Nucleic acids encoding human antibodies against sialyl Lewis a antigen

Abstract

This application relates to nucleic acids encoding human antibodies against sialyl Lewis a antigen. The present invention provides compositions for generating antibodies or functional fragments thereof against sialyl Lewis a antigen (sLe<supgt;a< / supgt;). The compositions of the present invention include polynucleotides encoding the heavy chain and / or light chain variable domains that bind to sLe<supgt;a< / supgt>. The present invention also provides isolated antibodies or functional fragments thereof and methods for treating or preventing diseases such as cancer or tumor formation, wherein the antibodies or functional fragments include heavy chain variable domains and light chain variable domains having the amino acid sequences provided herein. The present invention also provides conjugates of antibodies or functional fragments thereof conjugated or recombinantly fused with diagnostic reagents, detectable reagents or therapeutic agents, and methods for treating, preventing or diagnosing diseases in a subject in need thereof.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201480057851.8. The original application is the PCT international application PCT / US2014 / 052631 filed on August 26, 2014, which entered the Chinese national phase on April 21, 2016.

[0002] This patent application claims priority to U.S. Provisional Patent Application Serial No. 61 / 870,137, filed August 26, 2013, the entire contents of which are incorporated herein by reference.

[0003] This invention was made with government support under Grant No. CA-128362 awarded by the National Cancer Institute of the NIH. The government has certain rights in this invention. Technical Field

[0004] The present application relates to anti-sialyl-Lewis a antigen (Sialyl-Lewis a ,sLe a ) antibodies. Background Art

[0005] The present invention generally relates to anti-sialyl-Lewis a antigen (Sialyl-Lewis a ,sLe a ) antibodies, and more particularly, to antibodies encoding anti-sLe a Antibody polynucleotides and corresponding encoded antibodies or fragments thereof.

[0006] Passive administration of antibodies 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 against tumor-specific carbohydrates may be useful candidates in such cancer treatments. For example, a variety of tumor-restricted monoclonal antibodies generated by immunizing mice with human cancer cells have been shown to be directed against carbohydrate antigens expressed as glycolipids or glycoproteins on the cell surface. a It has been shown to be expressed on tumors of the gastrointestinal tract. a Expression of α-glucanol (α-glucan) influences and is associated with increased metastatic potential in human colon and pancreatic adenocarcinomas. However, the chemistry of carbohydrates is quite challenging, and the clinical development of antibodies that recognize these tumor-specific carbohydrates has been slow.

[0007] Pancreatic cancer is one of the most aggressive adenocarcinomas and is often associated with a poor prognosis. Pancreatic cancer ranks fourth among the leading causes of cancer death. Despite advances in screening for different cancers, the reliability of detecting malignant lesions arising from the pancreas remains poor. Fluorodeoxyglucose positron emission tomography (FDG-PET) has been shown to detect and grade pancreatic cancer. However, FDG-PET is not sensitive for pancreatitis differentiated from malignant tumors and has problems in grading small primary lesions (<7 mm) and liver metastases (<1 cm). One diagnostic screening method for monitoring the status of patients with pancreatic ductal adenocarcinoma (PDAC) involves the detection of circulating sLe in the serum. a Increased levels of antigen. Circulating sLe a Patients with antigen levels >37 U / ml showed recurrence of the cancer. However, the development of alternative diagnostic tools using these tumor-specific carbohydrates has been slow.

[0008] Therefore, it is necessary to identify and generate proteins that specifically recognize tumor-specific carbohydrates (such as sLe a The present invention satisfies this need and provides related advantages. Summary of the Invention

[0009] According to the present invention, there is provided a method for producing a sLe a The present invention also provides a composition of an antibody or functional fragment thereof. The composition comprises an isolated polynucleotide encoding an antibody or functional fragment thereof, wherein the antibody comprises a heavy chain (VH) variable domain having an amino acid sequence as provided herein. The isolated polynucleotide of the present invention may also comprise a nucleic acid sequence as provided herein, wherein the nucleic acid sequence encodes a VH domain of the antibody or functional fragment thereof.

[0010] In another embodiment of the present invention, the polynucleotide of described separation can encode antibody or its functional fragment, wherein said antibody comprises the light chain (VL) variable domain with the aminoacid sequence that this paper provides.The polynucleotide of separation of the present invention can also comprise the nucleotide sequence that this paper provides, wherein said nucleotide sequence encodes the VL domain of antibody or its functional fragment.

[0011] The composition of the present invention further comprises an isolated antibody or a functional fragment thereof, wherein the antibody binds to sLe a In some embodiments, the present invention provides a method for binding to sLe a The present invention provides an isolated antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises a VH domain having an amino acid sequence provided herein.

[0012] In some embodiments, the present invention provides a method for binding to sLe aThe present invention provides an isolated antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises a VL domain having an amino acid sequence provided herein.

[0013] In some embodiments, the present invention provides a method for binding to sLe a The present invention provides an isolated antibody or functional fragment thereof, wherein the antibody or functional fragment thereof comprises both a VH domain and a VL domain, wherein the VH domain and VL domain respectively comprise the amino acid sequences of the respective VH and VL domains of the clonal isolate provided herein.

[0014] In some embodiments, the present invention provides conjugates of antibodies or functional fragments provided herein that are conjugated or recombinantly fused to diagnostic agents, detectable agents, or therapeutic agents. In some aspects of the invention, the conjugates of the present invention comprising a detectable agent can be used in methods for detecting and / or diagnosing tumor formation in a subject. These methods may include administering an effective amount of the conjugate to a subject in need thereof.

[0015] In some embodiments, the present invention provides a pharmaceutical composition 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 a method for treating or preventing a disease in a subject in need thereof by administering a therapeutically effective amount of the pharmaceutical composition of the present invention. In another aspect, the present invention provides a second therapeutic agent administered simultaneously or sequentially with the antibody or functional fragment of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The nucleotide sequence and encoded amino acid sequence of the variable heavy chain (VH) domain of clone 5B1, along with a leader sequence that can be used for recombinant expression, are shown. The upper portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2. The three complementarity determining regions (CDR1, CDR2, and CDR3) are also indicated.

[0017] Figure 2 The nucleotide sequence and encoded amino acid sequence of the variable light chain (VL) domain of clone 5B1, along with a leader sequence that can be used for recombinant expression, are shown. The upper portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4. The three complementarity determining regions (CDR1, CDR2, and CDR3) are also indicated.

[0018] Figure 3The nucleotide sequence and encoded amino acid sequence of the variable heavy chain (VH) domain of clone 9H3, along with a leader sequence capable of recombinant expression, are shown. The upper portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 5 and the amino acid sequence of SEQ ID NO: 6. The three complementarity determining regions (CDR1, CDR2, and CDR3) are also indicated.

[0019] Figure 4 The nucleotide sequence and encoded amino acid sequence of the variable light chain (VL) domain of clone 9H3, along with a leader sequence that can be used for recombinant expression, are shown. The upper portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 8. The three complementarity determining regions (CDR1, CDR2, and CDR3) are also indicated.

[0020] Figure 5 The nucleotide sequence and encoded amino acid sequence of the variable heavy chain (VH) domain of clone 5H11, along with a leader sequence that can be used for recombinant expression, are shown. The upper portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10. The three complementarity determining regions (CDR1, CDR2, and CDR3) are also indicated.

[0021] Figure 6 The nucleotide sequence and encoded amino acid sequence of the variable light chain (VL) domain of clone 5H11, along with a leader sequence that can be used for recombinant expression, are shown. The upper portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 12. The three complementarity determining regions (CDR1, CDR2, and CDR3) are also indicated.

[0022] Figure 7 The nucleotide sequence and encoded amino acid sequence of the variable heavy chain (VH) domain of clone 7E3, as well as a leader sequence capable of recombinant expression, are shown. The upper portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 14. The three complementarity determining regions (CDR1, CDR2, and CDR3) are also indicated.

[0023] Figure 8 The nucleotide sequence and encoded amino acid sequence of the variable light chain (VL) domain of clone 7E3, along with a leader sequence that can be used for recombinant expression, are shown. The upper portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16. The three complementarity determining regions (CDR1, CDR2, and CDR3) are also indicated.

[0024] Figure 9The nucleotide sequence and encoded amino acid sequence of a diabody denoted 5B1CysDb are shown, which has the CDR1, CDR2, and CDR3 of both the variable heavy (VH) and variable light (VL) domains of clone 5B1. The upper portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO: 18. The three complementarity determining regions (CDR1, CDR2, and CDR3) of the VH and VL domains are also indicated in bold and underlined text. The linker sequence with added amino acids and the polyhistidine tag (Poly His-tag) are also indicated in italic and underlined text.

[0025] Figure 10 The nucleotide sequence and encoded amino acid sequence of a diabody, designated 7E3CysDb, is shown, which has the CDR1, CDR2, and CDR3 of both the variable heavy (VH) and variable light (VL) domains of clone 7E3. The upper portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO: 20. The three complementarity determining regions (CDR1, CDR2, and CDR3) of the VH and VL domains are also indicated in bold and underlined text. The linker sequence with added amino acids and the polyhistidine tag (Poly His-tag) are also indicated in italic and underlined text.

[0026] Figure 11, Parts AE, shows human anti-sLe analyzed by flow cytometry a Antibody Binding to Tumor Cells. Part A shows DMS-79 cells stained with recombinant (r) 5B1, 9H3, 5H11, and 7E3 antibodies. Parts BF show HT29, BxPC3, SW626, SK-MEL28, and Colo205-luc cells stained with 1-2 μg / mL r5B1 or r7E3 plus IgG or IgM-specific secondary antibodies, respectively, as described in Example 1.

[0027] Figure 12 , Parts A and B show the CDC activity of r5B1 and r7E3 antibodies compared to murine 121SLE (IgM) in the presence of human complement (Hu C') as measured on DMS-79 cells. Human isotype control antibodies, Hu IgG (◇) and Hu IgM (◆) showed <4% cytotoxicity. Dose responses for r5B1 IgG (■), r7E3 IgM (●), and 121SLE mIgM (▲) antibodies are shown in Part A. Calculated EC50 (μg / mL) for r5B1 (IgG), r7E3 (IgM), and 121SLE (mIgM) antibodies are shown in Part B.

[0028] Figure 13 , Parts AC show the antibody-dependent cell-mediated cytotoxicity (ADCC) of the r5B1 antibody. Part A shows r5B1-mediated ADCC by human PBMCs against DMS-79 cells. PBMCs were tested using DMS-79 tumor cells at an E:T ratio of 100:1 to 12.5:1 in the presence or absence of 2 μg / mL r5B1. Part B shows r5B1-mediated ADCC by primary human NK cells against DMS-79 cells. NK cells were tested using DMS-79 tumor cells at a lower E:T ratio of 5:1 to 0.6:1 in the presence or absence of 2 μg / mL r5B1. Part C shows ADCC of r5B1 at multiple concentrations using DMS-79 tumor cells at an E:T ratio of 1:100 using PBMCs from 2 donors in the presence or absence of specified concentrations of r5B1.

[0029] Figure 14 Shows sLe a Internalization into BxPC3 cells. In the presence of r5B1 (anti-sLe a BxPC3 pancreatic tumor cells were grown in the presence of either r1B7 (anti-GD2) or r1B7 antibodies. After 3 days, cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and sample values ​​were normalized to those of untreated cultures.

[0030] Figure 15 Figure 2 shows the activity of the r5B1 antibody in a xenograft model using Colo205-luc cells. On day 0, severe combined immunodeficient (SCID) mice (5 per group) received 500,000 Colo205-luc cells via tail vein injection. On days 1, 7, 14, and 21 (Experiment 1, Exp1) or on days 1, 4, 7, 10, 14, and 21 (Experiment 2, Exp2), mice received 100 μg of r5B1 via intraperitoneal injection for a total dose of 600 μg. Control animals received a mock injection of PBS.

[0031] Figure 16 The effect of r5B1 on Colo205-luc tumors in SCID mice is shown. As described in Example 1, mice received 100 μg of r5B1 per injection. , 300 μg (■) or 1 mg (◆) of r5b1 antibody. Control (■) animals received a mock injection of PBS.

[0032] Figure 17Fluorescence imaging of r5B1-treated Colo205-luc tumor-bearing mice, with 5 mice per group, at day 0 and week 5 are shown. Figure 16 The treatment regimen shown in and described in Example 1.

[0033] Figure 18, parts A and B, shows anti-tumor activity in a therapeutic subcutaneous xenograft model using DMS-79 cells. Part A shows the anti-tumor activity of 5B1-treated mice (5B1 alone (▲) or 5B1 + cRGD (●)) compared to human IgG (IgG alone (◆) or IgG + cRGD (●)) and PBS-injected controls (■). ) inhibition or regression. Arrows indicate the days of antibody or PBS injection. Part B shows representative images of treated mice. Arrows indicate the absence of any visible tumors.

[0034] Figure 19 , Parts A to F, show binding of 5B1 to various tumor types. Part A is a pancreatic ductal adenocarcinoma, a stage III tumor. Part B is a sigmoid colon cancer, a stage IIIB tumor. Part C is a lung adenocarcinoma, a stage IB tumor. Part D is a mucinous bladder adenocarcinoma, a stage IV tumor. Part E is an ovarian metastasis from a colon tumor. Part F is a lymph node metastasis, a stage IIIA tumor.

[0035] Figure 20 It shows that by 89 Zr radiolabeled 5B1 antibody ( 89 Serial PET maximum intensity projection (MIP) images acquired from 2 to 120 hours after Zr-5B1 was intravenously administered to female SCID mice bearing subcutaneous BxPC3 pancreatic tumors. PET-MIP imaging demonstrated high tumor uptake and clearance of nonspecifically bound tracer as early as 24 hours post-injection (h pi).

[0036] Figure 21 Shown with [[ID=…]]Figure 20 The biodistribution results were consistent with the PET data from the RT-PCR assay, with an observed tumor uptake of 84.73 ± 12.28% ID / g. Due to the small tumor weight, the inset shows the tumor uptake plot expressed as % ID relative to time. Tumor % ID is shown at all time points. 89 Zr-5B1 showed significant tumor uptake, and it was better than nonspecific 89 Zr-IgG was at least 7-fold higher.Competitive inhibition using cold 5B1 (200 μg) showed reduced tumor accumulation.

[0037] Figure 22, Parts AC, show PET-MIP images of mice with DMS79 (Part A) and Colo205-luc xenografts (Part B). 89 Schematic diagram of PET-MIP imaging of the tumor (T), heart (H), and liver (L) of Zr-5B1. Colorectal Colo205-luc xenograft model showing the appearance of 89 Zr-5B1 accumulated to a peak, which eventually decreased while showing an increase in nonspecific binding to the liver (Part C).

[0038] Figure 23 Figure 2 shows 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 5B1 treatment. Co-administration of the 5B1 antibody and Taxol significantly limited tumor growth and resulted in tumor regression compared to control human IgG (HuIgG) or 5B1 antibody and Taxol administered alone. Significant differences from control by two-way ANOVA are shown, p < 0.01 (**) and p < 0.001 (***). N = 5.

[0039] [[ID=…]]Figure 24 Shown is the inhibition of tumor growth in the BxPc3 pancreatic cancer xenograft model treated with the 5B1 antibody and Taxol (paclitaxel) continuous co-administration. The large arrow on the X-axis represents the Taxol+5B1 treatment, while the small arrow represents the 5B1 treatment alone. Compared with the control (PBS-Ctrl; human IgG-HuIgG) or the 5B1 antibody and Taxol administered alone, the co-administration of the 5B1 antibody and Taxol significantly limited tumor growth.

[0040] Figure 25 , Parts A and B, show representative images of mice implanted with orthotopically implanted BxPC3-luc pancreatic tumor xenografts. Part A: FDG-PET and computed tomography (CT) co-registration (left) and a planar section of FDG-PET alone (right) show minimal tumor detection of the tracer with high uptake in highly metabolic tissues (i.e., heart, H and bladder, B). Part B: Acquisition of the same mouse co-registered with CT 89 Zr radiolabeled-5B1 antibody ( 89 Zr-5B1) PET images showed superior tumor detection in BxPC3-luc tumor xenografts. DETAILED DESCRIPTION

[0041] Carbohydrates expressed on the surface of tumor cells can be targets for passive immunotherapy. The compositions provided herein are based, at least in part, on the production of sialyl-Lewis oligosaccharides α-keyhole limpet hemocyanin (sLe aIdentification and characterization of human antibodies produced by blood lymphocytes of individuals immunized with a sLe-KLH) conjugate vaccine. a Antibodies with high affinity (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 effective in complement dependent cytotoxicity (CDC) assays, and the 5B1 antibody was also highly active in antibody-dependent cytotoxicity assays. The in vivo efficacy of the antibodies was tested in two xenograft models using either Colo205 tumor cells or DMS-79 tumor cells transplanted into severe combined immunodeficiency (SCID) mice. The translational relevance of the invention provided herein is 2-fold: First, the methods provided herein show that sLe a The antibody response elicited by the KLH vaccine is useful as a vaccine itself. Second, the most effective antibodies generated in clinical trials can be preserved and ultimately used as therapeutic agents for the target cancer population or in the production of such therapeutic agents. The high avidity of the antibodies provided herein and their high effector function support this translational potential.

