Alkaline phosphatase antibodies and their use

Abstract

This invention relates to an antibody that binds to the enteric alkaline phosphatase (iAP) protein. The antibody can be used to detect and treat gastrointestinal diseases. TIFF2026516499000030.tif73170

Description

[Technical Field]

[0001] This application is an international application claiming priority from U.S. Provisional Application No. 63 / 465,361, filed on 10 May 2023, which is incorporated herein by reference in its entirety.

[0002] All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. To more fully describe the latest art known to those skilled in the art as of the date of the invention described herein and claimed herein, the disclosures of these publications are incorporated herein by reference in their entirety.

[0003] This patent disclosure includes materials that are protected by copyright. The copyright holder reserves all other rights, except that anyone may facsimile copies of the patent documents or patent disclosures as they appear in the patent files or records of the United States Patent and Trademark Office.

[0004] Sequence List This application includes an array listing submitted electronically in ASCII format, the entire listing of which is incorporated herein by reference. The ASCII copy of this listing, created in [], is named [] and has a size of [] bytes.

[0005] Field of Invention This invention relates to an antibody against intestinal alkaline phosphatase (iAP) and a method for using the same. [Background technology]

[0006] background Intestinal alkaline phosphatase (iAP) plays a crucial role in maintaining intestinal health and well-being. iAP is a major regulator of intestinal mucosal permeability and detoxifies harmful bacterial surface lipopolysaccharides (LPS) by cleaving inorganic phosphates. iAP is produced by intestinal cells in the small intestine and exhibits anti-inflammatory properties. [Overview of the project]

[0007] This invention provides an intestinal alkaline phosphatase (iAP) antibody composition and a method for using the same.

[0008] Aspects of the present invention relate to isolated antibodies or fragments thereof that bind to human intestinal alkaline phosphatase (hIAP), wherein the isolated antibody or fragment comprises a VH CDR1 containing the amino acid sequence of SEQ ID NO: 3, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 4, a VH CDR3 containing the amino acid sequence of SEQ ID NO: 5, or a sequence that is at least 80% identical thereto, and / or a VL CDR1 containing the amino acid sequence of SEQ ID NO: 6, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 7, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 8, or a sequence that is at least 80% identical thereto.

[0009] Aspects of the present invention also include isolated antibodies or fragments thereof that bind to human enteric alkaline phosphatase (hIAP), comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 22, VH CDR2 containing the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 31, VH CDR3 containing the amino acid sequence of SEQ ID NO: 24, or sequences that are at least 80% identical thereto, and / or VL CDR1 containing the amino acid sequence of SEQ ID NO: 25, VL CDR2 containing the amino acid sequence of SEQ ID NO: 26, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 27, or sequences that are at least 80% identical thereto.

[0010] Aspects of the present invention also include isolated antibodies or fragments thereof that bind to human enteric alkaline phosphatase (hIAP), comprising a heavy chain variable region containing the amino acid sequence described in SEQ ID NO: 1 or a sequence identical thereto by at least 80%, and / or a light chain variable region containing the amino acid sequence described in SEQ ID NO: 2 or a sequence identical thereto by at least 80%.

[0011] Aspects of the present invention also include isolated antibodies or fragments thereof that bind to human enteric alkaline phosphatase (hIAP), comprising a heavy chain variable region containing the amino acid sequence described in SEQ ID NO: 20 or a sequence identical thereto by at least 80%, and / or a light chain variable region containing the amino acid sequence described in SEQ ID NO: 21 or a sequence identical thereto by at least 80%.

[0012] In the embodiment, the isolated monoclonal antibody or antigen-binding fragment contains wild-type Fc or modified Fc.

[0013] In the embodiment, the isolated monoclonal antibody or antigen-binding fragment comprises a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody.

[0014] In the embodiment, the isolated monoclonal antibody or antigen-binding fragment further comprises a detectable portion.

[0015] Furthermore, aspects of the present invention relate to pharmaceutical compositions comprising an isolated monoclonal antibody or a fragment thereof as described herein, and a pharmaceutically acceptable carrier or excipient.

[0016] Aspects of the present invention also relate to nucleic acids encoding isolated monoclonal antibodies or fragments thereof as described herein.

[0017] In the embodiment, the nucleic acid includes the nucleic acid sequence described in SEQ ID NO: 9 and / or SEQ ID NO: 10, the nucleic acid sequence described in SEQ ID NO: 20 and / or SEQ ID NO: 21, or a sequence that is at least 80% identical thereto.

[0018] Aspects of the present invention also include vectors containing nucleic acids as described herein, and / or isolated cells containing the vectors.

[0019] Furthermore, aspects of the present invention also include isolated monoclonal antibodies or fragments thereof that bind to human enteric alkaline phosphatase (hIAP), comprising a heavy chain variable region encoded by the nucleic acid sequence described in SEQ ID NO: 9 or a sequence that is at least 80% identical thereto, and / or a light chain variable region encoded by the nucleic acid sequence described in SEQ ID NO: 10 or a sequence that is at least 80% identical thereto.

[0020] Aspects of the present invention also include isolated monoclonal antibodies or fragments thereof that bind to human enteric alkaline phosphatase (hIAP), comprising a heavy chain variable region encoded by the nucleic acid sequence described in SEQ ID NO: 20 or a sequence that is at least 80% identical thereto, and / or a light chain variable region encoded by the nucleic acid sequence described in SEQ ID NO: 21 or a sequence that is at least 80% identical thereto.

[0021] Furthermore, aspects of the present invention relate to isolated cells containing one or more polynucleotides encoding an isolated monoclonal antibody or a fragment thereof as described herein.

[0022] Aspects of the present invention relate to a method for detecting or diagnosing iAP-related gastrointestinal disorders in subjects requiring detection or diagnosis of iAP-related gastrointestinal disorders, the method comprising administering a therapeutically effective amount of the hIAP antibody described herein or a fragment thereof to the subject.

[0023] Aspects of the present invention also relate to methods for treating or preventing iAP-related gastrointestinal disorders in subjects requiring treatment or prevention of iAP-related gastrointestinal disorders, comprising administering a therapeutically effective amount of the hIAP antibody or a fragment thereof described herein to the subject.

[0024] In embodiments, iAP-related diseases include gastrointestinal diseases, and non-limiting examples of gastrointestinal diseases include colitis, inflammatory bowel disease (IBD), gastritis, gastroenteritis, pyloric stenosis, gastric cancer, gastrointestinal cancer, colon cancer, infectious diarrhea, fecal impaction, constipation, intestinal obstruction and pseudo-intestinal obstruction, or malabsorption.

[0025] Aspects of the present invention also include kits comprising isolated monoclonal antibodies or fragments thereof as described herein.

[0026] Other problems and advantages of the present invention will become readily apparent from the following description. [Brief explanation of the drawing]

[0027] This patent or application file includes at least one drawing finished in color. A copy of this patent or patent application publication containing the color drawing will be provided by the Patent Office after the application and payment of the required fees.

[0028] [Figure 1]This panel shows the affinity and specificity of the recombinant monoclonal antibody CDI005E8 against human intestinal alkaline phosphatase. Panel A: The Kd of rmAb CDI005E8 against 2 μg / mL peptide antigen 2-hsALPI coated on microwell plates was determined using absorbance measurements from horseradish peroxidase signals on the secondary antibody. Measurements were performed in triple replication. The mean and standard deviation are shown on log-joint curves. Panel B, upper panel: The monoclonal antibody CDI005E8 identified human intestinal alkaline phosphatase in infant stool and did not cross-react with purified human placenta-like (PL) alkaline phosphatase (lane 1), purified human tissue-nonspecific (TN) alkaline phosphatase (lane 3), or purified human germ cell (GC) alkaline phosphatase. A total of 50 ng of each alkaline phosphate protein (placental, tissue-nonspecific, and germ cell) was loaded into lanes 1, 3, and 6, respectively. Lanes 4 and 5 were loaded with 1 μg of infant stool sample or enteric lysate, respectively. The primary antibody concentration was 20 nM CDI005E8 (upper panel) or 5 nM polyclonal anti-intestinal alkaline phosphatase (panel B, lower panel; Heath et al. JAMA Netw Open 2(11), e19114996). The concentration of the goat anti-rabbit secondary antibody was 1 nM for both blots.

[0029] [Figure 2]The predicted three-dimensional models of the Fv region and its antigen-binding pocket are shown. (Panel A) Ribbon diagrams of the heavy and light chains of CDI005E8. The Fv structure of CDI005E8 was modeled using SAbPred. Antibody sequences were numbered using ANARCI. Templates for the VH and VL framework regions were selected from SAbDab and oriented relative to each other using ABangle. The orientation of VH and VL was found to be the same as that of PDB ID 5v6m. The CDR conformation of side chains whose coordinates could not be directly copied from the template structure was predicted using SCRWL4. (Panel B) The CDR regions (H1-H3 and L1-L3) are shown in different blue shades. Kabat guidelines were used for the shown CDR regions (see also Table 4). CDR conformation was predicted using FREAD along with a CDR-specific database. (Panel C) Predicted paratope residues are shown by both chain width and heatmap color, with the widest main chain diameter and red color associated with the highest-scoring residue. Ala65 in the heavy chain (H2) of the CDI005E8 sequence has the highest paratope score and is shown in red. The calculations were performed using the Antibody i-Patch portal on the SAbPred website. A complete description of this method can be found in Krawcyzk et al. Protein Engineering, Design & Selection 26, 621-629.

[0030] [Figure 3] This is a schematic diagram illustrating the outline of predictive epitope identification on antigen targets.

[0031] [Figure 4]The predicted human intestinal alkaline phosphatase epitopes for rmAb CDI05E8 are shown. (Panel A) Overlay of the top three ranked human iAP epitopes, as shown in the AlphaFold predicted iAP structure for rmAb CDI005E8. (Panel B) Epitope plank 1, olive. (Panel C) Epitope plank 2, yellow. This epitope, predicted without prior knowledge of the antigen, contains residues in the peptide used for antibody production. (Panel D) Epitope plank 3, orange. Calculations were performed on the EpiPred portal on the SAbPrep server. Reference for epitope identification is Krawczyk et al. Bioinformatics 30, 2288-2294.

[0032] [Figure 5] The three-dimensional structures of alkaline phosphatase are shown in (Panel A) monomeric form and (Panel B) dimeric form. (Panel A) Human intestinal alkaline phosphatase structure predicted from AlphaFold. Alphafold.ebi.ac.uk.entry / P09923 determined. Pymol file name-AF-P09923-F1-model-v2humaniAP.pse. (Panel B) Initial evaluation of alkaline phosphatase from existing structures of human intestinal alkaline phosphatase homologs. Human placental-like alkaline phosphatase (PDB 1ew2) and rat intestinal alkaline phosphatase (PDB 4kjg) are shown.

[0033] [Figure 6] This shows the spatial location of candidate antigen peptides in human enteric alkaline phosphatase. Panel A shows the first set of candidate peptides functioning as antigens for monoclonal antibodies, and Panel B shows the second set. Peptide sequences are shown as spherical representations on the modeled dimeric structures of predicted human enteric alkaline phosphatase (red diagram, determined Alphafold.ebi.ac.uk.entry / P09923) and human placental-like alkaline phosphatase (gray ribbon, PDB 1ew2) from Alphafold. Peptides have been omitted from human placental-like alkaline phosphatase for clarity.

[0034] [Figure 7] This shows the glycopeptide coverage map for rmAb CDI005E8. The amino acids (AA) in bold and underlined are the fragments selected for RA calculation. The red AAs represent N-glycosylation sites.

[0035] [Figure 8] The following shows a representative MS2 spectrum of the glycopeptide found in rmAb CDI005E8. The peak representing the HexNAc(3)Hex(3) structure on the peptide EQQFNSTIR (position 304) is shown. The glycoform structure is based on total mass and known biosynthetic pathways, and does not reflect sequencing or compositional analysis.

[0036] [Figure 9] The MALDI spectra of N-glycans identified in rmAb CDI005E8 are shown. For the major N-glycans, the m / z values ​​are shown along with a diagram of their structure.

[0037] [Figure 10] This shows the affinity and specificity of the recombinant monoclonal antibody CDI022E3 against human enteric alkaline phosphatase. [Modes for carrying out the invention]

[0038] Detailed explanation Abbreviations and Definitions A detailed description of one or more embodiments is provided herein. However, it will be understood that the invention can be embodied in various forms. Accordingly, the specific details disclosed herein should not be construed as limitations, but rather as representative grounds for the claims and for teaching those skilled in the art to use the invention in any suitable form.

[0039] The singular forms “a,” “an,” and “the” include multiple references unless the context explicitly indicates otherwise. The use of the words “a” or “an” in the claims and / or specification with the term “includes” may mean “one,” but also coincides with the meanings of “one or more,” “at least one,” and “one or two or more.”

[0040] Whenever any of the phrases "for example," "etc.," or "including" are used herein, it is understood that they are always accompanied by the phrase "without limitation" unless otherwise explicitly stated. Similarly, "for example," "exemplary," etc., are understood to be non-limiting.

[0041] The term "substantially" allows for deviations from descriptive terms that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term "substantially," even if the term is not explicitly enumerated.

[0042] The terms “comprising,” “including,” “having,” and “involving” (as well as “comprises,” “includes,” “has,” and “involves”) are used interchangeably and have the same meaning. Specifically, each term is defined in accordance with the general U.S. Patent Law definition of “comprising,” and is therefore interpreted as an open term meaning “at least the following,” and also as not excluding any additional features, limitations, aspects, etc. Thus, for example, “a process comprising steps a, b, and c” means that the process comprises at least steps a, b, and c. Whenever the terms “a” or “an” are used, they are understood to mean “one or more,” unless such interpretation is meaningless in the context.

[0043] As used herein, the term “approximately” is used to mean roughly, roughly, about, or within that range. When the term “approximately” is used with a numerical range, it modifies that range by extending the upper and lower boundaries of the stated numerical values. Generally, the term “approximately” is used herein to modify numerical values ​​that are above and below (higher or lower than) a 20 percent variance.

[0044] Enteric alkaline phosphatase (iAP) antibody Using single 100-150 mg stool samples from three healthy human donors, we identified 234 human proteins secreted from the gastrointestinal tract. Of these, we identified a core proteome of 57 proteins common to all three individuals.

[0045] Enteral alkaline phosphatase (iAP) is expressed in intestinal cells of the small intestine, co-secreted into the enteric lumen and systemic circulation, and plays an essential role in maintaining intestinal barrier function by detoxifying bacterial lipopolysaccharides and maintaining microbial homeostasis. As the major alkaline phosphatase in feces, iAP has been identified as one of 57 proteins in the core human fecal proteome.

[0046] Aspects of the present invention provide isolated antibodies specific to iAP. Where used herein in relation to cells, DNA, or RNA, the term “isolated” may refer to molecules separated from other DNA or RNA present in a natural source of macromolecules. The term “isolated” may also refer to nucleic acids or peptides that are substantially free from cellular material, viral material, or culture media when produced by recombinant DNA technology, or from chemical precursors or other chemical substances when chemically synthesized. For example, “isolated nucleic acid” may include nucleic acid fragments that do not exist naturally as fragments and would not be found in their natural state. “Isolated” may also refer to cells or polypeptides isolated from other cellular proteins or tissues. Isolated polypeptides may include both purified polypeptides and recombinant polypeptides. These antibodies represent a new class of monoclonal antibodies against iAP. For example, isolated cells may be mammalian cells such as HEK293 cells or CHO cells.

[0047] The embodiments described herein include recombinant monoclonal iAP antibodies. “Recombinant” in relation to polypeptides (such as antibodies) or polynucleotides can refer to forms of polypeptides or polynucleotides that do not exist in nature, and non-limiting examples thereof can be created by combining polynucleotides or polypeptides that would not normally be combined.

[0048] In addition to exemplary wild-type IgG constant regions useful in combination with the VH and VL sequences provided herein, amino acid sequences of monoclonal iAP antibodies are provided herein (see Tables 1A-B). The amino acid sequences of the heavy and light chain complementarity-determining regions (CDRs) of the iAP antibody are underlined (CDR1), underlined in bold (CDR2), or underlined in italicized bold (CDR3).

[0049] TIFF2026516499000002.tif123150

[0050] TIFF2026516499000003.tif123150

[0051] The amino acid sequences of the complementarity-determining regions of the heavy and light chains of the iAP antibody are shown in Tables 2A and 2B.

[0052] TIFF2026516499000004.tif53170

[0053] TIFF2026516499000005.tif53170

[0054] In addition to exemplary wild-type IgG constant regions useful in combination with the VH and VL sequences provided herein, nucleic acid sequences of monoclonal iAP antibodies are provided herein (see Tables 3A-B).

[0055] TIFF2026516499000006.tif166170

[0056] TIFF2026516499000007.tif166170

[0057] As described herein, iAP proteins, or their derivatives, fragments, analogs, homologs, or homologous species, can be used as immunogens in the production of antibodies that immunologically bind specifically to iAP. As described herein, immunogens used in the production of antibodies against iAP include PEYPADASQNGIRLDGK (SEQ ID NO: 11) and / or PGYVFNSGVRPDVNESESGSPDY (SEQ ID NO: 30). These sequences do not necessarily need to be presented as accessible epitopes on the native iAP protein, and antibodies that recognize these linear epitopes do not necessarily need to be produced at a recognizable level when the native iAP protein is used as an immunogen. Secondly, even if these epitopes are presented in an accessible manner on the surface of the native iAP, these sequences can be blocked in clinical samples by interactions with specific host antibodies or other host proteins. While we do not wish to be bound by theory, these confounding effects are eliminated when the diagnostic sample is digested to release the target peptide. Therefore, the use of peptide-specific antibodies to recognize target peptides produced by the specific digestion of diagnostic samples has a synergistic effect that increases the detection sensitivity and specificity of the target biomarker.

[0058] Those skilled in the art will recognize that, without excessive experimentation, it is possible to determine whether a monoclonal antibody has the same specificity as the monoclonal antibody of the present invention by checking whether the former prevents the latter from binding to the iAP protein or its fragments. While we do not wish to be bound by theory, if the monoclonal antibody being tested competes with the monoclonal antibody of the present invention, as indicated by the reduced binding by the monoclonal antibody of the present invention, the two monoclonal antibodies may bind to the same or closely related epitopes. Competitive assays are well known to those skilled in the art.

[0059] Another method for determining whether a monoclonal antibody has the specificity of the monoclonal antibody of the present invention is to pre-incubate the monoclonal antibody of the present invention with an iAP polypeptide that is normally reactive with it, then add the monoclonal antibody to be tested, and determine whether the monoclonal antibody to be tested is inhibited in its ability to bind to the iAP. If the monoclonal antibody to be tested is inhibited, it has the same or functionally equivalent epitope specificity as the monoclonal antibody of the present invention.

[0060] As used herein, the term “epitope” may include any protein determinant that can specifically bind to an immunoglobulin, a single-chain variable fragment (scFv), or a T cell receptor. Epitope determinants may consist of chemically active surface groups of molecules such as amino acids or sugar side chains and may have specific three-dimensional structural properties as well as specific charge properties. For example, antibodies can be produced against the N-terminal or C-terminal peptide of a polypeptide.

[0061] While we do not wish to be constrained by theory, the antibodies and fragments described herein bind to different epitopes. As described herein, peptides are used as immunogens in antibody production, and therefore the epitopes may include linear amino acid sequences. In embodiments, the linear amino acid sequence has an amino acid length of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20. In some embodiments, the linear amino acid sequence may have an amino acid length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In embodiments, the antibodies described herein may bind to the same or overlapping segments of the peptide immunogen. In other embodiments, the antibodies described herein may bind to distinct, non-overlapping peptide sequences.

[0062] When two or more antibodies bind to the same or overlapping segments of a peptide immunogen, they can be considered to compete with each other for binding to the peptide immunogen. The term “compete with” can refer to a pair of antibodies, for example, a first antibody that detectably competes with a second antibody (or other molecule) in a binding assay using recombinant immunogen or cell surface expression immunogen. For example, if a second antibody binds to an epitope that overlaps with the epitope to which a first antibody has bound, the first antibody may block or attenuate the binding of the second antibody. Furthermore, if the epitope of the second antibody is directly adjacent to the epitope to which the first antibody has bound, the first antibody may block or attenuate the binding of the second antibody by spatial exclusion or steric effect.

[0063] As used herein, the terms “immunological binding” and “immunological binding properties” may refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction can be expressed in terms of the dissociation constant (Kd) of the interaction, where a smaller Kd represents greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, which depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Thus, both “on-rate constants” (Kon) and “off-rate constants” (Koff) can be determined by calculating the concentrations and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). The Koff / Kon ratio allows for the cancellation of parameters unrelated to affinity and is equal to the dissociation constant Kd. (See, for example, Davies et al. (1990) Annual Rev Biochem 59:439-473). The antibodies of the present invention are said to bind specifically to iAP epitopes when their equilibrium binding constant (Kd) is ≤1 μM, ≤100 nM, ≤10 nM, or ≤100 pM to approximately 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art. For example, when using 2 μg / mL (1.1 μM) of the peptide antigen 2-hsALPI, the Kd of CDI005E8 Ab is 7.11 ± 1.01 pM. This was surprising considering that the hydrophobic iAP epitope and hydrophobic antibody paratope are an unusual combination for inducing picomolar binding affinity.

[0064] The monoclonal antibodies described herein may undergo affinity maturation, for example, to increase the strength or affinity of immunological binding. "Affinity maturation" can refer to the process by which an antibody evolves from a reference antibody (also called a parent antibody) through mutations in, for example, one or more amino acid residues, to possess increased activity against the target antigen compared to the corresponding form of the reference antibody against the same target antigen. Thus, the evolved antibody is optimized compared to the reference antibody or template antibody.

