US 20100272636A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0272636A1 Byrd et al. (43) Pub. Date: Oct. 28, 2010

(54) ANTI-CD74 IMMUNOCONJUGATES AND filed on Mar. 1, 2002, provisional application No. METHODS OF USE 61/265,999, filed on Dec. 2, 2009. (75) Inventors: John C. Byrd, Columbus, OH Publication Classification (US); David M. Goldenberg, (51) Int. Cl. Hansen, Picayune, MS (US) A 6LX 9/27 (2006.01) A6II 5L/12 (2006.01) SISNOSE RNc. A61K 38/19 (2006.01) 3OO AMERICAN ROAD A638/2A638/20 :08:2006.O1 MORRIS PLAINS, NJ 07950 (US) A6IP35/00 (2006.01) (73) Assignees: THE OHO STATE 39t. E: 308: UNIVERSITY, Columbus, OH A6IP37/06 (2006.015 (US); IMMUNOMEDICS, INC., Morris Plains, NJ (US) (52) U.S. Cl...... 424/1.21; 424/450; 424/178.1; 424/179.1; 424/183.1; 424/85.1; 424/85.2: (21) Appl. No.: 12/789,575 424/85.4; 424/85.5; 424/85.6; 424/85.7 (22) Filed: May 28, 2010 (57) ABSTRACT O O Disclosed are compositions that include anti-CD74 immuno Related U.S. Application Data conjugates and optionally a therapeutic and/or diagnostic (63) Continuation-in-part of application No. 10/706,852, agent. In preferred embodiments, the immunoconjugates filed on Nov. 12, 2003, which is a continuation-in-part comprise one or more anti-CD74 antibodies or antigen-bind of application No. 10,314 330, filed on Dec. 9, 2002 ing fragments thereof, conjugated to a liposome or micelle. which is a continuation of application No. 09/965,796, Also disclosed a methods for preparing the immunoconju filed on Oct. 1, 2001, which is a continuation of appli- gates and using the immunoconjugates in diagnostic and cation No. 09/307,816, filed on May 10, 1999, now therapeutic procedures. In certain preferred embodiments, Pat. No. 6 306 393. said application No. 10,706 852 is the therapeutic methods comprise administering to a subject a continuation-in-part of application No. 10/350,096, with a CD74-expressing disease al anti-CD74 immunocon filed on Jan. 24, 2003, which is a continuation of appli- jugate and thereby inducing apoptosis of CD74-expressing cation No. 09/590,284 filed on Jun. 9, 2000, now Pat. cells. In more preferred embodiments, the CD74 immuno No. 7074,403 said application No. 10,706 852 is a conjugate is capable of inducing cell death in the absence of continuation-in-part of application No. 10/377,122, any other therapeutic agent, although Such agents may be filed on Mar. 3, 2003, now Pat. No. 7,312.318 optionally administered prior to, together with or Subsequent • - s s • L vs - s- a 1- s- u Y- - to administration of the anti-CD74 immunoconjugate. The (60) Provisional application No. 60/478,830, filed on Jun. compositions may be part of a kit for administering the anti 17, 2003, provisional application No. 60/360,259, CD74 immunoconjugates or compositions. Patent Application Publication Oct. 28, 2010 Sheet 1 of 11 US 2010/0272636 A1

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ANT-CD74. IMMUNOCONUGATES AND bind to a known tumor associated antigen (TAA) or to an METHODS OF USE antigen associated with infectious diseases or other disease States. CROSS-REFERENCE TO RELATED 0006. One such tumor-associated antigen is CD74, which APPLICATIONS is an epitope of the major histocompatibility complex (MHC) 0001. This application is a continuation-in-part of appli class II antigen invariant chain, Ili, present on the cell Surface cation Ser. No. 10/706,852, filed Nov. 12, 2003, which and taken up in large amounts of up to 8x10" molecules per claimed the benefit under 35 U.S.C. 119(e) of provisional cell per day (Hansen et al., 1996, Biochem. J., 320: 293-300). application Ser. No. 60/478,830, filed on Jun. 17, 2003, and CD74 is present on the cell surface of B-lymphocytes, mono which was a continuation-in-part of application Ser. No. cytes and histocytes, human B-lymphoma cell lines, melano 10/314,330, filed on Dec. 9, 2002, which was a continuation mas, T-cell lymphomas and a variety of other tumor cell of application Ser. No. 09/965,796, filed on Oct. 1, 2001, types. (Hansen et al., 1996, Biochem.J.,320:293-300) CD74 which was a continuation of application Ser. No. 09/307,816, associates with C/B chain MHC II heterodimers to form MHC filed on May 10, 1999, now U.S. Pat. No. 6,306.393. U.S. Ser. II CBIi complexes that are involved in antigen processing and No. 10/706,852 was also a continuation-in-part of application presentation to T cells (Dixon et al., 2006, Biochemistry Ser. No. 10/350,096, filed on Jan. 24, 2003, which was a 45:5228-34; Loss et al., 1993, J Immunol 150:3187-97: continuation of application Ser. No. 09/590,284, filed on Jun. Cresswell et al., 1996: Cell 84:505-7). 9, 2000, now U.S. Pat. No. 7,074,403. U.S. Ser. No. 10/706, 0007 CD74 also plays an important role in cell prolifera 852 was also a continuation-in-part of application Ser. No. tion and Survival. Binding of the CD74 ligand, macrophage 10/377,122, filed on Mar. 3, 2003, now U.S. Pat. No. 7,312, migration inhibitory factor (MIF), to CD74 activates the 318, which claimed the benefit of provisional application Ser. MAP kinase cascade and promotes cell proliferation (Lenget No. 60/360,259, filed on Mar. 1, 2002. al., 2003, J Exp Med 197:1467–76). Binding of MIF to CD74 0002. This application claims the benefit under 35 U.S.C. also enhances cell survival through activation of NF-kB and 119(e) to provisional application Ser. No. 61/265,999, filed Bcl-2 (Lantner et al., 2007, Blood 110:4303-11). Dec. 2, 2009. All of the above-identified applications and 0008 Murine LL1 (mLL1 or murine anti-CD74 antibody) patents are incorporated herein by reference in their entire is a specific monoclonal antibody (mAb) reactive with CD74. ties. Cell surface-bound LL1 is rapidly internalized to the lysoso mal compartment and quickly catabolized, much faster than FEDERALLY FUNDED RESEARCH other antibodies, such as anti-CD19 or anti-CD22 (Hansen et 0003. This work was supported in part by National Cancer al., 1996, Biochem. J., 320: 293-300). LL1 was reported to Institute grants CA95426, CA103985 and CA81534-02. The exhibit the highest rate of accumulation inside B cells of any Federal Government may have certain rights in the invention. of the antibodies tested (Griffiths et al., 2003, Clin Cancer Res 9:6567-71). FIELD OF THE INVENTION 0009 Murine LL1 was developed by fusion of mouse myeloma cells with splenocytes from BALB/c mice immu 0004. The present invention concerns compositions and nized with preparations from the Raji B-lymphoma cell line methods of use of liposomes attached to targeting molecules, (called EPB-1 in Pawlak-Byczkowska et al., Can. Res., 49: Such as antibodies, antibody fragments or antibody fusion 4568 (1989)). The clinical use of mLL1, just as with most proteins. More particularly, the antibodies, fragments or other promising murine antibodies, has been limited by the fusion proteins may bind to a tumor associated antigen, infec development in humans of a human anti-mouse antibody tious disease associated antigen or other disease associated (HAMA) response. A HAMA response is generally not antigen. Even more particularly, the targeting molecule may observed following injection of mLL1 Fab', as evidenced in a bind to the invariant chain (Ii) of the HLA-DR complex, also bone marrow imaging study using an mLL1 Fab" labeled with known as CD74. An exemplary anti-CD74 antibody is mil "Tc. Juweidet al., Nuc. Med. Comm. 18: 142-148 (1997). atuZumab (hLL1). The antibody conjugated liposome is of However, in some therapeutic and diagnostic uses, a full use for the treatment of a variety of diseases, such as autoim length anti-CD74 antibody may be preferred. This use of the mune disease, immune dysregulation disease, cancer, lym full-length anti-CD74 antibody can limit the diagnostic and phoma, leukemia, chronic lymphocytic leukemia, follicular therapeutic usefulness of Such antibodies and antibody con lymphoma, diffused large B cell lymphoma, multiple jugates, not only because of the potential anaphylactic prob myeloma or non-Hodgkin’s lymphoma. lem, but also as a major portion of the circulating conjugate may be complexed to and sequestered by the circulating BACKGROUND anti-mouse antibodies. Although the use of antibody frag ments of mLL.1 may circumvent the problems of immunoge 0005 Liposomes have been used as carriers for therapeu nicity, there are circumstances in which whole IgG is more tic agents, where antibodies may be conjugated to the lipo desirable and the induction of cellular immunity is intended Some to provide specific targeting of the liposome/agent com for therapy or enhanced antibody Survival time. In general, plex to a diseased cell or tissue. (See, e.g., Xu et al., Mol. HAMA responses pose a potential obstacle to realizing the Cancer. Ther., 1:337-346 (2002); Torchilin, et al., Proc. Natl. full diagnostic and therapeutic potential of murine anti-CD74 Acad. Sci., 10: 6039 (2003); U.S. Pat. No. 6,165,440; U.S. antibodies. Therefore, the development of immunoconju Pat. No. 5,702,727; U.S. Pat. No. 5,620,708; U.S. Pat. No. gates that include chimeric, humanized and human anti 5,565,215; U.S. Pat. No. 6,530,944; U.S. Pat. No. 6,562,318; CD74 binding molecules, (e.g., antibodies and fragments U.S. Pat. No. 6,558,648; and U.S. Pat. No. 6,395.276). The thereof, antibody fusion proteins thereof, multivalent and/or antibody or antibodies of choice for targeting purposes may multispecific antibodies and fragments thereof), would be US 2010/0272636 A1 Oct. 28, 2010 extremely useful for therapy and diagnosis, with reduced cin, camptothecin, 10-hydroxycamptothecin, carmustine, production of human anti-mouse antibodies. celebrex, chlorambucil, cisplatin, irinotecan (CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide, cytara SUMMARY bine, dacarbazine, docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin, dexamethasone, diethylstil 0010 Disclosed is a composition that includes an immu bestrol, doxorubicin, doxorubicin glucuronide, epirubicin noconjugate, where the immunoconjugate includes an anti glucuronide, ethinyl estradiol, estramustine, etoposide, eto CD74 binding molecule conjugated to a liposome. In pre poside glucuronide, etoposide phosphate, floXuridine ferred embodiments, the anti-CD74 binding molecule is an (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, antibody, antigen-binding antibody fragment, antibody flutamide, fluorouracil, fluoxymesterone, gemcitabine, fusion protein or multispecific antibody or fragment thereof. hydroxyprogesterone caproate, hydroxyurea, idarubicin, In certain embodiments, the composition may also comprise ifosfamide, L-asparaginase, leucovorin, lomustine, mechlo one or more additional effector molecules, such as a radionu rethamine, medroprogesterone acetate, megestrol acetate, clide, a chemotherapeutic agent, a toxin, an enzyme, an melphalan, mercaptopurine, 6-mercaptopurine, methotrex immunomodulator or a second antibody or fragment thereof. ate, mitoxantrone, mithramycin, mitomycin, mitotane, phe The additional effector(s) may be contained within the lipo nylbutyrate, prednisone, procarbazine, paclitaxel, pentosta some or attached to the anti-CD74 antibody. However, the skilled artisan will realize that methods of therapy of common tin, PSI-341, Semustine Streptozocin, tamoxifen, taxanes, disease States, such as cancer, may involve the administration taxol, testosterone propionate, thalidomide, thioguanine, to a subject of two or more different therapeutic agents (effec thiotepa, teniposide, topotecan, uracil mustard, velcade, Vin tor molecules) concurrently, consecutively or separately. blastine, Vinorelbine, Vincristine, ricin, abrin, ribonuclease, 0011. The anti-CD74 binding molecule may be conju onconase, rap R1, DNase I, Staphylococcal enterotoxin-A, gated or linked to the liposome by a number of linkages pokeweed antiviral protein, gelonin, diphtheria toxin, including Sulfide linkages, hydrazone linkages, hydrazine Pseudomonas exotoxin, and Pseudomonas endotoxin, or linkages, ester linkages, amido linkages, amino linkages, combinations thereof. In certain embodiments, the effector imino linkages, thiosemicarbazone linkages, semicarbazone includes FUdR, or FUdR-dO. linkages, oxime linkages, and carbon-carbon linkages. A Sul 0016. The composition may also include one or more hard fide linkage may be preferred, where the binding molecule acid chelators or soft acid chelators. For example, the chelator may include disulfide linkages, which may be reduced to may include NOTA, DOTA, DTPA, TETA, Tscg-Cys, or provide free thiol groups. Tsca-Cys. In certain embodiments, the chelators may form 0012. The composition may include additional binding complexes with cations selected from Group II, Group III, molecules, (e.g., antibodies or fragments thereof that bind to Group IV, Group V, transition, lanthanide or actinide metal CD19, CD20, CD22, CD30, CD33, CD52, CD80, HLA-DR, cations, or mixtures thereof. Alternatively, the cations may be MUC1, TAC, IL-6, tenascin, VEGF, placental growth factor, covalently, non-covalently, or otherwise associated with any carbonic anhydrase IX, and mixtures thereof). The additional component of the complex. In certain embodiments, the com binding molecules may be covalently or non-covalently asso position includes cations selected from Tc, Re, Bi, Cu, AS, ciated with any component of the composition (e.g., the lipo Ag, Au, At, or Pb. Some). 0017. The composition may also include a nuclide (e.g., a 0013 To facilitate conjugation to the anti-CD74 binding radionuclide). The nuclide may be selected from a number of molecule, the lipid molecules making up the liposome may nuclides including F, P. P. Ti, Sc, Fe, Fe, Cu, contain one or more groups capable of reacting with the (Cu, 7Ga, 7Ga, Ga, 7Se, 77As, 86Y. 89Sr. 897r, 90Y, 99Tc, anti-CD74 binding molecule. Such as nucleophilic carbons, 99mTc, Mo, 99mTc, 105Pd, 103Rh, '''Ag, In, 123, 1241, (e.g., at a distal terminus). In one embodiment, the lipid is 125I. 13 II, 142Pr 143Pr 14°Pm, 'Sim, 154-158Gd, 16 Tb, 166Dy polyethyleneglycol (PEG)-maleimide and the anti-CD74 166Ho, 169Er, 175Lu, 177Lu, 186Re, 188Re, 189Re, 194Ir, 198Au, binding molecule reacts via free thiol groups with the male Au, 21 At 2. Pb, 212Bi, 212Pb, 213Bi, 223Ra O 225Ac. imide group. In addition to maleimide groups, other groups 0018 Where the effector is an enzyme, suitable enzymes for conjugating binding molecules may include vinylsul may include carboxylesterases, glucuronidases, carboxypep fones. tidases, beta-lactamases, phosphatases, and mixtures thereof. 0014. The composition may include therapeutic or diag Where the effector is an immunomodulator, suitable immu nostic agents, which may be covalently, non-covalently, or nomodulators may include IL-1, IL-2, IL-3, IL-6, IL-10, otherwise associated with any component of the composition. IL-12, IL-18, IL-21, interferon-C, interferon-B, interferon-Y, For example, the therapeutic or diagnostic agent may be G-CSF, GM-CSF, and mixtures thereof. The effector may covalently linked to the anti-CD74 binding molecule. Alter also include an anti-angiogenic agent, e.g., angiostatin, natively, the therapeutic or diagnostic agent may be endostatin, basculostatin, can statin, maspin, anti-VEGF covalently linked to the liposome or non-covalently or other binding molecules, anti-placental growth factor binding mol wise associated with the liposome. ecules, or anti-vascular growth factor binding molecules. 0015 The effector may comprise any number of therapeu (0019 Preferably, the anti-CD74 binding molecule may be tic or diagnostic agents. For example, the effector may LL1 or a fragment thereof, although any anti-CD74 binding include a drug, prodrug, toxin, enzyme, radioisotope, immu molecule is suitable. For example, production of monoclonal nomodulator, cytokine, hormone, binding molecule (e.g., an antibodies is well known in the art. See Harlow & Lane (eds), antibody), or an oligonucleotide molecule (e.g., an antisense Antibodies. A Laboratory Manual, Cold Spring Harbor Labo molecule or a gene). Antisense molecules may include anti ratory, NY. However, human, chimeric, or humanized deriva sense molecules that correspond to bcl-2 or p53. The effector tives of LL1 or fragments thereof may be particularly suit may include aplidin, azaribine, anastrozole, azacytidine, able. Derivatives of LL1 are described in U.S. Pat. No. 7,312, bleomycin, bortezomib. bryostatin-1, buSulfan, calicheamy 318 and U.S. Patent Application Ser. No. 60/360.259, filed US 2010/0272636 A1 Oct. 28, 2010

Mar. 1, 2002, which are incorporated herein by reference in the composition is administered to the patient intravenously, their entireties. The anti-CD74 binding molecule or fragment intramuscularly or subcutaneously at a dose of 20-5000 mg. thereof may be monoclonal. 0026. In preferred embodiments, the disease or disorder is 0020. The anti-CD74 binding molecule may include a associated with CD74-expressing cells and may be a cancer, an immune dysregulation disease, an autoimmune disease, an human, chimeric, or humanized anti-CD74 antibody or frag organ-graft rejection, a graft-versus-host disease, a solid ment thereof. For example, a binding molecule may contain tumor, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, the CDRs of the light and heavy chain variable regions of a multiple myeloma, a B-cell malignancy, or a T-cell malig murine anti-CD74 antibody. A humanized anti-CD74 anti nancy. A B-cell malignancy may-include indolent forms of body or fragment may include the complementarity-deter B-cell lymphomas, aggressive forms of B-cell lymphomas, mining regions (CDRs) of murine anti-CD74 (mLL1) and chronic lymphatic leukemias, acute lymphatic leukemias, human antibody constant and framework (FR) region and/or multiple myeloma. Solid tumors may include melano sequences, which may be substituted with at least one amino mas, carcinomas, sarcomas, and/or gliomas. A carcinoma acid from corresponding FRS of a murine antibody. An anti may include renal carcinoma, lung carcinoma, intestinal car body or fragment may include a humanized IgG1. cinoma, stomach carcinoma, breast carcinoma, prostate can 0021. An anti-CD74 binding molecule may be a chimeric cer, ovarian cancer, and/or melanoma. anti-CD74 antibody or fragment thereof and may include the 0027. In one embodiment, the composition may comprise light and heavy chain variable regions of a murine anti-CD74 an agent for photodynamic therapy, e.g., a photosensitizer antibody, attached to human antibody constant regions. A such as a benzoporphyrin monoacid ring A (BDP-MA), tin chimeric antibody or fragment thereof may include a chi etiopurpurin (SnET2), Sulfonated aluminum phthalocyanine meric IgG1 or fragment thereof. (AISPc), or lutetium texaphyrin (Lutex). The method may 0022. The anti-CD74 binding molecule may be selected also include administering an irradiating light source to the such that the binding of the molecule or fragment thereof to targeted cells or tissue. In certain embodiments, photody CD74 competes with or is blocked by an antibody or fragment namic therapy may be used diagnostically as well as thera thereof specific for CD74, such as the LL1 antibody. Alter peutically. natively, the binding molecule may be selected such that the 0028. The method may include administering a composi binding molecule binds to the same epitope of CD74 as an tion that includes a diagnostic nuclide, e.g., 'F, Fe, Cu, antibody or fragment thereof specific for CD74, such as the 46Cu, 67Cu, 7Ga, Ga, 86Y. 897r, 94Tc, 94mTc, 99mTc, 11 In, LL1 antibody. In still other alternatives, the binding molecule 'I, ‘I,’’I, ''I. Typically, the diagnostic nuclide will emit may be selected to be internalized by Raji lymphoma cells in 25-4000 keV gamma particles and/or positrons. culture. In another embodiment, an anti-CD74 binding mol 0029. The method may include administering a composi ecule, such as an antibody or fragment thereof, may be tion that includes a diagnostic agent, which can be used to selected Such that it induces apoptosis of Raji cells in cell perform positron emission tomography (PET), such as 'For culture when cross-linked with goat antisera reactive with the “Ga. As such, the method may include performing positron Fc of a murine IgG 1 antibody. emission tomography (PET). 0023 The anti-CD74 binding molecule may also include a 0030 The method may include administering a composi fragment which includes a F(ab'), Fab, scFv, Fv, or a fusion tion that includes one or more image enhancing agents, e.g., protein utilizing part or all of the light and heavy chains of the ions, lanthanum ions, manganese ions, iron, chro F(ab'), Fab, scfv, or Fv, such that the fragment is capable of mium, copper, cobalt, nickel, fluorine, dysprosium, rhenium, binding to CD74. The binding molecule may be selected or europium, terbium, holmium, neodymium, or mixtures designed to be multivalent, or multivalent and multispecific. thereof. As such, the method may include performing an The fragments may form a bispecific binding molecule or a imaging technique Such as magnetic resonance imaging diabody. In one embodiment, the binding molecule includes a (MRI). fusion protein that includes four or more FVs, or Fab's of the 0031. The method may include administering a composi antibodies or fragments thereof. In a further embodiment, the tion that includes one or more radioopaque agents or contrast binding molecule includes a fusion protein that includes one agents such as barium, , ethiodized oil, gallium or more FVs or Fab's of an anti-CD74 antibody or fragment citrate, , , , iodipamide, thereof, and one or more FVs or Fab's from an antibody , iogulamide, iohexyl, , iopanoic specific for a tumor cell marker that is not a CD74 antigen. For acid, ioprocemic acid, iosefamic acid, io.seric acid, ioSula example, the tumor cell marker may include a B-cell lineage mide , iosemetic acid, iotasul, iotetric acid, iotha antigen such as CD19, CD20, or CD22. Alternatively, the lamic acid, , , ioxotrizoic acid, ipo tumor cell marker may include HLA-DR, CD30, CD33, date, meglumine, , metrizoate, , CD52, MUC1 or TAC. thallous chloride, or combinations thereof. The method may 0024. The anti-CD 74 binding molecule may include include performing an X-ray or computed tomography (CT). human constant regions of IgG1, IgG2a, IgG3, or IgG4. 0032. The method may include administering a composi 0025. Also disclosed is a method for treating and/or diag tion that includes one or more ultrasound contrast agents, nosing a disease or disorder that includes administering to a Such as dextran or a liposome (e.g., a gas-filled liposome). patient a therapeutic and/or diagnostic composition. The The method may include performing an ultrasound proce therapeutic and/or diagnostic composition includes any of the dure. aforementioned compositions, generally a composition that 0033. In addition to the aforementioned procedures, the includes: (1) one or more anti-CD74 binding molecules or method may also include performing an operative, intravas fragments thereof conjugated to a liposome; (2) a pharma cular, laparoscopic, or endoscopic procedure, before, simul ceutically acceptable excipient; and (3) optionally an effector taneously with, or after the immunoconjugate or composition molecule (e.g., a therapeutic or diagnostic agent). Typically, is administered. US 2010/0272636 A1 Oct. 28, 2010

