AU2003295649A1

AU2003295649A1 - Methods of therapy and diagnosis

Info

Publication number: AU2003295649A1
Application number: AU2003295649A
Authority: AU
Inventors: Douglas Dedera; Peter C. R. Emtage
Original assignee: Nuvelo Inc
Current assignee: Nuvelo Inc
Priority date: 2002-11-22
Filing date: 2003-11-19
Publication date: 2004-06-18
Also published as: CA2506356A1; EP1567863A4; WO2004047612A3; EP1567863A2; WO2004047612A2

Description

WO 2004/047612 PCT/US2003/036971 1 METHODS OF THERAPY AND DIAGNOSIS 1. CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part application of U.S. Application Serial No. 5 10/641,222 filed on August 13, 2003, entitled "Methods of Therapy and Diagnosis Using Targeting of Cells that Express Toll-like Receptor Proteins," Attorney Docket No. HYS-49CP3, which in turn is a continuation-in-part application of U.S. Application Serial No. 10/327,491 filed on December 19, 2002, entitled "Methods of Therapy and Diagnosis Using Targeting of Cells that Express Toll-like Receptor Proteins," Attorney Docket No. HYS-49CP2, which in turn is a continuation-in-part 10 application of U.S. Application Serial No. 10/3 02,444 filed on November 22, 2002, entitled "Methods of Therapy and Diagnosis Using Targeting of Cells that Express Toll-like Receptor Proteins," Attorney Docket No. HYS-49CP. This and all other U.S. Patents and Patent Applications cited herein are hereby incorporated by reference in their entirety. 15 2. TECHNICAL FIELD This invention relates to compositions and methods for targeting Toll-like Receptor 10 (TLR10) protein-expressing cells and their use in the therapy and diagnosis of various pathological states, including cancer, autoimmune disease, organ transplant rejection, and allergic reactions. 20 2.1 BACKGROUND Antibody therapy for cancer involves the use of antibodies, or antibody fragments, against a tumor antigen to target antigen-expressing cells. Antibodies, or antibody fragments, may have direct or indirect cytotoxic effects or may be conjugated or fused to 25 cytotoxic moieties. Direct effects include the induction of apoptosis, the blocking of growth factor receptors, and anti-idiotype antibody formation. Indirect effects include antibody dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cellular cytotoxicity (CMCC). When conjugated or fused to cytotoxic moieties, the antibodies, or fragments thereof, provide a method of targeting the cytotoxicity towards the tumor antigen 30 expressing cells. (Green, et al., Cancer Treatment Reviews, 26:269-286 (2000), this and all other references cited herein are hereby incorporated by reference in their entirety.). Because antibody therapy targets cells expressing a particular antigen, there is a possibility of cross-reactivity with normal cells or tissue. Although some cells, such as hematopoietic cells, are readily replaced by precursors, cross-reactivity with many tissues WO 2004/047612 PCT/US2003/036971 2 can lead to detrimental results. Thus, considerable research has gone towards finding tumor specific antigens. Such antigens are found almost exclusively on tumors or are expressed at a greater level in tumor cells than the corresponding normal tissue. Tumor-specific antigens provide targets for antibody targeting of cancer, or other disease-related cells, expressing the 5 antigen. Antibodies specific to such tumor-specific antigens can be conjugated to cytotoxic compounds or can be used alone in immunotherapy. Immunotoxins target cytotoxic compounds to induce cell death. For example, anti-CD22 antibodies conjugated to deglycosylated ricin A may be used for treatment of B cell lymphoma that has relapsed after conventional therapy (Amlot, et at., Blood 82:2624-2633 (1993)) and has demonstrated 10 encouraging responses in initial clinical studies. Immunotherapy provides a method of harnessing the immune system to treat various pathological states, including cancer, autoimmune disease, transplant rejection, hyperproliferative conditions, and allergic reactions. The immune system functions to eliminate organisms or cells that are recognized as 15 non-self, including microorganisms, neoplasms and transplants. A cell-mediated host response to tumors includes the concept of immunologic surveillance, by which cellular mechanisms associated with cell-mediated immunity, destroy newly transformed tumor cells after recognizing tumor-associated antigens (antigens associated with tumor cells that are not apparent on normal cells). Furthermore, a humoral response to tumor-associated antigens 20 enables destruction of tumor cells through immunological processes triggered by the binding of an antibody to the surface of a cell, such as antibody-dependent cellular cytotoxicity (ADCC) and complement mediated lysis. Recognition of an antigen by the immune system triggers a cascade of events including cytokine production, B-cell proliferation, and subsequent antibody production. 25 Often tumor cells have reduced capability of presenting antigen to effector cells, thus impeding the immune response against a tumor-specific antigen. In some instances, the tumor-specific antigen may not be recognized as non-self by the immune system, preventing an immune response against the tumor-specific antigen from occurring. In such instances, stimulation or manipulation of the immune system provides effective techniques of treating 30 cancers expressing one or more tumor-specific antigens. For example, Rituximab (Rituxan@) is a chimeric antibody directed against CD20, a B cell-specific surface molecule found on >95% of B-cell non-Hodgkin's lymphoma (Press, et al., Blood 69:584-591 (1987); Malony, et al., Blood 90:2188-2195 (1997)). Rituximab WO 2004/047612 PCT/US2003/036971 3 induces ADCC and inhibits cell proliferation through apoptosis in malignant B cells in vitro (Maloney, et al., Blood 88:637a (1996)). Rituximab is currently used as a therapy for advanced stage or relapsed low-grade non-Hodgkin's lymphoma, which has not responded to conventional therapy. 5 Active immunotherapy, whereby the host is induced to initiate an immune response against its own tumor cells can be achieved using therapeutic vaccines. One type of tumor specific vaccine uses purified idiotype protein isolated from tumor cells, coupled to keyhole limpet hemocyanin (KLH) and mixed with adjuvant for injection into patients with low grade follicular lymphoma (Hsu, et al., Blood 89:3129-3135 (1997)). Another type of 10 vaccine uses antigen-presenting cells (APCs), which present antigen to naive T cells during the recognition and effector phases of the immune response. Dendritic cells, one type of APC, can be used in a cellular vaccine in which the dendritic cells are isolated from the patient, co-cultured with tumor antigen and then reinfused as a cellular vaccine (Hsu, et al., Nat. Med. 2:52-58 (1996)). Immune responses can also be induced by injection of naked 15 DNA. Plasmid DNA that expresses bicistronic mRNA encoding both the light and heavy chains of tumor idiotype proteins, such as those from B cell lymphoma, when injected into mice, are able to generate a protective, anti-tumor response (Singh, et al., Vaccine 20:1400 1411 (2002)). Thus, there exists a need in the art to identify and develop agents, such as peptide 20 fragments, nucleic acids, or antibodies that provide therapeutic compositions and diagnostic methods for treating and identifying cancer, hyperproliferative disorders, auto-immune diseases, and organ transplant rejection. 3. SUMMARY OF THE INVENTION 25 The invention provides therapeutic and diagnostic methods of targeting cells expressing the Toll-like Receptor 10 (TLR1 0) protein by using targeting elements such as TLR10 polypeptides, nucleic acids encoding TLR10 protein, and anti-TLR10 antibodies, including fragments or other modifications thereof, peptides, and small molecules. The TLR10 proteins are highly expressed in certain hematopoietic-based cancer cells relative to 30 their expression in healthy cells. Additionally, TLR10 mRNA is highly expressed in neutrophils. Thus, targeting of cells that express TLR1 0 will have a minimal effect on healthy tissues while destroying or inhibiting the growth of the hematopoietic-based cancer cells and/or neutrophils. Similarly, non-hematopoietic type tumors (solid tumors) can be WO 2004/047612 PCT/US2003/036971 4 targeted if they bear the TLRIO antigen. TLR1 0 protein is highly expressed in solid tumors, such as prostate, breast, colon and squamous cell carcinoma. For example, inhibition of growth and/or destruction of TLR10-expressing cancer cells results from targeting such cells with anti-TLR10 antibodies, respectively. One embodiment of the invention is a method of 5 destroying TLR1 0-expressing cells by conjugating anti-TLR1 0 antibodies with cytocidal materials such as radioisotopes or other cytotoxic compounds, respectively. The present invention provides a variety of targeting elements and compositions. One such embodiment is a composition comprising an anti-TLR10 antibody preparation. Exemplary antibodies include a single anti-TLR1 0 antibody, a combination of two or more 10 anti-TLR10 antibodies, a combination of an anti-TLR1O antibody with a non-TLR1O antibody, a combination of an anti-TLR1O antibody and a therapeutic agent, a combination of an anti-TLR1 0 antibody and a cytocidal agent, a bispecific anti-TLR1 0 antibody, Fab TLR10 antibodies or fragments thereof, including any fragment of an antibody that retains one or more complementary determining regions (CDRs) that recognize TLR10, humanized 15 anti-TLR1 0 antibodies that retain all or a portion of a CDR that recognizes TLR1 0, anti TLR10 conjugates, and anti-TLR1O antibody fusion proteins. Another targeting embodiment of the invention is a composition comprising a TLR 10 antigen, for example a TLR10 polypeptide, or a fragment thereof and optionally comprising a suitable adjuvant. 20 Yet another targeting embodiment of the invention is a preparation comprising a TLR10 polypeptide, or peptide fragment. A further targeting embodiment of the present invention is a non-TLRl 0 polypeptide or peptide that binds TLR1 0. Another targeting embodiment of the invention is a preparation comprising a small molecule that recognizes TLR1O. 25 Yet another targeting embodiment is a composition comprising a nucleic acid encoding TLR10, or a fragment or variant thereof, optionally within a recombinant vector. A further targeting embodiment of the present invention is a composition comprising an antigen-presenting cell transformed with a nucleic acid encoding TLR10, or a fragment or variant thereof, optionally within a recombinant vector. 30 The present invention further provides a method of targeting TLR1 0-expressing cells, which comprises administering a targeting element or composition in an amount effective to target TLR10-expressing cells. Any one of the targeting elements or compositions described herein may be used in such methods, including an anti-TLRIO antibody preparation, an WO 2004/047612 PCT/US2003/036971 5 antigen comprising a TLR1O polypeptide, or a fragment thereof or a composition of a nucleic acid encoding TLR1 0, or a fragment thereof, optionally within a recombinant vector or a composition of an antigen-presenting cell transformed with a nucleic acid encoding TLR1 0, or fragment or variant thereof, optionally within a recombinant vector, or a TLR1 0 5 polypeptide, peptide fragment, or a binding polypeptide, peptide or small molecule that binds TLR1O. The invention also provides a method of inhibiting the growth of hematopoetic based, TLR10-expressing cancer cells, which comprises administering a targeting element or a targeting composition in an amount effective to inhibit the growth of said hematopoetic 10 based cancer cells. Any one of the targeting elements or compositions described herein may be used in such methods, including an anti-TLR1O antibody preparation, an antigen comprising a TLR1 0 polyp eptide, or fragment thereof, composition of a nucleic acid encoding TLR1 0, or fragment or variant thereof, optionally within a recombinant vector, or a composition of an antigen-presenting cell transformed with a nucleic acid encoding TLR1 0, 15 or fragment or variant thereof, optionally within a recombinant vector, or a TLR10 polypeptide, peptide fragment, or a binding polypeptide, peptide or small molecule that binds TLR10. The present invention further provides a method of treating disorders associated with 20 the proliferation of TLR 10-expressing cells in a subject in need thereof, comprising the step of administering a targeting element or targeting composition in a therapeutically effective amount to treat disorders associated with TLR1 0-expressing cells. Any one of the targeting elements or compositions described herein may be used in such methods, including an anti TLR10 antibody preparation, an antigen comprising a TLR10 polypeptide or fragment 25 thereof, a composition of a nucleic acid encoding TLR10, or fragment thereof, optionally within a recombinant vector, or a composition of an antigen-presenting cell comprising a nucleic acid encoding TLR1O, or fragment thereof, optionally within a recombinant vector. Examples of disorders associated with the proliferation of TLR1 0-expressing cells include cancers, such as non-Hodgkin's B-cell lymphomas, B-cell leukemias, chronic 30 lymphocytic leukemia, multiple myeloma, acute and chronic myeloid leukemia; myelodysplastic syndromes; T cell lymphomas, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, lymphosarcoma leukemia, malignant lymphoma, B cell large cell lymphoma, X-linked WO 2004/047612 PCT/US2003/036971 6 lymphoproliferative disorders; Epstein Barr Virus-related conditions such as mononucleosis; and autoimmune disorders. Non-hematopoietic tumors that bear the TLR10 antigen, such as prostate, breast, colon, and squamous cell carcinoma, as well as other cancers of epithelial and squamous cell origin, can also be targeted. 5 Examples of disorders associated with neutrophils that could be treated with TLR10 targeting therapy include, but are not limited to, inflammatory disorders, autoimmune diseases, asthma, chronic pancreatitis, allergic dermatitis, idiopathic pulmonary fibrosis, upper and lower respiratory infection, acute pulmonary inflammation, wound healing, graft vs. host disease, chronic obstructive pulmonary disease, arthritis, periodontitis, and disorders 10 relating to oxidative stress, including hypertension pathogenesis and athlerosclerosis. The invention further provides a method of modulating the immune system by either suppression or stimulation of growth factors and cytokines, by administering the targeting elements or compositions of the invention. The invention also provides a method of modulating the immune system through activation of immune cells (such as natural killer 15 cells, T cells, B cells and myeloid cells), through the suppression of activation, or by stimulating or suppressing proliferation of these cells by TLR1 0 peptide fragments or TLR1O antibodies. The present invention thereby provides a method of treating immune-related disorders by suppressing the immune system in a subject in need thereof, by administering 20 the targeting elements or compositions of the invention. Such immune-related disorders include but are not limited to autoimmune disease and organ transplant rejection. The present invention also provides a method of diagnosing disorders associated with TLR10--expressing cells comprising the step of measuring the expression patterns of TLR10 protein and/or iRNA, respectively. Yet another embodiment of a method of 25 diagnosing disorders associated with TLR10-expressing cells comprising the step of detecting TLR10 expression using anti-TLR10 antibodies, respectively. Such methods of diagnosis include compositions, kits and other approaches for determining whether a patient is a candidate for TLR1 0 targeting therapy. The present invention also provides a method of enhancing the effects of therapeutic 30 agents and adjunctive agents used to treat and manage disorders associated with TLR10 expressing cells, by administering TLR10 preparations with therapeutic and adjuvant agents commonly used to treat such disorders.

WO 2004/047612 PCT/US2003/036971 7 4. BRIEF DESCRIPTION OF THE DRAWING Figure 1 depicts the relative expression of TLRl 0 mRNA (as determined by RT PCR) derived from leukocytes and leukocyte progenitors. 5 5. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to methods of targeting cells that express TLR10 using targeting elements, such as TLR10 polypeptides, nucleic acids encoding TLR10, anti-TLR10 antibodies, binding polypeptides, peptides, and small molecules, including fragments or other modifications of any of these elements. 10 The present invention provides a novel approach for diagnosing and treating diseases and disorders associated with TLR1O-expressing cells. The method comprises administering an effective dose of targeting preparations such as TLR1 0 antigens, antigen presenting cells, or pharmaceutical compositions comprising the targeting elements, TLR1O polypeptides, nucleic acids encoding TLR1O, anti-TLR1 0 antibodies, or binding polypeptides, peptides, 15 and small molecules, described below. Targeting of TLR10 on the cell membranes of TLR10-expressing cells, respectively, is expected to inhibit the growth of or destroy such cells. An effective dose will be the amount of such targeting TLR1 0 preparations necessary to target the TLR1 0 on the cell membrane and inhibit the growth of or destroy the TLR1 0 expressing cells and/or metastasis. 20 A further embodiment of the present invention is to enhance the effects of therapeutic agents and adjunctive agents used to treat and manage disorders associated with TLR1O expressing cells, by administering TLR10 preparations, respectively, with therapeutic and adjuvant agents commonly used to treat such disorders. Chemotherapeutic agents useful in treating neoplastic disease and antiproliferative agents and drugs used for 25 immunosuppression include alkylating agents, such as nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes; antimetabolites, such as folic acid analogs, pyrimidine analogs, and purine analogs; natural products, such as vinca alkaloids, epipodophyllotoxins, antibiotics, and enzymes; miscellaneous agents such as polatinum coordination complexes, substituted urea, methyl hydrazine derivatives, and adrenocortical suppressant; and honnones and 30 antagonists, such as adrenocorticosteroids, progestins, estrogens, androgens, and anti estrogens (Calebresi and Parks, pp. 1240-1306 in, Eds. A.G Goodman, L.S. Goodman, T.W. Rall, and F. Murad, The Pharmacological Basis of Therapeutics, Seventh Edition, MacMillan Publishing Company, New York, (1985)).

