AU2008200381B2 - Human monoclonal antibodies to dendritic cells - Google Patents

Description

AUSTRALIA PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT ORIGINAL Name of Applicant/s: Celldex Therapeutics, Inc. Actual Inventor/s: Yashwant M. Deo and Tibor Keler and John Treml Address for Service is: SHELSTON IP 60 Margaret Street Telephone No: (02) 9777 1111 SYDNEY NSW 2000 Facsimile No. (02) 9241 4666 CCN: 3710000352 Attomey Code: SW Invention Title: HUMAN MONOCLONAL ANTIBODIES TO DENDRITIC CELLS Details of Original Application No. 2005200250 dated 21 January 2005 The following statement is a full description of this invention, including the best method of performing it known to me/us: File: 37115AUP02 -la HUMAN MONOCLONAL ANTIBODIES TO DENDRITIC CELLS The present application is a divisional application of Australian Application No. 2005200250, which is incorporated in its entirety herein by reference. 5 Background of the Invention Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. 10 Dendritic cells are specialized cells of the immune system with the unique capacity for initiating primary and secondary T and B lymphocyte responses. Characterized as professional antigen presenting cells (APCs), dendritic cells express MHC and costimulatory molecules essential in priming naive T lymphocytes. This unique property of dendritic cells, also termed "nature's adjuvant", has led to a great interest in 15 their possible role in autoimmune diseases as well as their potential for exploitation in immunotherapy of various diseases. Recent advances in culturing of dendritic cells has greatly increased our understanding of this complex type of cells. There are several types of dendritic cells that are distinguished by their lineage, location in tissues, phenotype, and function. The 20 dendritic cell type that most prominently associates with T lymphocytes for the initiation of immune responses is of bone marrow origin. Bone marrow-derived dendritic cells can be further segregated into 1) thymic dendritic cells, which are of lymphoid origin and appear to be involved specifically in deletion of maturing T lymphocytes, 2) Langerhans cells, which are of myeloid lineage and have specialised APC functions in 25 the skin, and 3) myeloid lineage-derived dendritic cells found particularly in the blood, spleen and lymph nodes. The hallmarks of myeloid lineage-derived dendritic cells (including Langerhans cells) are the following: 1) capacity for antigen uptake and processing for presentation, 2) capacity for selective migration in tissues, and 3) capacity for direct stimulation of T 30 lymphocytes (both naive and primed). Despite the recent advances in characterization of dendritic cells, very little is known regarding dendritic cell specific receptors or molecules. There are numerous -2 dendritic cell-associated molecules that are shared with other myeloid and non-myeloid cells, however, very limited reagents are available which are specific to dendritic cells. Reagents, in particular antibodies, which react specifically or preferentially with dendritic cells have great potential as targeting agents to induce potent immune 5 responses to tumor or infectious disease antigens. These cell specific targeting agents could also be engineered to deliver toxins to eliminate potent APCs (e.g., dendritic cells) in bone marrow and organ transplantations or other autoimmune disorders. Accordingly, dendritic cell-specific binding agents would be of great therapeutic and diagnostic value. 10 Summary of the Invention According to a first aspect, the present invention provides a preparation of a monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising CDRI, CDR2, and CDR3 sequences and a light chain 15 variable region comprising CDR1, CDR2, and CDR3 sequences, wherein: (a) the heavy chain variable region CDR3 sequence comprises amino acid residues 99-105 of SEQ ID NO: 4, and conservative modifications thereof; (b) the light chain variable region CDR3 sequence comprises amino acid residues 89-97 of SEQ ID NO: 2, and conservative modifications thereof; and 20 (c) the antibody binds to the macrophage mannose receptor on dendritic cells. According to a second aspect, the present invention provides a preparation of a monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises a sequence that is at least 80% 25 homologous to SEQ ID NO: 4; (b) the light chain variable region comprises a sequence that is at least 80% homologous to SEQ ID NO: 2; and (c) the antibody binds to the macrophage mannose receptor on dendritic cells. According to a third aspect, the present invention provides a preparation of a 30 monoclonal antibody, or antigen binding portion thereof, that binds to dendritic cells wherein the monoclonal antibody, or antigen binding portion thereof, comprises: - 2a (a) a heavy chain variable region having CDRI, CDR2 and CDR3 sequences set forth as amino acids 31-35, amino acids 50-66 and amino acids 99-105 of SEQ ID NO: 4, respectively; and (b) a light chain variable region having CDRI, CDR2 and CDR3 sequences 5 set forth as amino acids 24-34, amino acids 50-56 and 89-97 of SEQ ID NO: 2, respectively. According to a fourth aspect, the present invention provides a preparation of a monoclonal antibody, or antigen binding portion thereof, wherein said antibody or antigen binding portion competes for binding to dendritic cells with the antibody of any 10 one of the first to third aspects. According to a fifth aspect, the present invention provides a composition comprising the monoclonal antibody, or antigen binding portion thereof, according to any one of the first to fourth aspects and a pharmaceutically acceptable carrier. According to a sixth aspect, the present invention provides a molecular complex 15 comprising the monoclonal antibody, or antigen binding portion thereof, of any one of the first to fourth aspects linked to an antigen. According to a seventh aspect, the present invention provides a bispecific molecule comprising the antibody, or antigen binding portion thereof, of any one of the first to third aspects and a second binding specificity for an antigen. 20 According to an eighth aspect, the present invention provides use of the composition of the fifth aspect to modulate dendritic cell activity. According to a ninth aspect, the present invention provides use of the composition of the fifth aspect to induce or enhance an immune response against an antigen in a subject in need thereof. 25 According to a tenth aspect, the present invention provides use of the composition of the fifth aspect to immunize a subject in need thereof According to an eleventh aspect, the present invention provides an expression vector comprising a nucleotide sequence encoding a antibody heavy or light chain variable region, wherein the nucleotide sequence is selected from the group consisting of 30 SEQ ID NO: 3, SEQ ID NO: I and conservative sequence modifications thereof. According to a twelfth aspect, the present invention provides an expression vector comprising a nucleotide sequence encoding a antibody heavy chain variable region comprising an amino acid sequence SEQ ID NO: 4, and conservative sequence - 2b modifications thereof, or a antibody light chain variable region comprising an amino acid sequence SEQ ID NO: 2, and conservative sequence modifications thereof According to a thirteenth aspect, the present invention provides a method of inducing or enhancing an immune response against an antigen in a subject in need 5 thereof comprising administering a composition according to the fifth aspect to said subject. According to a fourteenth aspect, the present invention provides a method of immunizing a subject in need thereof comprising administering a composition according to the fifth aspect to said subject. 10 According to a fifteenth aspect, the present invention provides use of: (i) a monoclonal antibody or antigen binding portion thereof according to any one the first to third aspects, or (ii) a molecular complex according to the sixth aspect, or (iii) an immunoconjugate comprising the antibody of any one of the first to 15 third aspects linked to a therapeutic agent, a cytoxin, or a radioisotype, or (iv) a bispecific molecule according to the seventh aspect, in the manufacture of a medicament for inducing or enhancing an immune response against an antigen. According to a sixteenth aspect, the present invention provides use of: 20 (i) a monoclonal antibody or antigen binding portion thereof according to any one the first to third aspects, or (ii) a molecular complex according to the sixth aspect, or (iii) an immunoconjugate comprising the antibody of any one of the first to third aspects linked to a therapeutic agent, a cytoxin, or a radioisotype, or 25 (iv) a bispecific molecule according to the seventh aspect, in the manufacture of a medicament for immunizing a subject. According to a seventeenth aspect, the present invention provides use of a composition according to the fifth aspect for the manufacture of a medicament for inducing or enhancing an immune response against an antigen in a subject in need 30 thereof. According to an eighteenth aspect, the present invention provides use of a composition according to the fifth aspect for the manufacture of a medicament for immunizing a subject in need thereof.

- 2c According to a nineteenth aspect, the present invention provides a method of inducing or enhancing an immune response against an antigen in a subject comprising administering to said subject: (i) a monoclonal antibody or antigen binding portion thereof according to any 5 one the first to third aspects, or (ii) a molecular complex according to the sixth aspect, or (iii) an immunoconjugate comprising the antibody of any one of the first to third aspects linked to a therapeutic agent, a cytoxin, or a radioisotype, or (iv) a bispecific molecule according to the seventh aspect. 10 According to a twentieth aspect, the present invention provides a method of immunizing a subject comprising administering to said subject: (i) a monoclonal antibody or antigen binding portion thereof according to any one the first to third aspects, or (ii) a molecular complex according to the sixth aspect, or 15 (iii) an immunoconjugate comprising the antibody of any one of the first to third aspects linked to a therapeutic agent, a cytoxin, or a radioisotype, or (iv) a bispecific molecule according to the seventh aspect. Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an 20 inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". The present invention provides isolated human monoclonal antibodies which specifically bind to antigen presenting cells (APCs) and, in particular, dendritic cells, as well as compositions containing such antibodies, either alone or combined (e.g., mixed 25 with or linked to other therapeutic or diagnostic reagents). Accordingly, the antibodies and compositions of the invention can be used in a variety of dendritic cell-targeted therapies, for example to affect antigen presentation or to treat APC-mediated diseases. In certain embodiments, the human antibodies are characterized by high affinity binding to dendritic cells, and by their ability to affect dendritic cell growth and/or 30 function by targeting molecules or cells with defined functions (e.g., a tumor cell, a bacterium, a virus, and effector cell) to dendritic cells. Accordingly, the human monoclonal antibodies of the invention can be used as diagnostic or therapeutic agents in vivo and in vitro.

