KR20070022219A - Humanized antibody - Google Patents

Abstract

New humanized and chimeric antibodies, humanized antibody fragments, polypeptide sequences of such antibodies, and derivatives thereof that specifically bind AF-20, as well as methods for their preparation are provided. Such humanized and chimeric antibodies, antibody fragments and polypeptide sequences are useful in the treatment of cancers expressing AF-20 as well as for diagnostic purposes, for example in vivo imaging of tumors or cancer cells expressing AF-20. .

Recombinant antibody molecules, humanized antibodies, chimeric antibodies, antibody fragments, polypeptides.

Description

Humanized Antibody {HUMANIZED ANTIBODY}

Embodiments of the invention relate to humanized and chimeric antibodies, fragments, polypeptides or derivatives thereof capable of binding to adenocarcinoma cell antigen AF-20, which are associated with carcinoma cells, in particular hepatocarcinoma cells and adenocarcinoma cells of the colon and lung.

Cancer is the second leading cause of death in the United States. Despite developments so far, the incidence of cancer per 100,000 US population has not decreased since 1950; In fact, it increased slightly. Therefore, there is an urgent need for effective cancer treatment and at the same time a new approach for the diagnosis, evaluation and monitoring of cancer conditions is still required.

What is particularly needed is a method of targeting metastases for prevention and / or treatment. One of the most embarrassing aspects of cancer is the tendency for cells from malignant neoplasms to spread from their original site and spread to organs far away. Despite advances in surgical treatment and aggressive therapy of major neoplasms, most cancer patients die as a result of disease metastasis. Animal testing indicates that about 0.01% of circulating cancer cells from solid tumors establish successful metastatic colonies (Fidler, 1993). Monoclonal antibodies specific for tumor-associated antigens (hereinafter “moAbs”) provide considerable hope in the treatment of cancer and in the targeting of metastasis.

Hepatocellular Carcinoma -Hepatocellular carcinoma ("HCC") is one of the leading causes of cancer-related deaths worldwide. HCC accounts for over 80% of liver cancers. There is a wide variation in the incidence of HCC in different regions of the world, and incidence in Asia and Africa is more than 10 times in the United States. Many factors have been identified as of potential importance in the etiology of these diseases. Chronic liver diseases such as chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection or cirrhosis make individuals susceptible to HCC. HCC is one of the most common human malignancies, possibly due to high rates of HBV and HCV infection in many parts of the world, particularly in Asia and Africa. In addition, aflatoxins present in certain foods such as tree nuts and peanuts are thought to make individuals susceptible to HCC. Since liver cancer occurs two to four times more frequently in men than in women, hormonal factors may be important in the etiology of HCC.

The prognosis for HCC is poor and often dies within three to six months. Only about 6% of patients diagnosed with HCC will survive for 5 years. Localized HCC may be treated surgically via partial liver resection or total liver resection by liver transplantation, or, if not resected, by radiologic excision, cryosurgery or transdermal ethanol infusion or hepatic arterial infusion or chemoembolization. Treatment may be through exfoliation therapy, such as chemotherapy being administered. Only 10-20% of all HCC surgeries succeed in completely removing cancerous tissue. Highly advanced HCC may also be treated with systemic chemotherapy or radiotherapy, but the effects are limited and rarely successful.

Symptoms of HCC are typically more pronounced only in the later stages of the disease, making treatment more difficult. Diagnostic tests include alpha-fetoprotein (AFP) blood tests. Only high AFP tests indicate the potential for liver cancer; This cannot confirm the diagnosis. 50-75% of people with primary liver cancer have high levels of AFP. In addition, other conditions, most notably cirrhosis, chronic hepatitis infection, and some other cancers cause high levels of AFP. In addition to the AFP blood test, many other tests that measure enzyme, bilirubin and protein levels can identify possible liver failure. Diagnostic imaging such as liver scans, computed tomography (CT) scans, ultrasound or magnetic resonance imaging (MRI) can identify potential liver tumors and biopsy sites. None of these tests can be used alone to diagnose liver cancer. Still, liver biopsy is considered the best method for the accurate diagnosis of HCC. Although some tumors are linked to multiple blood vessels, fatal bleeding can occur in less than 0.5% of cases, but the procedure is generally very safe.

Lung Cancer -Lung cancer is the leading cause of cancer deaths in the United States and one of the leading causes of death worldwide. The overall five-year survival rate (about 13%) of individuals with disease has not changed significantly over the past 25 years. The incidence of the disease has remained the same after years of dramatic increase. Tobacco smoking is responsible for about 90% of lung cancer symptoms in men and about 80% in women. The greater the amount and duration of smoking, the greater the risk of developing lung cancer. About 10 to 12% of all smokers eventually develop lung cancer.

Primary lung carcinoma is divided into two main types: non-small cell carcinoma and small cell carcinoma. Non-small cell carcinoma is more common and has a better prognosis than small cell carcinoma. There are three main classes of non-small cell lung carcinoma: squamous cell carcinoma (also called epidermal carcinoma), adenocarcinoma and large cell carcinoma. Adenocarcinoma is the most common type of lung cancer and accounts for 30-35% of all conditions.

The prognosis for primary lung cancer is poor. Less than 1% of individuals with small-cell carcinoma survive 5 years after diagnosis of the disease. In contrast, the prognosis of individuals with non-small cell carcinoma depends on the stage of the cancer, in particular the presence or absence of distant metastasis. Distant metastasis is associated with a 5 year survival rate of 5% or less. Liver tissue is the site where this metastasis often occurs.

Small cell carcinoma is currently treated by a combination of surgical resection, radiotherapy and chemotherapy. Despite this aggressive treatment, the prognosis of these diseases is very poor. Selected treatments for non-small cell carcinoma of the lung include surgical resection of cancerous lesions. Unfortunately, such surgery is only possible in the very early stages of the disease, and even with surgery the 5-year survival rate is on the order of 25% to 40%. Although radiotherapy may be applied to treat later stage non-small cell carcinoma, the prognosis of such therapy is poor. Chemotherapy has a limited effect for non-small cell carcinoma, but can significantly increase survival in metastatic non-small cell carcinoma.

Diagnosis and detection of lung cancer is particularly facilitated by chest X-rays, CT scans of the lungs, bronchoscopy and biopsy.

Colorectal cancer - colon and rectal cancer is cancer and the second leading cause of death related, account for approximately 20% of all cancer deaths in the United States. 5-year survival rate is approximately 63%; Distant metastasis is associated with much lower survival rates of less than about 10%. Approximately 60% of patients diagnosed with colorectal cancer will develop liver metastases, leaving the optimal therapeutic criteria for liver resection. Despite surgical treatment, most patients after liver resection will cause a relapse, and approximately 50% of relapses will occur in the liver. Almost all colorectal cancers are adenocarcinomas.

Delayed diagnosis significantly affects the prognosis of colorectal carcinoma. If detected early, colorectal cancer can often be treated successfully. In other words, patients whose tumors are limited to the visceral wall, for example, generally have excellent treatment opportunities after surgical resection (5-year survival> 95%). However, if the tumor has expanded to the mesenteric and mesenteric fats, the 5 year survival rate after resection is lowered to 80%. Lymph node metastasis lowers 5-year survival to 40%, while distant metastases (eg liver, lung, bone, brain) lower 5-year survival to less than 10%. Detection is often delayed because the symptoms of colorectal carcinoma in the initial state of the disease are often unclear and nonspecific. As a result, cancer is often settled until a positive diagnosis is made, resulting in difficult or impossible treatment. Colorectal carcinoma generally responds poorly to chemotherapy. Although alleviation of the disease may occur, chemotherapy does not appear to prolong the lives of patients diagnosed with colorectal cancer, especially when the disease is widespread.

The US Preventive Services Countermeasures Center (USPSTF) strongly recommends that clinicians screen men and women 50 and older for colorectal cancer. The USPSTF found good evidence that the periodic fecal occult blood test (FOBT) reduced mortality from colorectal cancer, and adequate evidence that the CT test alone or in combination with FOBT reduced mortality. However, screening tests commonly used for colorectal carcinoma can show false positives and delay the detection of the disease through false negatives. For example, FOBT detects occult blood in the toilet and defines that it has advanced to the bleeding stage before colon malignancies can be detected. Postmenopausal colonoscopy requires visible colorectal carcinoma and the diagnosis may be complicated by the presence of other lesions such as bleeding, polyps and proctitis. Colonoscopy also has similar drawbacks.

Monoclonal antibodies specific to tumor-associated antigens (“moAbs”) offer considerable hope in the study, diagnosis, surveillance and treatment of cancer. However, substantial practical problems exist for a wide range of in vivo uses in humans and other mammals.

It is a major concern that monoclonal antibodies of non-human origin are often immunogenic, thereby limiting their efficiency and in some cases causing dangerous allergic reactions. Most moAbs are of mouse origin and have been found to be generally immunogenic when injected into humans. The immune response to these foreign moAbs involves the generation of specific high affinity antibodies that bind to and carry out their removal, thereby facilitating removal from the body and binding to the target tumor-associated antigen. Suppressing significantly reduces the effect of moAb.

Many methods are known that may reduce the immunogenicity of non-human antibodies. These include:

US Patent 4,816,567 to Cabilly et al .; Morrison, S.L. Et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984); Boulianne, G.L. Et al., Nature 312; 643-646 (1984); Neuberger, M.S. Et al., Nature 314: 268-270 (1985), generation of chimeric antibodies by attaching variable regions in the heavy and light chains of non-human antibodies to the constant regions of human antibodies.

Winter, US Pat. No. 5,225,539, Jones, P.T. Et al., Nature 321: 522-525 (1986); Riechmann, L. Et al., Nature 332: 323-327 (1988); Verhoeyen, M. Et al., Science 239: 1534-1536 (1988), for the generation of humanized antibodies (also referred to as CDR grafting) by substituting non-human complementarity determining regions (CDRs) or CDR sequences with corresponding fragments of human antibodies. Known). This is described, for example, in US Pat. Nos. 5,530,101, 5,585,089, 5,693,762, 6,180,370 to Queen et al., US Pat. Nos. 6,054,297, 6,407,213 and 6,639,055 to Carter et al., US Pat. As such, some FR residues in human antibodies may be substituted with residues from similar sites in non-human antibodies to maintain antigen binding.

Selective selection of residues in the variable regions of non-human antibodies as described, for example, in US Pat. Nos. 5,639,641 to Pedersen et al., US Pat. Nos. 5,766,886 and 5,821,123 to Studnicka et al. And US Pat. Appl. No. 10/300215 to Carr et al. Generation of humanized antibodies by substitution (also known as veneering or resurfacing).

Binding of an antibody or antibody fragment to a self-antigen sequence, making the non-human antibody or antibody fragment low immunogenic, as described, for example, in US Pat. No. 6,652,863 to Jordna et al.

Many methods of generating chimeric or humanized antibodies represent the difficulties encountered in generating appropriate antibody candidates. That the resulting antibody has too low affinity or specificity for the target tumor-associated antigen, still elicits an undesirable immune response, or is too difficult or otherwise poorly expressed in a practical amount. It is not uncommon to find out.

Another major concern relates to the tumor-associated antigens that the antibody targets. The efficacy of treatment depends on the specificity of the antigen for the tumor, its role in tumor growth and expression by tumor cells. Tumor-associated antigens may be widely expressed in normal tissue, thereby requiring higher therapeutically effective amounts and increasing the risk of unwanted side effects. The antigen may be expressed by only a small percentage of the tumor, by the percentage of cells in either tumor, or both. Antigens may be secreted only by tumor cells and are not expressed on the tumor cell surface, which makes targeting of cytotoxic therapies more difficult if not impossible. Binding to the antigen expressed on the cell surface may not cause internalization of the cytotoxic agent into the cell or may not lead to the desired inhibition of function.

Despite these disorders, after many attempts, a small number of monoclonal antibodies have been approved for management. Approved antibody-based therapeutics include:

Trastuzmab (Herceptin®), a humanized monoclonal antibody (moAb) that binds to human epidermal growth factor receptor 2 (HER2), thereby leading to tumor cell proliferation and metastatic breast cancer overexpressing HER2 Suppresses movement It is currently indicated to be used alone or in combination with chemotherapeutic paclitaxel for the treatment of metastatic breast cancer overexpressing the HER2 protein. Herceptin® is under clinical trials for the treatment of non-metastatic breast cancer, and also HER-2 protein including bone sarcoma, non-small cell lung cancer and pancreas, salivary gland, colon, prostate, endometrium, bladder It is being studied in clinical trials for other types of cancer that may overexpress. It is also being investigated for use when conjugated to cytotoxins such as geldanamycin.

Alemtuzmab (Campath®), a humanized moAb that binds the CD52 antigen, which is currently indicated for use in the treatment of refractory B-cell chronic lymphocytic leukemia, and other chronic lymphoid and chronic myeloid It is under investigation to be used for the treatment of leukemia.

Gemtuzmab (Mylotarg®), a humanized moAb that binds CD33, is a protein expressed in about 90% of cases of acute myeloid leukemia (AML). Mylotarg® is conjugated to calicheamicin, a bacterial toxin that induces DNA strand breakage and cell death. This is indicated for use for the treatment of AML.

Rituximab (Rituxan®), a chimeric moAb that binds to CD20, an antigen found on the surface of mature B cells, thereby indicating that the cells are destroyed by the body's immune system. It is indicated for use for the treatment of relapsed or refractory low-grade or vesicle B-cell non-Hudkins lymphoma (NHL). In addition, it is under study for use for the treatment of B-cell lymphoma and chronic lymphocytic leukemia.

Ibritumomap (Zevalin®), a mouse moAb conjugated to beta-emitting radioisotope yttrium-90 ( ⁹⁰ Y), which binds CD20 and repairs cell damage in target and neighboring cells Induce. It is indicated for use in combination with Rituxan® for the treatment of NHL.

Tocitumomab (Bexxar®), a mouse moAb, which binds to CD20 and to other radioisotope iodine-131 ( ¹³¹ I). It is indicated for use for the treatment of NHL that recurs after chemotherapy with Rituxan®.

Edrecolomab (Panorex®), a mouse moAb that binds to epithelial cell adhesion molecules. It is approved for use in Europe for the treatment of colorectal cancer and is in Phase III clinical trials in the United States for colorectal and breast cancer.

Other therapeutic antibodies in phase III testing include:

Cetusimab (Erbitux®), chimeric moAb that binds to epidermal growth factor receptor (EGFR), head and neck, non-small cell lung cancer, colorectal cancer, breast cancer and pancreatic and prostate Investigated for the treatment of cancer.

Humanized moAb that binds to bevacizumab (Avastin®), vascular endothelial growth factor (VEGF) and is being investigated for the treatment of metastatic colorectal cancer, breast cancer and non-small cell lung cancer.

