AU2013201832C1 - Anti-extended type I glycosphingolipid antibody, derivatives thereof and use - Google Patents

Abstract

Human antibodies and antigen-binding portions of those antibodies that specifically bind extended Type I chain glycosphingolipids are provided. 41662091 (GHMatters) P6521AU 1 117

Description

TITLE "ANTI-EXTENDED TYPE I GLYCOSPHINGOLIPID ANTIBODY, DERIVATIVES THEREOF AND USE" 5 Tong-Hsuan Chang, Jerry Ting, Tsai-Hsia Hong, Mei-Chun Yang, Liahng-Yim Liu, Shu-Yen Chang, Ying-Zin Chen, Jaw-Yuan Wen, Kazuko Handa and Sen-itiroh Hakomori FIELD OF THE INVENTION [0001] The present invention relates to anti-extended Type I glycosphingolipid 10 antibodies and their use in the amelioration, treatment or prevention of diseases or disorders in mammals, including humans, resulting from improper activity or metabolism of same; resulting, causing or associated with; or the presence thereof, for example, in a cancer, such as colorectal cancer, or other pathology. An antibody of interest can be used for therapeutic purposes or diagnostic purposes. Thus, 15 prophylactic, immunotherapeutic and diagnostic compositions comprising the antibodies and derivatives thereof of interest and their use in methods for preventing or treating, or diagnosing diseases in mammals, including humans, caused by inappropriate metabolism and/or expression of extended Type I glycosphingolipid in and on cells, such as certain malignant cells, also are disclosed. 20 BACKGROUND [0002] Extended Type I glycosphingolipid is a cell surface molecule that can be associated with, for example, certain malignant states. [0003] Aberrant glycosylation has been observed to be a common feature of 25 many cancer types, Hakomori, PNAS 99:10231-10233, 2002. Some of the carbohydrate antigens used for the diagnosis of human cancers carry polylactosamine structures. Polylactosamines are usually classified into two categories according to the unit structure. A polylactosamine having the 4166209j IGHMatters) P86521.AU.1 Galpl-3GlcNAc structure is called a Type I chain, and that having the Galpl-4GlcNAc structure is referred to as a Type II chain. The most common tumor-associated antigens found in some human cancers have the lacto series Type II chain structure, which usually is sialylated and/or fucosylated. Type I chain 5 antigens are abundant in nonnal cells and tissues, and occasionally are associated with cancer, Stroud et al., JBC 266: 8439-8446, 1991. For example, 2-3 sialylated Le antigen (the CA 19-9 antigen defined by the N19-9 antibody) is a cancer-associated Type I chain antigen. However, cancer diagnostic methods based on the detection of those Type I antigens have been hampered by high false positive 10 and/or high false negative incidences, see, for example U.S. Patent Nos. 6,083,929 and 6,294,523. [0004] Two mouse monoclonal antibodies, NCC-ST421 and IMH2, were raised against extended Type I chain antigens. NCC-ST421 is specific for Lea-Lea. The NCC-ST421 antibody strongly induced antibody dependant cell cytotoxicity 15 (ADCC) using human peripheral blood leukocytes as effectors against a variety of human tumor cells, and induced complement dependent cytotoxicity (CDC) with a human complement source, Watanabe et al., Cancer Res. 51:2199-2204, 1991. The Lea-Le" antigen was found to be highly expressed in the human colon carcinoma cell line, Colo205. 20 [0005] IMH2 was also established against extended Type I chains. IMH2 bound to Le-Le", Ley-LeX, Leb and Le' based on 'H-NMR, FAB-MS and enzymatic degradation studies, Stroud et al., Eur. J. Biochem. 203:577-586, 1992. IMH2 showed strong lymphocyte-activated, as well as, complement-dependent killing of Colo205 cells in vitro, and inhibited Colo205 growth in vivo, 25 [0006] IMH2 reacted with carcinoma tissues derived from colon, pancreas, liver and endometrium. However, normal colon showed no reactivity with IMH2. Normal liver and pancreas showed weak or highly restricted reactivity in normal hepatocytes and islets of Langerhans cells. Immunochemical staining intensity was much stronger in endometrial carcinomas than in normal endometrium, Ito et al., 30 Cancer Res. 52:3739-3745, 1992. 41662091 (GHMatters) P86521.AU1 2 [0007] Both NCC-ST421 and IMH2 exhibit inhibition of tumor growth in nude mice after inoculation of human tumor cells expressing the extended Type I chain antigen, but no inhibition of growth occurred in tumor cells that did not express extended Type I chain antigen. 5 [0008] Because of the abundance of Type I structures on normal cells, the use of Type I antibodies for diagnostic and/or therapeutic purposes heretofore was not possible. [0009] Conventional cancer treatments, such as chemotherapy and radiotherapy, have shown some advantages in various cancer patients. Despite the benefits of 10 antitumor activity in conventional therapies, however, treatment-induced toxicity to nonnal tissues can substantially reduce the quality of life in cancer patients. Dose intensification for better antitumor activity is also limited. Monoclonal antibodies enable the promise of targeted cytotoxicity, focusing on tumor tissues, but not normal tissues. 15 [0010] Monoclonal antibodies (mAbs) can be developed with high specificity for antigens expressed on tumor cells and can elicit desired antitumor activities. The promise of mAbs was furthered by the development of mice that produce fully human mAbs. One such tool is the KM mouse, U.S. Pat No. 7,041,870 and Tomizuka et al., Nat. Genet. 16:133-143, 1997. In the KM mouse, the mouse genes 20 encoding immunoglobulins were inactivated and replaced with human antibody genes. Thus, the KM mouse expresses fully human antibodies. [0011] Several fully human antibodies have been successfully developed using the KM mouse. [0012] For example, Motoki et al. developed a human IgG (KMTR2) which 25 directed antibody dependent oligomerization of TRAIL-R2 and initiated efficient apoptotic signaling and tumor regression independent of host effector function (Clin. Cancer Res. 11 (8):3126-3135, 2005; and see U.S. Pat. No. 7,115,717 and Imakire et al., Int. J. Cancer 108:564-570, 2004). HD8, a fully human monoclonal antibody specific for human leukocyte antigen DR (HLA-DR), exerted antibody-dependent 30 cellular cytotoxicity (ADCC) as well as complement-dependent cytotoxicity (CDC) 4166209g1 (GHMatters) P6521.AU.1 3 in vitro, and extended the life span of immunocompromised mice inoculated with non-Hodgkin lymphoma cell lines, Tawara et al,, Cancer Sci. 98 (6) 921-928, 2007. [0013] Additionally, two human IgMs raised in KM mice and directed to carbohydrate antigens were reported. HMMC-1 specifically recognizes a novel 5 0-glycan structure, reacts positively with Mullerian duct-related carcinomas, and exhibits complement dependent cytotoxicity on a human uterine endometrial cancer cell line, SNG-S, Nozawa et al., Clin Cancer Res. 10:7071-7078, 2004. Another human monoclonal IgM, HMOCC-1, recognizing a glycoprotein located on the cell membrane, reacted with ovarian cancer (Suzuki et al., Gynecol. Oncol. 95:290-298, 10 2004). Since those two antibodies are IgMs, the application of those antibodies in cancer therapy should be limited by molecule size and restrictions in production. SUMMARY [0014] The present invention provides novel human antibodies, and fragments 15 and derivatives thereof, that specifically bind to extended Type I glycosphingolipid. [0015] The invention includes the amino acid sequences of the variable heavy and light chain of the antibodies and their corresponding nucleic acid sequences. [0016] Another embodiment of the invention includes the complementarity detennining regions (CDR) sequences of the antibodies of interest to obtain binding 20 molecules that comprise one or more CDR regions, or CDR-derived regions, that retain extended Type I glycosphingolipid-binding capacity of the parent molecule from which the CDR's were obtained. [0017] Another embodiment of the present invention includes the cell lines and vectors harboring the antibody sequences of the present invention. 25 [0018] Another embodiment of the present invention relates to the use of the antibodies for the preparation of a medicament or composition for the treatment of diseases and disorders associated with extended Type I glycosphingolipid function, metabolism and expression. 4166209_1 (GHMatters) P86521.AV1 4 [0019] Another embodiment of the present invention relates to the use of the antibodies in the diagnosis of disorders associated with atypical or abnormal extended Type I glycosphingolipid biology and expression. [0020] Those and other goals were met in the development of human 5 monoclonal antibodies against extended Type I chain carbohydrate antigens. For example, mAb GNX-8 is a human IgGI derived from a KM mouse. GNX-8 exhibits CDC and ADCC activity on several human colorectal cancer cell lines and inhibits Colo205 and DLD-1 tumor growth in vivo. GNX-8 reacts with primary and metastatic colorectal cancers, breast cancers, pancreas cancers as well as lung 10 cancers, but not with normal human tissues and blood cells. [0021] Additional features and advantages are described herein, and will be apparent from, the following Detailed Description. Brief description of the drawings 15 [0021a] The above and other objects, features and advantages of this invention will become apparent with reference to the following detailed description and the preferred embodiments taken in conjunction with the accompanying drawings, in which: [0021b] Figure 1 shows the TLC immunostaining results of mAbs GNX-8 and 20 GNX-10; [002 1c] Figure 2 shows the binding specificity of GNX-8 and GNX-10 to Colo205 neutral glycosphingolipids, in which mAbs GNX-8 and GNX-10 were examined by ELISA using mouse anti-human IgG (Fc)-HRP as the secondary antibody and 3,3',5,5'-tetramethylbenzidine (TMB) as the substrate for color 25 development, which was read by an ELISA reader at 450 nm; [002 1d] Figure 3 shows the respective binding specificities of mAbs GNX-8 and GNX-10 to Le -Le-Lac, in which mAbs GNX-8 and GNX-10 were added into the wells of 96-well ELISA plates sequentially coated with ImmunoPure Streptavidin and biotinylated Leb-Lea- Lac and then examined by ELISA using 6184551_1 (GHMatters) P86521.AU.1 KAROLA 5 mouse anti-human IgG (Fc)-HRP as the secondary antibody and TMB as the substrate for color development, which was read by an ELISA reader at 450 nm; [002 1e] Figure 4 shows the respective antibody-dependent cellular cytotoxicity (ADCC) activities of mAbs GNX-8 and GNX-10 as determined by 5 dehydrogenase (LDH) release assay described in Example 4, infra; and [0027f] Figure 5 shows the respective complement-dependent cytotoxicity (CDC) activities of mAbs GNX-8 and GNX-10 on human colon cancer cell line Colo205, as described in Example 4, infra., in which cancer cells treated with normal human IgGs served as a negative control. 10 DETAILED DESCRIPTION [0022] The invention is not limited to the particular methodology, protocols, polypeptides, polynucleotides, cell lines, vectors, or reagents described herein because variations can occur or can be used without departing from the spirit and 15 scope of the invention. Further, the terminology used herein is for the purpose of exemplifying particular embodiments only and is not intended to limit the scope of the present invention. Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Any method and 20 material similar or equivalent to those described herein can be used in the practice of the present invention and only exemplary methods, devices, and materials are described herein. [0023] All patents and publications mentioned herein are incorporated herein in entirety by reference for the purpose of describing and disclosing the proteins, 25 enzymes, vectors, host cells and methodologies reported therein that might be used with and in the present invention. However, nothing herein is to be construed as an 6184551_1 (GHMatters) P86521.AU.1 KAROLA 5a admission that the invention is not entitled to antedate such disclosures by virtue of prior invention. [0024] An "extended Type I glycosphingolipid disease" is a malady, disorder, disease, pathology, condition, abnormality and so on, which is characterized by, 5 associated with or caused by abnormal metabolism, overexpression or increased levels of extended Type I glycosphingolipid, for example, at the cell surface. [0025] The phrase "substantially identical" with respect to an antibody polypeptide sequence may be construed as an antibody chain exhibiting at least 70%, 80%, 90%, 95% or more sequence identity to a reference polypeptide 10 sequence. The term with respect to a nucleic acid sequence may be construed as a sequence of nucleotides exhibiting at least about 85%, 90%, 95%, 97% or more sequence identity to a reference nucleic acid sequence. [0026] The terms, "identity" or "homology" may mean the percentage of nucleotide bases or amino acid residues in the candidate sequence that is identical 15 with the residues of a corresponding sequence to which the candidate is compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. Neither N-terminal nor C-terminal extensions nor insertions shall be construed as reducing identity or 20 homology. Methods and computer programs for the alignment of sequences are available and are well known in the art. Sequence identity may be measured using sequence analysis software. [0027] The phrases and terms, "functional fragment, variant, derivative or analog" and the like, as well as forms thereof, of an antibody, nucleic acid or antigen 25 is a compound or molecule having qualitative biological activity in common with a full length antibody or antigen of interest. For example, a functional fragment or analog of an anti-extended Type I glycosphingolipid antibody is one which can bind to an extended Type I glycosphingolipid molecule, or is an agonistic or antagonistic antibody which binds to extended Type I glycosphingolipid. An example is an scFv 30 molecule. As to extended Type I glycosphingolipid, a variant or derivative thereof 41662091 (GHMaturs) PS6521.AU1 6 is a molecule that is not identical to a naturally occurring extended Type I glycosphingolipid and yet can be used for a purpose of the instant invention, such as, while not identical to a wild type extended Type I glycosphingolipid nevertheless can be used, for example, as immunogen to raise antibodies that selectively bind to 5 wild type extended Type I glycosphingolipid. [0028] "Substitutional" variants are those that have at least one amino acid residue in a native sequence removed and replaced with a different amino acid inserted in place at the same position. The substitutions may be single, where only one amino acid in the molecule is substituted, or may be multiple, where two or 10 more amino acids are substituted in the same molecule. The plural substitutions may be at consecutive sites. Also, one amino acid can be replaced with plural residues, in which case such a variant comprises both a substitution and an insertion. [0029] "Insertional" variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native sequence. 15 Immediately adjacent to an amino acid means connected to either the a-carboxyl or a-amino functional group of the amino acid. [0030] "Deletional" variants are those with one or more amino acids in the native amino acid sequence removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the molecule. 20 [0031] The terms, substitution, insertion and deletion variants also apply analogously to nucleic acids. [0032] The adaptive immune response has two major arms: the cellular immune response of T lymphocytes and the humoral immune response of antibody secreting B lymphocytes. B cell epitopes can be linear, contiguous amino acids, or can be 25 confonnational (Protein Science (2005) 14, 246). In contrast, T cell epitopes are short linear peptides that are cleaved from antigenic proteins that are presented in the context of major histocompatibility complex (MHC) proteins, or, in case of humans, human leukocyte antigen (HLA) class I or class II molecules. Epitope presentation depends on both MHC-peptide binding and T cell receptor (TCR) interactions. 30 MHC proteins are highly polymorphic, and each binds to a limited set of peptides. 4166209_1 (GHMalters) P86521AU.4 7 Thus, the particular combination of MHC alleles present in a host limits the range of potential epitopes recognized during an infection. [0033] Two fundamental types of T cells are distinguished by expression of CD8 and CD4 proteins, which dictate whether a T cell will recognize epitopes presented 5 by class I or class II molecules, respectively. CD4t T epitopes are processed after encapsulation by antigen presenting cells in membrane bound vesicles, where the antigen is degraded by proteases into peptide fragments that bind to MHC class II proteins. In contrast, CD8 T cells generally recognize viral or self antigens expressed from within a cell, proteins that are cleaved into short peptides in the 10 cytosol by the inununoproteasome. After cleavage, peptides are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum for loading onto HLA I antigens. CD4 T (helper) cell epitopes are critical in driving T cell-dependent immune responses to protein antigens. [0034] The term "antibody" is used in the broadest sense, and includes 15 monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments or synthetic polypeptides carrying one or more CDR or CDR-derived sequences so long as the polypeptides exhibit the desired biological activity. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural 20 characteristics. Generally, antibodies are considered Igs with a defined or recognized specificity. Thus, while antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity. [0035] The antibodies of the invention can be of any class (e.g., IgG, IgE, IgM, 25 IgD, IgA and so on), or subclass (e.g., IgGI, IgG2, IgG 2 a, IgG3, IgG 4 , IgA 1 , IgA 2 and so on) ("type" and "class," and "subtype" and ""subclass," are used interchangeably herein). Native or wildtype, that is, obtained from a non-artificially manipulated member of a population, antibodies and immunoglobulins, and monomers of polymeric antibodies, such as IgA and IgM, are usually heterotetrameric 30 glycoproteins of about 150,000 daltons, composed of two identical light (L) chains 416C2091 (GHMattam) P86521.AU 1 8 and two identical heavy (H) chains. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end. [0036] By "non-artificially manipulated" is meant not treated by non-natural 5 means, such as immunization or transformation, to contain or to express a foreign antigen binding molecule. Wildtype can refer to the most prevalent allele or species found in a population or to the antibody obtained from a non-artificially manipulated animal, as well as to naturally occurring alleles or polymorphisms which arise naturally and can be sustained in a population, or a variant or derivative arising 10 through natural means, such as a malignancy, as compared to that obtained by a fonn of manipulation, such as mutagenesis, use of recombinant methods and so on to change an amino acid of the antigen-binding molecule. The use of the term is readily inferred and understood by the artisan in the context of the sentence, paragraph, concept, thought, idea and so on in which the term is found, used and so 15 on. [0037] As used herein, "anti-extended Type I glycosphingolipid antibody" means an antibody or derived polypeptide which binds specifically to human extended Type I glycosphingolipid. [0038] The term "variable" in the context of a variable domain of antibodies, 20 refers to certain portions of a pertinent molecule which differ extensively in sequence between and among antibodies and can be integral in the specific recognition and binding of a particular antibody to a particular target. However, the variability is not evenly distributed through the variable domains of antibodies. [0039] The variability can be concentrated in three segments called 25 complementarity determining regions (CDRs; i.e., CDRl, CDR2 and CDR3) also known as hypervariable regions, both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework (FR) regions or sequences. The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a f sheet configuration, 30 connected by three CDRs, which form loops connecting, and in some cases forming 4166209_1 (GHMatters) PUUS21,AU.1 9 part of, the fl sheet structure. The CDRs in each chain are held together, often in proximity, by the FR regions and, with the CDRs from the other chain, contribute to the formation of the target (epitope or determinant) binding site of antibodies (see Kabat et al. Sequences of Proteins of Immunological Interest, National Institute of 5 Health, Bethesda, MD (1987)). One CDR, such as, CDR3 of the heavy chain, can alone carry the ability to bind specifically to the cognate epitope. [0040] As used herein, numbering of immunoglobulin amino acid residues is done according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated. 10 [0041] The term "antibody fragment" refers to a portion of an intact or a full length chain of an antibody, generally the target binding or variable region. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and F. fragments. A "functional fragment" or "analog of an anti-extended Type I glycosphingolipid antibody" is one which can bind a cognate antigen. As used 15 herein, functional fragment generally is synonymous with, "antibody fragment," and with respect to antibodies, can refer to fragments, such as Fy, Fab, F(ab')2 and so on which can bind a cognate antigen. [0042] An "F" fragment consists of a dimer of one heavy and one light chain variable domain in a non-covalent association (VH-VL dimer). That configuration of 20 the three CDR's of each variable domain interact to define a target binding site of the VH-VL dimer as in an intact antibody. Collectively, the six CDRs confer target binding specificity on the intact antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) can have the ability to recognize and to bind target. 25 [0043] "Single-chain F,," "sF," or "scAb" antibody fragments comprise the VH and VL domains of an antibody, wherein the domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker, often a flexible molecule, such as, an oligopeptides, which may be obtained from a naturally occurring molecule, derived from a naturally occurring molecule, is 30 an artificial sequence, such as polyglycine, and so on, between the VH and VL 4166209_1 (GHMattars)P86521.AU.1 10 domains, which enables the sFv to form the desired structure for target binding. Some molecules can include one or more constant domains or a portion thereof [0044] The term "diabodies" refers to antibody fragment constructs with two antigen binding sites, which fragments can comprise a heavy chain variable domain 5 (VH) connected to a light chain variable domain (VL) in the same polypeptide chain. By using a linker that is too short to allow pairing between the two variable domains on the same chain, the diabody domains are forced to pair with the binding domains of another chain to create an antigen binding site. [0045] The Fab fragment contains the variable and constant domains of the light 10 chain and the variable and first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl tenninus of the CHI domain to include one or more cysteines from the antibody hinge region. Fab, fragments can be produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional enzymatic and 15 chemical treatments of antibodies can yield other functional fragments of interest. [0046] The term "monoclonal antibody" (mAb or MAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. 20 [0047] Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass (type or subtype), with the remainder of the chain(s) identical with or homologous to corresponding sequences 25 in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as the chimeric antibodies exhibit the desired biological activity of binding to extended Type I glycosphingolipid or impacting extended Type I glycosphingolipid activity or metabolism (U.S. Pat. No. 4,816,567; and Morrison et al., Proc Natl Acad Sci USA 416620_1 (GHMatters) P86521.AU.1 11 81:6851 (1984)). Thus, CDR's from one class of antibody can be grafted into the FR of an antibody of different class or subclass. [0048] Monoclonal antibodies are specific, being directed against a single target site, epitope or detenninant. Furthennore, in contrast to conventional (polyclonal) 5 antibody preparations which typically include different antibodies directed against different detenninants (epitopes) of an antigen, each monoclonal antibody is directed against a single determinant on the target. In addition to their specificity, monoclonal antibodies are advantageous being synthesized by a host cell, uncontaminated by other immunoglobulins, and provide for cloning the relevant 10 gene and mRNA encoding the antibody chains thereof. