KR101141081B1 - Cancerous disease modifying antibodies - Google Patents

Abstract

The present invention is directed to methods of producing cancer disease modulating antibodies using the novel screening paradigm. The method allows the preparation of anti-cancer antibodies for therapeutic and diagnostic purposes by separating anti-cancer antibodies using cancer cell cytotoxicity as a endpoint. The antibodies can be used to aid in staging and diagnosis of cancer, and can be used to treat primary tumors and tumor metastases. The anti-cancer antibody may bind to toxins, enzymes, radioactive compounds, cytokines, interferons, target or reporter moieties and hematopoietic cells.

CDMAB, antibody, antibody-ligand, cancer, chemotherapeutic agent, binding assay

Description

Cancer Disease Control Antibody {CANCEROUS DISEASE MODIFYING ANTIBODIES}

The present invention relates to the isolation and preparation of cancerous disease modifying antibodies (CDMABs) and the use of these CDMABs alone or in combination of one or more CDMAB / chemotherapeutic agents in the course of treatment and diagnostics. The invention also relates to a binding assay using the CDMAB of the invention.

Monoclonal Antibodies as Cancer Therapies: Each individual who exhibits a cancer is unique and has a cancer that differs from other cancers as well as that individual's identity. Despite this, current treatments treat all patients with the same type of cancer at the same stage in the same way. At least 30% of these patients will fail in primary treatment, leading to the next order of treatment, which increases the likelihood of treatment failure, metastasis, and eventually death. A better approach to treatment would be tailored care for a particular individual. The only current treatment suitable for customization is surgery. Chemotherapy and radiation therapy cannot be tailored to the patient, and surgery is in itself insufficient to cause healing in most cases.

With the advent of monoclonal antibodies, the possibility of developing custom therapeutic methods has become more realistic because each antibody can be directed to only one epitope. Furthermore, it is possible to prepare antibody combinations directed to a group of epitopes that uniquely define a tumor of a particular individual.

Recognizing that the significant difference between cancer cells and normal cells is that the cancer cells have antigens specific for the modified cells, the scientific community has long identified specific modified cells by allowing them to specifically bind monoclonal antibodies to these cancer antigens. I thought it could be designed to be targeted with; This has led to the belief that monoclonal antibodies can function as "magic bullets" to kill cancer cells. However, it is now widely recognized that there is no single monoclonal antibody that can function in all cases of cancer, and that monoclonal antibodies can be developed as a class as targeted cancer therapy. Monoclonal antibodies isolated in accordance with the teachings of the present disclosure have been shown to modulate cancer disease progression in a manner beneficial to the patient, for example by reducing tumor burden, and they herein control cancer disease Various references will be made to antibodies (CDMAB) or "anti-cancer" antibodies.

Currently, cancer patients usually have few treatment options to choose from. A strictly controlled approach to cancer therapy has led to an improvement in overall survival and morbidity. However, for certain individuals, these improved statistics are not necessarily correlated with the improvement of their personal situation.

Thus, if a methodology was proposed that would allow physicians to treat each tumor independently of other patients in the same cohort, this would enable a unique tailored therapeutic approach for that very person. Such a course of treatment would ideally increase the healing rate and produce better results, thus satisfying the long perceived needs.

Historically, the use of polyclonal antibodies has shown limited success in treating human cancers. Lymphoma and leukemia were treated with human plasma but there was little long-term relief or response. In addition, there was a lack of reproducibility and no additional benefit compared to chemotherapy. Solid tumors such as breast cancer, melanoma and renal cell carcinoma were also treated with human blood, chimpanzee serum, human plasma and horse serum, but the results were likewise unpredictable and ineffective.

There have been many clinical trials using monoclonal antibodies against solid tumors. In the 1980s, there were at least four clinical trials for human breast cancer, using antibodies to specific antigens or based on tissue selectivity, with only one responder from at least 47 patients. It was only in 1998 that there was a successful clinical trial using humanized anti-Her2 / neu antibody (Herceptin ^® ) with CISPLATIN. In the experiment, 37 patients were evaluated for response, with about a quarter of these showing partial response rates and another quarter showing local or stable disease progression. The median time to disease progression in these responders was 8.4 months and the median duration of response was 5.3 months.

Herceptin ^® has been approved for use primarily used in combination with Taxol ^® in 1998. Clinical studies showed that in patients receiving antibody therapy + Taxol ^® , the median time to disease progression (6.9 months) was increased compared to the group receiving Taxol ^® alone (3.0 months). There was also a slight increase in median survival; 22 months versus 18 months for the Herceptin ^® + Taxol ^® treatment vs. Taxol ^® alone treatment. In addition, the number of both complete responders (8 vs. 2%) and partial responders (34 vs. 15%) increased in the antibody + Taxol ^® combination group compared to the Taxol ^® alone group. However, treatment with Herceptin ^® and Taxol ^® resulted in a higher incidence of cardiotoxicity compared to Taxol ^® alone treatment (13 vs 1%). In addition, Herceptin ^® therapy is available for patients overexpressing human epidermal growth factor receptor 2 (Her2 / neu) (as determined by immunohistochemistry (IHC) analysis), a receptor that is free of currently known functional or biologically important ligands. Only (approximately 25% of patients with metastatic breast cancer). Thus, great needs are not yet met in patients with breast cancer. Even patients who can benefit from Herceptin ^® treatment still need chemotherapy, so the side effects of this kind of treatment will still have to be addressed, at least to some extent.

Clinical trials studying colorectal cancer include antibodies to both glycoproteins and glycolipid targets. Antibodies such as 17-1A, with some specificity for adenocarcinoma, have undergone phase 2 clinical trials in over 60 patients, with only one patient responding partially. In other experiments, the use of 17-1A in a protocol further using cyclophosphamide resulted in only one complete response and two small responses in 52 patients. To date, phase III clinical trials for 17-1A have not demonstrated improved efficacy as adjuvant therapy for stage 3 colorectal cancer. The use of the first approved humanized murine monoclonal antibody for imaging also did not cause tumor degeneration.

Only recently have there been some positive results from colorectal cancer clinical studies using monoclonal antibodies. In 2004, ERBITUX ^® was approved for secondary treatment in patients with EGFR-expressing metastatic colorectal cancer, for which irinotecan-based chemotherapy was not effective. Was a 2-group (two-arm) phase II clinical studies and 23 and 15% The results from both the single group (single arm) study, ERBITUX ^® for each of irinotecan when used in combination with the response rate, the median time to disease progression It was shown that they were 4.1 and 6.5 months respectively. From the same 2-group II clinical study and another single-group study, treatment with ERBITUX ^® alone produced 11 and 9% response rates, respectively, with median time to disease progression being 1.5 and 4.2 months, respectively. appear.

As a result, both the Swiss and the United States, one in the ERBITUX ^® treatment in combination with irinotecan, and the United States, the ERBITUX ^® monotherapy, was approved as a secondary treatment for patients with primary colorectal cancer results failed irinotecan therapy. Thus, like Herceptin ^® , treatment in Switzerland is only approved as a combination of monoclonal antibody and chemotherapy. In addition, treatment in both Switzerland and the US is only approved as a secondary therapy for patients. Also, in 2004, AVASTIN ^® is uracil with intravenous 5-fluorouracil as a primary treatment for metastatic colorectal cancer - have been approved to use in combination with chemotherapy based. Phase III clinical studies demonstrated that the median survival of patients treated with AVASTIN ^® + 5-fluorouracil was extended compared to patients treated with 5-fluorouracil alone (20 months vs. 20 months). 16 months). However, as with Herceptin ^® and ERBITUX ^® , the treatment is only approved as a combination of monoclonal antibody and chemotherapy.

The results for lung-, brain-, ovary-, pancreas-, prostate- and gastric cancers are also consistently poor. The most promising recent results for non-small cell lung cancer have been described in the treatment of monoclonal antibodies (SGN-15; dox-BR96, anti-Sialyl-LeX) bound to the cell-killing drug doxorubicin in combination with the chemotherapeutic agent TAXOTERE ^® . From phase II clinical trials included. TAXOTERE ^® is the only FDA approved chemotherapy for secondary treatment of lung cancer. Initial data show an improvement in overall survival compared to TAXOTERE ^® alone. Of the 62 patients recruited for the study, two-thirds there was administered SGN-15 in combination with TAXOTERE ^® remaining one third was administered only TAXOTERE ^®. For patients receiving SGN-15 in combination with TAXOTERE ^® , the median overall survival was 7.3 months, compared to 5.9 months for patients receiving TAXOTERE ^® alone. Overall survival at 1 year and 18 months in patients receiving SGN-15 + TAXOTERE ^® was 29 and 18%, respectively, compared to 24 and 8% in patients receiving TAXOTERE ^® alone, respectively. Additional clinical trials are planned.

Preclinically, there has been some limited success in using monoclonal antibodies against melanoma. Few of these antibodies have reached clinical trials, and none of them have been approved or demonstrated positive results in phase III clinical trials to date.

The delay in finding new drugs to treat the disease lies in the failure to identify relevant targets among the products of the 30,000 known genes that may contribute to disease etiology. In oncological studies, potential drug targets are often selected simply because of the fact that they are overexpressed in tumor cells. The targets so identified are then screened for interaction with multiple compounds. In the case of potential antibody therapies, these candidate compounds are usually derived by traditional monoclonal antibody production according to the fundamental principles established by Kohler and Milstein (1975, Nature, 256, 495-497, Kohler and Milstein). Spleen cells are collected from mice immunized with antigens (such as whole cells, cell fractions, purified antigens) and fused with immortalized hybridoma counterparts. The resulting hybridomas are screened to secrete antibodies that bind most thermally to the target. Including Herceptin ^® and RITUXIMAB, a number of therapeutic and diagnostic antibodies against the cancer cell was prepared using this method, it was selected on the basis of their affinity. The drawback of this strategy is double. First, selecting appropriate targets for therapeutic or diagnostic antibody binding is achieved by extremely simple methods such as screening by overexpression, identifying these targets and, consequently, a lack of knowledge surrounding tissue specific carcinogenic processes. Limited. Second, the assumption that a drug molecule that binds with the greatest affinity to the receptor usually has the greatest potential to initiate or inhibit a signal may not always be.

Despite some progress in the treatment of breast- and colon cancer, either as a single agent or as a co-treatment, identification and development of effective antibody therapy has been inadequate for all types of cancer.

Prior patents:

U.S. Patent 5,750, 102 discloses a method of transfecting cells from a patient's tumor with an MHC gene, which may be cloned from the patient's cells or tissues. These transfected cells are then used for vaccination into the patient.

U.S. Patent 4,861,581 discloses monoclonal antibodies specific for the internal cellular components of mammalian neoplastic and normal cells but not for the external components, labeling the monoclonal antibodies and labeling the antibodies. Contacting tissue of a treated mammal that kills neoplastic cells and measuring the binding of the labeled antibody to internal cellular components of degenerative neoplastic cells to determine the effectiveness of the therapy. A method is disclosed. In preparing antibodies directed against human intracellular antigens, the patentee recognizes that malignant cells represent a convenient source for such antigens.