WO 2004/047612 PCT/US2003/036971 8 Adjunctive therapy used in the management of such disorders includes, for example, radiosensitizing agents, coupling of antigen with heterologous proteins, such as globulin or beta-galactosidase, or inclusion of an adjuvant during immunization. High doses may be required for some therapeutic agents to achieve levels to 5 effectuate the target response, but may often be associated with a greater frequency of dose related adverse effects. Thus, combined use of the therapeutic methods of the present invention with agents commonly used to treat TLRl 0 protein-related disorders allows the use of relatively lower doses of such agents resulting in a lower frequency of adverse side effects associated with long-term administration of the conventional therapeutic agents. 10 Thus another indication for the therapeutic methods of this invention is to reduce adverse side effects associated with conventional therapy of disorders associated with TLR1 0 expressing cells. 5.1 DEFINITIONS 15 The term "fragment" of a nucleic acid refers to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides. The fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 20 nucleotides, more preferably less than about 50 nucleotides and most preferably less than 30 nucleotides. Preferably the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules. A fragment or segment may uniquely identify each polynucleotide sequence of the present invention. Preferably the fragment comprises a 25 sequence substantially similar to a portion of SEQ ID NO: 1. A polypeptide "fragment " is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about 17 or more amino acids. The peptide preferably is not greater than about 200 amino acids, more preferably less than 150 amino acids and most preferably less than 100 amino acids. 30 Preferably the peptide is from about 5 to about 200 amino acids. To be active, any polypeptide must have sufficient length to display biological and/or immunological activity. The term "immunogenic" refers to the capability of the natural, recombinant or synthetic WO 2004/047612 PCT/US2003/036971 9 TLR1 0 peptide, or any peptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies. The term "variant"(or "analog") refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, 5 e g., recombinant DNA techniques. Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence. Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the "redundancy" in the genetic code. Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system. Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate. 10 The term "TLR1 0 antigen" refers to a molecule that when introduced into an animal is capable of stimulating an immune response in said animal specific to the TLR10 polypeptide or fragment thereof, of the present invention. The term "stringent" is used to refer to conditions that are commonly understood in the art as stringent. Stringent conditions can include highly stringent conditions (i.e., 15 hybridization to filter-bound DNA in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65'C, and washing in 0.1x SSC/0.1% SDS at 68'C), and moderately stringent conditions (i.e., washing in 0.2x SSC/0.1% SDS at 42'C). Other exemplary hybridization conditions are described herein in the examples. In instances of hybridization of deoxyoligonucleotides, additional exemplary 20 stringent hybridization conditions include washing in 6x SSC/0.05% sodium pyrophosphate at 37*C (for 14-base oligonucleotides), 48*C (for 17-base oligonucleotides), 55'C (for 20 base oligonucleotides), and 60'C (for 23-base oligonucleotides). 25 WO 2004/047612 PCT/US2003/036971 10 5.2 TARGETING OF TLR10 Toll and Toll-like receptors are type I transmembrane proteins with extracellular leucine-rich repeat motifs and an intracellular signaling domains. The Toll-like receptors make up a family of human receptors which have common structural features with the 5 Drosophila Toll (dToll) receptor molecule. They are found on the surface of several types of hematopoietic cells. Human Toll-like receptors are also expressed on antigen presenting cells, such as monocytes and dendritic cells (WO 01/55386 Al). Two human colon cancer cell lines (DLD and LoVo) showed expression of the toll-like receptor subtype TLR-2, whereas the toll-like receptor subtype TLR-4 was expressed in human hepatocellular 10 carcinoma (PLC/PRF/5) and acute myeloid leukemia (KG-1) cells (Yoshioka, et al., J. Int. Med. Res. 29:409-420 (2001)). Both dToll and human Toll-like receptors are thought to act as pattern recognition receptors for bacteria and other microorganisms, and play a role in immune surveillance mechanisms and innate immunity. Toll-like receptors can trigger pro-inflammatory 15 cytokine production and induce expression of cell surface co-stimulatory receptors for T-cell activation. Some human Toll-like receptors may be involved in co-ordination of interactions between immune cells resulting in an integrated immune response to infection. TLR2 and TLR4 have been shown to mediate host responses to gram positive and gram negative bacteria through recognition of specific bacterial wall components. TLR4 mediates 20 responses to certain viral proteins such as respiratory syncytial virus. Toll-like receptors may also form heterodimeric functional complexes and share in common signal transduction pathways with IL-I receptors. Activation of TLR2 and TLR4 leads to the activation of NFKB via an adapter protein MyD88 and recruitment of the IL-1 receptor - associated kinases (IiRAKs) (WO 01/55386 Al; Henneke and Golenbock, Nature munology 2:828 25 830 (2001)) Toll-like Receptor 9 (TLR9) was shown to mediate the cellular response to bacterial deoxy-cytidylate-phosphate-deoxyguanylate (CpG) DNA, suggesting that vertebrate systems have evolved a specific Toll-like receptor that distinguishes bacterial DNA from self-DNA (Hemml, et al., Nature 408:740-744 (2000); Bauer, et al., Proc. Nati. Acad. Sci. 98:9237-9242 (2001); Takeshita, et al., J. Immunol. 167:3555-3558 (2001); 30 Wagner, Immunity 14:499-502 (2001)). In addition, CpG DNA may be recognized by autoantibodies inducing an autoimmune response (Ichikawa et al., J. Immunol. 169:2781 2787 (2002); Leadbetter et al., Nature 416:603-607 (2002), both are herein incorporated by reference). TLR1O, a new member of the TLR family, is most closely related to TLR1 and WO 2004/047612 PCT/US2003/036971 11 TLR6 (Chuang and Ulevitch, Biochiin, Biophys, Acta 1518:157-161 (2001), herein incorporated by reference). The cytoplasmic domains of TLR1 and TLR6 have been demonstrated to form functional pairs with TLR2, thus TLR10 may also interact with TLR2 (Hornung et al., J Immunol. 168:4531-4535 (2002); Ozinsky et al., Proc. Natl. Acad. Sci. 5 USA 97:13766 (2000), both of which are herein incorporated by reference). These findings suggest that Toll and Toll-like receptors may play a role in immune defense mechanisms to counteract microbial infection. TLR10 polypeptides and polynucleotides encoding such polypeptides are disclosed in co-owned U.S. Patent Application Serial No. 09/488,725, which corresponds to PCT 10 Publication No. WO 01/53455, incorporated herein by reference, which relates, in general, to a collection or library of at least one novel nucleic acid sequences, specifically contigs, assembled from expressed sequence tags (ESTs). PCT International Publication No. WO 01/55386 (to Lewis et al.) discloses a novel Toll-like receptor and its use in screening for novel pharmacotherapeutic agents with 15 immunomodulatory activity. More specifically, WO 01/55386 discloses isolated Toll-like receptor polypeptides, polynucleotides encoding for the Toll-like receptor polypeptide, expression vectors comprising such polynucleotides, hosts cells comprising such expression vectors, antibodies specific for the Toll-like receptor polypeptide, methods for identification of compounds that modulate Toll-like receptor activity, and methods of treating disorders 20 responsive to toll-like receptor modulation. PCT International Publication No. WO 99/20756 (to Goddard et al.) discloses human Toll homolog polypeptides, polynucleotides encoding for the human Toll homologs, expression vectors comprising such polynucleotides, host cells comprising such expression vectors, antibodies specific for the human Toll homolog polypeptides, antibodies that specifically bind to a human TLR2 receptor, and 25 methods for treating septic shock using anti-Toll homolog antibodies. Table 1 shows that TLR10 was found to be expressed at high levels in the B cell lymphoma cell lines CA-46 and RAI, moderately expressed in the B cell lymphoma cell lines DB, HT, and RL. These results demonstrate that TLR10 mRNA is highly expressed in cell lines derived from B cell lymphomas. The results in Table 2 show that levels of mRNA 30 expression were high in follicular lymphoma tissue (L5856 and L5348), moderate in diffuse large B cell lymphoma tissue (L6879 and L22601), and anaplastic large T cell lymphoma tissue (L5664), and low in acute myeloid leukemia tissue (AML565). These findings demonstrate TLR1 0 mRNA expression in different Non-Hodgkin's B cell lymphoma tissues WO 2004/047612 PCT/US2003/036971 12 and cell lines, and indicate that TLR1 0 may be used as an immunotherapeutic antibody target and a diagnostic marker for certain cell types or disorders (e.g., B-cell lymphomas, T cell lymphomas, and myeloid leukemia). The results in Table 3 show that TLR1O is expressed in the following leukemias and lymphomas: acute myelogenous leukemia, acute 5 myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, and lymphosarcoma cell leukemia. Thus, targeting cells expressing TLRl 0 will be useful in treating these and other hematopoictic diseases. TLR1O is also expressed in solid tumors, such as prostate, breast, colon and squamous cell carcinoma (see Table 4). 10 Since TLR10 is expressed in these epithelial and squamous cell cancers, it is likely that TLR10 will be expressed in other cancers of epithelial and squamous cell origin. IHC analysis showed that TLR10 is also expressed in autoimmune disorders, including systemic lupus erythematosus; Hasimoto thyroiditis, Sjorgen's syndrome, and pericarditis lupus (see Table 5). Therefore, targeting TLRl0 may be useful in treating these and other autoimmune 15 disorders. Finally, IHC analysis showed that TLR10 is expressed in rejected transplanted heart, kidney and liver tissues, whereas normal tissues do not express TIR.10 (see Table 6). Thus, targeting TLR1O will be useful in reducing and/or eliminating tissue rejection after transplantation. Leukocytes, including neutrophils, are recruited to sites of infection and injury in the 20 early stages of the inflammatory pathway. Normally, unwanted effector cells are cleared at the end of the inflammatory response presumably due to the loss of survival signals. However, in situations of chronic inflammation, the resolution phase becomes prolonged and disordered leading to the persistence of inflammatory infiltrate (i.e. neutrophils), tissue hyperplasia, and ultimately tissue scarring (Buckley, Rheumatology 42:1-12 (2003); Simon, 25 inmunol. Rev. 193:101-110 (2003), both of which are herein incorporated by reference in their entirety). In addition, patients undergoing organ or tissue transplantation are at high risk for leukocyte-mediated morbidity due to activated neutrophils and oxygen free radicals (reviewed in Sievert, JECT 35:48-52 (2003) herein incorporated by reference in its entirety). During transplant surgery, reperfusion injury involves oxygen free radicals, increased 30 intracellular calcium concentrations, and neutrophil accumulation (Gravlee et al., eds. Surgical myocardial protection. In: Cardiopulmonary Bypass: Principles and Practice, 2 "d ed. Philadelphia, Lippincott, Williams and Wilkins; 214-264 (2000); Ambrosio et al., Am. Heart J 138:S69-S75 (1999) both of which are herein incorporated by reference in their entirety).

WO 2004/047612 PCT/US2003/036971 13 Upon activation, neutrophils generate oxidase thereby releasing oxygen metabolites. The neutrophils become more adhesive to the endothelium leading to edema and increased microvascular permeability to protein. Ultimately, edema can cause capillary plugging, thereby impeding blood flow resulting in the no-flow phenomenon (Gravlee 2000 supra; 5 Ambrosio 1999 supra; Byrne et al., J. Thorac. Cardiovasc. Surg. 104:1589-1596 (1992) all of which are herein incorporated by reference in their entirety). Thus, removal of the neutrophils would decrease the risk of reperfusion injury and transplantation rejection. In addition, disorders that are characterized by chronic inflammation, such as autoimmune diseases, allergic reactions, asthma, wound healing, graft vs. host disease, and the like would 10 benefit from therapies that target the excess neutrophils for clearance. As shown in Figure 1, TLR10 mRNA is highly expressed in neutrophils; therefore TLR1 0 targeting compositions may be effective in treating disorders which are characterized by aberrant levels of neutrophils. 15 5.3 TARGETING USING TLR10 ANTIGENS The use of Toll-like receptor proteins as adjuvants in vaccine preparations has been previously described (WOOI/55386; Kovarik and Siegrist, Arch. Inmunol. Ther. Exp. (Warsz) 49:209-215 (2001); Azuma and Seya, Int. Innunopharnacol. 1:1249-1259 (2001)). Thus one embodiment of the present invention provides a composition comprising a TLR 10 20 antigen, for example a polypeptide, to stimulate the immune system against TLR1O, thus targeting TLR10-expressing cells. Use of a tumor antigen in a composition for generating cellular and humoral immunity for the purpose of anti-cancer therapy is well known in the art. For example, one type of tumor-specific antigen composition uses purified idiotype protein isolated from tumor cells, coupled to keyhole limpet hemocyanin (KLH) and mixed 25 with adjuvant for injection into patients with low-grade follicular lymphoma (Hsu, et al., Blood 89: 3129-3135 (1997)). U.S. Patent No. 6,312,718 describes methods for inducing immune responses against malignant B cells, in particular lymphoma, chronic lymphocytic leukemia, and multiple myeloma. The methods described therein utilize vaccines that include liposomes having (1) at least one B-cell malignancy-associated antigen, (2) IL-2 30 alone, or in combination with at least one other cytokine or chemokine, and (3) at least one lipid molecule. Methods of targeting TLRI0 using a TLRI0 antigen typically employ a TLR10 polypeptide, including fragments, analogs and variants.

WO 2004/047612 PCT/US2003/036971 14 As another example, dendritic cells, one type of antigen-presenting cell, can be used in a cellular vaccine in which the dendritic cells are isolated from the patient, co-cultured with tumor antigen and then reinfused as a cellular vaccine (Hsu, et al., Nat. Med. 2:52-58 (1996)). 5 Combining this antigen therapy with other types of therapeutic agents or treatments, such as chemotherapy or radiation is also contemplated. 5.4 TARGETING USING TLR10 NUCLEIC ACIDS 5.4.1 DIRECT DELIVERY OF NUCLEIC ACIDS 10 However, in some embodiments, a nucleic acid encoding TLR10, or encoding a fragment, analog or variant thereof, within a recombinant vector is utilized. Such methods are known in the art. For example, immune responses can be induced by injection of naked DNA. Plasmid DNA that expresses bicistronic mRNA encoding both the light and heavy chains of tumor idiotype proteins, such as those from B cell lymphoma, when injected into 15 mice, are able to generate a protective, anti-tumor response (Singh, et al., Vaccine 20:1400 1411 (2002)). TLRIO viral vectors are particularly useful for delivering TLR1O-encoding nucleic acids to cells, respectively. Examples of vectors include those derived from influenza, adenovirus, vaccinia, herpes symplex virus, fowlpox, vesicular stomatitis virus, canarypox, poliovirus, adeno-associated virus, and lentivirus and sindbus virus. Of course, 20 non-viral vectors, such as liposomes or even naked DNA, are also useful for delivering TLR1 0-encoding nucleic acids to cells. Combining this type of therapy with other types of therapeutic agents or treatments such as chemotherapy or radiation is also contemplated. 25 5.4.2. TLR10 NUCLEIC ACIDS EXPRESSED IN CELLS In some embodiments, a vector comprising a nucleic acid encoding the TLR1O polypeptide (including a fragment, analog or variant) is introduced into a cell, such as a dendritic cell or a macrophage. When expressed in an antigen-presenting cell, TLR10 antigens are presented to T cells eliciting an immune response against TLR10. Such 30 methods are also known in the art. Methods of introducing tumor antigens into antigen presenting cells and vectors useful therefor are described in U.S. Patent No. 6,300,090. The vector encoding TLR1 0 may be introduced into the antigen presenting cells in vivo. Alternatively, antigen-presenting cells are loaded with TLR1O or a nucleic acid encoding WO 2004/047612 PCT/US2003/036971 15 TLR1 0 ex vivo and then introduced into a patient to elicit an immune response against TLR10, respectively. In another alternative, the cells presenting TLR10 antigen are used to stimulate the expansion of anti-TLR10 cytotoxic T lymphocytes (CTL) ex vivo followed by introduction of the stimulated CTL into a patient. (U.S. Patent No. 6,306,388) 5 Combining this type of therapy with other types of therapeutic agents or treatments such as chemotherapy or radiation is also contemplated. 5.4.3 ANTISENSE NUCLEIC ACIDS Another aspect of the invention pertains to isolated antisense nucleic acid molecules 10 that can hybridize to, or are complementary to, the nucleic acid molecule comprising the TLR10 nucleotide sequence, or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic 15 acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire TLR10 coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a TLR1 0 or antisense nucleic acids complementary to a TLR1 0 nucleic acid sequence of are additionally provided. 20 In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding a TLR1 0 protein. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "conceding region" of the coding strand of a nucleotide sequence 25 encoding the TLR10 protein. The term "conceding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3'untranslated regions). Given the coding strand sequences encoding the TLR1 0 protein disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and 30 Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of TLR10 mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of TLR10 mRNA. For example, the antisense oligonucleotide can be complementary to the WO 2004/047612 PCT/US2003/036971 16 region surrounding the translation start site of TLR1 0 mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense 5 nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex fonned between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used). 10 Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetyleytosine, 5-(carboxyhydroxylmethyl) uracil, 5 carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 15 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5 methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl 20 2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an 25 antisense orientation to a target nucleic acid of interest, described further in the following section). The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a TLR1O protein to thereby inhibit expression of the protein (e.g., 30 by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense WO 2004/047612 PCT/US2003/036971 17 nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed 5 on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens). The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred. 10 In yet another embodiment, the antisense nucleic acid molecule of the invention is an alpha-anomeric nucleic acid molecule. An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual alpha-units, the strands run parallel to each other. See, e.g., Gaultier, et al., Nucl. Acids Res. 15: 6625-6641 (1987). The antisense nucleic acid molecule can also comprise a 2'-o 15 methylribonucleotide (see, e.g., Inoue, et al., Nucl. Acids Res. 15: 6131-6148 (1987)) or a chimeric RNA-DNA analogue (see, e.g., Inoue, et al., FEBS Lett. 215: 327-330 (1987), all of which are herein incorporated by reference in their entirety. 5.4.4 RIBOZYMES AND PNA MOIETIES 20 In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave 25 mRNA transcripts to thereby inhibit translation of an mRNA. A ribozyme having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a DNA disclosed herein (i.e., SEQ ID NO: 1). For example, a derivative of Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a mRNA. See, e.g., Cech et al. 30 U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, mRNA of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.

WO 2004/047612 PCT/US2003/036971 18 Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells. See generally, Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene. et al. (1992) Ann. N Y. Acad. Sci. 5 660:27-36; and Maher (1992) Bioassays 14: 807-15. In various embodiments, the nucleic acids of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup 10 et al. (1996) Bioorg Med Chen 4: 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The 15 synthesis of PNA oligomers can be perfonned using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe et al. (1996) PNAS 93: 14670-675. PNAs of the invention can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation 20 of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of the invention can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S 1 nucleases (Hyrup B. (1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996), above; 25 Perry-O'Keefe (1996), above). In another embodiment, PNAs of the invention can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras can be generated that may 30 combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of WO 2004/047612 PCT/US2003/036971 19 base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, 5 and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA (Mag et al. (1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996) above). Alternatively, chimeric molecules can be synthesized with a 5' DNA 10 segment and a 3' PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124. In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Nati. Acad. Sci. U.S.A. 15 86:6553-6556; Lemaitre et al., 1987, Proc. Nat. Acad. Sci. 84:648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5: 53 9-549). To this end, the oligonucleotide may be conjugated to 20 another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc. 5.4.5 GENE THERAPY Mutations in the polynucleotides of the invention gene may result in loss of normal 25 function of the encoded protein. The invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention. Delivery of a functional gene encoding polypeptides of the invention to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo 30 by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). See, for example, Anderson, Nature, 392(Suppl):25-20 (1998). For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992), all of which are herein WO 2004/047612 PCT/US2003/036971 20 incorporated by reference in their entirety. Introduction of any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression). Cells may also be cultured ex vivo in the presence of 5 proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes. Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states. It is contemplated that antisense therapy or gene therapy could be 10 applied to negatively regulate the expression of polypeptides of the invention. Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated RNA sequences, by methods known in the art. Further, the polypeptides of the present invention can be inhibited by using targeted deletion methods, or the insertion of a negative 15 regulatory element such as a silencer, which is tissue specific. The present invention still further provides cells genetically engineered in vivo to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell. These methods can be used to increase or decrease the 20 expression of the polynucleotides of the present invention. Knowledge of DNA sequences provided by the invention allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide. Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a 25 heterologous promoter so that the cells express the protein at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences. See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No. WO 91/09955, all of which are incorporated by reference in their entirety. It is also 30 contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the desired protein coding WO 2004/047612 PCT/US2003/036971 21 sequence, amplification of the marker DNA by standard selection methods results in co amplification of the desired protein coding sequences in the cells. In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the 5 control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination. As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods. Such regulatory sequences may be comprised of promoters, 10 enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences. Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting. These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader 15 sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules. The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or 20 enhancer or both upstream of a gene. Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element. Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell type specificity than the naturally occurring elements. Here, the naturally occurring 25 sequences are deleted and new sequences are added. In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome. The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property 30 of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.

