BR122020012421B1 - ISOLATED POLYNUCLEOTIDES, ISOLATED ANTIBODY OR FUNCTIONAL FRAGMENT THEREOF THAT BINDS TO SIALYL-LEWISA, USES THEREOF, CONJUGATION AND COMPOSITIONS - Google Patents

Abstract

The present invention provides compositions for producing an antibody or functional fragment thereof directed against Sialyl-Lewisa (sLea). The compositions of the invention include polynucleotides encoding a heavy chain and/or light chain variable domain that binds to sLea. The invention also provides an isolated antibody or functional fragment thereof and methods of treating or preventing a disease, such as cancer or tumor formation, wherein the antibody or functional fragment includes a variable heavy chain domain and a variable light chain domain. which have an amino acid sequence provided in it. The invention also provides a conjugate of an antibody or functional fragment thereof conjugated or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent, and methods of treating, preventing or diagnosing a disease in an individual in need thereof.

Description

[001] This application claims the priority benefit of United States Provisional Application No. 61/870,137, filed August 26, 2013, the entirety of which is incorporated herein by reference.

[002] This invention was made with government support under grant number CA-128362 awarded by the National Cancer Institute, NIH. The government has certain rights in invention.

BACKGROUND OF THE INVENTION

[003] The present invention generally relates to antibodies directed against Sialyl-Lewisa (sLea), and more specifically to polynucleotides encoding anti-sLea antibodies and the corresponding encoded antibodies or fragments thereof.

[004] Passive administration of antibodies directed against specific tumor antigens can eliminate tumor cells and early metastasis during cancer development. This treatment may also have a significant impact on cancer recurrence. Antibodies directed against tumor-specific carbohydrates may be useful candidates in this cancer treatment. For example, many tumor-restricted monoclonal antibodies resulting from immunization of mice with human cancer cells have been shown to be directed against carbohydrate antigens expressed on the cell surface as glycolipids or glycoproteins. The carbohydrate sLea has been shown to be expressed in tumors of the gastrointestinal tract. Expression of sLea has also been shown to impact metastatic potential and correlates with increased metastatic potential in human colon cancer and pancreatic adenocarcinoma. However, carbohydrate chemistry has been quite challenging and the clinical development of antibodies that recognize such tumor-specific carbohydrates has been slow.

[005] Pancreatic carcinoma is one of the most aggressive adenocarcinomas and is often associated with a poor prognosis. Pancreatic carcinoma is classified as the fourth leading cause of cancer mortality. Despite advances in the evaluation of different carcinomas, the reliability of detecting malignant lesions arising from the pancreas remains poor. Positron emission tomography using fluorodeoxyglucase (FDG-PET) has been indicated for the detection and staging of pancreatic cancer. However, FDG-PET is insensitive to differentiating pancreatitis from malignancy and remains problematic in staging small primary lesions (<7 mm) and liver metastases (<1 cm). One diagnostic assessment method used to monitor the status of pancreatic ductal adenocarcinoma (PDAC) patients includes detecting elevated levels of circulating sLea antigen in sera. Patients with > 37 U/ml of circulating sLea antigen indicate cancer recurrence. However, the development of alternative diagnostic tools that utilize such tumor-specific carbohydrates has been slow.

[006] Therefore, in this context there is a need to identify and generate antibodies that specifically recognize tumor-specific carbohydrates, such as sLea, for the treatment of recurrent cancers and for detection of malignant lesions and metastases. This invention satisfies this need and provides related advantages.

SUMMARY OF THE INVENTION

[007] According to the present invention, compositions are provided here for the production of antibodies or functional fragments thereof, which bind to sLea. The compositions include an isolated polynucleotide encoding an antibody or a functional fragment thereof, wherein the antibody includes a variable heavy chain (VH) domain having an amino acid sequence provided herein. The isolated polynucleotide of the invention may also include a nucleic acid sequence provided herein, wherein the nucleic acid sequence encodes the VH domain of the antibody or functional fragment thereof.

[008] In another embodiment of the invention, the isolated polynucleotide may encode an antibody or a functional fragment thereof, wherein the antibody includes a variable light chain (VL) domain that has an amino acid sequence provided herein. The isolated polynucleotide of the invention may also include a nucleic acid sequence provided herein, wherein the nucleic acid sequence encodes the VL domain of the antibody or functional fragment thereof.

[009] The compositions of the invention also include an isolated antibody or functional fragment thereof, in which the antibody binds to sLea. In some embodiments, the invention provides an isolated antibody or functional fragment thereof that binds to sLea, wherein the antibody or functional fragment thereof includes a VH domain having an amino acid sequence provided herein.

[0010] In some embodiments, the invention provides an isolated antibody or functional fragment thereof that binds to sLea, wherein the antibody or functional fragment thereof includes a VL domain having an amino acid sequence provided herein.

[0011] In some embodiments, the invention provides an isolated antibody or functional fragment thereof that binds to sLea, wherein the antibody or functional fragment thereof includes both a VH domain and a VL domain, where the VH domain and the VL respectively include an amino acid sequence for the respective VH and VL domains of the clonal isolates provided here.

[0012] In some embodiments, the invention provides a conjugate having an antibody or functional fragment provided herein that is conjugated or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent. In some aspects of the invention, a conjugate of the invention that includes a detectable agent can be used in a method for detecting and/or diagnosing tumor formation is an object. Such methods may include administering an effective amount of the conjugate to an individual in need thereof.

[0013] In some embodiments, the invention provides pharmaceutical compositions having one or more antibodies or functional fragment of the invention and a pharmaceutically acceptable carrier. In some aspects, the invention also provides a method for treating or preventing a disease in an individual in need thereof by administering a therapeutically effective amount of a pharmaceutical composition of the invention. In yet another aspect, the invention provides for the administration of a second therapeutic agent concomitantly or successively with an antibody or functional fragment of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 shows the nucleotide sequence and the encoded amino acid sequence of the variable heavy chain (VH) domain of clone 5B1 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified.

[0015] FIG. 2 shows the nucleotide sequence and the encoded amino acid sequence of the variable light chain (VL) domain of clone 5B1 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified.

[0016] FIG. 3 shows the nucleotide sequence and the encoded amino acid sequence of the variable heavy chain (VH) domain of clone 9H3 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 5 and the amino acid sequence of SEQ ID NO: 6. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified.

[0017] FIG. 4 shows the nucleotide sequence and the encoded amino acid sequence of the variable light chain (VL) domain of clone 9H3 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 8. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified.

[0018] FIG. 5 shows the nucleotide sequence and the encoded amino acid sequence of the variable heavy chain (VH) domain of clone 5H11 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified.

[0019] FIG. 6 shows the nucleotide sequence and the encoded amino acid sequence of the variable light chain (VL) domain of clone 5H11 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 12. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified.

[0020] FIG. 7 shows the nucleotide sequence and the encoded amino acid sequence of the variable heavy chain (VH) domain of clone 7E3 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 14. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified.

[0021] FIG. 8 shows the nucleotide sequence and the encoded amino acid sequence of the variable light chain (VL) domain of clone 7E3 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified.

[0022] FIG. 9 shows the nucleotide sequence and the encoded amino acid sequence of a diabody designated 5B1CysDb having CDR1, CDR2 and CDR2 of both the variable heavy (VH) and variable light (VL) chain domains of clone 5B1. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO: 18. The three complementarity determining regions (CDR1, CDR2 and CDR3) for both VH domains and VL are identified in bold and underlined text. The linker sequence and polyhistidine tag (Poly His-Tag) with added amino acids are also indicated by italicized and underlined text.

[0023] FIG. 10 shows the nucleotide sequence and the encoded amino acid sequence of a diabody designated 7E3CysDb having CDR1, CDR2 and CDR2 of both the variable heavy (VH) and variable light (VL) chain domains of clone 7E3. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO: 20. The three complementarity determining regions (CDR1, CDR2 and CDR3) for both VH domains and VL are identified in bold and underlined text. The linker sequence and polyhistidine tag (Poly His-Tag) with added amino acids are also indicated by italicized and underlined text.

[0024] FIG. 11, panels A-E, shows the binding of anti-human sLea antibodies to tumor cells analyzed by flow cytometry. Panel A shows DMS-79 cells stained with recombinant antibodies (r) 5B1, 9H3, 5H11, and 7E3. Panels B-F respectively show HT29, BxPC3, SW626, SK-MEL28, and Colo205-luc cells stained with 1 to 2 µg/mL of r5B1 or r7E3 plus IgG or IgM-specific secondary antibody as described in Example I.

[0025] FIG. 12, panels A and B, shows CDC activity of r5B1 and r7E3 antibodies compared to murine 121SLE (IgM) in the presence of human complement (Hu C') when measured against DMS-79 cells. Human isotype control antibodies, Hu IgG (?) and Hu IgM (?) showed <4% cytotoxicity. A dose response for r5B1 IgG (¦), r7E3 IgM (•), and 121SLE mIgM (?) antibodies is shown in panel A. The calculated IC50 (µg/ml) for r5B1 (IgG), r7E3 (IgM), and 121SLE antibodies (mIgM) is shown in panel B.

[0026] FIG. 13, panels A-C, shows antibody-dependent cell-mediated cytotoxicity (ADCC) of r5B1 antibodies. Panel A shows r5B1-mediated ADCC with human PBMC against DMS-79 cells. PBMC were tested at E:T ratios of 100:1 to 12.5:1 with DMS-79 tumor cells in the presence or absence of 2 µg/mL r5B1. Panel B shows r5B1-mediated ADCC with primary human NK cells against DMS-79 cells. NK cells were tested at lower E:T ratios of 5:1 to 0.6:1 with DMS-79 tumor cells in the presence or absence of 2 µg/mL r5B1. Panel C shows ADCC of r5B1 at various concentrations with PBMCs from 2 donors at an E:T ratio of 1:100 with DMS-79 tumor cells in the presence of the indicated concentrations of r5B1.

[0027] FIG. 14 shows the internalization of sLea in BxPC3 cells. BxPC3 pancreatic tumor cells were cultured in the presence of r5B1 (anti-sLea) or r1B7 (anti-GD2) antibodies complexed with Hum-ZAP, an anti-human IgG conjugated to saporin. After 3 days, cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and sample values were normalized to culture values. untreated.

[0028] FIG. 15 shows r5B1 antibody activity in a xenograft model using Colo205-luc cells. Severe combined immunodeficient (SCID) mice (5 per group) received 0.5 million Colo205-luc cells by tail vein injection on day 0. Mice received 100 µg of r5B1 by intraperitoneal injection on days 1, 7, 14, and 21 (experiment 1, Exp1) or on days 1, 4, 7, 10, 14, and 21 (experiment 2, Exp2) for a total dose of 600 µg. Control animals (Ctrl) received mock injections of PBS.

[0029] FIG. 16 shows the effect of r5B1 on Colo205-luc tumors in SCID mice. Mice received 100 µg (?), 300 µg (¦) or 1 mg (?) r5B1 antibody per injection as described in example I. Control animals (¦) received mock injections of PBS.

[0030] FIG. 17 shows fluorescence imaging of five mice per group for r5B1-treated mice having Colo205-luc tumors on Day 0 and Week 5. The mice received the treatment regimen depicted in FIG. 16 and described in example I.

[0031] FIG. 18, panels A and B, shows antitumor activity in a therapeutic subcutaneous xenograft model using DMS-79 cells. Panel A shows suppression or regression of mice treated with 5B1 (5B1 alone (?) or 5B1 + cRGD (?) ) compared to human IgG (IgG alone (?) or IgG + cRGD (•)) and control injected with PBS (¦). Arrows indicate days of PBS or antibody injections. Panel B shows representative images from treated mice. Arrows indicate the absence of any visual tumor.

[0032] FIG. 19, panels A-F, shows binding of 5B1 to various tumor types. Panel A is a pancreas, ductal adenocarcinoma, stage III tumor. Panel B is a sigmoid colon, carcinoma stage IIIB tumor. Panel C is a lung, adenocarcinoma, stage IB tumor. Panel D is a urinary bladder, mucinous adenocarcinoma, stage IV tumor. Panel E is an ovarian, colon tumor metastatic carcinoma. Panel F is a lymph node, metastatic carcinoma, stage IIIA tumor.

[0033] FIG. 20 shows serial PET maximum intensity projection (MIP) images acquired from 2-120 h with 89Zr radiolabeled 5B1 antibody (89Zr-5B1) intravenously administered to female SCID mice subcutaneously implanted with BxPC3 pancreatic tumors. PET-MIP imaging demonstrates high tumor uptake with clearance of non-specifically bound tracer as early as 24 hours post injection (h p.i.)

[0034] FIG. 21 shows biodistribution results that are in agreement with the PET data of FIG. 20, with an observed tumor uptake of 84.73 ± 12.28% ID/g. Because of the small tumor weights, a plot of tumor uptake expressed as % ID versus time is shown by the inset graph. Tumor ID % shows significant tumor uptake by 89Zr-5B1 at all time points, and is at least seven times greater than non-specific 89Zr-IgG. Competitive inhibition with cold 5B1 (200 µg) shows a decrease in tumor accumulation.

[0035] FIG. 22, panels A-C, shows PET-MIP images of mice carrying DMS79 (Panel A) and Colo205-luc xenografts (Panel B). PET-MIP imaging delineation of tumor (T), heart (H), and liver (L) by 89Zr-5B1 is indicated. Colo205-luc model colorectal grafts show 89Zr-5B1 accumulation reaching a maximum at 24 hours, which eventually decreases, while an increase in nonspecific binding to the liver was displayed (Panel C).

[0036] FIG. 23 shows a dose-dependent inhibition and regression of tumor growth in a DMS-79 small cell lung carcinoma xenograft model treated with successive co-administration of 5B1 antibody and Taxol (Paclitaxel). Large arrows on the X axis indicate 5B1 treatment. Co-administration of 5B1 antibody and Taxol significantly limited tumor growth and resulted in tumor regression compared to control human IgG (HuIgG) or 5B1 antibody and Taxol administered individually. Significant differences from control by 2-way ANOVA at p<0.01 (**) and p<0.001 (***) are indicated. N=5.

[0037] FIG. 24 shows inhibition of tumor growth in a BxPc3 pancreatic carcinoma xenograft model treated with successive co-administration of antibody 5B1 and Taxol (Paclitaxel). Large arrows on the X axis indicate treatment with Taxol plus 5B1, whereas small arrows indicate treatment with 5B1 alone. Co-administration of 5B1 antibody and Taxol significantly limited tumor growth compared to controls (PBS - Ctrl; human IgG - HuIgG) or 5B1 antibody and Taxol administered individually.

[0038] FIG. 25, panels A and B, show representative images of mice that were orthotopically transplanted with BxPC3-luc pancreatic tumor xenografts. Panel A: Co-registration of FDG-PET and computed tomography (CT) (left) and flat sections of FDG-PET alone (right) showed minimal tumor detection of the tracer with a high uptake in highly metabolic tissues (i.e. heart, H and bladder, B). Panel B: PET image of 89Zr radiolabeled 5B1 antibody acquired (89Zr-5B1) from the same mouse co-registered with CT exhibited exceptional tumor detection from BxPC3-luc tumor xenografts.

DETAILED DESCRIPTION OF THE INVENTION

[0039] Carbohydrates expressed on the surface of tumor cells can be targets for passive immunotherapy. The compositions provided herein are based, at least in part, on the identification and characterization of human antibodies that were generated from blood lymphocytes of individuals immunized with a Sialyl-Lewisa-keyhole limpet hemocyanin (sLea-KLH) conjugate vaccine. At least four antibodies with high affinity for sLea (5B1, 9H3, 5H11 and 7E3) have been identified. Two of these antibodies were expressed as recombinant antibodies (r5B1 and r7E3) and also characterized in in vitro and in vivo models. Both antibodies were potent in complement-dependent cytotoxicity (CDC) assays, and the 5B1 antibody was also highly active in antibody-dependent cytotoxicity assays. The in vivo efficacy of the antibodies were tested using Colo205 tumor cells or DMS-79 tumor cells engrafted into severe combined immunodeficient (SCID) mice. The translational relevance of the invention provided here is 2-fold: First, the method provided here demonstrates that the antibody response elicited by a sLea-KLH vaccine is useful as a vaccine itself. Second, the most potent antibodies that were generated in a clinical trial can be preserved and ultimately used as therapeutic products, or in the generation of therapeutic products, for a target cancer population. The high affinity of the antibodies provided here and their high effector functions support this translational potential.

[0040] As used herein, the term "antibody" is intended to mean a B cell polypeptide product within the immunoglobulin class of polypeptides that is capable of binding to a specific molecular antigen and is composed of two identical pairs of chains of polypeptide, wherein each pair has a heavy chain (about 50-70 kDa) and a light chain (about 25 kDa) and each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids and each carboxy-terminal portion of each chain includes a constant region (see Borrebaeck (ed.) (1995) Antibody Engineering, Second Edition, Oxford University Press.; Kuby (1997) Immunology, Third Edition, W.H. Freeman and Company , New York). In the context of the present invention, the specific molecular antigen that can be bound by an antibody of the invention includes the target carbohydrate sLea.