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies for use with the present invention may be isolated from phage antibody 15 libraries using well known techniques or can be purified from a polyclonal prep. The parent monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant methods well known in the art. [0049] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric 20 immunoglobulins, immunoglobulin chains or fragments thereof (such as Fy, Fab, Fab, F(ab'>2 or other target-binding subsequences of antibodies) which contain sequences derived from non-human immunoglobulin, as compared to a human antibody. In general, the humanized antibody will comprise substantially all of one, and typically two, variable domains, in which all or substantially all of the CDR regions 25 correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin template sequence. [0050] The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of the human immunoglobulin template chosen. In general, the goal is to have an antibody molecule of certain 30 specificity that is minimally immunogenic in a human. Thus, it is possible that one 4166209.1 (GHMalter) P652.AU1 12 or more amino acids in one or more CDR's also can be changed to one that is less immunogenic to a human host, without substantially minimizing the specific binding function of the one or more CDR's to extended Type I glycosphingolipid. [0051] Alternatively, the FR can be non-human but those amino acids most 5 immunogenic are replaced with ones less immunogenic. Nevertheless, CDR grafting, as discussed above, is not the only way to obtain a humanized antibody. For example, modifying just the CDR regions may not be sufficient to optimize an antibody as it is not uncommon for framework residues to have a role in determining the overall three-dimensional structure of the CDR loops and the overall affinity of 10 the antibody for the ligand. [0052] Hence, any means can be practiced to reduce antibody immunogenicity so that the non-human parent antibody molecule is modified to be one that is less immunogenic to a human, and global sequence identity with a human antibody is not always a necessity. So, humanization also can be achieved, for example, by the 15 mere substitution of just a few residues, particularly those which are exposed on the antibody molecule surface and not buried within the molecule, and hence, not readily accessible to the host immune system. Such a method is taught herein with respect to substituting, for example, charged or certain other residues on the antibody molecule, the goal being to reduce or dampen the immunogenicity of the 20 resultant molecule without compromising the specificity of the antibody for the cognate epitope or determinant. See, for example, Studnicka et al., Prot Eng 7(6)805-814, 1994; Mol Imm 44:1986-1988, 2007; Sims et al., J Immunol 151:2296 (1993); Chothia et al., J Mol Biol 196:901 (1987); Carter et al., Proc Natl Acad Sci USA 89:4285 (1992); Presta et al., J imnunol 151:2623 (1993), WO 2006/042333 25 and U.S. Pat. No. 5,869,619. [0053] Strategies and methods for resurfacing antibodies, and other methods for reducing immunogenicity of antibodies within a different host, are disclosed, for example, in U.S. Pat. No. 5,639,641. Briefly, in a preferred method, (1) position alignments of a pool of antibody heavy and light chain variable regions are 30 generated to yield heavy and light chain variable region framework surface exposed 4166209_1(GHMatters) P86521AU,1 13 positions, wherein the alignment positions for all variable regions are at least about 98% identical; (2) a set of heavy and light chain variable region framework surface exposed amino acid residues is defined for a non-human, such as, a rodent antibody (or fragment thereof); (3) a set of heavy and light chain variable region framework 5 surface exposed amino acid residues that is most closely identical to the set of rodent surface exposed amino acid residues is identified; and (4) the set of heavy and light chain variable region framework surface exposed amino acid residues defined in step (2) is substituted with the set of heavy and light chain variable region framework surface exposed amino acid residues identified in step (3), except for 10 those amino acid residues that are within about 5 A of any atom of any residue of a CDR of the, for example, rodent antibody, to yield a humanized, such as, a rodent antibody retaining binding specificity. [0054] Antibodies can be humanized by a variety of other techniques including CDR grafting (EPO 0 239 400; WO 91/09967; and U.S. Pat. Nos. 5,530,101 and 15 5,585,089), veneering or resurfacing (EPO 0 592 106; EPO 0 519 596; Padlan, 1991, Molec Imm 28(4/5):489-498; Studnicka et al., 1994, Prot Eng 7(6):805-814; and Roguska et al., 1994, PNAS 91:969-973) and chain shuffling (U.S. Pat. No. 5,565,332). Human antibodies can be made by a variety of methods known in the art including, but not limited to, phage display methods, see U.S. Pat. Nos. 20 4,444,887, 4,716,111, 5,545,806 and 5,814,318; and WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741, using transgenic animals, such as rodents (Amgen, Kirin and Merdarex mice), using chimeric cells and so on. [0055] "Antibody homolog" or "homolog" refers to any molecule which 25 specifically binds extended Type I glycosphingolipid as taught herein. Thus, an antibody homolog includes native or recombinant antibody, whether modified or not, portions of antibodies that retain the biological properties of interest, such as binding extended Type I glycosphingolipid, such as an Fab or F, molecule, a single chain antibody, a polypeptide carrying one or more CDR regions and so on. The 30 amino acid sequence of the homolog need not be identical to that of the naturally 41662091 (GHMatters) PB6521.AU.1 14 occurring antibody but can be altered or modified to carry substitute amino acids, inserted amino acids, deleted amino acids, amino acids other than the twenty normally found in proteins and so on to obtain a polypeptide with enhanced or other beneficial properties. 5 [0056 Antibodies with homologous sequences are those antibodies with amino acid sequences that have sequence homology with the amino acid sequence of an extended Type I glycosphingolipid antibody of the present invention. Preferably, homology is with the amino acid sequence of the variable regions of an antibody of the present invention. "Sequence homology" as applied to an amino acid sequence 10 herein is defined as a sequence with at least about 90%, 91%, 92%, 93%, 94% or more sequence homology, and more preferably at least about 95%, 96%, 97%, 98% or 99% sequence homology to another amino acid sequence, as determined, for example, by the FASTA search method in accordance with Pearson & Lipman, Proc Natl Acad Sci USA 85, 2444-2448 (1988). 15 [0057] A chimeric antibody, as taught hereinabove, is one with different portions of an antibody derived from different sources, such as different antibodies, different classes of antibody, different animal species, for example, an antibody having a variable region derived from a murine monoclonal antibody paired with a human immunoglobulin constant region and so on. Thus, a humanized antibody is a 20 species of chimeric antibody. Methods for producing chimeric antibodies are known in the art, see, e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J Immunol Methods 125:191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567, and 4,816,397. [0058] Artificial antibodies include scFv fragments, chimeric antibodies, 25 diabodies, triabodies, tetrabodies and mru (see reviews by Winter & Milstein, 1991, Nature 349:293-299; and Hudson, 1999, Curr Opin Imm 11:548-557), each with antigen-binding or epitope-binding ability. In the single chain F, fragment (scF,), the VH and VL domains of an antibody are linked by a flexible peptide. Typically, the linker is a peptide of about 15 amino acids. If the linker is much smaller, for 30 example, 5 amino acids, diabodies are formed, which are bivalent scFv dimers. If 41662091 (GHMatters)POS$21.AU.1 15 the linker is reduced to less than three amino acid residues, trimeric and tetrameric structures are formed that are called triabodies and tetrabodies, respectively. The smallest binding unit of an antibody is a CDR, for example, CDR3 of the heavy chain which has sufficient specific recognition and binding capacity. Such a 5 fragment is called a molecular recognition unit or mru. Several such mrus can be linked together with short linker peptides, therefore forming an artificial binding protein with higher avidity than a single mru. [0059] Also included within the scope of the invention are functional equivalents of an antibody of interest. The tenn "functional equivalents" includes 10 antibodies with homologous sequences, antibody homologs, chimeric antibodies, antibody variants, antibody derivatives, artificial antibodies and modified antibodies, for example, wherein each functional equivalent is defined by the ability to bind to extended Type I glycosphingolipid. The skilled artisan will understand that there is an overlap in the group of molecules termed "antibody fragments" and the group 15 termed "functional equivalents." Methods of producing functional equivalents which retain extended Type I glycosphingolipid binding ability are known to the person skilled in the art and are disclosed, for example, in WO 93/21319, EPO No. 239,400, WO 89/09622, EPO No. 338,745 and EPO No. 332,424. [0060] The functional equivalents of the present application also include 20 modified antibodies, e.g., antibodies modified by the covalent attachment of any type of molecule to the antibody. For example, modified antibodies include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, deamidation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand, linkage 25 to a toxin or cytotoxic moiety or other protein etc. The covalent attachment need not yield an antibody that is immune from generating an anti-idiotypic response. The modifications may be achieved by known techniques, including, but not limited to, specific chemical cleavage, acetylation, fornylation, metabolic synthesis, chemical conjugation etc. Additionally, the modified antibodies may contain one or more 30 non-classical amino acids. 4166209_1 (GHMatters) P86521.AU_1 1 6 [0061] Many techniques are available to one of ordinary skill in the art which permit the optimization of binding affinity. Typically, the techniques involve substitution of various amino acid residues at the site of interest, followed by a screening analysis of binding affinity of the mutant polypeptide for the cognate 5 antigen or epitope. [0062] Once the antibody is identified and isolated, it is often useful to generate a variant antibody or mutant, or mutein, wherein one or more amino acid residues are altered, for example, in one or more of the hypervariable regions of the antibody. Alternatively, or in addition, one or more alterations (e.g., substitutions) of 10 framework residues may be introduced in the antibody where these result in an improvement in the binding affinity of the antibody mutant for extended Type I glycosphingolipid. [0063] Examples of framework region residues that can be modified include those which non-covalently bind antigen directly (Amit et al., Science 233:747-753 15 (1986)); interact with/affect the conformation of a CDR (Chothia et al., J. Mol. Biol. 196:901-917 (1987)); and/or participate in the VL-VH interface (EP 239 400). In certain embodiments, modification of one or more of such framework region residues results in an enhancement of the binding affinity of the antibody for the cognate antigen. For example, from about one to about five framework residues 20 may be altered in the particular embodiment of the invention. Sometimes, that may be sufficient to yield an antibody mutant suitable for use in preclinical trials, even where none of the hypervariable region residues have been altered. Normally, however, the antibody mutant can comprise one or more hypervariable region alteration(s). The constant regions also can be altered to obtain desirable or more 25 desirable effector properties. [0064] The hypervariable region residues which are altered may be changed randomly, especially where the starting binding affinity of the parent antibody is such that randomly-produced antibody mutants can be readily screened for altered binding in an assay as taught herein. 4162091 (GHMa'ttew) P86521.AU 1 17 [0065] One procedure for obtaining antibody mutants, such as, CDR mutants, is "alanine scanning mutagenesis" (Cunningham & Wells, Science 244:1081-1085 (1989); and Cunningham & Wells, Proc Nat. Acad Sci USA 84:6434-6437 (1991)). One or more of the hypervariable region residue(s) are replaced by alanine or 5 polyalanine residue(s). Those hypervariable region residue(s) demonstrating functional sensitivity to the substitutions then are refined by introducing further or other mutations at or for the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. Similar substitutions can be attempted 10 with other amino acids, depending on the desired property of the scanned residues. [0066J A more systematic method for identifying amino acid residues to modify comprises identifying hypervariable region residues involved in binding extended Type I glycosphingolipid and those hypervariable region residues with little or no involvement with extended Type I glycosphingolipid binding. An alanine scan of 15 the non-binding hypervariable region residues is performed, with each ala mutant tested for enhancing binding to extended Type I glycosphingolipid. In another embodiment, those residue(s) significantly involved in binding extended Type I glycosphingolipid are selected to be modified. Modification can involve deletion of a residue or insertion of one or more residues adjacent to a residue of interest. 20 However, normally the modification involves substitution of the residue by another amino acid. A conservative substitution can be a first substitution. If such a substitution results in a change in biological activity (e.g., binding affinity), then another conservative substitution can be made to determine if more substantial changes are obtained. 25 [0067] Even more substantial modification in an antibody range and presentation of biological properties can be obtained by selecting an amino acid that differs more substantially in properties from that nonnally resident at a site. Thus, such a substitution can be made while maintaining: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical 4166209_1 (GHMattem) P86521AU,1 1 8 conformation; (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. [0068] For example, the naturally occurring amino acids can be divided into groups based on common side chain properties: 5 [0069] (1) hydrophobic: methionine (M or met), alanine (A or ala), valine (V or val), leucine (L or leu) and isoleucine (I or ile); [0070] (2) neutral, hydrophilic: cysteine (C or cys), serine (S or ser), threonine (T or thr), asparagine (N or asn) and glutamine (Q or gln); [0071] (3) acidic: aspartic acid (D or asp) and glutamic acid (E or glu); 10 [0072] (4) basic: histidine (H or his), lysine (K or lys) and arginine (R or arg); [0073] (5) residues that influence chain orientation: glycine (G or gly) and proline (P or pro), and [0074] (6) aromatic: tryptophan (W or trp), tyrosine (Y or tyr) and phenylalanine (F or phe). 15 [0075] Non-conservative substitutions can entail exchanging an amino acid with an amino acid from another group. Conservative substitutions can entail exchange of one amino acid for another within a group. [0076] Preferred amino acid substitutions can include those which, for example: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, 20 (3) alter binding affinity and (4) confer or modify other physico-chemical or functional properties of such analogs. [0077] Analogs can include various muteins of a sequence other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the 25 naturally-occurring sequence (preferably in the portion of the polypeptide outside the domain (s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e. g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that 30 characterizes the parent sequence) unless of a change in the bulk or conformation of 41662091 (GHMattem)P6521.AU.1 19 the R group or side chain, Proteins, Structures and Molecular Principles (Creighton, ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (Branden & Tooze, eds., Garland Publishing, New York, N. Y. (1991)); and Thornton et al. Nature 354:105 (1991). 5 [0078] Ordinarily, the antibody mutant with improved biological properties will have an amino acid sequence having at least 75% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the parent anti-human extended Type I glycosphingolipid antibody, at least 80%, at least 85%, at least 90% and often at least 95% identity. Identity or 10 similarity with respect to parent antibody sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) or similar (i.e., amino acid residue from the same group based on common side-chain properties, supra) with the parent antibody residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum 15 percent sequence identity. [0079] Alternatively, antibody mutants can be generated by systematic mutation of the FR and CDR regions of the heavy and light chains, or the Fe region of the anti-extended Type I glycosphingolipid antibody. [0080] Another procedure for generating antibody mutants involves the use of 20 affinity maturation using phage display (Hawkins et al., J Mol Biol 254:889-896 (1992) and Lowman et al., Biochemistry 30(45):10832-10838(1991)). Bacteriophage coat-protein fusions (Smith, Science 228:1315 (1985); Scott & Smith, Science 249:386 (1990); Cwirla et al., Proc Natl Acad Sci USA 8:309 (1990); Devlin et al. Science 249:404 (1990); Wells & Lowman, Curr Opin Struct Biol 25 2:597 (1992); and U.S. Pat. No. 5,223,409) are known to be useful for linking the phenotype of displayed proteins or peptides to the genotype of bacteriophage particles which encode them. The Fab domains of antibodies have also been displayed on phage (McCafferty et al., Nature 348: 552 (1990); Barbas et al. Proc Natl Acad Sci USA 88:7978 (1991); and Garrard et al. Biotechnol 9:1373 (1991)). 41 6020 _1 (GHMatters) P86521.AU.1 20 [0081] Monovalent phage display consists of displaying a set of protein variants as fusions of a bacteriophage coat protein on phage particles (Bass et al., Proteins 8:309 (1990)). Affinity maturation, or improvement of equilibrium binding affinities of various proteins, has previously been achieved through successive 5 application of mutagenesis, monovalent phage display and functional analysis (Lowman & Wells, J Mol Biol 234:564 578 (1993); and U.S. Pat. No. 5,534,617), as well as using that approach with Fab domains of antibodies (Barbas et al., Proc Natl Acad Sci USA 91:3 809 (1994); and Yang et al., J Mol Biol 254:392 (1995)). [0082] Libraries of many (for example, 106 or more) protein variants, differing 10 at defined positions in the sequence, can be constructed on bacteriophage particles, each of which contains DNA encoding the particular protein variant. Thus, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. After cycles of affinity purification, using an immobilized antigen, individual bacteriophage clones are isolated, and the amino 15 acid sequence of the displayed protein is deduced from the DNA. [0083] Following production of the antibody mutant, the biological activity of that molecule relative to the parent antibody can be determined as taught herein. As noted above, that may involve detennining the binding affinity and/or other biological activities or physical properties of the antibody. In a preferred 20 embodiment of the invention, a panel of antibody mutants is prepared and is screened for binding affinity for the antigen. One or more of the antibody mutants selected from the screen are optionally subjected to one or more further biological activity assays to confirm that the antibody mutant(s) have new or improved properties. In preferred embodiments, the antibody mutant retains the ability to bind 25 extended Type I glycosphingolipid with a binding affinity similar to or better/higher than that of the parent antibody. [0084] Alternatively, multivalent phage (McCafferty et al. (1990) Nature 348:552-554; and Clackson et al. (1991) Nature 352:624-628) also can be used to express random point mutations (for example, generated by use of an error-prone 30 DNA polymerase) to generate a library of phage antibody fragments which then 41662091 (GHMatters) P 52AU.1 21 could be screened for affinity to extended Type I glycosphingolipid, Hawkins et al., (1992) J Mol Biol 254:889-896. [0085] Preferably, during the affinity maturation process, the replicable expression vector is under tight control of a transcription regulatory element, and the 5 culturing conditions are adjusted so the amount or number of particles displaying more than one copy of the fusion protein is less than about 1%. Also preferably, the amount of particles displaying more than one copy of the fusion protein is less than about 10% of the amount of particles displaying a single copy of the fusion protein. Preferably the amount is less than about 20%. 10 [0086] Another equivalent phrase used herein is an antigen binding portion, which relates to that portion of an antibody of interest which binds a Type I glycosphingolipid epitope. All of the phrases and terms used herein to describe various changes that can be made to an original antibody are considered to fall within the scope of the phrase, "antigen binding potion." Hence, for example, an 15 antibody fragment, such as, an Fab molecule, an F, an scAb, an mru, any such functional fragments, an antibody variant, such as an allele or a molecule containing a change in the primary amino acid sequence thereof, a derivative, such as a chimeric or humanized antibody, an analog and so on, including functional equivalents, which include genetically modified forms of an antibody of interest, 20 antibody homologs, as described herein, and so on are included in the phrase antigen binding portion. [0087] The antibody mutant(s) so selected may be subjected to further modifications, often depending on the intended use of the antibody. Such modifications may involve further alteration of the amino acid sequence, fusion to 25 heterologous polypeptide(s) and/or covalent modifications. For example, a cysteine residue not involved in maintaining the proper conformation of the antibody mutant may be substituted, generally with serine, to improve the oxidative stability of the molecule and to prevent aberrant cross-linking. Conversely, a cysteine may be added to the antibody to improve stability (particularly where the antibody is an 30 antibody fragment, such as an Fv fragment). 4166209_1 (GHMatter) P86521 AU. 22 [0088] Another type of antibody mutant has an altered glycosylation pattern. That may be achieved by adding or deleting one or more carbohydrate moieties found in the antibody and/or by adding or deleting one or more glycosylation sites that are not present in the antibody. Glycosylation of antibodies is typically either 5 N-linked to Asn or O-linked to Ser or Thr. The tripeptide sequences, asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are common recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. N-acetylgalactosamine, galactose, fucose or xylose, for example, are bonded to a hydroxyamino acid, most commonly 10 serine or threonine, although 5-hydroxyproline or 5-hydroxylysine also may be used. Addition or substitution of one or more serine or threonine residues to the sequence of the original antibody can enhance the likelihood of O-linked glycosylation. [0089] It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance the effectiveness of the antibody. For example, 15 cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in that region. The homodimeric antibody thus generated may have improved internalization capability and/or increased cell killing mediated by complement and antibody-dependent cellular cytotoxicity (ADCC), see Caron et al., J Exp Med 176:1191-1195 (1992) and Shopes, Immunol 148:2918-2922 20 (1993). Such an antibody derivative or analog also may be more resistant to degradation in vivo. [0090] Alternatively, an antibody can be engineered which has dual F, regions and may thereby have enhanced complement lysis and ADCC capabilities, see Stevenson et al., Anti-Cancer Drug Design 3: 219 230 (1989). 25 [0091] Covalent modifications of the antibody are included within the scope of the invention. Such may be made by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable. Other types of covalent modifications of the antibody are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is 416209_1 (GHMattem) PS6521AUA 23 capable of reacting with selected side chains or with the N-terminal or C-terminal residue. [0092] Cysteinyl residues can be reacted with a-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to yield carboxylmethyl or 5 carboxyamidomethyl derivatives. Cysteinyl residues also can be derivatized by reaction with bromotrifluoroacetone, a-bromo-#-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercura-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1,3-diazole, for example. 10 [0093] Histidyl residues can be derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0. p-bromophenacyl bromide also can be used, the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0. [0094] Lysinyl and a tenninal residues can be reacted with succinic or other carboxylic acid anhydrides to reverse the charge of the residues. Other suitable 15 reagents for derivatizing a-amino-containing residues include imidoesters, such as, methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, 0-methylisourea and 2,4-pentanedione, and the amino acid can be transaminase-catalyzed with glyoxylate. [0095] Arginyl residues can be modified by reaction with one or several 20 conventional reagents, such as, phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and ninhydrin. Derivatization of arginine residues often requires alkaline reaction conditions. Furthermore, the reagents may react with lysine as well as the arginine e-amino group. [0096] The specific modification of tyrosyl residues can be made with aromatic 25 diazonium compounds or tetranitromethane. For example, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues can be iodinated using 1251 or 131 to prepare labeled proteins for use in a radioimmunoassay. [0097] Carboxyl side groups (aspartyl or glutamyl) can be modified by reaction 30 with carbodiimides (R-N=C=C-R'), where R and R' can be different alkyl groups, 4166209_1 (GHMatters) P86521.AU.1 24 such as l-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4 azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. 5 [0098] Glutaminyl and asparaginyl residues are frequently dearnidated to the corresponding glutamyl and aspartyl residues, respectively, under neutral or basic conditions. The deamidated form of those residues falls within the scope of this invention. [0099] Other modifications include hydroxylation of proline and lysine, 10 phosphorylation of hydroxyl groups of serinyl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains (Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N terminal amine and amidation of any C terminal carboxyl group. 15 [001001 Another type of covalent modification involves chemically or enzymatically coupling carbohydrates and glycosides to the antibody. Those procedures do not require production of the antibody in a host cell that has glycosylation capabilities for N-linked or O-linked glycosylation. Depending on the coupling mode used, the sugar(s) may be attached to: (a) arginine and histidine; 20 (b) free carboxyl groups; (c) free sulfhydryl groups, such as those of cysteine; (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine or tryptophan; or (f) the amide group of glutamine. Such methods are described in WO 87/05330 and in Aplin & Wriston, CRC Crit Rev Biochem, pp. 259-306 (1981). 25 [00101] Removal of any carbohydrate moieties present on the antibody may be accomplished chemically or enzymatically. Chemical deglycosylation, for example, can require exposure of the antibody to the compound, trifluoromethanesulfonic acid, or an equivalent compound, resulting in cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the 30 antibody intact. Chemical deglycosylation is described, for example, in Hakimuddin 4166209_1 (GHMattrs) P86521.AU.1 25 et al., Arch Biochem Biophys 259:52 (1987) and in Edge et al., Anal Biochem 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by any of a variety of endoglycosidases and exoglycosidases as described, for example, in Thotakura et al., Meth Enzymol 138:350(1987). 5 [00102] Another type of covalent modification of the antibody comprises linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol or polyoxylalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. [00103] Functional equivalents may be produced by interchanging different 10 CDR's of different antibody chains within a framework or a composite FR derived from plural antibodies. Thus, for example, different classes of antibody are possible for a given set of CDRs by substitution of different heavy chains, for example, IgGJ, IgM, IgA 1

