Nucleic acids encoding human antibodies to sialyl-lewis a

Abstract

To provide nucleic acids encoding human antibodies to Sialyl-Lewis a.SOLUTION: 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. The antibody or functional fragment includes a variable heavy chain domain and a variable light chain domain that have 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.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 61 / 870,137, filed August 26, 2013, the entire contents of which are incorporated herein by reference.

[0002] This invention was made with government support under Grant No. CA-128362 awarded by the National Cancer Institute, NIH. The United States Government has certain rights in this application.

[0003] Background of the Invention The present invention generally relates to sialyl-Lewis a (sLe a ), more particularly anti-sLe a The present invention relates to polynucleotides encoding antibodies and the corresponding encoded antibodies or fragments thereof. [Background technology]

[0004] Passive administration of antibodies directed against tumor-specific antigens can eliminate tumor cells and early metastases during cancer development. This treatment can also have a significant impact on cancer recurrence. Antibodies directed against tumor-specific carbohydrates may be useful candidates for this cancer treatment. For example, many tumor-restricted monoclonal antibodies generated by immunizing mice with human cancer cells have been shown to target carbohydrate antigens expressed as glycolipids or glycoproteins on the cell surface. Carbohydrate sLe a has been shown to be expressed in tumors of the gastrointestinal tract. a Expression of α-carbohydrates has also been shown to affect metastatic potential and correlates with increased metastatic potential in human colon and pancreatic adenocarcinomas. However, carbohydrate chemistry is relatively challenging, and clinical development of antibodies that recognize such tumor-specific carbohydrates has been slow.

[0005] Pancreatic cancer is one of the most aggressive adenocarcinomas and is often associated with a poor prognosis. It is the fourth leading cause of cancer mortality. Despite advances in screening for various carcinomas, reliable detection of malignant lesions arising from the pancreas remains unreliable. Fluorodeoxyglucase-based positron emission tomography (FDG-PET) is used to detect and stage pancreatic cancer. However, FDG-PET is insensitive to pancreatitis during differentiation from malignant tumors, and staging of small primary lesions (<7 mm) and liver metastases (<1 cm) remains problematic. One diagnostic screening method used to monitor the status of patients with pancreatic ductal adenocarcinoma (PDAC) is serum circulating sLe. a Detecting an elevated level of the circulating sLe antigen. a Patients with antigen levels >37 U / ml indicate cancer recurrence. However, the development of alternative diagnostic tools that utilize such tumor-specific carbohydrates has been slow.

[0006] Therefore, sLe is useful for treating recurrent cancer and detecting malignant lesions and metastases. a There is a need to identify and generate antibodies that specifically recognize tumor-specific carbohydrates such as . The present invention fulfills this need and provides related advantages. Summary of the Invention [Means for solving the problem]

[0007] According to the present invention, sLe a Provided herein are compositions for producing antibodies or functional fragments thereof that bind to. The compositions include isolated polynucleotides encoding antibodies or functional fragments thereof comprising a variable heavy (VH) domain having an amino acid sequence provided herein. The isolated polynucleotides of the invention may also include nucleic acid sequences encoding the VH domain of the antibodies or functional fragments thereof provided herein.

[0008] In another embodiment of the present invention, an isolated polynucleotide can encode an antibody or functional fragment thereof comprising a variable light (VL) domain having an amino acid sequence provided herein. The isolated polynucleotide of the present invention can also comprise a nucleic acid sequence encoding the VL domain of an antibody or functional fragment thereof provided herein.

[0009] The composition of the present invention comprises sLe a In some embodiments, the present invention also includes an isolated antibody or functional fragment thereof that binds to sLe. a The present invention provides an isolated antibody or functional fragment thereof that binds to a VH domain having an amino acid sequence as provided herein.

[0010] In some embodiments, the present invention provides a method for treating sLe a The present invention provides an isolated antibody or functional fragment thereof that binds to a VL domain having an amino acid sequence as provided herein.

[0011] In some embodiments, the present invention provides a method for treating sLe a The present invention provides an isolated antibody or functional fragment thereof that binds to a clonal isolate provided herein, the antibody or functional fragment thereof comprising both a VH domain and a VL domain, wherein the VH domain and the VL domain, respectively, comprise the amino acid sequence of the VH domain and the VL domain, respectively, of a clonal isolate provided herein.

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

[0013] In some embodiments, the present invention provides pharmaceutical compositions comprising one or more antibodies or functional fragments of the present invention and a pharmaceutically acceptable carrier. In some aspects, the present invention also provides methods for treating or preventing disease in a subject in need thereof by administering a therapeutically effective amount of a pharmaceutical composition of the present invention. In yet other aspects, the present invention provides for administering a second therapeutic agent simultaneously or sequentially with the antibody or functional fragment of the present invention. In certain embodiments, for example, the following are provided: (Item 1) 1. An isolated polynucleotide encoding an antibody heavy chain or functional fragment thereof, wherein the antibody heavy chain or functional fragment thereof comprises a variable heavy (VH) domain having an amino acid sequence selected from the group consisting of residues 20 to 142 of SEQ ID NO: 2, residues 20 to 142 of SEQ ID NO: 6, residues 20 to 142 of SEQ ID NO: 10, and residues 20 to 145 of SEQ ID NO: 14. (Item 2) 2. The isolated polynucleotide according to Item 1, wherein the amino acid sequence of the VH domain is encoded by a nucleic acid sequence selected from the group consisting of residues 58 to 426 of SEQ ID NO: 1, residues 58 to 426 of SEQ ID NO: 5, residues 58 to 426 of SEQ ID NO: 9, and residues 58 to 435 of SEQ ID NO: 13. (Item 3) 1. An isolated polynucleotide encoding an antibody light chain or functional fragment thereof, wherein said antibody light chain or functional fragment thereof comprises a variable light (VL) domain having an amino acid sequence selected from the group consisting of residues 20 to 130 of SEQ ID NO:4, residues 20 to 129 of SEQ ID NO:8, residues 20 to 130 of SEQ ID NO:12, and residues 23 to 130 of SEQ ID NO:16. (Item 4) 4. The isolated polynucleotide according to Item 3, wherein the amino acid sequence of the VL domain is encoded by a nucleic acid sequence selected from the group consisting of residues 58 to 390 of SEQ ID NO: 3, residues 58 to 387 of SEQ ID NO: 7, residues 58 to 390 of SEQ ID NO: 11, and residues 67 to 390 of SEQ ID NO: 15. (Item 5) Cialis Lewis a 1. An isolated antibody or functional fragment thereof that binds to SEQ ID NO: 1, wherein the antibody or functional fragment thereof comprises a variable heavy chain (VH) domain, and the VH domain comprises an amino acid sequence selected from the group consisting of residues 20 to 142 of SEQ ID NO: 2, residues 20 to 142 of SEQ ID NO: 6, residues 20 to 142 of SEQ ID NO: 10, and residues 20 to 145 of SEQ ID NO: 14. (Item 6) Cialis Lewis a 1. An isolated antibody or functional fragment thereof that binds to SEQ ID NO: 4, wherein the antibody or functional fragment thereof comprises a variable light chain (VL) domain, and the VL domain comprises an amino acid sequence selected from the group consisting of residues 20 to 130 of SEQ ID NO: 4, residues 20 to 129 of SEQ ID NO: 8, residues 20 to 130 of SEQ ID NO: 12, and residues 23 to 130 of SEQ ID NO: 16. (Item 7) Cialis Lewis a 1. An isolated antibody or functional fragment thereof that binds to SEQ ID NO: 1, wherein the antibody or functional fragment thereof comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain and the VL domain comprise amino acid sequences selected from the group consisting of residues 20-142 of SEQ ID NO: 2 and residues 20-130 of SEQ ID NO: 4; residues 20-142 of SEQ ID NO: 6 and residues 20-129 of SEQ ID NO: 8; residues 20-142 of SEQ ID NO: 10 and residues 20-130 of SEQ ID NO: 12; and residues 20-145 of SEQ ID NO: 14 and residues 23-130 of SEQ ID NO: 16. (Item 8) 8. The isolated antibody or functional fragment thereof of any one of items 5 to 7, wherein the antibody is a human antibody. (Item 9) 8. The isolated antibody or functional fragment thereof of any one of items 5 to 7, wherein the antibody functional fragment is selected from the group consisting of Fab, Fab', F(ab')2, scFV, diabody, triabody, minibody and single domain antibody (sdAB). (Item 10) 10. The antibody or functional fragment thereof according to item 9, wherein the antibody functional fragment is a diabody. (Item 11) 11. The antibody or functional fragment of item 10, wherein the diabody comprises the amino acid sequence of SEQ ID NO: 18 or 20. (Item 12) 8. The isolated antibody or functional fragment thereof of any one of items 5 to 7, wherein the antibody is a monoclonal antibody. (Item 13) 8. The isolated antibody or functional fragment thereof of any one of items 5 to 7, wherein the antibody is an IgG or IgM isotype. (Item 14) 14. The isolated antibody or functional fragment thereof according to item 13, wherein the IgG antibody is an IgG1 subclass. (Item 15) 8. A conjugate comprising the isolated antibody or functional fragment of any one of items 5 to 7 conjugated or recombinantly fused to a diagnostic, detectable or therapeutic agent. (Item 16) 16. The conjugate according to item 15, comprising a detectable agent. (Item 17) The detectable agent is zirconium ( 89 17. The conjugate according to item 16, wherein Zr is a methyl group. (Item 18) 8. A pharmaceutical composition comprising the antibody or functional fragment according to any one of items 5 to 7 and a pharmaceutically acceptable carrier. (Item 19) 20. A method for treating or preventing a disease, comprising administering a therapeutically effective amount of the pharmaceutical composition of item 18 to a subject in need of such treatment or prevention. (Item 20) The disease is cancer or tumorigenesis, and the cells of the cancer or tumor are sLe a 20. The method of claim 19, wherein the vector expresses (Item 21) 21. The method of claim 20, wherein the cancer or tumor is selected from the group consisting of tumors of the gastrointestinal tract, colon cancer, colorectal adenocarcinoma, metastatic colon cancer, colorectal cancer, pancreatic cancer, pancreatic adenocarcinoma, small cell lung cancer, bladder adenocarcinoma, ovarian signet ring cell carcinoma, ovarian cancer, metastatic carcinoma, adenocarcinoma of the stomach, adenocarcinoma of the esophagus, adenocarcinoma of the throat, adenocarcinoma of the genitourinary tract, and adenocarcinoma of the breast. (Item 22) 20. The method of item 19, further comprising the step of simultaneously or sequentially administering a second therapeutic agent. (Item 23) 23. The method of item 22, wherein the second therapeutic agent is a chemotherapeutic agent or an immunotherapeutic agent. (Item 24) 17. A method for detecting a tumor in a subject, comprising administering to a subject in need thereof an effective amount of the conjugate of item 16. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows the nucleotide sequence and encoded amino acid sequence of the variable heavy (VH) chain domain and leader sequence of clone 5B1, which can be used for recombinant expression. The top of the figure shows an alignment of the nucleotide sequence of SEQ ID NO: 1 with the amino acid sequence of SEQ ID NO: 2. The three complementarity-determining regions (CDR1, CDR2, and CDR3) are also identified. [Figure 2] Figure 2 shows the nucleotide sequence and encoded amino acid sequence of the variable light (VL) chain domain and leader sequence of clone 5B1, which can be used for recombinant expression. The top of the figure shows an alignment of the nucleotide sequence of SEQ ID NO: 3 with the amino acid sequence of SEQ ID NO: 4. The three complementarity-determining regions (CDR1, CDR2, and CDR3) are also identified. [Figure 3]Figure 3 shows the nucleotide sequence and encoded amino acid sequence of the variable heavy (VH) chain domain and leader sequence of clone 9H3, which can be used for recombinant expression. The top of the figure shows an alignment of the nucleotide sequence of SEQ ID NO: 5 with the amino acid sequence of SEQ ID NO: 6. The three complementarity-determining regions (CDR1, CDR2, and CDR3) are also identified. [Figure 4] Figure 4 shows the nucleotide sequence and encoded amino acid sequence of the variable light (VL) chain domain and leader sequence of clone 9H3, which can be used for recombinant expression. The top of the figure shows an alignment of the nucleotide sequence of SEQ ID NO: 7 with the amino acid sequence of SEQ ID NO: 8. The three complementarity-determining regions (CDR1, CDR2, and CDR3) are also identified. [Figure 5] Figure 5 shows the nucleotide sequence and encoded amino acid sequence of the variable heavy (VH) chain domain and leader sequence of clone 5H11, which can be used for recombinant expression. The top of the figure shows an alignment of the nucleotide sequence of SEQ ID NO:9 with the amino acid sequence of SEQ ID NO:10. The three complementarity-determining regions (CDR1, CDR2, and CDR3) are also identified. [Figure 6] Figure 6 shows the nucleotide sequence and encoded amino acid sequence of the variable light (VL) chain domain and leader sequence of clone 5H11, which can be used for recombinant expression. The top of the figure shows an alignment of the nucleotide sequence of SEQ ID NO: 11 with the amino acid sequence of SEQ ID NO: 12. The three complementarity-determining regions (CDR1, CDR2, and CDR3) are also identified. [Figure 7] Figure 7 shows the nucleotide sequence and encoded amino acid sequence of the variable heavy (VH) chain domain and leader sequence of clone 7E3, which can be used for recombinant expression. The top of the figure shows an alignment of the nucleotide sequence of SEQ ID NO: 13 with the amino acid sequence of SEQ ID NO: 14. The three complementarity-determining regions (CDR1, CDR2, and CDR3) are also identified. [Figure 8]Figure 8 shows the nucleotide sequence and encoded amino acid sequence of the variable light (VL) chain domain and leader sequence of clone 7E3, which can be used for recombinant expression. The top of the figure shows an alignment of the nucleotide sequence of SEQ ID NO: 15 with the amino acid sequence of SEQ ID NO: 16. The three complementarity-determining regions (CDR1, CDR2, and CDR3) are also identified. [Figure 9] Figure 9 shows the nucleotide sequence and encoded amino acid sequence of a diabody designated 5B1CysDb, which has CDR1, CDR2, and CDR3 of both the variable heavy (VH) and variable light (VL) chain domains of clone 5B1. The top of the figure shows an alignment of the nucleotide sequence of SEQ ID NO: 17 with the amino acid sequence of SEQ ID NO: 18. The three complementarity determining regions (CDR1, CDR2, and CDR3) of both the VH and VL domains are identified in bold, underlined text. The linker sequence and the polyhistidine tag (polyHis tag) with added amino acids are also shown in italic, underlined text. [Figure 10] Figure 10 shows the nucleotide sequence and encoded amino acid sequence of a diabody designated 7E3CysDb, which has CDR1, CDR2, and CDR3 of both the variable heavy (VH) and variable light (VL) chain domains of clone 7E3. The top of the figure shows an alignment of the nucleotide sequence of SEQ ID NO: 19 with the amino acid sequence of SEQ ID NO: 20. The three complementarity determining regions (CDR1, CDR2, and CDR3) of both the VH and VL domains are identified in bold, underlined text. The linker sequence and the polyhistidine tag (polyHis tag) with added amino acids are also shown in italic, underlined text. [Figures 11A-11C]Panels A-E of Figure 11 show the binding of human anti-sLea antibodies to tumor cells analyzed by flow cytometry. Panel A shows DMS-79 cells stained with recombinant (r)5B1, 9H3, 5H11, and 7E3 antibodies. Panels B-F show HT29, BxPC3, SW626, SK-MEL28, and Colo205-luc cells stained with 1-2 μg / mL of r5B1 or r7E3 plus IgG- or IgM-specific secondary antibodies, respectively, as described in Example I. [Figures 11D-11F] Panels A-E of Figure 11 show the binding of human anti-sLea antibodies to tumor cells analyzed by flow cytometry. Panel A shows DMS-79 cells stained with recombinant (r)5B1, 9H3, 5H11, and 7E3 antibodies. Panels B-F show HT29, BxPC3, SW626, SK-MEL28, and Colo205-luc cells stained with 1-2 μg / mL of r5B1 or r7E3 plus IgG- or IgM-specific secondary antibodies, respectively, as described in Example I. [Figure 12] Panels A and B of Figure 12 show the CDC activity of the r5B1 and r7E3 antibodies compared to murine 121SLE (IgM) in the presence of human complement (Hu C') measured against DMS-79 cells. The human isotype control antibodies, Hu IgG (◇) and Hu IgM (◆), showed <4% cytotoxicity. Dose responses for the r5B1 IgG (■), r7E3 IgM (●), and 121SLE mIgM (▲) antibodies are shown in panel A. The calculated EC50 (μg / ml) for the r5B1 (IgG), r7E3 (IgM), and 121SLE (mIgM) antibodies are shown in panel B. [Figure 13]Panels A to C of Figure 13 show antibody-dependent cellular cytotoxicity (ADCC) of the r5B1 antibody. Panel A shows r5B1-mediated ADCC against DMS-79 cells using human PBMCs. PBMCs were tested with DMS-79 tumor cells at E:T ratios ranging from 100:1 to 12.5:1 in the presence or absence of 2 μg / mL of r5B1. Panel B shows r5B1-mediated ADCC against DMS-79 cells using primary human NK cells. NK cells were tested with DMS-79 tumor cells at low E:T ratios ranging from 5:1 to 0.6:1 in the presence or absence of 2 μg / mL of r5B1. Panel C shows ADCC of various concentrations of r5B1 using PBMCs from two donors at an E:T ratio of 1:100 with DMS-79 tumor cells in the presence of the indicated concentrations of r5B1. [Figure 14] Figure 14 shows the internalization of sLea into BxPC3 cells. BxPC3 pancreatic tumor cells were grown in the presence of r5B1 (anti-sLea) or r1B7 (anti-GD2) antibodies complexed with Hum-ZAP, an anti-human IgG conjugated to saporin. After 3 days, cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and sample values were normalized to those of untreated cultures. [Figure 15] Figure 15 shows the activity of the r5B1 antibody in a xenograft model using Colo205-luc cells. Severe combined immunodeficient (SCID) mice (5 per group) were injected via the tail vein with 500,000 Colo205-luc cells on day 0. Mice were injected intraperitoneally with r5B1 at a total dose of 600 μg on days 1, 7, 14, and 21 (Exp 1) or 100 μg on days 1, 4, 7, 10, 14, and 21 (Exp 2). Control (Ctrl) animals received sham injections of PBS. [Figure 16] Figure 16 shows the effect of r5B1 on Colo205-luc tumors in SCID mice. Mice were injected with 100 μg (▼), 300 μg (■), or 1 mg (◆) of r5B1 antibody per injection as described in Example I. Control (■) animals received a sham injection of PBS. [Figure 17] Figure 17 shows fluorescence imaging of r5B1-treated Colo205-luc tumor-bearing mice, 5 mice per group, at day 0 and week 5. Mice received the treatment regimen shown in Figure 16 and described in Example I. [Figure 18A] Panels A and B of Figure 18 show antitumor activity using DMS-79 cells in a therapeutic subcutaneous xenograft model. Panel A shows inhibition or regression for mice treated with 5B1 (5B1 alone (▲) or 5B1 + cRGD (▼)) compared with controls injected with human IgG (IgG alone (◆) or IgG + cRGD (●)) and PBS (■). Arrows indicate the days of antibody or PBS injection. [Figure 18B] Panels A and B of Figure 18 show antitumor activity using DMS-79 cells in a therapeutic subcutaneous xenograft model. Panel B shows a representative image of a treated mouse. The arrow indicates the absence of any visible tumor. [Figure 19] Panels A-F of Figure 19 show the binding of 5B1 to various tumor types. Panel A is a pancreatic 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 bladder mucinous adenocarcinoma, stage IV tumor. Panel E is an ovarian metastatic carcinoma from a colon tumor. Panel F is a lymph node metastatic carcinoma, stage IIIA tumor. [Figure 20] Figure 20 shows phased PET maximum intensity projection (MIP) images acquired at 2 to 120 hours using 89Zr-radiolabeled 5B1 antibody (89Zr-5B1) administered intravenously to female SCID mice implanted subcutaneously with BxPC3 pancreatic tumors. PET-MIP imaging demonstrates high tumor uptake accompanied by clearance of nonspecifically bound tracer as early as 24 hours post-injection (h p.i.). [Figure 21]Figure 21 shows the biodistribution results. This is consistent with the PET data in Figure 20, where a tumor uptake of 84.73 ± 12.28% ID / g was observed. Due to the small tumor weight, a plot of tumor uptake in % ID versus time is shown in the inset graph. The tumor % ID indicates significant tumor uptake by 89Zr-5B1 at all time points, at least 7-fold greater than that of nonspecific 89Zr-IgG. Competitive inhibition with non-radioactive 5B1 (200 μg) demonstrates reduced tumor accumulation. [Figure 22] Figure 22, panels A-C, show PET-MIP images of mice bearing DMS79 xenografts (panel A) and Colo205-luc xenografts (panel B). PET-MIP imaging of tumor (T), heart (H), and liver (L) with 89Zr-5B1 is shown. The colorectal Colo205-luc xenograft model showed 89Zr-5B1 accumulation that peaked at 24 hours and eventually decreased, indicating increased nonspecific binding to the liver (panel C). [Figure 23] Figure 23 shows dose-dependent inhibition of tumor growth and regression in a DMS-79 small lung cell carcinoma xenograft model treated with sequential co-administration of the 5B1 antibody and taxol (paclitaxel). The large arrow on the x-axis indicates treatment with 5B1. Co-administration of the 5B1 antibody and taxol significantly restricted tumor growth and led to tumor regression compared with control human IgG (HuIgG) or separate administration of the 5B1 antibody and taxol. Two-way ANOVA indicates significant differences at p<0.01 (**) and p<0.001 (***). N=5. [Figure 24] Figure 24 shows the inhibition of tumor growth in a BxPc3 pancreatic cancer xenograft model treated by sequential co-administration of the 5B1 antibody and taxol (paclitaxel). The large arrow on the x-axis indicates treatment with taxol and 5B1, and the small arrow indicates treatment with 5B1 alone. Co-administration of the 5B1 antibody and taxol significantly restricted tumor growth compared to control (PBS-Ctrl; human IgG-HuIgG) or separate administration of the 5B1 antibody and taxol. [Figure 25] Panels A and B of Figure 25 show representative images of a mouse orthotopically implanted with a BxPC3-luc pancreatic tumor xenograft. Panel A: Co-imaging of FDG-PET with computed tomography (CT) (left) and planar sections of FDG-PET alone (right) demonstrate minimal tumor detection by the tracer, with significant uptake in highly metabolic tissues (i.e., heart, H and bladder, B). Panel B: Co-imaging of CT with 89Zr-radiolabeled 5B1 antibody (89Zr-5B1) PET images acquired from the same mouse demonstrates excellent tumor detection of the BxPC3-luc tumor xenograft. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description of the Invention Carbohydrates expressed on the surface of tumor cells can be targets for passive immunotherapy. The compositions provided herein are, at least in part, sialyl-Lewis a -Keyhole limpet hemocyanin (sLe a The study is based on the identification and characterization of human antibodies generated from blood lymphocytes of individuals immunized with a sLe (KLH) conjugate vaccine. a At least four antibodies with high affinity for sLe were identified (5B1, 9H3, 5H11, and 7E3). Two of these antibodies were expressed as recombinant antibodies (r5B1 and r7E3) and further characterized in in vitro and in vivo models. Both antibodies were potent in complement-dependent cytotoxicity (CDC) assays, and the 5B1 antibody was also highly active in antibody-dependent cellular cytotoxicity assays. The in vivo efficacy of the antibodies was tested in two xenograft models using either Colo205 or DMS-79 tumor cells implanted in severe combined immunodeficient (SCID) mice. The relevance of the technology transfer of the invention provided herein is two-fold. First, the approach provided herein allows for the detection of sLe. aThis demonstrates that the antibody responses elicited by the KLH vaccine are useful as a vaccine itself. Second, the most potent antibodies generated in clinical trials can be preserved and ultimately used as therapeutics or in the generation of therapeutics for targeted cancer populations. The high affinity of the antibodies provided herein and their high effector functions support the potential for this technology transfer.