[0041] The term "human" when used in reference to an antibody or a functional fragment thereof refers to an antibody or functional fragment thereof that has a human variable region and/or a human constant region or a portion thereof corresponding to human germline immunoglobulin sequences. Such human germline immunoglobulin sequences are described by Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242. A human antibody, in the context of the present invention, may include an antibody that binds to sLeae and is encoded by a nucleic acid sequence that is a naturally occurring somatic variant of the human germline immunoglobulin nucleic acid sequence. Exemplary methods of producing human antibodies are provided in Example I, but any method well known to those skilled in the art can be used.

[0042] The term "monoclonal antibody" refers to an antibody that is the product of a single-cell hybridoma or clone or a population of cells derived from a single cell. A monoclonal antibody is also intended to refer to an antibody produced by recombinant methods of immunoglobulin genes encoding heavy and light chain to produce a single molecular immunoglobulin species. The amino acid sequence for the antibodies within a monoclonal antibody preparation is substantially homogeneous and the antibody binding activity within such a preparation exhibits substantially the same antigen binding activity. In contrast, polyclonal antibodies are obtained from different B cells within a population, which are a combination of immunoglobulin molecules that bind to a specific antigen. Each polyclonal antibody immunoglobulin can bind to a different epitope of the same antigen. Methods for producing both monoclonal antibodies and polyclonal antibodies are well known in the art (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) and Borrebaeck (ed.), Antibody Engineering: A Practical Guide, W.H. Freeman and Co., Publishers, New York, pp. 103-120 (1991)).

[0043] As used herein, the term "functional fragment" when used in reference to an antibody is intended to refer to a portion of the antibody including light or heavy chain polypeptides that retain some or all of the binding activities as the antibody of the which the fragment was derived from. Such functional fragments may include, for example, Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single-chain Fv (scFv), diabody, triabody, tetrabody and minibody. Other functional fragments may include, for example, light or heavy chain polypeptides, variable region polypeptides or CDR polypeptides or portions thereof, provided that such functional fragments maintain binding activity. Such antibody binding fragments can be found, described in, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publisher, Inc.; Huston et al., Cell Biophysics, 22:189-224 (1993); Plückthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E.D., Advanced Immunochemistry, Second Edition, Wiley-Liss, Inc., New York, NY (1990).

[0044] The term "heavy chain" when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids and a carboxy-terminal portion that includes a constant region. The constant region can be one of five distinct types, referred to as alpha (a), delta (d), epsilon (e), gamma (Y), and mu (µ), based on an amino acid sequence of the chain constant region. heavy. The distinct heavy chains differ in size: a, d, and y contain approximately 450 amino acids, although µ and e contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well-known classes of antibodies, IgA, IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, named IgG1, IgG2, IgG3 and IgG4. A heavy chain may be a human heavy chain.

[0045] The term "light chain" when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids and a carboxy-terminal portion that includes a constant region. The approximate size of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as lambda (A) coat (K) based on a constant domain amino acid sequence. The amino acid light chain sequence is well known in the art. A light chain may be a human light chain.

[046] The term "variable domain" or "variable region" refers to a portion of the light or heavy chains of an antibody that is generally located at the amino terminus of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are employed in the binding and specificity of each particular antibody for its particular antigen. The variable domains differ extensively in sequence between different antibodies. Variability in the sequence is concentrated in the CDRs although the less variable portions in the variable domain are referred to as framework regions (FR). The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with the antigen. The numbering of the amino acid positions used here is in accordance with the EU index, as in Kabat et al. (1991) Sequences of proteins of immunological interest. (U.S. Department of Health and Human Services, Washington, D.C.) 5aed. A variable region may be a human variable region.

[0046] A CDR refers to one of the three hypervariable regions (H1, H2 or H3) within the non-structural region of the immunoglobulin (Ig or antibody) ß VH sheet structure, or one of the three hypervariable regions (L1, L2 or L3) within a non-structural region of the antibody VL ß sheet structure. Consequently, CDRs are variable region sequences interspersed within structural region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the variable (V) domains of the antibody (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, Adv. Prot. Chem. 32:1-75 (1978)). The sequences of the CDR region were also structurally defined by Chotia as those residues that are not part of the conserved ß-sheet structure, and thus are capable of adapting different conformations (Chotia and Lesk, J. Mol. Biol. 196:901- 917 (1987)). Both terminologies are well recognized in the art. The positions of CDRs within a canonical antibody variable domain were determined by comparing the number of structures (Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); Morea et al. ), Methods 20:267-279 (2000)). Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to canonical positions are conventionally numbered with a, b, c, and so on next to the residue number in the canonical variable domain numbering scheme (Al- Lazikani and others, supra (1997)). Such nomenclature is similarly well known to those skilled in the art.

[0047] For example, CDRs defined according to the designations Kabat (hypervariable) or Chotia (structural), are presented in Table 1 below. TABLE 1: CDR Definitions 1 Residue numbering follows the nomenclature of Kabat and others, above 2 Residue numbering follows the nomenclature of Chotia and others, above

[0048] One or more CDRs can also be incorporated into a molecule covalently or non-covalently to make it an immunoadhesin. An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. CDRs allow the immunoadhesin to bind to a particular antigen of interest.

[0049] As used herein, the term "isolated" when used in reference to an antibody, functional antibody fragment or polynucleotide is intended to mean that the referenced molecule is free of at least one component as it is found in nature. The term includes an antibody, functional antibody fragment, or polynucleotide that is removed from some or all of the other components as it is found in its natural environment. Components of a natural antibody environment include, for example, erythrocytes, leukocytes, thrombocytes, plasma, proteins, nucleic acids, salts and nutrients. The components of a functional antibody fragment or natural polynucleotide environment include, for example, lipid membranes, cellular organelles, proteins, nucleic acids, salts and nutrients. An antibody, functional antibody fragment or polynucleotide of the invention may also be free or in any form be substantially free of all of these components or any other component of the cells from which it is isolated or recombinantly produced.

[0050] As used herein, "Isotype" refers to a class of antibody that is encoded by heavy chain constant region genes. The heavy chains of a provided antibody or functional fragment determine the class of that antibody or functional fragment: IgM, IgG, IgA, IgD or IgE. Each class can have K or À light chains. The term "subclass" refers to the minor differences in the amino acid sequence of the heavy chains that differentiate the subclasses. In humans there are two subclasses of IgA (subclasses IgA1 and IgA2) and there are four subclasses of IgG (subclasses IgG1, IgG2, IgG3 and IgG4). Such classes and subclasses are well known to those skilled in the art.

[0051] The terms "alloy" or "ligand" as used here refer to an interaction between molecules to form a complex. The interactions may be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex may also include the bond of two or more molecules held together by covalent or noncovalent bonds, interactions, or forces. Binding of an antibody or functional fragment thereof can be detected using, for example, an enzyme-linked immunosorbent assay, a method provided in Example I, or any of a variety of methods that are well known to those skilled in the art.

[0052] The strength of the total non-covalent interactions between a single antigen binding site on an antibody or functional fragment and a single epitope of a target molecule, such as sLea, is the affinity of the antibody or functional fragment for that epitope. The relationship of the Association (k1) to the dissociation (k-1) of an antibody or functional fragment thereof to a monovalent antigen (k1/ k-1) is the constant association K, which is a measure of affinity. The value of K varies for different antibody or functional fragment and antigen complexes and depends on both k1 and k-1. The constant association K for an antibody or functional fragment of the invention can be determined using any method provided herein or any other method well known to those skilled in the art.

[0053] The affinity at a binding site does not always reflect the strength of the actual interaction between an antibody or functional fragment and an antigen. When complex antigens containing multiple recurrent antigenic determinants, such as a polyvalent sLea, come into contact with antibodies containing multiple binding sites, interaction of the antibody or functional fragment with the antigen at one site will increase the likelihood of a reaction at a second site. The strength of such multiple interactions between an antigen and multivalent antibody is called the avidity. The avidity of an antibody or functional fragment may be a better measure of its binding capacity than is the affinity of its individual binding sites. For example, high avidity can compensate for low affinity as is sometimes discovered by pentameric IgM antibodies, which may have a lower affinity than IgG, but the high avidity of IgM, resulting from its multivalency, allows it to bind to the antigen effectively.

[0054] The specificity of an antibody or functional fragment thereof refers to the ability of an individual antibody or functional fragment thereof to react with only one antigen. An antibody or functional fragment can be considered specific when it can distinguish differences in the primary, secondary, or tertiary structure of an antigen or isomeric forms of an antigen.

[0055] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, deoxyribonucleotides or ribonucleotides or analogues thereof. The sequence of a polynucleotide is made up of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the terms "nucleotide sequence" or "nucleic acid sequence" is the alphabetical representation of a polynucleotide. A polynucleotide may include a gene or gene fragment (e.g., a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, polynucleotides branches, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, primers and nucleic acid probes. Polynucleotide also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide equally encompasses the double-stranded form and each of the two complementary single-stranded forms known or predicted to prepare the double-stranded form. It is understood that the isolated polynucleotides and Nucleic acids described herein are directed to non-naturally occurring polynucleotides and Nucleic acids. Non-naturally occurring polynucleotides and nucleic acids may include, but are not limited to, cDNA and chemically synthesized molecules.

[0056] The term "encode" or grammatical equivalents thereof as used in reference to polynucleotides refers to a polynucleotide in its native state or when manipulated by methods well known to those skilled in the art that can be transcribed to produce mRNA, which is then translated into a polypeptide and/or a fragment thereof. The antisense strand is the complement of such a polynucleotide, and the coding sequence can be deduced from it.

[0057] The phrase "therapeutic agent" refers to any agent that can be used in the treatment, management or improvement of a disease associated with the expression of sLeae/or a symptom related thereto. In certain embodiments, a therapeutic agent refers to an antibody or functional fragment of the invention. In other embodiments, a therapeutic agent refers to an agent other than an antibody or functional fragment of the invention. A therapeutic agent may be an agent that is well known to be useful for, or has been or is currently being used for the treatment, management or amelioration of a disease associated with the expression of sLeae/or one or more symptoms related thereto.

[0058] The phrase "diagnostic agent" refers to a substance administered to an individual that helps in diagnosing a disease. Such substances can be used to reveal, pinpoint, and/or define the location of a disease causing process. In certain embodiments, a diagnostic agent includes a substance that is conjugated to an antibody or functional fragment of the invention, which when administered to an individual or contacted with a sample from an individual helps in diagnosing cancer or tumor formation.

[0059] The phrase "detectable agent" refers to a substance that can be used to verify the existence or presence of a desired molecule, such as an antibody or functional fragment of the invention, in a sample or individual. A detectable agent may be a substance that is capable of being visualized or a substance that is otherwise capable of being determined and/or measured (e.g., by quantification).

[0060] An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount may be administered in one or more administrations, applications or dosages. Such release is dependent on several variables including the period of time for which the individual dosage unit must be used, the bioavailability of the agent, the administration routine, etc.

[0061] The phrase "therapeutically effective amount" as used herein refers to the amount of a therapeutic agent (e.g., an antibody or functional fragment provided herein or any other therapeutic agent provided herein) that is sufficient to reduce and/or improve the severity and/or duration of a given disease and/or a symptom related to it. A therapeutically effective amount of a therapeutic agent may be an amount necessary to reduce or ameliorate the advancement or progression of a given disease, reduce or ameliorate the recurrence, development or onset of a given disease, and/or improve or enhance the prophylactic effect. or therapeutic of another therapy (e.g., a therapy other than administration of an antibody or functional fragment provided herein).

[0062] The compound "Sialyl-Lewisa" (sLea), also known as sialyl Lea, Sialyl-Lewis A, Sialylated Lewis A and CA 19.9, is a tetrasaccharide with a molecular formula of C31H52N2O23 and a molar mass of 820.74 g /mol. The structure of sLea may include Neu5Aca2-3Galß1-3(Fuca1-4)GlcNAcß and Neu5Gca2-3Galß1-3(Fuca1-4)GlcNAcß. sLea is widely expressed in tumors of the gastrointestinal tract and is employed as a tumor marker in pancreatic and colon cancer. sLea is also a known ligand for E-selection, also known as endothelial leukocyte adhesion molecule (ELAM).

[0063] In some embodiments, the present invention provides an isolated polynucleotide encoding an antibody heavy or light chain or a functional fragment thereof, wherein an antibody or functional fragment thereof generated using the antibody heavy or light chain binds to sLea. Accordingly, in some embodiments, the invention provides an isolated polynucleotide encoding an antibody or a functional fragment thereof, wherein the antibody includes a VH domain having an amino acid sequence selected from the group consisting of residues 20-142 of SEQ ID NO : 2, residues 20-142 of SEQ ID NO: 6, residues 20-142 of SEQ ID NO: 10, and residues 20-145 of SEQ ID NO: 14. The isolated polynucleotide of the invention may also include a nucleic acid sequence of residues 58-426 of SEQ ID NO: 1, residues 58-426 of SEQ ID NO: 5, residues 58-426 of SEQ ID NO: 9 or residues 58-435 of SEQ ID NO: 13, wherein the sequence of nucleic acid encodes the VH domain of the antibody or functional fragment thereof.

[0064] In another embodiment of the invention, the isolated polynucleotide may encode an antibody or a functional fragment thereof, wherein the antibody includes a VL domain having an amino acid sequence selected from the group consisting of residues 20-130 of SEQ ID NO: 4, residues 20-129 of SEQ ID NO: 8, residues 20-130 of SEQ ID NO: 12, and residues 23-130 of SEQ ID NO: 16. The isolated polynucleotide of the invention may also include an acid sequence nucleic acid residues 58-390 of SEQ ID NO: 3, residues 58-387 of SEQ ID NO: 7, residues 58-390 of SEQ ID NO: 11 or residues 67-390 of SEQ ID NO: 15, wherein the sequence of nucleic acid encodes the VL domain of the antibody or functional fragment thereof.

[0065] In another embodiment, the invention provides an isolated polynucleotide encoding an antibody light or heavy chain or a functional fragment thereof, wherein the antibody light or heavy chain or functional fragment thereof encoded by the polynucleotide of the invention has one or more of the complementarity determining regions (CDRs) described in FIGS. 1-8 or listed in Table 2. An antibody or functional fragment thereof that includes one or more of the CDRs can specifically bind to sLea as described herein. Specific binding to sLea may include specificity, affinity and/or avidity as provided in Example I for any antibodies provided herein. In another aspect, an antibody or functional fragment thereof encoded by the polynucleotides of the invention may include complement-dependent cytotoxicity (CDC) activity and/or antibody-dependent cell-mediated cytotoxicity (ADCC) activity of any of the clonal isolates. 5B1, 9H3, 5H11 or 7E3 described here. Methods for evaluating the specificity, affinity and/or avidity of an antibody or functional fragment thereof are well known in the art and exemplary methods are provided herein. TABLE 2: CDRs of Clonal Isolates

[0066] In some embodiments, the antibody or functional fragment thereof of the invention includes fewer than six CDRs. In some embodiments, the antibody or functional fragment thereof includes one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3. In specific embodiments, the antibody or functional fragment thereof includes one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 from Clonal Isolates 5B1, 9H3, 5H11 or 7E3 described here.

[0067] In some embodiments, the invention provides an isolated polynucleotide encoding an antibody or functional fragment thereof, wherein the antibody or functional fragment includes a variable heavy chain (VH) domain having the amino acid sequence of CDR1, CDR2 and CDR3 of clonal isolate 5B1, 9H3, 5H11 or 7E3. Such VH domains may include amino acid residues 55-62, 70-77 and 116-131 of SEQ ID NO: 2, or alternatively amino acid residues 45-52, 70-77 and 116-131 of SEQ ID NO: 6, or alternatively amino acid residues 4552, 70-77 and 116-131 of SEQ ID NO: 10, or alternatively amino acid residues 45-52, 70-77 and 116-134 of SEQ ID NO: 14. In another aspect, the nucleotide sequence encoding the CDR1, CDR2 and CDR3 of the VH domain may respectively include the nucleotide sequence of residues 133-156, 208-231 and 346-393 of SEQ ID NO: 1, or alternatively the nucleotide sequence of residues 133-156, 208-231 and 346-393 of SEQ ID NO: 5, or alternatively the nucleotide sequence of residues 133-156, 208-231 and 346-393 of SEQ ID NO: 9, or alternatively the sequence of nucleotide residues 133-156, 208-231, 346-402 of SEQ ID NO: 13.