.

2 or IgD, to yield differing extended Type I glycosphingolipid antibody types and isotypes. Similarly, artificial antibodies within the scope of the 15 invention may be produced by embedding a given set of CDR's within an entirely synthetic framework. [00104] The antibody fragments and functional equivalents of the present invention encompass those molecules with a detectable degree of specific binding to extended Type I glycosphingolipid. A detectable degree of binding includes all 20 values in the range of at least 10-100%, preferably at least 50%, 60% or 70%, more preferably at least 75%, 80%, 85%, 90%, 95% or 99% of the binding ability of an antibody of interest. Also included are equivalents with an affinity greater than 100% that of an antibody of interest. [0015] The CDR's generally are of importance for epitope recognition and antibody 25 binding. However, changes may be made to residues that comprise the CDR's without interfering with the ability of the antibody to recognize and to bind the cognate epitope. For example, changes that do not impact epitope recognition, yet increase the binding affinity of the antibody for the epitope, may be made. Several studies have surveyed the effects of introducing one or more amino acid changes at 30 various positions in the sequence of an antibody, based on the knowledge of the 41662091 (GHMahier) PM21At,1 26 primary antibody sequence, on the properties thereof, such as, binding and level of expression (Yang et al., 1995, J Mol Biol 254:392-403; Rader et al., 1998, Proc Natl Acad Sci USA 95:8910-8915; and Vaughan et al., 1998, Nature Biotechnology 16, 535-539). 5 [00105] Thus, equivalents of an antibody of interest can be generated by changing the sequences of the heavy and light chain genes in the CDR1, CDR2 and/or CDR3, or in the framework regions, using methods such as oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling, amino acid modification or mutator-strains of E. coli (Vaughan et al., 1998, Nat 10 Biotech 16:535-539; and Adey et al., 1996, Chap. 16, pp. 277-291, in Phage Display of Peptides and Proteins, eds. Kay et al., Academic Press), for example. The methods of changing the nucleic acid sequence of the primary antibody can result in antibodies with improved affinity (Gram et al., 1992, Proc Natl Acad Sci USA 89:3576-3580; Boder et al., 2000, Proc Natl Acad Sci USA 97:10701-10705; Davies 15 & Riechmann, 1996, Immunotech 2:169-179; Thompson et al., 1996, J Mol Biol 256:77-88; Short et al., 2002, J Biol Chem 277:16365-16370; and Furukawa et al., 2001, J Biol Chem 276:27622-27628). [00106] Repeated cycles of "polypeptide selection" can be used to select for higher affinity binding by, for example, the selection of multiple amino acid changes 20 which are selected by multiple selection of cycles. Following a first round of selection, involving a first region of selection of amino acids in the ligand or antibody polypeptide, additional rounds of selection in other regions or amino acids of the ligand are conducted. The cycles of selection are repeated until the desired affinity properties are achieved. 25 [00107] Improved antibodies also include those antibodies having improved characteristics that are prepared by the standard techniques of animal immunization, hybridoma formation and selection for antibodies with specific characteristics. [00108] "Antagonist" refers to a molecule capable of inhibiting one or more biological activities associated with extended Type I glycosphingolipid. Antagonists 30 may interfere with the maintenance and the growth of a cell expensing a Type I 4162_1{GHMatter)P06521.AU 1 27 glycosphingolipid. All points of intervention by an antagonist are considered equivalent for purposes of the instant invention. Thus, included within the scope of the invention are antagonists, e.g., neutralizing antibodies that bind to extended Type I glycosphingolipid. 5 [00109] "Agonist" refers to an antibody, an antibody fragment, a conjugate and so on, which activates one or more biological activities of extended Type I glycosphingolipid or a cell expressing same. Agonists can act as a mitogen of cells expressing a Type I glycosphingolipid. All points of intervention by an agonist shall be considered equivalent for purposes of the instant invention. Thus, included 10 within the scope of the invention are antibodies that bind to extended Type I glycosphingolipid and enhance an activity, such as, differentiation, for example. [00110] The terms "cell," "cell line," and "cell culture" include progeny thereof It is also understood that all progeny may not be precisely identical, such as, in DNA content, due to deliberate or inadvertent mutation. Variant progeny that have the 15 same function or biological property of interest, as screened for in the original cell, are included in the scope of the invention. The "host cells" used in the present invention generally are prokaryotic or eukaryotic hosts, selected as a design choice. [00111] "Transformation" of a cellular organism, cell or cell line with a nucleic acid means introducing a nucleic acid into the target cell so that the nucleic acid is 20 replicable, either as an extrachromosomal element or by chromosomal integration, and, optionally, expressed. "Transfection" of a cell or organism with a nucleic acid refers to the taking up of the nucleic acid, e.g., an expression vector, by the cell or organism whether or not any coding sequences are in fact expressed. The terms "transfected host cell" and "transformed" refer to a cell in which a nucleic acid was 25 introduced. Typical prokaryotic host cells include various strains of E. coli. Typical eukaryotic host cells are mammal cells, such as Chinese hamster ovary, or cells of human origin. The introduced nucleic acid sequence may be from the same species as the host cell or of a different species from the host cell, or may be a hybrid nucleic acid sequence, containing some foreign and some homologous nucleic acids. 41662C9_1 (GHMalom) P86521AV,1 28 Transformation can also occur by transduction or infection with virus-derived elements or carriers. [00112] The term "vector" means a nucleic acid construct, a carrier, containing a nucleic acid, the transgene, the foreign gene or the gene of interest, which can be 5 operably linked to suitable control sequences for expression of the transgene in a suitable host. Such control sequences include, for example, a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites and sequences which control the termination of transcription and translation. The vector may be a plasmid, a phage 10 particle or just a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may in some instances, integrate into a host cell genome or other nucleic acid. In the present specification, "plasmid" and "vector" are used interchangeably, as a plasmid is a commonly used form of vector. However, the invention is intended to include such 15 other forms of vectors which serve equivalent carrier function as and which are, or become, known in the art, such as, viruses, phagemids, transposons, synthetic molecules that carry nucleic acids, liposomes and the like. [00113] "Mammal" for purposes of treatment refers to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and 20 zoo, sports or pet animals, such as dogs, horses, cats, cows etc. [00114] The antibodies of interest can be screened or can be used in an assay as described herein or as known in the art. Often, such assays require a reagent to be detectable, that is, for example, labeled. The word "label" when used herein refers to a detectable compound or composition which can be conjugated directly or 25 indirectly to a molecule or protein, e.g., an antibody. The label may itself be detectable (e.g., radioisotope labels, particles or fluorescent labels) or may be an instrument to obtain a detectable signal, such as, in the case of an enzymatic label, may catalyze a chemical alteration of a substrate compound or composition which then is detectable. 41662091 (GHMatters) P86521.AU.1 29 [00115] As used herein, "solid phase" means a matrix to which an entity or molecule, such as, the antibody of the instant invention, can adhere or bind. Example of solid phases encompassed herein include those fonned partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), 5 plastics, polypropylenes, polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others can be used in a purification column (e.g., an affinity chromatography column). Thus, the solid phase can be a paper, a bead, a plastic, a chip and so on, can be made from a variety of materials, such as 10 nitrocellulose, agarose, polystyrene, polypropylene, silicon and so on, and can be in a variety of configurations. [00116] Cells expressing extended Type I glycosphingolipid or glycans thereof, such as cell membrane preparations, as well as purified extended Type I glycosphingolipid can be used as immunogens for generating antibodies of interest. 15 The immunogen can be obtained or isolated from natural sources or can be made synthesized enzymatically or chemically. Whole cells, such as extended Type I glycosphingolipid expressing cells, cells derived from a natural source or from cancers, such as cancer cell lines, can be used. Cells that overexpress extended Type I glycosphingolipid may be used as the immunogen for making the antibodies 20 of interest. Also, membrane preparations carrying extended Type I glycosphingolipid can be used, as known in the art. Such cells and portions thereof can be used as the antigen source in a diagnostic assay. [00117] Nucleic acid molecules encoding amino acid sequence mutants can be prepared by a variety of methods known in the art. The methods include, but are not 25 limited to, oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis and cassette mutagenesis of an earlier prepared mutant or a non-mutant version of the molecule of interest, (see, for example, Kunkel, Proc Natl Acad Sci USA 82:488 (1985)). [00118] Recombinant expression of an antibody of the invention, or fragment, 30 derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the 4166209_1 (GHMatte ) P86521.AU.1 30 invention, a single chain antibody of the invention or an antibody mutein of the invention) includes construction of an expression vector containing a polynucleotide that encodes the antibody or a fragment of the antibody as described herein. Once a polynucleotide encoding an antibody molecule has been obtained, the vector for the 5 production of the antibody may be produced by recombinant DNA technology as known in the art. An expression vector is constructed containing antibody coding sequences and appropriate transcriptional and translational control signals. The methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. 10 [00119] The expression vector is transferred to a host cell by conventional techniques and the transfected cells then are cultured by conventional techniques to produce an antibody, or fragment, of the invention. In one aspect of the invention, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed herein. 15 [00120] A variety of host/expression vector systems may be utilized to express the antibody molecules of the invention. Such expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transfonned or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the 20 invention in situ. Bacterial cells, such as E. coli, and eukaryotic cells are commonly used for the expression of a recombinant antibody molecule, especially for the expression of whole recombinant antibody molecule. For example, mammal cells such as CHO cells, in conjunction with a vector, such as one carrying the major intennediate early gene promoter element from human cytomegalovirus, are an 25 effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); and Cockett et al., Bio/Technology 8:2 (1990)). Plants and plant cell culture, insect cells and so on also can be used to make the proteins of interest, as known in the art. [00121] In addition, a host cell is chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific 30 fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., 41662091 (GHMatters) P86521.AU.1 31 cleavage) of protein products may be important for the function of the protein. Different host cells can have the particular characteristic and specific mechanisms for the desired post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct 5 modification and processing of the expressed antibody of interest. Hence, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, COS, 293, 3T3 or myeloma cells. 10 [00122] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer sequences, 15 transcription tenninators, polyadenylation sites etc.) and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for one to two days in an enriched media, and then are moved to a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into a chromosome and be 20 expanded into a cell line. Alternatively, an extrachromosomal element can be maintained in the cells under selection. Such engineered cell lines not only are useful for antibody production but are useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule. [00123] A number of selection systems may be used, including but not limited to 25 using the Herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al., Proc Natl Acad Sci USA 48:202 (1992)), glutamate synthase, in the presence of methionine sulfoximide (Adv Drug Del Rev 58, 671, 2006 and see the website or literature of Lonza Group Ltd.) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 30 (1980)) genes in tk, hgprt or aprt cells, respectively. Also, antimetabolite resistance 4166209_1 (GHMaters) P86521.AU,1 32 can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proc Natl Acad Sci USA 77:357 (1980); O'Hare et al., Proc Nati Acad Sci USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan et al., Proc Natl Acad Sci USA 78:2072 5 (1981)); neo, which confers resistance to the aminoglycoside, G-418 (Wu et al., Biotherapy 3:87 (1991)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al., eds., Current 10 Protocols in Molecular Biology, John Wiley & Sons (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press (1990); Dracopoli et al., eds., Current Protocols in Human Genetics, John Wiley & Sons (1994); and Colberre-Garapin et al., J Mol Biol 150:1 (1981). [00124] The expression levels of an antibody molecule can be increased by vector 15 amplification (for example, see Bebbington et al., in DNA Cloning, Vol. 3. Academic Press (1987)). When a marker in the vector system expressing antibody is amplifiable, an increase in the level of inhibitor present in the culture will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., 20 Mol Cell Biol 3:257 (1983)). [00125] The host cell may be co-transfected with two or more expression vectors of the invention, for example, the first vector encoding a heavy chain-derived polypeptide and the second vector encoding a light chain-derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression 25 of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain can be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); and Kohler, Proc Natl Acad Sci USA 77:2197 (1980)). The coding sequences for 30 the heavy and light chains may comprise cDNA or genomic DNA. 4166209_1 (GHMatterx) P86521.AU 1 33 [00126] Once an antibody molecule of the invention has been produced by an animal, chemically synthesized or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for 5 extended Type I glycosphingolipid after Protein A and size-exclusion chromatography, and so on), centrifugation, differential solubility or by any other standard technique for the purification of proteins. In addition, the antibodies of the instant invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification. 10 [00127] The antibodies of the present invention may be generated by any suitable method known in the art. Thus, a purified extended Type I structure can be used as antigen, optionally, with an adjuvant, such as, complete or incomplete Freund's adjuvant. The antibodies of the present invention may comprise polyclonal antibodies, although because of the modification of antibodies to optimize use in 15 human, as well as to optimize the use of the antibody per se, monoclonal antibodies are preferred because of ease of production and manipulation of particular proteins. Methods of preparing polyclonal antibodies are known to the skilled artisan (Harlow et al., Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988)). 20 [00128] For example, an immunogen, as exemplified herein, may be administered to various host animals including, but not limited to, rabbits, mice, carnelids, rats etc., to induce the production of serum containing polyclonal antibodies specific for extended Type I glycosphingolipid. The administration of the immunogen may entail one or more injections of an immunizing agent and, if desired, an adjuvant. 25 Various adjuvants may be used to increase the immunological response, depending on the host species, and include, but are not limited to, Freund's (complete and incomplete), mineral oil, gels, alum (aluminum hydroxide), surface active substances, such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins (KLH), dinitrophenol and potentially useful 30 human adjuvants, such as BCG (bacille Calmette-Guerin) and Corynebacterium 41652091 (GHMatters) P86521.AU, 1 34 parvum. Additional examples of adjuvants which may be employed include the MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). Immunization protocols are well known in the art and may be performed by any method that elicit an immune response in the animal host chosen. 5 Thus, various administration routes can be used over various time periods as a design choice. [00129} Typically, the immunogen (with or without adjuvant) is injected into the mammal by multiple subcutaneous or intraperitoneal injections, or intramuscularly or intravenously. In certain circumstances, whole cells expressing extended Type I 10 glycosphingolipid can be used. Depending on the nature of the immunogen (i.e., percent hydrophobicity, percent hydrophilicity, stability, net charge, isoelectric point etc.), the extended Type I glycosphingolipid or portion thereof may be modified or conjugated to be immunogenic or more immunogenic in the animal, such as a mammal, being immunized. For example, extended Type I glycosphingolipid or a 15 portion thereof can be conjugated to a carrier. The conjugation includes either chemical conjugation by derivatizing active chemical functional groups on either or both the immunogen and the immunogenic protein to be conjugated such that a covalent bond is formed or other methods known to the skilled artisan. Examples of such carriers or immunogenic proteins include, but are not limited to, KLH, 20 ovalbumin, serum albumin, bovine thyroglobulin, soybean trypsin inhibitor and promiscuous T helper peptides. Various adjuvants may be used to increase the immunological response as described above. [00130] Once a suitable preparation is obtained, it is possible to isolate particular antibodies from the plural antibodies by known separation techniques, such as 25 affinity chromatography, panning, absorption and so on. In that way, an individual antibody species can be obtained for further study, for example, sequencing to obtain the amino acid sequences of one or more CDRs. [00131] The antibodies of the present invention preferably comprise monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma technology, 30 such as described by Kohler et al., Nature 256:495 (1975); U.S. Pat. No. 4,376,110; 41620OL_ {GHMatters)PRB521AU.1 35 Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988); and Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas, Elsevier (1981), recombinant DNA methods, for example, making and using transfectomas, or other methods known to the artisan. Other examples of 5 methods which may be employed for producing monoclonal antibodies include, but are not limited to, the human B-cell hybridoma technique (Kosbor et al., Immunology Today 4:72 (1983); and Cole et al., Proc Natl Acad Sci USA 80:2026 (1983)), and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss (1985)). Such antibodies may be of any 10 immunoglobulin class including IgG, IgM, IgE, IgA and IgD, and any subclass thereof. The hybridoma producing the mAb of the invention may be cultivated in vitro or in vivo. [00132] In the hybridoma model, a host such as a mouse, a humanized mouse, a transgenic mouse with human immune system genes, horse, sheep, hamster, rabbit, 15 rat, camel or any other appropriate host animal, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to extended Type I glycosphingolipid. [00133] Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as 20 polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)). [00134] Generally, in making antibody-producing hybridomas, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. 25 Iunortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine or human origin. Typically, a rat or mouse myeloma cell line is employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental 30 cells lack the enzyme, hypoxanthine guanine phosphoribosyl transferase (HGPRT or 41662091 (GHMattors) Pa6521AU1 36 HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin and thymidine ("HAT medium"), substances that prevent the growth of HGPRT-deficient cells. [00135] Preferred immortalized cell lines are those that fuse efficiently, support 5 stable high level production of antibody by the selected antibody-producing cells, and are sensitive to a medium, such as HAT medium. Among the myeloma cell lines are murine myeloma lines, such as those derived from the MOPC-21 and MPC-1 1 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. and SP2/0, FO or X63-Ag8-653 cells available from the American 10 Type Culture Collection, Manassas, VA. The mouse myeloma cell line NSO also may be used (European Collection of Cell Cultures, Salisbury, Wilshire, UK). [00136] Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J Immunol 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production 15 Techniques and Applications, Marcel Dekker, Inc, pp. 51-63 (1987)). [00137] Another alternative is to use electrical fusion rather than chemical fusion to form hybridomas. Instead of chemical fusion, a B cell can be immortalized using, for example, Epstein Barr Virus or another transfonning gene, see, e.g., Zurawaki et al., in Monoclonal Antibodies, ed., Kennett et al., Plenum Press, pp. 19-33. 20 (1980). Transgenic mice expressing immunoglobulins and severe combined immunodeficient (SCID) mice transplanted with human B lymphocytes also can be used. [00138] The culture medium in which hybridoma cells are grown is assayed for production of monoclonal antibodies directed against extended Type I 25 glycosphingolipid. The binding specificity of monoclonal antibodies produced by hybridoma cells may be detennined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA), fluorocytometric analysis (FACS) or enzyme-linked immunosorbent assay (ELISA). Such techniques are known in the art and are within the skill of the artisan. Also, the Biacore system can be used, as 30 known in the art. The binding affinity of the monoclonal antibody to extended 41662091 (GHMaters) P6521.A.1 37 Type I glycosphingolipid can, for example, be determined by a Scatchard analysis (Munson et al., Anal Biochem 107:220 (1980)). [001391 After hybridoma cells that produce antibodies of the desired specificity, affinity and/or activity are identified, the clones may be subcloned by limiting 5 dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)). Suitable culture medium includes, for example, Dulbecco's Modified Eagle's Medium (D-MEM) or RPMI-l 640. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal. 10 [00140] The monoclonal antibodies secreted by the subclones are suitably separated or isolated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures such as, for example, protein A Sepharose, protein G Sepharose, hydroxylapatite chromatography, gel exclusion chromatography, gel electrophoresis, dialysis or affinity chromatography. 15 [00141] A variety of methods exist in the art for the production of monoclonal antibodies and thus, the invention is not limited to their sole production in hybridomas. For example, the monoclonal antibodies may be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. Alternatively, human antibodies can be obtained from transgenic animals, such as the KM mouse, 20 discussed above. In that context, the tern "monoclonal antibody" refers to an antibody derived from a single eukaryotic, viral or prokaryotic clone. [00142] Thus, using human cancer cells known to express an extended Type I chain structure, such as Colo205 cells, or an extended Type I chain containing compound, such as glycosphingolipid, such as Le b/Lea, as antigen, inbred or 25 transgenic mice are immunized and boosted as known in the art. Spleens were obtained, cells fused to myeloma cells and hybridomas made and cultured. Cell supernatants were screened by ELISA using, for example, Leb/Lea as the capture reagent. Positive clones were amplified. IMH2 is an example of a mouse IgG 3 monoclonal antibody that binds specifically to an extended Type I chain structure. 