Antiglycan antibodies and their use

Abstract

The present disclosure provides antibodies or antigen-binding portions thereof that bind to sLeA and sLeC, as well as polynucleotides, vectors, host cells, pharmaceutical compositions, and methods related thereto.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits 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, which are each incorporated herein by reference in their entirety.

[0002] The present invention relates to anti-sialic acid-added glycan antibodies, including anti-sialyl Lewis A (sLeA) antibodies and anti-sialyl Lewis C (sLeC) antibodies, and to methods for using these antibodies.

[0003] Submission of sequence listings in ASCII text files. The contents of the following submission in ASCII text file are incorporated herein by reference in their entirety: a computer-readable format (CRF) sequence listing (filename: 203462000140SEQLIST.TXT, date: March 15, 2021, size: 14KB). [Background technology]

[0004] Glycans are carbohydrate-based polymers that can be free or bound to proteins (glycoproteins) or lipids (glycolipids). Glycans of glycoproteins, such as sialic acid-modified glycoproteins, are known to be involved in immunity and inflammation. High expression of sLeA has been reported in human pancreatic, colon, and gastric cell lines, as well as in adenocarcinomas of the colon, stomach, and pancreas. Furthermore, studies have shown that sLeA is upregulated in response to CD44v6 in inflammatory areas of the human colon of patients with ulcerative colitis. In addition, studies have demonstrated the functional role of sLeA in mucosal inflammation, suggesting that it may influence inflammatory bowel diseases (e.g., Crohn's disease or ulcerative colitis) by regulating sLeA.

[0005] In addition, abnormal glycosylation is a characteristic of cancer and has been reported to modulate the immune response. Similar to what has been observed in inflammatory bowel disease tissue, abnormal protein glycosylation leads to the overexpression of cancer-associated glycan antigens during malignant transformation. Studies have shown that cancer-associated glycan antigens contribute to various aspects of cancer development and progression, including growth, 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 ends of N-glycans, O-glycans, and glycosphingolipids. Within the range of sialic acid, diversity arises among the nine carbon skeletons through α-bonding, and a second diversity arises through modifications at these carbon positions. Examples of known sialic acid addition glycosylation include sLeA, sLeC, and sialyl Lewis X (the structures of each glycan are shown in Table 1).

[0007] These glycans are all examples of terminally sialic acid-added glycans. sLeC is a non-fucosylated precursor of sLeA, while sLeX is a stereoisomer of sLeA. Generally, sLeC is not typically found in normal human tissue but is present in mouse tissue. Therefore, antibodies that bind to both sLeA and sLeC would be useful in drug development, particularly for use in in vivo mouse models of disease.

[0008] Generally, antibodies suitable for development as pharmaceuticals to treat diseases and / or disorders possess high specificity, selectivity, and affinity. Therefore, anti-glycan antibodies are not usually suitable for pharmaceutical development. One reason for this is that glycan antigens have limited immunogenicity, and the affinity observed for anti-glycan antibodies is usually 10% higher than the affinity of antibodies specific to protein or peptide antigens. 3 ~10 5It can be twice as low (Ghassemi, Glycobiology, 25(9):920-952 (Sept 2015)). For generally weak carbohydrate binding, relatively fast k is usually required. on and k off This is accompanied by a rapid decrease in the overall K of antiglycan antibodies. D This will result.

[0009] This invention addresses the need for alternative antibody therapies targeting glycan epitopes in patients with gastrointestinal disorders, including inflammatory bowel disease and gastrointestinal cancers. [Overview of the project]

[0010] The present invention provides an antibody or its antigen-binding site, which includes a variable region that binds to a glycosylated antigen or glycosylated antigen fragment.

[0011] In one embodiment, the present specification provides for an antibody or antigen-binding site that binds to sialyl Lewis A (sLeA) and sialyl Lewis C (sLeC), wherein the binding affinity of such antibody or antigen-binding site to sLeA is 60 μM or less. D Therefore, K has a binding affinity of 100 μM or less for sLeC. D An antibody or its antigen-binding site is provided. In some embodiments, the antibody or antigen-binding site does not bind to sialyl Lewis X (sLeX). In some embodiments, the antibody or antigen-binding site is a humanized antibody or a humanized antigen-binding site.