0034. The method may also include administering a sec 0039 FIG. 3. Alignment of amino acid sequences of light ond or additional composition that includes a therapeutic or and heavy chain variable regions of a human antibody, cIL1 diagnostic agent, where the second or additional composition and hLL1. FIG. 3A shows the Vamino acid sequence align is administered before, simultaneously, or after the first com ment of the human antibody RF-TS3, cIL1 and hLL1. FIG. position is administered. The second or additional composi 3B shows the Vamino acid sequence alignment of the human tion may include any of the aforementioned compositions. In antibody HF-21/28, cIL1 and hLL1. Dots indicate the resi one embodiment the second or additional composition dues in cI L1 that are identical to the corresponding residues includes an anti-CD74 binding molecule conjugated to a in the human antibodies. Boxed regions represent the CDR therapeutic or diagnostic agent. The therapeutic or diagnostic regions. Both N- and C-terminal residues (underlined) of agent may comprise any of the aforementioned drugs, pro cLL1 are fixed by the staging vectors used and not compared drugs, toxins, enzymes, radioisotopes, immunomodulators, with the human antibodies. Kabat’s Ig molecule number cytokines, hormones, antibodies, binding molecules, oligo scheme is used as in FIG. 1. nucleotides, chelators, cations, therapeutic nuclides, agents 0040 FIG. 4. DNA and amino acid sequences of human for photodynamic therapy, diagnostic nuclides, image ized LL1 (hLL1) heavy and light chain variable regions. FIG. enhancing agents, radioopaque agents, and/or contrasting 4A shows the DNA and amino acid sequences of h L1 V. agents. The method may also include performing a PET, MRI, FIG. 4B shows the DNA and amino acid sequences of X-ray, CT, ultrasound, operative, intravascular, laparoscopic, hLL1V. Amino acid sequences encoded by the correspond or endoscopic procedure. ing DNA sequences are given as one letter codes. The amino 0035 Also disclosed are methods of preparing the afore acid residues in the CDR regions are shown in bold and mentioned compositions (e.g., an anti-CD74 immunoconju underlined. Kabat’s Ig molecule numbering scheme is used gate by mixing one or more amphiphilic lipids to form a for amino acid residues as in FIG. 1. liposome and contacting the liposome with an anti-CD74 0041 FIG. 5. Milatuzumab mediates direct cytotoxicity in binding molecule, (e.g., an antibody or fragment thereof)). In CLL cells. FIG.5A.Viability of CLL patient cells at 4, 24 and one example, the lipid contains nucleophilic carbons (e.g., 48 hours by propidium iodide (PI) staining. Cells were within a maleimide group), and the binding molecule con untreated, treated with goat anti-human Fc crosslinker alone, tains free thiol groups (e.g., disulfides treated with a reducing or treated with 5 lug/mL milatuzumab (Mita) or 10 gg/mL agent). The method may include mixing the composition with rituximab (Ritux) in the presence or absence of anti-Fc one or more therapeutic or diagnostic agents, which may be crosslinker (in five times excess of binding antibody) (N=26; covalently, non-covalently, or otherwise associated with any P<0.0001 for anti-Fc vs. mila+anti-Fc). Y-axis indicates the component of the composition. percent of PI positive cells and the active metabolite of flu 0036) Also disclosed is a kit that includes any of the afore darabine (2-F-ara A. Flud) was used as a positive control for mentioned compositions or the components Sufficient for pre cell death. FIG.5B. Viability of CLL patient cells at 24 hours paring any of the aforementioned compositions. Typically, by PI staining (N-26 from FIG. 5A; each line represents the kit includes instructions for administering the composi individual patient sample). FIG. 5C. Immunoblots for tions or, where applicable, instructions for preparing the com caspases-2, 3, 8, 6, 9 and PARP in whole cell lysates isolated positions. from CLL cells treated with anti-Fc. 5ug/mL mila--anti-Fc, or 5uM fludarabine in the presence or absence of 20 uM caspase BRIEF DESCRIPTION OF THE DRAWINGS inhibitor, QVD-OPH, for 24 hours. Jurkat cells treated with UV or staurosporine were used as controls for caspase cleav 0037 FIG. 1. DNA and amino acid sequences of the age. Pro-caspase (P) and cleaved (C) forms are indicated. murine LL1 heavy and light chain variable regions. FIG. 1A FIG.5D. Viability of CLL patient cells by PI staining treated shows DNA and amino acid sequences of LL1V. FIG. 1B with anti-Fc, 5 g/mL mila+anti-Fic, or 5 uM fludarabine in shows DNA and amino acid sequences of the LL1V. Amino the presence or absence of 20 uM caspase inhibitor, Q-VD acid sequences encoded by the corresponding DNA OPH, for 24 hours (N=5; P=0.03). FIG.5E. (paneli) Viability sequences are given as one-letter codes below the nucleotide of CLL patient cells treated with anti-Fc alone or 5ug/mL sequence. Numbering of the nucleotide sequence is on the mila--anti-Fc in the presence or absence of stroma co-culture. right side. The amino acid residues in the CDR regions are After 6 hours in drug, cells were washed and cultured in fresh shown in bold and underlined. Kabat’s Ig molecule number media alone or in the presence of HS-5 stromal cells. Viability ing is used for amino acid residues as shown by the number was determined by PI staining after 48 hours of co-culture ing above the amino acid residues. The residues numbered by (N=11; P=0.10). (panel ii) Viability of CLL patient cells a letter following a particular digit indicates the insertion treated with vehicle or 1 uM2-F-ara A in the presence or residues defined by Kabat numbering scheme. The insertion absence of stroma co-culture. After 6 hours in drug cells were residues numbered with a letter have the same preceding washed and cultured in fresh media alone or in the presence of digit. For example, residues 82A, 82B and 82C in FIG. 1A are HS-5 cells. Viability by PI staining was determined after 48 indicated as 82A, B, and C. hours of co-culture (N=6). (panel iii) Viability of CLL patient 0038 FIG. 2. DNA and amino acid sequences of chimeric cells treated with anti-Fc alone or 5ug/mL mila--anti-Fc in the LL1 (cLL1) heavy and light chain variable region (see, U.S. presence or absence of 500 ng/mL soluble CD40L. Viability Pat. No. 7,312.318). FIG. 2A shows DNA and amino acid by PI staining was determined at 48 hours (N=17: P=0.11). sequences of cIL1V. FIG. 2B shows double-stranded DNA 0042 FIG. 6. Milatuzumab immunoliposome increases and amino acid sequences of cIL1V. Amino acid sequences CD74 on the surface of CLL cells and induces cell death. FIG. encoded by the corresponding DNA sequences are given as 6A. Mean fluorescent intensity of surface CD74 in CLL one-letter codes. The amino acid residues in the CDR regions patient cells either untreated, treated with 25ug/mL anti-Fc. are shown in bold and underlined. The numbering of nucle or 5 g/mL milatuZumab with or without 25 ug/mL anti-Fc otides and amino acids is same as that in FIG. 1. for 1 hour. Histogram shown is representative of 14 patients. US 2010/0272636 A1 Oct. 28, 2010

FIG. 6B. Lett. Mean fluorescent intensity of CD74 in CLL domains of the antibody molecule are derived from those of a patient cells either untreated, treated with 25ug/mL anti-Fc. human antibody. For veterinary applications, the constant or 5 g/mL milatuZumab with or without 25 g/mL anti-Fc domains of the chimericantibody may be derived from that of for 1 hour (N=14; P=0.0003). Right. Mean fluorescent inten other species, such as a cat or dog. sity of CD20 in CLL patient cells either untreated, or treated 0050 A“humanized antibody' is a recombinant protein in with 5ug/mL milatuzumab with or without 25 g/mL anti-Fc which the CDRS from an antibody from one species; e.g., a for 1 hour (N=14; P=0.14). FIG. 6C. Mean fluorescent inten rodent antibody, are transferred from the heavy and light sity of CD74 in CLL patient cells either untreated, or treated variable chains of the rodent antibody into human heavy and with empty liposome or milatuZumabimmunoliposome light variable domains. The constant domains of the antibody (mila-IL) for 1 hour (N=4; P=0.0003). FIG. 6D. Viability by molecule are derived from those of a human antibody. PI staining in CLL patient cells either untreated, or treated 0051. A “human antibody' is an antibody obtained from with empty liposome, IgG-immunoliposome (IgG-IL), mila transgenic mice that have been genetically engineered to pro IL. 25ug/mL anti-Fc or 5ug/mL milatuZumab with 25ug/mL duce specific human antibodies in response to antigenic chal anti-Fc for 24 hours (N=11; P=0003). lenge. In this technique, elements of the human heavy and light chain locus are introduced into strains of mice derived DETAILED DESCRIPTION from embryonic stem cell lines that contain targeted disrup tions of the endogenous heavy chain and light chain loci. The Definitions transgenic mice can synthesize human antibodies specific for 0043. The following definitions are provided to facilitate human antigens, and the mice can be used to produce human understanding of the disclosure herein. Where a term is not antibody-secreting hybridomas. Methods for obtaining specifically defined, it is used in accordance with its plain and human antibodies from transgenic mice are described by ordinary meaning. Green et al., Nature Genet. 7:13 (1994), Lonberget al., Nature 0044 As used herein, the terms “a”, “an and “the may 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994). refer to either the singular or plural, unless the context other A fully human antibody also can be constructed by genetic or wise makes clear that only the singular is meant. chromosomal transfection methods, as well as phage display 0045. As used herein, the term “about” means plus or technology, all of which are known in the art. (See, e.g., minus ten percent (10%) of a value. For example, “about 100' McCafferty et al., Nature 348:552-553 (1990) for the produc would refer to any number between 90 and 110. tion of human antibodies and fragments thereof in vitro, from 0046. A “binding molecule, as used herein, is any mol immunoglobulin variable domain gene repertoires from ecule that can specifically or selectively bind to an antigen. A unimmunized donors). In this technique, antibody variable binding molecule may include an antibody or a fragment domain genes are cloned in-frame into eithera major or minor thereof. An anti-CD74 binding molecule is a molecule that coat protein gene of a filamentous bacteriophage, and dis binds to the CD74 antigen, such as an anti-CD74 antibody or played as functional antibody fragments on the Surface of the fragment thereof. Other anti-CD74 binding molecules may phage particle. Because the filamentous particle contains a also include multivalent molecules, multispecific molecules single-stranded DNA copy of the phage genome, selections (e.g., diabodies), fusion molecules, aptimers, avimers, or based on the functional properties of the antibody also result other naturally occurring or recombinantly created mol in selection of the gene encoding the antibody exhibiting ecules. those properties. In this way, the phage mimics Some of the 0047. An “antibody” refers to a full-length (i.e., naturally properties of the B cell. Phage display can be performed in a occurring or formed by normal immunoglobulin gene frag variety of formats, for their review, see, e.g. Johnson and ment recombinatorial processes) immunoglobulin molecule Chiswell, Current Opinion in Structural Biology 3:5564-571 (e.g., an IgG antibody) or an immunologically active (i.e., (1993). Human antibodies may also be generated by in vitro antigen-binding) portion of an immunoglobulin molecule, activated B cells. (See, U.S. Pat. Nos. 5,567,610 and 5.229, like an antibody fragment. 275). 0048. An “antibody fragment' is a portion of an antibody 0.052 An “effector” is an atom, molecule, or compound such as F(ab'), F(ab), Fab', Fab, Fv, scEv, single domain that brings about a chosen result. An effector may include a antibodies (DABs or VHHs) and the like, including half therapeutic agent and/or a diagnostic agent. molecules of IgG4 (van der Neut Kolfschoten et al. (Science 0053 A “therapeutic agent' is an atom, molecule, or com 2007; 317(14 September): 1554-1557). Regardless of struc pound that is useful in the treatment of a disease. Examples of ture, an antibody fragment binds with the same antigen that is therapeutic agents include but are not limited to antibodies, recognized by the intact antibody. For example, an anti-CD74 antibody fragments, drugs, toxins, enzymes, nucleases, hor antibody fragment binds with an epitope of CD74. The term mones, immunomodulators, antisense oligonucleotides, 'antibody fragment also includes isolated fragments con chelators, boron compounds, photoactive agents, dyes and sisting of the variable regions, such as the “Fv’ fragments radioisotopes. consisting of the variable regions of the heavy and light 0054 A“diagnostic agent” is an atom, molecule, or com chains, recombinant single chain polypeptide molecules in pound that is useful in diagnosing a disease. Useful diagnostic which light and heavy chain variable regions are connected by agents include, but are not limited to, radioisotopes, dyes, a peptide linker (“sclv proteins'), and minimal recognition contrast agents, fluorescent compounds or molecules and units consisting of the amino acid residues that mimic the enhancing agents (e.g., paramagnetic ions). Preferably, the hyperVariable region. diagnostic agents are selected from the group consisting of 0049. A “chimericantibody' is a recombinant protein that radioisotopes, enhancing agents, and fluorescent compounds. contains the variable domains including the complementarity In order to load an antibody component with radioactive determining regions (CDRs) of an antibody derived from one metals or paramagnetic ions, it may be necessary to react it species, preferably a rodent antibody, while the constant with a reagent having a long tail to which are attached a US 2010/0272636 A1 Oct. 28, 2010 multiplicity of chelating groups for binding the ions. Such a A “multivalent antibody' is an antibody that can bind simul tail can be a polymer Such as a polylysine, polysaccharide, or taneously to at least two targets that are of the same or differ other derivatized or derivatizable chain having pendant ent structure. Valency indicates how many binding arms or groups to which can be bound chelating groups such as, e.g., sites the antibody has to a single antigen or epitope; i.e., ethylenediaminetetraacetic acid (EDTA), diethylenetri monovalent, bivalent, trivalent or multivalent. The multiva aminepentaacetic acid (DTPA), DOTA, NOTA, NETA, por lency of the antibody means that it can take advantage of phyrins, polyamines, crown ethers, bis-thiosemicarbazones, multiple interactions in binding to an antigen, thus increasing polyoximes, and like groups known to be useful for this the avidity of binding to the antigen. Specificity indicates how purpose. Chelates are coupled to the peptide antigens using many antigens or epitopes an antibody is able to bind; i.e., standard chemistries. The chelate is normally linked to the monospecific, bispecific, trispecific, multispecific. Using antibody by a group which enables formation of a bond to the these definitions, a natural antibody, e.g., an IgG, is bivalent molecule with minimal loss of immunoreactivity and mini because it has two binding arms but is monospecific because mal aggregation and/or internal cross-linking. Other, more it binds to one epitope. Multispecific, multivalent antibodies unusual, methods and reagents for conjugating chelates to are constructs that have more than one binding site of differ antibodies are disclosed in U.S. Pat. No. 4,824,659. Particu ent specificity. For example, a diabody, where one binding larly useful metal-chelate combinations include 2-benzyl site reacts with one antigen and the other with another anti DTPA and its monomethyl and cyclohexyl analogs, used with gen. diagnostic isotopes in the general energy range of 60 to 4,000 0.058 A“bispecific antibody' is an antibody that can bind keV. Some useful diagnostic nuclides may include, such as simultaneously to two targets which are of different structure. 18F 52Fe, 62Cu, Cu, 67Cu, 7Ga, Ga, 86y, 89Zr, 9Tc, Bispecific antibodies (bsAb) and bispecific antibody frag "Tc, "Tc, or '''In. The same chelates, when complexed ments (bsFab) may have at least one arm that specifically with non-radioactive metals, such as manganese, iron and binds to, for example, a B-cell, T-cell, myeloid-, plasma-, and gadolinium are useful for MRI, when used along with the mast-cell antigen or epitope and at least one other arm that antibodies and liposomes described herein. Macrocyclic che specifically binds to a targetable conjugate that bears a thera lates such as NOTA, DOTA, and TETA are of use with a peutic or diagnostic agent. A variety of bispecific antibodies variety of metals and radiometals, most particularly with can be produced using molecular engineering. radionuclides of gallium, yttrium and copper, respectively. 0059 Preparation of Monoclonal Antibodies Such metal-chelate complexes can be made very stable by 0060. The immunoconjugates and compositions tailoring the ring size to the metal of interest. Other ring-type described herein may include monoclonal antibodies. Rodent chelates such as macrocyclic polyethers, which are of interest monoclonal antibodies to specific antigens may be obtained for stably binding nuclides, such as 'R' for RAIT may be by methods known to those skilled in the art. (See, e.g., used. In certain embodiments, chelating moieties may be Kohler and Milstein, Nature 256: 495 (1975), and Coligan et used to attach a PET imaging agent, such as an Al-F com al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, plex, to a targeting molecule for use in PET analysis (see, e.g., VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991)). U.S. Pat. Nos. 7,563,433 and 7,597,876, the Examples sec 0061 General techniques for cloning murine immunoglo tion of each of which is incorporated herein by reference.) bulin variable domains have been disclosed, for example, by 0055 An “immunoconjugate' is a conjugate of a binding the publication of Orlandi et al., Proc. Natl Acad. Sci. USA molecule (e.g., an antibody) with an atom, molecule, or a 86: 3833 (1989). Techniques for constructing chimeric anti higher-ordered structure (e.g., with a liposome), a therapeutic bodies are well known to those of skill in the art. As an agent, or a diagnostic agent. The diagnostic agent can com example, Leung et al., Hybridoma 13:469 (1994), disclose prise a radioactive or non-radioactive label, a how they produced an LL2 chimera by combining DNA (Such as for magnetic resonance imaging, computed tomog sequences encoding the V and V. domains of LL2 mono raphy or ultrasound), and the radioactive label can be a clonal antibody, an anti-CD22 antibody, with respective gamma-, beta-, alpha-, Auger electron-, or positron-emitting human and IgG constant region domains. This publication isotope. A "naked antibody' is an antibody that is not conju also provides the nucleotide sequences of the LL2 light and gated to any other agent. heavy chain variable regions, V and V, respectively. Tech 0056. As used herein, the term “antibody fusion protein' is niques for producing humanized antibodies are disclosed, for a recombinantly produced antigen-binding molecule in example, by Jones et al., Nature 321:522 (1986), Riechmann which an antibody or antibody fragment is linked to another et al., Nature 332: 323 (1988), Verhoeyen et al., Science 239: protein or peptide, such as the same or different antibody or 1534 (1988), Carter et al., Proc. Natl Acad. Sci. USA 89: antibody fragment or a DDD or AD peptide. The fusion 4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), and protein may comprise a single antibody component, a multi Singer et al., J. Immun. 150: 2844 (1993). valent or multispecific combination of different antibody 0062. A chimeric antibody is a recombinant protein that components or multiple copies of the same antibody compo contains the variable domains including the CDRs derived nent. The fusion protein may additionally comprise an anti from one species of animal. Such as a rodent antibody, while body or an antibody fragment and a therapeutic agent. the remainder of the antibody molecule; i.e., the constant Examples of therapeutic agents suitable for Such fusion pro domains, is derived from a human antibody. Accordingly, a teins include immunomodulators and toxins. One preferred chimeric monoclonal antibody can also be humanized by toxin comprises a ribonuclease (RNase), preferably a recom replacing the sequences of the murine FR in the variable binant RNase. domains of the chimeric antibody with one or more different 0057. A "multispecific antibody' is an antibody that can human FR. Specifically, mouse CDRs are transferred from bind simultaneously to at least two targets that are of different heavy and light variable chains of the mouse immunoglobulin structure, e.g., two different antigens, two different epitopes into the corresponding variable domains of a human antibody. on the same antigen, or a hapten and/or an antigen or epitope. As simply transferring mouse CDRs into human FRS often US 2010/0272636 A1 Oct. 28, 2010

