WO2022241236A2 - Procédés et réactifs pour l'analyse de protéines - Google Patents

Procédés et réactifs pour l'analyse de protéines Download PDF

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Publication number
WO2022241236A2
WO2022241236A2 PCT/US2022/029227 US2022029227W WO2022241236A2 WO 2022241236 A2 WO2022241236 A2 WO 2022241236A2 US 2022029227 W US2022029227 W US 2022029227W WO 2022241236 A2 WO2022241236 A2 WO 2022241236A2
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Prior art keywords
amino acid
seq
antibody
gcd59
acid sequence
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PCT/US2022/029227
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English (en)
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WO2022241236A3 (fr
Inventor
Michael Chorev
Jose A. Halperin
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Mellitus, Llc
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Publication of WO2022241236A2 publication Critical patent/WO2022241236A2/fr
Publication of WO2022241236A3 publication Critical patent/WO2022241236A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/02—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • Diabetes is characterized by elevated blood glucose levels. Sustained elevation of blood glucose levels may affect proteins by a process known as glycation. Glycation is the non-enzymatic attachment of glucose to proteins and is considered a major pathophysiological mechanism causing tissue damage in diabetic subjects. Glycation involves the reaction of glucose and/or other reducing sugars with amino groups in proteins resulting in the formation of a Schiff base or aldimine. This labile adduct can tautomerize via the Amadori rearrangement to the more stable ketoamine.
  • glycated proteins have been identified in diabetic subjects, including albumin, hemoglobin and others.
  • the function of glycated proteins may be impaired, depending on the location of the amino group(s) affected.
  • amino-terminal glycation of the b-chains of hemoglobin gives rise to the glycated hemoglobin (HbAlc) in which responsiveness to 2,3-diphosphoglycerate is decreased and oxygen affinity increased.
  • Glycation of the major thrombin inhibitor of the coagulation system, antithrombin III decreases its affinity for heparin, and has been postulated to contribute to the hypercoagulable state associated with diabetes.
  • GDM Gestational diabetes mellitus
  • GLT glucose load test
  • GCT glucose challenge test
  • GLT has been used for GDM screening routinely but has limitations.
  • a systematic review of the scientific literature reported GLT sensitivity estimates (relative to OGTT results) of 74% to 83% and specificity estimates of 72% to 85% depending on the study population or OGTT threshold value used to determine GDM status (van Leeuwen, M. et ah, 2012. BJOG, p393-401).
  • the present disclosure provides a method of detecting glycated CD59 (GCD59) in a sample, the method including: applying the sample to a substrate, wherein the substrate includes an immobilized capture antibody, wherein the capture antibody is directed to a glycated epitope of GCD59; applying a detection antibody to
  • the glycated epitope of GCD59 may include lysine residue 41 (K41) of mature human CD59, wherein K41 is glycated K41.
  • the glycated K41 may be Amadori-modified glycated K41.
  • the glycated epitope of GCD59 may include the amino acid sequence WKFEH (SEQ ID NO: 55).
  • the glycated epitope of GCD59 may include the amino acid sequence NKCWKFEHCNFNDV (SEQ ID NO: 54) or a fragment thereof.
  • the capture antibody may include an antibody light chain variable domain (VL) having a complementarity determining region (CDR)-L1 including the amino acid sequence of SEQ ID NO: 33; a CDR-L2 including the amino acid sequence of SEQ ID NO: 35; and a CDR-L3 including the amino acid sequence of SEQ ID NO: 36.
  • the capture antibody may include an antibody heavy chain variable domain (VH) having a CDR-H1 including the amino acid sequence of SEQ ID NO: 38; a CDR-H2 including the amino acid sequence of SEQ ID NO: 40; and a CDR-H3 including the amino acid sequence of SEQ ID NO: 42.
  • the capture antibody may include a VL including the amino acid sequence of SEQ ID NO: 29 and a VH including the amino acid sequence of SEQ ID NO: 30.
  • the capture antibody may be an antibody fragment.
  • the antibody fragment may be an antigen binding fragment (FAb).
  • the capture antibody may include an attachment agent.
  • the attachment agent may facilitate immobilization of the capture antibody to the substrate.
  • the attachment agent may include biotin.
  • the substrate may include a biotin-binding agent.
  • the biotin-binding agent may be selected from the group consisting of avidin, streptavidin, NeutrAvidin, and CAPTAVIDINTM.
  • the biotin-binding agent may be streptavidin.
  • the non-glycated epitope of GCD59 may not include K41.
  • the non-glycated epitope of GCD59 may include the amino acid sequence HCNFND VTTRLRENELT YY CCKK (SEQ ID NO: 48) or a fragment thereof.
  • the detection antibody may include a VL having: a CDR-L1 including the amino acid sequence of SEQ ID NO: 34; a CDR-L2 including the amino acid sequence of SEQ ID NO: 35; and a CDR-L3 including the amino acid sequence of SEQ ID NO: 37.
  • the detection antibody may include a VH having: a CDR-H1 including the amino acid sequence of SEQ ID NO: 39; a CDR-H2 including the amino acid sequence of SEQ ID NO: 41; and a CDR-H3 including the amino acid sequence of SEQ ID NO: 43.
  • the detection antibody may include a VL including the amino acid sequence of SEQ ID NO: 31 and a VH including the amino acid sequence of SEQ ID NO: 32.
  • the detection antibody may include a detectable label.
  • Detecting the detection antibody may include detecting the detection antibody detectable label.
  • the detection antibody detectable label may be selected from the group consisting of biotin, a biotin-binding agent, a fluorescent label, an enzymatic label, a luminescent label,
  • the detection antibody detectable label may include an enzymatic label, wherein the enzymatic label includes horse radish peroxidase. Detecting the detection antibody may include the use of a secondary detection agent.
  • the secondary detection agent may include a detectable label.
  • the secondary detection agent detectable label may be selected from the group consisting of biotin, a biotin-binding agent, a fluorescent label, an enzymatic label, a luminescent label, and a radioactive label.
  • the secondary detection agent may include a secondary detection antibody. Detecting the detection antibody may include detecting the secondary detection agent detectable label.
  • the secondary detection agent detectable label may be an enzymatic label, wherein the enzymatic label includes a peroxidase.
  • the peroxidase may be horse radish peroxidase.
  • the substrate may be selected from the group consisting of an assay surface, an assay plate, a bead, a membrane, a conducting surface, and a conducting nanoparticle.
  • the sample may be a mammalian sample.
  • the mammalian sample may be a human sample.
  • the sample may be a fluid sample.
  • the fluid sample may be selected from the group consisting of a urine sample, a blood sample, a plasma sample, a serum sample, a sweat sample, a lymph sample, and a saliva sample.
  • the substrate may be prepared by adhering a biotin-binding agent to the substrate; biotinylating the capture antibody; and contacting the adhered biotin-binding agent with the biotinylated capture antibody, thereby immobilizing the capture antibody on the substrate.
  • Adhering the biotin-binding agent to the substrate may include coating the substrate with the biotin-binding agent.
  • the biotin-binding agent may include streptavidin.
  • Biotinylating the capture antibody may include one or more of chemical biotinylation, carboxyl biotinylation, enzymatic biotinylation, sulfhydryl biotinylation, primary amine biotinylation, glycoprotein biotinylation, non-specific biotinylation, and long-arm biotinylation.
  • Detecting the detection antibody may include observing a detection signal associated with a detectable label. Observing the detection signal may include measuring the detection signal strength. Measuring the detection signal strength may be used to determine a GCD59 concentration or concentration equivalent for the sample.
  • the present disclosure provides a method of screening, diagnosing, and/or monitoring one or more diabetes-related indications in a subject by determining a GCD59 concentration or concentration equivalent for a subject sample according to any of the methods described herein and comparing the GCD59 concentration or concentration equivalent for the subject sample to a GCD59 concentration or concentration equivalent associated with at least one control sample or known threshold.
  • the one or more diabetes-related indications may include gestational diabetes mellitus (GDM).
  • Methods of the present disclosure include a method of assigning a diabetes-related indication risk level to a subject by determining a GCD59 concentration or concentration equivalent for a subject sample according to any of the methods disclosed herein; comparing the GCD59 concentration or concentration equivalent for the subject sample to a GCD59 concentration or concentration equivalent associated with at least one control sample or known threshold; and assigning the diabetes-related indication risk level to the subject based on GCD59 concentration or concentration equivalent variation between the subject sample and the at least one control sample or known threshold.
  • the diabetes-related indication risk level may include a risk level associated with developing one or more diabetes-related indications.
  • the one or more diabetes-related indications may include GDM. All or part of the method may be carried out at the point of care. At least part of the method may be carried out in conjunction with a patient monitoring device.
  • the patient monitoring device may be capable of receiving and/or transmitting an electronic signal.
  • the patient monitoring device may be a smart device selected from at least one of a smart phone and a smart watch.
  • the present disclosure provides a kit that includes a substrate; a capture antibody, wherein the capture antibody is directed to a glycated epitope of GCD59; a detection antibody, wherein the detection antibody is directed to a non-glycated epitope of GCD59; and instructions for use of the kit.
  • the glycated epitope of GCD59 may include glycated K41.
  • the glycated K41 may be Amadori-modified glycated K41.
  • the glycated epitope of GCD59 may include the amino acid sequence WKFEH (SEQ ID NO:
  • the glycated epitope of GCD59 may include the amino acid sequence NKCWKFEHCNFNDV (SEQ ID NO: 54) or a fragment thereof.
  • the capture antibody may include a VL having a CDR-L1 with the amino acid sequence of SEQ ID NO: 33; a CDR-L2 with the amino acid sequence of SEQ ID NO: 35; and a CDR-L3 with the amino acid sequence of SEQ ID NO: 36.
  • the capture antibody may include a VH having a CDR-H1 with the amino acid sequence of SEQ ID NO: 38; a CDR-H2 with the amino acid sequence of SEQ ID NO: 40; and a CDR-H3 with the amino acid sequence of SEQ ID NO: 42.
  • the capture antibody may include a VL including the amino acid sequence of SEQ ID NO: 29 and a VH including the amino acid sequence of SEQ ID NO: 30.
  • the capture antibody may be an antibody fragment.
  • the antibody fragment may be an antigen binding fragment (FAb).
  • the capture antibody may be immobilized on the substrate.
  • the capture antibody may be covalently or non-covalently immobilized on the substrate.
  • the non-glycated epitope of GCD59 may not include K41.
  • the non-glycated epitope of GCD59 may include the amino acid sequence HCNFND VTTRLRENELT YY CCKK (SEQ ID NO: 48) or a fragment thereof.
  • the detection antibody may include a VL having a CDR-L1 including the amino acid sequence of SEQ ID NO: 34; a CDR-L2 including the amino acid sequence of SEQ ID NO: 35; and a CDR-L3 including the amino acid sequence of SEQ ID NO: 37.
  • the detection antibody may include a VH having a CDR-H1 including the amino acid sequence of SEQ ID NO: 39; a CDR-H2 including the amino acid sequence of SEQ ID NO: 41; and a CDR-H3 including the amino acid sequence of SEQ ID NO: 43.
  • the detection antibody may include a VL with the amino acid sequence of SEQ ID NO: 31 and a VH with the amino acid sequence of SEQ ID NO: 32.
  • the detection antibody may include a detectable label. Detecting the detection antibody may include detecting the detection antibody detectable label.
  • the detection antibody detectable label may be selected from the group consisting of biotin, a biotin-binding agent, a fluorescent label, an enzymatic label, a luminescent label, and a radioactive label.
  • the detection antibody detectable label may include an enzymatic label, wherein the enzymatic label includes horse radish peroxidase.
  • Detecting the detection antibody may include the use of a secondary detection agent.
  • the secondary detection agent may include a detectable label.
  • the secondary detection agent detectable label may be selected from the group consisting of biotin, a biotin-binding agent, a fluorescent label, an enzymatic label, a luminescent label, and a radioactive label.
  • the secondary detection agent may include a secondary detection antibody. Detecting the detection antibody may include detecting the secondary detection agent detectable label.
  • the secondary detection agent detectable label may include an enzymatic label, wherein the enzymatic label includes a peroxidase.
  • the peroxidase may be horse radish peroxidase.
  • the substrate may be selected from the group consisting of an assay surface, an assay plate, a bead, a membrane, a conducting surface, and a conducting nanoparticle.
  • the substrate may include a biotin binding agent.
  • the biotin-binding agent may be selected from the group consisting of avidin, streptavidin, NeutrAvidin, and CAPTAVIDINTM.
  • the capture antibody may include biotin.
  • the capture antibody may be immobilized on the substrate.
  • the kit may include one or more control samples.
  • the one or more control samples may include a plasma assay control.
  • the kit may include at least one buffer.
  • the at least one buffer may include one or more of an assay diluent, a conjugate diluent, and a wash buffer.
  • the kit may include a compound for generation of a calibration curve.
  • the compound for generation of a calibration curve may include a first peptide, wherein the first peptide includes a glycated epitope of GCD59; a second peptide, wherein the second peptide includes a non-glycated epitope of GCD59; and a linker, wherein the linker joins the first peptide to the second peptide.
  • Fig. 1 A is a graph of a calibration curve generated using an Amadori-modified GCD59 surrogate and an anti-Amadori-modified GCD59 antibody (D2).
  • Fig. IB is a graph showing the correlation between the percent urine sample dilution and synthetic peptide units (SPU) detected using antibody D2.
  • FIG. 2 is an image from Western blot analysis showing detection of Amadori- modified hGCD59 with antibody D2.
  • the present disclosure provides compounds, compositions, reagents, methods, assays, kits, and devices for various therapeutic, diagnostic, monitoring, and/or research applications.
  • assays and kits of the present disclosure include diagnostic assays and kits. Some assays and kits may be used for detecting CD59 post-translational modifications.
  • the present disclosure includes antibodies that bind to modified and/or unmodified CD59. Such antibodies may be used in assays and kits to detect post-translationally modified CD59, e.g., for a variety of therapeutic applications. These and other antibody applications are described in further detail herein.
  • Proteins may exist as a whole polypeptide, a plurality of polypeptides or fragments of polypeptides, which independently may be encoded by one or more nucleic acids, a plurality of nucleic acids, fragments of nucleic acids or variants of any of the aforementioned.
  • polypeptide means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds.
  • polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
  • a polypeptide may be a single molecule or may be a multi- molecular complex such as a dimer, turner or tetramer. They may also include single chain or multichain polypeptides and may be associated or linked.
  • the term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.
  • polypeptide variant refers to molecules which differ in their amino acid sequence from a native or reference sequence.
  • the amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence.
  • variants will possess at least about 50% identity (homology) to a native or reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90% identical (homologous) to a native or reference sequence.
  • variant mimics are provided.
  • the term “variant mimic” is one which contains one or more amino acids which would mimic an activated sequence.
  • glutamate may serve as a mimic for phosphoro-threonine and/or phosphoro-serine.
  • variant mimics may result in deactivation or in an inactivated product containing the mimic, e.g., phenylalanine may act as an inactivating substitution for tyrosine; or alanine may act as an inactivating substitution for serine.
  • amino acid sequence variant refers to molecules with some differences in their amino acid sequences as compared to a native or starting sequence.
  • the amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence.
  • “Native” or “starting” sequence should not be confused with a wild type sequence.
  • a native or starting sequence is a relative term referring to an original molecule against which a comparison may be made.
  • “Native” or “starting” sequences or molecules may represent the wild-type (that sequence found in nature) but do not have to be the wild-type sequence.
  • Polypeptides of the present disclosure may include natural and/or unnatural amino acids.
  • variants will possess at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% at least 99.8%, or at least 99.9% sequence identity as compared to a native sequence.
  • Sequence identity as it applies to amino acid sequences is defined as the percentage of residues in the candidate amino acid sequence that are identical with the residues in the
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A.
  • the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package,
  • homologs as it applies to amino acid sequences is meant the corresponding sequence of other species having substantial identity to a second sequence of a second species.
  • Analogs is meant to include polypeptide variants which differ by one or more amino acid alterations, e.g., substitutions, additions or deletions of amino acid residues that still maintain the properties of the parent polypeptide.
  • Homology refers to the overall relatedness between polymeric molecules, e.g., between polypeptide molecules, nucleic acid molecules, and oligosaccharides. Determination of homology may take into account the sequence identity, sequence similarity, three dimensional conformation, and/or function of the polymeric molecules being compared.
  • polypeptides which are amino acid based including variants and derivatives. These include substitutional, insertional, deletion and covalent variants and derivatives. As such, included within the scope of this disclosure are polypeptides containing substitutions, insertions and/or additions, deletions and covalently modifications.
  • sequence tags or amino acids such as one or more lysines, can be added to the peptide sequences of the present disclosure (e.g., at the N- terminal or C-terminal ends). Sequence tags can be used for peptide purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation.
  • amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences.
  • Certain amino acids e.g., C-terminal or N-terminal residues
  • substitutional variants when referring to proteins are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position.
  • the substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.
  • conservative amino acid substitution refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity.
  • conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue.
  • conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue.
  • conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue.
  • substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine.
  • substitution of a basic residue such as lysine, arginine or histidine for another or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions.
  • non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.
  • a non-polar amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.