[0012] In another embodiment, the Specified Reference Indicators Provided are antibodies or antigen-binding sites that bind to sLeA and sLeC, 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 with 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 with 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 site does not bind to sLeX. In some embodiments, the heavy chain variable region comprises CDR-H1 containing the sequence of SEQ ID NO: 12, CDR-H2 containing the sequence of SEQ ID NO: 13, and CDR-H3 containing the sequence of SEQ ID NO: 14, and the light chain variable region comprises CDR-L1 containing a sequence selected from the group consisting of SEQ ID NOs: 15-17, CDR-L2 containing the sequence of SEQ ID NO: 18, and CDR-L3 containing the sequence of SEQ ID NO: 19. In some embodiments, the heavy chain variable region includes the sequence of SEQ ID NO: 3, and the light chain variable region includes a sequence selected from the group consisting of SEQ ID NOs: 5 to 8. In some embodiments, the heavy chain variable region includes the sequence of SEQ ID NO: 3, and the light chain variable region includes the sequence of SEQ ID NO: 8. In some embodiments, the heavy chain variable region includes the sequence of SEQ ID NO: 4, and the light chain variable region includes the sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0013] In another embodiment, the Specified Reference Non-Altered Antibody or Anti-Antibody Site is provided, comprising an antibody or antigen-binding site that binds to sLeA and sLeC, wherein the heavy chain variable region comprises a sequence having 90-100% sequence identity with SEQ ID NO: 3. In some embodiments, the antibody or antigen-binding site comprises a light chain variable region, wherein the light chain variable region comprises a sequence having 90-100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 5-11.

[0014] In another embodiment, the Specified Reference Indicators provide an antibody or antigen-binding site 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 containing the sequence of SEQ ID NO: 12, CDR-H2 containing the sequence of SEQ ID NO: 13, and CDR-H3 containing the sequence of SEQ ID NO: 14, and the light chain variable region comprises CDR-L1 containing a sequence selected from the group consisting of SEQ ID NOs: 15 to 17, CDR-L2 containing the sequence of SEQ ID NO: 18, and CDR-L3 containing the sequence of SEQ ID NO: 19. In some embodiments, the antibody or antigen-binding site comprises the heavy chain variable region and light chain variable region of a single antibody as shown in Table 6.

[0015] In another embodiment, this specification provides one or more polynucleotides encoding an antibody or its antigen-binding site according to any one of the embodiments described above. In another embodiment, this specification provides one or more vectors comprising one or more polynucleotides according to any one of the embodiments described above. In another embodiment, this specification provides a host cell comprising one or more polynucleotides according to any one of the embodiments described above, or a vector according to any one of the embodiments described above. In another embodiment, this specification provides a method for producing an antibody, comprising culturing a host cell according to any one of the embodiments described above so as to produce an antibody. In some embodiments, the method further includes recovering the antibody from the host cell. In another embodiment, this specification provides a pharmaceutical composition comprising an antibody or its antigen-binding site according to any one of the embodiments described above, and a pharmaceutically acceptable carrier.

[0016] In another aspect, provided herein is a method of treating or ameliorating the symptoms of a gastrointestinal disease or disorder, the method 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 Description of the Drawings

[0017] [Figure 1] A shows microscopic images at 0 hours and 24 hours after treatment with either human IgG control or antibody clone 4772 in an in vitro scratch assay using T84 cells. B shows the difference in wound healing activity observed 24 hours after treatment. [Figure 2A] Shows a schematic of the treatment protocol for an acute DSS-induced model of colitis. [Figure 2B] Shows the stool hardness of naive (untreated, no DSS), PBS-treated (administered on days 1 and 3 of the test, DSS), human IgG-treated (administered on days 1 and 3 of the test, DSS), and antibody clone 4772-treated (administered on days 1 and 3 of the test, DSS) mice on day 10 of the test. [Figure 2C] Shows the weight change of naive (untreated, no DSS), PBS-treated (administered on days 1 and 3 of the test, DSS), human IgG-treated (administered on days 1 and 3 of the test, DSS), and antibody clone 4772-treated (administered on days 1 and 3 of the test, DSS) mice, expressed as a percentage of the starting body weight at the start of the test. [Figure 2D]Shows the severity of inflammation from a histological perspective for mouse intestinal tissues after the final euthanasia of naive mice (untreated, without DSS), PBS-treated mice (administered on the 1st and 3rd days of the test, with DSS), human IgG-treated mice (administered on the 1st and 3rd days of the test, with DSS), and antibody clone 4772-treated mice (administered on the 1st and 3rd days of the test, with DSS). [Figure 2E] Shows the percentage of polymorphonuclear neutrophils (PMNs) present in distal colon sections of mouse tissues after the final euthanasia of naive mice (untreated, without DSS), PBS-treated mice (administered on the 1st and 3rd days of the test, with DSS), and 4772-treated mice (administered on the 1st and 3rd days of the test, with DSS).