WO 2004/047612 PCT/US2003/036971 22 Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene. The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Patent No. 5,272,071 5 to Chappel; U.S. Patent No. 5,578,461 to Sherwin et al.; International Application No. PCT/US92/09627 (WO 93/09222) by Selden et al.; and International Application No. PCT/US90/06436 (WO 91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety. 10 5.5 ANTI-TLR10 ANTIBODIES Alternatively, TLR1O targeting involves the administration of components of the immune system, such as antibodies, antibody fragments, or primed cells of the innune system against the target. Methods of targeting cancer cells using antibodies or antibody fragments are well known in the art. U.S. Patent No. 6,306,393 describes the use of anti 15 CD22 antibodies in the immunotherapy of B-cell malignancies, and U.S. Patent No. 6,329,503 describes immunotargeting of cells that express serpentine transmembrane antigens. TLR1 0 antibodies (including humanized or human monoclonal antibodies or fragments or other modifications thereof, optionally conjugated to cytotoxic agents) may be 20 introduced into a patient such that the antibody binds to TLR1 0 expressed by cancer cells and mediates the destruction of the cells and the tumor and/or inhibits the growth of the cells or the tumor. Without intending to limit the disclosure, mechanisms by which such antibodies can exert a therapeutic effect may include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity (ADCC), modulating the physiologic function of 25 TLR10, inhibiting binding or signal transduction pathways, modulating tumor cell differentiation, altering tumor angiogenesis factor profiles, modulating the secretion of immune stimulating or tumor suppressing cytokines and growth factors, modulating cellular adhesion, and/or by inducing apoptosis. TLR1O antibodies conjugated to toxic or therapeutic agents, such as radioligands or cytosolic toxins, may also be used therapeutically 30 to deliver the toxic or therapeutic agent directly to TLR10-bearing tumor cells. TLRIO antibodies may be used to suppress the immune system in patients receiving organ transplants or in patients with autoimmune diseases such as arthritis. Healthy immune WO 2004/047612 PCT/US2003/036971 23 cells would be targeted by these antibodies leading their death and clearance from the system, thus suppressing the immune system. TLR10 antibodies may be used as antibody therapy for solid tumors which express this action. Cancer immunotherapy using antibodies provides a novel approach to treating 5 cancers associated with cells that specifically express TLR10. As described above, human Toll-like receptors were shown to be expressed on antigen presenting cells, such as monocytes and dendritic cells (WO 01/55386 Al), human colon cancer cell lines (DLD and LoVo), human hepatocellular carcinoma (PLC/PRF/5) and acute myeloid leukemia (KG-1) cells (Yoshioka, et al., J. Int. Med. Res. 29:409-420 (2001)). Table 1 shows that TLR1O was 10 found to be expressed at high levels in the B cell lymphoma cell lines CA-46 and RA1, and moderately expressed in the B cell lymphoma cell lines DB, HT, and RL. These results demonstrate that TLR10 nRNA is highly expressed in cell lines derived from B cell lymphomas. The results in Table 2 show that levels of mRNA expression were high in follicular lymphoma tissue (L5856 and L5348), moderate in diffuse large B cell lymphoma 15 tissue (L6879 and L22601), and anaplastic large T cell lymphoma tissue (L5664), and low in acute myeloid leukemia tissue (AML565). These findings demonstrate TLR1O mRNA expression in different Non-Hodgkin's B cell lymphoma tissues and cell lines, and indicate that TLR10 may be used as an immunotherapeutic antibody target and a diagnostic marker for certain cell types or disorders (e.g., B-cell lymphomas, T cell lymphomas, and myeloid 20 leukemia). The results in Table 3 show that TLR1 0 is expressed in the following leukemias and lymphomas: acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, and lymphosarcoma cell leukemia. Thus, targeting cells expressing TLR10 will be useful in treating these diseases. 25 TLR10 is also expressed in solid tumors, such as prostate, breast, colon and squamous cell carcinoma (see Table 4); therefore, targeting TLR1 0 may be useful to treat these and other non-hematopoietic type tumors (solid tumors). Although TLR1 0 antibody therapy may be useful for all stages of the foregoing cancers, antibody therapy may be particularly appropriate in advanced or metastatic cancers. 30 Combining the antibody therapy method with a chemotherapeutic, radiation or surgical regimen may be preferred in patients that have not received chemotherapeutic treatment, whereas treatment with the antibody therapy may be indicated for patients who have received one or more chemotherapies. Additionally, antibody therapy can also enable the WO 2004/047612 PCT/US2003/036971 24 use of reduced dosages of concomitant chemotherapy, particularly in patients that do not tolerate the toxicity of the chemotherapeutic agent very well. Furthermore, treatment of cancer patients with TLR1O antibodies with tumors resistant to chemotherapeutic agents might induce sensitivity and responsiveness to these agents in combination. 5 Prior to TLR1O targeting, a patient may be evaluated for the presence and level of TLR10 expression by the cancer cells, preferably using immunohistochemical assessments of tumor tissue, quantitative TLR1O imaging, quantitative RT-PCR, or other techniques capable of reliably indicating the presence and degree of TLR10 expression. For example, a blood or biopsy sample may be evaluated by immunohistochemical methods to determine 10 the presence of TLR1 0-expressing cells or to determine the extent of TLR1 0 expression on the surface of the cells within the sample. Methods for immunohistochemical analysis of tumor tissues or released fragments of TLR1 0 in the serum are well known in the art. Anti-TLR1O antibodies useful in treating cancers include those, which are capable of initiating a potent immune response against the tumor and those, which are capable of direct 15 cytotoxicity. In this regard, anti-TLR10 mAbs may elicit tumor cell lysis by either complement-mediated or ADCC mechanisms, both of which require an intact Fe portion of the immunoglobulin molecule for interaction with effector cell Fc receptor sites or complement proteins. In addition, anti-TLR10 antibodies that exert a direct biological effect on tumor growth are useful in the practice of the invention. Potential mechanisms by which 20 such directly cytotoxic antibodies may act include inhibition of cell growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis. The mechanism by which a particular anti-TLR1 0 antibody exerts an anti tumor effect may be evaluated using any number of in vitro assays designed to determine ADCC, ADMMC, complement-mediated cell lysis, and so forth, as is generally known in 25 the art. The anti-tumor activity of a particular anti-TLR10 antibody, or combination of anti TLR1 0 antibody, may be evaluated in vivo using a suitable animal model. For example, xenogenic lymphoma cancer models wherein human lymphoma cells are introduced into immune compromised animals, such as nude or SCID mice. Efficacy may be predicted 30 using assays, which measure inhibition of tumor formation, tumor regression or metastasis, and the like. Anti-TLRI 0 antibodies can also be used to target non-cancer cells that express TLR10. For example, TLR10 is expressed in autoimmune disorders (see Table 5), in WO 2004/047612 PCT/US2003/036971 25 rejected transplanted tissue (see Table 6), and in neutrophils (see Figure 1). Therefore, targeting cells expressing TLR1 0 can be used in treating autoinimune disorders, reducing and/or eliminating tissue rejection after transplantation, and in neutrophil-associated disorders. 5 It should be noted that the use of murine or other non-human monoclonal antibodies, human/mouse chimeric mAbs may induce moderate to strong immune responses in some patients. In the most severe cases, such an immune response may lead to the extensive formation of immune complexes, which, potentially, can cause renal failure. Accordingly, preferred monoclonal antibodies used in the practice of the therapeutic methods of the 10 invention are those which are either fully human or humanized and which bind specifically to the target TLR1 0 antigen with high affinity but exhibit low or no antigenicity in the patient. The method of the invention contemplates the administration of single anti-TLR1 0 monoclonal antibodies (mAbs) as well as combinations, or "cocktails", of different mAbs. 15 Two or more monoclonal antibodies that bind to TLR10 may provide an improved effect compared to a single antibody. Alternatively, a combination of an anti-TLR1 0 antibody with an antibody that binds a different antigen may provide an improved effect compared to a single antibody. Such mAb cocktails may have certain advantages inasmuch as they contain mAbs, which exploit different effector mechanisms or combine directly cytotoxic mAbs 20 with mAbs that rely on immune effector functionality. Such mAbs in combination may exhibit synergistic therapeutic effects. In addition, the administration of anti-TLR10 mAbs may be combined with other therapeutic agents, including but not limited to various chemotherapeutic agents, androgen-blockers, and immune modulators (e.g., IL-2, GM-CSF). The anti-TLR10 mAbs may be administered in their "naked" or unconjugated form, or may 25 have therapeutic agents conjugated to them. Additionally, bispecific antibodies may be used. Such an antibody would have one antigenic binding domain specific for TLR1O and the other antigenic binding domain specific for another antigen (such as CD20 for example). Finally, Fab TLR10 antibodies or fragments of these antibodies (including fragments conjugated to other protein sequences or toxins) may also be used as therapeutic agents. 30 Antibodies that specifically bind TLR1 0 are useful in compositions and methods for immunotargeting cells expressing TLR1O and for diagnosing a disease or disorder wherein cells involved in the disorder express TLR10. Such antibodies include monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific WO 2004/047612 PCT/US2003/036971 26 antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds that include CDR and/or antigen-binding sequences, which specifically recognize TLR10. Antibody fragments, including Fab, Fab', F(ab')2, and Fy, are also useful. 5 The term "specific for" indicates that the variable regions of the antibodies recognize and bind TLR10 exclusively (i.e., able to distinguish TLR10 from other similar polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), but may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the 10 antibodies, and in particular, in the constant region of the molecule. Screening assays in which one can detennine binding specificity of an anti-TLR10 antibody are well known and routinely practiced in the art. (Chapter 6, Antibodies A Laboratory Manual, Eds. Harlow, et al., Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988)). TLRl 0 polypeptides can be used to immunize animals to obtain polyclonal and 15 monoclonal antibodies that specifically react with TLR10, respectively. Such antibodies can be obtained using either the entire protein or fragments thereof as an immunogen. The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides have been previously described (Merrifield, J Amer. Chem. Soc. 20 85, 2149-2154 (1963); Krstenansky, et al., FEBS Lett. 211:10 (1987)). Techniques for preparing polyclonal and monoclonal antibodies as well as hybridomas capable of producing the desired antibody have also been previously disclosed (Campbell, Monoclonal Antibodies Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984); St. Groth, et al., J. Immunol. 35:1 25 21 (1990); Kohler and Milstein, Nature 256:495-497 (1975)), the trioma technique, the human B-cell hybridoma technique (Kozbor, et al., Immunology Today 4:72 (1983); Cole, et al., in, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Ic., pp. 77-96 (1985)). Any animal capable of producing antibodies can be immunized with a TLR10 peptide or polypeptide. Methods for immunization include subcutaneous or intraperitoneal 30 injection of the polypeptide. The amount of the TLR1O peptide or polypeptide used for immunization depends on the animal that is immunized, antigenicity of the peptide and the site of injection. The TLR1O peptide or polypeptide used as an immunogen may be modified or administered in an adjuvant in order to increase the protein's antigenicity.

WO 2004/047612 PCT/US2003/036971 27 Methods of increasing the antigenicity of a protein are well known in the art and include, but are not limited to, coupling the antigen with a heterologous protein (such as globulin or p galactosidase) or through the inclusion of an adjuvant during immunization. For monoclonal antibodies, spleen cells from the immunized animals are removed, 5 fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells. Any one of a number of methods well known in the art can be used to identify the hybridoma cell that produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, Western blot analysis, or radioimmunoassay (Lutz, et al., Exp. Cell Res. 175:109-124 10 (1988)). Hybridomas secreting the desired antibodies are cloned and the class and subclass is determined using procedures known in the art (Campbell, A.M., Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984)). Techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies to 15 TLR1O (U.S. Patent 4,946,778). For polyclonal antibodies, antibody-containing antiserum is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures. Because antibodies from rodents tend to elicit strong immune responses against the 20 antibodies when administered to a human, such antibodies may have limited effectiveness in therapeutic methods of the invention. Methods of producing antibodies that do not produce a strong immune response against the administered antibodies are well known in the art. For example, the anti-TLR10 antibody can be a nonhuman primate antibody. Methods of making such antibodies in baboons are disclosed in WO 91/11465 and Losman et al., Int. J 25 Cancer 46:310-314 (1990). In one embodiment, the anti-TLRIO antibody is a humanized monoclonal antibody. Methods of producing humanized antibodies have been previously described. (U.S. Patent Nos. 5,997,867 and 5,985,279, Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323(1988); Verhoeyen et al., Science 239:1534-1536 (1988); Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285-4289 (1992); Sandhu, Crit. Rev. Biotech. 30 12:437-462 (1992); and Singer, et al., J hnmun. 150:2844-2857 (1993)). In another embodiment, the anti-TLR10 antibody is a human monoclonal antibody. Humanized antibodies are produced by transgenic mice that have been engineered to produce human antibodies. Hybridomas derived from such mice will secrete large amounts of human WO 2004/047612 PCT/US2003/036971 28 monoclonal antibodies. Methods for obtaining human antibodies from transgenic mice are described in Green, et al., Nature Genet. 7:13-21 (1994), Lonberg, et al., Nature 368:856 (1994), and Taylor, et al., Int. Innun. 6:579 (1994). The present invention also includes the use of anti-TLR10 antibody fragments. 5 Antibody fragments can be prepared by proteolytic hydrolysis of an antibody or by expression in E. coli of the DNA coding for the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 . This fragment can be further cleaved using a thiol reducing agent, 10 and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly. These methods have been previously described (U.S. Patent Nos. 4,036,945 and 4,331,647, Nisonoff, et al., Arch Biochem. Biophys. 89:230 (1960); Porter, Biochem. J. 73:119 (1959), 15 Edelman, et al., Meth. Enzymol. 1:422 (1967)). Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. For example, Fv fragments comprise an association of VH and VL chains, which can be 20 noncovalent (Inbar et al., Proc. Nat' A cad. Sci. USA 69:2659 (1972)). Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. In one embodiment, the Fv fragments comprise VH and VL chains that are connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by 25 constructing a structural gene comprising DNA sequences encoding the VH and VL domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs have been previously described (U.S. 30 Patent No. 4,946,778, Whitlow, et al., Methods: A Companion to Methods in Enzymology 2:97 (1991), Bird, et al., Science 242:423 (1988), Pack, et al., Bio/Technology 11:1271 (1993)).

WO 2004/047612 PCT/US2003/036971 29 Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the 5 variable region from RNA of antibody-producing cells (Larrick, et al., Methods: A Companion to Methods in Enymology 2:106 (1991); Courtenay-Luck, pp. 166-179 in, Monoclonal Antibodies Production, Engineering and Clinical Applications, Eds. Ritter et al., Cambridge University Press (1995); Ward, et al., pp. 137-185 in, Monoclonal Antibodies Principles and Applications, Eds. Birch et al., Wiley-Liss, Inc. (1995)). 10 The present invention further provides the above- described antibodies in detectably labeled form. Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishing such labeling have been previously 15 disclosed (Sternberger, et al., J. Histochen. Cytochem. 18:315 (1970); Bayer, et al., Meth. Enzym. 62:308 (1979); Engval, et al., linmunol. 109:129 (1972); Goding, J. Immunol. Meth. 13:215 (1976)). The labeled antibodies can be used for in vitro, in vivo, and in situ assays to identify cells or tissues in which TLR1 0 is expressed. Furthermore, the labeled antibodies can be 20 used to identify the presence of secreted TLR1 0 in a biological sample, such as a blood, urine, and saliva samples. 5.5.1 ANTIBODY CONJUGATES The present invention contemplates the use of "naked" anti-TLR1 0 antibodies, as 25 well as the use of immunoconjugates. Immunoconjugates can be prepared by indirectly conjugating a therapeutic agent such as a cytotoxic agent to an antibody component. Toxic moieties include, for example, plant toxins, such as abrin, ricin, modeccin, viscumin, pokeweed anti-viral protein, saporin, gelonin, momoridin, trichosanthin, barley toxin; bacterial toxins, such as Diptheria toxin, Pseudomonas endotoxin and exotoxin, 30 Staphylococcal enterotoxin A; fungal toxins, such as c-sarcin, restrictocin; cytotoxic RNases, such as extracellular pancreatic RNases; DNase I (Pastan, et al., Cell 47:641 (1986); Goldenberg, Cancer Journalfor Clinicians 44:43 (1994)), calicheamicin, and radioisotopes, such as 3P, 67 Cu, 77 As, 1 05 Rh, 1 09 Pd, "'Ag, mSn, m1 166 Ho, 1 77 Lu, 186Re, "Re, 194, 1 99 Au WO 2004/047612 PCT/US2003/036971 30 (Illidge, T.M. & Brock, S., Curr Pharm. Design 6: 1399 (2000)). In humans, clinical trials are underway utilizing a yttrium-90 conjugated anti-CD20 antibody for B cell lymphomas (Cancer Chenother Pharmacol 48(Suppl 1):S91-S95 (2001)). General techniques have been previously described (U.S. Patent Nos. 6,306,393 and 5 5,057,313, Shih, et al., Int. J. Cancer 41:832-839 (1988); Shih, et al., Int. J Cancer 46:1101-1106 (1990)). The general method involves reacting an antibody component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine function and that is loaded with a plurality of drug, toxin, chelator, boron addends, or other therapeutic agent. This reaction results in an initial Schiff base (imine) linkage, which 10 can be stabilized by reduction to a secondary amine to form the final conjugate. The carrier polymer is preferably an aminodextran or polypeptide of at least 50 amino acid residues, although other substantially equivalent polymer carriers can also be used. Preferably, the final immunoconjugate is soluble in an aqueous solution, such as mammalian serum, for ease of administration and effective targeting for use in therapy. 15 Thus, solubilizing functions on the carrier polymer will enhance the serum solubility of the final immunoconjugate. In particular, an aminodextran will be preferred. The process for preparing an inmnunoconjugate with an aminodextran carrier typically begins with a dextran polymer, advantageously a dextran of average molecular weight of about 10,000-100,000. The dextran is reacted with an oxidizing agent to affect a 20 controlled oxidation of a portion of its carbohydrate rings to generate aldehyde groups. The oxidation is conveniently effected with glycolytic chemical reagents such as NaIO 4 , according to conventional procedures. The oxidized dextran is then reacted with a polyamine, preferably a diamine, and more preferably, a mono- or polyhydroxy diamine. Suitable amines include ethylene diamine, propylene diamine, or other like polymethylene 25 diamines, diethylene triamine or like polyamines, 1,3-diamino-2-hydroxypropane, or other like hydroxylated diamines or polyamines, and the like. An excess of the amine relative to the aldehyde groups of the dextran is used to ensure substantially complete conversion of the aldehyde functions to Schiff base groups. A reducing agent, such as NaBH 4 , NaBH 3 CN or the like, is used to effect reductive stabilization of the resultant Schiff base intermediate. 30 The resultant adduct can be purified by passage through a conventional sizing column or ultrafiltration membrane to remove cross-linked dextrans. Other conventional methods of derivatizing a dextran to introduce amine functions can also be used, e.g., reaction with cyanogen bromide, followed by reaction with a diamine.

