserving the breast) and provides an overall survival close to that of adjuvant chemotherapy (Romez et al., 9U. SiIip. Opsoi. 13:547-52 ( 1995);

EizNeg eyi ai., u. Siip. Opsoi 16:2672-85 (1998)).

The main goal of this therapy is to provide an improved clinical effect due to the addition of bevacizumab to chemotherapy in patients with operable, indolent breast cancer. In particular, one of the primary measurements of the clinical effectiveness of neoadjuvant therapy can be the frequency of complete response to treatment (rSV). Other measures include overall response rate (OR), clinical complete response rate (CCR), disease-free survival (OE5) and overall survival (05). Moreover, the side effects of such treatment can be monitored, for example, by indicators such as the frequency of surgical complications, toxicity and adverse effects on cardiac function. The expression of certain genes or the activity of other biomarkers can also be used as markers of response to treatment.

Complete response to treatment (CRES) was used as a prognostic marker of treatment efficacy. rap characterizes the absence of residual histological signs of the tumor after neoadjuvant therapy during surgery.

The results of the conducted studies allow us to assume that patients who have achieved PCA have a significantly prolonged survival. However, there is no standard method for assessing response to treatment, and the use of roaring as a surrogate marker of efficacy remains controversial.

Patients eligible for neoadjuvant therapy with MESE-specific antagonists are selected based on predefined criteria and guidelines that vary depending on the specific type of cancer being treated. For example, patients can be selected on the basis of life expectancy, age, tumor histology, hematological function, liver and heart function, treatment dynamics, reproductive status and plans, as well as psychiatric status and harmful habits. More importantly, the primary tumor must be measurable and/or operable.

Treatment regimens include chemotherapy plus an MACE-specific antagonist such as bevacizumab.

Optionally, additional therapeutic drugs can also be used in this scheme. Typically, the chemotherapy regimen that is commonly used for a given type of cancer (eg, the standard treatment regimen) is implemented in combination with bevacizumab. For example, for neoadjuvant treatment of breast cancer, docetaxel, paclitaxel or doxorubicin/cyclophosphamide (AC) can be used in combination with bevacizumab. Alternatively, a docetaxel-based regimen or a paclitaxel-based regimen and an AC regimen may be used sequentially as chemotherapy combined with bevacizumab. For the treatment of non-small cell lung cancer, cisplatin (alone or in combination with other chemotherapy drugs such as gemcitabine, docetaxel or vinorelbine) can be used as the main chemotherapy drug in combination with bevacizumab. On the other hand, 5-fluorouracil-based regimens (such as ROI OX) can be used in combination with bevacizumab for the treatment of colorectal cancer.

Chemotherapy drugs and an MECE-specific antagonist, such as bevacizumab, are administered to patients in defined doses and at set intervals in the form of a series of cycles. For example, bevacizumab can be given at a dose of 15 mg/kg every three weeks for 4 cycles or 10 mg/kg every two weeks for 6 cycles. In another example

A MES-specific antagonist, such as bevacizumab, is administered at a dose of 7.5 mg/kg once every two or three weeks.

Patients' response to treatment is monitored. Upon completion of neoadjuvant therapy, patients undergo surgery to remove the primary tumor that was operable before neoadjuvant therapy or became operable after such therapy. After surgery, patients continue to receive therapy with an MACE-specific antagonist, such as bevacizumab, with or without chemotherapy, depending on the specific status of the patient. The patient's progress is monitored during and after treatment.

Adverse events associated with anti-MESE treatment should also be closely monitored with care. Major adverse events known from previous studies include hypertension, proteinuria, bleeding, thromboembolic events, gastrointestinal perforation/fistula, and wound complications. Certain adverse events, such as hypertension, can be treated with medication. If adverse events are severe and uncontrolled, treatment should be discontinued.

Example 6. Adjuvant therapy using bevacizumab

This example illustrates the use of bevacizumab combined with chemotherapy in the adjuvant therapy of patients with resected cancer.

Patients eligible for adjuvant bevacizumab therapy are selected based on predefined criteria and guidelines that vary depending on the specific type of cancer being treated. For example, patients may be selected based on life expectancy, age, cancer histology, hematological function, liver and heart function, lifestyle, treatment dynamics, reproductive status and plans, as well as psychiatric status and harmful habits. More importantly, patients should undergo complete resection of the cancer prior to adjuvant therapy. Acceptable types of resection depend on the specific type of tumor. In addition, sufficient biological material representative of the patient's tumor must be available for baseline analysis and to determine efficacy. At the time of such treatment, the surgical intervention must be quite recent and, however, this interval must be sufficient for the patient to fully recover from the operation. For example, patients should start taking adjuvant therapy no earlier than 3-6 weeks after surgery and no later than 8-12 weeks after surgery.