BRIEF DESCRIPTION OF THE DRAWINGS

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

[0019] The term “antibody” is intended to encompass antibodies, fragments, designated 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 can be produced in mice, rats, rabbits, or humans.

[0020] Antibodies can be full-length or, without limitation, Fab, Fab’, and F(ab’)2, facb, pFc 1 、Fd, dAb fragments, isolated CDRs, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and fragments (or multiple fragments) of antibodies having antigenic portions such as bispecific and multispecific antibodies formed from antibody fragments.

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

[0022] As used herein, the term “monoclonal antibody” is not limited to antibodies produced via hybridoma technology. Monoclonal antibodies are prepared by methods available or known in the art and are derived from a single clone, such as a clone of any eukaryote, prokaryote, or phage. The monoclonal antibodies of the present invention can be prepared using various techniques known in the art, such as the use of hybridomas, recombination and phage display techniques, or combinations thereof.

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

[0024] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin containing a minimal sequence derived from a non-human immunoglobulin. Generally, a humanized antibody contains at least one and usually two variable domains, in which all or almost all of the CDR region corresponds to that of a non-human immunoglobulin, and all or almost all of the framework region is from a human immunoglobulin sequence. A humanized antibody may also contain an immunoglobulin constant region (Fc), typically at least a portion of the constant region of the human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art.

[0025] As used herein, “effective dose” refers to a dose of an antibody or pharmaceutical composition sufficient to reduce the symptoms and signs of a gastrointestinal disease, such as inflammatory bowel disease or gastrointestinal cancer. Symptoms of such inflammatory bowel disease include diarrhea, weight loss, bloody diarrhea, bloody stools, pain, anemia, fatigue, rectal bleeding, and abdominal cramps. Symptoms of gastrointestinal cancer include weight loss, pain, and a clinically palpable mass or a mass detectable by radiology or other imaging techniques. The terms “effective dose” and “therapeutic effective dose” are used synonymously. In some embodiments, an effective dose of a drug, compound, or pharmaceutical composition may or may not be obtained in combination with another drug, compound, or pharmaceutical composition. Thus, “effective dose” may be considered in relation to the administration of one or more chemotherapeutic agents or other effective agents, and a monoagent may be considered administered in an effective dose if a desired result can or does occur when combined with one or more other agents. While individual needs differ, determining the optimal range of effective doses for each component is within the scope of the art. Typical dosages include 0.1–100 mg / kg body weight. Preferred dosages include 1–100 mg / kg body weight. Most preferred dosages include 10–100 mg / kg body weight.

[0026] The term "subject" or "individual" may refer to a vertebrate having a digestive system disorder, such as inflammatory bowel disease or a vertebrate having a digestive system cancer. The subject includes all warm-blooded animals, such as mammals, such as rodents, preferably primates or non-human primates, more preferably humans. The term "subject" also includes domesticated animals (e.g., cats, dogs), livestock (e.g., cattle, horses, pigs, sheep, goats), and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs). Therefore, veterinary use and pharmaceutical or pharmaceutically acceptable preparations are considered herein.

[0027] Those skilled in the art will understand that antibodies are inherently "modular." Throughout this disclosure, various specific embodiments of the various "modules" comprising the antibodies of the present invention are described. Various embodiments of variable heavy chain CDRs, variable heavy chains, variable light chain CDRs, and variable light chains are described as specific non-limiting examples. All specific embodiments are intended to be combined with one another, as if each specific combination were explicitly described individually.

[0028] The humanized antibody of the present invention may include heavy chain variable regions from Table 1 below, and may further include light chain variable regions from Table 2 below. Such variable regions can be incorporated into the human IgG1 backbone using methods well known in the art. [Table 1] [Table 2]

