Anti-glycan antibodies and uses thereof

Abstract

To provide an alternative antibody therapy targeting glycan epitopes.SOLUTION: Provided is an antibody or antigen-binding portion thereof that binds to sialyl Lewis A (sLeA) and sialyl Lewis C (sLeC), wherein the binding affinity of the antibody or antigen-binding portion to sLeA is a KD of 60 μ M or less and the binding affinity to sLeC is a KD of 100 μ M or less. Also provided are polynucleotides, vectors, host cells, pharmaceutical compositions, and methods related thereto.SELECTED DRAWING: Figure 2C

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 990,927, filed March 17, 2020, U.S. Provisional Application No. 63 / 037,374, filed June 10, 2020, and U.S. Provisional Application No. 63 / 074,956, filed September 4, 2020, each of which is incorporated by reference in its entirety.

[0002] The present invention relates to anti-sialylated glycan antibodies, including anti-sialyl Lewis A (sLeA) antibodies and anti-sialyl Lewis C (sLeC) antibodies, and methods of using them.

[0003] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 203462000140SEQLIST.TXT, Recorded: March 15, 2021, Size: 14KB). [Background technology]

[0004] Glycans are carbohydrate-based polymers that can be free or conjugated to proteins (glycoproteins) or lipids (glycolipids). Glycans on glycoproteins, such as sialylated glycoprotein glycans, are known to be involved in immunity and inflammation. High expression of sLeA has been reported in human pancreatic, colonic, and gastric cell lines, as well as in colonic, gastric, and pancreatic adenocarcinomas. Furthermore, studies have shown that sLeA is upregulated in response to CD44v6 in inflamed areas of the human colon in patients with ulcerative colitis. Furthermore, studies have demonstrated a functional role for sLeA in mucosal inflammation, thereby suggesting that modulating sLeA may impact inflammatory bowel diseases (e.g., Crohn's disease or ulcerative colitis).

[0005] In addition, aberrant glycosylation is also a hallmark of cancer and has been reported to regulate immune responses. Similar to what has been observed in inflammatory bowel disease tissues, aberrant protein glycosylation leads to the overexpression of cancer-associated carbohydrate antigens during malignant transformation. Studies have shown that cancer-associated carbohydrate antigens contribute to various aspects of cancer development and progression, including cancer proliferation, invasion, angiogenesis, and metastasis (Fuster, Nat Rev Cancer, 5:526-42 (2005) and Dube, Nat Rev Drug Discovery, 4:477-88 (2005)).

[0006] One type of glycosylation is sialic acid, which is typically found at the branched terminus of N-glycans, O-glycans, and glycosphingolipids. Within sialic acid, diversity is created among the nine carbon backbones through α-linkages, and a second level of diversity is created through modifications at these carbon positions. Examples of sialylated glycosylations that have been identified include sLeA, sLeC, and sialyl Lewis X (the structures of each glycan are listed in Table 1).

[0007] All of these glycans are examples of terminally sialylated glycans. sLeC is a nonfucosylated precursor of sLeA, while sLeX is a stereoisomer of sLeA. In general, sLeC is not commonly found in normal human tissues, but is found in mouse tissues. Therefore, antibodies that bind to both sLeA and sLeC would be useful for drug development, especially in in vivo mouse models of disease.

[0008] Generally, antibodies suitable for development as pharmaceuticals to treat diseases and / or disorders have high specificity, selectivity, and affinity. Therefore, anti-glycan antibodies are generally not suitable for development as pharmaceuticals. This is partly because carbohydrate antigens have limited immunogenicity, and the observed affinity of anti-glycan antibodies is typically 10 times lower than that of antibodies specific for protein or peptide antigens. 3 ~10 5The overall weak binding of carbohydrates is usually associated with a relatively fast k on and k off The overall low K D is brought about.

[0009] The present invention addresses the need for alternative antibody therapies that target glycan epitopes for patients with disorders affecting the digestive system, including inflammatory bowel disease and cancers of the digestive system. Summary of the Invention

[0010] The present invention provides antibodies or antigen-binding portions thereof comprising a variable region that binds to a carbohydrate antigen or a carbohydrate antigen fragment.