  • a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.
  • deletional variants when referring to proteins, are those with one or more amino acids in the native or starting amino acid sequence removed. Ordinarily, deletional variants will have one or more amino acids deleted in a particular region of the molecule.
  • derivatives are used synonymously with the term “variant” and refers to a molecule that has been modified or changed in any way relative to a reference molecule or starting molecule.
  • derivatives include native or starting proteins that have been modified with an organic proteinaceous or non-proteinaceous derivatizing agent, and post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of the protein with an organic derivatizing agent that is capable of reacting with selected side-chains or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant host cells.
  • the resultant covalent derivatives are useful in programs directed at identifying residues important for biological activity, for immunoassays, or for the preparation of anti-protein antibodies for immunoaffmity purification of the recombinant protein. Such modifications are within the ordinary skill in the art and are performed without undue experimentation.
  • Covalent derivatives specifically include fusion molecules in which proteins of the present disclosure are covalently bonded to a non-proteinaceous polymer.
  • the non- proteinaceous polymer ordinarily is a hydrophilic synthetic polymer, i.e. a polymer not otherwise found in nature.
  • polymers which exist in nature and are produced by recombinant or in vitro methods are useful, as are polymers which are isolated from nature.
  • Hydrophilic polyvinyl polymers fall within the scope of this disclosure, e.g. polyvinylalcohol and polyvinylpyrrolidone.
  • Particularly useful are polyvinylalkylene ethers such a polyethylene glycol, polypropylene glycol.
  • the proteins may be linked to various non- proteinaceous polymers, such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
  • Proteins when referring to proteins are defined as distinct amino acid sequence- based components of a molecule.
  • Features of the proteins of the present disclosure include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half domains, sites, termini or any combination thereof.
  • local conformational shape means a polypeptide based structural manifestation of a protein which is located within a definable space of the protein.
  • fold means the resultant conformation of an amino acid sequence upon energy minimization.
  • a fold may occur at the secondary or tertiary level of the folding process.
  • secondary level folds include beta sheets and alpha helices.
  • tertiary folds include domains and regions formed due to aggregation or separation of energetic forces. Regions formed in this way include hydrophobic and hydrophilic pockets, and the like.
  • the term "turn" as it relates to protein conformation means a bend which alters the direction of the backbone of a peptide or polypeptide and may involve one, two, three or more amino acid residues.
  • loop refers to a structural feature of a peptide or polypeptide which reverses the direction of the backbone of a peptide or polypeptide and includes four or more amino acid residues. Oliva et al. have identified at least 5 classes of protein loops (J. Mol Biol 266 (4): 814-830; 1997).
  • domain refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions.
  • sub-domains may be identified within domains or half-domains, these subdomains possessing less than all of the structural or functional properties identified in the domains or half domains from which they were derived. It is also understood that the amino acids that include any of the domain types herein need not be contiguous along the backbone of the polypeptide (i.e., nonadjacent amino acids may fold structurally to produce a domain, half-domain or subdomain).
  • site As used herein when referring to proteins the terms "site” as it pertains to amino acid based embodiments is used synonymous with "amino acid residue” and "amino acid side chain.”
  • a site represents a position within a peptide or polypeptide that may be modified, manipulated, altered, derivatized or varied within the polypeptide based molecules of the present disclosure.
  • terminal or terminus when referring to proteins refers to an extremity of a peptide or polypeptide. Such extremity is not limited only to the first or final site of the peptide or polypeptide but may include additional amino acids in the terminal regions.
  • the polypeptide based molecules of the present disclosure may be characterized as having both an N-terminus [terminated by an amino acid with a free amino group (NIL ⁇ )] and a C-terminus [terminated by an amino acid with a free carboxyl group (CO2H)].
  • Proteins of the present disclosure may be made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These sorts of proteins will have multiple N- and C-termini.
  • the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic conjugate.
  • any of several manipulations and/or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating.
  • manipulation of features may result in the same outcome as an alternative modification. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full length molecule would.
  • Modifications and manipulations may be accomplished by methods known in the art such as site-directed mutagenesis.
  • the resulting modified molecules may then be tested for sequence, structure, and/or activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.
  • Compounds of the present disclosure may contain one or more atoms that are isotopes.
  • isotope refers to a chemical element that has one or more additional neutron.
  • compounds of the present disclosure may be deuterated.
  • deuterated refers to a substance that has had one or more hydrogen atoms replaced by deuterium isotopes.
  • Deuterium isotopes are isotopes of hydrogen.
  • the nucleus of hydrogen contains one proton while deuterium nuclei contain both a proton and a neutron.
  • Compounds may be deuterated in order to change a physical property of the compound, such as stability, or to allow the compounds to be used in diagnostic and experimental applications.
  • target proteins refers to any protein of interest including proteins that are being analyzed as part of an assay, diagnostic method, or any other method described herein that involves protein detection and/or analysis.
  • Target proteins may also include synthetic constructs having one or more amino acid-based segments. Synthetic constructs may include one or more non-amino acid based elements such as linkers, chemical moieties, or chemical groups.
  • target proteins include moieties or tags, including, but not limited to detectable labels.
  • target proteins include CD59, fragments of CD59, variants of CD59, post-translationally modified versions of CD59, and/or synthetic constructs that include CD59, CD59 fragments, CD59 variants, post-translationally modified CD59, post- translationally modified CD59 fragments, and/or post-translationally modified variants of CD59.
  • Mature human CD59 hCD59; lacking the signal sequence
  • LQCYNCPNPT 10 ADCKTAVNCS 20 SDFDACLITK 30 AGLQVYNKCW 40 KFEHCNFNDV 50
  • Residue numbers of hCD59 referred to herein refer to residue numbers present in this sequence.
  • hCD59 fragments and variants may include any of those described in in European Patent Number EP2348050, International Publication Number WO2015084994, and US Patent Numbers US9068006, US9417248, the contents of each of which are herein incorporated by reference in their entireties.
  • antibodies of the present disclosure bind to non-glycated epitopes of CD59.
  • Such antibodies may include any of those described in International Publication Number WO2018195008, e.g., antibody H9 described therein.
  • Some capture antibodies bind to epitopes of hCD59 that do not include lysine 41 (referred to herein as “K41”) of hCD59 (SEQ ID NO: 1).
  • Some antibodies of the present disclosure bind to glycated epitopes of glycated CD59 (GCD59).
  • Such glycated epitopes may include K41- glycated human GCD59.
  • Some glycated epitopes include Amadori-modified K41.
  • Antibodies binding to GCD59 epitopes that include Amadori-modified K41 may include any of those described in International Publication Number WO2018195008, e.g., antibodies D2 or D3 described therein.
  • Glycated epitopes of hCD59 may include any of those described in in European Patent Number EP2348050, International Publication Number WO2015084994, and US Patent Numbers US9068006, US9417248, and US10732186 the contents of each of which are herein incorporated by reference in their entireties.
  • antibodies binding to glycated epitopes that include Amadori-modified K41 of GCD59 may bind with an equilibrium dissociation constant (KD) of from about 1.0 pM to about 5.0 x 10 8 pM. In some cases, the KD is from about 1.5 pM to about 150 pM.
  • KD equilibrium dissociation constant
  • Antibodies of the present disclosure may include capture antibodies.
  • Capture antibodies refer to antibodies that bind to target proteins for the purposes of isolation, immobilization, and/or further analysis of such target proteins.
  • Capture antibodies may bind CD59 or fragments and/or variants thereof. Some capture antibodies bind to CD59 regardless of the presence of one or more post-translational modification. Some capture antibodies bind to target epitopes of target proteins.
  • a “target epitope” refers to a specific sequence, feature, or moiety that an antibody or other molecule has a specific affinity for.
  • Target epitopes present on CD59, CD59 fragments, or CD59 variants may include, but are not limited to specific amino acids, amino acid sequences, and/or three-dimensional features, in some cases including glycated, non-glycated, glycosylated, and non-glycosylated features thereof.
  • Target epitopes of hCD59 may include any of the aforementioned CD59 epitopes or any of the hCD59 (human CD59) epitopes described in in European Patent Number EP2348050, International Publication Number WO2015084994, and US Patent Numbers US9068006, US9417248, and US10732186, the contents of each of which are herein incorporated by reference in their entireties.
  • Such epitopes may be captured or detected in a variety of ways including the use of antibodies or other biomolecules such as lectins, aptamers, other proteins or nucleic acids.
  • CD59 e.g., hCD59 epitopes such as N-glycosylated epitopes
  • Detection agents may be antibodies which bind to or recognize CD59 irrespective of its glycosylation state, e.g., glycated or non-glycated.
  • Detection antibodies are antibodies that bind target proteins for the purposes of determining the presence, absence, and/or level of such target proteins or for the characterization of a target protein. In some cases, detection antibodies may be used to
  • detection antibodies may target CD59.
  • detection antibodies may recognize an epitope of CD59 that may vary among CD59 species (e.g., different post- translationally modified CD59 species).
  • detection antibodies recognize post-translationally modified epitopes present on CD59.
  • detection antibodies recognize glycated CD59 (GCD59).
  • Glycated hCD59 may include K41 glycated hCD59.
  • detection antibodies may recognize K41 glycated CD59 that bears a sugar residue in Amadori form.
  • K41 Amadori-modified hCD59 are described, for example, in US Patent Number 9,068,006 and International Publication Number WO20 15/084994, the contents of each of which are herein incorporated by reference in their entirety.
  • Some detection antibodies bind only GCD59, but not non-glycated forms.
  • Antibodies described herein as “capture” or “detection” antibodies are not to be limited to capture and detection functions, respectively. In some cases, such antibodies may be useful for other purposes (e.g., therapeutic or manufacturing), including, but not limited to any of the purposes described herein.
  • antibodies of the present disclosure bind to target proteins presented in a solution-based format.
  • a “solution-based format” refers to a protein conformation that is present in a solution.
  • solutions may include, but are not limited to saline, buffer (e.g., phosphate buffered saline), serum, plasma, blood, lymph, sweat, urine, lymph, and saliva.
  • antibodies may be capable of binding target proteins in diluted serum or plasma.
  • serum may be diluted by from about 1 : 1 to about 1:100,000.
  • antibody is referred to in the broadest sense and specifically covers various embodiments including, but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies formed from at least two intact antibodies), and antibody fragments (e.g., diabodies) so long as they exhibit a desired biological activity (e.g., “functional”).
  • Antibodies are primarily amino-acid based molecules but may also include one or more modifications (including, but not limited to the addition of sugar moieties, fluorescent moieties, chemical tags, etc.).
  • antibody-based or “antibody-derived” compositions are monomeric or multi-meric polypeptides which include at least one amino-acid region derived from a known or parental antibody sequence and at least one amino acid region derived from a non-antibody sequence, e.g., mammalian protein.
  • Antibodies of the present disclosure may be therapeutic, diagnostic, industrial, or used for research purposes. Further, antibodies of the disclosure may include fragments of such antibodies or antibodies that have been developed to include one or more of such fragments (e.g., variable domains or complementarity determining regions (CDRs)).
  • CDRs complementarity determining regions
  • antibodies of the present disclosure may be used to capture CD59 and/or GCD59 in samples (e.g., clinical samples).
  • Antibodies of the present disclosure may be produced using methods known in the art. Such methods may include, but are not limited to immunization and display technologies (e.g., phage display, yeast display, and ribosomal display). Antibodies may be developed, for example, using naturally occurring or synthetic antigens.
  • an “antigen” is an entity which induces or evokes an immune response in an organism. An immune response is characterized by the reaction of the cells, tissues and/or organs of an organism to the presence of a foreign entity. Such an immune response typically leads to the production by the organism of one or more antibodies against the foreign entity, e.g., antigen or a portion of the antigen.
  • antigens also refer to binding partners for specific antibodies or binding agents in a display library.
  • Antibodies of the present disclosure may be derived from antibodies produced using hybridoma technology.
  • Host animals e.g. mice, rabbits, goats, and llamas
  • Lymphocytes may be collected and fused with immortalized cell lines to generate hybridomas which can be cultured in a suitable culture medium to promote growth.
  • the antibodies produced by the cultured hybridomas may be subjected to analysis to determine binding specificity of the antibodies for the target antigen. Once antibodies with desirable characteristics are identified, corresponding hybridomas may be subcloned through limiting dilution procedures and grown by standard methods. The antibodies produced by these cells may be isolated and purified using standard immunoglobulin purification procedures.
  • Recombinant antibodies are antibodies produced by expression of recombinant DNA.
  • Recombinant antibodies of the present disclosure may be generated using standard techniques known in the art.
  • Recombinant antibodies may be produced using variable domains obtained from hybridoma cell-derived antibodies produced according to methods described herein.
  • RNA may be extracted from antibody-producing hybridoma cells and converted to cDNA by reverse transcriptase (RT) polymerase chain reaction (PCR).
  • PCR reverse transcriptase
  • PCR amplification may be carried out on resulting cDNA to amplify variable region genes.
  • Such amplification may include the use of primers specific for amplification of heavy and light chain sequences.
  • recombinant antibodies may be produced using variable domains obtained from other sources. This includes the use of variable domains selected from one or more antibody fragment library, such as an scFv library used in antigen panning. Resulting PCR products may then be subcloned into plasmids and may be subjected to sequence analysis for verification and/or optimization.
  • Antibody coding sequences may be placed into expression vectors. For humanization, coding sequences for human heavy and light chain constant domains and/or variable domain framework regions may be used to substitute for homologous or otherwise corresponding murine sequences. The resulting constructs may then be transfected into cells (e.g., mammalian cells) for large scale translation. In some embodiments, recombinant antibodies and/or vectors encoding recombinant antibodies may be produced using one or more of any of the sequences presented herein.
  • Antibodies of the present disclosure may be generated using display technologies.
  • Display technologies used to generate polypeptides of the present disclosure may include any of the display techniques (e.g., display library screening techniques) disclosed in US Publication No. US2015/0284455, the contents of which are herein incorporated by reference in their entirety.
  • synthetic antibodies may be designed, selected or optimized by screening target antigens using display technologies (e.g. phage display technologies).
  • Display libraries may include millions to billions of display particles (e.g., phage or cellular display particles), each expressing unique antibody fragments on their surfaces (e.g., as part of viral coat proteins or cell surface molecules or proteins).
  • Such libraries may provide richly diverse resources that may be used to select potentially hundreds of antibody fragments with diverse levels of affinity for one or more antigens of interest (McCafferty, et al., 1990. Nature. 348:552-4; Edwards, B.M. et al., 2003. JMB. 334: 103-18; Schofield, D. et al., 2007. Genome Biol. 8, R254 and Pershad, K. et al., 2010. Protein Engineering Design and Selection. 23:279-88; the contents of each of which are herein incorporated by reference in their entirety).
  • Antibody fragments present in such libraries may be scFv antibody fragments, including a fusion protein of VH and VL antibody domains joined by a flexible linker.
  • scFvs may contain the same sequence with the exception of unique sequences encoding variable loops of the CDRs.
  • scFvs are
  • VL chains may be expressed separately for assembly with VH chains in the periplasm or intracellularly prior to complex incorporation into surface molecules.
  • Precipitated library members may be sequenced from the bound display particles to obtain cDNA encoding desired scFvs.
  • Antibody variable domains or CDRs from such sequences may be directly incorporated into antibody sequences for recombinant antibody production, or mutated and utilized for further optimization through affinity maturation.
  • antibodies may be produced using yeast surface display technology, wherein antibody variable domain sequences may be expressed on the cell surface of Saccharomyces cerevisiae.
  • Recombinant antibodies may be developed by displaying the antibody fragment of interest as a fusion to e.g. Aga2p protein on the surface of the yeast, where the protein interacts with proteins and small molecules in a solution.
  • scFvs with affinity for desired antigens may be isolated from the yeast surface using magnetic separation and flow cytometry.
  • yeast surface display and isolation may be done to attain scFvs with desired properties through directed evolution.
  • Yeast display may, for example, be carried out according to any of the methods taught in Chao, G. et al., Nat Protoc. 2006, l(2):755-68, the contents of which are herein incorporated by reference in their entirety.
  • antibodies of the present disclosure are IgG antibodies.
  • IgG antibodies e.g. IgGl, IgG2, IgG3 or IgG4
  • IgG antibodies may include one or more variable domain and/or CDR amino acid sequences presented herein (or fragment or variants thereof).
  • Such IgG antibodies may be synthesized for further testing and/or product development.
  • Some IgG antibodies may be produced by insertion of one or more segments of cDNA encoding desired amino acid sequences into expression vectors suited for IgG production.
  • Expression vectors may include mammalian expression vectors suitable for IgG expression in mammalian cells. Mammalian expression of IgGs may be carried out to ensure that antibodies produced include modifications (e.g. glycosylation) characteristic of mammalian proteins and/or to ensure that antibody preparations lack endotoxin and/or other contaminants that may be present in protein preparations from bacterial expression systems.
  • Antibodies of the present disclosure may include antibody fragments (e.g., antigen binding regions) from intact antibodies.
  • antibody fragments may include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; and single chain antibody molecules.
  • antibodies of the present disclosure include multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site. Also produced is a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen binding sites and is still capable of cross-linking antigen.
  • Antibodies of the present disclosure may include one or more of these fragments.
