NZ717428B2

NZ717428B2 - NUCLEIC ACIDS ENCODING HUMAN ANTIBODIES TO SIALYL-LEWISa

Info

Publication number: NZ717428B2
Application number: NZ717428A
Authority: NZ
Inventors: Ritsuko Sawada; Wolfgang Scholz; Shuman Sun; Shu Man Sun
Original assignee: Biontech Research And Development Inc
Priority date: 2013-08-26
Filing date: 2014-08-26
Publication date: 2021-01-06

Abstract

The present invention provides compositions for the production of an antibody or functional fragment thereof directed against Sialyl-Lewisa (sLea). The compositions of the invention include polynucleotides encoding a heavy chain and/or a 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 that has an amino acid sequence provided herein. The invention further 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 a subject in need thereof. The invention further provides co-administering a chemotherapeutic agent with the antibody or functional fragment thereof. 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 that has an amino acid sequence provided herein. The invention further 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 a subject in need thereof. The invention further provides co-administering a chemotherapeutic agent with the antibody or functional fragment thereof.

Description

Nucleic Acids ng Human Antibodies To Sialyl—Lewisa This application claims the beneﬁt of priority of United States Provisional Application Serial No. 61/870,137, ﬁled August 26, 2013, the entire contents of which is incorporated herein by reference.

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 the invention.

BACKGROUND OF THE ION The present invention relates generally to dies ed against Sialyl-Lewisa (sLea), and more speciﬁcally to polynucleotides encoding anti-sLea antibodies and the corresponding encoded antibodies or nts thereof.

Passive administration of antibodies directed against tumor speciﬁc antigens may eliminate tumor cells and early metastases 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 at the cell surface as glycolipids or glycoproteins. The carbohydrate sLea has been shown to be expressed on tumors of the intestinal tract. sion of sLea has also been shown to impact metastatic potential and correlates with increased metastatic potential in human colon cancer and pancreatic adenocarcinorna. However, carbohydrate chemistry has been rather nging and the clinical development of dies that recognize such tumor speciﬁc carbohydrates has been slow. atic carcinoma is one of the most aggressive adenocarcinomas and is often associated with a poor prognosis. Pancreatic carcinoma ranks as the fourth leading cause of cancer mortality. Despite advances in the screening for different carcinomas, the reliability of detecting malignant lesions stemming from the pancreas remains poor.

Positron emission aphy utilizing ﬂuorodeoxyglucase (FDG-PET) has been indicated for the detection and g of pancreatic cancer. However, FDG-PET is insensitive to differentiating pancreatitis from malignancy and remains problematic in staging small y lesions (< 7mm) and liver metastases (<1 cm). One diagnostic screening method used to monitor the state of pancreatic ductal adenocarcinoma (PDAC) patients includes detecting elevated levels of circulating sLea antigen in sera. ts with > 37 U/ml of circulating sLea antigen indicates cancer recurrence. However, development of alternative diagnostic tools that utilize such tumor specific carbohydrates has been slow.

Thus, there exists a need for identifying and generating antibodies that specifically ize tumor specific carbohydrates, such as sLea, for the treatment of recurring cancers and for detecting malignant lesions and metastases. This invention satisfies this need and provides related advantages, or at least to provide the public with a useful choice.

SUMMARY OF ION The present invention particularly relates to the treatment of cancer or tumor formations having cells sing Sialyl-Lewisa using an antibody or polypeptide comprising a functional fragment thereof, comprising a variable heavy chain (VH) domain and a le light chain (VL) domain, where said VH domain and said VL domain respectively comprise CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-142 of SEQ ID NO: 2 and CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-130 of SEQ ID NO: 4, or a polynucleotide encoding said antibody or polypeptide, and an agent of chemotherapy.

In one particular embodiment of the above aspect, provided is the use of the antibody, polypeptide or polynucleotide, for the preparation of a pharmaceutical ition for the treatment of a cancer or tumor formation having cells expressing Sialyl-Lewisa, said treatment comprising the stration of said composition and an agent of chemotherapy.

In another ular embodiment of the above aspect, provided is the use of an agent of chemotherapy, for the preparation of a pharmaceutical composition for the treatment of a cancer or tumor formation having cells expressing -Lewisa, said treatment sing the administration of said composition and said antibody, polypeptide or polynucleotide.

In certain embodiments said antibody is a human antibody. In other embodiments the polypeptide is selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a scFV, a diabody, a triabody, and a minibody, preferably y, still more preferably a diabody comprising the amino acid sequence of SEQ ID NO: 18.

In some embodiments the dy is a monoclonal antibody. In other embodiments the antibody is an IgG or IgM isotype. Preferably the IgG antibody is an IgG1 subclass.

In certain embodiments the antibody or polypeptide is conjugated or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent. In certain embodiments the detectable agent is a ctive material or a fluorescent material. Preferably the radioactive material is zirconium , iodine (131I, 125I, 124I, 123I, and , carbon (14C and 11C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In,), technetium , 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, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, and 117Sn, or the fluorescent material is selected from the group consisting of umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin. In certain embodiments the therapeutic agent is radioactive metal, for example an alpha-emitter, or an atin molecule, for example auristatin PHE, bryostatin 1, solastatin 10, monomethyl auristatin E (MMAE) and monomethylauristatin F (MMAF).

In certain embodiments said cancer or tumor is selected from the group consisting of a tumor of the gastrointestinal tract, colon cancer, colorectal arcinoma, atic colon cancer, colorectal cancer, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal cancer, small cell carcinoma of the lung, r adenocarcinoma, signet ring ovarian cancer, ovarian cancer, metastatic carcinoma, arcinoma of the stomach, adenocarcinoma of the esophagus, adenocarcinoma of the throat, adenocarcinoma of the urogenital tract, and adenocarcinoma of the breast.

In certain embodiments the treatment comprises concurrent or successive administration of the dy or polypeptide, or the polynucleotide ng said antibody or polypeptide, and the agent of chemotherapy.

In still further ments, the agent of chemotherapy is at least one agent selected from the group consisting of an anthracycline; a taxan; an antimetabolite; an alkylating agent, (followed by page 3A) lomustine, cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisdichlorodiamine platinum (II) (DDP), cisplatin; an otic; an Auristatin molecule; a hormone; a side analoge, a DNA-repair enzyme inhibitor, a kinase inhibitor; a cytotoxic agent, a topoisomerase inhibitor; a DNA minor groove binder; and adenosine deaminase inhibitors; or pharmaceutically acceptable salts, solvates, clathrates, or prodrugs f. In ular embodiments the agent of chemotherapy is a taxan selected from paclitaxel (Taxol) and docetaxel (Taxotere) or is selected from the group consisting of doxorubicin, daunorubicin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil and decarbazine, mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU), lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, actinomycin D, bleomycin, mithramycin, and anthramycin (AMC); auristatin PHE, bryostatin 1, solastatin 10, monomethyl auristatin E (MMAE) and monomethylauristatin F (MMAF), glucocorticoids, progestins, androgens, estrogens, gemcitabine, etoposide, topotecan, Gleevec, imatinib mesylate; maytansine, paclitaxel, alasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, cin, bicin, ubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, 1- dehydrotestosterone, ticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs or homologs thereof.

Further aspects and embodiments are described herein for teness. Certain of these aspects form the subject of the divisional application, NZ 756328.

Thus also provided are compositions for producing antibodies or functional fragments f that bind sLea. The itions include an isolated polynucleotide encoding an antibody or a functional fragment thereof, wherein the antibody includes a variable heavy chain (VH) domain that has an amino acid sequence provided herein. The isolated polynucleotide of the invention can also include a c acid sequence provided herein, wherein the nucleic acid sequence encodes the VH domain of the antibody or functional fragment f.

In another embodiment, the isolated polynucleotide can encode an antibody or a functional fragment f, wherein the antibody includes a variable light chain (VL) domain that has an amino acid sequence provided . The isolated polynucleotide can also include a nucleic acid sequence provided herein, wherein the c acid sequence encodes the VL domain of the antibody or functional fragment thereof. (followed by page 3B) In a particular embodiment provided is an isolated polynucleotide encoding an antibody or a polypeptide comprising a functional nt thereof that binds to Sialyl-Lewisa, said antibody or polypeptide comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, where said VH domain and said VL domain respectively comprise CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-142 of SEQ ID NO: 2 and CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-130 of SEQ ID NO: 4.

The compositions also include an isolated antibody or functional fragment thereof, wherein the antibody binds to sLea. In some embodiments, provided is an isolated antibody or functional fragment thereof that binds to sLea, wherein the antibody or onal nt thereof includes a VH domain having an amino acid sequence provided .

In some embodiments, provided is an isolated antibody or functional fragment thereof that binds to sLea, wherein the antibody or functional fragment f includes a VL domain having an amino acid sequence ed .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 domain respectively include an amino acid sequence for the respective VH and VL s of the clonal isolates provided .

In a particular embodiment provided is an isolated antibody or a polypeptide sing a functional fragment thereof that binds to Sialyl-Lewisa, said antibody or polypeptide comprising a variable heavy chain (VH) domain and a variable light chain (VL) domain, where said VH domain and said VL domain respectively comprise CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-142 of SEQ ID NO: 2 and CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-130 of SEQ ID NO: 4.

In some ments, provided is a conjugate having an antibody or onal nt provided herein that is conjugated or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent. In some aspects, a conjugate of the invention that es a detectable agent can be used in a method for detecting and/or diagnosing tumor formation is a subject. Such methods can include administering an effective amount of the conjugate to a (followed by page 3C) subject in need thereof. In particular embodiments the ion provides the use of such a ate in the manufacture of a medicament for the detection of a tumor which expresses sLea.

In particular embodiment ed is a fusion protein comprising a variable heavy chain (VH) domain or an antigen binding fragment thereof, a variable light chain (VL) domain or an antigen binding fragment thereof, and a heterologous protein, wherein said VH domain and said VL domain respectively se CDR1, CDR2 and CDR3 of an amino acid sequence of residues 20-142 of SEQ ID NO:2 and CDR1, CDR2 and CDR3 of an amino acid sequence of residues 20-130 of SEQ ID NO:4.

In some embodiments, the provided are pharmaceutical compositions having one or more antibody or onal fragment as provided herein and a pharmaceutically acceptable r.

In some aspects, also provided is a method for treating or preventing a disease in a subject in need thereof, by administering a therapeutically effective amount of a pharmaceutical composition as provided herein. In other aspects provided herein is the use of the antibody or polypeptide in the manufacture of a ment for treating or preventing a disease, wherein the disease is a cancer or a tumor formation having cells expressing sLea. In still another aspect, provided is the administering of a second therapeutic agent rently or sively with an antibody or onal fragment as provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS shows the nucleotide sequence and the encoded amino acid sequence of the variable heavy (VH) chain 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 amino acid sequence of SEQ ID NO: 2. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified. shows the nucleotide sequence and the encoded amino acid sequence of the variable light (VL) chain 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 (followed by page 4) 4 2014/052631 nucleotide sequence of SEQ ID NO: 3 and amino acid sequence of SEQ ID NO: 4. The three complementarity determining s (CDRl, CDR2 and CDR3) are also identiﬁed. shows the nucleotide sequence and the encoded amino acid sequence of the variable heavy (VH) chain 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 tide sequence of SEQ ID NO: 5 and amino acid sequence of SEQ ID NO: 6. The three complementarity determining regions (CDRl, CDR2 and CDR3) are also identiﬁed. shows the nucleotide sequence and the encoded amino acid sequence of the variable light (VL) chain 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 amino acid sequence of SEQ ID NO: 8. The three complementarity determining regions (CDRl, CDR2 and CDR3) are also identiﬁed. shows the nucleotide ce and the encoded amino acid sequence of the variable heavy (VH) chain domain of clone 5Hll and a leader sequence that can be used for recombinant expression. The top portion of the ﬁgure shows an ent between the nucleotide sequence of SEQ ID NO: 9 and amino acid sequence of SEQ ID NO: 10. The three complementarity ining regions (CDRl, CDR2 and CDR3) are also identiﬁed. shows the nucleotide sequence and the encoded amino acid sequence of the variable light (VL) chain domain of clone 5H1 l and a leader sequence that can be used for recombinant sion. The top portion of the ﬁgure shows an alignment between the nucleotide ce of SEQ ID NO: ll and amino acid sequence of SEQ ID NO: 12. The three complementarity determining regions (CDRl, CDR2 and CDR3) are also identiﬁed. shows the nucleotide sequence and the encoded amino acid sequence of the variable heavy (VH) chain 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 amino acid sequence of SEQ ID NO: 14. The three complementarity determining regions (CDRl, CDR2 and CDR3) are also identiﬁed. shows the tide sequence and the encoded amino acid ce of the variable light (VL) chain 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 amino acid ce of SEQ ID NO: 16. The three complementarity determining regions (CDRl, CDR2 and CDR3) are also identiﬁed. shows the nucleotide sequence and the d amino acid sequence of a y designated 5B1CysDb haVing CDRl, 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 amino acid sequence of SEQ ID NO: 18. The three mentarity determining regions (CDRl, CDR2 and CDR3) for both the VH and VL domains are identiﬁed in bold and underline text. The linker sequence and polyhistidine tag (Poly His-Tag) with added amino acids are also indicated by italic and underline text. shows the nucleotide sequence and the encoded amino acid sequence of a diabody designated 7E3CysDb haVing CDRl, CDR2 and CDR2 of both the le 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 amino acid sequence of SEQ ID NO: 20. The three complementarity determining regions (CDRl, CDR2 and CDR3) for both the VH and VL domains are identiﬁed in bold and underline text. The linker sequence and polyhistidine tag (Poly His-Tag) with added amino acids are also indicated by italic and underline text. , panels A-E, show the binding of human anti-sLea antibodies to tumor cells ed by ﬂow cytometry. Panel A shows DMS-79 cells stained with recombinant (r) 5B1, 9H3, 5H11, and 7E3 antibodies. Panels B-F respectively shows HT29, BxPC3, SW626, SK-MEL28, and ColoZOS-luc cells d with 1—2 ug/mL of r5B1 or r7E3 plus IgG or IgM-speciﬁc secondary antibody as described in Example I. , panels A and B, show CDC actiVity of rSBl and r7E3 antibodies in comparison to murine 121SLE (IgM) in the ce of human complement (Hu C’) as ed against DMS-79 cells. Human isotype control antibodies, Hu IgG (0) and Hu IgM (9) showed <4% cytotoxicity. A dose response for r5B1 IgG (I), r7E3 IgM (0) and 121SLE mIgM (A) antibodies is shown in panel A. The calculated ECSO (ug/ml) for rSBl (IgG), r7E3 (IgM) and 121SLE (mIgM) antibodies is shown in panel B. , panels A-C, show antibody-dependent cell-mediated xicity (ADCC) of r5B1 antibodies. Panel A shows rSBl-mediated ADCC with human PBMC against DMS- 79 cells. PBMC were tested at E:T ratios from 100:1 to 12.5:1 with DMS-79 tumor cells in the presence or e of 2 ug/mL r5Bl. Panel B shows r5Bl-mediated ADCC with primary human NK cells against DMS-79 cells. NK cells were tested at lower E:T ratios from 5:1 to 0.6:1 with DMS-79 tumor cells in the presence or absence of 2 ug/mL r5B1.

Panel C shows ADCC of r5Bl 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 r5Bl. shows internalization of sLea into BxPC3 cells. BxPC3 pancreatic tumor cells were grown in the presence of r5Bl (anti-sLea) or rlB7 (anti-GD2) antibodies complexed with P, a saporin-conjugated anti-human IgG. After 3 days, the viability of the cells was measured using an 3-(4,5-Dimethylthiazolyl)-2,5-diphenyltetrazolium e (MTT) assay and the sample values were normalized to the values of untreated cultures. shows activity of r5Bl antibody in a xenograft model using Colo205-luc cells.

Severe ed immunodeﬁcient (SCID) mice (5 per group) received 0.5 million Colo205-luc cells by tail vein injection on day 0. Mice received 100 ug r5B1 by intraperitoneal injection on days 1, 7, 14, and 21 (experiment 1, Expl) or on days 1, 4, 7, , 14, and 21 (experiment 2, Exp2) for a total dose of 600 ug. Control (Ctrl) animals received PBS mock injections. shows the effect of r5B1 on Colo205-luc tumors in SCID mice. Mice received 100 ug (V), 300 ug (I) or 1 mg (9) r5Bl antibody per injection as described in Example I.