- 2d In other embodiments, the human antibodies are characterized by binding to particular novel epitopes (e.g., receptors) on dendritic cells. In still other embodiments, the human antibodies are characterized by binding to the macrophage mannose receptor, or a related receptor. 5 Isolated human antibodies of the invention encompass various antibody isotypes, such as IgGI, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD, and IgE. Typically, they include IgGI (e.g., IgGIK) and IgM isotypes. The antibodies can be full-length (e.g., an IgGI or IgG4 antibody) or can include only an antigen-binding portion (e.g., a Fab, F(ab') 2 , Fv or a single chain Fv fragment). In one embodiment, the human 10 antibodies are recombinant human antibodies. In another embodiment, the human antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a human light chain transgene fused to an immortalized cell. 15 -3 Particular human antibodies of the invention include those produced by hybridomas referred to herein as A3, A5, A23, A24, A33, B9, BI 1, B33, B47, C8, CIO, C20, C28, C29, C30, C35, El, E8, El0, E18, E20, E21 and E24. In another embodiment, human antibodies of the present invention are 5 characterized by specific binding to dendritic cells and one or more of the following properties: a) a binding affinity constant of at least about 107 M-, preferably about 109 M-, and more preferably, about 10'0 M-' to 101" M~' or higher; c) an association constant (Kasso,) of at least about 103, more preferably 10 about 104 and most preferably about 105 M-'S'; d) a dissociation constant (Kdis) of about 10- s~', preferably about 10-4 s-, more preferably, 10~5 s-1, and most preferably, 10-6 s-; e) the ability to opsonize dendritic cells; f) the ability to internalize after binding to dendritic cells; 15 g) the ability to bind dendritic cells in situ (e.g., in human tissues); h) the ability to activate dendritic cells (e.g., induce cytokine release, expression of immunomodulatory surface molecules); or i) the ability to inhibit growth and/or mediate phagocytosis and killing of dendritic cells in the presence of human effector cells at a concentration of about 10 20 pg/ml or less (e.g., in vitro). In one embodiment, isolated human antibodies of the invention bind to dendritic cells with an affinity constant of at least about 107 M, preferably about 108 M-, more preferably, about 109 M-, and more preferably about 1010 to 10" M 1 or stronger. In another aspect, the invention provides nucleic acid molecules encoding the 25 antibodies, or antigen-binding portions, of the invention. Accordingly, recombinant expression vectors which include the antibody-encoding nucleic acids of the invention, and host cells transfected with such vectors, are also encompassed by the invention, as are methods of making the antibodies of the invention by culturing these host cells. In yet another aspect, the invention provides isolated B-cells from a transgenic 30 non-human animal, e.g., a transgenic mouse, which are capable of expressing various isotypes (e.g., IgG, IgA and/or IgM) of human monoclonal antibodies that specifically bind to dendritic cells. Preferably, the isolated B cells are obtained from a transgenic -4 non-human animal, e.g., a transgenic mouse, which has been immunized with a purified or enriched preparation of dendritic cells. Preferably, the transgenic non-human animal, e.g., a transgenic mouse, has a genome comprising a human heavy chain transgene and a human light chain transgene. The isolated B-cells are then immortalized to provide a 5 source (e.g., a hybridoma) of human monoclonal antibodies to dendritic cells. Accordingly, the present invention also provides a hybridoma capable of producing human monoclonal antibodies that specifically bind to dendritic cells. In one embodiment, the hybridoma includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain 10 transgene and a human light chain transgene, fused to an immortalized cell. The transgenic non-human animal can be immunized with a purified or enriched preparation of dentritic cells to generate antibody-producing hybridomas. Particular hybridomas of the invention include A3, A5, A23, A24, A33, B9, BI1, B33, B47, C8, C1O, C20, C28, C29, C30, C35, El, E8, ElO, E18, E20, E21 and E24. 15 In yet another aspect, the invention provides a transgenic non-human animal, such as a transgenic mouse (also referred to herein as a "HuMab"), which express human monoclonal antibodies that specifically bind to dendritic cells. In a particular embodiment, the transgenic non-human animal is a transgenic mouse having a genome comprising a human heavy chain transgene and a human light chain transgene. The 20 transgenic non-human animal can be immunized with a purified or enriched preparation of dendritic cells. Preferably, the transgenic non-human animal, e.g., the transgenic mouse, is capable of producing multiple isotypes of human monoclonal antibodies to dendritic cells (e.g., IgG, IgA and/or IgM) by undergoing V-D-J recombination and isotype switching. Isotype switching may occur by, e.g., classical or non-classical 25 isotype switching. In another aspect, the present invention provides methods for producing human monoclonal antibodies which specifically react with dendritic cells. In one embodiment, the method includes immunizing a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a human light 30 chain transgene, with a purified or enriched preparation of dendritic cells. B cells (e.g., splenic B cells) of the animal are then obtained and fused with myeloma cells to form immortal, hybridoma cells that secrete human monoclonal antibodies against dendritic cells. Isolated anti-dendritic cell human monoclonal antibodies of the invention, or antigen binding portions thereof, can be derivatized or linked to another functional 5 molecule, e.g., another peptide or protein (e.g., an Fab' fragment). For example, an antibody or antigen-binding portion of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as a or another antibody (e.g., a bispecific or a multispecific antibody). Accordingly, in one aspect, the present invention features a 10 human anti-dendritic cell antibody, or a fragment thereof, conjugated to a therapeutic moiety, e.g., a cytotoxic drug, an enzymatically active toxin, or .a fragment thereof, a radioisotope, or a small molecule, for example, an immunomodulatory (e.g., anti inflammatory) compound, or an anti-cancer drug. Human anti-dendritic cell antibodies of the invention also can be linked to an 15 antigen, such that the antigen is targeted to dendritic cells which internalize, process and present the antigen. The antigen can be, for example, a tumor cell antigen, a microbial antigen, a viral antigen or an autoantigen. In yet another aspect, the present invention provides a method for inducing or enhancing an immune response against an antigen (e.g., a tumor cell antigen, a microbial 20 antigen, or a viral antigen) in a subject, comprising administering to the subject a molecular complex comprising at least one binding specificity for a component on the surface of a dendritic cell linked to at least one antigen, wherein the component on the surface of the dendritic cell mediates internalization of the molecular complex when bound by the binding specificity. In one embodiment, the immune response comprises 25 antibodies that bind to the antigen. In another embodiment, the immune response comprises T cells that bind to the antigen as a component of an MHC-I or MHC-II complex. In one aspect, the anti-dendritic cell antibody, or a fragment thereof, can be used to target whole cells (e.g., a tumor cell, an effector cell) or pathogens to dendritic cells 30 for the induction of an immune response. In one embodiment, a cell can be transfected or transduced with a nucleic acid molecule encoding a human anti-dendritic cell antibody of the invention such that the anti-dendritic cell antibody is expressed on the -6 surface of the cell. In another embodiment, an human anti-dendritic cell antibody of the invention can be directly chemically or otherwise crosslinked, anchored or tagged to the cell surface of a cell (e.g., a tumor cell, a bacterium or a virus) such that the cell can be targeted to dendritic cells. 5 In a further aspect, the invention provides a method for immunizing a subject, comprising administering to the subject an effective amount of a molecular complex comprising at least one binding specificity for a component on the surface of a dendritic cell linked to at least one antigen, wherein the component on the surface of the dendritic cell mediates internalization of the molecular complex when bound by the binding 10 specificity. In another aspect, the present invention features a bispecific or multispecific molecule comprising at least one first binding specificity for dendritic cells and a second binding specificity an Fc receptor, e.g., human FcyRI or a human Fca receptor. In another aspect, the present invention provides a bispecific or multispecific molecule 15 comprising at least one first binding specificity for dendritic cells and a second binding sepcificity for an antigen on a target cell. A target cell is a cell whose elimination would be beneficial to the host, e.g., a tumor cell, a microbial pathogen, or a virus or virus infected cell. Multispecific molecules of the invention also include trispecific, tetraspecific 20 and other multispecific molecules. In one embodiment the multispecific molecule includes an anti-enhancement factor (EF) portion, e.g., a molecule which binds to a surface protein involved in cytotoxic activity. In a particular embodiment, bispecific and multispecific molecules of the invention comprise at least one antibody, or fragment thereof (e.g., an Fab, Fab', F(ab') 2 , 25 Fv, or a single chain Fv). In a particular embodiment, the antibody or fragment thereof is a completely human antibody or a portion thereof, or a "chimeric" or a "humanized" antibody or a portion thereof (e.g., has a variable region, or at least a complementarity determining region (CDR), derived from a non-human antibody (e.g., murine) with the remaining portion(s) being human in origin). 30 In one embodiment, the at least one antibody or fragment thereof of the bispecific or multispecific molecule binds to an Fc receptor, such as a human IgG receptor, e.g., an Fc-gamma receptor (FcyR), such as FcyRI (CD64), FcyRII(CD32), and -7 FcyRIII (CD 16). A preferred Fcy receptor is the high affinity Fcy receptor, FcyRI. However, other Fc receptors, such as human IgA receptors (e.g. FcaRI) also can be targeted. The Fc receptor is preferably located on the surface of an effector cell, e.g., a monocyte, macrophage or an activated polymorphonuclear cell. In a preferred 5 embodiment, the bispecific and multispecific molecules bind to an Fe receptor at a site which is distinct from the immunoglobulin Fc (e.g., IgG or IgA) binding site of the receptor. Therefore, the binding of the bispecific and multispecific molecules is not blocked by physiological levels of immunoglobulins. In another aspect, the present invention provides target-specific effector cells 10 which comprise an effector cell expressing an Fc receptor, e.g., a macrophage or an activated PMiN cell, linked to a bispecific or multispecific molecule of the invention, which binds to the effector cell via its Fc receptor, and also binds to a dendritic cell so that the effector cell is targeted to dendritic cells. In another aspect, the present invention provides compositions, e.g., 15 pharmaceutical and diagnostic compositions, comprising a pharmaceutically acceptable carrier and at least one human monoclonal antibody of the invention, or an antigen binding portion thereof, which specifically binds to dendritic cells. In one embodiment, the composition comprises a combination of the human antibodies or antigen-binding portions thereof, preferably each of which binds to a distinct epitope. For example, a 20 pharmaceutical composition comprising a human monoclonal antibody that mediates highly effective killing of dendritic cells in the presence of effector cells can be combined with another human monoclonal antibody that inhibits the growth of dendritic cells. Thus, the combination provides multiple therapies tailored to provide the . maximum therapeutic benefit. Compositions, e.g., pharmaceutical compositions, 25 comprising a combination of at least one human monoclonal antibody of the invention, or antigen-binding portions thereof, and at least one bispecific or multispecific molecule of the invention, or other therapeutic agents (e.g., cytotoxic agents) are also within the scope of the invention. In yet another aspect, the invention provides a method for inhibiting the 30 proliferation and/or differentiation of dendritic cells by inhibiting growth and/or by inducing phagocytosis and/or killing of dendritic cells by human effector cells, such as human polymorphonuclear cells (PMNs), monocytes and macrophages, using an -8 antibody, or antigen-binding portion thereof (or a bispecific or multispecific antibody) of the invention. In one embodiment, the method comprises contacting a dendritic cell either in vitro or in vivo with one or a combination of human monoclonal antibodies of the invention, or an antigen-binding portion thereof, in the presence of a human effector 5 cell. The method can be employed in culture, e.g. in vitro or ex vivo (e.g., cultures comprising dendritic cells and effector cells). For example, a sample containing dendritic cells and effector cells can be cultured in vitro, and combined with an antibody of the invention, or an antigen-binding portion thereof (or a bispecific or multispecific molecule of the invention). Alternatively, the method can be performed in a subject, 10 e.g., as part of an in vivo (e.g., therapeutic or prophylactic) protocol. For in vivo methods, the antibody, or antigen-binding portion thereof (or a bispecific or multispecific molecule of the invention), can be administered to a human subject suffering from a dendritic cell-mediated disease. These diseases include, for example, autoimmune disease, inflammatory disease, and graft versus host disease. 15 Exemplary autoimmune diseases that can be treated (e.g., ameliorated) or prevented using the methods and compositions of the invention include, but are not limited to rheumatoid arthritis, multiple sclerosis, diabetes mellitus, myasthenia gravis, pernicious anemia, Addison's disease, lupus erythematosus, Reiter's syndrome, and Graves disease. In one embodiment, the subject can be additionally treated with an agent that 20 modulates, e.g., enhances or inhibits, the expression or activity of Fc receptor, e.g., an Fca receptor or an Fcy receptor, by for example, treating the subject with a cytokine. Preferred cytokines for administration during treatment with the bispecific and multispecific molecule include granulocyte colony-stimulating factor (G-CSF), granulocyte- macrophage colony-stimulating factor (GM-CSF), interferon-y (IFN-y), 25 and tumor necrosis factor (TNF). Isolated human monoclonal antibody compositions of the invention also can be administered in combination with other known therapies, e.g., an anti-inflammatory or immunosuppressant therapies, or cytotoxins. In yet another aspect, the present invention provides a method for detecting in 30 vitro or in vivo the presence of dendritic cells in a sample, e.g., for diagnosing a dendritic cell-related disease. In one embodiment, this is achieved by contacting a sample to be tested, along with a control sample, with a human monoclonal antibody of -9 the invention, or an antigen-binding portion thereof (or a bispecific or multispecific molecule), under conditions that allow for formation of a complex between the antibody and a dendritic cell. Complex formation is then detected (e.g., using an ELISA) in both samples, and any statistically significant difference in the formation of complexes 5 between the samples is indicative the presence of dendritic cells in the test sample. Other features and advantages of the instant invention be apparent from the following detailed description and claims. 10 Brief Description of the Drawings Figure 1 shows the reactivity of human monoclonal antibodies B 11, C20 and E21 with dendritic cells and hematopoietic cell lines U937, CEM, THP-1 and L540, as assessed by flow cytometry. Binding was measured by mean fluorescence intensity. 15 Figure 2 shows the dose-dependent binding of human monoclonal antibodies B 11, C20 and E21 to dendritic cells, as assessed by flow cytometry. Binding was measured by mean fluorescence intensity. 20 Figure 3 shows the dose-dependent reactivity of human monoclonal antibody B 11 with CD34+ stem cell-derived dendritic cells, as assessed by flow cytometry. Binding was measured by mean fluorescence intensity. Figure 4 shows the binding of human monoclonal antibody B 11 to dendritic 25 cells and macrophages, as assessed by flow cytometry. Figure 5 shows the binding of human monoclonal antibody B 11 to THP-1 induced to differentiate into a dendritic cell phenotype, as assessed by flow cytometry. 30 Figure 6 shows binding of human monoclonal antibody B 11 to macaque dendritic cells, as assessed by flow cytometry. Binding was measured by mean fluorescence intensity.

-10 Figure 7 shows the percent internalization of human monoclonal antibody B 11 by dendritic cells over time at 37*C. 5 Figure 8 shows increased antigen presentation via antibody B I1 compared to antigen alone, as measured by stimulation of tetanus toxoid-specific T cells. Figure 9 shows the B 11 ScFv construct which was created by linking the VL (SEQ ID NO: 1 and 2) and VH (SEQ ID NO: 3 and 4) domains of human monoclonal 10 antibody B11. Figure 10 shows a binding comparison between whole human monoclonal antibody B 11 and F(ab')2 fragments of B 11 to dendritic cells, as measured by FACS analysis. 15 Figure 11 shows the percent of FITC-dextran internalization by dendritic cells. Figure 12 shows that conjugation of antigen to BI I enhances antigen presentation, as 10 to 100-fold lower amounts of antibody B 11-conjugated tetanus 20 toxoid are required to achieve the same level of T cell stimulation as with tetanus toxoid alone. Figure 13 shows the nucleotide and corresponding amino acid sequences of the variable light (VL) and variable heavy (VL) chains of antibody B 11. 25 - 11 Detailed Description of the Invention The present invention provides novel antibody-based therapies for modulating an immune response against an antigen, and for treating and diagnosing diseases mediated by dendritic cells. 5 Therapies of the invention employ isolated human monoclonal antibodies, or antigen-binding portions thereof, which bind to an epitope present on antigen presentic cells (APC), particularly dendritic cells and cells related thereto. In one embodiment, the human antibodies are produced in a non-human transgenic animal, e.g., a transgenic mouse, capable of producing multiple isotypes of human monoclonal antibodies to 10 dendritic cells (e.g., IgG, IgA and/or IgE) by undergoing V-D-J recombination and isotype switching. Accordingly, various aspects of the invention include human antibodies, antibody fragments and antibody mimetics, pharmaceutical compositions thereof, as well as non-human transgenic animals, and B-cells and hybridomas for making such monoclonal antibodies. Methods of using the antibodies of the invention 15 to detect a dendritic cells or a related cell type expressing a dendritic cell antigen, or to inhibit growth, differentiation and/or activity of a dendritic cell, either in vitro or in vivo, are also encompassed by the invention. In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description. 20 The term "dendritic cell" as used herein, includes immature and mature dendritic cells and related myeloid progenitor cells that are capable of differentiating into dendritic cells, or related antigen presenting cells (e.g., monocytes and macrophages) in that they express antigens in common with dendritic cells. As used herein, the term "related" includes a cell that is derived from a common progenitor cell or cell lineage. 25 In a preferred embodiment, binding of an antibody of the invention to a dendritic cell inhibits the growth of dendritic cells. In another preferred embodiment, binding of an antibody of the invention to dendritic cells mediates an effect on dendritic cell growth and/or function by targeting molecules or cells with defined functions (e.g., tumor cells, effector cells, microbial pathogens) to dendritic cells. In a further embodiment, binding 30 of an antibody of the invention to a dendritic cell results in internalization of the antibody by the dendritic cell.

-12 As used herein, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is 5 comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more 10 conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to 15 host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., an antigen on a dendritic cell). It has been 20 shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHi domains; (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the 25 hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate 30 genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) - 13 Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879 5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are 5 screened for utility in the same manner as are intact antibodies. The term "bispecific molecule" is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has two different binding specificities which bind to, or interact with, for example, (a) a dendritic cell and (b) an Fc receptor on the surface of an effector cell. In another embodiment, a bispecific molecule of the 10 invention has two different binding specificities which bind to, or interact with (a) a dendritic cell and (b) an antigen on a target cell (e.g., a tumor cell). The term "multispecific molecule" or "heterospecific molecule" is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has more than two different binding specificities which bind to, or interact with, for example, (a) a dendritic cell , (b) 15 an Fc receptor on the surface of an effector cell, and (c) at least one other component. Accordingly, the invention includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules which are directed to cell surface antigens, such as a dendritic cell antigen, and to Fc receptors on effector cells, or an antigen on a target cell (e.g., a tumor cell). The term "bispecific antibodies" further 20 includes diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Nal. Acad. Sci. USA 25 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123). As used herein, the term "heteroantibodies" refers to two or more antibodies, antibody binding fragments (e.g., Fab), derivatives therefrom, or antigen binding regions linked together, at least two of which have different specificities. These different specificities include a binding specificity for a dendritic cell, and a binding specificity 30 for an Fc receptor on an effector cell, or an antigen or epitope on a target cell, e.g., a tumor cell.

-14 The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by 5 random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "monoclonal antibody" or "monoclonal antibody composition" as used 10 herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. In one embodiment, 15 the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, fused to an immortalized cell. The term "recombinant human antibody", as used herein, is intended to include 20 all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (described further in Section I, below); antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, or antibodies 25 prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic 30 for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, - 15 while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. As used herein, a "heterologous antibody" is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody 5. having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal. As used herein, a "heterohybrid antibody" refers to an antibody having a light and heavy chains of different organismal origins. For example, an antibody having a 10 human heavy chain associated with a murine light chain is a heterohybrid antibody. Examples of heterohybrid antibodies include chimeric and humanized antibodies, discussed supra. An "isolated antibody", as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an 15 isolated antibody that specifically binds to dendritic cells and related myeloid derived antigen presenting cells (e.g., monocytes and macrophages), and is substantially free of antibodies that specifically bind cell types other than dendritic cells). An isolated antibody that specifically binds to a dendritic cell may, however, have cross-reactivity to other cells, e.g., cell types that express an antigen that is related to the cognate antigen 20 on a dendritic cell. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment of the invention, a combination of "isolated" monoclonal antibodies having different specificities are combined in a well defined composition. As used herein, the terms "specific binding" and "specifically binds to" refers to 25 specificity of antibody binding to a predetermined antigen or cell type. An antibody which "binds specifically to", or is "specific for" a particular antigen or cell type, binds to the antigen or cell type with selectivity over other antigens and cell types. While each antibody of the invention binds specifically to a particular target epitope (e.g., present on the surface of a dendritic cell), specific antibodies of the invention, in certain 30 embodiments, may exhibit some cross-reactivity with other APCs. In other embodiments, the specific antibodies react only with dendritic cells. Typically, the antibody binds with an affinity of at least about 1 x 107 M", and binds to the -16 predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The phrases "an antibody recognizing an antigen" and " an antibody specific for an antigen" are used interchangeably herein with the term 5 "an antibody which binds specifically to an antigen". As used herein, the term "high affinity" for an IgG antibody refers to a binding affinity of at least about 10 7 M', preferably at least about 10 9 M-, more preferably at least about 1' 0 "M ,10"M', 101 2 M- or greater, e.g., up tol0' 3 M' or greater. However, "high affinity" binding can vary for other antibody isotypes. For example, "high 10 affinity" binding for an IgM isotype refers to a binding affinity of at least about 1 x 10 7 M-1. The term "Kassoc", as used herein, is intended to refer to the association constant of a particular antibody-antigen interaction. The term "Kdis", as used herein, is intended to refer to the dissociation constant 15 of a particular antibody-antigen interaction. As used herein, "isotype" refers to the antibody class (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes. As used herein, "isotype switching" refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes. 20 As used herein, "nonswitched isotype" refers to the isotypic class of heavy chain that is produced when no isotype switching has taken place; the CH gene encoding the nonswitched isotype is typically the first CH gene immediately downstream from the functionally rearranged VDJ gene. Isotype switching has been classified as classical or non-classical isotype switching. Classical isotype switching occurs by recombination 25 events which involve at least one switch sequence region in the transgene. Non classical isotype switching may occur by, for example, homologous recombination between human a, and human 1. (8-associated deletion). Alternative non-classical switching mechanisms, such as intertransgene and/or interchromosomal recombination, among others, may occur and effectuate isotype switching. 30 As used herein, the term "switch sequence" refers to those DNA sequences responsible for switch recombination. A "switch donor" sequence, typically a i switch region, will be 5' (i.e., upstream) of the construct region to be deleted during the switch -17 recombination. The "switch acceptor" region will be between the construct region to be deleted and the replacement constant region (e.g., y, e, etc.). As there is no specific site where recombination always occurs, the final gene sequence will typically not be predictable from the construct. 5 As used herein, "glycosylation pattern" is defined as the pattern of carbohydrate units that are covalently attached to a protein, more specifically to an immunoglobulin protein. A glycosylation pattern of a heterologous antibody can be characterized as being substantially similar to glycosylation patterns which occur naturally on antibodies produced by the species of the nonhuman transgenic animal, when one of ordinary skill 10 in the art would recognize the glycosylation pattern of the heterologous antibody as being more similar to said pattern of glycosylation in the species of the nonhuman transgenic animal than to the species from which the CH genes of the transgene were derived. The term "naturally-occurring" as used herein as applied to an object refers to the 15 fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring. The term "rearranged" as used herein refers to a configuration of a heavy chain 20 or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete VH or VL domain, respectively. A rearranged immunoglobulin gene locus can be identified by comparison to germline DNA; a rearranged locus will have at least one recombined heptamer/nonamer homology element. 25 The term "unrearranged" or "germline configuration" as used herein in reference to a V segment refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment. The term "nucleic acid molecule", as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or 30 double-stranded, but preferably is double-stranded DNA. The term "isolated nucleic acid molecule", as used herein in reference to nucleic acids encoding antibodies or antibody portions (e.g., VH, VL, CDR3) that bind to -18 dendritic cells, is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies or antibody portions that bind cells other than dendritic cells, which other sequences may naturally flank the nucleic acid in human genomic 5 DNA. For nucleic acids, the term "substantial homology" indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98% 10 to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology =# of identical 15 positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below. 20 The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also determined using the algorithm of E. Meyers and W. Miller 25 (Comput. Apple. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. MoL. Biol. (48):444 453 (1970)) algorithm which has been incorporated into the GAP program in the GCG 30 software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

