AU2013202283A1 - Human monoclonal antibodies to fucosyl-GM1 and methods for using anti-fucosyl GM1 - Google Patents

Abstract

C:\NRPortb\CC\SCG(4659304 1.DOC-1,/520 12 The present disclosure provides isolated monoclonal antibodies, particularly human monoclonal antibodies that specifically bind to Fucosyl-GM1 with high affinity. Nucleic acid molecules encoding the antibodies of this disclosure, expression vectors, host cells and methods for expressing the antibodies of this disclosure are also provided. Immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies of this disclosure are also provided. This disclosure also provides methods for detecting Fucosyl-GMI, as well as methods for treating various diseases, including cancer, using anti-Fucosyl-GM1 antibodies.

Description

H:Xscg\Intonvoven\NRPonbIDCC\SCG\46593(4_ .doc-3/25/2013 UMAN MONOCLONAL ANTIBODIES TO FUCOSYL-GM1 AND METHODS FOR USING ANTI-FUCOSYL-GM1 ANTIBODIES Cross-Reference to Related Applications 5 This is a divisional of Australian Patent Application No. 2012241084, which is a divisional of Australian Patent Application No. 2006321554, the entire contents of which are incorporated herein by reference. This application claims priority of U.S. Provisional Application Serial No. 60/748,915, filed on December 8, 2005, which is herein incorporated by reference. 10 Background Fucosyl-GMI is a sphingolipid monosialoganglioside composed of a ceramide lipid component, which anchors the molecule in the cell membrane, and a carbohydrate component that is exposed at the cell surface. Carbohydrate antigens are the most abundantly expressed 15 antigens on the cell surface of cancers (Feizi T. (1985) Nature 314:53-7). In some tumor types, such as small cell lung cancer (SCLC), initial responses to chemotherapy are impressive, but chemo-refractory relapses rapidly follows. Intervention with novel immunotherapeutics may succeed in overcoming drug resistant relapse (Johnson DH. (1995) Lung Cancer 12 Suppl 3:S71-5). Several carbohydrate antigens, such as gangliosides GD3 and GD2, have been 20 shown to function as effective targets for passive immunotherapy with MAbs (Iric RF and Morton DL. (1986) PNAS 83:8694-8698; Houghton AN et al. (1985) PNAS 82:1242-1246). Ganglioside antigens have also been demonstrated to be effective targets for active immunotherapy with vaccines in clinical trials (Krug LM et al. (2004) Clinical Cancer Research 10:6094-6100; Dickler MN et al. (1999) Clinical Cancer Research 5:2773-2779; 25 Livingston PO et al. (1994) J Clin Oncol.12:1036-44). Indeed, serum derived from SCLC patients who developed antibody titers to Fucosyl-GMI following vaccination with KLH conjugated antigen, demonstrated specific binding to tumor cells and tumor specific complement dependant cytotoxicity (CDC). Anti-Fucosyl-GMl titer associated toxicities were mild and transient and three patients with limited-stage SCLC were relapse-free at 18, 24, and 30 30 months (Krug el al., supra; Dickler et al., supra). Fucosyl-GM1 expression has been shown in a high percentage of SCLC cases and unlike other ganglioside antigens, Fucosyl-GMl has little or no expression in normal tissues (Nilsson et WO 2007/067992 PCTIUS2006/061817 2 al. (1984) Glycoconjugate J 1:43-9; Krug et aL, supra; Brezicka el at (1989) Cancer Res 49:1300-5; Zhangyiet al (1997) IntJCancer 73:42-49; Brezicka et at (2000) Lung Cancer 28:29-36; Fredman et al. (1986) Biochim BiophysActa875: 316-23; Brezicka et at (1991) APMIS 99:797-802; Nilsson et al (1986) Cancer Res 46:1403-7). The presence of Fucosyl-GMl 5 has been demonstrated in culture media from SCLC cell lines, in tumor extracts and serum of nude mouse xenografts and in the serum of SCLC patients with extensive-stage disease (Vangsted el al (1991) Cancer Res 51:2879-84; Vangsted el al. (1994) Cancer Detect Prev 18:221-9)., These reports provide convincing evidence for Fucosyl-GM1 as a highly specific tumor antigen, which may be targeted by an immunotherapeutic. 10 Accordingly, agents that recognize Fucosyl-GM1, and methods of using such agents, are desired. Summary The present disclosure provides isolated monoclonal antibodies, in particular human monoclonal antibodies, that bind to Fucosyl-GM1 and that exhibit numerous desirable 15 properties. These properties include high affinity binding to Fucosyl-GMI and binding to the human small cell lung cancer cell line DMS-79 (Human SCLC ATCC # CRL-2049), Also provided are methods for treating a variety of Fucosyl-GM1 mediated diseases using the antibodies and compositions of this disclosure. In one aspect, this disclosure pertains to an isolated monoclonal antibody, or an antigen 20 binding portion thereof, wherein the antibody (a) specifically binds to Fucosyl-GM1; and (b) binds to the human small cell lung cancer cell line DMS-79 (Human SCLC ATCC # CRL-2049). Preferably the antibody is a human antibody, although in alternative embodiments the antibody can be a murine antibody, a chimeric antibody or humanized antibody. In another embodiment this disclosure provides an isolated monoclonal antibody, or 25 antigen binding protion thereof, wherein the antibody cross-competes for binding to Fucosyl GM1 with a reference antibody, wherein the reference antibody (a) specifically binds to Fucosyl GMI; and (b) binds to the human small cell lung cancer cell line DMS-79 (Human SCLC ATCC # CRL-2049). In certain embodiments, the reference antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1; and (b) a light chain WO 2007/067992 PCT/US2006/061817 3 variable region comprising the amino acid sequence of SEQ ID NO:7. In further embodiments, the reference antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:8. In other embodiments, the reference antibody comprises: (a) a heavy 5 chain variable region comprising the amino acid sequence of SEQ ID NO:3; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:9. In still further embodiments, the reference antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10. In yet further embodiments, the reference antibody [0 comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11. In even further embodiments, the reference antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12. l5 In one aspect, this disclosure pertains to an isolated monoclonal antibody, or an antigen binding portion thereof, comprising a heavy chain variable region that is the product of or derived from a human VH 3-48 gene, wherein the antibody specifically binds Fucosyl-GMI. This disclosure further provides an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a light chain variable region that is the product of or derived from a human 20 Vx L15 gene, wherein the antibody specifically binds Fucosyl-GMI. A preferred combination comprises: (a) a heavy chain variable region CDR1 -comprising SEQ ID NO:13; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 19; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:25; 25 (d) a light chain variable region CDR1 comprising SEQ ID NO:3 1; (e) a light chain variable region CDR2 comprising SEQ ID NO:37; and (f) a light chain variable region CDR3 comprising SEQ ID NO:43. Another preferred combination comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO:14; 30 (b) a heavy chain variable region CDR2 comprising SEQ ID NO:20; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:26; WO 2007/067992 PCT/US2006/061817 4 (d) a light chain variable region CDRI comprising SEQ ID NO:32; (e) a light chain variable region CDR2 comprising SEQ ID NO:38; and (0 a light chain variable region CDR3 comprising SEQ ID NO:44. Another preferred combination comprises: 5 (a) a heavy chain variable region CDRJ comprising SEQ ID NO: 15; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:2 1; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:27; (d) a light chain variable region CDR1 comprising SEQ ID NO:33; (e) a light chain variable region CDR2 comprising SEQ ID NO:39; and ) (f) a light chain variable region CDR3 comprising SEQ ID NO:45. Another preferred combination comprises: (a) a heavy chain variable region CDRI comprising SEQ ID NO:16; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:22; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:28; 5 (d) a light chain variable region CDR1 comprising SEQ LD NO:34; (e) a light chain variable region CDR2 comprising SEQ ID NO:40; and (f) a light chain variable region CDR3 comprising SEQ ID NO:46. Another preferred combination comprises: (a) a heavy chain variable region CDRI comprising SEQ ID NO:17; 0 (b) a heavy chain variable region CDR2 comprising SEQ ID NO:23; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:29; (d) a light chain variable region CDRI comprising SEQ ID NO:35; (e) a light chain variable region CDR2 comprising SEQ ID NO:41; and (f) a light chain variable region CDR3 comprising SEQ ID NO:47. 5 Another preferred combination comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO:I 8; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:24; (c) a heavy chain variable region CDR.3 comprising SEQ ID NO:30; (d) a light chain variable region CDRI comprising SEQ ID NO:36; 0 (e) a light chain variable region CDR2 comprising SEQ ID NO:42; and (f) a light chain variable region CDR3 comprising SEQ ID NO:48.

WO 2007/067992 PCT/US2006/06 817 Other preferred antibodies of this disclosure, or antigen binding portions thereof comprise: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:-1; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:7. 5 Another preferred combination comprises, (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:8. Another preferred combination comprises: 0 (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NQ:3; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:9. Another preferred combination comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4; 5 and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10. Another preferred combination comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5; and 20 (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:11. Another preferred combination comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:12. 25 The antibodies of this disclosure can be, for example, full-length antibodies, for example of an IgGI or JgG4 isotype. Alternatively, the antibodies can be antibody fragments, such as Fab or Fab'2 fragments, or single chain antibodies. This disclosure also provides an immunoconjugate comprising an antibody of this disclosure, or antigen-binding portion thereof, linked to a therapeutic agent, such as a cytotoxin 30 or a radioactive isotope, This disclosure also provides a bispecific molecule comprising an antibody, or antigen-binding portion thereof, of this disclosure, linked to a second functional WO 2007/067992 PCT/US2006/061817 6 moiety having a different binding specificity than said antibody, or antigen binding portion thereof. Compositions comprising an antibody, or antigen-binding portion thereoft or immunoconjugate or bispecific molecule of this disclosure and a pharmaceutically acceptable 5 carrier are also provided. Nucleic acid molecules encoding the antibodies, or antigen-binding portions thereof, of this disclosure are also encompassed by this disclosure, as well as expression vectors comprising such nucleic acids and host cells comprising such expression vectors. Moreover, this disclosure provides a transgenic mouse comprising human immunoglobulin heavy and light chain 3 transgenes, wherein the mouse expresses an antibody of this disclosure, as well as hybridomas prepared from such a mouse, wherein the hybridoma produces the antibody of this disclosure. In yet another aspect, this disclosure provides a method of treating or preventing a disease characterized by growth of tumor cells expressing Fucosyl-GM1, comprising administering to the subject the antibody, or antigen-binding portion thereof, of this disclosure in 5 an amount effective to treat or prevent the disease. The disease can be, for example, cancer, e.g., lung cancer (including small cell lung cancer). In a preferred embodiment, this disclosure provides a method of treating cancer in vivo using an anti-Fucosyl-GMI antibody. The anti-Fucosyl-GM1 antibody may be a murine, chimeric, humanized or human antibody. Examples of other cancers that may be treated using 0 the methods of this disclosure include lung cancer, including small cell lung cancer and non small cell lung cancer, renal cancer (e.g, renal cell carcinoma), glioblastoma, brain tumors, chronic or acute leukemias including acute lymphocytic leukemia (ALL), adult T-cell leukemia (T-ALL), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphomas (e.g, Hodgkin's and non-Hodgkin's lymphoma, lymphocytic lymphoma, 5 primary CNS lymphoma, T-cell lymphoma, BurkitVs lymphoma, anaplastic large-cell lymphomas (ALCL), cutaneous T-cell lymphomas, nodular small cleaved-cell lymphomas, peripheral T-cell lymphomas, Lennert's lymphomas, immunoblastic lymphomas, T-cell leukemia/lymphomas (ATLL), entroblastic/centrocytic (eb/ce) follicular lymphomas cancers, diffuse large cell lymphomas of B lineage, angioinimunoblastic lymphadenopathy (AILD)-like T 0 cell lymphoma and HIV associated body cavity based lymphomas), embryonal carcinomas, undifferentiated carcinomas of the rhino-pharynx (e.g., Schmincke's tumor), Castleman's WO 2007/067992 PCT/US2006/061817 7 disease, Kaposi's Sarcoma, multiple myeloma, Waldenstrom's macroglobulinemia and other B cell lymphomas, nasopharangeal carcinomas, bone cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, 5 carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central 0 nervous system (CNS), tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, epidenmoid cancer, squamous cell cancer, environmentally induced cancers including those induced by asbestos, e.g., mesothelioma and combinations of said cancers. Other features and advantages of the instant disclosure will be apparent from the following detailed description and examples which should not be construed as limiting. The 5 contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. Brief Description of the Drawings Figure IA shows the nucleotide sequence (SEQ ID NO:49) and amino acid sequence (SEQ ID NO: 1) of the heavy chain variable region of the 5B I human monoclonal antibody, The 20 CDRi (SEQ ID NO:13), CDR2 (SEQ ID NO:19) and CDR3 (SEQ ID NO:25) regions are delineated and the V, D and J germline derivations are indicated. Figure 1B shows the nucleotide sequence (SEQ ID NO:55) and amino acid sequence (SEQ ID NO:7) of the light chain variable region of the 5B 1 human monoclonal antibody. The CDRI (SEQ ID NO:3 1), CDR2 (SEQ ID NO:37) and CDR3 (SEQ ID NO:43) regions are 25 delineated and the V and J germline derivations are indicated. Figure 2A shows the nucleotide sequence (SEQ ID NO:50) and amino acid sequence (SEQ ID NO:2) of the heavy chain variable region of the 5Bla human monoclonal antibody. The CDRl (SEQ ID NO:14), CDR2 (SEQ ID NO:20) and CDR3 (SEQ ID NO:26) regions are delineated and the V, D and J germline derivations are indicated.