41662091 (GHMatrs) P86521.AU.1 3 8 [00143] Using a transgenic mouse model, human antibodies can be produced by immunizing the transgenic mice with a extended Type I chain immunogen. Such antibodies can be generated on a fee basis, for example, by Medarex, NJ and Amgen, CA. Using the KM mouse, the GNX-8 (IgG1) monoclonal antibody was 5 selected for further characterization and use. [00144] GNX-8 is a cytotoxic antibody. In assays using Colo205 colon cancer cells as targets, GNX-8 lysed the cancer cell line cells, and at least at 50 tg/ml, the antibody lysed all cells in the culture. GNX-8 does not bind to RBCs. The antibody binds to colorectal cancer cells, breast cancer cells and lung cancer cells. Unlike 10 IMH2, GNX-8 does not bind to LeYLeX or Ley. [00145] DNA encoding the monoclonal antibodies of the invention is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies, or such chains from human, humanized 15 or other sources) (Innis et al. in PCR Protocols. A Guide to Methods and Applications, Academic (1990) and Sanger et al., Proc Natl Acad Sci 74:5463 (1977)). The hybridoma cells can serve as the source of such DNA. [00146] Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, NSO cells, COS cells, Chinese 20 hamster ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; and 25 Morrison et al., Proc Natl Acad Sci USA 81:6851 (1984)) or by covalently joining to the immunoglobulin coding sequence, all or part of the coding sequence of a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one extended Type I 41662091 (GHMatters) P86521.AU.1 39 glycosphingolipid-combining site of an antibody of the invention to create a chimeric bivalent antibody. [00147] The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves 5 recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the F, region so as to prevent heavy chain cross-linking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent cross-linking. 10 [00148] Antibody fragments which recognize specific epitopes may be generated by known techniques. Traditionally, those fragments are derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., J Biochem Biophys Methods 24:107 (1992); and Brennan et al., Science 229:81 (1985)). For example, Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of 15 immunoglobulin molecules, using enzymes, such as, papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments). F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain. However, those fragments can be produced directly by recombinant host cells. For example, the antibody fragments can be isolated from an antibody phage 20 library. Alternatively, F(ab')2-SH fragments can be directly recovered from E. coli and chemically coupled to fonn F(ab')2 fragments (Carter et al., Bio/Technology 10:163 (1992). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other 25 embodiments, the antibody of choice is a single chain F, fragment (F,), see, for example, WO 93/16185. [00149] For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized or human antibodies. Methods for producing chimeric antibodies are known in the art, see 30 e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); 4166209_1 (GHMtver) PS6521.AU.1 40 Gillies et al., J Immunol Methods 125:191 (1989); and U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397. [00150] Humanized antibodies are derived from antibody molecules generated in a non-human species that bind extended Type I glycosphingolipid wherein one or 5 more CDR's therefrom are inserted into the FR regions from a human immunoglobulin molecule. Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR grafting (EPO 239,400; WO 91/09967; and U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EPO 592,106; EPO 519,596; Padlan, Molecular Immunology 28:489 10 (1991); Studnicka et al., Protein Engineering 7:805 (1994); and Roguska et al., Proc Nati Acad Sci USA 91:969 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). [00151] A humanized antibody has one or more amino acid residues from a source that is non-human. The non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. 15 Humanization can be essentially performed following the methods of Winter et al. (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); and Verhoeyen et al., Science 239:1534 (1988)), by substituting non-human CDR's or portions of CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 20 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possible some FR residues are substituted from analogous sites in rodent antibodies. The heavy chain constant region and hinge region can be from 25 any class or subclass to obtain a desired effect, such as a particular effector function. [00152] Often, framework residues in the human framework regions can be substituted with the corresponding residue from the CDR donor antibody to alter, and possibly improve, antigen binding. The framework substitutions are identified by methods known in the art, e.g., by modeling of the interactions of the CDR and 30 framework residues to identify framework residues important for antigen binding 41662091 (GHMatters) P86521 AU.1 41 and sequence comparison to identify unusual framework residues at particular positions, see, e.g., U.S. Pat. No. 5,585,089; and Riechmann et al., Nature 332:323 (1988). [00153] It is further preferable that humanized antibodies retain high affinity for 5 extended Type I glycosphingolipid, and retain or acquire other favorable biological properties. Thus, humanized antibodies can be prepared by a process by analyzing the parental sequences and various conceptual humanized antibody derivatives using three-dimensional models of the parental and humanized sequences. The hypothetical three-dimensional immunoglobulin models are commonly available and 10 are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional confornational structures of selected candidate immunoglobulin sequences. Inspection of the displays permits analysis of the likely role of certain residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of 15 the candidate immunoglobulin to bind extended Type I glycosphingolipid. In that way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is maximized, although it is the CDR residues that directly and most substantially influence extended Type I glycosphingolipid binding. The CDR 20 regions also can be modified to contain one or more amino acids that vary from that obtained from the parent antibody from which the CDR was obtained, to provide enhanced or different properties of interest, such as binding of greater affinity or greater avidity, for example. [00154] Certain portions of the constant regions of antibody can be manipulated 25 and changed to provide antibody homologs, derivatives, fragments and the like with properties different from or better than that observed in the parent antibody. Thus, for example, many IgG4 antibodies form intrachain disulfide bonds near the hinge region. The intrachain bond can destabilize the parent bivalent molecule forming monovalent molecules comprising a heavy chain with the associated light chain. 30 Such molecules can reassociate, but, on a random basis. 4166209_1 (GHMaitors) PB6521tAU.1 42 [00155] Another set of amino acids suitable for modification include amino acids in the area of the hinge which impact antibody functions, such as, binding of a molecule containing a heavy chain with binding to the Fe receptor and internalization of bound antibody. Such amino acids include, in IgG1 molecules, 5 residues from about 233 to about 237 (Glu-Leu-Leu-Gly-Gly, SEQ ID NO: 1); from about 252 to about 256 (Met-Ile-Ser-Arg-Thr, SEQ ID NO:2) and from about 318 (Glu) to about 331 (Pro), including, for example, Lys 3 20, Lys 322 and Pro 329 . [00156] Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of 10 methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences, see, U.S. Pat. Nos. 4,444,887 and 4,716,111; and WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741. The techniques of Cole et al. and Boerder et al. are also available for the preparation of human 15 monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss (1985); and Boerner et al., J Immunol 147:86 (1991)). [00157] Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which also express certain human immunoglobulin genes. For example, the human heavy and 20 light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region and diversity region may be introduced into mouse embryonic stem cells, in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be treated so as to be 25 non-functional separately or simultaneously with the introduction of the human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring 30 which express human antibodies, see, e.g., Jakobovitis et al., Proc Natl Acad Sci 41662091 (GHMatters) P86521AUA 43 USA 90:2551 (1993); Jakobovitis et al., Nature 362:255 (1993); Bruggermann et al., Year in Immunol 7:33 (1993); and Duchosal et al., Nature 355:258 (1992)). [00158] The transgenic mice are immunized in the normal fashion with an extended Type I glycosphingolipid, e.g., all or a portion of extended Type I 5 glycosphingolipid, or a membrane preparation containing same. Monoclonal antibodies directed against extended Type I glycosphingolipid can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human inununoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic 10 mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview, see Lonberg et al., Int Rev Immunol 13:65-93 (1995). For a discussion of producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., WO 98/24893; WO 92/01047; WO 96/34096; and WO 96/33735; EPO No. 0 598 15 877; and U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598. In addition, companies such as Amgen (Fremont, CA), Genpharm (San Jose, CA) and Medarex, Inc. (Princeton, NJ) can be engaged to provide human antibodies directed against extended Type I glycosphingolipid using technology similar to that described above. 20 [00159] Also, human mAbs could be made by immunizing mice transplanted with human peripheral blood leukocytes, splenocytes or bone marrow (e.g., trioma technique of XTL Biopharmaceuticals, Israel). [00160] Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In that approach, a 25 selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope (Jespers et al., Bio/Technology 12:899 (1988)). [00161] When using recombinant techniques, the antibody variant can be produced intracellularly, in the periplasmic space, or directly secreted into the 30 medium. If the antibody variant is produced intracellularly, as a first step, the 41652091 (GHMaters) P86521.AU.1 44 particulate debris, either host cells or lysed fragments, may be removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is exposed to sodium acetate 5 (pH 3.5) and EDTA. Cell debris can be removed by centrifugation. Where the antibody variant is secreted into the medium, supernatant from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor, such as, PMSF, may be included to inhibit proteolysis, 10 and antibiotics may be included to prevent growth of adventitious contaminants. [00162] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography. The suitability of protein A or protein G as an affinity ligand depends on the species and isotype of an immunoglobulin Fe domain that is 15 present in the antibody variant. Protein A can be used to purify antibodies that are based on human IgG1, IgG2 or IgG4 heavy chains (Lindmark et al., J Immunol Meth 62:1 (1983)). Protein G can be used for mouse isotypes and for human IgG3 (Guss et al., EMBO J 5:1567 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable 20 matrices, such as controlled pore glass or poly(styrenedivinyl)benzene, allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody variant comprises a CH3 domain, the Bakerbond ABXTM resin (JT Baker; Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as, fractionation on an ion-exchange column, ethanol precipitation, 25 reverse phase HPLC, chromatography on silica, chromatography on heparin agarose chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation are also available, depending on the antibody or variant to be recovered. [00163] Following any preliminary purification step(s), the mixture comprising 30 the antibody or variant of interest and contaminants may be subjected to low pH 41662091 (GHMaIIers) PS6521.AU1 45 hydrophobic interaction chromatography using an elution buffer at a pH of between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25 M salt). [00164] Further, antibodies of the invention can, in turn, be utilized to generate 5 anti-idiotype antibodies that "mimic" extended Type I glycosphingolipid using techniques well known to those skilled in the art (see, e.g., Greenspan et al., FASEB J 7:437 (1989); and Nissinoff, J Immunol 147:2429 (1991)). For example, antibodies which bind to and competitively inhibit multimerization and/or binding of a ligand to extended Type I glycosphingolipid can be used to generate anti-idiotypes 10 that "mimic" extended Type I glycosphingolipid. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic or diagnostic regimens. [00165] The antibodies of the present invention may be bispecific antibodies. Bispecific antibodies can be monoclonal, preferably human or humanized, 15 antibodies that have binding specificities for at least two different antigens. In the present invention, one of the binding specificities is directed towards extended Type I glycosphingolipid, whereas the other specificity may be for any other antigen, such as a cell-surface protein, receptor, receptor subunit, ligand, tissue-specific antigen, viral protein, virally-encoded envelope protein, 20 pharmacologically active agent, such as a drug, bacterially-derived protein, bacterial surface protein etc. [00166] Methods for making bispecific antibodies are well known. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain/light chain pairs, where the two heavy chains have 25 different specificities (Milstein et al., Nature 305:537 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, the hybridomas (quadromas) produce a potential mixture of about ten different antibody molecules, of which only about one might have the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity 30 chromatography steps. Similar procedures are disclosed in WO 93/08829 and in 4165209_ (GHMattem P6521.AU.1 46 Traunecker et al., EMBO J 10:3655 (1991). Other methods for making bispecific antibodies are provided in, for example, Kufer et al., Trends Biotech 22:238-244, 2004. [00167] Antibody variable domains with the desired binding specificities can be 5 fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2 and CH3 regions. It may have the first heavy chain constant region (CHI) containing the site necessary for light chain binding present in at least one of the fusions. DNA's encoding the immunoglobulin heavy chain fusions and, if desired, 10 the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transformed into a suitable host organism. For further details of generating bispecific antibodies see, for example Suresh et al., Meth Enzym 121:210 (1986). [00168] Heteroconjugate antibodies are also contemplated by the present invention. Heteroconjugate antibodies are composed of two covalently joined 15 antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980). It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming 20 a thioester bond. Examples of suitable reagents for that purpose include iminothiolate and methyl-4-mercaptobutyrimidate, and those disclosed, for example, in U.S. Pat. No. 4,676,980. [00169] In addition, one can generate single domain antibodies to extended Type I glycosphingolipid. Examples of that technology have been described in 25 W09425591 for antibodies derived from Camelidae heavy chain Ig, as well as in US20030130496 describing the isolation of single domain fully human antibodies from phage libraries. [00170] Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423 (1988); Huston et al., 30 Proc Natl Acad Sci USA 85:5879 (1988); and Ward et al., Nature 334:544 (1989)) 4166209 1 (GHMatters} PS6521.AU.1 47 can be practiced. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038 (1988)). Single chain antibodies 5 ("scFv") and a method of their construction are described in, for example, U.S. Pat. No. 4,946,778. Alternatively, Fab can be constructed and expressed by similar means. All of the wholly and partially human antibodies can be less immunogenic than wholly murine mAbs, and the fragments and single chain antibodies also can be less immunogenic. 10 [00171] The instant invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide. Fused or conjugated antibodies of the present invention may be used for ease in purification, see e.g., WO 93/21232; EP 439,095; Naramura et al., Immunol Lett 39:91 (1994); U.S. Pat. No. 5,474,981; Gillies et al., Proc Natl Acad 15 Sci USA 89:1428 (1992); and Fell et al., J Immunol 146:2446 (1991). [00172] The purification can be facilitated by using a recognition marker or tag. For example, the marker can be an amino acid sequence, such as, a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc., Chatsworth, CA), among others, many of which are commercially available, Gentz et al., Proc Natl 20 Acad Sci USA 86:821 (1989). Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the "flag" tag. [00173] Antibodies or antibody fragments can be isolated from antibody phage 25 libraries generated using the techniques described in McCafferty et al., Nature 348:552 (1990). Clarkson et al., Nature 352:624 (1991) and Marks et al., J Mol Biol 222:581 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., 30 Bio/Technology 10:779 (1992)), as well as combinatorial infection and in vivo 4156209_1 (GHMatter) P86521.AU.1 48 recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nucl Acids Res 21:2265 (1993)). Thus, the techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies. 5 [00174] Candidate anti-extended Type I glycosphingolipid antibodies can be tested by enzyme-linked immunosorbent assay (ELISA), FACS, Western iminunoblotting or other immunochemical techniques, as known in the art. Thus, B cells or cells expressing extended Type I glycosphingolipid can be used to detect antibody binding thereto using a known technique, or suitable membrane 10 preparations containing extended Type I glycosphingolipid or portion thereof, or purified or isolated extended Type I chain structures can be adhered to a solid phase and used as a capture element in an assay, configured as a design choice. [00175] To determine whether a particular antibody homolog binds to human extended Type I glycosphingolipid, any conventional binding assay may be used. 15 Useful extended Type I glycosphingolipid binding assays include FACS analysis, ELISA assays, radioimmunoassays and the like, which detect binding of antibody, and functions resulting therefrom, to human extended Type I glycosphingolipid. Full-length and soluble forms of human extended Type I glycosphingolipid taught herein are useful in such assays. The binding of an antibody or homolog to extended 20 Type I glycosphingolipid, or to soluble fragments thereof, may conveniently be detected through the use of a second antibody specific for immunoglobulins of the species from which the antibody or homolog is derived. The second antibody can carry a detectable label or configured to be detected. [00176] The ability of an antibody or homolog to bind to human extended Type I 25 glycosphingolipid can be evaluated by testing the ability thereof to bind to human extended Type I glycosphingolipid t cells. Suitable extended Type I glycosphingolipid* cells for use in determining whether a particular antibody or homolog binds to human extended Type I glycosphingolipid are available mammal tissue culture cells expressing extended Type I glycosphingolipid, such as, on the 30 cell surface. 4166209_1 (GHMattars) P86521.AU.1 49 [00177] Binding of the antibody or homolog to the extended Type I glycosphingolipid* cell can be detected by staining the cells with, for example, a fluorescently-labeled second antibody specific for immunoglobulins of the same species from which the antibody homolog being tested is derived. A fluorescence 5 activated cell sorter facesS" ) can be used to detect and to quantify any binding, see generally, Shapiro, Practical Flow Cytometry, Alan R. Liss, Inc., New York, N.Y. (1985). [00178] To determine whether a particular antibody or homolog causes no significant decrease in the number of circulating extended Type I glycosphingolipid t 10 cells in vivo, the number of circulating extended Type I glycosphingolipid* cells isolated from a mammal within 24 hours after administration of the antibody or homolog to a mammal having normal immune function is quantified, and compared to the pre-administration number or the number in a control mammal to whom an isotype-matched antibody or homolog of irrelevant specificity has been administered 15 instead of an antibody or homolog of the instant invention. Quantification of extended Type I glycosphingolipid* cells in animals dosed with an extended Type I glycosphingolipid antibody or functional portion or derivative thereof may be accomplished, for example, by staining obtained cells with fluorescently-labeled antibodies that bind the anti-extended Type I glycosphingolipid antibodies, as well 20 as labeled antibodies specific for T cells and B cells, followed by FACS analysis. [00179] Antibodies of the instant invention may be described or specified in terms of the epitope(s) or portion(s) of extended Type I glycosphingolipid to which the antibody recognizes or specifically binds. The epitope(s) may be specified as described herein, e.g., by physical means, such as mass spectrometry, compositional 25 analysis of the saccharides, the molecules to which the sugars bind, conformational epitopes and so on. [00180] Antibodies of the instant invention may also be described or specified in tens of cross-reactivity. Antibodies that bind extended Type I glycosphingolipids, which have at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at 30 least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as 4166209_1{GHMatters)PO5521.AU.1 50 calculated using methods known in the art and described herein) to extended Type I glycosphingolipid are also included in the instant invention. [00181] Antibodies of the instant invention also may be described or specified in terms of binding affinity to an extended Type I glycosphingolipid of interest. 5 Anti-extended Type I glycosphingolipid antibodies may bind with a KD of less than about 10-7 M, less than about 10~6 M, or less than about 10-5 M. Higher binding affinities in an antibody of interest can be beneficial, such as those with an equilibrium dissociation constant or KD of from about 10-8 to about 10-15 M or more, from about 10-8 to about 10-2 M, from about 10-9 to about 10[ M, or from about 10 10- to about 10-" M. The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the inmmunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 15 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%. [00182] The instant invention also includes conjugates comprising an antibody of interest. The conjugates comprise two primary components, an antibody of interest and a second component, which may be a cell-binding agent, a cytotoxic agent, a pharmacologically active agent, a drug and so on. 20 [00183] As used herein, the term "cell-binding agent" refers to an agent that specifically recognizes and binds to a molecule on the cell surface. Thus, the cell-binding agent can be one that binds a CD antigen, a pathogen antigen, such as a virus antigen, a differentiation antigen, a cancer antigen, a cell-specific antigen, a tissue-specific antigen, an Ig or Ig-like molecule and so on. 25 [00184] Cell-binding agents may be of any type as presently known, or that become known, and includes peptides, non-peptides, saccharides, nucleic acids, ligands, receptors and so on, or combinations thereof. The cell-binding agent may be any compound that can bind a cell, either in a specific or non-specific manner. Generally, the agent can be an antibody (especially monoclonal antibodies), 4166209_1 (GHMatt)e] P86521AU,1 51 lymphokines, hormones, growth factors, vitamins, nutrient-transport molecules (such as transferrin), or any other cell-binding molecule or substance. [00185] Other examples of cell-binding agents that can be used include: polyclonal antibodies; monoclonal antibodies; and fragments of antibodies such as 5 Fab, Fab., F(ab-)2 and F, fragments (Parham, J. immunol. 131:2895-2902 (1983); Spring et al., J. Immunol. 113:470-478 (1974); and Nisonoff et al., Arch. Biochem. Biophys. 89: 230-244 (1960)). [00186] The second component also can be a cytotoxic agent. The term "cytotoxic agent" as used herein refers to a substance that reduces or blocks the 10 function or growth, of cells and/or causes destruction of cells. Thus, the cytotoxic agent can be a taxol, a maytansinoid, such as, DM1 or DM4, CC-1065 or a CC-1065 analog, a ricin, a drug, mitomycin C and so on. In some embodiments, the cytotoxic agent, as with any binding agent of a conjugate of the instant invention, is covalently attached, directly or via a cleavable or non-cleavable linker, to an antibody of 15 interest. [00187] Examples of suitable maytansinoids include maytansinol and maytansinol analogs. Maytansinoids inhibit microtubule formation and are highly toxic to mammalian cells. [00188] Examples of suitable maytansinol analogues include those having a 20 modified aromatic ring and those having modifications at other positions. Such suitable maytansinoids are disclosed in U.S. Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545. 25 [00189] Examples of suitable analogues of maytansinol having a modified aromatic ring include: (1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared, for example, by LAH reduction of ansamytocin P2); (2) C-20-hydroxy (or C-20-demethyl)+/-C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared, for example, by demethylation using Streptomyces or Actinomyces or 30 dechlorination using lithium aluminum hydride (LAH)); and (3) C-20-demethoxy, 41662091 (GHMattrS)P852,AUI 52 C-20-acyloxy (-OCOR), +/-dechloro (U.S. Pat. No 4,294,757) (prepared by acylation using acyl chlorides). [00190] Examples of suitable analogues of maytansinol having modifications of other positions include: (1) C-9-SH (U.S. Pat. No. 4,424,219) (prepared by the 5 reaction of maytansinol with 12S or P 2