Humanized antibodies and antibody fragments are methods designed to improve their characteristics and performance, such as phage display, bacterial or yeast cell surface display (eg, US Pat. No. 6,300,065 to Kieke et al. And US Pat. No. 6,423,538 to Wittrup) and others. The application of designated molecular evolution techniques (eg, US Pat. No. 5,714,350 to Co et al.) Is useful for creating optimal new therapies.

Chimeric and humanized antibodies and antibody fragments have utility in the diagnosis, staging and therapeutic surveillance of cancer. Enhancement of half-life in vivo and reduction of immunogenicity make moAbs potentially more practical for in vivo immune imaging, which conjugates a detectable label, such as a radionuclide or resonance imaging reagent, to an antibody or antibody fragment. Such antibodies and antibody fragments have utility in determining whether a tumor expresses an antigen before initiating treatment, determining a site for topical administration of the antibody, and preventing relapse after treatment. Similar to other antibodies, such antibodies find use in immunohistochemistry and immunoassays.

The AF-20 tumor associated antigen is a rapidly internalized 180 kDa homodimeric cell-surface glycoprotein that is expressed in large quantities in distant metastases such as adenocarcinoma of human hepatocellular carcinoma (HCC) cells as well as lung cells and colorectal carcinoma cells. The AF-20 antigen was revealed by high affinity mouse monoclonal antibody (AF-20 moAb) found by immunizing mice with hepatocellular carcinoma cell line FOCUS and selecting hybridomas for antibody activity on a panel of cell lines. AF-20 antigen was found not to be expressed in normal liver tissue adjacent to HCC tissues, nor in other normal tissues except the adrenal gland. Low-level expression of the AF-20 antigen has been found on small populations of cells in the adrenal globules and follicular gland cells of the small intestine (Wands et al. US Pat. No. 5,703,213; Wilson et al., "Malignant Tumor Phenotype of Human Hepatocytes"). Cell-Surface Change Associated with Transformation "Proc Natl Acad Sci USA (1988 85: 3140-4); [Takahashi et al.," In vivo expression of two new tumor-associated antigens and in immunolocalization of human hepatocellular carcinoma Its use ", Hepatology (1989; 9: 625-34)," Moradpour et al., "Specific targeting of human hepatocellular carcinoma cells by immunoliposomes in vitro" Hepatology (1995; 22: 1527-37); [Wands et al., “Immunologic Approach to Hepatocellular Carcinoma” J Viral Hepat (1997; 4 Suppl 2: 60-74)]; [Mohr et al. “Hepatocellular Carcinoma in Vitro Using a New Monoclonal Antibody-Based Gene Delivery System” Targeted Gene Delivery to Cells "Hepatology (1999; 29: 82-9); [Yoon et al.," Bifunctional Fab-antigens " Targeting Recombinant Adenovirus Vectors to HCC Cells Using Conjugate Conjugates ", Biochem Biophys Res Commun. (2000; 272: 497-504); Palumbo et al.," Human Aspartyl (Asparaginyl) Beta-Hide Roxylase Monoclonal Antibodies: Potential Biomarkers for Pancreatic Carcinoma "Pancreas (2002; 25: 39-44)] [Yoon et al.," Chimera immunotoxin of AF-20 monoclonal antibody with Pseudomonas exotoxin for hepatocellular carcinoma Targeted Cancer Therapy Using "(June 2002, undisclosed excerpt); their disclosures are each incorporated by reference).

AF-20 moAb has shown potential as an immunotargeting agent and an immunoimaging agent. To localize hepatitis B virus-associated hepatocellular carcinoma cell line (FOCUS) grown into subcutaneous tumors, an AF-20 moAb labeled ¹²⁵ I was successfully used in a hairless mouse model for in vivo radioimaging. Nuclear imaging studies have shown clear visualization of tumor tissue and demonstrate good specificity and sensitivity of AF-20 moAb as a potential immunotargeting or immunoimaging agent.

AF-20 moAbs have been found to be rapidly internalized by HCC cells and are good candidates for targeted delivery of cytotoxic agents or gene therapies to tumor cells expressing AF-20 antigens. Antibodies to tumor-associated antigens that cannot be internalized in the tumor cells to which they bind are not generally useful for targeted delivery because such antibodies cannot reach the site of action within the cell. AF-20 moAb conjugated to Pseudomonas exotoxin in one study (Hoon, 2002 Hok.) Was found to have potent anti-tumor activity in in vitro tests on HCC cells and in vivo tests on hairless mice that received HCC xenografts. lost. In another approach (Moradpour, homology), AF-20 moAb was covalently bound to liposomes containing carboxyfluorescein. AF-20 immunoliposomes specifically bound to HCC and other human cancer cell lines expressing AF-20 antigen and rapidly internalized at 37 ° C. The interaction of these cell lines with AF-20-conjugated liposomes is 5-200 times greater than unconjugated liposomes, while no differences were observed between control liposomes and unconjugated liposomes with unrelated antibodies. Kinetic analysis showed rapid binding of the target cells with AF-20 immunoliposomes and reached saturation after 60 minutes.

AF-20 moAb was used to develop an experimental targeted gene delivery system. In this approach, a specific adenovirus gene delivery system consisting of bifunctional Fab-antibody conjugates (2Hx-2-AF-20) occurred via AF-20 moAb crosslinking to anti-hexone antibody Fab fragments. Conjugate complexes were found to internalize rapidly at 37 ° C., and high levels of reporter gene expression were observed in AF-20 antigen positive HCC cells, but not in AFD-20 antigen negative control cells. In another approach, AF-20 moAb was bound to the DNA-binding cationic amphiphile, cholesteryl-spermine, for gene delivery to hepatocellular carcinoma (HCC) cells. Binding and internalization of AF-20-cholesteryl-sperbin was demonstrated by fluorescence microscopy using fluorescein isothiocyanate (FITC) -labeled anti-mouse IgG antibody. Transfection of the luciferase or beta-galactosidase reporter gene complexed to AF-20-cholesteryl-sperbin resulted in high levels of gene expression in AF-20 antigen-positive tumor cells.

As indicated above, there is an urgent need for the treatment of cancer, in particular of hepatocellular carcinoma, lung adenocarcinoma and colorectal carcinoma. The mouse monoclonal antibody, AF-20 moAb, provides hope as a means to deliver targeted therapies to such tumors and their metastases, but to be effective, immunogenicity must be reduced or preferably not immunogenic. Thus, there is a need for chimeric and humanized antibodies derived from AF-20 moAbs that retain affinity and specificity for AF-20 antigens.

The description of the disadvantages and detrimental properties associated with the known compositions, methods and schemes described herein in no way exclusively limit the scope of the invention. Indeed, embodiments of the present invention may include some of one or more known compositions, methods, and systems as long as they do not have disadvantages and detrimental properties.

Summary of the Invention

Embodiments of the present invention are made available by surprisingly discovering and generating chimeric and humanized antibodies, which possess favorable affinity with AF-20 and provide an important new approach to the treatment of at least three embarrassing major cancers. It became.

One embodiment of the invention includes chimeric and humanized antibodies and fragments thereof (“AF-20 antibodies”) capable of recognizing AF-20 antigens associated with hepatocellular carcinoma, lung adenocarcinoma, colorectal carcinoma and other cancers. . Preferred embodiments of the invention relate to the chimeric and humanized antibodies described herein comprising the sequences of VRs, FRs and CDRs polypeptides and the polynucleotides encoding them.

Other embodiments of the present invention are directed to the VR, FR and CDR polypeptides described herein (“VRs, FRs and CDRs”), as well as to polynucleotides encoding AF-20, in non-human AF-20 antibodies and humanized AF-20 antibodies. The use of polynucleotides and polypeptides in the production of novel antibody and polypeptide compositions capable of recognizing antigens.

Further embodiments of the invention provide polynucleotides encoding AF-20 antibody polypeptides, VRs, FRs and CDRs. Also provided are various expression vectors comprising an AF-20 antibody operably linked to a promoter sequence and a polynucleotide encoding VRs, FRs and CDRs. Similarly, other embodiments of the invention include host cells transformed with expression vectors for the expression of AF-20 antibodies, VRs, FRs, and CDRs.

Embodiments of the invention relate to the use of AF-20 antibodies for the diagnosis, evaluation and treatment of hepatocellular carcinoma, lung adenocarcinoma, colorectal carcinoma and other cancers expressing the AF-20 antigen. A further embodiment of the present invention is directed to the use of an antibody as a targeted delivery system for cytotoxic drugs, such as chemotherapeutic drugs, peptides or radionuclides, for immunological response promoters, pro-drugs or gene therapies such as cytokines. It is about.

Another embodiment of the present invention provides humanized AF-20 antibodies in designated molecular evolution techniques such as phage display or bacterial or yeast cell surface display techniques to produce polypeptides with enhanced affinity, specificity, stability or other desirable characteristics. And the use of VRs, FRs and CDRs.

Other objects, features and features of the present invention will become apparent upon consideration of the following detailed description and claims.

1 shows the DNA and amino acid sequences of the NYR-1002 V _H chain with four FRs and three CDRs identified therein.

Figure 2 shows the DNA and amino acid sequences of the NYR-1002 variable light chain with four FRs and three CDRs identified therein.

3 shows antibody heavy chain expression vectors.

4 shows an antibody light chain expression vector.

5 shows potential human T cell epitopes identified in the heavy and trans variable regions of NYR-1002.

6 shows amino acid changes and potential epitopes generated in variants of the V _H region of NYR-1002.

7 shows amino acid changes and potential epitopes generated in variants of the V _K region of NYR-1002.

8 shows the DNA and amino acid sequences of primary NYDIVH1.

9 shows the DNA and amino acid sequences of the primary NYR-1002 V _K variant, NYDIVK1.

10 shows the yield of Protein A purified antibody from 1 liter of the modified antibody prepared and the culture supernatant.

11: Response of 20 donors to NYDIVH2 / NYDIVK2 antibody and NYR-1002 mouse antibody in human T cell assay.

In general, the following terms or phrases have the definitions indicated below when used in the description, examples, and claims.

The expression “AF-20” or “AF-20 antigen” is described in US Pat. No. 5,703,213, the disclosure of which is incorporated by reference in its entirety and is an American Type Culture Collection (“ATCC”) under the Budapest Treaty. Refers to an adenocarcinoma cell antigen capable of binding to mouse antibodies produced by a hybridoma cell line deposited with ATCC Accession No. HB 9687 on April 12, 1988.

The expression “AF-20 moAb”, “AF-20 antibody” or “AF-20 monoclonal antibody” refers to non-human antibodies and fragments thereof capable of binding to the AF-20 antigen. This expression is described in US Pat. No. 5,703,213 and claims for mouse antibodies generated by a hybridoma cell line deposited with ATCC Accession No. HB 9687 on April 12, 1988 in the American Type Culture Collection (ATCC) under the Budapest Treaty. Include.

As used herein, "ATCC" means the American Type Culture Collection located in Manassas University Boulevard 10801, USA 20110-2209. "NYR-1002" refers to a mouse antibody produced by the hybridoma cell line ATCC name HB 9687.

The expression “constant region” or “CR” refers to the constant domain of an antibody that is not directly involved in binding the antibody to the antigen but is involved in the involvement of the antibody in various effector functions, such as antibody dependent cellular cytotoxicity.

The general structure of antibodies in vertebrates is well understood (Edelman, G. M., Ann. N. Y. Acad. Sci., 190: 5 (1971)). An antibody consists of two identical polypeptide light chains ("light chains") having a molecular weight of 23,000 daltons and two identical heavy chains ("heavy chains") having a molecular weight of 53,000-70,000. The four chains are connected by disulfide bonds in the form of "Y", where the light chain starts at the mouth of the "Y" form and binds the heavy chains together. The “branch” portion of the “Y” type is named the Fab region, and the stem portion of the “Y” type is named the Fc region. The amino acid orientation extends from the N-terminus at the top of the "Y" form to the C-terminus at the bottom of each chain. The N-terminus has a variable region with specificity for the antigen that elicits it, is approximately 100 amino acids long and there are some modifications between the light and heavy chains and between antibodies.

The variable region in each chain is linked to a constant region extending over the rest of the chain and does not change depending on the specificity of the antibody (ie, the antigen leading to it) within a particular class of antibody. There are five known constant region classes that determine the class of immunoglobulin molecules (γ, μ, α, δ, and ε (gamma, mu, alpha, delta, or epsilon) corresponding to the heavy chain constant region IgG, IgM , IgA, IgD and IgE). The constant region or class may be a function of antibodies, including activation of complement [Kabat, EA, Structural Concepts in Immunology and Immunochemistry, 2nd Edition, p413-436, Holt, Rinehart, Winston (1976)] and other cellular responses [ Andrews, DW Et al., Clinical Immunobiology, pp 1-18, W. B. Sanders (1980); Kohl, S. Et al., Immunology, 48: 187 (1983); The variable region determines the antigen to which it responds. Light chains are classified as κ (kappa) or λ (lambda). Each heavy chain class can be prepared by kappa or lambda light chains. The light and heavy chains are covalently bound to each other, and the "tail" portions of the two heavy chains are bound to each other by covalent disulfide bonds when immunoglobulins are generated by hybridomas or by B cells.

The expression “variable region” or “VR” refers to a domain within each pair of light and heavy chains in an antibody directly associated with binding the antibody to the antigen. Each heavy chain has at one end a variable domain (V _H ) followed by a number of constant domains. Each light chain has a variable domain (V _L ) at one end and a constant domain at the other end; The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.

The expression “complementarity determining region”, “hypervariable region” or “CDR” refers to one or more hypervariable or complementarity determining regions (CDRs) found in the light or heavy chain variable regions of an antibody (Kabat, EA et al., See Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987). Such expressions are described in Kabat et al., "Sequences of Proteins of Immunological Interest", Kabat E. et al., US Dept. hypervariable loops in the hypervariable region or in the three-dimensional structure of an antibody as defined by of Health and Human Services, 1983 [Chothia and Lesk, J Mol. 196 901-917 (1987). CDRs in each chain are tightly anchored by framework regions and contribute to the formation of antigen binding sites with CDRs from other chains.

The expression “framework region” or “FR” refers to one or more framework regions within the light and heavy chain variable regions of an antibody (Kabat, EA et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987). Such expression includes amino acid sequence regions sandwiched between CDRs within the light and heavy chain variable regions of an antibody.

CDR and FR residues are defined by standard sequences defined by Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda Md. (1987)] and structural definitions [Chothia and Lesk, J. Mot. Biol. 196: 901-217 (1987). In the case where identification of CDRs is obtained slightly differently by these two methods, structural definition is preferred, but residues identified by the sequence definition method are regarded as important FR residues in order to determine the framework residues involved in the consensus sequence. .