[0065] The term "affinity-mature antibody" can refer to an antibody that exhibits increased activity against a target antigen compared to a reference antibody. For example, an affinity-mature antibody may show increased binding to a target antigen compared to a reference antibody or parent antibody. An "affinity-mature antibody" can bind to the same epitope as the reference antibody.

[0066] An optimized antibody may refer to an antibody or portion thereof that exhibits increased activity against a target protein or antigen compared to a reference antibody, for example, improved binding affinity to the target protein, and / or improved functional activity. An antibody can be optimized by one or more amino acid modifications (amino acid deletion, substitution, or insertion) compared to a parent antibody that does not contain these one or more amino acid modifications. Activity, such as binding affinity, can be increased by approximately 1.5 to 1000 times, for example, at least approximately 2 to 100 times, for example, approximately 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 times or more, compared to the activity of the parent antibody (e.g., a germline antibody that does not contain modifications).

[0067] Various procedures known within the art can be used to produce polyclonal or monoclonal antibodies directed against the proteins of the present invention, or against their derivatives, fragments, analogues, homologs, or homologous molecular species. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0068] Antibodies can be purified by well-known techniques such as affinity chromatography using protein A or protein G, which primarily provide the IgG fraction of immunoserum. Subsequently, or alternatively, the specific antigen or epitope that is the target of the desired immunoglobulin can be immobilized on a column, and immuno-specific antibodies can be purified by immunoaffinity chromatography. The purification of immunoglobulins has been discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0069] The antibodies described herein can be detected by appropriate assays, such as conventional immunoassays. For example, an assay can be performed in which iAP or fragments thereof are immobilized on a solid phase. The incubation is maintained for a sufficient time to bind the antibody in the sample to the immobilized polypeptide on the solid phase. After this initial incubation, the solid phase is separated from the sample. The solid phase is washed to remove unbound substances and interfering substances such as nonspecific proteins that may be present in the sample. Subsequently, the solid phase containing the antibody of interest bound to the immobilized polypeptide is incubated with a second labeled antibody or antibody conjugated to a coupling agent such as biotin or avidin. This second antibody may be another anti-iAP antibody or another antibody. Labels for antibodies are well known in the art and include radionuclides, enzymes (e.g., maleate dehydrogenase, horseradish peroxidase, glucose oxidase, catalase), fluorine (fluorescein isothiocyanate, rhodamine, phycocyanin, fluorescein camine), biotin, and others. Labeled antibodies are incubated with a solid, and the label bound to the solid phase is measured. These and other immunoassays can be easily performed by those skilled in the art.

[0070] As used herein, the terms “monoclonal antibody,” “MAb,” or “monoclonal antibody composition” may refer to a group of antibody molecules containing only one molecular species of antibody molecule, each consisting of a specific light chain gene product and a specific heavy chain gene product. For example, the complementarity-determining region (CDR) of a monoclonal antibody is identical across the molecules in the group. An MAb contains an antigen-binding site that can be immunoreactive with an antigen epitope characterized by a specific binding affinity to it.

[0071] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). Hybridoma methods involve immunizing mice, hamsters, or other suitable host animals with an immunizer to induce lymphocytes capable of producing antibodies that specifically bind to the immunizer. Alternatively, lymphocytes can be immunized in vitro.

[0072] The immunotherapy agent may include a protein antigen, a fragment thereof, or a fusion protein thereof. For example, if human-derived cells are required for the uses described herein, peripheral blood lymphocytes may be used, or if a non-human mammalian source is required for the uses described herein, spleen cells or lymph node cells may be used. The lymphocytes are then fused with the immortalized cell line using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59 103). The immortalized cell line may be transformed mammalian cells, such as myeloma cells of rodent, bovine, and human origin. For example, rat or mouse myeloma cell lines may be used. The hybridoma cells may be cultured in a suitable medium containing one or more substances that inhibit the proliferation or survival of non-fusioned immortalized cells. For example, if parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the hybridoma culture medium may contain hypoxanthine, aminopterin, and thymidine ("HAT (hypoxanthine, aminopterin, and thymidine) medium"), which prevent the growth of HGPRT-deficient cells.

[0073] Immortalized cell lines can efficiently fuse, maintain stable high levels of antibody expression by selected antibody-producing cells, and be sensitive to media such as HAT medium. Immortalized cell lines can be mouse myeloma lines available from the Salk Institute Cell Distribution Center (San Diego, California) and the American Type Culture Collection (Manassas, Virginia). Human myeloma and mouse-human heterozygous myeloma cell lines have also been described for human monoclonal antibody production (see Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

[0074] Next, the culture medium in which hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies against the antigen. The binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art.

[0075] "Affinity" or "binding affinity" can refer to the strength with which an antibody molecule or a portion thereof binds to an epitope on a target protein or antigen. Affinity is determined by the equilibrium binding constant (K). A ) or equilibrium dissociation constant (K D This can be measured by the equilibrium binding constant (K). Low affinity antibody-antigen interactions are weak, and molecules tend to separate rapidly, while high affinity antibody-antigen binding is strong, and molecules remain bound for a long period of time. For example, the affinity of an antibody to a target protein is measured by the equilibrium binding constant (K).A ) at about 10 6 M -1 or more, about 10 7 M -1 or more, about 10 8 M -1 or more, or about 10 9 M -1 10 10 M -1 or 10 11 M -1 or 10 12 M -1 is. Further, the antibody has an equilibrium dissociation constant (K -4 M to 10 -5 M, about 10 -5 M to 10 -6 M, about 10 -6 M to 10 -7 M, about 10 -7 M to 10 -8 M, about 10 -8 M, about 10 -9 M, about 10 -10 M, about 10 -11 M, about 10 -12 M or about 10 -13 M, or about -13 M or greater equilibrium dissociation constant (K D) can have a low dissociation constant. A low dissociation constant can also be a characteristic. While we do not wish to be bound by theory, a low dissociation constant may indicate that the antibody is characterized by a higher binding affinity. For example, antibodies with a dissociation constant of nM or less are considered high-affinity antibodies. Such affinities are known in the art and can be determined, for example, by equilibrium dialysis, by surface plasmon resonance (SPR) using a BIAcore instrument according to general procedures outlined by the manufacturer, by radioimmunoassay using a radiolabeled target antigen, or by other methods known to those skilled in the art. The analysis can be performed using the method of ScL, 51:660 (1949). Furthermore, the binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis in Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, for therapeutic applications of monoclonal antibodies, it is important to identify antibodies that have a high degree of specificity and high binding affinity to the target antigen.

[0076] After hybridoma cells are identified, clones can be subcloned using limiting dilution procedures and grown using standard methods (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986), pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle Medium and RPMI-1640 Medium. Alternatively, hybridoma cells can be grown in vivo as ascites in mammals.

[0077] Monoclonal antibodies secreted by subclones can be isolated or purified from culture media or ascites fluid by conventional immunoglobulin purification procedures such as protein A-Sepharose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0078] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibodies of the present invention can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the mouse antibody). Hybridoma cells of the present invention can serve as a source of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, to obtain the synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by substituting the coding sequences of human heavy and light chain constant domains for homologous mouse sequences (see U.S. Patent No. 4,816,567, Morrison, Nature 368,812 13 (1994)), or by covalently attaching all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can be used in place of the constant domains of the antibodies described herein, or in place of the variable domain of one antigen-binding site of the antibody of the present invention, to produce a chimeric bivalent antibody.

[0079] Fully human antibodies are antibody molecules in which the entire sequence of both the light and heavy chains, including the CDR, is derived from a human gene. Such antibodies are referred to herein as “human antibodies” or “fully human antibodies.” Human monoclonal antibodies can be prepared using trioma technology, human B-cell hybridoma technology (see Kozbor, et al, 1983 Immunol Today 4:72), and EBV hybridoma technology for producing human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77 96). Human monoclonal antibodies can be used and produced by using human hybridomas (see Cote, et al, 1983. Proc Natl Acad Sci USA 80:2026 2030) or by transforming human B cells with Epstein-Barr virus in vitro (see Cole, et al., 1985 "MONOCLONAL ANTIBODIES AND CANCER THERAPY", Alan R. Liss, Inc., pp. 77 96).

[0080] Humanized antibodies may be derived from non-human species (such as mice), and their amino acid sequences (e.g., in the CDR region) are modified to increase similarity to antibody variants naturally produced in humans. Antibodies can be humanized by methods known in the art, such as CDR transplantation. See also Safdari et al., (2013) Biotechnol Genet Eng Rev.; 29:175-86. In addition, humanized antibodies can be produced in transgenic plants as an inexpensive alternative to existing mammalian systems. For example, transgenic plants can be tobacco plants, namely Nicotiana benthamiana and Nicotiana tabaccum. Antibodies are purified from plant leaves. Stable transformation of plants can be achieved using Agrobacterium tumefaciens or particle gun methods. For example, nucleic acid expression vectors containing at least heavy and light chain sequences are expressed via transformation in bacterial cultures, i.e., A. tumefaciens strain BLA4404. Plant infiltration can be achieved by injection. Soluble leaf extracts can be prepared by grinding leaf tissue in a mortar and centrifugation. Antibody isolation and purification can be easily carried out by many methods known to those skilled in the art. Other methods of antibody production in plants are described, for example, in Fischer et al., Vaccine, 2003, 21:820-5, and Ko et al., Current Topics in Microbiology and Immunology, Vol.332, 2009, pp.55-78. Accordingly, the present invention further provides any cells or plants comprising a vector encoding the antibodies described herein or producing the antibodies of the present invention.

[0081] In addition, human antibodies can also be produced using additional techniques, including phage display libraries. (See Hoogenboom and Winter, J.Mol.Biol., 227:381 (1991), and Marks et al., J.Mol.Biol., 222:581 (1991)). Similarly, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, such as mice in which the endogenous immunoglobulin gene is partially or completely inactivated. After the challenge, human antibody production is observed, which is very similar to that found in humans, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patents No. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as Marks et al., Bio / Technology 10,779-783 (1992), Lonberg et al., Nature 368 856 859 (1994), Morrison, Nature 368 812 13 (1994), Fishwild et al, Nature Biotechnology 14,845 51 (1996), Neuberger, Nature Biotechnology 14,826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13 65 It is described in 93 (1995).

[0082] Human antibodies can also be produced using transgenic non-human animals that are modified to produce fully human antibodies rather than endogenous antibodies in response to antigenic challenge (see International Publication WO94 / 02602). Endogenous genes encoding heavy and light immunoglobulin chains in the non-human host are neutralized, and active loci encoding human heavy and light immunoglobulins are inserted into the host genome. Human genes are incorporated, for example, using a yeast artificial chromosome containing the required human DNA segment. The modified animal is then obtained as offspring by mating with an intermediate transgenic animal containing fewer complements than the complete complements of the modification. Embodiments of such non-human animals can be mice, which are called Xenomouse®, as disclosed in International Publications WO96 / 33735 and WO96 / 34096. This animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained, for example, as preparations of polyclonal antibodies, directly from animals after immunization with the immunogen of interest, or alternatively, from immortalized B cells of animal origin, such as hybridomas that produce monoclonal antibodies. In addition, genes encoding immunoglobulins with human variable regions can be recovered and expressed to directly obtain antibodies, or further modified to obtain antibody analogs, such as single-chain Fv(scFv) molecules.

[0083] An example of a method for producing a non-human host, exemplified by a mouse, lacking the expression of endogenous immunoglobulin heavy chains is disclosed in U.S. Patent No. 5,939,598. This can be obtained by a method comprising deleting a J-segment gene from at least one endogenous heavy chain locus in embryonic stem cells to prevent locus rearrangement and the formation of a transcript of the rearranged immunoglobulin heavy chain locus, wherein the deletion is carried out by a targeted vector containing a gene encoding a selectable marker, and producing a transgenic mouse from embryonic stem cells, wherein its somatic and germ cells contain a gene encoding a selectable marker.

[0084] One method for producing a target antibody, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. This method involves introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in a culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing both a heavy chain and a light chain.

[0085] Further improvements to this procedure include methods for identifying clinically relevant epitopes on an immunogen and correlation methods for selecting antibodies that bind immunospecifically to the relevant epitopes with high affinity, as disclosed in International Publication WO99 / 53049.

[0086] Antibodies can be expressed by vectors containing DNA segments encoding single-chain antibodies as described herein. A vector can refer to a system that can harbor foreign genes and be present within a host cell. These include plasmid vectors, recombinant viral vectors, recombinant bacterial vectors, pseudovirions, virus-like particles, and the like.

[0087] These include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, etc. Vectors include chemical conjugates such as those described in International Publication WO93 / 64701, which have a target-directing moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid-binding moiety (e.g., polylysine), viral vectors (e.g., DNA or RNA viral vectors), fusion proteins such as those described in International Application No. PCT / US95 / 02140 (International Publication WO95 / 22618), which contain a target moiety (e.g., an antibody specific to a target cell) and a nucleic acid-binding moiety (e.g., protamine), plasmids, phages, etc. Vectors can be chromosomal, non-chromosomal, or synthetic.

[0088] A "recombinant viral vector" refers to a vector that can carry foreign genes based on recombinant viruses, such as recombinant adenovirus vectors, recombinant poxvirus vectors, and baculovirus vectors.

[0089] A "recombinant bacterial vector" refers to a vector that can carry foreign genes constructed from recombinant bacteria. Examples include Listeria vectors and attenuated Salmonella vectors.

[0090] Vectors can include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney's mouse leukemia virus. DNA viral vectors can also be used. These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as herpes simplex I virus (HSV) vectors (see Geller, AI et al., J. Neurochem, 64:487 (1995), Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995), Geller, AI et al., Proc Natl. Acad. Sci.: USA 90, 7603 (1993), Geller, AI, et al., Proc Natl. Acad. Sci USA 87:1149 (1990)), and adenovirus vectors (see LeGal LaSalle et al., Science, 259:988 (1993), Davidson, et al. This includes al., Nat. Genet 3:219 (1993), Yang, et al., J. Virol. 69:2004 (1995), and adeno-associated virus vectors (see Kaplitt, MGet al., Nat. Genet. 8:148 (1994)).

[0091] Poxvirus vectors introduce genes into the cytoplasm of cells. Avipoxvirus vectors result in only short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors can be used to introduce nucleic acids into nerve cells. Adenovirus vectors result in shorter expression periods (about 2 months) than adeno-associated virus (about 4 months), and even shorter than HSV vectors. The choice of vector depends on the target cell. Introduction can be by standard techniques (e.g., infection, transfection, transduction, or transformation). Examples of gene transfer modes include, for example, naked DNA, CaPO4 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0092] Vectors can be used to target virtually any target cell. For example, stereotactic injection can be used to orient vectors (e.g., adenovirus, HSV) to the location required for the applications described herein. In addition, particles can be delivered by intracerebral (intrarebroventricular, ICV) injection using minipump infusion systems such as the SynchroMed Infusion System. Methods based on bulk flow, known as convection, have also proven effective in delivering large molecules to diastolic areas of the brain and may be useful for delivering vectors to target cells. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994), Morrison et al., Am. J. Physiol. 266:292-305 (1994)).

[0093] These vectors can be used to express large quantities of antibodies that can be used in various ways. For example, to detect the presence of iAP in a sample. Antibodies can also be used to attempt to bind to iAP and disrupt its function.

[0094] In one embodiment, the antibody of the present invention is a full-length antibody containing an Fc region similar to the wild-type Fc region that binds to the Fc receptor.

[0095] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies consist of two covalently bonded antibodies. Antibodies can be prepared in vitro using known methods in protein synthesis chemistry, including those containing crosslinking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolates and methyl-4 mercaptobutylimidate, as well as those disclosed, for example, in U.S. Patent No. 4,676,980.

[0096] In embodiments, the antibodies of the present invention have a certain percentage of identity or similarity to the amino acid or nucleotide sequence of the anti-iAP antibodies described herein. For example, “homology” or “identity” or “similarity” refers to sequence similarity between two peptides or two nucleic acid molecules. Homologousity can be determined by comparing the positions of each sequence, which can be aligned for comparison purposes. If the positions of the sequences being compared are occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. For example, an antibody may have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more amino acid sequence identity when compared to any one specific region or full length of any of the anti-iAP antibodies described herein. For example, an antibody may have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher nucleic acid identity when compared to any one specific region or full length of any of the anti-iAP antibodies described herein. The sequence identity or similarity of the nucleic acids and proteins of the present invention can be determined by sequence comparison and / or alignment by methods known in the art, using software programs known in the art, such as those described in Ausubel et al. (2007) Current Protocols in Molecular Biology. For example, the sequence identity or similarity percentage of the nucleic acids and proteins of the present invention can be determined using sequence comparison algorithms (i.e., BLAST or BLAST2.0), manual alignment, or visual inspection.

[0097] As used herein, “polypeptide” can encompass a single “polypeptide” as well as multiple “polypeptides,” and refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any one or more chains of two or more amino acids and does not refer to a specific length of the product. Thus, any other term used to refer to a peptide, dipeptide, tripeptide, oligopeptide, “protein,” “amino acid chain,” or one or more chains of two or more amino acids may, as herein, refer to a “polypeptide,” and the term “polypeptide” may be used in place of or interchangeably with any of these terms. “Polypeptide” may also refer to post-expression modified products of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification with amino acids not naturally occurring. Polypeptides may be derived from natural biological sources or produced by recombinant technology and do not necessarily have to be translated from nucleic acid sequences. Polypeptides can be produced in any manner, including chemical synthesis. With respect to amino acid sequences, those skilled in the art will readily recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences, which modify, add, delete, or substitute a single amino acid or a small percentage of amino acids in the encoded sequence, are collectively referred to herein as “conservatively modified variants.” In some embodiments, the modification involves substituting an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants of anti-iAP antibodies disclosed herein may exhibit increased cross-reactivity to iAP compared to unmodified iAP antibodies.

[0098] For example, a "conservative amino acid substitution" is one in which an amino acid residue is substituted with an amino acid residue having a similar side chain. In the art, families of amino acid residues having similar side chains are defined as basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, non-essential amino acid residues in immunoglobulin polypeptides are substituted with other amino acid residues derived from the same side chain family. In another embodiment, amino acid chains can be substituted with structurally similar chains that differ in the order and / or composition of their side chain family members.

[0099] As used herein, “antibody” or “antigen-binding polypeptide” may refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody may be a whole antibody, any antigen-binding fragment, or a single chain thereof. For example, an “antibody” may include any protein or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule having biological activity to bind to an antigen. Non-limiting examples include the complementarity-determining region (CDR) of a heavy or light chain or its ligand-binding portion, the variable region of a heavy or light chain, the constant region of a heavy or light chain, the framework (FR) region, or any portion thereof, or at least a portion of a binding protein. As used herein, the term “antibody” may refer to an immunoglobulin molecule and an immunoglobulin (Ig) molecule, i.e., the immunoactive portion of a molecule containing an antigen-binding site that specifically binds to (immunely reacts with) an antigen. “Specifically binding” or “immunely reacting” means that the antibody reacts with one or more antigenicity-determining sites of an antigen and not with other polypeptides.

[0100] As used herein, the terms “antibody fragment” or “antigen-binding fragment” are defined as F (ab’)2 F (ab)2 F ab ', F ab This refers to a portion of an antibody, such as Fv, scFv, etc. Regardless of its structure, an antibody fragment binds to the same antigen recognized by the complete antibody. The term "antibody fragment" can also encompass aptamers (such as Spiegelmer), minibodies, and diabodies. The term "antibody fragment" can also encompass any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen and forming a complex. The antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', F(ab')2, Fd, Fvs, single-chain Fv(scFv), single-chain antibodies, dAb (domain antibodies), minibodies, disulfide-bonded Fv(sdFv), fragments containing VL or VH domains, fragments generated by Fab expression libraries, and anti-idiotype (anti-Id) antibodies.

[0101] "Single-stranded variable fragment" or "scFv" refers to the heavy chain (V) of immunoglobulins. H ) and light chain (V L This refers to a fusion protein of the variable region of ). A single-chain Fv ("scFv") polypeptide molecule is a covalently bonded VH:VL heterodimer, which can be expressed from a gene fusion containing VH and VL coding genes linked by a peptide-coding linker. (Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). In some embodiments, the region is linked by a short linker peptide of 10 to about 25 amino acids. The linker may be rich in glycine for flexibility, and serine or threonine for solubility, and V H The N-terminus of V LIt may be linked to the C-terminus or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker. Numerous methods have been described for identifying the chemical structure to convert naturally aggregated but chemically separated light and heavy polypeptide chains from the antibody V region into scFv molecules that fold into a three-dimensional structure substantially similar to that of the antigen-binding site. See, for example, U.S. Patents 5,091,513, 5,892,019, 5,132,405, and 4,946,778 (each of which is incorporated herein by reference in whole).

[0102] Antibody molecules obtained from humans are classified into five classes of immunoglobulins: IgG, IgM, IgA, IgE, and IgD, based on the properties of their heavy chains. Those skilled in the art will understand that the heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and that there are several subclasses within these (e.g., γ1-γ4). Certain classes also have subclasses such as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, and IgG5, are well-characterized and known to confer functional specialization. In the case of IgG, a standard immunoglobulin molecule contains two identical light-chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy-chain polypeptides with molecular weights of 53,000-70,000. The four chains can be linked by disulfide bonds in a "Y" shape, with the light chain beginning at the mouth of the "Y" and surrounding the heavy chain that follows through the variable region. The immunoglobulin or antibody molecules described herein may be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a subclass.