  • an "antibody” may be made up of a heavy and light chain variable domain as well as an Fc region.
  • the Fc region may be a modified Fc region, as described in US Patent Publication US20150065690, wherein the Fc region may have a single amino acid substitution as compared to the corresponding sequence for the wild-type Fc region, wherein the single amino acid substitution yields an Fc region with preferred properties to those of the wild-type Fc region.
  • Fc properties that may be altered by the single amino acid substitution include binding properties and response to pH conditions (e.g., altered stability and/or target affinity).
  • the term "native antibody” refers to an usually heterotetrameric glycoprotein of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Genes encoding native antibody heavy and light chains are known and segments making up each have been well characterized and described (Matsuda, F. et ah, 1998. The Journal of Experimental Medicine. 188(11); 2151-62 and Li, A. et al., 2004.
  • Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes.
  • Each heavy and light chain typically have regularly spaced intrachain disulfide bridges.
  • Each heavy chain has at one end a heavy chain variable domain (VH) followed by a number of constant domains.
  • Each light chain has a light chain variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • variable domain refers to specific antibody domains found on both the antibody heavy and light chains that may differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. Variable domains typically include hypervariable regions.
  • hypervariable region refers to a region within a variable domain having amino acid residues responsible for antigen binding. The amino acids present within the hypervariable regions determine the structure of the complementarity determining regions (CDRs) that become part of the antigen-binding site of the antibody.
  • CDR refers to a region of an antibody having a structure that is complementary to its target antigen or epitope.
  • the antigen-binding site also known as the antigen combining site or paratope
  • the antigen-binding site typically includes amino acid residues necessary to interact with a particular antigen.
  • the exact residues making up antigen-binding sites are typically elucidated by co-crystallography with bound antigen, however computational assessments can also be used based on comparisons with other antibodies (Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 3, p47-54, the contents of which are herein incorporated by reference in their entirety).
  • Determining residues making up CDRs may include the use of numbering schemes including, but not limited to, those taught by Rabat [Wu, T.T. et ah, 1970, JEM, 132(2):211-50 and Johnson, G. et ah, 2000, Nucleic Acids Res. 28(1): 214-8, the contents of each of which are herein incorporated by reference in their entirety], Chothia [Chothia and Lesk, J. Mol. Biol. 196, 901 (1987), Chothia et ah, Nature 342, 877 (1989) and Al-Lazikani, B. et ah, 1997, J. Mol. Biol.
  • VH and VL domains typically have three CDRs each.
  • VL CDRS are referred to herein as CDR-L1, CDR-L2 and CDR-L3, in order of occurrence when moving from N- to C- terminus along the variable domain polypeptide.
  • VH CDRS are referred to herein as CDR- Hl, CDR-H2 and CDR-H3, in order of occurrence when moving from N- to C- terminus along the variable domain polypeptide.
  • Each of the CDRs typically have favored canonical structures with the exception of the CDR-H3, which may include amino acid sequences that may be highly variable in sequence and length between antibodies resulting in a variety of three-dimensional structures in antigen-binding domains (Nikoloudis, D. et al., 2014. Peer J.
  • CDR-H3s may be analyzed among a panel of related antibodies to assess antibody diversity.
  • Various methods of determining CDR sequences are known in the art and may be applied to known antibody sequences (Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 3, p47-54, the contents of which are herein incorporated by reference in their entirety).
  • antibodies of the present disclosure may be formatted as Fv fragments.
  • Fv refers to an antibody fragment including the minimum fragment on an antibody needed to form a complete antigen-binding site. These regions consist of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. Fv fragments can be generated by proteolytic cleavage, but are largely unstable.
  • light chain refers to a component of an antibody from any vertebrate species assigned to one of two clearly distinct types, called kappa and lambda based on amino acid sequences of constant domains.
  • antibodies of the present disclosure may be formatted as scFvs.
  • single chain Fv or “scFv” refers to a fusion protein of VH and VL antibody domains, wherein these domains are linked together into a single polypeptide chain by a flexible peptide linker.
  • the Fv polypeptide linker enables the scFv to form the desired structure for antigen binding.
  • scFvs are utilized in conjunction with antibody display methods (e.g., phage display, yeast display or other display format) where they may be expressed in association with a surface member (e.g. phage coat protein or cell surface molecule) and used in the identification of high affinity peptides for a given antigen.
  • Antibodies of the present disclosure may be formatted as bispecific antibodies.
  • the term “bispecific antibody” refers to an antibody capable of binding two different antigens. Such antibodies typically include regions from at least two different antibodies. Bispecific antibodies may include any of those described in Riethmuller, G. 2012. Cancer Immunity. 12:12-18, Marvin, J.S. et ah, 2005. Acta Pharmacol ogica Sinica.
  • antibodies of the present disclosure may be produced as diabodies.
  • the term "diabody” refers to a small antibody fragment with two antigen-binding sites.
  • Diabodies include a heavy chain variable domain VH connected to a light chain variable domain VL in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP404097; WO 1993011161; and Hollinger et al. (Hollinger, P. et al., “Diabodies”: Small bivalent and bispecific antibody fragments. PNAS. 1993. 90:6444-8) the contents of each of which are incorporated herein by reference in their entirety.
  • Antibodies of the present disclosure may be monoclonal antibodies.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous cells (or clones), i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibodies, such variants generally being present in minor amounts.
  • polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody is directed against a single determinant on the antigen
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.
  • antibodies of the present disclosure may be humanized antibodies.
  • the term "humanized antibody” refers to a chimeric antibody including a minimal portion from one or more non-human (e.g., murine) antibody source(s) with the remainder derived from one or more human immunoglobulin sources.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which
  • Antibodies of the present disclosure may include antibody mimetics.
  • antibody mimetic refers to any molecule which mimics the function or effect of an antibody and which binds specifically and with high affinity to their molecular targets.
  • antibody mimetics may be monobodies, designed to incorporate the fibronectin type III domain (Fn3) as a protein scaffold (US 6,673,901; US 6,348,584).
  • antibody mimetics may be those known in the art including, but are not limited to affibody molecules, affilins, affitins, anticalins, avimers, Centyrins, DARPINSTM, Fynomers and Kunitz and domain peptides.
  • antibody mimetics may include one or more non-peptide regions.
  • antibodies of the present disclosure may include antibody variants.
  • antibody variant refers to a modified antibody (in relation to a native or starting antibody) or a biomolecule resembling a native or starting antibody in structure and/or function (e.g., an antibody mimetic).
  • Antibody variants may be altered in their amino acid sequence, composition or structure as compared to a native antibody.
  • Antibody variants may include, but are not limited to, antibodies with altered isotypes (e.g., IgA, IgD, IgE, IgGi, IgG2, IgG3, IgG4, or IgM), humanized variants, optimized variants, multispecific antibody variants (e.g., bispecific variants), and antibody fragments.
  • isotypes e.g., IgA, IgD, IgE, IgGi, IgG2, IgG3, IgG4, or IgM
  • humanized variants e.g., optimized variants, multispecific antibody variants (e.g., bispecific variants), and antibody fragments.
  • antibodies of the present disclosure may include “unibodies,” in which the hinge region has been removed from IgG4 molecules. While IgG4 molecules are unstable and can exchange light-heavy chain heterodimers with one another, deletion of the hinge region prevents heavy chain-heavy chain pairing entirely, leaving highly specific monovalent light/heavy heterodimers, while retaining the Fc region to ensure stability and half-life in vivo. This configuration may minimize the risk of immune activation or oncogenic growth, as IgG4 interacts poorly with FcRs and monovalent unibodies fail to promote intracellular signaling complex formation. Other antibodies may be “miniaturized” antibodies, which are compacted 100 kDa antibodies (see, e.g., Nelson, A. U, MAbs., 2010. Jan-Feb; 2(l):77-83).
  • antibodies of the present disclosure may bind more than one epitope.
  • the terms “multibody” or “multispecific antibody” refer to an antibody wherein two or more variable regions bind to different epitopes. The epitopes may be on the same or different targets.
  • a multi-specific antibody is a "bispecific antibody,” which recognizes two different epitopes on the same or different antigens.
  • antibodies of the present disclosure may include antibodies developed for antibody-drug conjugate (ADC) therapeutic applications.
  • ADCs are antibodies in which one or more cargo (e.g., therapeutic agents) are attached (e.g. directly or via linker).
  • ADCs are useful for delivery of therapeutic agents (e.g., drugs) to one or more target cells or tissues (Panowski, S. et ah, 2014. mAbs 6:1, 34-45).
  • ADCs may be designed to bind to a surface antigen on a targeted cell. Upon binding, the entire antibody-antigen complex may be internalized and directed to a cellular lysosome. ADCs may then be degraded, releasing the bound cargo.
  • antibodies of the present disclosure may be produced using one or more of the translated polypeptides listed in Table 1. Signal peptides are marked in bold. In mature antibodies, the signal peptide may be removed prior to assembly of the full antibody.
  • Some antibodies may be produced using translated polypeptides having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to any of those listed in the previous table. Some antibodies may be produced using fragments of one or more of the amino acid sequences listed in the previous table.
  • antibodies of the present disclosure may be produced using one or more of the nucleic acids listed in Table 2.
  • Some antibodies may include heavy or light chains encoded by one or more nucleic acids having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to any of those listed.
  • Some antibodies may be encoded by nucleic acids that include fragments and/or codon-optimized variants of one or more of the nucleic acid sequences listed.
  • Antibodies may be produced from constructs carrying one or more of these nucleic acids, their variants, and/or fragments thereof.
  • antibodies of the present disclosure may include translated variable domains amino acid sequences having one or more of the translated leader amino acid sequences listed in Table 3. Some antibodies may have translated variable domain amino acid sequences having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to any of the amino acid sequences listed.
  • antibodies of the present disclosure may include framework region (FR) amino acid sequences that include one or more of the amino acid sequences
  • FR1, FR2, FR3, and FR4 refer to framework regions in order of location from the N-terminus of a variable domain to the C-terminus of a variable domain. Framework regions are typically followed by complementarity determining regions (CDRs) with the exception of FR4, which typically lies after the last CDR in a variable domain.
  • CDRs complementarity determining regions
  • antibodies of the present disclosure may include one or more of the heavy chain variable domain (VH) and light chain variable domain (VL) amino acid sequences presented in Table 5.
  • Some antibodies may include at least one variable domain with an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to any of those listed.
  • Some antibodies may include variable domains having fragments or variants of one or more of the amino acid sequences listed.
  • antibodies of the present disclosure may be produced using one or more of the translated CDR polypeptides listed in Table 6.
  • CDR-L1, CDR-L2, and CDR-L3 refer to CDRs present on the light chain variable domain in order of position from the N-terminus of the variable domain to the C-terminus of the variable domain.
  • CDR-H1, CDR-H2, and CDR-H3 refer to CDRs present on the heavy chain variable domain in order of position from the N-terminus of the variable domain to the C-terminus of the variable domain.
  • Some antibodies may be produced using translated polypeptides having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to any of those listed. Some antibodies may be produced using fragments of one or more of the amino acid sequences listed.
  • antibodies of the present disclosure include a heavy chain variable domain (VH) having a CDR having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity to an amino acid sequence selected from any of the CDR sequences presented.
  • VH heavy chain variable domain
  • the heavy chain CDR is a CDR-H3.
  • Some antibodies include VH domains, wherein the CDR-H1, CDR-H2, and CDR-H3 are all selected from any of the heavy chain CDRs presented or variants thereof having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to one or more of the heavy chain variable domains presented.
  • antibodies of the present disclosure include a light chain variable domain (VL) having a CDR having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity to an amino acid sequence selected from any of the CDR sequences presented.
  • VL light chain variable domain
  • the light chain CDR is a CDR-L3.
  • Some antibodies include VL domains, wherein the CDR-L1, CDR-L2, and CDR-L3 are all selected from any of the light chain CDRs presented or variants thereof having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to one or more of the light chain variable domains presented.
  • antibodies of the present disclosure may include one or more of the constant region amino acid sequences listed in Table 7. Some antibodies may include constant domain amino acid sequences having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to any of those listed. Some antibodies may include constant domains that include fragments or variants of one or more of the amino acid sequences listed.
  • capture antibodies bind to epitopes that include or are present on hCD59 fragments and/or variants described in US Patent Number US10732186.
  • Such fragments and/or variants may include: FEHCNFNDVTTRLRENELTYYCCKKDL (SEQ ID NO: 46); FEHCNFND VTTRLRENELTYYCCKK (SEQ ID NO: 47);
  • ACNFND VTTRLRENELT YY C AAK-NFh (SEQ ID NO: 50, Ac is an acetyl group and NFb is an amine group); AFEHCNFND VTTRLRENELT YY C AAKDL (SEQ ID NO: 51); AFEHCNFND VTTRLRENELT YY 0(b A)KDL (SEQ ID NO: 52, bA is b-alanine); or AFEHCNFND VTTRLRENELTYYC(Aib)AKDL (SEQ ID NO: 53, Aib is alpha-amino-
  • these fragments and/or variants may include disulfide bonds between cysteine residues.
  • these fragments and/or variants may include or exclude N- and/or C-terminal groups (e.g., hydroxyl groups “-OH,” acetyl groups “Ac-” or amine groups “-NH2”).
  • capture antibodies are generated by immunizing mammals with any of these fragments and/or variants and isolating resulting antibodies from immunized mammals or from antibody-producing cells obtained from immunized mammals.
  • capture antibodies are produced recombinantly or synthetically from antibody amino acid or nucleic acid sequences obtained from immunized mammals or from antibody-producing cells obtained from immunized mammals. (0095) In some embodiments, detection antibodies bind to epitopes that include or are present on glycated CD59 fragments and/or variants described in US Patent Number US10732186.
  • Such fragments and/or variants may include: NKCWKFEHCNFNDV (SEQ ID NO: 54); WKFEH (SEQ ID NO: 55); NKAWKFEHANFND (SEQ ID NO: 56); Ac- NKAWKFEHANFNDC-OH (SEQ ID NO:57, Ac is an acetyl group and OH is a hydroxyl group).
  • these fragments and/or variants may include or exclude N- and/or C-terminal groups (e.g., hydroxyl groups “-OH,” acetyl groups “Ac-” or amine groups “-NH2”).
  • the fragments and/or variants may include glycated lysine residues. Glycated residues may include different arrangements of chemical bonds and stereochemical structures. Such residues may include structures or intermediate forms occurring during lysine glycation and/or rearrangement. Glycated residues may include any of structures I- VII shown below.
  • detection antibodies are generated by immunizing mammals with any of the fragments and/or variants of glycated CD59 presented above and isolating resulting antibodies from immunized mammals or from antibody-producing cells obtained from immunized mammals. In some embodiments, detection antibodies are produced recombinantly or synthetically from antibody amino acid or nucleic acid sequences obtained from immunized mammals or from antibody-producing cells obtained from immunized mammals.
  • present disclosure provides compounds for modulating glycation of CD59, referred to herein as “glycation modulators.”
  • Glycation modulators may include, but are not limited to, small molecules, peptides, synthetic constructs, fusion proteins, aptamers, nucleic acids, and antibodies. Some glycation modulators may modulate glycation of residue K41 of hCD59. Some glycation modulators may modulate CD59 glycation without inhibiting the function of CD59 as a complement regulator.
  • K41 glycation inhibitors Glycation modulators inhibiting glycation of residue K41 of hCD59 are referred to herein as “K41 glycation inhibitors.”
  • K41 glycation inhibitors may, for example, bind to or otherwise interact with K41 or one or more residues near K41 (e.g., near K41 along the length of the polypeptide and/or near K41 due to secondary and tertiary protein structures).
  • K41 glycation inhibitors may physically block glycation at K41 or may alter the hCD59 protein conformation in a way that prevents K41 from becoming glycated.
  • Some K41 glycation inhibitors may bind to residue H44 of hCD59 and block chemical reactions necessary forK41 glycation.
  • K41 glycation inhibitors may include antibodies disclosed herein. Such antibodies may physically block glycation at K41 or may alter the hCD59 protein conformation in a way that prevents K41 from becoming glycated. Some antibodies may inhibit K41 glycation, for example, by targeting H44 of human hCD59. In some embodiments, such antibodies may block chemical reactions necessary for K41 glycation.
  • Methods of the disclosure include methods of detecting the presence, measurement of levels, and or changes in levels of various factors described herein. Such factors may include, but are not limited to, target proteins, post-translational modifications, and standard agents. “Levels” may refer to an actual number of factors detected, a concentration of a factor, or a relative level (e.g., through comparison of detection signals between a detected factor and a surrogate factor or standard agent). Such methods may include the use of capture agents to capture CD59.
  • the term “capture agent” refers to any compound capable of binding an assay component (e.g., assay target protein or compound). Capture agents may include capture antibodies. Capture antibodies may include any of the antibodies described herein. In some cases, capture agents are lectins capable of binding glycosylated assay components.
  • detection agents refers to an assay component capable of binding or otherwise indicating the presence and/or level of an analyte.
  • detection agents are antibodies (detection antibodies).
  • Detection agents may include antibodies used to detect GCD59.
  • Detection antibodies may include any antibodies described herein.
  • Further detection agents may include lectins capable of detecting glycosylated assay components.