[0011] In one aspect, the present invention provides an antibody or antigen-binding portion thereof that binds to sialyl Lewis A (sLeA) and sialyl Lewis C (sLeC), wherein the binding affinity of such antibody or antigen-binding portion to sLeA is 60 μM or less. D and has a K of 100 μM or less binding affinity to sLeC. D In some embodiments, the antibody or antigen-binding portion thereof is a humanized antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof is:

[0012] In another aspect, provided herein is an antibody or antigen-binding portion thereof that binds to sLeA and sLeC, the antibody or antigen-binding portion thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence having 90-100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the light chain variable region comprises a sequence having 90-100% sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In some embodiments, the antibody or antigen-binding portion thereof does not bind to sLeX. In some embodiments, the heavy chain variable region comprises CDR-H1 comprising the sequence of SEQ ID NO: 12, CDR-H2 comprising the sequence of SEQ ID NO: 13, and CDR-H3 comprising the sequence of SEQ ID NO: 14, and the light chain variable region comprises CDR-L1 comprising a sequence selected from the group consisting of SEQ ID NOs: 15-17, CDR-L2 comprising the sequence of SEQ ID NO: 18, and CDR-L3 comprising the sequence of SEQ ID NO: 19. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 3, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 5 to 8. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 3, and the light chain variable region comprises the sequence of SEQ ID NO: 8. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 4, and the light chain variable region comprises the sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0013] In another aspect, provided herein is an antibody or antigen-binding portion thereof that binds to sLeA and sLeC, the antibody or antigen-binding portion thereof comprising a heavy chain variable region, wherein the heavy chain variable region comprises a sequence having 90 to 100% sequence identity to SEQ ID NO: 3. In some embodiments, the antibody or antigen-binding portion thereof comprises a light chain variable region, wherein the light chain variable region comprises a sequence having 90 to 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 5 to 11.

[0014] In another aspect, provided herein is an antibody or antigen-binding portion thereof that binds to sLeA and sLeC, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1 comprising the sequence of SEQ ID NO: 12, CDR-H2 comprising the sequence of SEQ ID NO: 13, and CDR-H3 comprising the sequence of SEQ ID NO: 14, and the light chain variable region comprises CDR-L1 comprising a sequence selected from the group consisting of SEQ ID NOs: 15 to 17, CDR-L2 comprising the sequence of SEQ ID NO: 18, and CDR-L3 comprising the sequence of SEQ ID NO: 19. In some embodiments, the antibody or antigen-binding portion thereof comprises the heavy chain variable region and light chain variable region of a single antibody shown in Table 6.

[0015] In another aspect, provided herein are one or more polynucleotide(s) encoding an antibody, or antigen-binding portion thereof, according to any one of the above embodiments. In another aspect, provided herein are vector(s) comprising one or more polynucleotide(s) according to any one of the above embodiments. In another aspect, provided herein are host cells comprising one or more polynucleotide(s) according to any one of the above embodiments or vectors according to any one of the above embodiments. In another aspect, provided herein are methods of producing an antibody, the method comprising culturing a host cell according to any one of the above embodiments so that the antibody is produced. In some embodiments, the method further comprises recovering the antibody from the host cell. In another aspect, provided herein are pharmaceutical compositions comprising an antibody, or antigen-binding portion thereof, according to any one of the above embodiments and a pharmaceutically acceptable carrier.

[0016] In another aspect, provided herein are methods for treating or ameliorating symptoms of a gastrointestinal disease or disorder, comprising administering to an individual having the gastrointestinal disease or disorder an effective amount of an antibody, antigen-binding site, or pharmaceutical composition described herein. In some embodiments, the gastrointestinal disease or disorder is selected from inflammatory bowel disease, irritable bowel syndrome, pancreatic cancer, or colon cancer. In some embodiments, the gastrointestinal disease or disorder is inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is Crohn's disease or ulcerative colitis. In some embodiments, the individual is human. [Brief explanation of the drawings]

[0017] [Figure 1] A shows microscopic images of an in vitro scratch wound assay using T84 cells at 0 and 24 hours after treatment with either human IgG control or antibody clone 4772. B shows the difference in wound healing activity observed 24 hours after treatment. [Figure 2A] 1 shows a schematic representation of the treatment protocol for the acute DSS-induced model of colitis. [Figure 2B] Shown is the stool consistency on day 10 of the study for naive (untreated, no DSS), PBS-treated (administered on days 1 and 3 of the study, DSS), human IgG-treated (administered on days 1 and 3 of the study, DSS), and antibody clone 4772-treated mice (administered on days 1 and 3 of the study, DSS). [Figure 2C] Shown is the change in body weight of naive (untreated, no DSS), PBS-treated (administered on study days 1 and 3, DSS), human IgG-treated (administered on study days 1 and 3, DSS), and antibody clone 4772-treated (administered on study days 1 and 3, DSS) mice, expressed as a percentage of the body weight of the mice at the start of the study. [Figure 2D]The severity of inflammation from a histological perspective is shown for mouse intestinal tissues after terminal sacrifice for naive mice (untreated, no DSS), PBS-treated mice (administered on test days 1 and 3, DSS), human IgG-treated mice (administered on test days 1 and 3, DSS), and antibody clone 4772-treated mice (administered on test days 1 and 3, DSS). [Figure 2E] Shown are the percentages of polymorphonuclear neutrophils (PMNs) present in distal colon sections of mouse tissue after terminal sacrifice for naive mice (untreated, no DSS), PBS-treated mice (administered on study days 1 and 3, DSS), and 4772-treated mice (administered on study days 1 and 3, DSS). DETAILED DESCRIPTION OF THE INVENTION