WO 2004/047612 PCT/US2003/036971 31 The anminodextran is then reacted with a derivative of the particular drug, toxin, chelator, immunomodulator, boron addend, or other therapeutic agent to be loaded, in an activated form, preferably, a carboxyl-activated derivative, prepared by conventional means, e.g., using dicyclohexylcarbodiimide (DCC) or a water soluble variant thereof, to form an 5 intermediate adduct. Alternatively, polypeptide toxins such as pokeweed antiviral protein or ricin A-chain, and the like, can be coupled to aminodextran by glutaraldehyde condensation or by reaction of activated carboxyl groups on the protein with amines on the aminodextran. Chelators for radiometals or magnetic resonance enhancers are well-known in the art. Typical are derivatives of ethylenediaminetetraacetic acid (EDTA) and 10 diethylenetriaminepentaacetic acid (DTPA). These chelators typically have groups on the side chain by which the chelator can be attached to a carrier. Such groups include, e.g., benzylisothiocyanate, by which the DTPA or EDTA can be coupled to the amine group of a carrier. Alternatively, carboxyl groups or amine groups on a chelator can be coupled to a carrier by activation or prior derivatization and then coupling, all by well-known means. 15 Boron addends, such as carboranes, can be attached to antibody components by conventional methods. For example, carboranes can be prepared with carboxyl functions on pendant side chains, as is well known in the art. Attachment of such carboranes to a carrier, e.g., aminodextran, can be achieved by activation of the carboxyl groups of the carboranes and condensation with amines on the carrier to produce an intermediate conjugate. Such 20 intermediate conjugates are then attached to antibody components to produce therapeutically useful immunoconjugates, as described below. A polypeptide carrier can be used instead of aminodextran, but the polypeptide carrier should have at least 50 amino acid residues in the chain, preferably 100-5000 amino acid residues. At least some of the amino acids should be lysine residues or glutamate or 25 aspartate residues. The pendant amines of lysine residues and pendant carboxylates of glutamine and aspartate are convenient for attaching a drug, toxin, immunomodulator, chelator, boron addend or other therapeutic agent. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixed polymers of these amino acids and others, e.g., serines, to confer desirable solubility 30 properties on the resultant loaded carrier and immunoconjugate. Conjugation of the intermediate conjugate with the antibody component is effected by oxidizing the carbohydrate portion of the antibody component and reacting the resulting aldehyde (and ketone) carbonyls with amine groups remaining on the carrier after loading WO 2004/047612 PCT/US2003/036971 32 with a drug, toxin, chelator, inmunomodulator, boron addend, or other therapeutic agent. Alternatively, an intermediate conjugate can be attached to an oxidized antibody component via amine groups that have been introduced in the intermediate conjugate after loading with the therapeutic agent. Oxidation is conveniently effected either chemically, e.g., with NaIO 4 5 or other glycolytic reagent, or enzymatically, e.g., with neuraminidase and galactose oxidase. In the case of an aminodextran carrier, not all of the amines of the aminodextran are typically used for loading a therapeutic agent. The remaining amines of aminodextran condense with the oxidized antibody component to form Schiff base adducts, which are then reductively stabilized, normally with a borohydride reducing agent. 10 Analogous procedures are used to produce other immunoconjugates according to the invention. Loaded polypeptide carriers preferably have free lysine residues remaining for condensation with the oxidized carbohydrate portion of an antibody component. Carboxyls on the polypeptide carrier can, if necessary, be converted to amines by, e.g., activation with DCC and reaction with an excess of a diamine. 15 The final immunoconjugate is purified using conventional techniques, such as sizing chromatography on Sephacryl S-300 or affinity chromatography using one or more TLR10 epitopes. Alternatively, immunoconjugates can be prepared by directly conjugating an antibody component with a therapeutic agent. The general procedure is analogous to the 20 indirect method of conjugation except that a therapeutic agent is directly attached to an oxidized antibody component. It will be appreciated that other therapeutic agents can be substituted for the chelators described herein. Those of skill in the art will be able to devise conjugation schemes without undue experimentation. As a further illustration, a therapeutic agent can be attached at the hinge region of a 25 reduced antibody component via disulfide bond formation. For example, the tetanus toxoid peptides can be constructed with a single cysteine residue that is used to attach the peptide to an antibody component. As an alternative, such peptides can be attached to the antibody component using a heterobifunctional cross-linker, such as N-succinyl 3-(2 pyridyldithio)proprionate (SPDP) (Yu, et al., Int. J. Cancer 56:244 (1994)). General 30 techniques for such conjugation have been previously described (Wong, Chemistry of Protein Conjugation and Cross-linking, CRC Press (1991); Upeslacis, et al., pp. 187-230 in, Monoclonal Antibodies Principles and Applications, Eds. Birch et al., Wiley-Liss, Inc.

WO 2004/047612 PCT/US2003/036971 33 (1995); Price, pp. 60-84 in, Monoclonal Antibodies: Production, Engineering and Clinical Applications Eds. Ritter, et al., Cambridge University Press (1995)). As described above, carbohydrate moieties in the Fc region of an antibody can be used to conjugate a therapeutic agent. However, the Fe region may be absent if an antibody 5 fragment is used as the antibody component of the immunoconjugate. Nevertheless, it is possible to introduce a carbohydrate moiety into the light chain variable region of an antibody or antibody fragment (Leung, et al., J. Jmninunol. 154:5919-5926 (1995); U.S. Pat. No. 5,443,953). The engineered carbohydrate moiety is then used to attach a therapeutic agent. 10 In addition, those of skill in the art will recognize numerous possible variations of the conjugation methods. For example, the carbohydrate moiety can be used to attach polyethyleneglycol in order to extend the half-life of an intact antibody, or antigen-binding fragment thereof, in blood, lymph, or other extracellular fluids. Moreover, it is possible to construct a "divalent immunoconjugate" by attaching therapeutic agents to a carbohydrate 15 moiety and to a free sulfhydryl group. Such a free sulfhydryl group may be located in the hinge region of the antibody component. 5.5.2 ANTIBODY FUSION PROTEINS When the therapeutic agent to be conjugated to the antibody is a protein, the present 20 invention contemplates the use of fusion proteins comprising one or more anti-TLRI 0 antibody moieties and an immunomodulator or toxin moiety. Methods of making antibody fusion proteins have been previously described (U.S. Patent No. 6,306,393). Antibody fusion proteins comprising an interleukin-2 moiety have also been previously disclosed (Boleti, et al., Ann. Oncol. 6:945 (1995), Nicolet, et al., Cancer Gene Ther. 2:161 (1995), 25 Becker, et al., Proc. Nat'l Acad. Sci. USA 93:7826 (1996), Hank, et al., Clin. Cancer Res. 2:1951 (1996), Hu, et al., Cancer Res. 56:4998 (1996)). In addition, Yang, et al., Hum. Antibodies Hybridomas 6:129 (1995), describe a fusion protein that includes an F(ab') 2 fragment and a tumor necrosis factor alpha moiety. Methods of making antibody-toxin fusion proteins in which a recombinant molecule 30 comprises one or more antibody components and a toxin or chemotherapeutic agent also are known to those of skill in the art. For example, antibody-Pseudomnonas exotoxin A fusion proteins have been described (Chaudhary, et al., Nature 339:394 (1989), Brinkmann, et al., Proc. Nat'l Acad. Sci. USA 88:8616 (1991), Batra, et al., Proc. Nat!. Acad. Sci. USA 89:5867 WO 2004/047612 PCT/US2003/036971 34 (1992), Friedman, et al., J. Immunol. 150:3054 (1993), Wels, et al., lit. J Can. 60:137 (1995), Fominaya et al., J Biol. Chem. 271:10560 (1996), Kuan, et al., Biochemistry 35:2872 (1996), Schmidt, et al., it. J. Can. 65:538 (1996)). Antibody-toxin fusion proteins containing a diphtheria toxin moiety have been described (Kreitman, et al., Leukemia 7:553 5 (1993), Nicholls, et al., J. Biol. Chem. 268:5302 (1993), Thompson, et al., J. Biol. Chem. 270:28037 (1995), and Vallera, et al., Blood 88:2342 (1996). Deonarain et al. (Tumor Targeting 1:177 (1995)), have described an antibody-toxin fusion protein having an RNase moiety, while Linardou, et al. (Cell Biophys. 24-25:243 (1994)), produced an antibody-toxin fusion protein comprising a DNase I component. Gelonin and Staphylococcal enterotoxin-A 10 have been used as the toxin moieties in antibody-toxin fusion proteins (Wang, et al., Abstracts of the 209th ACS National Meeting, Anaheim, Calif., Apr. 2-6, 1995, Part 1, BIOT005; Dohlsten, et al., Proc. Nat'l Acad. Sci. USA 91:8945 (1994)). 5.5.3 Fab FRAGMENTS AND SINGLE CHAIN ANTIBODIES 15 According to the invention, techniques can be adapted for the production of single-chain antibodies specific to TLR10 (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., Science 246:1275-1281 (1989)) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or 20 homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F, fragments. 25 5.5.4 BISPECIFIC ANTIBODIES Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding 30 target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit. Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two WO 2004/047612 PCT/US2003/036971 35 immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the 5 correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., 1991 EMBO J., 10, 3655-3659. Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion 10 preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co 15 transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986). According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least 20 a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or 25 threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g. F(ab') 2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies 30 can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.

WO 2004/047612 PCT/US2003/036971 36 The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used 5 as agents for the selective immobilization of enzymes. Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical 10 coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies 15 have been produced using leucine zippers. Kostelny et al., J. Innunol. 148:1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. 20 The "diabody" technology described by Hollinger et al., Proc. Nat. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are 25 forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994). Antibodies with more than two valencies are contemplated. For example, trispecific 30 antibodies can be prepared. Tutt et al., J Immunol. 147:60 (1991). Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering WO 2004/047612 PCT/US2003/036971 37 molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcyR), such as Fc-yRI (CD64), FcyRII (CD32) and FcyRIII (CD 16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a 5 particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF). 10 5.5.5 HETEROCONJUGATE ANTIBODIES Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 15 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. 20 Patent No. 4,676,980. 5.5.6 EFFECTOR FUNCTION ENGINEERING It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For 25 example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond fonnation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J Inmunol., 148:2918-2922 (1992). 30 Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53:2560 2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can WO 2004/047612 PCT/US2003/036971 38 thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989). 5.6 TLR1O PEPTIDES 5 The TLR1O peptide itself may be used to target toxins or radioisotopes to tumor cells in vivo. TLR1O may be a homophilic adhesion protein which will bind to itself. In this case the extracellular domain of TLR10, or a fragment of this domain, may be able to bind to TLR10 expressed on tumor cells. This peptide fragment then may be used as a means to deliver cytotoxic agents to TLR10 bearing tumor cells. Much like an antibody, these 10 fragments may specifically target cells expressing this antigen. Targeted delivery of these cytotoxic agents to the tumor cells would result in cell death and suppression of tumor growth. An example of the ability of an extracellular fragment binding to and activating its intact receptor (by homophilic binding) has been demonstrated with the CD84 receptor (Martin, et al., J. Ininunol, 167:3668-3676 (2001)). 15 Cell surface molecules that bind TLRI 0 polypeptides, herein denoted TLrl 0 binding receptors, may be useful for TLR10 targeting therapy. Extracellular fragments of a TLR10 receptor may be used to modulate immune cells expressing the protein. On cells bearing a TLR10 binding receptor (such as neutrophils, NK cells, T cells, B cells and myeloid cells) this may result in stimulating the release of cytokines (such as interferon gamma for 20 example) that may enhance or suppress the immune system. Additionally, binding of these fragments to cells bearing a TLR1O binding receptor may result in the activation of these cells and also may stimulate proliferation. Some fragments may bind to an intact TLR1O binding receptor and block activation signals and cytokine release by immune cells. These fragments would then have an immunosuppressive effect. Fragments that activate and 25 stimulate the immune system may have anti-tumor properties. These fragments may stimulate an immunological response that can result in immune mediated tumor cell killing. The same fragments may result in stimulating the immune system to mount an enhance response to foreign invaders such as viruses and bacteria. Fragments that suppress the immune response may be useful in treating lymphoproliferative disorders, autoimmune 30 disease, graft-vs-host disease, and inflammatory disorders such as emphysema.

WO 2004/047612 PCT/US2003/036971 39 5.7 OTHER BINDING PEPTIDES OR SMALL MOLECULES Screening of organic compound or peptide libraries with recombinantly expressed TLRl 0 protein may be useful for identification of therapeutic molecules that function to specifically bind to or even inhibit the activity of TLR1 0. Synthetic and naturally occurring 5 products can be screened in a number of ways deemed routine to those of skill in the art. Random peptide libraries are displayed on phage (phage display) or on bacteria, such as on E. coli. These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or a polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. By 10 way of example, diversity libraries, such as random or combinatorial peptide or non-peptide libraries can be screened for molecules that specifically bind to TLR1O polypeptides. Many libraries are known in the art that can be used, i.e. chemically synthesized libraries, recombinant (i.e. phage display libraries), and in vitro translation-based libraries. Techniques for creating and screening such random peptide display libraries are known in 15 the art (Ladner et al., U.S. Patent No. 5,223, 409; Ladner et al., U.S. Patent No. 4,946,778; Ladner et al., U.S. Patent No. 5,403,484; Ladner et al., U.S. Patent No. 5,571,698, all of which are herein incorporated by reference in their entirety) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. 20 (Beverly, MA), and Pharmacia KLB Biotechnology Inc. (Piscataway, NJ). Random peptide display libraries can be screened using the TLR1O sequences disclosed herein to identify proteins which bind to TLR1 0. Examples of chemically synthesized libraries are described in Fodor et al., Science 251:767-773 (1991); Houghten et al., Nature 354:84-86 (1991); Lam et al., Nature 354:82 25 84 (1991); Medynski, Bio/Technology 12:709-710 (1994); Gallop et al., J Med. Chem. 37:1233-1251 (1994); Ohlmeyer et al., Proc. NatL. Acad. Sci. USA 90:10922-10926 (1993); Erb et al., Proc. Natl. Acad. Sci. USA 91:11422-11426 (1994); Houghten et al., Biotechniques 13:412 (1992); Jayawickreme et al., Proc. Natl. Acad. Sci. USA 91:1614-1618 (1994); Salmon et al., Proc. Natl. Acad. Sci. USA 90:11708-11712 (1993); PCT Publication 30 No. WO 93/20242; Brenner and Lerner, Proc. Nat. Acad. Sci. USA 89:5381-5383 (1992), all of which are herein incorporated by reference in their entirety. Examples of phage display libraries are described in Scott and Smith, Science 249:386-390 (1990); Devlin et al., Science 249:404-406 (1990); Christian et al., J Mol. Biol.