U.S. Patent 5,171,665 provides novel antibodies and methods for their preparation. Specifically, the patent teaches the formation of monoclonal antibodies that have the property of binding strongly to protein antigens to which human tumors, such as colon and lung tumors, are bound at a much lower level to normal cells.

U.S. Patent No. 5,484,596 surgically excises tumor tissue from a human cancer patient, treats the tumor tissue to obtain tumor cells, and irradiates the tumor cells to be viable but non-tumorigenic. And using these cells to produce a vaccine for the patient, which can inhibit the recurrence of the primary tumor while simultaneously inhibiting metastasis. The patent teaches the development of monoclonal antibodies reactive with surface antigens of tumor cells. As disclosed in column 4, line 45 and below, the patentee uses autologous tumor cells in the development of monoclonal antibodies that exhibit specific immunotherapy of activity in human neoplasia.

U.S. Patent 5,693,763 teaches glycoprotein antigens that are specific to human carcinoma and are independent of epithelial tissue of origin.

U.S. Patent No. 5,783,186 relates to an anti-Her2 antibody that induces apoptosis in Her2 expressing cells, a hybridoma cell line producing the antibody, a method for treating cancer using the antibody, and a pharmaceutical composition comprising the antibody. .

U.S. Patent 5,849,876 describes a new hybridoma cell line for the production of monoclonal antibodies against mucin antigens purified from tumor and non-tumor tissue sources.

U.S. Patent 5,869,268 relates to the production of human lymphocytes producing antibodies specific for the antigen of interest, to the preparation of monoclonal antibodies, and to monoclonal antibodies prepared by the above methods. The patent relates in particular to the preparation of anti-HD human monoclonal antibodies useful for the diagnosis and treatment of cancer.

U.S. Patent 5,869,045 relates to antibodies, antibody fragments, antibody conjugates and single chain immunotoxins that are reactive to human carcinoma cells. The mechanism by which these antibodies function is that the molecules are responsive to cell membrane antigens present on the surface of human carcinoma, and also have the ability to internalize into the carcinoma cells after binding, resulting in antibody-drug and antibody- It is dual in that it becomes particularly useful for forming toxin conjugates. In unmodified form the antibodies also exhibit cytotoxicity characteristics at certain concentrations.

U.S. Patent 5,780,033 discloses the use of autoantibodies for the treatment and prevention of tumors. However, these antibodies are antinuclear autoantibodies from older mammals. In such cases, the autoantibodies are called one type of natural antibody found in the immune system. Since the autoantibody is from an "elderly mammal", the autoantibody need not necessarily be from a patient to be treated. The patent also discloses natural and monoclonal antinuclear autoantibodies from older mammals, and hybridoma cell lines producing monoclonal antinuclear autoantibodies.

Summary of the Invention

The present application is directed to U.S. isolates that isolate hybridoma cell lines encoding cancer disease regulatory monoclonal antibodies. The methodology of making patient specific anti-cancer antibodies taught in the 6,180,357 patent is used. These antibodies can be prepared specifically for one tumor, thus allowing for customization of cancer therapies. Within the context of the present application, anti-cancer antibodies having either cell-killing (cytotoxicity) or cell-growth inhibition (cell proliferation inhibition) properties will be referred to as cytotoxic below. These antibodies can be used to aid in staging and diagnosis of cancer and can be used to treat tumor metastasis. These antibodies can also be used for the prevention of cancer as a prophylactic treatment. Unlike antibodies produced according to conventional drug development paradigms, antibodies generated in this manner can target molecules and pathways that have not previously appeared essential for the growth and / or survival of malignant tissue. Furthermore, the binding affinity of these antibodies is suitable for the requirement for initiation of cytotoxic events that may not conform to stronger affinity interactions. It is also within the scope of the present invention to combine standard chemotherapy modalities (such as radionuclides) with the CDMAB of the present invention to focus on the use of such chemotherapy. CDMAB can also be bound to toxins, cytotoxic residues, enzymes such as biotin binding enzymes, cytokines, interferons, target or reporter moieties or hematogenous cells to form antibody conjugates. CDMAB can be used alone or in combination with one or more CDMAB / chemotherapeutic agents.

The possibility of individualized anti-cancer therapies will lead to changes in the way patients are managed. A probable clinical scenario is to obtain and store tumor samples at the time of appearance. From the sample, the type of tumor can be determined from a group of previously existing cancer disease regulatory antibodies. The staging of the patient will be done in a conventional manner, but the available antibodies can be used for further staging for the patient. The patient can be immediately treated with the existing antibodies, and the antibody specific for the tumor can contain phage display libraries using the methods outlined herein or in conjunction with the screening methods disclosed herein. Can be prepared. All antibodies produced will be added to the anti-cancer antibody library because it is likely that other tumors contain some of the same epitopes as are being treated. Antibodies prepared according to the methods may be useful for treating cancer disease in any number of patients with cancer that binds to these antibodies.

In addition to anti-cancer antibodies, the patient may choose to receive the currently recommended therapy as part of a multiple therapy regimen. The fact that the antibodies isolated by this methodology are relatively non-toxic to non-cancer cells allows the combination of antibodies to be used in high doses, either alone or in combination with conventional therapies. The high therapeutic index also allows for short-term retreatment, which will reduce the likelihood of treatment resistant cells.

If the patient is refractory or metastasis occurs during the initial course of therapy, the method may be repeated to generate specific antibodies to the tumor for re-treatment. Furthermore, the anti-cancer antibody can be re-injected for the treatment of metastasis by binding to erythrocytes obtained from the patient. There has been little effective treatment for metastatic cancer, and metastasis is usually a precursor to a poor prognosis leading to death. However, metastatic cancer is usually well vascularized, so the delivery of anti-cancer antibodies by red blood cells can have the effect of focusing the antibodies on the tumor site. Even before metastasis, most cancer cells rely on the blood supply of the host for their survival, and anti-cancer antibodies bound to erythrocytes can be equally effective against in situ tumors. Alternatively, the antibodies may bind to other hematogenous cells such as lymphocytes, macrophages, monocytes, natural killer cells, and the like.

There are five classes of antibodies, each associated with a function conferred on its heavy chain. Cancer cell killing by naked antibodies is generally thought to be mediated through either antibody dependent cytotoxicity or complement dependent cytotoxicity. For example, murine IgM and IgG2a antibodies can activate human complement by binding to the C-1 component of the complement system, thereby activating the typical complement activation pathway that can lead to tumor lysis. The most effective complement activating antibodies for human antibodies are generally IgM and IgG1. The murine antibodies of the IgG2a and IgG3 isotypes are effective in collecting cytotoxic cells with Fc receptors, which cause cell killing by monocytes, macrophages, granulocytes and certain lymphocytes. Human antibodies of the IgG1 and IgG3 isotypes both mediate ADCC.

Cytotoxicity mediated through the Fc region requires the presence of effector cells and their corresponding receptors, or proteins such as NK cells, complement, and T-cells, respectively. In the absence of these effector mechanisms, the Fc portion of the antibody is inactive. The Fc portion of an antibody may confer properties that affect the pharmacokinetics of the antibody in vivo, but it has no functionality in vitro.

In the cytotoxicity assays in which we test antibodies, none of these effector mechanisms are present and performed in vitro. There are no effector cells (NK, macrophages, or T-cells) or complement in these assays. Since these assays are wholly defined by being added together, each component can be characterized. Assays used herein comprise only target cells, medium and serum. Because target cells are cancer cells or fibroblasts, they have no effector function. Without exogenous cells with effector functional properties, there are no cellular elements with such function. The medium does not contain complement or any cells. Serum used to support the growth of target cells does not have complement activity, as vendors have revealed. Furthermore, in our laboratory we confirmed the absence of complement activity in the serum used. Thus, our study demonstrates that the effect of these antibodies is due solely to the effect of antigen binding mediated through their Fab. In fact, since target cells do not have a receptor for Fc, only Fab recognizes and interacts with it. Although hybridomas secrete complete immunoglobulins tested as target cells, only a portion of the immunoglobulins that interact with the cells are Fabs, which act as antigen binding fragments.

With respect to the antibodies and antigen binding fragments claimed herein, the present application submitted as evidenced by the data in Table 1 demonstrated cellular cytotoxicity. As noted above, and as evidenced by objective evidence herein, such effects were solely due to the binding of Fab to tumor cells.

There is a wealth of evidence in the art regarding antibody mediated cytotoxicity due to direct binding of antibodies to target antigens, regardless of effector mechanisms convened by the Fc. The best evidence for this is in vitro experiments with no complementary cells or complement (and thus these mechanisms are formally excluded). This type of experiment was performed using antigen binding fragments such as complete immunoglobulins, or F (ab) '2 fragments. In this type of experiment, antibodies or antigen binding fragments directly induce apoptosis of target cells, as in the case of anti-Her2 and anti-EGFR antibodies with antibodies approved for sale in cancer treatment by the US FDA. can do.

Another possible mechanism of antibody mediated cancer killing may be through the use of antibodies, so-called catalytic antibodies, which function to catalyze the hydrolysis of various chemical bonds in the cell membrane and the glycoprotein or glycolipid bound thereto.

There are three additional mechanisms for antibody-mediated cancer cell killing. The first is to use antibodies as vaccines to induce the body to generate an immune response to putative antigens present on cancer cells. The second is to use antibodies that target growth receptors to interfere with their function or to down regulate the receptors so that their function is effectively lost. Third, these antibodies have an effect on direct ligation of cell surface parts that can lead to direct cell death, such as a death receptor such as TRAIL R1 or TRAIL R2, or integrin molecules such as alpha V beta 3.

The clinical utility of the anticancer agent is based on the benefit of the drug under an acceptable risk profile for that patient. Although survival has generally been the most sought benefit in cancer therapy, there are many other widely recognized benefits besides prolonging life. These other benefits when treatment does not adversely affect survival include symptomatic relief, protection against adverse events, prolonged time to relapse or disease free survival, and prolonged time to disease progression. These standards are generally accepted, and regulatory bodies such as the US Food and Drug Administration (FDA) approve drugs that bring these benefits (Hirschfeld et al., Critical Reviews in Oncology / Hematolgy 42: 137-143 2002). In addition to these criteria, it is well recognized that there are other endpoints that may be precursors of this type of benefit. As part of that, the accelerated approval process, approved by the USFDA, recognizes that there is a surrogate that is likely to predict patient benefits. As of the end of 2003, 16 drugs were approved under this process, of which 4 were fully approved, ie in subsequent studies, direct patient benefit predicted by surrogate endpoints was demonstrated. . One important endpoint in determining drug effects in solid tumors is assessing tumor loading by measuring response to treatment (Therasse et al., Journal of the National Cancer Institute 92 (3): 205-216 2000). The clinical criteria for evaluation (RECIST criteria) were published by the Response Evaluation Criteria in Solid Tumors Working Group, an international cancer expert group. Drugs that have demonstrated an effect on tumor loading as shown by objective responses according to RECIST criteria as compared to the appropriate controls ultimately tend to yield direct patient benefit. Oncological evaluation and recording in the preclinical environment is generally more straightforward. In that preclinical studies can be transferred to the clinical setting, drugs that lead to prolonged survival in the preclinical model are most likely to have clinical utility. Similar to causing a positive response to clinical treatment, drugs that reduce tumor loading in a preclinical environment may also have a significant direct impact on the disease. Although prolonged survival is the most sought clinical outcome from anticancer drug treatment, there are other benefits that are of clinical utility, and reduced tumor drop, prolonged survival, or both, which may be correlated with delayed disease progression, may also result in direct benefits. It is clear that Eckhardt et al., Developmental Therapeutics: Successes and Failures of Clinical Trial Designs of Targeted Compounds; ASCO Educational Book, 39 ^th Annual Meeting, 2003, pp. 209-219.