Throughout this description, Kabat, E.A. Et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991).) In this overview, Kabat is designed for each subclass. Many amino acid sequences of antibodies are described and the most commonly occurring amino acids for each residue position in this subclass are described Kabat uses a method of assigning residue numbers to each amino acid in the sequences described, This method of numbering has become the standard in this field, and this description also follows the Kabat numbering scheme.

For purposes of the present invention, to assign residue numbers to candidate antibody amino acid sequences not included in the Kabat intro, the following steps are followed. In general, candidate sequences are aligned with any immunoglobulin sequence or any consensus sequence within Kabat. Alignment can be performed by hand or by a computer using generally acceptable computer programs. Alignment may also be facilitated by using some amino acid residues that are common to most F _ab sequences. For example, light chains and heavy chains each typically have two cysteines with the same residue number; Two cysteines in the V _L domain are typically at residue numbers 23 and 88 and two cysteine residues in the V _H domain are typically numbered 22 and 92.

Framework residues are not always and generally have about the same number of residues, but CDRs may vary in size. For example, in the case of CDRs from candidate sequences that are longer than the CDRs in the sequences in Kabat aligned, a suffix is appended to the residue number to indicate the insertion of additional residues. For example, for a candidate sequence that is aligned with the Kabat sequence for residues 34 and 36 but does not have residues aligned with residue 35 therebetween, the number 35 is simply not assigned to the residue.

The term “antibody” is used in its broadest sense and specifically includes one monoclonal antibody (including agonist and antagonist antibodies) and antibody compositions with polyepitope specificity. The term “antibody” includes obvious variants, derivatives, analogs, fragments, mimetics, all of which substantially retain the binding characteristics and other properties of the antibodies mentioned.

As used herein, the expression “monoclonal antibody” (moAb) refers to an antibody obtained substantially from a population of homologous antibodies, that is to say that individual antibodies that make up the population are excluded except for possible naturally occurring mutations that may be present in small amounts. Same with each other. Monoclonal antibodies are highly specific and are directed against a single epitope region of the antigen. In addition, as opposed to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each moAb is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture and are not contaminated by other immunoglobulins.

The modifier “monoclonal” characterizes the antibody as obtained from a substantially homologous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be formed by hybridoma methods first described by Kohler et al., Nature, 256: 495 (1975), or by recombinant DNA methods (eg, , US Pat. No. 4,816,567, Cabilly et al.). "Monoclonal antibodies" are phage using techniques described in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991). Clones of antigen-recognition and binding-site containing antibody fragments isolated from antibody libraries (Fv clones).

As used herein, an “antibody fragment” and all modifications thereof are defined as part of a complete antibody or variable region of a complete antibody, including an antigen binding site, wherein that part is defined as a constant heavy chain domain of the Fc region of the complete antibody. That is, it does not have CH2, CH3 and CH4) depending on the antibody isotype. Examples of antibody fragments include Fab, Fab ', Fab'-SH, F (ab') ₂ and Fv fragments; Antibody fragments; Antibody fragments (herein referred to as "single chain antibody fragments" or "single chain polypeptides"), which are polypeptides having a major structure consisting of one uninterrupted sequence of contiguous amino acid residues (1) ) Single chain Fv (scFv) molecules; (2) a single chain polypeptide containing only one light chain variable domain, or a fragment thereof containing three CDRs of the light chain variable domain without having a linked heavy chain residue; And (3) a single chain polypeptide containing only one heavy chain variable region, or a fragment thereof containing three CDRs of the heavy chain variable region without having the light chain residues bound thereto. The antibody fragments of the present invention further comprise multispecific or multivalent structures formed from the above-mentioned antibody fragments. In antibody fragments comprising one or more heavy chains, the heavy chain (s) may contain any of the following:

One or more constant domain sequences found in the non-Fc region of a complete antibody (eg CH1 in the IgG isotype), and / or

Hinge region sequences found in intact antibodies, and / or

A leucine zipper sequence fused or located to the hinge region sequence or constant domain sequence of the heavy chain (s). Suitable leucine zipper sequences include the jun and fos leucine zippers taught in Kostelney et al., J. Immunol., 148: 1547-1553 (1992) and the GCN4 leucine zippers described in the Examples below.

The expression “chimeric antibody” refers to a polypeptide comprising a variable region of a non-human antibody that binds at least another portion of another protein, preferably an AF-20 linked to a constant region of a human antibody.

The term “humanization” refers to an “humanized” antibody in which a portion of the variable region, preferably substantially less than the fully human variable domain, is replaced by a corresponding sequence from a non-human species, and the modified variable region is It refers to a polypeptide comprising a modified variable region of a human antibody, which is linked to at least another portion of another protein, preferably to a constant region of a human antibody. The expression “humanized antibody” means that some or all CDR residues and / or possibly some FR residues are substituted by residues from similar sites in rodents or other non-human antibodies capable of binding to the AF-20 antigen. Human antibodies. The expression “humanized antibody” refers to an immunoglobulin amino acid sequence variant comprising a FR region capable of binding to an AF-20 antigen and comprising a FR region having substantially the amino acid sequence of human immunoglobulin and a CDR having substantially the amino acid sequence of non-human immunoglobulin or Includes fragments thereof.

In general, the humanized antibody comprises at least one, more preferably 2, wherein all or substantially all of the CDR regions correspond to non-human immunoglobulins and all or substantially all of the FR regions are regions of the human immunoglobulin consensus sequence. Substantially all of the two variable domains (Fab, Fab ', F (ab') ₂ , Fabc, Fv). Humanized antibodies will optimally comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a constant region of human immunoglobulin. Usually, the antibody will contain at least the variable domain of the heavy chain as well as the light chain. The antibody may comprise a heavy chain CH1, hinge, CH2, CH3 and CH4 region. Humanized antibodies may be selected from the class of immunoglobulins including IgM, IgG IgD, IgA and IgE and isotypes including IgG1, IgG2, IgG3 and IgG4. In general, where the humanized antibody exhibits cytotoxic activity, the constant domain is the complement fixed constant domain, and the class is typically IgG and preferably IgG1. If such cytotoxic activity is undesirable, the constant domain may be of IgG2 class. Humanized antibodies may comprise sequences from one or more classes or isotypes, and it is within the ordinary skill in the art to select particular constant domains to optimize the desired effector function. The FR and CDR regions of the humanized antibody do not have to correspond exactly to the parental sequence, for example, an endogenous CDR or consensus FR may be mutagenesis by substitution, insertion or deletion of one or more residues, resulting in CDR or FR residues of do not correspond to consensus or export antibodies. However, such mutations will not be widespread and will not have a dramatic effect on the binding of the antibody to the binding target.

The expression “humanized antibody” refers to a variable region of an anti-AF-20 antibody, irrespective of the species of origin, type of protein, immunoglobulin class or subclass indication, as long as the hybrid and recombinant antibodies and polypeptides exhibit the desired biological activity. Hybrid and recombinant antibodies and polypeptides generated by overlapping one or more CDRs with a heterologous protein (s).

The expression “humanized antibody” determines at least a portion of an amino acid sequence of an antibody or polypeptide (preferably part of a non-human origin such as the V _H or V _K region of a non-human antibody) and is one for human T-cells. These potential epitopes are identified in the amino acid sequence and modified to modify the amino acid sequence (s) of one or more epitopes to remove or reduce the immunogenicity of the protein or portion thereof when exposed to the human immune system. By a method of removing at least one, the method further comprises antibodies and polypeptides that are non-immunogenic to humans or have reduced immunogenic antibodies compared to native antibodies. In the production of humanized antibodies, preferably at least about 75%, more preferably at least about 90%, most preferably at least about 95% of the humanized antibody residues will correspond to the parent FR and CDR sequences.

The phrase “T-cell epitope” refers to a specific peptide sequence that exhibits the ability to bind MHC class II molecules with moderate efficiency or to stimulate T-cells through presence on MHC class II molecules from previous or other studies. However, it will be understood that all of these peptide sequences may not be delivered to the correct MHC class II cell compartment for MHC class II binding or may be properly released from larger cellular proteins for subsequent MHC class II binding. It is also understood that such peptides presented by MHC class II on the surface of antigen-presenting cells will elicit T cell responses for reasons including the lack of adequate T cell specificity and tolerance by the immune system for specific peptide sequences. .

The expression “bifunctional antibody” may have one arm with specificity for one antigenic site, such as a tumor associated antigen, while the other arm uses a hapten, which is a lethal drug for different targets, eg antigen-containing tumor cells. The antibody is to be recognized. Alternatively, the bifunctional antibody may be an antibody having specificity for different epitopes of tumor associated antigens of the cell in which each arm is to be therapeutically or biologically modified. In any case, the hybrid antibody has bispecificity and is preferably associated with one or more binding sites specific for the selected hapten or with one or more binding sites specific to the target antigen, eg, tumors, infectious organisms or other disease states. Has an antigen.

Biological bifunctional antibodies are described, for example, in European patent application EPA 0 105 360 (referenced by the person skilled in the art). Such hybrid or bifunctional antibodies may be derived biologically by cell fusion techniques or in particular by crosslinking agents or disulfide bridge forming reagents, and may consist of such antibodies and / or fragments thereof. Methods for obtaining such hybrid antibodies are described, for example, in International Application Publication WO 83/03679 published October 27, 1983 and European Patent Application EPA 0 217 577 published April 8, 1987. The entire contents of which are incorporated herein by reference. Particularly preferred bifunctional antibodies are other crosslinking agents that are biologically prepared from “polydomes” or “quadromas” or in combination with crosslinking agents such as bis- (maleimide) -methyl ether (“BMME”) or are familiar to those skilled in the art. It is prepared synthetically with.

The term "conjugated" means bonded directly or indirectly to another molecule by a number of means, for example by covalent, non-covalent, ionic or non-ionic bonding. Covalent bonds include bonds by various linkers, such as thioether linkers or thioester linkers. Direct bonding includes the attachment of one molecule to a major other molecule. Indirect bonds include attachment of one molecule to another, non-primary molecule that acts as a bridge, and then directly or indirectly to the primary molecule.

The expression "cytotoxic agent" means a drug that is harmful to cells. Examples thereof include antimetabolites such as methoxtrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine; Alkylating agents such as mechlorethamine, thioepa chlorambucil, melfaran, carmustine (BSNU), mitomycin C, romastin (CCNU), 1-methylnitrosourea, cyclotosamide, mechloreta Min, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin (paraplatin); Anthracyclines, including daunorubicin (formerly daunomycin), doxorubicin (adriamycin), detorrubicin, carminomycin, idarubicin, epirubicin, mitoxantrone and bisantrene; Antibiotics, including dactinomycin (actinomycin D), bleomycin, calicheamicin, mitramycin and anthracycin (AMC); And antimitotic agents such as vinca alkaloids, vincristine and vinblastine. Other cytotoxic agents include paclitaxel (taxol), lysine, pseudomonas exotoxin, gemcitabine, cytocarcin B, gramicidin D, ethidium bromide, emetine, etoposide, tenofoxide, colchicine, dihydroxy anthracene dione , 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroid, mitothene (O, P '-(DDD)) , Interferon and mixtures of these cytotoxic agents.

In particularly preferred embodiments, the cytotoxic agents are NTP peptides disclosed in US Application Serial Nos. 10 / 153,334, 10 / 198,070, 10 / 198,069 and 10 / 294,891, each disclosure of which is incorporated by reference in its entirety. It includes one or more of. NTP peptides, NTP peptide fragments, and the like may be conjugated to the humanized antibodies described herein to promote tumor cell necrosis.

As used herein, "oligonucleotides" include phospholisteres, phosphates or phosphoramidite chemistry using the solid phase technique described in known methods (e.g., EP 266,032, published May 4, 1988) or Froehler et al. , Via a deoxynucleoside H-phosphonate intermediate as described in Nucl. Acids Res., 14: 5399-2407 (1986)) Deoxynucleotides. These are purified on polyacrylamide gels.

As used herein, the technique of “polymerase chain reaction” or “PCR” is generally a procedure in which small amounts of specific nucleic acid fragment RNA and / or DNA are amplified as described in US Pat. No. 4,683,195 (July 28, 1987). Point to. In general, it is necessary to use sequence information from the ends of or beyond the main region, and thus oligonucleotide primers can be designed; Primers are identical or similar in sequence to opposite strands of the template to be amplified. The 5 'terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from whole genomic DNA, and cDNA transcribed from whole cell RNA, bacteriophage or plasmid sequences. In general, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51: 263 (1987); Erlich, ed., PCR Technology (Stockton Press, N.Y., 1989). As used herein, PCR uses nucleic acids known as primers (DNA or RNA) and nucleic acids to amplify or generate specific fragments of nucleic acids or to amplify or generate specific fragments of nucleic acids that are complementary to a particular nucleic acid. One example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, including using a polymerase, is considered to be not the only example.

As used herein, "treatment" refers to both treatment and prophylactic or preventative measures for treatment. Those in need of treatment are those already afflicted with the disease as well as those who are prone to the disease or whose disease is to be prevented.

Features of the embodiments described herein relate to humanized and chimeric antibodies, fragments, polypeptides or derivatives thereof capable of binding to adenocarcinoma cell antigen AF-20, which are associated with carcinoma cells, in particular hepatocellular carcinoma cells and adenocarcinoma cells of the rectum and lung. Related. More specifically, embodiments are humanized and chimeric antibodies, fragments, polypeptides derived from mouse monoclonal antibodies produced by the hybridoma cell line ATCC name HB 9687 or other non-human antibodies that specifically bind AF-20. Or derivatives thereof. Embodiments described herein include nucleic acid sequences expressing humanized and chimeric antibodies, fragments, polypeptides or derivatives thereof of the invention, methods for generating derivatives specific for such humanized and chimeric antibodies, fragments, polypeptides and AF-20, such Methods of using derivatives to produce humanized and chimeric antibodies, fragments, polypeptides and other polypeptides, variants and derivatives specific for AF-20, continuous hybridoma cell lines capable of secreting humanized and chimeric antibodies, such humanized or chimeric antibodies Or pharmaceutical and diagnostic compositions containing fragments or derivatives thereof, and methods of use thereof for the treatment or diagnosis of cancer.

Various embodiments described herein arise from the generation of chimeric and humanized antibodies from mouse SF-20 antibodies capable of binding AF-20. Surprisingly, the chimeric antibody chNYR-1002 and humanized antibody huNYR-1002 have been shown to be able to bind to AF-20 antigen.

One embodiment of the invention provides chimeric derivatives of the AF-20 antibody, chNYR-1002, wherein the mouse variable regions V _H and V _K of NYR-1002 are bound above a human IgG1 or K constant region, respectively. Other embodiments include other chimeric derivatives of NYR-1002 having different human constant regions, such as IgG2 or lambda constant regions, used to bind the mouse variable regions V _H and V _K of NYR-1002.