S

5 ); (2) C-14-alkoxymethyl (demethoxy/CH 2 OR) (U.S. Pat. No. 4,331,598); (3) C-14-hydroxymethyl or acyloxymethyl (CH 2 OH or CH 2 0Ac) (U.S. Pat. No. 4,450,254) (prepared from Nocardia); (4) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by the conversion of maytansinol by Streptomyces); (5) C-15-methoxy (U.S. Pat. Nos. 10 4,313,946 and 4,315,929) (isolated from Trewia nudiflora); (6) C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by the demethylation of maytansinol by Streptomyces); and (7) 4,5-deoxy (U.S. Pat. No 4,371,533) (prepared by the titanium trichloride/LAH reduction of maytansinol). [00191] The cytotoxic conjugates may be prepared by in vitro methods. To link a 15 cytotoxic agent, drug or prodrug to the antibody, commonly, a linking group is used. Suitable linking groups are known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. For example, conjugates can be constructed using a disulfide exchange reaction or by forming a thioether bond between an antibody of interest 20 and the drug or prodrug. [00192] The molecule conjugated to an antibody of interest can be a molecule with a pharmacologic activity, such as a drug, such as a small molecule or a biologic. Thus, the biologic can be a cytokine, for example. The molecule can be a prodrug, such as a drug ester. The molecule can be a radionuclide. 25 [00193] As discussed above, the instant invention provides isolated nucleic acid sequences encoding an antibody or functional variant thereof as disclosed herein, vector constructs comprising a nucleotide sequence encoding the extended Type I glycosphingolipid-binding polypeptides of the present invention, host cells comprising such a vector, and recombinant techniques for the production of the 30 polypeptide that binds extended Type I glycosphingolipids. 4166209_1 (GHMaters) P86521AU.1 53 [00194] The vector normally contains components known in the art and generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, a promoter, a polyA sequence, one or more marker or selection genes, sequences facilitating and/or enhancing translation, an enhancer element and 5 so on. Thus, the expression vectors include a nucleotide sequence operably linked to such suitable transcriptional or translational regulatory nucleotide sequences such as those derived from mammalian, microbial, viral or insect genes. Examples of additional regulatory sequences include operators, mRNA ribosomal binding sites, and/or other appropriate sequences which control transcription and translation, such 10 as initiation and termination thereof Nucleotide sequences are "operably linked" when the regulatory sequence functionally relates to the nucleotide sequence for the appropriate polypeptide. Thus, a promoter nucleotide sequence is operably linked to, e.g., the antibody heavy chain sequence if the promoter nucleotide sequence controls the transcription of that nucleotide sequence. 15 [00195] In addition, sequences encoding appropriate signal peptides that are not naturally associated with antibody heavy and/or light chain sequences can be incorporated into expression vectors. For example, a nucleotide sequence for a signal peptide (secretory leader) may be fused in-frame to the polypeptide sequence so that the antibody is secreted to the periplasmic space or into the medium. A 20 signal peptide that is functional in the intended host cells enhances extracellular secretion of the appropriate antibody or portion thereof. The signal peptide may be cleaved from the polypeptide on secretion of antibody from the cell. Examples of such secretory signals are well known and include, e.g., those described in U.S. Pat. Nos. 5,698,435; 5,698,417; and 6,204,023. 25 [00196] The vector may be a plasmid, a single-stranded or double-stranded viral vector, a single-stranded or double-stranded RNA or DNA phage vector, a phagemid, a cosmid or any other carrier of a transgene of interest. Such vectors may be introduced into cells as polynuclcotides by well known techniques for introducing DNA and RNA into cells. The vectors, in the case of phage and viral vectors also 30 may be introduced into cells as packaged or encapsulated virus, or a virus-like 4166209_1 (GHMatlors) P86521 AU.1 54 particle by well known techniques for infection and transduction. Viral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells and using plural vectors carrying the various virus components necessary to produce a particle. Cell 5 free translation systems may also be employed to produce the protein using RNAs derived from the present DNA constructs of interest (see, e.g., WO 86/05807 and WO 89/01036; and U.S. Pat. No. 5,122,464). [00197] The antibodies of the present invention can be expressed from any suitable host cell. Examples of host cells useful in the instant invention include 10 prokaryotic, yeast or eukaryotic cells and include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia and Shigella, as well as Bacilli, Pseudomonas and Streptomyces) transfonned with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the antibody 15 coding sequences of interest; yeast (e.g., Saccharomyces, Pichia, Actinomycetes, Kluyveromyces, Schizosaccharomyces, Candida, Trichoderma, Neurospora, and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium and Aspergillus) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors 20 (e.g., Baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; or tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293 or 3T3 cells) harboring 25 recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; or the vaccinia virus 7.5K promoter). [00198] Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes. A phenotypic selectable marker 30 gene is, for example, a gene encoding a protein that confers antibiotic resistance or 4166209_1 (GHMatters) P06521.A1 55 that supplies an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids, such as pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), pGEM1 (Promega Biotec, Madison, WI), pET (Novagen, Madison, WI) and the pRSET 5 (Invitrogen, Carlsbad, CA) series of vectors (Studier, J Mol Biol 219:37 (1991); and Schoepfer, Gene 124:83 (1993)). Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include T7 (Rosenberg et al., Gene 56:125 (1987)), 3-lactamase (penicillinase), lactose promoter (Chang et al., Nature 275:615 (1978); and Goeddel et al., Nature 281:544 (1979)), tryptophan (trp) 10 promoter system (Goeddel et al., Nuel Acids Res 8:4057 (1980)), and tac promoter (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory (1990)). [00199] Yeast vectors will often contain an origin of replication sequence, such as, from a 2p yeast plasmid, an autonomously replicating sequence (ARS), a 15 promoter region, sequences for polyadenylation, sequences for transcription termination and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J Biol Chem 255:2073 (1980)) or other glycolytic enzymes (Holland et al., Biochem 17:4900 (1978)) such as enolase, 20 glyceraldehydes-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in Fleer et al., Gene 107:285 (1991). Other suitable promoters and 25 vectors for yeast and yeast transformation protocols are well known in the art. Yeast transformation protocols are well known. One such protocol is described by Hinnen et al., Proc Natl Acad Sci 75:1929 (1978), which selects for Trp* transformants in a selective medium. [00200] Any eukaryotic cell culture is workable, whether from vertebrate or 30 invertebrate culture. Examples include plant and insect cells (Luckow et al., 4166209_1 (GHMatlrs) P86521 AU.1 56 Bio/Technology 6:47 (1988); Miller et al., Genetic Engineering, Setlow et al., eds., vol. 8, pp. 277-9, Plenum Publishing (1986); and Maeda et al., Nature 315:592 (1985)). For example, Baculovirus systems may be used for production of heterologous proteins. In an insect system, Autographa californica nuclear 5 polyhedrosis virus (AcNPV) may be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned under control of an AcNPV promoter (for example, the polyhedrin promoter). Other hosts that have been identified include Aedes, Drosophila melanogaster and Bombyx mori. A variety of viral strains for transfection are 10 publicly available, e.g., the -LI variant of AcNPV and the Bm-5 strain of Bombyx mori NPV. Moreover, plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, algae, duckweed and tobacco can also be utilized as hosts, as known in the art. (00201] Vertebrate cells, and propagation of vertebrate cells, in culture (tissue 15 culture) can be a routine procedure, although fastidious cell lines do exist which require, for example, a specialized medium with unique factors, feeder cells and so on, see Tissue Culture, Kruse et al., eds., Academic Press (1973). Examples of useful mammal host cell lines are monkey kidney; human embryonic kidney; baby hamster kidney; Chinese hamster ovary (CHO, Urlaub et al., Proc Natl Acad Sci 20 USA 77:4216 (1980)); mouse Sertoli; human cervical carcinoma (for example, HeLa); canine kidney;, human lung; human liver; mouse mammary tumor and NSO cells. [00202] Host cells are transformed with vectors for antibody production and cultured in conventional nutrient medium containing growth factors, vitamins, 25 minerals and so on, as well as inducers appropriate for the cells and vectors used. Commonly used promoter sequences and enhancer sequences are derived, for example, from polyoma virus, Adenovirus 2, Simian virus 40 (SV40) and human cytomegalovirus (CMV). DNA sequences derived from the SV40 viral genome may be used to provide other genetic elements for expression of a structural gene 30 sequence in a mammalian host cell, e.g., SV40 origin, early and late promoter, AtftBE6 _I (d&.fHo.)FM~JP 1 57 enhancer, splice and polyadenylation sites. Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment which may also contain a viral origin of replication. Exemplary expression vectors for use in mammalian host cells are commercially available. 5 [00203] Commercially available medium such as Ham's F10, Minimal Essential Medium (MEM), RPM -1640 and Dulbecco's Modified Eagle's Medium (DMEM) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth Enzymol 58:44 (1979) and Barnes et al., Anal Biochem 102:255 (1980), and in U.S. Pat. Nos. 4,767,704; 4,657,866; 4,560,655; 5,122,469; 5,712,163; or 10 6,048,728 may be used as a culture medium for the host cells. Any of those media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as chlorides, such as sodium, calcium or magnesium chloride; and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics, trace elements (which 15 may be defined as inorganic compounds usually present at final concentrations in the micromolar range) and glucose or an equivalent energy source. Any other necessary supplements may be included at appropriate concentrations, as a design choice. The culture conditions, such as temperature, pH and the like, are as known in the art appropriate for the cell and to enable the desired expression of the 20 transgene. [00204] The polynucleotides of interest may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized 25 oligonucleotides (e.g., as described in Kutmeier et al., Bio/Techniques 17:242 (1994)) and then amplifying the ligated oligonucleotides, for example, by PCR, [00205) Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid of a cell expressing same, If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody 30 molecule is known, a nucleic acid encoding the immunoglobulin may be obtained 4 _109 (GHMkAO PEMng ij. 58 from a suitable source, such as a library, which may be one specific for antibody-producing cells, such as hybridoma cells selected to express an antibody of the invention. Suitable primers can be configured for PCR amplification. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors 5 using any method known in the art. [00206] Once the nucleotide sequence and corresponding amino acid sequence of the antibody are determined, the nucleotide sequence of the antibody may be manipulated to obtain the equivalents of interest described herein using methods known in the art for manipulating nucleotide sequences, e.g., recombinant DNA 10 techniques, site directed mutagenesis, PCR etc. (see, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory (1990); and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons (1998)) to generate antibodies having a different amino acid sequence, for example, to create amino acid substitutions, deletions and/or insertions. 15 [00207] The amino acid sequence of the heavy and/or light chain variable domain may be inspected to identify the sequences of the CDR's by well known methods, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDR's may be inserted 20 within framework regions, e.g, into human framework regions to humanize a non-human antibody, as described supra. The polynucleotide of interest generated by the combination of the framework regions and one or more CDR's, encodes a molecule that specifically binds extended Type I glycosphingolipid, or at least the carbohydrate epitopes and structure recognized thereby. For example, such methods 25 may be used to make amino acid substitutions or deletions of one or more cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. [00208] The antibodies or antibody fragments of the invention can be used to detect extended Type I glycosphingolipid, and hence cells expressing extended 30 Type I glycosphingolipid, in a biological sample in vitro or in vivo. In one 41600DJ fQHfM"Wj.) J tU AU.1 59 embodiment, the anti-extended Type I glycosphingolipid antibody of the invention is used to determine presence of and the level of extended Type I glycosphingolipid in a tissue or in cells derived from the tissue. The levels of extended Type I glycosphingolipid in the tissue or biopsy can be determined, for example, in an 5 immunoassay with the antibodies or antibody fragments of the invention. The tissue or biopsy thereof can be frozen or fixed. The same or other methods can be used to determine other properties of extended Type I glycosphingolipid, such as the level thereof, cellular localization and so on. [00209] The above-described method can be used, for example, to diagnose a 10 cancer in a subject known to have or suspected to have a cancer, wherein the level of extended Type I glycosphingolipid measured in said patient is compared with that of a normal reference subject or standard. [00210] The instant invention further provides for monoclonal antibodies, humanized antibodies and epitope-binding fragments thereof that are further labeled 15 for use in research or diagnostic applications. In some embodiments, the label is a radiolabel, a fluorophore, a chromophore, an imaging agent or a metal ion, for example. [00211] A method for diagnosis is also provided in which said labeled antibodies or epitope-binding fragments thereof are administered to a subject suspected of 20 having a cancer, arthritis, autoimmune diseases or other diseases related to, caused by or associated with extended Type I glycosphingolipid expression and/or function, and the distribution of the label within the body of the subject is measured or monitored. [00212] The antibody and fragments thereof of the instant invention may be used 25 as affinity purification agents. In that process, the antibodies are immobilized on a solid phase, such as a dextran or agarose, resin or filter paper, using methods known in the art. The immobilized antibody is contacted with a sample containing extended Type I glycosphingolipid or cells carrying same to be purified, and thereafter the support is washed with a suitable solvent that will remove 30 substantially all the material in the sample except the extended Type I 419"2-I """HM"F""J"" 60 glycosphingolipid or cell to be purified, which is bound to the immobilized antibody of interest. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0, that will release the extended Type I glycosphingolipid or cell from the antibody of interest. 5 [00213] For diagnostic applications, the antibody of interest typically will be labeled with a detectable moiety or marker. Numerous labels are available which can be generally grouped into the following categories: (a) radioisotopes, such as 36 S, 1 4 C, 125I, 3 H and 13I (the antibody can be labeled with the radioisotope using a technique described in, for example, Current Protocols in Immunology, vol. 12, 10 Coligen et al., ed., Wiley-Interscience, New York (1991), and radioactivity can be measured using scintillation counting); (b) fluorescent labels, such as rare earth chelates (europium chelates), fluorescein and derivatives thereof, rhodamine and derivatives thereof, dansyl, lissamine, phycoerythrin and Texas Red, the fluorescent labels can be conjugated to the antibody using a technique disclosed in Current 15 Protocols in Immunology, supra, for example, where fluorescence can be quantified using a fluorimeter; and (c) various enzyme substrate labels are available (U.S. Pat. No. 4,275,149 provides a review), the enzyme generally catalyzes a chemical alteration of a chromogenic substrate which can be measured using various techniques, for example, the enzyme may catalyze a color change in a substrate, 20 which can be measured spectrophotometrically, or the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are known, for example, using a luminometer, or the label donates energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), 25 luciferin, 2,3-dihydrophthalazinediones, palate dehydrogenase, urease, peroxidase, such as horseradish peroxidase (HRPO), alkaline phosphatase, 3-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase and the like. 44Rk (OKMnat) PfllI AU 1 61 Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al., Meth Enz, ed. Langone & Van Vunakis, Academic Press, New York, 73 (1981). [002141 When such labels are used, suitable substrates are available, such as: (i) for horseradish peroxidase with hydrogen peroxidase as a substrate, wherein the 5 hydrogen peroxidase oxidizes a dye precursor (e.g., orthophenylene diarnine (OPD) or 3,3',5,5'-tetramethyl benzidine hydrochloride (TMB)); (ii) for alkaline phosphatase (AP) with p-nitrophenyl phosphate as the chromogenic substrate; and (iii) fi-D-galactosidase (j-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-#-D-galactosidase) or a fluorogenic substrate such as, 10 4-methylumbelliferyl--D-galactosidase. [00215] Other enzyme-substrate combinations are available to those skilled in the art. For a general review, see U.S. Pat. Nos. 4,275,149 and 4,318,980. [00216] Sometimes, the label is indirectly conjugated with the antibody. For example, the antibody can be conjugated with biotin and any of the reporters 15 mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in that indirect manner. Various avidins are known in the art. Alternatively, to achieve indirect conjugation of the label, the antibody can be conjugated with a small hapten (e.g., digoxin) and one of the different types of labels or reporters mentioned above 20 is conjugated with an anti-digoxin antibody. Thus, indirect conjugation of the label with the antibody or mutein can be achieved using a second antibody. [00217] In another embodiment of the invention, the antibody need not be labeled, and the presence thereof can be detected using a labeled antibody which binds to the antibody, another form of a second antibody. 25 [00218} The antibodies of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques (CRC Press, Inc. 1987). [00219] Competitive binding assays rely on the ability of a labeled standard to 30 compete with the test sample for binding with a limited amount of antibody. The 4IBE20_1 IHLMls)PB iH.AVl . 2 amount of antigen in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies generally are insolubilized before or after the competition. As a result, the standard and test sample that are bound to the 5 antibodies may conveniently be separated from the standard and test sample which remain unbound. [00220J Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, determinant or epitope, of the target to be detected. In a sandwich assay, the test sample to be analyzed is bound by a first 10 antibody which is immobilized directly or indirectly on a solid support, and thereafter a second antibody directly or indirectly labeled binds to the bound test sample, thus forming an insoluble three-part complex, see e.g., U.S. Pat. No. 4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-iumnunoglobulin 15 antibody or other suitable member of the binding pair (antibody/antigen, receptor/ligand, enzyme/substrate, for example) that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. [00221] For imnunahistochemistry, the cell or tissue sample may be fresh or 20 frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example. [00222] The antibodies may also be used for in vivo diagnostic assays. Generally, the antibody or variant thereof is labeled with a radionucleotide (such as "In "Tc, 'C, m1 'H, P or --S) so that the sites expressing extended Type I 25 glycosphingolipid can be localized using, for example, immunoscintigraphy and a gamma camera. [00223] The instant invention also includes kits, e.g., comprising an antibody, fragment thereof, homolog, derivative thereof and so on, such as a labeled or cytotoxic conjugate, and instructions for the use of the antibody, conjugate for 30 killing or labeling particular cell types and so on. The instructions may include d"""- 63 directions for using the antibody, conjugate and so on in vitro, in vivo or ex vivo. The antibody can be in liquid form or as a solid, generally lyophilized. The kit can contain suitable other reagents, such as a buffer, a reconstituting solution and other necessary ingredients for the intended use. A packaged combination of reagents in 5 predetermined amounts with instructions for use thereof, such as, for a therapeutic use for performing a diagnostic assay is contemplated. Where the antibody is labeled, such as with an enzyme, the kit can include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore). In addition, other additives may be included such as 10 stabilizers, buffers (e.g., a block buffer or lysis buffer) and the like. The relative amounts of the various reagents may be varied to provide for concentrates of a solution of a reagent, which provides user flexibility, economy of space, economy of reagents and so on. The reagents may be provided as dry powders, usually lyophilized, including excipients, which on dissolution provide a reagent solution 15 having the appropriate concentration. (00224) The antibodies of the present invention may be used to treat a mammal. In one embodiment, the antibody or equivalent of interest is administered to a nonhuman mammal for the purposes of obtaining preclinical data, for example. Exemplary nonhuman mammals to be treated include nonhuman primates, dogs, 20 cats, rodents and other mammals in which preclinical studies are performed. Such mammals maybe established animal models for a disease to be treated with the antibody, or may be used to study toxicity of the antibody of interest. In each of those embodiments, dose escalation studies may be performed in the mammal. The product of interest may have therapeutic use in those animals as well. 25 (00225] An antibody, with or without a second component, such as a therapeutic moiety conjugated to same, administered alone or in combination with a cytotoxic factors) can be used as a therapeutic. The present invention is directed to antibody-based therapies which involve administering antibodies of the invention to an animal, a mammal or a human, for treating an extended Type I 30 glycosphingolipid-mediated or associated disease, disorder or condition. The animal "1"2-"" (GKPM4-P64EAl 64 or subject may be a mammal in need of a particular treatment, such as a mammal having been diagnosed with a particular disorder, e.g., one relating to extended Type I glycosphingolipid, or associated with abnormal extended Type I chain structure expression and function . Antibodies directed against extended Type I 5 glycosphingolipid are useful, for example, for prophylaxis or treatment of cancer and autoimmune disorders, for example. For example, by administering a therapeutically acceptable dose of an anti-extended Type I glycosphingolipid antibody of the instant invention, or a cocktail of a plurality of the instant antibodies or equivalents thereof, or in combination with other antibodies of varying sources, or 10 in combination with a non-antibody drug, such as, an anti-inflammatory drug, a cytotoxic agent, an antibiotic and so on, such as, a platinum drug, methotrexate and so on, disease symptoms may be ameliorated or prevented in the treated mammal, particularly humans. [002261 Therapeutic compounds of the invention include, but are not limited to, 15 antibodies of the invention (including fragments, analogs, equivalents and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention as described herein (including fragments, analogs and derivatives thereof) and anti-idiotypic antibodies as described herein. The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated 20 with aberrant expression and/or activity of extended Type I glycosphingolipid, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders or conditions associated with aberrant expression and/or activity of extended Type I glycosphingolipid includes, but is not limited to, alleviating at least one symptom 25 associated with those diseases, disorders, or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein. The tern "physiologically acceptable," "pharmacologically acceptable," "pharmaceutically acceptable," and so on means approved by a regulatory agency of the Federal or a state government or listed in the U.S. "'""P 165 Pharmacopeia or other generally recognized pharmacopeia for use in animals and more particularly in humans. [00227] The anti-extended Type I glycosphingolipid antibody can be administered to a mammal in any acceptable manner. Methods of introduction 5 include, but are not limited to, parenteral, subcutaneous, intraperitoneal, intrapuhnonary, intranasal, epidural, inhalation and oral routes, and if desired for immunosuppressive treatment, intralesional administration. Parenteral infusions include intramuscular, intradermal, intravenous, intraarterial or intraperitoneal administration. The antibodies or compositions may be administered by any 10 convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the therapeutic antibodies or compositions of the invention into the central 15 nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Omnaya reservoir. In addition, the antibody can be suitably administered by pulse infusion, particularly with declining doses of the antibody. Preferably the dosing is given by injection, preferably 20 intravenous or subcutaneous injections, depending, in part, on whether the administration is brief or chronic. [00228] Various other delivery systems are known and can be used to administer an antibody of the present invention, including, e.g., encapsulation in liposomes, microparticles, microcapsules and so on (see Langer, Science 249:1527 (1990)); 25 expression of an antibody, mutein thereof or antigen-binding portion thereof, of interest on a liposome, particle, capsule and so on to yield a targeting vehicle, Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein et al., eds., p. 353-365 (1989); and Lopez-Berestein, ibid., p. 317-327; recombinant cells capable of expressing the compound, see, e.g., Wu A4'GSOL1 (QKM Mn~5IAV1 66 et aL, J Biol Chem 262:4429 (1987); construction of a nucleic acid as part of a retroviral or other vector; and so on. [00229] The active ingredients may also be entrapped in a microcapsule prepared, for example, by coascervation techniques or by interfacial polymerization, for 5 example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharnaceutical Sciences, 16th edition, A. Osal, Ed. 10 (1980). When the liposome or particle expresses an antibody of interest, any of a variety of compounds can be carried in the liposome, such as, a non-antibody drug, small molecule drug and so on. The instant antibody can thus serve a targeting function. [00230] Pulmonary administration can also be employed, e.g., by use of an 15 inhaler or nebulizer, and formulation with an aerosolizing agent. The antibody may also be administered into the lungs of a patient in the form of a dry powder composition, see e.g., U.S. Pat. No. 6,514,496. [00231] In a specific embodiment, it may be desirable to administer the therapeutic antibodies or compositions of the invention locally to the area in need of 20 treatment; that may be achieved by, for example, and not by way of limitation, local infusion, topical application, by injection, by means of a catheter, by means of a suppository or by means of an implant, said implant being of a porous, non-porous or gelatinous material, including membranes, such as sialastic membranes or fibers. Preferably, when administering an antibody of the invention, care is taken to use 25 materials to which the protein does not absorb or adsorb. [002321 In yet another embodiment, the antibody can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, Science 249:1527 (1990); Sefton, CRC Crit Ref Biomed Eng 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); and Saudek et al., N Eng J Med 321:574 (1989)). In 30 another embodiment, polymeric materials can be used (see Medical Applications of -1S0fli IGHFAd m)FIR2.AI. 67 Controlled Release, Langer et al., eds., CRC Press (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen et als, eds , Wiley (1984); Ranger et al., J Macromol Sci Rev Macromol Chem 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et aL, AnnNeural 25:351 (1989); and 5 Howard et al., J Neurosurg 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target. [00233] Sustained-release preparations maybe prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of 10 shaped articles, e.g., films or matrices. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethylmethacryl ate), poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as, injectable microspheres 15 composed of lactic acid-glycolic acid copolymer) and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain bydrogels release proteins for shorter time periods. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is 20 discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, amino acid substitution and developing specific polymer matrix compositions. 25 [00234] Therapeutic fonnulations of the polypeptide or antibody may be prepared for storage as lyophilized formulations or aqueous solutions by mixing the polypeptide having the desired degree of purity with optional "pharmaceutically acceptable" carriers, diluents, excipients or stabilizers typically employed in the art, ie., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic 30 detergents, antioxidants and other miscellaneous additives, see Remington's 41 -2U1 (Qt-LIr-pMtAai 68 Pharmaceutical Sciences, 16th ed., Osol, ed. (1980). Such additives are generally nontoxic to the recipients at the dosages and concentrations employed, hence, the excipients, diluents, carriers and so on are pharmaceutically acceptable. [00235] An "isolated" or "purified" antibody is substantially free of cellular 5 material or other contaminating proteins from the cell or tissue source or medium from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of an antibody in which the polypeptide/protein is separated from cellular components of the cells from which 10 same is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations of the antibody having less than about 30%, 20%, 10%, 5%, 2.5% or 1%, (by dry weight) of contaminating protein or cellular or subcellular material. When the antibody is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents 15 less than about 20%, 10%, 5%, 2.5% or 1% of the volume of the protein preparation. When antibody is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals and reagents, i.e, the antibody of interest is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly, such preparations of the antibody have less 20 than about 30%, 20%, 10%, 5% or 1% (by dry weight) of chemical precursors or compounds other than antibody of interest. In a preferred embodiment of the present invention, antibodies are isolated or purified. [00236] As used herein, the phrase "low to undetectable levels of aggregation" refers to samples containing no more than 5%, no more than 4%, no more than 3%, 25 no more than 2%, no more than 1% and often no more than 0.5% aggregation of antibody or variant thereof, that is, two or more antibody molecules or variants thereofjoined or coalesced together, by weight protein, as measured by, for example, high performance size exclusion chromatography (HPSEC). [00237] As used herein, the tenn "low to undetectable levels of fragmentation" 30 refers to samples containing equal to or more than 80%, 85%, 90%, 95%, 98% or 4'621L1 {GHMaivPOMAU1 69 99% of intact antibody molecule or variant thereof, of the total protein, for example, in a single peak, as determined by HPSEC, or in two (2) peaks (heavy chain and light chain) by, for example, reduced capillary gel electrophoresis (rCGE) and containing no other single peaks having more than 5%, more than 4%, more than 5 3%, more than 2%, more than 1% or more than 0.5% of the total protein, each. The rCGE as used herein refers to capillary gel electrophoresis under reducing conditions sufficient to reduce disulfide bonds in an antibody or antibody-type or derived molecule. [00238] The instant invention provides methods for preparing liquid formulations 10 of the antibody or extended Type I glycosphingolipid-binding fragment thereof, said methods comprising concentrating a fraction of purified antibody to a final concentration of about 15 mg/ml, about 20 mg/mI, about 30 mg/ml, about 40 mg/m, about 50 mg/mi, about 60 mg/ml, about 70 mg/mi, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 200 mg/ml, about 250 mg/ml, about 300 mg/ml or more 15 using, for example, a semi-permeable membrane with an appropriate molecular weight (mw) cutoff (e.g., 30 kD cutoff for F~bsg fragments thereof; and 10 kD cutoff for Fab fragments) and, optionally, diafiltering the concentrated antibody fraction into the formulation buffer using the same membrane. [00239] In addition, the present invention also encompasses stable liquid 20 formulations of the products of interest that can have improved half-life in vivo. Thus, the antibody of interest has a half-life in a subject, preferably a human, of greater than 3 days, greater than 7 days, greater than 10 days, greater than 15 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater 25 than 4 months, greater than 5 months or more. [00240] As used herein, the terms "stability" and "stable" in the context of a liquid formulation comprising an extended Type I glycosphingolipid antibody or binding fragment thereof refer to the resistance of the antibody or antigen-binding fragment thereof in the formulation to thermal and chemical unfolding, aggregation, 30 degradation or fragmentation under given manufacture, preparation, transportation 41629%1 Iai WIo 70 and storage conditions. The "stable fonnulations of the invention retain biological activity equal to or more than 80%, 85%, 90%, 95%, 98%, 99% or 99.5% under given manufacture, preparation, transportation and storage conditions. The stability of said antibody preparation can be assessed by degrees of aggregation, degradation 5 or fragmentation by methods known to those skilled in the art, including, but not limited to, rCGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and HPSEC, compared to a reference, for a predetermined period of time under selected storage conditions as a design choice. [00241] The instant invention encompasses liquid formulations having stability at 10 temperatures found in a commercial refrigerator or freezer found in the office of a physician or laboratory, such as from about -20" C to about 5" C., said stability assessed, for example, by high performance size exclusion chromatography (HPSEC), for storage purposes, such as, for about 60 days, for about 120 days, for about 180 days, for about a year, for about 2 years or more. The liquid formulations 15 of the present invention also exhibit stability, as assessed, for example, by HSPEC, at room temperatures, for at least a few hours, such as about one hour, about two hours or about three hours prior to use. [00242] The term, "carrier," refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic is administered. Such physiological carriers can be sterile 20 liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a suitable carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions also can be employed as liquid carriers, particularly for injectable solutions. Suitable 25 pharmaceutical recipients include starch, glucose, lactose, sucrose, gelatin, malt rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The compositions can take the form of 30 solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release I 3MiI(MOA PH621iU.i0 71 formulations, depots and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, 5 magnesium carbonate etc. flavorants, colorants, odorants and so on. Examples of suitable carriers are described in "Remington's Phannaceutical Sciences,' Martin. Such compositions will contain an effective amount of the antibody, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. As known in the art, the formulation will be 10 constructed to suit the mode of administration. [00243] Buffering agents help to maintain the pH in a range which approximates physiological conditions or conditions conducive to antibody stability. Buffers are preferably present at a concentration ranging from about 2 mM to about 50 mM. Suitable buffering agents for use with the instant invention include both organic and 15 inorganic acids, and salts thereof, such as, for example, citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture etc.), tartrate buffers (e.g., tartaric 20 acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture etc.), fumarate buffers (e.g., finnaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture etc.), gluconate buffers (e.g., gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxide 25 mixture, gluconic acid-potassium gluconate mixture etc.), oxalate buffers (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium 41"""l *MlflPtAu.1 72 hydroxide mixture etc.). Phosphate buffers, carbonate buffers, histidine buffers, trimethylamine salts such as Tris, HEPES and other such known buffers can be used. [00244] Preservatives may be added to retard microbial growth, and may be added in amounts ranging from about 0.2% to about 1% (w/v). Suitable 5 preservatives for use with the present invention include phenol, benzyl alcohol, m-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzylconiurn halides (e.g., chloride, bromide and iodide), hexamethonium chloride, alkyl parabens, such as, methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol. 10 [00245] Isotonicifiers are present to ensure physiological isotonicity of liquid compositions of the instant invention and include polhydric sugar alcohols, such as, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Polyhydric alcohols can be present in an amount of between about 0.1% to about 25%, by weight, preferably about 1% to about 5% taking into 15 account the relative amounts of the other ingredients. [00246] Stabilizers refer to a broad category of recipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can be polyhydric sugar alcohols; amino acids, such as arginine, lysine, 20 glycine, glutamine, asparagine, histidine, alanine, ornithine, L leucine, 2-phenylalanine, glutamic acid, threonine etc.; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, arabitol, erythritol, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur containing reducing 25 agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monotbioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins, such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone, saccharides, monosaccharides, such as xylose, mannose, fructose, glucose; 30 disaccharides, such as lactose, maltose and sucrose; trisaccharides such as raffinose; 416620] [HMae) PM2I00 73 polysaccharides such as dextran and so on. Stabilizers can be present in the range from about 0.1 to about 10,000 w/w per part of active protein. [00247] Additional miscellaneous excipients can include bulking agents, (e.g., agar, gelatin, starch and so on), chelating agents (e.g., EDTA), antioxidants (e.g, 5 ascorbic acid, metbionine or vitamin E) and cosolvents. [00248] As used herein, the term "surfactant" refers to organic substances having amphipathic structures, namely, are composed of groups of opposing solubility tendencies, typically an oil soluble hydrocarbon chain and a water soluble ionic group. Surfactants can be classified, depending on the charge of the surface active 10 moiety, into anionic, cationic and nonionic surfactants. Surfactants often are used as wetting, emulsifying, solubilizing and dispersing agents for various pharmaceutical compositions and preparations of biological materials as those discussed herein. [00249] Non-ionic surfactants or detergents (also known as "wetting agents") may be added to help solubilize the therapeutic agent, as well as to protect the therapeutic 15 protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stresses without causing denaturation of the protein. Suitable non-ionic surfactants include polysorbates (20, 80 etc.), polyoxamers (184, 188 etc.), Pluronic polyols and polyoxyethylene sorbitan monoethers (TWEEN-20, TWEEN-80 etc.). Non-ionic surfactants may be present in a range 20 of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/mL. [00250] As used herein, the term, "inorganic salt," refers to any compound, containing no carbon, that results from replacement of part or all of the acid hydrogen or an acid by a metal or a group acting like a metal, and often are used as a 25 tonicity adjusting compound in pharmaceutical compositions and preparations of biological materials. The most common inorganic salts are NaCl, KCl, NaH 2