The present invention describes the development and use of AR81A410.7 found to be effective in cytotoxicity assays and in animal models of human cancer. The present invention describes reagents that specifically bind to epitopes or epitopes present on a target molecule and also have in vitro cytotoxic properties against malignant tumor cells as pure antibodies but not for normal cells. They also directly mediate tumor growth inhibition as pure antibodies. Another advance is to achieve tumor growth inhibition, and other positive outcomes for cancer treatment, using such anti-cancer antibodies that target tumors expressing cognate antigen markers.

In summary, the present invention teaches the use of the AR81A410.7 antigen as a target for therapeutic agents that can reduce the tumor loading of cancers that express the AR81A410.7 antigen in mammals upon administration. The invention also uses CDMAB (AR81A410.7), and derivatives thereof, and antigen binding fragments thereof, and their cytotoxic inducing ligands, to target these antigens and to express the antigens in mammals. Teaches to reduce tumor drip. Furthermore, the present invention also teaches the use of AR81A410.7 antigen detection in cancer cells, which may be useful for the diagnosis, treatment, and prognosis of mammals with tumors expressing the antigen.

Accordingly, it is an object of the present invention to isolate a hybridoma cell line and a corresponding isolated monoclonal antibody and antigen binding fragment thereof encoding the hybridoma cell line, from a specific individual, or from one or more specific cancer cell lines. It occurs by using a method of producing a cancer disease regulatory antibody (CDMAB) which occurs against one cancer cell, which is cytotoxic to cancer cells and at the same time relatively non-toxic to non-cancer cells.

A further object of the present invention is to teach cancer disease modulating antibodies, their ligands and antigen binding fragments.

Another object of the present invention is to prepare cancer disease regulatory antibodies having cytotoxicity mediated through antibody dependent cytotoxicity.

Another additional object of the present invention is to prepare cancer disease modulating antibodies having cytotoxicity mediated through complement dependent cytotoxicity.

It is another object of the present invention to prepare cancer disease modulating antibodies with cytotoxicity as a function of the ability to catalyze the hydrolysis of cellular chemical bonds.

Another object of the present invention is to prepare cancer disease modulating antibodies useful in binding assays for diagnosis, prognosis, and monitoring for cancer.

Other objects and advantages of the invention will be apparent from the following detailed description, in which specific embodiments of the invention are disclosed as examples and examples.

Brief description of the drawings

1 compares the cytotoxicity percentage and binding level of hybridoma supernatants against A549, NCI-H23, NCI-H460, MDA-MB-231 and Hs888.Lu cell lines.

2 shows the binding of AR81A410.7 to cancer and normal cell lines. The data showing the mean fluorescence intensity as an increase fold compared to the isotype control are summarized in a table.

3 contains representative FACS histograms of AR81A410.7 and anti-EGFR antibodies directed against several cancer and non-cancer cell lines.

4 shows the effect of AR81A410.7 on tumor growth in a prophylactic BxPC-3 pancreatic cancer model. Vertical dotted lines indicate the period of time the antibody was administered. Data points represent mean +/- SEM.

5 shows the effect of AR81A410.7 on body weight in a prophylactic BxPC-3 pancreatic cancer model. Data points represent mean +/- SEM.

DETAILED DESCRIPTION OF THE INVENTION

In general, the following words or phrases have the definitions shown below when used in the present summary, detailed description, examples, and claims.

The term “antibody” is used in its broadest sense and specifically includes, for example, monoclonal antibodies alone (including agonists, antagonists, and neutralizing antibodies, de-immunized, murine, chimeric or humanized antibodies), Antibody compositions with polyepitopic specificity, single chain antibodies, diabodies, triabodies, immunoconjugates and antibody fragments (see below).

As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homologous antibodies, ie the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in small amounts. Do. Monoclonal antibodies are highly specific, directed to a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations comprising different antibodies directed at different determinants (antigenic determinants), each monoclonal antibody is directed against a single determinant on the antigen of interest. In addition to their specificity, such monoclonal antibodies are advantageous in that they can be synthesized uncontaminated with other antibodies. The modifier of “monoclonal” indicates the nature of the antibody as obtained from a population of substantially homologous antibodies, and should not be construed as producing the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be prepared by the hybridoma (Bu or human) method first described in Kohler et al ., Nature , 256: 495 (1975), or recombinant DNA It may also be prepared by the method (see, eg, US Pat. No. 4,816,567). "Monoclonal antibodies" are also described, eg, in Clackson et al ., Nature , 352: 624-628 (1991) and in Marks et al . , J. Mol. Biol. , 222: 581-597 (1991) may be used to isolate from phage antibody libraries.

"Antibody fragments" include portions of intact antibodies, preferably those comprising antigen-binding or variable regions thereof. Examples of antibody fragments include: antibodies shorter than full length, Fab, Fab ', F (ab') ₂ , and Fv fragments; Diabody; Linear antibodies; Single chain antibody molecules; Multispecific antibodies formed from single chain antibodies, single domain antibody molecules, fusion proteins, recombinant proteins and antibody fragment (s).

A "complete" antibody is one comprising the C- _H 1, C _H 2 and C _H 3, which are light chain constant domains (C _L ) and heavy chain constant domains as well as antigen-binding variable regions. The constant domains may be constant domains of the original sequence (eg human native sequence constant domains) or amino acid sequence variants thereof. Preferably, a complete antibody has one or more effector functions.

Depending on the amino acid sequence of the constant domain of the heavy chains, complete antibodies can be assigned to different “classes”. There are five major classes of complete antibodies, IgA, IgD, IgE, IgG, and IgM, some of which are referred to as "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. It can be divided further. Heavy chain constant domains corresponding to different antibody classes are called α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional shapes of different immunoglobulin classes are well known.

Antibody “effector function” refers to a biological activity that may be attributable to the Fc region of an antibody (the Fc region of an original sequence or the Fc region of an amino acid sequence variant). Examples of antibody effector functions include C1q binding; Complement dependent cytotoxicity; Fc receptor binding; Antibody-dependent cell-mediated cytotoxicity (ADCC); Phagocytic action; Down regulation of cell surface receptors (eg B cell receptor; BCR) and the like.

"Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to binding of nonspecific cytotoxic cells (eg, natural killer (NK) cells, neutrophils, and macrophages) that express Fc receptors (FcRs) on target cells. Refers to a cell-mediated response that recognizes the antibody and subsequently causes lysis of the target cell. NK cells, primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is described by Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991)] is summarized in Table 3 on page 464. To assess ADCC activity of the molecule of interest, in vitro ADCC assays, such as those described in US Pat. No. 5,500,362 or 5,821,337, may be performed. Useful effector cells for this assay include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, such as in an animal model as disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

An “effector cell” is a white blood cell that expresses one or more FcRs to perform effector functions. Preferably, the cells express at least FcγRIII to perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include: peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; PBMC and NK cells are preferred. Effector cells may be isolated from their original source, such as from blood or PBMCs as described herein.

The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to the Fc region of an antibody. Preferred FcRs are human FcRs of the original sequence. Furthermore, preferred FcRs bind to IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII, and Fcγ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Included. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibiting receptor”), which have similar amino acid sequences that differ primarily in cytoplasmic domain portions. FcγRIIA, an activating receptor, has an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. FcγRIIB, an inhibitory receptor, has an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, eg, M. in Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcR is reviewed below: Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); And de Haas et al . , J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, including those to be identified in the future, are also included herein by the term "FcR". The term also includes FcRn, a neonatal receptor responsible for delivery of maternal IgGs to the fetus (Guyer et al. , J. Immunol. 117: 587 (1976) and Kim et al. , Eur. J. Immunol. 24: 2429 ( 1994)).

"Complement dependent cytotoxicity" or "CDC" refers to the ability of a molecule to dissolve a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component (C1q) of the complement system to a molecule (eg an antibody) complexed with a cognate antigen. To assess complement activation, see, eg, Gazzano-Santoro et al. , J. Immunol. Methods 202: 163 (1996), can be carried out a CDC assay.

The term “variable” refers to the fact that the sequences of certain portions of the variable domains vary widely between antibodies, so that they are used for binding and specificity of each specific antibody to the corresponding specific antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions that exist in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework regions (FR). The variable domains of the original heavy and light chains each comprise four FRs, which predominantly take a β-sheet arrangement that is linked to three hypervariable regions, some of which link the β-sheet structure. In this case, loops forming part of the structure are formed. The hypervariable regions in each chain are very closely bound by the FRs and contribute to the formation of antigen-binding sites of antibodies with the hypervariable regions from other chains (see Kabat et al., Sequences of Proteins). of Immunological Interest , 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The constant domains are not directly involved in binding the antibody to the antigen, but exhibit various effector functions such as the involvement of the antibody in antibody dependent cytotoxicity (ADCC).

The term "hypervariable region" as used herein refers to an amino acid residue of an antibody that is responsible for antigen-binding. Hypervariable regions generally include: amino acid residues from “complementarity determining regions” or “CDRs” (eg, residues 24-34 (L1), 50-56 (L2) and 89-97 (in the light chain variable domain); L3) and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest , 5th edition, Public Health Service, National Institutes of Health , Bethesda, Md. (1991)) and / or amino acid residues from an "overvariable loop" (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain) and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). “Framework Region” or “FR” residues are those variable domain residues that are not hypervariable region residues as herein defined. Cleavage of the antibodies with papain yields two identical antigen-binding fragments, each with a single antigen-binding site called "Fab" fragments, and the remaining "Fc" fragments whose name reflects their ability to readily crystallize. Pepsin treatment produces an F (ab ') ₂ fragment that has two antigen-binding sites and is still able to crosslink with the antigen.

