Anti-receptor tyrosine kinase-related protein (Ryk) antibodies and uses thereof

Abstract

Provided herein are, inter alia, antibodies that bind to receptor tyrosine kinase-associated protein (Ryk) with high efficiency and specificity. The antibodies and antibody compositions provided herein include, for example, novel light and heavy chain domain CDRs and framework regions, and are useful, inter alia, for the diagnosis and treatment of cancer and other RYK-associated diseases. In embodiments, the anti-RYK antibodies provided herein can bind to human RYK protein, but not to mouse RYK protein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 234,527, filed August 18, 2021, which is incorporated by reference in its entirety for all purposes.

[0002] STATEMENT AS TO RIGHTS TO INVETIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under CA236361 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Sequence Listing The attached sequence listing material is incorporated herein by reference in its entirety. The attached file named "048537-648001WO_SL_ST26.xml" was created on August 15, 2022 and is 120,584 bytes. This file can be accessed using Microsoft Word on a computer using the Windows OS. [Background technology]

[0004] Related-to-receptor tyrosine kinase (Ryk) is a kinase that mediates the synthesis and function of Wingless-int (Wnt) ligands (e.g., Wnt1 1 , Wnt3a 1 , Wnt5a 2 , and possibly Wnt5b 3 ) is a highly conserved single-transmembrane receptor. The extracellular domain contains a WIF (Wnt inhibitory factor-1-like) domain, which may initiate complex formation with Frizzled (Frz) receptors for crosstalk in the Wnt signaling network, affecting the activation of β-catenin-dependent and β-catenin-independent (e.g., non-canonical) Wnt signaling pathways.1、4、5 The cytoplasmic domain contains a tyrosine kinase domain that lacks apparent kinase activity. 5~7 However, the cytoplasmic domain of Ryk can be cleaved by gamma secretase and released for nuclear translocation. 8 , suggesting that it may have an alternative role in regulating cell signaling.

[0005] Ryk plays a major role in embryonic development, regulating axonal outgrowth, cardiovascular and craniofacial development, and fetal hepatic hematopoiesis. 9~11、12 Genomic disruption of RYK results in perinatal lethality. Despite the importance of Ryk in early development, its expression appears to be attenuated during development and is not critically expressed in tissues postnatally. However, postnatal expression of Ryk has not been well studied due to the lack of a high-affinity, highly specific anti-Ryk monoclonal antibody (mAb) that reacts with live cells expressing Ryk.

[0006] Notably, however, there are reports describing the expression of Ryk by a variety of cancers. 5 For example, Ryk is significantly expressed in glioblastoma and promotes the "stemness" of glioblastoma cells through its ability to regulate the Wnt / β-catenin pathway. 13 In addition, Ryk may be expressed in breast cancer, where it has been shown to promote the proliferation of breast cancer tumor-initiating cells and enhance breast cancer cell proliferation. 14、15 Ryk is also involved in gastric cancer tumorigenesis. 16 Again, evaluation of Ryk expression by various cancers has been difficult due to the lack of highly specific anti-Ryk mAbs to examine the relative expression of Ryk in cancer cells and normal postpartum tissues.

[0007] For example, there is a need in the art for mAbs that are highly specific to cells expressing Ryk, for studying the functional significance of Ryk in early development and neoplasia. Thus, there is a need in the art for mAbs that target cells expressing Ryk for detection, elimination, functional inhibition, immune-mediated destruction, or targeted drug delivery. Commercially available reagents used to assess the expression of Ryk on cells are antibodies derived from heterologous antisera, e.g., sheep anti-Ryk (R&D systems). These anti-Ryk antibodies were selected to bind to Ryk. However, they are not specific to human Ryk and cross-react with cells that do not express human Ryk. There is one commercially available mAb that claims to be specific to human Ryk (e.g., SAP18, LSBio 2401 Fourth Avenue Suite 900, Seattle WA 98121). However, this mAb reacts with apparently denatured human Ryk. Thus, although this mAb can be used to detect Ryk in immunoblot or ELISA assays, it has not been validated for use in flow cytometry due to its apparent lack of specific binding to cell surface Ryk on live cells.In summary, previously generated anti-Ryk antibodies do not appear suitable for potential use in therapy of patients with Ryk-expressing cancers.

[0008] Provided herein, among other things, are solutions to these and other needs in the art. Summary of the Invention

[0009] In one aspect, an anti-RYK antibody is provided comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 1, CDR H2 as set forth in SEQ ID NO: 2, and CDR H3 as set forth in SEQ ID NO: 3, and the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 4, CDR L2 as set forth in SEQ ID NO: 5, and CDR L3 as set forth in SEQ ID NO: 6.

[0010] In another aspect, an anti-RYK antibody is provided comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 17, CDR H2 as set forth in SEQ ID NO: 18, and CDR H3 as set forth in SEQ ID NO: 19, and the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 20, CDR L2 as set forth in SEQ ID NO: 21, and CDR L3 as set forth in SEQ ID NO: 22.

[0011] In another aspect, an anti-RYK antibody is provided comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 33, CDR H2 as set forth in SEQ ID NO: 34, and CDR H3 as set forth in SEQ ID NO: 35, and the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 36, CDR L2 as set forth in SEQ ID NO: 37, and CDR L3 as set forth in SEQ ID NO: 38.

[0012] In another aspect, an anti-RYK antibody is provided comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 49, CDR H2 as set forth in SEQ ID NO: 50, and CDR H3 as set forth in SEQ ID NO: 51, and the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 52, CDR L2 as set forth in SEQ ID NO: 53, and CDR L3 as set forth in SEQ ID NO: 54.

[0013] In another aspect, an anti-RYK antibody is provided, which binds to the same epitope as an antibody comprising a heavy chain variable domain comprising CDR H1 set forth in SEQ ID NO:1, CDR H2 set forth in SEQ ID NO:2, and CDR H3 set forth in SEQ ID NO:3, and a light chain variable domain comprising CDR L1 set forth in SEQ ID NO:4, CDR L2 set forth in SEQ ID NO:5, and CDR L3 set forth in SEQ ID NO:6.

[0014] In another aspect, an anti-RYK antibody is provided, which binds to the same epitope as an antibody comprising a heavy chain variable domain comprising CDR H1 set forth in SEQ ID NO: 17, CDR H2 set forth in SEQ ID NO: 18, and CDR H3 set forth in SEQ ID NO: 19, and a light chain variable domain comprising CDR L1 set forth in SEQ ID NO: 20, CDR L2 set forth in SEQ ID NO: 21, and CDR L3 set forth in SEQ ID NO: 22.

[0015] In another aspect, an anti-RYK antibody is provided, which binds to the same epitope as an antibody comprising a heavy chain variable domain comprising CDR H1 set forth in SEQ ID NO: 33, CDR H2 set forth in SEQ ID NO: 34, and CDR H3 set forth in SEQ ID NO: 35, and a light chain variable domain comprising CDR L1 set forth in SEQ ID NO: 36, CDR L2 set forth in SEQ ID NO: 37, and CDR L3 set forth in SEQ ID NO: 38.

[0016] In another aspect, an anti-RYK antibody is provided, which binds to the same epitope as an antibody comprising a heavy chain variable domain comprising CDR H1 set forth in SEQ ID NO:49, CDR H2 set forth in SEQ ID NO:50, and CDR H3 set forth in SEQ ID NO:51, and a light chain variable domain comprising CDR L1 set forth in SEQ ID NO:52, CDR L2 set forth in SEQ ID NO:53, and CDR L3 set forth in SEQ ID NO:54.

[0017] In another aspect, there is provided an isolated nucleic acid encoding the anti-RYK antibodies provided herein, including embodiments thereof.

[0018] In another aspect, a cell is provided that comprises an anti-RYK antibody provided herein (including embodiments thereof), or a nucleic acid provided herein (including embodiments thereof).

[0019] In another aspect, there is provided a pharmaceutical composition comprising a therapeutically effective amount of an antibody provided herein (including embodiments thereof) and a pharma- ceutically acceptable excipient.

[0020] In another aspect, a method of generating an antibody capable of binding to a RYK protein is provided, the method comprising immunizing a mammal with a peptide comprising the sequence of SEQ ID NO:129.

[0021] In another aspect, a method for detecting a RYK-expressing cell is provided, the method comprising: (i) contacting a RYK-expressing cell with an antibody (including embodiments thereof) provided herein; and (ii) detecting binding of the antibody to the RYK protein expressed by the cell.

[0022] In another aspect, a method of treating cancer in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an anti-RYK antibody provided herein (including embodiments thereof).

[0023] In another aspect, a method for identifying an anti-RYK antibody is provided, the method comprising: (i) contacting an antibody with a first RYK polypeptide comprising an amino acid sequence corresponding to amino acid residues 48-57 of SEQ ID NO: 129; (ii) detecting an antibody that binds to the first RYK polypeptide; (iii) contacting the antibody with a second RYK polypeptide that does not comprise an amino acid sequence corresponding to amino acid residues 48-57 of SEQ ID NO: 129; and (iv) detecting an antibody that does not bind to the second RYK polypeptide, thereby identifying an anti-RYK antibody. [Brief description of the drawings]

[0024] [Figure 1] 1 shows a comparison of the extracellular domains of human RYK and mouse RYK. An alignment of the amino acid sequences of the extracellular domains of human RYK (hRYK, top sequence; SEQ ID NO: 132) and mouse RYK (mRYK, bottom sequence, SEQ ID NO: 131) is shown. Dots indicate homology at that position, differences are indicated by single letter amino acid codons. The signal peptide and WIF domains are labeled and indicated by lines above the sequence (SEQ ID NO: 132). [Figure 2A]Shown are the amino acid sequences shown for each of the four mouse anti-human RYK hybridomas, designated 2-D11 (FIG. 2A, SEQ ID NO: 15 and SEQ ID NO: 16), 7-H10 (FIG. 2B, SEQ ID NO: 31 and SEQ ID NO: 32), 11-E9 (FIG. 2C, SEQ ID NO: 47 and SEQ ID NO: 48), and 3-C12 (FIG. 2D, SEQ ID NO: 63 and SEQ ID NO: 64), and their alignment with the closest mouse IGHV (top sequence) or IGKV (bottom sequence) germline gene. For each alignment, the top sequence shows the amino acid sequence of the heavy or light chain variable region starting from the first codon of the first framework region and ending with the last codon of the fourth framework region. The bottom sequence shows the amino acid sequence of the heavy or light chain variable region of the most homologous mouse IGHV or IGKV germline gene. A dot indicates homology at that position, and differences are indicated by single letter amino acid codons. The framework (FR) and complementarity determining (CDR) regions are marked above the sequences. [Figure 2B] Shown are the amino acid sequences shown for each of the four mouse anti-human RYK hybridomas, designated 2-D11 (FIG. 2A, SEQ ID NO: 15 and SEQ ID NO: 16), 7-H10 (FIG. 2B, SEQ ID NO: 31 and SEQ ID NO: 32), 11-E9 (FIG. 2C, SEQ ID NO: 47 and SEQ ID NO: 48), and 3-C12 (FIG. 2D, SEQ ID NO: 63 and SEQ ID NO: 64), and their alignment with the closest mouse IGHV (top sequence) or IGKV (bottom sequence) germline gene. For each alignment, the top sequence shows the amino acid sequence of the heavy or light chain variable region starting from the first codon of the first framework region and ending with the last codon of the fourth framework region. The bottom sequence shows the amino acid sequence of the heavy or light chain variable region of the most homologous mouse IGHV or IGKV germline gene. A dot indicates homology at that position, and differences are indicated by single letter amino acid codons. The framework (FR) and complementarity determining (CDR) regions are marked above the sequences. [Figure 2C]Shown are the amino acid sequences shown for each of the four mouse anti-human RYK hybridomas, designated 2-D11 (FIG. 2A, SEQ ID NO: 15 and SEQ ID NO: 16), 7-H10 (FIG. 2B, SEQ ID NO: 31 and SEQ ID NO: 32), 11-E9 (FIG. 2C, SEQ ID NO: 47 and SEQ ID NO: 48), and 3-C12 (FIG. 2D, SEQ ID NO: 63 and SEQ ID NO: 64), and their alignment with the closest mouse IGHV (top sequence) or IGKV (bottom sequence) germline gene. For each alignment, the top sequence shows the amino acid sequence of the heavy or light chain variable region starting from the first codon of the first framework region and ending with the last codon of the fourth framework region. The bottom sequence shows the amino acid sequence of the heavy or light chain variable region of the most homologous mouse IGHV or IGKV germline gene. A dot indicates homology at that position, and differences are indicated by single letter amino acid codons. The framework (FR) and complementarity determining (CDR) regions are marked above the sequences. [Figure 2D] Shown are the amino acid sequences shown for each of the four mouse anti-human RYK hybridomas, designated 2-D11 (FIG. 2A, SEQ ID NO: 15 and SEQ ID NO: 16), 7-H10 (FIG. 2B, SEQ ID NO: 31 and SEQ ID NO: 32), 11-E9 (FIG. 2C, SEQ ID NO: 47 and SEQ ID NO: 48), and 3-C12 (FIG. 2D, SEQ ID NO: 63 and SEQ ID NO: 64), and their alignment with the closest mouse IGHV (top sequence) or IGKV (bottom sequence) germline gene. For each alignment, the top sequence shows the amino acid sequence of the heavy or light chain variable region starting from the first codon of the first framework region and ending with the last codon of the fourth framework region. The bottom sequence shows the amino acid sequence of the heavy or light chain variable region of the most homologous mouse IGHV or IGKV germline gene. A dot indicates homology at that position, and differences are indicated by single letter amino acid codons. The framework (FR) and complementarity determining (CDR) regions are marked above the sequences. [Diagram 3]1 shows a comparison of the extracellular domain of hROR1 (SEQ ID NO: 129) with mutant forms of hRYK used to map the binding region epitopes hRYK bound by each of the anti-human RYK mAbs of the present disclosure. The name of the protein represented by the amino acid sequence is at the left margin. The amino acids are indicated by the single letter amino acid code. The numbers provided at the right margin or above the sequence are the numbers of the amino acid residue positions below. A dot within the sequence indicates sequence homology with hRYK at that position. A letter indicates an amino acid in the mutant RYK that differs from that present in hRYK at that position. The WIF domain of the RYK extracellular domain is shown above the amino acid sequence underlined. [Figure 4A] Figure 4A shows the amino acid sequence required for binding of anti-human RYK mAbs to the extracellular domain of human RYK (SEQ ID NO: 129). Figure 4A shows an experiment in which a recombinant human RYK protein (SEQ ID NO: 129) in which one amino acid that differs between human RYK and mouse RYK in the extracellular domain is replaced with the corresponding amino acid in mouse RYK was used to evaluate the binding of 2-D11, 7-H10, 3-C12, 11-E9, 6-B5, 6-D10, and sheep anti-RYK mAbs. Each recombinant protein was transferred to a nylon membrane and probed with the indicated anti-RYK mAb or sheep anti-RYK Ab, and detected with anti-mouse IgG or donkey anti-sheep antibody conjugated with horseradish peroxidase. The rabbit anti-IgG blotting is positive for protein blotting and detection because the recombinant protein has a rabbit IgG tag fpr purification. An alignment of the protein sequences of the extracellular domains of human and mouse RYK is shown in the lower panel, with boxed amino acids indicating the amino acid changes made to each recombinant protein. [Figure 4B]Figure 4B shows the amino acid sequence required for binding of anti-human RYK mAb to the extracellular domain of human RYK (SEQ ID NO: 129). Figure 4B shows additional blotting with 2-D11 antibody to further evaluate binding, where substitutions were made within the leader peptide or within the coding region adjacent to the WIF domain of the human RYK protein (SEQ ID NO: 129). hRYK with mRYK 48-57 is human RYK with substitutions of mouse amino acids at positions 48-57, which confers loss of binding to human RYK, as does the substitution of the mouse leader region fused to human RYK. [Figure 5A] Figure 5 shows the variable region sequence of mAb 2-D11. Figure 5A shows the Ig heavy chain variable region sequence of mAb 2-D11. Figure 5B shows the Ig kappa chain variable region sequence of mAb 2-D11. [Figure 5B] Figure 5B shows the variable region sequence of mAb 2-D11. Figure 5B shows the Ig kappa chain variable region sequence of mAb 2-D11. [Figure 6A] Figure 6A shows the variable region sequence of mAb 7-H10. Figure 6A shows the Ig heavy chain variable region sequence of mAb 7-H10. [Figure 6B] Figure 6B shows the variable region sequence of mAb 7-H10. Figure 6B shows the Ig kappa chain variable region sequence of mAb 7-H10. [Figure 7A] Figure 7 shows the affinity measurement of binding of 2-D11 and 7-H10 mAbs to recombinant human RYK2 protein. Analysis was performed using a KinExA3200 instrument. Figure 7A: Percentage of anti-human RYK mAb bound to particles coated with RYK protein (y-axis) in the presence of increasing molar (M) concentrations of soluble RYK competitor (x-axis) for 2-D11 mAb (top left panel) and 7-H10 mAb (bottom left panel). [Figure 7B] Figure 7B shows the binding affinity measurement of 2-D11 and 7-H10 mAb to recombinant human RYK2 protein. Analysis was performed using a KinExA3200 instrument. Figure 7B: 95% confidence intervals of the measured KD of 2-D11 mAb (upper right panel) and 7-H10 mAb (lower right panel) for binding to human RYK. [Figure 8] Figure 1 shows that 2-D11 anti-human RYK mAb specifically binds human RYK. Binding of 2-D11 mAb to human RYK was assessed by flow cytometry staining and analysis of several cell lines. Cells were stained with 10ug / ml of 2-D11 anti-human RYK-Alexa647 conjugated mAb (shaded histograms) or an equivalent amount of isotype-matched control mAb (open histograms) for 20 minutes on ice, washed, and analyzed. The histograms show the relative fluorescence intensity (x-axis) of live cells as determined by light scatter properties. ++, +, and neg correspond to the staining levels shown in Table 1 for these and other cell lines, based on the ratio of median fluorescence intensity (MFI) of stained cells to the MFI of isotype control stained cells. [Figure 9] Examples of 2-D11 staining of lymphoid cells from adult blood, umbilical cord blood (N=2), tonsil (N-2), or spleen are shown. [Figure 10] First passage breast cancer patient-derived xenografts (PDXs) are shown. The first passage (T1) breast cancer PDX (M0026) is derived from estrogen / progesterone receptor-negative and HER2-negative breast cancer (triple-negative breast cancer, TNBC). Human TNBC cells from M0026 were dissociated into single cells, which were stained with fluorochrome-conjugated isotype control mAb (Cont mAb, dark grey histogram) or fluorochrome-conjugated 2-D11 (light grey shaded histogram) and then analyzed on a flow cytometer. Cont mAb-stained cells had the same fluorescence as unstained cells (not shown). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] While various embodiments and aspects of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used in the practice of the invention.

[0026] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and papers, are expressly incorporated herein by reference in their entirety for any purpose.

[0027] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulas set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0028] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. See, for example, Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J.Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the present invention. The following definitions are provided to facilitate understanding of certain terms frequently used herein and are not intended to limit the scope of the present disclosure.

[0029] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof or their complements, or nucleosides (e.g., deoxyribonucleosides or ribonucleosides) in either single-stranded, double-stranded, or multiple-stranded form. In embodiments, "nucleic acid" does not include nucleosides. The terms "polynucleotide," "oligonucleotide," "oligo," and the like refer to a linear sequence of nucleotides in the usual and customary sense. The term "nucleoside" refers to a glycosylamine containing a nucleobase and a five-carbon sugar (ribose or deoxyribose) in the usual and customary sense. Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. The term "nucleotide" refers to a single polynucleotide unit (i.e., monomer) in the usual and customary sense. A nucleotide can be a ribonucleotide, a deoxyribonucleotide, or a modified version thereof. Examples of polynucleotides contemplated herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules having a mixture of single-stranded and double-stranded DNA and RNA. Examples of nucleic acids (e.g., polynucleotides) contemplated herein include all kinds of RNA (e.g., mRNA, siRNA, miRNA, and guide RNA), and all kinds of DNA (genomic DNA, plasmid DNA, and minicircle DNA), and any fragments thereof. The term "duplex" in the context of a polynucleotide refers to double-strandedness in the usual and customary sense. Nucleic acids can be linear or branched. For example, nucleic acids can be linear chains of nucleotides, or nucleic acids can be branched, e.g., nucleic acids include one or more arms or branches of nucleotides. Optionally, branched nucleic acids are repeatedly branched to form higher order structures, such as dendrimers.