[0068] In another embodiment, the invention provides an isolated polynucleotide encoding an antibody or functional fragment thereof, wherein the antibody includes a variable light chain (VL) domain having the amino acid sequence of CDR1, CDR2 and CDR3 of the clonal isolate 5B1, 9H3, 5H11 or 7E3. Such a VL domain may include amino acid residues 45-52, 70-72 and 109-120 of SEQ ID NO: 4, or alternatively amino acid residues 45-52, 70-72 and 109-119 of SEQ ID NO: 8 , or alternatively amino acid residues 45-52, 70-72 and 109-120 of SEQ ID NO: 12, or alternatively amino acid residues 49-53, 72-74 and 111-120 of SEQ ID NO: 16. In In another aspect, the nucleotide sequence encoding the CDR1, CDR2 and CDR3 of the VH domain may respectively include the nucleotide sequence of residues 133-156, 208-216 and 325-360 of SEQ ID NO: 3, or alternatively the nucleotide sequence of residues 133-156, 208-216 and 325-357 of SEQ ID NO: 7, or alternatively the nucleotide sequence of residues 134-156, 208-216 and 325-360 of SEQ ID NO: 11, or alternatively the sequence of residues 145-162, 214-222 and 331-360 of SEQ ID NO: 15

[0069] In another embodiment, the invention provides a variant of the polynucleotides provided here. A variant when used in reference to a polynucleotide includes a polynucleotide having one or more modified nucleotides, such as, but not limited to, a methylated nucleotide or a nucleotide analog. Additionally, a variant polynucleotide may include a polynucleotide that is interrupted by non-nucleotide components. Modifications to a polynucleotide can be provided before or after assembly of the polynucleotide using methods well known to those skilled in the art. For example, a polynucleotide can be modified after polymerization by conjugation with a tagging component using enzymatic or chemical techniques (e.g., as described in Gottfried and Weinhold, 2011, Biochem. Soc. Trans., 39(2):523- 628; Paredes et al., 2011, Methods, 54(2):251-259).

[0070] Polynucleotides can be obtained, and the nucleotide sequence of the polynucleotides determined by any method well known in the art. Since the amino acid sequence of the variable heavy and light chain domains of 5B1, 9H3, 5H11 and 7E3 are known (see, for example, SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14 and 16) , antibodies encoding nucleotide sequences and modified versions of these antibodies can be determined using methods well known in the art, i.e., nucleotide codons known to encode particular amino acids are assembled in such a way to generate a nucleic acid that encodes the antibody. Such an antibody-encoding polynucleotide can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, fragments, or variants thereof, annealing and ligating those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0071] A polynucleotide encoding an antibody or a functional fragment thereof of the invention can be generated using the nucleic acid sequence of the variable heavy and/or light chain domains of isolated 5B1, 9H3, 5H11 or 7E3 (e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 and 15). A nucleic acid encoding the antibody or functional fragment may be chemically synthesized or obtained from a suitable source (e.g., cDNA isolated from cells expressing the antibody or functional fragment thereof, such as hybridoma cells selected to express the antibody or functional fragment thereof). same) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific to the particular nucleic acid sequence. The amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0072] In some embodiments, the present invention provides an isolated antibody or functional fragment thereof, in which the antibody binds to sLea. Accordingly, in some aspects, the invention provides an isolated antibody or functional fragment thereof that binds to sLea, wherein the antibody or functional fragment thereof includes a VH domain having an amino acid sequence selected from the group consisting of residues 20- 142 of SEQ ID NO: 2, residues 20-142 of SEQ ID NO: 6, residues 20-142 of SEQ ID NO: 10, and residues 20-145 of SEQ ID NO: 14.

[0073] In some embodiments, the invention provides an isolated antibody or functional fragment thereof that binds to sLea, wherein the antibody or functional fragment thereof includes a VL domain having an amino acid sequence selected from the group consisting of residues 20 -130 of SEQ ID NO: 4, residues 20-129 of SEQ ID NO: 8, residues 20-130 of SEQ ID NO: 12, and residues 23-130 of SEQ ID NO: 16.

[0074] In some embodiments, the invention provides an isolated antibody or functional fragment thereof that binds to sLea, wherein the antibody or functional fragment thereof includes both a VH domain and a VL domain, where the VH domain and the VL respectively include an amino acid sequence selected from the group consisting of residues 20-142 of SEQ ID NO: 2 and residues 20-130 of SEQ ID NO: 4; residues 20-142 of SEQ ID NO: 6 and residues 20-129 of SEQ ID NO: 8; residues 20-142 of SEQ ID NO: 10 and residues 20-130 of SEQ ID NO: 12; and residues 20-145 of SEQ ID NO: 14 and residues 23-130 of SEQ ID NO: 16.

[0075] In some embodiments, in order to bind the sLea, the antibody or functional fragment thereof of the invention has one or more of the CDRs described in FIGS. 1-8 or listed in Table 2. An antibody or functional fragment thereof that includes one or more of the CDRs, in particular CDR3, can specifically bind to sLea as described herein. Specific binding to sLea may include specificity and affinity as provided in Example I for any of the antibodies provided herein. In some aspects, an antibody or functional fragment thereof of the invention may include the CDC activity and/or ADCC activity of any of the 5B1, 9H3, 5H11 or 7E3 clonal isolates described herein.

[0076] In some embodiments, the invention provides an isolated antibody or functional fragment thereof, wherein the antibody includes a VH chain domain having the amino acid sequence of CDR1, CDR2 and CDR3 of the clonal isolate 5B1, 9H3, 5H11 or 7E3 . Such VH domains may include amino acid residues 55-62, 70-77 and 116-131 of SEQ ID NO: 2, or alternatively amino acid residues 45-52, 70-77 and 116-131 of SEQ ID NO: 6 , or alternatively amino acid residues 45-52, 70-77 and 116-131 of SEQ ID NO: 10, or alternatively amino acid residues 4552, 70-77 and 116-134 of SEQ ID NO: 14.

[0077] In some embodiments, the invention provides an isolated antibody or functional fragment thereof, wherein the antibody includes a VL chain domain having the amino acid sequence of CDR1, CDR2 and CDR3 of the clonal isolate 5B1, 9H3, 5H11 or 7E3 . Such a VL domain may include amino acid residues 45-52, 70-72 and 109-120 of SEQ ID NO: 4, or alternatively amino acid residues 45-52, 70-72 and 109-119 of SEQ ID NO: 8 , or alternatively amino acid residues 45-52, 70-72 and 109-120 of SEQ ID NO: 12, or alternatively amino acid residues 49-53, 72-74 and 111-120 of SEQ ID NO: 16.

[0078] In some aspects of the invention, the isolated antibody or functional fragment thereof is a monoclonal antibody. In some aspects of the invention, the isolated antibody or functional fragment thereof provided herein is an IgG or IgM isotype. In another aspect of the invention, the antibody or functional fragment thereof is an IgG1 subclass antibody.

[0079] In some embodiments, the functional antibody fragment of the invention may be, but is not limited to, Fab, Fab', F(ab')2, Fabc, scFV, a diabody, a triabody, minibody or a single domain antibody (sdAB). In some aspects, the invention provides a diabody that includes the amino acid sequence of SEQ ID NO: 18 or 20. Such diabodies of the invention may be, in some aspects, encoded by a polynucleotide having the nucleic acid sequence of SEQ ID NO: 17 or 19. With respect to antibodies and functional fragments thereof, various forms, changes and modifications are well known in the art. The specific antibody fragments of this invention can include any of various antibody forms, changes and modifications. Examples of such various forms and terms as they are known in the art are presented below.

[0080] In some embodiments, the invention provides a method of producing an antibody or functional fragment thereof of the invention. The method of the invention may include introducing a polynucleotide of the invention into a host cell, culturing the host cell under conditions and for a period of time sufficient to produce the encoded heavy and/or light chain of an antibody or functional fragment thereof. invention, and purify the heavy and/or light chain of an antibody or functional fragment.

[0081] Recombinant expression of an antibody or functional fragment thereof of the invention that binds to the sLea antigen may include the construction of an expression vector containing a polynucleotide encoding the heavy and/or light chain of an antibody or functional fragment of the invention . Once a polynucleotide encoding an antibody or functional fragment thereof (preferably, but not necessarily, containing a heavy and/or light chain variable domain) of the invention has been obtained, the vector for producing the antibody or functional fragment can be produced. by recombinant DNA technology using techniques well known in the art. Methods for preparing a protein by expressing a polynucleotide containing an antibody or a functional fragment thereof encoding the nucleotide sequence are described herein.

[0082] Methods that are well known to those skilled in the art can be used to construct expression vectors containing the antibody or functional fragments thereof encoding appropriate transcriptional and translational control sequences and signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention thus provides replicable vectors including a nucleotide sequence encoding an antibody or functional fragment thereof of the invention operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, for example, International Publication Nos. WO 86/05807 and WO 89/01036; and United States Patent No. 5,122,464) and the variable domain of the antibody molecule. Antibody can be cloned into such a vector for expression of the entire light chain, entire heavy chain or both the entire light and heavy chains.

[0083] The expression vector can be transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody or functional fragment thereof of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody or functional fragment thereof of the invention operably linked to a heterologous promoter. In some embodiments for the expression of double-chain antibodies, vectors encoding equally heavy and light chains can be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

[0084] A variety of host expression vector systems can be used to express the antibody or functional fragments thereof of the invention (see, for example, United States Patent No. 5,807,715). Such host expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but they also represent cells that can then be transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule. of the invention in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the antibody coding sequences; plant cell systems infected with the recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with the recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the mammalian cell genome (e.g., metallothionein promoter) or mammalian viruses (e.g., the recent adenovirus promoter; the vaccinia virus 7.5K promoter). In some aspects, bacterial cells such as Escherichia coli, or eukaryotic cells, especially for the expression of the entire recombinant antibody, are employed for the expression of a recombinant antibody or functional fragment. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, in conjunction with a vector such as the human cytomegalovirus major intermediate early gene promoter element, is an effective expression system for antibodies (Foecking and others, 1986, Gene 45:101; and Cockett et al., 1990, Bio/Technology 8:2). In some embodiments, the antibodies or fragments thereof of the invention are produced in CHO cells. In one embodiment, the expression of nucleotide sequences encoding antibodies or functional fragments thereof of the invention that bind to sLea is regulated by a constitutive promoter, inducible promoter, or tissue-specific promoter.

[0085] In bacterial systems, several expression vectors can be advantageously selected depending on the intended use of the antibody molecule being expressed. For example, when a large quantity of such an antibody must be produced, to generate pharmaceutical compositions from an antibody molecule, vectors that direct the expression of high levels of fusion protein products that are easily purified may be desirable. Such vectors include, but are not limited to, E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791), wherein the antibody coding sequence can be individually ligated into the vector framed with the Z lac coding region so that a fusion protein is produced; the pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and similar(s). pGEX vectors can also be used to express external polypeptides such as glutathione 5-transferase (GST) fusion proteins. In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to include factor Xa or thrombin protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0086] In an insect system, the Autographa californica nuclear polyhedrosis virus (AcNPV) is employed as a vector to express external genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence or functional fragment can be individually cloned into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0087] In mammalian host cells, several viral-based expression systems can be used. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest can be linked to an adenovirus transcription/translation control complex, for example, the late promoter and the tripartite guide sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., E1 or E3 region) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, 1984, Proc Natl. Acad. Sci. USA 8 1:355-359). Specific initiation signals can also be used for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the complete insertion. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. Expression efficiency can be enhanced by including appropriate transcription enhancer elements, transcription terminators, etc. (see, for example, Bittner et al., 1987, Methods in Enzymol. 153:51-544).

[0088] Additionally, a host cell line can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific desired model. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the antibody or functional fragment. Different host cells have characteristic and specific mechanisms for the post-translational modification and processing of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the expressed foreign protein. At this end, eukaryotic host cells that possess the cellular machinery for the proper processing of primary transcription, glycosylation, and phosphorylation of the gene product can be employed. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells.

[0089] During long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the antibody or functional fragment of the invention can be designed. Instead of using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) ), and a selectable marker. After introducing the foreign DNA, the constructed cells can be allowed to grow for 1-2 days in an enriched medium, and are then switched to a selective medium. The selectable marker on recombinant plasmids confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that can be successively cloned and expanded into cell lines. This method can advantageously be used to construct cell lines that express the antibody molecule.

[0090] Various selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthineguanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes can be employed in tk, hgprt or aprt cells, respectively. Furthermore, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. U S A. 77( 6):3567-70; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); glutamine synthetase (GS), which is an enzyme responsible for the biosynthesis of glutamine using glutamate and ammonia (Bebbington et al., 1992, Biuotechnology 10:169); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926 -932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods well known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in chapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, which are incorporated herein by reference in their entirety.

[0091] Expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). When a marker in the vector system expressing an antibody or functional fragment thereof is amplifiable, increasing the level of inhibitor present in host cell culture will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, antibody production will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

[0092] The host cell can be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain-derived polypeptide and the second vector encoding a light chain-derived polypeptide. The two vectors can contain identical selectable markers that allow equal expression of heavy and light chain polypeptides. Alternatively, a single vector can be used that 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, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197 -2199). The coding sequences for the heavy and light chains may include cDNA or genomic DNA.

[0093] Additionally, polynucleotides encoding the heavy and/or light chains of the antibody or functional fragment of the invention can be subjected to codon optimization using techniques well known in the art to obtain optimized expression of an antibody or functional fragment of the invention in a host cell desired. For example, in a codon optimization method, a native codon is replaced by the most frequent codon from a group of reference genes, where the codon translation rate for each amino acid is designed to be high. Additional exemplary methods for generating codon-optimized polynucleotides for expression of a desired protein, which can be applied to heavy and/or light chains of the antibody or functional fragment of the invention, are described in Kanaya et al., Gene, 238:143- 155 (1999), Wang et al., Mol. Biol. Evol., 18(5):792-800 (2001), United States Patent 5,795,737, United States Publication 2008/0076161 and WO 2008/000632.

[0094] Since an antibody molecule of the invention has been produced by recombinant expression, it can be purified by any method known in the art for purifying an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Furthermore, antibodies or functional fragments of the present invention may be fused to heterologous polypeptide sequences provided herein or otherwise known in the art to facilitate purification. For example, an antibody or functional fragment of the invention can be purified recombinantly by adding a polyhistidine tag (His-tag), FLAG tag, hemagglutinin tag (HA-tag) or myc-tag among others that are commercially available and using methods purification methods well known to those skilled in the art.

[0095] A Fab fragment refers to a monovalent fragment consisting of the VL, VH, CL and CH1 domains; an F(ab')2 fragment is a bivalent fragment including two Fab fragments linked by a disulfide bridge in the hinge region; an Fd fragment consists of the VH and CH1 domains; an Fv fragment consists of the VL and VH domains of a single branch of an antibody; and a dAb fragment (Ward et al., Nature 341:544-546, (1989)) consists of a VH domain.

[0096] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to each other or they may be different. For example, a naturally occurring immunoglobulin has two identical binding sites, a single-chain antibody or antibody fragment has one binding site, while a "bispecific" or "bifunctional" antibody has two different binding sites.

[0097] A single-chain antibody (scFv) refers to an antibody in which a VL and a VH region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a polypeptide chain continuous in which the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird et al., Science 242:42326 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-83 (1988)). Diabodies refer to bivalent antibodies including two polypeptide chains, wherein each polypeptide chain includes VH and VL domains linked to a linker that is too short to permit pairing between two domains on the same chain, thereby allowing each domain to pair with a complementary domain on another polypeptide chain (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993), and Poljak et al., Structure 2:1121-23 (1994 )). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen-binding sites. Polypeptide chains having different sequences can be used to prepare a diabody with two different antigen binding sites. Similarly, tribodies and tetrabodies are antibodies including three and four polypeptide chains, respectively, and forming three and four antigen-binding sites, respectively, which may be the same or different.

[0098] The present invention also provides an antibody or functional fragment thereof derived from 5B1, 9H3, 5H11 and/or 7E3, wherein the antibody or functional fragment binds to sLea. Standard techniques well known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding an antibody or functional fragment thereof of the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis that results in amino acid substitutions. . In some aspects, the derivative includes fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original molecule.

[0099] In some embodiments, the invention provides an antibody or functional fragment thereof having modified forms of naturally occurring amino acids, conservative substitutions, non-naturally occurring amino acids, amino acid analogues and mimetics as long as such antibody or functional fragment retains functional activity as defined here. In one embodiment, the derivative has conservative amino acid substitutions that are made at one or more predicted non-essential amino acid residues. A conservative amino acid substitution is one in which the amino acid residue is replaced by an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and side chains aromatics (e.g. tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly throughout all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be evaluated for biological activity to identify mutants that retain activity. After mutagenesis, the encoded antibody or functional fragment thereof can be expressed and the activity of the antibody or functional fragment can be determined.