results in a reduction or even loss of antibody affinity, addi Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), tional modification might be required in order to restore the and Taylor et al., Int. Immun. 6:579 (1994). A non-limiting original affinity of the murine antibody. This can be accom example of such a system is the XenoMouse R (e.g., Green et plished by the replacement of one or more some human al., 1999, J. Immunol. Methods 231:11-23, incorporated residues in the FR regions with their murine counterparts to herein by reference) from Abgenix (Fremont, Calif.). In the obtain an antibody that possesses good binding affinity to its XenoMouse Rand similar animals, the mouse antibody genes epitope. (See, e.g., Tempest et al., Biotechnology 9:266 have been inactivated and replaced by functional human anti (1991) and Verhoeyen et al., Science 239: 1534 (1988)). body genes, while the remainder of the mouse immune sys 0063 A fully human antibody, i.e., human anti-CD74 anti tem remains intact. bodies or other human antibodies, such as anti-CD22, anti 0067. The XenoMouseR) was transformed with germline CD19, anti-CD23, anti-CD20 or anti-CD21 antibodies for configured YACs (yeast artificial chromosomes) that con combination therapy with humanized, chimeric or human tained portions of the human IgE and Igkappaloci, including anti-CD74 antibodies, can be obtained from a transgenic non the majority of the variable region sequences, along acces human animal. (See, e.g., Mendez et al., Nature Genetics, 15: sory genes and regulatory sequences. The human variable 146-156, 1997: U.S. Pat. No. 5,633,425.) Methods for pro region repertoire may be used to generate antibody producing ducing fully human antibodies using either combinatorial B cells, which may be processed into hybridomas by known approaches or transgenic animals transformed with human techniques. A XenoMouse Rimmunized with a target antigen immunoglobulin loci are known in the art (e.g., Mancini et al., will produce human antibodies by the normal immune 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005, response, which may be harvested and/or produced by stan Comb. Chem. High Throughput Screen. 8:117-26: Brekke dard techniques discussed above. A variety of strains of Xen and Loset, 2003, Curr. Opin. Phamacol. 3:544-50; each oMouse(R) are available, each of which is capable of produc incorporated herein by reference). Such fully human antibod ing a different class of antibody. Transgenically produced ies are expected to exhibit even fewer side effects than chi human antibodies have been shown to have therapeutic poten meric or humanized antibodies and to function in Vivo as tial, while retaining the pharmacokinetic properties of normal essentially endogenous human antibodies. In certain embodi human antibodies (Green et al., 1999). The skilled artisan will ments, the claimed methods and procedures may utilize realize that the claimed compositions and methods are not human antibodies produced by Such techniques. limited to use of the XenoMouse(R) system but may utilize any 0064. In one alternative, the phage display technique may transgenic animal that has been genetically engineered to be used to generate human antibodies (e.g., Dantas-Barbosa produce human antibodies. et al., 2005, Genet. Mol. Res. 4:126-40, incorporated herein 0068. In various embodiments, the claimed methods and by reference). Human antibodies may be generated from nor compositions may utilize any of a variety of antibodies known mal humans or from humans that exhibit a particular disease in the art. Antibodies of use may be commercially obtained state, such as cancer (Dantas-Barbosa et al., 2005). The from a number of known sources. For example, a variety of advantage to constructing human antibodies from a diseased antibody secreting hybridoma lines are available from the individual is that the circulating antibody repertoire may be AmericanType Culture Collection (ATCC, Manassas, Va.). A biased towards antibodies against disease-associated anti large number of antibodies against various disease targets, genS. including but not limited to tumor-associated antigens, have 0065. In one non-limiting example of this methodology, been deposited at the ATCC and/or have published variable Dantas-Barbosa et al. (2005) constructed a phage display region sequences and are available for use in the claimed library of human Fab antibody fragments from osteosarcoma methods and compositions. See, e.g., U.S. Pat. Nos. 7.312, patients. Generally, total RNA was obtained from circulating 318; 7.282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509; blood lymphocytes (Id.) Recombinant Fab were cloned from 7,049,060; 7,045,132; 7,041,803; 7,041802; 7,041,293; the L, Y and K chain antibody repertoires and inserted into a 7,038,018; 7,037,498; 7,012,133; 7,001,598; 6,998,468; phage display library (Id.) RNAs were converted to cDNAs 6,994,976; 6,994,852: 6,989,241; 6,974,863; 6,965,018; and used to make Fab cDNA libraries using specific primers 6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924: against the heavy and light chain immunoglobulin sequences 6,949,244; 6,946,129; 6,943,020; 6,939,547; 6,921,645; (Marks et al., 1991, J. Mol. Biol. 222:581-97). Library con 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475; struction was performed according to Andris-Widhopfet al. 6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; (2000. In: Phage Display Laboratory Manual, Barbas et al. 6,884,594; 6,881405; 6,878,812: 6,875,580; 6,872,568: (eds), 1 edition, Cold Spring Harbor Laboratory Press, Cold 6,867,006; 6,864,062: 6,861,511; 6,861,227; 6,861,226; Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporated herein by 6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778: reference). The final Fab fragments were digested with 6,812,206: 6,793,924; 6,783,758; 6,770,450; 6,767,711; restriction endonucleases and inserted into the bacteriophage 6,764,688: 6,764,681; 6,764,679; 6,743,898; 6,733,981; genome to make the phage display library. Such libraries may 6,730,307; 6,720,15; 6,716,966: 6,709,653; 6,693,176: be screened by standard phage display methods. The skilled 6,692,908; 6,689,607; 6,689,362; 6,689,355; 6,682,737; artisan will realize that this technique is exemplary only and 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852; any known method for making and screening human antibod 6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441: ies or antibody fragments by phage display may be utilized. 6,605,279; 6,596,852: 6,592,868; 6,576,745; 6,572,856; 0066. In another alternative, transgenic animals that have 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058: been genetically engineered to produce human antibodies 6,528,625; 6,528,269; 6,521,227; 6,518.404: 6,511,665; may be used to generate antibodies against essentially any 6,491,915; 6,488,930; 6,482,598; 6,482.408; 6,479,247; immunogenic target, using standard immunization protocols 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356; as discussed above. Methods for obtaining human antibodies 6,455,044; 6,455,040, 6,451,310; 6,444,206, 6,441,143: from transgenic mice are described by Green et al., Nature 6,432,404; 6,432,402; 6,419,928; 6,413,726; 6,406,694; US 2010/0272636 A1 Oct. 28, 2010

6,403,770; 6,403,091; 6,395,276; 6,395.274; 6,387,350; structed (Huse et al., 1989, Science, 246:1274-1281) to allow 6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; rapid and easy identification of monoclonal Fab' fragments 6,355,481; 6,355,444; 6,355,245; 6,355,244; 6,346,246; with the desired specificity. 6,344, 198; 6,340,571; 6,340,459; 6,331,175; 6,306,393; 0072 A single chain Fv molecule (sclv) comprises a VL 6,254,868; 6,187,287: 6,183,744; 6,129,914; 6,120,767; domain and a VH domain. The VL and VH domains associate 6,096,289; 6,077,499; 5,922,302; 5,874,540; 5,814,440; to form a target binding site. These two domains are further 5,798,229; 5,789,554; 5,776,456; 5,736,119, 5,716,595; covalently linked by a peptide linker (L). Methods for making 5,677, 136; 5,587,459; 5,443,953, 5,525,338, the Examples ScFv molecules and designing Suitable peptide linkers are section of each of which is incorporated herein by reference. disclosed in U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, These are exemplary only and a wide variety of other anti R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB Vol bodies and their hybridomas are known in the art. The skilled 9:73-80 (1995) and R. E. Bird and B. W. Walker, “Single artisan will realize that antibody sequences or antibody-se Chain Antibody Variable Regions.” TIBTECH, Vol 9: 132 137 (1991). creting hybridomas against almost any disease-associated 0073. An antibody fragment can be prepared by known antigen may be obtained by a simple search of the ATCC, methods, for example, as disclosed by Goldenberg, U.S. Pat. NCBI and/or USPTO databases for antibodies against a Nos. 4,036,945 and 4,331,647 and references contained selected disease-associated target of interest. The antigen therein. Also, see Nisonoffet al., Arch Biochem. Biophys. 89: binding domains of the cloned antibodies may be amplified, 230 (1960); Porter, Biochem. J. 73: 119 (1959), Edelman et excised, ligated into an expression vector, transfected into an al., in METHODS IN ENZYMOLOGY VOL. 1, page 422 adapted host cell and used for protein production, using stan (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 dard techniques well known in the art. and 2.10.-2.10.4. 0069 Particular antibodies that may be of use for therapy 0074. A single complementarity-determining region of cancer within the scope of the claimed methods and com (CDR) is a segment of the variable region of an antibody that positions include, but are not limited to, LL1 (anti-CD74), is complementary in structure to the epitope to which the LL2 and RFB4 (anti-CD22), RS7 (anti-epithelial glycopro antibody binds and is more variable than the rest of the vari tein-1 (EGP-1)), PAM4 and KC4 (both anti-mucin), MN-14 able region. Accordingly, a CDR is sometimes referred to as (anti-carcinoembryonic antigen (CEA, also known as hyperVariable region. A variable region comprises three CD66e), Mu-9 (anti-colon-specific antigen-p), Immu 31 (an CDRs. CDR peptides can be obtained by constructing genes anti-alpha-fetoprotein), TAG-72 (e.g., CC49), Tn. J591 or encoding the CDR of an antibody of interest. Such genes are HuJ591 (anti-PSMA (prostate-specific membrane antigen)), prepared, for example, by using the polymerase chain reac AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), tion to synthesize the variable region from RNA of antibody G250 (an anti-carbonic anhydrase IXMAb) and hL243 (anti producing cells. (See, e.g., Larrick et al., Methods: A Com HLA-DR). Such antibodies are known in the art (e.g., U.S. panion to Methods in Enzymology 2: 106 (1991); Courtenay Pat. Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306, Luck, "Genetic Manipulation of Monoclonal Antibodies.” in 393; 6,653,104; 6,730.300; 6,899,864; 6,926,893; 6,962,702: MONOCLONAL ANTIBODIES: PRODUCTION, ENGI 7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004; NEERING AND CLINICAL APPLICATION, Ritter et al. 7,282,567; 7,300,655; 7,312,318; 7,585,491; 7,612, 180; (eds.), pages 166-179 (Cambridge University Press 1995); 7,642,239; and U.S. Patent Application Publ. No. and Ward et al., “Genetic Manipulation and Expression of 20040202666 (now abandoned); 20050271671; and Antibodies, in MONOCLONAL ANTIBODIES: PRIN 20060193865; the Examples section of each incorporated CIPLES AND APPLICATIONS, Birch et al., (eds.), pages herein by reference.) Specific known antibodies of use 137-185 (Wiley-Liss, Inc. 1995). include hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. 0075 Another form of an antibody fragment is a single No. 7,251,164), ha19 (U.S. Pat. No. 7,109,304), hIMMU31 domain antibody (dAb), sometimes referred to as a single (U.S. Pat. No. 7,300,655), h L1 (U.S. Pat. No. 7,312.318), chain antibody. Techniques for producing single-domain hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S. Pat. No. 7,387, antibodies are well known in the art (see, e.g., Cossins et al., 773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat. Protein Expression and Purification, 2007, 51:253-59; No. 6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hR1 Shuntao et al., Molec Immunol 2006, 43:1912-19: Tanha et (U.S. Provisional Patent Application 61/145,896), hRS7 al., J. Biol. Chem. 2001, 276:24774-780). (U.S. Pat. No. 7,238,785), hMN-3 (U.S. Pat. No. 7,541,440), 0076. In certain embodiments, the sequences of antibod AB-PG1-XG1-026 (U.S. patent application Ser. No. 1 1/983, ies, such as the Fc portions of antibodies, may be varied to 372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B optimize the physiological characteristics of the conjugates, (WO 2009/130575) the text of each recited patent or applica such as the half-life in serum. Methods of substituting amino tion is incorporated herein by reference with respect to the acid sequences in proteins are widely known in the art, Such as Figures and Examples sections. by site-directed mutagenesis (e.g. Sambrook et al., Molecular 0070 Production of Antibody Fragments Cloning. A laboratory manual, 2" Ed, 1989). In preferred 0071 Antibody fragments which recognize specific embodiments, the variation may involve the addition or epitopes can be generated by known techniques. The anti removal of one or more glycosylation sites in the Fc sequence body fragments are antigen binding portions of an antibody, (e.g., U.S. Pat. No. 6,254,868, the Examples section of which such as F(ab), Fab', Fab, Fv, sclv and the like. Other anti is incorporated herein by reference). In other preferred body fragments include, but are not limited to: the F(ab'), embodiments, specific amino acid Substitutions in the Fc fragments which can be produced by pepsin digestion of the sequence may be made (e.g., Hornicket al., 2000, JNucl Med antibody molecule and the Fab' fragments, which can be 41:355-62; Hinton et al., 2006, J Immunol 176:346-56; Pet generated by reducing disulfide bridges of the F(ab')2 frag kova et al. 2006, Int Immunol 18:1759-69; U.S. Pat. No. ments. Alternatively, Fab' expression libraries can be con 7.217,797). US 2010/0272636 A1 Oct. 28, 2010

0077 Anti-CD74 Antibodies chain variable region of FIG. 2B. The chimeric antibody or 0078. The anti-CD74 binding molecules of the present fragment thereofmay beachimeric IgG1 or fragment thereof. immunoconjugates and compositions may contain specific I0081. Where the anti-CD74 binding molecule is a human murine CDRs that have binding affinity for the CD74 antigen. anti-CD74 antibody, the human anti-CD74 antibody or frag For example, the anti-CD74 binding molecules may be ment thereof may include a light chain variable region of the humanized, chimeric or human antibodies, and they may human anti-CD74 antibody (e.g., a CDR1 having an amino contain the amino acids of the CDRs of a murine anti-CD74 acid sequence RSSQSLVHRNGNTYLEH (SEQID NO:1); a antibody, (e.g., the murine anti-CD74 antibody, LL1). CDR2 having an amino acid sequence TVSNRFS (SEQ ID Humanized, chimeric, and human anti-CD74 antibody or NO:2); and a CDR3 having an amino acid sequence SQSSH fragments thereof are described in U.S. Pat. No. 7.312,318, VPPT (SEQID NO:3)). In one embodiment, the human anti the Examples section of which is incorporated herein by CD74 antibody or fragment thereof may include a heavy reference. chain variable region of the human antibody which may 0079. Where the anti-CD74 antibody is humanized, it may include CDRs of a heavy chain variable region of a murine contain CDRs of a light chain variable region of a murine anti-CD74 antibody (e.g., a CDR1 having an amino acid anti-CD74 antibody (e.g., a CDR1 having an amino acid sequence NYGVN (SEQID NO:4); a CDR2 having an amino sequence RSSQSLVHRNGNTYLEH (SEQ ID NO:1); a acid sequence WINPNTGEPTFDDDFKG (SEQ ID NO:5); CDR2 having an amino acid sequence TVSNRFS (SEQ ID and a CDR3 having an amino acid sequence SRGKNEAW NO:2); and a CDR3 having an amino acid sequence SQSSH FAY (SEQ ID NO:6)). The human antibody or fragment VPPT (SEQID NO:3)). The humanized anti-CD74 antibody thereof may be a human IgG1. or fragment may include the heavy chain variable region of I0082 Anti-CD74 antibodies of use may include murine, the humanized antibody, which may include CDRs of a heavy chimeric, humanized or human antibodies that contain CDRs chain variable region of a murine anti-CD74 antibody (e.g., a other than the CDRs of the LL1 antibody recited above, but CDR1 having an amino acid sequence NYGVN (SEQ ID which still block or compete for binding to CD74 with a NO:4); a CDR2 having an amino acid sequence WINPNT murine, chimeric or humanized LL1 antibody. For example, GEPTFDDDFKG (SEQ ID NO:5); and a CDR3 having an such anti-CD74 antibodies may bind to the same epitope of amino acid sequence SRGKNEAWFAY (SEQ ID NO:6)). CD74 as the LL1 antibody. Antibody binding competition The humanized anti-CD74 antibody or fragment thereof may experiments are well known in the art and may be performed include light and heavy chain variable regions including using any standard techniques, described in more detail complementarity-determining regions (CDRs) of murine below. anti-CD74 (mLL1) and human antibody constant and frame I0083. Multispecific and Multivalent Antibodies work (FR) region sequences, which may be substituted with I0084. The anti-CD74 binding molecule of the present at least one amino acid from the corresponding FRS of the immunoconjugates and compositions, as well as other bind murine antibody. In one embodiment, the Substituted amino ing molecules with different specificities for use in combina acid may be selected from amino acid residue 2, 3, 4, 46,87 tion therapy, can also include multispecific antibodies (com and 100 of the murine LL1 light chain variable region (FIG. prising at least one binding site to a CD74 epitope or antigen 3B), and amino acid residues 5,37,38,46, 68.91 and 93 of the and at least one binding site to another epitope on CD74 or murine heavy chain variable region (FIG. 3A). In another another antigen), and multivalent antibodies (comprising embodiment, the antibody or fragment thereof comprises a multiple binding sites to the same epitope or antigen), or the heavy chain variable region of FIG. 4A and a light chain antibodies can be both multivalent and multispecific. variable region of FIG. 4B. In a further embodiment, the I0085. A preferred binding molecule of the present immu antibody or fragment thereofmay comprise a light and heavy noconjugates or compositions is a fusion protein, which con chain constant region of a human antibody or a portion tains two or more FVs, Fab's or other antigen-binding frag thereof. The antibody or fragment may include a humanized ments of a humanized, chimeric, human or murine anti-CD74 IgG1. antibody. Another preferred antibody fusion protein contains 0080 Where the anti-CD74 binding molecule includes a one or more FVs, Fab's or other antigen-binding fragments of chimeric anti-CD74 antibody, the chimeric anti-CD74 anti a humanized, chimeric, human or murine anti-CD74 antibody body or fragment thereof may include a light chain variable and one or more FVs, Fab's or other fragments from antibod region of a murine anti-CD74 antibody (e.g., a CDR1 having ies specific for another antigen that is a tumor cell marker an amino acid sequence RSSQSLVHRNGNTYLEH (SEQ ID other than CD74. For example, the non-CD74 antigen may be NO:1); a CDR2 having an amino acid sequence TVSNRFS expressed by the CD74-expressing cells and may include a (SEQID NO:2); and a CDR3 having an amino acid sequence tumor marker selected from a B-cell lineage antigen, (e.g., SQSSHVPPT (SEQID NO:3)). In another embodiment, the CD19, CD20, or CD22 for the treatment of B-cell malignan chimeric anti-CD74 antibody or fragment thereof may cies). The non-CD74 antigen may also be expressed on other include a heavy chain variable region of a murine anti-CD74 CD74 positive cells that cause other types of malignancies, antibody (e.g., a CDR1 having an amino acid sequence such as S100 in melanoma, etc. Further, the tumor cell marker NYGVN (SEQ ID NO:4); a CDR2 having an amino acid may be a non-B-cell lineage antigen selected from the group sequence WINPNTGEPTFDDDFKG (SEQID NO:5); and a consisting of HLA-DR, CD30, CD33, CD52 MUC1 and CDR3 having an amino acid sequence SRGKNEAWFAY TAC. Other useful antigens may include carbonic anhydrase (SEQID NO:6)). In a further embodiment, the chimericanti IX, B7, CCCL19, CCCL21, CSAp, HER-2/neu, BrE3, CD1, CD74 antibody or fragment thereof may include the frame CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, work (FR) regions of a murine anti-CD74 antibody and the CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5 MAbs), light and heavy chain constant regions of a human antibody. CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, Alternatively, the chimericantibody or fragment thereof may CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, include a heavy chain variable region of FIG. 2A and a light CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, US 2010/0272636 A1 Oct. 28, 2010

CD80, CD83, CD95, CD126, CD133, CD138, CD147, cytokines and other functional agents may be conjugated to CD154, CEACAM5, CEACAM-6, alpha-fetoprotein (AFP), the anti-CD74 antibody or fragment, preferably through VEGF (e.g. AVASTINR, fibronectin splice variant), ED-B covalent attachments to the side chains of the amino acid fibronectin (e.g., L19), EGP-1, EGP-2 (e.g., 17-1A), EGF residues of the anti-CD74 antibody or fragment, for example receptor (ErbB1) (e.g., ERBITUX(R), ErbB2, ErbB3, Factor amine, carboxyl, phenyl, thiol or hydroxyl groups. Various H, FHL-1, Flt-3, folate receptor, Ga 733, GROB, HMGB-1, conventional linkers may be used for this purpose, for hypoxia inducible factor (HIF), HM1.24, HER-2/neu, insu example, diisocyanates, disothiocyanates, bis(hydroxysuc lin-like growth factor (ILGF), IFN-y, IFN-O, IFN-B, IL-2R, cinimide) esters, carbodiimides, maleimide-hydroxysuccin IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, imide esters, glutaraldehyde and the like. Conjugation of IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, IGF-1R, Ia, agents to the anti-CD74 antibody or fragment thereof prefer HM1.24, gangliosides, HCG, the HLA-DR antigen to which ably does not significantly affect the protein's binding speci L243 binds, CD66 antigens, i.e., CD66a-d or a combination ficity or affinity to its target. thereof, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, mac 0091. In still other embodiments, antibody-directed deliv rophage migration-inhibitory factor (MIF), MUC1, MUC2, ery of therapeutics or prodrug polymers to in Vivo targets can MUC3, MUC4, MUC5, placental growth factor (P1GF), PSA be combined with antibody delivery of radionuclides, such (prostate-specific antigen), PSMA, pancreatic cancer mucin, that combination chemotherapy and radioimmunotherapy is PAM4 antigen, NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1, achieved. Each therapeutic agent can be conjugated to a tar Le(y), mesothelin, S100, tenascin, TAC. Tnantigen, Thomas getable conjugate and administered simultaneously, or the Friedenreich antigens, tumor necrosis antigens, tumorangio nuclide can be given as part of a first targetable conjugate and genesis antigens, TNF-C. TRAIL receptor (R1 and R2), the drug given in a later step as part of a second targetable VEGFR, RANTES, T101, as well as cancer stem cell anti conjugate. gens, complement factors C3, C3a, C3b, C5a, C5, and an 0092 Avimers oncogene product. 0093. In certain embodiments, the binding moieties I0086 Methods for producing bispecific antibodies described herein may comprise one or more avimer include engineered recombinant antibodies which have addi sequences. Avimers are a class of binding proteins somewhat tional cysteine residues so that they crosslink more strongly similar to antibodies in their affinities and specificities for than the more common immunoglobulin isotypes. (See, e.g., various target molecules. They were developed from human FitzGerald et al. Protein Eng. 10(10): 1221-1225, (1997)). extracellular receptor domains by in vitro exon shuffling and Another approach is to engineer recombinant fusion proteins phage display. (Silverman et al., 2005, Nat. Biotechnol. linking two or more different single-chain antibody or anti 23:1493-94; Silverman et al., 2006, Nat. Biotechnol. 24:220.) body fragment segments with the needed dual specificities. The resulting multidomain proteins may comprise multiple (See, e.g., Coloma et al., Nature Biotech. 15:159-163, independent binding domains, that may exhibit improved (1997)). A variety of bispecific antibodies can be produced affinity (in some cases Sub-nanomolar) and specificity com using molecular engineering. In one form, the bispecific anti pared with single-epitope binding proteins. (Id.) In various body may consist of for example, an ScFV with a single embodiments, avimers may be attached to, for example, DDD binding site for one antigen and a Fab fragment with a single and/or AD sequences for use in the claimed methods and binding site for a second antigen. In another form, the bispe compositions. Additional details concerning methods of con cific antibody may consist of for example, an IgG with two struction and use of avimers are disclosed, for example, in binding sites for one antigen and two scEv with two binding U.S. Patent Application Publication Nos. 20040175756 (now sites for a second antigen. abandoned), 20050048512 (now abandoned), 20050053973 I0087 Conjugated Anti-CD74 Antibodies (now abandoned), 2005.0089932 and 200502.21384 (now 0088. In various embodiments, a conjugated anti-CD74 abandoned), the Examples section of each of which is incor antibody or fragment thereof may be used to prepare an porated herein by reference. immunoconjugate or composition. In certain embodiments, (0094 Phage Display additional amino acid residues may be added to either the N 0.095 Certain embodiments of the claimed compositions or C-terminus of the anti-CD74 antibody or fragment. The and/or methods may concern binding peptides and/or peptide additional amino acid residues may comprise a peptide tag, a mimetics of various target molecules or target-binding mol signal peptide, a cytokine, an enzyme (for example, a pro ecules. Binding peptides may be identified by any method drug activating enzyme), a hormone, a peptide toxin, such as known in the art, including but not limiting to the phage Pseudomonas exotoxin, a peptide drug, a cytotoxic protein or display technique. Various methods of phage display and other functional proteins. As used herein, a functional protein techniques for producing diverse populations of peptides are is a protein that has a biological function. well known in the art. For example, U.S. Pat. Nos. 5.223,409: 0089. In another embodiment, a liposome may be conju 5,622,699 and 6,068,829 disclose methods for preparing a gated to the anti-CD74 antibody or fragment thereof. See, phage library. The phage display technique involves geneti e.g., Ryser et al., Proc. Natl. Acad. Sci. USA, 75:3867-3870, cally manipulating bacteriophage so that Small peptides can 1978, U.S. Pat. No. 4,699,784 and U.S. Pat. No. 4,046,722, be expressed on their surface (Smith and Scott, 1985, Science which are incorporated herein by reference. Conjugation 228:1315-1317; Smith and Scott, 1993, Meth. Enzymol. preferably does not significantly affect the binding specificity 21:228-257). In addition to peptides, larger protein domains or affinity of the anti-CD74 antibody or fragment thereof. In Such as single-chain antibodies may also be displayed on the certain embodiments, the liposome may be covalently or surface of phage particles (Arap et al., 1998, Science 279: non-covalently attached to one or more therapeutic and/or 377-380). diagnostic agents. 0096 Targeting amino acid sequences selective for a given 0090. In one embodiment, drugs, toxins, radioactive com organ, tissue, cell type or target molecule may be isolated by pounds, enzymes, hormones, cytotoxic proteins, chelates, panning (Pasqualini and Ruoslahti, 1996, Nature 380:364 US 2010/0272636 A1 Oct. 28, 2010