[0030] For example, a nucleic acid, including a nucleic acid having a phosphothioate backbone, may contain one or more reactive moieties. As used herein, the term reactive moiety includes any group that can react with another molecule (e.g., a nucleic acid or a polypeptide) through a covalent bond, a non-covalent bond, or other interaction. As an example, a nucleic acid may contain an amino acid reactive moiety that reacts with an amino acid on a protein or polypeptide through a covalent bond, a non-covalent bond, or other interaction.

[0031] This term also includes nucleic acids that contain known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, and non-naturally occurring nucleic acids that have similar binding properties as the reference nucleic acid and are metabolized in the same manner as the reference nucleotide.Examples of such analogs include, but are not limited to, for example, phosphoramidates, phosphorodiamidates, phosphorothioates (also known as phosphothioates, in which the oxygen in the phosphoric acid is replaced with a double bond sulfur), phosphorodithioates, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acids, phosphonoformic acids, methylphosphonates, boronphosphonates, or phosphodiester derivatives that contain O-methylphosphoramidite bonds (Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, Oxford University Press), as well as modifications to nucleotide bases such as 5-methylcytidine or pseudouridine, and peptide nucleic acid backbones and bonds. Other analog nucleic acids include those with cationic backbones, non-ionic backbones, modified sugars, and non-ribose backbones (e.g., phosphorodiamidate morpholino oligos or locked nucleic acids (LNAs) as known in the art), including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and in Chapters 6 and 7 of ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acid. Modifications of the ribose-phosphate backbone can be made for a variety of reasons, including increasing the stability and half-life of such molecules in physiological environments or as probes on biochips. Mixtures of naturally occurring nucleic acids and analogs can be made, or mixtures of different nucleic acid analogs and mixtures of naturally occurring nucleic acids and analogs can be made. In embodiments, the internucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

[0032] A nucleic acid may include a non-specific sequence. As used herein, the term "non-specific sequence" refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary or only partially complementary to any other nucleic acid sequence. By way of example, a non-specific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism. In an embodiment, a non-specific nucleic acid sequence does not code for a biological function. In an embodiment, a non-specific nucleic acid sequence is a scrambled nucleic acid sequence. A "scrambled nucleic acid sequence" provided herein is a recombinant nucleic acid sequence that comprises nucleotides randomly linked together in vitro. Scrambled nucleic acid sequences are commonly used in the art as control or reference sequences to compare the activity (biological function) of a test nucleic acid sequence.

[0033] A polynucleotide is typically composed of a specific sequence of the four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T) (if the polynucleotide is RNA, uracil (U) is substituted for thymine (T)). Thus, the term "polynucleotide sequence" is an alphabetical representation of a polynucleotide molecule, or the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be entered into a database of a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. A polynucleotide may optionally include one or more non-standard nucleotide(s), nucleotide analog(s), and / or modified nucleotides.

[0034] The term "complement" as used herein refers to a nucleotide (e.g., RNA or DNA) or sequence of nucleotides that can base pair with a complementary nucleotide or sequence of nucleotides. As described herein and generally known in the art, the complementary (matching) nucleotide of adenosine is thymidine, and the complementary (matching) nucleotide of guanidine is cytosine. Thus, a complement can include a sequence of nucleotides that base pair with the corresponding complementary nucleotides of a second nucleic acid sequence. The nucleotides of the complement can partially or completely match the nucleotides of the second nucleic acid sequence. If the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, the complement will base pair with each nucleotide of the second nucleic acid sequence. If the nucleotides of the complement partially match the nucleotides of the second nucleic acid sequence, only a portion of the nucleotides of the complement will base pair with the nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding sequences and non-coding sequences, where the non-coding sequence includes complementary nucleotides to the coding sequence, thus forming the complement of the coding sequence. Further examples of complementary sequences include sense and antisense sequences, where the sense sequence comprises complementary nucleotides to the antisense sequence, thus forming the complement of the antisense sequence.

[0035] As described herein, the complementarity of sequences can be partial, where only some of the nucleic acids match according to base pairing, or all of the nucleic acids match completely according to base pairing. Thus, two sequences that are complementary to each other can have a certain percentage of nucleotides that are the same (i.e., about 60% identity over a particular region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity).

[0036] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an alpha carbon attached to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium). Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. The terms "non-naturally occurring amino acid and unnatural amino acid" refer to amino acid analogs, synthetic amino acids, and amino acid mimetics that are not found in nature.

[0037] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted single-letter codes.

[0038] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, which in embodiments may be conjugated to a moiety that is not composed of amino acids. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers. A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0039] The "position" of an amino acid or nucleotide base is indicated by a number that sequentially identifies each amino acid (or nucleotide base) of the reference sequence based on its position relative to the N-terminus (or 5'-terminus). Due to deletions, insertions, truncations, fusions, etc. that must be considered when determining optimal alignment, in general, the number of amino acid residues in a test sequence, determined by simple counting from the N-terminus, is not necessarily the same as the number at its corresponding position in the reference sequence. For example, if a variant has a deletion relative to the aligned reference sequence, there is no variant amino acid that corresponds to the reference sequence position at the site of the deletion. If there is an insertion in the aligned reference sequence, the insertion does not correspond to a numbered amino acid position in the reference sequence. In the case of a truncation or fusion, there may be a stretch of amino acids in the reference or aligned sequence that does not correspond to any amino acid in the corresponding sequence.

[0040] The term "numbered with reference to" or "corresponding to", when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of residues in a particular reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. An amino acid residue of a protein "corresponds" to a given residue if it occupies the same essential structural position in the protein as the given residue. A person skilled in the art will readily recognize the identity and position of a residue that corresponds to a particular position in a protein (e.g., RYK) in other proteins with different numbering systems. For example, by performing a simple sequence alignment with a protein (e.g., RYK), the identity and position of a residue that corresponds to a particular position in a protein is identified in other protein sequences that align to the protein. For example, if the selected residue occupies the same essential spatial or other structural relationship as the glutamic acid at position 138, then the selected residue in the selected protein corresponds to the glutamic acid at position 138. In some embodiments, when a selected protein is aligned for maximum homology with a protein, the position in the aligned selected protein that aligns with glutamic acid 138 is the position corresponding to glutamic acid 138. Also, instead of a primary sequence alignment, a three-dimensional structural alignment can be used, for example, where the structures of the selected proteins are aligned to maximize the correspondence with glutamic acid at position 138, and the overall structures are compared. In this case, the amino acid occupying the same essential position as glutamic acid 138 in the structural model corresponds to the glutamic acid 138 residue.

[0041] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to nucleic acids that code for the same or essentially the same amino acid sequence. Due to the degeneracy of the genetic code, several nucleic acid sequences will code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variation. Every nucleic acid sequence herein that codes for a polypeptide also describes every possible silent variation of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, every silent variation of a nucleic acid that codes for a polypeptide is implicit in each sequence described.

[0042] With respect to amino acid sequences, one of skill in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, or delete a single amino acid or a small percentage of the amino acids in the encoded sequence are "conservatively modified variants" in which the change results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables that result in functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, the polymorphic variants, interspecies homologs, and alleles of the present disclosure.

[0043] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine ​​(C), Methionine (M) (See, e.g., Creighton, Proteins (1984)).

[0044] The term "identical" or "percentage of identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that contain the same or identical amino acid residues or nucleotides in a specified percentage (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for closest correspondence over a comparison window or designated region) when measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters as described below, or by manual alignment and visual inspection (see, e.g., the NCBI website http: / / www.ncbi.nlm.nih.gov / BLAST / , etc.). Such sequences are then said to be "substantially identical." This definition may also refer to or be applied to the complement of a test sequence. This definition also includes sequences that have deletions and / or additions, as well as sequences that have substitutions. As explained below, preferred algorithms can take into account gaps, etc. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0045] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where an identical nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0046] For sequence comparison, typically, one sequence serves as a reference sequence, to which test sequence is compared.When using sequence comparison algorithm, test and reference sequences are input into computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity for test sequence compared to reference sequence based on program parameters.

[0047] A "comparison window", as used herein, includes a reference to a full length sequence or any one segment of a number of contiguous positions selected from the group consisting of 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides, in which a sequence may be compared to a reference sequence for the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0048] Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or meet some positive-valued threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence as far as the cumulative alignment score can be increased. The cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of the word hits in each direction is stopped when the cumulative alignment score falls by a quantity X from its maximum achieved value, when the cumulative score falls below zero due to the accumulation of one or more negatively scoring residue alignments, or when either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. For example, the BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) of alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0049] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability in the comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0050] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with an antibody raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, when the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

[0051] "RYK" as referred to herein includes any recombinant or naturally occurring receptor tyrosine kinase-associated (RYK) protein or variant or homolog thereof that maintains RYK activity (e.g., within the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to RYK). In some aspects, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to a naturally occurring RYK protein. In embodiments, the RYK protein is substantially identical to a protein identified by UniProt reference number P34925 or a variant or homolog having substantial identity thereto.

[0052] Antibodies are large, complex molecules (molecular weight of about 150,000 or about 1320 amino acids) with a complex internal structure. Natural antibody molecules contain two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light and heavy chain in turn consists of two regions: a variable ("V") region, which is responsible for binding the target antigen, and a constant ("C") region, which interacts with other components of the immune system. The light and heavy chain variable regions (also referred to herein as light chain variable (VL) domain and heavy chain variable (VH) domain, respectively) come together in three-dimensional space to form a variable region that binds to antigens (e.g., receptors on cell surfaces). Within each light or heavy chain variable region, there are three short segments (average 10 amino acids long) called complementarity determining regions ("CDRs"). The six CDRs in an antibody variable domain (three from the light chain and three from the heavy chain) fold together in three-dimensional space to form the actual antibody binding site that docks to the target antigen. The position and length of the CDRs are precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, 1983, 1987. The parts of the variable region that are not included in the CDRs are called the framework ("FR"), which forms the environment for the CDRs.

[0053] An "antibody variant" as provided herein refers to a polypeptide that can bind to an antigen and includes one or more structural domains of an antibody or fragment thereof (e.g., a light chain variable domain, a heavy chain variable domain). Non-limiting examples of antibody variants include single domain antibodies or nanobodies, monospecific Fabs, and the like. 2 , bispecific Fab 2 , trispecific Fab 3, monovalent IgG, scFv, bispecific antibodies, bispecific diabodies, trispecific triabodies, scFv-Fc, minibodies, IgNAR, V-NAR, hcIgG, VhH, or peptibodies. As provided herein, "peptibody" refers to a peptide moiety attached (via a covalent or non-covalent linker) to the Fc domain of an antibody. Further non-limiting examples of antibody variants known in the art include antibodies produced by cartilaginous fish or camelids. A general description of camelid-derived antibodies and their variable regions, as well as methods for producing, isolating, and using them, can be found in references WO97 / 49805 and WO97 / 49805, which are incorporated herein by reference in their entirety for all purposes. Similarly, antibodies and variable regions thereof derived from cartilaginous fish, as well as methods for producing, isolating, and using them, can be found in WO2005 / 118629, the entirety of which is incorporated by reference herein for all purposes.

[0054] The terms "CDR L1", "CDR L2" and "CDR L3" as provided herein refer to complementarity determining regions (CDRs) 1, 2 and 3 of the variable light (L) chain of an antibody. In embodiments, the variable light chain provided herein comprises, from the N-terminus to the C-terminus, CDR L1, CDR L2 and CDR L3. Similarly, the terms "CDR H1", "CDR H2" and "CDR H3" as provided herein refer to complementarity determining regions (CDRs) 1, 2 and 3 of the variable heavy (H) chain of an antibody. In embodiments, the variable heavy chain provided herein comprises, from the N-terminus to the C-terminus, CDR H1, CDR H2 and CDR H3.

[0055] The terms "FR L1", "FR L2", "FR L3", and "FR L4" provided herein are used according to their general meaning in the art and refer to framework regions (FR) 1, 2, 3, and 4 of the variable light (L) chain of an antibody. In embodiments, the variable light chain provided herein comprises, from N-terminal to C-terminal, FR L1, FR L2, FR L3, and FR L4. Similarly, the terms "FR H1", "FR H2", "FR H3", and "FR H4" provided herein are used according to their general meaning in the art and refer to framework regions (FR) 1, 2, 3, and 4 of the variable heavy (H) chain of an antibody. In embodiments, the variable heavy chain provided herein comprises, from N-terminal to C-terminal, FR H1, FR H2, FR H3, and FR H4.

[0056] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL), variable light chain (VL) domain, or light chain variable region, and variable heavy chain (VH), variable heavy chain (VH) domain, or heavy chain variable region refer to these light chain and heavy chain regions, respectively. The terms variable light chain (VL), variable light chain (VL) domain, and light chain variable region referred to herein may be used interchangeably. The terms variable heavy chain (VH), variable heavy chain (VH) domain, and heavy chain variable region referred to herein may be used interchangeably. The Fc (i.e., fragment crystallizable region) is the "base" or "tail" of an immunoglobulin and is typically composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. The Fc region ensures that each antibody generates an appropriate immune response to a given antigen by binding to specific proteins. The Fc region also binds to various cellular receptors, such as Fc receptors, and other immune molecules, such as complement proteins.

[0057] The term "antibody" is used according to its commonly known meaning in the art. Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies under disulfide bonds in the hinge region and separates V-terminal fragments by disulfide bonds. H -C H1 F(ab)' is a dimer of Fab, a light chain linked to 2 Generate F(ab)' 2 is reduced under mild conditions to cleave the disulfide bond in the hinge region, thereby forming F(ab)' 2 The dimers can be converted to Fab' monomers. A Fab' monomer is essentially a Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993)). Although various antibody fragments are defined in terms of the digestion of an intact antibody, one skilled in the art will understand that such fragments can be synthesized chemically or de novo using recombinant DNA methodology. Thus, as used herein, the term antibody also includes antibody fragments either produced by the modification of whole antibodies, synthesized de novo using recombinant DNA methodology (e.g., single chain Fv), or identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)). The term "antibody" as referred to herein further includes antibody variants such as single domain antibodies. Thus, in embodiments, an antibody comprises a single monomeric variable antibody domain. Thus, in embodiments, the antibody comprises a variable light (VL) domain or a variable heavy (VH) domain. In embodiments, the antibody is a variable light (VL) domain or a variable heavy (VH) domain.

[0058] Any technique known in the art can be used for preparation of monoclonal or polyclonal antibodies (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pp.77-96 in Monoclonal Antibodies and Cancer Therapy (1985)). A "monoclonal" antibody (mAb) refers to an antibody derived from a single clone. Techniques for producing single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies against polypeptides of the invention. Also, transgenic mice or other organisms, such as other mammals, can be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to a selected antigen (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)).

[0059] Single chain variable fragments (scFv) are typically fusion proteins of the variable regions of immunoglobulin heavy (VH) and light (VL) chains, connected with a short linker peptide of 10 to about 25 amino acids. The linker is usually glycine-rich for flexibility and may be serine- or threonine-rich for solubility. The linker may connect the N-terminus of VH to the C-terminus of VL, or vice versa.

[0060] The epitope of a mAb is the region of its antigen to which the mAb binds. Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other antibody by at least 30%, but preferably 50%, 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0061] Many techniques known in the art can be used for the preparation of suitable antibodies of the invention (e.g., recombinant, monoclonal, or polyclonal antibodies) and for use according to the invention (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). Genes encoding the heavy and light chains of the antibody of interest can be cloned from cells, for example, genes encoding monoclonal antibodies can be cloned from hybridomas and used to produce recombinant monoclonal antibodies. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be made from hybridoma cells or plasma cells. Random combination of heavy and light chain gene products generates a large pool of antibodies with different antigen specificities (see, for example, Kuby, Immunology (3rd ed. 1997)). Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Pat. No. 4,946,778, U.S. Pat. No. 4,816,567) can be adapted to produce antibodies against the polypeptides of the invention.Also, transgenic mice or other organisms (e.g., other mammals) can be used to express humanized or human antibodies (see, e.g., U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & (See Huszar, Intern. Rev. Immunol. 13, 65-93 (1995)). Alternatively, phage display techniques can be used to identify antibodies and heteromeric Fab fragments that specifically bind to a selected antigen (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can also be made bispecific, i.e., capable of recognizing two different antigens (see, e.g., WO 93 / 08829; Traunecker et al., EMBOJ. 10:3655-3659 (1991); and Suresh et al. Methods in Enzymology 121:210 (1986)). The antibody can also be a heteroconjugate, such as two covalently linked antibodies, or an immunotoxin (see, for example, US Pat. No. 4,676,980, WO 91 / 00360, WO 92 / 200373, and EP 03089).

[0062] Methods for humanizing or primatizing non-human antibodies are well known in the art (e.g., U.S. Pat. Nos. 4,816,567, 5,530,101, 5,859,205, 5,585,089, 5,693,761, 5,693,762, 5,777,085, 6,180,370, 6,210,671, and 6,329,511, WO 87 / 02671, European Patent Application No. 0173494, Jones et al. (1986) Nature 321:522, and Verhoyen et al. (1988) Science 239:1534). Humanized antibodies are further described in, for example, Winter and Milstein (1991) Nature 349:293. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues, often referred to as introduced residues, are usually removed from the introduced variable domain. Humanization can be performed essentially according to the method of Winter et al. (e.g., Morrison et al., PNAS USA, 81:6851-6855 (1984); Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen et al., Science 34:65-92 (1988)) by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. 239:1534-1536 (1988); Presta, Curr. Op. Struct. Biol. 2:593-596 (1992); Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3):169-217 (1994). Such humanized antibodies thus are chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced with residues from analogous sites in rodent antibodies. For example, a polynucleotide comprising a first set of sequences encoding a humanized immunoglobulin framework region and a second set of sequences encoding the desired immunoglobulin complementarity determining regions can be produced synthetically or by combining appropriate cDNA and genomic DNA segments. Human constant region DNA sequences can be isolated from a variety of human cells according to well-known procedures.

[0063] A "chimeric antibody" is (a) an antibody molecule in which the constant region or a portion thereof has been altered, substituted or replaced such that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function, and / or species, or to an entirely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc., or (b) an antibody molecule in which the variable region or a portion thereof has been altered, substituted or replaced with a variable region having a different or altered antigen specificity. Preferred antibodies of the invention and for use in accordance with the invention include humanized and / or chimeric monoclonal antibodies.

[0064] When referring to a protein or peptide, the phrases "specifically (or selectively) bind to an antibody" or "specifically (or selectively) immunoreact with" refer to a binding reaction that determines the presence of a protein, often in a heterogeneous population of proteins and other biologics. Thus, under specified immunoassay conditions, a specified antibody will bind to a particular protein at least twice background, and more typically more than 10-100 times background. Specific binding to an antibody under such conditions requires that the antibody be selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with a selected antigen and not with other proteins. This selection can be accomplished by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats can be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0065] "Ligand" refers to an agent (eg, a polypeptide or other molecule) that can bind to a receptor or to an antibody, antibody variant, antibody region, or fragment thereof.

[0066] Techniques for conjugating therapeutic agents to antibodies are well known (e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2 nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe et al., "The Preparation and Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982). As used herein, the term "antibody-drug conjugate" or "ADC" refers to a therapeutic agent that is conjugated or otherwise covalently attached to an antibody.

[0067] For certain proteins described herein, the named proteins include any naturally occurring form, variant or homologue of the protein that maintains the protein transcription factor activity (e.g., in the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the naturally occurring protein). In some embodiments, the variant or homologue has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to the naturally occurring form. In other embodiments, the protein is a protein identified by its NCBI sequence reference. In other embodiments, the protein is a protein identified by its NCBI sequence reference, a homologue or a functional fragment thereof.