8 Figure 2B shows the nucleotide sequence (SEQ ID NO:56) and amino acid sequence (SEQ ID NO:8) of the light chain variable region of the 5Bla human monoclonal antibody. The CDR] (SEQ ID NO:32), CDR2 (SEQ ID NO:38) and CDR3 (SEQ ID NO:44) regions are delineated and the V and J germline derivations are indicated. Figure 3A shows the nucleotide sequence (SEQ ID NO:51) and amino acid sequence (SEQ ID NO:3) of the heavy chain variable region of the 7D4 human monoclonal antibody. The CDR1 (SEQ ID NO: 15), CDR2 (SEQ ID NO:21) and CDR3 (SEQ ID NO:27) regions are delineated and the V, D and J germline derivations are indicated. Figure 3B shows the nucleotide sequence (SEQ ID NO:57) and amino acid sequence (SEQ ID NO:9) of the light chain variable region of the 7D4 human monoclonal antibody. The CDR1 (SEQ ID NO:33), CDR2 (SEQ ID NO:39) and CDR3 (SEQ ID NO:45) regions are delineated and the V and J germline derivations are indicated. Figure 4A shows the nucleotide sequence (SEQ ID NO:52) and amino acid sequence (SEQ ID NO:4) of the heavy chain variable region of the 7E4 harnan monoclonal antibody. The CDR I (SEQ ID NO: 16), CDR2 (SEQ ID NO:22) and CDR3 (SEQ ID NO:28) regions are delineated and the V, D and J germline derivations are indicated. Figure 4B shows the nucleotide sequence (SEQ ID NO:58) and amino acid sequence (SEQ ID NO:10) of the light chain variable region of the 7E4 human monoclonal antibody. The CDRI (SEQ ID NO:34), CDR2 (SEQ ID NO:40) and CDR3 (SEQ ID NO:46) regions are delineated and the V and J germline derivations are indicated. Figure 5A shows the nucleotide sequence (SEQ ID NO:53) and amino acid sequence (SEQ ID NO:5) of the heavy chain variable region of the 13138 human monoclonal antibody. The CDR1 (SEQ ID NO:17), CDR2 (SEQ ID NO:23) and CDR3 (SEQ ID NO:29) regions are delineated and the V, D and J germline derivations are indicated. Figure 5B shows the nucleotide sequence (SEQ ID NO:59) and amino acid sequence (SEQ ID NO:1) of the light chain variable region of the 13B8 human monoclonal antibody. The CDR1 (SEQ ID NO:35), CDR2 (SEQ ID NO:4 1) and CDR3 (SEQ ID NO:47) regions are delineated and the V and J germline derivations are indicated. Figure 6A shows the nucleotide sequence (SEQ ID NO:54) and amino acid sequence (SEQ ID NO:63) of the heavy chain variable region of the 3C4 human monoclonal antibody. C:\Norb\DCC\KLLJ93609I DOC - 19/10/11 9 The CDR1 (SEQ IDNO:65), CDR2 (SEQ IDNO:66) and CDR3 (SEQ ID No:67) regions are delineated and the V, D and J germline derivations are indicated. Figure 6B shows the nucleotide sequence (SEQ ID NO:60) and amino acid sequence (SEQ ID NO:64) of the light chain variable region of the 3C4 human monoclonal antibody. The CDRI (SEQ ID NO:68), CDR2 (SEQ ID NO:69) and CDR3 (SEQ ID NO:70) regions are delineated and the V and J germline derivations are indicated. Figure 7 shows the alignment of the amino acid sequences of the heavy chain variable regions of 5B1, 5B Ia, 7D4, 7E4, 13B8 and 18D5 with the human germline VH 3-48 amino acid sequence (SEQ ID NO:6 1). Figure 8 shows the alignment of the amino acid sequences of the light chain variable regions of 5B], 5B la, 7D4, 7E4, 13B8 and 18D5 with the human germline Vk L15 amino acid sequence (SEQ ID NO:62). Figures 9A-C show the results of ELISA experiments demonstrating that human monoclonal antibodies against Fucosyl-GM1 specifically bind to Fucosyl-GM1. Figures 1 OA-C show the results of whole-cell ELISA experiments demonstrating that human monoclonal antibodies against Fucosyl-GM1 specifically bind to cells expressing Fucosyl-GMI. Figures I1 IA-C show the results of flow cytometry experiments demonstrating that (A and B) the human monocional'antibodies against Fucosyl-GM1 bind the cell surface of cell lines DMS79 and H-4-II-E expressing Fucosyl-GM1, and that (C) the DMS79 cells were found to continue expressing Fucosyl-GM1 in vivo (i.e., after implantation in a mouse). Figures 12A and B show the results of Hum-Zap internalization experiments demonstrating that human monoclonal antibodies against Fucosyl-GMI can internalize into Fucosyl-GM1+ cells. Figures 13A and B show the results of a complement dependent cytotoxicity (CDC) cell proliferation assay demonstrating that human monoclonal anti-Fucosy]-GMI antibodies kill cell lines (A) DMS79 and (B) H-4-II-E that express Fucosyl-GMI. Figures 14A and B show the results of an antibody dependent cell cytotoxicity (ADCC) cell proliferation assay demonstrating that human monoclonal anti-Fucosyl-GM I antibodies kill cell lines expressing Fucosyl-GMI in the absence of CD16 blockade. C:\NRPOMb\DCC\KjLj936809.I.DOC - 910111 WO 2007/067992 PCT/US2006/061817 10 Figures 15A-C show the tumor volume over time in individual SCID mice that were implanted with DMS79 small cell lung cancer tumor cells (Fucosyl-GM I'). After a tumor was established, the mice were treated five times with one of the following therapies: (A) PBS (vehicle control); (B) human IgGi (isotype control) at 30 mg/kg per mouse; (C) anti-Fucosyl GMI monoclonal antibody 5B1 at 10 mg/kg per mouse; (D) anti-Fucosyl-GMI monoclonal antibody 5B1 at 30 mg/kg per mouse; (E) anti-Fucosyl-GM1 monoclonal antibody 7E4 at 10 mg/kg per mouse; or (F) anti-Fucosyl-GMI monoclonal antibody 7E4 at 30 mg/kg per mouse. The tumor volume on the first day of treatment was about 200 mm 3 . Figures 16A and B show the mean and median tumor volume, respectively, of the mice shown in Figure 15. Figure 17 shows the mean group weight of the mice shown in Figure 15. Detailed Description In one aspect, the present disclosure relates generally to isolated monoclonal antibodies, particularly human monoclonal antibodies that bind specifically to Fucosyl-GMI, In certain embodiments, the antibodies of this disclosure exhibit one or more desirable functional properties, such as high affinity binding to Fucosyl-GMI and/or the ability to inhibit growth of tumor cells in vitro or in vivo. In certain embodiments, the antibodies of this disclosure are derived from particular heavy and light chain gernline sequences and/or comprise particular structural features such as CDR regions comprising particular amino acid sequences. This 0 disclosure provides isolated antibodies, methods of making such antibodies, immunoconjugates and bispecific molecules comprising such antibodies and pharmaceutical compositions containing the antibodies, immunconjugates or bispecific molecules of this disclosure. This disclosure also relates to methods of using the antibodies, such as to treat diseases such as cancer. 5 In order that the present disclosure may be more readily understood, certain terms are first defied. Additional definitions are set forth throughout the detailed description. The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in WO 2007/067992 PCT/US2006/06817 11 selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. A "signal transduction pathway" refers to the biochemical relationship between a variety 5 of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. As used herein, the phrase "cell surface receptor" includes, for example, molecules and complexes of molecules capable of receiving a signal and the transmission of such a signal across the plasma membrane of a cell. An example of a,"cell surface receptor" of the present disclosure is the Fucosyl-GMI receptor. 0 The term "antibody" as referred to herein includes whole antibodies and any antigen binding fragment (i.e., "antigen-binding portion") or single chains thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain 5 constant region. The heavy chain constant region is comprised of three domains, CHi, Cm and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, 20 termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR 1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the inmunoglobulin to host tissues or factors, including various cells of the immune 25 system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., Fucosyl-GM1). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding 30 fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, C and CHI domains; (ii) a WO 2007/067992 PCT/US206/0061817 12 F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546), which consists of a Vu domain; and (vi) an isolated 5 complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and.VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables there to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et aL. (1988) Science 242:423-426; and Huston et al (1988) Proc. Natl. Acad. Sci. USA ) 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. An "isolated antibody", as used herein, is intended to refer to an antibody that is 5 substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds Fucosyl-GM I is substantially free of antibodies that specifically bind antigens other than Fucosyl-GMI). Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein 0 refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, 5 the constant region also is derived from human gerniline immunoglobulin sequences. The human antibodies of this disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the WO 2007/067992 PCT/US2006/061817 13 germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are 5 derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. The term "recombinant human antibody", as used herein, includes all human antibodies 0 that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, 5 and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal 20 transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. As used herein, "isotype" refrs to the antibody class (e.g., IgM or IgGl) that is encoded 25 by the heavy chain constant region genes. The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen." The term "human antibody derivatives" refers to any modified form of the human 30 antibody, e.g., a conjugate of the antibody and another agent or antibody.

WO 2007/067992 PCT/US2006/061817 14 The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences. The term "chimeric antibody" is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody. As used herein, an antibody that "specifically binds to Fucosyl-GM1" is intended to refer ) to an antibody that binds to Fucosyl-GM1 with a K 0 of 1 x 10- 7 M or less, more preferably 5 x 10 4 M or less, more preferably 1 x 10- M or less, more preferably 5 x 10- 9 M or less, The term "Kassoo" or "KB", as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term "Kdis" or "Kd," as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term 5 "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of K 0 to Ka (i.e,. K/K) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art. A preferred method for determining the KD of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore@ system. 0 As used herein, the term "high affinity" for an IgG antibody refers to an antibody having a Kpof 10.8 M or less, more preferably I0 M or less and even more preferably 1010 M or less for a target antigen. However, "high affinity" binding can vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to an antibody having a KD of 104 M or less, more preferably 10"8 M or less, even more preferably 10'9 M or less. 5 As used herein, the term "subject" includes any human or nonhuman animal. The term "nonhuman animal" includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Various aspects of this disclosure are described in further detail in the following subsections. 0 WO 2007/067992 PCT/US2006/06 1817 15 Anti-Fucosyl-GMI Antibodies The antibodies of this disclosure are characterized by particular functional features or properties of the antibodies. For example, the antibodies bind specifically to Fucosyl-GML, preferably Fucosyl-GMI. Preferably, an antibody of this disclosure binds to Fucosyl-GM1 with 5 high affinity, for example with a KD of 1 x 10-7 M or less. The anti-Fucosyl-GMI antibodies of this disclosure preferably exhibit one or more of the following characteristics: (a) specifically binds to Fucosyl-GMI; and (b) binds to the human small cell lung cancer cell line DMS-79 (Human SCLC ATCC # CRL-2049). 10 Preferrably, the antibody binds to Fucosyl-GMI with a KD of 5 x 10~ 8 M or less, binds to Fucosyl-GM1 with a KD of I x 10'8 M or less, binds to Fucosyl-GMI with a KD of 5 x 10 9 M or less, or binds to Fucosyl-GMI with a Kt of between 1 x I0-'M and 1 x 10-' M or less. Standard assays to evaluate the binding ability of the antibodies toward Fucosyl-GMI are known in the art, including for example, ELISAs, Western blots and RIAs. The binding kinetics (e.g., 15 binding affinity) of the antibodies also can be assessed by standard assays known in the art, such as by'ELISA, Scatchard and Biacore analysis. Monoclonal Antibodies 5B1, 5Bla, 7D4, 7E4, 13B8 and 18D5 Preferred antibodies of this disclosure are the human monoclonal antibodies 5B1, 5B 1 a, 20 7D4, 7E4, 13B8 and 18D5, isolated and structurally characterized as described in Examples 1 and 2. The Vsj amino acid sequences of 5B1, 5Bl a, 7D4, 7E4, 13B8 and 18D5 are shown in SEQ ID NOs:1, 2,3, 4, 5 and 6, respectively. The VL amino acid sequences of 5B1, 5Bla, 7D4, 7E4, 13B8 and 18D5 are shown in SEQ ID NOs:7, 8, 9, 10, 11 and 12, respectively. Given that each of these antibodies can bind to Fucosyl-GM1, the VH and VL sequences 25 can be "mixed and matched" to create other anti-Fucosyl-GMI binding molecules of this disclosure. Fucosyl-GMI binding of such "mixed and matched" antibodies can be tested using the binding assays described above and in the Examples (e.g., ELISAs). Preferably, when VH and VL chains are mixed and matched, a VH sequence from a particular VHNL pairing is replaced with a structurally similar VH sequence. Likewise, preferably a V sequence from a particular 30 VHNL pairing is replaced with a structurally similar Vt sequence.

WO 2007/067992 PCT/US2006/061817 16 Accordingly, in one aspect this disclosure provides an isolated monoclonal antibody, -or antigen binding portion thereof comprising: (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 4, 5 and 6; and go (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;,7, 8, 9, 10, 11 and 12; wherein the antibody specifically binds Fucosyl-GM1, preferably Fucosyl-GMl. Preferred heavy and light chain combinations include (a) a heavy chain variable region comprising the amino acid sequence of ) SEQ ID NO:1; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:7; or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:8; or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:9; or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4; and (b) a light chain variable region comprising the amino acid sequence of SEQ 0 ID NO:10; or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:11; or (a) a heavy chain variable region comprising the amino acid sequence of 5 SEQ ID NO:6; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:12. In another aspect, this disclosure provides antibodies that comprise the heavy chain and light chain CDRIs, CDR2s and CDR3s of5B1, 5Bla, 7D4, 7E4, 13118 and 18D5, or combinations thereof. The amino acid sequences of the VH CDRls of 5B1, 5Bl a, 7D4, 7E4, 0 13B8 and 18D5 are shown in SEQ IDNOs:13, 14,15,16,17 and 18, respectively. The amino acid sequences of the VH CDR2s of 5B1, 5BIa, 7D4, 7E4, 13B8 and 18D5 are shown in SEQ ID WO 2007/067992 PCTIUS2006/061817 17 NOs: 19, 20, 21, 22, 23 and 24, respectively. The amino acid sequences of the VH CDR3s of 51, 5Bla, 7D4, 7E4, 13B8 and 18D5 are shown in SEQ ID NOs:25, 26, 27, 28, 29 and 30, respectively. The amino acid sequences of the Vk CDR1s of 5B1, 5B la, 7D4, 7E4, 13B8 and 18D5 are shown in SEQ ID NOs:31, 32, 33, 34, 35 and 36, respectively. The amino acid 5 sequences of the Vk CDR2s of 5B1, 5Bla, 7D4, 7E4, 13B8 and 18D5 are shown in SEQ ID NOs:37, 38,39,40,41 and 42, respectively. The amino acid sequences of the Vk CDR3s of 5B1, 5Bla, 7D4, 7E4, 13B8 and 18D5 are shown in SEQ ID NOs:43, 44, 45, 46,47 and 48, respectively. The CDR regions are delineated using the Kabat system (Kabat, E. A., et a! (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and 0 Human Services, NIH Publication No. 91-3242). Given that each of these antibodies can bind to Fucosyl-GMI and that antigen-binding specificity is provided primarily by the CDR1, CDR2, and CDR3 regions, the VH CDR1, CDR2, and CDR3 sequences and Vk CDRL, CDR2, and CDR3 sequences can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and match, although each antibody must 5 contain a VH 1 CDRl, CDR2, and CDR3 and a Vk CDRI, CDR2, and CDR3) to create other anti Fucosyl-GMi binding molecules of this disclosure. Fucosyl-GMI binding of such "mixed and matched" antibodies can be tested using the binding assays described above and in the Examples (e.g., ELISAs, Biacore analysis). Preferably, when VH CDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequence from a particular VH sequence is replaced with a 20 structurally similar CDR sequence(s). Likewise, when Vk CDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequence from a particular Vk sequence preferably is replaced with a structurally similar CDR sequence(s). It will be readily apparent to the ordinarily skilled artisan that novel VIi and VL sequences can be created by substituting one or more Vii and/or VL CDR region sequences with structurally similar sequences from the CDR sequences 25 disclosed herein for monoclonal antibodies antibodies 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5. Accordingly, in another aspect, this disclosure provides an isolated monoclonal antibody, or antigen binding portion thereof comprising: (a) a heavy chain variable region CDRI comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:13, 14, 15,16,17 and 18; 30 (b) a heavy chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:19, 20, 21, 22, 23 and 24; WO 2007/067992 PCT/US2006/061817 18 (c) a heavy chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:25, 26, 27,28, 29 and 30; (d) a light chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:31, 32, 33, 34, 35 and 36; (e) a light chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:37, 38, 39, 40, 41 and 42; and (f) a light chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:43, 44, 45, 46, 47 and 48; wherein the antibody specifically binds Fucosyl-GM1, preferably Fucosyl-GM1. In a preferred embodiment, the antibody comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 13; (b) aheavy chain variable region CDR2 comprising SEQ ID NO:19; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:25; (d) a light chain variable region CDR] comprising SEQ ID NO:31; (e) a light chain variable region CDR2 comprising SEQ ID NO:37; and (f a light chain variable region CDR3 comprising SEQ ID NO:43. In another preferred embodiment, the antibody comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO:14; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:20; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:26; (d) a light chain variable region CDR1 comprising SEQ ID NO:32; (e) a light chain variable region CDR2 comprising SEQ ID NO:38; and (f) a light chain variable region CDR3 comprising SEQ ID NO:44. In another preferred embodiment, the antibody comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 15; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:21; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:27; (d) a light chain variable region CDRI comprising SEQ ID NO:33; (e) a light chain variable region CDR2 comprising SEQ ID NO:39; and (f) a light chain variable region CDR3 comprising SEQ ID NO:45. In another preferred embodiment, the antibody comprises: WO 2007/067992 PCT/US2006/061817 19 (a) a heavy chain variable region CDR1 comprising SEQ ID NO:I6; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:22; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:28; (d) a light chain variable region CDR1 comprising SEQ ID NO:34; 5 (e) a light chain variable region CDR2 comprising SEQ ID NO:40; and (0 a light chain variable region CDR3 comprising SEQ ID NO:46. In another preferred embodiment, the antibody comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO:17; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:23; .0 (c) a heavy chain variable region CDR3 comprising SEQ ID NO:29; (d) a light chain variable region CDR1 comprising SEQ ID NO:35; (e) a light chain variable region CDR2 comprising SEQ ID NO:41; and (f) a light chain variable region CDR3 comprising SEQ ID NO:47. In another preferred embodiment, the antibody comprises: 5 (a) a heavy chain variable region CDR1 comprising SEQ ID NO:1 8; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:24; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:30; (d) a light chain variable region CDRI comprising SEQ ID NO:36; (e) a light chain variable region CDR2 comprising SEQ U) NO:42; and 20 (f) a light chain variable region CDR3 comprising SEQ ID NO:48. It is well known in the art that the CDR3 domain, independently from the CDR1 and/or CDR2 domain(s), alone can determine the binding specificity of an antibody for a cognate antigen and that multiple antibodies can predictably be generated having the same binding specificity based on a common CDR3 sequence. See, for example, Klimka et aL, British J of 25 Cancer j3(2):252-260 (2000) (describing the production of a humanized anti-CD30 antibody using only the heavy chain variable domain CDR3 of urine anti-CD30 antibody Ki-4); 3eiboer el al, J MoL Biol 296:833-849 (2000) (describing recombinant epithelial glycoprotein-2 (EGP 2) antibodies using only the heavy chain CDR3 sequence of the parental murine MOC-31 anti EGP-2 antibody); Rader et al, Proc. Natl Acad. Si. U.SA. 95:8910-8915 (1998) (describing a 30 panel of humanized anti-integrin cxp3 antibodies using a heavy and light chain variable CDR3 WO 2007/067992 PCT/US2006/06]1817 20 domain of a murine anti-integrin avPs antibody LM609 wherein each member antibody comprises a distinct sequence outside the CDR3 domain and capable of binding the same epitope as the parent during antibody with affinities as high or higher than the parent murine antibody); Barbas et al., J Am. Chem. Soc. 116:2161-2162 (1994) (disclosing that the CDR3 domain provides the most significant contribution to antigen binding); Barbas et al., Proc NatL. Acad. Sci U.SA. 92:2529-2533 (1995) (describing the grafting of heavy chain CDR3 sequences of three Fabs (SI-1, SI-40, and SI-32) against human placental DNA onto the heavy chain of an anti-tetanus toxoid Fab thereby replacing the existing heavy chain CDR3 and demonstrating that the CDR3 domain alone conferred binding specificity); and Ditzel et al., J Immunol. 157:739 749 (1996) (describing grafting studies wherein transfer of only the heavy chain CDR3 of a parent polyspecific Fab LNA3 to a heavy chain of a monospecific IgG tetanus toxoid-binding Fab p 3 13 antibody was sufficient to retain binding specificity of the parent Fab). Each of these references is hereby incorporated by reference in its entirety. Accordingly, within certain aspects, the present disclosure provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domain from a non-human antibody, such as a mouse or rat antibody, wherein the monoclonal antibody is capable of specifically binding to Fucosyl-GMI. Within some embodiments, such antibodies comprising one or more heavy and/or light chain CDR3 domain from a non-human antibody (a) are capable of competing for binding with; (b) retain the functional characteristics; (c) bind to the same 0 epitope; and/or (d) have a similar binding affinity as the corresponding parental non-human antibody. Within other aspects, the present disclosure provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domain from a first human antibody, such as, for example, a human antibody obtained from a non-human animal, wherein the first human 5 antibody is capable of specifically binding to Fucosyl-GM] and wherein the CDR3 domain from the first human antibody replaces a CDR3 domain in a human antibody that is lacking binding specificity for Fucosyl-GMI to generate a second human antibody that is capable of specifically binding to Fucosyl-GM1. Within some embodiments, such inventive antibodies comprising one or more heavy and/or light chain CDR3 domain from the first human antibody (a) are capable of 0 competing for binding with; (b) retain the functional characteristics; (c) bind to the same epitope; and/or (d) have a similar binding affinity as the corresponding parental first human antibody.