-19 The nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. 5 BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for 10 comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially 15 purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. Current 20 Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). The nucleic acid compositions of the present invention, while often in a native sequence (except for modified restriction sites and the like), from either cDNA, genomic or mixtures may be mutated, thereof in accordance with standard techniques to provide 25 gene sequences. For coding sequences, these mutations, may affect amino acid sequence as desired. In particular, DNA sequences substantially homologous to or derived from native V, D, J, constant, switches and other such.sequences described herein are contemplated (where "derived" indicates that a sequence is identical or modified from another sequence). 30 The term "operatively linked" or "operably linked" is intended to mean that molecules are functionally coupled to each other in that the change of activity or state of one molecule is affected by the activity or state of the other molecule. A nucleic acid is -20 "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcription regulatory sequences, operably linked means that the DNA sequences being linked are 5 contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. For switch sequences, operably linked indicates that the sequences are capable of effecting switch recombination. Typically, two polypeptides that are operably linked are covalently attached through peptide bonds. The term "vector", as used herein, is intended to refer to a nucleic acid molecule 10 capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are 15 introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)- can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are 20 referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral 25 vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the 30 particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, -21 such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Various aspects of the invention are described in further detail in the following subsections. 5 I. Production of Human Antibodies to Dendritic Cells While particularly preferred methods of generating human monoclonal antibodies (mAbs) of the invention are described in detail herein, a variety of other techniques, including conventional monoclonal antibody methodology e.g., the standard 10 somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975) also can be used. Although somatic cell hybridization procedures are preferred, other techniques for producing monoclonal antibody can be employed e.g., viral or oncogenic transformation of B lymphocytes. The preferred animal system for preparing hybridomas is the murine system. 15 Hybridoma production in murine systems is a well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are well known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also well known. In a preferred embodiment, human monoclonal antibodies directed against 20 dendritic cells are generated using transgenic mice carrying parts of the human immune system rather than the mouse system. These transgenic mice, referred to herein as "HuMAb" mice, contain a human immunoglobulin gene miniloci that encodes unrearranged human heavy (pt and y) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous L and K chain loci 25 (Lonberg, N. et al. (1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or K, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGK monoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook ofExperimental Pharmacology 113:49-101; 30 Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. NY Acad Sci 764:536-546). The preparation of HuMab mice is described in detail Section II below and in Taylor, L. et al. (1992) - 22 Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad Sci USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993)EMBOJ. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Lonberg et al., (1994) Nature 5 368(6474): 856-859; Lonberg, N. (1994) Handbook ofExperimental Pharmacology 113:49-101; Taylor, L. et al. (1994) International Immunology 6: 579-591; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93; Harding, F. and Lonberg, N. (1995) Ann. N. Y A cad. Sci 764:536-546; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-85 1, the contents of all of which are hereby incorporated by 10 reference in their entirety. See further, U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay, and GenPharm International; U.S. Patent No. 5,545,807 to Surani et al.; International Publication Nos. WO 98/24884, published on June 11, 1998; WO 94/25585, published November 10, 1994; WO 93/1227, published 15 June 24, 1993; WO 92/22645, published December 23, 1992; WO 92/03918, published March 19, 1992, the disclosures of all of which are hereby incorporated by reference in their entity. HuMab Immunizations 20 To generate fully human monoclonal antibodies to dendritic cells, HuMab mice can be immunized with a purified or enriched preparation of dendritic cells, as described by Lonberg, N. et al. (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851 and WO 98/24884. Preferably, the mice will be 6-16 weeks of age upon the first immunization. For example, a purified or enriched 25 preparation of dendritic cells (1-10 million cells) can be used to immunize the HuMab mice intraperitoneally. In the event that immunizations using a purified or enriched preparation of dendritic cells do not result in antibodies, mice can also be immunized with a dendritic cell lysate to promote immune responses. Cumulative experience with various antigens has shown that the HuMAb 30 transgenic mice respond best when initially immunized intraperitoneally (IP) with antigen in complete Freund's adjuvant, followed by every other week IP immunizations (up to a total of 6) with antigen in incomplete Freund's adjuvant. The immune response - 23 can be monitored over the course of the immunization protocol with plasma samples being obtained by retroorbital bleeds. The plasma can be screened, for example by ELISA or flow cytometry (as described below), and mice with sufficient titers of anti dendritic cell human immunoglobulin can be used for fusions. Mice can be boosted 5 intravenously with antigen 3 days before sacrifice and removal of the spleen. It is expected that 2-3 fusions for each antigen may need to be performed. Several mice will be immunized for each antigen. For example, a total of twelve HuMAb mice of the HC07 and HC012 strains can be immunized. 10 Generation of Hybridomas Producing Human Monoclonal Antibodies to Dendritic Cells The mouse splenocytes can be isolated and fused with PEG to a mouse myeloma cell line based upon standard protocols. The resulting hybridomas are then screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice are fused to one-sixth the number of P3X63 15 Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2 x 105 in flat bottom microtiter plate, followed by a two week incubation in selective medium containing 20% fetal Clone Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM L-glutamine, 1 mM sodium pyruvate, 5mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 20 50 mg/ml streptomycin, 50 mg/ml gentamycin and 1X HAT (Sigma; the HAT is added. 24 hours after the fusion). After two weeks, cells are cultured in medium in which the HAT is replaced with HT. Individual wells are then screened by ELISA for human anti dendritic cell monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, medium is observed usually after 10-14 days. The antibody secreting 25 hybridomas are replated, screened again, and if still positive for human IgG, anti dendritic cell monoclonal antibodies, can be subcloned at least twice by limiting dilution. The stable subclones are then cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.

-24 Characterization of Binding of Human Monoclonal Antibodies to Dendritic Cells To characterize binding of human monoclonal dendritic cell antibodies of the invention, hybridomas can be screened, for example, for positive reactivy with dendritic cells by flow cytometry. 5 Briefly, dendritic cells are harvested and washed, then added to 96 well plates and incubated with dilutions of hybridoma supertatants (or monoclonal antibodies in PBS containing 0.1% Tween 80 and 20% mouse serum) at 4*C for 1 hour. The plates are then washed, and further incubated with secondary antibodies (e.g. FITC or PE labeled anti-human IgG) for 1 hour at 4"C. After washing the cells are fixed with 1% 10 paraformaldehyde, and analyzed. The samples can be analyzed by FACScan instrument using light and side scatter properties to gate on single cells. An alternative assay using fluorescence microscopy may be used (in addition to or instead of) the flow cytometry assay. Cells can be stained exactly as described above and examined by fluorescence microscopy. This method allows visualization of individual cells, but may have 15 diminished sensitivity depending on the density of the antigen. Hybridomas that bind with high avidity to dendritic cells will be subcloned and further characterized. One clone from each hybridoma, which retains the reactivity of the parent cells (by flow cytometry), can be chosen for making a 5-10 vial cell bank stored at -140 'C, and for antibody purification. 20 To purify human anti-dendritic cell antibodies, selected hybridomas can be grown in two-liter spinner-flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated before affinity chromatography with protein A sepharose (Pharmacia, Piscataway, NJ). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The 25 buffer solution can be exchanged into PBS, and the concentration can be determined by

OD

280 using 1.43 extinction coefficient. The monoclonal antibodies can be aliquoted and stored at -80 *C. To determine if the selected human anti-dendritic cell monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available 30 reagents (Pierce, Rockford, IL). Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using flow - 25 cytometry as described above. Biotinylated monoclonal antibody binding can be detected with a strep-avidin-alkaline phosphatase probe. To determine the isotype of purified antibodies, isotype ELISAs can be performed. Wells of microtiter plates can be coated with 10 pig/ml of anti-human Ig 5 overnight at 4 0 C. After blocking with 5% BSA, the plates are reacted with 10 pig/ml of monoclonal antibodies or purified isotype controls, at ambient temperature for two hours. The wells can then be reacted with either human IgGl or human IgM-specific alkaline phosphatase-conjugated probes. After washing, the plates are developed with pNPP substrate (1 mg/ml), and analyzed at OD of 405-650. 10 Anti-dendritic cell human IgGs can be further tested for reactivity with dendritic cells by western blotting. Briefly, cell extracts from dendritic cells can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes, blocked with 20% mouse serum, and probed with the monoclonal antibodies to be 15 tested. Human IgG binding can be detected using anti-human IgG alkaline phosphatase and developed with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, MO). Phagocytic and Cell Killing Activities of Human Monoclonal Antibodies to Dendritic Cells 20 In addition to binding specifically to dendritic cells, human monoclonal anti dendritic cell antibodies can be tested for their ability to mediate phagocytosis and killing of dendritic cells. The testing of monoclonal antibody activity in vitro can provide an initial screening prior to testing in vivo models. Briefly, polymorphonuclear cells (PMN), or other effector cells, from healthy donors can be purified by Ficoll 25 Hypaque density centrifugation, followed by lysis of contaminating erythrocytes. Washed PMNs, can be suspended in RPMI supplemented with 10% heat-inactivated fetal calf serum and mixed with 51 Cr labeled dendritic cells, at various ratios of effector cells to dendritic cells(effector cells: dendritic cells). Purified human anti-dendritic cell IgGs can then be added at various concentrations. Irrelevant human IgG can be used as 30 negative control. Assays can be carried out for 0-120 minutes at 37'C. Samples can be assayed for cytolysis by measuring 5 1Cr release into the culture supernatant. Anti- - 26 dendritic cell monoclonal can also be tested in combinations with each other to determine whether cytolysis is enhanced with multiple monoclonal antibodies. Human monoclonal antibodies which bind to dendritic cells also can be tested in an in vivo model (e.g., in mice) to determine their efficacy in mediating phagocytosis 5 and killing of dendritic cells. These antibodies can be selected, for example, based on the following criteria, which are not intended to be exclusive: 1.) binding to live dendritic cells; 2.) high affinity of binding to dendritic cells; 10 3.) binding to a unique epitope on dendritic cells (to eliminate the possibility that monoclonal antibodies with complimentary activities when used in combination would compete for binding to the same epitope); 4.) opsonization of dendritic cells; 5.) mediation of growth inhibition, phagocytosis and/or killing of dendritic cells 15 in the presence of human effector cells; 6.) internalization after binding to dendritic cells; 7.) binding to dendritic cells in situ (e.g., in human tissues); 8.) activation of dendritic cells (e.g., induce cytokine release, expression of immunomodulatory surface molecules (e.g.,CD80 (B7.1), CD86 (B7.2), CD40, and 20 CD54 (ICAM)); 9.) binding to the human mannose receptor on dendritic cells; and 10.) binding to a dendritic cell antigen which is conserved among primates. Preferred human monoclonal antibodies of the invention meet one or more, and 25 preferably all, of these criteria. In a particular embodiment, the human monoclonal antibodies are used in combination, e.g., as a pharmaceutical composition comprising two or more anti-dendritic cell monoclonal antibodies or fragments thereof. For example, human anti-dendritic cell monoclonal antibodies having different, but complementary activities can be combined in a single therapy to achieve a desired 30 therapeutic or diagnostic effect. An illustration of this would be a composition containing an anti-dendritic cell human monoclonal antibody that is rapidly internalized by dendritic cells, combined with another human anti-dendritic cell monoclonal - 27 antibody that induces antigen presenting cell activities of dendritic cells, e.g., release of immunostimulatory cytokines. II. Production of Transgenic Nonhuman Animals Which Generate Human Monoclonal 5 Anti-Dendritic Cell Antibodies In yet another aspect, the invention provides transgenic non-human animals, e.g., a transgenic-mice, which are capable of expressing human monoclonal antibodies that specifically bind to dendritic cells, preferably with high affinity. In a preferred embodiment, the transgenic non-human animals, e.g., the transgenic mice (HuMab 10 mice), have a genome comprising a human heavy chain transgene and a light chain transgene. In one embodiment, the transgenic non-human animals, e.g., the transgenic mice, have been immunized with a purified or enriched preparation of dendritic cells and/or a dendritic cell lysate. Preferably, the transgenic non-human animals, e.g., the transgenic mice, are capable of producing multiple isotypes of human monoclonal 15 antibodies to dendritic cells (e.g., IgG, IgA and/or IgE) by undergoing V-D-J recombination and isotype switching. Isotype switching may occur by, e.g., classical or non-classical isotype switching. The design of a transgenic non-human animal that responds to foreign antigen stimulation with a heterologous antibody repertoire, requires that the heterologous 20 immunoglobulin transgenes contain within the transgenic animal function correctly throughout the pathway of B-cell development. In a preferred embodiment, correct function of a heterologous heavy chain transgene includes isotype switching. Accordingly, the transgenes of the invention are constructed so as to produce isotype switching and one or more of the following: (1) high level and cell-type specific 25 expression, (2) functional gene rearrangement, (3) activation of and response to allelic exclusion, (4) expression of a sufficient primary repertoire, (5) signal transduction, (6) somatic hypermutation, and (7) domination of the transgene antibody locus during the immune response. Not all of the foregoing criteria need be met. For example, in those 30 embodiments wherein the endogenous immunoglobulin loci of the transgenic animal are functionally disrupted, the transgene need not activate allelic exclusion. Further, in those embodiments wherein the transgene comprises a functionally rearranged heavy - 28 and/or light chain immunoglobulin gene, the second criteria of functional gene rearrangement is unnecessary, at least for that transgene which is already rearranged. For background on molecular immunology, see, Fundamental Immunology, 2nd edition (1989), Paul William E., ed. Raven Press, N.Y., which is incorporated herein by 5 reference. In certain embodiments, the transgenic non-human animals used to generate the human monoclonal antibodies of the invention contain rearranged, unrearranged or a combination of rearranged and unrearranged heterologous immunoglobulin heavy and light chain transgenes in the germline of the transgenic animal. Each of the heavy chain 10 transgenes comprises at least one CH gene. In addition, the heavy chain transgene may contain functional isotype switch sequences, which are capable of supporting isotype switching of a heterologous transgene encoding multiple CH genes in the B-cells of the transgenic animal. Such switch sequences may be those which occur naturally in the germline immunoglobulin locus from the species that serves as the source of the 15 transgene Ca genes, or such switch sequences may be derived from those which occur in the species that is to receive the transgene construct (the transgenic animal). For example, a human transgene construct that is used to produce a transgenic mouse may produce a higher frequency of isotype switching events if it incorporates switch sequences similar to those that occur naturally in the mouse heavy chain locus, as 20 presumably the mouse switch sequences are optimized to function with the mouse switch recombinase enzyme system, whereas the human switch sequences are not. Switch sequences may be isolated and cloned by conventional cloning methods, or may be synthesized de novo from overlapping synthetic oligonucleotides designed on the basis of published sequence information relating to immunoglobulin switch region 25 sequences (Mills et al., NucL. Acids Res. 15:7305-7316 (1991); Sideras et al., Intl. Immunol. 1:631-642 (1989), which are incorporated herein by reference). For each of the foregoing transgenic animals, functionally rearranged heterologous heavy and light chain immunoglobulin transgenes are found in a significant fraction of the B-cells of the transgenic animal (at least 10 percent). 30 The transgenes used to generate the transgenic animals of the invention include a heavy chain transgene comprising DNA encoding at least one variable gene segment, one diversity gene segment, one joining gene segment and at least one constant region -29 gene segment. The immunoglobulin light chain transgene comprises DNA encoding at least one variable gene segment, one joining gene segment and at least one constant region gene segment. The gene segments encoding the light and heavy chain gene segments are heterologous to the transgenic non-human animal in that they are derived 5 from, or correspond to, DNA encoding immunoglobulin heavy and light chain gene segments from a species not consisting of the transgenic non-human animal. In one aspect of the invention, the transgene is constructed such that the individual gene segments are unrearranged, i.e., not rearranged so as to encode a functional immunoglobulin light or heavy chain. Such unrearranged transgenes support 10 recombination of the V, D, and J gene segments (functional rearrangement) and preferably support incorporation of all or a portion of a D region gene segment in the resultant rearranged immunoglobulin heavy chain within the transgenic non-human animal when exposed to dendritic cells. In an alternate embodiment, the transgenes comprise an unrearranged "mini 15 locus". Such transgenes typically comprise a substantial portion of the C, D, and J segments as well as a subset of the V gene segments. In such transgene constructs, the various regulatory sequences, e.g. promoters, enhancers, class switch regions, splice donor and splice-acceptor sequences for RNA processing, recombination signals and the like, comprise corresponding sequences derived from the heterologous DNA. Such 20 regulatory sequences may be incorporated into the transgene from the same or a related species of the non-human animal used in the invention. For example, human immunoglobulin gene segments may be combined in a transgene with a rodent immunoglobulin enhancer sequence for use in a transgenic mouse. Alternatively, synthetic regulatory sequences may be incorporated into the transgene, wherein such 25 synthetic regulatory sequences are not homologous to a functional DNA sequence that is known to occur naturally in the genomes of mammals. Synthetic regulatory sequences are designed according to consensus rules, such as, for example, those specifying the permissible sequences of a splice-acceptor site or a promoter/enhancer motif. For example, a minilocus comprises a portion of the genomic immunoglobulin locus having 30 at least one internal (i.e., not at a terminus of the portion) deletion of a non-essential DNA portion (e.g., intervening sequence; intron or portion thereof) as compared to the naturally-occurring germline Ig locus.