366; Pasqualini, 1999, The Quart. J. Nucl. Med. 43:159-162). P(O)R, P(O)OR, CO, or CNR, wherein R is H or alkyl In brief, a library of phage containing putative targeting pep (1-20C) and R' is alkyl (1-20C); in addition, this group may be tides is administered to an intact organism or to isolated attached to adjacent nucleotides through O or S. Not all organs, tissues, cell types or target molecules and samples linkages in an oligomer need to be identical. containing bound phage are collected. Phage that bind to a 0103 Use of Humanized, Chimeric and Human Antibody target may be eluted from a target organ, tissue, cell type or for Treatment and Diagnosis target molecule and then amplified by growing them in host 0104 Compositions and/or immunoconjugates compris bacteria. ing humanized, chimeric or human monoclonal antibodies, 0097. In certain embodiments, the phage may be propa i.e., anti-CD74 antibodies and other antibodies described gated in host bacteria between rounds of panning. Rather than herein, are suitable for use in the therapeutic methods and being lysed by the phage, the bacteria may instead secrete diagnostic methods as described herein. Accordingly, the multiple copies of phage that display a particular insert. If immunoconjugates or compositions may include naked desired, the amplified phage may be exposed to the target humanized, chimeric or human antibodies or may comprise organs, tissues, cell types or target molecule again and col antibodies that have been conjugated to a liposome, a thera lected for additional rounds of panning. Multiple rounds of peutic agent, or a diagnostic agent. The immunoconjugates panning may be performed until a population of selective or may be administered as a multimodal therapy. For example, specific binders is obtained. The amino acid sequence of the additional therapeutic or diagnostic agents may be adminis peptides may be determined by sequencing the DNA corre tered before, simultaneously, or after administration of the sponding to the targeting peptide insert in the phage genome. immunoconjugate or composition. The identified targeting peptide may then be produced as a 0105. The efficacy of the immunoconjugates may be synthetic peptide by Standard protein chemistry techniques enhanced by Supplementing the anti-CD74 binding mol (Arap et al., 1998, Smith et al., 1985). ecules with one or more other binding molecules, (e.g., anti 0098. In some embodiments, a subtraction protocol may bodies to antigens such as carbonic anhydrase IX, CCCL19, be used to further reduce background phage binding. The CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, purpose of Subtraction is to remove phage from the library CD1 1A, CD14, CD15, CD16, CD18, CD19, IGF-1R, CD20, that bind to targets other than the target of interest. In alter CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, native embodiments, the phage library may be prescreened CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, against a control cell, tissue or organ. For example, tumor CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, binding peptides may be identified after prescreening a CD80, CD83, CD95, CD126, CD133, CD138, CD147, library against a control normal cell line. After subtraction the CD154, AFP, PSMA, CEACAM5, CEACAM-6, B7, ED-B of library may be screened against the molecule, cell, tissue or fibronectin, Factor H, FHL-1, Flt-3, folate receptor, GROB, organ of interest. Other methods of subtraction protocols are HMGB-1, hypoxia inducible factor (HIF), HM1.24, insulin known and may be used in the practice of the claimed meth like growth factor-1 (ILGF-1), IFN-y, IFN-C, IFN-B, IL-2, ods, for example as disclosed in U.S. Pat. Nos. 5,840.841, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, 5,705,610, 5,670,312 and 5,492.807. IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, MAGE, mCRP, 0099. Aptamers MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, 0100. In certain embodiments, a targeting moiety of use MUC4, MUC5, PAM4 antigen, NCA-95, NCA-90, Ia, HM1. may be anaptamer. Methods of constructing and determining 24, EGP-1, EGP-2, tenascin, Le(y), RANTES, T101, TAC, the binding characteristics of aptamers are well known in the Tn antigen, Thomson-Friedenreich antigens, tumor necrosis art. For example, such techniques are described in U.S. Pat. antigens, TNF-ct, TRAIL receptor (R1 and R2), VEGFR, Nos. 5,582,981, 5,595,877 and 5,637,459, the Examples sec EGFR, PIGF, complement factors C3, C3a, C3b, C5a, C5, tion of each incorporated herein by reference. Methods for and an oncogene product or HLA-DR, preferably mature preparation and screening of aptamers that bind to particular HLA-DR dimer). Preferred B-cell-associated antigens targets of interest are well known, for example U.S. Pat. No. include CD19, CD20, CD21, CD22, CD23, CD46, CD52, 5,475,096 and U.S. Pat. No. 5,270,163, the Examples section CD74, CD80 and CD5. Preferred T-cell antigens include of each incorporated herein by reference. CD4, CD8 and CD25 (the IL-2 receptor). HLA-DR antigen 0101 Aptamers may be prepared by any known method, can be used in treatment of both B-cell and T-cell disorders. including synthetic, recombinant, and purification methods, Particularly preferred B-cell antigens are CD19, CD22, and may be used alone or in combination with other ligands CD21, CD23, CD74, CD80 and HLA-DR. Particularly pre specific for the same target. In general, a minimum of ferred T-cell antigens are CD4, CD8 and CD25. CD46 is an approximately 3 nucleotides, preferably at least 5 nucle antigen on the Surface of cancer cells that block complement otides, are necessary to effect specific binding. Aptamers of dependent lysis (CDC). Preferred malignant melanoma asso sequences shorter than 10 bases may be feasible, although ciated antigens are MART-1, TRP-1, TRP-2 and gp100. Fur aptamers of 10, 20, 30 or 40 nucleotides may be preferred. ther, preferred multiple myeloma-associated antigens are 0102 Aptamers may be isolated, sequenced, and/or MUC1 and CD38. amplified or synthesized as conventional DNA or RNA mol 0106 The supplemental binding molecule may be naked ecules. Alternatively, aptamers of interest may comprise or conjugated with a liposome, a therapeutic agent, or a diag modified oligomers. Any of the hydroxyl groups ordinarily nostic agent, including drugs, toxins, immunomodulators, present in aptamers may be replaced by phosphonate groups, hormones, enzymes and therapeutic radionuclides. The phosphate groups, protected by a standard protecting group, Supplemental binding molecule may be administered concur or activated to prepare additional linkages to other nucle rently, sequentially, or according to a prescribed dosing regi otides, or may be conjugated to Solid Supports. One or more men, with the anti-CD74 immunoconjugate. phosphodiester linkages may be replaced by alternative link 0107 Further contemplated herein is the administration of ing groups, such as P(O)O replaced by P(O)S, P(O)NR, an immunoconjugate for diagnostic and therapeutic uses in B US 2010/0272636 A1 Oct. 28, 2010 12 cell lymphomas and other disease or disorders. An immuno positions comprising photoactive agents or dyes, and the conjugate is a molecule comprising a binding molecule con present diagnostic/therapeutic methods may include the diag jugated to a liposome. The immunoconjugate may be used to nostic or therapeutic use of anti-CD74 immunoconjugate form a composition that further includes a therapeutic or compositions comprising photoactive agents or dyes. diagnostic agent, which may include a peptide that may bear 0110. Also contemplated is the use of radioactive and non the diagnostic or therapeutic agent. An immunoconjugate radioactive agents as diagnostic agents in the anti-CD74 retains the immunoreactivity of the binding molecule, (i.e., immunoconjugate compositions as described herein. A Suit the antibody moiety has about the same or slightly reduced able non-radioactive diagnostic agent is a contrast agent Suit ability to bind the cognate antigen after conjugation as before able for magnetic resonance imaging, computed tomography conjugation). or ultrasound. Magnetic imaging agents include, for example, 0108. A wide variety of diagnostic and therapeutic non-radioactive metals, such as manganese, iron and gado reagents can be used to form the immunoconjugates and linium, complexed with metal-chelate combinations that compositions as described herein. Therapeutic agents include 2-benzyl-DTPA and its monomethyl and cyclohexyl include, for example, chemotherapeutic drugs such as Vinca analogs, when used along with the antibodies described alkaloids, anthracyclines, epidophyllotoxins, taxanes, anti herein. (See U.S. Ser. No. 09/921.290 filed on Oct. 10, 2001, metabolites, alkylating agents, antibiotics, Cox-2 inhibitors, which is incorporated in its entirety by reference). antimitotics, antiangiogenic and apoptotic agents, particu 0111. Furthermore, the anti-CD74 immunoconjugate larly doxorubicin, methotrexate, taxol. CPT-11, camptoth compositions may include a radioisotope or a positron-emit ecans, and others from these and other classes of anticancer ter useful for diagnostic imaging. Suitable radioisotopes may agents, and the like. Other useful cancer chemotherapeutic include those in the energy range of 60 to 4,000 keV. Suitable drugs for the preparation of immunoconjugates and antibody radioisotopes may include F, Fe, Cu, Cu, 'Cu, 'Ga, fusion proteins include nitrogen mustards, alkyl Sulfonates, Ga, 86Y. 89Zr, 9Tc, 94mTc, 99mTc, In, 123, 1241, 125I. 13 II, nitrosoureas, triaZenes, folic acid analogs, COX-2 inhibitors, and like. pyrimidine analogs, purine analogs, platinum coordination 0112 A toxin, Such as Pseudomonas exotoxin, may also complexes, hormones, and the like. Suitable chemotherapeu be present in the anti-CD74 immunoconjugate compositions tic agents are described in REMINGTON'S PHARMACEU as described herein. For example, the toxin may be com TICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), plexed to or form the therapeutic agent portion of an antibody and in GOODMAN AND GILMANS THE PHARMACO fusion protein of an anti-CD74 antibody described herein. LOGICAL BASIS OF THERAPEUTICS, 7th Ed. (Mac Other toxins include ricin, abrin, ribonuclease (RNase), Millan Publishing Co. 1985), as well as revised editions of DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral these publications. Other Suitable chemotherapeutic agents, protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, Such as experimental drugs, are known to those of skill in the and Pseudomonas endotoxin. (See, e.g., Pastan et al., Cell art 47:641 (1986), and Goldenberg, CA A Cancer Journal for 0109 Additionally, a chelator such as DTPA, DOTA, Clinicians 44:43 (1994)). Additional toxins suitable for use TETA, or NOTA can be conjugated to one or more compo herein are known to those of skill in the art and are disclosed nents of the compositions as described herein. Alternatively, in U.S. Pat. No. 6,077.499, the Examples section of which is a Suitable peptide including a detectable label, (e.g., a fluo incorporated herein by reference. rescent molecule), or a cytotoxic agent, (e.g., aheavy metal or 0113 An immunomodulator, such as a cytokine may also radionuclide), can be covalently, non-covalently, or otherwise be present in the administered anti-CD74 immunoconjugate associated with more components of the compositions as compositions as described herein. For example, an immuno described herein. For example, a therapeutically useful modulator may be conjugated to, or form the therapeutic immunoconjugate can be obtained by incorporating a photo agent portion of an antibody fusion protein or be administered active agent or dye in the composition as described herein. as part of the anti-CD74 immunoconjugate compositions as Fluorescent compositions, such as fluorochrome, and other described herein. Suitable cytokines include, but are not lim chromogens, or dyes, such as porphyrins sensitive to visible ited to, interferons and interleukins, as described below. light, have been used to detect and to treatlesions by directing 0114 Preparation of Immunoconjugates the suitable light to the lesion. In therapy, this has been termed 0115 The immunoconjugates described hereincan be pre photoradiation, phototherapy, or photodynamic therapy (Joni pared by known methods of linking antibodies with lipids, et al. (eds.), PHOTODYNAMIC THERAPY OF TUMORS carbohydrates, protein, or other atoms and molecules. For AND OTHER DISEASES (Libreria Progetto 1985); van den example, the binding molecules described herein can be con Bergh, Chem. Britain 22:430 (1986)). Moreover, monoclonal jugated with a liposome to form an immunoconjugate. In antibodies have been coupled with photoactivated dyes for certain embodiments, the immunoconjugate can incorporate achieving phototherapy. Mew et al., J. Immunol. 130:1473 a therapeutic or diagnostic agent either covalently, non-co (1983); idem. Cancer Res. 45:4380 (1985); Oseroff et al., valently. Further, any of the binding molecules described Proc. Natl. Acad. Sci. USA 83:8744 (1986); idem. Photo herein can be further conjugated with one or more therapeutic chem. Photobiol. 46:83 (1987); Hasan et al., Prog. Clin. Biol. or diagnostic agents described herein. Generally, one thera Res. 288:471 (1989); Tatsuta et al., Lasers Surg. Med. 9:422 peutic or diagnostic agent may be attached to each binding (1989); Pelegrin et al., Cancer 67:2529 (1991). Endoscopic molecule but more than one therapeutic agent or diagnostic applications are also contemplated. Endoscopic methods of agent can be attached to the same binding molecule. The detection and therapy are described in U.S. Pat. Nos. 4,932, antibody fusion proteins contemplated herein comprise two 412; 5,525,338; 5,716,595;5,736,119; 5,922,302; 6,096,289; or more antibodies or fragments thereof and each of the and 6,387.350, the Examples section of each of which is antibodies that comprises this fusion protein may be conju incorporated herein by reference. Thus, contemplated herein gated with a liposome. Additionally, one or more of the anti is the therapeutic use of anti-CD74 immunoconjugate com bodies of the antibody fusion protein may have one or more US 2010/0272636 A1 Oct. 28, 2010 therapeutic of diagnostic agent attached. Further, the thera No. 6,254,868, the Examples section of each incorporated peutic do not need to be the same but can be different thera herein by reference). The engineered carbohydrate moiety peutic agents. For example, the compositions described may be used to attach a liposome or a therapeutic or diagnos herein may include a drug and a radioisotope. tic agent. 0116 For example, an IgG can be radiolabeled with 13 II I0120 Liposomes and conjugated to a lipid, such that the IgG-lipid conjugate I0121 The formation of liposomes and micelles is known can form a liposome. The liposome may incorporate one or in the art. (See, e.g., Wrobel et al., Biochimica et Biophysica more therapeutic or diagnostic agents, (e.g., a drug Such as Acta, 1235:296 (1995); Lundberg et al., J. Pharm. Pharma FUdR-dO). Alternatively, in addition to the liposome, the IgG col. 51:1099-1105 (1999); Lundberg et al., Int. J. Pharm., may be conjugated to 'I (e.g., at a tyrosine residue) and a 205:101-108 (2000); Lundberg, J. Pharm. Sci., 83:72-75 drug (e.g., at the epsilon amino group of alysine residue), and (1994); Xu et al., Molec. Cancer Ther., 1:337-346 (2002); the liposome may incorporate an additional therapeutic or Torchilin et al., Proc. Natl. Acad. Sci., 100:6039-6044 diagnostic agent. Therapeutic and diagnostic agents may be (2003). See also U.S. Pat. No. 5,565,215; U.S. Pat. No. 6,379, covalently associated with the binding molecule, (e.g., con 698; and U.S. Pat. No. 6,858,226, the Examples section of jugated to reduced disulfide groups, carbohydrate side chains, each of which is incorporated herein by reference). or any other reactive group on the binding molecule. How 0.122 Immunoliposomes ever, the skilled artisan will realize that the liposome-conju I0123. The conjugation of antibodies or binding molecules gated antibodies may be used without any additional thera to liposomes to form a targeted carrier for therapeutic or peutic or diagnostic agents to induce apoptosis, as further diagnostic agents has been described. (See, e.g., Bendas, described in the Examples below. Biodrugs, 15:215-224 (2001); Xu et al., Molec. Cancer Ther. 0117. A liposome, therapeutic agent, or diagnostic agent 1:337-346 (2002); Torchilin et al., Proc. Natl. Acad. Sci., can be attached at the hinge region of a reduced antibody 100:6039-6044 (2003); Bally, et al., J. Liposome Res., 8:299 component via disulfide bond formation. As an alternative, 335 (1998); Lundberg, Int. J. Pharm., 109:73-81 (1994); Lun peptides or other molecules containing reactive moieties can dberg, J. Pharm. Pharmacol. 49:16-21 (1997); Lundberg, be attached to an antibody component using a heterobifunc Anti-cancer Drug Design, 13:453-461 (1998). See also U.S. tional cross-linker, such as N-succinyl 3-(2-pyridyldithio) Pat. Nos. 6,306,393 and 7,074,403: U.S. Ser. No. 10/350,096; proprionate (SPDP). Yu et al., Int. J. Cancer 56: 244 (1994). U.S. Ser. No. 60/138,284, filed Jun. 9, 1999, the Examples General techniques for Such conjugation are well known in section of each of which is incorporated herein by reference.) the art. (See, e.g., Wong, CHEMISTRY OF PROTEIN CON Further details concerning exemplary methods for conjuga JUGATION AND CROSS-LINKING (CRC Press 1991); tion of antibodies or binding molecules to liposomes are Upeslacis et al., “Modification of Antibodies by Chemical disclosed in the Examples below. Methods, in MONOCLONAL ANTIBODIES: PRIN 0.124 Pharmaceutically Acceptable Excipients CIPLES AND APPLICATIONS, Birch et al. (eds.), pages 0.125 The immunoconjugates or compositions may 187-230 (Wiley-Liss, Inc. 1995); Price, “Production and include one or more pharmaceutically suitable excipients, Characterization of Synthetic Peptide-Derived Antibodies.” one or more additional ingredients, or some combination of in MONOCLONAL ANTIBODIES: PRODUCTION, these. ENGINEERING AND CLINICAL APPLICATION, Ritteret 0.126 The immunoconjugate or compositions disclosed al. (eds.), pages 60-84 (Cambridge University Press 1995)). herein can be formulated according to known methods to Alternatively, the liposome, therapeutic agent, or diagnostic prepare pharmaceutically useful compositions, whereby the agent can be conjugated via a carbohydrate moiety in the Fc immunoconjugate or compositions are combined in a mixture region of an antibody. The carbohydrate group can be used to with a pharmaceutically suitable excipient. Sterile phos increase the loading of the same peptide that is bound to a phate-buffered saline is one example of a pharmaceutically thiol group, or the carbohydrate moiety can be used to bind a suitable excipient. Other suitable excipients are well known different peptide. to those in the art. (See, e.g., Ansel et al., PHARMACEUTI 0118 Methods for conjugating peptides to antibody com CAL DOSAGE FORMS AND DRUG DELIVERY SYS ponents via an antibody carbohydrate moiety are well known TEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.), to those of skill in the art. (See, e.g., Shih et al., Int. J. Cancer REMINGTON'S PHARMACEUTICAL SCIENCES, 18th 41: 832 (1988); Shihet al., Int. J. Cancer 46: 1101 (1990); and Edition (Mack Publishing Company 1990), and revised edi Shih et al., U.S. Pat. No. 5,057,313, all of which are incorpo tions thereof. rated in their entirety by reference). Similar chemistry can be 0127. The immunoconjugate or compositions disclosed used to conjugate one or more anti-CD74 binding molecules herein can be formulated for intravenous administration via, to one or more liposomes, therapeutic agents, or diagnostic for example, bolus injection or continuous infusion. Formu agents. The general method involves reacting an antibody lations for injection can be presented in unit dosage form, e.g., component having an oxidized carbohydrate portion with a in ampules or in multi-dose containers, with an added preser liposome that has at least one free amine function and that is Vative. The compositions can take Such forms as Suspensions, loaded with a plurality of peptide. This reaction results in an Solutions or emulsions in oily or aqueous vehicles, and can initial Schiffbase (imine) linkage, which can be stabilized by contain formulatory agents such as Suspending, stabilizing reduction to a secondary amine to form the final conjugate. and/or dispersing agents. Alternatively, the active ingredient 0119) The Fc region may be absent if the anti-CD74 bind can be in powderform for constitution with a suitable vehicle, ing molecule is an antibody fragment. However, it is possible e.g., sterile pyrogen-free water, before use. to introduce a carbohydrate moiety into the light chain vari I0128. Additional pharmaceutical methods may be able region of a full-length antibody or antibody fragment. employed to control the duration of action of the therapeutic (See, e.g., Leung et al., J. Immunol. 154: 5919 (1995); Hansen or diagnostic conjugate or naked antibody. Control release et al., U.S. Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat. preparations can be prepared through the use of polymers to US 2010/0272636 A1 Oct. 28, 2010 complex or adsorb the immunoconjugate or naked antibody. bucil and cyclophosphamide), purine analogs (e.g., fludara For example, biocompatible polymers include matrices of bine), and most recently monoclonal antibodies (mAb). Anti poly(ethylene-co-vinyl acetate) and matrices of a polyanhy bodies such as rituximab that target the CD20 antigen dride copolymer of a stearic acid dimer and sebacic acid. selectively expressed on CLL cells augment the cytotoxicity Sherwoodet al., Bio/Technology 10: 1446 (1992). The rate of of traditional chemotherapy agents, and are associated with release of an immunoconjugate or antibody from Such a improved response rates and progression-free Survival matrix depends upon the molecular weight of the immuno (Robak et al., ASHAnnual Meeting Abstracts. Nov. 16, 2008; conjugate or antibody, the amount of immunoconjugate or 112(11):lba; Hallek et al., ASH Annual Meeting Abstracts. antibody within the matrix, and the size of dispersed particles. Nov. 16, 2008 2008; 112(11):325; Keating et al., J Clin Saltzman et al., Biophys.J. 55: 163 (1989); Sherwood et al., Oncol. 2005, 23:4079-4088: Byrd et al., Blood, 2005, 105: Supra. Other solid dosage forms are described in Ansel et al., 49-53). However, nearly all patients eventually relapse after PHARMACEUTICAL DOSAGE FORMS AND DRUG Such treatments, indicating a need for novel and specific DELIVERY SYSTEMS. 5th Edition (Lea & Febiger 1990), therapeutic agents for CLL. and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL I0134) CD74 is a type II transmembrane protein expressed SCIENCES, 18th Edition (Mack Publishing Company 1990), on B cells which has recently been pursued as a target for and revised editions thereof. antibody mediated therapy (Stein et al., Clin Cancer Res. 0129. The immunoconjugate or compositions may also be 2007, 13:5556s-5563s). It associates with the C. and B chains administered to a mammal Subcutaneously or even by other of HLA-DR, and normally functions as an MHC class II parenteral routes. Moreover, the administration may be by chaperone. However, signaling through CD74 is also impli continuous infusion or by single or multiple boluses. In gen cated in B-cell proliferation, NF-kB activation, and cell sur eral, the dosage of an administered immunoconjugate, fusion vival (Binsky et al., Proc Natl Acad Sci USA. 2007, 104: protein or naked antibody for humans will vary depending 13408-13413: Starlets et al., Blood, 2006, 107:48.07-4816). upon Such factors as the patient's age, weight, height, sex, CD74 expression is increased on the surface of leukemic B general medical condition and previous medical history. cells, making it an attractive target for CLL and other B-cell Typically, it is desirable to provide the recipient with a dosage malignancies. CD74 signaling is initiated following engage of immunoconjugate or composition including the immuno ment with macrophage migration-inhibitory factor (MIF), conjugate that is in the range of from about 1 mg/kg to 20 and Subsequent activation of Survival pathways to inhibit mg/kg as a single intravenous infusion, although a lower or apoptosis and stimulate proliferation (Gore et al., J Biol higher dosage also may be administered as circumstances Chem, 2008, 283:2784-2792: Leng et al., J Exp Med. 03, dictate. This dosage may be repeated as needed, for example, 7:1467-1476). Additionally, a recent study demonstrates that once per week for 4-10 weeks, preferably once per week for CD74 signaling induces Tap63 and VLA-3 to enhance CLL 8 weeks, and more preferably, once per week for 4 weeks. It cell survival and homing to the bone marrow (Binsky et al., J may also be given less frequently, such as every other week Immunol Mar. 31, 2010 epub). Therefore, disruption of CD74 for several months. The dosage may be given through various signaling represents a potential therapeutic option in CLL and parenteral routes, with appropriate adjustment of the dose and other CD74-expressing malignancies (Stein et al., Clin Can schedule. cer Res, 2007, 13:5556s-5563s). 0130 For purposes of therapy, the immunoconjugate, or 0.135 Described herein is an antagonistic humanized anti composition including the immunoconjugate, is administered body to CD74, milatuzumab (hLL1). Milatuzumab has dem to a mammal in a therapeutically effective amount. A suitable onstrated anti-proliferative activity in non-Hodgkin lym Subject for the therapeutic and diagnostic methods disclosed phoma (NHL) and multiple myeloma (MM) cell lines and herein is usually a human, although a non-human animal extended the survival of SCID mice injected with NHL and Subject is also contemplated. An antibody preparation is said MM cells (Stein et al., Clin Cancer Res, 2007, 13:5556s to be administered in a “therapeutically effective amount” if 5563s; Starlets et al., Blood, 2006, 107:48.07-4816; Griffiths the amount administered is physiologically significant. An et al., Clin Cancer Res, 2003,9:6567-6571). However, little is agent is physiologically significant if its presence results in a known about the effect of milatuzumab in CLL. The detectable change in the physiology of a recipient mammal. Examples below demonstrate that milatuZumab mediates In particular, an antibody preparation is physiologically sig direct cytotoxicity in CLL cells by a mechanism involving nificant if its presence invokes an antitumor response or miti aggregation of CD74 on the cell Surface. Furthermore, incor gates the signs and symptoms of an autoimmune disease state. poration of milatuZumab into a liposome potentiates the cyto A physiologically significant effect could also be the evoca toxic effect of this antibody, Suggesting a novel therapeutic tion of a humoral and/or cellular immune response in the formulation. recipient mammal. 0.136 Methods of Treatment 0131 Chronic Lymphocytic Leukemia 0.137 Contemplated herein is the use of immunoconju 0.132. In a preferred embodiment, the compositions or gates or compositions including immunoconjugates for treat immunoconjugates described herein are of use for therapy of ment of a CD74 expressing disease. The disease or disorder chronic lymphocytic leukemia (CLL). CLL is a presently may be selected from the group consisting of cancer, neopla incurable progressive disease for which new therapies are sia, an immune dysregulation disease, an autoimmune dis required. Therapy with monoclonal antibodies (mAb) has ease, organ graft rejection, and graft versus host disease. The improved the outcome of patients with CLL, making further CD74 expressing disease may be selected from the group investigation of novel antibodies directed against alternative consisting of a Solid tumor, non-Hodgkin’s lymphoma, and specific targets on B cells an important area of research. Hodgkin's lymphoma, multiple myeloma, a B-cell disease 0133. CLL is the most common adult leukemia, and is a and/or a T-cell disease. The solid tumor may be selected from progressive and incurable disease. Present CLL treatments the group consisting of a melanoma, carcinoma and sarcoma. include alkylating chemotherapeutic drugs (e.g., chloram The carcinoma may be selected from the group consisting of US 2010/0272636 A1 Oct. 28, 2010