[0103] Light chains are classified as kappa or lambda (κ, λ). Each heavy chain class can be bound to a kappa or lambda light chain. Generally, light and heavy chains are covalently linked to each other, and when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. In heavy chains, the amino acid sequence extends from the N-terminus of the Y-branched ends to the C-terminus at the bottom of each chain.

[0104] Both the light and heavy chains are divided into structurally and functionally homologous regions. The terms “constant” and “variable” are used in a functional sense. The variable domains (VL and VH) of both the light and heavy chain portions determine antigen recognition and specificity. Conversely, the constant domains (CL, as well as CH1, CH2, or CH3) of the light and heavy chains confer important biological characteristics such as secretion, transplacental mobility, Fc receptor binding, and complement binding. The term “antigen-binding site” or “binding region” can refer to the portion of the immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly distinct segments within the V regions of the heavy and light chains, referred to as the “hypervariable region,” are inserted between more conserved adjacent segments known as the “framework region” or “FR.” Thus, the term “FR” can refer to the naturally occurring amino acid sequences between and adjacent to the hypervariable region of immunoglobulins. In antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of the heavy chain and the three hypervariable regions of the light chain are called "complementarity-determining regions" or "CDRs." Table 4 shows the VH and VL regions containing the CDRs and framework (FR) of the iAP antibody.

[0105] The six CDRs in each antigen-binding domain are short, discontinuous sequences of amino acids that are specifically positioned to form the antigen-binding domain when the antibody takes its three-dimensional configuration in an aqueous environment. The remaining amino acids of the antigen-binding domain, the FR region, exhibit less intermolecular variation. The framework region primarily conforms to a β-sheet structure, where the CDRs link together to form loops and, in some cases, form part of the β-sheet structure. The framework region functions to form a scaffold for positioning the CDRs in the correct orientation through non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs provides a complementary surface to the epitope on the antigen in the immune response, promoting non-covalent binding of the antibody to the congeneral epitope. Since the amino acids containing the CDR and framework regions have been previously defined (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., USD Department of Health and Human Services, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)), those skilled in the art can readily identify them for the heavy chain or light chain variable region.

[0106] Where there are two or more definitions for a term used and / or permitted in the art, the definitions used herein are intended to include all such meanings unless explicitly stated otherwise. A specific example is the use of the term “complementarity-determining region” (“CDR”) to describe non-adjacent antigen-binding sites found within the variable regions of both heavy-chain and light-chain polypeptides. These regions are described by Kabat et al., USDept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and Chothia et al., J.Mol.Biol.196:901-917 (1987), which are incorporated herein by reference in their entirety. The definitions of CDR by Kabat and Chothia include overlaps or subsets of amino acid residues when compared to one another. Nevertheless, it is intended that applying the definitions to refer to the CDR of an antibody or its variant is also within the scope of the terms defined and used herein. For comparison, the appropriate amino acid residues encompassing the CDR as defined by each of the references cited herein are listed in the table herein. The exact residue numbers containing the CDR vary depending on the sequence and size of the CDR. Those skilled in the art can systematically determine which residues constitute the CDR simply by knowing the amino acid sequence of the antibody's variable region.

[0107] TIFF2026516499000008.tif33128

[0108] Kabat et al. defined a numbering system for variable domain sequences applicable to any antibody. Those skilled in the art can clearly assign this “Kabat numbering” system to any variable domain sequence without relying on other experimental data of the sequence itself. As used herein, “Kabat numbering” refers to the numbering system presented by Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0109] In addition to the tables described herein, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins around amino acid 31 (i.e., about 9 residues after the first cysteine ​​residue), contains about 5-7 amino acids, and ends with the following tryptophan residue. CDR-H2 begins at the 15th residue after the end of CDR-H1, contains about 16-19 amino acids, and ends with the following arginine or lysine residue. CDR-H3 begins at approximately the 33rd amino acid residue after the end of CDR-H2, contains 3-25 amino acids, and ends with the sequence WGXG (where X is any amino acid). CDR-L1 begins at approximately residue 24 (i.e., after the cysteine ​​residue), contains about 10-17 residues, and ends with the following tryptophan residue. CDR-L2 begins at approximately the 16th residue after the end of CDR-L1, contains about 7 residues. CDR-L3 begins approximately 33 residues after the end of CDR-L2 (i.e., after the cysteine ​​residue), contains about 7 to 11 residues, and ends with the sequence F or WGXG (where X is any amino acid).

[0110] As used herein, the term “epitope” may include any protein determinant that can specifically bind to an immunoglobulin, scFv, or T cell receptor. The variable region allows an antibody to selectively recognize and specifically bind to an epitope on an antigen. For example, a combination of the VL and VH domains of an antibody, or a subset of complementarity-determining regions (CDRs), forms a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites at the ends of each arm of the Y. Epitope determinants can consist of chemically active surface classifications of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural and specific charge characteristics. For example, antibodies can be produced against the N-terminal or C-terminal peptide of a polypeptide. More specifically, the antigen-binding site is defined by three CDRs on the VH and VL chains, respectively (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In one embodiment, the antibody may target human enteric alkaline phosphatase (iAP) (528 amino acid residues in length) having NCBI reference number NP_001622.2, which includes the amino acid sequence of SEQ ID NO: 12. TIFF2026516499000009.tif46134

[0111] As used herein, the terms “immunological binding” and “immunological binding properties” may refer to a type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (K) of the interaction. d ) can be expressed in terms of K d A smaller value indicates greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, whose rates depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Thus, both "on-rate constants" (K) are... on) and "off-speed constant" (K off ) can be determined by calculating the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). K off / K on The ratio allows for the cancellation of all parameters unrelated to affinity, and the equilibrium coupling constant K D This is equivalent to (see, for example, Davies et al. (1990) Annual Rev Biochem 59:439-473). The antibodies of the present invention can be measured by dynamic assays, e.g., radioligand binding assays or similar assays known to those skilled in the art, e.g., BIAcore or Octet (BLI), to obtain an equilibrium binding constant (K D When the ratio is ≤1 μM, ≤10 μm, ≤10 nM, ≤10 pM, or ≤100 pM to approximately 1 pM, it can specifically bind to the iAP epitope. For example, in some embodiments, K D It is approximately 1E -12 M~K D Approximately 1E -11 M is the case in some embodiments. D It is approximately 1E -11 M~K D Approximately 1E -10 M is the case in some embodiments. D It is approximately 1E -10 M~K D Approximately 1E -9 M is the case in some embodiments. D It is approximately 1E -9 M~K D Approximately 1E -8 M is the case in some embodiments. D It is approximately 1E -8 M~K D Approximately 1E -7 M is the case in some embodiments. D It is approximately 1E -7 M~K D Approximately 1E -6 M is M. For example, in some embodiments, K D It is approximately 1E -12 M is M, and in other embodiments, K D It is approximately 1E -11is M. In some embodiments, K D is about 1E -10 M, and in other embodiments, K D is about 1E -9 M. In some embodiments, K D is about 1E -8 M, and in other embodiments, K D is about 1E -7 M. In some embodiments, K D is about 1E -6 M, and in other embodiments, K D is about 1E -5 M. In some embodiments, for example, K D is about 3E -11 M, and in other embodiments, K D is about 3E -12 M. In some embodiments, K D is about 6E -11 M. "Specifically binds to" or "has specificity for" can refer to an antibody that binds to an epitope via its antigen-binding domain, where the binding involves some complementarity between the antigen-binding domain and the epitope. For example, an antibody is said to "specifically bind to" an epitope when it binds to that epitope via its antigen-binding domain more readily than it would bind to a random unrelated epitope.

[0112] For example, an iAP antibody can be monovalent or bivalent and can include single-stranded or double-stranded. Functionally, the binding affinity of an iAP antibody is in the range of 10 -5 M to 10 -12 M. For example, the binding affinity of an iAP antibody is 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10 -5 M to 10 -11 M, 10 -6 M to 10 -11 M, 10-7 M~10 -11 M, 10 -8 M~10 -11 M, 10 -9 M~10 -11 M, 10 -10 M~10 -11 M, 10 -5 M~10 -10 M, 10 -6 M~10 -10 M, 10 -7 M~10 -10 M, 10 -8 M~10 -10 M, 10 -9 M~10 -10 M, 10 -5 M~10 -9 M, 10 -6 M~10 -9 M, 10 -7 M~10 -9 M, 10 -8 M~10 -9 M, 10 -5 M~10 -8 M, 10 -6 M~10 -8 M, 10 -7 M~10 -8 M, 10 -5 M~10 -7 M, 10 -6 M~10 -7 M, or 10 -5 M~10 -6 It is M.

[0113] iAP proteins, or their derivatives, fragments, analogs, homologs, or homologous molecular species, can be used as immunogens in the production of antibodies that immunologically bind specifically to these protein components, for example, amino acid residues including SEQ ID NO: 12. iAP proteins, or their derivatives, fragments, analogs, homologs, or orthologs coupled to proteoliposomes, can be used as immunogens in the production of antibodies that immunospecifically bind to these protein components.

[0114] Those skilled in the art will recognize that, without excessive experimentation, it is possible to determine whether a human monoclonal antibody has the same specificity as the human monoclonal antibody of the present invention by determining whether the former prevents the latter from binding to iAPs. For example, if the human monoclonal antibody under test competes with the human monoclonal antibody of the present invention, as indicated by the reduced binding by the human monoclonal antibody of the present invention, the two monoclonal antibodies can bind to the same or closely related epitopes.

[0115] Another method for determining whether a human monoclonal antibody has the specificity of the human monoclonal antibody of the present invention is to pre-incubate the human monoclonal antibody of the present invention with an iAP protein that is normally reactive with it, then add the human monoclonal antibody to be tested, and determine whether the human monoclonal antibody to be tested is inhibited in its ability to bind to the iAP. If the human monoclonal antibody to be tested is inhibited, it likely has the same or functionally equivalent epitope specificity as the monoclonal antibody of the present invention. Screening of the human monoclonal antibody of the present invention can also be carried out by utilizing the iAP and determining whether the monoclonal antibody to be tested can neutralize the iAP.

[0116] Various procedures known within the art can be used to produce polyclonal or monoclonal antibodies directed against the proteins of the present invention, or against their derivatives, fragments, analogues, homologs, or homologous molecular species. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0117] Antibodies can be purified by well-known techniques such as affinity chromatography using protein A or protein G, which primarily provide the IgG fraction of immunoserum. Subsequently, or alternatively, the specific antigen or epitope that is the target of the desired immunoglobulin can be immobilized on a column, and immuno-specific antibodies can be purified by immunoaffinity chromatography. The purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0118] As used herein, the terms “monoclonal antibody,” “mAb,” “Mab,” or “monoclonal antibody composition” may refer to a group of antibody molecules containing only one species of antibody molecule, consisting of a specific light chain gene product and a specific heavy chain gene product. For example, the complementarity-determining region (CDR) of a monoclonal antibody is identical for all molecules in the group. A MAb contains an antigen-binding site that can react immunologically with an antigen epitope characterized by a specific binding affinity to it.

[0119] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). Hybridoma methods involve immunizing mice, hamsters, or other suitable host animals with an immunizer to induce lymphocytes that produce antibodies that specifically bind to the immunizer, or lymphocytes capable of producing such antibodies. Alternatively, lymphocytes can be immunized in vitro.

[0120] The immunotherapy agent may include protein antigens, their fragments, or fusion proteins. For example, for human-derived cells, it may be peripheral blood lymphocytes, or for non-human mammalian sources, it may be spleen cells or lymph node cells. The lymphocytes are then fused with the immortalized cell line using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). The immortalized cell line may be transformed mammalian cells, such as myeloma cells of rodent, bovine, and human origin. For example, rat or mouse myeloma cell lines may be used. The hybridoma cells can be cultured in a suitable medium containing one or more substances that inhibit the proliferation or survival of non-fusioned immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium may contain hypoxanthine, aminopterin, and thymidine ("HAT medium"), which prevent the growth of HGPRT-deficient cells.

[0121] Useful immortalized cell lines are those that efficiently fuse, maintain stable high levels of antibody expression by selected antibody-producing cells, and are sensitive to culture media such as HAT medium. Immortalized cell lines include, for example, mouse myeloma lines available from the Salk Institute Cell Distribution Center (San Diego, California) and the American Type Culture Collection (Manassas, Virginia). Human myeloma and mouse-human heterozygous myeloma cell lines have also been described for the production of human monoclonal antibodies. (See Kozbor, J. Immunol, 133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

[0122] Next, the culture medium in which hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies against the antigen. For example, the binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosolvent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis as described in Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, for therapeutic applications of monoclonal antibodies, it is important to identify antibodies that have high specificity and high binding affinity to the target antigen.

[0123] After hybridoma cells are identified, clones can be subcloned using limiting dilution procedures and grown using standard methods (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986), pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle Medium and RPMI-1640 Medium. Alternatively, hybridoma cells can be grown in vivo as ascites in mammals.

[0124] Monoclonal antibodies secreted by subclones can be isolated or purified from culture media or ascites fluid by conventional immunoglobulin purification procedures such as protein A-Sepharose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0125] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567 (which is incorporated herein by reference in its entirety). The DNA encoding the monoclonal antibodies of the present invention can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can bind to the genes encoding the heavy and light chains of the mouse antibody). Hybridoma cells of the present invention serve as a source of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, to obtain the synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by substituting the coding sequences of human heavy and light chain constant domains for homologous mouse sequences (see U.S. Patent No. 4,816,567, Morrison, Nature 368,812-13 (1994)), or by covalently bonding all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can be used in place of the constant domain of the antibody of the present invention, or in place of the variable domain of one antigen-binding site of the antibody of the present invention, to produce a chimeric bivalent antibody.

[0126] Fully human antibodies are antibody molecules in which the entire sequence of both the light and heavy chains, including, for example, the CDR, is derived from a human gene. Such antibodies are referred to herein as “humanized antibodies” or “fully human antibodies.” Human monoclonal antibodies, such as fully human antibodies and humanized antibodies, can be prepared using trioma technology, human B-cell hybridoma technology (see Kozbor, et al, 1983 Immunol Today 4:72), and EBV hybridoma technology for producing human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies can be used and produced by using human hybridomas (see Cote, et al, 1983. Proc Natl Acad Sci USA80:2026-2030) or by transforming human B cells with Epstein-Barr virus in vitro (see Cole, et al., 1985 "MONOCLONAL ANTIBODIES AND CANCER THERAPY", Alan R. Liss, Inc., pp.77-96).

[0127] Humanized antibodies may be derived from non-human species (such as mice), and their amino acid sequences (e.g., in the CDR region) are modified to increase similarity to antibody variants produced in humans. Antibodies can be humanized by methods known in the art, such as CDR transplantation. See also Safdari et al., (2013) Biotechnol Genet Eng Rev.; 29:175-86. In addition, humanized antibodies can be produced in transgenic plants as an inexpensive alternative to existing mammalian systems. For example, transgenic plants can be tobacco plants, namely Nicotiana benthamiana and Nicotiana tabaccum. Antibodies are purified from plant leaves. Stable transformation of plants can be achieved using Agrobacterium tumefaciens or particle gun methods. For example, nucleic acid expression vectors containing at least heavy and light chain sequences are expressed via transformation in bacterial cultures, i.e., A. tumefaciens strain BLA4404. Plant infiltration can be achieved by injection. Soluble leaf extracts can be prepared by grinding leaf tissue in a mortar and centrifugation. Antibody isolation and purification can be easily carried out by many methods known to those skilled in the art. Other methods of antibody production in plants are described, for example, in Fischer et al., Vaccine, 2003, 21:820-5, and Ko et al., Current Topics in Microbiology and Immunology, Vol.332, 2009, pp.55-78. Accordingly, the present invention further provides any cells or plants comprising a vector encoding or producing the antibody of the present invention.

[0128] Antibodies are used, for example, in CDR transplantation (European Patent No. 239,400, International Publication No. WO91 / 09967, U.S. Patents No. 5,225,539, 5,530,101, and 5,585,089), veneering, or resurfacing (European Patent No. 592,106, European Patent No. 519,596, Padlan, Molecular Immunology 28(4 / 5):489-498 (1991), Studnicka et al., Protein Engineering 7(6):805-814 (1994), Roguska et al., Proc. Natl. Sci. USA). Humanization can be performed using various techniques known in the art, including chain shuffling (US Patent No. 5,565,332, which is incorporated in whole by reference). "Humanization" (also called reshaping or CDR transplantation) is a well-established technique understood by those skilled in the art to reduce the immunogenicity of monoclonal antibodies (mAbs) derived from xenogeneic sources (such as rodents) and improve the activation of the human immune system (see, for example, Hou S, Li B, Wang L, Qian W, Zhang D, Hong X, Wang H, Guo Y (July 2008). "Humanization of an anti-CD34 monoclonal antibody by complementarity-determining region grafting based on computer-assisted molecular modeling." J Biochem. 144(1):115-20).

[0129] In addition, antibodies (such as human antibodies) can also be produced using other techniques, including phage display libraries. (See Hoogenboom and Winter, J.Mol.Biol, 227:381 (1991), Marks et al., J.Mol.Biol, 222:581 (1991)). Similarly, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, such as mice in which the endogenous immunoglobulin gene is partially or completely inactivated. After the challenge, human antibody production is observed, which is very similar in all aspects to that seen in humans, including gene rearrangement, assembly, and antibody repertoire. This approach is, for example, described in U.S. Patents No. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as Marks et al., Bio / Technology 10,779-783 (1992), Lonberg et al., Nature 368 856-859 (1994), Morrison, Nature 368 812-13 (1994), Fishwild et al., Nature Biotechnology 14,845-51 (1996), Neuberger, Nature Biotechnology 14,826 (1996), and Lonberg et al. This is described in Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0130] Human antibodies can also be produced using transgenic non-human animals that are modified to produce fully human antibodies rather than endogenous antibodies in response to antigenic challenge (see International Publication WO94 / 02602 and U.S. Patent No. 6,673,986). Endogenous genes encoding heavy and light chain immunoglobulin chains in the non-human host are neutralized, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host genome. Human genes are incorporated, for example, using a yeast artificial chromosome containing the required human DNA segment. Animals providing all modifications are then obtained as offspring by mating with intermediate transgenic animals containing fewer complements than the complete complements of the modifications. A preferred embodiment of such a non-human animal is the mouse, referred to as Xenomouse®, as disclosed in International Publication WO96 / 33735 and International Publication WO96 / 34096. This animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained, for example, as preparations of polyclonal antibodies, directly from animals after immunization with the immunogen of interest, or alternatively, from immortalized B cells of animal origin, such as hybridomas that produce monoclonal antibodies. In addition, genes encoding immunoglobulins with human variable regions can be recovered and expressed to directly obtain antibodies, or further modified to obtain antibody analogs, such as single-chain Fv(scFv) molecules.

[0131] Therefore, such techniques can be used to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technique for producing human antibodies, see Lonberg and Huszar Int. Rev. Immunol. 73:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies, and protocols for producing such antibodies, see, for example, International Publication Nos. WO98 / 24893, WO96 / 34096, WO96 / 33735, U.S. Patents Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, all of which are incorporated herein by reference. In addition, companies such as Creative BioLabs (Shirley, NY) can offer services to provide human antibodies against selected antigens using technologies similar to those described above.

[0132] An example of a method for producing a non-human host, exemplified by a mouse, lacking the expression of endogenous immunoglobulin heavy chains is disclosed in U.S. Patent No. 5,939,598. This can be obtained by a method comprising deleting a J-segment gene from at least one endogenous heavy chain locus in embryonic stem cells to prevent locus rearrangement and the formation of a transcript of the rearranged immunoglobulin heavy chain locus, wherein the deletion is carried out by a targeted vector containing a gene encoding a selectable marker, and producing a transgenic mouse from embryonic stem cells, wherein its somatic and germ cells contain a gene encoding a selectable marker.

[0133] One method for producing a target antibody, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. This method involves introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in a culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing both a heavy chain and a light chain.

[0134] Further improvements to this procedure include methods for identifying clinically relevant immunogenic epitopes and correlational methods for selecting antibodies that bind immunospecifically to these relevant epitopes with high affinity, as disclosed in International Publication No. WO99 / 53049.

[0135] The antibody of interest can also be expressed by a vector containing a DNA segment encoding the single-chain antibody described herein. Examples of vectors include, but are not limited to, chemical conjugates such as those described in International Publication WO93 / 64701, having a target-directing moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid-binding moiety (e.g., polylysine); viral vectors (e.g., DNA or RNA viral vectors); fusion proteins such as those described in International Application No. PCT / US95 / 02140 (International Publication WO95 / 22618), which include a target moiety (e.g., an antibody specific to a target cell) and a nucleic acid-binding moiety (e.g., protamine); plasmids; phages; and viral vectors. Vectors can be chromosomal, non-chromosomal, or synthetic. Retroviral vectors can also be used, such as Moloney's mouse leukemia virus. DNA viral vectors can also be used, including pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as herpes simplex virus I (HSV) vectors (see Geller, AI et al, J. Neurochem, 64:487 (1995), Lim, F., et al, DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995), Geller, AI et al, Proc Natl. Acad. Sci.: USA 90, 7603 (1993), Geller, AI et al, Proc Natl. Acad. Sci USA 87:1149 (1990)), and adenovirus vectors (see LeGal LaSalle et al, Science, 259:988 (1993), Davidson, et al, Nat. Genet This includes 3:219 (1993), Yang, et al, J. Virol. 69:2004 (1995), and adeno-associated virus vectors (see Kaplitt, MG. et al, Nat. Genet. 8:148 (1994)).