[0068] The term "gene" refers to a segment of DNA involved in producing a protein, including the coding regions (leader and trailer) between individual coding segments (exons) as well as the regions preceding and following the intervening sequences (introns). The leader, trailer, and introns contain regulatory elements required during transcription and translation of a gene. Furthermore, a "protein gene product" is a protein expressed from a particular gene.

[0069] The term "plasmid," "vector," or "expression vector" refers to a nucleic acid molecule that encodes genes and / or regulatory elements necessary for expression of genes. Expression of genes from a plasmid can occur in cis or trans. When a gene is expressed in cis, the gene and regulatory elements are encoded by the same plasmid. Expression in trans refers to the case where the gene and regulatory elements are encoded by separate plasmids.

[0070] The terms "transfection", "transduction", "transfecting" or "transducing" may be used interchangeably and are defined as the process of introducing a nucleic acid molecule or protein into a cell. The nucleic acid is introduced into the cell using a non-viral or virus-based method. The nucleic acid molecule may be a genetic sequence encoding a complete protein or a functional portion thereof. Non-viral methods of transfection include any suitable transfection method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell. Exemplary non-viral transfection methods include calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, heat shock transfection, magnetifection, and electroporation. In some embodiments, the nucleic acid molecule is introduced into the cell using electroporation, following standard procedures well known in the art. In the case of viral-based methods of transfection, any useful viral vector can be used in the methods described herein. Examples of viral vectors include, but are not limited to, retroviral vectors, adenoviral vectors, lentiviral vectors, and adeno-associated viral vectors. In some embodiments, the nucleic acid molecule is introduced into the cell using a retroviral vector, according to standard procedures well known in the art. The term "transfection" or "transduction" also refers to the introduction of a protein into a cell from the external environment. Typically, protein transduction or transfection relies on the binding of a peptide or protein capable of passing through the cell membrane to the protein of interest. See, for example, Ford et al. (2001) Gene Therapy 8:1-4 and Prochiantz (2007) Nat. Methods 4:119-20.

[0071] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in ELISA), biotin, digoxigenin, or haptens, as well as proteins or other entities that can be made detectable, for example, by incorporating a radioactive label into a peptide or an antibody that specifically reacts with the peptide. Any suitable method known in the art for conjugating an antibody to a label can be used, for example, the methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

[0072] If the label or detectable moiety is a radioactive metal or a paramagnetic ion, the agent may be reacted with another long-tailed reagent having a long tail with one or more chelating groups attached to the long tail for binding these ions. The long tail may be a polymer (e.g., polylysine, polysaccharide, or other derivatized or derivatizable chain with pendant groups to which metals or ions may be added for binding). Examples of chelating groups that can be used in accordance with the present disclosure include, but are not limited to, groups such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA, NOTA, NETA, TETA, porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes, and the like. The chelate is typically linked to the PSMA antibody or functional antibody fragment by a group that allows for the formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal cross-linking. The same chelating agents, when complexed with non-radioactive metals such as manganese, iron, and gadolinium, are useful for MRI when used with the antibodies and carriers described herein. Macrocyclic chelators such as NOTA, DOTA, and TETA are used with a variety of metals and radiometals, including but not limited to gallium, yttrium, and copper, respectively. 223 Other ring-type chelators, such as macrocyclic polyethers, intended for stably binding nuclides such as Ra, are encompassed herein. In certain embodiments, the chelating moiety is a PET imaging agent (e.g., Al-) for use in PET analysis. 18 F complex) to a targeting molecule.

[0073] "Contacting" is used according to its plain and ordinary meaning to refer to a process that allows at least two different species (e.g., an antibody and an antigen) to come into sufficient proximity to react, interact, or come into physical contact. However, it should be understood that the resulting reaction product may be produced directly from the reaction between the added reagents, or from an intermediate from one or more added reagents that may be produced in the reaction mixture.

[0074] The term "contacting" can include allowing two species to react, interact, or come into physical contact, where the two species can be, for example, a pharmaceutical composition provided herein and a cell. In embodiments, contacting includes, for example, allowing a pharmaceutical composition described herein to interact with a cell.

[0075] As used herein, "cell" refers to a cell that performs metabolic or other functions sufficient to preserve or replicate its genomic DNA. Cells can be identified by methods well known in the art, including, for example, the presence of an intact membrane, staining with a particular dye, the ability to produce progeny, or, in the case of gametes, the ability to combine with a second gamete to produce viable progeny. Cells can include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells of plant and animal origin, such as mammalian, insect (e.g., Spodoptera) and human cells.

[0076] The term "recombinant," when used with reference to, for example, a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found in the native (non-recombinant) form of the cell, or expresses native genes that are otherwise abnormally expressed, under-expressed, or not expressed at all. Transgenic cells and plants are typically those that express heterologous genes or coding sequences as a result of recombinant methods.

[0077] The term "isolated," as applied to a nucleic acid or protein, indicates that the nucleic acid or protein is essentially free from other cellular components with which it is naturally associated. It can be, for example, in a homogeneous state, either a dry solution or an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0078] The term "heterologous," when used in reference to a portion of a nucleic acid, indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, a nucleic acid is typically produced recombinantly, having two or more sequences from unrelated genes arranged to create a new functional nucleic acid, such as a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more sequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0079] The term "exogenous" refers to a molecule or substance (e.g., a compound, nucleic acid, or protein) that originates from outside a given cell or organism. For example, an "exogenous promoter" as referred to herein is a promoter that is not native to the cell or organism in which it is expressed. Conversely, the terms "endogenous" or "endogenous promoter" refer to a molecule or substance that is native to or derived from a given cell or organism.

[0080] As defined herein, the terms "inhibit", "inhibit", "inhibiting" and the like with respect to cell proliferation (e.g., cancer cell proliferation) refer to adversely affecting a cell (e.g., decreasing proliferation) or killing a cell. In embodiments, inhibition refers to the alleviation of a disease or disease symptom (e.g., cancer, cancer cell proliferation). Thus, inhibition includes, at least in part, partially or completely blocking a stimulus, reducing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating a signal transduction or enzyme activity or amount of a protein. Similarly, an "inhibitor" is a compound or protein that inhibits a receptor or another protein, for example, by binding to, partially or completely blocking, reducing, preventing, delaying, inactivating, desensitizing, or downregulating an activity (e.g., receptor activity or protein activity).

[0081] As defined herein, the terms "inhibit," "inhibit," "inhibiting," and the like, with respect to protein-inhibitor interactions, mean to adversely affect (e.g., decrease) the activity or function of a protein relative to the activity or function of the protein in the absence of an inhibitor.

[0082] The terms "inhibitor", "repressor", or "antagonist", or "downregulator" interchangeably refer to a substance that can detectably reduce the expression or activity of a given gene or protein. Antagonists can reduce expression or activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control in the absence of the antagonist. In certain cases, expression or activity is 1.5-fold or less, 2-fold or less, 3-fold or less, 4-fold or less, 5-fold or less, 10-fold or less than the expression or activity in the absence of the antagonist.

[0083] The term "expression" includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be detected using conventional techniques for detecting proteins (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0084] "Biological sample" or "sample" refers to a material obtained from or derived from a subject or patient. Biological samples can also include sections of tissue, such as biopsy and autopsy samples, as well as frozen sections taken for histological purposes. Such samples include bodily fluids, such as blood and blood fractions or blood products (e.g., serum, plasma, platelets, red blood cells, etc.), sputum, tissues, cultured cells (e.g., primary cultures, explants, and transformed cells), stool, urine, synovial fluid, articular tissue, synovial tissue, synovial cells, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, and the like. Biological samples are typically obtained from eukaryotic organisms, such as mammals, such as primates (e.g., chimpanzees or humans), cows, dogs, cats, rodents (e.g., guinea pigs, rats, mice), rabbits, or birds, reptiles, or fish.

[0085] "Control" or "standard control" refers to a sample, measurement, or value that serves as a reference (usually a known reference) for comparison with a test sample, measurement, or value. For example, a test sample may be taken from a patient suspected of having a given disease (e.g., cancer) and compared to a known normal (non-disease) individual (e.g., standard control subject). A standard control may also represent an average measurement or mean value collected from a population of similar individuals (e.g., standard control subjects) who do not have a given disease (i.e., a standard control population), e.g., healthy individuals with a similar medical background, the same age, weight, etc. A standard control value may also be obtained from the same individual, e.g., from a sample previously obtained from the patient before the onset of the disease. For example, controls can be devised to compare therapeutic benefits (e.g., comparing side effects) based on pharmacological data (e.g., half-life) or therapeutic measures. Controls are also useful for determining the significance of data. For example, if the value of a given parameter varies widely in the control, the variation in the test sample is not considered significant. One of skill in the art will recognize that standard controls can be designed for evaluation of any number of parameters (e.g., RNA levels, protein levels, particular cell types, particular body fluids, particular tissues, etc.).

[0086] Those skilled in the art can understand which standard control is most appropriate in a given situation and can analyze data based on comparison with the standard control value.Standard controls are also useful for determining the significance (e.g., statistical significance) of data.For example, if the value of a given parameter varies greatly with the standard control, the variation of the test sample is not considered significant.

[0087] "Patient" or "subject in need thereof" refers to a living organism suffering from or susceptible to a disease or condition that can be treated by administration of a composition or pharmaceutical composition provided herein. Non-limiting examples include humans, other mammals, cows, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammalian animals. In some embodiments, the patient is a human.

[0088] The term "disease" or "condition" refers to a state or condition of a patient or subject that can be treated with the compounds or methods provided herein. The disease can be cancer. Cancer can refer to solid tumor malignancies. Solid tumor malignancies include malignancies that may not have fluid or cysts. For example, solid tumor malignancies can include breast cancer, ovarian cancer, pancreatic cancer, cervical cancer, gastric cancer, renal cancer, head and neck cancer, bone cancer, skin cancer, or prostate cancer. In some further examples, "cancer" refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias (including solid cancers and cancers of the lymphatic system), kidney cancer, breast cancer, lung cancer, bladder cancer, colon cancer, ovarian cancer, prostate cancer, pancreatic cancer, stomach cancer, brain cancer, head and neck cancer, skin cancer, uterine cancer, testicular cancer, gliomas, esophageal cancer, and liver cancer (including hepatocellular carcinoma), lymphomas (including B acute lymphoblastic lymphoma), non-Hodgkin's lymphomas (e.g., Burkitt's lymphoma, small cell lymphoma, and large cell lymphoma), Hodgkin's lymphoma, leukemias (including acute myeloid leukemia (AML), ALL, and CML), or multiple myeloma.

[0089] As used herein, the term "cancer" refers to all types of cancer, neoplasms, or malignant tumors found in mammals, including leukemia, lymphoma, neuroendocrine tumors, carcinomas, and sarcomas. Exemplary cancers that may be treated with the compounds, pharmaceutical compositions, or methods provided herein include lymphoma (e.g., mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, Burkitt's lymphoma), sarcoma, bladder cancer, bone cancer, brain cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g., triple negative, ER positive, ER negative, chemotherapy resistant, Herceptin resistant, HER2 positive, doxorubicin resistant, tamoxifen resistant, ductal carcinoma, lobular carcinoma, bladder cancer, bone cancer, brain cancer, cervical cancer, colon cancer, esophageal cancer, stomach cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g., triple negative, ER positive, ER negative, chemotherapy resistant, Herceptin resistant, HER2 positive, doxorubicin resistant, tamoxifen resistant, ductal carcinoma, lobular carcinoma, bladder cancer, bone cancer, sarcoma, bladder cancer, bone cancer, bladder ... cancer, primary, metastatic), ovarian cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer, squamous cell lung carcinoma, adenocarcinoma, large cell lung cancer, small cell lung carcinoma, carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple-negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck, or esophageal), colorectal cancer, leukemia (e.g., lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia), acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma. Further examples include thyroid cancer, endocrine system cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, stomach cancer, uterine cancer or medulloblastoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulinoma, and the like. cancer, malignant carcinoid, bladder cancer, premalignant skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical carcinoma, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, Paget's disease of the breast, phyllodes tumor, lobular carcinoma, ductal carcinoma, pancreatic stellate cell carcinoma, hepatic stellate cell carcinoma, or prostate cancer.

[0090] The term "leukemia" refers broadly to progressive, malignant diseases of the blood-forming organs and is generally characterized by the distorted proliferation and development of white blood cells and their precursor cells in the blood and bone marrow. Leukemias are generally classified clinically based on (1) the duration and character of the disease-acute or chronic, (2) the type of cells involved-myeloid (myeloid), lymphocytic (lymphatic), or monocytic, and (3) the increased or non-increased number of abnormal cells in the blood-leukemic or nonleukemic (subleukemic). Exemplary leukemias that may be treated with the compounds or methods provided herein include, for example, acute myeloid leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, leukocytic leukemia, basophilic leukemia, blastic leukemia, bovine leukemia, chronic myeloid leukemia, leukemia cutis, embryonic leukemia, eosinophilic leukemia, Gross' leukemia, hairy cell leukemia, hemoblastic leukemia, hemoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, leukemia), lymphoblastic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, small myeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloblastic leukemia, myelogranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling leukemia, stem cell leukemia, subleukemic leukemia, or anaplastic cell leukemia.

[0091] The term "sarcoma" generally refers to tumors composed of embryonic connective tissue-like material and generally composed of tightly packed cells embedded in a fibrous or homogeneous substance. Sarcomas that may be treated with the compounds or methods provided herein include chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, liposarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic ... sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple chromatohemorrhagic sarcoma, B cell immunoblastic sarcoma, lymphoma, T cell immunoblastic sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukemia sarcoma, malignant mesenchymal sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, or telangiectatic sarcoma.

[0092] The term "melanoma" is intended to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas that may be treated with the compounds or methods provided herein include, for example, acral lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungual melanoma, or superficial spreading melanoma.

[0093] The term "carcinoma" refers to a malignant neoplasm composed of epithelial cells that tend to invade surrounding tissues and give rise to metastases. Exemplary cancers that may be treated with the compounds or methods provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, adenocystic carcinoma, acinar carcinoma, acinous carcinoma, adenoid cystic carcinoma, adenomatous carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basal cell carcinoma ... carcinoma), basal squamous cell carcinoma, bronchiolocarcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocarcinoma, choriocarcinoma, mucinous carcinoma, comedocarcinoma, uterine carcinoma, cribriform carcinoma, armor carcinoma, skin cancer, columnar carcinoma, columnar cell carcinoma, ductal carcinoma, compact carcinoma, embryonal carcinoma, cerebriform carcinoma, epidermoid carcinoma, adenoid epithelial carcinoma, exophytic carcinoma, ulcer carcinoma, fibrous carcinoma, gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma, adenocarcinoma, granulosa cell carcinoma, hair matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hürthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, embryonal carcinoma of childhood, intraepithelial carcinoma, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher carcinoma, Klutzycki cell carcinoma, large cell carcinoma, lenticular carcinoma carcinoma, lenticular carcinoma, lipomatous carcinoma, lymphoepithelial carcinoma, medullary carcinoma, melanoma, soft carcinoma, mucinous carcinoma, mucinous adenocarcinoma, mucinous cell carcinoma, mucoepidermoid carcinoma, carcinoma mucosum, mucouscarcinoma), myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, squamous cell carcinoma, atheromatous carcinoma, renal cell carcinoma, reserve cell carcinoma, sarcomatoid carcinoma, Schneider's carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, solanoid carcinoma, globular cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, squamous cell carcinoma, ligamentous carcinoma, telangiectasia-like carcinoma, transitional cell carcinoma, nodular carcinoma (carcinoma tuberosum), nodular carcinoma (tuberous carcinoma), verrucous carcinoma, or choriocarcinoma.

[0094] As used herein, the terms "metastasis" and "metastatic cancer" can be used interchangeably and refer to the spread of a proliferative disease or disorder (e.g., cancer) from one organ or another non-adjacent organ or part of the body. Cancer begins at a site of origin (e.g., breast), which is referred to as the primary tumor (e.g., primary breast cancer). Some cancer cells in the primary tumor or site of origin acquire the ability to penetrate and invade the normal tissues surrounding the local area, and / or penetrate the walls of the lymphatic or vascular system and circulate through the system to other sites and tissues in the body. A second clinically detectable tumor formed from the cancer cells of the primary tumor is called a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the breast site will consist of abnormal lung cells, not abnormal breast cells. A secondary tumor in the breast is referred to as a metastatic lung cancer. Thus, the phrase metastatic cancer refers to a disease in which the subject has or has had a primary tumor, and has one or more secondary tumors.The phrase non-metastatic cancer or the subject with non-metastatic cancer can refer to a disease in which the subject has a primary tumor, but does not have one or more secondary tumors.For example, metastatic lung cancer refers to a disease in a subject who has or has a history of a primary lung tumor, and has one or more secondary tumors in a second location or multiple locations (e.g., breast).

[0095] In the context of a substance or substance activity or function associated with a disease (e.g., a protein-related disease, a cancer associated with RKY activity, an RKY-related cancer, an RKY-related disease (e.g., cancer, an inflammatory disease, an autoimmune disease, or an infectious disease)), the term "associated" or "associated with" means that the disease (e.g., cancer, an inflammatory disease, an autoimmune disease, or an infectious disease) is caused (in whole or in part) or the symptoms of the disease are caused (in whole or in part) by the substance or substance activity or function. As used herein, what is described as being associated with a disease may be a target for treating the disease if it is a causative agent. For example, if an increase in RKY activity or function (e.g., signal transduction pathway activity) causes the disease (e.g., cancer, an inflammatory disease, an autoimmune disease, or an infectious disease), the cancer or RKY-related disease (e.g., cancer, an inflammatory disease, an autoimmune disease, or an infectious disease) associated with Ryk activity or function may be treated with an RKY modulator or an RKY inhibitor. For example, an inflammatory disease associated with RKY activity or function or an RKY-associated inflammatory disease can be treated with an RKY modulator or an RKY inhibitor, where increased RKY activity or function (e.g., signal transduction pathway activity) causes the disease.

[0096] As used herein, the term "signal transduction pathway" refers to a series of interactions between cellular and optionally extracellular components (e.g., proteins, nucleic acids, small molecules, ions, lipids) that can transmit a change in one component to one or more other components, which can in turn transmit changes to additional components, optionally which are propagated to other signal transduction pathway components.

[0097] As used herein, the term "aberrant" refers to something that is different from normal. Abnormal, when used to describe enzyme activity, refers to activity that is greater or less than the average of a normal control or normal non-disease control sample. Abnormal activity may refer to an amount of activity that results in disease, and restoring the abnormal activity to a normal or non-disease associated amount (e.g., by using the methods described herein) results in the alleviation of the disease or one or more disease symptoms.

[0098] A "therapeutic agent" as referred to herein is a composition useful in the treatment or prevention of a disease such as cancer (e.g., leukemia). In some embodiments, the therapeutic agent is an anti-cancer agent. "Anti-cancer agent" is used according to their plain and ordinary meaning to refer to compositions (e.g., compounds, drugs, antagonists, inhibitors, modulators) that have anti-neoplastic properties or the ability to inhibit cell growth or proliferation. In embodiments, the anti-cancer agent is a chemotherapeutic agent. In embodiments, the anti-cancer agent is an agent identified herein that has utility in methods of treating cancer. In embodiments, the anti-cancer agent is an agent approved by the FDA or similar regulatory agency in a country other than the United States to treat cancer.