Control (I) animals received PBS mock injections. shows the ﬂuorescence g of five mice per group for r5Bl treated mice having Colo205-luc tumors at Day 0 and Week 5. The mice ed the treatment regiment depicted in and described in Example I. , panels A and B, shows the anti-tumor activity in a therapeutic subcutaneous xenograft model using DMS-79 cells. Panel A shows the ssion or regression of 5B1 treated mice (5B1 alone (A) or 5B1 + cRGD (V)) in comparison to Human IgG (IgG alone (9) or IgG + cRGD (0)) and PBS injected control (I). Arrows te days of antibody or PBS injections. Panel B shows representative images of treated mice. Arrows indicate absence of any visual tumor. , panels A-F, show 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 a ovary, atic carcinoma from colon tumor. Panel F is a lymph node, metastatic carcinoma, stage IIIA tumor. shows serial PET maximum intensity projection (MIP) images acquired from 2- 120 h with 89Zr radiolabed-SBl antibody (89Zr-5B l) intravenously administered to female SCID mice subcutaneously implanted with BxPC3 atic tumors. PET-MIP g demonstrates high tumor uptake with clearance of non-speciﬁcally bound tracer as early as 24 hours post ion (h pi.) shows biodistribution results that are in ent with the PET data of , with an observed tumor uptake of 84.73::12.28%ID/g. Because of the small tumor s, a plot of tumor uptake sed as %ID versus time is displayed by the inset graph. The tumor %ID display signiﬁcant tumor uptake by 89Zr-SBl at all time points, and, is at least seven-fold greater than non-speciﬁc 89Zr—IgG. Competitive inhibition with cold 5B1 (200 ug) show a decrease in tumor accumulation. , panels A-C, show PET-MIP images of mice-bearing DMS79 (Panel A) and Colo205-luc xenografts (Panel B). P imaging delineation of tumor (T), heart (H) and liver (L) by 89Zr—SBl are indicated. The colorectal Colo205-luc xenografts model displays 89Zr-SBl accumulation peaking at 24 h, which eventually decreases while an increase in non-speciﬁc binding to the liver was exhibited (Panel C). shows a dose dependent inhibition and regression of tumor growth in a DMS-79 small lung cell carcinoma xenograft model treated with sive 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 in comparison to control human IgG (HngG) or 5B1 antibody and Taxol administered individually. Signiﬁcantly differences from control by 2-way ANOVA at p<0.01 (**) and p<0.001 (***) are indicated. N=5. shows the inhibition of tumor growth in a BxPc3 pancreatic carcinoma xenograft model treated with sive co-administration of 5B1 antibody and Taxol (Paclitaxel).

Large arrows on the X axis indicate Taxol plus 5B1 ent, whereas the small arrows indicate 5B1 alone treatment. Co-administration of 5B1 antibody and Taxol signiﬁcantly limited tumor growth in ison to controls (PBS - Ctrl; human IgG — HngG) or 5Bl dy and Taxol administered individually. , panels A and B, show entative images of mice that were orthotopically transplanted with BxPC3-luc pancreatic tumor xenografts. Panel A: The co-registration of FDG-PET and computed tomography (CT) (left) and planar sections of FDG-PET only (right) displayed minimal tumor detection of the tracer with a high uptake in highly metabolic tissues (i.e. heart, H and bladder, B). Panel B: Acquired 89Zr radiolabed-5Bl antibody (89Zr-5B 1) PET image of the same mouse co-registered with CT exhibited exceptional tumor detection of the BxPC3-luc tumor xenografts.

DETAILED DESCRIPTION OF THE INVENTION Carbohydrates expressed on the tumor cell surface can be targets for e 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 y for sLea (5Bl, 9H3, 5Hll and 7E3) were identiﬁed. Two of these antibodies were expressed as recombinant antibodies (r5Bl and r7E3) and further characterized in in vitro and in vivo models. Both antibodies were potent in complement-dependent cytotoxicity (CDC) assays, and the 5Bl antibody was also highly active in antibody-dependent cytotoxicity assays. The in viva efﬁcacy of the antibodies were tested in two xenograft models using either Colo205 tumor cells or DMS-79 tumor cells engrafted into severe combined deﬁcient (SCID) mice. The translational relevance of the invention provided herein is 2 fold: First, the ch provided herein demonstrates that the antibody response elicited by a sLea-KLH vaccine is useful as a vaccine itself. Second, the most potent antibodies that were ted in a clinical trial can be preserved and ultimately used as therapeutics, or in the generation of eutics, for a target cancer population.

The high afﬁnity of the antibodies provided herein and their high effector ons support this translational potential.

As used herein, the term “antibody” is intended to mean a ptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a speciﬁc molecular n and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa) and each amino-terminal portion of each chain includes a le region of about 100 to about 130 or more amino acids and each carboxy-terminal portion of each chain includes a constant region (See aeck (ed.) (1995) Antibody Engineering, Second Edition, Oxford sity Press; Kuby (1997) Immunology, Third Edition, W.H. Freeman and Company, New York). In the context of the present invention, the c molecular antigen that can be bound by an antibody of the invention includes the target carbohydrate sLea.

The term “human” when used in reference to an antibody or a functional fragment thereof refers an dy or ﬁanctional fragment thereof that has a human variable region and/or a human nt region or a portion f 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, US. Department of Health and Human Services, NIH Publication No. 91-3242.

A human antibody, in the context of the present invention, can include an antibody that binds to sLea and is d by a nucleic acid sequence that is a naturally occurring c 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.

The term “monoclonal antibody” refers to an antibody that is the product of a single cell clone or hybridoma or a population of cells derived from a single cell. A monoclonal antibody also is intended to refer to an antibody produced by inant methods from heavy and light chain encoding immunoglobulin genes to produce a single molecular immunoglobulin species. Amino acid sequences for antibodies within a monoclonal antibody preparation are substantially homogeneous and the binding activity of antibodies within such a preparation t substantially the same antigen binding activity. In contrast, polyclonal antibodies are obtained from ent B cells within a population, which are a combination of immunoglobulin molecules that bind a specific antigen. Each immunoglobulin of the polyclonal antibodies can bind 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 tory Press (1989) and Borrebaeck (ed.), dy Engineering: A cal Guide, W.H. Freeman and Co., Publishers, New York, pp. 103-120 (1991)).

W0 2015/053871 10 PCT/USZOl4/052631 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 heavy or light chain polypeptides that retains some or all of the binding activity as the antibody from which the fragment was derived. Such onal fragments can include, for example, an Fd, Fv, Fab, F(ab’), F(ab)2, F(ab’)2, single chain Fv (scFv), diabody, triabody, tetrabody and minibody. Other functional fragments can include, for example, heavy or light chain polypeptides, variable region polypeptides or CDR polypeptides or portions thereof so long as such onal fragments retain binding activity. Such dy 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 her, Inc.; Huston et al., Cell sics, 22:189-224 (1993); Pliickthun and Skerra, Meth. l., 178:497-515 (1989) and in Day, E.D., Advanced Immunochemistg, Second Ed., Wiley- Liss, Inc., New York, NY (1990).

The term “heavy chain” when used in reference to an antibody refers to a ptide chain of about 50-70 kDa, wherein the 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 (5), epsilon (a), gamma (y) and mu (u), based on the amino acid sequence of the heavy chain constant region. The ct heavy chains differ in size: or, 5 and y contain approximately 450 amino acids, while u and 8 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, namely IgG1, IgG2, IgG3 and IgG4. A heavy chain can be a human heavy chain.

The term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, n the amino-terminal n 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 length of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as kappa (K) of lambda (7») based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. A light chain can be a human light chain.

W0 2015/053871 11 PCT/USZOl4/052631 The term ble domain” or “variable ” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal 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 used in the binding and speciﬁcity of each particular antibody for its particular antigen. The variable domains differ extensively in sequence between different antibodies. The variability in sequence is concentrated in the CDRs while the less variable portions in the variable domain are referred to as ork regions (FR). The CDRs of the light and heavy chains are ily responsible for the interaction of the dy with antigen. Numbering of amino acid positions used herein is according to the EU Index, as in Kabat et al. (1991) Sequences of proteins of immunological interest. (US. Department of Health and Human Services, Washington, DC.) 5th ed. A variable region can be a human variable region.

A CDR refers to one of three hypervariable regions (Hl H2 or H3) within the non- framework region of the immunoglobulin (Ig or antibody) VH B-sheet framework, or one of three hypervariable regions (Ll L2 or L3) within the non-framework region of the antibody VL B-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework 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 antibody variable (V) domains (Kabat et al., J. Biol.

Chem. 09-6616 (1977); Kabat, Adv. Prot. Chem. 32: 1-75 (1978)). CDR region sequences also have been deﬁned structurally by Chothia as those residues that are not part of the conserved B-sheet framework, and thus are able to adapt different conformations (Chothia 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 have been determined by comparison of numerous 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 ent antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the cal variable domain numbering scheme (Al- Lazikani et al., supra (1997)). Such nomenclature is similarly well known to those skilled in the art.

For example, CDRs deﬁned ing to either the Kabat variable) or Chothia tural) designations, are set forth in the Table 1 below.

W0 2015/053871 12 PCT/USZOl4/052631 Table l: CDR Deﬁnitions Kabat1 Chothia2 Loop Location VH CDRl 31-35 26-32 linking B and C strands VH CDR2 50-65 53-55 linking C’ and C” strands VH CDR3 95-102 96-101 linking F and G strands VL CDRl 24-34 26-32 linking B and C strands VL CDR2 50-56 50-52 linking C’ and C” strands VL CDR3 89-97 91-96 linking F and G strands 1 Residue numbering s the nomenclature of Kabat et al., supra 2 Residue numbering follows the nomenclature of Chothia et al., supra One or more CDRs also can be incorporated into a molecule either ntly or noncovalently to make it an immunoadhesin. An immunoadhesin can incorporate the CDR(s) as part of a larger polypeptide chain, can covalently link the CDR(s) to r polypeptide chain, or can incorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesin to bind to a particular antigen of interest.

As used herein, the term “isolated” when used in reference to an dy, antibody onal fragment or polynucleotide is ed to mean that the referenced molecule is free of at least one component as it is found in nature. The term includes an antibody, antibody ﬁanctional fragment or polynucleotide that is removed from some or all other components as it is found in its natural environment. Components of an antibody’s natural environment include, for example, erythrocytes, leukocytes, thrombocytes, plasma, proteins, nucleic acids, salts and nts. Components of an antibody functional fragment’s or polynucleotide’s natural environment include, for example, lipid membranes, cell organelles, proteins, nucleic acids, salts and nutrients. An dy, antibody ﬁanctional nt or cleotide of the invention can also be free or all the way to substantially free from all of these components or any other component of the cells from which it is isolated or recombinantly produced.

W0 2015/053871 13 PCT/USZOl4/052631 As used herein, "isotype" refers to the antibody class that is encoded by heavy chain constant region genes. The heavy chains of a given antibody or onal nt determine the class of that antibody or functional fragment: IgM, IgG, IgA, IgD or IgE.

Each class can have either K or 9» light chains. The term “subclass” refers to the minor differences in amino acid sequences of the heavy chains that differentiate the subclasses.

In humans there are two subclasses of IgA (subclasses IgAl and IgA2) and there are four subclasses of IgG asses IgGl, IgG2, IgG3 and IgG4). Such s and subclasses are well known to those skilled in art.

The terms “binds” or “binding” as used herein refer to an interaction between molecules to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions.

A complex can also include the g of two or more molecules held together by covalent or non-covalent bonds, interactions or forces. Binding of an antibody or functional fragment thereof can be ed using, for e, an -linked immunosorbant assay, a method provided in Example I or any one of a number of methods that are well known to those skilled in the art.

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 ﬁanctional fragment for that epitope. The ratio of ation (k1) to dissociation (k_1) of an antibody or ﬁanctional fragment thereof to a monovalent n (k1/ k_1) is the association constant K, which is a measure of affinity.

The value of K varies for different complexes of dy or onal fragment and antigen and depends on both k; and k_1. The association constant K for an antibody or onal nt of the invention can be determined using any method provided herein or any other method well known to those skilled in the art.

The affinity at one binding site does not always reﬂect the true strength of the interaction between an antibody or functional fragment and an antigen. When complex antigens containing multiple, repeating antigenic determinants, such as a polyvalent sLea, come in contact with antibodies containing multiple binding sites, the interaction of antibody or ﬁanctional fragment with antigen at one site will increase the probability of a reaction at a second site. The strength of such multiple interactions n a multivalent antibody and antigen is called the avidity. The avidity of an antibody or ﬁanctional fragment can be a better measure of its binding capacity than is the affinity of its individual binding sites.

W0 2015/053871 14 PCT/USZOl4/052631 For example, high avidity can compensate for low afﬁnity as is sometimes found for eric IgM antibodies, which can have a lower afﬁnity than IgG, but the high y of IgM, resulting from its alence, enables it to bind antigen effectively.

The speciﬁcity of an antibody or onal fragment thereof refers to the y of an individual dy or ﬁlnctional fragment thereof to react with only one antigen. An antibody or ﬁlnctional fragment can be considered speciﬁc when it can distinguish differences in the primary, ary or tertiary structure of an antigen or isomeric forms of an antigen.

The term "polynucleotide" refers to a polymeric form of nucleotides of any , either deoxyribonucleotides or ribonucleotides or analogs thereof. The sequence of a polynucleotide is composed 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 can include a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, s, ger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched cleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotide also refers to both double- and single-stranded molecules. Unless otherwise speciﬁed or required, any embodiment of this invention that is a cleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up 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 can include, but are not limited to, cDNA and chemically synthesized molecules.

The term “encode” or grammatical equivalents thereof as it is used in reference to polynucleotides refers to a polynucleotide in its native state or when manipulated by s 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 encoding sequence can be deduced therefrom.

The phrase “therapeutic agent” refers to any agent that can be used in the treatment, management or amelioration of a e associated with expression of sLea and/or a symptom related thereto. In certain embodiments, a eutic agent refers to an antibody or ﬁanctional nt 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 can be an agent which is well known to be useful for, or has been or is currently being used for the ent, ment or ration of a disease associated with expression of sLea and/or one or more symptoms related thereto.

The phrase “diagnostic agent” refers to a substance administered to a subject that aids in the diagnosis of a disease. Such substances can be used to reveal, pinpoint, and/or deﬁne the zation 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, that when administered to a subject or contacted to a sample from a subject aids in the diagnosis of cancer or tumor formation.

The phrase “detectable agent” refers to a substance that can be used to ascertain the existence or presence of a desired molecule, such as an antibody or ﬁanctional fragment of the invention, in a sample or subject. A detectable agent can be a nce that is capable of being visualized or a nce that is otherwise able to be determined and/or measured (e.g., by quantitation).

An "effective amoun " is an amount sufﬁcient to effect beneﬁcial or desired s. An effective amount can be stered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the agent, the route of administration, etc.

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) which is ient to reduce and/or ameliorate the severity and/or duration of a given disease and/or a symptom related thereto. A therapeutically effective amount of a therapeutic agent can be an amount necessary for the reduction or amelioration of the advancement or progression of a given e, reduction or amelioration of the recurrence, development or onset of a given disease, and/or to improve or enhance the prophylactic or therapeutic effect of r therapy (e.g., a W0 53871 16 PCT/USZOl4/052631 therapy other than the administration of an antibody or ﬁanctional fragment provided herein).

The compound “Sialyl-Lewisa” , also known as sialyl Lea, Sialyl-Lewis A, Sialylated Lewis a and CA 19.9, is a tetrasaccharide with a molecular formula of C31H52N2023 and a molar mass of 820.74 g/mol. The structure of sLea can include NeuSAcu2-3GalB l -3(Fuc0Ll -4)GlcNAcB and NeuSch2-3GalB l -3(Fuc0Ll -4)GlcNAcB. sLea is widely expressed on tumors of the gastrointestinal tract and is used as a tumor marker in pancreatic and colon cancer. sLea is also a known ligand for E-selection, also known as elial leukocyte adhesion molecule .

In some embodiments, the present ion 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 that has an amino acid sequence selected from the group ting of residues 20-142 of SEQ ID NO: 2, residues 20-142 of SEQ ID NO: 6, es 20-142 of SEQ ID NO: 10, and residues 20-145 of SEQ ID NO: 14. The isolated polynucleotide of the invention can 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 nucleic acid sequence encodes the VH domain of the antibody or ﬁanctional fragment thereof.

In another embodiment of the invention, the isolated polynucleotide can encode an antibody or a functional fragment thereof, n the antibody includes a VL domain that has an amino acid ce ed 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 can also include a nucleic acid sequence of residues 58-390 of SEQ ID NO: 3, es 58-387 of SEQ ID NO: 7, residues 58-390 of SEQ ID NO: 11 or residues 67-390 of SEQ ID NO: 15, n the nucleic acid ce encodes the VL domain of the antibody or ﬁanctional fragment thereof.