[0136] Poxvirus vectors introduce genes into the cytoplasm of cells. Avidoxvirus vectors result in short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors can be used to introduce nucleic acids into nerve cells. Adenovirus vectors result in shorter expression periods (approximately 2 months) than adeno-associated virus vectors (approximately 4 months), and consequently shorter than HSV vectors. The choice of vector will depend on the target cell and the condition being treated. Introduction may be by standard techniques (e.g., infection, transfection, transduction, or transformation). Examples of gene introduction methods include, for example, naked DNA, CaP04 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0137] Vectors can be used to target any target cell. For example, stereotactic injection can be used to orient the vector (e.g., adenovirus, HSV) to a specific location. In addition, particles can be delivered by intraventricular (icv) injection using minipump infusion systems such as the SynchroMed Infusion System. Methods based on bulk flow called convection have also been proven effective in delivering large molecules to dilated areas of the brain and may be useful for delivering vectors to target cells. (See Bobo et al, Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994), Morrison et al, Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used include catheter, intravenous, parenteral, intraperitoneal, and subcutaneous injections, as well as oral or other known routes of administration.

[0138] These vectors can be used to express large quantities of antibodies that can be used in various ways, for example, to detect the presence of iAP in a sample.

[0139] In one embodiment, the antibody described herein may be a full-length antibody containing an Fc region similar to the wild-type Fc region that binds to the Fc receptor.

[0140] The technique can be adapted for the production of single-chain antibodies specific to the antigenic protein of the present invention (see, for example, U.S. Patent No. 4,946,778). In addition, the method can be adapted for monoclonal F having specificity to the protein or its derivatives, fragments, analogs or homologs. ab To enable rapid and effective identification of fragments, F ab The method can be adapted for the construction of expression libraries (see, for example, Huse, et al, 1989 Science 246:1275-1281). Antibody fragments containing idiotypes for protein antigens can be produced by techniques known in the art, including, but not limited to, the following: (i) F produced by pepsin digestion of antibody molecules. (ab’)2 Fragment, (ii)F (ab’)2 F is produced by reducing the disulfide bridges of the fragments. ab (iii) F produced by treating the fragment and antibody molecule with papain and a reducing agent. ab Fragments, and (iv)F v piece.

[0141] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies consist of two covalently bound antibodies. Such antibodies can, for example, target immune system cells to undesirable cells (see U.S. Patent No. 4,676,980) and may be used for the treatment of HIV infection (see International Publications WO91 / 00360 and WO92 / 20373). Antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those containing crosslinking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolates and methyl-4-mercaptobutylimidate, as disclosed, for example, in U.S. Patent No. 4,676,980.

[0142] The antibodies of the present invention can be modified with respect to effector function, for example, to enhance the efficacy of the antibody in the detection or treatment of gastrointestinal diseases. For example, a cysteine ​​residue can be introduced into the Fc region, thereby enabling the formation of interchain disulfide bonds in this region. The homodimeric antibodies thus produced may have improved internalization ability and / or increased complement-mediated cell killing and antibody-dependent cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, antibodies having a double Fc region, thereby having enhanced complement lysis and ADCC ability, can be manipulated. (See Stevenson et al, Anti-Cancer Drug Design, 3:219-230 (1989)). In one embodiment, the antibody of the present invention has a modification of the Fc region such that the Fc region does not bind to the Fc receptor. For example, the Fc receptor is the Fcγ receptor. Antibodies having a modified Fc region in which the Fc region does not bind to Fcγ but still binds to the neonatal Fc receptor are useful, as described herein.

[0143] In certain embodiments, the antibodies of the present invention may include Fc variants that modify the antigen-independent effector function of the antibody, for example, by amino acid substitutions that alter the circulating half-life of the antibody. Such antibodies exhibit increased or decreased binding to FcRn when compared to antibodies lacking these substitutions, and therefore have increased or decreased serum half-lives, respectively. Fc variants with improved affinity for FcRn may have a longer serum half-life, and such molecules have use applications in methods of treating mammals where a longer half-life of the administered antibody is beneficial, for example, to treat chronic diseases or disorders. In contrast, Fc variants with reduced FcRn binding affinity may have a shorter half-life, and such molecules are also useful for administration to mammals where a shortened circulating time may be advantageous, for example, for in vivo diagnostic imaging, or in situations where the starting antibody has toxic side effects if it is circulating for a long period of time. Fc variants with reduced FcRn binding affinity are less likely to cross the placenta and are therefore useful in the treatment of diseases or disorders in pregnant women. In addition, other applications of reduced FcRn binding affinity include localizing antibodies to the brain, kidneys, and / or liver. In one embodiment, an Fc variant-containing antibody can exhibit reduced transport from the vascular system across the renal glomerular epithelium. In another embodiment, an Fc variant-containing antibody can exhibit reduced transport from the brain across the blood-brain barrier (BBB) ​​into the vascular space. In one embodiment, an antibody with modified FcRn binding contains an Fc domain having one or more amino acid substitutions within the "FcRn binding loop" of the Fc domain. The FcRn binding loop consists of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions that modify FcRn binding activity are disclosed in International Publication WO05 / 047327, incorporated herein by reference. In certain exemplary embodiments, the antibody or fragment thereof of the present invention comprises an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering).

[0144] In some embodiments, mutations are introduced into the constant region of an mAb so as to alter the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the mAb. For example, the mutation is an LALA mutation in the CH2 domain. In one embodiment, the antibody (e.g., a human mAb, or a bispecific Ab) contains a mutation on one scFv unit of a heterodimeric mAb that reduces ADCC activity. In another embodiment, the mAb contains mutations on both strands of a heterodimeric mAb that completely eliminate ADCC activity. For example, a mutation introduced into one or both scFv units of an mAb is an LALA mutation in the CH2 domain. These mAbs with variable ADCC activity can be optimized so that the mAb exhibits maximum selective killing toward cells expressing one antigen recognized by the mAb, but minimum killing toward a second antigen recognized by the mAb.

[0145] In other embodiments, antibodies for use in the diagnostic and therapeutic methods described herein have a constant region, such as a heavy chain constant region of IgG1 or IgG4, which can be modified to reduce or eliminate glycosylation. For example, the antibodies of the present invention may also include Fc variants that include amino acid substitutions that modify the glycosylation of the antibody. For example, in the Fc variant, glycosylation (e.g., N-linked or O-linked glycosylation) can be reduced. In some embodiments, the Fc variant includes reduced glycosylation of an N-linked glycan commonly found at amino acid position 297 (EU numbering). In some embodiments, the antibody has an N-linked glycosylation site in the heavy chain at amino acid position 304 (EU numbering). In some embodiments, the antibody has an N-linked glycosylation site in the light chain at amino acid position 202 (EU numbering). In another embodiment, the antibody has amino acid substitutions near or within a glycosylation motif, such as an N-linked glycosylation motif containing the amino acid sequence NXT or NXS. In one embodiment, the antibody comprises an Fc variant having an amino acid substitution at amino acid position 228 or 299 (EU numbering). In a more specific embodiment, the antibody comprises an IgG1 or IgG4 constant region containing S228P and T299A mutations (EU numbering). In some embodiments, the N-linked glycosylation site of the antibody described herein is removed. For example, a deglycosylation variant of the heavy chain of the antibody described herein may include the N304A mutation (EU numbering). For example, a deglycosylation variant of the light chain of the antibody described herein may include the N202A mutation (EU numbering).

[0146] Exemplary amino acid substitutions for reducing or altering glycosylation are disclosed in International Publication No. WO05 / 018572, incorporated herein by reference. In some embodiments, the antibody or fragment thereof of the present invention is modified to eliminate glycosylation. Such an antibody or fragment thereof may be referred to as an "agly" antibody or fragment thereof (e.g., an "agly" antibody). While not wishing to be bound by theory, an "agly" antibody or fragment thereof may have an improved safety and stability profile in vivo. An exemplary agri antibody or fragment thereof includes a deglycosylated Fc region of an IgG4 antibody that lacks Fc effector function, thereby eliminating the possibility of Fc-mediated toxicity to normal living tissues and cells expressing iAP. In yet another embodiment, the antibody or fragment thereof of the present invention includes a modified glycan. For example, the antibody may have a reduced number of fucose residues on the N-glycan at Asn297 in the Fc region, i.e., it is defucosylated. In another embodiment, the antibody may have a modified number of sialic acid residues on the N-glycan at Asn297 in the Fc region.

[0147] The present invention also covers cytotoxic agents such as toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or immunoconjugates (the latter being radioconjugates) comprising antibodies conjugated to radioisotopes.

[0148] Enzymatically active toxins and their fragments that can be used include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modexin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, geronin, mitogenin, restrictoctocin, phenomycin, enomycin, and trichothecenes. Various radionuclides are available for the production of radioconjugated antibodies. Non-limiting examples include: 212 Bi, 131 I, 131 In, 90 Y, and 186 Re is one example.

[0149] Antibody and cytotoxic agent conjugates are prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutarelaldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., triene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al, Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radioactive nucleotides to antibodies. (See International Publication WO94 / 11026 and U.S. Patents 5,736,137).

[0150] Those skilled in the art will understand that a wide variety of parts can be coupled to the resulting antibody or other molecules of the present invention. (See, for example, “Conjugate Vaccines,” Contributions to Microbiology and Immunology, JMCruse and RELewis, Jr (eds), Carger Press, New York, (1989), which is incorporated herein by reference in its entirety.)

[0151] Coupling can be achieved by any chemical reaction that will bind two molecules together, insofar as the antibody and the other part retain their respective activities. This binding can include many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordination bonding, and complex formation. In one embodiment, the binding is covalent. Covalent bonding can be achieved by direct condensation of existing side chains or by the incorporation of external crosslinking molecules. Many divalent or polyvalent binding agents are useful for coupling protein molecules, such as the antibody of the present invention, to other molecules. For example, typical coupling agents can include organic compounds such as thioesters, carbodiimides, succinimides, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to encompass all classes of coupling agents known in the art, but rather to be illustrative of coupling agents. (See Killen and Lindstrom, Jour.Immun.133:1335-2549 (1984), Jansen et al., Immunological Reviews 62:185-216 (1982), and Vitetta et al, Science 238:1098 (1987)). Non-limiting examples of linkers are described in the literature. (For example, Ramakrishnan, S. et al., Cancer Res.44:201-208 (1984) describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester).) See also U.S. Patent No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives coupled to antibodies via oligopeptide linkers.Some non-limiting examples of useful linkers that can be used with the antibodies of the present invention include: (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene) (Pierce Chem. Co., Cat. (21558G)), (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio)propionamide] hexanoate) (Pierce Chem. Co., Cat #21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6 [3-(2-pyridyldithio)-propianamide (propianamide)] hexanoate) (Pierce Chem. Co. Examples include (v) Sulfo-NHS (-hydroxysulfosuccinimide: Pierce Chem.Co., Cat.#24510) conjugated to EDC.

[0152] The linkers described herein contain components with different attributes, resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylate salts. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, linker SMPTs can contain sterically hindered disulfide bonds and form conjugates with improved stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in fewer available conjugates. For example, sulfo-NHS can enhance the stability of carbodimide coupling. When used in combination with sulfo-NHS, carbodimide coupling (such as EDC) forms esters that are more resistant to hydrolysis than carbodimide coupling alone.

[0153] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies are prepared by methods known in the art, such as those described in Epstein et al, Proc. Natl. Acad. Sci. USA, 82:3688 (1985), Hwang et al, Proc. Natl. Acad. Sci. USA, 77:4030 (1980), and U.S. Patents 4,485,045 and 4,544,545. Liposomes with extended circulation times are disclosed in U.S. Patent 5,013,556. Non-limiting examples of useful liposomes can be produced by reverse-phase evaporation using lipid compositions containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through a filter of a defined pore size to produce liposomes having a given diameter. The Fab' fragment of the antibody of the present invention can be conjugated to liposomes via a disulfide exchange reaction, as described in Martin et al, J. Biol. Chem., 257:286-288 (1982).

[0154] A multispecific antibody is an antibody capable of recognizing two or more different antigens. For example, a bispecific antibody (bsAb) is an antibody containing two variable domains or scFv units so that the resulting antibody recognizes two different antigens. For example, a trispecific antibody (tsAb) is an antibody containing two variable domains or scFv units so that the resulting antibody recognizes three different antigens. The present invention provides multispecific antibodies, such as bispecific antibodies and trispecific antibodies, that recognize iAP and a second and / or third antigen. In one embodiment, the multispecific antibody (e.g., bispecific and trispecific antibodies) may include an iAP-specific fusion protein encompassing the antibody described herein. In one embodiment, the bispecific and trispecific antibodies include an iAP fusion protein. For example, the fusion protein includes an antibody containing a variable domain or scFv unit as described herein, and a ligand or antigen and / or a third ligand or antigen, so that the resulting antibody recognizes the antigen and binds to a ligand-specific receptor. In one embodiment, the fusion protein further includes a constant region and / or a linker as described herein. Different formats of multispecific antibodies (e.g., bispecific and trispecific antibodies such as fusion proteins containing an antibody and ligand that recognize iAP) are described herein. Various formats of bispecific or trispecific antibodies are also provided herein. In some embodiments, each of the anti-iAP fragment and the second and / or third antigen-specific fragments is independently selected from Fab fragments, single-chain variable fragments (scFv), or single-domain antibodies. The bispecific or trispecific antibodies of the present invention may include combinations of heavy and light chains or scFv of the iAP antibody described herein.

[0155] The multispecific antibodies (e.g., bispecific and tripspecific antibodies) of the present invention (e.g., anti-iAP-scFv fusion proteins) can be constructed using methods known in the art. In some embodiments, the bispecific antibody is a single polypeptide in which two scFv fragments are linked by a long linker polypeptide of sufficient length to allow intramolecular association between the two scFv units to form an antibody. In other embodiments, the bispecific antibody is two or more polypeptides linked by covalent or non-covalent bonds. In some embodiments, the amino acid linker (GGGGSGGGGS; "(G4S)2") shown herein can be produced using a longer G4S linker to improve flexibility. For example, the linker may also be "(G4S)3" (e.g., GGGGSGGGGSGGGGS), "(G4S)4" (e.g., GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 13), "(G4S)5" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 14), "(G4S)6" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGGSGGGGGS) (SEQ ID NO: 15), "(G4S)7" (e.g., GGGGSGGGGSGGGGGSGGGGGSGGGGGSGGGGS) (SEQ ID NO: 16), and so on. For example, using the (G4S)5 linker can provide greater flexibility to the ligands described herein and can improve expression. In some embodiments, the linker may also be (GS) n (GGS) n (GGGS) n (GGSG) n (GGSGG) n , or (GGGGS) n This can be done, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Non-limiting examples of linkers known to those skilled in the art that can be used to construct the fusions described herein can be found in U.S. Patent No. 9,708,412, U.S. Publication Nos. 2018,013,4789 and 2020,0148,771, and International Publication No. WO2019,051,122 (each of which is incorporated by reference in whole).

[0156] In another embodiment, multispecific antibodies (e.g., bispecific and trispecific antibodies such as anti-iAP-scFv fusions) can be constructed using the "knob-into-hole" method (Ridgway et al., Protein Eng 7:617-621 (1996)). In this method, Ig heavy chains of two different variable domains are reduced to selectively cleave the heavy chain pairing while retaining the heavy-light chain pairing. Two heavy-light heterodimers recognizing two or three different antigen / ligands are mixed to facilitate heteroligand binding pair formation mediated by the manipulated "knob-into-hole" action of the CH3 domain.

[0157] In another embodiment, multispecific antibodies (e.g., bispecific and trispecific antibodies such as anti-iAP-scFv fusions) can be constructed by exchanging heavy-light chain dimers from two or more different antibodies to produce hybrid antibodies, where the first heavy-light chain dimer recognizes iAP and the second heavy-light chain dimer recognizes a second and / or third antigen. The mechanism of heavy-light chain dimerization is analogous to the formation of human IgG4, which also functions as a bispecific molecule. Dimerization of IgG heavy chains is facilitated by intramolecular forces such as pairing of the CH3 domain of each heavy chain with disulfide crosslinks. The presence of a specific amino acid (R409) in the CH3 domain has been shown to facilitate dimerization and the construction of the IgG4 molecule. Heavy chain pairing is also further stabilized by inter-heavy chain disulfide crosslinks in the hinge region of the antibody. Specifically, in IgG4, the hinge region contains the amino acid sequence Cys-Pro-Ser-Cys at amino acids 226-230 (compared to the stable IgG1 hinge region containing the sequence Cys-Pro-Pro-Cys). This difference in the serine sequence at position 229 is related to IgG4's tendency to form intrachain disulfides in the hinge region (Van der Neut Kolfschoten, M. et al, 2007, Science 317:1554-1557 and Labrijn, AF et al, 2011, Journal of Immunol 187:3238-3246).

[0158] The multispecific antibodies of the present invention (e.g., bispecific and trispecific antibodies such as anti-iAP-scFv fusions) can be produced by introducing the R409 residue in the CH3 domain and the Cys-Pro-Ser-Cys sequence in the hinge region of the antibody that recognizes iAP or a second and / or third antigen, thereby replacing the heavy-light dimer to produce an antibody molecule having one heavy-light dimer that recognizes iAP and a second heavy-light dimer that recognizes a second and / or third antigen, where the second and / or third antigen (or ligand) is any antigen (or ligand) disclosed herein. Known IgG4 molecules can also be modified so that the heavy and light chains recognize iAP or a second and / or third antigen, as disclosed herein. The use of this method for constructing multispecific antibodies (e.g., bispecific and triplicate antibodies such as anti-AP-scFv fusions) of the present invention can be advantageous due to the unique characteristics of the IgG4 molecule, where the Fc region differs from other IgG subtypes in that it has insufficient interaction with effector systems of the immune response, such as complement and Fc receptors expressed by specific leukocytes. This particular property makes these IgG4-based multispecific antibodies (e.g., bispecific and triplicate antibodies such as anti-iAP-scFv fusions) attractive for therapeutic applications where the antibody needs to bind to a target and functionally modify a target-related signaling pathway, but without inducing effector activity.

[0159] The multispecific antibodies described herein (e.g., bispecific and triplicate antibodies such as anti-iAP-scFv fusions) can be manipulated with non-depleted heavy chain isotypes, such as IgG1-LALA, stabilized IgG4, or one of other non-depleted variants. In some embodiments, the mutation is introduced into the constant region of bsAb so as to alter the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of bsAb. For example, the mutation is an LALA mutation in the CH2 domain. In one embodiment, the multispecific antibody (e.g., bispecific and triplicate antibodies such as anti-iAP-scFv fusions) contains a mutation on one scFv unit of the heterodimer multispecific antibody that reduces ADCC activity. In another embodiment, the multispecific antibody (e.g., bispecific and triplicate antibodies such as anti-iAP-scFv fusions) contains a mutation on both chains of the heterodimer multispecific antibody that completely eliminates ADCC activity. For example, a mutation introduced into one or both scFv units of a multispecific antibody (e.g., bispecific and trispecific antibodies such as anti-iAP-scFv fusions) is an LALA mutation in the CH2 domain. These multispecific antibodies with variable ADCC activity (e.g., bispecific and trispecific antibodies such as anti-iAP-scFv fusions) can be optimized so that the multispecific antibody exhibits maximum selective cell death toward cells expressing one antigen recognized by the multispecific antibody, but minimum cell death toward a second antigen recognized by the multispecific antibody.

[0160] The multispecific antibodies disclosed herein (e.g., bispecific and trispecific antibodies such as anti-iAP-scFv fusions) may be useful in the treatment of chronic infections, diseases, or medical conditions, such as gastrointestinal disorders.

[0161] Pharmaceutical composition The antibody that specifically binds to the iAP protein or fragment thereof according to the present invention can be administered in the form of a pharmaceutical composition for the treatment of gastrointestinal diseases. Principles and considerations involved in preparing a therapeutic pharmaceutical composition containing the antibody, as well as guidance in the selection of components, are provided, for example, in Remington: The Science And Practice Of Pharmacy 20th ed. (Alfonso R. Gennaro, et al, editors) Mack Pub. Co., Easton, Pa., 2000; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0162] The specific dosage and treatment plan for a patient depends on various factors, including the antibody, its variant or derivative used, the patient's age, weight, general health, sex, and diet, as well as the timing of administration, excretion frequency, concomitant drug use, and the severity of the disease being treated. The assessment of such factors by healthcare professionals is within the scope of the skills of those skilled in the art. The dosage also depends on the individual patient being treated, the route of administration, the type of formulation, the properties of the compound used, the severity of the disease, and the intended effect. The dosage used can be determined by pharmaceutical and pharmacokinetic principles well known in the art.

[0163] The therapeutically effective dose of the antibody of the present invention can be the amount necessary to achieve the therapeutic objective. As described herein, this can be the binding interaction between the antibody and its target antigen that, in particular, interferes with the function of the target. The amount to be administered further depends on the binding affinity of the antibody to its specific antigen, and also on the rate at which the administered antibody is depleted from the free volume of the other target to which it is administered. The dose administered to a target (e.g., a patient) of the antigen-binding polypeptide described herein can be 0.1 mg / kg to 100 mg / kg patient body weight, 0.1 mg / kg to 20 mg / kg patient body weight, or 1 mg / kg to 10 mg / kg patient body weight. Human antibodies have a longer half-life in the human body than antibodies from other species due to the immune response to exogenous polypeptides. Therefore, lower doses and lower frequencies of administration of human antibodies can be used. Furthermore, the dose and frequency of administration of the antibodies of this disclosure can be reduced by enhancing antibody uptake and penetration into tissues (e.g., the brain) through modifications such as lipidization. The therapeutically effective dosage range for the antibody or antibody fragment of the present invention can be, as a non-limiting example, about 0.1 mg / kg body weight to about 50 mg / kg body weight. The dosage frequency can be, for example, in the range of twice a day to once a week.

