WO2007059300A2 - Anti-alk antagonist and agonist antibodies and uses thereof - Google Patents

Abstract

This disclosure provides ALK antagonists and agonists (e.g., antibodies and antigen-binding portions thereof that bind to ALK), and methods for modulating ALK activity. In other embodiments, the present disclosure provides methods and compositions for using ALK antagonists and agonists to treat various diseases.

Description

ALK ANTAGONISTS, AGONISTS AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional

Application No. 60/737324, filed on November 15, 2005, the content of which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE DISCLOSURE Pleiotrophin (PTN) is a 136-amino acid, secreted, heparin-binding cytokine that has diverse functions including a role in angiogenesis. PTN has been shown to specifically bind to a receptor tyrosine kinase, Anaplastic Lymphoma Kinase (ALK), and such binding leads to auto-phosphorylation of the receptor and subsequent phosphorylation of a number of signal transduction molecules such as IRS-I, PLC-gamma, PI3 kinase, and She, and activates a cell survival pathway. See PCT Pat. App. Pub. No. WO 01/96364.

Angiogenesis, the development of new blood vessels from the endothelium of a preexisting vasculature, is a critical process implicated in the pathogenesis of a variety of disorders. These include solid tumors, intraocular neovascular syndromes such as proliferative retinopathies or age-related macular degeneration (AMD), rheumatoid arthritis, and psoriasis. See Folkman et al. J. Biol. Chem. 267:10931- 10934 (1992); Klagsbrun et al. Annu. Rev. Physiol. 53:217-239 (1991); and Garner A, Vascular diseases. In: Pathobiology of ocular disease. A dynamic approach. Garner A, Klintworth G K, Eds. 2nd Edition Marcel Dekker, New York, pp 1625- 1710 (1994). In the case of solid tumors, the neovascularization allows the tumor cells to acquire a growth advantage and proliferative autonomy compared to the normal cells. Accordingly, a correlation has been observed between density of microvessels in tumor sections and patient survival in breast cancer as well as in several other tumors. Weidner et al. N Engl J Med 324:1-6 (1991); Horak et al. Lancet 340:1120-1124 (1992); and Macchiarini et al. Lancet 340:145-146 (1992). Agents that inhibit angiogenesis have proven to be effective in treating a variety of disorders. Avastin^® (bevacizumab), a monoclonal antibody that binds to Vascular Endothelial Growth Factor (VEGF), has proven to be effective in the treatment of a variety of cancers. Macugen^®, an aptamer that binds to VEGF has proven to be effective in the treatment of neo vascular (wet) age-related macular degeneration. Antagonists of the SDF/CXCR4 signaling pathway inhibit tumor neovascularization and are effective against cancer in mouse models (Guleng et al. Cancer Res. 2005 JuI 1;65(13):5864-71). The isocoumarin 2-(8-hydroxy-6- methoxy-l-oxo-1 H-2-benzopyran-3-yl) propionic acid (NM-3) has completed phase I clinical evaluation as an orally bioavailable angiogenesis inhibitor. NM-3 directly kills both endothelial and tumor cells in vitro and is effective in the treatment of diverse human tumor xenografts in mice (Agata et al. Cancer Chemother Pharmacol. 2005 Jun 10; [Epub ahead of print]). Thalidomide and related compounds have shown beneficial effects in the treatment of cancer, and although the molecular mechanism of action is not clear, the inhibition of angiogenesis appears to be an important component of the anti-tumor effect (see, e.g., Dredge et al. Microvasc Res. 2005 Jan;69(l-2):56-63). The success of TNF-alpha antagonists in the treatment of rheumatoid arthritis is partially attributed to anti-angiogenic effects on the inflamed joint tissue (Feldmann et al. Annu Rev Immunol. 2001 ; 19 : 163 -96) . Anti-angiogenic therapies are widely expected to have beneficial effects on other inflammatory diseases, particularly psoriasis.

Accordingly, the present disclosure provides agents and therapeutic treatments that regulate ALK-mediated signal transduction pathways and affect one or more ALK-regulated functions, including, for example, angiogenesis.

SUMMARY OF THE DISCLOSURE

In certain aspects, the disclosure provides agents that either inhibit or promote one or more ALK-mediated biological functions, referred to as ALK antagonists or agonists, respectively. In certain aspects, the disclosure provides mechanisms by which an ALK antagonist or agonist may affect ALK and by which ALK antagonists and agonists may be identified. As shown herein, ALK participates in various disease states, including cancers and diseases related to unwanted or excessive angiogenesis. Additionally, the disclosure demonstrates that ALK participates in a desirable way in certain processes, such as wound healing. Accordingly, ALK antagonists and agonists may be used, as appropriate, to treat various disorders. In further aspects, the disclosure relates to the discovery that ALK and/or PTN are expressed, often at high levels, in a variety of tumors. Therefore, agents that downregulate ALK and/or PTN function may affect tumors by a direct effect on the tumor cells, an indirect effect on the angiogenic processes recruited by the tumor, or a combination of direct and indirect effects. In certain embodiments, the disclosure provides the identity of tumor types particularly suited to treatment with an agent that downregulates ALK and/or PTN function.

In certain aspects, the disclosure provides polypeptide agents that bind to ALK and function as antagonists of ALK. Polypeptide agents may be antibodies (including antigen-binding portions thereof), soluble extracellular domains of ALK and other polypeptides that bind to and affect ALK. In certain aspects, the disclosure provides small molecules, peptidomimetics and other agents that bind to ALK and function as antagonists of ALK. In certain aspects, the disclosure provides agents that effectively antagonize ALK by reducing or downregulating ALK expression; such ALK antagonists include but are not limited to nucleic acid agents, such as siRNAs, antisense molecules, and ribozymes.

In certain aspects, the disclosure provides agents that bind to ALK and function as agonists of ALK. Such agonists may be, for example, polypeptide agents, small molecules or nucleic acids that encode ALK or otherwise increase ALK expression. ALK agonists may act by one or more of a variety of mechanisms. ALK agonists may bind to the extracellular portion of the ALK protein, such as the ligand binding domain. Alternatively, such agonists may bind to a portion of the ALK protein that does not interact with PTN and nonetheless cause increased ALK signaling or one or more ALK-mediated biological functions. It is generally expected that certain agonists of ALK will stimulate the kinase activity of ALK, which leads to increased phosphorylation of one or more ALK substrates and/or autophosphorylation of the ALK protein. However, agonists (as well as antagonists) may also act through alternative mechanisms, such as by promoting interaction with other proteins that mediate or modulate ALK signaling. In certain aspects, an antagonist of the present disclosure may be considered as an agonist of ALK because it may stimulate an ALK kinase activity such as the autokinase activity; however, such an antagonist is generally expected to have an inhibitory effect on ALK-mediated signaling or one or more ALK-mediated biological functions. For example, an "agonist" of ALK that stimulates an ALK kinase activity may lead to increased endocytosis and degradation of ALK, thereby having an overall inhibitory effect on ALK-mediated signaling or one or more ALK-regulated biological functions. In certain embodiments, the disclosure provides an isolated antibody or antigen-binding portion thereof that binds to an epitope situated in the extracellular portion of ALK and inhibits one or more ALK-mediated biological functions. The anti-ALK antibody or antigen-binding portion thereof may inhibit an angiogenic activity, for example in an endothelial cell. The isolated anti-ALK antibody or antigen-binding portion thereof may inhibit cell growth or proliferation and/or invasiveness of cancer cells, for example by changing cell adhesion properties of the cancer cells. The isolated anti-ALK antibody or antigen-binding portion thereof may induce apoptosis, for example by altering cell signaling. The isolated anti-ALK antibody or antigen-binding portion thereof may inhibit the formation of tubes by cultured endothelial cells, the vascularization of a tissue in vivo, the vascularization of tissue implanted in the cornea of an animal, the vascularization of a Matrigel tissue plug implanted in an animal, and/or the growth of a human tumor xenograft in a mouse. The isolated anti-ALK antibody or antigen-binding portion thereof may inhibit wound healing. The isolated anti-ALK antibody or antigen-binding portion thereof may induce tumor regression, for example by reducing angiogenesis of a tumor. The isolated anti-ALK antibody or antigen-binding portion thereof may induce tumor regression in an angiogenesis-independent manner, for example by preventing cell proliferation and/or by inducing apoptosis. In certain embodiments, an anti-ALK antibody or antigen-binding portion thereof will reduce an ALK- regulated biological function by at least 5%, or by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 100%. An anti-ALK antibody or antigen-binding portion thereof that causes a decrease in an ALK-regulated biological function may act by one or more of a variety of biochemical mechanisms. For example, such an antibody may inhibit a biochemical activity of ALK, such as an ALK autokinase or other kinase activity, or a binding or interacting activity including ALK dimerization or multimerization with other proteins. For example, the disclosure demonstrates that ALK physically interacts with certain proteins, such as for example, another receptor protein (e.g., UNC5 which is a receptor for netrins), another receptor tyrosine kinase (e.g., LTK receptor), an actin-based cytoskeleton molecule (e.g., tensin or actinin), or an adaptor molecule (e.g., IRS). Therefore, an anti-ALK antibody or antigen-binding portion thereof may affect one or more of such interactions. As another example, an anti-ALK antibody or antigen-binding portion thereof may cause a decrease in the levels of ALK protein at the cell surface (or other membrane surface that is relevant for signaling) and may in fact decrease the overall levels of ALK protein. Antibodies that decrease ALK protein levels may, in some instances, cause an increase in the kinase activity or phosphorylation state of ALK but nonetheless have an overall antagonistic effect on ALK-regulated biological activities. In certain embodiments, an anti-ALK antibody or antigen-binding portion thereof will reduce an ALK biochemical activity by at least 5%, or by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 100%.

In certain embodiments, an isolated anti-ALK antibody or antigen-binding portion thereof binds to an epitope situated within amino acids 368-447 of the ALK sequence of SEQ ID NO: 1. For example, the epitope may be situated within amino acids 391-401 of the ALK sequence of SEQ ID NO: 1; the isolated antibody or antigen-binding portion thereof may inhibit the binding of PTN or Midkine, to the extracellular portion of ALK. As another example, the epitope may be situated within an 80 amino acid portion the ALK sequence (also referred to as the ALK- LBD herein), as shown in SEQ ID NO: 6. In certain embodiments, the isolated anti- ALK antibody or antigen-binding portion thereof may bind to an epitope situated within another portion of the extracellular domain (ECD) of ALK. For example, the isolated antibody or antigen-binding portion thereof may bind to one or more MAM domains with the ECD of ALK. Anti-ALK antibody or antigen-binding portion may inhibit ALK dimerization or multimerization or kinase activity.

In certain aspects, the disclosure provides an isolated anti-ALK antibody or antigen-binding portion thereof that binds to an epitope situated in the extracellular portion of ALK and stimulates one or more ALK-mediated biological function or activity. Such anti-ALK antibodies may also have an agonistic effect on an ALK kinase activity, and for example, can cause increased ALK autophosphorylation. In some instances, an antibody that stimulates an ALK kinase activity will also stimulate ALK endocytosis, degradation and/or inactivation, in which case the cellular and physiological effect of the antibody will be that of an antagonist. In other instances, an antibody that stimulates an ALK kinase activity will cause genuine ALK activation, with cellular and physiological consequences that are consistent with an ALK agonist effect.

In certain embodiments, the disclosure provides an isolated antibody or antigen-binding portion thereof that binds to an epitope situated in the extracellular portion of ALK and stimulates one or more ALK-regulated biological functions. The anti-ALK antibody or antigen-binding portion thereof may stimulate an angiogenic activity, for example in an endothelial cell. The isolated anti-ALK antibody or antigen-binding portion thereof may stimulate cell growth or proliferation and/or invasiveness of cells, for example by changing cell adhesion properties of the cells. The isolated anti-ALK antibody or antigen-binding portion thereof may inhibit apoptosis, for example by altering cell signaling. The isolated anti-ALK antibody or antigen-binding portion thereof may stimulate the formation of tubes by cultured endothelial cells, the vascularization of a tissue in vivo, the vascularization of tissue implanted in the cornea of an animal, the vascularization of a Matrigel tissue plug implanted in an animal, and/or the growth of cells in a soft agar assay. The isolated anti-ALK antibody or antigen-binding portion thereof may promote wound healing, for example by increasing cell proliferation and/or angiogenesis. In certain embodiments, an anti-ALK antibody or antigen-binding portion thereof will increase an ALK-regulated biological function by at least 5%, or by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 100%.

An anti-ALK antibody or antigen-binding portion thereof that causes a increase in an ALK-regulated biological function may act by one or more of a variety of biochemical mechanisms. For example, such an antibody may stimulate a biochemical activity of ALK, such as an ALK autokinase or other kinase activity, or a binding or interacting activity including ALK dimerization or multimerization with other proteins. For example, the disclosure demonstrates that ALK physically interacts with certain proteins, such as for example, another receptor protein (e.g., UNC5 which is a receptor for netrins), another receptor tyrosine kinase (e.g., LTK receptor), an actin-based cytoskeleton molecule (e.g., tensin or actinin), or an adaptor molecule (e.g., IRS). Therefore, an anti-ALK antibody or antigen-binding portion thereof may affect one or more of such interactions. As another example, an anti-ALK antibody or antigen-binding portion thereof may cause a increase in the levels of ALK protein at the cell surface (or other membrane surface that is relevant for signaling) and may in fact increase the overall levels of ALK protein. Antibodies that increase ALK protein levels may, in some instances, also increase in the kinase activity or phosphorylation state of ALK. In certain embodiments, an anti-ALK antibody or antigen-binding portion thereof will increase an ALK biochemical activity by at least 5%, or by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 100%.

The disclosure provides human and humanized versions of any of the antibodies disclosed herein, as well as antibodies and antigen-binding portions thereof that comprise at least one CDR portion derived from an antibody disclosed herein. In specific embodiments, the antibody is a monoclonal antibody that is immunocompatible with the subject to which it is to be administered, and preferably is clinically acceptable for administration to a human being.

In certain aspects, the disclosure provides a hybridoma or other cell type that produces an anti-ALK antibody disclosed herein, and particularly a hybridoma that produces an antibody denoted as 8B10, 16G2-3 and 9Cl 0-5 (also referred to as 19C 10) herein.

In further aspects, the disclosure provides methods and compositions for producing and isolating the anti-ALK antibodies disclosed herein. In specific embodiments, methods and compositions suitable for producing and isolating fully human antibodies, in particular the human antibodies denoted as 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El and E5, are provided. Such methods and compositions may include recombinant expression systems including antibody- expressing cell lines. In other aspects, the disclosure provides fully human anti-ALK antibodies as disclosed herein, in particular the human antibodies denoted as 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El and E5.

Certain embodiments provide an anti-ALK antibody comprising a heavy chain variable region that has an amino acid sequence at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to the heavy chain variable region of 8B10, 16G2-3, 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El or E5.

Certain embodiments provide an anti-ALK antibody comprising a light chain variable region that has an amino acid sequence at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to the light chain variable region of 8B10, 16G2-3, 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El or E5.

In specific embodiments, one or more CDRs of an anti-ALK antibody have an identical sequence to the corresponding CDRs of 8B10, 16G2-3, 9Cl 0-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El or E5.

Accordingly, the disclosure provides antibodies having at least one, at least two, at least three, at least four, at least five, or all six of the CDRs of the anti-ALK antibodies such as those antibodies denoted as 8B10, 16G2-3, 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El or E5. Isolated polynucleotides or nucleic acids that encode these antibodies (and fragments thereof) are also embodiments of the disclosure. Antibodies that bind to the same epitopes as these antibodies are also embodiments of the disclosure, as are antibodies that compete for binding with any of the antibodies denoted as 8B10, 16G2-3, 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El and E5. In particular the present invention encompasses an antibody that competes with the antibody denoted as 8B10, 16G2-3, 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El or E5, or an antigen-binding fragment thereof for binding to ALK or a fragment thereof (e.g., the ECD or the LBD fragment of ALK).

The disclosure provides a method of treating cancer, the method comprising administering to a patient in need thereof an effective amount of an ALK antagonist. In one embodiment, the patient is diagnosed with a glioblastoma or a hormone- independent breast cancer. Optionally, the cancer to be treated does not exhibit ALK overexpression. Optionally, the cancer is an angiogenesis independent cancer (i.e., the cancer is one for which anti-angiogenic therapies are not generally used or recommended). Optionally, the cancer is a pre-metastatic cancer. In certain embodiments, the ALK antagonist is an anti-ALK antibody, particularly an isolated antibody or antigen-binding portion thereof that binds to an epitope situated in the extracellular portion of ALK. The isolated anti-ALK antibody or antigen-binding portion thereof may inhibit ALK that is present in the cancer cells, ALK that is present in non-cancer cells that support tumor growth or survival (such as those that participate in tumor-induced angiogenesis) or a combination thereof. The isolated antibody or antigen-binding portion thereof may be administered systemically or locally.

Additionally, the disclosure provides methods of modulating (inhibiting or stimulating) angiogenesis in a patient, the method comprising administering to a patient in need thereof an effective amount of an isolated antibody or antigen- binding portion thereof that binds to an epitope situated in the extracellular portion of ALK. Optionally, the patient is diagnosed with macular degeneration, and the isolated antibody or antigen-binding portion thereof inhibits angiogenesis through binding to ALK. Alternatively, the patient is diagnosed with an angiogenesis- associated condition such as, for example, rheumatoid arthritis, psoriasis, or a risk of restenosis. In other embodiments, the patient is diagnosed with a condition that angiogenesis is desirable (also included as an angiogenesis-associated condition), such as for example, to promote wound healing.

In certain aspects, the disclosure provides a pharmaceutical preparation comprising any of the ALK antagonists disclosed herein. For the prevention of restenosis, ALK antagonists may be formulated into a coated stent or drug-eluting stent.

In certain aspects, the disclosure describes the use of ALK antagonists for preparing a medicament for treating cancer. In a specific embodiment, the cancer is a glioblastoma or a hormone-independent breast cancer. Optionally, the cancer to be treated does not exhibit ALK overexpression. Optionally, the cancer is an angiogenesis independent cancer (i.e., the cancer is one for which anti-angiogenic therapies are not generally used or recommended). Optionally, the cancer is a pre- metastatic cancer. In certain embodiments, treatment with the ALK antagonist will result in tumor regression. In specific embodiments, treatment with the ALK antagonist results in at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, by at least 90%, or at least 100% tumor regression. In one embodiment, the ALK antagonist is an anti-ALK antibody, particularly an isolated antibody or antigen- binding portion thereof that binds to an epitope situated in the extracellular portion of ALK. The isolated anti-ALK antibody or antigen-binding portion thereof may inhibit ALK that is present in the cancer cells, ALK that is present in non-cancer cells that support tumor growth or survival (such as those that participate in tumor- induced angiogenesis) or a combination thereof. The isolated antibody or antigen- binding portion thereof may be administered systemically or locally. In certain aspects, the disclosure provides a pharmaceutical preparation comprising any of the ALK agonists disclosed herein. In certain aspects, the disclosure describes the use of ALK antagonists for preparing a medicament for promoting angiogenesis where it is desirable, for example, wound healing.

In certain aspects, the antibodies or antigen-binding portions thereof disclosed herein may be covalently linked (or otherwise stably associated with) an additional functional moiety, such as for example, a label or a moiety that confers desirable pharmacokinetic and/or pharmaceutical properties. Exemplary labels include those that are suitable for detection by a method selected from the group consisting of fluorescence detection methods, positron emission tomography detection methods, and nuclear magnetic resonance detection methods. Labels may, for example, be selected from the group consisting of a fluorescent label, a radioactive label, and a label having a distinctive nuclear magnetic resonance signature. Moieties such as a polyethylene glycol (PEG) moiety may be affixed to an antibody or antigen-binding portion thereof to increase serum half-life. Moieties such as cytotoxic and/or chemotherapeutic agents may be affixed to an antibody or antigen-binding portion thereof to facilitate targeted cell killing. Labeled anti-ALK antibodies may be used to detect sites of angiogenic activity and/or tumors, and may also be used to direct therapeutic agents, such as cytotoxic or chemotherapeutic agents to the site of angiogenic activity.

In certain aspects, the antibodies or antigen-binding portions thereof disclosed herein may be as diagnostic agents, for example, to detect the presence or absence and/or to quantify the amount of ALK in a sample. The sample may be a biological sample, for example, tumor sections or cells, or blood vessels. Diagnostic kits including an antibody of the present disclosure are also provided.

An article of manufacture containing a container with an antibody or antigen- binding portion thereof and a label is also provided. Such articles include an active agent that is anti-ALK antibody or antigen-binding portion thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the inhibitory effect of anti-ALK monoclonal antibody 8B10 on tumor size using the murine MDA-MB231 breast cancer model.

Figures 2 A and 2B show the inhibitory effect of anti-ALK monoclonal antibody 8B10 on final tumor size and necrotic tumor size, respectively.