  • Detection agents may include detectable labels. Detectable labels may include, but are not limited to biotin, streptavidin, avidin, fluorescent labels, enzymatic labels, luminescent labels, and radioactive labels.
  • detection agents may be detected using a secondary antibody.
  • methods of the present disclosure may include the use of other antibodies known in the art for capturing or detecting CD59 and/or GCD59.
  • Such antibodies may include any of those described and/or claimed in European Patent Number EP2348050, International Publication Number WO2015084994, and US Patent Numbers US9068006, US9417248, and US10732186, the contents of each of which are herein incorporated by reference in their entireties.
  • Methods may include standard immunological assay formats for capturing proteins and detecting one or more specific epitopes present on such proteins. Such epitopes may include post-translationally modified epitopes. Post-translationally modified epitopes may include glycated and/or glycosylated epitopes. 0103 Methods may include immunological assays. As used herein, an “immunological assay” refers to any assay which utilizes at least one antibody for detection of an analyte.
  • Immunological assays may include, but are not limited to, enzyme linked immunosorbent assays (ELISAs), immunoprecipitation assays, immunofluorescence assays, enzyme immunoassays (EIA), radioimmunoassays (RIA), and Western blot analysis.
  • Methods of the present disclosure may include the use of surface-associated formats or solution-based formats.
  • surface-associated format refers to a method that includes immobilization of one or more assay components on a surface. Such surfaces may include, but are not limited to assay plates, membranes, sensors, or other substrates that include a surface.
  • the surface-associated format may include a magnetic interaction surface, bead or coated magnetic beads. In some embodiments, they may include the use of ferromagnetic labels.
  • solution-based format refers to a method that includes immobilization of one or more assay components on a substrate that is able to move freely in
  • Substrates suitable for solution-based formats may include beads or other particles that are mobile in liquid media. Methods utilizing solution-based formats may be analyzed by forcing liquid media through tubes, channels, or other passageways where detection of bound analytes may be carried out. Assay components may be immobilized on surface or solution-based format substrates in varying levels and/or coating densities. Immobilization may be facilitated through the formation of any number of interactions that include, but are not limited to, covalent bonds, non-covalent bonds, hydrogen bonds, and hydrophobic bonds.
  • attachment agent refers to any substance used to adhere an assay component to a substrate.
  • attachment agents include biotin or biotin-binding agents.
  • Biotin is a small water-soluble compound that is known to form specific high affinity bonds with avidin and streptavidin (Barat, B. et al. 2007. Biomol Eng. 24(3): 283-91).
  • biotin-binding agent refers to any substance capable of forming strong and specific interactions with biotin.
  • Biotin-binding agents may include, but are not limited to, avidin, streptavidin, NeutrAvidin, and CAPTAVIDINTM. Substances may be conjugated with one or more biotin molecules (a process referred to as “biotinylation”). Biotinylation may be carried out through chemical or enzymatic processes. Chemical processes utilize activated biotin derivatives to form covalent bonds with target compounds. Enzymatic biotinylation utilizes biotin conjugating enzymes referred to as “biotin ligases” to link biotin with proteins capable of interacting with such ligases.
  • biotinylation includes one or more of carboxyl biotinylation, enzymatic biotinylation, sulfhydral biotinylation, primary amine biotinylation, glycoprotein biotinylation, non-specific biotinylation, and long-arm biotinylation.
  • methods of the disclosure include the use of a capture antibody to immobilize CD59 to a surface or substrate. Such methods may further include the use of a detection agent (e.g., lectin or detection antibody) to detect a specific epitope present on CD59.
  • a detection agent e.g., lectin or detection antibody
  • Such epitopes may include glycated and/or glycosylated epitopes.
  • Glycated epitopes may include glycated K41 of hCD59.
  • Such epitopes may include Amadori -modified K41 of GCD59.
  • Glycosylated epitopes may include N-glycosylated epitopes on hCD59 and/or GCD59.
  • Detection of N-glycosylated epitopes may be carried out using a lectin as a detection agent, wherein the lectin binds to such N-glycosylated eptitopes (e.g., an N- glycosylated residue of GCD59).
  • detection of N-glycosylated epitopes may be carried out using a detection antibody.
  • anti-Amadori -modified GCD59 antibodies may be used as a capture antibody to immobilize GCD59 to a surface or substrate. Such methods may further include the use of a detection antibody that recognizes a non-glycated epitope of GCD59. Some methods may include the use of a detection antibody that recognizes a glycosylated epitope of GCD59. Such epitopes may include an N- glycosylated epitope of GCD59.
  • Methods of the disclosure may include the use of one or more internal controls.
  • Such internal controls may include plasma assay controls.
  • Some methods include one or more of an assay diluent, a conjugate diluent, and a wash buffer.
  • methods include at least one buffer having one or more of reducing agents, oxidizing agents, dithiothreitol (DTT), and/or beta-mercaptoethanol (BME).
  • methods of the present disclosure may include the use of one or more of the antibodies described herein to carry out any of the methods described in any of European Patent Number EP2348050, International Publication Number WO2015084994, and US Patent Numbers US6835545, US9068006, US9417248, and US10732186, the contents of each of which are herein incorporated by reference in their entireties.
  • Methods of determining concentration levels may include comparison to a standard curve to determine concentrations of a target protein.
  • a standard curve is a set of data obtained from samples having a range of concentrations of a standard agent.
  • a “standard agent” is an agent that is the same as or representative of a target protein being detected in an assay.
  • standard agents are surrogate compounds.
  • a “surrogate compound” is a compound that exhibits one or more features of a target protein or other molecule. Such features may include epitopes present on one or more target proteins.
  • surrogate compounds include CD59 fragments or variants thereof.
  • surrogate compounds include fragments that include SEQ ID NO: 50.
  • capture agents bind to such fragments.
  • surrogate compounds include glycated CD59 fragments or variants thereof. Glycated fragments may include SEQ ID NO: 57. Such fragments may include glycated lysine residues. In some embodiments, the glycated lysine residue includes glucitollysine. In some embodiments, the glycated lysine residue includes Amadori-modified glycated lysine.
  • detection agents bind to such fragments.
  • Surrogate compounds may include one or more non-protein components.
  • Such non-protein components may include linkers, labels (e.g., detectable labels), moieties, or other features not present on native or
  • surrogate compounds may include any of those disclosed in U.S. Patent No. 9,417,248 or U.S. Publication No. US2016/0299150, the contents of each of which are herein incorporated by reference in their entirety. Such surrogate compounds may include the compound depicted in Fig. 27 of U.S. Patent No. 9,417,248.
  • concentration equivalents of a target protein are used to indicate the concentration of a target protein.
  • a “concentration equivalent” is an indicator of concentration that is based on a comparison to a standard curve generated using a surrogate compound.
  • methods of diagnosing, screening, and/or monitoring described herein may include comparison of factor levels from a subject to a threshold value or receiver operating characteristic (ROC) curve.
  • ROC receiver operating characteristic
  • methods of the present disclosure include methods of detecting GCD59 in samples by: (1) applying the samples to a substrate, wherein the substrate includes immobilized capture antibodies directed to a glycated epitope of GCD59; (2) applying detection antibodies to the substrate, wherein the detection antibodies are directed to a non-glycated epitope of GCD59; and (3) detecting the detection antibodies.
  • the samples may include, but are not limited to one or more cell, tissue, tissue section, or body fluid (e.g., urine, blood, sweat, serum, plasma, lymph, and saliva).
  • the glycated epitope may include glycated K41.
  • the glycated K41 may be Amadori-modified glycated K41.
  • the glycated K41 may be reduced glycated K41.
  • the glycated epitope may include the amino acid sequence WKFEH (SEQ ID NO: 55), or a portion thereof.
  • the glycated epitope may include the amino acid NKCWKFEHCNFNDV (SEQ ID NO: 54), or a fragment thereof.
  • Capture antibodies may include antibodies D2 or D3 or include one or more variable domain or CDR thereof.
  • Capture antibodies may be antibody fragments.
  • Antibody fragments may be antigen binding fragments (Fab).
  • Capture antibodies may be immobilized to substrates through one or more attachment agents. Such attachment agents may include biotin or biotin binding agents. Capture agents may be biotinylated.
  • Capture antibodies may be biotinylated with one or multiple biotin molecules.
  • Biotinylated capture antibodies may be immobilized on substrates, wherein the substrates are associated with one or more biotin-binding agents.
  • the biotin binding agents may include one or more of avidin, streptavidin, NeutrAvidin and CAPTAVIDINTM.
  • substrates are biotinylated and capture antibodies are associated with one or more biotin-binding agents. The non-glycated epitope detected
  • Detection antibodies may include H9, H9 fragments, or antibodies with one or more variable domains or CDRs of H9. Detection antibodies may be detected directly or be detected using a secondary detection agent (e.g., a secondary antibody). Detection antibodies or secondary detection agents may include detectable labels. Detectable labels may include, but are not limited to biotin, biotin-binding agents, fluorescent labels, enzymatic labels, or radioactive labels. Enzymatic labels may include horse radish peroxidase.
  • Substrates with immobilized capture antibodies directed to glycated GCD59 epitopes may be prepared by adhering biotin-binding agents to the substrates; biotinylating the capture antibodies; and contacting the adhered biotin-binding agents with the biotinylated capture antibodies for substrate immobilization.
  • Substrates used for capture antibody immobilization may include, but are not limited to, assay surfaces, assay plates, beads, membranes, conducting surfaces, and conducting nanoparticles. Substrates may be coated with biotin-binding agents.
  • the biotin-binding agent is streptavidin.
  • Capture antibody biotinylation may be carried out by one or more of chemical biotinylation, enzymatic biotinylation, sulfhydral biotinylation, primary amine biotinylation, glycoprotein biotinylation, non-specific biotinylation, and long-arm biotinylation.
  • Detection antibodies may be detected through observation of detection signals associated with detectable labels. Detection may include measurement of detection signal strength. Detection signal strength measurements may be used to determine GCD59 concentration or concentration equivalent in samples.
  • the present disclosure provides methods for therapeutic, diagnostic, and monitoring applications. Such methods may include the use of one or more antibodies described herein. Such antibodies may include, but are not limited to, anti- Amadori-modified GCD59 antibodies.
  • methods of the present disclosure include therapeutic applications that may be used to treat, prevent, and/or reduce the occurrence or symptoms of one or more diseases and/or disorders in a subject.
  • a “subject” is any person, individual, fetus, newborn, infant, animal, and/or patient. Subjects may include gestational subjects.
  • a “gestational subject” is a subject residing within a womb, although the term may be used to refer to a subject over a period of time that includes both periods
  • a subject is within and outside of a womb.
  • diagnosis of a gestational subject may refer to a diagnosis that is confirmed at the time of or after birth.
  • a “pregnant subject” or “gestational carrier” refers to an individual that bears a gestational subject in the subject’s womb and includes individuals with or without a biological relationship with the gestational subject.
  • the term “infant subject” refers to subjects who are infants and embraces subjects from birth to about 1 year of age.
  • Subjects may be undergoing treatment, may be in need of treatment, may have undergone treatment, or may be screened or stratified for possible or potential treatments.
  • Subjects may include animals, e.g., primates, non-human primates or any animals such as farm animals and the like.
  • methods of the present disclosure may be used to treat, prevent, and/or reduce the occurrence or symptoms of one or more diabetes-related indications.
  • diabetes-related indication refers to any disease, disorder, complication, and/or condition related to elevated blood glucose levels, decreased cellular glucose uptake, reduced insulin levels, or reduced insulin sensitivity. Diabetes-related indications may include, but are not limited to, diabetes and pre-diabetes.
  • the present disclosure provides methods of diagnosing, monitoring, screening, and/or treating one or more diabetes-related indications that may include, but are not limited to, indications related to complement dysfunction, hemolytic disease, paroxysmal nocturnal hemoglobinuria, atypical hemolytic-uremic syndrome, wound healing, complications associated with organ transplantation, vascular indications, and age- related macular degeneration.
  • diabetes-related indications may include a pre-clinical diabetes-related indication.
  • Pre-clinical diabetes-related indications may include, but are not limited to, predisposition to an organ specific complication of diabetes, pre-clinical diabetic peripheral neuropathy, pre-clinical diabetic nephropathy, pre-clinical diabetic retinopathy, and pre-clinical diabetic vascular disease.
  • Diabetes is a disease characterized by elevated blood glucose levels, also referred to as hyperglycemia.
  • Insulin along with other hormones including, but not limited to glucagon and epinephrine, is critical for maintenance of normal glucose levels in the blood. Insulin binding to cellular receptors facilitates cellular uptake of glucose, providing an energy source for cells and lowering glucose levels in the blood (Rodger, W., CMAJ. 1991.
  • Insulin is expressed by pancreatic b cells and its expression is upregulated
  • Type II diabetes insulin-dependent diabetes
  • Type II diabetes insulin-independent diabetes
  • Type I diabetes is less common and typically brought on by autoimmune destruction of b cells, the primary source of insulin. 90% or more of those with diabetes suffer from Type II diabetes.
  • This form of the disease is characterized by reduced insulin secretion and/or reduced sensitivity to insulin (e.g., reduced ability of insulin to stimulate glucose uptake in cells) (Rodger, W., Non-insulin-dependent (Type II) diabetes mellitus.
  • diabetes refers to an individual with one or more types of insulin deficiency (e.g., reduced insulin levels and/or reduced insulin sensitivity).
  • diabetes includes, but is not limited to, individuals with juvenile diabetes (Type I diabetes), adult-onset diabetes (Type II diabetes), gestational diabetes mellitus (GDM), and any other conditions of insulin deficiency.
  • diabetic is a term of art, known and understood by those practicing in the medical profession, a formal definition of which can be found in Harrison's Principles of Medicine (Harrisons, Vol 14, Principles of Internal Medicine, Eds. Fauci, A. S., E. Braunwald, K. J. Isselbacher, J. D. Wilson, J. B. Martin, D. L. Kasper, S. L. Hauser, D. L. Longo, McGraw-Hill, New York, 1999).
  • GDM Gestational diabetes mellitus
  • GDM gestational diabetes mellitus
  • the terms “gestational diabetes mellitus” or “GDM” refer to a diabetes-related indication characterized by elevated blood glucose levels, carbohydrate intolerance, and/or reduced insulin sensitivity that is brought on by pregnancy.
  • GDM diagnosis in each country may rely on different standards set by the professional bodies from such countries that issue recommendations to physicians practicing there. GDM may affect up to 18% of pregnancies with adverse outcomes that affect both the mother and offspring, including both short term and long term effects.
  • GDM is one of the most prevalent disorders affecting pregnant women and carries with it a greater risk for complications during pregnancy, at the time of birth and even after birth. Additionally, such complications may affect both mother and offspring. Individuals with GDM lack the ability to adequately break down carbohydrates into energy (Okun, N., Can. Fam. Physician. 1997. 43:88-93). In some cases, GDM diagnosis may be carried out through the detection of high blood glucose levels and/or through the observation of a decreased ability to respond to a glucose challenge during pregnancy. Such diagnosis occurs most often in the third trimester. Although mechanisms leading to GDM are still unclear, in some cases, it is believed that hormones that become elevated during pregnancy may interfere with normal insulin signaling, including, but not limited to insulin resistance. This insulin signaling dysfunction leads to decreased cellular glucose uptake and elevated blood glucose levels. GCD59 levels may be elevated in subjects displaying different levels of glucose intolerance including those diagnosed with GDM.
  • GDM is one of the most common medical complications of pregnancy. In each country, GDM diagnosis may be determined by standards set by professional bodies responsible for issuing recommendations to practicing physicians. There is ongoing debate among professional bodies within the United States as well as between professional bodies in the United States and those abroad as to how to approach GDM diagnosis.
  • Such US professional bodies may include, but are not limited to, the National Institutes of Health (NIH), the American Diabetes Association (ADA) and the American Congress of Obstetricians and Gynecologists (ACOG).
  • Such International bodies may include, but are not limited to, the International Association of Diabetes and Pregnancy Study Groups (IADPSG). Professional bodies in the US and abroad may tailor their approaches based on different studies, different analysis of such studies and may be affected by health care and economic pressures.
  • the ACOG recommends screening all pregnant women for GDM by measuring blood glucose levels (Committee on Practice Bulletins - Obstetrics, 2017).
  • factors for defining GDM and criteria for diagnosis may change over time.
  • methods of the present disclosure, described herein may be conducted in accordance with the most current recommendations issued by professional bodies in each country involved in the health of pregnant women, fetuses, and newborns.
  • glucose challenge or “glucose load” refers to a testing component characterized by the administration of glucose to a subject.
  • Glucose challenge testing typically assesses the response in subjects to a glucose challenge. This may include analyzing blood glucose levels. The amount of glucose administered during a glucose challenge may vary. Typical tests include the administration of from about 50 g to about 100 g of glucose. In other embodiments, 75 g of glucose are administered. In some cases, plasma glucose levels may be assessed after one or more glucose challenge or after a meal.
  • methods of the present disclosure related to GCD59 detection may be carried out with subjects with or without fasting. Some methods may be carried out with subjects with or without glucose challenge.
  • screening is typically performed at gestation weeks 24 to 28 by glucose challenge.