[0164] When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein can be used. For example, based on the variable region sequence of the antibody, a peptide molecule that retains the ability to bind to the target protein sequence can be designed. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, for example, Marasco et al, Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)). The formulation may also contain two or more active compounds necessary for the indication being treated, for example, those having complementary activity that does not adversely affect each other. Alternatively, or in addition, the composition may include agents that enhance its function (e.g., cytotoxic agents, cytokines (e.g., IL-15), chemotherapeutic agents, or growth inhibitors). Such molecules are preferably combined in amounts effective for the intended purpose.

[0165] The active ingredient can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions, respectively.

[0166] Preparations used for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filtration membrane.

[0167] Sustained-release preparations can be prepared. Preferred examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, the matrices in the form of molded articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, e.g., LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow for molecular release over more than 100 days, while certain hydrogels release proteins over shorter periods.

[0168] The antibodies or drugs of the present invention (also referred to herein as “active compounds”), as well as their derivatives, fragments, analogs, and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such pharmaceutical compositions typically include the antibody or drug and a pharmaceutically acceptable carrier.

[0169] As used herein, “pharmaceutically acceptable carrier” can include any solvent, dispersion medium, coating, antimicrobial and antifungal agent, isotonic agent and absorption retarder, etc., suitable for pharmaceutically acceptable administration. Preferred carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the art, incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, physiological saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixing oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. The use of any conventional media or agent in a composition is included in this application, except insofar as any conventional media or agent is incompatible with the active compound. Auxiliary active compounds can also be incorporated into the composition.

[0170] The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents, e.g., water for injection, physiological saline, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and agents for adjusting tonicity, such as sodium chloride or dextrose. pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0171] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In embodiments, the composition is sterile and fluid enough to allow easy passage through an injection needle. It may be stable under manufacturing and storage conditions and can be preserved against microbial contamination such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, isotonic agents, such as sugars, polyhydric alcohols such as mannitol and sorbitol, and sodium chloride can be included in the composition. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents in the composition, such as aluminum monostearate and gelatin.

[0172] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent having, as necessary, one or a combination thereof of the components listed herein, followed by filtration sterilization. For example, a dispersion can be prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion solvent and other necessary components listed herein. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and lyophilization, which yield powders of the active component and any additional components from its previously sterile filtered solution.

[0173] Oral compositions include an inert diluent or an edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, in which case the compound in the fluid carrier is applied orally, swirled in the mouth, spit out, or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or sterotes; flow promoters such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring, or compounds with similar properties.

[0174] For administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas like carbon dioxide, or a nebulizer.

[0175] Systemic administration may also be carried out by mucosal or percutaneous means. For mucosal or percutaneous administration, a penetrating agent suitable for the barrier to penetration is used in the formulation. Such penetrating agents are known in the art and, for example, for mucosal administration, include cleansing agents, bile salts, and fusidic acid derivatives. Mucosal administration can be achieved through the use of nasal sprays or suppositories. For percutaneous administration, the active compound is formulated into ointments, plasters, gels, or creams known in the art.

[0176] The compounds can also be prepared in the form of suppositories (e.g., those having a conventional suppository base such as cocoa butter and other glycerides) or retained enemas for rectal delivery.

[0177] In one embodiment, the active compound is prepared on a carrier that will protect the compound from rapid elimination from the body, such as a controlled-release formulation, which includes implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are apparent to those skilled in the art. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0178] For ease of administration and uniformity of dosage, oral or parenteral compositions can be formulated in dose unit forms. As used herein, dose unit forms refer to physically distinct units suitable as unit doses for the subject to be treated, each unit containing a predetermined amount of the active compound calculated to produce a therapeutic effect in conjunction with the necessary pharmaceutical carrier. The specifications of the dose unit forms of the present invention are determined and directly depend on the specific characteristics of the active compound, the therapeutic effect to be achieved, and the limitations inherent in the techniques for formulating such active compounds for the treatment of an individual.

[0179] The pharmaceutical composition may be included in a container, pack, or dispenser along with instructions for administration.

[0180] Method for detecting iAP-related gastrointestinal disorders The antibodies of the present invention, which specifically bind to the iAP protein or fragments thereof, can be administered for the detection of iAP-related gastrointestinal diseases or disorders. "iAP-related diseases or disorders" include conditions and / or symptoms associated with conditions characterized by elevated levels of iAP and / or activation of cellular signaling pathways involving iAP. Exemplary iAP-related diseases or disorders include, but are not limited to, diseases involving the gastrointestinal tract or its regions, i.e., the esophagus, stomach, small intestine, large intestine, or rectum, as well as organs and tissues related to digestion, such as the pancreas, gallbladder, and liver. In some embodiments, gastrointestinal diseases may include colitis, inflammatory bowel disease (IBD), gastritis, gastroenteritis, pyloric stenosis, gastric cancer, infectious diarrhea, fecal impaction, constipation, intestinal obstruction and pseudo-intestinal obstruction, or malabsorption. In addition to necrotizing enterocolitis (NEC), non-limiting examples of enteritis types include adult necrotizing enterocolitis (ANEC), pseudomembranous colitis, infectious colitis, very early-onset inflammatory bowel disease, ulcerative colitis, Crohn's disease, ischemic colitis, and radiation colitis. In some embodiments, gastrointestinal disorders may be caused by microorganisms such as Gram-positive bacteria, Gram-negative bacteria, protozoa, fungi, DNA viruses, RNA viruses, or reverse transcription viruses.

[0181] Non-exclusive examples of bacteria that cause disease include Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, and Campylobacter jejuni. Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularemia Haemophilus influenzae, Helicobacter pylori, Legionella pneumophilaPseudomonas aeruginosa) aeruginosa), Rickettsia, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Ureaplasma urea Vibrio cholera, Yersinia pestis, Yersinia enterocoliticaExamples include Yersinia enterocolitica and Yersinia pseudotuberculosis.

[0182] Non-exclusive examples of protozoa that cause disease include Plasmodium falciparum (malaria), Toxoplasma gondii (toxoplasmosis), Leishmania species (leishmaniasis), Trypanosoma brucei (African sleeping disorder), Trypanosoma cruzi (Chagas disease), and Giardia intestinalis (giardiasis).

[0183] Non-exclusive examples of fungi that cause disease include Candida albicans, Aspergillus fumigatus, Aspergillus flavus, Cryptococcus neoformans, Cryptococcus gattii, Histoplasma capsulatum, Pneumocystis carinii, and Stachybotrys chartarum.

[0184] Non-exclusive examples of viruses that cause disease include adenovirus, coxsackievirus, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 1, herpes simplex virus type 2, cytomegalovirus, human herpesvirus type 8, HIV, influenza virus, measles virus, mumps virus, human papillomavirus, parainfluenza virus, poliovirus, rabies virus, respiratory syncytial virus, rubella virus, varicella-zoster virus, and coronaviruses (such as human coronavirus NL63 (HCoV-NL63), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), or severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)).

[0185] In embodiments, the antibody may include a detectable label. The antibody may be polyclonal or monoclonal. A intact antibody or a fragment thereof (e.g., F ab , scFv, or F (ab)2) can be used. With respect to probes or antibodies, the term “labeled” can include direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include the detection of a primary antibody using a fluorescently labeled secondary antibody, and the end labeling of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. The term “biological sample” can include tissues, cells, and bodily fluids isolated from a subject, as well as tissues, cells, and bodily fluids within a subject. Thus, the use of the term “biological sample” can include feces, e.g., stool, tissues (e.g., blood), cells (e.g., hematopoietic cells, stem cells, or plasma cells such as hematopoietic stem cells, leukocytes, or reticulocytes), vesicles, biomolecular aggregates, or platelets from a subject. In other words, the detection method of the present invention can be used to detect subject mRNA, proteins, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro techniques for detecting subject mRNA include Northern hybridization and in-situ hybridization. In vitro techniques for detecting subject proteins include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include Southern hybridization.

[0186] Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology," Vol. 42, JRCrowther (Ed.), Human Press, Totowa, NJ, 1995; "Immunoassay," E. Diamandis and T. Christophorus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays," P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detecting subject proteins include introducing labeled anti-subject protein antibodies into the target. For example, the antibody can be labeled with a radiomarker whose presence and location in the target can be detected by standard imaging techniques.

[0187] Antibodies against iAP protein (or fragments thereof) can be used in methods known in the art related to the localization and / or quantification of iAP protein (e.g., in measuring the level of iAP protein in a suitable physiological sample, in diagnostic methods, or in protein imaging). In a given embodiment, an antibody containing an antigen-binding domain derived from the antibody, which is specific to iAP protein, or its derivatives, fragments, analogs, or homologs, is used as a pharmaceutically active compound (hereinafter referred to as "therapeutic agent").

[0188] Using antibodies specific to the iAP protein of the present invention, iAP polypeptides can be isolated by standard techniques such as immunoaffinity assay, chromatography, or immunoprecipitation. Antibodies against iAP protein (or fragments thereof) can be used diagnostically, for example, to monitor protein levels in tissues as part of a clinical trial procedure, or to determine the effectiveness of a given treatment regimen.

[0189] Detection can be facilitated by coupling (i.e., physically linking) antibodies to detectable substances. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include 125 I, 131 I, 35 S, 32 P, or 3 H can be mentioned.

[0190] The present invention also relates to immunoconjugates comprising antibodies conjugated with cytotoxic agents such as toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof) or radioconjugates (i.e., radioconjugates). The immune complex may also include antibodies conjugated with a label. In some embodiments, the label comprises a chromophor. Each listed chromophor is reactive with the corresponding enzyme and produces a signal reporting the presence of the immunoconjugate. Superscript notation ( * This indicates that the chromophor emits fluorescence rather than producing a color change. Non-exclusive examples of chromophores and their corresponding enzymes are listed herein.

[0191] TIFF2026516499000010.tif97170

[0192] In some embodiments, the label comprises a binding moiety (e.g., biotin-avidin, biotin-streptavidin, or sugar-lectin, myc tag, his tag, etc.). In some embodiments, the label comprises a coloring compound such as a fluorescent dye. Non-limiting examples of fluorescent dye compounds include fluorescein, ethidium bromide, rhodamine, Texas Red, phycoerythrin (RPE), and cyanine. In some embodiments, the label comprises a fluorescent compound (e.g., GFP, RFP, YFP, BFP, etc.). In some embodiments, the label comprises nanoparticles, the size, composition, and shape of which can determine the absorption and emission properties of the nanoparticles, which can be modified by interaction with other particles having overlapping emission and absorption spectra, which can be subject to other interactions (e.g., plasmon resonance). In some embodiments, the label comprises a chemiluminescent compound (e.g., N-(4-aminobutyl)-N-ethylisoluminol, 4-aminophthalhydrazide, disodium 2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2'-(5-chlorotricyclo[3.3.1.13.7]decane])-4-yl]-1-phenylphosphate). In some embodiments, the label comprises an enzyme compound (e.g., β-D-galactosidase or peroxidase).

[0193] Enzymatically active toxins and their fragments that can be used include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modexin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, geronin, mitogenin, restrictoctocin, phenomycin, enomycin, and trichothecenes. Various radionuclides are available for the production of radioconjugated antibodies. Examples include, 212 Bi, 131 I, 131 In, 90 Y, and 186 The word "Re" is included.

[0194] Antibody and cytotoxic agent conjugates are prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutarelaldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., triene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al, Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radionucleotides to antibodies. (See International Publication WO94 / 11026).

[0195] Those skilled in the art will recognize that a wide variety of parts can be coupled to the resulting antibody or other molecules of the present invention. (See, for example, “Conjugate Vaccines,” Contributions to Microbiology and Immunology, JMCruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), the entire content of which is incorporated herein by reference.)

[0196] For example, antibodies can be coupled (i.e., physically linked) to detectable substances for the detection of iAP or fragments thereof. Non-limiting examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Suitable enzyme examples include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; suitable radioactive materials include... 125 I, 131 I, 35 S, or 3 H can be mentioned.

[0197] Coupling can be achieved by any chemical reaction that will bind two molecules together, insofar as the antibody and the other part retain their respective activities. This binding can include many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordination bonding, and complex formation. Covalent bonding can be used, which can be achieved, for example, by direct condensation of existing side chains or by the incorporation of external crosslinking molecules. Many divalent or polyvalent binding agents are useful for coupling protein molecules, such as the antibodies of the present invention, to other molecules. For example, typical coupling agents can include organic compounds such as thioesters, carbodiimides, succinimides, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to encompass all classes of coupling agents known in the art, but rather to be illustrative of coupling agents. (See Killen and Lindstrom, Jour.Immun.133:1335-2549 (1984), Jansen et al., Immunological Reviews 62:185-216 (1982), and Vitetta et al., Science 238:1098 (1987)).

[0198] Linkers are described in the literature. (For example, see Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Patent No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives coupled to antibodies via an oligopeptide linker. The linkers used are (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene) (Pierce Chem. Co., Cat. (21558G)), (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio)propionamide] hexanoate) (Pierce Chem. Co., Cat #21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6 [3-(2-pyridyldithio)-propianamide (propianamide)] hexanoate) (Pierce Chem. Co., Cat. #2165-G), and (v) sulfo-NHS (N-hydroxysulfosuccinimide) conjugated to EDC: Pierce Chem.Co., Cat.#24510 is one example.

[0199] The linkers described herein contain components with different attributes, resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylate salts. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, linker SMPTs can contain sterically hindered disulfide bonds and form conjugates with improved stability. Disulfide bonds are generally less stable than other bonds because they are more easily broken in vitro, resulting in fewer available conjugates. For example, sulfo-NHS can enhance the stability of carbodimide coupling. When used in combination with sulfo-NHS, carbodimide coupling (such as EDC) forms esters that are more resistant to hydrolysis than carbodimide coupling alone.

[0200] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies are prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985), Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980), and U.S. Patents No. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

[0201] Useful liposomes can be produced by reverse-phase evaporation in a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a defined pore size to produce liposomes having the diameter required for the applications described herein. The Fab' fragment of the antibody of the present invention can be conjugated into liposomes via a disulfide exchange reaction, as described in Martin et al., J. Biol. Chem., 257:286-288 (1982).

[0202] Aspects of the present invention include measuring or detecting biomarkers of gastrointestinal diseases in biological samples. Gastrointestinal diseases can refer to diseases affecting the gastrointestinal tract, i.e., the esophagus, stomach, small intestine, large intestine and rectum, as well as the digestive appendages, liver, gallbladder and pancreas. For example, such diseases may arise from infectious conditions, autoimmune conditions and physiological conditions. Non-limiting examples of gastrointestinal diseases include colitis, inflammatory bowel disease (IBD), gastritis, gastroenteritis, pyloric stenosis, gastric cancer, infectious diarrhea, fecal impaction, constipation, intestinal obstruction and pseudo-intestinal obstruction, or malabsorption. In addition to necrotizing enterocolitis (NEC), non-limiting examples of types of enteritis include adult necrotizing enterocolitis (ANEC), pseudomembranous colitis, infectious colitis, very early-onset inflammatory bowel disease, ulcerative colitis, Crohn's disease, ischemic colitis, and radiation colitis.

[0203] In this embodiment, the gastrointestinal disorder is necrotizing enterocolitis (NEC). NEC is an acquired gastrointestinal disorder that is frequently seen in premature infants. In NEC, bacteria invade the intestinal wall, causing local infection and inflammation. NEC is characterized by a high mortality rate and long-term morbidity (including short bowel syndrome, recurrent infections, malnutrition, and neurodevelopmental delay). Despite an overall net decrease in premature infant mortality, there has been an increase in NEC-related deaths. NEC is often difficult to diagnose and manage due to its early, nonspecific overall symptoms and rapid deterioration.

[0204] In addition to necrotizing enterocolitis seen in neonates and premature infants, necrotizing enterocolitis can also affect non-neonatal individuals. For example, necrotizing enterocolitis in non-neonatal individuals such as adults may be caused by inflammatory mediators; nutritional deficiencies, e.g., anorexia or significant weight loss; functional gastrointestinal disorders; alcoholism; malabsorption; drugs that block intestinal proteases; smoking; circulatory disorders, e.g., decreased mesenteric blood flow, intestinal ischemia, atherosclerosis of the intestinal arteries; cholelithiasis; drug administration; immunodeficiency such as IgA secretory components or intestinal T lymphocytes, accompanied by poor antibody response; fecal impaction or constipation; or infectious agents such as bacterial infections, foodborne infections, and foodborne diseases.

[0205] Non-exclusive examples of such drugs include drugs with anticholinergic properties, such as nerve relaxants or phenothiazine-based nerve relaxants, anesthetics, inflammatory mediators, antidepressants, iron supplements, laxatives, or antacids.

[0206] Non-exclusive examples of such infectious agents include bacteria (e.g., Klebsiella, Escherichia coli, Enterobacter, Pseudomonas, Clostridia, and Staphylococcus epidermidis), viruses (e.g., coronaviruses, rotaviruses, and enteroviruses), and, rarely, fungi (e.g., Candida albicans). Enteropathogenic viruses can infect epithelial cells, potentially leading to cell destruction, necrosis, and intestinal perforation.

[0207] Constipation or impacted stool can have many different causes known in the art, not limited to, antacids containing calcium or aluminum, changes in diet or activity, colon cancer, dairy products, eating disorders, neurological conditions, inactivity, dehydration, fiber intake, laxative abuse, pregnancy, digestive disorders, resistance to the urge to defecate, medication, stress, or hypothyroidism.

[0208] Embodiments may include measuring or detecting biomarkers for gastrointestinal diseases in biological samples. The biomarkers of the present invention can be measured in different types of biological samples. Non-limiting examples of biological samples that can be used in the methods of the present invention include, but are not intended to be limiting, feces, plasma, umbilical cord blood, neonatal blood, cerebrospinal fluid, tears, vomit, saliva, urine, stool, and meconium. In embodiments, samples may be prepared to enhance the detection ability of the biomarker. For example, a sample can be fractionated from a subject. Any method for concentrating the biomarker polypeptide of interest can be used. Sample preparation, such as a preliminary fractionation protocol, is optional and may or may not be necessary to enhance the detection ability of the biomarker, depending on the detection method used. For example, if the presence of a biomarker in a sample is detected using an antibody that specifically binds to the biomarker, sample preparation may not be necessary. Sample preparation may involve fractionation of the sample and collection of the fraction determined to contain the biomarker. Methods for preliminary fractionation include, for example, size exclusion chromatography, ion exchange chromatography, heparin chromatography, affinity chromatography, serial extraction, gel electrophoresis, mass spectrometry, and liquid chromatography.

[0209] The methods described herein may involve obtaining biological samples from subjects such as infants. As used herein, the phrase “obtain a biological sample” refers to any process for directly or indirectly obtaining a biological sample from a subject. For example, a biological sample may be obtained by taking a tissue or fluid sample (e.g., blood, bone marrow, spinal puncture) from a subject (e.g., at a point-of-care facility, e.g., a clinic, hospital, or laboratory). Alternatively, a biological sample may be obtained by receiving a biological sample from one or more people who have obtained a sample directly from the subject (e.g., at a laboratory). A biological sample may be, for example, feces, stool, tissue (e.g., blood), cells (e.g., hematopoietic stem cells, leukocytes, or reticulocytes, or other hematopoietic cells, stem cells, or plasma cells), vesicles, biomolecular aggregates, or platelets derived from the subject.

[0210] The term “sample” can refer to any sample that may contain the analyte of interest, such as iAP or fragments thereof. Samples may be non-biological or biological samples and include clinical specimens (diagnostic specimens collected as part of standard clinical procedures). Non-biological samples include those prepared in vitro, containing the target molecule of interest at various concentrations in a solution. Biological samples include, but are not limited to, whole blood, lymph, serum, plasma, urine, saliva, sputum, exhaled breath extracts (meaning exhaled breath captured in solution), bone marrow, aspirates (nose, lung, bronchi, trachea), eye fluid, amniotic fluid, feces, other body fluids and secretions, cells, and tissue specimens and their dilutions. As used herein, the term “body fluid” means any fluid that can be isolated from the body of an individual. For example, “body fluid” may include blood, plasma, serum, bile, saliva, urine, tears, sweat, etc. In embodiments, a sample includes a human-derived body fluid sample, for example, plasma or serum. For example, bodily fluids can be obtained from vertebrate mammals, such as humans who have, are suspected of having, and / or are at risk of developing an infection. Any suitable biological sample can be used. For example, a biological sample may be a specimen obtained from or derived from such a subject. A subject can provide a number of biological samples, including, for example, solid biological samples from biopsies or tissues. In some cases, a sample may be a tissue section or cells placed in or adapted to tissue culture. A biological sample may also be a biological fluid (e.g., urine, blood, plasma, serum, saliva, tears, or mucus), or such a sample absorbed onto paper or a polymer substrate. A biological sample may be further fractionated, for example, into fractions containing cell types. In some embodiments, a sample may be a combination of samples from a subject (e.g., a combination of tissue and fluid samples). In some cases, serum or plasma may be obtained from a subject using techniques known in the art.

[0211] The term “biological sample” can refer to tissues, cells, and bodily fluids isolated from a subject, as well as tissues, cells, and bodily fluids within a subject. Therefore, the use of the term “biological sample” includes blood, and fractions or components of blood, including serum, plasma, or lymph. This definition may also include samples that have been manipulated in any way after acquisition, such as by treatment with reagents, solubilization, or concentration of specific components, such as proteins or polynucleotides.