Figure 3 shows that the stimulatory effect of FGF-2 and conditioned media of MDA-MB231 breast cancer cells on ALK expression in human endothelial cells. Figures 4 A and 4B show the presence of ALK in new blood vessels in control sample and FGF-2 treated sample, respectively. Figure 5 shows the inhibitory effect of anti-ALK monoclonal antibody 8B10 on the quantity of new blood vessels induced by PTN or FGF-2 treatment.

Figure 6 shows overexpression of ALK in various cancers.

Figure 7 shows selective overexpression of ALK in colon cancer. Figure 8 shows expression of ALK in tumor cells and vasculature.

Figure 9A shows PTN and ALK expression levels in breast cancer; Figure 9B shows prognostic significance of PTN expression in breast cancer.

Figure 10 shows expression of PTN and ALK in brain cancers.

Figure 11 shows that the level of ALK expression correlates with malignant transformation in the murine U87 glioblastoma tumor model.

Figure 12 shows the inhibitory effect of anti-ALK monoclonal antibody 8B10 on wound healing in a murine model.

Figure 13 illustrates a Myc/His-tagged ALK molecule used in certain assays of the disclosure. Figure 14 shows the result of immunoprecipitation assays using HEK293 cells expressing the Myc/His-tagged ALK molecule.

Figure 15 illustrates structure prediction of the ECD of ALK using the 3D- PSSM program available at www.sbg.bio.ic.ac.uk (last accessed November 1, 2005). The ligand binding domain is predicted to be on the surface of the receptor. In addition, the ligand binding domain as well as several other regions are predicted to be involved in receptor dimerization.

Figure 16 shows the inhibitory effect of a monoclonal anti-ALK LBD antibody on the spreading of MDA-MB231 cells.

Figure 17 illustrates the ECIS method. ECIS: electric cell-substrate impedance sensing.

Figure 18 shows the inhibitory effect of an anti-ALK antibody on the invasion of an endothelial layer by breast cancer cells (MCF-7 and MDA-MB231 cells) measured by the ECIS method.

Figure 19 shows the analysis of the antibody 19C10 (also known as 9Cl 0-5) by ECIS. MDA-MB-231 cells were used in the analysis. Top panel shows all treatments (except the 100 μg/ml 19C10 sample) done in duplicate, thus 2 traces for each sample. Bottom panel shows a repeat study of the same analysis, and all samples were done in duplicate in this study.

Figure 20 shows the different cell morphology between the cells in the control samples and the cells treated with the antibody 19C10 in the ECIS studies. The cells in the 19C10-treated samples were growing in clumps, rather than attaching well and spreading.

Figures 21 A and 21 B show that an anti-ALK IgM antibody (19C10) inhibits soft agar colony formation. The data plotted in Figure 21 A show that the antibody 6A6 has little effect on colony formation while 19C10 at either 50 μg/ml or 100 μg/ml reduces colony formation nearly as well as the positive control taxol. Figure 2 IB shows representative photographs of cells treated with 50 μg/ml or 100 μg/ml of 6A6 (top middle and right, respectively); 50 μg/ml or 100 μg/ml of 19C10 (bottom left and middle, respectively); 10 nM taxol (bottom right); and untreated cells (top left). Figure 22 shows that the antibody 19C 10 does not bind to ALK-ECD (right) as well as it does to an ALK-LBD-Fc fusion protein (left).

Figure 23 shows the result of the ALK-ECD panning experiments; 67 unique clones were identified. The ALK-ECD-Myc/His expression vector was transfected into 293F cells and the peptide was purified from the conditioned media for using in the panning experiment.

Figure 24 shows the phylogenic tree for 24 unique λ chains identified by the panning experiments.

Figures 25A-25N show the nucleotide and corresponding amino acid sequences of the variable regions of the heavy (V_H) and the light chains (VL) of the human anti-ALK antibodies of the invention. A) 3Al 1 V_H (nucleotide sequence: SEQ ID NO: 7; amino acid sequence: SEQ ID NO: 8) and V_L (nucleotide sequence: SEQ ID NO: 9; amino acid sequence: SEQ ID NO: 10); B) 6A2 V_H (nucleotide sequence: SEQ ID NO: 11; amino acid sequence: SEQ ID NO: 12) and V_L (nucleotide sequence: SEQ ID NO: 13; amino acid sequence: SEQ ID NO: 14); C) Al V_H (nucleotide sequence: SEQ ID NO: 15; amino acid sequence: SEQ ID NO: 16) and V_L (nucleotide sequence: SEQ ID NO: 17; amino acid sequence: SEQ ID NO: 18); D) A2 V_H (nucleotide sequence: SEQ ID NO: 19; amino acid sequence: SEQ ID NO: 20) and V_L (nucleotide sequence: SEQ ID NO: 21; amino acid sequence: SEQ ID NO: 22); E) A7 V_H (nucleotide sequence: SEQ ID NO: 23; amino acid sequence: SEQ ID NO: 24) and V_L (nucleotide sequence: SEQ ID NO: 25; amino acid sequence: SEQ ID NO: 26); F) B2 V_H (nucleotide sequence: SEQ ID NO: 27; amino acid sequence: SEQ ID NO: 28) and V_L (nucleotide sequence: SEQ ID NO: 29; amino acid sequence: SEQ ID NO: 30); G) B6 V_H (nucleotide sequence: SEQ ID NO: 31 ; amino acid sequence: SEQ ID NO: 32) and V_L (nucleotide sequence: SEQ ID NO: 33; amino acid sequence: SEQ ID NO: 34); H) B8 V_H (nucleotide sequence: SEQ ID NO: 35; amino acid sequence: SEQ ID NO: 36) and V_L (nucleotide sequence: SEQ ID NO: 37; amino acid sequence: SEQ ID NO: 38); I) Cl VH (nucleotide sequence: SEQ ID NO: 39; amino acid sequence: SEQ ID NO: 40) and V_L (nucleotide sequence: SEQ ID NO: 41; amino acid sequence: SEQ ID NO: 42); J) C6 V_H (nucleotide sequence: SEQ ID NO: 43; amino acid sequence: SEQ ID NO: 44) and V_L (nucleotide sequence: SEQ ID NO: 45; amino acid sequence: SEQ ID NO: 46); K) D5 V_H (nucleotide sequence: SEQ ID NO: 47; amino acid sequence: SEQ ID NO: 48) and VL (nucleotide sequence: SEQ ID NO: 49 ; amino acid sequence: SEQ ID NO: 50); L) DlO VH (nucleotide sequence: SEQ ID NO: 51; amino acid sequence: SEQ ID NO: 52) and V_L (nucleotide sequence: SEQ ID NO: 53; amino acid sequence: SEQ ID NO: 54); M) El V_H (nucleotide sequence: SEQ ID NO: 55; amino acid sequence: SEQ ID NO: 56) and V_L

(nucleotide sequence: SEQ ID NO: 57; amino acid sequence: SEQ ID NO: 58); and N) E5 V_H (nucleotide sequence: SEQ ID NO: 59; amino acid sequence: SEQ ID NO: 60) and V_L (nucleotide sequence: SEQ ID NO: 61; amino acid sequence: SEQ ID NO: 62). Figure 26 shows the ELISA study that compares binding to ALK-LBD-Fc and ALK-ECD by selected phage clones. The ELISA plate surface was coated with 5ug/ml ALK-LBD or ALK-ECD protein, and then blocked with 4% milk. 15 μl of selected phage Fab supernatants was mixed with 10 μl of 4% milk and added to the coated surface. The plate was then washed and HRP labeled anti-M13 antibody at 1 :3000 dilution was then added. Clones were divided into four groups on the basis of their binding specificity. Figure 27 shows binding by 2 selected phage clones the surface of ALK- expressing cells.

Figure 28 shows binding to ALK-LBD and ALK-ECD by 5 of the human IgGs converted from selected phage clones. ALK-ECD-My c/His was at 10 μg/ml; blocking was done with milk; human IgGs were added as indicated; detection was through 1 : 1000 anti human IgG-HRP.

Figure 29 also shows binding to ALK-LBD and ALK-ECD by the remaining 9 human IgGs converted from selected phage clones.

Figure 30 shows binding of the converted human IgGs to the surface of ALK-expressing cells, as determined by FACS. DlO and Al showing binding while several other antibodies, C6, B 8 and A2 do not in this assay.

Figure 31 shows the binding kinetics between the Al antibody (top) and DlO antibody (bottom) and the ALK-ECD-Myc/His as determined by BIAcore.

Figure 32 shows that the antibody DlO stimulates cell growth as examined by soft agar assays.

Figure 33 also shows the agonistic activity of the antibody DlO. The Western Blot shows the level of ALK autophorylation as detected by the antibody 4G10 that specifically binds to phosphorylated ALK. As shown in the bottom panel, the total amount (phosphorylated or unphosphorylated) of ALK is comparable between the untreated sample, the PTN-treated samples, and the DlO-treated samples. However, the DlO-treated samples have significantly higher level of phosphorylated ALK as compared to the untreated sample and the PTN-treated samples.

DETAILED DESCRIPTION OF THE DISCLOSURE /. ALK Antibodies

The disclosure provides, in part, defined portions of the ALK molecule that can be effectively targeted by polypeptide binding agents, such as antibodies, antigen-binding portions of antibodies, and non-immunoglobulin antigen-binding scaffolds. The ALK polypeptide binding agents described herein may be used to treat a variety of disorders, particularly angiogenesis-associated conditions. An angiogenesis-associated condition includes a condition associated with unwanted angiogenesis, such as, for example, cancers or AMD. Alternatively, an angiogenesis-associated condition can be a condition for which angiogenesis is desirable, such as, for example, wound healing.

ALK belongs to a family of transmembrane receptor protein tyrosine kinases. ALK is composed of three principal portions, an extracellular region or domain (ECD), a transmembrane domain (TM), and an intracellular domain (ICD). The ECD of ALK contains a PTN-binding site, also termed ligand-binding domain (LBD). In human ALK, the LBD encompasses amino acids 368-447 of the ALK sequence of SEQ ID NO:1, with the actual contact with PTN predicted to be located at amino acids 391-401. More specifically, a 16 amino acid sequence is predicted to be responsible for ligand binding as discussed below. Within the ECD of ALK are also the MAM domains, typical signature patterns for ECDs.

SEQ ID NO: 1— Human ALK

>gi I 24541681 gb| AAB71619.11 anaplastic lymphoma kinase [Homo sapiens] MGΆIGLLWLLPLLLSTAAVGSGMGTGQRΆGSPAΆGSPLQPREPLSYSRLQRKSLAVDFVVPSLFRVYA

RD

LLLPPSSSELKAGRPEARGSLALDCAPLLRLLGPAPGVSWTAGSPAPAEARTLSRVLKGGSVRKLRRA

KQ

LVLELGEEAILEGCVGPPGEAAVGLLQFNLSELFSWWIRQGEGRLRIRLMPEKKASEVGREGRLSAAI RA

SQPRLLFQIFGTGHSSLESPTNMPSPSPDYFTWNLTWIMKDSFPFLSHRSRYGLECSFDFPCELEYSP

PL

HDLRNQSWSWRRIPSEEASQMDLLDGPGAERSKEMPRGSFLLLNTSADSKHTILSPWMRSSSEHCTLA

VS VHRHLQPSGRYIAQLLPHNEAAREILLMPTPGKHGWTVLQGRIGRPDNPFRVALEYISSGNRSLSAVD

FF

ALKNCSEGTSPGSKMALQSSFTCWNGTVLQLGQACDFHQDCAQGEDESQMCRKLPVGFYCNFEDGFCG

WT

QGTLSPHTPQWQVRTLKDARFQDHQDHALLLSTTDVPASESATVTSATFPAPIKSSPCELRMSWLIRG VL

RGNVSLVLVENKTGKEQGRMVWHVAAYEGLSLWQWMVLPLLDVSDRFWLQMVAWWGQGSRAIVAFDNI

SI

SLDCYLTISGEDKILQNTAPKSRNLFERNPNKELKPGENSPRQTPIFDPTVHWLFTTCGASGPHGPTQ

AQ CNNAYQNSNLSVEVGSEGPLKGIQIWKVPATDTYSISGYGAAGGKGGKNTMMRSHGVSVLGIFNLEKD

DM

LYILVGQQGEDACPSTNQLIQKVCIGENNVIEEEIRVNRSVHEWAGGGGGGGGATYVFKMKDGVPVPL

II

AAGGGGRAYGAKTDTFHPERLENNSSVLGLNGNSGAAGGGGGWNDNTSLLWAGKSLQEGATGGHSCPQ AM

KKWGWETRGGFGGGGGGCSSGGGGGGYIGGNAASNNDPEMDGEDGVSFISPLGILYTPALKVMEGHGE

VN

IKHYLNCSHCEVDECHMDPESHKVICFCDHGTVLAEDGVSCIVSPTPEPHLPLSLILSVVTSALVAAL

VL AFSGIMIVYRRKHQELQAMQMELQSPEYKLSKLRTSTIMTDYNPNYCFAGKTSSISDLKEVPRKNITL

IR GLGHGAFGEVYEGQVSGMPNDPSPLQVΆVKTLPEVCSEQDELDFLMEΆLIISKFNHQNIVRCIGVSLQ

SL

PRFILLELMAGGDLKSFLRETRPRPSQPSSLAMLDLLHVARDIACGCQYLEENHFIHRDIAARNCLLT

CP GPGRVAKIGDFGiyiARDIYRASYYRKGGCAMLPVKWMPPEAFMEGIFTSKTDTWSFGVLLWEIFSLGYM

PY

PSKSNQEVLEFVTSGGRMDPPKNCPGPVYRIMTQCWQHQPEDRPNFAIILERIEYCTQDPDVINTALP

IE

YGPLVEEEEKVPVRPKDPEGVPPLLVSQQAKREEERSPAAPPPLPTTSSGKAAKKPTAAEVSVRVPRG PA

VEGGHVNMAFSQSNPPSELHKVHGSRNKPTSLWNPTYGSWFTEKPTKKNNPIAKKEPHDRGNLGLEGS

CT

VPPNVATGRLPGASLLLEPSSLTANMKEVPLFRLRHFPCGNVNYGYQQQGLPLEAATAPGAGHYEDTI

LK SKNSMNQPGP

SEQ ID NO:2— mouse ALK

>gi| 6680680 I ref |NP_031465.11 anaplastic lymphoma kinase isoform 1 [Mus musculus] MGAAGFLWLLPPLLLAAASYSGAATDQRAGSPASGPPLQPREPLSYSRLQRKSLAVDFVVPSLFRVYA RD

LLLPQPRSPSEPEAGGLEARGSLALDCEPLLRLLGPLPGISWADGASSPSPEAGPTLSRVLKGGSVRN VR RAKQLVLELGEETILEGCIGPPEEVAAVGILQFNLSELFSWWILHGEGRLRIRLMPEKKASEVGREGR LS

SAIRASQPRLLFQIFGTGHSSLESPSETPSPPGTFMWNLTWTMKDSFPFLSHRSRYGLECSFDFPCEL EY

SPPLHNHGNQSWSWRHVPSEEASRMNLLDGPEAEHSQEMPRGSFLLLNTSADSKHTILSPWMRSSSDH CT LAVSVHRHLQPSGRYVAQLLPHNEAGREILLVPTPGKHGWTVLQGRVGRPANPFRVALEYISSGNRSL SA

VDFFALKNCSEGTSPGSKMALQSSFTCWNGTVLQLGQACDFHQDCAQGEDEGQLCSKLPAGFYCNFEN GF CGWTQSPLSPHMPRWQVRTLRDAHSQGHQGRALLLSTTDILASEGATVTSATFPAPMKNSPCELRMSW Li

RGVLRGNVSLVLVENKTGKEQSRTVWHVATDEGLSLWQHTVLSLLDVTDRFWLQIVTWWGPGSRATVG FD

NISISLDCYLTISGEEKMSLNSVPKSRNLFEKNPNKESKSWANISGPTPIFDPTVHWLFTTCGASGPH GP TQAQCNNAYQNSNLSVVVGSEGPLKGVQIWKVPATDTYSISGYGAAGGKGGKNTMMRSHGVSVLGIFN LE

KGDTLYILVGQQGEDACPRANQLIQKVCVGENNVIEEEIRVNRSVHEWAGGGGGGGGATYVFKMKDGV PV PLIIAAGGGGRAYGAKRETFHPERLESNSSVLGLNGNSGAAGGGGGWNDNTSLLWAGKSLLEGAAGGH sc

PQAMKKWGWETRGGFGGGGGGCSSGGGGGGYIGGNAASNNDPEMDGEDGVSFISPLGILYTPALKVME

GH

GEVNIKHYLNCSHCEVDECHMDPESHKVICFCDHGTVLADDGVSCIVSPTPEPHLPLSLILSVVTSAL

VA ALVLAFSGIMIVYRRKHQELQAMQIQLQSPEYKLSKLRTSTIMTDYNPNYCFAGKTSSISDLKEVPRK

NI

TLIRGLGHGAFGEVYEGQVSGMPNDPSPLQVAVKTLPEVCSEQDELDFLMEALIISKFNHQNIVRCIG

VS

LQALPRFILLELMAGGDLKSFLRETRPRPNQPTSLAMLDLLHVARDIACGCQYLEENHFIHRDIAARN CL LTCPGAGRIAKIGDFGMARDIYRASYYRKGGCAMLPVKWMPPEAFMEGIFTSKTDTWSFGVLLWEIFS LG

YMPYPSKSNQEVLEFVTSGGRMDPPKNCPGPVYRIMTQCWQHQPEDRPNFAIILERIEYCTQDPDVIN TA LPIEYGPVVEEEEKVPMRPKDPEGMPPLLVSPQPAKHEEASAAPQPAALTAPGPSVKKPPGAGAGAGA GA

GAGPVPRGAADRGHVNMAFSQPNPPPELHKGPGSRNKPTSLWNPTYGSWFTAKPAKKTHPPPGAEPQA RA

GAAEGGWTGPGAGPRRAEAALLLEPSALSATMKEVPLFRLRHFPCPNVNYGYQQQGLPLEATAAPGDT ML

KSKNKVTQPGP

As used herein, the term "ALK" refers to an ALK polypeptide from a mammal including humans. In one embodiment, the antibodies (immunoglobulins) are raised against an isolated and/or recombinant mammalian ALK or portion thereof (e.g., peptide) or against a host cell which expresses recombinant mammalian ALK or portion thereof. In certain aspects, antibodies of the disclosure specifically bind to an extracellular portion of an ALK protein. As used herein, the ALK proteins or polypeptides include fragments, functional variants, and modified forms of ALK polypeptide.

In one aspect, this disclosure provides anti-ALK antibodies, which include, without limitation, intact monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two antigen-binding domains, and antibody fragments, so long as they exhibit the desired biological activity. The immunoglobulin molecules of the invention can be of any type {e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

Native IgG antibodies and native immunoglobulins (which may include a variety of structurally related proteins that are not necessarily antibodies) are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (V_L) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.

Certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework region (FR). The variable domains of native heavy and light chains each comprise four FRs (FRl, FR2, FR3 and FR4, respectively), largely adopting a beta- sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), pages 647-669). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

Hypervariable region usually includes the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (i.e. amino acid residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop" (i.e. amino acid residues 26-32 (Ll), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. MoI. Biol. 196:901-917 (1987)). "Framework" or "FR" residues typically include those variable domain residues other than the hypervariable region residues. Generally, CDRs vary considerably from antibody to antibody (and by definition will not exhibit homology with the Kabat consensus sequences). Maximal alignment of FR residues frequently requires the insertion of "spacer" residues in the numbering system, to be used for the Fv region. It will be understood that the CDRs referred to herein correspond to those of Kabat et al., supra. In addition, the identity of certain individual residues at any given Kabat site number may vary from antibody chain to antibody chain due to interspecies or allelic divergence.

This disclosure also provides fragments of ALK antibodies, which may comprise a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')₂, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng., 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')₂ fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

"Fv" usually refers to the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V_H-V_L dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although likely at a lower affinity than the entire binding site. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')₂ antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

This disclosure also provides monoclonal ALK antibodies. A monoclonal antibody can be obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are often synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. Monoclonal antibodies may also be produced in transfected cells, such as CHO cells and NSO cells. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and does not require production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al, Nature, 256: 495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. Nos. 4,816,567 and 6,331,415). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. MoI. Biol, 222: 581-597 (1991), for example. Other antibodies specifically contemplated are "oligoclonal" antibodies. As used herein, the term "oligoclonal" antibodies" refers to a predetermined mixture of distinct monoclonal antibodies. See, e.g., PCT publication WO 95/20401; U.S. Pat. Nos. 5,789,208 and 6,335,163. In one embodiment, oligoclonal antibodies consist of a predetermined mixture of antibodies against one or more epitopes are generated in a single cell. In other embodiments, oligoclonal antibodies comprise a plurality of heavy chains capable of pairing with a common light chain to generate antibodies with multiple specificities (e.g., PCT publication WO 04/009618). Oligoclonal antibodies are particularly useful when it is desired to target multiple epitopes on a single target molecule (e.g., ALK). In view of the assays and epitopes disclosed herein, those skilled in the art can generate or select antibodies or mixture of antibodies that are applicable for an intended purpose and desired need. In certain embodiments that include a humanized and/or chimeric antibody, one or more of the CDRs are derived from an anti-human ALK antibody. In a specific embodiment, all of the CDRs are derived from an anti-human ALK antibody. In another specific embodiment, the CDRs from more than one anti- human ALK antibodies are mixed and matched in a chimeric antibody. For instance, a chimeric antibody may comprise a CDRl from the light chain of a first anti-human ALK antibody may be combined with CDR2 and CDR3 from the light chain of a second anti-human ALK antibody, and the CDRs from the heavy chain may be derived from a third anti-human ALK antibody. Further, the framework regions may be derived from one of the same anti-human ALK antibodies, from one or more different antibodies, such as a human antibody, or from a humanized antibody.