[0099] As used herein, an "anti-cancer agent" refers to a molecule (e.g., a compound, peptide, protein, nucleic acid, etc.) used to treat cancer by destruction or inhibition of cancer cells or tissues. An anti-cancer agent may be selective for a particular cancer or a particular tissue. In embodiments, anti-cancer agents herein may include epigenetic inhibitors and multikinase inhibitors, and "anti-cancer agent" and "anticancer agent" are used according to their plain and ordinary meaning to refer to compositions (e.g., compounds, drugs, antagonists, inhibitors, modulators) that have anti-neoplastic properties or the ability to inhibit cell growth or proliferation. In some embodiments, an anti-cancer agent is a chemotherapeutic agent. In some embodiments, an anti-cancer agent is an agent identified herein that has utility in methods of treating cancer. In some embodiments, an anti-cancer agent is an agent approved by the FDA or similar regulatory agency of a country other than the United States to treat cancer. Examples of anticancer drugs include MEK (e.g., MEK1, MEK2, or MEK1 and MEK2) inhibitors (e.g., XL518, CI-1040, PD035901, selumetinib / AZD6244, GSK1120212 / trametinib, GDC-0973, ARRY-162, ARRY-300, AZD8330, PD0325901, U0126, PD98059, TAK-733, PD318088, AS703026, BAY869766), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, , mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogen mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, meiphalan), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, semustine, streptozocin), triazenes (decarbazine), antimetabolites (e.g., 5-azathioprine, leucovorin,Capecitabine, fludarabine, gemcitabine, pemetrexed, raltitrexed, folic acid analogues (e.g., methotrexate), or pyrimidine analogues (e.g., fluorouracil, floxuridine, cytarabine), purine analogues (e.g., mercaptopurine, thioguanine, pentostatin, etc.), plant alkaloids (vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel, docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan, amsacrine, etoposide (VP16 ), etoposide phosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin, etc.), platinum compounds (e.g., cisplatin, oxaloplatin, carboplatin), anthracenediones (e.g., mitoxantrone), substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine), adrenal cortex suppressants (e.g., mitotane, aminoglutethimide), epipodophy rotoxins (e.g., etoposide), antibiotics (e.g., daunorubicin, doxorubicin, bleomycin), enzymes (e.g., L-asparaginase), inhibitors of mitogen-activated protein kinase signaling (e.g., U0126, PD98059, PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY43-9006, wortmannin, or LY294002), Syk inhibitors, mTOR inhibitors, antibodies (such as Rituxan), gossyphol, genasense, Polyphenol E, chlorofusin, all-trans retinoic acid (ATRA), bryostatin, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), 5-aza-2'-deoxycytidine, all-trans retinoic acid, doxorubicin, vincristine, etoposide, gemcitabine, imatinib (Gleevec.RTM.), geldanamycin, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), flavopiridol, LY294002, bortezomib, trastuzumab, BAY11-7082, PKC412,These include, but are not limited to, PD184352, 20-epi-1,25 dihydroxyvitamin D3, or 5-ethynyluracil.

[0100] Further examples of anti-cancer drugs include abiraterone, aclarubicin, acylfulvene, adecypenol, adozelesin, aldesleukin, ALL-TK antagonists, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, anti-dorsal morphogenetic protein-1, antiandrogens, prostate cancer, antiestrogens, antineoplastons, antisense oligonucleotides, and the like. cleotide, aphidicolin glycinate, apoptosis gene regulator, apoptosis regulator, apurinic acid, ara-CDP-DL-PTBA, arginine deaminase, asulaculin, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivative, balanol, batimastat, BCR / ABL antagonist, benzochlorine, benzoylstaurosporine, beta-lactam derivative, beta-arretin, beta-clamycin B, betulinic acid, bF GF inhibitors, bicalutamide, bisantrene, bisaziridinylspermine, bisnafide, bisstraten A, bizelesin, brefulate, bropirimine, budotitane, buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivatives, canarypox IL-2, capecitabine, carboxamido-amino-triazole, carboxyamidotriazole, CaRestM3, CARN700, cartilage-derived inhibitor, carzelesin, casein kinase inhibitor (ICOS), castanospermine, cecropin B, cetrorelix, chloramphenicol , chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, clomiphene analogue, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analogue, conagenin, crambecidin 816, crisnatol, cryptophycin 8, cryptophycin A derivative, curacin A, cyclopentaanthraquinone, cycloplatam, sipemycin, cytarabine ocfosfate, cytolytic factor, cytostatin, dacliximab, decitabine, dehydrodidemnin B, deslorelin,Dexamethasone, dexphosphamide, dexrazoxane, dexverapamil, diaziquone, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, 9-dioxamycin, diphenylspiromustine, docosanol, dolasetron, doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, elemene, emiteflu, epirubicin, epristeride, estramustine analogs, estrogen agonists, estrogen antagonists, etanidazole, etoposide phosphate, or exemestane.

[0101] Further examples of anticancer drugs include fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezerastine, fluasterone, fludarabine, fluorodaunorhysine hydrochloride, forphenimex, formestane, fostriecin, fotemustine, gadolinium texaphyrin, gallium nitrate, gallocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfame, heregulin, hexamethylene bisacetamide, hypericin, and ibandronate. , idarubicin, idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridone, imiquimod, immunostimulatory peptides, insulin-like growth factor-1 receptor inhibitors, interferon agonists, interferons, interleukins, iobenguane, iododoxorubicin, ipomeanol, 4-, iropract, irsogladine, isobengazole, isohomohalichondrin B, itasetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lenograstim lanin sulfate, leptolstatin, letrozole, leukemia inhibitory factor, leukocyte alpha interferon, leuprolide + estrogen + progesterone, leuprorelin, levamisole, liarozole, linear polyamine analogues, lipophilic disaccharide peptides, lipophilic platinum compounds, lysocrine amide 7, lobaplatin, lombricine, lometrexol, lonidamine, losoxantrone, lovastatin, loxoribine, lurtotecan, lutetium texaphyrin, lysofylline, lytic peptides, maytansine , mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors, menogaril, mervalone, meterelin, methioninase, metoclopramide, MIF inhibitors, mifepristone, miltefosine, millimostim, mismatched double-stranded RNA, mitoguazone, mitolactol, mitomycin analogues, mitonafide, mitotoxin fibroblast growth factor saporin, mitoxantrone, mofalotene, molgramostim, monoclonal antibodies, human chorionic gonadotrophin,Monophosphoryl lipid A + myobacterium cell wall sk, mopidamol, multidrug resistance gene inhibitors, multiple tumor suppressor 1-based therapy, mustard anticancer drugs, mycaperoxide B, mycobacterial cell wall extract, myriaporone, N-acetyldinaline, N-substituted benzamides, nafarelin, nagressip, naloxone + pentazocine, napavine, naphterpine, nartograstim, nedaplatin, nemorubicin, neridronic acid, neutral Endopeptidases, nilutamide, nisamycin, nitric oxide regulators, nitroxide antioxidants, nitrulline, O6-benzylguanine, octreotide, oxenone, oligonucleotides, onapristone, ondansetron, ondansetron, oracin, oral cytokine inducers, ormaplatin, osateron, oxaliplatin, oxaunomycin, parauamine, palmitoyl rhizoxin, pamidronate, panaxytriol, panomyphen, parabactin, These include, but are not limited to, pazeriptin, pegaspargase, perdecin, pentosan polysulfate sodium, pentostatin, pentrozole, perflubron, perfosfamide, perillyl alcohol, phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pirarubicin, piritrexim, prasetin A, prasetin B, plasminogen activator inhibitors, platinum complexes, platinum compounds, platinum-triamine complexes, porfimer sodium salt, porfiromycin, prednisone, propyl bis-acridone, prostaglandin J2, proteasome inhibitors, protein A-based immunomodulators, protein kinase C inhibitors, protein kinase C inhibitors, microalgal, protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, purpurin, pyrazoloacridine, or pyridoxylated hemoglobin polyoxyethylene conjugates.

[0102] Further examples of anticancer drugs include raf antagonists, raltitrexed, ramosetron, ras farnesyl protein transferase inhibitors, ras inhibitors, ras-GAP inhibitors, reteriptin demethylation, rhenium Re186 etidronate, rhizoxin, ribozyme, RII retinamide, logretimide, rohitukin, romurtide, roquinimex, rubiginone B1, ruboxil, safingol, centopin, SarCNU, sarcophytol A, sargramostim, Sdi1 mimetics, semustine, senescence derived inhibitor factor 1, sense oligonuclear nucleotides, signal transduction inhibitors, signal transduction modulators, single-chain antigen binding proteins, schizofuran, sobuzoxane, sodium borocaptate, sodium phenylacetate, sorberol, somatomedin binding proteins, sonermin, sparfosic acid, spicamycin D, spiromustine, splenopentin, spongiostatin 1, squalamine, stem cell inhibitors, stem cell division inhibitors, stipiamide, stromelysin inhibitors, sulfinosine, superactive vasoactive intestinal peptide antagonists, slajista, suramin, swainsonine, synthetic glycosaminoglycans thrombin, talimustine, tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium, tegafur, tellupyrium, telomerase inhibitors, temoporfin, temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine, thaliblastine, thiocoraline, thrombopoietin, thrombopoietin mimetics, thymalfasin, thymopoietin receptor agonist, thymotrin, thyrotropin, thyrotropin stimulating hormone, ethyl etiopurinse, tirapazamine, titanocene dichloride, topsentin, toremifene, totipotent stem cell factor, translation inhibitors, tor Retinoin, triacetyluridine, triciribine, trimetrexate, triptorelin, tropisetron, turosteride, tyrosine kinase inhibitors, tyrophostin, UBC inhibitors, ubenimex, urogenital sinus-derived growth inhibitor, urokinase receptor antagonists, vapreotide, variolin B, vector systems, red blood cell gene therapy, veraresol, veramine, verdine, verteporfin, vinorelbine, vinxartin, vitaxin, vorozole, zanoteron, zeniplatin, zilascorub, zinostatin stimalamer, adriamycin,Dactinomycin, bleomycin, vinblastine, cisplatin, acivicin, aclarubicin, acodazole hydrochloride, acronine, adzelesin, aldesleukin, altretamine, ambomycin, amethanthrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamine Examples of the anti-inflammatory drugs include, but are not limited to, bisantrene hydrochloride, visnafide dimesylate, bizelesin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan, cactinomycin, calsterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, ciloremycin, cladribine, crisnatol mesylate, cyclophosphamide, and cytarabine.

[0103] Further examples of anticancer drugs include dacarbazine, daunorubicin hydrochloride, decitabine, dexorumaplatin, desaguanine, desaguanine mesylate, diaziquone, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin, enpromate, epipropizine, epirubicin hydrochloride, elbrozole, esorubicin hydrochloride, estramustine, estramustine phosphate sodium salt, Etanidazole, etoposide, etoposide phosphate, etopurine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, fluorocitabine, fosquidone, fostriecin sodium salt, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, i.m.foscin, interleukin I1 (including recombinant interleukin II, i.e., rlL.sub.2), interferon alpha-2a, interferon alpha-2b, interferon alpha-n1, interferon alpha-n3, interferon beta-1a, interferon gamma-1b, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liarozole hydrochloride, lometrexol sodium salt, lomustine, losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium salt, metoprine, meturede Pa, mitindomide, mitocalcin, mitochromine, mitogillin, mitomarcine, mitomycin, mitosper, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nocodazoie, nogalamycin, ormaplatin, oxisuran, pegaspargase, periomycin, pentamustine, peplomycin sulfate, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, promestane, porfimer sodium salt, porfiromycin, prednimustine, procarbazine hydrochloride,Puromycin, puromycin hydrochloride, pyrazofurin, ribopurin, rogletimide, safingol, safingol hydrochloride, semustine, simtrazene, sparphosate sodium salt, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, tallysomycin, tecogalan sodium salt, tegafur, teroxantrone hydrochloride, temoporfin, teni Poside, teroxylon, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, toremifene citrate, trestron acetate, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tubrozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, binepidizine vinorelbine sulfate, vinglisinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrocidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, zinostatin, zorubicin hydrochloride, agents that arrest cells in the G2-M phase and / or modulate the formation or stability of microtubules (e.g., Taxol™ (i.e., paclitaxel), Taxotere™, compounds containing a taxane skeleton, erbrozole (i.e., R-5510, 4), dolastatin 10 (i.e., DLS-10 and NSC-376128), mibobrine isethionate (i.e., as CI-980), vincristine, NSC-639829, discodermolide (i.e., as NVP-XX-A-296), ABT-751 (Abbott, i.e., E-7010), or altorhyrtin (e.g., altorhyrtin A and altorhyrtin C).

[0104] Further examples of anti-cancer agents include spongistatins (e.g., spongistatin 1, spongistatin 2, spongistatin 3, spongistatin 4, spongistatin 5, spongistatin 6, spongistatin 7, spongistatin 8, and spongistatin 9), cemadotin hydrochloride (i.e., LU-103793 and NSC-D-669356), epothilones (e.g., epothilone A, epothilone B, epothilone C (i.e., desoxyepothilone A or dEpoA), epothilone D (i.e., KOS-862, dEpoB, and desoxyepothilone B), epothilone E, epothilone F, epothilone B N-oxide, epothilone AN-oxide, 16-aza-epothilone B, 21-aminoepothilone B (i.e., BMS-31070, 5), 21-hydroxyepothilone D (i.e., desoxyepothilone F and dEpoF), 26-fluoroepothilone, auristatin PE (i.e., NSC-654663), sobridotin (i.e., TZT-1027), LS-4559-P (Pharmacia, i.e., LS-4577), LS-4578 (Pharmacia, i.e., LS-477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR-112378 (Aventis), vincristine sulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, i.e., WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2 (Hungarian Academy of Sciences), BSF-223651 (BASF, i.e. ILX-651 and LU-223651), SAH-49960 (Lilly / Novartis), SDZ-268970 (Lilly / Novartis), AM-97 (Armad / Kyowa Hakko), AM-132 (Armad), AM-138 (Armad / KyowaHakko), IDN-5005 (Indena), cryptophycin 52 (i.e., LY-355703), AC-7739 (Ajinomoto, i.e., AVE-8063A and CS-39.HCl), AC-7700 (Ajinomoto, i.e., AVE-8062, AVE-8062A, CS-39-L-Ser.HCl, and RPR-258062A), bitilebuamide, tubulysin A, canadensol, centaureydin (i.e., NSC-106969), T-138067 (Tularik, i.e., T-67, TL-138067, and TI-138067), COBRA-1 (Parker Hughes Institute, i.e., DDE-261 and WHI-261), H10 (Kansas State University), H16 (Kansas State University), oncocidin A1 (i.e., BTO-956 and DIME), DDE-313 (Parker Hughes Institute), physianolide B, laulimalide, SPA-2 (Parker Hughes Institute), SPA-1 (Parker Hughes Institute, i.e., SPIKET-P), 3-IAABU (Cytoskeleton / Mt. Sinai School of Medicine, i.e., MF-569), narcosine (also known as NSC-5366), nascapine, D-24851 (Asta Medica), A-105972 (Abbott), hemiasterin, 3-BAABU (Cytoskeleton / Mt. Sinai School of Medicine, i.e., MF-191), TMPN (Arizona State University), vanadocene acetylacetonate, T-138026 (Tularik), Monsatrol, inanosin (i.e., NSC-698666), 3-IAABE (Cytoskeleton / Mt. Sinai School of Medicine),Medicine), A-204197 (Abbott), T-607 (Tuiarik, i.e., T-900607), RPR-115781 (Aventis), eleutherobin (e.g., desmethyleleutherobin, desacetyleleutherobin, isoeleutherobin A, Z-eleutherobin), carbaeoside, carbaeolin, halichondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica), diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), taccalonolide A, TUB-245 (Aventis), A-259754 (Abbott), diozostatin, (-)-phenylahistine (i.e., NSCL-96F037), D-68838 (Asta Medica), D-68836 (Asta Medica), myoseverin B, D-43411 (Zentaris, i.e., D-81862), A-289099 (Abbott), A-318315 (Abbott), HTI-286 (i.e., SPA-110, trifluoroacetate salt) (Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC-12983 (NCI), resbellastatin phosphate sodium salt, BPR-OY-007 (National Health ResearchInstitutes, and SSR-250411 (Sanofi)), steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, gonadotropin releasing hormone agonists (GnRH) (e.g., goserelin or leuprolide), corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate), estrogens (e.g., diethlystilbestrol, ethinyl estradiol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogens (e.g., flutamide), immunostimulants (e.g., bacillus Calmette-Guerin (BCG), levamisole, interleukin 2, alpha-interferon, etc.), monoclonal antibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, and anti-VEGF monoclonal antibodies), immunotoxins (anti-CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonal antibody-Pseudomonas exotoxin conjugate, etc.), radioimmunotherapy (e.g., 111 In, 90 Y, or 131I-conjugated anti-CD20 monoclonal antibodies, triptolide, homoharringtonine, dactinomycin, doxorubicin, epirubicin, topotecan, itraconazole, vindesine, cerivastatin, vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan, clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib, gefitinib, EGFR inhibitors, epidermal growth factor receptor (EGFR) targeted therapies or therapeutics (e.g., gefitinib (Iressa™), erlotinib (Tarceva™), cetuximab (Erbitux™), lapatinib (Tykerb™), panitum Mab (Vectibix™), vandetanib (Caprelsa™), afatinib / BIBW2992, CI-1033 / canertinib, neratinib / HKI-272, CP-724714, TAK-285, AST-1306, ARRY334543, ARRY-380, AG-1478, dacomitinib / PF299804, OS I-420 / desmethylerlotinib, AZD8931, AEE788, pelitinib / EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib, sunitinib, dasatinib, and the like.

[0105] As used herein, "treating" or "treatment" of a condition, disease or disorder, or a symptom associated with a condition, disease or disorder, refers to an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether partial or total, reduction in the extent of a condition, disorder or disease, stabilization of the condition, disorder or disease state, prevention of the onset of a condition, disorder or disease, prevention of the spread of a condition, disorder or disease, delay or slowing of the progression of a condition, disorder or disease, delay or slowing of the onset of a condition, disorder or disease, improvement or alleviation of a pathology, disorder or disease, and remission. "Treating" may also mean prolonging survival compared to expected survival in the absence of treatment. "Treating" may also mean inhibiting the progression of a condition, disorder or disease, slowing the progression of a condition, disorder or disease temporarily, but in some cases involving permanently halting the progression of a condition, disorder or disease. As used herein, the terms treatment, therapy, or therapy refer to a method of reducing the effects of one or more symptoms of a disease or condition characterized by the expression of a protease, or a method of reducing the symptoms of a disease or condition characterized by the expression of a protease. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or a symptom of the disease or condition. For example, a method for treating a disease is considered to be therapeutic if one or more symptoms of the disease are reduced by 10% in a subject compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to a native or control level. It is understood that treatment does not necessarily refer to a cure or complete elimination of a disease, condition, or a symptom of a disease or condition.Furthermore, as used herein, references to reduce, decrease, or inhibit include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control level, and such terms can include, but do not necessarily include, a complete elimination.

[0106] The terms "dose" and "dosage" are used interchangeably herein. Dose refers to the amount of active ingredient given to an individual at each administration. Dosage varies depending on several factors, including the usual dose range for a given treatment, frequency of administration, size and tolerance of the individual, severity of the condition, risk of side effects, and route of administration. Those skilled in the art will recognize that dosage may be altered depending on the above factors or based on the progress of treatment. The term "dosage form" refers to the specific format of a medicament or pharmaceutical composition and depends on the route of administration. For example, the dosage form may be in the form of a liquid for nebulization, such as a tablet or liquid for inhalation, such as for oral delivery, or saline, such as for injection.

[0107] As used herein, a "therapeutically effective dose or amount" refers to a dose that produces the desired effect (e.g., treatment or prevention of a disease) for which it is administered. The exact dose and formulation will depend on the purpose of the treatment and can be ascertained by one of ordinary skill in the art using known techniques (Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003); and Pickar, Dosage Calculations (1999)). For example, for a given parameter, a therapeutically effective amount will exhibit at least a 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least a 100% increase or decrease. The therapeutic efficacy can also be expressed as a "-fold" increase or decrease. For example, a therapeutically effective amount can have at least 1.2, 1.5, 2, 5 or more times the efficacy of a standard control. A therapeutically effective dose or amount can improve one or more symptoms of a disease. A therapeutically effective dose or amount can prevent or delay the onset of a disease or one or more symptoms of a disease when the effect for which it is administered is to treat a person at risk of developing a disease.