WO 2004/047612 PCT/US2003/036971 40 227:711-718 (1992); Lenstra, J. Immunol Meth. 152:149-157 (1992); Kay et al., Gene 128:59-65 (1993); PCT Publication No. WO 94/18318, all of which are herein incorporated by reference in their entirety. In vitro translation-based libraries include but are not limited to those described in 5 PCT Publication No. WO 91105058, and Mattheakis et al., Proc. Nat. Acad. Sci. USA 91:9022-9026 (1994), both of which are herein incorporated by reference in their entirety. By way of examples of non-peptide libraries, a benzodiazepine library (see for example, Bunin et al., Proc. Nat!. A cad. Sci. USA 91:4708-4712 (1994), herein incorporated by reference in its entirety) can be adapted for use. Peptoid libraries (Simon et al., Proc. 10 Nat!. A cad. Sci. USA 89:9367-9371 (1992), herein incorporated by reference in its entirety) can also be used. Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (Proc. Natd. Acad. Sci. USA 91:11138 11142 (1994), herein incorporated by reference in its entirety). 15 Screening the libraries can be accomplished by any of a variety of commonly known methods. See, for example, the following references which disclose screening of peptide libraries: Parmley and Smith, Adv. Exp. Med. Biol. 251:215-218 (1989); Scott and Smith, Science 249:386-390 (1990); Fowlkes et al., Biotechniques 13:422-427 (1992); Oldenburg et al., Proc. Natl. Acad. Sci. USA 89:5393-5397 (1992); Yu et al., Cell 76:933-945 (1994); 20 Staudt et al., Science 241:577-580 (1988); Bock et al., Nature 355:564-566 (1992); Tuerk et al., Proc. NatL. Acad. Sci. USA 89:6988-6992 (1992); Ellington et al., Nature 355:850-852 (1992); Rebar and Pabo, Science 263:671-673 (1993); and PCT Publication No. WO 94/18318, all of which are herein incorporated by reference in their entirety. In a specific embodiment, screening can be carried out by contacting the library 25 members with a TLR1O protein (or nucleic acid or derivative) immobilized on a solid phase and harvesting those library members that bind to the protein (or nucleic acid or derivative). Examples of such screening methods, termed "panning" techniques are described by way of example in Parmley and Smith, Gene 73:305-318 (1988); Fowlkes et al., Biotechniques 13:422-427 (1992); PCT Publication No. WO 94/18318, all of which are herein incorporated 30 by reference in their entirety, and in references cited hereinabove. In another embodiment, the two-hybrid system for selecting interacting protein in yeast (Fields and Song, Nature 340:245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA WO 2004/047612 PCT/US2003/036971 41 88:9578-9582 (1991), both of which are herein incorporated by reference in their entirety) can be used to identify molecules that specifically bind to TLR1O or a TLR1O derivative. These "binding polypeptides" or small molecules which interact with TLR1O polypeptides can be used for tagging or targeting cells; for isolating homolog polypeptides 5 by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding polypeptides or small molecules can also be used in analytical methods such as for screening expression libraries and neutralizing activity, i.e., for blocking interaction between ligand and receptor, or viral binding to a receptor. The binding polypeptides or small molecules can also be used for diagnostic assays for 10 determining circulating levels of TLR10 polypeptides of the invention; for detecting or quantitating soluble TLR10 polypeptides as marker of underlying pathology or disease. These binding polypeptides or small molecules can also act as TLRIO "antagonists" to block TLRIO binding and signal transduction in vitro and in vivo. These TLR1O binding polypeptides or small molecules would be useful for inhibiting TLR10 activity or protein 15 binding. Binding polypeptides can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. Binding peptides can also be fused to other polypeptides, for example an immunoglobulin constant chain or portions thereof, to enhance their half-life, and can be 20 made multivalent (through, e.g. branched or repeating units) to increase binding affinity for TLR10. For instance, binding polypeptides of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance). More specifically, binding polypeptides or bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules 25 and delivered to a mammal having cells, tissues or organs that express the anti complementary molecule. Suitable detectable molecules may be directly or indirectly attached to the binding polypeptide, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, cheniluminescent markers, magnetic particles and the like, Suitable 30 cytotoxic molecules may be directly or indirectly attached to the binding polypeptide, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudononas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-13 1, rhenium-188, or yttrium-90 (either directly attached to the binding polypeptide, or indirectly attached through WO 2004/047612 PCT/US2003/036971 42 a means of a chelating moiety, for instance). Binding polypeptides may also be conjugated to cytotoxic drags, such as adriamycin. For indirect attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated with a member of a complementary/anticomplementary pair, where the other member is bound to the binding 5 polypeptide. For these purposes, biotin/streptavidin is an exemplary complementary/anticomplementary pair. In another embodiment, binding polypeptide-toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues). Alternatively, if the binding polypeptide has multiple functional domains (i.e., an activation 10 domain or a ligand binding domain, plus a targeting domain), a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule, or a complementary molecule to a cell or tissue type of interest. In instances where the domain only fusion protein includes a complementary molecule, the anti complementary molecule can be conjugated to a detectable or cytotoxic molecule. Such 15 domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue-specific delivery of generic anti-complementary-detectable/cytotoxic molecule conjugates. 5.8 DISEASES AMENABLE TO TLR10 TARGETING THERAPY 20 In one aspect, the present invention provides reagents and methods useful for treating diseases and conditions wherein cells associated with the disease or disorder express TLR1O. These diseases can include cancers, and other hyperproliferative conditions, such as hyperplasia, psoriasis, contact dermatitis, immunological disorders, and infertility. Whether the cells associated with a disease or condition express TLR1 0 can be detennined using the 25 diagnostic methods described herein. Comparisons of TLR10 mRNA and protein expression levels between diseased cells, tissue or fluid (blood, lymphatic fluid, etc.) and corresponding normal samples are made to determine if the patient will be responsive to TLR10 targeting therapy. Methods for detecting and quantifying the expression of TLR10 mRNA or protein use standard nucleic 30 acid and protein detection and quantitation techniques that are well known in the art and are described in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989) or Ausubel, et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY (1989), both of which are incorporated herein by WO 2004/047612 PCT/US2003/036971 43 reference in their entirety. Standard methods for the detection and quantification of TLR10 mRNA include in situ hybridization using labeled TLR1O riboprobes (Gemou-Engesaeth, et al., Pediatrics 109: E24-E32 (2002)), Northern blot and related techniques using TLR10 polynucleotide probes (Kunzli, et al., Cancer 94: 228 (2002)), RT-PCR analysis using 5 TLR1 0-specific primers (Angchaiskisiri, et al., Blood 99:130 (2002)), and other amplification detection methods, such as branched chain DNA solution hybridization assay (Jardi, et al., J Viral Hepat. 8:465-471 (2001)), transcription-mediated amplification (Kimura, et al., J. Clin. Microbiol. 40:439-445 (2002)), microarray products, such as oligos, cDNAs, and monoclonal antibodies, and real-time PCR (Simpson, et al., Molec. Vision, 10 6:178-183 (2000)). Standard methods for the detection and quantification of TLR1O protein include western blot analysis (Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989), Ausubel, et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY (1989)), immunocytochemistry (Racila, et al., Proc. NatL. A cad. Sci. USA 95:4589-4594 (1998)), and a variety of immunoassays, including 15 enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), and specific enzyme immunoassay (HTA) (Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989), Ausubel, et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY (1989)). Peripheral blood cells can also be analyzed for TLR1 0 expression using flow cytometry using, for example, immunomagnetic 20 beads specific for TLR1O (Racila, et al., Proc. Natl. Acad. Sci. USA 95:4589-4594 (1998)) or biotinylated TLR10 antibodies (Soltys, et al., J. Immunol. 168:1903 (2002)). Tumor aggressiveness can be gauged by determining the levels of TLR10 protein or mRNA in tumor cells compared to the corresponding normal cells (Orlandi, et al., Cancer Res. 62:567 (2002)). hi one embodiment, the disease or disorder is a cancer. 25 The cancers treatable by methods of the present invention preferably occur in mammals. Mammals include, for example, humans and other primates, as well as pet or companion animals such as dogs and cats, laboratory animals such as rats, mice and rabbits, and farm animals such as horses, pigs, sheep, and cattle. Tumors or neoplasms include growths of tissue cells in which the multiplication of 30 the cells is uncontrolled and progressive. Some such growths are benign, but others are termed "malignant" and may lead to death of the organism. Malignant neoplasms or "cancers" are distinguished from benign growths in that, in addition to exhibiting aggressive cellular proliferation, they may invade surrounding tissues and metastasize. Moreover, WO 2004/047612 PCT/US2003/036971 44 malignant neoplasms are characterized in that they show a greater loss of differentiation (greater "dedifferentiation"), and greater loss of their organization relative to one another and their surrounding tissues. This property is also called "anaplasia." Neoplasms treatable by the present invention also include solid phase 5 tumors/malignancies, i.e., carcinomas, locally advanced tumors and human soft tissue sarcomas. Carcinomas include those malignant neoplasms derived from epithelial cells that infiltrate (invade) the surrounding tissues and give rise to metastastic cancers, including lymphatic metastases. Adenocarcinomas are carcinomas derived from glandular tissue, or which form recognizable glandular structures. Another broad category or cancers includes 10 sarcomas, which are tumors whose cells are embedded in a fibrillar or homogeneous substance like embryonic connective tissue. As seen in Table 4, TLR10 is expressed in breast, colon, prostate, and squamous cell carcinoma. It is likely that other epithelial and squamous cell cancers will express TLR1O as well. In addition, other solid phase tumors may express TLR1 0. The invention also enables treatment of cancers of the myeloid or 15 lymphoid systems, including leukemias, lymphomas and other cancers that typically do not present as a tumor mass, but are distributed in the vascular or lymphoreticular systems. The type of cancer or tumor cells that may be amenable to treatment according to the invention include, for example, acute lymphocytic leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, cutaneous T-cell 20 lymphoma, hairy cell leukemia, acute myeloid leukemia, erythroleukemia, chronic mycloid (granulocytic) leukemia, Hodgkin's disease, and non-Hodgkin's lymphoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer, polyps associated with colorectal neoplasms, pancreatic cancer and gallbladder cancer, cancer of the adrenal cortex, ACTH-producing tumor, bladder cancer, brain cancer including intrinsic 25 brain tumors, neuroblastomas, astrocytic brain tumors, gliomas, and metastatic tumor cell invasion of the central nervous system, Ewing's sarcoma, head and neck cancer including mouth cancer and larynx cancer, kidney cancer including renal cell carcinoma, liver cancer, lung cancer including small and non-small cell lung cancers, malignant peritoneal effusion, malignant pleural effusion, skin cancers including malignant melanoma, tumor progression 30 of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, and hemangiopericytoma, mesothelioma, Kaposi's sarcoma, bone cancer including osteomas and sarcomas such as fibrosarcoma and osteosarcoma, cancers of the female reproductive tract including uterine cancer, endometrial cancer, ovarian cancer, ovarian (germ cell) cancer and WO 2004/047612 PCT/US2003/036971 45 solid tumors in the ovarian follicle, vaginal cancer, cancer of the vulva, and cervical cancer; breast cancer (small cell and ductal), penile cancer, prostate cancer, retinoblastoma, testicular cancer, thyroid cancer, trophoblastic neoplasms, and Wilms' tumor. The invention is particularly illustrated herein in reference to treatment of certain 5 types of experimentally defined cancers. In these illustrative treatments, standard state-of the-art in vitro and in vivo models have been used. These methods can be used to identify agents that can be expected to be efficacious in in vivo treatment regimens. However, it will be understood that the method of the invention is not limited to the treatment of these tumor types, but extends to any cancer derived from any organ system. As demonstrated in the 10 Examples, TLR9 and TLR10 are highly expressed in B-cell related disorders. Leukemias can result from uncontrolled B cell proliferation initially within the bone marrow before disseminating to the peripheral blood, spleen, lymph nodes and finally to other tissues. Uncontrolled B cell proliferation also may result in the development of lymphomas that arise within the lymph nodes and then spread to the blood and bone marrow. Immunotargeting of 15 TLR9 or TLRI 0 is used in treating B cell malignancies, leukemias, lymphomas and myelomas including but not limited to multiple myeloma, Burkitt's lymphoma, cutaneous B cell lymphoma, primary follicular cutaneous B cell lymphoma, B lineage acute lymphoblastic leukemia (ALL), B cell non-Hodgkin's lymphoma (NHL), B cell chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia, hairy cell leukemia (HCL), 20 acute myelogenous leukemia, acute myelomonocytic leukemia, chronic myelogenous leukemia, lymphosarcoma cell leukemia, B cell large cell lymphoma, malignant lymphoma, splenic marginal zone lymphoma, diffuse large B cell lymphoma, prolymphocytic leukemia (PLL), lymphoplasma cytoid lymphoma, mantle cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, primary thyroid lymphoma, intravascular malignant 25 lymphomatosis, splenic lymphoma, Hodgkin's Disease, and intragraft angiotropic large-cell lymphoma. Expression of TLR10 has also been demonstrated in Examples 1-2 in myeloid leukemia cell lines and tissue, T cell leukemia cell lines and T cell lymphoma tissues and may be treated with TLR 10 antibodies. Other diseases that may be treated by the methods of the present invention include multicentric Castleman's disease, primary amyloidosis, 30 Franklin's disease, Seligmann's disease, primary effusion lymphoma, post-transplant lymphoproliferative disease (PTLD) [associated with EBV infection.], paraneoplastic pemphigus, chronic lymphoproliferative disorders, X-linked lymphoproliferative syndrome (XLP), acquired angioedema, angioimmunoblastic lymphadenopathy with dysproteinemia, WO 2004/047612 PCT/US2003/036971 46 Herman's syndrome, post-splenectomy syndrome, congenital dyserythropoietic anemia type III, lymphoma-associated hemophagocytic syndrome (LAHS), necrotizing ulcerative stomatitis, Kikuchi's disease, lymphomatoid granulomatosis, Richter's syndrome, polycythemic vera (PV), Gaucher's disease, Gougerot-Sjogren syndrome, Kaposi's sarcoma, 5 cerebral lymphoplasmocytic proliferation (Bind and Neel syndrome), X-linked lymphoproliferative disorders, pathogen associated disorders such as mononucleosis (Epstein Barr Virus), lymphoplasma cellular disorders, post-transplantational plasma cell dyscrasias, and Good's syndrome. Autoimmune diseases can be associated with hyperactive B cell activity that results 10 in autoantibody production. Inhibition of the development of autoantibody-producing cells or proliferation of such cells may be therapeutically effective in decreasing the levels of autoantibodies in autoimmune diseases including but not limited to systemic lupus erythematosus, Crohn's Disease, graft-verses-host disease, Graves' disease, myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, asthma, 15 cryoglubulinemia, primary biliary sclerosis, pernicious anemia, Waldenstrom macroglobulinemia, hyperviscosity syndrome, macroglobulinemia, cold agglutinin disease, monoclonal gammopathy of undetermined origin, anctoderma and POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, M component, skin changes), connective tissue disease, multiple sclerosis, cystic fibrosis, rheumatoid arthritis, autoimniune 20 pulmonary inflammation, psoriasis, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, autoimmune inflammatory eye disease, Goodpasture's disease, Rasmussen's encephalitis, dermatitis herpetiformis, thyoma, autoimmune polyglandular syndrome type 1, primary and secondary membranous nephropathy, cancer associated retinopathy, autoimmune hepatitis type 1, mixed cryoglobulinemia with renal 25 involvement, cystoid macular edema, endometriosis, IgM polyneuropathy (including Hyper IgM syndrome), demyclinating diseases, angiomatosis, and monoclonal gammopathy. As shown in Table 5, IHC analysis showed TLR1 0 expression on tissues from the following autoimmune disorders: systemic lupus erythematosus, Hasimoto thyroiditis, Sjurgen's syndrome, and pericarditis lupus; thus targeting TLR1 0 may be useful therapies for these 30 and other autoimmune disorders. Targeting of TLR10 may also be useful in the treatment of allergic reactions and conditions e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, WO 2004/047612 PCT/US2003/036971 47 eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis, allergic gastroenteropathy, inflammatory bowel disorder (IBD), and contact allergies, such as asthma (particularly allergic asthma), or other 5 respiratory problems. Targeting TLR1 0 may also be useful in the management or prevention of transplant rejection in patients in need of transplants such as stem cells, tissue or organ transplant. Thus, one aspect of the invention may find therapeutic utility in various diseases (such as those usually treated with transplantation, including without limitation, aplastic anemia and 10 paroxysmal nocturnal hemoglobinuria) as wells in repopulating the stem cell compartment post irridiation/chemotherapy, either in vivo or ex vivo (i.e. in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous) as normal cells or genetically manipulated for gene therapy. As shown in Tables 6, IHC analysis showed TLR1 0 is expressed in rejected heart, liver and kidney tissue 15 after transplantation as opposed to normal tissue. Thus, targeting of TLR10 may be useful to prevent and/or reduce tissue rejection after transplantation. Targeting of TLR10 may also be possible to modulate immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune 20 response. Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, TLR1 0)), e.g., modulating or preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue 25 transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells (i.e. cytotoxic T lymphocytes), followed by an immune reaction that destroys the transplant. The administration of a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, a lack of costimulation may also be sufficient to anergize 30 the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.

WO 2004/047612 PCT/US2003/036971 48 The efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been 5 used to examine the immnunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Nat. Acad. Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of therapeutic compositions of the invention on the development of that 10 disease. Targeting TLR1 0 may also be useful in the treatment of chronic inflammation. As shown in Figure 1, TLR10 is highly expressed in neutrophils. Leukocytes, including neutrophils, are recruited to sites of infection or injury and are subsequently cleared at the end of the inflammatory response. However, in situations of chronic inflammation, removal 15 of such neutrophils is impaired or inhibited; therefore, destruction of these neutrophils by means of TLr1O targeting can be used in the management of chronic inflammation. Conditions that may benefit from such therapy include, but are not limited to, inflammatory disorders, autoimmune diseases, asthma, chronic pancreatitis, allergic dermatitis, idiopathic pulmonary fibrosis, upper and lower respiratory infection, acute pulmonary inflammation, 20 wound healing, graft vs. host disease, chronic obstructive pulmonary disease, arthritis, periodontitis, and disorders relating to oxidative stress, including hypertension pathogenesis and athlerosclerosis. 5.9 ADMINISTRATION 25 The anti-TLR1 0 monoclonal antibodies used in the practice of a method of the invention may be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material which when combined with the anti-TLR1 0 antibodies retain the anti-tumor function of the antibody and are nonreactive with the subject's immune systems. Examples include, but are not limited to, 30 any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like. The anti-TLR10 antibody formulations may be administered via any route capable of delivering the antibodies to the tumor site. Potentially effective routes of administration WO 2004/047612 PCT/US2003/036971 49 include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like. The preferred route of administration is by intravenous injection. A preferred formulation for intravenous injection comprises anti-TLR10 mAbs in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in 5 polyvinylchloride or polyethylene bags containing 0.9% sterile sodium chloride for Injection, USP. The anti-TLR1O mAb preparation may be lyophilized and stored as a sterile powder, preferably under vacuum, and then reconstituted in bacteriostatic water containing, for example, benzyl alcohol preservative, or in sterile water prior to injection. Treatment will generally involve the repeated administration of the anti-TLR1 0 10 antibody preparation via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1 to about 10 mg/kg body weight; however other exemplary doses in the range of 0.01 mg/kg to about 100 mg/kg are also contemplated. Doses in the range of 10-500 mg mAb per week may be effective and well tolerated. Rituximab (Rituxan@), a chimeric CD20 antibody used to treat B-cell lymphoma, non 15 Hodgkin's lymphoma, and relapsed indolent lymphoma, is typically administered at 375 mg/m 2 by IV infusion once a week for 4 to 8 doses. Sometimes a second course is necessary, but no more than 2 courses are allowed. An effective dosage range for Rituxan@ would be 50 to 500 mg/m 2 (Maloney, et al., Blood 84: 2457-2466 (1994); Davis, et al., J. Clin. Oncol. 18: 3135-3143 (2000)). Based on clinical experience with Trastuzumab 20 (Herceptin@), a humanized monoclonal antibody used to treat HER2 (human epidermal growth factor 2)-positive metastatic breast cancer (Slamon, et al., Mol Cell Biol. 9: 1165 (1989)), an initial loading dose of approximately 4 mg/kg patient body weight IV followed by weekly doses of about 2 mg/kg IV of the anti-TLR10 mAb preparation may represent an acceptable dosing regimen (Slamon, et al., N. Engl. J. Med. 344: 783(2001)). Preferably, the 25 initial loading dose is administered as a 90 minute or longer infusion. The periodic maintenance dose may be administered as a 30 minute or longer infusion, provided the initial dose was well tolerated. However, as one of skill in the art will understand, various factors will influence the ideal dose regimen in a particular case. Such factors may include, for example, the binding affinity and half life of the mAb or mAbs used, the degree of TLR10 30 overexpression in the patient, the extent of circulating shed TLR1O antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic agents used in combination with the treatment method of the invention.

WO 2004/047612 PCT/US2003/036971 50 Treatment can also involve anti-TLR10 antibodies conjugated to radioisotopes. Studies using radiolabeled-anticarcinoembryonic antigen (anti-CEA) monoclonal antibodies, provide a dosage guideline for tumor regression of 2-3 infusions of 30-80 mCi/m 2 (Behr, et al. Clin, Cancer Res. 5(10 Suppl.): 3232s-3242s (1999), Juweid, et al., J Nucl. Med. 39:34 5 42 (1998)). Alternatively, dendritic cells transfected with mRNA encoding TLR1O can be used as a vaccine to stimulate T-cell mediated anti-tumor responses. Studies with dendritic cells transfected with prostate-specific antigen mRNA suggest 3 cycles of intravenous administration of 1xi07 - 5x 107 cells for 2-6 weeks concomitant with an intradermal 10 injection of 10 7 cells may provide a suitable dosage regimen (Heiser, et al., J Clin. Invest. 109:409-417 (2002); Hadzantonis and O'Neill, Cancer Biother. Radiopharm. 1:11-22 (1999)). Other exemplary doses of between 1x10 5 to 1x10 9 or 1x10 6 to 1x10 8 cells are also contemplated. Naked DNA vaccines using plasmids encoding TLR10 can induce an immunologic 15 anti-tumor response. Patent application W0200188200-A2 teaches the use of naked DNA vaccines by particle bombardment technique where Ly-9 (a member of the immunoglobulin (Ig) superfamily) DNA-coated microprojectiles at high velocity are used to pierce cell membranes and enter cells without killing the cells. Administration of naked DNA by direct injection into the skin and muscle is not associated with limitations encountered using viral 20 vectors, such as the development of adverse immune reactions and risk of insertional mutagenesis (Hengge, et al., J Invest. Dermatol. 116:979 (2001)). Studies have shown that direct injection of exogenous cDNA into muscle tissue results in a strong immune response and protective immunity (Ilan, Curr. Opin. Mol. Ther. 1:116-120 (1999)). Physical (gene gun, electroporation) and chemical (cationic lipid or polymer) approaches have been 25 developed to enhance efficiency and target cell specificity of gene transfer by plasmid DNA (Nishikawa and Huang, Hum. Gene Ther. 12:861-870 (2001)). Plasmid DNA can also be administered to the lungs by aerosol delivery (Densmore, et al., Mol. Other. 1:180-188 (2000)). Gene therapy by direct injection of naked or lipid-coated plasmid DNA is envisioned for the prevention, treatment, and cure of diseases such as cancer, acquired 30 immunodeficiency syndrome, cystic fibrosis, cerebrovascular disease, and hypertension (Prazeres, et al., Trends Biotechnol. 17:169-174 (1999); Weihl, et al., Neurosurgery 44:239 252 (1999)). HIV-1 DNA vaccine dose-escalating studies indicate administration of 30-300 pg/dose as a suitable therapy (Weber, et al., Eur. J. Clin. Microbiol. Infect. Dis. 20: 800).

WO 2004/047612 PCT/US2003/036971 51 Naked DNA injected intracerebrally into the mouse brain was shown to provide expression of a reporter protein, wherein expression was dose-dependent and maximal for 150 pg DNA injected (Schwartz, et al., Gene Ther. 3:405-411 (1996)) Gene expression in mice after intramuscular injection of nanospheres containing 1 microgram of beta-galactosidase 5 plasmid was greater and more prolonged than was observed after an injection with an equal amount of naked DNA or DNA complexed with Lipofectamine (Truong, et al., Hum. Gene Ther. 9:1709-1717 (1998)). In a study of plasmid-mediated gene transfer into skeletal muscle as a means of providing a therapeutic source of insulin, wherein four plasmid constructs comprising a mouse furin cDNA transgene and rat proinsulin cDNA were injected 10 into the calf muscles of male Balb/c mice, the optimal dose for most constructs was 100 micrograms plasmid DNA (Kon, et al. J Gene Med. 1:186-194 (1999)). Other exemplary doses of 1-1000 Ag/dose or 10-500 pg/dose are also contemplated. Optimally, patients should be evaluated for the level of circulating shed TLR10 antigen in serum in order to assist in the determination of the most effective dosing regimen 15 and related factors. Such evaluations may also be used for monitoring purposes throughout therapy, and may be useful to gauge therapeutic success in combination with evaluating other parameters. 5.9.1 TLR10 TARGETING COMPOSITIONS 20 Compositions for targeting TLR10-expressing cells are within the scope of the present invention. Pharmaceutical compositions comprising antibodies are described in detail in, for example, US Patent No. 6,171,586, to Lam et al., issued January 9, 2001. Such compositions comprise a therapeutically or prophylactically effective amount an antibody, or a fragment, variant, derivative or fusion thereof as described herein, in admixture with a 25 pharmaceutically acceptable agent. Typically, the TLR10 targeting agent will be sufficiently purified for administration to an animal. The pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of 30 the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, other organic acids); bulking agents (such as WO 2004/047612 PCT/US2003/036971 52 mannitol or glycine), chelating agents [such as ethylenediamine tetraacetic acid (EDTA)]; complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, 5 gelatin or immunoglobulins); coloring; flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene 10 glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides (preferably sodium or potassium chloride, mannitol sorbitol); delivery 15 vehicles; diluents; excipients and/or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, Ed. A.R. Gennaro, Mack Publishing Company, (1990)). The optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. Such 20 compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the TLR10 targeting agent. The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly 25 supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor. In one embodiment of the present invention, TLR10 targeting agent compositions 30 may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Reinington's Pharmaceutical Sciences, supra) in the fonn of a lyophilized cake or an aqueous solution. Further, the binding agent product may be formulated as a lyophilizate using appropriate excipients such as sucrose.