-30 In a preferred embodiment of the invention, the transgenic animal used to generate human antibodies to dendritic cells contains at least one, typically 2-10, and sometimes 25-50 or more copies of the transgene described in Example 12 of WO 98/24884 (e.g., pHC1 or pHC2) bred with an animal containing a single copy of a light 5 chain transgene described in Examples 5, 6, 8, or 14 of WO 98/24884, and the offspring bred with the JH deleted animal described in Example 10 of WO 98/24884, the contents of which are hereby expressly incorporated by reference. Animals are bred to homozygosity for each of these three traits. Such animals have the following genotype: a single copy (per haploid set of chromosomes) of a human heavy chain unrearranged 10 mini-locus (described in Example 12 of WO 98/24884), a single copy (per haploid set of chromosomes) of a rearranged human K light chain construct (described in Example 14 of WO 98/24884), and a deletion at each endogenous mouse heavy chain locus that removes all of the functional JH segments (described in Example 10 of WO 98/24884). Such animals are bred with mice that are homozygous for the deletion of the JH 15 segments (Examples 10 of WO 98/24884) to produce offspring that are homozygous for the JH deletion and hemizygous for the human heavy and light chain constructs. The resultant animals are injected with antigens and used for production of human monoclonal antibodies against these antigens. B cells isolated from such an animal are monospecific with regard to the human 20 heavy and light chains because they contain only a single copy of each gene. Furthermore, they will be monospecific with regards to human or mouse heavy chains because both endogenous mouse heavy chain gene copies are nonfunctional by virtue of the deletion spanning the JH region introduced as described in Example 9 and 12 of WO 98/24884. Furthermore, a substantial fraction of the B cells will be monospecific with 25 regards to the human or mouse light chains because expression of the single copy of the rearranged human Y light chain gene will allelically and isotypically exclude the rearrangement of the endogenous mouse K and lambda chain genes in a significant fraction of B-cells. The transgenic mouse of the preferred embodiment will exhibit immunoglobulin 30 production with a significant repertoire, ideally substantially similar to that of a native mouse. Thus, for example, in embodiments where the endogenous Ig genes have been inactivated, the total immunoglobulin levels will range from about 0.1 to 10 mg/ml of -31 serum, preferably 0.5 to 5 mg/ml, ideally at least about 1.0 mg/ml. When a transgene capable of effecting a switch to IgG from IgM has been introduced into the transgenic mouse, the adult mouse ratio of serum IgG to IgM is preferably about 10:1. The IgG to IgM ratio will be much lower in the immature mouse. In general, greater than about 5 10%, preferably 40 to 80% of the spleen and lymph node B cells express exclusively human IgG protein. . The repertoire will ideally approximate that shown in a non-transgenic mouse, usually at least about 10% as high, preferably 25 to 50% or more. Generally, at least about a thousand different immunoglobulins (ideally IgG), preferably 104 to 106 or 10 more, will be produced, depending primarily on the number of different V, J and D regions introduced into the mouse genome. These immunoglobulins will typically recognize about one-half or more of highly antigenic proteins, e.g., dendritic cell proteins. Typically, the immunoglobulins will exhibit an affinity for preselected antigens of at least about 10 7 M-, preferably at least about 10 9

M~

1 , more preferably at 15 least about 10' 0 M',10"M~1, 101 2 M-, or greater, e.g., up tol0' 3 M' or greater. In some embodiments, it may be preferable to generate mice with predetermined repertoires to limit the selection of V genes represented in the antibody response to a predetermined antigen type. A heavy chain transgene having a predetermined repertoire may comprise, for example, human VH genes which are preferentially used in antibody 20 responses to the predetermined antigen type in humans. Alternatively, some VH genes may be excluded from a defined repertoire for various reasons (e.g., have a low likelihood of encoding high affinity V regions for the predetermined antigen; have a low propensity to undergo somatic mutation and affinity sharpening; or are immunogenic to certain humans). Thus, prior to rearrangement of a transgene containing various heavy 25 or light chain gene segments, such gene segments may be readily identified, e.g. by hybridization or DNA sequencing, as being from a species of organism other than the transgenic animal. The transgenic mice of the present invention can be immunized with a purified or enriched preparation of dendritic cells and/or a dendritic cells lysate as described 30 previously. The mice will produce B cells which undergo class-switching via intratransgene switch recombination (cis-switching) and express immunoglobulins reactive with dendritic cells. The immunoglobulins can be human sequence antibodies, -32 wherein the heavy and light chain polypeptides are encoded by human transgene sequences, which may include sequences derived by somatic mutation and V region recombinatorial joints, as well as germline-encoded sequences; these human sequence immunoglobulins can be referred to as being substantially identical to a polypeptide 5 sequence encoded by a human VL or VH gene segment and a human JL or JL segment, even though other non-germline sequences may be present as a result of somatic mutation and differential V-J and V-D-J recombination joints. With respect to such human sequence antibodies, the variable regions of each chain are typically at least 80 percent encoded by human germline V, J, and, in the case of heavy chains, D, gene 10 segments; frequently at least 85 percent of the variable regions are encoded by human germline sequences present on the transgene; often 90 or 95 percent or more of the variable region sequences are encoded by human germline sequences present on the transgene. However, since non-germline sequences are introduced by somatic mutation and VJ and VDJ joining, the human sequence antibodies will frequently have some 15 variable region sequences (and less frequently constant region sequences) which are not encoded by human V, D, or J gene segments as found in the human transgene(s) in the germline of the mice. Typically, such non-germline sequences (or individual nucleotide positions) will cluster in or near CDRs, or in regions where somatic mutations are known to cluster. 20 The human sequence antibodies which bind to the predetermined antigen can result from isotype switching, such that human antibodies comprising a human sequence y chain (such as yl, y2a, y2B, or y3) and a human sequence light chain (such as K) are produced. Such isotype-switched human sequence antibodies often contain one or more somatic mutation(s), typically in the variable region and often in or within about 10 25 residues of a CDR) as a result of affinity maturation and selection of B cells by antigen, particularly subsequent to secondary (or subsequent) antigen challenge. These high affinity human sequence antibodies may have binding affinities of at least 1 x 10' M-, typically at least 5 x 109 M-, frequently more than 1 x 1010 M-, and sometimes 5 x 10' M- to 1 x 10" M- or greater. 30 Another aspect of the invention pertains to the B cells from such mice which can be used to generate hybridomas expressing human monoclonal antibodies which bind with high affinity (e.g., greater than 2 x 109 M-) to dendritic cells. Thus, in another -33 embodiment of the invention, these hybridomas are used to generate a composition comprising an immunoglobulin having an affinity constant (Ka) of at least 2 x 10 9 M for binding dendritic cells, wherein said immunoglobulin comprises: a human sequence light chain composed of (1) a light chain variable region 5 having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human VL gene segment and a human JL segment, and (2) a light chain constant region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human CL gene segment; and a human sequence heavy chain composed of a (1) a heavy chain variable region 10 having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human VH gene segment, optionally a D region, and a human JH segment, and (2) a constant region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human CH gene segment. The development of high affinity human monoclonal antibodies against dendritic 15 cells is facilitated by a method for expanding the repertoire of human variable region gene segments in a transgenic mouse having a genome comprising an integrated human immunoglobulin transgene, said method comprising introducing into the genome a V gene transgene comprising V region gene segments which are not present in said integrated human immunoglobulin transgene. Often, the V region transgene is a yeast 20 artificial chromosome comprising a portion of a human VH or VL (VK) gene segment array, as may naturally occur in a human genome or as may be spliced together separately by recombinant methods, which may include out-of-order or omitted V gene segments. Often at least five or more functional V gene segments are contained on the YAC. In this variation, it is possible to make a transgenic mouse produced by the V 25 repertoire expansion method, wherein the mouse expresses an immunoglobulin chain comprising a variable region sequence encoded by a V region gene segment present on the V region transgene and a C region encoded on the human Ig transgene. By means of the V repertoire expansion method, transgenic mice having at least 5 distinct V genes can be generated; as can mice containing at least about 24 V genes or more. Some V 30 gene segments may be non-functional (e.g., pseudogenes and the like); these segments may be retained or may be selectively deleted by recombinant methods available to the skilled artisan, if desired.

-34 Once the mouse germline has been engineered to contain a functional YAC having an expanded V segment repertoire, substantially not present in the human Ig transgene containing the J and C gene segments, the trait can be propagated and bred into other genetic backgrounds, including backgrounds where the functional YAC 5 having an expanded V segment repertoire is bred into a mouse germline having a different human Ig transgene. Multiple functional YACs having an expanded V segment repertoire may be bred into a germline to work with a human Ig transgene (or multiple human Ig transgenes). Although referred to herein as YAC transgenes, such transgenes when integrated into the genome may substantially lack yeast sequences, 10 such as sequences required for autonomous replication in yeast; such sequences may optionally be removed by genetic engineering (e.g., restriction digestion and pulsed-field gel electrophoresis or other suitable method) after replication in yeast in no longer necessary (i.e., prior to introduction into a mouse ES cell or mouse prozygote). Methods of propagating the trait of human sequence immunoglobulin expression, include 15 breeding a transgenic mouse having the human Ig transgene(s), and optionally also having a functional YAC having an expanded V segment repertoire. Both VH and VL gene segments may be.present on the YAC. The transgenic mouse may be bred into any background desired by the practitioner, including backgrounds harboring other human transgenes, including human Ig transgenes and/or transgenes encoding other human 20 lymphocyte proteins. The invention also provides a high affinity human sequence immunoglobulin produced by a transgenic mouse having an expanded V region repertoire YAC transgene. Although the foregoing describes a preferred embodiment of the transgenic animal of the invention, other embodiments are contemplated which have been classified in four categories: 25 1. Transgenic animals containing an unrearranged heavy and rearranged light immunoglobulin transgene; II. Transgenic animals containing an unrearranged heavy and unrearranged light immunoglobulin transgene; III. Transgenic animal containing rearranged heavy and an unrearranged light 30 immunoglobulin transgene; and IV. Transgenic animals containing rearranged heavy and rearranged light immunoglobulin transgenes.

-35 Of these categories of transgenic animal, the preferred order of preference is as follows II> I > III> IV where the endogenous light chain genes (or at least the K gene) have been knocked out by homologous recombination (or other method) and I> II> III >IV where the endogenous light chain genes have not been knocked out and must be 5 dominated by allelic exclusion. III. Bispecific/ Multispecific Molecules Which Bind to Dendritic Cells In yet another embodiment of the invention, human monoclonal antibodies to dendritic cells, or antigen-binding portions thereof, can be derivatized or linked to 10 another functional molecule, e.g., another peptide or protein (e.g., an Fab' fragment) to generate a bispecific or multispecific molecule which binds to multiple binding sites or target epitopes. For example, an antibody or antigen-binding portion of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another 15 antibody, antibody fragment, peptide or binding mimetic. Accordingly, the present invention includes bispecific and multispecific molecules comprising at least one first binding specificity for dendritic cells and a second binding specificity for a second target epitope. In a preferred embodiment of the invention, the second target epitope is an antigen on a target cell, e.g. a tumor cell 20 antigen, a microbial antigen, a viral antigen or an autoantigen. These bispecific and multispecific molecules target dendritic cells to target cells such that the dendritic cells can modulate an immune response against such a target cell or target cell antigen. In another embodiment of the invention, the second target epitope is an Fc receptor, e.g., human FcyRI (CD64) or a human Fca receptor (CD89). Therefore, the 25 invention includes bispecific and multispecific molecules capable of binding both to Fcy R, FcaR or FcR expressing effector cells (e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)), and to dendritic cells. These bispecific and multispecific molecules target dendritic cells to effector cells and, like the human monoclonal antibodies of the invention, may trigger Fc receptor-mediated effector cell 30 activities, such as phagocytosis of dendritic cells, antibody dependent cell-mediated cytotoxicity (ADCC), cytokine release, or generation of superoxide anion - 36 Bispecific and multispecific molecules of the invention can further include a third binding specificity, in addition to an anti-Fc binding specificity or an anti-target cell antigen, and an anti-dendritic cell binding specificity. In one embodiment, the third binding specificity is an anti-enhancement factor (EF) portion, e.g., a molecule which 5 binds to a surface protein involved in cytotoxic activity and thereby increases the immune response against the target cell. The "anti-enhancement factor portion" can be an antibody, functional antibody fragment or a ligand that binds to a given molecule, e.g., an antigen or a receptor, and thereby results in an enhancement of the effect of the binding determinants for the Fc receptor, target cell antigen or dendritic cell. The "anti 10 enhancement factor portion" can bind an Fc receptor, target cell antigen, or dendritic cell. Alternatively, the anti-enhancement factor portion can bind to an entity that is different from the entity to which the first and second binding specificities bind. For example, the anti-enhancement factor portion can bind a cytotoxic T-cell (e.g. via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1) or other immune cell that results in an 15 increased immune.response against the target cell. In one embodiment, the bispecific and multispecific molecules of the invention comprise as a binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., an Fab, Fab', F(ab') 2 , Fv, or a single chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a 20 single chain construct as described in Ladner et al. U.S. Patent No. 4,946,778, issued August 7, 1990, the contents of which is expressly incorporated by reference. In one embodiment, bispecific and multispecific molecules of the invention comprise a binding specificity for an antigen on a target cell, e.g. a tumor cell antigen, a microbial antigen, a viral antigen or an autoantigen, and a second binding sepcificity for 25 dendritic cells. In another embodiment bispecific and multispecific molecules of the invention comprise a binding specificity for an FcyR or an FcaR present on the surface of an effector cell, and a second binding specificity for dendritic cells. In one embodiment, the binding specificity for an Fc receptor is provided by a 30 human monoclonal antibody, the binding of which is not blocked by human immunoglobulin G (IgG. As used herein, the term "IgG receptor" refers to any of the eight y-chain genes located on chromosome 1. These genes encode a total of twelve -37 transmembrane or soluble receptor isoforms which are grouped into three Fcy receptor classes: FcyRI (CD64), FcyRII(CD32), and FeyRIII (CD16). In one preferred embodiment, the Fey receptor a human high affinity FcyRI. The human FcyRI is a 72 kDa molecule, which shows high affinity for monomeric IgG (108 - 109M-1). 5 The production and characterization of these preferred monoclonal antibodies are described by Fanger et al. in PCT application WO 88/00052 and in U.S. Patent No. 4,954,617, the teachings of which are fully incorporated by reference herein. These antibodies bind to an epitope of FcyRI, FcyRII or FcyRIII at a site which is distinct from the Fcy binding site of the receptor and, thus, their binding is not blocked substantially 10 by physiological levels of IgG. Specific anti-FcyRI antibodies useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. The hybridoma producing mAb 32 is available from the American Type Culture Collection, ATCC Accession No. HB9469. Anti-FcyRI mAb 22, F(ab') 2 fragments of mAb 22, and can be obtained from Medarex, Inc. (Annandale, N.J.). In other embodiments, the anti-Fcy receptor antibody 15 is a humanized form of monoclonal antibody 22 (1122). The production and, characterization of the H22 antibody is described in Graziano, R.F. et al. (1995) J. Immunol 155 (10): 4996-5002 and PCT/US93/10384. The H22 antibody producing cell line was deposited at the American Type Culture Collection on November 4, 1992 under the designation HA022CLI and has the accession no. CRL 11177. 20 In still other preferred embodiments, the binding specificity for an Fc receptor is provided by an antibody that binds to a human IgA receptor, e.g., an Fe-alpha receptor (FcaRI (CD89)), the binding of which is preferably not blocked by human immunoglobulin A (IgA). The term "IgA receptor" is intended to include the gene product of one a-gene (FcaRI) located on chromosome 19. This gene is known to 25 encode several alternatively spliced transmembrane isoforms of 55 to 110 kDa. FcaRI (CD89) is constitutively expressed on monocytes/macrophages, eosinophilic and neutrophilic granulocytes, but not on non-effector cell populations. FcaRl has medium affinity (~ 5 x 107 M-1) for both IgAl and IgA2, which is increased upon exposure to cytokines such as G-CSF or GM-CSF (Morton, H.C. et al. (1996) Critical Reviews in 30 Immunology 16:423-440). Four FcaRI-specific monoclonal antibodies, identified as A3, A59, A62 and A77, which bind FcaRI outside the IgA ligand binding domain, have been described (Monteiro, R.C. et al., 1992, J Immunol. 148:1764).