[0016] As used herein, the term "antibody" refers to a polypeptide product of B cells that is capable of binding to a specific molecular antigen and that is comprised of a pair of two identical polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids, and the carboxy-terminal portion of each chain containing a constant region (see Borrebaeck (ed.) (1995) Antibody Engineering, 2nd ed., Oxford University Press; Kuby (1997) Immunology, 3rd ed., W.H. Freeman and Company, New York). In the context of the present invention, the specific molecular antigen to which the antibodies of the present invention can bind includes the target carbohydrate sLe. a Examples include:

[0017] The term "human," when used with respect to an antibody or functional fragment thereof, refers to an antibody or functional fragment thereof having a human variable region and / or a human constant region or portion thereof that corresponds to human germline immunoglobulin sequences. Such human germline immunoglobulin sequences are described by Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242. A human antibody, in the context of the present invention, is defined as a sLe aand encoded by a nucleic acid sequence that is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence. An exemplary method for making a human antibody is provided in Example I, although any method known to one of skill in the art can be used.

[0018] 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. Monoclonal antibodies also refer to antibodies produced by recombinant methods from immunoglobulin genes encoding heavy and light chains, so that a single immunoglobulin species is produced. The amino acid sequences of antibodies within a monoclonal antibody preparation are substantially uniform, and the binding activity of antibodies within such a preparation exhibits substantially the same antigen-binding activity. In contrast, polyclonal antibodies are a combination of immunoglobulin molecules that bind to a specific antigen obtained from different B cells within a population. Each immunoglobulin in a polyclonal antibody can bind to a different epitope of the same antigen. Methods for producing both monoclonal and polyclonal antibodies are well known in the art (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) and Borrebaeck (ed.), Antibody Engineering: A Practical Guide, W.H. Freeman and Co., Publishers, New York, pp. 103-120 (1991)).

[0019] As used herein, the term "functional fragment," when used with respect to an antibody, refers to a portion of an antibody, including a heavy or light chain polypeptide, that retains some or all of the binding activity of the antibody from which the fragment is derived. Such functional fragments include, for example, Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, and minibodies. Other functional fragments include, for example, heavy or light chain polypeptides, variable region polypeptides, or CDR polypeptides, or portions thereof, so long as such functional fragments retain binding activity. Binding fragments of such antibodies can be found, for example, in 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 Publishers, Inc.; Huston et al., Cell Biophysics, 22:189-224 (1993); Plueckthun and Skerra, Meth. Enzymol., 178:497-515 (1989), and Day, ED, Advanced Immunochemistry, 2nd ed., Wiley-Liss, Inc., New York, NY (1990).

[0020] The term "heavy chain," when used in reference to an antibody, refers to a polypeptide chain of approximately 50 to 70 kDa, the amino-terminal portion of which contains a variable region of approximately 120 to 130 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The constant region can be one of five distinct types, designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains vary in size, with α, δ, and γ containing approximately 450 amino acids, and μ and ε containing approximately 550 amino acids. These distinct types of heavy chains, when combined with light chains, give rise to the five well-known classes of antibodies: IgA, IgD, IgE, IgG, and IgM, including the four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4. The heavy chain can be a human heavy chain.

[0021] The term "light chain," when used in reference to an antibody, refers to a polypeptide chain of approximately 25 kDa, the amino-terminal portion of which contains a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. Two distinct types exist, designated kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. The light chain may be a human light chain.

[0022] The term "variable domain" or "variable region" refers to the portion of an antibody light or heavy chain, generally located at the amino terminus of the light or heavy chain, approximately 120-130 amino acids in length for heavy chains and approximately 100-110 amino acids in length for light chains, that is used for binding and specificity of a particular antibody to its specific antigen, respectively. Variable domains vary extensively in sequence among different antibodies. Sequence variability is concentrated in the CDRs, while the less variable portions of variable domains are referred to as framework regions (FRs). The CDRs of the light and heavy chains are primarily responsible for antibody-antigen interactions. The numbering of amino acid positions used herein follows 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. The variable region may be a human variable region.

[0023] CDR refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH β-sheet framework, or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of the antibody VL β-sheet framework. Thus, CDRs are variable region sequences interspersed within framework region sequences. CDR regions are well known to those skilled in the art, and have been defined, for example, by Kabat as the most hypervariable region within an antibody variable (V) domain (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, Adv. Prot. Chem. 32:1-75 (1978)). CDR region sequences have also been structurally defined by Chothia as residues that are not part of the conserved β-sheet framework and therefore can adopt 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 standard antibody variable domains have been determined by comparing multiple 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 hypervariable regions varies among different antibodies, standard variable domain numbering schemes conventionally assign a, b, c, etc., number adjacent to the residue number for additional residues relative to the standard position (Al-Lazikani et al., supra (1997)). Such nomenclature is also well known to those skilled in the art.

[0024] For example, CDRs defined according to either the Kabat (hypervariable) or Chothia (structural) nomenclature are set forth in Table 1 below.

[0025] [Table 1]

[0026] One or more CDRs can be incorporated into a molecule, either covalently or noncovalently, to create an immunoadhesin. An immunoadhesin can incorporate the CDR(s) as part of a larger polypeptide chain, can covalently link the CDR(s) to another polypeptide chain, or can incorporate the CDR(s) noncovalently. The CDRs enable the immunoadhesin to bind to a specific antigen of interest.

[0027] As used herein, the term "isolated," when used with respect to an antibody, antibody functional fragment, or polynucleotide, means that the referenced molecule is free from at least one component with which it is found in nature. This term encompasses antibodies, antibody functional fragments, or polynucleotides that have been removed from some or all of the other components found in their natural environment. Components of an antibody's natural environment include, for example, red blood cells, white blood cells, platelets, plasma, proteins, nucleic acids, salts, and nutrients. Components of an antibody functional fragment or polynucleotide's natural environment include, for example, lipid membranes, organelles, proteins, nucleic acids, salts, and nutrients. An antibody, antibody functional fragment, or polynucleotide of the invention may also be free or substantially free from all of these or any other components of the cells from which it is isolated or recombinantly produced.

[0028] As used herein, "isotype" refers to the antibody class encoded by the heavy chain constant region genes. The heavy chain of a given antibody or functional fragment determines the class of that antibody or functional fragment: IgM, IgG, IgA, IgD, or IgE. Each class may have either a kappa or lambda light chain. The term "subclass" refers to minor differences in the amino acid sequence of the heavy chain that distinguish the subclass. In humans, there are two subclasses of IgA (subclasses IgA1 and IgA2) and four subclasses of IgG (subclasses IgG1, IgG2, IgG3, and IgG4). Such classes and subclasses are well known to those skilled in the art.

[0029] The terms "binds" or "binding," as used herein, refer to an interaction between molecules that results in the formation of a complex. The interaction may be a non-covalent interaction, including, for example, hydrogen bonding, ionic bonding, hydrophobic interactions, and / or van der Waals interactions. A complex may also include the binding of two or more molecules that are held together by covalent or non-covalent bonds, interactions, or forces. Binding of an antibody or functional fragment thereof can be detected using, for example, an enzyme-linked immunosorbent assay, a method presented in Example I, or any one of several methods well known to those of skill in the art.

[0030] A single antigen-binding site on an antibody or functional fragment and sLe a The strength of the overall non-covalent interactions between an antibody or functional fragment and a single epitope of a target molecule, such as a nucleotide, is the affinity of the antibody or functional fragment for that epitope. The association (k1) and dissociation (k2) of an antibody or functional fragment with a monovalent antigen are expressed as -1 ) ratio (k1 / k -1 ) is the association constant, K, which is a measure of affinity. The value of K varies for different complexes of antibody or functional fragment with antigen, and the values of k and k -1 The association constant K for an antibody or functional fragment of the invention can be determined using any of the methods provided herein or any other method known to one of skill in the art.

[0031] The affinity at one binding site does not necessarily reflect the true strength of the interaction between the antibody or functional fragment and the antigen. aFor antibodies containing multiple binding sites, such as those containing multiple repeating antigenic determinants that contact an antibody, interaction of the antibody or functional fragment with the antigen at one site increases the probability of reaction at a second site. The strength of the multiple interactions between such a multivalent antibody and the antigen is referred to as avidity. The avidity of an antibody or functional fragment may be a better measure of its binding capacity than the affinity of its individual binding sites. For example, high avidity can compensate for low affinity, as is sometimes found with pentameric IgM antibodies, which may have lower affinity than IgG but whose high avidity of IgM, resulting from their multivalency, allows them to bind effectively to antigens.

[0032] The specificity of an antibody or functional fragment thereof refers to the ability of an individual antibody or functional fragment thereof to react with only one antigen. An antibody or functional fragment can be considered specific if it can distinguish differences in the primary, secondary, or tertiary structure of an antigen or isomeric forms of an antigen.

[0033] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The sequence of a polynucleotide is composed of the four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and, if the polynucleotide is RNA, uracil (U) instead of thymine. Thus, the term "nucleotide sequence" or "nucleic acid sequence" is the alphabetical representation of a polynucleotide. A polynucleotide can include a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotide also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the present invention that is a polynucleotide includes both double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute double-stranded form.It is understood that the isolated polynucleotide and nucleic acid described herein are intended for non-naturally occurring polynucleotide and nucleic acid.Non-naturally occurring polynucleotide and nucleic acid can include, but are not limited to, cDNA and chemically synthesized molecules.

[0034] The term "encoding," or grammatical equivalents thereof, when used with respect to a polynucleotide refers to a polynucleotide that, in its native state or when manipulated by methods well known to those of skill in the art, can be transcribed to produce mRNA, which is then translated into polypeptides and / or fragments thereof. The antisense strand is the complement of such a polynucleotide, from which a coding sequence can be deduced.

[0035] The phrase "therapeutic agent" refers to aTherapeutic agents refer to any agent that can be used to treat, manage, or ameliorate a disease associated with the expression of sLe and / or symptoms associated therewith. In certain embodiments, therapeutic agents refer to antibodies or functional fragments of the invention. In other embodiments, therapeutic agents refer to agents other than antibodies or functional fragments of the invention. Therapeutic agents include agents that inhibit the expression of sLe. a The agent may be an agent that is known to be useful for, or has been used or is currently being used to treat, manage, or ameliorate a disease associated with the development of, and / or one or more symptoms associated therewith.

[0036] The phrase "diagnostic agent" refers to a substance administered to a subject that aids in the diagnosis of disease. Such substances can be used to localize, pinpoint, and / or define the disease-causing process. In certain embodiments, a diagnostic agent comprises a substance conjugated to an antibody or functional fragment of the invention that, when administered to a subject or contacted with a sample from a subject, aids in the diagnosis of cancer or tumorigenesis.

[0037] The phrase "detectable agent" refers to a substance that can be used to confirm the existence or presence of a desired molecule, such as an antibody or functional fragment of the invention, in a sample or subject. A detectable agent can be a substance that can be visualized or otherwise determined and / or measured (e.g., by quantification).

[0038] An "effective amount" is an amount sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages. Such delivery depends on several variables, including the period for which the individual dosage units are used, the bioavailability of the drug, the route of administration, etc.

[0039] The phrase "therapeutically effective amount," as used herein, refers to an amount of a therapeutic agent (e.g., an antibody or functional fragment provided herein or any other therapeutic agent provided herein) sufficient to reduce and / or ameliorate the severity and / or duration of a given disease and / or its associated symptoms. A therapeutically effective amount of a therapeutic agent can be the amount necessary to reduce or ameliorate the advancement or progression of a given disease, reduce or ameliorate the recurrence, occurrence, or onset of a given disease, and / or improve or enhance the prophylactic or therapeutic effects of another therapy (e.g., a therapy other than administering an antibody or functional fragment provided herein).

[0040] "Cialil-Lewis a ” (sLe a ) is a compound called sialyl Le a , also known as sialyl-Lewis A, sialylated Lewis a, and CA19.9, and has the molecular formula C 31 H 52 N2O 23 and a tetrasaccharide with a molar mass of 820.74 g / mol. a The structure of sLe may include Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ and Neu5Gcα2-3Galβ1-3(Fucα1-4)GlcNAcβ. a sLe is widely expressed on tumors of the gastrointestinal tract and is used as a tumor marker for pancreatic and colon cancer. a is also a known ligand for E-selection, also known as endothelial leukocyte adhesion molecule (ELAM).

[0041] In some embodiments, the present invention provides an isolated polynucleotide encoding an antibody heavy or light chain or functional fragment thereof, wherein the antibody or functional fragment thereof produced using the antibody heavy or light chain is sLe aThus, in some embodiments, the invention provides isolated polynucleotides encoding antibodies, or functional fragments thereof, comprising 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. Isolated polynucleotides of the invention can also comprise the 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 an antibody, or functional fragment thereof.