"Single-chain Fv" or "scFv" antibody fragments comprise the V_H and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and V_L domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, eds. (Springer- Verlag: New York, 1994), pp. 269-315.

The term "diabodies" refers to small antibody fragments with two antigen- binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (V_L) in the same polypeptide chain (VH- V_L). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In specific embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, or greater than 99% by weight, (2) to a degree that complies with applicable regulatory requirements for administration to human patients (e.g., substantially pyrogen-free); (3) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (4) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step, for example, an affinity chromatography step, an ion (anion or cation) exchange chromatography step, or a hydrophobic interaction chromatography step.

Certain embodiments provide an ALK activating antibody or agonist antibody that promotes one or more ALK-regulated biological functions. ALK activating antibodies may promote an ALK-regulated biological function by at least about 10% when added to a cell, tissue or organism expressing ALK. In one embodiment, the antibody promotes an ALK-regulated biological function by at least 20%, or by at least 40%, or by at least 60%, or by at least 80%, or by at least 85%. An ALK activating antibody may affect ALK through one or more biochemical mechanisms, such as by increasing ALK autokinase activity. In a specific embodiment, the antibody activates ALK kinase activity by at least 20%, or by at least 40%, or by at least 60%, or by at least 80%, or by at least 85%. ALK kinase activity may be measured as the phosphorylation state of ALK itself (tyrosine auto-phosphorylation) or its substrate proteins. Alternatively, ALK kinase activity may be determined by measuring the phosphorylation state of a substrate for ALK, such as, for example, the peptide substrates as described in Donella-Deana et al. ((2005) Biochemistry 44:8533-8542). An ALK activating antibody or agonist antibody also includes an antibody that potentiates the binding between ALK and its ligand and/or ALK dimerization and/or the specific interaction between activated (or phosphorylated) ALK with another protein (e.g., UNC5, IRS, actinin, or tensin).

Certain embodiments provide an ALK inhibiting antibody or antagonist antibody, which include an antibody that inhibits an ALK-regulated biological function by at least about 10% when added to a cell, tissue or organism expressing ALK. In one embodiment, the antibody inhibits an ALK-mediated biological function by at least 20%, or by at least 40%, or by at least 60%, or by at least 80%, or by at least 85%. An ALK inhibiting antibody may affect ALK through one or more biochemical mechanisms, such as by decreasing ALK kinase activity. In a specific embodiment, the antibody inhibits ALK kinase activity by at least 20%, or by at least 40%, or by at least 60%, or by at least 80%, or by at least 85%. ALK kinase activity may be measured as the phosphorylation state of ALK itself (tyrosine auto-phosphorylation) or its substrate proteins. Certain ALK inhibiting antibodies may inhibit the binding of ALK to one or more of its ligands, e.g., PTN. This type of antibody is commonly referred to in the art as a neutralizing antibody. An ALK neutralizing antibody may, when varying amounts of the anti-ALK antibody are used, reduce the amount of ALK bound to PTN by at least about 20% or by at least 40%, or by at least 60%, or by at least 80%, or by at least 85%. The binding reduction may be measured by any means, for example, as measured in an in vitro competitive binding assay. It is contemplated that an ALK inhibiting antibody may inhibit the binding of ALK to one ligand and not to another ligand. For example, two naturally occurring forms of PTN (18 and 15 kDa) have been reported (Lu et al. (2005) J. Biol. Chem. 280:26953-26964). In certain embodiments, an ALK inhibiting antibody inhibits the binding of the 15 kDa form of PTN, or the 18 kDa form of PTN, or of both forms of PTN. An "ALK inhibiting antibody" or antagonist antibody also includes an antibody that reduces the binding between ALK and its ligand and/or ALK dimerization and/or the specific interaction between ALK with another protein (e.g., UNC5, IRS, actinin, or tensin). It is contemplated that an ALK inhibiting antibody may bind and induce activation of an ALK biochemical activity, such as for example, kinase activity, wherein the activation results in the down regulation of ALK, for example by internalization and degradation. By down regulating the receptor, such an ALK antibody would have the net effect of an antagonist (i.e., cause a decrease in one or more ALK-regulated biological functions.).

As used herein, the terms "label" or "labeled" refers to incorporation of another molecule in the antibody. In one embodiment, the label is a detectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In another embodiment, the label or marker can be therapeutic, e.g., a drug conjugate or toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵1, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents, such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

As shown in the Figures and Examples below, Applicants have generated monoclonal antibodies against ALK as well as hybridoma cell lines producing ALK monoclonal antibodies 8B10 (deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, Va. 20110-2209) and assigned ATCC Deposit No. PTA-7429), 16G2-3 and 9Cl 0-5 (also referred to herein as "19C10"). These antibodies were characterized in many ways, such as, their ability to modulate angiogenesis, their ability to modulate invasion by cancer cells, their ability to modulate wound healing, their ability to modulate cell adhesion and their ability to modulate tumor growth.

Further, Applicants have generated fully human antibodies against ALK. 14 examples of these fully human antibodies, denoted as 3Al 1, 6A2, Al₃ A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El and E5, are described in Figures 25A-25N, which provide nucleotide and amino acid sequences (SEQ ID NOS: 7-62) for each heavy and light chain of these 14 fully human anti-ALK antibodies. These antibodies were initially selected from a phage library of human antibodies by panning with ALK or ALK fragments (e.g., ECD or LBD) and then converted into fully human IgGs. These antibodies were also characterized in many ways (before or after the conversion into full IgGs), such as, their ability to bind ALK or different fragments of ALK (e.g., ECD or LBD) in vitro, their ability to bind ALK expressed on cell surface, and other assays. For example, the antibody DlO was examined by the soft agar assays, which results are shown in Figure 32 and indicate DlO's agonistic activities. DlO was also analyzed for its ability to modulate an ALK kinase activity. As illustrated in Figure 33, DlO enhanced autophosphorylation of ALK at a level even greater than PTN.

In certain aspects, the disclosure provides anti-ALK antibodies that bind to the same epitope as a monoclonal anti-ALK antibody or a human anti-ALK antibody described herein, such as the antibody denoted as 8B10, 16G2-3 and 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl₅ C6, D5, DlO, El, or E5.

In certain aspects, the disclosure provides anti-ALK antibodies that bind to an epitope or an ALK protein (including fragments thereof) competitively against a monoclonal anti-ALK antibody or a human anti-ALK antibody described herein, such as the antibody denoted as 8B10, 16G2-3 and 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El, or E5. In certain aspects, the disclosure provides anti-ALK antibodies that comprise one or more regions or domains of a monoclonal anti-ALK antibody or a human anti-ALK antibody described herein, such as the antibody denoted as 8B10, 16G2-3 and 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El, or E5. The one or more regions or domains may include the variable region of a heavy chain, the variable region of a light chain, one or more variable domains of a heavy chain, one or more variable domains of a light chain, or one or more CDRs of any of the antibodies.

In certain aspects, an anti-ALK antibody of the disclosure includes an amino acid sequence that is at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to the amino acid sequence of a variable region of a monoclonal anti-ALK antibody or a human anti-ALK antibody described herein, such as the antibody denoted as 8B10, 16G2-3 and 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El, or E5. Thus, certain aspects of the disclosure provide anti-ALK antibodies that have an amino acid sequence that is homologous or similar to the amino acid sequence of a variable region of the antibody denoted as 8B10, 16G2-3 and 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El, or E5.

In certain aspects, the disclosure provides a nucleic acid or polynucleotide encoding an anti-ALK antibody. Such a nucleic acid may include a nucleotide sequence that is at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to the nucleotide sequence of a variable region of a monoclonal anti-ALK antibody or a human anti- ALK antibody described herein, such as the antibody denoted as 8B10, 16G2-3 and 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El, or E5. Thus, certain aspects of the disclosure provide isolated nucleic acids having a nucleotide sequence that is homologous or similar to the nucleotide sequence of a variable region of the antibody denoted as 8B10, 16G2-3 and 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El, or E5. Certain embodiments provides a nucleic acid that encodes an anti-ALK antibody. The encoded antibody may include an amino acid sequence that is at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to the amino acid sequence of a variable region of a monoclonal anti-ALK antibody or a human anti-ALK antibody described herein, such as the antibody denoted as 8B10, 16G2-3 and 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El, or E5. Thus, certain aspects of the disclosure provide isolated nucleic acids encoding anti-ALK antibodies that have an amino acid sequence that is homologous or similar to the amino acid sequence of a variable region of the antibody denoted as 8B10, 16G2-3 and 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El, or E5. As used herein, "homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology and identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology/similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. In comparing two sequences, the absence of residues (amino acids or nucleic acids) or presence of extra residues may also decrease the identity and homology/similarity.

The term "homology" describes a mathematically based comparison of sequence similarities which is used to identify genes or proteins with similar functions or motifs. The nucleic acid and protein sequences of the present application may be used as a "query sequence" to perform a search against public databases to, for example, identify other family members, related sequences or homologs. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J MoI. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the application. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the application. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and BLAST) can be used.

As used herein, "identity" means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988, Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-3402 ( 1997)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. MoI. Biol. 215: 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity.

In certain aspects, antibodies of the disclosure specifically bind to an ECD of an ALK protein, particularly an LBD of ALK. Exemplary ALK proteins or polypeptides are provided in SEQ ID NOs: 1 and 2. As used herein, the ALK proteins or polypeptides include fragments, functional variants, and modified forms of ALK polypeptide.

Examples of antibodies include, but are not limited to, monoclonal anti-ALK antibodies 8B10, 16G2-3 and 9Cl 0-5, and human antibodies A311, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El and E5. Optionally, the immunoglobulins can bind to ALK with a dissociation constant or K_d (k_off/k_on) of less than IxIO^"6, of less than IxIO^"7, of less than Ix 10^"8, of less than IxIO^"9 M, of less than IxIO^"11 M, of less than IxIO^"12 M or of less than IxIO^'13 M or less. Optionally, the immunoglobulins can bind to ALK with a K_off of less than IxIO^"3 s^"1, less than 5x10^{" 3} S^"1, less than IxIO^-4 S^"1, less than 5XlO^-4 S^"1, less than Ix IO^-5 S^"1, less than 5XlO^-5 S^"1, less than Ix 10^"6 S^"1, less than 5XlO^-6 S^"1, less than IxIO^-7 S^"1, less than 5XlO^-7 S^"1, less than IxIO^-8S^"1, less than 5xlθ^-8 s^-1, less than IxIO^-9S^"1, less than 5XlO^-9S^"1, or less than 10^"10 s^"1. Optionally, the immunoglobulins can bind to ALK with an association rate constant or k_on rate of at least IxIO⁵M^-1S^-1, at least 5 x 10⁵ M^-1S^-1, at least IxIO⁶ M^-1S^-1, at least 5 x 10⁶M^-1S^-1, at least IxIO⁷M^-1S^"1, at least 5 x 10⁷M^-1S^-1, or at least 1 x 10⁸ M- ' S- ', or at least 10⁹ M^" ' s^" 1. Optionally, antibodies and portions thereof bind to ALK with an affinity that is roughly equivalent to that or substantially better than that of a ligand (e.g., PTN) of ALK.

Antibodies disclosed herein will preferably be specific for ALK, with minimal binding to other members of receptor tyrosine kinase families. In another aspect, the anti-ALK antibody demonstrates both species and molecule selectivity. In one embodiment, the anti-ALK antibody binds to human ALK. In certain embodiments, the anti-ALK antibody does not bind to mouse, rat, guinea pig, dog, or rabbit ALK. Optionally, the antibody does bind to multiple different ALKs from different species, such as human and mouse. Following the teachings of the specification, one may determine the species selectivity for the anti-ALK antibody using various methods. For instance, one may determine species selectivity using Western blot, FACS, ELISA or RIA. In one embodiment, one may determine the species selectivity using Western blot. In another embodiment, the anti-ALK antibody has a tendency to bind ALK that is at least 50 times greater than its tendency to bind other members of the receptor tyrosine kinase family from the same species, and preferably 100 or 200 times greater. One may determine selectivity using methods well known in the art following the teachings of the specification. For instance, one may determine the selectivity using Western blot, FACS, ELISA or RIA. In one embodiment, one may determine the molecular selectivity using Western blot. The anti-ALK antibody may be an IgG, an IgM, an IgE, an IgA or an IgD molecule. In one embodiment, the antibody is an IgG and is an IgGl, IgG2, IgG3, or IgG4 subtype. In another embodiment, the anti-ALK antibody is subclass IgG2. The class and subclass of ALK antibodies may be determined by any method known in the art. In general, the class and subclass of an antibody may be determined using antibodies that are specific for a particular class and subclass of antibody. Such antibodies are available commercially. The class and subclass can be determined by ELISA, Western Blot as well as other techniques. Alternatively, the class and subclass may be determined by sequencing all or a portion of the constant domains of the heavy and/or light chains of the antibodies, comparing their amino acid sequences to the known amino acid sequences of various class and subclasses of immunoglobulins, and determining the class and subclass of the antibodies. An anti- ALK antibody of the disclosure may include portions from different classes or subclasses of immunoglobulins. For example, one or more CDRs from an anti-ALK IgM antibody can be grafted onto an IgG molecule to create an anti-ALK IgG antibody.

In certain embodiments, single chain antibodies, and chimeric, humanized or primatized (CDR-grafted) antibodies, as well as chimeric or CDR-grafted single chain antibodies, comprising portions derived from different species, are also encompassed by the present disclosure as antigen-binding portions of an antibody. The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., U.S. Pat. Nos. 4,816,567 and 6,331,415; U.S. Pat. No. 4,816,397; European Patent No. 0,120,694; WO 86/01533; European Patent No. 0,194,276 Bl; U.S. Pat. No.

5,225,539; and European Patent No. 0,239,400 Bl. See also, Newman, R. et al, BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody. See, e.g., Ladner et al., U.S. Pat. No. 4,946,778; and Bird, R. E. et al., Science, 242: 423-426 (1988)), regarding single chain antibodies.

In addition, functional fragments of antibodies, including fragments of chimeric, humanized, primatized or single chain antibodies, can also be produced. Functional fragments of the subject antibodies retain at least one binding function and/or modulation function of the full-length antibody from which they are derived. Preferred functional fragments retain an antigen-binding function of a corresponding full-length antibody (e.g., specificity for an ALK). Certain preferred functional fragments retain the ability to inhibit one or more functions characteristic of an ALK, such as a binding activity, a signaling activity, and/or stimulation of a cellular response. For example, in one embodiment, a functional fragment of an ALK antibody can inhibit the interaction of ALK with its ligand (e.g., PTN) and/or can inhibit one or more receptor-mediated functions, such as auto-phosphorylation of the receptor itself, phosphorylation of substrates of the receptor, cell proliferation, angiogenesis, cell adhesion or invasion, and/or tumor growth.

For example, antibody fragments capable of binding to an ALK receptor or portion thereof, including, but not limited to, Fv, Fab, Fab' and F(ab')₂ fragments are encompassed by the disclosure. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons has been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab')₂ heavy chain portion can be designed to include DNA sequences encoding the CHi domain and hinge region of the heavy chain.

A humanized antibody is an antibody that is derived from a non-human species, in which certain amino acids in the framework and constant domains of the heavy and light chains have been mutated so as to avoid or abrogate an immune response in humans. Alternatively, a humanized antibody may be produced by fusing the constant domains from a human antibody to the variable domains of a non-human species. Accordingly, the present disclosure relates to a humanized immunoglobulin having binding specificity for an ALK (e.g., human ALK), the immunoglobulin comprising an antigen-binding region of nonhuman origin (e.g., rodent) and at least a portion of an immunoglobulin of human origin (e.g., a human framework region, a human constant region or portion thereof). For example, the humanized antibody can comprise portions derived from an immunoglobulin of nonhuman origin with the requisite specificity, such as a mouse, and from immunoglobulin sequences of human origin (e.g., a chimeric immunoglobulin), joined together chemically by conventional techniques (e.g., synthetic) or prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of the chimeric antibody can be expressed to produce a contiguous polypeptide chain).

Another example of a humanized antibody of the present disclosure is an immunoglobulin containing one or more immunoglobulin chains comprising a CDR of nonhuman origin (e.g., one or more CDRs derived from an antibody of nonliuman origin) and a framework region derived from a light and/or heavy chain of human origin (e.g., CDR-grafted antibodies with or without framework changes). In one embodiment, the humanized immunoglobulin can compete with murine monoclonal antibody for binding to an ALK polypeptide. Chimeric or CDR-grafted single chain antibodies can also be humanized antibodies.

In certain embodiments, the present disclosure provides neutralizing or antagonist antibodies. As described herein, the term "antagonist antibody" refers to an antibody that can inhibit one or more functions of an ALK, such as a binding activity (e.g., ligand binding, dimerization or specific interaction with another protein such as, for example, UNC5) and a signaling activity (e.g., auto- phosphorylation of ALK, stimulation of a cellular response, such as stimulation of cell proliferation, change of cell adhesion, or angiogenesis). For example, an antagonist antibody can inhibit (reduce or prevent) the interaction of an ALK receptor with a natural ligand (e.g., PTN or fragments thereof) or the specific interaction of an activated or phosphorylated ALK with another protein (e.g., UNC5). In one embodiment, antagonist antibodies directed against ALK can inhibit functions mediated by ALK, including endothelial cell migration, angiogenesis, cell adhesion, cell proliferation, and/or tumor growth. Optionally, the antagonist antibody binds to the ECD of ALK or a portion thereof (e.g., the LBD of ALK). In other embodiments, the present disclosure provides agonist antibodies that stimulate an ALK-mediated biological function. An agonist antibody may enhance an ALK kinase activity, even independent of any ligand, e.g., PTN. An agonist antibody may potentiate the interaction between ALK and its natural ligand or the specific interaction between activated ALK and another protein, such as for example, UNC5.

In certain embodiments, anti-idiotypic antibodies are also provided. Anti- idiotypic antibodies recognize antigenic determinants associated with the antigen- binding site of another antibody. Anti-idiotypic antibodies can be prepared against a first antibody by immunizing an animal of the same species, and preferably of the same strain, as the animal used to produce the first antibody. See, e.g., U.S. Pat. No. 4,699,880. In one embodiment, antibodies are raised against receptor (e.g., ALK) or a portion thereof (e.g., extracellular domain of ALK), and these antibodies are used in turn to produce an anti-idiotypic antibody. The anti-idiotypic antibodies produced thereby can bind compounds which bind receptor, such as ligands of receptor function, and can be used in an immunoassay to detect or identify or quantify such compounds. Such an anti-idotypic antibody can also be an inhibitor of an ALK receptor function, although it does not bind receptor itself. Such an anti-idotypic antibody can also be called an antagonist antibody.

//. Methods of Antibody Production

In certain aspects, the present disclosure provides the cell lines, as well as to the monoclonal antibodies produced by these cell lines. The cell lines of the present disclosure have uses other than for the production of the monoclonal antibodies. For example, the cell lines of the present disclosure can be fused with other cells (such as suitably drug-marked human myeloma, mouse myeloma, human-mouse heteromyeloma or human lymphoblastoid cells) to produce additional hybridomas, and thus provide for the transfer of the genes encoding the monoclonal antibodies. In addition, the cell lines can be used as a source of nucleic acids encoding the anti- ALK immunoglobulin chains, which can be isolated and expressed (e.g., upon transfer to other cells using any suitable technique (see e.g., Cabilly et al, U.S. Pat. Nos. 4,816,567 and 6,331,415; Winter, U.S. Pat. No. 5,225,539)). For instance, clones comprising a rearranged anti-ALK light or heavy chain can be isolated (e.g., by PCR) or cDNA libraries can be prepared from mRNA isolated from the cell lines, and cDNA clones encoding an anti-ALK immunoglobulin chain can be isolated. Thus, nucleic acids encoding the heavy and/or light chains of the antibodies or portions thereof can be obtained and used in accordance with recombinant DNA techniques for the production of the specific immunoglobulin, immunoglobulin chain, or variants thereof (e.g., humanized immunoglobulins) in a variety of host cells or in an in vitro translation system. For example, the nucleic acids, including cDNAs, or derivatives thereof encoding variants such as a humanized immunoglobulin or immunoglobulin chain, can be placed into suitable prokaryotic or eukaryotic vectors (e.g., expression vectors) and introduced into a suitable host cell by an appropriate method (e.g., transformation, transfection, electroporation, infection), such that the nucleic acid is operably linked to one or more expression control elements (e.g., in the vector or integrated into the host cell genome). For production, host cells can be maintained under conditions suitable for expression

(e.g., in the presence of inducer, suitable media supplemented with appropriate salts, growth factors, antibiotic, nutritional supplements, etc.), whereby the encoded polypeptide is produced. If desired, the encoded protein can be recovered and/or isolated (e.g., from the host cells or medium). It will be appreciated that the method of production encompasses expression in a host cell of a transgenic animal (see e.g., WO 92/03918, GenPharm International, published Mar. 19, 1992).