[0212] In embodiments, biological samples can be isolated from or obtained from a subject. As used herein, the terms “obtain a biological sample” or “isolate a biological sample” can refer to any process for directly or indirectly obtaining a biological sample from a subject. For example, a biological sample can be obtained by taking a tissue or fluid sample (e.g., blood, bone marrow, spinal puncture) from a subject (e.g., in a point-of-care facility, e.g., a clinic, hospital, or laboratory facility). Alternatively, a biological sample can be obtained by receiving a biological sample from one or more people who have obtained a sample directly from the subject (e.g., in a laboratory facility). Furthermore, biological samples can be obtained, for example, directly or indirectly from animals for the management efforts or veterinary care of livestock or wildlife.

[0213] In one embodiment, the sample contains protein molecules from the subject. In one embodiment, the biological sample is a peripheral blood leukocyte sample isolated from the subject by conventional means. In another embodiment, the biological sample is serum isolated from the subject by conventional means.

[0214] As used herein, the terms “purify,” “purify,” and “concentrate” do not refer to the removal of materials other than the analyte of interest from a sample. Instead, these terms refer to a procedure that concentrates the amount of one or more analytes of interest compared to other components in the sample that may interfere with the detection of the analyte of interest.

[0215] Purification of a sample by various means can allow for the relative reduction of one or more interfering substances, such as one or more substances that can interfere with or cannot interfere with the detection of a selected analyte by mass spectrometry. Relative reduction, as used in this term, does not require that any substance containing the analyte of interest in the material being purified be completely removed by the purification.

[0216] The terms “immunopurification” or “to immunopurify” can refer to a purification procedure that utilizes antibodies, including polyclonal or monoclonal antibodies, such as those described herein, to concentrate one or more analytes of interest. Immunopurification can be carried out using any of the immunopurification methods known in the art. Often, immunopurification procedures utilize antibodies that are bound to, conjugated to, or otherwise attached to a solid support (e.g., beads, columns, wells, tubes, gels, capsules, particles, etc.).

[0217] Immunopurification may include, but is not limited to, procedures often referred to in the art as immunoprecipitation, and procedures often referred to in the art as affinity chromatography or immunoaffinity chromatography.

[0218] The term "immune particle" can refer to capsules, beads, gel particles, etc., having antibodies bound, conjugated, or otherwise attached to their surface (e.g., on and / or within the particle). In certain embodiments, the immune particle is a cephalos or agarose bead. In alternative embodiments, the immune particle includes glass, plastic or silica beads, or silica gel.

[0219] As used herein, the term “anti-iAP antibody” may refer to any polyclonal or monoclonal antibody having affinity for iAP, its epitope, or its peptide fragment. In various embodiments, the specificity of the antibody to chemical species other than iAP may vary. For example, in certain embodiments, the anti-iAP antibody is specific to iAP and therefore has little or no affinity for chemical species other than iAP, while in other embodiments, the anti-iAP antibody is nonspecific and therefore binds to specific chemical species other than iAP.

[0220] The antibodies and fragments described herein can be used in conjunction with any solid-phase enrichment approach. The terms “solid-phase enrichment,” “solid-phase extraction,” or “SPE” can refer to a process in which a chemical mixture is separated into its components as a result of the affinity of components dissolved or suspended in a solution (i.e., mobile phase) to a solid (i.e., solid phase) through which the solution passes. In some examples, as the mobile phase passes through or around the solid phase, the components of the mobile phase can be retained by the solid phase, resulting in the purification of the analyte in the mobile phase. In other examples, the analyte, such as iAP, can be retained by the solid phase, allowing the components of the mobile phase to pass through or around the solid phase. In these examples, a second mobile phase is then used to elute the retained analyte from the solid phase for further processing or analysis. SPEs, including TFLCs, can operate by a single-mode mechanism or a mixed-mode mechanism. The mixed-mode mechanism utilizes ion exchange and hydrophobic retention within the same column. For example, the solid phase of a mixed-mode SPE column can exhibit strong anion exchange and hydrophobicity retention, or strong cation exchange and hydrophobicity retention.

[0221] To facilitate the separation of antibody-protein complexes from unbound proteins in a sample, the antibody can be linked to a separation facilitating agent, such as a binding partner (e.g., biotin, oligonucleotide, aptamer), a solid support (such as beads or a matrix including microarrays or multiwell plates), or any other agent known in the art. The linkage can be covalent or non-covalent and can be direct or indirect. Methods for linking antibodies to such agents are well known in the art. See, for example, Kennedy et al. (1976) Clin. Chim.Acta 70:1-31, and Schurs et al. (1977) Clin. Chim.Acta 81:1-40, which describe coupling techniques including the glutaraldehyde method, the periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method, each of which is incorporated herein by reference).

[0222] Methods for separating antibody-protein complexes from a sample are known in the art and include the use of binding partners (e.g., avidin to capture biotinylated antibodies, a capture agent that binds an oligonucleotide to capture an oligonucleotide linked to an antibody). Physical separations such as precipitation, filtration, FACS (e.g., using beads labeled with a spectral signature), and magnetic separation (when the antibody is linked to a matrix having magnetic properties such as magnetic beads) can also be used.

[0223] Many binding partners that can be used in the present invention are known in the art (e.g., dinitrophenyl group, digoxigenin, fluorophore, Oregon Green dye, Alexa Fluor 488 (Molecular Probes), fluorescein, dansyl group, Marina Blue (Molecular Probes), tetramethylrhodamine, Texas Red (Molecular Probes), BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, U.S. Patent No. 4,774,339) dyes, etc.). Antibodies that can be used as capture reagents and can specifically bind to binders are commercially available from suppliers such as Molecular Probes, Eugene, Oreg. These antibodies include antibodies that can specifically bind to dinitrophenyl group, digoxigenin, fluorophore, Oregon Green dye, Alexa Fluor 488 (Molecular Probes), fluorescein, dansyl group, Marina Blue (Molecular Probes), tetramethylrhodamine, Texas Red (Molecular Probes), and BODIPY dyes (Molecular Probes). Any suitable ligand and anti-ligand can also be used.

[0224] Oligonucleotides can be used as binding partners and capture reagents. Oligonucleotides include nucleic acids such as DNA, RNA, and hybrid RNA / DNA molecules. Oligonucleotides used as affinity labels can hybridize to the sequence of oligonucleotides on the capture reagent. Those skilled in the art will recognize that many different oligonucleotide sequences that hybridize to each other can be designed. Important considerations for designing such oligonucleotide pairs include the actual nucleotide sequence, the length of the oligonucleotide, hybridization conditions (e.g., temperature, salt concentration, presence of organic chemicals, etc.), and the melting temperature of the oligonucleotide.

[0225] Solid supports (and modifications to make solid supports suitable for antibody immobilization) for immobilizing (linking) antibodies or proteins derived from a sample are well known in the art. Examples of solid supports include beads (including magnetized beads), microwell plates, and protein microarrays (see, for example, U.S. Patent No. 6,365,418). For example, CdSe-CdS core-shell nanocrystals encapsulated in silica shells can be readily derivatized for coupling to biological molecules. Bruchez et al. (1998) Science 281:2013-2016. Similarly, highly fluorescent quantum dots (cadmium selenide capped with zinc sulfide) are covalently bonded to biomolecules for use in ultra-sensitive biological detection. Warren and Nie (1998) Science 281:2016-2018. Fluorescently labeled beads are commercially available from Luminex and Quantum Dot.

[0226] The bound protein (or, in some embodiments, a polypeptide fragment) can be released from the antibody-protein complex using conventional immunoaffinity elution conditions such as acidic pH, ionic strength, surfactant, or a combination thereof. In embodiments, the peptide or protein is desalted for subsequent fractionation, characterization, or other analysis.

[0227] In more than one embodiment, the antibody or antigen can be immobilized, for example, to facilitate the separation of the complexed form from one or both of the uncomplexed forms. Observation of the antibody-antigen complex can be achieved in any container suitable for containing the reactants. Examples of such containers include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided to which a domain is added that allows one or both of the proteins to bind to the matrix. For example, a GST-antibody fusion protein or a GST-antigen fusion protein can be adsorbed onto glutathione Sepharose beads (Sigma Chemical, St. Louis, MO) or a glutathione derivatized microtiter plate, and the mixture is incubated under conditions that lead to complex formation (e.g., physiological conditions of salt and pH). After incubation, the bead or microtiter plate wells are washed to remove unbound components, and in the case of beads, the matrix is ​​immobilized, and the complex is determined directly or indirectly. Alternatively, the complex can be dissociated from the matrix, and the level of antibody-antigen complex formation can be determined using standard techniques.

[0228] Other techniques for immobilizing proteins on a matrix can also be used in the assay of the present invention. For example, an antibody or antigen (e.g., 1593 peptide or 65D3-1 antibody) can be immobilized using a biotin and streptavidin conjugation. Biotinylated antibody or antigen molecules can be prepared from biotin NHS (N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kits, Pierce Chemicals, Rockford, Ill) and immobilized in the wells of a streptavidin-coated 96-well plate (Pierce Chemical). Alternatively, other antibodies that are reactive with the antibody or antigen of interest but do not interfere with the formation of the antibody-antigen complex of interest can be derivatized into the wells of the plate, and unbound antibodies or antigens can be captured in the wells by antibody conjugation. Methods for detecting such complexes include immunodetection of the complexes using such other antibodies that are reactive with the antibody or antigen, in addition to the methods described herein for GST-immobilized complexes.

[0229] In some embodiments, the method of the present invention further comprises the step of treating the sample with a protein cleavage agent, thereby generating polypeptide fragments. In one embodiment, the sample is contacted with the protein cleavage agent before contacting the sample with an anti-iAP antibody. In another embodiment, the protein is contacted with the protein cleavage agent after the separation of the protein from the antibody-protein complex.

[0230] Protein cleavage agent treatment generates protein cleavage fragments (such as polypeptides), which can facilitate subsequent mass spectral analysis of the amount and identity of protein in the sample. Treatment with protein cleavage agents can facilitate the analysis of proteins with molecular weights greater than 25 kDa. Protein cleavage reagent treatment can also facilitate the accessibility and / or approach of antibodies to congeneral epitopes. Protein cleavage agents are well known in the art and are further discussed herein. In some embodiments, one protein cleavage agent is used. In other embodiments, more than one protein cleavage reagent is used. In some embodiments, more than one type of protein cleavage agent is used for a single sample (e.g., two or more proteases, two or more chemical cleavage agents, or a combination of one or more proteases and one or more chemical cleavage agents). The conditions for treatment with protein cleavage agents are well known in the art.

[0231] In one embodiment, the protein cleavage agent is a protease. Examples of proteases that can be used as protein cleavage agents include, but are not limited to, chymotrypsin, trypsin (arg, lys cleavage sequence), thermolysin (phe, leu, iso, val cleavage sequence), V8 protease, endoproteinase Glu-C, endoproteinase Asp-N, endoproteinase Lys-C, endoproteinase Arg-C, endoproteinase Arg-N, factor Xa protease, thrombin, enterokinase, V5 protease, and tobacco etch virus protease. Proteases useful in the methods of the present invention can be genetically modified and / or chemically modified to prevent autolysis. It is understood that enzyme protein cleavage agents (such as proteases) can be modified to facilitate the removal of the protease from the polypeptide cleavage product after polypeptide cleavage. Such modifications are known in the art and include: (1) bead-bound (e.g., latex, silica, or magnetic beads) proteases, (2) haptenized proteases, (3) protease affinity depletion (e.g., using bead-bound antiproteases or bead-bound non-cleavable substrates), and / or (4) size exclusion chromatography. Protease activity can be inhibited by treatment with, for example, heat, protease inhibitors, or metal chelating agents (e.g., EGTA, EDTA).

[0232] In another embodiment, the protein cleavage agent is a chemical cleavage agent such as a chemical substance or compound that cleaves polypeptide and peptide bonds. Non-limiting examples of chemical cleavage agents include cyanogen bromide (cleaving at methionine residues), hydroxylamine (cleaving between Asn and Gly residues), and acidic pH (which can cleave Asp-Pro bonds) (see, for example, Ausubel et al. (cited above)).

[0233] In further embodiments, phosphatases (e.g., alkaline phosphatase, acid phosphatase, serine phosphatase, tyrosine phosphatase, threonine phosphatase, etc.), lipases, and other enzymes can be used as protein cleavage agents.

[0234] In another aspect, the present invention provides a method for characterizing a protein using mass spectrometry, comprising (a) reducing the complexity of a sample using any of the methods described herein, thereby enriching and / or purifying the protein, and (b) analyzing the isolated, purified, prepared and / or separated protein (interchangeably referred to as “products”) using any of the methods described herein, wherein the analysis is by mass spectrometry. Mass spectrometry is well known in the art. For example, in some embodiments, the mass spectrometry is matrix-assisted laser desorption / ionization ("MALDI") mass spectrometry; surface-enhanced laser desorption ionization ("SELDI"); and / or tandem mass spectrometry (e.g., MS / MS, MS / MS / MS, ESI-MS / MS). In some embodiments, tandem mass spectrometry is performed using a laser desorption / ionization mass spectrometer further coupled to a quadrupole time-of-flight mass spectrometer (QqTOF MS) (see, e.g., Krutchmsky et al., International Publication WO99 / 38185). Methods, e.g., MALDI-QqTOFMS (Krutchmsky et al., International Publication WO99 / 38185, Shevchenko et al. (2000) Anal. Chem. 72:2132-2141), ESI-QqTOFMS (Figeys et al. (1998) Rapid Comm'ns. Mass Spec. 12-1435-144), and tip capillary electrophoresis (tip-CE)-QqTOFMS (Li et al. (2000) Anal. Chem. 72:599-609) have been previously described. The mass spectrometers and the techniques for using them in the methods of the present invention are well known to those skilled in the art. Those skilled in the art will understand that any of the components of the mass spectrometer (e.g., desorption source, mass spectrometer, detector, etc.) can be combined with other suitable components described herein or those known in the art.For further information on mass spectrometers, see, for example, Principles of Instrumental Analysis, 3rd ed., Skoog, Saunders College Publishing, Philadelphia, 1985 and Kirk-Othmer Encyclopedia of Chemical Technology, 4th ed. Vol.15 (John Wiley & Sons, New York 1995), pp.1071–1094.

[0235] Using antibodies specific to iAP, iAP polypeptides can be isolated by standard techniques such as immunoaffinity assay, chromatography, or immunoprecipitation. Antibodies against iAP protein (or fragments thereof) can be used diagnostically as part of a clinical trial procedure to monitor protein levels in biological samples, e.g., tissues or fluids, to determine the effectiveness of a given treatment regimen, or to diagnose a given disease or infection.

[0236] In embodiments, detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances are described herein.

[0237] The antibodies of the present invention, including polyclonal, monoclonal, humanized, and fully human antibodies, can be used as diagnostic agents. Such agents can be used to diagnose iAP-related diseases or pathologies in subjects.

[0238] The therapeutically effective dose of the antibody of this invention may refer to the amount required to achieve the objective. As described herein, this may be due to the binding interaction between the antibody and its target antigen. The required effective dose further depends on the binding affinity of the antibody to its specific antigen.

[0239] The antibody according to the present invention can be used as a drug for detecting the presence of iAP (or its protein or protein fragment) in a sample. For example, the antibody includes a detectable label. The antibody may be polyclonal or monoclonal. In embodiments, the antibody is an intact antibody. With respect to the probe or antibody, the term “labeled” is intended to include direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include the detection of a primary antibody using a fluorescently labeled secondary antibody, and end-labeling a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin.

[0240] The detection methods of the present invention can be used in vitro and in vivo to detect analytes in biological samples. For example, in vitro techniques for detecting subject proteins include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology," Vol. 42, JRCrowther (Ed.), Human Press, Totowa, NJ, 1995; "Immunoassay," E. Diamandis and T. Christophorus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays," P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detecting subject proteins include targeting with labeled anti-subject protein antibodies. For example, antibodies can be labeled with radioactive markers whose presence and location in a target can be detected by standard imaging techniques.

[0241] Treatment methods for iAP-related gastrointestinal disorders As used herein, the terms “to treat” or “treatment” can refer to both therapeutic measures and preventive or protective measures, the purpose of which is to prevent or slow (mitigate) physiological changes or impairments, such as the progression of cancer. Clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, a stable (i.e., non-worsening) state of the disease, delay or slowing of disease progression, improvement or relief of the condition, and remission (partial or total), whether detectable or not. “Treatment” can refer to extending survival time compared to the survival rate without treatment. Those who require treatment include those who already have a condition or impairment, those who are susceptible to a condition or impairment, or those who need to prevent a condition or impairment.

[0242] The present invention provides both prophylactic and therapeutic methods for treating subjects at risk (or susceptible) of gastrointestinal diseases (for example, when identifying early detection biomarkers for gastrointestinal diseases in such subjects), or other cell proliferation-related diseases or disorders. Such diseases or disorders include, but are not limited to, diseases or disorders associated with the abnormal expression of iAP. For example, the method is used to treat, prevent, or alleviate the signs of gastrointestinal diseases. In one embodiment, the method is used to treat, prevent, or alleviate the symptoms of gastrointestinal diseases. Non-limiting examples of gastrointestinal diseases include colitis, inflammatory bowel disease (IBD), gastritis, gastroenteritis, pyloric stenosis, gastric cancer, infectious diarrhea, fecal impaction, constipation, intestinal obstruction and pseudo-intestinal obstruction, or malabsorption. In addition to necrotizing enterocolitis (NEC), non-specific examples of enteritis types include adult necrotizing enterocolitis (ANEC), pseudomembranous colitis, infectious colitis, very early-onset inflammatory bowel disease, ulcerative colitis, Crohn's disease, ischemic colitis, and radiation colitis.

[0243] Thus, in one aspect, the present invention provides a method for preventing, treating, or alleviating the symptoms of gastrointestinal diseases in a subject by administering to the subject a monoclonal antibody, scFv antibody, or bispecific antibody of the present invention. For example, an anti-iAP antibody can be administered in a therapeutically effective amount.

[0244] Subjects at risk of gastrointestinal diseases or disorders can include patients with a family history of gastrointestinal diseases, or subjects exposed to agents known or suspected to cause gastrointestinal diseases. Administration of a prophylactic agent can be carried out before the symptoms of a gastrointestinal disease so that the disease is prevented or alternatively, its progression is delayed.

[0245] subject As described herein, embodiments of the present invention include methods of diagnosing, prognosticating, and / or treating a subject having a gastrointestinal disease. The term "subject" or "patient" can refer to any organism to which an embodiment of the present invention can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Subjects to whom a compound can be administered include mammals such as primates, particularly humans. For veterinary use, a wide variety of subjects are suitable, such as livestock animals such as cows, sheep, goats, female cows, pigs, poultry such as chickens, ducks, geese, turkeys, and domesticated animals such as pets, for example, dogs and cats. For diagnostic or research use, a wide variety of mammals including rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs such as inbred pigs would be suitable subjects. The term "biological subject" refers to the above subjects or another living organism. The term "biological subject" refers not only to a part excised from a biological subject (e.g., a liver or other organ) but also to the entire subject or organism.

[0246] In embodiments of this specification, the subject matter includes humans and mammals such as vertebrates. Such examples include, but are not limited to, dogs, cats, horses, cattle, pigs, sheep, goats, chickens, primates such as monkeys, fish (aquaculture species) such as salmon, rats, and mice. Humans include premature infants, infants, children, adolescents, adults, or elderly individuals.

[0247] While the embodiments of the present invention described herein relate to human gastrointestinal disorders, embodiments of the present invention are also applicable to other non-human vertebrates. Embodiments of the present invention are applicable to veterinary use in livestock and other animals. Generally, embodiments vary according to the type of use and mode of administration, as well as the requirements of individual subjects.

[0248] In embodiments, the subject may be receiving an antibiotic regimen. The term “antibiotic regimen” refers to a method for treating or preventing a disease such as an infection, or for achieving a change such as a reduction or prevention of infection, and such treatment includes administering an antibiotic to the subject to effectively treat the disease or to produce a physiological change. An antibiotic regimen may include variations known to those skilled in the art, such as the selection of the antibiotic (e.g., correct drug selection, route of administration, and drug schedule), the timing of administration, and the duration. Non-limiting examples of such antibiotics include vancomycin, ampicillin, Zosyn (a combination of piperacillin and tazobactam), gentamicin, Flagyl (a metronidazole generic), meropenem, metronidazole, cefotaxime, clindamycin, or any combination thereof. In some embodiments, an antifungal agent may be further administered. In other embodiments, the antifungal agent may be fluconazole, terconazole, voriconazole, posaconazole, pentamidine, itraconazole, or ketoconazole.

[0249] Screening assay and method The present invention provides screening assays and methods for identifying therapeutic and / or prophylactic agents, i.e., candidate or test compounds or drugs (e.g., peptides, peptide mimes, small molecules, or other drugs), that can be used to prevent or treat iAP-related disorders. The present invention also encompasses candidate and / or compounds identified using the screening assays described herein.

[0250] The term “screening” may refer to determining whether a candidate and / or test compound has the ability or characteristics to prevent or delay (mitigate) the targeted disease conditions described herein. In some embodiments, the peptide antibodies described herein can be used in immunoassays to evaluate approaches that specifically inhibit the expression or secretion of iAPs. For example, the antibodies can be used in assays that utilize denatured proteins (e.g., Western blotting). In other embodiments, the peptide antibodies described herein can rapidly quantify the levels of iAP samples and may therefore be useful in preclinical studies to evaluate the efficacy of candidate drugs. While we do not wish to be constrained by theory, iAP levels can be analyzed sequentially during treatment in preclinical animal models. The ability to examine long-term responses to treatment in the same animals can be extremely valuable. This ability can be extremely useful in studies designed to evaluate dose response and treatment intervals.

