BR112015015465B1 - BETA1 ANTI-INTEGRIN HUMANIZED ANTIBODY, VH REGION, VL REGION, NUCLEIC ACID MOLECULE, IMMUNOCONJUGATE, USE OF THE FOREGOING, VECTOR, PROKARYOTIC HOST CELL, PHARMACEUTICAL COMPOSITION, AND IN VITRO METHOD TO DETECT A CELL PROLIFERATING DISEASE OR INFINITE DISEASE LAMATORY - Google Patents

Abstract

HUMANIZED ANTI-INTEGRIN BETA1 ANTIBODY, VH REGION, VL REGION, NUCLEIC ACID MOLECULE, IMMUNOCONJUGATE, USE OF THE FOREGOING, VECTOR, HOST CELL, PHARMACEUTICAL COMPOSITION, AND METHOD FOR DETECTING A DISEASE. The present invention relates to framework regions of human variable chain and humanized antibody comprising the framework regions, the antibodies being specific for integrin (Beta)1. The invention also relates to methods of using the antibodies, for example, to treat diseases such as cancer.

Description

CROSS REFERENCE TO RELATED ORDER(S)

[001] This application claims benefit under 35 USC § 119(e) to United States Application Serial No. 61/746,023, filed December 26, 2012. The description of the foregoing application is considered part of, and is incorporated by reference in its entirety in the description of this order.

INCORPORATION OF SEQUENCE LISTING

[002] The material in the accompanying Sequence Listing is hereby incorporated by reference into this application. The accompanying sequence listing text file, name Sequence Listing ONCO1100_1WO, was created on December 23, 2013, and is 81 KB. The file can be evaluated using Microsoft Word on a computer using Windows OS.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

[003] The present invention relates generally to the field of immunology, and more specifically to anti-integrin antibodies, and methods of using them.

BACKGROUND INFORMATION

[004] Cancer is one of the leading causes of death in the developed world, resulting in more than 500,000 deaths a year in the United States alone. More than one million people are diagnosed with cancer in the United States each year, and overall, it is estimated that more than 1 in 3 people will develop some form of cancer during their lifetime. Although there are more than 200 different types of cancer, four of them, including breast, lung, colorectal, and prostate, account for more than half of all new cases.

[005] Breast cancer is the most common cancer in women, with an estimated 12% of women at risk of developing the disease during their lifetime. Although mortality rates have declined due to earlier detection and improved treatments, breast cancer remains a leading cause of death in middle-aged women. Furthermore, metastatic breast cancer is still an incurable disease. Upon presentation, many patients with metastatic breast cancer have only one or two organ systems affected, but as the disease progresses, multiple sites usually become involved. The most common sites of metastatic involvement are locoregional recurrences in the skin and soft tissues of the chest wall, as well as in the axillary and supraclavicular areas. The most common site for distant metastasis is bone (30 - 40% of distant metastases), followed by the lungs and liver. Although only approximately 1-5% of women with newly diagnosed breast cancer have distant metastases at the time of diagnosis, approximately 50% of patients with local disease eventually relapse with metastases within five years. At present, the median survival from the onset of distant metastasis is about three years.

[006] Current methods of diagnosing and staging breast cancer include the tumor-node metastasis (TNM) system which depends on the size of the tumor, presence of the tumor in lymph nodes, and the presence of distant metastasis as described in the American Joint Committee on Cancer: AJCC Cancer Staging Manual. Philadelphia, Pa.: Lippincott-Raven Publishers, 5th ed., 1997, pp 171180, and in Harris, JR: "Staging of breast carcinoma" in Harris, J.R., Hellman, S., Henderson, I.C., Kinne D.W. ( eds.): Breast Diseases. Philadelphia, Lippincott, 1991. These parameters are used to provide a prognosis, and to select an appropriate therapy. The morphological appearance of the tumor can also be evaluated, but because tumors with similar histopathological appearance can exhibit significant clinical variability, this approach has serious limitations. Finally, assays for cell surface markers can be used to subclass certain tumor types. For example, a factor considered in the prognosis and treatment of breast cancer is the presence of estrogen receptor (ER) as ER positive breast cancers typically respond more readily to hormonal therapies such as tamoxifen or aromatase inhibitors than tumors. of ER negative. Yet these analyses, while useful, are only partially predictive of the clinical behavior of breast tumors, and there is much phenotypic diversity present in breast cancers that current diagnostic tools fail to detect and current therapies fail to treat.

[007] Prostate cancer is the most common cancer in men in the developed world, accounting for an estimated 33% of all new cases in the United States, and is the second most frequent cause of death. Since the introduction of the prostate-specific antigen (PSA) blood test, early detection of prostate cancer has dramatically improved survival rates, and the five-year survival rate for patients with local and regional stage prostate cancers. at the time of diagnosis it is close to 100%. Furthermore, more than 50% of patients will eventually develop locally advanced or metastatic disease.

[008] Currently, radical prostatectomy and radiation therapy provide curative treatment for most localized prostate tumors. However, therapeutic options are very limited for advanced cases. For metastatic disease, androgen ablation with a luteinizing hormone-releasing hormone (LHRH) agonist alone, or in combination with antiandrogens, is the standard of care. Yet despite maximum androgen blockade, the disease almost always progresses with the majority developing androgen-independent disease. At present, there is no uniformly accepted treatment for hormone-refractory prostate cancer, and chemotherapeutic regimens are commonly used.

[009] Lung cancer is the most common cancer worldwide, the third most commonly diagnosed cancer in the United States, and by far the most frequent cause of death. Cigarette smoking is believed to be responsible for an estimated 87% of all lung cancers, making it the most deadly preventable disease. Lung cancer is divided into two major types that account for over 90% of all lung cancers: small cell lung cancer (SCLC), and non-small cell lung cancer (NSCLC). SCLC accounts for 15-20% of cases, and is characterized by its origin in large central airways and histological composition of sheets of small cells with little cytoplasm. SCLC is more aggressive than NSCLC, growing rapidly and metastasizing early and frequently. NSCLC accounts for 80-85% of all cases, and is further divided into three major subtypes based on histology: adenocarcinoma, squamous cell carcinoma (squamous cell carcinoma), and undifferentiated large cell carcinoma.

[0010] Lung cancer is typically delayed in its course, and thus has a median survival of only 6-12 months after diagnosis, and a 5-year overall survival rate of only 5-10%. Although surgery offers the best chance of a cure, only a small fraction of lung cancer patients are eligible, with most depending on chemotherapy and radiotherapy. Despite attempts to manipulate the timing and dose intensity of these therapies, survival rates have increased little over the last 15 years.

[0011] Colorectal cancer is the third most common cancer, and the fourth most frequent cause of cancer deaths around the world. Approximately 5-10% of all colorectal cancers are hereditary, with one of the main forms being familial adenomatous polyposis (FAP), an autosomal dominant disease in which approximately 80% of affected individuals carry a germline mutation in the polyposis coli gene adenomatous (APC). Colorectal carcinoma has a tendency to invade locally by circumferential growth, and elsewhere by lymphatic, hematogenous, transperitoneal, and perineural diffusion. The most common site of extralymphatic involvement is the liver, with the lungs the most frequently affected extra-abdominal organ. Other sites of hematogenous spread include the bones, kidneys, adrenal glands, and brain.

[0012] The current staging system for colorectal cancer is based on the degree of tumor penetration through the bowel wall, and the presence or absence of nodal involvement. This staging system is defined by the three major Duke classifications: Duke A disease is confined to submucosal layers of the colon or rectum; Duke's disease B has tumors that invade through the muscularis propria, and may penetrate the wall of the colon or rectum; and Duke C disease includes any degree of bowel wall invasion with regional lymph node metastasis. While surgical resection is highly effective for early-stage colorectal cancers, providing 95% cure rates in Duke A patients, the rate is reduced to 75% in Duke B patients, and the presence of positive lymph node in C disease of Duke predicts a 60% probability of recurrence within five years. Treatment of Duke C patients with a post-surgical course of chemotherapy reduces the recurrence rate to 40%-50%, and is now the standard of care for these patients.

[0013] Epithelial carcinomas of the head and neck occur from the mucosal surfaces in the head and neck area, and are typically squamous cell in origin. This category includes tumors of the paranasal sinuses, oral cavity, and nasopharynx, oropharynx, hypopharynx, and larynx.

[0014] The annual number of new cases of head and neck cancers in the United States is approximately 40,000 per year, accounting for about 5 percent of adult malignancies. Head and neck cancers are more common in some other countries, and the worldwide incidence probably exceeds half a million cases annually. In North America and Europe, tumors usually arise from the oral cavity, oropharynx, or larynx, so nasopharyngeal cancer is more common in Mediterranean countries and the Far East.

[0015] Traditional modes of therapy (radiation therapy, chemotherapy, and hormone therapy), while useful, have been limited by the emergence of treatment-resistant cancer cells. Clearly, new approaches are needed to identify targets for treatment of head and neck cancer and cancer generally.

[0016] Pancreatic cancer is a malignant neoplasm that originates from transformed cells that occur in tissues that form the pancreas. The most common type of pancreatic cancer, accounting for 95% of these tumors, is adenocarcinoma (tumors that exhibit glandular architecture on light microscopy) that occur within the exocrine component of the pancreas. A minority occur from islet cells, and are classified as neuroendocrine tumors. Pancreatic cancer is the fourth most common cause of cancer-related deaths in the United States, and the eighth most common worldwide.

[0017] Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults with a median survival of less than one year with maximal therapy. So far, only three drugs have been approved by the FDA to treat GBM, and overall survival has not improved in over 25 years.

[0018] Integrins are cell adhesion molecules that are responsible for mechanosensing the microenvironment, and inducing extracellular matrix (ECM)-induced signaling in both normal and pathological states, such as inflammation and cancer. Importantly, integrins occur at the interface of the cell and microenvironment, playing a role in tumor progression and regulation of growth and survival pathways. Upregulation of many types of integrins has been associated with epithelial malignancies, particularly during the processes of invasion, metastasis, and angiogenesis. Importantly, β1 integrins, which coordinate many broader functional activities such as inflammation, proliferation, adhesion, and invasion, have recently been implicated in therapeutic resistance in multiple solid cancer models and hematopoietic malignancies. Importantly, this β1 integrin-mediated resistance is to occur at the level of the tumor cells themselves. In addition to the above, β1 integrin has important functions during tumor vascularization, such as VEGF-dependent and VEGF-independent angiogenesis by promoting vascular endothelial cell migration. β1 integrin inhibition overcomes resistance to anti-angiogenesis therapy via multiple potential mechanisms: (1) prevention of vessel co-option (and/or growth/invasion following any classical ECM substrate; (2) reduction of tumor cell viability after insults, such as ionizing radiation; (3) inhibition directly of tumor cell proliferation; (4) inhibition directly of the process of vascularization; and (5) inhibition of the aggressive mesenchymal phenotype, including spheroidal growth, typically seen after establishment resistance to therapy.

[0019] Anti-β1 integrin compositions, such as antibodies directed to β1 integrin, may also be important in immunological diseases/inflammatory diseases and disorders give the role of β1 integrin in broader functional activities as discussed above. Additionally, diseases and disorders that can be targeted through the β1 integrin pathway include multiple sclerosis, Crohn's disease, rheumatoid arthritis, inflammatory bowel disease, and the like. Similarly, it is to be expected that certain eye-related diseases may be targeted including wet age-related macular degeneration (AMD).

SUMMARY OF THE INVENTION

[0020] The present invention is based on the generation of humanized antibody having human framework sequences that specifically bind β1 integrin. β1 integrin is known to be an overexpressed protein in solid tumors, and thus as a useful cancer cell marker in the characterization, study, diagnosis, and treatment of cancer. In addition, β1 integrin has been shown to trigger cancer resistance to both conventional therapies (eg, chemotherapy and ionizing radiation) and targeted therapies (eg, trastuzumab, bevicizumab, lapatinib) by orchestrating growth and survival signals from the cancer microenvironment. tumor. Humanization efforts have been limited in the past by the number of human structures available. This invention meets the need for additional human framework sequences for an antibody to specifically bind β1 Integrin.

[0021] In one embodiment, the present invention provides a humanized antibody that specifically binds β1 integrin. The antibody includes a heavy chain (VH) variable region and a light chain (VL) variable region, in which the VH region has less than about 97%, 96%, or 95% identity to a VH region having an amino acid sequence as set forth in SEQ ID NO:2, and in which the VL region has less than about 97%, 96% or 95% identity to a VL region having an amino acid sequence as set forth in SEQ ID NO:4.

[0022] In another embodiment, the VH region has greater than 75%, 80%, 85%, 90%, 95% or 99% identity to a VH region having an amino acid sequence as set forth in SEQ ID NO :6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NOs:29-43 and SEQ ID NOs:91-100. In embodiments, the VL region has greater than 75%, 80%, 85%, 90%, 95% or 99% identity to a VL region having an amino acid sequence as set forth in SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NOs:44-57 and SEQ ID NOs:107-116.

[0023] In another embodiment, the antibody has CDRs from the VH and VL regions of a donor antibody, such as OS2966. In embodiments, the VH region CDRs have amino acid sequences as set forth in SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25, and the VL region CDRs have amino acid sequences as set forth in SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28.

[0024] In one aspect, the invention provides an antibody that includes VH and VL regions of the present invention.

[0025] In another aspect, the invention provides a nucleic acid molecule encoding the antibody of the present invention.

[0026] In another aspect, the invention provides a vector that includes a nucleic acid molecule of the present invention.

[0027] In another aspect, the invention provides an isolated host cell that includes the vector of the present invention.

[0028] In another aspect, the invention provides pharmaceutical compositions. In one embodiment, the pharmaceutical composition includes the antibody of the present invention, and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition includes the nucleic acid molecule of the present invention, and a pharmaceutically acceptable carrier.

[0029] In another aspect, the invention provides an immunoconjugate including the antibody of the present invention linked to a detection or therapeutic moiety.

[0030] In another aspect, the invention provides a chimeric protein including the antibody of the present invention operably linked to a separate peptide, such as cytokine.

[0031] In another embodiment, the invention provides a method of treating a disease in an individual. In embodiments, the method includes administering to the subject the antibody of the present invention, the nucleic acid molecule of the present invention, the pharmaceutical composition of the present invention, the immunoconjugate of the present invention, or the chimeric protein of the present invention, thereby treating the disease. In one embodiment, the disease is a cell proliferative disorder, such as cancer.

[0032] In another embodiment, the invention provides a method of detecting a disease in an individual. The method includes contacting the antibody or immunoconjugate of the present invention with a sample from the subject; detect the level of β1 integrin, via specific binding with β1 integrin; and comparing the detected level of β1 integrin to that of β1 integrin in a normal sample, in which an increased level of β1 integrin in the sample from the subject as compared to the normal sample is indicative of a disease.

[0033] In another embodiment, the invention provides a VH region having greater than 75%, 80%, 85%, 90%, 95% or 99% identity to a VH region having an amino acid sequence as placed in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NOs:29-43 and SEQ ID NOs:91-100.