  • the testing may include 1-hour venous glucose measurement after consumption of an oral glucose solution with 50 g of sugar.
  • “Low risk” pregnant subjects at the lowest risk of developing GDM, include those who are less than 25 years old, have normal body mass index, have no family history of diabetes (at the level of first-degree relatives,) do not have a history of abnormal glucose metabolism, do not have a history of poor obstetric outcome and are not of a high risk ethnicity (e.g. Hispanic, Native American, African American and South Asian).
  • Pregnant subject risk assessment is typically carried out during a first prenatal visit. Women at higher risk of developing GDM (e.g., obese, personal history of GDM, glycosuria, family history of diabetes, etc.) typically undergo testing as soon as possible.
  • High plasma glucose levels may be indicative of GDM in the absence of a glucose challenge.
  • Some methods disclosed herein include fasting glucose tests. According to such tests, fasting plasma glucose (FPG) levels may be determined.
  • FPG fasting plasma glucose levels refer to levels of glucose measured directly after a period of fasting. Periods of fasting may be from about 1 hour to about 24 hours. In certain embodiments, FPG levels may be measured after about 12 hours of fasting.
  • methods described herein for assessing GCD59 in subjects may be carried out in combination with assessments of FPG. Some methods of the present disclosure may be carried out prior to or in response to determinations of FPG levels falling within or outside of GDM diagnosis levels recognized by the ADA and/or IADPSG.
  • random glucose tests may be conducted. Such tests measure random plasma glucose levels (also referred to as casual plasma glucose levels). Random glucose levels refer to glucose levels obtained without any consumption restrictions and/or requirements (e.g., fasting). In pregnant subjects, random plasma glucose levels that are greater than 200 mg/dl indicate GDM in such subjects. In certain embodiments, a second measurement is required the next day for both FGP levels and random plasma levels to confirm diagnosis of GDM. In some embodiments of the present disclosure, GCD59 levels may also be obtained without consumption restrictions and/or requirements (e.g., fasting). [0129] In cases where hyperglycemia is subtle, other approaches may be necessary for diagnosis. In pregnant subjects identified as being high risk, the one-step approach may be sufficient.
  • diagnosis may be carried out by oral glucose tolerance testing (OGTT) without any prior blood glucose screening.
  • OGTT oral glucose tolerance testing
  • the two-step approach is typically carried out.
  • an initial screening including a glucose challenge is carried out.
  • Recommendations by the American College of Obstetricians made in 2001 call for a 50 g, one-hour oral glucose challenge test (GCT) to be used (Committee on Obstetric Practice, The American College of Obstetricians and Gynecologists: Committee Opinion. 2011).
  • GCT oral glucose challenge test
  • the one hour oral GCT measures blood glucose concentrations 1 hour after the oral administration of 50 g of glucose.
  • cut-off value refers to a value or level at which an indication may be made with regard to a diagnostic determination or other type of determination, wherein a level below a given cut-off leads to a determination that is different from a determination based on a level above a given cut-off (American Diabetes Association, Diabetes Care. 31(1): S62-S67).
  • a 100 g OGTT may be carried out.
  • oral glucose tolerance test or “OGTT” refers to a test that measures the ability of the body to utilize glucose. Such testing typically begins in the morning,
  • a baseline concentration is established based on an initial blood sample.
  • baseline when referring to measurements, levels or values refers to an initial measurement, level or value to which subsequent measurements, levels or values may be compared.
  • subjects are given a glucose solution to drink with a measured concentration of glucose.
  • 100 g of glucose is administered in the glucose solution.
  • Subjects are typically required to finish the drink within a 5-minute time frame.
  • OGTTs include the obtaining of subsequent blood samples to monitor blood glucose and/or insulin levels.
  • diagnosis of GDM in pregnant subjects may be made when subject blood glucose levels exceed a cut-off value of 95 mg/dl for baseline readings, a cut-off value of 180 mg/dl one hour after glucose administration, a cut-off value of 155 mg/dl two hours after glucose administration and/or a cut-off of 140 mg/dl three hours after glucose administration.
  • a diagnosis of GDM may require that two out of four tests yield elevated blood glucose levels(e.g., according to any of the assessments described herein).
  • a 75 g OGTT is carried out in the second step of the two step approach to GDM diagnosis.
  • the 75 g OGTT may be carried out according to the 100 g OGTT with the exception that only 75 g of glucose is administered.
  • diagnosis of GDM in pregnant subjects may be made when subject blood glucose levels exceed a cut-off value of 95 mg/dl for baseline readings, a cut-off value of 180 mg/dl one hour after glucose administration and/or a cut-off value of 155 mg/dl two hours after glucose administration.
  • a diagnosis of GDM may require that two out of four tests yield elevated blood glucose levels.
  • the 2-hour postprandial glucose test is carried out during GDM screening.
  • 2-hour postprandial glucose testing includes the analysis of blood glucose levels 2 hours after a meal.
  • 1,5-anhydroglucitol testing may be carried out during GDM screening. Levels of 1,5-anhydroglucitol levels are reduced during periods of hyperglycemia (wherein blood glucose levels are above 180 mg/dl), requiring up to 2 weeks to return to normal after hyperglycemic conditions have ended (McGill, J.B. et ah, Diabetes Care. 2004. 27(8): 1859-65). 1,5-anhydroglucitol testing may be done to determine whether subject have endured extended periods of hyperglycemia.
  • hemoglobin A1 c (HbAlc) testing may be carried out during GDM screening. Such testing measures the level of a glycated version of hemoglobin, HbAlc in
  • HbAlc levels increase during periods of hyperglycemia. HbAlc remains in the blood from about 8 to about 12 weeks until red blood cells with HbAlc are replaced, making HbAlc a good longer term reading of overall blood glucose levels during that period (http://medweb.bham.ac.uk/easdec/prevention/what_is_the_hbalc.htm).
  • fructosamine testing may be carried out during GDM screening.
  • Fructosamine levels become elevated under hyperglycemic conditions. Elevated levels of fructosamine remain elevated for two to three weeks after hyperglycemic conditions subside, making them a good longer term indicator of high blood glucose levels (Delpierre,
  • subject samples may be obtained and analyzed prior to pregnancy.
  • the positive and negative predictive values of GCD59 to identify pregnant women with normal or abnormal GLT were 99.3% (95% Cl: 97.9, 99.8) and 87.5% (95% Cl: 84.5, 90.1), respectively, when using a cut-off that maximized sensitivity and specificity.
  • some methods of the present disclosure include analyzing subject samples for GCD59 levels to determine a level of risk for developing GDM and/or pre eclampsia in a current and/or future pregnancy.
  • methods of the present disclosure may be combined with any of the tests described herein. Such tests may include, but are not limited to glucose challenge testing, oral glucose tolerance testing, fasting glucose testing, random glucose testing, 2-hour postprandial glucose testing, hemoglobin Ale (HbAlc) testing, fructosamine testing and 1,5-anhydroglucitol testing. In some embodiments, methods of the present disclosure may be combined with such tests for the purposes of diagnosis, prognosis and/or monitoring of GDM or other diabetic conditions. In some embodiments, methods of the present disclosure may be used for post-partum screening for type-2 diabetes.
  • methods of the present disclosure may be combined with detection of other glycated proteins.
  • Many other proteins present within bodily fluids include amino groups that may be capable of being glycated. Such proteins may include glycated albumin, glycated hemoglobin, glycated immunoglobulins, glycated hemopexin, glycated vitamin D binding protein, glycated fibrinogen alpha chain, glycated apolipoprotein Al, glycated transferrin, glycated macroglobulin alpha 2, glycated complement component 4A, glycated fibrinogen beta chain, glycated fibrinogen alpha chain, glycated abhydrolase
  • GCD59 may be detected as part of a panel or array of biomarkers including any of the glycated proteins listed above.
  • pregnant subjects may be placed into different subcategories of disease based on certain criteria.
  • Two such categories include those with impaired glucose tolerance (IGT) and those with impaired fasting glucose (IFG). These categories are designated for subjects whose glucose levels are above normal, but do not rise to the level of GDM or that fall short of the requirement for GDM diagnosis. Factors determining placement of subjects into such categories may be different for each country and may be controlled by professional bodies in such countries responsible for providing recommendations to physicians practicing in such countries.
  • pregnant subjects may be diagnosed with IFG when fasted glucose levels in such subjects include from about 100 mg/dl to about 125 mg/dl as compared to those with normal fasted glucose levels (e.g., less than 92 mg/dL according to assessments described previously) and those whose levels lead to a provisional diagnosis of GDM [in some cases with levels > 92 mg/dL (5.1 mmol/L), or with levels > 92 mg/dL (5.1 mmol/L) but ⁇ 126 mg/dL (7.0 mmol/L)]. In some cases, pregnant subjects may be diagnosed with IGT after OGTT results.
  • pregnant subjects with IGT may have blood glucose levels from about 140 mg/dl to about 199 mg/dl two hours after glucose administration as compared to those with normal levels (in some cases with levels less than 140 mg/dl) and those whose levels lead to a provisional diagnosis of GDM (in some cases with levels greater than 200 mg/dl).
  • pre-diabetes refers to a condition characterized by high risk for developing diabetes (American Diabetes Association, Diabetes Care. 2008. 31(1): S62-S67). Factors determining designation of subjects into the category of pre-diabetes may be different for each country and may be controlled by professional bodies in such countries responsible for providing recommendations to physicians practicing in such countries.
  • pregnant subjects suffering from GDM may be assigned to a category that includes a class of GDM developed by Dr. Priscilla White, referred to herein as “White’s GDM class” (Dunn, P.M., Dr. Priscilla White (1900-1989) of Boston and pregnancy diabetes. Arch Dis Child Fetal Neonatal Ed. 2004 May; 89(3): F276-8, herein incorporated by reference in its entirety).
  • GDM classes may include any of those listed in Table 8.
  • White’s GDM classes include Class Al, Class A2, Class B, Class C, Class D,
  • GDM may be categorized according to two or more levels of GDM severity.
  • level of GDM severity refers to a category of disease characterized by different levels of complications or negative outcomes, typically from less severe to more severe. GDM severity may be assigned based on the level of one or more factors that correlate with such complications or negative outcomes. In other embodiments, GDM severity may be assigned based on the metabolism of blood glucose. GDM severity may also be determined by levels of GCD59. In such embodiments, mild, moderate and severe GDM levels may be assigned to subjects depending on where
  • GDM Gdid fever
  • symptoms typically include, but are not limited to thirst, fatigue, nausea, vomiting, bladder infection, yeast infection and blurred vision (http://www.nlm.nih.gov/medlineplus/ency/article/000896.htm).
  • Preliminary indications of disease typically involve test results (e.g., elevated glucose levels, elevated levels of glycated proteins).
  • Risk factors for GDM may include, but are not limited to elevated body mass index (BMI), family history of diabetes or GDM, advanced maternal age, a history of polycystic ovary syndrome, a history of smoking, a history of obstetric issues, high cholesterol, short stature and ethnicity (Ross, G., Australian Family Physician. 2006. 35(6):392-6; Bjorge, T. et ah, Am J Epid. 2004. 160(12): 1168-76; Ma, R.M. at ah, Diabetes Care. 2007. 30(11):2960-1). In some cases, the presence or absence of risk factors may influence one or more course of action with regard to testing and/or treatment of female subjects.
  • BMI body mass index
  • body mass index refers to a number calculated from a subject’s weight and height that correlates with the level of body fat of a given subject. This value is obtained from a subject by dividing the weight of the subject in kilograms by (height) 2 in meters.
  • BMI values may be interpreted as follows: below 18.5 kg/m 2 - underweight; 18.5 kg/m 2 - 24.9 kg/m 2 - normal; 25.0 kg/m 2 - 29.9 kg/m 2 - overweight; 30.0 kg/m 2 - 34.9 kg/m 2 - grade I obesity; 35.0 kg/m 2 - 39.9 kg/m 2 - grade II obesity and above 40 kg/m 2 - grade III obesity.
  • subjects who are overweight have a 2.14-fold increased risk of developing GDM (Yessoufou, A. et ah, Experimental Diabetes Research. 2011. 2011:1-12).
  • Subjects who are obese have a 3.56- fold increased risk of developing GDM and subjects who are severely obese have an 8.56- fold increased risk of developing GDM.
  • BMI interpretations may be different in each country and may be determined by professional bodies responsible for setting guidelines for physicians practicing within such countries and/or governing bodies.
  • Pregnant subjects with a history of pre-diabetes and/or GDM have a higher risk of developing GDM. Additionally, pregnant subjects with a family history of diabetes, pre diabetes and/or GDM have a higher risk of developing GDM. Subject history and/or family history are typically reviewed during the first prenatal appointment. In some embodiments,
  • 55 subject history and/or family history may be used to make decisions about subject testing and/or treatment.
  • 10.149 j Advanced maternal age is also a risk factor for developing GDM.
  • the percent of pregnant subjects with GDM varies among different age groups (Ross, G., Australian Family Physician. 2006. 35(6):392-6).
  • About 1% of pregnant subjects under 20 develop GDM during pregnancy, while about 1.8% of pregnant subjects from ages 20 to 24 develop GDM, about 2.5% of pregnant subjects from ages 25 to 29 develop GDM, about 4.1% of pregnant subjects from ages 30 to 34 develop GDM, about 6.5% of pregnant subjects from ages 35 to 39 develop GDM, about 9.8% of pregnant subjects from ages 40 to 45 develop GDM and about 12.8% of pregnant subjects over 45 develop GDM.
  • Rates of GDM are also influenced by ethnicity with higher incidence in pregnant subjects who are African American, Native American, Hispanic and South Asian (including, but not limited to Pacific Islanders) (Kim, S.Y. et ah, Prev Chronic Dis. 2012. 9: E88).
  • GDM is a major cause of peri- and post-natal complications for mothers and their offspring.
  • Gestational carriers afflicted with GDM may have or be at risk for developing one or more GDM-related conditions.
  • a “GDM-related condition” is any disorder in a gestational carrier or gestational subject associated with or resulting from GDM.
  • GDM- related conditions in gestational carriers may include, but are not limited to complications at delivery, pre-term birth, increased number of C-sections, risk of pre-eclampsia/eclampsia, miscarriage and/or post-pregnancy diabetes.
  • Gestational subjects and/or infant subjects bom to gestational carriers afflicted with GDM may have or be at risk for developing one or more GDM-related conditions that include, but are not limited to, macrosomia, large for gestational age (LGA), birth defects, birth trauma, hyperbilirubinemia, hypoglycemia, respiratory distress syndrome, future diabetes, cardiometabolic disease, seizures, and still birth.
  • methods of the present disclosure used to diagnose, assess, monitor, or stratify risk e.g., assigning a level of risk to one or more subjects
  • for GDM in a gestational carrier may be used to identify gestational subjects and/or infant subjects at risk for developing one or more GDM-related conditions.
  • GDM-related conditions affecting gestational and/or infant subjects may include macrosomia.
  • macrosomia refers to a condition in infant subjects characterized by large birth weight.
  • Large birth weight refers to birth weights above about 8 pounds, 13 ounces or roughly above 4 kg.
  • GDM-related conditions may include large for gestational age (LGA).
  • LGA Large for gestational age refers to a condition characterized by birthweights that exceed the 90 th percentile at a given gestational age. Although threshold weights may vary for this, generally, babies larger than about 8 pounds and 13 ounces when born at full term fall into the category of LGA. Where a gestational carrier has GDM, LGA may result from excess insulin produced by the fetus in response to high blood glucose levels in the gestational carrier that leads to greater than normal growth and subsequently elevated birth weight. In some embodiments, methods of the present disclosure used to diagnose, assess, monitor, or stratify risk for GDM may be used to identify gestational subjects at risk for developing LGA.
  • Pregnancy-related hypertensive disorders such as pre-eclampsia have also been shown to be related to GDM (Feig, D.S. et al., PLoS Med. 2013. 10(4): el001425.)
  • GDM GDM
  • Pre eclampsia is a serious medical condition in pregnant subjects characterized by elevated blood pressure and proteinuria (protein in the urine).
  • the risk of pre-eclampsia increases with intolerance to glucose.
  • pregnant subjects with pre-eclampsia have been shown to have a higher incidence of insulin resistance.
  • methods of the present disclosure used to diagnose, assess, monitor, or stratify risk for GDM may be used to diagnose, assess, monitor and/or stratify risk for developing pre-eclampsia.
  • the length of pregnancy may be divided into two or more gestational windows.
  • the term “gestational window” refers to any temporally, developmentally and/or physiologically defined period of a pregnancy. Gestational windows may include weeks of pregnancy. The typical term of a human pregnancy is from about 40 to about 42 weeks (but may extend beyond 42 weeks in some cases) and is calculated starting with the end of the last menstrual cycle of a pregnant subject.
  • gestational windows may include from about 0 to about 46, from about 0 to about 42, from about 2 to about 42, from about 4 to about 42, from about 8 to about 42, from about 12 to about 42, from about 16 to about 42, from about 20 to about 42, from about 24 to about 42, from about 28 to about 42, from about 32 to about 42, from about 36 to about 42, from about 12 to about 36, from about 16 to about 36, from about 20 to about 36, from about 24 to about 36, from about 10 to about 28, from about 16 to about 28, from about 20 to about 28, from about 16 to about 24, or from about 18 to about 24 weeks of pregnancy.