Preparation of immunizing antigen, and polyclonal and monoclonal antibody production can be performed as described herein, or using other suitable techniques. A variety of methods have been described. See e.g., Kohler et al., Nature, 256: 495- 497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al., Nature 266:

550-552 (1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N. Y.); Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, N. Y.), Chapter 11, (1991). Generally, a hybridoma can be produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as SP2/0) with antibody-producing cells. The antibody-producing cell, preferably those of the spleen or lymph nodes, are obtained from animals immunized with the antigen of interest. The fused cells (hybridomas) can be isolated using selective culture conditions, and cloned by limiting dilution. Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA). To illustrate, immunogens derived from an ALK polypeptide (e.g., an ALK polypeptide or an antigenic fragment thereof which is capable of eliciting an antibody response, or an ALK fusion protein) can be used to immunize a mammal, such as a mouse, a hamster, or rabbit. See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988). Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of an ALK polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies. In certain embodiments, antibodies of the disclosure are specific for an extracellular portion of the ALK protein (e.g., an extracellular portion of SEQ ID NO: 1 or 2), particularly the LBD of ALK (e.g., the ALK-LBD as shown in SEQ ID NO: 6 below). In other embodiments, antibodies of the disclosure are specific for the intracellular portion or the transmembrane portion of the ALK protein.

Following immunization of an animal with an antigenic preparation of an ALK polypeptide, antisera can be obtained and, if desired, polyclonal antibodies can be isolated from the serum. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art, and include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar et al, (1983) Immunology Today, 4: 72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with an ALK polypeptide and monoclonal antibodies isolated from a culture comprising such hybridoma cells.

Other suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, for example, methods which select recombinant antibody from a library, or which rely upon immunization of transgenic animals (e.g., mice) capable of producing a full repertoire of human antibodies. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (J_H) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258(1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and 5,545,807. Human antibodies can also be derived from phage-display libraries. See Hoogenboom et al., J. MoI. Biol, 227:381 (1991); Marks et al., J. MoI. Biol., 222:581-597 (1991); and U.S. Pat. Nos. 5,565,332 and 5,573,905. Human antibodies may also be generated by in vitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275.

In certain embodiments, antibodies of the present disclosure can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab')₂ fragments can be generated by treating antibody with pepsin. The resulting F(ab') ₂ fragment can be treated to reduce disulfide bridges to produce Fab fragments.

In certain embodiments, antibodies of the present disclosure are further intended to include bispecific, single-chain, and chimeric and humanized molecules having affinity for an ALK polypeptide conferred by at least one CDR region of the antibody. Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can also be adapted to produce single chain antibodies. Also, transgenic mice or other organisms including other mammals, may be used to express humanized and/or human antibodies. Methods of generating these antibodies are known in the art. See, e.g., U.S. Pat. Nos. 4,816,567 and 6,331,415; European Patent No. 0,125,023; European Patent No. 0,451,216; U.S. Pat. No. 4,816,397; European Patent No. 0,120,694; WO 86/01533; European Patent No. 0,194,276; U.S. Pat. No. 5,225,539; European Patent No. 0,239,400; European Patent Application No. 0,519,596 Al. See also, U.S. Pat. No. 4,946,778; U.S. Pat. No. 5,476,786; and Bird, R. E. et al., Science, 242: 423-426 (1988)).

Humanized immunoglobulins can be produced using synthetic and/or recombinant nucleic acids to prepare genes (e.g., cDNA) encoding the desired humanized chain. For example, nucleic acid (e.g., DNA) sequences coding for humanized variable regions can be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously humanized variable region (see e.g., Kamman, M., et al., Nucl. Acids Res., 17: 5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993); Daugherty, B. L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991); and Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302 (1991)). Using these or other suitable methods, variants can also be readily produced. In one embodiment, cloned variable regions can be mutagenized, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al., U.S. Pat. No. 5,514,548; Hoogenboom et al., WO 93/06213, published Apr. 1, 1993)).

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate(TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab '-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. In yet a further embodiment, Fab '-SH fragments directly recovered from E. coli can be chemically coupled in vitro to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992).

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (V_H) connected to a light-chain variable domain (V_L) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and V_L domains of one fragment are forced to pair with the complementary V_L and V_H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152:5368 (1994). Alternatively, the bispecific antibody may be a "linear antibody" produced as described in Zapata et al. Protein Eng. 8(10):1057-1062 (1995).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991). Variants of antibodies disclosed herein are also provided. Amino acid sequence variants of an anti-ALK antibody (e.g., 8B10, 16G2-3 and 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El, or E5) are prepared by introducing appropriate nucleotide changes into the anti-ALK antibody DNA, or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the anti-ALK antibodies of the examples herein. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of the humanized or variant anti-ALK antibody, such as changing the number or position of glycosylation sites.

A useful method for identification of certain residues or regions of the anti- ALK antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells Science, 244:1081- 1085 (1989). To illustrate, a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and GIu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with an ALK antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is conducted at the target codon or region and the expressed anti-ALK antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an anti-ALK antibody with an N- terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of the anti-ALK antibody molecule include the fusion to the N- or C- terminus of the anti-ALK antibody of an enzyme or a polypeptide which increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the anti-ALK antibody molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated.

One contemplated type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., 8B10, 16G2-3 and 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El, or E5). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6- 7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M 13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity for the antigen, or the ability to modulate angiogenesis, cell adhesion, cancer cell invasion, or tumor growth) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identified hypervariable region residues contributing significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure or a predicted structure (e.g., using the 3D-PSSM program as disclosed herein) of the antigen-antibody complex to identify contact points between the antibody and an ALK protein. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development. Another type of amino acid variant of the antibody alters the glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5 -hydroxy Iy sine may also be used.

Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above- described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

Additionally or alternatively, an antibody of the invention can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting

GIcNAc structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. See, for example, Shields, RX. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1; Davies et al., 20017 Biotechnol Bioeng 74:288-294; Shinkawa et al., 2003, J Biol Chem 278:3466-3473); U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; as well as, European Patent No: EP 1,176,195; PCT Publications WO 03/035835; WO 99/54342; WO 00/61739; PCT WO 01/292246; PCT WO 02/311140; PCT WO 02/30954 and WO 00/061739.

In certain embodiments, the antibodies are further attached to a functional moiety. Functional moieties include a label or active moiety that can be detected. The label can be a radioisotope, fluorescent compound, enzyme, or enzyme co- factor. The label can be amino acid insertions as described above. The label may be a radioactive agent, such as: radioactive heavy metals such as iron chelates, radioactive chelates of gadolinium or manganese, positron emitters of oxygen, nitrogen, iron, carbon, or gallium, ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸ Ga, ¹²³1, ¹²⁵1, ¹³¹I, ¹³²I, or ⁹⁹Tc. A binding agent affixed to such a moiety may be used as an imaging agent and is administered in an amount effective for diagnostic use in a mammal such as a human and the localization and accumulation of the imaging agent is then detected. The localization and accumulation of the imaging agent may be detected by radioscintigraphy, nuclear magnetic resonance imaging, computed tomography, or positron emission tomography. Immunoscintigraphy using antibodies or other binding polypeptides directed at ALK may be used to detect and/or diagnose cancers and vasculature. For example, monoclonal antibodies against the ALK marker labeled with "Technetium, ⁿ indium or ¹²⁵Iodine may be effectively used for such imaging. As will be evident to the skilled artisan, the amount of radioisotope to be administered is dependent upon the radioisotope. The amount of the imaging agent to be administered is formulated based upon the specific activity and energy of a given radionuclide used as the active moiety. Typically 0.1-100 millicuries per dose of imaging agent, preferably 1-10 millicuries, most often 2-5 millicuries are administered. Thus, compositions according to the present disclosure useful as imaging agents comprising a targeting moiety conjugated to a radioactive moiety comprise 0.1-100 millicuries, in some embodiments preferably 1-10 millicuries, in some embodiments preferably 2-5 millicuries, in some embodiments more preferably 1-5 millicuries.

Other modifications of a humanized or variant anti-ALK antibody are also contemplated. For example, it may be desirable to modify the antibody of the disclosure with respect to effector function, so as to enhance the effectiveness of the antibody in treating cancer, for example. For example cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992).

Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). Also contemplated are anti-ALK antibodies comprising modifications/substations and/or novel amino acids within their Fc domains that modulate effector function. For example, those disclosed in Ghetie et al., 1997, Nat Biotech. 15:637-40; Duncan et al, 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol 147:2657-2662; Lund et al, 1992, MoI Immunol 29:53-59; Alegre et al, 1994, Transplantation 57:1537- 1543; Hutchins et al., 1995, Proc Natl. Acad Sci U S A 92:11980-11984; Jefferis et al, 1995, Immunol Lett. 44: 111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al, 1996, Immunol Lett 54:101-104; Lund et al, 1996, J Immunol 157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, J Immunol 164:4178-4184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu et al., 2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol 166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferis et al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490); U.S. Patent Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. Patent Application

Nos. 10/370,749 and PCT Publications WO 94/2935; WO 99/58572; WO 00/42072; WO 02/060919, WO 04/029207. Other modifications/substitutions of the Fc domain will be readily apparent to one skilled in the art

The disclosure also pertains to immunoconjugates comprising the antibody described herein conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). Chemotherapeutic agents are useful in the generation of such immuno- conjugates. Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, neomycin, and the tricothecenes.

A variety of radionuclides are available for the production of radio conjugated anti-ALK antibodies. Examples include ²¹²Bi, ¹³¹1, ¹¹¹In, ⁹⁰Y and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p- azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6- diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4- dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon- 14-labeled 1-isothiocyanatobenzyl- 3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pretargeting wherein the antibody- receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a radionuclide).

The anti-ALK antibodies disclosed herein may also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556, such as for example, pegylated liposomes.

Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present disclosure can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem. 257:286- 288 (1982) via a disulfide interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst.81(19):1484 (1989).

The antibody of the present disclosure may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g., apeptidyl chemotherapeutic agent, see WO81/01145) to an active anti-cancer drug. See, e.g., WO 88/07378 and U.S. Pat. No. 4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its . more active, cytotoxic form. Enzymes that are useful in the method of this disclosure include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide- containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as beta-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; beta-lactamase useful for converting drugs derivatized with beta-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes," can be used to convert the prodrugs of the disclosure into free active drugs. See, e.g., Massey, Nature 328:457-458 (1987). Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.

The enzymes can be covalently bound to the anti-ALK antibodies by techniques such as the use of the heterobifunctional crosslinking reagents discussed above. Alternatively, fusion proteins comprising at least the antigen-binding region of an antibody of the disclosure linked to at least a functionally active portion of an enzyme of the disclosure can be constructed using recombinant DNA techniques well known in the art. See, e.g., Neuberger et al., Nature 312:604-608 (1984).

In certain embodiments, it may be desirable to use an antibody fragment, rather than an intact antibody, to increase tumor penetration, for example. In this case, it may be desirable to modify the antibody fragment in order to increase its serum half life. This may be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment (e.g., by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle, e.g., by DNA or peptide synthesis). See, e.g., WO96/32478. The salvage receptor binding epitope generally constitutes a region wherein any one or more amino acid residues from one or two loops of a Fc domain are transferred to an analogous position of the antibody fragment. In certain embodiments, three or more residues from one or two loops of the Fc domain are transferred. In still other embodiments, the epitope is taken from the C_H2 domain of the Fc region (e.g., of an IgG) and transferred to the C_HI, C_H3, or V_H region, or more than one such region, of the antibody. Alternatively, the epitope is taken from the C_H2 domain of the Fc region and transferred to the C_L region or VL region, or both, of the antibody fragment.

Alternatively, or optionally, antibodies having increased in vivo half-lives can be generated for example, by modifying (e.g., substituting, deleting or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor (see, e.g., WO 97/34631; WO 04/029207; U.S. Pat. No. 6,737,056 and U.S. Pat. App. Publication No. 2003/0190311).

Covalent modifications of an anti-ALK antibody or a variant anti-ALK antibody are also included within the scope of this disclosure. They may be made by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable. Other types of covalent modifications of the antibody are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues. Exemplary covalent modifications of polypeptides are described in U.S. Pat. No. 5,534,615. One type of covalent modification of the antibody comprises linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337. In certain embodiments, an antibody of the disclosure is a monoclonal antibody, and in certain embodiments the disclosure makes available methods for generating novel antibodies. For example, a method for generating a monoclonal antibody that binds specifically to an ALK polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the ALK polypeptide effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify a hybridoma that produces a monoclonal antibody that binds specifically to the ALK polypeptide. Once obtained, a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody that binds specifically to ALK polypeptide. The monoclonal antibody may be purified from the cell culture.

The disclosure also provides isolated nucleic acid encoding an anti-ALK antibody, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the antibody. For recombinant production of the antibody, the nucleic acid encoding it may be isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. In another embodiment, the antibody may be produced by homologous recombination, e.g., as described in U.S. Pat. No. 5,204,244.

Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram- positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniforniis (e.g., B. licheniformis 41P disclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One specific E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-ALK antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger. Suitable host cells for the expression of glycosylated anti-ALK antibody are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-I variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present disclosure, particularly for transfection of Spodoptera frugiperda cells. Certain plants, including duckweed (Lemna spp.) (e.g., Biolex, Inc.), cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts, and cell suspensions thereof may also be used.

Examples of useful mammalian host cell lines of the disclosure are monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cellsADHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; mouse myeloma cells (NSO, ATCC CRL-11177; GS-NSO, Bebbington, Biotechnol. 10:169-175 (1992)) and a human hepatoma line (Hep G2).

Host cells are transformed with the above-described expression or cloning vectors for anti-ALK antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The host cells used to produce the anti-ALK antibody of this disclosure may be cultured in a variety of media. Commercially available media such as Ham's FlO (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI- 1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem.102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl-fluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Protein A can be used to purify antibodies that are based on human gamma heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human gamma 3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH₃ domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).

In addition, the techniques used to screen antibodies in order to identify a desirable antibody may influence the properties of the antibody obtained. For example, an antibody to be used for certain therapeutic purposes will preferably be able to target a particular cell type. Accordingly, to obtain antibodies of this type, it may be desirable to screen for antibodies that bind to cells that express the antigen of interest (e.g., by fluorescence activated cell sorting). Likewise, if an antibody is to be used for binding an antigen in solution, it may be desirable to test solution binding. A variety of different techniques are available for testing antibody:antigen interactions to identify particularly desirable antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (e.g., the Biacore binding assay, Bia-core AB, Uppsala, Sweden), sandwich assays (e.g., the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Maryland), western blots, immunoprecipitation assays, and immunohistochemistry. Antibody binding studies may be carried out in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques (CRC Press, Inc., 1987), pp. 147-158.

Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody. The amount of target protein in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies preferably are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte that remain unbound.

Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody that is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an antiimmunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.

//. Functional and Biological Assays

Antibodies (or polypeptide binding agents) of the present disclosure can be identified and evaluated based on their effects on an ALK activity, which can be an ALK binding activity (e.g., binding to PTN₅ actinin, tensin, IRS, or UNC5), ALK- regulated angiogenesis, cell growth or proliferation, cell adhesion, wound healing, cancer cell invasion, tumor growth, or any combination thereof.

A test antibody's effect on an ALK binding activity may be determined by measuring its ability to modulate the interaction between ALK and its natural ligand. Alternatively, as depicted in Figure 14, immunoprecipitation assays can be employed to determine the test antibody's ability to modulate the specific interaction between an ALK protein and another protein of interest (e.g., actinin, tensin, IRS, or UNC5).

As depicted in Figure 16, the effect of a polypeptide binding agent of the disclosure on spreading of cancer cells can be evaluated. As depicted in Figures 17 and 18, the effect of a polypeptide binding agent on invasion of the endothelial cells by cancer cells can be evaluated. The ability of the antibodies of the invention to inhibit cancer cell formation in soft agar may be assayed (such assays may be carried out, e.g., as described in Zelinski et al., 2001, Cancer Res. 61:2301). Cell adhesion and cell rounding assays can also be performed as described in

Miao, et al. (Nature Cell Biol. 2:62, 2000). To study cell adhesion, briefly, cells are plated in triplicate onto 96-well plates previously coated with various ECM proteins or poly-L-lysine. Cells are plated at a density of Ix 10⁵ cells per well in the presence or absence of the polypeptide binding agent and allowed to adhere for 30 minutes at 37° C. Non-adherent cells are washed from the wells, and adherent cells are fixed, stained, and quantified by measuring absorbance on an enzyme-linked immunosorbent assay (ELISA) reader. Cells treated with the polypeptide binding agent will exhibit a change in attachment to ECM protein-treated wells relative to control cells allowed to adhere in the absence of the polypeptide binding agent. For cell rounding assays, briefly, cells are plated onto ECM protein coated six-well dishes, or ECM protein-coated coverslips in 24-well dishes. Cells are allowed to adhere for 48 hours, then treated with media with or without the polypeptide binding agent for 10 minutes. Plates or coverslips are washed, fixed and stained and visualized by microscopy. Cells treated with the polypeptide binding agent protein may exhibit a difference in cell rounding relative to cells treated with media lacking the polypeptide binding agent, indicate decreased attachment to the ECM matrix. Various assays can be used to test the polypeptide binding agents herein for their endothelial and/or angiogenic activity. Assays for measuring angiogenic activity include a Matrigel™ (BD Biosciences) plug assay, such as that depicted in Figure 5. In a typical Matrigel plug assay, test agents such as angiogenesis-inducing compounds (e.g., bFGF) or tumor cells are introduced into cold liquid Matrigel which, after subcutaneous injection, solidifies and permits penetration by host cells and the formation of new blood vessels. Assessment of angiogenesis in the Matrigel plug is achieved either by measuring hemoglobin or by scoring selected regions of histological sections for vascular density (e.g., vessel count as shown in Figure 5). Improved or optimized Matrigel-based assays are also available, such as those described in Akhtar et al., Angiogenesis. 2002;5(l-2):75-80; Kragh et al., Int J Oncol. 2003 Feb;22(2):305-l l.

Assays for wound-healing activity include, for example, those described in Winter, Epidermal Wound Healing, Maibach, H I and Rovee, D T, eds. (Year Book Medical Publishers, Inc., Chicago), pp. 71-112, as modified by the article of Eaglstein and Mertz, J. Invest. Dermatol, 71: 382-384 (1978). Figure 12 of the present disclosure also depicts the assay result using a murine model of wound healing.

Other assays can also be employed to evaluate the ALK agonists and antagonists of the disclosure. For example, the soft agar assays described herein can be employed to determine whether an anti-ALK antibody has agonistic activities as indicated by promoting colony formation (e.g., Figure 32), or whether an anti-ALK antibody has antagonistic acitvities as indicated by inhibiting colony formation (Figures 21 A and 21B). As another example, an anti-ALK antibody can also be evaluated for its ability to modulate an ALK kinase activity, such as an autophorylation activity; Figure 33 shows the results of an example of such an assay based on ALK autophosphorylation activity. A further example may employ the ECIS method as illustrated in Figure 17, which measures invasiveness of tumor cells; thus, an antagonist of ALK can be evaluated using the ECIS method to determine whether it would inhibit the invasion by metastatic cells. For cancer, a variety of well-known animal models can be used to test the efficacy of candidate polypeptide binding agents of the disclosure. The in vivo nature of such models makes them particularly predictive of responses in human patients. Animal models of tumors and cancers (e.g., breast cancer, colon cancer, prostate cancer, lung cancer, etc.) include both non-recombinant and recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodent, e.g., murine models. Such models can be generated by introducing tumor cells (e.g., MDA-MB231 breast cancer cells or U87 glioblastoma cells) into syngeneic mice using standard techniques, e.g., subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, or orthopin implantation, e.g., colon cancer cells implanted in colonic tissue. See, e.g., WO 97/33551.

Probably the most often used animal species in oncology studies are immunodeficient mice and, in particular, nude mice. The observation that the nude mouse with thymic hypo/aplasia could successfully act as a host for human tumor xenografts has lead to its widespread use for this purpose. The autosomal recessive nu gene has been introduced into a very large number of distinct cόngenic strains of nude mouse, including, for example, ASW, A/He, AKR, BALB/c, B 10.LP, C 17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII, and SJL. In addition, a wide variety of other animals with inherited immunological defects other than the nude mouse have been bred and used as recipients of tumor xenografts. For further details see, e.g., The Nude Mouse in Oncology Research, E. Boven and B. Winograd, eds. (CRC Press, Inc., 1991).