[0034] In another embodiment, the invention provides a VL region having greater than 75%, 80%, 85%, 90%, 95% or 99% identity to a VL region having an amino acid sequence as placed in SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NOs:44-57 and SEQ ID NOs:107-116.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a pictorial representation depicting nucleic acid and amino acid sequences of the VH region of antibody OS2966 produced by hybridoma OS2966.

[0036] Figure 2 is pictorial representation depicting nucleic acid and amino acid sequences of VL region of antibody OS2966 produced by hybridoma OS2966.

[0037] Figure 3 is a pictorial representation depicting analysis of OS2966 antibody CDRs produced by OS2966 hybridoma that are used in the VH and VL regions of the antibody of the present invention.

[0038] Figure 4 is a pictorial representation depicting the amino acid sequences of hybridoma-produced OS2966 antibody CDRs that are used in the VH and VL regions of the antibody of the present invention.

[0039] Figure 5 is a pictorial representation depicting nucleic acid and amino acid sequences of the VH region of the antibody of the present invention in one embodiment. Conserved CDRs are underlined.

[0040] Figure 6 is a pictorial representation depicting nucleic acid and amino acid sequences of the VH region of the antibody of the present invention in one embodiment. Conserved CDRs are underlined.

[0041] Figure 7 is a pictorial representation depicting nucleic acid and amino acid sequences of the VH region of the antibody of the present invention in one embodiment. Conserved CDRs are underlined.

[0042] Figure 8 is a pictorial representation depicting nucleic acid and amino acid sequences of the VH region of the antibody of the present invention in one embodiment. Conserved CDRs are underlined.

[0043] Figure 9 is a pictorial representation depicting nucleic acid and amino acid sequences of the VH region of the antibody of the present invention in one embodiment. Conserved CDRs are underlined.

[0044] Figure 10 is a pictorial representation depicting nucleic acid and amino acid sequences of the VL region of the antibody of the present invention in one embodiment. Conserved CDRs are underlined.

[0045] Figure 11 is a pictorial representation depicting nucleic acid and amino acid sequences of the VL region of the antibody of the present invention in one embodiment. Conserved CDRs are underlined.

[0046] Figure 12 is a pictorial representation depicting nucleic acid and amino acid sequences of the VL region of the antibody of the present invention in one embodiment. Conserved CDRs are underlined.

[0047] Figure 13 is a pictorial representation depicting nucleic acid and amino acid sequences of the VL region of the antibody of the present invention in one embodiment. Conserved CDRs are underlined.

[0048] Figure 14 is a schematic diagram of vectors.

[0049] Figure 15 is a pictorial representation depicting sequence analysis and alignment of humanized VH chains of the invention against the VH of antibody OS2966 (SEQ ID NO:2). The sequences of Figure 15 are as follows: OS2966 is SEQ ID NO:2; Variant 1 is SEQ ID NO:6; Variant 2 is SEQ ID NO:8; Variant 3 is SEQ ID NO:10; Variant 4 is SEQ ID NO:12; Variant 5 is SEQ ID NO:14; Variant 6 is SEQ ID NO:29; Variant 7 is SEQ ID NO:30; Variant 8 is SEQ ID NO:31; Variant 9 is SEQ ID NO:32; Variant 10 is SEQ ID NO:33; Variant 11 is SEQ ID NO:34; Variant 12 is SEQ ID NO:35; Variant 13 is SEQ ID NO:36; Variant 14 is SEQ ID NO:37; Variant 15 is SEQ ID NO:38; VH2 is SEQ ID NO:91; Variant 16 is SEQ ID NO:92; VH4 is SEQ ID NO:93; Variant 17 is SEQ ID NO:94; VH5 is SEQ ID NO:95; Variant 18 is SEQ ID NO:96; VH6 is SEQ ID NO:97; Variant 19 is SEQ ID NO:98; VH7 is SEQ ID NO:99; and Variant 20 is SEQ ID NO:100.

[0050] Figure 16 is a pictorial representation depicting sequence analysis and alignment of humanized J regions (VH) of the invention corresponding to the alignments shown in Figure 15. The sequences are as follows: J1 is SEQ ID NO:101; J2 is SEQ ID NO:102; J3 is SEQ ID NO:103; J4 is SEQ ID NO:104; J5 is SEQ ID NO:105; and J6 is SEQ ID NO:106.

[0051] Figure 17 is a pictorial representation depicting sequence analysis and alignment of humanized VL chains of the invention against VL of antibody OS2966 (SEQ ID NO:4). The sequences are as follows: OS2966 is SEQ ID NO:4; Variant 1 is SEQ ID NO:16; Variant 2 is SEQ ID NO:18; Variant 3 is SEQ ID NO:20; Variant 4 is SEQ ID NO:22; Variant 5 is SEQ ID NO:44; Variant 6 is SEQ ID NO:45; Variant 7 is SEQ ID NO:46; Variant 8 is SEQ ID NO:47; Variant 9 is SEQ ID NO:48; Variant 10 is SEQ ID NO:49; Variant 11 is SEQ ID NO:50; Variant 12 is SEQ ID NO:51; Variant 13 is SEQ ID NO:52; VkI is SEQ ID NO:107; Variant 14 is SEQ ID NO:108; VkII is SEQ ID NO:109; Variant 15 is SEQ ID NO:110; VkIII is SEQ ID NO:111; Variant 16 is SEQ ID NO:112; VkIV is SEQ ID NO:113; Variant 17 is SEQ ID NO:114; VkVI is SEQ ID NO:115; and Variant 18 is SEQ ID NO:116.

[0052] Figure 18 is a pictorial representation depicting sequence analysis and alignment of humanized J regions (elevated kappa chains) of the invention corresponding to the alignments shown in Figure 17. The sequences are as follows: J1 is SEQ ID NO:117; J2 is SEQ ID NO:118; J3 is SEQ ID NO:119; J4 is SEQ ID NO:120; and J5 is SEQ ID NO:121.

[0053] Figure 19 is a pictorial representation depicting codon usage of the VH and VL chains of the present invention.

[0054] Figure 20 is a graphical representation depicting relative affinity of compound human antibody variants of the present invention (those shown in Table 2).

[0055] Figure 21 is a graphical representation depicting relative affinity of composite human antibody variants of the present invention (those shown in Table 2).

[0056] Figure 22 is a graphical representation depicting relative affinity of compound human antibody variants of the present invention (those shown in Table 2).

[0057] Figure 23 is a graphical representation depicting relative affinity of compound human antibody variants of the present invention (those shown in Table 2).

[0058] Figure 24 is a graphical representation depicting relative affinity of composite human antibody variants of the present invention (those shown in Table 2).

[0059] Figure 25A-D is a series of graphical representations illustrating functional validation in extracellular matrix (ECM) adhesion assays of composite human antibody variants of the present invention. Functional inhibition of the β1 integrin subunit with composite human antibody variants of the present invention (from Table 2) was evaluated in a microplate adhesion assay on multiple ECMs and on multiple cancer cell lines. Figure 25A uses PANC-1 human pancreatic cancer. Figure 25B uses PANC-1 human pancreatic cancer. Figure 25B uses PANC-1 human pancreatic cancer. Figure 25C uses MDA-MB-231 triple negative human breast cancer. Figure 25D uses AsPC-1 human pancreatic cancer.

[0060] Figure 26A-B is a series of pictorial and graphical representations illustrating functional validation in extracellular matrix (ECM) migration assays of composite human antibody variants of the present invention. Functional inhibition of the β1 integrin subunit with composite human antibody variants of the present invention (from Table 2) was evaluated in a microplate migration assay on ECM component fibronectin with triple negative breast cancer cells ( MDA-MB-231). Figure 26A is a series of 10x magnification images of plaques demonstrating wound migration attenuation in OS2966, and composite human variants (H1, H2, H3) treated in wells. Figure 26B is a plot of quantification of cell free area for each condition (performed in triplicate and repeated).

[0061] Figure 27A-B is a series of pictorial and graphical representations illustrating functional validation in human umbilical vein endothelial cell (HUVEC) tube formation angiogenesis assays of composite human antibody variants of the present invention. Functional inhibition of the β1 integrin subunit with composite human antibody variants of the present invention (from Table 2), was evaluated in an in vitro model of angiogenesis; the human umbilical vein endothelial cell tube forming assay (HUVEC). Figure 27A is a series of 10x magnification images of plaques demonstrating attenuation of vascular tube formation in OS2966 and composite human variant (H1, H2, H3) treated in wells. Figure 27B is a series of quantification plots of closed unit formation for each condition (performed in at least triplicate and repeated with endothelial progenitor cells).

[0062] Figure 28A-B is a series of pictorial and graphical representations illustrating functional validation in a human orthotopic xenograft model of triple negative breast cancer of composite human antibody variants of the present invention. Functional inhibition of the β1 integrin subunit with composite human antibody variants of the present invention (from Table 2), was evaluated in an in vivo model of human triple negative breast cancer with the MDA-MB-231 cell line. Figure 28A is a graph showing mean tumor volume group by time. Figure 28B is an image of a western blot.

[0063] Figure 29 is a series of pictorial representations illustrating functional validation in a human orthotopic xenograft model of spontaneous lung metastasis from triple negative breast cancer of composite human antibody variants of the present invention.

[0064] Figure 30A-C is a series of pictorial and graphical representations illustrating functional validation in a human xenograft model of pancreatic cancer of composite human antibody variants of the present invention. Functional inhibition of the β1 integrin subunit with composite human antibody variants of the present invention (from Table 2), was evaluated in an in vivo model of human gemcitabine-resistant pancreatic cancer with the PANC1-GEMR cell line. Figure 30A is a graph showing mean tumor volume group by time. Figure 30B is a graph showing mean tumor volume group by time. Figure 30C is an image of a western blot.

[0065] Figure 31A-B is a series of pictorial and graphical representations illustrating functional validation in an established human glioblastoma xenograft model of composite human antibody variants of the present invention. Functional inhibition of the β1 integrin subunit with composite human antibody variants of the present invention (from Table 2), was evaluated in an in vivo model of human glioblastoma established with the U87MG cell line. Figure 31A is a graph showing mean tumor volume group by time. Figure 3B is an image of a blob.

[0066] Figure 32A-B depicts results of an immunogenicity scoring assay (EpiScreen™). Figure 32A is a histogram. Figure 32B is a summary of healthy donor T cell proliferation responses to the donor cohort.

DETAILED DESCRIPTION OF THE INVENTION

[0067] The present invention provides human VH and VL framework sequences, and nucleic acid sequences encoding the same. Such sequences are used, for example, to provide frameworks for grafting CDRs from a donor antibody, for example a rodent antibody. In that way, an antibody comprising a VH and/or VL structure of the invention with the binding specificity of a donor antibody can be generated.

[0068] The invention also provides humanized antibody having the specificity of an antibody called OS2966, for example, specific binding to β1 integrin, via murine OS2966 CDRs, provided in the framework regions of human VH and VL placed in SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 29-57, 91100 and 107-116.

[0069] The humanized antibodies of the invention are used for a variety of therapeutic and diagnostic purposes, as described herein. Uses include diagnosing and treating diseases, such as cancer.

[0070] Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular device, methods, and experimental conditions described, as devices, methods, and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0071] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references, unless the context clearly indicates otherwise. Thus, for example, references to "the composition" or "the method" include one or more compositions and methods, and/or steps of the type described herein which will become apparent to those skilled in the art upon reading this description, and so on.

[0072] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention pertains. While any methods and materials similar or equivalent to those described herein may be used in practicing or testing the invention, preferred methods and materials are now described.

[0073] The term "β1 integrin" is synonymous with CD29, and includes reference to a protein encoded by the ITGB1 gene. β1 integrin is associated with posterior antigen receptors, and is known to bind with a number of alpha subunits including alpha-1 to alpha-9, for example, complexing the alpha-3 subunit creates α3β1 complex that reacts to such molecules such as netrin-1 and reelin.

[0074] As used herein, the term "anti-β1 integrin" in reference to an antibody, refers to an antibody that specifically binds β1 integrin.

[0075] The term "antibody" refers to a polypeptide encoded by an immunoglobulin gene, or functional fragments thereof, that specifically bind and recognize an antigen. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.

[0076] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having a "light" chain (about 25 kDa) and a "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

[0077] Examples of functional antibody fragments include, but are not limited to, whole antibody molecules, antibody fragments such as Fv, single chain Fv (scFv), Complementarity Determining Regions (CDRs), VL (Region light chain variable region), VH (heavy chain variable region), Fab, F(ab)2', and any combination thereof, or any other functional portion of an immunoglobulin peptide capable of binding the target antigen (see, for For example, Fundamental Immunology (Paul ed., 3d ed. 1993) As appreciated by one skilled in the art, various antibody fragments can be obtained by a variety of methods, for example, digestion of an intact antibody with an enzyme, such as pepsin; or de novo synthesis. Antibody fragments are often synthesized de novo, either chemically, or by the use of recombinant DNA methodology. Thus, the term antibody, as used herein, includes antibody fragments, or produced by modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (eg, single-chain Fv), or those identified using phage display libraries. The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are known in the art.

[0078] References to "VH" or to "VH" refer to the variable region of an immunoglobulin heavy chain, including an Fv, scFv, a disulfide-stabilized Fv (dsFv) or Fab. References to "VL" or to "VL" refer to the variable region of an immunoglobulin light chain, including of an Fv, scFv, dsFv or Fab.

[0079] CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the antibody heavy chain variable domain in which it is found, whereby a VL CDR1 CDR1 is the CDR1 from the antibody light chain variable domain in which it is found. The numbering of the heavy and light chain variable regions here is according to Kabat (see, for example, Johnson et al., (2001) "Kabat Database and its applications: future directions" Nucleic Acids Research, 29: 205 -206; and the Kabat Database of Sequences of Proteins of Immunological Interest, Feb. 22, 2002 Dataset), unless otherwise indicated.

[0080] The positions of the CDRs and framework regions can be determined using various definitions well known in the art, for example, Kabat, Chothia, international ImMunoGeneTics database (IMGT), and AbM (see, for example, Johnson et al., supra Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, structural repertoire of the human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J. Mol. Biol. 1997, 273(4)). Definitions of antigen-combining sites are also described in the following: (Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc, M.-P. IMGT , the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1;29(l):207-9 (2001); MacCallum et al., Antibodyantigen interactions: Contact analysis and binding site topography, J Mol. Biol., 262 (5) , 732-745 (1996), and Martin et al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989), Martin, et al, Methods Enzymol., 203, 121-153, (1991); Pedersen et al, Immunomethods, 1, 126, (1992) and Rees et al., In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996).

[0081] Exemplary structure and CDR sequences for human VH and VL regions disclosed herein are shown in Figure 4.

[0082] "OS2966" refers to a murine IgG1 antibody that specifically binds human β1 integrin. OS2966 is commercially available (under a different designation) from several sources, such as the Developmental Studies Hybridoma Bank of the University of Iowa. OS2966 heavy and light chains were cloned. The nucleotide sequences and amino acid sequences of the VH region of OS2966 are placed in SEQ ID NO:1 and SEQ ID NO:2, respectively. The nucleotide sequences and amino acid sequences of the VL region of OS2966 are placed in SEQ ID NO:3 and SEQ ID NO:4, respectively. The OS2966 CDRs as designated for the exemplary humanized antibody described herein are placed in SEQ ID NOs:23-28, and shown in Figure 4.