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

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

Table 5

[0031] Antibodies against carbohydrates / glycans can be unsuitable for pharmaceutical development because they usually have low binding affinity. Methods for determining the binding affinity of antibodies are known in the art. Generally, the binding affinity for a specific target or substrate is determined by the relationship between the association rate (k D ), which leads to the equilibrium dissociation constant (K a (M -1 s -1 )) and the dissociation rate (k d (s -1 )). The lower the K D , the higher the affinity. Examples of methods for determining the affinity of antibodies include assays using an Octet system (Fortebio), or a Biacore system (GE Healthcare), or other system measuring instruments for determining association and dissociation constants. Accordingly, the present invention includes antibodies having a K D of about 500 μM or less for binding to sLeA, a K D of about 500 μM or less for binding to sLeC, and that do not bind to sLeX. In non-limiting examples, the antibodies of the present invention have a K D for binding to 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.The binding affinity to sLeC is 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 of K. D The present invention includes antibodies that do not bind to sLeX. In one embodiment, the antibodies useful in the present invention are those with a binding affinity to a glycan target of 41 μM or less for sLeA. D Therefore, for sLeC, K is less than 70 μM. D It does not bind to sialyl Lewis X (sLeX). In some embodiments, the antibody of this disclosure binds to a specific target (e.g., sLeX) with a binding affinity of approximately 1 mM or more for the antibody to the target. D If this is the case, do not combine.

[0032] The antibodies of the present invention may also be used in methods to aid in the diagnosis of diseases, for example, cancer in an individual. Such cancers include gastrointestinal cancers, such as pancreatic cancer or colon cancer. Such methods include determining the binding level of sLeA and / or sLeC in an individual or specific tissue of an individual using the antibodies of the present invention. As used herein, “methods to aid in diagnosis” means that these methods help in making clinical decisions regarding classification, or character, or cancer, and may or may not be definitive in relation to a definitive diagnosis. Thus, a method to aid in the diagnosis of cancer may include the steps of 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. Furthermore, antibodies that recognize an epitope or part thereof may be used to create diagnostic immunoassays for detecting antigenic determinants released into body fluids, non-limited to blood, saliva, urine, pulmonary fluid, or ascites fluid, etc. Similarly, this type of immunoassay using the antibodies of the present invention may be used to monitor the effectiveness of treatment and / or remission of the disease. In such cases, the presence and / or levels of antigenic determinants reactive to the antibodies of the present invention may be useful as biomarkers for monitoring disease activity and / or progression.

[0033] In some embodiments, the antibodies of this disclosure may be used in methods for detecting the presence of sLeA and / or sLeC in an individual or specific tissue of an individual, and / or for measuring the levels of sLeA and / or sLeC.

[0034] Certain aspects of this disclosure relate to polynucleotides (e.g., isolated polynucleotides) and / or vectors (e.g., expression vectors) encoding the antibodies of this disclosure, as well as host cells (e.g., isolated host cells) containing the polynucleotides or vectors. In some embodiments, the host cell is a prokaryotic host cell, e.g., 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 yeast, fungi, insects, plants, animals, humans, or other multicellular organisms. For example, filamentous fungi or yeasts are suitable hosts for cloning or expression of antibody-coding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used lower eukaryotic host microorganism. Host cells suitable for glycosylated antibody expression 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, for example, SV40-transformed monkey kidney cell line CV1 (COS-7, ATCC CRL 1651), human embryonic kidney cell line (293 cells, or 293 cells subcloned in suspension culture for proliferation, 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 cancer cells (HELA, ATCC CCL 2), canine kidney-derived cells (MDCK, ATCC CCL 34), and buffalo rat hepatocytes (BRL 3A, ATCC CRL Examples include Chinese hamster ovary (CHO) cells such as 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary cancer cells (MMT 060562, ATCC CCL 51), TRI cells (Mather et al., Annals NYAcad.Sci.383:44-68 (1982)), MRC 5 cells, FS4 cells, human hepatocellular carcinoma-derived cell lines (Hep G2), 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 this disclosure relate to methods of production using host cells of this disclosure. Antibodies or their antigen-binding sites may be produced using recombinant methods. Recombinant production of antibodies or antigen-binding sites involves isolating the nucleic acid encoding the antibody / site and inserting it into a vector for further cloning (DNA amplification) or expression. The DNA encoding the antibody may be isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody). Vector components generally include, but are not limited to, one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.

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

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

[0038] Various formulations of the antibodies or fragments thereof of the present invention may be used for administration. In some embodiments, the antibodies or fragments thereof of the present invention may be administered undiluted. In addition to the pharmacological agent, the composition of the present invention may contain a suitable pharmaceutically acceptable carrier. This carrier may include excipients and adjuvants, which are well known in the art and are relatively inert substances, and which facilitate the processing of the active compound into the pharmaceutically usable preparation for the administration of the pharmacologically effective substance or for delivery to the site of action. For example, excipients can provide moldability or uniformity, or act as diluents. Suitable excipients include, but are not limited to, stabilizers, wetting agents and emulsifiers, salts for altering osmotic pressure, encapsulating agents, buffers and skin penetration enhancers.