[0042] As used herein, the term "antibody" is intended to refer to polypeptide products of B cells within the immunoglobulin class of polypeptides that are capable of binding to specific molecular antigens and comprised of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), and each amino-terminal portion of each chain comprises a variable region of about 100 to about 130 or more amino acids and each carboxyl-terminal portion of each chain comprises a constant region (see Borrebaeck (ed.) (1995) Antibody Engineering , 2nd edition, Oxford University Press.; Kuby (1997) Immunology , 3rd edition, WH Freeman and Company, New York). In the context of the present invention, specific molecular antigens that can be bound by the antibodies of the present invention include target carbohydrates sLe a .

[0043] The term "human" when used to refer to an antibody or a functional fragment thereof refers to an antibody or a functional fragment thereof having a human variable region and / or a human constant region or portions thereof corresponding to human germline immunoglobulin sequences. Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 describes these human germline immunoglobulin sequences. In the context of the present invention, human antibodies may include those that bind to sLe a And the antibody encoded by the nucleic acid sequence that is a naturally occurring somatic variant of the human germline immunoglobulin nucleic acid sequence.Example 1 provides an exemplary method for producing human antibodies, but any method known to those skilled in the art can be used.

[0044] The term "monoclonal antibody" refers to an antibody that is a product of a single cell clone or hybridoma or a cell colony derived from a single cell. Monoclonal antibodies are also intended to represent antibodies produced by recombinant methods from heavy and light chain encoding immunoglobulin genes that produce a single molecule immunoglobulin substance. The amino acid sequence of the antibody in a monoclonal antibody preparation is substantially uniform, and the binding activity of the antibody in the preparation shows substantially the same antigen-binding activity. In contrast, polyclonal antibodies are derived from different B cells in a colony, and are a combination of immunoglobulin molecules that bind to specific antigens. Each immunoglobulin of a polyclonal antibody can bind to different epitopes of the same antigen. Methods for producing 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 Enaineering:A Practical Guide , WH Freeman and Co., Publishers, New York, pp. 103-120 (1991)).

[0045] As used herein, when used to refer to an antibody, the term "functional fragment" is intended to mean a portion of an antibody comprising a heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. These functional fragments may 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 may include, for example, heavy or light chain polypeptides, variable region polypeptides, or CDR polypeptides, or portions thereof, as long as these functional fragments retain binding activity. For example, in Harlow and Lane, Antibodies:A Laboratory Manual,C0ld 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); Plückthun and Skerra, Meth. Enzymol., 178: 497-515 (1989); and Day, ED, Advanced Immunochemistry A description of these antibody binding fragments is found in Antibody Binding Fragments, 2nd ed., Wiley-Liss, Inc., New York, NY (1990).

[0046] When used to refer to an antibody, the term "heavy chain" refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids and the carboxyl-terminal portion contains a constant region. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types called α, δ, ε, γ and μ. Different heavy chains have different sizes: α, δ and γ contain about 450 amino acids, while μ and ε contain about 550 amino acids. When combined with a light chain, these different types of heavy chains give rise to the five well-known classes of antibodies, IgA, IgD, IgE, IgG and IgM, respectively, including the four subclasses of IgG, namely IgG1, IgG2, IgG3 and IgG4. The heavy chain can be a human heavy chain.

[0047] When used to refer to an antibody, the term "light chain" refers to a polypeptide chain of about 25 kDa, wherein the amino terminal portion comprises a variable region of about 100 to about 110 or more amino acids and the carboxyl terminal portion comprises a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, referred to as kappa and lambda. Light chain amino acid sequences are well known in the art. The light chain can be a human light chain.

[0048] The term "variable domain" or "variable region" refers to the portion of an antibody's light or heavy chain that is typically located at the amino terminus of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and is used in the binding or specificity of each particular antibody for its particular antigen. The variable domains vary widely in sequence between different antibodies. The degree of sequence variability is concentrated in the CDRs, while the less variable portions of the variable domain are called framework regions (FRs). The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with the antigen. Amino acid positions as used herein are numbered according to the EU index, as in Kabat et al. (1991). Sequences of proteins ofimmunological interest (USDepartment of Health and Human Services, Washington, DC) 5th Edition. The variable region may be a human variable region.

[0049] CDR refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the VH β-sheet framework of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of the VL β-sheet framework of an antibody. Thus, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the most highly variable regions within the variable (V) domain of an antibody (Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat, Adv. Prot. Chem. 32: 1-75 (1978)). Chothia also structurally defined CDR region sequences as those residues that are not part of the conserved β-sheet framework and are therefore able to fit into different conformations (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). Both terms are well known in the art. The CDR positions within canonical antibody variable domains were determined by comparing multiple structures (Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); Morea et al., Methods 20:267-279 (2000)). Since the number of residues within the hypervariable region varies among different antibodies, other residues are generally numbered with a, b, c, etc., adjacent to the residue number in the canonical variable domain numbering scheme relative to the canonical position (Al-Lazikani et al., supra (1997)). This nomenclature is similarly well-known to those skilled in the art.

[0050] For example, defined CDRs according to Kabat (hypervariable) or Chothia (structural) nomenclature are shown in Table 1 below.

[0051] Table 1: CDR Definition

[0052]

[0053] 1 Residue numbering according to the nomenclature of Kabat et al. (supra)

[0054] 2 Residue numbering according to the nomenclature of Chothia et al. (supra)

[0055] One or more CDRs can also be covalently or non-covalently introduced into a molecule to make it an immunoadhesin. An immunoadhesin can incorporate a CDR as part of a larger polypeptide chain, can be covalently linked to another polypeptide chain, or can be non-covalently introduced. The CDR enables the immunoadhesin to bind to a specific antigen of interest.

[0056] As used herein, when used to represent an antibody, antibody functional fragment or polynucleotide, the term "isolated" is intended to mean that the molecule mentioned does not contain at least one component that exists in nature. The term comprises antibodies, antibody functional fragments or polynucleotides removed from some or all other components present in their natural environment. The components of the antibody natural environment include, for example, red blood cells, white blood cells, platelets, plasma, proteins, nucleic acids, salts and nutrients. The components of the natural environment of the antibody functional fragment or polynucleotide include, for example, lipid membranes, organelles, proteins, nucleic acids, salts and nutrients. The antibodies, antibody functional fragments or polynucleotides of the present invention can also be free of or completely to substantially free of all these components or any other components of the cell from which they are separated or recombinantly produced.

[0057] As used herein, "isotype" refers to the antibody species encoded by the heavy chain constant region gene. The heavy chain of a given antibody or functional fragment determines the species of the antibody or functional fragment: IgM, IgG, IgA, IgD or IgE. Each class can have a kappa or lambda light chain. The term "subclass" refers to the lesser differences in the heavy chain amino acid sequence that distinguish subclasses. In humans, there are two IgA subclasses (subclass IgA1 and IgA2) and four IgG subclasses (subclass IgG1, IgG2, IgG3 and IgG4). These species and subclasses are well known to those skilled in the art.

[0058] As used herein, the term "binding" refers to the interaction between molecules to form a complex. The interaction can be, for example, a non-covalent interaction, including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. The complex can also include two or more molecules being bound together by covalent or non-covalent bonds, interactions, or forces. The binding of the antibody or its functional fragment can be detected using, for example, an enzyme-linked immunosorbent assay (method provided in Example 1) or any of several methods well known to those skilled in the art.

[0059] Single antigen binding site on antibody or functional fragment and target molecule (such as sLe a The strength of the total non-covalent interaction between the single epitopes of the antibody or functional fragment is the affinity of the antibody or functional fragment for the epitope. The binding (k1) and dissociation (k -1 ) ratio (k1 / k -1) is the binding constant, which is a measure of affinity. For different complexes of antibody or functional fragment and antigen, the K value is different and depends on k1 and k -1 The binding constant K of the antibodies or functional fragments of the present invention can be determined using any of the methods provided herein or any other method known to those skilled in the art.

[0060] The affinity of a binding site does not always reflect the true strength of the interaction between the antibody or functional fragment and the antigen. a ) When an antibody containing multiple binding sites comes into contact, the interaction of the antibody or functional fragment with the antigen at one site will increase the likelihood of a reaction at a second site. The strength of these multiple interactions between the multivalent antibody and the antigen is called avidity. The avidity of an antibody or functional fragment can be a better measure of its binding ability than the avidity of its individual binding sites. For example, high affinity can compensate for low affinity, as sometimes found for pentameric IgM antibodies, which may have lower affinity than IgG, but the high affinity of the IgM generated by its multivalency enables it to effectively bind to the antigen.

[0061] The specificity of an antibody or its functional fragment refers to the ability of each antibody or its functional fragment to react with only one antigen. An antibody or functional fragment is considered specific when it can distinguish differences in the primary, secondary or tertiary structure of the antigen or in isomeric forms of the antigen.

[0062] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, which can be deoxyribonucleotides or ribonucleotides or their analogs. A polynucleotide sequence is composed of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) that replaces thymine when the polynucleotide is RNA. Therefore, the term "nucleotide sequence" or "nucleic acid sequence" is an alphabetic 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. Polynucleotides also refer to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the polynucleotides of the present invention encompasses each of the double-stranded form and the two complementary single-stranded forms known or predicted to constitute the double-stranded form. It should be understood that the isolated polynucleotides and nucleic acids described herein refer to non-naturally occurring polynucleotides and nucleic acids. Non-naturally occurring polynucleotides and nucleic acids can include, but are not limited to, cDNA and chemically synthesized molecules.

[0063] When used in reference to a polynucleotide, the term "encoding" or its grammatical equivalents refers to a polynucleotide in its native state or a polynucleotide that, when manipulated by methods known to those skilled in the art, can be transcribed to produce mRNA and then translated into a polypeptide and / or fragment thereof. The antisense strand is the complementary strand of the polynucleotide, and the coding sequence can be deduced therefrom.

[0064] The phrase "therapeutic agent" refers to a therapeutic agent that can be used in combination with sLe a Any agent used in the treatment, control or improvement of diseases related to the expression of sLe and / or symptoms associated therewith. In certain embodiments, the therapeutic agent refers to the antibody or functional fragment of the present invention. In other embodiments, the therapeutic agent refers to an agent other than the antibody or functional fragment of the present invention. The therapeutic agent can be for a The invention relates to an agent that is known to be useful for the treatment, control or improvement of a disease related to the expression of β-lactamase and / or one or more symptoms related thereto or that has been or is currently used for such treatment, control or improvement.

[0065] The phrase "diagnostic agent" refers to a substance administered to a subject that aids in the diagnosis of a disease. These substances can be used to reveal, pinpoint, and / or localize the process that leads to the disease. In certain embodiments, the diagnostic agent comprises a substance conjugated to an antibody or functional fragment of the present invention that, when administered to a subject or in contact with a sample from a subject, aids in the diagnosis of cancer or tumor formation.

[0066] The phrase "detectable agent" refers to a substance that can be used to determine the presence of a desired molecule in a sample or subject, such as an antibody or functional fragment of the present invention. A detectable agent can be a substance that can be imaged or a substance that can be determined and / or measured (e.g., by quantification).

[0067] An "effective amount" is an amount sufficient to cause a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages. Such delivery depends on a number of variables, including the time period over which a single dosage unit is to be used, the bioavailability of the agent, the route of administration, and the like.

[0068] As used herein, the phrase "therapeutically effective amount" 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 improve the severity and / or duration of a given cause of disease and / or symptoms associated therewith. A therapeutically effective amount of a therapeutic agent can be an amount required to slow down or improve the progression or development of a given disease, reduce or improve the recurrence, development or occurrence of a given disease, and / or improve or enhance the preventive or therapeutic effect of another therapy (e.g., a therapy other than administering an antibody or functional fragment provided herein).

[0069] Compound "Sialyl-Lewis a antigen (Sialyl-Lewis a )”(sLe a ), also known as sialylated Le a , Sialyl-Lewis A, Sialyl-Lewis a, and CA 19.9, which is a tetrasaccharide with the molecular formula C 31 H 52 N2O 23 , the molar mass is 820.74g / mol. a The structure of sLe can include Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ and Neu5Gcα2-3Galβ1-3(Fucα1-4)GlcNAcβ. a It is widely expressed on gastrointestinal tumors and is used as a tumor marker in pancreatic and colon cancer. a It is also a known ligand for E-selectin, also known as endothelial leukocyte adhesion molecule (ELAM).

[0070] In some embodiments, the present invention provides an isolated polynucleotide encoding an antibody heavy chain or light chain or a functional fragment thereof, wherein the polynucleotide is used to bind to the sLe aThe present invention provides an isolated polynucleotide encoding an antibody heavy chain or light chain comprising an antibody or a functional fragment thereof. Therefore, in some embodiments, the present invention provides an isolated polynucleotide encoding an antibody or a functional fragment thereof, wherein the antibody comprises a VH domain having an amino acid sequence selected from the group consisting of residues 20-142 of SEQ ID NO: 2, residues 20-142 of SEQ ID NO: 6, residues 20-142 of SEQ ID NO: 10, and residues 20-145 of SEQ ID NO: 14. The isolated polynucleotide of the present invention may also comprise a nucleic acid sequence of residues 58-426 of SEQ ID NO: 1, residues 58-426 of SEQ ID NO: 5, residues 58-426 of SEQ ID NO: 9, or residues 58-435 of SEQ ID NO: 13, wherein the nucleic acid sequence encodes the VH domain of the antibody or a functional fragment thereof.

[0071] In another embodiment of the present invention, the isolated polynucleotide may encode an antibody or a functional fragment thereof, wherein the antibody comprises a VL domain having an amino acid sequence selected from the group consisting of residues 20-130 of SEQ ID NO: 4, residues 20-129 of SEQ ID NO: 8, residues 20-130 of SEQ ID NO: 12, and residues 23-130 of SEQ ID NO: 16. The isolated polynucleotide of the present invention may also comprise a 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 the VL domain of the antibody or a functional fragment thereof.

[0072] In another embodiment, the present invention provides an isolated polynucleotide encoding an antibody heavy chain or light chain or a functional fragment thereof, wherein the antibody heavy chain or light chain or a functional fragment thereof encoded by the polynucleotide of the present invention has Figures 1-8 One or more complementarity determining regions (CDRs) as shown in or listed in Table 2. Antibodies or functional fragments thereof comprising one or more CDRs can specifically bind to sLe as described herein. a . With sLe aSpecific binding can include specificity, affinity and / or avidity, as provided in Example 1 for any of the antibodies provided herein. In another aspect, the antibodies encoded by the polynucleotides of the present invention or their functional fragments can include complement dependent cytotoxicity (CDC) activity and / or antibody dependent cell-mediated cytotoxicity (ADCC) activity of any of the clonal isolates 5B1, 9H3, 5H11 or 7E3 described herein. Methods for evaluating the specificity, affinity and / or avidity of antibodies or their functional fragments are well known in the art and exemplary methods are provided herein.

[0073] Table 2: CDRs of cloned isolates

[0074]

[0075]

[0076] In some embodiments, the antibodies or functional fragments thereof of the present invention comprise less than 6 CDRs. In some embodiments, the antibodies or functional fragments thereof comprise 1, 2, 3, 4, or 5 CDRs selected from VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and / or VL CDR3. In specific embodiments, the antibodies or functional fragments thereof comprise 1, 2, 3, 4, or 5 CDRs selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of the clonal isolates 5B1, 9H3, 5H11, or 7E3 described herein.

[0077] In some embodiments, the present invention provides an isolated polynucleotide encoding an antibody or a functional fragment thereof, wherein the antibody or functional fragment comprises a variable heavy chain (VH) domain having the CDR1, CDR2 and CDR3 amino acid sequences of clonal isolates 5B1, 9H3, 5H11 or 7E3. These VH domains 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 aspect, the nucleotide sequences encoding CDR1, CDR2, and CDR3 of the VH domain can include 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, 346-402 of SEQ ID NO: 13.

[0078] In another embodiment, the present invention provides an isolated polynucleotide encoding an antibody or a functional fragment thereof, wherein the antibody comprises a variable light chain (VL) domain having the CDR1, CDR2 and CDR3 amino acid sequences of clonal isolates 5B1, 9H3, 5H11 or 7E3. These VL domains 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 aspect, the nucleotide sequences encoding CDR1, CDR2, and CDR3 of the VH domain can include 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.

[0079] In another embodiment, the present invention provides variants of the polynucleotides provided herein. When used to represent polynucleotides, variants include polynucleotides having one or more modified nucleotides (such as, but not limited to, methylated nucleotides or nucleotide analogs). In addition, variant polynucleotides may include polynucleotides interrupted by non-nucleotide components. Before or after polynucleotide assembly, polynucleotides can be modified using methods well known to those skilled in the art. For example, polynucleotides can be modified after polymerization using enzymatic or chemical techniques by conjugation with labeling components (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).

[0080] Polynucleotides can be obtained and their nucleotide sequences determined by any method known in the art. Since the amino acid sequences of the variable heavy and light chain domains of 5B1, 9H3, 5H11, and 7E3 are known (see, e.g., SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16), the nucleotide sequences encoding the antibodies and modified forms of these antibodies can be determined using methods known in the art, i.e., assembling nucleotide codons known to encode specific amino acids in a manner that produces nucleic acids encoding the antibodies. Such polynucleotides encoding the antibodies can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, Biotechniques 17: 242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, fragment, or variant thereof, annealing and ligation of those oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0081] Polynucleotides encoding the antibodies of the present invention or their functional fragments can be generated using nucleic acid sequences of the variable heavy and / or 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 thereof can be obtained from a suitable source (e.g., cDNA isolated from cells expressing the antibody or its functional fragment, such as hybridoma cells selected to express the antibody or its functional fragment) by chemical synthesis or PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for a specific nucleic acid sequence. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method known in the art.