WO 2007/067992 PCT/US2006/061817 21 Antibodies Having Particular Germline Sequences In certain embodiments, an antibody of this disclosure comprises a heavy chain variable region from a particular germline heavy chain immunoglobulin gene and/or a light chain variable region from a particular germline light chain immunoglobulin gene. 5 For example, in a preferred embodiment, this disclosure provides an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region that is the product of or derived from a human VH 3-48 gene, wherein the antibody specifically binds Fucosyl-GMi, preferably Fucosyl-GMI. In another preferred embodiment, this disclosure provides an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a 0 light chain variable region that is the product of or derived from a human VK Li 5 gene, wherein the antibody specifically binds Fucosyl-GM1, preferably Fucosyl-GMI. In yet another preferred embodiment, this disclosure provides an isolated monoclonal antibody, or antigen-binding portion thereof, wherein the antibody: (a) comprises a heavy chain variable region that is the product of or derived from a 5 human VH 3-48 gene (which gene encodes the amino acid sequence set forth in SEQ ID NO: 61); (b) comprises a light chain variable region that is the product of or derived from a human VK L 15 gene (which gene encodes the amino acid sequence set forth in SEQ ID NO:62); and (c) specifically binds to Fucosyl-GMI. Examples of antibodies having VH and VK of VH 3-48 and VK L15, respectively, are 5B 1, 0 5Bla, 7D4, 7E4, 13B8 and 18D5. As used herein, a human antibody comprises heavy or light chain variable regions that is "the product of' or "derived from" a particular germline sequence if the variable regions of the antibody are obtained from a system that uses human geriline immunoglobulin genes. Such systems include immunizing a transgenic mouse carrying human inununoglobulin genes with the 5 antigen of interest or screening a human immunoglobulin gene library displayed on phage with the antigen of interest. A human antibody that is "the product of' or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in 0 sequence (i.e., greatest % identity) to the sequence of the human antibody. A human antibody that is "the product of' or "derived from" a particular human germline immunoglobulin sequence WO 2007/067992 PCT/US2006/061817 22 may contain amino acid differences as compared to the germline sequence, due to, for example, naturally-occurring somatic mutations or intentional introduction of site-directed mutation, However, a selected human antibody typically is at least 90% identical in amino acids sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as being human when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine genline sequences). In certain cases, a human antibody may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody may display no more than 5, or even no more than 4, 3, 2, or ] amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene. Homologous Antibodies In yet another embodiment, an antibody of this disclosure comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the preferred antibodies described herein, and wherein the antibodies retain the desired functional properties of the anti-Fucosyl-GMI antibodies of this disclosure. ) For example, this disclosure provides an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to an amino acid sequence selected from the group consisting of SEQ ID 5 NOs: 1, 2, 3, 4, 5 and 6; (b) the light chain variable region comprises an amino acid sequence that is at least 80% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs:7, 8, 9, 10, 11 and 12; and the antibody exhibits one or more of the following properties: (c) the antibody binds to Fucosyl-GM1 with a Kp of 1 x 104 M or less; WO 2007/067992 PCT/US2006/061817 23 (d) the antibody binds to the human small cell lung cancer cell line DMS-79 (Human SCLC ATCC # CRL-2049). In other embodiments, the VH and/or VL amino acid sequences may be 85%, 9 0 %O, 95%, 96%, 97%, 98% or 99% homologous to the sequences set forth above. An antibody having VH 5 and VL regions having high (i.e., 80% or greater) homology to the Vs and Vt regions of the sequences set forth above, can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ ID NOs:49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60, followed by testing of the encoded altered antibody for retained function (i.e., the functions set forth in (c) and (d) above) using the functional assays described herein. 0 As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. 5 The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below. The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Apple. Biosci., 4:11-17 (1988)) which has been 20 incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoL BioL 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, 25 and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Additionally or alternatively, the protein sequences of the present disclosure can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, etal. (1990)J MoL. BioL 215:403-10. BLAST protein searches can be 30 performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the antibody molecules of this disclosure. To obtain gapped WO 2007/067992 PCT/US2006/061817 24 alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997)Nucleic Acids Res. L5(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. (See www.ncbi.nlm.nih.gov). 5 Antibodies with Conservative Modifications In certain embodiments, an antibody of this disclosure comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein one or more of these CDR sequences [0 comprise specified amino acid sequences based on the preferred antibodies described herein (e.g, 531, 5Bl a, 7D4, 7E4, 1388 or 18D5), or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the anti-Fucosyl-GM1 antibodies of this disclosure. Accordingly, this disclosure provides an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising CDRI, CDR2, and .5 CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein: (a) the heavy chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs:25, 26, 27, 28, 29 and 30, and conservative modifications thereof; 20 (b) the light chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequence of SEQ ID NOs:43, 44, 45, 46, 47 and 48, and conservative modifications thereof; and the antibody exhibits one or more of the following properties: (c) specifically binds to Fucosyl-GMI; and 25 (d) the antibody binds to the human small cell lung cancer cell line DMS-79 (Human SCLC ATCC # CRL-2049). In a preferred embodiment, the heavy chain variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs:19, 20, 21, 22, 23 and 24, and conservative modifications thereof; and the light chain 30 variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs:37, 38, 39, 40, 41 and 42, and conservative WO 2007/067992 PCT/US2006/061817 25 modifications thereof. In another preferred embodiment, the heavy chain variable region CDR] sequence comprises an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs:13, 14, 15, 16, 17 and 18, and conservative modifications thereof; and the light chain variable region CDRl sequence comprises an amino acid sequence selected from 5 the group consisting of amino acid sequences of SEQ ID NOs:31, 32, 33, 34, 35 and 36, and conservative modifications thereof. As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino 10 acid substitutions, additions and deletions. Modifications can be introduced into an antibody of this disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families 15 include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g, threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, 20 histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of this disclosure can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the functions set forth in (c) and (d) above) using the functional assays described herein. 25 Antibodies that Bind to the Same Epitope as Anti-Fucosyl-GMI Antibodies of this disclosure In another embodiment this disclosure provides antibodies that bind to the same epitope on Fucosyl-GMI as any of the Fucosyl-GMI monoclonal antibodies of this disclosure (i.e., antibodies that have the ability to cross-compete for binding to Fucosyl-GMl with any of the monoclonal antibodies of this disclosure). In preferred embodiments, the reference antibody for 30 cross-competition studies can be the monoclonal antibody 5B1 (having VH and VL sequences as shown in SEQ ID NOs: I and 7, respectively), or the monoclonal antibody 5BIa (having VH and WO 2007/067992 PCT/US2006/06]l817 26 V, sequences as shown in SEQ ID NOs:2 and 8, respectively), or the monoclonal antibody 7D4 (having V4 and V sequences as shown in SEQ ID NOs:3 and 9, respectively), or the monoclonal antibody 7E4 (having V4 and VL sequences as shown in SEQ ID NOs:4 and 10, respectively), or the monoclonal antibody 13B8 (having VH and VL sequences as shown in SEQ 5 ID NOs:5 and 11, respectively), or or the monoclonal antibody 18D5 (having VH and VL sequences as shown in SEQ ID NOs:6 and 12, respectively), Such cross-competing antibodies can be identified based on their ability to cross-compete with 5B 1, 5Bla, 7D4, 7E4, 13B8 or 18D5 in standard Fucosyl-GMI binding assays, For example, BlAcore analysis, ELISA assays or flow cytometry may be used to demonstrate cross-competition with the antibodies of the 10 current disclosure. The ability of a test antibody to inhibit the binding of, for example, 5B 1, 5Bla, 7D4, 7E4, 31B8 or 18D5, to Fucosyl-GM1 demonstrates that the test antibody can compete with 5B1, 5Bla, 7D4, 7E4, 13B8 or 18D5 for binding to Fucosyl-GMI and thus binds to the same epitope on Fucosyl-GM I as 5B1, 5Bla, 7D4, 7E4, 13B8 or 18D5. In a preferred embodiment, the antibody that binds to the same epitope on Fucosyl-GM1 as 5B1, 5BIa, 7D4, 15 7E4, 31B8 or 18D5 is a human monoclonal antibody. Such human monoclonal antibodies can be prepared and isolated as described in the Examples. Engineered and Modified Antibodies An antibody of this disclosure further can be prepared using an antibody having one or 20 more of the VH and/or VL sequences disclosed herein as starting material to engineer a modified antibody, which modified antibody may have altered properties from the starting antibody. An antibody can be engineered by modifying one or more residues within one or both variable regions (i.e., VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by 25 modifying residues within the constant region(s), for example to alter the effector function(s) of the antibody. One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this 30 reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody- WO 2007/067992 PCT/US2006/061817 27 antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 5 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. et al. (1989) Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.) Accordingly, another embodiment of this disclosure pertains to an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region 10 comprising CDR1, CDR2, and CDR3 sequences comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:13, 14, 15, 16,17 and 18, SEQ ID NOs:19, 20,21, 22, 23 and 24 and SEQ ID NOs:25, 26, 27, 28, 29 and 30, respectively, and a light chain variable region comprising CDRI, CDR2, and CDR3 sequences comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:31,32, 33, 34, 35 and 36, SEQ IDNOs:37, 15 38, 39, 40,41 and 42 and SEQ ID NOs:43, 44,45, 46,47 and 48, respectively. Thus, such antibodies contain the VH and VL CDR sequences of monoclonal antibodies 5B1, 5B la, 7D4, 7E4, 13B8 or I 8D5 yet may contain different framework sequences from these antibodies. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA 20 sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam,ac.uk/vbase.), as well as in Kabat, E. A., et al (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al. (1992) "The Repertoire of Human Germline VH Sequences Reveals 25 about Fifty Groups of VH Segments with Different Hypervariable Loops" J Mol. Biol. 227:776 798; and Cox, J. P. L et al. (1994) "A Directory of Human Germ-line VH Segments Reveals a Strong Bias in their Usage" Eur. J Immunol. 24:827-836; the contents of each of which are expressly incorporated herein by reference. As another example, the germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database. 30 For example, the following heavy chain gemline sequences found in' the HCo7 HuMAb mouse are available in the accompanying Genbank accession numbers: 1-69 (NG_0010109, WO 2007/067992 PCT/US2006/06] 817 28 NT_024637 and BC070333), 3-33 (NG_0010109 and NT_024637) and 3-7 (NG 00i0109 and NT_024637). As another example, the following heavy chain germline sequences found in the HCol2 HuMAb mouse are available in the accompanying Genbank accession numbers: 1-69 (NO 0010109, NT 024637 and BC070333), 5-51 (NG0010109 and NT_024637), 4-34 5 (NO_0010109 and NT_024637), 3-30.3 (?) and 3-23 (AJ406678), Preferred framework sequences for use in the antibodies of this disclosure are those that are structurally similar to the framework sequences used by selected antibodies of this disclosure, e.g., similar to the V 1 3-48 framework sequences (SEQ ID NO:61) and/or the Vc L 15 framework sequences (SEQ ID NO:62) used by preferred monoclonal antibodies of this 10 disclosure. The VH CDR1, CDR2, and CDR3 sequences, and the VK CDR1, CDR2, and CDR3 sequences, can be grafted onto framework regions that have the identical sequence as that found in the germline immunoglobulin gene from which the framework sequence derive, or the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the gernline sequences. For example, it has been found that in certain instances it 15 is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al). Another type of variable region modification is to mutate amino acid residues within the V and/or VK CDR1, CDR2 and/or CDR3 regions to thereby improve one or more binding 20 properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as described herein and provided in the Examples. Preferably conservative modifications (as discussed above) are introduced. The mutations may be amino acid substitutions, additions or deletions, 25 but are preferably substitutions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered. Accordingly, in another embodiment, this disclosure provides isolated anti-Fucosyl-GMI monoclonal antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising: (a) a V CDRI region comprising an amino acid sequence selected from the 30 group consisting of SEQ ID NOs-13, 14, 15, 16, 17 and 18, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ WO 2007/067992 PCT/US20061817 29 ID NOs: 13, 14, 15, 16, 17 and 18; (b) a VJ CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:19, 20, 21, 22, 23 and 24, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NOs:19, 20, 21, 22, 23 and 24; (c) a VH CDR3 region comprising an amino 5 acid sequence selected from the group consisting of SEQ ID NOs:25, 26, 27, 28, 29 and 30, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NOs:25, 26, 27, 28, 29 and 30; (d) a VK CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 32, 33, 34, 35 and 36, or an amino acid sequence having one, two, three, four or five amino acid 10 substitutions, deletions or additions as compared to SEQ ID NOs:31, 32, 33, 34, 35 and 36; (e) a VK CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:37, 38, 39, 40, 41 and 42, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NOs:37, 38, 39, 40, 41 and 42; and (f) a VK CDR3 region comprising an amino acid sequence selected from the group 15 consisting of SEQ ID NOs:43, 44, 45,46, 47 and 48, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NOs:43, 44, 45, 46, 47 and 48. Engineered antibodies of this disclosure include those in which modifications have been made to framework residues within VH and/or Vx, e.g. to improve the properties of the antibody. 20 Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding gernline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework 25 sequences to the germline sequences from which the antibody is derived. For example, for 7E4, amino acid residue #11 (within FRI) of VH is a serine whereas this residue in the corresponding VH 3-48 germline sequence is a leucine. To return the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis 30 (e.g., residue #11 of FRI of the Vii of 7E4 can be "backmutated" from serine to leucine).

WO 2007/067992 PCT/US2006/061817 30 As another example, for 5B1, 5Bla, 7D4, 7E4, 13B8 and 18D5, amino acid residue #16 (within FRI) Of VH is a glutamic acid whereas this residue in the corresponding VH 3-48 germline sequence is a glycine. To return the framework region sequences to their gernline configuration, for example, residue #16 of the VH of5BI, 5Bla, 7D4, 7E4, 13B8 and 18D5 can 5 be "backnutated" from glutamic acid to glycine. Such "backinutated" antibodies are also intended to be encompassed by this disclosure. As another example, for 5B1, 5Bla, 7D4, 7E4, 13B8 and 18D5, amino acid residue #23 (within FRI) of V is a valine whereas this residue in the corresponding VH 3-48 germline sequence is an alanine. To return the framework region sequences to their germline 10 configuration, for example, residue #23 of the V 8 of5B1, 5Bla, 7D4, 7E4, 13B8 and 18D5 can be "backmutated" from valine to alanine. Such "backmutated" antibodies are also intended to be encompassed by this disclosure. As another example, for 7D4, amino acid residue #24 (within FRI) of V is a valine whereas this residue in the coresponding VH 348 germline sequence is an alanine. To return 15 the framework region sequences to their gennline configuration, for example, residue #24 of the

V

1 of 7D4 can be "backmutated" from valine to alanine. Such "backnutated" antibodies are also intended to be encompassed by this disclosure. As another example, for 13B8, amino acid residue #29 (within FRI) of VH is a leucine whereas this residue in the corresponding VH 3-48 germline sequence is an phenylalanine. To 20 return the framework region sequences to their germline configuration, for example, residue #29 of the VH of 13B8 can be "backinutated" from leucine to phenylalanine. Such "backmutated" antibodies are also intended to be encompassed by this disclosure. As another example, for 7D4, 13B8 and 18D5, amino acid residue #48 (within FR2) of VH is an isoleucine whereas this residue in the corresponding VH 3-48 germline sequence is a 25 valine. To return the framework region sequences to their germline configuration, for example, residue #48 (residue #13within FR2) of the VH of 7D4, 13B8 and 18D5 can be "backmutated" from isoleucineto valine. Such "backmutated" antibodies are also intended to be encompassed by this disclosure. As another example, for 7D4 and 18D5, amino acid residue #84 (within FR3) Of VH is a 30 serine whereas this residue in the corresponding VH 3-48 germline sequence is an asparagine. To return the framework region sequences to their germline configuration, for example, residue #84 WO 2007/067992 PCT/US2006/061817 31 (residue #18 within FR3) of the VH of 7D4 and 18D5 can be "backmutated" from serine to asparagine. Such "backmutated" antibodies are also intended to be encompassed by this disclosure. Another type of framework modification involves mutating one or more residues within 5 the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in father detail in U.S. Patent Publication No. 20030153043 by Carr et al. In addition or alternative to modifications made within the framework or CDR regions, [0 antibodies of this disclosure may be engineered to include modifications within the Fe region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, FEc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody of this disclosure may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again .5 to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat. In one embodiment, the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g,, increased or decreased. This approach is described 20 further in U.S. PatentNo. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half life of the antibody, More specifically, one or more amino acid mutations are 25 introduced into the CH2-CH3 domain interface region of the Fe-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al. In another embodiment, the antibody is modified to increase its biological half life. 30 Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 to Ward.

WO 2007/067992 PCT/US2006/06817 32 Alternatively, to increase the biological half life, the antibody can be altered within the CH I or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fe region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 by Presta et al. 5 In yet other embodiments, the Fe region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent .0 antibody. The effector ligand to which affinity is altered can be, for example, an Fe receptor or the CI component of complement. This approach is described in father detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al. In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered 5 Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6,194,551 by Idusogie et aL In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et at 20 In yet another example, the Fe region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 25 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is described further in PCT Publication WO 00/42072 by Presta. Moreover, the binding sites on human IgGI for FeyRI, FcyRIJ, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R1. et al. (2001) J Biol. Chem. 276:6591-6604). Specific mutations at 30 positions 256, 290, 298, 333, 334 and 339 were shown to improve binding to FEyRIII.