[0083] A "humanized antibody" refers to an antibody that comprises a donor antibody binding specificity, i.e., the CDR regions of a donor antibody, typically a mouse monoclonal antibody, grafted onto human framework sequences. A "humanized antibody", as used herein, binds the same epitope as the donor antibody and typically has at least 25% of the binding affinity. An exemplary assay for binding affinity is described in Example 5. Methods for determining whether the antibody binds the same epitope are well known in the art, see, for example, Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which discloses techniques for epitope mapping, or alternatively competition experiments, to determine whether an antibody binds the same epitope as the donor antibody. A humanized antibody comprising a novel framework region provided in the invention.

[0084] A "VH" or "VL" "region" or "structure" of the invention refers to the VH or VL amino acid sequence that has at least 70% identity, often at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, to an amino acid sequence placed in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NOs:29-57, SEQ ID NOs:91-100 , or SEQ ID NOs:107-116. A "framework" of a VH or VL chain refers to the regions of chain structure not including the CDRs. The term as applied to each chain encompasses all framework regions.

[0085] A "humanized anti-β1 integrin antibody" refers to a humanized antibody comprising a human framework sequence that has the binding specificity of murine OS2966 grafted to that framework. A CDR of a humanized anti-β1 integrin antibody of the invention has at least 85%, more typically, at least 90%, 95%, 96%, 97%, 98%, or 99% identity, to a CDR of the sequences light and heavy chain placed in SEQ ID NO:2 and SEQ ID NO:4, respectively. The VH region CDRs are placed in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25. The VH region CDRs are placed in SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.

[0086] In one aspect, the invention provides compound humanized antibody. Composite human antibody technology generates humanized non-immunogenic antibodies by avoiding T cell epitopes (deimmunization) in variable region (V region) sequences (EP 2,388,871). Unlike other humanization technologies that use single human V region structures as 'acceptors' for complementarity determining regions (CDRs) from the starting (typically murine) antibody, Composite Human Antibodies™ comprise multiple sequence segments (' compounds') derived from V regions of unrelated human antibodies. The key properties of Compound Human Antibodies are as follows:

[0087] Sequence segments derived from unrelated human V region databases are selected after determination of amino acids that are considered critical for antigen binding of the starting antibody. All selected sequence segments derived from human V region databases are filtered for the presence of potential T cell epitopes using Antitope's in silico tools. Composite Human Antibodies™ retain better affinity and specificity than standard humanized antibody due to close fit of human sequence segments with all sections of the starting antibody V regions. Composite Human Antibodies™ are depleted of T cell epitopes and therefore considered both humanized and deimmunized.

[0088] In one embodiment, antibodies of the invention are prepared by identifying candidate residues in the framework region that are mutated at specific sites within T cell epitopes. Antibodies of the invention may exhibit altered binding affinity, and/or immunogenicity altered as compared to donor antibodies.

[0089] Methods known in the art can be used to map T cell epitopes within a protein sequence. For example, EpiScreen™ (EP1989544, Antitope, UK) is used to map T cell epitopes within a protein sequence to determine potential for immunogenicity, which is based on the number and potency of T cell epitopes within a sequence. EpiScreen™ T cell epitope mapping typically uses CD8+ T cell depleted PBMCs from a minimum of 50 HLA-typed donors (selected to represent the human population of interest). Typically, 15-mer peptides of 12 amino acids that largely overlap a protein sequence are analyzed in large numbers of replicate cultures for in vitro CD4+ T cell stimulation by incorporation of 3H TdR. CD4+ T cell stimulation is often detected in two or three adjacent and overlapping peptides, as the core 9mer that bridges the MHC class II binding groove will be present in more than one peptide sequence. After precisely identifying peptides that stimulate CD4+ T cells in vitro, in silico technologies can be used to designate epitope-depleted (de-immunized) variants by determining the precise location of core 9mer sequences, and the location of class II anchor residues of key MHC.

[0090] The phrase "single-chain Fv" or "scFv" refers to an antibody in which the heavy chain and light chain variable domains of a traditional two-chain antibody have been joined together to form one chain. Typically, a linker peptide is inserted between the two strands to allow for stabilization of the variable domains without interfering with correct folding and creation of an active binding site. A single-chain humanized antibody of the invention, for example, humanized anti-β1 integrin antibody, can bind as a monomer. Other exemplary single chain antibodies can form diabodies, triabodies, and tetrabodies. (See, for example, Hollinger et al., 1993, supra). Additionally, the humanized antibody of the invention, e.g., humanized anti-β1 integrin antibody, may also form a component of a "reconstituted" antibody or antibody fragment, e.g., a Fab, a Fab' monomer, a Fab' dimer, F(ab)'2, or an entire immunoglobulin molecule. Thus, a humanized antibody of the present invention can additionally comprise a human Fc region.

[0091] "Joining" or "linking" or "conjugating" refers to any method known in the art to functionally connect protein domains, including, without limitation, recombinant fusion with or without intervening domains, intein-mediated fusion, association non-covalent, and covalent bonding, for example, disulfide bonding, peptide bonding; hydrogen bonding; electrostatic bonding; and conformational binding, for example, antibody-antigen, and biotin-avidin associations. In the context of the present invention, the terms include reference to linking an antibody moiety to an effector molecule (EM). The linkage can be either chemically or recombinantly. The chemical means refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.

[0092] The term "effector fraction" means the portion of an immunoconjugate intended to have an effect on a targeted cell by targeting the fraction, or to identify the presence of the immunoconjugate. Thus, the effector moiety can be, for example, a therapeutic moiety, such as a cytotoxic agent or drug, or a detectable moiety, such as a fluorescent label.

[0093] A "therapeutic fraction" is that portion of an immunoconjugate intended to act as a therapeutic agent.

[0094] The term "therapeutic agent" includes any number of compounds currently known, or later developed, to act as chemotherapeutic agents, antineoplastic compounds, anti-inflammatory compounds, anti-infective compounds, enzyme activators or inhibitors, modifiers allosterics, antibiotics, or other agents administered to induce a desired therapeutic effect in a patient. The therapeutic agent may also be a toxin, or a radioisotope, where the intended therapeutic effect is, for example, the killing of a cancer cell.

[0095] The terms "effective amount" or "effective amount for", or "therapeutically effective amount" refer to an amount sufficient to elicit a detectable therapeutic response in the subject. Preferably, the therapeutic response is effective in reducing the proliferation of cancer cells, or inhibiting the growth of cancer cells present in a subject. Assays for determining therapeutic responses are well known in the art.

[0096] The term "immunoconjugate" refers to a composition comprising an antibody linked to a second molecule, such as a detectable tag or effector molecule. Often, the antibody is linked to the second molecule by covalent bonding.

[0097] In the context of an immunoconjugate, a "detectable tag" or "detectable fraction" refers to a portion of the immunoconjugate that has a property, making its presence detectable. For example, the immunoconjugate can be labeled with a radioactive isotope that allows cells in which the immunoconjugate is present to be detected in immunohistochemical assays. A "detectable label", or a "detectable fraction", is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include radioisotopes (eg, 3H, 35S, 32P, 51Cr, or 125I), fluorescent dyes, electron-dense reagents, enzymes (eg, alkaline phosphatase, horseradish peroxidase, or others commonly used in a ELISA), biotin, dioxygenin, or haptens and proteins that can be made detectable, for example, by incorporating a radiolabel into the peptide, or used to detect antibodies specifically reactive with the peptide.

[0098] The term "immunologically reactive conditions" includes reference to conditions that allow an antibody generated on a particular epitope to bind to that epitope to a detectably greater degree than, and/or to the substantial exclusion of, binding to substantially all others epitopes. Immunologically reactive conditions are dependent on the format of the antibody binding reaction and typically are those used in immunoassay protocols, or those conditions encountered in vivo. See Harlow & Lane, supra, for a description of immunoassay formats and conditions. Preferably, the immunologically reactive conditions employed in the methods of the present invention are "physiological conditions" which include reference to conditions (e.g., temperature, osmolarity, pH) that are typical within a living mammal or mammalian cell. While it is recognized that some organs are subjected to extreme conditions, the intraorganism and intracellular environment is normally around pH 7 (i.e. pH 6.0 to pH 8.0, more typically pH 6.5 to 7, 5), contains water as the predominant solvent, and exists at a temperature above 0 degrees C and below 50 degrees C. The osmolarity is within the range that is favorable for cell viability and proliferation.

[0099] The term "specifically binds", "binding specificity", "specifically binds to an antibody", or "specifically immunoreactive with", when referring to an epitope, refers to a binding reaction that is determinative of the presence of the epitope in a heterogeneous population of proteins and other biologicals. Thus, under designated immunoassay conditions, the specified antibodies bind a particular epitope at least twice the background, and more typically, more than 10 to 100 times the background. A variety of immunoassay formats can be used to select for antibodies specifically immunoreactive with a particular protein or carbohydrate. For example, solid phase ELISA immunoassays are routinely used to screen for antibodies specifically immunoreactive with a protein or carbohydrate. See, Harlow & Lane, ANTIBODOIES, A LABORATORY MANUAL, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, USING ANTIBODOIES, A LABORATORY MANUAL, Cold Spring Harbor Press, New York (1999), for a description of formats immunoassay methods and conditions that can be used to determine specific immunoreactivity. As used herein, "specifically binds" means that an antibody binds to a protein with a Kd of at least about 0.1 mM, at least about 1 µM, at least about 0.1 µM, or better or 0.01 µM or better.

[00100] "Nucleic acid" and "polynucleotide" are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof, in either single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs, or modified support residues or linkages, that are synthetic, that are naturally and non-naturally occurring, that have similar binding properties as the reference nucleic acid, and that are metabolized into in a similar way to reference nucleotides. Examples of such analogues include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs). As appreciated by one skilled in the art, the complement of one nucleic acid sequence can readily be determined from the sequence of the other strand. Thus, any particular nucleic acid sequence placed here also reveals the complementary strand.

[00101] "Polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid.

[00102] "Amino acid" refers to naturally occurring amino acids, and synthetic amino acids, as well as amino acid analogues and amino acid mimetics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. "Amino acid analogues" refer to compounds that have the same fundamental chemical structure as a naturally occurring amino acid, that is, an alpha carbon that is bonded to a hydrogen, a carboxyl group, an amino group, and an R group, for example, homoserine, norleucine, methionine sulfoxide, methionine methyl sulphonium. Such analogues have modified R groups (eg, norleucine), or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetics" refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but which functions in a similar manner to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature.

[00103] "Conservatively modified variants" apply to both nucleic acid sequences and amino acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical, or essentially identical, amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, essentially identical sequences. Due to the degeneracy of the genetic code, large numbers of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, in every position where an alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations", which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One skilled in the art will recognize that every codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan), can be modified to produce a functionally identical molecule. Consequently, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

[00104] With regard to amino acid sequences, a person skilled in the art will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence, which alter, add, or delete a single amino acid, or a small percentage of amino acids in the encoded sequence, is a "conservatively modified variant", where the alteration results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables that provide for functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the invention.

[00105] For example, substitutions can be made in which an aliphatic amino acid (G, A, I, L, or V) is replaced with another member of the group, or substitution such as replacing one polar residue with another, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine. Each of the following eight groups contains other exemplary amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, for example, Creighton, Proteins (1984)).

[00106] Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, for example, Alberts et al., Molecular Biology of the Cell (3rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I. The Conformation of Biological Macromolecules (1980). "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to locally ordered three-dimensional structures within a polypeptide. "Tertiary structure" refers to the complete three-dimensional structure of a polypeptide monomer. Domains are portions of a polypeptide that form a compact unit of the polypeptide, and are typically 50 to 350 amino acids long. Typical domains are composed of smaller organizing sections, such as β-sheet stretches and a-helices. "Quaternary structure" refers to the three-dimensional structure formed by non-covalent association of independent tertiary units.

[00107] The terms "isolated" or "substantially purified", when applied to a nucleic acid or protein, denote that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state, although it may be in solution, or dry, or aqueous. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis, or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[00108] The terms "identical" or "percent identity", in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same, or have a specified percentage of residues from amino acid or nucleotides that are the same (i.e. about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum match over a designated comparison window or region), as measured using the string comparison algorithms BLAST or BLAST 2.0 with fault parameters described below, or by manual alignment and visual inspection. Such sequences are then referred to as being "substantially identical". This definition also refers to, or may apply to, the fulfillment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described above, preferred algorithms can rely on slack and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or, more preferably, over a region that is 50-100 amino acids or nucleotides in length.

[00109] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, fault program parameters can be used, or alternative parameters can be assigned. The sequence comparison algorithm then calculates the percentage of sequence identities for the test sequences relative to the reference sequences, based on program parameters.

[00110] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, more usually about from 100 to about 150, where a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local alignment algorithm of Smith & Waterman, Adv. app. Math. 2:482 (1981), by the global alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), for research into the similarity method of Pearson & Lipman, Proc. Nat'l. academic Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)). Smith & Waterman alignment with failure parameters are often used when comparing sequences as described herein.

[00111] Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403410 (1990), respectively. BLAST and BLAST 2.0 are used, typically with the default parameters, to determine percent sequence identity for nucleic acids and proteins of the invention. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high ranking sequence pairs (HSPs) by identifying short words of length W in the search string, which either match or satisfy some threshold ranking of positive value T when aligned with a word of the same length in a database string. T is referred to as the neighbor word ranking threshold. These initial neighborhood word hits act as initial seeds for searchers to find longer HSPs containing them. The word hits are extended in both directions along each sequence so that the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a ranking matrix is used to calculate the cumulative ranking. Word length in each direction stops when: the cumulative alignment score decreases by the amount X from its maximum achieved value; the cumulative score goes to zero or below due to the accumulation of one or more negative ranking residual alignments; or the end of your sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both braided. For amino acid (protein) sequences, the BLASTP program defaults to a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 sorting matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad.Sci.USA 89:10915)). For the purpose of this invention, the BLAST2.0 algorithm is used with the fault parameters.

[00112] A "phage description library" refers to a "library" of bacteriophages on the surface of which exogenous peptides or proteins are expressed. Foreign peptides or polypeptides are revealed on the outer surface of the phage capsid. The foreign peptide can be revealed as recombinant fusion proteins incorporated as part of a phage coat protein, as recombinant fusion proteins which are not normally phage coat proteins but which are capable of becoming incorporated on the outer surface of the phage. capsid, or as proteins or peptides that become attached, covalently or not, to such proteins. This is accomplished by inserting an exogenous nucleic acid sequence into a nucleic acid that can be packaged into phage particles. Such exogenous nucleic acid sequences can be inserted, for example, into the coding sequence of a phage coat protein gene. If the foreign sequence is cloned into the framework, the protein it encodes will be expressed as part of the coat protein. In this way, libraries of nucleic acid sequences, such as those of an antibody repertoire produced from the gene segments encoding the entire B cell repertoire of one or more individuals, can be inserted into phages in this way to create "phage libraries". As peptides and proteins representative of those encoded by the nucleic acid library are displayed by the phage, a "peptide description library" is generated. While a variety of bacteriophage are used in such library constructs, typically, filamentous phage are used (Dunn (1996) Curr. Opin. Biotechnol. 7:547-553). See, for example, description of phage description libraries, below.