a renal carcinoma, lung carcinoma, intestinal carcinoma and TRAIL receptor (R1 and R2), VEGFR, EGFR, PIGF, comple stomach carcinoma. The B-cell disease may be selected from ment factors C3, C3a, C3b, C5a, C5, and an oncogene prod the group consisting of indolent forms of B-cell lymphomas, uct. aggressive forms of B-cell lymphomas, chronic lymphatic 0.139. Also contemplated herein is the treatment of a dis leukemias, acute lymphatic leukemias, multiple myeloma, ease comprising administering to a subject with a CD74 anti B-cell disorders and other diseases. In particular, the compo gen-positive disease other than lymphoma or leukemia, a sitions described herein are particularly useful for treatment therapeutic composition that includes: (1) an immunoconju of various autoimmune as well as indolent forms of B-cell gate of an anti-CD74 binding molecule and a liposome; (2) lymphomas, aggressive forms of B-cell lymphomas, chronic optionally, an effector moiety; and (3) a pharmaceutically lymphatic leukemias, acute lymphatic leukemias, multiple acceptable excipient. The immunoconjugate or composition myeloma, and Waldenstrom's macroglobulinemia. For may be administered intravenously, intramuscularly or Sub example, humanized anti-CD74 antibody components and cutaneously at a dose of 20-5000 mg. Further, the immuno immunoconjugates can be used to treat both indolent and conjugate may be administered before, simultaneously with, or after the administration of at least one additional therapeu aggressive forms of non-Hodgkin's lymphoma. tic agent or diagnostic agent. Therapeutic agents, as described 0138 More specifically, the method for treating a B-cell above and throughout the specification, may include an disease may include administering to a Subject with a B-cell immunomodulator, a hormone, a cytotoxic agent, or a binding related disease, a therapeutic composition comprising an molecule (either naked or conjugated to at least one immu immunoconjugate including an anti-CD74 binding molecule, nomodulator, radioactive label, enzyme, hormone, cytotoxic (e.g., a humanized, chimeric, or human anti-CD74 antibody agent, antisense oligonucleotide, or a combination thereof, or fragment thereof or antibody fusion protein thereof), a where the immunomodulator preferably is a cytokine and the pharmaceutically acceptable carrier, and optionally a thera cytotoxic agent preferably is a drug or toxin). A therapeutic peutic agent, wherein the B-cell disease is a lymphoma or agent or diagnostic agent may include the compositions or leukemia. More specifically, the B-cell disease may be immunoconjugates as disclosed herein. When an antibody is selected from indolent forms of B-cell lymphomas, aggres administered in combination with the therapeutic and/or sive forms of B-cell lymphomas, multiple myeloma, chronic diagnostic composition to treat a malignancy that is not a lymphatic leukemias, or acute lymphatic leukemias. The B-cell malignancy, it should be reactive with a tumor marker immunoconjugate or composition comprising the immuno other than CD74, which is expressed by the cells that com conjugate may be administered intravenously, intramuscu prise the malignancy that is treated, and the antibody should larly or subcutaneously at a dose of 20-2000 mg. The method be formulated in a pharmaceutically acceptable vehicle. may further comprise administering the immunoconjugate or Examples of antibodies that can be administered for malig nant melanoma associated antigens are those antibodies reac composition before, simultaneously with, or after the admin tive with MART-1, TRP-1, TRP-2 and gp100. Further, pre istration of at least one additional therapeutic agent or diag ferred antibodies to multiple myeloma-associated antigens nostic agent used to treat the B-cell disease. The additional are those reactive with MUC1 and CD38. agent may include an additional immunoconjugate as 0140. The compositions for treatment contain at least one described herein, including a therapeutic or diagnostic agent. immunoconjugate, which typically includes a humanized, A therapeutic agent may be selected from the group consist chimeric or human monoclonal anti-CD74 antibody alone or ing of a naked antibody, an immunomodulator, a hormone, a in combination with other antibodies. Such as other human cytotoxic agent, an enzyme, and/oran antibody conjugated to ized, chimeric, or human antibodies. In particular, combina at least one immunomodulator, radioactive label, hormone, tion therapy wherein the immunoconjugate includes a fully enzyme, or cytotoxic agent, or a combination thereof. The human antibody is also contemplated. immunomodulator preferably is a cytokine and the cytotoxic 0.141. The compositions also may include an immuno agent preferably is a drug or toxin. The antibody that is modulator as an effector. As used herein, the term “immuno administered in combination as a naked antibody or as a modulator” includes may be selected from a cytokine, a stem Supplemental immunoconjugate preferably is reactive with cell growth factor, a lymphotoxin, an hematopoietic factor, a an antigen selected from the group consisting of carbonic colony stimulating factor (CSF), an interferon (IFN), eryth anhydrase IX, CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, ropoietin, thrombopoietin and a combination thereof. Spe CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18. cifically useful are lymphotoxins such as tumor necrosis fac CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, tor (TNF), hematopoietic factors, such as interleukin (IL), CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, colony stimulating factor, Such as granulocyte-colony stimu CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, lating factor (G-CSF) or granulocyte macrophage-colony CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, stimulating factor (GM-CSF), interferon, such as interferons CD138, CD147, CD154, AFP, PSMA, CEACAM5, C.-Bor-Y, and stem cell growth factor, Such as that designated CEACAM-6, B7, ED-B of fibronectin, Factor H, FHL-1, “S1 factor. Included among the cytokines are growth hor Flt-3, folate receptor, GROB, HMGB-1, hypoxia inducible mones such as human growth hormone, N-methionyl human factor (HIF), HM1.24, insulin-like growth factor-1 (ILGF-1), growth hormone, and bovine growth hormone; parathyroid IFN-y, IFN-C, IFN-?3, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, hormone; thyroxine; insulin; proinsulin; relaxin, prorelaxin; IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, glycoprotein hormones Such as follicle stimulating hormone IL-25, IP-10, MAGE, mCRP MCP-1, MIP-IA, MIP-1B, (FSH), thyroid stimulating hormone (TSH), and luteinizing MIF, MUC1, MUC2, MUC3, MUC4, MUC5, PAM4 antigen, hormone (LH), hepatic growth factor, prostaglandin, fibro NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR, blast growth factor; prolactin; placental lactogen, OB protein; tenascin, Le(y), RANTES, T101, TAC. Tn antigen, Thom tumor necrosis factor-C. and -3; mullerian-inhibiting Sub son-Friedenreich antigens, tumor necrosis antigens, TNF-C. stance; mouse gonadotropin-associated peptide; inhibin; US 2010/0272636 A1 Oct. 28, 2010

activin; vascular endothelial growth factor; integrin, throm tered with an antibody, immunoconjugate or fusion protein. bopoietin (TPO); nerve growth factors such as NGF-?3; plate The cytokines, chemotherapeutic drugs and antibody or let-growth factor; transforming growth factors (TGFs) such immunoconjugate can be administered in any order, or as TGF-C. and TGF-B; insulin-like growth factor-I and -II; together. erythropoietin (EPO); osteoinductive factors; interferons 0144. In a preferred embodiment, NHL is treated with 4 Such as interferon-C, -f, and -y, colony Stimulating factors weekly infusions of a humanized anti-CD74 immunoconju (CSFs) such as macrophage-CSF (M-CSF); interleukins gate (e.g., atherapeutic emulsion) at a dose of 200-400 mg/m (ILs) such as IL-1, IL-1C., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, weekly for 4 consecutive weeks or every-other week (iv over IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, 2-8 hours), repeated as needed over next months/yrs. Also IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand or FLT-3, preferred, NHL is treated with 4 semi-monthly infusions as angiostatin, thrombospondin, endostatin, tumor necrosis fac above, but combined with epratuzumab (anti-CD22 human tor and LT. As used herein, the term cytokine includes pro ized antibody) on the same days, at a dose of 360 mg/m. teins from natural sources or from recombinant cell culture given as an iv infusion over 1 hour, either before, during or and biologically active equivalents of the native sequence after the anti-CD74 immunoconjugate infusion. Still pre cytokines. The immunomodulator may be present in the com ferred, NHL is treated with 4 weekly infusions of the anti position, or alternatively, the immunomodulator can be CD74 immunoconjugate as above, combined with one or administered before, simultaneously with, or after adminis more injections of CD22 antibody radiolabeled with a thera tration of the therapeutic and/or diagnostic compositions. As peutic isotope such as yttrium-90 (at dose of 'Y between 5 discussed Supra, the anti-CD74 antibody may also be conju and 35 mCi/meter-square) as one or more injections over a gated to the immunomodulator. The immunomodulator may period of weeks or months. also be conjugated to a hybrid antibody consisting of one or 0145. In addition, a therapeutic composition as contem more antibodies binding to different antigens. plated herein can contain a mixture or hybrid molecules of 0142 Multimodal therapies contemplated herein further monoclonal anti-CD74 immunoconjugates directed to differ include immunotherapy with immunoconjugates that include ent, non-blocking CD74 epitopes. Accordingly, contem anti-CD74 binding molecules supplemented with administra plated herein are therapeutic compositions comprising a mix tion of additional binding molecules, (e.g., anti-CD22, anti ture of monoclonal anti-CD74 immunoconjugates that bind at CD19, anti-CD21, anti-CD20, anti-CD80, anti-CD23, anti least two CD74 epitopes. Additionally, the immunoconju CD46 or HLA-DR, preferably the mature HLA-DR dimer gates described herein may contain a mixture of anti-CD74 antibodies in the form of naked antibodies, fusion proteins, or antibodies with varying CDR sequences. as immunoconjugates). Further, a liposome as described 0146 AS discussed Supra, the immunoconjugates can be herein, may include binding molecules in addition to anti used for treating B cell lymphoma and leukemia, and other B CD74 binding molecules. Useful antibodies may be poly cell diseases or disorders as well as other malignancies in clonal, monoclonal, chimeric, human or humanized antibod which affected or associated malignant cells are reactive with ies that recognize at least one epitope on the above-noted CD74. For example, anti-CD74 immunoconjugates can be antigenic determinants. For example, anti-CD19 and anti used to treat immune dysregulation disease and related CD22 antibodies are known to those of skill in the art. (See, autoimmune diseases, including Class-III autoimmune dis e.g., Ghetie et al., Cancer Res. 48:2610 (1988); Hekman et al., eases Such as immune-mediated thrombocytopenias. Such as Cancer Immunol. Immunother. 32:364 (1991); Longo, Curr. acute idiopathic thrombocytopenic purpura and chronic idio Opin. Oncol. 8:353 (1996) and U.S. Pat. Nos. 5,798,554 and pathic thrombocytopenic purpura, dermatomyositis, 6,187,287.) Sjogren's syndrome, multiple Sclerosis, Sydenham's chorea, 0143. In another form of multimodal therapy, subjects myasthenia gravis, systemic lupus erythematosus, lupus receive anti-CD74 immunoconjugates, in conjunction with nephritis, rheumatic fever, polyglandular syndromes, bullous standard cancer chemotherapy. For example, "CVB (1.5 pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, g/m cyclophosphamide, 200-400 mg/metoposide, and 150 post-Streptococcal nephritis, erythema nodosum, Takayasu's 200 mg/m carmustine) is a regimen used to treat non arteritis, Addison's disease, rheumatoid arthritis, Sarcoidosis, Hodgkin's lymphoma. Patti et al., Eur. J. Haematol. 51: 18 ulcerative colitis, erythema multiforme, IgA nephropathy, (1993). Other suitable combination chemotherapeutic regi polyarteritis nodosa, ankylosing spondylitis, Goodpasture's mens are well known to those of skill in the art. (See, e.g., syndrome, thromboangitis ubiterans, primary biliary cirrho Freedman et al., “Non-Hodgkin's Lymphomas, in CANCER sis, Hashimoto's thyroiditis, thyrotoxicosis, Scleroderma, MEDICINE, VOLUME 2, 3rd Edition, Holland et al. (eds.), chronic active hepatitis, polymyositisfdermatomyositis, pages 2028-2068 (Lea & Febiger 1993)). As an illustration, polychondritis, pamphigus Vulgaris, Wegener's granuloma first generation chemotherapeutic regimens for treatment of tosis, membranous nephropathy, amyotrophic lateral sclero intermediate-grade non-Hodgkin’s lymphoma (NHL) sis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious include C-MOPP (cyclophosphamide, Vincristine, procarba anemia, rapidly progressive glomerulonephritis and fibrosing zine and prednisone) and CHOP (cyclophosphamide, doxo alveolitis. rubicin, Vincristine, and prednisone). A useful second-gen 0147 In particular, immunoconjugates including human eration chemotherapeutic regimen is m-BACOD ized, chimeric or human anti-CD74 antibodies or fragments (methotrexate, bleomycin, doxorubicin, cyclophosphamide, thereof or antibody fusion proteins thereof are administered Vincristine, dexamethasone and leucovorin), while a Suitable to a Subject with one or more of these autoimmune diseases. third generation regimen is MACOP-B (methotrexate, doxo The anti-CD74 immunoconjugates disclosed herein are par rubicin, cyclophosphamide, Vincristine, prednisone, bleomy ticularly useful in the method of treating autoimmune disor cin and leucovorin). Additional useful drugs include phenyl ders, disclosed in U.S. Pat. No. 7,074,403, the Figures and butyrate and bryostatin-1. In a preferred multimodal therapy, Examples section of which is incorporated herein by refer both chemotherapeutic drugs and cytokines are co-adminis ence. Preferably the anti-CD74 immunoconjugate is admin US 2010/0272636 A1 Oct. 28, 2010