In another embodiment, the invention provides an isolated polynucleotide encoding an antibody heavy or light chain or a ﬁanctional fragment thereof, wherein the antibody heavy 17 2014/052631 or light chain or functional fragment thereof encoded by the polynucleotide of the invention has one or more of the complementarity determining s (CDRs) depicted in FIGS. 1-8 or listed in Table 2. An antibody or ﬁlnctional fragment thereof that includes one or more of the CDRs can speciﬁcally bind to sLea as described herein. Speciﬁc g to sLea can include the speciﬁcity, afﬁnity and/or y as provided in Example I for any of the antibodies provided herein. In another aspect, an antibody or functional fragment thereof encoded by the polynucleotides of the invention can include the complement dependent cytotoxicity (CDC) activity and/or antibody-dependent cell- mediated cytotoxicity (ADCC) activity of any one of the clonal isolates 5B1, 9H3, 5H11 or 7E3 described herein. Methods for assessing the speciﬁcity, afﬁnity and/or y of an antibody or ﬁlnctional fragment f are well known in the art and ary methods are provided herein.

Table 2: CDRs of Clonal Isolates Nucleic Acid Residues Amino Acid Residues (SEQ ID N0:) (SEQ ID N0:) ““2"?” CDRI CDR2 CDR3 CDRI CDR2 CDR3 Domaln 5B1 VH 133- 156 208-231 3 55-62 70-77 116- 131 5B1 VL 133- 156 208-216 325-360 45-52 70-72 109- 120 9H3 VH 133- 156 1 346-393 45-52 70-77 116- 131 0:5 NO: 5 0:5 0:6 0:6 0:6 9H3 VL 133-156 208-216 325-357 45-52 70-72 109-119 0:7 NO: 7 0:7 0:8 0:8 0:8 5H11 VH 133- 156 208-231 346-393 45-52 70-77 1 5H11 VL 134-156 208-216 325-360 45-52 70-72 109-120 NO:11 0:11 NO:11 NO: 12 NO: 12 NO: 12 7E3 VH 133-156 208-231 346-402 45-52 70-77 116-134 NO: 13 0:13 NO: 13 NO: 13 NO: 13 NO: 14 7E3 VK 145-162 214-222 331-360 49-53 72-74 111-120 NO: 15 0:15 NO: 15 NO: 16 NO: 16 NO: 16 In some embodiments, the antibody or ﬁlnctional fragment thereof of the invention includes less than six CDRs. In some embodiments, the antibody or ﬁlnctional fragment thereof includes one, two, three, four, or ﬁve CDRs ed 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 ﬁlnctional fragment thereof includes one, two, three, four, or ﬁve CDRs selected from the group consisting ofVH CDR1, VH CDR2, VH CDR3, VL W0 2015/053871 18 PCT/USZOl4/052631 CDRl, VL CDR2, and/or VL CDR3 of clonal isolates 5B1, 9H3, 5Hll or 7E3 described herein.

In some embodiments, the invention provides an isolated polynucleotide that encodes an antibody or ﬁanctional fragment thereof, wherein the antibody or functional fragment includes a variable heavy (VH) chain domain having the CDRl CDR2 and CDR3 amino acid sequence of the clonal e 5B1, 9H3, 5Hll or 7E3. Such VH domains can e the amino acid residues 55-62, 70-77 and 116-131 of SEQ ID NO: 2, or alternatively the amino acid residues 45-52, 70-77 and 116-131 of SEQ ID NO: 6, or atively the amino acid residues 45-52, 70-77 and 116-131 of SEQ ID NO: 10, or alternatively the amino acid residues 45-52, 70-77 and 116-134 of SEQ ID NO: 14. In another aspect, the nucleotide sequence encoding the CDRl, CDR2 and CDR3 of the VH domain can 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 6, 208-231 and 346-393 of SEQ ID NO: 9, or alternatively the nucleotide sequence of residues 133-156, 208-231, 346-402 of SEQ ID NO: 13.

In another embodiment, the invention provides an isolated polynucleotide encoding an antibody or ﬁanctional fragment f, wherein the antibody es a variable light (VL) chain domain having the CDRl , CDR2 and CDR3 amino acid ce of the clonal isolate 5B1, 9H3, 5H1 l or 7E3. Such VL domain can include the amino acid residues 45- 52, 70-72 and 109-120 of SEQ ID NO: 4, or alternatively the amino acid residues 45-52, 70-72 and 109-1 19 of SEQ ID NO: 8, or alternatively the amino acid es 45-52, 70- 72 and 109-120 of SEQ ID NO: 12, or alternatively the amino acid residues 49-53, 72-74 and 111-120 of SEQ ID NO: 16. In another aspect, the tide ce encoding the CDRl, CDR2 and CDR3 of the VH domain can respectively include the nucleotide sequence of residues 133-156, 208-216 and 325-360 of SEQ ID NO: 3, or alternatively the nucleotide sequence ofresidues 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: ll, or alternatively the nucleotide sequence of residues 145-162, 214-222 and 0 of SEQ ID NO: 15 In another embodiment, the invention es a t of the polynucleotides provided herein. A variant when used in reference to a cleotide includes a polynucleotide having one or more modif1ed nucleotides, such as, but not limited to, a methylated W0 2015/053871 19 PCT/USZOl4/052631 nucleotide or a nucleotide analog. Additionally, a variant polynucleotide can include a cleotide that is interrupted by non-nucleotide ents. Modiﬁcations to a polynucleotide can be imparted 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 labeling component using either enzymatic or chemical ques (e.g, as described in Gottfried and Weinhold, 2011, Biochem. Soc. Trans., 523-628; Paredes et al., 2011, Methods, 54(2):251-259).

The polynucleotides can be obtained, and the nucleotide sequence of the polynucleotides determined, by any method well known in the art. Since the amino acid sequences of the variable heavy and light chain domains of 5B1, 9H3, 5H11 and 7E3 are known (see, e.g., SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14 and 16), nucleotide sequences encoding dies and modiﬁed versions of these antibodies can be determined using methods well known in the art, z'.e., nucleotide codons known to encode particular amino acids are led in such a way to te a c acid that encodes the antibody. Such a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, Bio Techniques 17242), which, brieﬂy, involves the synthesis of overlapping oligonucleotides containing portions of the sequence ng the antibody, fragments, or variants thereof, annealing and ligating of those oligonucleotides, and then ampliﬁcation of the ligated oligonucleotides by PCR.

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 ofisolates 5B1, 9H3, 5H11 or 7E3 (e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 and ). A nucleic acid encoding the antibody or ﬁlnctional fragment can be chemically synthesized or obtained from a suitable source (e.g., cDNA isolated from cells expressing the antibody or onal fragment thereof, such as hybridoma cells selected to express the antibody or functional fragment f) by PCR ampliﬁcation using synthetic primers hybridizable to the 3’ and 5’ ends of the sequence or by cloning using an oligonucleotide probe speciﬁc for the particular nucleic acid ce. Ampliﬁed nucleic acids generated by PCR can then be cloned into able cloning vectors using any method well known in the art.

In some embodiments, the present invention provides an isolated antibody or ﬁlnctional fragment thereof, wherein the antibody binds to sLea. Accordingly, in some s, the invention provides an isolated dy or ﬁlnctional fragment thereof that binds to sLea, W0 2015/053871 20 PCT/USZOl4/052631 wherein the antibody or ﬁanctional fragment thereof includes a VH domain having an amino acid sequence ed 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 some embodiments, the invention provides an isolated antibody or ﬁanctional fragment thereof that binds to sLea, wherein the antibody or ﬁanctional fragment thereof es a VL domain having an amino acid sequence selected from the group consisting of residues -130 of SEQ ID NO: 4, residues 20-129 of SEQ ID NO: 8, es 20-130 of SEQ ID NO: 12, and residues 23-130 of SEQ ID NO: 16.

In some embodiments, the invention provides an isolated dy or ﬁanctional fragment thereof that binds to sLea, n the dy or ﬁanctional fragment thereof includes both a VH domain and a VL domain, where the VH domain and the VL domain 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: 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.

In some embodiments, in order to bind sLea, the antibody or onal fragment thereof of the invention has one or more of the CDRs depicted in FIGS. 1-8 or listed in Table 2. An dy or onal 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 can include the specificity and affinity as provided in Example I for any of the antibodies provided herein. In some aspects, an antibody or ﬁanctional fragment thereof of the invention can include the CDC activity and/or ADCC activity of any one of the clonal isolates 5B1, 9H3, 5Hll or 7E3 described herein.

In some ments, the invention provides an isolated antibody or ﬁanctional nt thereof, wherein the antibody includes a VH chain domain having the CDRl, CDR2 and CDR3 amino acid sequence of the clonal isolate 5B1, 9H3, 5Hll or 7E3. Such VH domains can include the amino acid residues 55-62, 70-77 and 116-131 of SEQ ID NO: 2, or alternatively the amino acid residues 45-52, 70-77 and 116-131 of SEQ ID NO: 6, or alternatively the amino acid residues 45-52, 70-77 and 116-131 of SEQ ID NO: 10, or alternatively the amino acid es 45-52, 70-77 and 116-134 of SEQ ID NO: 14.

W0 2015/053871 21 PCT/USZOl4/052631 In some embodiments, the invention provides an isolated antibody or ﬁanctional fragment thereof, wherein the antibody includes a VL chain domain having the CDRl, CDR2 and CDR3 amino acid sequence of the clonal isolate 5B1, 9H3, 5Hll or 7E3. Such VL domain can include the amino acid residues 45-52, 70-72 and 109-120 of SEQ ID NO: 4, or alternatively the amino acid residues 45-52, 70-72 and 109-1 19 of SEQ ID NO: 8, or alternatively the amino acid residues 45-52, 70-72 and 109-120 of SEQ ID NO: 12, or alternatively the amino acid residues 49-53, 72-74 and 111-120 of SEQ ID NO: 16.

In some aspects of the invention, the isolated antibody or functional nt f is a monoclonal dy. In some aspects of the invention, the isolated antibody or functional nt thereof provided herein is an IgG or IgM isotype. In a further aspect of the invention, the antibody or function fragment f is an antibody of the IgGl subclass.

In some embodiments, the antibody functional fragment of the invention can be, but is not limited to, a Fab, a Fab’, a F(ab’)2, a Fabc, a 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 ce of SEQ ID NO: 18 or 20. Such diabodies of the invention can be, in some aspects, d by a polynucleotide having the nucleic acid sequence of SEQ ID NO: 17 or 19. With t to antibodies and ﬁanctional fragments thereof, various forms, alterations and modiﬁcations are well known in the art. The sLea speciﬁc antibody fragments of the invention can include any of such various antibody forms, alterations and ations. Examples of such various forms and terms as they are known in the art are set forth below.

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

Recombinant expression of an antibody or functional fragment thereof of the invention that binds to a sLea n can include construction of an expression vector containing a polynucleotide that encodes 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 the heavy and/or light chain W0 2015/053871 22 PCT/USZOl4/052631 variable domain) of the invention has been obtained, the vector for the production of the antibody or ﬁanctional fragment can be produced by recombinant DNA technology using techniques well known in the art. Methods for preparing a protein by expressing a polynucleotide ning an antibody or a functional nt thereof encoding nucleotide sequence are described herein.

Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody or functional fragments thereof coding sequences and appropriate transcriptional and translational control signals. These methods e, for example, in vitro recombinant DNA techniques, synthetic techniques, and in viva genetic ination. The ion, 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 can include the nucleotide sequence encoding the constant region of the dy molecule (see, e.g, ational Publication Nos. WO 86/05807 and WO 36; and US. Patent No. 464) and the variable domain of the antibody can be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.

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

A variety of host-expression vector systems can be utilized to express the antibody or functional fragments thereof of the invention (see, e.g., US. Patent No. 5,807,715). Such host-expression systems represent vehicles by which the coding sequences of interest can be ed and subsequently puriﬁed, but also represent cells which can, when transformed or transfected with the appropriate tide coding sequences, s 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 sion s containing antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed W0 2015/053871 23 with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression s (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliﬂower mosaic virus, CaMV; tobacco mosaic virus, TMV) or ormed with recombinant plasmid sion vectors (e.g, Ti plasmid) containing antibody coding ces; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring recombinant expression constructs ning promoters derived from the genome alian cells (e.g, metallothionein promoter) or from mammalian viruses (e.g, the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In some aspects, bacterial cells such as Escherichia coli, or eukaryotic cells, especially for the expression of whole recombinant dy, are used for the expression of a recombinant antibody or functional fragment. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene er t from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990, Bio/Technology 8:2). In some embodiments, 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 which bind to sLea is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.

In bacterial systems, a number of expression vectors can be advantageously ed depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such an antibody is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12: 1791), in which the antibody coding sequence can be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13 :3 101-3 109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors can also be used to s foreign polypeptides as fusion proteins with hione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be puriﬁed from lysed cells by adsorption and binding to matrix glutathione e beads followed by n in the presence of free hione. The pGEX vectors are ed to include thrombin or W0 2015/053871 24 PCT/USZOl4/052631 factor Xa protease ge sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa calz'form'ca nuclear polyhedrosis virus (AcNPV) is used as a vector to s foreign genes. The virus grows in teraﬁugz’perda cells. The antibody or ﬁanctional fragment coding sequence can be cloned individually into non- essential regions (for e the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of based expression systems can be utilized. In cases where an irus is used as an expression vector, the antibody coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in viva recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) 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 1355-359). Speciﬁc initiation signals can also be used for efﬁcient ation of inserted antibody coding sequences. These s 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 entire insert. These exogenous translational control signals and initiation codons can be of a variety of s, both natural and synthetic. The efﬁciency of expression can be enhanced by the inclusion of appropriate ription enhancer elements, transcription ators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153 :5 1-544).

In addition, a host cell strain can be chosen which modulates the sion of the inserted ces, or modif1es and processes the gene product in the speciﬁc fashion desired.

Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the ﬁanction of the antibody or functional fragment.

Different host cells have characteristic and specific mechanisms for the post-translational processing and modiﬁcation of proteins and gene products. riate cell lines or host systems can be chosen to ensure the correct modiﬁcation and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene t can be used. Such mammalian host cells include but are not limited to CH0, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (a murine myeloma cell line that does not endogenously produce any globulin chains), CRL703O and HsS78Bst cells.

For long-term, high-yield tion of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody or functional fragment of the invention can be engineered. Rather than using expression s which n viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression l elements (e.g, promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express the antibody molecule.

A number of selection systems can be used, including but not limited to, the herpes x virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthineguanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine oribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite ance can be used as the basis of selection for the ing 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, Biaotechnology 10:169); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 2); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. T0xic0l. 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 logy can be ely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current W0 2015/053871 26 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 by reference herein in their entireties.

The expression levels of an dy molecule can be increased by vector ampliﬁcation (for a review, see Bebbington and Hentschel, The use of vectors based on gene ampliﬁcation for the sion 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 ﬁanctional nt thereof is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also se (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

The host cell can be co-transfected with two sion vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector ng a light chain derived polypeptide. The two vectors can contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector can be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain can be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197-2199). The coding sequences for the heavy and light chains can include cDNA or genomic DNA.

Additionally, polynucleotides ng the heavy and/or light chains of the dy or functional fragment of the invention can be subjected to codon optimization using techniques well known in the art to achieve optimized expression of an dy or onal fragment of the invention in a d host cell. For example, in one method of codon optimization, a native codon is substituted by the most frequent codon from a reference set of genes, wherein the rate of codon ation 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 the heavy and/or light chains of the antibody or functional fragment of the invention, are described in Kanaya et al., Gene, 3-155 (1999), Wang et al., Mol. Biol. Evol., 18(5):792-800 (2001), US. Patent 5,795,737, US. Publication 2008/0076161 and .

Once an antibody molecule of the invention has been produced by recombinant expression, it can be puriﬁed by any method known in the art for puriﬁcation of an immunoglobulin molecule, for example, by chromatography (e.g, ion exchange, afﬁnity, ularly by y for the speciﬁc antigen after Protein A, and sizing column chromatography), ﬁlgation, differential solubility, or by any other standard technique for the puriﬁcation of proteins. Further, the antibodies or functional fragments of the present invention can be fused to logous polypeptide sequences ed herein or otherwise known in the art to facilitate puriﬁcation. For example, an antibody or functional fragment of the invention can be puriﬁed through inantly adding a poly- ine tag (His-tag), FLAG-tag, hemagglutinin tag (HA-tag) or myc-tag among others that are commercially available and ing puriﬁcation methods well known to those skilled in the art.