Another embodiment provides that a potential human T-cell epitope (in amino acid sequences of the V _H and V _K regions of chNYR-1002) is identified and that the amino acid of the putative T-cell epitope is removed to remove or reduce the immunogenicity of the antibody. The sequence is modified to provide a humanized derivative of the chNYR-1002 chimeric antibody, huNYR-1002, wherein one or more of the identified T-cell epitopes have been removed. Embodiments incorporate other humanized derivatives of NYR-1002 in which different human constant regions, such as IgG2 or lambda constant regions, are used to link the humanized variable regions, V _H and V _K of NYR-1002 contained in huNYR-1002. Include.

Other embodiments include amino acids and corresponding nucleic acid sequences of CDRs identified at V _H and V _K of NYR-1002: light chain CDR1 (SEQ ID No.), CDR2 (SEQ ID No.) and CDR3 (SEQ ID No. ) And the heavy chain CDR1 (SEQ ID No.), CDR2 (SEQ ID No.) and CDR3 (SEQ ID No.). However, the amino acid sequence of CDRs may be modified. The amino acid sequence of each CDR is preferably changed by up to 10%, more preferably up to 20%, more preferably up to 30%, even more preferably up to 40% by amino acid substitutions, insertions and / or deletions However, the obtained humanized antibody comprising an amino acid sequence retains binding specificity for the binding target. Thus, each CDR may comprise one, two or more amino acid substitutions, insertions and / or deletions. Preferably, the amino acid sequence of each CDR is substantially homologous to a particular CDR disclosed herein. Those skilled in the art will appreciate that a list of specific amino acid sequences may be included in all lists of such modifications so long as the activity and utility of the sequences are substantially maintained.

The polynucleotides and polypeptides provided herein are used in the generation of other humanized antibodies, for example by substituting the CDR regions of the variable regions of human antibodies. If the human variable region shares significant homology with the mouse variable region of NYR-1002, this sequence has utility in the generation of other humanized variable regions by substituting CDR regions in the variable regions of human antibodies. Such sequences also have utility in the generation of polypeptides capable of binding to the AF-20 antigen. The polynucleotide and polypeptide sequences provided herein have utility in the identification of highly homologous CDRs of human or humanized antibodies, such as CDRs contained in libraries or banks of such antibodies. Embodiments also include humanized antibodies and antibody fragments in which one or more original CDRs in a humanized antibody have been replaced with the CDRs described herein. Embodiments also include bispecific antibodies, antibody fragments and polypeptides containing one or more CDRs of the invention.

Embodiments disclosed herein further include variants and equivalents that are substantially homologous to humanized antibodies, antibody fragments, polypeptides, variable regions, and CDRs. They may, for example, contain conservative substitution mutations (ie, substitution of one or more amino acids by similar amino acids). For example, a conservative substitution may be made by substituting an amino acid with another amino acid within the same class, for example by replacing one acidic amino acid with another acidic amino acid, replacing one basic amino acid with another basic amino acid, or by another neutral amino acid. Refers to the substitution of one neutral amino acid. What is intended by conservative amino acid substitutions is well known in the art.

The phrase “substantially homologous” is used with regard to the similarity of the subject amino acid sequence (of oligo- or poly-peptide or protein) to the associated reference amino acid sequence. Such phrases are typically defined as " matches " of at least about 75%-in other words, when the sequences are "aligned" (ie, in order to maximize the number of bases that coincide between the sequences, the minimum of the subject and / or reference sequence is maximized). When inserting a number of "null" bases) the state of the same amino acid residue is located in parallel between the subject and reference sequence. "Null" bases are not part of the subject and reference sequences; The minimum number of "null" bases inserted into the subject sequence may also differ from the minimum number inserted into the reference sequence. In this definition, when both amino acid sequences form a portion of a protein or protein that is an AF-20 antibody, antibody fragment or polypeptide having the ability to bind AF-20, the reference sequence is considered to be "associated" with the subject sequence. . Each protein comprising an AF-20 antibody, antibody fragment or polypeptide is independently an antibody, antibody fragment, polypeptide or bi- or multi-functional protein, eg, a fusion protein, a bi- and multi-specific antibody, a single chain antibody. Or a multimer thereof.

Another aspect of the invention provides amino acids and corresponding nucleic acid sequences of the humanized V _H and V _K regions of huNYR-1002. Such sequences have utility in the development of other humanized antibodies, eg in substitutions for the corresponding variable regions of human antibodies. Such sequences also have utility in identifying highly homologous variable regions of human or humanized antibodies, such as those contained in libraries or banks of such antibodies. Such sequences have utility in the generation of antibody fragments and polypeptides capable of binding to the AF-20 antigen. Embodiments further include humanized antibodies and antibody fragments in which one or more original variable regions of a humanized antibody are replaced with variable region (s) described herein. Embodiments include bispecific antibodies, antibody fragments, and polypeptides containing one or more humanized variable regions.

Embodiments of the present invention include the use of humanized antibodies, antibody fragments, CDRs and humanized variable regions described herein in designated molecular evolution techniques such as phage display techniques and bacterial and yeast cell surface display techniques. Phage display technology (McCafferty et al., Nature 348: 552 (1990)) can be used to generate novel human antibodies and antibody fragments in vitro from variable region genes or genes encoding humanized antibodies or antibody fragments. According to this technique, antibody variable region genes are cloned in the main or sub coat protein genes of filamentous bacteriophages, such as M13 and fd, in frame, and expressed as functional antibody fragments on the surface of phage particles. Since filamentous particles contain single-stranded NDA copies of the phage genome, selection based on the functional properties of the antibody selects the genes encoding the antibodies exhibiting these properties. That is, the phage mimics some of the properties of B-cells.

Phage display can be performed in a variety of ways, see Johnson et al., Current Opinion in Structural Biology 3: 564 (1993) for review. Variable gene segments can be used for phage display. Clackson et al., Nature 352: 624 (1991) isolated various arrays of anti-oxazolone antibodies from a small random combinatorial library of variable region genes derived from the spleen of immunized mice. In the natural immune response, antibody genes accumulate mutations at high rates (body cavity overmutation). Some of the changes introduced impart higher affinity and B cells exhibiting high-affinity surface immunoglobulins preferentially replicate and differentiate during subsequent antigenic challenge. This natural process can be mimicked by using techniques to introduce small random mutations in antibody genes. In this method, affinity, specificity, immunogenicity or other characteristics of humanized antibodies can be improved and new humanized antibodies, antibody fragments and polypeptides capable of binding to the AF-20 antigen were identified.

Other embodiments include chimeric and humanized antibodies and antibody fragments derived from other non-human monoclonal antibodies that bind to AF-20. Increases of monoclonal antibodies against the desired antigen are known in the art. US Pat. No. 5,703,213 describes one method for generating mouse monoclonal antibodies to AF-20 antigens by immunizing mice with FOCUS HCC cells. This method can be applied to the development of other AF-20 antibodies from mice and other non-human host animals.

Other methods for generating non-human antibodies are well known in the art and can be applied for immunization with FOCUS HCC cells or if isolated with the AF-20 antigen itself. Monoclonal antibodies may be made using the hybridoma methods described in Kohler et al., Nature, 256: 495 (1975), or by recombinant DNA methods (US Pat. No. 4,816,567). Multiple subcutaneous (sc) or intraperitoneal (ip) of antigens and adjuvant such as monophosphoryl lipid A (MPL) / trehalose dicrinomycolate (TDM) (Ribi Immunochem. Research, Inc. Montana Hamilton, USA) at various sites By injection) hybridoma methods, mice or other suitable host animals such as hamsters or macaques are immunized. After 2 weeks, the animals are further immunized and after 7-14 days the animals are bled and serum is analyzed for anti-antigen titers. Animals are further immunized to titer stability levels. Serum was collected from the animals and polyclonal antibodies were isolated from the serum by conventional immunoglobulin purification procedures such as protein A-sepharose chromatography, hydroxylapatite chromatography, gel filtration, permeation or antigen affinity chromatography.

The methods described above are used to generate lymphocytes that can produce or can produce antibodies that specifically bind to an immunizing agent. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes can be fused with myeloma cells using suitable fusing agents such as polyethylene glycol to form hybridoma cells. Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)].

The hybridoma cells thus prepared can be seeded and grown in a suitable culture medium containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas typically uses hypoxanthine, aminopterin and thymidine (HAT medium). Will include, and this material prevents the growth of HGPRT-deficient cells.

Preferred myeloma cells are cells that fuse efficiently, support stable high-level production of antibodies by selected antibody-producing cells, and are sensitive to media such as HAT medium. Among these, preferred myeloma cell lines are obtained from mouse myeloma cell lines such as ATCC and cell lines derived from MOP-21 and MC-11 mouse tumors available from Salk Institute Cell Distribution Center (San Diego, CA, USA). Possible SP-2 or X-63-Ag8-653 cells. Human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc. New York, 1987)].

For production of monoclonal antibodies directed against the antigen, the culture medium in which hybridoma cells grow can be analyzed. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). do. The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis (Munson et al., Anal. Biochem., 107: 220 (1980)). One method for determining binding affinity for AF-20 antigens is described in US Pat. No. 5,703,213.

Antibodies of embodiments of the invention may be described or defined in terms of binding affinity for AF-20 polypeptide. Preferred binding affinity includes those having a dissociation constant of less than 1 μM or Kd, or more preferably less than about 100 nM, most preferably less than about 1 nM.

After identifying hybridoma cells that produce antibodies of the desired specificity, affinity and / or activity, clones can be subcloned by restriction dilution procedures and grown by standard methods. [Goding, Monoclonal Antibodies: Principles and Practice , pp. 59-103 (Academic Press, 1986)]. Suitable culture media for this purpose include D-MEM or RPMI-1640 medium. Hybridoma cells may also be grown in vivo as ascites tumors in an animal.

Monoclonal antibodies secreted by the subclone may be cultured media, ascites fluid or by conventional immunoglobulin purification procedures such as, for example, protein A-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. May be appropriately separated from serum.

DNA encoding monoclonal antibodies can easily be isolated and isolated using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of monoclonal antibodies). Can be sequenced. Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA is placed in an expression vector and then introduced into a host cell, such as E. coli cells, Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin proteins, thereby recombining host cells. The synthesis of monoclonal antibodies can be obtained. Review articles on recombinant expression of antibody-encoded DNA in bacteria are described in Skerra et al., Curr. Opinion in Immunolo., 5: 256 (1993) and Pluckthun, Immunol. Revs., 130: 151 (1992). Other methods also exist for generating non-human monoclonal antibodies that can bind to AF-20 antigens.

According to the methods described herein, or according to other methods known to those skilled in the art, monoclonal antibodies and DNAs encoding such antibodies can be used to generate chimeric antibodies, humanized antibodies and antibody fragments.

Preferred methods determine at least a portion of an amino acid sequence of an antibody of one or more potential epitopes against human T-cells (preferably a portion of non-human origin such as the V _H or V _K region of a non-human antibody) and Modifying the amino acid sequence to remove at least one of the putative T-cell epitopes, thereby removing or reducing the immunogenicity of the protein or portion thereof when exposed to the human immune system, thereby making non-human antibodies non-human. -To be immunogenic or less immunogenic. According to this method, a panel of modified antibodies can be generated. Modified antibodies obtained in terms of expression level, immunogenicity and affinity and specificity for AF-20 antigen are selected to select the best candidate (s).

Other methods of generating chimeric and humanized antibodies from non-human antibodies or reducing the immunogenicity of non-human antibodies are known to those skilled in the art, including but not limited to:

US Patent 4,816,567 to Cabilly et al .; Morrison, S.L. Et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984); Boulianne, G.L. Et al., Nature 312; 643-646 (1984); Neuberger, M.S. Et al., Nature 314: 268-270 (1985), generation of chimeric antibodies by attaching variable regions within the heavy and light chains of non-human antibodies to the constant regions of human antibodies.

Winter, US Pat. No. 5,225,539, Jones, P.T. Et al., Nature 321: 522-525 (1986); Riechmann, L. Et al., Nature 332: 323-327 (1988); Verhoeyen, M. Et al., Science 239: 1534-1536 (1988), generation of humanized antibodies by substituting non-human complementarity determining regions (CDRs) or CDR sequences for corresponding fragments of human antibodies. For example, as described in US Pat. Nos. 5,530,101, 5,585,089, 5,693,762, 6,180,370, Carter et al., US Pat. Nos. 6,054,297, 6,407,213 and 6,639,055, Adair, US Pat. Nos. 6,632,927 and Winter, US Pat. FR residues may be substituted with residues from similar sites in non-human antibodies to maintain antigen binding.

Humanization by selective substitution of residues within the variable regions of non-human antibodies, as described, for example, in US Pat. No. 5,639,641 to Pedersen et al., US Pat. Nos. 5,766,886 and 5,821,123 to Studnicka et al. And US Patent Application 10/300215 to Carr et al. Development of antibodies.

L Linking the antibody or antibody fragment to a self-antigen sequence that makes the non-human antibody or antibody fragment less immunogenic, as described in US Pat. No. 6,652,863 to Jordan et al.

As described above, DNA encoding major monoclonal antibodies or antibody fragments can be isolated from hybridomas or phage display clones of origin and then manipulated to generate humanized and / or affinity matured constructs. In addition, amino acid residues or residues are introduced into cysteine residues located at desired positions on the polypeptide backbone of the antibody fragment, eg, in the hinge region of the heavy chain, thereby providing a site for specific attachment of the polymer molecule (s). Known techniques can also be used. In one embodiment, the antibody fragment generally forms a disulfide bridge that connects the light and heavy chains in order to leave the partner cysteine residue in the opposite chain with free sulfhydryl for specific attachment of the polymer molecule. Natural cysteine residues in the chain or heavy chain are replaced with other amino acids such as serine.

In constructing the desired antibody or antibody fragment-encoded clone, the clone can be used for recombinant production of the antibody or antibody fragment using methods known to those skilled in the art. Finally, the antibody or antibody fragment product can be recovered from the host cell culture and purified using methods known to or skilled in the art. For embodiments using antibody fragments engineered to remove cysteine residues as described above, preferred recombinant preparation systems include bacterial expression and product recovery procedures known to or described herein. If full length antibodies are prepared, the desired antibody fragment can be obtained from this by enzymatic digestion of the complete antibody according to methods known in the art.

Known methods can be used to make chimeric and humanized antibodies, fragments and polypeptides of embodiments. One detailed manufacturing method is described in the Examples. Those skilled in the art should understand that conventional techniques known for the purpose of achieving the same or similar results may be substituted for those described below. How to:

a DNA sequence encoding the antibody heavy chain, wherein (a) at least a portion of the variable domain framework region necessary for maintaining (1) operon and (2) antibody binding specificity is derived from a non-human antibody, and (3) an expression vector containing the rest of the antibody chain derived from a human antibody is constructed by conventional techniques, thereby producing a vector of the present invention;

(b) encoding a complementary antibody light chain, wherein at least a portion of the variable domain framework region and CDRs necessary to maintain donor antibody binding specificity are derived from a non-human antibody and the rest of the antibody chain is from a human antibody. An expression vector containing a DNA sequence and an operon is constructed by conventional techniques, thereby producing a vector of the present invention;

(c) importing the expression vector into the host cell by conventional techniques to generate an imported host cell of the invention;

(d) Humanized antibodies of the embodiments described herein may also be prepared by methods of culturing introduced host cells by conventional techniques to produce the modified antibodies of the invention.