istered intravenously, intramuscularly or Subcutaneously at a and wherein the liposome can be detected by an ultrasound dose of 20-5000 mg. Further, the anti-CD74 immunoconju scanning device. As such, the immunoconjugate may form a gate may be administered before, during or after the admin liposome, and/or the diagnostic agent may comprise a second istration of at least one therapeutic agent or diagnostic agent. liposome. The therapeutic agent, as described above and throughout the 0150. The internalization of the immunoconjugate into specification, may include an antibody, an immunomodula target cells can be followed by fluorescence labeling, essen tor, a hormone, an enzyme, a cytotoxic agent, an antibody tially according to the procedure of Pirker et al., J. Clin. conjugated to at least one immunomodulator, radioactive Invest. 76: 1261 (1985). Further, a method for screening/ label, hormone, enzyme, or cytotoxic agent, antisense oligo diagnosing bone cancers as described in Juweid et al., 1999, nucleotide or a combination thereof, where the immuno could benefit from the immunoconjugates disclosed herein. modulator may be a cytokine and said cytotoxic agent may be Accordingly, a method comprising '"Tc-labeled humanized a drug or toxin. The therapeutic agent may include an immu or chimeric anti-CD74 antibody immunoconjugates is con noconjugate as described herein. Antibodies that may be templated. administered in combination as a naked antibody or as a Supplemental immunoconjugate include antibodies that react EXAMPLES with carbonic anhydrase IX, B7, CCCL19, CCCL21, CSAp, HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, Example 1 CD1 1A, CD14, CD15, CD16, CD18, CD19, CD20 (e.g., Preparation of Anti-CD74 Immunoliposomes Carry C2B8, hA20, 1 F5 MAbs), CD21, CD22, CD23, CD25, ing FUdR-dO CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, 0151 Triolein (TO), egg phosphatidylcholine (EPC), CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, dipalmitoyl phosphatidylethanolamine (DPPE), cholesterol CD133, CD138, CD147, CD154, CEACAM5, CEACAM-6, (CHOL), 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS), poly alpha-fetoprotein (AFP), VEGF (e.g. AVASTINR), fibronec oxyethylenesorbitan monooleate (sorbitan 80), methoxy tin splice variant), ED-B fibronectin (e.g., L19), EGP-1, polyethyleneglycol (mean mol. wt 2000), oleoyl chloride, EGP-2 (e.g., 17-1A), EGF receptor (ErbB1) (e.g., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro ERBITUX(R), ErbB2, ErbB3, Factor H, FHL-1, Flt-3, folate mide (MTT) and DL-dithiotreitol (DTT) were obtained from receptor, Ga 733, GROB, HMGB-1, hypoxia inducible factor Sigma Chemical Co. (St. Louis, Mo.). Poly(ethylene glycol)- (HIF), HM 1.24. HER-2/neu, insulin-like growth factor maleimide-N-hydroxy-1-succinimidyl eSter (MAL (ILGF), IFN-y, IFN-C, IFN-B, IL-2R, IL-4R, IL-6R, IL-13R, PEGooo-NHS) was purchased from Shearwater Polymers IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, Europe (Enschede. The Netherlands). HCholesteryl oleoyl IL-17, IL-18, IL-25, IP-10, IGF-1R, Ia, HM1.24, ganglio ether (COE) and 'Cldipalmitoyl phosphatidylcholine were sides, HCG, the HLA-DR antigen to which L243 binds, obtained from Amersham International plc (Amersham, UK). CD66 antigens, i.e., CD66a-d or a combination thereof, A PEGoo derivative of DPPE with a maleimide group at the MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, macrophage distal terminus of the PEG chain (DPPE-PEG-MAL) was migration-inhibitory factor (MIF), MUC1, MUC2, MUC3, synthesized by reacting 25 umol NHS-PEG-MAL with 23 MUC4, MUC5, placental growth factor (PIGF), PSA (pros umol DPPE and 50 umol triethylamine in chloroform for 6 h tate-specific antigen), PSMA, pancreatic cancer mucin, at 40°C. The product was purified by preparative silica gel PAM4 antigen, NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1, TLC. 3',5'-O-dioleoyl-FUdR (FUdR-dO) was synthesized by Le(y), mesothelin, S100, tenascin, TAC. Tnantigen, Thomas adding 20 umol oleoyl chloride and 50 ul N,N-diisopropyl Friedenreich antigens, tumor necrosis antigens, tumorangio ethylamine to 10 Limol FUdR in dimethylacetamide. The genesis antigens, TNF-C. TRAIL receptor (R1 and R2), mixture was incubated overnight at 40°C. and then water was VEGFR, RANTES, T101, as well as cancer stem cell anti added to the mixture and the fatty acid derivative of FUdR gens, complement factors C3, C3a, C3b, C5a, C5, an onco was extracted with chloroform. The prodrug was purified by gene product and mature HLA-DR, preferably a mature preparative silica gel TLC with chloroform/methanol (95:5) HLA-DR dimer, formulated in a pharmaceutically acceptable as eluent. vehicle. 0152 The Burkitt's lymphoma cell lines, Raji and Ramos, 0148 Method of Diagnosis Jurkat acute lymphoblastic leukemia T-cells and HL-60 0149. Also provided is a method of diagnosing a disease in myelomonocytic leukemia cells, obtained from American a Subject, diagnosed with or Suspected of having at least one Type Culture Collection (Rockville, Md.), were grown in of the diseases selected from the groups consisting of lym RPMI 1640 medium with 10% heat-inactivated fetal calf phoma, leukemia, myeloma, other CD74-expressing malig serum. Cells were maintained at 37° C. and gassed with 5% nancies, immune dysregulation disease, autoimmune disease CO in air. and a combination thereof, comprising administering to said 0153. The anti-CD74. Ab, LL1, was obtained from Immu Subject a diagnostically effective amount of a composition nomedics, Inc. (Morris Plains, N.J.). It was labeled with fluo that includes (1) an immunoconjugate including at least one rescein (FITC) for quantitation. anti-CD74 binding molecule conjugated to a liposome, (2) a 0154 Submicron lipid emulsions were prepared as diagnostic agent, and (3) a pharmaceutically acceptable described in detail elsewhere. (See, Lundberg, J. Pharm. Sci., excipient. The diagnostic agent may be covalently, non-co 83:72-75 (1994); Lundberget al., Int. J. Pharm., 134:119-127 Valently, or otherwise associated with one or more compo (1996)). The composition of the drug-loaded emulsions was nents of the composition. A useful diagnostic agent may TO, EPC, polysorbate 80, DPPE-PEG-MAL, FUdR-dO include a radioisotope, wherein the photons of the radioiso 2:2:0.8:0.6:0.3 (w/w). The components were dispensed into tope are detected by radioscintigraphy or PET, or a metal that vials from Stock solutions and the solvent was evaporated to can be detected by MRI, or a liposome or gas filled liposome, dryness under reduced pressure. Phosphate-buffered saline US 2010/0272636 A1 Oct. 28, 2010

(PBS) was added and the mixture was heated to 50° C., vortex COE, were taken up about 50 times faster than unconjugated mixed for 30s, and sonicated with a Branson probe sonicator emulsions by Raji cells in culture (not shown). The fast and for 2 min. massive uptake of immunoemulsions is demonstrated by the 0155 Drug loaded liposomes were composed of EPC, fact that under standard incubation conditions about 30% of DPPE-PEG-MAL FUdR-dO 1:0.2:0.1 (w/w). In experi the added preparation was associated with cells after 24h (not ments involving HPTS-encapsulated liposomes the compo shown). The corresponding association of emulsions without sition was EPC, CHOL, DPPE-PEG2000-MAL 2:0.5:0.4. coupled Ab was about 0.6% (not shown). The uptake values When required, the lipids were labeled with trace amounts of for Ramos cells were considerably lower but still about 30 HICOE. Dried lipid films were hydrated in 25 mM HEPES times higher for LL1-complexes than for uncomplexed emul and 140 mMNaCl buffer (pH 7.4), (containing 35 mMHPTS sions (not shown). The time-dependent association of tar when appropriate) Subjected to five freezing-thawing cycles geted liposomes was fairly linear up to 24-h, but at prolonged and Subsequent Sonication for 2 min with a Branson probe incubation times the curve declined (not shown). Sonicator. The phospholipid concentration was quantitated by 'CDPPC. MAL 2:0.5:04. Coupling of LL1 to liposomes Example 3 was performed by reaction between the maleimide (MAL) groups at the distal PEG termini on the surface of the lipo Specificity of Immunoconjugates Somes and free thiol groups on the Ab. Before the coupling 0158. The cell specificity of the preparations was tested on reaction LL1 was reduced with 50 mM dithiotreitol for 1 hat HL-60 and Jurkat cells. The cellular association obtained 4° C. in 0.2 M tris buffer (pH6.5). The reduced Ab was after 24h was between 1 and 2% for both cell types with no separated from excess dithiotreitol by use of Sephadex G-25 evident difference between conjugates and plain emulsions spin-columns, equilibrated with 50 mM sodium acetate buff (not shown). This extent of cellular association clearly repre ered 0.9% saline (pH 5.3). The conjugation was performed in sent unspecific uptake. The specificity of the interaction was HEPES-buffered saline (pH 7.4) for 16hat room temperature further studied by measuring the cellular association of H under argon. Excess maleimide groups were blocked with 2 COE-labeled LL1-emulsions versus the amount of LL.1 per mM2-mercaptoethanol for 30 min, whereafter excess Aband emulsion EPC (not shown). These experiments demonstrated 2-mercaptoethanol were removed on a Sepharose CL-4B col that association of the LL1-emulsions was dependent on the umn. The immunoliposomes were collected near the Void concentration of LL1 (not shown). The specificity of the volume of the column, passed through a 0.22 um sterile filter interaction of immunoemulsions with cells was also studied and stored at 4°C. The coupling efficiency was estimated by by displacement experiments. Free LLI competed effectively use of fluorescein labeled LL1. with the LL 1-emulsion complexes and at high concentrations the cellular association was practically abolished (not Example 2 shown). These findings strongly indicate that LL1 preserves its immunoreactivity after binding to liposomes. Cellular Uptake and of the Anti-CD74 Immunoliposomes Example 4 0156 Liposomes containing the non-exchangeable Endocytosis of HPTS-Containing Immunoliposomes marker HICOE were used to study the cellular uptake of drug carrier. After completed incubation, the cells were thor 0159. The intracellular fate of LL1-liposomal complexes oughly washed three times with cold PBS and the radioactiv was studied by use of the pH-sensitive probe HPTS. The ity measured by liquid-scintillation counting. The pH-sensi spectral shifts of the probe with changes in pH make it a tive probe HPTS was used to study the internalization of useful marker of the uptake and fate of the encapsulated dye. liposomes to low pH compartments. HPTS exhibits two Internalization of LL1-liposomes to low-pH compartments major fluorescence excitation maxima: a peak at 403 maxi was demonstrated by the fluorescence ratio. 454/413. Val mal at low pH values and a peak at 454 maximal at high pH ues near 0.6 were obtained which corresponds to a pH value values, while the fluorescence is independent of pH at 413 nm of 6.5 (not shown). This value is near those obtained by other (isobestic point). (See, Straubinger, et al., Biochemistry, authors with HPTS-immunoliposomes. See Kirpotin, et al., 29:4929-4939 (1990)). The ratio between the fluorescence at Biochemistry 36 (1997) 66-75; Lopes de Menezes, et al., J. 454 nm and 413 nm can be used to study the internalization of Liposome Res. 9 (1999) 199-228. HPTS-liposomes without the HPTS-liposomes to intracellular acidic compartments. ligand gave values near 0.8, which corresponds to a pH value HPTS-liposomes were diluted to 80 uM phospholipid in of about 7.0. See Lundberg, et al., Int. J. Pharm. 205 (2000) HEPES buffer and added to culture dishes (4x10 cells) at 37° 101-108. It thus seems very likely that the LL1-drug-lipo C. After incubation for 6 h the cells were washed twice with some complexes are delivered to and catabolized by the lyso cold PBS and the fluorescence was measured in a stirred SOS. cuvette at 20° C. Peak heights were measured at 510 nm emission at the two excitation wavelengths (413 and 454 nm) Example 5 and corrected for appropriate background fluorescence. Cytotoxicity Assays 0157. The concentration-dependent cellular association of liposomes with coupled LL1 and liposomes without coupled 0160 Comparison of the in vitro cytotoxicity of free LL1 was examined (not shown). Association of liposomes FUdR and FUdR-dO-loaded emulsions and liposomes with with and without coupled LL1 was concentration dependent and without coupled LL1 was performed on Raji human (not shown). The Burkitt's lymphoma cells, Raji, showed a B-cell lymphoma lines with a proliferation assay utilizing massive interaction with LL1-liposome complexes as com tetrazolium dye, MTT. (See, Mosmann, J. Immun. Meth. pared to untargeted preparations (not shown). LL1-emulsion 65:55-63 (1983)). To begin, 4x10 cells were plated in conjugates, labeled with the nontransferable compound H 24-well plates and incubated with drug containing prepara US 2010/0272636 A1 Oct. 28, 2010

tions. Control experiments included free LL1 and drug free 27–39; Bornet al., J. Controlled Release 74 (2001) 325-333; emulsions and liposomes. Cells were incubated for 24hat 37° and Maranhao et al., Cancer. Chemother. Pharmacol. 49 C. in an atmosphere of 95% humidity and 5% CO. At the 24 (2002) 487-498. h time point, the cells were washed twice before replacing 0166 An explanation for such a favorable effect appears to with fresh media and incubated for an additional 48 h. At the be that the half-life of the drug increases and the tolerability is end of the incubation time, tetrazolium dye was added, the improved so that high doses can be administered. A recent in formed reduction product was spun down, dissolved in EtOH: vivo study with nude mice demonstrated specific Ab local DMSO 1:1 and read at 570 nm. ization of LL1 to Ramos xenografts. See Shih et al., Cancer Immunol. Immunother. 49 (2000) 208-216. The present study 0161 The cytotoxic activity of FUdR-dO in LL1 conju shows an improved cytotoxic activity of the targeted prodrug gated emulsions and liposomes was tested and compared with compared to the parent drug. Immunoliposomes generally the activity of unconjugated drug-liposomes on Raji lym show lower or similar activity compared to the untargeted phoma cells (not shown). The effect of free FUdR (in PBS) drug, but still demonstrate improved efficacy in in vivo was also recorded. The cells were incubated with the various experiments. See Moase et al., Biochim. Biophys. Acta 1510 preparations for 24-h, followed by an additional 48-h in fresh (2001) 43-55; Lopes de Menezes et al., Cancer Res. 58 (1998) medium. From the dose-response curves it could be seen that 3320-333O. FUdR-dO is somewhat more efficacious in emulsions than in liposomes (not shown). However, the activity of FUdR-dO Example 6 administered in both LL1-emulsions and LL1-liposomes exceeded that of FUdR (not shown). The IC70 values Therapy of Chronic Lymphocytic Leukemia obtained were 0.45, 1.25, 5.3 and 7.3 uM for FUdR-dO 0.167 As disclosed herein, milatuzumab, a humanized loaded LL1-emulsions, LL1-liposomes, emulsions and lipo antibody to CD74, used with an in vitro crosslinking antibody somes, respectively. The corresponding value for FUdR was induced cytotoxicity in CLL cells in a caspase- and stromal calculated to 4.35uM. The ICs values were 2.5, 5.3 and 7.0 independent manner associated with aggregation of CD74 on uM for LL1-emulsions, LL1-liposomes and FudR, respec the cell Surface. Incorporation of milatuZumab into an immu tively (FUdR-dO in plain emulsions and liposomes did not noliposome induced even more of a cytotoxic response as in reach that level (not shown). vitro crosslinking, representing a novel therapeutic formula 0162 The prodrug FUdR-dO employed in this study tion for this antibody. These results demonstrate the feasibil shows several advantageous features for administration in ity of using milatuzumab-immunoliposome as a therapeutic liposomes. It is amphiphilic and will be situated in the phos agent for CLL. pholipid monolayer and bilayer of lipid emulsions and lipo (0168 Materials and Methods 0169 Patients, cell separation, culture conditions, and Somes, respectively. This makes the preparation of drug-lipo reagents. For in vitro studies, written, informed consent was Some very convenient; the components are just mixed obtained to procure cells from patients with previously diag together and Sonicated. An alternative method, which is more nosed CLL, as defined by the modified NC1 criteria (Cheson Suited for large scale production, would be the use of high et al., Blood, 1996, 87:4990-4997). Isolated mononuclear pressure homogenization. cells were negatively B-cell selected and placed in culture, as 0163 A prerequisite for site-specific delivery of the pro previously described (Lucas et al., Blood, 2009, 113:4656 drug to the target cells is the stable entrapment of the prodrug 4666). HS-5 stromal cells were obtained from the ATCC. in the drug-liposome. The unspecific transfer of FUdR-dO CD40L was purchased from Peprotech (Rocky Hill, N.J.). from liposome to cells was not actually measured but the Milatuzumab was provided by Immunomedics, Inc. (Morris much higher cytotoxic activity of the LL1-conjugated prepa Plains, N.J.). Goat anti-human IgG antibody (Fc gamma frag rations indicated that unspecific transfer of prodrug to cells is ment-specific, anti-Fc) was purchased from Jackson Immu relatively low (not shown). That some degree of surface trans noResearch Laboratories (West Grove, Pa.). Q-VD-OPH fer probably occurs finds Support by a study of Koning et al., pan-caspase inhibitor was purchased from MP Biomedicals Biochim. Biophys. Acta 1420 (1999) 153-167. They found (Solon, Ohio). that dipalmitoyl-FUdR immunoliposomes, without internal 0170 Flow cytometry assays. Viability was determined by ization, could deliver the prodrug to target cells more efficient flow cytometery using propidium iodide (PI). For surface than liposomes without antibody. staining, CLL cells were washed in PBS and stained with 0164. The prodrug concept comprises a pharmacologi antibodies to CD20 or CD74 (BD Biosciences, San Jose, cally inactive compound that is activated when exposed into Calif.; anti-CD74. MAb-BD catalog #55540/clone M-B741). the target cells. In this respect FUdR-dO may fulfill the cri 0171 Immunoblot analysis. Immunoblots of whole cell teria of a good prodrug. It has been shown that FUdR fatty lysates were performed as described (Johnson et al., Blood, acid esters are hydrolyzed fast in cells, apparently in lysos 2006, 108:1334-1338). Antibodies used included PARP (Cal omes. Seeid. An efficient intracellular liberation of the parent biochem, Gibbstown, N.J.), caspase 3 and 9 (R&D Systems, drug FUdR is also indirectly supported by the high cytotoxic Minneapolis, Minn.), and tubulin (Santa Cruz Biotechnology, efficacy of the FUdR-dO preparations. Santa Cruz, Calif.). 0.165. This in vitro study demonstrates the potential for 0172 Preparation of Immunoliposome (IT). Immunolipo site-specific delivery of anti-cancer drugs by use of liposomes somes (ILS) were Prepared as previously described (Sapra with LL1 as targeting ligand. Several recent studies also show and Allen, Cancer Res, 2002, 62:7190-7194). A post-inser that liposomes, even without attached ligand, can give in vivo tion method was used to incorporate milatuZumab into pre advantage as administration vehicles for lipophilic and formed liposomes, and targeted milatuZumab-IL were pre amphiphilic drugs. See Constantinides et al., Pharm. Res. 17 pared with an antibody-to-lipid ratio of 1:1000. Lipids Chol: (2000) 175-182; Perkins et al., Int. J. Pharm. 200 (2000) Egg-PC:PEG-DSPE (molar ratio=33.5:65:1.5) were US 2010/0272636 A1 Oct. 28, 2010 20 dissolved in ethanol and a thin lipid layer was formed under diminished by co-culture with stroma despite the variation in ROTAVAPORR). Lipids were rehydrated in PBS and particle fludarabine responsiveness among individual samples (FIG. size was reduced by high pressure extrusion with nuclear 5E (panel ii); 19.3% more cell death with fludarabine vs. polycarbonate membranes (pore sizes 0.2 and 0.1 mm, North vehicle control in the absence of HS-5; 2.2% more cell death ern Lipids). Liposome size distribution was analyzed on a with fludarabine vs. vehicle control in the presence of HS-5). NICOMPR Particle Sizer Model 370 (Particle Sizing Sys Furthermore, treatment with CD40L, commonly found to tems, Santa Barbara, Calif.). Volume weighted analysis protect CLL cells from cell death, was unable to prevent showed particle size of approximately 100 nm. milatuzumab-induced cytotoxicity (FIG. 5E (panel iii); 23% 0173 A post-insertion method was adopted to incorporate more cell death with mila--anti-Fc vs. anti-Fc alone in the antibody (milatuZumab) into preformed liposomes. In this absence of CD40L; 29% more cell death with mila+anti-Fc method, milatuZumab was reacted with 10x Traut’s reagent in vs. anti-Fc alone in the presence of CD40L, N=17: P=0. PBS (pH 8.0) for 2 hr at room temperature to yield sulhydryl 09N=17; P=0.11). Together, these data indicate that milatu modified antibodies. Separation of milatuzumab-SH from Zumab may be effective in vivo despite the presence of an unreacted 2-iminothiolane was performed with a SEPHA intact microenvironment. DEX(R) PD-10 desalting column eluted with PBS (pH 6.5). 0177. We next investigated whether milatuzumab was The milatuZumab-SH was added to Mal-PEG-DSPE at a ratio able to mediate antibody-dependant cellular cytotoxicity of 10:1 (Mal-PEG-DSPE:milatuzumab-SH), and coupled (ADCC). Similar to other reports in lymphoma cell lines overnight at room temperature. This antibody mixture was (Stein et al., Blood, 2004, 104:3705-3711), no ADCC was then incubated with liposomes for 1 hr at 37° C. to form detected with either mononuclear cells or granulocytes at any targeted ILP with antibody-to-lipid ratios of 1:1000. Non effector to CLL target cell ratio (data not shown). These coupled antibody was separated on a SEPHAROSER CL-4B findings indicate that direct apoptosis via CD74 ligation is column using PBS (pH 7.4). All liposomal suspensions were likely the principal contributor to milatuzumab efficacy. filtered through 0.22 mM PES syringe filters to ensure steril 0.178 We next sought to determine how CD74 ligation by ity. Milatuzumab-ILP was stored at 4°C. until use. milatuzumab promoted cell death. We observed that upon 0.174 Statistical analysis. All reported statistical evalua milatuZumab treatment in the presence of crosslinking, CLL tions were performed by the Center for Biostatistics at Ohio cells aggregated in culture (not shown) and the mean fluores State University. Since the observations from the same patient cent intensity (MFI) of CD74 on the cell surface increased are correlated, linear mixed effects models were used for significantly (FIGS. 6A and 6B left; 1.50 vs. 5.03 MFI (anti analysis to take account of this within patient correlation. Fc vs. mile anti-Fe); N=14; P=0.0003). No significant Treatment differences were estimated and tested from these change in surface expression of CD74 was observed follow models. The Holm's step-down procedure was used to adjust ing treatment with anti-Fc alone, while milatuZumab alone for multiple comparisons or multiple endpoints when neces led to a slight decrease in surface CD74, potentially due to sary. P-values.<0.05 for single comparisons or after adjust increased receptor internalization in the absence of ment for multiple comparisons were considered significant. crosslinker. In addition, significant surface retention of CD20 was not observed following in vitro crosslinking with milatu Results and Discussion Zumab (FIG. 6B, right; 6.36 vs. 5.56 MFI (anti-Fc vs. mila+ (0175 We first determined the in vitro survival of primary anti-Fc) N=14; P=0.14), indicating that the increased surface CLL cells aftermilatuzumab treatment. As shown in FIG. 5A CD74 is antigen-specific and not due to nonspecific antibody and FIG. 5B, milatuzumab (mila) with anti-Fc crosslinker trapping between clustered cells. These data Suggest that rapidly induced significant cell death compared to anti-Fc milatuZumab promotes the maintenance and/or accumulation alone (difference of 18% averaged across time points, N=26, of CD74 on the cell surface, which likely initiates down P<0.0001). This result was verified by MTT assay (not stream signaling pathway(s) leading to cell death. The lack of shown). This effect was dependent on crosslinking, since this effect following treatment with milatuzumab alone indi milatuzumab alone had no cytotoxic effect on the cells. Fur cates that crosslinking is necessary for milatuZumab-induced thermore, the induction of apoptosis was significantly greater cell death. However, association with Fc receptors on other (P<0.0001) than that of rituximab, and the effect of mila--anti cells in the microenvironment may not be sufficient to medi Fc was greater than that of rituximab-anti-Fc. Milatuzumab ate this effect, as indicated by the lack of ADCC with milatu mediated killing appeared to be caspase-independent, since Zumab (Steinet al., Blood, 2004, 104:3705-11). We therefore treatment does not increase cleavage of caspases 3, 6, 8 or 9 or investigated whether other methods to promote receptor the caspase substitute PARP relative to Fc crosslinker control, accumulation with milatuZumab induce a similar in vitro although processing of caspase 2 was observed (FIG. 5C). cytotoxic effect as crosslinking antibody. While the pan-caspase inhibitor Q-VD-OPH is able to block (0179 Similar to previous studies with the anti-CD22 cleavage of both PARP and caspase 2, it had no significant mAb, epratuzumab (Carnahan et al., Mol Immunol, 2007, effect on milatuzumab-induced cell death (FIG. 5D; N+5; 44: 1331-41), we found that milatuzumab immobilized on a P=0.03). plastic cell culture plate increased cell death compared to the 0176 The role of microenvironmental factors in the sur non-immobilized antibody, but this was not as active in cell vival and drug resistance of CLL cells is becoming increas killing compared to using soluble anti-Fc (data not shown: ingly studied (Kurtova et al., Blood, 2009, 114:4441-50). The 18.2% vs. 43.2% PI positive, immobilized mila vs. mila--anti effect of milatuzumab was not significantly diminished by Fc averaged across time points; N=6; P<0.0001). These co-culture with a stromal cell line (FIG. 5E (panel i); 37.3% results suggest that association of multiple receptors may be more cell death with mila--anti-Fc vs. anti-Fc alone in the required to initiate a death signal, which is limited when absence of HS-5; 48.5% more cell death with mila+anti-Fc antibodies are fixed on cell culture plates. Enhanced cell vs. anti-Fc alone in the presence of HS-5; N=11; P=0.10). In death through Fas signaling has been described for CD44 contrast, fludarabine-induced cytotoxicity was noticeably (Mielgo et al., Apoptosis, 2007, 12:2051-61), a binding part US 2010/0272636 A1 Oct. 28, 2010