-38 FcaRI and FcyRI are preferred trigger receptors for use in the invention because they are (1) expressed primarily on immune effector cells, e.g., monocytes, PMNs, macrophages and dendritic cells; (2) expressed at high levels (e.g., 5,000-100,000 per cell); (3) mediators of cytotoxic activities (e.g., ADCC, phagocytosis); (4) mediate 5 enhanced antigen presentation of antigens, including self-antigens, targeted to them. In other embodiments, bispecific and multispecific molecules of the invention further comprise a binding specificity which recognizes, e.g., binds to, dendritic cells e.g., an antigen on a dendritic cell. In a preferred embodiment, the binding specificity is provided by a human monoclonal antibody of the present invention. 10 An "effector cell specific antibody" as used herein refers to an antibody or functional antibody fragment that binds the Fc receptor of effector cells. Preferred antibodies for use in the subject invention bind the Fc receptor of effector cells at a site which is not bound by endogenous immunoglobulin. As used herein, the term "effector cell" refers to an immune cell which is 15 involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, e.g., lymphocytes (e.g., B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and 20 basophils. Some effector cells express specific Fc receptors and carry out specific immune functions. In preferred embodiments, an effector cell is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC. For example, monocytes, macrophages, which express FcR are involved in specific killing of target cells and presenting antigens to other components 25 of the immune system, or binding to cells that present antigens. In other embodiments, an effector cell can phagocytose a target antigen, target cell, or microorganism. The expression of a particular FcR on an effector cell can be regulated by humoral factors such as cytokines. For example, expression of FcyRI has been found to be up-regulated by interferon gamma (IFN-y). This enhanced expression increases the cytotoxic activity 30 of FcyRI-bearing cells against targets. An effector cell can phagocytose or lyse a target antigen or a target cell.

-39 "Target cell" shall mean any undesirable cell in a subject (e.g., a human or animal) that can be targeted by a composition (e.g., a human monoclonal antibody, a bispecific or a multispecific molecule) of the invention. In one embodiment, the target cell is a dendritic cell. In other embodiments, a target cell includes a tumor cell, a 5 microbial pathogen, a virus, or a virus infected cell. While human monoclonal antibodies are preferred, other antibodies which can be employed in the bispecific or multispecific molecules of the invention are murine, chimeric and humanized monoclonal antibodies. Chimeric mouse-human monoclonal antibodies (i.e., chimeric antibodies) can be 10 produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted. (see Robinson et al., International Patent Publication PCT/US86/02269; 15 Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 20 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natli Cancer Inst. 80:1553-1559). The chimeric antibody can be further humanized by replacing sequences of the Fv variable region which are not directly involved in antigen binding with equivalent 25 sequences from human Fv variable regions. General reviews of humanized chimeric antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207 and by Oi et al., 1986, Bio Techniques 4:214. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid 30 are well known to those skilled in the art and, for example, may be obtained from 7E3, an anti-GPIbIlIa antibody producing hybridoma. The recombinant DNA encoding the chimeric antibody, or fragment thereof, can then be cloned into an appropriate -40 expression vector. Suitable humanized antibodies can alternatively be produced by CDR substitution U.S. Patent 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; and Beidler et al. 1988 J. Immunol. 141:4053 4060. 5 All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to the Fc receptor. An antibody can be humanized by any method, which is capable of replacing at 10 least a portion of a CDR of a human antibody with a CDR derived from a non-human antibody. Winter describes a method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on March 26, 1987), the contents of which is expressly incorporated by reference. The human CDRs may be replaced with non-human CDRs using oligonucleotide site 15 directed mutagenesis as described in International Application WO 94/10332 entitled, Humanized Antibodies to Fc Receptors for Immunoglobulin G on Human Mononuclear Phagocytes. Also within the scope of the invention are chimeric and humanized antibodies in which specific amino acids have been substituted, deleted or added. In particular, 20 preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, in a humanized antibody having mouse CDRs, amino acids located in the human framework region can be replaced with the amino acids located at the corresponding positions in the mouse antibody. Such substitutions are known to improve binding of humanized antibodies to 25 the antigen in some instances. Antibodies in which amino acids have been added, deleted, or substituted are referred to herein as modified antibodies or altered antibodies. The term modified antibody is also intended to include antibodies, such as monoclonal antibodies, chimeric antibodies, and humanized antibodies which have been modified by, e.g., deleting, adding, or substituting portions of the antibody. For 30 example, an antibody can be modified by deleting the constant region and replacing it with a constant region meant to increase half-life, e.g., serum half-life, stability or affinity of the antibody. Any modification is within the scope of the invention so long -41 as the bispecific and multispecific molecule has at least one antigen binding region specific for an FcyR and triggers at least one effector function. Bispecific and multispecific molecules of the present invention can be made using chemical techniques (see e.g., D. M. Kranz et al. (1981) Proc. Nat?. Acad. Sci. 5 USA 78:5807), "polydoma" techniques (See U.S. Patent 4,474,893, to Reading), or recombinant DNA techniques. In particular, bispecific and multispecific molecules of the present invention can be prepared by conjugating the constituent binding specificities, e.g., the anti-FcR and anti-dendritic cell binding specificities, using methods known in the art and described in 10 the examples provided herein. For example, each binding specificity of the bispecific and multispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 15 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N maleimidomethyl) cyclohaxane-l-carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus (Behring Ins. Mitt. (1985) 20 No. 78, 118-132); Brennan et al. (Science (1985) 229:81-83), and Glennie et al. (J. Immunol. (1987) 139: 2367-2375). Preferred conjugating agents are SATA and sulfo SMCC, both available from Pierce Chemical Co. (Rockford, IL). When the binding specificities are antibodies (e.g., two humanized antibodies), they can be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the 25 two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation. Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific and multispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2 or 30 ligand x Fab fusion protein. A bispecific and multispecific molecule of the invention, e.g., a bispecific molecule can be a single chain molecule, such as a single chain bispecific antibody, a single chain bispecific molecule comprising one single chain - 42 antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific and multispecific molecules can also be single chain molecules or may comprise at least two single chain molecules. Methods for preparing bi- and multspecific molecules are described for example in U.S. Patent 5 Number 5,260,203; U.S. Patent Number 5,455,030; U.S. Patent Number 4,881,175; U.S. Patent Number 5,132,405; U.S. Patent Number 5,091,513; U.S. Patent Number 5,476,786; U.S. Patent Number 5,013,653; U.S. Patent Number 5,258,498; and U.S. Patent Number 5,482,858. Binding of the bispecific and multispecific molecules to their specific targets can 10 be confirmed by enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), FACS analysis, a bioassay (e.g., growth inhibition), or a Western Blot Assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, the FcR-antibody complexes can be detected using 15 e.g., an enzyme-linked antibody or antibody fragment which recognizes and specifically binds to the antibody-FcR complexes. Alternatively, the complexes can be detected using any of a variety of other immunoassays. For example, the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on 20 Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a y counter or a scintillation counter or by autoradiography. IV. Antibody Conjugates/Iminunotoxins 25 In another aspect, the present invention features a human anti-dendritic cell monoclonal antibody, or a fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug or a radioisotope. When conjugated to a cytotoxin, these antibody conjugates are referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Examples include taxol, cytochalasin 30 B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, - 43 glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, 5 melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and 10 vinblastine). An antibody of the present invention can be conjugated to a radioisotope, e.g., radioactive iodine, to generate cytotoxic radiopharmaceuticals for treating a dendritic-related disorder, such as an autoimmune or inflammatory disease, or graft versus host disease. The antibody conjugates of the invention can be used to modify a given 15 biological response, and the drug moiety is not to he construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or 20 interferon-y; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors. Techniques for conjugating such therapeutic moiety to antibodies are well 25 known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer 30 Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in -44 Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982). In another aspect, human antibodies specific for dendritic cells can be used to 5 directly target whole cells, e.g., a tumor cell, an effector cell or a microbial pathogen, to dendritic cells. Anti-dendritic cell antibodies or antigen binding fragments thereof can be directly expressed on the surface of a cell, for example, by transfection or transduction of a cell with a vector containing nucleic acid sequences encoding a human dendritic cell-specific antibody of the invention, or antigen binding fragment thereof. 10 This can be done, for example, by transfecting the target cell with a nucleic acid encoding a fusion protein containing a transmembrane domain and a human anti dendritic cell antibody, or antigen binding fragment thereof. Methods for generating such nucleic acids, fusion proteins, and cells expressing such fusion proteins are described, for example, in U.S. Patent Application Serial No: 09/203,958, incorporated 15 herein in its entirety by this reference. Alternatively, anti-dendritic cell antibodies, or antigen binding fragments thereof, can be bound to a cell or a pathogen by the use of chemical linkers, lipid tags, or other related methods (deKruif, J. et al. (2000) Nat. Med. 6:223-227; Nizard, P. et al. (1998) FEBS Lett. 433:83-88). Cells with surface-anchored anti-dendritic cell antibodies, or an antigen binding fragments thereof, may be used to 20 induce specific immune responses against the cell, e.g., a tumor cell or microbial pathogen. V. Pharmaceutical Compositions In another aspect, the present invention provides therapeutic compositions, e.g., 25 pharmaceutical compositions, containing one or a combination of human monoclonal antibodies, or antigen-binding portion(s) thereof, of the present invention, formulated together with a pharmaceutically acceptable carrier. Such compositions can additionally include other therapeutic reagents, such as other antibodies, cytotoxins or drugs (e.g., immunosuppressants), and can be administered alone or in combination with other 30 therapies, such as radiation. In one embodiment, human anti-dendritic cell monoclonal antibodies having complementary activities are used in combination, e.g., as a pharmaceutical - 45 composition, comprising two or more human anti-dendritic monoclonal antibodies. For example, a human monoclonal antibody that mediates highly effective killing of dendritic cells in the presence of effector cells can be combined with another human monoclonal antibody that inhibits the growth of dendritic cells. In another embodiment, 5 a human monoclonal antibody that is rapidly internalized by dendritic cells can be combined with another human monoclonal antibody that induces antigen presenting cell activities of dendritic cells, e.g., release of immunostimulatory cytokines. In another embodiment, the composition comprises one or a combination of bispecific or multispecific molecules of the invention (e.g., which contains at least one 10 binding specificity for an Fc receptor and at least one binding specificity for dendritic cells). In yet another embodiment, the composition comprises at least one binding specificity for dendritic cells functionally linked to another molecular entity, for example, a cytotoxin, or an antigen, e.g., an antigen on a target cell. 15 As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of 20 administration, the active compound, i.e., antibody, bispecific and multispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired 25 toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl 30 substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as - 46 well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N methylglucamine, chloroprocaine, choline, diethanolamine, ethylenedianine, procaine and the like. A composition of the present invention can be administered by a variety of 5 methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible 10 polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. 15 To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. 20 Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al. (1984) J Neuroimmunol. 7:27). Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active 25 substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions. Therapeutic compositions typically must be sterile and stable under the 30 conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for - 47 example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In 5 many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. 10 Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from 15 those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Dosage regimens are adjusted to provide the optimum desired response (e.g., a 20 therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to 25 physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular 30 therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

-48 Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the [ike; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, 5 propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. For the therapeutic compositions, formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal 10 and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be 15 combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, preferably from about 0.1 per cent to about 70 per cent, most preferably from about 1 per cent to about 30 per cent. 20 Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forms for the topical or transdermal administration of compositions of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and 25 inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, 30 usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, - 49 intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols 5 (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. 10 These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include 15 isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. When the compounds of the present invention are administered as 20 pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.01 to 99.5% (more preferalily, 0.1 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier. Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical 25 compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a 30 particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention -50 employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general 5 health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the 10 invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a compositions of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the 15 factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the site of the target. If desired, the effective daily dose of a therapeutic compositions may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is 20 possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as 25 the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering 30 medicants through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for - 51 continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to 5 those skilled in the art. In certain embodiments, the human monoclonal antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for 10 example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade (1989)J Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to 15 Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J Physiol. 1233:134), different species of which may comprise the formulations of the inventions, as well as components of the invented molecules; p120 20 (Schreier et al. (1994) J Bio. Chem. 269:9090); see also K. Keinanen; M.L. Laukkanen (1994) FEBSLett. 346:123; J.J. Killion; I.J. Fidler (1994) Immunomethods 4:273. In one embodiment of the invention, the therapeutic compounds of the invention are formulated in liposomes; in a more preferred embodiment, the liposomes include a targeting moiety. In a most preferred embodiment, the therapeutic compounds in the 25 liposomes are delivered by bolus injection to a site proximal to the tumor or infection. The composition must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. A "therapeutically effective dosage" preferably modulates dendritic cell growth 30 and/or activity by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to modulate dendritic cell growth -52 and/or activity can be evaluated in an animal model system predictive of efficacy in antigen presentation and/or immunomodulation. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to modulate immune cell stimulation by dendritic cells, such as in in vitro by assays described herein 5 and known to the skilled practitioner. In one embodiment, a therapeutically effective amount of a therapeutic compound can inhibit dendritic cell growth and/or activity, or otherwise ameliorate symptoms, e.g., symptoms of autoimmunity, in a subject. In another embodiment, a therapeutically effective amount of a therapeutic compound can enhance antigen processing and presentation by dendritic cells, and thus enhance 10 immune responses against a immunogen or target antigen. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, immune activity in the subject, and the particular composition or route of administration selected. The composition must be sterile and fluid to the extent that the composition is 15 deliverable by syringe. In addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is 20 preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin. When the active compound is suitably protected, as described above, the 25 compound may be orally administered, for example, with an inert diluent or an assimilable edible carrier. VI. Uses and Methods of the Invention The compositions (e.g., human monoclonal antibodies to dendritic cells and 30 derivatives/conjugates thereof) of the present invention have in vitro and in vivo diagnostic and therapeutic utilities.