[0018] As used herein, the articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0019] The term "antibody" is intended to encompass antibodies, fragments, specified portions, and variants thereof, such as single chain antibodies and fragments thereof derived from the antibodies of the present invention. Antibodies include antibody fragments, antibody variants, monoclonal antibodies, polyclonal antibodies, and recombinant antibodies. Antibodies may be produced in mice, rats, rabbits, or humans.

[0020] Antibodies can be full length or include, but are not limited to, Fab, Fab' and F(ab')2, facb, pFc 1 The antibody fragments may include a fragment (or multiple fragments) of an antibody having an antigen portion such as an Fd, FdAb fragment, isolated CDR, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, bispecific antibodies and multispecific antibodies formed from antibody fragments.

[0021] In some embodiments, the antibody comprises all or a portion of an antibody constant region. The constant region is of an isotype selected from IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), or IgM. As used herein, the "constant region" of an antibody includes a native constant region, an allotype, or a natural variant, such as D356E and L358M or A431G in human IgG1 (see, e.g., Jefferies and Lefranc, MAbs, 1(4):332-338 (July-August 2009)).

[0022] The term "monoclonal antibody," as used herein, is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies are made by methods available or known in the art and are derived from a single clone, such as any eukaryotic, prokaryotic, or phage clone. The monoclonal antibodies of the present invention can be prepared using a variety of techniques known in the art, such as the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0023] As used herein, the term "chimeric antibody" refers to an antibody having alterable sequences derived from a non-human immunoglobulin, e.g., a rat or mouse antibody, and a human immunoglobulin constant region, usually selected from a human immunoglobulin template. Methods for producing chimeric antibodies are known in the art.

[0024] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins that contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody will comprise substantially all of at least one, and usually two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art.

[0025] As used herein, "effective amount" refers to a dose of an antibody or pharmaceutical composition sufficient to reduce the symptoms and signs of a digestive system disease, such as inflammatory bowel disease or digestive system cancer. Symptoms of inflammatory bowel disease include diarrhea, weight loss, bloody diarrhea, bloody stools, pain, anemia, fatigue, rectal bleeding, and abdominal cramps. Symptoms of digestive system cancer include weight loss, pain, and a mass that is clinically palpable or detectable radiologically or through other imaging techniques. The terms "effective amount" and "therapeutically effective amount" are used interchangeably. In some embodiments, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in relation to the administration of one or more chemotherapeutic agents or other effective agents, and a single agent may be considered administered in an effective amount if a desired result is likely or is achieved in combination with one or more other agents. While individual needs vary, determining the optimal range of effective amounts of each component is within the skill of the art. Typical dosages include 0.1-100 mg / kg body weight, preferred dosages include 1-100 mg / kg body weight, and most preferred dosages include 10-100 mg / kg body weight.

[0026] The term "subject" or "individual" can refer to a vertebrate with a digestive system disease, such as a vertebrate with inflammatory bowel disease or digestive system cancer.Subjects include all warm-blooded animals, such as mammals, such as rodents, preferably primates or non-human primates, more preferably humans.The term "subject" also includes domestic animals (e.g., cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, etc.).Therefore, veterinary use and pharmaceutical or pharmaceutical preparations are contemplated herein.

[0027] Those skilled in the art will understand that antibodies are "modular" in nature. Throughout this disclosure, various specific embodiments of the various "modules" that comprise the antibodies of the invention are described. As specific, non-limiting examples, various embodiments of variable heavy chain CDRs, variable heavy chains, variable light chain CDRs, and variable light chains are described. It is intended that all specific embodiments can be combined with each other just as if each specific combination were explicitly described individually.