[00100] In some embodiments, the invention provides an antibody or functional fragment thereof having modified phycosylation, galactosylation and/or sialylation of an Fc fragment contained within an antibody or functional fragment of the invention. Such modifications of an Fc fragment can execute Fc receptor-mediated activity as described in Peipp et al., Blood, 112(6):2390-2399 (2008). For example, glycoconstructed therapeutic antibodies lacking core fucose residues of the Fc N-glycans exhibit strong ADCC at lower concentrations with much greater efficacy compared to fucosylated counterparts. Shields et al., J. Biol. Chem., 277(30):26733-40 (2002); Okazaki et al., J Mol Biol., 336:1239-1249 (2004); Natsume et al., J. Immunol. Methods., 306:93-103 (2005). Methods for modifying the fucosylation, galactosylation and/or sialylation of an antibody to a functional fragment thereof are well known in the art. For example, defucosylation methods can be grouped into three methodologies (1) conversion of the non-mammalian cell N-glycosylation reaction series into the ‘humanized’ non-fucosylation reaction series; (2) inactivation of the N-glycan fucosylation reaction series of mammalian cells and (3) in vitro chemical synthesis of non-fucosylated N-glycoprotein or enzymatic modification of N-glycans to non-fucosylated forms, as described in Yamane-Ohnuki et al. al., MAbs., 1(3):230-236 (2009). It is understood that any of these methods or any other method that is well known in the art can be used to produce an antibody or functional fragment thereof having modified fucosylation, galactosylation and/or sialylation.

[00101] Antibodies or functional fragments thereof of the invention, which bind to sLea, can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression techniques. The practice of the invention employs, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields included in technical experience. These techniques are described in the references cited here and are fully explained in the literature. See, for example, Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren et al. (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Borrebaeck (ed.) (1995) Antibody Engineering, Second Edition, Oxford University Press; Lo (ed.) (2006) Antibody Engineering: Methods and Protocols (Methods in Molecular Biology); Vol. 248, Humana Press, Inc; each of which is incorporated herein by reference in its entirety.

[00102] Monoclonal antibodies can be prepared using a wide range of techniques known in the art, including the use of hybridoma and recombinant technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd Edition 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981), each of which is incorporated herein by reference in its entirety. A monoclonal antibody is not limited to antibodies produced using hybridoma technology. Other exemplary methods of producing monoclonal antibodies are known in the art. Additional exemplary methods of producing monoclonal antibodies are provided in Example I herein.

[00103] Functional antibody fragments that bind to sLea can be generated by any technique well known to those skilled in the art. For example, Fab and F(ab')2 fragments of the invention can be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments. ). Fragments of F(ab')2 contain the variable region, the light chain constant region, and the heavy chain CH1 domain.

[00104] The functional antibody fragments of the invention can also be generated using various phage display methods known in the art. For example, in phage display methods, functional antibody domains, such as the heavy and/or light chain variable regions having one, two, three, four, five or six CDRs provided herein, are displayed on the particle surface. of phage that carry the polynucleotide sequences encoding them. DNA encoding the VH and VL domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli and the E. coli is infected with helper phage. The phage used in these methods are typically filamentous phages including fd and M13 and the VH and VL domains are generally recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen-binding domain that binds to a particular antigen, such as sLea, can be selected or identified with antigen, for example, using labeled antigen or antigen bound or captured to a bead or solid surface. Examples of phage display methods that can be used to prepare the functional antibody fragment of the present invention include those described in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177186; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; PCT Application No. PCT/GB91/01134; International Publications Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401, and WO97/13844; and United States Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733. 743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

[00105] As described in the aforementioned references, after phage selection, the antibody coding regions of the phage can be isolated and used to generate entire antibodies, including human antibodies, or any other desired antigen-binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described herein.

[00106] Techniques for recombinantly producing Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art, such as those described in PCT publication No. WO 92/22324; Mullinax et al., 1992, BioTechniques 12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and Better et al., 1988, Science 240:1041-1043, each of which is incorporated herein by reference in its entirety.

[00107] To generate full-length antibodies, PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones . Using cloning techniques well known to those skilled in the art, the PCR-amplified VH domains can be cloned into vectors expressing a VH constant region, e.g., the human gamma 1 constant region, and the PCR-amplified VL domains can be cloned in vectors expressing a VL constant region, for example, human kappa or lambda constant regions. The VH and VL domains can also be cloned into a vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then cotransfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques well known to those skilled in the art.

[00108] In some embodiments, an antibody or functional fragment of the invention is conjugated (covalent or non-covalent conjugations) or recombinantly fused to one or more diagnostic agent, detectable agent or therapeutic agent or any other desired molecule. The conjugated or recombinantly fused antibody or functional fragment may be useful for monitoring or diagnosing the onset, development, progression and/or severity of a disease associated with sLea expression, such as cancer or tumor formation, as part of a screening procedure. clinical test, such as determining the effectiveness of a particular therapy.

[00109] Detection and diagnostics can be carried out, for example, by coupling the antibody or functional fragment of the invention to detectable substances including, but not limited to, radioactive materials, such as, but not limited to, zirconium (89Zr), iodine (131I , 125I, 124I, 123I, and 121I,), carbon (14C, 11C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In,), technetium (99Tc), thallium (201Ti ), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 15O, 13N, 64Cu, 94mTc, 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho , 86Y, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, and 117Sn ; and positron emission metals using various positron emission tomography scans, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as, but not limited to, luciferase, luciferin, and aequorin, and non-radioactive paramagnetic metal ions.

[00110] The present invention also covers therapeutic uses of an antibody or functional fragment of the invention conjugated (covalent or non-covalent conjugations) or recombinantly fused to one or more therapeutic agent. In this context, for example, the antibody may be conjugated or recombinantly fused to a therapeutic agent, such as a cytotoxin, e.g., a cytostatic or cytocidal agent, or a radioactive metal ion, e.g., alpha-emitters. A cytotoxin or cytotoxic agent includes any agent that is harmful to cells. A therapeutic agent may be a chemotherapeutic such as, but not limited to, an anthracycline (e.g., doxorubicin and daunorubicin (formerly daunomycin)); a taxane (e.g., paclitaxel (Taxol) and docetaxel (Taxotere); an antimetabolite (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, and decarbazine); or an alkylating agent (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BCNU), lomustine (CCNU), cyclotosfamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisdichlorodiamine (II) platinum (DDP), and cisplatin); an antibiotic (e.g., actinomycin D, bleomycin, mithramycin, and anthramycin (AMC)); an Auristatin molecule (e.g., auristatin PHE, bryostatin 1, solastatin 10, monomethyl auristatin E (MMAE), and monomethylauristatin F (MMAF)); a hormone (e.g., glucocorticoids, progestins, androgens, and estrogens); a nucleoside analog (e.g., Gemcitabine), a DNA repair enzyme inhibitor (e.g., etoposide and topotecan), a kinase inhibitor (e.g., compound ST1571, also known as Gleevec or imatinib mesylate); a cytotoxic agent (e.g., maytansine, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colcycin, doxorubicin, daunorubicin, dihydroxy anthracine dione, mitoxantrone, mithramycin, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogues or homologues thereof, and those compounds described in United States Patent Nos. 6,245,759, 6,399,633, 6,383,790, 6,335,156, 6,271. 242, 6,242,196, 6,218,410, 6,218,372, 6,057,300, 6,034,053, 5,985,877, 5,958,769, 5,925,376, 5,922,844, 5,911,995, 5,872. 223, 5,863,904, 5,840,745, 5,728,868, 5,648,239, 5,587,459); a farnesyl transferase inhibitor (e.g., R115777, BMS-214662, and those described by, e.g., United States Patent Nos: 6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959, 6,420 .387, 6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615, 6,387,905, 6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342 .487, 6,300,501 , 6,268,363, 6,265,422, 6,248,756, 6,239,140, 6,232,338, 6,228,865, 6,228,856, 6,225,322, 6,218,406, 6,211,193, 6,187,786 , 6,169,096, 6,159 ,984, 6,143,766, 6,133,303, 6,127,366, 6,124,465, 6,124,295, 6,103,723, 6,093,737, 6,090,948, 6,080,870, 6,077,853, 6,071 ,935, 6,066,738 , 6,063,930, 6,054,466, 6,051,582, 6,051,574, and 6,040,305); a topoisomerase inhibitor (e.g., camptothecin, irinotecan, SN-38, topotecan, 9-aminocamptothecin, GG-211 (GI 147211), DX-8951f, IST-622, rubitecan, pyrazoloacridine, XR-5000, saintopine, UCE6, UCE1022, TAN-1518A, TAN 1518B, KT6006, KT6528, ED-110, NB-506, ED-110, NB-506, fagaronin, corallin, beta-lapachone and rebecamycin); a DNA minor groove binder (e.g., Hoescht dye 33342 and Hoechst dye 33258); adenosine deaminase inhibitors (e.g., fludarabine phosphate and 2-chlorodeoxyadenosine); or pharmaceutically acceptable salts, solvates, clathrates, or prodrugs thereof. A therapeutic agent may be an immunotherapeutic, such as, but not limited to, cetuximab, bevacizumab, heceptin, rituximab).

[00111] Furthermore, an antibody or functional fragment of the invention can be conjugated to a therapeutic agent, such as a radioactive metal ion, such as alpha-emitters such as 213Bi or macrocyclic chelators useful for the conjugation of radiometal ions , including, but not limited to, 131In, 131LU, 131Y, 131Ho, 131Sm; or a macrocyclic chelator, such as 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) that can be linked to the antibody or functional fragment via a molecule connector. Such binding molecules are commonly known in the art and described in Denardo et al., 1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol. 26(8):943-50.

[00112] Furthermore, an antibody or functional fragment of the invention can be conjugated (covalent or non-covalent conjugations) or recombinantly fused to a therapeutic agent that modifies a biological response provided. Therefore, therapeutic agents should not be construed as limited to classical chemical therapeutic agents. For example, the therapeutic agent may be a protein, peptide, or polypeptide having a desired biological activity. Such proteins may include, for example, a toxin (e.g., abren, ricin A, pseudomonas exotoxin, cholera toxin and diphtheria toxin); a protein such as tumor necrosis factor, interferon-Y, α-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, an apoptotic agent (e.g., TNF-Y, AIM I , AIM II, Fas ligand and VEGF), an anti-angiogenic agent (e.g., angiostatin, endostatin and a component of the coagulation reaction series, such as tissue factor); a biological response modifier (e.g., a cytokine such as interferon gamma, interleukin-1, interleukin-2, interleukin-5, interleukin-6, interleukin-7, interleukin-9, interleukin-10, interleukin-12, interleukin- 15, interleukin-23, granulocyte macrophage colony-stimulating factor, and granulocyte colony-stimulating factor); a growth factor (e.g., growth hormone), or a clotting agent (e.g., calcium, vitamin K, tissue factor, such as but not limited to, Hageman factor (factor XII), high-weight kininogen molecular (HMWK), prekallikrein (PK), coagulation protein factors II (prothrombin), factor V, XIIa, VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipid, and fibrin monomer).

[00113] The present invention encompasses antibodies or functional fragments of the invention recombinantly fused or chemically conjugated (covalent or non-covalent conjugations) to a heterologous protein or polypeptide to generate fusion proteins. In some aspects, such a polypeptide may be about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90 or about 100 amino acids in length. In some aspects, the invention provides fusion proteins having a functional fragment of an antibody of the invention (e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VH CDR, a VL or a VL CDR) and the heterologous protein or polypeptide. In one embodiment, the heterologous protein or polypeptide to which the antibody or functional fragment is fused is useful for targeting the antibody or functional fragment to a particular cell type, such as a cell that expresses sLea.

[00114] A fusion protein or conjugate of the invention includes any antibody or functional fragment of the invention provided herein conjugated (covalent or non-covalent conjugations) or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent. In one embodiment, a fusion protein or conjugate of the invention includes a 5B1, 9H3, 5H11 or 7E3 antibody, and a diagnostic agent, detectable agent or therapeutic agent. In another embodiment, a fusion protein or conjugate of the invention includes a functional fragment of antibodies 5B1, 9H3, 5H11 or 7E3, and a diagnostic agent, detectable agent or therapeutic agent. In another embodiment, a fusion or conjugated protein of the invention includes a VH domain having the amino acid sequence of any of the VH domains depicted at residues 20-142 of SEQ ID NO: 2, residues 20-142 of SEQ ID NO: 6 , residues 20-142 of SEQ ID NO: 10, or residues 20-145 of SEQ ID NO: 14, and/or a VL domain having the amino acid sequence of any of the VL domains depicted in residues 20-130 of SEQ ID NO: 4, residues 20-129 of SEQ ID NO: 8, residues 20-130 of SEQ ID NO: 12, or residues 23-130 of SEQ ID NO: 16, and a diagnostic agent, detectable agent or therapeutic agent. In another embodiment, a fusion protein or conjugate of the present invention includes one or more VH CDRs having the amino acid sequence of any of the VH CDRs depicted in SEQ ID NOS: 2, 6, 10 or 14, and a diagnostic agent, detectable agent or therapeutic agent. In another embodiment, a fusion or conjugated protein includes one or more VL CDRs having the amino acid sequence of any of the VL CDRs depicted in SEQ ID NOS: 4, 8, 12 or 16, and a diagnostic agent, detectable agent or therapeutic agent. In another embodiment, a fusion or conjugate protein of the invention includes at least one VH domain and at least one VL domain depicted in residues 20-142 of SEQ ID NO: 2 and residues 20-130 of SEQ ID NO: 4; residues 20-142 of SEQ ID NO: 6 and residues 20-129 of SEQ ID NO: 8; residues 20-142 of SEQ ID NO: 10 and residues 20130 of SEQ ID NO: 12; or residues 20-145 of SEQ ID NO: 14 and residues 23-130 of SEQ ID NO: 16, respectively, and a diagnostic agent, detectable agent or therapeutic agent.

[00115] Methods for fusing or conjugating diagnostic agents, detectable agents or therapeutic agents (including polypeptides) to antibodies are well known, see, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Edition), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies 84: Biological and Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, and Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection and Therapy, Baldwin et al (eds.), pp. 303-16 (Academic Press 1985), Thorpe et al., 1982, Immunol. Rev. 62:11958; United States Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, 5,112,946, 7,981,695, 8,039,273, 8,142. 784; United States publications 2009/0202536, 2010/0034837, 2011/0137017, 2011/0280891, 2012/0003247; EP 307,434; EP 367,166; EP 394,827; PCT publications WO 91/06570, WO 96/04388, WO 96/22024, WO 97/34631, and WO 99/04813; Ashkenazi et al., Proc. Natl. Academic. Sci. USA, 88: 10535-10539, 1991; Traunecker et al., Nature, 331:84-86, 1988; Zheng et al., J. Immunol., 154:5590-5600, 1995; Vil and another, Proc. Natl. Academic. Sci. USA, 89:11337-11341, 1992; and Senter, Current Opinion in Chemical Biology, 13:235-244 (2009), which are incorporated herein by reference in their entirety.

[00116] In another aspect, a diagnostic agent, detectable agent or therapeutic agent can be linked to the hinge region of a reduced antibody component through disulfide bond formation. Alternatively, such agents can be linked to the antibody component using a heterobifunctional crosslink, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56:244 (1994). General techniques for such conjugation are well known in the art. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et al., "Modification of Antidodies by Chemical Methods," in MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84 (Cambridge University Press 1995).

[00117] Alternatively, a diagnostic agent, detectable agent or therapeutic agent can be conjugated via a carbohydrate moiety in the Fc region of the antibody. Methods for conjugating peptides to antibody components via an antibody carbohydrate moiety are well known to those skilled in the art. See, for example, Shih et al., Int. J. Cancer. 41:832-839 (1988); Shih et al., Int. J. Cancer. 46:1101-1106 (1990); and Shih et al., United States Patent No. 5,057,313, all of which are incorporated in their entirety by reference. The general method involves reacting an antibody component having an oxidized carbohydrate moiety with a carrier polymer that has at least one free amine function and that is loaded with a plurality of peptides. This reaction results in an initial Schiff base bond (imine), which can be stabilized by reduction to a secondary amine to form the final conjugate.

[00118] However, if the Fc region is absent, for example, if a functional antibody fragment as provided here is desirable, it is still possible to bind a diagnostic agent, a detectable agent or a therapeutic agent. A carbohydrate moiety can be introduced into the light chain variable region of a full-sized antibody or antibody fragment. See, for example, Leung et al., J. Immunol., 154: 5919 (1995); United States Patent Nos. 5,443,953 and 6,254,868, all of which are incorporated in their entirety by reference. The constructed carbohydrate moiety is used to bind the diagnostic agent, detectable agent or therapeutic agent.

[00119] The therapeutic agent conjugated or recombinantly fused to a functional antibody fragment of the invention that binds to sLea can be chosen to activate the desired therapeutic or prophylactic effect(s). It is understood that it is within the level of experience of a clinician or other medical personnel to consider the following when deciding which therapeutic agent to conjugate or recombinantly fuse to an antibody or functional fragment of the invention: the nature of the disease, the severity of the disease, and the condition of the individual.