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

An antibody can have one or more binding sites. If there is more than one binding site, the binding sites can be identical to one r or can be different. For example, a naturally occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a “bispeciﬁc” or “biﬁlnctional” antibody has two different binding sites.

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 continuous polypeptide chain n the linker is long enough to allow the n chain to fold back on itself and form a monovalent antigen g site (see, e. g., Bird et al., Science 242:423-26 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-83 (1988)).

Diabodies refer to bivalent antibodies including two polypeptide chains, n each polypeptide chain includes VH and VL domains joined by a linker that is too short to allow for pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993), and Poljak et al., Structure 2: 1 121-23 (1994)). If the two polypeptide chains of a diabody are cal, then a diabody resulting W0 2015/053871 28 PCT/USZOl4/052631 from their pairing will have two identical antigen binding sites. Polypeptide chains having different sequences can be used to make a y 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 can be the same or different.

The present invention also provides an antibody or ﬁanctional fragment f derivative of 5B1, 9H3, 5H1 1 and/or 7E3, wherein the antibody or functional fragment binds to sLea.

Standard techniques well known to those of skill in the art can be used to introduce mutations in the nucleotide ce ng an antibody or functional fragment thereof of the invention, including, for example, site-directed mutagenesis and diated mutagenesis which results in amino acid substitutions. In some s, the derivative includes less than 25 amino acid substitutions, less than 20 amino acid tutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original molecule.

In some embodiments, the ion provides an antibody or ﬁanctional fragment thereof having modified forms of naturally ing amino acids, conservative substitutions, non- naturally occurring amino acids, amino acid analogues and mimetics so long as such the dy or onal fragment retains functional activity as defined herein. 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 with 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., ic acid, glutamic acid), uncharged polar side chains (e.g., glycine, gine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., ine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, phan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain W0 2015/053871 29 PCT/USZOl4/052631 activity. Following mutagenesis, the encoded antibody or functional fragment thereof can be expressed and the actiVity of the antibody or functional fragment can be determined.

In some embodiments, the invention provides an antibody or ﬁanctional fragment thereof haVing modified fucosylation, galactosylation and/or sialylation of an Fc fragment contained within an antibody or onal fragment of the invention. Such cations of an Fc fragment can effect Fc receptor—mediated actiVity as discussed in Peipp et al., Blood, :2390-2399 (2008). For example, glycoengineered eutic dies lacking core fucose residues from the Fc N—glycans exhibit strong ADCC at lower concentrations with much higher efficacy compared to fucosylated counterparts. Shields et al., J. Biol. Chem, 277(30):26733-40 (2002); Okazaki et al., JMol Bz'ol., 336:1239— 1249 (2004); Natsume et al., J. Immunol. Methods, 306:93—103 (2005). Methods for modifying the ﬁlcosylation, galactosylation and/or sialylation of an dy for functional fragment thereof are well known in the art. For example, defucosylation approaches can be grouped into three ologies (1) conversion of the N—glycosylation pathway of nonmammalian cells to the ‘humanized’ non-fucosylation pathway; (2) inactivation of the N—glycan lation pathway of ian cells and (3) in vitro chemical synthesis of non-ﬁlcosylated N—glycoprotein or enzymatic modification ofN—glycans to nonfucosylated forms, as described in Yamane-Ohnuki et al., MAbs., 1(3):230-236 (2009). It is tood that any one of these methods or any other method that is well known in the art can be used to produce an antibody or functional fragment f having modified fucosylation, galactosylation and/or ation.

Antibodies or functional fragments thereof of the invention that bind to sLea can be ed 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 y, microbiology, c analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and cation, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described in the references cited herein and are fully explained in the literature. See, e.g., , Maniatis et al. (1982) Molecular Cloning: A tory 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 Laboratog Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; W0 2015/053871 30 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 sis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) ucleotides and Analogues: A Practical Approach, IRL Press; Birren et al. (eds.) (1999) Genome Analysis: A Laboratopy Manual, Cold Spring Harbor Laboratory Press; Borrebaeck (ed.) (1995) dy Engineering, Second Edition, Oxford University Press; Lo (ed.) (2006) Antibody ering: Methods and Protocols (Methods in Molecular y); Vol. 248, Humana Press, Inc; each of which is incorporated herein by reference in its entirety.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of oma and recombinant logies, 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 tory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y., 198 1), each of which is incorporated herein by reference in its ty. A monoclonal antibody is not limited to antibodies produced through hybridoma technology.

Other exemplary methods of producing onal antibodies are known in the art.

Additional exemplary methods of producing monoclonal dies are provided in e I herein.

Antibody functional fragments which bind sLea can be generated by any technique well known to those of skill 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).

F(ab’)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.

The dy functional fragments of the invention can also be generated using various phage display s 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 surface of phage particles which carry the polynucleotide sequences encoding them. The 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 in E. coli and the E. coli W0 2015/053871 31 2014/052631 is infected with helper phage. Phage used in these s are typically ﬁlamentous phage including fd and M13 and the VH and VL domains are usually recombinantly ﬁlsed 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, e.g, using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the dy functional fragments of the t invention include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184: 177-186; 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 Publication Nos. W0 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and WO97/13844; and US.

Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, ,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 108; each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, the dy coding regions from the phage can be ed and used to generate whole dies, including human antibodies, or any other desired n binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g, as described herein.

Techniques to recombinantly produce Fab, Fab’ and F(ab’)2 fragments can also be employed using methods known in the art such as those disclosed in PCT ation No.

WO 92/22324; Mullinax et al., 1992, BioTechnz'ques 12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and Better et al., 1988, Science 240: 1041-1043, each h is incorporated by reference in its entirety.

To generate whole dies, PCR primers ing VH or VL nucleotide sequences, a restriction site, and a ﬂanking ce to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones. Utilizing cloning techniques well known to those of skill 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 s can be cloned into vectors expressing a VL constant region, e.g., human kappa or lambda constant regions. The VH and VL domains can also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion W0 53871 32 PCT/USZOl4/052631 vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques well known to those of skill in the art.

In some embodiments, an dy or functional fragment of the invention is conjugated (covalent or non-covalent conjugations) or recombinantly fused to one or more stic agent, detectable agent or therapeutic agent or any other desired molecule. The ated or recombinantly fused antibody or ﬁlnctional fragment can be useful for monitoring or diagnosing the onset, pment, progression and/or severity of a disease associated With the expression of sLea, such as cancer or tumor formation, as part of a clinical testing procedure, such as determining the efﬁcacy of a particular therapy.

Detection and diagnosis can be accomplished, for example, by coupling the antibody or functional fragment of the invention to detectable nces including, but not limited to, radioactive materials, such as, but not limited to, ium (89Zr), iodine ( 131 125 124 1, 1 1 9 9 1231, and 1211,), carbon ( 14C, 11C), sulfur (355), tritium (3H), indium (“51m “3m, “2m, and 1111a,), technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (”313(1), molybdenum (99Mo), xenon (133Xe), e (18F), 15O, 13N, 64Cu, 94mTc, 153 Sm, 177Lu, 159Gd, 149F111, 140La, 175Yb, 166HO, 86Y, 90Y, 4780, 186R6, 188R6, 142PI', IOSRh, 97Ru, 68G6, 57 65 85 32 153 169Yb, 51 54 75 113 Co, Zn, Sr, P, Gd, Cr, Mn, Se, Sn, anden; andpos1tronem1tt1ng- .- metals using various positron on tomographies, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, avidin/biotin and avidin/biotin; cent materials, such as, but not limited to, umbelliferone, ﬂuorescein, ﬂuorescein ocynate, rhodamine, dichlorotriazinylamine ﬂuorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not d to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin, and non-radioactive paramagnetic metal ions.

The present invention ﬁlrther asses 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 atic or cytocidal agent, or a radioactive metal ion, e.g., alpha- emitters. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. A therapeutic agent can be a chemotherapeutic such as, but is not limited to, an anthracycline W0 2015/053871 33 (e.g, doxorubicin and daunorubicin (formerly daunomycin)); a taxan (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), cyclothosphamide, busulfan, omannitol, streptozotocin, mitomycin C, cisdichlorodiamine um (II) (DDP) and tin); an antibiotic (e.g., mycin D, bleomycin, mithramycin, and anthramycin (AMC)); an Auristatin molecule (e.g., auristatin PHE, bryostatin 1, solastatin 10, monomethyl atin E (MMAE) and monomethylauristatin F (MMAF)); a hormone (e.g, glucocorticoids, progestins, androgens, and estrogens); a nucleoside analoge (e.g. Gemcitabine), a pair 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, idin D, ethidium bromide, emetine, cin, etoposide, tenoposide, Vincristine, Vinblastine, colchicin, doxorubicin, ubicin, oxy anthracin dione, mitoxantrone, mithramycin, 1-dehydrotestosterone, glucorticoids, procaine, tetracaine, lidocaine, nolol, puromycin and analogs or homologs thereof, and those compounds disclosed in US. Patent Nos. 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, ,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, 459); a famesyl transferase inhibitor (e.g, R115777, EMS-214662, and those disclosed by, for example, US. 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, 747, 034, 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, 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, can, 9-aminocamptothecin, GG-211 (GI 147211), DX-8951f, IST-622, rubitecan, pyrazoloacridine, XR-5000, saintopin, UCE6, UCE1022, 18A, TAN 1518B, KT6006, KT6528, ED-110, NB-506, ED-110, NB- 506, fagaronine, coralyne, beta-lapachone and rebeccamycin); a DNA minor groove binder (e.g., Hoescht dye 33342 and Hoechst dye 33258); adenosine ase inhibitors (e.g., Fludarabine phosphate and 2-Chlorodeoxyadenosine); or pharmaceutically able salts, solvates, clathrates, or prodrugs thereof. A therapeutic agent can be a W0 2015/053871 34 PCT/USZOl4/052631 immunotherapeutic such as, but is not limited to, cetuximab, bevacizumab, heceptin, rituximab) .

In addition, 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 213B1- or macrocyclic chelators useful for conjugating radiometal ions, including but not limited to, 131In, 131LU, 131Y, 131Ho, 131Sm; or a macrocyclic chelator, such as l,4,7,lO- tetraazacyclododecane-N,N’,N’ ’,N’ ’ ’-tetraacetic acid (DOTA) which can be attached to the antibody or functional fragment via a linker molecule. Such linker molecules are commonly known in the art and described in Denardo et al., 1998, Clin Cancer Res. 2483-90; Peterson et al., 1999, jug. Chem. lO(4):553-7; and man et al., 1999, Nucl. Med. Biol. 26(8):943-50. r, an antibody or ﬁanctional fragment of the ion may be conjugated (covalent or non-covalent conjugations) or recombinantly fused to a therapeutic agent that s a given biological response. Thus, therapeutic agents are not to be construed as limited to classical chemical therapeutic agents. For example, the therapeutic agent can be a protein, peptide, or polypeptide possessing a desired ical activity. Such proteins can include, for example, a toxin (e.g., abrin, ricin A, pseudomonas exotoxin, cholera toxin and diphtheria ; a protein such as tumor necrosis , y-interferon, (x-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 pathway such as tissue factor); a biological response modifier (e.g., a cytokine such as interferon gamma, interleukin-l, interleukin-2, interleukin-5, interleukin-6, interleukin-7, interleukin-9, interleukin-10, interleukin-l2, eukin-15, interleukin-23, ocyte macrophage colony stimulating factor, and granulocyte colony stimulating factor); a growth factor (e.g., growth hormone), or a coagulation agent (e.g., calcium, vitamin K, tissue factors, such as but not limited to, Hageman factor (factor XII), high-molecular-weight kininogen (HMWK), prekallikrein (PK), coagulation proteins-factors II (prothrombin), factor V, XIIa, VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipid, and ﬁbrin r).

The present invention encompasses dies or ﬁanctional 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 can be about 10, about 20, about 30, about 40, about 50, about 60, about 70, WO 53871 35 about 80, about 90 or about 100 amino acids in length. In some s, the invention provides ﬁJsion 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 domain or a VL CDR) and a heterologous protein or polypeptide. In one embodiment, the heterologous n or polypeptide that the antibody or ﬁanctional fragment is fused to is useful for targeting the antibody or ﬁanctional fragment to a particular cell type, such as a cell that expresses sLea.

A conjugated or ﬁJsion n of the invention includes any dy or ﬁanctional 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 conjugated or ﬁJsion protein of the invention includes a 5B1, 9H3, 5Hll or 7E3 antibody, and a diagnostic agent, detectable agent or therapeutic agent.

In another embodiment, a conjugated or fusion n of the invention includes a functional fragment of 5B1, 9H3, 5Hll or 7E3 dies, and a diagnostic agent, detectable agent or therapeutic agent. In another embodiment, a conjugated or ﬁJsion protein of the invention es a VH domain having the amino acid sequence of any one of the VH domains depicted in 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 one of the VL domains depicted in residues 20-130 of SEQ ID NO: 4, residues 20-129 of SEQ ID NO: 8, residues -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 conjugated or ﬁJsion protein of the present invention includes one or more VH CDRs having the amino acid sequence of any one 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 conjugated or fusion n includes one or more VL CDRs having the amino acid sequence of any one of the VL CDRs ed in SEQ ID NOS: 4, 8, 12 or 16, and a diagnostic agent, detectable agent or therapeutic agent. In another embodiment, a conjugated or fusion 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 es 20-129 of SEQ ID NO: 8; residues 20-142 of SEQ ID NO: 10 and residues 20-130 of SEQ ID NO: 12; or residues -145 of SEQ ID NO: 14 and residues 23-130 of SEQ ID NO: 16, tively, and a diagnostic agent, detectable agent or therapeutic agent.

W0 2015/053871 36 PCT/USZOl4/052631 Methods for fusing or conjugating diagnostic agents, able agents or therapeutic agents (including polypeptides) to antibodies are well known, see, e.g., Amon et al., lonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R.

Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in onal 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 y”, in Monoclonal Antibodies For Cancer ion And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), Thorpe et al., 1982, Immunol. Rev. 62:119-58; US. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, ,349,053, 5,447,851, 5,723,125, 181, 5,908,626, 5,844,095, 5,112,946, 7,981,695, 8,039,273, 8,142,784; US. Publications 2009/0202536, 2010/0034837, 2011/0137017, 2011/0280891, 2012/0003247; EP 307,434; EP 367,166; EP 394,827; PCT publications WO 70, WO 96/04388, WO 96/22024, WO 97/34631, and WO 99/04813; Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-10539, 1991; Traunecker et al., Nature, 331:84-86, 1988; Zheng et al., J. Immunol., 154:5590-5600, 1995; Vil et al., Proc.

Natl. Acad. Sci. USA, 89: 1 1337-1 1341, 1992; and Senter, Current Opinion in Chemical Biology, 13 :235-244 , which are incorporated herein by reference in their entireties.

In another , a diagnostic agent, detectable agent or therapeutic agent can be attached at the hinge region of a reduced antibody component via ide bond ion.

Alternatively, such agents can be attached to the antibody component using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyldithio)proprionate (SPDP).

Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for such conjugation are well known in the art. See, for e, Wong, TRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modiﬁcation of Antibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages 187-230 -Liss, Inc. 1995); Price, “Production and Characterization of Synthetic Peptide-Derived dies,” in MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84 (Cambridge University Press 1995). 37 2014/052631 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 of skill in the art. See, for example, Shih et al., Int. J. Cancer. 41 :832-839 (1988); Shih et al., Int. J. Cancer. 46: 1 101-1 106 (1990); and Shih et al., US. Patent No. 313, all of which are incorporated in their entirety by reference. The l method es reacting an antibody component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine ﬁanction and that is loaded with a plurality of e. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the ﬁnal conjugate.

However, if the Fc region is absent, for example, if an antibody ﬁanctional fragment as provided herein is desirable, it is still possible to attach a diagnostic agent, a detectable agent or a therapeutic agent. A ydrate moiety can be introduced into the light chain variable region of a ﬁJll-length dy or antibody fragment. See, for example, Leung et al., J. Immunol., 154: 5919 (1995); US. Patent Nos. 5,443,953 and 6,254,868, all ofwhich are incorporated in their entirety by reference. The engineered carbohydrate moiety is used to attach the diagnostic agent, detectable agent or therapeutic agent.

The therapeutic agent conjugated or recombinantly fused to an antibody ﬁanctional fragment of the invention that binds to sLea can be chosen to achieve the desired prophylactic or eutic effect(s). It is understood that it is within the skill level 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 ﬁanctional fragment of the invention: the nature of the disease, the ty of the disease, and the ion of the subject.