-53 For example, these molecules can be administered to cells in culture, e.g. in vitro or ex vivo, or in a subject, e.g, in vivo, to treat, prevent or diagnose a variety of disorders. As used herein, the term "subject" is intended to include human and non human animals. The term "non-human animals" of the invention includes all 5 vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc. In a particular embodiment, the human antibodies and derivatives thereof are used in vivo to treat, prevent or diagnose a variety of dendritic cell-mediated or dendritic cell-related diseases. 10 In one embodiment, preferred human animals include a human patient having dendritic cell-mediated or a dendritic cell-related disease. For example, the methods and compositions of the present invention can be used to treat a subject with an autoimmune, immune system, or inflammatory disorder, e.g., a disorder characterized by aberrant or unwanted immune activity associated with immunomodulation by dendritic cells. 15 Autoimmune, immune system, and inflammatory disorders that may benefit from treatment with the human anti-dendritic cells of the invention include rheumatoid arthritis, multiple sclerosis, diabetes mellitus, myasthenia gravis, pernicious anemia, Addison's disease, lupus erythematosus, Reiter's syndrome, and Graves disease. For example, a subject suffering from an autoimmune disorder may benefit from inhibition 20 of dendritic cell mediated presentation of an autoantigen. Other examples of diseases that can be treated using the human anti-dendritic cell antibodies of the invention include transplant rejection and graft versus host disease. Transplant Rejection Over recent years there has been a considerable improvement in the efficiency of 25 surgical techniques for transplanting tissues and organs such as skin, kidney, liver, heart, lung, pancreas and bone marrow. Perhaps the principal outstanding problem is the lack of satisfactory agents for inducing immune-tolerance in the recipient to the transplanted allograft or organ. When allogeneic cells or organs are transplanted into a host (i.e., the donor and donee are different individual from the same species), the host immune 30 system is likely to mount an immune response to foreign antigens in the transplant (host versus-graft disease) leading to destruction of the transplanted tissue. CD8+ cells, CD4+ cells and monocytes are all involved in the rejection of transplant tissues. The - 54 therapeutic agents of the present invention are useful to inhibit dendritic cell mediated alloantigen-induced immune responses in the donee thereby preventing such cells from participating in the destruction of the transplanted tissue or organ. Graft Versus Host Disease 5 A related use for the therapeutic agents of the present invention is in modulating the immune response involved in "graft versus host" disease (GVHD). GVHD is a potentially fatal disease that occurs when immunologically competent cells are transferred to an allogeneic recipient. In this situation, the donor's immunocompetent cells may attack tissues in the recipient. Tissues of the skin, gut epithelia and liver are 10 frequent targets and may be destroyed during the course of GVHD. The disease presents an especially severe problem when immune tissue is being transplanted, such as in bone marrow transplantation; but less severe GVHD has also been reported in other cases as well, including heart and liver transplants. The therapeutic agents of the present invention are used to inhibit the activity of host antigen presenting cells, e.g., dendritic 15 cells. In another embodiment, the methods and compositions of the invention can be used to modulate an immune response in a subject towards an antigen. The human anti dendritic cell antibodies of the invention can be used to target an antigen to a dendritic cell and thereby modulate antigen presentation and processing, such that an immune 20 response to the antigen is induced. The antigen can be a tumor antigen, or an antigen from a pathogen, e.g., a microbial pathogen. The pathogen can be a virus (e.g., HIV), a bacterium, a fungus, or a parasite. The antigen can also be a component of an amyloid deposit in a patient, such as a patient suffering from Alzheimer's disease and the antigen is As peptide. 25 For example, a molecular complex comprising at least one binding specificity for a component on the surface of a dendritic cell linked to an antigen, wherein binding of the complex to the dendritic cell mediates internalization of the molecular complex, can be administered to a subject to induce or enhance an immune response against the antigen. The immune response generated against the antigen includes antibodies that 30 bind to the antigen and T cells that bind to the antigen as a component of an MHC-I or MHC-II complex. Accordingly, the human anti-dendritic cell antibodies of the invention can also be used to mediate dendritic cell-targeted immunization of a subject.

- 55 For example, a subject can be immunized with a molecular complex comprising at least one binding specificity for a component on the surface of a dendritic cell linked to an antigen, wherein binding of the complex to the dendritic cell mediates internalization of the molecular complex, and, for example, enhances processing and presentation of the 5 antigen. In another aspect, human antibodies specific for dendritic cells can be used to directly target whole cells, e.g., a tumor cell, an effector cell or a microbial pathogen, to dendritic cells. Anti-dendritic cell antibodies or antigen binding fragments thereof can be directly expressed on the surface of a cell, for example, by transfection or 10 transduction of a cell with a vector containing nucleic acid sequences encoding a human dendritic cell-specific antibody of the invention. Alternatively, anti-dendritic cell antibodies, or antigen binding fragments thereof, can be bound to a cell or a pathogen by the use of chemical linkers, or lipid tags, or other related methods. Cells with surface anchored anti-dendritic cell antibodies, or an antigen binding fragments thereof, may be 15 used to induce specific immune responses against the cell, e.g., a tumor cell or microbial pathogen. Thus, the antibodies of the invention can be used to stimulate the immune response to pathogens, toxins, and self-antigens. Examples of pathogens for which this therapeutic approach may be particularly useful, include pathogens for which there is 20 currently no effective vaccine, or pathogens for which conventional vaccines are less than completely effective. These include, but are not limited to HIV, Hepatitis (A, B, & C), Influenza, Herpes, Giardia, Malaria, Leishmania, Staphylococcus Aureus, Pseudononas aeruginosa. The compositions (e.g., human antibodies, multispecific and bispecific 25 molecules) of the invention can be initially tested for binding activity associated with therapeutic or diagnostic use in vitro. For example, compositions of the invention can be tested using the flow cytometric and internalization assays described in the Examples below. Moreover, the activity of these molecules in triggering at least one effector-' mediated effector cell activity, including cytolysis of dendritic cells can be assayed. 30 Protocols for assaying for effector cell-mediated phagocytosis and cytolysis are known in the art.

-56 The compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention have additional utility in therapy and diagnosis of dendritic cell-mediated or dendritic cell-related diseases. For example, the human monoclonal antibodies, the multispecific or bispecific molecules can be used, for example, to elicit 5 in vivo or in vitro one or more of the following biological activities: to opsonize a dendritic cell; to mediate phagocytosis or cytolysis of a dendritic cell in the presence of human effector cells; to inhibit the growth of a dendritic cell; to be internalized by a dendritic cell; or to target an antigen to a dendritic cell. Methods of administering the compositions (e.g., human antibodies, 10 multispecific and bispecific molecules) of the invention are known in the art. Suitable dosages of the molecules used will depend on the age and weight of the subject and the particular drug used. The molecules can be coupled to radionuclides, such as D00I, 90 Y, 1sRh, etc., as described in Goldenberg, D.M. et al. (1981) Cancer Res. 41: 4354-4360, and in EP 0365 997. The compositions (e.g., human antibodies, multispecific and 15 bispecific molecules) of the invention can also be coupled to immunomodulatory agents. Target-specific effector cells, e.g., effector cells linked to compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can also be used as therapeutic agents. Effector cells for targeting can be human leukocytes such as macrophages, neutrophils or monocytes. Other cells include eosinophils, natural killer 20 cells and other IgG- or IgA-receptor bearing cells. If desired, effector cells can be obtained from the subject to be treated. The target-specific effector cells, can be administered as a suspension of cells in a physiologically acceptable solution. The number of cells administered can be in the order of 10 8 -10 9 but will vary depending on the therapeutic purpose. In one embodiment, the amount will be sufficient to obtain 25 localization at, for example, a dendritic cell, and to effect cell killing by, e.g., phagocytosis. In another embodiment, target-specific dendritic cells, e.g., dendritic cells linked to compositions of the invention can be used as therapeutic agents for localization at a target cell, e.g., a tumor cell, microbial pathogen, virus, or virus infected cell, or for targeting an antigen, and to effect an immune response against the target cell or a target 30 antigen, by, e.g., antigen processing and presentation. Routes of administration can also vary.

-57 Therapy with target-specific effector cells or target-specific dendritic cells can be performed in conjunction with other techniques for removal of targeted cells. For example, anti-dendritic cell therapy using the compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention and/or effector cells armed with 5 these compositions can be used in conjunction with chemotherapy or immunomodulatory therapy, e.g., anti-inflammatory or immunosuppressive therapy. Additionally, combination immunotherapy may be used to direct two distinct cytotoxic effector populations toward, for example, dendritic cells. For example, anti-dendritic cell antibodies linked to anti-Fc-gammaRI or anti-CD3 may be used in conjunction with 10 IgG- or IgA-receptor specific binding agents. In another embodiment, dendritic cells targeted with, for example, human antibodies, multispecific and bispecific molecules of the invention, can be used in conjunction with immunomodulatory therapy, e.g., immunstimulation, to enhance an immune response against a target cell or a target antigen. A dendritic cell targeted 15 therapy can be combined with other forms of immunotherapy such as cytokine treatment (e.g. interferons, TNFa, GM-CSF, G-CSF, IL-2). Bispecific and multispecific molecules of the invention can also be used to modulate dendritic cell activities, e.g., antigen processing and presentation, as well as to modulate the level of a cognate antigen on a dendritic cell, such as by capping and 20 elimination of receptors on the cell surface. The compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention which have complement binding sites, such as portions from IgG1, -2, or -3 or IgM which bind complement, can also be used in the presence of complement. In one embodiment, ex vivo treatment of a population of cells comprising 25 target cells with a binding agent of the invention and appropriate effector cells can be supplemented by the addition of complement or serum containing complement. Phagocytosis of target cells coated with a binding agent of the invention can be improved by binding of complement proteins. In another embodiment target cells coated with the compositions (e.g., human antibodies, multispecific and bispecific 30 molecules) of the invention can also be lysed by complement. The compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can also be administered together with complement.

-58 Accordingly, within the scope of the invention are compositions comprising human antibodies, multispecific or bispecific molecules and serum or complement. These compositions are advantageous in that the complement is located in close proximity to the human antibodies, multispecific or bispecific molecules. Alternatively, the human 5 antibodies, multispecific or bispecific molecules of the invention and the complement or serum can be administered separately. In other embodiments, the subject can be additionally treated with an agent that modulates, e.g., enhances or inhibits, immune cell activity and/or the expression or activity of FcO or FcO receptors, by for example, treating the subject with a cytokine. 10 Preferred cytokines for administration during treatment with the multispecific molecule include of granulocyte colony-stimulating factor (G-CSF), granulocyte- macrophage colony-stimulating factor (GM-CSF), interferon-y (IFN-y), and tumor necrosis factor (NFa). The compositions (e.g., human antibodies, multispecific and bispecific 15 molecules) of the invention can also be used to target, for example, dendritic cells, e.g., for labeling such cells. For such use, the binding agent can be linked to a molecule that can be detected. Thus, the invention provides methods for localizing ex vivo or in vitro dendritic cells. The detectable label can be, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. 20 In one embodiment, the invention provides methods for detecting the presence of dendritic cells or a dendritic cell antigen in a sample, or measuring the amount of dendritic cells or a dendritic cell antigen, comprising contacting the sample, and a control sample, with a human monoclonal antibody, or an antigen binding portion thereof, which specifically binds to dendritic cells or a dendritic cell antigen, under 25 conditions that allow for formation of a complex between the antibody or portion thereof and dendritic cells or a dendritic cell antigen. The formation of a complex is then detected, wherein a difference complex formation between the sample compared to the control sample is indicative the presence of dendritic cells or a dendritic cell antigen in the sample. 30 In still another embodiment, the invention provides a method for detecting the presence or quantifying the amount of dendritic cells in vivo or in vitro. The method comprises (i) administering to a subject a composition (e.g., a multi- or bispecific - 59 molecule) of the invention or a fragment thereof, conjugated to a detectable marker; (ii) exposing the subject to a means for detecting said detectable marker to identify areas containing dendritic cells. Also within the scope of the invention are kits comprising the compositions (e.g., 5 human antibodies, multispecific and bispecific molecules) of the invention and instructions for use. The kit can further contain a least one additional reagent, such as a cytokine or complement, or one or more additional human antibodies of the invention (e.g., a human antibody having a complementary activity which binds to an epitope on dendritic cells distinct from the first human antibody). 10 The present invention is further illustrated by the following examples which should not be construed as further limiting. The contents of all figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. 15 - 60 1. G.Kobler and Milstein C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495-497. 2. G.L. Boulianne. Hozum N., and Shulman M.J. (1984) Production of functional 5 chimeric mouse/human antibody. Nature 312: 643-646. 3. P.T. Jones. Dear P.H., Foote J., Neuberger M.S., and Winter G. (1989) Replacing the complementarity-determining regions in a human antibody with those of a mouse. Nature 321: 522-525. 10 4. J.D. Marks et al.(1991) By-passing Immunization Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222: 581-597. 5. N. Lonberg, et al. 1994. Antigen-specific human antibodies from mice 15 comprising four distinct genetic modifications. Nature 368(6474): 856-859. 6. G.Gafie, Howe S.C., Butcher M.C. C. W., and Howard H.C. (1997) Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 266:550-552. 20 - 61 EXAMPLES Example 1 Production of Human Monoclonal Antibodies Against Dendritic Cells 5 Human anti-dendritic cell monoclonal antibodies were generated by immunizing the HCO7 strain of HuMAb mice with preparations of dendritic cells. HCO7 HuMAb mice were generated as described in U.S. Patent Nos. 5,770,429 and 5,545,806, the entire disclosures of which are hereby incorporated by reference. 10 In particular, HC07 mice were immunized four times with intraperitoneal injections of human dendritic cells emulsified in Freund's Adjuvant. Briefly, dendritic cells were prepared as follows. Human peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation of whole blood or Leukopak platelet apheresis preparations. Monocytes were isolated by adherance to tissue culture flasks 15 for two hours, and then differentiated into dendritic cells by incubation with 2 ng/ml GM-CSF and 10 ng/ml IL-4 in macrophage serum free media (Gibco) for 5 to 9 days. Cells for immunizations were used fresh or stored frozen at -80*C. Mice were immunized every 2-3 weeks. Finally, an intravenous injection of dendritic cells in phosphate buffered saline (PBS) was performed prior to splenectomy. The spleens from 20 responding mice were harvested and dispersed into single cells. To generate hybridomas producing anti-dendritic cell antibodies, splenocytes from mice with plasma containing anti-dendritic cell antibodies were fused with P3X63-Ag8.653 myeloma cells (deposited with the ATCC under designation ATCC CRL 1580 nonsecreting mouse myeloma cells) and PEG. Hybridomas were selected by 25 growth in HAT containing media. After hybridomas grew out (about 10-14 days) each well containing hybridomas was screened for the production of human IgG using an anti-human IgG ELISA. Positive hybridomas were screened for and selected based on the following properties: (1) production of human IgG antibodies, and (2) binding to dendritic cells. 30 The hybridomas secreting human IgG were tested for reactivity with various types of blood cells by flow cytometry. Dendritic cells were prepared from adherent mononuclear cells by culturing for 5-7 days in media supplemented with GM-CSF and -62 IL-4. Granulocytes (PMN), monocytes and lymphocytes were obtained from heparanized whole blood. The cells were incubated with hybridoma supernatants from IgG-positive clones at 4*C. Binding was detected with a FITC-labeled goat anti-human IgG(Fc) probe. The cell associated fluorescence was determined by analysis using a 5 FACScalibur instrument. Several hybridomas that were screened produced human IgGlIc antibodies that demonstrated reactivity with dendritic cells as assessed by flow cytometry (e.g., A3, A5, A23, A24, A33, B9, B1 1, B33, B47, C8, C1O, C20, C28, C29, C30, C35, El, E8, E10, El 8, E20, E21 and E24), as shown in Table 1 below. Some of the human antibodies 10 demonstrated very high and preferential reactivity with dendritic cells as compared to other blood cell types.