"Fv" is the minimum antibody fragment with a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in a strong noncovalent state. In this arrangement, three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the V _H -V _L dimer. Collectively, six hypervariable regions confer antigen-binding specificity to the antibody in question. However, even a single variable domain (or half of the Fv containing only three hypervariable regions specific for the antigen), although less affinity than the entire binding site, has the ability to recognize and bind antigen. The Fab fragment also has the constant domain of the light chain and the first constant domain of the heavy chain (CH1). Fab 'fragments differ from Fab fragments by the addition of several residues, including one or more cysteines from the antibody hinge region, at the carboxy terminus of the heavy chain CH1 domain. Fab'-SH refers to Fab 'herein in which the cysteine residue (s) of the constant domains have at least one free thiol group. F (ab ') ₂ antibody fragments originally were produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The “light chain” of an antibody of any vertebrate species can be classified into one of two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

"Single-chain Fv" or "scFv" antibody fragments comprise the V _H and V _L domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the V _H and V _L domains, which allows the scFv to form the desired structure for antigen binding. For an overview of scFv, reference may be made to Pluckthun, The Pharmacology of Monoclonal Antibodies , vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term "diabody" is a small antibody fragment with two antigen-binding sites in which the variable heavy domain (V _H ) is linked to the variable light domain (V _L ) within the same polypeptide chain (V _H -V _L ). Refers to the intercepted section. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are promoted to pair with the complementary domain of another chain to create two antigen-binding sites. Diabodies are described, for example, in EP 404,097; WO 93/11161; And Hollinger et al . , Proc. Natl. Acad. ScL USA , 90: 6444-6448 (1993).

The term "tribody" or "trivalent trimer" refers to a combination of three single chain antibodies. Triabodies are constructed at the amino acid terminus of the V _L or V _H domain, ie without any linker sequence. Triabodies have three Fv heads in which three polypeptides are arranged in a cyclic, head-to-tail fashion. A possible form of the tribody is a plane in which three binding sites are located at an angle of 120 degrees to each other in one plane. Triabodies may be monospecific, bispecific or trispecific.

An "isolated" antibody is one that has been identified and isolated and / or recovered from components of its natural environment. Contaminant elements of its natural environment are materials that interfere with diagnostic or therapeutic use of the antibody, which may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. Isolated antibodies include antibodies present in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

An antibody that "binds" an antigen of interest is one capable of binding the antigen with sufficient affinity such that the antibody is useful as a therapeutic or diagnostic agent in targeting cells expressing the antigen. If the antibody is to bind to the antigenic moiety, it will usually preferentially bind to the antigenic moiety as opposed to other receptors, including accidental binding, such as non-specific Fc contact, Or binding to post-translational modifications common to other antigens, which may not be significantly cross-reacted with other proteins. Methods of detecting antibodies that bind the antigen of interest are well known in the art and include, but are not limited to, assays such as FACS, cellular ELISA, and western blot.

As used herein, the expressions "cell", "cell line", and "cell culture" are used interchangeably and all such notations include progeny. It will also be apparent that due to intentional or unintentional mutations, the DNA content of all progeny may not be exactly the same. Mutant progeny that have the same function or biological activity as screened for in the first modified cell are included. Where other meanings are intended, it will be apparent from the context.

"Treating or treating" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or attenuate (or weaken) the progression of the target pathological condition or disorder. Those in need of treatment include those already with the disorder, as well as those prone to have the disorder or those in which the disorder is to be prevented. Thus, a mammal to be treated herein may be diagnosed with the disorder or may be susceptible to or vulnerable to the disorder.

The terms "cancer" and "cancerous" refer to or describe physiological conditions in mammals that are typically characterized by unregulated cell growth or death. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell cancer (such as squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer, lung cancer including lung adenocarcinoma and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer Gastric or stomach cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney- or Renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, and head and neck cancer.

A "chemotherapeutic agent" is a chemical compound useful for the treatment of cancer. Examples of chemotherapeutic agents include the following: alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN ^™ ); Alkyl sulfonates such as busulfan, improsulfan and piposulfan; Aziridine such as benzodopa, carboquone, meturedopa, and uredopa; Methylmelamine and ethyleneimine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; Nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine Retamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; Nitrosurea such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; Antibiotics such as aclacinomycin, actinomycin, anthracycin, azaserrine, bleomycin, cactinomycin, calicheamicin, Carabicin, carnomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-dia Crude-5-oxo-L-norleucine, doxorubicin, epirubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid ( mycophenolic acid, nogalamycin, olivomycin, peplomycin, potpyromycin, puromycin, quelamycin, rodorubicin , Streptonigreen (stre ptonigrin), streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; Anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogs such as ancitabine, azaciitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, Doxifluridine, enocitabine, floxuridine, 5-FU; Androgens such as calusterone, dromostanolone propionate, epipitanstanol, mepitiostane, testolactone; Anti-adrenal agents such as aminoglutethimide, mitotane, trilostane; Folic acid supplements such as folinic acid; Aceglatone; Aldophosphamide glycoside; Aminolevulinic acid; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Elformithine; Elliptinium acetate; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Ronidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Phennamet; Pyrarubicin; Podophyllinic acid; 2-ethylhydrazide; Procarbazine; PSK®; Razoxane; Sizofiran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2 ', 2''-trichlorotriethylamine;Urethane;Vindesine;Dacarbazine;Mannomustine;Mitobronitol;Mitoractol;Pipobroman;Gacytosine; Arabinoside ("Ara-C");Cyclophosphamide;Thiotepa; Taxanes such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Aventis, Rhone-Poulenc Rorer, Antony, France); Chlorambucil; Gemcitabine; 6-thioguanine; Mercaptopurine; Methotrexate; Platinum analogs such as cisplatin and carboplatin; Vinblastine; platinum; Etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxanthrone; Vincristine; Vinorelbine; Navelbine; Novantron; Teniposide; Daunomycin; Aminopterin; Xeloda; Ibandronate; CPT-11; Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoic acid; Esperamicin; Capecitabine; And pharmaceutically acceptable salts, acids or derivatives of any of the above. The definition also includes: anti-hormonal agents that act to modulate or inhibit hormonal action on tumors, such as anti-estrogens such as tamoxifen, raloxifene, aromatase Inhibitory 4 (5) -imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); And anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; And pharmaceutically acceptable salts, acids or derivatives of any of the above.

“Mammals” for therapeutic purposes include humans, mice, SCID or nude mouse or mouse strains, livestock and breeding animals, and zoos, sports, or pets such as sheep, dogs, horses, cats, cows, and the like. , Refers to any animal classified as a mammal. Preferably, the mammal herein is a human.

"Oligonucleotides" are short-length, single- or double-stranded polydeoxynucleotides chemically synthesized by known methods (e.g., using solid phase techniques such as those described in EP 266,032 published May 4, 1988 or Or phosphoester, phosphite, or phosphoramidite, via a deoxynucleoside H-phosphonate intermediate as described in Froehler et al. , Nucl.Acids Res. , 14: 5399-5407, 1986. chemistry). These are then purified on polyacrylamide gels.

According to the present invention, the “humanized” and / or “chimeric” forms of non-human (eg, murine) immunoglobulins are human anti-mouse antibody (HAMA), human anti-chimeric, compared to the original antibody. Specific chimeric immunoglobulins, their immunoglobulin chains or fragments thereof (eg, Fv, Fab, Fab ', F (ab') ₂ or antibodies that cause a decrease in antibody (HACA) or human anti-human antibody (HAHA) response Essential parts (eg CDR (s), antigen binding region (s), variable domains) derived from said non-human immunoglobulin, including other antigen-binding subsequences, and necessary to reproduce the desired effect (S) and the like), while at the same time retaining binding properties comparable to the non-human immunoglobulin. For the most part, humanized antibodies are those residues of a non-human species (donor antibody) such as mouse, rat or rabbit CDRs in which the residues of the complementarity determining regions (CDRs) of the recipient antibody have the desired specificity, affinity and ability. Human immunoglobulin (receptor antibody). In some cases, Fv structural region (FR) residues of human immunoglobulins are replaced by corresponding non-human FR residues. Furthermore, the humanized antibody may comprise residues which are not found in the recipient antibody or in the imported CDR or FR sequences. These modifications are made to further refine and optimize antibody performance. In general, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, wherein all or substantially all CDR regions are of non-human immunoglobulin and all Or substantially all FR residues are residues of the human immunoglobulin consensus sequence. The humanized antibody will optimally also comprise at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin.

“De-immunized” antibodies are immunoglobulins that are non-immunogenic or less immunogenic for a given species. De-immunization can be accomplished by making structural alterations to the antibody. Any de-immunization technique known to those skilled in the art can be used. One suitable technique for de-immunizing an antibody is described, for example, in WO 00/34317 published June 15, 2000.

Antibodies that induce “apoptosis” may be by any means, including but not limited to binding of annexin V, caspase activity, DNA fragmentation, cell contraction, endoplasmic reticulum, cell fragmentation, and / or Or antibodies that induce predetermined cell death, exemplified by the formation of membrane vesicles (called apoptotic bodies).

As used herein, “antibody inducible cytotoxicity” refers to a cytotoxic effect derived from a hybridoma supernatant or an antibody produced by a hybridoma deposited with IDAC 051206-01. It should be understood that it is not necessarily related to the degree of coupling.

Throughout this specification, hybridoma cell lines, and the isolated monoclonal antibodies generated therefrom, are otherwise referred to by their internal designation, AR81A410.7 or deposit name, IDAC 051206-01.

As used herein, “antibody-ligand” includes moieties that exhibit binding specificity for at least one epitope of a target antigen, which is an isolated monoclonal antibody produced by a hybridoma cell line designated IDAC 051206-01. Complete antibody molecules, antibody fragments, and at least their antigen-binding regions or portions (ie, variable of antibody molecules) that specifically recognize and bind to at least one epitope of the antigen bound by (IDAC 051206-01 antigen) Moiety), such as a Fv molecule, a Fab molecule, a Fab 'molecule, an F (ab') ₂ molecule, a bispecific antibody, a fusion protein, or any genetically engineered molecule.

As used herein, a "cancer disease modulating antibody" (CDMAB) is a monoclonal antibody that modulates a cancer disease process in a manner that is beneficial to a patient, for example by reducing tumor loading or prolonging the survival of an individual with the tumor. And antibody-ligands thereof.

"CDMAB related binding agent" is, in the broadest sense, competitively binding to at least one CDMAB target epitope, including but not limited to any form of human or non-human antibody, antibody fragment, antibody It is to be understood to include ligands and the like.

A “competitive binder” should be understood to include any form of human or non-human antibody, antibody fragment, antibody ligand, etc., having a binding affinity for at least one CDMAB target epitope.

Tumors to be treated include primary and metastatic tumors, and refractory tumors. Refractory tumors include tumors that do not respond to or are resistant to treatment with chemotherapeutic agents alone, antibodies alone, radiation alone, or a combination thereof. Refractory tumors include tumors that appear to be inhibited by treatment with such agents, but recur after up to 5 years, sometimes up to 10 years or more after discontinuation of treatment.

Treatable tumors include tumors in which the blood vessels are not distributed or in which the blood vessels are not yet substantially distributed and tumors in which the blood vessels are distributed. Examples of solid tumors thus treatable are breast carcinoma, lung carcinoma, colorectal carcinoma, pancreatic carcinoma, glioma and lymphoma. Some examples of such tumors include epidermal tumors, squamous epithelial tumors such as head and neck tumors, colorectal tumors, prostate tumors, breast tumors, lung tumors including small cell and non-small cell lung tumors, pancreatic tumors, thyroid tumors, Ovarian tumors, and liver tumors. Other examples include Kaposi's sarcoma, CNS neoplasia, neuroblastoma, capillary blastoma, meningiomas and brain metastases, melanoma, gastrointestinal and renal carcinoma and sarcoma, rhabdomyosarcoma, glioblastoma, preferably polymorphic glioblastoma, and smooth muscle There is sarcoma.