[00120] A conjugated or fusion antibody or functional fragment of the invention that is detectably labeled as provided herein and binds to sLea may be used for diagnostic purposes to detect, diagnose, or monitor disease, in which cells causing or are associated with the disease express sLea. For example, as provided herein, cancer cells and tumors have been shown to express sLea, such as, but not limited to, gastrointestinal tract tumors, breast cancer, ovarian cancer, colon cancer, colorectal adenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, small cell lung carcinoma, bladder adenocarcinoma, metastatic colon cancer, colorectal cancer, signet ring ovarian cancer and metastatic carcinoma. Accordingly, the invention provides methods for detecting cancer or tumor formation in an individual by administering an effective amount of a fusion antibody or conjugate or functional fragment of the invention to an individual in need thereof. In some aspects, the detection method may also include assaying the expression of a sLea on cells or a tissue sample from an individual using one or more fusion antibodies or conjugates or functional fragments of the invention that bind to sLea; and comparing the level of the sLea with a control level, e.g., levels in normal tissue samples (e.g., from an individual not having the disease, or from the same individual before the onset of the disease), whereby an increase in the tested level of sLeacompared to the control level of sLeais indicative of the disease. Such diagnostic methods may allow healthcare professionals to employ preventive measures or aggressive treatment earlier than otherwise possible, thereby preventing the development or progression of the disease.

[00121] An antibody or functional fragment of the invention can also be used to assay sLea antigen levels in a biological sample using classical immunohistological methods as provided herein or as well known to those skilled in the art (for example, see Jalkanen et al., 1985 , J. Cell. Biol. 101:976-985; and Jalkanen et al., 1987, J. Cell. Biol. 105:30873096). Other antibody-based methods useful for detecting sLeain include immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

[00122] In one aspect, the invention provides for the detection and diagnosis of disease in a human. In one embodiment, diagnostics include: a) administering (e.g., parenterally, subcutaneously, or intraperitoneally) to an individual an effective amount of a fusion or conjugate protein of the invention that binds to sLea; b) waiting for a period of time following administration to allow the fusion or conjugated protein to preferentially concentrate at sites in the individual where sLea is expressed (and, in some aspects, for the unbound fusion or conjugated protein to be ascertained to level base); c) determine base level; and d) detecting a fusion or conjugate protein in the subject, such that detection of the fusion or conjugate protein above the background level indicates that the subject has a disease. Baseline level can be determined by various methods including, comparing the amount of fusion or conjugated protein detected to a previously determined standard value for a particular system.

[00123] It is understood that the size of the subject and the imaging system used will determine the amount of image portion required to produce diagnostic images and can be readily determined by one skilled in the art. For example, in the case of a radioisotope conjugated to an antibody or functional fragment of the invention, for a human subject, the amount of radioactivity injected will typically range from about 5 to 20 millicuries of 99Tc. The conjugate will then preferentially accumulate at the location of sLea-expressing cells. In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B.A. Rhodes, eds., Masson Publishing Inc. (1982 ).

[00124] Depending on several variables, including the type of detectable agent used and the mode of administration, the time interval following administration to allow the conjugate to preferentially concentrate at sites in the individual and for the unbound conjugate to be determined to a level base, it is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment, the time interval following administration is 5 to 20 days or 5 to 10 days. In one embodiment, monitoring of a disease is performed by repeating the method for diagnosis as provided herein, for example, one month after initial diagnoses, six months after initial diagnoses, one year after initial diagnoses, or greater.

[00125] The presence of the fusion or conjugated protein can be detected in the individual using methods known in the art for in vivo scanning. These methods depend on the type of detectable agent used. A skilled artisan will be able to determine the appropriate method for detecting a particular detectable agent. Methods and devices that can be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scanning such as positron emission tomography (PET), magnetic resonance imaging (MRI), and sonography. In one embodiment, an antibody or functional fragment of the invention is conjugated to a radioisotope and is detected in the subject using a radiation-responsive surgical instrument. In another embodiment, an antibody or functional fragment of the invention is conjugated to a fluorescent compound and is detected in the subject using a fluorescence-responsive scanning instrument. In another embodiment, an antibody or functional fragment of the invention is conjugated to a positron-emitting metal, such as zirconium (89Zr) or any other positron-emitting metal provided herein or which is well known in the art to be detectable by emission tomography. positron, and is detected in the individual using positron emission tomography. In yet another embodiment, an antibody or functional fragment of the invention is conjugated to a paramagnetic label and is detected in an individual using magnetic resonance imaging (MRI).

[00126] In one embodiment, the invention provides a pharmaceutical composition having an antibody or functional fragment of the invention and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier that can be used in the pharmaceutical compositions of the invention includes any of the standard pharmaceutical carriers known in the art, such as phosphate-buffered saline, water, and emulsions, such as an oil-water emulsion, and various types. of wetting agents. These pharmaceutical compositions can be prepared in liquid unit dose forms or any other dosage form that is sufficient to deliver the antibody or functional fragment of the invention to the target area of the individual in need of treatment. For example, the pharmaceutical compositions may be prepared in any manner appropriate to the chosen mode of administration, e.g., intravascular, intramuscular, subcutaneous, intraperitoneal, etc. Other optional components, for example, pharmaceutical grade stabilizers, buffers, preservatives, excipients and the like, can be readily selected by one skilled in the art. The preparation of a pharmaceutical composition, paying due attention to pH, isotonicity, stability and the like, is included in the level of experience in the art.

[00127] Pharmaceutical formulations containing one or more antibodies or functional fragments of the invention provided herein can be prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co ., Easton, PA), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) of polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn protein complexes); and/or non-ionic surfactants, such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[00128] Thus, in some embodiments, the invention provides a method for treating or preventing a disease in an individual in need thereof. The methods of the invention may include administering a therapeutically effective amount of a pharmaceutical composition provided herein to the subject. For example, the pharmaceutical composition may include one or more antibodies or functional fragment provided herein. Diseases that can be treated or prevented using the methods of the invention include cancer, tumor formation and/or metastasis. In particular, the methods of the invention are useful for treating cancers or tumor formation in which the cancer cells or tumor express the carbohydrate sLea. Non-limiting examples of cancers or tumors that can be treated or prevented using the methods of the invention include tumors of the gastrointestinal tract, for example, colon cancer, colorectal adenocarcinoma, metastatic colon cancer, colorectal cancer, pancreatic cancer, or pancreatic adenocarcinoma; small cell lung carcinoma; bladder adenocarcinoma; signet ring ovarian cancer; ovarian cancer, metastatic carcinoma; and adenocarcinoma of the stomach, esophagus, throat, urogenital tract, or breast.

[00129] Accordingly, in some aspects, the invention provides a method for treating cancer or preventing tumor metastasis in an individual in need thereof by administering a therapeutically effective amount of a pharmaceutical composition having an antibody or functional fragment thereof. , wherein the antibody or functional fragment binds to sLeae includes a VH domain having an amino acid sequence selected from the group consisting of residues 20-142 of SEQ ID NO: 2, residues 20-142 of SEQ ID NO: 6, residues 20-142 of SEQ ID NO: 10, and residues 20-145 of SEQ ID NO: 14. In another aspect, the invention provides a method for treating cancer or preventing tumor metastasis in an individual in need of the even administering a therapeutically effective amount of a pharmaceutical composition having an antibody or functional fragment thereof, wherein the antibody or functional fragment binds to sLeae includes a VL domain having an amino acid sequence selected from the group consisting of residues 20-130 of SEQ ID NO: 4, residues 20-129 of SEQ ID NO: 8, residues 20-130 of SEQ ID NO: 12, and residues 23-130 of SEQ ID NO: 16. In yet another aspect, the invention provides a method for treating cancer or preventing tumor metastasis in an individual in need thereof by administering a therapeutically effective amount of a pharmaceutical composition having an antibody or functional fragment thereof, wherein the antibody or functional fragment binds to sLeae includes either a VH domain and a VL domain, wherein the VH domain and the VL domain respectively include an amino acid sequence selected from the group consisting of residues 20142 of SEQ ID NO: 2 and residues 20-130 of SEQ ID NO: 4; residues 20-142 of SEQ ID NO: 6 and residues 20-129 of SEQ ID NO: 8; residues 20-142 of SEQ ID NO: 10 and residues 20-130 of SEQ ID NO: 12; and residues 20-145 of SEQ ID NO: 14 and residues 23-130 of SEQ ID NO: 16.

[00130] Formulations, such as those described here, may also contain more than one active compound as necessary for the particular disease being treated. In certain embodiments, formulations include an antibody or functional fragment of the invention and one or more active compounds with complementary activities that do not adversely affect each other. Such molecules are suitably presented in combination in amounts that are effective for the intended purpose. For example, an antibody or functional fragment of the invention can be combined with one or more other therapeutic agents. Such combined therapy may be administered to the individual concomitantly or successively.

[00131] Thus, in some aspects, the invention provides a method for treating or preventing a disease by administering a therapeutically effective amount of a pharmaceutical composition provided herein to an individual in need thereof, wherein the pharmaceutical composition includes an antibody or fragment functional of the invention and a second therapeutic agent. The appropriate second therapeutic agent can be readily determined by one skilled in the art as discussed herein. As provided herein in Example IV, in some aspects of the invention, the second therapeutic agent may be Taxol.

[00132] The pharmaceutical compositions provided herein contain therapeutically effective amounts of one or more of the antibodies of the invention provided herein, and optionally one or more additional therapeutic agents, in a pharmaceutically acceptable carrier. Such pharmaceutical compositions are useful in preventing, treating, managing or ameliorating a disease, such as cancer or tumor formation, or one or more symptoms thereof.

[00133] Pharmaceutical compositions may contain one or more antibodies or functional fragments of the invention. In one embodiment, the functional antibodies or fragments are formulated into suitable pharmaceutical preparations, such as sterile solutions or suspensions for parenteral administration. In one embodiment, the antibodies or functional fragments provided herein are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, for example, Ansel (1985) Introduction to Pharmaceutical Dosage Forms, 4th Edition, p. 126).

[00134] An antibody or functional fragment of the invention can be included in a pharmaceutical composition in a therapeutically effective amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the treated individual. The therapeutically effective concentration can be determined empirically by testing the compounds in in vitro and in vivo systems using routine methods and then extrapolated from them to human dosages. The concentration of an antibody or functional fragment in a pharmaceutical composition will depend, for example, on the physicochemical characteristics of the antibody or functional fragment, the dosage schedule, and amount administered, as well as other factors well known to those skilled in the art.

[00135] In one embodiment, a therapeutically effective dosage produces a serum concentration of an antibody or functional fragment of about 0.1 ng/ml to about 50-100 µg/ml. The pharmaceutical compositions, in another embodiment, provide a dosage of about 0.001 mg to about 500 mg of antibody per kilogram of body weight per day. Pharmaceutical dosage unit forms can be prepared to provide from about 0.01 mg, 0.1 mg, or 1 mg to about 30 mg, 100 mg, or 500 mg, and in one embodiment from about 10 mg to about 500 mg. mg of the antibody or functional fragment and/or a combination of other optional essential ingredients per unit dosage form.

[00136] The antibody or functional fragment of the invention can be administered all at once, or can be divided into several smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It should be noted that concentrations and dosage values may also vary with the severity of the condition being alleviated. It should also be understood that for any particular individual, specific dosage regimens may be adjusted from time to time according to individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that concentration ranges depicted herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

[00137] After mixing or adding the antibody or functional fragment of the invention, the resulting mixture can be a solution, suspension or the like. The form of the resulting mixture depends on several factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient to improve symptoms of the disease, disorder or condition treated and can be empirically determined.

[00138] Pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as sterile parenteral solutions or suspensions containing suitable amounts of the compounds or pharmaceutically acceptable derivatives thereof. The antibody or functional fragment can be, in one embodiment, formulated and administered in unit dosage forms or multiple dosage forms. Unit dosage forms refer to physically discrete units suitable for human and animal subjects and individually packaged as is known in the art. Each unit dose contains a predetermined amount of the antibody or functional fragment of the invention sufficient to produce the desired therapeutic effect, in association with the necessary pharmaceutical carrier, vehicle or diluent. Examples of unit dosage forms include ampoules and syringes. Unit dosage forms may be administered in fractions or multiples thereof. A multiple dose form is a plurality of identical unit dosage forms packaged in a single container to be administered in segregated unit dose form. Examples of multiple dosage forms include pint or gallon bottles or bottles. Consequently, multiple dosage form is a multiple of unit doses that are not segregated in packaging.

[00139] In one embodiment, one or more antibodies or functional fragment of the invention in a liquid pharmaceutical formulation. Administerable liquid pharmaceutical compositions may, for example, be prepared by dissolving, dispersing, or otherwise admixing an antibody or functional fragment as provided herein and optional pharmaceutical adjuvants in a vehicle, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to form a solution. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrin derivatives. , sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents. Current methods for preparing such dosage forms are known, or will be apparent, to those skilled in the art; for example, see Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA.

[00140] Methods for administering a pharmaceutical composition of the invention are well known in the art. It is understood that the appropriate administration routine of a pharmaceutical composition can be easily determined by a skilled practitioner. Exemplary administration routines include intravenous injection, intramuscular injection, intradermal injection, or subcutaneous injection. Furthermore, it is understood that the formulation of the pharmaceutical composition can be easily adjusted to adapt to routine administration. The invention also provides that following administration of a pharmaceutical composition of the invention, delayed, successive and/or repeated dosages of one or more pharmaceutical compositions, as provided herein, may be administered to the subject.

[00141] The methods of the invention for treating a disease are intended to include (1) preventing the disease, that is, causing the clinical symptoms of the disease not to develop in an individual who may be predisposed to the disease but does not yet experience it. or presents symptoms of the disease; (2) inhibiting the disease, that is, stopping or reducing the development of the disease or its clinical symptoms; or (3) alleviating the disease, that is, causing regression of the disease or its clinical symptoms. The methods of the invention for preventing a disease are intended to include warning of a clinical symptom indicative of cancer or tumor formation. Such warning includes, for example, maintaining normal physiological indicators in an individual. Therefore, prevention may include prophylactic treatment of an individual to protect them from the occurrence of tumor metastasis.

[00142] The therapeutically effective amount of the pharmaceutical composition used in the methods of the invention will vary depending on the pharmaceutical composition used, the disease and its severity, and the age, weight, etc., of the individual being treated, all of which are included in the experiment. of the attending physician. An individual who can be treated by the methods of the invention includes a vertebrate, preferably a mammal, more preferably a human.

[00143] It is understood that modifications that do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided here. Therefore, the following examples are intended to illustrate but not limit the present invention.

EXAMPLE I Human Monoclonal Antibodies to sLeat Have Potent Antitumor Activity

[00144] The carbohydrate antigen sLea is widely expressed in epithelial tumors of the gastrointestinal tract, breast, and pancreas and in small cell lung cancers. Since overexpression of sLea appears to be a key event in the invasion and metastasis of many tumors and results in susceptibility to antibody-mediated lysis, sLea is an attractive molecular target for tumor therapy. Therefore, as described herein, fully human monoclonal antibodies (mAb) from blood lymphocytes of individuals immunized with an sLea-KLH vaccine were generated and characterized. Several mAbs were selected based on ELISA and FACS including two mAbs with high affinity for sLea (5B1 and 7E3, binding affinities 0.14 and 0.04 nmol/L, respectively) and also characterized. Both antibodies were specified for Neu5Aca2-3Galß1-3(Fuca1-4)GlcNAcß and Neu5Gca2-3Galß1-3(Fuca1-4)GlcNAcß as determined by glycan arrangement analysis. Complement-dependent cytotoxicity against DMS-79 cells was higher (EC50 0.1 µg/mL vs. 1.7 µg/mL) for r7E3 (IgM) than for r5B1 (IgG1). Furthermore, r5B1 antibodies showed high level of antibody-dependent cell-mediated cytotoxicity activity on DMS-79 cells, with human NK cells or peripheral blood mononuclear cells. To evaluate in vivo efficacy, antibodies were tested in a xenograft model with Colo205 tumor cells or DMS-79 tumor cells engrafted into severe combined immunodeficient (SCID) mice. In the Colo205 xenograft model, treatment during the first 21 days with four doses of r5B1 (100 µg per dose) doubled the median survival time to 207 days, and three of five animals survived on six doses. In the DSM-79 xenograft model, growth of established DMS-79 tumors was suppressed or repressed in animals treated with r5B1 antibody. On the basis of sLea's potential as a target for immune system attack and its affinity, specificity, and effector functions, 5B1 and 7E3 have clinical utility in the treatment of cancer. Materials, cells, and antibodies

[00145] DMS-79 (Pettengill et al., Cancer, 45:906-18 (1980)), SW626, EL4, HT29, BxPC3, SK-MEL28, and P3 x 63Ag8.653 Cell lines were obtained from American Type Culture Collection (ATCC). Colo205-luc cells (Bioware ultra) were obtained from Caliper Life Sciences. Murine control mAb 121SLE (IgM) was obtained from GeneTex. Tetrasaccharide sLea (Cat # S2279) was obtained from Sigma-Aldrich. sLea-HSA (human serum albumin) conjugate (Catalog #07-011), monovalent sLeabiotinylated (sLea-sp-biotin; Catalog #02-044), polyvalent biotinylated sLea-PAA (Catalog #01-044), Lea- Biotin-labeled PAA (Catalog #01-035), and sLex-PAA-biotin (Catalog #01-045) were obtained from GlycoTech. In the polyvalent presentation, the tetrasaccharide is incorporated into a polyacrylamide (PAA) matrix, thus creating a 30-kDa multivalent polymer with approximately every fifth amide group of the polymer chain N-substituted with biotin in a ratio of 4 to 1 and approximately 20% of carbohydrate content. Other HSA or BSA glycoconjugates used in this study were prepared in-house using sLeapentenyl glycoside as described. Ragupathi et al., Cancer Immunol Immunother, 58:1397-405 (2009). GD3, fucosyl-GM1, GM2, and GM3 were obtained from Matreya, and GD2 was obtained from Advanced ImmunoChemical.