WO 2007/067992 PCT/US2006/061817 33 Additionally, the following combination mutants were shown to improve FcyRIII binding: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A. In still another embodiment the glycosylation of an antibody is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can 5 be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the 0 antibody for antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al. Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns 5 have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of this disclosure to thereby produce an antibody with altered glycosylation. For example, the cell 20 lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8' cell lines were created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 by Yamane et aL and Yamane-Ohnuki et 25 al. (2004) BiotechnolBioengL8:614-22). As another example, EP 1,176,195 by Hanai et aL. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the alpha 1,6 bond-related enzyme. Hanai et al. also describe cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the 30 Fc region of the antibody or does not have the enzyme activity, for example the rat myelorna cell WO 2007/067992 PCT/US2006/061817 34 line YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lec 3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al. (2002) J. Biol, Chem. 277:26733-26740). PCT Publication WO 5 99/54342 by Umana et al describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTLID) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GleNac structures which results in increased ADCC activity of the antibodies (see also Umana et al (1999) Nat. Biotech. 17:176-180). Alternatively, the fucose residues of the antibody may be cleaved off 10 using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies (Tarentino, A.L. et al. (1975) Biochem. 14:5516-23). Another modification of the antibodies herein that is contemplated by this disclosure is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g, serum) half life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is 15 reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG 20 that have been used to derivatize other proteins, such as mono (Cl-C10) alkoxy- or aryloxy polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of this disclosure. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al. 25 Methods of Engineering Antibodies As discussed above, the anti-Fucosyl-GMI antibodies having Vsj and VK sequences disclosed herein can be used to create new anti-Fucosyl-GMI antibodies by modifying the VH and/or VK sequences, or the constant region(s) attached thereto. Thus, in another aspect of this 30 disclosure, the structural features of an anti-Fucosyl-GM1 antibody of this disclosure, e.g. 5B1, 5B1 a, 7D4, 7E4, 13B8 or 18D5, are used to create structurally related anti-Fucosyl-GMI WO 2007/067992 PCT/US2006/061817 35 antibodies that retain at least one functional property of the antibodies of this disclosure, such as binding to Fucosyl-GMI. For example, one or more CDR regions of SB], 5Bla, 7D4, 7E4, 13B8 or I 8D5, or mutations thereof, can be combined recombinantly with known framework regions and/or other CDRs to create additional, recombinantly-engineered, anti-Fucosyl-GM1 5 antibodies of this disclosure, as discussed above. Other types of modifications include these described in the previous section. The starting material for the engineering method is one or more of the VH and/or VK sequences provided herein, or one or more CDR regions thereof. To create the engineered antibody, it is not necessary to actually prepare (i.e., express as a protein) an antibody having one or more of the VH and/or VK sequences provided herein, or one or more [0 CDR regions thereof. Rather, the information contained in the sequence(s) is used as the starting material to create a "second generation" sequence(s) derived from the original sequence(s) and then the "second generation" sequence(s) is prepared and expressed as a protein. Accordingly, in another embodiment, this disclosure provides a method for preparing an anti-Fucosyl-GM1 antibody comprising: .5 (a) providing: (i) a heavy chain variable region antibody sequence comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs:13, 14, 15, 16, 17 and 18, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, 22, 23 and 24, and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs:25, 26, 27, 28, 29 and 30; and/or (ii) a light chain variable region antibody sequence comprising a CDRI sequence 20 selected from the group consisting of SEQ ID NOs:31, 32, 33, 34, 35 and 36, a CDR2 sequence selected from the group consisting of SEQ ID NOs:37, 38, 39, 40, 41 and 42, and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs:43, 44, 45, 46, 47 and 48; (b) altering at least one amino acid residue within the heavy chain variable region antibody sequence and/or the light chain variable region antibody sequence to create at least one 25 altered antibody sequence; and (c) expressing the altered antibody sequence as a protein. Standard molecular biology techniques can be used to prepare and express the altered antibody sequence. Preferably, the antibody encoded by the altered antibody sequence(s) is one that retains 30 one, some or all of the functional properties of the anti-Fucosyl-GM1 antibodies described herein, which functional properties include, but are not limited to: WO 2007/067992 PCT/US2006/061817 36 (a) the antibody binds to Fucosyl-GM1 with a Kr, of 1 x 10 7 M or less; (b) binds to the human small cell lung cancer cell line DMS-79 (Human SCLC ATCC # CRL-2049). 5 The functional properties of the altered antibodies can be assessed using standard assays available in the art and/or described herein, such as those set forth in the Examples (e.g, flow cytometry, binding assays). In certain embodiments of the methods of engineering antibodies of this disclosure, mutations can be introduced randomly or selectively along all or part of an anti-Fucosyl-GM1 0 antibody coding sequence and the resulting modified anti-Fucosyl-GM1 antibodies can be screened for binding activity and/or other functional properties as described herein. Mutational methods have been described in the art. For example, PCT Publication WO 02/092780 by Short describes methods for creating and screening antibody mutations using saturation mutagenesi s, synthetic ligation assembly, or a combination thereof. Alternatively, PCT Publication WO 5 03/074679 by Lazar et al. describes methods of using computational screening methods to optimize physiochemical properties of antibodies. Nucleic Acid Molecules Encoding Antibodies of this disclosure Another aspect of this disclosure pertains to nucleic acid molecules that encode the .0 antibodies of this disclosure. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsC1 banding, column chromatography, agarose gel electrophoresis and others well 5 known in the art. See, F. Ausubel, et at, ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. A nucleic acid of this disclosure can be, for example, DNA or RNA and may or may not contain intronic sequences, In a preferred embodiment, the nucleic acid is a cDNA molecule, Nucleic acids of this disclosure can be obtained using standard molecular biology 0 techniques. For antibodies expressed by hybridomas (e.g, hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cDNAs encoding the 37 light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acid encoding the antibody can be recovered from the library. Preferred nucleic acids molecules of this disclosure are those encoding the VH and VL sequences of the 5B1, 5Bla, 7D4, 7E4, 13B8 or 3C4 monoclonal antibodies. DNA sequences encoding the VH sequences of 5B1, 5Bla, 7D4, 7E4, 13B8 and 3C4 are shown in SEQ ID NOs:49, 50, 51, 52, 53 and 54, respectively. DNA sequences encoding the VL sequences of 5B 1, 5Bla, 7D4, 7E4, 13B8 and 3C4 are shown in SEQ ID NOs:55, 56, 57, 58, 59 and 60, respectively. Once DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible tinker. The term "operatively linked", as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame. The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CHI, CH2 and CH3). The sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG 4 , IgA, IgE, IgM or IgD constant region, but most preferably is an IgGi or IgG4 constant region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CHlI constant region. The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light C:1NRcnbl\DCC\KLL\39368O9_ DOC- 19/1011 WO 2007/067992 PCT/US2006/06 1817 38 chain constant region genes are known in the art (see e,g., Kabat, E. A., et al (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Humran Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or 5 lambda constant region, but most preferably is a kappa constant region. To create a scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4 Ser) 3 , such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al, (1988) Science 0 242:423-426; Huston et al. (1988) Proc. Nat, Acad Sc. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554). Production of Monoclonal Antibodies of this disclosure Monoclonal antibodies (mAbs) of the present disclosure can be produced by a variety of 5 techniques, including conventional monoclonal antibody methodology e.g., the standard somatic cell hybridization technique of Kohler and Milstein (1975) Nature 256: 495. Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed e.g., viral or oncogenic transformation of B lymphocytes. The preferred animal system for preparing hybridomas is the murine system. Hybridoma 20 production in the mouse is a very well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. Chimeric or humanized antibodies of the present disclosure can be prepared based on the sequence of a murine monoclonal antibody prepared as described above. DNA encoding the 25 heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, the murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Patent No. 4,816,567 to Cabilly et aL.). To create a humanized antibody, the murine CDR 0 regions can be inserted into a human framework using methods known in the art (see e.g., U.S.

WO 2007/067992 PCT/US2006/061817 39 Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al). In a preferred embodiment, the antibodies of this disclosure are human monoclonal antibodies. Such human monoclonal antibodies directed against Fucosyl-GMI can be generated 5 using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include mice referred to herein as HuMAb mice and KM miceTM, respectively, and are collectively referred to herein as "human Ig mice." The HuMAb mouse (Medarex, Inc.) contains human immunoglobulin gene miniloci 10 that encode unrearranged human heavy (p and y) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous p and r chain loci (see e.g., Lonberg, et al (1994) Nature 168(6474): 856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or c, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high 15 affinity human IgGc monoclonal (Lonberg, N. et al (1994), supra; reviewed in Lonberg, N. (1994) Handbook ofExperimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N. (1995)Ann. N.Y Acad Sci. 764:536-546). The preparation and use of HuMab mice, and the genomic modifications carried by such mice, is further described in Taylor, L. et al. (1992) Nucleic Acids Research 20 20:6287-6295; Chen, J. et al. (1993) International immunology 5: 647-656; Tuaillon et aL (1993) Proc. Natl Acad. Sci. USA 90:3720-3724; Choi et al (1993) Nature Genetics 4:117-123; Chen, J. et al (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J Immunol 52:2912-2920; Taylor, L. et al (1994) International Immunology§: 579-591; and Fishwild, D. et al (1996) Nature Biotechnology 1L: 845-851, the contents of all of which are hereby specifically 25 incorporated by reference in their entirety. See further, U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay; U.S. Patent No. 5,545,807 to Surani et al; PCT Publication Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 to Korman et al WO 2007/067992 PCT/US2006/061817 40 In another embodiment, human antibodies of this disclosure can be raised using a mouse that carries human immunoglobulin sequences on transgenes and transchomosomes, such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as "KM miceTm", are described in detail in PCT Publication WO 5 02/43478 to Ishida et al. Still further, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-Fucosyl-GMI antibodies of this disclosure. For example, an alternative transgenic system referred to as the Xenomouse (Abgenix, Inc.) can be used; such mice are described in, for example, U.S. Patent Nos. 0 5,939,598; 6,075,181; 6,114,598; 6, 150,584 and 6,162,963 to Kucherlapati et aL. Moreover, alternative transchromosomic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-Fucosyl-GM1 antibodies of this disclosure. For example, mice carrying both a human heavy chain transchromosome and a human light chain tranchromosome, referred to as "TC mice" can be 5 used; such mice are described in Tomizuka et aL (2000) Proc. NatL Acad. Sci. USA 97:722-727. Furthermore, cows carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be used to raise anti-Fucosyl-GM1 antibodies of this disclosure. Human monoclonal antibodies of this disclosure can also be prepared using phage display 2O methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are established in the art. See for example: U.S. Patent Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; U.S. Patent Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Patent Nos. 5,969,108 and 6,172,197 to McCafferty etal.; and U.S. Patent Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al 25 Human monoclonal antibodies of this disclosure can also be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. Such mice are described in, for example, U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al. 30 Immunization of Human Ig Mice WO 2007/067992 PCT/US2006/061817 41 When human Ig mice are used to raise human antibodies of this disclosure, such mice can be immunized with a purified or enriched preparation of Fucosyl-GMI antigen and/or . recombinant Fucosyl-GMI, or an Fucosyl-GMI fusion protein, as described by Lonberg, N. et al. (1994) Nature 3 (6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845 5 851; and PCT Publication WO 98/24884 and WO 01/14424. Preferably, the mice will be 6-16 weeks of age upon the first infusion. For example, a purified or recombinant preparation (5-50 ILg) of Fucosyl-GMI antigen can be used to immunize the human Ig mice intraperitoneally. Detailed procedures to generate fully human monoclonal antibodies to Fucosyl-GMI are described in Example 1 below. Cumulative experience with various antigens has shown that the [0 transgenic mice respond when initially immunized intraperitoneally (IP) with antigen in complete Freund's adjuvant, followed by every other week IP immunizations (up to a total of 6) with antigen in incomplete Freund's adjuvant However, adjuvants other than Freund's are also found to be effective. In addition, whole cells in the absence of adjuvant are found to be highly immunogenic. The immune response can be monitored over the course of the immunization .5 protocol with plasma samples being obtained by retroorbital bleeds. The plasma can be screened by ELISA (as described below), and mice with sufficient titers of anti-Fucosyl-GM 1 human immunoglobulin can be used for fusions. Mice can be boosted intravenously with antigen 3 days before sacrifice and removal of the spleen. It is expected that 2-3 fusions for each immunization may need to be performed. Between 6 and 24 mice are typically immunized for each antigen. 20 Usually both HCo7 and HCoJ2 strains are used. In addition, both HCo7 and HCol2 transgene can be bred together into a single mouse having two different human heavy chain transgenes (HCo7/HCol2). Alternatively or additionally, the KM mouses strain can be used, as described in Example 1. 25 Generation of Hybridomas Producing Human Monoclonal Antibodies of this disclosure To generate hybridomas producing human monoclonal antibodies of this disclosure, splenocytes and/or lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. For example, single cell 30 suspensions of splenic lymphocytes from immunized mice can be fused to one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG.

WO 2007/067992 PCT/US2006/061817 42 Alternatively, the single cell suspensions of splenic lymphocytes from immunized mice can be fused using an electric field based electrofusion method, using a Cyto Pulse large chamber cell fusion electroporator (Cyto Pulse Sciences, Inc., Glen Burnie, MD). Cells are plated at approximately 2 x 10 5 in flat bottom microtiter plate, followed by a one week incubation in 5 DMEM high glucose medium with L-glutamine and sodium pyruvate (Mediatech, Inc.;Herndon, VA) and further containing 20% fetal Bovine Serum (Hyclone, Logan, UT), 18% P3 88DI conditional media, 5% Origen Hybridoma cloning factor (BioVeris, Gaithersburg, VA), 4 mM L glutamine, 5mM HEPES, 0.055 mM p-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin and 1X Hypoxanthine-aminopterin-thymidine (HAT) media (Sigma; the HAT is ) added 24 hours after the fusion). After one week, cells cultured in medium in which HAT was used was replaced with HT. Individual wells can then be screened by ELISA for human monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, medium can be observed usually after 10-14 days. The antibody secreting hybridomas can be replated, screened again, and if still positive for human IgG, the monoclonal antibodies can be subcloned at least 5 twice by limiting dilution. The stable subclones can then be cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization. To purify human monoclonal antibodies, selected hybridomas can be grown in two-liter spinner-flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated before affinity chromatography with protein A-sepharose (Pharmacia, Piscataway, 0 N.J.). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using 1.43 extinction coefficient. The monoclonal - antibodies can be aliquoted and stored at -80' C. 5 Generation of Transfectomas Producing Monoclonal Antibodies of this disclosure Antibodies of this disclosure also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (e.g., Morrison, S. (1985) Science 229:1202). For example, to express the antibodies, or antibody fragments thereof, DNAs encoding 0 partial or full-length light and heavy chains, can be obtained by standard molecular biology WO 2007/067992 PCT/US2006/061837 43 techniques (e.g., PCR amplification or cDNA cloning using a hybridoma that expresses the antibody of interest) and the DNAs can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term "operatively linked' is intended to mean that an antibody gene is ligated into a vector such 5 that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector. The .0 antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). .The light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain .5 constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment(s) within the vector and the VK segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino 20 terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein). In addition to the antibody chain genes, the recombinant expression vectors of this disclosure carry regulatory sequences that control the expression of the antibody chain genes in a 25 host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). It will be appreciated by those skilled in the art that the design of the 30 expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desird, etc.

WO 2007/067992 PCT/US2006/06 1817 44 Preferred regulatory sequences for marmalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP) and polyoma. Alternatively, nonviral regulatory 5 sequences may be used, such as the ubiquitin promoter or p-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SRox promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type I (Takebe, Y. et al (1988) Mol. Cell. Biol. 8:466-472). In addition to the antibody chain genes and regulatory sequences, the recombinant 10 expression vectors of this disclosure may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., US. Pat. Nos. 4,399,216,4,634,665 and 5,179,017, all by Axel et aL). For example, typically the selectable marker gene confers resistance to drugs, such as G418, l5 hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection). For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the 20 term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the antibodies of this disclosure in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, and most 25 preferably mammalian host cells, is the most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody. Prokaryotic expression of antibody genes has been reported to be ineffective for production of high yields of active antibody (Boss, M. A. and Wood, C. R. (1985) Immunology Today :12-13). 30 Preferred mammalian host cells for expressing the recombinant antibodies of this WO 2007/067992 PCT/US2006/061817 45 disclosure include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO-cells, described in Urlaub and Chasin, (1980) Proc. Natd Acad Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) MoL BioL 159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO 5 myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which 0 the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods, Characterization of Antibody Binding to Antigen Antibodies of this disclosure can be tested for binding to Fucosyl-GM1 by, for example, 5 standard ELISA. Briefly, microtiter plates are coated with purified Fucosyl-GM1 at 0.25 g/rnl in PBS, and then blocked with 5% bovine serum albumin in PBS. Dilutions of antibody (e.g., dilutions of plasma from Fucosyl-GM1-inunized mice) are added to each well and incubated for 1-2 hours at 37"C. The plates are washed with PBS/Tween and then incubated with secondary reagent (eg., for human antibodies, a goat-anti-human IgG Fc-specific polyclonal 20 reagent) conjugated to alkaline phosphatase for 1 hour at 37"C. After washing, the plates are developed with pNPP substrate (1 mg/ml), and analyzed at OD of 405-650. Preferably, mice which develop the highest titers will be used for fusions. An ELISA assay as described above can also be used to screen for hybridomas that show positive reactivity with Fucosyl-GMI immunogen. Hybridomas that bind with high avidity to 25 Fucosyl-GMi are subcloned and further characterized. One clone from each hybridoma, which retains the reactivity of the parent cells (by ELISA), can be chosen for making a 5-10 vial cell bank stored at -140 'C, and for antibody purification. To purify anti-Fucosyl-GMI antibodies, selected hybridomas can be grown in two-liter spinner-flasks for monoclonal antibody purification. Supenatants can be filtered and 30 concentrated before affinity chromatography with protein A-sepharose (Pharmacia, Piscataway, WO 2007/067992 PCT/US2006/061817 46 NJ). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD28o using 1.43 extinction coefficient. The monoclonal antibodies can be aliquoted and stored at -80 *C. 5 To determine if the selected anti-Fucosyl-GM1 monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using connercially available reagents (Pierce, Rockford, IL). Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using Fucosyl-GMI coated-ELISA plates as described above. Biotinylated mAb binding can be detected with a strep-avidin-alkaline phosphatase 10 probe. To determine the isotype of purified antibodies, isotype ELISAs can be performed using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, wells of microtiter plates can be coated with ] pg/ml of anti human immunoglobulin ovemight at 4' C. After blocking with 1% BSA, the plates are reacted 15 with I pg /ml or less of test monoclonal antibodies or purified isotype controls, at ambient temperature for one to two hours. The wells can then be reacted with either human IgG1 or human IgM-specific alkaline phosphatase-conjugated probes. Plates are developed and analyzed as described above. Anti-Fucosyl-GM1 human IgGs can be further tested for reactivity with Fucosyl-GM1 20 antigen by Western blotting. Briefly, Fucosyl-GMI can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked with 10% fetal calf serum, and probed with the monoclonal antibodies to be tested. Human IgG binding can be detected using anti-human IgG alkaline phosphatase and developed with BCIP/NBT substrate tablets (Sigma Chem. Co., St. 25 Louis, Mo.). Immunoconjugates In another aspect, the present disclosure features an anti-Fucosyl-GM I antibody, or a fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g., an 30 inmunosuppressant) or a radiotoxin. Such conjugates are referred to herein as WO 2007/067992 PCT/US2006/061817 47 "immunoconjugates". Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, 5 daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mecblorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) 0 and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (I) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). 5 Other preferred examples of therapeutic cytotoxins that can be conjugated to an antibody of this disclosure include duocarmycins, calicheamicins, maytansines and auristatins, and derivatives thereof. An example of a calicheamicin antibody conjugate is commercially available (MylotargTM; Wyeth-Ayerst). Examples of therapeutic cytotoxins may be found, for example, in US Patent Nos: 6548530 and 6281354 and US Patent application Nos: US 20 2003/0064984, US 2003/0073 852 and US 2003/0050331. Cytotoxins can be conjugated to antibodies of this disclosure using linker technology available in the art. Examples of linker types that have been used to conjugate a cytotoxin to an antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide containing linkers. A linker can be chosen that is, for example, susceptible to cleavage by low 25 pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D). For further discussion of types of cytotoxins, linkers and methods for conjugating therapeutic agents to antibodies, see also Saito, G, et al. (2003) Adv. Drug Deliv Rev. 55:1 99 215; Trail, PA. et al. (2003) Cancer Immunol. Immunother: 52:328-337; Payne, G. (2003) 30 Cancer Cell 3:207-212; Allen, T.M. (2002) Nat. Rev. Cancer 2:750-763; Pastan, I. and WO 2007/067992 PCT/US2006/061817 48 Kreitman, R. J. (2002) Curr. Opin Investig. Drugs 3:1089-1091; Senter, P.D. and Springer, C.J. (2001) Adv. Drug Deliv. Rev. 53:247-264. Antibodies of the present disclosure also can be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated to antibodies for use diagnostically or therapeutically include, but are not limited to, iodine 13 1 , indium"', yttrium9 and lutetium . Method for preparing radioimmunconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including ZevalinTM (IDEC Pharmaceuticals) and BexxarTM (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of this disclosure. The antibody conjugates of this disclosure can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-y; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1 "), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors. 0 Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Amon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers 5 Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et at. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et at (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al, "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982).