As used herein, "antigen-binding region" refers to a portion of a molecule that recognizes a target antigen.

As used herein, “competitively inhibit” is directed to a monoclonal antibody (IDAC 051206-01 antibody) generated by a hybridoma cell line designated IDAC 051206-01 using conventional reciprocal antibody competition assays. It means that can recognize and bind to the determinant site. (Belanger L., Sylvestre C. and Dufour D. (1973), Enzyme linked immunoassay for alpha fetoprotein by competitive and sandwich procedures. Clinica Chimica Acta 48, 15).

As used herein, a “target antigen” is an IDAC 051206-01 antigen or portion thereof.

As used herein, “immunoconjugate” refers to an antibody, such as an antibody, that is chemically or biologically linked to a cytotoxin, radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, toxin, anti-tumor drug or therapeutic agent. Any molecule or CDMAB. The antibody or CDMAB can be linked to a cytotoxin, radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, toxin, anti-tumor drug or therapeutic agent at any position on the molecule as long as it can bind the target of interest. Can be. Examples of immunoconjugates include antibody toxin chemical conjugates and antibody-toxin fusion proteins.

) 이다. 상기 항체는 상기 종양에 대한 사이토카인을 표적으로 하여, 사이토카인이 다른 조직에 영향을 주지 않으면서 상기 종양의 손상 또는 파괴를 매개하게 한다. 상기 사이토카인을, 통상의 재조합 DNA 기술을 사용하여 DNA 수준에서 상기 항체에 융합시킨다. 인터페론 또한 사용가능하다.Radioactive agents suitable for use as antitumor agents are known to those skilled in the art. For example, 131I or 211At is used. These isotopes are attached to the antibody by conventional techniques (eg Pedley et al ., Br. J. Cancer 68, 69-73 (1993)). Alternatively, the antitumor agent attached to the antibody is an enzyme that activates the prodrug. Prodrugs that remain in inactive form may be administered until reaching the tumor site, where it is converted to its cytotoxin form when the antibody complex is administered. In practice, the antibody-enzyme conjugate is administered to the patient and allowed to be localized into the area of tissue to be treated. Thereafter, the prodrug is administered to the patient, such that conversion to a cytotoxic drug occurs in the area of the tissue to be treated. Alternatively, antitumor agents conjugated to the antibody may be cytokines such as interleukin-2 (IL-2), interleukin-4 (IL-4) or tumor necrosis factor alpha (TNF-

) to be. The antibody targets a cytokine against the tumor, allowing the cytokine to mediate damage or destruction of the tumor without affecting other tissues. The cytokine is fused to the antibody at the DNA level using conventional recombinant DNA techniques. Interferons can also be used.

As used herein, “fusion protein” refers to any molecule to which an antigen binding region is linked to a biologically active molecule, such as a toxin, enzyme, fluorescent protein, luminescent marker, polypeptide tag, cytokine, interferon, target or reporter moiety, or protein drug. Means chimeric protein.

The present invention further contemplates the CDMAB of the present invention to which the target or reporter moiety is linked. The target moiety is the first member of the binding pair. Antitumor agents, for example, are conjugated to the second member of such a pair and directed to the site where the antigen-binding protein bound. Common examples of such binding pairs are avidin and biotin. In a preferred embodiment, biotin is bound to the target antigen of the CDMAB of the invention to be a target for an antitumor or other moiety bound to avidin or streptavidin. Alternatively, biotin or another such portion may be linked to a target antigen of the CDMAB of the invention, for example in a diagnostic system in which a detectable signal-producing agent is bound to avidin or streptavidin. , Use it as a reporter.

Detectable signal-generating agents are useful for diagnostic purposes in vivo and in vitro. Signal generators produce measurable signals detectable by external means, usually by measurement of electromagnetic radiation. In most cases, the signal generator is an enzyme or chromophore, or emits light by fluorescence, phosphorescence or chemiluminescence. Chromophores include dyes that absorb light in the ultraviolet or visible region, which may be substrates or degradation products of enzymatic catalysis.

Further, the scope of the present invention includes the use of the CDMAB of the present invention in vivo and in vitro for research or diagnostic methods well known in the art. To carry out the diagnostic methods contemplated herein, the invention may further comprise a kit comprising the CDMAB of the invention. Such kits will be useful for identifying such individuals by detecting overexpression of the target antigen of CDMAB on cells of individuals at risk of certain types of cancer.

Diagnostic assay kit

It is contemplated to use the CDMAB of the present invention in the form of a diagnostic assay kit that determines the presence of a tumor. Tumors in such patients based on the presence of one or more tumor-specific antigens, such as proteins and / or polynucleotides encoding such proteins, in a biological sample, such as blood, serum, urine and / or tumor biopsies, which will generally be obtained from the patient Will be detected.

Proteins function as markers indicating the presence or absence of certain tumors, such as colon, breast, lung or prostate tumors. It is further contemplated to use such antigens for detection of other cancerous tumors. When the diagnostic assay kit includes a binding agent consisting of the CDMAB of the present invention, or a CDMAB related binding agent, the level of antigen binding to the agent in the biological sample can be detected. Polynucleotide primers and probes can also be used to detect levels of mRNA encoding tumor proteins, which also indicates the presence or absence of cancer. In order for the binding assay to be diagnostic, data relating statistically significant antigen levels with respect to what is present in normal tissues can be generated so that the recognition of the binding can clearly diagnose the presence of cancerous tumors. Will be. In detecting polypeptide markers in a sample using binding agents, it is contemplated that many forms will be useful in the diagnostic assays of the present invention, as is known to those skilled in the art. See, eg, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapters 9-14, 1988. Any and all combinations, modifications or variations of the diagnostic assay forms described above are further contemplated.

The presence or absence of cancer in a patient typically results in (a) contacting a biological sample taken from the patient with a binding agent; (b) detecting the level of polypeptide binding to the binding agent in the sample; (c) will be determined by comparing the level of the polypeptide with a predetermined cut-off value.

In an exemplary embodiment, it is contemplated to include in the assay that the CDMAB based binding agent immobilized on a solid support causes binding to and removal of the polypeptide in the residue of the sample. The bound polypeptide can then be detected using a detection reagent that includes a reporter group and specifically binds the binding agent / polypeptide complex. Exemplary detection reagents include CDMAB based binding agents that specifically bind to the polypeptide or antibodies or other agents that specifically bind to the binding agents, such as anti-immunoglobulins, protein G, protein A or lectins. . In alternative embodiments, it is contemplated that competitive assays may be employed that label a polypeptide with a reporter group and bind it to an immobilized binding agent incubated with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample to the immobilized binding agent. Suitable polypeptides for use in such assays include full-length tumor-specific proteins and / or portions thereof in which the binding agent has a binding affinity for them.

The diagnostic kit will be equipped with a solid support, which can be in the form of any substance known to those skilled in the art to which the protein can be attached. Suitable examples include test wells or nitrocellulose or other suitable membranes in microtiter plates. Alternatively, the support may be beads or discs such as glass, fiberglass, latex or plastic materials such as polystyrene or polyvinylchloride. The support is also described, for example, in U.S. Magnetic particles or optical fiber sensors, such as those disclosed in patent 5,359,681.

It is contemplated to immobilize the binding agent on the solid support using various techniques known to those skilled in the art, which techniques are described in detail in the patent and scientific literature. The term "immobilization" refers to both non-covalent association and covalent attachment, such as adsorption, which may, in the context of the present invention, be a direct link between the agent and the functional group on the support, or a linkage using a crosslinking agent. It may be. In a preferred, but non-limiting embodiment, immobilization by adsorption to wells or membranes in microtiter plates is preferred. Adsorption can be achieved by contacting the binding agent with the solid support in a suitable buffer for a suitable time. The contact time may vary depending on the temperature and will generally be in the range of about 1 hour to about 1 day.

Covalent attachment of a binding agent to a solid support is usually accomplished by first reacting the support with a bifunctional reagent that reacts with both the support and a functional group on the binding agent (eg, a hydroxyl or amino group). For example, the binding agent can be covalently attached to the support with the appropriate polymer coating, either by using benzoquinone or by condensing an aldehyde group on the support with an amine and active hydrogen on the other side of the binding.

It is further contemplated that the diagnostic assay kit takes the form of a double-antibody sandwich assay. Such assays may be performed by first contacting a sample with a CDMAB disclosed herein immobilized on a well of an antibody, such as a solid support, usually a microtiter plate, to allow the polypeptide in the sample to bind to the immobilized antibody. . The unbound sample is then removed from the immobilized polypeptide-antibody complex and a detection reagent containing a reporter group (preferably a secondary antibody capable of binding other sites on the polypeptide) is added. The amount of detection reagent that remains bound to the solid support is then measured using methods appropriate to the particular reporter group.

In certain embodiments, when the antibody is immobilized on a support as described above, the protein binding site remaining on the support is selected from bovine serum albumin or Tween 20 ^™ (Sigma Chemical Co., St. Louis, Mo.). It is contemplated to block using any suitable blocker known to those skilled in the art. The immobilized antibody is then incubated with the sample to allow polypeptide to bind to the antibody. Prior to incubation, the sample may be diluted with a suitable diluent such as phosphate buffered saline (PBS). In general, an appropriate contact time (ie, incubation time) will be selected to correspond to a time period sufficient to detect the presence of a polypeptide in a sample obtained from an individual with a specifically selected tumor. Preferably, the contact time is sufficient to achieve a level of at least about 95% of the binding that takes place at equilibrium between bound and unbound polypeptides. Those skilled in the art will appreciate that by analyzing the level of binding occurring over a period of time, one can readily determine the time needed to achieve equilibrium.

Thereafter, it is further contemplated to remove the unbound sample by washing the solid support with a suitable buffer. Then, a secondary antibody containing a reporter group is added to the solid support. Thereafter, the detection reagent is incubated with the immobilized antibody-polypeptide complex for a time sufficient to detect the bound polypeptide. The unbound detection reagent is then removed and the bound detection reagent is detected using a reporter group. The method used to detect reporter groups must be specific to the type of reporter group selected, for example for radioactive groups, scintillation counting or autoradiography is generally appropriate. Spectroscopy can be used to detect dyes, luminescent groups and fluorescent groups. Biotin can be detected using avidin coupled to different reporter groups (usually radioactive or fluorescent groups or enzymes). Enzyme reporter groups can generally be detected by addition of a substrate (typically for a certain period of time), followed by spectroscopic or other analysis of the reaction product.