Host cells can also be co-introduced into two vectors of the invention: the first vector containing the operon encoding the light chain derived polypeptide and the second vector containing the operon encoding the heavy chain derived polypeptide. The two vectors contain different selectable markers, but apart from the antibody heavy and light chain coding sequences, the same is preferred to achieve the same expression of the heavy and light chain polypeptides. Alternatively, a single vector may be used that is a vector comprising sequences encoding light and heavy chain polypeptides. The light and heavy chain coding sequences may comprise cDNA, genomic DNA or both.

The host cell used to express the altered antibody of the invention may be a bacterial cell such as Escherichia coli or eukaryotic cell. In a particularly preferred embodiment of the invention, mammalian cells of a defined type, such as myeloma cells or Chinese hamster ovary (CHO) cells, may be used for this purpose.

General methods by which the vectors of the invention can be constructed, the import methods necessary for producing host cells of the invention, and the culturing methods necessary for producing the antibodies of the invention from such host cells include conventional techniques. The following examples provide one such method. Preferably the cell line used to produce the humanized antibody is a mammalian cell line, but other suitable cell lines such as bacterial cell lines or yeast cell lines may alternatively be used. In particular, it is contemplated that E. coli-derived bacterial strains may be used.

Similarly, once purified, humanized antibodies of embodiments of the invention may be purified according to standard procedures in the art, including cross-flow filtration, ammonium sulfate precipitation, affinity column chromatography, gel electrophoresis, and the like.

It should be understood that humanized antibodies of the embodiments are performed in the same or substantially similar fashion as non-humanized variants of the same antibody. However, preferably, humanized antibodies are more advantageously used in humans as compared to non-humanized variants of the same antibody. Humanized antibodies of this embodiment may also be used for the design and synthesis of peptides or non-peptide compounds (mimetics) that may be useful for the same therapies as antibodies [Saragobi et al., Science 253: 792-795 (1991)]. (The contents of which are hereby incorporated by reference in their entirety).

Embodiments of the invention include fragments of humanized antibodies capable of binding to an AF-20 antigen. Antibody fragments can provide significant advantages over complete antibodies, in particular the fact that recombinant antibody fragments can be formed in bacterial cell expression systems. Bacterial cell expression systems offer several advantages over mammalian cell expression systems, including reduced time and cost at both the research and development and manufacturing stages of the product.

Antibody fragments can be prepared by methods known in the art or described herein. In general, antibody fragments are derived from a parent intact antibody. Preferred antibody fragments can be generated from antibody preparations purified by conventional enzymatic methods, for example F (ab ') ₂ fragments are prepared by pepsin cleavage of the complete antibody, and Fab fragments short the complete antibody with papain. Prepared by digestion.

Particular embodiments include the use of bispecific and heteroconjugated antibody fragments having specificity for at least two different antigens. Bispecific and heteroconjugate antibodies can be prepared as full length antibodies or as antibody fragments (eg, F (ab ') ₂ bispecific antibody fragments). For use herein, antibody fragments having two or more valences (eg, trivalent or higher valence antibody fragments) are contemplated. Bispecific antibodies, heteroconjugate antibodies, and multivalent antibodies can be prepared by methods known to or skilled in the art.

Embodiments of the present invention include therapeutic compositions containing the humanized antibodies, antibody fragments, and polypeptides described herein. For example, the humanized antibodies, antibody fragments and polypeptides described herein can be conjugated to effector residues having therapeutic activity and used to selectively target cells expressing the AF-20 antigen. Such conjugates will have the advantage of internalization of the AF-20 antibody when bound to the AF-20 antigen. Many such effector residues are known in the art and include cytotoxic agents, immunological response modifiers, oligonucleotides, genes, viral vectors containing therapeutic genes, liposomes containing genes or cytotoxic agents, or prodrugs. Or enzymes.

Many cytotoxic agents are known to those skilled in the art. These include chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine and bleomycin do. To generate cell-type-specific-killing reagents, toxic enzymes from plants and bacteria, such as lysine, diphtheria toxin and pseudomonas toxin, can be conjugated to the humanized antibodies, antibody fragments and polypeptides of the invention [Youle et al., Proc. Nat'l Acad.Sci. USA 77: 5483 (1980); Gilliland et al., Proc. Nat'l Acad Sci. USA 77: 4539 (1980); Krolick et al., Proc. Nat'l Acad. Sci. USA 77: 5419 (1980). Other cytotoxic agents include cytotoxic ribonucleases described by Goldenberg in US Pat. No. 6,653,104.

Embodiments of the invention relate to radioimmunoconjugates in which radionuclides that release alpha or beta particles are stably bound to an antibody, antibody fragment or polypeptide, with or without complex-forming agents. Such radionuclides include beta-releasing agents such as phosphorus-32 ( ³² P), scandium-47 ( ⁴⁷ Sc), copper-67 ( ⁶⁷ Cu), gallium-67 ( ⁶⁷ Ga), yttrium- ⁸⁸ ( ⁸⁸ Y), Yttrium-90 ( ⁹⁰ Y), Iodine-125 ( ¹²⁵ I), Iodine-131 ( ¹³¹ I), Samarium-153 ( ¹⁵³ Sm), Lutetium- ¹⁷⁷ ( ¹⁷⁷ Lu), Rhenium- ¹⁸⁶ ( ¹⁸⁶ Re) or Rhenium- 188 ( ¹⁸⁸ Re) and alpha-releasing agents such as asstatin-211 ( ²¹¹ At), lead-212 ( ²¹² Pb), bismuth-212 ( ²¹² Bi) or 213 ( ²¹³ Bi) or actinium-225 ( ²²⁵ Ac) It includes.

Particularly preferred embodiments include conjugation of NTP peptides or derivatives thereof to humanized antibodies and should be considered useful in embodiments.

Therapeutic compositions may also be used to introduce immunological response modifiers into tumor cells expressing AF-20 antigens, thereby marking tumor cells directly or indirectly for destruction by the patient's immune system. Therapeutic compositions may also be used to introduce gene sequences into tumor cells that express AF-20 antigens, thereby allowing expression of genes in tumor cells. Genes may replace or supplement genes that are impaired or absent in tumor cells, thereby inducing cell death through apoptosis or other mechanisms, inhibiting or preventing tumor cell proliferation or migration, Mark tumor cells for destruction by the immune system, or have similar or other therapeutic effects. The gene may be exogenous to the tumor cell genome. Such genes may also express enzymes capable of cleaving prodrugs with cytotoxic proteins or cytotoxic residues. Such gene sequences may also be delivered to target cell (s) by gene delivery systems, such as viral vectors or liposomes, conjugated to the therapeutic compositions of the invention.

Similarly, therapeutic compositions may be used to introduce oligonucleotides into tumor cells that express AF-20 antigens. Such oligonucleotides include antisense oligonucleotides that inhibit the function of targeting mRNA in tumor cells; Short interfering ribonucleic acid (siRNA) that inhibits expression of proteins in tumor cells required for viability, proliferation or migration; Ribozyme; Agents that increase the sensitivity of tumor cells to other anticancer therapies; And triple helix-forming oligonucleotides.

Embodiments envision different therapeutic compositions, such as pharmaceutical compositions, each containing two or more different antibody conjugates with different antibodies or different effector residues. Agonist residues, when introduced into tumor cells as a result of internalization of the antibody therapeutic, induce cell death; Replace dysfunctional genes; It may also include genes or other oligonucleotides that provide an advantageous therapeutic response, such as by expressing a therapeutically beneficial protein. The gene may be sealed in liposomes or attached to a suitable vector such as a virus.

In other embodiments of the invention, the specificity and reduced immunogenicity for AF-20 are detectable for the detection of different cancer types, such as hepatocellular carcinoma, adenocarcinoma of the lung and colorectal carcinoma, as described herein. When conjugated to a label, it is made suitable for use as a diagnostic reagent. Such compositions may be useful for diagnosis, judgment of appropriate treatment, and prognostic assessment of cancer characterized by AF-20 expression (based on AF-20 expression level); These may be useful as tumor imaging reagents or radioimmunoguide surgery. Useful as radiolabeled antibody in the RTM system (RIGS.RTM). See Hinkle et al., Antibody, Immunoconjugates and Radiopharmaceuticals, 4 (3): 339-358 (1991).

Many detectable labels that may be conjugated to an antibody or polypeptide are known in the art. Detectable labels include radionuclides such as ³ H, ¹¹ C, ¹⁴ C, ¹⁸ F, ⁶⁴ Cu, ⁷⁶ Br, ⁸⁶ Y, ^{99 m} Tc, ¹¹¹ In, ¹²³ I, ¹²⁵ I or ¹⁷⁷ Lu. Methods of detecting such labels include PET scans and immunosynthesis. Detectable labels for in vitro assays include enzymes such as horseradish peroxidase; Fluorophores; Chromophores; Chemiluminescent reagents; Radionuclide; Chelated complexes; dyes; Colloidal gold or latex particles.

Diagnostic compositions of this embodiment may be used in in vitro assays to determine whether a human or animal has a cancer that expresses an AF-20 antigen. Such assays have utility in cancer diagnosis, stage determination and therapeutic evaluation. Preferably, this assay is used to determine whether the patient or animal has cancer that is sensitive to treatment with a therapeutic composition capable of binding to tumor cells expressing the AF-20 antigen. Most preferably, this assay is used to determine how and whether to treat a patient or animal with the therapeutic composition of the present invention.

Methods of developing such assays are known in the art. Assay types include, but are not limited to, immunohistochemical analysis of biopsy tissue with a diagnostic composition of the present invention, and immunoassay for contacting a tissue or body fluid sample with the diagnostic composition of the present invention.

Many methods for conjugating agonist residues or detectable labels are known to those skilled in the art. The attachment of antibodies to preferred agents is well known. See, eg, US Pat. No. 5,435,990 to Cheng et al., The disclosure of which is incorporated by reference in its entirety. In addition, bifunctional linkers for promoting such attachment are known in the art and can be widely used. In addition, chelators (chelants and chelates) that provide for attachment of radionuclides are known in the art and are readily available.

Therapeutic and Diagnostic Compositions may have utility in the diagnosis and treatment of cancer. Preferably, such cancer is adenocarcinoma, most preferably such cancer is hepatocellular carcinoma, lung adenocarcinoma and colorectal carcinoma.

Therapeutic compositions have utility in the treatment of cancer and other tumors that express AF-20 antigens. They may be used alone or in combination with other anticancer or antitumor therapies such as chemotherapy, immunotherapy, radiation, medical resection or excision.

Those skilled in the art will be able to determine (by routine experimentation) the amount of an effective, non-toxic antibody, antibody fragment or polypeptide for the purpose of treating a particular cancer. Generally, however, the effective dosage will range from about 0.05 to 100 milligrams per kilogram of body weight per day, preferably from about 0.5 to about 25 milligrams per kilogram of body weight per day.

According to the therapeutic methods described above, the chimeric or humanized antibodies, antibody fragments or polypeptides described herein may be administered to a human or other animal in an amount sufficient to produce a therapeutic or prophylactic effect. According to known techniques, antibodies can also be administered to humans or other animals in conventional dosage forms prepared by combining conventional pharmaceutically acceptable carriers, diluents and / or excipients. Those skilled in the art will understand that the form and characteristics of pharmaceutically acceptable carriers, diluents and / or excipients are dictated by the amount of active ingredient that can be combined, the route of administration, and other known variables.

Pharmaceutically acceptable formulations may, for example, include suitable solvents, preservatives such as benzyl alcohol, if desired, and buffers. Useful solvents may include, for example, water, aqueous alcohols, glycols, and phosphonates and carbonate esters. Such aqueous solutions contain up to 50% by volume of organic solvents. Suspension type preparations may comprise liquid suspending media as carriers, for example aqueous polyvinylpyrrolidone, inert oils such as vegetable oils or highly refined mineral oils, or aqueous cellulose ethers such as aqueous carboxymethylcellulose. Thickeners such as gelatin or alginate may be present, one or more natural or synthetic surfactants or antifoams may be used, or one or more suspending agents such as sorbitol or other sugars may be used. Such formulations may contain one or more adjuvants.

The route of administration of the antibody, fragment or polypeptide of the invention may be oral, parenteral, inhaled or topical. The term “parenteral” as used herein includes intrathrombotic, intravenous, intramuscular, subcutaneous, rectal, vaginal, or intraperitoneal administration. Intravenous, intravenous and intramuscular forms of parenteral administration are preferred routes of administration.

The daily parenteral and oral administration regimens for the prophylactic or therapeutic use of the humanized antibodies of the invention generally range from about 0.005 to 100, preferably from about 0.5 to 10 milligrams per kilogram of body weight per day.

The antibody may be administered by inhalation. "Inhalation" refers to intranasal and oral inhalation administration. Suitable dosage forms, such as aerosol formulations or metered dose inhalers, for such administration may be prepared by conventional techniques. Preferred dosages of the compounds of the invention used are generally in the range of about 0.1 to about 100, more preferably about 10 to 100 milligrams per kilogram of body weight.

The antibody may be administered topically. Topical administration refers to non-systemic administration. This involves externally administering a humanized antibody (or humanized antibody fragment) preparation to the epidermis or oral cavity and injecting the antibody anywhere in the ear, eye or nose and where it does not significantly enter the bloodstream. Systemic administration refers to oral, intravenous, intraperitoneal, subcutaneous and intramuscular administration. The amount of antibody required for a therapeutic, prophylactic or diagnostic effect varies depending upon the antibody selected, the nature and severity of the condition being treated, and the animal being treated, and ultimately at the physician's discretion. Suitable topical dosages of the antibodies of the invention are generally in the range of about 1 to 100 milligrams per kilogram of body weight per day.

Although it is possible for the antibodies, fragments or polypeptides described herein to be administered alone, it is preferred that they exist as pharmaceutical agents. The active ingredient may comprise as much as 10% w / w of the formulation, preferably not more than 5% w / w, more preferably from 0.1% to 1% w / w, but for topical administration Of 0.001% to 10% w / w, for example 1% to 2% by weight.

Topical formulations may include the active ingredient in combination with one or more acceptable carrier (s) and optionally other therapeutic ingredient (s). The carrier (s) is typically "acceptable" in the sense of being affinity with the other ingredients of the formulation and not harmful to its recipient. Formulations suitable for topical administration may be suitable liquid or semi-liquid formulations such as topical medications, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose, for penetration through the skin to the area in need of treatment. Include the first.