WO 2007/067992 PCT/US2006/061817 49 Bispecific Molecules In another aspect, the present disclosure features bispecific molecules comprising an anti Fucosyl-GM I antibody, or a fragment thereof, of this disclosure. An antibody of this disclosure, 5 or antigen-binding portions thereof, can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody of this disclosure may in fact be derivatized or linkd to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites 0 and/or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule of this disclosure, an antibody of this disclosure can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule .5 results. Accordingly, the present disclosure includes bispecific molecules comprising at least one first binding specificity for Fucosyl-GMI and a second binding specificity for a second target epitope. In a particular embodiment of this disclosure, the second target epitope is an Fc receptor, e.g., human FcyRI (CD64) or a human Fca receptor (CD89). Therefore, this disclosure 20 includes bispecific molecules capable of binding both to FeyR or FcaR expressing effector cells (e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)), and to target cells expressing Fucosyl-GM1. These bispecific molecules target Fucosyl-OMI expressing cells to effector cell and trigger Fe receptor-mediated effector cell activities, such as phagocytosis of an Fucosyl-GMI expressing cells, antibody dependent cell-mediated cytotoxi city (ADCC), cytokine 25 release, or generation of superoxide anion. In an embodiment of this disclosure in which the bispecific molecule is multispecific, the molecule can further include a third binding specificity, in addition to an anti-Fc binding specificity and an anti-Fucosyl-GM1 binding specificity. In one embodiment, the third binding specificity is an anti-enhancement factor (EF) portion, e.g., a molecule which binds to a surface 30 protein involved in cytotoxic activity and thereby increases the immune response against the WO 2007/067992 PCT/US2006/061817 50 target cell. The "anti-enhancement factor portion" can be an antibody, functional antibody fragment or a ligand that binds to a given molecule, e.g., an antigen or a receptor, and thereby results in an enhancement of the effect of the binding determinants for the Fc receptor or target cell antigen. The "anti-enhancement factor portion" can bind an Fe receptor or a target cell 5 antigen. Alternatively, the anti-enhancement factor portion can bind to an entity that is different from the entity to which the first and second binding specificities bind. For example, the anti enhancement factor portion can bind a cytotoxic T-cell (e.g. via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell that results in an increased immune response against the target cell). 0 In one embodiment, the bispecific molecules of this disclosure comprise as a binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., an Fab, Fab', F(ab') 2 , Fv, or a single chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in Ladner et al. U.S. Patent No. 4,946,778, the contents of which is expressly incorporated by reference. 5 In one embodiment, the binding specificity for an Fey receptor is provided by a monoclonal antibody, the binding of which is not blocked by human immunoglobulin G (IgG). As used herein, the term "IgG receptor" refers to any of the eight y-chain genes located on chromosome 1. These genes encode a total of twelve transmembrane or soluble receptor isoforms which are grouped into three Fcy receptor classes: FcyRI (CD64), FeyRII(CD32), and 20 FcyRIII (CD16). In one preferred embodiment, the Fey receptor a human high affinity FcyRI. The human FeyRI is a 72 kDa molecule, which shows high affinity for monomeric IgG (101 - 109 M). The production and characterization of certain preferred anti-Fey monoclonal antibodies are described by Fanger et al, in PCT Publication WO 88/00052 and in U.S. Patent No. 25 4,954,617, the teachings of which are fully incorporated by reference herein. These antibodies bind to an epitope of FcyRI, FEcyRII or FcyRIII at a site which is distinct from the Fey binding site of the receptor and, thus, their binding is not blocked substantially by physiological levels of IgG. Specific anti-FeyRI antibodies useful in this disclosure are mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. The hybridoma producing mAb 32 is available from the American Type 0 Culture Collection, ATCC Accession No. HB9469. In other embodiments, the anti-Fcy receptor WO 2007/067992 PCT/US20061061817 51 antibody is a humanized form of monoclonal antibody 22 (H22). The production and characterization of the H22 antibody is described in Graziano, R.F. et al. (1995) J Immunol 155 (10): 4996-5002 and PCT Publication WO 94/10332. The H22 antibody producing cell line was deposited at the American Type Culture Collection under the designation HA022CL1 and has 5 the accession no. CRL 11177. In still other preferred embodiments, the binding specificity for an Fc receptor is provided by an antibody that binds to a human IgA receptor, e.g., an Fc-alpha receptor (FoaRI (CD89)), the binding of which is preferably not blocked by human immunoglobulin A (IgA). The term "IgA receptor" is intended to include the gene product of one a-gene (FcaRI) located on t0 chromosome 19. This gene is known to encode several alternatively spliced transmembrane isoforms of 55 to 110 kDa. FcaRI (CD89) is constitutively expressed on monocytes/macrophages, eosinophilic and neutrophilic granulocytes, but not on non-effector cell populations. FEcaRI has medium affinity (~ 5 x 107 M-1) for both IgAl and IgA2, which is increased upon exposure to cytokines such as G-CSF or GM-CSF (Morton, H.C. et al. (1996) 15 Critical Reviews in Immunology L6:423-440). Four FcaM-specific monoclonal antibodies, identified as A3, A59, A62 and A77, which bind FcaRI outside the IgA ligand binding domain, have been described (Monteiro, R.C. et al. (1992) J Immunol. 148:1764), FcaRI and FcyRI are preferred trigger receptors for use in the bispecific molecules of this disclosure because they are (1) expressed primarily on immune effector cells, e.g., mono cytes, 20 PMNs, macrophages and dendritic cells; (2) expressed at high levels (e.g., 5,000-100,000 per cell); (3) mediators of cytotoxic activities (e.g, ADCC, phagocytosis); (4) mediate enhanced antigen presentation of antigens, including self-antigens, targeted to them. While human monoclonal antibodies are preferred, other antibodies which can be employed in the bispecific molecules of this disclosure are murine, chimeric and humanized 25 monoclonal antibodies. The bispecific molecules of the present disclosure can be prepared by conjugating the constituent binding specificities, e.g., the anti-FcR and anti-Fucosyl-GMI binding specificities, using methods known in the art, For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to one another. When the binding 30 specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for WO 2007/067992 PCT/US2006/061817 52 covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N succinimidyl-S-acetyl-thioacet ate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate (sulfo-SMCC) (see e.g,, 5 Karpovsky et al (1984) J. Exp. Med. 160:1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie et al. (1987)] Jimmunol. 139: 2367-2375). Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL). .0 When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation. Alternatively, both binding specificities can be encoded in the same vector and expressed .5 and assembled in the same host cell. This method is particularly useful where the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab') 2 or ligand x Fab fusion protein. A bispecific molecule of this disclosure can be a single chain molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific molecules may comprise at least two single chain molecules. 20 Methods for preparing bispecific molecules are described for example in U.S. Patent Number 5,260,203; U.S. Patent Number 5,455,030; U.S. Patent Number 4,881,175; U.S. Patent Number 5,132,405; U.S. Patent Number 5,091,513; U.S. Patent Number 5,476,786; U.S. Patent Number 5,013,653; U.S, Patent Number 5,258,498; and U.S. Patent Number 5,482,858. Binding of the bispecific molecules to their specific targets can be confirmed by, for 25 example, enzyme-linked immunosorbent assay (ELISA), radioinununoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, the FcR antibody complexes can be detected using e.g., an enzyme-linked antibody or antibody fragment 30 which recognizes and specifically binds to the antibody-FcR complexes. Alternatively, the WO 2007/067992 PCT/US2006/061817 53 complexes can be detected using any of a variety of other immnunoassays. For example, the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference 5 herein). The radioactive isotope can be detected by such means as the use of a y counter or a scintillation counter or by autoradiography. Pharmaceutical Compositions In another aspect, the present disclosure provides a composition, e.g., a pharmaceutical 0 composition, containing one or a combination of monoclonal antibodies, or antigen-binding portion(s) thereof, of the present disclosure, formulated together with a pharmaceutically acceptable carrier. Such compositions may include one or a combination of (e.g., two or more different) antibodies, or immunoconjugates or bispecific molecules of this disclosure. For example, a pharmaceutical composition of this disclosure can comprise a combination of 5 antibodies (or immunoconjugates or bispecifics) that bind to different epitopes on the target antigen or that have complementary activities. Pharmaceutical compositions of this disclosure also can be administered in combination therapy, i.e., combined with other agents. For example, the combination therapy can include an anti-Fucosyl-GMI antibody of the present disclosure combined with at least one other anti .0 inflammatory or immunosuppressant agent. Examples oftherapeutic agents that can be used in combination therapy are described in greater detail below in the section on uses of the antibodies of this disclosure. As used herein, "phannaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying .5 agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, immunoconjuage, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the 0 compound.

WO 2007/067992 PCT/US2006/061817 54 The pharmaceutical compounds of this disclosure may include one or more pharmaceutically acceptable salts. A pharmaceuticallyy acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et a. (1977) J. Pharm. Sci. 66:1-19). Examples of 5 such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts 10 include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as NN' dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like, A pharmaceutical composition of this disclosure also may include a pharmaceutically 15 acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, 20 ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of this disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be 25 maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and 30 antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the WO 2007/067992 PCT/US2006/061817 55 compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be bought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and 5 sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of this disclosure is contemplated. Supplementary active compounds can also be incorporated into the compositions. 0 Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol- and liquid polyethylene glycol, and the like), and suitable mixtures 5 thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition Z0 an agent that delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion Z5 medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The amount of active ingredient which can be combined with a carrier material to 0 produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with WO 2007/067992 PCT/US2006/061817 56 a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, preferably from about 0.1 per cent to about 70 per cent, most preferably from about I per cent to 5 about 30 per cent of active ingredient in combination with a pharmaceutically acceptable carrier. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to .0 formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages fbr the subjects to be treated; each unit contains a predetennined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of this disclosure are dictated 5 by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals, For administration of the antibody, the dosage ranges from about 0.000 1 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 0.3 20 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months. Preferred dosage regimens for an anti-Fucosyl-GM1 antibody of this disclosure include I mg/kg body weight or 3 25 mg/kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks. In some methods, two or more monoclonal antibodies with different binding specificities 30 are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. Antibody is usually administered on multiple occasions. Intervals WO 2007/067992 PCT/US2006/061817 57 between single dosages can be, for example, weeldy, monthly, every three monthgs or yearly, Intervals can also be irregular as indicated by measuring blood levels of antibody to the target antigen in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 pLg /mJ and in some methods about 25-300 pig /ml, 5 Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half life of the antibody in the patient. In general, human antibodies show the longest half life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or 10 therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. 15 Thereafter, the patient can be administered a prophylactic regime. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will 20 depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, or the ester, salt or aide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general 25 health and prior medical history of the patient being treated, and like factors well known in the medical arts. A therapeuticallyy effective dosage" of an anti-Fucosyl-GMI antibody of this disclosure preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the 30 disease affliction. For example, for the treatment of tumors, a therapeuticallyy effective WO 2007/067992 PCT/US2006/06] 817 58 dosage" preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors. 5 Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's .0 symptoms, and the particular composition or route of administration selected. A composition of the present disclosure can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Preferred routes of administration for antibodies of this disclosure 5 include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, 20 transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, an antibody of this disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. 25 The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are 30 patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