- 63 TABLE 1 Human Monoclonal Antibodies with Reactivity to Dendritic Cells Human MAb Lymphocytes Monocytes PMNs Dendritic Cells A3 -+- +/- + A5 +/- - +/ A23 -+ - + A24 - ++ + ++ A33 -+- - +/ B9 +/- ++ + . B11l +/- - + B33 +/- - +/ B47 +/- +/- +/ C8 +/- - +/ C1O +- +/- + C20 +/- +- ++ C28 +/- - +/. C29 +/- +/- ++ C30 - - +/ C35 -+- - ++ El -+- - + E8 -+ + + E10 + + E18 -+ + ++ E20 + ++ +/ E21 +/- ++ +/ E24 +/- - +/ Key: - no binding detected +/- weak/equivocal binding + low/significant binding ++ high binding +++ extremely high binding -64 Example 2 Characterization of Human Monoclonal Antibodies Against Dendritic Cells 5 I. Binding specificity of purified human anti-dendritic cell antibodies to dendritic cells Several hybridomas that secreted human IgG antibodies with specificity for 10 -dendritic cells were subcloned and expanded for purification. Monoclonal antibodies were isolated from supernatants of hybridoma cultures grown in spinner flasks in a humidified incubator containing 5% C02. Antibodies were purified by chromatography on a Protein A-agarose column according to the manufacturer's specifications (Pierce, Rockford IL). 15 The purified human antibodies were then tested for reactivity with dendritic cells and cell lines representing various other hematopoetic cell types using flow cytometry. Briefly, dendritic cells were prepared from adherent mononuclear cells by culturing for 5-7 days in media supplemented with GM-CSF and IL-4, as described above. The U937, CEM, THP-1 and L540 cell lines were cultured in media 20 supplemented with 10% fetal bovine serum. The cells were harvested, washed, and incubated with saturating concentrations of human monoclonal antibodies B 11, C20, E21 or an isotype control (human IgG1) at 4"C with shaking for 1 hour. Antibody binding was detected by further incubation with a FITC-labeled goat anti-human IgG(Fe) probe for 1 hour at 4*C. The cells were washed, fixed with 1% 25 paraformaldehyde, and cell associated fluorescence was analyzed using a FACScalibur (Beckton Dickinson) instrument with CellQuest software. As shown in Figure 1, human monoclonal antibody B 11 bound exclusively to dendritic cells. Monoclonal antibody C20 bound specifically to dendritic cells, and also demonstrated low level reactivity with the monocyte-like cell lines U-937 and THP-1, 30 and the Hodgkin's lymphoma cell line L540. Similarly, human monoclonal antibody E21 bound preferentially to dendritic cells, but also reacted at a low level with L540 cells and THP-l cells. These data demonstrate that human monoclonal antibodies BI 1, - 65 C20 and E21 recognize different antigens, and that they preferentially bind to dendritic cells compared to other cells of hematopoetic lineage. II. Dose-dependent binding of purified human anti-dendritic cell antibodies to 5 dendritic cells The dose-dependent reactivity of purified human anti-dendritic cell monoclonal antibodies B 11, C20, and E21 with dendritic cells was examined by flow cytometry. Dendritic cells were prepared from adherent mononuclear cells as described 10 above. The cells were harvested and incubated with varying concentrations of the monoclonal antibodies B 11, C20, E21 or an isotype control at 4*C. Antibody binding was detected with a FITC-labeled goat anti-human IgG(Fc) probe, and cell associated fluorescence was determined using a FACScalibur instrument with CellQuest software. Each monoclonal antibody demonstrated dose-dependent binding to dendritic 15 cells as compared to an isotype matched control IgG antibody, as shown in Figure 2. These data demonstrate that the purified human monoclonal antibodies B 11, C20 and E2 bind in a concentration-dependent manner to dendritic cells. The varying intensity of binding between the anti-dendritic cell antibodies indicates that they recognize unique molecules or epitopes on the dendritic cells. 20 III. Binding of human antibody B 11 to CD34+ stem cell-derived dendritic cells Due to their availability, dendritic cells differentiated from circulating blood monocytes are the most commonly used type of dendritic cell for both research and 25 clinical applications. However, dendritic cells derived from progenitor stem cells also can be used and may more accurately represent dendritic cells in human tissues. Accordingly, in the following study, dendritic cells differentiated from CD34+ progenitor cells were evaluated for reactivity with human monoclonal antibody B 11 by flow cytometry. 30 Purified monoclonal antibody B I was extensively dialyzed against 0.3 M sodium carbonate buffer, pH 9.5, for labeling with fluorescence isothiocyanate (FITC). A stock FITC solution was prepared by dissolving 1 mg solid FITC in I ml of DMSO.

- 66 Stock FITC was added dropwise with constant mixing in an amount to provide 50 pg FITC per mg of antibody protein. Following the addition of FITC, the solution was incubated in the dark for 1-3 hours at room temperature. FITC labeled antibody was isolated by gel filtration on a Sephadex G-10 column equilibrated in PBS. 5 CD34+ progenitor cells and dendritic cell differentiation media were obtained from Poetic Technologies, Inc. (Gaithersburg, MD). The cells were differentiated according to the manufacturer's instructions. Dendritic cells were harvested and incubated with varying concentrations of B11 -FITC or an isotype control antibody (human IgG-FITC) at 4*C. The cell associated fluorescence was determined by analysis 10 using a FACScalibur instrument with CellQuest software. The results are shown in Figure 3 and demonstrate that human monoclonal antibody B 11 binds to dendritic cells differentiated from CD34+ stem cells in a dose dependent manner. Therefore, the B 11 target antigen is expressed on dendritic cells that are derived from monocytes and from progenitor stem cells. 15 IV. Binding of human antibody B 11 to macrophages and dendritic cells The ability of human anti-dendritic cell monoclonal antibody B 11 to bind to macrophages compared dendritic cells was assessed by flow cytometry. 20 Dendritic cells were prepared from adherent mononuclear cells as described above. Macrophages were prepared from adherent mononuclear cells by culturing for 5 7 days with M-CSF. The cells were harvested and incubated with 10 pg/ml of monoclonal antibody B 11 or an isotype control antibody at 4*C. Human antibody binding was detected with a FITC-labeled goat anti-human IgG(Fc) probe. The cell 25 associated fluorescence was determined by analysis using a FACScalibur instrument with CellQuest software. The results are shown in Figure 4 and demonstrate that human antibody B 11 binds to macrophages to a lesser extent than to dendritic cells. Thus, the B 11 target antigen is also expressed on macrophages, although the level of expression is lower than 30 that observed on dendritic cells. The reactivity of monoclonal antibody B 11 with macrophages is not surprising due to the similarity between dendritic cells and macrophages. Since these two cell types share both structural and functional properties, - 67 including the ability to stimulate T and B lymphocyte responses, the cross-reactivity of antibody B 11 with macrophages may be beneficial for targeting antigen presenting cells. V. Induction of human antibody B 11 target antigen on THP-1 cells 5 Human monoclonal antibody B 11 was tested using flow cytometry for binding to THP-1 cells, a monocyte-like cell line derived from a human monocytic leukemia, before and after the cells had been induced to differentiate towards a dendritic cell phenotype. 10 Briefly, THP- 1 cells were grown in standard culture media or in media supplemented with GM-CSF and IL-4. The cells were incubated with 10 ptg/mil of monoclonal antibody B 11 -FITC or an isotype control antibody (human IgG-FITC) at 4*C. The cell associated fluorescence was determined by analysis using a FACScalibur instrument with CellQuest software. The results are shown in Figure 5. 15 These data demonstrate that, under normal growth conditions, TIP-I cells do not express the B 11 target antigen. However, when THP-1 cells are driven towards a dendritic cell phenotype by culturing in media containing GM-CSF and IL-4, the expression of the B 11 target antigen is concomitantly induced. Accordingly, these results further confirm the specificity of the B 11 human antibody for a target antigen 20 (B 11) associated specifically with dendritic cells. VI. Binding of human antibody B 11 to macaque dendritic cells The animal (monkey) model of cynomolgus macaques can provide relevant 25 information regarding the clinical application of antibodies, provided that the target antigen is conserved among primates. Accordingly, the cross-reactivity of human monoclonal antibody B 11 with dendritic cells from cynomolgus monkey was evaluated by flow cytometry. Fresh cynomolgus blood was obtained from Sierra Biomedicals, and dendritic 30 cells were prepared from adherent mononuclear cells by culturing with GM-CSF and IL 4. Dendritic cells were incubated with 10 ptg/ml of monoclonal antibody B 11 -FITC or - 68 an isotype control antibody (human IgG-FITC) at 4*C. The cell associated fluorescence was determined by analysis using a FACScalibur instrument. As shown in Figure 6, human monoclonal antibody B 11 binds to dendritic cells derived from cynomolgus macaques, suggesting that the B 11 target antigen is conserved 5 in primates. VII. Binding of human antibody B1 Ito dendritic cells in human tissues The reactivity of human monoclonal antibody B 11 with dendritic cells from 10 human tissues was evaluated by immunohistochemistry. These experiments were also designed to evaluate any potential cross-reactivity of antibody B 11 with other cells or antigens of human tissues. Cryosections of human tissues were obtained via autopsy or surgical biopsy and embedded in Tissue-Tek O.C.T. medium and stored frozen below -70*C. Tissues were 15 sectioned at 5mm, fixed for 10 minutes with acetone, and dried overnight. Slides were fixed with 10% neutral-buffered formalin for 10 seconds prior to staining. An indirect immunoperoxidase technique was employed. Sections were first stained with the B11 FITC or isotype-matched-FITC antibody diluted in PBS containing heat aggregated IgG to block Fc-dependent binding. Primary antibodies were detected using a rabbit anti 20 FITC antibody followed by a peroxidase labeled anti-rabbit reagent. Each slide was read by a certified pathologist and binding was rated according to the following key: + (equivocal), 1+ (weak), 2+ (moderate), 3+ (strong), 4+ (intense), Neg. (negative). The results shown in Table 2 below demonstrate clear staining of dendritic cells and some macrophages in all tissues examined. No specific staining was observed with the 25 isotype control antibody. These data demonstrate that human monoclonal antibody B 11 binds to dendritic cells in human tissues, as well as macrophages in human tissues albeit to a lesser extent. The minimal binding of Bi to mononuclear cells in the spleen and progenitor cells of the bone marrow may represent binding to immature dendritic cells. Human antibody 30 B 11 did not cross-react with other cell types or tissues in the samples tested, further demonstrating the specificity of this antibody for dendritic cells and, to a lesser extent, macrophages.

- 69 TABLE 2 Immunohistochemistry of Human Monoclonal Antibody B11 Binding to Human Tissues Antibody Tissue and reactivity B11-FITC (2 pg/ml) Skin: Dermal dendritic cells 3+, all other elements negative. Tonsil: Interstitial and/or subepithelial dendritic cells 2+, all other elements negative. Liver: Interstitial dendritic cells 2+, Kupffer cells 2+, all other elements negative. Breast: Dermal/subcutaneous/interstitial dendritic cells 3-4+, all other elements negative. Spleen: Interstitial dendritic cells 3-4+, Reticulotendothelial cells lining cords of Billiroth 2+, occasional mononuclear cells in marginal zone 2+, rare to occasional mononuclear cells in PALS/follicles 2+, other elements negative. Kidney: Interstitial dendritic cells 3-4+, all other elements negative. Lymph node: Capsular dendritic cells 3-4+, subcapsular dendritic cells/macrophages 3+, follicular dendritic cells 2-3+, paracortical dendritic cells 2-3+, medullary sinus dendritic cell s/macrophages 1-2+, other elements negative. Brain: Meningeal/peritheleal dendritic cells 3-4+, all other elements negative. Testis: Interstitial dendritic cells 3-4+, all other elements negative. Pancreas: Interstitial dendritic cells 3-4+, all other elements negative. Heart: Interstitial dendritic cells 3-4+, all other elements negative. Small intestine: Interstitial dendritic cells 3-4+, Lamina propria dendritic cells/macrophages 2-3+, Peyers patch dendritic cells/macrophages 2-3+, other elements negative.

- 70.

Bone Marrow: Interstitial dendritic cells 3-4+, Hematopoetic progenitors 2+, other elements negative. Lung: Interstitial dendritic cells 3-4+, Alveolar macrophages 3 4+, other elements negative. IgGl-FITC (2 pg/ml) All tested tissues: All elements negative. Tissue cross-reactivity studies were conducted at Pathology Associates International, Frederick, MD study# IM598. VIII. Binding of single chain Fv (ScFv) fragments of human antibody B111 to human 5 dendritic cells. The reactivity of a single chain Fv fragment of human monoclonal antibody B 11 with dendritic cells from human tissues was evaluated by flow cytometry. The B 11 ScFv was constructed by linking the VL (SEQ ID NO:1 and 2) and VH 10 (SEQ ID NO: 3 and 4) domains of human monoclonal antibody B 11 as shown in Figure 9. EGF sequences were incorporated in order to detect the binding of the ScFv to dendritic cells using anti-EFG antibodies. Dendritic cells were incubated with BI 1 ScFv for one hour at 4*C, then washed before incubation with anti-EFG-FITC probe for 1 hour at 4"C. The samples were analyzed using FACS analysis. 15 The results of the FACS analysis demonstrated that the B 11 ScFv fragment of human monoclonal antibody B11 bound to human dendritic cells. Accordingly, the ScFv can be used as a vaccine or as an immunotoxin by linking the ScFv to a selected antigen or toxin, respectively. 20 IX. Binding of F(ab')2 fragments of human antibody B Ito human dendritic cells The reactivity of F(ab')2 fragments of human monoclonal antibody B 11 with dendritic cells from human tissues was evaluated by flow cytometry. These experiments were also designed to evaluate whether the Fe portion of human antibody B 1 is 25 significantly involved in the binding of B 11 to dendritic cells. F(ab')2 fragments were prepared by digestion of purified B 11 mAb with pepsin under standard conditions. The F(ab')2 fragments were purified by protein L - 71 chromatography. Dendritic cells, prepared fresh from human monocytes by culture in GM-CSF and IL-4, were incubated with whole antibody or F(ab')2 antibody fragments for 1 hour at 4*C. The cells were washed before incubation with anti-human IgG F(ab')2-PE probe for 1 hour at 4*C. The cells were washed again prior to analysis using 5 a FACScalibur instrument and Cellquest software. As shown in Figure 10, the results of the FACS analysis demonstrated that whole human monoclonal antibody B 11 and F(ab')2 fragments of B 1 bound to dendritic cells with similar kinetics, indicating that the Fc portion of the whole monoclonal antibody does not contribute significantly to binding of B Ito dendritic cells. 10 Example 3 Characterization of B11 Target Antigen I. Immunoprecipitation of the human monoclonal antibody B 11-target antigen from dendritic cells 15 Human monoclonal antibody B 11 was used to immunoprecipitate its cognate target antigen from dendritic cells. Briefly, cell lysates from dendritic cells were prepred and incubated with monoclonal antibody B 11 or an isotype control IgG antibody at 4*C. Antibody-antigen 20 complexes were captured with anti-human IgG-agarose, and separated by SDS polyacrylamide gel electrophoresis. A band corresponding to a molecular weight of approximately 180 kilodaltons was evident in antibody B 11 immunoprecipitates from two different preparations of dendritic cell lysates, but not in the control sample. Accordingly, these 25 immunoprecipitation studies showed that human monoclonal antibody B 11 recognizes a target antigen on dendritic cells with an approximate molecular weight of 180 kilodaltons, as analyzed by SDS-PAGE. II. N-terminal sequencing of the human monoclonal antibody B II target antigen 30 from dendritic cells - 72 Following immunoprecipitation as described above, the BI 1 target antigen was subjected to N-terminal amino acid sequencing to determine homology with known proteins. Cell lysates were prepared from dendritic cells and allowed to incubate with 5 monoclonal antibody B 11 at 4*C. Antibody-antigen complexes were captured with anti human IgG-agarose, and separated by SDS polyacrylamide gel electrophoresis. Proteins from the gel were transferred to nitrocellulose and the band corresponding to the monoclonal antibody BI 1 target antigen was eluted for N-terminal amino acid sequencing. The N-terminal sequencing and data base search were conducted at 10 Midwest Analytical, Inc. (St. Louis, MO). Sequencing of the N-terminal 15 amino acid residues of the monoclonal antibody B 11 target antigen revealed protein sequence homology with the human macrophage mannose receptor, as follows: 15 DDXXQFLDXEDXKR (SEQ ID NO:5) B11 antigen LDTRQFLIYNEDHKR (SEQ ID NO:6) Macrophage mannose receptor A computer search of the human protein database did not identify any other proteins as having significant homology to B 11 antigen. 20 In a further study, dendritic cell lysates were cleared of non-specifically binding proteins by overnight incubation with anti-mouse IgG-charged agarose The agarose was spun out, and the cleared supernatant was recovered and incubated overnight with anti human IgG -agarose previously charged with antibody B 11. The agarose was washed with PBS and boiled with reducing loading buffer. Finally, the agarose was spun out 25 and the supernatant was loaded onto a gel. Antibody B 11 immunoprecipitated a protein of approximately 150-180 kD. This protein was blotten onto a PVDF membrane and sent to Midwest Analytical, Inc. for microsequencing. The N-terminal microsequencing results of the monoclonal antibody B 11 target antigen revealed the following protein sequence: LLDTR QFLIY LEDTK RCVDA 30 (SEQ ID NO:7). This sequence again matched that of the human macrophage mannose receptor (GenBank Accession # NP 002429) with 100% identity over 20 amino acids as determined using the BLAST algorithm at the National Center for Biotechnology - 73 Information web site (http://www.ncbi.nlm.nih.gov/. These data indicate that the target molecule on dendritic cells recognized by human monoclonal antibody B 11 is the macrophage mannose receptor. 5 III. B 11 inhibits FITC-dextran uptake by dendritic cells The following experiment was designed to test whether antibody B 11 blocks the mannose receptor and thus can be used, for example, to prevent or inhibit interaction of pathogens with the mannose -receptor (e.g., cellular infection). Dendritic cells were incubated with FITC-Dextran (500 pg/ml) and either 10 isotype control human IgG or B 11 HuMAb (25 tg/ml) for 30 minutes at the temperatures indicated in Figure 11. Dextran molecules are known to be specifically internalized by dendritic cells through the mannose receptor (F. Sallusto et. al. J. Exp. Med. 1995, 185:389-400). Labelled (FITC)-dextran uptake was determined by FACS analysis (i.e., fluorescence intensity of the dendritic cells samples using a FACScalibur 15 instrument). The percent of FITC-dextran uptake was set at 100% at 37*C and 0% at 4 0 C. As shown in Figure 11, antibody B 11 blocked the uptake of FITC-dextran by 61.5% under these conditions. These results suggest that human monoclonal antibody B II can be used to block the interaction of pathogens with the mannose receptor. 20 Example 4 Activity of Human Anti-Dendritic Cell Antibodies I. Internalization of human monoclonal antibody B11 by dendritic cells 25 The extent to which human antibody B 11 is internalized following binding to dendritic cells was evaluated by flow cytometry. Dendritic cells were prepared from adherent mononuclear cells as described above. The cells were incubated with a saturating concentration of monoclonal antibody B 11 -FITC at 4*C for 1 hour to allow maximum surface binding of the antibody, washed 30 with cold PBS to remove excess antibody, and then incubated at 37 0 C for various periods of time to allow for antibody internalization. The samples were then washed with cold PBS to stop the reaction, futher washed with 0.1% PBA, pH 2.5 to strip -74 surface bound antibody from the cells. The remaining fluorescence is antibody B 11 FTIC that has been internalized. Control cells were immediately washed with 0.1% PBA, pH 2.5 and kept at 4*C to represent minial internalization, or washed only with 0.1% PBA, pH 7 to represent maximal loading of antibody B 11 -FITC. The percent 5 antibody internalization was calculated by the following formula: % Internalization= (mean fluorescence intensity of sample - mean fluorescence intensity of acid washed 4*C control )/ (mean fluorescence intensity of PBS washed 4*C control - mean fluorescence intensity of acid washed 4*C control) 10 The results, shown in Figure 7, demonstrate that human monoclonal antibody B 11 is efficiently internalized after binding to dendritic cells. This unexpected property of human anti-dendritic cell monoclonal antibody B 11 indicates that the antibody can be used to deliver agents, such as antigens and toxins, intracellularly to dendritic cells. 15 II. B 11 -FITC Internalization by Dendritic Cells Microscopic visualization was also used to confirm that antibody B 11 is internalized following binding to dendritic cells. Dendritic cells were generated by incubating monocytes with GM-CSF (10 20 ng/ml) and IL-13 (50 ng/ml) under non-adherent conditions for 7 days. These were combined with mAb B 11-FITC (1 pg) in the presence of human IgG (600 pg) and anti CD32 mAb IV.3 (10 jpg) to block non-specific uptake and incubated at 37 0 C. Control cells were incubated on ice during the entire procedure. After 15 and 60 minutes at 37 0 C, dendritic cells were placed on ice and stained with anti-CD 1 Ic-PE (1 jig). 25 Internalization of mAb B 11 was examined by confocal microscopy using a BioRad NlRC1024 laser scanning confocal microscope. Cells were scanned for fluorescence using the 488 nmi line from a 15 mW Kr/Ar laser and two photodetectors (522/35 nm dichroic for FITC fluorescence and 605/32 nm dichroic for PE fluorescence). A 63X Plan-APO 1.4 NA objective (Carl Zeiss, Inc., Thornwood, NY) was used in conjunction 30 with an iris setting of 2.1 which allowed for detection of optical sections of the fluorescence image that were approximately 1.0 pm thick. Representative images were selected from the slices through the center of the cells after sectioning the entire cell.