In order to measure the presence or absence of cancer, such as prostate cancer, using the diagnostic assay kit of the present invention, a signal corresponding to a predetermined cut-off value of a signal detected from a reporter group which is generally bound to a solid support and Compare. For example, an exemplary cut-off value for cancer detection may be the average mean signal obtained when incubating the immobilized antibody with a sample from a patient who does not have cancer. In general, a sample that produces a signal with a standard deviation of about 3 higher than a predetermined cut-off value is considered positive for that cancer. In an alternative embodiment, the cut-off value can be determined using the Receiver Operator Curve according to Sackett et al. (Clinical Epidemiology. A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7). have. In one such embodiment, the cut-off values are true positive rate (ie sensitivity) and false positive rate (100% -specific) corresponding to each possible cut-off value for the diagnostic test result. Can be determined from the graph of the pair of. The cut-off value closest to the upper left corner (i.e., the value surrounding the widest area) on the graph is the most accurate cut-off value and a sample producing a signal larger than the cut-off value determined by the method. It can be considered positive. Alternatively, the cut-off value can be moved to the left along the graph to minimize false positive rate, or to the right to minimize false negative rate. In general, samples that produce a signal greater than the cut-off value determined by the method are considered positive for cancer.

It is contemplated that a diagnostic assay that can be made with the kit is performed in either a flow-through or strip test format, wherein the binding agent is immobilized on a membrane such as nitrocellulose. In the flow test, the polypeptide in the sample binds to the immobilized binding agent as the sample passes through the membrane. Thereafter, when the solution containing the second binding agent flows through the membrane, the second labeled binding agent binds to the binding agent-polypeptide complex. Detection of the bound second binding agent can then be performed as described above. In the strip test format, one end of the membrane to which the binding agent is bound is immersed in the solution containing the sample. The sample travels along the membrane through the region containing the second binding agent to the region of the immobilized binding agent. The concentration of said second binding agent in the region of the immobilized antibody indicates the presence of cancer. At the binding site, the generation of a visually readable pattern, such as a line, will indicate a positive test. Absence of this pattern indicates negative results. In general, in the form discussed above, the amount of binding agent immobilized on the membrane results in a visually identifiable pattern when the biological sample contains a level of polypeptide that will be sufficient to produce a positive signal in a double-antibody sandwich assay. Selected to create. Preferred binding agents for use in this diagnostic assay are the antibodies disclosed herein, antigen-binding fragments thereof, and any CDMAB related binding agents described herein. The amount of antibody immobilized on the membrane will be any amount effective to enable a diagnostic assay, which may range from about 25 nanograms to about 1 microgram. Typically such tests can be performed using very small amounts of biological samples.

In addition, the CDMAB of the present invention may be used in the laboratory for research due to its ability to identify its target antigen.

In order that the present invention described herein may be understood in more detail, the following description is set forth.

The present invention provides a CDMAB (ie, IDAC 051206-01 CDMAB) that specifically recognizes and binds to an IDAC 051206-01 antigen.

The CDMAB of an isolated monoclonal antibody produced by hybridoma deposited with IDAC 051206-01 was immunospecific to its target antigen of the isolated monoclonal antibody produced by hybridoma IDAC 051206-01. It may be in any form as long as it has an antigen-binding region that competitively inhibits binding. Thus, any recombinant protein having the same binding specificity as the IDAC 051206-01 antibody (eg, a fusion protein in which the antibody is bound to a second protein such as lymphokine or tumor inhibiting growth factor) is within the scope of the present invention. .

In one embodiment of the invention, the CDMAB is an IDAC 051206-01 antibody.

In other embodiments, CDMAB is an Fv molecule (eg, a single chain Fv molecule), an Fab molecule, a Fab 'molecule, an F (ab') ₂ molecule, a fusion protein, bispecific with an antigen-binding region of an IDAC 051206-01 antibody. Antigen binding fragments which may be antibodies, heterologous antibodies or any recombinant molecule. The CDMAB of the present invention is directed to epitopes directed by IDAC 051206-01 monoclonal antibodies.

The CDMAB of the present invention can be modified, i.e., modified by amino acid modification in the molecule, to produce derivative molecules. Chemical modifications may also be possible. Modification by direct mutations, methods of affinity maturation, phage display or chain shuffling may also be possible.

Affinity and specificity can be modified or improved by mutating CDR and / or phenylalanine tryptophan (FW) residues to screen antigen binding sites with the desired properties (eg, Yang et al., J. Mol. Biol., (1995) 254: 392-403). One method is to randomize individual residues or combinations of residues such that a subset of 2 to 20 amino acids is found at a particular position, in the remainder of the same antigen binding site population. Alternatively, error prone PCR methods (eg, Hawkins et al., J. Mol. Biol., (1992) 226: 889-96) can induce mutations for a series of residues. In another example, phage display vectors with heavy and light chain variable region genes can be propagated to mutator strains of E. coli (eg, Low et al., J. Mol. Biol., (1996). 250: 359-68). Such mutagenesis methods are an example of many methods known to those skilled in the art.

Another way to increase the affinity of the antibodies of the invention is to perform chain shuffling, which randomly pairs heavy or light chains with other heavy or light chains to produce antibodies with higher affinity. Various CDRs of such antibodies can also be shuffled with the corresponding CDRs of other antibodies.

Derivative molecules will retain the functional properties of the polypeptide, ie molecules with such substitutions will still allow binding of the polypeptide to the IDAC 051206-01 antigen or portion thereof.

Such amino acid substitutions include, but are not necessarily limited to, amino acid substitutions known in the art as "conservative".

For example, it is a well established principle in protein chemistry that certain amino acid substitutions, often referred to as “conservative amino acid substitutions,” that do not alter the shape or function of the protein in question, can often be made within the protein.

Such alterations include any of isoleucine (I), valine (V), and leucine (L) with any other of these hydrophobic amino acids; Aspartic acid (D) to glutamic acid (E) and vice versa; Glutamine (Q) to asparagine (N) and vice versa; And substitution of serine (S) with threonine (T) and vice versa. Other substitutions may also be considered conservative, depending on the environment of the particular amino acid and its role within the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) are frequently interchangeable, as are alanine and valine (V). Methionine (M) is relatively hydrophobic and can be frequently interchanged with leucine and isoleucine and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable at positions where the significant characteristic of that amino acid residue is their charge and the different pK of these two amino acid residues is not significant. Still other changes may be considered "conservative" in certain circumstances.

Example 1

Hybridoma production-hybridoma cell line AR81A410.7

The hybridoma cell line AR81A410.7, in accordance with the Budapest Treaty, was released by the Canadian Depository of the International Depository Authority of the Canadian Department of Health Microbiology at 1015, Arlington Street 1015, Manitoba, Winnipeg, Manitoba, Canada, on December 5, 2006. Canada, IDAC) under the registration number 051206-01. In accordance with 37 CFR 1.808, our depositors guarantee that all restrictions imposed on the public's availability of deposited materials will be removed at the time of patent approval. The deposit will be replaced if the deposit institution is unable to provide a viable sample.

To prepare hybridomas that produce anti-cancer antibody AR81A410.7, a single cell suspension of frozen lung adenocarcinoma tumor tissue (Genomics Collaborative, Cambridge, Mass.) Was prepared in PBS. IMMUNEASY ^™ (Qiagen, Venlo, Netherlands) adjuvant was prepared for use by moderate mixing. BALB / c mice 5-7 weeks of age were immunized with subcutaneous injections of 2 million cells in 50 microliters of the antigen-adjuvant. Two and five weeks after the initial immunization, the immunized mice were boosted intraperitoneally with 2 million cells in 50 microliters using recently prepared antigen-adjuvant. Three days after the last immunization, the spleen was fused. Hybridomas were prepared by fusing the isolated splenocytes with an NSO-1 myeloma counterpart. Supernatants from the fusions were tested from subclones of the hybridomas.

To determine whether the antibodies secreted by the hybridoma cells are either IgG or IgM isotypes, an ELISA assay was used. ELISA plate at 100 microliters / well of goat anti-mouse IgG + IgM (H + L) at a concentration of 2.4 micrograms / mL in coating buffer (0.1 M carbonate / bicarbonate buffer, pH 9.2-9.6) at 4 ° C. It was added to the field and left overnight. The plates were washed three times in wash buffer (PBS + 0.05% Tween). 100 microliters / well of blocking buffer (5% milk in wash buffer) was added to the plate and allowed to stand at room temperature for 1 hour and then washed three times in wash buffer. 100 microliters / well of hybridoma supernatant were added and the plates were incubated for 1 hour at room temperature. The plates were washed three times with wash buffer and 1 / 100,000 dilutions of goat anti-mouse IgG or IgM horseradish peroxidase conjugate (diluted in PBS containing 1% milk) at 100 microliters / well Added. After incubating the plate for 1 hour at room temperature, the plate was washed three times with wash buffer. 100 microliters / well of TMB solution was incubated for 1-3 minutes at room temperature. 50 microliters / well of 2M H ₂ SO ₄ was added to terminate the color reaction and the plates were read at 450 nm with a Perkin-Elmer HTS7000 plate reader. As shown in FIG. 1, AR81A410.7 hybridomas secreted antibodies of mainly IgG isotype.

In order to determine the subclass of antibodies secreted by the hybridoma cells, isotyping experiments were performed using the Mouse Monoclonal Antibody Isotyping Kit (HyCult Biotechnology, Frontstraat, The Netherlands). 500 microliters of buffer was added to a test strip containing rat anti-mouse subclass specific antibodies. 500 microliters of hybridoma supernatant were added to the test tube and immersed in gentle shaking. Captured mouse immunoglobulins were detected directly with secondary rat monoclonal antibodies coupled to colloidal particles. The combination of these two proteins produces visual cues that are used for isotype analysis. The anti-cancer antibody AR81A410.7 is an IgG2a, kappa isotype.

After one limit dilution of the hybridoma supernatants, the cells were tested for antibodies bound to target cells in a cell ELISA assay. The following three human lung cancer cell lines, one human breast cancer cell line and one human non-cancer lung cell line were tested: A549, NCI-H23, NCI-H460, MDA-MB-231 and Hs888.Lu, respectively. All cell lines were obtained from American Type Tissue Collection (ATCC; Manassas, VA). The plated cells were fixed before use. The plates were washed three times at room temperature with PBS containing MgCl ₂ and CaCl ₂ . 100 microliters of 2% paraformaldehyde diluted in PBS was added to each well for 10 minutes at room temperature, and then discarded. The plates were again washed three times at room temperature with PBS containing MgCl ₂ and CaCl ₂ . Blocking was performed for 1 hour at room temperature with 100 microliters / well of 5% milk in wash buffer (PBS + 0.05% Tween). The plates were washed three times with wash buffer and hybridoma supernatant was added at 100 microliters / well for 1 hour at room temperature. The plates were washed three times with wash buffer and 100 microliters / well of 1 / 25,000 dilution (diluted in PBS containing 1% milk) of goat anti-mouse IgG antibody conjugated to horseradish peroxidase was added. It was. After 1 hour incubation at room temperature, the plates were washed three times with wash buffer and 100 microliters / well of TMB substrate was incubated for 1-3 minutes at room temperature. The reaction was terminated with 50 microliters / well of 2M H ₂ SO ₄ and the plate was read at 450 nm with a Perkin-Elmer HTS7000 plate reader. The results shown in the table in FIG. 1 were expressed in multiples of the background compared to the in-house IgG isotype control, which previously showed no binding to the tested cell lines. Antibodies from hybridoma AR81A410.7 showed strong binding to MDA-MB-231 breast cancer cell lines and weak binding to lung cancer cell lines NCI-H23 and NCI-H460 and normal lung cell line Hs888.Lu.