[0042] In another embodiment of the invention, an isolated polynucleotide may encode an antibody, or functional fragment thereof, comprising 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. An isolated polynucleotide of the invention may also comprise the nucleic acid sequence of residues 58-390 of SEQ ID NO: 3, residues 58-387 of SEQ ID NO: 7, residues 58-390 of SEQ ID NO: 11, or residues 67-390 of SEQ ID NO: 15, wherein the nucleic acid sequence encodes a VL domain of an antibody, or functional fragment thereof.

[0043] In another embodiment, the present invention provides isolated polynucleotides encoding antibody heavy or light chains or functional fragments thereof, wherein the antibody heavy or light chains or functional fragments thereof encoded by the polynucleotides of the present invention have one or more of the complementarity determining regions (CDRs) shown in Figures 1-8 or listed in Table 2. Antibodies or functional fragments thereof comprising one or more of the CDRs can be used in conjunction with sLe as described herein. a It can specifically bind to sLe aSpecific binding to may include the specificity, affinity, and / or avidity set forth in Example I for any of the antibodies provided herein. In another embodiment, an antibody or functional fragment thereof encoded by a polynucleotide of the invention may comprise the complement-dependent cytotoxicity (CDC) activity and / or antibody-dependent cellular cytotoxicity (ADCC) activity of any one of clonal isolates 5B1, 9H3, 5H11, or 7E3 described herein. Methods for assessing the specificity, affinity, and / or avidity of an antibody or functional fragment thereof are well known in the art, and exemplary methods are provided herein.

[0044] [Table 2]

[0045] In some embodiments, the antibodies or functional fragments thereof of the present invention comprise fewer than six CDRs. In some embodiments, the antibodies or functional fragments thereof comprise one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3. In specific embodiments, the antibodies or functional fragments thereof comprise one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of clonal isolates 5B1, 9H3, 5H11, or 7E3 described herein.

[0046] In some embodiments, the invention provides an isolated polynucleotide encoding an antibody or functional fragment thereof comprising a variable heavy (VH) chain domain having the amino acid sequence of CDR1, CDR2, and CDR3 of clonal isolate 5B1, 9H3, 5H11, or 7E3. Such a VH domain may comprise amino acid residues 55-62, 70-77, and 116-131 of SEQ ID NO:2, or alternatively, amino acid residues 45-52, 70-77, and 116-131 of SEQ ID NO:6, or alternatively, amino acid residues 45-52, 70-77, and 116-131 of SEQ ID NO:10, or alternatively, amino acid residues 45-52, 70-77, and 116-134 of SEQ ID NO:14. In another embodiment, the nucleotide sequence encoding CDR1, CDR2 and CDR3 of the VH domain may comprise the nucleotide sequence of residues 133-156, 208-231 and 346-393 of SEQ ID NO:1, respectively, or alternatively the nucleotide sequence of residues 133-156, 208-231 and 346-393 of SEQ ID NO:5, or alternatively the nucleotide sequence of residues 133-156, 208-231 and 346-393 of SEQ ID NO:9, or alternatively the nucleotide sequence of residues 133-156, 208-231 and 346-402 of SEQ ID NO:13.

[0047] In another embodiment, the invention provides an isolated polynucleotide encoding an antibody or functional fragment thereof comprising a variable light (VL) chain domain having the amino acid sequences of CDR1, CDR2, and CDR3 of clonal isolate 5B1, 9H3, 5H11, or 7E3. Such a VL domain may comprise amino acid residues 45-52, 70-72, and 109-120 of SEQ ID NO:4, or alternatively, amino acid residues 45-52, 70-72, and 109-119 of SEQ ID NO:8, or alternatively, amino acid residues 45-52, 70-72, and 109-120 of SEQ ID NO:12, or alternatively, amino acid residues 49-53, 72-74, and 111-120 of SEQ ID NO:16. In another embodiment, the nucleotide sequence encoding CDR1, CDR2 and CDR3 of the VH domain may comprise the nucleotide sequence of residues 133-156, 208-216 and 325-360 of SEQ ID NO:3, respectively, or alternatively the nucleotide sequence of residues 133-156, 208-216 and 325-357 of SEQ ID NO:7, or alternatively the nucleotide sequence of residues 134-156, 208-216 and 325-360 of SEQ ID NO:11, or alternatively the nucleotide sequence of residues 145-162, 214-222 and 331-360 of SEQ ID NO:15.

[0048] In another embodiment, the present invention provides variants of the polynucleotides provided herein. Variant, when used in reference to a polynucleotide, includes polynucleotides having one or more modified nucleotides, such as, but not limited to, methylated nucleotides or nucleotide analogs. Furthermore, variant polynucleotides can include polynucleotides interleaved with non-nucleotide components. Modifications to polynucleotides can be added before or after assembly of the polynucleotides using methods well known to those skilled in the art. For example, polynucleotides can be modified after polymerization by conjugation with a labeling component using either enzymatic or chemical techniques (e.g., as described in Gottfried and Weinhold, 2011, Biochem. Soc. Trans., 39(2):523-628; Paredes et al., 2011, Methods, 54(2):251-259).

[0049] Polynucleotides can be obtained and the nucleotide sequence of the polynucleotides can be determined by any method known in the art. Since the amino acid sequences of the variable heavy and light domains of 5B1, 9H3, 5H11, and 7E3 are known (see, for example, SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16), the nucleotide sequences encoding antibodies and modified versions of these antibodies can be determined using methods known in the art: nucleotide codons known to encode specific amino acids are assembled to generate nucleic acids encoding the antibodies. Polynucleotides encoding such antibodies can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which briefly involves synthesizing overlapping oligonucleotide-containing portions of the sequence encoding the antibody, fragment, or variant thereof, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0050] Polynucleotides encoding antibodies or functional fragments thereof of the present invention can be generated using the nucleic acid sequences of the variable heavy and / or variable light chain domains of isolates 5B1, 9H3, 5H11, or 7E3 (e.g., SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15). Nucleic acids encoding antibodies or functional fragments can be chemically synthesized or obtained from a suitable source (e.g., cDNA isolated from cells expressing the antibody or functional fragment, such as hybridoma cells selected to express the antibody or functional fragment) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular nucleic acid sequence. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0051] In some embodiments, the present invention provides a method for treating sLe a Thus, in some aspects, the present invention provides an isolated antibody or functional fragment thereof that binds to sLe. a The present invention provides an isolated antibody or functional fragment thereof that binds to SEQ ID NO: 2, wherein the antibody or functional fragment thereof comprises a VH domain having an amino acid sequence selected from the group consisting of residues 20 to 142 of SEQ ID NO: 2, residues 20 to 142 of SEQ ID NO: 6, residues 20 to 142 of SEQ ID NO: 10, and residues 20 to 145 of SEQ ID NO: 14.

[0052] In some embodiments, the present invention provides a method for treating sLe a The present invention provides an isolated antibody or functional fragment thereof that binds to SEQ ID NO: 4, wherein the antibody or functional fragment thereof comprises a VL domain having an amino acid sequence selected from the group consisting of residues 20 to 130 of SEQ ID NO: 4, residues 20 to 129 of SEQ ID NO: 8, residues 20 to 130 of SEQ ID NO: 12, and residues 23 to 130 of SEQ ID NO: 16.

[0053] In some embodiments, the present invention provides a method for treating sLe aThe present invention provides an isolated antibody or functional fragment thereof that binds to SEQ ID NO: 1, wherein the antibody or functional fragment thereof comprises both a VH domain and a VL domain, wherein the VH domain and the VL domain respectively comprise amino acid sequences selected from the group consisting of residues 20-142 of SEQ ID NO: 2 and residues 20-130 of SEQ ID NO: 4; residues 20-142 of SEQ ID NO: 6 and residues 20-129 of SEQ ID NO: 8; residues 20-142 of SEQ ID NO: 10 and residues 20-130 of SEQ ID NO: 12; and residues 20-145 of SEQ ID NO: 14 and residues 23-130 of SEQ ID NO: 16.

[0054] In some embodiments, sLe a For binding to sLe, the antibodies or functional fragments thereof of the present invention have one or more of the CDRs shown in Figures 1-8 or listed in Table 2. Antibodies or functional fragments thereof comprising one or more of the CDRs, particularly CDR3, can be used to bind to sLe as described herein. a It can specifically bind to sLe a Specific binding to can include the specificities and affinities set forth in Example I for any of the antibodies provided herein. In some embodiments, an antibody or functional fragment thereof of the invention can comprise the CDC activity and / or ADCC activity of any one of clonal isolates 5B1, 9H3, 5H11, or 7E3 described herein.

[0055] In some embodiments, the invention provides an isolated antibody or functional fragment thereof comprising a VH chain domain having the amino acid sequences of CDR1, CDR2, and CDR3 of clonal isolate 5B1, 9H3, 5H11, or 7E3. Such a VH domain may comprise amino acid residues 55-62, 70-77, and 116-131 of SEQ ID NO:2, or alternatively, amino acid residues 45-52, 70-77, and 116-131 of SEQ ID NO:6, or alternatively, amino acid residues 45-52, 70-77, and 116-131 of SEQ ID NO:10, or alternatively, amino acid residues 45-52, 70-77, and 116-134 of SEQ ID NO:14.

[0056] In some embodiments, the invention provides an isolated antibody or functional fragment thereof comprising a VL chain domain having the amino acid sequences of CDR1, CDR2, and CDR3 of clonal isolate 5B1, 9H3, 5H11, or 7E3. Such a VL domain may comprise amino acid residues 45-52, 70-72, and 109-120 of SEQ ID NO:4, or alternatively, amino acid residues 45-52, 70-72, and 109-119 of SEQ ID NO:8, or alternatively, amino acid residues 45-52, 70-72, and 109-120 of SEQ ID NO:12, or alternatively, amino acid residues 49-53, 72-74, and 111-120 of SEQ ID NO:16.

[0057] In some embodiments of the present invention, the isolated antibody or functional fragment thereof is a monoclonal antibody. In some embodiments of the present invention, the isolated antibody or functional fragment thereof provided herein is an IgG or IgM isotype. In further embodiments of the present invention, the antibody or functional fragment thereof is an IgG1 subclass antibody.

[0058] In some embodiments, the antibody functional fragment of the present invention may be, but is not limited to, a Fab, Fab', F(ab')2, Fabc, scFV, diabody, triabody, minibody, or single domain antibody (sdAB). In some aspects, the present invention provides a diabody comprising the amino acid sequence of SEQ ID NO: 18 or 20. Such a diabody of the present invention may, in some aspects, be encoded by a polynucleotide having the nucleic acid sequence of SEQ ID NO: 17 or 19. Various forms, variations, and modifications of antibodies and functional fragments thereof are well known in the art. The sLe of the present invention a A specific antibody fragment may include any of the various such antibody forms, variations and modifications. Examples of such various forms and terms known in the art are described below.

[0059] In some embodiments, the invention provides methods of making an antibody or functional fragment thereof of the invention, which may include introducing a polynucleotide of the invention into a host cell, culturing the host cell under conditions and for a period of time sufficient to produce the encoded heavy and / or light chain of the antibody or functional fragment of the invention, and purifying the heavy and / or light chain of the antibody or functional fragment.

[0060] sLe a Recombinant expression of an antigen-binding antibody or functional fragment thereof of the present invention can involve the construction of an expression vector containing a polynucleotide encoding the heavy and / or light chain of the antibody or functional fragment thereof of the present invention. Once a polynucleotide encoding an antibody or functional fragment thereof (preferably, but not necessarily, containing a heavy chain variable domain and / or a light chain variable domain) of the present invention is obtained, a vector for producing the antibody or functional fragment can be generated by recombinant DNA technology using techniques well known in the art. Methods for preparing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody or functional fragment thereof are described herein.

[0061] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of an antibody or functional fragment thereof and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic methods, and in vivo genetic recombination. Thus, the present invention provides replicable vectors containing a nucleotide sequence encoding the antibody of the present invention or a functional fragment thereof operably linked to a promoter. Such vectors may contain nucleotide sequences encoding the constant region of an antibody molecule (see, e.g., International Publication Nos. WO86 / 05807 and WO89 / 01036; and U.S. Patent No. 5,122,464), and antibody variable domains can be cloned into such vectors to express the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

[0062] The expression vector can be transferred into a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention or a functional fragment thereof. Thus, the invention encompasses host cells containing a polynucleotide encoding an antibody of the invention or a functional fragment thereof operably linked to a heterologous promoter. In some embodiments for expression of double-chain antibodies, vectors encoding both the heavy and light chains can be co-expressed in a host cell to express the entire immunoglobulin molecule, as described in more detail below.

[0063] A variety of host-expression vector systems can be utilized to express the antibodies of the invention or functional fragments thereof (see, e.g., U.S. Patent No. 5,807,715). Such host-expression systems refer not only to vehicles in which a coding sequence of interest can be produced and subsequently purified, but also to cells which, when transformed or transfected with the appropriate nucleotide coding sequence, are capable of expressing the antibody molecules of the invention in situ. These include, but are not limited to, bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequences; microorganisms such as yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). In some embodiments, bacterial cells such as Escherichia coli, particularly for the expression of whole recombinant antibodies, or eukaryotic cells are used to express recombinant antibodies or functional fragments. For example, mammalian cells such as Chinese hamster ovary cells (CHO) are effective expression systems for antibodies, in conjunction with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus (Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990, Bio / Technology 8:2).In some embodiments, the antibodies or fragments thereof of the invention are produced in CHO cells. a Expression of the nucleotide sequence encoding the antibody of the present invention or a functional fragment thereof that binds to is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0064] In bacterial systems, several expression vectors can be advantageously selected depending on the intended use of the antibody molecule being expressed. For example, when producing large quantities of such antibodies to generate pharmaceutical compositions of the antibody molecule, vectors directing the expression of high-level fusion protein products that are easily purified may 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 antibody coding sequences can be individually ligated into the vector in frame with the lac Z coding region, thus producing a fusion protein; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509). pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to glutathione-agarose beads, followed by elution in the presence of free glutathione. The pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0065] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus is grown in Spodoptera frugiperda cells. The coding sequence for the antibody or functional fragment can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0066] Several virus-based expression systems are available for mammalian host cells. When adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated into an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., the E1 or E3 region) generates recombinant virus that is viable in infected hosts and capable of expressing the antibody molecule (see, e.g., Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals can also be used for efficient translation of the inserted antibody coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, to ensure translation of the entire insert, the initiation codon must be in phase with the reading frame of the desired coding sequence. These exogenous translational control signals and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, eg, Bittner et al., 1987, Methods in Enzymol. 153:51-544).

[0067] Additionally, a host cell strain can be selected that modulates the expression of the inserted sequences, or modifies 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 function of the antibody or functional fragment. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO cells, VERY cells, BHK cells, Hela cells, COS cells, MDCK cells, 293 cells, 3T3 cells, W138 cells, BT483 cells, Hs578T cells, HTB2 cells, BT2O cells and T47D cells, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O cells and HsS78Bst cells.

[0068] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines can be engineered that stably express the antibodies or functional fragments of the present invention. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, engineered cells can be grown in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form nests, which can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express antibody molecules.

[0069] Several selection systems can be used, including, but not limited to, the herpes simplex virus thymidine kinase gene (Wigler et al., 1977, Cell 11:223), the hypoxanthine guanine phosphoribosyltransferase gene (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and the adenine phosphoribosyltransferase gene (Lowy et al., 1980, Cell 22:8-17), which can be used in tk-cells, hgprt-cells, or aprt-cells, respectively. Additionally, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77(6):3567-70; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); glutamine synthetase (GS), an enzyme involved in the biosynthesis of glutamine, in which glutamate and ammonia are used (Bebbington et al., 1992, Biotechnology 10:169); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:1527). 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May 1993, TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).To select the desired recombinant clone, methods well known in the art of recombinant DNA technology can be routinely applied, such as those described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Dracopoli et al. (eds.), Current Protocols in Human Genetics, Chapters 12 and 13, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, which are incorporated herein by reference in their entireties.

[0070] Expression levels of antibody molecules can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells, DNA cloning, vol. 3 (Academic Press, New York, 1987)). If the marker in the vector system expressing the antibody or its functional fragment is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, antibody production will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

[0071] Host cells can be co-transfected with two expression vectors of the present invention: a first vector encoding heavy chain-derived polypeptides and a second vector encoding light chain-derived polypeptides. The two vectors may contain identical selectable markers that allow for equal expression of heavy and light chain polypeptides. Alternatively, a single vector can be used that encodes and expresses both heavy and light chain polypeptides. In such situations, the light chain can precede the heavy chain to avoid excess 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 comprise cDNA or genomic DNA.

[0072] Furthermore, polynucleotides encoding the heavy and / or light chains of the antibodies or functional fragments of the present invention can be subjected to codon optimization using techniques well known in the art to achieve optimized expression of the antibodies or functional fragments of the present invention in desired host cells. For example, one method of codon optimization involves replacing native codons with the most frequently occurring codons from a reference gene set, designed to increase the rate of codon translation for each amino acid. Additional exemplary methods for generating codon-optimized polynucleotides for expressing desired proteins, which can be applied to the heavy and / or light chains of the antibodies or functional fragments of the present invention, are described in Kanaya et al., Gene, 238:143-155 (1999); Wang et al., Mol. Biol. Evol., 18(5):792-800 (2001); U.S. Patent No. 5,795,737; U.S. Patent Publication No. 2008 / 0076161; and WO2008 / 000632.

[0073] Once an antibody molecule of the present invention is produced by recombinant expression, it can be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly for specific antigens, followed by protein A chromatography, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. Furthermore, the antibodies or functional fragments of the present invention can be fused to heterologous polypeptide sequences provided herein or otherwise known in the art to facilitate purification. For example, the antibodies or functional fragments of the present invention can be purified by recombinantly adding a poly-histidine tag (His tag), a FLAG tag, a hemagglutinin tag (HA tag), or a myc tag, among others, which are commercially available, and utilizing purification methods well known to those of skill in the art.

[0074] A Fab fragment refers to a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; an F(ab')2 fragment is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consists of the VH and CH1 domains; an Fv fragment consists of the VL and VH domains of a single arm of an antibody; and a dAb fragment (Ward et al., Nature 341:544-546, (1989)) consists of the VH domain.