  • gestational windows may include week 1, week 2, week 3, week 4, week 5, week 6, week 7, week 8, week 9, week 10, week 11, week 12, week 13, week 14, week 15, week 16, week 17, week 18, week 19, week 20, week 21, week 22, week 23, week 24, week 25, week 26, week 27, week 28, week 29, week 30, week 31, week 32, week 33, week 34, week 35, week 36, week 37, week 38, week 39, week 40, week 41, week 42, week 43, week 44, week 45, week 46 or after week 46 of pregnancy.
  • Gestational windows may also include months of pregnancy. The typical term of a pregnancy is 9-10 months.
  • gestational windows may include from about month 1 to about month 10, from about month 2 to about month 10, from about month 3 to about month 10, from about month 4 to about month 10, from about month 5 to about month 10, from about month 6 to about month 10, from about month 7 to about month 10, from about month 8 to about month 10, from about month 9 to about month 10, from about month 1 to about month 9, from about month 2 to about month 9, from about month 3 to about month 9, from about month 4 to about month 9, from about month 5 to about month 9, from about month 6 to about month 9, from about month 7 to about month 9, from about month 8 to about mount 9, from about month 1 to about month 6, from about month 1 to about month 4, from about month 1 to about month 3, from about month 3 to about month 9, from about month 3 to about month 6, from about month 4 to about month 6, from about month 3 to about month 7, from about month 2 to about month 7 or from about month 2 to about month 6 of pregnancy.
  • gestational windows may include
  • gestational windows may include trimesters.
  • the term of a pregnancy may be divided into three trimesters.
  • the first trimester may include from about 1 month to about 3 months of pregnancy and/or from about week 1 to about week 12 of pregnancy.
  • typical development includes fetal growth to a weight of about 28 g (or about 1 ounce) and a length from about 7.6 cm to about 10 cm (or from about 3 to about 4 inches) long.
  • the second trimester may include from about 4 months to about 6 months of pregnancy and/or from about 13 weeks to about 28 weeks of pregnancy.
  • typical development includes fetal growth to a weight of about 910 g (or about 2 pounds) and length from about 23 cm to about 31 cm (or from about 9 inches to about 12 inches) long.
  • the third trimester may include from about 7 months to about 9 months of pregnancy and/or from about 29 to about 40 weeks of pregnancy.
  • typical development includes fetal growth to a weight of about 3.2 kg (or about 7 pounds) and length from about 45 cm to about 51 cm (or from about 18 to about 20 inches) long.
  • gestational windows may include stages of fetal development. Such stages may include, but are not limited to blastocyst formation, placental formation, embryo formation, heart development, lung development, liver development, kidney development, gastrointestinal development and nervous system development.
  • Analyses disclosed herein may include use of a single sample obtained from a subject. Such samples may include bodily fluid samples. Bodily fluid samples may include, but are not limited to blood, urine, mucous, amniotic fluid, saliva, lymph, sweat, and/or other sample types disclosed herein. Biomarker levels may be analyzed in a single subject sample or in multiple subject samples. GCD59 levels, for example, may be monitored over time. (0158) In some embodiments, values for the purposes of monitoring may include concentration values or concentration equivalent values. Monitoring is typically carried out by obtaining initial or baseline values by which subsequent values may be compared. During monitoring, one or more subsequent values may be obtained and compared to baseline values and/or any other previously obtained values.
  • Short-term comparisons may be used to monitor one or more biomarker levels in subject samples in response to a particular challenge (e.g., a glucose challenge) to the subject.
  • a particular challenge e.g., a glucose challenge
  • subject samples may be obtained every 10, 20, 30, 40, 50, 75
  • Subject samples for short-term comparisons may be obtained every 1, 2, 3, 4, 5, 10, 12 and/or 24 hours for the generation of subsequent values.
  • Such subject samples may include, but are not limited to, blood, urine, mucous, amniotic fluid, saliva, lymph, sweat, and/or any other bodily fluids.
  • Glucose levels and/or levels of glycated proteins may also be obtained from such samples.
  • levels of GCD59 (including, but not limited to concentration values) may be obtained from subject samples for short-term comparisons.
  • long-term comparisons may be used to monitor one or more biomarker levels/concentrations in subjects. Subsequent values obtained for long-term comparisons may be obtained each week, each month, each quarter, each year and/or at least each year. Long-term comparisons may include subsequent values obtained from subject samples obtained about 2 weeks to about 2 months apart. Such subject samples may include bodily fluids samples. Such bodily fluid samples may include, but are not limited to, blood, urine, mucous, amniotic fluid, saliva, lymph, sweat, and/or any other bodily fluid samples.
  • glucose levels may be obtained from samples for long-term comparisons.
  • levels of glycated proteins may be obtained from samples for long term comparisons.
  • GCD59 levels e.g. GCD59 concentration levels
  • observance, evaluation and/or measurement values may include, but are not limited to values reflecting weight, blood glucose levels, levels of glycated proteins (e.g., GCD59), biomarker levels, fetal weight, fetal size and BMI.
  • Monitoring of GDM may be carried out through repeated tests and/or observations. Baseline values may be obtained prior to pregnancy, upon a first prenatal medical examination or within a given interval of pregnancy.
  • Baseline values may, for example, be obtained from about 12 weeks to about 36 weeks of pregnancy, from about 20 weeks to about 36 weeks of pregnancy and/or from about 24 to about 28 weeks of pregnancy (including during week 24 of pregnancy, during week 25 of pregnancy, during week 26 of pregnancy, during week 27 of pregnancy and/or during week 28 of pregnancy).
  • Some baseline values may be concentration values. Such concentration values may be obtained from a variety of sources.
  • Baseline concentration values may also be obtained from bodily fluids including, but not limited to blood, urine, mucous, amniotic fluid, lymph, saliva and/or any other bodily fluid disclosed herein. Subject sample levels may be compared to baseline levels and/or previously obtained levels to determine changes in the level of one or more
  • subject sample levels are compared to a threshold value for one or more factors being analyzed.
  • baseline values may include the results of one or more tests used to evaluate one or more factors related to GDM.
  • tests may include, but are not limited to, glucose challenge testing (GCT,) the OGTT, the fasting glucose test, the opportunistic glucose test, the 2-hour postprandial glucose test, the HbAlc test, the fructosamine test and the 1,5-anhydroglucitol test.
  • one or more subsequent values may be obtained and compared to baseline values and/or any other previously obtained values. Some subsequent values may be obtained for the purposes of short-term comparisons, long-term comparisons, weekly comparisons, monthly comparisons, trimester comparisons, transpartum comparisons (e.g., comparisons between pre- and post-delivery), transgestational comparisons (e.g., comparisons between pre-, peri- and/or post-pregnancy) and interpregnancy comparisons (e.g., between a first pregnancy and a second, third and/or fourth pregnancy). Short-term comparisons may be used to monitor one or more biomarker levels in response to a particular challenge (e.g., a glucose challenge) to the subject.
  • a particular challenge e.g., a glucose challenge
  • Bodily fluid samples obtained for short term comparisons may be obtained every 10, 20, 30, 40, 50, 75 and/or 150 minutes and analyzed to generate subsequent values for comparison.
  • bodily fluids for short-term comparisons may be obtained every 1, 2, 3, 4, 5, 10, 12 and/or 24 hours for the generation of subsequent values.
  • Some bodily fluid samples for short-term comparisons may include blood, urine, mucous, amniotic fluid, lymph, saliva, sweat, and/or any other bodily fluid.
  • glucose levels may be obtained from such samples.
  • Levels of glycated proteins may also be obtained from bodily fluids for short-term comparisons. Such levels may include GCD59 levels.
  • long-term comparisons may be used to monitor one or more biomarker levels/concentrations in pregnant subjects. Subsequent values obtained for long term comparisons may be obtained each week, each month, each trimester, each pregnancy and/or in each of pre-gestational, peri-gestational and post-gestational periods. Some long term comparisons may include subsequent values obtained from subject samples obtained about 2 weeks to about 2 months apart. Some such subject samples may include bodily fluids samples. Such bodily fluid samples may include blood, urine, mucous, amniotic fluid, lymph, saliva and/or any other bodily fluids disclosed herein. In some cases, glucose levels may be obtained from samples for long-term comparisons. In other embodiments, levels of
  • GCD59 levels e.g., GCD59 concentration levels
  • GCD59 concentration levels may be obtained.
  • Monitoring may be carried out to observe the onset of one or more conditions and/or diseases. Some monitoring may be carried out to determine the onset of GDM and/or pre-eclampsia. In such embodiments, baseline values obtained may not indicate GDM and/or pre-eclampsia; however, subsequent values obtained may indicate onset. Onset may be determined by monitoring subject samples, including, but not limited to bodily fluids. Such bodily fluids may include blood, urine, mucous, amniotic fluid, lymph, saliva and/or any other bodily fluids disclosed herein. In some subject samples, glucose levels may be monitored to determine onset of GDM and/or pre-eclampsia.
  • levels of glycated proteins may be monitored to determine onset of GDM and/or pre-eclampsia.
  • GCD59 levels e.g., GCD59 concentration levels
  • GCD59 concentration levels may be monitored to determine onset of GDM and/or pre-eclampsia.
  • monitoring may be carried out to observe or assess the progression or regression of one or more conditions and/or diseases. Some monitoring may be carried out to observe or assess the progression or regression of GDM and/or pre eclampsia.
  • baseline values obtained may indicate GDM and/or pre eclampsia; however, subsequent values obtained may indicate progression or regression of disease.
  • Progression or regression may be assessed by monitoring subject samples, including, but not limited to bodily fluids. Such bodily fluids may include blood, urine, mucous, amniotic fluid, lymph, saliva and/or any other bodily fluids disclosed herein.
  • glucose levels may be monitored to assess progression or regression of GDM and/or pre-eclampsia.
  • levels of glycated proteins may be monitored to assess progression or regression of GDM and/or pre-eclampsia.
  • GCD59 levels e.g., GCD59 concentration levels
  • GCD59 concentration levels may be monitored to assess progression or regression of GDM and/or pre-eclampsia.
  • monitoring may be carried out to observe or assess the progression of a diabetic condition in a postpartum subject.
  • postpartum subject refers to a subject that has recently given birth. Postpartum subjects may include subjects that have given birth in about the last hour, about the last day, about the last month, about the last 3 months and/or about the last year.
  • methods of the present disclosure may be used to determine GCD59 levels in one or more samples obtained from postpartum subjects. Such GCD59 levels obtained from one or more
  • samples obtained from postpartum subjects may be used to diagnose, prognose or otherwise analyze one or more diabetic condition in such postpartum subjects.
  • evaluation of subject samples may be carried out in order to apply an appropriate form of therapy.
  • subject samples may be obtained from pregnant subjects with GDM.
  • Therapeutic strategies for GDM may include diet modulation, increased activity, increased exercise, periodic blood glucose monitoring and/or insulin therapy. Selecting one or more therapeutic strategies based on evaluation of samples from a pregnant subject and applying one or more of the selected therapeutic strategies to the pregnant subject may prevent GDM-related conditions that effect infant subjects born to such pregnant subjects. Samples obtained from such pregnant subjects may be evaluated for levels of one or more biomarkers in order to select one or more therapeutic strategies.
  • biomarkers may include glycated proteins, including, but not limited to GCD59.
  • GCD59 concentration values obtained from pregnant subject samples may be used to select one or more therapies for the treatment of GDM.
  • one or more GDM-related conditions in infant subjects bom to such pregnant subjects may be reduced, reversed and/or prevented.
  • methods of the present disclosure may be used to monitor subjects undergoing treatment for GDM. Such methods may include adjusting treatment dosages and/or types of therapy based on insights obtained from any of the types of monitoring described herein.
  • methods of the present disclosure may be used as companion diagnostics.
  • the term “companion diagnostic” refers to an assay, the results of which aid in the diagnosis or treatment of subjects. Companion diagnostics may be useful for stratifying patient disease, disorder or condition severity levels, allowing for modulation of treatment regimen and dose to reduce costs, shorten the duration of clinical trial, increase safety and/or increase effectiveness. Companion diagnostics may be used to predict the development of a disease, disorder or condition and aid in the prescription of preventative therapies. Some companion diagnostics may be used to select subjects for one or more clinical trials. In some cases, companion diagnostic assays may go hand-in-hand with a specific treatment to facilitate treatment optimization.
  • methods of the present disclosure may be useful as companion diagnostics for diseases, disorders and/or conditions related to glycemic levels. Some companion diagnostics of the present disclosure may be useful for predicting and/or
  • Some companion diagnostics of the present disclosure may be used to stratify subjects by risk of developing diabetic complications. Such diabetic complications may include, but are not limited to diabetic ketoacidosis, hyperglycemia, hypoglycemia, hyperglycemia hyperosmolar state, diabetic coma, infections of the respiratory tract, gum disease, heart damage, kidney damage, decreased sensation, vision loss, cardiovascular disease, muscle deterioration and stroke. Some companion diagnostics of the present disclosure may be used to facilitate and expedite drug development for anti-diabetic and metabolic disease drugs.
  • point-of-care testing refers to medical testing that is carried out at or near a site where a subject is receiving medical care. Point-of-care testing may facilitate shorter intervals between testing, review of test results and treatment. Point-of-care testing may also allow for patients to be tested and receive treatments determined by the results of such testing during the same day and/or during the same medical visit.
  • a “platform technology” refers to an instrument or system configured to standardize one or more steps of a method or assay. Platform technologies may be used, for example, to carry out immunological assays (e.g., ELISAs, immunoprecipitation assays, immunofluorescence assays, EIAs, RIAs, and Western blot analysis).
  • immunological assays e.g., ELISAs, immunoprecipitation assays, immunofluorescence assays, EIAs, RIAs, and Western blot analysis.
  • Platform technologies may include one or more commercially available instruments or systems used to carry out immunological assays, for example, one or more Bio-Rad (Hercules, CA) instruments (including, but not limited to an IMARKTM Microplate Absorbance Reader, an XMARKTM Microplate Absorbance Spectrophotometer, a PW 41 Microplate Washer, and/or an IMMUNOWASHTM 1575 Microplate Washer); one or more BioTek (Winooski, VT) instruments (including, but not limited to an ELX800TM Absorbance Reader, a SYNERGYTM HT Multi-mode Microplate Reader, an ELX50TM Microplate Strip Washer, a PRECISIONTM XS Automated Pipetting System, a MULTIFLOTM Automated Dispenser, an EL406TM Washer Dispenser, a MULTIFLOTM FX Multi-mode Dispenser, a
  • Thermo-Fisher (Waltham, MA) instruments (including, but not limited to a MULTISKANTM FC Microplate Photometer, a MULTISKANTM GO Microplate Spectrophotometer, a MEILTIDROPTM Combi Reagent Dispenser, a MULTIDROPTM 384 Reagent Dispenser, a MULTIDROPTM DW Reagent Dispenser, and/or a WELLWASHTM Microplate Washer); one or more Molecular Devices (Sunnyvale, CA) instruments (including, but not limited to a GENEPIX® 4300 A Microarray Scanner, a GENEPIX® 4400A Microarray Scanner, a GENEPIX® 4000B Microarray Scanner, a AQUAMAX® Micro
  • platform technologies include automated platform technologies.
  • Automated platform technologies are automated instruments or systems configured to carry out one or more steps of a method or assay in a standardized manner and independent of human manipulation, but may carry out such steps based on human input or guidance.
  • Automated platform technologies may include one or more commercially available systems used to carry out immunological assays, for example, one or more Bio-Rad (Hercules, CA) systems (including, but not limited to a BIO-PLEX® 200 System and/or a BIO-PLEX® 3D Suspension Array System); one or more Tecan (Mannedorf, Switzerland) systems (including, but not limited to a FREEDOM EVO® series system); a TRITURUS® system (Grifols, Los Angeles, CA); one or more Hamilton Laboratory Solutions (Manitowac, WI) systems (including, but not limited to a Microlab STARTM Line System, a Microlab NIMBUSTM system, and/or a Microlab VANTAGETM Liquid Handling System); one or more Abbott Laboratories (Abbott Park, IL) systems (including, but not limited to an ARCHITECTTM i2000SR immunoassay analyzer, an ARCHITECTTM ilOOOSR immunoassay analyzer, and/or
  • DIMENSION® EXLTM systems DIMENSION VISTA® systems, DIMENSION® XPAND® Plus systems, DIMENSION® RXL MAX® systems
  • AD VIA Centaur XPT Immunoassay systems AD VIA Centaur XP Immunoassay systems, AD VIA Centaur CP Immunoassay systems, IMMULITE 2000 XPi Immunoassay systems, IMMULITE 1000 Immunoassay systems, VersaCell systems, and/or VersaCell X3 systems
  • Luminex Austin, TX
  • Luminex including, but not limited to a MAGPIX® System and/or a LUMINEX® 100/200TM System.
  • platform technologies used to carry out immunological assays may include one or modules capable of performing one or more desired steps. Such modules may be configured to carry out one or more of sample distribution, reagent addition and/or removal, incubation, mixing, signal detection, signal analysis, analyte quantification, rinsing, and sample and/or substrate disposal.