A conjugate or ﬁasion antibody or functional fragment of the invention that is detectably labeled as provided herein and binds to sLea can be used for diagnostic purposes to , diagnose, or monitor a e, wherein the cells that cause 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, tumors of the gastrointestinal tract, breast cancer, ovarian cancer, colon cancer, colorectal adenocarcinoma, atic cancer, pancreatic adenocarcinoma, small cell carcinoma of the lung, bladder adenocarcinoma, metastatic colon cancer, colorectal cancer, signet ring ovarian cancer and metastatic carcinoma. Accordingly, the invention provides methods for detecting cancer or a tumor W0 2015/053871 38 ZOl4/052631 formation in a subject by administering an effective amount of a conjugate or fusion antibody or ﬁanctional fragment of the invention to a subject in need thereof In some aspects, the detection method can further include assaying the expression of a sLea on the cells or a tissue sample of a t using one or more conjugates or fusion antibodies or functional fragments of the invention that bind to sLea; and comparing the level of the sLea with a l level, e.g., levels in normal tissue samples (e.g., from a subject not having a disease, or from the same subject before disease , whereby an increase in the d level of sLea compared to the l level of the sLea is indicative of the disease.

Such diagnostic methods can allow health professionals to employ preventative measures or aggressive treatment r than otherwise possible thereby preventing the pment or ﬁarther ssion of the disease.

An antibody or functional fragment of the invention can also be used to assay sLea n levels in a biological sample using classical immunohistological s as provided herein or as well known to those of skill in the art (e.g., see Jalkanen et al., 1985, J. Cell.

Biol. 101 85; and Jalkanen et al., 1987, J. Cell . Biol. 105:3087-3096). Other antibody-based methods useful for detecting sLea e immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, e oxidase; radioisotopes, such as iodine (1251, 121I), carbon (14C), sulfur (35 S), tritium (3H), indium ( In), and technetium (99Tc); luminescent labels, such as luminol; and ﬂuorescent labels, such as ﬂuorescein and rhodamine, and biotin.

In one aspect, the ion provides for the detection and diagnosis of disease in a human.

In one embodiment, diagnosis includes: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a conjugate or fusion protein of the invention that binds to sLea; b) waiting for a time interval following the administering for permitting the conjugate or fusion protein to preferentially concentrate at sites in the subject where sLea is sed (and, in some aspects, for unbound conjugate or fusion protein to be cleared to background level); c) determining background level; and d) detecting the conjugate or fusion protein in the subject, such that detection of conjugate or fusion protein above the background level indicates that the subject has a disease. Background level can be determined by various methods including, comparing the amount of conjugate or fusion protein detected to a standard value previously determined for a particular system.

W0 2015/053871 39 PCT/USZOl4/052631 It is understood that the size of the subject and the imaging system used will ine the quantity of imaging moiety needed to produce stic images and can be readily determined by one of skill 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 quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99Tc. The conjugate will then preferentially accumulate at the location of cells which express sLea.

In vivo tumor imaging is described in S.W. Burchiel et al., opharmacokinetics of abeled Antibodies and Their Fragments.” er 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and BA. , eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of able agent used and the mode of administration, the time interval ing the stration for permitting the conjugate to preferentially trate at sites in the subject and for unbound conjugate to be cleared to background level 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 carried out by ing the method for diagnosing as provided herein, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, or longer.

The presence of the conjugate or fusion protein can be detected in the subject using methods known in the art for in viva scanning. These methods depend upon the type of detectable agent used. A d artisan will be able to determine the appropriate method for detecting a particular detectable agent. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography. In one embodiment, an antibody or ﬁanction 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 function fragment of the ion is conjugated to a ﬂuorescent compound and is detected in the subject using a ﬂuorescence responsive scanning instrument. In another embodiment, an antibody or function fragment of the invention is conjugated to a positron emitting metal, such as zirconium (”Zr) or any other positron emitting metal ed herein or that is well known in the art to be detectable by positron emission-tomography, and is detected in the subject using positron emission-tomography. In yet r embodiment, an antibody or W0 2015/053871 40 ZOl4/052631 function fragment of the ion is conjugated to a paramagnetic label and is detected in a subject using magnetic resonance imaging (MRI).

In one embodiment, the invention es a pharmaceutical composition having an dy or a functional fragment of the invention and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier that can be used in the pharmaceutical compositions of the ion include any of the standard pharmaceutical carriers known in the art, such as phosphate buffered saline on, water and emulsions such as an oil and water emulsion, and various types of wetting agents. These pharmaceutical compositions can be prepared in liquid unit dose forms or any other dosing form that is sufficient for delivery of the dy or functional fragment of the invention to the target area of the subject in need of treatment. For example, the pharmaceutical compositions can be prepared in any manner appropriate for the chosen mode of administration, e.g., intravascular, intramuscular, taneous, intraperitoneal, etc. Other optional components, e.g., pharmaceutical grade stabilizers, buffers, preservatives, excipients and the like can be readily selected by one of skill in the art. The preparation of a pharmaceutically composition, having due regard to pH, isotonicity, stability and the like, is within the level of skill in the art.

Pharmaceutical formulations containing one or more antibodies or ﬁanctional fragments of the ion 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 ents at the dosages and concentrations employed, and include s such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as cyldimethylbenzyl ammonium chloride; hexamethonium de; 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) polypeptides; proteins, such as serum albumin, n, or immunoglobulins; hydrophilic polymers such as nylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; ccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, W0 2015/053871 41 PCT/USZOl4/052631 mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).

Thus, in some embodiments, the invention provides a method for treating or ting a disease in a subject in need thereof. The methods of the ion can include administering a therapeutically effective amount of a pharmaceutical composition provided herein to the subject. For example, the pharmaceutical composition can e one or more antibody or functional fragment provided herein. Diseases that can be treated or prevented using the methods of the invention include cancer, tumor formation and/or asis. In particular, the methods of the invention are useful for treating cancers or tumor formation wherein the cancer cells or tumor expresses the carbohydrate sLea. Non- limiting es 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, ctal cancer, pancreatic cancer, or pancreatic arcinoma; small cell carcinoma of the lung; bladder adenocarcinoma; signet ring ovarian cancer; ovarian cancer, metastatic carcinoma; and arcinoma of the stomach, esophagus, throat, ital tract, or .

Accordingly, in some aspects, the invention provides a method for treating cancer or preventing tumor metastasis in a subject 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 sLea and 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 , the invention es a method for treating cancer or preventing tumor metastasis in a subject in need thereof by administering a therapeutically effective amount of a pharmaceutical composition having an antibody or ﬁanctional nt thereof, wherein the dy or functional fragment binds to sLea and 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 a subject in need thereof by stering a therapeutically ive amount of a pharmaceutical composition having an antibody or W0 2015/053871 42 PCT/USZOl4/052631 functional fragment thereof, wherein the antibody or ﬁanctional fragment binds to sLea and includes both a VH domain and a VL domain, where the VH domain and the VL domain 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: and es 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.

Formulations, such as those described herein, can also contain more than one active compound as necessary for the particular disease being treated. In certain ments, formulations e an antibody or ﬁanctional fragment of the invention and one or more active compounds with complementary activities that do not ely affect each other.

Such molecules are suitably present in combination in amounts that are effective for the e intended. For example, an antibody or functional fragment of the invention can be combined with one or more other therapeutic agents. Such combined therapy can be stered to the subject concurrently or successively.

Thus, in some aspects, invention provides a method for treating or preventing a disease by administering a eutically effective amount of a pharmaceutical composition ed herein to a t in need thereof, n the pharmaceutical composition includes an antibody or functional fragment of the invention and a second therapeutic agent. The appropriate second therapeutic agent can be readily determined by one of ordinary skill in the art as discussed herein. As provided herein in Example IV, in some aspects of the invention, the second eutic agent can be Taxol.

The pharmaceutical compositions provided herein n therapeutically effective amounts of one or more of the antibodies of the invention provided herein, and ally one or more additional therapeutic agents, in a pharmaceutically acceptable carrier. Such pharmaceutical compositions are useful in the prevention, treatment, ment or amelioration of a disease, such cancer or tumor formation, or one or more of the symptoms thereof.

The pharmaceutical compositions can contain one or more antibodies or functional fragments of the invention. In one embodiment, the antibodies or onal fragments are formulated into suitable pharmaceutical preparations, such as sterile solutions or suspensions for parenteral administration. In one embodiment, the antibodies or W0 2015/053871 43 PCT/USZOl4/052631 functional fragments provided herein are ated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel (1985) Introduction to Pharmaceutical Dosage Forms, 4th Ed., p. 126).

An antibody or functional fragment of the invention can be included in the pharmaceutical composition in a eutically effective amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the subject treated. The therapeutically effective concentration can be determined empirically by testing the compounds in in vitro and in viva systems using routine methods and then extrapolated therefrom for dosages for humans. The concentration of an dy or functional fragment in the pharmaceutical composition will depend on, e.g., the physicochemical characteristics of the antibody or functional fragment, the dosage schedule, and amount administered as well as other factors well known to those of skill in the art.

In one embodiment, a therapeutically effective dosage produces a serum concentration of an antibody or ﬁanctional nt of from about 0.1 ng/ml to about 50-100 ug/ml. The pharmaceutical compositions, in another embodiment, provide a dosage of from about 0.001 mg to about 500 mg of antibody per kilogram of body weight per day. ceutical 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 of the antibody or functional fragment and/or a combination of other optional essential ingredients per dosage unit form.

The antibody or onal fragment of the invention can be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a ﬁanction 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 is to be noted that concentrations and dosage values can also vary with the ty of the condition to be alleviated. It is to be further understood that for any ular subject, speciﬁc dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the tration ranges set forth herein are exemplary only and are not intended to limit the scope or ce of the claimed compositions.

W0 2015/053871 44 PCT/USZOl4/052631 Upon mixing or addition of the antibody or onal fragment of the invention, the ing e can be a solution, suspension or the like. The form of the resulting mixture s upon a number of factors, including the intended mode of stration and the solubility of the nd in the selected carrier or vehicle. The effective concentration is ient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined.

The pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as sterile parenteral ons or suspensions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives f. The antibody or ﬁanctional fragment can be, in one embodiment, formulated and administered in unit-dosage forms or multiple-dosage forms. Unit-dose forms refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the antibody or functional fragment of the invention sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit- dose forms include ampoules and syringes. Unit-dose forms can be administered in fractions or multiples thereof. A multiple-dose form is a plurality of identical osage forms packaged in a single container to be administered in segregated unit-dose form.

Examples of le-dose forms include vials or bottles of pints or gallons. Hence, le dose form is a multiple of unit-doses which are not ated in packaging.

In one embodiment, one or more antibody or functional fragment of the ion is in a liquid pharmaceutical formulation. Liquid pharmaceutically strable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an antibody or functional fragment as provided herein and optional pharmaceutical adjuvants in a carrier, such as, for e, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution. If desired, the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, lizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents. Actual methods of ing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA.

Methods for administering a pharmaceutical composition of the invention are well known in the art. It is understood that the appropriate route of administration of a pharmaceutical composition can be readily determined by a skilled clinician. Exemplary routes of administration include intravenous injection, intramuscular injection, intradermal injection or subcutaneous injection. Moreover, it is understood that the ation of the pharmaceutical composition can be readily adjusted to accommodate the route of administration. The invention also provides that following administration of a pharmaceutical composition of the invention, delayed, sive and/or repeated dosages of one or more ceutical ition as provided herein may be administered to the t.

The methods of the invention for treating a disease is intended to include (1) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop in a subject that may be predisposed to the disease but does not yet experience or display symptoms of the disease; (2) inhibiting the e, i.e., arresting or reducing the development of the disease or its clinical symptoms; or (3) ing the disease, i.e., causing regression of the disease or its al symptoms. The s of the invention for preventing a disease is ed to include forestalling of a clinical symptom tive of cancer or tumor formation. Such forestalling includes, for example, the maintenance of normal physiological indicators in a subject. Therefore, preventing can include the prophylactic treatment of a subject to guard them from the occurrence of tumor metastasis.

The therapeutically effective amount of the pharmaceutical composition used in the methods of the ion will vary depending on the pharmaceutical composition used, the disease and its severity and the age, weight, etc., of the subject to be treated, all of which is within the skill of the attending clinician. A subject that that can be treated by the methods of the invention include a vertebrate, preferably a mammal, more preferably a human.

It is tood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the ion provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.

EXAMPLE I Human Monoclonal Antibodies to sLea have Potent mor Activity W0 2015/053871 46 PCT/USZOl4/052631 The carbohydrate antigen sLea is widely sed on epithelial tumors of the intestinal tract, breast, and pancreas and on small-cell lung cancers. Since over- expression of sLea appears to be a key event in invasion and metastasis ofmany tumors and results in susceptibility to antibody-mediated lysis, sLea is an attractive molecular target for tumor therapy. Accordingly, as described herein, fully human monoclonal antibodies (mAb) from blood lymphocytes from individuals immunized with a sLea-KLH vaccine were generated and characterized. l 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 further terized. Both antibodies were speciﬁc for Neu5Acu2-3GalBl-3(Fucu1-4)GlcNAcB and Neu5ch2-3GalBl- 3(Fuc0L1-4)GlcNAcB as determined by glycan array analysis. Complement-dependent cytotoxicity against DMS-79 cells was higher (ECSO 0.1 ug/mL vs. 1.7 ug/mL) for r7E3 (IgM) than for r5B1 (IgGl). In addition, r5B1 antibodies showed high level of antibody- dependent ediated cytotoxicity activity on DMS-79 cells with human NK cells or peripheral blood mononuclear cells. To evaluate in vivo efﬁcacy, the 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 5 xenograft model, treatment during the ﬁrst 21 days with four doses of r5B1 (100 ug per dose) doubled the median survival time to 207 days, and three of ﬁve animals survived with six doses. In the DSM-79 xenograft model, growth of established DMS-79 tumors was suppressed or regressed in animals treated with r5B1 antibody. On the basis of the potential of sLea as a target for immune attack and their afﬁnity, speciﬁcity, and effector ﬁlnctions, 5B1and 7E3 have clinical utility in the treatment of cancer. als, cells, and antibodies DMS-79 (Pettengill et al., Cancer, —18 (1980)), SW626, EL4, HT29, BxPC3, SK- MEL28, and P3 >< 63Ag8.653 cell lines were sed from an Type Culture Collection (ATCC). Colo205-luc cells (Bioware ultra) were obtained from Caliper Life es. The murine control mAb 121SLE (IgM) was purchased from GeneTex. sLea tetrasaccharide (Cat # S2279) was purchased from Sigma-Aldrich. sLea-HSA (human serum albumin) conjugate (Cat # 07-011), monovalent biotinylated sLea (sLea-sp-biotin; Cat # 02-044), polyvalent biotinylated sLea- PAA (Cat # 01-044), biotin-labeled Lea-PAA (Cat # 01-035), and AA-biotin (Cat # 01-045) were purchased from GlycoTech. In the polyvalent presentation, the tetrasaccharide is incorporated into a polyacrylamide matrix (PAA), thereby creating a 30-kDa alent polymer with approximately every ﬁfth amide group of the polymer chain N—substituted with biotin in a 4:1 ratio and approximately 20% carbohydrate content. Other HSA or BSA glycoconjugates used in this study were prepared in-house using sLea pentenyl glycoside as described. Ragupathi et al., Cancer Immunol Immunother, 58:1397—405 (2009). GD3, fucosyl-GMl, GM2, and GM3 were purchased from Matreya, and GD2 was purchased from Advanced ImmunoChemical.

Generation of anti-sLea oducing hybridomas Blood samples were obtained from 3 patients in an ongoing trial with LH conjugate e in patients with breast cancer initiated at MSKCC under an MSKCC- and FDA-approved IRB protocol and IND. Blood specimens were selected from 2 ts after 3 or 4 vaccinations, which showed dy 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 clear cells (PBMC) were isolated from approximately 80 to 90 mL of blood by nt centrifugation on Histopaque-1077 (Sigma-Aldrich).

PBMCs were cultured in RPMI-1640 medium supplemented with amine, nonessential amino acids, sodium pyruvate, vitamin, penicillin/streptomycin, 10% FBS (Omega Scientiﬁc), 10 ng/mL IL-21 (Biosource), and 1 ug/mL anti-CD40 mAb (G28-5 hybridoma supernatant; ATCC). Cells were fused by electrofusion to P3 >< 63Ag8.653 myeloma cells. sLe" ELISA For the sLea ELISA, plates were coated either with 1 ug/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 lly detected with horseradish peroxidase (HRP)- labeled goat uman IgA + G + M (Jackson ImmunoResearch), and positive wells were subsequently probed with IgG-Fc- or IgM-speciﬁc secondary antibodies to determine isotypes.