WO 2007/067992 PCT/US2006/061817 59 Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of this disclosure can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. PatentNos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 5 4,596,556. Examples of well-known implants and modules useful in the present disclosure include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S, Patent No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; 10 U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,43 9,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art. 15 In certain embodiments, the human monoclonal antibodies of this disclosure can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of this disclosure cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811; 5,374,548; and 20 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade (1 989) J Clin. Pharmacol. 29:685), Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low et al); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P.G. Blo eman et al (1995) FEBS Lett. 357:140; M. Owais 25 et aL (1995)Antimicrob. Agents Chemother. 3 9:180); surfactant protein A receptor (Briscoe et al (1995) Am. J. Physiol. 1233:134); p120 (Schreier et aL (1994) J Bo Chem, 269:9090); see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346:123; J.J. Killion; I.J. Fidler (1994) Immunomethods 4:273. 30 Uses and Methods of this disclosure WO 2007/067992 PCTIUS2006/061817 60 The antibodies, antibody compositions and methods of the present disclosure have numerous in vitro and in vivo diagnostic and therapeutic utilities involving the diagnosis and treatment of Fucosyl-GMI mediated disorders. In a preferred embodiment, the antibodies of the present disclosure are human antibodies. For example, these molecules can be administered to 5 cells in culture, in vitro or ex vivo, or to human subjects, e.g., in vivo, to treat, prevent and to diagnose a variety of disorders. As used herein, the term "subject" is intended to include human and non-human animals. Non-human animals includes all vertebrates, e.g., mammals and non manunals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. Preferred subjects include human patients having disorders mediated by Fucosyl 10 GMI activity or having disorders coincident with a Fucosyl-GM1 mediated activity. The methods are particularly suitable for treating human patients having a disorder associated with aberrant Fucosyl-GMI expression or increased Fucosyl-GM1 presence. When antibodies to Fucosyl-GMi are administered together with another agent, the two can be administered in either order or simultaneously. 15 Given the specific binding of the antibodies of this disclosure for Fucosyl-GMI, the antibodies of this disclosure can be used to specifically detect Fucosyl-GMI expression on the surface of cells and, moreover, can be used to purify Fucosyl-GM1 via immunoaffinity purification. This disclosure further provides methods for detecting the presence of Fucosyl-GM 1 20 antigen in a sample, or measuring the amount of Fucosyl-GM1 antigen, comprising contacting the sample, and a control sample, with a human monoclonal antibody, or an antigen binding portion thereof, which specifically binds to Fucosyl-GM1, under conditions that allow for formation of a complex between the antibody or portion thereof and Fucosyl-GM1. The formation of a complex is then detected, wherein a difference complex formation between the 25 sample compared to the control sample is indicative the presence of Fucosyl-GM1 antigen in the sample. Fucosyl-GMI is expressed in small cell lung cancer, but not detected in normal lung or other tissues (Nilsson et al. (1984) Glycoconjugate J1:43-9; Krug et al. (2004) Clin Cancer Res .0:6094-100). An anti-Fucosyl-GMI antibody may be used alone to inhibit the growth of 30 cancerous tumors. Alternatively, an anti-Fucosyl-GM I antibody may be used in conjunction WO 2007/067992 PCT/US2006/061817 61 with other immunogenic agents, standard cancer treatments or other antibodies, as described below. Anti-fucosyl GMI monoclonal antibodies have been demonstrated to mediate potent CDC directed against fucosyl GM1 positive cell lines (Livingston PO et al. (1994) J Clin Oncol 12:1036-44; Brezicka et al. (2000) Cancer Immunol Immunother 42:235-42). Further, it has been reported that the complement activation, induced by monoclonal antibodies specific to fucosyl-GMI, in combination with cytostatic drugs result in synergistic cytotoxic effects on fucosyl-GMI expressing cells lines (Brezicka and Einbeigi (2001) Tumour Biol 22:97-103). Finally, anti-fucosyl-GM1 monoclonal antibodies have also been reported to inhibit the engraftment of fucosyl-GMl expressing tumor cells in nude mice (Brezicka et a. (1991) Int J Cancer 49:911-8). These data support the development of a fully human monoclonal antibody to fucosyl-GMI as an immunotherapeutic for the treatment of SCLC either alone or in combination with chemotherapeutic agents. Preferred cancers whose growth may be inhibited using the antibodies of this disclosure include cancers typically responsive to immunotherapy. Non-limiting examples of preferred cancers for treatment include lung cancer (including small cell lung cancer and non-small cell lung cancer). Examples of other cancers that may be treated using the methods of this disclosure include colon cancer (including small intestine cancer), lung cancer, breast cancer, pancreatic cancer, melanoma (e.g., metastatic malignant melanoma), acute myeloid leukemia, 0 kidney cancer, bladder cancer, ovarian cancer and prostate cancer, renal cancer (e.g., renal cell carcinoma), glioblastoma, brain tumors, chronic or acute leukemias including acute lymphocytic leukemia (ALL), adult T-cell leukemia (T-ALL), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphomas (e.g., Hodgkin's and non-Hodgkin's lymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma, Burkitt's 5 lymphoma, anaplastic large-cell lymphomas (ALCL), cutaneous T-cell lymphomas, nodular small cleaved-cell lymphomas, peripheral T-cell lymphomas, Lennert's lymphomas, immunoblastic lymphomas, T-cell leukemia/lymphomas (ATLL), entroblastic/centrocytic (cb/cc) follicular lymphomas cancers, diffuse large cell lymphomas of B lineage, angioimmunoblastic lymphadenopathy (AILD)-like T cell lymphoma and HIV associated body cavity based 0 lymphomas), embryonal carcinomas, undifferentiated carcinomas of the rhino-pharynx (e.g., Schmincke's tumor), Castleman's disease, Kaposi's Sarcoma, multiple myeloma, Waldenstrom's WO 2007/067992 PCT/US2006/061817 62 macroglobulinemia and other B-cell lymphomas, nasopharangeal carcinomas, bone cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, 5 carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), tumor angiogenesis, spinal 10 axis tumor, brain stem glioma, pituitary adenoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers including those induced by asbestos, e.g., mesothelioma and combinations of said cancers, Furthermore, given the expression of Fucosyl-GMI on various tumor cells, the human antibodies, antibody compositions and methods of the present disclosure can be used to treat a [5 subject with a tumorigenic disorder, e.g., a disorder characterized by the presence of tumor cells expressing Fucosyl-GM1 including, for example, lung cancer (including small cell lung cancer and non-small cell lung cancer), colon cancer (including small intestine cancer), melanoma (e.g,, metastatic malignant melanoma), acute myeloid leukemia, lung cancer, breast cancer, bladder cancer, pancreatic cancer, ovarian cancer and prostate cancer. Examples of other subjects with a 20 tumorigenic disorder include subjects having renal cancer (e.g., renal cell carcinoma), glioblastoma, brain tumors, chronic or acute lenkemias including acute lymphocytic leukemia (ALL), adult T-cell leukemia (T-ALL), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphomas (e.g., Hodgkin's and non-Hodgkin's lymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma, Burkitt's 25 lymphoma, anaplastic large-cell lymphomas (ALCL), cutaneous T-cell lymphomas, nodular small cleaved-cell lymphomas, peripheral T-cell lymphomas, Lennert's lymphomas, immunoblastic lymphomas, T-cell leukemia/lymphomas (ATLL), entroblastic/centrocytic (cb/cc) follicular lymphomas cancers, diffuse large cell lymphomas of B lineage, angioimmunoblastic lymphadenopathy (AILD)-like T cell lymphoma and HIV associated body cavity based 30 lymphomas), embryonal carcinomas, undifferentiated carcinomas of the rhino-pharynx (e.g., Schmincke's tumor), Castleman's disease, Kaposi's Sarcoma, multiple myeloma, Waldenstrorn's WO 2007/067992 PCTIUS20606161817 63 macroglobulinemia and other B-cell lymphomas, nasopharangeal carcinomas, bone cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometirium, carcinoma of the cervix, 5 carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), tumor angiogenesis, spinal 10 axis tumor, brain stem glioma, pituitary adenoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers including those induced by asbestos, e.g., mesothelioma and combinations of said cancers. Accordingly, in one embodiment, this disclosure provides a method of inhibiting growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective 15 amount of an anti-Fucosyl-GM1 antibody or antigen-binding portion thereof. Preferably, the antibody is a human anti-Fucosyl-GM1 antibody (such as any of the human anti-Fucosyl-GMI antibodies described herein). Additionally or alternatively, the antibody may be a chimeric or humanized anti-Fucosyl-GMI antibody. In one embodiment, the antibodies (e.g,, human monoclonal antibodies, multispecific and 20 bispecific molecules and compositions) of this disclosure can be used to detect levels of Fucosyl GMi or levels of cells which contain Fucosyl-GM1 on their membrane surface, which levels can then be linked to certain disease symptoms. Alternatively, the antibodies can be used to inhibit or block Fucosyl-GM1 function which, in turn, can be linked to the prevention or amelioration of certain disease symptoms, thereby implicating Fucosyl-GMl as a mediator of the disease. This 25 can be achieved by contacting an experimental sample and a control sample with the anti Fucosyl-GM 1 antibody under conditions that allow for the formation of a complex between the antibody and Fucosyl-GM1. Any complexes formed between the antibody and Fucosyl-GM 1 are detected and compared in the experimental sample and the control. In another embodiment, the antibodies (e.g., human antibodies, multispecific and 30 bispecific molecules and compositions) of this disclosure can be initially tested for binding WO 2007/067992 PCT/US2006/061817 64 activity associated with therapeutic or diagnostic use in vitro. For example, compositions of this disclosure can be tested using the flow cytometric assays described in the Examples below. The antibodies (e.g., human antibodies, multispecific and bispecific molecules, immunoconjugates and compositions) of this disclosure have additional utility in therapy and 5 diagnosis of Fucosyl-GMI-related diseases. For example, the human monoclonal antibodies, the multispecific or bispecific molecules and the immunoconjugates can be used to elicit in vivo or in vitro one or more of the following biological activities: to inhibit the growth of and/or kill a cell expressing Fucosyl-GMi; to mediate phagocytosis or ADCC of a cell expressing Fucosyl GM1 in the presence of human effector cells; or to block Fucosyl-GMI ligand binding to 0 Fucosyl-GMl. In a particular embodiment, the antibodies (e.g., human antibodies, multispecific and bispecific molecules and compositions) are used in vivo to treat, prevent or diagnose a variety of Fucosyl-GMI-related diseases. Examples of Fucosyl-GM1 -related diseases include, among others, lung cancer (including small cell lung cancer and non-small cell lung cancer). 5 Suitable routes of administering the antibody compositions (e.g., human monoclonal antibodies, multispecific and bispecific molecules and immunoconjugates ) of this disclosure in vivo and in vitro are well known in the art and can be selected by those of ordinary skill. For example, the antibody compositions can be administered by injection (e.g., intravenous or subcutaneous). Suitable dosages of the molecules used will depend on the age and weight of the 20 subject and the concentration and/or formulation of the antibody composition. As previously described, human anti-Fucosyl-GM1 antibodies of this disclosure can be co-administered with one or other more therapeutic agents, e.g., a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent. The antibody can be linked to the agent (as an immunocomplex) or can be administered separate from the agent. In the latter case (separate 25 administration), the antibody can be administered before, after or concurrently with the agent or can be co-administered with other known therapies, e.g., an anti-cancer therapy, e.g., radiation. Such therapeutic agents include, among others, anti-neoplastic agents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil and cyclophosphamide hydroxyurea which, by themselves, are only effective at levels which are toxic or subtoxic to a 30 patient. Cisplatin is intravenously administered as a 100 mg/ dose once every four weeks and adriamycin is intravenously administered as a 60-75 mg/ml dose once every 21 days. Co- WO 2007/067992 PCT/US2006/061817 65 administration of the human anti-Fucosyl-GM1 antibodies or antigen binding fragments thereof, of the present disclosure with chemotherapeutic agents provides two anti-cancer agents which operate via different mechanisms which yield a cytotoxic effect to human tumor cells. Such co administration can solve problems due to development of resistance to drugs or a change in the 5 antigenicity of the tumor cells which would render them unreactive with the antibody. In one embodiment, immunoconjugates of this disclosure can be used to target compounds (e.g., therapeutic agents, labels, cytotoxins, radiotoxoins immunosuppressants, etc.) to cells which have Fucosyl-GMI cell surface receptors by linking such compounds to the antibody. For example, an anti-Fucosyl-GMI antibody can be conjugated to any of the toxin 10 compounds described in US Patent Nos. 6,281,354 and 6,548,530, US patent publication Nos. 20030050331, 20030064984, 20030073852 and 20040087497 or published in WO 03/022806, which are hereby incorporated by reference in their entireties. Thus, this disclosure also provides methods for localizing ex vivo or in vivo cells expressing Fucosyl-GM1 (e.g., with a detectable label, such as a radioisotope, a fluorescent compound, an enzyme or an enzyme co-factor). 15 Alternatively, the immunoconjugates can be used to kill cells which have Fucosyl-GM1 cell surface receptors by targeting cytotoxins or radiotoxins to Fucosyl-GM 1, Target-specific effector cells, e.g., effector cells linked to compositions (e.g., human antibodies, multispecific and bispecific molecules) of this disclosure can also be used as therapeutic agents. Effector cells for targeting can be human leukocytes such as macrophages, 20 neutrophils or monocytes. Other cells include eosinophils, natural killer cells and other IgG- or IgA-receptor bearing cells. If desired, effector cells can be obtained from the subject to be treated. The target-specific effector cells can be administered as a suspension of cells in a physiologically acceptable solution. The number of cells administered can be in the order of 108 10 9 but will vary depending on the therapeutic purpose. In general, the amount will be sufficient 25 to obtain localization at the target cell, e.g., a tumor cell expressing Fucosyl-GMI and to effect cell killing by, e.g., phagocytosis. Routes of administration can also vary. Therapy with target-specific effector cells can be performed in conjunction with other techniques for removal of targeted cells. For example, anti-tumor therapy using the compositions (e.g., human antibodies, multispecific and bispecific molecules) of this disclosure 30 and/or effector cells armed with these compositions can be used in conjunction with chemotherapy. Additionally, combination immunotherapy may be used to direct two distinct WO 2007/067992 PCT/US2006/061817 66 cytotoxic effector populations toward tumor cell rejection. For example, anti-Fucosyl-GMl antibodies linked to anti-Fe-gamma RI or anti-CD3 may be used in conjunction with IgG- or IgA-receptor specific binding agents. Bispecific and multispecific molecules of this disclosure can also be used to modulate 5 FcyR or FcyR levels on effector cells, such as by capping and elimination of receptors on the cell surface. Mixtures of anti-Fc receptors can also be used for this purpose. The compositions (e.g., human antibodies, multispecific and bispecific molecules and immunoconjugates) of this disclosure which have complement binding sites, such as portions from IgG1, -2 or -3 or IgM which bind complement, can also be used in the presence of .0 complement. In one embodiment, ex vivo treatment of a population of cells comprising target cells with a binding agent of this disclosure and appropriate effector cells can be supplemented by the addition of complement or serum containing complement. Phagocytosis of target cells coated with a binding agent of this disclosure can be improved by binding of complement proteins. In another embodiment target cells coated with the compositions (e,g., human .5 antibodies, multispecific and bispecific molecules) of this disclosure can also be lysed by complement. In yet another embodiment, the compositions of this disclosure do not activate complement. The compositions (e.g., human antibodies, multispecific and bispecific molecules and immunoconjugates) of this disclosure can also be administered together with complement. 20 Accordingly, within the scope of this disclosure are compositions comprising human antibodies, maultispecific or bispecific molecules and serum or complement. These compositions are advantageous in that the complement is located in close proximity to the human antibodies, multispecific or bispecific molecules. Alternatively, the human antibodies, multispecific or bispecific molecules of this disclosure and the complement or serum can be administered 25 separately. Accordingly, patients treated with antibody compositions of this disclosure can be additionally administered (prior to, simultaneously with or following administration of a hurnan antibody of this disclosure) with another therapeutic agent, such as a cytotoxic or radiotoxic agent, which enhances or augments the therapeutic effect of the human antibodies.

WO 2007/067992 PCT/US2006/061817 67 In other embodiments, the subject can be additionally treated with an agent that modulates, e.g., enhances or inhibits, the expression or activity of Fey or Fcy receptors by, for example, treating the subject with a cytokine. Preferred cytokines for administration during treatment with the multispecific molecule include of granulocyte colony-stimulating factor (G 5 CSF), granulocyte- macrophage colony-stimulating factor (GM-CSF), interferon-y (IFN-7) and tumor necrosis factor (TNF). The compositions (e.g., human antibodies, multispecific and bispecific molecules) of this disclosure can also be used to target cells expressing FcyR or Fucosyl-GMI, for example for labeling such cells, For such use, the binding agent can be linked to a molecule that can be .0 detected. Thus, this disclosure provides methods for localizing ex vivo or in vitro cells expressing Fc receptors, such as FeyR or Fucosyl-GM1. The detectable label can be, e.g., a radioisotope, a fluorescent compound, an enzyme or an enzyme co-factor. Also within the scope of the present disclosure are kits comprising the antibody compositions of this disclosure (e.g., human antibodies, bispecific or multispecific molecules, or .5 immunoconjugates) and instructions for use. The kit can further contain one more more additional reagents, such as an immunosuppressive reagent, a cytotoxic agent or a radiotoxic agent or one or more additional human antibodies of this disclosure (e.g., a human antibody having a complementary activity which binds to an epitope in the Fucosyl-OM1 antigen distinct from the first human antibody). Kits typically include a label indicating the intended use of the 20 contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit. The present disclosure is further illustrated by the following examples which should not be construed as further limiting. The contents of all figures and all references, patents and 25 published patent applications cited throughout this application are expressly incorporated herein by reference.

WO 2007/067992 PCT/US2006/061817 68 Examples Example 1: Generation of human monoclonal antibodies against Fucosyl-GM1 Antigen Immunization protocols utilized as antigen Salmonella minnesota adsorbed fucosyl-GMJ 5 (Northwest Biotherapeutics, Inc.). Transgenic HuMab and KM miceM Fully human monoclonal antibodies to Fucosyl-GMI were prepared using the HCo7, HCol 2, HCo7-+HCol2 strains of HuMab transgenic mice and the KM strain of transgenic transchromosomic mice, each of which express human antibody genes. In each of these mouse 10 strains, the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al. (1993) EMBO J 12:811-820 and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of PCT Publication WO 01/09187. Each of these mouse strains carries a human kappa light chain transgene, KCo5, as described in Fishwild et al. (1996) Nature Biotechnology j4:845-85 1. The HCo7 strain carries 15 the HCo7 human heavy chain transgene as described in U.S. Patent Nos. 5, 770,429; 5,545,806; 5,625,825; and 5,545,807. The HCo12 strain carries the HCol2 human heavy chain transgene as described in Example 2 of WO 01/09187 or example 2 WO 01/14424. The HCo7-+HCol2 strain carries both the HCo7 and the HCoI2 heavy chain transgenes. The KM strain contains the SC20 transchromosome as described in PCT Publication WO 02/43478. All of these strains are 20 referred to herein as HuMAb mice. HuMab and KM Immunizations: To generate fully human monoclonal antibodies to Fucosyl-GM I, HuMab mice and KM miceTM were immunized with Fucosyl-GM1 adsorbed by lyophilization onto the surface of acid treated Salmonella Minnesota (Northwest Biotherapuetics, Inc.). General immunization schemes 25 for HuMab mice are described in Lonberg, N. et al (1994) Nature 368(6474): 856-859; Fishwild, D. et al, (1996) Nature Biotechnology 14: 845-851 and PCT Publication WO 98/24884. The mice were 6-16 weeks of age upon the first infusion of antigen.

WO 2007/067992 PCT/US2006/061817 69 Transgenic mice were immunized intraperitonealy (JP) or subcutaneously (Sc) with antigen in complete Freund's adjuvant twice, followed by 2-4 weeks IP immunization (up to a total of 8 immunizations) with the antigen in incomplete Freund's adjuvant. The immune response was monitored by ELISA (described below) of sera obtained from retro-orbital bleeds. 5 Mice with sufficient titers of anti-fucosyl-GM 1 human immunogolobulin were used for fusions. Mice were boosted intravenously with antigen 3 and 2 days before sacrifice and removal of the spleen. Selection of HuMab or KM MiceTh Producing Anti-Fucosyl-GM1 Antibodies: To select HuMab or KM miceTm producing antibodies that bound Fucosyl-GMI, sera 0 from immunized mice were tested by ELISA as described by Fishwild, D. et al. (1996). Briefly, natural fucosyl-OMI purified from either fucosyl Iransferase transfectant cell lines (Northwest Biotherapeutics, Inc.) or from bovine brain (Matreya, Inc) was solubilized in methanol at 1 mg/mil and passively adsorbed onto polypropylene microtiter plates, 50 ul/well, by air-drying at room temperature for 1-2 hrs. Similarly, plates coated with related control antigens such as GMI 5 were prepared as a counter-screen for cross reactive antibodies. Assay plates were then blocked with 250 pl/well of 1% ovalbumin in PBS for 1 hr at room temperature. Dilutions of plasma from fucosyl-GMI immunized mice were added to each well and incubated for 1-2 hours at ambient temperature. The plates were washed with PBS and then incubated for 1 hour at room temperature with either a goat-anti-human IgG FEc or with goat-anti-human IgM Fe polyclonal 20 antibodies each conjugated to horseradish peroxidase (HRP). After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450nm. Mice that developed the highest titers of anti-fucosyl-GMI antibodies were used for fusions. Fusions were performed as described below and hybridoma supernatants were tested for anti-fucosyl-GMI activity by ELISA. 25 Generation of Hybridomas Producing Human Monoclonal Antibodies to Fucosyl-GMI: Splenocytes were isolated from the HuMab@ mice and KM-miceTM and were fused to a mouse myeloma cell line either using PEG based upon standard protocols or electric field based electrofision using a Cyto Pulse large chamber cell fusion electroporator (Cyto Pulse Sciences, Inc., Glen Bumie, MD). The resulting hybridomas were then screened for the production of 0 antigen-specific antibodies.