[00113] The term "chimeric antibodies" refers to antibodies in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light chains and heavy chains correspond to the variable region of antibodies derived from a mammalian species (e.g., mouse, rat, rabbit, and the like) with the desired specificity, affinity, and ability, while the constant regions are homologous to sequences in mutually derived antibodies (usually human) to avoid inducing an immune response in that species.

[00114] The term "epitope", or "antigenic determinant", or "antigen determination region", are used interchangeably herein, and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed of both contiguous and non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained after protein denaturation, whereas epitopes formed by tertiary folding are typically lost after protein denaturation. An epitope typically includes at least 3, and more usually at least 5 or 8-10 amino acids in a unique spatial conformation. An antigenic determinant can compete with intact antigen (ie, the "immunogen" used to elicit the immune response) for binding to an antibody.

[00115] Competition between antibodies is determined by an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen. Numerous types of competitive binding assays are known, for example: direct or indirect solid phase radioimmunoassay (RIA), direct or indirect solid phase enzyme immunoassay (EIA), interleaved competition assay (see Stahli et al., Methods in Enzymology 9:242-253 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619 (1986)); solid phase direct labeled assay, solid phase direct interleaved assay (see Harlow and Lane, "Antibodies, A Laboratory Manual," Cold Spring Harbor Press (1988)); Solid phase direct tag RIA using 1-125 tags (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solid phase direct biotin-avidin EIA ( Cheung et al., Virology 176:546-552 (1990 )); and direct-labeled RIA ( Moldenhauer et al., Scand. J. Immunol. 32:77-82 (1990 )). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin, and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to a solid surface, or cells in the presence of the test immunoglobulin. Usually, the test immunoglobulin is present in excess. Antibodies identified by competition assay (competing antibodies) include binding of antibodies to the same epitope as the reference antibody, and binding of antibodies to an adjacent epitope sufficiently close to the epitope bound by the reference antibody for steric hindrance to occur. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50 or 75%.

[00116] The antibodies of the present invention, for example, VH polypeptides, VL polypeptides, or single chain antibodies, can be generated using routine techniques in the field of recombinant genetics. Basic texts revealing the general methods in this invention include Sambrook & Russell, Molecular Cloning, A Laboratory Manual (3d ed. 2001) and Current Protocols in Molecular Biology (Ausubel et al., eds., 1999).

[00117] The humanized antibody of the invention can be generated by grafting the specificity, i.e. the antigen-binding loops, of a donor antibody, typically a murine antibody, to a human framework. The framework regions of the human light chains and heavy chains provided herein can readily be determined by the practitioner. The position numbers of the heavy and light chains are assigned according to common numbering schemes, for example the numbering scheme of Kabat and Chothia. Chothia's number scheme is identical to Kabat's scheme, but places the insertions in CDR-L1 and CDR-H1 in structurally different positions. Unless otherwise noted, Kabat's numbering scheme is used herein in reference to sequence positions. The position of an amino acid residue in a particular VH or VL sequence does not refer to the number of amino acids in a particular sequence, but rather refers to the position as designated with reference to a numbering scheme.

[00118] The positions of the CDRs and, consequently, the positions of the framework regions of human heavy chains and light chains are determined using definitions that are standards in the field. For example, the following four definitions are commonly used. Kabat's definition is based on sequence variability, and is the most commonly used. Chothia's definition is based on the location of the structural loop regions. The AbM definition is a compromise between the two AbM antibody modeling software used by Oxford Molecular. The contact definition was recently introduced, and is based on an analysis of the complex crystal structures available. The following are the loop positions, ie CDRs, using the four different definitions.

[00119] A VH or VL sequence of the invention comprises a heavy chain or light chain that typically has at least 70% identity, more typically 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identity to a sequence comprised by SEQ ID NOs: 5-22, 29-57, 91-100 or 107-116.

[00120] Furthermore, a number of important residues have been identified outside of the OS2966 CDRs that are preferably used in the humanized VH and VL chains. For example, in one embodiment, one or more amino acid residues in the VH chain are identical to those of OS2966 (SEQ ID NO:2), including residues 48, 67, 69, 73, 76, 80, 89, 91 and 93. In one embodiment, one or more amino acid residues in the VL chain are identical to those of OS2966 (SEQ ID NO:4), including residues 36 and 71.

[00121] A humanized antibody of the invention binds to the same epitope as the donor antibody, for example, binds to the same β1 integrin epitope, or competes for binding to the same β1 integrin epitope, which OS2966 binds to, for example. Methods for determining whether the antibody binds to the same epitope are well known in the art, see, for example, Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which discloses techniques for epitope mapping or, alternatively, competition experiments, to determine whether an antibody binds the same epitope as the donor antibody.

[00122] A stable humanized antibody of the invention may exhibit altered affinity when compared to the donor antibody. For example, in some embodiments, the affinity of a humanized single chain anti-β1 integrin, for example, may be decreased compared to a single chain antibody comprising the VH and VL regions of OS2966. Such a decrease can be by more than 10-fold in comparison, but typically a humanized antibody of the invention has an affinity that is at least 25%, more often at least 50%, of that of the comparable wild-type antibody. (A "comparable wild-type antibody" refers to an antibody of the same embodiment, e.g., scFv, which comprises the VH and VL regions of the donor antibody). In some embodiments, the affinity for the epitope is increased, such that a humanized antibody of the invention has an affinity that is 2 times, and sometimes 5, 10, 50, or 100 times the affinity of the comparable wild-type antibody.

[00123] The heavy and light chain regions of the invention are typically obtained using recombinant DNA technology. The recombinant DNA methodologies that are commonly employed to accomplish this are well known to those skilled in the art. Typically, nucleic acid sequences encoding the donor antibody frameworks and CDRs are generated by PCR, for example by overlap extension. In this technique, donor antibody antigen-binding sequences are typically joined to human framework regions by incorporating the desired sequences into oligonucleotides, and creating a series of products using PCR that comprise the desired human and donor sequences. The products can then be joined together, typically using additional PCR reactions, in the correct orientation, to create VH and VL chains comprising human framework regions with donor antibody CDRs. The VL and VH DNA sequences can be linked together, either directly, or through a DNA sequence encoding a peptide linker, using techniques well known to those skilled in the art. These techniques include PCR as well as techniques such as in vitro ligation. The VL and VH sequences can be joined in any orientation.

[00124] Examples of techniques sufficient to direct those skilled in the art through in vitro amplification methods are well known in the art.

[00125] Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automatic synthesizer as described in Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).

[00126] The sequence of cloned genes and synthetic oligonucleotides can be verified after cloning using, for example, sequencing.

[00127] The PCR products are subcloned into suitable cloning vectors that are well known to those skilled in the art, and commercially available. The nucleotide sequence of the heavy or light chain coding regions is then determined.

[00128] One skilled in the art will appreciate that, using the sequence information provided for the variable regions, nucleic acids encoding these sequences are obtained using any number of additional methods well known to those skilled in the art. Thus, the DNA encoding the Fev. is prepared by any suitable method, including, for example, other amplification techniques, such as ligase chain reaction (LCR), transcription amplification, and self-sustaining sequence replication, or cloning and restriction of appropriate sequences.

[00129] The nucleic acids encoding the antibodies and antibody fragments of the invention can also be generated by direct chemical synthesis using methods such as the phosphotriester method; the phosphodiester method; the diethylphosphoramidite method; and the solid support method of US Patent No. 4,458,066. If the DNA sequence is chemically synthesized, a single stranded oligonucleotide will result. This can be converted to double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. While it is possible to chemically synthesize an entire single-stranded region of Fv, it is preferable to synthesize a number of shorter sequences (about 100 to 150 bases) that are typically later spliced together, for example using overlapping extension PCR.

[00130] For nucleic acids, sizes are given in kilobases (kb), or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa), or acid residue numbers. Protein sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from derived protein sequences.

[00131] The VH and VL domains of an antibody of the invention can be directly linked, or can be separated by a linker, for example, to stabilize the antibody variable domains of the light chain and heavy chain, respectively. Suitable linkers are well known to those skilled in the art, and include the well known GlyGlyGlyGlySer linker (SEQ ID NO:122), or a variant thereof. For example, a typical linker is (Gly4Ser)3 (SEQ ID NO:123). Other linkers, including hinge regions, that can be used in the invention include those described, for example, in Alfthan et al., Protein Eng. 8(7), 725-31; Choi et al., Eur. J Immunol. 31(1), 94-106; Hu et al, Cancer Res. 56(13), 3055-61; Kipriyanov, et al., Protein Eng. 10(4), 445-53; Pack, et al., Biotechnology (NY) 11(11), 1271-7; and Roovers, et al., Cancer Immunol. Immunother. 50(l):51-9.

[00132] To obtain high-level expression of a cloned gene or nucleic acid, such as those cDNAs encoding the humanized antibody, for example, a humanized antibody of the invention, or an immunoconjugate or chimeric antibody comprising a humanized antibody of the invention, typically subclones a nucleic acid encoding the antibody or immunoconjugate into an expression vector that contains an appropriate promoter to direct transcription, a transcription/translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for initiation translational. Suitable bacterial promoters are well known in the art, and described, for example, in Sambrook et al. and Ausubel et al. Bacterial expression systems for protein expression are available in, for example, E. coli, Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235 (1983); Mosbach et al., Nature 302:543 -545 (1983) Kits for such expression systems are commercially available Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art, and are also commercially available.

[00133] Often, in order to express a protein at high levels in a cell, the codon preference for the expression system is considered in constructing the nucleic acid sequence to be expressed. In this way, a nucleic acid from an organism, for example a human or mouse, can be designed to accommodate the codon preference of the expression system.

[00134] The promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter is optionally positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.

[00135] In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all of the additional elements required for expression of the nucleic acid-encoding protein in host cells. A typical expression cassette, therefore, contains a promoter operably linked to the nucleic acid sequence encoding the protein to be expressed, and signals required for efficient transcriptional polyadenylation, ribosome binding sites, and translation termination. The nucleic acid sequence encoding a protein can typically be linked to a cleavable signal peptide sequence to promote secretion of the encoded protein by the transformed cell. Such signal peptides would include, among others, the signal peptides of tissue plasminogen activator, insulin, and neuron growth factor, and juvenile hormone esterase from Heliothis virescens. Additional cassette elements may include enhancers and, if genomic DNA is used as the structural gene, functionally spliced donor introns and acceptor sites.

[00136] In addition to a promoter sequence, the expression cassette must also contain a transcription termination region downstream of the structural gene to provide efficient termination. The termination region can be obtained from the same gene as the promoter sequence, or it can be obtained from different genes.

[00137] Expression control sequences that are suitable for use in a particular host cell are often obtained by cloning a gene that is expressed in that cell. Commonly used prokaryotic control sequences, which are defined herein to include promoters for initiating transcription, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase ) and lactose (lac) (Change et al., Nature (1977) 198: 1056), the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. (1980) 8: 4057), the tac (DeBoer, et al., Proc. Natl. Acad. Sci. U.S.A. (1983) 80:21-25); and the lambda-derived P.sub.L promoter and N-gene ribosome binding site (Shimatake et al., Nature (1981) 292: 128). The particular promoter system is not critical to the invention, any available promoter that functions in prokaryotes can be used.

[00138] Standard bacterial expression vectors include plasmids, such as pBR322-based plasmids, for example, pBLUESCRIPTTM, pSKF, pET23D, vectors derived from phage-.lamda., and fusion expression systems, such as GST and LacZ . Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, for example, c-myc, HA-tag, 6-His tag (SEQ ID NO:124), maltose binding protein, VSV-G tag, anti-DYKDDDDK tag (SEQ ID NO:125), or any such tag, most of which are well known to those skilled in the art.

[00139] Eukaryotic expression systems for mammalian cells, yeast, and insect cells, are well known in the art, and are also commercially available. In yeast, vectors include Yeast Integrating plasmids (eg, YIp5) and Yeast Replicating plasmids (the YRp-series plasmids) and pGPD-2. Expression vectors containing eukaryotic virus regulatory elements are typically used in eukaryotic expression vectors, for example, SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr viruses. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 promoter, metallothionein, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[00140] Some expression systems have markers that provide gene amplification, such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. Alternatively, high-throughput expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a sequence encoding GPCR under the direction of the polyhedrin promoter, or other baculovirus promoters. strong.

[00141] Elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene that encodes antibiotic resistance to allow selection of bacteria that harbor recombinant plasmids, and unique restriction sites in non- essential parts of the plasmid, to allow insertion of eukaryotic sequences. The particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable. Prokaryotic sequences are optionally chosen such that they do not interfere with DNA replication in eukaryotic cells, if necessary.

[00142] Standard transfection methods are used to produce bacterial, mammalian, yeast, or insect cell lines that express large amounts of protein, which are then purified using standard techniques (see, for example, Colley et al., J. Biol.Chem., 264:17619-17622 (1989), Guide to Protein Purification, in Methods in Enzymology, vol.182 (Deutscher, ed., 1990)). Transformations of eukaryotic and prokaryotic cells are performed according to standard techniques (see, for example, Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 ( Wu et al., eds, 1983).

[00143] Any of the well-known procedures for introducing foreign nucleotide sequences into host cells can be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors, and any of the well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material in a host cell (see, for example, Sambrook and Russell, supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing a polypeptide of the invention.

[00144] After the expression vector is introduced into the cells, the transfected cells are cultured under conditions that favor expression of the protein, which is recovered from the culture using standard techniques identified below.

[00145] One skilled in the art would recognize that modifications can be made to a nucleic acid encoding a polypeptide of the present invention (i.e., an antibody, a tag, or effector, or an immunoconjugate formed using the antibody) without diminishing its biological activity . Some modifications can be made to facilitate cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those skilled in the art and include, for example, stop codons, a methionine added at the amino terminus to provide additional initiation site amino acids placed at either terminus to create conveniently located restriction sites, or additional amino acids (such as a poly His) to aid in purification steps.

[00146] Once expressed, the recombinant antibodies, immunoconjugates, and/or effector molecules of the present invention, can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, R. Scopes, PROTEIN PURIFICATION, Springer-Verlag, N.Y. (1982)). Substantially pure compositions of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity is more preferred for pharmaceutical uses. Once purified, partially or to homogeneity as desired, if to be used therapeutically, polypeptides must be substantially free of endotoxin.

[00147] Methods for expressing single-chain antibodies and/or refolding to an appropriate active form, including single-chain antibodies, from bacteria, such as E. coli, have been described, and are well known and are applicable to the antibodies of this invention . See, Buchner, et al., Anal Biochem. 205:263-270 (1992); Pluckthun, Biotechnology 9:545 (1991); Huse, et al., Science 246:1275 (1989) and Ward, et al., Nature 341:544 (1989), all incorporated by reference herein.