[0108] As used herein, the term "administering" refers to oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or implantation of a sustained release device, such as a mini-osmotic pump, into a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. "Concomitant administration" means that the compositions described herein are administered simultaneously with, immediately prior to, or immediately following the administration of one or more additional therapies, such as cancer therapies, such as chemotherapy, hormonal therapy, radiation therapy, or immunotherapy. The compounds of the present invention can be administered alone or can be co-administered to a patient. Co-administration is meant to include simultaneous or sequential administration of compounds individually or in combination (more than one compound).The preparation can therefore also be combined with other active substances as necessary (e.g., to reduce metabolic degradation).The compositions of the present invention can be delivered by topical route, transdermally, or formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0109] Formulations suitable for oral administration may consist of (a) a liquid solution, such as an effective amount of an antibody provided herein suspended in a diluent (e.g., water, saline, or PEG 400), (b) capsules, sachets, or tablets, each containing a predetermined amount of the active ingredient as a liquid, solid, granules, or gelatin, (c) a suspension in a suitable liquid, or (d) a suitable emulsion. Tablet forms may contain one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrants, and pharma- ceutically compatible carriers. Lozenge forms may include the active ingredient in a flavoring (e.g., sucrose), as well as pastilles containing the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia emulsion, gels, etc., which contain, in addition to the active ingredient, carriers known in the art.

[0110] Pharmaceutical compositions may also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex-functionalized Sepharose™, agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Additionally, these carriers can function as immunostimulating agents (i.e., adjuvants).

[0111] The preparation suitable for rectal administration includes, for example, suppositories that are made of packaged nucleic acid with suppository base.Suitable suppository base includes natural or synthetic triglyceride or paraffin hydrocarbon.In addition, it is also possible to use gelatin rectal capsules that are made of the combination of selected compound and base, including, for example, liquid triglyceride, polyethylene glycol, and paraffin hydrocarbon.

[0112] Suitable formulations for parenteral administration, such as intra-articular (intra-articular), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In the practice of the present invention, the compositions may be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally. Parenteral, oral, and intravenous administration are preferred methods of administration. The formulations of the compounds may be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.

[0113] Injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described. Cells transduced with nucleic acid for ex vivo therapy may also be administered intravenously or parenterally as described above.

[0114] Co-administration contemplates simultaneous administration using separate formulations or a single pharmaceutical formulation, and sequential administration in either order, preferably with a period during which both (or all) active agents exert their biological activity simultaneously.

[0115] The compositions of the present invention may further comprise components that provide sustained release and / or comfort. Such components include high molecular weight, anionic mucus-mimetic polymers, gelling polysaccharides, and finely divided drug carrier substrates. These components are described in more detail in U.S. Patent Nos. 4,911,920, 5,403,841, 5,212,162, and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present invention may also be delivered as microspheres for sustained release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres for subcutaneous sustained release (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995), as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995), or as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49, 669-674, 1997). In embodiments, formulations of the compositions of the present invention can be delivered by the use of liposomes that fuse with cell membranes or are endocytosed, i.e., by using receptor ligands attached to the liposomes that bind to cell surface membrane protein receptors that result in endocytosis. The use of liposomes can focus the delivery of the compositions of the present invention to target cells in vivo, especially when the liposome surface carries receptor ligands specific to the target cells or is otherwise preferentially directed to a particular organ. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46, 1576-1587, 1989.) The compositions of the invention can also be delivered as nanoparticles.

[0116] As used herein, the term "pharmacologically acceptable" is used interchangeably with "physiologically acceptable" and "pharmacologically acceptable." Pharmaceutical compositions generally include agents for buffering and preservation during storage and, depending on the route of administration, can include buffers and carriers for appropriate delivery.

[0117] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to substances that aid in the administration and absorption of active agents to and by a subject and can be included in the compositions of the present invention without causing significant adverse toxic effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (such as Ringer's solution), alcohols, oils, carbohydrates such as gelatin, lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and coloring agents. Such preparations can be sterilized and mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, coloring agents, and / or aromatic substances that do not adversely react with the compounds of the present invention. Those skilled in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0118] The term "pharmaceutically acceptable salt" refers to salts derived from a variety of organic and inorganic counterions well known in the art, including, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, and the molecule includes salts of organic or inorganic acids such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, and the like.

[0119] The term "preparation" is intended to include the formulation of active compound with encapsulating material as a carrier to provide a capsule. In a capsule, the active ingredient is surrounded by a carrier, with or without other carriers, and is therefore associated with the carrier. Also included are cachets and lozenges. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0120] The pharmaceutical preparation is optionally in unit dosage form. In such dosage form, the preparation is subdivided into unit doses containing appropriate amounts of active ingredient. The unit dosage form can be a packaged preparation, the package containing discrete amounts of the preparation, such as packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, tablet, cachet, or lozenge itself, or the appropriate number of any of these packaged dosage forms. The unit dosage form can be a cryodispersion.

[0121] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes thereto will be suggested to those skilled in the art and are within the spirit and scope of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0122] Anti-RYK antibody Provided herein are, inter alia, antibodies (e.g., chimeric antibodies, monoclonal antibodies, antibody fragments (e.g., scFv)) that bind to human associated tyrosine receptor kinase (RYK) with high efficiency and specificity. The antibodies and antibody compositions provided herein include, for example, novel light and heavy chain domain CDRs and framework regions and are useful, inter alia, for the diagnosis and treatment of cancer and other RYK-related diseases. In embodiments, the anti-RYK antibodies provided herein can bind to human RYK protein, but not to mouse RYK protein.

[0123] In one aspect, an anti-RYK antibody is provided comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 1, CDR H2 as set forth in SEQ ID NO: 2, and CDR H3 as set forth in SEQ ID NO: 3, and the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 4, CDR L2 as set forth in SEQ ID NO: 5, and CDR L3 as set forth in SEQ ID NO: 6.

[0124] In an embodiment the heavy chain variable domain comprises the sequence of SEQ ID NO: 15. In an embodiment the heavy chain variable domain is the sequence of SEQ ID NO: 15. In an embodiment the light chain variable domain comprises the sequence of SEQ ID NO: 16. In an embodiment the light chain variable domain is the sequence of SEQ ID NO: 16. In an embodiment the heavy chain variable domain comprises the sequence of SEQ ID NO: 15 and the light chain variable domain comprises the sequence of SEQ ID NO: 16. In an embodiment the heavy chain variable domain is the sequence of SEQ ID NO: 15 and the light chain variable domain is the sequence of SEQ ID NO: 16.

[0125] In embodiments, the monoclonal antibody has an equilibrium dissociation constant (K D In embodiments, the anti-RYK antibody binds to RYK with a K of about 2 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 3 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 4 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 5 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 6 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 7 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 8 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 9 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 10 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 50 pM to about 2 nM. DIn embodiments, the anti-RYK antibody has a K of about 100 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 200 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 300 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 400 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 500 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 600 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 700 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 800 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 900 pM to about 2 nM. D In embodiments, the anti-RYK antibody has a K of about 1 nM to about 2 nM. D has.

[0126] In embodiments, the monoclonal antibody has an equilibrium dissociation constant (K D In embodiments, the anti-RYK antibody binds to RYK with a K of about 2 pM to about 1 nM. D In embodiments, the anti-RYK antibody has a K of about 2 pM to about 900 pM. D In embodiments, the anti-RYK antibody has a K of about 2 pM to about 800 pM. D In embodiments, the anti-RYK antibody has a K of about 2 pM to about 700 pM. D In embodiments, the anti-RYK antibody has a K of about 2 pM to about 600 pM. D In embodiments, the anti-RYK antibody has a K of about 2 pM to about 500 pM. D In embodiments, the anti-RYK antibody has a K of about 2 pM to about 400 pM. D In embodiments, the anti-RYK antibody has a K of about 2 pM to about 300 pM. D In embodiments, the anti-RYK antibody has a K of about 2 pM to about 200 pM. DIn embodiments, the anti-RYK antibody has a K of about 2 pM to about 100 pM. D In embodiments, the anti-RYK antibody has a K of about 2 pM to about 50 pM. D In embodiments, the anti-RYK antibody has a K of about 2 pM to about 10 pM. D In embodiments, the anti-RYK antibody has a K of about 2 pM to about 9 pM. D In embodiments, the anti-RYK antibody has a K of about 2 pM to about 8 pM. D In embodiments, the anti-RYK antibody has a K of about 2 pM to about 7 pM. D In embodiments, the anti-RYK antibody has a K of about 2 pM to about 6 pM. D In embodiments, the anti-RYK antibody has a K of about 2 pM to about 5 pM. D In embodiments, the anti-RYK antibody has a K of about 2 pM to about 4 pM. D In embodiments, the anti-RYK antibody has a K of about 2 pM to about 3 pM. D has.

[0127] In embodiments, the monoclonal antibody has an equilibrium dissociation constant (K D In embodiments, the anti-RYK antibody binds to RYK with a K of 2 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 3 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 4 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 5 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 6 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 7 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 8 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 9 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 10 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 50 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 100 pM to 2 nM.D In embodiments, the anti-RYK antibody has a K of 200 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 300 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 400 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 500 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 600 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 700 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 800 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 900 pM to 2 nM. D In embodiments, the anti-RYK antibody has a K of 1 nM to 2 nM. D has.

[0128] In embodiments, the monoclonal antibody has an equilibrium dissociation constant (K D In embodiments, the anti-RYK antibody binds to RYK with a K of 2 pM to 1 nM. D In embodiments, the anti-RYK antibody has a K of 2 pM to 900 pM. D In embodiments, the anti-RYK antibody has a K of 2 pM to 800 pM. D In embodiments, the anti-RYK antibody has a K of 2 pM to 700 pM. D In embodiments, the anti-RYK antibody has a K of 2 pM to 600 pM. D In embodiments, the anti-RYK antibody has a K of 2 pM to 500 pM. D In embodiments, the anti-RYK antibody has a K of 2 pM to 400 pM. D In embodiments, the anti-RYK antibody has a K of 2 pM to 300 pM. D In embodiments, the anti-RYK antibody has a K of 2 pM to 200 pM. D In embodiments, the anti-RYK antibody has a K of 2 pM to 100 pM. D In embodiments, the anti-RYK antibody has a K of 2 pM to 50 pM. DIn embodiments, the anti-RYK antibody has a K of 2 pM to 10 pM. D In embodiments, the anti-RYK antibody has a K of 2 pM to 9 pM. D In embodiments, the anti-RYK antibody has a K of 2 pM to 8 pM. D In embodiments, the anti-RYK antibody has a K of 2 pM to 7 pM. D In embodiments, the anti-RYK antibody has a K of 2 pM to 6 pM. D In embodiments, the anti-RYK antibody has a K of 2 pM to 5 pM. D In embodiments, the anti-RYK antibody has a K of 2 pM to 4 pM. D In embodiments, the anti-RYK antibody has a K of 2 pM to 3 pM. D In embodiments, the anti-RYK antibody has a K of about 513 pM. D In embodiments, the anti-RYK antibody has a K D In embodiments, the anti-RYK antibody is referred to herein as 2-D11.

[0129] In another aspect, an anti-RYK antibody is provided comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 17, CDR H2 as set forth in SEQ ID NO: 18, and CDR H3 as set forth in SEQ ID NO: 19, and the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 20, CDR L2 as set forth in SEQ ID NO: 21, and CDR L3 as set forth in SEQ ID NO: 22.

[0130] In an embodiment the heavy chain variable domain comprises the sequence of SEQ ID NO: 31. In an embodiment the heavy chain variable domain is the sequence of SEQ ID NO: 31. In an embodiment the light chain variable domain comprises the sequence of SEQ ID NO: 32. In an embodiment the light chain variable domain is the sequence of SEQ ID NO: 32. In an embodiment the heavy chain variable domain comprises the sequence of SEQ ID NO: 31 and the light chain variable domain comprises the sequence of SEQ ID NO: 32. In an embodiment the heavy chain variable domain is the sequence of SEQ ID NO: 31 and the light chain variable domain is the sequence of SEQ ID NO: 32.

[0131] In embodiments, the monoclonal antibody has an equilibrium dissociation constant (K D In embodiments, the anti-RYK antibody binds to RYK with a K of about 6 nM to about 17 nM. D In embodiments, the anti-RYK antibody has a K of about 7 nM to about 17 nM. D In embodiments, the anti-RYK antibody has a K of about 8 nM to about 17 nM. D In embodiments, the anti-RYK antibody has a K of about 9 nM to about 17 nM. D In embodiments, the anti-RYK antibody has a K of about 10 nM to about 17 nM. D In embodiments, the anti-RYK antibody has a K of about 11 nM to about 17 nM. D In embodiments, the anti-RYK antibody has a K of about 12 nM to about 17 nM. D In embodiments, the anti-RYK antibody has a K of about 13 nM to about 17 nM. D In embodiments, the anti-RYK antibody has a K of about 14 nM to about 17 nM. D In embodiments, the anti-RYK antibody has a K of about 15 nM to about 17 nM. D In embodiments, the anti-RYK antibody has a K of about 16 nM to about 17 nM. D has.

[0132] In embodiments, the monoclonal antibody has an equilibrium dissociation constant (K D In embodiments, the anti-RYK antibody binds to RYK with a K of 6 nM to 16 nM. D In embodiments, the anti-RYK antibody has a K of about 6 nM to about 15 nM. D In embodiments, the anti-RYK antibody has a K of about 6 nM to about 14 nM. D In embodiments, the anti-RYK antibody has a K of about 6 nM to about 13 nM. D In embodiments, the anti-RYK antibody has a K of about 6 nM to about 12 nM. D In embodiments, the anti-RYK antibody has a K of about 6 nM to about 11 nM. D In embodiments, the anti-RYK antibody has a K of about 6 nM to about 10 nM. DIn embodiments, the anti-RYK antibody has a K of about 6 nM to about 9 nM. D In embodiments, the anti-RYK antibody has a K of about 6 nM to about 8 nM. D In embodiments, the anti-RYK antibody has a K of about 6 nM to about 7 nM. D has.

[0133] In embodiments, the monoclonal antibody has an equilibrium dissociation constant (K D In embodiments, the anti-RYK antibody binds to RYK with a K of 6 nM to 17 nM. D In embodiments, the anti-RYK antibody has a K of 7 nM to 17 nM. D In embodiments, the anti-RYK antibody has a K of 8 nM to 17 nM. D In embodiments, the anti-RYK antibody has a K of 9 nM to 17 nM. D In embodiments, the anti-RYK antibody has a K of 10 nM to 17 nM. D In embodiments, the anti-RYK antibody has a K of 11 nM to 17 nM. D In embodiments, the anti-RYK antibody has a K of 12 nM to 17 nM. D In embodiments, the anti-RYK antibody has a K of 13 nM to 17 nM. D In embodiments, the anti-RYK antibody has a K of about 14 nM to about 17 nM. D In embodiments, the anti-RYK antibody has a K of 15 nM to 17 nM. D In embodiments, the anti-RYK antibody has a K of 16 nM to 17 nM. D has.

[0134] In embodiments, the monoclonal antibody has an equilibrium dissociation constant (K D In embodiments, the anti-RYK antibody binds to RYK with a K of 6 nM to 16 nM. D In embodiments, the anti-RYK antibody has a K of 6 nM to 15 nM. D In embodiments, the anti-RYK antibody has a K of 6 nM to 14 nM. D In embodiments, the anti-RYK antibody has a K of 6 nM to 13 nM. DIn embodiments, the anti-RYK antibody has a K of 6 nM to 12 nM. D In embodiments, the anti-RYK antibody has a K of 6 nM to about 11 nM. D In embodiments, the anti-RYK antibody has a K of 6 nM to 10 nM. D In embodiments, the anti-RYK antibody has a K of 6 nM to 9 nM. D In embodiments, the anti-RYK antibody has a K of 6 nM to 8 nM. D In embodiments, the anti-RYK antibody has a K of about 6 nM to 7 nM. D In embodiments, the anti-RYK antibody has a K of about 10 nM. D In embodiments, the anti-RYK antibody has a K D In embodiments, the anti-RYK antibody is referred to herein as 7-D10.

[0135] In another aspect, an anti-RYK antibody is provided comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 33, CDR H2 as set forth in SEQ ID NO: 34, and CDR H3 as set forth in SEQ ID NO: 35, and the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 36, CDR L2 as set forth in SEQ ID NO: 37, and CDR L3 as set forth in SEQ ID NO: 38.

[0136] In embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 47. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO: 47. In embodiments, the light chain variable domain comprises the sequence of SEQ ID NO: 48. In embodiments, the light chain variable domain is the sequence of SEQ ID NO: 48. In embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 47 and the light chain variable domain comprises the sequence of SEQ ID NO: 48. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO: 47 and the light chain variable domain is the sequence of SEQ ID NO: 48. In embodiments, the anti-RYK antibody antibody is referred to herein as 11-E9.

[0137] In another aspect, an anti-RYK antibody is provided comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 49, CDR H2 as set forth in SEQ ID NO: 50, and CDR H3 as set forth in SEQ ID NO: 51, and the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 52, CDR L2 as set forth in SEQ ID NO: 53, and CDR L3 as set forth in SEQ ID NO: 54.

[0138] In embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 63. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO: 63. In embodiments, the light chain variable domain comprises the sequence of SEQ ID NO: 64. In embodiments, the light chain variable domain is the sequence of SEQ ID NO: 64. In embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 63 and the light chain variable domain comprises the sequence of SEQ ID NO: 64. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO: 63 and the light chain variable domain is the sequence of SEQ ID NO: 64. In embodiments, the anti-RYK antibody antibody is referred to herein as 3-C12.

[0139] In embodiments, the anti-RYK antibody is a chimeric antibody. In embodiments, the anti-RYK antibody is a Fab' fragment. In embodiments, the anti-RYK antibody is an IgG. In embodiments, the light chain variable domain and the heavy chain variable domain form part of an scFv.

[0140] In embodiments, the anti-RYK antibody is capable of binding to a RYK protein. In embodiments, the anti-RYK antibody binds to an extracellular RYK domain. In embodiments, the anti-RYK antibody binds to a human extracellular RYK domain. In embodiments, the anti-RYK antibody binds to an extracellular RYK domain comprising an amino acid sequence of SEQ ID NO: 129. In embodiments, the anti-RYK antibody binds to an extracellular RYK domain that is an amino acid sequence of SEQ ID NO: 129. In embodiments, the anti-RYK antibody binds to an amino acid sequence corresponding to amino acid residues 48-57 of SEQ ID NO: 129. In embodiments, the anti-RYK antibody is bound to a RYK protein. In embodiments, the RYK protein is a human RYK protein. In embodiments, the RYK protein comprises a sequence of SEQ ID NO: 130. In embodiments, the RYK protein is a sequence of SEQ ID NO: 130. In embodiments, the RYK protein is a sequence of SEQ ID NO: 129. In embodiments, the RYK protein does not bind to a mouse RYK protein. In embodiments, the anti-RYK antibody does not bind to a RYK protein comprising an amino acid sequence corresponding to amino acid residues 32-41 of SEQ ID NO: 131. In embodiments, the anti-RYK antibody does not bind to a RYK protein comprising the sequence of SEQ ID NO: 131. In embodiments, the anti-RYK antibody does not bind to the RYK protein of SEQ ID NO: 131. In embodiments, the anti-RYK antibody does not bind to the mouse extracellular RYK domain. In embodiments, the RYK protein forms part of a cell. In some embodiments, the RYK protein is expressed on the surface of a cell.

[0141] In another aspect, an anti-RYK antibody is provided, which binds to the same epitope as an antibody comprising a heavy chain variable domain comprising CDR H1 set forth in SEQ ID NO:1, CDR H2 set forth in SEQ ID NO:2, and CDR H3 set forth in SEQ ID NO:3, and a light chain variable domain comprising CDR L1 set forth in SEQ ID NO:4, CDR L2 set forth in SEQ ID NO:5, and CDR L3 set forth in SEQ ID NO:6.