PO

4 etc. [00251] The present invention provides liquid formulations of an anti-extended Type I glycosphingolipid-binding compound or fragment thereof, having a pH ranging from about 5.0 to about 7.0, or about 5.5 to 6.5, or about 5.8 to about 6.2, or 30 about 6.0. AlMgftfli "W AWd r~ 1J 74 (00252] The formulation herein also may contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely impact each other. For example, it may be desirable to father provide an immunosuppressive agent. Such molecules 5 suitably are present in combination in amounts that are effective for the purpose intended. The formulation also can contain another drug, or a small molecule, a pharmacologic agent, such as an anti-neoplastic drug, such as, cisplatin. [00253] The term "small molecule" and analogous terms include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogues, organic 10 compounds, pharmacologically active agents, such as drugs, polynucleotides, polynucleotide analogues, nucleotides, nucleotide analogues, organic or inorganic compounds (i.e., including heterorganic and/organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, 15 organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. [00254] Thus, in the case of cancer, the antibodies of the invention may be 20 administered alone or in combination with other types of cancer treatments, including conventional chemotherapeutic agents (paclitaxel, carboplatin, cisplatin and doxorubicin), anti-EGFR agents (gefitinib, erlotinib and cetuximab), anti-angiogenesis agents (bevacizumab and sunitinib), as well as immunomodulating agents, such as interferon-a and thalidomide. 25 [00255] As used herein, the terms "therapeutic agent" and "therapeutic agents" refer to any agent(s) which can be used in the treatment, management or amelioration of a disease, disorder, malady and the like associated with aberrant extended Type I glycosphingolipid expression, and metabolism in general, and activity. Also included are known compounds with a phannacologic effect in 411BE2 (J9 (H lk rP1 l521AU.i 75 treating a disorder and so on that is associated with aberrant extended Type I glycosphingolipid expression, metabolism and activity. [00256] The antibody or variant, optionally, is formulated with one or more agents currently used to prevent or treat the disorder in question. The effective 5 amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment and other factors discussed above. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages. (00257] The formulations to be used for in vivo administration must be sterile. 10 That can be accomplished, for example, by filtration through sterile filtration membranes. For example, the liquid formulations of the present invention may be sterilized by filtration using a 0.2 pm or a 0.22 pm filter. [00258] In addition, the antibodies of the instant invention may be conjugated to various effector molecules such as heterologous polypeptides, drugs, 15 radionucleotides or toxins, see, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EPO 396,387. An antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agent or a radioactive metal ion (e.g, a emitters, such as, for example, 213 Bi). A cytotoxin or cytotoxic agent includes any agent that is 20 detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and analogs or 25 homologues thereof Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil and decarbazine), alkylating agents (e.g., mechlorethamine, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and 30 cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., 410MLI ~GKH*AarOPM2sti 76 daunorubicin, daunomycin and doxorubicin), antibiotics (e.g., dactinomycin, actinomycin, bleomycin, mithramycin and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). [00259] To prolong the serum circulation of an antibody in vivo, various 5 techniques can be used. For example, inert polymer molecules, such as high molecular weight polyethylene glycol (PEG), can be attached to an antibody with or without a multifunctional linker either through site-specific conjugation of the PEG to the N-terminus or to the C-terminus of the antibody or via e amino groups present on lysine residues. Linear or branched polymer derivatization that results in 10 minimal loss of biological activity can be used. The degree of conjugation can be closely monitored by SD S-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by size-exclusion or by ion exchange chromatography. PEG-derivatized antibodies can be tested for binding activity as 15 well as for in vivo efficacy using methods known to those of skilled in the art, for example, by inmunoassays described herein. 100260] An antibody having an increased half-life in vivo can also be generated by introducing one or more amino acid modifications (i.e., substitutions, insertions or deletions) into an IgG constant domain, or FeR binding fragment thereof (such as 20 an Fe or hinge Fe domain fragment), see, e.g., WO 98/23289; WO 97/34631; and U.S. Pat. No. 6,277,375. [00261] Further, an antibody can be conjugated to albumin to make an antibody more stable in vivo or to have a longer half life in vivo. The techniques are known in the art, see e.g., WO 93/15199, WO 93/15200 and WO 01/77137; and 25 EPO 413622. The antibody also can be modified, for example, by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein and so on. [00262] Techniques for conjugating such a therapeutic moiety to antibodies are 30 well known, see, e.g, Arnon et al., in Monoclonal Antibodies and Cancer Therapy, A1G602K 1 C 21A 77 Reisfeld et at (eds.), Alan R. Liss (1985); Hellstrom et al, in Controlled Drug Delivery, 2nd ed., Robinson et al., eds, Marcel Dekker (1987); Thorpe, in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al., eds. (1985); Monoclonal Antibodies For Cancer Detection and Therapy, Baldwin 5 et al., eds., Academic Press (1985); and Thorpe et al., Immunol Rev 62:119 (1982). Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate, such as a bifunctional antibody, see, e.g., U.S. Pat, No. 4,676,980. (00263] The conjugates of the invention can be used for modifying a given 10 biological response, the therapeutic agent or 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, fbr example, a toxin such as abrin, ricin A, pseudomonas exotoxin or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, #-interferon, 15 nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-or TNF-0, AIM I (WO 97/33899), AIM II (WO 97/34911), Fas ligand (Takahashi et al., Int Immunol, 6:1567 (1994)), VEGF (WO 99/23105); a thrombotic agent; an anti-angiogenic agent, e.g., angiostatin or endostatin; or biological response modifiers such as, for example, lymphokines, 20 interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (GCSF) or other growth factors. [00264 The antibody or variant composition will be formulated, dosed and administered in a manner consistent with good medical practice. Factors for 25 consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent the method of administration, the scheduling of administration, and other factors known to medical practitioners. The "therapeutically effective amount" of the antibody or variant to be administered 30 will be governed by such considerations, and can be die minimum amount necessary 4t0ZtI 4GHMAh Pl52I,.I 78 to prevent, ameliorate or treat an extended Type I glycosphingolipid disease, condition or disorder. [00265] As used herein, the term "effective amount" refers to the amount of a therapy (e.g., a prophylactic or therapeutic agent), which is sufficient to reduce the 5 severity and/or duration of an extended Type I glycosphingolipid related or associated disease, ameliorate one or more symptoms thereof, prevent the advancement of an extended Type I glycosphingolipid related or associated disease or cause regression of an extended Type I glycosphingolipid related or associated disease, or which is sufficient to result in the prevention of the development, 10 recurrence, onset, or progression of an extended Type I glycosphingolipid related or associated disease or one or more symptoms thereof, or enhance or improve the prophylactic and/or therapeutic effect(s) of another therapy (e.g., another therapeutic agent) useful for treating an extended Type I glycosphingolipid disease related or associated. For example, a treatment of interest can reduce a symptom, based on 15 baseline or a nonnal level, by at least about 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. In one other embodiment, an effective amount of a therapeutic or a prophylactic agent 20 reduces a symptom of an extended Type I glycosphingolipid related or associated disease, such as a cancer, by at least about 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. Also used 25 herein as an equivalent is the term, therapeuticallyy effective amount." [00266] The amount of therapeutic polypeptide, antibody or fragment thereof which will be effective in the use or treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Where possible, a dose-response curve and the 30 pharmaceutical compositions of the invention can be first derived in vitro. If a 41903R1 qUAMt.ruPa6R~Ail. 79 suitable animal model system is available, again a dose-response curve can be obtained and used to extrapolate a suitable human dose practicing methods known in the art. However, based on common knowledge of the art, a pharmaceutical composition effective in promoting a diminution of an inflammatory effect, for 5 example, may provide a local therapeutic agent concentration of between about 5 and about 20 ng/ml, and, preferably, between about 10 and about 20 ng/ml [00267] In a preferred embodiment, an aqueous solution of therapeutic polypeptide, antibody or fragment thereof can be administered by subcutaneous injection. Each dose may range from about 0.5 mg to about 50 mg per kilogram of 10 body weight, or more preferably, from about 3 mg to about 30 mg per kilogram body weight. The dosage can be ascertained empirically for the particular disease, patient population , mode of administration and so on, practicing pharmaceutic methods known in the art. [00268] The dosing schedule for subcutaneous administration may vary from 15 once a week to daily to multiple times a day depending on a number of clinical factors, including the type of disease, severity of disease and the sensitivity of the subject to the therapeutic agent. [00269] In one embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous 20 administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine or other "caine" anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage fonm, for 25 example, as a dry lyophilized powder or a concentrate in a sealed container, such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be 412LAW9- ) PMMIAU. 80 provided, for example, in a kit, so that the ingredients may be mixed prior to administration. [00270] The invention also provides that a liquid formulation of the present invention is packaged in a sealed container such as an ampule or sachet indicating 5 the quantity of the product of interest. The liquid formulations of the instant invention can be in a sealed container indicating the quantity and concentration of the antibody or antibody fragment. The liquid formulation of the instant invention can be supplied in a sealed container with at least about 15 mg/ml, 20 mg/m1, 30 mg/mI, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/mfl, 80 mg/ml, 90 mg/ml, 10 100 mg/ml, 150 mg/ml, 200 mg/ml 250 mg/ml, or 300 mg/ml of extended Type I glycosphingolipid antibody in a quantity of about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml or 20 ml, for example. [00271] An article of manufacture containing materials useful for the treatment of the disorders described above is provided. The article of manufacture comprises a 15 container and a label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for diagnosing, preventing or treating an extended Type I glycosphingolipid condition or disease and may have a sterile access port (for example, the container may be an 20 intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label on or associated with the container indicates that the composition is used for treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. 25 It may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes and package inserts with instructions for use. (00272] In another aspect of the invention, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, 30 inhibit or prevent a disease or disorder associated with aberrant expression and/or 06QII 'M_ 1 I )P IAWt 81 activity of extended Type I glycosphingolipid, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid of interest. Alternatively, cells manipulated to carry gene sequences of interest are administered to a host. In an embodiment of the 5 invention, the nucleic acids produce the encoded protein in and by target host cells that mediate a therapeutic effect. Any of the methods for gene therapy available can be used according to the instant invention. [00273] For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488 (1993); Wu et al, Biotherapy 3:87 (1991); Tolstoshev, 10 Ann Rev Pharmacol Toxicol 32:573 (1993); Mulligan, Science 260:926 (1993); Morgan et al., Ann Rev Biochem 62:191 (1993); and May, TIBTECH 11:155 (1993). [00274] In one aspect, the compound comprises nucleic acid sequences encoding an antibody, or functional binding fragments thereof, said nucleic acid sequences 15 being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody or antigen-binding coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific, as well as other regulatory sequences. 20 [00275] In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for integration and intrachromosomal expression of the antibody-encoding nucleic acids (Koller at al., Proc NatI Acad Sci USA 86:8932 25 (1989); Zijlstra et al., Nature 342:435 (1989)). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody. Alternative methods for integration include using particular transcription factors that recognize specific nucleic acid sequences, 30 zinc fingers and so on. 4120U{ GHMln rPIM2AJ1 82 [00276] Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. 5 [00277] In one embodiment, the nucleic acid sequences are directly administered in vivo and are expressed to produce the encoded product. That can be accomplished by any of numerous methods known in the art, e.g, by constructing the antibody encoding sequences as part of an appropriate nucleic acid expression vector and administering same so that the vectors become intracellular, e.g., by 10 infection using defective or attenuated retroviral or other viral vectors (see U.S. Pat. No. 4,980,286), by direct injection of naked DNA, by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), using non-viral vectors, such as synthetic compositions comprising an amphipathic compound that binds the hydrophilic nucleic acid and has the ability to fuse with cells, generally thus 15 containing a hydrophobic portion for combining with membranes, coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, by administering the vector in linkage with a peptide which is known to enter the nucleus, by administering the vector in linkage with a ligand subject to receptor-mediated endocytosis (see, e.g,, Wu et al, J Biol 20 Chem 262:4429 (1987)) (which can be used to target cell types specifically expressing the receptors) etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell-specific uptake 25 and expression, by targeting a specific receptor (see, e.g., WO 92/06180; WO 92122635; W092/20316; W09314188 and WO 93/20221). [00278] Regarding vectors, for example, a lentiviral vector can be used as known in the art. The lentiviral vectors contain components for packaging the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the 30 antibody to be used in gene therapy are cloned into one or more vectors, which -iOIl " 83 facilitate the delivery of the gene into a patient. For example, a lentiviral vector can be used to deliver a transgene to hematopoietic stem cells. References illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J Clin Invest 93:644 (1994); Kiem et al., Blood 83:1467 (1994); Salmons et at, Human Gene Therapy 5 4:129 (1993); and Grossman et al, Curr Opin Gen and Dev 3:110 (1993). [00279] Adenoviruses also may be used in the instant invention. Targets for adenovirus-based delivery systems include liver, the central nervous system, endothelial cells and muscle, for example. Adenoviruses infect non-dividing cells, an advantage over early retroviral vectors. Kozarsky et al., Curr Opin Gen Dev 10 3:499 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al, Science 252:431 (1991); Rosenfeld et al., Cell 68:143 (1992); Mastrangeli et al., I Clin 15 Invest 91:225 (1993); W094/12649; and Wang et al., Gene Therapy 2:775 (1995). [00280] Adeno-associated virus (AAV) also can be used in gene therapy (Walsh et al., Proc Soc Exp Biol Med 204:289 (1993); and U.S. Pat. Nos. 5,436,146; 6,632,670; and 6,642,051). [00281] Another approach to gene therapy involves transferring a gene to cells in 20 tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated transfection or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells then are placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells then are delivered to a patient. 25 [00282] Thus, the nucleic acid can be introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene 30 transfer, microcell-mediated gene transfer, spheroplast fusion etc. Numerous 4i02fl. P&Bhrl2.AtMl 84 techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler et al., Meth Enzymol 217:599 (1993); Cohen et al., Meth Enzymol 217:618 (1993); and Cline, Pharm Ther 29:69 (1985)) and may be used in accordance with the present invention, provided that the necessary developmental 5 and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressed by the cell, heritable and expressed by the cell progeny. [00283] The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g, hematopoietic stem or 10 progenitor cells) are preferably administered intravenously, for example, as known in the bone marrow transplantation art. The amount of cells envisioned for use depends on the desired effect, patient state etc., and can be determined by one skilled in the art. [00284) Cells into which a nucleic acid can be introduced for purposes of gene 15 therapy encompass any desired, available cell type, and include, but are not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells, such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes and granulocytes, various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g, as 20 obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver etc. [00285] In one embodiment, the cell used for gene therapy is autologous to the patient. Nucleic acid sequences encoding an antibody of the instant invention are introduced into the cells such that the transgene is expressed by the cells or their progeny, and the recombinant cells then are administered in vivo for therapeutic 25 effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with the embodiment of the instant invention (see e.g., WO 94/08598; Sternple et al., Cell 71:973 (1992); Rheinwald Meth Cell Bio 21 A:229 (1980); and Pittelkow et al., Mayo Clinic Proc 61:771 (1986)). Because 30 extended Type I glycosphingolipid is expressed on, for example, B cells, blood cells 4 i&M (tGHjHkW mPM2uA. 85 and bone marrow cells are suitable host cells. However, the scope of the instant invention regarding the use of stem cell hosts does not contemplate the making and using of a transgene to make a transgenic organism by administering the transgene of interest to embryos and/or embryonic stem cells. 5 [00286] The invention thus provides methods of treatment, prophylaxis and amelioration of extended Type I glycosphingolipid related and associated diseases or one or more symptoms thereof by administering to a subject an effective amount of, for example, a liquid formulation, an antibody or variant thereof of the invention. The subject is preferably a mammal such as non-primate (e.g., cows, pigs, horses, 10 cats, dogs, rats etc.) and a primate (e.g., monkey, such as a cynomolgus monkey, and a human). In a preferred embodiment, the subject is a human. [00287] Extended Type I glycosphingolipid also is expressed on certain cancer cells, such as pancreas, colon and bladder, as well as on T cell leukemias (Qinping et al, Oncogene 24:573-584, 2005), and stimulation of extended Type I 15 glycosphingolipid correlated with proliferation of carcinoma cells, Meijer et al, Canc Res 66:9576-9582, 2006. [00288] Thus, the antibody or derivative thereof of interest can be used to control proliferation of cancer cells expressing extended Type I glycosphingolipid, which cancers are identified by determining presence of extended Type I glycosphingolipid 20 expression by a diagnostic assay taught herein. The antibody of interest can reduce infiltration of malignant cells, reduce resistance to apoptosis and minimize proliferation. Such patients then are administered a cancer cell proliferation inhibiting amount of an antibody, or derivative thereof, of interest as provided herein. As taught herein, an antibody or antigen binding portion thereof can be 25 administered to a patient in a number of ways, including administering a polypeptide, a polynucleotide and so on. Essentially any cancer that expresses a Type I epitope of interest can be detected and/or treated with an antibody of interest. For example, the malignant cell can be an epithelial cell. The epithelial cell can be found in any malignant cell of any organ or tissue origin, such as, colon, rectum, 30 esophagus, lung, prostate, breast, pancreas, the oral cavity, vagina, the 410MILI "GJ44arekm"F"1 A" 86 gastrointestinal tract in general, urinary tract and so on. However, the cancer need not be limited to an epithelial cell, so long as the malignant cell expresses a Type I epitope of interest. [00289] The invention now will be exemplified for the benefit of the artisan by 5 the following non-limiting examples that depict some of the embodiments by and in which the instant invention can be practiced. EXAMPLES EXAMPLE 1: GENERATION OF IMMUNOGEN [00290 Colo2O5 cells (ATCC) (Semple et al., Cancer Res 38: 1345-1355, 1978) are grown in RPMI 1640 medium containing 10%, fetal calf serum. Harvested cells are washed twice with PBS and stored at -20 0 C until needed. Cell pellets are 10 extracted with isopropanol-hexane-water (IHW) (55:25:20) followed by Folch partition, DEAE Sephadex chromatography and HPLC on an Latrobead 6RS-801 0 column. Gradient elution of the upper-phase neutral fraction is performed in IHW from 55:40:5 to 55:25:20 over 200 minutes. Fractions are collected and pooled according to IPTLC migration in chloroform-methanol-water (50:40:10). The 15 extended Type I chain glycosphingolipids are purified further by preparative TLC on Merck HPTLC plates (Silica Gel 60, Merck, Darmstadt, Germany), see U.S. Pat No. 6,083,929. [00291] A positive band (by immunostaining with mAb IMH2) which migrated just below dimeric Le antigen is purified as taught herein. 20 [00292] The colorectal adenocarcinoma cells Colo205 (ATCC CCL-222) and DLD-1 (ATCC CCL-22 1) are cultured in RPMI 1640 medium (Invitrogen Co., Cat. No. 31800) supplemented with 1 mM sodium pyruvate (Invitrogen Co., Cat. No. 11360). Other colorectal adenocarcinoma cells, SW 116 (ATCC CCL-233) and HT-29 (ATCC HTB-38), and lung-derived T84 cells (ATCC, CCL-248) are 25 separately maintained in Leibovitz's L-15 medium (Invitrogen Co., Cat. No. 41300), McCoy's 5a medium (Invitrogen Co., Cat. No. 12330) and DMSM/F12 medium (Invitrogen Co., Cat. No. 12400). The KATO III gastric carcinoma cells (ATCC 41""-1 87 HTB-103) are cultured in IMDM medium (Invitrogen Co., Cat. No. 12200). All media used in the studies are supplemented with 10% fetal calf serum. EXAMPLE 2: GENERATION OF ANTI-EXTENDED TYPE I GLYCOSPHINGOLIPID MABS [00293] KM mice (Kirin Brewery Co., Ltd.) are generated by cross breeding double transchromosomic mice and transgenic mice. KM mice possess human 5 chromosome fragments containing the entire human immunoglobulin heavy chain loci and a YAC transgene for half of the human immunoglobulin kappa light chain loc1 KM mice are engineered to express neither endogenous immunoglobulin heavy chain nor kappa light chain. All of the animals are maintained and handled according to the rules and regulations accepted in the art. 10 [00294] Colo205 cells are injected intraperitoneally into KM mice every 3 weeks (5 x 106 cells/injection) for a total of 4 injections, followed by injection of extended Type I chain glycosphingolipids which are isolated from Colo205 cells and adsorbed on lipopolysaccharide (Sigma, L-7011) (Young et al., J. Exp. Med. 150:1008-1019, 1979) every week for 8 injections. The anti-Colo205 neutral glycosphingolipid 15 titers of immunized mice are monitored by ELISA, using anti-human kappa-HRP (Southern Biotechnology Associates, Cat. No. 9220-05) as the secondary antibody until the titer reached 1:6000. Three days after the final injection, splenocytes from the boosted mouse are fused with P3/NS 1/1-Ag4-1 (NS-1) mouse myeloma cells (ATCC TIB-18) practicing methods known in the art. Hybridomas are screened by 20 ELISA using 96-well ELISA plates (Corstar, Cat. No. 2592) coated with Colo205 neutral glycolipid. Mouse anti-human IgG antibodies conjugated with HRP? are used as the secondary antibody (Southern Biotechnology Associates, Cat. No. 9040-05) and teramethylbenzidene (TMB) (Kem-Zn-Tec Diagnostics, Cat. No. 4390) is used as the substrate. Hybridoma supematants showing high reactivity with Colo205 25 neutral glycolipids are further confirmed by HPTLC innunostaining and by flow cytometry. Clones strongly staining the extended Type I chain glycolipids and 41in21Th1 GHPIflflJPS& 1.AU1 88 showing high binding on the surface of Colo2O5 cells are repeatedly subeloned by limiting dilution until stable clones are established. One stable clone is GNX-8. EXAMPLE 3: GNX-8 ANTIBODY 5 [002951 Monoclonal antibody is purified from culture supernatants using protein A Sepharose (GE Healthcare 17-129-79-02) with pH gradient elution according to the manufacturer's suggested procedures. Each fraction is collected and the presence of antibody is examined by ELISA. Fractions with Colo205 neutral glycolipid binding activity are pooled and dialyzed against PBS (pH 7.4). Purified 10 antibodies are aliquot and stored at -20* C. [00296] The concentration of monoclonal antibody is determined with the Bio-Rad Protein Assay kit (Bio-Rad, Cat. No. 500-0006) using IgG as the standard according to the manufacturer's recommended procedures. (00297] The isotype of GNX-8 is determined using an ELISA. GNX-8 is a 15 human IgG1 and the light chain is kappa. [00298] Purified GNX-8 is applied to 10% SDS-polyacrylamide gels after being boiled in 2X SDS gel-loading buffer with (reducing condition) or without (non-reducing condition) fl-mercaptoethanol. Electrophoresis is conducted using the Minutesi-PROTEAN3 Electrophoresis System (BIO-RAD) according to the 20 manufacturer's recommendations. [00299] GNX-8 separated on reducing SDS-PAGE gels is transferred onto nitrocellulose (NC) membranes (Amersham) and blocked with 3% skim milk in PBS. The membrane is incubated with secondary antibody for 1 hour at room temperature. HRP-labeled goat anti-human IgG(y) antibody (Zyned, 62-8420) at 25 1:5000 dilution and HRP-labeled rabbit anti-human kappa chain IgG antibody (DAKO, P0129) at 1:2000 dilution are separately used to detect the heavy chain and light chain of GNX-8. Western Lightning m Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Cat, No. NEL105) is used to develop the signal on BioMax Light Film (KODAK, Cat. No. 1788207). 4t4#k&J ICHMUsp P8lZN.M 89 [00300] Under reducing conditions, the molecular weight of the GNX-8 light chain and heavy chain are as expected for an IgG. GNX-8 is a human monoclonal antibody by Western blot with goat anti-human IgG(y)-HRP and rabbit anti-human kappa chain-HRP as secondary antibodies, separately. GNX-8 is a human antibody 5 by ELISA isotyping, £00301] The pI analysis of GNX-8 is determined by the PhastSystem (Pharnacia). Briefly, an antibody sample and p1 standard are applied on an IEF PhastGel 3-9 using a PhastGel Sample applicator 8/1 comb and are separated according to the manufacturer's protocol. The gel subsequently is silver stained in 10 the PhastSystem Development Unit (Pharnacia) according to the manufacturer's protocol. [00302] The p1 analysis reveals multiple bands ranging from pH 8.15 to 8.65 indicating the possibility of post-translational modifications of the antibody. The high pI indicates that GNX-8 will be soluble at physiologic pH, 15 [00303] For detecting cell binding activity, 2 x 10 5 cells are washed with PBS and incubated with various concentrations of antibody for 30 minutes at room temperature. After a PBS wash, FITC-labeled goat anti-human IgG (Fc) antibodies at 1:3000 dilution (ICN, Cat no. 55198) are added to each cell sample for an additional 30 minutes at room temperature. For CDC studies, cells after antibody 20 treatment are washed three times with PBS and incubated with 1 pl of propidium iodide solution (Sigma-Aldrich, P4846) for 30 minutes. After a final PBS washing, cells are analyzed on a flow cytometer (BD, FACSort). The results are processed with CELLQuest 3.3 (BD). EXAMPLE 4: CYTOTOXICITY ASSAY 25 [00304) Human colon cancer cell lines, SWI 116, Colo205 and DLD-1, are seeded in 48-well plates (Coming Costar) at a density of 2 x 104 cells/well. After being cultured overnight, the cells are incubated in 500 gl of medium with 25% non-inactivated human serum at various antibody concentrations for 2 hr. After a PBS wash, the remaining live cells are quantified by propidium iodide (PI) solution 4 PUMSk.4 4StetM IJI pwazl~o90 (Sigma-Aldrich, P4846) staining and are analyzed by flow cytometry. Normal human Ig~s purified from normal human serum are used as a negative control. [00305] In an alternative assay, target cells are labeled by incubation with about 100 p of 51 Cr fbr about 90 min. at about 37" C. After washing (3x) and incubation 5 (about 1 hr at 37" C), cells (about 1 x 106 ml) are suspended in RPMJ-1640 supplemented with about 25 mM HEPES buffer and about 3% bovine serum albumin. About 20 d of labeled cells, about 100 pl of mAb and 25% heat inactivated human serum are mixed in the wells of microtiter U bottom plates (Corning, N.Y.). Non-specific mouse Ig (Sigma, St. Louis, MO) can be used as a 10 negative control. After about 4 hr incubation, the plates are centrifuged (500 x g, 2 min) with a hanging plate holder assembled in a centrifuge, and radioactivity in about 100 s1 supernatant in each well is measured with a gamma counter. Each experimental group can be tested in triplicate. Percent specific lysis can be calculated according to the formula ([A-B] x 100)/C, where A=cpm in lysed 15 experimental cells; B=cpm in unlysed target cells; and C=cpm in total target cells. Spontaneous release preferably should not exceed 15% of maximally releasable labeled radioactivity. [00306] ADCC assays are performed by the lactate dehydrogenase (LDH) release assay (Promega, CytoTox 96 Non-Radioactive Cytotoxicity Assay) using human 20 peripheral blood mononuclear cells (PBMC) as effector cells prepared from healthy donors using Ficoll-Paque (GE, 71-7167-00). The assay quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released on cell lysis. Released LDH in culture supematants is measured with a 30 minute coupled enzymatic assay, which results in the conversion of a tetrazolium salt (INT) into a 25 red formazan product. The amount of color formed is proportional to the number of lysed cells. [00307] Colo205 cells used as target cells are distributed into 96-well U bottom plates (2 x 104 cells/well) and are incubated with antibodies in the presence of the PBMC with various EIT ratios for 4 hours at 37" C. The LDH activity in the 30 supernatant was measured by CytoTox 9 6 a Non-Radioactive Cytotoxicity Assay. 4 ima 1 (Q~kftwno PDK5I.Av1 91 The percent specific cytolysis is calculated according to the following formula: % specific lysis 100 x (E-SE -ST)/ (M -ST ) where E is the experimental release (activity in the supernatant from target cells incubated with antibody and effector cells), SE is the spontaneous release in the presence of effector cells (activity in the 5 supernatant from effector cells with medium alone), Sr is the spontaneous release of target cells (activity in the supernatant from target cells incubated with medium alone), and M is the maximum release of target cells (activity released from target cells lysed with 9% Triton X-1 00). [00308] The in vitro antitumor activity of GNX-8 is evaluated by CDC assay. 10 Treatment of human colorectal cancer cells, SWI 116, Colo205 and DLD-1, with GNX-8 in the presence of 25% human serum, results in substantial cell lysis in a dose dependent manner. The results indicate that GNX-8 kills target cells through complement-dependent cytolysis. [00309] The CDC effect on SW 1116 and Colo205 cells in some experiments is 15 stronger than that on DLD- 1 cells. The viability of cells is inversely proportional proportion to the level of expression of GNX-8 antigen. The CDC effect of GNX-8 and levels of GNX-S antigen expression on the three colorectal cancer lines demonstrate that cancer cells with higher GNX-8 antigen expression are more susceptible to cytotoxicity while those with lower GNX-8 antigen expression have 20 higher viability. The results lead to the conclusion that the antitumor activity of GNX-8 can depend on the expression level of GNX-8 antigen. Patients with high GNX-8 antigen expression on tumor cells might be treated with GNX-8 alone, while tumors expressing lower levels of GNX-S antigen, may benefit from a combination therapy with one or more other cancer drugs in addition to GNX-8 antibody. 25 (00310] ADCC activity of human peripheral blood mononuclear cells (PBMC) is evaluated against human colorectal cancer Colo205 in the presence of GNX-8. ADCC activity with IMH2 is used as the positive control and human IgG as the negative control. [00311] GNX-8 induces strong ADCC activity against Colo205 cells. The 30 cytotoxic effect is correlated positively with both E/T ratio and GNX-8 4iKfl._i jsa&iumjPMI2AMi 92 concentration. One hundred per cent cell lysis is observed at E/T of about 20/1. A maximal ADCC effect, and a trend observed for about 50% lysis as well, is observed at about 5 pg/mi of GNX-8 and at about 50 Rg/ml for IMH2, respectively. Control human IgG shows no cytotoxic effect regardless of FJT ratio or IgG concentration. 5 The dose of GNX-8 to reach 50% lysis is less than 1/10 needed for IMH2. EXAMPLE 5: BIACORE AFFINITY ANALYSIS [00312] Type I glycosphingolipids are affixed to a chip. Then the mAb's are exposed to the chip for kinetic measurements and epitope sequence analysis 10 surrounding the antibody-antigen binding reaction, following the manufacturer's recommendations (GE Healthcare, Pistcataway, NI). EXAMPLE 6: IN VIVO ASSAY (00313] Antitumor activity of GNX-8 is evaluated in a Colo205 xenograft model. 15 Colo205 cells are washed twice with PBS and reconstituted at a cell density of 5 x 106/100 pl in PBS. Female nude mice of age 6-8 weeks are inoculated s.c. with 100 p1 of the Colo2O5 cell suspension in the flank region. Tumor sizes are measured three times a week with a vernier caliper and tumor weights (mg) are estimated as (widtl 2 x length)/2. GNX-8 or normal human IgG is i.p. injected in tumor-bearing 20 nude mice acconing to designed doses and schedules. [00314] To evaluate the in vivo antitumor efficacy of GNX-8, cancer cells (5 x 106 cells/mouse) are injected in nude mice and treated with either GNX-8 (treatment group, 8 mice/group) or normal human IgG (control group, 7 mice/group) 24 hours after tumor inoculation. Five doses (300 jg/mouse) at 24-hour intervals 25 and subsequently four doses (600 pg/mouse) at 48-hour intervals are injected in both groups. [00315] Tumor growth is significantly inhibited in GNX-8-treated mice. The treatment group reaches a median tumor weight of T/C (Treatment/Control) of about 23% on day II and continues at that approximate level to the end of the study. A 30 T/C measure of : 4 2% is considered significant in demonstrating antitumor activity. i44200,,j (CGniai uMA2-AU, 1 93 [00316] Half (4/8) of the mice in the GNX-8 treatment group achieve long term tumor-free survival over 50 days. On the other hand, tumor size of the control group animals continually increases during the study. [00317] A similar study is conducted in a Colo2O5 xenograft nude mice model, 5 The first dose of GNX-8 is given at a tumor size of 80 to 100 mg. GNX-8 (treatment group) and normal human IgG (control group) are injected once (300 jig/mouse) daily for five days, and with two similar doses at days 17 and 21. [00318] Significant tumor inhibition also is observed in the treatment group, although the treatments are discontinued after only 5 doses, The median tumor 10 weight of T/C (Treatment/Control) is lower than 42% after day 10 and through to the end of the study. [00319] To determine whether host effector function contributed to GNX-8 efficacy, SCID mice bearing Colo205 xenografts are treated with GNX-8 or normal human IgG at 600 pg/mouse twice weekly for three weeks. Tumor size is measured 15 twice every week until the tumor size reached 10% of the body weight, which is considered the endpoint of the study. (00320] Survivability is prolonged in the treatment group. (00321) To explore the occurrence of the GNX-8 epitope on human colorectal cancers, several human colorectal cancer cell lines are analyzed. 20 [00322] For example, the in vivo inhibition of DLD-1 by GNX-8 is significant. EXAMPLE 7: GNX-8 ANTIGEN [00323] For the analysis of cell glycoprotein, cultured cells are scraped from the T-75 flasks and washed twice with PBS, followed by lysis buffer (50 mM Tris-HC1 25 pH 75, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 1 mM PMSF). The lysates are passed through a 26 gauge needle several times to disperse any large aggregates. Protein concentration is determined by Protein Assay Kit (Bio-Rad). The lysates containing the same amount of proteins are separated on a gel and analyzed by Western blot with GNX-8 as the primary antibody and mouse 30 anti-human IgG (Fc) labeled with HRP (Southern Biotechnology Associates, 4161Ml-. GH *AOt) PBMZ1AIJ,1 94 #9040-05) as the secondary antibody. Western Lightningsv Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Cat. No. NEL105) is used to develop the signal on BioMax Light Fihn (KODAK, Cat. No. 1788207). [00324] Neutral glycolipids (2 p1/sample) are spotted on a HPTLC plate (Merck, 5 1.05642, silica gel 60 F 2 s4), and developed with a mobile phase containing chloroform:methanol:water at a ratio of 50:40:10 (V:V:V). For glycan staining of glycolipids, 0.2% orcinol (Sigma, 0-1875) in 10% H 2 S0 4 is sprayed onto a HPTLC plate and incubated for 10 minutes in a 110" C in an oven. For imrnunostaining, the HPTLC plate is first fixed with 0.5% poly(isobutyl metbacrylate) (Aldrich, 181544) 10 in eblorofbnn:hexane, 1:9 (V:V) for 45 seconds, followed by blocking for 10 minutes in 3% BSA/PBS. The plates then are washed with PBS and incubated with the primary antibody at room temperature for I hr, followed by biotinylated secondary antibody at room temperature for 1 hr. An Avidin-Biotin Complex kit (Vector Laboratories, Burlingame, CA) is used to amplify signals from the 15 secondary antibody. The plate is incubated at room temperature for 30 minutes, followed by color development with an Immunostaining HRP-1000 kit (Konica Minolta, 130990) according to the manufacturer's protocol. [00325] To a lyophilized sample, 20 pl of 48% hydrogen fluoride (HF) (Merck) are added, and then the mixture is incubated at 4* C for 48 h. At the end of the 20 reaction, HF is removed with N 2 gas. The defucosylated glycolipids are used for specificity study of GNX-8. (00326] The neutral glycolipids of Colo205 are treated with HF and analyzed by MALDI-TOF MS to confmn the removal of fucose. TLC immunostaining is performed to analyze the specificity of GNX-8 directed to Colo205 neutral 25 glycolipids before and after HF treatment. (00327] GNX-8 recognizes uncleaved glycolipids but not the defucosylated forms, suggesting that the epitope of GNX-8 is a carbohydrate moiety and fucose is an essential component of the structure, [00328] The specificity of GNX-8 is characterized further by HPTLC 30 immunostaining on neutral and monosialyl glycolipids isolated from Colo205 cells. 4l""4dHM.MPHU2iAV.1 95 One hundred grams of Colo205 cells are collected, and glycolipid fractions are extracted. Colo205 glycolipids separated by TLC are stained for carbohydrate with orcinal/H 2