[0082] In some embodiments, the present invention provides an isolated antibody or a functional fragment thereof, wherein the antibody binds to sLe a Thus, in some aspects, the present invention provides a method for binding to sLe a The invention relates to an isolated antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises 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.

[0083] In some embodiments, the present invention provides a method for binding to sLe aThe invention relates to an isolated antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises 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.

[0084] In some embodiments, the present invention provides a method for binding to sLe a The invention relates to an isolated antibody or a functional fragment thereof, 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 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.

[0085] In some embodiments, in order to combine sLe a The antibody or functional fragment thereof of the present invention has Figures 1-8 One or more CDRs shown in Table 2 or listed in Table 2. Antibodies or functional fragments thereof comprising one or more CDRs (particularly CDR3) can specifically bind to sLe as described herein. a . With sLe a Specific binding can include specificity and affinity, as provided for any of the antibodies provided herein in Example 1. In some aspects, the antibodies of the present invention or their functional fragments can include the CDC activity and / or ADCC activity of any of the clonal isolates 5B1, 9H3, 5H11 or 7E3 described herein.

[0086] In some embodiments, the present invention provides an isolated antibody or functional fragment thereof, wherein the antibody comprises a VH chain domain having the CDR1, CDR2, and CDR3 amino acid sequences of clonal isolates 5B1, 9H3, 5H11, or 7E3. These VH domains 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.

[0087] In some embodiments, the present invention provides an isolated antibody or functional fragment thereof, wherein the antibody comprises a VL chain domain having the CDR1, CDR2, and CDR3 amino acid sequences of clonal isolates 5B1, 9H3, 5H11, or 7E3. These VL domains 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.

[0088] In some aspects of the invention, the isolated antibody or its functional fragment is a monoclonal antibody. In some aspects of the invention, the isolated antibody or its functional fragment provided herein is an IgG or IgM isotype. In other aspects of the invention, the antibody or its functional fragment is an antibody of the IgG1 subclass.

[0089] In some embodiments, the functional fragment of an antibody of the present invention may be (but not limited to) Fab, Fab', F(ab')2, Fabc, scFv, a diabody, a triabody, a minibody, or a 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. In some aspects, such a diabody of the present invention may be encoded by a polynucleotide having a nucleic acid sequence of SEQ ID NO: 17 or 19. Various forms, changes, and modifications of antibodies and their functional fragments are well known in the art. The sLe of the present invention aSpecific antibody fragments may include any of these various antibody forms, alterations and modifications. Examples of these various forms and terms as they are known in the art are described below.

[0090] In some embodiments, the present invention provides a method for producing an antibody of the present invention or a functional fragment thereof. The method described herein may include introducing a polynucleotide of the present invention into a host cell, culturing the host cell under conditions and for a time sufficient to produce the encoded heavy and / or light chains of the antibody of the present invention or the functional fragment, and purifying the heavy and / or light chains of the antibody or the functional fragment.

[0091] Bind to sLe a Recombinant expression of the antibodies of the present invention or their functional fragments of antigens can include constructing expression vectors containing polynucleotides encoding the heavy and / or light chains or functional fragments of the antibodies of the present invention. Once polynucleotides encoding the antibodies of the present invention or their functional fragments (preferably, but not necessarily, containing heavy and / or light chain variable domains) are obtained, vectors for producing the antibodies or functional fragments can be generated by DNA recombination technology using techniques well known in the art. Methods for preparing proteins by expressing polynucleotides containing nucleotide sequences encoding antibodies or their functional fragments are described herein.

[0092] Methods well known to those skilled in the art can be used to construct expression vectors containing antibody or its functional fragment coding sequence and appropriate transcription and translation control signals. These methods include (for example) in vitro recombinant DNA technology, synthetic technology and in vivo gene recombination. Therefore, the invention provides reproducible vectors, which include nucleotide sequences encoding antibodies of the present invention or their functional fragments that are operably connected to promoters. These vectors can include nucleotide sequences encoding antibody molecule constant regions (see, for example, International Patent Publication No. WO 86 / 05807 and No. WO 89 / 01036; and U.S. Patent No. 5,122,464), and the variable domains of the antibody can be cloned into the vector for expression of the entire heavy chain, the entire light chain, or both the heavy chain and the light chain.

[0093] The expression vector can be transferred to a host cell by conventional methods, and the transfected cells are then cultured by conventional methods to produce an antibody of the present invention or its functional fragment. Therefore, the present invention includes a host cell containing a polynucleotide encoding an antibody of the present invention or its functional fragment that is operably linked to a heterologous promoter. In some embodiments, for the expression of double-chain antibodies, a vector encoding both heavy and light chains can be co-expressed in a host cell for expressing the entire immunoglobulin molecule, as described below.

[0094] A variety of host-expression vector systems can be used to express the antibodies of the present invention or their functional fragments (see, for example, U.S. Patent No. 5,807,715). These host-expression systems represent coding sequences of interest that can be produced and subsequently purified, and they also represent cells that can express the antibody molecules of the present invention in situ when transformed or transfected with the appropriate nucleotide coding sequences. These host-expression systems include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequence; yeast (e.g., Pichia pastoris) transformed with recombinant yeast expression vectors containing the antibody coding sequence; Pichia); insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) containing antibody coding sequences or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) with recombinant expression constructs containing promoters derived from mammalian cell genomes (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). In some aspects, particularly for the expression of complete recombinant antibodies, bacterial cells (e.g., Escherichia coli) or eukaryotic cells are used to express recombinant antibodies or functional fragments. For example, mammalian cells such as Chinese hamster ovary cells (CHO) used with vectors such as the major intermediate early gene promoter element from human cytomegalovirus are an efficient expression system for antibodies (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. In one embodiment, the antibody encoding the invention binds to sLe a The expression of the nucleotide sequence of the antibody or its functional fragment is regulated by a constitutive promoter, an inducible promoter or a tissue-specific promoter.

[0095] In bacterial systems, some expression vectors may be advantageously selected depending on the intended use of the expressed antibody molecule. For example, when large quantities of such antibodies are to be produced for the purpose of generating pharmaceutical compositions of the antibody molecule, vectors that direct the expression of high levels of fusion protein products that are easily purified may be desirable. These vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791), in which the antibody coding sequence can be ligated separately into the vector in-frame with the lacZ coding region to produce a fusion protein; the pIN vector (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors can also be used to express exogenous polypeptides as fusion proteins with glutathione 5-transferase (GST). Typically, these fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to a glutathione-agarose bead matrix followed by elution in the presence of free glutathione. pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0096] In insect systems, the Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes. The virus is grown in Spodoptera frugiperda cells. The antibody or functional fragment coding sequence can be cloned separately into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0097] In mammalian host cells, some viral-based expression systems can be used. When adenovirus is used as an expression vector, the antibody coding sequence of interest can be connected to an adenovirus transcription / translation control complex (e.g., a late promoter and a tripartite leader sequence). The chimeric gene can then be inserted into the adenoviral genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., E1 or E3 region) will produce a recombinant virus that survives and is able to express antibody molecules in the infected host (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81: 355-359). Specific initiation signals can also be used for the efficient translation of the inserted antibody coding sequence. These signals include the ATG initiation codon and adjacent sequences. In addition, the initiation codon must be consistent with the reading frame of the desired coding sequence to ensure translation of the entire inserted portion. These exogenous translation control signals and initiation codons can be of various origins, both natural and synthetic. Expression efficiency can be improved by including appropriate transcription enhancer elements, transcription terminators, etc. (See, e.g., Bittner et al., 1987, Methods in Enzymol. 153:51-544).

[0098] In addition, a host cell strain can be selected that regulates the expression of the inserted sequence or modifies and processes the gene product in a desired specific manner. These modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product can be important for the function of the antibody or functional fragment. Different host cells have characteristics and specific mechanisms for post-translational processing and modification of proteins and gene products. A suitable cell line or host system can be selected to ensure the correct modification and processing of the expressed exogenous protein. For this purpose, eukaryotic host cells can be used, which have a cell system capable of properly processing the primary transcript, glycosylation, and phosphorylation of the gene product. These mammalian host cells include (but are not limited to) CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, and HsS78Bst cells.

[0099] For the long-term, high-yield preparation of recombinant protein, stable expression is preferred.For example, the cell line of antibody of the present invention or functional fragment can be stably expressed by through engineering design.Can be with the DNA and the selectable marker that are controlled by suitable expression control element transformed host cell, and do not use the expression vector that comprises viral replication origin, described expression control element is such as promoter, enhancer, sequence, transcription terminator, polyadenylation site etc.After introducing foreign DNA, can allow engineered cell to grow 1-2 days in enrichment medium, then be switched to selective culture medium.The selectable marker in the recombinant plasmid has given selection resistance, and allows cell to stably integrate plasmid and grow to form focus in its chromosome, and it can be cloned and expanded into cell line then.This method can be advantageously used for the engineered cell line of expressing antibody molecule.

[0100] Several selection systems are available, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), xanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes, which can be used for tk-, hgprt-, or aprt- cells, respectively. In addition, antimetabolite resistance can be used as the basis for 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), which is the enzyme responsible for the biosynthesis of glutamine from glutamate and ammonia (Bebbington et al., 1992, Biotechnology 10:169); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215); and hygro, which confers hygromycin resistance (Santerre et al., 1984, Gene 30:147). Methods well known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clones and are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology , John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression , A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (Eds.), Current Protocols in Human Genetics., John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1, the entire contents of which are incorporated herein by reference.

[0101] The expression level of the antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). When the marker in the vector system expressing the antibody or its functional fragment is amplifiable, an increase in the level of inhibitors 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, the production of the antibody will also be increased (Crouse et al., 1983, Mol. Cell. Biol. 3: 257).

[0102] Host cells can be co-transfected with two expression vectors of the present invention, wherein the first vector encodes a polypeptide derived from a heavy chain and the second vector encodes a polypeptide derived from a light chain. The two vectors can contain the same selectable marker, which allows the heavy and light chain polypeptides to be expressed equally. Alternatively, a single vector can be used that encodes and is capable of expressing both heavy and light chain polypeptides. In this case, the light chain can be placed before the heavy chain to avoid an excess of non-toxic heavy chain (Proudfoot, 1986, Nature 322: 52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77: 2197-2199). The coding sequences of the heavy and light chains can include cDNA or genomic DNA.

[0103] In addition, the polynucleotides encoding the heavy and / or light chains or functional fragments of the antibodies of the present invention can be codon-optimized using techniques well known in the art to achieve optimized expression of the antibodies of the present invention or functional fragments in the desired host cells. For example, in one method of codon optimization, the natural codons are replaced by the most common codons from a set of reference genes, wherein the codon translation rate of each amino acid is designed to be high. Other exemplary methods for generating codon-optimized polynucleotides for expressing the desired protein 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 WO 2008 / 000632, which can be applied to the heavy and / or light chains or functional fragments of the antibodies of the present invention.

[0104] Once the antibody molecules of the present invention have been produced by recombinant expression, they can be purified by any method known in the art for purification of immunoglobulin molecules, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography, specifically, by affinity for specific antigens after protein A, and column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. In addition, the antibodies or functional fragments of the present invention can be fused to heterologous polypeptide sequences provided herein or known in the art to facilitate purification. For example, commercially available polyhistidine tags (His-tags), FLAG-tags, hemagglutinin tags (HA-tags), or myc-tags can be added by recombination and purification methods well known to those skilled in the art can be used to purify the antibodies or functional fragments of the present invention.

[0105] A Fab fragment refers to a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; a F(ab′)2 fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bond located in the hinge region; an Fd fragment consists of the VH and CH1 domains; an Fv fragment consists of the VL and VH domains of a single arm of an antibody; and a dAb fragment (Ward et al., Nature 341:544-546, (1989)) consists of the VH domain.

[0106] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical or different. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, and "bispecific" or "bifunctional" antibodies have two different binding sites.

[0107] A single-chain antibody (scFv) is an antibody in which the VL and VH regions are connected by a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous polypeptide chain, wherein the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen-binding site (see, e.g., Bird et al., Science 242:423-26 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-83 (1988)). Diabodies are bivalent antibodies comprising two polypeptide chains, each comprising a VH and a VL domain connected by a linker that is too short to allow pairing of the two domains on the same chain, thereby allowing each domain to pair with the complementary domain on the other 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 diabody produced by pairing them will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to prepare diabodies with two different antigen-binding sites. Similarly, triabodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen-binding sites, respectively, which may be the same or different.

[0108] The present invention also provides antibodies or functional fragment derivatives thereof of 5B1, 9H3, 5H11 and / or 7E3, wherein the antibodies or functional fragments bind to sLe a Standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding the antibodies of the present invention or their functional fragments, including, for example, directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions. In some aspects, the derivative comprises less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original molecule.

[0109] 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, as long as such antibodies or functional fragments retain functional activity as defined herein. In one embodiment, the derivatives have conservative amino acid substitutions at one or more predicted nonessential amino acid residues. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similarly charged side chain. Families of amino acid residues with similarly charged side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence by, for example, saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. Following mutation, the encoded antibody or functional fragment thereof can be expressed and the activity of the antibody or functional fragment can be determined.

[0110] In some embodiments, the present invention provides antibodies or functional fragments thereof having modified fucosylation, galactosylation and / or sialylation of the Fc fragment contained within the antibody or functional fragment of the invention. Such modifications of the Fc fragment can affect Fc receptor-mediated activity, as discussed in Peipp et al., Blood, 112(6):2390-2399 (2008). For example, glycoengineered therapeutic antibodies lacking core fucose residues in the Fc N-glycans exhibit potent ADCC at lower concentrations with much higher potency 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 or functional fragments thereof are well known in the art. For example, defucosylation methods can be divided into three approaches: (1) conversion of the N-glycosylation pathway of non-mammalian cells to a "humanized" non-fucosylation 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 a non-fucosylated form, as described in Yamane-Ohnuki et al., MAbs., 1(3): 230-236 (2009). It will be appreciated that any of these methods, or any other methods known in the art, can be used to generate antibodies or functional fragments thereof with modified fucosylation, galactosylation and / or sialylation.

[0111] Antibodies binding to sLe can be produced by any method known in the art for antibody synthesis, in particular, by chemical synthesis or by recombinant expression techniques. a Unless otherwise indicated, the practice of the present invention employs conventional methods in molecular biology, microbiology, gene 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) 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 updated annually); Current Protocols in Immunology , John Wiley & Sons (1987 and updated annually) Gait (ed.) (1984) Oligonucleotide Synthesis:A Practical Approach , IRL Press; Eckstein (ed.) (1991) Oligonucleotides andAnalogues:A Practical Approach , IRL Press; Birren et al. (eds.) (1999) Genome Analysis:A Laboratory Manual , Cold Spring Harbor Laboratory Press; Borrebaeck (Editor-in-Chief) (1995) Antibody Engineering , 2nd edition, Oxford University Press; Lo (editor) (2006) Antibody Engineering:Methods and Protocols (Methods in Molecular Biology); Vol. 248, Humana Press, Inc.; each of the above documents is incorporated herein by reference in its entirety.

[0112] 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 technology, including those known in the art and taught in Harlow et al., Antibodies:A Laboratory Manual , (Cold Spring Harbor Laboratory Press, 2nd edition, 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563681 (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. Other exemplary methods for producing monoclonal antibodies are provided in Example 1 herein.

[0113] The binding sLe can be produced by any technique known to those skilled in the art. a Functional fragments of antibodies. 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). The F(ab')2 fragment contains the variable region, the light chain constant region, and the CH1 domain of the heavy chain.

[0114] Various phage display methods known in the art can also be used to produce functional antibody fragments of the present invention. For example, in a phage display method, functional antibody domains are displayed on the surface of phage particles having polynucleotide sequences encoding them, such as heavy and / or light chain variable regions with 1, 2, 3, 4, 5 or 6 CDRs provided herein. The DNA encoding the VH and VL domains is recombined with an scFv linker by PCR and cloned into a phagemid vector. In Escherichia coli (E. coli), the vector is electroporated and infected with a helper phage. The phage used in these methods is typically a filamentous phage comprising fd and M13, and the VH and VL domains are typically recombinantly fused to phage gene III or gene VIII. Antigens, for example, labeled antigens or antigens bound or captured to a solid surface or a bead are selected or differentiated for expression and binding to a specific antigen (such as scFv). a ) of the phage antigen-binding domain. Examples of phage display methods that can be used to prepare functional fragments of the antibodies of the present invention include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; PCT Patent Application No. PCT / GB91 / 01134; International Patent Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401 and WO 97 / 13844; and 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; each of which is incorporated herein by reference in its entirety.

[0115] As described in the above references, following phage selection, the antibody coding regions from the phage can be isolated and used to produce whole antibodies, including human antibodies, or any other desired antigen-binding fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described herein.