Generation of anti-sLea mAb-producing hybridomas

[00146] Blood samples were obtained from 3 patients in an ongoing trial with sLea-KLH conjugate vaccine in patients with breast cancer initiated at MSKCC under an IRB protocol approved by MSKC and FDA and IND. Blood samples were selected from 2 patients after 3 or 4 vaccinations, which showed antibody titers of 1/160 and 1/320, respectively, against sLea. These sera (and murine mAb 19.9) react well with sLea-positive cell lines in FACS assays and mediate potent CDC. Ragupathi et al., Cancer Immunol Immunother, 58:1397-405 (2009). Peripheral blood mononuclear cells (PBMC) were isolated from approximately 80 to 90 mL of blood by gradient centrifugation on Histopaque-1077 (Sigma-Aldrich).

[00147] PBMCs were cultured in RPMI-1640 medium supplemented with L-glutamine, non-essential amino acids, sodium pyruvate, vitamin, penicillin/streptomycin, 10% FBS (Omega Scientific), 10 ng/mL IL-21 (Biosource ), and 1 µg/mL anti-CD40 mAb (G28-5 hybridoma supernatant; ATCC). Cells were fused by electrofusion to P3 x 63Ag8.653 myeloma cells.

sLea ELISA

[00148] For the sLea ELISA, plates were coated with 1 µg/mL of sLea-HSA conjugate, monovalent biotinylated sLea, or with polyvalent biotinylated sLea-PAA captured on Neutr-Avidin-coated plates. Uncoated wells (PBS) and HSA-coated wells were used as controls. Bound antibodies were initially detected with horseradish peroxidase (HRP)-labeled goat anti-human IgA+G+M (Jackson ImmunoResearch), and positive wells were subsequently probed with IgG-Fc- or IgM-specific secondary antibodies to determine isotypes.

Carbohydrate specificity analysis

[00149] Cross-reactivity against the closely related antigens, Leae sLex, was assessed by surface plasmon resonance (SPR) and confirmed by ELISA using biotin-labeled Lea-PAA and biotin-sLex-PAA. Binding to gangliosides GD2, GD3, fucosyl-GM1, GM2, and GM3 was tested by ELISA. A competition ELISA was used to evaluate the specificity of the mAbs against several other related carbohydrate moieties. Briefly, 2 µg/mL of sLea-HSA conjugate was coated onto plates followed by blocking with 3% BSA in PBS. Then, 30 µL of different carbohydrate moieties (40 µg/mL in PBS prepared from 1 mg/mL stock solutions) unconjugated or conjugated to HSA or BSA were mixed separately with 30 µL of test antibody and incubated at room temperature in a sample plate. After 30 minutes 50 µL of the mixture was transferred to the coated assay plate and incubated for 1 hour, followed by incubation with HRP-labeled goat anti-human IgA+G+M, washing and colorimetric detection of bound antibody using a Versamax spectrofluorometer. (all steps were carried out at room temperature). Carbohydrate moieties tested included globe H, Lewis Y, Lewis sLea-HSA conjugate. To determine the fine specificity of the antibodies, glycan arrangement analysis was performed by the Consortium for Functional Glycomics Core H Group. Antibodies 5B1 and 7E3 were tested at 10 µg/mL using version 4.1 of the printed array consisting of 465 glycans in replication of 6.

Immunoglobulin cDNA Cloning and Recombinant Antibody Expression

[00150] Variable region of human mAb heavy and light chain cDNA was recovered by RT-PCR from the individual hybridoma cell line and subcloned into IgG1 or IgM heavy chain or IgK or IgL light chain expression vector as described previously. Sawada-Hirai et al., J. Immune Based Ther. Vaccines, 2:5 (2004). Heavy chain or light chain expression vector was double digested with Not I and Sal I, and then both fragments were ligated to form a dual gene expression vector. CHO cells in 6-well plates were transfected with the dual gene expression vector using Lipofectamine 2000 (Invitrogen). After 24 hours, transfected cells were transferred to a 10 cm dish with media selection [DMEM supplemented with 10% dialyzed FBS (Invitrogen), 50 µmol/L L-methionine sulfoximine (MSX), GS supplement (Sigma -Aldrich), and penicillin/streptomycin (Omega Scientific)]. Two weeks later MSX-resistant transfectants were isolated and expanded. High anti-sLeae antibody-producing clones were selected by measuring antibody levels in supernatants in an expanded sLeae-specific ELISA assay for large-scale mAb production.

Purification of human mAb

[00151] Antibodies were purified using the Ãkta Explorer system (GE Healthcare) running Unicorn 5.0 software. Briefly, stable clones of 5B1 or 7E3 were grown in serum-free culture medium in a Wave bioreactor, and the harvested supernatant was clarified by centrifugation and filtration and stored refrigerated until use. Human IgG antibodies were purified on appropriately sized protein A columns using 10 mmol/L PBS and 150 mmol/L NaCl running buffer. Human IgM antibodies were purified on a hydroxyapatite column, and IgM was eluted with a 500 mmol/L phosphate gradient. Antibody concentrations were determined by OD280 using an E1% of 1.4 and 1.18 for IgG and IgM, respectively, to calculate the concentration. The purity of each preparation was assessed by SDS-PAGE analysis (15μg per column) under reducing conditions, and the purity was greater than 90% based on the sum of heavy and light chains.

Flow Cytometry

[00152] sLea positive or negative tumor cell lines (0.5 x 106 cells per condition) were washed in PBS / 2% FBS (PBSF). Test or control human mAb was then added (1 to 2 µg/mL in complete medium) and incubated on ice for 30 minutes. Gilewski et al., Clin Cancer Res, 6:1693-701 (2000); Gilewski et al. Proc. Natl. Academic. Sci. U.S.A., 98:3270-5 (2001). After washing in PBSF, cells were incubated with anti-human IgG-Fcy or anti-human IgM-µ (Invitrogen) Alexa-488 for 30 minutes on ice. Cells were washed twice in PBSF and analyzed by flow cytometry using the Guava Personal Cell Analysis-96 (PCA-96) System (Millipore). Colo205-luc cells were incubated with 2 µg/mL primary antibody, followed by staining with secondary antibodies from SouthernBiotech, and analyzed on a Becton Dickinson FACS Advantage IV instrument using FlowJo 7.2.4 software.

Affinity determination

[00153] Affinity constants were determined using the SPR principle with the Biacore 3000 (GE Healthcare). Biotin-labeled univalent sLea (Catalog #02-044) or polyvalent sLea-PAA-biotin (Catalog #01-044) were coupled to separate flow cells of a SPA biosensor chip according to the manufacturer's instructions. A flow cell blocked with HSA and culture medium containing free biotin was used as a reference cell. Binding kinetic parameters were determined from various known concentrations of the antibody diluted in HBS-EP buffer (10 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 3.4 mmol/L EDTA, 0.005% of surfactant P20) using the flow cell coated with sLea-PAA-biotin. Curve fitting software provided by Biacore instrument was used to generate estimates of association and dissociation rates from which affinities are calculated.

CDC Assay

[00154] sLea antigen positive and negative cell lines were used for a 90 minute cytotoxicity assay (Guava PCA-96 Cell-Toxicity kit; Millipore; Cat # 4500-0200) using human complement (Quidel; Cat # A113 ) and purified human mAbs at various dilutions (0.1-25 µg/mL) or with positive control mAbs as previously described (Ragupathi et al. Clin Cancer Res 2003, 9:5214; Ragupathi et al. Int J Cancer 2000, 85:659; Dickler et al. Cancer Res1999, 5:2773). Briefly, 2.5 x 106 target cells were stained with carboxyfluorescein diacetate succinimyl ester (CSFE) to produce green/yellow fluorescent target cells. The stained cells (1 x 105/50 µL sample) were incubated for 40 minutes with 100 µL of antibodies on ice. Next, 50 µL of human complement diluted 1:2 in complete medium (RPMI-1640, 10% FCS) or medium alone was added to triplicate samples and incubated for 90 minutes at 37°C. Therefore, the final complement dilution in the assay was 1:8. Cells that were killed during this incubation time were labeled by adding the membrane-impermeable pigment 7-amino-actinomycin D (7-AAD), and samples were analyzed by dual-color immunofluorescence using the Guava CellToxicity software module. Control samples receiving NP40 were used to determine maximum killing and samples receiving complement alone served as baseline. The percentage of dead cells was determined by appropriate closure and calculated according to the following formula: % dead = [(% sample - % complement alone) / (% NP40 - % complement alone)] x 100.

Antibody-dependent cell-mediated cytotoxicity assay

[00155] PBMC effector cells were isolated by Ficoll-Hypaque density centrifugation of blood samples obtained under an MSKCC IRB-approved protocol. Target cells were incubated at 5 x 106 cells/mL in complete growth medium with 15 µL of 0.1% calcein-AM solution (Sigma-Aldrich) for 30 minutes at 37°C in the presence of 5% CO2. Cells were washed twice with 15 mL of PBS-0.02% EDTA and resuspended in 1 mL of complete growth medium. Fifty microliters (10,000 cells) of labeled target cells were seeded in a 96-well plate in the presence or absence of antibodies at the concentrations described in FIG. 13, and incubated with 50 µL of mononuclear cells recently isolated from peripheral blood (effector cells, in a ratio of 100:1 E/T) accordingly. After 2 hours of incubation, the plate was centrifuged at 300 x g for 10 minutes, and 75 µL of supernatant was transferred into a new 96-well flat-bottom plate. Fluorescence in the supernatant was measured at 485 nm excitation and 535 nm emission on a Fluoroskan Ascent (Thermo Scientific). Spontaneous release was determined from target cells in RPMI-1640 medium with 30% FBS without effector cells and maximal release was determined from target cells in RPMI-1640 medium with 30% FBS and 6% Triton X-100 without effector cells. Percent cytotoxicity was calculated as [(sample counts - spontaneous release)/(maximum counts - spontaneous release)] x 100.

mAb internalization assay

[00156] Internalization of the 5B1 antibody was assessed by measuring the cytotoxic activity of r5B1 and Hum-ZAP secondary conjugate complex (Advanced Targeting Systems) against BxPC3 cells expressing sLea, which were seeded in a 96-well plate (2,000 cells/ 90 µL/well) and incubated overnight in duplicates. Various concentrations of 5B1 antibody were incubated with Hum-ZAP secondary conjugates at RT according to the manufacturer's instruction. Then, 10 µL/well of r5B1 and Hum-ZAP complex were added to the cells and incubated for 3 days. Twenty-five microliters of thiazolyl blue tetrazolium bromide solution (Sigma-Aldrich) (5 mg/mL in PBS) was added to each well and incubated at 37°C. After 2 hours of incubation, 100 µL/well of solubilization solution (20% SDS / 50% N,N-dimethylformamide) was added to each well and incubated for a further 16 hours at 37°C. OD was measured at 570/690 nm, and values obtained with medium alone were used for base plate subtraction. Eight parallel cultures without antibody were used to normalize sample values (untreated sample/mean × 100).

Xenograft Transplant Model

[00157] Female SCID CB17 mice (5 to 8 weeks old) were purchased from Taconic. For the Colo205 xenograft model, Colo205-luc cells (0.5 x 106) in 0.1 mL of complete growth medium were injected via the tail vein on day 0 using a BD insulin syringe with needle. 28G (Becton Dickinson & Co). For the first study, one hundred mAb 5B1 microorganisms were injected intraperitoneally on days 1, 7, 14, and 21 (experiment 1) or on days 1, 4, 7, 10, 14, and 21 (experiment 2). For the second study, 100 µg, 300 µg, or 1 mg of mAb 5B1 was injected intraperitoneally on day 4 after tumor cell injection, then twice a week for the first two weeks and once a week for the next 7 weeks. The mice were monitored for tumor development. For the DMS-79 xenograft model, DMS-79 cells (1 x 106) were injected subcutaneously into female SCID CB17 mice, and the mice started treatment on day 19 after the tumor length had reached 5 mm (~20 mm). ). Animals were then treated with human IgG or 5B1 antibodies administered by intraperitoneal injection at 200 µg per dose, plus cRGD by intravenous injection to increase vascular permeability initially at 80 µg, then 5 days a week at 40 µg per dose until the day 37.

[00158] All procedures were performed under a protocol approved by the Memorial Sloan Kettering Cancer Center Institutional Animal Care and Use Committee. Kaplan-Meier survival curves were generated using GraphPad Prism 5.1 (GraphPad Software) and analyzed using the Mantel-Haenszel log rank test.

RESULTS Identification of human monoclonal antibodies by ELISA and generation of recombinant antibodies

[00159] Blood samples from 3 vaccinated patients were used for efforts and hybridoma generation and many positive wells were detected in the antigen-specific ELISA assays (Table 3). Extensive evaluation was used to eliminate antibodies that showed inferior or nonspecific binding. Eight hybridoma cells expressing human antibody (1 IgM and 7 IgG) with strong reactivity against sLea were initially selected, expanded, and subcloned for further characterization. Two antibodies (9H1 and 9H3) showed strong binding to sLea-HSA conjugates, but not to sLea-PAA-coated plates. Three antibodies (5B1, 5H11, and 7E3) showed strong binding to monovalent and polyvalent sLea, and sLea-HSA conjugates were measured by ELISA assays (Table 4). TABLE 3: Binding of candidate hybridoma supernatants containing IgG or IgM monoclonal antibodies to sLea-acetylphenylenediamine (APD) human serum albumin (HSA) conjugate (sLea-HuSA). * subtracted void from isotype control. HuSA indicates human serum albumin control. PBS indicates phosphate-buffered saline control. TABLE 4: Binding of selected antibodies to sLea presented as univalent (mono-) sLea, multivalent (poly-) sLea, or sLea-HSA. HuSA indicates human serum albumin control. PBS indicates phosphate-buffered saline control. NAV indicates Neutral Avidin control.

[00160] The heavy and light chain variable regions of 4 selected antibodies were recovered by RT-PCR and cloned into our full-size IgG1 or IgM expression vectors. Molecular sequence analysis using IMGT/V-Quest (Brochet et al., Nucleic Acids Res., 36:W503-8 (2008)) revealed that the 3 selected IgG antibodies 5B1 (IgG/À), 9H3 (IgG/À), and 5H11 (IgG/À) were derived from the same VH family and all lambda light chains used. These IgG1 antibodies showed different CDR sequences with 16, 5, or 3 germline deviating mutations, respectively (FIGS. 1-6; Table 5). The IgM antibody (7E3) uses the kappa light chain and has 6 heavy chain mutations (FIGS. 7-8; Table 5). Increased mutations in 5B1 are indicative of affinity maturation. Recombinant antibodies were produced in CHO cell lines in a wave bioreactor system and purified using protein A or hydroxyapatite chromatography for IgG and IgM, respectively. The purified recombinant antibodies maintained the properties of the original hybridoma-derived antibodies with respect to ELISA binding and specificity. TABLE 5: cDNA classification of selected human anti-sLea antibodies derived from vaccinated blood donors.

Tumor cell linkage analysis

[00161] Cell surface binding is crucial for cytotoxic activity and was therefore tested next. Flow cytometry showed strong binding of recombinant 5B1, 9H3, 5H11, and 7E3 antibodies to DMS-79 cells, a small cell lung cancer suspension cell line (FIG. 11A). Binding of r5B1 and r7E3 was also confirmed in HT29 colon cancer cells (FIG. 11B), BxPC3 pancreatic cancer cells (FIG. 11C), SW626 ovarian cancer cells (FIG. 11D), and Colo205 colon cancer cells -luc (FIG. 11F). These antibodies failed to bind to sLea-negative (SLE121-negative) SK-MEL28 melanoma cells (FIG. 11E) or EL4 mouse lymphoma cells (data not shown).