WO 2007/067992 PCT/US2006/061817 70 Single cell suspensions of splenic lymphocytes from immunized mice were fused to one fourth the number of P3X63-Ag8.653 non-secreting mouse myeloma cells (ATCC, CRL 1580) or SP2/0 non-secreting mouse myeloma cells (ATCC, CRL 1581) using 50% PEG (Sigma). Cells were plated at a density of about lx1 0 5/well in flat bottom microtiter plates and incubated 5 approximately 2 weeks in selective medium containing 10% fetal bovine serum, 10% P388D1 (ATCC, CRL TIB-63) conditioned medium, 3-5% origen (IGEN) in DMEM (Mediatech, CRL 10013, with high glucose, L-glutamine and sodium pyruvate) plus 5 mM HEPES, 0.055 mM 2 mercaptoethanol, 50 mg/ml gentamicin and lx HAT (Sigma, CRL P-7185). After 1-2 weeks, cells were cultured in medium in which the HAT was replaced with HT. Individual wells were 0 then screened by ELISA (described above) for human anti-fucosyl-GM1 IgG and IgM antibodies. The hybridomas with specific binding activity in the ELISA assay screen were further tested by FACS (described below) for specific binding to fucosyl-GM1 adsorbed to mammalian cells. Hybridomas exhibiting the highest specific binding by ELISA and FACS were sub 5 cloned at least twice by limiting dilution. The resulting stable sub-clones were then cultured in vitro to generate small amounts of monoclonal antibody in tissue culture medium. The ELISA screen was repeated to confirm the activity of the sub-clones, The sub-clones with highest activity in the ELISA were scaled up to produce sufficient conditioned medium (typically 1 L) for purification of monoclonal anti-fucosyl-GM 1 for further characterization. 20 Hybridoma clones 5B1, 5Bla, 7D4, 7E4, 13B8,13B8a and 18D5 were selected for further analysis. Example 2: Structural characterization of human monoclonal antibodies 5B1, 5BMa, 7D4, 7E4, 13B8 and 18D5 The cDNA sequences encoding the heavy and light chain variable regions of the 5B1, 25 5B1la, 7D4, 7E4, 13B8 and 18D5 monoclonal antibodies were obtained from the 5B1, 5B1 a, 7D4, 7E4, 13B8 and 18D5 hybridomas, respectively, using standard PCR techniques and were sequenced using standard DNA sequencing techniques. The nucleotide and amino acid sequences of the heavy chain variable region of 5B I are shown in Figure 1A and in SEQ ID NO:49 and 1, respectively.

WO 2007/067992 PCT/US2006/061817 71 The nucleotide and amino acid sequences of the light chain variable region of 5B 1 are shown in Figure lB and in SEQ ID NO:55 and 7, respectively. Comparison of the 5B1 heavy chain immunoglobulin sequence to the known human germline immunoglobulin heavy chain sequences demonstrated that the 5B1 heavy chain utilizes 5 a VH segment from human germline VH 3-48, a D segment from the human germline 1-1, and a JH segment from human germline JH 6b. The alignment of the 5B1 VH sequence to the germline VH 3-48 sequence is shown in Figure 7. Further analysis of the 5B1 VH sequence using the Kabat system of CDR region determination led to the delineation of the heavy chain CDR1, CDR2 and CD3 regions as shown in Figures IA and 7, and in SEQ ID NOs:13, 19 and 0 25, respectively. Comparison of the 5B1 light chain immunoglobulin sequence to the known human germline immunoglobulin light chain sequences demonstrated that the 5B1 light chain utilizes a VL segment from human germline VK LI 5 and a JK segment from human gernnline JX 4. The alignment of the 5B1 VL sequence to the germline VK L15 sequence is shown in Figure 8. 5 Further analysis of the 5B1 VL sequence using the Kabat system of CDR region determination led to the delineation of the light chain CDRI, CDR2 and CD3 regions as shown in Figures 1B and 8, and in SEQ ID NOs;31, 37 and 43, respectively. The nucleotide and amino acid sequences of the heavy chain variable region of 5Bla are shown in Figure 2A and in SEQ ID NO:50 and 2, respectively. The nucleotide and amino acid sequences of the light chain variable region of 5B 1 a are shown in Figure 2B and in SEQ ID NO:56 and 8, respectively. Comparison of the 5Bla heavy chain immunoglobulin sequence to the known human gernline immunoglobulin heavy chain sequences demonstrated that the 5B I a heavy chain utilizes a VH segment from human germline VH 3-48, a D segment from the human germline I 5 1, and a JH segment from human germline JH 6b. The alignment of the 5Bla VH sequence to the germline VH 3-48 sequence is shown in Figure 7. Further analysis of the 5Bla VH sequence using the Kabat system of CDR region determination led to the delineation of the heavy chain CDR I, CDR2 and CD3 regions as shown in Figures 2A and 7, and in SEQ ID NOs:14, 20 and 26, respectively. 0 Comparison of the 5Bla light chain immunoglobulin sequence to the known human germline immunoglobulin light chain sequences demonstrated that the 5B 1 a light chain utilizes a WO 2007/067992 PCT/US2006/061817 72 VL segment from human germline VK L15 and a JK segment from human germline JX 4. The alignment of the 5Bla VL sequence to the gernine VK L15 sequence is shown in Figure 8. Further analysis of the 5Bla VL sequence using the Kabat system of CDR region determination led to the delineation of the light chain CDRI, CDR2 and CD3 regions as shown in Figures 2B 5 and 8, and in SEQ ID NOs:32, 38 and 44, respectively. The nucleotide and amino acid sequences of the heavy chain variable region of 7D4 are shown in Figure 3A and in SEQ ID NO:51 and 3, respectively. The nucleotide and amino acid sequences of the light chain variable region of 7D4 are shown in Figure 3B and in SEQ ID NO:57 and 9, respectively. .0 Comparison of the 7D4 heavy chain immunoglobulin sequence to the known human germline immunoglobulin heavy chain sequences demonstrated that the 7D4 heavy chain utilizes a VH segment from human germline VH 3-48, a D segment from the human germline 1-1, and a JfH segment from human germline JH 6b. The alignment of the 7D4 VH sequence to the germline VH 3-48 sequence is shown in Figure 7. Further analysis of the 7D4 VH sequence 5 using the Kabat system of CDR region determination led to the delineation of the heavy chain CDRI, CDR2 and CD3 regions as shown in Figures 3A and 7, and in SEQ ID NOs:15, 21 and 27, respectively. Comparison of the 7D4 light chain immunoglobulin sequence to the known human gernline immunoglobulin light chain sequences demonstrated that the 7D4 light chain utilizes a 20 VL segment from human germline VK L15 and a JK segment from human germline JK 4. The alignment of the 7D4 VL sequence to the germlineVKLl5 sequence is shown in Figure 8. Further analysis of the 7D4 VL sequence using the Kabat system of CDR region determination led to the delineation of the light chain CDR1, CDR2 and CD3 regions as shown in Figures 3B and 8, and in SEQ ID NOs:33, 39 and 45, respectively. 25 The nucleotide and amino acid sequences of the heavy chain variable region of 7E4 are shown in Figure 4A and in SEQ ID NO:52 and 4, respectively. The nucleotide and amino acid sequences of the light chain variable region of 7E4 are shown in Figure 4B and in SEQ ID NO:58 and 10, respectively. Comparison of the 7E4 heavy chain immunoglobulin sequence to the known human 30 gernline immunoglobulin heavy chain sequences demonstrated that the 7E4 heavy chain utilizes a VH segment from human germline VIH 3-48, a D segment from the human germline 1-1, and a WO 2007/067992 PCT/US2006/061817 73 JH segment from human germline JH 6b. The alignment of the 7E4 VH sequence to the germline VH 3-48 sequence is shown in Figure 7. Further analysis of the 7E4 VH sequence using the Kabat system of CDR region determination led to the delineation of the heavy chain CDR1, CDR2 and CD3 regions as shown in Figures 4A and 7, and in SEQ ID NOs:16, 22 and 5 28, respectively. Comparison of the 7B4 light chain immunoglobulin sequence to the known human germline immunoglobulin light chain sequences demonstrated that the 7B4 light chain utilizes a VL segment from human germline VK L15 and a JK segment from human germline JK 4. The alignment of the 7E4 VL sequence to the germline VK L15 sequence is shown in Figure 8. 10 Further analysis of the 7E4 VL sequence using the Kabat system of CDR region determination led to the delineation of the light chain CDRI, CDR2 and CD3 regions as shown in Figures 4B and 8, and in SEQ ID NOs:34, 40 and 46, respectively. The nucleotide and amino acid sequences of the heavy chain variable region of 13B8 are shown in Figure 5A and in SEQ ID NO:53 and 5, respectively. 15 The nucleotide and amino acid sequences of the light chain variable region of 13B8 are shown in Figure 5B and in SEQ ID NO:59 and 11, respectively. Comparison of the 13B8 heavy chain immunoglobulin sequence to the known human germline immunoglobulin heavy chain sequences demonstrated that the 13B8 heavy chain utilizes a VH segment from human germline VH 3-48, a D segment from the human germline 1 20 1, and a JH segment from human germline JH 6b. The alignment of the 13B8 VH sequence to the germline VH 3-48 sequence is shown in Figure 7. Further analysis of the 1338 VH sequence using the Kabat system of CDR region determination led to the delineation of the heavy chain CDR1, CDR2 and CD3 regions as shown in Figures 5A and 7, and in SEQ ID NOs:11, 17 and 23, respectively. 25 Comparison of the 13B8 light chain immunoglobulin sequence to the known human germline immunoglobulin light chain sequences demonstrated that the 13B8 light chain utilizes a VL segment from human germline VK L15 and a JK segment from human germline JK 4. The alignment of the 13B8 VL sequence to the germline VK L15 sequence is shown in Figure 8. Further analysis of the 13B8 VL sequence using the Kabat system of CDR region determination 30 led to the delineation of the light chain CDR1, CDR2 and CD3 regions as shown in Figures 5B and 8, and in SEQ ID NOs:35, 41 and 47, respectively.