[0075] An antibody can have one or more binding sites. When two or more binding sites are present, the binding sites may be identical to one another or may be different. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, while "bispecific" or "bifunctional" antibodies have two different binding sites.

[0076] A single-chain antibody (scFv) refers to an antibody in which the VL and VH domains are connected by a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous polypeptide chain, which linker is long enough to enable the protein chain to fold and form a monovalent antigen-binding 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)). A diabody refers to a bivalent antibody comprising two polypeptide chains, each of which comprises a VH domain and a VL domain connected by a linker that is too short to allow pairing between the two domains on the same chain, and therefore allows 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 2:1121-23 (1994)). If the two polypeptide chains of a diabody are identical, the resulting diabody will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to create diabodies with two different antigen-binding sites. Similarly, tribodies and tetrabodies are antibodies that contain three and four polypeptide chains, respectively, forming three and four antigen-binding sites, respectively, which may be the same or different.

[0077] The present invention is directed to sLe aAlso provided are antibodies or functional fragments thereof that are derivatives of 5B1, 9H3, 5H11, and / or 7E3 and bind to the antibody of the present invention. Standard techniques well known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding the antibody or functional fragment thereof, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions. In some embodiments, the derivative contains fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions compared to the original molecule.

[0078] In some embodiments, the present invention provides antibodies or functional fragments thereof having modified forms of naturally occurring amino acids, conservative substitutions, non-naturally occurring amino acids, amino acid analogs, and mimetics, so long as such antibodies or functional fragments retain the functional activity defined herein. In one embodiment, the derivatives have conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues with side chains with similar charges are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly throughout all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain 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.

[0079] In some embodiments, the present invention provides antibodies or functional fragments thereof in which the fucosylation, galactosylation, and / or sialylation of the Fc fragment contained within the antibody or functional fragment of the present invention has been modified. As discussed in Peipp et al., Blood, 112(6):2390-2399 (2008), such Fc fragment modifications can result in Fc receptor-mediated activity. For example, glycoengineered therapeutic antibodies lacking core fucose residues from the Fc N-glycan exhibit potent ADCC at low concentrations and are much more effective than their fucosylated counterparts. Shields et al., J. Biol. Chem., 277(30):26733-40 (2002); Okazaki et al., J. Mol. Biol., 336:1239-1249 (2004); Natsume et al., J. Immunol. Methods., 306:93-103 (2005). Methods for modifying the fucosylation, galactosylation, and / or sialylation of antibodies with respect to functional fragments thereof are well known in the art. For example, defucosylation techniques can be grouped into three methodologies, as described in Yamane-Ohnuki et al., MAbs., 1(3):230-236 (2009): (1) conversion of the N-glycosylation pathway of non-mammalian cells to a "humanized" non-fucosylated pathway; (2) inactivation of the N-glycan fucosylation pathway of mammalian cells; and (3) in vitro chemical synthesis of non-fucosylated N-glycoproteins or enzymatic modification of N-glycans to non-fucosylated forms. It is understood that any one of these methods, or any other method known in the art, can be used to produce antibodies or functional fragments thereof with altered fucosylation, galactosylation, and / or sialylation.

[0080] sLe aThe antibodies or functional fragments thereof of the present invention that bind to can be produced by any method known in the art for synthesizing antibodies, particularly by chemical synthesis or by recombinant expression techniques. The practice of the present invention employs, unless otherwise specified, conventional techniques of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, 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, for example, Maniatis et al. (1982), each of which is incorporated herein by reference in its entirety. Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press; Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual , 2nd edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al. Current Protocols in Molecular Biology , John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology , John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach , IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach , IRL Press; Birren et al. (eds.) (1999) Genome Analysis: A Laboratory Manual , Cold Spring Harbor Laboratory Press; Borrebaeck (ed.) (1995) Antibody Engineering , 2nd edition, Oxford University Press; Lo (ed.) (2006) Antibody Engineering: Methods and Protocols (Methods in Molecular Biology); Vol. 248, Humana Press, Inc.

[0081] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridoma and recombinant technology, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques, including those taught in Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas, pp. 563-681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety. Monoclonal antibodies are not limited to antibodies produced by hybridoma technology. Other exemplary methods for producing monoclonal antibodies are known in the art. Additional exemplary methods for producing monoclonal antibodies are presented in Example I of this specification.

[0082] sLe a Functional antibody fragments that bind to can be produced by any technique known to those skilled in the art. For example, Fab and F(ab')2 fragments of the present 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 heavy chain variable region, light chain constant region, and CH1 domain.

[0083] The antibody functional fragments of the present invention can also be produced using various phage display methods known in the art. For example, in phage display methods, functional antibody domains, such as 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 carrying the polynucleotide sequences encoding them. DNA encoding the VH and VL domains is reassembled together with an scFv linker by PCR and cloned into a phagemid vector. The vector is introduced into E. coli by electroporation, and the E. coli is infected with helper phage. The phages used in these methods are typically filamentous phages, including fd and M13, and the VH and VL domains are usually recombinantly fused to either phage gene III or gene VIII. sLe aPhage expressing an antigen-binding domain that binds to a particular antigen, such as, can be selected or identified using the antigen, for example, using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to generate antibody functional fragments of the present invention include those described in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186; 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. WO 90 / 02809, WO 91 / 10737, and WO 91 / 10738, each of which is incorporated herein by reference in its entirety. 92 / 01047, WO 92 / 18619, WO 93 / 1 1236, WO 95 / 15982, WO 95 / 20401, and WO 97 / 13844; and those disclosed in U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

[0084] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, which can be expressed in any desired host, including, for example, mammalian cells, insect cells, plant cells, yeast, and bacteria, as described herein.

[0085] Techniques for recombinantly producing Fab, Fab' and F(ab')2 fragments can also be utilized using methods known in the art, such as those disclosed in PCT Publication No. WO 92 / 22324; Mullinax et al., 1992, BioTechniques 12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and Better et al., 1988, Science 240:1041-1043, each of which is incorporated by reference in its entirety.

[0086] To generate a whole antibody, the VH or VL sequence can be amplified in an scFv clone using PCR primers containing the VH or VL nucleotide sequence, a restriction site, and a flanking sequence to protect the restriction site. Using cloning techniques well known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a VH constant region, such as the human gamma 1 constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a VL constant region, such as the human kappa or lambda constant region. The VH and VL domains can also be cloned into a vector expressing the necessary constant regions. Then, using techniques well known to those skilled in the art, the heavy chain conversion vector and the light chain conversion vector are co-transfected into a cell line to generate a stable or transient cell line expressing a full-length antibody, such as an IgG.

[0087] In some embodiments, the antibodies or functional fragments of the invention are conjugated (covalently or non-covalently) or recombinantly fused to one or more diagnostic, detectable, or therapeutic agents or any other desired molecule. The conjugated or recombinantly fused antibodies or functional fragments can be used to assess the efficacy of sLe as part of a clinical trial procedure, such as to determine the efficacy of a particular therapy. a These may be useful for monitoring or diagnosing the onset, development, progression and / or severity of a disease associated with expression of, for example, cancer or tumorigenesis.

[0088] Detection and diagnosis can be achieved, for example, by combining an antibody or functional fragment of the invention with a radioactive material, for example, but not limited to, zirconium ( 89 Zr), iodine ( 131 I, 125 I, 124 I, 123 I, and 121 I), carbon ( 14 C. 11 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115 In, 113 In, 112 In, and 111 In), technetium ( 99 Tc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), 15 O. 13 N, 64 Cu, 94m Tc, 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 86 Y, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn, and 117and positron-emitting metals using various positron emission tomography techniques, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as, but not limited to, luciferase, luciferin, and aequorin, and non-radioactive paramagnetic metal ions.

[0089] The present invention further encompasses therapeutic uses of antibodies or functional fragments of the present invention conjugated (covalently or non-covalently) or recombinantly fused to one or more therapeutic agents. In this case, for example, the antibody can be conjugated or recombinantly fused to a therapeutic agent such as a cytotoxin, e.g., a cytostatic or cytocidal agent, or a radioactive metal ion, e.g., an alpha-emitter. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. The therapeutic agent may be a chemotherapeutic agent, such as, but not limited to, anthracyclines (e.g., doxorubicin and daunorubicin (formerly daunomycin)); taxanes (e.g., paclitaxel (Taxol) and docetaxel (Taxotere); antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, and decanoate); or alkylating agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BCNU), lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisplatin, cyclosulfonyl 3-phosphate dehydrogenase, cyclosulfonyl 4-phosphate dehydrogenase, cyclosulfonyl 5 ... dichlorodiamineplatinum(II) (DDP) and cisplatin; antibiotics (e.g., actinomycin D, bleomycin, mithramycin, and anthramycin (AMC)); auristatin molecules (e.g., auristatin PHE, bryostatin 1, solastatin 10, monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF)); hormones (e.g., glucocorticoids, progestins, androgens, and estrogens); nucleoside analogs (e.g., gemcitabine), DNA repair enzyme inhibitors (e.g., etoposide and topotecan), kinase inhibitors (e.g., the compound ST1571, also known as Gleevec or imatinib mesylate);Cytotoxic agents (e.g., maytansine, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs or homologs thereof, and those described in U.S. Pat. Nos. 6,245,759, 6,399, No. 633, No. 6,383,790, No. 6,335,156, No. 6,271,242, No. 6,242,196, No. 6,218,4 No. 10, No. 6,218,372, No. 6,057,300, No. 6,034,053, No. 5,985,877, No. 5,958,76 9, 5,925,376, 5,922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745, 5,728,868, 5,648,239, and 5,587,459);Farnesyltransferase inhibitors (e.g., R115777, BMS-214662, and the like) are described in, for example, U.S. Patent Nos. 6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959, 6,420,387, 6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615, 6,387,905, 6,37 No. 2,747, No. 6,369,034, No. 6,362,188, No. 6,342,765, No. 6,342,487, No. 6,300,501, No. 6,268,363, No. 6,265,422, No. 6,248,75 6, 6,239,140, 6,232,338, 6,228,865, 6,228,856, 6,225,322, 6,218,406, 6,211,193, 6,187,786, No. 6,169,096, No. 6,159,984, No. 6,143,766, No. 6,133,303, No. 6,127,366, No. 6,124,465, No. 6,124,295, No. 6,103,723, No. 6,0 No. 93,737, No. 6,090,948, No. 6,080,870, No. 6,077,853, No. 6,071,935, No. 6,066,738, No. 6,063,930, No. 6,054,466, No. 6,051,5 82, 6,051,574, and 6,040,305); topoisomerase inhibitors (e.g., camptothecin, irinotecan, SN-38, topotecan, 9-aminocamptothecin, GG-211 (GI147211), DX-8951f, IST-622, rubitecan, pyrazoloacridine, XR-5000, saintopin, UCE6, UCE1022, TAN-1518A, TAN 1518B, KT6006, KT6528, ED-110, NB-506, ED-110, NB-506, fagaronine, coralyne, beta-lapachone, and rebeccamycin);The therapeutic agent may be, for example, a DNA minor groove binder (e.g., Hoescht dye 33342 and Hoechst dye 33258); an adenosine deaminase inhibitor (e.g., fludarabine phosphate and 2-chlorodeoxyadenosine); or a pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof. The therapeutic agent may be, for example, an immunotherapeutic agent such as, but not limited to, cetuximab, bevacizumab, heceptin, rituximab, etc.;

[0090] Additionally, the antibodies or functional fragments of the present invention may be used in combination with, for example, radioactive metal ions, e.g., 213 Alpha-emitters such as Bi, or, but not limited to: 131 In, 131 LU, 131 Y, 131 Ho, 131 Macrocyclic chelators useful for conjugating radiometal ions, including Sm; or macrocyclic chelators that can be conjugated to therapeutic agents, such as 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), which can be attached to antibodies or functional fragments by linker molecules. Such linker molecules are generally known in the art and are described in Denardo et al., 1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol. 26(8):943-50.

[0091] Furthermore, the antibodies or functional fragments of the present invention can be conjugated (covalently or non-covalently conjugated) or recombinantly fused to therapeutic agents that modify a given biological response. Thus, the therapeutic agent should not be construed as limited to classical chemical therapeutic agents. For example, the therapeutic agent can be a protein, peptide, or polypeptide with a desired biological activity. Such proteins include, for example, toxins (e.g., abrin, ricin A, pseudomonas exotoxin, cholera toxin, and diphtheria toxin); tumor necrosis factor, gamma-interferon, alpha-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptotic agents (e.g., TNF-γ, AIM I, AIM II, Fas ligand, and VEGF), anti-angiogenic agents (e.g., components of the coagulation pathway such as angiostatin, endostatin, and tissue factor); biological response modifiers (e.g., interferon gamma, interleukin-1, interleukin-2, interleukin-5, interleukin-6, interleukin-7, interleukin-9, interleukin-10, interleukin-12, interleukin-15, interleukin-23, granulocyte-macrophage colony-stimulating factor, and granulocyte cytokines such as colony-stimulating factors); growth factors (e.g., growth hormones), or coagulation agents (e.g., calcium, vitamin K, tissue factor, including, but not limited to, Hageman factor (factor XII), high molecular weight kininogen (HMWK), prekallikrein (PK), coagulation proteins—factor II (prothrombin), factor V, factor XIIa, factor VIII, factor XIIIa, factor XI, factor XIa, factor IX, factor IXa, factor X, phospholipids, and fibrin monomers).

[0092] The present invention encompasses antibodies or functional fragments of the present invention that are recombinantly fused or chemically conjugated (covalently or non-covalently) to a heterologous protein or polypeptide to generate a fusion protein. In some aspects, such polypeptides can be about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids in length. In some aspects, the present invention provides fusion proteins having a functional fragment of an antibody of the present invention (e.g., a Fab fragment, an Fd fragment, an Fv fragment, an 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 protein or polypeptide fused to the antibody or functional fragment is a heterologous protein or polypeptide that binds the antibody or functional fragment to a sLe a These are useful for targeting to specific cell types, such as cells expressing the .

[0093] The conjugated or fusion proteins of the invention comprise any of the antibodies or functional fragments of the invention provided herein conjugated (covalently or non-covalently) or recombinantly fused to a diagnostic, detectable, or therapeutic agent. In one embodiment, the conjugated or fusion protein of the invention comprises a 5B1, 9H3, 5H11, or 7E3 antibody and a diagnostic, detectable, or therapeutic agent. In another embodiment, the conjugated or fusion protein of the invention comprises a functional fragment of a 5B1, 9H3, 5H11, or 7E3 antibody and a diagnostic, detectable, or therapeutic agent. In another embodiment, a conjugated or fusion protein of the invention comprises a VH domain having the amino acid sequence of any one of the VH domains set forth 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 set forth in residues 20-130 of SEQ ID NO:4, residues 20-129 of SEQ ID NO:8, residues 20-130 of SEQ ID NO:12, or residues 23-130 of SEQ ID NO:16, and a diagnostic, detectable, or therapeutic agent. In another embodiment, a conjugated or fusion protein of the invention comprises one or more VH CDRs having the amino acid sequence of any one of the VH CDRs set forth in SEQ ID NOs:2, 6, 10, or 14, and a diagnostic, detectable, or therapeutic agent. In another embodiment, the conjugated or fusion protein comprises one or more VL CDRs having the amino acid sequence of any one of the VL CDRs set forth in SEQ ID NOs: 4, 8, 12, or 16, and a diagnostic, detectable, or therapeutic agent.In another embodiment, a conjugated or fusion protein of the invention comprises at least one VH domain and at least one VL domain set forth in residues 20-142 of SEQ ID NO:2 and residues 20-130 of SEQ ID NO:4; residues 20-142 of SEQ ID NO:6 and residues 20-129 of SEQ ID NO:8; residues 20-142 of SEQ ID NO:10 and residues 20-130 of SEQ ID NO:12; or residues 20-145 of SEQ ID NO:14 and residues 23-130 of SEQ ID NO:16, respectively, and a diagnostic, detectable, or therapeutic agent.

[0094] Methods for fusing or conjugating antibodies to diagnostic, detectable or therapeutic agents, including polypeptides, are well known. See, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies Vol. 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); and "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibodies In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection, all of which are incorporated by reference herein in their entireties. And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), Thorpe et al., 1982, Immunol. Rev.62:119-58; U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, Nos. 5,112,946, 7,981,695, 8,039,273, and 8,142,784; U.S. Application Publication Nos. 2009 / 0202536, 2010 / 0034837, 2011 / 0137017, 2011 / 0280891, and 2012 / 0003247; EP 307,434; EP 367,166; EP 394,827; PCT Publication Nos. WO 91 / 06570, 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, 89:11337-11341, 1992; and Senter, Current Opinion in Chemical Biology, 13:235-244 (2009).

[0095] In another embodiment, diagnostic, detectable, or therapeutic agents can be attached by disulfide bond formation at the hinge region of a reduced antibody component. Alternatively, such agents can be attached to the antibody component using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56:244 (1994). General techniques for such conjugation are well known in the art. See, e.g., Wong, CHEMISTRY 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.), pp. 187-230 (Wiley-Liss, Inc. 1995); Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pp. 60-84 (Cambridge University Press 1995).

[0096] Alternatively, diagnostic, detectable, or therapeutic agents can be conjugated via carbohydrate moieties in the Fc region of an antibody. Methods for conjugating peptides to antibody components via antibody carbohydrate moieties are well known to those skilled in the art. See, for example, Shih et al., Int. J. Cancer. 41:832-839 (1988); Shih et al., Int. J. Cancer. 46:1101-1106 (1990); and Shih et al., U.S. Pat. No. 5,057,313, all of which are incorporated by reference in their entireties. A typical method involves reacting an antibody component bearing an oxidized carbohydrate moiety with a carrier polymer bearing at least one free amine function and carrying multiple peptides. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized to a secondary amine by reduction to form the final conjugate.

[0097] However, even if the Fc region is absent, for example, when the antibody functional fragments provided herein are desired, it is possible to attach a diagnostic, detectable, or therapeutic agent. Carbohydrate moieties can be introduced into the light chain variable region of a full-length antibody or antibody fragment. See, for example, Leung et al., J. Immunol., 154:5919 (1995); U.S. Patent Nos. 5,443,953 and 6,254,868, all of which are incorporated by reference in their entirety. An engineered carbohydrate moiety is used to attach a diagnostic, detectable, or therapeutic agent.