The cells introduced into such animals can be derived from known tumor/cancer cell lines, such as any of the above-listed tumor cell lines, and, for example, the MDA-MB231 cell line (hormone-independent breast cancer); the U87 glioblastoma cell line; the B 104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene); ras-transfected NIH-3T3 cells; Caco-2 (ATCC HTB- 37); or a moderately well-differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38); or from tumors and cancers. Samples of tumor or cancer cells can be obtained from patients undergoing surgery, using standard conditions involving freezing and storing in liquid nitrogen. Karmali et al., Br. J. Cancer, 48: 689-696 (1983).

Tumor cells can be introduced into animals such as nude mice by a variety of procedures. The subcutaneous (s.c.) space in mice is very suitable for tumor implantation. Tumors can be transplanted s.c. as solid blocks, as needle biopsies by use of a trocar, or as cell suspensions. For solid-block or trochar implantation, tumor tissue fragments of suitable size are introduced into the s.c. space. Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously. Tumor cells can also be injected as subdermal implants. In this location, the inoculum is deposited between the lower part of the dermal connective tissue and the s.c. tissue.

Animal models of breast cancer can be generated, for example, by implanting rat neuroblastoma cells (from which the neu oncogene was initially isolated), or neu-transformed NIH-3T3 cells into nude mice, essentially as described by Drebin et al. Proc. Nat. Acad. Sci. USA, 83: 9129-9133 (1986).

Similarly, animal models of colon cancer can be generated by passaging colon cancer cells in animals, e.g., nude mice, leading to the appearance of tumors in these animals. An orthotopic transplant model of human colon cancer in nude mice has been described, for example, by Wang et al., Cancer Research, 54: 4726-4728 (1994) and Too et al., Cancer Research, 55: 681-684 (1995). This model is based on the so-called METAMOUSE™ sold by Anticancer, Inc. (San Diego, Calif.).

Tumors that arise in animals can be removed and cultured in vitro. Cells from the in vitro cultures can then be passaged to animals. Such tumors can serve as targets for further testing or drug screening. Alternatively, the tumors resulting from the passage can be isolated and RNA from pre-passage cells and cells isolated after one or more rounds of passage analyzed for differential expression of genes of interest. Such passaging techniques can be performed with any known tumor or cancer cell lines.

For example, Meth A, CMS4, CMS5, CMS21, and WEHI- 164 are chemically induced fibrosarcomas of BALB/c female mice (DeLeo et al., J. Exp. Med., 146: 720 (1977)), which provide a highly controllable model system for studying the anti-tumor activities of various agents. Palladino et al., J. Immunol., 138: 4023-4032 (1987). Briefly, tumor cells are propagated in vitro in cell culture. Prior to injection into the animals, the cell lines are washed and suspended in buffer, at a cell density of about 10⁶ to 10⁸ cells/ml. The animals are then infected subcutaneously with 10 to 100 μl of the cell suspension, allowing one to three weeks for a tumor to appear.

In addition, the Lewis lung (3LL) carcinoma of mice, which is one of the most thoroughly studied experimental tumors, can be used as an investigational tumor model. Efficacy in this tumor model has been correlated with beneficial effects in the treatment of human patients diagnosed with small-cell carcinoma of the lung (SCCL). This tumor can be introduced in normal mice upon injection of tumor fragments from an affected mouse or of cells maintained in culture. Zupi et al., Br. J. Cancer, 41: suppl. 4, 30 (1980). Evidence indicates that tumors can be started from injection of even a single cell and that a very high proportion of infected tumor cells survive. For further information about this tumor model see Zacharski, Haemostasis, 16: 300-320 (1986).

Other animal models such as hamsters, rabbits, etc. are known in the art and described in Relevance of Tumor Models for Anticancer Drug Development (1999, eds. Fiebig and Burger); Contributions to Oncology (1999, Karger); The Nude Mouse in Oncology Research (1991, eds. Boven and Winograd); and Anticancer Drug Development Guide (1997 ed. Teicher).

One way of evaluating the efficacy of a test polypeptide binding agent in an animal model with an implanted tumor is to measure the size of the tumor before and after treatment. Traditionally, the size of implanted tumors has been measured with a slide caliper in two or three dimensions. The measure limited to two dimensions does not accurately reflect the size of the tumor; therefore, it is usually converted into the corresponding volume by using a mathematical formula. However, the measurement of tumor size is very inaccurate. The therapeutic effects of a drug candidate can be better described as treatment-induced growth delay and specific growth delay. Another important variable in the description of tumor growth is the tumor volume doubling time. Computer programs for the calculation and description of tumor growth are also available, such as the program reported by Rygaard and Spang- Thomsen, Proc. 6th Int. Workshop on Immune-Deficient Animals Wu and Sheng eds. (Basel, 1989), p. 301. It is noted, however, that necrosis and inflammatory responses following treatment may actually result in an increase in tumor size, at least initially. Therefore, these changes need to be carefully monitored, by a combination of a morphometric method and flow cytometric analysis.

Cell-based assays and animal models for angiogenic disorders, such as tumors, can be used to evaluate the polypeptide agents described herein. The role of gene products identified herein in the development and pathology of undesirable angiogenic cell growth, e.g., tumor cells, can be tested by using cells or cells lines that have been identified as being stimulated or inhibited by the anti-ALK antibodies herein.

In a different approach, cells of a cell type known to be involved in a particular angiogenic disorder are transfected with the cDNAs herein, and the ability of these cDNAs to induce excessive growth or inhibit growth is analyzed. If the angiogenic disorder is cancer, suitable tumor cells include, for example, stable tumor cells lines such as the B 104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and ras-transfected NIH-3T3 cells, which can be transfected with the desired gene and monitored for tumorigenic growth. Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody agents to inhibit tumorigenic cell growth by exerting cytostatic or cytotoxic activity on the growth of the transformed cells, or by mediating antibody-dependent cellular cytotoxicity (ADCC). In addition, primary cultures derived from tumors in transgenic animals (as described above) can be used in the cell-based assays herein, although stable cell lines may be preferred. Techniques to derive continuous cell lines from transgenic animals are well known in the art. See, e.g., Small et al., MoI. Cell. Biol. 5: 642-648 (1985). The antibodies of the present disclosure are useful in a variety of applications, including research, diagnostic, and therapeutic applications. For instance, they can be used to isolate and/or purify receptor or portions thereof, and to study receptor structure (e.g., conformation) and function.

III. Diagnostic Applications

In certain aspects, the various antibodies of the present disclosure can be used to detect or measure the expression of ALK receptor, for example, on tumor tissue or endothelial cells (e.g., venous endothelial cells), or on cells transfected with an ALK receptor gene. Thus, they also have utility in applications such as cell sorting and imaging (e.g., flow cytometry, and fluorescence activated cell sorting), for diagnostic or research purposes. In certain embodiments, the antibodies or antigen-binding fragments of the antibodies can be labeled or unlabeled for diagnostic purposes. Typically, diagnostic assays entail detecting the formation of a complex resulting from the binding of an antibody to ALK. The antibodies can be directly labeled. A variety of labels can be employed, including, but not limited to, radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (e.g., biotin, haptens). Numerous appropriate immunoassays are known to the skilled artisan (see, for example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654; and 4,098,876). When unlabeled, the antibodies can be used in assays, such as agglutination assays. Unlabeled antibodies can also be used in combination with another (one or more) suitable reagent which can be used to detect antibody, such as a labeled antibody (e.g., a second antibody) reactive with the first antibody (e.g., anti-idiotype antibodies or other antibodies that are specific for the unlabeled immunoglobulin) or other suitable reagent (e.g., labeled protein A). An ALK antibody may also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, e.g., to increase serum half-life or to increase tissue binding.

In one embodiment, the antibodies of the present disclosure can be utilized in enzyme immunoassays, wherein the subject antibodies, or second antibodies, are conjugated to an enzyme. When a biological sample comprising an ALK protein is combined with the subject antibodies, binding occurs between the antibodies and ALK protein. In one embodiment, a sample containing cells expressing an ALK protein (e.g., endothelial cells) is combined with the subject antibodies, and binding occurs between the antibodies and cells bearing an ALK protein comprising an epitope recognized by the antibody. These bound cells can be separated from unbound reagents and the presence of the antibody-enzyme conjugate specifically bound to the cells can be determined, for example, by contacting the sample with a substrate of the enzyme which produces a color or other detectable change when acted on by the enzyme. In another embodiment, the subject antibodies can be unlabeled, and a second, labeled antibody can be added which recognizes the subject antibody.

In certain aspects, kits for use in detecting the presence of an ALK protein in a biological sample can also be prepared. Such kits will include an antibody which binds to an ALK protein or portion of said receptor, as well as one or more ancillary reagents suitable for detecting the presence of a complex between the antibody and ALK or portion thereof. The antibody compositions of the present disclosure can be provided in lyophilized form, either alone or in combination with additional antibodies specific for other epitopes. The antibodies, which can be labeled or unlabeled, can be included in the kits with adjunct ingredients (e.g., buffers, such as Tris, phosphate and carbonate, stabilizers, excipients, biocides, and/or inert proteins, e.g., bovine serum albumin). For example, the antibodies can be provided as a lyophilized mixture with the adjunct ingredients, or the adjunct ingredients can be separately provided for combination by the user. Generally these adjunct materials will be present in less than about 5% weight based on the amount of active antibody, and usually will be present in a total amount of at least about 0.001% weight based on antibody concentration. Where a second antibody capable of binding to the monoclonal antibody is employed, such antibody can be provided in the kit, for instance in a separate vial or container. The second antibody, if present, is typically labeled, and can be formulated in an analogous manner with the antibody formulations described above. Similarly, the present disclosure also relates to a method of detecting and/or quantifying expression of an ALK or portion of the receptor by a cell, wherein a composition comprising a cell or fraction thereof (e.g., membrane fraction) is contacted with an antibody which binds to an ALK or portion of the receptor under conditions appropriate for binding of the antibody thereto, and antibody binding is monitored. Detection of the antibody, indicative of the formation of a complex between antibody and ALK or a portion thereof, indicates the presence of the receptor. Binding of antibody to the cell can be determined by standard methods, such as those described in the working examples. The method can be used to detect expression of ALK on cells from an individual. Optionally, a quantitative expression of ALK on the surface of endothelial cells can be evaluated, for instance, by flow cytometry, and the staining intensity can be correlated with disease susceptibility, progression, or risk.

The present disclosure also relates to a method of detecting the susceptibility of a mammal to certain diseases. To illustrate, the method can be used to detect the susceptibility of a mammal to diseases which progress based on the amount of ALK present on cells and/or the number of ALK-positive cells in a mammal. In one embodiment, the disclosure relates to a method of detecting susceptibility of a mammal to a tumor. In this embodiment, a sample to be tested is contacted with an antibody which binds to an ALK or portion thereof under conditions appropriate for binding of said antibody thereto, wherein the sample comprises cells which express ALK in normal individuals. The binding of antibody and/or amount of binding is detected, which indicates the susceptibility of the individual to a tumor, wherein higher levels of receptor correlate with increased susceptibility of the individual to a tumor. The disclosure presents data showing that expression of ALK and/or its ligand PTN has a correlation with tumor growth and progression, and cancer prognosis. The antibodies of the present disclosure can also be used to further elucidate the correlation of ALK expression with progression of angiogenesis- associated diseases in an individual.

IV. Other ALK Antagonists

In addition to antibodies, ALK antagonists include compounds such as soluble ALK polypeptides and nucleic acids (e.g, RNAi, antisense, aptamers, ribozymes). Soluble ALK polypeptides will generally comprise a functional portion of the extracellular domain of the ALK kinase. In one embodiment, the soluble ALK polypeptide comprises the LBD, which encompasses amino acids 368-447 of the human ALK sequence (SEQ ID NO: 1). A soluble ALK polypeptide may be fused to additional polypeptides, such as Fc domains or serum albumin (HSA). A soluble ALK polypeptide may also modified so as to improve pharmacokinetics, e.g., by covalent attachment to one or more polyalkylene glycol moieties, particularly polyethylene glycol (PEG).

In certain aspects, the disclosure provides enzymatic nucleic acids including for example, ribozymes or DNA enzymes. Methods for generating ribozymes specific for ALK have been described. See, for example, Powers et al., JBC 277:14153-14158 (2002). In certain aspects, the disclosure provides isolated nucleic acid compounds comprising at least a portion that hybridizes to an ALK transcript under physiological conditions and decreases the expression of ALK in a cell. Such nucleic acids may be used as ALK antagonists, as described herein. The ALK transcript may be any pre-splicing transcript (i.e., including introns), post-splicing transcript, as well as any splice variant. In certain embodiments, the ALK transcript has a sequence corresponding to the cDNA set forth below in SEQ ID NO:3, and particularly the coding portion thereof. Homo sapiens anaplastic lymphoma kinase (ALK), cDNA, NM_004304

(SEQ ID NO:3):

1 gggggcggca gcggtggtag cagctggtac ctcccgccgc ctctgttcgg agggtcgcgg 61 ggcaccgagg tgctttccgg ccgccctctg gtcggccacc caaagccgcg ggcgctgatg 121 atgggtgagg agggggcggc aagatttcgg gcgcccctgc cctgaacgcc ctcagctgct 181 gccgccgggg ccgctccagt gcctgcgaac tctgaggagc cgaggcgccg gtgagagcaa

241 ggacgctgca aacttgcgca gcgcgggggc tgggattcac gcccagaagt tcagcaggca

301 gacagtccga agccttcccg cagcggagag atagcttgag ggtgcgcaag acggcagcct 361 ccgccctcgg ttcccgccca gaccgggcag aagagcttgg aggagccaaa aggaacgcaa

421 aaggcggcca ggacagcgtg cagcagctgg gagccgccgt tctcagcctt aaaagttgca

481 gagattggag gctgccccga gaggggacag accccagctc cgactgcggg gggcaggaga

541 ggacggtacc caactgccac ctcccttcaa ccatagtagt tcctctgtac cgagcgcagc

601 gagctacaga cgggggcgcg gcactcggcg cggagagcgg gaggctcaag gtcccagcca

661 gtgagcccag tgtgcttgag tgtctctgga ctcgcccctg agcttccagg tctgtttcat 721 ttagactcct gctcgcctcc gtgcagttgg gggaaagcaa gagacttgcg cgcacgcaca

781 gtcctctgga gatcaggtgg aaggagccgc tgggtaccaa ggactgttca gagcctcttc

841 ccatctcggg gagagcgaag ggtgaggctg ggcccggaga gcagtgtaaa cggcctcctc

901 cggcgggatg ggagccatcg ggctcctgtg gctcctgccg ctgctgcttt ccacggcagc 961 tgtgggctcc gggatgggga ccggccagcg cgcgggctcc ccagctgcgg ggccgccgct

1021 gcagccccgg gagccactca gctactcgcg cctgcagagg aagagtctgg cagttgactt 1081 cgtggtgccc tcgctcttcc gtgtctacgc ccgggaccta ctgctgccac catcctcctc

1141 ggagctgaag gctggcaggc ccgaggcccg cggctcgcta gctctggact gcgccccgct

1201 gctcaggttg ctggggccgg cgccgggggt ctcctggacc gccggttcac cagccccggc

1261 agaggcccgg acgctgtcca gggtgctgaa gggcggctcc gtgcgcaagc tccggcgtgc 1321 caagcagttg gtgctggagc tgggcgagga ggcgatcttg gagggttgcg tcgggccccc

1381 cggggaggcg gctgtggggc tgctccagtt caatctcagc gagctgttca gttggtggat 1441 tcgccaaggc gaagggcgac tgaggatccg cctgatgccc gagaagaagg cgtcggaagt

1501 gggcagagag ggaaggctgt ccgcggcaat tcgcgcctcc cagccccgcc ttctcttcca 1561 gatcttcggg actggtcata gctccttgga atcaccaaca aacatgcctt ctccttctcc 1621 tgattatttt acatggaatc tcacctggat aatgaaagac tccttccctt tcctgtctca

1681 tcgcagccga tatggtctgg agtgcagctt tgacttcccG tgtgagctgg agtattcccc 1741 tccactgcat gacctcagga accagagctg gtcctggcgc cgcatcccct ccgaggaggc 1801 ctcccagatg gacttgctgg atgggcctgg ggcagagcgt tctaaggaga tgcccagagg 1861 ctcctttctc cttctcaaca cctcagctga ctccaagcac accatcctga gtccgtggat 1921 gaggagcagc agtgagcact gcacactggc cgtctcggtg cacaggcacc tgcagccctc

1981 tggaaggtac attgcccagc tgctgcccca caacgaggct gcaagagaga tcctcctgat

2041 gcccactcca gggaagcatg gttggacagt gctccaggga agaatcgggc gtccagacaa 2101 cccatttcga gtggccctgg aatacatctc cagtggaaac cgcagcttgt ctgcagtgga

2161 cttctttgcc ctgaagaact gcagtgaagg aacatcccca ggctccaaga tggccctgca 2221 gagctccttc acttgttgga atgggacagt cctccagctt gggcaggcct gtgacttcca 2281 ccaggactgt gcccagggag aagatgagag ccagatgtgc cggaaactgc ctgtgggttt 2341 ttactgcaac tttgaagatg gcttctgtgg ctggacccaa ggcacactgt caccccacac 2401 tcctcaatgg caggtcagga ccctaaagga tgcccggttc caggaccacc aagaccatgc

2461 tctattgctc agtaccactg atgtccccgc ttctgaaagt gctacagtga ccagtgctac 2521 gtttcctgca ccgatcaaga gctctccatg tgagctccga atgtcctggc tcattcgtgg 2581 agtcttgagg ggaaacgtgt ccttggtgct agtggagaac aaaaccggga aggagcaagg 2641 caggatggtc tggcatgtcg ccgcctatga aggcttgagc ctgtggcagt ggatggtgtt 2701 gcctctcctc gatgtgtctg acaggttctg gctgcagatg gtcgcatggt ggggacaagg

2761 atccagagcc atcgtggctt ttgacaatat ctccatcagc ctggactgct acctcaccat 2821 tagcggagag gacaagatcc tgcagaatac agcacccaaa tcaagaaacc tgtttgagag 2881 aaacccaaac aaggagctga aacccgggga aaattcacca agacagaccc ccatctttga 2941 ccctacagtt cattggctgt tcaccacatg tggggccagc gggccccatg gccccaccca

3001 ggcacagtgc aacaacgcct accagaactc caacctgagc gtggaggtgg ggagcgaggg

3061 ccccctgaaa ggcatccaga tctggaaggt gccagccacc gacacctaca gcatctcggg

3121 ctacggagct gctggcggga aaggcgggaa gaacaccatg atgcggtccc acggcgtgtc

3181 tgtgctgggc atcttcaacc tggagaagga tgacatgctg tacatcctgg ttgggcagca 3241 gggagaggac gcctgcccca gtacaaacca gttaatccag aaagtctgca ttggagagaa

3301 caatgtgata gaagaagaaa tccgtgtgaa cagaagcgtg catgagtggg caggaggcgg

3361 aggaggaggg ggtggagcca cctacgtatt taagatgaag gatggagtgc cggtgcccct

3421 gatcattgca gccggaggtg gtggcagggc ctacggggcc aagacagaca cgttccaccc

3481 agagagactg gagaataact cctcggttct agggctaaac ggcaattccg gagccgcagg 3541 tggtggaggt ggctggaatg ataacacttc cttgctctgg gccggaaaat ctttgcagga

3601 gggtgccacc ggaggacatt cctgccccca ggccatgaag aagtgggggt gggagacaag 3661 agggggtttc ggagggggtg gaggggggtg ctcctcaggt ggaggaggcg gaggatatat

3721 aggcggcaat gcagcctcaa acaatgaccc cgaaatggat ggggaagatg gggtttcctt 3781 catcagtcca ctgggcatcc tgtacacccc agctttaaaa gtgatggaag gccacgggga 3841 agtgaatatt aagcattatc taaactgcag tcactgtgag gtagacgaat gtcacatgga 3901 ccctgaaagc cacaaggtca tctgcttctg tgaccacggg acggtgctgg ctgaggatgg

3961 cgtctcctgc attgtgtcac ccaccccgga gccacacctg ccactctcgc tgatcctctc 4021 tgtggtgacc tctgccctcg tggccgccct ggtcctggct ttctccggca tcatgattgt 4081 gtaccgccgg aagcaccagg agctgcaagc catgcagatg gagctgcaga gccctgagta

4141 caagctgagc aagctccgca cctcgaccat catgaccgac tacaacccca actactgctt 4201 tgctggcaag acctcctcca tcagtgacct gaaggaggtg ccgcggaaaa acatcaccct 4261 cattcggggt ctgggccatg gcgcctttgg ggaggtgtat gaaggccagg tgtccggaat

4321 gcccaacgac ccaagccccc tgcaagtggc tgtgaagacg ctgcctgaag tgtgctctga 4381 acaggacgaa ctggatttcc tcatggaagc cctgatcatc agcaaattca accaccagaa 4441 cattgttcgc tgcattgggg tgagcctgca atccctgccc cggttcatcc tgctggagct