[0142] In another aspect, an anti-RYK antibody is provided, which binds to the same epitope as an antibody comprising a heavy chain variable domain comprising CDR H1 set forth in SEQ ID NO: 17, CDR H2 set forth in SEQ ID NO: 18, and CDR H3 set forth in SEQ ID NO: 19, and a light chain variable domain comprising CDR L1 set forth in SEQ ID NO: 20, CDR L2 set forth in SEQ ID NO: 21, and CDR L3 set forth in SEQ ID NO: 22.

[0143] In another aspect, an anti-RYK antibody is provided, which binds to the same epitope as an antibody comprising a heavy chain variable domain comprising CDR H1 set forth in SEQ ID NO: 33, CDR H2 set forth in SEQ ID NO: 34, and CDR H3 set forth in SEQ ID NO: 35, and a light chain variable domain comprising CDR L1 set forth in SEQ ID NO: 36, CDR L2 set forth in SEQ ID NO: 37, and CDR L3 set forth in SEQ ID NO: 38.

[0144] In another aspect, an anti-RYK antibody is provided, which binds to the same epitope as an antibody comprising a heavy chain variable domain comprising CDR H1 set forth in SEQ ID NO:49, CDR H2 set forth in SEQ ID NO:50, and CDR H3 set forth in SEQ ID NO:51, and a light chain variable domain comprising CDR L1 set forth in SEQ ID NO:52, CDR L2 set forth in SEQ ID NO:53, and CDR L3 set forth in SEQ ID NO:54.

[0145] In embodiments, the anti-RYK antibody is conjugated to a therapeutic or diagnostic moiety. In embodiments, the anti-RYK antibody is conjugated to a therapeutic moiety. In embodiments, the anti-RYK antibody is conjugated to a diagnostic moiety.

[0146] Nucleic acid composition The compositions provided herein (including embodiments thereof) include nucleic acid molecules encoding the anti-RYK antibodies or portions thereof provided herein. Antibodies encoded by the isolated nucleic acids provided herein are described in detail throughout this application (including in the Description and Examples sections above). Thus, in one aspect, isolated nucleic acids encoding the anti-RYK antibodies provided herein (including embodiments thereof) are provided.

[0147] antibody composition The light and heavy chains of the antibodies provided herein can form part of recombinant proteins (e.g., chimeric antigen receptors (CARs) or bispecific antibodies (BiTes)) using, inter alia, conventional methods well known in the art. Through the recruitment of effector cells, the anti-RYK antibodies provided herein induce cell killing of RYK-expressing cells and are useful for therapeutic purposes by themselves or when used in the context of a CAR or BiTe.

[0148] cell composition Compositions provided herein (including embodiments thereof) include cell compositions comprising an anti-RYK antibody provided herein. Thus, in one aspect, a cell is provided that comprises an anti-RYK antibody provided herein (including embodiments thereof) or a nucleic acid provided herein (including embodiments thereof).

[0149] Pharmaceutical Compositions Compositions provided herein (including embodiments thereof) include pharmaceutical compositions comprising the anti-RYK antibodies provided herein. Thus, in one aspect, a pharmaceutical composition is provided, comprising a therapeutically effective amount of an antibody provided herein (including embodiments thereof) and a pharma- ceutical acceptable excipient.

[0150] method In one aspect, a method of generating an antibody capable of binding to a RYK protein is provided, the method comprising immunizing a mammal with a peptide comprising the sequence of SEQ ID NO:129.

[0151] In another aspect, a method for detecting a RYK-expressing cell is provided, the method comprising: (i) contacting a RYK-expressing cell with an antibody (including embodiments thereof) provided herein; and (ii) detecting binding of the antibody to the RYK protein expressed by the cell.

[0152] In embodiments, the antibody is conjugated to a detectable moiety. In embodiments, the biological sample is whole blood, a blood fraction or blood product, tissue, or cultured cells. In embodiments, the biological sample is whole blood. In embodiments, the biological sample is a blood fraction or blood product. In embodiments, the biological sample is a blood fraction. In embodiments, the biological sample is a blood product. In embodiments, the biological sample is a tissue. In embodiments, the biological sample is cultured cells.

[0153] In embodiments, the RYK expressing cell is a cancer cell. In embodiments, the cancer cell is a bladder cancer cell, a brain cancer cell, a breast cancer cell, a chronic myelogenous leukemia (CML) cell, a colon cancer cell, a Ewing's sarcoma cell, a lung cancer cell, a mantle cell lymphoma cell, an ovarian cancer cell, a pancreatic cancer cell, a skin cancer cell, or a melanoma cell. In embodiments, the cancer cell is a bladder cancer cell. In embodiments, the cancer cell is a brain cancer cell. In embodiments, the cancer cell is a breast cancer cell. In embodiments, the cancer cell is a chronic myelogenous leukemia (CML) cell. In embodiments, the cancer cell is a colon cancer cell. In embodiments, the cancer cell is a Ewing's sarcoma cell. In embodiments, the cancer cell is a lung cancer cell. In embodiments, the cancer cell is a mantle cell lymphoma cell. In embodiments, the cancer cell is an ovarian cancer cell. In embodiments, the cancer cell is a pancreatic cancer cell. In embodiments, the cancer cell is a skin cancer cell. In embodiments, the cancer is a melanoma cell.

[0154] In another aspect, a method of treating cancer in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an anti-RYK antibody provided herein (including embodiments thereof).

[0155] In embodiments, the cancer is bladder cancer, brain cancer, breast cancer, chronic myelogenous leukemia (CML), colon cancer, Ewing's sarcoma, lung cancer, mantle cell lymphoma, ovarian cancer, pancreatic cancer, skin cancer, or melanoma. In embodiments, the cancer is bladder cancer. In embodiments, the cancer is brain cancer. In embodiments, the cancer is chronic myelogenous leukemia (CML). In embodiments, the cancer is colon cancer. In embodiments, the cancer is Ewing's sarcoma. In embodiments, the cancer is lung cancer. In embodiments, the cancer is mantle cell lymphoma. In embodiments, the cancer is ovarian cancer. In embodiments, the cancer is pancreatic cancer. In embodiments, the cancer is skin cancer. In embodiments, the cancer is melanoma.

[0156] In another aspect, a method for identifying an anti-RYK antibody is provided, the method comprising: (i) contacting an antibody with a first RYK polypeptide comprising an amino acid sequence corresponding to amino acid residues 48-57 of SEQ ID NO: 129; (ii) detecting an antibody that binds to the first RYK polypeptide; (iii) contacting the antibody with a second RYK polypeptide that does not comprise an amino acid sequence corresponding to amino acid residues 48-57 of SEQ ID NO: 129; and (iv) detecting an antibody that does not bind to the second RYK polypeptide, thereby identifying an anti-RYK antibody.

[0157] In embodiments, the antibody is a chimeric antibody. In embodiments, the antibody is a Fab' fragment. In embodiments, the antibody is a single chain antibody.

[0158] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes thereto will be suggested to those skilled in the art and are within the spirit and scope of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. EXAMPLES

[0159] Example 1: Applicants have generated mAbs that are highly specific for the extracellular domain of human Ryk, but not the highly homologous mouse Ryk. These mAbs apparently bind to epitopes found in the amino terminus of the mature human Ryk protein. Moreover, these mAbs have very high affinity for Ryk and, surprisingly, do not appear to react with normal human postpartum tissues. For example, Applicants have found that these mAbs do not bind to hematopoietic cells in normal human umbilical cord blood or postpartum blood, tonsils, spleen, or bone marrow. However, surprisingly, these mAbs react with cancer cell lines derived from a variety of different solid tumors or hematological cancers. Because these mAbs do not react with all cancers derived from the same tissue type, we speculate that these mAbs do not react with all postpartum cells derived from that tissue type. Surprisingly, Applicants have found that the mAbs react very strongly with primary tumor cells from patients with aggressive cancers, such as triple-negative metastatic breast cancer cells. Furthermore, applicants have found that these mAbs can react strongly with cancer cells in malignant pleural or ascites effusions and with circulating tumor cells in the blood. Preliminary studies also indicate that these mAbs react most strongly with cancer cells with cancer stem cell or mesenchymal characteristics, which are associated with high metastatic pathways and poor prognosis.

[0160] These antibodies can be used to bind to cancer cells expressing Ryk. Since Ryk is apparently low or negligible in normal postpartum tissues, these mAbs can be targeted to destroy Ryk-expressing cancer cells via antibody-dependent cellular cytotoxicity (ADCC) or by inhibiting an as yet undefined function of Ryk that apparently promotes cancer cell migration, proliferation, and / or cancer stem cell renewal. Furthermore, these mAbs can be conjugated with toxins, allowing specific delivery of anti-Ryk mAb-bound toxins to tumor cells expressing Ryk. Because of their high affinity, these mAbs can be "humanized" to assimilate human antibodies and / or used to generate single-chain Fv (scFv) domains that bind Ryk. This can be used to create bispecific antibodies that target Ryk and another molecule (e.g., CD3) that may enable cellular immune activation of anti-tumor immune responses against tumor cells expressing Ryk. Furthermore, these anti-Ryk scFvs can be used to generate chimeric antigen receptors (CARs). Expression of anti-Ryk CAR by T cells or NK cells can enable anti-Ryk CAR T / NK cell therapy against Ryk-expressing cancers.

[0161] As described herein, applicants have generated anti-Ryk antibodies and measured their affinity to the antigen. Furthermore, the Ryk binding site has been sequenced and the amino acid sequences of the heavy and light chains of two disclosed anti-Ryk mAbs, 2-D11 and 7-H10 (coming soon), have been determined. The antibodies have been tested on various cancer cell lines, including triple-negative breast cancer xenografts. Furthermore, the 2-D11 antibody has been used to stain cancer cell lines derived from various cancer tissue types, including primary tumor cells from xenografts (e.g., triple-negative metastatic breast cancer) derived from breast cancer patients.

[0162] Example 3: In a study using anti-RYK antiserum, data suggest that RYK is expressed in chronic lymphocytic leukemia (CLL) cells, so an anti-RYK mAb was generated that binds to leukemia cells in patients with CLL. However, when a high affinity mAb specific for RYK was generated, applicant was surprised to find that the anti-RYK antiserum apparently had a spurious binding activity to CLL cells, which in fact do not express RYK and do not react with the disclosed anti-Ryk mAb described herein. Applicant also found that RYK is expressed in tumor cells of many different human cancers, but not in all non-cancer cells tested.

[0163] Example 3: The extracellular protein sequence of human Ryk is highly homologous to that of mouse RYK (Figure 1). An anti-human RYK mAb was generated that binds to the mature extracellular protein domain of human RYK, consisting of the amino acids shown in Figure 2, but does not bind to the highly homologous mouse RYK, consisting of the amino acids shown in Figure 3.

[0164] The anti-human RYK mAbs, 2-D11 and 7-H10, appear to be specific for an amino-terminal epitope (residues 46-57) of human Ryk that is distinct from mouse Ryk (Figure 3). These mAbs also bind to mutant forms of Ryk that contain amino acid substitutions in human Ryk at positions distinct from the highly homologous mouse Ryk, assimilating mouse Ryk at the site of the substitution, as shown by immunoblot analysis of each of the human or mouse recombinant extracellular proteins or various mutant forms of human Ryk, with the amino acid sequence shown in Figure 3 (Figure 4). To this end, binding of 2-D11, 7-H10, or sheep anti-RYK mAbs was assessed using keyed recombinant extracellular RYK protein. Each recombinant protein was transferred to a nylon membrane and probed with 2-D11, 7-H10, or sheep anti-RYK and detected with anti-mouse IgG or donkey anti-sheep antibodies conjugated with horseradish peroxidase. Black dots represent positive signals of antibody binding. As shown in Figure 4, sheep anti-Ryk reacted with either human or mouse Ryk, whereas 2-D11 or 7-H10 reacted with human but not mouse Ryk, respectively. Furthermore, 2-D11 or 7-H10 reacted with each of the mutant forms of human Ryk that have amino acid substitution(s) at specific sites where the human Ryk sequence differs from the mouse Ryk sequence. The amino acid sequences of each of these recombinant proteins are provided in Figure 3.

[0165] The above-mentioned mAb 2-D11 has a heavy chain variable region sequence shown in Figure 5A, and the light chain variable region sequence of 2-D11 is provided in Figure 5B. The above-mentioned mAb 7-H10 has a heavy chain variable region sequence shown in Figure 6A, and the light chain variable region sequence of 2-D11 is provided in Figure 6B. Also shown in these figures are the mouse germline heavy chain variable region genes (Figures 5A, 6A) or light chain variable region genes (Figures 5B, 6B) that have the closest sequence homology to 2-D11 or 7-H10, respectively.

[0166] KinExA binding data for 2-D11 or 7-H10 for human Ryk is provided in Figure 7. The calculated Kd of 2-D11 for human Ryk is 512.9 pM and the calculated Kd of 7-H10 for human Ryk is 10.56 nM.

[0167] 2-D11 mAb was conjugated with a fluorescent dye (Alexa647) and the conjugated mAb was used to stain established tumor cell lines. Open histograms show the fluorescence intensity of cell lines stained with a control fluorescent dye-conjugated mAb of irrelevant specificity (Figure 8). Shaded histograms show the fluorescence of cells stained with 2-D11 fluorescent dye-conjugated mAb. Representative staining of tumor cell lines is provided, showing "++" staining (for BT549), "+" staining (for HT29), or no staining or "neg" (for SK-MES-1) compared to cells treated with a fluorescent dye-conjugated "control mAb" of irrelevant specificity that does not bind to these cells. These control mAb-stained cells had the same fluorescence as unstained cells.

[0168] Table 1 provides flow cytometry data for various cancer cell lines stained with fluorochrome-conjugated 2-D11 shown in FIG. 8. The name of each cell line is listed in the first column, and the tissue of origin is listed in the second column. Binding of 2-D11 mAb to human RYK was assessed by flow cytometry staining and analysis by staining with 5 μg / ml of 2-D11 anti-human RYK-Alexa647 conjugated mAb or an equivalent amount of isotype-matched control mAb for 20 minutes on ice, washing, and analysis. The median fluorescence intensity (MFI) of 2-D11 stained cells is listed in the middle column, and the MFI of isotype control stained cells is listed in the adjacent column. Cell lines were scored as "++", "+", or neg (not marked with "+") lines based on the ratio of the MFI of stained cells to the median fluorescence intensity (MFI) of isotype control stained cells (MFIR).

[0169] FIG. 9 provides flow cytometry data for fluorochrome-conjugated 2-D11 staining of lymphocytes found in adult blood, umbilical cord blood (N=2), tonsils (N=2), or spleen, as indicated at the top of each column. Open histograms represent cells stained with a fluorochrome-conjugated mAb of irrelevant specificity (control staining) instead of 2-D11. Shaded histograms are of cells stained with 2-D11. Cells were also stained with mAbs conjugated to different colored fluorochromes that were specific for CD19 or CD3. The top row provides data for gated CD19+ B cells, the middle row provides data for gated CD3+ T cells, and the bottom row provides data for cells lacking binding to mAbs specific for CD19 or CD3 (NK cells). As can be seen from these data, 2-D11 does not react with normal human lymphoid cells.

[0170] Figure 10 shows representative staining of 2-D11 of primary human breast cancer cells. The cells stained in Figure 10 were dissociated single cells prepared from patient-derived xenografts (PDXs), which were generated by transplanting triple-negative (ER / PR-, HER2-) breast adenocarcinoma tissue removed from a patient with metastatic breast cancer into an immune-deficient mouse model.

[0171] table Table 1. Flow cytometry staining analysis of cancer cell lines using anti-human RYK mAb. The name of each cell line is listed in the first column and the tissue of origin is listed in the second column. Binding of 2-D11 mAb to human RYK was assessed by flow cytometry staining and analysis by staining with 5ug / ml of 2-D11 anti-human RYK-Alexa647 conjugated mAb or an equivalent amount of isotype-matched control mAb for 20 minutes on ice, washing, and analysis. The median fluorescence intensity (MFI) of 2-D11 stained cells is listed in the middle column and the MFI of isotype control stained cells is listed in the adjacent column. Cell lines were scored as "++", "+", or neg (not marked with "+") lines based on the ratio of the MFI of stained cells to the median fluorescence intensity (MFI) of isotype control stained cells (MFIR). [Table 1-1] [Table 1-2] [Table 2]

[0172] References 1.Lu, W., Yamamoto, V., Ortega, B. & Baltimore, D. Mammalian Ryk is a Wnt coreceptor required for stimulation of neurite outgrowth.Cell 119,97-108(2004). 2.Keeble, TR, et al.The Wnt receptor Ryk is required for Wnt5a-mediated axon guidance on the contralateral side of the corpus callosum.J Neurosci 26,5840-5848(2006). 3.Chakravadhanula,M.,et al.Wnt pathway in atypical teratoid rhabdoid tumors.Neuro Oncol 17,526-535(2015). 4.Anastas,J.N.Functional Crosstalk Between WNT Signaling and Tyrosine Kinase Signaling in Cancer.Semin Oncol 42,820-831(2015). 5.Roy,J.P.,Halford,M.M.& Stacker,S.A.The biochemistry,signalling and disease relevance of RYK and other WNT-binding receptor tyrosine kinases.Growth Factors 36,15-40(2018). 6.Mikels,A.J.& Nusse,R.Purified Wnt5a protein activates or inhibits beta-catenin-TCF signaling depending on receptor context.PLoS Biol 4,e115(2006). 7.Wu,C.H.& Nusse,R.Ligand receptor interactions in the Wnt signaling pathway in Drosophila.J Biol Chem 277,41762-41769(2002). 8.Lyu,J.,Yamamoto,V.& Lu,W.Cleavage of the Wnt receptor Ryk regulates neuronal differentiation during cortical neurogenesis.Dev Cell 15,773-780(2008). 9.Halford,M.M.,et al.Ryk-deficient mice exhibit craniofacial defects associated with perturbed Eph receptor crosstalk.Nat Genet 25,414-418(2000). 10.Blakely,B.D.,et al.Ryk,a receptor regulating Wnt5a-mediated neurogenesis and axon morphogenesis of ventral midbrain dopaminergic neurons.Stem Cells Dev 22,2132-2144(2013). 11.Kugathasan,K.,et al.Deficiency of the Wnt receptor Ryk causes multiple cardiac and outflow tract defects.Growth Factors 36,58-68(2018). 12.Famili,F.,et al.The non-canonical Wnt receptor Ryk regulates hematopoietic stem cell repopulation in part by controlling proliferation and apoptosis.Cell Death Dis 7,e2479(2016). 13.Adamo,A.,et al.RYK promotes the stemness of glioblastoma cells via the WNT / beta-catenin pathway.Oncotarget 8,13476-13487(2017). 14.Borcherding,N.,et al.Paracrine WNT5A Signaling Inhibits Expansion of Tumor-Initiating Cells.Cancer Res 75,1972-1982(2015). 15.Kessenbrock, K., et al.Diverse regulation of mammary epithelial growth and branching morphogenesis through noncanonical Wnt signaling.Proc Natl Acad Sci USA 114,3121-3126(2017). 16.Fu, Y., Chen, Y., Huang, J., Cai, Z. & Wang, Y. RYK, a receptor of noncanonical Wnt ligand Wnt5a, is positively correlated with gastric cancer tumorigenesis and potential of liver metastasis.Am J Physiol Gastrointest Liver Physiol 318,G352-G360(2020).