Treatment regimens include chemotherapy plus bevacizumab. Optionally, additional therapeutic drugs can also be used in this scheme. Typically, chemotherapy drugs in combination with bevacizumab are used in the first stage of treatment, and then bevacizumab is used as maintenance monotherapy. For example, patients are treated with chemotherapy drugs and bevacizumab for about 4-12 cycles, and then bevacizumab alone for 1-2 years. The duration of each cycle of treatment with the chemotherapy drug bevacizumab depends on the specific drugs and doses used.

For example, bevacizumab can be given at a dose of 15 mg/kg every 3 weeks as a single cycle or at a dose of 5 mg/kg every two weeks as a single cycle. In another example, bevacizumab can be given at a dose of 7.5 mg/kg or 10 mg/kg every two or every three weeks.

Typically, the chemotherapy regimen that is commonly used for a given type of cancer (eg, the standard treatment regimen) is implemented in combination with bevacizumab. For example, for the adjuvant treatment of breast cancer, docetaxel, paclitaxel or doxorubicin/cyclophosphamide (AC) can be used in combination with bevacizumab. Alternatively, a docetaxel-based regimen or a paclitaxel-based regimen and an AC regimen may be used sequentially as chemotherapy combined with bevacizumab. For the treatment of non-small cell lung cancer, cisplatin (alone or in combination with other chemotherapy drugs such as gemcitabine, docetaxel or vinorelbine) can be used as the main chemotherapy drug in combination with bevacizumab. On the other hand, for the treatment of colorectal cancer, regimens based on 5-fluorouracil (such as ROG ROX) can be used in combination with bevacizumab.

The goal of adjuvant treatment with bevacizumab is to improve patient survival, preferably disease-free. In the meantime, any adverse events associated with bevacizumab should be carefully monitored, especially because adjuvant therapy is long-term. Survival can be estimated by the Kaplan-Meier method, and any difference in survival is calculated using the stratified log-rank test. Multivariate analyzes using the Cox proportional hazards model were used to assess the simultaneous effects of prognostic factors on survival. The 5A5 statistical software package was used for all calculations. Data were considered statistically significant when the P value was 0.05 or less. All statistical tests were two-sided.

Adverse events associated with anti-MESE treatment should also be closely monitored with appropriate care. Major adverse events known from previous studies include hypertension, proteinuria, bleeding, thromboembolic events, gastrointestinal perforation/fistula, and wound complications. Certain adverse events, such as hypertension, can be treated with medication. If adverse events are severe and uncontrolled, treatment should be discontinued.

Other implementation options

It should be apparent from the foregoing description that variations and modifications of this invention may be made to adapt it to various applications and conditions. Such variants of implementation are also included in the scope of the invention, as defined by the following formula.

All publications, patent applications and patents mentioned in this description, including previous US patent applications

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Fig. 12A

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Fig.

LIST OF SEQUENCES

"10" GENENTEC, INC. "1205 MESOR. SPECIFIC ANTAGONISTS FOR ADJUVANT AND NEOADJUVANT

THERAPIES AND TREATMENT OF TUMORS AT THE EARLY STAGE

"1302 BO4TADIYAAUOV "10" This is 08397 "1812 2007-11-20 "18505 8 ОЯА For "151" 2007-07-05 "150" ОВ 8(у819,638 kіі5Б1" 2007-03-22 "18502 58087755 "15i" 2008-12-27 "1505 5 850/870,745 "1512 2008-12-19 "Т5О0з Bad "1512 ДО0Б-12-19 "1805 5 "1705 Patent in version 33 "r 1 "rii" 24 "122 DNA "2132 Artificial sequence "ОО" "028 synthetic spascisasasi vysovaisa yus. 24 "in"

"rii" 23

"212" DNA

"genus" An artificial sequence

"share"

keoa" is synthetic

"400"

passive vacuum cleaners 959 23 "2105 3

"her". 25

"217" DNA

"213" Artificial sequence di"

"223" is synthetic

"4005 3 ayuchesavi aidasissasesisasa o "oh 4

"visa 25

"2125 DNA

"913" Artificial sequence

"at

"ve" is synthetic

"00" 4 "10" 5

"211" 25

"Weigh DNA

"213" Artificial sequence

"Oh

"ve" is synthetic

"400" 5 avossoais ssvisnssa dozosdatsa. 23