4501 catggcgggg ggagacctca agtccttcct ccgagagacc cgccctcgcc cgagccagcc

4561 ctcctccctg gccatgctgg accttctgca cgtggctcgg gacattgcct gtggctgtca 4621 gtatttggag gaaaaccact tcatccaccg agacattgct gccagaaact gcctcttgac 4681 ctgtccaggc cctggaagag tggccaagat tggagacttc gggatggccc gagacatcta 4741 cagggcgagc tactatagaa agggaggctg tgccatgctg ccagttaagt ggatgccccc 4801 agaggccttc atggaaggaa tattcacttc taaaacagac acatggtcct ttggagtgct

4861 gctatgggaa atcttttctc ttggatatat gccatacccc agcaaaagca accaggaagt 4921 tctggagttt gtcaccagtg gaggccggat ggacccaccc aagaactgcc ctgggcctgt 4981 ataccggata atgactcagt gctggcaaca tcagcctgaa gacaggccca actttgccat 5041 cattttggag aggattgaat actgcaccca ggacccggat gtaatcaaca ccgctttgcc 5101 gatagaatat ggtccacttg tggaagagga agagaaagtg cctgtgaggc ccaaggaccc

5161 tgagggggtt cctcctctcc tggtctctca acaggcaaaa cgggaggagg agcgcagccc 5221 agctgcccca ccacctctgc ctaccacctc ctctggcaag gctgcaaaga aacccacagc 5281 tgcagagatc tctgttcgag tccctagagg gccggccgtg gaagggggac acgtgaatat 5341 ggcattctct cagtccaacc ctccttcgga gttgcacaag gtccacggat ccagaaacaa 5401 gcccaccagc ttgtggaacc caacgtacgg ctcctggttt acagagaaac ccaccaaaaa

61 5461 gaataatcct atagcaaaga aggagccaca cgacaggggt aacctggggc tggagggaag

5521 ctgtactgtc ccacctaacg ttgcaactgg gagacttccg ggggcctcac tgctcctaga 5581 gccctcttcg ctgactgcca atatgaagga ggtacctctg ttcaggctac gtcacttccc 5641 ttgtgggaat gtcaattacg gctaccagca acagggcttg cccttagaag ccgctactgc

5701 ccctggagct ggtcattacg aggataccat tctgaaaagc aagaatagca tgaaccagcc 5761 tgggccctga gctcggtcgc acactcactt ctcttccttg ggatccctaa gaccgtggag 5821 gagagagagg caatggctcc ttcacaaacc agagaccaaa tgtcacgttt tgttttgtgc 5881 caacctattt tgaagtacca ccaaaaaagc tgtattttga aaatgcttta gaaaggtttt 5941 gagcatgggt tcatcctatt ctttcgaaag aagaaaatat cataaaaatg agtgataaat

6001 acaaggccca gatgtggttg cataaggttt ttatgcatgt ttgttgtata cttccttatg 6061 cttctttcaa attgtgtgtg ctctgcttca atgtagtcag aattagctgc ttctatgttt 6121 catagttggg gtcatagatg tttccttgcc ttgttgatgt ggacatgagc catttgaggg 6181 gagagggaac ggaaataaag gagttatttg taatgactaa aa Examples of categories of nucleic acid compounds include antisense nucleic acids, RNAi constructs, and catalytic nucleic acid constructs. A nucleic acid compound may be single or double stranded. A double stranded compound may also include regions of overhang or non-complementarity, where one or the other of the strands is single stranded. A single stranded compound may include regions of self-complementarity, meaning that the compound forms a so-called "hairpin" or "stem-loop" structure, with a region of double helical structure. A nucleic acid compound may comprise a nucleotide sequence that is complementary to a region consisting of no more than 1000, no more than 500, no more than 250, no more than 100, or no more than 50 nucleotides of the ALK nucleic acid sequence. In certain embodiments, the region of complementarity will be at least 8 nucleotides, and optionally at least 10 or at least 15 nucleotides. A region of complementarity may fall within an intron, a coding sequence or a noncoding sequence of the target transcript, such as the coding sequence portion. Generally, a nucleic acid compound will have a length of about 8 to about 500 nucleotides or base pairs in length, and optionally the length will be about 14 to about 50 nucleotides. A nucleic acid may be a DNA (particularly for use as an antisense), RNA, or RNA:DNA hybrid. Any one strand may include a mixture of DNA and RNA, as well as modified forms that cannot readily be classified as either DNA or RNA. Likewise, a double stranded compound may be DNA:DNA, DNArRNA or RNA:RNA, and any one strand may also include a mixture of DNA and RNA, as well as modified forms that cannot readily be classified as either DNA or RNA. A nucleic acid compound may include any of a variety of modifications, including one or modifications to the backbone (the sugar-phosphate portion in a natural nucleic acid, including internucleotide linkages) or the base portion (the purine or pyrimidine portion of a natural nucleic acid). An antisense nucleic acid compound will generally have a length of about 15 to about 30 nucleotides and will often contain one or more modifications to improve characteristics such as stability in the serum, in a cell or in a place where the compound is likely to be delivered, such as the stomach in the case of orally delivered compounds and the lung for inhaled compounds. In the case of an RNAi construct, the strand complementary to the target transcript will generally be RNA or modifications thereof. The other strand may be RNA, DNA, or any other variation. The duplex portion of double stranded or single stranded "hairpin" RNAi construct will generally have a length of 18 to 40 nucleotides in length and optionally about 21 to 23 nucleotides in length, so long as it serves as a Dicer substrate. Catalytic or enzymatic nucleic acids may be ribozymes or DNA enzymes and may also contain modified forms. Nucleic acid compounds may inhibit expression of the target by about 50%, 75%, 90% or more when contacted with cells under physiological conditions and at a concentration where a nonsense or sense control has little or no effect. Contemplated concentrations for testing the effect of nucleic acid compounds are 1, 5 and 10 micromolar. Nucleic acid compounds may also be tested for effects on, for example, angiogenesis.

In certain aspects, the disclosure provides isolated nucleic acid compounds known in the art as aptamers. Aptamers are macromolecules composed of nucleic acid (e.g., RNA, DNA) that bind tightly to a specific molecular target (e.g., ALK, extracellular domain of ALK (e.g., ECD or LBD) and/or ALK polypeptides as described herein). A particular aptamer may be described by a linear nucleotide sequence and an aptamer is typically about 15-60 nucleotides in length. The chain of nucleotides in an aptamer form intramolecular interactions that fold the molecule into a complex three-dimensional shape, and this three-dimensional shape allows the aptamer to bind tightly to the surface of its target molecule. Given the extraordinary diversity of molecular shapes that exist within the universe of all possible nucleotide sequences, aptamers may be obtained for a wide array of molecular targets, including proteins and small molecules. In addition to high specificity, aptamers have very high affinities for their targets (e.g., affinities in the picomolar to low nanomolar range for proteins). Aptamers are chemically stable and can be boiled or frozen without loss of activity. Because they are synthetic molecules, they are amenable to a variety of modifications, which can optimize their function for particular applications. For in vivo applications, aptamers can be modified to dramatically reduce their sensitivity to degradation by enzymes in the blood. In addition, modification of aptamers can also be used to alter their biodistribution or plasma residence time.

Selection of apatmers that can bind ALK or a fragment there of (e.g., ECD or a fragment thereof) can be achieved through methods known in the art. For example, aptamers can be selected using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method (Tuerk, C, and Gold, L., Science 249:505-510 (1990)). In the SELEX method, a large library of nucleic acid molecules (e.g., 10¹⁵ different molecules) is produced and/or screened with the target molecule (e.g., ALK, extracellular domain of ALK (e.g., ECD or LBD) and/or ALK polypeptides as described herein). The target molecule is allowed to incubate with the library of nucleotide sequences for a period of time. Several methods can then be used to physically isolate the aptamer target molecules from the unbound molecules in the mixture and the unbound molecules can be discarded. The aptamers with the highest affinity for the target molecule can then be purified away from the target molecule and amplified enzymatically to produce a new library of molecules that is substantially enriched for aptamers that can bind the target molecule. The enriched library can then be used to initiate a new cycle of selection, partitioning, and amplification. After 5-15 cycles of this selection, partitioning and amplification process, the library is reduced to a small number of aptamers that bind tightly to the target molecule. Individual molecules in the mixture can then be isolated, their nucleotide sequences determined, and their properties with respect to binding affinity and specificity measured and compared. Isolated aptamers can then be further refined to eliminate any nucleotides that do not contribute to target binding and/or aptamer structure (i.e., aptamers truncated to their core binding domain). See Jayasena, S.D. Clin. Chem. 45:1628-1650 (1999) for review of aptamer technology.

In certain embodiments, the aptamers of the invention have the binding specificity and/or functional activity described herein for the anti-ALK antibodies. Thus, for example, in certain embodiments, the present invention is drawn to aptamers that have the same or similar binding specificity as described herein for the anti-ALK antibodies (e.g., binding specificity, agonistic or antagonistic activity). In particular embodiments, the aptamers of the invention can bind to an ALK polypeptide and inhibit one or more activity of the ALK polypeptide as described herein.

V. Therapeutic Applications ~ ALK Antagonists

Based on the information presented herein, ALK antagonists are generally suitable for administration to patients having a disorder that is associated with or caused by angiogenesis. The most prominent of these disorders are various cancers (particularly solid tumors), many inflammatory disorders, and age-related macular degeneration. Additionally, as demonstrated herein, ALK antagonists can inhibit tumor growth and survival in a manner that is independent of angiogenesis. Thus, ALK antagonists may be used to treat cancers for which anti-angiogenic therapy is not generally recognized as an attractive approach to disease management. Furthermore, as demonstrated herein, ALK antagonists inhibit wound healing in vivo. Wound healing involves substantial scar formation, and therefore, ALK antagonists may be used as inhibitors of scar formation in a variety of tissues. Given the effects of ALK antagonists on angiogenesis and wound healing, ALK antagonists may be used in the prevention of arterial restenosis. These methods involve administering to the individual in need thereof, a therapeutically effective amount of one or more ALK antagonists. These methods are particularly aimed at therapeutic and prophylactic treatments of animals, and more particularly, humans.

A variety of non-cancer disorders that are associated with angiogenesis may be treated with ALK antagonists. For example, ovarian hyperstimulation, endometriosis associated with neovascularization, inflammatory disorders such as immune and non-immune inflammation; chronic articular rheumatism, rheumatoid arthritis, Crohn's disease and psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osier-Webber Syndrome; atherosclerosis; plaque neovascularization; telangiectasia; hemophiliac joints and angiofibroma.

Age-related macular degeneration (AMD) is a leading cause of severe vision loss in the elderly population. The exudative form of AMD is characterized by choroidal neovascularization and retinal pigment epithelial cell detachment. Because choroidal neovascularization is associated with a dramatic worsening in prognosis, an ALK antagonist can be useful in reducing the severity of AMD.

Restenosis occurs subsequent to various invasive techniques that affect an arterial vessel wall, including balloon angioplasty and stent implantation. ALK antagonists may be administered to patients who have undergone such procedures so as to decrease the risk of restenosis. The desired effect may be accomplished by single or repeat systemic administration of the ALK antagonist to the at-risk patient. However, it is contemplated that ALK antagonists are incorporated into a stent. Coated or drug-eluting stents have been highly successful in reducing the risk of restenosis. ALK antagonists may be used to inhibit scar tissue overproduction. For example, the formation of keloid after surgery, fibrosis after myocardial infarction, or fibrotic lesions associated with pulmonary fibrosis may be prevented by local or systemic administration of ALK antagonists.

With respect to cancer, the present disclosure demonstrates that ALK antagonists have a significant overall effect on tumors, resulting in complete or near- complete tumor elimination in well-established animal models. Certain ALK antagonists affect tumors, in part, through an anti-angiogenic mechanism. Because ALK is expressed in the developing vessels, such ALK antagonists can be used to inhibit tumor angiogenesis regardless of whether tumor cells themselves express ALK (i.e., both ALK-positive and ALK-negative tumors). Certain ALK antagonists affect tumors, in part, through a mechanism that is independent of angiogenesis. While not wishing to be bound to a particular mechanism, it is expected that ALK is a survival factor for many tumor cells, particularly ALK-positive tumor cells, and that inhibition of ALK causes tumor cell apoptosis. Furthermore, ALK antagonists inhibit the development of metastatic phenotypes in tumor cells. Thus, ALK antagonists are particularly suited to treatment of pre-metastatic cancers or cancers at a relatively early stage of metastasis, such as, in many cancers, involvement of the regional lymph nodes.

Therefore, while ALK antagonists are proposed for wide anti-cancer use, the present disclosure indicates that there are several surprising cancer subgroups in which ALK antagonists are likely to have desirable effects. These include cancers that are ALK negative (which term includes cancers with relatively low or undetectable ALK expression) but nonetheless exhibit substantial angiogenesis, generally solid tumors. Cancers that are not expected to respond to anti-angiogenic therapy but that do have substantial ALK expression are also a desirable group for treatment with ALK antagonists. Cancers that are pre-metastatic or, less preferably, at an early stage of metastasis, are particularly suited to treatment with ALK antagonists, as the tendency of these cancers to become metastatic will be inhibited, in addition to whatever anti-angiogenic effects and pro-apoptotic (or other cell autonomous) effects that an ALK antagonist may have. ALK antagonists may be used to treat a variety of cancers that are associated with tumor angiogenesis. In general, solid tumors are associated with tumor angiogenesis. Particular examples of such cancers include squamous cell cancer (of various tissues including esophagus, head and neck, vulva, nasopharyngeal), lung cancers, such as small-cell lung cancer and non-small cell lung cancer, gastrointestinal cancer, including pancreatic cancer, brain cancer such as glioblastoma, soft tissue sarcomas, cervical cancer, ovarian cancer, liver cancer such as hepatic carcinoma and hepatoma, bladder cancer, breast cancer (particularly hormone-independent breast cancer), colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer such as renal cell carcinoma and Wilms 's tumors, basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, adrenomedullary tumors (and others occurring in Von Hippel Lindau syndrome) and various types of head and neck cancer, and certain benign tumors, such as hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas. Specific cancers contemplated for treatment herein include brain, breast, colon, lung, melanoma, ovarian, and others involving vascular tumors as noted above. Additional cancers contemplated for treatment herein also include those cancers which are resistant to Herceptin®, Rituxan® or related therapies, cancers which do not respond to Herceptin®, Rituxan® or related therapies, cancers which express or overexpress of PTN and/or ALK. Hormone-independent breast cancer is a specific target for ALK antagonist therapy. These types of breast cancers are characterized by the absence or reduced levels of estrogen and/or progesterone receptors, and are generally refractory to treatment with antihormonal (especially antiestrogenic) therapies.

ALK antagonists are also useful for treating cancers that are not generally expected to be treatable with anti-angiogenic therapies (also referred to herein as "angiogenesis-independent cancer"). For example, hepatic metastases are thought to co-opt existing hepatic blood vessels, thereby avoiding the need for substantial angiogenesis. The effectiveness of anti-angiogenic therapy in hematologic cancers, such as many lymphomas and leukemias, remains uncertain. Adrenal cortical carcinomas and pituitary tumors often appear to have little increase in vascularization relative to normal tissue. Further examples of such cancers include the following: leukemias, such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias, such as, myeloblasts, promyelocytic, myelomonocytic, monocytic, and erythroleukemia leukemias and myelodysplastic syndrome; chronic leukemias, such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullar plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas such as but not limited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget' s disease, and inflammatory breast cancer; adrenal cancer such as but not limited to pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer such as but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin- secreting tumor, and carcinoid or islet cell tumor; pituitary cancers such as but limited to Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipius; eye cancers such as but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, and cilliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers such as but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancers such as but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers such as but not limited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers; liver cancers such as but not limited to hepatocellular carcinoma and hepatoblastoma; gallbladder cancers such as adenocarcinoma; cholangiocarcinomas such as but not limited to pappillary, nodular, and diffuse; lung cancers such as non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; testicular cancers such as but not limited to germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such as but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and verrucous; skin cancers such as but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancers such as but not limited to renal cell carcinoma, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and/ or uterer); Wilms' tumor; bladder cancers such as but not limited to transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A., Inc., United States of America). In certain embodiments of such methods, one or more ALK antagonists can be administered, together (simultaneously) or at different times (sequentially). In addition, antibodies can be administered with another agent for treating cancer or for inhibiting angiogenesis. In a specific embodiment, the subject antagonists of the present disclosure can also be used with other therapeutics.

In certain embodiments, the subject ALK antagonists of the disclosure can be used alone. Alternatively, the subject antagonists may be used in combination with other conventional anti-cancer therapeutic approaches directed to treatment or prevention of proliferative disorders (e.g., tumor). For example, such methods can be used in prophylactic cancer prevention, prevention of cancer recurrence and metastases after surgery, and as an adjuvant of other conventional cancer therapy. The present disclosure recognizes that the effectiveness of conventional cancer therapies (e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced through the use of one or more ALK antagonists of the disclosure.

A wide array of conventional compounds have been shown to have antineoplastic activities. These compounds have been used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastases and further growth, or decrease the number of malignant cells in leukemic or bone marrow malignancies. Although chemotherapy has been effective in treating various types of malignancies, many anti-neoplastic compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, the treatments may work synergistically and allow reduction of dosage of each of the treatments, thereby reducing the detrimental side effects exerted by each compound at higher dosages. In other instances, malignancies that are refractory to a treatment may respond to a combination therapy of two or more different treatments.

When a subject ALK antagonist is administered in combination with another conventional anti-neoplastic agent, either concomitantly or sequentially, such antagonist may enhance the therapeutic effect of the anti-neoplastic agent or overcome cellular resistance to such anti-neoplastic agent. This allows decrease of dosage of an anti-neoplastic agent, thereby reducing the undesirable side effects, or restores the effectiveness of an anti-neoplastic agent in resistant cells.

Pharmaceutical compounds that may be used for combinatory anti-cancer or anti-tumor therapy include, merely to illustrate: aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

These chemotherapeutic anti-tumor compounds may be categorized by their mechanism of action into, for example, the following groups: anti-metabolites/anti- cancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, Cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP 16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes - dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts, and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (TNP -470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (e.g., trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors. In certain embodiments, pharmaceutical compounds that may be used for combinatory anti-angiogenesis therapy include: (1) inhibitors of release of "angiogenic molecules," such as bFGF (basic fibroblast growth factor); (2) neutralizers of angiogenic molecules, such as an anti-bFGF antibodies; and (3) inhibitors of endothelial cell response to angiogenic stimuli, including collagenase inhibitor, basement membrane turnover inhibitors, angiostatic steroids, fungal- derived angiogenesis inhibitors, platelet factor 4, thrombospondin, arthritis drugs such as D-penicillamine and gold thiomalate, vitamin D₃ analogs, alpha-interferon, and the like. For additional proposed inhibitors of angiogenesis, see Blood et al., Bioch. Biophys. Acta., 1032:89-118 (1990), Moses et al., Science, 248:1408-1410 (1990), Ingber et al., Lab. Invest, 59:44-51 (1988), and U.S. Pat. Nos. 5,092,885; 5,112,946; 5,192,744; 5,202,352; and 6573256. In addition, there are a wide variety of compounds that can be used to inhibit angiogenesis, for example, peptides or agents that block the VEGF-mediated angiogenesis pathway, Vitaxin™, Eph Receptor Kinase antibodies (e.g., anti-EphB4 and/or Ephrin B2 antibodies), endostatin protein or derivatives, lysine binding fragments of angiostatin, melanin or melanin-promoting compounds, plasminogen fragments (e.g., Kringles 1-3 of plasminogen), tropoin subunits, antagonists of vitronectin α_vβ₃, peptides derived from Saposin B, antibiotics or analogs (e.g., tetracycline, or neomycin), dienogest- containing compositions, compounds comprising a MetAP-2 inhibitory core coupled to a peptide, the compound EM- 138, chalcone and its analogs, and naaladase inhibitors. See, for example, U.S. Pat. Nos. 6,590,079, 6,531,580, 6,395,718, 6,462,075, 6,465,431, 6,475,784, 6,482,802, 6,482,810, 6,500,431, 6,500,924, 6,518,298, 6,521,439, 6,525,019, 6,538,103, 6,544,758, 6,544,947, 6,548,477, 6,559,126, and 6,569,845.

Depending on the nature of the combinatory therapy, administration of ALK antagonists may be continued while the other therapy is being administered and/or thereafter. Administration of the antagonists may be made in a single dose, or in multiple doses. In some instances, administration of the antagonist is commenced at least several days prior to the conventional therapy, while in other instances, administration is begun either immediately before or at the time of the administration of the conventional therapy. VI. Therapeutic Applications - ALK Agon ists

In certain aspects, the disclosure provides methods of using ALK agonists for therapeutic purposes. There are a variety of disorders where pro-angiogenic agents may be useful. In particular, wound healing, bone formation, and collateral blood vessel formation (to treat ischemic conditions) are promoted by stimulating angiogenesis.