ner for CD74, and is a Suggested mechanism for milatuZumab presently representative of preferred embodiments are exem (Berkova et al., Expert Opin Investig Drugs, 2010, 19:141 plary and are not intended as limitations on the scope of the 49). invention. Changes therein and other uses will occur to those 0180 Incorporation of internalizing antibodies into lipo skilled in the art, which are encompassed within the inven Some particles has been described as a method for targeted tion. drug delivery in B-cell malignancies (Sapra et al., Cancer 0184. It will be readily apparent to one skilled in the art that varying Substitutions and modifications may be made to Res, 2002, 62:7190-7194; Clin Cancer Res, 2005, 11:5257 the invention disclosed herein without departing from the 5264). We found that incorporation of milatuzumab into a Scope and spirit of the invention. Thus, such additional liposome (mila-IL) was able to mediate the same receptor embodiments are within the scope of the present invention. aggregation on the cell Surface as milatuZumab with anti-Fc 0185. The invention illustratively described herein suit (FIG. 6C), an effect which was not evident with liposome ably may be practiced in the absence of any element or ele alone (mila-IL vs. liposome; 4.23 vs. 1.39 MFI; N=4; P=0. ments, limitation or limitations which is not specifically dis 0003). Furthermore, mila-IL induced significantly 6 more closed herein. Thus, for example, in each instance herein any cell death in CLL cells compared to IgG incorporated lipo of the terms "comprising”, “consisting essentially of and somes (FIG. 6D; mila-IL vs. IgG-IL: 36% vs. 4.8% PI posi “consisting of may be replaced with either of the other two tive: N=11; P-0.0001). Cell death induced by mila-IL was terms. The terms and expressions which have been employed significantly higher than that caused by milatuZumab plus are used as terms of description and not of limitation, and crosslinking in vitro (FIG. 6D; mila-IL vs. mila+anti-Fc. 36% there is no intention that in the use of Such terms and expres vs. 25.5% PI positive: N=11; P=0.0003). This cytotoxicity sions of excluding any equivalents of the features shown and was evident even without packaging the immunoliposome described orportions thereof, but it is recognized that various with a chemotherapeutic drug, such as doxorubicin, which modifications are possible within the scope of the invention. has been described previously with this antibody (Sapra et al., Thus, it should be understood that although the present inven Clin Cancer Res, 2005, 11:5257-5264). tion has been specifically disclosed by preferred embodi 0181. Here, we provide evidence that the incorporation of ments and optional features, modification and variation of the milatuZumab into a liposome may induce cell death without concepts herein disclosed may be resorted to by those skilled the dependence on other cell types in the microenvironment. in the art, and that Such modifications and variations are Together, these Support the clinical application of mila-IL considered to be within the scope of this invention. based therapy in CLL and other CD74-positive malignancies. 0186. In addition, where features or aspects of the inven 0182 All patents and other references cited in the speci tion are described interms of Markush groups or other group fication are indicative of the level of skill of those skilled in ing of alternatives, those skilled in the art will recognize that the art to which the invention pertains, and are incorporated the invention is also thereby described in terms of any indi herein by reference, including any Tables and Figures, to the vidual member or subgroup of members of the Markush same extent as if each reference had been incorporated by group or other group. reference individually. 0187. Also, unless indicated to the contrary, where various 0183) One skilled in the art would readily appreciate that numerical values are provided for embodiments, additional the present invention is well adapted to obtain the ends and embodiments are described by taking any 2 different values advantages mentioned, as well as those inherent therein. The as the endpoints of a range. Such ranges are also within the methods, variances, and compositions described herein as scope of the described invention.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 2O

<21 Os SEQ ID NO 1 &211s LENGTH: 16 212s. TYPE: PRT <213> ORGANISM: Mus sp. <4 OOs SEQUENCE: 1 Arg Ser Ser Glin Ser Lieu Val His Arg Asn Gly Asn. Thir Tyr Lieu. His 1. 5 1O 15

<21 Os SEQ ID NO 2 &211s LENGTH: 7 212s. TYPE: PRT <213> ORGANISM: Mus sp. <4 OOs SEQUENCE: 2 Thr Val Ser Asn Arg Phe Ser 1. 5

US 2010/0272636 A1 Oct. 28, 2010 23

- Continued ttg cag atc agc aac ct c aaa aat gag gac atg ggt aca tat titc tigt 288 Lieu. Glin Ile Ser Asn Lieu Lys Asn. Glu Asp Met Gly Thr Tyr Phe Cys 85 90 95 tca aga. tcg agg ggit aaa aac gala gcc tig titt gct t at tig ggc caa 336 Ser Arg Ser Arg Gly Lys Asn. Glu Ala Trp Phe Ala Tyr Trp Gly Glin 1OO 105 11 O ggg act ctg gtC act gtc. tct gala 360 Gly Thr Lieu Val Thr Val Ser Glu 115 12 O

<210s, SEQ ID NO 8 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Mus sp. <4 OOs, SEQUENCE: 8 Glin Ile Glin Lieu Val Glin Ser Gly Pro Glu Lieu Lys Llys Pro Gly Glu 1. 5 1O 15 Thr Val Llys Val Thr Cys Llys Thr Ser Gly Tyr Thr Phe Thr Asn Tyr 2O 25 3O Gly Val Asn Trp Ile Lys Glin Thr Pro Gly Glu Gly Lieu. Glin Trp Met 35 4 O 45 Gly Trp Ile Asin Pro Asn Thr Gly Glu Pro Thr Phe Asp Asp Asp Phe SO 55 6 O Lys Gly Arg Phe Ala Phe Ser Lieu. Glu Ser Ser Ala Ser Thr Ala Phe 65 70 7s 8O Lieu. Glin Ile Ser Asn Lieu Lys Asn. Glu Asp Met Gly Thr Tyr Phe Cys 85 90 95 Ser Arg Ser Arg Gly Lys Asn. Glu Ala Trp Phe Ala Tyr Trp Gly Glin 1OO 105 11 O Gly Thr Lieu Val Thr Val Ser Glu 115 12 O

<210s, SEQ ID NO 9 &211s LENGTH: 337 &212s. TYPE: DNA <213> ORGANISM: Mus sp. 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) ... (333)

<4 OOs, SEQUENCE: 9 gat gtt gtg atg acc caa act coa ct c toc ctd cct gtc agt citt gga 48 Asp Val Val Met Thr Glin Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1. 5 1O 15 gat caa goc toc atc. tct tcc aga tot agt cag agc citt gta cac aga 96 Asp Glin Ala Ser Ile Ser Cys Arg Ser Ser Glin Ser Lieu Val His Arg 2O 25 3O aat gga aac acc tat tta cat tdg tac ct g cag aag cca ggc cag tot 144 Asn Gly Asn Thr Tyr Lieu. His Trp Tyr Lieu Gln Lys Pro Gly Glin Ser 35 4 O 45 cca aag ct c ct g atc tac aca gtt to c aac cqa titt tot ggg gtc. cca 192 Pro Llys Lieu. Lieu. Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro SO 55 6 O gac agg tt C agt ggc agt gga to a ggg aca gat titc aca ct c aag at C 24 O Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu Lys Ile 65 70 7s 8O agt aga gtg gag gct gag gat Ctg gga citt tat titc tic tot caa agt 288 US 2010/0272636 A1 Oct. 28, 2010 24

- Continued Ser Arg Val Glu Ala Glu Asp Lieu. Gly Lieu. Tyr Phe Cys Ser Glin Ser 85 90 95 tca cat gtt cot CCC acg titc ggit gct ggg acc aag Ctg gag at C taac 337 Ser His Val Pro Pro Thr Phe Gly Ala Gly Thr Lys Lieu. Glu Ile 1OO 105 11 O

<210s, SEQ ID NO 10 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Mus sp. <4 OOs, SEQUENCE: 10 Asp Val Val Met Thr Glin Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1. 5 1O 15 Asp Glin Ala Ser Ile Ser Cys Arg Ser Ser Glin Ser Lieu Val His Arg 2O 25 3O Asn Gly Asn Thr Tyr Lieu. His Trp Tyr Lieu Gln Lys Pro Gly Glin Ser 35 4 O 45 Pro Llys Lieu. Lieu. Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro SO 55 6 O Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu Lys Ile 65 70 7s 8O Ser Arg Val Glu Ala Glu Asp Lieu. Gly Lieu. Tyr Phe Cys Ser Glin Ser 85 90 95 Ser His Val Pro Pro Thr Phe Gly Ala Gly Thr Lys Lieu. Glu Ile 1OO 105 11 O

<210s, SEQ ID NO 11 &211s LENGTH: 360 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polynucleotide 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) ... (360)

<4 OOs, SEQUENCE: 11 Cag gt C caa citg cag cag tict gga cct gag ctgaag aag cct gga gag 48 Glin Val Glin Lieu. Glin Glin Ser Gly Pro Glu Lieu Lys Llys Pro Gly Glu 1. 5 1O 15 aca gttcaag gtc acc togc aag act tct gga tat acc titc aca aac tat 96 Thr Val Llys Val Thr Cys Llys Thr Ser Gly Tyr Thr Phe Thr Asn Tyr 2O 25 3O gga gtgaac tog ata aag cag act coa gga gag ggit tta cag tog atg 144 Gly Val Asn Trp Ile Lys Glin Thr Pro Gly Glu Gly Lieu. Glin Trp Met 35 4 O 45 ggc tigg at a aac C cc aac act gga gag cca acattt gat gat gaC titc 192 Gly Trp Ile Asin Pro Asn Thr Gly Glu Pro Thr Phe Asp Asp Asp Phe SO 55 6 O aag gga cqa titt gcc titc. tct ttg gaa toc toit gcc agc act gcc titt 24 O Lys Gly Arg Phe Ala Phe Ser Lieu. Glu Ser Ser Ala Ser Thr Ala Phe 65 70 7s 8O ttg cag atc agc aac ct c aaa aat gag gac atg ggt aca tat titc tigt 288 Lieu. Glin Ile Ser Asn Lieu Lys Asn. Glu Asp Met Gly Thr Tyr Phe Cys 85 90 95 tca aga. tcg agg ggit aaa aac gala gcc tig titt gct t at tig ggc caa 336 Ser Arg Ser Arg Gly Lys Asn. Glu Ala Trp Phe Ala Tyr Trp Gly Glin US 2010/0272636 A1 Oct. 28, 2010 25

- Continued

1OO 105 11 O ggg act ctg gtC acc gtc. tcc to a 360 Gly Thr Lieu Val Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 12 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 12 Glin Val Glin Lieu. Glin Glin Ser Gly Pro Glu Lieu Lys Llys Pro Gly Glu 1. 5 1O 15 Thr Val Llys Val Thr Cys Llys Thr Ser Gly Tyr Thr Phe Thr Asn Tyr 2O 25 3O Gly Val Asn Trp Ile Lys Glin Thr Pro Gly Glu Gly Lieu. Glin Trp Met 35 4 O 45 Gly Trp Ile Asin Pro Asn Thr Gly Glu Pro Thr Phe Asp Asp Asp Phe SO 55 6 O Lys Gly Arg Phe Ala Phe Ser Lieu. Glu Ser Ser Ala Ser Thr Ala Phe 65 70 7s 8O Lieu. Glin Ile Ser Asn Lieu Lys ASn Glu Asp Met Gly Thr Tyr Phe Cys 85 90 95 Ser Arg Ser Arg Gly Lys Asn. Glu Ala Trp Phe Ala Tyr Trp Gly Glin 1OO 105 11 O Gly Thr Lieu Val Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 13 &211s LENGTH: 339 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polynucleotide 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) ... (339)

<4 OOs, SEQUENCE: 13 gac at C cag citg acc caa act coa ct c toc ctd cct gtc agt citt gga 48 Asp Ile Glin Lieu. Thr Glin Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1. 5 1O 15 gat caa goc toc atc. tct tcc aga tot agt cag agc citt gta cac aga 96 Asp Glin Ala Ser Ile Ser Cys Arg Ser Ser Glin Ser Lieu Val His Arg 2O 25 3O aat gga aac acc tat tta cat tdg tac ct g cag aag cca ggc cag tot 144 Asn Gly Asn Thr Tyr Lieu. His Trp Tyr Lieu Gln Lys Pro Gly Glin Ser 35 4 O 45 cca aag ct c ct g atc tac aca gtt to c aac cqa titt tot ggg gtc. cca 192 Pro Llys Lieu. Lieu. Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro SO 55 6 O gac agg tt C agt ggc agt gga to a ggg aca gat titc aca ct c aag at C 24 O Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu Lys Ile 65 70 7s 8O agt aga gtg gag gct gag gat Ctg gga citt tat titc tic tot caa agt 288 US 2010/0272636 A1 Oct. 28, 2010 26

- Continued

Ser Arg Val Glu Ala Glu Asp Lieu. Gly Lieu. Tyr Phe Cys Ser Glin Ser 85 90 95 toa cat gtt CCt c cc acg titc ggit gct ggg acc aag Ctg gag at C aaa. 336 Ser His Val Pro Pro Thr Phe Gly Ala Gly Thr Lys Lieu. Glu Ile Llys 105 11 O cgt 339 Arg

<210s, SEQ ID NO 14 &211s LENGTH: 113 212. TYPE: PRT ORGANISM: Artificial Sequence 22 Os. FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 14

Asp Ile Glin Lieu. Thir Glin. Thir Pro Luell Ser Luell Pro Wall Ser Luell Gly 1. 5 15

Asp Glin Ala Ser Ile Ser Cys Arg Ser Ser Glin Ser Lell Wall His Arg 2O 25

Asn Gly Asn Thr Tyr Lieu. His Trp Luell Glin Pro Gly Glin Ser 35 4 O 45

Pro Llys Lieu. Lieu. Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Wall Pro SO 55 6 O

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thir Asp Phe Thir Luell Ile 65 70

Ser Arg Val Glu Ala Glu Asp Lieu Gly Luell Tyr Phe Ser Glin Ser 85 90 95

Ser His Val Pro Pro Thr Phe Gly Ala Gly Thir Lell Glu Ile 1OO 105 11 O Arg

<210s, SEQ ID NO 15 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 15

Glin Val Glin Leu Val Glin Ser Gly Ser Glu Luell Pro Gly Ala 1. 5 1O 15

Ser Val Llys Val Ser Cys Lys Ala Ser Gly Tyr Thir Phe Thir Ser Tyr 2O 25

Ala Met Asn Trp Val Arg Glin Ala Pro Gly Glin Gly Lell Glu Trp Met 35 4 O 45

Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thir Tyr Ala Glin Gly Phe SO 55 6 O

Thir Gly Arg Phe Val Phe Ser Lieu. Asp Thir Ser Wall Ser Thir Ala Tyr 65 70

Lell Glin Ile Ser Ser Lieu Lys Ala Asp Asp Thir Ala Wall Tyr Cys 85 90 95

Ala Arg Glu Asp Ser Asn Gly Tyr Lys Ile Phe Asp Trp Gly Glin 1OO 105 11 O

Gly Ser Lieu Val Thir Wal Ser Ser 115 12 O US 2010/0272636 A1 Oct. 28, 2010 27

- Continued

<210s, SEQ ID NO 16 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 16 Asp Val Val Met Thr Glin Ser Pro Leu Ser Leu Pro Val Thr Lieu. Gly 1. 5 1O 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Glin Ser Lieu Val His Ser 2O 25 3O Asp Gly Asn Thr Tyr Lieu. Asn Trp Phe Glin Glin Arg Pro Gly Glin Ser 35 4 O 45 Pro Arg Arg Lieu. Ile Tyr Llys Val Ser Asn Arg Asp Ser Gly Val Pro SO 55 6 O Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu Lys Ile 65 70 7s 8O Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Glin Gly 85 90 95 Thr His Trp Pro Phe Thr Phe Gly Glin Gly Thr Arg Lieu. Glu Ile 1OO 105 11 O

<210s, SEQ ID NO 17 &211s LENGTH: 360 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polynucleotide 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) ... (360)

<4 OOs, SEQUENCE: 17 Cag gt C caa citg cag caa tict ggg tot gag titg aag aag cct ggg gCC 48 Glin Val Glin Lieu. Glin Glin Ser Gly Ser Glu Lieu Lys Llys Pro Gly Ala 1. 5 1O 15 t ca gtg aag gtt toc togc aag got tot gga tac acc titc act aac tat 96 Ser Val Llys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 2O 25 3O gga gtgaac tog ata aag cag gCC cct gga caa ggg Ctt cag tog atg 144 Gly Val Asn Trp Ile Llys Glin Ala Pro Gly Glin Gly Lieu. Glin Trp Met 35 4 O 45 ggc tigg at a aac C cc aac act gga gag cca acattt gat gat gaC titc 192 Gly Trp Ile Asin Pro Asn Thr Gly Glu Pro Thr Phe Asp Asp Asp Phe SO 55 6 O aag gga cqa titt gcc titc. tcc titg gac acc tot gtc agc acg gca tat 24 O Lys Gly Arg Phe Ala Phe Ser Lieu. Asp Thr Ser Val Ser Thr Ala Tyr 65 70 7s 8O

Ct c cag at C agc agc cta aag gct gac gac act gcc gtg tat tt C tit 288 Lieu. Glin Ile Ser Ser Lieu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 tca aga. tcg agg ggit aaa aac gala gcc tig titt gct t at tig ggc caa 336 Ser Arg Ser Arg Gly Lys Asn. Glu Ala Trp Phe Ala Tyr Trp Gly Glin 1OO 105 11 O ggg agc ctg gtC acc gtc. tcc to a 360 Gly Ser Leu Val Thr Val Ser Ser 115 12 O US 2010/0272636 A1 Oct. 28, 2010 28

- Continued <210s, SEQ ID NO 18 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 18 Glin Val Glin Lieu. Glin Glin Ser Gly Ser Glu Lieu Lys Llys Pro Gly Ala 1. 5 1O 15 Ser Val Llys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 2O 25 3O Gly Val Asn Trp Ile Llys Glin Ala Pro Gly Glin Gly Lieu. Glin Trp Met 35 4 O 45 Gly Trp Ile Asin Pro Asn Thr Gly Glu Pro Thr Phe Asp Asp Asp Phe SO 55 6 O Lys Gly Arg Phe Ala Phe Ser Lieu. Asp Thr Ser Val Ser Thr Ala Tyr 65 70 7s 8O Lieu. Glin Ile Ser Ser Lieu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ser Arg Ser Arg Gly Lys Asn. Glu Ala Trp Phe Ala Tyr Trp Gly Glin 1OO 105 11 O Gly Ser Leu Val Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 19 &211s LENGTH: 339 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polynucleotide 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) ... (339)

<4 OOs, SEQUENCE: 19 gac at C cag citg act cag tot coa ct c toc ctd ccc gtc acc citt gga 48 Asp Ile Glin Lieu. Thr Glin Ser Pro Leu Ser Leu Pro Val Thr Lieu. Gly 1. 5 1O 15 cag ccg gcc toc atc. tcc tdc aga to a agt cag agc citt gta cac aga 96 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Glin Ser Lieu Val His Arg 2O 25 3O aat gga aac acc tat tta cat tdg titt cag cag agg cca ggc caa tot 144 Asn Gly Asn Thr Tyr Lieu. His Trp Phe Glin Glin Arg Pro Gly Glin Ser 35 4 O 45 cca agg ct c ct g atc tac aca gtt to c aac cqa titt tot ggg gtc. cca 192 Pro Arg Lieu. Lieu. Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro SO 55 6 O gac aga tt C agc ggc agt ggg to a ggc act gat titc aca citg aaa at C 24 O Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu Lys Ile 65 70 7s 8O agc agg gtg gag gct gag gat gtt ggg gtt tat titc tic tot caa agt 288 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Glin Ser 85 90 95 tca cat gtt cot CCC acg titc ggit gct ggg aca Ca Ctg gag at C aaa 336 Ser His Val Pro Pro Thr Phe Gly Ala Gly Thr Arg Lieu. Glu Ile Llys 1OO 105 11 O US 2010/0272636 A1 Oct. 28, 2010

- Continued cgt 339 Arg

<210s, SEQ ID NO 2 O &211s LENGTH: 113 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 2O Asp Ile Glin Lieu. Thr Glin Ser Pro Leu Ser Leu Pro Val Thr Lieu. Gly 1. 5 1O 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Glin Ser Lieu Val His Arg 2O 25 3O Asn Gly Asn Thr Tyr Lieu. His Trp Phe Glin Glin Arg Pro Gly Glin Ser 35 4 O 45 Pro Arg Lieu. Lieu. Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro SO 55 6 O Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu Lys Ile 65 70 7s 8O Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Glin Ser 85 90 95 Ser His Val Pro Pro Thr Phe Gly Ala Gly Thr Arg Lieu. Glu Ile Llys 1OO 105 11 O Arg