[0116] Techniques for recombinant production of Fab, Fab′ and F(ab′)2 fragments may also be used using methods known in the art, such as those disclosed in PCT Patent 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.

[0117] In order to produce complete antibodies, PCR primers, restriction enzyme cutting sites and flanking sequences protecting restriction enzyme cutting sites comprising VH or VL nucleotide sequences can be used to amplify the VH or VL sequences in scFv clones. Using cloning techniques well known to those skilled in the art, the VH domains amplified by PCR can be cloned into vectors expressing VH constant regions, for example, human γ1 constant regions, and the VL domains amplified by PCR can be cloned into vectors expressing VL constant regions, for example, human κ or λ constant regions. 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 produce a stable or transient cell line expressing a full-length antibody (e.g., IgG).

[0118] In some embodiments, the antibodies or functional fragments of the present invention are conjugated (covalently or non-covalently) or recombinantly fused to one or more diagnostic agents, detectable agents or therapeutic agents or any other desired molecule. For monitoring or diagnosis as part of a clinical trial program (e.g., to determine the efficacy of a particular therapy) and sLe a Conjugated or recombinantly fused antibodies or functional fragments may be useful in the development, development, progression and / or severity of a disease related to the expression of a gene or protein, such as cancer or tumor formation.

[0119] Detection and diagnosis can be accomplished, for example, by coupling the antibodies or functional fragments of the invention to a detectable substance, including but not limited to a radioactive substance, such as but not limited to zirconium ( 89 Zr), iodine ( 131 I. 125 I. 124 I. 123I 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 Such as 68 Ge, 57 C o 、 65 Zn, 85 Sr. 32 P. 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn and 117Sn; and positron-emitting metals for positron emission tomography, various enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; prosthetic groups such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent substances such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinamine 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.

[0120] The present invention also encompasses the therapeutic use of antibodies or functional fragments of the present invention that are conjugated (covalently or non-covalently conjugated) or recombinantly fused to one or more therapeutic agents. In this regard, 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. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. The therapeutic agent can be a chemotherapeutic agent, such as, but not limited to, an anthracycline (e.g., doxorubicin and daunorubicin (formerly daunomycin)); a taxan (e.g., Taxol and Taxotere); an antimetabolite (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, and decarbazine); or an alkylating agent (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BCNU), lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP), and cisplatin); an antibiotic (e.g., actinomycin D, bleomycin, plicamycin, and anthramycin (AMC)); an auristatin molecule (e.g., auristatin PHE, bryostatin 1, dolastatin 1, or 0, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF)); hormones (e.g., glucocorticoids, progesterone, androgen and estrogen); nucleoside analogs (e.g., gemcitabine), DNA-repair enzyme inhibitors (e.g., etoposide and topotecan), kinase inhibitors (e.g., compound ST1571, also known as Gleevec or imatinib mesylate); cytotoxic agents (e.g., maytansine, paclitaxel, cytokine B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, plicamycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs or homologs thereof, and those compounds disclosed in the following patents: U.S. Patent No.6,245,759、6,399,633、6,383,790、6,335,156、6,271,242、6,242,196、6,218,410、6,218,372、6,057,300、6,034,053、5,985,877、5,958,769、5,925,376、5, 922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745, 5,728,868, 5,648,239, 5,587,459); farnesyltransferase inhibitors (e.g., R115777, BMS-214662, and those disclosed, for example, in U.S. Pat. No.6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959, 6,420,387, 6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615, 6,387,905, 6,372,747, 6, 369,034, 6,362,188, 6,342,765, 6,342,487, 6,300,501, 6,268,363, 6,265,422, 6,248,756, 6,239,140, ​​6,232,338, 6,228,865, 6,228,856, 6,225,322, 6,218 ,406、6,211,193、6,187,786、6,169,096、6,159,984、6,143,766、6,133,303、6,127,366、6,124,465、6,124,295、6,103,723、6,093,737、6,090,948、6,080,8 70, 6,077,853, 6,071,935, 6,066,738, 6,063,930, 6,054,466, 6,051,582, 6,051,574, and 6,040,305; topoisomerase inhibitors (e.g., camptothecin, irinotecan, SN-38, topotecan, 9-aminocamptothecin, GG-211 (GI 147211), DX-8951f, IST-622, rubitecan, pyrazoline acridine, XR-5000, saintopin, UCE6, UCE1022, TAN-1518A, TAN 1518B, KT6006, KT6528, ED-110, NB-506, ED-110, NB-506, fargroin, corylanine, β-lapachone, and pterostilbecin); DNA minor groove binders (e.g., Hoescht dye 33342 and Hoechst dye 33258); adenosine deaminase inhibitors (e.g., fludarabine phosphate and 2-chlorodeoxyadenosine); or pharmaceutically acceptable salts, solvates, clathrates, or prodrugs thereof. The therapeutic agent can be an immunotherapeutic agent, such as, but not limited to, cetuximab, bevacizumab, Herceptin, or rituximab.

[0121] Additionally, the antibodies or functional fragments of the present invention may be conjugated to therapeutic agents, such as radioactive metal ions, such as alpha emitters, such as 213 Bi or a macrocyclic chelator for conjugating radiometal ions, including, but not limited to, 131 In, 131 LU, 131 Y. 131 Ho,131 Sm; or a macrocyclic chelator, such as 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-ethylene glycol bisethylamine ether (DOTA), which can be linked to the antibody or a functional fragment thereof via a linker molecule. These 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.

[0122] In addition, the antibodies or functional fragments of the present invention can be conjugated (covalently or non-covalently conjugated) or recombinantly fused to therapeutic agents that alter a given biological response. Therefore, therapeutic agents are not considered to be limited to classical chemotherapeutic agents. For example, therapeutic agents can be proteins, peptides or polypeptides having the desired biological activity. These proteins can include, for example, toxins (e.g., abrin, ricin A, Pseudomonas exotoxin, cholera toxin and diphtheria toxin); proteins such as 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., angiostatins, endostatin, and coagulation pathway components, such as tissue factor); bioresponse-modifying substances (e.g., cytokines, such as interferon gamma, interleukin-1, interleukin-2, interleukin-5, interleukin-6, interleukin-7, interleukin-9, interleukin-10, interleukin-12, interleukin-15, interleukin-23, granulocyte macrophage colony-stimulating factor, and granulocyte colony-stimulating factor); growth factors (e.g., growth hormone), or coagulants (e.g., calcium, vitamin K, tissue factor, such as, but not limited to, Hagemann factor (factor XII), high molecular weight kininogen (HMWK), prekallikrein (PK), coagulation proteins - factor II (prothrombin), factors V, XIIa, VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipids, and fibrin monomers).

[0123] The present invention encompasses antibodies or functional fragments of the present invention that are recombinantly fused or chemically conjugated (covalently or non-covalently) to heterologous proteins or polypeptides to produce fusion proteins. In some aspects, the length of 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 some aspects, the present invention provides fusion proteins having a functional fragment of an antibody of the present invention (e.g., a Fab fragment, a Fd fragment, a Fv fragment, a 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 to which the antibody or functional fragment is fused is useful for targeting the antibody or its functional fragment to a specific cell type, such as cells expressing sLe a cells.

[0124] The conjugate or fusion protein of the present invention includes any antibody or functional fragment of the present invention provided herein conjugated (covalently or non-covalently conjugated) or recombinantly fused to a diagnostic reagent, detectable agent, or therapeutic agent. In one embodiment, the conjugate or fusion protein of the present invention includes a 5B1, 9H3, 5H11, or 7E3 antibody, and a diagnostic reagent, detectable agent, or therapeutic agent. In another embodiment, the conjugate or fusion protein of the present invention includes a functional fragment of a 5B1, 9H3, 5H11, or 7E3 antibody, and a diagnostic reagent, detectable agent, or therapeutic agent. In another embodiment, the conjugate or fusion protein of the invention comprises a VH domain having the amino acid sequence of any one 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, or residues 20-145 of SEQ ID NO: 14, and / or a VL domain having the amino acid sequence of any one 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, or residues 23-130 of SEQ ID NO: 16, and a diagnostic agent, detectable agent, or therapeutic agent. In another embodiment, the conjugate 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 NO: 2, 6, 10, or 14, and a diagnostic agent, detectable agent, or therapeutic agent. In another embodiment, the conjugate 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, the conjugate 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.

[0125] Methods for fusing or conjugating diagnostic, detectable, or therapeutic agents, including polypeptides, to antibodies are well known; see, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies F or Drug Delivery”, 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”, Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibodies In Cancer Therapy”, Monoclonal Antibodies For Cancer Detection 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, 5,112,946, 7,981,695, 8,039,273, 8,142,784; U.S. Patent Publication Nos. 2009 / 0202536, 2010 / 0034837, 2011 / 0137017, 2011 / 0280891, 2012 / 0003247; EP 307,434; EP 367,166; EP 394,827; PCT Patent 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), the entire contents of which are incorporated herein by reference.

[0126] In another aspect, diagnostic, detectable, or therapeutic agents can be attached to the hinge region of a reduced antibody component via disulfide bond formation. Alternatively, heterobifunctional cross-linkers such as N-hydroxysuccinimide 3-(2-pyridyldithiol)propionate (SPDP) can be used to attach these agents to the antibody component. 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).

[0127] Alternatively, diagnostic agents, detectable agents, or therapeutic agents can be conjugated via a carbohydrate moiety in the Fc region of an antibody. Methods for conjugating peptides to antibodies 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. Patent No. 5,057,313, all of which are incorporated herein by reference in their entirety. The general method involves reacting an antibody component having an oxidized carbohydrate moiety with a carrier polymer having at least one free amine function and loaded with a plurality of peptides. The reaction results in the production of an initial Schiff base (imine) bond, which can be stabilized by reduction to a secondary amine to form the final conjugate.

[0128] However, if the Fc region is absent, for example, if a functional fragment of an antibody as provided herein is desired, it is still possible to attach a diagnostic agent, a detectable agent, or a therapeutic agent. A carbohydrate moiety can be introduced into the light chain variable region of a full-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 herein by reference in their entirety. Engineered carbohydrate moieties are used to attach diagnostic agents, detectable agents, or therapeutic agents.

[0129] It can be selected to be conjugated or recombinantly fused to the binding to sLe of the present invention a It should be understood that when deciding which therapeutic agent to conjugate or recombinantly fuse to the antibody or functional fragment of the present invention, the following factors should be considered within the skill level of the clinician or other medical personnel: the nature of the disease, the severity of the disease, and the condition of the subject.

[0130] detectably labeled and bound to sLe as provided herein a The conjugated or fusion antibodies or functional fragments of the present invention can be used for diagnostic purposes to detect, diagnose or monitor diseases, wherein the cells causing the disease or associated with the disease express sLe a For example, as provided herein, cancer cells and tumors have been shown to express sLe a, such as, but not limited to, gastrointestinal tumors, breast cancer, ovarian cancer, colon cancer, colorectal adenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, small cell lung cancer, bladder adenocarcinoma, metastatic colon cancer, colorectal cancer, signet ring cell ovarian cancer and metastatic cancer. Therefore, the present invention provides a method for detecting cancer or tumor formation in a subject by administering an effective amount of a conjugated or fusion antibody or functional fragment of the present invention to a subject in need thereof. In some aspects, the detection method can also include using a conjugated or fusion antibody or functional fragment to detect cancer or tumor formation in a subject. a One or more conjugates or fusion antibodies or functional fragments of the present invention are used to determine the sLe on the subject's cells or tissue samples a and comparing the level of sLea with a control level, e.g., a normal tissue sample (e.g., from a non-diseased subject, or from the same subject before the onset of the disease), thereby a The control level was compared with sLe a An increase in the measured level of is indicative of disease. These diagnostic methods can enable medical professionals to use preventative measures or aggressive treatment before otherwise possible, thereby preventing the development or further progression of the disease.

[0131] The antibodies or functional fragments of the present invention can also be used to determine sLe in biological samples using classical immunohistological methods as provided herein or known to those skilled in the art. a Antigen levels (see, for example, 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 include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase; radioisotopes such as iodine ( 125 I. 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In) and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

[0132] In one aspect, the present invention provides disease detection and diagnosis in humans. In one embodiment, the diagnosis comprises: a) administering to a subject (e.g., parenterally, subcutaneously, or intraperitoneally) an effective amount of aa b) after administration, waiting for a certain time interval to allow the conjugate or fusion protein to preferentially express sLe in the subject a The method further comprises the steps of: collecting the conjugate or fusion protein at the site of infection (and, in some aspects, waiting for unbound conjugate or fusion protein to be cleared 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 the background level indicates that the subject is diseased. Background levels can be determined by a variety of methods, including comparing the amount of conjugate or fusion protein detected to a standard value previously determined for a particular system.

[0133] It will be understood that the size of the subject and the imaging system used will determine the amount of imaging moiety required to produce a diagnostic image and this can be readily determined by one skilled in the art. For example, in the case of a radioisotope conjugated to an antibody or functional fragment of the invention, for a human subject, the amount of radioactive material injected will typically be between about 5 and 20 millicuries. 99 The conjugate will then preferably be expressed in the a In vivo tumor imaging is described in SW Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, SW Burchiel and BARhodes, eds., Masson Publishing Inc. (1982)).

[0134] Based on a number of variables, including the type of detectable agent used and the mode of administration, the time interval after administration that allows the conjugate to preferably concentrate at the site in the subject and for unbound conjugate to be cleared 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, for example, one month after the initial diagnosis, six months after the initial diagnosis, one year after the initial diagnosis, or more, the disease is monitored by repeating the method for diagnosis as provided herein.

[0135] The presence of the conjugate or fusion protein in a subject can be detected using methods known in the art for in vivo scanning. These methods depend on the type of detectable agent used. A skilled person will be able to determine the appropriate method for detecting a particular detectable agent. The methods and devices that can be used in the diagnostic methods of the present invention include, but are not limited to, computed tomography (CT), whole-body scans such as positron emission tomography (PET), magnetic resonance imaging (MRI), and sonography. In one embodiment, the antibody or functional fragment of the present invention is conjugated to a radioisotope and detected in a subject using a radioactive 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 scanner. In another embodiment, the antibody or functional fragment of the present invention is conjugated to a positron-emitting metal, such as zirconium ( 89 In 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).

[0136] In one embodiment, the present invention provides a pharmaceutical composition with an antibody or functional fragment of the present invention and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier that can be used in the pharmaceutical composition of the present invention includes any standard pharmaceutical carrier known in the art, such as phosphate buffered saline solution, water and emulsions, such as oil-water emulsions, and various types of wetting agents. These pharmaceutical compositions can be prepared in liquid unit dosage form or in any other dosage form sufficient to deliver the antibody or functional fragment of the present invention to the target area of ​​the subject in need of treatment. For example, the pharmaceutical composition can be prepared in any manner suitable for the selected form of administration (e.g., intravascular, intramuscular, subcutaneous, intraperitoneal, etc.). Those skilled in the art can easily select other optional components, such as pharmaceutical grade stabilizers, buffers, preservatives, excipients, etc. Taking into full consideration pH, isotonicity, stability, etc., the preparation of pharmaceutical compositions is within the technical level of this area.

[0137] Antibodies having the desired degree of purity can be prepared by mixing them with optional physiologically acceptable carriers, excipients or stabilizers ( Remington′s Pharmaceutical Sciences(1990) Mack Publishing Co., Easton, PA) are mixed and prepared for storage in the form of a freeze-dried formulation or an aqueous solution containing one or more antibodies or functional fragments of the present invention provided herein. Useful carriers, excipients or stabilizers are non-toxic to the recipient at the dosages and concentrations used and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residual proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0138] Therefore, in some embodiments, the present invention also provides methods for treating or preventing a disease in a subject in need thereof. The methods provided herein 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 may be treated or prevented using the methods provided herein include cancer, tumor formation, and / or metastasis. Specifically, the methods provided herein are useful for treating cancer or tumor formation, wherein the cancer cells or tumors express carbohydrate sLe a Non-limiting examples of cancers or tumors that can be treated or prevented using the methods described herein include gastrointestinal tumors, e.g., colon cancer, colorectal adenocarcinoma, metastatic colon cancer, colorectal cancer, pancreatic cancer, or pancreatic adenocarcinoma; small cell lung cancer; bladder adenocarcinoma; signet ring cell ovarian cancer; ovarian cancer, metastatic cancer; and stomach, esophagus, larynx, genitourinary tract, or breast adenocarcinoma.

[0139] Therefore, 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 a functional fragment thereof, wherein the antibody or functional fragment binds to sLe aand 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 a functional fragment thereof, wherein the antibody or functional fragment binds to sLe a and 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. 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 a functional fragment thereof, wherein the antibody or functional fragment binds to sLe a and includes both a VH domain and a VL domain, wherein the VH domain and the VL domain each include an amino acid sequence selected from the group consisting of residues 20-142 of SEQ ID NO:2 and residues 20-130 of SEQ ID NO:4; residues 20-142 of SEQ ID NO:6 and residues 20-129 of SEQ ID NO:8; residues 20-142 of SEQ ID NO:10 and residues 20-130 of SEQ ID NO:12; and residues 20-145 of SEQ ID NO:14 and residues 23-130 of SEQ ID NO:16.