[00148] Often, functional heterologous proteins from E. coli, or other bacteria, are isolated from inclusion bodies, and require solubilization using strong denaturants, and subsequent refolding. During solubilization, as is well known in the art, a reducing agent must be present to separate disulfide bonds. An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena, et al., Biochemistry 9: 5015-5021 (1970), incorporated by reference herein, and especially as described by Buchner, et al., supra.

[00149] Renaturation is typically accomplished by diluting (eg, 100-fold) the denatured and reduced protein in refolding buffer. An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.

[00150] As a modification to the two-chain antibody purification protocol, the heavy and light chain regions are separately solubilized and reduced, and then combined in the refolding solution. A preferred yield is obtained when these two proteins are mixed in a molar ratio such that a 5-fold molar excess of one protein over the other is not exceeded. It is desirable to add oxidized glutathione or other low molecular weight oxidation compounds to the refolding solution after the redox mixing is complete.

[00151] In addition to recombinant methods, the antibodies and immunoconjugates of the invention can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of polypeptides of the present invention of less than about 50 amino acids in length can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany & Merrifield, THE PEPTIDES: ANALYSIS, SYNTHESIS, BIOLOGY. VOL. 2: SPECIAL METHODS IN PEPTIDESYNTHESIS, PART A. pp. 3-284; Merrifield, et al. J. Am. chem. Soc. 85:2149-2156 (1963), and Stewart, et al., SOLID PHASE PEPTIDESYNTHESIS, 2ND ED., Pierce Chem. Co., Rockford, Ill. (1984). Longer length proteins can be synthesized by condensing the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activating a carboxyl terminus (for example, by using the N,N'-dicylohexylcarbodiimide coupling reagent) are known to those skilled in the art.

[00152] Conservatively modified variants of antibodies of the present invention have at least 80% sequence similarity, often at least 85% sequence similarity, 90% sequence similarity, or at least 95%, 96%, 97%, 98%, or 99% sequence similarity, at the amino acid level, with the protein of interest, such as a humanized antibody of the invention.

[00153] As noted, the term "conservatively modified variants" can be applied to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acid sequences that encode identical, or essentially identical, amino acid sequences, or if the nucleic acid does not encode an amino acid sequence, essentially identical nucleic acid sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, in every position where an alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations", which are a kind of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One skilled in the art will recognize that every codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to produce a functionally identical molecule. Consequently, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

[00154] As for amino acid sequences, one skilled in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence, which alter, add, or delete a single amino acid, or a small percentage of amino acids in the encoded sequence, is a "conservatively modified variant" where the alteration results in the replacement of an amino acid with a chemically similar amino acid.

[00155] One embodiment of the present invention provides an immunoconjugate comprising a humanized antibody of the invention, linked to an effector molecule, or detectable tag. Preferably, the effector molecule is a therapeutic molecule, such as, for example, a toxin, a chemotherapeutic agent, a small molecule, a cytokine, or a chemokine, an enzyme, or a radiolabel. Exemplary toxins include, but are not limited to, Pseudomonas exotoxin or diphtheria toxin. Suitable toxins are described in, for example, Chaudhary, et al. (1987) Proc Natl Acad Sci US A 84:4538, Chaudhary, et al. (1989) Nature 339:394, Batra, et al. (1991) Mol Cell Biol 11:2200. Brinkmann, et al. (1991) Proc Natl Acad Sci USA 88:8616, Siegall, (1995) Semin Cancer Biol 6:289. Examples of small molecules include, but are not limited to, chemotherapeutic compounds, such as taxol, doxorubicin, etoposide, and bleiomycin. Exemplary cytokines include, but are not limited to, IL-1, IL-2, IL-4, IL-5, IL-6, and IL-12. Suitable cytokines and chemokines are described in, for example, Rosenblum et al. (2000) Int J Cancer 88:267 and Xu et al. (2000) Cancer Res 60:4475 and Biragyn et al. (1999) Nat Biotechnol 17:253. Suitable enzymes include, but are not limited to, RNAses, DNases, proteases, kinases, and caspases. Suitable proteases are described in, for example, Bosslet et al. (1992) Br J Cancer 65:234, Goshom et al. (1993) Cancer Res 53:2123, Rodrigues et al. (1995) Cancer Res 55:63, Michael et al. (1996) Immunotechnology 2:47, Haisma et al. (1998) Blood 92:184. Exemplary radioisotopes include, but are not limited to, 32P and 125I. Suitable radionuclides are also described in, for example, Colcher et al. (1999) Ann NY Acad Sci 880:263. Additional exemplary effector fractions are, for example, Fc fragments from homologous or heterologous antibodies.

[00156] It will be appreciated by those skilled in the art that the sequence of any effector protein molecule can be altered in a manner that does not substantially affect the functional advantages of the effector protein. For example, glycine and alanine are typically considered to be interchangeable as are aspartic acid and glutamic acid and asparagine and glutamine. One skilled in the art will recognize that many different variations of effector sequences will encode effectors with roughly the same activity as the native effector.

[00157] The effector molecule and the antibody can be conjugated by chemical means, or by recombinant means, as described above. Chemical modifications include, for example, derivation to the proposed binding of the effector molecule and the antibody to each other, either directly, or through the binding compound, by methods that are well known in the art of protein chemistry. Both covalent and non-covalent attachment means can be used with the humanized antibody of the present invention.

[00158] The procedure for attaching an effector molecule to an antibody will vary according to the chemical structure of the fraction to be attached to the antibody. Polypeptides typically contain a variety of functional groups; for example, carboxylic acid (COOH), free amine (--NH.sub.2), or sulfhydryl (--SH) groups, which are available for reaction with a suitable functional group on an antibody to result in binding of the effector molecule .

[00159] Alternatively, the antibody is derivatized to expose or to attach additional reactive functional groups. Derivatization can involve attachment of any of a number of linker molecules, such as those available from the Pierce Chemical Company, Rockford Ill.

[00160] The linker is capable of forming covalent bonds to both the antibody and to the effector molecule. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight or branched chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where both the antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids through side groups (eg, through a disulphide bond to cysteine). However, in a preferred embodiment, the linkers will be attached to the alpha carbon amino and carboxyl groups of the terminal amino acids.

[00161] In some circumstances, it is desirable to rid the effector molecule of the antibody when the immunoconjugate has reached its target site. Therefore, under these circumstances, the immunoconjugates will comprise linkages that are cleavable in the vicinity of the target site. Cleavage of the linker to release the antibody effector molecule can be promoted by enzymatic activity, or conditions to which the immunoconjugate is subjected, either within the target cell, or in the vicinity of the target site. When the target site is a tumor, a linker that is cleavable under conditions present at the tumor site (eg, when exposed to tumor-associated enzymes or acidic pH) can be used.

[00162] In the currently preferred chemical conjugation embodiment, the means for linking the effector molecule and the antibody comprises a hetero-bifunctional coupling reagent which finally contributes to the formation of an intermolecular disulfide bond between the effector molecule and the antibody. Other types of coupling reagents that are useful in this capacity for the present invention are described, for example, in U.S. Patent No. 4,545,985. Alternatively, an intermolecular disulfide can conveniently be formed between cysteines on the effector molecule and the antibody that are naturally occurring, or are inserted by genetic engineering. The means for linking the effector molecule and the antibody can also use thioether linkages between hetero-bifunctional crosslinking reagents, or specific low pH cleavable crosslinkers, or specific cleavable protease linkers, or other cleavable or non-cleavable chemical linkers. The means for linking the effector molecule and the antibody may also comprise a peptidyl linkage formed between the effector molecule and the antibody that are separately synthesized by standard peptide synthetic chemistry or recombinant means.

[00163] Exemplary chemical modifications of the effector molecule and the antibody of the present invention also include derivatization with polyethylene glycol (PEG) for prolonged residence time in the circulatory system and reduction of immunogenicity, according to well known methods (See, for example, Lisi , et al., Applied Biochem., 4:19 (1982); Beauchamp, et al., Anal Biochem., 131:25 (1982); and Goodson, et al., Bio/Technology 8:343 (1990)).

[00164] The antibodies of the present invention may optionally be covalently or non-covalently linked to a detectable label. Detectable labels suitable for such use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Useful tags of the present invention include magnetic beads (e.g., DYNABEADS), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like), radio tags (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (eg, horseradish peroxidase, alkaline phosphatase, and others commonly used in an ELISA), and colorimetric labels, such as colloidal gold or glass or colored plastic beads (eg, polystyrene, polypropylene, latex , etc.).

[00165] Means of detecting such tags are well known to those skilled in the art. Thus, for example, radiolabels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate, and detecting the reaction product produced by the enzyme's action on the substrate, and colorimetric labels are detected by simply viewing the colored label.

[00166] The antibody and/or immunoconjugate compositions of this invention are particularly useful for parenteral administration, such as intravenous administration, or administration into a body cavity.

[00167] Compositions for administration will commonly comprise a solution of the antibody and/or immunoconjugate dissolved in a pharmaceutically acceptable vehicle, preferably an aqueous vehicle. A variety of aqueous vehicles can be used, for example, buffered saline, and the like. These solutions are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required for approximate physiological conditions, such as pH adjusting agents and buffering agents, toxicity adjusting agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride , calcium chloride, sodium lactate, and the like. The concentration of fusion protein in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the needs of the patient.

[00168] Thus, a typical pharmaceutical composition of the present invention for intravenous administration would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 to about 100 mg per patient per day can be used. Current methods for preparing manageable compositions will be known or apparent to those skilled in the art, and are described in more detail in such publications as REMINGTON'S PHARMACEUTICAL SCIENCE, 19TH ED., Mack Publishing Company, Easton, Pa. (1995).

[00169] The compositions of the present invention can be administered for therapeutic treatments. In therapeutic applications, the compositions are administered to a patient suffering from a disease, in an amount sufficient to cure or at least partially arrest the disease and its complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose". Effective amounts for this use will depend on the severity of the illness and the general health of the patient. An effective amount of the compound is that which provides either subjective relief of a symptom(s) or objectively identifiable improvement as noted by the clinician or other qualified observer.

[00170] Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition must provide a sufficient amount of the proteins of this invention to effectively treat the patient. Preferably, the dosage is administered once, but may be applied periodically until a therapeutic result is achieved, or until side effects warrant discontinuation of therapy. Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.

[00171] Controlled-release parenteral formulations of the immunoconjugate compositions of the present invention can be produced as implants, oily injections, or as particulate systems. For a broader overview of protein delivery systems, see, Banga, A.J., THERAPEUTIC PEPTIDES AND PROTEINS: FORMULATION, PROCESSING, AND DELIVERY SYSTEMS, Technomic Publishing Company, Inc., Lancaster, Pa., (1995), incorporated herein by reference. Particulate systems include microbeads, microparticles, microcapsules, nanocapsules, nanobeads, and nanoparticles. The microcapsules contain the therapeutic protein as a central core. In microbeads, the therapeutic is dispersed throughout the particle. Particles, microbeads, and microcapsules smaller than about 1 µm are generally referred to as nanoparticles, nanobeads, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 µm, so only nanoparticles are administered intravenously. Microparticles are typically around 100 µm in diameter, and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., COLLOIDAL DRUG DELIVERY SYSTEMS, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp. 219-342 (1994); and Tice & Tabibi, TREATISE ON CONTROLLED DRUG DELIVERY, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp.315-339, (1992), both of which are incorporated herein by reference.

[00172] The polymers can be used for controlled ion release from immunoconjugate compositions of the present invention. Various degradable and non-degradable polymeric matrices for use in controlled drug release are known in the art (Langer, R., Accounts Chem. Res. 26:537-542 (1993)). For example, the block copolymer, polyxamer 407, exists as a viscous liquid that is still mobile at low temperatures, but forms a semi-solid gel at body temperature. It has been shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston, et al., Pharm. Res. 9:425-434 (1992); and Pec, et al., J. Parent .Sci.Tech.44(2):58-65 (1990)). Alternatively, hydroxyapatite has been used as a microcarrier for controlled protein release ( Ijntema, et al., Int. J. Pharm. 112:215-224 (1994 )). In yet another aspect, liposomes are used for controlled release as well as drug targeting of lipid-encapsulated drugs (Betageri, et al., LIPOSOME DRUG DELIVERY SYSTEMS, Technomic Publishing Co., Inc., Lancaster, Pa. (1993)) . Numerous additional systems for controlled delivery of therapeutic proteins are known. See, for example, US Patent Nos. 5,055,303, 5,188,837, 4,235,871, 4,501,728, 4,837,028, 4,957,735, 5,019,369, 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206, 5,271,961; 5,254,342 and 5,534,496, each of which is incorporated herein by reference.

[00173] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells are characterized by up-regulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, benign or malignant tumors. More particular examples of such cancers include squamous cell cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma , cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, brain cancer, liver carcinoma, and various types of head and neck cancer, neurofibromatosis type I or II. Other examples of such cancers include those that are resistant to therapy, refractory, or metastatic.

[00174] Other diseases or disorders that may be treated include "inflammatory diseases or disorders." "Inflammatory disease or disorder", as used herein, include, and are not limited to, pruritis, skin inflammation, psoriasis, multiple sclerosis, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, Hashimoto's thyroid, myasthenia gravis, type I diabetes or II, asthma, inflammatory lung damage, inflammatory liver damage, inflammatory glomerular damage, atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, seborrheic dermatitis, Soerem's syndrome, keratoconjunctivitis, uveitis, inflammatory bowel disease, Crohn's disease , ulcerative colitis, an inflammatory disease of the joints, skin, or muscle, acute or chronic idiopathic inflammatory arthritis, myositis, a disease of demyelination, chronic obstructive pulmonary disease, interstitial lung disease, interstitial nephritis, and chronic active hepatitis.

[00175] "Metastasis", as used herein, refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion in the new location. A "metastatic" or "metastatic" cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures.

[00176] As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[00177] As used herein, the term "individual suspected of having cancer" refers to an individual who exhibits one or more symptoms indicative of a cancer (e.g., a noticeable lump or mass), or is being screened for a cancer ( for example, during a routine physics). An individual suspected of having cancer may also have one or more risk factors. An individual suspected of having cancer has not usually been tested for cancer. However, an "individual suspected of having cancer" involves an individual who has received an initial diagnosis, but for whom the stage of cancer is not known. The term additionally includes people who have had cancer once (eg, an individual in remission).

[00178] As used herein, the term "individual at risk for cancer" refers to an individual with one or more risk factors for developing a specific cancer. Risk factors include, but are not limited to, gender, age, genetic predisposition, environmental exposure, previous incidents of cancer, pre-existing non-cancer diseases, and lifestyle.

[00179] As used herein, the term "characterization of cancer in an individual" refers to the identification of one or more properties of a cancer specimen in an individual, including, but not limited to, the presence of precancerous tissue or benign cancer, the stage of the cancer, and the prognosis of the individual. Cancers can be characterized by identifying the expression of one or more cancer marker genes, including, but not limited to, the cancer markers disclosed herein.