Affinity Measurements

[00162] The relative affinity/avidity of binding to sLea was probed by SPR using a streptavidin-coated biosensor chip to capture biotinylated sLea-PPA. As shown in Table 6, r5B1 and r7E3 rapidly bind to sLea-PPA and show a significantly slower off-rate compared to 121SLE, a commercially available murine IgM anti-sLeade antibody that was used for comparison. The affinity of 5B1 was measured at 0.14 nmol/L, and the apparent affinity/avidity of 7E3 was approximately 4 times higher (Table 6). Determination of 9H3 affinity was hampered as 9H3 antibodies (native and recombinant) failed to bind to the sLea-PAA coated biosensor chip. TABLE 6: Determination of kinetic parameters of anti-sLeapor SPR antibodies.

Specificity analysis

[00163] Preliminary assays to investigate carbohydrate specificity showed that 5B1, 9H3, and 7E3 did not bind to the closely related antigens sLeX, Lea, or LeY or the gangliosides GD2, GD3, fucosyl-GM1, GM2, and GM3 as measured by ELISA or SPR. Additional analysis of 7E3, 5B1, and 121SLE binding to sLea-PAA-biotin or sLea-sp-biotin captured on a Biacore avidin chip showed that all three antibodies bound to the polyvalent form of sLea, whereas 7E3 and 5B1 were found to bind to the monovalent form. Binding of 5B1 to sLea-PAA was also inhibited by sLeade tetrasaccharide in a dose-dependent manner in a Biacore concentration analysis series (data not shown). These results are consistent with previous observations that sera with high anti-sLea antibody titers were found to be specific for sLea, that is, nonreactive with gangliosides GM2, GD2, GD3, fucosyl GM1, or the neutral glycolipids globo H and Ley by ELISA. Ragupathi et al., Cancer Immunol Immunother 58:1397-405 (2009). In a competition assay with 9 distinct related carbohydrate moieties in various presentations (e.g., as ceramide, or conjugated to BSA or HSA), only sLeae tetrasaccharide conjugated sLea-HSA were able to inhibit binding to the sLea-HSA conjugate. (Table 7). TABLE 7: Binding to sLeA-PAA-HSA in the presence of several related glycoconjugates.

[00164] To examine carbohydrate specificity in more detail, antibodies 5B1 and 7E3 were also tested by glycan array analysis performed by the Consortium for Functional Glycomics Core H Group. Both antibodies were tested at 10 µg/mL on printed arrays consisting of 465 glycans in 6 replicates. The results confirmed the high specificity of both antibodies with selective recognition of the sLea tetrasaccharide, Neu5Aca2-3Galß1-3(Fuca1-4)GlcNAcß and Neu5Gca2-3Galß1-3(Fuca1- 4)GlcNAcß and virtual absence of binding to closely related antigens. who were present in the matrix, including sLex, Lea, Lex, and Ley. The results are summarized in Table 8, which shows the top 5 of 465 Glycan Structures that were recognized by the respective antibodies. TABLE 8: Carbohydrate specificity analysis by glycan microdisposition assessment. A. 5B1 CDC Activity

[00165] To evaluate the functional activity of 5B1 and 7E3, we tested cytotoxic activity with DMS-79 cells in the presence of human serum as a source of complement. Both antibodies showed in some assays close to 100% killing activity at 10 µg/mL, although a control antibody with different specificity (1B7, anti-GD2 IgG1 mAb) had no effect at the same concentrations (data not shown). . CDC activity is concentration dependent, and 7E3 was significantly more active than 5B1 in this assay (FIG. 12), which is expected since IgM antibodies are known to be more effective in complement-mediated cytotoxicity assays. The EC50 (50% cytotoxicity) was 1.7 µg/mL for 5B1 and 0.1 µg/mL for 7E3, which translates into approximately 85-fold higher potency for 7E3 on a molar basis (FIG. 12).

ADCC activity

[00166] Although 7E3 is significantly more potent in the CDC assay, IgG antibodies are known to have antibody-dependent cell-mediated cytotoxicity (ADCC) activity, which is considered to be important for tumor killing in vivo. High levels of cytotoxicity were measured using 5B1 antibody with human PBMC and DMS-79 target cells at various E:T ratios (FIG. 13A). Similar levels of cytotoxicity were observed at lower E:T ratios with primary NK cells (FIG. 13B). A dose response experiment with PBMC from 2 donors measured at an E/T ratio of 100:1 showed similar efficacy, and more than 85% cytotoxicity was achieved at concentrations of 0.5 µg/mL or more of 5B1 ( FIG. 13C). 5B1-mediated cytotoxicity requires FcyRIII receptors as it can be blocked with anti-CD16 3G8 antibodies. High levels of cytotoxicity were also measured using 5B1 antibody with human PBMC against Colo205-luc cells at an E:T ratio of 100:1. The ADCC activity obtained with 1 µg/mL of 5B1 antibodies was superior to the activity observed with antibodies to GM2, fucosyl-GM1, globe H, or polysialic acid. As expected, murine 7E3 and 121SLE (both are IgM) were inactive in this assay.

5B1 internalization assay

[00167] Antibody conjugates targeting "closely related to" Lewis Y antigen have previously been shown to be rapidly internalized and very effective in animal models. Hellstrom et al., Cancer Res50:2183-90 (1990); Trail et al., Science 261:212-5 (1993). To examine whether sLea is internalized, we incubated the pancreatic cell line, BxPC3 with 5B1, and then added Hum-ZAP, an anti-human IgG conjugated to the ribosome inactivation protein saporin. Kohls et al., Biotechniques 28:162-5 (2000). Cells that internalize the saporin-containing complex die, although the uninternalized saporin leaves the cells unharmed. As shown in FIG. 14, BxPC3 cells are effectively killed in the presence of increased doses of 5B1 although the presence of an isotype-matched IgG1 antibody directed against GD2, which is not expressed in these cells, does not kill the cells.

Activity in a xenograft animal model for metastasis

[00168] To evaluate the activity of 5B1 in vivo, antibodies were tested in two xenograft models using Colo205-luc tumor cells or DMS-79 tumor cells in SCID mice. For the xenograft model using Colo205-luc tumor cells, five mice per group were injected with 0.5 x 106 cells into the tail vein on day 0, and successful injection of the cells was verified by imaging the animals using the in vivo imaging IVIS 200 (Caliper Life Sciences). One day later, animals were treated with 5B1 antibodies administered by intraperitoneal or mock PBS injection. In experiment 1, 100 µg of 5B1 was administered on days 1, 7, 14, and 21 (400 µg total dose), and in experiment 2 the animals received 100 µg of 5B1 on days 1, 4, 7, 10, 14 , and 21 (600 µg total dose). The average survival of untreated animals was 102 days in the 2 experiments, and all untreated animals died within 155 days (FIG. 15). Animal treatment improved survival significantly: median survival was doubled to 207 days in the group that received 4 doses of 5B1 and 2 of the 5 animals survived to the end of the experiment after 301 days (log rank test, P = 0. 0499; HR = 3.46). The proportion of survivors also increased for 3 of 5 mice when 6 doses were administered (log rank test, P = 0.0064; HR = 6.375). The second experiment was completed after 308 days, and surviving animals did not reveal Colo205-luc tumors at the highest sensitivity of the imaging system (data not shown).

[00169] In a second study, mice similarly injected with Colo205-luc tumor cells as described above, were treated with increasing doses of 5B1 or 7E3 antibodies (100 µg, 300 µg or 1 mg). All animals initially received interperitoneal or sham PBS (control) injection of 5B1 or 7E3 antibody on day 4 after tumor cell injection, then twice a week for the first two weeks and once a week for the next 7 weeks. . Delayed treatment with various doses of 5B1 showed dose-dependent protection until complete cure in SCID mice engrafted with Colo205-luc tumor cells (FIGS. 16 and 17). Treatment with 7E3 antibodies did not show greater protection, despite the apparent increased affinity (data not shown).

[00170] In a xenograft model using DMS-79 cells, five mice per group were injected subcutaneously with 1 x 106 cells on day 0, and started treatment on day 19 after the tumor length had reached 5 mm (~20 mm2 ). Animals were then treated with human IgG or 5B1 antibodies administered by intraperitoneal injection at 200 µg per dose, plus cRGD by intravenous injection initially at 80 µg, then 5 days a week at 40 µg per dose until day 37. Growth of established DMS-79 tumors was suppressed or regressed in animals treated with 5B1 or a combination of 5B1 plus cRGD (FIG. 18A and 18B). Treatment of animals with 5B1 on the day of engraftment with DMS-79 cells in a subcutaneous model completely prevented tumor growth (data not shown).

[00171] The above data demonstrate a significant ability to suppress or regress stabilized tumors and provide a survival benefit using 5B1 antibody treatment.

EXAMPLE II Immuno-PET Detection and Diagnosis of Pancreatic Cancer and Other sLea-Positive Adenocarcinomas Using Radiolabeled Monoclonal 5B1 Antibody.

[00172] Adenocarcinomas are a death-inducing cause of cancer. Detection of pancreatic cancer remains especially difficult with diagnosis often made at a late stage. Methods for early detection of primary and metastatic pancreatic cancers can have significant clinical impact. In clinical practice, elevated sLeason antigen levels are monitored to identify suspected occult malignancy in patients with pancreatic cancer. As described here, the potential of a novel immunoPET imaging probe targeting sLea in preclinical models of pancreatic cancer and other sLea-positive adenocarcinomas was investigated. The human anti-sLea monoclonal antibody 5B1 stained positive in human adenocarcinomas known to be sLea-positive malignancies but not in sLea-negative or most normal tissues. 89Zr radiolabeled 5B1 (89Zr-5B1) exhibited high labeling (>80%) and purification yields (>95%). Imaging with 89Zr-5B1 was investigated in subcutaneous, orthotopic and metastatic pancreatic cancer xenografts in female SCID mice. Acquired PET images and biodistribution studies demonstrated exceptional specificity and localization of 89Zr-5B1 to BxPC3 xenografts overexpressing sLea with minimal nonspecific binding to healthy tissues. Another analysis in subcutaneous colon and small cell lung cancer xenograft models resulted in excellent tumor delineation by 89Zr-5B1 as well. Therefore, these results show that 89Zr-5B1 can be used as a molecular probe for early detection of clinical sLeana-expressing malignancies.

Tissue Culture and Cell Lines

[00173] All tissue culture manipulations were performed following sterile techniques. DMS79 small cell lung cancer and BxPC3 pancreatic cancer cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Colo205-luc colorectal cancer cells (Bioware Ultra) were purchased from Caliper Life Sciences (CLS, Hopkinton, MA). All cells were cultured according to ATCC and CLS recommendations under 37°C with a humidified atmosphere of 5% CO2.

In vitro evaluation of sLea expression levels through FACS

[00174] Flow cytometry with the cultured cancer cell lines was performed as described here in example I. In summary, single cell suspensions of 1x106 cultured tumor cells per tube were washed in PBS with 3% fetal bovine serum ( FBS). Human monoclonal antibodies r5B1 (IgG against sLea) were then added at 20 ug/ml per tube, and incubated on ice for 30 minutes. After washing in PBS with 3% FBS, 20 µl of goat anti-human IgG diluted 1:25 labeled with fluorescein-isothiocyanate (FITC, Southern Biotechnology, Birmingham, AL) was added, and the mixture incubated for an additional 30 minutes on ice. After a final wash, the positive population and median fluorescence intensity of strained cells were differentiated using FACS Scanning (Becton & Dickinson, San Jose, CA). Cells stressed only with goat anti-human IgG labeled with fluorescein-isothiocyanate were used to establish the 1% FACScan result as a basis for comparison to the percentage of positive cells stained with primary mAb.

Preparation of 89Zr-labeled antibodies

[00175] Recombinant 5B1 antibodies were prepared and purified as described here. 5B1 antibodies and a nonspecific human IgG were functionalized with p-isothiocyanatobenzyldesferrioxamine (DFO-Bz-NCS, Macrocyclics, Inc., Dallas, TX) with a 1:4 mAb:DFO-Bz-NCS ratio. For example, to 300 µL of 5B1 (1.23 mg in PBS, pH~9), a volume of 7.2 µL of DFO-Bz-NCS (4.25 mM in DMSO) was added. The reaction was incubated at 37°C for 1-1.5 hours. Functionalized antibodies were purified using either a PD10 desalting column (GE Healthcare) or a 10 kDa centrifugal filter (Amicon).

[00176] Zr-89 was produced by means of a proton beam bombardment of yttrium thin foil and isolated in high purity as Zr-89 oxalate at MSKCC according to the previously established procedure. Holland et al., Nuclear Medicine and Biology 36:729-39 (2009). Antibody labeling proceeded using methods as described by Holland et al., Journal of Nuclear Medicine official publication, Society of Nuclear Medicine 51:1293-300 (2010). In general, Zr-89 oxalate was neutralized to pH 7.0-7.2 with 1 M Na2CO3. DFO antibodies were then added. The reaction was incubated at room temperature for 1 to 2 hours. Subsequent purification was conducted using either a PD10 desalting column with 0.9% saline.

In vitro experiments

[00177] 89Zr-5B1 was investigated for in vitro stability in 0.9% saline and 1% bovine serum albumin for 5 days at 37°C. Changes in radiochemical purity were monitored at t = 0-5 days by radio iTLC with 50 mM DTPA as the mobile phase. In vitro immunoreactivity assays were performed according to the protocol described by Lindmo et al., Journal of Immunological Methods 72:77-89 (1984), to demonstrate the integrity of the Zr-89 radiolabeled antibodies.

Animal Models

[00178] All animal studies were conducted in accordance with standards established by the Institutional Animal Care and Use Committee. Female SCID CB17SC-F mice (Jackson Laboratories, 6-8 weeks, 20-22 g) or athymic nude mice (nu/nu) were induced with hind leg tumors. All cell lines were inoculated subcutaneously into 200 µL of 1:1 medium: Matrigel solution (BD Biosciences) and cultured to a maximum tumor volume of 250 mm before use.

Biodistribution Studies

[00179] Biodistribution studies were performed on several cohorts of mice carrying separate Colo205-luc colorectal, BxPC3 pancreas and DMS79 small cell lung xenografts (n=3-5). Zr-89 mAbs (10-20 µCi, 1-2 µg) in 100 µL of 0.9% saline were administered intravenously into the lateral vein. Additional unlabeled mAb (10-50 µg) was co-injected along with the tracer. A blocking study with 250 µg excess unlabeled mAb was performed to address the specificity of the antibody to sLea in a cohort of mice. After each time point (t=24, 48, 120 h p.i.), mice were euthanized by CO2 asphyxiation. Blood was collected immediately through cardiac puncture, although the tumor along with chosen organs was harvested. The wet weight of each tissue was measured, and the radioactivity bound to each organ was counted using a Wizard22480 gamma recorder (Perkin Elmer). Percent tracer uptake expressed as % injected dose per gram (%ID/g) was calculated as tissue-bound activity per organ weight per decrease in actual injected dose corrected for counting time.

Small animal immuno-PET

[00180] Imaging experiments were performed with a microPET Focus 120 or R4 scanner (Concorde Microsystems). Mice (n = 3-5) were administered Zr-89-labeled antibodies (200-300 µCi, 15-25 µg) in 100-200 µL of 0.9% saline formulations via lateral tail vein injections. . Whole-body PET acquisitions were recorded in mice at 24-96 h p.i. while anesthetized with 1.5-2.0% isofluorane (Baxter Healthcare) in oxygen. The images were analyzed using the ASIPro VMTM software (Concorde Microsystems). Regions-of-interest (ROI) were drawn and plotted as a function of time.

Immunohistochemistry

[00181] Biotinylated 5B1 was prepared by incubating 20x molar excess of Sulfo-NHS-LC-biotin (Thermo Scientific/Pierce cat. #21327) for 30 minutes at room temperature. Free biotin was removed with Zebra™ spin desalting columns (Thermo Scientific/Pierce, cat # 89889) according to the manufacturer's instructions. Antibodies were buffer exchanged into PBS containing 0.01% sodium azide at a concentration of 1.1 mg/ml. Binding to DMS79 cells was confirmed by FACS and was comparable to the parental antibody 5B1.

[00182] Preliminary immunohistochemical staining conditions were determined using Colo205 cells as a positive control and SK-MEL28 cells as a negative control. Cell pellets were prepared, formalin fixed and paraffin embedded. Slides were incubated with 5B1 diluted in 10% (v/v) normal human serum in PBS (Jackson ImmunoResearch Labs; cat# 009-000-121). Staining was performed by Ventana automation (Discovery XT platform-Ventana Medical Systems, Inc, Tucson, AZ) with the standard streptavidin-biotin immunoperoxidase method and the DAB detection system as a staining method. Antigen retrieval was conducted using heat and Ventana’s CC1 conditioning solution. Monoclonal mouse CA 19.9 (clone 116-NS-19-9) from Signet (Covance) provided comparable results in the pilot study. Colo205 cells are strongly positive with biotinylated 5B1 used at 10 µg/ml, although SKMEL28 cells were completely negative. Histo-Array™ tissue microarrays were purchased from Imgenex (San Diego, CA). The following slides containing tumor biopsy cores as well as some normal tissue cores were used: IMH-327 (Common Cancers, 59 samples), IMH-359 (colorectal: cancer-metastasis-normal; 59 samples), and IMH- 324 (Metastatic ovarian cancer). Pancreatic tumor tissue cores were present in IMH-327.