W0 2015/053871 48 PCT/USZOl4/052631 Carbohydrate speciﬁcity analysis Cross-reactivity against the y related antigens, Lea and sLeX, was evaluated by surface plasmon resonance (SPR) and conﬁrmed by ELISA using biotin-labeled Lea-PAA and biotin-sLeX-PAA. Binding to gangliosides GD2, GD3, fucosyl-GMl, 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. In brief, 2 ug/mL SA conjugate was coated onto plates followed by blocking with 3% BSA in PBS. Next, 30 uL of different ydrate moieties (40 ug/mL in PBS prepared from 1 mg/mL stock solutions) either unconjugated or conjugated to HSA or BSA was mixed separately with 30 uL of test antibody and incubated at room temperature in a sample plate. After 30 minutes 50 uL 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 ion of bound dy using a Versamax spectroﬁuorometer (all steps were carried out at room temperature). The tested carbohydrate moieties included globo H, Lewis Y, Lewis X, -Thomson-nouveaux (sTn), red sTn, Thomson Friedenreich (TF), Tighe Leb/LeY mucin, porcine submaxillary mucin (PSM), and sLea tetrasaccharide and sLea-HSA conjugate. To determine the ﬁne speciﬁcity of the antibodies, glycan array analysis was done by the Consortium for Functional Glycomics Core H group. 5Bl and 7E3 antibodies were tested at 10 ug/mL using version 4.1 of the printed array consisting of 465 glycans in replicates of 6.

Immunoglobulin cDNA cloning and recombinant antibody expression Variable region of human mAb heavy and light chain cDNA was recovered by RT-PCR from the individual hybridoma cell line and subcloned into IgGl or IgM heavy chain or IgK or IgL light chain expression vector as described before. -Hirai et al., J.

Immune Based Ther. Vaccines, 2:5 (2004). Ig heavy chain or light chain expression vector was -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 ctamine 2000 (Invitrogen). After 24 hours, transfected cells were transferred to a 10-cm dish with ion medium [DMEM supplemented with 10% dialyzed FBS rogen), 50 umol/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-sLea W0 2015/053871 49 PCT/USZOl4/052631 dy-producing clones were selected by measuring the antibody levels in supematants in a sLea-speciﬁc ELISA assay and expanded for large-scale mAb production.

Human mAb puriﬁcation Antibodies were puriﬁed using the Akta Explorer (GE Healthcare) system running Unicorn 5.0 software. In brief, 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 used. Human IgG antibodies were puriﬁed on appropriate-sized protein A s using 10 mmol/L PBS and 150 mmol/L NaCl running buffer. Human IgM antibodies were puriﬁed on a yapatite column, and IgM was eluted with a gradient of 500 mmol/L phosphate. The antibody trations 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 evaluated by SDS-PAGE analysis (1-5 ug per lane) under reducing conditions, and the purity was more than 90% based on the sum of heavy and light chains.

Flow cytometry ositive 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 (1-2 ug/mL in complete medium) and incubated on ice for 30 minutes. Gilewski et al., Clin Cancer Res, —701 (2000); Gilewski et al. Proc. Natl. Acad. Sci. U.S.A., 98:3270—5 (2001). After washing in PBSF, the cells were incubated with Alexa-488 anti-human IgG-Fcy or anti- human IgM-u (Invitrogen) for 30 minutes on ice. Cells were washed twice in PBSF and analyzed by ﬂow cytometry using the Guava Personal Cell is-96 (PCA-96) System (Millipore). 5-luc cells were incubated with 2 ug/mL of primary antibody, followed by staining with secondary antibodies from SouthemBiotech, and analyzed on a Becton Dickinson FACS Advantage IV instrument using FlowJo 7.2.4 software.

Afﬁnity determination Afﬁnity constants were determined using the principle of SPR with a Biacore 3000 (GE Healthcare). Biotin-labeled univalent sLea (Cat # 02-044) or polyvalent sLea-PAA-biotin (Cat # 01-044) were coupled to te ﬂow cells of an SPA biosensor chip according to the cturer’s instructions. A ﬂow cell blocked with HSA and culture medium ning free biotin was used as a reference cell. The binding kinetic parameters were determined from several known concentrations of antibody diluted in HBS-EP buffer (10 50 2014/052631 mmol/L HEPES, pH 7.4, 150 mmol/ L NaCl, 3.4 mmol/L EDTA, 0.005% surfactant P20) using the sLea-PAA-biotin-coated ﬂow cell. The curve-ﬁtting software provided by the Biacore instrument was used to generate estimates of the association and dissociation rates from which affinities are calculated.

CDC assay 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 l; Cat # Al 13) and purified human mAbs at various dilutions (0.1-25 ug/mL) or with positive l mAbs as previously described (Ragupathi et al. Clin Cancer Res 2003, 9:5214; Ragupathi et al. IntJ Cancer 2000, 85:659; Dickler et al. Cancer Res 1999, ). In brief, 2.5 X 106 target cells were painted with carboxyﬂuorescein diacetate succinymyl ester (CSFE) to yield green/yellow ﬂuorescent target cells. The painted cells (1 X 105/50 uL sample) were incubated for 40 minutes with 100 uL of antibodies on ice.

Next, 50 uL 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 s at 37°C. Thus, 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 dye 7- amino-actinomycin D (7-AAD), and samples were ed by dual-color immunoﬂuorescence utilizing the Guava CellToxicity software module. Control samples that received NP40 were used to determine maximal killing and samples ing complement alone served as baseline. The percentage of killed cells was determined by appropriate gating and calculated ing to the following formula: % killed = [(% sample — % complement alone)/(% NP40 — % complement ] X 100.

Antibody-dependent cell-mediated cytotoxicity assay PBMC effector cells were isolated by Ficoll-Hypaque density centriﬁagation from blood samples ed under an MSKCC IRB-approved protocol. The target cells were incubated at 5 x 106 cells/mL in complete growth media with 15 uL of 0.1% calcein-AM solution (Sigma-Aldrich) for 30 s at 37°C, in the presence of 5% C02. The cells were washed twice with 15 mL of PBS-0.02% EDTA and resuspended in 1 mL complete growth medium. Fifty microliters (10,000 cells) of labeled target cells was plated into a 96-well plate in the presence or absence of antibodies at the concentrations described in , and incubated with 50 u L of freshly isolated eral blood mononuclear cells (effector cells, at 100:1 E/T ratio) accordingly. After 2 hours of incubation, the plate was 51 2014/052631 centrifuged at 300 X g for 10 minutes, and 75 uL of supernatant was transferred into a new ﬂat-bottomed 96-well plate. The ﬂuorescence in the supernatant was measured at 485-nm tion and 535-nm emission in Fluoroskan Ascent (Thermo Scientiﬁc). Spontaneous release was determined from target cells in RPMI-l640 medium with 30% FBS without effector cells and maximum release was determined from target cells in RPMI-l640 medium with 30% FBS and 6% Triton X-100 without effector cells. t xicity was calculated as [(counts in sample — spontaneous e)/(maximum counts — spontaneous release)] x 100. mAb internalization assay Intemalization of 5Bl antibody was evaluated by measuring the cytotoxic activity of r5Bl and Hum-ZAP secondary conjugate (Advanced Targeting Systems) complex against sLea expressing BxPC3 cells, which were plated into a 96-well plate (2,000 cells/ 90 uL/well) and incubated overnight in duplicates. Various concentrations of 5Bl antibody were incubated with Hum-ZAP secondary ates at RT according to the manufacturer’s instruction. Next, 10 l of r5Bl and Hum-ZAP complex was added to the cells and incubated for 3 days. Twenty-ﬁve microliters of Thiazolyl Blue Tetrazolium Bromide (Sigma-Aldrich) solution (5 mg/mL in PBS) was added to each well and ted at 37°C. After 2 hours of incubation, 100 uL/well of lization solution (20% SDS/50% N,N—dimethylformamide) was added to each well and incubated for another 16 hours at 37°C. The OD was measured at 570/690 nm, and values obtained with medium alone were used for plate background subtraction. Eight parallel cultures without antibody were used to normalize the sample values (sample/mean untreated X 100).

Xenograft transplantation model Female CBl7 SCID mice (5-8 weeks old) were purchased from Taconic. For the 5 xenograft model, 5-luc cells (0.5 X 106) in 0.1 mL complete growth media were injected via the tail vein on day 0 using a BD insulin syringe with 28G needle (Becton Dickinson & Co). For the first study, one hundred micrograms ofmAb 5Bl was injected intraperitoneally on days 1, 7, l4, and 21 (experiment 1) or on days 1, 4, 7, 10, 14, and 21 (experiment 2). For the second study, 100 ug, 300 ug or 1 mg ofmAb 5Bl was ed 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. Mice were monitored for tumor development. For the DMS-79 xenograft model, DMS-79 cells (l X 106) were injected aneously into Female CB17 SCID mice, and the mice began treatment on Day 19 after the tumor length reached 5 mm (~20 mmz). The animals were then treated with human IgG or 5B1 antibodies given by intraperitoneal injection at 200 ug per dose, plus cRGD by intravenous injection to increase vascular permeability initially at 80 ug, then 5 days per week, 40 ug per dose until day 37.

All procedures were done 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 Blood samples from 3 vaccinated patients were used for hybridoma generation efforts and many positive wells were ed in the antigen-speciﬁc ELISA assays (Table 3).

Extensive screening was used to ate antibodies that showed inferior or nonspecific binding. Eight human antibody-expressing hybridoma cells (1 IgM and 7 IgG) with strong reactivity against sLea were lly selected, expanded, and subcloned for further characterization. Two antibodies (9H1 and 9H3) showed strong g to sLea-HSA conjugates but not to AA-coated plates. Three dies (5B1, 5H11, and 7E3) showed strong binding to lent and polyvalent sLea and sLea-HSA conjugates when measured by ELISA assays (Table 4).

Table 3: Binding of candidate hybridoma supematants containing IgG or IgM monoclonal antibodies to sLea-acetylphenylenediamine(APD)-human serum albumin(HSA) conjugate (sLea-HuSA).

OD (490 nm)* Supernatant Isotypem HuSA EF41-5B1 G 0.000 2.240 0.020 H11 G 0.020 2.180 -0.010 EF41-6F7 G 0.010 0.480 -0.010 EF41-9H1 G 0.010 0.730 -0.020 EF41-9H3 G 0.010 1.100 -0.020 EF41-9A10 G 0.010 2.140 -0.010 EF41-10C1 G 0.000 0.040 -0.020 EF40-3C4 G 0.000 0.500 0.000 EF40-10H3 G 0.000 0.130 0.000 EF41-7E3 M -0.020 2.130 0.010 EF41-9A7 M 2.700 2.540 2.610 EF40-5B7 M 0.070 0.070 0.080 * isotype control blank subtracted. HuSA tes human serum albumin control. PBS tes phosphate ed saline control.

Table 4: Binding of the select antibodies to sLea presented as univalent (mono-) sLea, multivalent (poly-) sLea, or sLea-HSA form.

OD (490 nm) Supernatant PBS NAV NAV+ NAV+ SLeA- Lea poly-sLea HSA" EF41-5B1(G) 0.050 0.050 0.900 2.280 1.740 EF41-5H11(G) 0.040 0.050 1.280 2.130 1.900 EF41-6F7(G) 0.050 0.050 0.050 0.080 0.100 EF41-9H1(G) 0.050 0.050 0.050 0.060 0.300 EF41-9H3 (G) 0.050 0.050 0.050 0.050 0.750 EF41-9A10 (G) 0.040 0.040 0.170 0.870 1.330 EF40-3C4 (G) 0.040 0.050 0.040 0.050 0.070 EF41-7E3 (M) 0.050 0.050 0.970 0.920 1.310 HuSA indicates human serum albumin control. PBS indicates phosphate buffered saline control. NAV indicates Neutral Avidin l.

The heavy and light chain variable regions from 4 selected antibodies were recovered by RT-PCR and cloned into our full-length 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/9t), 9H3 (IgG/9t), and 5H11 t) were derived from the same VH family and all used lambda light chains. These IgG1 antibodies showed different CDR sequences with 16, 5, or 3 mutations deviating from the germ line, respectively (FIGS. 1-6; Table 5). The IgM antibody (7E3) utilizes the kappa light chain and has 6 heavy chain mutations (FIGS. 7-8; Table 5). The increased mutations in 5B1 are indicative of affinity maturation. Recombinant antibodies were ed 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 retained the properties of the original oma-derived antibodies with respect to ELISA binding and speciﬁcity.

Table 5: cDNA Classiﬁcation of selected human anti-sLea antibodies derived from vaccinated blood donors.

VH VL Muta. Muta.

Clone from CDR from CDR ID VH _ermline DH (RF) JH len_th VL ne JL len_th ——--————— Analysis of tumor cell g Cell surface binding is crucial for cytotoxic activity and was therefore tested next. Flow cytometry showed strong binding of 5B1, 9H3, 5H11, and 7E3 recombinant antibodies to DMS-79 cells, a small-cell lung cancer sion cell line (A). Binding of r5B1 and r7E3 was also conﬁrmed on HT29 colon cancer cells (B), BxPC3 pancreatic cancer cells (C), SW626 ovarian cancer cells (D), and ColoZOS-luc colon cancer cells (F). These antibodies failed to bind to egative (SLE121- negative) SK-MEL28 melanoma cells (E) or EL4 mouse lymphoma cells (data not shown).

Afﬁnity measurements The relative y/avidity of the 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 bind rapidly to sLea-PPA and show a cantly slower off-rate compared with 1218LE, a commercially available murine IgM anti-sLea dy 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 since 9H3 antibodies (native and recombinant) failed to bind to the sLea-PAA-coated biosensor chip.

Table 6: ination of kinetic parameters of anti-sLea antibodies by SPR. mAb Afﬁnity, Kd, Ka, Association Dissociation Isotype nmol/L mol/L l/mol/L ka, l/mol/L s) kd, l/s rSBl 0.14 1.4 x10"Iij 7.0 x10; 1.1 x 105 1.6 x10" IgGl/Qt r7E3 0.04 3.6 x 10‘11 2.8 x1010 8.8 x 105 3.2 x 10-5 IgM/K IZISLE 0.35 3.5 x 10‘10 2.8 x109 2.7 x 106 9.4 x 10-4 m-IgM W0 2015/053871 55 Speciﬁcity analysis Preliminary assays to probe carbohydrate speciﬁcity showed that 5B1, 9H3, and 7E3 did not bind to the closely related sLeX, Lea, or LeY antigens or the osides GD2, GD3, ﬁlcosyl-GMl, 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 lent form of sLea, whereas 7E3 and 5B1 were found to bind the rnonovalent form. The binding of 5B1 to sLea-PAA was also inhibited by sLea 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, not reactive with gangliosides GM2, GD2, GD3, ﬁlcosyl GMl, or the l 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 cerarnide, or conjugated to BSA or HSA), only sLea tetrasaccharide and sLea-HSA conjugate were able to inhibit binding to sLea-HSA ate (Table 7).

Table 7: Binding to sLeA-PAA-HSA in the presence of s related glycoconjugates. r5B1 r9H3 r7E3 Antigens Exp 1 Exp 2 Exp 1 Exp 2 Exp 1 Exp 2 Sialyl Tn-HSA 1.866 1.981 1.882 1.970 2.218 2.259 GloboH-cerarnide 1.866 1.852 1.906 1.821 2.098 2.201 sTn(c)—HSA (direct) 1.896 1.864 1.947 1.883 2.131 2.136 sTn-M2-HSA (rnono) 1.937 1.857 1.843 1.826 2.040 2.066 LeX-gal-cer 1.893 1.863 1.791 1.810 2.173 2.175 dPSM 1.897 1.890 1.757 1.700 2.218 2.110 Tn-rnono allyl M2-HSA 1.837 1.905 2.041 1.991 2.083 2.107 Tighe Y rnucin 1.808 1.837 1.951 1.964 2.106 2.065 LeX-PAA 1.830 1.873 2.053 2.036 2.099 2.108 LeY-cerarnide 1.824 1.821 1.940 1.980 2.143 2.085 Lewis Y cerarnide 1.833 1.844 1.941 1.874 2.090 2.111 Tn(c)—HSA 1.881 1.711 1.893 1.917 2.146 2.030 T-serine-BSA 1.809 1.830 2.128 2.089 2.137 2.039 TF(c) HSA 1.874 1.909 2.031 2.032 2.119 2.094 Tn LY-BSA 1.901 1.863 1.944 1.959 2.084 2.118 NPrGBMP-HSA 1.892 1.797 1.944 1.964 2.090 2.111 sLeA - HSA 1.329 1.298 1.373 1.266 1.542 1.621 sLeA tetrasaccharide 0.371 0.312 0.797 0.814 2.114 2.041 None 1.809 1.809 1.993 1.993 2.096 2.096 Blank 0.101 0.093 0.093 0.092 0.108 0.100 To examine the carbohydrate speciﬁcity in further detail, 5B1 and 7E3 antibodies were also tested by glycan array is done by the Consortium for Functional Glycomics Core H group. Both antibodies were tested at 10 ug/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, Neu5Acu2-3GalBl-3(Fucul- 4)GlcNAcB and Neu5ch2-3GalBl-3(Fuc0Ll-4)GlcNAcB and virtual absence of binding to closely related antigens that were present in the array, ing sLeX, Lea, Lex, and Ley.