74 The nucleotide and amino acid sequences of the heavy chain variable region of 3C4 are shown in Figure 6A and in SEQ ID NO:54 and 63, respectively. The nucleotide and amino acid sequences of the light chain variable region of 3C4 are shown in Figure 6B and in SEQ ID NO:60 and 64, respectively. Comparison of the I8DS heavy chain immunoglobulin sequence to the known human germline immunoglobulin heavy chain sequences demonstrated that the 18D5 heavy chain utilizes a VH segment from human germline VH 3-48, a D segment from human germline 1-1, and a JH segment from human germline JH 6b. The alignment of the 18D5 VH sequence to the germ line VH 3-48 sequence is shown in Figure 7. Further analysis of the 18D5 VH sequence using the Kabat system of CDR region determination led to the delineation of the heavy chain CDR], CDR2 and CD3 regions as shown in.Figure 7, and in SEQ ID NOs:18, 24 and 30, respectively. Comparison of the 18D5 light chain immunoglobulin sequence to the known human germline immunoglobulin light chain sequences demonstrated that the I 8D5 light chain utilizes a VL segment from human gen line VK L15 and a JK segment from human germline JK 4. The alignment of the 18D5 VL sequence to the germline VK L15 sequence is shown in Figure 8. Further analysis of the 18D5 VL sequence using the Kabat system of CDR region determination led to the delineation of the light chain CDR1, CDR2 and CD3 regions as shown in Figure 8, and in SEQ ID NOs:36, 42 and 48, respectively. Example 3. Preparation of cels doped with Fucosyl-GMI Cells were prepared that contained fucosyl-GM] embedded in the plasma membrane for use in binding assays. Purified fucosyl-GM1 was solubilized in a 1:1 mixture of chloroform:methanol in a glass tube and evaporated to dryness under nitrogen. PBS without Ca or Mg was added to the dry fucosyl-GMI such that the concentration of antigen was 200 kg/ml; an emulsion was created by vortexing for 5 minutes followed by sonication for 5 minutes. A suspension of target cells, typically HEK 293 (human kidney, ATCC #CRL- 1573) or Daudi (human Burkitt's lymphoma, ATCC # CCL-213) was prepared in PBS at 2 x 106 cells/ml. Equal volumes of fucosyl-GML emulsion and cell suspension were mixed to a final concentration of 100 jg antigen/I0 6 cells/ml. Cells plus antigen were incubated to allow fucosyl-GMI CANRPorbI\DC\KtLU936809_I.DOC - 19/J I WO 2007/067992 PCT/US2006/061817 75 intercalation into the membrane for 15 minutes at 37*C, followed by 30 minutes at room temperature with occasional mixing throughout. Cells were centrifuged at 1000 rpm for 10 minutes. The supemate was discarded and the "doped"cells were resuspended at a density of 4 x 106 cells/ml in FACS buffer (PBS without Ca or Mg + 1% human serum +2% FBS + 2mM 5 EDTA), Similarly, control cells were prepared "doped" with a related antigen such as GMI or with no antigen. Example 4. Characterization of binding specificity and binding kinetics of anti-Fueosyl-GM1 human monoclonal antibodies Binding specificity by ELISA .0 Purified, monoclonal, human anti-Fucosyl-GM 1 antibodies were tested for specific binding to purified fucosyl-GMI by ELISA, by both recombinant antigen and doped cells. All procedures were performed on ice. Conditioned medium from ELISA positive hybridoma cultures were mixed with antigen or cells in "V" bottom plates and were incubated 1 hour. Cells were washed and bound antibody was detected with an HRP conjugated mouse anti-human IgG 5 Fe secondary antibody. After washing, the plates were developed with a colorimetric substrate, pelleted by centrifugation and supernate was transferred to a flat-bottom microtiter assay plate for analysis by spectrophotometer. The results are shown in Figures 9 (antigen ELISA) and 10 (whole cell ELISA). The anti-fucosyl-GMI antibodies were shown to bind specifically to fucosyl-GMI. 20 Binding specificity by flow cytometry Naturally expressing fucosy-GMI positive cells lines such as H-4-II-E (Rat hepatorna ATCC# CRL-1 548) or DMS-79 (Human SCLC ATCC # CRL-2049) were used to determine the specificity of Fucosyl-GMl human monoclonal antibodies by flow cytometry, All staining procedures were performed on ice. Binding of an anti-Fucosyl-GMI human monoclonal 25 antibody was assessed by incubating the transfected cells with the anti-Fucosyl-GM 1 human monoclonal antibody at a concentration of 10 yg/ml. The cells were washed and binding was detected with a FITC-labeled anti-human IgG Ab. Flow cytometric analyses were performed using a FACScan flow cytometry (Becton Dickinson, San Jose, CA). The results are depicted in WO 2007/067992 PCT/US2006/061817 76 Figure 11. The anti-Fucosyl-GM1 human monoclonal antibody bound to the 1-4-11-E and DMS 79 cell lines. This data demonstrates the specificity of anti-Fucosyl-GMI human monoclonal antibodies for Fucosyl-GM 1. Example 5. Internalization of anti-Fucosyl-GM1 monoclonal antibody 5 Anti-Fucosyl-GM1 HuMAbs were tested for the ability to internalize into Fucosyl-GM1 expressing cell lines using a Hum-Zap internalization assay. The Hum-Zap assay tests for internalization of a primary human antibody through binding of a secondary antibody with affinity for human IgG conjugated to the toxin saporin. Cell lines expressing fucosyl-GM1 (H-4-II-E or DMS 79) were suspended in culture 10 medium and dispensed into microtiter cell culture plates at 7500 cells/well. Serial dilutions of test antibodies and isotype controls are prepared in culture medium and mixed with a 2:1 molar excess of saporin conjugated secondary antibody (Hum-Zap T m , Advanced Targeting Systems, San Diego, CA, IT-22-25) for 1 hour on ice. Antibody plus saporin conjugate mixtures were then mixed with cells and incubated at 370C, 48-72 hours. Cell viability was measured with the 15 addition of MTS (Promega) O.D. is read after a further 4 hour incubation, with absorbance inversely proportional to internalization. The results are shown in Figures 12. This data demonstrates that the human anti-fucosyl GM1 antibodies can internalize into cell lines expressing fucosyl-GMJ. Example 6 . Complement dependent cytotoxicity effect of anti-Fucosyl GMI antibodies 20 Target cells, either "doped" or naturally expressing cell lines were suspended to 10 6 /mL in CDC buffer (RPM11640). Human complement (HC) was diluted 1:3 in cell line growth medium. Serial dilutions of test antibodies and isotype controls were prepared. Cells, complement and antibodies were mixed in equal volumes in a microtiter assay plate and incubated at 37"C for 2 hours. Alamar blue was added to each well and the plates were 25 incubated for an additional 21 hrs at 37"C. The plates were read on a fluorescent plate reader using a 530 nm absorption/590 rn emission profile, with cell viability being proportional to fluorescence units. The results are shown in Figure 13. Human and mouse control antibodies WO 2007/067992 PCT/US2006/061817 77 exhibit robust, dose dependant CDC activity on both DMS 79 and H-4-Il-E cells. Isotype control antibodies show no significant cytotoxicity. CDC activity on fucosyl-GMI negative cell lines, such as Ramos and ARH77 is negligible Example 7. Assessment of ADCC activity of anti-Fucosyl GM1 antibodies 5 In this example, anti-fucosyl GM1 monoclonal antibodies were tested for the ability to kill fucosyl GMi expressing cell lines in the presence of effector cells via antibody dependent cellular cytotoxicity (ADCC) in a fluorescence cytotoxicity assay. ADCC activity is measured using the Delfia System (Perkin-Elmer). Briefly, effector cells are cultured from human peripheral blood mononuclear cells (PBMC) by overnight 10 stimulation with 200 u/ml IL-2 and are resuspended to 2 x 10 7 /ml. Target cells are diluted to 106/ml and are loaded with a fluorescence enhancing ligand (BATDA) by incubating 20 minutes and are diluted to 2 x 107 cells/ml. Effector and target cells are combined at a 100; 1 ratio in a microtiter assay plate and mixed with a serial dilution of test antibody and isotype control. Plates are incubated 1 hour at 37"C, centrifuged to pellet cells and 20 ul of supernate is removed [5 and mixed with Eu solution (Perkin-Elmer). Fluorescence resulting from released ligand combined with Eu is measured on a Fusion plate reader (Perkin-Elmer) and is proportional to cell lysis. Assay wells with effector cells in the absence of antibody and with detergent control for background lysis and complete lysis respectively allowing antibody specific lysis to be calculated. The results are shown in Figure 14. This data demonstrates that the anti-fucosyl 20 GMI antibodies are cytotoxic to cells expressing fucosyl-GMI on the cell surface. Example . Immunohistochemistry of lung carcinoma The ability of the anti-fucosyl-GMI HuMAb 7E4 to recognize fucosyl-GM1 by IHC was examined using clinical biopsies of small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). 25 For immunohistochemistry, 5mm frozen sections (Ardais Inc, USA) prepared from clinical biopsies of small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). After drying for 30 minutes, sections were fixed with methanol (at room temperature WO 2007/067992 PCT/US2006/061817 78 for 10 minutes) and air-dried for 5 minutes. Slides were rinsed in PBS and then pre-incubated with 10% normal goat serum in PBS for 20 min and subsequently incubated with 10 mg/mI biotinylated 7E4 in PBS with 10% normal goat serum for 30 nin at room temperature. Slides were washed three times with PBS and then incubated for 30 min with streptavidin-FITC ( 5 DAKO) at room temperature. Slides were washed again with PBS and incubated with Goat anti FITC HRP conjugate (DAKO) for 30 minutes at room temperature, Slides were washed again 3x with PBS. Diaminobenzidine (Sigma) was used as an HRP substrate, resulting in brown staining of tissues positive for 7E4 binding. After washing with distilled water, slides were counter stained with hematoxyllin for 1 min to show tissue structure. Subsequently, slides were washed 10 for 10 seconds in running distilled water and mounted in glycergel (DAKO). Clinical biopsy immunohistochemical staining displayed positive staining in both the SCLC and NSCLC sections. Only malignant cells were positive in each case, adjacent normal lung tissue was not stained. Overall prevalence in lung carcinoma was 5/13 samples tested (2/6 SCLC and 3/7 NSCLC). IHC was negative for 7E4 binding to normal lung tissue. 15 Example 9. Production of nonfucosylated HuMAbs Antibodies with reduced amounts of fucosyl residues have been demonstrated to increase the ADCC ability of the antibody. In this example, an anti-Fucosyl GMI HuMAb is produced that is lacking in fucosyl residues. The CHO cell line Ms704-PF, which lacks the fucosyltransferase gene, FUT 8 (Biowa, 20 Inc,, Princeton, NJ) is electroporated with a vector which expresses the heavy and light chains of an anti-Fucosyl GMI HuMAb. Drug-resistant clones are selected by growth in Ex-Cell 325-PF CHO media (JRH Biosciences, Lenexa, KS) with 6 mM L-glutamine and 500 g/ml G41 8 (Invitrogen, Carlsbad, CA). Clones are screened for IgG expression by standard ELISA assay. Example 10. In vivo Efficacy of anti-Fucosyl-GM1 human monoclonal antibodies 25 DMS79 small cell lung cancer cells (Fucosyl-GM1 were subcutaneously implanted in in male SCID mice (5 x 106 cells/mouse) for a time sufficient (about 8 days) to permit the formation of tumors. On day 8 post-implantation, tumor measurements were taken and mice WO 2007/067992 PCT/US2006/061817 79 were randomized based on mean tumor volume (about 200 mm 3 ) into six groups of eight mice each for subsequent antibody therapy. On days 8 11, 15, 18, and 22 post-implantation, mice were injected intraperitoneally (i.p.) as the following groups: (A) PBS (vehicle control); (B) human IgGI (isotype control) at 30 mg/kg per mouse; (C) anti-Fucosyl-GM1 monoclonal 5 antibody 5B1 at 10 mg/kg per mouse; (D) anti-Fucosyl-GMI monoclonal antibody 51 at 30 mg/kg per mouse; (E) anti-Fucosyl-GM1 monoclonal antibody 7E4 at 10 mg/kg per mouse; or (F) anti-Fucosyl-OMI monoclonal antibody 7E4 at 30 mg/kg per mouse. The monoclonal antibody compositions used in these experiments had low levels of endotoxin and did not significantly aggregate. Using an electronic precision caliper, tumors were measured three 10 dimensionally (height x width x length) and tumor volume was calculated. Tumor measurements were taken twice a week for the duration of the experiment (61 days). Mice were euthanized when the tumors reached a designated tumor end-point (a particular tumor volume such as 1500 mm3 and/or when the mice showed sign of discomfort or greater than about 15% weight loss). To examine whether the anti-Fucosyl-GMI monoclonal antibodies delayed tumor 15 growth, the day tumor reached a volume of 1000 mm was monitored, Both of the 7E4 and 5B1 anti-Fucosyl-GM1 monoclonal antibodies significantly delayed tumor growth compared to vehicle and isotype controls (see Table 1). The antibody efficacy appears to be dose-dependant with the 30 mg/kg treatment groups showing a greater response in each case. Moreover, tumor volumes in mice treated with the 7E4 antibody at 30 mg/kg never reached 1000 mm 3 and had a 20 mean tumor volume of 600 mm3 at the termination of the study on day 61. Table 1. Treatment . Day Tumor Volume at 1000 mm 3 PBS (vehicle control) 34 h-IgG1 (isotype control) 32 Anti-5B1 (10 mg/kg) 56 Anti-5B1 (30 mg/kg) 60 Anti-7E4 (10 mg/kg) 57 Anti-7E4 (30 mg/kg) >61 WO 2007/067992 PCT/US2006/061817 80 To examine whether Fucosyl-GM1 continued to be expressed by the DMS79 cells in vivo, binding of monoclonal antibodies 7114 and 5B1 to DMS79 cells was analyzed by FACS prior to implantation and after recovery of DMS79 cells from untreated tumors. The antibodies bound pre- and post-implantation of DMS79 cells demonstrated that Fucosyl-GM1 expression 5 levels were maintained in vivo (Figure 11C). Figure ISA shows that all control mice (Groups A and B) except one reached the tumor end-point well before day 61. Figure 15B shows that the group treated with 10 mg/kg of anti Fucosyl-GM1 5B 1 antibody (Group C) had two mice that reached the tumor end-point and six mice with tumors having a volume ranging from about 600 mm 3 to 1000 mm 3 , while none of the [0 eight mice in the group treated with 30 mg/kg of anti-Fucosyl-GM1 5B1 antibody (Group D) reached the tumor end-point by day 61 (having a volume of 1000 mm 3 or less). Figure 1 5C shows that none of the eight mice in the group treated with 10 mg/kg anti-Fucosyl-GMl monoclonal antibody 7E4 (Group E) reached the tumor end-point by day 61 (having a volume of about 1200 mm 3 or less, and one mouse being tumor free). Figure 15C also shows that none of .5 the eight mice in the group treated with 30 mg/kg anti-Fucosyl-GMI monoclonal antibody 7E4 (Group F) reached the tumor end-point by day 61 (having a volume of about 800 mm 3 or less, and two mice being tumor free). Figure 16 shows the (A) mean and (B) median tumor volumes measured at day 61. The antibody efficacy appears to be dose-dependant with the 30 mg/kg treatment groups showing a greater response in each case as compared to the controls. 20 Table 2. Tumor Growth Inhibition (TGI) Tumor Free, Day 40 Group (Day 32) % (#/total) Mean Vol.* %a MedianVol.* % PBS 897 - 882 h-IgGl 1189 - 1066 Anti-5B 1 (10mg/kg) 227 81 201 81 Anti-5B1 (30mg/kg) 174 85 172 84 Anti-7E4 (10mg/kg) 177 85 175 84 12.5 % (1/8) Anti-7E4 (30mg/kg) 116 90 141 87 25 % (2/8) This study indicates tt, in a mutine tunor model treatment with ati-Fueosyl-GMI anybody functions in a dose dependent m ner and has a signiacantly greatereffecton tumor growth thn the vehicle and isotype controls. The an&Fucosyl-&MI antibody treatment also did not cause the mice to lose weight or result in any other sigiiflcant sidets, indicating that these andbodies are safe and well tolerated (Figure 1 ). Indeed, the anti-Fucosyl-GMI antibodies showed a percent tumor growth inhibition (TOI %) ranging from 81 to 90 % on day 32 (Table 2). In addon, Ireatment with antibody 7E4 at a 30 mg/kg dose resulted in 25% of the mice being tunor free. The present disclosure is not to be limited in scope by the spcific embodiments described herein. Indeed, various modifications of this disclosure, in addition to those described herein, will become apparent to those sidled in the art from the foregoing deseiption and the accompanying figures. Such modifications are intended to fall within the scope ofthe appended claims. This disclosure is, therefore, to be limited only by the terms of the appended claims along with the full scope of equivalents to which the claims are entitled. The reference in this specification to any prior publication (or iiiformation derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to Nthich this specification relates. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

82 SEQUENCE LISTING SEQ ID NO: SEQUENCE SEQ ID NO; SEQUENCE 1 VH a.a. 5B1 32 VK CDR1 a.a. 5BI a 2 VH a.a. 5Bla 33 VK CDR1 a.a. 7D4 3 VH a.a. 7D4 34 VK CDR1 a.a. 7E4 4 VH a.a.7E4 35 VK CDR1 a.a. 13B8 5 VH a.a. 13B8 36 VK CDRI a.a. 18D5 6 VH a.a. 18D5 37 VK CDR2 a.a. 5B1 7 VK a.a. 5B1 38 VK CDR2 a.a. 5BI a 8 VK a.a. 5B Ia 39 VK CDR2 a.a. 7D4 9 VK a.a. 7D4 40 VK CDR2 a.a. 7E4 10 VK a.a. 7E4 41 VK CDR2 a.a. 13B8 11 VK a.a. 13B8 42 VK CDR2 a.a. 18D5 12 VK a.a. 18D5 43 VK CDR3 a.a. 5BI 13 VH CDR1 a.a. 5B1 44 VK CDR3 aa. 5BIa 14 VH CDR1 a.a. 5Bla 45 VK CDR3 a.a. 7D4 15 VH CDR1 a.a. 7D4 46 VK CDR3 a.a. 7E4 16 VH CDR1 a.a.7E4 47 VK CDR3 a.a. 13B8 17 VH CDR1 a.a. 13B8 48 VK CDR3 aa. 18D5 18 VH CDR1 a.a. 18D5 49 VH n.t. 5B1 19 VH CDR2 a.a. 5B1 50 VH n.t. 5B I a 20 VH CDR2 a.a. 5Bl a 51 VH n-t. 7D4 21 VH CDR2 a.a. 7D4 52 VH n.t. 7E4 22 VH CDR2 a.a. 7E4 53 VH n.t. 13B8 23 VH CDR2 a.a. 13B8 54 VH n.t. 3C4 24 VH CDR2 a.a. 18D5 55 VK n.t. 5BI 25 VH CDR3 a.a. 5B1 56 VK nt. 5BI a 26 VH CDR3 a.a. 5Bla 57 VK n-t. 7D4 27 VH CDR3 a.a. 7D4 58 VK n.t. 7E4 28 VHCDR3 a.a. 7E4 59 VK n.t. 13B8 29 VH CDR3 a.a. 13B8 60 VK n.t. 3C4 30 VH CDR3 a.a, 18D5 61 VH 3-48 germlinea.a. 31 VK CDR1 a.a. 5B1 62 VK L 15 germline a.a. C:NRPonblI\DCCULL393 6809 1.DOC - 19/) 111

Claims (39)

1. An isolated human monoclonal antibody, or an antigen-binding portion thereof, wherein the antibody: (a) specifically binds to Fucosyl-GMI; and (b) binds to the human small cell lung cancer cell line DMS-79 (Human SCLC ATCC # CRL-2049).

2. The antibody of claim 1 which is full-length antibody of an IgGI or IgG4 isotype.

3. The antibody of claim I which is an antibody fragment or a single chain antibody.

4. An isolated human monoclonal antibody, or an antigen-binding portion thereof, wherein the antibody cross-competes for binding to Fucosyl-GMI with a reference antibody comprising: (a) a human heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 4, 5 and 6; and (b) a human light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:7, 8, 9, 10, 11 and 12.

5. The antibody of claim 4, wherein the human heavy chain variable region comprises the amino acid sequence of SEQ ID NO:1 and the human light chain variable region comprises the amino acid sequence of SEQ ID NO:7.

6. The antibody of claim 4, wherein the human heavy chain variable region comprises the amino acid sequence of SEQ ID NO:2 and the human light chain variable region comprises the amino acid sequence of SEQ ID NO:8.

7. The antibody of claim 4, wherein the human heavy chain variable region comprises the amino acid sequence of SEQ ID NO:3 and the human light chain variable region comprises the amino acid sequence of SEQ ID NO:9. WO 2007/067992 PCT/US2006/061817 84

8. The antibody of claim 4, wherein the human heavy chain variable region comprises the amino acid sequence of SEQ ID NO:4 and the human light chain variable region comprises the amino acid sequence of SEQ ID NO:10.

9. The antibody of claim 4, wherein the human heavy chain variable region comprises the amino acid sequence of SEQ ID NO:5 and the human light chain variable region comprises the amino acid sequence of SEQ ID NO:11.

10. The antibody of claim 4, wherein the human heavy chain variable region comprises the amino acid sequence of SEQ ID NO:6 and the human light chain variable region comprises the amino acid sequence of SEQ ID NO:12.

11. An isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region that is the product of or derived from a human VH 3-48 gene, wherein the antibody specifically binds to Fucosyl-GM1.

12. An isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a light chain variable region that is the product of or derived from a human VK L15 gene, wherein the antibody specifically binds to Fucosyl-GMI.

13. The isolated monoclonal antibody or an antigen-binding portion thereof of claim 12 further comprising a heavy chain variable region that is the product of or derived from a human VH 3-48 gene.

14. The antibody of claim 1, which comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO:13; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:19; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:25; (d) a light chain variable region CDR1 comprising SEQ ID NO:31; (e) a light chain variable region CDR2 comprising SEQ ID NO:37; and WO 2007/067992 PCT/US2006/06] 817 85 (f) a light chain variable region CDR3 comprising SEQ ID NO:43.

15. The antibody of claim 1, which comprises: (a) a heavy chain variable region CDR comprising SEQ ID NO: 14; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:20; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:26; (d) a light chain variable region CDR1 comprising SEQ ID NO:32; (e) alight chain variable region CDR2 comprising SEQ ID NO;38; and (f) alight chain variable region CDR3 comprising SEQ ID NO:44.

16. The antibody of claim 1, which comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO:I 5; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:21; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:27; (d) a light chain variable region CDR1 comprising SEQ ID NO:33; (e) a light chain variable region CDR2 comprising SEQ ID NO:39; and (f) a light chain variable region CDR3 comprising SEQ ID NO:45.

17. The antibody of claim 1, which comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 16; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:22; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:28; (d) a light chain variable region CDRI comprising SEQ ID NO:34; (e) a light chain variable region CDR2 comprising SEQ ID NO:40; and (f) a light chain variable region CDR3 comprising SEQ ID NO:46.

18. The antibody of claim 1, which comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 17; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:23; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:29; (d) a light chain variable region CDR1 comprising SEQ ID NO:35; WO 2007/067992 PCT/US2006/061817 86 (e) a light chain variable region CDR2 comprising SEQ ID NO:41; and (f) a light chain variable region CDR3 comprising SEQ ID NO:47,

19. The antibody of claim 1, which comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO:1 8; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:24; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:30; (d) a light chain variable region CDR1 comprising SEQ ID NO:36; (e) a light chain variable region CDR2 comprising SEQ ID NO:42; and (f) a light chain variable region CDR3 comprising SEQ ID NO:48.

20. An isolated monoclonal antibody or antigen binding portion thereof comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:7.

21. An isolated monoclonal antibody or antigen binding portion thereof comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:8.

22. An isolated monoclonal antibody or antigen binding portion thereof comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:9.

23. An isolated monoclonal antibody or antigen binding portion thereof comprising: WO 20071067992 PCT/US2006/06] 817 87 (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:10.

24. An isolated monoclonal antibody or antigen binding portion thereof comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:11.

25. An isolated monoclonal antibody or antigen binding portion thereof comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:12.

26. A composition comprising the antibody or antigen-binding portion thereof, of claim I and a pharmaceutically acceptable carrier.

27. An immunoconjugate comprising the antibody or antigen-binding portion thereof, of claim 1, linked to a therapeutic agent.

28. A composition comprising the immunoconjugate of claim 27 and a pharmaceutically acceptable carrier.

29. The immunoconjugate of claim 27, wherein the therapeutic agent is a cytotoxin.

30. A composition comprising the immunoconjugate of claim 29 and a phannaceutically acceptable carrier. WO 2007/067992 PCT/US2006/061817 88

31. The immunoconjugate of claim 27, wherein the therapeutic agent is a radioactive isotope.

32. A composition comprising the immunoconjugate of claim 31 and a pharmaceutically acceptable carrier,

33. An isolated nucleic acid molecule encoding the antibody or antigen-binding portion thereof, of claim 1.

34. An expression vector comprising the nucleic acid molecule of claim 33.

35. A host cell comprising the expression vector of claim 34.

36. A method for preparing an anti-Fucosyl--GMI antibody which comprises expressing the antibody in the host cell of claim 35 and isolating the antibody from the host cell.

37. A method of treating or preventing a disease characterized by growth of tumor cells expressing Fucosyl-GMi, comprising administering to a subject the antibody or antigen binding portion thereof, of claim 1 in an amount effective to treat or prevent the disease.

38. The method of claim 37, wherein the disease is cancer.

39. The method of claim 38, wherein the cancer is lung cancer.