[0098] sLe a Therapeutic agents conjugated or recombinantly fused to an antibody functional fragment of the invention that binds to can be selected to achieve the desired prophylactic or therapeutic effect(s). It is understood that it is within the level of skill of the clinician or other medical professional to consider the nature of the disease, the severity of the disease, and the condition of the subject when determining which therapeutic agent to conjugate or recombinantly fused to an antibody or functional fragment of the invention.

[0099] Detectably labeled sLe as provided herein a The conjugates or fusion antibodies or functional fragments of the invention that bind to sLe can be used for diagnostic purposes to detect, diagnose, or monitor disease, where the disease-causing or disease-associated cells are sLe. a For example, as provided herein, cancer cells and tumors, such as, but not limited to, tumors of the gastrointestinal tract, breast cancer, ovarian cancer, colon cancer, colorectal adenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, small cell lung cancer, bladder adenocarcinoma, metastatic colon cancer, colorectal cancer, ovarian signet ring cell carcinoma, and metastatic cancer, express sLe a Thus, the present invention provides methods for detecting cancer or tumor formation in a subject by administering to a subject in need thereof an effective amount of a conjugate or fusion antibody or functional fragment of the present invention. In some embodiments, the detection method comprises administering to a subject in need thereof an effective amount of a conjugate or fusion antibody or functional fragment of the present invention. a One or more conjugates or fusion antibodies or functional fragments of the invention that bind to sLe in a cell or tissue sample of a subject can be used to detect sLe. a and assaying the expression of sLe a The level of the assayed sLe is compared to a control level, e.g., a level in a normal tissue sample (e.g., from a subject without the disease or from the same subject before the onset of the disease), thereby determining the level of the assayed sLe. a The level of sLe a The method may further include the step of detecting elevated levels of the antibody as compared to a control level, thereby indicating disease. Such diagnostic methods may enable medical professionals to employ preventative measures or invasive treatments earlier than would otherwise be possible, thereby preventing the onset or further progression of disease.

[0100] The antibodies or functional fragments of the present invention can be used to detect sLe in biological samples using classical immunohistological methods provided herein or well known to those skilled in the art. aIt can also be used to assay antigen levels (see, e.g., Jalkanen et al., 1985, J. Cell. Biol. 101:976-985; and Jalkanen et al., 1987, J. Cell. Biol. 105:3087-3096). a Other antibody-based methods useful for detecting include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and include enzyme labels, such as glucose oxidase; radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium ( 99 luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

[0101] In one aspect, the present invention provides for the detection and diagnosis of disease in humans. In one embodiment, diagnosis is achieved by: a) detecting sLe a a) administering to a subject (e.g., parenterally, subcutaneously, or intraperitoneally) an effective amount of a conjugate or fusion protein of the invention that binds to sLe; b) administering to the subject (e.g., parenterally, subcutaneously, or intraperitoneally) an effective amount of a conjugate or fusion protein of the invention that binds to sLe; aThe methods include: c) allowing a time interval after administration to allow the conjugate or fusion protein to preferentially concentrate at sites where the conjugate or fusion protein is expressed (and, in some embodiments, to remove unbound conjugate or fusion protein to background levels); c) determining the background level; and d) detecting the conjugate or fusion protein in the subject, such that detection of the conjugate or fusion protein above background levels indicates that the subject has the disease. The background level can be determined by a variety of methods, including comparing the amount of conjugate or fusion protein detected to a predetermined standard value for a particular system.

[0102] It is understood that the size of the subject and the imaging system used will determine the number of imaging moieties required to generate a diagnostic image, and can be readily determined by one of skill in the art. For example, for a human subject, in the case of a radioisotope conjugated to an antibody or functional fragment of the present invention, the quantity of radioactivity injected will typically be about 5-20 millicuries. 99 The conjugate then binds to sLe a In vivo tumor imaging is described in SW Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments." (Tumor Imaging: The Radiochemical Detection of Cancer, Chapter 13, edited by SW Burchiel and BA Rhodes, Masson Publishing Inc. (1982)).

[0103] Depending on several variables, including the type of detectable agent used and the mode of administration, the time interval after administration for the conjugate to preferentially concentrate at the site in the subject and for unbound conjugate to clear to background levels is 6 to 48 hours, or 6 to 24 hours, or 6 to 12 hours. In another embodiment, the time interval after administration is 5 to 20 days, or 5 to 10 days. In one embodiment, disease monitoring is performed by repeating the diagnostic methods provided herein, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, or more.

[0104] The presence of the conjugate or fusion protein can be detected in a subject using methods known in the art of in vivo scanning. These methods depend on the type of detectable agent used. One skilled in the art can determine a suitable method for detecting a particular detectable agent. Methods and devices that can be used in the diagnostic methods of the present invention include, but are not limited to, whole-body scans such as computed tomography (CT), position emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound. In one embodiment, the antibody or functional fragment of the present invention is conjugated to a radioisotope and detected in a subject using a radiation-responsive surgical instrument. In another embodiment, the antibody or functional fragment of the present invention is conjugated to a fluorescent compound and detected in a subject using a fluorescence-responsive scanning instrument. In another embodiment, the antibody or functional fragment of the present invention is conjugated to a zirconium ( 89 In yet another embodiment, the antibody or functional fragment of the present invention is conjugated to a positron-emitting metal such as Zr or any other positron-emitting metal provided herein or known in the art to be detectable by positron emission tomography, and detected in a subject using positron emission tomography. In yet another embodiment, the antibody or functional fragment of the present invention is conjugated to a paramagnetic label and detected in a subject using magnetic resonance imaging (MRI).

[0105] In one embodiment, the present invention provides pharmaceutical compositions comprising an antibody or functional fragment of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that can be used in pharmaceutical compositions of the present invention include any of the standard pharmaceutical carriers known in the art, such as phosphate-buffered saline solution, water, emulsions such as oil-and-water emulsions, and various types of wetting agents. These pharmaceutical compositions can be prepared in a unit dose form or any other dosage form of a liquid sufficient to deliver the antibody or functional fragment of the present invention to the target area of a subject in need of treatment. For example, pharmaceutical compositions can be prepared in any manner appropriate for the selected mode of administration, e.g., intravenous, intramuscular, subcutaneous, intraperitoneal, etc. Other optional ingredients, such as pharmaceutical-grade stabilizers, buffers, preservatives, excipients, etc., can be readily selected by those skilled in the art. Preparation of pharmaceutical compositions taking into account pH, isotonicity, stability, etc., is within the skill of those in the art.

[0106] Pharmaceutical formulations containing one or more antibodies or functional fragments of the invention provided herein can be prepared for storage in the form of a lyophilized formulation or aqueous solution by mixing antibodies having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; These include proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0107] Thus, in some embodiments, the present invention provides a method for treating or preventing a disease in a subject in need thereof. The method of the present invention may comprise administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein. For example, the pharmaceutical composition may comprise one or more antibodies or functional fragments provided herein. Diseases that can be treated or prevented using the method of the present invention include cancer, tumor formation, and / or metastasis. In particular, the method of the present invention is directed to treating or preventing a disease in a subject in need thereof, wherein cancer cells or tumors express the carbohydrate sLe a The methods of the present invention are useful for treating cancers or tumors that express the IL-11 receptor agonist (IL-11). Non-limiting examples of cancers or tumors that can be treated or prevented using the methods of the present invention include tumors of the gastrointestinal tract, such as colon cancer, colorectal adenocarcinoma, metastatic colon cancer, colorectal cancer, pancreatic cancer, or pancreatic adenocarcinoma; small cell lung cancer; bladder adenocarcinoma; ovarian signet ring cell carcinoma; ovarian cancer, metastatic carcinoma; and adenocarcinoma of the stomach, esophagus, throat, genitourinary tract, or breast.

[0108] Thus, in some aspects, the present 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 is sLe a and comprising a VH domain having an amino acid sequence selected from the group consisting of residues 20-142 of SEQ ID NO: 2, residues 20-142 of SEQ ID NO: 6, residues 20-142 of SEQ ID NO: 10, and residues 20-145 of SEQ ID NO: 14. In another aspect, the present 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 sLe aand the VL domain has 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 present 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 is sLe a and the VH domain and VL domain each comprise an amino acid sequence selected from the group consisting of residues 20-142 of SEQ ID NO:2 and residues 20-130 of SEQ ID NO:4; residues 20-142 of SEQ ID NO:6 and residues 20-129 of SEQ ID NO:8; residues 20-142 of SEQ ID NO:10 and residues 20-130 of SEQ ID NO:12; and residues 20-145 of SEQ ID NO:14 and residues 23-130 of SEQ ID NO:16.

[0109] Formulations such as those described herein may contain two or more active compounds as needed for the particular disease being treated. In certain embodiments, the formulation comprises an antibody or functional fragment of the present invention and one or more active compounds with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts effective for the intended purpose. For example, the antibody or functional fragment of the present invention can be combined with one or more other therapeutic agents. Such combination therapy can be administered to a subject simultaneously or sequentially.

[0110] Thus, in some aspects, the present invention provides a method for treating or preventing a disease by administering a therapeutically effective amount of a pharmaceutical composition provided herein to a subject in need thereof, wherein the pharmaceutical composition comprises an antibody or functional fragment of the present invention and a second therapeutic agent. Suitable second therapeutic agents can be readily determined by those skilled in the art as discussed herein. As provided herein in Example IV, in some aspects of the present invention, the second therapeutic agent may be taxol.

[0111] The pharmaceutical compositions provided herein contain a therapeutically effective amount of one or more of the antibodies of the invention provided herein, and optionally one or more additional therapeutic agents, in a pharmaceutically acceptable carrier. Such pharmaceutical compositions are useful in the prevention, treatment, management, or amelioration of a disease, such as cancer or tumorigenesis, or one or more symptoms thereof.

[0112] Pharmaceutical compositions may contain one or more antibodies or functional fragments of the present invention. In one embodiment, the antibodies or functional fragments are formulated into a suitable pharmaceutical preparation, such as a sterile solution or suspension for parenteral administration. In one embodiment, the antibodies or functional fragments provided herein are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel (1985) Introduction to Pharmaceutical Dosage Forms, 4th ed., p. 126).

[0113] The antibody or functional fragment of the present invention can be included in the pharmaceutical composition in a therapeutically effective amount sufficient to exert a therapeutically useful effect on the treated subject without undesirable side effects. The therapeutically effective concentration can be determined empirically by testing the compound in in vitro and in vivo systems using conventional methods and then extrapolating the dosage for humans therefrom. The concentration of the antibody or functional fragment in the pharmaceutical composition depends, for example, on the physicochemical properties of the antibody or functional fragment, the dosing schedule and dosage, and other factors well known to those skilled in the art.

[0114] In one embodiment, a therapeutically effective dosage provides a serum concentration of antibody or functional fragment from about 0.1 ng / ml to about 50-100 μg / ml. In another embodiment, the pharmaceutical composition provides a dosage of from about 0.001 mg to about 500 mg of antibody per kilogram of body weight per day. Pharmaceutical dosage unit forms can be prepared to provide from about 0.01 mg, 0.1 mg, or 1 mg to about 30 mg, 100 mg, or 500 mg, and in one embodiment, from about 10 mg to about 500 mg, of antibody or functional fragment and / or other optional basic component combinations per unit dosage form.

[0115] The antibody or functional fragment of the present invention can be administered at once or divided into several smaller doses administered at intervals. It is understood that the exact dosage and duration of treatment can be determined empirically depending on the disease being treated, using known testing protocols or by extrapolating in vivo or in vitro test data. It should be noted that concentrations and dosage values may also vary depending on the severity of the condition to be alleviated. It should be further understood that for any particular subject, specific dosage regimens can be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition, and that the concentration ranges described herein are merely exemplary and do not limit the scope or practice of the claimed compositions.

[0116] The mixture that results when the antibody or functional fragment of the present invention is mixed or added may be a solution, suspension, etc. The form of the resulting mixture depends on several factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the disease, disorder, or condition being treated and can be empirically determined.

[0117] Pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as sterile parenteral solutions or suspensions containing an appropriate amount of the compound or a pharmaceutically acceptable derivative thereof. In one embodiment, the antibody or functional fragment can be formulated and administered in unit dosage or multiple dosage forms. A unit-dose form refers to a physically discrete unit suitable for human and animal subjects and packaged individually as known in the art. Each unit dose contains a predetermined quantity of the antibody or functional fragment of the present invention sufficient to produce the desired therapeutic effect, along with the necessary pharmaceutical carrier, vehicle, or diluent. Examples of unit dosage forms include ampoules and syringes. Unit dosage forms can be administered in fractions or multiples thereof. A multiple-dose form is a plurality of identical unit dosage forms packaged in a single container for separate administration of the unit dosage forms. Examples of multiple dosage forms include pint or gallon vials or bottles. Hence, multiple-dosage form is a multiple of unit doses in unsegregated packaging.

[0118] In one embodiment, one or more antibodies or functional fragments of the present invention are in a liquid pharmaceutical formulation. Pharmaceutically administrable liquid compositions can be prepared, for example, by dissolving, dispersing, or otherwise mixing an antibody or functional fragment provided herein and an optional pharmaceutical adjuvant in a carrier, such as water, saline, aqueous dextrose, glycerol, glycol, ethanol, or the like, thereby forming a solution. If desired, the administered pharmaceutical composition may also contain minor amounts of non-toxic auxiliary substances, such as wetting agents, emulsifiers, solubilizers, pH buffers, and the like, such as acetic acid, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents. Actual methods for preparing such dosage forms are known or will become apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA.

[0119] The method for administering the pharmaceutical composition of the present invention is well known in the art.It is understood that the appropriate administration route of the pharmaceutical composition can be easily determined by a skilled clinician.Exemplary administration routes include intravenous injection, intramuscular injection, intradermal injection or subcutaneous injection.In addition, it is understood that the formulation of the pharmaceutical composition can be easily adjusted to suit the administration route.The present invention also provides that after administration of the pharmaceutical composition of the present invention, the subject can be administered delayed, continuous and / or repeated administration of one or more pharmaceutical compositions provided herein.

[0120] The methods of the present invention for treating disease include (1) preventing disease, i.e., preventing the development of clinical symptoms of disease in a subject who may be predisposed to the disease but who has not yet experienced or manifested symptoms of the disease; (2) inhibiting disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; or (3) alleviating disease, i.e., causing regression of the disease or its clinical symptoms. The methods of the present invention for preventing disease include preventing clinical symptoms indicative of cancer or tumor formation. Such prevention includes, for example, maintaining normal physiological indicators in the subject. Thus, prevention can include prophylactic treatment of a subject to protect the subject from the appearance of tumor metastasis.

[0121] The therapeutically effective amount of the pharmaceutical composition used in the method of the present invention 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 are within the skill of the attending clinician. Subjects treated by the method of the present invention include vertebrates, preferably mammals, and more preferably humans.

[0122] It is understood that modifications that do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate, but not limit, the present invention. [Example]

[0123] Example I sLe a Human monoclonal antibodies against α-amyloid have potent antitumor activity

[0124] Carbohydrate antigen sLe a sLe is widely expressed on epithelial tumors of the gastrointestinal tract, breast, and pancreas, as well as on small cell lung cancer. a Overexpression of sLe appears to be a key event in the invasion and metastasis of many tumors, rendering them susceptible to antibody-mediated lysis. ais an attractive molecular target for tumor therapy. Therefore, as described herein, sLe a We generated and characterized fully human monoclonal antibodies (mAbs) from blood lymphocytes from individuals immunized with the sLe-KLH vaccine. Based on ELISA and FACS, a Several mAbs, including two with high affinity for r5B1 and r5B1 (5B1 and 7E3, with binding affinities of 0.14 and 0.04 nmol / L, respectively), were selected and further characterized. Both antibodies were specific for Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ and Neu5Gcα2-3Galβ1-3(Fucα1-4)GlcNAcβ, as determined by glycan array analysis. Complement-dependent cytotoxicity against DMS-79 cells was higher with r7E3 (IgM) than with r5B1 (IgG1) (EC50, 0.1 μg / mL vs. 1.7 μg / mL). Furthermore, the r5B1 antibody demonstrated high levels of antibody-dependent cellular cytotoxicity against DMS-79 cells using human NK cells or peripheral blood mononuclear cells. To assess in vivo efficacy, the antibodies were tested in xenograft models using Colo205 or DMS-79 tumor cells implanted in severe combined immunodeficient (SCID) mice. In the Colo205 xenograft model, treatment with four doses of r5B1 (100 μg per dose) over the first 21 days doubled median survival to 207 days, with three of five animals surviving six doses. In the DMS-79 xenograft model, the growth of established DMS-79 tumors was inhibited or regressed in animals treated with the r5B1 antibody. sLe as a target of immune attack a Based on their potency and their affinity, specificity, and effector functions, 5B1 and 7E3 have clinical utility in the treatment of cancer.

[0125] Materials, cells, and antibodies DMS-79 (Pettengill et al., Cancer, 45:906-18 (1980)), SW626, EL4, HT29, BxPC3, SK-MEL28, and P3x63Ag8.653 cell lines were purchased from the American Type Culture Collection (ATCC). Colo205-luc cells (Bioware ultra) were obtained from Caliper Life Sciences. Murine control mAb 121SLE (IgM) was purchased from GeneTex. sLe a Tetrasaccharide (Cat. No. S2279) was purchased from Sigma-Aldrich. a -HSA (human serum albumin) conjugate (Cat. No. 07-011), monovalent biotinylated sLe a (sLe a -sp-biotin; Cat. No. 02-044), polyvalent biotinylated sLe a -PAA (Cat. No. 01-044), biotin-labeled Le a -PAA (Cat. No. 01-035), and sLe x -PAA-biotin (Cat. No. 01-045) was purchased from GlycoTech. For multivalent presentation, the tetrasaccharide was incorporated into a polyacrylamide matrix (PAA), thereby creating a 30 kDa multivalent 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 sLe as described. a Prepared in-house using pentenyl glycosides. Ragupathi et al., Cancer Immunol Immunother, 58:1397-405 (2009). GD3, fucosyl-GM1, GM2, and GM3 were purchased from Matreya, and GD2 was purchased from Advanced ImmunoChemical.