The results are summarized in Table 8, which shows the top 5 of 465 glycan ures that were ized by the respective antibodies.

Table 8: Analysis of carbohydrate speciﬁcity by glycan array screening.

A. 5B1 Chart Common Glycan Structure Average StDev %CV Number Name 237 sLea Neu5Acu2-3GalB1-3(Fucu1-4)GlcNAcB-Sp8 3 8,851 2,797 7 278 sLea Neu5Gca2-3GalBl-3(Fucu1-4)GlcNAcB-Sp0 32,714 2,624 8 329 sLeaLea Neu5Acu2-3GalB1-3(Fuc011-4)GlcNAcB1-3GalB1- 6,477 399 9 3(Fuc011-4)GlcNAcB-Sp0 238 sLeaLex Neu5Aca2-3GalB1-3(Fuca1 -4)GlcNAcB1-3GalB1- 1,344 131 10 4(Fuc011-3)GlcNAc[3-Sp0 349 4GlcNAcB1-2Man011-3(Man011 -6)ManB1- 129 62 48 4GlcNAc NAc -S 12 B. 7E3 Glycan Structure Average StDev %CV Number Name Neu5A662—3Ga151—3 Fuca1-4 GlcNAcB-Sn8 40,920 4,676 329 sLeaLea Neu5Acu2-3GalB1-3(Fucu1-4)GlcNAcBl-3GalBl- 40,210 2,095 5 3 Fucal -4 GlcNAc 3 -S 0 0 238 sLeaLex Neu5Acu2-3GalB1-3(Fucu1-4)GlcNAcBl-3GalBl- 39,848 3,621 4 Fucu1-3 GlcNAc 3 -S 0 0 62—3Ga151—3 Fuca1-4 GlcNAcB-Sno 36,707 2,733 349 GalBl-4GlcNAcB1-2Man011-3(Man011 -6)ManB1- 692 52 4GlcNAcB 1-4GlcNAc B -S .12 CDC activity To evaluate the nal activity of 5B1 and 7E3, we tested the 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 ug/mL, while a control antibody with different speciﬁcity (1B7, anti-GD2 IgGl mAb) had no effect at the same concentrations (data not . The CDC activity is concentration dependent, and W0 53871 57 7E3 was signiﬁcantly more active than 5B1 in this assay (), which is expected since IgM antibodies are known to be more effective in complement-mediated xicity assays. The EC50 (50% cytotoxicity) was 1.7 ug/mL for 5B1 and 0.1 ug/mL for 7E3, which ates to roughly 85-fold higher potency for 7E3 on a molar basis ().

ADCC activity While 7E3 is signiﬁcantly more potent in the CDC assay, IgG antibodies are known to have antibody-dependent cell-mediated cytotoxicity (ADCC) activity, which is thought to be important for tumor killing in viva. High levels of cytotoxicity were measured using 5B1 antibody with human PBMC and DMS-79 target cells at various E:T ratios ( 13A). Similar levels of cytotoxicity were observed at lower E:T ratios with primary NK cells (B). A dose-response experiment with PBMC from 2 donors measured at an E/T ratio of 100:1 showed similar y, and more than 85% cytotoxicity was reached at trations of 0.5 ug/mL or more of 5B1 (C). The cytotoxicity mediated by 5B1 requires FcyRIII receptors since it can be blocked with 3G8 anti-CD16 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 achieved with 1 ug/mL of 5B1 antibodies was superior to the activity observed with antibodies to GM2, fucosyl-GMl, globo H, or polysialic acid. As expected, 7E3 and murine 121SLE (both are IgM) were inactive in this assay. 5B1 alization assay Antibody conjugates directed at antigen “closely related to” Lewis Y were previously shown to be rapidly alized and very effective in animal models. rom et al., Cancer Res 50:2183—90 (1990); Trail et al., Science 261 :212—5 (1993). To examine whether sLea is alized, 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, while nonintemalized saporin leaves the cells unharmed. As shown in , BxPC3 cells are effectively killed in the presence of increasing doses of 5B1 while the presence of an isotype-matched IgG1 antibody directed against GD2, which is not expressed on these cells, does not kill the cells.

Activity in xenograft animal model for metastasis To evaluate the activity of 5B1 in vivo, the dies were tested in two xenograft models using either Colo205-luc tumor cells or DMS-79 tumor cells in SCID mice. For the xenograft model using 5-luc tumor cells, ﬁve mice per group were injected with 0.5 X 106 cells into the tail vein on day 0, and sful injection of the cells was veriﬁed by imaging the animals using the IVIS 200 in viva imaging system (Caliper Life Sciences). One day later, animals were treated with 5B1 antibodies given intraperitoneal or PBS mock injection. In experiment 1, 100 ug of 5B1 was given on days 1, 7, 14, and 21 (400 ug total dose), and in ment 2 the animals received 100 ug 5B1 on days 1, 4, 7, 10, 14, and 21 (600 ug total dose). The average median al of untreated animals was 102 days in the 2 experiments, and all untreated animals died within 155 days (). Treatment of s improved survival signiﬁcantly: the median survival was doubled to 207 days in the group that received 4 doses of 5B1 and 2 of 5 animals ed until termination of the experiment after 301 days (log-rank test, P = 0.0499; HR = 3.46).

The proportion of survivors further increased to 3 of 5 mice when 6 doses were administered (log-rank test, P = 0.0064; HR = . The second experiment was terminated after 308 days, and the surviving animals failed to reveal 5-luc tumors at the highest sensitivity of the imaging system (data not shown).

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 ug, 300 ug or 1 mg). All animals initially received interperitoneal or PBS mock injection (control) of the 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. The delayed treatment with various doses of 5B1 showed a dose dependent protection up to complete cure in SCID mice engrafted with Colo205-luc tumor cells (FIGS. 16 and 17). Treatment with 7E3 antibodies did not show higher protection despite increased apparent afﬁnity (data not shown).

In a xenograft model using DMS-79 cells, ﬁve mice per group were injected aneously with 1 X 106 cells on day 0, and began treatment on day 19 after the tumor length reached 5 mm (~20 mmz). The animals were then treated with human IgG or 5B1 antibodies given by intraperitoneal injection at 200 ug per dose, plus cRGD by intravenous injection initially at 80 ug, then 5 days per week, 40 ug per dose until day 37.

The growth of established DMS-79 tumors was suppressed or regressed in animals treated with 5B1 or a ation of 5B1 plus cRGD (A 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).

The above data trates a signiﬁcant ability to suppress or regress established 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 Antibody 5B1 Adenocarcinomas are a leading cause of death from cancer. Detection of pancreatic cancer remains especially difﬁcult with sis often made at a late stage. Approaches for earlier ion of primary and atic pancreatic s could have signiﬁcant clinical impact. In clinical practice, elevated levels of sLea antigen are red to identify suspected occult malignancy in patients with pancreatic cancer. As described herein, 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 showed positive staining on human adenocarcinomas known to be sLea positive but not on sLea negative malignancies or most normal tissues. 89Zr-radiolabeled 5B1 (”Zr—531) displayed high labeling (>80%) and puriﬁcation yields (>95%). g with 89Zr-SBl was investigated in subcutaneous, orthotopic and metastatic pancreatic cancer xenografts in female SCID mice. Acquired PET images and biodistribution studies demonstrated ional speciﬁcity and localization of 89Zr—SBl for the sLea overexpressing BxPC3 xenografts with minimal non- speciﬁc binding to healthy tissues. Further analysis in colon and small cell lung cancer subcutaneous xenograft models resulted in excellent tumor delineation by 89Zr-SBl as well. Accordingly, these results show that 89Zr-SBl can be used as a molecular probe for early detection of sLea sing malignancies in the clinic.

Cell lines and Tissue Culture All tissue culture lations were performed following sterile techniques. The small cell lung cancer DMS79 and BXE’CB pancreas cancer cells were obtained from the an Type Culture tion (ATCC, Manassas, VA). ColoZOS-luc colorectal cancer cells (Bioware Ultra) were purchased from Caliper Life Sciences (CLS, Hopkinton, MA).

All cells were grown according to the recommendations ofATCC and CLS under 37°C with 5% C02 humidiﬁed atmosphere.

W0 53871 60 PCT/USZOl4/052631 In vitro evaluation of sLea expression levels through FACS Flow cytometry with the indicated cultured cancer cell lines was performed as described herein in Example I. In brief, single cell suspensions of 1x106 culture tumor cells per tube were washed in PBS with 3% fetal bovine serum (FB S). Human monoclonal antibodies r5B1 (IgG against sLea) was then added at 20 ug/ml per tube, and incubated on ice for 30min. After wash in PBS with 3% PBS, 20 ul of 1 :25 diluted goat anti-human IgG labeled with ﬁuorescein-isothiocyanate (FITC, Southern Biotechnology, Birmingham, AL) was added, and the mixture incubated for r 30 minutes on ice. After a ﬁnal wash, the positive tion and median ﬂuorescence intensity of d cells were differentiated using FACS Scan (Becton & Dickinson, San Jose, CA). Cells stained only with goat antihuman IgG labeled with ﬁuorescein-isothiocyanate were used to set the FACScan result at 1% as background for comparison to percent positive cells stained with primary mAb. ation of 89Zr-labeled antibodies Recombinant 5B1 antibodies was prepared and puriﬁed as bed . The 5B1 antibodies and a non-speciﬁc human IgG were ﬁlnctionalized with p- isothiocyanatobenzyl-desferrioxamine (DFO-Bz-NCS, Macrocyclics, Inc., Dallas, TX) with a 1:4 mAb:DFO-Bz-NCS ratio. For example, to 300 uL of 5B1 (1.23 mg in PBS, pH~9), a volume of 7.2 uL -NCS (4.25 mM in DMSO) was added. The reaction was incubated at 37 0C for 1-1.5 h. The ﬁlnctionalized antibodies were puriﬁed via either PD10 desalting column (GE Healthcare) or a 10 kDa centrifugal ﬁlter n).

Zr—89 was produced through proton beam bombardment of yttrium foil and isolated in high purity as Zr-89 oxalate at MSKCC according to previously established procedure.

Holland et al., Nuclear ne and Biology 36:729-39 (2009). Labeling of the antibodies ded via methods as described by Holland et al., Journal ofNuclear Medicine oﬁicialpublication, Society ofNuclear Medicine 51 :1293-300 (2010). In general, Zr—89 oxalate was neutralized to pH 7.0-7.2 with 1 M N32C03. The DFO- antibodies were then added. The reaction was incubated at room temperature for 1-2 hours. Subsequent puriﬁcation was conducted using either a PD10 desalting column with 0.9% saline.

In vitro experiments W0 2015/053871 61 PCT/USZOl4/052631 89Zr-SBl was investigated for stability in vitro in 0.9% saline and in 1% bovine serum albumin for 5 days at 37°C. Changes in radiochemical purity were monitored at t=0-5 days via radio iTLC with 50 mM DTPA as 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 All animal studies were conducted in accordance with the guidelines set by the Institutional Animal Care and Use Committee. Female CBl7SC-F SCID mice (Jackson Laboratories, 6-8 weeks, 20-22 g) or nude athymic (nu/nu) mice were induced with tumors on hind legs. All cell lines were inoculated subcutaneously in 200 uL of l :l media:Matrigel (BD Biosciences) solution and grown to a maximum tumor volume of 250 mm3 before use. tribution Studies Biodistribution s were performed on several cohorts of mice bearing separate ColoZOS-luc colorectal, BxPC3 pancreas and DMS79 small cell lung xenografts ).

Zr—89 mAbs (10-20 uCi, 1-2 ug) in 100 uL 0.9 % saline were administered enously in the lateral vein. Additional unlabeled mAb (10-50 ug) was co-injected along with the tracer. A blocking study with 250 ug excess of unlabeled mAb was performed to address specificity of the antibody to sLea in a cohort of mice. After each time point (t=24, 48, 120 h p.i.), the mice were euthanized by asphyxiation with C02. Blood was collected ately via cardiac puncture while the tumor along with chosen organs was ted. The wet weight of each tissue was measured, and the radioactivity bound to each organ was counted using a Wizard2 2480 gamma counter (Perkin Elmer). The percentage of tracer uptake expressed as % injected dose per gram (%ID/g) was ated as the activity bound to the tissue per organ weight per actual injected dose decay- corrected to the time of counting.

Small animal immuno-PET g experiments were accomplished with a microPET Focus 120 or R4 scanner (Concorde Microsystems). Mice (n=3-5) were administered Zr-89 labeled antibodies (200-300 uCi, 15-25 ug) in 0 LLL 0.9% saline formulations via lateral tail vein W0 2015/053871 62 injections. PET whole body acquisitions were recorded on mice at 24-96 h pi. while esized with l.5-2.0% isoﬂuorane (Baxter Healthcare) in oxygen. The images were analyzed using ASIPro VMTM software (Concorde Microsystems). Regions-of—interest (ROI) were drawn and plotted vs. time.

Immunohistochemistry Biotinylated 5B1 was prepared by incubating 20x molar excess Sulfo-NHS-LC-biotin (Thermo Scientific/Pierce cat#2l327) for 30 minutes at room temperature. Free biotin was removed with ZebraTM desalt spin columns (Thermo Scientific/Pierce, cat # 89889) according to the manufacturer’s instructions. The antibodies were buffer exchanged to PBS ning 0.01% sodium azide at a concentration of 1.1 mg/ml. The binding on DMS79 cells was conﬁrmed by FACS and was comparable to the parent 5B1 antibody.

Preliminary histochemistry staining conditions were determined using Colo205 cells as positive control and SK-MEL28 cells as ve l. Cell pellets were prepared, formalin fixed and paraffin embedded. The slides were incubated with biotinylated 5Bl diluted in 10 % (v/v) normal human serum in PBS (Jackson ImmunoResearch Labs; cat# 009l2l). The staining was performed by Ventana tion (Discovery XT platform-Ventana Medical Systems, Inc, Tucson, AZ) with standard streptavidin-biotin immunoperoxidase method and DAB detection system as a staining method. Antigen recovery was conducted using heat and Ventana’s CCl conditioning on. CA 19.9 mouse monoclonal (clone ll6-NS-l9-9) from Signet (Covance) gave comparable results in the pilot study. Colo205 cells are strongly positive with biotinylated 5B1 used at 10 ug/ml while SKMEL28 cells were completely negative.

Histo-ArrayTM tissue microarrays were sed from Imgenex (San Diego, CA). The ing slides containing tumor biopsy cores as well as some normal tissue cores were used: IMH-327 n Cancers, 59 samples), IMH-359 (colorectal: -metastasis- normal; 59 samples), and IMH-324 (Metastatic cancer to ovary). atic tumor tissue cores were present on IMH-327. sLea serum concentration in vivo Mice bearing xenografts of Colo205, BxPC3 and DMS79 were exsanguinated for sLea antigen assays. A group of mice with no tumor served as a control. The sLea levels in the sera of mice were measured using the ST AIA-PACK CAl9.9 kit (Cat# , TOSOH Bioscience Inc, South San Francisco, CA). The principle of the assay is based on the two W0 2015/053871 63 PCT/USZOl4/052631 site immunoenzyme-metric assay. The analysis was performed as bed in the manufacturer’s instruction manual. The optical density of immunoassay plates were measured by TOSOH AIA2000 Automated immunoassay analyzer (TOSOH Bioscience, Inc, San sco, CA).

Statistical Analysis Data values were expressed as the mean :: SD unless otherwise stated. Statistical analysis was performed using GraphPad Prism version 5.03 software using y ANOVA followed by Dunnett test. A P value of <0.05 is considered statistically signiﬁcant.

Results The binding icity of 5Bl was probed by staining selected ant and normal tissue microarrays. 5Bl reactivity was restricted to malignancies and occasional normal tissues previously known to overexpress sLea (; Table 9). Most normal tissues were completely negative (Table 9). In contrast, strong ve staining was found in 21/34 colon adenocarcinomas (62%), 33/57 arcinoma metastases to the ovary (58%), and 7/9 pancreatic ductal cancers (66%) at various stages (Table 10). As shown in , typical reactivity was diffuse cytoplasmic ng with some tumor cells clearly showing ct staining of the cell membrane. In addition, some signet ring n cancers, and some cancers of the lung and breast were also 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: Survey of 5Bl binding to normal tissues.