[0028] The humanized antibodies of the invention can comprise a heavy chain variable region from Table 1 below, and can further comprise a light chain variable region from Table 2 below. Such variable regions can be incorporated into a human IgG1 framework using methods well known in the art. [Table 1] [Table 2]

[0029] In other embodiments, the humanized antibodies of the invention may comprise heavy chain and light chain CDR sequences selected from Tables 3 and 4 below. [Table 3] [Table 4]

[0030] The present invention encompasses antibodies and fragments that specifically bind to sLeA and sLeC but not to sialyl Lewis X (sLeX), compositions containing the antibodies, polynucleotides encoding anti-sLeA / sLeC but not anti-sLeX antibodies, polynucleotides encoding such antibodies, methods and compositions useful for making such antibodies and binding fragments, and various methods of using them. The glycan structures of sLeA, sLeC, and sLeX are shown in Table 5 below. [Table 5]

[0031] Antibodies against carbohydrates / glycans typically have low binding affinity, which may make them unsuitable for drug development. Methods for determining the binding affinity of antibodies are known in the art. Generally, the binding affinity for a particular target or substrate is determined by the equilibrium dissociation constant (K D ) to derive the binding rate (k a (M -1 s -1 )) and dissociation rate (k d (s -1 It is determined by the relationship between K D The lower the K, the higher the affinity. Exemplary methods for determining antibody affinity include assays utilizing the Octet system (Fortebio), or the Biacore system (GE Healthcare), or other systems that determine association and dissociation constants. Thus, the present invention provides antibodies with a binding affinity for sLeA of about 500 μM or less. D and a K of approximately 500 μM or less binding affinity for sLeC. D and does not bind to sLeX. In a non-limiting example, the antibodies of the present invention have a binding affinity for sLeA of 100 μM or less, 90 μM or less, 80 μM or less, 70 μM or less, 60 μM or less, 50 μM or less, 40 μM or less, 30 μM or less, 20 μM or less, 10 μM or less, 1 μM or less, or 0.1 μM or less. Dand a K binding affinity for sLeC of 100 μM or less, 90 μM or less, 80 μM or less, 70 μM or less, 60 μM or less, 50 μM or less, 40 μM or less, 30 μM or less, 20 μM or less, 10 μM or less, 1 μM or less, or 0.1 μM or less. D and does not bind to sLeX. In one embodiment, antibodies useful in the present invention have a binding affinity for a glycan target of 41 μM or less for sLeA. D and a K of 70 μM or less for sLeC. D and does not bind to sialyl Lewis X (sLeX). In some embodiments, the antibodies of the disclosure bind to a particular target (e.g., sLeX) with a K of about 1 mM or greater. D If so, do not combine.

[0032] The antibodies of the present invention may also be used in methods to aid in the diagnosis of disease, e.g., cancer, in an individual. Such cancers include cancers of the digestive system, e.g., pancreatic or colon cancer. Such methods include using the antibodies of the present invention to determine the level of sLeA and / or sLeC binding in an individual or in specific tissues of an individual. As used herein, "aiding in diagnosis" means that the method aids in making a clinical decision regarding the classification, nature, or nature of cancer and may or may not be definitive regarding a definitive diagnosis. Thus, methods to aid in the diagnosis of cancer may include detecting the level of sLeA and / or sLeC in a biological sample from an individual and / or determining the level of sLeA and / or sLeC in the sample. Antibodies recognizing epitopes or portions thereof may also be used in the development of diagnostic immunoassays to detect antigenic determinants shed in body fluids, such as, but not limited to, blood, saliva, urine, pulmonary fluid, or ascites fluid. Similarly, immunoassays of this type using the antibodies of the invention may be used to monitor the effectiveness of treatment and / or disease remission, and in such cases the presence and / or level of antigenic determinants reactive to the antibodies of the invention may be useful as biomarkers for monitoring disease activity and / or progression.

[0033] In some embodiments, the antibodies of the present disclosure may be used in methods to detect the presence of and / or measure levels of sLeA and / or sLeC in an individual or in specific tissues of an individual.

[0034] Certain aspects of the present disclosure relate to polynucleotides (e.g., isolated polynucleotides) and / or vectors (e.g., expression vectors) encoding the antibodies of the present disclosure, as well as host cells (e.g., isolated host cells) comprising the polynucleotides or vectors. In some embodiments, the host cell is a prokaryotic host cell, such as a bacterial host cell (e.g., E. coli). Suitable prokaryotic host cells include, but are not limited to, eubacteria, e.g., gram-negative or gram-positive organisms, e.g., Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella. In some embodiments, the host cell is a eukaryotic host cell, e.g., a nucleated cell from a yeast, fungus, insect, plant, animal, human, or other multicellular organism. For example, filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells.Examples of vertebrate or mammalian cells include SV40-transformed monkey kidney-derived cell line CV1 (COS-7, ATCC CRL 1651), human embryonic kidney cell line (293 cells, or 293 cells subcloned in suspension culture for growth, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney-derived cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney-derived cells (MDCK, ATCC CCL 34), and buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary carcinoma (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)), MRC 5 cells, FS4 cells, a human hepatoma-derived cell line (Hep G2), Chinese hamster ovary (CHO) cells such as DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines such as NS0 and Sp2 / 0.