SO

4 . The positions of Le', Let, Le"-Lea and Lebiet are identified according to Stroud et al. (1992) supra. Sialyl Le (SLe') is indicated by staining 5 with nAb NKH3 (U.S. Pat. No. 5,240,833) and was later identified with MALDI-MS. HPTLC immunostaining of the same glycolipid fractions are conducted with CF4C4 (U.S. Pat. No. 5,011,920) (anti-Le"), T218 (Abcam, Cambridge, MA) (anti-Leb), IMH2 (Stroud et al., 1992, supra) (anti-LebLe ), and GNX-8 antibodies. 10 [00329] The results indicate that GNX-8 strongly reacts with extended Type I chain glycolipids. GNX-8 did not bind to Le' extended Type I chains. The monosialyl glycolipids of Colo205 cells are not recognized by GNX-8. GNX-8 shows very slight cross-reactivity with Leb at higher concentration (0.6 pg/ml). Unlike IMH2, GNX-8 did not bind to Lea or to Le'. GNX-8 binds to an extended 15 chain containing Leo. GNX-8 binds to Le-Le. [00330] In addition to TLC immunostaining, the epitope of GNX-8 is characterized by competitive ELISA using synthetic glycans Le 5 , LeaLe, Le1Le' and LeXLeX as inhibitors, and the LebL8/Le3aLe glycolipid mixture as a positive control. 20 [00331] The results indicate that GNX-8 slightly cross-reacts with Leb-LeX at high inhibitor concentration, but has no reactivity with other tested synthetic glycans including synthetic Leb glycans. The binding activity of GNX-8 to extended Let is 1000 times higher than that to simple L.C? [00332] Based on the results, the epitope of GNX-8 likely is an Leb structure on 25 an extended Type I chain with fucosylation, but it is not a simple Leb. EXAMPLE 8: CELL AND TISSUE DISTRIBUTION [00333] Formalin-fixed paraffin-embedded specimens of human normal and cancer tissues are obtained, for example, from US Biomax. A*MW MHOM409 FlMJU.1 96 [00334] The fonnalin-fixed, paraffin-embedded tissue arrays of normal and malignant human tissues are blocked with 0.1% skim milk in PBS for 30 minutes. After an additional 10 minutes incubation with 3% H 2 0 2 , the tissue arrays are washed thrice with PBS before samples are incubated with 0.1% BSA/PBS-diluted 5 biotinylated GNX-8 for 1 hour. Then the tissue samples are reacted with biotin-streptavidin-peroxidase complex (ABC kit, Vector, #PK-61 00) for 30 minutes for signal amplification. The DAB PLUS Substrate Kit (Zymed #00-2020) is used to visualize immunoreactive staining according to the manufacturer's protocol. Counterstaining is performed using hematoxylin. The results are determined by 10 visualization under a light microscope. [00335] The expression of GNX-8 antigen on human cancer cells is evaluated by flow cytometry. A number of human colorectal and gastric tumor cell lines, such as, Colo205, HT-29, DLD-1, SW 1116, T84 and KATO I, are separately examined for the expression of GNX-8 antigen by flow cytometry. 15 (00336] Flow cytometric analyses demonstrate that GNX-8 exhibits binding activity to all tested cancer cell lines. However, the binding is significantly stronger to SW 1116, Colo205 and DLD-l cells than to the other tested human cancer cell lines. (00337] In addition, GNX-8 antigen expression is tested on HL60 (a 20 promyelocytic cell line), MCF-7 (a breast cancer cell line) and PANC-1 (a pancreas cancer cell line), as well as on a mouse colon cancer cell line, CT26. GNX-8 does not bind to those four cell lines, [00338] Two colorectal cancer cell lines, Colo205 and SW1 116, are analyzed by Western blot. The two cell lines demonstrated strong binding with GNX-8 in flow 25 cytometry analyses. [00339] The results show presence of GNX-8 antigen on glycoprotein of Colo205 and SWi 116 over a molecular weight range from 32 to > 175 kDa, Accodingly, GNX-8 antigens are not only in glycolipids but also in glycoproteins. [00340] A variety of specimens from various organs, including both normal 30 tissues and cancer tissues, are separately stained with GNX-8. Staining patterns in 41eS~llfl "G-ln* Pt5021u 1U 97 tissue specimens are evaluated by staining intensity and frequency of positive cells. Staining is graded on a scale of 1+ (10-20%), 2+ (20-50%) or 3+ (> 50%), whereas frequency is classified based on the percentage of positive cells in each section. [00341] A strong correlation is observed between GNX-8 antigen expression in 5 primary and metastatic colorectal carcinomas. An immunohistochemical staining of a panel of tissue sections from a colorectal cancer patient is conducted. [00342) GNX-8 antigen is expressed not only on colorectal cancer tissues but also on adjacent tissues. For example, a polyp next to a cancer region is stained by GNX-8. However, no staining is observed on distal normal tissues. Hence, it can be 10 concluded GNX-8 identifies transfonned cells or cells undergoing transformation before recognizable cell morphology changes occur. [00343] GNX-8 antigen expression is also studied on various grades of cancer. [00344] GNX-8 antigen is expressed in each cancer stage. [00345] GNX-8 does not bind to normal colon, rectum, stomach, small intestine, 15 liver, esophagus, lung, prostate or breast. 100346] Fifty-eight percent (44/76) of colon cancer samples are stained with GNX-8; 47% of rectum cancer samples; 57% of metastatic colon cancer samples; 53% of stomach cancer samples; 29% of esophageal cancer samples; 22% of lung cancer samples; 4% of prostate cancer samples; 17% of breast cancer samples; and 20 67% of pancreatic samples are stained with GNX-8. GNX-8 does not bind to small intestine, liver and kidney cancer samples. 25 Table 1 Specificity of GNX-8 on Human Normal Tissues Normal Tissues Human (issues Incidence (No. posiive/No. tested) Colon 14/102 Esophagus 3/4* A002l_ 4GHuAr) PMZI.A .1 98 Breast 13/56 Pancreas 4/12" Kidney 11163" Rectum 1/106 Small intestine 0/2 Liver 0/4 Lung 0/3 Prostate 0/6 * stained on keratinization of stratified squamous epithelium § stained on epithelial cells of duct systen/lactiferous ducts # stained on epithelial cells of duct system 5 Table 2 Specificity of GNX-8 on Hunan Cancer Tissues 10 Human cancer tissues Incidence (No. Staining intensity and positive/No. location tested) Colon 44/76 3+(5), 2+(12), 1+(27) Rectum 50/107 3+(1), 2+(20), 1+(17) Small intestine 0/10 Liver 0112 Kidney 0/3 Colon (metastatic) 27/47 2+(9), 1+(18) Esophagus 4114 2+(1), 1+(3) Long 10/45 3+(1), 2+(5), 1+(4) Prostate 2/45 2+(2) Breast 7/45 3+(1), 2+(4), 1+(2) Pancreas 8112 2+(3), 1+(5) 41m ~AunMimu PU99 EXAMPLE 9: CLONING AND SEQUENCING OF GNX-8 [00347] GNX-8 producing hybridoma cells are routinely cultured in IMDM (Invitrogen) containing 10% low IgG fetal bovine serum (HyClone). To prepare RNA for cDNA synthesis, 1 x 100 hybridoma cells are first harvested by low speed centrifugation (1000 rpm, 5 in). Total RNA then is isolated from the cell pellet 5 using TRIZOL reagent (Invitrogen) according to the manufacturer's protocol. First strand cDNAs are synthesized from the purified RNA sample using the SMART RACE cDNA Amplification Kit (BD Biosciences-Clontech). Briefly, 1 pg total RNA is incubated with 1 pI 5-CDS and I pl SMART II A oligo primers at 70* C for 2 minutes. After addition of 2 gl 5x first-strand buffer, 1 p1 20 mM DTT, I p1 10 10 mM dNTP and I pl of PowerScript RT are added to the RNA/primer mixture. The sample is incubated further at 42* C for 1.5 hours. The first strand cDNA synthesis reaction is terminated by adding 100 M 1 Tricine buffer and incubated at 72* C for 7 minutes. [00348] The cDNA encoding the heavy chain fragment of GNX-8 is amplified by 15 PCR using UPM (BD SMART RACE cDNA Amplication Kit) and a primer of the 3' end of the heavy chain, CHI, SEQ ID NO:3. The PCR reaction is carried out at 94"C for 30 seconds, followed by 58* C for 30 seconds, 72* C for 3 minutes, and that cycle is repeated 26 times. (00349] The variable region of the heavy chain cDNA is re-amplified from 1 p 20 of the above reaction product in the presence of NUP (SMART RACE amplification kit) and a primer of the middle of the heavy chain CHI, SEQ ID NO:4. The PCR reaction is performed at 94*C for 15 seconds, 68" C for 30 seconds and that cycle is repeated 25 times. The amplified product is purified using a PCR purification kit (GeneMark) and the nucleotide sequence is determined using a primer of the 5' end 25 of the heavy chain CHI, SEQ ID NO:5. (00350] Based on the sequence information, the full length heavy chain cDNA is specifically amplified by PCR from previously prepared first strand cDNAs with newly synthesized primers, SEQ ID NO:6, and for the end of the heavy chain gene, 4 -00W93( GH4kWt n =Pal 100 SEQ ID NO:7, with the BD Advantagj M 2 PCR Enzyme System (BD Biosciences), The PCR reaction is set at 94" C for 40 seconds, 60" C for 30 seconds and 720 C for 100 seconds, and that cycle is repeated 35 times. [00351] The amplified full length heavy chain cDNA is first double digested with 5 EcoRi and Xbal. After gel purification, the recovered heavy chain CDNA then is ligated into the pCIneo vector (Promega) at the same sites to obtain the expression vector pCI-GNX-8.H3. The inserted eDNA sequence is confirmed using a primer that hybridizes upstream of the multiple cloning site, SEQ ID NO:8, and a primer downstream of the multiple cloning site, SEQ ID NO:9. GNX-8 heavy chain cDNA 10 and the deduced amino acid sequence are shown in Table 3 as SEQ ID NOS:14 and 15, respectively. [00352 To identify the light chain eDNA sequences, the light chain peptide of GNX-8 is subjected to mass spectrometry analysis and database search. According to the protein identification infonnation, the light chain of GNX-8 is homologous to 15 the mouse X chain. A primer flanking the 5' end of mouse lambda gene constant region, SEQ ID NO:10, is synthesized. A cDNA including the variable region and a part of the constant region of the light chain gene is amplified from the first strand cDNAs described previously by touchdown PCR. The PCR reaction is carried out first for 5 cycles of 30 seconds at 940 C, 90 seconds at 720 C, followed by 5 cycles of 20 30 seconds at 940 C, 30 seconds at 66" C, 90 seconds at72" C, and then the cycle of 30 seconds at 94" C, 30 seconds at 63* C, and 90 seconds at 72* C is repeated 27 times. The amplified PCR fragments then are introduced into the yT&A vector (Yeastern Biotech) for positive clone identification. [00353] Four clones with the expected size are selected for sequence 25 determination with primer, SEQ ID NO:11. [00354] The results indicate that all four clones have the identical cDNA with a structure homologous to the 5' end of known light chain genes. [003553 To reconstitute the full length light chain cDNA, a new set of primers, SEQ ID NO:12 and SEQ ID NO:13, are synthesized and the cDNA described above 30 is used to prepare only the variable region of the light chain gene by PCR. The PCR 41SEZ2kOlJ(HMaMs2F5 PIM.A,1 101 reaction includes 30 cycles at 940 C for 30 seconds, 60" C for 30 seconds and 720 C for 1 minute. {00356] The amplified light chain variable region cDNA with incorporated EcoRI and BsiWI restriction enzyme sites is digested with the respective enzymes. After 5 agarose gel purification, the amplified variable region cDNA fragment is ligated into the same sites of the pCIck vector (a pCIneo based expression vector with the insertion of a human 1c constant region at the XbaI and Noti sites) to give the light chain expression vector pCIck-GNX-8.mX Sequencing confirmation is performed and the deduced nucleotide, as well as amino acid sequences of GNX-8 light chain 10 are shown as SEQ ID NOS:16 and 17, respectively. [00357] A single vector that expresses both heavy chain and light chain genes of the recombinant GNX-8 antibody is constructed with either the neomycin gene (pCIck-GNX-8 neo) or the DHFR gene (pCIck-GNX-8 DHFR) as a selection marker. The light chain vector pClck-GNX-8.mX is linearized with BglI followed 15 by 5' end dephosphorylation with calf intestine phosphatase (CIP). The heavy chain vector pCI-GNX-8.H3 is cleaved with BglIl and NgoMIV. The BglII-NgoMIV fragment containing the CMV promoter, full length heavy chain cDNA and SV40 polyA is recovered by gel extraction and introduced into the linearized light chain vector by blunt end ligation to form pCIck-GNX-8 neo. Subsequently, the 20 pCIck-GNX-8 DHFR vector is generated by removing the neomycin gene with NgoMIV/ClaI cleavage from pCIck-GNX-8 neo and replacement with the DHFR gene. The DHFR minigene is cleaved from the pdhfr3.2 vector (ATCC No. 37166) by HindIll/SalI digestion, and is separated and recovered by gel extraction. Both fragments are treated with Kienow to give blunt ends, then the DHFR gene is ligated 25 into the NgoMIV/ClaI-cleaved pCIck-GNX-8 neo fragment by blunt end ligation. 30 41Sjbk.1 HMM-P F.iLNVJ 102 TABLE 3 Primers and Sequences 5 Sequence (5' to 3') SEQ ID NO ELLGG I MISRT 2 GCA TOT ACT AGT TTT GTC ACA 3 10 AGA TTT OGG GTG CAC GCC GCT GOT CAG GGC 4 GCC TO GOT GCC AGG GGG AAG ACC 5 GAT GG 15 CGA ATT CAC CAT GGC TOT CTC 6 CTTCCTC OCT CTA GAT CAT TTA CCC GGA 7 GAC AGG ACT CCC AGT TCA ATT ACA GC 8 20 TOG TTT GTC CAA ACT CAT C 9 GCA TOT ACT AGT TTT GTC ACA 10 AGA TTT GGG OTT TTC CCA GTC ACO AC 11 25 GCG AAT TCA CCA TGG CCT GGA 12 CTT CAC GCC GTA COT AGO ACA GTO ACC 13 TTG GTT C GDSVSSKSVA 18 30 GGGGACAGTGTCTCTAGCAA 19 GAGTGTTGCT TYYRSKWYN 20 ACATACTACAGGTCCAAGT 21 GGTATAAT 35 ARNFDY 22 GCAAGAAACTTTGACTAC 23 TGAVTTNNY 24 ACTGOGGGCTGTTACAACT 25 AATAACTAT 40 ATS 26 GCTACCAOC 27 ALWYNTHFV 28 GCTCTATGGTACAACACCC 29 ATTTTGTT 45 mes~w~wame103 SEQ IDNO:14 Length: 318 Type: DNA (heavy chain, variable region) 5 GGACTOGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGG GGACAGTGTCTCTAGCAAGAGTGTTOCTTGGAACTGGATCAGGCAGTCC CCATTGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGT GGTATAATGAATATGCAGTATCTGTGAAAAGTCGAATAACCATCAATCC 10 AGACACATCCAAGAACCAGTTCTCCCTGCACCTGAACTCTGTGACTCCCG AGGACACGGCTGTGTATTACTGTGCAAGAAACTTTGACTACTGGGGCCA GGGAACCCTGGTCACCGTCTCC 15 SEQ ID NO:15 Length: 106 Type: amino acid (heavy chain, variable region) 20 GLVKPSQTLSLTCAISGDSVSSKSVAWNWIRQSPLRGLEWLGRTYYRSKWY NEYAVSVKSRITINPDTSKNQFSLHLNSVTPEDTAVYYCARNFDYWGQGTL VTVS 25 SEQ ID NO:16 Length: 300 Type: DNA (light chain, variable region) 30 CTCACCACAGCACCTGGTGGAACAGTCATACTCACTTGTCGCTCAAGTAC TGGGGCTOTTACAACTAATAACTATGCCAACTGGGTCCAAGAAAAACCA GATCATTTATTCACTGGTCTAATAGATGCTACCAGCAACCGAGTTCCAGG TGTTCCTGTCAGATTCTCCGGCTCCCTGATTGGAGACAAGOCTGCCCTCA CCATCACAGGGGCACAGACTGAGGATGATGCAATGTATTTCTGTGCTCT 35 ATGGTACAACACCCATTTTGTTTTCGGCGGTGGAACCAAGGTCACTQTCC TA SEQ ID NO:17 40 Length: 100 Type: amino acid (light chain, variable region) LTTAPGGTVILTCRSSTGAVTTNNYANWVQEKPDHLFTGLIDATSNRVPGVP 45 VRFSGSLIGDKAALTITGAQTEDDAMYFCALWYNTHFVFGGGTKVTVL 41M0nL (r*Mff~jqp FPNkZ1M 1 104 [00358] Once the light and heavy chains are sequenced, the nucleic acids can be recoded to optimize expression in, for example, specific human host cells. EXAMPLE 10: TRANSFECTOMAS 5 [00359] NSO cells are grown to a density of I x I 0 cells/ml. The cells are maintained in exponential growth phase and medium is changed the day before transfection. The day of transfection, 40 x 10 cells are washed. Then, 10 pg of linearized nucleic acid containing, for example, light chain DNA and linearized heavy chain DNA are added to the cell suspension (the total DNA volume should be 10 less than 50 sl) and the culture incubated on ice for 15 min. The DNA and cell mixture is transferred to a chilled euvette (0.4 cm) and an electric pulse (750 V and 25 pF) is applied. The cuvette is placed on ice immediately after the electric pulse and kept on ice for 10-15 min. The cells are collected and plated. The cells axe incubated in a 5% CO 2 incubator for 12-16 days or until colonies appear. The 15 supernatant of the cell colonies or cells grown in suspension culture is tested by ELISA and positive transfectomas are cloned in fresh medium. To further screen the positive transfectomas, either titration ELISA or the Biacore assay is conducted, Expanded transfectomas are maintained in shaker flasks and antibody or derivative thereof collected from the supernatant. 20 EXAMPLE 11: PHARMACOKINETICS [00360] Rats are randomized into two groups (four rats/group). Animals receive 1 or 10 mg/kg of GNX-8 as a single iLv. bolus injection via a tail vein. Blood samples are collected at 0, 5, 15 and 30 minutes, and 1, 2, 4, 6, 24, 30, 72, 144, 168, 25 216, 240, 312, 336 and 360 hours. Serum is harvested and is stored at -20" C until the GNX-8 concentration assay. GNX-8 concentration is determined by ELISA. [00361] GNX-8 is radiolabeled with 13j using the IODO-Gen method. [00362] Nude mice are used for in vivo biodistribution and imaging studies. Five million Colo205 cells are inoculated subcutaneously. When the tumor reaches a size 30 of 0.5 g, the biodistribution study is performed. For the biodistribution study, mice ii1ezisI (GW.H..HaZIrP .M 1 105 are injected in the tail vein with 10 pCi/2.3 pg "'-labeled GNX-8. Five mice are sacrificed at 6, 24, 48, 72 and 96 hours after injection. Blood samples are taken just before the mice are sacrificed. Tumors and organs (brain, skin, muscle, bone, heart, lung, pancreas, eye, adrenal, tail, spleen, kidney, liver, bladder, stomach, small 5 intestine and large intestine) are removed immediately and weighed. Presence of radiolabeled antibody in tumor, organ and blood are separately counted in a y counter (Packard). Standards are counted each time with the tissues and tumors. Tissue radioactivities are expressed as the percentage of injected dose per gram of organ (%ID/g). 10 [003631 Imaging studies are performed on a microSPECT single photon emission computerized tomography X-SPECT animal imaging system (Gamma Medica Inc. USA) and microCT X-ray computerized tomography. Nude mice bearing Colo205 tumors are Lv.-injected with 200 mCi/5.5mg/ 100ml ' 31 1-labeled GNX-8 via tail vein. Images are acquired at 1, 6, 24, 48, 72 and 96 hours. Pharmacokinetic (PK) studies 15 are conducted for GNX-8 in rats, SCID mice and nude mice following a single Lv. administration. Serum concentration of GNX-8 is analyzed by ELISA. (00364] A two-compartment model provides a good fit to the data and generates the PK parameters summarized in Tables 4 and 5. A dose-related increase in Cmax is observed following a single i.v. administration of 1 .0 and 10 mg/kg GNX-8 in 20 rats. GNX-8 is cleared from the serum in a terminal half-life of 3.81 and 4.98 days at a dose of 1 and 10 mg/kg, respectively. [00365] The pharmacokinetic parameters of GNX-8 after administration in nude mice and SCID mice with or without Colo205 tumor-bearing mice presented with a Ti, that is about one day in both species with tumor. For non-tumor-bearing 25 animals, T 1 m values are 58.09 hr in SCID mice and 98.31 hr in nude mice, respectively. From the pharmacokinetic parameters, the T 1