[00180] As used herein, the terms "cancer cell marker(s)," "cancer cell marker(s),", "tumor cell marker(s)," or "solid tumor cell marker ", refer to a gene or genes or a protein, polypeptide, or peptide, expressed by the gene or genes whose level of expression, alone or in combination with other genes, is correlated with the presence of tumorigenic cancer cells compared to non- tumorigenic, such as β1 integrin. The correlation can relate to either increased or decreased expression of the gene (eg, increased or decreased levels of mRNA, or the peptide encoded by the gene).

[00181] As used herein, the term "detection of an increased or decreased expression relative to non-cancerous control" refers to measuring the level of expression of a gene (for example, the level of mRNA or protein) relative to the level in a sample non-cancerous control. Gene expression can be measured using any suitable method, including, but not limited to, those described herein.

[00182] As used herein, the term "detecting a change in gene expression in a cell sample in the presence of said test compound relative to the absence of said test compound", refers to measuring an altered level of expression (e.g., increased or decreased) in the presence of a test compound relative to the absence of the test compound. Gene expression can be measured using any suitable method.

[00183] As used herein, the term "instructions for using said kit to detect cancer in said subject" includes instructions for using the reagents contained in the kit for detecting and characterizing cancer in a sample from a subject.

[00184] As used herein, "provision of a diagnosis", or "diagnostic information", refers to any information that is useful in determining whether a patient has a disease or condition, and/or in classifying the disease or condition into a phenotypic category, or any category having significance with respect to the prognosis of or likely response to treatment (or treatment in general, or any particular treatment) of the disease or condition. Similarly, diagnosis refers to the provision of any type of diagnostic information, including, but not limited to, whether an individual is likely to have a condition (such as a tumor), information relating to the nature or classification of a tumor such as , a high-risk tumor, or a low-risk tumor, information related to prognosis, and/or information helpful in selecting an appropriate treatment. Treatment selection may include choosing a particular chemotherapeutic agent, or another treatment modality, such as surgery or radiation, or a choice about withholding or delivering therapy.

[00185] As used herein, the terms "providing a prognosis", "prognostic information", or "predicted information", refer to the provision of information related to the impact of the presence of cancer (e.g., as determined by diagnostic methods of the present invention) on an individual's future health (e.g., expected morbidity or mortality, likelihood of getting cancer, and risk of metastasis).

[00186] As used herein, the term "post-surgical tumor tissue" refers to cancerous tissue (eg, biopsy tissue) that has been removed from an individual (eg, during surgery).

[00187] As used herein, the term "individual diagnosed with a cancer" refers to an individual who has been tested and found to have cancerous cells. Cancer can be diagnosed using any suitable method, including, but not limited to, biopsy, x-ray, blood test, and the diagnostic methods of the present invention.

[00188] As used herein, the terms "biopsy tissue", "patient sample", "tumor sample", and "cancer sample", refer to a sample of cells, tissue or fluid that is removed from a individual for the purpose of determining whether the sample contains cancerous tissue, including cancer cells, or for determining the gene expression profile of that cancerous tissue. In some embodiments, biopsy tissue or fluid is obtained because an individual is suspected of having cancer. The biopsy tissue or fluid is then examined for the presence or absence of cancer, cancer cells, and/or cancer cell gene signature expression.

[00189] In certain embodiments of the present invention, cancer cell expression, such as pancreatic cancer, colon cancer, breast cancer, liver cancer, kidney cancer, brain cancer, GBM and the like, comprises elevated levels of β1 integrin compared to non-tumorigenic colon tumor cells. Integrins are heterodimeric extracellular matrix (ECM) cell surface proteins consisting of both an alpha chain and a beta chain with chains associating with multiple partners to form different integrins. Integrins function in cell adhesion and migration to reversibly connect cells to the extracellular matrix, or to receptors on other cells, and thus may play a critical role in cancer invasion and metastasis. Integrin-mediated adhesion also affects intracellular signaling, and may thereby regulate cell survival, proliferation, and differentiation.

[00190] Beta 1 integrin can form functional receptors with the greatest diversity of alpha integrins known, resulting in the ability to interact with a diverse range of ECM environments, and has been implicated in cancer. For example, increased beta 1 integrin signaling is associated with malignant progression of breast cancer, both clinically, and in breast cancer cell lines.

[00191] β1 integrin has been identified as directly affecting tumor growth. Specifically, treatment of tumor cells with anti-β1 integrin antibodies reduces tumor size, and inhibits metastasis.

[00192] In some embodiments, the present invention provides methods for detecting β1 integrin expression as a marker for cancer. In some embodiments, expression is measured directly (e.g., at the protein level), in some embodiments, expression is detected in tissue samples (e.g., biopsy tissue). In other embodiments, expression is detected in body fluids (e.g., including, but not limited to, plasma, serum, whole blood, mucus, and urine). The present invention additionally provides kits for detecting markers, in some embodiments, the presence of a cancer cell marker is used to provide a prognosis for an individual. The information provided is also used to direct the course of treatment. For example, if an individual is found to have a marker indicative of a solid tumor cell, additional therapies (e.g., hormone or radiation therapies) can be started at an earlier point when they are most likely to be effective (e.g., before metastasis). In addition, if an individual is found to have a tumor that is unresponsive to hormone therapy, the cost and inconvenience of such therapies can be avoided.

[00193] In embodiments, β1 integrin gene expression is detected by measuring the level of the protein. Protein expression can be detected by any suitable method. In some embodiments, proteins are detected by immunohistochemistry using the antibodies described herein.

[00194] Antibody binding is detected by techniques known in the art (e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "interleaved" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays , in situ immunoassays (e.g. using colloidal gold, enzyme or radioisotope tags, for example), Western blots, precipitation reactions, agglutination assays (e.g. gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.

[00195] In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting the binding of a secondary antibody, or reagent, to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many methods are known in the art for detecting binding in an immunoassay, and are within the scope of the present invention.

[00196] In some embodiments, an automated detection assay is used. Methods for automating immunoassays include those described in US Patents 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which is incorporated herein by reference. In some embodiments, the analysis and presentation of the results are also automatic. For example, in some embodiments, software that generates a prognosis based on the presence or absence of a series of proteins corresponding to cancer markers is used.

[00197] In other embodiments, the immunoassay described in United States Patents 5,599,677 and 5,672,480, each of which is incorporated herein by reference, can be used.

[00198] In some embodiments, a computer-based analysis program is used to translate the raw data generated by the detection assay (e.g., the presence, absence, or amount of a given marker or markers) into predicted value data to a clinician. The clinician can evaluate the predicted data using any suitable means. Thus, in some embodiments, the present invention additionally provides the benefit that the clinician, who is likely not trained in genetics or molecular biology, does not need to understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician is then able to immediately use the information to optimize the individual's care.

[00199] The present invention contemplates any method capable of receiving, processing, and transmitting information to and from laboratories that conduct the tests, information providers, medical personnel, and individuals. For example, in some embodiments of the present invention, a sample (e.g., a biopsy, or a serum or urine sample) is obtained from an individual, and submitted to an evaluation service (e.g., clinical laboratory at a medical facility). , genomic evaluation company, etc.), located anywhere in the world (for example, in a different country than the country where the individual resides, or where the information is ultimately used) to generate raw data. Where the sample comprises a tissue or other biological sample, the individual may visit a medical center to have the sample obtained and sent to the evaluation center, or individuals may collect the sample themselves, and send it directly to an evaluation center. Where the sample comprises predetermined biological information, the information can be directly sent to the assessment service by the individual (for example, an information card containing the information can be scanned by a computer, and the data transmitted to a computer at the assessment center using an electronic communication system). Once received by the assessment service, the sample is processed, and a profile is produced (eg expression data), specific to the desired diagnostic or prognostic information for the individual.

[00200] The profile data is then prepared in a format suitable for interpretation by a treatment clinic. For example, rather than providing raw expression data, the prepared format may represent a diagnosis or risk assessment for the individual, along with recommendations for particular treatment options. The data may be disclosed to the clinician by any suitable method. For example, in some embodiments, the assessment service generates a report that can be printed for the clinician (eg, at the point of care), or displayed to the clinician on a computer monitor.

[00201] In some embodiments, the information is first analyzed at the point of care, or at a regional facility. The raw data is then sent to a central processing facility for further analysis and/or conversion of the raw data into useful information for a clinician or patient. The central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis. The central processing facility can then control the destination of the data following the processing of the individual. For example, using an electronic communication system, the central facility can provide data to the clinician, individual, or researchers.

[00202] In some embodiments, the individual is able to directly access the data using the electronic communication system. The individual may additionally choose outcome-based intervention or counseling. In some embodiments, the data is used for research purposes. For example, the data can be used to further optimize the inclusion or elimination of markers as useful indicators of a particular condition or disease stage.

[00203] In still other embodiments, the present invention provides kits for the detection and characterization of cancer. In some embodiments, kits contain antibodies specific to a cancer marker, such as antibodies of the present invention, in addition to detection reagents and buffers. In other embodiments, kits contain specific reagents for detecting mRNA or cDNA (e.g., probes or oligonucleotide primers). In some embodiments, kits contain all necessary and/or sufficient components to perform a detection assay, including all controls, directions for performing assays, and any software necessary for analysis and presentation of results.

[00204] Another embodiment of the present invention comprises a kit for testing the presence of proteins, such as β1 integrin, for example, in a tissue sample, or in a body fluid. The kit may comprise, for example, an antibody for detecting a polypeptide. In addition, the kit may comprise a reference sample or control sample; instructions for processing the samples, performing the test and interpreting the results; and buffers and other reagents needed to perform the test.

[00205] In some embodiments, in vivo imaging techniques are used to visualize the expression of cancer markers in an animal (e.g., a human mammal or non-human mammal). For example, in some embodiments, the β1 integrin is labeled using a labeled antibody of the present invention. A specifically bound antibody and labeled antibody can be detected in an individual using an in vivo imaging method, including, but not limited to, radionuclide imaging, positron emission tomography, computed tomography imaging, X-ray or magnetic resonance, fluorescence detection, and cheluminescent detection.

[00206] The in vivo imaging methods of the present invention are useful in diagnosing cancers that express solid tumor cell cancer markers of the present invention (e.g., in breast cancer). In vivo imaging is used to visualize the presence of a marker indicative of cancer. Such techniques allow diagnosis without the use of an unpleasant biopsy. The in vivo imaging methods of the present invention are also useful for providing prognosis for cancer patients. For example, the presence of a marker indicative of cancer cells can be detected. The in vivo imaging methods of the present invention can additionally be used to detect metastatic cancers elsewhere in the body.

[00207] In some embodiments, reagents (e.g., antibodies) specific to the cancer markers of the present invention are fluorescently labeled. The labeled antibodies are introduced into an individual (eg, orally or parenterally). Fluorescently labeled antibodies are detected using any suitable method (for example, using the apparatus described in US Patent 6,198,107, incorporated herein by reference).

[00208] In other embodiments, the antibodies are radioactively labeled. The use of antibodies for in vivo diagnosis is well known in the art.

[00209] In some embodiments, the present invention provides therapies for diseases and disorders, including immune diseases/inflammatory diseases and disorders, given the role of β1 integrin in broader functional activities, as discussed above. Additionally, diseases and disorders that can be targeted by the compositions of the present invention include multiple sclerosis, Crohn's disease, rheumatoid arthritis, inflammatory bowel disease, and the like. Similarly, it is to be expected that certain eye-related diseases can be targeted, including wet age-related macular degeneration (AMD).

[00210] In some embodiments, the present invention provides therapies for diseases such as cancer, for example, breast cancer, brain cancer, prostate cancer, and colon cancer. In some embodiments, the therapies target cancer markers, such as β1 integrin.

[00211] In some embodiments, the present invention provides antibodies that target tumors that express a cancer cell marker, for example, β1 integrin.

[00212] In some embodiments, therapeutic antibodies comprise an antibody of the present invention conjugated to a cytotoxic agent as discussed above.

[00213] The following example is provided to further illustrate the advantages and features of the present invention, but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art, could alternatively be used.

EXAMPLE 1 VARIABLE REGION GENE SEQUENCING

[00214] The genes encoding the OS2966 anti-human β1 integrin monoclonal antibody were subjected to variable region (V) sequence analysis. Total RNA was extracted from 3 to 10x106 hybridoma cells using the RNAqueous-4PCR KitTM (Ambion, Warrington, UK) and used to synthesize cDNA. The murine immunoglobulin heavy and kappa light chain V-region fragments were amplified by PCR using degenerate mouse leader sequence primers (Sigma) and single constant domain primers (Sigma) as shown in Table 1. The PCR fragments The resulting inserts were subcloned into the pGEM-T Easy ITM vector system (Promega, Southampton, UK), and inserts were sequenced using the vector-specific primer, M13Forward (Sigma). All DNA sequencing was performed by Geneservice Ltd, Cambridge, UK). Unique V-region nucleotide sequences were obtained for OS2966 (SEQ ID NOs: 1 (VH) and 3 (VL)). Table 1.

EXAMPLE 2 GENERATION OF CHIMERIC ANTIBODIES

[00215] The heavy and light chain variable domain sequences of the monoclonal antibody OS2966 were amplified by PCR, and subcloned into pANT antibody expression vectors (Figure 14) with heavy and light chain V-regions cloned into pANT17 and pANT13, respectively. The heavy chain V-region genes were cloned into pANT17, via MluI and HindIII sites, in frame, with either the human heavy chain gene D1 (G1m3 (G1m(f)) allotype), or the human heavy chain gene4 , and light chain V-region genes, were cloned into pANT13 via BssHII and BamHI sites in-frame with the human kappa light chain constant region gene (Km3 allotype). Transcription of both heavy and light chain genes was under the control of the CMV I/E promoter (US5168062 and US5385839, University of Iowa) and the pANT17 plasmid contained a dhfr minigene mutant (Simonsen & Levinson 1983, PNAS 80:2495- 2499) under the control of an SV40 promoter, and polyA sequence for selection in eukaryotic cells. Both pANT17 and pANT13 contained a β-lactamase (ApR) gene for prokaryotic selection and a pMB1 origin of replication for propagation in prokaryotic cells. All plasmids were propagated in E. coli XL1-blue (Stratagene Cat. No. 200130).

[00216] The heavy and light chain expression constructs were then co-transfected, either transiently into HEK293 cells by calcium phosphate-based transfection, or stably transfected into NS0 cells by electroporation. Secreted antibody was purified from cell culture supernatants by Protein A chromatography.