[0126] anti-sLe a Generation of mAb-producing hybridomas A study of sLeu in patients with breast cancer initiated at MSKCC under an MSKCC- and FDA-approved IRB protocol and IND. a Blood samples were obtained from three patients in an ongoing clinical trial using the sLe-KLH conjugate vaccine. Blood samples were selected from two patients after three or four vaccinations. These were sLe a These sera (and murine mAb 19.9) showed antibody titers of 1 / 160 and 1 / 320 against sLe in FACS assays. a It reacts well with positive cell lines and mediates potent CDC. Ragupathi et al., Cancer Immunol Immunother, 58:1397-405 (2009). Peripheral blood mononuclear cells (PBMCs) were isolated from approximately 80-90 mL of blood by gradient centrifugation on Histopaque-1077 (Sigma-Aldrich).

[0127] PBMCs were cultured in RPMI-1640 medium supplemented with L-glutamine, non-essential amino acids, sodium pyruvate, vitamins, penicillin / streptomycin, 10% FBS (Omega Scientific), 10 ng / mL IL-21 (Biosource), and 1 μg / mL anti-CD40 mAb (G28-5 hybridoma supernatant; ATCC). Cells were fused with P3x63Ag8.653 myeloma cells by electrofusion.

[0128] sLe a ELISA sLe a For ELISA, plates were pre-filled with 1 μg / mL sLe a -HSA conjugate, monovalent biotinylated sLe a or polyvalent biotinylated sLe captured on Neutr-avidin coated plates aThe wells were coated with HSA-PAA. Uncoated wells (PBS) and wells coated with HSA were used as controls. Bound antibodies were first detected with horseradish peroxidase (HRP)-conjugated goat anti-human IgA+G+M (Jackson ImmunoResearch), and positive wells were subsequently probed with IgG-Fc or IgM-specific secondary antibodies to determine isotype.

[0129] Carbohydrate specificity analysis A closely related antigen, Le a and sLe x Cross-reactivity to biotin-labeled Le was assessed by surface plasmon resonance (SPR). a -PAA and biotin-sLe x Binding to gangliosides GD2, GD3, fucosyl-GM1, GM2, and GM3 was tested by ELISA. Competitive ELISA was used to assess the specificity of the mAb for several other related carbohydrate moieties. Briefly, 2 μg / mL of sLe a The plate was coated with a β-HSA conjugate and then blocked with 3% BSA in PBS. Next, 30 μL of different carbohydrate moieties (40 μg / mL in PBS, prepared from a 1 mg / mL stock solution) either unconjugated or conjugated to HSA or BSA were mixed with 30 μL of test antibody separately and incubated at room temperature in a sample plate. After 30 minutes, 50 μL of the mixture was transferred to the coated assay plate and incubated for 1 hour, followed by incubation with HRP-labeled goat anti-human IgA+G+M, washing, and colorimetric detection of bound antibody using a Versamax spectrofluorometer (all steps performed at room temperature). The carbohydrate moieties tested included globo H, Lewis Y, Lewis X, sialyl-Thomson-nouveaux (sTn), clustered sTn, Thomson-Friedenreich (TF), and Tighe Le. b / Le YMucin, porcine submaxillary mucin (PSM), and sLe a Tetrasaccharide and sLe a The 5B1 and 7E3 antibodies were tested at 10 μg / mL using a 465-glycan version 4.1 printed array in sextuplicate.

[0130] Immunoglobulin cDNA cloning and recombinant antibody expression Human mAb heavy and light chain variable region cDNAs were recovered from individual hybridoma cell lines by RT-PCR and subcloned into IgG1 or IgM heavy chain expression vectors or IgK or IgL light chain expression vectors as previously described (Sawada-Hirai et al., J. Immune Based Ther. Vaccines, vol. 2:5 (2004)). The Ig heavy or light chain expression vector was double-digested with Not I and Sal I, and then both fragments were ligated to form a double-gene expression vector. CHO cells in 6-well plates were transfected with the double-gene expression vector using Lipofectamine 2000 (Invitrogen). After 24 hours, the transfected cells were transferred to a 10-cm dish containing selective medium (DMEM supplemented with 10% dialyzed FBS (Invitrogen), 50 μmol / L L-methionine sulfoximine (MSX), GS supplement (Sigma-Aldrich), and penicillin / streptomycin (Omega Scientific)). After two weeks, MSX-resistant transfectants were isolated and expanded. a High anti-sLeu antibody levels were measured in the supernatant using a specific ELISA assay. a Antibody-producing clones were selected and expanded for large-scale mAb production.

[0131] Human mAb purification Antibodies were purified using an Aekta Explorer (GE Healthcare) system running Unicorn 5.0 software. Briefly, stable clones of 5B1 or 7E3 were grown in a Wave bioreactor in serum-free culture medium, and the harvested supernatant was clarified by centrifugation and filtration and stored refrigerated until use. Human IgG antibodies were purified on an appropriately sized Protein A column using 10 mmol / L PBS and 150 mmol / L NaCl running buffer. Human IgM antibodies were purified on a hydroxyapatite column, and IgM was eluted using a 500 mmol / L phosphate gradient. IgG and IgM were purified using E. coli, respectively. 1% OD using 1.4 and 1.18 280 The antibody concentration was determined and calculated by The purity of each preparation was assessed by SDS-PAGE analysis under reducing conditions (1–5 μg per lane), and was greater than 90% based on the combined heavy and light chains.

[0132] Flow cytometry sLe a Positive or negative tumor cell lines (0.5 × 10 cells per condition) 6Cells (800 cells) were washed in PBS / 2% FBS (PBSF). Test or control human mAbs were then added (1-2 μg / mL in complete medium) and incubated on ice for 30 minutes. Gilewski et al., Clin Cancer Res, 6:1693-701 (2000); Gilewski et al., Proc. Natl. Acad. Sci. USA, 98:3270-5 (2001). After washing in PBSF, cells were incubated with Alexa-488 anti-human IgG-Fcγ or anti-human IgM-μ (Invitrogen) for 30 minutes on ice. Cells were washed twice in PBSF and analyzed by flow cytometry using a Guava Personal Cell Analysis-96 (PCA-96) System (Millipore). Colo205-luc cells were incubated with 2 μg / mL of primary antibody, then stained with secondary antibody from SouthernBiotech and analyzed on a Becton Dickinson FACS Advantage IV instrument using FlowJo 7.2.4 software.

[0133] Affinity determination The affinity constant was determined using the SPR principle on a Biacore 3000 (GE Healthcare). a (Cat. No. 02-044) or multivalent sLe a HSA-PAA-biotin (Cat. No. 01-044) was coupled to separate flow cells of an SPA biosensor chip according to the manufacturer's instructions. A flow cell blocked with culture medium containing HSA and free biotin was used as a reference cell. sLe was measured from several known concentrations of antibodies diluted in HBS-EP buffer (10 mmol / L HEPES, pH 7.4, 150 mmol / L NaCl, 3.4 mmol / L EDTA, 0.005% surfactant P20). aBinding kinetic parameters were determined using a -PAA-biotin coated flow cell. Curve fitting software provided by the Biacore instrument was used to generate estimates of association and dissociation rates for affinity calculations.

[0134] CDC assay sLe a Antigen-positive and -negative cell lines were used in a 90-minute cytotoxicity assay (Guava PCA-96 Cell-Toxicity Kit; Millipore; Cat. No. 4500-0200) using human complement (Quidel; Cat. No. A113) and various dilutions (0.1–25 μg / mL) of purified human mAbs or a positive control mAb, as previously described (Ragupathi et al. Clin Cancer Res 2003;9:5214; Ragupathi et al. Int J Cancer 2000;85:659; Dickler et al. Cancer Res 1999;5:2773). Briefly, 2.5 × 10 target cells were incubated at 4°C for 1 hour. 6 Each well was coated with carboxyfluorescein diacetate succinimidyl ester (CSFE) to obtain green / yellow fluorescent target cells. The coated cells (1 x 10 per 50 μL of sample) were 5(100 μL) were incubated with 100 μL of antibody on ice for 40 minutes. Next, 50 μL of human complement diluted 1:2 in complete medium (RPMI-1640, 10% FCS) or medium alone was added to triplicate samples and incubated at 37°C for 90 minutes. Therefore, the final complement dilution in the assay was 1:8. Cells killed during this incubation period were labeled by adding the membrane-impermeable dye 7-amino-actinomycin D (7-AAD), and the samples were analyzed by two-color immunofluorescence using the Guava CellToxicity software module. Control samples with NP40 were used to determine maximum killing, and samples with complement alone served as the baseline. The percentage of killed cells was determined by appropriate gating and calculated according to the following formula: % killing = [(sample % - complement alone %) / (NP40% - complement alone %)] × 100.

[0135] Antibody-dependent cellular cytotoxicity assay PBMC effector cells were isolated by Ficoll-Hypaque density centrifugation from blood samples obtained under an MSKCC IRB-approved protocol. Target cells were cultured at 5 x 10 cells per mL in complete growth medium. 6The cells were incubated with 15 μL of 0.1% calcein-AM solution (Sigma-Aldrich) at 37°C for 30 minutes in the presence of 5% CO2. Cells were washed twice with 15 mL of PBS-0.02% EDTA and resuspended in 1 mL of complete growth medium. Fifty microliters of labeled target cells (10,000 cells) were plated into a 96-well plate in the presence or absence of antibody at the concentrations indicated in Figure 13 and incubated appropriately with 50 μL of freshly isolated peripheral blood mononuclear cells (effector cells, E / T ratio 100:1). After 2 hours of incubation, the plate was centrifuged at 300 × g for 10 minutes, and 75 μL of the supernatant was transferred to a new flat-bottom 96-well plate. Fluorescence in the supernatant was measured using a Fluoroskan Ascent (Thermo Scientific) with excitation at 485 nm and emission at 535 nm. Spontaneous release was determined from target cells in RPMI-1640 medium with 30% FBS and no effector cells, and maximum release was determined from target cells in RPMI-1640 medium with 30% FBS and 6% Triton X-100 and no effector cells. Percent cytotoxicity was calculated as [(counts in sample - spontaneous release) / (maximum counts - spontaneous release)] x 100.

[0136] mAb internalization assay Internalization of the 5B1 antibody was measured using sLe cells plated in duplicate in 96-well plates (2,000 cells / 90 μL / well) and incubated overnight. aThe cytotoxic activity of r5B1 and a Hum-ZAP secondary conjugate (Advanced Targeting Systems) was assessed against BxPC3 cells expressing r5B1. Various concentrations of r5B1 antibody were incubated with the Hum-ZAP secondary conjugate at room temperature according to the manufacturer's instructions. Next, 10 μL / well of the r5B1 and Hum-ZAP conjugate 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 incubated at 37°C. After 2 hours of incubation, 100 μL / well of solubilization solution (20% SDS / 50% N,N-dimethylformamide) was added to each well and incubated for an additional 16 hours at 37°C. OD was measured at 570 / 690 nm, and the value obtained using medium alone was used for plate background subtraction. Eight parallel cultures without antibody were used to normalize sample values (sample / untreated mean × 100).

[0137] Xenograft model Female CB17 SCID mice (5–8 weeks old) were purchased from Taconic. For the Colo205 xenograft model, Colo205-luc cells (0.5 × 10) were cultured in 0.1 mL of complete growth medium. 6 1 × 10 cells) were injected into the tail vein using a BD insulin syringe (Becton Dickinson & Co.) with a 28G needle on day 0. For the first study, 100 micrograms of mAb 5B1 were injected intraperitoneally on days 1, 7, 14, and 21 (Experiment 1) or days 1, 4, 7, 10, 14, and 21 (Experiment 2). For the second study, 100 μg, 300 μg, or 1 mg of mAb 5B1 were injected intraperitoneally 4 days after tumor cell injection, then twice a week for the first 2 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 (1 × 10 cells) were injected intraperitoneally. 6) was subcutaneously injected into female CB17 SCID mice, and tumors were grown to a length of 5 mm (approximately 20 mm 2 Treatment of mice began on day 19 after reaching a normal serum albumin (NA) level. Animals were then treated with human IgG or 5B1 antibody at 200 μg per dose intraperitoneally, plus cRGD to increase vascular permeability by intravenous injection initially at 80 μg, then at 40 μg per dose, 5 days per week, until day 37.

[0138] All procedures were performed under protocols 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.

[0139] result Identification of human monoclonal antibodies by ELISA and generation of recombinant antibodies Blood samples from three vaccinated patients were used for hybridoma generation trials, and many positive wells were detected in antigen-specific ELISA assays (Table 3). Extensive screening was used to eliminate antibodies that showed poor or nonspecific binding. a Eight human antibody-expressing hybridoma cells (one IgM and seven IgG) with strong reactivity against sLe were initially selected, expanded, and subcloned for further characterization. Two antibodies (9H1 and 9H3) were identified. a showed strong binding to sLe-HSA conjugates, but a Three antibodies (5B1, 5H11, and 7E3) showed no binding to plates coated with sLe-PAA. a , multivalent sLe a and sLe a -HSA conjugates (Table 4).

[0140] [Table 3]

[0141] [Table 4]

[0142] The heavy and light chain variable regions from the four 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 three selected IgG antibodies, 5B1 (IgG / λ), 9H3 (IgG / λ), and 5H11 (IgG / λ), were derived from the same VH family and all used lambda light chains. These IgG1 antibodies displayed distinct CDR sequences with 16, 5, or 3 off-germline mutations, respectively (Figures 1-6; Table 5). The IgM antibody (7E3) utilized a kappa light chain and had six heavy chain mutations (Figures 7-8; Table 5). The increased mutations in 5B1 indicate affinity maturation. Recombinant antibodies were produced in CHO cell lines in a wave bioreactor system and purified using protein A or hydroxyapatite chromatography for IgG and IgM, respectively. The purified recombinant antibodies retained the properties of the original hybridoma-derived antibodies with respect to binding and specificity in ELISA.

[0143] [Table 5]

[0144] Analysis of tumor cell binding Cell surface binding is crucial for cytotoxic activity, and therefore was examined next. Flow cytometry demonstrated strong binding of the 5B1, 9H3, 5H11, and 7E3 recombinant antibodies to DMS-79 cells, a small cell lung cancer suspension cell line (Figure 11A). Binding of r5B1 and r7E3 was also confirmed to HT29 colon cancer cells (Figure 11B), BxPC3 pancreatic cancer cells (Figure 11C), SW626 ovarian cancer cells (Figure 11D), and Colo205-luc colon cancer cells (Figure 11F). These antibodies inhibited the binding of sLe a It was unable to bind to negative (SLE121-negative) SK-MEL28 melanoma cells (Fig. 11E) or cells of the EL4 mouse lymphoma (data not shown).

[0145] Affinity measurement sLe a The relative affinity / avidity of binding to biotinylated sLe a The r5B1 and r7E3 proteins were probed by SPR using a streptavidin-coated biosensor chip to capture sLe-PPA. As shown in Table 6, r5B1 and r7E3 proteins were probed by SPR using a streptavidin-coated biosensor chip to capture sLe-PPA. a A commercially available murine IgM anti-sLe antibody rapidly binds to -PPA and was used for comparison. a The affinity of 5B1 was measured at 0.14 nmol / L, and the apparent affinity / avidity of 7E3 was approximately 4-fold higher (Table 6). The 9H3 affinity determinations demonstrated that the 9H3 antibody (native and recombinant) binds sLe a -Abstructed by its inability to bind to a PAA-coated biosensor chip.

[0146] [Table 6]

[0147] Specificity analysis Preliminary assays to probe carbohydrate specificity revealed that 5B1, 9H3, and 7E3 are closely related sLeX , Le a , and Le Y It was shown not to bind to the antigen or to gangliosides GD2, GD3, fucosyl-GM1, GM2, and GM3. a -PAA-biotin or sLe a Further analysis of the binding of 7E3, 5B1, and 121SLE to -sp-biotin demonstrated that all three antibodies bind to the multivalent form of sLe. a sLe of 5B1 was shown to bind to 5B1, whereas 7E3 and 5B1 were found to bind to 5B1 in a monovalent form. a Binding to -PAA is sLe a The tetrasaccharide inhibited the activity of the anti-sLe tetrasaccharide in a dose-dependent manner (data not shown). a Serum with high antibody titers is sLe a Specific for gangliosides GM2, GD2, GD3, fucosyl GM1, or the neutral glycolipids globo H and Le by ELISA. y This is consistent with previous observations that found no reaction with sLe. Ragupathi et al., Cancer Immunol Immunother 58:1397-405 (2009). In competition assays using nine distinct related carbohydrate moieties in various presentation formats (e.g., as ceramides or conjugated to BSA or HSA), sLe a Tetrasaccharide and sLe a -HSA conjugates are the only a -HSA conjugates (Table 7).

[0148] [Table 7]

[0149] To further explore carbohydrate specificity, the 5B1 and 7E3 antibodies were also tested by glycan array analysis performed by the Consortium for Functional Glycomics Core H group. Both antibodies were tested in sextuplicate at 10 μg / mL on a printed array of 465 glycans. Results confirmed the high specificity of both antibodies and demonstrated a significant reduction in sLe a The tetrasaccharides Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ and Neu5Gcα2-3Galβ1-3(Fucα1-4)GlcNAcβ are selectively recognized. x , Le a , Le x , and Le y There was virtually no binding to closely related antigens present on the array, including . The results are summarized in Table 8, which shows the top five recognized by each antibody out of 465 glycan structures.

[0150] [Table 8]

[0151] CDC activity To evaluate the functional activity of 5B1 and 7E3, cytotoxic activity was tested using DMS-79 cells in the presence of human serum as a complement source. Both antibodies demonstrated nearly 100% killing activity at 10 μg / mL in some assays, whereas a control antibody with a different specificity (1B7, an anti-GD2 IgG1 mAb) had no effect at the same concentration (data not shown). CDC activity was concentration-dependent, with 7E3 being significantly more active than 5B1 in this assay (Figure 12), which is expected since IgM antibodies have been shown to be more effective in complement-mediated cytotoxicity assays. 50 (50% cytotoxicity) was 1.7 μg / mL for 5B1 and 0.1 μg / mL for 7E3, which translates to approximately 85-fold greater potency of 7E3 on a molar basis (FIG. 12).