[0173] Unofficial sequence listing SEQ ID NO: 1 2-D11 CDR H1 GFSLNDYG SEQ ID NO:2 2-D11 CDR H2 IWGDGVT SEQ ID NO:3 2-D11 CDR H3 QGSGVWFAH SEQ ID NO: 4 2-D11 CDR L1 QTIVHSNGNTY SEQ ID NO:5 2-D11 CDR L2 KVS SEQ ID NO:6 2-D11 CDR L3 FQGSHVPYT SEQ ID NO:7 2-D11 FR H1 QVQLKESGPGLVAPSQSLSITCSVS SEQ ID NO:8 2-D11 FR H2 VNWVRQPPGKDLEWLGM SEQ ID NO: 9 2-D11 FR H3 EYNSTLKSRLSISKDNSKSQVFLKMNNLQTEDTARYYCVR SEQ ID NO: 10 2-D11 FR H4 WGQGTLVSVSS SEQ ID NO: 11 2-D11 FR L1 DVLVTQTPLSLPVSLGDQASISCRSS SEQ ID NO:12 2-D11 FR L2 LEWYLQKPGQSPKLLIY SEQ ID NO:13 2-D11 FR L3 NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYC SEQ ID NO: 14 2-D11 FR L4 FGGGTKLEIK SEQ ID NO: 15 2-D11 VH QVQLKESGPGLVAPSQSLSITCSVGFSLNDYGVNWVRQPPGKDLEWLGMIWGDGVTEYNSTLKSRLSISKDNSKSQVFLKMNNLQTEDTARYYCVRQGSGVWFAHWGQGTLVSVSS SEQ ID NO: 16 2-D11 VL DVLVTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPYTFGGGTKLEIK SEQ ID NO: 17 7-H10 CDR H1 GYIFTNYD SEQ ID NO:18 7-H10 CDR H2 IFPGDDST SEQ ID NO: 19 7-H10 CDR H3 YHYYGSSLGWSFDV SEQ ID NO: 20 7-H10 CDR L1 SRISIN SEQ ID NO: 21 7-H10 CDR L2 GTS SEQ ID NO:22 7-H10 CDR L3 QQWSSYPYT SEQ ID NO:23 7-H10 FR H1 QVQLQQSGAELAKPGTSVKLSCKAS SEQ ID NO:24 7-H10 FR H2 INWVRQRPEQGLEWIGW SEQ ID NO:25 7-H10 FR H3 KYNEKFEGKAALTTDKSSNTAYIQLSRLTSGDSAVYFCTR SEQ ID NO:26 7-H10 FR H4 WGAGTSVTVSS SEQ ID NO:27 7-H10 FR L1 EIVLTQSPALMAASPGEKVTITCSVS SEQ ID NO:28 7-H10 FR L2 LHWYQQKSETSPKTWIY SEQ ID NO:29 7-H10 FR L3 NLASGVPSRFSGSGSGTSYSLTISNMEAEDAATYYC SEQ ID NO:30 7-H10 FR L4 FGGGTKVEIK SEQ ID NO:31 7-H10 VH QVQLQQSGAELAKPGTSVKLSCKASGYIFTNYDINWVRQRPEQGLEWIGWIFPGDDSTKYNEKFEGKAALTTDKSSNTAYIQLSRLTSGDSAVYFCTRYHYYGSSLGWSFDVWGAGTSVTVSS SEQ ID NO:32 7-H10 VL EIVLTQSPALMAASPGEKVTITCSSVSSRISSINLHWYQQKSETSPKTWIYGTSNLASGVPSRFSGSGSGTSYSLTISNMEAEDAATYYCQQWSSYPYTFGGGTKVEIK SEQ ID NO:33 11-E9 CDR H1 GFSLNGYG SEQ ID NO:34 11-E9 CDR H2 IWGDGIT SEQ ID NO:35 11-E9 CDR H3 QGSGVWFAY SEQ ID NO:36 11-E9 CDR L1 QTIVHSNGNTY SEQ ID NO:37 11-E9 CDR L2 KVS SEQ ID NO:38 11-E9 CDR L3 FQGSHVPYT SEQ ID NO:39 11-E9 FR H1 QVQLKESGPGLVAPSQSLSITCTVS SEQ ID NO: 40 11-E9 FR H2 VNWVRQPPGKDLEWLGM SEQ ID NO: 41 11-E9 FR H3 EFNSALKSRLSISKDNSKSQVFLKMNSLQTEDTARYYCVR SEQ ID NO:42 11-E9 FR H4 WGQGTLS SEQ ID NO: 43 11-E9 FR L1 DVLVTQTPLSLPVSLGDQASISCRSS SEQ ID NO:44 11-E9 FR L2 LEWYLQKPGQSPKLLIY SEQ ID NO:45 11-E9 FR L3 NRFCGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC SEQ ID NO:46 11-E9 FR L4 FGGGTKLEIK SEQ ID NO: 47 11-E9 VH QVQLKESGPGLVAPSQSLSITCTVSGFSLNGYGVNWVRQPPGKDLEWLGMIWGDGITEFNSALKSRLSISKDNSKSQVFLKMNSLQTEDTARYYCVRQGSGVWFAYWGQGTLS SEQ ID NO: 48 11-E9 VL DVLVTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFCGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK SEQ ID NO: 49 3-C12 CDR H1 GFSLNGYG SEQ ID NO:50 3-C12 CDR H2 IWGDGIT SEQ ID NO:51 3-C12 CDR H3 QGSGVWFAY SEQ ID NO:52 3-C12 CDR L1 QTIVHSNGNTY SEQ ID NO:53 3-C12 CDR L2 KVS SEQ ID NO:54 3-C12 CDR L3 FQGSHVPYT SEQ ID NO:55 3-C12 FR H1 QVQLKESGPGLVAPSQSLSITCTVS SEQ ID NO:56 3-C12 FR H2 VNWVRQPPGKDLEWLGM SEQ ID NO:57 3-C12 FR H3 EFNSALKSRLSISKDNSKSQVFLKMNSLQTEDTARYYCVR SEQ ID NO:58 3-C12 FR H4 WGQGTLVS SEQ ID NO:59 3-C12 FR L1 DVLVTQTPLSLPVSLGDQASISCRSS SEQ ID NO:60 3-C12 FR L2 LEWYLQKPGQSPKLLIY SEQ ID NO:61 3-C12 FR L3 NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC SEQ ID NO:62 3-C12 FR L4 FGGGTKLEIK SEQ ID NO:63 3-C12 VH QVQLKESGPGLVAPSQSLSITCTVSGFSLNGYGVNWVRQPPGKDLEWLGMIWGDGITEFNSALKSRLSISKDNSKSQVFLKMNSLQTEDTARYYCVRQGSGVWFAYWGQGTLVS SEQ ID NO:64 3-C12 VL DVLVTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK SEQ ID NO: 65 2-D11 CDR H1 nucleotide GGGTTCTCATTAAACGACTATGGT SEQ ID NO:66 2-D11 CDR H2 nucleotide ATTTGGGGTGATGGAGTCACA SEQ ID NO:67 2-D11 CDR H3 nucleotide GTCAGACAGGGGTCTGGTGTCTGGTTTGCTCAC SEQ ID NO:68 2-D11 CDR L1 nucleotide CAGACCATTGTACATAGTAATGGAAACACGTAT SEQ ID NO:69 2-D11 CDR L2 nucleotide AAAGTTTCC SEQ ID NO: 70 2-D11 CDR L3 nucleotide TTTCAAGGTTCACATGTTCCGTACACG SEQ ID NO: 71 2-D11 FR H1 nucleotide CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGTTCCGTCTCA SEQ ID NO:72 2-D11 FR H2 nucleotide GTAAATTGGGTTCGCCAGCCTCCAGGAAAGGATCTGGAGTGGCTGGGAATG SEQ ID NO:73 2-D11 FR H3 nucleotide GAGTATAATTCAACTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAAATGAACAATCTGCAAACTGAAGACACAGCCAGGTACTACTGT SEQ ID NO:74 2-D11 FR H4 nucleotide TGGGGCCAAGGGACTCTGGTCAGTGTCTCTTCA SEQ ID NO: 75 2-D11 FR L1 nucleotide GATGTTTTGGTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGT SEQ ID NO:76 2-D11 FR L2 nucleotide TTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTAATCTAC SEQ ID NO:77 2-D11 FR L3 nucleotide AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTACTGC SEQ ID NO:78 2-D11 FR L4 nucleotide TTCGGAGGGGGGACCAAGCTGGAAAATAAAA SEQ ID NO:79 2-D11 VH nucleotide CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGTTCCGTCTCAGGGTTCTCATTAAACGACTATGGTGTAAATTGGGTTCGCCAGGCCTCCAGGAAAGGATCTGGAGTGGCTGGGAATGATTTGGGGTGATGGAGTCACAGAGT ATAATTCAACTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAAATGAACAATCTGCAAACTGAAGACACAGCCAGGTACTACTGTGTCAGACAGGGGTCTGGTGTCTGGTTTGCTCACTGGGGCCAAGGGACTCTGGTCAGTGTCTCTTCA SEQ ID NO: 80 2-D11 VL nucleotide GATGTTTTGGTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGACCATTGTACATAGTAATGGAAACACGTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTAATCTACAAAGTT TCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA SEQ ID NO: 81 7-H10 CDR H1 nucleotide GGCTACATCTTCACAAACTATGAT SEQ ID NO:82 7-H10 CDR H2 nucleotides ATTTTTCCTGGAGATGATAGTACT SEQ ID NO:83 7-H10 CDR H3 nucleotides ACAAGATATCATTACTACGGTAGTTCCTTGGGGTGGTCCTTCGATGTC SEQ ID NO:84 7-H10 CDR L1 nucleotide TCAAGAATAAGTTCCATTAAC SEQ ID NO: 85 7-H10 CDR L2 nucleotide GGCACATCC SEQ ID NO:86 7-H10 CDR L3 nucleotide CAACAGTGGAGTAGTTATCCGTACACG SEQ ID NO:87 7-H10 FR H1 nucleotide CAGGTTCAGCTGCAGCAGTCTGGAGCTGAACTGGCAAAGCCTGGGACTTCAGTGAAATTGTCCTGCAAGGCTTCT SEQ ID NO:88 7-H10 FR H2 nucleotide ATAAACTGGGTGAGGCAGAGGCCTGAACAGGGACTTGAGTGGATTGGATGG SEQ ID NO:89 7-H10 FR H3 nucleotide AAGTACAATGAGAAATTCGAGGGCAAGGCCGCACTGACTACAGACAAGTCCTCCAACACAGCCTACATACAACTCAGCAGACTGACATCTGGGGACTCAGCTGTCTATTTCTGT SEQ ID NO: 90 7-H10 FR H4 nucleotide TGGGGCGCAGGGACCTCGGTCACCGTCTCCTCA SEQ ID NO: 91 7-H10 FR L1 nucleotide GAAATTGTGCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGAGAAGGTCACCATCACCTGCAGTGTCAGT SEQ ID NO:92 7-H10 FR L2 nucleotide TTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAAACCTGGATTTAT SEQ ID NO:93 7-H10 FR L3 nucleotide AACCTGGCTTCTGGAGTCCCTAGTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTCTCACAATCAGCAACATGGAGGCTGAAGATGCTGCCACTTATTACTGT SEQ ID NO:94 7-H10 FR L4 nucleotide TTCGGAGGGGGGACCAAAGGTGGAAAATAAAA SEQ ID NO: 95 7-H10 VH nucleotides CAGGTTCAGCTGCAGCAGTCTGGAGCTGAACTGGCAAAGCCTGGGACTTCAGTGAAATTGTCCTGCAAGGCTTCTGGCTACATCTTCACAAACTATGATATAAACTGGGTGAGGCAGAGGCCTGAACAGGGACTTGAGTGGATTGGATGGATTTTTCCTGGAGATGATAGTACTAAGTACAATG AGAAATTCGAGGGCAAGGCCGCACTGACTACAGACAAGTCCTCCAACACAGCCTACATACAACTCAGCAGACTGACATCTGGGGACTCAGCTGTCTATTTCTGTACAAGATATCATTACTACGGTAGTTCCTTGGGGTGGTCCTTCGATGTCTGGGGCGCAGGGACCTCGGTCACCGTCTCCTCA SEQ ID NO:96 7-H10 VL nucleotide GAAATTGTGCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGAGAAGGTCACCATCACCTGCAGTGTCAGTTCAAGAATAAGTTCCATTAACTTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAAACCTGGATTTATGGCACATCCAAC CTGGCTTCTGGAGTCCCTAGTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTCTCACAATCAGCAACATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTGGAGTAGTTATCCGTACACGTTCGGAGGGGGGACCAAGGTGGAAATAAAA SEQ ID NO:97 11-E9 CDR H1 nucleotide GGGTTCTCATTAAACGGCTATGGT SEQ ID NO: 98: 11-E9 CDR H2 nucleotide ATCTGGGGTGATGGAATCACA SEQ ID NO:99 11-E9 CDR H3 nucleotide GTCAGACAGGGGTCTGGTGTCTGGTTTGCTTAC SEQ ID NO:100 11-E9 CDR L1 nucleotide CAGACCATTGTACATAGTAATGGAAACACCTAT SEQ ID NO: 101 11-E9 CDR L2 nucleotide AAAGTTTCC SEQ ID NO:102 11-E9 CDR L3 nucleotide TTTCAAGGTTCACATGTTCCGTACACG SEQ ID NO:103 11-E9 FR H1 nucleotide CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGTACCGTCTCA SEQ ID NO:104 11-E9 FR H2 nucleotide GTAAACTGGGTTCGCCAGCCTCCAGGAAAGGATCTGGAGTGGCTGGGAATG SEQ ID NO: 105 11-E9 FR H3 nucleotide GAGTTTAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGAAGACACAGCCAGGTACTACTGT SEQ ID NO:106 11-E9 FR H4 nucleotide TGGGGCCAAGGGACTCTGTCA SEQ ID NO:107 11-E9 FR L1 nucleotide GATGTTTTGGTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGT SEQ ID NO:108 11-E9 FR L2 nucleotide TTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCTTGATTTAC SEQ ID NO:109 11-E9 FR L3 nucleotide AACCGATTTTGTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGC SEQ ID NO:110 11-E9 FR L4 nucleotide TTCGGAGGGGGGACCAAGCTGGAAAATAAAA SEQ ID NO:111 11-E9 VH nucleotide CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGTACCGTCTCAGGGTTCTCATTAAACGGCTATGGTGTAAACTGGGTTCGCCAGCCTCCAGGAAAGGATCTGGAGTGGCTGGGAATGATCTGGGGTGATGGAATCA CAGAGTTTAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGAAGACACAGCCAGGTACTACTGTGTCAGACAGGGGTCTGGTGTCTGGTTTGCTTACTGGGGCCAAGGGACTCTGTCA SEQ ID NO:112 11-E9 VL nucleotide GATGTTTTGGTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGACCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCTTGATTTACAAAGTT TCCAACCGATTTTGTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA SEQ ID NO:113 3-C12 CDR H1 nucleotide GGGTTCTCATTAAACGGCTATGGT SEQ ID NO:114 3-C12 CDR H2 nucleotide ATCTGGGGTGATGGAATCACA SEQ ID NO: 115 3-C12 CDR H3 nucleotide CAGGGGTCTGGTGTCTGGTTTGCTTAC SEQ ID NO:116 3-C12 CDR L1 nucleotide CAGACCATTGTACATAGTAATGGAAACACCTAT SEQ ID NO:117 3-C12 CDR L2 nucleotide AAAGTTTCC SEQ ID NO:118 3-C12 CDR L3 nucleotide TTTCAAGGTTCACATGTTCCGTACACG SEQ ID NO:119 3-C12 FR H1 nucleotide CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGTACCGTCTCA SEQ ID NO:120 3-C12 FR H2 nucleotide GTAAACTGGGTTCGCCAGCCTCCAGGAAAGGATCTGGAGTGGCTGGGAATG SEQ ID NO:121 3-C12 FR H3 nucleotide GAGTTTAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGAAGACACAGCCAGGTACTACTGTGTCAGA SEQ ID NO:122 3-C12 FR H4 nucleotide TGGGGCCAAGGGACTCTGGTCAGT SEQ ID NO:123 3-C12 FR L1 nucleotide GATGTTTTGGTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGT SEQ ID NO:124 3-C12 FR L2 nucleotide TTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATTTAC SEQ ID NO:125 3-C12 FR L3 nucleotide AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGC SEQ ID NO:126 3-C12 FR L4 nucleotide TTCGGAGGGGGGACCAAGCTGGAAAATAAAA SEQ ID NO: 127 3-C12 VH nucleotide CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGTACCGTCTCAGGGTTCTCATTAAACGGCTATGGTGTAAACTGGGTTCGCCAGCCTCCAGGAAAGGATCTGGAGTGGCTGGGAATGATCTGGGGTGATGGAATCACA GAGTTTAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGAAGACACAGCCAGGTACTACTGTGTCAGACAGGGGTCTGGTGTCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCAGT SEQ ID NO:128 3-C12 VL nucleotide GATGTTTTGGTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGACCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATTTACAAAGTT TCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA SEQ ID NO: 129 Amino acids 1-181 of the extracellular domain of human RYK MRGAARLGRPGRSCLPGARGLRAPPPPPLLLLLALLPLLPAPGAAAAPAPRPPELQSASAGPSVSLYLSEDEVRRLIGLDAELYYVRNDLISHYALSFSLLVPSETNFLHFTWHAKSKVEYKLGFQVDNVLAMDMPQVNISVQGEVPRTLSVFRVELSCTGKVDSEVMILMQLNLTVNSSK SEQ ID NO: 130 Amino acids 48-57 of the extracellular domain of human RYK PAPRPPELQS SEQ ID NO: 131 Mouse RYK extracellular domain MRAGRGGVPGSGGLRAPPPPLLLLLLAMLPAAAPRSPALAAAPAGPSVSLYLSEDEVRRLLGLDAELYYVRNDLISHYALSFNLLVPSETNFLHFTWHAKSKVEYKL GFQVDNFVAMGMPQVNISAQGEVPRTLSVFRVELSCTGKVDSEVMILMQLNLTVNSSKNFTVLNFKRRKMCYKKLEEVKTSALDKNTSRTIYDPVHAAPTTSTRVFY SEQ ID NO: 132 Human RYK extracellular domain mrgaarlgrpgrsclpgarglrapppppllllllallpllpapgaaaapaprppelqsasagpsvslylsedevrrligldaelyyvrndlishyalsfsllvpsetnflhftwha kskveyklgfqvdnvlamdmpqvnisvqgevprtlsvfrvelsctgkvdsevmilmqlnltvnssknftvlnfkrrkmcykkleevktsaldkntsrtiydpvhaapttstrvfy SEQ ID NO: 133 Mouse IGHV2-6 QVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCAR SEQ ID NO: 134 Mouse IGKV1-117 DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP SEQ ID NO: 135 Mouse IGHV1-85 QVQLQQSGPELVKPGASVKLSCKASGYTFTSYDINWVKQRPGQGLEWIGWIYPRDGSTKYNEKFKGKATLTVDTSSSTAYMELHSLTSEDSAVYFCAR SEQ ID NO: 136 Mouse IGKV4-53 EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTSNLASGVPVRFSGSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPL

[0174] P embodiment P embodiment 1. An anti-receptor tyrosine kinase associated protein (Ryk) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO:1, CDR H2 as set forth in SEQ ID NO:2, and CDR H3 as set forth in SEQ ID NO:3, and the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO:4, CDR L2 as set forth in SEQ ID NO:5, and CDR L3 as set forth in SEQ ID NO:6. P embodiment 2. The antibody of P embodiment 1, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:21. P embodiment 3. The antibody of P embodiment 1 or 2, wherein the light chain variable domain comprises the sequence of SEQ ID NO:22. P embodiment 4. An anti-receptor tyrosine kinase associated protein (Ryk) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO:7, CDR H2 as set forth in SEQ ID NO:8, and CDR H3 as set forth in SEQ ID NO:9, and the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO:10, CDR L2 as set forth in SEQ ID NO:11, and CDR L3 as set forth in SEQ ID NO:12. P embodiment 5. The antibody of P embodiment 4, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:31. P embodiment 6. The antibody of P embodiment 4 or 5, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 32. P embodiment 7. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody described in any one of P embodiments 1 to 6.