  • the compounds or compositions of the present disclosure can be formulated using one or more excipients to: (1) increase stability; (2) increase cell permeability; (3) permit the sustained or delayed release (e.g., from a formulation); and/or (4) alter the biodistribution (e.g., target the composition to specific tissues or cell types).
  • formulations of the present disclosure can include, without limitation, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the compounds or compositions of the present disclosure (e.g., for transplantation into a subject) and combinations thereof.
  • excipient refers to any substance combined with a compound and/or composition of the present disclosure before use. In some embodiments, excipients are inactive and used primarily as a carrier, diluent or vehicle for a compound and/or composition of the present disclosure.
  • excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21 st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference).
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients.
  • a pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered.
  • a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
  • an excipient is approved for use in humans and for veterinary use.
  • an excipient is approved by United States Food and Drug Administration.
  • an excipient is pharmaceutical grade.
  • an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical compositions.
  • Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc ., and/or combinations thereof.
  • Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation- exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinyl pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM ® ), sodium lauryl sulfate, quaternary ammonium compounds, etc ., and/or combinations thereof.
  • crospovidone cross-linked polyvinyl pyrrolidone
  • Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGEIM ® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g.
  • natural emulsifiers e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin
  • colloidal clays e.g. bentonite [aluminum si
  • stearyl alcohol cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g.
  • polyoxyethylene monostearate [MYRJ ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL' * ), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR ® ), polyoxyethylene ethers, (e.g.
  • polyoxyethylene lauryl ether [BRIJ ® 30]), poly(vinyl-pyrrolidone), di ethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC l F 68, POLOXAMER ® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.
  • Exemplary binding agents include, but are not limited to, starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish
  • Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives.
  • Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabi sulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabi sulfite, and/or sodium sulfite.
  • Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • EDTA ethylenediaminetetraacetic acid
  • citric acid monohydrate disodium edetate
  • dipotassium edetate dipotassium edetate
  • edetic acid fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chi oroxy lend, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal.
  • Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid.
  • Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol.
  • Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid.
  • preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabi sulfite, potassium sulfite, potassium metabi sulfite, GLYDANT PLUS ⁇ PHENONIP ® , methylparaben, GERMALL ® 115, GERMABEN ® II, NEOLONE TM , KATHON TM , and/or EUXYL ® .
  • Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,
  • Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc. , and combinations thereof.
  • oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl my ri state, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana
  • oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyl dodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.
  • Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
  • compounds or compositions of the present disclosure may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents.
  • Compounds or compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • the present disclosure encompasses the delivery of pharmaceutical, prophylactic, diagnostic, and/or imaging compositions in combination with agents that may improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
  • the present disclosure provides methods for diagnosing, monitoring, and/or screening subjects for one or more diabetes-related indications (e.g., GDM).
  • Such methods may include GCD59 detection in samples obtained from subjects being evaluated.
  • GCD59 detection may include one or more of: (1) obtaining subject samples; (2) applying subject samples to substrates having capture antibodies; (3) applying detection antibodies; and (4) detecting the detection antibodies.
  • Substrates may be prepared by immobilizing capture antibodies on the substrates. Capture antibodies may be immobilized through association between biotin and biotin-binding agents. Capture antibodies may be biotinylated. Substrates may have biotin-binding agents. Such biotin-binding agents may include streptavidin.
  • capture antibodies bind to CD59 and detection antibodies bind only to glycated CD59 (e.g., K41-glycated CD59, reduced and/or Amadori- modified form). In some embodiments, capture antibodies bind only to glycated CD59 (e.g., K41 -glycated CD59, reduced and/or Amadori -modified form) and detection antibodies bind to CD59.
  • Subject samples may include, but are not limited to, cells, tissues, tissue sections, and body fluids (e.g., urine, blood, sweat, serum, plasma, lymph, and saliva).
  • samples are used without pretreatment.
  • samples may be diluted prior to analysis.
  • Such dilutions may be at least 10: 1, at least 5:1, at least 2:1, at least 1:1, at least 1:10, at least 1:100, at least 1:1,000, at least 1:10,000, at least 1:100,000, at least 1:1,000,000, at least 1 : 10 9 , at least 1 : 10 12 , or at least 1 : 10 15 .
  • samples are diluted from about 1:200 to about 1:300.
  • samples are treated with reducing agents, oxidizing agents, dithiothreitol (DTT), and/or beta- mercaptoethanol (BME) prior to analysis.
  • DTT dithiothreitol
  • BME beta- mercaptoethanol
  • reducing agent refers to any electron donor chemical or compound involved in or used for oxidation-reduction reactions.
  • reducing agents are selected from sodium borohydride (NaBEB) and sodium cyanoborohydride (NaCNBEb).
  • Sample pretreatment with reducing agents may be conducted to modify glycated side chain structure, e.g., for detection agent recognition of GCD59 with reduced K41.
  • NaCNBEb treatment reduces Schiff s base glycated K41 to glucitollysine.
  • NaBH4 treatment reduces both Schiff s base glycated K41 and Amadori -modified glycated K41 to glucitollysine.
  • Reducing agents may be used alone or in solution (referred to herein as “reducing agent solution”).
  • Reducing agent solutions may include any of a variety of solvents.
  • reducing agent solutions include water.
  • reducing agent solutions include organic solvents.
  • Organic solvents are solvents that include carbon-based components.
  • organic solvents may include, but are not limited to, 1,1- dichloroethane, 1,2-dichloroethane, 1,2-dimethoxy-ethane, 1 -butanol, 1-heptanol, 1-hexanol, 1-octanol, 1-pentanol, 1 -propanol, 2-aminoethanol, 2-butanol, 2-butanone, bis(2- m ethoxy ethyl) ether, 2-pentanol, 2-pentanone, 2-propanol, 3-pentanol, 3-pentanone, acetic acid, acetone, acetonitrile, acetyl acetone, aniline, anisole, benzene, benzonitrile, benzyl alcohol, carbon disulfide, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, cyclohexanol, cyclohex
  • reducing agent solutions may include triethylene glycol dimethyl ether, bis(2-methoxy ethyl) ether and/or tetraglyme solvents.
  • Reducing agent solutions with organic solvents may include NaBEB solutions.
  • Such solutions may include or be prepared from commercially available preparations (for
  • Such commercial preparations may include, but are not limited to, 99% Sodium Borohydride Solution 0.5 M in bis(2-methoxy ethyl) ether; 99% Sodium Borohydride Solution 3 M in tetraglyme ether; 99% Sodium Borohydride Solution 2.0 M in triethylene glycol dimethyl ether; 99.5% Sodium Borohydride Solution 0.5 M in bis(2-methoxy ethyl) ether; 99.5% Sodium Borohydride Solution 3 M in tetraglyme ether; 99.5% Sodium Borohydride Solution 2.0 M in triethylene glycol dimethyl ether; 99.9% Sodium Borohydride Solution 3 M in tetraglyme ether; 99.9% Sodium Borohydride Solution 2.0 M in triethylene glycol dimethyl ether; 99.95% Sodium Borohydride Solution 0.5 M in bis(2-methoxy ethyl) ether; 99.95% Sodium Borohydride Solution 0.5 M in bis(2-methoxy ethyl
  • Reducing agent solutions may include reducing agent concentrations of from about 0.1 M to about 10 M.
  • stock solutions of reducing agent solutions are used that require dilution prior to use.
  • Reducing agent stock solutions may include greater than 1 M reducing agent concentrations (e.g., from about 1.1 to about 1.5 M, from about 1.2 M to about 2 M, from about 1.7 M to about 3 M, from about 2.5 M to about 4 M, from about 3.5 M to about 5 M, from about 4.5 M to about 8 M, or from about 6 M to about 10 M).
  • Reducing agent stock solutions may be diluted to from about 0.5 M to about 1 M reducing agent concentrations prior to sample treatment. Dilution may be carried out using aqueous and/or organic solvents.
  • the anti-Amadori-modified GCD59 detection antibodies used in methods of the disclosure may include detectable labels.
  • detectable labels may include, but are not limited to biotin, streptavidin, avidin, fluorescent labels, enzymatic labels, luminescent labels, and radioactive labels.
  • anti-Amadori-modified GCD59 detection antibodies may be detected using a secondary antibody.
  • 1 M reducing agent solutions may be combined with subject samples at a ratio of from about 1 : 1 (sample reducing agent solution) to about 1 : 1000.
  • the ratio may be 1:5, 1:10, 1:20, 1:100 or 1:500.
  • Samples may be incubated with reducing agent solution for from about 20 minutes to about 2 hours, from about 1 hour
  • microtiter plates are used.
  • methods of the disclosure may be used for monitoring, detecting, and/or screening for diabetes in subjects. In some embodiments, methods of the disclosure may be used for assessing and/or stratifying risk in diabetic subjects. Some methods may be used as part of a companion diagnostic for diabetes-related indications. 10203
  • Diabetes-related indication risk levels may include a range of levels that correlate with subject GCD59 concentration. Levels may be based on GCD59 concentrations or concentration equivalents (e.g., where concentration is determined in relation to a GCD59 surrogate compound) known to be associated with disease, disease risk, disease outcome, or disease severity. The levels may be determined according to any of the methods described herein. Such methods may include ELISA assays utilizing anti-GCD59 antibodies as capture or detection antibodies.
  • Methods of assigning diabetes-related indication risk levels to subjects may include one or more of: (1) determining GCD59 concentration or concentration equivalent in subject samples; (2) comparing the GCD59 concentration or concentration equivalent obtained to that associated with at least one control sample or known threshold; and assigning a diabetes-related indication risk levels to subjects based on variation between subject samples and control samples or known threshold.
  • Diabetes-related indication risk levels assigned may include risk levels associated with developing one or more diabetes-related indications (e.g., GDM). All or part of such methods may be carried out at the point of care. All or part of such methods may be carried out in conjunction with patient monitoring devices.
  • methods of the disclosure may be used for drug development. Such embodiments may include testing and/or developing therapeutics for diabetes-related indications. In some cases, methods of the disclosure may be used to conduct clinical trials.
  • methods of the disclosure include remote monitoring of subjects. Such methods may incorporate all or part of other methods taught herein.
  • methods of the present disclosure include methods of assessing GCD59 for the presence of at least one N-glycosylation.
  • plasma, serum, and/or urine may be assessed to identify the organ/tissue most affected by glycation and therefore most susceptible for vascular complications of diabetes.
  • detection of kidney-specific N-glycosylated GCD59, the predominant form of GCD59 in blood and urine samples of pre diabetic or diabetic patients may indicate patient susceptibility for development of diabetes nephropathy. In some cases, this detection may be carried out before the onset of actual kidney damage that may otherwise go undetected with current diagnostic methods. Such early detection may be used to raise patient awareness and indicate the need for life style changes. In some cases, early detection may direct a physician to initiate early preventive therapeutic intervention.
  • the present disclosure provides methods of assessing GCD59 that include the steps of isolating GCD59 with an antibody capable of binding K41 Amadori-modified GCD59 and detecting at least one N-glycosylation present on GCD59 (e.g., N-glycosylation on residue N18 of GCD59).
  • Methods of the present disclosure may include methods of identifying at least one of: (1) predisposition to an organ specific complication of diabetes; (2) pre-clinical diabetic peripheral neuropathy; (3) pre-clinical diabetic nephropathy; (4) pre-clinical diabetic retinopathy; and (5) pre-clinical diabetic vascular disease.
  • Such methods may include the steps of: (1) isolating GCD59 with an antibody specific for K41 Amadori-modified GCD59 and (2) detecting at least one N-glycosylation present on GCD59 (e.g., N-glycosylation on residue N18 of GCD59).
  • Detection of GCD59 N-glycosylation may be carried out by any methods known in the art. In some cases, GCD59 N-glycosylation is detected using immunological methods. In other embodiments, GCD59 N-glycosylation may be detected using mass spectrometry.
  • methods of the present disclosure include methods of screening, diagnosing, and/or monitoring one or more diabetes related indications in a subject by determining GCD59 concentration or concentration equivalent in a subject sample and comparing the concentration or concentration equivalent to that of a control sample or known threshold.
  • the GCD59 concentration or concentration equivalent may be determined according to a method utilizing immobilized capture antibodies directed to a glycated epitope of GCD59. Such methods may include any of those described herein.
  • the one or more diabetes-related indications may include GDM.
  • Kits and methods (including all or part of such methods) disclosed may be formatted for point of care use.
  • point of care use may include the use of one or more patient monitoring device.
  • patient monitoring devices may include a device capable of receiving and/or transmitting an electronic signal.
  • patient monitoring devices may be or be combined with smart devices (e.g., a smart phone or smart watch).
  • the present disclosure provides CD59 glycation inhibitors and methods of developing such inhibitors.
  • Human CD59 is a major complement regulator and its normal function may be reduced or blocked by glycation which can occur under hyperglycemic conditions.
  • hCD59 contains a putative glycation motif that includes residues K41 and H44. These residues fall within the proximity of W40 considered to be an integral part of the active site of the protein.
  • Glycation of K41 requires H44 to provide acid-base catalysis for the chemical reaction between glucose and the epsilon amino group of K41.
  • Glycation of hCD59 at K41 may increase the risk of developing diabetes-related indications, including, but not limited to vascular diabetes.
  • Methods of the present disclosure may include methods of inhibiting CD59 glycation.
  • agents may be administered that target CD59 and block K41 glycation.
  • Such agents may target, for example, H44 of human CD59 and block glycation of K41.
  • anti-Amadori-modified GCD59 antibodies may be used in screening procedures to identify agents targeting CD59 and blocking K41 glycation.
  • Methods of the present disclosure may include methods of developing inhibitors of hCD59 K41 glycation. Such methods may include the use of one or more antibodies capable of binding K41 -glycated CD59, including, but not limited to any of the anti-K41 Amadori-
  • such methods include the steps of: (1) contacting a sample with at least one test compound, wherein the sample includes hCD59 (purified or recombinant) and conditions suitable for K41 gly cation and (2) evaluating the level of K41 -glycated hCD59 in the sample using a GCD59 detection antibody.
  • the evaluation may be carried out according to any of the methods described herein or previously in any of U.S. Patent 6,835,545; U.S. Patent 7,049,082; U.S. Patent 7,439,330; U.S. Patent 9,068,006; U.S. Patent 9,417,248; and U.S. Publication No.
  • test compounds may include, but are not limited to small molecules, peptides, synthetic constructs, fusion proteins, aptamers, nucleic acids, and antibodies.
  • kits for determining the concentration of GCD59 in a subject sample may include one or more of (1) a capture antibody, (2) a detection antibody, and (3) instructions for use.
  • Some kits include compounds for the generation of a standard curve.
  • Such compounds may include standard agents (e.g., surrogate compounds).
  • surrogate compounds may include any of those disclosed in U.S. Patent No. 9,417,248 (e.g., the surrogate compound presented in Figure 27, therein) or U.S. Publication No. US2016/0299150, the contents of each of which are herein incorporated by reference in their entirety.
  • kits include at least one buffer. Such buffers may be used to contact subject samples.
  • Buffers may include reducing agents, oxidizing agents, dithiothreitol (DTT), and/or beta-mercaptoethanol (BME).
  • Reducing agents may be provided as part of a reducing agent solution.
  • Reducing agent solutions may include water and/or organic solvent.
  • the organic solvent may include bis(2- methoxy ethyl) ether.
  • Kits of the disclosure may be used for detecting the presence, level, and/or changes in GCD59 levels.
  • Such kits may include one or more capture antibody to capture CD59 and/or GCD59.
  • Capture antibodies may include any of the antibodies described herein (e.g., antibody H9, encoded by SEQ ID NOs: 9 and 10; or antibodies D2 or D3, encoded by SEQ ID NOs: 6 and 7 for D2 or SEQ ID NOs: 6 and 8 for D3).
  • Additional kits may include one or
  • kits may include other antibodies known in the art for capturing or detecting CD59 and/or GCD59.
  • Such antibodies may include any of those described and/or claimed in European Patent Number EP2348050, International Publication Number WO20 15084994, US Patent Number US9068006, US9417248, or US 10732186, the contents of each of which are herein incorporated by reference in their entireties.
  • Kits of the disclosure may include one or more internal controls. Such internal controls may include plasma assay controls and/or controls based on any other bodily fluids. Some kits include one or more of an assay diluent, a conjugate diluent, and a wash buffer. Kit reagents may be lyophilized.
  • kits may be used to determine the presence, absence, or quantity of glycosylated protein epitope.
  • glycosylated protein epitopes may include glycosylated CD59 epitopes.
  • N-glycosylated CD59 and/or GCD59 may be detected.
  • kits may be used, for example, to detect cell and/or tissue-specific N- glycosylated epitopes.
  • Kits may include any of those described and/or claimed in European Patent Number EP2348050, International Publication Number WO2015084994, and US Patent Numbers US9068006, US9417248, and US10732186, the contents of each of which are herein incorporated by reference in their entireties.
  • Such kits may have one or more antibody described herein as a substitute for one or more of the antibodies described in such kits.
  • such kits may include an anti-Amadori-modified GCD59 antibody described herein in place of a detection antibody described therein.
  • These kits may be assembled without a reducing agent and may be used for detection and/or quantitation of GCD59 in samples without the need for carrying out a sample reducing step.