[0035] Other aspects of the present disclosure relate to production methods using the host cells of the present disclosure. Antibodies or antigen-binding portions thereof can be produced using recombinant methods. For recombinant production of antibodies or antigen-binding portions, nucleic acid encoding the antibody / portion is isolated and inserted into a vector for further cloning (amplification of the DNA) or expression. DNA encoding the antibody can be isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the antibody heavy and light chains). Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0036] The antibodies of the invention may be used for therapeutic purposes in individuals with diseases or disorders of the digestive system, including, but not limited to, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), irritable bowel syndrome, and cancers of the digestive system, including pancreatic and colon cancer.

[0037] In some embodiments, the antibodies of the invention may be used alone to treat or ameliorate one or more disease symptoms, hi other embodiments, the antibodies of the invention may be used in combination with other therapeutic agents or drugs to treat or ameliorate one or more disease symptoms.

[0038] Various formulations of the antibodies of the present invention or their fragments may be used for administration. In some embodiments, the antibodies of the present invention or their fragments may be administered undiluted. The compositions of the present invention may contain a suitable pharmaceutically acceptable carrier in addition to the pharmacologically active agent. Carriers include excipients and auxiliary agents, which are well known in the art and are relatively inert substances that facilitate the administration of pharmacologically active agents or facilitate the processing of active compounds into pharmaceutically usable preparations for delivery to the site of action. For example, excipients can provide compactness or uniformity or act as diluents. Suitable excipients include, but are not limited to, stabilizing agents, wetting agents and emulsifying agents, salts for varying osmotic pressure, encapsulating agents, buffers, and skin penetration enhancers.

[0039] Preparations suitable for parenteral administration include aqueous solutions of the active compound in water-soluble form, for example, water-soluble salts. In addition, suitable suspensions of the active compound in oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. In some cases, the suspension may contain stabilizers. Liposomes can also be used to encapsulate drugs for intracellular delivery.

[0040] Pharmaceutical formulations for systemic administration according to the present invention may be prepared for enteral, parenteral, or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient. Excipients and formulations for parenteral and non-parenteral drug delivery are described in Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing (2000).

[0041] Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets such as coated tablets, elixirs, suspensions, syrups or inhalants, and controlled release forms thereof.

[0042] Generally, these agents are prepared for administration by injection (e.g., intraperitoneal, intravenous, subcutaneous, intramuscular, etc.), although other administration modes (e.g., oral, mucosal, etc.) are also available. Thus, the antibodies of the present invention are preferably combined with a pharmaceutically acceptable vehicle such as saline, Ringer's solution, dextrose solution, etc.

[0043] The individual dosing regimen, i.e., dosage, timing, and repetition, will be determined by the particular individual and their medical history. Generally, a dose of at least about 0.1 mg / kg body weight, or more preferably at least about 1 mg / kg body weight, or at least about 5 mg / kg body weight, more preferably at least about 10 mg / kg body weight, or at least about 20 mg / kg body weight, is administered.

[0044] In some embodiments, the antibodies of the present invention or their fragments are administered in one or more doses over the course of treatment. Empirical considerations such as half-life will typically contribute to determining the dosage. Antibodies, such as humanized or fully human antibodies, are compatible with the human immune system and can be used to extend the half-life of the antibody or to protect the antibody from attack by the host's immune system. The frequency of administration can be determined and adjusted over the course of treatment and can be based on the reduction of one or more clinical symptoms of the disease. Alternatively, sustained, continuous-release formulations of the antibodies of the present invention may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0045] The following examples are offered to illustrate, but not to limit, the present invention. [Example]

[0046] Example 1: Generation of humanized antibody clones Humanized antibodies having a heavy chain variable region comprising one of the heavy chain variable regions from Table 1 above and a light chain variable region comprising one of the light chain variable regions from Table 2 above were synthesized and incorporated into an IgG1 framework. The heavy chain and light chain variable region pairs synthesized and incorporated into an IgG1 framework were those in Table 6 below. [Table 6]

[0047] Humanized antibodies having the above combinations of heavy and light chain variable region pairs were produced by transfecting mammalian cells with the plasmids and purified using affinity chromatography.