[0039] Suitable formulations for parenteral administration include water-soluble forms, such as aqueous solutions of the active compound in the form of a water-soluble salt. In addition, a suspension of the active ingredient compound suitable for an oily injectable suspension may be administered. Suitable lipophilic solvents or vehicles include oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. The aqueous injectable suspension may contain a substance that increases the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. In some cases, the suspension may contain a stabilizer. Furthermore, the drug may be encapsulated using liposomes to deliver the drug into cells.

[0040] The pharmaceutical formulations for systemic administration according to the present invention may be prepared for enteral, parenteral, or topical administration. In fact, all three formulations may be used simultaneously to achieve systemic delivery 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 gelatin capsules or soft gelatin capsules, pills, 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.), but other forms of administration (e.g., oral administration, mucosal administration, etc.) are also available. Therefore, the antibodies of the present invention are preferably combined with a pharmaceutically acceptable vehicle, such as physiological saline, Ringer's solution, or dextrose solution.

[0043] Individual medication plans, i.e., dosage, timing, and repetition, shall be determined by the specific individual and its medical history. Generally, a dose of at least about 0.1 mg / kg body weight, 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 during the course of treatment. Empirical considerations, such as half-life, will usually contribute to the determination of the dosage. Antibodies such as humanized antibodies or fully human antibodies are compatible with the human immune system and can be used to extend the half-life of antibodies or to prevent them from being attacked by the host immune system. The frequency of administration may be determined and adjusted during the course of treatment and may be based on the reduction of one or more clinical symptoms of the disease. Alternatively, a sustained, continuous-release formulation 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 provided to illustrate the present invention and are not limiting. [Examples]

[0046] Example 1: Generation of humanized antibody clones Humanized antibodies were synthesized that contained a heavy chain variable region including one of the heavy chain variable regions from Table 1 above, and a light chain variable region including one of the light chain variable regions from Table 2 above, and incorporated into the IgG1 skeleton. The pairs of heavy chain variable regions and light chain variable regions synthesized and incorporated into the IgG1 skeleton were those shown in Table 6 below. [Table 6]

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

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

[0049] Example 2: Binding affinity to sialyl Lewis A (sLeA) and sialyl Lewis C (sLeC) The binding affinity of humanized antibodies from Example 1 to sLeA and sLeC was evaluated in vitro at 25 degrees Celsius using a Biocore T100 SPR biosensor with a CM7 sensor chip and PBS-p (0.005% Tween-20) running buffer. The antibodies were bound to an NHS / EDC activated surface at a concentration of 50 ug / ml and approximately 30,000 RU in 10 mM sodium acetate at pH 5.0. A reference surface was activated and blocked to function 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. Subsequently, each sample was prepared in a 2-fold dilution series at a maximum concentration of 300 μm, and each concentration series was tested on the antibody surface. Reaction data was processed by subtracting the reactions from the reference surface and buffer injection. The binding constant at 25 degrees Celsius was determined.

[0050] The binding affinity of the mouse antibody (GM35) (described in Brazil, J Immunol 191:4804-4817 (2013)) to sLeA, sLeC, and sLeX was tested using the method described above. The results for this antibody showed no binding to sLeX, but it showed binding to sLeA at concentrations of 41.5 μM to 46.8 μM. D , and 68.8 μM to 78.2 μM of K for sLeC D It had [this characteristic]. For comparison, another antibody, NS19-9 (Dako, Carpenteria, CA), was tested. This antibody is known to bind to sLeA, but did not bind to sLeX, and showed binding to sLeA with 39.5 μM K D , and 1.5 mM K for sLeC D It had.

[0051] Example 3: Binding affinity of humanized antibody clones to sialyl Lewis A (sLeA) and sialyl Lewis C (sLeC) The binding affinity of sLeA, sLeC, and sLeA was tested for seven humanized antibody clones from Example 1. The same method as in Example 2 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 measured. D The values ​​are shown in Table 7 below. [Table 7]