[0152] ADCC activity Although 7E3 was significantly more potent in the CDC assay, IgG antibodies are known to possess antibody-dependent cellular cytotoxicity (ADCC) activity, which is thought to be important for tumor killing in vivo. High levels of cytotoxicity were measured using the 5B1 antibody with human PBMCs and DMS-79 target cells at various E:T ratios (Figure 13A). Similar levels of cytotoxicity were observed with primary NK cells at lower E:T ratios (Figure 13B). Dose-response experiments using PBMCs from two donors measured at an E / T ratio of 100:1 demonstrated similar efficacy, with cytotoxicity reaching >85% at 5B1 concentrations of 0.5 μg / mL or higher (Figure 13C). 5B1-mediated cytotoxicity requires the FcγRIII receptor, as it can be blocked with the 3G8 anti-CD16 antibody. Similar high levels of cytotoxicity were measured using the 5B1 antibody and human PBMCs against Colo205-luc cells at an E:T ratio of 100:1. The ADCC activity achieved with 1 μg / mL of the 5B1 antibody was superior to that observed with antibodies to GM2, fucosyl-GM1, globo H, or polysialic acid. As expected, 7E3 and murine 121SLE (both of which are IgM) were inactive in this assay.

[0153] 5B1 internalization assay Antibody conjugates directed against antigens "closely related" to Lewis Y have previously been shown to be rapidly internalized and highly effective in animal models. Hellstrom et al., Cancer Res 50:2183-90 (1990); Trail et al., Science 261:212-5 (1993). aTo investigate whether 5B1 is internalized, the pancreatic cell line BxPC3 was incubated with 5B1 and then Hum-ZAP, an anti-human IgG conjugated to the ribosome-inactivating protein saporin, was added. Kohls et al., Biotechniques 28:162-5 (2000). Cells that internalized the saporin-containing complex were killed, whereas failure to internalize saporin left the cells intact. As shown in Figure 14, BxPC3 cells were effectively killed in the presence of increasing doses of 5B1, but not in the presence of an isotype-matched IgG1 antibody directed against GD2, which is not expressed in these cells.

[0154] Activity in xenograft animal models of metastasis To assess the activity of 5B1 in vivo, the antibody was tested in two xenograft models using either Colo205-luc or DMS-79 tumor cells in SCID mice. For the xenograft model using Colo205-luc tumor cells, 5 mice per group were inoculated with 0.5 × 10 cells on day 0. 6Cells were injected into the tail vein, and successful cell injection was verified by imaging the animals using an IVIS 200 in vivo imaging system (Caliper Life Sciences). One day later, animals were treated with 5B1 antibody given intraperitoneally or with a PBS sham injection. In experiment 1, 100 μg of 5B1 was given on days 1, 7, 14, and 21 (total dose of 400 μg), and in experiment 2, animals were given 100 μg of 5B1 on days 1, 4, 7, 10, 14, and 21 (total dose of 600 μg). The mean median survival time of untreated animals in the two experiments was 102 days, and all untreated animals died within 155 days (Figure 15). Treating animals significantly improved survival; median survival in the group receiving four doses of 5B1 doubled to 207 days, with 2 of 5 animals surviving until the end of the study after 301 days (log-rank test, P = 0.0499; HR = 3.46). The proportion of surviving animals further increased to 3 of 5 mice after six doses (log-rank test, P = 0.0064; HR = 6.375). A second study was terminated after 308 days, and Colo205-luc tumors could not be detected in surviving animals using the highest sensitivity of the imaging system (data not shown).

[0155] In a second study, mice similarly injected with Colo205-luc tumor cells as described above were treated with increasing doses of 5B1 or 7E3 antibodies (100 μg, 300 μg, or 1 mg). All animals received intraperitoneal injections of 5B1 or 7E3 antibodies or PBS sham injections (control), first 4 days after tumor cell injection, then twice weekly for the first 2 weeks and once weekly for the next 7 weeks. In SCID mice implanted with Colo205-luc tumor cells, delayed treatment with various doses of 5B1 demonstrated dose-dependent protection leading to complete cure (Figures 16 and 17). Treatment with 7E3 antibody did not provide greater protection despite its apparent increased affinity (data not shown).

[0156] In the xenograft model using DMS-79 cells, 5 mice per group were inoculated with 1 × 10 cells on day 0. 6 The tumor was injected subcutaneously until it reached a length of 5 mm (approximately 20 mm 2 Treatment began on day 19, after the tumor reached a normal tumor size (T1 / T2). Animals were then treated with human IgG or 5B1 antibody at 200 μg per dose intraperitoneally, plus cRGD at 80 μg initially, then 40 μg per dose, 5 days per week, by intravenous injection until day 37. Growth of established DMS-79 tumors was suppressed or regressed in animals treated with 5B1 or the combination of 5B1 plus cRGD (Figures 18A and 18B). Treating animals with 5B1 on the day of DMS-79 cell implantation in a subcutaneous model completely prevented tumor growth (data not shown).

[0157] The above data demonstrate a significant ability to suppress or regress established tumors and provide a survival advantage using 5B1 antibody treatment.

[0158] Example II Radiolabeled monoclonal antibody 5B1 was used to investigate the efficacy and safety of pancreatic cancer and other sLeu a Immuno-PET detection and diagnosis of positive adenocarcinoma Adenocarcinoma is the leading cause of cancer death. Detection of pancreatic cancer remains particularly challenging, with diagnosis often occurring at a late stage. Techniques for early detection of primary and metastatic pancreatic cancer could have significant clinical impact. In clinical practice, sLE is used to identify suspicious subclinical malignancies in patients with pancreatic cancer. a The elevated levels of the antigen are monitored. As described herein, pancreatic cancer and other sLeu a sLe in preclinical models of sLe-positive adenocarcinoma a We investigated the potential of a novel immunoPET imaging probe targeting human anti-sLe a Monoclonal antibody 5B1 inhibits sLe a Positive staining was observed in known human adenocarcinomas, but sLe aNegative malignant tumors and most normal tissues showed no positive staining. 89 Zr radiolabeled 5B1 ( 89 Zr-5B1) showed high labeling yields (>80%) and purification yields (>95%) in subcutaneous orthotopic and metastatic pancreatic cancer xenografts in female SCID mice. 89 Imaging with Zr-5B1 was investigated. PET images and biodistribution studies showed minimal nonspecific binding to healthy tissues and a significant increase in sLe a against BxPC3 xenografts overexpressing 89 Excellent specificity and localization of Zr-5B1 were demonstrated. Further analysis in subcutaneous colon cancer xenograft models and small cell lung cancer xenograft models also demonstrated 89 Zr-5B1 provided excellent tumor delineation. Therefore, these results 89 Zr-5B1 was administered to sLe a It is shown that these proteins can be used as molecular probes for the early detection of malignant tumors.

[0159] Cell lines and tissue cultures All tissue culture procedures were performed according to sterile technique. Small cell lung cancer DMS79 cells and BxPC3 pancreatic cancer cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Colo205-luc colorectal cancer cells (Bioware Ultra) were purchased from Caliper Life Sciences (CLS, Hopkinton, MA). All cells were grown at 37°C in a 5% CO2 humidified atmosphere according to ATCC and CLS recommendations.

[0160] sLe by FACS a In vitro evaluation of expression levels Flow cytometry using the indicated cultured cancer cell lines was performed as described in Example I herein. Briefly, culture 1 x 10 tumor cells per tube. 6The single cell suspension was washed in PBS with 3% fetal bovine serum (FBS). Then, human monoclonal antibody r5B1 (sLe a IgG against goat anti-human IgG (anti-IgG) was added at 20 μg / ml per tube and incubated on ice for 30 minutes. After washing in PBS with 3% FBS, 20 μl of a 1:25 dilution of fluorescein-isothiocyanate (FITC, Southern Biotechnology, Birmingham, AL)-labeled goat anti-human IgG was added, and the mixture was incubated on ice for an additional 30 minutes. After the final wash, the positive population and median fluorescence intensity of stained cells were identified using a FACS Scan (Becton & Dickinson, San Jose, CA). Cells stained only with fluorescein-isothiocyanate-labeled goat anti-human IgG were used to set 1% as background for comparing the percent positive cells stained with the primary mAb.

[0161] 89 Preparation of Zr-labeled antibodies Recombinant 5B1 antibody was prepared and purified as described herein. 5B1 antibody and nonspecific human IgG were functionalized with p-isothiocyanate benzyl-desferrioxamine (DFO-Bz-NCS, Macrocyclics, Inc., Dallas, TX) at a mAb:DFO-Bz-NCS ratio of 1:4. For example, to 300 μL of 5B1 (1.23 mg in PBS, pH ∼9), a volume of 7.2 μL of DFO-Bz-NCS (4.25 mM in DMSO) was added. The reaction was incubated at 37°C for 1–1.5 hours. The functionalized antibody was purified using either a PD10 desalting column (GE Healthcare) or a 10 kDa centrifugal filter (Amicon).

[0162] Zr-89 was generated by proton irradiation of yttrium foil at MSKCC according to a previously established procedure and isolated in high purity as Zr-89 oxalate (Holland et al., Nuclear Medicine and Biology 36:729-39 (2009)). Antibody labeling proceeded as described by Holland et al., Journal of Nuclear Medicine official publication, Society of Nuclear Medicine 51:1293-300 (2010). In general, Zr-89 oxalate was neutralized to pH 7.0-7.2 with 1 M Na2CO3. DFO-antibody was then added. The reaction was incubated at room temperature for 1-2 hours. Subsequent purification was performed using a PD10 desalting column with 0.9% saline.

[0163] In vitro experiments 89 Zr-5B1 was investigated for in vitro stability in 0.9% saline and 1% bovine serum albumin at 37°C for 5 days. Changes in radiochemical purity were monitored by radio-iTLC using 50 mM DTPA as the mobile phase from t = 0 to 5 days. 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 antibody.

[0164] Animal models All animal studies were performed in accordance with the guidelines established by the Institutional Animal Care and Use Committee. Tumors were induced in the hind limbs of female CB17SC-F SCID mice (Jackson Laboratories, 6-8 weeks, 20-22 g) or nude athymic (nu / nu) mice. All cell lines were inoculated subcutaneously in 200 μL of a 1:1 medium:Matrigel (BD Biosciences) solution and allowed to grow to a maximum tumor volume of 250 mm before use. 3 It grew to.

[0165] Biodistribution study Biodistribution studies were performed in several cohorts of mice (n = 3–5) bearing separate Colo205-luc colorectal xenografts, BxPC3 pancreatic xenografts, and DMS79 small cell lung xenografts. Zr-89 mAb (10–20 μCi, 1–2 μg) in 100 μL of 0.9% saline was administered intravenously into the lateral vein. Additional unlabeled mAb (10–50 μg) was co-injected with the tracer. Blocking studies using 250 μg excess unlabeled mAb were performed in cohorts of mice to assess sLe a The specificity of the antibody against the tumor was addressed. After each time point (t = 24, 48, 120 h pi), mice were euthanized by CO2 asphyxiation. Blood was immediately collected by cardiac puncture, and tumors were collected along with selected organs. The wet weight of each tissue was measured and analyzed by Wizard. 2 The radioactivity bound to each organ was counted using a 2480 gamma counter (Perkin Elmer). The percentage of tracer uptake, expressed as % injected dose per gram (%ID / g), was calculated as tissue-bound activity per organ weight per actual injected dose, decay-corrected for the counting time.

[0166] small animal immuno-PET Imaging experiments were performed using a microPET Focus 120 or R4 scanner (Concorde Microsystems). Mice (n = 3–5) were administered Zr-89-labeled antibody (200–300 μCi, 15–25 μg) in 100–200 μL of 0.9% saline via lateral tail vein injection. PET whole-body acquisitions were recorded on mice while anesthetized with 1.5–2.0% isofluorane in oxygen (Baxter Healthcare) at 24–96 h postinjection. Images were analyzed using ASIPro VM™ software (Concorde Microsystems). Regions of interest (ROIs) were drawn and plotted against time.

[0167] Immunohistochemistry Biotinylated 5B1 was prepared by incubating a 20x molar excess of sulfo-NHS-LC-biotin (Thermo Scientific / Pierce, cat. no. 21327) at room temperature for 30 minutes. Free biotin was removed using a Zebra™ Desalt spin column (Thermo Scientific / Pierce, cat. no. 89889) according to the manufacturer's instructions. The antibody buffer was exchanged with PBS containing 0.01% sodium azide at a concentration of 1.1 mg / ml. Binding to DMS79 cells was confirmed by FACS and was comparable to that of the parental 5B1 antibody.

[0168] Preliminary immunohistochemical staining conditions were determined using Colo205 cells as a positive control and SK-MEL28 cells as a negative control. Cell pellets were prepared, formalin-fixed, and paraffin-embedded. Slides were incubated with biotinylated 5B1 diluted in 10% (v / v) normal human serum in PBS (Jackson ImmunoResearch Labs; cat. no. 009-000-121). Staining was performed by Ventana automation (Discovery XT platform - Ventana Medical Systems, Inc., Tucson, AZ) using the standard streptavidin-biotin immunoperoxidase method and the DAB detection system. Antigen retrieval was performed using heat and Ventana's CC1 conditioning solution. Comparable results were obtained in a pilot study with the CA19.9 mouse monoclonal antibody (clone 116-NS-19-9) from Signet (Covance). Colo205 cells were strongly positive for biotinylated 5B1 used at 10 μg / ml, whereas SKMEL28 cells were completely negative. Histo-Array™ tissue microarrays were purchased from Imgenex (San Diego, CA). The following slides containing tumor biopsy cores as well as several normal tissue cores were used: IMH-327 (Common Cancers, 59 samples), IMH-359 (Colorectal: Cancer-Metastasis-Normal; 59 samples), and IMH-324 (Ovarian Metastatic Cancer). A pancreatic tumor tissue core was present in IMH-327.

[0169] In vivo sLe a Serum concentration sLe mice bearing Colo205, BxPC3, and DMS79 xenografts aBlood was exsanguinated for antigen assay. A group of tumor-free mice served as a control. sLe in mouse serum was measured using the ST AIA-PACK CA19.9 kit (Cat. No. 025271, TOSOH Bioscience Inc., South San Francisco, CA). a The levels were measured. The principle of this assay is based on a two-site immunoenzyme measurement assay. The analysis was performed as described in the manufacturer's instructions. The optical density of the immunoassay plate was measured using a TOSOH AIA2000 Automated immunoassay analyzer (TOSOH Bioscience, Inc., San Francisco, CA).

[0170] statistical analysis Data values were expressed as mean ± SD unless otherwise specified. Statistical analysis was performed using GraphPad Prism version 5.03 software using one-way ANOVA followed by Dunnett's test. A P value <0.05 was considered statistically significant.

[0171] result The binding specificity of 5B1 was probed by staining microarrays of selected malignant and normal tissues. 5B1 reactivity was observed in malignant tumors and sLe a The reactivity was restricted to specific cases of normal tissue previously known to overexpress IL-1 (Figure 19; Table 9). Most normal tissues were completely negative (Table 9). In contrast, strong positive staining was found in 21 / 34 (62%) colon adenocarcinomas, 33 / 57 (58%) ovarian adenocarcinoma metastases, and 7 / 9 (66%) pancreatic ductal carcinomas of various stages (Table 10). As shown in Figure 19, typical reactivity was diffuse cytoplasmic staining, with distinct membrane staining evident in some tumor cells. Furthermore, some ovarian signet ring cell carcinomas and some lung and breast carcinomas were also found to be strongly positive. In contrast, only 4 / 43 prostate cancer samples were positive, and 0 / 51 GIST cases were positive (data not shown).

[0172] [Table 9]

[0173] [Table 10]

[0174] 5B1 immunostaining a The high specificity for cancer tissues expressing sLe was the basis for the use of this mAb as a PET probe. Modification of 5B1 with a benzyl-isothiocyanate analog of desferrioxamine (DFO-Bz-NCS) was performed at a 4:1 (chelate:mAb) ratio, followed by purification by centrifugal filtration using saline as the wash buffer. After adjusting the pH to 7.0-7.2, facile radiolabeling with Zr-89 proceeded at room temperature. A narrow, near-neutral pH range is required to achieve optimal radiolabeling yields of >80%. Free, unbound Zr-89 was removed using a PD10 desalting column. Product concentration was performed using a centrifugal filter (MWCO: 10 kDa). A relatively high specific activity of 12.1 ± 1.1 mCi / mg was established. Radiochemical purity of >95% was ensured before use. Immunoreactivity assays demonstrated the activity of sLe. a The retention of activity against ribonucleotides was demonstrated (72.4 ± 1.1%, n = 3). Stability in bovine serum albumin at 37°C was maintained at >95% over 5 days (data not shown). In saline, demetallation was observed as early as 24 hours (>85% complexation), with >75% of the radiometal bound after 120 hours at 37°C.

[0175] Small animal PET imaging and biodistribution studies were performed using female SCID mice implanted with BxPC3 pancreatic cancer xenografts subcutaneously in the left hind leg. 89 Tumor-associated sLe caused by Zr-5B1 aSubstantial delineation of the intravenously administered radiotracer was confirmed. From the maximum intensity projection (MIP) in Figure 20, BxPC3 xenografts (n=3) showed excellent adhesion of the intravenously administered radiotracer. Regions of interest (ROIs) drawn for the tumor from the PET images showed uptake of 5.0±0.4%ID / g (2 hr), 16.2±2.5%ID / g (24 hr), 23.8±4.7%ID / g (48 hr), 36.8±6.1%ID / g (96 hr), and 49.5±7.7%ID / g (120 hr). Binding activity in the blood pool and normal tissues appeared to disappear 24 hr after injection. Results from biodistribution experiments are consistent with the PET data. At 24 hr 89 High tumor localization of Zr-5B1 (84.7 ± 12.3% ID / g, n = 4) was observed, and increased uptake was observed at 120 h postinjection (114.1 ± 23.1% ID / g, n = 4) (Figure 21). Tumor uptake exceeded 100% due to the low weight (62.4 ± 0.03 mg). The %ID at 24 h postinjection was found to be 10-fold higher than that of nonspecific IgG at the same time point (Figure 21 inset). Competitive inhibition with 250 μg of non-radiolabeled 5B1 at 24 h postinjection blocked tracer accumulation, which defines the specificity of uptake. 89 Minimal binding of Zr-5B1 to normal pancreas and the rest of the harvested normal tissue was observed, resulting in high tumor-to-tissue contrast at all time points.

[0176] In accordance with the above results, in the orthotopic BxPC3 pancreatic tumor model 89 Zr-5B1 was assayed. The orthotopic model is clinically relevant and provides a clinically acceptable test of the efficacy of the PET probe. After inoculation into the pancreas, tumor growth was monitored weekly by optical imaging with bioluminescence. PET imaging experiments were performed once tumors were palpable. FDG-PET and 89 A comparison of probe tumor delineation was performed between Zr-5B1 and Zr-5B1 (Figure 25). PET and tandem computed tomography (CT) provided enhanced visualization of the anatomical region of interest.