WO 2004/047612 PCT/US2003/036971 53 The pharmaceutical compositions can be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. The formulation components are present in 5 concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at slightly lower pH, typically within a pH range of from about 5 to about 8. When parenteral administration is contemplated, the therapeutic compositions for use in this invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the TLR10 targeting 10 agent in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which a TLR1 0 targeting agent is formulated as a sterile, isotonic solution, properly preserved. Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (polylactic acid, polyglycolic acid), beads, or liposomes, 15 that provides for the controlled or sustained release of the product which may then be delivered via a depot injection. Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices. In another aspect, pharmaceutical formulations suitable for parenteral administration 20 may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable 25 lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions. 30 In another embodiment, a pharmaceutical composition may be formulated for inhalation. For example, a TLR1 0 targeting agent may be formulated as a dry powder for inhalation. Polypeptide or nucleic acid molecule inhalation solutions may also be formulated with a propellant for aerosol delivery. In yet another embodiment, solutions may WO 2004/047612 PCT/US2003/036971 54 be nebulized. Pulmonary administration is further described in PCT Application No. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins. It is also contemplated that certain formulations may be administered orally. In one embodiment of the present invention, TLR1O targeting agents that are administered in this 5 fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of the binding agent 10 molecule. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed. Pharmaceutical compositions for oral administration can also be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as 15 tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. Pharmaceutical preparations for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be added, if desired. 20 Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the 25 cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate. Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, tale, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic 30 solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

WO 2004/047612 PCT/US2003/036971 55 Pharmaceutical preparations that can be used orally also include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as tale or magnesium stearate, and, 5 optionally, stabilizers. In soft capsules, the TLR1 0 targeting agent may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers. Another pharmaceutical composition may involve an effective quantity of TLR10 targeting agent in a mixture with non-toxic excipients that are suitable for the manufacture of 10 tablets. By dissolving the tablets in sterile water, or other appropriate vehicle, solutions can be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc. 15 Additional pharmaceutical compositions will be evident to those skilled in the art, including formulations involving TLR10 targeting agents in sustained- or controlled-delivery formulations. Tecimiques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See, for example, PCT/US93/00829 20 that describes controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides (U.S. Patent No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate 25 (Sidman et al., Biopolymers, 22:547-556 (1983)), poly (2-hydroxyethyl-methaerylate) (Langer et al., JBiomedMater Res, 15:167-277, (1981)) and (Langer et al., Chem Tech, 12:98-105(1982)), ethylene vinyl acetate (Langer et al., supra) or poly-D (-)-3 hydroxybutyric acid (EP 133,988). Sustained-release compositions also include liposomes, which can be prepared by any of several methods known in the art. See e.g., Epstein, et al., 30 Proc NatlAcad Sci (USA), 82:3688-3692 (1985); EP 36,676; EP 88,046; EP 143,949. The pharmaceutical composition to be used for in vivo administration typically must be sterile. This may be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method may be conducted WO 2004/047612 PCT/US2003/036971 56 either prior to or following lyophilization and reconstitution. The composition for parenteral administration may be stored in lyophilized form or in solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection 5 needle. Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration. 10 In a specific embodiment, the present invention is directed to kits for producing a single-dose administration unit. The kits may each contain both a first container having a dried TLR1 0 targeting agent and a second container having an aqueous formulation. Also included within the scope of this invention are kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes). 15 5.9.2 DOSAGE An effective amount of a pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in 20 part, upon the molecule delivered, the indication for which the TLR10 targeting agent is being used, the route of administration, and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. A typical dosage may range from about 0.1 mg/kg to up to about 100 mg/kg or more, 25 depending on the factors mentioned above. In other embodiments, the dosage may range from 0.1 mg/kg up to about 100 mg/kg; or 0.01 mg/kg to 1 g/kg; or 1 mg/kg up to about 100 mg/kg or 5 mg/kg up to about 100 mg/kg. In other embodiments, the dosage may range from 10 mCi to 100 mCi per dose for radioimmunotherapy, from about 1xi07 to 5x107 cells or 1x10 5 to 1x10 9 cells or 1x10 6 to 1x10 8 cells per injection or infusion, or from 30 yg to 30 300 ig naked DNA per dose or 1-1000 pg/dose or 10-500 pg/dose, depending on the factors listed above. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, or pigs. An WO 2004/047612 PCT/US2003/036971 57 animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The exact dosage will be determined in light of factors related to the subject 5 requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active compound or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical 10 compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation. The frequency of dosing will depend upon the pharmacokinetic parameters of the TLRIO targeting agent in the formulation used. Typically, a composition is administered until a dosage is reached that achieves the desired effect. The composition may therefore be 15 administered as a single dose or as multiple doses (at the same or different concentrations/dosages) over time, or as a continuous infusion. Further refinement of the appropriate dosage is routinely made. Appropriate dosages may be ascertained through use of appropriate dose-response data. 20 5.9.3 ROUTES OF ADMINISTRATION The route of administration of the phannaceutical composition is in accord with known methods, e.g. orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intra-arterial, intraportal, intralesional routes, intramedullary, intrathecal, intraventricular, transdermal, 25 subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, urethral, vaginal, or rectal means, by sustained release systems or by implantation devices. Where desired, the compositions may be administered by bolus injection or continuously by infusion, or by implantation device. Alternatively or additionally, the composition may be administered locally via 30 implantation of a membrane, sponge, or another appropriate material on to which the TLR1O targeting agent has been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the TLR1 0 targeting agent may be via diffusion, timed-release bolus, or continuous administration.

WO 2004/047612 PCT/US2003/036971 58 In some cases, it may be desirable to use pharmaceutical compositions in an ex vivo manner. In such instances, cells, tissues, or organs that have been removed from the patient are exposed to the pharmaceutical compositions after which the cells, tissues and/or organs are subsequently implanted back into the patient. 5 In other cases, a TLR10 targeting agent can be delivered by implanting certain cells that have been genetically engineered to express and secrete the polypeptide. Such cells may be animal or human cells, and may be autologous, heterologous, or xenogeneic. Optionally, the cells may be immortalized. In order to decrease the chance of an immunological response, the cells may be encapsulated to avoid infiltration of surrounding tissues. The 10 encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues. 15 5.10 COMBINATION THERAPY TLR10 targeting agents of the invention can be utilized in combination with other therapeutic agents. These other therapeutics include, for example radiation treatment, chemotherapeutic agents, as well as other growth factors. In one embodiment, anti-TLR10 antibody is used as a radiosensitizer. In such 20 embodiments, the anti-TLR1 0 antibody is conjugated to a radiosensitizing agent. The term "radiosensitizer," as used herein, is defined as a molecule, preferably a low molecular weight molecule, administered to animals in therapeutically effective amounts to increase the sensitivity of the cells to be radiosensitized to electromagnetic radiation and/or to promote the treatment of diseases that are treatable with electromagnetic radiation. Diseases that are 25 treatable with electromagnetic radiation include neoplastic diseases, benign and malignant tumors, and cancerous cells. The terms "electromagnetic radiation" and "radiation" as used herein include, but are not limited to, radiation having the wavelength of 10-20 to 100 meters. Preferred embodiments of the present invention employ the electromagnetic radiation of: gamma 30 radiation (1020 to 10-13 in), X-ray radiation (1012 to 10- in), ultraviolet light (10 nm to 400 in), visible light (400 nm to 700 nm), infrared radiation (700 nm to 1.0 mm), and microwave radiation (1 mm to 30 cm).

WO 2004/047612 PCT/US2003/036971 59 Radiosensitizers are known to increase the sensitivity of cancerous cells to the toxic effects of electromagnetic radiation. Many cancer treatment protocols currently employ radiosensitizers activated by the electromagnetic radiation of X-rays. Examples of X-ray activated radiosensitizers include, but are not limited to, the following: metronidazole, 5 misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, E09, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IIdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives of the same. Photodynamic therapy (PDT) of cancers employs visible light as the radiation 10 activator of the sensitizing agent. Examples of photodynamic radiosensitizers include the following, but are not limited to: hematoporphyrin derivatives, Photofrin(r), benzoporphyrin derivatives, NPe6, tin etioporphyrin (SnET2), pheoborbide-a, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanine, and therapeutically effective analogs and derivatives of the same. 15 Chemotherapy treatment can employ anti-neoplastic agents including, for example, alkylating agents including: nitrogen mustards, such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan and chlorarnbucil; nitrosoureas, such as carmustine (BCNJ), lomustine (CCNU), and semustine (methyl-CCNU); ethylenimines/methylmelamine such as thriethylenemelamine (TEM), triethylene, 20 thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, 25 azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; ppipodophylotoxins such as etoposide and teniposide; antibiotics such as actimomycin D, 30 daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin), mitomycinC, and actinomycin; enzymes such as L-asparaginase; biological response modifiers such as interferon-alpha, IL-2, G-CSF and GM-CSF; miscellaneous agents including platinium coordination complexes such as cisplatin and carboplatin, WO 2004/047612 PCT/US2003/036971 60 anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (o,p'-DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and 5 equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; 10 and non-steroidal antiandrogens such as flutamide. Combination therapy with growth factors can include cytokines, lymphokines, growth factors, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL 17, IL-18, IFN, TNFO, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell 15 factor, and erythropoietin. Other compositions can include known angiopoietins, for example, vascular endothelial growth factor (VEGF). Growth factors include angiogenin, bone morphogenic protein-1, bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, 20 bone morphogenic protein- 10, bone morphogenic protein-11, bone morphogenic protein- 12, bone morphogenic protein-13, bone morphogenic protein-14, bone morphogenic protein-15, bone morphogenic protein receptor IA, bone morphogenic protein receptor IB, brain derived neurotrophic factor, ciliary neutrophic factor, cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil chemotactic factor 2, endothelial cell growth factor, 25 endothelin 1, epidermal growth factor, epithelial-derived neutrophil attractant, fibroblast growth factor 4, fibroblast growth factor 5, fibroblast growth factor 6, fibroblast growth factor 7, fibroblast growth factor 8, fibroblast growth factor 8b, fibroblast growth factor 8c, fibroblast growth factor 9, fibroblast growth factor 10, fibroblast growth factor acidic, fibroblast growth factor basic, glial cell line-derived neutrophic factor receptor 1, glial cell 30 line-derived neutrophic factor receptor 2, growth related protein, heparin binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, WO 2004/047612 PCT/US2003/036971 61 leukemia inhibitory factor receptor, nerve growth factor, nerve growth factor receptor, neurotrophin-3, neurotrophin-4, placenta growth factor, placenta growth factor 2, platelet derived endothelial cell growth factor, platelet derived growth factor, platelet derived growth factor A chain, platelet derived growth factor AA, platelet derived growth factor AB, platelet 5 derived growth factor B chain, platelet derived growth factor BB, platelet derived growth factor receptor, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor, transforming growth factor 1, transforming growth factor 1.2, transforming growth factor 2, transforming growth factor 3, transforming growth factor 5, latent transforming growth factor 1, transforming growth factor binding protein I, 10 transforming growth factor binding protein II, transforming growth factor binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase type plasminogen activator receptor, vascular endothelial growth factor, and chimeric proteins and biologically or immunologically active fragments thereof. 15 5.11 DIAGNOSTIC USES OF TLR10 5.11.1 ASSAYS FOR DETERMINING EXPRESSION STATUS Determining the status of TLR1 0 expression patterns in an individual may be used to diagnose cancer, or other disorder characterized by TLR1 0 expression, and may provide prognostic information useful in defining appropriate therapeutic options. Similarly, the 20 expression status of TLRl 0 may provide information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining TLR10 expression status and diagnosing diseases that express TLR10. In one aspect, the invention provides assays useful in determining the presence of 25 cancer in an individual, comprising detecting a significant increase in TLR10 mRNA or protein expression in a test cell or tissue or fluid sample relative to expression levels in the corresponding normal cell or tissue. In one embodiment, the presence of TLR1 0 mRNA is evaluated in tissue samples of a lymphoma. The presence of significant TLR1O expression may be useful to indicate whether the lymphoma is susceptible to TLR10 targeting therapy. 30 In a related embodiment, TLR1 0 expression status may be determined at the protein level rather than at the nucleic acid level. For example, such a method or assay would comprise determining the level of TLRl 0 expressed by cells in a test tissue sample and comparing the level so determined to the level of TLR1O expressed in a corresponding normal sample. In WO 2004/047612 PCT/US2003/036971 62 one embodiment, the presence of TLR1 0 is evaluated, for example, using immunohistochemical methods. TLR10 antibodies capable of detecting TLR10 expression may be used in a variety of assay formats well known in the art for this purpose. Peripheral blood may be conveniently assayed for the presence of cancer cells, 5 including lymphomas and leukemias, using RT-PCR to detect TLR10 expression. The presence of RT-PCR amplifiable TLR1O mRNA provides an indication of the presence of one of these types of cancer. A sensitive assay for detecting and characterizing carcinoma cells in blood may be used (Racila, et al., Proc. Natl. Acad. Sci. USA 95: 4589-4594 (1998)). This assay combines immunomagnetic enrichment with multiparameter flow cytometric and 10 immunohistochemical analyses, and is highly sensitive for the detection of cancer cells in blood, reportedly capable of detecting one epithelial cell in 1 ml of peripheral blood. A related aspect of the invention is directed to predicting susceptibility to developing cancer in an individual. In one embodiment, a method for predicting susceptibility to cancer comprises detecting TLR 10 mRNA or protein in a tissue sample, its presence indicating 15 susceptibility to cancer, wherein the degree of TLR1 0 mRNA expression present is proportional to the degree of susceptibility. Yet another related aspect of the invention is directed to methods for assessment of tumor aggressiveness (Orlandi, et al., Cancer Res. 62:567 (2002)). In one embodiment, a method for gauging aggressiveness of a tumor comprises determining the level of TLR1 0 20 mRNA or protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of TLR10 mRNA or protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of TLR1 0 mRNA or protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness. 25 Methods for detecting and quantifying the expression of TLR10 mRNA or protein are described herein and use standard nucleic acid and protein detection and quantification technologies well known in the art. Standard methods for the detection and quantification of TLR1O mRNA include in situ hybridization using labeled TLRIO riboprobes (Gemou Engesaeth, et al., Pediatrics, 109:E24-E32 (2002)), Northern blot and related techniques 30 using TLR10 polynucleotide probes (Kunzli, et al., Cancer 94:228 (2002)) , RT-PCR analysis using primers specific for TLR10 (Angchaiskisiri, et aL., Blood 99:130 (2002)), and other amplification type detection methods, such as, for example, branched DNA (Jardi, et al., J Viral Hepat. 8:465-471 (2001)), SISBA, TMA (Kimura, et al., J. Clin. Microbiol.

WO 2004/047612 PCT/US2003/036971 63 40:439-445 (2002)), and microarray products of a variety of sorts, such as oligos, cDNAs, and monoclonal antibodies. In a specific embodiment, real-time RT-PCR may be used to detect and quantify TLR10 mRNA expression (Simpson, et al., Molec. Vision 6:178-183 (2000)). Standard methods for the detection and quantification of protein may be used for 5 this purpose. In a specific embodiment, polyclonal or monoclonal antibodies specifically reactive with the wild-type TLR1O may be used in an immunohistochemical assay of biopsied tissue (Ristimaki, et al., Cancer Res. 62:632 (2002)). Diagnosis of other diseases characterized by TLR10 expression, such as autoimmune diseases or inflammatory diseases, or any of the aforementioned diseases is also 10 contemplated using the assays outlined in this section. 5.11.2 MEDICAL IMAGING TLR1O antibodies and fragments thereof are useful in medical imaging of sites expressing TLR1 0, respectively. Such methods involve chemical attachment of a labeling or 15 imaging agent, such as a radioisotope, which include 67 Cu, 90 y 125 131 18Re, 188Re, 21At, and 212 Bi, administration of the labeled antibody and fragment to a subject in a pharmaceutically acceptable carrier, and imaging the labeled antibody and fragment in vivo at the target site. Radiolabelled anti-TLR1O antibodies or fragments thereof may be particularly useful in in vivo imaging of TLR1 0 expressing cancers, such as lymphomas or 20 leukemias. Such antibodies may provide highly sensitive methods for detecting metastasis of TLR10-expressing cancers. Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention. Accordingly, it is intended that the broader aspects of the present invention not be 25 limited to the disclosure of the following examples. 6. EXAMPLES EXAMPLE 1 30 TLR10 IS EXPRESSED IN CELL LINES OF LYMPHOMA ORIGIN Expression of TLR10 was determined in various lymphoid and myeloid cell lines. Poly-A messenger RNA was isolated from the cell lines listed in Table 1 and subjected to quantitative, real-time PCR analysis (Simpson et al., Molec. Vision 6:178-183 (2000)) to determine the relative copy number of TLR10 mRNA expressed per cell in each line.