[0052] In three independent tests at a carbohydrate concentration of 300 μM, none of the antibody clones mentioned above showed binding to sLeX. The binding affinity data above indicates that the humanized antibody clone with 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 antibodies was tested using an in vitro scratch assay. Human colon epithelial cells, T84 (CCL-248, ATCC, Manassas, Virginia), were grown to confluence in a 6-well plate. Under each condition, scratches were created using a pipette tip, and the cells were treated with either human IgG (hIgG) control (Sigma-Aldrich), 10 ug / ml, or the humanized antibody from Example 3, 10 ug / ml. 24 hours after treatment, wound healing activity was measured by visual inspection and by measuring the size of the scratches using ImageJ-based image analysis. The humanized antibody showed higher 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 from Example 3, is shown in Figures 1A and 1B. The increased wound healing activity shown in Figures 1A and 1B indicates that the humanized antibody 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 day 1 of the study, all animals were randomized by body weight. Animals weighing between 18 and 22 g were enrolled in the study. Starting on day 0, all 10-week-old male C57BL / 6 mice enrolled in the study and not belonging to the "naive" group were given 2.5% dextran sulfate sodium (DSS) dissolved in sterile water as needed for 7 days. The DSS water was replaced on day 3 of the study. On day 7 of the study, after all measurements were completed, all animals were switched to drinking water only. Mice in the "naive" group were given acidic water as needed and were not treated with the test substance.

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

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

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

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

Claims

1. An antibody or its antigen-binding site that binds to sialyl Lewis A (sLeA) and sialyl Lewis C (sLeC), The antibody or its antigen-binding site includes a heavy chain variable region and a light chain variable region, The heavy chain variable region includes CDR-H1 consisting of the sequence of sequence number 12, CDR-H2 consisting of the sequence of sequence number 13, and CDR-H3 consisting of the sequence of sequence number 14, and the light chain variable region includes CDR-L1 consisting of the sequence of sequence number 15, CDR-L2 consisting of the sequence of sequence number 18, and CDR-L3 consisting of the sequence of sequence number 19. The antibody or its antigen-binding site.

2. The antibody or antigen-binding site according to claim 1, which is a humanized antibody or its antigen-binding site.

3. It does not bind to sialyl Lewis X (sLeX); and / or The binding affinity of the antibody or antigen-binding site to sLeA is 60 μM or less. Ｄ Therefore, K has a binding affinity of 100 μM or less to sLeC. Ｄ That is, The antibody or antigen binding site according to claim 1 or 2.

4. (a) The heavy chain variable region includes a sequence having 90-100% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, and the light chain variable region includes a sequence having 90-100% sequence identity with SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9; (b) The heavy chain variable region includes the sequence of sequence number 3, and the light chain variable region includes a sequence selected from the group consisting of sequence numbers 5 to 8; or (c) The heavy chain variable region includes the sequence of sequence number 4, and the light chain variable region includes the sequence of sequence number 9. The antibody or antigen binding site according to any one of claims 1 to 3.

5. The antibody or antigen binding site according to claim 4, wherein the heavy chain variable region includes the sequence of SEQ ID NO: 3, and the light chain variable region includes the sequence of SEQ ID NO:

8.

6. The antibody or antigen binding site according to any one of claims 1 to 3, wherein the heavy chain variable region includes a sequence having 90 to 100% sequence identity with SEQ ID NO:

3.

7. The antibody or antigen binding site according to claim 6, wherein the light chain variable region includes a sequence having 90-100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 5-9.

8. The antibody or antigen binding site according to any one of claims 1 to 7, wherein the heavy chain variable region is a portion of a heavy chain polypeptide further comprising a human IgG1 constant region.

9. A polynucleotide encoding an antibody or its antigen-binding site according to any one of claims 1 to 8.

10. A vector comprising the polynucleotide described in claim 9.

11. A host cell comprising the polynucleotide described in claim 9 or the vector described in claim 10.

12. A method for producing an antibody or its antigen-binding site, comprising culturing the host cells described in claim 11 so that the antibody or its antigen-binding site is produced.

13. The method according to claim 12, further comprising recovering the antibody or its antigen-binding site from the host cell.

14. A pharmaceutical composition comprising an antibody or its antigen-binding site according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14, for use in the treatment or improvement of symptoms of digestive disorders or conditions in an individual having symptoms of a digestive disorder or condition.

16. The pharmaceutical composition according to claim 15, wherein the digestive system disease or disorder is selected from the group consisting of inflammatory bowel disease, irritable bowel syndrome, and cancer of the digestive system.

17. The pharmaceutical composition according to claim 16, wherein the cancer of the digestive system is pancreatic cancer or colon cancer.

18. The pharmaceutical composition according to claim 16, wherein the disease or disorder of the digestive system is inflammatory bowel disease.

19. The pharmaceutical composition according to claim 18, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

20. The pharmaceutical composition according to any one of claims 15 to 19, wherein the individual is a human.

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