[0140] Formulations such as those described herein may also contain more than one active compound necessary for the specific disease to be treated. In certain embodiments, the formulation comprises an antibody or functional fragment of the present invention and one or more active compounds having complementary activities that do not adversely affect each other. These molecules are suitably present in combination in an amount effective for the intended use. For example, the antibody or functional fragment of the present invention may be mixed with one or more other therapeutic agents. These combination therapies may be administered to a subject simultaneously or sequentially.

[0141] Therefore, in some aspects, the invention provides a method for treating or preventing a disease by administering a therapeutically effective amount of a pharmaceutical composition provided herein to a subject in need thereof, wherein the pharmaceutical composition comprises an antibody of the present invention or a functional fragment and a second therapeutic agent. As discussed herein, those skilled in the art can easily determine that the second therapeutic agent is suitable. As provided herein in Example IV, in some aspects of the invention, the second therapeutic agent can be Taxol.

[0142] The pharmaceutical compositions provided herein contain a therapeutically effective amount of one or more antibodies of the present invention provided herein, and optionally one or more other therapeutic agents in a pharmaceutically acceptable carrier. These pharmaceutical compositions are useful in the prevention, treatment, control or improvement of diseases (such as cancer or tumor formation or one or more symptoms thereof).

[0143] Pharmaceutical compositions may contain one or more antibodies or functional fragments of the 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 a pharmaceutical composition using techniques and procedures well known in the art (see, e.g., Ansel (1985)). Introduction to Pharmaceutical It seems there is an ellipsis in the original text at "Figure 20 " and "Figure 24 " which is not clear in the context. The translation above tries to maintain the consistency as much as possible while keeping the original tags intact. If you have any further clarification about those parts, it would be helpful for a more accurate translation. Dosage Forms , 4th ed., p.126).

[0144] The antibodies or functional fragments of the present invention can be included in a pharmaceutical composition in a therapeutically effective amount sufficient to produce a therapeutically effective effect without causing undesirable side effects on the treated subject. 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 dose for use in humans. The concentration of the antibody or functional fragment in the pharmaceutical composition will depend on, for example, the physicochemical properties of the antibody or functional fragment, the dosage schedule and the amount administered, as well as other factors well known to those skilled in the art.

[0145] In one embodiment, the therapeutically effective dose produces a serum concentration of the antibody or functional fragment of about 0,1 ng / ml to about 50-100 μg / ml. In another embodiment, the pharmaceutical composition provides a dosage of about 0.001 mg to about 500 mg of antibody / kg body weight / day. Pharmaceutical dosage unit forms can be prepared to provide about 0.01 mg, 0.1 mg or 1 mg to about 30 mg, 100 mg or 500 mg, and in one embodiment, about 10 mg to about 500 mg of the antibody or functional fragment and / or a combination of other optional essential ingredients of each dosage unit form.

[0146] The antibody or functional fragment of the present invention can be administered once, or it can be divided into some smaller doses administered at certain time intervals. It should be understood that the precise dosage and duration of treatment are functions of the disease to be treated, and can be determined empirically using known test procedures or by extrapolation of in vivo or in vitro test data. It should be noted that concentrations and dosage values ​​can also vary with the severity of the condition to be alleviated. It should also be understood that for any particular subject, specific dosage regimens can be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the compound, and the concentration ranges described herein are merely exemplary and are not intended to limit the scope and practical application of the claimed composition.

[0147] Once the antibodies or functional fragments of the present invention are mixed or added, the resulting mixture can be a solution, a suspension, etc. The form of the resulting mixture depends on several factors, including the intended form of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient to ameliorate the symptoms of the disease, disorder or condition to be treated and can be determined empirically.

[0148] Pharmaceutical compositions for human and animal administration are provided in unit dose form, such as sterile parenteral solutions or suspensions containing a suitable amount of a compound or a pharmaceutically acceptable derivative thereof. In one embodiment, the antibody or functional fragment can be formulated and administered in unit dose form or in multiple dose form. A unit dose form refers to a physically separate unit suitable for human and animal subjects and individually packaged, as known in the art. Each unit dose contains a predetermined amount of the antibody or functional fragment of the present invention in combination with a desired pharmaceutical carrier, vehicle, or diluent sufficient to produce the desired therapeutic effect. Examples of unit dose forms include ampoules and syringes. Unit dose forms can be administered in part or multiple times. A multiple dose form is a plurality of identical unit dose forms packaged in a single container for administration in separate unit dose forms. Examples of multiple dose forms include pint or gallon vials or bottles. Thus, a multiple dose form is a plurality of unit doses that are not separated in the packaging.

[0149] In one embodiment, one or more antibodies or functional fragments of the present invention are in a liquid pharmaceutical formulation. Liquid pharmaceutically administrable compositions can be prepared, for example, by dissolving, dispersing or mixing the antibodies or functional fragments as provided herein and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous glucose solution, glycerol, ethylene glycol, ethanol, etc., thereby forming a solution. If desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic auxiliary substances, such as wetting agents, emulsifiers, solubilizers, pH buffers, etc., for example, hydrochloric acid, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents. The actual methods for preparing these dosage forms are known or obvious to those skilled in the art; for example, see Remington's Pharmaceutical Sciences (1990)Mack Publishing Co., Easton, PA.

[0150] The method of administering the pharmaceutical composition of the present invention is well known in the art. It should be understood that suitable routes of administration of the pharmaceutical composition can be easily determined by a skilled clinician. Exemplary routes of administration include intravenous injection, intramuscular injection, intradermal injection, or subcutaneous injection. In addition, it should be understood that the preparation of the pharmaceutical composition can be easily adjusted to be suitable for the route of administration. The present invention also provides that after the pharmaceutical composition of the present invention is administered, delayed, continuous, and / or repeated doses of one or more pharmaceutical compositions as provided herein can be administered to a subject.

[0151] The methods of the present invention for treating a disease are intended to include (1) preventing the disease, i.e., causing clinical symptoms of the disease to not appear in a subject who may be susceptible to the disease but has not yet experienced or displayed symptoms of the disease; (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; or (3) alleviating the disease, i.e., causing regression of the disease or its clinical symptoms. The methods of the present invention for preventing a disease are intended to include hindering clinical symptoms indicative of cancer or tumor formation. Such hindering includes, for example, the maintenance of normal physiological indicators in a subject. Thus, prevention can include prophylactic treatment of a subject to protect them from developing tumor metastasis.

[0152] The therapeutically effective amount of the pharmaceutical composition used in the methods described herein 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 that can be treated by the methods described herein include vertebrates, preferably mammals, and more preferably humans.

[0153] It is to be understood that modifications that do not substantially affect the activity of the various embodiments of the invention are also provided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but not to limit the invention.

[0154] Example 1

[0155] Anti-sLe a Human monoclonal antibodies have potent anti-tumor activity

[0156] Carbohydrate antigen sLe a It is widely expressed in gastrointestinal, breast, and pancreatic epithelial cell tumors and small cell lung cancer. a Overexpression of sLe appears to be an important event in the invasion and metastasis of various tumors and leads to sensitivity to antibody-mediated lysis. aare attractive molecular targets in tumor therapy. Therefore, as described herein, we generated and characterized a - Fully human monoclonal antibodies (mAbs) against blood lymphocytes of individuals immunized with KLH vaccine. Based on ELISA and FACS, some mAbs were selected, including those against sLe a Two mAbs with high affinity (5B1 and 7E3, with binding affinities of 0.14 and 0.04 nmol / L, respectively) were further characterized. As determined by glycan array analysis, both antibodies are specific for Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ and Neu5Gcα2-3Galβ1-3(Fucα1-4)GlcNAcβ. r7E3 (IgM) exhibited higher complement-dependent cytotoxicity against DMS-79 cells than r5B1 (IgG1) (EC50 0.1 μg / mL versus 1.7 μg / mL). In addition, the r5b1 antibody demonstrated high levels of antibody-dependent cell-mediated cytotoxicity against DMS-79 cells using human NK cells or peripheral blood mononuclear cells. To evaluate in vivo efficacy, the antibodies were tested in xenograft models using Colo205 tumor cells or DMS-79 tumor cells transplanted into severe combined immunodeficient (SCID) mice. In the Colo205 xenograft model, treatment doubled the median survival time to 207 days during the first 21 days using four r5B1 doses (100 μg per dose), and three of five animals survived through six doses. In the DSM-79 xenograft model, the growth of established DMS-79 tumors was inhibited or regressed in animals treated with the r5B1 antibody. a The potential of 5B1 and 7E3 as targets of immune attack, along with their affinity, specificity, and effector functions, have clinical applications in cancer therapy.

[0157] Materials, cells, and antibodies

[0158] DMS-79 (Pettengill et al., Cancer, 45:906-18 (1980)), SW626, EL4, HT29, BxPC3, SK-MEL28, and P3×63Ag8.653 cell lines were purchased from the American Type Culture Collection (ATCC). Colo205-luc cells (Bioware ultra) were obtained from Caliper Life Sciences. Mouse control mAb 121SLE (IgM) was purchased from GeneTex. a Tetrasaccharide (Cat#S2279) was purchased from Sigma-Aldrich. a-HSA (human serum albumin) conjugate (Cat#07-011), monovalent biotinylated sLe a (sLe a -sp-biotin; Cat#02-044), multivalent biotinylated sLe a -PAA (Cat#01-044), biotin-labeled Lea-PAA (Cat#01-035) and sLe x -PAA-biotin (Cat#01-045) was purchased from GlycoTech. In a polyvalent presentation, the tetrasaccharide was incorporated into a polyacrylamide matrix (PAA), thereby generating a 30-kDa multivalent polymer in which approximately every five amide groups of the polymer chain were N-substituted with biotin in a 4:1 ratio and a carbohydrate content of approximately 20%. As described, sLe was used. a Pentenyl glycosides were prepared in the laboratory for the other HSA or BSA glycoconjugates used in this study. 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.

[0159] Hybridoma generation of anti-sLea mAb

[0160] The ongoing sLe initiated at MSKCC is conducted in accordance with MSKCC- and FDA-approved IRB procedures and IND. a - In breast cancer patients in the KLH conjugate vaccine trial, blood samples were obtained from 3 patients. Blood samples were selected from 2 patients after 3 or 4 vaccinations who had sLe a These sera (and mouse mAb 19.9) showed antibody titers of 1 / 160 and 1 / 320, respectively. a -positive cell lines responded well and mediated efficient CDC. Ragupathi et al., Cancer Immunol Immunother, 58: 1397-405 (2009). Peripheral blood mononuclear cells (PBMCs) were isolated from approximately 80 to 90 mL of blood by gradient centrifugation on Histopaque-1077 (Sigma-Aldrich).

[0161] 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 to P3×63Ag8.653 myeloma cells by electrofusion.

[0162] sLe a ELISA

[0163] For sLe a ELISA, using 1 μg / mL sLe a -HSA conjugate, monovalent biotinylated sLe a or multivalent biotinylated sLe captured on Neutr-avidin-coated plates a -PAA coated plates. Uncoated wells (PBS) and HSA-coated wells served as controls. Initially, bound antibodies were detected with horseradish peroxidase (HRP)-labeled goat anti-human IgA+G+M (Jackson ImmunoResearch), and positive wells were subsequently tested with IgG-Fc- or IgM-specific secondary antibodies to determine isotype.

[0164] Carbohydrate-specific analysis

[0165] The surface plasmon resonance (SPR) was used to evaluate the a -PAA and biotin-sLe x -PAA confirms the presence of a closely related antigen Le a and sLe x Cross-reactivity was tested by ELISA for binding to gangliosides GD2, GD3, fucosyl-GM1, GM2, and GM3. Competitive ELISA was used to evaluate the specificity of mAbs for some other related carbohydrate moieties. Briefly, 2 μg / mL sLe a-HSA conjugates were coated onto the plates and subsequently blocked with 3% BSA in PBS. 30 μL of different carbohydrate moieties (40 μg / mL in PBS, prepared from 1 mg / mL stock solutions) either unconjugated or conjugated to HSA or BSA were then mixed with 30 μL of the test antibody and incubated in the sample plate at room temperature. 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 antibodies using a Versamax spectrofluorometer (all steps were performed at room temperature). The carbohydrate moieties tested included globo H, Lewis Y, Lewis X, sialyl-Thomson-nouveaux (sTn), clustered sTn, Thomson Friedenreich (TF), Tighe Le b / Le Y Mucin, porcine submandibular mucin (PSM) and sLe a Tetrasaccharide and sLe a To determine the fine specificity of the antibodies, glycan array analysis was performed by the Consortium for Functional Glycomics Core H group. 5B1 and 7E3 antibodies were tested at 10 μg / mL using a version 4.1 printed array consisting of 465 glycans, and the experiment was repeated six times.

[0166] Immunoglobulin cDNA cloning and recombinant antibody expression

[0167] 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 or IgK or IgL light chain expression vectors as previously described. Sawada-Hirai et al., J. Immune Based Ther. Vaccines, 2:5 (2004). The Ig heavy or light chain expression vectors were double-digested with Not I and Sal I, and the two fragments were then ligated to form a dual-gene expression vector. CHO cells were transfected with the dual-gene expression vectors in 6-well plates using Lipofectamine 2000 (Invitrogen). After 24 hours, the transfected cells were transferred to 10-cm dishes with selection medium [DMEM supplemented with 10% dialyzed FBS (Invitrogen), 50 μmol / l 1-methionine sulfoxime (MSX), GS supplement (Sigma-Aldrich), and penicillin / streptomycin (Omega Scientific). Two weeks later, MSX-resistant transfectants were isolated and expanded. a -specific ELISA assay to measure antibody levels in the supernatant and select anti-sLe a High-yielding antibody clones were isolated and expanded for large-scale mAb production.

[0168] Purification of human mAbs

[0169] Antibodies were purified using an Akta Explorer (GE Healthcare) system running Unicorn 5.0 software. Briefly, stable 5B1 or 7E3 clones were grown in serum-free medium in a Wave bioreactor, the harvested supernatant clarified by centrifugation and filtration, and stored frozen until use. Human IgG antibodies were purified on a protein A column of appropriate size using a running buffer of 10 mmol / L PBS and 150 mmol / L NaCl. Human IgM antibodies were purified on a hydroxyapatite column and the IgM was eluted with a 500 mmol / L phosphate gradient. The OD values ​​were calculated using the OD values. 280 Determine the antibody concentration using an E of 1.4 for IgG and 1.18 for IgM. 1％ The purity of each preparation was assessed by SDS-PAGE analysis (1-5 μg / lane) under reducing conditions and was greater than 90% based on the sum of heavy and light chains.

[0170] Flow cytometry

[0171] Wash sLe in PBS / 2% FBS (PBSF) a -positive or -negative tumor cell lines (0.5 × 10 6cells). Then, test or control human mAb (1-2 μg / mL in complete medium) was added 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 on ice for 30 minutes with Alexa-488 anti-human IgG-Fcγ or anti-human IgM-μ (invitrogen). Cells were washed twice in PBSF and analyzed by flow cytometry using the Guava Personal Cell Analysis 96 (PCA-96) system (Millipore). Colo205-luc cells were incubated with 2 μg / mL 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.

[0172] Affinity determination

[0173] Affinity constants were determined using the principle of SPR by Biacore 3000 (GE Healthcare). a (Cat#02-044) or multivalent sLe a -PAA-biotin (Cat#01-044) was coupled to a separate flow cell of the SPA biosensor chip. The flow cell was blocked with HSA and the culture medium containing free biotin was used as a reference cell. a Binding kinetic parameters were determined using a PAA-biotin-coated flow cell from several known antibody concentrations 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). Estimated association and dissociation rates were generated using the curve fitting software provided with the Biacore instrument, from which affinity was calculated.

[0174] CDC assay

[0175] sLe was diluted with human supplement (Quidel; Cat#A113) and various dilutions of purified human mAb (0.1-25 μg / mL) or with a positive control mAb as described previously. aAntigen-positive and -negative cell lines were used in a 90-minute cytotoxicity assay (Guava PCA-96 cytotoxicity kit; Millipore; Cat# 4500-0200) (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 6 Target cells were stained to obtain green / yellow fluorescent target cells. The stained cells (1×105 / 50 μL sample) were incubated with 100 μL of antibody on ice for 40 minutes. Then, 50 μL of human supplement or culture medium alone diluted 1:2 in complete culture medium (RPMI-1640, 10% FCS) was added to three replicate samples and incubated at 37°C for 90 minutes. Therefore, the final supplement dilution in the assay was 1:8. Cells killed during incubation were labeled by adding the membrane-impermeable dye 7-amino-actinomycin D (7-AAD), and samples were analyzed by two-color immunofluorescence using the Guava CellToxicity software module. Control samples receiving NP40 were used to determine maximum killing, and samples receiving the supplement alone were used as baseline. The percentage of cells killed was determined by appropriate gating and calculated according to the following formula: % Kill = [(% Sample - % Supplement Alone) / (% NP40 - % Supplement Alone)] × 100.