WO 2004/047612 PCT/US2003/036971 64 Elongation factor 1 mRNA expression was used as a positive control and normalization factor in all samples. All assays were performed in duplicate with the resulting values averaged and expressed as "-" for samples with no detectable TLR10 miRNA in that sample to "+++" for 5 samples with the highest mRNA copy number for TLR1 0. The following estimated quantitation scale for the real-time PCR experiments was used: "-" 0 copies/cell; "+" = approximately 1-10 copies/cell; "++"= approximately 11-50 copies/cell; and "+++"= approximately >50 copies/cell. The results are indicated in Table 1. Table 1 Cell Line TLR10 mRNA Expression Burkitt's Lymphoma (CA-46) ++ B cell Lymphoma (RAl) +++ Diffuse B cell Lymphoma (HT) ++ B cell Lymphoma (DB) ++ B cell Lymphoma (RL) ++ Acute Myeloid Leukemia (AML-193) Pro-myelomonocitic Leukemia (HL-60) Multiple Myeloma (RPMI) 10 The results shown in Table 1 demonstrate that the B cell lymphoma cell lines CA-46 and RA1 had high levels of TLR10 expression. Additionally, the B cell lymphoma cell lines HT, DB, and RL were observed to have medium TLR1 0 expression levels, whereas the myeloid cell lines had no observed TLR10 expression. These results demonstrate that 15 TLR10 mRNA is highly expressed in cell lines derived from B cell lymphomas. EXAMPLE 2 TLR1 0 MRNA IS EXPRESSED IN PATIENT TISSUES OF LYMPHOMA ORIGIN Expression of TLR1O was determined in various healthy and tumor tissues (Table 2). 20 Poly-A mRNA was isolated from the lymphoma and acute myeloid leukemia tissue samples obtained from the Cooperative Human Tissue Network (CHTN, National Cancer Institute). All other RNAs were purchased from Clontech (Palo Alto, CA), Ambion (Austin, TX), and Stratagene (La Jolla, CA). Tissues 5348 and 5856 were B cell follicular lymphomas and samples 22601 and 6879 were diffuse large B cell lymphoma samples. Lymphoma patient 25 tissue samples were snap frozen immediately after surgical removal. Poly-A* mRNA was subjected to quantitative, real-time PCR analysis, as described in Example 1, to determine the relative expression of TLR1O mRNA in the sample. All assays were performed in duplicate with the resulting values averaged and expressed as "-" for samples with no WO 2004/047612 PCT/US2003/036971 65 detectable TLR1O mRNA in that sample to "+++" for samples with the highest mRNA copy number for TLR10. The following quantitation scale for the real-time PCR experiments was used: "-" = 0 copies/cell; "+" = approximately 1-10 copeis/cell; "+-+"= approximately 11-50 copies/cell; and "+++" approximately >50 copies/cell. The results are indicated in Table 5 2. Table 2 Patient Tissue TLR10 mRNA Expression Follicular Lymphoma 5348 ++ Follicular Lymphoma 5856 ++ Diffuse Large B cell Lymphoma 6879 ++ Diffuse Large B cell Lymphoma 22601 ++ Anaplastic Large T cell Lymphoma 5664 ++ Acute Myeloid Leukemia 565 + Normal Ileum + Ileum B cell lymphoma +++ CD14+ cells + Adult spleen tissue ++ Thymus tissue + Testicle tissue Small Intestine tissue ++ Adult brain tissue + Adult liver tissue Heart tissue Kidney tissue Lung tissue + Prostate tissue Adrenal tissue Bladder tissue Breast tissue + Cervix tissue + Colon tissue Skeletal muscle tissue + Mammary gland tissue Ovary tissue Pancreas tissue Placenta tissue The results in Table 2 demonstrate that B cell and T cell lymphoma tissue expressed moderate to high levels of TLR10 mRNA. Additionally, ileum B cell lymphoma tissue 10 expressed high levels of TLR10 mRNA as compared to normal ileum tissue. All other non cancerous and healthy tissues showed low levels of or no TLR10 expression. The results demonstrate TLR10 mRNA expression in different Non-Hodgkin's B cell lymphomas, T cell lymphoma, acute myeloid leukemia, and ileum B cell lymphoma tissues and indicate that TLR10 may be used as an immunotherapeutic antibody target or as a diagnostic marker for 15 these types of disorders.' WO 2004/047612 PCT/US2003/036971 66 EXAMPLE 3 NEUTROPHILS EXPRESS HIGH LEVELS OF TLR10 MRNA Expression of TLR10 was determined in various leukocytic cells. Poly-A messenger RNA generated from cDNA isolated from the cells listed in Figure 1 (from AllCells, 5 Berkeley, CA) and subjected to quantitative, real-time PCR analysis (Simpson, et al., Molec. Vision. 6: 178-183 (2000)) to determine the relative copy number of TLR10 mRNA expressed per cell in each line. DNA encoding elongation factor 1 mRNA was used as a positive control and normalization factors in all samples. All assays were performed in duplicate with the resulting values averaged. The y-axis shows the relative expression of 10 TLR1 0 mRNA, wherein the lowest expression was set as equal to 1 and the rest of the values are expressed as relative to 1. Figure 1 shows that TLR10 mRNA is highly expressed in neutrophils as compared to other leukocytes and their precursors, indicating that targeting TLR1O may be useful as a therapeutic or as a diagnostic for neutrophil-related disorders, such as inflammatory 15 disorders. EXAMPLE 4 DIAGNOSTIC METHODS USING TLR10-SPECIFIC ANTIBODIES TO DETECT TLR10 EXPRESSION 20 Expression of TLR10 in tissue samples (normal or diseased) was detected using anti TLR10 antibodies (see Table 3). Samples were prepared for immunohistochemical (IHC) analysis (Clinomics Biosciences, Inc., Pittsfield, MA) by fixing tissues in 10% formalin embedding in paraffin, and sectioning using standard techniques. Sections were stained using the TLR10-specific antibody followed by incubation with a secondary horse radish 25 peroxidase (HRP)-conjugated antibody and visualized by the product of the HRP enzymatic reaction. Data as seen in Table 3 shows that TLR1O is highly expressed on cell surface of tumor tissues. No expression of TLR10 was observed on the cell surface of normal tissues; however a low level of TLR10 expression could be detected in tonsils. In addition to TLR1O expression in leukemia and lymphoma tissues, TLR1 0 expression was found in solid tumors 30 from prostate, breast, colon, and squamous cell carcinoma (see Table 4). Based on this expression pattern, it is likely that other cancers of epithelial and squamous cell origin will also express TLR1O.

WO 2004/047612 PCT/US2003/036971 67 Table 3 Tissue Positive Total Acute myelogenous leukemia 19 23 Acute myelomonocytic leukemia 11 11 Acute lymphoblastic leukemia 3 4 Chronic myelogenous leukemia 6 13 Chronic lymphocytic leukemia 13 15 B cell large cell lymphoma 3 7 Malignant lymphoma 6 8 Acute leukemia 1 2 Lymphosarcoma cell leukemia 1 2 Normal prostate 0 4 Normal breast 0 4 Nonnal colon 0 4 Normal thyroid 0 4 Normal adrenal gland 0 4 Normal placenta 0 4 Normal tonsil 0 4 Normal lymph node 0 4 Normal spleen 0 4 Normal heart 0 4 Normal skeletal muscle 0 4 Normal liver 0 4 Normal pancreas 0 4 Normal ovary 0 4 Normal myometrium 0 4 Normal endometrium 0 4 Normal endocervical 0 4 Normal kidney 0 4 Normila seminal vesicle 0 4 Normal testis 0 4 Normal brain 0 4 Normal lung 0 4 Normal cerebellum 0 4 Normal cerebrum_ 0 4 Table 4 Tumor Tissue TLR9 Expression Prostate, Gleason scores 7, 8 and 9 +++ Breast, stage 3 and 4 Colon, Dukes B and C +++ Squamous cell carcinoma +++ 5 IHC analysis was also performed on tissues derived from autoinmune diseases. Table 5 shows that TLR1 0 was overexpressed in systemic lupus erythematosus, Hasimoto thyroiditis, Sj6rgen's syndrome, and pericarditis lupus. 10 WO 2004/047612 PCT/US2003/036971 68 Table 5 Tissue Positive Total Systemic lupus erythematosus 18 20 Hasimoto thyroiditis 14 15 Sjbrgen's syndrome 5 5 Pericarditis lupus 17 17 lHC was also performed on tissues derived from rejected organ transplants. Table 6 shows that TLR10 was overexpressed in rejected heart, liver, and kidney, whereas TLR1 0 5 was not present on healthy tissues. Table 6 Tissue Positive Total Rejected heart 24 27 Rejected liver 30 35 Rejected kidney 13 18 Normal heart 0 4 Normal liver 0 4 Normal kidney 0 4 Expression of TLR1 0 on the surface of cells within a blood sample is detected by flow cytometry. Peripheral blood mononuclear cells (PBMC) are isolated from a blood 10 sample using standard techniques. The cells are washed with ice-cold PBS and incubated on ice with the TLR10-specific polyclonal antibody for 30 min. The cells are gently pelleted, washed with PBS, and incubated with a fluorescent anti-rabbit antibody for 30 min. on ice. After the incubation, the cells are gently pelleted, washed with ice cold PBS, and resuspended in PBS containing 0.1% sodium azide and stored on ice until analysis. Samples 15 are analyzed using a FACScalibur flow cytometer (Becton Dickinson) and CELLQuest software (Becton Dickinson). Instrument settings are determined using FACS-Brite calibration beads (Becton-Dickinson). Tumors expressing TLR1 0 are imaged using TLR1 0-specific antibodies conjugated to a radionuclide, such as 1231, and injected into the patient for targeting to the tumor 20 followed by X-ray or magnetic resonance imaging. EXAMPLE 5 PRODUCTION OF TLR1O-SPECIFIc ANTIBODIES Cells expressing TLR1 0 are identified using antibodies to TLR1 0, respectively. 25 Polyclonal antibodies are produced by DNA vaccination or by injection of peptide antigens into rabbits or other hosts. An animal, such as a rabbit, is immunized with a peptide from the extracellular region of TLR1 0 conjugated to a carrier protein, such as BSA (bovine WO 2004/047612 PCT/US2003/036971 69 serum albumin) or KLH (keyhole limpet hemocyanin). The rabbit is initially immunized with conjugated peptide in complete Freund's adjuvant, followed by a booster shot every two weeks with injections of conjugated peptide in incomplete Freund's adjuvant. Anti TLR10 antibodies are affinity purified from rabbit serum using a TLR10 peptide, 5 respectively, coupled to Affi-Gel 10 (Bio-Rad), and stored in phosphate-buffered saline (PBS) with 0.1% sodium azide. To determine that the polyclonal antibodies are TLRl 0 specific, an expression vector encoding TLR10 is introduced into mammalian cells. Western blot analysis of protein extracts of non-transfected cells and the TLR1 0-containing cells is performed using the polyclonal antibody sample as the primary antibody and a horseradish 10 peroxidase-labeled anti-rabbit antibody as the secondary antibody. Detection of an approximately 89 kD band in the TLR1O-containing cells and lack thereof in the control cells indicates that the polyclonal antibodies are specific for TLR10. Monoclonal antibodies are produced by injecting mice with a TLR10 peptide, with or without adjuvant. Subsequently, the mouse is boosted every 2 weeks until an appropriate 15 immune response has been identified (typically 1-6 months), at which point the spleen is removed. The spleen is minced to release splenocytes, which are fused (in the presence of polyethylene glycol) with murine myeloma cells. The resulting cells (hybridomas) are grown in culture and selected for antibody production by clonal selection. The antibodies are secreted into the culture supernatant, facilitating the screening process, such as screening 20 by an enzyme-linked immunosorbent assay (ELISA). Alternatively, humanized monoclonal antibodies are produced either by engineering a chimeric murine/human monoclonal antibody in which the murine-specific antibody regions are replaced by the human counterparts and produced in mammalian cells, or by using transgenic "knock out" mice in which the native antibody genes have been replaced by human antibody genes and 25 immunizing the transgenic mice as described above. EXAMPLE 6 IN VITRO ANTIBODY-DEPENDENT CYTOTOXICITY ASSAY The ability of a TLR1 0-specific antibody to induce antibody-dependent cell-mediated 30 cytoxicity (ADCC) is determined in vitro. ADCC is performed using the CytoTox 96 Non Radioactive Cytoxicity Assay (Promega; Madison, WI) (Hornick et al., Blood 89:4437 4447, (1997)) as well as effector and target cells. Peripheral blood mononuclear cells (PBMC) or neutrophilic polymorphonuclear leukocytes (PMN) are two examples of effector WO 2004/047612 PCT/US2003/036971 70 cells that can be used in this assay. PBMC are isolated from healthy human donors by Ficoll-Paque gradient centrifugation, and PMN are purified by centrifugation through a discontinuous percoll gradient (70% and 62%) followed by hypotonic lysis to remove residual erythrocytes. RA1 B cell lymphoma cells (for example) are used as target cells. 5 RA1 cells are suspended in RPMI 1640 medium supplemented with 2% fetal bovine serum and plated in 96-well V-bottom microtitier plates at 2x 104 cells/well. TLR1O-specific antibodies are added in triplicate to individual wells at 1 tg/ml, and effector cells are added at various effector:target cell ratios (12.5:1 to 50:1). The plates are incubated for 4 hours at 37 0 C. The supernatants are then harvested, lactate dehydrogenase release determined, and 10 percent specific lysis calculated using the manufacture's protocols. EXAMPLE 7 TOXIN-CONJUGATED TLR10-SPECIFIC ANTIBODIES Antibodies to TLR1 0 are conjugated to toxins and the effect of such conjugates in 15 animal models of cancer is evaluated. Chemotherapeutic agents, such as calicheamycin and carboplatin, or toxic peptides, such as ricin toxin, are used in this approach. Antibody-toxin conjugates are used to target cytotoxic agents specifically to cells bearing the antigen. The antibody-toxin binds to these antigen-bearing cells, becomes internalized by receptor mediated endocytosis, and subsequently destroys the targeted cell. In this case, the 20 antibody-toxin conjugate targets TLR1 0-expressing cells, such as B cell lymphomas, and deliver the cytotoxic agent to the tumor resulting in the death of the tumor cells. One such example of a toxin that may be conjugated to an antibody is carboplatin. The mechanism by which this toxin is conjugated to antibodies is described in Ota et al., Asia-Oceania J. Obstet. Gynaecol. 19: 449-457 (1993). The cytotoxicity of carboplatin 25 conjugated TLR9- or TLR10-specific antibodies is evaluated in vitro, for example, by incubating TLR10-expressing target cells (such as the RA1 B cell lymphoma cell line) with various concentrations of conjugated antibody, medium alone, carboplatin alone, or antibody alone. The antibody-toxin conjugate specifically targets and kills cells bearing the TLR10 antigen, whereas, cells not bearing the antigen, or cells treated with medium alone, 30 carboplatin alone, or antibody alone, show no cytotoxicity. The antitumor efficacy of carboplatin-conjugated TLR10-specific antibodies is demonstrated in in vivo murine tumor models. Five to six week old, athymic nude mice are engrafted with tumors subcutaneously or through intravenous injection. Mice are treated WO 2004/047612 PCT/US2003/036971 71 with the TLR1 0-carboplatin conjugate or with a non-specific antibody-carboplatin conjugate. Tumor xenografts in the mouse bearing the TLR10 antigen are targeted and bound to by the TLR1 0-carboplatin conjugate, respectively. This results in tumor cell killing as evidenced by tumor necrosis, tumor shrinkage, and increased survival of the treated mice. 5 Other toxins are conjugated to TLR10-specific antibodies using methods known in the art. An example of a toxin conjugated antibody in human clinical trials is CMA-676, an antibody to the CD33 antigen in AML which is conjugated with calicheamicin toxin (Larson, Semin. Henatol. 38(Suppl 6):24-31 (2001)). 10 EXAMPLE 8 RADIO-IMMUNOTHERAPY USING TLR1 0-SPECIFIC ANTIBODIES Animal models are used to assess the effect of antibodies specific to TLR10 as vectors in the delivery of radionuclides in radio-immunotherapy to treat lymphoma, hematological malignancies, and solid tumors. Human tumors are propagated in 5-6 week 15 old athymic nude mice by injecting a carcinoma cell line or tumor cells subcutaneously. Tumor-bearing animals are injected intravenously with radio-labeled anti-TLR1O antibody (labeled with 30-40 1 Ci of 1311, for example) (Behr, et al., Int. J Cancer 77: 787-795 (1988)). Tumor size is measured before injection and on a regular basis (i.e. weekly) after injection and compared to tumors in mice that have not received treatment. Anti-tumor 20 efficacy is calculated by correlating the calculated mean tumor doses and the extent of induced growth retardation. To check tumor and organ histology, animals are sacrificed by cervical dislocation and autopsied. Organs are fixed in 10% formalin, embedded in paraffin, and thin sectioned. The sections are stained with hematoxylin-eosin. 25 EXAMPLE 9 IMMUNOTHERAPY USING TLR1 0-SPECIFIC ANTIBODIES Animal models are used to evaluate the effect of TLR10-specific antibodies as targets for antibody-based immunotherapy using monoclonal antibodies. Human myeloma cells are injected into the tail vein of 5-6 week old nude mice whose natural killer cells have been 30 eradicated. To evaluate the ability of TLR10-specific antibodies in preventing tumor growth, mice receive an intraperitoneal injection with TLR1 0-specific antibodies either 1 or 15 days after tumor inoculation followed by either a daily dose of 20 p[g or 100 p/g once or twice a week, respectively (Ozaki, et al., Blood 90:3179-3186 (1997)). Levels of human IgG WO 2004/047612 PCT/US2003/036971 72 (from the immune reaction caused by the human tumor cells) are measured in the murine sera by ELISA. The effect of TLR10-specific antibodies on the proliferation of myeloma cells is examined in vitro using a 3 H-thymidine incorporation assay (Ozaki et al., supra). Cells are 5 cultured in 96-well plates at 1x 105 cells/ml in 100 pl/well and incubated with various amounts of TLR9 or TLR10 antibody or control IgG (up to 100 yg/ml) for 24 h. Cells are incubated with 0.5 pCi 3 H-thymidine (New England Nuclear, Boston, MA) for 18 h and harvested onto glass filters using an automatic cell harvester (Packard, Meriden, CT). The incorporated radioactivity is measured using a liquid scintillation counter. 10 The cytotoxicity of the TLR1 0 monoclonal antibody is examined by the effect of complements on myeloma cells using a 51 Cr-release assay (Ozaki et al., supra). Myeloma cells are labeled with 0.1 mCi 51 Cr-sodium chromate at 374C for 1 h. 5 1 Cr-labeled cells are incubated with various concentrations of TLR10 monoclonal antibody or control IgG on ice for 30 min. Unbound antibody is removed by washing with medium. Cells are distributed 15 into 96-well plates and incubated with serial dilutions of baby rabbit complement at 37'C for 2 h. The supernatants are harvested from each well and the amount of 5 1 Cr released is measured using a gamma counter. Spontaneous release of 5 Cr is measured by incubating cells with medium alone, whereas maximum 51 Cr release is measured by treating cells with 1% NP-40 to disrupt the plasma membrane. Percent cytotoxicity is measured by dividing 20 the difference of experimental and spontaneous 51 Cr release by the difference of maximum and spontaneous 5 1 Cr release. Antibody-dependent cell-mediated cytotoxicity (ADCC) for the TLR10 monoclonal antibody is measured using a standard 4 h 5 Cr-release assay (Ozaki et al., supra). Splenic mononuclear cells from SCID mice are used as effector cells and cultured with or without 25 recombinant interleukin-2 (for example) for 6 days. 51 Cr-labeled target myeloma cells (lx104 cells) are placed in 96-well plates with various concentrations of anti-TLRIO monoclonal antibody or control IgG. Effector cells are added to the wells at various effector to target ratios (12.5:1 to 50:1). After 4 h, culture supernatants are removed and counted in a gamma counter. The percentage of cell lysis is determined as above. 30 WO 2004/047612 PCT/US2003/036971 73 EXAMPLE 10 TLR10-spEciFIc ANTIBODIES As IMMUNOSUPPRESSANTS Animal models are used to assess the effect of TLR10-specific antibodies on blocking signaling through the TLR10 receptor to suppress autoimmune diseases, such as 5 arthritis or other inflammatory conditions, or rejection of organ transplants. Immunosuppression is tested by injecting mice with horse red blood cells (HRBCs) and assaying for the levels of HRBC-specific antibodies (Yang, et al., Int. linmunopharm. 2:389 397 (2002)). Animals are divided into five groups, three of which are injected with anti TLR10 antibodies for 10 days, and 2 of which receive no treatment. Two of the 10 experimental groups and one control group are injected with either Earle's balanced salt solution (EBSS) containing 5-10x107 HRBCs or EBSS alone. Anti-TLR10 antibody treatment is continued for one group while the other groups receive no antibody treatment. After 6 days, all animals are bled by retro-orbital puncture, followed by cervical dislocation and spleen removal. Splenocyte suspensions are prepared and the serum is removed by 15 centrifugation for analysis. Immunosupression is measured by the number of B cells producing HRBC-specific antibodies. The Ig isotype (for example, IgM, IgGl, IgG2, etc.) is determined using the IsoDetectTM Isotyping kit (Stratagene, La Jolla, CA). Once the Ig isotype is known, murine antibodies against HRBCs are measured using an ELISA procedure. 96-well plates are 20 coated with HRBCs and incubated with the anti-HRBC antibody-containing sera isolated from the animals. The plates are incubated with alkaline phosphatase-labeled secondary antibodies and color development is measured on a microplate reader (SPECTRAmax 250, Molecular Devices) at 405 nm usingp-nitrophenyl phosphate as a substrate. Lymphocyte proliferation is measured in response to the T and B cell activators 25 concanavalin A and lipopolysaccharide, respectively (Jiang, et al., . Immunol. 154:3138 3146 (1995). Mice are randomly divided into 2 groups, 1 receiving anti-TLR1O antibody therapy for 7 days and I as a control. At the end of the treatment, the animals are sacrificed by cervical dislocation, the spleens are removed, and splenocyte suspensions are prepared as above. For the ex vivo test, the same number of splenocytes are used, whereas for the in vivo 30 test, the anti-TLR1 0 antibody is added to the medium at the beginning of the experiment. Cell proliferation is also assayed using the 3 H-thymidine incorporation assay described above (Ozaki, et al., Blood 90: 3179 (1997)).