[0175] Embodiment Embodiment 1. An anti-RYK antibody comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO:1, CDR H2 as set forth in SEQ ID NO:2, and CDR H3 as set forth in SEQ ID NO:3, and the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO:4, CDR L2 as set forth in SEQ ID NO:5, and CDR L3 as set forth in SEQ ID NO:6. Embodiment 2. An anti-RYK antibody described in embodiment 1, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 15. Embodiment 3. An anti-RYK antibody described in embodiment 1 or 2, wherein the light chain variable domain comprises the sequence of SEQ ID NO: 16. Embodiment 4. An anti-RYK antibody described in any one of embodiments 1 to 3, wherein the anti-RYK antibody has a KD of about 2 pM to about 2 nM. Embodiment 5. An anti-RYK antibody described in any one of embodiments 1 to 4, wherein the anti-RYK antibody has a KD of approximately 513 pM. Embodiment 6. An anti-RYK antibody comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 17, CDR H2 as set forth in SEQ ID NO: 18, and CDR H3 as set forth in SEQ ID NO: 19, and the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 20, CDR L2 as set forth in SEQ ID NO: 21, and CDR L3 as set forth in SEQ ID NO: 22. Embodiment 7. An anti-RYK antibody described in embodiment 6, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:31. Embodiment 8. An anti-RYK antibody described in embodiment 6 or 7, wherein the light chain variable domain comprises the sequence of SEQ ID NO: 32. Embodiment 9. An anti-RYK antibody described in any one of embodiments 6 to 8, wherein the anti-RYK antibody has a KD of approximately 6 nM to approximately 17 nM. Embodiment 10. An anti-RYK antibody described in any one of embodiments 6 to 9, wherein the anti-RYK antibody has a KD of approximately 10 nM. Embodiment 11. An anti-RYK antibody comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 33, CDR H2 as set forth in SEQ ID NO: 34, and CDR H3 as set forth in SEQ ID NO: 35, and the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 36, CDR L2 as set forth in SEQ ID NO: 37, and CDR L3 as set forth in SEQ ID NO: 38. Embodiment 12. An anti-RYK antibody described in embodiment 11, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:47. Embodiment 13. An anti-RYK antibody described in embodiment 11 or 12, wherein the light chain variable domain comprises the sequence of SEQ ID NO: 48. Embodiment 14. An anti-RYK antibody comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 49, CDR H2 as set forth in SEQ ID NO: 50, and CDR H3 as set forth in SEQ ID NO: 51, and the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 52, CDR L2 as set forth in SEQ ID NO: 53, and CDR L3 as set forth in SEQ ID NO: 54. Embodiment 15. An anti-RYK antibody described in embodiment 14, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:63. Embodiment 16. An anti-RYK antibody described in embodiment 14 or 15, wherein the light chain variable domain comprises the sequence of SEQ ID NO: 64. Embodiment 17. An anti-RYK antibody described in any one of embodiments 1 to 16, wherein the anti-RYK antibody is a chimeric antibody. Embodiment 18. An anti-RYK antibody described in any one of embodiments 1 to 17, wherein the anti-RYK antibody is a Fab' fragment. Embodiment 19. An anti-RYK antibody described in any one of embodiments 1 to 18, wherein the anti-RYK antibody is an IgG. Embodiment 20. An anti-RYK antibody described in any one of embodiments 1 to 17, wherein the light chain variable domain and the heavy chain variable domain form part of an scFv. Embodiment 21. An anti-RYK antibody described in any one of embodiments 1 to 20, wherein the anti-RYK antibody is capable of binding to RYK protein. Embodiment 22. An anti-RYK antibody described in any one of embodiments 1 to 21, wherein the anti-RYK antibody binds to the extracellular RYK domain. Embodiment 23. An anti-RYK antibody described in any one of embodiments 1 to 22, wherein the anti-RYK antibody binds to an extracellular RYK domain comprising the amino acid sequence of sequence number 129. Embodiment 24. An anti-RYK antibody described in any one of embodiments 1 to 23, wherein the anti-RYK antibody binds to an amino acid sequence corresponding to amino acid residues 48 to 57 of SEQ ID NO: 129. Embodiment 25. An anti-RYK antibody described in any one of embodiments 1 to 21, wherein the anti-RYK antibody is bound to a RYK protein. Embodiment 26. An anti-RYK antibody described in any one of embodiments 21 to 25, wherein the RYK protein is a human RYK protein. Embodiment 27. An anti-RYK antibody described in any one of embodiments 21 to 26, wherein the RYK protein comprises the sequence of sequence number 130. Embodiment 28. An anti-RYK antibody described in any one of embodiments 21 to 27, wherein the RYK protein does not bind to mouse RYK protein. Embodiment 29. An anti-RYK antibody described in any one of embodiments 21 to 28, wherein the anti-RYK antibody does not bind to an RYK protein comprising an amino acid sequence corresponding to amino acid residues 32 to 41 of SEQ ID NO: 131. Embodiment 30. An anti-RYK antibody described in any one of embodiments 25 to 28, wherein the RYK protein forms part of a cell. Embodiment 31. An anti-RYK antibody described in any one of embodiments 21 to 30, wherein the RYK protein is expressed on the surface of a cell. Embodiment 32. An anti-RYK antibody, wherein the anti-RYK antibody binds to the same epitope as an antibody comprising a heavy chain variable domain comprising CDR H1 set forth in SEQ ID NO:1, CDR H2 set forth in SEQ ID NO:2, and CDR H3 set forth in SEQ ID NO:3, and a light chain variable domain comprising CDR L1 set forth in SEQ ID NO:4, CDR L2 set forth in SEQ ID NO:5, and CDR L3 set forth in SEQ ID NO:6. Embodiment 33. An anti-RYK antibody, wherein the anti-RYK antibody binds to the same epitope as an antibody comprising a heavy chain variable domain comprising CDR H1 as set forth in SEQ ID NO: 17, CDR H2 as set forth in SEQ ID NO: 18, and CDR H3 as set forth in SEQ ID NO: 19, and a light chain variable domain comprising CDR L1 as set forth in SEQ ID NO: 20, CDR L2 as set forth in SEQ ID NO: 21, and CDR L3 as set forth in SEQ ID NO: 22. Embodiment 34. An anti-RYK antibody, wherein the anti-RYK antibody binds to the same epitope as an antibody comprising a heavy chain variable domain comprising CDR H1 set forth in SEQ ID NO: 33, CDR H2 set forth in SEQ ID NO: 34, and CDR H3 set forth in SEQ ID NO: 35, and a light chain variable domain comprising CDR L1 set forth in SEQ ID NO: 36, CDR L2 set forth in SEQ ID NO: 37, and CDR L3 set forth in SEQ ID NO: 38. Embodiment 35. An anti-RYK antibody, wherein the anti-RYK antibody binds to the same epitope as an antibody comprising a heavy chain variable domain comprising CDR H1 set forth in SEQ ID NO: 49, CDR H2 set forth in SEQ ID NO: 50, and CDR H3 set forth in SEQ ID NO: 51, and a light chain variable domain comprising CDR L1 set forth in SEQ ID NO: 52, CDR L2 set forth in SEQ ID NO: 53, and CDR L3 set forth in SEQ ID NO: 54. Embodiment 36. An anti-RYK antibody described in any one of embodiments 1 to 35, wherein the anti-RYK antibody is conjugated to a therapeutic or diagnostic moiety. Embodiment 37. An isolated nucleic acid encoding an anti-RYK antibody described in any one of embodiments 1 to 36. Embodiment 38. A cell comprising an anti-RYK antibody described in any one of embodiments 1 to 36, or a nucleic acid described in embodiment 37. Embodiment 39. A pharmaceutical composition comprising a therapeutically effective amount of an antibody according to any one of embodiments 1 to 36, and a pharma- ceutically acceptable excipient. Embodiment 40. A method for generating an antibody capable of binding to a RYK protein, the method comprising immunizing a mammal with a peptide comprising the sequence of SEQ ID NO:129. Embodiment 41. A method for detecting a RYK-expressing cell, the method comprising: (i) contacting a RYK-expressing cell with an antibody described in any one of embodiments 1 to 36; and (ii) detecting binding of the antibody to the RYK protein expressed by the cell. Embodiment 42 The method of embodiment 41, wherein the antibody is conjugated to a detectable moiety. Embodiment 43. The method of embodiment 41 or 42, wherein the RYK-expressing cell is in a biological sample. Embodiment 44. The method of embodiment 41, wherein the biological sample is whole blood, a blood fraction or blood product, tissue, or cultured cells. Embodiment 45. The method of any one of embodiments 41 to 44, wherein the RYK-expressing cell is a cancer cell. Embodiment 46. The method of embodiment 45, wherein the cancer cells are bladder cancer cells, brain cancer cells, breast cancer cells, chronic myeloid leukemia (CML) cells, colon cancer cells, Ewing's sarcoma cells, lung cancer cells, mantle cell lymphoma cells, ovarian cancer cells, pancreatic cancer cells, skin cancer cells, or melanoma cells. Embodiment 47. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an anti-RYK antibody described in any one of embodiments 1 to 36. Embodiment 48. The method of embodiment 47, wherein the cancer is bladder cancer, brain cancer, breast cancer, chronic myeloid leukemia (CML), colon cancer, Ewing's sarcoma, lung cancer, mantle cell lymphoma, ovarian cancer, pancreatic cancer, skin cancer, or melanoma. Embodiment 49. A method for identifying an anti-RYK antibody, the method comprising: (i) contacting an antibody with a first RYK polypeptide comprising an amino acid sequence corresponding to amino acid residues 48-57 of SEQ ID NO: 129; (ii) detecting the antibody that binds to the first RYK polypeptide; (iii) contacting the antibody with a second RYK polypeptide that does not comprise an amino acid sequence corresponding to amino acid residues 48-57 of SEQ ID NO: 129; and (iv) detecting the antibody that does not bind to the second RYK polypeptide, thereby identifying an anti-RYK antibody. Embodiment 50. The method of embodiment 49, wherein the antibody is a chimeric antibody. Embodiment 51 The method of embodiment 49 or 50, wherein the antibody is a Fab' fragment. Embodiment 52 The method of embodiment 49 or 50, wherein the antibody is a single chain antibody.

Claims

1. An anti-RYK antibody, comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises CDR H1 set forth in SEQ ID NO: 17, CDR H2 set forth in SEQ ID NO: 18, and CDR H3 set forth in SEQ ID NO: 19; An anti-RYK antibody, wherein the light chain variable domain comprises CDR L1 set forth in SEQ ID NO:20, CDR L2 set forth in SEQ ID NO:21, and CDR L3 set forth in SEQ ID NO:

22.

2. The anti-RYK antibody of claim 1, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:

31.

3. The anti-RYK antibody of claim 1, wherein the light chain variable domain comprises the sequence of SEQ ID NO:

32.

4. The anti-RYK antibody has a K Ｄ The anti-RYK antibody of claim 1, having the following structure:

5. The anti-RYK antibody has a K Ｄ The anti-RYK antibody of claim 1, having the following structure:

6. An anti-RYK antibody, comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises CDR H1 set forth in SEQ ID NO: 1, CDR H2 set forth in SEQ ID NO: 2, and CDR H3 set forth in SEQ ID NO: 3; An anti-RYK antibody, wherein the light chain variable domain comprises CDR L1 set forth in SEQ ID NO:4, CDR L2 set forth in SEQ ID NO:5, and CDR L3 set forth in SEQ ID NO:

6.

7. The anti-RYK antibody of claim 6, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:

15.

8. The anti-RYK antibody of claim 6, wherein the light chain variable domain comprises the sequence of SEQ ID NO:

16.

9. The anti-RYK antibody has a K Ｄ The anti-RYK antibody of claim 6, having the following structure:

10. The anti-RYK antibody has a K Ｄ The anti-RYK antibody of claim 6, having the following structure:

11. An anti-RYK antibody, comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises a CDR H1 set forth in SEQ ID NO: 33, a CDR H2 set forth in SEQ ID NO: 34, and a CDR H3 set forth in SEQ ID NO: 35; An anti-RYK antibody, wherein the light chain variable domain comprises CDR L1 set forth in SEQ ID NO:36, CDR L2 set forth in SEQ ID NO:37, and CDR L3 set forth in SEQ ID NO:

38.

12. The anti-RYK antibody of claim 11, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:

47.

13. The anti-RYK antibody of claim 11, wherein the light chain variable domain comprises the sequence of SEQ ID NO:

48.

14. An anti-RYK antibody, comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises a CDR H1 set forth in SEQ ID NO: 49, a CDR H2 set forth in SEQ ID NO: 50, and a CDR H3 set forth in SEQ ID NO: 51; An anti-RYK antibody, wherein the light chain variable domain comprises CDR L1 set forth in SEQ ID NO:52, CDR L2 set forth in SEQ ID NO:53, and CDR L3 set forth in SEQ ID NO:

54.

15. The anti-RYK antibody of claim 14, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:

63.

16. The anti-RYK antibody of claim 14, wherein the light chain variable domain comprises the sequence of SEQ ID NO:

64.

17. The anti-RYK antibody of claim 1 , 6, 11, or 14, wherein the anti-RYK antibody is a chimeric antibody.

18. The anti-RYK antibody of claim 1, 6, 11, or 14, wherein the anti-RYK antibody is a Fab' fragment.

19. The anti-RYK antibody of claim 1 , 6, 11, or 14, wherein the anti-RYK antibody is an IgG.

20. 15. The anti-RYK antibody of claim 1, wherein the light chain variable domain and the heavy chain variable domain form part of an scFv.

21. The anti-RYK antibody of claim 1, 6, 11 or 14, wherein the anti-RYK antibody is capable of binding to a RYK protein.

22. The anti-RYK antibody of claim 1 , 6, 11, or 14, wherein the anti-RYK antibody binds to the extracellular RYK domain.

23. The anti-RYK antibody of claim 1 , 6, 11, or 14, wherein the anti-RYK antibody binds to an extracellular RYK domain comprising the amino acid sequence of SEQ ID NO:

129.

24. The anti-RYK antibody of any one of claims 1, 6, 11 or 14, wherein the anti-RYK antibody binds to an amino acid sequence corresponding to amino acid residues 48-57 of SEQ ID NO:

129.

25. The anti-RYK antibody of claim 1 , 6, 11, or 14, wherein the anti-RYK antibody is bound to a RYK protein.

26. The anti-RYK antibody of claim 21 , wherein the RYK protein is a human RYK protein.

27. 22. The anti-RYK antibody of claim 21, wherein the RYK protein comprises the sequence of SEQ ID NO:

130.

28. The anti-RYK antibody of claim 21 , wherein the RYK protein does not bind to a mouse RYK protein.

29. The anti-RYK antibody of claim 21, wherein the anti-RYK antibody does not bind to a RYK protein comprising an amino acid sequence corresponding to amino acid residues 32 to 41 of SEQ ID NO:

131.

30. 26. The anti-RYK antibody of claim 25, wherein the RYK protein forms part of a cell.

31. The anti-RYK antibody of claim 21 , wherein the RYK protein is expressed on the surface of a cell.

32. An anti-RYK antibody, wherein the anti-RYK antibody binds to the same epitope as an antibody comprising a heavy chain variable domain comprising CDR H1 set forth in SEQ ID NO: 1, CDR H2 set forth in SEQ ID NO: 2, and CDR H3 set forth in SEQ ID NO: 3, and a light chain variable domain comprising CDR L1 set forth in SEQ ID NO: 4, CDR L2 set forth in SEQ ID NO: 5, and CDR L3 set forth in SEQ ID NO:

6.

33. An anti-RYK antibody, wherein the anti-RYK antibody binds to the same epitope as an antibody comprising a heavy chain variable domain comprising CDR H1 set forth in SEQ ID NO: 17, CDR H2 set forth in SEQ ID NO: 18, and CDR H3 set forth in SEQ ID NO: 19, and a light chain variable domain comprising CDR L1 set forth in SEQ ID NO: 20, CDR L2 set forth in SEQ ID NO: 21, and CDR L3 set forth in SEQ ID NO:

22.

34. An anti-RYK antibody, wherein the anti-RYK antibody binds to the same epitope as an antibody comprising a heavy chain variable domain comprising CDR H1 set forth in SEQ ID NO: 33, CDR H2 set forth in SEQ ID NO: 34, and CDR H3 set forth in SEQ ID NO: 35, and a light chain variable domain comprising CDR L1 set forth in SEQ ID NO: 36, CDR L2 set forth in SEQ ID NO: 37, and CDR L3 set forth in SEQ ID NO:

38.

35. An anti-RYK antibody, wherein the anti-RYK antibody binds to the same epitope as an antibody comprising a heavy chain variable domain comprising CDR H1 set forth in SEQ ID NO:49, CDR H2 set forth in SEQ ID NO:50, and CDR H3 set forth in SEQ ID NO:51, and a light chain variable domain comprising CDR L1 set forth in SEQ ID NO:52, CDR L2 set forth in SEQ ID NO:53, and CDR L3 set forth in SEQ ID NO:

54.

36. 15. The anti-RYK antibody of any one of claims 1, 6, 11 or 14, wherein the anti-RYK antibody is conjugated to a therapeutic or diagnostic moiety.

37. 16. An isolated nucleic acid encoding an anti-RYK antibody of any one of claims 1, 6, 11 or 14.

38. A cell comprising the anti-RYK antibody of any one of claims 1, 6, 11 or 14.

39. A cell containing the nucleic acid described in claim 37.

40. A pharmaceutical composition comprising a therapeutically effective amount of an antibody of any one of claims 1, 6, 11 or 14 and a pharmaceutically acceptable excipient.

41. 1. A method for generating antibodies capable of binding to a RYK protein, said method comprising immunizing a mammal with a peptide comprising the sequence of SEQ ID NO:

129.

42. A method for detecting a RYK-expressing cell, the method comprising: (i) contacting a RYK-expressing cell with an antibody described in any one of claims 1, 6, 11 or 14; and (ii) detecting binding of the antibody to a RYK protein expressed by the cell.

43. 43. The method of claim 42, wherein the antibody is conjugated to a detectable moiety.

44. 43. The method of claim 42, wherein the RYK-expressing cell is in a biological sample.

45. 45. The method of claim 44, wherein the biological sample is whole blood, a blood fraction or blood product, tissue, or cultured cells.

46. 43. The method of claim 42, wherein the RYK-expressing cell is a cancer cell.

47. 47. The method of claim 46, wherein the cancer cells are bladder cancer cells, brain cancer cells, breast cancer cells, chronic myelogenous leukemia (CML) cells, colon cancer cells, Ewing's sarcoma cells, lung cancer cells, mantle cell lymphoma cells, ovarian cancer cells, pancreatic cancer cells, skin cancer cells, or melanoma cells.

48. 20. Use of an anti-RYK antibody according to any one of claims 1, 6, 11 or 14 in the manufacture of a medicament for the treatment of cancer.

49. 49. The use of claim 48, wherein the cancer is bladder cancer, brain cancer, breast cancer, chronic myeloid leukemia (CML), colon cancer, Ewing's sarcoma, lung cancer, mantle cell lymphoma, ovarian cancer, pancreatic cancer, skin cancer, or melanoma.

50. 1. A method for identifying an anti-RYK antibody, said method comprising: (i) contacting the antibody with a first RYK polypeptide comprising an amino acid sequence corresponding to amino acid residues 48-57 of SEQ ID NO: 129; (ii) detecting the antibody that binds to the first RYK polypeptide; (iii) contacting the antibody with a second RYK polypeptide that does not contain an amino acid sequence corresponding to amino acid residues 48-57 of SEQ ID NO: 129; (iv) detecting said antibody that does not bind to said second RYK polypeptide, thereby identifying an anti-RYK antibody.

51. The antibody of claim 1, 6, 11, or 14, wherein the RYK antibody comprises a human IgG1, IgG2, IgG3, or IgG4 constant region.

52. The antibody of claim 51, wherein the RYK antibody comprises a human IgG1 constant region.

53. The antibody of claim 51, wherein the RYK antibody comprises a human IgG2 constant region.

54. The antibody of claim 51, wherein the RYK antibody comprises a human IgG3 constant region.

55. The antibody of claim 51, wherein the RYK antibody comprises a human IgG4 constant region.

Images