[0176] Antibody-dependent cell-mediated cytotoxicity assay

[0177] PBMC effector cells were isolated by Ficoll-Hypaque density centrifugation from blood samples obtained according to MSKCC IRB-approved protocols. Target cells were plated at 5 × 10 6 Cells / mL were incubated in complete growth medium with 15 μL of 0.1% calcein-AM solution (Sigma-Aldrich) for 30 minutes. Cells were washed twice with 15 mL of PBS-0.02% EDTA and resuspended in 1 mL of complete growth medium. Figure 13With the antibody concentrations described in [ 15 ], 50 μL (10,000 cells) of labeled target cells were plated into a 96-well plate and incubated with 50 μL of freshly isolated peripheral blood mononuclear cells (effector cells, 100:1 E / T ratio). After a 2-hour 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 in a Fluoroskan Ascent (ThermoScientific) at 485 nm excitation and 535 nm emission. Spontaneous release was determined for target cells in RPMI-1640 medium with 30% FBS and without effector cells, and maximal release was determined for target cells in RPMI-1640 medium with 30% FBS and 6% Triton X-100 and without effector cells. Percent cytotoxicity was calculated as [(counts in sample - spontaneous release) / (maximum counts - spontaneous release)] × 100.

[0178] mAb internalization assay

[0179] For cells expressing sLe, plated in 96-well plates (2000 cells / 90 μl / well) and incubated overnight a The BxPC3 cells (repeated twice) were used to evaluate the internalization of the 5B1 antibody by measuring the cytotoxic activity of the second conjugate of r5B1 and Hum-ZAP (Advanced Targeting Systems) complex. According to the manufacturer's instructions, different concentrations of 5B1 antibody were incubated together with the second conjugate of Hum-ZAP at RT. Then, 10 μl / well of r5B1 and Hum-ZAP complex were added to the cells and incubated for 3 days. 25 microlitres of thiazolyl blue tetrazolium bromide (Sigma-Aldrich) solution (5 mg / mL, solution in PBS) were added to each well and incubated at 37°C. After incubation for 2 hours, 100 μL / well of lysate (20% SDS / 50% N, N-dimethylformamide) were added to each well and incubated for another 16 hours at 37°C. OD was measured at 570 / 690nm, and the value obtained using a separate culture medium was used for plate background subtraction. Eight replicate cultures without antibody were used to normalize sample values ​​(average value of sample / untreated x 100).

[0180] Xenograft models

[0181] Female CB17 SCID mice (5-8 weeks old) were purchased from Taconic. For the Colo205 xenograft model, Colo205-luc cells (0.5 × 10 cells / ml) in 0.1 mL complete growth medium were injected via the tail vein on day 0 using a BD insulin syringe with a 28G needle (Becton Dickinson & Co). 6For the first study, 100 μg of mAb 5B1 was injected intraperitoneally on days 1, 7, 14, and 21 (experiment 1) or on days 1, 4, 7, 10, 14, and 21 (experiment 2). For the second study, 100 μg, 300 μg, or 1 mg of mAb 5B1 was injected intraperitoneally on day 4 after tumor cell injection, then twice weekly for the first two weeks and once weekly for the next 7 weeks. Mice were monitored for tumor development. For the DMS-79 xenograft model, DMS-79 cells (1×10 6 ) were injected subcutaneously into female CB17 SCID mice and the tumors were grown to 5 mm (~20 mm 2 ) were initiated on day 19 after 48 h. Animals were then treated with human IgG or 5B1 antibody administered intraperitoneally at 200 μg per dose plus cRGD initially at 80 μg and then intravenously at 40 μg per dose five days a week until day 37 to increase vascular permeability.

[0182] All procedures were performed under protocols approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. Kaplan-Meier survival curves were generated using GraphPad Prism 5.1 (GraphPad Software) and analyzed using the Mantel-Haenszel log-rank test.

[0183] result

[0184] Identification of human monoclonal antibodies and recombinant antibody production by ELISA

[0185] Blood samples from 3 vaccinated patients were used for hybridoma production and multiple positive wells were detected in the antigen-specific ELISA assay (Table 3). Extensive screening was used to eliminate antibodies that showed poor or nonspecific binding. Initially, antibodies to sLe were selected. a Eight hybridoma cells expressing human antibodies with stable reactivity (1 IgM and 7 IgG) were expanded and subcloned for further characterization. Two antibodies (9H1 and 9H3) showed a -HSA conjugates have strong binding but are not very effective for sLe a -PAA coated plates were not. Three antibodies (5B1, 5H11 and 7E3) showed activity against monovalent and multivalent sLe3 when measured by ELISA assay. a and sLe a -HSA conjugates had strong binding (Table 4).

[0186] Table 3: Candidate hybridoma supernatants containing IgG or IgM monoclonal antibodies and sLe a -acetylphenylenediamine (APD)-human serum albumin (HSA) conjugate (sLe a -HuSA).

[0187]

[0188] *Isotype control blank subtracted. HuSA denotes human serum albumin control.

[0189] PBS indicates phosphate-buffered saline control.

[0190] Table 4: Selected antibodies and their use as monovalent (mono-) sLe a , multivalent (poly-)sLe a or sLe a -HSA provided in the form of sLe a combination.

[0191]

[0192] HuSA denotes human serum albumin control. PBS denotes phosphate-buffered saline control. NAV denotes neutravidin control.

[0193] 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)) showed 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 λ light chains. These IgG1 antibodies showed different CDR sequences, which had 16, 5 or 3 mutations ( Figure 1-6 ; Table 5). The IgM antibody (7E3) uses a kappa light chain and has six heavy chain mutations ( Figure 7-8 ; Table 5). The increase in mutations in 5B1 is an indication of affinity maturation. Recombinant antibodies were produced in a CHO cell line in a shake bag bioreactor system and purified using either protein A or hydroxyapatite chromatography for IgG and IgM, respectively. For ELISA binding and specificity, the purified recombinant antibodies retained the properties of the original hybridoma-derived antibodies.

[0194] Table 5: Selected human anti-sLe derived from vaccinated blood donors a cDNA classification of antibodies.

[0195]

[0196] Analysis of tumor cell binding

[0197] Cell surface binding is crucial for cytotoxic activity and was therefore subsequently determined. Flow cytometry showed strong binding of the 5B1, 9H3, 5H11, and 7E3 recombinant antibodies to DMS-79 cells, a small cell lung cancer suspension cell line ( FIG. 11A ). Binding of r5B1 and r7E3 was also confirmed for HT29 colon cancer cells ( FIG. 11B ), BxPC3 pancreatic cancer cells ( FIG. 11C ), SW626 ovarian cancer cells ( FIG. 11D ), and Colo205-luc colon cancer cells ( FIG. 11F ). These antibodies were unable to bind to sLe a -negative (SLE121-negative) SK-MEL28 melanoma cells (Figure 11E) or EL4 mouse lymphoma cells (data not shown).

[0198] Affinity measurement

[0199] Use capture biotinylated sLe a -ppA streptavidin-coated biosensor chip, and the interaction with sLe was examined by SPR. a As shown in Table 6, r5B1 and r7E3 bind to sLe a -pPA rapidly binds and reacts with 121SLE (commercially available murine IgM anti-sLe for comparison) a The affinity of 5B1 was measured to be 0.14 nmol / L, and the apparent affinity / avidity of 7E3 was approximately 4 times higher (Table 6). a -PAA-coated biosensor chip, thus hampering the determination of 9H3 affinity.

[0200] Table 6: Anti-sLe by SPR a Determination of kinetic parameters of antibodies.

[0201]

[0202] Specificity analysis

[0203] Initial assays exploring carbohydrate specificity showed that 5B1, 9H3, and 7E3 did not bind to the closely related sLe x 、Le a or Le YAntigens or gangliosides GD2, GD3, fucosyl-GM1, GM2 and GM3. 7E3, 5B1 and 121SLE were bound to sLe captured on a Biacore avidin chip. a -PAA-biotin or sLe a Additional analysis of -sp-biotin binding showed that all three antibodies bound to sLe a The multivalent form of sLe was found, while 7E3 and 5B1 were found to bind to the monovalent form. a The tetrasaccharide also inhibited the expression of 5B1 and sLe in a dose-dependent manner. a -PAA binding (data not shown). These results are consistent with the above observations: high anti-sLe by ELISA a Antibody titers of serum against sLe a It is specific, that is, it binds to gangliosides GM2, GD2, GD3, fucosyl-GM1 or neutral glycolipids globo H and Le y No reaction. Ragupathi et al., Cancer Immunol Immunother 58: 1397-405 (2009). In various presentations (e.g., as ceramide or conjugated to BSA or HSA), only sLe a Tetrasaccharide and sLe a -HSA conjugates can inhibit the a -HSA conjugates (Table 7).

[0204] Table 7: sLe in the presence of various related glycoconjugates a -PAA-HSA binding

[0205]

[0206]

[0207] To further examine carbohydrate specificity in detail, the 5B1 and 7E3 antibodies were also tested by glycan array analysis using the Consortium for Functional Glycomics Core H group. Both antibodies were tested at 10 μg / ml on a printed array consisting of 465 glycans, with six replicates. The results confirmed the high specificity of both antibodies, which selectively recognized sLe aThe tetrasaccharides Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ and Neu5Gcα2-3Galβ1-3(Fucα1-4)GlcNAcβ did not bind substantially to closely related antigens present in the array, including sLe x 、Le a 、Le x and Le y The results are summarized in Table 8, which shows the top 5 structures among the 465 glycan structures recognized by each antibody.

[0208] Table 8: Carbohydrate specificity analysis by glycan array screening.

[0209] A.5B1

[0210]

[0211] B.7E3

[0212]

[0213]

[0214] CDC activity

[0215] To evaluate the functional activity of 5B1 and 7E3, we tested their cytotoxic activity on DMS-79 cells in the presence of human serum as a supplement source. In some assays, both antibodies showed close to 100% killing activity at 10 μg / mL, whereas control antibodies with different specificities (1B7, anti-GD2 IgG1 mAb) had no effect at the same concentration (data not shown). CDC activity was concentration-dependent, and in this assay, 7E3 was significantly more active than 5B1 ( Figure 12 ), which is expected since IgM antibodies are known to be more effective in complement-mediated cytotoxicity assays. The EC50 (50% cytotoxicity) for 5B1 is 1.7 μg / mL and the EC50 for 7E3 is 0.1 μg / mL, which is approximately 85-fold more potent than 7E3 on a molar basis ( Figure 12 ).

[0216] ADCC activity

[0217] Although 7E3 was significantly more effective in the CDC assay, IgG antibodies are known to have antibody-dependent cell-mediated cytotoxicity (ADCC) activity, which is thought to be important for tumor killing in vivo. Using the 5B1 antibody, high cytotoxicity levels were measured at multiple E:T ratios using human PBMCs and DMS-79 target cells ( Figure 13A). Similar levels of cytotoxicity were observed at lower E:T ratios using primary NK cells ( Figure 13 B). Dose-response experiments using PBMCs from two donors at an E / T ratio of 100:1 showed similar potency, with greater than 85% cytotoxicity achieved at concentrations of 5B1 of 0.5 μg / mL or above ( Figure 13 C). 5B1-mediated cytotoxicity requires the FcγRIII receptor, as it can be blocked by the 3G8 anti-CD16 antibody. High levels of cytotoxicity were also measured using the 5B1 antibody against Colo205-luc cells using human PBMCs at an E:T ratio of 100:1. ADCC activity achieved at 1 μg / mL of the 5B1 antibody was superior to that observed using antibodies against GM2, Fucosyl-GM1, Globo H, or polysialic acid. As expected, 7E3 and murine 121SLE (both IgM) were inactive in this assay.

[0218] 5B1 internalization assay

[0219] It has been previously shown that antibody conjugates against antigens "closely related" to Lewis Y are rapidly internalized and very potent in animal models. Hellstrom et al., Cancer Res 50:2183-90 (1990); Trail et al., Science 261:212-5 (1993). To detect sLe a To determine whether 5B1 is internalized, we incubated the pancreatic cell line BxPC3 with 5B1 and then added Hum-ZAP, an anti-human IgG conjugated to the ribosome-inactivating protein saporin. Kohls et al., Biotechniques 28:162-5 (2000). Cells that internalized the saporin-containing complex died, while saporin that was not internalized did not damage the cells. Figure 14 As shown, BxPC3 cells were efficiently killed in the presence of high doses of 5B1, whereas cells were not killed in the presence of an isotype-matched IgG1 antibody against GD2 (which is not expressed on these cells).

[0220] Metastatic activity in xenograft animal models

[0221] To evaluate the in vivo activity of 5B1, the antibody was tested in two xenograft models using either Colo205-luc tumor cells or DMS-79 tumor cells in SCID mice. For the xenograft model using Colo205-luc tumor cells, 5 mice per group were injected into the tail vein on day 0 with 0.5 × 10 6Cells were injected into the animals and the successful injection of cells was verified by imaging the animals using an IVIS200 in vivo imaging system (Caliper Life Sciences). One day later, the animals were treated with 5B1 antibody or PBS mock injection administered intraperitoneally. In Experiment 1, 100 μg of 5B1 was administered on days 1, 7, 14, and 21 (400 μg total dose), and in Experiment 2, animals received 100 μg of 5B1 (600 μg total dose) on days 1, 4, 7, 10, 14, and 21. In Experiment 2, the average median survival time of untreated animals was 102 days, and all untreated animals died within 155 days ( Figure 15 ). Treatment of the animals significantly improved survival: in the group that received four doses of 5B1, the median survival time doubled to 207 days, and two of the five animals survived until the end of the experiment after 301 days (log-rank test, P = 0.0499; HR = 3.46). When six doses were administered, the proportion of survivors further increased to three of five mice surviving (log-rank test, P = 0.0064; HR = 6.375). The second group of experiments was terminated after 308 days, and the surviving animals could not show Colo205-luc tumors on the most sensitive imaging system (data not shown).

[0222] In a second study, mice injected with Colo205-luc tumor cells similarly as described above were treated with high doses of 5B1 or 7E3 antibodies (100 μg, 300 μg, or 1 mg). Initially, all animals received intraperitoneal injections of 5B1 or 7E3 antibodies or PBS mock injections (controls) on day 4 after tumor cell injection, then twice a week for the first 2 weeks and once a week for the next 7 weeks. In SCID mice implanted with Colo205-luc tumor cells, delayed treatment with multiple 5B1 doses showed dose-dependent protection up to complete cure ( Figure 16 and Figure 17 Treatment with the 7E3 antibody did not show increased protection despite the apparent increase in avidity (data not shown).

[0223] In the xenograft model using DMS-79 cells, 5 mice per group were injected subcutaneously on day 0 with 1 × 10 6 cells, and when the tumor reaches 5 mm (~20 mm) in length 2 Treatment was initiated on day 19 after 48 hours of follow-up. Animals were then treated with either human IgG or 5B1 antibody administered intraperitoneally at 200 μg per dose, plus cRGD injected intravenously (80 μg initially, then 40 μg per dose five days a week until day 37). In animals treated with 5B1 or the combination of 5B1 plus cRGD, the growth of established DMS-79 tumors was inhibited or regressed ( Figure 18A and Figure 18B Treatment of animals with 5B1 completely prevented tumor growth in the subcutaneous model on the day of DMS-79 cell implantation (data not shown).

[0224] The above data demonstrate the remarkable ability of treatment with the 5B1 antibody to inhibit or regress established tumors and provide a survival benefit.

[0225] Example II

[0226] Pancreatic cancer and other sLeukemia using radiolabeled monoclonal antibody 5B1 a Immuno-PET detection and diagnosis of positive adenocarcinoma

[0227] Adenocarcinoma is the leading cause of cancer death. Detection of pancreatic cancer remains particularly difficult, with diagnosis often occurring at an advanced stage. Early detection of primary and metastatic pancreatic cancer could have a significant clinical impact. In clinical practice, monitoring of sLe a Elevated antigen levels are used to identify suspected occult malignancies in patients with pancreatic cancer. a Targeting sLe in preclinical models of sLe a The potential of a new immunoPET imaging probe. a Monoclonal antibody 5B1 showed activity against a known sLe a Positive staining for human adenocarcinoma, but not for sLe a Negative staining of malignant tumors or most normal tissues. 89 Zr radiolabeled 5B1 ( 89 Zr-5B1) showed high labeling (>80%) and purification yield (>95%). The use of Zr-5B1 was studied in subcutaneous, orthotopic, and metastatic pancreatic cancer xenografts in female SCID mice. 89 Imaging of Zr-5B1. The acquired PET images and biodistribution studies showed 89 Zr-5B1 vs sLe a Xenografts overexpressing BxPC3 showed excellent specificity and localization, with minimal nonspecific binding to healthy tissue. Further analysis in subcutaneous xenograft models of colon and small cell lung cancer was also confirmed by 89 Thus, these results indicate that Zr-5B1 has an excellent tumor profile. 89 Zr-5B1 can be used as sLe in clinical practice a Expression of molecular probes for early detection of malignant tumors.

[0228] Cell lines and tissue culture

[0229] All tissue culture procedures were performed using aseptic technique. Small cell lung cancer DMS79 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 humidified atmosphere of 5% CO according to the recommendations of ATCC and CLS.