[0048] Of the nine humanized antibody clones in Table 6, two antibody clones (4764 and 4767) showed weak expression, resulting in less than 1 mg of antibody after purification. The production levels of these two clones were deemed too low for practical cell line production optimization. The remaining seven clones were subsequently screened for binding affinity to sLeA, sLeC, and sLeX.

[0049] Example 2: Binding affinity to sialyl Lewis A (sLeA) and sialyl Lewis C (sLeC) In vitro evaluation of the binding affinity of the humanized antibodies from Example 1 to sLeA and sLeC was performed at 25°C using a Biocore T100 SPR biosensor with a CM7 sensor chip using PBS-p (0.005% Tween-20) running buffer. Antibodies were coupled at 50 μg / ml, approximately 30,000 RU, in 10 mM sodium acetate, pH 5.0, on an NHS / EDC-activated surface. A reference surface was activated and blocked to serve as a control. Carbohydrate samples (sLeA, sLeC, and sialyl Lewis X (all purchased from Dextra Labs)) were dissolved in PBS-p running buffer to a stock concentration of 10 mM. Each sample was then prepared in a two-fold dilution series with a maximum concentration of 300 μM, and each concentration series was tested on the antibody surface. Response data were processed by subtracting the response from the reference surface and buffer injections. Binding constants at 25°C were determined.

[0050] A murine antibody (GM35) (described in Brazil, J Immunol 191:4804-4817 (2013)) was tested for binding affinity to sLeA, sLeC, and sLeX using the methods described above. Results for this antibody showed no binding to sLeX and a K of 41.5 μM to 46.8 μM for sLeA. D , and a K of 68.8 μM to 78.2 μM for sLeC. D For comparison, another antibody, NS19-9 (Dako, Carpenteria, CA), was tested. This antibody is known to bind to sLeA, but shows no binding to sLeX, and has a K of 39.5 μM for sLeA. D , and 1.5 mM K for sLeC D had.

[0051] Example 3: Binding affinity of humanized antibody clones to sialyl Lewis A (sLeA) and sialyl Lewis C (sLeC) The seven humanized antibody clones from Example 1 were tested for binding affinity to sLeA, sLeC, and sLeA. The same method as in Example 2 above was used, except that only one concentration of sLeX, 300 μM, was used. The average binding K for each of the seven humanized antibody clones to sLeA and sLeC was D The values ​​are shown in Table 7 below. [Table 7]

[0052] In three independent experiments at a carbohydrate concentration of 300 μM, none of the above antibody clones showed binding to sLeX. The above binding affinity data indicated that the humanized antibody clone containing the HvCh3 variable region had the highest affinity for both sLeA and sLeC.

[0053] Example 4: In vitro wound healing activity The wound healing mechanism of humanized antibody action was tested in an in vitro scratch wound assay. Human colonic epithelial cells, T84 (CCL-248, ATCC, Manassas, Virginia), were grown to confluency in 6-well plates. For each condition, a scratch was made using a pipette tip, and the cells were treated with either a human IgG (hIgG) control (Sigma-Aldrich) at 10 μg / ml or the humanized antibody described in Example 3 at 10 μg / ml. Twenty-four hours after treatment, wound healing activity was measured by visual inspection and by measuring the dimensions of the scratch wound using ImageJ-based image analysis. The humanized antibody exhibited enhanced wound healing activity compared to the hIgG control. As a non-limiting example, the wound healing activity of clone 4772, one of the humanized antibody clones described in Example 3, is shown in Figures 1A-1B. The enhanced wound healing activity seen in Figures 1A and 1B indicates that the humanized antibodies of Example 3, including antibody clone 4772, may be effective in treating gastrointestinal disorders such as inflammatory bowel disease.

[0054] Example 5: In vivo efficacy in an acute model of DSS-induced colitis The efficacy of antibody clone 4772 was tested in an in vivo mouse acute model of dextran sulfate sodium (DSS)-induced colitis. On study day 1, all animals were randomized by body weight. Animals weighing 18–22 g were enrolled in the study. Starting on study day 0, all 10-week-old male C57BL / 6 mice enrolled in the study who were not part of the "naive" group were given 2.5% dextran sulfate sodium (DSS) dissolved in sterile water ad libitum for 7 days. The DSS water was refreshed on study day 3. On study day 7, after all measurements were completed, all animals were switched to drinking water only. Mice in the "naive" group received acidified water ad libitum and were not treated with test substances.