,

2 is much longer in the non-tumor-bearing mice than in tumor-bearing mice. 30 106 Table 4 Pharmacokinetic parameters in rats Parameter 1 mg/kg Vi. 10 mg/kg iv. T. (minutes) 5 5 Cm(.ug/m1) 9.49 102.08 Tia terminal (day) 3.81 4.98 Table 5 Pharmacokinetic parameters in SCID mice and Nude mice 5 (iv. administration of 5 mg/kg GNX-8) SCID mice Nude mice Tumor-bearing + - + Cm.. (ug/ml) 76.45 72.52 76.5 84.4 Tm (min) 5 5 5 30 Tia (elimination) (hr) 22.99 58.09 26.73 98.31 [00366] Biodistribution studies are done in nude mice bearing Colo205 xenografts to assess the in vivo tumor targeting activity and specificity of GNX-8. 10 [00367] The highest level of 'I-GNX-8 radioactivity is detected in plasma at all time points, 6 h , 24 h, 48 h, 72 h and 96 h. At 6 h, about 60% of the injected does per gram (%ID/g) is detected in plasma. At 48, 72 and 96 hours, the %ID/g for plasma was about 20%. The plasma level is significantly higher than in other organs, brain, skin, muscle, bone, heart, lung, pancreas, eye, adrenal gland, tail, 15 spleen, kidney, liver, bladder, stomach, small intestine and large intestine, where the %ID/g at all time points in all organs does not exceed 5%ID/g. Radioactivity 4ISW.. !|HMAm PUS21AU.1 107 decreases over time in plasma and in the other organs, except the tumor. Plasma radioactivity decreases by about 70% between 6 and 96 hours. [00368] Radioactivity of the tumor is initially higher than in normal organs. The highest tumor uptake is observed at 48 hours after "'l-GNX-8 injection, and 5 maintains a steady state while the radioactivity of other organs decreases. Therefore, the tumor/organ ratios increase in other organs. Rapidly decreasing radioactivity is observed in plasma, heart, lung, adrenal gland, tail, spleen, kidney and liver between 6 and 24 hours. On the other hand, tumor/plasma ratios increase about 4 times from 6 to 96 hours after injection. No accumulation of 'I-GNX-8 in 10 kidney is observed. [00369] The in vivo tumor targeting activity in Colo205 tumor bearing nude mice is studied by imaging analysis. A time course experiment is done to monitor the distribution of m 3 I-GNX-8. The result also indicates that the majority of m1-GNX-8 is located in blood, and tumor targeting is clearly visible from 24 to 72 hours after 15 injection, (00370] The in vivo data indicate that most GNX-8 is retained in blood after injection. There is no significant non-specific binding in various normal organs. Moreover, GNX-8 targets the Colo205 tumor rapidly after i.v. injection and maintains labeling at a steady state level over 96 hours. 20 EXAMPLE 12: TOXICITY [00371] Single dose toxicity is performed in male and female BALB/c AnN Cr BR mice (6 mice/group) at 8-9 weeks of age. Mice are i.v. injected with GNX-8 at a dose of 150 mg/kg or with vehicle alone (PBS). Body weight for all mice is 25 measured on study days 1, 8 and 16 prior to sacrifice. Mice are observed daily for signs of morbidity or mortality. [00372] Repeat dose toxicity is carried out in male Sprague-Dawley Crl CD (SD) rats (6 rats/group) at 8 weeks of age. Each group is administered vehicle (PBS) or 3, 15 or 75 mg/kg/dose of GNX-8 twice weekly for 4 weeks. All animals are checked 30 daily for mortality and any finding is recorded individually. Rats are weighed -4G4WteO P* AA 108 weekly during the pretreatment and treatment periods, and a final ovemight fasting body weight is obtained at terminal sacrifice. Blood samples are collected at terminal sacrifice and evaluated for hematology and clinical chemistry parameters. The significance of differences in body weight and all tested parameters is 5 detennined by Student's t test. [00373] To determine the cross reactivity of GNX-8 on nonnal human tissues, a very high dose (150 pg/ml) of biotinylated GNX-8 is used, A tissue array with 72 human tissues (24 types of normal organs taken from 3 normal human individuals) (US Biomax FDA 801-1) are stained with GNX-8. Based on the results from the 10 C,,, of the pharmacokinetic study, GNX-8 is used at 150 pg/ml to ensure that GNX-8 at C.x would have no serious cross reactivity with normal human tissues. That concentration is much higher than regularly used in immunohistochemical studies. [00374] Weak to moderate staining of GNX-8 is observed on several human 15 tissues of epithelial origin, including mucosal epithelium of the gastrointestinal tract, epithelium cells of lactiferous ducts and keratinized cells of stratified squamous epithelium. According to the previous findings of Finstad et at. (Clin. Cancer Res, 3:1433-1442, 1997), antibodies introduced into circulating blood show specific localization to carcinoma cells and did not accumulate in the antigen-positive, 20 adjacent normal epithelial cells. Also, antibodies do not traverse the basement membrane. Therefore, the staining in epithelial cells of ducts in normal tissue by GNX-8 is not considered a detriment. [00375] Two additional studies are conducted for examining the cross reactivity of GNX-8 on normal human blood cells. All tested blood cells from the four various 25 blood type (ABO) donors show negative response with GNX-$. The results support that GNX-8 does not bind to blood cells. Therefore, GNX-8 administered to the circulatory system should not cause damage to blood cells. [00376) To address the safety of GNX-8 in vivo, two studies are conducted to determine the acute and subacute toxicity effects. J16MflL1 (GIW4M iw)FMlAa.1 109 [00377] The single dose toxicity of GNX-8 is tested in BALB/c mice at a dose of 150 mg/kg. Mice are observed once daily and no deaths are found before scheduled sacrifice. All mice gain weight over the duration of the study and there are no significant differences in mean body weight gain between the GNX-8 treatment 5 group and the control group. [00378] The repeat dose toxicity of GNX-8 is performed in Sprague Dawley Cr1 CD rats. Six animals of 8 weeks age are allocated to the groups. One group is administered vehicle (PBS) twice weekly for four weeks. Groups 2, 3 and 4 receive GNX-8 in PBS at 3, 15 and 75 mg/kg/dose, respectively, twice weekly for four 10 weeks. All animals are examined daily for mortality and all findings are recorded. Rats are weighed weekly during the pretreatment and treatment periods and a final overnight fasting body weight is obtained at terminal sacrifice. Blood samples are collected at sacrifice and evaluated for hematology and clinical chemistry parameters. Significance of differences in body weight and all other measured 15 parameters are analyzed by Student's t test. [00379] There are no clinical signs of toxicity related to GNX-8 treatment. Analysis of final body weight gain indicates no difference between treatment and control groups. Blood samples of the control group and the high dose group are taken prior to euthanasia following an overnight fast, and are analyzed for 20 hematology and clinical chemistry profiles. [00380] There are no statistically significant differences between the high dose and the control groups for hemoglobin amount, hematocrit, RBC number, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. The results indicate no hematology toxicity is induced 25 by repeated high-dose GNX-8 administration. [00381] For clinical chemistry analyses, mean values for the parameters for the control and the high-dose groups are indicated in Table 6. A statistically significant increase in total protein is noted in the high dose group that might result from the repeated injections of high dose antibody. Additionally, a slight increase in albumin 30 also is observed. However, the value is still within the range of normal limits for 1fMaI{CMkWmO PMMLIAU.1 110 rats. The clinical chemistry data show no notable injury to metabolism and excretion function after repeated injection of high dose GNX-8. [00382] Both hematology and clinical chemistry analyses verify the safety of repeated high dose GNX-8 administration over a four-week duration. 5 Table 6 Clinical Chemistry Analyses Control High dose Items Mean SD mean SD albumin (g/dl) 3.3 0.4 3.9 0.2 ALT(GPT) (U/L) 118.6 50.0 67.6 6.0 AST(GOT) (U/L) 372.0 203.8 323.3 118.2 total bilirubin (ing/dI) 0.3 0.1 0.6 0.2 BUN (mg/dl) 12.3 5.8 11.2 1.5 Ca (mg/dl) 11.0 0,2 11.2 0.2 CF (mnol/L) 108.0 2,1 106.0 2.2 cholesterol (mg/dl) 96.6 9.2 86.4 18.7 creatinine (mg/dl) 12 0.7 1.1 0.5 K* (mmol/L) 4.9 0.6 4.9 0.5 Na* (mmol/L) 144.6 27 148.2 4.2 phosphorous (mg/dl) 8.4 0.6 7.4 0.9 Total protein (g/dl) 6.3 0.3 7.1 0.2 10 EXAMPLE 13: SCALE-UP [00383) To obtain a stable cell line for large scale production of GNX-8, NSO and CHO cell lines expressing recombinant GNX-8 (rGNX-8) are obtained. Briefly, cDNAs encoding both the heavy and light chains of GNX-8 are cloned from the 5 original hybridoma. The isolated antibody genes then are reassembled in expression vectors. The molecular weight of rGNX-8 purified from media conditioned by transfected NSO cells is confirmed by SDS-PAGE. The specificity, binding activity and efficacy of rGNX-8 and the original GNX-8 hybridoma are compared by HPTLC, immunostaining, flow cytometry and CDC assay. 10 [00384] There are no differences between the original and recombinant GNX-8 antibodies. [00385] The N-glycosylation profiles of rGNX-8 and GNX-8 then are analyzed by MALDI-MS. [00386] The data illustrate a highly similar N-linked sugar pattern between the 15 two antibodies. Almost all N-glycans of the two antibodies contain the core fucosylation structure, but no tenninal sialic acids. [003 87] Dihydrofolate reductase deficient Chinese hamster ovary (CHO/dhfr-) cells (ATCC CRL-9096) are maintained in IMDM containing 5% FBS and supplemented with 100 nM hypoxanthine and 16 pM thymidine. To prepare 20 recombinant GNX-8 production cell lines, expression vector pCIck-GNX-8 DHFR is linearized by BamHI and the concentration of recovered DNA in solution is determined by OD 26 0 absorbance. Approximately 1.2 x 106 CHO/dhff cells are transfected with 10 pg linearized DNA and 30 p±l of Fugene6 transfection reagent (Roche) according to the manufacturer's instructions. After 48 hours, the culture 25 medium is replaced with 5% dialyzed fetal bovine serum containing IMDM for transfectant selection. The selection is continued for approximately two weeks until stable colonies are obtained. Multiple colonies are picked and cultured under the same selective medium in 48-well plates. Individual CHO clones are screened for rGNX-8 expression by antigen-specific ELISA using HRP-labeled anti-human 30 IgG(Fe) antibodies as the second antibody. AIMfl* (* ~ai W~I AJ I112 [00388] A CHO clone that expressed high levels of antibody is selected for subsequent gene amplification with methotrexate (MTX). Gene amplification in 10 nM of MTX results in more than a 30-fold increase of rGNX-8 secretion. The stable CHO clone is named CHO-rGNX-8.5M10. 5 CHO-rGNX-8.5M10 cells are later adapted to serum-free culture in shaking flasks and achieve a maximum yield of approximately 120 [tg/ml of GNX-8 over a 14-day culture. The culture supernatant is collected and purified by Protein A chromatography. The rGNX-8 antibody purified from the CHO culture supernatant by Protein A chromatography reveals the expected light chain and heavy chain 10 peptide bands on SDS-PAGE. EXAMPLE 14 - MONOCLONAL ANTIBODY GNX-10 [00389] Monoclonal antibody GNX-10 was generated from the same KM mouse as mAb GNX-8 described above, except for using different mouse myeloma cell 15 lines as the fusion partner, namely, NS-1 and FO cell lines were used in generating the antibody-producing hybridoma clones GNX-8 and GNX-10, respectively. [00390] The epitope of mAb GNX-10 was specified as extended Type I chain Le -Le' based on the results of HPTLC immunostaining and ELISA. 20 1. Sequence analysis of mAb GNX-10 [00391] The amino acid sequences of both the heavy and light chain variable regions of mAb GNX-10 are very similar to those of mAb GNX-8. The amino acid sequences of the heavy and light chains of mAb GNX-10 are shown in SEQ ID 25 NOS: 30 and 34, respectively, and the CDR regions contained therein are shown in SEQ ID NOS:31-33 and SEQ ID NOS:35-37. [00392] The amino acid sequence of the heavy chain variable region of mAb GNX-10 (SEQ ID NO:30) 30 GLVKPSQTLSLTCTISGDSVSSNTVAWNWIRQSPSRGL 6184551_1 (GHMatters) P86521.AU.1 KAROLA 113 EWLGRTYYRSKWYNDYAIFVKSRITFNPDTSKNQF SL HLNS VTPED TAVYYC ARNFDS W GQ GTLVT VS [00393] (CDR regions are underlined and marked with bold-letter.) 5 [00394] The amino acid sequence of the light chain variable region of mAb GNX-10 (SEQ ID NO:34) LTT SP GGT VILT CRS S TGA VT TD NYANW VQEKPDHLF T GLIDAT SNR VPG VP VRF S GS LIGDKAALT IT GAQ TED 10 DAMYFCALWYNTHFVFGGGTKVTVL [00395] (CDR regions are underlined and marked with bold-letter.) [00396] Alignments of the amino acid sequences of the heavy and light chain variable regions of mAbs GNX-8 and GNX-10 are shown below, in which 9 amino 15 acid differences (i.e., the amino acid residues that are marked by box and bold-letter) are in the heavy chain variable region and 2 amino acid differences in the light chain variable region. There are 4 amino acid differences located in predicted CDR regions of the heavy chain variable region and only one amino acid difference in predicted CDR region (i.e., the underlined regions) of the light chain variable region. 20 [00397] Alignment of heavy chain variable region (mAb GNX-8 vs. mAb GNX 10) GNX-10 GLVKPSQTLSLTGISGDSVSSIVAWNWIRQSPGLEWLGRTYYRSKWYNA S GNX-8 GLVKPSQTLSLTGISGDSVSS SVAWNWIRQSPGLEWLGRTYYRSKWYNA S 25 GNX-10 RIT NPDTSKNQFSLHLNSVTPEDTAVYYCARND GQGTLVTVSSASTKGP GNX-8 RIT NDTSKNQFSLHLNSVTPEDTAVYYCARND GQGTLVTVSSASTKGP 30 61845511 (GHMatters) P86521.AU.1 KAROLA 114 [00398] Alignment of light chain variable region (mAb GNX-8 vs. mAb GNX 10) GNX-10 LTI TVILTCRSSTGAvTr1NYANWVQEKPDHLFTGLIDATSNRVPG 5 GNX-8 LTIcGTVILTCRSSTGAVTINWANWVQEKPDHLFTGLIDATSNRVPG GNX-10 VPVRFSGSLIGDKAALTITGAQTEDDAMYFCALWYNTHFVFGGGTKVTVL GNX-8 VPVRFSGSLIGDKAALTITGAQTEDDAMYFCALWYNTHFVFGGGTKVTVL 10 2. Cloning and expression of cDNA encoding mAb GNX-10 in FreeStyle 293F cell [00399] The heavy and light chain variable region cDNAs of mAb GNX-10 were 15 cloned using SMART RACE cDNA amplification kit (Clontech), in which primers GNX8hVH5Eco (5'-cgaattcaccatggctgtctccttcctc-3')(SEQ ID NO: 38) and hh-3 (5' gtgcacgccgctggtcagggcgcctg-3')(SEQ ID NO: 39) were used to amplify the heavy chain variable region cDNA, and primers GNX8mvlSEco (5' gcgaattcaccatggcctggacttcac-3')(SEQ ID NO: 40) and GNX8mvlASBwi (5' 20 gccgtacgtaggacagtgaccttggtta-3')(SEQ ID NO: 41) were used to amplify the light chain variable region cDNA. [00400] The heavy and light chain variable regions cDNAs thus obtained were inserted to heavy and light chain expression vectors, respectively. The expression vectors were derived from GNX8 expression vectors pCI-GNX-8.H3 and pCIck 25 GNX-8.mk. Both expression vectors were co-transfected into FreeStyle 293F cells (Invitrogen) to transiently express recombinant GNX-10 (rGNX-10) antibody. Dual vector of GNX8 pCI-GNX-8.H3 was included as a control. Culture supernatants were harvested and directly used for characterization or purified by Protein A. Antibody concentrations were determined by human IgG-specific ELISA. 6184551_1 (GHMatters) P86521.AU.1 KAROLA 115 [00401] Since the sequences of mAb GNX-8 and mAb GNX-10 are very similar, the applicants further generated 2 cross-matched antibodies (i.e., hybrid antibody "GNX8H/GNX1OL" constituted of GNX-8 heavy chain and GNX-10 light chain, and hybrid antibody "GNX1OH/GNX8L" constituted of GNX-10 heavy chain and 5 GNX-8 light chain) and found that these two hybrid antibodies exhibited binding specificity substantially to that of mAbs GNX-8 and GNX-10 (data not shown). [00402] 3. Comparison of mAbs GNX-8 and GNX-10 [00403] A. TLC immunostaining [00404] Colo205 neutral glycosphigolipids (2 ptL/sample) were spotted on 10 HPTLC plate (Merck, 1.05642, silica gel 60 F254), and developed with a mobile phase containing chloroform: methanol: water with a ratio of 50:40:10 (V: V: V). For glycan staining of glycolipids, 0.2% orcinol (Sigma, 0-1875) in 10% H 2 SO4 were sprayed onto a HPTLC plate and incubated for 10 minutes at 1 10C in oven. For immunostaining, the HPTLC plate was first fixed with 0.5% poly(isobutyl 15 methacrylate)(Aldrich, 181544) in chloroform: hexane (1:9, V:V) for 45 seconds, followed by blocking for 10minutes in 3% BSA/PBS. The plates were then washed with PBS and incubated with the primary antibody at room temperature for lhr, followed by HRP-labeled secondary antibody at room temperature for lhr. Immunostaining HRP-1000 kit (Konica Minolta, #130990) was used for color 20 development according to manufacturer's protocol. [00405] Referring to Figure 1, mAbs GNX-8 and GNX-10 bind to substantially the same epitope band. [00406] B. Antigen specific ELISA [00407] BI) Using Colo205 neutral GSLs as antigen 25 [00408] 96-well ELISA plates (Costar, Corning, NY) coated with 1 [tg/well of Colo205 neutral glycosphigolipids (GSLs). GSLs were dissolved in ethanol and directly dried on wells. Wells were blocked with 1% bovine serum. Diluted mAbs GNX-8 and GNX-10 were added into wells (100 [pL/well) in duplicate, incubated at 37'C for 30 min. Antibodies bound to the coated GSLs were detected by mouse anti 30 human IgG (Fc)-HRP (Southern Biotech, #9040-05) at 1:5000 dilution (100 6184551_1 (GHMatters) P86521.AU.1 KAROLA 116 [tL/well), incubated at 37 0 C for 30 min, followed by color development with TMB (KPL, Cat# 53-00-03). The reaction was stopped by 0.5N HCl and was read by ELISA reader at 450 nm. [00409] It can be seen from Figure 2 that the binding behavior of mAb GNX-10 5 to Colo205 neutral glycosphigolipids is closely similar to that of mAb GNX-8. [00410] B2) Using biotinylated Leb-Lea as antigen [00411] 96-well ELISA plates were sequentially coated with 0.3 [tg/well ImmunoPure Streptavidin (Thermo scientific, Cat.# 21125) and biotinylated Leb Lea-Lac prepared by the applicants' lab. Wells were blocked with 0.1% skim milk. 10 MAbs GNX-8 and GNX-10 were added into wells (100 [pL/well) in duplicate and incubated at 37 0 C for 30 min, followed by incubation with mouse anti-human IgG (Fc)-HRP (Southern Biotech, #9040-05) at 1:5000 dilution (100 [pL/well) for 30 min. Color development with TMB (KPL, Cat# 53-00-03) was stopped by 0.5N HCl and read by ELISA reader at 450 nm. 15 [00412] Like Figure 2, the binding behavior of mAb GNX-10 to Le -Lea-Lac as shown in Figure 3 is closely similar to that of mAb GNX-8. [00413] B3) ADCC assay [00414] The ADCC assay was performed substantially according to the procedures described in Example 4, in which Colo205 cells were used as target cells, and 20 human peripheral blood mononuclear cells (PBMC) as effector cells. Blood donors were selected from healthy volunteers of GlycoNex's employees. [00415] It can be seen from Figure 4 that the ADCC activity of mAb GNX-10 to Colo205 cells is similar to that of mAb GNX-8. [00416] B4) CDC assay 25 [00417] The CDC assay was performed substantially according to the procedures described in Example 4, in which Colo205 cells were used as target cells, and cells treated with normal human IgGs purified from normal human serum served as a negative control. [00418] It can be seen from Figure 5 that the CDC activity of mAb GNX-10 to 30 Colo205 cells is similar to that of mAb GNX-8. 6184551_1 (GHMatters) P86521.AU.1 KAROLA 117 [00419] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. 6184551_1 (GHMatters) P86521.AU.1 KAROLA 118

Claims (21)

1. An isolated binding moiety which specifically binds to an epitope comprising an extended Type I chain comprising Leb, wherein the binding moiety comprises: 5 (a) a heavy chain variable region (VH) that includes: (i) a VH CDR1 sequence: Gly-Asp-Ser-Val-Ser-Ser-X 1 ,-X 2 -Val Ala (SEQ ID NO:42), wherein X 1 is selected from the group consisting of Asn and Lys, and X 2 is selected from the group consisting of Thr and Ser; (ii) a VH CDR2 sequence: Thr-Tyr-Tyr-Arg-Ser-Lys-Trp-Tyr 10 Asn (SEQ ID NO:32); and (iii) a VH CDR3 sequence: Ala-Arg-Asn-Phe-Asp-X 3 (SEQ ID NO:43), wherein X 3 is selected from the group consisting of Ser and Tyr; and (b) a light chain variable region (VL) that includes: (i) a VL CDR1 sequence: Thr-Gly-Ala-Val-Thr-Thr-X 4 -Asn-Tyr 15 (SEQ ID NO:44), wherein X 4 is Asp; (ii) a VL CDR2 sequence: Ala-Thr-Ser (SEQ ID NO:36); and (iii) a VL CDR3 sequence: Ala-Leu-Trp-Tyr-Asn-Thr-His-Phe-Val (SEQ ID NO:37); wherein the binding moiety is an antibody or an antigen-binding fragment thereof. 20

2. An isolated binding moiety according to claim 1, wherein the epitope comprises an extended Type I chain Leb-Le.

3. An isolated binding moiety according to claim 1 or 2, wherein the epitope is 25 expressed on a cancer cell of primary and metastatic colorectal cancers, breast cancers, pancreas cancers, lung cancers, stomach cancers, oesophagus cancers, and prostate cancers.

4. An isolated binding moiety according to claim 3, wherein the cancer cell is 30 an epithelial cell. 6184551_1 (GHMatters) P86521.AU.1 KAROLA 119

5. An isolated binding moiety according to any one of claims I to 4, wherein the binding moiety binds to glycosphigolipids, glycoproteins or glycans present on or isolated from cancer cells expressing the epitope. 5

6. An isolated binding moiety according to any one of claims 1 to 5, wherein the binding moiety is an antibody or an antigen-binding fragment thereof, said antibody being selected from the group consisting of polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies. 10

7. An isolated binding moiety according to claim 6, wherein the binding moiety is an antigen-binding fragment of the antibody, said fragment being selected from the group consisting of Fab fragments, Fab' fragments, F(ab')2 fragments, F' fragments, disulfide-linked Fv (dsFv) fragments, single domain antibodies, and 15 single chain Fv (scFv) molecules.

8. An isolated binding moiety according to claim 7, wherein the binding moiety is a scFv molecule. 20

9. An isolated binding moiety according to any one of claims 1 to 8, wherein the binding moiety comprises a VH CDR1 sequence of SEQ ID NO:3 1, a VH CDR2 sequence of SEQ ID NO:32, a VH CDR3 sequence of SEQ ID NO:33, a VL CDR1 sequence of SEQ ID NO:35, a VL CDR2 sequence of SEQ ID NO:36 and a VL CDR3 sequence of SEQ ID NO:37. 25

10. An isolated binding moiety according to any one of claims I to 8, wherein the binding moiety comprises a VH CDR1 sequence of SEQ ID NO: 18, a VH CDR2 sequence of SEQ ID NO:20, a VH CDR3 sequence of SEQ ID NO:22, a VL CDR1 sequence of SEQ ID NO:35, a VL CDR2 sequence of SEQ ID NO:36 and a VL 30 CDR3 sequence of SEQ ID NO:37.

11. An isolated binding moiety according to any one of claims 1 to 8, wherein the binding moiety comprises a heavy chain variable region of SEQ ID NO:30 and a light chain variable region of SEQ ID NO:34. 6635744_1 (GHMatters) P86521.AU.1 KAROLA 120

12. An isolated binding moiety according to any one of claims I to 8, wherein the binding moiety comprises a heavy chain variable region of SEQ ID NO: 15 and a light chain variable region of SEQ ID NO:34. 5

13. A pharmaceutical or diagnostic composition comprising an isolated binding moiety according to any one of claims I to 12.

14. A pharmaceutical or diagnostic composition according to claim 13, wherein 10 the binding moiety is labelled.

15. A pharmaceutical or diagnostic composition according to claim 13 or 14 and a pharmacologically active agent. 15

16. A pharmaceutical or diagnostic composition according to any one of claims 13 to 15 and a detectable moiety.

17. A pharmaceutical or diagnostic composition according to any one of claims 13 to 16 and a pharmaceutically acceptable carrier, excipient or diluent. 20

18. A method of treating diseases or disorders associated with extended Type 1 glycosphingolipid function, metabolism or expression comprising administering to a subject in need thereof an isolated binding moiety according to any one of claims 1 to 12 or a pharmaceutical or diagnostic composition according to any one of claims 25 13 to 17.

19. Use of an isolated binding moiety according to any one of claims I to 12 or a pharmaceutical or diagnostic according to any one of claims 13 to 17 in the manufacture of a medicament for treating or diagnosing diseases or disorders 30 associated with extended Type 1 glycosphingolipid function, metabolism or expression. 7215902_1 (GHMatters) P86521.AU.1 KAROLA 121

20. A method according to claim 18 or use according to claim 19 in which the disease or disorder is cancer. 5

21. A method or use according to claim 20 in which the disease or disorder is colon, rectal, esophageal, lung, prostate, breast, pancreatic, oral or vaginal cancer, or cancer of the gastrointestinal or urinary tract. 6635744_1 (GHMatters) P86521.AU.1 KAROLA 122