Accordingly, an ALK agonist may be used to treat patients suffering from vascular disease, hypertension, Reynaud's disease and Reynaud's phenomenon, aneurysms, wounds and burns, tissue damage, ischemia reperfusion injury, angina, myocardial infarctions such as acute myocardial infarctions, chronic heart conditions, heart failure such as congestive heart failure, stroke, ischemic limb, osteoporosis and fractures.

As demonstrated herein, ALK participates in wound healing. Accordingly, in certain embodiments, ALK agonists are used to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.

VII. Pharmaceutical Compositions and Modes of Administration

In certain embodiments, the subject antibodies of the present disclosure are formulated with a pharmaceutically acceptable carrier. Such antibodies can be administered alone or as a component of a pharmaceutical formulation

(composition). The compounds may be formulated for administration in any convenient way for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

In other embodiments formulations of the subject antibodies are pyrogen-free formulations which are substantially free of endotoxins and/or related pyrogenic substances. Endotoxins include toxins that are confined inside a microorganism and are released when the microorganisms are broken down or die. Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, it is advantageous to remove even low amounts of endotoxins from intravenously administered pharmaceutical drug solutions. The Food & Drug Administration ("FDA") has set an upper limit of 5 endotoxin units (EU) per dose per kilogram body weight in a single one hour period for intravenous drug applications (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). When therapeutic proteins are administered in amounts of several hundred or thousand milligrams per kilogram body weight, as can be the case with monoclonal antibodies, it is advantageous to remove even trace amounts of endotoxin. In a specific embodiment, endotoxin and pyrogen levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg. Formulations of the subject antibodies include those suitable for oral, dietary, topical, parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous injection), ophthalmologic (e.g., topical or intraocular), inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops), rectal, and/or intravaginal administration. Other suitable methods of administration can also include rechargeable or biodegradable devices and controlled release polymeric devices. Stents, in particular, may be coated with a controlled release polymer mixed with a subject ALK antagonist or agonist. The pharmaceutical compositions of this disclosure can also be administered as part of a combinatorial therapy with other agents (either in the same formulation or in a separate formulation). The amount of the formulation which will be effective can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. The dosage of the compositions to be administered can be determined by the skilled artisan without undue experimentation in conjunction with standard dose-response studies. Relevant circumstances to be considered in making those determinations include the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms. For example, the actual patient body weight may be used to calculate the dose of the formulations in milliliters (mL) to be administered. There may be no downward adjustment to "ideal" weight. In such a situation, an appropriate dose may be calculated by the following formula:

Dose (mL) = [patient weight (kg) x dose level (mg/kg)/ drug concentration (mg/mL)]

For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. In certain embodiments, the dosage administered to a patient is between about 0.1 mg/kg and about 20 mg/kg of the patient's body weight, or is between about 1 mg/kg to about 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.

In certain embodiments, methods of preparing these formulations or compositions include combining another type of anti-tumor or anti-angiogenesis agent and a carrier and, optionally, one or more accessory ingredients. In general, the formulations can be prepared with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

Formulations for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of one or more subject antibodies as an active ingredient. In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, and the like), one or more antibodies of the present disclosure may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. i

Methods of the disclosure can be administered topically, either to skin, eye, or to mucosal membranes such as those on the cervix and vagina. This offers the greatest opportunity for direct delivery to tumor with the lowest chance of inducing side effects. The topical formulations may further include one or more of the wide variety of agents known to be effective as skin or stratum corneum penetration enhancers. Examples of these are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl or isopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents may further be included to make the formulation cosmetically acceptable. Examples of these are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surface active agents. Keratolytic agents such as those known in the art may also be included. Examples are salicylic acid and sulfur. Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The subject antibodies may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams, and gels may contain, in addition to an antibody, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an antibody, excipients sμch as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Pharmaceutical compositions suitable for parenteral administration may comprise one or more antibodies in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin. Injectable depot forms are made by forming microencapsule matrices of one or more antibodies in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.

Formulations for intravaginal or rectally administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the disclosure with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

Formulations particularly useful for antibody-based therapeutic agents are described in U.S. Pat. App. Publication Nos. 20030202972, 20040091490 and 20050158316. It is contemplated that certain formulations are liquid formulations. In a specific embodiment, the liquid formulations are substantially free of surfactant and/or inorganic salts. In another specific embodiment, the liquid formulations have a pH ranging from about 5.0 to about 7.0, about 5.5 to about 6.5, about 5.8 to about 6.2, and about 6.0. In yet another specific embodiment, the liquid formulations comprise histidine at a concentration ranging from about 1 mM to about 100 mM, from about 5 mM to about 50 mM, about 10 mM to about 25 mM. In still another specific embodiment, the liquid formulations comprise histidine at a concentration ranging from 1 mM to 100 mM, from 5 mM to 50 mM, 10 mM to 25 mM. It is contemplated that the liquid formulations have a concentration of one or more antibodies of the invention is about 50 mg/ml, about 75 mg/ml, about 100 mg/ml, about 125 mg/ml, about 150 mg/ml, about 175 mg/ml, about 200 mg/ml, about 225 mg/ml, about 250 mg/ml, about 275 mg/ml, or about 300 mg/ml. It is also contemplated that the liquid formulations may further comprise one or more excipients such as a saccharide, an amino acid (e.g., arginine, lysine, and methionine) and a polyol. Additional descriptions and methods of preparing and analyzed liquid formulations can be found, for example, in PCT publications WO 03/106644; WO 04/066957; WO 04/091658. Antibodies of the disclosure may also be prepared as a stable lyophilized protein formulation, such as those described in U.S. Pat. No. 6,685,940. For example, such a formulation may include an antibody and a lyoprotectant. A lyoprotectant can prevent or reduce chemical or physical instability of the antibody upon lyophilization and subsequent storage. Optionally, a lyophilized formulation includes a mixture of a non-reducing sugar, an antibody, and histidine.

Antibodies of the disclosure may also be prepared as a stable isotonic reconstituted formulation, for example, as described in U.S. Pat. No. 6,267,958. Such a reconstituted formulation can be prepared from a lyophilized mixture of the antibody and a lyoprotectant.

For treatment of lung disorders, including lung cancers, an anti-ALK antibody may delivered by inhalation. The pulmonary drug delivery compositions are useful for treating a pulmonary disease or condition. For example, aerosol compositions are provided for the delivery of an antibody or an antibody combined with an additional active agent to the respiratory tract. The respiratory tract includes the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchioli. The upper and lower airways are called the conductive airways. The terminal bronchioli then divide into respiratory bronchioli which then lead to the ultimate respiratory zone, the alveoli, or deep lung.

Pulmonary drug delivery may be achieved by inhalation, and administration by inhalation herein may be oral and/or nasal. Examples of pharmaceutical devices for pulmonary delivery include metered dose inhalers (MDIs) and dry powder inhalers (DPIs). Exemplary delivery systems by inhalation which can be adapted for delivery of the subject antibody and/or active agent are described in, for example, U.S. Pat. Nos. 5,756,353; 5,858,784; and PCT applications WO98/31346; WO98/10796; WO00/27359; WO01/54664; WO02/060412. Other aerosol formulations that may be used for delivering the antibody and/or active agent are described in U.S. Pat. Nos. 6,294,153; 6,344,194; 6,071,497, and PCT applications WO02/066078; WO02/053190; WO01/60420; WO00/66206. Pressurized metered dose inhalers (pMDIs) are the most commonly used inhaler worldwide. The aerosol is created when a valve is opened (usually by pressing down on the propellant canister), allowing liquid propellant to spray out of a canister. Typically, a drug or therapeutic is contained in small particles (usually a few microns in diameter) suspended in the liquid propellant, but in some formulations the drug or therapeutic may be dissolved in the propellant. The propellant evaporates rapidly as the aerosol leaves the device, resulting in small drug or therapeutic particles that are inhaled. Propellants typically used in such pMDIs include but are not limited to hydrofluoroalkanes (HFAs). A surfactant may also be used, for example, to formulate the drug or therapeutic, with pMDIs. Other solvents or excipients may also be employed with pMDIs, such as ethanol, ascorbic acid, sodium metabisulfate, glycerin, chlorobutanol, and cetylpyridium chloride. Such pMDIs may further include add-on devices such as, for example, spacers, holding chambers and other modifications. The third type of inhaler is the dry powder inhaler (DPI). In DPIs, the aerosol is usually a powder, contained within the device until it is inhaled. The therapeutic or drug is manufactured in powder form as small powder particles (usually a few millionths of a meter, or micrometers, in diameter). In many DPIs, the drug or therapeutic is mixed with much larger sugar particles (e.g., lactose monohydrate), that are typically 50-100 micrometers in diameter. The increased aerodynamic forces on the lactose/drug agglomerates improve entrainment of the drug particles upon inhalation, in addition to allowing easier filling of small individual powder doses. Upon inhalation, the powder is broken up into its constituent particles with the aid of turbulence and/or mechanical devices such as screens or spinning surfaces on which particle agglomerates impact, releasing the small, individual drug powder particles into the air to be inhaled into the lung. The sugar particles are usually intended to be left behind in the device and/or in the mouth-throat.

One aspect of the application provides an aerosol composition comprising an antibody that is an ALK antagonist. An aerosol antibody composition can be a composition comprising aerosolized antibody or a composition comprising an antibody in a formulation suitable for aerosolization. The antibody may be formulated in combination with an additional active agent, and the combination formulation is suitable for aerosolization. Alternatively, the antibody and an additional active agent may be formulated separately, such that they will be combined after aerosolization occurs or after being administered to a subject. An example of formulation suitable for aerosolization of an antibody is in physiologic osmolarity (e.g., between 280 and 320 mM) at a suitable pH (e.g., pH 6 to 8). A formulation of the present application may further comprise an excipient, for example polysorbate 80 which can be used at 0.0015 to 0.02%.

U.S. Pat. No. 5,474,759 discloses aerosol formulations that are substantially free of chlorofluorocarbons, and having particular utility in medicinal applications. The formulations contain a propellant (such as 1,1,1,2,3,3,3,-heptafluoropropane), a medium-chain fatty acid propylene glycol diester, a medium-chain triglyceride, optionally a surfactant, and optionally auxiliary agents such as antioxidants, preservatives, buffers, sweeteners, and taste masking agents. Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present application with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present application with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

"Aerosol composition" means an antibody and/or an active agent described herein in a form or formulation that is suitable for pulmonary delivery. The aerosol composition may be in the dry powder form, it may be a solution, suspension, or it may be in admixture with a suitable low boiling point, highly volatile propellant. It is to be understood that more than one antibody and optionally other active agents or ingredients may be incorporated into the aerosolized formulation or aerosol composition and that the use of the term "antibody" or "active agent" in no way excludes the use of two or more such antibodies or other agents or ingredients.

In certain embodiments, an antibody or active agent retains more than about 50% of its activity after formulation, preferably more than about 70%. In certain embodiments, an antibody or active agent retains more than about 50% of its purity after formulation, preferably more than about 70%.

Active agent formulations suitable for use in the present application include dry powders, solutions and particles suspended or dissolved within a propellant. Dry powders suitable for use in the present application include amorphous active agents, crystalline active agents and mixtures of both amorphous and crystalline active agents. The dry powder active agents have a particle size selected to permit penetration into the alveoli of the lungs, that is, preferably 10 μm mass median diameter (MMD), preferably less than 7.5 μm, and most preferably less than 5 μm, and usually being in the range of 0.1 μm to 5 μm in diameter. The delivered dose efficiency (DDE) of these powders is >30%, usually >40%, preferably >50 and often >60% and the aerosol particle size distribution is about 1.0-5.0 μm mass median aerodynamic diameter (MMAD), usually 1.5-4.5 μm MMAD and preferably 1.5-4.0 μm MMAD. These dry powder active agents have a moisture content below about 10% by weight, usually below about 5% by weight, and preferably below about 3% by weight. Such active agent powders are described in WO 95/24183 and WO 96/32149.

Dry powder active agent formulations may be prepared by spray drying under conditions which result in a substantially amorphous powder. Bulk active agent, usually in crystalline form, is dissolved in a physiologically acceptable aqueous buffer, typically a citrate buffer having a pH range from about 2 to 9. The active agent is dissolved at a concentration from 0.01% by weight to 1% by weight, usually from 0.1% to 0.2%. The solutions may then be spray dried in a conventional spray drier available from commercial suppliers such as Niro A/S (Denmark), Buchi (Switzerland) and the like, resulting in a substantially amorphous powder. These amorphous powders may also be prepared by lyophilization, vacuum drying, or evaporative drying of a suitable active agent solution under conditions to produce the amorphous structure. The amorphous active agent formulation so produced can be ground or milled to produce particles within the desired size range. Dry powder active agents may also be in a crystalline form. The crystalline dry powders may be prepared by grinding or jet milling the bulk crystalline active agent. The active agent powders of the present application may optionally be combined with pharmaceutical carriers or excipients which are suitable for respiratory and pulmonary administration. Such carriers may serve simply as bulking agents when it is desired to reduce the active agent concentration in the powder which is being delivered to a patient, but may also serve to improve the dispersability of the powder within a powder dispersion device in order to provide more efficient and reproducible delivery of the active agent and to improve handling characteristics of the active agent such as flowability and consistency to facilitate manufacturing and powder filling. Such excipients include but are not limited to (a) carbohydrates, e.g., monosaccharides such as fructose, galactose, glucose, D- mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, cellobiose, and the like; cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such as raffmose, maltodextrins, dextrans, and the like; (b) amino acids, such as glycine, arginine, aspartic acid, glutamic acid, cysteine, lysine, and the like; (c) organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, magnesium gluconate, sodium gluconate, tromethamin hydrochloride, and the like; (d) peptides and proteins such as aspartame, human serum albumin, gelatin, and the like; and (e) alditols, such as mannitol, xylitol, and the like. A specific group of carriers includes lactose, trehalose, raffmose, maltodextrins, glycine, sodium citrate, human serum albumin and mannitol.

The dry powder active agent formulations may be delivered using Inhale Therapeutic Systems's dry powder inhaler as described in WO 96/09085, but adapted to control the flow rate at a desirable level or within a suitable range. The dry powders may also be delivered using a metered dose inhaler as described by Laube et al. in U.S. Pat. No. 5,320,094.

Propellant systems may include an active agent dissolved in a propellant or particles suspended in a propellant. Both of these types of formulations are described in Rubsamen et al., U.S. Pat. No. 5,672,581. EXEMPLIFICATION

The disclosure now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the disclosure.

Example 1: Generation of Anti-ALK Antibodies

Glutathion S-transferase (GST) fusion protein with a fragment of the human ALK ECD (amino acids 368-447, GenBank™ NP_004295) was described in Stoica et al. (2001), J. Biol. Chem. 276:16772-16779. That fragment of the ALK ECD contains the putative LBD (amino acids 396-406 of the human ALK sequence). The GST fusion protein was used to raise rabbit antibodies, and IgGs were purified from the antiserum using protein G affinity chromatography (Pierce).

Preparation of anti-ALK LBD murine monoclonal antibodies was described in Stoica et al. (2002) J. Biol. Chem. 277:35990-35998. The monoclonal antibodies to the LBD of ALK were prepared by immunizing mice with the LBD in the form of a GST fusion protein as described above (Stoica et al., 2001, supra). A different protein produced in mammalian cells was used for the screening of mouse sera and hybridoma superaatants; the complete ECD of ALK was expressed as a secreted protein in SW- 13 cells by subcloning the ECD (Iwahara et al. (1997) Oncogene 14:439-449) into the eukaryotic expression vector pCDNA with a C-terminal

Myc/His tag. This construct, including the original secretory signal sequence at the N terminus, was stably transfected and expressed in SW- 13 cells, and the secreted ECD protein was isolated from the cell supernatants. To generate hybridoma cells producing antibodies of interest, spleen cells from an immunized mouse showing a positive enzyme-linked immunosorbent assay signal with the ECD protein was used for fusion with FOX-NY myeloma cells based on a protocol of Koehler and Milstein ((1976) Eur. J. Immunol. 6:511-519). The complete ECD of ALK was then used to screen individual hybridoma clones by enzyme-linked immunosorbent assay for the presence of anti-LBD antibodies. The antibodies used in Stoica et al. (2002) were derived from supernatants of two different hybridoma, 16G2-3 and 9Cl 0-5 (also referred to as 19C10 herein).

Example 2: Effect of Anti-ALK Antibody on Tumor Growth/Size— Murine Models

Figures 1 and 2 show the inhibitory effect of anti-ALK antibody 8B10 on tumor growth as determined by tumor sizes in murine models of MDA-MB231 hormone-independent breast adenocarcinoma. The anti-ALK antibody caused marked decrease in tumor size, nearly eliminating the tumor in some animals.

Example 3: Angiogenesis and ALK Gene Expression

As shown in Figure 3, FGF-2 is a potent inducer of ALK expression. This effect may be relatively indirect. FGF-2 is an important early-acting pro-angiogenic factor. Thus, this data indicates that ALK expression is substantially increased in endothelial cells that are subjected to pro-angiogenic signals.

Figures 4A and 4B show that ALK is expressed in new blood vessels using Matrigel plug assays. The new vessels formed in the Matrigel stain positive when an antisense ALK probe is used to detect gene expression. When bFGF is included in the Matrigel plug, the production of ALK mRNA increases. Figure 5 shows an inhibitory effect of the 8B10 antibody on new vessel formation based on vessel counts obtained by Matrigel plug assays.

Example 4: Angiogenesis and ALK Gene Expression

ALK is expressed, and overexpressed, in various human cancers. Figure 6 shows the expression of ALK in various human clinical tumor samples. Prostate, breast, colon, lung, ovarian, bladder, and soft tissue cancers all show ALK overexpression in some percentage of samples. As shown in Figure 7, ALK and PTN expression are both present in an increasing proportion of colon cancer samples as the cancer stage progresses. ALK and PTN are present and overexpressed in a high percentage of colon cancer metastases. ALK is expressed in tumor cells and vasculature (Figure 8). Clinical breast cancer samples, taken from patients with known outcome, were used to detect PTN levels. PTN expression is associated with increased likelihood of patient death in stage I-III breast cancers and a substantially shorter time to death in stage IV cancers (Figure 9B). In glioblastomas, PTN and ALK are expressed in a very high percentage of tumors (Figure 10). Lu et al. ((2005) J. Biol. Chem. 280:26953-26964) also reported two naturally occurring forms of PTN (18 and 15 kDa) that differentially promoted glioblastoma migration and proliferation. U87 glioblastoma cells have low to high levels of ALK expression (see, Powers et al., JBC 277:14153-14158 (2002)) and transplanted into mice. Tumors containing cells expressing higher levels of ALK tended to become far larger than those expressing little or no ALK (Figure 11).

Example 5: Effect of Anti-ALK Antibody on Angiogenesis — Wound Healing Assay

Figure 12 depicts an inhibitory effect of the 8B10 antibody on wound healing using a murine model. A skin punch was used to generate full thickness wounds on a mouse of even size. Starting at day four, mice were sacrificed and the wound analyzed histologically to determine the extent of wound closure. At both four and six days post- wounding, Anti-ALK treated wounds showed less closure than controls.

Example 6: Effect of Anti-ALK Antibody on Spreading of Cancer Cells

MDS-MB231 cells were tested for spreading, a feature associated with metastasis, in the presence and absence of an anti-ALK antibody that binds to the ligand binding domain (LBD) of ALK. Anti-ALK antibody treatment caused dose- dependent inhibition of cell spreading, reaching complete inhibition (Figure 16).

Example 7: Effect of Anti-ALK Antibody on Invasion of A Endothelial Layer by Cancer Cells

Figure 17 illustrates the ECIS method used to measure invasiveness of cancer cells. A cell layer is grown on a electrode. The current passing through the cell layer can be measured in real time. A metastatic cell is then added to the well, and when the cancer cell penetrates/invades the preexisting cell layer, there is a net change in the conductivity that can be measured. Figure 18 (in color) shows the inhibitory effect of the 8B10 antibody on MDA-MB231 breast cancer cells' ability to invade an endothelial layer. The blue line demonstrates the ability of the MDA- MB231 cells to invade an endothelial layer. The addition of an anti-ALK antibody reverses the invasion of the endothelial layer by the MDA-MB231 cells. Figure 19 shows the inhibitory effect of the 19C10 antibody on MDA-MB231 breast cancer cells' ability to invade an endothelial layer at both 100 μg/ml and 50 μg/ml. As shown in Figure 20, differences are seen between the cells treated with the control antibody (6 AS) and the cells treated with the anti-ALK antibody 19C10 in the ECIS studies. The cells in the 19C10-treated samples were growing in clumps, rather than attaching well and spreading.

Example 8: Effect of Anti-ALK Antibody in Soft Agar Colony Formation Assay

Figure 21 shows the inhibitory effects of 19Cl on colony formation in Soft Agar assays conducted using 24- well plates with Cell Titer GIo. 19C10 was seen to inhibit colony formation at antibody concentrations of 100 μg/ml or 50 μg/ml by about 5 fold.