[0230] sLe by FACS a In vitro evaluation of expression levels

[0231] Flow cytometry was performed as described herein using the indicated cultured cancer cell lines in Example 1. Briefly, 1×10 cells per tube were washed in PBS with 3% fetal bovine serum (FBS). 6 Then, human monoclonal antibody r5B1 (anti-sLe a IgG) and incubated on ice for 30 minutes. After washing in PBS with 3% FBS, 20 μl of 1:25 diluted goat anti-human IgG labeled with fluorescein-isothiocyanate (FITC, Southern Biotechnology, Birmingham, AL) was added and the mixture was incubated on ice for another 30 minutes. After the final wash, FACS scanning (Becton & Dickinson, San Jose, CA) was used to distinguish the positive population and median fluorescence intensity of stained cells. Cells stained only with fluorescein-isothiocyanate labeled goat anti-human IgG were used as background. The FACScan results were set to 1% for comparison with the percentage of positive cells stained with the primary mAb.

[0232] 89 Preparation of Zr-labeled antibodies

[0233] Recombinant 5B1 antibody was prepared and purified as described herein. 5B1 antibody and nonspecific human IgG were functionalized with p-isothiocyanate benzyl-deferoxamine (DFO-Bz-NCS, Macrocyclics, Inc., Dallas, TX) at a 1:4 mAb:DFO-Bz-NCS ratio. For example, a volume of 7.2 μL of DFO-Bz-NCS (4.25 mM in DMSO) was added to 300 μL of 5B1 (1.23 mg in PBS, pH ~9). The reaction was incubated at 37°C for 1-1.5 hours. The functionalized antibody was purified using a PD10 desalting column (GE Healthcare) or a 10 kDa centrifugal filter (Amicon).

[0234] Zr-89 was produced by proton beam bombardment of yttrium foil and isolated at high purity as Zr-89 oxalate at MSKCC according to previously established procedures. Holland et al., Nuclear Medicine and Biology 36:729-39 (2009). Antibody labeling was performed by the method described in Holland et al., Journal of Nuclear Medicine official publication, Society of Nuclear Medicine 51:1293-300 (2010). Typically, Zr-89 oxalate was neutralized to pH 7.0-7.2 with 1 M Na2CO3. Then, DFO-antibody was 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.

[0235] In vitro experiments

[0236] At 37 °C, in 0.9% saline and 1% bovine serum albumin, the 89 The in vitro stability of Zr-5B1 was 5 days. The changes in radiochemical purity were monitored by radioactive iTLC using 50 mM DTPA as the mobile phase from t = 0 to 5 days. In vitro immunoreactivity assays were performed to demonstrate the integrity of the Zr-89 radiolabeled antibody according to the protocol described by Lindmo et al., Journal of Immunological Methods 72: 77-89 (1984).

[0237] Animal models

[0238] All animal studies were performed in accordance with the guidelines set by the Institutional Animal Care and Use Committee. Female CB17SC-F SCID mice (Jackson Laboratories, 6-8 weeks, 20-22 g) or athymic (nu / nu) nude mice were induced with tumors in the hind legs. All cell lines were inoculated subcutaneously in 200 μL of a 1:1 culture medium:Matrigel (BD Biosciences) solution and allowed to grow to a maximum tumor volume of 250 mm before use. 3 .

[0239] Biodistribution studies

[0240] Biodistribution studies were performed on groups of mice (n=3-5) bearing individual Colo205-luc colorectal, BxPC3 pancreatic, and DMS79 small cell lung xenografts. Zr-89 mAb (10-20 μCi, 1-2 μg) was administered intravenously in a lateral vein in 100 μl of 0.9% saline. Additional unlabeled mAb (10-50 μg) was co-injected with the tracer. Blocking studies were performed using a 250 μg excess of unlabeled mAb to account for antibody resistance to sLe in the mouse group. a Specificity. After each time point (t = 24 hours, 48 ​​hours, 120 hours, pi), mice were euthanized by asphyxiation with CO2. Blood was immediately collected by cardiac puncture and tumors were harvested simultaneously with the selected organs. The wet weight of each tissue was measured and the radioactivity bound to each organ was counted using a Wizard2 2480 gamma counter (Perkin Elmer). The percentage of tracer expressed as % injected dose / gram (% ID / g) was calculated as the decrease in activity per actual injected dose per unit organ weight - corrected to the counting time.

[0241] Small Animal Immunization-PET

[0242] Imaging experiments were performed using a microPET Focus 120 or R4 scanner (Concorde Microsystems). Zr-89 labeled antibodies (200-300 μCi, 15-25 μg) were administered to mice (n=3-5) in a 100-200 μL 0.9% saline formulation via lateral tail vein injection. At 24-96 hours, pi, PET whole-body acquisition was recorded for mice under anesthesia with 1.5-2.0% isoflurane (Baxter Healthcare) in oxygen. Images were analyzed using ASIPro VM™ software (Concorde Microsystems). Regions of interest (ROIs) were plotted and plotted relative to time.

[0243] Immunohistochemistry

[0244] Biotinylated 5B1 was prepared by incubating with a 20× molar excess of Sulfo-NHS-LC-Biotin (Thermo Scientific / Pierce cat# 21327) for 30 minutes at room temperature. Free biotin was removed using Zebra desalting spin columns (Thermo Scientific / Pierce, cat# 89889) according to the manufacturer's instructions. The antibody buffer was exchanged into PBS containing 0.01% sodium azide (concentration 1.1 mg / ml). Binding to DMS79 cells was confirmed by FACS and was comparable to that of the parental 5B1 antibody.

[0245] Basic 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 a solution of 10% (v / v) normal human serum in PBS (Jackson ImmunoResearch Labs; cat#009-000-121). Standard streptavidin-biotin immunoperoxidase was used and the DAB detection system was used as the staining method using a Ventana automated instrument (Discovery XT platform-Ventana Medical Systems, Inc, Tucson, AZ). Antigen retrieval was performed using heat and Ventana's CC1 conditioning solution. In preliminary studies, the CA 19.9 mouse monoclonal clone (clone 116-NS-19-9) from Signet (Covance) provided comparable results. When biotinylated 5B1 was used at 10 μg / ml, Colo205 cells were strongly positive, while SKMEL28 cells were completely negative. TM Tissue microarrays were purchased from Imgenex (San Diego, CA). The following slides containing tumor biopsy cores and some normal tissue cores were used: IMH-327 (common cancer, 59 samples), IMH-359 (colorectal: cancer-metastasis-normal; 59 samples), and IMH-324 (cancer metastatic to the ovary). Pancreatic tumor tissue cores were present on IMH-327.

[0246] sLe a Serum concentration in vivo

[0247] Mice bearing Colo205, BxPC3, and DMS79 xenografts were bled for sLe a Antigen determination. A group of tumor-free mice was used as a control. sLe in mouse serum was measured using the ST AIA-PACK CA19.9 kit (Cat#025271, TOSOH Bioscience Inc, South San Francisco, CA). aThe assay principle is based on a two-site immunoenzyme-metric assay. The assay was performed as described in the manufacturer's instruction manual. The optical density of the immunoassay plate was measured using a TOSOH AIA2000 automated immunoassay analyzer (TOSOH Bioscience, Inc, San Francisco, CA).

[0248] Statistical analysis

[0249] Unless otherwise indicated, data values ​​are expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism version 5.03 software using one-way analysis of variance followed by Dunnett's test. A P value of < 0.05 was considered statistically significant.

[0250] result

[0251] The binding specificity of 5B1 was explored by staining selected malignant and normal tissue microarrays. 5B1 reactivity was limited to malignant tumors and occasionally some cells previously known to overexpress sLe a Normal tissue ( Figure 19 ; Table 9). Most normal tissues were completely negative (Table 9). In contrast, strong positive staining was found in 21 / 34 colon adenocarcinomas (62%), 33 / 57 adenocarcinomas metastatic to the ovary (58%), and 7 / 9 pancreatic ductal carcinomas (66%) at all stages (Table 10). Figure 19 Shown in Figure 2, typical reactivity is diffuse cytoplasmic staining, and some of these tumor cells clearly demonstrate the obvious staining of cell membrane. In addition, also find that some signet ring cell ovarian cancers and some lung cancers and breast cancers are strongly positive. On the contrary, only 4 / 43 routine prostate cancer samples and 0 / 51 routine GIST case are positive (data not shown).

[0252] Table 9: Investigation of 5B1 binding to normal tissues.

[0253]

[0254] Table 10: 5B1 staining of pancreatic ductal adenocarcinoma

[0255]

[0256]

[0257] 5B1 expression of sLe aThe high specificity of immunostaining of cancer tissues was demonstrated using this mAb as a PET probe. 5B1 was modified with the benzyl-isothiocyanate analog of deferoxamine (DFO-Bz-NCS) in a ratio of 4:1 (chelate:mAb) and subsequently purified by centrifugal filtration using saline as a washing buffer. Simple radiolabeling with Zr-89 was performed at room temperature after pH adjustment to 7.0-7.2. A narrower pH range closer to neutral was necessary to achieve an optimal radiolabeling yield of >80%. Free, unbound Zr-89 was removed by a PD10 desalting column. The product was concentrated using a centrifugal filter (MWCO: 10 kDa). A relatively high specific activity of 12.1±1.1 mCi / mg was established. Before use, a radiochemical purity of greater than 95% was ensured. Immunoreactivity assays showed a strong affinity for sLe a Activity was retained (72.4 ± 1.1%, n = 3). Stability remained > 95% in bovine serum albumin at 37°C over 5 days (data not shown). In saline, demetallation (> 85% complexation) was observed as early as 24 hours, with approximately > 75% of the radiometal bound after 120 hours at 37°C.

[0258] Small animal PET imaging and biodistribution studies were performed using female SCID mice implanted subcutaneously with BxPC3 pancreatic cancer xenografts in the left hind leg. 89 PET images acquired with Zr-5B1 confirmed tumor-associated sLe a According to the obvious outline of Figure 20 Maximum intensity projections (MIPs) in BxPC3 xenografts (n=3) showed superior accumulation of intravenously administered radiotracer. Regions of interest (ROIs) drawn on tumors from PET images showed uptake of 5.0±0.4% ID / g (2h), 16.2±2.5% ID / g (24h), 23.8±4.7% ID / g (48h), 36.8±6.1% ID / g (96h), and 49.5±7.7% ID / g (120h). After 24h pi, binding activity to blood mixtures and normal tissues appeared clear. The results of the biodistribution experiments were consistent with the PET data. At 24 hours, 89 Zr-5B1 was highly tumor localized (84.7±12.3% ID / g, n=4); at 120 h pi, it further showed enhanced uptake (114.1±23.1% ID / g, n=4) ( Figure 21Due to the small body weight (62.4 ± 0.03 mg), tumor uptake exceeded 100%. The %ID at 24 h pi was found to be 10-fold higher than that of nonspecific IgG at similar time points ( Figure 21 Competitive inhibition by 250 μg of non-radiolabeled 5B1 at 24 h pi blocked the accumulation of the tracer, which defined the uptake specificity. 89 Zr-5B1 had minimal binding to normal pancreas and other harvested normal tissues, providing high tumor-to-tissue contrast at all time points.

[0259] Based on the above results, the BxPC3 pancreatic tumor model was used to determine 89 Zr-5B1. The orthotopic model is clinically relevant and provides a clinically acceptable test of the efficacy of PET probes. After inoculation in the pancreas, tumor growth was monitored weekly by bioluminescent optical imaging. Once tumors were palpable, PET imaging experiments were performed. 89 Comparison of the schematic properties of the probe tumor profile between Zr-5B1 ( Figure 25 Computed tomography (CT) coupled with PET provides enhanced imaging of anatomical regions of interest.

[0260] In order to a Evaluation of PET probes in adenocarcinomas expressing 89 Zr-5B1 was tested in lung and colon cancer models. 89 Small animal experiments were conducted using DMS79 small cell lung cancer cells and Colo205-luc colon cancer cells injected subcutaneously into the right hind leg of female SCID mice. PET MIP images were acquired 24-120 h pi after intravenous injection of 200-300 μCi (16-25 μg). Heterogeneous DMS79 tumor uptake (38.15 ± 2.12% ID / g) was demonstrated as early as 24 h pi, with excellent signal to background ( Figure 22 A). After 48 h pi, the tracer tumor accumulation increased (44.60 ± 6.47% ID / g), which was still retained at 120 h pi (41.97 ± 12.23% ID / g). Nonspecific binding was rapidly cleared from normal tissues. 89 Zr-5B1, which had minimal to no background at 48 h pi. Figure 22As shown in B, tumor contours were observed in Colo205-luc xenografts at 24-120 h p.i. ROIs were shown at 2, 24, 48, 96, and 120 h, with tumor accumulation of 10.5 ± 0.76, 23.5 ± 2.7, 24.8 ± 4.0, 18.4 ± 4.7, and 16.5 ± 2.3% ID / g, respectively. As shown by the region of interest plotted in the PET images, liver accumulation observably increased over time, followed by a decrease in tumor uptake ( Figure 22 C) Data generated from the biodistribution studies correlated well with the observed PET results (data not shown).

[0261] As the tumor develops, the serum levels of sLe a Levels were quantified. Blood was drawn from SCID mice with Colo205, DMS79, and BxPC3 xenografts, and a tumor-free group was used as a control. a The values ​​showed that sLe in Colo205-challenged mice were significantly higher than those in pancreatic BxPC3- and DSM79-implanted mice. a The level was relatively high (Table 11).

[0262] Table 11: sLe in mice bearing colorectal (Colo205), pancreatic (BxPC3) and small cell lung (DMS79) tumor xenografts compared to controls a Serum values.

[0263]

[0264]

[0265] ND = not detected.

[0266] These results indicate that radiolabeled anti-sLe a Antibody( 89 Zr-5B1) for pancreatic adenocarcinoma and other sLe a The detection and diagnosis of positive adenocarcinoma is specific. It is prepared with excellent yield and purity as well as high specific activity and retained immunoreactivity. 89 Zr-5B1. in subcutaneous, orthotopic, and metastatic pancreatic tumor models 89 Evaluation of Zr-5B1 provided excellent tumor delineation and diagnosis. Preclinical evaluation of this radiotracer in small animals with colon and small cell lung tumors demonstrated that this tracer is effective for tumors expressing sLe a Universal use for the treatment of malignant tumors.

[0267] Example III

[0268] Anti-sLe aDiabody binds to multiple cancer cell lines

[0269] Two diabodies were generated using the VH and VL domains of the 5B1 and 7E3 clonal isolates described herein and are designated 5B1CysDb and 7E3CysDb, respectively. Figure 9 and Figure 10 Both diabodies contain a 5-amino acid linker region between the VL and VH domains. Both diabodies also include a C-terminal polyhistidine tag for purification and detection.

[0270] The binding of 5B1CysDb and 7E3CysDb to three cancer cell lines was determined by incubating 250,000 cells in 0.2 mL with 10 μg / mL of either 5B1CysDb or 7E3CysDb, respectively: (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. The cells and the diabody combination were incubated in PBS / 2% FBS on ice for 40 minutes.

[0271] After washing, the cells were incubated with 0.2 mL of a 1:1000 dilution of ALEXA-488-labeled anti-His antibody (Life Technologies, Cat# A21215) for 40 minutes. After a second wash, the cells were analyzed using a Guava flow cytometer. Both 5B1CysDb and 7E3CysDb showed significant binding to DMS-79, Capan-2, and BxPC3 cells (Table 12).

[0272] Table 12: Binding of 5B1CysDb and 7E3CysDb to cell lines.

[0273]

[0274] MFI - mean fluorescence intensity

[0275] Example IV

[0276] Administration of 5B1 and Taxol inhibits tumor growth

[0277] Co-administration of anti-sLe was evaluated in xenograft models of pancreatic cancer and small cell lung cancer. a Antitumor activity of antibody (5B1) and chemotherapeutic agent Taxol (paclitaxel). As described above, 1 million BxPc3 cells (pancreatic tumor cells) or 5 million DMS-79 cells (small cell lung cancer cells) were injected into the flank of 6-week-old female CB17 SCID mice (day 0; N=5). DMS79 tumors were grown for 21 days until the average tumor size was 193±64 mm. 3Human IgG or 5B1 (0.5 or 1 mg) was administered ip twice weekly (starting on day 21), and Taxol (0.2 mg / dose) was administered iv on days 23, 30, 37, and 44. In the DMS-79 xenograft model, co-administration of 5B1 antibody and Taxol significantly limited tumor growth and resulted in tumor regression compared to control human IgG or 5B1 antibody and Taxol administered alone ( Figure 23 ).

[0278] In the BxPc3 xenograft model, tumors grew for 14 days, at which time they reached an average of 126 ± 30 mm 3 Taxol was administered iv on days 14, 21, 28, and 34 (weekly), and 5B1 was administered twice weekly starting on day 14. Co-administration of 5B1 antibody and Taxol significantly limited tumor growth compared to control or 5B1 antibody and Taxol administered alone ( Figure 24 These results suggest that anti-sLe a Synergistic effect of antibodies and chemotherapeutic agents in preventing tumor growth and / or reducing tumor size.