Drops may include sterile aqueous or oily solutions or suspensions, and may be prepared by dissolving the active ingredient in a suitable aqueous solution and / or other suitable preservative of the bactericidal and / or fungicide, preferably including a surface active agent. . The resulting solution may be clarified and sterilized by filtration or transferred to the container by antiseptic technique. Examples of suitable bactericidal and fungicides for inclusion in drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and clohexidine acetate (0.01%). Suitable solvents for the preparation of oily solutions include glycerol, dilute alcohol and propylene glycol.

Lotions include those suitable for application to the skin or eyes. The eye lotion may comprise a sterile aqueous solution optionally containing a fungicide, or may be prepared by a method similar to that for the preparation of a drop. Lotions or application agents for application to the skin may include agents for promoting drying and agents for cooling the skin, such as alcohols or acetone, and / or moisturizers such as glycerol or oils such as castor oil or peanut oil. have.

Creams, ointments or pastes are typically semi-solid preparations of the active ingredient for external application. These may be made by mixing the active ingredient alone or in micro-separated or powdered form in a solution or suspension in an aqueous or non-aqueous fluid using a greasy or non-greasy substrate with the aid of a suitable machine. Substrates may be hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, metallic soaps; Mucus; It may also comprise fatty acids such as stearic acid or oleic acid with oils of natural origin such as almonds, corn, peanuts, oils of castor or olive oil, wool fat or derivatives thereof, or alcohols such as propylene glycol or macrogel. The formulations may also comprise suitable surface active agents such as anionic, cationic or non-ionic surface actives such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents may also be included, such as natural rubber, cellulose derivatives or inorganic materials such as silica silica, and other ingredients such as lanolin.

Kits according to embodiments are frozen or lyophilized chimeric or humanized antibodies, antibody fragments or polypeptides, which are each reconstituted by thawing (then optionally optionally further diluted) or by suspension in a liquid excipient (preferably buffered). It includes. The kits also contain buffer and / or excipient powder formulations that are reconstituted with buffered and / or excipient solutions (in liquid or frozen form)-or water for mixing with humanized antibodies or humanized antibody fragments to prepare a suitable formulation for administration. It may also include-. That is, kits containing chimeric or humanized antibodies, antibody fragments or polypeptides are preferably effective for in vivo or in vitro use in the treatment or diagnosis of cancer by adding a predetermined amount of heat, water or solution provided in the kit. To a concentration sufficient to achieve such a concentration and pH, the preparation is frozen, lyophilized, pre-diluted or pre-mixed.

Preferably such kits will include instructions for reconstructing and using chimeric or humanized antibodies, antibody fragments or polypeptide compositions to treat or detect cancer. The kit may also include two or more component parts for the reconstituted active composition. For example, the second component moiety-in addition to the chimeric or humanized antibody, antibody fragment or polypeptide-may be a therapeutic agent such as a bifunctional chelant, a bifunctional chelate, or a radionuclide, which is mixed with a humanized antibody or humanized antibody fragment. Form a junction system with it. The buffers, excipients, and other component parts described above can be sold separately or together with the kit.

Optimal amounts of chimeric or humanized antibodies, antibody fragments or polypeptides and respective intervals of administration of embodiments described herein are determined by the nature and extent of the condition being treated, the dosage form, the route and site, and the particular animal being treated. It will be understood by those skilled in the art that such optimization can be determined by conventional techniques. Those skilled in the art will understand that the optimal course of treatment, i.e., the number of administrations of the chimeric or humanized antibody, antibody fragment or polypeptide of the present invention per day for a limited number of days, can be ascertained by one skilled in the art using conventional therapeutic determination test procedures. will be.

Subject chimeric or humanized antibodies, antibody fragments or polypeptides may also be administered in combination with other anticancer agents, eg, other antibodies or drugs.

Further embodiments include recombinant antibody molecules having antigen binding regions derived from the heavy or light chain variable regions of an antibody capable of binding AF-20. Embodiments include chimeric antibodies, including variable regions and human constant regions obtained from non-human antibodies that bind to AF-20. The variable regions of chimeric antibodies are obtained from mouse antibodies and human constant regions, more preferably mouse monoclonal antibodies (moAbs) and human constant regions, prepared by the hybridoma cell line ATCC name HB 9686.

Other embodiments include chimeric antibodies comprising the variable heavy chain sequence of SEQ ID No. or the variable light chain region of SEQ ID No., or both. Preferably the chimeric antibody is chNYR-1002.

Another embodiment includes a humanized antibody or humanized antibody fragment (collectively referred to as "humoAb") that binds to AF-20, wherein the non-human antibody to which the humanized antibody or humanized antibody fragment binds to AF-20 Is derived from. Preferably, humoAb is derived from mouse monoclonal antibody (moAb) that binds to AF-20, more preferably humoAb is derived from mouse moAb prepared by the hybridoma cell line ATCC name HB 9686. Particularly preferred humanized antibody is huNYR-1002.

A further embodiment comprises a humanized antibody or humanized binding to AF-20 comprising a complementarity determining region (CDR) amino acid residue and a human framework region (FRs) amino acid residue obtained from a non-human antibody binding to AF-20. Antibody fragments. Preferably, the CDRs are obtained from mouse moAb and human framework region (FRs) amino acid residues that bind to AF-20, and more preferably the CDRs are mouse moAb and human produced by the hybridoma cell line ATCC name HB 9686. Framework region (FRs) are obtained from amino acid residues.

Particularly preferred humanized antibodies or fragments comprise binding to AF-20, wherein the complementarity determining regions (CDR1, CDR2 and CDR3) of the light chain variable region and the complementarity determining regions (CDR1, CDR2 and CDR3) of the heavy chain variable region Has the following amino acid sequence:

Light chain:

CDR1 (SEQ ID No.) [RASQSIGTSIH];

CDR2 (SEQ ID No.) [YASESIS]; And

CDR3 (SEQ ID No.) [QQSSSWPFT];

Heavy chain:

CDR1 (SEQ ID No.) [GYTFAGHYVH];

CDR2 (SEQ ID No.) [WIFPGKVNTKYNEKFKG] and

CDR3 (SEQ ID No.) [VGYDYFYYFDY]

Embodiments described herein include the humanized monoclonal antibodies or antibody fragments described above wherein one or more amino acid residues in the variable or constant regions are replaced with other amino acid residues. Preferably, one or more amino acid residues in CDRs or FRs are replaced with other amino acid residues. In addition, embodiments include one or more additions, substitutions, or deletions of amino acid residues made in human framework regions (FRs).

Additional embodiments described herein include the humanized monoclonal antibodies described above or antibodies thereof wherein the potential human helper T-cell epitopes identified in the variable regions are removed by substitution, addition or deletion of amino acid residues. Preferably, potential human helper T-cell epitopes identified in CDRs or FRs have been removed by substitution, addition or deletion of amino acid residues.

The humanized antibodies or humanized antibody fragments described herein (“humoAb”) preferably have an antigen binding affinity for AF-20, which is at least 10% of the antibody from which humoAb is derived. Particularly preferred humoAbs are SEQ ID No. Humanized variable heavy chain sequence of SEQ ID No. Humanized variable light chain sequences, or both. More preferably humoAb is a variant of the humanized variable heavy chain sequence of SEQ ID No. A variant of the humanized variable light chain, or both.

Further embodiments include polypeptide sequences comprising one or more of the following polypeptides:

SEQ ID No. [GYTFAGHYVH];

SEQ ID No. [WIFPGKVNTKYNEKFKG];

SEQ ID No. [VGYDYFYYFDY];

SEQ ID No. [RASQSIGTSIH];

SEQ ID No. [YASESIS]; And / or

SEQ ID No. [QQSSSWPFT].

Embodiments described herein include DNA encoding the antibodies, polypeptides or antibody fragments described above, and fragments, variants or derivatives thereof. Preferably, the DNA molecule encodes the amino acid sequence of a humanized antibody or fragment thereof, whereby the antibody or fragment specifically binds to AF-20, wherein the CDRs of the light chain variable region and the CDRs of the heavy chain variable region are Has the following amino acid sequence:

Light chain:

CDR1 (SEQ ID No:) [RASQSIGTSIH];

CDR2 (SEQ ID No:) [YASESIS]; And

CDR3 (SEQ ID No:) [QQSSSWPFT];

Heavy chain:

CDR1 (SEQ ID No:) [GYTFAGHYVH];

CDR2 (SEQ ID No:) [WIFPGKVNTKYNEKFKG]; And

CDR3 (SEQ ID No.) [VGYDYFYYFDY].

Embodiments include DNA molecules encoding the light or heavy chains of humoAbs described above. Preferred DNA molecules encode the amino acid sequence of a humanized antibody or fragment thereof whereby the antibody or fragment specifically binds AF-20, wherein the CDRs of the light chain variable region have the following amino acid sequence.

CDR1 (SEQ ID No:) [RASQSIGTSIH];

CDR2 (SEQ ID No:) [YASESIS]; And

CDR3 (SEQ ID No:) [QQSSSWPFT].

Other preferred DNA molecules encode the heavy chain of the antibody or fragment thereof and the nucleotide sequence of the heavy chain CDRs is as follows:

CDR1 (SEQ ID No:) [GYTFAGHYVH];

CDR2 (SEQ ID No:) [WIFPGKVNTKYNEKFKG]; And

CDR3 (SEQ ID No:) [VGYDYFYYFDY].

Preferably, the DNA molecule is in the form of an expression vector. In this regard, embodiments further comprise a host transformed with the expression vector. Embodiments also include host cells comprising recombinant expression systems encoding the light and heavy chains of the humanized antibodies or humanized antibody fragments described above.

Other embodiments of the invention include nucleic acid sequences in which the chimeric antibodies described herein can be expressed. Embodiments also include vectors comprising nucleic acid sequences. The vector is preferably a nucleic acid segment, carrier-binding nucleic acid segment, nucleoprotein, plasmid, virus, viroid or translocation element. Other preferred embodiments include hybridoma cell lines that have produced chimeric antibodies described herein.

Further embodiments include nucleic acid sequences in which the humanized antibodies, humanized antibody fragments or polypeptides described above may be expressed. In this regard, embodiments further include vectors comprising nucleic acid sequences. Preferably, the vector is a nucleic acid segment, carrier-binding nucleic acid segment, nucleoprotein, plasmid, virus, viroid or translocation element. Other preferred embodiments include hybridoma cell lines that produce the humanized antibodies, humanized antibody fragments or polypeptides described herein.

Particular embodiments include compositions for treating cancer comprising a therapeutically effective amount of a humanized or chimeric antibody, humanized antibody fragment or polypeptide described herein. Preferably, the humanized or chimeric antibody, humanized antibody fragment or polypeptide is associated or linked directly or indirectly to an effector residue having therapeutic activity. More preferably, the effector residue is an anticancer drug, chemotherapeutic agent, cytotoxin, radionuclide, therapeutic enzyme, prodrug, cytokine or anti-proliferative agent. Preferred radionuclides are ³² P, ⁴⁷ Sc, ⁶⁷ Cu, ⁹⁰ Y, ¹⁰⁵ Rh, ¹²⁵ I, ¹³¹ I, ^{117 m} Sn, ¹⁵³ Sm, ¹⁶⁶ Dy, ¹⁷⁵ Yb, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁴ Os, ²¹¹ At, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac or mixtures or combinations thereof.

Another embodiment includes a method of in vivo treatment of a mammal with an AF20-expressing cancer comprising administering to the mammal a therapeutically effective amount of the composition described above. Preferably, the composition is administered after surgery.

Further embodiments include compositions suitable for in vivo or in vitro detection of cancer, including a diagnostically effective amount of a humanized or chimeric antibody, humanized antibody fragment or polypeptide described herein. Preferably, the humanized or chimeric antibody, humanized antibody fragment or polypeptide is linked or linked directly or indirectly to a detectable label. More preferably, the detectable label is a radionuclide, phosphor, enzyme, enzyme substrate, enzyme cofactor, enzyme inhibitor or ligand. Preferred radionuclides include ³ H, ¹¹ C, ¹⁴ C, ¹⁸ F, ⁶⁴ Cu, ⁷⁶ Br, ⁸⁶ Y, ^{99 m} Tc, ¹¹¹ In, ¹²³ I, ¹⁷⁷ Lu and mixtures and combinations thereof.

Further embodiments include methods of in vitro immunodetection of AF-20-expressing cancer cells, comprising contacting the cancer cells with the composition described above. In this embodiment, a humanized or chimeric antibody, humanized antibody fragment or polypeptide of the composition is bound to a solid support.

Another preferred method includes a method for in vivo immunodetection of AF-20 expressing cancer cells in a mammal, comprising administering to the mammal a diagnostically effective amount of a composition as described above suitable for detection of cancer. Preferably, the immunodetection procedure is in vivo tumor imaging.

Further embodiments include (i) intravenous administration of a radionuclide-labeled antibody, antibody fragment or polypeptide, (ii) detection of tumor cells using a radionuclide activity probe, and (iii) subsequent surgical resection. In vivo treatment methods of cancer, comprising removing tumor cells detected by In this method, the antibody or polypeptide is preferably a humanized or chimeric antibody, humanized antibody fragment or polypeptide as described above. Preferably, the radionuclide is ³ H, ¹¹ C, ¹⁴ C, ¹⁸ F, ⁶⁴ Cu, ⁷⁶ Br, ⁸⁶ Y, ^{99 m} Tc, ¹¹¹ In, ¹²³ I, ¹⁷⁷ Lu or mixtures and combinations thereof.

Embodiments of the present invention provide methods of using the humanized or chimeric antibodies, humanized antibody fragments or polypeptides described above, or the DNA molecules described above to produce antibodies, fragments or variants or derivatives of the polypeptides that bind to the polypeptides or AF-20. It further includes. Preferably, the method of generating a polypeptide, variant or derivative is a phage or yeast display technique.

Embodiments of the present invention will be described with reference to the following non-limiting examples.

Example 1 Sequencing Mouse Mouse Genes

Revive mouse hybridomas AD20D4 and supplement with 20% fetal bovine serum and 1 mM sodium pyruvate (list number 11360-039, lot number 3069371) of North American origin (Invotrogen Corporation Listing No. 16000-044, Batch No. 1137907) Cultured as indicated in Dulbecco's Modified Eagles Medium with Glutamax I (Invitrogen Corporation, List No. 61965-026, Lot No. 3070663).