Materials used in these assays include: 2.4% low, melting temp agarose (SeaPlaque Agarose, Cambrex, #50100), autoclaved; 1 X IMEM + 10% FBS; 24- well tissue culture plate; 40 ⁰C water bath. Autoclaved agar was melted by micro waving and cooled slightly before being mixed with the IMEM +10% FBS pre-warmed in the water bath. A stock of 0.6% agarose can be made this way.

300 μl of 0.6% agarose was added to each well of the 24-well plate and left at room temperature to solidify. Cells were trypsinized and resuspended in IMEM+10% FBS. About 3500 cells per well/tube were used in the assays.

Treatments as desired were added to each tube. Additional 0.6% agarose mixture was added to each tube. The mixture of cells, and treatments, and agarose mixture was immediately added to the solidified bottom agarose layer in the plate, which was then placed into an incubator for an extended period of time (e.g., for 7-10 days for SWl 3 cells). Analysis using Cell Titer GIo was carried out using the standard protocol, and the plate was then read using a plate reader (e.g., Wallac/Victor).

Example 9: Binding Specificity of 19C10 by ELISA Analysis

Microtiter wells were coated with either ALK-LBD-Fc fusion protein or recombinant ALK-ECD-myc/his tag at 10 μg/mL and the wells were then washed and blocked with milk. Next the wells were washed and then incubated with increasing concentrations of 19C10 (-1-5 ng/mL). After washing anti-mouse IgM- HPR was used for detection. Figure 22 shows that 19C10 binds well to an ALK- LBD-Fc fusion protein but not to a recombinantly expressed ALK-ECD protein.

Example 10: Immunoprecipitation Assays to Identify ALK-Interacting

Proteins

Figure 13 illustrates a Myc/His-tagged ALK molecule useful in immunoprecipitation assays. The Myc/His-tagged ALK molecule, either a kinase active form or a kinase inactive mutant, is expressed in HEK293 cells. Anti-myc antibody or anti-phosphotyrosine antibody is used to immunoprecipitate ALK- containing complexes. As shown in Figure 14, certain protein bands were found to only associate with the (tyrosine) phosphorylated ALK complexes. These protein bands were subjected to mass spectrometry sequencing and identification. The following proteins were identified as interacting with ALK: IJNC5, a netrin receptor; LTK receptor; tensin and actinin, both actin-based cytoskeleton molecules; and the IRS adaptor protein.

Example 11: Preparation of Additional Antibodies

Figure 15 shows structural models of the ECD of ALK as compared to the extracellular domain of another receptor tyrosine kinase. As depicted, the LBD is predicted to be on the surface of ALK and contribute to dimerization of ALK. Other regions (e.g., the portion of the ECD in yellow) are also predicted to contribute to dimerization of ALK. Accordingly, the LBD as well as these other regions can be used to generate antibodies, in particular, antibodies capable of disrupting dimerization of ALK. For example, GST fusion proteins comprising the LBD or another region of ALK that contributes to dimerization may be generated and used to immunize a host animal, as described above.

Similarly, regions of ALK may be identified by structural modeling as contributing to heterodimerization or heteromultimerization, such as, for example, forming a complex with LTK. Antibodies that recognize those regions of ALK can also be made, and such antibodies may disrupt heterodimerization or heteromultimerization of ALK with another protein (e.g., LTK).

Example 12: Identification of Fully-Human Anti-ALK Antibodies by Phage Panning

Phage display technology was used to identify Fabs that bind to the ECD region or the smaller LBD of the ALK protein. Phage library fab310 from Dyax was used for solid and soluble phase panning. Soluble phase panning: Streptavidin coated dynabeads (Dynal Biotech) was blocked in 1% milk. Phage library was blocked with 2% milk and deselected on biotin-streptavidin beads. Blocked and deselected phage was exposed to 3 μg of biotinylated ALK-ECD or the ALK-LBD and the ALK-phage complex was captured by blocked dynabeads. Bound phage was eluted using 1 niL of 100 mM triethylamine (Sigma) and elute was neutralized by adding 0.5 mL of IM Tris-HCL. Solid Phase panning: The phage library was blocked with 2% milk. Blocked phage library was transferred to ALK-ECD or the ALK-LBD coated immunotube

(20 μg coated in 1 mL PBS 7.4) that was blocked with 2% milk. After two hours incubation, the immunotube was washed with PBST (PBS + 0.1% Tween) 10-20 times then with PBS 10-20 times to remove the unbound phage. The phage bound to the immunotube was eluted with 1 ml of 100 mM triethylamine (Sigma) and neutralized by adding 0.5 mL of IM Tris-HCL.

Infection was carried out by mixing 1 volume of phage elute with 5 volumes of TGl (Novagen) at log phase and four volumes of 2YT (Teknova). This mix was incubated for 30 minutes at 37 °C water bath. After infection, it was spun down at

400Og for 5 minutes and pellet resuspended in 2YT. TG-I cells were plated on 2YT plates containing 50ug/ml carbenicillin and 2% glucose (Teknova) and were incubated at 30 ⁰C overnight. On the second day, bacterial colonies were collected and infected with helper phage (Invitrogen). The infected cells were grown overnight in 2YT medium containing carbenicillin (Invitrogen) and kanamycin(Sigma) to generate high titer. The phage was concentrated from overnight culture by PEG precipitation. PEG precipitation was done using PEG/NaCl solution at one fifth volume of culture (PEG from Fluka). After precipitation, phage pellet was resuspended in 1 mL PBS (pH 7.4, Invitrogen) and was used for next round panning. Three rounds of solid and soluble phase panning were done.

The entire ECD domain tagged with Myc/His was expressed in 293 cells and purified from the conditioned media by affinity chromatography and biotin labeled.

An ALK-LBD polypeptide labeled with biotin via a aminohexanolic acid linker (abbreviated as Biot-K(Ahx) as shown below) was also used for the panning process described above. The protein contains a 16 amino acid ligand binding region (see below). In addition a similarly label control peptide was used as a negative control for binding assays.

Biot-K(Ahx)GRIGRPDNPFRVALEY-LBD16-LBDi₆ (SEQ ID NO: 4)

Biot-K(Ahx)APVGRPEILRYRGNDF-Control (SEQ ID NO: 5)

96 clones were identified using the ALK-LBD polypeptide. None of the 96 cloned bound to the ALK-ECD-myc/his tag protein (data not shown). 67 clones were identified using the ALK-ECD protein (Figure 23). None of them bound to the 16 amino acid ALK-LBD polypeptide (Figure 23), but 60 of them bound to an ALK fragment-Fc fusion protein containing the 16 amino acid LBD sequence (ALK- LBD-Fc). The ALK fragment of the fusion protein has the following 80 amino acid sequence (and the underlined portion corresponds to the 16 amino acid LBD):

HNEAAREILLMPTPGKHGWTVLQGRIGRPDNPFRVALEYISSGNRSLS AVDFFALKNCSEGTSPGSKMALQSSFTCWNGT (SEQ ID NO: 6) In addition, 2 of these 67 clones bound to ALK expressed on the cell surface. The isolated clones represent 24 unique λ light chains (Figure 24) and 45 unique K light chains (data not shown). 14 of these clones have been converted to fully human IgGs. The nucleotide and corresponding amino acid sequences of the variable regions of the heavy (V_H) and the light chains (V_L) of the Human anti-ALK clones converted to IgGs are shown in Figure 25A-N.

Example 13: Characterization of the Fully Human Anti-ALK Antibodies

Fab supernatant from isolated phage clones was examined by ELISA for binding to the ALK-ECD-myc/his tag as well as the ALK-LBD-Fc fusion protein. As shown in Figure 26, the clones could be classified into four classes: those that bind equally well to both ALK-LBD-Fc and ALK-ECD (top left); those that bind better to ALK-ECD (top right); those that bind better to ALK-LBD-Fc (bottom left); and those that bind minimally to ALK-LBD-Fc (bottom right).

Clones were also examined for their ability to bind to the ALK protein expressed on the cell surface. 293 cells transiently expressing ALK on their surface were blocked with FBS and a 1:2 dilution of phage Fab supernatant was added to the cells and detected with a 1 :200 dilution of FITC conjugated anti-M13 antibody. Figure 27 shows the results for two clones, 3Al 1 and 3E8, which bind to the ALK- expressing 293 cells in this assay as indicated by the surface staining.

As described above, 14 clones were converted into full length IgGs. These full length IgGs were assayed for binding to the ALK-ECD as well as the ALK- LBD-Fc fusion protein as follows, microtiter wells were coated with either ALK- LBD-Fc fusion protein or recombinant ALK-ECD-myc/his tag at 10 μg/mL and the wells were then washed and blocked with milk. Next the wells were washed and then incubated with increasing concentrations of the full length IgG clones. After washing anti-human IgG-HPR (at 1:1000 dilution) was used for detection. Figures 28 and 29 show the binding curves for all 14 clones. Each clone had similar binding specificity as a full IgG as they had as a Fab fragment. The EC_5O Values for ECD (Table 1) were determined for several clones, Table 1: EC₅₀ Values for ECD (ng/ml)

The full length IgGs were also examined by FACS for their ability to bind to the cell surface of ALK expressing cells as follows, 200,000 to 500,000 ALK positive cells were used per well. The cells were blocked with 2% FBS and then stained with 1 to 20 μg/mL of primary human anti-ALK IgG antibody. The cells were washed and stained with FITC conjugated anti-human IgG, 1 :200 dilution then the cells were washed and sorted. Figure 30 shows some representative FACS data. Antibody clones DlO and Al strongly bind the surface of ALK expressing cells.

Example 14: Binding Kinetics as Determined by Surface Plasmon

Resonance Measurements

All experiments were performed on a BIAcore 3000 instrument (BIAcore, Inc., Uppsala, Sweden). The ligand, ALK-ECD, was prepared at 50 nM in 10 mM NaOAc, pH4 buffer, then injected onto an EDC/NHS-activated CM5 sensor chip (BIAcore, Inc. Uppsala, Sweden) using a standard immobilization protocol.

Following this, any unreacted active ester moieties were quenched using IM Et- NH2 (ethanolamine). These coupling reagents were also purchased from the manufacturer (BIAcore, Inc.). A total of 604 RUs worth of ALK-ECD remained bound to the surface. Separately, a blank surface was prepared using the identical protocol, minus the protein. This surface was used as a reference cell throughout the experiment, and served to correct for both non-specific binding and some housekeeping artifacts.

For the kinetic measurements, IgGs Al and DlO were prepared at 200 nM in HBS-EP buffer (BIAcore, Inc., consisting of the following: 10 mM HEPES buffer, pH7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% P20), then serially diluted, 1:1, down to 1.56 nM in this same buffer. Duplicate injections of each concentration of IgG were then made over the ALK-ECD and reference cell surfaces, which are connected in series. Between injections, surfaces were regenerated with 3 consecutive, 1 -minute injections of 3 M MgC12.

Raw binding data was corrected in the manner described by Myszka (D.G. Myszka, Improving biosensor analysis. J. MoI. Recognit. 12 (1999), pp. 279-284). Fully corrected binding data was then globally fit using a 1 : 1 binding model (BIAevaluation 4.1 software, BIAcore, Inc, Uppsala, Sweden), that included a term to correct for mass transport-limited binding, should it be detected.

Representative BIAcore curves for binding of full length IgG Al and DlO to ALK-ECD-myc/his are shown in Figure 31 (top and bottom panels, respectively). The binding constants are summarized in Table 2.

Table 2. Binding Constants For Al and DlO

Example 15: Agonist Activity of DlO

The activity of the fully human anti-ALK IgG DlO was measured using the soft agar colony formation assay as described above (see Example 8). As shown in Figure 32, DlO demonstrated a strong agonist effect, greatly enhancing colony formation. The ALK ligand, PTN, also enhances colony formation in a soft agar assay (data not shown).

The activity of DlO was also examined in a kinase stimulation assay. ALK expressing cells (SK-N-SH, serum starved cells) were either untreated or stimulated with either PTN as a positive control or DlO. Cell extracts are then generated and incubated with mixture of 3 Ab: mAb (BD), 4ug/sample; pAb (Santa Cruz), lug/sample; pAb (Zymed), lug/sample at 4 ⁰C overnight. ALK was then immunoprecipitated by incubation with 100 μl 50% slurry of a combination of protein A and protein G Sepharose beads for 3-4 hours at 4 ⁰C with mixing followed by 5 washes in immunoprecipitation (IP) buffer and elution from the beads with LDS buffer and heated at 95 ⁰C for 10 minutes. The immunoprecipitated protein was subjected to SDS-PAGE on 3-8% Tris-Acetate gels and blotted to nitrocellulose. Phosphorylated ALK was then detected with pTyr: 4G10 (Upstate), 1:500 dilution; all is detected with ALK: rabbit pAb mixture (Cell Sig. Tech + Zymed), 1 : 1J300 each. 800 μg protein per sample of SK-N-SH was used for IP. Extract from 293 cells expressing ALK was used directly for IB as a positive control for the Western Blots.

As shown in Figure 33, DlO stimulates phosphorylation of ALK more strongly then even the ALK ligand PTN.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

While specific embodiments of the subject disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the disclosure will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

CLAIMS:

1. An isolated antibody or antigen-binding portion thereof that binds to an epitope situated in the extracellular portion of ALK and inhibits an ALK- mediated biological function selected from the group consisting of: angiogenesis, tumor growth, spreading of cancer cells, kinase activity, ligand binding, cell proliferation, cell adhesion, wound healing and invasion of an endothelial layer by cancer cells.

2. A hybridoma that produces an antibody of claim 1.

3. A method of treating cancer, the method comprising administering to a patient in need thereof an effective amount of an ALK antagonist.

4. The method of claim 3, wherein the ALK antagonist is an isolated antibody or antigen-binding portion thereof that binds to an epitope situated in the extracellular portion of ALK and inhibits an ALK-regulated biological function selected from the group consisting of: angiogenesis, tumor growth, spreading of cancer cells, and invasion of an endothelial layer by cancer cells.

5. The method of claim 3, wherein the patient is diagnosed with a glioblastoma or a hormone-independent breast cancer.

6. The method of claim 3, wherein the cancer does not exhibit ALK overexpression.

7. The method of claim 3, wherein the cancer is an angiogenesis independent cancer.

8. The method of claim 3, wherein the cancer is a pre-metastatic cancer.

9. The method of claim 4 wherein the isolated antibody or antigen-binding portion thereof is administered systemically.

10. The method of claim 4, wherein the isolated antibody is administered locally.

11. A method of inhibiting angiogenesis in a patient, the method comprising administering to a patient in need thereof an effective amount of an isolated antibody or antigen-binding portion thereof that binds to an epitope situated in the extracellular portion of ALK and inhibits angiogenesis.

12. The method of claim 11, wherein the patient is diagnosed with macular degeneration.

13. A method of inducing tumor regression in a patient, the method comprising administering to a patient in need thereof an effective amount of an isolated antibody or antigen-binding portion thereof that binds to an epitope situated in the extracellular portion of ALK and induces tumor regression.

14. The method of claim 13, wherein the tumor regression is angiogenesis independent.

15. The method of claim 13, wherein the tumor regression is angiogenesis dependent.

16. A pharmaceutical preparation comprising the isolated antibody or antigen- binding portion thereof of claim 1.

17. Use of an ALK antagonist to make a pharmaceutical preparation for treating cancer in a patient.

18. The use of claim 17, wherein the patient is diagnosed with a glioblastoma or a hormone-independent breast cancer.

19. The use of claim 17, wherein the cancer does not exhibit ALK overexpression.

20. The use of claim 17, wherein the cancer is an angiogenesis independent cancer.

21. The use of claim 17, wherein the cancer is a pre-metastatic cancer.

22. The use of claim 17, wherein the ALK antagonist is an antibody of claim 1.

23. An isolated antibody or antigen-binding portion thereof of claim 1 , wherein the isolated antibody or antigen-binding portion thereof is covalently linked to an additional functional moiety.

24. The isolated antibody or antigen-binding portion thereof of claim 23, wherein the additional functional moiety is a label.

25. The isolated antibody or antigen-binding portion thereof of claim 23, wherein the label is suitable for detection by a method selected from the group consisting of: fluorescence detection methods, positron emission tomography detection methods and nuclear magnetic resonance detection methods.

26. The isolated antibody or antigen-binding portion thereof of claim 23, wherein the label is selected from the group consisting of: a fluorescent label, a radioactive label, and a label having a distinctive nuclear magnetic resonance signature.

27, The isolated antibody or antigen-binding portion thereof of claim 23, wherein the additional functional moiety confers increased serum half-life on the antibody or antigen-binding portion thereof.

28. The isolated antibody or antigen-binding portion thereof of claim 27, wherein the additional functional moiety comprises a polyethylene glycol (PEG) moiety.

29. The monoclonal antibody designated 8B10.

30. The monoclonal antibody produced by the 8B10 hybridoma (ATCC Deposit No. PTA-7429).

31. A humanized anti-ALK antibody comprising one or more variable regions of 8B10.

32. A humanized anti-ALK antibody comprising one of more CDRs of 8B10.

33. An isolated antibody or antigen-binding portion thereof that binds to an epitope situated in the extracellular portion of ALK and activates an ALK- mediated biological function selected from the group consisting of: angiogenesis, kinase activity, ligand binding, cell proliferation, cell adhesion, wound healing.

34. The antibody of claim 33, wherein said antibody or antigen-binding portion thereof binds to the same epitope as DlO.

35. Use of an ALK agonist to make a pharmaceutical preparation for treating a patient suffering from a condition selected from the group consisting of: vascular disease, hypertension, Reynaud's disease, Reynaud's phenomenon, aneurysms, wounds, burns, tissue damage, ischemia reperfusion injury, angina, myocardial infarctions, chronic heart conditions, heart failure, ischemic limb, osteoporosis and fractures.

36. The use of claim 35, wherein the ALK agonist is an antibody of claim 33.

37. An isolated human anti-ALK antibody that inhibits an ALK-mediated biological function selected from the group consisting of: angiogenesis, tumor growth, spreading of cancer cells, kinase activity, ligand binding, cell proliferation, cell adhesion, wound healing and invasion of an endothelial layer by cancer cells.

38. An isolated human anti-ALK antibody that stimulates an ALK-mediated biological function selected from the group consisting of: angiogenesis, kinase activity, ligand binding, cell proliferation, cell adhesion, and wound healing.

39. A human anti-ALK antibody selected from the group consisting of: 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El and E5.

40. The human anti-ALK antibody of claim 39, wherein the antibody comprises a full-length IgG.

41. An anti-ALK antibody comprising a V_H that has an amino acid sequence at least 80% identical to a V_H selected from the group consisting of the VH regions of 8B10, 16G2-3, 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, D10, El and E5.

42. An anti-ALK antibody comprising a V_L that has an amino acid sequence at least 80% identical to a V_L selected from the group consisting of the V_L regions of 8B10, 16G2-3, 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, D10, El and E5.

43. The anti-ALK antibody of any of claims 41 and 42, wherein the antibody comprises a CDR having the same amino acid sequence as a CDR of an antibody selected from the group consisting of 8B10, 16G2-3, 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El and E5.

44. The anti-ALK antibody of claim 43, wherein the antibody comprises at least two CDRs that have the same amino acid sequences as at least two CDRs of an antibody selected from the group consisting of 8B10, 16G2-3 , 9C 10-5 ,

3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El and E5.

45. An anti-ALK antibody that binds to the same epitope as the epitope for an antibody selected from the group consisting of 8B10, 16G2-3, 9Cl 0-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El and E5.

46. The anti-ALK antibody of claim 45 that competes for binding to the epitope against the antibody selected from the group consisting of 8B10, 16G2-3, 9C10-5, 3A11, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El and E5..

47. An isolated nucleic acid encoding an anti-ALK antibody, wherein the nucleic acid comprises a nucleotide sequence encoding a peptide that has an amino acid sequence at least 80% identical to the amino acid sequence of a variable region selected from the group consisting of the variable regions of 8B10, 16G2-3, 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El and E5.

48. An isolated nucleic acid encoding an anti-ALK antibody, wherein the nucleic acid comprises a nucleotide sequence at least 70% identical to a nucleotide sequence of a variable region selected from the group consisting of the variable regions of 8B10, 16G2-3, 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El and E5.

49. An isolated nucleic acid encoding an anti-ALK antibody, wherein the nucleic acid comprises a nucleotide sequence encoding a CDR of a variable region selected from the group consisting of the variable regions of 8B10, 16G2-3, 9C10-5, 3Al 1, 6A2, Al, A2, A7, B2, B6, B8, Cl, C6, D5, DlO, El and E5.

50. The isolated nucleic acid of claim 49, wherein the nucleic acid comprises a nucleotide sequence encoding at least 2 CDRs of a variable region selected from the group consisting of the variable regions of 8B10, 16G2-3, 9C10-5, 3Al 1, 6A2, Al, A2, Kl, B2, B6, B8, Cl, C6, D5, DlO₅ El and E5.