In addition to testing for antibody binding, the cytotoxic effects (antibody induced cytotoxicity) of the hybridoma supernatants were tested in the following cell lines: A549, NCI-H23, NCI-H460, MDA-MB-231 and Hs888. Lu. Calcein AM was obtained from Molecular Probes (Eugene, OR) and the assay was performed as outlined below. Cells were plated at appropriate predetermined density prior to the assay. After 2 days, 100 microliters of supernatant from the hybridoma microtiter plate was transferred to the cell plates and incubated for 5 days in a 5% CO ₂ incubator. Sodium azide (NaN ₃ , 0.01%, Sigma, Oakville, ON) or cycloheximide (CHX, 0.5 micromolar, Sigma, Oakville) was aspirated until emptyed and dissolved in culture medium as a positive control , ON) 100 microliters were added. After 5 days of treatment, the plates were then inverted and blotted dry with blotting dry. Room temperature DPBS (Dulbecco Phosphate Buffered Saline) containing MgCl ₂ and CaCl ₂ was dispensed into each well from a multichannel squeeze bottle, pounded three times, inverted and emptied and dried on a blotter paper. 50 microliters of fluorescent calcein dye diluted in DPBS containing MgCl ₂ and CaCl ₂ were added to each well and incubated for 30 minutes at 37 ° C. in a 5% CO ₂ incubator. The plates were read in a Perkin-Elmer HTS7000 fluorescent plate reader and the data analyzed with Microsoft Excel. The results are shown in the table in FIG. Supernatants from AR81A410.7 hybridomas produced 43% specific cytotoxicity against NCI-H23 lung cancer cells. This was 86 and 391% of the cytotoxicity obtained using the positive controls sodium azide and cycloheximide, respectively. AR81A410.7 also caused 12% specific cytotoxicity against NCI-H460 lung cancer cells, 17 and 52% of the cytotoxicity obtained using the positive controls sodium azide and cycloheximide, respectively.

The results from FIG. 1 demonstrate that the cytotoxic effect of AR81A410.7 was not directly related to the level of binding to these cancer cell types. Highest binding was detected for MDA-MB-231 breast cancer cells, but cytotoxicity was detected for NCI-H23 and NCI-H460 lung cancer cells. As shown in the table in FIG. 1, AR81A410.7 did not cause cytotoxicity in Hs888.Lu normal human lung cell line. Known non-specific cytotoxic agents, cycloheximide and NaN ₃ , generally produced the expected cytotoxicity.

Example 2

In vitro bonding

The hybridomas were cultured in a CL-1000 flask (BD Biosciences, Oakville, ON) with collection and reseeding twice a week to prepare AR81A410.7 monoclonal antibody. Standard antibody purification procedures were performed using Protein G Sepharose 4 Fast Flow (Amersham Biosciences, Baie d'Urfe, QC). It is within the scope of the present invention to use monoclonal antibodies that are de-immunized, humanized, chimeric or murine.

Lungs (A549, NCI-H23, NCI-H322M, NCI-H460, and NCI-H520), Colon (Lovo), Breast (MDA-MB-231), Pancreas (BxPC-3), Prostate (PC-3) and Binding of AR81A410.7 to ovarian (OVCAR-3) cancer and non-cancer cell lines from skin (CCD-27sk) and lung (Hs888.Lu) was evaluated by flow cytometry (FACS). . All cell lines were obtained from American Type Tissue Collection (ATCC; Manassas, VA), except lung cancer cell line NCI-H322M. NCI-H322M was obtained from NCI-Frederick Cancer DCTD Tumor / Cell Line Repository (Frederick, MD).

Cells were initially prepared for FACS by washing the cell monolayer with DPBS (without Ca ⁺⁺ and Mg ⁺⁺ ). Cells were then separated from their cell culture plates at 37 ° C. using cell isolation buffer (Invitrogen, Burlington, ON). After collection by centrifugation, cells are resuspended in DPBS (stained medium) at 4 ° C. containing MgCl ₂ , CaCl ₂ and 2% fetal bovine serum, counted, aliquoted to the appropriate cell density, and spin down cells were pelleted and resuspended in 4 ° C. staining medium in the presence of test antibody (AR81A410.7) or control antibodies (isotype control, anti-EGFR). Isotype controls and the test antibody were evaluated at 20 micrograms / mL and anti-EGFR at 5 micrograms / mL for 30 minutes on ice. The cells were washed once with staining medium before the addition of Alexa Fluor 546-conjugated secondary antibody. Thereafter, Alexa Fluor 546-conjugated antibody in the staining medium was added at 4 ° C. for 30 minutes. Thereafter, the cells were finally washed and resuspended in a fixing medium (staining medium containing 1.5% paraformaldehyde). FACSarray ^TM System Software using (BD Biosciences, Oakville, ON) by the gel sample on a FACSarray ^TM was evaluated by flow cytometric analysis obtained value (flow cytometric acquisition) of the cell. The voltage and amplitude gains were adjusted for forward scatter (FSC) and lateral scatter (SSC) detectors to establish the FSC and SSC of these cells. Unstained cells were run to adjust the detectors for the fluorescence (Alexa-546) channel so that the cells had a constant peak with a median fluorescence intensity of approximately 1-5 units. For each sample, approximately 10,000 gated events (stained fixed cells) were obtained for analysis and the results are shown in FIG. 2.

2 shows the mean fluorescence intensity fold increase for isotype controls. Representative histograms of AR81A410.7 antibodies were collected and shown in FIG. 3. AR81A410.7 is lung cancer cell line NCI-H23 (3.5 times), breast cancer cell line MDA-MB-231 (1.6 times), pancreatic cancer cell line BxPC-3 (2.6 times), ovarian cancer cell line OVCAR-3 (2.5 times), prostate cancer cell line Detectable binding to PC-3 (2.5 fold), skin non-cancer cell line CCD-27sk (1.9 fold) and lung non-cancer cell line Hs888.Lu (2.7 fold) was demonstrated. These data also demonstrate that AR81A410.7 binds to several different cell lines with varying antigen expression levels.

Example 3

In Vivo Tumor Experiments with BxPC-3 Cells

Examples 1 and 2 demonstrated that AR81A410.7 has anti-cancer properties against human cancer cell lines by detectably binding to several different cancer indications. With reference to FIGS. 4 and 5, female SCID mice from 8 to 10 weeks of age were injected by injection of 5 million human pancreatic cancer cells (BxPC-3) in 100 microliter PBS solution subcutaneously into the nape of the neck. The mice were randomly divided into two treatment groups of six animals. On the day of injection, the stock concentration of 20 mg / kg of AR81A410.7 test antibody or buffer control contained 2.7 mM KCl, 1 mM KH ₂ PO ₄ , 137 mM NaCl and 20 mM Na ₂ HPO ₄ . After dilution with a diluent, it was administered intraperitoneally to each cohort in a volume of 300 microliters. The antibody and control samples were then administered once weekly in the same manner during the study period. Tumor growth was measured by calipers approximately every seven days. The study was completed after eight doses of antibody. Animal weights were recorded once a week for the duration of the study. At the end of the study all animals were euthanized according to CCAC guidelines.

AR81A410.7 reduced tumor growth in a BxPC-3 in vivo prophylactic model of human pancreatic cancer. Treatment of ARIUS antibody AR81A410.7 reduced the growth of BxPC-3 tumors by 71.5% on day 6 after the last antibody administration compared to the buffer treatment control measured on day 56 (FIG. 4). Due to the small number of mice in each group, this result was not significant (p = 0.14, t-test), but the tumor size of the antibody-treated group was smaller at each time point compared to the vehicle control. On day 41 all mice were still alive, with tumor growth 75% inhibited (p = 0.104, t-test).

There were no signs of clinical toxicity throughout this study. Body weight measured at weekly intervals was a proxy for health and growth failure. There was no significant difference in body weight in each group from the beginning to the end of the study (control, p = 1.000, t-test; AR81A410.7, p = 0.2094, t-test). However, at day 62 there was a significant difference between the groups at the end of the study (p = 0.0396, t-test) (FIG. 5).

In short, AR81A410.7 was satisfactorily tolerated in this human pancreatic cancer xenograft model and reduced tumor loading.

Example 4

Separation of Competitive Joiners

Given an antibody, one of ordinary skill in the art can produce competitively inhibiting CDMABs, eg, competing antibodies, which are antibodies that recognize the same epitope (Belanger L et al., Clinica Chimica Acta 48: 15-18 (1973)). One method involves immunizing with an immunogen that expresses an antigen recognized by the antibody. Samples may include, but are not limited to, tissue, isolated protein (s) or cell line (s). The resulting hybridomas can be screened using a competitive assay, which identifies antibodies that inhibit binding of test antibodies, such as ELISA, FACS or Western blotting. Alternatively, panning can be used for phage display antibody libraries and antibodies that recognize at least one epitope of the antigen (Rubinstein JL et al . Anal Biochem 314: 294-300 (2003)). . In both cases, antibodies are selected based on their ability to replace the binding of the original labeled antibody to at least one epitope of its target antigen. Such antibodies will thus have the property of recognizing at least one epitope of the antigen as the original antibody.

Example 5

Cloning of Variable Regions of AR81A410.7 Monoclonal Antibody

The sequences of the variable regions from the heavy (V _H ) and light (V _L ) monoclonal antibodies produced by the AR81A410.7 hybridoma cell line can be determined. RNA encoding the heavy and light chains of immunoglobulins can be extracted from subject hybridomas using standard methods involving cell lysis with guanidinium isothiocyanates (Chirgwin et al. Biochem. 18: 5294-5299). (1979)). The mRNA can be used to prepare cDNAs for subsequent isolation of V _H and V _L genes by PCR methodologies known in the art (Sambrook et al., Eds., Molecular Cloning, Chapter 14, Cold Spring Harbor laboratories Press). , NY (1989). The N-terminal amino acid sequences of the heavy and light chains can be determined independently by automated Edman sequencing. Additional sequences for CDRs and flanking FRs can be determined by amino acid sequencing for V _H and V _L fragments. Synthetic primers can then be designed for isolation of V _H and V _L genes from AR81A410.7 monoclonal antibody, and the isolated genes can be ligated into appropriate sequencing vectors. To generate chimeric and humanized IgG, the variable light and variable heavy domains can be subcloned into appropriate expression vectors.

In another embodiment, the nucleic acid encoding the antibody is expressed in a transgenic animal to allow the antibody to be expressed and recovered to produce AR81A410.7 or a de-immunized, chimeric or humanized form thereof. For example, the antibody can be expressed in a tissue specific manner that facilitates recovery and purification. In one such embodiment, the antibodies of the invention are expressed in the mammary gland to be secreted during lactation. Transgenic animals include, but are not limited to, mice, goats, and rabbits.