What is claimed is: 7. The method of claim 1, wherein the anti-CD74 antibody 1. A method of treating a disease associated with cells that or fragment thereof is a naked anti-CD74 antibody or frag express CD74 comprising ment thereof. a. administering to an individual with the disease an immu 8. The method of claim 7, further comprising administer noconjugate comprising at least one anti-CD74 binding ing at least one therapeutic agent to said individual. molecule attached to a liposome, wherein said adminis 9. The method of claim 1, wherein the anti-CD74 antibody tration results in cross-linking of CD74 on the surface of or fragment thereof is conjugated to at least one therapeutic or CD74-expressing cells; and diagnostic agent. b. inducing apoptosis of the CD74 expressing cells. 10. The method of claim 1, wherein the one or more anti CD74 binding molecules are conjugated to the liposome by a 2. The method of claim 1, wherein the anti-CD74 binding linkage selected from the group consisting of a sulfide link molecule is selected from the group consisting of a mono age, a hydrazone linkage, a hydrazine linkage, an ester link clonal antibody, an antigen-binding fragment of a mono age, an amido linkage, an amino linkage, an imino linkage, a clonal antibody, a bispecific antibody, a multispecific anti thiosemicarbazone linkage, a semicarbazone linkage, an body and an antibody fusion protein. Oxime linkage, a carbon-carbon linkage, or a combination 3. The method of claim 1, wherein the immunoconjugate thereof. further comprises at least one therapeutic or diagnostic agent. 11. The method of claim 3, wherein the therapeutic or 4. The method of claim 1, wherein the anti-CD74 antibody diagnostic agent is selected from the group consisting of a is milatuZumab. drug, a prodrug, a toxin, an enzyme, a radionuclide, an immu 5. The method of claim 1, wherein the anti-CD74 antibody nomodulator, a cytokine, a hormone, a second binding mol comprises the light chain variable region complementarity ecule, an antisense oligonucleotide, an RNAi, an anti-angio determining region (CDR) sequences CDR1 (RSSQS genic agent, a pro-apoptosis agent, a dye, a fluorescent agent, LVHRNGNTYLH: SEQID NO:1), CDR2 (TVSNRFS: SEQ a contrast agent, a paramagnetic ion and a photodynamic ID NO:2), and CDR3 (SQSSHVPPT: SEQID NO:3) and the agent. heavy chain variable region CDR sequences CDR1 12. The method of claim 11, wherein the second binding (NYGVN; SEQ ID NO:4), CDR2 (WINPNTGEPTFD molecule binds to an antigen selected from the group consist DDFKG; SEQ ID NO:5), and CDR3 (SRGKNEAWFAY; ing of carbonic anhydrase IX, CCCL19, CCCL21, CSA.p. SEQID NO:6). CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, 6. The method of claim 1, wherein the anti-CD74 antibody CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, is a chimeric, humanized or human antibody. CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, US 2010/0272636 A1 Oct. 28, 2010 30

CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, 154-158Gd, 161Tb, 166Dy 16Ho, 16°Er, 175Lu, 177Lu, 186Re, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, 188Re, 189Re, 194Ir, 198Au, 199Au, 21 At 21 Pb, 212 Bi, 212Pb, CD95, CD126, CD133, CD138, CD147, CD154, AFP, 213Bi, 22-Ra, or 225Ac. PSMA, CEACAM5, CEACAM-6, B7, ED-B of fibronectin, 20. The method of claim 11, wherein the enzyme is Factor H, FHL-1, Flt-3, folate receptor, GROB, HMGB-1, selected from the group consisting of carboxylesterase, glu hypoxia inducible factor (HIF), HM1.24, insulin-like growth coronidase, carboxypeptidase, beta-lactamase and phos factor-1 (ILGF-1), IFN-y, IFN-C, IFN-B, IL-2, IL-4R, IL-6R, phatase. IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, 21. The method of claim 11, wherein the immunomodula IL-17, IL-18, IL-25, IP-10, MAGE, mCRP, MCP-1, MIP-1A, tor is selected from the group consisting of a cytokine, a stem MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, cell growth factor, a lymphotoxin, a hematopoietic factor, a PAM4 antigen, NCA-95, NCA-90, Ia, HM1.24, EGP-1, colony stimulating factor (CSF), an interleukin (IL) and an EGP-2, HLA-DR, tenascin, Le(y), RANTES, T101, TAC, Tn. interferon (IFN). antigen, Thomson-Friedenreich antigens, tumor necrosis 22. The method of claim 21, wherein the immunomodula antigens, TNF-ct, TRAIL receptor (R1 and R2), VEGFR, tor is selected from the group consisting of nerythropoietin, EGFR, PIGF, complement factors C3, C3a, C3b, C5a, C5, thrombopoietin tumor necrosis factor-O. (TNF). TNF-B, and an oncogene product. granulocyte-colony stimulating factor (G-CSF), granulocyte 13. The method of claim 11, wherein the therapeutic agent macrophage-colony stimulating factor (GM-CSF), inter is selected from the group consisting of aplidin, azaribine, feron-C., interferon-?3, interferon-Y, stem cell growth factor anastroZole, azacytidine, bleomycin, bortezomib. bryostatin designated “S1 factor', human growth hormone, N-methio 1, buSulfan, calicheamycin, camptothecin, 10-hydroxycamp nyl human growth hormone, bovine growth hormone, par tothecin, carmustine, celebrex, chlorambucil, cisplatin, irino athyroid hormone, thyroXine, insulin, proinsulin, relaxin, tecan (CPT-11), SN-38, carboplatin, cladribine, prorelaxin, follicle stimulating hormone (FSH), thyroid cyclophosphamide, cytarabine, dacarbazine, docetaxel, dac stimulating hormone (TSH), luteinizing hormone (LH), tinomycin, daunomycin glucuronide, daunorubicin, dexam hepatic growth factor, prostaglandin, fibroblast growth factor, ethasone, diethylstilbestrol, doxorubicin, doxorubicin glucu prolactin, placental lactogen, OB protein, mullerian-inhibit ronide, epirubicin glucuronide, ethinyl estradiol. ing Substance, mouse gonadotropin-associated peptide, estramustine, etoposide, etoposide glucuronide, etoposide inhibin, activin, vascular endothelial growth factor, integrin, phosphate, floxuridine (FUdR). 3',5'-O-dioleoyl-FudR NGF-13, platelet-growth factor, TGF-C. TGF-B, insulin-like (FUdR-dO), fludarabine, flutamide, fluorouracil, fluoxymes growth factor-I, insulin-like growth factor-II, macrophage terone, gemcitabine, hydroxyprogesterone caproate, hydrox CSF (M-CSF), IL-1, IL-1C, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, yurea, idarubicin, ifosfamide, L-asparaginase, leucovorin, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, lomustine, mechlorethamine, medroprogesterone acetate, IL-17, IL-18, IL-21, IL-25, LIF, FLT-3, angiostatin, throm megestrol acetate, melphalan, mercaptopurine, 6-mercap bospondin, endostatin and LT. topurine, methotrexate, mitoxantrone, mithramycin, mito mycin, mitotane, phenylbutyrate, prednisone, procarbazine, 23. The method of claim 11, wherein the anti-angiogenic paclitaxel, pentostatin, PSI-341, Semustine Streptozocin, agent is selected from the group consisting of angiostatin, tamoxifen, taxanes, taxol, testosterone propionate, thalido endostatin, basculostatin, can statin, maspin, anti-VEGF mide, thioguanine, thiotepa, teniposide, topotecan, uracil binding molecules, anti-placental growth factor binding mol mustard, velcade, vinblastine, Vinorelbine, Vincristine, ricin, ecules and anti-vascular growth factor binding molecules. abrin, ribonuclease, onconase, rapLR1, DNase I, Staphylo 24. The method of claim 1, wherein the antibody fragment coccal enterotoxin-A, pokeweed antiviral protein, gelonin, is selected from the group consisting of F(ab'), F(ab), Fab, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas Fab' and scFv fragments. endotoxin. 25. The method of claim 6, wherein the human, chimeric, 14. The method of claim 13, wherein the therapeutic agent or humanized anti-CD74 antibody or fragment thereof further comprises FUdR, FUdR-dO, or mixtures thereof. comprises human constant regions selected from the group 15. The method of claim 1, wherein the immunoconjugate consisting of IgG1, IgG2a, IgG3 and IgG4. further comprises one or more hard acid chelators or soft acid 26. The method of claim 1, wherein the disease is selected chelators. from the group consisting of an immune dysregulation dis 16. The method of claim 15, wherein the immunoconjugate ease, an autoimmune disease, an organ-graft rejection, a further comprises one or more cations selected from Group II. graft-Versus-host disease and cancer. Group III, Group IV, Group V, transition, lanthanide or 27. The method of claim 26, wherein the cancer is selected actinide metal cations, or mixtures thereof, wherein said cat from the group consisting of a Solid tumor, leukemia, lym ion is attached to the one or more hard or soft acid chelators. phoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, 17. The method of claim 16, wherein the cation is selected multiple myeloma, a B-cell malignancy and a T-cell malig from the group consisting of Tc, Re, Bi, Cu, AS, Ag, Au, At nancy. and Pb. 28. The method of claim 27, wherein the solid tumor is 18. The method of claim 15, wherein said chelator is selected from the group consisting of a melanoma, carci selected from the group consisting of NOTA, DOTA, DTPA, noma, Sarcoma, and glioma. TETA, Tscg-Cys and Tsca-Cys. 29. The method of claim 28, wherein the carcinoma is 19. The method of claim 11, wherein the radionuclide is selected from the group consisting of a renal carcinoma, lung selected from the group consisting of F. P. P. "Ti, 'Sc, carcinoma, intestinal carcinoma, stomach carcinoma, breast 52Fe, Fe, 62Cu, Cu, 7Ga, 7Ga, Ga, 7Se, 77As, 86Y. carcinoma, prostate cancer and ovarian cancer. 89Sr. 897r, 90Y. 99Tc, 99mTc, Mo, 99mTc, 105Pd, 105Rh, 30. The method of claim 27, wherein the B-cell malignancy '''Ag, In, 123, 1241, 125I, 13 II, 142Pr 14.Pr 14°Pm, 'Sim, is selected from the group consisting of indolent forms of US 2010/0272636 A1 Oct. 28, 2010

B-cell lymphomas, aggressive forms of B-cell lymphomas, 43. The composition of claim 37, wherein the anti-CD74 chronic lymphatic leukemias, acute lymphatic leukemias and antibody or fragment thereof is conjugated to at least one multiple myeloma therapeutic or diagnostic agent. 31. The method of claim 11, wherein the photodynamic 44. The composition of claim 36, wherein the one or more agentis selected from the group consisting of benzoporphyrin anti-CD74 binding molecules are conjugated to the liposome monoacid ring A (BDP-MA), tin etiopurpurin (SnET2), sul by a linkage selected from the group consisting of a sulfide fonated aluminum phthalocyanine (AISPc) and lutetium linkage, a hydraZone linkage, a hydrazine linkage, an ester texaphyrin (Lutex). linkage, an amido linkage, an amino linkage, an imino link 32. The method of claim 11, wherein the diagnostic agent age, a thiosemicarbazone linkage, a semicarbazone linkage, is a radionuclide selected from the group consisting of F, an oxime linkage, a carbon-carbon linkage, or a combination 52Fe,6°Cu, Cu, 67Cu, 67Ga Ga, Y, Zr, 9Tc, 97Tc, thereof. 99mTc, 11 In, 123, 1241, 125 and 13 II. 45. The composition of claim 38, wherein the therapeutic 33. The method of claim 11, wherein the diagnostic agent or diagnostic agent is selected from the group consisting of a is a contrast agent selected from the group consisting of drug, a prodrug, a toxin, an enzyme, a radionuclide, an immu gadolinium ions, lanthanum ions, manganese ions, iron, chro nomodulator, a cytokine, a hormone, a second binding mol mium, copper, cobalt, nickel, fluorine, dysprosium, rhenium, ecule, an antisense oligonucleotide, an RNAi, an anti-angio europium, terbium, holmium, neodymium, barium, diatri genic agent, a pro-apoptosis agent, a dye, a fluorescent agent, Zoate, ethiodized oil, gallium citrate, iocarmic acid, iocetamic a contrast agent, a paramagnetic ion and a photodynamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide, agent. iohexyl, iopamidol, , ioprocemic acid, iose 46. The composition of claim 45, wherein the second bind famic acid, io.seric acid, ioSulamide meglumine, iosemetic ing molecule binds to an antigen selected from the group acid, iotasul, iotetric acid, iothalamic acid, iotroXic acid, ioxa consisting of carbonic anhydrase IX, CCCL19, CCCL21, glic acid, ioxotrizoic acid, ipodate, meglumine, metrizamide, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, metrizoate, propyliodone and thallous chloride. CD14, CD15, CD16, CD18, CD19, IGF-1R, CD2O, CD21, 34. The method of claim 1, further comprising performing CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, an operative, intravascular, laparoscopic, or endoscopic pro CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, cedure. CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, 35. The method of claim 1, wherein the immunoconjugate CD83, CD95, CD126, CD133, CD138, CD147, CD154, AFP, is administered by intravenous, intramuscular, intraperito PSMA, CEACAM5, CEACAM-6, B7, ED-B of fibronectin, neal, intravascular, parenteral or subcutaneous administra Factor H, FHL-1, Flt-3, folate receptor, GROB, HMGB-1, tion. hypoxia inducible factor (HIF), HM1.24, insulin-like growth 36. A therapeutic composition comprising: factor-1 (ILGF-1), IFN-y, IFN-C, IFN-B, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, a. an immunoconjugate comprising at least one anti-CD74 IL-17, IL-18, IL-25, IP-10, MAGE, mCRP, MCP-1, MIP-1A, binding molecule; and MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, b. a liposome attached to the at least one anti-CD74 binding PAM4 antigen, NCA-95, NCA-90, Ia, HM1.24, EGP-1, molecule: EGP-2, HLA-DR, tenascin, Le(y), RANTES, T101, TAC, Tn wherein exposing cells expressing CD74 to the composition antigen, Thomson-Friedenreich antigens, tumor necrosis results in cross-linking of CD74 on the surface of CD74 antigens, TNF-ct, TRAIL receptor (R1 and R2), VEGFR, expressing cells. EGFR, P1GF, complement factors C3, C3a, C3b, C5a, C5, 37. The composition of claim 36, wherein the anti-CD74 and an oncogene product. binding molecule is selected from the group consisting of a 47. The composition of claim 45, wherein the therapeutic monoclonal antibody, an antigen-binding fragment of a agent is selected from the group consisting of aplidin, azarib monoclonal antibody, a bispecific antibody, a multispecific ine, anastroZole, azacytidine, bleomycin, bortezomib. bry antibody and an antibody fusion protein. ostatin-1, buSulfan, calicheamycin, camptothecin, 10-hy 38. The composition of claim 36, wherein the composition droxycamptothecin, carmustine, celebrex, chlorambucil, further comprises at least one therapeutic or diagnostic agent. cisplatin, irinotecan (CPT-11), SN-38, carboplatin, cladrib ine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, 39. The composition of claim 37, wherein the anti-CD74 dactinomycin, daunomycin glucuronide, daunorubicin, dex antibody is milatuZumab. amethasone, diethylstilbestrol, doxorubicin, doxorubicin 40. The composition of claim 37, wherein the anti-CD74 glucuronide, epirubicin glucuronide, ethinyl estradiol, estra antibody comprises the light chain variable region comple mustine, etoposide, etoposide glucuronide, etoposide phos mentarity-determining region (CDR) sequences CDR1 phate, floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR (RSSQSLVHRNGNTYLH: SEQID NO:1), CDR2 (TVSN dO), fludarabine, flutamide, fluorouracil, fluoxymesterone, RFS: SEQ ID NO:2), and CDR3 (SQSSHVPPT, SEQ ID gemcitabine, hydroxyprogesterone caproate, hydroxyurea, NO:3) and the heavy chain variable region CDR sequences idarubicin, Ifosfamide, L-asparaginase, leucovorin, lomus CDR1 (NYGVN; SEQ ID NO:4), CDR2 (WINPNTGEPT tine, mechlorethamine, medroprogesterone acetate, mege FDDDFKG; SEQID NO:5), and CDR3 (SRGKNEAWFAY; strol acetate, melphalan, mercaptopurine, 6-mercaptopurine, SEQID NO:6). methotrexate, mitoxantrone, mithramycin, mitomycin, mito 41. The composition of claim 37, wherein the anti-CD74 tane, phenylbutyrate, prednisone, procarbazine, paclitaxel, antibody is a chimeric, humanized or human antibody. pentostatin, PSI-341, Semustine Streptozocin, tamoxifen, tax 42. The composition of claim 37, wherein the anti-CD74 anes, taxol, testosterone propionate, thalidomide, thiogua antibody or fragment thereof is a naked anti-CD74 antibody nine, thiotepa, teniposide, topotecan, uracil mustard, velcade, or fragment thereof. vinblastine, vinorelbine, Vincristine, ricin, abrin, ribonu US 2010/0272636 A1 Oct. 28, 2010 32 clease, onconase, rapLR1, DNase I, Staphylococcal entero thyroid stimulating hormone (TSH), luteinizing hormone toxin-A, pokeweed antiviral protein, gelonin, diphtheria (LH), hepatic growth factor, prostaglandin, fibroblast growth toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. factor, prolactin, placental lactogen, OB protein, mullerian 48. The composition of claim 47, wherein the therapeutic inhibiting Substance, mouse gonadotropin-associated pep agent comprises FUdR, FUdR-dO, or mixtures thereof. tide, inhibin, activin, vascular endothelial growth factor, inte 49. The composition of claim 36, further comprising one or grin, NGF-?3, platelet-growth factor, TGF-C. TGF-B, insulin like growth factor-I, insulin-like growth factor-II, more hard acid chelators or soft acid chelators. macrophage-CSF (M-CSF), IL-1, IL-1C., IL-2, IL-3, IL-4, 50. The composition of claim 49, further comprising one or IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, more cations selected from Group II, Group III, Group IV. IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, FLT-3, Group V, transition, lanthanide or actinide metal cations, or angiostatin, thrombospondin, endostatin and LT. mixtures thereof, wherein said cation is attached to the one or 57. The composition of claim 45, wherein the anti-angio more hard or soft acid chelators. genic agent is selected from the group consisting of angiosta 51. The composition of claim 50, wherein the cation is tin, endostatin, basculostatin, can statin, maspin, anti-VEGF selected from the group consisting of Tc, Re, Bi, Cu, AS, Ag, binding molecules, anti-placental growth factor binding mol Au, At and Pb. ecules and anti-vascular growth factor binding molecules. 52. The composition of claim 49, wherein said chelator is 58. The composition of claim 37, wherein the antibody selected from the group consisting of NOTA, DOTA, DTPA, fragment is selected from the group consisting of F(ab'), TETA, Tscg-Cys and Tsca-Cys. F(ab), Fab, Fab' and scFv fragments. 53. The composition of claim 45, wherein the radionuclide 59. The composition of claim 41, wherein the human, is selected from the group consisting of F, ‘P. P. "Ti, chimeric, or humanized anti-CD74 antibody or fragment 7Sc, 52Fe, Fe, 62Cu, Cu, 7Ga, 7Ga, Ga, 7Se, 77As, thereof further comprises human constant regions selected 86Y. 89Sr. 897r, 90Y. 99Tc, 99mTc, Mo, 99mTc, 105Pd, 105Rh, from the group consisting of IgG1, IgG2a, IgG3 and IgG4. '''Ag, In, 123, 1241, 125I, 13 II, 142Pr 14.Pr 14°Pm, 'Sim, 60. The composition of claim 45, wherein the photody 154-158Gd, 161Tb, 166Dy, 166Ho, 169Er, 175Lu, 177Lu, 186Re, namic agent is selected from the group consisting of ben 188Re, 18°Re, 194Ir, Au, Au, 2. At, 2. Pb, 212Bi, 212Pb, Zoporphyrin monoacid ring A (BDP-MA), tin etiopurpurin 213Bi, 223Ra or 225Ac. (SnRT2), sulfonated aluminum phthalocyanine (AISPc) and 54. The composition of claim 45, wherein the enzyme is lutetium texaphyrin (Lutex). selected from the group consisting of carboxylesterase, glu 61. The composition of claim 45, wherein the diagnostic coronidase, carboxypeptidase, beta-lactamase and phos agent is a radionuclide selected from the group consisting of phatase. 18F 52Fe, 62Cu, 6.Cu, 67Cu, 7Ga, Ga, 86Y. 897r, 94Tc, 55. The composition of claim 45, wherein the immuno 94mTc, 99mTc, In, 123, 1241, 125I and 131I. modulator is selected from the group consisting of a cytokine, 62. The composition of claim 45, wherein the diagnostic a stem cell growth factor, a lymphotoxin, a hematopoietic agent is a contrast agent selected from the group consisting of factor, a colony stimulating factor (CSF), an interleukin (IL) gadolinium ions, lanthanum ions, manganese ions, iron, chro and an interferon (IFN). mium, copper, cobalt, nickel, fluorine, dysprosium, rhenium, 56. The composition of claim 55, wherein the immuno europium, terbium, holmium, neodymium, barium, diatri modulator is selected from the group consisting of nerythro Zoate, ethiodized oil, gallium citrate, iocarmic acid, iocetamic poietin, thrombopoietin tumor necrosis factor-O. (TNF). acid, iodamide, iodipamide, iodoxamic acid, iogulamide, TNF-B, granulocyte-colony stimulating factor (G-CSF), iohexyl, iopamidol, iopanoic acid, ioprocemic acid, iose granulocyte macrophage-colony stimulating factor (GM famic acid, io.seric acid, ioSulamide meglumine, iosemetic CSF), interferon-C., interferon-B, interferon-Y, stem cell acid, iotasul, iotetric acid, iothalamic acid, iotroXic acid, ioxa growth factor designated “S1 factor', human growth hor glic acid, ioxotrizoic acid, ipodate, meglumine, metrizamide, mone, N-methionyl human growth hormone, bovine growth metrizoate, propyliodone and thallous chloride. hormone, parathyroid hormone, thyroxine, insulin, proinsu lin, relaxin, prorelaxin, follicle stimulating hormone (FSH), c c c c c