[0251] Embodiments of the present invention may include measuring or detecting such biomarkers of iAP-related diseases using assays known in the art. Non-limiting examples of assays include immunoassays, colorimetric assays, fluorescence assays, or combinations thereof. Non-limiting examples of immunoassays include Western blot assays, enzyme-linked immunosorbent assays (ELISA), immunoprecipitation, or combinations thereof. For example, a biological sample collected from a subject may be incubated with a biomarker-specific antibody, such as an anti-iAP antibody or a fragment thereof, and the binding of the antibody to the biomarker in the sample may be detected or measured.

[0252] In embodiments, the assay may be a multiplex assay. A "multiplex assay" can refer to a procedure that measures multiple analytes (dozens or more) simultaneously in a single assay, and is distinct from procedures that measure one or a few analytes at a time. Multiplex assays can be used to detect or assay a given class of molecules in a biological sample, for example, to diagnose a disease or to determine the effectiveness of a treatment.

[0253] Candidate and / or test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries, spatially addressable parallel solid-phase or liquid-phase libraries, synthetic library methods requiring deconvolution, "one bead, one compound" library methods, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, but the other four approaches are applicable to small molecule libraries of peptides, non-peptide oligomers, or compounds. (See, for example, Lam, 1997. Anticancer Drug Design 12:145).

[0254] The methods described herein may include obtaining biological samples from a subject. Where used herein, the phrase “obtain a biological sample” may refer to any process for directly or indirectly obtaining a biological sample from a subject. For example, a biological sample may be obtained by taking a sample (e.g., a tissue sample, a fluid sample, or a waste sample) from a subject (e.g., in a point-of-care facility, e.g., a clinic, hospital, or laboratory). Alternatively, a biological sample may be obtained by receiving a biological sample from one or more people who have taken a sample directly from the subject (e.g., in a laboratory). A biological sample may be, for example, feces derived from a subject, e.g., stool, tissue (e.g., blood), cells (e.g., hematopoietic stem cells, leukocytes, or reticulocytes, or other hematopoietic cells, stem cells, or plasma cells), vesicles, biomolecular aggregates, or platelets.

[0255] As used herein, “small molecule” means a composition having a molecular weight of less than about 5 kDa, for example, less than about 4 kDa. Small molecules can be, for example, nucleic acids, peptides, polypeptides, peptide mimes, carbohydrates, lipids, or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened using any of the assays of the present invention.

[0256] Examples of methods for synthesizing molecular libraries can be found in the art, for example, in DeWitt, et al., 1993. Proc. Natl. Acad. Sci. USA 90:6909, Erb, et al., 1994. Proc. Natl. Acad. Sci. USA 91:11422, Zuckermann, et al., 1994. J. Med. Chem. 37:2678, Cho, et al., 1993. Science 261:1303, Carrell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33:2059, Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33:2061, and Gallop, et al., 1994. J. Med. Chem. 37 / 1233.

[0257] The compound library can be obtained in solution (see, e.g., Houghten, 1992. Biotechniques 13:412-421), on beads (see, Lam, 1991. Nature 354:82-84), on tips (see, Fodor, 1993. Nature 364:555-556), in bacteria (see, U.S. Patent No. 5,223,409), in spores (see, U.S. Patent No. 5,233,409), on plasmids (see, Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89:1865-1869), or on phages (see, Scott and Smith, 1990. Science 249:386-390, Devlin, 1990. Science 249:404-406, Cwirla, et al.). See al., 1990. Proc. Natl. Acad. Sci. USA 87:6378-6382, Felici, 1991. J. Mol. Biol. 222:301-310, and U.S. Patent No. 5,233,409.

[0258] Those skilled in the art will recognize that in any of the screening methods disclosed herein, the antibody can be an iAP-specific antibody. In addition, the antigen can be the iAP protein or a portion or fragment thereof.

[0259] The screening methods disclosed herein can be performed as cell-based assays or cell-free assays. The cell-free assays of the present invention are suitable for use in both soluble and membrane-bound forms of proteins and their fragments. In the case of cell-free assays involving membrane-bound proteins, solubilizers can be used to maintain the membrane-bound proteins in solution. Examples of such solubilizers include nonionic surfactants, such as n-octyl glucoside, n-dodecyl glucoside, n-dodecyl maltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, and isotridecypoly(ethylene glycol ether). n Examples include N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, 3-(3-collamidopropyl)dimethylamino-1-propanesulfonate (CHAPS), or 3-(3-collamidopropyl)dimethylamino-2-hydroxy-1-propanesulfonate (CHAPSO).

[0260] The present invention further relates to novel agents identified by any of the aforementioned screening assays, and their use for the treatments described herein.

[0261] Diagnostic assay Anti-iAP antibodies can be used, for example, as part of a clinical trial procedure to determine the effectiveness of a given therapeutic and / or prophylactic regimen, or diagnostically to monitor the onset or progression of gastrointestinal disease.

[0262] In some embodiments, for diagnostic purposes, the anti-iAP antibody of the present invention is linked to a detectable portion, for example, to provide a method for detecting gastrointestinal diseases in subjects at risk of or suffering from gastrointestinal diseases.

[0263] The detectable portion can be conjugated directly to the antibody or fragment, or indirectly, for example, by using a fluorescent secondary antibody. Direct conjugation can be achieved, for example, by standard chemical coupling of a fluorophore to an antibody or antibody fragment, or through genetic engineering. Chimeras, or fusion proteins containing antibodies or antibody fragments coupled to fluorescent or bioluminescent proteins, can be constructed. For example, Casadei et al. (Proc Natl Acad Sci USA. 1990 Mar; 87(6): 2047-51) describe a method for constructing a vector construct capable of expressing aequorin and antibody fusion protein genes in mammalian cells. For example, the detectable portion can be europium-labeled, SULFO-TAG-labeled, HRP, or Alexa Fluor.

[0264] As used herein, the term “labeled” with respect to a probe or antibody may include direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include the detection of a primary antibody using a fluorescently labeled secondary antibody, and the end labeling of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. The term “biological sample” is intended to include tissues, cells, and biological fluids isolated from a subject (e.g., biopsy), as well as tissues, cells, and fluids within a subject. That is, the detection methods of the present invention can be used in vitro and in vivo to detect cells expressing iAP in a biological sample. For example, in vitro techniques for the detection of iAP include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. Furthermore, in vivo techniques for the detection of iAP include introducing a labeled anti-iAP antibody into the subject. For example, antibodies can be labeled with radioactive markers whose presence and location in a target can be detected by standard imaging techniques.

[0265] In the case of a “target-oriented” conjugate, i.e., a conjugate containing a target-oriented moiety—a molecule or feature designed to localize the conjugate to one or more sites within a subject or animal—localization can refer to a state in which an equilibrium is essentially achieved between the bound “localized” entities and the unbound “free” entities within the subject. The rate at which such equilibrium is achieved depends on the route of administration. For example, a conjugate administered by intravenous injection may achieve localization within minutes of injection. On the other hand, a conjugate administered orally may take several hours to achieve localization. Alternatively, localization can refer to the location of the entities within the subject or animal over a selected period after administration. Another example is when localization is achieved when the moiety becomes distributed after administration.

[0266] A reasonable estimate of the time required to achieve localization can be made by those skilled in the art. Furthermore, the state of localization as a function of time can be tracked by imaging a detectable portion (e.g., a light-emitting conjugate) according to the method of the present invention, such as with a photodetector device. The “photodetector device” used can image faint light from within a mammal over a reasonable time and can have sufficient sensitivity to construct an image using the signal from such a device.

[0267] When extremely bright photogenerating portions can be used, and / or when detecting photogenerating fusion proteins localized near the surface of the object or animal being imaged, "night vision" goggles or standard high-sensitivity video cameras such as Silicon Intensified Tube (SIT) cameras (e.g., Hammamatsu Photonic Systems (Bridgewater, NJ)) can be used. In embodiments, more sensitive photodetection methods can be used.

[0268] At extremely low light levels, the photon flux per unit area becomes so low that the imaged scene no longer appears continuous. Instead, it is represented by individual photons distinct from one another, both in time and space. When viewed on a monitor, such an image appears as a series of shimmering points of light, each representing a single detected photon. By accumulating these detected photons over time in a digital image processor, an image can be acquired and constructed. In contrast to conventional cameras where an intensity value is assigned to the signal at each image point, the amplitude of the signal is irrelevant in photon counting imaging. The goal is to detect the presence of a signal (photon) and count the occurrence of the signal relative to its location over time.

[0269] Photodetector devices, such as those described herein, can detect individual photons and generate signals that can be analyzed by an image processor. Noise-reducing photodetectors achieve sensitivity not by amplifying the photon signal, but by reducing the background noise of the photon detector. Noise is reduced primarily by cooling the detector array. Devices include charge-coupled device (CCD) cameras, referred to as "back-thinning" cooled CCD cameras. In more sensitive instruments, cooling is achieved using liquid nitrogen, for example, to raise the temperature of the CCD array to about -120°C. "Back-thinning" refers to an ultra-thin backplate that reduces the path length that photons travel until detected, thereby increasing quantum efficiency. A high-sensitivity back-thinning cryogenic CCD camera is the "TECH 512," Series 200 camera, available from Photometries, Ltd. (Tucson, Arizona).

[0270] A "photon amplification device" amplifies photons before they strike the detection screen. This class includes CCD cameras equipped with enhancement tubes, such as microchannel enhancement tubes. A microchannel enhancement tube contains a metal array of channels perpendicular to and extending toward the camera's detection screen. The microchannel array is placed between the sample, object, or animal being imaged and the camera. Most photons entering the channels of the array contact the sides of the channels before exiting. A voltage applied across the array results in the emission of many electrons from each photon collision. Electrons from such collisions exit their channels of origin in a "shotgun" pattern and are detected by the camera.

[0271] By arranging enhanced microchannel arrays in series, even higher sensitivity can be achieved, resulting in electrons generated in the first stage leading to an amplified signal of electrons in the second stage. However, this increase in sensitivity is achieved at the expense of spatial resolution, which decreases with each additional stage of amplification. An exemplary microchannel enhancement tube-based single-photon detection device is the C2400 series, available from Hamamatsu.

[0272] An image processor processes signals generated by a photodetector device that counts photons to construct an image that can be displayed on a monitor or printed by a video printer. Such image processors are available for sale as part of a system including the high-sensitivity photon counting camera described herein, and are therefore available from the same source. The image processor can be connected to a personal computer such as an IBM-compatible PC or an Apple Macintosh (Apple Computer, Cupertino, Calif), which may or may not be included as part of a purchased imaging system. Once the images are in the form of digital files, they can be manipulated and printed by various image processing programs ("ADOBE PHOTOSHOP", Adobe Systems, Mt.View, Calif, etc.).

[0273] In one embodiment, the biological sample contains protein molecules from the subject of test. One exemplary biological sample is a fecal sample isolated from the subject by conventional means.

[0274] The present invention also encompasses a kit for detecting the presence of iAP or iAP-expressing cells in a biological sample. For example, the kit may include a labeled compound or drug (e.g., anti-iAP scFv or monoclonal antibody) capable of detecting gastrointestinal diseases in a biological sample, means for determining the amount of iAP in the sample, and means for comparing the amount of iAP in the sample with a standard. The compound or drug may be packaged in a suitable container. The kit may further include instructions for using the kit to detect biomarkers of gastrointestinal diseases in a sample.

[0275] In embodiments, the diagnostic assay includes a disposable article for detecting or measuring biomarkers of gastrointestinal diseases. The disposable article may include a biosensor and may optionally include other components known in the art. In embodiments, the biosensor may comprise at least one biorecognition element.

[0276] In some embodiments, the biosensor can detect or measure iAP in a sample. In other embodiments, the biosensor can detect or measure iAP enzyme activity, total fecal protein, iAP dimerization / dissociation, post-translational modified iAP, or a combination thereof. Non-limiting examples of post-translational modifications and samples are described herein.

[0277] In embodiments, the biosensor may be an immunosensor and may further include a detection signal. Non-limiting examples of detection signals include radioactive signals, colorimetric signals, fluorescent signals, chemiluminescent signals, or combinations thereof. For example, the biosensor may produce a new color or a change in spectral absorption. In embodiments, the biosensor of the present invention includes a biorecognition element or molecular recognition element that provides highly specific binding or detection selectivity to an analyte such as iAP. The biorecognition element or system may be a biologically derived material such as an enzyme or sequence of enzymes, an antibody or fragment thereof, a membrane receptor protein, DNA, an organelle, a native or synthetic cell membrane, intact or partially viable or non-viable bacteria, a plant or animal cell, or a plant or mammalian tissue fragment, and may function to interact specifically with the target biological analyte. The biorecognition element is responsible for the selective recognition of the analyte and a physicochemical signal that provides the basis for the output signal. The physicochemical signal generated by the biorecognition element may be communicated visually (i.e., by a change in color visible to the human eye) to the wearer or caregiver. Other embodiments can generate optical signals, which may require other equipment to enhance the signals. These include fluorescence, bioluminescence, total internal reflection resonance, surface plasmon resonance, Raman spectroscopy, and other laser-based methods.

[0278] Alternatively, the signal can be processed via an associated transducer capable of generating an electrical signal (e.g., current, potential, inductance, or impedance) that can be displayed (e.g., on a reading device such as an LED or LCD display), or that can trigger an audible or tactile (e.g., vibration) signal, or that can trigger an actuator as described herein. The signal can be qualitative (e.g., indicating the presence of a target biological analyte) or quantitative (i.e., a measurement of the amount or concentration of a target biological analyte). In such embodiments, the transducer can optionally generate an optical, thermal, or acoustic signal.

[0279] In either case, the signal can be durable (i.e., stable and readable over a certain period of time, and can be at least as large as the lifespan of the article) or transient (i.e., recording real-time measurements). In addition, the signal can be transmitted to a remote indicator site, including inside or on the article or remote device (e.g., via a wire or a transmitter such as an infrared or RF transmitter). Furthermore, the biosensor or any of its components can be adapted to detect and / or signal only the concentration of a target biological analyte above a predetermined threshold level (e.g., when the target biological analyte is normally found in excrement, or when the concentration of the analyte is below a known "hazard" level).

[0280] In one embodiment, the disposable item may be a diaper worn by the subject. Non-limiting examples of additional disposable items include wipes, dipsticks, spoons, spatulas, filter paper, or swabs for cleaning the subject.

[0281] In aspects of the present invention, the disposable articles described herein can be components of a kit useful for diagnosing subjects with gastrointestinal diseases. Additional components of the kit of the present invention may include biorecognition elements, a support structure, and instructions for use thereof. For example, iAP biorecognition elements such as antibodies described herein can be immobilized on a solid support structure.

[0282] Non-limiting examples of the composition of the solid support structure include plastic, cardboard, glass, plexiglass, tin, paper, or combinations thereof. The solid support may also include dipsticks, spoons, spatulas, filter paper, or swabs.

[0283] kit Aspects of the present invention further relate to a diagnostic kit for molecular biomarkers for identifying subjects that exhibit or have a predisposition to developing gastrointestinal diseases. In embodiments, the kit comprises means for determining total fecal protein concentration, means for determining intestinal alkaline phosphatase (iAP) activity, and at least one iAP biorecognition element, together representing a molecular signature indicating the presence of gastrointestinal disease or predisposition to developing gastrointestinal disease in a human subject. In embodiments, the signature includes a total protein concentration at least two standard deviations above the mean of a control sample, an intestinal alkaline phosphatase protein concentration at least two standard deviations above the mean of a control sample, or intestinal alkaline phosphatase activity at least two standard deviations below the mean of a control sample. In yet another embodiment, the signature is selected from at least two of the group including a total protein concentration at least two standard deviations above the mean of a control sample, an intestinal alkaline phosphatase protein concentration at least two standard deviations above the mean of a control sample, and intestinal alkaline phosphatase activity at least two standard deviations below the mean of a control sample. In embodiments, the control sample may include two or more control samples.

[0284] In one embodiment, the kit includes (a) a container comprising the components and support structure described herein, and optionally (b) informational material. The informational material may be explanatory, instructional, marketing, or other material, including the use of the methods and / or agents for diagnostic purposes described herein. In one embodiment, the kit also includes therapeutic agents such as antibiotics, probiotics, or iAP alternative compositions.

[0285] The informational materials of the kit are not limited in form. In one embodiment, the informational materials may include information about the manufacturing of the kit components, such as molecular weight, concentration, expiration date, batch, or place of manufacture. In one embodiment, the informational materials may include how to use the kit components (for example, to diagnose a subject with a GI disorder). The information may be provided in various forms, including printed text, computer-readable materials, video recordings, or audio recordings, or information providing links or addresses to substantial materials.

[0286] The kit may include other components such as solvents or buffers, stabilizers, or preservatives. Optionally, the kit may include therapeutic agents such as iAP substitute compositions or antibiotics, which can be provided in any form, e.g., liquid, dry, or lyophilized, preferably substantially pure and / or sterile. If the agent is provided in solution, the liquid solution may be an aqueous solution. If the agent is provided in dry form, reconstitution may be by adding a suitable solvent. A solvent, e.g., sterile water or a buffer, may optionally be provided in the kit. In one embodiment, the kit includes other components for detecting the presence of iAP or iAP-expressing cells in a biological sample. For example, the kit may include a labeled compound or agent (e.g., anti-iAP scFv or monoclonal antibody) capable of detecting gastrointestinal disease in a biological sample, means for determining the amount of iAP in the sample, and means for comparing the amount of iAP in the sample to a standard. The compound or agent may be packaged in a suitable container. The kit may further include instructions for using the kit to detect biomarkers of gastrointestinal disease in a sample.

[0287] In embodiments, the kit may include means for obtaining a sample from a subject and / or a container for storing the collected sample for a period of time, a drug capable of detecting iAP in the sample (e.g., an anti-iAP monoclonal antibody), means for determining the amount of iAP in the sample, and means for comparing the amount of iAP in the sample with a standard. The compound or drug may be packaged in a suitable container. The kit may further include instructions for detecting iAP in a sample using the kit.

[0288] In one embodiment, the kit can be used with a primary instrument for detecting iAP (e.g., a mass spectrometer). The assay kit may include reagents for use in sample preparation. These reagents may include, but are not limited to, commercially available materials such as nanoparticles, digestion compounds, and washing buffers for rinsing. Other components in the kit may include pipette tips, aliquot tubes (sometimes referred to as "vials" or "Eppendorf tubes"), aliquot stands for tube management, sample identification labels, and device labels such as Instructions for Use (IFU). The kit may further consist of reusable primary components, including magnets for nanoparticle separation and / or additional disposable components.

[0289] Other Embodiments The present invention is described in detail, but the foregoing description is intended to illustrate, not limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0290] The present invention will be further described by the following embodiments, but these will not limit the scope of the invention as described in the claims. [Examples]

[0291] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary forms of constructing and carrying out the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, and similar results can be obtained by using alternative methods; therefore, these examples are for illustrative purposes only.

[0292] Example 1 Example 1: Selected diagnostic INC monoclonal antibody IP Recombinant monoclonal antibodies with the following characteristics were generated using peptides synthesized from human sequences of enteric alkaline phosphatase.

[0293] Human intestinal alkaline phosphatase epitope: PEYPADASQNGIRLDGK (Sequence No. 11)

[0294] >CDI005E8_H (heavy chain) (Sequence ID 9)

[0295] >CDI005E8_L (light chain) (SEQ ID NO: 10) ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGATGTTGTGATGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTGGCACTGCATTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTACAAGGCATCCACTCTGACATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAATGTACTGTTGATGATAGTAGTAGTGGTAGTAGTTATGGTAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAGAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG

[0296] CDI0O5E8 heavy chain (SEQ ID NO: 1) METGLRWLLLVAVLKGVQCQSLEESGGGLVQPGASLTLTCTASGFSFTNNYWIYWVRQAPGKGLEWIGSVIISSSSAYYASWAKGRFTITKPSSTTVTLQVTSLTAADTATYF CVTGGILWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVA PSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIE KTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0297] CDI005E8 Light Chain (Sequence ID 2) MDTRAPTQLLGLLLLWLPGARCDVVMTQTPASVEAAVGGTVTIKCQASQSIGTALAWYQQKPGQPPKRLIYKASTLTSGVPSRFKGSGSGTDFTLTISDLECADAATYYCQCTVDDSSSG SSYGNAFGGGTEVVVRGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0298] CDI005E8 Heavy-chain CDR1 (SEQ ID NO: 3) CTASGFSFTNNYWIYWV

[0299] CDI005E8 Heavy-chain CDR2 (SEQ ID NO: 4) LEWIGSVIISSSSAYYASWAKGRFT

[0300] CDI005E8 Heavy-chain CDR3 (SEQ ID NO: 5) CVTGGILWGPGT

[0301] CDI005E8 Light Chain CDR1 (Sequence ID 6) CQASQSIGTALAWYQ

[0302] CDI005E8 Light Chain CDR1 (Sequence ID 7) IYKASTLTS

[0303] CDI005E8 Light Chain CDR1 (Sequence ID 8) CTVDDSSSGSSYGNAFGGG

[0304] Example 2 Antibody development file Peptide antigen: 2-hsALPI=PEYPADASQNGIRLDGK (Sequence ID 11, Human Enteral Alkaline Phosphatase Protein aa234~aa250), Calculated MW=1830.9725g / mol

[0305] Chain identification CDI005E8 heavy chain antibody sequence (SEQ ID NO: 1): METGLRWLLLVAVLKGVQCQSLEESGGGLVQPGASLTLTCTASGFSFTNNYWIYWVRQAPGKGLEWIGSVIISSSSAYYASWAKGRFTITKPSSTTVTLQVTSLTAADTATYF CVTGGILWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVA PSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIE KTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0306] CDI005E8 light chain antibody sequence (SEQ ID NO: 2): MDTRAPTQLLGLLLLWLPGARCDVVMTQTPASVEAAVGGTVTIKCQASQSIGTALAWYQQKPGQPPKRLIYKASTLTSGVPSRFKGSGSGTDFTLTISDLECADAATYYCQCTVDDSSSG SSYGNAFGGGTEVVVRGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0307] Determination of affinity and specificity: K d When using 2 μg / mL (1.1 μM) of the peptide antigen 2-hsALPI, the CDI005E8 Ab was 7.11 ± 1.01 pM, which was surprising considering that the hydrophobic iAP epitope and hydrophobic antibody paratope are an unusual combination for inducing picomolar binding affinity.