(i) monoclonal antibodies

DNA encoding a monoclonal antibody (as outlined in Example 1) is an oligonucleotide capable of specifically binding to genes encoding the heavy and light chains of monoclonal antibodies, eg, using conventional procedures. By using a probe) is readily isolated and sequenced. The hybridoma cells serve as a preferred source for such DNA. Once the DNA is isolated, it can be placed in an expression vector, which is then placed in a host cell that otherwise does not produce immunoglobulin proteins, such as E. coli cells, monkey COS cells, Chinese hamsters. Transfection into ovarian (CHO) cells, or myeloma cells, achieves the synthesis of monoclonal antibodies in recombinant host cells. In addition, the DNA can be modified by, for example, replacing homologous murine sequences with coding sequences for human heavy and light chain constant domains. Chimeric or hybrid antibodies can also be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose are iminothiolates and methyl-4-mercaptobutyrimidadate.

(ii) humanized antibodies

Humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken in an "import" variable domain. Humanization can be performed by Winter and colleagues' methods by replacing the CDRs or CDR sequences of human antibodies with the corresponding rodent sequences (Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323). -327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988); overviewed by Clark, Immunol. Today 21: 397-402 (2000).

Humanized antibodies can be prepared by methods for analyzing parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and are well known to those skilled in the art. Computer programs are available that illustrate and represent possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. By reviewing these representations, it becomes possible to analyze the possible role of the residues in the action of the candidate immunoglobulin sequence, ie, the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from consensus and import sequences so that desired antibody properties, such as increased affinity for the target antigen (s), are achieved. In general, CDR residues are directly and most substantially involved in affecting antigen binding.

(iii) antibody fragments

Various techniques have been developed for the preparation of antibody fragments. These fragments can be produced by recombinant host cells (Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today 21: 364-370 (2000)). For example, Fab'-SH fragments can be recovered directly from E. coli and chemically coupled to form F (ab ') ₂ fragments (Carter et al., Biotechnology 10: 163-167 (1992)). In another embodiment, F (ab ') ₂ is formed using leucine zipper GCN4 which facilitates assembly of F (ab') ₂ molecules. According to another approach, Fv, Fab or F (ab ') ₂ fragments can be isolated directly from recombinant host cell culture.

Example 6

Compositions Containing Antibodies Of The Invention

Antibodies of the invention can be used in compositions for preventing / treating cancer. Compositions for the prevention / treatment of cancer, containing the antibodies of the present invention, are low toxic and can be used in human or mammalian (eg, rat, rabbit, sheep, pig, bovine, Cats, dogs, monkeys, etc.) orally or parenterally (eg, intravascularly, intraperitoneally, subcutaneously, etc.). The antibody of the present invention may be administered by itself or may be administered as a suitable composition. The composition used for such administration may contain a pharmacologically acceptable carrier, diluent or excipient with the antibody or salt thereof of the invention. Such compositions are provided in the form of pharmaceutical preparations suitable for oral or parenteral administration.

Examples of compositions for parenteral administration are injections, suppositories, and the like. Injectables include dosage forms such as intravenous, subcutaneous, intradermal and intramuscular injections, instillation, intraarticular injections, and the like. These injections can be prepared by known methods. For example, injectables can be prepared by dissolving, suspending or emulsifying the antibody or salt thereof of the invention in sterile aqueous or oily media commonly used in injectables. Aqueous media for injection include, for example, isotonic solutions containing physiological saline, glucose and other auxiliaries, including alcohols such as ethanol, polyhydric alcohols such as propylene glycol, polyethylene glycol, and nonionic surfactants. (E.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mole) adduct of hardened castor oil)) and the like. As the oily medium, for example, sesame oil, soybean oil and the like are used, which can be used in combination with a solubilizer such as benzyl benzoate, benzyl alcohol and the like. Injectables so prepared are usually filled in appropriate ampoules. Suppositories used for rectal administration can be prepared by blending the antibodies of the invention or salts thereof with conventional suppository bases. Compositions for oral administration include solid or liquid formulations, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions Etc. Such compositions are prepared by known methods and may contain vehicles, diluents or excipients commonly used in the pharmaceutical formulation art. Examples of vehicles or excipients for purification are lactose, starch, sucrose, magnesium stearate and the like.

Advantageously, the compositions for oral or parenteral use described above are prepared in pharmaceutical formulations with unit doses adapted to suit the doses of the active ingredients. Such unit dose formulations include, for example, tablets, pills, capsules, injections (ampoules), suppositories, and the like. The content of the above-mentioned compound is generally 5 to 500 mg per dosage unit form; The antibodies described above are particularly preferably contained in about 5 to about 100 mg in the injection form, and 10 to 250 mg in the other forms.

The dose of the prophylactic / treatment agent or modulator comprising the antibody of the present invention may vary depending on the subject to be administered, the target disease, condition, route of administration and the like. For example, when used to treat / prevent breast cancer in an adult, for example, the antibody of the present invention may contain from about 0.01 to about 20 mg / kg body weight, preferably from about 0.1 to about 10 mg / kg body weight and more It is advantageously administered intravenously at a dose of about 0.1 to about 5 mg per kg of body weight, about 1 to 5 times a day, preferably about 1 to 3 times a day. In other parenteral and oral administrations, the formulations may be administered in doses corresponding to those given above. If the condition is particularly severe, the dose may be increased depending on the condition.

Antibodies of the invention can be administered as such or in the form of a suitable composition. The composition used for such administration may contain a pharmacologically acceptable carrier, diluent or excipient with the above-described antibody or salt thereof. Such compositions are provided in the form of pharmaceutical preparations suitable for oral or parenteral administration (eg, endovascular injection, subcutaneous injection, etc.). Each composition described above may further contain other active ingredients. Furthermore, the antibodies of the present invention may be further formulated with other drugs such as alkylating agents (eg, cyclophosphamide, ifosfamide, etc.), metabolic antagonists (eg, methotrexate, 5-fluorouracil, etc.), anti-tumor antibiotics ( For example, it can be used in combination with mitomycin, adriamycin, etc., plant-derived anti-tumor agents (e.g., vincristine, bindecine, Taxol, etc.), cisplatin, carboplatin, etoposide, irinotecan and the like. Antibodies of the invention and the drugs described above may be administered to a patient at the same time or at staggered times.

The methods of treatment described herein, particularly for cancer, may also be carried out by administering other antibodies or chemotherapeutic agents. For example, especially in the treatment of colon cancer, antibodies to EGFR such as ERBITUX® (cetuximab; cetuximab) can also be administered. ERBITUX® has also been shown to be effective in treating psoriasis. Other antibodies to be used in combination is, in particular breast cancer, treatment of Herceptin® (trastuzumab; trastuzumab) particularly when treating colon cancer AVASTIN ^®, and in particular non-(非) - a SGN-15 small cell lung cancer during treatment. Antibodies and other antibodies / chemotherapeutic agents of the invention can be administered via the same or different routes, simultaneously or separately.

The chemotherapeutic / other antibody regimens used include any regimen that is considered best suited for the treatment of the patient's condition. Different malignancies may require the use of specific anti-tumor antibodies and specific chemotherapeutic agents, which will be determined on a patient-by-patient basis. In a preferred embodiment of the invention, chemotherapy is performed concurrently with, or more preferably, after antibody therapy. However, it should be emphasized that the present invention is not limited to any particular method or route of administration.

Predominant evidence shows that AR81A410.7 mediates anti-cancer effects through ligation of epitopes present on cancer cell lines. Furthermore, it can be revealed that the AR81A410.7 antibody can be used to detect cells expressing epitopes specifically bound by the antibody using techniques exemplified by, but not limited to, FACS, cellular ELISA or IHC. there was.

All patents and publications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All patents and publications are incorporated herein by reference to the same extent as each individual publication is indicated to be specifically and individually incorporated by reference.

While specific forms of the invention have been described, it should be understood that it is not intended to limit the invention to the specific forms or arrangements of parts described and indicated herein. It will be apparent to those skilled in the art that various changes may be made without departing from the scope of the present invention and that the present invention should not be considered limited to what is shown and described herein.

Those skilled in the art will readily recognize that the present invention is well suited to carry out the above objects and achieve the stated objects and advantages, as well as the objects and advantages inherent therein. Any of the oligonucleotides, peptides, polypeptides, biologically related compounds, methods, procedures, and techniques described herein are representative of the presently preferred embodiments, intended to be exemplary, and intended to limit the scope of the present invention. no. Such modifications and other uses will occur to those skilled in the art which are included in the spirit of the invention and defined by the scope of the appended claims. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications apparent to those skilled in the art to the described modes of carrying out the invention are intended to be within the scope of the following claims.

Claims (48)

An isolated monoclonal antibody produced by hybridoma deposited with IDAC 051206-01. A Fab, Fab ', F (ab') ₂ or Fv fragment of the isolated monoclonal antibody of claim 1. Humanized forms of isolated monoclonal antibodies produced by hybridomas deposited with IDAC 051206-01 in IDAC or Fab, Fab ', F (ab') ₂ or Fv fragments generated from such humanized antibodies. delete A chimeric form of an isolated monoclonal antibody produced by a hybridoma deposited with IDAC 051206-01 in IDAC or a Fab, Fab ', F (ab') ₂ or Fv fragment generated from said chimeric antibody. delete Claims 1, 2 and 3, combined with a member selected from the group consisting of cytotoxic moieties, enzymes, radioactive compounds, cytokines, interferons, target or reporter moieties and hematogenous cells Or the isolated antibody of any one of claims 5 or a Fab, Fab ', F (ab') ₂ or Fv fragment thereof. An isolated hybridoma cell line deposited with IDAC at 051206-01. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete Isolated monoclonal antibody produced by hybridoma cell line AR81A410.7 with IDAC Accession No. 051206-01, Humanized antibody of isolated monoclonal antibody produced by hybridoma deposited with ID 051206-01 in IDAC Or as a binding assay to determine the presence of cancer cells in a tissue sample selected from human tumors specifically bound by chimeric antibodies of isolated monoclonal antibodies produced by hybridomas deposited with IDAC 051206-01 in IDAC. , Assay comprising the following steps: Providing a tissue sample from the human tumor; Providing at least one of said isolated monoclonal antibody, said humanized antibody, said chimeric antibody, or Fab, Fab ', F (ab') ₂ or Fv fragments thereof; Contacting at least one of the provided antibodies, or Fab, Fab ', F (ab') ₂ or Fv fragments thereof with the tissue sample; And Measuring the binding of said at least one provided antibody, or Fab, Fab ', F (ab') ₂ or Fv fragment, to said tissue sample; This indicates the presence of the cancer cells in the tissue sample. delete delete delete delete delete delete delete delete delete delete delete delete delete delete Compositions effective for the treatment of human cancerous tumors, comprising the following combinations: The antibody or Fab, Fab ', F (ab') ₂ or Fv fragment of any one of claims 1, 2, 3 or 5; A member selected from the group consisting of said antibody or Fab, Fab ', F (ab') ₂ or Fv fragment thereof, a cytotoxic moiety, an enzyme, a radioactive compound, a cytokine, an interferon, a target or reporter moiety and a hematopoietic cell; Conjugates of; And Required amount of pharmaceutically acceptable carrier; Wherein said composition is effective for treating said human cancerous tumor. delete

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