EXAMPLE 3 GENERATION OF HUMANIZED ANTIBODY

[00217] Humanized antibodies were generated using methods described in EP1844074 (Antitope Ltd). Structural models of the mouse V-regions were produced using Swiss PDB and analyzed in order to identify important amino acids from the OS2966 V-regions that were likely to be important for the antibody's β1 integrin binding properties ('residues of restriction'). A database of human V-region sequences was used to identify segments of human V-region sequences containing each of the restriction residues to be used in humanized antibody design. Typically, two or more alternative V-region sequence segments were used to provide each restriction residue, resulting in a large range of possible sequences of humanized anti-integrin B1 V-region sequences. These sequences were then analyzed for the prediction of non-germline MHC class II peptide binding by in silico analysis as described in Fothergill et al. (WO9859244, assignee Eclagen Ltd), and also for CD4+ T-cell epitopes using databases including "The Immune Epitope Database and Analysis Resource", available on the worldwide web at URL: immuneepitope.org/. V-region sequences with predicted MHC class II binding peptides, or with significant hits against T cell epitope databases, were discarded. This resulted in a reduced fit of V-Region Sequences. Selected combinations of V-region sequence segments were then combined to produce humanized heavy and light chain variable region amino acid sequences. Five heavy chain sequences and four light chain sequences (designated VH1 to VH5, and VK1 to VK4, respectively) were selected (SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22) to gene synthesis. In addition, an additional set of heavy and light chain V-region sequences (designated VH6 to VH15, and V5 to V13, respectively), were designated (SEQ ID NOs: 29 to 38 and 44 to 52, respectively).

[00218] In addition to the above, humanized antibodies were designed using the general methods of Winter (US 6,548,640B2) as modified in Winter, Carr, Harris (EP0629240B1), whereby sequences were designed using the CDR sequences of OS2966 (SEQ ID NOs: 23 to 28) to replace the CDRs in a set of human germline V-region sequences with added human J-region sequences. In addition, the restriction residues as used in the humanized antibody of the above paragraph were introduced into the germline V-region framework sequences. The resulting sequences for the heavy and light chain V-regions were designated VH16 to VH20, and V14 to V18, respectively, and are listed as SEQ ID NOs: 39-43 and 53-57.

[00219] In addition to the above, de-immunized antibodies were assigned using the general methods of Carr et al. (US7465572 B2), whereby CD4+ T cell epitopes within the OS2966 V-region sequences (SEQ ID NOs: 2 and 4) were identified, and mutations introduced in order to potentially remove one or more of these epitopes.

[00220] DNAs encoding humanized OS2966 variant regions were synthesized and subcloned into pANT17 and pANT13 expression vectors, as described in Example 2. All combinations of humanized VH and VK chains (i.e., combinations of VH1 to VH5, and VK1 to VK4, (i.e. a total of 20 pairings) were transiently transfected into HEK293 and also transfected into NS0 cells, and the antibody was purified by protein A chromatography from culture supernatants as described in Example 2.

EXAMPLE 4 HUMANIZED ANTIBODY ANALYSIS

[00221] The binding of HEK-derived and NS0-derived humanized variants of OS2966 to human β1 integrin was evaluated in a competition ELISA against the chimeric antibody of Example 2. The chimeric antibody OS2966 was biotinylated using the Biotin TagTM Micro Biotinylation kit ( Sigma-Aldrich). MaxiSorp 96-well plates (Nunc) were coated with 0.5 µg/ml human β1 integrin (100 µl final volume) at 4°C overnight. Plates were washed with wash buffer (0.05% Tween 20 in Dulbecco's-PBS), and blocked with Dulbecco's PBS-2% BSA for 1 hour at room temperature. Plates were then washed 3 times with wash buffer. Test humanized antibody at various concentrations was pre-mixed with biotinylated chimeric antibody (0.02 µg/ml final concentration) and then added to the human β1 integrin-coated plate (100 µl final volume). Plates were incubated for 1 hour at room temperature, and washed 3 times with wash buffer. 100 µl of 1 in 500 dilution of Streptavidin HRP (Sigma-Aldrich) was added and incubated for 1 hour at room temperature. Plates were washed 3 times with wash buffer, and 100 µl of SigmaFast OPD substrate (Sigma-Aldrich, Cat# P9187) was added and incubated at room temperature in the dark for 4 minutes. The reaction was stopped by adding 50 µL of 3M HCl. Plates were read at 490 nm using a Dynex plate read.

EXAMPLE 5 SCFv's and Fab's GENERATION

[00222] The humanized OS2966 anti-human integrin variants of Example 3 were converted into scFv's and cloned into M13 phage display vectors, as described in Benhar I. and Reiter Y., Current Protocols in Immunology, Unit 10.19B, Wiley Online Library, May 2002 (available on the worldwide web at URL: currentprotocols.com/protocol/im1019b) using the pCANTAB5E RPAS Expression Module vector (Amersham Pharmacia Biotech, Little Chalfont, UK). The humanized VH and VK genes were amplified using primers that provide SfiI and NotI terminal restriction sites, a Gly4Ser internal linker, and a his6 C-terminal tag. The scFv constructs were inserted into the pCANTAB5E vector as SfiI-NotI fragments and transformed into E.coli HB2151, resulting in scFv exported to the periplasm, and partially to the growth medium. The scFv's were purified from the growth medium by nickel-chelate affinity chromatography using HIS-Select HF Cartridges (Sigma-Aldrich). The purified scFv's were tested in the competition assay as detailed in Example 4. The humanized OS2966 variants from Example 3 were also converted to Fab's using the method used for scFv's, except that the humanized amplified VH and VK genes were further amplified with genes from CH1 and Cn constant region to form VH-CH1 and VK-C fragments that were further amplified with primers to join these fragments with a 22 amino acid pelB leader sequence (Lei S.P., Lin H.C., Wang S.S., Callaway J., and Wilcox G., J Bacteriol. 169 (1987) p4379-4383) between the upstream VH-CH1 and downstream VK-CK gene fragments, resulting in a dicistronic Fab gene. Fab's of humanized antibody variants were generated and purified as above for scFv's, and tested in the human β1 integrin competition assay, as detailed in Example 4.

EXAMPLE 6 ANALYSIS OF CD4+ T CELL RESPONSES

[00223] The potential immunogenicity of the humanized antibody of Examples 3 and 5 was performed in comparison to humanized human anti-β1 integrin K20 (Poul et al., Molecular Immunology 32 (1995) p102116). PBMCs (peripheral blood mononuclear cells) were isolated from healthy community donor buffy coats (from blood drawn within 24 hours) obtained from the UK National Blood Transfusion Service (Addenbrooke's Hospital, Cambridge, UK) and, according to with approval secured by Addenbrooke's Hospital Local Research Ethics Committee. PBMCs were isolated from buffy coat density centrifugation by Lymphoprep (Axis-shield, Dundee, UK), and CD8+ T cells were depleted using CD8+ RosetteSep™ (StemCell Technologies Inc, London, UK). Donors were characterized by identifying HLA-DR haplotypes using an HLA-based SSP-PCR tissue typing kit (Biotest, Solihull, UK). T cell responses to control antigens including the withdrawal antigen tetanus toxin were also determined (KLH Pierce, CramLingtom, UK, and peptides derived from Influenza A and Epstein Barr viruses). The PBMC was then frozen and stored in liquid nitrogen until required.

[00224] To prepare monocyte-derived dendritic cells (DC), 50 different donor PBMCs were selected to provide a distribution with HLA-DR and HLA-DQ allotype frequencies similar to frequencies in the total world population. PBMCs were revived in AIM-V® culture medium and CD14+ cells isolated using Miltenyi CD14 Microbeads and LS columns (Miltenyi Biotech, Oxford, UK). Monocytes were resuspended in AIM-V® supplemented with 1000U/ml IL-4 and 1000U/ml GM-CSF ("DC culture medium") at 4-6x106 PBMC/ml and then distributed in 24-well plates (2 ml final culture volume). Cells were fed on day 2 by half a DC volume of culture medium change. By day 3, monocytes differentiated to semi-mature DC that were pre-incubated with either 40 ug/ml humanized test antibody, or chimeric antibody, 100 ug/ml KLH, or medium alone. Semi-mature DCs were incubated with antigen for 24 hours after which excess test antibody was removed by washing the cells twice and resuspension in DC culture medium supplemented with 50 ng/ml TNF-α (Peprotech, London, UK ). DCs were fed on day 7 by a larger volume change of DC culture medium (supplemented with 50 ng/ml TNFα) prior to collection of mature DC on day 8. Collected mature DCs were counted, and viability assessed using trypan blue dye exclusion. DCs were then Y-irradiated (4000 rads) and resuspended at 2x10 5 cells per ml in AIM-V medium before use in ELISpot and proliferation assays. Additionally, on day 8, fresh CD4+ T cells were also prepared. To purify CD4+ T cells, PBMCs were revived in AIM-V® culture medium and CD4+ cells isolated using Miltenyi CD4 Microbeads and LS columns (Miltenyi Biotech, Oxford, UK), and resuspended in AIM-V® medium in 2x106 cells/ mL.

[00225] On day 8, T cell proliferation assays were established whereby 1x10 5 autologous CD4+ T cells were added to 1x10 4 DC loaded with humanized antibody or chimeric antibody (10:1 ratio) in 96-well plates of U-shaped bottom, with AIM-V® medium added to a final volume of 200ul/well). On day 14, assay plates were pulsed with 1uCi[3H] (Perkin Elmer, Beaconsfield, UK) per well in 25ul of AIMV for 6 hours before collection on filter mats (Perkin Elmer) using a TomTec Mach cell harvester III (Hamden CT, USA). All antibody preparations were tested in sextuplet cultures. Counts per minute (cpm) for each well were determined by Meltilex™ scintillation counting (Perkin Elmer) in a 1450 Microbeta Wallac Trilux Liquid Scintillation Counter (Perkin Elmer) on low background count, paralux. Counts per minute for each antibody sample were normalized to the mean for control only.

[00226] For ELISpot assays, ELISpot plates (Millipore, Watford, UK) were coated with 100ul/well of IL-2 capture antibody (R&D Systems, Abingdon, UK) in PBS. Plates were then washed twice in PBS, incubated overnight in block buffer (1% BSA (Sigma) in PBS), and washed in AIM V® medium. On day 8, 1x10 5 autologous CD4+ T cells were added to 1x10 4 antigen-loaded DC (10:1 ratio) in 96-well ELISpot plates. All antibody preparations were tested in sextuplet cultures. For each donor PBMC, a negative control (AIM V® medium alone), no cell control, and a PHA positive control (10 ug/mL), were also included.

[00227] After an additional 7 day incubation period, the ELISpot plates were developed by three sequential washes in dH2O and PBS before the addition of 100 ultrafiltered biotinylated detection antibodies (R&D Systems, Abingdon, UK) in PBS/1% BSA. Following incubation at 37°C for 1.5 hours, the plates were further washed three times in PBS and 100 ul filtered streptavidin-AP (R&D Systems) in PBS/1% BSA was added for 1 hour (incubation at room temperature). Streptavidin-AP was discarded, and the plates were washed four times in PBS. BCIP/NBT (R&D Systems) was added to each well and incubated for 30 minutes at room temperature. Spot development was stopped by washing the wells and the well returns washed three times with dH2O. Dried plates were scanned on an Immunoscan™ Analyzer and points per well (spw) were determined using Immunoscan™ Version 4 software.

[00228] For both proliferation and IL-2 ELISpot assays, results were expressed as a Stimulation Index (SI) defined as the ratio of cpm (proliferation assay) or points (ELISpot assay) for the test antibody against a control-only medium using an SI cutoff equal to or greater than 2 (SI > 2.0) for positive T cell responses.

EXAMPLE 7 ANIMAL TUMOR MODEL

[00229] An animal tumor model can be used for in vivo analysis of humanized anti-β1 integrin antibodies in inhibiting tumor growth. For example, an orthotic model of breast cancer using MDA-MB-231 cells can be used as a model of primary tumor growth and spontaneous metastasis in mice primed with human β1 integrin, or immune deficient mice (e.g., nu/nu , severe combined immune deficiency [SCID]).

[00230] β1 integrin shaken mice (7-10 weeks old, male and female, equally distributed across groups), can be injected subcutaneously into the mammary fat pad with MDA-MB-231 cells in 0.1 mL of volume. The anti-β1 integrin antibody of the present invention, OS2966, or an isotype matched control antibody, can be injected at 1 mg/kg-20 mg/kg, such as 5 mg/kg or 10 mg/kg doses ( dosing volume of 10 mL/kg) weekly, starting the day after tumor cell administration ("Day 2"), or when the tumor is palpable, and the average tumor volume is approximately 100 mm 3 . Tumor measurements can be taken bimonthly during the course of the experiment by caliper measurement. Animals can be followed to determine results.

EXAMPLE 8 SEQUENCE ANALYSIS

[00231] Sequence analysis was performed on the heavy and light chains generated in the Examples to determine important VH and VL amino acid residues outside the CDRs from the OS2966 sequence that are preferably included in the humanized sequences. Such residues are identical to those of OS2966, in addition to those of CDRs. Such residues are identified as "c" residues in Figures 15 and 16. As shown, for VH chain residues 48, 67, 69, 73, 76, 80, 89, 91 and 93 are preferably identical to residues corresponding OS2966 (SEQ ID NO:2). Also, for the VL chain, residues 36 and 71 are preferably identical to the corresponding residues of OS2966 (SEQ ID NO:4).

[00232] The codon usage corresponding to the VH and VL sequences is provided in Figure 17, while Figure 18 provides a summary of the amino acid sequences of the VH and VL chains.

EXAMPLE 9 RADIATION SENSITIVITY INCREASE METHOD BY INTEGRIN β1 INHIBITION

[00233] The humanized antibody of the present invention can be used to increase radiation sensitivity by inhibiting β1 integrin, as disclosed in United States Patent Application No. 20070237711, Park et al., Cancer Res 2008; 68:(11)4398, and Yao et al., Cancer Res 2007; 67:(2)659, all of which are hereby incorporated in their entirety. Antibodies of the present invention can be used in a method of co-administering the antibody, or antibody-containing compositions described herein, in combination with ionizing radiation that cause increased apoptosis in tumor cells, notably breast cancer tumor cells.

EXAMPLE 10 VALIDATION AND ANALYSIS OF COMPOUND HUMAN ANTIBODY VARIANTS

[00234] The composite humanized antibodies were generated as discussed in Example 3. Table 2 provides a list of the various antibodies generated showing which VH and Vk sequences were used. Table 2. Variant designations according to region ID V.

[00235] Binding of composite human antibody variants (from Table 2) was evaluated in a competition FACS assay with the murine chimeric antibody OS2966 (Murine OS2966 sliced human Fc Fab). Briefly, a dilution series (three times) of chimeric antibody or humanized antibody starting at 25.0 μg/mL, was pre-mixed with a constant concentration of murine OS2966 (0.1875 μg/mL) in FACS buffer (1% BSA in 1x PBS pH 7.4) before mixing with 3x10 5 Jurkat cells. After incubation on ice for 1 hour, the cells were washed, and murine OS2966 binding was detected with PE-tagged goat antimurine Fc (Jackson ImmunoResearch, Cat. No. 115-116-071). After incubation on ice for 45 min, cells were washed, resuspended in 300 µL of FACS buffer, and analyzed on a Beckton Dickinson FACScaliburTM. Geometric mean fluorescence intensity was plotted against antibody concentration (Figures 20-24). These data were used to calculate IC50 values for each antibody, and these values were normalized to the IC50 of the chimeric antibody that was included in each FACS assay (Table 3). Table 3. Relative affinity of the human variant. Relative IC50 was calculated by dividing the IC50 of the test antibody by that of the chimeric antibody assayed in the same way.