[0308] CDI005E8 is specific to the intestinal isoform and does not bind to purified placenta, tissue-nonspecific or germ cell APs.

[0309] Identification of Complementarity Determination Regions (CDRs) Table 4. Key regions and residues in the CDI005E8 antigen-binding fragment, although we do not wish to be constrained by theory. To identify the CDR region, we analyzed the sequences of the CDI005E8 heavy chain (VH) and light chain (VL) of the variable region (Fv) using two different methods: (i) Genscript website guidance or (ii) ANARCI calculation. For ANARCI calculation on the SAbPred server, PDB 5v6m was used for the VH and VL framework. READ was used with a CDR-specific database to predict the CDR conformation. Where knowledge-based prediction was not possible, MODELLER was used to predict the CDR loop. The following templates, methods, and scores were obtained: CDR H1: PDB ID 6cjk(D), fread score=71, CDR H2: PDB ID 2xlo(B), fread score=30, CDR H3: PDB ID 4okv(C), sequence similarity score=9, CDR L1: PDB ID 5vkd(L), fread score=44, CDR L2: PDB ID 5v6m(L), fread score=43, and CDR L3: PDB ID 6cjk(C), modeler ab initio score=25. Amino acids common to both manual determination and ANARCI server calculations are underlined. Paratopes, or antigen-binding regions, were calculated using the i-Patch algorithm. The top 10 paratope residues in the VH and VL chains are shown along with their scores. The scores describe the frequency with which the residue type in its local environment (patch) is involved in antigen binding in the known structure. The paratope residues with the highest four scores are shown in bold in the CDR prediction. The paratope residues with the highest scores are shown in bold and highlighted in gray. Abbreviations: Ka=Kabat; Ch=Chothia; IM=IMGT; No=North.

[0310] TIFF2026516499000011.tif129149 * H3-CDR can be the most important component of an antibody molecule in conferring binding activity and specificity. This specific target binding is the result of unique V and D rearrangements and VL pairing. HCDR3 is necessary, though insufficient, for specific antibody binding.

[0311] EpiPred uses geometric matching of antibody-antigen interfaces in combination with potentials based on antigen-antibody specific knowledge. Computational B-cell epitope prediction provides information about the region of the antigen to which the antibody binds, but does not directly contribute to knowledge of antibody residues that need to be mutated to modify its function. This problem can be approached by antibody-antigen docking, which can provide a list of putative orientations of the two molecules relative to each other. Antibody-antigen docking requires a different methodology than that used for the corresponding problem in non-antibody targets. This is because, in part, antibodies use different residues at their binding sites compared to both common proteins and antigens, and therefore an asymmetric scoring system is needed to account for these mismatches. The use of EpiPred has been shown to enrich top decoys with more native poses.

[0312] Human enteric alkaline phosphatase protein sequence: UniProt P09923, NCBI reference sequence: NP_001622.2 Enteric alkaline phosphatase precursor [Homo sapiens] and NCBI reference sequence: NM_001631.5

[0313] Structure of human intestinal alkaline phosphatase No experimental structures are available in the Human Enteral Alkaline Phosphatase-Protein Databank (Protein Databank, PDB). Assumption: A single static X-ray structure is sufficient to identify the determinants of antibody-antigen recognition.

[0314] CDRs are parts of the variable chains in immunoglobulins (antibodies) and T cell receptors, respectively, produced by B cells and T cells, and these molecules bind to their specific antigens. Precise identification of CDRs is crucial for understanding and manipulating antigen interactions. One way to do this is by marking residues on antibodies that interact with B cell epitopes on antigens. This requires the identification of B cell epitopes and CDRs. The diversity of sequences and structures is concentrated in six hypervariable loops, or CDRs. A set of CDRs constitutes a paratope. Existing tools for identifying CDRs are based on sequence analysis or general biophysical principles. Here, we identified CDR regions using two different methods: (i) Genscript website guidance or (ii) ANARCI server.

[0315] In the case of Antigen Receptor Numbering and Receptor Classification (ANARCI) software, this tool provides amino acid numbering for antigen and T cell receptor variable domains. ANACI aligns a given sequence against a database of Hidden Markov models describing germline sequences of antigen and TCR domain types. The most important alignment predicts the domain type and species of the input sequence. Domain identification and their annotation can be performed using IMGT, Chothia, Kabat, Martin (Enhanced Chothia), or AHo numbering schemes. A complete description of the pipeline is publicly available [Dunbar J and Deane CM. ANARCI: Antigen receptor numbering and receptor classification. Bioniformatics (2016)]. Alternatively, a web interface program can be found at opig.stats.ox.ac.uk / webapps / anarci.

[0316] Determination of peptide antigens overview Epitope discovery was performed using Discotope 2.0 and Discotope 1.0 for human intestinal alkaline phosphatase residues. Discotope 1.0 showed 75% specificity and 47.2% sensitivity using a score threshold of -7.7. Discotope 2.0 showed improved performance in both cross-validation and independent evaluation. For the 15aa peptide, it showed 82% sensitivity using a score threshold of -3.7.

[0317] Round 2 Candidate human iAP peptide antigens (P09923(PPBI_HUMAN)) were identified using DiscoTope 2.0. ● aa234~250 between α11 and α12 17-mer PEYPADASQNGIRLDGK (Sequence No. 11)

[0318] Round 1 Candidate human iAP peptide antigens (P09923(PPBI_HUMAN)) were identified using DiscoTope 1.0 [Figure 5]. ● aa234~250 between α11 and α12 (sequence number 18) 17-mer PEYPADASQNGIRLDGK (sequence number 11)

[0319] DiscoTope is a method for predicting discontinuous B-cell epitopes from the 3D structure of proteins in PDB format.

[0320] https: / / services.healthtech.dtu.dk / service.php?DiscoTope-2.0

[0321] Reliable B Cell Epitope Predictions: Impacts of Method Development and Improved Benchmarking Jens Vindahl Kringelum,Claus Lundegaard,Ole Lund,and Morten Nielsen.Plos Computational Biology,2012

[0322] The method utilizes surface accessibility (estimated in relation to the number of contacts) and the calculation of a novel epitope tendency amino acid score. The final score is calculated by combining the propensity scores and contact numbers of spatially adjacent residues.

[0323] Table 5: Discotope 2.0 score for human intestinal alkaline phosphatase: TIFF2026516499000012.tif34128TIFF2026516499000013.tif22180TIFF2026516499000014.tif22180TIFF202 6516499000015.tif21888TIFF2026516499000016.tif22180TIFF2026516499000017.tif22181TIFF20265164990 00018.tif22188TIFF2026516499000019.tif22181TIFF2026516499000020.tif22181TIFF2026516499000021.t if22188TIFF2026516499000022.tif22182TIFF2026516499000023.tif22181TIFF2026516499000024.tif172128

[0324] Example 3 Recombinant monoclonal antibodies with the following characteristics were generated using peptides synthesized from human sequences of enteric alkaline phosphatase.

[0325] Peptide antigen: Human intestinal alkaline phosphatase epitope (6-hsALPI; also known as peptide 6): PGYVFNSGVRPDVNESESGSPDY (Sequence ID 30, Human Enteral Alkaline Phosphatase Protein aa416~aa438) The calculated molecular weight of 6-hsALPI is 2472.53 g / mol.

[0326] CDI022E3 heavy chain amino acid sequence: (SEQ ID NO: 20) METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTVSGIDLSTYAMGWVRQAPGEGLEWIGTIGVSGSTYYASWAKGRFTISKTSTTVDLKMTSPATEDTATYFCA RGSVWGPGTLVTVSLGQPKAPSVFPLAPCCGDTPSSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPS TCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEK TISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK *

[0327] CDI022E3 light chain amino acid sequence: (SEQ ID NO: 21) MDTRAPTQLLGLLLLWLPGATFAQVLTQTPASVEAAVGGTVTIKCQASQSISTWLAWYQQKPGQAPKRLIYSASTLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCRCSYGKS YGEAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC *

[0328] Heavy chain CDR1-CDI022E3 (Sequence ID 22) TVSGIDLSTYAMG

[0329] Heavy-chain CDR2-CDI022E3 (SEQ ID NO: 23 or 31) TIGVSGSTY (Sequence ID 23), or TIGVSGSTYYASWAKG (Sequence ID 31)

[0330] Heavy-chain CDR3-CDI022E3 (Sequence ID 24) ARGSV

[0331] Light chain CDR1-CDI022E3 (Sequence ID 25) QASQSISTWLA

[0332] Light chain CDR2-CDI022E3 (Sequence ID 26) YSASTLAS

[0333] Light chain CDR3-CDI022E3 (Sequence ID 27) RCSYGKSYGEA

[0334] CDI022E3_H (heavy-chain DNA sequence): (SEQ ID NO: 28)

[0335] CDI022E3_L (light chain DNA sequence): (Sequence ID 29) ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGCACCTGGTTAGCCTGGTATCAG CAGAAACCAGGGCAGGCTCCCAAGCGCCTGATCTATTCTGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCGATGTAGTTATGGTAAAAGTTAT GGTGAGGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACC CAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG

[0336] Determination of affinity and specificity for CDI022E3 [See Figure 10]: When using 2ug / mL (0.8μM) peptide antigen 6-hsALPI, K D,CDI022E3 Ab =200pM. When using 2 μg / mL (1.1 μM) peptide antigen 2-hsALPI, K D,CDI022E3 Ab= 21 μM.

[0337] Example 4 Glycopeptide analysis Antibodies are known to undergo glycosylation, and this post-translational modification is crucial to their antibody effector properties. Generally, antibody glycosylation defines the structure of the antibody Fc region and determines which Fc receptors it can bind to in order to recruit effector cells. Antibody glycosylation can be utilized to improve the efficacy of monoclonal therapeutics. Antibody glycosylation can be modulated by vaccination, and we have shown that rational immunogen design can be explored to induce a specific antibody glycosylation response.

[0338] Glycosylation is a common post-translational modification for IgG antibodies produced by mammalian cells, specifically Chinese hamster ovary (CHO) cells, which are frequently used for production. The IgG1 molecule contains a single N-linked glycan at Asn297 within the CH2 domain of each of the two heavy chain Fc regions. During the synthesis of the N-glycan, multiple sugar moieties can be added to form different glycoforms, e.g., G0, G1, G2, and afucosylated complexes. These glycoforms may be necessary to achieve therapeutic efficacy. The observed high heterogeneity of glycoforms can affect the antibody stability, pharmacokinetics (PK), efficacy, and immunogenicity of mAbs. In addition, glycoform diversity plays a crucial role in complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) functions by regulating binding to the Fcγ receptor. Targeting specific glycoforms can be carried out via molecular glycosylation, but this can also be influenced by cell culture conditions.

[0339] Terminal sugar residues, such as mannose, sialic acid, fucose, or galactose, can positively or negatively affect therapeutic mAbs. Mannosylation, which has a significantly undesirable effect on glycoprotein PK, can lead to a reduction in the half-life of mAbs. Furthermore, terminal galactose residues can enhance CDC activity and Fc-C1q interactions, while core fucose can decrease ADCC and Fc-FcγR binding.

[0340] Glycopeptide analysis of monoclonal antibody samples Materials and methods: Protein digestion was performed. Briefly, the provided proteins were treated with dithiothreitol (DTT) and iodoacetamide (IAA) and reduced and alkylated before overnight trypsin digestion. LC-MS / MS analysis was performed using a Thermo Eclipse mass spectrometer coupled to an Ultimate 3000 RSLCnano system. Data were analyzed using Byonic ver5 software and manual interpretation, including searching for diagnostic ions of NeuAc2 (Medzihradszky et al. (2015) Anal. Chem). Delta mod scores greater than 10 were used for typical glycopeptides, and delta mod scores greater than 1 were used for acetylation searches.

[0341] result: Nano LC-MS / MS analysis confirmed the presence of monoclonal antibody proteins in the provided samples. The overall quality of the recovered peptides was high, and the peptide analysis was reproducible with independent antibody samples. Bionic searching revealed one glycosylation site in the heavy chain and one glycosylation site on the light chain.

[0342] Table 6. Coverage and glycosylated sites present in the provided samples. TIFF2026516499000025.tif28146

[0343] The reagents used for the analysis were purchased from Sigma Aldrich. Data acquisition was performed on a Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer tandem NanoFlow-LC system. Data analysis was performed using FreeStyle 1.8, Xcalibur 3.0, and Byonic v4.0.12 software. Peptide analysis and site mapping were performed using LC-MS / MS. Two provided samples were reduced with DTT, alkylated with iodoacetamide, and the proteins were digested with 0.5 μg / μl sequencing-grade trypsin and chymotrypsin (post-dilution) at 37°C for 16 hours. Peptides were analyzed on an Ultimate 3000 RSLCnano connected to a Thermo Eclipse mass spectrometer. A 15 cm long nanoLC column with a 75 μm inner diameter, packed with 3 μm C18 reversed-phase material, was used for chromatographic separation of the samples. The separation conditions were low to high acetonitrile in a solution containing 0.1% formic acid, with a separation time of 60 minutes. Precursor ion scanning was acquired at a resolution of 120,000 using an Orbitrap analyzer, and precursors within a 3-second timeframe were selected for subsequent fragmentation using HCD. Charge state screening was enabled to exclude precursors with unknown charge states or a +1 charge state. Dynamic exclusion was enabled (exclusion period of 30 seconds). Fragment ions were analyzed on Orbitrap for HCD at a resolution of 30,000. The obtained glycoproteome data were processed with Byonic (v4.0.12) and searched against the provided sequences as well as a catalog of over 70 mammalian N-glycans and 9 common O-glycans. Precursor mass tolerances and fragment mass tolerances were set to 5 ppm and 10 ppm, respectively. Further modifications, including deamidation of N and Q, carboxymethylation of C, and oxidation of precursors, were also included in the search. Assignments were made using Byonic software and manual interpretation.

[0344] Referring to Figure 7, the glycopeptide coverage map of rmAb CDI005E8 is shown. The amino acids (AA) in bold and underlined are the fragments selected for RA calculation. The red AAs are N-glycosylation sites.

[0345] TIFF2026516499000026.tif104146

[0346] JPEG2026516499000027.jpg46170

[0347] JPEG2026516499000028.jpg57170

[0348] Referring to Figure 8, a representative MS of glycopeptides found in rmAb CDI005E8 2 The spectrum is shown. The peaks represent the HexNAc(3)Hex(3) structure on the peptide EQQFNSTIR (position 304). The glycoform structure is based on total mass and known biosynthetic pathways. The structure does not reflect sequencing or compositional analysis.

[0349] Glycomics analysis of monoclonal antibody samples Materials and methods: Glycoprotein processing and N-glycan release: Each 100 μg sample was transferred to a clean Eppendorf tube, an equal volume of 25 mM DTT was added, and the sample was incubated at 50°C for 45 minutes to denaturate it. The sample was then gradually desalted by centrifugation at 14,000 × g for 15 minutes using a 10 kDa cutoff spin filter. (Note: The 10 kDa cutoff spin filter was washed twice with 400 μL of 50 mM ammonium bicarbonate before use). The sample was then washed with 400 μL of 50 mM ammonium bicarbonate and centrifuged again at 14,000 × g for 15 minutes. This step was repeated once more. The sample remaining in the filter was transferred to a clean Eppendorf tube, PNGase F was added, and the N-glycan was released from the protein by incubation at 37°C for 18 hours. The released N-glycan was separated from the O-glycoprotein using a 10kDa cutoff spin filter, purified using a C18 cartridge, freeze-dried, and then permethylated.

[0350] Referring to Figure 9, the MALDI spectra of the N-glycans identified in rmAb CDI005E8 are shown. For the major N-glycans, the m / z values ​​are shown along with a diagram of the structure. The glycoforms are provided in Table 9.

[0351] JPEG2026516499000029.jpg97159

[0352] Equal parts Those skilled in the art will recognize or be able to identify numerous equivalents to the specific substances and procedures described herein without using anything beyond routine experiments. Such equivalents are considered to fall within the scope of the present invention and are covered by the following claims.

Claims

1. An isolated antibody or fragment thereof that binds to human enteric alkaline phosphatase (hIAP), a. VH CDR1 containing the amino acid sequence of SEQ ID NO: 3, VH CDR2 containing the amino acid sequence of SEQ ID NO: 4, VH CDR3 containing the amino acid sequence of SEQ ID NO: 5, or sequences that are at least 80% identical thereto, and / or VL CDR1 containing the amino acid sequence of SEQ ID NO: 6, VL CDR2 containing the amino acid sequence of SEQ ID NO: 7, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 8, or sequences that are at least 80% identical to them, b. VH CDR1 containing the amino acid sequence of SEQ ID NO: 22, VH CDR2 containing the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 31, VH CDR3 containing the amino acid sequence of SEQ ID NO: 24, or sequences that are at least 80% identical thereto, and / or VL CDR1 containing the amino acid sequence of SEQ ID NO: 25, VL CDR2 containing the amino acid sequence of SEQ ID NO: 26, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 27, or sequences that are at least 80% identical to them. An isolated antibody or fragment thereof containing [the specified substance].

2. An isolated antibody or fragment thereof that binds to human enteric alkaline phosphatase (hIAP), a. A heavy chain variable region containing the amino acid sequence described in SEQ ID NO: 1 or a sequence that is at least 80% identical thereto, and / or a light chain variable region containing the amino acid sequence described in SEQ ID NO: 2 or a sequence that is at least 80% identical thereto, b. A heavy chain variable region containing the amino acid sequence described in SEQ ID NO: 20 or a sequence that is at least 80% identical thereto, and / or a light chain variable region containing the amino acid sequence described in SEQ ID NO: 21 or a sequence that is at least 80% identical thereto. An isolated antibody or fragment thereof containing [the specified substance].

3. The isolated monoclonal antibody or antigen-binding fragment according to claim 1 or 2, wherein the isolated monoclonal antibody or antigen-binding fragment comprises wild-type Fc or modified Fc.

4. The isolated monoclonal antibody or antigen-binding fragment according to claim 1 or 2, wherein the fragment comprises a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody.

5. The isolated monoclonal antibody or antigen-binding fragment according to claim 1 or 2, further comprising a detectable portion.

6. A pharmaceutical composition comprising an isolated monoclonal antibody or a fragment thereof according to claim 1 or 2, and a pharmaceutically acceptable carrier or excipient.

7. A nucleic acid encoding the antibody according to claim 1 or 2.

8. The nucleic acid according to claim 7, comprising the nucleic acid sequence described in SEQ ID NO: 9 and / or SEQ ID NO: 10, the nucleic acid sequence described in SEQ ID NO: 28 and / or SEQ ID NO: 29, or a sequence that is at least 80% identical thereto.

9. A vector comprising the nucleic acid described in claim 8.

10. Isolated cells containing the vector according to claim 9.

11. An isolated monoclonal antibody or fragment thereof that binds to human enteric alkaline phosphatase (hIAP), a. A heavy chain variable region encoded by the nucleic acid sequence described in Sequence ID No. 9 or a sequence that is at least 80% identical thereto, and / or a light chain variable region encoded by the nucleic acid sequence described in Sequence ID No. 10 or a sequence that is at least 80% identical thereto, b. A heavy chain variable region encoded by the nucleic acid sequence described in Sequence ID No. 28 or a sequence that is at least 80% identical thereto, and / or a light chain variable region encoded by the nucleic acid sequence described in Sequence ID No. 29 or a sequence that is at least 80% identical thereto. An isolated monoclonal antibody or fragment thereof containing [the specified substance].

12. An isolated cell comprising one or more polynucleotides encoding an isolated monoclonal antibody or a fragment thereof according to claim 1 or 2.

13. A method for detecting or diagnosing iAP-related gastrointestinal disorders in subjects requiring detection or diagnosis of iAP-related gastrointestinal disorders, comprising administering a therapeutically effective amount of the hIAP antibody or a fragment thereof according to claim 1, 2, or 4 to the subject.

14. A method for treating or preventing iAP-related gastrointestinal disease in a subject requiring treatment or prevention of iAP-related gastrointestinal disease, comprising administering a therapeutically effective amount of the hIAP antibody or a fragment thereof according to claim 1, 2, or 4 to the subject.

15. The method according to claim 13 or 14, wherein the iAP-related disease includes gastrointestinal diseases.

16. The method according to claim 15, wherein the gastrointestinal disorder includes colitis, inflammatory bowel disease (IBD), gastritis, gastroenteritis, pyloric stenosis, stomach cancer, gastrointestinal cancer, colon cancer, infectious diarrhea, fecal impaction, constipation, intestinal obstruction and pseudo-intestinal obstruction, or malabsorption.

17. A kit comprising the isolated monoclonal antibody according to claim 1 or 2.

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