- 75 The results confirmed that B 11 mAb was rapidly internalized by dendritic cells after binding to the mannose receptor, again suggesting this antibody can efficiently deliver antigens or toxins into antigen presenting cells. Consistent with the FACS data, the internalization was evident within 15 minutes, and nearly complete within one hour. 5 III. Enhanced antigen presentation by dendritic cells following targeted antigen delivery with human monoclonal antibody B11 In order to determine whether human monoclonal antibody B I1 can be used to 10 enhance the processing and presentation of antigens by dendritic cells, the antibody was conjugated to the tetanus toxoid (TT) antigen using the chemical cross-linking reagent SMCC. Dendritic cells were prepared from adherent mononuclear cells as described above, with the exception that cells were cultured in Teflon containers. Dendritic cells 15 were harvested and replated in 96 well microtiter plates at 5000 cells per well in macrophage serum-free medium with 10% fetal calf serum. Monoclonal antibody B 11 conjugated to tetanus toxoid or tetanus toxoid alone was added at various concentrations to the dendritic cells. Autologous tetanus toxoid-specifc T cells generated by incubation of mononuclear cells with tetanus toxoid followed by IL-2 were added to each well 20 containing dendritic cells at 50,000 cells per well. Cells were cultured together for 7 days at 37*C and assayed for the number of living cells using a MTT based assay according to the manufacturer's instructions (Promega, Madison, WI). The ability to induce dendritic cells to specifically stimulate tetanus toxoid-specific T lymphocytes was compared after exposing cells to tetanus toxoid or antibody B 11 -tetanus toxoid. 25 The results (Figure 8) showed that conjugating tetanus toxoid as a model antigen to B 11 leads to significantly more efficient antigen presentation as measured by antigen specific T cell proliferation. In another experiment, 3 H-Thymadine was used as the readout for T cell proliferation following loading of dendritic cells with either B 11 conjugated with 30 tetanus toxoid (TT) or with tetanus toxoid mixed with B 11 antibody (unconjugated control). As an additional control, a blocking antibody to FcyRII (CD32), mAb IV-3, was added to some wells containing the B1 1-TT conjugate to determine whether this Fc -76 receptor contributed to enhanced antigen processing and presentation by B 11-TT. The following day, cells were combined with freshly thawed, previously established tetanus toxoid-specific T-cells for 4 days. Cells were co-incubated with 'H-Thymadine at 37 0 C during the final day. The amount of 3 H incorporated into the T cells was assayed. 5 As with the previous experiment, the results (Figure 12) demonstrated that conjugating tetanus toxoid as a model antigen to B 11 leads to significantly more efficient antigen presentation as measured by antigen-specific T cell proliferation. Addition of excess amounts of mAb IV.3 did not significantly alter the enhanced antigen presentation by B I1-TT conjugate, showing that interaction of Bl -TT with FcyRII is 10 not required for this activity. Overall, the results of these studies, shown in Figures 8 and 12, indicate that 10 to 100-fold lower amounts of antibody B 11-conjugated tetanus toxoid are required to achieve the level of T cell stimulation compared with tetanus toxoid alone. In addition, the absolute degree of T cell stimulation as shown in Figure 8 was 2-fold greater when 15 tetanus toxoid was targeted to dendritic cells with antibody B 11. Thus, data demonstrate that an antigen can be conjugated to human monoclonal antibody B 11 and that the antibody targeted antigen is processed and presented more efficiently than non-targeted antigen, leading to enhanced antigen-specific T cell responses. 20 Example 5 Characterization of Human Monoclonal Antibody E21 Against Dendritic Cells I. Molecular weight analysis of human antibody E21 antigen Dendritic cell lysates were prepared from cultured human dendritic cells. 25 Briefly, dendritic cells were washed and resuspended in Triton X-100 containing lysis buffer at 4 0 C. The unfractionated lysate was loaded onto a 4-15% SDS-polyacrylamide gel and then the protein were transferred to nitrocellulose. The blot was incubated with 10 ug/ml E21 followed by anti-human IgG-alkaline phosphatase probe and visualized using horseradish peroxidase. 30 The results of this experiment demonstrated that the human monoclonal antibody E21 antigen has an approximate molecular weight of 36-40 kilodaltons.

-77 Il. Binding of human antibody E21 to human dermal and epidermal human dendritic cells As shown in figure 1, human monoclonal Ab E21 bound preferentially to dendritic cells. This experiment was designed to test the reactivity of human antibody 5 E21 to dermal and epidermal dendritic cells by immunohistochemistry analysis of frozen skin with E21. Frozen sections of human skin were stained with FITC-E21 or FITC-huIgG control, and detected using rabbit anti-FITC probe. The results of the immunohistochemistry analysis demonstrate that human 10 antibody E21 reacts with both dermal dendritic cells/macrophages and epidermal dendritic cells (Langerhan cells) in human skin sections. III. Binding of human antibody E21 to macaque dendritic cells The animal (monkey) model of cynomolgus macaques can provide relevant 15 information regarding the clinical application of antibodies, provided that the target antigen is conserved among primates. Accordingly, the cross-reactivity of human monoclonal antibody E21 with dendritic cells from cynomolgus monkey was evaluated by flow cytometry. Cynomolgus monocytes were differentiated into dendritic cells with GM-CSF 20 and IL-4 treatment, and tested for E21 binding by flow cytometry. The dendritic cells were incubated with E21 for 1 hour at 4*C, then washed before incubation with anti human IgG-FITC probe for 1 hour at 4*C. The samples were analyzed using a FACScalibur instrument. 25 Conclusion The foregoing Examples demonstrate the generation of human monoclonal antibodies that specifically react with high affinity to dendritic cells. In particular, human monoclonal antibody B 11 specifically recognizes the human macrophage mannose receptor on dendritic cells. In addition, human 30 monoclonal antibody B 11 is efficiently internalized by dendritic cells, and enhances antigen processing and presentation by dendritic cells.

-78 Human monoclonal antibody E21 binds to a different antigen than B 11 on human dendritic cells. The E21 antibody also cross-reacts with dendritic cells derived from cynomolgus macaques (monkey), suggesting that the E21 antigen is conserved in primates and therefore provides a relevant animal model for further development of the 5 antibody. These results support the conclusion that the fully human monoclonal antibodies of the present invention, including fragments, conjugates and bispecific molecules thereof, can be used are for the diagnosis and treatment of dendritic cell related disorders. 10 Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention 15 described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (4)

1. A preparation of a monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 5 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein: (a) the heavy chain variable region CDR3 sequence comprises amino acid residues 99-105 of SEQ ID NO: 4, and conservative modifications thereof; (b) the light chain variable region CDR3 sequence comprises amino acid 10 residues 89-97 of SEQ ID NO: 2, and conservative modifications thereof; and (c) the antibody binds to the macrophage mannose receptor on dendritic cells.

2. The preparation of the antibody, or antigen binding portion thereof, of claim 1, wherein the heavy chain variable region CDR2 sequence comprises amino acid residues 15 50-66 of SEQ ID NO: 4, and conservative modifications thereof; and the light chain variable region CDR2 sequence comprises amino acid residues 50-56 of SEQ ID NO: 2, and conservative modifications thereof

3. The preparation of the antibody, or antigen binding portion thereof, of claim 2, 20 wherein the heavy chain variable region CDR1 sequence comprises amino acid residues

31-35 of SEQ ID NO: 4, and conservative modifications thereof; and the light chain variable region CDR1 sequence comprises amino acid residues 24-34 of SEQ ID NO: 2, and conservative modifications thereof. 25 4. A preparation of a monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises a sequence that is at least 80% homologous to SEQ ID NO: 4; (b) the light chain variable region comprises a sequence that is at least 80% 30 homologous to SEQ ID NO: 2; and (c) the antibody binds to the macrophage mannose receptor on dendritic cells. -80 5. A preparation of a monoclonal antibody, or antigen binding portion thereof, that binds to dendritic cells wherein the monoclonal antibody, or antigen binding portion thereof, comprises: (a) a heavy chain variable region having CDRI, CDR2 and CDR3 sequences 5 set forth as amino acids 31-35, amino acids 50-66 and amino acids 99-105 of SEQ ID NO: 4, respectively; and (b) a light chain variable region having CDRI, CDR2 and CDR3 sequences set forth as amino acids 24-34, amino acids 50-56 and 89-97 of SEQ ID NO: 2, respectively. 10 6. A preparation of a monoclonal antibody, or antigen binding portion thereof, wherein said antibody or antigen binding portion competes for binding to dendritic cells with the antibody of any one of claims I to 5. 15 7. A composition comprising the monoclonal antibody, or antigen binding portion thereof, according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier. 8. A molecular complex comprising the monoclonal antibody, or antigen binding portion thereof, of any one of claims 1 to 6 linked to an antigen. 20 9. The molecular complex of claim 8, wherein the antigen comprises a tumor antigen. 10. A bispecific molecule comprising the antibody, or antigen binding portion 25 thereof, of any one of claims 1 to 5 and a second binding specificity for an antigen. 11. Use of the composition of claim 7 to modulate dendritic cell activity. 12. Use of the composition of claim 7 to induce or enhance an immune response 30 against an antigen in a subject in need thereof. -81 13. The use according to claim 12, wherein the immune response comprises presentation of the antigen as a component of an MHC-I or MHC-lI complex. 14. Use of the composition of claim 7 to immunize a subject in need thereof 5 15. An expression vector comprising a nucleotide sequence encoding a antibody heavy or light chain variable region, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: I and conservative sequence modifications thereof. 10 16. An expression vector comprising a nucleotide sequence encoding a antibody heavy chain variable region comprising an amino acid sequence SEQ ID NO: 4, and conservative sequence modifications thereof, or a antibody light chain variable region comprising an amino acid sequence SEQ ID NO: 2, and conservative sequence 15 modifications thereof 17. A method of inducing or enhancing an immune response against an antigen in a subject in need thereof comprising administering a composition according to claim 7 to said subject. 20 18. The method according to claim 17, wherein the immune response comprises presentation of the antigen as a component of an MHC-I or MHC-II complex. 19. A method of immunizing a subject in need thereof comprising administering a 25 composition according to claim 7 to said subject. 20. Use of: (i) a monoclonal antibody or antigen binding portion thereof according to any one of claims I to 5, or 30 (ii) a molecular complex according to claim 8 or claim 9, or (iii) an immunoconjugate comprising the antibody of any one of claims I to 5 linked to a therapeutic agent, a cytoxin, or a radioisotype, or - 82 (iv) a bispecific molecule according to claim 10, in the manufacture of a medicament for inducing or enhancing an immune response against an antigen. 5 21. The use according to claim 20, wherein the immune response comprises presentation of the antigen as a component of an MHC-I or MHC-1I complex. 22. Use of: (i) a monoclonal antibody or antigen binding portion thereof according to any 10 one of claims I to 5, or (ii) a molecular complex according to claim 8 or claim 9, or (iii) an immunoconjugate comprising the antibody of any one of claims I to 5 linked to a therapeutic agent, a cytoxin, or a radioisotype, or (iv) a bispecific molecule according to according to claim 10, 15 in the manufacture of a medicament for immunizing a subject. 23. Use of a composition according to claim 7 for the manufacture of a medicament for inducing or enhancing an immune response against an antigen in a subject in need thereof 20 24. Use of a composition according to claim 7 for the manufacture of a medicament for immunizing a subject in need thereof 25. A method of inducing or enhancing an immune response against an antigen in a 25 subject comprising administering to said subject: (i) a monoclonal antibody or antigen binding portion thereof according to any one of claims I to 5, or (ii) a molecular complex according to claim 8 or claim 9, or (iii) an immunoconjugate comprising the antibody of any one of claims I to 5 30 linked to a therapeutic agent, a cytoxin, or a radioisotype, or (iv) a bispecific molecule according to according to claim 10. - 83 26. A method of immunizing a subject comprising administering to said subject: (i) a monoclonal antibody or antigen binding portion thereof according to any one of claims I to 5, or (ii) a molecular complex according to claim 8 or claim 9, or 5 (iii) an immunoconjugate comprising the antibody of any one of claims I to 5 linked to a therapeutic agent, a cytoxin, or a radioisotype, or (iv) a bispecific molecule according to according to claim 10. 27. A preparation according to any one of claims I or 4 to 6; a composition 10 according to claim 7; a molecular complex according to claim 8; a bispecific molecule according to claim 10; use according to any one of claims 11, 12, 14, 20 or 22 to 24; or a method according to any one of claims 17, 19, 25 or 26, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.