[0177] Other sLea As a PET probe in expressing adenocarcinoma 89 To evaluate Zr-5B1 in lung and colon cancer models 89 Zr-5B1 was assayed. Small animal studies were performed using DMS79 small cell lung cancer cells and Colo205-luc colon cancer cells injected subcutaneously into the right hind limb of female SCID mice. PET MIP images were acquired at 24-120 hours after intravenous injection of 200-300 μCi (16-25 μg). As early as 24 hours post-injection, uptake into heterologous DMS79 tumors was demonstrated at 38.15 ± 2.12% ID / g with excellent signal-to-background (Figure 22A). Tracer tumor accumulation increased at 48 hours post-injection (44.60 ± 6.47% ID / g) and was maintained at 120 hours post-injection (41.97 ± 12.23% ID / g). Nonspecific binding was observed. 89 Zr-5B1 was rapidly cleared from normal tissues, with minimal to no background uptake at 48 h post-injection. Furthermore, as shown in Figure 22B, tumor delineation was observed in Colo205-luc xenografts at 24–120 h post-injection. ROIs showed tumor accumulation at 2, 24, 48, 96, and 120 h, respectively, with %ID / g of 10.5 ± 0.76, 23.5 ± 2.7, 24.8 ± 4.0, 18.4 ± 4.7, and 16.5 ± 2.3. As shown in regions of interest drawn from PET images, an observable increase in liver accumulation occurred over time, resulting in a decrease in tumor uptake (Figure 22C). Data generated from the biodistribution study correlated well with the observed PET results (data not shown).

[0178] sLe in mouse serum as tumors progress a The levels were quantified. Exsanguination was performed on SCID mice bearing Colo205 xenografts, SCID mice bearing DMS79 xenografts, and SCID mice bearing BxPC3 xenografts, along with a group of tumor-free mice serving as controls. aThe values showed that mice challenged with Colo205 had higher levels of sLe compared with mice implanted with pancreatic BxPC3 and mice implanted with DSM79. a The results are shown in Table 11.

[0179] [Table 11]

[0180] These results suggest that the radiolabeled anti-sLea antibody ( 89 Zr-5B1) in pancreatic adenocarcinoma and other sLe a It is demonstrated to be specific for the detection and diagnosis of positive adenocarcinoma. 89 Zr-5B1 was produced in excellent yield and purity, with high specific activity and retention of immunoreactivity. 89 Evaluation of Zr-5B1 has resulted in excellent tumor delineation and diagnosis. Preclinical evaluation of this radiotracer in small animals with colon tumors and small cell lung tumors has demonstrated the sLe a Its universal usefulness against malignant tumors expressing HIV-1 has been demonstrated.

[0181] Example III anti-sLe a Diabodies bind to various cancer cell lines Two diabodies were generated using the VH and VL domains of the 5B1 and 7E3 clonal isolates described herein, designated 5B1CysDb and 7E3CysDb, respectively (Figures 9 and 10). Both diabodies contained a five amino acid linker region between the VL and VH domains. Both diabodies also contained a polyhistidine tag on the C-terminus, which was utilized for purification and detection.

[0182] The binding of 5B1CysDb and 7E3CysDb to three cancer cell lines: (1) DMS-79 cells, a small cell lung cancer suspension cell line; (2) Capan-2 cells, pancreatic adenocarcinoma cells; and (3) BxPC3 cells, pancreatic cancer cells, was assayed by incubating 250,000 cells in 0.2 ml with 10 μg / ml of 5B1CysDb or 7E3CysDb, respectively. The cell and diabody combination was incubated on ice for 40 minutes in PBS / 2% FBS.

[0183] After washing, the cells were incubated with 0.2 ml of 1:1000 diluted ALEXA-488-labeled anti-His antibody (Life Technologies, Cat. No. A21215) for 40 minutes. After a second wash, the cells were analyzed using a Guava flow cytometer. Both the 5B1CysDb and 7E3CysDb demonstrated significant binding to DMS-79, Capan-2, and BxPC3 cells (Table 12).

[0184] [Table 12]

[0185] Example IV Administration of 5B1 and Taxol inhibits tumor growth anti-sLe a The antitumor activity of coadministering the antibody (5B1) with the chemotherapeutic agent Taxol (paclitaxel) was evaluated in xenograft models of pancreatic and small cell lung cancer. As previously described herein, one million BxPc3 cells (pancreatic tumor cells) or five million DMS-79 cells (small cell lung cancer cells) were injected into the hind flank of 6-week-old female CB17 SCID mice (day 0; N=5). DMS79 tumors were grown to a mean tumor size of 193±64 mm. 3The tumors were grown for 21 days until tumor size reached 100%. Human IgG or 5B1 (0.5 or 1 mg) was given intraperitoneally twice a week (starting on day 21), and taxol (0.2 mg / dose) was administered intravenously on days 23, 30, 37, and 44. In a DMS-79 xenograft model, coadministration of 5B1 antibody and taxol significantly restricted tumor growth and led to tumor regression compared with control human IgG or administration of 5B1 antibody and taxol separately (Figure 23).

[0186] In the BxPc3 xenograft model, tumors were allowed to grow for 14 days, at which point they averaged 126 ± 30 mm 3 Taxol was administered intravenously on days 14, 21, 28, and 34 (once a week), and 5B1 was given twice a week starting on day 14. Coadministration of the 5B1 antibody with taxol significantly restricted tumor growth compared to control or separate administration of the 5B1 antibody and taxol (Figure 24). These results demonstrate the potential of anti-sLeu antibodies in preventing tumor growth and / or reducing tumor size in pancreatic and small cell lung cancers. a A synergistic effect between the antibody and the chemotherapeutic agent is demonstrated.

[0187] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. While the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.

[0188] (Item 1) 1. An isolated polynucleotide encoding an antibody heavy chain or functional fragment thereof, wherein the antibody heavy chain or functional fragment thereof comprises a variable heavy (VH) domain having an amino acid sequence selected from the group consisting of residues 20 to 142 of SEQ ID NO: 2, residues 20 to 142 of SEQ ID NO: 6, residues 20 to 142 of SEQ ID NO: 10, and residues 20 to 145 of SEQ ID NO: 14. (Item 2) 2. The isolated polynucleotide according to Item 1, wherein the amino acid sequence of the VH domain is encoded by a nucleic acid sequence selected from the group consisting of residues 58 to 426 of SEQ ID NO: 1, residues 58 to 426 of SEQ ID NO: 5, residues 58 to 426 of SEQ ID NO: 9, and residues 58 to 435 of SEQ ID NO: 13. (Item 3) 1. An isolated polynucleotide encoding an antibody light chain or functional fragment thereof, wherein said antibody light chain or functional fragment thereof comprises a variable light (VL) domain having an amino acid sequence selected from the group consisting of residues 20 to 130 of SEQ ID NO:4, residues 20 to 129 of SEQ ID NO:8, residues 20 to 130 of SEQ ID NO:12, and residues 23 to 130 of SEQ ID NO:16. (Item 4) 4. The isolated polynucleotide according to Item 3, wherein the amino acid sequence of the VL domain is encoded by a nucleic acid sequence selected from the group consisting of residues 58 to 390 of SEQ ID NO: 3, residues 58 to 387 of SEQ ID NO: 7, residues 58 to 390 of SEQ ID NO: 11, and residues 67 to 390 of SEQ ID NO: 15. (Item 5) Cialis Lewis a 1. An isolated antibody or functional fragment thereof that binds to SEQ ID NO: 1, wherein the antibody or functional fragment thereof comprises a variable heavy chain (VH) domain, and the VH domain comprises an amino acid sequence selected from the group consisting of residues 20 to 142 of SEQ ID NO: 2, residues 20 to 142 of SEQ ID NO: 6, residues 20 to 142 of SEQ ID NO: 10, and residues 20 to 145 of SEQ ID NO: 14. (Item 6) Cialis Lewis a 1. An isolated antibody or functional fragment thereof that binds to SEQ ID NO: 4, wherein the antibody or functional fragment thereof comprises a variable light chain (VL) domain, and the VL domain comprises an amino acid sequence selected from the group consisting of residues 20 to 130 of SEQ ID NO: 4, residues 20 to 129 of SEQ ID NO: 8, residues 20 to 130 of SEQ ID NO: 12, and residues 23 to 130 of SEQ ID NO: 16. (Item 7) Cialis Lewisa 1. An isolated antibody or functional fragment thereof that binds to SEQ ID NO: 1, wherein the antibody or functional fragment thereof comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain and the VL domain comprise amino acid sequences selected from the group consisting of residues 20-142 of SEQ ID NO: 2 and residues 20-130 of SEQ ID NO: 4; residues 20-142 of SEQ ID NO: 6 and residues 20-129 of SEQ ID NO: 8; residues 20-142 of SEQ ID NO: 10 and residues 20-130 of SEQ ID NO: 12; and residues 20-145 of SEQ ID NO: 14 and residues 23-130 of SEQ ID NO: 16. (Item 8) 8. The isolated antibody or functional fragment thereof of any one of items 5 to 7, wherein the antibody is a human antibody. (Item 9) 8. The isolated antibody or functional fragment thereof of any one of items 5 to 7, wherein the antibody functional fragment is selected from the group consisting of Fab, Fab', F(ab')2, scFV, diabody, triabody, minibody and single domain antibody (sdAB). (Item 10) 10. The antibody or functional fragment thereof according to item 9, wherein the antibody functional fragment is a diabody. (Item 11) 11. The antibody or functional fragment of item 10, wherein the diabody comprises the amino acid sequence of SEQ ID NO: 18 or 20. (Item 12) 8. The isolated antibody or functional fragment thereof of any one of items 5 to 7, wherein the antibody is a monoclonal antibody. (Item 13) 8. The isolated antibody or functional fragment thereof of any one of items 5 to 7, wherein the antibody is an IgG or IgM isotype. (Item 14) 14. The isolated antibody or functional fragment thereof according to item 13, wherein the IgG antibody is an IgG1 subclass. (Item 15) 8. A conjugate comprising the isolated antibody or functional fragment of any one of items 5 to 7 conjugated or recombinantly fused to a diagnostic, detectable or therapeutic agent. (Item 16) 16. The conjugate according to item 15, comprising a detectable agent. (Item 17) The detectable agent is zirconium ( 89 17. The conjugate according to item 16, wherein Zr is a methyl group. (Item 18) 8. A pharmaceutical composition comprising the antibody or functional fragment according to any one of items 5 to 7 and a pharmaceutically acceptable carrier. (Item 19) 20. A method for treating or preventing a disease, comprising administering a therapeutically effective amount of the pharmaceutical composition of item 18 to a subject in need of such treatment or prevention. (Item 20) The disease is cancer or tumorigenesis, and the cells of the cancer or tumor are sLe a 20. The method of claim 19, wherein the vector expresses (Item 21) 20. The method of claim 19, wherein the cancer or tumor is selected from the group consisting of tumors of the gastrointestinal tract, colon cancer, colorectal adenocarcinoma, metastatic colon cancer, colorectal cancer, pancreatic cancer, pancreatic adenocarcinoma, small cell lung cancer, bladder adenocarcinoma, ovarian signet ring cell carcinoma, ovarian cancer, metastatic carcinoma, adenocarcinoma of the stomach, adenocarcinoma of the esophagus, adenocarcinoma of the throat, adenocarcinoma of the genitourinary tract, and adenocarcinoma of the breast. (Item 22) 20. The method of item 19, further comprising the step of simultaneously or sequentially administering a second therapeutic agent. (Item 23) 23. The method of item 22, wherein the second therapeutic agent is a chemotherapeutic agent or an immunotherapeutic agent. (Item 24) 17. A method for detecting a tumor in a subject, comprising administering to a subject in need thereof an effective amount of the conjugate of item 16.

Claims

1. Sialyl-Lewis ａ 1. A polypeptide comprising an isolated antibody or antigen-binding fragment thereof that binds to a polypeptide of claim 1, wherein the antibody or polypeptide comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain has a VH CDR1, a VH CDR2, and a VH CDR3 amino acid sequence, and the VL domain has a VL CDR1, a VL CDR2, and a VL CDR3 amino acid sequence; the VH CDR1 amino acid sequence is residues 45-52 of SEQ ID NO:2; the VH CDR2 amino acid sequence is residues 70-77 of SEQ ID NO:2; the VH CDR3 amino acid sequence is residues 116-131 of SEQ ID NO:2; the VL CDR1 amino acid sequence is residues 45-52 of SEQ ID NO:4; the VL CDR2 amino acid sequence is residues 70-72 of SEQ ID NO:4; An isolated antibody or polypeptide wherein said VL CDR3 amino acid sequence is residues 109-120 of SEQ ID NO:

4.

2. The isolated antibody or polypeptide of claim 1, wherein the antibody is a human antibody.

3. The polypeptide comprising the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') ２ 3. The isolated antibody or polypeptide of claim 1 or 2, which is selected from the group consisting of: an scFv, a diabody, a triabody and a minibody.

4. 4. The isolated antibody or polypeptide of claim 3, wherein the polypeptide comprising the antigen-binding fragment is a diabody.

5. 5. The isolated antibody or polypeptide of any one of claims 1 to 4, wherein the antibody is a monoclonal antibody.

6. 6. The isolated antibody or polypeptide of any one of claims 1 to 5, wherein the antibody is an IgG or IgM isotype.

7. 7. The isolated antibody or polypeptide of claim 6, wherein the IgG antibody is of the IgG1 subclass.

8. An isolated polynucleotide encoding the antibody or polypeptide of any one of claims 1 to 7.

9. The isolated polynucleotide of claim 8, wherein the VH domain of the antibody or polypeptide is encoded by the nucleic acid sequence shown in SEQ ID NO: 1, and the VL domain of the antibody or polypeptide is encoded by the nucleic acid sequence shown in SEQ ID NO:

3.

10. A conjugate comprising the isolated antibody or polypeptide of any one of claims 1 to 7 conjugated or recombinantly fused to a diagnostic, detectable or therapeutic agent.

11. The conjugate of claim 10 comprising a detectable agent.

12. The conjugate of claim 11 , wherein the detectable agent is a radioactive material.

13. The radioactive material is zirconium ( ８９ Zr), iodine ( １３１ I, １２５ I, １２４ I, １２３ I, and １２１ I), carbon ( １４ C. １１ C), sulfur ( ３５ S), tritium ( ３ H), indium ( １１５ In, １１３ In, １１２ In, and １１１ In), technetium ( ９９ Tc), thallium ( ２０１ Ti), gallium ( ６８ Ga, ６７ Ga), palladium ( １０３ Pd), molybdenum ( ９９ Mo), xenon ( １３３ Xe), fluorine ( １８ F), １５ O. １３ N. ６４ Cu, ９４ｍ Tc, １５３ Sm, １７７ Lu, １５９ Gd, １４９ Pm, １４０ La, １７５ Yb, １６６ Ho, ８６ Y. ９０ Y. ４７ Sc, １８６ Re, １８８ Re, １４２ Pr, １０５ Rh, ９７ Ru, ６８ Ge, ５７ Co, ６５ Zn, ８５ Sr, ３２ P. １５３ Gd, １６９ Yb, ５１ Cr, ５４ Mn, ７５ Se, １１３ Sn, and １１７ 13. The conjugate of claim 12, wherein the conjugate is selected from the group consisting of Sn.

14. The conjugate of claim 11 , wherein the detectable agent is a fluorescent material.

15. 15. The conjugate of claim 14, wherein the fluorescent material is selected from the group consisting of umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin.

16. The conjugate of claim 10 comprising a therapeutic agent.

17. 17. The conjugate of claim 16, wherein the therapeutic agent is a radioactive metal.

18. 18. The conjugate of claim 17, wherein the radioactive metal is an alpha-emitter.

19. 17. The conjugate of claim 16, wherein the therapeutic agent is an auristatin molecule.

20. 20. The conjugate of claim 19, wherein the auristatin molecule is selected from the group consisting of auristatin PHE, bryostatin 1, solastatin 10, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

21. A pharmaceutical composition comprising an antibody or polypeptide described in any one of claims 1 to 7, a conjugate described in any one of claims 10 to 11, or an isolated polynucleotide described in claim 8 or 9, and a pharmaceutically acceptable carrier.

22. 22. Use of a therapeutically effective amount of the pharmaceutical composition of claim 21 for the manufacture of a medicament for treating a disease in a subject in need thereof, wherein the disease is cancer or tumorigenesis, and the cells of the cancer or tumor are sLe. ａ and the cancer or tumor is selected from the group consisting of colon cancer, colorectal adenocarcinoma, metastatic colon cancer, colorectal cancer, pancreatic cancer, pancreatic adenocarcinoma, and small cell lung cancer.

23. 23. The use according to claim 22, wherein the medicament is adapted for simultaneous or sequential administration with a second therapeutic agent.

24. 24. The use of claim 23, wherein the second therapeutic agent is a chemotherapeutic agent or an immunotherapeutic agent.

25. Use of an effective amount of the conjugate of any one of claims 10 to 15 for the manufacture of a medicament for detecting a tumor in a subject, wherein the tumor in the subject is sLe. ａ and the cancer is selected from the group consisting of colon cancer, colorectal adenocarcinoma, metastatic colon cancer, colorectal cancer, pancreatic cancer, pancreatic adenocarcinoma, and small cell lung cancer.

26. 22. The pharmaceutical composition of claim 21 for treating a disease in a subject in need thereof, wherein the disease is cancer or tumorigenesis, and the cells of the cancer or tumor are sLe. ａ and the cancer or tumor is selected from the group consisting of colon cancer, colorectal adenocarcinoma, metastatic colon cancer, colorectal cancer, pancreatic cancer, pancreatic adenocarcinoma, and small cell lung cancer.

27. 27. The pharmaceutical composition of claim 26, wherein a second therapeutic agent is administered simultaneously or sequentially with the pharmaceutical composition.

28. 28. The pharmaceutical composition of claim 27, wherein the second therapeutic agent is a chemotherapeutic agent or an immunotherapeutic agent.

29. 16. A composition for detecting a tumor in a subject, comprising the conjugate of any one of claims 10 to 15, wherein the tumor in the subject is sLe. ａ and wherein the cancer is selected from the group consisting of colon cancer, colorectal adenocarcinoma, metastatic colon cancer, colorectal cancer, pancreatic cancer, pancreatic adenocarcinoma, and small cell lung cancer.

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