  • the present disclosure provides a kit that includes one or more of: (1) a substrate; (2) a capture antibody directed to a glycated epitope of GCD59; (3) a detection antibody directed to a non-glycated epitope of GCD59; and (4) instructions for use of the kit.
  • Glycated epitopes may include glycated K41 of GCD59.
  • the glycated K41 may be Amadori-modified K41.
  • Capture antibodies may be antibody fragments (e.g., FAb fragments).
  • the capture antibody may be antibody D2 or a fragment of D2 (e.g., an FAb fragment).
  • Capture antibodies may be immobilized on substrates. Immobilization may be by covalent or non-covalent bonding.
  • Non-glycated epitopes may include regions of GCD59 that
  • Detection antibodies may be antibody fragments (e.g., FAb fragments). Detection antibodies may be antibody H9 or a fragment of H9 (e.g., an FAb fragment). Detection antibodies may include detectable labels or may be detected using secondary detection agents that include detectable labels. Detectable labels may include, but are not limited to, biotin, biotin-binding agents, fluorescent labels, enzymatic labels, luminescent labels, and radioactive labels. Enzymatic labels may include peroxidase labels (e.g., horse radish peroxidase). Substrates may include, but are not limited to, assay surfaces, assay plates, beads, membranes, conducting surfaces, and conducting nanoparticles.
  • Substrates may include biotin-binding agents.
  • Biotin-binding agents may include, but are not limited to, avidin, streptavidin, NeutrAvidin, and CAPTAVIDINTM.
  • Capture antibodies may be biotinylated to facilitate substrate immobilization.
  • Substrates may be provided with capture antibodies immobilized through association between antibody biotin and substrate-associated biotin-binding agents.
  • Kits may further include one or more control samples (e.g., plasma assay controls).
  • Kits may further include one or more buffers. Such buffers may include, but are not limited to, assay diluents, conjugate diluents, and wash buffers. Kits may further include compounds for calibration curve generation.
  • Such compounds may include: (1) a first peptide that includes a glycated epitope of GCD59; (2) a second peptide having a non- glycated epitope of GCD59; and (3) a linker joining the first peptide to the second peptide.
  • the present disclosure provides devices. Such devices may be used to carry out methods disclosed herein or may be used in combination or coordination with such methods or kits or kit components disclosed herein.
  • Devices may include patient monitoring devices.
  • Patient monitoring devices may be used to monitor one or more patient characteristics. Such characteristics may include patient vital signs and/or biomarker levels. Such biomarkers may include, but are not limited to, glucose, insulin, and GCD59.
  • patient monitoring devices are wearable, meaning that they are configured for attachment to a patient, a patient garment, or other patient accessory through use of an adhesive, tape, strap, band, clip, magnet, or other form of attachment.
  • Some patient monitoring devices may be capable of receiving or transmitting electronic signals.
  • patient monitoring devices may include smart devices, including, but not limited to, smart phones or smart watches.
  • the term “detectable label” refers to an observable marker, signal, or moiety that is attached, incorporated, or otherwise associated with an entity.
  • Detectable labels may include, but are not limited to, radioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands, biotin, avidin, streptavidin, haptens, quantum dots, polyhistidine tags, myc tags, flag tags, human influenza hemagglutinin (HA) tags, and the like.
  • Detectable labels may be located at any position in or on the entity with which they are attached, incorporated, or associated. For example, when attached, incorporated in, or associated with a peptide or protein, they may be within the amino acids, the peptides, or proteins, or located at the N- or C-termini.
  • Detectable labels may be observed or quantified through observation of associated detection signals.
  • the term “detection signal” refers to any transmission or activity that facilitates direct or indirect observation of an object. Observation may include any form of sensing the presence of a detectable label.
  • technologies for observing detectable labels may include, but are not limited to, radiography, fluorescence detection, chemiluminescence detection, spectrophotometry, immunological detection, enzymatic activity detection, and the like.
  • Detection signals may include, but are not limited to, visual signals, audio signals, radioactive signals, electronic signals, thermal signals, magnetic signals, electromagnetic signals, and chemical signals. Visual signals may include light that is emitted or reflected off of a detectable label.
  • the light may be fluorescent light emitted by excitation of a detectable label fluorophore.
  • Visual signals may include luminescent light (e.g., light given off by chemiluminescent reaction).
  • Visual signals may include changes in light absorbance detected by spectrophotometry that are associated with a detectable label.
  • detectable labels that include enzymes may be detected and/or quantified by exposure to enzyme substrate.
  • Resulting detectable signals may be in the form of enzymatic product accumulation.
  • Detection and/or quantification of accumulated product may be carried out by detecting and/or measuring changes in absorbance of light passed through an area surrounding the detectable labels (e.g., by spectrophotometry). Measuring detectable label levels or levels of associated analytes may be carried out by measuring the strength or intensity of associated detection signals.
  • epitope refers to a region or surface on a molecule that is capable of interacting with one or more components of the immune system, including, but not limited to antibodies.
  • epitopes may include linear stretches of
  • Epitopes may include post-translational protein modifications (e.g., glycation moieties, lipid moieties, glycosylation moieties, etc.).
  • monitoring refers to the act of observing, evaluating, and/or measuring over time. Observing, evaluating, and/or measuring may be recorded in the form of one or more amounts or values. Monitoring may include tracking one or more factors, analytes, or indicators of a disease, disorder, or condition over time to evaluate progression or regression.
  • patient monitoring device refers to any tool used to evaluate one or more patient characteristics over time. Such characteristics may include biomarker levels (e.g., insulin, blood sugar, or GCD59 levels).
  • patient monitoring devices are wearable, meaning that they are configured for attachment to a patient, a patient garment, or other patient accessory through use of an adhesive, tape, strap, band, clip, magnet, or other form of attachment. Some patient monitoring devices may be capable of receiving or transmitting electronic signals.
  • patient monitoring devices may include smart devices, including, but not limited to, smart phones or smart watches.
  • ROC curve refers to a statistical tool that generates a graphical plot to illustrate the performance of a binary classifier system as its discrimination cut-off (threshold) is varied.
  • An ROC curve may serve as a tool for diagnostic test evaluation and may be used to quantify how accurately a medical diagnostic test can discriminate between two patient states.
  • a ROC curve may be used to demonstrate a tradeoff between sensitivity and specificity in an analysis. Analysis of an ROC curve, employing familiar statistical methods, may be used to identify an optimal diagnostic cut-off for the level of a biomarker. The optimal diagnostic cut-off value may then be used to make a medical decision.
  • sample refers to a portion or aliquot obtained or derived from a source (e.g., a biological source). Samples may be used for analyses intended to characterize the source or otherwise provide information about the source. Samples may be of any phase (e.g., solid, fluid, or gas). Samples may be biological samples.
  • biological sample refers to any sample obtained or derived from one or more living organisms. Living organisms may include, but are not limited to, prokaryotic organisms, eukaryotic organisms, plants, animals, mammals, and/or humans. In some embodiments, samples may include histological or cytological specimens, tissues, bodily fluids, biopsies,
  • Body fluids samples may include, but are not limited to, blood, plasma, urine, mucous, amniotic fluid, lymph, saliva, sweat, lymphatic fluid, synovial fluid, cerebrospinal fluid, amniotic cord blood, vaginal fluid and semen).
  • Some samples may be homogenates, lysates, or extracts prepared from whole organisms or subsets of organism tissues, cells, component parts, or fractions or portion thereof.
  • homogenates, lysates, or extracts include, but are not limited to, plasma, serum, spinal fluid, lymph fluid, external skin sections, respiratory samples, intestinal samples, genitourinary tracts, tears, saliva, milk, blood cells, tumors, or organs.
  • Some samples may include media, such as a nutrient broth or gel, which contains cellular components (e.g., proteins or nucleic acid molecules).
  • screening refers to the evaluation of one or more entities or subjects for one or more properties, characteristics, or conditions. Screening may include evaluating one or more subjects for disease, disorders, or conditions, or for one or more factors or analytes indicating disease, disorder, or condition risk.
  • threshold value refers to a value used to divide continuous results into categories (e.g., positive and negative) for purposes of characterizing an agent used to generate the results, to determine the placement of an agent into one or more such categories, or to segregate subjects into categories based on a corresponding value (falling within the range of continuous results) associated with one or more such subjects.
  • a threshold value may be a level of a biomarker or other indicator used to categorize one or more subjects with biomarker or other indicator levels falling above, falling below, or equal to the threshold value.
  • a threshold value may be a concentration of a factor present in a subject or subject sample that is used to divide subjects into disease categories.
  • the threshold value is a level of GCD59 in a subject
  • the threshold value may be used to assign subjects into diabetic or non-diabetic categories based on whether GCD59 levels in such subjects fall above or below the threshold value, respectively.
  • a threshold value may be used to make a medical decision based on whether values associated with one or more patients fall above, fall below, or are equal to the threshold value.
  • a diagnostic threshold value also referred to as a “cut-off point” for a biomarker that yields continuous real values may be used to delineate tests positives and test negatives.
  • a training set of blood plasma samples may be used to generate a Receiver Operating Characteristic (ROC) curve that is a plot of the true positive rate against the false positive rate for different possible cut-off points.
  • ROC Receiver Operating Characteristic
  • the optimal diagnostic cut-off value may be used to arrive at a medical decision.
  • articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the invention includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.
  • compositions of the invention e.g., any nucleic acid or protein encoded thereby; any method of production; any method of use; etc.
  • compositions of the invention can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.
  • Antibodies to CD59 and K41 Amadori-modified GCD59 were each generated using CD59 fragments and their analogs.
  • CD59 fragments and their analogs having Amadori-modified K41 were used.
  • Antibodies were prepared by mouse immunization and development of hybridoma cells from animals exhibiting successful expression of antibodies with high affinity and specificity.
  • Clone H9 was developed as a capture antibody, binding to both glycated and non-glycated CD59.
  • Clones D2 and D3 were developed as detection antibodies, binding to K41 Amadori- modified GCD59.
  • Antibodies were sequenced and analyzed to identify antibody regions. Resulting sequences are provided in Tables 1-7.
  • Antibodies D2 and D3 were found to have heavy and light chains with identical amino acid sequences, but with heavy chain nucleotide sequences differing by a single nucleotide.
  • GCD59 detection in urine samples was carried out using detection antibody D2, described above, specific for Amadori-modified GCD59. Assay plates were first coated with CD59 capture antibody, H9. Urine samples were obtained from two subjects, one known to have a high level of HbAlC (HbAlC-H) and one known to have a low level of HbAlC (HbACl-L). High levels of HbAlC are a known indicator of long-term elevated blood glucose. Samples were prepared by first adding beta-mercaptoethanol (BME) to a final concentration of 5%. Samples were then boiled at 100°C for 5 min and centrifuged to pellet any debris. Supernatants were then isolated for the assay.
  • BME beta-mercaptoethanol
  • Amadori-modified surrogate compound standard as shown in Fig. 27 of US Patent No. 9,417,248, was used to prepare a dilution series for generating a calibration curve.
  • the Amadori-modified GCD59 surrogate included synthetic compounds comprised of capture and detection peptide antigens linked by a flexible linker.
  • Urine samples serially diluted surrogate compound standard samples, as well as control plasma samples [plasma dilution buffer (50 mM Acetic acid/Na acetate; 2% Nonldet P40 Substitute; 0.15% Tween 20; 0.1% Triton X-100; 50 ppm ProClin 300) only] were added
  • Urine samples were further diluted using plasma dilution buffer to create a series of conditions ranging from 6% v/v urine sample to 0.2% urine sample in plasma dilution buffer.
  • Assay plates were placed on a shaker for one hour at room temperature. Plates were then washed with wash buffer [phosphate buffered saline (PBS) with 0.05% TWEEN®-20] before the addition of 100 m ⁇ of antibody D2 solution [1.25 pg/ml in PBS with 10% protein-free blocking buffer (Thermo Fisher, Waltham, MA)]. Plates were then incubated for 2 hours at room temperature with shaking.
  • wash buffer phosphate buffered saline (PBS) with 0.05% TWEEN®-20
  • FIG. 1 A A calibration curve (Fig. 1 A) was generated based on wells using the surrogate compound standard. Results from urine samples were converted into standard protein units (SPU) based on the calibration curve and plotted against percent urine values from each of the samples (Fig. IB). Values obtained from HbAlC-H samples indicated concentration dependent antibody D2 binding with around 2.5 SPU in high concentration samples. HbAlC- L sample values were low indicating little to no binding of antibody D2. These results demonstrate the ability of H9 to capture glycated and non-glycated forms of CD59 and D2 to detect Amadori-modified GCD59 in biofluid samples using an ELISA format in a concentration-dependent manner.
  • SPU standard protein units
  • the protein samples were separated using SDS-PAGE (Sodium Dodecyl sulfate- Polyacrylamide gel electrophoresis) and immunoblotted with anti-Amadori modified hGCD59 antibody (D2) and anti hCD59 antibody (Fig. 2). Consistent with the elevation of glycated CD59 in diabetes, the anti-
  • Amadori modified hGCD59 antibody showed an intense band in the diabetic mice and only a faint band in the control non-diabetic mice. Both the diabetic and control non-diabetic mice showed similar levels of hCD59, demonstrating the specificity of the anti-Amadori modified hGCD59 antibody.
  • a sandwich-type enzyme-linked immunosorbent assay is carried out using an anti-GCD59 capture antibody, wherein the anti-GCD59 antibody is specific for an epitope of mature human CD59 that includes an Amadori-modified glycated lysine residue at residue K41 (e.g., according to antibody D2 or D3 described herein).
  • An anti-human CD59 antibody is used as a detection antibody, wherein the detection antibody binds equally to glycated and non-glycated CD59 formats and to an epitope that is non-overlapping with the capture antibody (e.g., according to antibody H9 described herein).
  • Capture antibodies are biotinylated and immobilized on ELISA assay plates coated with NeutraAvidin or other biotin-binding agent.
  • a surrogate compound (as shown in Fig. 27 of US Patent No.
  • the surrogate compound includes a capture antigen that includes the Amadori-modified K41- glycated GCD59 epitope (recognized by the capture antibody) and a detection peptide antigen that includes the detection antibody epitope, with the two antigens linked by a flexible linker.
  • Wells coated with capture antibody are incubated with the biological samples before washing with wash buffer [phosphate buffered saline (PBS) with 0.05% TWEEN®-20] and treatment with horseradish peroxidase (HRP) labeled detection antibody.
  • Bound detection antibody is detected using TMB substrate reagents A and B (Thermo Fisher Scientific, Waltham, MA).
  • the ensuing colorimetric detection reaction generates a colored product, which is quenched after a short time by treating plates with H2SO4 solution.
  • Optical density values at 450 nm are obtained spectrophotometrically for each well.
  • Optical density values obtained with the surrogate compound dilution series are used to prepare a calibration curve.
  • GCD59 concentrations for biological samples are calculated by extrapolation using the calibration curve. Results are used to identify subjects associated with biological samples having elevated GCD59 concentration and with or at risk for diabetes-related indications.
  • Example 5 ELISA assay with anti-Amadori-modified GCD59 capture antibody [0247] Sandwich-type ELISA assays were carried out using anti-Amadori-modified GCD59 antibody D2 for capture and H9 antibody for detection. D2 antibody, highly specific
  • 86 to the K41-Amadori-modified hCD59 was immobilized on ELISA assay plates.
  • Human plasma samples from three different subjects were diluted with sample dilution buffer and added to the prepared assay plates to promote capture of GCD59 based on D2 affinity for the Amadori-modified form of K41 -glycated CD59.
  • a surrogate compound standard as shown in Fig. 27 of US Patent No. 9,417,248, was used to prepare a dilution series for generating a calibration curve.
  • the Amadori-modified GCD59 surrogate/calibrator was comprised of the K41 -Amadori-modified capture antigen and detection peptide antigen linked by a flexible linker.
  • Wells coated with the capture antibody D2 were incubated with human plasma samples followed by washing them with wash buffer [phosphate buffered saline (PBS) with 0.05% TWEEN®-20] and treatment with a solution of biotinylated detection antibody H9.
  • wash buffer phosphate buffered saline (PBS) with 0.05% TWEEN®-20
  • H9 binds to both glycated and non-glycated forms of CD59 and is specific for an epitope that does not overlap with the epitope recognized by antibody D2.
  • Wells were again washed followed by treatment with streptavidin-labeled horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • Bound biotinylated H9 detection antibody was detected using TMB substrate reagents A and B (Thermo Fisher Scientific, Waltham, MA).
  • the ensuing colorimetric detection reaction generated a colored product, which was quenched after a short time by treating the plates with H2SO4 solution.
  • Optical density values at 450 nm were obtained spectrophotometrically for each well.
  • Optical density values obtained with the surrogate compound dilution series were used to prepare a calibration curve.

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Abstract

La divulgation concerne des méthodes de détection de CD59 glyqués dans des échantillons à l'aide d'anticorps de capture et de détection sur des épitopes de CD59 glyqués et non glyqués, respectivement, suivie de la détection de complexes résultants. La divulgation concerne également des méthodes associées de sélection, de surveillance et de diagnostic de sujets présentant des indications liées au diabète et des kits pour la mise en œuvre de telles méthodes.
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