Total RNA was prepared from 10 ⁷ hybridoma cells with care to avoid contamination with RNAase. Certain RNAase free reagents were used, including nuclease-free water. The cells were spun by MSE 2000R cooling bench centrifugation at 1500 rpm for 5 minutes at 4 ° C. and then washed three times in ice cold PBS. Cells were resuspended in 6 mL in ice cold RNA cytolysis buffer (0.14 M NaCl, 1.5 mM MgCl ₂ , 10 mM Tris pH 8.6, 0.5% NP-40) with 5 μL RNAase OUT and vortexed for 10 seconds. This solution was superimposed on an equal volume of 24% (w / v) sucrose and 1% NP-40 and stored on ice for 5 minutes. The solution was centrifuged at 4000 rpm for 30 minutes at 4 ° C. in a cooled bench centrifuge. The upper cytosolic layer was removed in equal volume of 2X PK buffer (0.3M NaCl, 0.025M EDTA, 0.2M Tris pH 7.5, 2% SDS) and 200 μg / ml of proteinase K (Life Technologies List No. 25530-049) Was added to a final concentration of. The solution was incubated at 37 ° C. for 30 minutes.

The solution was extracted with equal volume of phenol / chloroform (1: 1 w / v). 2.5 volumes of 100% ethanol was added to the aqueous phase and the solution was stored overnight at -20 ° C. RNA was collected by centrifugation (400 rpm, 30 minutes) and then dried in a vacuum desiccator. The RNA is dissolved in H ₂ O (Promega List No. P119C) and the concentration is determined by spectrophotometry which assumes A260 1 = 40 μg / mL. The quality of RNA was confirmed by flowing 1-2 μg over 1.2% agarose gel in TAE: Good quality RNA shows a sharp ribosome band without evidence of degradation.

V _H and V _K cDNAs were prepared using reverse transferase with mouse IgG constant region and mouse K constant region primers, with care to avoid contamination with RNAase. 5 μg RNA, 10 μl 5 × reverse transtase buffer (Promega List No. M351A), 1 μl Primer (25 pmol / μl in H ₂ O (Promega List No. P119C) in a microcentrifuge tube, MuIgGVH3 for the heavy chain '(Oligo no. 152); using MuIgKVL3' (oligo no. 160) for light chains, 2 μl from dATP, dCTP, dGTP, dTTP 10 mM, 2 μL 10 mM dNTPs solution (100 mM stock solution (Life Technologies List No. 10297018), respectively) The first strand variable region cDNA was prepared by mixing RNAase OUT (Life Technologies List No. 10777019) and up to 50 μl of H ₂ O (Promega List No. P119C). The solution was heated to 70 ° C. for 10 minutes and then slowly cooled to 37 ° C. 100 units of M-MLV reverse transcriptase (Promega List No. M530A) were added and the solution was incubated at 37 ° C. for 1 hour, heated to 70 ° C. for 15 minutes and stored at −20 ° C. until needed.

Subsequently, amplification and cloning of the variable region genes were performed. 5 μl first strand cDNA, 5 μl 10 Taq polymerase buffer (Life Technologies Listing No. 402028) in a microcentrifuge tube, 1 μl 3 ′ primer (25 pmol / μl in H ₂ O; MuIgGVH3 ′ for heavy chains (oligo number 152); use MuIgKVL3 'for oligonucleotides (oligo number 160), 1 μl 5' leader primer mix (25 pmol / μl of each primer in the mixture), 1 μl 10 mM dNTPs solution, 0.5 μl Taq polymerase buffer (life Technologies List No. 10342-020) and up to 50 μl of H ₂ O were mixed. All reagents except Taq enzyme were mixed in a 0.5 mL thin wall PCR tube and heated to 94 ° C. on a PCR block. Taq enzyme was added and the enzyme was circulated: 94 ° C./2 minutes, 40 cycles of 94 ° C./30 seconds, 50 ° C./30 seconds, 72 ° C./2 minutes, finishing at 5 ° C. for 5 minutes. 5 μl of each reaction was run on agarose gel to check if PCR provided a product of expected size (approximately 350 bp). The remainder of the product was loaded onto a 1.5% low-melting agarose gel and the DNA strips were cut and purified. Gel purified V by mixing with 2 μl 10 × T4 DNA ligase buffer (Promega List No. C126B) and 1 μl T4 DNA ligase (Promega List No. M180A) and then incubating overnight at 15-20 ° C. for 2 hours. Region DNA was ligated into 1 μl pGem T-easy clone vector (Promega List No. A1360). Vectors were transformed with capacity E. coli TG1 and plated on LB + IPTG + XGAL + ampicillin plates. White colonies were picked from 3 mL of LB + ampicillin in a general container and incubated at 37 ° C. After 2-4 hours, the inserts were examined by test PCR. 50 μl of culture was transferred to a microcentrifuge tube and heated at 95 ° C. for 5 minutes. It was then spun for 5 minutes in a microcentrifuge tube and the supernatant transferred to a new tube. 10 μl of supernatant was mixed with 5 μl 10 × Taq polymerase buffer, 1 μl M13 positive primer, 1 μl M13 reverse primer, 1 μl 10 mM dNTPs, 0.5 μl Taq polymerase and up to 50 μl of H ₂ O.

All reagents except Taq enzyme were mixed in a 0.5 mL thin wall PCR tube and heated to 94 ° C. on a PCR block. Taq enzyme was added and then the sample was cycled in the following cycles: 94 ° C./2 minutes, 40 cycles of 94 ° C./30 sec, 50 ° C./30 sec, 72 ° C./2 min, 5 ° C. at 72 ° C. 10 μl of each reaction was run on agarose gel to check the insert (band at 500 bp). Cultures were incubated overnight and DNA was prepared for DNA sequencing.

DNA sequences of selected clones (V _H and V _K clones selected for inserts of the size expected by PCR) were determined by automated DNA sequencing. Plasmid DNA was prepared from selected bacterial stocks and 5 mL cultures were added to 50 μg / ml (or as needed) ampicillin stock (Sigma List No. A-0166) (50 mg / mL in H ₂ O) in a normal container. (LB) (10 g NaCl, 10 g tryptone, 5 g yeast extract per liter of H ₂ O). Cultures were grown overnight or with shaking for at least 5 hours. The culture was spun by microcentrifugation and DNA was purified using the Wizard Plus SV Mini-Prep Kit (Promega List No. A1460) according to the manufacturer's instructions. Purified DNA was resuspended in 100 μl H ₂ O and sequenced using an automated DNA sequencing device.

DNA and amino acid sequences for the NYR-1002 heavy chain V region are shown in FIG. 1. No productive heavy chain gene was isolated from the first batch of cells. From the second batch, 15 independent clones provided the same complete heavy chain sequence. The location of the CDRs was confirmed with reference to other antibody sequences (Kabat EA et al., 1991). CDR is SEQ ID No. Provided by (CDR1), (CDR2) and (CDR3). The NYR-1002 V _H amino acid sequence was compared with the consensus sequence for mouse kappa chain subgroup V and designated as this subpopulation.

DNA and amino acid sequences for the NYR-1002 light chain V region are shown in FIG. 2. Five independent clones from each batch of cells provided the same sequence. CDR positions were determined with reference to other antibody sequences (Kabat EA et al., 1991). CDR is SEQ ID No. Provided by (CDR1), (CDR2) and (CDR3). The NYR-1002 V _H amino acid sequence was compared with the consensus sequence for mouse heavy chain subgroup IIB and designated as this subgroup.

Example 2 chimeric antibody genes and chimera  Construction of antibodies

Chimeric antibodies were constructed by binding the mouse variable regions identified in Example 1 to human constant regions. Mouse variable regions were attached by overlapping methods of PCR recombination as described in Orlandi et al. (1989). See also Daughterty BL et al. (1991). The cloned mouse V _H and V _K genes were amplified. The vectors VH-PCR1 and VK-PCR1 (Riechmann et al., 1988) were used as templates to introduce 5 'contiguous sequences, leader introns and mouse immunoglobulin promoters and 3' contiguous sequences including splice sites and intron sequences. The prepared V _H and V _K expression cassettes were cloned into pUC19 and the entire DNA sequence was verified to be correct by sequencing. PCR amplification was performed as follows: Mutagenized oligonucleotide sets were all synthesized at 25 pmol /. This set included sites where the DNA sequence was mutated to be amplified as a series of fragments. Adjacent oligonucleotides were designed depending on the number of sites to be mutated.

PCR amplification was set up for each primer pair: 1 μl template DNA was added to 5 μl 10 × Pfu polymerase buffer (stratogen list number 600153-82 or promega list number M776A), 1 μl (25 pmol / μl) tablets. Primer, 1 μl (25 pmol / μl) reverse primer, 2 μl 10 mM dNTPs, 0.5 μl (1 unit) Pfu DNA polymerase (Stratagen List No. 600252-51 or Promega List No. M774A) and up to 50 μl H ₂ Mixed with O. These 5 'and 3' primers included the terminal 18 bp of the random sequence. All reagents except Pfu enzyme were mixed in a 0.5 mL thin wall PCR tube and heated to 94 ° C. on a PCR block. Pfu enzyme was added and samples were circulated at the following cycles: 94 ° C./2 minutes, 94 ° C./30 seconds, 15 ° C./30 seconds, 50 ° C./30 seconds, 72 ° C./1 minute (depending on the required extension length) ), 5 minutes finish at 72 ℃. The annealing temperature was adjusted higher or lower than 50 ° C. depending on the Tm of the oligo. 5 μl of the reaction was run over an agarose gel to check the product of expected size. If not, the annealing temperature was lowered by 5 ° C., the number of cycles of PCR was increased and / or the MgCl ₂ concentration was increased to 5 mM. If this PCR cycle produced multiple bands, it was necessary to gel-purify the bands of the correct size.

The product was linked in the second PCR using the second cycle 5 'and 3 primers containing the terminal 18 bp added in the first cycle of the PCR. The template for the second ligation PCR is a fragment generated in the first cycle with an adjusted amount to add approximately equal amounts. 5 μl 10 × Pfu polymerase buffer (Stratagen List No. 600153-82 or Promega List No. M776A), 2 μl (50 pmol / μl) 5 ′ second circulating primer, 2 μl (50 pmol) / Μl) 3 'second circulating primer, 2μl 10mM dNTPs, 0.5μl (1 unit) Pfu DNA polymerase (Stratagen List No. 600252-51 or Promega List No. M774A) and up to 50 μl H ₂ O . All reagents except Pfu enzyme were mixed in a 0.5 mL thin wall PCR tube and heated to 94 ° C. on a PCR block. After adding Pfu enzyme, the samples were circulated: 94 ° C./2 min, 15 cycles of 94 ° C./30 sec, 50 ° C./30 sec, 72 ° C./1 min (depending on the required extension length), 72 ° C. Finish for 5 minutes at. 5 μl of each reaction was run on agarose gel to check the product of expected size (about 820 bp for V _H expression cassette and 650 bp for V _K expression cassette). If not, the second cycle of PCR was repeated to lower the annealing temperature by 5 ° C. and / or to increase the number of cycles of the PCR. PCR products were extracted and precipitated using phenol / chloroform and ethanol or Qiagen MiniElute PCR purification kit (List No. 28004). The product obtained was digested with the required enzymes (HindIII and BamHI for expression cassettes) and loaded onto a 1.5% low-melting agarose gel. DNA strips of the correct size were cut out and purified. DNA was sequenced to verify that it was correct and that no pseudo mutations were introduced.

Heavy and light chain V-region genes were imported into expression vectors pSVgpt and pSVhyg containing markers for selection in human IgG1 or K constant regions and mammalian cells, respectively. The antibody heavy chain expression vector is shown in FIG. The part shown in angular brackets was removed from the figure. The heavy chain expression vector pSVgptHuIgG1 is based on pSV ₂ gpt (Mulligan and Berg, Science (1980; 209: 1422-1427)). This includes ampicillin resistance genes for selection in bacterial cells, gpt genes for selection in mammalian cells, mouse heavy chain immunoglobulin enhancer regions, genomic sequences encoding human IgG1 constant region genes, and SV40 polyA sequences. . The heavy chain variable region for expression is inserted into the BamH1 fragment as HindIII. Such expression cassettes include mouse heavy chain promoters, signal peptide coding sequences and signal sequences introns, V _H genes, VC splice donor sequences and intron sequences.

The antibody light chain expression vector is shown in FIG. The area in angular brackets has been removed from the figure. There is 3EcoR1 internal to HuC _K. The light chain expression vector pSVgptHuC _k is based on the vector pSVhyg. It encodes a genomic sequence comprising an ampicillin resistance gene for selection in bacterial cells, a hyg gene for selection in mammalian cells, a mouse heavy chain immunoglobulin enhancer region, a human kappa constant region gene and comprising a kappa enhancer and an SV40 polyA sequence. Include. The light chain variable region for expression is inserted into the BamH1 fragment as HindIII. Such expression cassettes include mouse heavy chain promoters, signal peptide coding sequences and signal sequences introns, V _K genes, VC splice donor sequences and intron sequences. DNA sequences have proven to be correct for V _H and V _K in chimeric expression vectors.

Heavy and light chain expression vectors were co-introduced into NS0 cells (European Collection of Animal Cell Culture, Porton, England, ECACC No. 85110503) by electroporation. PvuI digested about 3-6 μg of pSVgptHuIgG1 and pSVgptHuC plasmids, respectively. Digested DNA was ethanol precipitated and dissolved in 20 μl dH ₂ O. Cells were resuspended from a semi-fused 75 cm ³ flask and collected for 5 minutes by centrifugation at 1000 rpm. The supernatant was discarded. Cells were resuspended in 0.5 mL Dulbecco's Modified Eagle's Medium (DMEM) and transferred to Gene Pulser Couvet (Bio-Rad). The DNA was mixed with the cells by slow pipetting and left for 5 minutes under ice cooling. A cuvette was inserted between the electrodes of the bio-rad gin pulser and a single pulse of 170 V, 960 μF was applied. The Kuvet returned to ice for 20 minutes. Cell suspensions were transferred to 75 cm ³ flasks containing 20 mL DMEM and recovered for 1-2 days. Cells were collected and resuspended in 80 mL selected DMEM and divided into 200 μl into each well of a 96-well plate.

After approximately 10 days, 20 μl of media from each well was analyzed for the presence of human antibodies and wells were selected for expansion based on antibody production levels and the number of cells in the wells. Cells from selected wells were resuspended by rubbing across the surface with the tip of the Gilson P00 pipette (yellow tip) and transferred to the wells of a 24-well tissue culture plate containing 1.5 mL of fresh selected DMEM. Colonies expressing the gpt gene were selected from DMEM supplemented with 10% fetal bovine serum, 0.8 μg / mL mycophenolic acid and 250 μg / mL xanthine. Imported clones were selected for production of human antibodies by ELISA against human IgG. Cell lines secreting antibodies were expanded, the best producers were selected and frozen in liquid nitrogen. Chimeric antibodies were purified using Prosep®-A (Millipore Corporation). Concentrations were determined by ELISA for human IgG1 _K.