[00236] These variants (H1, H2, H5) demonstrated slightly higher relative affinities, and four variants (H3, H4, H10, H13) have significantly lower relative affinities than murine OS2966.

[00237] Functional inhibition of the β1 integrin subunit with the human variant antibodies was evaluated in a microplate adhesion assay in multiple ECM, and in multiple cancer cell lines of PANC-1 human pancreatic cancer (Figures 25A and 25B ), MDA-MB-231 human triple negative breast cancer (Figure 25C), and AsPC-1 human pancreatic cancer (Figure 25D). Soon, ECM adhesion assays were performed with 10 μg/mL of ECM components or BSA (control). Cells were pre-incubated with 10 μg/mL of antibodies in serum-free DMEM/F12 medium for 30 min. Adherence was tested for 30 min to 1 hour. Non-adherent cells were removed from dishes, cells fixed with 1% paraformaldehyde, and plates stained with crystal violet for 30 min. After extensive rinsing, crystal violet was solubilized in Triton X100 for 1 hour, and the plates read for absorbance at A590.

[00238] All 19 human variants were functionally active in attenuating cell adhesion to all ECM with some significant variation depending on ECM protein and cell line. Relative affinities (Table 3) demonstrate no obvious correlation to function in this assay regardless of ECM type, or cell line. These data were normalized to negative IgG control (BSA, bovine serum albumin as negative control; IgG, isotype control; murine OS2966, OS2966; TS2/16, positive control β1 integrin activating antibody; FN, fibronectin; LN, laminin; C1, collagen type I; C4, collagen type IV).

[00239] Functional inhibition of β1 integrin subunit with human variant antibodies was evaluated in a microplate migration assay on ECM component fibronectin with human triple negative breast cancer cells (MDA-MB -231). Briefly, the plates were coated with 10 µg/ml of fibronectin, and seeded with tumor cells. A yellow pipette tip was used to scratch confluent cell monolayers and substituted medium including 10 µg/ml of the above antibodies. Plates were fixed after 8 hours of incubation at 37C and images were obtained. 10x magnification images of plaques demonstrated scratch migration attenuation in OS2966 and composite human variant (H1, H2, H3) treated in wells ( Figure 26A ). Cell free area quantification for each condition (performed in triplicate and repeated) is shown in Figure 26B.

[00240] Migration of triple negative breast cancer cells was significantly attenuated by treatment with composite human variants of OS2966.

[00241] The functional inhibition of the β1 integrin subunit with the human variant antibodies was evaluated in an in vitro model of angiogenesis; the human umbilical vein endothelial cell tube forming assay (HUVEC). Briefly, plates were coated with Matrigel ECM, and seeded with HUVEC cells. Plaques were imaged after 8 hours of incubation at 37°C, and quantified for vascular tube formation (closed unit formation). 10x magnification images of plaques demonstrated attenuation of vascular tube formation in OS2966 and composite human variant (H1, H2, H3) treated in wells ( Figure 27A ). Quantitation of closed unit formation for each condition (performed in at least triplicate and repeated with endothelial progenitor cells) is shown in Figure 27B.

[00242] Compound human variants completely block angiogenesis in the in vitro tube formation assay.

[00243] The functional inhibition of the β1 integrin subunit with the human variant antibodies was evaluated in an in vivo model of human triple negative breast cancer with the MDA-MB-231 cell line. Briefly, 106 cells were injected into the 4th mammary fat pad in 5 to 6 week old female athymic nu/nu mice. Mice were randomized into treatment groups after tumors were established (mean subcutaneous volume = 80-100 mm3). Control and experimental antibodies were administered at 5 mg/kg intraperitoneally (IP) twice weekly. Human IgG at equivalent doses served as the control (Sigma). Orthotopic tumors were measured with calipers twice weekly. As shown in Figure 28A, human H1-H3 variants significantly attenuated MDA-MB-231 tumor growth in vivo compared to IgG control. A trend towards superior efficacy for the composite human variants was apparent compared to murine OS2966, particularly for H3. Western blot pharmacodynamic analysis demonstrated a reduction of activities in critical pro-growth signaling pathways (Extracellular Phosphorylated Related Kinase, ERK, and Phosphorylated Focal Adhesion Kinase, FAK) in treated tumors compared to control that may have contributed to reduced proliferation and elevation of apoptosis ( Figure 28B ).

[00244] The functional inhibition of the β1 integrin subunit with the human variant antibodies was evaluated in an in vivo model of spontaneous lung metastasis with human triple negative breast cancer with the MDA-MB-231 cell line. Briefly, 106 cells were injected into the 4th mammary fat pad in 5 to 6 week old female athymic nu/nu mice. Mice were randomized into treatment groups after tumors were established (mean subcutaneous volume = 80-100 mm3). Control and experimental antibodies were administered at 5 mg/kg intraperitoneally (IP) twice weekly. Human IgG at equivalent doses served as the control (Sigma). After 7 weeks, the mice were perfused, the lungs collected, and sectioned coronally for H&E analysis. Spontaneous lung metastasis was seen in 50% (3 of 6) of mice given IgG control antibody. Spontaneous hand lung metastases were observed in either mice given OS2966 or H1-H3 composite human variants (0%, 0/28 mice). The results are shown in Figure 29.

[00245] Composite human variants completely prevent spontaneous lung metastasis in an orthotopic model of triple negative breast cancer.

[00246] The functional inhibition of the β1 integrin subunit with the human variant antibodies was evaluated in an in vivo model of human gemcitabine-resistant pancreatic cancer with the PANC1-GEMR cell line. Briefly, 106 cells were injected subcutaneously into 5 to 6 week old male nude athymic nu/nu mice. Mice were randomized into treatment groups after tumors were established (mean subcutaneous volume = 80-100 mm3). Control and experimental antibodies were administered at 5 mg/kg intraperitoneally (IP) twice weekly, or gemcitabine at 50 mg/kg. Human IgG at equivalent doses served as the control (Sigma). Subcutaneous tumors were measured with calipers twice weekly. Figure 30A shows in vivo verification of gemcitabine resistance in the PANC1-GEMR line. Figure 30B shows that the H3 composite human variant significantly attenuated the growth of PANC1-GEMR tumors in vivo compared to the IgG control. Figure 30C shows Western Blot pharmacodynamic analysis of critical pro-growth signaling pathways (phosphorylated Extracellular Related Kinase, ERK, and Phosphorylated Focal Adhesion Kinase, FAK) in treated compared to control tumors, which may have contributed to reduced proliferation and elevation of apoptosis.

[00247] The functional inhibition of the β1 integrin subunit with the human variant antibodies was evaluated in an in vivo model of human glioblastoma established with the U87MG cell line. Briefly, 107 cells were injected subcutaneously into 5 to 6 week old male athymic nu nu/nu mice. Mice were randomized into treatment groups after tumors were well established (mean subcutaneous volume = ~200-250 mm3). Control and experimental antibodies were administered at 5 mg/kg intraperitoneally (IP) twice weekly. Human IgG at equivalent doses served as the control (Sigma). Subcutaneous tumors were measured with calipers twice weekly. Figure 31A demonstrates that the H3 composite human variant significantly attenuates the growth of established glioblastoma tumors in vivo compared to IgG control. The efficacy of H3 was equivalent to murine OS2966. Western blot pharmacodynamic analysis demonstrated a reduction of activities in critical pro-growth signaling pathways (Phosphorylated Extracellular Related Kinase, ERK, and Phosphorylated Focal Adhesion Kinase, FAK) in tumors treated with H3 compared to controls, which may have contributed to reduced proliferation and elevation of apoptosis, as shown in Figure 31B.

[00248] The EpiScreen™ time course T cell proliferation assay was used to determine the potential for clinical immunogenicity of H3 variant antibody. Fully humanized and chimeric antibodies were tested for their ability to induce CD4+ T cell responses as measured by proliferation against a panel of 12 HLA-typed donors. Healthy donor T cell proliferation responses to chimeric IS2966 antibody (murine/human), humanized H3 antibody, and a positive control humanized antibody are shown in Figure 32A. CD4+ T cells were incubated with autologous mature DC loaded with the samples and evaluated for proliferation after 7 days of incubation. Proliferation responses with an SI > 2.00 (indicated by the red dotted line) that were significant (p < 0.05) using two unpaired student's t test samples were considered positive. Figure 32B provides a summary of healthy donor T cell proliferation responses to the donor cohort. Positive (SI > 2.00, significant p < 0.05). T cell responses to proliferation ("P") are shown. Borderline responses (significant p < 0.05 with SI > 1.90) are shown (*). Frequencies of positive responses to the proliferation assay are shown as a percentage at the bottom of the columns. A33 (humanized A33) is the mAb clinical bench control that shows high levels of immunogenicity in the clinic, and routinely induces 20-30% T cell responses in EpiScreen assay. For each donor, an immunogenic reproducibility control (cells incubated with 100 μg/mL KLH) was also included.

[00249] A composite H3 human variant demonstrated significantly reduced immunogenicity (0% responses) compared to that murine/human OS2966 chimera (25% responses). It is concluded that the human H3 antibody exhibits a clinically acceptable immunogenicity profile from the EpiScreen™ assay providing confirmation of reduced immunogenicity as a result of Antitope Composite Human Antibody technology.

[00250] Although the invention has been described with reference to the above example, it will be understood that modifications and variations are involved within the spirit and scope of the invention. Accordingly, the invention is limited only by the claims that follow.

Claims (16)

1. Humanized antibody, characterized in that it specifically binds β1 integrin, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH region and the VL region have a sequence of amino acids, respectively, as set forth in SEQ ID NOs: 2 and 4, SEQ ID NOs: 6 and 16, SEQ ID NOs: 6 and 18, SEQ ID NOs: 6 and 20, SEQ ID NOs: 8 and 16, SEQ ID NOs : 8 and 18, SEQ ID NOs: 8 and 20, SEQ ID NOs: 8 and 22, SEQ ID NOs: 10 and 16, SEQ ID NOs: 10 and 18, SEQ ID NOs: 10 and 20, SEQ ID NOs: 10 and 22, SEQ ID NOs: 12 and 16, SEQ ID NOs: 12 and 18, SEQ ID NOs: 12 and 20, SEQ ID NOs: 12 and 22, SEQ ID NOs: 14 and 16, SEQ ID NOs: 14 and 18 , SEQ ID NOs: 14 and 20 and SEQ ID NOs: 14 and 22. 2. Antibody according to claim 1, characterized in that the complementarity determining regions (CDRs) of the VH and VL regions are from a donor antibody, such as OS2966. 3. Antibody according to claim 2, characterized in that the CDRs of the VH region have amino acid sequences as shown in SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25, or in which the VL region CDRs have amino acid sequences as shown in SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28. 4. Antibody according to claim 1, characterized in that it competes with OS2966 for specific binding to β1 integrin. 5. Antibody according to any one of claims 1 to 4, characterized in that when tested in vitro for induction of CD4+ helper T cell responses in blood samples with a distribution of HLA-DR allotypes from the population human, produces less than 10% T cell responses. 6. Multispecific antibody, characterized in that it comprises one or more antibodies, as defined in any one of claims 1 to 5. 7. VH region, characterized in that it has an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NOs:29-43, SEQ ID NOs:91-99 and SEQ ID NO:100. 8. VL region, characterized in that it has an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NOs:44-57, SEQ ID NOs:107-115 and SEQ ID NO:116. 9. Nucleic acid molecule, characterized in that it comprises nucleic acid sequences as set out in SEQ ID NOs: 1 and 3, SEQ ID NOs: 5 and 15, SEQ ID NOs: 5 and 17, SEQ ID NOs: 5 and 19, SEQ ID NOs:7 and 15, SEQ ID NOs:7 and 17, SEQ ID NOs:7 and 19, SEQ ID NOs:7 and 21, SEQ ID NOs:9 and 15, SEQ ID NOs:9 and 17, SEQ ID NOs:9 and 20, SEQ ID NOs: 9 and 21, SEQ ID NOs:11 and 15, SEQ ID NOs:11 and 17, SEQ ID NOs:1 and 19, SEQ ID NOs:11 and 21, SEQ ID NOs:13 and 15, SEQ ID NOs:13 and 17, SEQ ID NOs:13 and 19 and SEQ ID NOs:13 and 21. 10. Expression vector, characterized in that it comprises the nucleic acid molecule, as defined in claim 9. 11. Isolated prokaryotic host cell, characterized by the fact that it comprises the vector, as defined in claim 10. 12. Pharmaceutical composition, characterized in that it comprises the antibody, as defined in any one of claims 1 to 5, or the nucleic acid molecule, as defined in claim 9, and a pharmaceutically acceptable vehicle. 13. Immunoconjugate, characterized in that it comprises the antibody, as defined in any one of claims 1 to 5, linked to a detectable fraction such as a fluorescent fraction or a therapeutic fraction, for example a cytotoxic fraction or a chemotherapeutic agent. 14. Antibody, according to any one of claims 1 to 5, nucleic acid molecule, according to claim 9, pharmaceutical composition, according to claim 12, or immunoconjugate, according to claim 13, characterized by the fact that it is for use in treating a disease, such as a cell proliferative disorder or an inflammatory disease. 15. In vitro method for detecting a cell proliferation disease or an inflammatory disease, as defined in claim 14, in an individual, characterized in that it comprises: a) contacting the antibody, as defined in any one of claims 1 to 5, with a sample from the individual; b) detecting the level of β1 integrin via specific binding of the antibody with β1 integrin; and c) comparing the detected level of β1 integrin to that of β1 integrin in a normal sample, where an increased level of β1 integrin in the subject's sample as compared to the normal sample is indicative of a disease. 16. Use of an antibody, as defined in any one of claims 1 to 5, a nucleic acid molecule, as defined in claim 9, or an immunoconjugate, as defined in claim 13, characterized in that it is for the manufacture of a pharmaceutical composition for the treatment of a cell proliferation disorder selected from lymphoma, leukemia, squamous cell cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, cancer of peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, carcinoma salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, brain, hepatic carcinoma, head and neck cancer, neurofibromatosis type I or II or an inflammatory disease selected from pruritus, skin inflammation, psoriasis, multiple sclerosis, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, Hashimoto's thyroid, myasthenia gravis, type I or II diabetes, asthma, inflammatory lung injury, inflammatory liver injury, inflammatory glomerular injury, atopic dermatitis, dermatitis allergic contact, irritant contact dermatitis, seborrheic dermatitis, Sjogren's syndrome, keratoconjunctivitis, uveitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, an inflammatory disease of the joints, skin or muscle, acute or chronic idiopathic inflammatory arthritis, myositis, a demyelinating disease, chronic obstructive pulmonary disease, interstitial lung disease, interstitial nephritis, and chronic active hepatitis.

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