Live sLeain serum concentration

[00183] Mice carrying Colo205, BxPC3 and DMS79 xenografts were exsanguinated for sLea antigen assays. A group of mice without any tumor served as a control. Mouse sera sLean levels were measured using the ST AIA-PACK CA19.9 kit (Catalog #025271, TOSOH Bioscience Inc, South San Francisco, CA). The assay principle is based on two-site enzyme immunoassay metric. The analysis was performed as described in the manufacturer's instruction manual. The optical density of immunoassay plates was measured by TOSOH AIA2000 automated immunoassay analyzer (TOSOH Bioscience, Inc, San Francisco, CA).

Statistical analysis

[00184] Data values were expressed as the mean ± SD, unless otherwise stated. Statistical analysis was performed using GraphPad Prism software version 5.03 using one-way ANOVA followed by Dunnett's test. A P value of <0.05 is considered statistically significant.

Results

[00185] The binding specificity of 5B1 was probed by staining microarrays of malignant and normal tissue. 5B1 reactivity was restricted to malignancies and occasional normal tissues previously known to overexpress sLea (FIG. 19; Table 9). The majority of normal tissues were completely negative (Table 9). In contrast, strong positive staining was found in 21/34 colon adenocarcinomas (62%), 33/57 adenocarcinoma metastases to the ovary (58%), and 7/9 pancreatic ductal cancers (66%) at various stages (Table 10). As shown in FIG. 19, typical reactivity was diffuse cytoplasmic staining with some tumor cells clearly showing distinct cell membrane staining. In addition, some signet ring ovarian cancers, and some lung and breast cancers have also been found to be strongly positive. In contrast, only 4/43 prostate cancer samples and 0/51 GIST cases were positive (data not shown). TABLE 9: Research on 5B1 binding to normal tissues.

[00186] The high immunostaining specificity of 5B1 for cancer tissues expressing sLea was the basis for the use of this mAb as a PET probe. Modification of 5B1 with the benzyl isothiocyanate analog desferrioxamine (DFO-Bz-NCS) was done in a 4:1 ratio (chelate:mAb) with subsequent purification via centrifugal filtration using saline as the wash buffer. . Facile radiolabeling with Zr-89 proceeded at room temperature after pH adjustment to 7.0-7.2. A narrow pH range, close to neutral, is necessary to obtain optimal radiolabeling yields of >80%. Free, unbound Zr-89 was removed via PD10 desalting column. Product concentration was done using a centrifugal filter (MWCO: 10 kDa). A relatively high specific activity of 12.1±1.1 mCi/mg was established. Radiochemical purities of over 95% were assured prior to use. Immunoreactivity assays showed retention of activity for sLea (72.4 ± 1.1%, n=3). Stability in bovine serum albumin at 37°C was maintained at >95% for 5 days (data not shown). In saline, demetallation was observed as early as 24 hours (>85% complexed) with about >75% radiometal bound after 120 hours at 37°C.

[00187] Small animal PET imaging and biodistribution studies were conducted using female SCID mice subcutaneously implanted with BxPC3 pancreatic cancer xenografts in the left hind leg. Acquired PET images confirmed substantial delineation of the 89Zr-5B1 tumor-associated sLea. From the maximum intensity projections (MIP) in FIG. 20, BxPC3 xenografts (n=3) showed exceptional accretion of the intravenously administered radiotracer. Intersection regions traced over the tumor from PET images showed an uptake of 5.0 ± 0.4% ID/g (2 h), 16.2 ± 2.5% ID/g (24 h), 23 .8 ± 4.7 %ID/g (48 h), 36.8 ± 6.1% ID/g (96 h) and 49.5 ± 7.7% ID/g (120 h). Binding activity of normal tissue and blood lake appeared to clear after 24 h p.i. The results of the biodistribution experiments are consistent with the PET data. High tumor localization of 89Zr-5B1 at 24 hours (84.7±12.3%ID/g, n=4) was observed; Increased uptake was further exhibited at 120 h p.i. (114.1 ± 23.1% ID/g, n=4) (FIG. 21). Tumor uptake exceeds 100% due to the small weight (62.4 ± 0.03 mg). The %ID at 24 h p.i. was found to be ten times greater than that of nonspecific IgG at similar time points (FIG. 21 Inset). Competitive inhibition with 250 µg of non-radiolabeled 5B1 at 24 h p.i. blocked tracer accumulation, defining uptake specificity. Minimal binding of 89Zr-5B1 to normal pancreas and the rest of the harvested normal tissues was observed, providing high tumor-to-tissue contrast at all time points.

[00188] Following the above results, 89Zr-5B1 was tested in an orthotopic BxPC3 pancreas tumor model. Orthotopic models are clinically relevant and offer a clinically accepted test of PET probe efficacy. After inoculation into the pancreas, tumor growth was monitored weekly using bioluminescent optical imaging. PET imaging experiments were conducted once tumors are palpable. A comparison of probe tumor delineation properties was made between FDG-PET and 89Zr-5B1 (FIG. 25). Computed tomography (CT) in tandem with PET gave rise to an enhanced visualization of the anatomical region of interest.

[00189] To evaluate 89Zr-5B1 as a PET probe in other sLea-expressing adenocarcinomas, 89Zr-5B1 was tested in lung and colon cancer models. Small animal experiments were conducted using DMS79 small cell lung cancer cells and Colo205-luc colon cancer cells injected subcutaneously into the right hind leg of female SCID mice. PET MIP images were acquired after 24-120 h p.i. of 200-300 µCi (16-25 µg) injected intravenously. Heterogeneous DMS79 tumor uptake was demonstrated with 38.15 ± 2.12 %ID/g as early as 24 h p.i with excellent signal against background (FIG. 22A). An increase in tumor tracer accumulation resulted after 48 h p.i. (44.60 ± 6.47 %ID/g) with retention at 120 h p.i. (41.97±12.23 %ID/g). Nonspecifically bound 89Zr-5B1 cleared rapidly from normal tissues with minimal to no base uptake at 48 h p.i. Furthermore, tumor delineation was observed in the Colo205-luc xenografts as shown in FIG. 22B at 24-120 h p.i. The ROIs showed tumor accumulation with 10.5 ± 0.76, 23.5 ± 2.7, 24.8 ± 4.0, 18.4 ± 4.7, 16.5 ± 2.3 %ID/g a 2, 24, 48, 96 and 120 hours, respectively. An observable increase in liver accumulation resulted in prolonged time with consequent decrease in tumor uptake as shown in the regions of interest traced from the PET images (FIG. 22C).

[00190] Data generated from biodistribution studies correlate well with observed PET results (data not shown).

[00191] The level of sLea in mouse serum when the tumors progressed was quantified. Exsanguination of SCID mice carrying Colo205, DMS79 and BxPC3 xenografts with a non-tumor carrying group serving as control was performed. sLea values showed high levels of sLea in Colo205-challenged mice compared to pancreatic BxPC3 and DSM79 implanted mice (Table 11). TABLE 11: Serum values for mice carrying colorectal (Colo205), pancreas (BxPC3) and small cell lung (DMS79) tumor xenografts compared to control. ND = Not detected.

[00192] These results demonstrate that a radiolabeled anti-sLea antibody (89Zr-5B1) is specific for the detection and diagnosis of pancreatic adenocarcinoma and other sLea-positive adenocarcinomas. 89Zr-5B1 was produced with excellent yields and purity, along with high specific activity and retained immunoreactivity. Evaluation of 89Zr-5B1 in subcutaneous, orthotopic, and metastatic pancreatic tumor models provided excellent tumor delineation and diagnosis. Preclinical evaluation of this radiotracer in small animals carrying colon and small cell lung tumors demonstrated the universal utility of this tracer for sLea-expressing malignancies.

EXAMPLE III Anti-sLea Diabodies Bind to Multiple Cancer Cell Lines

[00193] Two diabodies were generated using the VH and VL domains of 5B1 and 7E3 clonal isolates described here, designated 5B1CysDb and 7E3CysDb, respectively (FIGS. 9 and 10). Both diabodies contained a five-amino acid linker region between the VL and VH domains. A polyhistidine tag at the C terminus, which was used for purification and detection, was also included for both diabodies.

[00194] Binding of 5B1CysDb and 7E3CysDb to three cancer cell lines: (1) DMS-79 cells, a small cell lung cancer suspension cell line; (2) capan- 2 cells, pancreatic adenocarcinoma cells; and (3) BxPC3 cells, pancreatic cancer cells, were assayed by incubating 0.25 million cells in 0.2 ml with 10 µg/ml of 5B1CysDb or 7E3CysDb, respectively. Cell and diabody combinations were incubated for 40 minutes on ice in PBS/2% FBS.

[00195] After washing, the cells were incubated for 40 minutes with 0.2 ml of ALEXA-488-labeled anti-His antibody diluted 1:1000 (Life Technology, Catalog # A21215). After a second wash, cells were analyzed with a Guava Flow Cytometer. Both 5B1CysDb and 7E3CysDb demonstrated significant binding to DMS-79, Capan-2 and BxPC3 cells (Table 12). TABLE 12: Binding of 5B1CysDb and 7E3CysDb to Cell Lines MFI - mean fluorescent intensity

EXAMPLE IV Administration of 5B1 and Taxol Inhibits Tumor Growth

[0195] The anti-tumor activity of co-administration of an anti-sLea(5B1) antibody and the chemotherapy agent Taxol (Paclitaxel) was evaluated in xenograft models of pancreatic cancer and small cell lung cancer. As previously described here, 1 million BxPc3 cells (pancreatic tumor cells) or 5 million DMS-79 cells (small cell lung cancer cells) were injected into the hind flank of 6-week-old female SCID CB17 mice ( Day 0; N=5). DMS79 tumors were allowed to grow for 21 days until the average tumor size was 193 ± 64 mm3. Human IgG or 5B1 (0.5 or 1 mg) was administered ip twice weekly (starting on day 21), and Taxol (0.2 mg/dose) was administered iv on days 23,30, 37, and 44. In the DMS-79 xenograft model, co-administration of 5B1 antibody and Taxol significantly limited tumor growth and resulted in tumor regression compared to control human IgG or 5B1 antibody and Taxol administered individually (FIG. 23).

[0196] In the BxPc3 xenograft model, tumors were cultured for 14 days, in which they reached an average of 126 ± 30 mm3. Taxol was administered iv on days 14, 21, 28, and 34 (weekly) and 5B1 was administered twice weekly starting on day 14. Coadministration of 5B1 antibody and Taxol significantly limited tumor growth compared to controls or 5B1 antibody. and Taxol administered individually (FIG. 24). These results demonstrate a synergistic effect of an anti-sLea antibody and a chemotherapeutic agent in preventing tumor growth and/or reducing tumor size for pancreatic and small cell lung cancers.

[0197] Throughout this application several publications were referenced. The descriptions of these publications in their entirety are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention belongs. Although the invention has been described with reference to the examples given above, it is to be understood that various modifications may be made without departing from the spirit of the invention.

Claims (26)

1. Isolated polynucleotide, characterized by the fact that it encodes a heavy chain variable domain (VH) and a light chain variable domain (VL) of an antibody or a polypeptide comprising a functional fragment thereof that binds to Sialyl-Lewisa, said polynucleotide comprising the nucleic acid sequence of residues 58 to 435 of SEQ ID NO:13 and comprising the nucleic acid sequence of residues 67 to 390 of SEQ ID NO:15. 2. Isolated polynucleotide, characterized by the fact that it comprises the nucleic acid sequence of residues 58 to 435 of SEQ ID NO:13. 3. Isolated polynucleotide, characterized by the fact that it comprises the nucleic acid sequence of residues 67 to 390 of SEQ ID NO:15. 4. Isolated polynucleotide, characterized by the fact that it comprises the nucleic acid sequence of residues 133 to 156, 208-231 and 346 to 402 of SEQ ID NO:13. 5. Isolated polynucleotide, characterized by the fact that it comprises the nucleic acid sequence of residues 145 to 162, 214 to 222 and 331 to 360 of SEQ ID NO:15. 6. Polynucleotide according to any one of claims 1 to 5, characterized in that the polynucleotide is RNA or mRNA. 7. Isolated antibody or polypeptide comprising a functional fragment thereof that binds to Sialyl-Lewisa, said antibody or polypeptide characterized in that it comprises a heavy chain variable domain (VH) and a light chain variable domain (VL ), wherein said VH domain and said VL domain respectively comprise CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-145 of SEQ ID NO: 14 and CDR1, CDR2 and CDR3 of an amino acid sequence comprising the residues 23-130 of SEQ ID NO: 16. 8. Isolated antibody or functional fragment thereof, according to claim 7, characterized by the fact that said antibody is a human antibody. 9. Isolated antibody or polypeptide according to claim 7, characterized in that said polypeptide is selected from the group consisting of a Fab, a Fab', an F(ab')2, a scFV, a diabody , a tribody and a minibody. 10. Antibody or polypeptide according to claim 9, characterized in that said polypeptide is a diabody. 11. Antibody or polypeptide according to claim 10, characterized in that said diabody comprises the amino acid sequence of SEQ ID NO: 20. 12. Isolated antibody or polypeptide according to claim 7, characterized in that said antibody is a monoclonal antibody. 13. Isolated antibody or polypeptide according to any one of claims 7 to 12, characterized in that said antibody is an IgG or IgM isotype. 14. Isolated antibody or polypeptide according to claim 13, characterized in that said IgG antibody is an IgG1 subclass. 15. Conjugate, characterized by the fact that it comprises an isolated antibody or polypeptide, as defined in any one of claims 7 to 14, conjugated or fused in recombinant form to a diagnostic agent, detectable agent or therapeutic agent. 16. Conjugate, according to claim 15, characterized by the fact that said detectable agent is a radioactive material or a fluorescent material. 17. Conjugate, according to claim 16, characterized by the fact that said radioactive material is zirconium (89Zr), iodine (131I, 125I, 124I, 123I, and 121I,), carbon (14C and 11C), sulfur ( 35S), tritium (3H), indium (115In, 113In, 112In, and 111In,), technetium (99Tc), thallium (201Ti), gallium (68Ga and 67Ga), palladium (103Pd), molybdenum (99Mo), xenon ( 133Xe), fluorine (18F), 15O, 13N, 64Cu, 94mTc, 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 86Y, 90Y, 47Sc, 186Re, 188Re, 142Pr, 1 05Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn and 117Sn, or wherein said fluorescent material is selected from the group consisting of umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, fluorescein dichlorotriazinylamine, fluorescein chloride of dansil and phytoerythrin. 18. Conjugate, according to claim 15, characterized by the fact that said therapeutic agent is a radioactive metal or an Auristatin molecule. 19. Conjugate, according to claim 18, characterized by the fact that said radioactive metal is an alpha emitter, or in which said Auristatin molecule is selected from the group consisting of auristatin PHE, bryostatin 1, solastatin 10, monomethylauristatin E (MMAE) and monomethylauristatin F (MMAF). 20. Pharmaceutical composition, characterized by the fact that it comprises the antibody or polypeptide, as defined in any one of claims 7 to 14, and a pharmaceutically acceptable carrier. 21. Use of the antibody or polypeptide, as defined in any one of claims 7 to 14, characterized by the fact that it is for the preparation of a medicament for the treatment or prevention of a disease, wherein the disease is a cancer or a formation of tumor with cells expressing sLea. 22. Use according to claim 21, characterized in that said cancer or tumor is selected from the group consisting of a tumor of the gastrointestinal tract, colon cancer, colorectal adenocarcinoma, metastatic colon cancer, colorectal cancer, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal cancer, small cell carcinoma of the lung, adenocarcinoma of the bladder, signet ring ovarian cancer, ovarian cancer, metastatic carcinoma, adenocarcinoma of the stomach, adenocarcinoma of the esophagus, adenocarcinoma of the throat, adenocarcinoma of the urogenital tract and breast adenocarcinoma. 23. Use according to claim 21 or 22, characterized by the fact that said treatment further comprises the simultaneous or successive administration of a second therapeutic agent. 24. Use according to claim 23, characterized by the fact that said second therapeutic agent is a chemotherapeutic agent or an immunotherapeutic agent. 25. Composition, characterized by the fact that it comprises nucleic acid sequences that encode the antibody or polypeptide as defined in any one of claims 7 to 14. 26. Use of the isolated polynucleotide as defined in any one of claims 1 to 6, characterized in that it is for the preparation of a medicament for treating or preventing a disease, wherein the disease is a cancer or tumor formation with cells expressing sLea.