[0055] Animals receiving antibody clone 4772 or human IgG were injected via intraperitoneal (IP) injection with the appropriate drug at 10 mg / kg body weight (4772) or 25 mg / kg body weight (human IgG) on two separate days, study day 1 and study day 3. Mice in the group designated to receive PBS received PBS IP injections on study days 1 and 3.

[0056] Body weights were measured daily starting on study day 1, and stool consistency assessments were performed on study days 0, 2, 4, 6, 8, 9, 10, and 13. The results of this study are shown in Figures 2A-2E. Figure 2A is a schematic diagram of the study method. Figure 2B shows the results of stool consistency measurements taken on study day 10. As shown, mice treated with antibody clone 4772 had better stool consistency (lower y-axis values) compared to the PBS and hIgG controls. Similarly, Figure 2B shows that mice treated with antibody clone 4772 experienced less weight loss compared to the PBS and hIgG controls. Stool consistency and weight loss are two factors that suggest symptoms of digestive disorders, including inflammatory bowel disease. These data indicate that the humanized antibodies of Example 3, including antibody clone 4772, may be effective in treating digestive disorders, including inflammatory bowel disease.

[0057] Intestinal tissue samples collected after terminal sacrifice were evaluated for the severity of inflammation (the percent of necrosis, erosion, hyperplasia, polymorphonuclear neutrophil (PMN) infiltration, and edema observed in the colonic tissue). As shown in Figure 2D, mice receiving antibody clone 4772 showed reduced signs of inflammation compared to PBS and hIgG controls. Furthermore, mice receiving antibody clone 4772 had more than 50% less PMN infiltration in the distal colon compared to mice treated with PBS (see Figure 2E). These data indicate that the humanized antibodies of Example 3, including antibody clone 4772, can reduce intestinal tissue inflammation after DSS-induced injury and may be effective in treating gastrointestinal disorders, including inflammatory bowel disease.

[0058] Sequence Listing SEQ ID NO: 1: Amino acid sequence of HvCh1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSTNAMSWVRQAPGKGLEWVSRLRPKSDNYATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTGFWGQGTTVTVSS SEQ ID NO: 2: Amino acid sequence of HvCh2 EVQLVESGGGLVQPGGSLRLSCAASGFTFSTNAMSWVRQAPGKGLEWVARLRPKSDNYATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTGTGFWGQGTTLTVSS SEQ ID NO: 3: Amino acid sequence of HvCh3 EVQLVESGGGLVQPGGSLRLSCAASGFTFSTNAMSWVRQAPGKGLEWVARLRPKSDNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVTGTGFWGQGTTLTVSS SEQ ID NO: 4: Amino acid sequence of HvCh4 EVQLVESGGGLVQPGGSLRLSCAASGFTFSTNAMSWVRQAPGKGLEWVARLRPKSDNYATYYADSVKGRFTISRDDSTSTLYLQMNSLRAEDTAVYYCVTGTGFWGQGTTLTVSS SEQ ID NO: 5: Amino acid sequence of LiCh1 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTSPYTFGQGTKLEIK SEQ ID NO: 6: Amino acid sequence of LiCh2 DIVMTQSPDSLAVSLGERVTMNCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQNDYTSPYTFGQGTKLEIK SEQ ID NO: 7: Amino acid sequence of LiCh3 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLFYWTSTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTSPYTFGQGTKLEIK SEQ ID NO: 8: Amino acid sequence of LiCh4 DIVMTQSPDSLAVSLGERVTMNCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLFYWTSTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQNDYTSPYTFGQGTKLEIK SEQ ID NO: 9: Amino acid sequence of LiCh5 DIVMTQSPDSLAVSLGERVTMNCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLFYWTSTRESGVPDRFSGSGSGTDFTLTISSVQAEDLAVYYCQNDYTSPYTFGQGTKLEIK SEQ ID NO: 10: Amino acid sequence of LiCh6 DIVMTQSPDSLAVSLGERVTMNCKSSQSLLQSGNQKNYLTWYQQKPGQPPKLLFYWTSTRESGVPDRFSGSGSGTDFTLTISSVQAEDLAVYYCQNDYTSPYTFGQGTKLEIK SEQ ID NO: 11: Amino acid sequence of LiCh7 DIVMTQSPDSLAVSLGERVTMNCKSSQSLLSSGNQKNYLTWYQQKPGQPPKLLFYWTSTRESGVPDRFSGSGSGTDFTLTISSVQAEDLAVYYCQNDYTSPYTFGQGTKLEIK SEQ ID NO: 12: Amino acid sequence of heavy chain CDR-1 GFTFSTNAMS SEQ ID NO: 13: Amino acid sequence of heavy chain CDR-2 RLRPKSDNYATY SEQ ID NO: 14: Amino acid sequence of heavy chain CDR-3 VTGTGF SEQ ID NO: 15: Amino acid sequence of light chain CDR-1 KSSQSLLNSGNQKNYLT SEQ ID NO: 16: Amino acid sequence of light chain CDR-1B KSSQSLLQSGNQKNYLT SEQ ID NO: 17: Amino acid sequence of light chain CDR-1C KSSQSLLSSGNQKNYLT SEQ ID NO: 18: Amino acid sequence of light chain CDR-2 WTSTRES SEQ ID NO: 19: Amino acid sequence of light chain CDR-3 QNDYTSPYT