Example  3-mice NYR Human contained within the variable region of -1002 helper  T cell Epi Toffee's Compassion

The amino acid sequences determined in Example 1 were analyzed to generate human T cell epitope maps of variable regions using peptide threading software (biotechnology). 5 shows the results of this analysis. The analysis showed a total of 17 potential human T cell epitopes in NYR-1002, 9 in V _H and 8 in V _K. None of the potential T cell epitopes occurred in complete agreement with the CDRs.

Example  4-Design of Modified Antibody Sequences

To remove potential human T cell epitopes while maintaining important amino acids if necessary, major V _H and V _K variant sequences (NYDIVH1, NYDIVK1) were designed by substituting amino acid residues in the variable region of the mouse AF-20 antibody: See 6, 7, 8 and 9. Amino acid sequences for DNA and major V _H regions are shown in FIG. 8 and amino acid sequences for major V _K regions are shown in FIG. 9.

Since the development of major V _H and V _K variant sequences requires a small number of amino acid substitutions that may affect the binding of the final polypeptide, six different variants V _H s (NYDIVH1A, NYDIVH2, NYDIVH3, NYDIVH4, NYDIVH5 and the named NYDIVH6) and four different V _K s (named NYDIVK2, NYDIVK3, NYDIVK4 and NYDIVK5) were designed. Comparative amino acid sequences of mice and de-immunized V regions are shown in FIG. 6 for V _H and FIG. 7 for V _K. The altered amino acid sequence for variants V _H and V _K s reintroduced some potential T cell epitopes (Table 1 in FIGS. 6 and 7, respectively).

Example  5-Construction of Modified Antibody Sequences

The variable region was constructed by an overlap PCR recombination method as described by Orlandi et al. (1989) and detailed in Example 2 above. Cloned mouse V _H as a template for mutagenesis of the framework region with the required sequence And the V _K gene was used. A set of mutagenic primer pairs comprising the region to be altered was synthesized. The vectors VH-PCR1 and VK-PCR1 (Riechmann et al., For introducing a 5 'contiguous sequence comprising a leader signal peptide sequence, a leader intron and a mouse immunoglobulin promoter, and a 3' contiguous sequence comprising a splice site and an intron sequence 1988) was used as a template. The resulting modified V _H and V _K expression cassettes were cloned into pUC19, demonstrating that the entire DNA sequence was correct for each modified V _H and V _K sequence.

The modified heavy and light chain V-region genes were cut out from pUC19 as HindIII to BamH1 expression cassettes. They were transferred to expression vectors pSVgpt and pSVhyg (FIGS. 3 and 4, respectively), which contain markers for selection in human IgG1 or κ constant regions and mammalian cells, respectively. DNA sequences have proven to be correct for the modified V _H and V _K sequences in expression vectors.

The host cell line for antibody expression was NS0, non-immunoglobulin producing mouse myeloma obtained from European Collection of Animal Cell Cultures (Portton, England, ECACC N0 85110503). Heavy and light chain expression vectors were co-introduced by electroporation into NS0 cells (see Example 2 above). Colonies expressing the gpt gene were selected in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum, 0.8 μg / ml mycophenolic acid and 250 μg / ml xanthine. Imported cell clones were selected for production of human antibodies by ELISA for human IgG. Cell lines secreting antibodies were expanded, the best producers were selected and frozen in liquid nitrogen. Modified antibodies were purified using Prosep®-A (Bioprocessing Limited). Concentrations were determined by ELISA for human IgG1 _K. Antibodies were analyzed by SDS-PAGE.

Example  6-expression of modified antibodies

Infected cell clones were tested for antibody expression. Most combinations of heavy and light chains were poor producers (FIG. 10). NYDIVH5 is the best producer when combined with NYDIVK2, resulting in 3.6 mg of purified antibody. For comparison, the chimeric antibody produced 0.66 mg of purified protein. NYDIVH1A did not produce any antibody when combined with any light chain.

Antibodies containing NYDIVH2 / NYDIVK2, NYDIVH2 / NYDIVK3 performed best in the analysis, and the antibodies containing NYDIVH4 / NYDIVK5, NYDIVH4 / NYDIVK3 were slightly weak. NYDIVH2 / NYDIVK2 was chosen as the leading humanized antibody.

Example  7-modified antibody and chimera  Human T Cell Analysis of Antibodies

Humanized antibody NYDIVH2 / NYDIVK2 (huNYR-1002) along with chimeric antibody (chNYR-1002) was tested in human T cell proliferation assay. Tan coats from healthy donors were used to isolate peripheral blood mononuclear cells (PBMCs) containing antigen presenting cells (APCs) and T cells. MHC class II star specimens of these donors were determined and 20 donors were selected to obtain greater than 80% HLA DRB1 star sample coverage for the cocacian population. As shown in Table 1 of FIG. 11, none of the 20 donors provided a significant response (stimulation index SI> 2) to the humanized antibody, huNYR-1002 in the human T cell proliferation assay. In contrast, although five of these donors showed a borderline response with SI 2 to 2.5, 12 donors responded to mouse IgG, chNYR-1002 with more than 2 SI (Table 2 in FIG. 11). Clearly distinct responses to humanized antibodies demonstrated the effect of amino acid substitutions in the murine variable regions in reducing T cell immunogenicity.

Example  8-with cytotoxic agents Spliced  Determination of Cytotoxic Efficacy of Modified Antibodies

Antibodies (huNYR-1002) were conjugated to various known cytotoxic molecules and tested for cytotoxic effects in culture of cancer cells. Cytotoxic compounds (such as methotrexate and doxorubicin) are antibodies by maleimide activation and size exclusion chromatography purification using sulfosuccinimidyl-4- [N-maleimidomethyl] -cyclohexane-1-carboxylate. Conjugated to. Substitution ratios of 2 to 10 moles of compound per mole of antibody were used.

In the embodiments disclosed herein, modifications in the conjugation methods are planned, which can be achieved by one skilled in the art using the instructions provided herein;

In the embodiments disclosed herein, modifications in the selection of conjugated cytotoxic molecules are planned, which are within the skill of the skilled person using the instructions provided herein.

96 well plates were inoculated with 10 ³ to 10 ⁵ cells / well of cells (eg CCL-185 cells). The surrounding wells did not have cells. Blank well controls were filled with compound alone, medium alone, and compound + medium. Controls included cells + medium alone and cells alone. Compounds were tested in the 0.01 to 0.25 μg / mL concentration range. Plates were incubated for 4 days and tested by various viability assays according to the manufacturer's instructions (eg, Celtiter 96 Aquarus One Solution (MTS)). After staining or reagents were added to the cell wells, the plates were incubated at 37 ° C. for 1-4 days, mixed for a few seconds and then the absorbance on the plate reader was read.

result:

Newly prepared compounds (conjugates of antibody to cytotoxic molecules) have been found to be more toxic than unconjugated cytotoxic compounds (per mole of cytotoxic compounds). Increased cytotoxicity of compounds conjugated to antibodies ranged from 10 to 10 ⁴ x potency (for the same cytotoxic effect, nonconjugated compounds require 10 to 10 ⁴ x or more compound molecules compared to compounds conjugated to antibodies). ). Maximum effect was observed after 4 days of culture. These results suggest that 1) antibody conjugates provide the same cytotoxicity at low concentrations of cytotoxic compounds, suggesting low toxicity to non-carcinoma cells and tissues when the antibodies bind less; 2) the antibody conjugate is more cytotoxic at low concentrations, suggesting greater efficacy in killing cancer cells; 3) Cytotoxic compounds are sequestered and conjugated to monoclonal antibodies, suggesting low toxicity to non-carcinoma cells and tissues when the antibodies bind less.

Claims (44)

A recombinant antibody molecule comprising an antigen binding region derived from a heavy or light chain variable region of an antibody capable of binding AF-20. A chimeric antibody comprising a variable region obtained from a non-human antibody binding AF-20 and a human constant region. The chimeric antibody of claim 2 comprising a variable region obtained from a mouse antibody that binds AF-20 and a human constant region. The chimeric antibody of claim 3, wherein the mouse antibody is a mouse monoclonal antibody (moAb) produced by the hybridoma cell line ATCC name HB 9686 and a human constant region. Chimeric Antibody chNYR-1002. The humanized antibody or humanized antibody fragment that binds AF-20, wherein the humanized antibody or humanized antibody fragment is derived from a non-human antibody that binds AF-20. The humanized antibody or humanized antibody fragment of claim 6, wherein the humanized antibody or humanized antibody fragment (humoAb) is derived from a mouse monoclonal antibody (moAb) that binds AF-20. 8. The humanized antibody or humanized antibody fragment of claim 7, wherein humoAb is derived from mouse moAb generated by the hybridoma cell line ATCC name HB 9686. Humanized antibody huNYR-1002. A humanized antibody or humanized antibody fragment that binds AF-20, comprising AF-20 and complementary determining region (CDRs) amino acid residues obtained from non-human antibodies that bind human framework region (FRs) amino acid residues. The humanized antibody or humanized antibody fragment of claim 10, wherein the complementarity determining regions (CDRs) amino acid residues are obtained from a mouse moAb that binds AF-20. The humanized antibody or humanized antibody fragment of claim 11, wherein the mouse moAb that binds AF-20 is produced by the hybridoma cell line ATCC name HB 9686 and human framework region (FRs) amino acid residues. A humanized antibody binding to AF-20, wherein the complementarity determining regions (CDR1, CDR2 and CDR3) of the light chain variable region and the complementarity determining regions (CDR1, CDR2 and CDR3) of the heavy chain variable region are composed of the following amino acid sequences snippet. Light chain: CDR1 (SEQ ID No.) [RASQSIGTSIH] CDR2 (SEQ ID No.) [YASESIS] CDR3 (SEQ ID No.) [QQSSSWPFT] Heavy chain: CDR1 (SEQ ID No.) [GYTFAGHYVH] CDR2 (SEQ ID No.) [WIFPGKVNTKYNEKFKG] CDR3 (SEQ ID No.) [VGYDYFYYFDY]. The humanized antibody or humanized antibody fragment of claim 13, wherein one or more additions, substitutions, or deletions of amino acid residues are done in human framework regions (FRs). The humanized antibody or humanized antibody fragment of claim 13, wherein the potential human helper T-cell epitope identified in the variable region is removed by substitution, addition or deletion of amino acid residues. The humanized antibody or humanized antibody fragment of claim 6, wherein the antibody has an antigen binding affinity for AF-20 of at least 10% of the antibody from which the humanized antibody or humanized antibody fragment is derived. a) SEQ ID No. [GYTFAGHYVH]; b) SEQ ID No. [WIFPGKVNTKYNEKFKG]; c) SEQ ID No. [VGYDYFYYFDY]; d) SEQ ID No. [RASQSIGTSIH]; e) SEQ ID No. [YASESIS]; And f) SEQ ID No. [QQSSSWPFT] A polypeptide sequence comprising a sequence selected from one or more of the group consisting of. DNA encoding the polypeptide sequence of claim 17. A DNA molecule encoding the amino acid sequence of the humanized antibody or fragment of claim 13. A DNA molecule encoding the light chain of the antibody or fragment as claimed in claim 6. The DNA molecule of claim 20, wherein the nucleotide sequence of the light chain CDRs is as follows. CDR1 (SEQ ID No.) [RASQSIGTSIH]; CDR2 (SEQ ID No.) [YASESIS]; And CDR3 (SEQ ID No.) [QQSSSWPFT]. A DNA molecule encoding the heavy chain of the antibody or fragment as claimed in claim 6. The DNA molecule of claim 22, wherein the nucleotide sequence of the heavy chain CDRs is as follows. CDR1 (SEQ ID No.) [GYTFAGHYVH]; CDR2 (SEQ ID No.) [WIFPGKVNTKYNEKFKG]; And CDR3 (SEQ ID No.) [VGYDYFYYFDY]. The DNA molecule of claim 19 in the form of an expression vector. A host transformed with the expression vector of claim 24. A host cell comprising a recombinant expression system encoding the light and heavy chains of the antibody or antibody fragment of claim 13. A hybridoma cell line producing the chimeric antibody of claim 2. A hybridoma cell line producing the humanized antibody or antibody fragment of claim 6. A composition for the treatment of cancer comprising a therapeutically effective amount of a humanized antibody or humanized antibody fragment according to claim 6. The composition of claim 29, wherein the humanized antibody or humanized antibody fragment is directly or indirectly linked to or linked to agonist residues having therapeutic activity. 31. The agent of claim 30, wherein the effector residue is selected from the group consisting of anticancer drugs, chemotherapeutic agents, cytotoxins, radionuclides, therapeutic enzymes, prodrugs, cytokines, anti-proliferative agents, and mixtures thereof. Composition. ^32. The method of claim 31, wherein the radionuclide is ³² P, ⁴⁷ Sc, ⁶⁷ Cu, ⁹⁰ Y, ¹⁰⁵ Rh, ¹²⁵ I, ¹³¹ I, ^{117 m} Sn, ¹⁵³ Sm, ¹⁶⁶ Dy, ¹⁷⁵ Yb, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁴ Os, ²¹¹ At, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac and mixtures thereof. A method of treating a mammal with an AF-20-expressing cancer comprising administering to the mammal a therapeutically effective amount of the composition of claim 29. The method of claim 33, wherein the composition is administered after surgery. A composition for detecting cancer comprising a diagnostically effective amount of the humanized antibody or humanized antibody fragment of claim 6. The composition of claim 35, wherein the humanized antibody or humanized antibody fragment is directly or indirectly linked to or linked to a detectable label. The composition of claim 36, wherein the detectable label is selected from the group consisting of radionuclides, phosphors, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands, and mixtures thereof. The method of claim 37, wherein the radionuclide is in a group consisting of ³ H, ¹¹ C, ¹⁴ C, ¹⁸ F, ⁶⁴ Cu, ⁷⁶ Br, ⁸⁶ Y, ^{99 m} Tc, ¹¹¹ In, ¹²³ I, ¹⁷⁷ Lu, and mixtures thereof The composition selected. A method of immunodetection of AF-20-expressing cancer cells, comprising contacting the composition of claim 35 with cancer cells. The method of claim 39, wherein the humanized antibody or humanized antibody fragment of the composition is bound to a solid support. 36. A method for immunodetection of AF-20-expressing cancer cells in a mammal comprising administering to the mammal a diagnostically effective amount of the composition according to claim 35. 42. The method of claim 41, wherein said immunodetection is tumor imaging in vivo. (i) intravenously administering the radionuclide-labeled humanized antibody, or humanized antibody fragment of claim 6; (ii) detecting tumor cells using a radionuclide activity probe; (iii) a method of treating cancer comprising removing tumor cells detected by surgical resection. The method of claim 43, wherein the radionuclide is in a group consisting of ³ H, ¹¹ C, ¹⁴ C, ¹⁸ F, ⁶⁴ Cu, ⁷⁶ Br, ⁸⁶ Y, ^{99 m} Tc, ¹¹¹ In, ¹²³ I, ¹⁷⁷ Lu, and mixtures thereof Which method is chosen.

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