[0279] Throughout the present application, reference is made to a number of documents. The disclosures of these documents are incorporated herein by reference in their entirety to more fully describe the current state of the art in the field to which the present invention pertains. Although the present invention has been described with reference to the embodiments provided above, it will be appreciated that various modifications may be made without departing from the spirit of the present invention. Sequence Listing <110> Biotechnology R&D Company <120> Nucleic acid encoding human antibodies against sialyl Lewis a antigen <130> 12967-033-228 <140> PCT / US2014 / 052631 <141> 2014-08-26 <150> US 61 / 870,137 <151> 2013-08-26 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Clone 5B1 VH chain domain <400> 1 atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggcgt acagtgccag 60 gtgcagctgg tggagtctgg gggaggctcg gtgcagcctg gcaggtccct gagactctcc 120 tgtgaagcct ctggattcac ctttgaggcc tatgccatgc actgggtccg gcaacctcca 180 gggaagggcc tggagtgggt ctcaagtatt aattggaata gtggtcgcat agcctatgcg 240 gactctgtga agggccgatt caccatctcc agagacaacg ccaggaattc cctgtatctg 300 caaatgaaca gtctgagact tgaggacacg gccttctatt actgtgcaaa agatatacgg 360 aggtttagta ccgggggggc ggagtttgag tactggggcc agggaaccct ggtcaccgtc 420 tcctca 426 <210> 2 <211> 142 <212> PRT <213> Artificial Sequence <220> <223> VH domain of clone 5B1 <400> 2 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe 35 40 45 Glu Ala Tyr Ala Met His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Ser Ile Asn Trp Asn Ser Gly Arg Ile Ala Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn 85 90 95 Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Leu Glu Asp Thr Ala Phe 100 105 110 Tyr Tyr Cys Ala Lys Asp Ile Arg Arg Phe Ser Thr Gly Gly Ala Glu 115 120 125 Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 130 135 140 <210> 3 <211> 390 <212> DNA <213> Synthetic sequence <220> <223> VL domain of clone 5B1 <400> 3 atggccggct tccctctcct cctcaccctc ctcactcact gtgcagggtc ttgggcccag 60 tctgtgctga ctcagccgcc ctcagcgtct gggacccccg ggcagagggt caccatctct 120 tgttctggaa gcagctccaa catcggaagt aattttgtat actggtacca gcagctccca 180 ggaacggccc ccaaactcct catatatagg aataatcagc ggccctcagg ggtccctgac 240 cgattctctg gctccaggtc tggcacctca gcctccctgg ccatcagtgg actccggtcc 300 gaggatgagg ctgattatta ctgtgcagca tgggatgaca gcctgggagg ccattatgtc 360 ttcggaactg ggaccaaggt caccgtcctt 390 <210> 4 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> VL domain of clone 5B1 <400> 4 Met Ala Gly Phe Pro Leu Leu Leu Thr Leu Leu Thr His Cys Ala Gly 1 5 10 15 Ser Trp Ala Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 20 25 30 Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile 35 40 45 Gly Ser Asn Phe Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 50 55 60 Lys Leu Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp 65 70 75 80 Arg Phe Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 85 90 95 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp 100 105 110 Asp Ser Leu Gly Gly His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr 115 120 125 Val Leu 130 <210> 5 <211> 426 <212> DNA <213> Artificial sequence <220> <223> VH chain domain of clone 9H3 <400> 5 atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggcgt acagtgcgaa 60 gtgcagctgt tggagtctgg gggaggcttg gtacagcctg gcaggtccct gagactctcc 120 tgtgcggcct ctggatttac ctttgatgat tatgtcatgc actgggtccg gcaagctcca 180 gggaagggcc tggagtgggt ctcaagtatt agttggaata gtggtagcat aggctatgcg 240 gactctgtga agggccgatt catcatctcc agagacaacg ccaagaactc cctgtatctg 300 caaatgaaca gtctgagagc tgaggacacg gccttgtatt actgtgcaaa agatcgtcgt 360 attaggggtg actcggggtt cgagggtgac tactggggcc agggaaccct ggtcaccgtc 420 tcctca 426 <210> 6 <211> 142 <212> PRT <213> Artificial sequence <220> <223> VH domain of clone 9H3 <400> 6 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Asp Asp Tyr Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Ser Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu 100 105 110 Tyr Tyr Cys Ala Lys Asp Arg Arg Ile Arg Gly Asp Ser Gly Phe Glu 115 120 125 Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 130 135 140 <210> 7 <211> 387 <212> DNA <213> Synthetic sequence <220> <223> VL domain of clone 9H3 <400> 7 [[ID=2...]] atggccggct tccctctcct cctcaccctc ctcactcact gtgcagggtc ttgggcccag 60 tctgtgttga cgcagccgcc ctcagcgtct gggacccccg ggcagagggt caccatctct 120 tgttctggaa gcagctccaa catcggaagt aattatgtat actggtacca gcagctccca 180 ggaacggccc ccaaactcct catctatagg aataatcagc ggccctcagg ggtccctgac 240 cgattctctg gctccaagtc tggcacctca gcctccctgg ccatcagtgg gctccggtcc 300 gaggatgagg ctgattatta ctgtgcagca tgggatgcca gcctgagtgg tgtggtattc 360 ggcggaggga ccaagctgac cgtccta 387 <210> 8 <211> 129 <212> PRT <213> Synthetic sequence <220> <223> Cloning of the VL chain domain of 9H3 <400> 8 Met Ala Gly Phe Pro Leu Leu Leu Thr Leu Leu Thr His Cys Ala Gly 1 5 10 15 Ser Trp Ala Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 20 25 30 Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile 35 40 45 Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 50 55 60 Lys Leu Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp 65 70 75 80 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 85 90 95 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp 100 105 110 Ala Ser Leu Ser Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val 115 120 125 Leu <210> 9 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Clone the VH chain domain of 5H11 <400> 9 atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggcgt acagtgccag 60 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly Val Gln Cys Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln gtgcagctgt tggagtctgg gggaggcttg gtacagcctg gcaggtccct gagactctcc 120 tgtgcagcct ctggattcac ctttgatgaa tatgccatgc actgggtccg gcaagctcca 180 gggaagggcc tggagtgggt ctcaagtgtt agttggaata gtggtagcat aggctatgcg 240 gactctgtga agggccgatt caccatctcc agagacaacg ccaagaactc cctgtatcta 300 caaatgaaca gtctgagagc tgaggacacg gccttgtatt actgtgcaaa agatatacgg 360 acctatagca ccgggggggc ggagtttgcc tcctggggcc agggaaccct ggtcaccgcc 420 tcctca 426 <210> 10 <211> 142 <212> PRT <213> Artificial Sequence <220> <223> VH domain of clone 5H11 <400> 10 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Asp Glu Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Ser Val Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu 100 105 110 Tyr Tyr Cys Ala Lys Asp Ile Arg Thr Tyr Ser Thr Gly Gly Ala Glu 115 120 125 Phe Ala Ser Trp Gly Gln Gly Thr Leu Val Thr Ala Ser Ser 130 135 140 <210> 11 ''<211> 390 <212> DNA <213> Artificial sequence <220> <223> Clone the VL domain of 5H11 <400> 11 atggccggct tccctctcct cctcaccctc ctcactcact gtgcagggtc ttgggcccag 60 tctgtgttga cgcagccgcc ctcagcgtct gggacccccg ggcagagggt caccatctct 120 tgttctggaa gcagctccaa catcggaagt aattatgtat actggtacca gcaggtccca 180 tgttctggaa gcagctccaa catcggaagt aattatgtat actggtacca gcaggtccca 180 ggaacggccc ccaaactcct catctatagg aataatcagc ggccctcagg ggtccctgac 240 ggaacggccc ccaaactcct catctatagg aataatcagc ggccctcagg ggtccctgac 240 cgattctctg gctccaagtc tggcacctca gcctccctgg ccatcagtgg gctccggtcc 300 cgattctctg gctccaagtc tggcacctca gcctccctgg ccatcagtgg gctccggtcc 300 gaggatgagg ctgattatta ctgtgcagca tgggatgaca gcctgagtgg ccattatgtc 360 gaggatgagg ctgattatta ctgtgcagca tgggatgaca gcctgagtgg ccattatgtc 360 ttcggaactg ggaccaaggt caccgtccta 390 ttcggaactg ggaccaaggt caccgtccta 390 <210> 12<210> 12 <211> 130<211> 130 <212> PRT<212> PRT <213> Artificial sequence<213> Artificial sequence <220><220> <223> Clone the VL domain of 5H11<223> Clone the VL domain of 5H11 <400> 12<400> 12 Met Ala Gly Phe Pro Leu Leu Leu Thr Leu Leu Thr His Cys Ala Gly Met Ala Gly Phe Pro Leu Leu Leu Thr Leu Leu Thr His Cys Ala Gly 1 5 10 15 1 5 10 15 Ser Trp Ala Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Ser Trp Ala Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 20 25 30 20 25 30 Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile 35 40 45 35 40 45 Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Val Pro Gly Thr Ala Pro Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Val Pro Gly Thr Ala Pro 50 55 60 50 55 60 Lys Leu Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Lys Leu Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp 65 70 75 80 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 85 90 95 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp 100 105 110​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​caaatgaaca gcctgagagc tgaggacacg gctgtgtatt actgtgcgaa aaggcccaac 360 caattttatt gtagtgatgg tagatgctac tccattgact actggggcca gggaaccctg 420 gtcaccgtct cctca 435 <210> 14 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> VH domain of cloned 7E3 <400> 14 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Phe Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu<00012�1>50 55 60 Glu Trp Val Ala Ala Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala ​​​​​​Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Lys Arg Pro Asn Gln Phe Tyr Cys Ser Asp Gly Arg 115 120 125 Cys Tyr Ser Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 130 135 140 Ser 145 <210> 15 <211> 390 <212> DNA <213> Artificial Sequence <220> <223> VL domain of clone 7E3 <400> 15 atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc 60 cggtgtgaaa ttgtaatgac gcagtctcca gccaccctgt ctgtgtctcc aggggagaga 120 gccaccctct cctgcagggc cagtcagagt gttagcagca acttagcctg gtaccagcag 180 aaacctggcc aggctcccag gctcctcatc tatggtgcat ccaccagggc cactggtatc 240 ccagccaggt tcagtggcag tgggtctggg acagacttca ctctcaccat cagcagcctg 300 cagtctgtag attctgcagt ttattactgt cagcagtata ataactggcc tccgtacact 360 tttggccagg ggaccaagct ggagatcaaa 390 <210> 16 <211> 130 <212> PRT <213> artificial sequence <220> <223> clone7E3's VL chain structure <400> 16 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Glu Ile Val Met Thr Gln Ser Pro Ala Thr 20 25 30 Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser 35 40 45 Gln Ser Val Ser Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60 Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile 65 70 75 80 Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Ser Val Asp Ser Ala Val Tyr Tyr Cys Gln Gln 100 105 110 Tyr Asn Asn Trp Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu 115 120 125 Ile Lys 130 <210> 17 <211> 750 <212> DNA <213> Artificial sequence <220> <223> Double-chain antibody 5B1CysDb sequence <400> 17 cagtctgtgc tgacgcagcc gccctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgttctg gaagcagctc caacatcgga agtaattttg tatactggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatatat aggaataatc agcggccctc aggggtccct 180 gaccgattct ctggctccag gtctggcacc tcagcctccc tggccatcag tggactccgg 240 tccgaggatg aggctgatta ttactgtgca gcatgggatg acagcctggg aggccattat 300 gtcttcggaa ctgggaccaa ggtcaccgtc ctttctggtg gtggtggtca ggtgcagctg 360 gtggagtctg ggggaggctc ggtgcagcct ggcaggtccc tgagactctc ctgtgaagcc 420 tctggattca cctttgaggc ctatgccatg cactgggtcc ggcaacctcc agggaagggc 480 ctggagtggg tctcaagtat taattggaat agtggtcgca tagcctatgc ggactctgtg 540 aagggccgat tcaccatctc cagagacaac gccaggaatt ccctgtatct gcaaatgaac 600 agtctgagac ttgaggacac ggccttctat tactgtgcaa aagatatacg gaggtttagt 660 accggggggg cggagtttga gtactggggc cagggaaccc tggtcaccgt ctcctcaggt 720 tctcaccatc accatcacca tggcggttgc 750 <210> 18 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> Double - chain antibody 5B1CysDb sequence <400> 18 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Phe Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Gly Gly His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Ser 100 105 110 Gly Gly Gly Gly Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val 115 120 125 Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr 130 135 140 Phe Glu Ala Tyr Ala Met His Trp Val Arg Gln Pro Pro Gly Lys Gly 145 150 155 160 Leu Glu Trp Val Ser Ser Ile Asn Trp Asn Ser Gly Arg Ile Ala Tyr 165 170 175 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg 180 185 190 Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Leu Glu Asp Thr Ala 195 200 205 Phe Tyr Tyr Cys Ala Lys Asp Ile Arg Arg Phe Ser Thr Gly Gly Ala 210 215 220 Glu Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 225 230 235 240 Ser His His His His His His Gly Gly Cys 245 250 <210> 19 <211> 750 <212> DNA <213> Artificial sequence <220> <223> Bispecific antibody 7E3CysDb sequence <400> 19 gatgttgtgc tgacgcagtc tccagccacc ctgtctgtgt ctccagggga gagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcaacttag cctggtacca gcagaaacct 120 ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc 180 aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctgcagtct 240 gaagattctg cagtttatta ctgtcagcag tataataact ggcctccgta cacttttggc 300 caggggacca aggtggatat caaatctggt ggtggtggtg aagtgcagct ggtggagtct 360 gggggaggcg tggtccagcc tgggaggtcc ctgagactct cctgtgcagc ctctggattc 420 accttcagtt tctatggcat gcactgggtc cgccaggctc caggcaaggg gctggagtgg 480 gtggcagcta tatcatatga tggaagtaat aaatactatg cagactccgt gaagggccga 540 ttcaccatct ccagagacaa ttccaagaac acgctgtatc tgcaaatgaa cagcctgaga 600 gctgaggaca cggctgtgta ttactgtgcg aaaaggccca accaatttta ttgtagtgat 660 ggtagatgct actccattga ctactggggc cagggaaccc tggtcaccgt ctcctcaggt 720 tctcaccatc accatcacca tggcggttgc 750 <210> 20 <211> 249 <212> PRT <213> Artificial sequence <220> <223> Double - chain antibody 7E3CysDb sequence <400> 20 Asp Val Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu 65 70 75 80 Asp Ser Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Ser Gly Gly Gly Gly 100 105 110 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 115 120 125 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr 130 135 140 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 145 150 155 160 Ala Ala Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 165 170 175 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 180 185 190 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 195 200 205 Ala Lys Arg Pro Asn Gln Phe Tyr Cys Ser Asp Gly Arg Cys Tyr Ser 210 215 220 Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser 225 230 235 240 His His His His His His Gly Gly Cys 245

Claims

1. An isolated antibody conjugated to sialyl Lewis a or a polypeptide comprising an antigen-binding fragment thereof, said antibody or polypeptide comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, said VH domain having VH CDR1, VH CDR2 and VH CDR3 amino acid sequences, and said VL domain having VL CDR1, VL CDR2 and VL CDR3 amino acid sequences, wherein The VH CDR1 amino acid sequence is residues 55 - 62 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; and The VL CDR3 amino acid sequence is residues 109 - 120 of SEQ ID NO:

4.

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

3. The isolated antibody or polypeptide according to claim 1 or 2, wherein the polypeptide comprising an antigen - binding fragment is selected from Fab, Fab', F(ab')2, scFV, diabody, triabody, and minibody.

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

5. The isolated antibody or polypeptide according to claim 1, wherein the antibody is a monoclonal antibody.

6. The isolated antibody or polypeptide according to claim 5, wherein the antibody is of IgG or IgM isotype.

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

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

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

10. The conjugate according to claim 9, wherein the conjugate comprises a detectable reagent.

11. The conjugate according to claim 10, wherein the detectable reagent is a radioactive substance.

12. The conjugate according to claim 11, wherein the radioactive substance is selected from 89 Zr, 131 I, 125 I, 124 I, 123 I, 121 I, 14 C, 11 C, 35 S, 3 H, 115 In, 113 In, 112 In, 111 In, 99 Tc, 201 Ti, 68 Ga, 67 Ga, 103 Pd, 99 Mo, 133 Xe, 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 Sn.

13. The conjugate according to claim 10, wherein the detectable reagent is a fluorescent substance.

14. The conjugate according to claim 13, wherein the fluorescent substance is selected from umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin.

15. The conjugate according to claim 9, wherein the conjugate comprises a therapeutic agent.

16. The conjugate according to claim 15, wherein the therapeutic agent is a radioactive metal.

17. The conjugate according to claim 16, wherein the radioactive metal is an α - emitter.

18. The conjugate according to claim 15, wherein the therapeutic agent is an auristatin molecule.

19. The conjugate according to claim 18, wherein the auristatin molecule is selected from auristatin PHE, bryostatin 1, dolastatin 10, monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF).

20. A pharmaceutical composition comprising the antibody or polypeptide according to any one of claims 1-7, the conjugate according to any one of claims 9-10, or the isolated polynucleotide according to claim 8, and a pharmaceutically acceptable carrier.

21. Use of a therapeutically effective amount of the pharmaceutical composition according to claim 20 in the preparation of a medicament for treating a disease in a subject in need thereof, wherein the disease is cancer or tumor formation, and wherein the cells of the cancer or the tumor express sLe a , and wherein the cancer or tumor is selected from colon cancer, pancreatic adenocarcinoma, and small cell lung cancer. Use of an effective amount of a conjugate according to any one of claims 9 - 14 in the preparation of a medicament for detecting cancer in a subject, said cancer expressing sLe a , wherein said cancer is selected from colon cancer, pancreatic adenocarcinoma and small cell lung cancer.

Images