Claims

1. An antibody or antigen-binding portion thereof that binds to sialyl Lewis A (sLeA) and sialyl Lewis C (sLeC), wherein the antibody or antigen-binding portion thereof has a binding affinity for sLeA of 60 μM or less. Ｄ and a binding affinity for sLeC of 100 μM or less K Ｄ The antibody or antigen-binding portion thereof,

2. 2. The antibody or antigen-binding portion of claim 1, which does not bind to sialyl Lewis X (sLeX).

3. 3. The antibody or antigen-binding site of claim 1 or claim 2, which is a humanized antibody or humanized antigen-binding site.

4. An antibody or antigen-binding portion thereof that binds to sLeA and sLeC, comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises a sequence having 90-100% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; The antibody or antigen-binding portion thereof, wherein the light chain variable region comprises a sequence having 90 to 100% sequence identity with SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:

11.

5. 5. The antibody or antigen-binding site of claim 4, which does not bind to sLeX.

6. 6. The antibody or antigen-binding site of claim 4 or claim 5, wherein the heavy chain variable region comprises CDR-H1 comprising the sequence of SEQ ID NO: 12, CDR-H2 comprising the sequence of SEQ ID NO: 13, and CDR-H3 comprising the sequence of SEQ ID NO: 14, and the light chain variable region comprises CDR-L1 comprising a sequence selected from the group consisting of SEQ ID NOs: 15 to 17, CDR-L2 comprising the sequence of SEQ ID NO: 18, and CDR-L3 comprising the sequence of SEQ ID NO:

19.

7. 5. The antibody or antigen-binding site of claim 4, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 3 and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 5-8.

8. The antibody or antigen-binding site of claim 7, wherein the light chain variable region comprises the sequence of SEQ ID NO:

8.

9. 5. The antibody or antigen-binding site of claim 4, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 4 and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 9-10.

10. An antibody or antigen-binding site thereof that binds to sLeA and sLeC, comprising a heavy chain variable region, wherein the heavy chain variable region comprises a sequence having 90 to 100% sequence identity with SEQ ID NO:

3.

11. The antibody or antigen-binding site of claim 10, wherein the antibody or antigen-binding site comprises a light chain variable region, and the light chain variable region comprises a sequence having 90 to 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 5 to 11.

12. An antibody or antigen-binding site thereof that binds to sLeA and sLeC, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1 comprising the sequence of SEQ ID NO: 12, CDR-H2 comprising the sequence of SEQ ID NO: 13, and CDR-H3 comprising the sequence of SEQ ID NO: 14, and the light chain variable region comprises CDR-L1 comprising a sequence selected from the group consisting of SEQ ID NOs: 15 to 17, CDR-L2 comprising the sequence of SEQ ID NO: 18, and CDR-L3 comprising the sequence of SEQ ID NO:

19.

13. A polynucleotide encoding the antibody or antigen-binding portion of any one of claims 1 to 12.

14. A vector comprising the polynucleotide of claim 13.

15. A host cell comprising the polynucleotide of claim 13 or the vector of claim 14.

16. A method for producing an antibody, comprising culturing the host cell of claim 15 so that the antibody is produced.

17. 17. The method of claim 16, further comprising recovering the antibody from the host cell.

18. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

19. 19. A method of treating or ameliorating a symptom of a gastrointestinal disease or disorder, the method comprising administering to an individual having said gastrointestinal disease or disorder an effective amount of the antibody or antigen-binding site of any one of claims 1 to 12, or the pharmaceutical composition of claim 18.

20. 20. The method of claim 19, wherein the gastrointestinal disease or disorder is selected from the group consisting of inflammatory bowel disease, irritable bowel syndrome, pancreatic cancer, and colon cancer.

21. 21. The method of claim 20, wherein the gastrointestinal disease or disorder is inflammatory bowel disease.

22. 22. The method of claim 21, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

23. The method of any one of claims 19 to 22, wherein the individual is a human.

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