WO 2004/047612 PCT/US2003/036971 74 EXAMPLE 11 CYTOKINE SECRETION IN RESPONSE To TLR10 PEPTIDE FRAGMENTS Assays are carried out to assess activity of fragments of the TLR1 0 protein, such as the Ig domain, to stimulate cytokine secretion and to stimulate immune responses in NK 5 cells, B cells, T cells, and myeloid cells. Such immune responses can be used to stimulate the immune system to recognize and/or mediate tumor cell killing or suppression of growth. Similarly, this immune stimulation can be used to target bacterial or viral infections. Alternatively, fragments of the TLR10 that block activation through the TLR10 receptor, respectively, may be used to block immune stimulation in natural killer (NK), B, T, and 10 myeloid cells. Fusion proteins containing fragments of the TLR1 0, such as the Ig domain (TLR1 0 Ig), are made by inserting a CD33 leader peptide, followed by a TLR10 domain fused to the Fc region of human IgG1 into a mammalian expression vector, which is stably transfected into NS- 1 cells, for example. The fusion proteins are secreted into the culture supernatant, 15 which is harvested for use in cytokine assays, such as interferon-y (IFN-y) secretion assays (Martin, et al., J fnmunol. 167:3668-3676 (2001)). PBMCs are activated with a suboptimal concentration of soluble CD3 and various concentrations of purified, soluble anti-TLR10 monoclonal antibody or control IgG. For TLR10-Ig cytokine assays, anti-human Fc Ig at 5 or 20 ptg/ml is bound to 96-well plates and 20 incubated overnight at 4C. Excess antibody is removed and either TLR1 0-Ig or control Ig is added at 20-50 ptg/inl and incubated for 4 h at room temperature. The plate is washed to remove excess fusion protein before adding cells and anti-CD3 to various concentrations. Supernatants are collected after 48 h of culture and IFN--y levels are measured by sandwich ELISA, using primary and biotinylated secondary anti-human IFN-y antibodies as 25 recommended by the manufacturer. EXAMPLE 12 TUMOR IMAGING USING TLR1 0-SPECIFIC ANTIBODIES TLR10-specific antibodies are used for imaging TLR10-expressing cells in vivo. Six-week-old athymic nude mice are irradiated with 400 rads from a cesium source. Three 30 days later the irradiated mice are inoculated with 4x 10 7 RAl cells and 4x 106 human fetal lung fibroblast feeder cells subcutaneously in the thigh. When the tumors reach approximately 1 cm in diameter, the mice are injected intravenously with an inoculum containing 100 pCi/10 tg of 31 1-labeled TLR10-specific antibody. At 1, 3, and 5 days WO 2004/047612 PCT/US2003/036971 75 postinjection, the mice are anesthetized with a subcutaneous injection of 0.8 mg sodium pentobarbital. The immobilized mice are then imaged in a prone position with a Spectrum 91 camera equipped with a pinhole collimator (Raytheon Medical Systems; Melrose Park, IL) set to record 5,000 to 10,000 counts using the Nuclear MAX Plus image analysis 5 software package (MEDX Inc.; Wood Dale, IL) (Hornick, et al., Blood 89:4437-4447 (1997)). EXAMPLE 13 PHASE I CLINICAL TRIAL USING ESCALATING SINGLE-DOSE INFUSION OF CHIMERIC 10 ' ANTI-TLR10 MONOCLONAL ANTIBODIES IN PATIENTS This example describes an exemplary clinical study in patients with recurrent B cell lymphoma. Similar studies are also contemplated to be performed on patients with other cancers and diseases relating to TLR10 expression. 15 A. PATIENT SELECTION This study is directed to patients with relapsed non-Hodgkin's lymphoma with measurable disease after at least one prior course of standard therapy. A tumor biopsy is performed to document tumor cell expression of the TLR10 antigen of the invention and reactivity with an antibody that reacts with said antigen using flow cytometry. In addition, 20 baseline hematologic function (1500 granulocytes and 50,000 platclets/l), renal function (serum creatine of <2.5 mg/dl), quantitative serum IgG of greater than 600 mg/dl, a negative serology to human immunodeficiency virus (HIV), a negative hepatitis B surface antigen, a life expectancy of at least 3 months without other serious illness, and between the ages of 18 and 75 years. Other exclusion criteria are previous treatment with murine antibodies, active 25 opportunistic infections, any other severe infection not controlled by medical or surgical therapy, or major organ dysfunction. Patients who are pregnant or lactating or those who had participated in other trials during the past 12 weeks of this study are also excluded. B. PROTOCOL DESIGN 30 This is a phase I clinical trial of single-dose anti-TLR1 0 chimeric monoclonal antibody (mAb) administered to patients with relapsed non-Hodgkin's lymphoma (for protocol detail, see Maloney et al., Blood 84:2457-2466 (1994), herein incorporated by reference in its entirety). Detailed informed consent is obtained from all patients in WO 2004/047612 PCT/US2003/036971 76 accordance with the human subjects institutional review board of the institution. Three patients are treated at each dose level of 10, 50, 100, 250, or 500 mg/m 2 (based on Rituximab dosing) of TLR10 mAb. Patients are evaluated for infusional related toxicity and effect on peripheral blood B cells, T cells, neutrophils and platelets, serum chemistries, Ig, and 5 complement levels. In patients treated at the upper three doses, tumor biopsies are obtained 2 weeks after treatment and examined for evidence of antibody binding and B- and T-cell content. All patients are evaluated for anti-tumor activity. C. FLow CYTOMETRY 10 TLR10 expression is determined on all cases before antibody treatment by flow cytometry of fresh or cryopreserved tumor cell suspensions. Tumor cells are obtained from excisional biopsies or from fine needle tumor aspirations and stained for said TLR10 expression with fluorescently-labeled anti-TLR10 antibodies. Tumor cells are also analyzed for expression of surface Ig light chains (fluorescein (FITC)-goat F(ab) 2 -anti-human K or A; 15 Tago, Burlingame, CA), CD19, CD4, CD3, CD8 (FITC- or phycoerythrin (PE)-conjugated Leul2, Leu3, Leu4, and Leu2;Becton Dickinson), and CD37 (MBI clone 6A4). Peripheral blood samples are analyzed for the number of cells expressing the TLR1 0 antigen using two color flow cytometry using PE or FITC conjugates of the above reagents. Two week post-treatment tumor biopsies are also evaluated for B- and T- cell content 20 using the same reagents described above. Antibody bound to tumor cells from in vivo administration is detected by a combination of two different methods. In the first method, cells are stained using FITC-labeled anti-TLR10 antibodies. The presence of the unlabeled antibody blocks the binding of the labeled antibody, resulting in decreased immunostaining of the B-cell tumor population (as identified using antibodies to additional B-cell antigens 25 CD19, CD37, IgM, IgG, K or X). Second, the bound chimeric antibody is detected directly by looking for IgM K-or X-positive tumor cells now bearing the human IgG (K) constant regions of the chimeric antibody using a FITC labeled goat F(ab )2 anti-human IgG y-chain specific reagent (Tago). An estimate of the percentage of tumor cells with the chimeric antibody attached is obtained by comparing the staining of the pretreatment and the post 30 treatment biopsies for human IgG constant regions.

WO 2004/047612 PCT/US2003/036971 77 D. ANTI-TLR10 ANTIBODY PHARMACOKINETICS Serum levels of the chimeric antibody are determined using an enzyme-linked immunosorbant assay (ELISA). Microtiter plates are coated with a purified polyclonal anti TLR1 0 idiotype antiserum. After washing and blocking, post-treatment sera are serially 5 diluted. Bound human IgG is detected using an HRP-conjugated polyclonal anti-human IgG reagent, and the plates are developed with the substrate 2,2-azinobis(3-ethylbenzthiazoline sulfonic acid) (ABTS). Antibody concentration is determined by comparison of the signal from the patients sera with that obtained from known concentrations of purified chimeric antibody diluted into normal human serum. 10 E. MEASUREMENT OF HOST ANTI-TLR10 ANTIBODY RESPONSE Post-treatment sera from evaluations at 1, 2, and 3 months are analyzed for evidence of a host anti-chimeric antibody immune response using a sandwich ELISA with microtiter plates coated with anti-TLR10 antibody, the murine anti-TLR10 antibody, or normal murine 15 IgG. Dilutions of the patients sera are added and, after washing, are detected with biotin labeled chimeric antibody followed by Avidin-HRP and the substrate ABTS. This assay has a level of quantification of 5 tg/ml. F. STUDY MEASUREMENTS 20 Patients are evaluated for infusional related toxicity using the National Cancer Institute's Common Toxicity (NCICT) criteria. Hematologic, renal, and hepatic function is monitored before and after infusion and during monthly intervals after therapy. Sera for evaluation of antibody levels and pharmacokinetics, serum IgG and IgM levels, and TLR1 0 expression on peripheral blood B cells is obtained at each follow-up visit. Tumnor response 25 is assessed by evaluation of tumor measurements from physical examination and from radiologic imaging studies. For 3 months after therapy, patients are evaluated at monthly intervals and then followed at 1- to 3-month intervals until disease progression is observed. A complete remission (CR) requires complete resolution of all detectable disease. A partial remission (PR) requires a greater than 50% reduction in measurable disease persisting more 30 than 30 days. A minor response (MR) is defined as a 25% to 50% reduction in disease. Stable disease (SD) is defined as no significant change in tumor measurements without progression over the period of observation. Progressive disease (PD) is noted when there is a 25% increase in measurable disease or the appearance of any new lesion.

WO 2004/047612 PCT/US2003/036971 78 EXAMPLE 14 PHASE 2 STUDY OF RELAPSED B-CELL LYMPHOMA USING ANTI-TLR10 ANTIBODIES This example describes an exemplary clinical study in patients with recurrent B cell 5 lymphoma. Similar studies are also contemplated to be performed on patients with other cancers and diseases relating to TLR10 expression. A. PATIENTS Eligibility criteria include TLR1 0-positive B-cell lymphoma at first or higher relapse 10 or progressive disease after at least one standard treatment. Lesions are classified as TLR10 positive when the TLR1O antigen of the invention is expressed on more than 30% of malignant cells. All histologic slides are reviewed by an independent expert panel consisting of 6 reference pathologists. For enrollment into the study, patients also have to meet the following requirements: have a bidimensionally measurable disease, at least one lesion 15 larger than 1.0 cm in its greatest diameter, and a World Health Organization performance status of 0, 1, or 2. In addition, patients have to be at least 18 years of age, neither pregnant nor lactating, using accepted birth control methods, and have to have a life expectancy of 3 months or longer. Patients with major organ dysfunction or active infections are excluded from this study. Prior treatment with anti-TLR1O antibodies is also an exclusion criterion. 20 Concurrent therapeutic use of corticosteroids is not allowed. B. SAMPLE SIZE This phase 2 trial is designed to evaluate the feasibility of single-dose anti-TLR10 antibody in patients with B-cell lymphoma and to document possible anti-tumor effects (for 25 protocol details see Rehwald et al., Blood 101:420-424 (2003), herein incorporated by reference in its entirety). Patients are evaluated when completing at least 2 infusions of anti TLR10 antibody. For the efficacy analysis the best response achieved from the start of treatment to progressive disease is recorded. The response rates (overall objective and complete response rates) are reported in rates with 95% confidence intervals (Pearson 30 Clopper intervals).

Claims

1. A pharmaceutical composition comprising an anti-Toll-like receptor 10 (TLR1 0), antibody, wherein said antibody specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 2 or immunogenic fragment thereof. 5

2. The pharmaceutical composition of claim 1, wherein said antibody is a monoclonal anti-TLR1 0 antibody or antibody fragment thereof.

3. The pharmaceutical composition of claim 1, wherein said antibody is labeled with 10 a radioisotope.

4. The pharmaceutical composition of claim 1, wherein said antibody is labeled with a toxin. 15

5. The pharmaceutical composition of claim 1, wherein said antibody is administered in an amount effective to kill or inhibit the growth of cells that cause a disease selected from the group consisting of B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's Disease, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic 20 lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, solid phase cancer, autoimmune disorder, inflammatory disease, allergic reaction, and rejection of transplanted tissues or organs.

6. A method of targeting a TLR10 protein on cells that cause disease selected 25 from the group consisting of B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's Disease, acute myelogenous leukemia, acute myclomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, solid phase cancer, autoimmune disorder, inflammatory disease, allergic reaction, 30 and rejection of transplanted tissues or organs, comprising the step of administering a composition to said cells in an amount effective to target said cells, wherein said composition is an anti-TLR1 0 antibody that specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 2, or immunogenic fragment thereof. WO 2004/047612 PCT/US2003/036971 80

7. A method of killing or inhibiting the growth of TLR10-expressing cells, that cause disease selected from the group consisting of B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's Disease, acute myelogenous leukemia, acute 5 myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, solid phase cancer, autoimmune disorder, inflammatory disease, allergic reaction, and rejection of transplanted tissues or organs, comprising the step of administering a composition to said cells in an amount effective to 10 kill or inhibit the growth of said cells, wherein said composition is an anti-TLR1O antibody that specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 2, or immunogenic fragment thereof.

8. A method of killing or inhibiting the growth of TLR1O-expressing cells that 15 cause a disease selected from the group consisting of B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's Disease, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, solid phase cancer, autoimmune disorder, 20 inflammatory disease, allergic reaction, and rejection of transplanted tissues or organs, comprising the step of administering a composition to said cells, wherein said composition comprises a TLR10 antigen, in an amount effective to kill or inhibit the growth of said cells.

9. A method of killing or inhibiting the growth of TLRI0-expressing cells that 25 cause a disease selected from the group consisting of B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's Disease, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, solid phase cancer, autoimmune disorder, 30 inflammatory disease, allergy, and rejection of transplanted tissues or organs, comprising the step of administering a composition to said cells in an amount effective to kill or inhibit the growth of said cells, wherein said composition comprises a nucleic acid encoding TLR10, or immunogenic fragment thereof, within a recombinant vector. WO 2004/047612 PCT/US2003/036971 81

10. A method of killing or inhibiting the growth of TLR1 0-expressing cells that cause a disease selected from the group consisting of B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's Disease, acute myelogenous leukemia, acute 5 myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, solid phase cancer, autoinunue disorder, inflammatory disease, allergic reaction, and rejection of transplanted tissues or organs, comprising the step of administering a composition to said cells in an amount effective to 10 kill or inhibit the growth of said cells, wherein said composition comprises an antigen presenting cell comprising a nucleic acid encoding TLRl 0, or immunogenic fragment thereof, within a recombinant vector.

11. A method of killing or inhibiting the growth of TLR10-expressing cells that cause 15 a disease selected from the group consisting of B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's Disease, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, solid phase cancer, autoimmune disorder, inflammatory disease, allergic reaction, 20 and rejection of transplanted tissues or organs, comprising the step of administering a composition to said cells in an amount effective to kill or inhibit the growth of said cells, wherein said composition comprises a small molecule that specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 2 or immunogenic fragment thereof. 25

12. A method of killing or inhibiting the growth of TLR10-expressing cells that cause a disease selected from the group consisting of B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's Disease, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma 30 cell leukemia, solid phase cancer, autoimmune disorder, inflammatory disease, allergic reaction, and rejection of transplanted tissues or organs, comprising the step of administering a composition to said cells in an amount effective to kill or inhibit the growth of said cells, wherein said composition comprises a non-TLR1 0 polypeptide that specifically binds to a WO 2004/047612 PCT/US2003/036971 82 polypeptide having an amino acid sequence of SEQ ID NO: 2 or immunogenic fragment thereof.

13. The method according to any one of claims 6-12, wherein said cells are 5 contacted with as second therapeutic agent.

14. The method according to any of claims 6-12, wherein said anti-TLR10 antibody composition is administered in an amount effective to achieve a dosage range from about 0.1 to about 10 mg/kg body weight. 10

15. The method according to any one of claims 6-12, wherein said pharmaceutical composition is administered in a sterile preparation together with a pharmaceutically acceptable carrier therefore. 15

16. A method of diagnosing a disease selected from the group consisting of B cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's Disease, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, solid phase cancer, 20 autoimmune disorder, inflammatory disease, allergic reaction, and rejection of transplanted tissues or organs, comprising the steps of: (a) detecting or measuring the expression of TLR1 0 protein on a cell; and (b) comparing said expression to a standard indicative of said disease. 25

17. A method of diagnosing a disease selected from the group consisting of B cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's Disease, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, solid phase cancer, 30 autoimmune disorder, inflammatory disease, allergic reaction, and rejection of transplanted tissues or organs, comprising the steps of: (a) detecting or measuring the expression of TLR1 0 protein on a cell; and (b) comparing said expression to nonnal tissue. WO 2004/047612 PCT/US2003/036971 83

18. The method according to claim 16 or 17, wherein said expression is TLR10 mRNA expression. 5

19. The method according to claim 16 or 17, wherein said expression is detected or measured using anti-TLR1 0 antibodies.

20. Use of an anti-TLR1 0 antibody, in preparation of a medicament for killing or inhibiting the growth of TLR10-expressing cells that cause a disease selected from the group 10 consisting of B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's Disease, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, solid phase cancer, autoimmune disorder, inflammatory disease, allergic reaction, and rejection of 15 transplanted tissues or organs, wherein said antibody specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 2, or immunogenic fragment thereof.

21. Use of a TLR1O antigen in preparation of a medicament for killing or inhibiting the growth of TLR1 0-expressing cells that cause a disease selected from the group 20 consisting of B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's Disease, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, solid phase cancer, autoimmune disorder, inflammatory disease, allergic reaction, and rejection of 25 transplanted tissues or organs.

22. Use of a nucleic acid encoding TLR1 0, or immunogenic fragment thereof, within a recombinant vector, in preparation of a medicament for killing or inhibiting the growth of TLR10-expressing cells that cause a disease selected from the group consisting of 30 B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's Disease, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, solid phase cancer, WO 2004/047612 PCT/US2003/036971 84 autoimmune disorder, inflammatory disease, allergic reaction, and rejection of transplanted tissues or organs.

23. Use of an antigen-presenting cell comprising a nucleic acid encoding TLRIO 5 or immunogenic fragment thereof, within a recombinant vector, in preparation of a medicament for killing or inhibiting the growth of TLR10-expressing cells that cause a disease selected from the group consisting of B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's Disease, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic 10 lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, solid phase cancer, autoimmune disorder, inflammatory disease, allergic reaction, and rejection of transplanted tissues or organs.

24. Use of a small molecule that specifically binds a TLR1O polypeptide having an 15 amino acid sequence of SEQ ID NO: 2 or immunogenic fragment thereof, in preparation of a medicament for killing or inhibiting the growth of TLR1 0 expressing cells that cause a disease selected from the group consisting of B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's Disease, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, 20 B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, solid phase cancer, autoimmune disorder, inflammatory disease, allergic reaction, and rejection of transplanted tissues or organs.

25. Use of a non-TLR10 polypeptide that specifically binds the TLR10 polypeptide 25 having an amino acid sequence of SEQ ID NO: 2 or immunogenic fragment thereof, in preparation of a medicament for killing or inhibiting the growth of TLRI 0 expressing cells that cause a disease selected from the group consisting of B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's Disease, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic 30 leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, solid phase cancer, autoinmune disorder, inflammatory disease, allergic reaction, and rejection of transplanted tissues or organs.