Normal Tissue Stain Brain negative Breast positive Colon positive Kidney negative Liver negative Lung negative Lymph node negative Muscle negative Pancreas positive Placenta negative Skin negative Spleen negative Stomach ne ative W0 2015/053871 64 PCT/USZOl4/052631 Table 10: ng of Pancreatic Ductal Adenocarcinomas with 5B1.

[HC 531 Sta e A e Sex Histolo neg II 71 VI moderately differentiated p0s++ III 68 VI tely differentiated neg III 64 F moderately differentiated p0s++ III 46 VI moderately differentiated p0s++ III 54 VI moderately differentiated p0s++ III 40 VI moderately differentiated pos+/- IVA 66 VI tely differentiated p0s++ IVA 45 VI moderately differentiated poor tissue IVA 64 F moderately differentiated os++ IVA 69 VI oorl differentiated The high speciﬁcity of 5B1 immunostaining for cancer tissues expressing sLea was the basis for using this mAb as a PET probe. Modiﬁcation of 5B1 with the benzyl- isothiocyanate analog of desferrioxamine z-NCS) was made at a ratio of 4:1 (chelate:mAb) with subsequent puriﬁcation via centrifugal ﬁltration using saline as the washing buffer. Facile radiolabeling with Zr—89 proceeded at room temperature after pH adjustment to 7.0-7.2. A narrower pH range closer to neutral is ary to achieve optimum radiolabeling yields of > 80%. Free, unbound Zr-89 was removed via PDlO desalting column. Concentration of the product was made using a centrifugal ﬁlter (MWCO: 10 kDa). A relatively high speciﬁc activity of 12. 1:1 .1 mCi/mg was established. Radiochemical purities of more than 95% were ensured prior to use.

Immunoreactivity assays displayed retention of activity for sLea (72.4::l .l%, n=3).

Stability in bovine serum albumin at 37°C was ined at > 95% over 5 days (data not shown). In saline, de-metallation was observed as early as 24 h (>85% complexed) with about >75% radiometal bound after 120 h at 37°C.

Small animal PET imaging and biodistribution s were conducted using female SCID mice subcutaneously ted with BxPC3 pancreas cancer xenografts on the left hind leg. Acquired PET images conﬁrmed substantial delineation of the tumor-associated sLea by 89Zr—5Bl. From the maximum ity projections (MIP) in , the BxPC3 xenografts (n=3) showed exceptional ion of the radiotracer administered intravenously. Regions-of—interest (ROI) drawn on the tumor from the PET images displayed an uptake of 50:04 %ID/g (2 h), l6.2::2.5 %ID/g (24 h), 23.8::4.7 %ID/g (48 h), 36.8::6.l %ID/g (96 h) and 49.5::7.7 %ID/g (120 h). Blood pool and normal tissue binding activity ed to clear after 24 h pi. Results from the biodistribution experiments are consistent with the PET data. High tumor localization of 89Zr-SBl at 24 h (84.7::12.3%ID/g, n=4) was observed; increased uptake was exhibited ﬁarther at 120 h pi. (114.1::23.1%ID/g, n=4) (). The tumor uptake exceeds 100% due to the small weight :0.03 mg). The %ID at 24 h pi. was found to be ten-fold higher than that of the non-speciﬁc IgG at similar time points ( Inset). Competitive inhibition with 250 ug of non-radiolabeled 5B1 at 24 h pi. blocked the tracer accumulation deﬁning the speciﬁcity of uptake. Minimal binding of the 89Zr-SBl to normal pancreas and the rest of the ted normal tissues was observed, providing a high to-tissue contrast at all time points.

Following the above results, 89Zr-SBl was assayed in an orthotopic BxPC3 pancreas tumor model. Orthotopic models are clinically relevant and offer an clinically accepted test of the efﬁcacy of the PET probe. After inoculation in the pancreas, the tumor growth was red weekly via bioluminescent optical imaging. PET imaging experiments were conducted once the tumors are palpable. A comparison of probe tumor delineation properties were made n FDG-PET and 89Zr-5B1 (). ed tomography (CT) in tandem with PET afforded an enhanced visualization of the anatomic region of interest.

To evaluate 89Zr-SBl as a PET probe in other sLea expressing adenocarcinomas, 89Zr-SBl was assayed in lung and colon cancer models. Small animal ments were conducted using DMS79 small cell lung cancer cells and 5-luc colon cancer cells injected subcutaneously on the right hind leg of female SCID mice. PET MIP images were acquired after 24-120 h pi. of 200-300 uCi (16-25 ug) injected intravenously.

Heterogeneous DMS79 tumor uptake was demonstrated with 38. 15:2. 12 %ID/g as early as 24 h pi with excellent signal against ound (A). An increase in tracer tumor accumulation resulted after 48 h pi. (44.60::6.47 %ID/g) with retention at 120 h pi. (41 .97::12.23 %ID/g). Non-speciﬁc bound 89Zr-5B1 d rapidly from normal tissues with minimal to no background uptake at 48 h pi. In addition, tumor delineation was observed in the Colo205-luc xenografts as shown in B at 24-120 h pi. The ROIs displayed tumor accumulation with 10.5::0.76, 23.5::2.7, 24.8::4.0, 4.7, l6.5::2.3 %ID/g at 2, 24, 48, 96 and 120 h tively. An observable increase in liver accumulation resulted over time with consequent decrease in tumor uptake as shown in the regions-of-interest drawn from the PET images (C). Data generated from the biodistribution studies correlate well with the observed PET results (data not shown).

W0 2015/053871 66 The sLea level in mouse serum as tumors progressed was ﬁed. uination of SCID mice bearing Colo205, DMS79 and BxPC3 xenografts with a mor bearing group serving as control was performed. sLea values showed high levels of sLea in mice challenged with Colo205 in comparison to the pancreatic BxPC3 and DSM79 implanted mice (Table ll).

Table 11: sLea serum values from mice bearing ctal (Colo205), pancreas (BxPC3) and small cell lung (DMS79) tumor xenografts compared to control.

Tumor type Animal # Tumor volume, mm3 sLe”, U/ml C010205-luc V11 269.5 3227 V12 257.3 2957 V13 281.3 1318 BxPC3 V11 232.38 ND.

V12 320.00 ND.

V13 220.50 ND.

DMS79 V11 288.0 ND.

V12 245.0 ND.

V13 232.4 ND.

Control V11 - 3 V12 - 3 V13 - 3 ND. = Not detected.

These s demonstrate that a radiolabeled anti-sLea antibody (89Zr-5Bl) is speciﬁc for the detection and diagnosis of pancreatic adenocarcinoma and other sLea positive adenocarcinomas. 89Zr—5Bl was produced with excellent yields and purity, along with high speciﬁc activity and retained immunoreactivity. Evaluation of 89Zr—5Bl in subcutaneous, orthotopic and metastatic as tumor models afforded excellent tumor delineation and diagnosis. Pre-clinical evaluation of this radiotracer in colon and small cell lung bearing small animals demonstrated the universal utility of this tracer for malignancies expressing sLea.

EXAMPLE III Anti-sLea Diabodies Bind to Various Cancer Cell Lines Two diabodies were generated using the VH and VL domains of 5B1 and 7E3 clonal isolates described herein, designated 5B1CysDb and 7E3CysDb, respectively (FIGS. 9 and ). Both diabodies contained a ﬁve amino acid linker region between the VL and VH domains. A poly histidine tag on the C-terminal, which was utilized for puriﬁcation and ion, was also included for both diabodies.

The g 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, was assayed by incubating 0.25 million cells in 0.2 ml with 10 ug/ml 5B1CysDb or 7E3CysDb, respectively. The cell and diabody combinations were incubated for 40 minutes on ice in PBS/2% PBS.

After washing, the cells were incubated for 40 s with 0.2 ml ALEXAlabeled anti-His antibody diluted 1:1000 (Life Technology, Cat # A212l5). Following a second wash, the cells were analyzed with a Guava Flow Cytometer. Both 5B1CysDb and 7E3CysDb trated signiﬁcant binding to DMS-79, Capan-2 and BXPC3 cells (Table 12).

Table 12: Binding of 5B1CysDb and 7E3CysDb to Cell Lines 5B1CysDb 7E3CysDb Cell line Percent (+) MFI Percent (+) MFI DMS-79 98.1 113.0 93.8 124.6 Capan-2 63.8 98.5 65.9 235.3 BXPC3 51.3 39.9 50.2 49.7 MFI — mean ﬂuorescent ity EXAMPLE IV stration of 5B1 and Taxol Inhibits Tumor Growth The anti-tumor ty of inistrating an anti-sLea antibody (5B1) and the chemotherapeutic agent Taxol (Paclitaxel) was assessed in xenograft models of pancreatic cancer and small cell lung cancer. As described previously herein, 1 n BXPc3 cells (pancreatic tumor cells) or 5 million DMS-79 cells (small cell lung cancer cells) were injected into the hind ﬂank of 6 weeks old female CB17 SCID mice (Day 0; N=5).

W0 2015/053871 68 ZOl4/052631 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 given ip twice a week (strating 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 in comparison to control human IgG or 5B1 antibody and Taxol administered individually ().

In the BXPc3 xenograft model, tumors were grown for 14 days, at which they reached an average of 126::30 mm3. Taxol was administered iv on days 14, 21, 28 and 34 (weekly) and 5B1 was given twice per week starting on day 14. Co-administration of 5B1 antibody and Taxol signiﬁcantly limited tumor growth in comparison to controls or 5B1 antibody and Taxol administered individually (). These results demonstrate a synergistic effect of an anti-sLea antibody and a chemotherapeutic agent in ting tumor growth and/or reducing tumor size for atic and small cell lung cancers.

Throughout this application various publications have been referenced. The disclosures of these ations in their entireties are hereby incorporated by reference in this application in order to more ﬁllly describe the state of the art to which this invention pertains. Although the invention has been bed with reference to the examples provided above, it should be understood that various ations can be made without departing from the spirit of the invention.

Claims

CLAIMS 1.:

1. Use of a polynucleotide for the preparation of a pharmaceutical composition for the treatment of a cancer or tumor formation having cells sing Sialyl-Lewisa, said treatment comprising the administration of said composition and an agent of chemotherapy, the polynucleotide encoding an antibody or a polypeptide comprising a functional fragment thereof that binds to -Lewisa, said antibody or polypeptide ses a variable heavy chain (VH) domain and a variable light chain (VL) domain, where said VH domain and said VL domain respectively comprise CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-142 of SEQ ID NO: 2 and CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-130 of SEQ ID NO: 4.

2. Use of an antibody or a polypeptide comprising a functional fragment f that binds to -Lewisa for the preparation of a pharmaceutical composition for the treatment of a cancer or tumor formation having cells expressing Sialyl-Lewisa, said treatment sing the administration of said composition and an agent of chemotherapy, said antibody or polypeptide comprising a variable heavy chain (VH) domain and a variable light chain (VL) domain, where said VH domain and said VL domain respectively comprise CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-142 of SEQ ID NO: 2 and CDR1, CDR2 and CDR3 of an amino acid ce comprising residues 20-130 of SEQ ID NO: 4.

3. The use of an agent of chemotherapy, for the preparation of a pharmaceutical composition for the treatment of a cancer or tumor formation having cells expressing Sialyl-Lewisa, said treatment comprising the administration of said composition and a cleotide encoding an antibody or a polypeptide sing a functional fragment thereof that binds to Sialyl-Lewisa, said antibody or polypeptide comprising a variable heavy chain (VH) domain and a le light chain (VL) domain, where said VH domain and said VL domain respectively comprise CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-142 of SEQ ID NO: 2 and CDR1, CDR2 and CDR3 of an amino acid sequence sing es 20-130 of SEQ ID NO: 4.

4. The use of an agent of chemotherapy, for the preparation of a pharmaceutical composition for the treatment of a cancer or tumor formation having cells expressing Sialyl-Lewisa, said treatment comprising the administration of said composition and an antibody or polypeptide comprising a functional fragment thereof that binds to Sialyl- Lewisa, said antibody or ptide comprising a variable heavy chain (VH) domain and a variable light chain (VL) , where said VH domain and said VL domain tively comprise CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-142 of SEQ ID NO: 2 and CDR1, CDR2 and CDR3 of an amino acid sequence comprising residues 20-130 of SEQ ID NO: 4.

5. The use of any one of claims 1 to 4, wherein said antibody is a human antibody.

6. The use of any one of claims 1 to 4, wherein said polypeptide is selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a scFV, a diabody, a triabody, and a minibody.

7. The use of claim 6, wherein said polypeptide is a diabody.

8. The use of claim 7, wherein said diabody comprises the amino acid sequence of SEQ ID NO: 18.

9. The use of any one of claims 1 to 4, wherein said dy is a monoclonal antibody.

10. The use of any one of claims 1-9, wherein said antibody is an IgG or IgM isotype.

11. The use of claim 10, wherein said IgG antibody is an IgG1 subclass.

12. The use of any one of claims 1 to 11, wherein the antibody or polypeptide is conjugated or inantly fused to a diagnostic agent, detectable agent or therapeutic agent.

13. The use of claim 12, wherein said detectable agent is a radioactive material or a fluorescent material.

14. The use of claim 13, wherein said radioactive material is zirconium (89Zr), iodine (131I, 125I, 124I, 123I, and 121I,), carbon (14C and 11C), sulfur (35S), tritium (3H), indium , 113In, 112In, and ), technetium (99Tc), thallium (201Ti), gallium (68Ga and 67Ga), palladium (103Pd), molybdenum (99Mo), xenon ), 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, or wherein said fluorescent material is selected from the group consisting of umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, rotriazinylamine fluorescein, dansyl chloride and phycoerythrin.

15. The use of claim 12, wherein said therapeutic agent is radioactive metal or an Auristatin molecule.

16. The use of claim 15, wherein said radioactive metal is an alpha-emitter, or wherein said Auristatin molecule is selected from the group consisting of auristatin PHE, bryostatin 1, solastatin 10, monomethyl auristatin E (MMAE) and thylauristatin F (MMAF).

17. The use of any one of claims 1 to 16, wherein 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 , pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal cancer, small cell carcinoma of the lung, bladder adenocarcinoma, signet ring ovarian cancer, ovarian cancer, metastatic carcinoma, adenocarcinoma of the stomach, adenocarcinoma of the esophagus, adenocarcinoma of the , adenocarcinoma of the urogenital tract, and adenocarcinoma of the breast.

18. The use of any one of claims 1 to 17, wherein the treatment comprises concurrent or successive administration of the antibody or polypeptide, or the polynucleotide ng said dy or polypeptide, and the agent of chemotherapy.

19. The use of any one of claims 1 to 18, wherein the agent of chemotherapy is at least one agent ed from the group consisting of an cycline; a taxan; an antimetabolite; an alkylating agent, lomustine, cyclothosphamide, busulfan, dibromomannitol, ozotocin, mitomycin C, cisdichlorodiamine platinum (II) (DDP), cisplatin; an antibiotic; an Auristatin le; a hormone; a nucleoside analoge, a DNA- repair enzyme inhibitor, a kinase inhibitor; a cytotoxic agent, a topoisomerase inhibitor; a DNA minor groove binder; and adenosine deaminase tors; or pharmaceutically acceptable salts, solvates, clathrates, or prodrugs f.

20. The use of claim 19, wherein the agent of chemotherapy is a taxan selected from paclitaxel (Taxol) and docetaxel (Taxotere).

21. The use of claim 19, wherein the agent of chemotherapy is selected from the group consisting of doxorubicin, ubicin, methotrexate, 6-mercaptopurine, 6- thioguanine, bine, 5-fluorouracil and decarbazine, mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU), lomustine , cyclothosphamide, busulfan, omannitol, streptozotocin, actinomycin D, bleomycin, mithramycin, and anthramycin (AMC); auristatin PHE, bryostatin 1, solastatin 10, monomethyl auristatin E (MMAE) and monomethylauristatin F (MMAF), glucocorticoids, progestins, ens, ens, gemcitabine, etoposide, topotecan, Gleevec, imatinib mesylate; maytansine, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, e, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, oxy anthracin dione, mitoxantrone, mithramycin, 1- dehydrotestosterone, glucorticoids, ne, tetracaine, lidocaine, propranolol, puromycin and analogs or homologs thereof.

22. A use according to claim 1, substantially as herein described and exemplified.

23. A use according to claim 2, substantially as herein described and exemplified.

24. A use according to claim 3, substantially as herein described and exemplified.

25. A use according to claim 4, substantially as herein described and exemplified. 1 /27 W0